KR20130116858A - Extraction solvents derived from oil for alcohol removal in extractive fermentation - Google Patents

Abstract

In alcohol fermentation processes, oils derived from biomass are chemically converted into extractants available for in situ collection of product alcohols such as butanol from fermentation broth. Glycerides in oils may be chemically converted into reaction products such as fatty acids, fatty alcohols, fatty amides, fatty acid methyl esters, fatty acid glycol esters, and hydroxylated triglycerides, and mixtures thereof, which may be used in biomass oils. A fermentation product extractant is formed having a partition coefficient for the product alcohol that is greater than the partition coefficient for the product alcohol. The oil from the feedstock of the alcoholic fermentation process can be chemically converted to the fermentation product extractant. The oil can be separated from the feedstock before the feedstock is fed to the fermentation vessel, and the separated oil can be chemically converted to a fermentation product extractant, which is then contacted with the fermentation product comprising the product alcohol, thereby producing the fermentation product. Product alcohol is separated from.

Description

Extract solvent derived from oil for alcohol collection in extract fermentation {EXTRACTION SOLVENTS DERIVED FROM OIL FOR ALCOHOL REMOVAL IN EXTRACTIVE FERMENTATION}

This application discloses US Provisional Application No. 61 / 356,290, filed June 18, 2010; US Provisional Application No. 61 / 368,451, filed July 28, 2010; US Provisional Application No. 61 / 368,436, filed July 28, 2010; US Provisional Application No. 61 / 368,444, filed July 28, 2010; US Provisional Application No. 61 / 368,429, filed July 28, 2010; US Provisional Application No. 61 / 379,546, filed September 2, 2010; And US Provisional Application No. 61 / 440,034, filed February 7, 2011; Claiming the benefit of US patent application Ser. No. 13 / 160,766, filed June 15, 2011; The entire contents of which are incorporated herein by reference.

The Sequence Listing linked to this application is filed in electronic form via the EFS-web, and is therefore incorporated by reference in its entirety.

The present invention relates to the production of fermentation alcohols such as butanol, and in particular to extraction solvents for extractive fermentation, and processes for converting oils derived from biomass into extraction solvents.

Butanol is an important industrial chemical with a variety of applications including use as a fuel additive, as a feedstock chemical in the plastics industry, and as a food-grade extractant in the food and flavor industry. Thus, along with a high need for butanol, there is a high need for an efficient and environmentally friendly production method.

Production of butanol using fermentation by microorganisms is an environmentally friendly production method. Some microorganisms that produce butanol in high yields also have a low butanol toxicity threshold, so it is necessary to collect it from the fermentation vessel as butanol is produced. In situ product removal (ISPR) (also referred to as extractive fermentation) allows the microorganism to produce butanol in high yield by collecting it from the fermentation vessel as it is produced. One method for ISPR described in the art is liquid-liquid extraction (US Patent Application Publication No. 2009/0305370). In order to be technically and economically practical, ideally, liquid-liquid extraction should include good contact between the extractant and the fermentation broth for efficient mass transfer of the product alcohol into the extractant; Good phase separation of the extractant from the fermentation broth (during fermentation and after fermentation); Efficient recovery and reuse of extractant; Minimization of the lowering of the extractant capacity to extract the product alcohol (e.g., by preventing the partition coefficient of the product alcohol into the extractant lowered), and extractant with lipids that lower the partition coefficient over long periods of operation. Requires minimization of contamination.

In particular, by the accumulation of lipids present in the biomass fed to the fermentation vessel, for example as feedstock of hydrolysable starch, the extractant may be contaminated with lipids over time due to the respective reuse. For example, liquified corn mash, loaded into a fermentation vessel with 30% by weight dry corn solids, is co-glycosylated (using saccharification of liquefied mash that occurs during fermentation to produce glucose by addition of glucoamylase). Simultaneous saccharification and fermentation (SSF) can result in fermentation broth containing about 1.2% by weight corn oil during the conversion of glucose to butanol. The dissolution of corn oil lipids in oleyl alcohol (OA), which acts as an extractant in ISPR, causes accumulation of lipid concentrations due to the respective OA reuses, resulting in an increase in lipid concentrations in the OA due to each OA reuse. The partition coefficient for the product alcohols in the OA can be reduced.

In addition, the presence of undissolved solids during extractive fermentation can negatively affect the efficiency of alcohol production. For example, the presence of undissolved solids lowers the mass transfer coefficient inside the fermentation vessel, hinders phase separation in the fermentation vessel, and causes accumulation of corn oil from the undissolved solids in the extractant over time. Resulting in decreased extraction efficiency, ultimately increasing solvent loss due to entrapment of solvent in solids removed as Dried Distillers' Grains with Solubles (DDGS), Delay and / or cause lower fermentation vessel volume efficiency.

Several approaches to reduce the lowering of the partition coefficient of extractant used in extractive fermentation have included wet milling, fractionation, and removal of solids. Wet milling is a costly, multistage process that separates biomass (eg corn) into all its major components (embryonic, pericarp fiber, starch, and gluten) to gain value separately from each byproduct. to be. This process provides a purified starch stream; It involves separating the biomass into its non-starch component which is expensive and unnecessary for fermentation alcohol production. Fractionation removes most of the lipid-bearing fibers and embryos present in ground corn, resulting in corn with higher starch (endosperm) content. Since dry fractionation does not separate embryos and fibers, it is less expensive than wet milling. However, fractionation does not remove all fibers or embryos and does not cause complete removal of solids. In addition, there is some loss of starch in fractionation. Wet milling of corn is more expensive than dry fractionation, but dry fractionation is more expensive than dry grinding of unfractionated corn. For example, as described in US Provisional Application No. 61 / 356,290, filed June 18, 2010, which is concurrently ongoing and commonly owned, includes embryos containing lipids from a liquefied mash prior to use in fermentation. Removing the solids can substantially remove undissolved solids. However, it would be advantageous if the degradation of the partition coefficient of the extractant could be reduced without even fractionation or removal of undissolved solids. Thus, there is a continuing need to develop more efficient methods and systems for producing product alcohols such as butanol through extractive fermentation with reduced degradation of the partition coefficient of the extractant.

In addition, the extractant (eg, oleyl alcohol) is typically added to the process instead of being produced at a stage in the process, so the extractant is a raw material cost. Since the extractant can be lost by adsorption of non-fermentable solid phase and diluted with lipids introduced into the process, the economics of the alcohol production process can be influenced by the efficiency of extractant recovery and reuse. Thus, there is a continuing need for alternative extractants for ISPR that can result in more economical processes by reducing capital and / or operating costs.

Summary of the Invention

The present invention relates to product alcohols, such as butanol, wherein the lipids in the biomass are converted into extractants that can be used for ISPR, and the amount of lipids supplied to the fermentation vessel with the feedstock and / or upon reuse of the extractant is reduced. By providing a production method, this need is met. The present invention provides related advantages of additional degrees which will be apparent by the description of the following embodiments.

Of lipids derived from biomass into extractants comprising fatty acids, fatty alcohols, fatty amides, fatty acid esters, fatty acid glycol esters, and triglycerides, and mixtures thereof (collectively referred to herein as "fatty acid extractants"). Chemical conversion can reduce the amount of lipids present in the ISPR extractant. Triglycerides can be hydroxylated or alkoxylated (eg, methoxylated, ethoxylated). Fatty acid extractants will not be expected to reduce the partition coefficient of product alcohols such as isobutanol into the extractant phase as much as lipids. In addition, fatty acid extractants may be used as ISPR extractants. The fatty acid extractant may be derived from biomass which supplies fermentable carbon which is fed to the fermentation vessel. Therefore, the fatty acid extractant may be produced at a stage in the alcohol production process and may be used instead of or in addition to exogenous supplied ISPR extractant (eg, externally supplied oleyl alcohol) that is not produced during the process. As such, it reduces or even eliminates raw material costs for ISPR extractants.

Extractants may also be produced by converting oils from biomass or feedstock into fatty acids using high temperature and / or high pressure conditions. In addition, oils derived from biomass or feedstock may be treated with one or more lipases to produce fatty acids or hydrogenated to produce fatty alcohols.

The present invention provides a recombinant microorganism capable of producing alcohol from a feedstock; Alcohol; And at least one extractant selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof; The extractant here relates to a composition produced from the feedstock. In one embodiment, the extractant is a mixture of fatty amides, and in further embodiments, the mixture of fatty amides includes linoleamide, oleamide, palmitamide, or stearamide. In another embodiment, the extractant is a mixture of fatty amides and fatty acids, and in further embodiments, the mixture of fatty amides and fatty acids is linoleamide, linoleic acid, oleamide, oleic acid, palmitamide, palmitic acid, stearamide Or stearic acid. In one embodiment, the extractant is selected from hydroxylated triglycerides, alkoxylated triglycerides, hydroxylated fatty acids, alkoxylated fatty acids, hydroxylated fatty alcohols, and alkoxylated fatty alcohols. In one embodiment, the triglycerides may be methoxylated or ethoxylated. In another embodiment, the extractant is selected from saturated fatty acids, unsaturated fatty acids, saturated fatty alcohols, unsaturated fatty alcohols, saturated fatty amides, unsaturated fatty amides, saturated fatty esters, unsaturated fatty esters, and mixtures thereof. In one embodiment, the extractant may be liquid or solid. In further embodiments, the extractant may be in the form of beads. In one embodiment, the alcohol is a C ₁ to C ₈ alkyl alcohol. In another embodiment, the feedstock comprises rye, wheat, corn, cane, barley, cellulose based material, lignocellulosic based material, or mixtures thereof.

In one embodiment, the extractant comprises one or more fatty amides of the formula R (C═O) N (R ′) (R ″), wherein

R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,

R ′ and R ″ are independently selected from the group consisting of hydrogen and C ₁ to C ₆ alkyl groups optionally containing one or more hydroxyl groups.

In another embodiment, the extractant comprises one or more fatty esters of the formula R- (C = 0) -OCHR'CHR "-OH, wherein

R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,

R ′ and R ″ are independently selected from the group consisting of hydrogen and C ₁ to C ₄ alkyl groups.

In one embodiment, the extractant comprises one or more fatty esters of the formula R- (C = 0) -OR ', wherein

R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,

R 'is an alkyl group of 8 carbons or less.

The present invention provides a method for producing a biomass comprising oil; Converting at least a portion of the oil into an extractant by contacting the oil with one or more substances that can chemically convert the oil into an extractant selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof It relates to a method for producing an extract, comprising the step of. In one embodiment, triglycerides may be hydroxylated or alkoxylated (eg, methoxylated, ethoxylated). In one embodiment, the extractant may be liquid or solid. In further embodiments, the extractant may be in the form of beads. The method further includes, in one embodiment, separating the oil from the biomass before contacting the oil with the one or more materials. In one embodiment, the one or more materials are selected from aqueous ammonium hydroxide, anhydrous ammonia, ammonium acetate, ammonia water, hydrogen peroxide, toluene, glacial acetic acid, lipase, and cation exchange resins. In another embodiment, the partition coefficient of the extractant for the product alcohol is greater than the partition coefficient of oil for the product alcohol before the oil is converted to the extractant. In one embodiment, the product alcohol is a C ₁ to C ₈ alkyl alcohol. In another embodiment, the biomass is corn grain, corncob, crop residues, such as corn husk, corn cobs, grass, wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass. , Waste paper, sugarcane bagasse, sorghum, sugarcane, soybeans, ingredients obtained from milling of grains, cellulosic materials, lignocellulosic materials, trees, branches, roots, leaves, wood chips, sawdust, shrubs and Bushes, vegetables, fruits, flowers, animal compost, or mixtures thereof.

The present invention also provides a method of preparing a biomass comprising: (a) providing a biomass comprising oligosaccharides and oils; (b) contacting the biomass with a glycosylating enzyme capable of converting the oligosaccharide into a monosaccharide; (c) separating the oil from the biomass of (a) or (b); (d) contacting the separated oil with one or more reactants or solvents to form an extractant; (e) producing the product alcohol by contacting the biomass with fermentation broth comprising recombinant microorganisms capable of converting monosaccharides to product alcohols; And (f) contacting the product alcohol with the extractant, wherein the partition coefficient for the product alcohol of the extractant is greater than the partition coefficient for the product alcohol of the oil of the biomass. will be. In one embodiment, the one or more reactants or solvents are selected from aqueous ammonium hydroxide, anhydrous ammonia, ammonium acetate, aqueous ammonia, hydrogen peroxide, toluene, glacial acetic acid, lipase, and cation exchange resins. In one embodiment, the extractant is selected from fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof. In one embodiment, the extractant is hydroxylated triglycerides, alkoxylated triglycerides (eg, methoxylated, ethoxylated), hydroxylated fatty acids, alkoxylated fatty acids, hydroxylated fatty alcohols, and alkoxylated Selected from fatty alcohols. In another embodiment, the extractant is selected from saturated fatty acids, unsaturated fatty acids, saturated fatty alcohols, unsaturated fatty alcohols, saturated fatty amides, unsaturated fatty amides, saturated fatty esters, unsaturated fatty esters, and mixtures thereof. In one embodiment, the extractant may be liquid or solid. In further embodiments, the extractant may be in the form of beads. In one embodiment, the alcohol is a C ₁ to C ₈ alkyl alcohol. In another embodiment, the oil is tallow oil, corn oil, canola oil, capric / caprylic triglycerides, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, flax seed oil, angled oil One selected from Otisica oil, palm oil, peanut oil, rapeseed oil, rice oil, safflower oil, soybean oil, sunflower oil, liquid oil, jatropha oil, wheat oil, rye oil, barley oil, and vegetable oil blend It contains the above oil.

In one embodiment, the extractant comprises one or more fatty amides of the formula R (C═O) N (R ′) (R ″), wherein

R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,

R ′ and R ″ are independently selected from the group consisting of hydrogen and C ₁ to C ₆ alkyl groups optionally containing one or more hydroxyl groups.

In another embodiment, the extractant comprises one or more fatty esters of the formula R- (C = 0) -OCHR'CHR "-OH, wherein

R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,

R ′ and R ″ are independently selected from the group consisting of hydrogen and C ₁ to C ₄ alkyl groups.

In one embodiment, the extractant comprises one or more fatty esters of the formula R- (C = 0) -OR ', wherein

R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,

R 'is an alkyl group of 8 carbons or less.

The present invention comprises the steps of (a) producing the product alcohol by providing a fermentation broth comprising a recombinant microorganism capable of producing the product alcohol in the fermentation vessel; (b) contacting the fermentation broth with an extractant to form a two-phase mixture comprising an aqueous phase and an organic phase, wherein the product alcohol and oil are partitioned into the organic phase such that the organic phase comprises the product alcohol and oil; (c) separating the organic phase from the aqueous phase; (d) separating the product alcohol from the organic phase; And optionally, steps (b) and (c) occur simultaneously. In one embodiment, the method includes producing a feedstock slurry; Separating the feedstock slurry to produce (i) an aqueous layer, (ii) an oil layer, and (iii) a solid layer; And feeding the aqueous layer to the fermentation vessel. In another embodiment, the method comprises oils of the oil layer and one or more substances capable of chemically converting the oil into an extractant selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof. By contacting, further comprising the step of converting at least a portion of the oil into an extractant.

In one embodiment, the extractant is selected from fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof. In another embodiment, the extractant is hydroxylated triglycerides, alkoxylated triglycerides (eg, methoxylated, ethoxylated), hydroxylated, fatty acids, alkoxylated fatty acid hydroxylated fatty alcohols, and alkoxylated Selected from fatty alcohols. In one embodiment, the extractant is selected from saturated fatty acids, unsaturated fatty acids, saturated fatty alcohols, unsaturated fatty alcohols, saturated fatty amides, unsaturated fatty amides, saturated fatty esters, unsaturated fatty esters, and mixtures thereof. In one embodiment, the extractant may be liquid or solid. In further embodiments, the extractant may be in the form of beads. In one embodiment, the product alcohol is a C ₁ to C ₈ alkyl alcohol. In another embodiment, the partition coefficient of the extractant for the product alcohol is greater than the partition coefficient of oil of the oil layer for the product alcohol. In one embodiment, the product alcohol is a C ₁ to C ₈ alkyl alcohol. In another embodiment, the oil is tallow oil, corn oil, canola oil, capric / caprylic triglycerides, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, flax seed oil, angled oil One selected from Otisica oil, palm oil, peanut oil, rapeseed oil, rice oil, safflower oil, soybean oil, sunflower oil, liquid oil, jatropha oil, wheat oil, rye oil, barley oil, and vegetable oil blend It contains the above oil.

In some embodiments, a method of harvesting biomass-derived oil from a fermentation process comprises extracting a biomass feedstream comprising a predetermined amount of oil from fatty acids, fatty alcohols, fatty amides, fatty acid methyl esters, fatty acid glycol esters, Converting at least a portion of the oil into the extractant by contacting at least one substance that can be chemically converted to an extractant selected from the group consisting of triglycerides and mixtures thereof. Triglycerides can be hydroxylated or alkoxylated (eg, methoxylated, ethoxylated). The partition coefficient of the extractant for fermentation alcohol is greater than the partition coefficient of oil for fermentation alcohol. In some embodiments the biomass feedstream is milled corn and the oil is corn oil. The method also, in some embodiments, contacts the biomass feedstream with extractant with the fermentation broth, wherein the fermentation broth comprises fermentation alcohol, wherein the fermentation alcohol is dispensed into the extractant.

The present invention provides a method for producing a biomass comprising oil; And converting at least a portion of the oil into an extractant selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof. In one embodiment, converting the oil to the extractant comprises incubating the oil in the presence of Tetrahydrofuran and lithium aluminum hydride; Incubating the oil with sodium hydroxide; Incubating the oil with sulfuric acid and methanol; Incubating the oil with anhydrous ammonia in the presence of ammonium acetate; Incubating the oil with aqueous ammonia; Contacting the oil with toluene, cation exchange resin, glacial acetic acid, lipase, and hydrogen peroxide; Incubating the oil under high temperature conditions, or incubating the oil under high pressure conditions.

In some embodiments, an in-situ production method of extractant for in situ collection of product alcohols comprises: (a) providing a biomass comprising fermentable sugars and an oil, the oil comprising triglycerides; (b) separating the oil of (a) from the biomass; And (c) chemically reacting the isolated oil with triglycerides to form fatty acids, fatty alcohols, fatty amides, fatty amides and mixtures of fatty acids, fatty acid methyl esters, fatty acid glycol esters, triglycerides, and mixtures thereof. Converting the triglyceride in the oil to the reaction product by contacting with one or more reactants or solvents from which the selected reaction product can be obtained. Triglycerides can be hydroxylated or alkoxylated (eg, methoxylated, ethoxylated). The reaction product forms a fermentation product extractant wherein the partition coefficient for the product alcohol is greater than the partition coefficient for the oil of the biomass for the product alcohol.

In some embodiments, a method of producing butanol comprises: (a) providing a biomass comprising oligosaccharides and an oil, wherein the oil comprises glycerides; (b) contacting the biomass with a glycosylating enzyme capable of converting the oligosaccharide into a monosaccharide; (c) separating the oil from the biomass of (a) or (b); (d) contacting the separated oil with a composition comprising one or more reactants or solvents so that the glycerides in the oil form an extractant; (e) producing a fermentation product comprising butanol by contacting the biomass with a recombinant microorganism capable of converting monosaccharides to butanol; And (f) separating the butanol from the fermentation product by contacting the fermentation product with the extractant of (d). The partition coefficient of the extractant to butanol is greater than the partition coefficient of the oil of biomass to butanol. In some embodiments, the extractant of step (d) is selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty amides and mixtures of fatty acids, fatty acid methyl esters, fatty acid glycol esters, triglycerides, and mixtures thereof. Triglycerides can be hydroxylated or alkoxylated (eg, methoxylated, ethoxylated).

In some embodiments, in a step in the process of producing the product alcohol from the feedstock, the method further comprises the step of dispensing at least a portion of the plant-derived oil into a product-derived oil with respect to the product alcohol with a partition coefficient of the extractant to the product alcohol. Converting to an extractant larger than the coefficient. In some embodiments, the plant-derived oil is derived from a feedstock.

In some embodiments, the product alcohol is isobutanol and the extractant partition coefficient is at least about 0.28. In some embodiments, the extractant partition coefficient for isobutanol is at least about 1. In some embodiments, the extractant partition coefficient for isobutanol is at least about 2.

In some embodiments, the process of producing the product alcohol from the feedstock comprises: (a) producing a feedstock slurry; (b) separating the feedstock slurry of (a) to produce a product comprising (i) an aqueous layer, (ii) an oil layer, and (iii) a solid layer; And (c) feeding the aqueous layer of (b) to the fermentation vessel. In some embodiments, separating the feedstock slurry occurs by centrifugation. In some embodiments, the oil layer is a plant-derived oil layer. The method further includes, in some embodiments, obtaining at least a portion of the plant-derived oil from the plant-derived oil layer.

The process further includes, in some embodiments, dispersing the product alcohol into the product alcohol-containing organic phase by adding an extractant to the fermentation vessel to form a two-phase mixture comprising an aqueous phase and a product alcohol-containing organic phase. do.

The process further includes, in some embodiments, dispersing the product alcohol into the product alcohol-containing organic phase by fermenting the sugar in the aqueous phase to produce the product alcohol.

In some embodiments, a method of harvesting biomass-derived oil from a fermentation process comprises (a) providing a fermentation broth comprising product alcohol and biomass-derived oil, the oil comprising glycerides; (b) contacting the fermentation broth with an extractant to form a two-phase mixture comprising an aqueous phase and an organic phase, wherein the product alcohol and oil are partitioned into the organic phase such that the organic phase comprises the product alcohol and oil; (c) separating the organic phase from the aqueous phase; (d) separating the product alcohol from the organic phase; And (e) contacting the organic phase with a composition comprising at least one reactant or solvent to form glycerides in the oil to form additional extractants; And (f) repeating step (b) by contacting the fermentation broth with the additional extractant of step (e).

In some embodiments, the additional extractant is selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty acid methyl esters, fatty acid glycol esters, triglycerides, and mixtures thereof. Triglycerides can be hydroxylated or alkoxylated (eg, methoxylated, ethoxylated).

In some embodiments, the product alcohol is butanol.

In some embodiments, the in-situ fermentation extractant-forming composition comprises (a) an oil derived from biomass; (b) one or more substances capable of chemically converting the oil into one or more products selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty acid methyl esters, fatty acid glycol esters, and triglycerides; And (c) one or more products, wherein the one or more products are in an amount of about 50% to about 99% by weight of the composition. Triglycerides can be hydroxylated or alkoxylated (eg, methoxylated, ethoxylated).

In some embodiments, the composition comprises one or more solvents selected from the group consisting of recombinant microorganisms, butanol, and fatty acids, fatty alcohols, fatty amides, fatty acid methyl esters, fatty acid glycol esters, triglycerides, and mixtures thereof capable of producing butanol. Include. Triglycerides can be hydroxylated or alkoxylated (eg, methoxylated, ethoxylated).

In some embodiments, the composition comprises recombinant microorganisms, butanol, and fatty alcohols capable of producing butanol.

In some embodiments, the composition comprises a mixture of recombinant microorganisms, butanol, and fatty amides capable of producing butanol, wherein the mixture of fatty amides comprises linoleamide, oleamide, palmitamide, and stearamide .

In some embodiments, the composition comprises a composition comprising recombinant microorganisms, butanol, and corn oil capable of producing butanol, wherein the corn oil is about 28% to about 67% hydroxylated.

Brief description of the drawings

The accompanying drawings, which are incorporated in and form a part of the specification, illustrate the invention, and together with the description further serve to explain the principles of the invention and to enable those skilled in the art to understand and use the invention. Do it.

1 schematically illustrates an exemplary method and system of the present invention wherein lipids are collected from liquefied biomass prior to fermentation, and the collected lipids are converted into extractants and fed into a fermentation vessel.

FIG. 2 schematically illustrates an exemplary method and system of the present invention wherein lipids are collected prior to fermentation from liquefied and glycated biomass, and the collected lipids are converted into extractants and fed into a fermentation vessel.

FIG. 3 schematically illustrates an exemplary method and system of the present invention wherein lipids are collected from biomass and converted into extractants supplied to fermentation vessels.

4 schematically illustrates an exemplary method and system of the present invention wherein lipids in the biomass feedstream are converted to extractant and fed to the fermentation vessel.

FIG. 5 schematically illustrates an exemplary method and system of the present invention wherein lipids present in the first extractant from the fermentation vessel are separated from the first extractant and converted to a second extractant supplied to the fermentation vessel. .

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Also, unless otherwise required by context, a singular term will include the plural, and the plural terms will include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes.

To further define the present invention, the following terms and definitions are provided herein.

As used herein, the terms "comprise", "comprising", "comprise", "constituting", "have", "having", "contains", or "contains", or their Any other variant is to be understood to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers. For example, a composition, mixture, process, method, article, or apparatus comprising a list of elements is not necessarily limited to such elements, and is not specifically listed or such compositions, mixtures, processes, methods, articles, or apparatuses May contain other elements inherent in Moreover, unless expressly stated to the contrary, "or" does not mean " comprehensive " or " exclusive " For example, condition A or B is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (Or present), both A and B are true (or present).

Also, the indefinite articles "a" and "an" before an element or component of the present invention are intended to be non-limiting in the case of the element or component, ie, the number of appearances. Therefore, the indefinite article "a" or "an" should be understood to include one or more than one, and the singular form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term "invention" or "invention" as used herein is a non-limiting term and is not intended to refer to any single embodiment of a particular invention, and is intended to refer to all possible embodiments as described in the application. Include.

As used herein, the term "about," which modifies the amount of a component or reactant of the invention employed, includes, for example, routine measurement and liquid handling procedures used to prepare concentrates or solutions in practice; Accidental errors in these processes; Differences in the manufacture, source, or purity of ingredients employed to make the compositions or practice the methods; And the like, and the like. The term “about” also includes amounts that vary due to different equilibrium conditions for the composition resulting from the particular initial mixture. Whether or not modified by the term "about", the claims include equivalents in amounts. In one embodiment, the term “about” means within 10% of the reported value, alternatively within 5% of the reported value.

As used herein, "biomass" refers to any natural product containing hydrolysable polysaccharides that provide fermentable sugars, such as any sugars and starches derived from natural sources such as corn, cane, wheat, cellulose based Or lignocellulosic materials and materials comprising cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides and / or monosaccharides, and mixtures thereof. The biomass may also include additional components such as proteins and / or lipids. The biomass may be derived from a single source or the biomass may comprise a mixture derived from more than one source. For example, the biomass may comprise corncobs and mixtures of corncobs, or mixtures of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacturing, yard waste, wood and woodland waste. Examples of biomass include corn grains, corncobs, crop residues, for example corn husks, corn stalks, grass, wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane Ingredients derived from milling of bagasse, sorghum, sugar cane, beans, grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal compost, and mixtures thereof It is not limited. For example, the mash, juice, molasses, or hydrolyzate can be formed from the biomass by any processing known in the art for biomass processing for fermentation purposes, such as milling, processing, and / or liquefaction. And fermentable sugars, and may include water. For example, the cellulose and / or lignocellulosic biomass can be processed by any method known to those skilled in the art to obtain hydrolysates containing fermentable sugars. Low ammonia pretreatmen is disclosed in US Patent Application Publication No. 2007 / 0031918A1, which is incorporated herein by reference. Enzymatic glycosylation of cellulose and / or lignocellulosic biomass typically uses an enzyme consortium that degrades cellulose and hemicellulose to produce hydrolysates containing sugars including glucose, xylose, and arabinose. do. (Saccharification enzymes suitable for cellulose and / or lignocellulosic biomass are reviewed in Lynd, et al., Microbiol. Mol. Biol. Rev. 66: 506-577, 2002).

As described herein, the mash, juice, molasses, or hydrolyzate may comprise feedstock 12 and feedstock slurry 16. The aqueous feedstream may be formed or derived from the biomass by any processing known in the art for biomass processing for fermentation purposes, such as milling, processing, and / or liquefaction, and may include, for example, fermentable carbon substrates (eg, , Sugar) and water. As described herein, the aqueous feedstream may comprise feedstock 12 and feedstock slurry 16.

As used herein, a "feedstock" means a raw material in a fermentation process, a raw material containing a fermentable carbon source with or without an undissolved solid, and, where applicable, where the fermentable carbon source is liquefied, saccharified, Or a raw material containing a fermentable carbon source, either before or after being released from the decomposition of complex sugars or released from starch by further processing, such as by other processes. The feedstock includes or is derived from biomass. Suitable feedstocks include, but are not limited to, rye, wheat, corn, cane, barley, cellulose based materials, lignocellulosic based materials, or mixtures thereof. When referring to "corn oil", it will be appreciated that the term includes oil produced from a feedstock given in another embodiment of the present invention.

As used herein, "fermentation broth" means a mixture of water, sugars, dissolved solids, optionally microorganisms producing alcohol, product alcohols, and all other components of the material contained in the fermentation vessel, where the product Alcohol is produced by the reaction of sugars with alcohol, water, and carbon dioxide (CO ₂ ) by the microorganisms present. Sometimes, as used herein, the terms "fermentation medium" and "fermented mixture" may be used synonymously with "fermentation broth".

As used herein, "fermentable carbon source" or "fermentable carbon substrate" means a carbon source that can be metabolized by the microorganisms disclosed herein for the production of fermentation alcohol. Suitable fermentable carbon sources include monosaccharides such as glucose or fructose; Disaccharides such as lactose or sucrose; Oligosaccharides; Polysaccharides such as starch or cellulose; One-carbon substrates; And mixtures thereof.

As used herein, "fermentable sugar" refers to a sugar that can be metabolized by the microorganisms disclosed herein for the production of fermentation alcohol.

As used herein, “fermentation vessel” means a vessel in which a fermentation reaction is conducted in which a product alcohol such as butanol is prepared from sugars.

As used herein, "liquefaction container" means a container in which liquefaction is performed. Liquefaction is a process in which oligosaccharides are liberated from a feedstock. In some embodiments where the feedstock is corn, oligosaccharides are liberated from the corn starch content during liquefaction.

As used herein, "glycosylation vessel" means a vessel in which saccharification (ie, oligosaccharides are broken down to monosaccharides) is performed. If fermentation and saccharification occur simultaneously, the saccharification vessel and the fermentation vessel may be one in the same vessel.

As used herein, "sugar" refers to oligosaccharides, disaccharides, and / or monosaccharides.

As used herein, “glycosylation enzyme” means one or more enzymes capable of hydrolyzing the alpha-1,4-glucoside bonds of polysaccharides and / or oligosaccharides, eg, starches, or glycogen. . Glycosylating enzymes may include enzymes that can also hydrolyze cellulosic or lignocellulosic materials.

As used herein, "undissolved solid" means a non-fermentable portion of a feedstock, such as embryos, fibers, and gluten.

As used herein, “product alcohol” refers to any alcohol that can be produced by a microorganism in a fermentation process that uses biomass as a source of fermentable carbon substrate. Product alcohols include, but are not limited to, C ₁ to C ₈ alkyl alcohols. In some embodiments, the product alcohol is a C ₂ to C ₈ alkyl alcohol. In another embodiment, the product alcohol is a C ₂ to C ₅ alkyl alcohol. It will be appreciated that C ₁ to C ₈ alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, and pentanol. Likewise, C ₂ to C ₈ alkyl alcohols include, but are not limited to, ethanol, propanol, butanol, and pentanol. "Alcohol" is also used herein with reference to the product alcohol.

As used herein, "butanol" specifically refers to butanol isomer 1-butanol (1-BuOH), 2-butanol (2-BuOH), and / or isobutanol (iBuOH or I-BUOH, also 2- Also known as methyl-1-propanol) or individually as a mixture thereof.

As used herein, “propanol” refers to the propanol isopropanol or 1-propanol.

As used herein, “pentanol” refers to pentanol isomer 1-pentanol, 3-methyl-1-butanol,,,,,, or.

As used herein, the term "alcohol equivalent" refers to the weight of the alcohol to be obtained by complete hydrolysis and recovery of a predetermined amount of alcohol ester.

As used herein, the term “aqueous phase titer” refers to the concentration of a particular alcohol (eg, butanol) in a fermentation broth.

As used herein, the term "effective titer" refers to the alcohol equivalent of an alcohol ester produced by alcohol esterification per liter of fermentation medium or the total amount of a specific alcohol (eg butanol) produced by fermentation. do. For example, an effective titer of butanol in the unit volume of fermentation may include (i) the amount of butanol in the fermentation medium; (ii) the amount of butanol recovered from the organic extractant; (iii) the use of gas stripping, the amount of butanol recovered from the gas phase, and (iv) the alcohol equivalent of the butanol ester in the organic or aqueous phase.

As used herein, “in situ product collection (ISPR)” means the selective collection of specific fermentation products from a biological process, such as fermentation, to control product concentration in a biological process as the product is produced. .

As used herein, "extractant" or "ISPR extractant" means an organic solvent used to extract any product alcohol, such as butanol isomers. The extractant may be solid or liquid at fermentation temperature. Sometimes, as used herein, the term "solvent" may be used synonymously with "extractant".

As used herein, a "fatty acid extractant" refers to fatty acids, fatty alcohols, fatty amides, fatty acid methyl esters, fatty acid glycol esters, triglycerides by chemically reacting glycerides in natural oils with one or more solvents or reactants. Means an extractant derived from a natural oil by obtaining one or more reaction products selected from the group consisting of, and mixtures thereof. Triglycerides can be hydroxylated or alkoxylated (eg, methoxylated, ethoxylated).

As used herein, "natural oil" refers to a lipid obtained from a plant (eg biomass) or animal. As used herein, "plant-derived oil" refers, in particular, to lipids obtained from plants. Sometimes, "lipid" can be used synonymously with "oil" and "glyceride". Natural oils include tallow, corn, canola, capric / caprylic triglycerides, castor, coconut, cottonseed, fish, jojoba, lard, linseed, carpentry, otissica, palm, peanut, rapeseed, rice, Safflower, soybean, sunflower, rhesus, jatropha, and vegetable oil blends.

As used herein, the term “fatty acid” refers to a carboxylic acid (eg, aliphatic) having a saturated or unsaturated C ₄ to C ₂₈ carbon atom (most commonly C ₁₂ to C ₂₄ carbon atom). Monocarboxylic acid). Fatty acids may also be branched or unbranched. Fatty acids may be derived from animal or vegetable fats, oils or waxes, or may be contained in esterified form. Fatty acids appear naturally in the form of glycerides in fats and fatty oils, or can be obtained by hydrolysis of fats or by synthesis. The term fatty acid may describe a single species or a mixture of fatty acids. The term fatty acid also includes free fatty acids.

As used herein, the term "fatty alcohol" refers to an alcohol having an aliphatic long chain of saturated or unsaturated C ₄ to C ₂₂ carbon atoms.

As used herein, the term “fatty aldehyde” refers to an aldehyde having an aliphatic long chain of saturated or unsaturated C ₄ to C ₂₂ carbon atoms.

As used herein, the term “fatty amide” refers to an amide having an aliphatic long chain of saturated or unsaturated C ₄ to C ₂₂ carbon atoms.

As used herein, the term "fatty ester" refers to an ester having an aliphatic long chain of saturated or unsaturated C ₄ to C ₂₂ carbon atoms.

The term “water immiscible” refers to the inability of a chemical component, such as an extractant or solvent, to be mixed with an aqueous solution, such as fermentation broth, in such a way as to form one liquid phase.

As used herein, the term "aqueous phase" refers to the aqueous phase of a biphasic mixture obtained by contacting fermentation broth with a water-immiscible organic extractant. At this point, in one embodiment of the process described herein, including fermentation extraction, the term “fermentation broth” specifically refers to the aqueous phase in two-phase fermentation extraction.

As used herein, the term "organic phase" refers to the non-aqueous phase of a biphasic mixture obtained by contacting fermentation broth with a water-immiscible organic extractant.

As used herein, the term “separation” is synonymous with “recovery” and refers to the step of collecting a chemical compound from the initial mixture to obtain the compound at a concentration or purity higher than the purity or concentration of the compound in the initial mixture. .

As used herein, a "recombinant microorganism" is modified by the use of recombinant DNA technology, such as by manipulating host cells to include biosynthetic pathways such as, for example, biosynthetic pathways that produce alcohols such as butanol. Refers to microorganisms such as bacteria or yeast.

The present invention provides an extractant obtained by chemical conversion of oil derived from biomass, and a method for producing the extractant. In particular, glycerides in oils are one or more products comprising fatty acids, fatty alcohols, fatty amides, fatty acid methyl esters, fatty acid glycol esters, and triglycerides, collectively referred to herein as fatty acid extractants, and mixtures thereof. Can be converted chemically. Triglycerides can be hydroxylated or alkoxylated (eg, methoxylated, ethoxylated). The fatty acid extractant may serve as an extractant for in situ collection of product alcohols such as butanol from the fermentation broth. Accordingly, the present invention also provides a method for producing a product alcohol such as butanol via extractive fermentation using an extractant produced from biomass oil. The present invention also provides a method for collecting oil from an alcoholic fermentation process by separating oil from a feedstock. The feedstock may be liquefied prior to oil collection to produce a slurry. Thus, the slurry comprises a fermentable carbon source, an oil, and an undissolved solid. The oil, and in some embodiments undissolved solids, may be removed from the slurry before feeding the slurry to the fermentation vessel. Removal of the oil and in some embodiments the undissolved solids may reduce the loss, loss of partition coefficient of the extractant over time due to the presence of oil (and in some embodiments solids) in the fermentation vessel. . In addition, the separated oil can be chemically converted to a fatty acid extractant, which can be fed to a fermentation vessel. The partition coefficient of the fatty acid extractant for fermentation alcohol may be greater than the partition coefficient of oil for fermentation alcohol. In addition, fatty acid extractants may be used in place of, or in addition to, commercial exogenous extractants such as oleyl alcohols that have not been chemically converted from the feedstock in accordance with the methods of the present invention. Thus, the process of the present invention can reduce raw material costs associated with exogenous extractants by producing extractants at the stage of the fermentation process through chemical conversion of oils from the feedstock.

The present invention will be described with reference to the drawings. 1 illustrates an exemplary process flow diagram for the production of fermented alcohol according to an embodiment of the invention. As shown, feedstock 12 may be introduced at the inlet of liquefaction vessel 10 and liquefied to produce feedstock slurry 16. Feedstock 12 contains hydrolyzable starch that supplies a fermentable carbon source (eg, fermentable sugars such as glucose), rye, wheat, corn, cane, barley, cellulose based material, lignocellulosic based It may be a biomass such as, but not limited to, materials, or mixtures thereof, or may be derived from other methods from biomass. In some embodiments, feedstock 12 may be one or more components of fractionated biomass, and in other embodiments, feedstock 12 may be milled unfractionated biomass. In some embodiments, feedstock 12 may be corn, such as dry milled unfractionated corn kernels, and the undissolved particles may include embryos, fibers, and gluten. Undissolved solids are a non-fermentable part of the feedstock 12. For the purpose of the discussion herein with reference to the embodiments shown in the figures, feedstock 12 will often be described as constituting milled unfractionated corn in which undissolved solids are not separated therefrom. However, as will be apparent to those skilled in the art, it should be understood that the exemplary methods and systems described herein may be modified for different feedstocks, whether or not fractionated. In some embodiments, feedstock 12 may be high-oleic corn, so the corn oil derived therefrom is high-oleic corn oil having an oleic acid content of at least about 55% by weight oleic acid. In some embodiments, the oleic acid content in the high-oleic corn oil may be up to about 65% by weight, compared to the oleic acid content in the normal corn oil. High-oleic oils can provide some advantages in use in the process of the present invention, since the hydrolysis of the oil can produce fatty acid extractants with high oleic acid content for contacting the fermentation broth. In some embodiments, the fatty acids or mixtures thereof include unsaturated fatty acids. The presence of unsaturated fatty acids reduces the melting point, providing a handling advantage. Monounsaturated (ie, having a single carbon-carbon double bond) among unsaturated fatty acids can provide an advantage for the melting point without sacrificing suitable thermal and oxidative stability in process considerations.

Feedstock 12 liquefaction process involves hydrolyzing starch in feedstock 12 to sugars, including, for example, dextrin and oligosaccharides, which is a conventional process. Any known liquefaction process commonly used in the industry, including but not limited to an acid process, an acid-enzyme process, or an enzyme process, can be used with the corresponding liquefaction vessels. These processes can be used alone or in combination. In some embodiments, an enzymatic process may be used, and an appropriate enzyme 14, for example alpha-amylase, is introduced at the inlet of the liquefaction vessel 10. Water may also be introduced into the liquefaction vessel 10.

Feedstock slurry 16 produced from liquefaction of feedstock 12 includes sugars, oils, and undissolved solids from the feedstock. Feedstock slurry 16 may be withdrawn from the outlet of liquefaction vessel 10. In some embodiments, feedstock 12 is corn or corn kernels, so feedstock slurry 16 is a corn mash slurry.

Feedstock slurry 16 is introduced into the inlet of separator 20 configured to collect a portion, or preferably substantially all, of the oil present in feedstock slurry 16. The collected oil is provided to the reaction vessel 40 as stream 26 and the remaining feedstock comprising sugar and water is discharged to fermentation vessel 30 as aqueous stream 22. The aqueous stream 22 may comprise the undissolved solids of the slurry 16, but since the oil 26 was collected through the separator 20, the fermentation broth in the fermentation vessel 30 still has a reduced amount of oil. Has The oil stream 26 discharged from the separator 20 has a predetermined amount of glycerides, in particular triglycerides, and contacts them with one or more substances 42 in the reaction vessel 40. Material 42 is a reactant or solvent capable of chemically converting at least a portion of the glycerides in oil 26 to fatty acid extractant 28. In some embodiments, the amount of fatty acid extractant 28 in the oil from the chemical conversion of glycerides through material 42 is at least about 17 wt%, at least about 20 wt%, at least about 30 wt%, at least about 40 wt% At least about 50 wt%, at least about 60 wt%, at least about 65 wt%, at least about 70 wt%, at least about 75 wt%, at least about 80 wt%, at least about 85 wt%, at least about 90 wt%, Or at least about 95 weight percent, or at least about 99 weight percent.

Separator 20 may be any suitable separator known in the art for collecting oil from an aqueous feedstream, including siphoning, aspiration decantation, centrifugation, gravity settler (gravity settler), membrane-assisted phase splitting, and the like. In some embodiments, separator 20 may also remove undissolved solids in feedstock slurry 16 and discharge the undissolved solids as a solid phase or wet cake 24. For example, in some embodiments, separator 20 may include a filter press, vacuum filtration, or centrifuge for separating undissolved solids from feedstock slurry 16. For example, in some embodiments, separator 20 is stirred or rotated feedstock slurry 16 to aqueous layers containing sugar and water (ie stream 22), solids containing undissolved solids. A tricanter centrifuge 20 that produces a centrifuge product comprising a layer (ie, wet cake 24), and an oil layer (ie, oil stream 26). If slurry 16 is a corn mash slurry, then oil 26 is free corn oil. As used herein, the term free corn oil means corn oil liberated from corn embryos. For the corn mash slurry as feedstock slurry 16, the wet cake 24 comprises at least about 50% by weight of the undissolved particles present in the feedstock slurry and about 55% by weight of the undissolved particles present in the feedstock slurry. At least%, at least about 60% by weight of the undissolved particles present in the feedstock slurry, at least about 65% by weight of the undissolved particles present in the feedstock slurry, about 70% of the undissolved particles present in the feedstock slurry. At least% by weight, at least about 75% by weight of the undissolved particles present in the feedstock slurry, at least about 80% by weight of the undissolved particles present in the feedstock slurry, at least about undissolved particles present in the feedstock slurry. At least 85% by weight, at least about 90% by weight of the undissolved particles present in the feedstock slurry, present in the feedstock slurry That undissolved undissolved present in about 95 wt.% Or more, or the feed slurry of particles comprises about 99% by weight or more of the particles.

When removing the wet cake 24 through the centrifuge 20, in some embodiments, a portion of the oil from the feedstock 12, such as corn oil, is in the wet cake 24 when the feedstock is corn. Remains. In this case, the wet cake 24 includes corn oil in an amount less than about 20% by weight of the dry solids content of the wet cake 24. The wet cake 24 may be discharged out of the outlet located near the bottom of the centrifuge 20. Wet cake 24 may also include some of the water and fermentable carbon. Once the aqueous solution 22 is discharged from the centrifuge 20, the wet cake 24 may be washed with additional water in the centrifuge. Washing the wet cake 24 will recover the sugars (eg, oligosaccharides) present in the wet cake, and the recovered sugars and water can be reused in the liquefaction vessel 10. After washing, the wet cake 24 may be dried through any suitable known process to form dry corn flakes (DDGS). DDGS formation from the wet cake 24 formed in the centrifuge 20 has several benefits. Since the undissolved solids do not go to the fermentation vessel, the DDGS does not have entrapped extractant and / or product alcohol, for example butanol, which is not subject to the conditions of the fermentation vessel, which is in contact with the microorganisms present in the fermentation vessel. I never do that. These benefits make it easier to process and sell DDGS, for example as animal feed. Methods and systems for removing undissolved solids from feedstock 16 via centrifugation are described in detail in US Provisional Application No. 61 / 356,290, filed June 18, 2010, which is concurrently ongoing and commonly owned. Which are hereby incorporated by reference in their entirety.

In some embodiments, oil 26 is not separately discharged from wet cake 24, but instead oil 26 is included as part of wet cake 24 and ultimately present in DDGS. In this case, the oil can be separated from the DDGS and converted to a fatty acid extractant for later use in the same or different alcohol fermentation process. In either case, the collection of the oil component of the feedstock is advantageous for alcohol production, such as butanol production, where the oil present in the fermentation vessel can dilute the ISPR extractant and reduce the partition coefficient of the fermentation alcohol into the organic phase. Because there is. In addition, the oil can be broken down into fatty acids and glycerin, which can accumulate in water and reduce the amount of water available for reuse throughout the system. Thus, the collection of the oil component of the feedstock can also increase the efficiency of product alcohol production by increasing the amount of water that can be reused through the system.

Aqueous stream 22 and microorganism 32 are introduced into fermentation vessel 30 to be included in the fermentation broth contained in fermentation vessel 30. Fermentation vessel 30 is configured to ferment aqueous stream 22 to produce product alcohols such as butanol. In particular, the microorganism 32 metabolizes the fermentable sugar in the slurry 16 to secrete the product alcohol. The microorganism 32 is selected from the group of bacteria, cyanobacteria, filamentous fungi, and yeast. In some embodiments, microorganism 32 may be a bacterium such as E. coli. In some embodiments, microorganism 32 may be a fermentative recombinant microorganism. The aqueous solution 22 may include, for example, sugars in the form of oligosaccharides, and water, and may contain less than about 20 g / L monomeric glucose, more preferably less than about 10 g / L, or about 5 g. And less than / L monomeric glucose. Suitable techniques for determining the amount of monomeric glucose are well known in the art. Such suitable methods known in the art include HPLC.

In some embodiments, a glycosylation process is applied to aqueous stream 22 to break down complex sugars (eg, oligosaccharides) into monosaccharides that can be readily metabolized by microorganisms 32 in stream 22. . Any known glycosylation process commonly used in the industry may be used, including, but not limited to, acid processes, acid-enzyme processes, or enzyme processes. In some embodiments, simultaneous saccharification and fermentation (SSF) may occur within fermentation vessel 30. In some embodiments, an enzyme 38 such as glucoamylase may be introduced at the inlet of fermentation vessel 30 to break down the starch into glucose that can be metabolized by microorganism 32.

As the product alcohol is produced by the microorganism 32, the product alcohol can be collected from the fermentation vessel 30 using in situ product harvesting (ISPR). In extractive fermentation, these ISPRs include liquid-liquid extraction. Liquid-liquid extraction can be performed according to the process described in US Patent Application Publication No. 2009/0305370, the disclosure of which is incorporated herein in its entirety. US Patent Application Publication No. 2009/030537 describes a method for producing and recovering butanol from fermentation broth using extractive fermentation, comprising contacting the fermentation broth with a water immiscible extractant. Typically extractants are fatty acids, fatty alcohols, fatty amides, mixtures of fatty amides and fatty acids, esters of fatty acids, fatty aldehydes, fatty acid methyl esters, fatty acid glycols, for forming a two-phase mixture comprising an aqueous phase and an organic phase. Organic extractant selected from the group consisting of esters, triglycerides and mixtures thereof. Referring to the embodiment of FIG. 1, fermentation vessel 30 has one or more inlets for receiving one or more water immiscible ISPR extractants, eg, fatty acid extractant 28 from vessel 40. The fatty acid extractant 28 is contacted with the fermentation broth to form a two-phase mixture comprising an aqueous phase and an organic phase. The product alcohol present in the fermentation broth is partitioned into the organic phase. The biphasic mixture can be collected from fermentation vessel 30 as stream 39 and introduced into vessel 35, where separation of the aqueous and organic phases is carried out for alcohol-containing organic phase 36 and aqueous phase 34. To produce. Aqueous phase 34 of biphasic mixture 39 using methods known in the art, including but not limited to siphoning, decantation, centrifugation, use of gravity settler, membrane-assisted phase separation, and the like. The alcohol-containing organic phase 36 is separated from it. All or part of the aqueous phase 34 may be reused (as shown) into the fermentation vessel 30 as fermentation medium, otherwise discarded and replaced with fresh medium, or treated for collection of any remaining product alcohol and then fermented. The container 30 can be reused. After the alcohol-containing organic phase 36 is treated to recover the product alcohol, the resulting alcohol-lean extractant is usually combined with a new supplemental extractant 28 for further extraction of the product alcohol. Can be reused back into fermentation vessel 30 (not shown). Alternatively, fresh extractant 28 may be added continuously to the fermentation vessel to replace the extractant removed in the biphasic mixture stream 39.

In some embodiments, one or more additional ISPR extractants, such as the extractant 29 illustrated in the embodiments of FIGS. 3 to 5, are introduced into the fermentation vessel 30 to include an aqueous phase and an organic phase and the product alcohol comprises an organic phase. It is possible to form a two-phase mixture that is dispensed into. One or more such additional extractants 29 may be prepared by using other fatty acid extractants and / or exogenous organic extractants such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof. In some embodiments, ISPR extractant 29 may be a carboxylic acid, and in some embodiments, ISPR extractant 29 may be a free fatty acid. In some embodiments, the carboxylic acid or free fatty acid is 4 to 28 carbons, 4 to 22 carbons in other embodiments, 8 to 22 carbons in other embodiments, 10 to 28 carbons in other embodiments, other 7 to 22 carbons in an embodiment, 12 to 22 carbons in another embodiment, 4 to 18 carbons in another embodiment, 12 to 22 carbons in another embodiment, and 12 to 18 carbons in another embodiment It can have a chain of.

In some embodiments, the ISPR extractant 29 is one or more of the following fatty acids: azaleic acid, capric acid, caprylic acid, castor fatty acid, coconut fatty acid (ie, lauric acid, myristic acid, arm) As a naturally occurring combination of fatty acids, including myrtic acid, caprylic acid, capric acid, stearic acid, caproic acid, arachidic acid, oleic acid, and linoleic acid), dimer acid, isostearic acid, lauric acid, flax seed Fatty Acids, Myristic Acid, Oleic Acid, Palm Oil Fatty Acids, Palmitic Acid, Palm Kernel Fatty Acids, Pelagonic Acid, Lysinoleic Acid, Sebacic Acid, Soy Fatty Acids, Stearic Acid, Tall Oil Fatty Acids, Tallow Fatty Acids, and # 12 Hydroxy Stear mountain. In some embodiments, ISPR extractant 29 is one or more diacids, such as azelaic acid and sebacic acid. Thus, in some embodiments, ISPR extractant 29 may be a mixture of two or more different fatty acids. For example, as described in concurrent, commonly owned US Provisional Application No. 61 / 368,444, filed Jul. 28, 2010, in some embodiments, the ISPR extractant 29 is comprised of biomass lipids and It may be a free fatty acid produced from enzymatic hydrolysis of the same natural oil. In such embodiments, the biomass lipids for producing the extractant 29 may be derived from the same or different biomass source as the biomass source from which the feedstock 12 is obtained. For example, in some embodiments, the biomass lipids for producing extractant 29 may be derived from soybean, while the biomass source of feedstock 12 is corn. As will be apparent to those skilled in the art, any possible combination of different biomass sources may be used for the extractant 29 versus the feedstock 12.

In the embodiment of FIG. 1, the product alcohol is extracted in situ from the fermentation broth and the separation of the biphasic mixture 39 takes place in a separate vessel 35. In-situ extraction fermentation may be carried out in batch mode or continuous mode in fermentation vessel 30. In in situ extraction fermentation, the organic extractant may contact the fermentation medium at the beginning of the fermentation to form a two phase fermentation medium. Alternatively, the organic extractant may be contacted with the fermentation medium after the microorganism has achieved the desired amount of growth, which may be determined by measuring the optical density of the culture. In addition, the organic extractant may be contacted with the fermentation medium at a time when the product alcohol level in the fermentation medium reaches a preselected level. In the case of butanol production, for example, the ISPR extractant may be contacted with the fermentation medium at a time before the butanol concentration reaches toxic levels. After contacting the fermentation medium with the ISPR extractant, the butanol product is dispensed into the extractant to reduce the concentration of butanol in the aqueous phase containing the microorganism, thereby limiting the exposure of the producing microorganisms to the inhibitory butanol product.

The volume of ISPR extractant to be used depends on a number of factors including the volume of the fermentation medium, the size of the fermentation vessel, the partition coefficient of the extractant to the butanol product, and the fermentation mode selected, as follows. The volume of extractant may be from about 3% to about 60% of the fermentation vessel working volume. Depending on the extraction efficiency, the aqueous phase titer of butanol in the fermentation medium may be, for example, from about 5 g / L to about 85 g / L, from about 10 g / L to about 40 g / L, from about 10 g / L to about 20 g / L, about 15 g / L to about 50 g / L, or about 20 g / L to about 60 g / L. Without wishing to be bound by theory, in part, higher butanol titers can be obtained by extractive fermentation methods by collecting toxic butanol products from the fermentation medium and thereby keeping the levels below those toxic to microorganisms. It is thought to be.

In the batch mode of in situ extraction fermentation, a predetermined volume of organic extractant is added to the fermentation vessel and no extractant is removed during the process. This mode requires a larger volume of organic extractant to minimize the concentration of inhibitory butanol product in the fermentation medium. As a result, the volume of the fermentation medium is smaller and the amount of product produced is less than that obtained using the continuous mode. For example, the volume of extractant in batch mode may be from 20% to about 60% of the fermentation vessel working volume in one embodiment, and from about 30% to about 60% in another embodiment.

The product alcohol can be collected from the fermentation medium using gas stripping (not shown) simultaneously with the organic extractant.

As shown in the embodiments of FIGS. 4 and 5 described below, in some embodiments, separation of the biphasic mixture may occur within the fermentation vessel. In particular in a continuous mode of in situ extraction fermentation, in one embodiment, extractant 28 may be introduced into fermentation vessel 30 to obtain a biphasic mixture therein, and alcohol-containing organic-phase stream 36 Directly from fermentation vessel 30. The aqueous phase stream 34 may also come directly from the fermentation vessel 30 or may be processed and reused for removal of any remaining product alcohol, discarded and replaced with fresh fermentation medium. Extraction of the alcohol product by the ISPR extractant can be performed with or without removing the microorganism 32 from the fermentation broth. The microorganism 32 can be removed from the fermentation broth by means known in the art, including but not limited to filtration or centrifugation. For example, aqueous phase stream 34 may comprise microorganisms 32 such as yeast. For example, microorganisms 32 can be easily separated from the aqueous phase stream in a centrifuge (not shown). The microorganism 32 can then be reused into the fermentation vessel 30, which can lead to an increase in the alcohol production efficiency by increasing the production rate of the alcohol product over time.

The volume of extractant in a continuous mode of in situ extraction fermentation is, in one embodiment, from about 3% to about 50% of the fermentation vessel working volume, in other embodiments from about 3% to about 30%, in other embodiments from 3% to about 20%; And from 3% to about 10% in other embodiments. As the product continues to be collected from the reactor, a smaller volume of extractant is required, allowing the use of larger volumes of fermentation medium.

As an alternative to in situ extraction fermentation, the product alcohol can be extracted from the fermentation broth downstream of fermentation vessel 30. In this case, the fermentation broth may be collected from the fermentation vessel 30 and introduced into the vessel 35. Thereafter, the extractant 28 may be introduced into the vessel 35 and contacted with the fermentation broth to obtain a biphasic mixture 39 in the vessel 35, which is then followed by an organic phase 36 and an aqueous phase 34. Separated by. Alternatively, extractant 28 may be added to the fermentation broth in a separate vessel (not shown) prior to introduction into vessel 35.

The partition coefficient for the product alcohol of the fatty acid extractant 28 is greater than the partition coefficient for the product alcohol of the oil 26. For example, if feedstock 12 is corn, the partition coefficient for the product alcohol of corn oil 26 (if present in the fermentation broth) may be less than about 0.28, while fatty acid extraction from corn oil 26 The dispensing factor of the twenty eighth may be at least about 0.28. In one embodiment, the partition coefficient of fatty acid extractant 28 to a product alcohol such as butanol is about 1 or more, about 2 or more in other embodiments, about 2.5 or more in other embodiments, about 2.75 or more in other embodiments, And about 3 or more in other embodiments. Thus, removal of the oil component of the feedstock not only reduces the threat of lowering the partition coefficient of the ISPR extractant, but also allows fatty acids from which the product alcohol can be dispensed more from the aqueous phase than is distributed to the oil itself. By acting as a raw material for the production of extractant, it increases the efficiency of product alcohol production in extractive fermentation. In addition, by using the fatty acid extractant 28 derived from the oil 26 in the feedstock 12 alone or in combination with an exogenous extractant (for example, an externally supplied oleyl alcohol), the exogenous extractant The cost associated with this can be reduced or eliminated.

In addition, in the case where the fatty acid extractant 28 includes free fatty acids, the consumption rate of glucose by the microorganisms 32 in the fermentation vessel 30 will be higher when these free fatty acids are present than when these free fatty acids are absent. Can be. Thus, in some embodiments of the present invention, a fatty acid extractant having free fatty acids may be contacted with a fermentation broth, whereby ISPR extractant free of free fatty acids (eg, oleyl alcohol) is used for extractive fermentation. The free fatty acid can increase glucose uptake by the microorganism 32 as compared to glucose uptake in the case of the above. For example, as exemplified in Table 1 of Example 2 below, the fatty amide / fatty acid mixture used as the fatty acid extractant for extractive fermentation, compared to the case where oleyl alcohol is used as the extractant, is Saccharomyces. It can provide higher rates of glucose uptake by Saccharomyces butanologen. A method for producing product alcohol from a fermentation process, in which free fatty acids are produced at an in-process stage and contacted with microbial cultures in a fermentation vessel to improve microbial growth rate and glucose consumption, filed on July 28, 2010, Concurrent and commonly owned US Provisional Application No. 61 / 368,451, which is incorporated herein by reference in its entirety.

As will be apparent to those skilled in the art, in some embodiments, the simultaneous saccharification and fermentation in fermentation vessel 30 are replaced by a separate saccharification vessel 60 between separator 20 and fermentation vessel 30. The system and process can be modified.

In another embodiment, for example, saccharification may occur in a separate saccharification vessel 60 located between the separator 20 and the liquefaction vessel 10, as shown in the embodiment of FIG. FIG. 2 is substantially the same as FIG. 1, except that a separate saccharification vessel 60 containing the enzyme 38 is included and the oil stream 26 is separated from the liquefied, saccharified feedstock stream 62. Do. By introducing the feedstock slurry 16 into the saccharification vessel 60 together with an enzyme 38 such as glucoamylase, the oligosaccharide in the slurry 16 can be broken down into monosaccharides. Liquefaction, saccharification feedstock stream 62 exits saccharification vessel 60 and is introduced into separator 20. Feedstock stream 62 includes monosaccharides, oils, and undissolved solids from the feedstock. In separator 20, feedstock stream 62 is separated into oil stream 26 and substantially aqueous stream 23, which is fed to fermentation vessel 30. In the embodiment shown, aqueous stream 23 comprises undissolved solids. Alternatively, as described in connection with the embodiment of FIG. 1, solids may be removed as wet cake 24 in separator 20. The oil stream 26 discharged from the separator 20 has a predetermined amount of glycerides, in particular triglycerides, and contacts them with one or more substances 42 in the reaction vessel 40. Material 42 chemically converts at least a portion of the glycerides from oil 26 to fatty acid extractant 28, which is supplied to fermentation vessel 30. The remaining process operation of the embodiment of FIG. 2 is the same as that of FIG. 1 and will not be described again in detail.

In some embodiments of the invention, for example, extractive fermentation, as shown in the embodiment of FIG. 3, is derived from a biomass source that is the same as or different from the biomass source of feedstock 12, but is applied to feedstock 12. A fatty acid extractant 28 'that is not derived from the actual oil contained can be employed. For example, where feedstock 12 is corn, fatty acid extractant 28 'may be derived from corn oil, while corn oil producing extractant 28' is provided in the embodiment of FIG. It is not a corn oil contained in the feedstock 12 and separated in the reaction vessel 40 as will be. As another example, the fatty acid extractant 28 'may be derived from soybean (or soybean) as a biomass source, while the biomass source of feedstock 12 is corn. As will be apparent to those skilled in the art, any possible combination of different biomass sources for extractant 28 'versus feedstock 12 may be used.

In some embodiments, the fatty acid extractant 28 'is derived from any natural oil and thus may be from a biomass source or alternatively from an animal source.

In the embodiment of FIG. 3, liquefied aqueous stream 22 is fed to fermentation vessel 30 together with saccharifying enzyme 38 and microorganism 32 to produce product alcohol by simultaneous saccharification and fermentation (SSF). In some embodiments, saccharification may occur, for example, in a separate container as described in connection with the embodiment of FIG. 2. In some embodiments, liquefied aqueous stream 22 preferably has oil 26 removed at least through separator 20 (see FIG. 1), and in some embodiments, is introduced into fermentation vessel 30. It also has an undissolved solid that is removed as a wet cake 24 before it is turned on (see FIG. 1). A reaction vessel (a stream 26 ') of natural oil, such as plant-derived oil, with the substance (s) 42 for chemically converting at least a portion of the oil 26' to the fatty acid extractant 28 ' 40) Introduce into. Oil stream 26 ′ is not oil 26 derived from feedstock slurry 16 upstream (see FIG. 1). Any plant-derived oil or other natural oil that can be chemically converted to fatty acid extractant 28 'for ISPR can be a source of oil stream 26'. Thereafter, the fatty acid extractant 28 'from the reaction vessel 40 is introduced into the fermentation vessel 30 so that the product alcohol, if present in the fermentation vessel, is to a greater extent that the product alcohol is to be dispensed into the oil 26'. The alcohol is dispensed into fatty acid extractant 28 '.

Thus, in some embodiments, the product alcohol is extracted using fatty acid extractant 28 'obtained from plant-derived oil 26' rather than the same oil 26 originally introduced into the process via feedstock 12. Extract. Optionally, one or more additional extractant 29 may be introduced into fermentation vessel 30 to preferentially distribute the product alcohol from the aqueous phase. The one or more additional extractants 29 may be exogenous extractants such as oleyl alcohol supplied exogenously which may not be produced in the process and / or may be other fatty acid extractants. In some embodiments, such other fatty acid extractant 29 may be produced from oil derived from the same or different biomass source as either the fermentation vessel feed stream 22 or the biomass source of the oil stream 26 '. Can be.

Except for the aqueous phase 34, which is not shown to be fed back to the fermentation vessel 30, the remaining process operations of the embodiment of FIG. 3 are the same as in FIGS. 1 and 2 and will not be described again in detail. However, in any of the embodiments provided herein, all or part of the aqueous phase 34 may be reused and / or discarded and / or additionally to collect the product alcohol as described above with respect to FIG. 1. It should be understood that this can be done.

In some embodiments of the present invention, the oil from feedstock 12 is not separated in separator 20, but instead instead of slurry 16 in, for example, feedstock 12 before or during liquefaction. In situ, or chemically in situ with fatty acid extractant in saccharification stream 62 (see FIG. 2). For example, in the embodiment of FIG. 4, in order to produce hydrolyzed feedstock by hydrolyzing starch in feedstock 12, feedstock 12 may be combined with an appropriate enzyme 14, for example alpha-amylase. It is supplied to the liquefaction container 10 together. One or more substances 42 also liquefied vessel 10 for chemically converting oil present in feedstock 12 to fatty acid extractant 28 before, during or after liquefaction of feedstock 12. Is introduced into. The substance 42 can be introduced into the liquefaction vessel 10 before or after the addition of the enzyme 14, and oil in the feedstock 12 before liquefaction, during or after liquefaction of the feedstock 12 May be converted to extractant 28. In either case, the oil in the feedstock 12 is converted to the fatty acid extractant 28 so that the biphasic stream 18 exits from the liquefaction vessel 10. Two-phase stream 18 comprises both fatty acid extractant 28 with sugar, water, and undissolved solids forming liquefied aqueous phase 22. In some embodiments in which the fatty acid extractant comprises fatty acids, the aqueous phase 22 of the biphasic stream 18 may comprise glycerol (glycerine) resulting from the conversion of glycerides in the oil to fatty acids. In some embodiments, such glycerol, if present, may be removed from stream 18 prior to introduction into fermentation vessel 30.

Referring to FIG. 4, the biphasic stream 18 (ie, streams 22, 28) is contacted with fermentation broth in fermentation vessel 30 to form a biphasic mixture. In fermentation vessel 30, the product alcohol produced by SSF is dispensed into an organic phase comprising fatty acid extractant 28. Alternatively, in some embodiments, the process may be modified to include a separate saccharification container as discussed in connection with FIG. 2. Separation of the biphasic mixture takes place in fermentation vessel 30, whereby alcohol-containing organic phase stream 36 and aqueous phase stream 34 exit directly from fermentation vessel 30. Alternatively, the separation of the biphasic mixture may be performed in a separate vessel 35 as provided in the embodiments of FIGS. Optionally, one or more additional extractant 29 may be introduced into fermentation vessel 30 to form an organic phase in which product alcohol is preferentially distributed from the aqueous phase. The remaining process operations of the embodiment of FIG. 4 are the same as the previously described drawings and will not be described again in detail.

In some embodiments of the present invention, biomass oils present in feedstock 12 may be separated from the process stream in a step following alcoholic fermentation. The post-fermentation separated oil can then be converted to a fatty acid extractant and introduced into the fermentation vessel as an ISPR extractant. For example, in the embodiment of FIG. 5, the feedstock 12 is liquefied to produce a feedstock slurry 16 comprising oil 26 derived from the feedstock. Feedstock slurry 16 may also include undissolved solids from the feedstock. Alternatively, undissolved solids may be separated from slurry 16 via a separator (not shown), such as a centrifuge. Feedstock slurry 16 containing oil 26 is introduced directly into fermentation vessel 30 containing fermentation broth containing saccharifying enzyme 38 and microorganism 32. Product alcohol is produced in fermentation vessel 30 by SSF. Alternatively, in some embodiments, the process may be modified to include a separate saccharification container as discussed in connection with FIG. 2.

 ISPR extractor 29 is introduced into fermentation vessel 30 to form a biphasic mixture and collected by dispensing the product alcohol into the organic phase of ISPR extractor 29. Oil 26 is also dispensed into the organic phase. ISPR extractant 29 may be one or more fatty acid extractants and / or exogenous organic extractants (eg, oleyl alcohol) not derived from natural oils. If the extractant 29 is a fatty acid extractant, the extractant 29 may be a fatty acid extractant 28 produced from a natural oil, such as plant-derived oil, rather than the same oil originally introduced into the process via feedstock 12. ') (See FIG. 3).

Separation of the biphasic mixture takes place in fermentation vessel 30, whereby alcohol-containing organic phase stream 36 and aqueous phase stream 34 exit directly from fermentation vessel 30. Alternatively, the separation of the biphasic mixture may be performed in a separate vessel 35 as provided in the embodiments of FIGS. An organic phase stream 36 comprising oil 26 is introduced into separator 50 to recover product alcohol 54 from extractant 29. The resulting alcohol-low extractant 27 includes recovered extractant 29 and oil 26. One or more substances 42 (eg, for introducing an extractant 27 comprising oil 26 into the reaction vessel 40 and chemically converting at least a portion of the oil 26 into a fatty acid extractant 28) For example, reactants and / or solvents).

The extractant 27 comprising the fatty acid extractant 28 can then be reused back into the fermentation vessel 30. This reused extractant stream 27 may be a separate stream or a stream combined with a new supplemental extractant stream 29. The subsequent recovery of the alcohol-containing organic phase 36 may then comprise a fatty acid extractant 28 and an ISPR extractant 29 in addition to the oil 26 and the product alcohol. The organic phase 36 is then treated to recover the product alcohol, the oil is reacted to form a fatty acid extractant, and fermented as the resulting alcohol-low fatty acid extractant 28 and alcohol-low extractor 29. It can be reused back into the container 30. In some embodiments, the use of the extractant 29 may be phased out as the fermentation process is run over time, which may result in a sufficient amount of fatty acid extractant 28 to extract the product alcohol. Because it can produce. Thus, the ISPR extractant may be an extractant 27 and a fatty acid extractant 28 reused as a supplemental ISPR extractant through the reaction vessel 40.

Alternatively, in some embodiments, organic phase stream 36 comprising oil 26 may be introduced into reaction vessel 40 prior to product alcohol recovery 54 in separator 50. In such embodiments, the organic phase stream 36 may be introduced into the reaction vessel 50 and contacted with one or more materials 42 for producing the fatty acid extractant 28. Thereafter, the resulting organic phase stream 36 comprising fatty acid extractant 28 may be introduced into separator 50 to recover product alcohol 54, and the resulting alcohol-low extractant is then subjected to fatty acid. An extractant stream 27 comprising extractant 28 may be reused back into fermentation vessel 30. In another embodiment, oil 26 is extracted from organic phase stream 36 or extractant stream 27 prior to contacting oil 26 with material (s) 42 for producing fatty acid extractant 28. Can be separated. The fatty acid extractant 28 is then used as fermentation vessel 30, an ISPR extractant supplied to a different fermentation vessel (e.g., operated in parallel or in series with fermentation vessel 30 in an alcohol production plant), You can save it for later use.

Accordingly, FIGS. 1-5 illustrate fatty acid extractants 28 produced from fermentation processes and biomass-derived oils 26, and plant-derived oils 26 ′ that can be used to harvest product alcohols in extractive fermentation. Various non-limiting embodiments of methods and systems comprising fatty acid extractants 28 'produced from natural oils, such as), are provided. These fatty acid extractants 28 and 28 'may be selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty acid methyl esters, fatty acid glycol esters, triglycerides, and mixtures thereof. Triglycerides can be hydroxylated or alkoxylated (eg, methoxylated, ethoxylated). The chemical conversion of glycerides from the natural oils described herein to fatty acid extractants can be performed using any reaction scheme known in the art. With respect to plant-derived oils, such as corn oil, for example, in some embodiments, the corn oil as oil 26 or 26 'is contacted with various reactants and solvents 42 to show the hydroxides shown in Scheme I below. By achieving misfire, one can produce hydroxylated triglycerides as fatty acid extractant (28 or 28 ') (see Example 1 below for details):

(I)

See, eg, Roe, et al., Am. Oil Chem. Soc. 29: 18-22, 1952 and in some embodiments, reacting corn oil triglycerides as oil 26 or 26 'with aqueous ammonium hydroxide as reactant 42 Fatty amides and fatty acids, which can be used together or separately as fatty acid extractants (28 or 28 '):

(II)

In some embodiments, the aqueous ammonium hydroxide is about 28% by weight ammonia water. In some embodiments, a mixture of corn oil fatty amide and corn oil fatty acid produced according to Scheme (II) may be used to produce a single fatty acid extractant (28 or 28 '). In some embodiments, the mixture of fatty amides and fatty acids may include linoleamide, linoleic acid, oleamide, oleic acid, palmiamide, palmitic acid, stearamide, and stearic acid. In such embodiments, such mixtures comprise about 37 weight percent linoleamide, about 18% linoleic acid, about 19 weight percent oleamide, about 9 weight percent oleic acid, about 8.7 weight percent palmitamide, about 4.3 weight percent palmitic acid, About 1.2 wt% stearamide, and about 0.7 wt% stearic acid. It is to be understood that other composition amounts are possible and may depend on the naturally occurring amounts of linoleic acid, oleic acid, palmitic acid, and stearic acid in the corn oil used. For example, high-oleic corn oil as oil 26 or 26 ', which may have an oleic acid content of, for example, up to about 65% by weight, may be used using ordinary corn oil having an oleic acid content of about 24% by weight. It is expected that a mixture with higher oleamide and oleic acid content than would be produced in accordance with the reaction of Scheme (II). In some embodiments, corn oil fatty amides and corn oil fatty acids produced according to Scheme (II) may be mixed with fatty acids to vary the ratio of fatty amides to fatty acids in the fatty acid extractant 28 or 28 '. In some embodiments, the mixture of fatty amides and fatty acids may be in a ratio of about 2: 1 to about 1: 2 mixture.

See, eg, Kohlhase, et al., J. Am. Oil Chem. Soc. 48: 265-270, 1971, in some embodiments, fatty acid extraction from corn oil as oil 26 or 26 'using anhydrous ammonia as reactant as material 42 together with ammonium acetate as catalyst. Pure corn oil fatty amides as the agent (28 or 28 ') can be synthesized. In some embodiments, pure corn oil fatty amides may include linoleamide, oleamide, palmitamide, and stearamide. In such embodiments, the pure corn oil fatty amide may consist of about 55% linoleamide, about 28% oleamide, about 13% palmitamide, and about 2% stearamide. As mentioned above, it should be understood that other composition amounts are possible and may depend on the naturally occurring amounts of linoleic acid, oleic acid, palmitic acid, and stearic acid in the corn oil used.

In some embodiments, fatty acid extractant 28 or 28 'may comprise a fatty amide of formula R (C = 0) N (R') (R "), wherein R is optionally substituted by one or more double bonds. Are independently selected from the group consisting of intervening C ₃ to C ₂₇ alkyl groups, and R ′ and R ″ are independently selected from the group consisting of hydrogen and C ₁ to C ₆ alkyl groups optionally containing one or more hydroxyl groups.

In some embodiments, the oil (26 or 26 ') can be reacted, for example, by base hydrolysis using NaOH and water as material 42 (see, eg, Example 4 below), according to the reaction of Scheme III. Corn oil fatty acid as fatty acid extractant (28 or 28 ') can be synthesized from corn oil as

(III)

In some embodiments, pure corn oil fatty amides and pure corn oil fatty acids may be mixed to produce a single fatty acid extractant 28 or 28 '. Such a mixture of fatty amides to fatty acids may be a 2: 1 mixture, and in other embodiments may be for example 1: 2.

In some embodiments, for example, by reducing with tetrahydrofuran (THF) and LiAlH ₄ as substance 42 (see, eg, Example 3 below), according to the reaction of Scheme IV below, oil ( Fatty oil as fatty acid extractant 28 or 28 'from corn oil as 26 or 26'):

(IV)

In some embodiments, corn oil as oil 26 or 26 ′ may be contacted with methanol as acid 42 and an acid catalyst to produce fatty acid esters as fatty acid extractant 28 or 28 ′. For example, according to the reaction of Scheme V below, fatty acid esters can be obtained by reacting corn oil with alcohols including but not limited to alcohols of up to 8 carbons in the presence of sulfuric acid (e.g. See example 4):

(V)

In some embodiments, for example, according to the reaction of Scheme VI, fatty acid methyl ester (FAME) is produced (see Scheme V above) and FAME is further added to ethylene glycol as additional material 42 By reacting (see, eg, Example 5 below), corn oil as oil 26 or 26 'can be converted to corn oil ethylene glycol ester (FAGE) as fatty acid extractant 28 or 28':

(VI)

R- (C = O) -OMe + HO-CH 2 CH 2 - OH → R- (C = O) -O-CH 2 CH 2 - OH

In some embodiments, fatty alcohols can be hydroxylated and used as extractant. For example, as shown in Scheme VII, fatty alcohols can be reacted with peracetic acid followed by reaction with an aqueous acid to hydroxylate double bonds along the chain (see, eg, Example 8):

In some embodiments, the extractant may be a liquid or a solid, for example beads. Extractants available in solid form such as beads will be readily handled during the manufacturing process. In addition, product alcohols (eg butanol) may be recovered from such extractants by, for example, gas stripping, dissolution in other solvents, or any other applicable method known to those skilled in the art.

In some embodiments, including any of the electrical embodiments described in connection with FIGS. 1-5, the fermentation broth in fermentation vessel 30 is adapted to produce butanol via a biosynthetic route from one or more fermentable carbon sources to butanol. One or more recombinant microorganisms 32 that are wholly modified (ie, genetically engineered). In particular, recombinant microorganisms can be grown in fermentation broths containing suitable carbon substrates. Further carbon substrates include monosaccharides such as fructose; Oligosaccharides such as lactose maltose, or sucrose; Polysaccharides such as starch or cellulose; Or mixtures thereof, and crude mixtures from renewable feedstocks such as cheese whey permeate, corn steep liquor, beet molasses, and barley malt. Other carbon substrates may include ethanol, lactate, succinate or glycerol.

In addition, the carbon substrate may also be a monoatomic-carbon substrate, such as carbon dioxide or methanol, for which metabolic conversion to major biochemical intermediates has been demonstrated. Methyltrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine, and various amino acids in metabolic activity in addition to monoatomic and diatomic carbon substrates. For example, methyltrophic yeast is known to form trehalose or glycerol using carbon from methylamine (Bellion, et al., Microb. Growth C1 Compd., Int. Symp., 7th). (1993), 415-32, Editor (s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK]. Likewise, Candida of various species will metabolize alanine or oleic acid (Sulter, et al., Arch. Microbiol. 153: 485-489, 1990). Therefore, the carbon source used in the present invention may include a wide range of carbon containing substrates and is believed to be limited only by the choice of organism.

Although all of the aforementioned carbon substrates and mixtures thereof are considered suitable, in some embodiments, the carbon substrate is xylose and / or arabinose for yeast cells modified to use glucose, fructose and sucrose, or C5 sugars. Such as C5 sugars and mixtures thereof. Sucrose may be derived from renewable sugar sources such as sugar cane, sugar beet, cassava, sweet sorghum and mixtures thereof. Glucose and dextrose may be derived from renewable grain sources through the saccharification of feedstocks of starch systems including grains such as corn, wheat, rye, barley, oats and mixtures thereof. In addition, as described, for example, in US Patent Application Publication No. 2007/0031918 A1, which is incorporated herein by reference, the fermentable sugar is a cellulosic or lignocellulosic biorenewable through a process of pretreatment and saccharification. Can be derived from the mass. In addition to an appropriate carbon source (from aqueous stream 22), the fermentation broth is known to those skilled in the art, suitable for the promotion of enzymatic pathways and the growth of cultures comprising dihydroxyacid dehydratase (DHAD). It should contain suitable minerals, salts, cofactors, buffers, and other ingredients.

Recombinant microorganisms that produce butanol through biosynthetic pathways include Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, and Kleb. Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella , Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarois (Yarrowia), Pichia, Candida, Hansenula, or Saccharomyces. In one embodiment, the recombinant microorganism can be selected from the group consisting of Escherichia coli, Lactobacillus plantarum, and Saccharomyces cerevisiae. In one embodiment, the recombinant microorganisms are Saccharomyces, Zygosaccharomyces, Schizocarcinomyces, Dekkera, Torulopsis, Bretanomyces, and Crabtree-positive yeast selected from some species of Candida. Crabtree-positive yeast species include Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Saccharomyces bayanus, and Saccharomyces bayanus. Saccharomyces mikitae, Saccharomyces paradoxus, Zygosaccharomyces rouxii, and Candida glabrata. . For example, in addition to the production of butanol using fermentation using microorganisms, microorganisms producing butanol are disclosed and known, for example, in US Patent Application Publication No. 2009/0305370, which is incorporated herein by reference. . In some embodiments, the microorganism comprises a butanol biosynthetic pathway. Suitable isobutanol biosynthesis routes are known in the art (see, eg, US Patent Application Publication No. 2007/0092957, incorporated herein by reference). In some embodiments, at least one, at least two, at least three, or at least four polypeptides that catalyze substrate to product conversion of the pathway are encoded by heterologous polynucleotides in the microorganism. In some embodiments, all polypeptides that catalyze substrate to product conversion of the pathway are encoded by heterologous polynucleotides in the microorganism. In some embodiments, the microorganism comprises reducing or eliminating pyruvate decarboxylase activity. Microorganisms substantially free of pyruvate decarboxylase activity are described in US Patent Application Publication No. 2009/0305363, which is incorporated herein by reference.

Provided herein is the construction of certain strains, including those used in the Examples.

Construction of Saccharomyces cerevisiae strain BP1064 and isobutanologen BP1083 (NGCI-070)

Strain BP1064 is derived from CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Center, Netherlands) and contains deletions of the following genes: URA3, HIS3, PDC1, PDC5, PDC6 and GPD2. . BP1064 was transfected with plasmid pYZ090 (SEQ ID NO: 1, its construction is described in US Patent Application No. 61 / 246,844, filed September 29, 2009) and pLH468 (SEQ ID NO: 2) The conversion resulted in isobutanologen strain NGCI-070 (BP1083).

Deletion in which the entire coding sequence is completely removed by homologous recombination with a G418 resistance marker or URA3 gene for selection of transformants and PCR fragments containing regions of homology upstream and downstream of the target gene. Generated. G418 resistance markers flanked by loxP sites were removed using Cre recombinase. To generate Scarris deletions, the URA3 gene was removed by homologous recombination, or by Cre recombinase if loxP sites were flanked.

Scarris deletion procedures were employed from Akada, et al., Yeast 23: 399-405, 2006. Generally, a PCR cassette for each scarless deletion was made by combining four fragments, A-B-U-C, by overlapping PCR. The PCR cassette consisting of the native CEN.PK 113-7D URA3 gene, along with a promoter (250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the URA3 gene), provides a selectable / counter-selectable marker, URA3 (fragment U). It contained. Fragments A and C, each 500 bp in length, correspond to 500 bp upstream of the target gene (fragment A) and 3 '500 bp of the target gene (fragment C). Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. Fragment B (500 bp in length) corresponds to 500 bp adjacent downstream of the target gene, and since direct repetition of the sequence corresponding to Fragment B was generated upon incorporation of the cassette into the chromosome, from homologous recombination chromosome It was used for the exclusion of the URA3 marker and fragment C. Using the PCR product ABUC cassette, the URA3 marker was first integrated into the chromosome by homologous recombination and then excised from the chromosome. Early integration resulted in the deletion of genes other than 3 '500 bp. Upon ablation, the 3 '500 bp region of the gene was also deleted. For integration of genes using this method, the gene to be integrated was included in a PCR cassette between fragments A and B.

URA3 deletion

To delete the endogenous URA3 coding region, the ura3 :: loxP-kanMX-loxP cassette was PCR amplified from pLA54 template DNA (SEQ ID NO: 3). pLA54 contains the K. lactis TEF1 promoter and kanMX marker, and the loxP site is flanked to allow recombination and removal of the marker using Cre recombinase. PCR using Fusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) And primers BK505 and BK506 (SEQ ID NOs: 4 and 5) Was run. The URA3 portion of each primer was derived from the 5 'region upstream of the URA3 promoter and the 3' region downstream of the coding region, resulting in the replacement of the URA3 coding region by integration of the loxP-kanMX-loxP marker. PCR products were transformed into CEN.PK 113-7D using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), and The converter was selected on YPD containing G418 (100 μg / mL) at 30 ° C. The transformants were screened to confirm correct integration by PCR using primers LA468 and LA492 (SEQ ID NOs 6 and 7, respectively) and named CEN.PK 113-7D Δura3 :: kanMX.

HIS3 fruit

Fusion® High Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA), and Gentra® Puregene (registered) Amplify four fragments for PCR cassettes for Scarris HIS3 deletion using CEN.PK 113-7D genomic DNA as a template made from East / Back, a kit (Qiagen, Valencia, CA) I was. HIS3 fragment A was amplified with primer oBP453 (SEQ ID NO: 15) containing a primer oBP452 (SEQ ID NO: 14) and a 5 'tail homologous to the 5' end of HIS3 fragment B. Primer oBP454 containing the 5 'tail homologous to the 3' end of HIS3 fragment A (SEQ ID NO: 16), and primer oBP455 containing the 5 'tail homologous to the 5' end of HIS3 fragment U ( SEQ ID NO: 17) to amplify HIS3 fragment B. Primer oBP456 containing a 5 'tail homologous to the 3' end of HIS3 fragment B (SEQ ID NO: 18), and primer oBP457 containing a 5 'tail homologous to the 5' end of HIS3 fragment C ( SEQ ID NO: 19) amplified HIS3 fragment U. HIS3 fragment C was amplified with primer oBP458 (SEQ ID NO: 20), and primer oBP459 (SEQ ID NO: 21) containing a 5 'tail homologous to the 3' end of HIS3 fragment U. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). HIS3 fragment AB was generated by overlapping PCR by mixing HIS3 fragment A and HIS3 fragment B and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP455 (SEQ ID NO: 17). HIS3 fragment UC was generated by overlapping PCR by mixing HIS3 fragment U and HIS3 fragment C and amplifying with primers oBP456 (SEQ ID NO: 18) and oBP459 (SEQ ID NO: 21). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, CA). The HIS3 ABUC cassette was generated by overlapping PCR by mixing HIS3 fragment AB and HIS3 fragment UC and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP459 (SEQ ID NO: 21). PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA).

Receptor cells of CEN.PK 113-7D Δura3 :: kanMX were prepared and HIS3 ABUC PCR cassettes using the Frozen-EZ East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA). And transformed. Transformation mixtures were plated on synthetic complete medium supplemented with 2% glucose at 30 ° C. and lacking uracil. Gentra (registered trademark) fue (registered trademark) yeast / bag. By PCR using genomic DNA made from the kit (Qiagen, Valencia, Calif.), Transformants with his3 knockout were screened with primers oBP460 (SEQ ID NO: 22) and oBP461 (SEQ ID NO: 23). The correct transformants were selected as strain CEN.PK 113-7D Δura3 :: kanMX Δhis3 :: URA3.

Removal of the KanMX marker from the Δura3 site and removal of the URA3 marker from the Δhis3 site

CEN.PK 113-7D Δura3 :: kanMX Δhis3 :: URA3 using the Frozen-EZ East Transformation II (trademark) kit (Zymo Research Corporation, Irvine, Calif.), And pRS423 :: PGAL1-cre (SEQ ID NO: 66). KanMX markers were removed by transformation, and described on US Provisional Application No. 61 / 290,639) and plating on synthetic complete medium supplemented with 2% glucose at 30 ° C. and lacking histidine and uracil. Transformants were grown for 6 hours at 30 ° C. in YP supplemented with 1% galactose to induce Cre recombinase and KanMX marker ablation and plated on YPD (2% glucose) plates at 30 ° C. for recovery. The isolates were grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (5-FOA, 0.1%) at 30 ° C. to select isolates that lost the URA3 marker. 5-FOA resistant isolates were grown and plated on YPD to remove the pRS423 :: PGAL1-cre plasmid. For loss of KanMX marker, URA3 marker, and pRS423 :: PGAL1-cre plasmid by assay growth on YPD + G418 plate, synthetic complete medium plate lacking uracil, and synthetic complete medium plate lacking histidine. Isolates were identified. The correct isolate, sensitive to G418 and nutritional to uracil and histidine, was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 and named BP857. Gentra (registered trademark) fue (registered trademark) yeast / bag. Using genomic DNA made from the kit (Qiagen, Valencia, CA), primers oBP450 (SEQ ID NO: 24) and oBP451 (SEQ ID NO: 25) and primers oBP460 (SEQ ID NO: 22) and oBP461 (SEQ ID NO: 22) for Δura3 Deletion and marker removal were confirmed by sequencing and PCR using SEQ ID NO: 23).

PDC6 deletion

Fusion® High Fidelity PCR Master Mix (New England Biolabs, Ipswich, Mass.) And Gentra® Fused® East / Back. CEN.PK 113-7D genomic DNA as a template made with the kit (Qiagen, Valencia, CA) was used to amplify four fragments for the PCR cassette for Scaris PDC6 deletion. PDC6 fragment A was amplified with primer oBP440 (SEQ ID NO: 26) and primer oBP441 (SEQ ID NO: 27) containing a 5 'tail homologous to the 5' end of PDC6 fragment B. Primer oBP442 containing a 5 'tail with homology to the 3' end of PDC6 fragment A (SEQ ID NO: 28), and primer oBP443 containing a 5 'tail with homology to the 5' end of PDC6 fragment U ( SEQ ID NO: 29) to amplify PDC6 fragment B. Primer oBP444 containing a 5 'tail with homology to the 3' end of PDC6 fragment B (SEQ ID NO: 30), and primer oBP445 containing a 5 'tail with homology to the 5' end of PDC6 fragment C ( SEQ ID NO: 31) to amplify PDC6 fragment U. PDC6 fragment C was amplified with primer oBP446 (SEQ ID NO: 32), and primer oBP447 (SEQ ID NO: 33) containing a 5 'tail with homology to the 3' end of PDC6 fragment U. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). PDC6 fragment AB was generated by overlapping PCR by mixing PDC6 fragment A and PDC6 fragment B and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP443 (SEQ ID NO: 29). PDC6 fragment UC was generated by overlapping PCR by mixing PDC6 fragment U and PDC6 fragment C and amplifying with primers oBP444 (SEQ ID NO: 30) and oBP447 (SEQ ID NO: 33). The resulting PCR product was purified on an agarose gel followed by a gel extraction kit (Qiagen, Valencia, CA). The PDC6 ABUC cassette was generated by overlapping PCR by mixing PDC6 fragment AB and PDC6 fragment UC and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP447 (SEQ ID NO: 33). PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA).

Receptor cells of CEN.PK 113-7D Δura3 :: loxP Δhis3 were made and transformed with a PDC6 ABUC PCR cassette using the Frozen-EZ East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA). . The transformation mixture was plated on synthetic complete medium supplemented with 2% glucose at 30 ° C. and lacking uracil. Gentra (registered trademark) fue (registered trademark) yeast / bag. Using genomic DNA prepared from the kit (Qiagen, Valencia, Calif.), Transformants with pdc6 knockout were screened by PCR using primers oBP448 (SEQ ID NO: 34) and oBP449 (SEQ ID NO: 35). The correct transformants were selected with strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 :: URA3.

CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 :: URA3 were grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C. The isolates that lost markers were selected. Gentra (registered trademark) fue (registered trademark) yeast / bag. Using genomic DNA made from the kit (Qiagen, Valencia, CA), deletions and marker removal were confirmed by sequencing and PCR using primers oBP448 (SEQ ID NO: 34) and oBP449 (SEQ ID NO: 35). The negative PCR results using primers specific for the coding sequence of PDC6, oBP554 (SEQ ID NO: 36) and oBP555 (SEQ ID NO: 37), demonstrated the absence of the PDC6 gene from the isolate. The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 and named BP891.

PDC1 deletion ilvDSm integration

PDC1 gene was deleted and Streptococcus mutans ATCC No. Replaced with the ilvD coding region from 700610. Following the A fragment, the ilvD coding region from Streptococcus mutans for the PCR cassette for PDC1 deletion-ilvDSm integration was incorporated into a Fusion® High Fidelity PCR Master Mix (New England Biolabs Inc., Massachusetts). Switch material), and Gentra (trademark) fused (trademark) yeast / bag. Amplified using genomic DNA NYLA83 (as described herein and in US Provisional Application No. 61 / 246,709) as a template made with the kit (Qiagen, Valencia, Calif.). PDC1 fragment A-ilvDSm (SEQ ID NO: 138) was amplified with primer oBP515 (SEQ ID NO: 39) containing a primer oBP513 (SEQ ID NO: 38) and a 5 'tail homologous to the 5' end of PDC1 fragment B. Fusion® High Fidelity PCR Master Mix (New England Biolabs, Ipswich, Mass.), And Gentra® Fused® East / Back. CEN.PK 113-7D genomic DNA as a template made with the kit (Qiagen, Valencia, CA) was used to amplify the B, U, and C fragments for the PCR cassette for PDC1 deletion-ilvDSm integration. Primer oBP516 (SEQ ID NO: 40) containing a 5 'tail with homology to the 3' end of PDC1 fragment A-ilvDSm, and a primer containing a 5 'tail with homology to the 5' end of PDC1 fragment U PDC1 fragment B was amplified with oBP517 (SEQ ID NO: 41). Primer oBP518 containing a 5 'tail with homology to the 3' end of PDC1 fragment B (SEQ ID NO: 42), and primer oBP519 containing a 5 'tail with homology to the 5' end of PDC1 fragment C ( SEQ ID NO: 43) to amplify PDC1 fragment U. PDC1 fragment C was amplified with primer oBP520 (SEQ ID NO: 44), and primer oBP521 (SEQ ID NO: 45) containing a 5 'tail with homology to the 3' end of PDC1 fragment U. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). PDC1 fragment A-ilvDSm-B was generated by overlapping PCR by mixing PDC1 fragment A-ilvDSm and PDC1 fragment B and amplifying with primers oBP513 (SEQ ID NO: 38) and oBP517 (SEQ ID NO: 41). PDC1 fragment UC was generated by overlapping PCR by mixing PDC1 fragment U and PDC1 fragment C and amplifying with primers oBP518 (SEQ ID NO: 42) and oBP521 (SEQ ID NO: 45). The resulting PCR product was purified on an agarose gel followed by a gel extraction kit (Qiagen, Valencia, CA). PDC1 A-ilvDSm-BUC cassette (SEQ ID NO: 139) by overlapping PCR by mixing PDC1 fragment A-ilvDSm-B and PDC1 fragment UC and amplifying with primers oBP513 (SEQ ID NO: 38) and oBP521 (SEQ ID NO: 45) Generated. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA).

Receptor cells of CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 were prepared and PDC1 A-ilvDSm-BUC PCR using the Frozen-EZ East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA). The cassette was transformed. Transformation mixtures were plated on synthetic complete medium supplemented with 2% glucose at 30 ° C. and lacking uracil. Gentra (registered trademark) fue (registered trademark) yeast / bag. Transformants with pdc1 knockout ilvDSm integration were generated by PCR using primers oBP511 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47) using genomic DNA made from the kit (Qiagen, Valencia, CA). Screened. Negative PCR results using primers specific for the coding sequence of PDC1, oBP550 (SEQ ID NO: 48) and oBP551 (SEQ ID NO: 49), demonstrated the absence of the PDC1 gene from the isolate. The correct transformants were selected with strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm-URA3.

CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm-URA3 were grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C. Thus, isolates that lost the URA3 marker were selected. Gentra (registered trademark) fue (registered trademark) yeast / bag. Deletion of PDC1, integration of ilvDSm by sequencing and PCR using primers oBP511 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47), using genomic DNA made from the kit (Qiagen, Valencia, CA), And marker removal was confirmed. The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm and named BP907.

PDC5 elimination sadB integration

The PDC5 gene was deleted and replaced with the sadB coding region from Achromobacter xylosoxidans. First, a segment of the PCR cassette for PDC5 deletion-sadB integration was cloned into the plasmid pUC19-URA3MCS.

pUC19-URA3MCS is based on pUC19 and contains the sequence of the URA3 gene from Saccharomyces cerevisiae located within a multiple cloning site (MCS). pUC19 contains pMB1 replicon and a gene encoding beta-lactamase for replication and selection in Escherichia coli. In addition to the coding sequences for URA3, sequences from upstream and downstream of these genes were included for expression of the URA3 gene in yeast. The vector may be used for cloning purposes and may be used as a yeast integration vector.

URA3 coding region from Saccharomyces cerevisiae CEN.PK 113-7D genomic DNA using Fusion® High Fidelity PCR Master Mix (New England Biolabs, Ipswich, Mass.) DNA comprising the URA3 coding region with 250 bp upstream and 150 bp downstream of primer oBP438 (SEQ ID NO: 12) containing BamHI, AscI, PmeI, and FseI restriction sites, and XbaI, PacI, and NotI restriction sites Amplified with oBP439 (SEQ ID NO: 13) containing. Gentra (registered trademark) fue (registered trademark) yeast / bag. Genomic DNA was prepared using a kit (Qiagen, Valencia, CA). The PCR product and pUC19 (SEQ ID NO: 140) were digested with BamHI and XbaI and then ligated with T4 DNA ligase to generate the vector pUC19-URA3MCS. Vectors were identified by sequencing and PCR using primers oBP264 (SEQ ID NO: 10) and oBP265 (SEQ ID NO: 11).

The coding sequence for sadB and PDC5 fragment B was cloned into pUC19-URA3MCS to generate the sadB-BU portion of the PDC5 A-sadB-BUC PCR cassette. primer oBP530 containing the AscI restriction site (SEQ ID NO: 50) using pLH468-sadB (SEQ ID NO: 67) as a template, and primer oBP531 (containing 5 'tail with homology to the 5' end of PDC5 fragment B) SEQ ID NO: 51) amplified the coding sequence of sadB. PDC5 fragment B was amplified with primer oBP532 (SEQ ID NO: 52) containing a 5 'tail with homology to the 3' end of sadB, and primer oBP533 (SEQ ID NO: 53) containing a PmeI restriction site. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). sadB-PDC5 fragment B was generated by overlapping PCR by mixing the sadB and PDC5 fragment B PCR products and amplifying with primers oBP530 (SEQ ID NO: 50) and oBP533 (SEQ ID NO: 53). The resulting PCR product was digested with AscI and PmeI and ligated into the corresponding sites of pUC19-URA3MCS after digestion with appropriate enzymes using T4 DNA ligase. The resulting plasmid was amplified of sadB-fragment B-fragment U using primers oBP546 (SEQ ID NO: 55) and oBP536 (SEQ ID NO: 54) containing 5 'tails having homology to the 5' end of PDC5 fragment C. Used as a template for. PDC5 fragment C was amplified with primer oBP547 (SEQ ID NO: 56), and primer oBP539 (SEQ ID NO: 57) containing a 5 'tail with homology to the 3' end of PDC5 sadB-fragment B-fragment U. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). PDC5 sadB-fragment B-fragment U-fragment C was synthesized by overlapping PCR by mixing PDC5 sadB-fragment B-fragment U and PDC5 fragment C and amplifying with primers oBP536 (SEQ ID NO: 54) and oBP539 (SEQ ID NO: 57). Generated. The resulting PCR product was purified on an agarose gel followed by a gel extraction kit (Qiagen, Valencia, CA). PDC5 sadB-fragment B-fragment U-fragment C was replaced with primers oBP542 (SEQ ID NO: 58), and oBP539 (SEQ ID NO: 57) containing a 5 'tail with homology to 50 nucleotides just upstream of the native PDC5 coding sequence. Amplification produced the PDC5 A-sadB-BUC cassette (SEQ ID NO: 141). PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA).

Receptor cells of CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm were made and PDC5 A-sadB using the Frozen-EZ East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA). Transformed with -BUC PCR cassette. Transformation mixtures were plated on synthetic complete medium supplemented with 1% ethanol (no glucose) at 30 ° C. and lacking uracil. Gentra (registered trademark) fue (registered trademark) yeast / bag. Screening for transformants with pdc5 knockout sadB integration by PCR using primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60) using genomic DNA made from the kit (Qiagen, Valencia, CA) It was. The negative PCR results using primers specific for the coding sequence of PDC5, oBP552 (SEQ ID NO: 61) and oBP553 (SEQ ID NO: 62), demonstrated the absence of the PDC5 gene from the isolate. The correct transformants were selected with strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3.

CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3 are grown overnight in YPE (1% ethanol), supplemented with ethanol (no glucose) and 5-fluoro-orotic acid Plated at 30 ° C. on synthetic complete medium containing (0.1%) to select isolates that lost the URA3 marker. Gentra (registered trademark) fue (registered trademark) yeast / bag. Deletion of PDC5, integration of sadB, and marker removal by PCR using primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60) using genomic DNA made from the kit (Qiagen, Valencia, CA) It was confirmed. The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB and named BP913.

GPD2 Fruit

To delete the endogenous GPD2 coding region, the gpd2 :: loxP-URA3-loxP cassette (SEQ ID NO: 142) was PCR-amplified using loxP-URA3-loxP (SEQ ID NO: 68) as template DNA. loxP-URA3-loxP contains a URA3 marker from (ATCC No. 77107) flanked by loxP recombinase sites. PCR was performed using Fusion® DNA polymerase (New England Biolabs, Ipswich, Mass.) And primers LA512 and LA513 (SEQ ID NOs: 8 and 9). The GPD2 portion of each primer was derived from the 5 'region upstream of the GPD2 encoding region and the 3' region downstream of the encoding region, resulting in the replacement of the GPD2 encoding region by integration of the loxP-URA3-loxP marker. PCR products were transformed into BP913 and transformants were selected on synthetic complete medium supplemented with 1% ethanol (no glucose) and lacking uracil. Transformants were screened to confirm correct integration by PCR using primers oBP582 and AA270 (SEQ ID NOs 63 and 64).

URA3 markers were reused by transforming with pRS423 :: PGAL1-cre (SEQ ID NO: 66) and plating on synthetic complete medium supplemented with 1% ethanol at 30 ° C. and lacking histidine. Transformants were streaked on synthetic complete medium supplemented with 1% ethanol and containing 5-fluoro-orotic acid (0.1%) and incubated at 30 ° C. to select isolates that lost the URA3 marker. For removal of the pRS423 :: PGAL1-cre plasmid, 5-FOA resistant isolates were grown in YPE (1% ethanol). Deletion and marker removal were confirmed by PCR using primers oBP582 (SEQ ID NO: 63) and oBP591 (SEQ ID NO: 65). The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB Δgpd2 :: loxP and named PNY1503 (BP1064).

BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1) and pLH468 (SEQ ID NO: 2) to generate strain NGCI-070 (BP1083; PNY1504).

Strain NYLA74  And NYLA83 of Construction

Insertion-inactivation of the endogenous PDC1 and PDC6 genes of S. cerevisiae. The PDC1, PDC5, and PDC6 genes encoding the three major isozymes of pyruvate decarboxylase are described as follows:

Construction of pRS425 :: GPM-sadB

A DNA fragment encoding a butanol dehydrogenase (SEQ ID NO: 70) from Acromobacter xyloxidans (disclosed in US Patent Application Publication 2009/0269823) was cloned. Using Gentra® Purified® Kit (Qiagen, Valencia, CA) according to the recommended protocol for Gram negative organisms using forward and reverse primers N473 and N469 (SEQ ID NOs: 74 and 75), respectively The coding region (SEQ ID NO: 69) of this gene, called sadB for secondary alcohol dehydrogenase, was amplified using standard conditions from A. xyloxyidans genomic DNA prepared. PCR products were cloned into TOPO (R) -blunt into pCR (R) 4 Blunt (Invitrogen (R), Carlsbad, CA) to generate pCR4 blunt :: sadB And transformed into E. coli Match-1 cells. The plasmid was then isolated from four clones and the sequence confirmed.

The sadB coding region was PCR amplified from pCR4Blunt :: sadB. PCR primers contained an additional 5 'sequence to be overlapped with the yeast GPM1 promoter and ADH1 terminators (N583 and N584 provided as SEQ ID NOs: 76 and 77). Thereafter, PCR products were cloned into Saccharomyces cerevisiae using a "gap repair" technique (Ma, et al., Gene 58: 201-216, 1987). . GPM1 promoter (SEQ ID NO: 72), kivD coding region from Lactococcus lactis (SEQ ID NO: 71), and ADH1 terminator (SEQ ID NO: 73) (US Patent Application Publication No. 2007/0092957 A1, Example 17) Yeast-E. E. coli shuttle vector pRS425 :: GPM :: kivD :: ADH was digested with BbvCI and PacI restriction enzymes to free the kivD coding region. Approximately 1 μg of the remaining vector fragment was transformed into S. cerevisiae strain BY4741 with 1 μg of sadB PCR product. Transformants were selected in synthetic complete medium lacking leucine. PCR using primers N142 and N459 (SEQ ID NOs 108 and 109) confirmed the appropriate recombination event to generate pRS425 :: GPM-sadB (SEQ ID NO: 124).

pDC6 :: P _GPM1- Construction of the sadB Integrated Cassette and Deletion of PDC6

The pdc6 :: PGPM1-sadB-ADH1t-URA3r integration cassette was prepared by linking the GPM-sadB-ADHt segment (SEQ ID NO: 79) from pRS425 :: GPM-sadB (SEQ ID NO: 78) to the URA3r gene from pUC19-URA3r. It was. To enable in vivo homologous recombination and removal of the URA3 marker, pUC19-URA3r (SEQ ID NO: 80) contains a URA3 marker from pRS426 (ATCC No. 77107) flanked by 75 bp homologous repeat sequences. . Fusion® DNA polymerase (New England Biolabs, Ipswich, Mass.) And primers 114117-11A through 114117- using pRS425 :: GPM-sadB and pUC19-URA3r plasmid DNA as templates. SOE PCR (Horton, et al., Gene 77: 61-68, 1989) with 11D (SEQ ID NOs 81, 82, 83, and 84), and 114117-13A and 114117-13B (SEQ ID NOs 85 and 86). Two DNA segments were linked) as described above.

External primers for SOE PCR (114117-13A and 114117-13B) contained approximately 50 bp regions of 5 'and 3' homology to the upstream and downstream regions of the PDC6 promoter and terminator, respectively. Using standard gene techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), the completed cassette PCR fragments were incorporated into BY4700 (ATCC No. 200866). Transformations and transformants were maintained at 30 ° C. on synthetic complete medium lacking uracil and supplemented with 2% glucose. The transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs: 87 and 88), and 112590-34F and 112590-49E (SEQ ID NOs: 89 and 90) to determine the deletion of the PDC6 coding region. Together, integration at the PDC6 locus was confirmed. URA3r markers were reused by plating on synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Marker removal was confirmed by patching colonies from 5-FOA plates onto SD-URA medium to confirm the absence of growth. The resulting identified strains have the following genotypes: BY4700 pdc6 :: PGPM1-sadB-ADH1t.

pdc1 :: Construction of the PPDC1-ilvD Integration Cassette and PDC1 Deletion

Fusion® DNA polymerase (New England Biolabs, Ipswich, Mass.) And primers 114117-27A to 114117-27D (SEQ ID NOs: 110) using pLH468 and pUC19-URA3r plasmid DNA as templates. , 111, 112, and 113) ilvD-FBA1t segment from pLH468 (SEQ ID NO: 2) by SOE PCR (as described in Horton, et al., Gene 77: 61-68, 1989). Pdc1 :: PPDC1-ilvD-FBA1t-URA3r integration cassette was prepared by linking SEQ ID NO: 91) to the URA3r gene from pUC19-URA3r.

The external primers for SOE PCR (114117-27A and 114117-27D) contained approximately 50 bp regions of 5 'and 3' homology to the downstream region of the PDC1 promoter and the downstream region of the PDC1 coding sequence. Using standard gene techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), the completed cassette PCR fragment was BY4700 pdc6 :: PGPM1-sadB- Transformed into ADH1t and transformants were maintained at 30 ° C. on synthetic complete media lacking uracil and supplemented with 2% glucose. The transformants were screened by PCR using primers 114117-36D and 135 (SEQ ID NOs: 92 and 93), and primers 112590-49E and 112590-30F (SEQ ID NOs: 90 and 94), along with deletion of the PDC1 coding sequence. Integration at the PDC1 locus was confirmed. URA3r markers were reused by plating on synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Marker removal was confirmed by patching colonies from 5-FOA plates onto SD-URA medium to confirm the absence of growth. The resulting identified strain “NYLA67” has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t.

HIS3 fruit

To delete the endogenous HIS3 coding region, the his3 :: URA3r2 cassette was PCR-amplified from the URA3r2 template DNA (SEQ ID NO: 95). To enable in vivo homologous recombination and removal of the URA3 marker, URA3r2 contains a URA3 marker from pRS426 (ATCC No. 77107) flanked by 500 bp homologous repeat sequences. PCR was performed using Fusion® DNA polymerase (New England Biolabs, Ipswich, Mass.) And primers 114117-45A and 114117-45B (SEQ ID NOs: 96 and 97). A 2.3 kb PCR product was generated. The HIS3 portion of each primer was derived from the 5 'region upstream of the HIS3 promoter, and the 3' region downstream of the coding region, so that the integration of the URA3r2 marker resulted in the replacement of the HIS3 coding region. PCR products were transformed into NYLA67 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and at 30 ° C. Transformants were selected on synthetic complete media lacking and supplemented with 2% glucose. Transformants were screened by replicating the transformants on synthetic complete medium lacking histidine at 30 ° C. and supplemented with 2% glucose to confirm correct integration. The URA3r marker was reused by plating on synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Marker removal was confirmed by patching colonies from 5-FOA plates onto SD-URA medium to confirm the absence of growth. The resulting identified strain, called NYLA73, has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3.

Construction of the pdc5 :: kanMX Integrated Cassette and PDC5 Deletion

Strain YLR134W chromosomal DNA (SEQ ID NOs: 98 and 99) using Fusion® DNA polymerase (New England Biolabs, Ipswich, Mass.) And primers PDC5 :: KanMXF and PDC5 :: KanMXR (SEQ ID NOs: 98 and 99) Pdc5 :: kanMX4 cassette from ATCC No. 4034091) was PCR-amplified to produce a ˜2.2 kb PCR product. The PDC5 portion of each primer was derived from the 5 'region upstream of the PDC5 promoter, and from the 3' region downstream of the coding region, allowing the integration of the kanMX4 marker to replace the PDC5 coding region. PCR products were transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), 1% ethanol and Geneti Transformants were selected at 30 ° C. on YP medium supplemented with shin (200 μg / mL). Transformants were screened by PCR using primers PDC5kofor and N175 (SEQ ID NOs: 100 and 101) to confirm correct integration at the PDC locus with replacement of the PDC5 coding region. The exact transformant identified has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3 pdc5 :: kanMX4. The strain was named NYLA74.

Deletion of HXK2 (hexokinase II)

Hxk2 :: URA3r cassette from URA3r2 template (above) using Fusion® DNA polymerase (New England Biolabs, Ipswich, Mass.) And primers 384 and 385 (SEQ ID NOs: 102 and 103) Was PCR-amplified to produce ˜2.3 kb PCR product. The HXK2 portion of each primer was derived from the 5 'region upstream of the HXK2 promoter, and the 3' region downstream of the coding region, so that integration of the URA3r2 marker caused replacement of the HXK2 coding region. PCR products were transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), lacking uracil and 2% Transformants were selected at 30 ° C. on synthetic complete medium supplemented with glucose. Transformants were screened by PCR using primers N869 and N871 (SEQ ID NOs: 104 and 105) to confirm correct integration at the HXK2 locus with replacement of the HXK2 coding region. The URA3r2 marker was reused by plating on synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Confirmation of marker removal by patching colonies from 5-FOA plates onto SD-URA medium and confirming the absence of growth and confirming correct marker removal by PCR using primers N946 and N947 (SEQ ID NOs 106 and 107). It was. The resulting identified strain named NYLA83 has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3 Δhxk2.

NYLA93's work

Insertion-inactivation of the endogenous PDC1, PDC5, and PDC6 genes of S. cerevisiae is described below. The PDC1, PDC5, and PDC6 genes encode three major isozymes of pyruvate decarboxylase. The resulting PDC inactivated strain was used as a host for the expression vectors pYZ067 (SEQ ID NO: 129) and pYZ090 (SEQ ID NO: 1), the construction of which was incorporated by reference in the United States, filed on September 29, 2009. Provisional application 61 / 246,844.

Deletion of NAD-dependent Glycerol 3-Phosphate Dehydrogenase

From pUC19 :: loxP-URA3-loxP plasmid template using Fusion® DNA polymerase (New England Biolabs, Ipswich, Mass.) And primers LA512 and LA513 (SEQ ID NOs: 8 and 9) The gpd2 :: loxP-URA3-loxP cassette was PCR-amplified to produce ˜1.6 kb PCR product. pUC19 :: loxP-URA3-loxP (SEQ ID NO: 130) contains the URA3 marker from flanked by loxP recombinase sites (ATCC No. 77107). The GPD2 portion of each primer was derived from the 5 'region upstream of the GPD2 promoter and the 3' region downstream of the coding region, so that integration of the loxP-URA3-loxP marker caused replacement of the GPD2 coding region. PCR products were transformed into NYLA83 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), lacking uracil and 2% Transformants were selected at 30 ° C. on synthetic complete medium supplemented with glucose. Transformants were screened by PCR using primers LA516 and N175 (SEQ ID NOs 132 and 101) to confirm correct integration at the GPD2 locus with replacement of the HXK2 coding region. URA3 markers are reused by transformation with pRS423 :: P _GAL1- cre (SEQ ID NO: 131) and plating at 30 ° C. on synthetic complete media lacking histidine and supplemented with 2% glucose. Colonies are patched at 30 ° C. on YP (1% galactose) plates to induce URA3 marker ablation and transfer on YPD plates at 30 ° C. for recovery. Removal of the URA3 marker is confirmed by patching colonies from YPD plates onto synthetic complete media lacking uracil to confirm the absence of growth. The exact clones identified have the following genotype: BY4700 pdc6 :: P _GPM1- sadB-ADH1t pdc1 :: P _PDC1- ilvD-FBA1t Δhis3 Δhxk2 Δgpd2 :: loxP. The strain was named NYLA92.

Construction of the pdc5 :: loxP-kanMX-loxP Integrated Cassette and PDC5 Deletion

Plasmid pUC19 :: loxP-kanMX-loxP (SEQ ID NO.) Using Fusion® DNA polymerase (New England Biolabs, Ipswich, Mass.) And primers LA249 and LA397 (SEQ ID NOs: 136 and 137) Pdc5 :: loxP-kanMX-loxP cassette from No. 135) was PCR-amplified to produce a ˜2.2 kb PCR product. pUC19 :: loxP-kanMX-loxP (SEQ ID NO: 135) is the K. lactis TEF1 promoter and terminator flanked by loxP recombinase sites and the kanMX gene from pFA6 (Wach, et al., Yeast 10: 1793 -1808, 1994). The PDC5 portion of each primer was derived from the 5 'region upstream of the PDC5 promoter and the 3' region downstream of the coding region, so that integration of the loxP-kanMX-loxP marker caused replacement of the PDC5 coding region. PCR products were transformed into NYLA92 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), 1% ethanol and Geneti Transformants were selected at 30 ° C. on YP medium supplemented with shin (200 μg / ml). Transformants were screened by PCR using primers LA363 and LA364 (SEQ ID NOs 133 and 134) to confirm correct integration at the PDC5 locus with replacement of the PDC5 coding region. The exact transformants identified have the following genotypes: BY4700 pdc6 :: P _{GPM1 -} sadB-ADH1t pdc1 :: P _{PDC1 -} ilvD-FBA1t Δhis3 Δhxk2 Δgpd2 :: loxP Δpdc5: loxP-kanMX-loxP. The strain was named NYLA93.

NYLA93 (pYZ067 / pYZ090)

Using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), plasmid vectors pYZ067 and pYZ090 were used to isolate strain NYLA93 (BY4700 pdc6 :: P _GPM1- sadB-ADH1t pdc1: : P _PDC1- ilvD-FBA1t Δhis3 Δhxk2 Δgpd2 :: loxP Δpdc5: loxP-kanMX-loxP) and simultaneously transformed the resulting strains (isobutanologen NYLA93, also called NGCI-065) lacking histidine and uracil It was kept at 30 ° C. on synthetic complete medium supplemented with 1% ethanol.

Expression vector pLH468

PLH468 plasmid (SEQ ID NO: 2) was constructed for the expression of DHAD, ketoisovaleric decarboxylase (KivD) and horse liver alcohol dehydrogenase (HADH) in yeast.

Coding for Lactococcus lactis ketoisovalerate decarboxylase (KivD) and hepatic alcohol dehydrogenase (HADH) by DNA2.0 based codon optimized for expression in Saccharomyces cerevisiae Regions were synthesized (SEQ ID NOs 71 and 117, respectively) and provided within plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0. The encoded proteins are SEQ ID NOs: 116 and 118, respectively. Individual expression vectors for KivD and HADH were constructed. To assemble pLH467 (pRS426 :: P _TDH3- kivDy-TDH3t), US patent application publication no. 2008/0182308, also referred to herein, also referred to herein as the vector pNY8 (SEQ ID NO: 120; pRS426.GPD-ald-GPDt) The GPD promoter and ald coding region were excised by digesting No., described in Example 17) with AscI and SfiI enzymes. PCR amplification of the TDH3 promoter fragment (SEQ ID NO: 121) from pNY8 using 5 'primer OT1068 and 3' primer OT1067 (SEQ ID NOs: 122 and 123), resulting in the AscI site at the 5 'end and the SpeI site at the 3' end. Added. The pNY8 vector fragment digested with AscI / SfiI was ligated with the TDH3 promoter PCR product digested with AscI and SpeI, and the SpeI-SfiI fragment containing the codon optimized kivD coding region was isolated from the vector pKivD-DNA2.0. By triple ligation, the vector pLH467 (pRS426 :: P _TDH3- kivDy-TDH3t) was produced. PLH467 was confirmed by restriction enzyme mapping and sequencing.

pLH435 (pRS425 :: P _GPM1- Hadhy-ADH1t) was derived from the vector pRS425 :: GPM-sadB (SEQ ID NO: 78) described in US Provisional Application No. 61/058970, Example 3, which is incorporated herein by reference. . pRS425 :: GPM-sadB is the coding region from the butanol dehydrogenase of the GPM1 promoter (SEQ ID NO: 72), Acromobacter xylloxidans (sadB; DNA SEQ ID NO: 69; Protein SEQ ID NO: 70: US Patent Application Publication No. 2009 / 0269823), and a pRS425 vector (ATCC No. 77106) with a chimeric gene containing the ADH1 terminator (SEQ ID NO: 73). pRS425 :: GPMp-sadB contains the BbvI and PacI sites at the 5 'and 3' ends of the sadB coding region, respectively. The NheI site was added to the 5 'end of the sadB coding region by site-directed mutations using primers OT1074 and OT1075 (SEQ ID NOs: 125 and 126) to generate the vector pRS425-GPMp-sadB-NheI as demonstrated by sequencing I was. Decompose pRS425 :: P _GPM1- sadB-NheI into NheI and PacI to drop out the sadB coding region and contain a NheI-PacI fragment containing codon optimized HADH coding region from vector pHadhy-DNA2.0 Ligation to generate pLH435.

To combine the KivD and HADH expression cassettes in a single vector, the yeast vector pRS411 (ATCC No. 87474) was digested with SacI and NotI and in a triple ligation reaction from pLH435 containing the P _GPM1- Hadhy-ADH1t cassette. Along with the SalI-NotI fragment, a SacI-SalI fragment from pLH467 containing the P _TDH3- kivDy-TDH3t cassette was ligated. This resulted in the vector pRS411 :: P _TDH3- kivDy-P _GPM1- Hadhy (pLH441), which was confirmed by restriction enzyme mapping.

To generate co-expression vectors for all three genes of the lower isobutanol pathway, ilvD, kivDy and Hadhy, pRS423 FBA ilvD (Strep) (SEQ ID NO: 127) was used, which is US Provisional Application No. 61 / 100,792. Is described as the source of the IlvD gene. This shuttle vector contains an F1 replication origin (nt 1423-1879) for maintenance in E. coli, and a 2 micron origin (nt 8082-9426) for replication in yeast. The vector has FBA1 promoters (nt 2111 to 3108; SEQ ID NO: 119) and FBA terminators (nt 4861 to 5860; SEQ ID NO: 128). It also has His markers (nt 504-1163) for selection in yeast, and ampicillin resistance markers (nt 7092-7949) for selection in E. coli. The ilvD coding region (nt 3116-4828; SEQ ID NO: 1154 protein SEQ ID NO: 115) from Streptococcus mutans UA159 (ATCC No. 700610) is present between the FBA promoter and the FBA terminator to form a chimeric gene for expression. There is also a lumino tag fused to the ilvD coding region (nt 4829 to 4849).

The first step consists of pRS423 FBA ilvD (Strep) (pRS423-FBA (SpeI) -IlvD (also known as Streptococcus mutans) -Lumio) with SacI and SacII (blunt-ended SacII sites using T4 DNA polymerase). Linearization together provided a vector with a total length of 9,482 bp. The second step was to isolate the kivDy-hADHy cassette from pLH441 with SacI and KpnI (KpnI site blunt ended using T4 DNA polymerase) to provide a 6,063 bp fragment. This segment was linked with a 9,482 bp vector segment from pRS423-FBA (SpeI) -IlvD (Streptococcus mutans) -Lumio. This produced the vector pLH468 (pRS423 :: P _FBA1- ilvD (Strep) Lumio-FBA1t-PTDH3-kivDy--TDH3tP _GPM1- hadhy-ADH1t), which was confirmed by restriction enzyme mapping and sequencing.

pYZ090  And pYZ067

AlsS gene from Bacillus subtilis expressed from yeast CUP1 promoters (nt 2 to 449) followed by CYC1 terminators (nt 2181 to 2430) for expression of acetolactate synthase (ALS) A yeast ILV5 promoter (2433 to 3626) followed by an ILV5 terminator (nt) for expression of a chimeric gene having a coding region of (nt positions 457 to 2172), and keto-acid reductoisomerase (KARI) PYZ090 was constructed to contain a chimeric gene having the coding region (nt 3634-4656) of the ilvC gene from Lactococcus lactis expressed from 4670-5292).

PYZ067 was constructed to contain the following chimeric genes: 1) ilvD from S. mutans UA159 expressed from yeast FBA1 promoters (nt 1161 to 2250) followed by FBA terminators (nt 4005 to 4317) for expression of DHAD. Coding region for genes (nt positions 2260 to 3971), 2) coding region for HADH expressed from yeast GPM promoters (nt 5819 to 6575) followed by ADH1 terminators (nt 4356 to 4671) for expression of alcohol dehydrogenases ( nt 4680 to 5807), and 3) Lacrococcus expressed from the yeast TDH3 promoter (nt 8830 to 9493) followed by the TDH3 terminator (nt 5682 to 7161) for the expression of ketoisovalerate decarboxylase. coding region of the KivD gene from lactis (nt 7175 to 8821).

In addition, while various embodiments of the invention have been described above, it should be understood that they are provided by way of example only, and not limitation. It will be apparent to those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. Accordingly, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the claims and their equivalents.

All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention relates, and each individual publication, patent, or patent application is specifically and individually incorporated by reference. Inc. is incorporated herein by reference to the same extent as set forth.

Example

The following non-limiting examples will further illustrate the present invention, where the partition coefficient for butanol of fatty acid extractant is demonstrated. The above chemical conversion and the following examples include corn oil as plant-derived oil for producing fatty acid extractant, but other natural oils such as plant-derived oil can be used without departing from the present invention. Should understand. From the foregoing discussion and these examples, those skilled in the art can identify the essential features of the present invention and can make various changes and modifications to the present invention without departing from the invention to suit a variety of uses and conditions.

As used herein, the meaning of the abbreviations used was as follows: "g" means gram (s), "kg" means kilogram (s), and "L" means liter (s) "ML" means milliliter (s), "mL / L" means milliliter (s) per liter, "mL / min" means milliliter (s) per minute, and "μl" means Means microliter (s), “DI” means deionization, “uM” means micrometer (s), “nM” means nanometer (s), and “w / v” Means weight / volume, "GC" means gas chromatograph, "OD" means optical density, "OD ₆₀₀ " means optical density at a wavelength of 600 nM, and "dcw" Means dry cell weight, “rpm” means revolutions per minute, “° C.” means degrees Celsius (s), “° C./min” means degrees Celsius per minute, and “slpm” is standard per minute Liter (s), where "ppm" is millions of minutes "Pdc" means pyruvate decarboxylase followed by the enzyme number.

Examples 1-6 describe exemplary methods of chemically converting corn oil into the following fatty acid extractants: hydroxylated triglycerides (Example 1), mixtures of fatty amides and fatty acids (Example 2), Fatty alcohols (Example 3), fatty acids (Example 4), fatty acid methyl esters (Example 5); And fatty acid glycol esters (Example 6). Example 7 provides a series of comparative examples of extractive fermentation experiments performed using the water-immiscible extractants listed in Table 3 and Tables 7-10, with performance data summarized in Table 11.

Example 1

Hydroxylated triglycerides from corn oil

A. Corn Oil hydroxylation (63% hydroxylation)

To a 3-neck 500 mL flask equipped with a mechanical stirrer and addition funnel, add corn oil (50.0 g), toluene (25.0 mL), Amberlyte IR-120 resin (12.5 g), and glacial acetic acid (7.5 g) It was. The resulting mixture was heated to 60 ° C. and then hydrogen peroxide (41.8 g of 30% H ₂ O _{2 in} water) was added dropwise over 1 hour. The mixture was stirred at 60 ° C. for 2 hours, at which time the reaction mixture was worked up: the resin was removed by filtration and the filtrate was partitioned between ethyl acetate (75 mL) and water (50 mL). After separating the layers, the organic layer was washed with saturated aqueous NaHCO ₃ (50 mL), and brine (50 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to give 48.9 g of a yellow oil. ¹ H NMR analysis of the crude reaction product showed that 63% of the double bonds were epoxidized.

To a 500 mL round bottom flask was added epoxidized corn oil (20.0 g), tetrahydrofuran (THF) (100.0 mL), and sulfuric acid (50 mL of 1.7 M aqueous solution). The cloudy mixture was stirred for 2 h at 50 ° C. and then worked up by partitioning between water (100 mL) and ethyl acetate (200 mL). The organic layer was washed with water (3x50 mL) and then brine (50 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to yield 19.9 g of dark yellow oil (63% hydroxylated corn oil).

B. Corn Oil hydroxylation (47% hydroxylation)

To a 3-neck 500 mL flask equipped with a mechanical stirrer and addition funnel was added corn oil (50.0 g), toluene (25.0 mL), Amberlite IR-120 resin (12.5 g), and glacial acetic acid (7.5 g). The resulting mixture was heated to 60 ° C. and then hydrogen peroxide (41.8 g of 30% H ₂ O _{2 in} water) was added dropwise over 1 hour. The mixture was stirred at 60 ° C. for 1 hour, at which time the reaction mixture was worked up: the resin was removed by filtration and the filtrate was partitioned between ethyl acetate (75 mL) and water (50 mL). After separating the layers, the organic layer was washed with saturated aqueous NaHCO ₃ (50 mL), and brine (50 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to give 49.8 g of yellow oil. ¹ H NMR analysis of the crude reaction product showed that 47% of the double bonds were epoxidized.

To a 500 mL round bottom flask was added epoxidized corn oil (20.0 g), THF (100.0 mL), and sulfuric acid (50 mL of 1.7 M aqueous solution). The cloudy mixture was stirred for 2 h at 50 ° C. and then worked up by partitioning between water (100 mL) and ethyl acetate (200 mL). The organic layer was washed with water (3x50 mL) and then brine (50 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to yield 19.2 g of dark yellow oil (47% hydroxylated corn oil).

C. Corn Oil hydroxylation (28% hydroxylation)

To a 3-neck 500 mL flask equipped with a mechanical stirrer and addition funnel was added corn oil (50.0 g), toluene (25.0 mL), Amberlite IR-120 resin (12.5 g), and glacial acetic acid (7.5 g). The resulting mixture was heated to 60 ° C. and then hydrogen peroxide (41.8 g of 30% H ₂ O _{2 in} water) was added dropwise over 1 hour. The mixture was stirred at 60 ° C. for 2 hours, at which time the reaction mixture was worked up: the resin was removed by filtration and the filtrate was partitioned between ethyl acetate (75 mL) and water (50 mL). After separating the layers, the organic layer was washed with saturated aqueous NaHCO ₃ (50 mL), and brine (50 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to give 47.2 g of yellow oil. ¹ H NMR analysis of the crude reaction product showed that 28% of the double bonds were epoxidized.

To a 500 mL round bottom flask was added epoxidized corn oil (20.0 g), THF (100.0 mL), and sulfuric acid (50 mL of 1.7 M aqueous solution). The cloudy mixture was stirred for 2 h at 50 ° C. and then worked up by partitioning between water (100 mL) and ethyl acetate (200 mL). The organic layer was washed with water (3x50 mL) and then brine (50 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to yield 20.3 g of dark yellow oil (28% hydroxylated corn oil).

Distribution factor measurement

To a 5 mL vial was added 0.910 g of 67% hydroxylated corn oil and 0.910 mL of 3 wt% iBuOH aqueous solution. The biphasic mixture was stirred vigorously for 10 minutes using Vortex Genie®. Once mixed, layer separation was aided by centrifuging the mixture for 10 minutes using a Fisher Scientific Centrific 228 centrifuge (3300 rpm). 0.100 g of both layers were taken. The organic, upper layer was diluted to 1.00 mL with toluene solution of ethylene glycol diethyl ether (10.1 mg / mL), and the aqueous layer was diluted to 1.00 mL with methanol solution (10.2 mg / mL) of ethylene glycol diethyl ether. The concentration of i-BuOH was measured in both phases using a calibrated gas chromatograph (GC). The same procedure was repeated for 47% and 28% hydroxylated corn oil. The partition coefficient thus measured was 3.2 for 67% hydroxylated corn oil, 2.3 for 47% hydroxylated corn oil and 2.1 for 28% hydroxylated corn oil.

The procedure outlined above was repeated with 6% i-BuOH aqueous solution. The partition coefficients for 67%-, 47%-, and 28% -hydroxylated corn oil were 2.9, 2.9, and 2.0, respectively.

Example 2

Pure fatty amides from corn oil, and fatty amides + fatty acids

Roe, et al., J. Am. Oil Chem. Soc. 29: 18-22, 1952, corn oil was reacted with aqueous ammonium hydroxide. Mazola® corn oil (0.818 L, 755 g) is placed in a 1 gallon stainless steel reactor, to which 1.71 L (1540 g) of aqueous ammonium hydroxide (28% as NH ₃ ) is added. It was. The reactor was heated to 160 ° C. with stirring and held at that temperature for 7 hours with stirring, during which time the pressure reached 400 psi (2.76 kPa). The reactor was cooled down, the product creamy white solid was collected and the reactor was rinsed with ethyl acetate. The product was dissolved in 5 L ethyl acetate and washed five times with 500 mL of water each, which was neutralized with H ₂ SO ₄ . The ethyl acetate was then dried over anhydrous Na ₂ SO ₄ and the solvent removed on a rotary evaporator, leaving a pale brown soft solid.

¹³ C NMR in CDCl ₃ showed that the product contained approximately 2: 1 ratios of fatty amides to fatty acids and the conversion of corn oil to the product was quantitative. The melting point of the product was 57-58 ° C., but dropped about 11 ° C. when saturated with water.

Kohlhase, et al., J. Am. Oil Chem. Soc. 48: 265-270, 1971] pure corn oil fatty amides were synthesized from corn oil using anhydrous ammonia with ammonium acetate as catalyst.

3 grams of ammonium acetate was placed in a 400 mL stainless steel shaker tube, to which 51.8 g of corn oil was added. Anhydrous ammonia (89.7 g) was then added and the reactor was sealed and heated at 125 ° C. for 7 hours during which time the pressure reached 8.96 MPa (1300 psi). The reactor was cooled down, the light solid was collected and the reactor was rinsed with ethyl acetate. The product dissolved in ethyl acetate was then worked up as in the case of the fatty amide / fatty acid mixture.

Fatty acids were synthesized from corn oil by base hydrolysis using NaOH as described in Example 4 below.

The following three formulations were all tested for their ability to extract isobutanol from a 3% aqueous solution: (1) 2: 1 mixture of corn oil fatty amides and corn oil fatty acids from aqueous ammonia, (2 A) pure corn oil fatty amide: a 2: 1 mixture of pure corn oil fatty acids, and (3) a pure corn oil fatty amide: a 1: 2 mixture of corn oil fatty acids. 700 mg of each formulation was added to 2.1 mL of water containing 3% isobutanol in a 20 mL scintillation vial and placed on a rotary shaker at 30 ° C. overnight. In all three cases, the organic phase became liquid at this temperature, suggesting that the melting point is further lowered by the absorption of isobutanol. 50 microliters of the top phase were diluted with 200 μl of toluene containing ethylene glycol diethyl ether (10.068 mg / mL) as the GC standard, or 200 μl isopropanol containing the same concentration of ethylene glycol diethyl ether. 50 microliters of the lower phase was diluted with 50 μl of isopropanol and 150 μl of methanol containing the same concentration of ethylene glycol diethyl ether. Calibrated GC was used to determine the concentration of isobutanol in both phases. The partition coefficients measured are as follows: 3.81 for (1), 4.31 for (2) and 3.58 for (3).

Saccharomyces formulation (1a) (equivalent to 1: 2 fatty amides: fatty acid) consisting of a fatty amide / fatty acid aqueous ammonia formulation (1), and a formulation (1) mixed 1: 1 with pure corn oil fatty acid. Incubation at a ratio of 3 parts broth to 1 part amide / acid mixture in a shake flask with fermentation broth containing butanologen NGCI-070. At 30 ° C., Formulation (1) was a soft solid, whereas Formulation (1a) was a liquid. Starting at a glucose concentration of 8.35 g / L, the shake flask was then incubated on an incubator shaker for 25 hours, followed by consumption of glucose as a function of time. Table 1 shows that at both ratios the fatty amide / fatty acid mixture was not toxic to butanologen and even showed higher glucose absorption rates than when using oleyl alcohol.

Example 3

Fatty alcohol from corn oil

Regarding the reaction of Scheme IV above to produce fatty alcohol from corn oil, a 22 L round bottom flask equipped with a mechanical stirrer, reflux condenser with N ₂ source, addition funnel, internal thermocouple, and rubber septum was flamed under nitrogen. -Flame-dry. 132 g (3.30 moles) of 95% lithium aluminum hydride powder, weighed in a dry box and charged into a solid addition funnel, was charged to the flask. The 22 L flask was cooled in an ice bath and 9.0 liters of anhydrous THF were added through the cannula into the reactor. The resulting slurry was cooled to 0-5 ° C. and a solution of 956 g (1.10 moles) of Wesson Δ corn oil in 1.00 liter of dry THF was maintained while maintaining the reaction temperature at 5-20 ° C. Dropwise added over 3 h. After the addition of corn oil, the slurry was stirred overnight at ambient temperature. As confirmed by TLC chromatography, when the reaction was complete, it was quenched by dropwise addition of a solution of 130 g of water dissolved in 370 mL of THF. Then 130 g of 15% aqueous NaOH solution were added followed by 400 g of water. The mixture was stirred vigorously while warming to room temperature, yielding a white granular solid. The solid was filtered off using a sinter-glass filter funnel and washed with additional THF. THF was removed on a rotary evaporator and the residue was dissolved in 3.00 liters of ethyl acetate. The product solution was washed with 2 × 1.00 L water, 1 × 1.00 L brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo to give 836 g (97%) of fatty alcohol as a yellow oil. The crude fatty alcohol mixture was then distilled (1.05 ° C./Pascal (140 ° C./1 mmHg)) and used for the following partition coefficient experiments.

Distribution coefficient experiment

To each of the five 5-mL vials was added 1 mL of fatty alcohol mixture, and 1 mL of 3 wt% iBuOH aqueous solution. The two-phase mixture was vigorously stirred for 10, 20, 30, 40, and 60 minutes using Vortex Genie®. Once mixed, the separation of layers was aided by centrifuging the mixture for 10 minutes using a Fisher Scientific Centripic 228 centrifuge (3300 rpm). 0.100 mL of both layers were taken. The organic and upper layers were diluted to 1.00 mL with a toluene solution of ethylene glycol diethyl ether and the aqueous layer was diluted to 1.00 mL with a methanol solution of ethylene glycol diethyl ether. The concentration of i-BuOH was measured in both phases using calibrated GC. The partition coefficient thus measured was 2.70.

The same partition coefficient measurements were performed as above for a 6 wt% i-BuOH concentration. The partition coefficient thus measured was 3.06.

In Examples 4 to 6 below, the method used to determine the partition coefficient of the extractant was a quiescent method. In the stationary method, 5 mL of 3% or 6% (w / v) isobutanol aqueous solution was placed in a vial and 5 mL of the solvent of interest was carefully added on top of the aqueous solution so that the physical phases were not mixed. After a given period of time, samples of clear portions of the solvent and aqueous phases were collected and analyzed for isobutanol content by GC. Any emulsion layer was ignored in this analysis. For GC analysis, 100 uL of sample was added to 400 uL of isopropanol. 500 uL of diethylene glycol diethyl ether (internal standard) solution was added and the solution was injected into a Carbowax® column with FID detector to determine the concentration of isobutanol.

In order to determine the partition coefficient of the extractant according to the invention, other methods known in the art can also be used. For example, a shaking method can be used. As an example of the shaking method, 5 mL of 3% or 6% (w / v) isobutanol aqueous solution can be added to the centrifuge tube with 5 mL of the solvent of interest. The tube can be shaken vigorously for 1 minute. The tube can then be rotated in a centrifuge at approximately 12500 G for 15 minutes. Samples of the clear solvent layer and clear aqueous layer can be collected and analyzed for isobutanol content by this method.

Example 4

Corn oil fatty acid

The round bottom flask (5 L) was equipped with a mechanical stirrer, thermocouple, heating mantle, condenser, and nitrogen tee. 500 g of food grade corn oil, 1 L of water, and 75 g of sodium hydroxide were added. The mixture was heated to 90 ° C. and maintained for 3 hours during which time the mixture became a single, thick emulsion-like single phase. At the end of this time, TLC indicates that no corn oil remains in the mixture. The mixture was then cooled to 72 ° C. and acidified by adding 500 mL of 25% sulfuric acid. Then it was cooled to room temperature and 2 L of diethyl ether was added. The ether layer was washed with 3 × 1 L of 1% sulfuric acid, 1 × 1 L saturated brine, dried over MgSO ₄ and filtered. After ether removal by rotary evaporator, the oil was purged with nitrogen overnight to yield 470 g of yellow oil partially crystallized overnight. Titration for free fatty acids via AOCS method Ca 5a-40 shows a fatty acid content of 95%, expressed as oleic acid. The samples were silanized by reacting 104 mg in 1 mL of dry pyridine with 100 uL of N-methyl-N- (trimethylsilyl) trifluoroacetamide. Gas chromatography-mass spectrometry (GCMS) analysis of silanated products indicates the presence of TMS derivatives of 16: 0, 18: 2, 18: 1, 18: 0, and 20: 0 acids.

As determined by the stationary method, the partition coefficient of isobutanol in the COFA / water system is 2.8 after 168 hours at the initial 6% I-BuOH concentration.

Example 5

Corn oil fatty acid methyl ester (FAME)

The round bottom flask (5 L) was equipped with a mechanical stirrer, thermocouple, heating mantle, condenser, and nitrogen tee. 1500 g food grade corn oil, 1500 g methanol, and 30 g concentrated sulfuric acid were added. After the mixture was refluxed for 24 hours, thin layer chromatography was performed. The reaction mixture was then cooled and the layers separated. The organic layer was washed with 1 × 1 L of water, 1 × 1 L of saturated sodium bicarbonate, 2 × 1 L of water, 1 × 1 L of saturated brine, and then dried over MgSO ₄ . 1416 g of pale yellow oil were obtained. GCMS analysis shows the presence of methyl esters of acids 16: 0, 18: 2, 18: 1, 18: 0, 20: 1, and 20: 0. Titration to free fatty acids via AOCS method Ca 5a-40 shows a fatty acid content of 0.2%, expressed as oleic acid.

As determined by the stationary method, the partition coefficient of isobutanol in the FAME / water system is 1.06 after 236 hours at the initial 6% I-BuOH concentration.

Example 6

Corn oil ethylene glycol ester (FAGE)

A round bottom flask 3L was equipped with a mechanical stirrer, thermocouple, heating mantle, Dean-Stark trap, condenser, nitrogen purge, and nitrogen tee; 1000 g corn oil fatty acid methyl ester (FAME) and 1000 g ethylene glycol were charged. 2 g of clean sodium is added to the mixture and it is heated to 60 ° C. After 90 minutes, the temperature is increased to 100 ° C. and nitrogen is slowly subsurfaced into the reaction mixture. Collect methanol in the Dean-Stark trap. The temperature is slowly increased to 160 ° C. over 3 hours and methanol continues to distill from the reaction mixture. After an additional 2 hours, a total of 100 mL of methanol was collected. The reaction mixture was cooled to room temperature and neutralized with 20 g of 25% sulfuric acid. The layers were separated and the top layer was washed with 4 × 200 ml of 10% calcium chloride solution. An emulsion was formed but separated over time. The organic layer was washed with 250 mL of saturated brine, dried over MgSO ₄ and filtered to give 916 g of a clear yellow oil. Titration to free fatty acids via AOCS method Ca 5a-40 indicates 2.9% of the acid present (denoted as oleic acid). The sample was silanized by reacting 109 mg in 1 ml of dry pyridine with 100 uL of N-methyl-N- (trimethylsilyl) trifluoroacetamide. GCMS analysis of the silanated products shows the presence of TMS derivatives of 16: 0, 18: 2, and 18: 1 acids with 16: 0, 18: 2, 18: 1, and 18: 0 ethylene glycol monoesters.

As determined by the stationary method, the partition coefficient of isobutanol in the FAGE / water system after 2.3 hours at the initial 6% I-BuOH concentration is 2.3.

Example 7

Fermentation Comparative Example

The materials listed in Table 2 were used in the comparative example of Example 7. All commercial reagents were used as received. Solvents synthesized from corn oil were also used as received.

General method

Optical density was measured using an Amersham Biosciences Ultrospec 2100 Pro spectrophotometer. Measurements were typically performed at a wavelength of 600 nanometers.

Glucose concentrations were measured using a YSI Life Sciences 2700 Select Biochemistry Analyzer. Fermentation samples were centrifuged in 1.7 mL microcentrifuge tubes for 2 minutes at 13,200 rpm and the aqueous supernatant was analyzed for glucose concentration.

Fermentation conditions: 30% pO ₂ ; Temperature: 30 ° C .; pH 5.5 (unless otherwise noted); Initial batch glucose: 20 g / L, maintained during production.

Both HPLC and GC analysis were used for the quantization of isobutanol in the aqueous and solvent phases, respectively. After filtration through a 0.2 um nylon filter, isobutanol in aqueous was measured by HPLC (Agilent 1100, Agilent, Santa Clara, CA) under the following conditions:

Column: Bio-Rad, Aminex HPX-87H, no. 125-0143

Mobile phase: 0.01 M Na ₂ HPO ₄ , pH = 8.0

Injection volume: 10 uL

Flow rate: 0.6 mL / min

Run time: 22.5 minutes

Column temperature: 65 ° C

Detector: Refractive Index

Detector temperature: 40 ℃

UV detection: 210 nm, 4 nm bandwidth, reference 360 nm, 100 nm bandwidth.

The solvent phase was measured by GC (HP6890, Agilent, Santa Clara, Calif.) Under the following conditions:

Column: J & W Scientific DB Waxter (50 m X 0.32 mm ID, 1 um film)

Gas carrier: helium 4 mL / min

Injection volume: 2 uL

Fill Flow Rate: 40 mL / min

Run time: 29 minutes

Oven temperature: 40 ° C. for 5 minutes at 10 ° C./min from 40 ° C. to 230 ° C.,

5 minutes 230 ℃

Injector Split: 1: 5 at 250 ° C

Flame ionization detection: 250 ℃

Comparative Example: GLNOR635A-640A of an extractant derived from corn oil

And its components

A series of comparative examples were performed using the water-immiscible extractants listed in Table 3. At time zero, an extractant was added to the fermentor broth to expose the culture to the solvent for continued fermentation. Isobutanol concentrations were measured in both the aqueous and organic phases to calculate the partition coefficient of the extraction solvent. Glucose use was used to determine the biocompatibility of the microorganisms for the extractant.

Fermentation was performed as described using strain NGCI-065. Inoculum was prepared in two steps and incubated at 30 ° C. and 250 rpm in an incubator shaker (Innova 4200, New Brunswick Scientific, Edison, NJ). The first step or pre-seed was inoculated from frozen glycerol spawn stock, placed two vials into a 250 mL flask with 30 mL of filtered sterilized pre-seed medium (Table 4) for approximately 24 hours. Grow up to an OD of 2. Thereafter, 15 mL of pre-seed was transferred to a 2 L flask with 270 mL of sterile seed media (Table 5) for the second step, which was incubated for 24 hours. Then 30 mL of filtered sterilized 10X YEP and 300 mL of filtered sterilized 90-95% oleyl alcohol were added and incubated for an additional 24 hours until a final OD of approximately 5-10 in the aqueous phase of the seed culture. In Examples GLNOR639 and GLNOR640, the pH was 4.5.

Fermentation vessels (Applikon AD1010 Bioreactor, Applikon Biotechnology, Dover, NJ) were sterilized for 30 minutes with deionized H ₂ O (Amsco Renaissance 3033 Levi's Steam Steril) Amsco Renaissance 3033 Revas Steam Sterilizer, Steris Corporation, Mentor, Ohio. Once sterilization of the vessel in the autoclave was completed and cooled to 30 ° C., sterile deionized H ₂ O was removed and filtered sterilized fermentation medium was added to a volume of 280 mL. 70 mL of the aqueous phase from the second stage seed flask were added to the fermentation vessel in a final aqueous volume of 350 mL. Immediately after inoculation, 100 mL of 10 × YEP medium supplement was added with 450 mL of each extraction solvent for a final 1: 1 solvent to broth ratio. Fermentation setpoint conditions were temperature 30 ° C., pO ₂ 30%, pH 5.5 at GLNOR635A-638A and pH 4.5 at GLNOR639A-640A. Fermentation was sampled approximately every 8 hours from inoculation time to monitor glucose concentration and maintained at 5-20 g / L through addition of 50% w / w glucose solution and isobutanol in both aqueous and extractive phases Accumulation was analyzed. After taking sufficient sample volume at each time point to obtain samples from both phases, those samples were centrifuged to ensure each clean cut.

Comparative Example: GLNOR661A-666A and its components of extractant derived from corn oil

Comparative Example GLNOR661A-666A was performed using the water-immiscible extractants listed in Table 7. Except for supplemental addition, pH, and strain at time 0, this set of examples was performed as in the previous examples GLNOR635A-640A. In this series of examples strain NGCI-070 was used, the pH setting was 5.5, and 4 mL nicotinic acid / thiamine medium supplement was added in place of 100 mL yeast extract peptone.

Comparative example: GLNOR690A-695A and its components of extractant derived from corn oil

Comparative Example GLNOR690A-695A was performed using the water-immiscible extractants listed in Table 8. This set of examples was performed in the same manner as in the previous examples GLNOR661A-666A. Strain NGCI-070 was used.

Comparative Example of a Extract Derivative from Corn Oil GLNOR721A-726A and Its Components

Comparative Example GLNOR721A-726A was performed using the water-immiscible extractants listed in Table 9. This set of examples was performed in the same manner as in the previous examples GLNOR690A-695A. Strain NGCI-070 was used.

Comparative Example: GLNOR749A-754A and its components of extractant derived from corn oil

Comparative Example GLNOR749A-754A was performed using the water-immiscible extractants listed in Table 10. This set of examples was performed in the same manner as in the previous examples GLNOR721A-726A. Strain NGCI-070 was used.

The performance data for the fermentation examples GLNOR635A-640A, GLNOR661A-666A, GLNOR690A-695A, GLNOR721A-726A, GLNOR749A-754A are summarized in Table 11, which is combined with the conventional commercial solvents of oleyl alcohol and isopol®. In comparison, an aqueous isobutanol concentration (g / L), solvent isobutanol concentration (g / L), and solvent partition coefficient (K _p ) are provided for exemplary corn oil derived extractants and components thereof.

Example 8

Fatty alcohol hydroxylation (65% hydroxylation)

In a 3-neck 250 mL flask equipped with a mechanical stirrer and addition funnel, a mixture of fatty alcohol (43 g, 0.16 mmol), toluene (25.0 mL), Amberlite IR-120 resin (12.5 g), and glacial acetic acid (7.5 g) Added. The resulting mixture was heated to 60 ° C. and then hydrogen peroxide (41.8 g of 30% H ₂ O _{2 in} water) was added dropwise over 30 minutes. The mixture was stirred at 60 ° C. for 2 hours, at which time the reaction mixture was worked up: the resin was removed by filtration and the filtrate was partitioned between ethyl acetate (75 mL) and water (50 mL). After separating the layers, the organic layer was washed with saturated aqueous NaHCO ₃ (50 mL), and brine (50 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to yield 50 g of yellow oil. ¹ H NMR analysis of the crude reaction product showed that 65% of the double bonds were epoxidized. The resulting mixture was used for next step without purification.

To a 500 mL round bottom flask was added epoxidized fatty alcohol (14.5 g), THF (200.0 mL), and sulfuric acid (100 mL of 1.7 M aqueous solution). The cloudy mixture was stirred at 50 ° C. overnight and then worked up by partitioning between water (100 mL) and ethyl acetate (200 mL). The organic layer was washed with water (2 × 100 mL) and then saturated aqueous NaHCO ₃ (100 mL) followed by brine (100 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to give 14.7 g of a dark clear liquid and white solid mixture.

Measurement of distribution factor

1 mL of a hydroxylated fatty alcohol mixture was added to a 1 mL solution of 3% i-BuOH aqueous solution, and the resulting two-phase mixture was stirred vigorously using vortex for 10 minutes. The experiment was run in duplicate on 6% i-BuOH solution. After mixing, the layers were separated and samples were taken from both layers to determine i-BuOH concentration using GC (Table 12). A partition coefficient of 3.7 was observed.

While various embodiments of the invention have been described above, it should be understood that they are provided by way of example only and not of limitation. It will be apparent to those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. Accordingly, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention relates, and each individual publication, patent, or patent application is specifically and individually incorporated by reference. Inc. is incorporated herein by reference to the same extent as set forth.

                         SEQUENCE LISTING <110> Butamax (TM) Advanced Biofuels   <120> EXTRACTION SOLVENTS DERIVED FROM OIL FOR ALCOHOL REMOVAL IN        EXTRACTIVE FERMENTATION <130> CL5000USNA <150> US 61 / 356,290 <151> 2010-06-18 <150> US 61 / 368,451 <151> 2010-07-28 <150> US 61 / 368,436 <151> 2010-07-28 <150> US 61 / 368,444 <151> 2010-07-28 <150> US 61 / 368,429 <151> 2010-07-28 <150> US 61 / 379,546 <151> 2010-09-02 <150> US 61 / 440,034 <151> 2011-02-07 <150> US 13 / 160,766 <151> 2011-06-15 <160> 143 <170> PatentIn version 3.5 <210> 1 <211> 11844 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 1 tcccattacc gacatttggg cgctatacgt gcatatgttc atgtatgtat ctgtatttaa 60 aacacttttg tattattttt cctcatatat gtgtataggt ttatacggat gatttaatta 120 ttacttcacc accctttatt tcaggctgat atcttagcct tgttactagt tagaaaaaga 180 catttttgct gtcagtcact gtcaagagat tcttttgctg gcatttcttc tagaagcaaa 240 aagagcgatg cgtcttttcc gctgaaccgt tccagcaaaa aagactacca acgcaatatg 300 gattgtcaga atcatataaa agagaagcaa ataactcctt gtcttgtatc aattgcatta 360 taatatcttc ttgttagtgc aatatcatat agaagtcatc gaaatagata ttaagaaaaa 420 caaactgtac aatcaatcaa tcaatcatcg ctgaggatgt tgacaaaagc aacaaaagaa 480 caaaaatccc ttgtgaaaaa cagaggggcg gagcttgttg ttgattgctt agtggagcaa 540 ggtgtcacac atgtatttgg cattccaggt gcaaaaattg atgcggtatt tgacgcttta 600 caagataaag gacctgaaat tatcgttgcc cggcacgaac aaaacgcagc attcatggcc 660 caagcagtcg gccgtttaac tggaaaaccg ggagtcgtgt tagtcacatc aggaccgggt 720 gcctctaact tggcaacagg cctgctgaca gcgaacactg aaggagaccc tgtcgttgcg 780 cttgctggaa acgtgatccg tgcagatcgt ttaaaacgga cacatcaatc tttggataat 840 gcggcgctat tccagccgat tacaaaatac agtgtagaag ttcaagatgt aaaaaatata 900 ccggaagctg ttacaaatgc atttaggata gcgtcagcag ggcaggctgg ggccgctttt 960 gtgagctttc cgcaagatgt tgtgaatgaa gtcacaaata cgaaaaacgt gcgtgctgtt 1020 gcagcgccaa aactcggtcc tgcagcagat gatgcaatca gtgcggccat agcaaaaatc 1080 caaacagcaa aacttcctgt cgttttggtc ggcatgaaag gcggaagacc ggaagcaatt 1140 aaagcggttc gcaagctttt gaaaaaggtt cagcttccat ttgttgaaac atatcaagct 1200 gccggtaccc tttctagaga tttagaggat caatattttg gccgtatcgg tttgttccgc 1260 aaccagcctg gcgatttact gctagagcag gcagatgttg ttctgacgat cggctatgac 1320 ccgattgaat atgatccgaa attctggaat atcaatggag accggacaat tatccattta 1380 gacgagatta tcgctgacat tgatcatgct taccagcctg atcttgaatt gatcggtgac 1440 attccgtcca cgatcaatca tatcgaacac gatgctgtga aagtggaatt tgcagagcgt 1500 gagcagaaaa tcctttctga tttaaaacaa tatatgcatg aaggtgagca ggtgcctgca 1560 gattggaaat cagacagagc gcaccctctt gaaatcgtta aagagttgcg taatgcagtc 1620 gatgatcatg ttacagtaac ttgcgatatc ggttcgcacg ccatttggat gtcacgttat 1680 ttccgcagct acgagccgtt aacattaatg atcagtaacg gtatgcaaac actcggcgtt 1740 gcgcttcctt gggcaatcgg cgcttcattg gtgaaaccgg gagaaaaagt ggtttctgtc 1800 tctggtgacg gcggtttctt attctcagca atggaattag agacagcagt tcgactaaaa 1860 gcaccaattg tacacattgt atggaacgac agcacatatg acatggttgc attccagcaa 1920 ttgaaaaaat ataaccgtac atctgcggtc gatttcggaa atatcgatat cgtgaaatat 1980 gcggaaagct tcggagcaac tggcttgcgc gtagaatcac cagaccagct ggcagatgtt 2040 ctgcgtcaag gcatgaacgc tgaaggtcct gtcatcatcg atgtcccggt tgactacagt 2100 gataacatta atttagcaag tgacaagctt ccgaaagaat tcggggaact catgaaaacg 2160 aaagctctct agttaattaa tcatgtaatt agttatgtca cgcttacatt cacgccctcc 2220 ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat 2280 ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt 2340 ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt 2400 tttgggacgc tcgaaggctt taatttgcgg gcggccgcac ctggtaaaac ctctagtgga 2460 gtagtagatg taatcaatga agcggaagcc aaaagaccag agtagaggcc tatagaagaa 2520 actgcgatac cttttgtgat ggctaaacaa acagacatct ttttatatgt ttttacttct 2580 gtatatcgtg aagtagtaag tgataagcga atttggctaa gaacgttgta agtgaacaag 2640 ggacctcttt tgcctttcaa aaaaggatta aatggagtta atcattgaga tttagttttc 2700 gttagattct gtatccctaa ataactccct tacccgacgg gaaggcacaa aagacttgaa 2760 taatagcaaa cggccagtag ccaagaccaa ataatactag agttaactga tggtcttaaa 2820 caggcattac gtggtgaact ccaagaccaa tatacaaaat atcgataagt tattcttgcc 2880 caccaattta aggagcctac atcaggacag tagtaccatt cctcagagaa gaggatataca 2940 taacaagaaa atcgcgtgaa caccttatat aacttagccc gttattgagc taaaaaacct 3000 tgcaaaattt cctatgaata agaatacttc agacgtgata aaaatttact ttctaactct 3060 tctcacgctg cccctatctg ttcttccgct ctaccgtgag aaataaagca tcgagtacgg 3120 cagttcgctg tcactgaact aaaacaataa ggctagttcg aatgatgaac ttgcttgctg 3180 tcaaacttct gagttgccgc tgatgtgaca ctgtgacaat aaattcaaac cggttatagc 3240 ggtctcctcc ggtaccggtt ctgccacctc caatagagct cagtaggagt cagaacctct 3300 gcggtggctg tcagtgactc atccgcgttt cgtaagttgt gcgcgtgcac atttcgcccg 3360 ttcccgctca tcttgcagca ggcggaaatt ttcatcacgc tgtaggacgc aaaaaaaaaa 3420 taattaatcg tacaagaatc ttggaaaaaa aattgaaaaa ttttgtataa aagggatgac 3480 ctaacttgac tcaatggctt ttacacccag tattttccct ttccttgttt gttacaatta 3540 tagaagcaag acaaaaacat atagacaacc tattcctagg agttatattt ttttacccta 3600 ccagcaatat aagtaaaaaa ctgtttaaac agtatggcag ttacaatgta ttatgaagat 3660 gatgtagaag tatcagcact tgctggaaag caaattgcag taatcggtta tggttcacaa 3720 ggacatgctc acgcacagaa tttgcgtgat tctggtcaca acgttatcat tggtgtgcgc 3780 cacggaaaat cttttgataa agcaaaagaa gatggctttg aaacatttga agtaggagaa 3840 gcagtagcta aagctgatgt tattatggtt ttggcaccag atgaacttca acaatccatt 3900 tatgaagagg acatcaaacc aaacttgaaa gcaggttcag cacttggttt tgctcacgga 3960 tttaatatcc attttggcta tattaaagta ccagaagacg ttgacgtctt tatggttgcg 4020 cctaaggctc caggtcacct tgtccgtcgg acttatactg aaggttttgg tacaccagct 4080 ttgtttgttt cacaccaaaa tgcaagtggt catgcgcgtg aaatcgcaat ggattgggcc 4140 aaaggaattg gttgtgctcg agtgggaatt attgaaacaa cttttaaaga agaaacagaa 4200 gaagatttgt ttggagaaca agctgttcta tgtggaggtt tgacagcact tgttgaagcc 4260 ggttttgaaa cactgacaga agctggatac gctggcgaat tggcttactt tgaagttttg 4320 cacgaaatga aattgattgt tgacctcatg tatgaaggtg gttttactaa aatgcgtcaa 4380 tccatctcaa atactgctga gtttggcgat tatgtgactg gtccacggat tattactgac 4440 gaagttaaaa agaatatgaa gcttgttttg gctgatattc aatctggaaa atttgctcaa 4500 gatttcgttg atgacttcaa agcggggcgt ccaaaattaa tagcctatcg cgaagctgca 4560 aaaaatcttg aaattgaaaa aattggggca gagctacgtc aagcaatgcc attcacacaa 4620 tctggtgatg acgatgcctt taaaatctat cagtaaggcc ctgcaggcct atcaagtgct 4680 ggaaactttt tctcttggaa tttttgcaac atcaagtcat agtcaattga attgacccaa 4740 tttcacattt aagatttttt ttttttcatc cgacatacat ctgtacacta ggaagccctg 4800 tttttctgaa gcagcttcaa atatatatat tttttacata tttattatga ttcaatgaac 4860 aatctaatta aatcgaaaac aagaaccgaa acgcgaataa ataatttatt tagatggtga 4920 caagtgtata agtcctcatc gggacagcta cgatttctct ttcggttttg gctgagctac 4980 tggttgctgt gacgcagcgg cattagcgcg gcgttatgag ctaccctcgt ggcctgaaag 5040 atggcgggaa taaagcggaa ctaaaaatta ctgactgagc catattgagg tcaatttgtc 5100 aactcgtcaa gtcacgtttg gtggacggcc cctttccaac gaatcgtata tactaacatg 5160 cgcgcgcttc ctatatacac atatacatat atatatatat atatatgtgt gcgtgtatgt 5220 gtacacctgt atttaatttc cttactcgcg ggtttttctt ttttctcaat tcttggcttc 5280 ctctttctcg agcggaccgg atcctccgcg gtgccggcag atctatttaa atggcgcgcc 5340 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 5400 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 5460 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 5520 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 5580 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 5640 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 5700 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 5760 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 5820 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 5880 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 5940 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 6000 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 6060 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 6120 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 6180 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 6240 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 6300 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 6360 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 6420 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 6480 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 6540 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 6600 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 6660 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 6720 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 6780 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 6840 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 6900 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 6960 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 7020 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 7080 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 7140 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 7200 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 7260 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 7320 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 7380 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 7440 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 7500 tgattacgcc aagctttttc tttccaattt tttttttttc gtcattataa aaatcattac 7560 gaccgagatt cccgggtaat aactgatata attaaattga agctctaatt tgtgagttta 7620 gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat cttctcaaat 7680 atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc cttccctttg 7740 caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca tcatccacgg 7800 ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg ggtgtcataa 7860 tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata aagccgataa 7920 caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct ccagtagata 7980 gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc tttgttactt 8040 cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg tgtgcattcg 8100 taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat ttgactgtat 8160 taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg gcggataatg 8220 cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc acatgtgttt 8280 ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc ttggtggtac 8340 gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta aatagcttgg 8400 cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc gacatgattt 8460 atcttcgttt cctgcaggtt tttgttctgt gcagttgggt taagaatact gggcaatttc 8520 atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc tccttccttc 8580 gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaggaaac cgaaatcaaa 8640 aaaaagaata aaaaaaaaat gatgaattga aaagcttgca tgcctgcagg tcgactctag 8700 tatactccgt ctactgtacg atacacttcc gctcaggtcc ttgtccttta acgaggcctt 8760 accactcttt tgttactcta ttgatccagc tcagcaaagg cagtgtgatc taagattcta 8820 tcttcgcgat gtagtaaaac tagctagacc gagaaagaga ctagaaatgc aaaaggcact 8880 tctacaatgg ctgccatcat tattatccga tgtgacgctg catttttttt tttttttttt 8940 tttttttttt tttttttttt tttttttttt ttttgtacaa atatcataaa aaaagagaat 9000 ctttttaagc aaggattttc ttaacttctt cggcgacagc atcaccgact tcggtggtac 9060 tgttggaacc acctaaatca ccagttctga tacctgcatc caaaaccttt ttaactgcat 9120 cttcaatggc tttaccttct tcaggcaagt tcaatgacaa tttcaacatc attgcagcag 9180 acaagatagt ggcgataggg ttgaccttat tctttggcaa atctggagcg gaaccatggc 9240 atggttcgta caaaccaaat gcggtgttct tgtctggcaa agaggccaag gacgcagatg 9300 gcaacaaacc caaggagcct gggataacgg aggcttcatc ggagatgata tcaccaaaca 9360 tgttgctggt gattataata ccatttaggt gggttgggtt cttaactagg atcatggcgg 9420 cagaatcaat caattgatgt tgaactttca atgtagggaa ttcgttcttg atggtttcct 9480 ccacagtttt tctccataat cttgaagagg ccaaaacatt agctttatcc aaggaccaaa 9540 taggcaatgg tggctcatgt tgtagggcca tgaaagcggc cattcttgtg attctttgca 9600 cttctggaac ggtgtattgt tcactatccc aagcgacacc atcaccatcg tcttcctttc 9660 tcttaccaaa gtaaatacct cccactaatt ctctaacaac aacgaagtca gtacctttag 9720 caaattgtgg cttgattgga gataagtcta aaagagagtc ggatgcaaag ttacatggtc 9780 ttaagttggc gtacaattga agttctttac ggatttttag taaaccttgt tcaggtctaa 9840 cactaccggt accccattta ggaccaccca cagcacctaa caaaacggca tcagccttct 9900 tggaggcttc cagcgcctca tctggaagtg gaacacctgt agcatcgata gcagcaccac 9960 caattaaatg attttcgaaa tcgaacttga cattggaacg aacatcagaa atagctttaa 10020 gaaccttaat ggcttcggct gtgatttctt gaccaacgtg gtcacctggc aaaacgacga 10080 tcttcttagg ggcagacatt acaatggtat atccttgaaa tatatataaa aaaaaaaaaa 10140 aaaaaaaaaa aaaaaaatgc agcttctcaa tgatattcga atacgctttg aggagataca 10200 gcctaatatc cgacaaactg ttttacagat ttacgatcgt acttgttacc catcattgaa 10260 ttttgaacat ccgaacctgg gagttttccc tgaaacagat agtatatttg aacctgtata 10320 ataatatata gtctagcgct ttacggaaga caatgtatgt atttcggttc ctggagaaac 10380 tattgcatct attgcatagg taatcttgca cgtcgcatcc ccggttcatt ttctgcgttt 10440 ccatcttgca cttcaatagc atatctttgt taacgaagca tctgtgcttc attttgtaga 10500 acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac 10560 agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt 10620 gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt 10680 tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct 10740 tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt 10800 actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag 10860 gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc 10920 tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat 10980 aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa 11040 gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt 11100 ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg 11160 aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat 11220 gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag 11280 ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt 11340 attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg 11400 tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagagaata 11460 ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac 11520 gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt gttgcctgta 11580 tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc gtacttatat 11640 gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta tcccattcca 11700 tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat gctgccactc 11760 ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg gatcatatgc 11820 atagtaccga gaaactagag gatc 11844 <210> 2 <211> 15539 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 2 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat 420 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag 480 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca 540 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac 600 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg 660 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat 720 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc 780 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt 840 ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg 900 gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta 960 ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga 1020 attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg 1080 ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt 1140 ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat 1200 atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta 1260 tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg 1320 ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg 1380 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt 1440 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 1500 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 1560 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 1620 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 1680 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 1740 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 1800 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 1860 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc 1920 gcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 1980 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag 2040 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac 2100 ggcgcgccac tggtagagag cgactttgta tgccccaatt gcgaaacccg cgatatcctt 2160 ctcgattctt tagtacccga ccaggacaag gaaaaggagg tcgaaacgtt tttgaagaaa 2220 caagaggaac tacacggaag ctctaaagat ggcaaccagc cagaaactaa gaaaatgaag 2280 ttgatggatc caactggcac cgctggcttg aacaacaata ccagccttcc aacttctgta 2340 aataacggcg gtacgccagt gccaccagta ccgttacctt tcggtatacc tcctttcccc 2400 atgtttccaa tgcccttcat gcctccaacg gctactatca caaatcctca tcaagctgac 2460 gcaagcccta agaaatgaat aacaatactg acagtactaa ataattgcct acttggcttc 2520 acatacgttg catacgtcga tatagataat aatgataatg acagcaggat tatcgtaata 2580 cgtaatagct gaaaatctca aaaatgtgtg ggtcattacg taaataatga taggaatggg 2640 attcttctat ttttcctttt tccattctag cagccgtcgg gaaaacgtgg catcctctct 2700 ttcgggctca attggagtca cgctgccgtg agcatcctct ctttccatat ctaacaactg 2760 agcacgtaac caatggaaaa gcatgagctt agcgttgctc caaaaaagta ttggatggtt 2820 aataccattt gtctgttctc ttctgacttt gactcctcaa aaaaaaaaat ctacaatcaa 2880 cagatcgctt caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa 2940 ttttatccct catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga 3000 cataatactt ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc 3060 tttttctttt gtcatatata accataacca agtaatacat attcaaacta gtatgactga 3120 caaaaaaact cttaaagact taagaaatcg tagttctgtt tacgattcaa tggttaaatc 3180 acctaatcgt gctatgttgc gtgcaactgg tatgcaagat gaagactttg aaaaacctat 3240 cgtcggtgtc atttcaactt gggctgaaaa cacaccttgt aatatccact tacatgactt 3300 tggtaaacta gccaaagtcg gtgttaagga agctggtgct tggccagttc agttcggaac 3360 aatcacggtt tctgatggaa tcgccatggg aacccaagga atgcgtttct ccttgacatc 3420 tcgtgatatt attgcagatt ctattgaagc agccatggga ggtcataatg cggatgcttt 3480 tgtagccatt ggcggttgtg ataaaaacat gcccggttct gttatcgcta tggctaacat 3540 ggatatccca gccatttttg cttacggcgg aacaattgca cctggtaatt tagacggcaa 3600 agatatcgat ttagtctctg tctttgaagg tgtcggccat tggaaccacg gcgatatgac 3660 caaagaagaa gttaaagctt tggaatgtaa tgcttgtccc ggtcctggag gctgcggtgg 3720 tatgtatact gctaacacaa tggcgacagc tattgaagtt ttgggactta gccttccggg 3780 ttcatcttct cacccggctg aatccgcaga aaagaaagca gatattgaag aagctggtcg 3840 cgctgttgtc aaaatgctcg aaatgggctt aaaaccttct gacattttaa cgcgtgaagc 3900 ttttgaagat gctattactg taactatggc tctgggaggt tcaaccaact caacccttca 3960 cctcttagct attgcccatg ctgctaatgt ggaattgaca cttgatgatt tcaatacttt 4020 ccaagaaaaa gttcctcatt tggctgattt gaaaccttct ggtcaatatg tattccaaga 4080 cctttacaag gtcggagggg taccagcagt tatgaaatat ctccttaaaa atggcttcct 4140 tcatggtgac cgtatcactt gtactggcaa aacagtcgct gaaaatttga aggcttttga 4200 tgatttaaca cctggtcaaa aggttattat gccgcttgaa aatcctaaac gtgaagatgg 4260 tccgctcatt attctccatg gtaacttggc tccagacggt gccgttgcca aagtttctgg 4320 tgtaaaagtg cgtcgtcatg tcggtcctgc taaggtcttt aattctgaag aagaagccat 4380 tgaagctgtc ttgaatgatg atattgttga tggtgatgtt gttgtcgtac gttttgtagg 4440 accaaagggc ggtcctggta tgcctgaaat gctttccctt tcatcaatga ttgttggtaa 4500 agggcaaggt gaaaaagttg cccttctgac agatggccgc ttctcaggtg gtacttatgg 4560 tcttgtcgtg ggtcatatcg ctcctgaagc acaagatggc ggtccaatcg cctacctgca 4620 aacaggagac atagtcacta ttgaccaaga cactaaggaa ttacactttg atatctccga 4680 tgaagagtta aaacatcgtc aagagaccat tgaattgcca ccgctctatt cacgcggtat 4740 ccttggtaaa tatgctcaca tcgtttcgtc tgcttctagg ggagccgtaa cagacttttg 4800 gaagcctgaa gaaactggca aaaaatgttg tcctggttgc tgtggttaag cggccgcgtt 4860 aattcaaatt aattgatata gttttttaat gagtattgaa tctgtttaga aataatggaa 4920 tattattttt atttatttat ttatattatt ggtcggctct tttcttctga aggtcaatga 4980 caaaatgata tgaaggaaat aatgatttct aaaattttac aacgtaagat atttttacaa 5040 aagcctagct catcttttgt catgcactat tttactcacg cttgaaatta acggccagtc 5100 cactgcggag tcatttcaaa gtcatcctaa tcgatctatc gtttttgata gctcattttg 5160 gagttcgcga ttgtcttctg ttattcacaa ctgttttaat ttttatttca ttctggaact 5220 cttcgagttc tttgtaaagt ctttcatagt agcttacttt atcctccaac atatttaact 5280 tcatgtcaat ttcggctctt aaattttcca catcatcaag ttcaacatca tcttttaact 5340 tgaatttatt ctctagctct tccaaccaag cctcattgct ccttgattta ctggtgaaaa 5400 gtgatacact ttgcgcgcaa tccaggtcaa aactttcctg caaagaattc accaatttct 5460 cgacatcata gtacaatttg ttttgttctc ccatcacaat ttaatatacc tgatggattc 5520 ttatgaagcg ctgggtaatg gacgtgtcac tctacttcgc ctttttccct actcctttta 5580 gtacggaaga caatgctaat aaataagagg gtaataataa tattattaat cggcaaaaaa 5640 gattaaacgc caagcgttta attatcagaa agcaaacgtc gtaccaatcc ttgaatgctt 5700 cccaattgta tattaagagt catcacagca acatattctt gttattaaat taattattat 5760 tgatttttga tattgtataa aaaaaccaaa tatgtataaa aaaagtgaat aaaaaatacc 5820 aagtatggag aaatatatta gaagtctata cgttaaacca cccgggcccc ccctcgaggt 5880 cgacggtatc gataagcttg atatcgaatt cctgcagccc gggggatcca ctagttctag 5940 agcggccgct ctagaactag taccacaggt gttgtcctct gaggacataa aatacacacc 6000 gagattcatc aactcattgc tggagttagc atatctacaa ttgggtgaaa tggggagcga 6060 tttgcaggca tttgctcggc atgccggtag aggtgtggtc aataagagcg acctcatgct 6120 atacctgaga aagcaacctg acctacagga aagagttact caagaataag aattttcgtt 6180 ttaaaaccta agagtcactt taaaatttgt atacacttat tttttttata acttatttaa 6240 taataaaaat cataaatcat aagaaattcg cttactctta attaatcaaa aagttaaaat 6300 tgtacgaata gattcaccac ttcttaacaa atcaaaccct tcattgattt tctcgaatgg 6360 caatacatgt gtaattaaag gatcaagagc aaacttcttc gccataaagt cggcaacaag 6420 ttttggaaca ctatccttgc tcttaaaacc gccaaatata gctcccttcc atgtacgacc 6480 gcttagcaac agcataggat tcatcgacaa attttgtgaa tcaggaggaa cacctacgat 6540 cacactgact ccatatgcct cttgacagca ggacaacgca gttaccatag tatcaagacg 6600 gcctataact tcaaaagaga aatcaactcc accgtttgac atttcagtaa ggacttcttg 6660 tattggtttc ttataatctt gagggttaac acattcagta gccccgacct ccttagcttt 6720 tgcaaatttg tccttattga tgtctacacc tataatcctc gctgcgcctg cagctttaca 6780 ccccataata acgcttagtc ctactcctcc taaaccgaat actgcacaag tcgaaccctg 6840 tgtaaccttt gcaactttaa ctgcggaacc gtaaccggtg gaaaatccgc accctatcaa 6900 gcaaactttt tccagtggtg aagctgcatc gattttagcg acagatatct cgtccaccac 6960 tgtgtattgg gaaaatgtag aagtaccaag gaaatggtgt ataggtttcc ctctgcatgt 7020 aaatctgctt gtaccatcct gcatagtacc tctaggcata gacaaatcat ttttaaggca 7080 gaaattaccc tcaggatgtt tgcagactct acacttacca cattgaggag tgaacagtgg 7140 gatcacttta tcaccaggac gaacagtggt aacaccttca cctatggatt caacgattcc 7200 ggcagcctcg tgtcccgcga ttactggcaa aggagtaact agagtgccac tcaccacatg 7260 gtcgtcggat ctacagattc cggtggcaac catcttgatt ctaacctcgt gtgcttttgg 7320 tggcgctact tctacttctt ctatgctaaa cggctttttc tcttcccaca aaactgccgc 7380 tttacactta ataactttac cggctgttga catcctcagc tagctattgt aatatgtgtg 7440 tttgtttgga ttattaagaa gaataattac aaaaaaaatt acaaaggaag gtaattacaa 7500 cagaattaag aaaggacaag aaggaggaag agaatcagtt cattatttct tctttgttat 7560 ataacaaacc caagtagcga tttggccata cattaaaagt tgagaaccac cctccctggc 7620 aacagccaca actcgttacc attgttcatc acgatcatga aactcgctgt cagctgaaat 7680 ttcacctcag tggatctctc tttttattct tcatcgttcc actaaccttt ttccatcagc 7740 tggcagggaa cggaaagtgg aatcccattt agcgagcttc ctcttttctt caagaaaaga 7800 cgaagcttgt gtgtgggtgc gcgcgctagt atctttccac attaagaaat ataccataaa 7860 ggttacttag acatcactat ggctatatat atatatatat atatatgtaa cttagcacca 7920 tcgcgcgtgc atcactgcat gtgttaaccg aaaagtttgg cgaacacttc accgacacgg 7980 tcatttagat ctgtcgtctg cattgcacgt cccttagcct taaatcctag gcgggagcat 8040 tctcgtgtaa ttgtgcagcc tgcgtagcaa ctcaacatag cgtagtctac ccagtttttc 8100 aagggtttat cgttagaaga ttctcccttt tcttcctgct cacaaatctt aaagtcatac 8160 attgcacgac taaatgcaag catgcggatc ccccgggctg caggaattcg atatcaagct 8220 tatcgatacc gtcgactggc cattaatctt tcccatatta gatttcgcca agccatgaaa 8280 gttcaagaaa ggtctttaga cgaattaccc ttcatttctc aaactggcgt caagggatcc 8340 tggtatggtt ttatcgtttt atttctggtt cttatagcat cgttttggac ttctctgttc 8400 ccattaggcg gttcaggagc cagcgcagaa tcattctttg aaggatactt atcctttcca 8460 attttgattg tctgttacgt tggacataaa ctgtatacta gaaattggac tttgatggtg 8520 aaactagaag atatggatct tgataccggc agaaaacaag tagatttgac tcttcgtagg 8580 gaagaaatga ggattgagcg agaaacatta gcaaaaagat ccttcgtaac aagattttta 8640 catttctggt gttgaaggga aagatatgag ctatacagcg gaatttccat atcactcaga 8700 ttttgttatc taattttttc cttcccacgt ccgcgggaat ctgtgtatat tactgcatct 8760 agatatatgt tatcttatct tggcgcgtac atttaatttt caacgtattc tataagaaat 8820 tgcgggagtt tttttcatgt agatgatact gactgcacgc aaatataggc atgatttata 8880 ggcatgattt gatggctgta ccgataggaa cgctaagagt aacttcagaa tcgttatcct 8940 ggcggaaaaa attcatttgt aaactttaaa aaaaaaagcc aatatcccca aaattattaa 9000 gagcgcctcc attattaact aaaatttcac tcagcatcca caatgtatca ggtatctact 9060 acagatatta catgtggcga aaaagacaag aacaatgcaa tagcgcatca agaaaaaaca 9120 caaagctttc aatcaatgaa tcgaaaatgt cattaaaata gtatataaat tgaaactaag 9180 tcataaagct ataaaaagaa aatttattta aatgcaagat ttaaagtaaa ttcacggccc 9240 tgcaggcctc agctcttgtt ttgttctgca aataacttac ccatcttttt caaaacttta 9300 ggtgcaccct cctttgctag aataagttct atccaataca tcctatttgg atctgcttga 9360 gcttctttca tcacggatac gaattcattt tctgttctca caattttgga cacaactctg 9420 tcttccgttg ccccgaaact ttctggcagt tttgagtaat tccacatagg aatgtcatta 9480 taactctggt tcggaccatg aatttccctc tcaaccgtgt aaccatcgtt attaatgata 9540 aagcagattg ggtttatctt ctctctaatg gctagtccta attcttggac agtcagttgc 9600 aatgatccat ctccgataaa caataaatgt ctagattctt tatctgcaat ttggctgcct 9660 agagctgcgg ggaaagtgta tcctatagat ccccacaagg gttgaccaat aaaatgtgat 9720 ttcgatttca gaaatataga tgaggcaccg aagaaagaag tgccttgttc agccacgatc 9780 gtctcattac tttgggtcaa attttcgaca gcttgccaca gtctatcttg tgacaacagc 9840 gcgttagaag gtacaaaatc ttcttgcttt ttatctatgt acttgccttt atattcaatt 9900 tcggacaagt caagaagaga tgatatcagg gattcgaagt cgaaattttg gattctttcg 9960 ttgaaaattt taccttcatc gatattcaag gaaatcattt tattttcatt aagatggtga 10020 gtaaatgcac ccgtactaga atcggtaagc tttacaccca acataagaat aaaatcagca 10080 gattccacaa attccttcaa gtttggctct gacagagtac cgttgtaaat ccccaaaaat 10140 gagggcaatg cttcatcaac agatgattta ccaaagttca aagtagtaat aggtaactta 10200 gtctttgaaa taaactgagt aacagtcttc tctaggccga acgatataat ttcatggcct 10260 gtgattacaa ttggtttctt ggcattcttc agactttcct gtattttgtt cagaatctct 10320 tgatcagatg tattcgacgt ggaattttcc ttcttaagag gcaaggatgg tttttcagcc 10380 ttagcggcag ctacatctac aggtaaattg atgtaaaccg gctttctttc ctttagtaag 10440 gcagacaaca ctctatcaat ttcaacagtt gcattctcgg ctgtcaataa agtcctggca 10500 gcagtaaccg gttcgtgcat cttcataaag tgcttgaaat caccatcagc caacgtatgg 10560 tgaacaaact taccttcgtt ctgcactttc gaggtaggag atcccacgat ctcaacaaca 10620 ggcaggttct cagcatagga gcccgctaag ccattaactg cggataattc gccaacacca 10680 aatgtagtca agaatgccgc agcctttttc gttcttgcgt acccgtcggc catataggag 10740 gcatttaact cattagcatt tcccacccat ttcatatctt tgtgtgaaat aatttgatct 10800 agaaattgca aattgtagtc acctggtact ccgaatattt cttctatacc taattcgtgt 10860 aatctgtcca acagatagtc acctactgta tacattttgt ttactagttt atgtgtgttt 10920 attcgaaact aagttcttgg tgttttaaaa ctaaaaaaaa gactaactat aaaagtagaa 10980 tttaagaagt ttaagaaata gatttacaga attacaatca atacctaccg tctttatata 11040 cttattagtc aagtagggga ataatttcag ggaactggtt tcaacctttt ttttcagctt 11100 tttccaaatc agagagagca gaaggtaata gaaggtgtaa gaaaatgaga tagatacatg 11160 cgtgggtcaa ttgccttgtg tcatcattta ctccaggcag gttgcatcac tccattgagg 11220 ttgtgcccgt tttttgcctg tttgtgcccc tgttctctgt agttgcgcta agagaatgga 11280 cctatgaact gatggttggt gaagaaaaca atattttggt gctgggattc tttttttttc 11340 tggatgccag cttaaaaagc gggctccatt atatttagtg gatgccagga ataaactgtt 11400 cacccagaca cctacgatgt tatatattct gtgtaacccg ccccctattt tgggcatgta 11460 cgggttacag cagaattaaa aggctaattt tttgactaaa taaagttagg aaaatcacta 11520 ctattaatta tttacgtatt ctttgaaatg gcagtattga taatgataaa ctcgaactga 11580 aaaagcgtgt tttttattca aaatgattct aactccctta cgtaatcaag gaatcttttt 11640 gccttggcct ccgcgtcatt aaacttcttg ttgttgacgc taacattcaa cgctagtata 11700 tattcgtttt tttcaggtaa gttcttttca acgggtctta ctgatgaggc agtcgcgtct 11760 gaacctgtta agaggtcaaa tatgtcttct tgaccgtacg tgtcttgcat gttattagct 11820 ttgggaattt gcatcaagtc ataggaaaat ttaaatcttg gctctcttgg gctcaaggtg 11880 acaaggtcct cgaaaatagg gcgcgcccca ccgcggtgga gctccagctt ttgttccctt 11940 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 12000 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 12060 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 12120 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 12180 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 12240 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 12300 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 12360 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 12420 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 12480 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 12540 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 12600 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 12660 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 12720 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 12780 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 12840 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 12900 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 12960 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 13020 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 13080 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 13140 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 13200 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 13260 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 13320 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 13380 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 13440 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 13500 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 13560 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 13620 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 13680 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 13740 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 13800 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 13860 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 13920 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 13980 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 14040 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 14100 cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt ttgtagaaca 14160 aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga 14220 acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt catttttgta 14280 aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt 14340 acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta tacttctttt 14400 ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc ttagattact 14460 ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc actgtaggtc 14520 cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa aaaagcctga 14580 ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt tcaagataaa 14640 ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga acagaaagtg 14700 atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct attttgtctc 14760 tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca ctctatgaat 14820 agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat aaaaaatgta 14880 gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt tatataggga 14940 tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg aagcggtatt 15000 cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga aagtgcgtct 15060 tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta gagaatagga 15120 acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa atgcaacgcg 15180 agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt gcctgtatat 15240 atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta cttatatgcg 15300 tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc cattccatgc 15360 ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct gccactcctc 15420 aattggatta gtctcatcct tcaatgctat catttccttt gatattggat catactaaga 15480 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc 15539 <210> 3 <211> 4586 <212> DNA <213> Template <400> 3 gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 60 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 120 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 180 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 240 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 300 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 360 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 420 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 480 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 540 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 600 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 660 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 720 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 780 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 840 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 900 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 960 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 1020 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 1080 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 1140 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 1200 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 1260 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 1320 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 1380 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 1440 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 1500 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 1560 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 1620 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 1680 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 1740 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 1800 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 1860 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 1920 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 1980 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 2040 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 2100 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 2160 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 2220 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 2280 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 2340 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 2400 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 2460 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 2520 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 2580 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 2640 attacgccaa gcttgcatgc ctgcaggtcg actctagagg atccccgcat tgcggattac 2700 gtattctaat gttcagataa cttcgtatag catacattat acgaagttat ctagggattc 2760 ataaccattt tctcaatcga attacacaga acacaccgta caaacctctc tatcataact 2820 acttaatagt cacacacgta ctcgtctaaa tacacatcat cgtcctacaa gttcatcaaa 2880 gtgttggaca gacaactata ccagcatgga tctcttgtat cggttctttt ctcccgctct 2940 ctcgcaataa caatgaacac tgggtcaatc atagcctaca caggtgaaca gagtagcgtt 3000 tatacagggt ttatacggtg attcctacgg caaaaatttt tcatttctaa aaaaaaaaag 3060 aaaaattttt ctttccaacg ctagaaggaa aagaaaaatc taattaaatt gatttggtga 3120 ttttctgaga gttccctttt tcatatatcg aattttgaat ataaaggag atcgaaaaaa 3180 tttttctatt caatctgttt tctggtttta tttgatagtt tttttgtgta ttattattat 3240 ggattagtac tggtttatat gggtttttct gtataacttc tttttatttt agtttgttta 3300 atcttatttt gagttacatt atagttccct aactgcaaga gaagtaacat taaaactcga 3360 gatgggtaag gaaaagactc acgtttcgag gccgcgatta aattccaaca tggatgctga 3420 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg 3480 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc 3540 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc 3600 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc 3660 cggcaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga 3720 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa 3780 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga 3840 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat 3900 gcataagctt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga 3960 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat 4020 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc 4080 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca 4140 gtttcatttg atgctcgatg agtttttcta agtttaactt gatactacta gattttttct 4200 cttcatttat aaaatttttg gttataattg aagctttaga agtatgaaaa aatccttttt 4260 tttcattctt tgcaaccaaa ataagaagct tcttttattc attgaaatga tgaatataaa 4320 cctaacaaaa gaaaaagact cgaatatcaa acattaaaaa aaaataaaag aggttatctg 4380 ttttcccatt tagttggagt ttgcattttc taatagatag aactctcaat taatgtggat 4440 ttagtttctc tgttcgtttt tttttgtttt gttctcactg tatttacatt tctatttagt 4500 atttagttat tcatataatc tataacttcg tatagcatac attatacgaa gttatccagt 4560 gatgatacaa cgagttagcc aaggtg 4586 <210> 4 <211> 80 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 4 ttccggtttc tttgaaattt ttttgattcg gtaatctccg agcagaagga gcattgcgga 60 ttacgtattc taatgttcag 80 <210> 5 <211> 81 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 5 gggtaataac tgatataatt aaattgaagc tctaatttgt gagtttagta caccttggct 60 aactcgttgt atcatcactg g 81 <210> 6 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 6 gcctcgagtt ttaatgttac ttctcttgca gttaggga 38 <210> 7 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 7 gctaaattcg agtgaaacac aggaagacca g 31 <210> 8 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 8 gtattttggt agattcaatt ctctttccct ttccttttcc ttcgctcccc ttccttatca 60 gcattgcgga ttacgtattc taatgttcag 90 <210> 9 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 9 ttggttgggg gaaaaagagg caacaggaaa gatcagaggg ggaggggggg ggagagtgtc 60 accttggcta actcgttgta tcatcactgg 90 <210> 10 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 10 tcggtgcggg cctcttcgct a 21 <210> 11 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 11 aatgtgagtt agctcactca t 21 <210> 12 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 12 aattggatcc ggcgcgccgt ttaaacggcc ggccaatgtg gctgtggttt cagggtc 57 <210> 13 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 13 aatttctaga ttaattaagc ggccgcaagg ccatgaagct ttttctttc 49 <210> 14 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 14 ttctcgacgt gggccttttt cttg 24 <210> 15 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 15 tgcagcttta aataatcggt gtcactactt tgccttcgtt tatcttgcc 49 <210> 16 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 16 gagcaggcaa gataaacgaa ggcaaagtag tgacaccgat tatttaaag 49 <210> 17 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 17 tatggaccct gaaaccacag ccacattgta accaccacga cggttgttg 49 <210> 18 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 18 tttagcaaca accgtcgtgg tggttacaat gtggctgtgg tttcagggt 49 <210> 19 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 19 ccagaaaccc tatacctgtg tggacgtaag gccatgaagc tttttcttt 49 <210> 20 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 20 attggaaaga aaaagcttca tggccttacg tccacacagg tatagggtt 49 <210> 21 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 21 cataagaaca cctttggtgg ag 22 <210> 22 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 22 aggattatca ttcataagtt tc 22 <210> 23 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 23 ttcttggagc tgggacatgt ttg 23 <210> 24 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 24 tgatgatatt tcataaataa tg 22 <210> 25 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 25 atgcgtccat ctttacagtc ctg 23 <210> 26 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 26 tacgtacgga ccaatcgaag tg 22 <210> 27 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 27 aattcgtttg agtacactac taatggcttt gttggcaata tgtttttgc 49 <210> 28 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 28 atatagcaaa aacatattgc caacaaagcc attagtagtg tactcaaac 49 <210> 29 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 29 tatggaccct gaaaccacag ccacattctt gttatttata aaaagacac 49 <210> 30 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 30 ctcccgtgtc tttttataaa taacaagaat gtggctgtgg tttcagggt 49 <210> 31 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 31 taccgtaggc gtccttagga aagatagaag gccatgaagc tttttcttt 49 <210> 32 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 32 attggaaaga aaaagcttca tggccttcta tctttcctaa ggacgccta 49 <210> 33 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 33 ttattgtttg gcatttgtag c 21 <210> 34 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 34 ccaagcatct cataaaccta tg 22 <210> 35 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 35 tgtgcagatg cagatgtgag ac 22 <210> 36 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 36 agttattgat accgtac 17 <210> 37 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 37 cgagataccg taggcgtcc 19 <210> 38 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 38 ttatgtatgc tcttctgact tttc 24 <210> 39 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 39 aataattaga gattaaatcg ctcatttttt gccagtttct tcaggcttc 49 <210> 40 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 40 agcctgaaga aactggcaaa aaatgagcga tttaatctct aattattag 49 <210> 41 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 41 tatggaccct gaaaccacag ccacattttt caatcattgg agcaatcat 49 <210> 42 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 42 taaaatgatt gctccaatga ttgaaaaatg tggctgtggt ttcagggtc 49 <210> 43 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 43 accgtaggtg ttgtttggga aagtggaagg ccatgaagct ttttctttc 49 <210> 44 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 44 ttggaaagaa aaagcttcat ggccttccac tttcccaaac aacacctac 49 <210> 45 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 45 ttattgctta gcgttggtag cag 23 <210> 46 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 46 tttttggtgg ttccggcttc c 21 <210> 47 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 47 aaagttggca tagcggaaac tt 22 <210> 48 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 48 gtcattgaca ccatct 16 <210> 49 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 49 agagataccg taggtgttg 19 <210> 50 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 50 aattggcgcg ccatgaaagc tctggtttat cac 33 <210> 51 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 51 tgaatcatga gttttatgtt aattagctca ggcagcgcct gcgttcgag 49 <210> 52 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 52 atcctctcga acgcaggcgc tgcctgagct aattaacata aaactcatg 49 <210> 53 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 53 aattgtttaa acaagtaaat aaattaatca gcat 34 <210> 54 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 54 acacaataca ataacaagaa gaacaaaatg aaagctctgg tttatcacg 49 <210> 55 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 55 agcgtataca tctgttggga aagtagaagg ccatgaagct ttttctttc 49 <210> 56 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 56 ttggaaagaa aaagcttcat ggccttctac tttcccaaca gatgtatac 49 <210> 57 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 57 ttattgttta gcgttagtag cg 22 <210> 58 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 58 cataatcaat ctcaaagaga acaacacaat acaataacaa gaagaacaaa atgaaagctc 60 tggtttatca cg 72 <210> 59 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 59 taggcataat caccgaagaa g 21 <210> 60 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 60 aaaatggtaa gcagctgaaa g 21 <210> 61 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 61 agttgttaga actgttg 17 <210> 62 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 62 gacgatagcg tatacatct 19 <210> 63 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 63 cttagcctct agccatagcc at 22 <210> 64 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 64 ttagttttgc tggccgcatc ttc 23 <210> 65 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 65 cccattaata tactattgag a 21 <210> 66 <211> 7523 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 66 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc 60 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatagga gccggaagca 120 taaagtgtaa agcctggggt gcctaatgag tgaggtaact cacattaatt gcgttgcgct 180 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 240 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 300 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 360 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 420 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 480 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 540 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 600 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 660 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 720 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 780 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 840 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 900 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 960 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1020 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1080 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1140 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1200 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1260 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1320 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 1380 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1440 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1500 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1560 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1620 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1680 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1740 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1800 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 1860 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 1920 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 1980 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2040 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2100 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2160 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaacga agcatctgtg 2220 cttcattttg tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg 2280 agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc tattttacca acgaagaatc 2340 tgtgcttcat ttttgtaaaa caaaaatgca acgcgagagc gctaattttt caaacaaaga 2400 atctgagctg catttttaca gaacagaaat gcaacgcgag agcgctattt taccaacaaa 2460 gaatctatac ttcttttttg ttctacaaaa atgcatcccg agagcgctat ttttctaaca 2520 aagcatctta gattactttt tttctccttt gtgcgctcta taatgcagtc tcttgataac 2580 tttttgcact gtaggtccgt taaggttaga agaaggctac tttggtgtct attttctctt 2640 ccataaaaaa agcctgactc cacttcccgc gtttactgat tactagcgaa gctgcgggtg 2700 cattttttca agataaaggc atccccgatt atattctata ccgatgtgga ttgcgcatac 2760 tttgtgaaca gaaagtgata gcgttgatga ttcttcattg gtcagaaaat tatgaacggt 2820 ttcttctatt ttgtctctat atactacgta taggaaatgt ttacattttc gtattgtttt 2880 cgattcactc tatgaatagt tcttactaca atttttttgt ctaaagagta atactagaga 2940 taaacataaa aaatgtagag gtcgagttta gatgcaagtt caaggagcga aaggtggatg 3000 ggtaggttat atagggatat agcacagaga tatatagcaa agagatactt ttgagcaatg 3060 tttgtggaag cggtattcgc aatattttag tagctcgtta cagtccggtg cgtttttggt 3120 tttttgaaag tgcgtcttca gagcgctttt ggttttcaaa agcgctctga agttcctata 3180 ctttctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct 3240 tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct 3300 gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa 3360 atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat 3420 attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct 3480 atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat 3540 attggatcat ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 3600 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 3660 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 3720 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 3780 ttgtactgag agtgcaccat aaattcccgt tttaagagct tggtgagcgc taggagtcac 3840 tgccaggtat cgtttgaaca cggcattagt cagggaagtc ataacacagt cctttcccgc 3900 aattttcttt ttctattact cttggcctcc tctagtacac tctatatttt tttatgcctc 3960 ggtaatgatt ttcatttttt tttttcccct agcggatgac tctttttttt tcttagcgat 4020 tggcattatc acataatgaa ttatacatta tataaagtaa tgtgatttct tcgaagaata 4080 tactaaaaaa tgagcaggca agataaacga aggcaaagat gacagagcag aaagccctag 4140 taaagcgtat tacaaatgaa accaagattc agattgcgat ctctttaaag ggtggtcccc 4200 tagcgataga gcactcgatc ttcccagaaa aagaggcaga agcagtagca gaacaggcca 4260 cacaatcgca agtgattaac gtccacacag gtatagggtt tctggaccat atgatacatg 4320 ctctggccaa gcattccggc tggtcgctaa tcgttgagtg cattggtgac ttacacatag 4380 acgaccatca caccactgaa gactgcggga ttgctctcgg tcaagctttt aaagaggccc 4440 tactggcgcg tggagtaaaa aggtttggat caggatttgc gcctttggat gaggcacttt 4500 ccagagcggt ggtagatctt tcgaacaggc cgtacgcagt tgtcgaactt ggtttgcaaa 4560 gggagaaagt aggagatctc tcttgcgaga tgatcccgca ttttcttgaa agctttgcag 4620 aggctagcag aattaccctc cacgttgatt gtctgcgagg caagaatgat catcaccgta 4680 gtgagagtgc gttcaaggct cttgcggttg ccataagaga agccacctcg cccaatggta 4740 ccaacgatgt tccctccacc aaaggtgttc ttatgtagtg acaccgatta tttaaagctg 4800 cagcatacga tatatataca tgtgtatata tgtataccta tgaatgtcag taagtatgta 4860 tacgaacagt atgatactga agatgacaag gtaatgcatc attctatacg tgtcattctg 4920 aacgaggcgc gctttccttt tttctttttg ctttttcttt ttttttctct tgaactcgac 4980 ggatctatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa 5040 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt 5100 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag 5160 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg 5220 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat 5280 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc 5340 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga 5400 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac 5460 ccgccgcgct taatgcgccg ctacagggcg cgtcgcgcca ttcgccattc aggctgcgca 5520 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 5580 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 5640 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtaccgggc 5700 cccccctcga ggtattagaa gccgccgagc gggcgacagc cctccgacgg aagactctcc 5760 tccgtgcgtc ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca 5820 ctgctccgaa caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt 5880 ggcagtaacc tggccccaca aaccttcaaa ttaacgaatc aaattaacaa ccataggatg 5940 ataatgcgat tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt 6000 ttgatctatt aacagatata taaatggaaa agctgcataa ccactttaac taatactttc 6060 aacattttca gtttgtatta cttcttattc aaatgtcata aaagtatcaa caaaaaattg 6120 ttaatatacc tctatacttt aacgtcaagg agaaaaatgt ccaatttact gcccgtacac 6180 caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg 6240 gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct tctgtccgtt 6300 tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc cgcagaacct 6360 gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt aaaaactatc 6420 cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc acgaccaagt 6480 gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt 6540 gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc 6600 atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg gattgcttat 6660 aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat ctcacgtact 6720 gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag caccgcaggt 6780 gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat ttccgtctct 6840 ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa tggtgttgcc 6900 gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt tgaagcaact 6960 catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc ctggtctgga 7020 cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc aataccggag 7080 atcatgcaag ctggtggctg gaccaatgta aatattgtca tgaactatat ccgtaacctg 7140 gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattagga gtaagcgaat 7200 ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa 7260 ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg 7320 taggtcaggt tgctttctca ggtatagcat gaggtcgctc ttattgacca cacctctacc 7380 ggcatgccga gcaaatgcct gcaaatcgct ccccatttca cccaattgta gatatgctaa 7440 ctccagcaat gagttgatga atctcggtgt gtattttatg tcctcagagg acaacacctg 7500 tggtccgcca ccgcggtgga gct 7523 <210> 67 <211> 15456 <212> DNA <213> Artificial sequence <220> <223> Template <400> 67 aaagagtaat actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca 60 aggagcgaaa ggtggatggg taggttatat agggatatag cacagagata tatagcaaag 120 agatactttt gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca 180 gtccggtgcg tttttggttt tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag 240 cgctctgaag ttcctatact ttctagagaa taggaacttc ggaataggaa cttcaaagcg 300 tttccgaaaa cgagcgcttc cgaaaatgca acgcgagctg cgcacataca gctcactgtt 360 cacgtcgcac ctatatctgc gtgttgcctg tatatatata tacatgagaa gaacggcata 420 gtgcgtgttt atgcttaaat gcgtacttat atgcgtctat ttatgtagga tgaaaggtag 480 tctagtacct cctgtgatat tatcccattc catgcggggt atcgtatgct tccttcagca 540 ctacccttta gctgttctat atgctgccac tcctcaattg gattagtctc atccttcaat 600 gctatcattt cctttgatat tggatcatac taagaaacca ttattatcat gacattaacc 660 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa 720 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 780 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac 840 tatgcggcat cagagcagat tgtactgaga gtgcaccata aattcccgtt ttaagagctt 900 ggtgagcgct aggagtcact gccaggtatc gtttgaacac ggcattagtc agggaagtca 960 taacacagtc ctttcccgca attttctttt tctattactc ttggcctcct ctagtacact 1020 ctatattttt ttatgcctcg gtaatgattt tcattttttt ttttccacct agcggatgac 1080 tctttttttt tcttagcgat tggcattatc acataatgaa ttatacatta tataaagtaa 1140 tgtgatttct tcgaagaata tactaaaaaa tgagcaggca agataaacga aggcaaagat 1200 gacagagcag aaagccctag taaagcgtat tacaaatgaa accaagattc agattgcgat 1260 ctctttaaag ggtggtcccc tagcgataga gcactcgatc ttcccagaaa aagaggcaga 1320 agcagtagca gaacaggcca cacaatcgca agtgattaac gtccacacag gtatagggtt 1380 tctggaccat atgatacatg ctctggccaa gcattccggc tggtcgctaa tcgttgagtg 1440 cattggtgac ttacacatag acgaccatca caccactgaa gactgcggga ttgctctcgg 1500 tcaagctttt aaagaggccc taggggccgt gcgtggagta aaaaggtttg gatcaggatt 1560 tgcgcctttg gatgaggcac tttccagagc ggtggtagat ctttcgaaca ggccgtacgc 1620 agttgtcgaa cttggtttgc aaagggagaa agtaggagat ctctcttgcg agatgatccc 1680 gcattttctt gaaagctttg cagaggctag cagaattacc ctccacgttg attgtctgcg 1740 aggcaagaat gatcatcacc gtagtgagag tgcgttcaag gctcttgcgg ttgccataag 1800 agaagccacc tcgcccaatg gtaccaacga tgttccctcc accaaaggtg ttcttatgta 1860 gtgacaccga ttatttaaag ctgcagcata cgatatatat acatgtgtat atatgtatac 1920 ctatgaatgt cagtaagtat gtatacgaac agtatgatac tgaagatgac aaggtaatgc 1980 atcattctat acgtgtcatt ctgaacgagg cgcgctttcc ttttttcttt ttgctttttc 2040 tttttttttc tcttgaactc gacggatcta tgcggtgtga aataccgcac agatgcgtaa 2100 ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa 2160 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa 2220 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact 2280 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc 2340 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa 2400 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc 2460 ggaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt 2520 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat 2580 tcgccattca ggctgcgcaa ctgttgggaa gggcgcggtg cgggcctctt cgctattacg 2640 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 2700 ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg 2760 cgaattgggt accgggcccc ccctcgaggt cgacggcgcg ccactggtag agagcgactt 2820 tgtatgcccc aattgcgaaa cccgcgatat ccttctcgat tctttagtac ccgaccagga 2880 caaggaaaag gaggtcgaaa cgtttttgaa gaaacaagag gaactacacg gaagctctaa 2940 agatggcaac cagccagaaa ctaagaaaat gaagttgatg gatccaactg gcaccgctgg 3000 cttgaacaac aataccagcc ttccaacttc tgtaaataac ggcggtacgc cagtgccacc 3060 agtaccgtta cctttcggta tacctccttt ccccatgttt ccaatgccct tcatgcctcc 3120 aacggctact atcacaaatc ctcatcaagc tgacgcaagc cctaagaaat gaataacaat 3180 actgacagta ctaaataatt gcctacttgg cttcacatac gttgcatacg tcgatataga 3240 taataatgat aatgacagca ggattatcgt aatacgtaat agctgaaaat ctcaaaaatg 3300 tgtgggtcat tacgtaaata atgataggaa tgggattctt ctatttttcc tttttccatt 3360 ctagcagccg tcgggaaaac gtggcatcct ctctttcggg ctcaattgga gtcacgctgc 3420 cgtgagcatc ctctctttcc atatctaaca actgagcacg taaccaatgg aaaagcatga 3480 gcttagcgtt gctccaaaaa agtattggat ggttaatacc atttgtctgt tctcttctga 3540 ctttgactcc tcaaaaaaaa aaatctacaa tcaacagatc gcttcaatta cgccctcaca 3600 aaaacttttt tccttcttct tcgcccacgt taaattttat ccctcatgtt gtctaacgga 3660 tttctgcact tgatttatta taaaaagaca aagacataat acttctctat caatttcagt 3720 tattgttctt ccttgcgtta ttcttctgtt cttctttttc ttttgtcata tataaccata 3780 accaagtaat acatattcaa actagtatga ctgacaaaaa aactcttaaa gacttaagaa 3840 atcgtagttc tgtttacgat tcaatggtta aatcacctaa tcgtgctatg ttgcgtgcaa 3900 ctggtatgca agatgaagac tttgaaaaac ctatcgtcgg tgtcatttca acttgggctg 3960 aaaacacacc ttgtaatatc cacttacatg actttggtaa actagccaaa gtcggtgtta 4020 aggaagctgg tgcttggcca gttcagttcg gaacaatcac ggtttctgat ggaatcgcca 4080 tgggaaccca aggaatgcgt ttctccttga catctcgtga tattattgca gattctattg 4140 aagcagccat gggaggtcat aatgcggatg cttttgtagc cattggcggt tgtgataaaa 4200 acatgcccgg ttctgttatc gctatggcta acatggatat cccagccatt tttgcttacg 4260 gcggaacaat tgcacctggt aatttagacg gcaaagatat cgatttagtc tctgtctttg 4320 aaggtgtcgg ccattggaac cacggcgata tgaccaaaga agaagttaaa gctttggaat 4380 gtaatgcttg tcccggtcct ggaggctgcg gtggtatgta tactgctaac acaatggcga 4440 cagctattga agttttggga cttagccttc cgggttcatc ttctcacccg gctgaatccg 4500 cagaaaagaa agcagatatt gaagaagctg gtcgcgctgt tgtcaaaatg ctcgaaatgg 4560 gcttaaaacc ttctgacatt ttaacgcgtg aagcttttga agatgctatt actgtaacta 4620 tggctctggg aggttcaacc aactcaaccc ttcacctctt agctattgcc catgctgcta 4680 atgtggaatt gacacttgat gatttcaata ctttccaaga aaaagttcct catttggctg 4740 atttgaaacc ttctggtcaa tatgtattcc aagaccttta caaggtcgga ggggtaccag 4800 cagttatgaa atatctcctt aaaaatggct tccttcatgg tgaccgtatc acttgtactg 4860 gcaaaacagt cgctgaaaat ttgaaggctt ttgatgattt aacacctggt caaaaggtta 4920 ttatgccgct tgaaaatcct aaacgtgaag atggtccgct cattattctc catggtaact 4980 tggctccaga cggtgccgtt gccaaagttt ctggtgtaaa agtgcgtcgt catgtcggtc 5040 ctgctaaggt ctttaattct gaagaagaag ccattgaagc tgtcttgaat gatgatattg 5100 ttgatggtga tgttgttgtc gtacgttttg taggaccaaa gggcggtcct ggtatgcctg 5160 aaatgctttc cctttcatca atgattgttg gtaaagggca aggtgaaaaa gttgcccttc 5220 tgacagatgg ccgcttctca ggtggtactt atggtcttgt cgtgggtcat atcgctcctg 5280 aagcacaaga tggcggtcca atcgcctacc tgcaaacagg agacatagtc actattgacc 5340 aagacactaa ggaattacac tttgatatct ccgatgaaga gttaaaacat cgtcaagaga 5400 ccattgaatt gccaccgctc tattcacgcg gtatccttgg taaatatgct cacatcgttt 5460 cgtctgcttc taggggagcc gtaacagact tttggaagcc tgaagaaact ggcaaaaaat 5520 gttgtcctgg ttgctgtggt taagcggccg cgttaattca aattaattga tatagttttt 5580 taatgagtat tgaatctgtt tagaaataat ggaatattat ttttatttat ttatttatat 5640 tattggtcgg ctcttttctt ctgaaggtca atgacaaaat gatatgaagg aaataatgat 5700 ttctaaaatt ttacaacgta agatattttt acaaaagcct agctcatctt ttgtcatgca 5760 ctattttact cacgcttgaa attaacggcc agtccactgc ggagtcattt caaagtcatc 5820 ctaatcgatc tatcgttttt gatagctcat tttggagttc gcgattgtct tctgttattc 5880 acaactgttt taatttttat ttcattctgg aactcttcga gttctttgta aagtctttca 5940 tagtagctta ctttatcctc caacatattt aacttcatgt caatttcggc tcttaaattt 6000 tccacatcat caagttcaac atcatctttt aacttgaatt tattctctag ctcttccaac 6060 caagcctcat tgctccttga tttactggtg aaaagtgata cactttgcgc gcaatccagg 6120 tcaaaacttt cctgcaaaga attcaccaat ttctcgacat catagtacaa tttgttttgt 6180 tctcccatca caatttaata tacctgatgg attcttatga agcgctgggt aatggacgtg 6240 tcactctact tcgccttttt ccctactcct tttagtacgg aagacaatgc taataaataa 6300 gagggtaata ataatattat taatcggcaa aaaagattaa acgccaagcg tttaattatc 6360 agaaagcaaa cgtcgtacca atccttgaat gcttcccaat tgtatattaa gagtcatcac 6420 agcaacatat tcttgttatt aaattaatta ttattgattt ttgatattgt ataaaaaaac 6480 caaatatgta taaaaaaagt gaataaaaaa taccaagtat ggagaaatat attagaagtc 6540 tatacgttaa accacccggg ccccccctcg aggtcgacgg tatcgataag cttgatatcg 6600 aattcctgca gcccggggga tccactagtt ctagagcggc cgctctagaa ctagtaccac 6660 aggtgttgtc ctctgaggac ataaaataca caccgagatt catcaactca ttgctggagt 6720 tagcatatct acaattgggt gaaatgggga gcgatttgca ggcatttgct cggcatgccg 6780 gtagaggtgt ggtcaataag agcgacctca tgctatacct gagaaagcaa cctgacctac 6840 aggaaagagt tactcaagaa taagaatttt cgttttaaaa cctaagagtc actttaaaat 6900 ttgtatacac ttattttttt tataacttat ttaataataa aaatcataaa tcataagaaa 6960 ttcgcttact cttaattaat caggcagcgc ctgcgttcga gaggatgatc ttcatcgcct 7020 tctccttggc gccattgagg aatacctgat aggcgtgctc gatctcggcc agctcgaagc 7080 gatgggtaat catcttcttc aacggaagct tgtcggtcga ggcgaccttc atcagcatgg 7140 gcgtcgtgtt cgtgttcacc agtcccgtgg tgatcgtcag gttcttgatc cagagcttct 7200 gaatctcgaa gtcaaccttg acgccatgca cgccgacgtt ggcgatgtgc gcgccgggct 7260 tgacgatctc ctggcagatg tcccaagtcg ccggtatgcc caccgcctcg atcgcaacat 7320 cgactccctc tgccgcaatc ctatgcacgg cttcgacaac gttctccgtg ccggagttga 7380 tggtgtgcgt tgccccgagc tccttggcga gctggaggcg attctcgtcc atgtcgatca 7440 cgatgatggt cgagggggag tagaactggg cggtcaacag tacggacatg ccgacggggc 7500 ccgcgccgac aatagccacc gcatcgcccg gctggacatt cccatactgg acgccgattt 7560 cgtggccggt gggcaggatg tcgctcagca ggacggcgat ttcgtcgtca attgtctggg 7620 ggatcttgta gaggctgttg tcggcatgcg ggatgcggac gtattcggcc tgcacgccat 7680 cgatcatgta acccaggatc cacccgccgt cgcggcaatg ggagtaaagc tgcttcttgc 7740 agtagtcgca cgagccgcaa gaagtgacgc aggaaatcag gaccttgtcg cctttcttga 7800 actgcgtgac actctcgccc acttcctcga tgacgcctac cccttcatgg cccaggatgc 7860 gcccgtcggc gacctctgga ttcttgcctt tgtagatgcc gagatccgtg ccgcagatcg 7920 tggtcttcaa aacccgtact actacatccg tgggcttttg aagggtgggc ttgggcttgt 7980 cttcaagcga gatcttgtgg tcaccgtgat aaaccagagc tttcatcctc agctattgta 8040 atatgtgtgt ttgtttggat tattaagaag aataattaca aaaaaaatta caaaggaagg 8100 taattacaac agaattaaga aaggacaaga aggaggaaga gaatcagttc attatttctt 8160 ctttgttata taacaaaccc aagtagcgat ttggccatac attaaaagtt gagaaccacc 8220 ctccctggca acagccacaa ctcgttacca ttgttcatca cgatcatgaa actcgctgtc 8280 agctgaaatt tcacctcagt ggatctctct ttttattctt catcgttcca ctaacctttt 8340 tccatcagct ggcagggaac ggaaagtgga atcccattta gcgagcttcc tcttttcttc 8400 aagaaaagac gaagcttgtg tgtgggtgcg cgcgctagta tctttccaca ttaagaaata 8460 taccataaag gttacttaga catcactatg gctatatata tatatatata tatatatgta 8520 acttagcacc atcgcgcgtg catcactgca tgtgttaacc gaaaagtttg gcgaacactt 8580 caccgacacg gtcatttaga tctgtcgtct gcattgcacg tcccttagcc ttaaatccta 8640 ggcgggagca ttctcgtgta attgtgcagc ctgcgtagca actcaacata gcgtagtcta 8700 cccagttttt caagggttta tcgttagaag attctccctt ttcttcctgc tcacaaatct 8760 taaagtcata cattgcacga ctaaatgcaa gcatgcggat cccccgggct gcaggaattc 8820 gatatcaagc ttatcgatac cgtcgactgg ccattaatct ttcccatatt agatttcgcc 8880 aagccatgaa agttcaagaa aggtctttag acgaattacc cttcatttct caaactggcg 8940 tcaagggatc ctggtatggt tttatcgttt tatttctggt tcttatagca tcgttttgga 9000 cttctctgtt cccattaggc ggttcaggag ccagcgcaga atcattcttt gaaggatact 9060 tatcctttcc aattttgatt gtctgttacg ttggacataa actgtatact agaaattgga 9120 ctttgatggt gaaactagaa gatatggatc ttgataccgg cagaaaacaa gtagatttga 9180 ctcttcgtag ggaagaaatg aggattgagc gagaaacatt agcaaaaaga tccttcgtaa 9240 caagattttt acatttctgg tgttgaaggg aaagatatga gctatacagc ggaatttcca 9300 tatcactcag attttgttat ctaatttttt ccttcccacg tccgcgggaa tctgtgtata 9360 ttactgcatc tagatatatg ttatcttatc ttggcgcgta catttaattt tcaacgtatt 9420 ctataagaaa ttgcgggagt ttttttcatg tagatgatac tgactgcacg caaatatagg 9480 catgatttat aggcatgatt tgatggctgt accgatagga acgctaagag taacttcaga 9540 atcgttatcc tggcggaaaa aattcatttg taaactttaa aaaaaaaagc caatatcccc 9600 aaaattatta agagcgcctc cattattaac taaaatttca ctcagcatcc acaatgtatc 9660 aggtatctac tacagatatt acatgtggcg aaaaagacaa gaacaatgca atagcgcatc 9720 aagaaaaaac acaaagcttt caatcaatga atcgaaaatg tcattaaaat agtatataaa 9780 ttgaaactaa gtcataaagc tataaaaaga aaatttattt aaatgcaaga tttaaagtaa 9840 attcacggcc ctgcaggcct cagctcttgt tttgttctgc aaataactta cccatctttt 9900 tcaaaacttt aggtgcaccc tcctttgcta gaataagttc tatccaatac atcctatttg 9960 gatctgcttg agcttctttc atcacggata cgaattcatt ttctgttctc acaattttgg 10020 acacaactct gtcttccgtt gccccgaaac tttctggcag ttttgagtaa ttccacatag 10080 gaatgtcatt ataactctgg ttcggaccat gaatttccct ctcaaccgtg taaccatcgt 10140 tattaatgat aaagcagatt gggtttatct tctctctaat ggctagtcct aattcttgga 10200 cagtcagttg caatgatcca tctccgataa acaataaatg tctagattct ttatctgcaa 10260 tttggctgcc tagagctgcg gggaaagtgt atcctataga tccccacaag ggttgaccaa 10320 taaaatgtga tttcgatttc agaaatatag atgaggcacc gaagaaagaa gtgccttgtt 10380 cagccacgat cgtctcatta ctttgggtca aattttcgac agcttgccac agtctatctt 10440 gtgacaacag cgcgttagaa ggtacaaaat cttcttgctt tttatctatg tacttgcctt 10500 tatattcaat ttcggacaag tcaagaagag atgatatcag ggattcgaag tcgaaatttt 10560 ggattctttc gttgaaaatt ttaccttcat cgatattcaa ggaaatcatt ttattttcat 10620 taagatggtg agtaaatgca cccgtactag aatcggtaag ctttacaccc aacataagaa 10680 taaaatcagc agattccaca aattccttca agtttggctc tgacagagta ccgttgtaaa 10740 tccccaaaaa tgagggcaat gcttcatcaa cagatgattt accaaagttc aaagtagtaa 10800 taggtaactt agtctttgaa ataaactgag taacagtctt ctctaggccg aacgatataa 10860 tttcatggcc tgtgattaca attggtttct tggcattctt cagactttcc tgtattttgt 10920 tcagaatctc ttgatcagat gtattcgacg tggaattttc cttcttaaga ggcaaggatg 10980 gtttttcagc cttagcggca gctacatcta caggtaaatt gatgtaaacc ggctttcttt 11040 cctttagtaa ggcagacaac actctatcaa tttcaacagt tgcattctcg gctgtcaata 11100 aagtcctggc agcagtaacc ggttcgtgca tcttcataaa gtgcttgaaa tcaccatcag 11160 ccaacgtatg gtgaacaaac ttaccttcgt tctgcacttt cgaggtagga gatcccacga 11220 tctcaacaac aggcaggttc tcagcatagg agcccgctaa gccattaact gcggataatt 11280 cgccaacacc aaatgtagtc aagaatgccg cagccttttt cgttcttgcg tacccgtcgg 11340 ccatatagga ggcatttaac tcattagcat ttcccaccca tttcatatct ttgtgtgaaa 11400 taatttgatc tagaaattgc aaattgtagt cacctggtac tccgaatatt tcttctatac 11460 ctaattcgtg taatctgtcc aacagatagt cacctactgt atacattttg tttactagtt 11520 tatgtgtgtt tattcgaaac taagttcttg gtgttttaaa actaaaaaaa agactaacta 11580 taaaagtaga atttaagaag tttaagaaat agatttacag aattacaatc aatacctacc 11640 gtctttatat acttattagt caagtagggg aataatttca gggaactggt ttcaaccttt 11700 tttttcagct ttttccaaat cagagagagc agaaggtaat agaaggtgta agaaaatgag 11760 atagatacat gcgtgggtca attgccttgt gtcatcattt actccaggca ggttgcatca 11820 ctccattgag gttgtgcccg ttttttgcct gtttgtgccc ctgttctctg tagttgcgct 11880 aagagaatgg acctatgaac tgatggttgg tgaagaaaac aatattttgg tgctgggatt 11940 cttttttttt ctggatgcca gcttaaaaag cgggctccat tatatttagt ggatgccagg 12000 aataaactgt tcacccagac acctacgatg ttatatattc tgtgtaaccc gccccctatt 12060 ttgggcatgt acgggttaca gcagaattaa aaggctaatt ttttgactaa ataaagttag 12120 gaaaatcact actattaatt atttacgtat tctttgaaat ggcagtattg ataatgataa 12180 actcgaactg aaaaagcgtg ttttttattc aaaatgattc taactccctt acgtaatcaa 12240 ggaatctttt tgccttggcc tccgcgtcat taaacttctt gttgttgacg ctaacattca 12300 acgctagtat atattcgttt ttttcaggta agttcttttc aacgggtctt actgatgagg 12360 cagtcgcgtc tgaacctgtt aagaggtcaa atatgtcttc ttgaccgtac gtgtcttgca 12420 tgttattagc tttgggaatt tgcatcaagt cataggaaaa tttaaatctt ggctctcttg 12480 ggctcaaggt gacaaggtcc tcgaaaatag ggcgcgcccc accgcggtgg agctccagct 12540 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc 12600 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 12660 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 12720 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 12780 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 12840 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 12900 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 12960 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 13020 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 13080 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 13140 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 13200 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 13260 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 13320 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 13380 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 13440 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 13500 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 13560 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 13620 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 13680 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 13740 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 13800 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 13860 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 13920 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 13980 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 14040 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 14100 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 14160 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 14220 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 14280 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 14340 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 14400 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 14460 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 14520 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 14580 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 14640 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 14700 tgcccctga tttgtagaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc 14820 atttttacag aacagaaatg caacgcgaaa gcgctatttt accaacgaag aatctgtgct 14880 tcatttttgt aaaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga 14940 gctgcatttt tacagaacag aaatgcaacg cgagagcgct attttaccaa caaagaatct 15000 atacttcttt tttgttctac aaaaatgcat cccgagagcg ctatttttct aacaaagcat 15060 cttagattac tttttttctc ctttgtgcgc tctataatgc agtctcttga taactttttg 15120 cactgtaggt ccgttaaggt tagaagaagg ctactttggt gtctattttc tcttccataa 15180 aaaaagcctg actccacttc ccgcgtttac tgattactag cgaagctgcg ggtgcatttt 15240 ttcaagataa aggcatcccc gattatattc tataccgatg tggattgcgc atactttgtg 15300 aacagaaagt gatagcgttg atgattcttc attggtcaga aaattatgaa cggtttcttc 15360 tattttgtct ctatatacta cgtataggaa atgtttacat tttcgtattg ttttcgattc 15420 actctatgaa tagttcttac tacaattttt ttgtct 15456 <210> 68 <211> 1559 <212> DNA <213> Artificial sequence <220> <223> Template <400> 68 gcattgcgga ttacgtattc taatgttcag taccgttcgt ataatgtatg ctatacgaag 60 ttatgcagat tgtactgaga gtgcaccata ccaccttttc aattcatcat ttttttttta 120 ttcttttttt tgatttcggt ttccttgaaa tttttttgat tcggtaatct ccgaacagaa 180 ggaagaacga aggaaggagc acagacttag attggtatat atacgcatat gtagtgttga 240 agaaacatga aattgcccag tattcttaac ccaactgcac agaacaaaaa cctgcaggaa 300 acgaagataa atcatgtcga aagctacata taaggaacgt gctgctactc atcctagtcc 360 tgttgctgcc aagctattta atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt 420 ggatgttcgt accaccaagg aattactgga gttagttgaa gcattaggtc ccaaaatttg 480 tttactaaaa acacatgtgg atatcttgac tgatttttcc atggagggca cagttaagcc 540 gctaaaggca ttatccgcca agtacaattt tttactcttc gaagacagaa aatttgctga 600 cattggtaat acagtcaaat tgcagtactc tgcgggtgta tacagaatag cagaatgggc 660 agacattacg aatgcacacg gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc 720 ggcagaagaa gtaacaaagg aacctagagg ccttttgatg ttagcagaat tgtcatgcaa 780 gggctcccta tctactggag aatatactaa gggtactgtt gacattgcga agagcgacaa 840 agattttgtt atcggcttta ttgctcaaag agacatgggt ggaagagatg aaggttacga 900 ttggttgatt atgacacccg gtgtgggttt agatgacaag ggagacgcat tgggtcaaca 960 gtatgaacc gtggatgatg tggtctctac aggatctgac attattattg ttggaagagg 1020 actatttgca aagggaaggg atgctaaggt agagggtgaa cgttacagaa aagcaggctg 1080 ggaagcatat ttgagaagat gcggccagca aaactaaaaa actgtattat aagtaaatgc 1140 atgtatacta aactcacaaa ttagagcttc aatttaatta tatcagttat taccctatgc 1200 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaaacgt 1260 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 1320 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 1380 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 1440 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagataact 1500 tcgtataatg tatgctatac gaacggtacc agtgatgata caacgagtta gccaaggtg 1559 <210> 69 <211> 1047 <212> DNA <213> Achromobacter xylosoxidans <400> 69 atgaaagctc tggtttatca cggtgaccac aagatctcgc ttgaagacaa gcccaagccc 60 acccttcaaa agcccacgga tgtagtagta cgggttttga agaccacgat ctgcggcacg 120 gatctcggca tctacaaagg caagaatcca gaggtcgccg acgggcgcat cctgggccat 180 gaaggggtag gcgtcatcga ggaagtgggc gagagtgtca cgcagttcaa gaaaggcgac 240 aaggtcctga tttcctgcgt cacttcttgc ggctcgtgcg actactgcaa gaagcagctt 300 tactcccatt gccgcgacgg cgggtggatc ctgggttaca tgatcgatgg cgtgcaggcc 360 gaatacgtcc gcatcccgca tgccgacaac agcctctaca agatccccca gacaattgac 420 gacgaaatcg ccgtcctgct gagcgacatc ctgcccaccg gccacgaaat cggcgtccag 480 tatgggaatg tccagccggg cgatgcggtg gctattgtcg gcgcgggccc cgtcggcatg 540 tccgtactgt tgaccgccca gttctactcc ccctcgacca tcatcgtgat cgacatggac 600 gagaatcgcc tccagctcgc caaggagctc ggggcaacgc acaccatcaa ctccggcacg 660 ggaacgttg tcgaagccgt gcataggatt gcggcagagg gagtcgatgt tgcgatcgag 720 gcggtgggca taccggcgac ttgggacatc tgccaggaga tcgtcaagcc cggcgcgcac 780 atcgccaacg tcggcgtgca tggcgtcaag gttgacttcg agattcagaa gctctggatc 840 aagaacctga cgatcaccac gggactggtg aacacgaaca cgacgcccat gctgatgaag 900 gtcgcctcga ccgacaagct tccgttgaag aagatgatta cccatcgctt cgagctggcc 960 gagatcgagc acgcctatca ggtattcctc aatggcgcca aggagaaggc gatgaagatc 1020 atcctctcga acgcaggcgc tgcctga 1047 <210> 70 <211> 348 <212> PRT <213> Achromobacter xylosoxidans <400> 70 Met Lys Ala Leu Val Tyr His Gly Asp His Lys Ile Ser Leu Glu Asp 1 5 10 15 Lys Pro Lys Pro Thr Leu Gln Lys Pro Thr Asp Val Val Arg Val             20 25 30 Leu Lys Thr Thr Ile Cys Gly Thr Asp Leu Gly Ile Tyr Lys Gly Lys         35 40 45 Asn Pro Glu Val Ala Asp Gly Arg Ile Leu Gly His Glu Gly Val Gly     50 55 60 Val Ile Glu Glu Val Gly Glu Ser Val Thr Gln Phe Lys Lys Gly Asp 65 70 75 80 Lys Val Leu Ile Ser Cys Val Thr Ser Cys Gly Ser Cys Asp Tyr Cys                 85 90 95 Lys Lys Gln Leu Tyr Ser His Cys Arg Asp Gly Gly Trp Ile Leu Gly             100 105 110 Tyr Met Ile Asp Gly Val Gln Ala Glu Tyr Val Arg Ile Pro His Ala         115 120 125 Asp Asn Ser Leu Tyr Lys Ile Pro Gln Thr Ile Asp Asp Glu Ile Ala     130 135 140 Val Leu Leu Ser Asp Ile Leu Pro Thr Gly His Glu Ile Gly Val Gln 145 150 155 160 Tyr Gly Asn Val Gln Pro Gly Asp Ala Val Ala Ile Val Gly Ala Gly                 165 170 175 Pro Val Gly Met Ser Val Leu Leu Thr Ala Gln Phe Tyr Ser Ser Ser             180 185 190 Thr Ile Ile Val Ile Asp Met Asp Glu Asn Arg Leu Gln Leu Ala Lys         195 200 205 Glu Leu Gly Ala Thr His Thr Ile Asn Ser Gly Thr Glu Asn Val Val     210 215 220 Glu Ala Val His Arg Ile Ala Ala Glu Gly Val Asp Val Ala Ile Glu 225 230 235 240 Ala Val Gly Ile Pro Ala Thr Trp Asp Ile Cys Gln Glu Ile Val Lys                 245 250 255 Pro Gly Ala His Ile Ala Asn Val Gly Val His Gly Val Lys Val Asp             260 265 270 Phe Glu Ile Gln Lys Leu Trp Ile Lys Asn Leu Thr Ile Thr Thr Gly         275 280 285 Leu Val Asn Thr Asn Thr Thr Pro Met Leu Met Lys Val Ala Ser Thr     290 295 300 Asp Lys Leu Pro Leu Lys Lys Met Ile Thr His Arg Phe Glu Leu Ala 305 310 315 320 Glu Ile Glu His Ala Tyr Gln Val Phe Leu Asn Gly Ala Lys Glu Lys                 325 330 335 Ala Met Lys Ile Ile Leu Ser Asn Ala Gly Ala Ala             340 345 <210> 71 <211> 1644 <212> DNA Artificial sequence <220> <223> codon optimized L. lactis kivD coding region for S. cerevisiae        expression <400> 71 atgtatacagag taggtgacta tctgttggac agattacacg aattaggtat agaagaaata 60 ttcggagtac caggtgacta caatttgcaa tttctagatc aaattatttc acacaaagat 120 atgaaatggg tgggaaatgc taatgagtta aatgcctcct atatggccga cgggtacgca 180 agaacgaaaa aggctgcggc attcttgact acatttggtg ttggcgaatt atccgcagtt 240 aatggcttag cgggctccta tgctgagaac ctgcctgttg ttgagatcgt gggatctcct 300 acctcgaaag tgcagaacga aggtaagttt gttcaccata cgttggctga tggtgatttc 360 aagcacttta tgaagatgca cgaaccggtt actgctgcca ggactttatt gacagccgag 420 aatgcaactg ttgaaattga tagagtgttg tctgccttac taaaggaaag aaagccggtt 480 tacatcaatt tacctgtaga tgtagctgcc gctaaggctg aaaaaccatc cttgcctctt 540 aagaaggaaa attccacgtc gaatacatct gatcaagaga ttctgaacaa aatacaggaa 600 agtctgaaga atgccaagaa accaattgta atcacaggcc atgaaattat atcgttcggc 660 ctagagaaga ctgttactca gtttatttca aagactaagt tacctattac tactttgaac 720 tttggtaaat catctgttga tgaagcattg ccctcatttt tggggattta caacggtact 780 ctgtcagagc caaacttgaa ggaatttgtg gaatctgctg attttattct tatgttgggt 840 gtaaagctta ccgattctag tacgggtgca tttactcacc atcttaatga aaataaaatg 900 atttccttga atatcgatga aggtaaaatt ttcaacgaaa gaatccaaaa tttcgacttc 960 gaatccctga tatcatctct tcttgacttg tccgaaattg aatataaagg caagtacata 1020 gataaaaagc aagaagattt tgtaccttct aacgcgctgt tgtcacaaga tagactgtgg 1080 caagctgtcg aaaatttgac ccaaagtaat gagacgatcg tggctgaaca aggcacttct 1140 ttcttcggtg cctcatctat atttctgaaa tcgaaatcac attttattgg tcaacccttg 1200 tggggatcta taggatacac tttccccgca gctctaggca gccaaattgc agataaagaa 1260 tctagacatt tattgtttat cggagatgga tcattgcaac tgactgtcca agaattagga 1320 ctagccatta gagagaagat aaacccaatc tgctttatca ttaataacga tggttacacg 1380 gttgagaggg aaattcatgg tccgaaccag agttataatg acattcctat gtggaattac 1440 tcaaaactgc cagaaagttt cggggcaacg gaagacagag ttgtgtccaa aattgtgaga 1500 acagaaaatg aattcgtatc cgtgatgaaa gaagctcaag cagatccaaa taggatgtat 1560 tggatagaac ttattctagc aaaggagggt gcacctaaag ttttgaaaaa gatgggtaag 1620 ttatttgcag aacaaaacaa gagc 1644 <210> 72 <211> 753 <212> DNA <213> Saccharomyces cerevisiae <400> 72 gcatgcttgc atttagtcgt gcaatgtatg actttaagat ttgtgagcag gaagaaaagg 60 gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg ttgagttgct 120 acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct aagggacgtg 180 caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa cttttcggtt 240 aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata tatagccata 300 gtgatgtcta agtaaccttt atggtatatt tcttaatgtg gaaagatact agcgcgcgca 360 cccacacaca agcttcgtct tttcttgaag aaaagaggaa gctcgctaaa tgggattcca 420 ctttccgttc cctgccagct gatggaaaaa ggttagtgga acgatgaaga ataaaaagag 480 agatccactg aggtgaaatt tcagctgaca gcgagtttca tgatcgtgat gaacaatggt 540 aacgagttgt ggctgttgcc agggagggtg gttctcaact tttaatgtat ggccaaatcg 600 ctacttgggt ttgttatata acaaagaaga aataatgaac tgattctctt cctccttctt 660 gtcctttctt aattctgttg taattacctt cctttgtaat tttttttgta attattcttc 720 ttaataatcc aaacaaacac acatattaca ata 753 <210> 73 <211> 316 <212> DNA <213> Saccharomyces cerevisiae <400> 73 gagtaagcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata 60 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt 120 aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac 180 cacacctcta ccggcatgcc gagcaaatgc ctgcaaatcg ctccccattt cacccaattg 240 tagatatgct aactccagca atgagttgat gaatctcggt gtgtatttta tgtcctcaga 300 ggacaacacc tgtggt 316 <210> 74 <211> 30 <212> DNA Artificial sequence <220> <223> primer <400> 74 ggaattcaca catgaaagct ctggtttatc 30 <210> 75 <211> 28 <212> DNA Artificial sequence <220> <223> primer <400> 75 gcgtccaggg cgtcaaagat caggcagc 28 <210> 76 <211> 55 <212> DNA Artificial sequence <220> <223> primer <400> 76 aaacaaacac acatattaca atagctgagg atgaaagctc tggtttatca cggtg 55 <210> 77 <211> 55 <212> DNA Artificial sequence <220> <223> primer <400> 77 atcataagaa attcgcttac tcttaattaa tcaggcagcg cctgcgttcg agagg 55 <210> 78 <211> 8994 <212> DNA Artificial sequence <220> &Lt; 223 > constructed plasmid <400> 78 ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg 60 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 120 ctcacaattc cacacaacat aggagccgga agcataaagt gtaaagcctg gggtgcctaa 180 tgagtgaggt aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 240 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 300 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 360 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 420 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 480 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 540 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 600 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 660 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 720 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 780 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 840 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 900 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 960 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 1020 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 1080 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 1140 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 1200 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 1260 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 1320 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 1380 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 1440 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 1500 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 1560 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 1620 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 1680 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 1740 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 1800 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 1860 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 1920 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 1980 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 2040 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 2100 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 2160 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 2220 ccccgaaaag tgccacctga acgaagcatc tgtgcttcat tttgtagaac aaaaatgcaa 2280 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg 2340 caacgcgaaa gcgctatttt accaacgaag aatctgtgct tcatttttgt aaaacaaaaa 2400 tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag 2460 aaatgcaacg cgagagcgct attttaccaa caaagaatct atacttcttt tttgttctac 2520 aaaaatgcat cccgagagcg ctatttttct aacaaagcat cttagattac tttttttctc 2580 ccttgtgcgc tctataatgc agtctcttga taactttttg cactgtaggt ccgttaaggt 2640 tagaagaagg ctactttggt gtctattttc tcttccataa aaaaagcctg actccacttc 2700 ccgcgtttac tgattactag cgaagctgcg ggtgcatttt ttcaagataa aggcatcccc 2760 gattatattc tataccgatg tggattgcgc atactttgtg aacagaaagt gatagcgttg 2820 atgattcttc attggtcaga aaattatgaa cggtttcttc tattttgtct ctatatacta 2880 cgtataggaa atgtttacat tttcgtattg ttttcgattc actctatgaa tagttcttac 2940 tacaattttt ttgtctaaag agtaatacta gagataaaca taaaaaatgt agaggtcgag 3000 tttagatgca agttcaagga gcgaaaggtg gatgggtagg ttatataggg atatagcaca 3060 gagatatata gcaaagagat acttttgagc aatgtttgtg gaagcggtat tcgcaatatt 3120 ttagtagctc gttacagtcc ggtgcgtttt tggttttttg aaagtgcgtc ttcagagcgc 3180 ttttggtttt caaaagcgct ctgaagttcc tatactttct agagaatagg aacttcggaa 3240 taggaacttc aaagcgtttc cgaaaacgag cgcttccgaa aatgcaacgc gagctgcgca 3300 catacagctc actgttcacg tcgcacctat atctgcgtgt tgcctgtata tatatataca 3360 tgagaagaac ggcatagtgc gtgtttatgc ttaaatgcgt acttatatgc gtctatttat 3420 gtaggatgaa aggtagtcta gtacctcctg tgatattatc ccattccatg cggggtatcg 3480 tatgcttcct tcagcactac cctttagctg ttctatatgc tgccactcct caattggatt 3540 agtctcatcc ttcaatgcta tcatttcctt tgatattgga tcatactaag aaaccattat 3600 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt 3660 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct 3720 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg 3780 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatcga 3840 ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc accattatgg 3900 gaaatgcttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca ttgagtgttt 3960 tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat taggaatcgt 4020 agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc ttgtcaatat 4080 taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc aatttgctta 4140 cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt agattgcgta 4200 tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg tttctattat 4260 gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct ttttaagcaa 4320 ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg ttggaaccac 4380 ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct tcaatggcct 4440 taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac aagatagtgg 4500 cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat ggttcgtaca 4560 aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc aacaaaccca 4620 aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg ttgctggtga 4680 ttataatacc atttaggtgg gttgggttct taactaggat catggcggca gaatcaatca 4740 attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc acagtttttc 4800 tccataatct tgaagaggcc aaaagattag ctttatccaa ggaccaaata ggcaatggtg 4860 gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact tctggaacgg 4920 tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc ttaccaaagt 4980 aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca aattgtggct 5040 tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt aagttggcgt 5100 acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca ctaccggtac 5160 cccatttagg accagccaca gcacctaaca aaacggcatc aaccttcttg gaggcttcca 5220 gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca attaaatgat 5280 tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga accttaatgg 5340 cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc ttcttagggg 5400 cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata tattgctgaa 5460 atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat tggaaaaaac 5520 aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat ttagtcatga 5580 acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct ttttctccca 5640 atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca aaaaatttcc 5700 agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa 5760 aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga gtattcccac 5820 agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg gccaaacaac 5880 caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt ttgaacacac 5940 atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg atgtaattgt 6000 tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta gaagttctcc 6060 tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 6120 aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 6180 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 6240 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 6300 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 6360 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 6420 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 6480 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca 6540 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg ccattcaggc 6600 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 6660 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 6720 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta 6780 ccgggccccc cctcgaggtc gacggtatcg ataagcttga tatcgaattc ctgcagcccg 6840 ggggatccgc atgcttgcat ttagtcgtgc aatgtatgac tttaagattt gtgagcagga 6900 agaaaaggga gaatcttcta acgataaacc cttgaaaaac tgggtagact acgctatgtt 6960 gagttgctac gcaggctgca caattacacg agaatgctcc cgcctaggat ttaaggctaa 7020 gggacgtgca atgcagacga cagatctaaa tgaccgtgtc ggtgaagtgt tcgccaaact 7080 tttcggttaa cacatgcagt gatgcacgcg cgatggtgct aagttacata tatatatata 7140 tatatatata tagccatagt gatgtctaag taacctttat ggtatatttc ttaatgtgga 7200 aagatactag cgcgcgcacc cacacacaag cttcgtcttt tcttgaagaa aagaggaagc 7260 tcgctaaatg ggattccact ttccgttccc tgccagctga tggaaaaagg ttagtggaac 7320 gatgaagaat aaaaagagag atccactgag gtgaaatttc agctgacagc gagtttcatg 7380 atcgtgatga acaatggtaa cgagttgtgg ctgttgccag ggagggtggt tctcaacttt 7440 taatgtatgg ccaaatcgct acttgggttt gttatataac aaagaagaaa taatgaactg 7500 attctcttcc tccttcttgt cctttcttaa ttctgttgta attaccttcc tttgtaattt 7560 tttttgtaat tattcttctt aataatccaa acaaacacac atattacaat agctagctga 7620 ggatgaaggc attagtttat catggggatc acaaaatttc gttagaagac aaaccaaaac 7680 ccactctgca gaaaccaaca gacgttgtgg ttagggtgtt gaaaacaaca atttgcggta 7740 ctgacttggg aatatacaaa ggtaagaatc ctgaagtggc agatggcaga atcctgggtc 7800 atgagggcgt tggcgtcatt gaagaagtgg gcgaatccgt gacacaattc aaaaaggggg 7860 ataaagtttt aatctcctgc gttactagct gtggatcgtg tgattattgc aagaagcaac 7920 tgtattcaca ctgtagagac ggtggctgga ttttaggtta catgatcgac ggtgtccaag 7980 ccgaatacgt cagaatacca catgctgaca attcattgta taagatcccg caaactatcg 8040 atgatgaaat tgcagtacta ctgtccgata ttttacctac tggacatgaa attggtgttc 8100 aatatggtaa cgttcaacca ggcgatgctg tagcaattgt aggagcaggt cctgttggaa 8160 tgtcagtttt gttaactgct caattttact cgcctagtac cattattgtt atcgacatgg 8220 acgaaaaccg tttacaatta gcgaaggagc ttggggccac acacactatt aactccggta 8280 ctgaaaatgt tgtcgaagct gtgcatcgta tagcagccga aggagtggat gtagcaatag 8340 aagctgttgg tatacccgca acctgggaca tctgtcagga aattgtaaaa cccggcgctc 8400 atattgccaa cgtgggagtt catggtgtta aggtggactt tgaaattcaa aagttgtgga 8460 ttaagaatct aaccatcacc actggtttgg ttaacactaa tactacccca atgttgatga 8520 aggtagcctc tactgataaa ttgcctttaa agaaaatgat tactcacagg tttgagttag 8580 ctgaaatcga acacgcatat caggttttct tgaatggcgc taaagaaaaa gctatgaaga 8640 ttattctatc taatgcaggt gccgcctaat taattaagag taagcgaatt tcttatgatt 8700 tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga 8760 ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt 8820 gctttctcag gtatagcatg aggtcgctct tattgaccac acctctaccg gcatgccgag 8880 caaatgcctg caaatcgctc cccatttcac ccaattgtag atatgctaac tccagcaatg 8940 agttgatgaa tctcggtgtg tattttatgt cctcagagga caacacctgt ggta 8994 <210> 79 <211> 2145 <212> DNA Artificial sequence <220> <223> constructed chimeric gene <400> 79 gcatgcttgc atttagtcgt gcaatgtatg actttaagat ttgtgagcag gaagaaaagg 60 gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg ttgagttgct 120 acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct aagggacgtg 180 caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa cttttcggtt 240 aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata tatatatata 300 tatagccata gtgatgtcta agtaaccttt atggtatatt tcttaatgtg gaaagatact 360 agcgcgcgca cccacacaca agcttcgtct tttcttgaag aaaagaggaa gctcgctaaa 420 tgggattcca ctttccgttc cctgccagct gatggaaaaa ggttagtgga acgatgaaga 480 ataaaaagag agatccactg aggtgaaatt tcagctgaca gcgagtttca tgatcgtgat 540 gaacaatggt aacgagttgt ggctgttgcc agggagggtg gttctcaact tttaatgtat 600 ggccaaatcg ctacttgggt ttgttatata acaaagaaga aataatgaac tgattctctt 660 cctccttctt gtcctttctt aattctgttg taattacctt cctttgtaat tttttttgta 720 attattcttc ttaataatcc aaacaaacac acatattaca atagctagct gaggatgaag 780 gcattagttt atcatgggga tcacaaaatt tcgttagaag acaaaccaaa acccactctg 840 cagaaaccaa cagacgttgt ggttagggtg ttgaaaacaa caatttgcgg tactgacttg 900 ggaatataca aaggtaagaa tcctgaagtg gcagatggca gaatcctggg tcatgagggc 960 gttggcgtca ttgaagaagt gggcgaatcc gtgacacaat tcaaaaaggg ggataaagtt 1020 ttaatctcct gcgttactag ctgtggatcg tgtgattatt gcaagaagca actgtattca 1080 cactgtagag acggtggctg gattttaggt tacatgatcg acggtgtcca agccgaatac 1140 gtcagaatac cacatgctga caattcattg tataagatcc cgcaaactat cgatgatgaa 1200 attgcagtac tactgtccga tattttacct actggacatg aaattggtgt tcaatatggt 1260 aacgttcaac caggcgatgc tgtagcaatt gtaggagcag gtcctgttgg aatgtcagtt 1320 ttgttaactg ctcaatttta ctcgcctagt accattattg ttatcgacat ggacgaaaac 1380 cgtttacaat tagcgaagga gcttggggcc acacacacta ttaactccgg tactgaaaat 1440 gttgtcgaag ctgtgcatcg tatagcagcc gaaggagtgg atgtagcaat agaagctgtt 1500 ggtatacccg caacctggga catctgtcag gaaattgtaa aacccggcgc tcatattgcc 1560 aacgtgggag ttcatggtgt taaggtggac tttgaaattc aaaagttgtg gattaagaat 1620 ctaaccatca ccactggttt ggttaacact aatactaccc caatgttgat gaaggtagcc 1680 tctactgata aattgccttt aaagaaaatg attactcaca ggtttgagtt agctgaaatc 1740 gaacacgcat atcaggtttt cttgaatggc gctaaagaaa aagctatgaa gattattcta 1800 tctaatgcag gtgccgccta attaattaag agtaagcgaa tttcttatga tttatgattt 1860 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg 1920 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc 1980 aggtatagca tgaggtcgct cttattgacc acacctctac cggcatgccg agcaaatgcc 2040 tgcaaatcgc tccccatttc acccaattgt agatatgcta actccagcaa tgagttgatg 2100 aatctcggtg tgtattttat gtcctcagag gacaacacct gtggt 2145 <210> 80 <211> 4280 <212> DNA Artificial sequence <220> <223> vector <400> 80 ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg 60 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 120 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 180 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 240 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 300 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 360 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 420 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 480 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 540 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 600 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 660 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 720 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 780 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 840 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 900 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 960 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 1020 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 1080 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 1140 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 1200 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 1260 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 1320 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1380 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1440 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1500 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1560 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1620 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1680 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1740 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1800 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1860 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1920 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1980 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 2040 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 2100 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 2160 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 2220 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt 2280 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt 2340 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 2400 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg 2460 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca 2520 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag 2580 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag 2640 tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg gtacccccgg ctctgagaca 2700 gtagtaggtt agtcatcgct ctaccgacgc gcaggaaaag aaagaagcat tgcggattac 2760 gtattctaat gttcagcccg cggaacgcca gcaaatcacc acccatgcgc atgatactga 2820 gtcttgtaca cgctgggctt ccagtgtact gagagtgcac cataccacag cttttcaatt 2880 caattcatca tttttttttt attctttttt ttgatttcgg tttctttgaa atttttttga 2940 ttcggtaatc tccgaacaga aggaagaacg aaggaaggag cacagactta gattggtata 3000 tatacgcata tgtagtgttg aagaaacatg aaattgccca gtattcttaa cccaactgca 3060 cagaacaaaa acctgcagga aacgaagata aatcatgtcg aaagctacat ataaggaacg 3120 tgctgctact catcctagtc ctgttgctgc caagctattt aatatcatgc acgaaaagca 3180 aacaaacttg tgtgcttcat tggatgttcg taccaccaag gaattactgg agttagttga 3240 agcattaggt cccaaaattt gtttactaaa aacacatgtg gatatcttga ctgatttttc 3300 catggagggc acagttaagc cgctaaaggc attatccgcc aagtacaatt ttttactctt 3360 cgaagacaga aaatttgctg acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt 3420 atacagaata gcagaatggg cagacattac gaatgcacac ggtgtggtgg gcccaggtat 3480 tgttagcggt ttgaagcagg cggcagaaga agtaacaaag gaacctagag gccttttgat 3540 gttagcagaa ttgtcatgca agggctccct atctactgga gaatatacta agggtactgt 3600 tgacattgcg aagagcgaca aagattttgt tatcggcttt attgctcaaa gagacatggg 3660 tggaagagat gaaggttacg attggttgat tatgacaccc ggtgtgggtt tagatgacaa 3720 gggagacgca ttgggtcaac agtatagaac cgtggatgat gtggtctcta caggatctga 3780 cattattatt gttggaagag gactatttgc aaagggaagg gatgctaagg tagagggtga 3840 acgttacaga aaagcaggct gggaagcata tttgagaaga tgcggccagc aaaactaaaa 3900 aactgtatta taagtaaatg catgtatact aaactcacaa attagagctt caatttaatt 3960 atatcagtta ttaccctatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 4020 gcatcaggaa attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat 4080 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ttcagcccgc ggaacgccag 4140 caaatcacca cccatgcgca tgatactgag tcttgtacac gctgggcttc cagtgatgat 4200 acaacgagtt agccaaggtg agcacggatg tctaaattag aattacgttt taatatcttt 4260 ttttccatat ctagggctag 4280 <210> 81 <211> 30 <212> DNA Artificial sequence <220> <223> primer <400> 81 gcatgcttgc atttagtcgt gcaatgtatg 30 <210> 82 <211> 54 <212> DNA Artificial sequence <220> <223> primer <400> 82 gaacattaga atacgtaatc cgcaatgcac tagtaccaca ggtgttgtcc tctg 54 <210> 83 <211> 54 <212> DNA Artificial sequence <220> <223> primer <400> 83 cagaggacaa cacctgtggt actagtgcat tgcggattac gtattctaat gttc 54 <210> 84 <211> 28 <212> DNA Artificial sequence <220> <223> primer <400> 84 caccttggct aactcgttgt atcatcac 28 <210> 85 <211> 100 <212> DNA Artificial sequence <220> <223> primer <400> 85 ttttaagccg aatgagtgac agaaaaagcc cacaacttat caagtgatat tgaacaaagg 60 gcgaaacttc gcatgcttgc atttagtcgt gcaatgtatg 100 <210> 86 <211> 98 <212> DNA Artificial sequence <220> <223> primer <400> 86 cccaattggt aaatattcaa caagagacgc gcagtacgta acatgcgaat tgcgtaattc 60 acggcgataa caccttggct aactcgttgt atcatcac 98 <210> 87 <211> 29 <212> DNA Artificial sequence <220> <223> primer <400> 87 caaaagccca tgtcccacac caaaggatg 29 <210> 88 <211> 26 <212> DNA Artificial sequence <220> <223> primer <400> 88 caccatcgcg cgtgcatcac tgcatg 26 <210> 89 <211> 28 <212> DNA Artificial sequence <220> <223> primer <400> 89 tcggtttttg caatatgacc tgtgggcc 28 <210> 90 <211> 22 <212> DNA Artificial sequence <220> <223> primer <400> 90 gagaagatgc ggccagcaaa ac 22 <210> 91 <211> 2745 <212> DNA Artificial sequence <220> <223> constructed coding region-terminator segment <400> 91 atgactgaca aaaaaactct taaagactta agaaatcgta gttctgttta cgattcaatg 60 gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta tgcaagatga agactttgaa 120 aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca caccttgtaa tatccactta 180 catgactttg gtaaactagc caaagtcggt gttaaggaag ctggtgcttg gccagttcag 240 ttcggaacaa tcacggtttc tgatggaatc gccatgggaa cccaaggaat gcgtttctcc 300 ttgacatctc gtgatattat tgcagattct attgaagcag ccatgggagg tcataatgcg 360 gatgcttttg tagccattgg cggttgtgat aaaaacatgc ccggttctgt tatcgctatg 420 gctaacatgg atatcccagc catttttgct tacggcggaa caattgcacc tggtaattta 480 gacggcaaag atatcgattt agtctctgtc tttgaaggtg tcggccattg gaaccacggc 540 gatatgacca aagaagaagt taaagctttg gaatgtaatg cttgtcccgg tcctggaggc 600 tgcggtggta tgtatactgc taacacaatg gcgacagcta ttgaagtttt gggacttagc 660 cttccgggtt catcttctca cccggctgaa tccgcagaaa agaaagcaga tattgaagaa 720 gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa aaccttctga cattttaacg 780 cgtgaagctt ttgaagatgc tattactgta actatggctc tgggaggttc aaccaactca 840 acccttcacc tcttagctat tgcccatgct gctaatgtgg aattgacact tgatgatttc 900 aatactttcc aagaaaaagt tcctcatttg gctgatttga aaccttctgg tcaatatgta 960 ttccaagacc tttacaaggt cggaggggta ccagcagtta tgaaatatct ccttaaaaat 1020 ggcttccttc atggtgaccg tatcacttgt actggcaaaa cagtcgctga aaatttgaag 1080 gcttttgatg atttaacacc tggtcaaaag gttattatgc cgcttgaaaa tcctaaacgt 1140 gaagatggtc cgctcattat tctccatggt aacttggctc cagacggtgc cgttgccaaa 1200 gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta aggtctttaa ttctgaagaa 1260 gaagccattg aagctgtctt gaatgatgat attgttgatg gtgatgttgt tgtcgtacgt 1320 tttgtaggac caaagggcgg tcctggtatg cctgaaatgc tttccctttc atcaatgatt 1380 gttggtaaag ggcaaggtga aaaagttgcc cttctgacag atggccgctt ctcaggtggt 1440 acttatggtc ttgtcgtggg tcatatcgct cctgaagcac aagatggcgg tccaatcgcc 1500 tacctgcaaa caggagacat agtcactatt gaccaagaca ctaaggaatt acactttgat 1560 atctccgatg aagagttaaa acatcgtcaa gagaccattg aattgccacc gctctattca 1620 cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg cttctagggg agccgtaaca 1680 gacttttgga agcctgaaga aactggcaaa aaatgttgtc ctggttgctg tggttaagcg 1740 gccgcgttaa ttcaaattaa ttgatatagt tttttaatga gtattgaatc tgtttagaaa 1800 taatggaata ttatttttat ttatttattt atattattgg tcggctcttt tcttctgaag 1860 gtcaatgaca aaatgatatg aaggaaataa tgatttctaa aattttacaa cgtaagatat 1920 ttttacaaaa gcctagctca tcttttgtca tgcactattt tactcacgct tgaaattaac 1980 ggccagtcca ctgcggagtc atttcaaagt catcctaatc gatctatcgt ttttgatagc 2040 tcattttgga gttcgcgatt gtcttctgtt attcacaact gttttaattt ttatttcatt 2100 ctggaactct tcgagttctt tgtaaagtct ttcatagtag cttactttat cctccaacat 2160 atttaacttc atgtcaattt cggctcttaa attttccaca tcatcaagtt caacatcatc 2220 ttttaacttg aatttattct ctagctcttc caaccaagcc tcattgctcc ttgatttact 2280 ggtgaaaagt gatacacttt gcgcgcaatc caggtcaaaa ctttcctgca aagaattcac 2340 caatttctcg acatcatagt acaatttgtt ttgttctccc atcacaattt aatatacctg 2400 atggattctt atgaagcgct gggtaatgga cgtgtcactc tacttcgcct ttttccctac 2460 tccttttagt acggaagaca atgctaataa ataagagggt aataataata ttattaatcg 2520 gcaaaaaaga ttaaacgcca agcgtttaat tatcagaaag caaacgtcgt accaatcctt 2580 gaatgcttcc caattgtata ttaagagtca tcacagcaac atattcttgt tattaaatta 2640 attattattg atttttgata ttgtataaaa aaaccaaata tgtataaaaa aagtgaataa 2700 gt; <210> 92 <211> 27 <212> DNA Artificial sequence <220> <223> primer <400> 92 gacttttgga agcctgaaga aactggc 27 <210> 93 <211> 20 <212> DNA Artificial sequence <220> <223> primer <400> 93 cttggcagca acaggactag 20 <210> 94 <211> 26 <212> DNA Artificial sequence <220> <223> primer <400> 94 ccaggccaat tcaacagact gtcggc 26 <210> 95 <211> 2347 <212> DNA Artificial sequence <220> <223> constructed URA3 marker with flanking homologous repeat sequences        for HIS gene replacement and marker excision <400> 95 gcattgcgga ttacgtattc taatgttcag gtgctggaag aagagctgct taaccgccgc 60 gcccagggtg aagatccacg ctactttacc ctgcgtcgtc tggatttcgg cggctgtcgt 120 ctttcgctgg caacgccggt tgatgaagcc tgggacggtc cgctctcctt aaacggtaaa 180 cgtatcgcca cctcttatcc tcacctgctc aagcgttatc tcgaccagaa aggcatctct 240 tttaaatcct gcttactgaa cggttctgtt gaagtcgccc cgcgtgccgg actggcggat 300 gcgatttgcg atctggtttc caccggtgcc acgctggaag ctaacggcct gcgcgaagtc 360 gaagttatct atcgctcgaa agcctgcctg attcaacgcg atggcgaaat ggaagaatcc 420 aaacagcaac tgatcgacaa actgctgacc cgtattcagg gtgtgatcca ggcgcgcgaa 480 tcaaaataca tcatgatgca cgcaccgacc gaacgtctgg atgaagtcat ggtacctact 540 gagagtgcac cataccacag cttttcaatt caattcatca tttttttttt attctttttt 600 ttgatttcgg tttctttgaa atttttttga ttcggtaatc tccgaacaga aggaagaacg 660 aaggaaggag cacagactta gattggtata tatacgcata tgtagtgttg aagaaacatg 720 aaattgccca gtattcttaa cccaactgca cagaacaaaa acctgcagga aacgaagata 780 aatcatgtcg aaagctacat ataaggaacg tgctgctact catcctagtc ctgttgctgc 840 caagctattt aatatcatgc acgaaaagca aacaaacttg tgtgcttcat tggatgttcg 900 taccaccaag gaattactgg agttagttga agcattaggt cccaaaattt gtttactaaa 960 aacacatgtg gatatcttga ctgatttttc catggagggc acagttaagc cgctaaaggc 1020 attatccgcc aagtacaatt ttttactctt cgaagacaga aaatttgctg acattggtaa 1080 tacagtcaaa ttgcagtact ctgcgggtgt atacagaata gcagaatggg cagacattac 1140 gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt ttgaagcagg cggcagaaga 1200 agtaacaaag gaacctagag gccttttgat gttagcagaa ttgtcatgca agggctccct 1260 atctactgga gaatatacta agggtactgt tgacattgcg aagagcgaca aagattttgt 1320 tatcggcttt attgctcaaa gagacatggg tggaagagat gaaggttacg attggttgat 1380 tatgacaccc ggtgtgggtt tagatgacaa gggagacgca ttgggtcaac agtatagaac 1440 cgtggatgat gtggtctcta caggatctga cattattatt gttggaagag gactatttgc 1500 aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct gggaagcata 1560 tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg catgtatact 1620 aaactcacaa attagagctt caatttaatt atatcagtta ttaccctatg cggtgtgaaa 1680 taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaaacg ttaatatttt 1740 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat 1800 cggcaaaatc tctagagtgc tggaagaaga gctgcttaac cgccgcgccc agggtgaaga 1860 tccacgctac tttaccctgc gtcgtctgga tttcggcggc tgtcgtcttt cgctggcaac 1920 gccggttgat gaagcctggg acggtccgct ctccttaaac ggtaaacgta tcgccacctc 1980 ttatcctcac ctgctcaagc gttatctcga ccagaaaggc atctctttta aatcctgctt 2040 actgaacggt tctgttgaag tcgccccgcg tgccggactg gcggatgcga tttgcgatct 2100 ggtttccacc ggtgccacgc tggaagctaa cggcctgcgc gaagtcgaag ttatctatcg 2160 ctcgaaagcc tgcctgattc aacgcgatgg cgaaatggaa gaatccaaac agcaactgat 2220 cgacaaactg ctgacccgta ttcagggtgt gatccaggcg cgcgaatcaa aatacatcat 2280 gatgcacgca ccgaccgaac gtctggatga agtcatccag tgatgataca acgagttagc 2340 caaggtg 2347 <210> 96 <211> 80 <212> DNA Artificial sequence <220> <223> primer <400> 96 cttcgaagaa tatactaaaa aatgagcagg caagataaac gaaggcaaag gcattgcgga 60 ttacgtattc taatgttcag 80 <210> 97 <211> 80 <212> DNA Artificial sequence <220> <223> primer <400> 97 cttcgaagaa tatactaaaa aatgagcagg caagataaac gaaggcaaag gcattgcgga 60 ttacgtattc taatgttcag 80 <210> 98 <211> 26 <212> DNA Artificial sequence <220> <223> primer <400> 98 gacttgaata atgcagcggc gcttgc 26 <210> 99 <211> 30 <212> DNA Artificial sequence <220> <223> primer <400> 99 ccaccctctt caattagcta agatcatagc 30 <210> 100 <211> 25 <212> DNA Artificial sequence <220> <223> primer <400> 100 aaaaattgat tctcatcgta aatgc 25 <210> 101 <211> 20 <212> DNA Artificial sequence <220> <223> primer <400> 101 ctgcagcgag gagccgtaat 20 <210> 102 <211> 90 <212> DNA Artificial sequence <220> <223> primer <400> 102 atggttcatt taggtccaaa aaaaccacaa gccagaaagg gttccatggc cgatgtgcca 60 gcattgcgga ttacgtattc taatgttcag 90 <210> 103 <211> 91 <212> DNA Artificial sequence <220> <223> primer <400> 103 ttaagcaccg atgataccaa cggacttacc ttcagcaatt cttttttggg ccaaagcagc 60 caccttggct aactcgttgt atcatcactg g 91 <210> 104 <211> 24 <212> DNA Artificial sequence <220> <223> primer <400> 104 ctaggatgag tagcagcacg ttcc 24 <210> 105 <211> 26 <212> DNA Artificial sequence <220> <223> primer <400> 105 ccaattccgt gatgtctctt tgttgc 26 <210> 106 <211> 20 <212> DNA Artificial sequence <220> <223> primer <400> 106 gtgaacgagt tcacaaccgc 20 <210> 107 <211> 22 <212> DNA Artificial sequence <220> <223> primer <400> 107 gttcgttcca gaattatcac gc 22 <210> 108 <211> 28 <212> DNA Artificial sequence <220> <223> primer <400> 108 ggatccgcat gcttgcattt agtcgtgc 28 <210> 109 <211> 20 <212> DNA Artificial sequence <220> <223> primer <400> 109 gggatgcgga cgtattcggc 20 <210> 110 <211> 99 <212> DNA Artificial sequence <220> <223> primer <400> 110 tcctttctca attattattt tctactcata acctcacgca aaataacaca gtcaaatcaa 60 tcaaagtatg actgacaaaa aaactcttaa agacttaag 99 <210> 111 <211> 77 <212> DNA Artificial sequence <220> <223> primer <400> 111 gaacattaga atacgtaatc cgcaatgctt ctttcttttc cgtttaacgt atagacttct 60 aatatatttc tccatac 77 <210> 112 <211> 45 <212> DNA Artificial sequence <220> <223> primer <400> 112 aaacggaaaa gaaagaagca ttgcggatta cgtattctaa tgttc 45 <210> 113 <211> 88 <212> DNA Artificial sequence <220> <223> primer <400> 113 tatttttcgt tacataaaaa tgcttataaa actttaacta ataattagag attaaatcgc 60 caccttggct aactcgttgt atcatcac 88 <210> 114 <211> 1713 <212> DNA <213> Streptococcus mutans <400> 114 atgactgaca aaaaaactct taaagactta agaaatcgta gttctgttta cgattcaatg 60 gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta tgcaagatga agactttgaa 120 aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca caccttgtaa tatccactta 180 catgactttg gtaaactagc caaagtcggt gttaaggaag ctggtgcttg gccagttcag 240 ttcggaacaa tcacggtttc tgatggaatc gccatgggaa cccaaggaat gcgtttctcc 300 ttgacatctc gtgatattat tgcagattct attgaagcag ccatgggagg tcataatgcg 360 gatgcttttg tagccattgg cggttgtgat aaaaacatgc ccggttctgt tatcgctatg 420 gctaacatgg atatcccagc catttttgct tacggcggaa caattgcacc tggtaattta 480 gacggcaaag atatcgattt agtctctgtc tttgaaggtg tcggccattg gaaccacggc 540 gatatgacca aagaagaagt taaagctttg gaatgtaatg cttgtcccgg tcctggaggc 600 tgcggtggta tgtatactgc taacacaatg gcgacagcta ttgaagtttt gggacttagc 660 cttccgggtt catcttctca cccggctgaa tccgcagaaa agaaagcaga tattgaagaa 720 gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa aaccttctga cattttaacg 780 cgtgaagctt ttgaagatgc tattactgta actatggctc tgggaggttc aaccaactca 840 acccttcacc tcttagctat tgcccatgct gctaatgtgg aattgacact tgatgatttc 900 aatactttcc aagaaaaagt tcctcatttg gctgatttga aaccttctgg tcaatatgta 960 ttccaagacc tttacaaggt cggaggggta ccagcagtta tgaaatatct ccttaaaaat 1020 ggcttccttc atggtgaccg tatcacttgt actggcaaaa cagtcgctga aaatttgaag 1080 gcttttgatg atttaacacc tggtcaaaag gttattatgc cgcttgaaaa tcctaaacgt 1140 gaagatggtc cgctcattat tctccatggt aacttggctc cagacggtgc cgttgccaaa 1200 gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta aggtctttaa ttctgaagaa 1260 gaagccattg aagctgtctt gaatgatgat attgttgatg gtgatgttgt tgtcgtacgt 1320 tttgtaggac caaagggcgg tcctggtatg cctgaaatgc tttccctttc atcaatgatt 1380 gttggtaaag ggcaaggtga aaaagttgcc cttctgacag atggccgctt ctcaggtggt 1440 acttatggtc ttgtcgtggg tcatatcgct cctgaagcac aagatggcgg tccaatcgcc 1500 tacctgcaaa caggagacat agtcactatt gaccaagaca ctaaggaatt acactttgat 1560 atctccgatg aagagttaaa acatcgtcaa gagaccattg aattgccacc gctctattca 1620 cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg cttctagggg agccgtaaca 1680 gacttttgga agcctgaaga aactggcaaa aaa 1713 <210> 115 <211> 571 <212> PRT <213> Streptococcus mutans <400> 115 Met Thr Asp Lys Lys Thr Leu Lys Asp Leu Arg Asn Arg Ser Ser Val 1 5 10 15 Tyr Asp Ser Met Val Lys Ser Pro Asn Arg Ala Met Leu Arg Ala Thr             20 25 30 Gly Met Gln Asp Glu Asp Phe Glu Lys Pro Ile Val Gly Val Ile Ser         35 40 45 Thr Trp Ala Glu Asn Thr Pro Cys Asn Ile His Leu His Asp Phe Gly     50 55 60 Lys Leu Ala Lys Val Gly Val Lys Glu Ala Gly Ala Trp Pro Val Gln 65 70 75 80 Phe Gly Thr Ile Thr Val Ser Asp Gly Ile Ala Met Gly Thr Gln Gly                 85 90 95 Met Arg Phe Ser Leu Thr Ser Arg Asp Ile Ile Ala Asp Ser Ile Glu             100 105 110 Ala Ala Met Gly Gly His Asn Ala Asp Ala Phe Val Ala Ile Gly Gly         115 120 125 Cys Asp Lys Asn Met Pro Gly Ser Val Ile Ala Met Ala Asn Met Asp     130 135 140 Ile Pro Ala Ile Phe Ala Tyr Gly Gly Thr Ile Ala Pro Gly Asn Leu 145 150 155 160 Asp Gly Lys Asp Ile Asp Leu Val Ser Val Phe Glu Gly Val Gly His                 165 170 175 Trp Asn His Gly Asp Met Thr Lys Glu Glu Val Lys Ala Leu Glu Cys             180 185 190 Asn Ala Cys Pro Gly Pro Gly Gly Cys Gly Gly Met Tyr Thr Ala Asn         195 200 205 Thr Met Ala Thr Ala Ile Glu Val Leu Gly Leu Ser Leu Pro Gly Ser     210 215 220 Ser Ser His Pro Ala Glu Ser Ala Glu Lys Lys Ala Asp Ile Glu Glu 225 230 235 240 Ala Gly Arg Ala Val Val Lys Met Leu Glu Met Gly Leu Lys Pro Ser                 245 250 255 Asp Ile Leu Thr Arg Glu Ala Phe Glu Asp Ala Ile Thr Val Thr Met             260 265 270 Ala Leu Gly Gly Ser Thr Asn Ser Thr Leu His Leu Leu Ala Ile Ala         275 280 285 His Ala Ala Asn Val Glu Leu Thr Leu Asp Asp Phe Asn Thr Phe Gln     290 295 300 Glu Lys Val Pro His Leu Ala Asp Leu Lys Pro Ser Gly Gln Tyr Val 305 310 315 320 Phe Gln Asp Leu Tyr Lys Val Gly Gly Val Pro Ala Val Met Lys Tyr                 325 330 335 Leu Leu Lys Asn Gly Phe Leu His Gly Asp Arg Ile Thr Cys Thr Gly             340 345 350 Lys Thr Val Ala Glu Asn Leu Lys Ala Phe Asp Asp Leu Thr Pro Gly         355 360 365 Gln Lys Val Ile Met Pro Leu Glu Asn Pro Lys Arg Glu Asp Gly Pro     370 375 380 Leu Ile Ile Leu His Gly Asn Leu Ala Pro Asp Gly Ala Val Ala Lys 385 390 395 400 Val Ser Gly Val Lys Val Arg Arg His Val Gly Pro Ala Lys Val Phe                 405 410 415 Asn Ser Glu Glu Glu Ala Ile Glu Ala Val Leu Asn Asp Asp Ile Val             420 425 430 Asp Gly Asp Val Val Val Val Arg Phe Val Gly Pro Lys Gly Gly Pro         435 440 445 Gly Met Pro Glu Met Leu Ser Leu Ser Ser Met Ile Val Gly Lys Gly     450 455 460 Gln Gly Glu Lys Val Ala Leu Leu Thr Asp Gly Arg Phe Ser Gly Gly 465 470 475 480 Thr Tyr Gly Leu Val Val Gly His Ile Ala Pro Glu Ala Gln Asp Gly                 485 490 495 Gly Pro Ile Ala Tyr Leu Gln Thr Gly Asp Ile Val Thr Ile Asp Gln             500 505 510 Asp Thr Lys Glu Leu His Phe Asp Ile Ser Asp Glu Glu Leu Lys His         515 520 525 Arg Gln Glu Thr Ile Glu Leu Pro Pro Leu Tyr Ser Arg Gly Ile Leu     530 535 540 Gly Lys Tyr Ala His Ile Val Ser Ser Ala Ser Arg Gly Ala Val Thr 545 550 555 560 Asp Phe Trp Lys Pro Glu Glu Thr Gly Lys Lys                 565 570 <210> 116 <211> 548 <212> PRT <213> Lactococcus lactis <400> 116 Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu             20 25 30 Asp Gln Ile Ile Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn         35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys     50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile                 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His             100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu         115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val     130 135 140 Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro                 165 170 175 Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln             180 185 190 Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro         195 200 205 Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr     210 215 220 Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile                 245 250 255 Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser             260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr         275 280 285 Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn     290 295 300 Ile Asp Glu Gly Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe 305 310 315 320 Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys                 325 330 335 Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala             340 345 350 Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln         355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala     370 375 380 Ser Ser Ile Phe Leu Lys Ser Lys Ser His Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile                 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu             420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn         435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu     450 455 460 Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser                 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala             500 505 510 Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys         515 520 525 Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu     530 535 540 Gln asn lys ser 545 <210> 117 <211> 1125 <212> DNA Artificial sequence <220> <223> horse ADH coding region codon optimized for S. cerevisiae        expression <400> 117 atgtcaacag ccggtaaagt tattaagtgt aaagcggcag ttttgtggga agagaaaaag 60 ccgtttagca tagaagaagt agaagtagcg ccaccaaaag cacacgaggt tagaatcaag 120 atggttgcca ccggaatctg tagatccgac gaccatgtgg tgagtggcac tctagttact 180 cctttgccag taatcgcggg acacgaggct gccggaatcg ttgaatccat aggtgaaggt 240 gttaccactg ttcgtcctgg tgataaagtg atcccactgt tcactcctca atgtggtaag 300 tgtagagtct gcaaacatcc tgagggtaat ttctgcctta aaaatgattt gtctatgcct 360 agaggtacta tgcaggatgg tacaagcaga tttacatgca gagggaaacc tatacaccat 420 ttccttggta cttctacatt ttcccaatac acagtggtgg acgagatatc tgtcgctaaa 480 atcgatgcag cttcaccact ggaaaaagtt tgcttgatag ggtgcggatt ttccaccggt 540 tacggttccg cagttaaagt tgcaaaggtt acacagggtt cgacttgtgc agtattcggt 600 ttaggaggag taggactaag cgttattatg gggtgtaaag ctgcaggcgc agcgaggatt 660 ataggtgtag acatcaataa ggacaaattt gcaaaagcta aggaggtcgg ggctactgaa 720 tgtgttaacc ctcaagatta taagaaacca atacaagaag tccttactga aatgtcaaac 780 ggtggagttg atttctcttt tgaagttata ggccgtcttg atactatggt aactgcgttg 840 tcctgctgtc aagaggcata tggagtcagt gtgatcgtag gtgttcctcc tgattcacaa 900 aatttgtcga tgaatcctat gctgttgcta agcggtcgta catggaaggg agctatattt 960 ggcggtttta agagcaagga tagtgttcca aaacttgttg ccgactttat ggcgaagaag 1020 tttgctcttg atcctttaat tacacatgta ttgccattcg agaaaatcaa tgaagggttt 1080 gatttgttaa gaagtggtga atctattcgt acaattttaa ctttt 1125 <210> 118 <211> 375 <212> PRT <213> Equus caballus <400> 118 Met Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15 Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro             20 25 30 Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg         35 40 45 Ser Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val     50 55 60 Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly 65 70 75 80 Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro                 85 90 95 Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys             100 105 110 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr         115 120 125 Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr     130 135 140 Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys 145 150 155 160 Ile Asp Ala Ser Ser Leu Glu Lys Val Cys Leu Ile Gly Cys Gly                 165 170 175 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln             180 185 190 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val         195 200 205 Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp     210 215 220 Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235 240 Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr                 245 250 255 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg             260 265 270 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly         275 280 285 Val Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met     290 295 300 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe 305 310 315 320 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe                 325 330 335 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro             340 345 350 Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser         355 360 365 Ile Arg Thr Ile Leu Thr Phe     370 375 <210> 119 <211> 643 <212> DNA <213> Saccharomyces cerevisiae <400> 119 gaaatgaata acaatactga cagtactaaa taattgccta cttggcttca catacgttgc 60 atacgtcgat atagataata atgataatga cagcaggatt atcgtaatac gtaatagttg 120 aaaatctcaa aaatgtgtgg gtcattacgt aaataatgat aggaatggga ttcttctatt 180 tttccttttt ccattctagc agccgtcggg aaaacgtggc atcctctctt tcgggctcaa 240 ttggagtcac gctgccgtga gcatcctctc tttccatatc taacaactga gcacgtaacc 300 aatggaaaag catgagctta gcgttgctcc aaaaaagtat tggatggtta ataccatttg 360 tctgttctct tctgactttg actcctcaaa aaaaaaaaat ctacaatcaa cagatcgctt 420 caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa ttttatccct 480 catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga cataatactt 540 ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc tttttctttt 600 gtcatatata accataacca agtaatacat attcaaatct aga 643 <210> 120 <211> 9089 <212> DNA Artificial sequence <220> &Lt; 223 > constructed plasmid <400> 120 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 1860 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 1920 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat 1980 acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac tggccattaa 2040 tctttcccat attagatttc gccaagccat gaaagttcaa gaaaggtctt tagacgaatt 2100 acccttcatt tctcaaactg gcgtcaaggg atcctggtat ggttttatcg ttttatttct 2160 ggttcttata gcatcgtttt ggacttctct gttcccatta ggcggttcag gagccagcgc 2220 agaatcattc tttgaaggat acttatcctt tccaattttg attgtctgtt acgttggaca 2280 taaactgtat actagaaatt ggactttgat ggtgaaacta gaagatatgg atcttgatac 2340 cggcagaaaa caagtagatt tgactcttcg tagggaagaa atgaggattg agcgagaaac 2400 attagcaaaa agatccttcg taacaagatt tttacatttc tggtgttgaa gggaaagata 2460 tgagctatac agcggaattt ccatatcact cagattttgt tatctaattt tttccttccc 2520 acgtccgcgg gaatctgtgt atattactgc atctagatat atgttatctt atcttggcgc 2580 gtacatttaa ttttcaacgt attctataag aaattgcggg agtttttttc atgtagatga 2640 tactgactgc acgcaaatat aggcatgatt tataggcatg atttgatggc tgtaccgata 2700 ggaacgctaa gagtaacttc agaatcgtta tcctggcgga aaaaattcat ttgtaaactt 2760 taaaaaaaaa agccaatatc cccaaaatta ttaagagcgc ctccattatt aactaaaatt 2820 tcactcagca tccacaatgt atcaggtatc tactacagat attacatgtg gcgaaaaaga 2880 caagaacaat gcaatagcgc atcaagaaaa aacacaaagc tttcaatcaa tgaatcgaaa 2940 atgtcattaa aatagtatat aaattgaaac taagtcataa agctataaaa agaaaattta 3000 tttaaatgca agatttaaag taaattcacg gccctgcagg ccctaacctg ctaggacaca 3060 acgtctttgc ctggtaaagt ttctagctga cgtgattcct tcacctgtgg atccggcaat 3120 tgtaaaggtt gtgaaaccct cagcttcata accgacacct gcaaatgact ttgcattctt 3180 aacaaagata gttgtatcaa tttcacgttc gaatctatta aggttatcga tgttcttaga 3240 ataaatgtag gcggaatgtt ttctattctg ctcagctatc ttggcgtatt taatggcttc 3300 atcaatgtcc ttcactctaa ctataggcaa aattggcatc atcaactccg tcataacgaa 3360 cggatggttt gcgttgactt cacaaataat acactttaca ttacttggtg actctacatc 3420 tatttcatcc aaaaacagtt tagcgtcctt accaacccac ttcttattaa tgaaatattc 3480 ttgagtttca ttgttctttt gaagaacaag gtctatcagc ttggatactt ggtcttcatt 3540 gataatgacg gcgttgtttt tcaacatgtt agagatcaga tcatctgcaa cgttttcaaa 3600 cacgaacact tctttttccg cgatacaagg aagattgttg tcaaacgaac aaccttcaat 3660 aatgcttctg ccggccttct cgatatctgc tgtatcgtct acaataaccg gaggattacc 3720 cgcgccagct ccgatggcct ttttaccaga attaagaagg gtttttacca tacccgggcc 3780 acccgtaccg cacaacaatt ttatggatgg atgtttgata atagcgtcta aactttccat 3840 agttgggttc tttatagtag tgacaaggtt ttcaggtcca ccacagctaa ttatggcttt 3900 gtttatcatt tctactgcga aagcgacaca ctttttggcg catgggtgac cattaaatac 3960 aactgcattc cccgcagcta tcatacctat agaattgcag ataacggttt ctgttggatt 4020 cgtgcttgga gttatagcgc cgataactcc gtatggactc atttcaacca ctgttagtcc 4080 attatcgccg gaccatgctg ttgttgtcag atcttcagtg cctggggtat acttggccac 4140 taattcatgt ttcaagattt tatcctcata ccttcccatg tgggtttcct ccaggatcat 4200 tgtggctaag acctctttat tctgtaatgc ggcttttctt atttcggtga ttattttctc 4260 tctttgttcc tttgtgtagt gtagggaaag aatcttttgt gcatgtactg cagaagaaat 4320 ggcattctca acattttcaa atactccaaa acatgaagag ttatctttgt aattctttaa 4380 gttgatgttt tcaccattag tcttcacttt caagtctttg gtggttggga ttaaggtatc 4440 tttatccatg gtgtttgttt atgtgtgttt attcgaaact aagttcttgg tgttttaaaa 4500 ctaaaaaaaa gactaactat aaaagtagaa tttaagaagt ttaagaaata gatttacaga 4560 attacaatca atacctaccg tctttatata cttattagtc aagtagggga ataatttcag 4620 ggaactggtt tcaacctttt ttttcagctt tttccaaatc agagagagca gaaggtaata 4680 gaaggtgtaa gaaaatgaga tagatacatg cgtgggtcaa ttgccttgtg tcatcattta 4740 ctccaggcag gttgcatcac tccattgagg ttgtgcccgt tttttgcctg tttgtgcccc 4800 tgttctctgt agttgcgcta agagaatgga cctatgaact gatggttggt gaagaaaaca 4860 atattttggt gctgggattc tttttttttc tggatgccag cttaaaaagc gggctccatt 4920 atatttagtg gatgccagga ataaactgtt cacccagaca cctacgatgt tatatattct 4980 gtgtaacccg ccccctattt tgggcatgta cgggttacag cagaattaaa aggctaattt 5040 tttgactaaa taaagttagg aaaatcacta ctattaatta tttacgtatt ctttgaaatg 5100 gcagtattga taatgataaa ctcgaactga aaaagcgtgt tttttattca aaatgattct 5160 aactccctta cgtaatcaag gaatcttttt gccttggcct ccgcgtcatt aaacttcttg 5220 ttgttgacgc taacattcaa cgctagtata tattcgtttt tttcaggtaa gttcttttca 5280 acgggtctta ctgatgaggc agtcgcgtct gaacctgtta agaggtcaaa tatgtcttct 5340 tgaccgtacg tgtcttgcat gttattagct ttgggaattt gcatcaagtc ataggaaaat 5400 ttaaatcttg gctctcttgg gctcaaggtg acaaggtcct cgaaaatagg gcgcgcccca 5460 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa 5520 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 5580 ggagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgaggta actcacatta 5640 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 5700 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5760 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 5820 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 5880 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 5940 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6000 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6060 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6120 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6180 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6240 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6300 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6360 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6420 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6480 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6540 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6600 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 6660 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 6720 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 6780 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 6840 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 6900 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 6960 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 7020 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 7080 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 7140 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 7200 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 7260 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 7320 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 7380 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 7440 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 7500 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7560 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 7620 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa 7680 cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc taatttttca 7740 aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag cgctatttta 7800 ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag agcgctaatt 7860 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gagagcgcta 7920 ttttaccaac aaagaatcta tacttctttt ttgttctaca aaaatgcatc ccgagagcgc 7980 tatttttcta acaaagcatc ttagattact ttttttctcc tttgtgcgct ctataatgca 8040 gtctcttgat aactttttgc actgtaggtc cgttaaggtt agaagaaggc tactttggtg 8100 tctattttct cttccataaa aaaagcctga ctccacttcc cgcgtttact gattactagc 8160 gaagctgcgg gtgcattttt tcaagataaa ggcatccccg attatattct ataccgatgt 8220 ggattgcgca tactttgtga acagaaagtg atagcgttga tgattcttca ttggtcagaa 8280 aattatgaac ggtttcttct attttgtctc tatatactac gtataggaaa tgtttacatt 8340 ttcgtattgt tttcgattca ctctatgaat agttcttact acaatttttt tgtctaaaga 8400 gtaatactag agataaacat aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag 8460 cgaaaggtgg atgggtaggt tatataggga tatagcacag agatatatag caaagagata 8520 cttttgagca atgtttgtgg aagcggtatt cgcaatattt tagtagctcg ttacagtccg 8580 gtgcgttttt ggttttttga aagtgcgtct tcagagcgct tttggttttc aaaagcgctc 8640 tgaagttcct atactttcta gagaatagga acttcggaat aggaacttca aagcgtttcc 8700 gaaaacgagc gcttccgaaa atgcaacgcg agctgcgcac atacagctca ctgttcacgt 8760 cgcacctata tctgcgtgtt gcctgtatat atatatacat gagaagaacg gcatagtgcg 8820 tgtttatgct taaatgcgta cttatatgcg tctatttatg taggatgaaa ggtagtctag 8880 tacctcctgt gatattatcc cattccatgc ggggtatcgt atgcttcctt cagcactacc 8940 ctttagctgt tctatatgct gccactcctc aattggatta gtctcatcct tcaatgctat 9000 catttccttt gatattggat catactaaga aaccattatt atcatgacat taacctataa 9060 aaataggcgt atcacgaggc cctttcgtc 9089 <210> 121 <211> 1023 <212> DNA <213> Saccharomyces cerevisiae <400> 121 caccgcggtg gggcgcgccc tattttcgag gaccttgtca ccttgagccc aagagagcca 60 agatttaaat tttcctatga cttgatgcaa attcccaaag ctaataacat gcaagacacg 120 tacggtcaag aagacatatt tgacctctta acaggttcag acgcgactgc ctcatcagta 180 agacccgttg aaaagaactt acctgaaaaa aacgaatata tactagcgtt gaatgttagc 240 gtcaacaaca agaagtttaa tgacgcggag gccaaggcaa aaagattcct tgattacgta 300 agggagttag aatcattttg aataaaaaac acgctttttc agttcgagtt tatcattatc 360 aatactgcca tttcaaagaa tacgtaaata attaatagta gtgattttcc taactttatt 420 tagtcaaaaa attagccttt taattctgct gtaacccgta catgcccaaa atagggggcg 480 ggttacacag aatatataac atcgtaggtg tctgggtgaa cagtttattc ctggcatcca 540 ctaaatataa tggagcccgc tttttaagct ggcatccaga aaaaaaaaga atcccagcac 600 caaaatattg ttttcttcac caaccatcag ttcataggtc cattctctta gcgcaactac 660 agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc tcaatggagt gatgcaacct 720 gcctggagta aatgatgaca caaggcaatt gacccacgca tgtatctatc tcattttctt 780 acaccttcta ttaccttctg ctctctctga tttggaaaaa gctgaaaaaa aaggttgaaa 840 ccagttccct gaaattattc ccctacttga ctaataagta tataaagacg gtaggattattg 900 attgtaattc tgtaaatcta tttcttaaac ttcttaaatt ctacttttat agttagtctt 960 ttttttagtt ttaaaacacc aagaacttag tttcgaataa acacacataa actagtaaac 1020 aaa 1023 <210> 122 <211> 21 <212> DNA Artificial sequence <220> <223> primer <400> 122 caaaagctga gctccaccgc g 21 <210> 123 <211> 44 <212> DNA Artificial sequence <220> <223> primer <400> 123 gtttactagt ttatgtgtgt ttattcgaaa ctaagttctt ggtg 44 <210> 124 <400> 124 000 <210> 125 <211> 39 <212> DNA Artificial sequence <220> <223> primer <400> 125 cacacatatt acaatagcta gctgaggatg aaagctctg 39 <210> 126 <211> 39 <212> DNA Artificial sequence <220> <223> primer <400> 126 cagagctttc atcctcagct agctattgta atatgtgtg 39 <210> 127 <211> 9491 <212> DNA Artificial sequence <220> &Lt; 223 > constructed plasmid <400> 127 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt cccctagcgg atgactcttt ttttttctta gcgattggca ttatcacata 420 atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc 480 aggcaagata aacgaaggca aagatgacag agcagaaagc cctagtaaag cgtattacaa 540 atgaaaccaa gattcagatt gcgatctctt taaagggtgg tcccctagcg atagagcact 600 cgatcttccc agaaaaagag gcagaagcag tagcagaaca ggccacacaa tcgcaagtga 660 ttaacgtcca cacaggtata gggtttctgg accatatgat acatgctctg gccaagcatt 720 ccggctggtc gctaatcgtt gagtgcattg gtgacttaca catagacgac catcacacca 780 ctgaagactg cgggattgct ctcggtcaag cttttaaaga ggccctactg gcgcgtggag 840 taaaaaggtt tggatcagga tttgcgcctt tggatgaggc actttccaga gcggtggtag 900 atctttcgaa caggccgtac gcagttgtcg aacttggttt gcaaagggag aaagtaggag 960 atctctcttg cgagatgatc ccgcattttc ttgaaagctt tgcagaggct agcagaatta 1020 ccctccacgt tgattgtctg cgaggcaaga atgatcatca ccgtagtgag agtgcgttca 1080 aggctcttgc ggttgccata agagaagcca cctcgcccaa tggtaccaac gatgttccct 1140 ccaccaaagg tgttcttatg tagtgacacc gattatttaa agctgcagca tacgatatat 1200 atacatgtgt atatatgtat acctatgaat gtcagtaagt atgtatacga acagtatgat 1260 actgaagatg acaaggtaat gcatcattct atacgtgtca ttctgaacga ggcgcgcttt 1320 ccttttttct ttttgctttt tctttttttt tctcttgaac tcgacggatc tatgcggtgt 1380 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata 1440 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 1500 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 1560 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 1620 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt 1680 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 1740 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 1800 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 1860 cgccgctaca gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc 1920 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 1980 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 2040 agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc ctcgaggtcg 2100 acggcgcgcc actggtagag agcgactttg tatgccccaa ttgcgaaacc cgcgatatcc 2160 ttctcgattc tttagtaccc gaccaggaca aggaaaagga ggtcgaaacg tttttgaaga 2220 aacaagagga actacacgga agctctaaag atggcaacca gccagaaact aagaaaatga 2280 agttgatgga tccaactggc accgctggct tgaacaacaa taccagcctt ccaacttctg 2340 taaataacgg cggtacgcca gtgccaccag taccgttacc tttcggtata cctcctttcc 2400 ccatgtttcc aatgcccttc atgcctccaa cggctactat cacaaatcct catcaagctg 2460 acgcaagccc taagaaatga ataacaatac tgacagtact aaataattgc ctacttggct 2520 tcacatacgt tgcatacgtc gatatagata ataatgataa tgacagcagg attatcgtaa 2580 tacgtaatag ttgaaaatct caaaaatgtg tgggtcatta cgtaaataat gataggaatg 2640 ggattcttct atttttcctt tttccattct agcagccgtc gggaaaacgt ggcatcctct 2700 ctttcgggct caattggagt cacgctgccg tgagcatcct ctctttccat atctaacaac 2760 tgagcacgta accaatggaa aagcatgagc ttagcgttgc tccaaaaaag tattggatgg 2820 ttaataccat ttgtctgttc tcttctgact ttgactcctc aaaaaaaaaa aatctacaat 2880 caacagatcg cttcaattac gccctcacaa aaactttttt ccttcttctt cgcccacgtt 2940 aaattttatc cctcatgttg tctaacggat ttctgcactt gatttattat aaaaagacaa 3000 agacataata cttctctatc aatttcagtt attgttcttc cttgcgttat tcttctgttc 3060 ttctttttct tttgtcatat ataaccataa ccaagtaata catattcaaa ctagtatgac 3120 tgacaaaaaa actcttaaag acttaagaaa tcgtagttct gtttacgatt caatggttaa 3180 atcacctaat cgtgctatgt tgcgtgcaac tggtatgcaa gatgaagact ttgaaaaacc 3240 tatcgtcggt gtcatttcaa cttgggctga aaacacacct tgtaatatcc acttacatga 3300 ctttggtaaa ctagccaaag tcggtgttaa ggaagctggt gcttggccag ttcagttcgg 3360 aacaatcacg gtttctgatg gaatcgccat gggaacccaa ggaatgcgtt tctccttgac 3420 atctcgtgat attattgcag attctattga agcagccatg ggaggtcata atgcggatgc 3480 ttttgtagcc attggcggtt gtgataaaaa catgcccggt tctgttatcg ctatggctaa 3540 catggatatc ccagccattt ttgcttacgg cggaacaatt gcacctggta atttagacgg 3600 caaagatatc gatttagtct ctgtctttga aggtgtcggc cattggaacc acggcgatat 3660 gaccaaagaa gaagttaaag ctttggaatg taatgcttgt cccggtcctg gaggctgcgg 3720 tggtatgtat actgctaaca caatggcgac agctattgaa gttttgggac ttagccttcc 3780 gggttcatct tctcacccgg ctgaatccgc agaaaagaaa gcagatattg aagaagctgg 3840 tcgcgctgtt gtcaaaatgc tcgaaatggg cttaaaacct tctgacattt taacgcgtga 3900 agcttttgaa gatgctatta ctgtaactat ggctctggga ggttcaacca actcaaccct 3960 tcacctctta gctattgccc atgctgctaa tgtggaattg acacttgatg atttcaatac 4020 tttccaagaa aaagttcctc atttggctga tttgaaacct tctggtcaat atgtattcca 4080 agacctttac aaggtcggag gggtaccagc agttatgaaa tatctcctta aaaatggctt 4140 ccttcatggt gaccgtatca cttgtactgg caaaacagtc gctgaaaatt tgaaggcttt 4200 tgatgattta acacctggtc aaaaggttat tatgccgctt gaaaatccta aacgtgaaga 4260 tggtccgctc attattctcc atggtaactt ggctccagac ggtgccgttg ccaaagtttc 4320 tggtgtaaaa gtgcgtcgtc atgtcggtcc tgctaaggtc tttaattctg aagaagaagc 4380 cattgaagct gtcttgaatg atgatattgt tgatggtgat gttgttgtcg tacgttttgt 4440 aggaccaaag ggcggtcctg gtatgcctga aatgctttcc ctttcatcaa tgattgttgg 4500 taaagggcaa ggtgaaaaag ttgcccttct gacagatggc cgcttctcag gtggtactta 4560 tggtcttgtc gtgggtcata tcgctcctga agcacaagat ggcggtccaa tcgcctacct 4620 gcaaacagga gacatagtca ctattgacca agacactaag gaattacact ttgatatctc 4680 cgatgaagag ttaaaacatc gtcaagagac cattgaattg ccaccgctct attcacgcgg 4740 tatccttggt aaatatgctc acatcgtttc gtctgcttct aggggagccg taacagactt 4800 ttggaagcct gaagaaactg gcaaaaaatg ttgtcctggt tgctgtggtt aagcggccgc 4860 gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 4920 gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 4980 tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 5040 caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 5100 gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 5160 ttggagttcg cgattgtctt ctgttattca caactgtttt aatttttatt tcattctgga 5220 actcttcgag ttctttgtaa agtctttcat agtagcttac tttatcctcc aacatattta 5280 acttcatgtc aatttcggct cttaaatttt ccacatcatc aagttcaaca tcatctttta 5340 acttgaattt attctctagc tcttccaacc aagcctcatt gctccttgat ttactggtga 5400 aaagtgatac actttgcgcg caatccaggt caaaactttc ctgcaaagaa ttcaccaatt 5460 tctcgacatc atagtacaat ttgttttgtt ctcccatcac aatttaatat acctgatgga 5520 ttcttatgaa gcgctgggta atggacgtgt cactctactt cgcctttttc cctactcctt 5580 ttagtacgga agacaatgct aataaataag agggtaataa taatattatt aatcggcaaa 5640 aaagattaaa cgccaagcgt ttaattatca gaaagcaaac gtcgtaccaa tccttgaatg 5700 cttcccaatt gtatattaag agtcatcaca gcaacatatt cttgttatta aattaattat 5760 tattgatttt tgatattgta taaaaaaacc aaatatgtat aaaaaaagtg aataaaaaat 5820 accaagtatg gagaaatata ttagaagtct atacgttaaa ccaccgcggt ggagctccag 5880 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt 5940 tcctgtgtga aattgttatc cgctcacaat tccacacaac ataggagccg gaagcataaa 6000 gtgtaaagcc tggggtgcct aatgagtgag gtaactcaca ttaattgcgt tgcgctcact 6060 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 6120 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 6180 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 6240 cilgaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 6300 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 6360 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 6420 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 6480 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 6540 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 6600 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 6660 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 6720 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 6780 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 6840 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 6900 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 6960 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 7020 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 7080 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 7140 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 7200 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 7260 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 7320 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 7380 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 7440 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 7500 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 7560 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 7620 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 7680 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 7740 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 7800 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 7860 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 7920 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 7980 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 8040 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaacgaagca tctgtgcttc 8100 attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct 8160 gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg 8220 cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct 8280 gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat 8340 ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc 8400 atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt 8460 tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat 8520 aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt 8580 ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg 8640 tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct 8700 tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat 8760 tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa 8820 cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta 8880 ggttatatag ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg 8940 tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt 9000 tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt 9060 ctagagaata ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg 9120 aaaatgcaac gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt 9180 gttgcctgta tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc 9240 gtacttatat gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta 9300 tcccattcca tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat 9360 gctgccactc ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg 9420 gatcatctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 9480 gccctttcgt c 9491 <210> 128 <211> 1000 <212> DNA <213> Saccharymoces cerevisiae <400> 128 gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 60 gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 120 tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 180 caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 240 gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 300 ttggagttcg cgattgtctt ctgttattca caactgtttt aatttttatt tcattctgga 360 actcttcgag ttctttgtaa agtctttcat agtagcttac tttatcctcc aacatattta 420 acttcatgtc aatttcggct cttaaatttt ccacatcatc aagttcaaca tcatctttta 480 acttgaattt attctctagc tcttccaacc aagcctcatt gctccttgat ttactggtga 540 aaagtgatac actttgcgcg caatccaggt caaaactttc ctgcaaagaa ttcaccaatt 600 tctcgacatc atagtacaat ttgttttgtt ctcccatcac aatttaatat acctgatgga 660 ttcttatgaa gcgctgggta atggacgtgt cactctactt cgcctttttc cctactcctt 720 ttagtacgga agacaatgct aataaataag agggtaataa taatattatt aatcggcaaa 780 aaagattaaa cgccaagcgt ttaattatca gaaagcaaac gtcgtaccaa tccttgaatg 840 cttcccaatt gtatattaag agtcatcaca gcaacatatt cttgttatta aattaattat 900 tattgatttt tgatattgta taaaaaaacc aaatatgtat aaaaaaagtg aataaaaaat 960 accaagtatg gagaaatata ttagaagtct atacgttaaa 1000 <210> 129 <211> 13114 <212> DNA Artificial sequence <220> &Lt; 223 > constructed plasmid <400> 129 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat 420 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag 480 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca 540 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac 600 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg 660 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat 720 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc 780 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt 840 ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg 900 gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta 960 ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga 1020 attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg 1080 ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt 1140 ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat 1200 atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta 1260 tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg 1320 ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg 1380 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt 1440 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 1500 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 1560 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 1620 aaaaccgtct atcagggcga tggcccacta cgtggccggc ttcacatacg ttgcatacgt 1680 cgatatagat aataatgata atgacagcag gattatcgta atacgtaata gctgaaaatc 1740 tcaaaaatgt gtgggtcatt acgtaaataa tgataggaat gggattcttc tatttttcct 1800 ttttccattc tagcagccgt cgggaaaacg tggcatcctc tctttcgggc tcaattggag 1860 tcacgctgcc gtgagcatcc tctctttcca tatctaacaa ctgagcacgt aaccaatgga 1920 aaagcatgag cttagcgttg ctccaaaaaa gtattggatg gttaatacca tttgtctgtt 1980 ctcttctgac tttgactcct caaaaaaaaa aatctacaat caacagatcg cttcaattac 2040 gccctcacaa aaactttttt ccttcttctt cgcccacgtt aaattttatc cctcatgttg 2100 tctaacggat ttctgcactt gatttattat aaaaagacaa agacataata cttctctatc 2160 aatttcagtt attgttcttc cttgcgttat tcttctgttc ttctttttct tttgtcatat 2220 ataaccataa ccaagtaata catattcaaa cacgtgagta tgactgacaa aaaaactctt 2280 aaagacttaa gaaatcgtag ttctgtttac gattcaatgg ttaaatcacc taatcgtgct 2340 atgttgcgtg caactggtat gcaagatgaa gactttgaaa aacctatcgt cggtgtcatt 2400 tcaacttggg ctgaaaacac accttgtaat atccacttac atgactttgg taaactagcc 2460 aaagtcggtg ttaaggaagc tggtgcttgg ccagttcagt tcggaacaat cacggtttct 2520 gatggaatcg ccatgggaac ccaaggaatg cgtttctcct tgacatctcg tgatattatt 2580 gcagattcta ttgaagcagc catgggaggt cataatgcgg atgcttttgt agccattggc 2640 ggttgtgata aaaacatgcc cggttctgtt atcgctatgg ctaacatgga tatcccagcc 2700 atttttgctt acggcggaac aattgcacct ggtaatttag acggcaaaga tatcgattta 2760 gtctctgtct ttgaaggtgt cggccattgg aaccacggcg atatgaccaa agaagaagtt 2820 aaagctttgg aatgtaatgc ttgtcccggt cctggaggct gcggtggtat gtatactgct 2880 aacacaatgg cgacagctat tgaagttttg ggacttagcc ttccgggttc atcttctcac 2940 ccggctgaat ccgcagaaaa gaaagcagat attgaagaag ctggtcgcgc tgttgtcaaa 3000 atgctcgaaa tgggcttaaa accttctgac attttaacgc gtgaagcttt tgaagatgct 3060 attactgtaa ctatggctct gggaggttca accaactcaa cccttcacct cttagctatt 3120 gcccatgctg ctaatgtgga attgacactt gatgatttca atactttcca agaaaaagtt 3180 cctcatttgg ctgatttgaa accttctggt caatatgtat tccaagacct ttacaaggtc 3240 ggaggggtac cagcagttat gaaatatctc cttaaaaatg gcttccttca tggtgaccgt 3300 atcacttgta ctggcaaaac agtcgctgaa aatttgaagg cttttgatga tttaacacct 3360 ggtcaaaagg ttattatgcc gcttgaaaat cctaaacgtg aagatggtcc gctcattatt 3420 ctccatggta acttggctcc agacggtgcc gttgccaaag tttctggtgt aaaagtgcgt 3480 cgtcatgtcg gtcctgctaa ggtctttaat tctgaagaag aagccattga agctgtcttg 3540 aatgatgata ttgttgatgg tgatgttgtt gtcgtacgtt ttgtaggacc aaagggcggt 3600 cctggtatgc ctgaaatgct ttccctttca tcaatgattg ttggtaaagg gcaaggtgaa 3660 aaagttgccc ttctgacaga tggccgcttc tcaggtggta cttatggtct tgtcgtgggt 3720 catatcgctc ctgaagcaca agatggcggt ccaatcgcct acctgcaaac aggagacata 3780 gtcactattg accaagacac taaggaatta cactttgata tctccgatga agagttaaaa 3840 catcgtcaag agaccattga attgccaccg ctctattcac gcggtatcct tggtaaatat 3900 gctcacatcg tttcgtctgc ttctagggga gccgtaacag acttttggaa gcctgaagaa 3960 actggcaaaa aatgttgtcc tggttgctgt ggttaagcgg ccgcgttaat tcaaattaat 4020 tgatatagtt ttttaatgag tattgaatct gtttagaaat aatggaatat tatttttatt 4080 tatttattta tattattggt cggctctttt cttctgaagg tcaatgacaa aatgatatga 4140 aggaaataat gatttctaaa attttacaac gtaagatatt tttacaaaag cctagctcat 4200 cttttgtcat gcactatttt actcacgctt gaaattaacg gccagtccac tgcggagtca 4260 tttcaaagtc atcctaatcg atctatcgtt tttgatagct cattttggag ttcgcgagga 4320 tccactagtt ctagagcggc cgctctagaa ctagtaccac aggtgttgtc ctctgaggac 4380 ataaaataca caccgagatt catcaactca ttgctggagt tagcatatct acaattgggt 4440 gaaatgggga gcgatttgca ggcatttgct cggcatgccg gtagaggtgt ggtcaataag 4500 agcgacctca tgctatacct gagaaagcaa cctgacctac aggaaagagt tactcaagaa 4560 taagaatttt cgttttaaaa cctaagagtc actttaaaat ttgtatacac ttattttttt 4620 tataacttat ttaataataa aaatcataaa tcataagaaa ttcgcttact cttaattaat 4680 caaaaagtta aaattgtacg aatagattca ccacttctta acaaatcaaa cccttcattg 4740 attttctcga atggcaatac atgtgtaatt aaaggatcaa gagcaaactt cttcgccata 4800 aagtcggcaa caagttttgg aacactatcc ttgctcttaa aaccgccaaa tatagctccc 4860 ttccatgtac gaccgcttag caacagcata ggattcatcg acaaattttg tgaatcagga 4920 ggaacaccta cgatcacact gactccatat gcctcttgac agcaggacaa cgcagttacc 4980 atagtatcaa gacggcctat aacttcaaaa gagaaatcaa ctccaccgtt tgacatttca 5040 gtaaggactt cttgtattgg tttcttataa tcttgagggt taacacattc agtagccccg 5100 acctccttag cttttgcaaa tttgtcctta ttgatgtcta cacctataat cctcgctgcg 5160 cctgcagctt tacaccccat aataacgctt agtcctactc ctcctaaacc gaatactgca 5220 caagtcgaac cctgtgtaac ctttgcaact ttaactgcgg aaccgtaacc ggtggaaaat 5280 ccgcacccta tcaagcaaac tttttccagt ggtgaagctg catcgatttt agcgacagat 5340 atctcgtcca ccactgtgta ttgggaaaat gtagaagtac caaggaaatg gtgtataggt 5400 ttccctctgc atgtaaatct gcttgtacca tcctgcatag tacctctagg catagacaaa 5460 tcatttttaa ggcagaaatt accctcagga tgtttgcaga ctctacactt accacattga 5520 ggagtgaaca gtgggatcac tttatcacca ggacgaacag tggtaacacc ttcacctatg 5580 gattcaacga ttccggcagc ctcgtgtccc gcgattactg gcaaaggagt aactagagtg 5640 ccactcacca catggtcgtc ggatctacag attccggtgg caaccatctt gattctaacc 5700 tcgtgtgctt ttggtggcgc tacttctact tcttctatgc taaacggctt tttctcttcc 5760 cacaaaactg ccgctttaca cttaataact ttaccggctg ttgacatcct cagctagcta 5820 ttgtaatatg tgtgtttgtt tggattatta agaagaataa ttacaaaaaa aattacaaag 5880 gaaggtaatt acaacagaat taagaaagga caagaaggag gaagagaatc agttcattat 5940 ttcttctttg ttatataaca aacccaagta gcgatttggc catacattaa aagttgagaa 6000 ccaccctccc tggcaacagc cacaactcgt taccattgtt catcacgatc atgaaactcg 6060 ccgtcagctg aaatttcacc tcagtggatc tctcttttta ttcttcatcg ttccactaac 6120 ctttttccat cagctggcag ggaacggaaa gtggaatccc atttagcgag cttcctcttt 6180 tcttcaagaa aagacgaagc ttgtgtgtgg gtgcgcgcgc tagtatcttt ccacattaag 6240 aaatatacca taaaggttac ttagacatca ctatggctat atatatatat atatatatat 6300 gtaacttagc accatcgcgc gtgcatcact gcatgtgtta accgaaaagt ttggcgaaca 6360 cttcaccgac acggtcattt agatctgtcg tctgcattgc acgtccctta gccttaaatc 6420 ctaggcggga gcattctcgt gtaattgtgc agcctgcgta gcaactcaac atagcgtagt 6480 ctacccagtt tttcaagggt ttatcgttag aagattctcc cttttcttcc tgctcacaaa 6540 tcttaaagtc atacattgca cgactaaatg caagcgacgt cagggaaaga tatgagctat 6600 acagcggaat ttccatatca ctcagatttt gttatctaat tttttccttc ccacgtccgc 6660 gggaatctgt gtatattact gcatctagat atatgttatc ttatcttggc gcgtacattt 6720 aattttcaac gtattctata agaaattgcg ggagtttttt tcatgtagat gatactgact 6780 gcacgcaaat ataggcatga tttataggca tgatttgatg gctgtaccga taggaacgct 6840 aagagtaact tcagaatcgt tatcctggcg gaaaaaattc atttgtaaac tttaaaaaaa 6900 aaagccaata tccccaaaat tattaagagc gcctccatta ttaactaaaa tttcactcag 6960 catccacaat gtatcaggta tctactacag atattacatg tggcgaaaaa gacaagaaca 7020 atgcaatagc gcatcaagaa aaaacacaaa gctttcaatc aatgaatcga aaatgtcatt 7080 aaaatagtat ataaattgaa actaagtcat aaagctataa aaagaaaatt tatttaaatg 7140 caagatttaa agtaaattca cggccctgca ggcctcagct cttgttttgt tctgcaaata 7200 acttacccat ctttttcaaa actttaggtg caccctcctt tgctagaata agttctatcc 7260 aatacatcct atttggatct gcttgagctt ctttcatcac ggatacgaat tcattttctg 7320 ttctcacaat tttggacaca actctgtctt ccgttgcccc gaaactttct ggcagttttg 7380 agtaattcca cataggaatg tcattataac tctggttcgg accatgaatt tccctctcaa 7440 ccgtgtaacc atcgttatta atgataaagc agattgggtt tatcttctct ctaatggcta 7500 gtcctaattc ttggacagtc agttgcaatg atccatctcc gataaacaat aaatgtctag 7560 attctttatc tgcaatttgg ctgcctagag ctgcggggaa agtgtatcct atagatcccc 7620 acaagggttg accaataaaa tgtgatttcg atttcagaaa tatagatgag gcaccgaaga 7680 aagaagtgcc ttgttcagcc acgatcgtct cattactttg ggtcaaattt tcgacagctt 7740 gccacagtct atcttgtgac aacagcgcgt tagaaggtac aaaatcttct tgctttttat 7800 ctatgtactt gcctttatat tcaatttcgg acaagtcaag aagagatgat atcagggatt 7860 cgaagtcgaa attttggatt ctttcgttga aaattttacc ttcatcgata ttcaaggaaa 7920 tcattttatt ttcattaaga tggtgagtaa atgcacccgt actagaatcg gtaagcttta 7980 cacccaacat aagaataaaa tcagcagatt ccacaaattc cttcaagttt ggctctgaca 8040 gagtaccgtt gtaaatcccc aaaaatgagg gcaatgcttc atcaacagat gatttaccaa 8100 agttcaaagt agtaataggt aacttagtct ttgaaataaa ctgagtaaca gtcttctcta 8160 ggccgaacga tataatttca tggcctgtga ttacaattgg tttcttggca ttcttcagac 8220 tttcctgtat tttgttcaga atctcttgat cagatgtatt cgacgtggaa ttttccttct 8280 taagaggcaa ggatggtttt tcagccttag cggcagctac atctacaggt aaattgatgt 8340 aaaccggctt tctttccttt agtaaggcag acaacactct atcaatttca acagttgcat 8400 tctcggctgt caataaagtc ctggcagcag taaccggttc gtgcatcttc ataaagtgct 8460 tgaaatcacc atcagccaac gtatggtgaa caaacttacc ttcgttctgc actttcgagg 8520 taggagatcc cacgatctca acaacaggca ggttctcagc ataggagccc gctaagccat 8580 taactgcgga taattcgcca acaccaaatg tagtcaagaa tgccgcagcc tttttcgttc 8640 ttgcgtaccc gtcggccata taggaggcat ttaactcatt agcatttccc acccatttca 8700 tatctttgtg tgaaataatt tgatctagaa attgcaaatt gtagtcacct ggtactccga 8760 atatttcttc tatacctaat tcgtgtaatc tgtccaacag atagtcacct actgtataca 8820 tgtttaaact ttgtttacta gtttatgtgt gtttattcga aactaagttc ttggtgtttt 8880 aaaactaaaa aaaagactaa ctataaaagt agaatttaag aagtttaaga aatagattta 8940 cagaattaca atcaatacct accgtcttta tatacttatt agtcaagtag gggaataatt 9000 tcagggaact ggtttcaacc ttttttttca gctttttcca aatcagagag agcagaaggt 9060 aatagaaggt gtaagaaaat gagatagata catgcgtggg tcaattgcct tgtgtcatca 9120 tttactccag gcaggttgca tcactccatt gaggttgtgc ccgttttttg cctgtttgtg 9180 cccctgttct ctgtagttgc gctaagagaa tggacctatg aactgatggt tggtgaagaa 9240 aacaatattt tggtgctggg attctttttt tttctggatg ccagcttaaa aagcgggctc 9300 cattatattt agtggatgcc aggaataaac tgttcaccca gacacctacg atgttatata 9360 ttctgtgtaa cccgccccct attttgggca tgtacgggtt acagcagaat taaaaggcta 9420 attttttgac taaataaagt taggaaaatc actactatta attatttacg tattctttga 9480 aatggcagta ttggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg 9540 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 9600 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 9660 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 9720 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 9780 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 9840 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 9900 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 9960 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 10020 cgacaggact ataaatatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 10080 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 10140 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 10200 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 10260 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 10320 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 10380 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 10440 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 10500 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 10560 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 10620 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 10680 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 10740 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 10800 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 10860 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 10920 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 10980 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 11040 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 11100 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 11160 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 11220 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 11280 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 11340 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 11400 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 11460 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 11520 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 11580 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 11640 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 11700 ctgaacgaag catctgtgct tcattttgta gaacaaaaat gcaacgcgag agcgctaatt 11760 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gaaagcgcta 11820 ttttaccaac gaagaatctg tgcttcattt ttgtaaaaca aaaatgcaac gcgagagcgc 11880 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgagag 11940 cgctatttta ccaacaaaga atctatactt cttttttgtt ctacaaaaat gcatcccgag 12000 agcgctattt ttctaacaaa gcatcttaga ttactttttt tctcctttgt gcgctctata 12060 atgcagtctc ttgataactt tttgcactgt aggtccgtta aggttagaag aaggctactt 12120 tggtgtctat tttctcttcc ataaaaaaag cctgactcca cttcccgcgt ttactgatta 12180 ctagcgaagc tgcgggtgca ttttttcaag ataaaggcat ccccgattat attctatacc 12240 gatgtggatt gcgcatactt tgtgaacaga aagtgatagc gttgatgatt cttcattggt 12300 cagaaaatta tgaacggttt cttctatttt gtctctatat actacgtata ggaaatgttt 12360 acattttcgt attgttttcg attcactcta tgaatagttc ttactacaat ttttttgtct 12420 aaagagtaat actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca 12480 aggagcgaaa ggtggatggg taggttatat agggatatag cacagagata tatagcaaag 12540 agatactttt gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca 12600 gtccggtgcg tttttggttt tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag 12660 cgctctgaag ttcctatact ttctagagaa taggaacttc ggaataggaa cttcaaagcg 12720 tttccgaaaa cgagcgcttc cgaaaatgca acgcgagctg cgcacataca gctcactgtt 12780 cacgtcgcac ctatatctgc gtgttgcctg tatatatata tacatgagaa gaacggcata 12840 gtgcgtgttt atgcttaaat gcgtacttat atgcgtctat ttatgtagga tgaaaggtag 12900 tctagtacct cctgtgatat tatcccattc catgcggggt atcgtatgct tccttcagca 12960 ctacccttta gctgttctat atgctgccac tcctcaattg gattagtctc atccttcaat 13020 gctatcattt cctttgatat tggatcatac taagaaacca ttattatcat gacattaacc 13080 tataaaaata ggcgtatcac gaggcccttt cgtc 13114 <210> 130 <211> 4236 <212> DNA Artificial sequence <220> <223> construct <400> 130 gatccgcatt gcggattacg tattctaatg ttcagataac ttcgtatagc atacattata 60 cgaagttatg cagattgtac tgagagtgca ccataccaca gcttttcaat tcaattcatc 120 attttttttt tattcttttt tttgatttcg gtttctttga aatttttttg attcggtaat 180 ctccgaacag aaggaagaac gaaggaagga gcacagactt agattggtat atatacgcat 240 atgtagtgtt gaagaaacat gaaattgccc agtattctta acccaactgc acagaacaaa 300 aacctgcagg aaacgaagat aaatcatgtc gaaagctaca tataaggaac gtgctgctac 360 tcatcctagt cctgttgctg ccaagctatt taatatcatg cacgaaaagc aaacaaactt 420 gtgtgcttca ttggatgttc gtaccaccaa ggaattactg gagttagttg aagcattagg 480 tcccaaaatt tgtttactaa aaacacatgt ggatatcttg actgattttt ccatggaggg 540 cacagttaag ccgctaaagg cattatccgc caagtacaat tttttactct tcgaagacag 600 aaaatttgct gacattggta atacagtcaa attgcagtac tctgcgggtg tatacagaat 660 agcagaatgg gcagacatta cgaatgcaca cggtgtggtg ggcccaggta ttgttagcgg 720 tttgaagcag gcggcagaag aagtaacaaa ggaacctaga ggccttttga tgttagcaga 780 attgtcatgc aagggctccc tatctactgg agaatatact aagggtactg ttgacattgc 840 gaagagcgac aaagattttg ttatcggctt tattgctcaa agagacatgg gtggaagaga 900 tgaaggttac gattggttga ttatgacacc cggtgtgggt ttagatgaca agggagacgc 960 attgggtcaa cagtatagaa ccgtggatga tgtggtctct acaggatctg acattattat 1020 tgttggaaga ggactatttg caaagggaag ggatgctaag gtagagggtg aacgttacag 1080 aaaagcaggc tgggaagcat atttgagaag atgcggccag caaaactaaa aaactgtatt 1140 ataagtaaat gcatgtatac taaactcaca aattagagct tcaatttaat tatatcagtt 1200 attaccctat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga 1260 aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt 1320 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat 1380 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa 1440 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta 1500 atcaagataa cttcgtatag catacattat acgaagttat ccagtgatga tacaacgagt 1560 tagccaaggt gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 1620 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 1680 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga 1740 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca 1800 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg 1860 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 1920 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg 1980 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt 2040 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 2100 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 2160 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 2220 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 2280 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 2340 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 2400 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 2460 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 2520 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 2580 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 2640 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 2700 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 2760 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 2820 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 2880 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 2940 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 3000 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 3060 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 3120 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 3180 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 3240 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 3300 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 3360 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 3420 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 3480 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 3540 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 3600 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 3660 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 3720 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 3780 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 3840 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 3900 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 3960 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 4020 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 4080 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 4140 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 4200 acgccaagct tgcatgcctg caggtcgact ctagag 4236 <210> 131 <211> 7523 <212> DNA <213> Artificial sequence <220> <223> construct <400> 131 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc 60 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatagga gccggaagca 120 taaagtgtaa agcctggggt gcctaatgag tgaggtaact cacattaatt gcgttgcgct 180 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 240 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 300 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 360 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 420 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 480 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 540 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 600 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 660 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 720 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 780 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 840 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 900 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 960 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1020 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1080 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1140 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1200 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1260 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1320 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 1380 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1440 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1500 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1560 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1620 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1680 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1740 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1800 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 1860 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 1920 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 1980 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2040 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2100 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2160 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaacga agcatctgtg 2220 cttcattttg tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg 2280 agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc tattttacca acgaagaatc 2340 tgtgcttcat ttttgtaaaa caaaaatgca acgcgagagc gctaattttt caaacaaaga 2400 atctgagctg catttttaca gaacagaaat gcaacgcgag agcgctattt taccaacaaa 2460 gaatctatac ttcttttttg ttctacaaaa atgcatcccg agagcgctat ttttctaaca 2520 aagcatctta gattactttt tttctccttt gtgcgctcta taatgcagtc tcttgataac 2580 tttttgcact gtaggtccgt taaggttaga agaaggctac tttggtgtct attttctctt 2640 ccataaaaaa agcctgactc cacttcccgc gtttactgat tactagcgaa gctgcgggtg 2700 cattttttca agataaaggc atccccgatt atattctata ccgatgtgga ttgcgcatac 2760 tttgtgaaca gaaagtgata gcgttgatga ttcttcattg gtcagaaaat tatgaacggt 2820 ttcttctatt ttgtctctat atactacgta taggaaatgt ttacattttc gtattgtttt 2880 cgattcactc tatgaatagt tcttactaca atttttttgt ctaaagagta atactagaga 2940 taaacataaa aaatgtagag gtcgagttta gatgcaagtt caaggagcga aaggtggatg 3000 ggtaggttat atagggatat agcacagaga tatatagcaa agagatactt ttgagcaatg 3060 tttgtggaag cggtattcgc aatattttag tagctcgtta cagtccggtg cgtttttggt 3120 tttttgaaag tgcgtcttca gagcgctttt ggttttcaaa agcgctctga agttcctata 3180 ctttctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct 3240 tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct 3300 gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa 3360 atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat 3420 attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct 3480 atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat 3540 attggatcat ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 3600 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 3660 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 3720 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 3780 ttgtactgag agtgcaccat aaattcccgt tttaagagct tggtgagcgc taggagtcac 3840 tgccaggtat cgtttgaaca cggcattagt cagggaagtc ataacacagt cctttcccgc 3900 aattttcttt ttctattact cttggcctcc tctagtacac tctatatttt tttatgcctc 3960 ggtaatgatt ttcatttttt tttttcccct agcggatgac tctttttttt tcttagcgat 4020 tggcattatc acataatgaa ttatacatta tataaagtaa tgtgatttct tcgaagaata 4080 tactaaaaaa tgagcaggca agataaacga aggcaaagat gacagagcag aaagccctag 4140 taaagcgtat tacaaatgaa accaagattc agattgcgat ctctttaaag ggtggtcccc 4200 tagcgataga gcactcgatc ttcccagaaa aagaggcaga agcagtagca gaacaggcca 4260 cacaatcgca agtgattaac gtccacacag gtatagggtt tctggaccat atgatacatg 4320 ctctggccaa gcattccggc tggtcgctaa tcgttgagtg cattggtgac ttacacatag 4380 acgaccatca caccactgaa gactgcggga ttgctctcgg tcaagctttt aaagaggccc 4440 tactggcgcg tggagtaaaa aggtttggat caggatttgc gcctttggat gaggcacttt 4500 ccagagcggt ggtagatctt tcgaacaggc cgtacgcagt tgtcgaactt ggtttgcaaa 4560 gggagaaagt aggagatctc tcttgcgaga tgatcccgca ttttcttgaa agctttgcag 4620 aggctagcag aattaccctc cacgttgatt gtctgcgagg caagaatgat catcaccgta 4680 gtgagagtgc gttcaaggct cttgcggttg ccataagaga agccacctcg cccaatggta 4740 ccaacgatgt tccctccacc aaaggtgttc ttatgtagtg acaccgatta tttaaagctg 4800 cagcatacga tatatataca tgtgtatata tgtataccta tgaatgtcag taagtatgta 4860 tacgaacagt atgatactga agatgacaag gtaatgcatc attctatacg tgtcattctg 4920 aacgaggcgc gctttccttt tttctttttg ctttttcttt ttttttctct tgaactcgac 4980 ggatctatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa 5040 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt 5100 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag 5160 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg 5220 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat 5280 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc 5340 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga 5400 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac 5460 ccgccgcgct taatgcgccg ctacagggcg cgtcgcgcca ttcgccattc aggctgcgca 5520 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 5580 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 5640 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtaccgggc 5700 cccccctcga ggtattagaa gccgccgagc gggcgacagc cctccgacgg aagactctcc 5760 tccgtgcgtc ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca 5820 ctgctccgaa caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt 5880 ggcagtaacc tggccccaca aaccttcaaa ttaacgaatc aaattaacaa ccataggatg 5940 ataatgcgat tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt 6000 ttgatctatt aacagatata taaatggaaa agctgcataa ccactttaac taatactttc 6060 aacattttca gtttgtatta cttcttattc aaatgtcata aaagtatcaa caaaaaattg 6120 ttaatatacc tctatacttt aacgtcaagg agaaaaatgt ccaatttact gcccgtacac 6180 caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg 6240 gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct tctgtccgtt 6300 tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc cgcagaacct 6360 gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt aaaaactatc 6420 cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc acgaccaagt 6480 gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt 6540 gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc 6600 atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg gattgcttat 6660 aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat ctcacgtact 6720 gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag caccgcaggt 6780 gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat ttccgtctct 6840 ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa tggtgttgcc 6900 gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt tgaagcaact 6960 catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc ctggtctgga 7020 cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc aataccggag 7080 atcatgcaag ctggtggctg gaccaatgta aatattgtca tgaactatat ccgtaacctg 7140 gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattagga gtaagcgaat 7200 ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa 7260 ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg 7320 taggtcaggt tgctttctca ggtatagcat gaggtcgctc ttattgacca cacctctacc 7380 ggcatgccga gcaaatgcct gcaaatcgct ccccatttca cccaattgta gatatgctaa 7440 ctccagcaat gagttgatga atctcggtgt gtattttatg tcctcagagg acaacacctg 7500 tggtccgcca ccgcggtgga gct 7523 <210> 132 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 132 ctcgaaacaa taagacgacg atggctctg 29 <210> 133 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 133 gtctagacca gaagttaaga aggctgtag 29 <210> 134 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 134 tagcacccaa tagagcgccg actgtg 26 <210> 135 <211> 4519 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 135 caccttggct aactcgttgt atcatcactg gataacttcg tataatgtat gctatacgaa 60 gttatcgaac agagaaacta aatccacatt aattgagagt tctatctatt agaaaatgca 120 aactccaact aaatgggaaa acagataacc tcttttattt ttttttaatg tttgatattc 180 gagtcttttt cttttgttag gtttatattc atcatttcaa tgaataaaag aagcttctta 240 ttttggttgc aaagaatgaa aaaaaaggat tttttcatac ttctaaagct tcaattataa 300 ccaaaaattt tataaatgaa gagaaaaaat ctagtagtat caagttaaac ttagaaaaac 360 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt 420 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca 480 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc 540 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt 600 gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc 660 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg 720 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg 780 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat 840 acctggaatg ctgttttgcc ggggatcgca gtggtgagta accatgcatc atcaggagta 900 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc 960 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc 1020 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga 1080 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcgaaacg 1140 tgagtctttt ccttacccat ctcgagtttt aatgttactt ctcttgcagt tagggaacta 1200 taatgtaact caaaataaga ttaaacaaac taaaataaaa agaagttata cagaaaaacc 1260 catataaacc agtactaatc cataataata atacacaaaa aaactatcaa ataaaaccag 1320 aaaacagatt gaatagaaaa attttttcga tctcctttta tattcaaaat tcgatatatg 1380 aaaaagggaa ctctcagaaa atcaccaaat caatttaatt agatttttct tttccttcta 1440 gcgttggaaa gaaaaatttt tctttttttt tttagaaatg aaaaattttt gccgtaggaa 1500 tcaccgtata aaccctgtat aaacgctact ctgttcacct gtgtaggcta tgattgaccc 1560 agtgttcatt gttattgcga gagagcggga gaaaagaacc gatacaagag atccatgctg 1620 gtatagttgt ctgtccaaca ctttgatgaa cttgtaggac gatgatgtgt atttagacga 1680 gtacgtgtgt gactattaag tagttatgat agagaggttt gtacggtgtg ttctgtgtaa 1740 ttcgattgag aaaatggtta tgaatcccta gataacttcg tataatgtat gctatacgaa 1800 gttatctgaa cattagaata cgtaatccgc aatgcgggga tcctctagag tcgacctgca 1860 ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 1920 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 1980 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 2040 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 2100 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 2160 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 2220 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 2280 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 2340 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 2400 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 2460 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 2520 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 2580 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 2640 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 2700 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 2760 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 2820 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 2880 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 2940 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 3000 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 3060 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 3120 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 3180 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 3240 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 3300 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 3360 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 3420 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 3480 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 3540 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 3600 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 3660 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 3720 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 3780 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 3840 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 3900 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 3960 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 4020 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 4080 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 4140 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 4200 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 4260 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg 4320 taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag 4380 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 4440 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 4500 gtgaattcga gctcggtac 4519 <210> 136 <211> 86 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 136 aggataaaaa aagcttgtga ataaaaatct ttcgctaaaa atcaatataa gaaaatggta 60 caccttggct aactcgttgt atcatc 86 <210> 137 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 137 tgcatacttt atgcgtttat gcgttttgcg ccccttggaa aaaaattgat tctcatcgta 60 gcattgcgga ttacgtattc taatgttcag 90 <210> 138 <211> 2237 <212> DNA <213> Artificial Sequence <220> <223> PDC1 Fragment A-ilvDSm <400> 138 ttatgtatgc tcttctgact tttcgtgtga tgaggctcgt ggaaaaaatg aataatttat 60 gaatttgaga acaattttgt gttgttacgg tattttacta tggaataatc aatcaattga 120 ggattttatg caaatatcgt ttgaatattt ttccgaccct ttgagtactt ttcttcataa 180 ttgcataata ttgtccgctg cccctttttc tgttagacgg tgtcttgatc tacttgctat 240 cgttcaacac caccttattt tctaactatt ttttttttag ctcatttgaa tcagcttatg 300 gtgatggcac atttttgcat aaacctagct gtcctcgttg aacataggaa aaaaaaatat 360 ataaacaagg ctctttcact ctccttgcaa tcagatttgg gtttgttccc tttattttca 420 tatttcttgt catattcctt tctcaattat tattttctac tcataacctc acgcaaaata 480 acacagtcaa atcaatcaaa atgactgaca aaaaaactct taaagactta agaaatcgta 540 gttctgttta cgattcaatg gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta 600 tgcaagatga agactttgaa aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca 660 caccttgtaa tatccactta catgactttg gtaaactagc caaagtcggt gttaaggaag 720 ctggtgcttg gccagttcag ttcggaacaa tcacggtttc tgatggaatc gccatgggaa 780 cccaaggaat gcgtttctcc ttgacatctc gtgatattat tgcagattct attgaagcag 840 ccatgggagg tcataatgcg gatgcttttg tagccattgg cggttgtgat aaaaacatgc 900 ccggttctgt tatcgctatg gctaacatgg atatcccagc catttttgct tacggcggaa 960 caattgcacc tggtaattta gacggcaaag atatcgattt agtctctgtc tttgaaggtg 1020 tcggccattg gaaccacggc gatatgacca aagaagaagt taaagctttg gaatgtaatg 1080 cttgtcccgg tcctggaggc tgcggtggta tgtatactgc taacacaatg gcgacagcta 1140 ttgaagtttt gggacttagc cttccgggtt catcttctca cccggctgaa tccgcagaaa 1200 agaaagcaga tattgaagaa gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa 1260 aaccttctga cattttaacg cgtgaagctt ttgaagatgc tattactgta actatggctc 1320 tgggaggttc aaccaactca acccttcacc tcttagctat tgcccatgct gctaatgtgg 1380 aattgacact tgatgatttc aatactttcc aagaaaaagt tcctcatttg gctgatttga 1440 aaccttctgg tcaatatgta ttccaagacc tttacaaggt cggaggggta ccagcagtta 1500 tgaaatatct ccttaaaaat ggcttccttc atggtgaccg tatcacttgt actggcaaaa 1560 cagtcgctga aaatttgaag gcttttgatg atttaacacc tggtcaaaag gttattatgc 1620 cgcttgaaaa tcctaaacgt gaagatggtc cgctcattat tctccatggt aacttggctc 1680 cagacggtgc cgttgccaaa gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta 1740 aggtctttaa ttctgaagaa gaagccattg aagctgtctt gaatgatgat attgttgatg 1800 gtgatgttgt tgtcgtacgt tttgtaggac caaagggcgg tcctggtatg cctgaaatgc 1860 tttccctttc atcaatgatt gttggtaaag ggcaaggtga aaaagttgcc cttctgacag 1920 atggccgctt ctcaggtggt acttatggtc ttgtcgtggg tcatatcgct cctgaagcac 1980 aagatggcgg tccaatcgcc tacctgcaaa caggagacat agtcactatt gaccaagaca 2040 ctaaggaatt acctttgat atctccgatg aagagttaaa acatcgtcaa gagaccattg 2100 aattgccacc gctctattca cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg 2160 cttctagggg agccgtaaca gacttttgga agcctgaaga aactggcaaa aaatgagcga 2220 tttaatctct aattatt 2237 <210> 139 <211> 4420 <212> DNA <213> Artificial Sequence <220> <223> PDC1A-ilVDSm-BUC cassette <400> 139 ttatgtatgc tcttctgact tttcgtgtga tgaggctcgt ggaaaaaatg aataatttat 60 gaatttgaga acaattttgt gttgttacgg tattttacta tggaataatc aatcaattga 120 ggattttatg caaatatcgt ttgaatattt ttccgaccct ttgagtactt ttcttcataa 180 ttgcataata ttgtccgctg cccctttttc tgttagacgg tgtcttgatc tacttgctat 240 cgttcaacac caccttattt tctaactatt ttttttttag ctcatttgaa tcagcttatg 300 gtgatggcac atttttgcat aaacctagct gtcctcgttg aacataggaa aaaaaaatat 360 ataaacaagg ctctttcact ctccttgcaa tcagatttgg gtttgttccc tttattttca 420 tatttcttgt catattcctt tctcaattat tattttctac tcataacctc acgcaaaata 480 acacagtcaa atcaatcaaa atgactgaca aaaaaactct taaagactta agaaatcgta 540 gttctgttta cgattcaatg gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta 600 tgcaagatga agactttgaa aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca 660 caccttgtaa tatccactta catgactttg gtaaactagc caaagtcggt gttaaggaag 720 ctggtgcttg gccagttcag ttcggaacaa tcacggtttc tgatggaatc gccatgggaa 780 cccaaggaat gcgtttctcc ttgacatctc gtgatattat tgcagattct attgaagcag 840 ccatgggagg tcataatgcg gatgcttttg tagccattgg cggttgtgat aaaaacatgc 900 ccggttctgt tatcgctatg gctaacatgg atatcccagc catttttgct tacggcggaa 960 caattgcacc tggtaattta gacggcaaag atatcgattt agtctctgtc tttgaaggtg 1020 tcggccattg gaaccacggc gatatgacca aagaagaagt taaagctttg gaatgtaatg 1080 cttgtcccgg tcctggaggc tgcggtggta tgtatactgc taacacaatg gcgacagcta 1140 ttgaagtttt gggacttagc cttccgggtt catcttctca cccggctgaa tccgcagaaa 1200 agaaagcaga tattgaagaa gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa 1260 aaccttctga cattttaacg cgtgaagctt ttgaagatgc tattactgta actatggctc 1320 tgggaggttc aaccaactca acccttcacc tcttagctat tgcccatgct gctaatgtgg 1380 aattgacact tgatgatttc aatactttcc aagaaaaagt tcctcatttg gctgatttga 1440 aaccttctgg tcaatatgta ttccaagacc tttacaaggt cggaggggta ccagcagtta 1500 tgaaatatct ccttaaaaat ggcttccttc atggtgaccg tatcacttgt actggcaaaa 1560 cagtcgctga aaatttgaag gcttttgatg atttaacacc tggtcaaaag gttattatgc 1620 cgcttgaaaa tcctaaacgt gaagatggtc cgctcattat tctccatggt aacttggctc 1680 cagacggtgc cgttgccaaa gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta 1740 aggtctttaa ttctgaagaa gaagccattg aagctgtctt gaatgatgat attgttgatg 1800 gtgatgttgt tgtcgtacgt tttgtaggac caaagggcgg tcctggtatg cctgaaatgc 1860 tttccctttc atcaatgatt gttggtaaag ggcaaggtga aaaagttgcc cttctgacag 1920 atggccgctt ctcaggtggt acttatggtc ttgtcgtggg tcatatcgct cctgaagcac 1980 aagatggcgg tccaatcgcc tacctgcaaa caggagacat agtcactatt gaccaagaca 2040 ctaaggaatt acctttgat atctccgatg aagagttaaa acatcgtcaa gagaccattg 2100 aattgccacc gctctattca cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg 2160 cttctagggg agccgtaaca gacttttgga agcctgaaga aactggcaaa aaatgagcga 2220 tttaatctct aattattagt taaagtttta taagcatttt tatgtaacga aaaataaatt 2280 ggttcatatt attactgcac tgtcacttac catggaaaga ccagacaaga agttgccgac 2340 agtctgttga attggcctgg ttaggcttaa gtctgggtcc gcttctttac aaatttggag 2400 aatttctctt aaacgatatg tatattcttt tcgttggaaa agatgtcttc caaaaaaaaa 2460 accgatgaat tagtggaacc aaggaaaaaa aaagaggtat ccttgattaa ggaacactgt 2520 ttaaacagtg tggtttccaa aaccctgaaa ctgcattagt gtaatagaag actagacacc 2580 tcgatacaaa taatggttac tcaattcaaa actgccagcg aattcgactc tgcaattgct 2640 caagacaagc tagttgtcgt agatttctac gccacttggt gcggtccatg taaaatgatt 2700 gctccaatga ttgaaaaatg tggctgtggt ttcagggtcc ataaagcttt tcaattcatc 2760 tttttttttt ttgttctttt ttttgattcc ggtttctttg aaattttttt gattcggtaa 2820 tctccgagca gaaggaagaa cgaaggaagg agcacagact tagattggta tatatacgca 2880 tatgtggtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg cacagaacaa 2940 aaacctgcag gaaacgaaga taaatcatgt cgaaagctac atataaggaa cgtgctgcta 3000 ctcatcctag tcctgttgct gccaagctat ttaatatcat gcacgaaaag caaacaaact 3060 tgtgtgcttc attggatgtt cgtaccacca aggaattact ggagttagtt gaagcattag 3120 gtcccaaaat ttgtttacta aaaacacatg tggatatctt gactgatttt tccatggagg 3180 gcacagttaa gccgctaaag gcattatccg ccaagtacaa ttttttactc ttcgaagaca 3240 gaaaatttgc tgacattggt aatacagtca aattgcagta ctctgcgggt gtatacagaa 3300 tagcagaatg ggcagacatt acgaatgcac acggtgtggt gggcccaggt attgttagcg 3360 gtttgaagca ggcggcggaa gaagtaacaa aggaacctag aggccttttg atgttagcag 3420 aattgtcatg caagggctcc ctagctactg gagaatatac taagggtact gttgacattg 3480 cgaagagcga caaagatttt gttatcggct ttattgctca aagagacatg ggtggaagag 3540 atgaaggtta cgattggttg attatgacac ccggtgtggg tttagatgac aagggagacg 3600 cattgggtca acagtataga accgtggatg atgtggtctc tacaggatct gacattatta 3660 ttgttggaag aggactattt gcaaagggaa gggatgctaa ggtagagggt gaacgttaca 3720 gaaaagcagg ctgggaagca tatttgagaa gatgcggcca gcaaaactaa aaaactgtat 3780 tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa ttatatcagt 3840 tattacccgg gaatctcggt cgtaatgatt tctataatga cgaaaaaaaa aaaattggaa 3900 agaaaaagct tcatggcctt ccactttccc aaacaacacc tacggtatct ctcaagtctt 3960 atggggttcc attggtttca ccactggtgc taccttgggt gctgctttcg ctgctgaaga 4020 aattgatcca aagaagagag ttatcttatt cattggtgac ggttctttgc aattgactgt 4080 tcaagaaatc tccaccatga tcagatgggg cttgaagcca tacttgttcg tcttgaacaa 4140 cgatggttac accattgaaa agttgattca cggtccaaag gctcaataca acgaaattca 4200 aggttgggac cacctatcct tgttgccaac tttcggtgct aaggactatg aaacccacag 4260 agtcgctacc accggtgaat gggacaagtt gacccaagac aagtctttca acgacaactc 4320 taagatcaga atgattgaaa tcatgttgcc agtcttcgat gctccacaaa acttggttga 4380 acaagctaag ttgactgctg ctaccaacgc taagcaataa 4420 <210> 140 <211> 2686 <212> DNA <213> Artificial Sequence <220> <223> pUC19 <400> 140 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat 420 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct 480 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 780 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 840 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 900 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1200 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1260 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1320 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1560 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 1740 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 1920 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 2640 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 2686 <210> 141 <211> 3521 <212> DNA <213> Artificial Sequence <220> <223> PDC5A-sADB-BUC cassette <400> 141 aaggaaataa agcaaataac aataacacca ttattttaat tttttttcta ttactgtcgc 60 taacacctgt atggttgcaa ccaggtgaga atccttctga tgcatacttt atgcgtttat 120 gcgttttgcg ccccttggaa aaaaattgat tctcatcgta aatgcatact acatgcgttt 180 atgggaaaag cctccatatc caaaggtcgc gtttctttta gaaaaactaa tacgtaaacc 240 tgcattaagg taagattata tcagaaaatg tgttgcaaga aatgcattat gcaatttttt 300 gattatgaca atctctcgaa agaaatttca tatgatgaga cttgaataat gcagcggcgc 360 ttgctaaaag aacttgtata taagagctgc cattctcgat caatatactg tagtaagtcc 420 tttcctctct ttcttattac acttatttca cataatcaat ctcaaagaga acaacacaat 480 acaataacaa gaagaacaaa atgaaagctc tggtttatca cggtgaccac aagatctcgc 540 ttgaagacaa gcccaagccc acccttcaaa agcccacgga tgtagtagta cgggttttga 600 agaccacgat ctgcggcacg gatctcggca tctacaaagg caagaatcca gaggtcgccg 660 acgggcgcat cctgggccat gaaggggtag gcgtcatcga ggaagtgggc gagagtgtca 720 cgcagttcaa gaaaggcgac aaggtcctga tttcctgcgt cacttcttgc ggctcgtgcg 780 actactgcaa gaagcagctt tactcccatt gccgcgacgg cgggtggatc ctgggttaca 840 tgatcgatgg cgtgcaggcc gaatacgtcc gcatcccgca tgccgacaac agcctctaca 900 agatccccca gacaattgac gacgaaatcg ccgtcctgct gagcgacatc ctgcccaccg 960 gccacgaaat cggcgtccag tatgggaatg tccagccggg cgatgcggtg gctattgtcg 1020 gcgcgggccc cgtcggcatg tccgtactgt tgaccgccca gttctactcc ccctcgacca 1080 tcatcgtgat cgacatggac gagaatcgcc tccagctcgc caaggagctc ggggcaacgc 1140 acaccatcaa ctccggcacg gagaacgttg tcgaagccgt gcataggatt gcggcagagg 1200 gagtcgatgt tgcgatcgag gcggtgggca taccggcgac ttgggacatc tgccaggaga 1260 tcgtcaagcc cggcgcgcac atcgccaacg tcggcgtgca tggcgtcaag gttgacttcg 1320 agattcagaa gctctggatc aagaacctga cgatcaccac gggactggtg aacacgaaca 1380 cgacgcccat gctgatgaag gtcgcctcga ccgacaagct tccgttgaag aagatgatta 1440 cccatcgctt cgagctggcc gagatcgagc acgcctatca ggtattcctc aatggcgcca 1500 aggagaaggc gatgaagatc atcctctcga acgcaggcgc tgcctgagct aattaacata 1560 aaactcatga ttcaacgttt gtgtattttt ttacttttga aggttataga tgtttaggta 1620 aataattggc atagatatag ttttagtata ataaatttct gatttggttt aaaatatcaa 1680 ctattttttt tcacatatgt tcttgtaatt acttttctgt cctgtcttcc aggttaaaga 1740 ttagcttcta atattttagg tggtttatta tttaatttta tgctgattaa tttatttact 1800 tgtttaaacg gccggccaat gtggctgtgg tttcagggtc cataaagctt ttcaattcat 1860 cttttttttt tttgttcttt tttttgattc cggtttcttt gaaatttttt tgattcggta 1920 atctccgagc agaaggaaga acgaaggaag gagcacagac ttagattggt atatatacgc 1980 atatgtggtg ttgaagaaac atgaaattgc ccagtattct taacccaact gcacagaaca 2040 aaaacctgca ggaaacgaag ataaatcatg tcgaaagcta catataagga acgtgctgct 2100 actcatccta gtcctgttgc tgccaagcta tttaatatca tgcacgaaaa gcaaacaaac 2160 ttgtgtgctt cattggatgt tcgtaccacc aaggaattac tggagttagt tgaagcatta 2220 ggtcccaaaa tttgtttact aaaaacacat gtggatatct tgactgattt ttccatggag 2280 ggcacagtta agccgctaaa ggcattatcc gccaagtaca attttttact cttcgaagac 2340 agaaaatttg ctgacattgg taatacagtc aaattgcagt actctgcggg tgtatacaga 2400 atagcagaat gggcagacat tacgaatgca cacggtgtgg tgggcccagg tattgttagc 2460 ggtttgaagc aggcggcgga agaagtaaca aaggaaccta gaggcctttt gatgttagca 2520 gaattgtcat gcaagggctc cctagctact ggagaatata ctaagggtac tgttgacatt 2580 gcgaagagcg acaaagattt tgttatcggc tttattgctc aaagagacat gggtggaaga 2640 gattgaaggtt acgattggtt gattatgaca cccggtgtgg gtttagatga caagggagac 2700 gcattgggtc aacagtatag aaccgtggat gatgtggtct ctacaggatc tgacattatt 2760 attgttggaa gaggactatt tgcaaaggga agggatgcta aggtagaggg tgaacgttac 2820 agaaaagcag gctgggaagc atatttgaga agatgcggcc agcaaaacta aaaaactgta 2880 ttataagtaa atgcatgtat actaaactca caaattagag cttcaattta attatatcag 2940 ttattacccg ggaatctcgg tcgtaatgat ttctataatg acgaaaaaaa aaaaattgga 3000 aagaaaaagc ttcatggcct tctactttcc caacagatgt atacgctatc gtccaagtct 3060 tgtggggttc cattggtttc acagtcggcg ctctattggg tgctactatg gccgctgaag 3120 aactggcc aaagaagaga gttattttat tcattggtga cggttctcta caattgactg 3180 ttcaagaaat ctctaccatg attagatggg gtttgaagcc atacattttt gtcttgaata 3240 acaacggtta caccattgaa aaattgattc acggtcctca tgccgaatat aatgaaattc 3300 aaggttggga ccacttggcc ttattgccaa cttttggtgc tagaaactac gaaacccaca 3360 gagttgctac cactggtgaa tgggaaaagt tgactcaaga caaggacttc caagacaact 3420 ctaagattag aatgattgaa gttatgttgc cagtctttga tgctccacaa aacttggtta 3480 aacaagctca attgactgcc gctactaacg ctaaacaata a 3521 <210> 142 <211> 1685 <212> DNA <213> Artificial Sequence <220> <223> gpd2 :: loxP-URA3-loxP cassette <400> 142 gtattttggt agattcaatt ctctttccct ttccttttcc ttcgctcccc ttccttatca 60 gcattgcgga ttacgtattc taatgttcag ataacttcgt atagcataca ttatacgaag 120 ttatgcagat tgtactgaga gtgcaccata ccacagcttt tcaattcaat tcatcatttt 180 ttttttattc ttttttttga tttcggtttc tttgaaattt ttttgattcg gtaatctccg 240 aacagaagga agaacgaagg aaggagcaca gacttagatt ggtatatata cgcatatgta 300 gtgttgaaga aacatgaaat tgcccagtat tcttaaccca actgcacaga acaaaaacct 360 gcaggaaacg aagataaatc atgtcgaaag ctacatataa ggaacgtgct gctactcatc 420 ctagtcctgt tgctgccaag ctatttaata tcatgcacga aaagcaaaca aacttgtgtg 480 cttcattgga tgttcgtacc accaaggaat tactggagtt agttgaagca ttaggtccca 540 aaatttgttt actaaaaaca catgtggata tcttgactga tttttccatg gagggcacag 600 ttaagccgct aaaggcatta tccgccaagt acaatttttt actcttcgaa gacagaaaat 660 ttgctgacat tggtaataca gtcaaattgc agtactctgc gggtgtatac agaatagcag 720 aatgggcaga cattacgaat gcacacggtg tggtgggccc aggtattgtt agcggtttga 780 agcaggcggc agaagaagta acaaaggaac ctagaggcct tttgatgtta gcagaattgt 840 catgcaaggg ctccctatct actggagaat atactaaggg tactgttgac attgcgaaga 900 gcgacaaaga ttttgttatc ggctttattg ctcaaagaga catgggtgga agagatgaag 960 gttacgattg gttgattatg acacccggtg tgggtttaga tgacaaggga gacgcattgg 1020 gtcaacagta tagaaccgtg gatgatgtgg tctctacagg atctgacatt attattgttg 1080 gaagaggact atttgcaaag ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag 1140 caggctggga agcatatttg agaagatgcg gccagcaaaa ctaaaaaact gtattataag 1200 taaatgcatg tatactaaac tcacaaatta gagcttcaat ttaattatat cagttattac 1260 cctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg 1320 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 1380 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt 1440 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 1500 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa 1560 gataacttcg tatagcatac attatacgaa gttatccagt gatgatacaa cgagttagcc 1620 aaggtgacac tctccccccc cctccccctc tgatctttcc tgttgcctct ttttccccca 1680 accaa 1685 <210> 143 <211> 4506 <212> DNA <213> Artificial Sequence <220> <223> Sc Hap1 DNA <400> 143 atgtcgaata ccccttataa ttcatctgtg ccttccattg catccatgac ccagtcttcg 60 gtctcaagaa gtcctaacat gcatacagca actacgcccg gtgccaacac cagctctaac 120 tctccaccct tgcacatgtc ttcagattcg tccaagatca agaggaagcg taacagaatt 180 ccgctcagtt gcaccatttg tcggaaaagg aaagtcaaat gtgacaaact cagaccacac 240 tgccagcagt gcactaaaac tggggtagcc catctctgcc actacatgga acagacctgg 300 gcagaagagg cagagaaaga attgctgaag gacaacgaat taaagaagct tagggagcgc 360 gtaaaatctt tagaaaagac tctttctaag gtgcactctt ctccttcgtc taactccttg 420 aaaagttaca acactcccga gagcagcaac ctgtttatgg gtagcgatga acacaccacc 480 cttgttaatg caaatacagg ctctgcttcc tctgcctcgc atatgcatca gcaacaacag 540 caacagcagc aacaggaaca acaacaagac ttttccagaa gtgcgaacgc caacgcgaat 600 tcctcgtccc tttctatctc aaataaatat gacaacgatg agctggactt aactaaggac 660 tttgatcttt tgcatatcaa aagtaacgga accatccact taggtgccac ccactggttg 720 tctatcatga aaggtgaccc gtacctaaaa cttttgtggg gtcatatctt cgctatgagg 780 gaaaagttaa atgaatggta ctaccaaaaa aattcgtact ctaagctgaa gtcaagcaaa 840 tgtcccatca atcacgcgca agcgccgcct tctgccgctg ccgccgctac cagaaaatgt 900 cctgttgatc actccgcgtt ttcgtctggc atggtggccc caaaggagga gactcctctt 960 cctaggaaat gtccagttga ccacaccatg ttctcttcgg gaatgattcc tcccagagag 1020 gacacttcgt cccagaagag gtgtcccgtt gaccacacca tgtattccgc aggaatgatg 1080 ccgcccaagg acgagacacc ttccccattt tccactaaag ctatgataga ccataacaag 1140 catacaatga atccgcctca gtcaaaatgt cctgtggacc atagaaacta tatgaaggat 1200 tatccctctg acatggcaaa ttcttcttcg aacccggcaa gtcgttgccc cattgaccat 1260 tcaagcatga aaaatacagc ggccttacca gcttcaacgc acaataccat cccacaccac 1320 caaccacagt ccggatctca tgctcgttcg catcccgcac aaagcaggaa acatgattcc 1380 tacatgacag aatctgaagt cctcgcaaca ctttgtgaga tgttgccacc aaagcgcgtc 1440 atcgcattat tcatcgagaa attcttcaaa catttatacc ctgccattcc aatcttagat 1500 gaacagaatt tcaaaaatca cgtgaatcaa atgctttcgt tgtcttcgat gaatcccaca 1560 gttaacaact ttggtatgag catgccatct tcatctacac tagagaacca acccataaca 1620 caaatcaatc ttccaaaact ttccgattct tgtaacttag gtattctgat aataatcttg 1680 agattgacat ggctatccat accttctaat tcctgcgaag tcgacctggg agaagaaagt 1740 ggctcatttt tagtgcccaa cgaatctagc aatatgtctg catctgcatt gacctcgatg 1800 gctaaagaag aatcacttct gctaaagcat gagacaccgg tcgaggcact ggagctatgt 1860 caaaaatact tgattaaatt cgatgaactt tctagtattt ccaataacaa cgttaattta 1920 accacggtgc agtttgccat tttttacaac ttctatatga aaagtgcctc taatgatttg 1980 actaccttga caaataccaa caacactggc atggccaatc ctggtcacga ttccgagtct 2040 caccagatcc tattgtccaa tattactcaa atggccttta gttgtgggtt acacagagac 2100 cctgataatt ttcctcaatt aaacgctacc attccagcaa ccagccagga cgtgtctaac 2160 aacgggagca aaaaggcaaa ccctagcacc aatccaactt tgaataacaa catgtctgct 2220 gccactacca acagcagtag cagatctggc agtgctgatt caagaagtgg ttctaaccct 2280 gtgaacaaga aggaaaatca ggttagtatc gaaagattta aacacacttg gaggaaaatt 2340 tggtattaca ttgttagcat ggatgttaac caatctcttt ccctggggag ccctcgacta 2400 ctaagaaatc tgagggattt cagcgataca aagctaccaa gtgcgtcaag gattgattat 2460 gttcgcgata tcaaagagtt aatcattgtg aagaatttta ctcttttttt ccaaattgat 2520 ttgtgtatta ttgctgtatt aaatcacatt ttgaatgttt ctttagcaag aagcgtgaga 2580 aaatttgaac tggattcatt gattaattta ttgaaaaatc tgacctatgg tactgagaat 2640 gtcaatgatg tagtgagctc cttgatcaac aaagggttat taccaacttc ggaaggtggt 2700 tctgtagatt caaataatga tgaaatttac ggtctaccga aactacccga tattctaaac 2760 catggtcaac ataaccaaaa cttgtatgct gatggaagaa atacttctag tagtgatata 2820 gataagaaat tggaccttcc tcacgaatct acaacgagag ctctattctt ttccaagcat 2880 atgacaatta gaatgttgct gtacttattg aactacattt tgtttactca ttatgaacca 2940 atgggcagtg aagatcctgg tactaatatc ctagctaagg agtacgctca agaggcatta 3000 aattttgcca tggatggcta cagaaactgc atgattttct tcaacaatat cagaaacacc 3060 aattcactat tcgattacat gaatgttatc ttgtcttacc cttgtttgga cattggacat 3120 cgttctttac aatttatcgt ttgtttgatc ctgagagcta aatgtggccc attgactggt 3180 atgcgtgaat catcgatcat tactaatggt acatcaagtg gatttaatag ttcggtagaa 3240 gatgaggacg tcaaagttaa acaagaatct tctgatgaat tgaaaaaaga cgatttcatg 3300 aaagatgtaa atttggattc aggcgattca ttagcagaga ttctaatgtc aagaatgctg 3360 ctatttcaaa aactaacaaa acaactatca aagaagtaca actacgctat tcgtatgaac 3420 aaatccactg gattctttgt ctctttacta gatacacctt caaagaaatc agactcgaaa 3480 tcgggtggta gttcattcat gttgggtaat tggaaacatc caaaggtttc aaacatgagc 3540 ggatttcttg ctggtgacaa agaccaatta cagaaatgcc ccgtgtacca agatgcgctg 3600 gggtttgtta gtccaaccgg tgctaatgaa ggttctgctc cgatgcaagg catgtcctta 3660 cagggctcta ctgctaggat gggagggacc cagttgccac caattagatc atacaaacct 3720 atcacgtaca caagtagtaa tctacgtcgt atgaatgaaa cgggtgaggc agaagctaag 3780 agaagaagat ttaatgatgg ctatattgat aataatagta acaacgatat acctagagga 3840 atcagcccaa aaccttcaaa tgggctatca tcggtgcagc cactattatc gtcattttcc 3900 atgaaccagc taaacggggg taccattcca acggttccat cgttaaccaa cattacttca 3960 caaatgggag ctttaccatc tttagatagg atcaccacta atcaaataaa tttgccagac 4020 ccatctagag atgaagcatt tgacaactcc atcaagcaaa tgacgcctat gacaagtgca 4080 ttcatgaatg ctaatactac aattccaagt tcaactttaa acgggaatat gaacatgaat 4140 ggagctggaa ctgcgaatac agatacaagt gccaacggca gtgctttatc gacactgaca 4200 agcccacaag gctcagactt agcatccaat tctgctacac agtataaacc tgacttagaa 4260 gactttttga tgcaaaattc taactttaat gggctaatga taaatccttc cagtctggta 4320 gaagttgttg gtggatacaa cgatcctaat aaccttggaa gaaatgacgc ggttgatttt 4380 ctacccgttg ataatgttga aattgatggt gttggaataa aaatcaacta tcatctacta 4440 actagtattt acgttactag tatattatca tatacggtgt tagaagatga cgcaaatgat 4500 gagaaa 4506

Claims (38)

Recombinant microorganisms capable of producing alcohol from a feedstock;
Alcohol; And
At least one extractant selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof;
Wherein the extractant is produced from a feedstock. The method of claim 1, wherein the extractant comprises one or more fatty amides of the formula R (C═O) N (R ′) (R ″),
Wherein R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,
R ′ and R ″ are independently selected from the group consisting of hydrogen and C ₁ to C ₆ alkyl groups optionally containing one or more hydroxyl groups. The compound of claim 1, wherein the extractant comprises one or more fatty esters of the formula R— (C═O) —OCHR′CHR ″ —OH,
Wherein R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,
R ′ and R ″ are independently selected from the group consisting of hydrogen and C ₁ to C ₄ alkyl groups. The compound of claim 1, wherein the extractant comprises at least one fatty ester of formula R— (C═O) —OR ′,
Wherein R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,
R 'is an alkyl group of 8 carbons or less. The composition of claim 1, wherein the extractant is a mixture of fatty amides, wherein the mixture of fatty amides comprises linoleamide, oleamide, palmitamide, or stearamide. The method of claim 1, wherein the extractant is a mixture of fatty amides and fatty acids, wherein the mixture of fatty amides and fatty acids is linoleamide, linoleic acid, oleamide, oleic acid, palmitamide, palmitic acid, stearamide, or stearic acid. Composition comprising a. The composition of claim 1 wherein the extractant is selected from hydroxylated triglycerides, alkoxylated triglycerides, hydroxylated fatty acids, alkoxylated fatty acids, hydroxylated fatty alcohols, and alkoxylated fatty alcohols. The composition of claim 1 wherein the extractant is selected from saturated fatty acids, unsaturated fatty acids, saturated fatty alcohols, unsaturated fatty alcohols, saturated fatty amides, unsaturated fatty amides, saturated fatty esters, unsaturated fatty esters, and mixtures thereof. The composition of claim 1 wherein the alcohol is a C ₁ to C ₈ alkyl alcohol. The composition of claim 1 wherein the feedstock comprises rye, wheat, corn, cane, barley, cellulose based material, lignocellulosic based material, or mixtures thereof. Providing a biomass comprising oil;
Converting at least a portion of the oil into an extractant by contacting the oil with one or more substances that can chemically convert the oil into an extractant selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof Method for producing an extract, comprising the step of. The method of claim 11, wherein the one or more materials are selected from aqueous ammonium hydroxide, anhydrous ammonia, ammonium acetate, ammonia water, hydrogen peroxide, toluene, glacial acetic acid, lipase, and cation exchange resins. The method of claim 11, wherein the partition coefficient of the extractant for the product alcohol is greater than the partition coefficient of oil for the product alcohol before the oil is converted to the extractant. The method of claim 12, wherein the product alcohol is a C ₁ to C ₈ alkyl alcohol. The method of claim 11, further comprising separating the oil from the biomass prior to contacting the oil with at least one substance. The method of claim 11, wherein the biomass is corn grain, corncob, crop residues, for example corn husk, corn cobs, grass, wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass. ), Waste paper, sugar cane bagasse, sorghum, sugar cane, soybean, ingredients obtained from milling of grains, cellulose based materials, lignocellulosic materials, trees, branches, roots, leaves, wood chips, sawdust, shrubs And bushes, vegetables, fruits, flowers, animal compost, or mixtures thereof. (a) providing a biomass comprising oligosaccharides and oils;
(b) contacting the biomass with a glycosylating enzyme capable of converting the oligosaccharide into a monosaccharide;
(c) separating the oil from the biomass of (a) or (b);
(d) contacting the separated oil with one or more reactants or solvents to form an extractant;
(e) producing the product alcohol by contacting the biomass with fermentation broth comprising recombinant microorganisms capable of converting monosaccharides to product alcohols; And
(f) contacting the product alcohol with an extractant,
Wherein the partition coefficient for the product alcohol of the extractant is greater than the partition coefficient for the product alcohol of the oil of the biomass. 18. The method of claim 17, wherein the extractant is selected from fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof. 19. The extract of claim 18, wherein the extractant comprises one or a fatty amide of the formula R (C = 0) N (R ') (R "),
Wherein R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,
R ′ and R ″ are independently selected from the group consisting of hydrogen and C ₁ to C ₆ alkyl groups optionally containing one or more hydroxyl groups. 19. The method of claim 18, wherein the extractant comprises one or more fatty esters of the formula R- (C = 0) -OCHR'CHR "-OH,
Wherein R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,
R ′ and R ″ are independently selected from the group consisting of hydrogen and C ₁ to C ₄ alkyl groups. 19. The extract of claim 18, wherein the extractant comprises at least one fatty ester of the formula R- (C = 0) -OR ',
Wherein R is independently selected from the group consisting of C ₃ to C ₂₇ alkyl groups optionally interrupted by one or more double bonds,
R 'is an alkyl group of 8 carbons or less. The method of claim 17, wherein the extractant is selected from hydroxylated triglycerides, alkoxylated triglycerides, hydroxylated fatty acids, alkoxylated fatty acids, hydroxylated fatty alcohols, and alkoxylated fatty alcohols. 18. The method of claim 17, wherein the extractant is selected from saturated fatty acids, unsaturated fatty acids, saturated fatty alcohols, unsaturated fatty alcohols, saturated fatty amides, unsaturated fatty amides, saturated fatty esters, unsaturated fatty esters, and mixtures thereof. The method of claim 17, wherein the one or more reactants or solvents are selected from aqueous ammonium hydroxide, anhydrous ammonia, ammonium acetate, aqueous ammonia, hydrogen peroxide, toluene, glacial acetic acid, lipase, and cation exchange resins. 18. The method of claim 17, wherein the product alcohol is a C ₁ to C ₈ alkyl alcohol. 18. The oil of claim 17, wherein the oil is tallow oil, corn oil, canola oil, capric / caprylic triglycerides, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, flaxseed oil, right angle Oils, otissica oil, palm oil, peanut oil, rapeseed oil, rice oil, safflower oil, soybean oil, sunflower oil, liquid oil, jatropha oil, wheat oil, rye oil, barley oil, and vegetable oil blends A method comprising one or more oils. (a) producing the product alcohol by providing a fermentation broth comprising recombinant microorganisms capable of producing the product alcohol in a fermentation vessel;
(b) contacting the fermentation broth with an extractant to form a two-phase mixture comprising an aqueous phase and an organic phase, wherein the product alcohol and oil are partitioned into the organic phase such that the organic phase comprises the product alcohol and oil;
(c) separating the organic phase from the aqueous phase;
(d) separating the product alcohol from the organic phase;
Optionally, steps (b) and (c) occur simultaneously. The method of claim 27, wherein the extractant is selected from fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof. The method of claim 28, wherein the extractant is selected from hydroxylated triglycerides, alkoxylated triglycerides, hydroxylated fatty acids, alkoxylated fatty acids, hydroxylated fatty alcohols, and alkoxylated fatty alcohols. The method of claim 28, wherein the extractant is selected from saturated fatty acids, unsaturated fatty acids, saturated fatty alcohols, unsaturated fatty alcohols, saturated fatty amides, unsaturated fatty amides, saturated fatty esters, unsaturated fatty esters, and mixtures thereof. The method of claim 27, wherein the product alcohol is a C ₁ to C ₈ alkyl alcohol. 28. The method of claim 27,
Producing a feedstock slurry;
Separating the feedstock slurry to produce (i) an aqueous layer, (ii) an oil layer, and (iii) a solid layer; And
Supplying an aqueous layer to the fermentation vessel. 28. The method of claim 27,
At least a portion of the oil is brought into contact by contacting the oil of the oil layer with at least one substance that can be chemically converted into an extractant selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof. The method further comprises the step of switching to an extractant. The method of claim 27, wherein the partition coefficient of the extractant for the product alcohol is greater than the partition coefficient for the product alcohol of the oil of the oil layer. The method of claim 27, wherein the product alcohol is a C ₁ to C ₈ alkyl alcohol. The oil of claim 27 wherein the oil is tallow oil, corn oil, canola oil, capric / caprylic triglycerides, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, flaxseed oil, embossed Oils, otissica oil, palm oil, peanut oil, rapeseed oil, rice oil, safflower oil, soybean oil, sunflower oil, liquid oil, jatropha oil, wheat oil, rye oil, barley oil, and vegetable oil blends A method comprising one or more oils. Providing a biomass comprising oil; And
Converting at least a portion of the oil into an extractant selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty esters, triglycerides, and mixtures thereof. 38. The method of claim 37, wherein converting the oil to the extractant comprises: incubating the oil in the presence of tetrahydrofuran and lithium aluminum hydride; Incubating the oil with sodium hydroxide; Incubating the oil with sulfuric acid and methanol; Incubating the oil with anhydrous ammonia in the presence of ammonium acetate; Incubating the oil with aqueous ammonia; Contacting the oil with toluene, cation exchange resin, glacial acetic acid, lipase, and hydrogen peroxide; Incubating the oil under high temperature conditions, or incubating the oil under high pressure conditions.

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