WO2010092924A1 - Pentose-assimilating recombinant escherichia coli that produce ethanol and method for manufacturing ethanol using same - Google Patents

Abstract

Disclosed are Escherichia coli that improve or resolve the diauxy problems that were a flaw in the KO11 strain of recombinant ethanol-producing Escherichia coli, a method for manufacturing ethanol using the same, and an ethanol-containing solution produced thereby. Disclosed are recombinant Escherichia coli obtained by destroying or eliminating the ptsG gene of the KO11 strain of Escherichia coli.

Description

Pentose assimilating ethanol-producing recombinant Escherichia coli and method for producing ethanol using the same

The present invention relates to an ethanol-producing recombinant Escherichia coli, more specifically, a recombinant Escherichia coli capable of efficiently producing ethanol etc. from a saccharified liquid of biomass such as lignocellulose containing a polysaccharide that generates pentose by hydrolysis, and It relates to a method for producing ethanol and the like.

In order to prevent global warming due to an increase in the concentration of CO _{2 in} the atmosphere, there is an urgent need to develop new energy that does not rely on fossil fuels. Among such new energies, attention has been focused on bioethanol as a renewable energy, particularly bioethanol made from lignocellulose-derived saccharide, which is a so-called “carbon neutral” feedstock.

リ Lignocellulose itself cannot be used directly as food. Therefore, lignocellulose-derived carbohydrates are carbohydrates that “do not compete with food”, unlike food-derived carbohydrates such as sucrose and starch. Effective utilization of lignocellulose-derived carbohydrates is important for the future development of mankind.

However, there are also problems with carbohydrates derived from lignocellulose that are carbon neutral and do not compete with food. For example, the problem of “pentose fermentation”. The lignocellulose-derived sugar solution contains sugars derived from cellulose and hemicellulose which constitute the cell wall of plants, and unlike sugar solutions derived from sugarcane juice or starch, C5 sugars such as xylose and arabinose produced from hemicellulose. That is, it contains pentose.

The conventional general ethanol production method is S.H. Utilizes ethanol fermentation using cerevisiae . S. Although cerevisiae has high fermentative ability of C6 sugars such as glucose and fructose, that is, hexose, it hardly lacks fermentative ability of pentose. Therefore, the conventional S.I. When lignocellulose sugar solution was used in the ethanol production method using cerevisiae , hexose was converted to ethanol, but pentose such as xylose coexisting in the solution was not converted to ethanol.

In order to solve this problem, Ingram and Ota et al. At the University of Florida are able to use ethanol fermentation in Escherichia coli by giving the ability of alcohol production to E. coli that can be used regardless of whether it is hexose or pentose. We thought that hexose and pentose in lignocellulose-derived sugar solution could be converted to ethanol. Then, by introducing the Zymomonas mobilis gene involved in ethanol production into Escherichia coli B strain, an alcohol-producing recombinant Escherichia coli was successfully produced (Patent Documents 1 and 2 and Non-Patent Document 1).

This recombinant Escherichia coli with the ability to produce alcohol created by the idea of Ingram and Ota et al. Is called KO11 strain and is used worldwide as a useful microorganism capable of producing ethanol from lignocellulose sugar solution containing hexose and pentose. Has been.

However, the KO11 strain included several problems. One of them is the problem of diauxie. That is, microorganisms such as Escherichia coli have the property that when hexose and pentose coexist, hexose is used first and then pentose is used from the advantage of energy production. Due to dioxy, the use of pentose is delayed from the beginning of fermentation. Therefore, ethanol production in a mixed system of hexose and pentose has a long total fermentation time, and a big problem remains in economic efficiency.

In Escherichia coli, a phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports glucose. Glucose phosphotransferase (PtsG) is known as a membrane protein involved in this system (Non-patent Document 2).

Eiteman et al. Performed an operation to destroy the ptsG gene using normal E. coli that does not produce ethanol, and obtained recombinant E. coli that can efficiently use xylose in the presence of glucose (Non-patent Document 3).

However, the substance produced by recombinant E. coli of Eiteman et al. Is lactic acid, not ethanol. In addition, E. coli used here by Eiteman et al. Is completely phenotypically different from the KO11 strain, which disrupts genes that produce organic acids such as lactic acid and acetic acid and distributes most of the ability to produce organic acids to ethanol production. Is different. Furthermore, in this culture, anaerobic fermentation is performed from the middle.

Nichols et al. Show that Escherichia coli having the disrupted ptsG gene has the ability to ferment glucose, arabinose, and xylose alone or in a mixture to produce ethanol, and that glucose and pentose are simultaneously used by this Escherichia coli. (Non-Patent Document 4).

However, the mutant strain used here is one in which the gene of Zymomonas mobilis involved in ethanol production has been introduced by transformation with a plasmid. Is essential. This strain is cultured under absolute anaerobic conditions in which the gas phase is replaced with CO ₂ and nitrogen. These culture conditions are not suitable for mass culture on an industrial scale in terms of cost and equipment.

US Patent No. 5000000 US Pat. No. 5,424,202

Ohta et al. (1991) Appl. Environ. Microbiol. 57: 893-900 Gosset, Microbial Cell Factories 2005, 4:14 Eiteman, MA et al., “A Substrate-Selective Co-Fermentation Strategy with Escherichia coli Produces Lactate by Simultaneously Consuming Xylose and Glucose,” Biotechnology and Bioengineering, published online 29 August 29 Nichols, NN, et al., Appl. Microbiol. Biotechnol. (2001) 56: 120-125

The present invention provides Escherichia coli in which the problem of dioxy, which is a drawback of the KO11 strain which is an ethanol-producing genetically modified Escherichia coli, is improved or solved, an ethanol production method using the same, and an ethanol-containing solution produced thereby. With the goal.

The present inventors produced ethanol-producing genetically modified Escherichia coli with suppressed glucose uptake and relatively enhanced xylose utilization, and by making a difference in carbohydrate utilization in E. coli, I thought it would be possible to release it. A novel recombinant Escherichia coli (KO11ΔP strain; KO11 delta (KO11 delta)) that efficiently utilizes xylose, which is pentose in the presence of glucose, by suppressing the uptake of glucose, which is a typical hexose, in a mixed solution of hexose and pentose. delta) P strain).

Specifically, the inventors of the present invention have modified the KO11 strain (KO11ΔP strain) that has been modified so that glucose is not taken up by PtsG by destroying a gene ( ptsG ) encoding PtsG, which is a membrane protein that takes in glucose. And the utilization of carbohydrates was compared with the original strain KO11. As a result, it was found that the utilization rate of xylose in a mixed solution of hexose and pentose was increased, and the present invention was completed.

That is, according to the present invention, [1] a recombinant Escherichia coli that is a variant of Escherichia coli KO11 strain in which the ptsG gene is disrupted or deleted;
[2] The recombinant Escherichia coli according to the above [1], which is pentose assimilating under aerobic conditions;
[3] The recombinant Escherichia coli according to [1] or [2], wherein ethanol productivity and vitamin B2 productivity are high as compared to Escherichia coli KO11 strain;
[4] Recombinant Escherichia coli having accession number FERM P-21758 (FERM BP-11221);
[5] A method for producing ethanol and / or vitamin B2, comprising a step of fermenting a biomass saccharified solution using the recombinant Escherichia coli according to any one of [1] to [4];
[6] The method according to [5] above, wherein the fermentation step is performed under aerobic conditions;
[7] The method according to [5] or [6] above, wherein the biomass saccharified solution is a saccharified solution in which hexose and pentose coexist.
[8] The method according to any one of [5] to [7] above, comprising a step of saccharifying biomass before the fermentation step;
[9] The method according to any one of [5] to [8] above, comprising a solid-liquid separation step of the mash after the fermentation step, and a distillation step and / or a dehydration step of ethanol;
[10] An ethanol-containing solution produced using the method according to any one of [5] to [9],
Is provided.

The Escherichia coli of the present invention is a genetically modified Escherichia coli in which pentose utilization is relatively enhanced by suppressing glucose uptake, and the pentose is used for ethanol production.

The Escherichia coli of the present invention retains the advantages of the KO11 strain in addition to the pentose availability. For example, like the KO11 strain, the Escherichia coli of the present invention has a gene related to alcohol production in the chromosome rather than in the plasmid, so that it is not necessary to use antibiotics for maintaining the trait and stable ethanol production is possible. For this reason, there are advantages such as cost advantage during mass production. Furthermore, as with the KO11 strain, the strain of the present invention produces ethanol well under aerobic conditions and does not require anaerobic conditions, so it should be cultured under normal conditions with a general fermenter. Can do. Therefore, the Escherichia coli of the present invention has very high industrial production suitability.

According to the present invention, since the utilization of pentose in a mixed solution of hexose and pentose is improved by modifying the ptsG gene, ethanol can be produced in a shorter time than before. Therefore, it has a higher ethanol production capacity than the KO11 strain.

Furthermore, it was also found that the Escherichia coli of the present invention has all the advantages of the KO11 strain, but unexpectedly has additional advantages that the KO11 strain did not originally have. For example, the Escherichia coli of the present invention is excellent in reusability because the cells are robust and strong compared to other Escherichia coli such as the KO11 strain which is the original strain. That is, the number of times the cells used in the fermentation process are collected and used in the fermentation process of a new sugar solution can be increased. Surprisingly, it was also found that the Escherichia coli of the present invention produces a large amount of vitamin B2 in parallel with ethanol production and releases it into the culture medium. Therefore, it is also possible to collect and use vitamin B2 from the culture solution.

Due to the above-mentioned various advantages, by using the Escherichia coli of the present invention, very efficient ethanol production, in particular, more efficient ethanol production than conventional using lignocellulose-derived sugar solution becomes possible. Therefore, the size of the ethanol production plant using the lignocellulose-derived raw material can be made smaller than the conventional one, the capital investment is reduced, and the production efficiency per hour is greatly increased by shortening the fermentation time. Can be improved. Furthermore, since more vitamin B2 is produced than KO11 strain, ethanol and vitamin B2 can be produced simultaneously.

FIG. 1 is a diagram showing xylose consumption in an M9 sugar mixed medium of KO11 strain (white circle; ◯) and KO11ΔP strain (black square; ■). FIG. 2 is a diagram showing glucose consumption in the M9 sugar mixed medium of the KO11 strain (white circle; ◯) and the KO11ΔP strain (black square; ■). FIG. 3 is a diagram showing ethanol production in the M9 sugar mixed medium of the KO11 strain (white circle; ◯) and the KO11ΔP strain (black square; ■). FIG. 4 shows ethanol production in a jar fermenter (Panel (A)) in a medium containing 7% (W / V) glucose and 3% (W / V) glucose of KO11 strain and KO11ΔP strain, and the amount of residual sugar in the medium. It is a figure which shows (panel (B)). In panel (A), KO11 = white circle; ○, KO11ΔP (“ΔP” (delta P)) = black square; ■; in panel (B), glucose KO11 = black rhombus; ◆, xylose KO11 = black square; Glucose KO11ΔP (“glucose ΔP”) = white triangle; Δ, xylose KO11ΔP (“xylose ΔP”) = white circle; FIG. 5, Panels (A) and (B) show the ethanol production (panels) of the KO11 strain (white circle; ○, black circle; ●) and KO11ΔP strain (white triangle; Δ, black triangle; ▲) after repeated fermentation. It is a figure which respectively shows (A)) and a microbial cell weight (panel (B)). KO11-1 and KO11-2 represent the results of two experiments conducted independently for the KO11 strain, and ΔP-1 and ΔP-2 for the KO11ΔP strain, respectively. FIG. 5, Panels (C) and (D) are diagrams showing the remaining amounts of glucose (panel (C)) and xylose (panel (D)) of the KO11 strain and the KO11ΔP strain when repeatedly fermented, respectively. . KO11-1 and KO11-2 represent the results of two experiments conducted independently for the KO11 strain, and ΔP-1 and ΔP-2 for the KO11ΔP strain, respectively. The black bar is the result of the first day (first time), and the shaded bar is the result of the second day (second time). FIG. 6 is a graph showing vitamin B2 (riboflavin) production of the KO11 strain (black bar) and the KO11ΔP strain (shaded bar). FIG. 7 is a diagram for explaining the ptsG gene that is disrupted by homologous recombination shown in the Examples.

Recombinant Escherichia coli The recombinant Escherichia coli of the present invention is characterized in that a gene ( ptsG ) encoding PtsG that is a glucose uptake membrane protein is disrupted or deleted, is pentose- assimilating , and has ethanol-producing ability. To do.

In the context of the present invention, “disruption” of a gene means that the gene itself is present but that the protein encoded by the gene is not expressed and that the protein encoded by the gene does not function normally. Means that a functional expression product of the gene is not produced in a cell having the gene. For example, the disruption of the gene may be an amino acid modification of the coding region, may interfere with the movement of the gene product to the membrane, or may be inactivation of the promoter region.
In the present invention, a “deficiency” of a gene refers to a state in which the gene itself does not exist.

The recombinant Escherichia coli of the present invention is transformed into, for example, a commonly available Escherichia coli so as to have a genotype similar to that of the KO11 strain at least with respect to sugar metabolism and organic acid metabolism. It can be obtained by destroying or deleting a gene ( ptsG ) encoding a PtsG protein.

The KO11 strain is already commercially available as the standard strain ATCC 55124 strain and can be easily obtained. Use of this strain as the original strain is advantageous because the Escherichia coli of the present invention can be easily produced by disrupting or deleting the ptsG gene.

The amino acid and base sequence of the ptsG gene of Escherichia coli is known, and examples thereof include base numbers 1159446 to 1160879 (SEQ ID NO: 1 in the sequence listing) of GenBank AC — 000091 (Escherichia coli K-12 strain substr. W3110).

The ptsG gene is disrupted or deleted by methods such as homologous recombination and the transposon method (see, for example, Kimata et al., Proc. Natl. Acad. Sci. USA, vol. 94, pp. 12914-12919, Nov. 1997). It can be carried out.

A method for confirming whether the ptsG gene is disrupted or deleted in a recombinant is known and can be arbitrarily selected by those skilled in the art. For example, the genomic DNA of the recombinant as a template, PCR was performed using primers specific for the ptsG gene, genomic PCR method to confirm that no product is amplified product of ptsG gene, DIG (Digoxigenin-deoxyuridine monophosphate) For example, a method of performing Southern blotting using a probe specific to the ptsG gene labeled with, for example. It can also be confirmed that no PtsG protein is detected in the recombinant.

The recombinant E. coli of the present invention can be cultured well under the same conditions as general E. coli. Specific examples of the culture conditions include the same conditions as in the fermentation step in ethanol production described below.

Ethanol production method The ethanol production method of the present invention is characterized by using the above-described recombinant Escherichia coli of the present invention as a fermentation microorganism in the fermentation step. The fermentation process may be any of a batch system, a continuous system, or a modified system thereof. Therefore, in the framework of a conventionally known ethanol production process, the method using the recombinant E. coli of the present invention in the fermentation step is the method of the present invention.

The raw material for producing ethanol using the recombinant Escherichia coli of the present invention may be a biological raw material (biomass), and may be plant or animal. These biomass contain carbohydrates. Carbohydrates are generally classified into monosaccharides, oligosaccharides and polysaccharides, but the raw materials used are pentose or hexose and biomass containing monosaccharides including pentose, or pentose or hexose by hydrolysis and Any of the oligosaccharide which produces | generates the monosaccharide containing a pentose, or the biomass containing a polysaccharide may be sufficient. Furthermore, the extract and / or decomposition product from these biomass, and an extraction and / or decomposition residue can also be used as a raw material.

The method of the present invention is characterized in that pentose can be efficiently converted to ethanol even in the presence of hexose. When a raw material such as a saccharified liquid of lignocellulose containing pentose is used, the method of the present invention is used. The advantage is particularly noticeable.

Specific examples of biomass that can be used as a raw material include terrestrial plant biomass, aqueous plant biomass, and animal biomass. Terrestrial plant biomass includes, for example, woody lignocelluloses such as hardwoods and conifers; herbaceous lignocelluloses (including palms and bamboos) such as switchgrass and cedar; cereals such as corn and rice, cassava, potatoes, etc. Examples include starch raw materials containing potatoes, and sugar raw materials containing sugarcane, sugar beet, molasses, etc., and biomass residues such as rice straw and bagasse after the production of these starches and sugars are also included. Also included are wood pulp, waste paper, building waste, municipal waste, pruning material, food waste and the like.
Aquatic plant biomass includes aquatic plants such as water hyacinth; seaweeds such as kombu and seaweed. Also included are chlorella and phytoplankton, which are plant microorganisms.
Animal biomass includes shrimp and crab shells and whey.

The method for producing ethanol from biomass varies slightly depending on the characteristics of the raw materials used, but it can basically be composed of a pretreatment process, a saccharification process, and a fermentation process, and in general, a concentration (distillation) process and a dehydration process. Etc. Steps other than the fermentation step can be omitted in some cases.

The method of the present invention requires a fermentation process, but other processes may or may not be present as necessary. For example, the method of the present invention can include a pretreatment step suitable for each raw material as required. In general, biomass is stored after being collected from the production area and subjected to a treatment such as drying in order to prevent alteration and decomposition until it is used. In most cases, a biomass pulverization step is performed after collection or just before ethanol production. As the grinding method, a dry grinding method or a wet grinding method is adopted as necessary.

When using biomass other than saccharide raw materials and monosaccharide raw materials as raw materials, a process of saccharifying biomass is required. In general, the saccharification step is carried out by acid treatment, alkali treatment, neutral salt treatment, hydrothermal treatment, enzyme treatment or the like alone or in combination.

For example, when starchy raw materials are used, starch can be liquefied and saccharified using amylase and pullulanase.

As pretreatment of lignocellulosic biomass, concentrated sulfuric acid treatment (Peoria method, Scholtani-Leone method, Hokkaido method, etc.), dilute sulfuric acid treatment (Scholer method, Madison method, improved Madison method, Soviet method, two-stage decomposition method, etc.) , Acid decomposition such as sulfurous acid treatment, concentrated hydrochloric acid treatment (Pergius-Ryinau method, new Reinau method, etc.), hydrochloric acid gas treatment (Brodle method, Dalbuofen method, Elan method, Noguchi Laboratory method, etc.), and hydrolysis by enzymes Etc. Cellulases, hemicellulases, and the like are used as enzymes for pretreatment of lignocellulosic biomass. After completion of saccharification, the used chemicals and insoluble biomass components are separated as necessary. By these treatments, a saccharified solution containing monosaccharide, oligosaccharide, solubilized lignin and the like is obtained.

Details of these pretreatment methods and saccharification methods are described in various documents, and those skilled in the art can appropriately design and select according to the raw materials used.

The method of the present invention requires a fermentation process. In the fermentation process, theoretically, 100 g of saccharide becomes about 50 g of ethanol and about 50 g of CO ₂ (in this case, the fermentation rate is 100%. Here, the saccharide refers to pentose and hexose). The material used for the fermentation process is the above-described biomass itself (a saccharide raw material, a monosaccharide raw material, etc.) or a saccharified liquid obtained through a pretreatment step and / or a saccharification step as necessary. The content of pentose and / or hexose in these materials reaches several percent (W / V) to 80% (W / V) or more, but the material used in the present invention is pentose and / or hexose content. A relatively high value is desirable. More preferably, the content of pentose and / or hexose is 10% (W / V) or more, and most preferably 30% (W / V) or more. If the initial content is less than 10% (W / V), it can be concentrated and used as 10% (W / V) or more.

The fermentation process can be suitably performed at pH 4 or higher, particularly at pH 5.5 or higher, and fermentation proceeds well at least up to pH 8. Considering contamination prevention, fermentation rate, and a small change in conditions with other processes, it is advantageous to carry out at pH 5-7, especially at pH 5.5-6.5.

Moreover, temperature conditions should just be 20 degreeC or more, and a fermentation process can be performed suitably to 40 degreeC. Considering the fermentation rate and the small change in conditions with other processes, it is advantageous to carry out the reaction at 30 ° C. to 40 ° C., particularly 35 ° C. to 40 ° C.

Therefore, the optimum pH and temperature conditions for the fermentation process are pH 5.5 to 6.5 and 35 to 40 ° C. The reaction time is generally 6 to 72 hours, but can be appropriately selected according to the characteristics of the biomass or saccharified solution used. According to the method of the present invention, a high fermentation rate of generally 85 to 98% can be realized.

After the fermentation process, the fermented liquor contains the produced ethanol. Ethanol produced in the fermentation process is generally recovered and purified from the fermentation broth through a distillation process and a dehydration process. The implementation conditions of these steps are well known. From the fermentation liquid containing ethanol to the ethanol purified to high purity obtained through the subsequent additional steps, such as a distillation step and a dehydration step, are collectively referred to as an ethanol-containing solution for convenience.

In the method of the present invention, it is preferable to perform a mash solid-liquid separation step after the fermentation step. Moromi's solid-liquid separation step is known, and examples of the method include filtration (filter cloth, screen, membrane, etc.), centrifugation, decantation and the like.
Such various steps of ethanol production are described in, for example, Bioresource Technology 98 (2007) 241-2457.

Here, “purified ethanol” refers to “ethanol” by the Japanese Pharmacopoeia, that is, an aqueous solution containing 95.1 to 95.6% (V / V) ethanol at 15 ° C. This is industrially useful as a raw material for the production of ethyl acetate and the like. “Anhydrous ethanol” according to the Japanese Pharmacopoeia is an aqueous solution containing 99.5% (V / V) or more of ethanol at 15 ° C., and is industrially used as a fuel.

Ethanol obtained by the method of the present invention can be used as fuel (petroleum alternative energy, gasoline additive, etc.), industrial raw material (chemical raw material), food additive, etc. Can also be used in the manufacture of pharmaceuticals and hygiene products.

Recovery of vitamin B2 By carrying out the ethanol production method of the present invention, a large amount of vitamin B2 (riboflavin) is simultaneously produced in the fermentation broth. Vitamin B2 produced from the KO11ΔP strain accumulates even when dissolved in the culture solution, but accumulates as crystals in the culture solution when the production amount increases. The culture solution containing vitamin B2 derived from the KO11ΔP strain may be used as a pharmaceutical raw material, a food additive, or a feed additive as vitamin B2 crystals, or can be used as it is for a feed or the like after the cells are completely sterilized.

Vitamin B2 accumulated as crystals in the culture solution can be recovered and purified by a general purification method. For example, the culture solution is heat-treated (60 ° C., 30 minutes) to sterilize the KO11ΔP strain, and then the crystal fraction is centrifuged to collect the precipitate. This precipitate is further acid-treated (hydrochloric acid or sulfuric acid about 2%) for DNA degradation (depurination) to obtain about 96% vitamin B2. It can be further crystallized with an acid solvent to obtain a product with a content of about 98%.

1. Preparation of KO11ΔP strain by disruption of ptsG on the chromosome of KO11 strain (1) Preparation of homologous recombination fragment The homologous recombination fragment is FRT of “Quick & Easy E. coli Gene Deletion kit” (trade name, Gene Bridges GmbH). A PGK-gb2-neo-FRT cassette was used as a template, and a homologous sequence (50base) of the target gene ptsG gene (SEQ ID NO: 1) was added to both ends of this cassette by PCR. The homologous sequence (50base) was added by the three-step PCR method shown in (a) to (c). Table 1 lists the primer sequences used.

The following were used.
・ 10x KOD-plus-buffer (trade name "Buffer for restriction enzyme / modification enzyme reaction buffer KOD-Plus- (10x)", Toyobo)
・ MgSO ₄ (25 mM; Toyobo)
・ DNTPs (each 10 mM; Toyobo)
・ KOD-plus-DNA polymerase (trade name “KOD-Plus-”, 1 unit / μL; Toyobo)
FRT-PGK-gb2-neo-FRT PCR template (trade name “Quick & Easy E. coli Gene Deletion kit”, Gene Bridges GmbH)
-Forward primer 1st time: ptsG1 (P1)
Second time: ptsG2 (P3)
Third time: ptsG3 (P5)
・ Reverse primer 1st time: ptsG1 (P2)
Second time: ptsG2 (P4)
Third time: ptsG3 (P6)
6 × Loading Dye: 25 mg (0.25%) bromophenol blue (Wako Pure Chemicals), 25 mg (0.25%) xylene cyanol FF (Wako Pure Chemicals), 3 mL (3%) glycerol and 100 μL After mixing 0.5M EDTA (Wako Pure Chemical Industries, Ltd.) and pH 8.0 (final 5 mM), the total volume was made up to 10 mL with distilled water.
・ Thermal cycler (Takara Bio Inc.)

(I) Addition of homologous sequence to FRT-PGK-gb2-neo-FRT cassette (first time)
In a 200 μL PCR tube, add 31 μL Milli Q water, 5 μL 10 × KOD-plus-buffer, 2 μL MgSO ₄ , 5 μL dNTPs, 2.5 μL primers [ptsG1 (P1) and ptsG1 (P2)], respectively 1.0 μL (50 ng) of FRT-PGK-gb2-neo-FRT PCR template was added as a template, and finally 0.5 μL (0.25 U) of KOD-plus-DNA polymerase was added to make a total volume of 50 μL. This reaction solution was set on a thermal cycler. After warming at 94 ° C. for 2 minutes to activate KOD-plus-DNA polymerase, 30 cycles of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 2 minutes were performed.

1 μL of 6 × Loading Dye was mixed with this PCR reaction solution (5 μL). Using 6 μL of this solution, electrophoresis was performed on a 1% TAE agarose gel at 60 V for 60 minutes. At this time, a 1 kbp marker was simultaneously run in another lane as an index for confirming the position of the amplified fragment.

After electrophoresis, the gel was immersed in a cyber-safe staining solution (trade name “Cyber-safe DNA gel staining reagent (1 × TAE)”, Invitrogen), and stained for 20 minutes while gently stirring. Ultraviolet light was irradiated, and the band of the amplified fragment was confirmed at a position near 1662 bp. The remaining 45 μL of the PCR reaction solution was subjected to CIA treatment, ethanol precipitation, and rinsing. Thereafter, the pellets dried under reduced pressure were dissolved in 20 μL of Milli Q water.

(Ii) Addition of homologous sequence to FRT-PGK-gb2-neo-FRT cassette (second time)
The same operation as in (i) was repeated using the PCR amplified fragment obtained in (i) above as a template and ptsG2 (P3) and ptsG2 (P4) as primers.
(Iii) Addition of homologous sequence to FRT-PGK-gb2-neo-FRT cassette (third time)
The same operation as in (i) was repeated using the PCR amplified fragment obtained in (ii) as a template and ptsG3 (P5) and ptsG3 (P6) as primers.

(2) Gel extraction of homologous recombination fragments The following were used.
50 × TAE: 2.0M Tris-HCl, 20M acetic acid (Wako Pure Chemical Industries), 0.5M EDTA
1 × TAE: 50 × TAE was diluted 50 times with distilled water.
• 1.0% agarose gel: Agarose S (Nippon Gene) was dissolved in 1 × TAE buffer so as to be 1.0% (W / V).
・ Cyber-safe stain (supra, Invitrogen)
・ QIAquick Gel Extraction Kit (trade name, Qiagen)
-PCR amplified fragment obtained in (1)-(iii) above

The PCR-amplified fragment (50 μL) obtained in (1)-(iii) above was mixed with 10 μL of 6 × Loading Dye, and this solution (60 μL) was mixed on a 1% TAE agarose gel at 60 V for 60 minutes. Electrophoresis was performed. At this time, a 1 kbp marker was simultaneously run in another lane as an index for confirming the position of the amplified fragment to be extracted. After the electrophoresis, the gel was immersed in a cyber-safe staining solution and stained for 20 minutes with gentle stirring. The band of the amplified fragment was confirmed at a position near 1747 bp by irradiating with ultraviolet rays, and the gel containing the band was recovered. The gel containing the recovered amplified fragment was extracted from the gel using “QIAquick Gel Extraction Kit”. Hereinafter, the extracted fragment is referred to as “ ptsG homologous recombination fragment”.

(3) KO11 strain homologous recombination wherein to target gene on the chromosome using the ptsG homologous recombination fragment was extracted with (2), was disruption of ptsG gene. In order to increase the efficiency of homologous recombination, Red / ET-derived recombination-inducing protein is expressed in the KO11 strain and is homologous using the fact that it specifically reacts with the FRT-PGK-gb2-neo-FRT cassette. Recombination was induced.

(I) Introduction of Red / ET into KO11 strain The following were used.
LB / Cm liquid medium Competent cells: E. coli KO11 strain • pRedET (amp) (20 μg / μL) (Gene Bridges GmbH)
・ Electroporation cuvette (0.2cm; BIO-RAD)
・ Gene Pulsar II (BIO-RAD)
・ Pulse controller PLUS (BIO-RAD)
・ LB liquid medium ・ LB / Amp, Cm plate medium

To the LB liquid medium, 1.5% (W / V) agar was added and sterilized by autoclave. After cooling to about 60 ° C., ampicillin (Wako Pure Chemical Industries) was added to a final concentration of 100 μg / mL and chloramphenicol to a final concentration of 600 μg / mL, and dispensed into a petri dish.

The KO11 strain was inoculated into an LB / Cm liquid medium, and cultured with shaking at 37 ° C. and 60 rpm until the turbidity reached OD 660≈0.6. After transferring 1.4 mL of this culture solution to a tube and centrifuging at 6,000 rpm for 5 minutes, the supernatant was discarded, and the remaining pellet was resuspended in 1 mL of sterile water.

The centrifugation and resuspension operations were performed a total of three times to wash the cells, and finally resuspended in 40 μL of sterilized water. To this, 1 μL of pRed / ET (amp) was added and mixed, and transferred to an electroporation cuvette. After electroporation with Gene Pulser II and Pulse Controller PLUS at a voltage of 2.5 kV, a resistance of 200 Ω, and a capacitance of 25 μF, immediately add 400 μL of LB liquid medium and transfer to a 1.5 mL tube. The culture was shaken at 150 ° C. for 1 hour (150 rpm). 150 μL of the culture solution was applied to an LB / Amp, Cm plate medium and cultured at 30 ° C. for 16 hours. The colony formed on the plate was designated as “KO11 (Red / ET)”, and the plate was stored at 4 ° C.

(Ii) Introduction of homologous recombination fragment into KO11 strain and induction of homologous recombination The following were used.
LB / Cm liquid medium 10% (W / V) L-arabinose: Distilled water was added so that L-arabinose (Wako Pure Chemical Industries) was 10% (W / V).
-PtsG homologous recombination fragment-Competent cell: KO11 (Red / ET)
・ LB / kan, Cm plate medium

To the LB liquid medium, 1.5% (W / V) agar was added and sterilized by autoclave. After cooling to about 60 ° C., kanamycin sulfate (Wako Pure Chemical Industries) was added to a final concentration of 30 μg / mL and chloramphenicol to a final concentration of 600 μg / mL, and dispensed into a petri dish.

KO11 (Red / ET) was inoculated into an LB / Cm liquid medium, and cultured under shaking at 30 ° C. and 150 rpm until the turbidity reached OD660≈0.3.

To induce recombination with the Red / ET-derived recombinant protein, 50 μL of 10% (W / V) L-arabinose was inoculated into each test tube. After culturing at 37 ° C. and 60 rpm until the turbidity reaches OD660≈0.6 , 1 μL (100 to 100 μg ) of ptsG homologous recombination fragment was obtained by the electroporation method described in (3)-(i) above. 400 ng) was introduced, followed by shaking culture (60 rpm) at 37 ° C. for 3 hours. 150 μL of the culture solution was applied to LB / kan, Cm plate medium and cultured at 37 ° C. for 16 hours. Colonies generated on the plate were used as transformants, and the plate was stored at 4 ° C.

By this homologous recombination, the fragment (1068 bp) shown in lowercase letters with an underline in FIG. 7 is destroyed in the ptsG gene. In FIG. 7, the uppercase letters indicate the portions corresponding to the sequences of the primers P1, P3, and P5, and the underlined uppercase letters indicate the portions that correspond to the sequences of the primers P2, P4, and P6.

(Iii) Confirmation of homologous recombination fragment introduction by colony direct PCR The following were used. In order to confirm the destruction of the ptsG gene, a primer that anneals to the outside of the homologous recombination fragment was used.
・ 10 × Reaction buffer (BIOLINE)
・ 25mM dNTPs solution (BIOLINE)
・ 50 mM MgCl ₂ solution (BIOLINE)
・ HibriPol ™ DNA polymerase (BIOLINE)
-Forward primer: ptsG4 (P7)
・ Reverse primer: ptsG4 (P8)
1.0% (W / V) agarose gel Transformant obtained in (3)-(ii) above

To a 200 μL PCR tube, add 2 μL of 10 × Reaction buffer, 2 μL of 25 mM dNTPs solution, 1 μL of 50 mM MgCl ₂ solution, 1 μL of each forward and reverse primer, 12.9 μL of Milli Q water, and finally 0.1 μL of HibriPol ™. DNA polymerase was added to make a total volume of 20 μL.

A white colony of the transformant obtained in the above (3)-(ii) was picked with a sterilized toothpick and added as a template to the reaction solution to prepare a PCR reaction solution. After setting in a thermal cycler and heating at 94 ° C. for 5 minutes to activate the HibriPol ™ DNA polymerase, denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, extension reaction at 72 ° C. for 30 seconds, These three stages were repeated 32 cycles. After completion of PCR, electrophoresis was carried out on a 1.0% (W / V) agarose gel at 100 V for 30 minutes.
A clone that was confirmed to be amplified in the vicinity of 1970 bp in the ptsG- disrupted strain was designated as “KO11ΔP” strain, which was glycerol stock (−84 ° C.).

The KO11ΔP strain was deposited on January 26, 2009 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, Tsukuba City, Ibaraki Prefecture, Central 6th), with accession number FERM P-21758 was given. In addition, on January 4, 2010, it was transferred to FERM BP-11221 and transferred to an international deposit under the “Budapest Convention on the International Approval of Deposits of Microorganisms in Patent Procedures”.

2. Evaluation of Sugar Consumption and Ethanol Production in Recombinant Escherichia coli KO11ΔP Saccharified liquid of lignocellulosic biomass is rich in glucose (hexose) and xylose (pentose). For the purpose of evaluating the sugar consumption of the KO11 strain and KO11ΔP strain with respect to the lignocellulosic biomass saccharified solution, a fermentation test was conducted in an M9 medium (M9 mixed sugar medium) containing glucose and xylose.

(1) Medium preparation The following were used.
・ Glucose (Wako Pure Chemical Industries)
・ Xylose (Wako Pure Chemical Industries)
・ 20 × M9 salts: NH ₄ Cl (Wako Pure Chemical) 20 g / L, KH ₂ PO ₄ (Wako Pure Chemical) 60 g / L, NaHPO ₄ (Wako Pure Chemical) 120 g / L, NaCl (Wako Pure Chemical) 10 g / L L was dissolved in distilled water so as to be L, and autoclaved (120 ° C., 15 minutes).
・ Casamino acid (Nippon Pharmaceutical)
・ MgSO ₄ (Wako Pure Chemical Industries)
1M phosphate buffer solution: 1M Na ₂ HPO ₄ · 12H ₂ O (Wako Pure Chemical Industries) solution, 1M KH ₂ PO ₄ solution added at 30 ° C. until pH 6.7, and autoclaved Using.

The M9 mixed sugar medium was prepared as follows. A 100 mL Erlenmeyer flask to which 58.2 mL of an aqueous solution containing 4.8 g of glucose and 3.2 g of xylose was added was autoclaved. To this 100 mL Erlenmeyer flask, add ₄ mL of 20 × M9 salts, 0.8 mL of 100 mM MgSO ₄ , 16 mL of 1M phosphate buffer, and 1 mL of 20% (W / V) casamino acid to make a total volume of 80 mL. Medium with concentrations of 6% (W / V) and 4% (W / V), respectively, was designated as M9 mixed sugar medium.

(2) Fermentation test The following were used.
KO11 strain, KO11ΔP strain: Glycerol stock (30% (W / V)) was used.
LB liquid medium: 1% (W / V) NaCl, 1% (W / V) Bacto ™ Peptone (Nippon Becton Dickinson), 0.5% (W / V) East Extract (Nippon Pharmaceutical), It was dissolved in distilled water so as to be 5% agar (Wako Pure Chemical Industries), and sterilized by autoclave.
LB / Cm plate: 1.5% (W / V) agar was added to the LB liquid medium, and autoclaved. After cooling to about 60 ° C., chloramphenicol (Wako Pure Chemical Industries) was added to a final concentration of 600 μM and dispensed into a petri dish.
LB / Cm liquid medium: After autoclaving 5 mL of LB liquid medium, chloramphenicol was added to a final concentration of 600 μg / mL.
・ M9 mixed sugar medium ・ Concentrated sulfuric acid (Wako Pure Chemical Industries)
・ Incubator (Tokyo Rika Kikai Co., Ltd.)
-Toothpick: Placed in a 100 mL beaker and sterilized by autoclave.

In a clean bench, the KO11 strain and the KO11ΔP strain were streaked using a toothpick on an LB / Cm plate (LB / Cm and Km plates were used in the case of the KO11ΔP strain), and then statically cultured at 37 ° C. for 16 hours. Thereafter, a single colony was inoculated into 5 mL of LB / Cm liquid medium in a clean bench, and cultured with shaking at 37 ° C. and 60 rpm for 16 hours. Furthermore, 100 μL of the culture solution was inoculated into 100 mL of LB liquid medium in a clean bench, and cultured with shaking at 30 ° C. and 150 rpm for 16 hours. This culture solution was used as a preculture solution.

40 mL of the preculture was dispensed into a 50 mL sterilized tube and centrifuged at 25 ° C. and 6,000′G for 10 minutes. The supernatant was discarded by decantation, and the wet weight of the cells was measured with an electronic balance. After the measurement, M9 mixed sugar medium was inoculated so as to have a dry weight of 1 g / L, and a fermentation stopper was applied. Further, about 2 mL of concentrated sulfuric acid was added to the fermentation stopper using a 10 mL measuring pipette. In this state, a fermentation test was conducted for 8 days under the conditions of 30 ° C. and 150 rpm using an incubator.

(3) Medium sugar concentration analysis by HPLC The following were used.
・ Glucose ・ Xylose ・ Acetonitrile (Wako Pure Chemical Industries)
・ HPLC equipment Degasser DGU-12 (SHIMADZU)
Column oven CTO-10ASvp (SHIMADZU)
Detector RID-10A (SHIMADZU)
Chromatopack C-R6A (SHIMADZU)
System controller SCL-10Avp (SHIMADZU)
Autosampler SIL-20A (SHIMADZU)
Dual pump LC-10AD (SHIMADZU)
Analysis conditions Column Wakosil 5NH2 Diameter 4.0 mm, Length 150 mm (Wakopak (registered trademark), Wako Pure Chemical Industries)
Solvent acetonitrile: water = 80: 20 (v / v)
Column temperature 40 ° C
Flow rate 1mL / min

A sample for liquid chromatography was prepared by sterilizing 600 μL of the culture supernatant collected by sampling with a microsyringe. In addition, glucose solutions and xylose solutions having concentrations of 0, 2, 4, 6, 8, and 10% (W / V) were used as standards.

Using an autosampler SIL-20A, 10 μL of each sample was injected into the dual pump LC-10AD, and the sugar concentration in the culture supernatant was calculated from the ratio of peak areas detected by Chromatopack CR-6A.

(4) Analysis of ethanol concentration by GC The following were used.
・ Isopropyl alcohol (Wako Pure Chemical Industries)
・ Ethanol (Wako Pure Chemical Industries)
・ GC system Gas chromatograph GC-2014 (SHIMADZU)
Chromatopack C-R6A
Detector FID-2014 (SHIMADZU)
・ Analysis conditions Column temperature 150 ℃
Detector temperature 250 ° C
Vaporization chamber temperature 250 ℃
Column Liquid phase 5% Thermon 1000 (trade name; Shinwa Kako Co., Ltd.)
Carrier Sunpak-A Diameter 3.2mm, Length 2.1m (Trade name; Shinwa Kako Co., Ltd.)
Flow rate 60mL / min
Carrier gas N ₂

300 μL of the culture supernatant collected by sampling and an equal amount of 5% (v / v) isopropyl alcohol (internal standard substance) were mixed to obtain a sample for gas chromatography. Further, 5% (v / v) isopropyl alcohol and 0, 2, 4, 6, 8, 10% (v / v) ethanol solutions of equal concentration were mixed in equal amounts to obtain a standard.

5 μL of each sample was injected into the gas chromatograph GC-2014, and the ethanol concentration in the culture supernatant was calculated from the ratio of the peak areas detected by Chromatopack CR-6A.

The results are shown in FIGS.
FIG. 1 shows the change in xylose concentration in the culture of the KO11 strain and the KO11ΔP strain in an M9 sugar mixed medium. In the presence of glucose and xylose, the KO11 strain (white circle; ◯) hardly used xylose, whereas the KO11ΔP strain (black square; ■) was able to use xylose.
Comparing the consumption of xylose on the third day of culture, KO11 strain consumed 16% of the initial xylose concentration, whereas KO11ΔP strain consumed 55%. That is, it was found that the xylose consumption of the KO11ΔP strain is three times or more that of the KO11 strain, and the KO11ΔP strain suppresses glucose consumption and efficiently consumes xylose in the presence of glucose.

FIG. 2 shows the change in glucose concentration in the culture of the KO11 strain and the KO11ΔP strain in an M9 sugar mixed medium. On the third day of culture, KO11 strain (white circle; ○) consumed 97% of the initial glucose concentration, while KO11ΔP strain (black square; ■) showed 43% consumption, destroying the ptsG gene. It was found that glucose consumption was suppressed.

FIG. 3 shows the change in ethanol concentration in the culture of the KO11 strain and KO11ΔP strain in the M9 sugar mixed medium. It was found that the KO11ΔP strain (black square; ■) produced ethanol with a production rate of 0.8%, although the ethanol production rate was slightly inferior to that of the KO11 strain (white circle; ○).

3. Ethanol fermentation test with jar fermenter Fermentation test with jar fermenter was performed using Escherichia coli KO11 strain and KO11ΔP strain.

The following were used.
YPD medium (Difco, Lot No. 7242780)
・ Glucose (Wako Pure Chemical Industries, special grade, Lot No. ALP4694)
・ Xylose (Wako Pure Chemical Industries, special grade, Lot No. PEH5709)
・ 99.5% ethanol (Kanto Chemical Co., Ltd., special grade, Lot No. 002XI222)
・ Rotary shake incubator (Takasugi Mfg. Co., Ltd., model TS-RS12L (24))
・ Rotary shake incubator (Takasugi Seisakusho, Model TS-RS12L)
・ High-speed cooling centrifuge (Model Kubota, Model 6500)
・ Centrifuge (TOMY model MX-305)
・ Jar Fermenter (Model Takasugi Seisakusho, Model TS-M5L)

(1) Preparation of bacterial cells For cultivation, YPD medium (manufactured by Difco, Lot No. 7242780; containing 3% (W / V) glucose) and xylose (manufactured by Wako Pure Chemical Industries, Ltd., special grade, Lot No. YPD medium containing 3% glucose (W / V) and 3% xylose (W / V) prepared by adding PEH5709) to 3% (W / V) was used.

In a 500 mL Erlenmeyer flask, 100 mL of the aforementioned YPD medium containing 3% glucose (W / V) and 3% xylose (W / V) was dispensed, and one colony of each strain was aseptically inoculated from the agar medium. An aluminum cap was used for the lid of the Erlenmeyer flask. Using a rotary shaking incubator (Model TS-RS12L (24), manufactured by Takasugi Seisakusho Co., Ltd.), the cells were cultured at 37 ° C. and 120 rpm for 28 hours.

The culture solution was centrifuged at 5,000 rpm for 5 minutes in a high-speed cooling centrifuge (model 6500, manufactured by Kubota Corporation) to separate the cells and the medium. The obtained cell pellet was washed with physiological saline and used for a fermentation test.

(2) Jar culture fermentation test Glucose 7% (W / V) (Wako Pure Chemical Industries, Special grade, Lot No. ALP4694), Xylose 3% (W / V) (Wako Pure Chemical) Glucose and xylose were dissolved in distilled water so that it would be made by Kogyo Co., Ltd., special grade, Lot No. PEH5709), and 1000 mL of this medium was added to a jar fermenter-attached glass fermenter (Takasugi Seisakusho, Model TS-101) Then, autoclaved at 121 ° C. for 15 minutes. After completion of the autoclave, 5 g of YPD medium (Difco, Lot No. 7242780; containing 3% (W / V) glucose) was added as a nitrogen source.

850 mg of the bacterial cell pellet obtained in the pre-culture was collected by wet weight, and the whole amount was inoculated into the medium in the jar fermenter. Thereafter, the cells were cultured in an aerobic state at 37 ° C. and 180 rpm, and the pH was automatically controlled in the pH range of 5.5 to 6.0 with NaOH. The cells were continuously cultured for 134 hours from the start of the culture. Sampling was performed at an appropriate time. The sampled culture solution was centrifuged at 8,000 rpm for 5 minutes, and the ethanol concentration and the residual sugar (glucose and xylose) concentrations that were not used were measured by the following method.

For the measurement of ethanol, the sample is diluted 1 to 20 times so that the ethanol concentration in the sample is within the range of 0.2 to 1.0% (V / V), and the gas chromatograph for ethanol analysis (GL Sciences) (The column is “Porapak Type R” (trade name; manufactured by Waters) column, the carrier gas is nitrogen, and the flow rate is 60 mL / min). Separately, ethanol (manufactured by Kanto Chemical Co., Ltd., special grade, Lot No. 002XI222) was separated under the same conditions, and conditions were set so that the measurement area and the weight of ethanol were standard curves with excellent linearity. Isopropanol was used as an internal standard, and a conversion coefficient was obtained from a linear regression analysis of ethanol with respect to the internal standard.

For the measurement of sugar concentration, the sample was diluted 10 to 100 times so that the sugar concentration of the sample was in the range of 10 to 300 ppm, and subjected to separation and quantification using an ion column chromatograph for sugar analysis (DIONEX). The column used was PA-1 (trade name; manufactured by DIONEX), and elution was performed with water alone at a rate of 1 mL / min. Separately, 5 types of monosaccharides (glucose, mannose, galactose, xylose, arabinose) are separated under the same conditions for quantification of eluted sugars, and a standard curve with excellent linearity in the measurement area and the weight of each sugar is obtained. A condition was set. Deoxyglucose was used as an internal standard, and a conversion coefficient was obtained from a linear regression analysis of each sugar relative to the internal standard. The content of monosaccharide contained in the sample was calculated from the conversion factor of each obtained sugar and the measured area.

The results are shown in FIG. As can be seen from the figure, the KO11ΔP strain (labeled “ΔP”; black square; ■) has a final ethanol concentration (panel (A)) of 4.59% even under aerobic conditions. Residual sugar at the time (panel (B)) is 1.02% for glucose (displayed as “glucose ΔP”; white triangle; Δ), and 0.59% for xylose (displayed as “xylose ΔP”; white circle; ○) there were. On the other hand, KO11 strain (white circle; ○) has a final ethanol concentration (panel (A)) of 3.77%, and residual sugar (panel (B)) at this time is indicated by glucose (“glucose KO11”; black) The diamond shape (♦) was 0.01%, and the xylose (labeled “xylose KO11”; black square; ■) was 2.21%. Therefore, it can be seen that the final ethanol production is higher in the KO11ΔP strain than in the KO11 strain.

4). Evaluation of cell robustness of recombinant Escherichia coli KO11ΔP strain Using the Escherichia coli KO11ΔP strain and KO11 strain, the robustness of the cells of each strain was compared.

(1) Preparation of bacterial cells For cultivation, YPD medium (manufactured by Difco, Lot No. 7242780; containing 3% (W / V) glucose) and xylose (manufactured by Wako Pure Chemical Industries, Ltd., special grade, Lot No. PEH5709) YPD medium containing 3% glucose (W / V) and 3% xylose (W / V) prepared by adding 3% (W / V) was used.

In a 500 mL Erlenmeyer flask, 100 mL of the aforementioned YPD medium containing 3% glucose (W / V) and 3% xylose (W / V) was dispensed, and one colony was aseptically inoculated from the agar medium. An aluminum cap was used for the lid of the Erlenmeyer flask. Using a rotary shake incubator (Model TS-RS12L (24), manufactured by Takasugi Seisakusho Co., Ltd.), the cells were cultured at 37 ° C. and 120 rpm for 20 hours.

The culture solution was centrifuged at 5,000 rpm for 5 minutes in a high-speed cooling centrifuge (model 6500, manufactured by Kubota Corporation) to separate the cells and the medium. The obtained cell pellet was washed with physiological saline and used for a fermentation test.

(2) Repeated fermentation test As substrate for fermentation test, glucose 2% (W / V) (manufactured by Wako Pure Chemical Industries, special grade, Lot No. ALP4694), xylose 2% (W / V) (Wako Pure Chemical Industries) Glucose and xylose were dissolved in distilled water so as to be a special grade, Lot No. PEH5709, and a mixed sugar solution filtered through a membrane having a pore size of 0.2 μm (NALGENE bottle top filter filter) was used.

20 mL of glucose 2% (W / V) / xylose 2% (W / V) mixed sugar solution is placed in a 50 mL polypropylene centrifuge tube (CORNING), and 1.1 g wet weight (in terms of dry cell weight) 1% (W / V)) of the aforementioned microbial cells was dispersed in the sugar solution. Using a rotary shaking incubator (Model TS-RS12L, manufactured by Takasugi Seisakusho Co., Ltd.), the culture was performed at 37 ° C. and 120 rpm for 24 hours as one cycle.

After completion of one cycle, the supernatant and the cells were separated by centrifugation at 5,000 rpm for 5 minutes using a centrifuge (Model MX-305, manufactured by TOMY). The cells were centrifuged again at 5,000 rpm for 1 minute to completely remove the liquid, and then the wet cell weight was measured. The cells were again dispersed in 20 mL of a new mixed sugar solution, and the next cycle of fermentation was performed. This was repeated a total of 4 times.

The supernatant collected after each cycle was further centrifuged at 8,000 rpm for 5 minutes, and the concentrations of ethanol, glucose and xylose were measured.

For the measurement of ethanol, the sample is diluted 1 to 20 times so that the ethanol concentration in the sample is within the range of 0.2 to 1.0% (V / V), and the gas chromatograph for ethanol analysis (GL Sciences) (“Porapak® Type®” (trade name; manufactured by Waters) column was used, the carrier gas was nitrogen, and the flow rate was 60 mL / min). Separately, ethanol (manufactured by Kanto Chemical Co., Ltd., special grade, Lot No. 002XI222) was separated under the same conditions, and conditions were set so that the measurement area and the weight of ethanol were standard curves with excellent linearity. Isopropanol was used as an internal standard, and a conversion coefficient was obtained from a linear regression analysis of ethanol with respect to the internal standard.

For the measurement of the sugar concentration, the sample was diluted 10 to 100 times so that the sugar concentration of the sample was in the range of 10 to 300 ppm, and subjected to separation and quantification using an ion column chromatograph for sugar analysis (manufactured by DIONEX Corporation). The column used was PA-1, and elution was performed with water alone at a rate of 1 mL / min. Separately, 5 types of monosaccharides (glucose, mannose, galactose, xylose, arabinose) are separated under the same conditions for quantification of eluted sugars, and a standard curve with excellent linearity in the measurement area and the weight of each sugar is obtained. A condition was set. Deoxyglucose was used as an internal standard, and a conversion coefficient was obtained from a linear regression analysis of each sugar relative to the internal standard. The content of monosaccharide contained in the sample was calculated from the conversion factor of each obtained sugar and the measured area.

The results are shown in FIG. In FIG. 5, KO11-1 and KO11-2 represent the results of two experiments conducted independently for the KO11 strain and ΔP-1 and ΔP-2 for the KO11ΔP strain, respectively. When the KO11 strain and the KO11ΔP strain were subjected to a fermentation test under the same conditions, the KO11 strain could not be repeatedly fermented, but the KO11ΔP strain could be repeatedly fermented three times (panel (A): ethanol production). Amount; Panel (B): Cell weight). Since PtsG that is deficient in the KO11ΔP strain is a membrane protein, it is considered that in the KO11ΔP strain, the properties of the cell membrane changed and the robustness of the cells increased.

Residual sugars were the same for glucose the first time, but the second time the KO11ΔP strain had higher consumption and less residual sugar (panel (C)). Regarding xylose, it was confirmed that the KO11ΔP strain had higher consumption and higher utilization efficiency in both the first and second rounds (panel (D)).

5). The following vitamin production tests were used.
YPD medium (Difco, Lot No. 7242780)
・ Glucose (Wako Pure Chemical Industries, special grade, Lot No. ALP4694)
・ Xylose (Wako Pure Chemical Industries, special grade, Lot No. PEH5709)
・ 99.5% ethanol (Kanto Chemical Co., Ltd., special grade, Lot No. 002XI222)
・ 50mL polypropylene centrifuge tube (CORNING)
・ Rotary shake incubator (Takasugi Mfg. Co., Ltd., model TS-RS12L (24))
・ Rotary shake incubator (Takasugi Seisakusho, Model TS-RS12L)
・ High-speed cooling centrifuge (Model Kubota, Model 6500)
・ Centrifuge (TOMY model MX-305)

For culture, YPD medium (Difco, Lot No. 7724780; containing 3% (W / V) glucose) and xylose (Wako Pure Chemical Industries, special grade, Lot No. PEH5709) 3% (W / A YPD medium containing 3% glucose (W / V) and 3% xylose (W / V) prepared by adding V) was used.

A 500 mL Erlenmeyer flask was dispensed with 100 mL of the aforementioned YPD medium containing 3% glucose (W / V) and 3% xylose (W / V), and aseptically inoculated with 30 mg wet cell weight. An aluminum cap was used for the lid of the Erlenmeyer flask. The culture was carried out using a rotary shake incubator (model TS-RS12L (24), manufactured by Takasugi Seisakusho Co., Ltd.) at 37 ° C. and 120 rpm for 15 hours and 24 hours.

50 mL of each culture solution was centrifuged at 5,000 rpm for 5 minutes with a centrifuge (Model MX-305, manufactured by TOMY) to obtain a supernatant. Vitamin B2 content in the obtained supernatant was quantified by requesting the Japan Food Analysis Center to detect vitamin B2 using a fluorescence detector by high performance liquid chromatography.
Specifically, 5 mL of a 2.5% Takadiastase solution was added to 50 mL of a culture solution sample adjusted to pH 4.5 using a 4 mol / L sodium acetate solution, followed by enzymatic degradation at 38 ° C. for 24 hours. Water was added to make a constant volume of 100 mL, and after filtration through a 0.45 μm membrane filter, 10 μL was subjected to high performance liquid chromatography. The excitation wavelength of the fluorescence detector was measured at 445 nm and the measurement wavelength was 530 nm.

The results are shown in FIG. The vertical axis represents the amount of vitamin B2 (mg) in 100 g of the culture solution. The KO11ΔP strain (“ΔP”) produced vitamin B2 in a larger amount than the KO11 strain by culturing in the YPD medium.

6). Fermentation test using real liquid derived from biomass Using actual liquid obtained from an actual factory, the amount of ethanol produced by KO11 strain and KO11ΔP strain was evaluated.

Coniferous wood chips and hardwood wood chips were used as biomass. The black liquor produced when these biomasses were pulped by sulfite treatment was used as biomass sugar liquid (saccharified liquid). Calcium hydroxide was appropriately added to the biomass sugar solution to adjust to pH 6.0, and then heated at 80 ° C. for 3 hours to decompose furfural in the sugar solution. The biomass sugar solution was produced three times each on different days.

The sugar in the biomass sugar solution thus obtained was quantified according to the sugar analysis method described above, and the sugar composition was evaluated. Table 2 shows the concentration in each sugar solution.

The sugar solution derived from hardwood is a sugar solution rich in xylose due to the decomposition of xylan, which is hemicellulose in wood. On the other hand, the sugar solution derived from conifers is a sugar solution rich in mannose because hemicellulose is mannan.

Fermentation tests were conducted using these sugar solutions. Both KO11 strain and KO11ΔP strain were cultured according to the method described above. The obtained microbial cells were collected by centrifugation. Thereafter, the cells are added to the biomass sugar solution so that the dry weight becomes 2% (W / V), and cultured at 30 ° C. and 120 rpm for 5 days, and the amount of ethanol produced is evaluated as described above. At the same time, the residual sugar in the biomass sugar liquid was quantified and evaluated.
Table 3 shows the evaluation results.

As can be seen from Table 3, the ethanol concentration produced by the use of biomass sugar solution was 1.00% for conifers and 0.03% for hardwoods for the KO11 strain, while 0.99% for conifers for the KO11ΔP strain. %, Broad-leaved tree was 0.26%. Therefore, compared to the original KO11 strain, the KO11ΔP strain had the same ethanol productivity using the coniferous-derived biomass sugar solution, and when the hardwood-derived biomass sugar solution was used, It was found that ethanol productivity was higher than that of a certain KO11 strain.

This application is based on Japanese Patent Application No. 2009-029041 filed on Feb. 10, 2009, and all the contents described in the specification and claims of Japanese Patent Application No. 2009-029041 Included in the application specification.

FERM P-21758 (FERM BP-11221)

Claims (10)

Recombinant E. coli, which is a variant of E. coli KO11 strain, in which the ptsG gene is disrupted or deleted. The recombinant Escherichia coli according to claim 1, which is pentose assimilating under aerobic conditions. 3. Recombinant E. coli according to claim 1 or 2, which has higher ethanol productivity and vitamin B2 productivity than E. coli KO11 strain. Recombinant E. coli having the deposit number FERM P-21758 (FERM BP-11221). A method for producing ethanol and / or vitamin B2, comprising a step of fermenting a biomass saccharified solution using the recombinant Escherichia coli according to any one of claims 1 to 4. The method according to claim 5, wherein the fermentation step is performed under aerobic conditions. The method according to claim 5 or 6, wherein the biomass saccharified solution is a saccharified solution in which hexose and pentose coexist. The method according to any one of claims 5 to 7, comprising a step of saccharifying the biomass before the fermentation step. The method according to any one of claims 5 to 8, further comprising a solid-liquid separation step of the mash and a distillation step and / or a dehydration step of ethanol after the fermentation step. An ethanol-containing solution produced using the method according to any one of claims 5 to 9.

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