KR20140001165A - Kluyveromyces marxianus deficient in the ethanol fermentation and use thereof - Google Patents

Abstract

The present invention provides a variety of organic acids by introducing various exogenous genes into Kluyveromyces marxianus strain, which is a Krebtri negative yeast inactivated pyruvate decarboxylase to prevent ethanol fermentation. It relates to a yeast strain. The present invention also relates to a method for producing lactic acid by culturing the strain. The mutant strain in which the pyruvate decarboxylase activity of the present invention is inactivated does not produce ethanol during lactic acid production, and relates to a method for effectively producing an organic acid through a strain capable of producing an organic acid by introducing an exogenous gene into the strain.

Description

Kluyveromyces marxianus deficient in the ethanol fermentation and use etc.

The present invention is Kluyveromyces Kluyveromyces , a Krebtri negative yeast inactivated pyruvate decarboxylase marxianus ) and yeast strains that produce various organic acids by introducing various exogenous genes into the strain. The present invention also relates to a method for producing lactic acid by culturing the strain.

As the depletion of petroleum resources approaches, research in the field of renewable energy and biochemistry, which produces various compounds extracted from petroleum from renewable raw materials, is competitive.

Among the various compounds extracted from petroleum, organic products such as lactic acid (Lactic acid, Lactate, lactate) are of industrial importance. For example, organic acids can be used to synthesize plastic materials and other products. In response to the increasing demand for such organic products, more efficient and cost-effective production methods have been developed. One such method is to use bacteria. Specifically, some bacteria can produce large quantities of particular organic products under certain fermentation conditions. The use of live bacteria as producers has the disadvantage of limiting the growth of bacteria since organic products accumulate in the growth medium. To overcome this drawback, various product purification techniques have been used in the synthesis. In addition, the use of microorganisms other than bacteria has also been attempted.

Lactic acid, which is one of the most representative compounds among the organic products, has been proved to function as a biodegradable polymer, and a production method using microorganisms has been researched and developed. Lactobacillus, a microorganism producing lactic acid, is already known in many kinds, but when lactic acid is produced by culturing these microorganisms, two optical isomers (L-lactate) are produced.

Due to this, recently, a method of producing lactic acid by manipulating a gene of yeast which does not produce lactic acid in recent years has been developed. To produce lactic acid in yeast, selective introduction of lactate dehydrogenase can produce pure D- or L-lactic acid. In order to prepare a biodegradable polymer having high thermal stability, an equivalent amount of D-lactic acid is required together with L-lactic acid, but research on the production of D-lactic acid is relatively insufficient compared to L-lactic acid. When a general yeast strain is used as a lactic acid producing strain, ethanol is produced together with lactic acid as a by-product, thereby reducing the productivity of lactic acid.

In order to alleviate this drawback, a method of blocking the ethanol pathway by inactivating pyruvate decarboxylase is used. However, strains in which pyruvate decarboxylase is inactivated have the disadvantage of slow growth compared to wild strains. Therefore, there is an urgent need to develop yeasts that selectively produce high concentrations of pure optical isomer lactic acid, do not produce ethanol as a by-product, maintain growth ability even by inactivation of pyruvatetal carbonate, and maintain physiological properties even at low pH.

In order to improve the problems of the conventional strains, the present inventors selected the optimal strains by comparing the ability to use the carbon source and the resistance to high temperature, and produced a strain in which pyruvate decarboxylase was inactivated and a Cluiberomyces membrane By selecting the D or L-lactate dehydrogenase gene showing high activity in the cyanus strain, the optimal expression strain was prepared, and the growth was recovered to a level similar to that of the wild type strain, and a strain capable of selectively producing D or L-lactic acid was produced. It was. Using the strain produced in this way, it was confirmed that high-density pure D or L-lactic acid could be selectively produced from inexpensive biomass pork potato powder.

One object of the present invention is to provide a Kluyveromyces Marxian mutant strain in which pyruvate decarboxylase is inactivated in the parent strain Kluyveromyces marxianus . .

Another object of the present invention is to provide a method for producing lactic acid, comprising culturing the strain.

As one embodiment for achieving the above object, the present invention is a Kluyveromyces marxianus , the parent strain, Pluuvate Decarboxylase inactivated Pluuvate Decarboxylase It provides a loose mutation strain. The Kluyveromyces maxianus strain of the present invention is a kind of Krebtree negative strain.

The crabtree is the same as the crabtree effect, meaning that the respiration of cells is suppressed by the addition of glucose. In the present invention, the term "crebtree negative strain" refers to a strain having a trait which does not occur the Krebtri phenomenon, that is, the phenomenon that the respiration of the cell by the addition of glucose does not occur.

In the present invention, the parent strain may be preferably Kluyveromyces marxianus . Kluyveromyces maxilians are yeast strains with high growth capacity, high growth rate and low propensity to produce ethanol when exposed to excess sugar (Creptree negative type). In particular, because it secretes an excess of Inulin degrading enzymes, it is an excellent yeast capable of using porcine potatoes containing excessive amounts of Inulin as a nutrient source. Unlike common yeast strains, Kluyveromyces maximus has been reported a lot of strains, there is a characteristic that shows a variety of physiological characteristics depending on the strain. Even the same strains have reported different results between different study groups. Therefore, in the present invention, it is an important process to select specific strains of Cluyveromyces maximians with the required characteristics.

The parent strain of the present invention Kluyveromyces maxilianus is preferably Accession No. KCTC 7001, KCTC 7118, KCTC 7149, KCTC 7150, KCTC 7155, KCTC 7524, KCTC 17212, KCTC 17544, KCTC 17555, KCTC 17631, It may be one selected from the group consisting of KCTC 17694, KCTC 17724, KCTC 17725, KCTC 17759 (Genetic Bank of Korea Biotechnology Institute), BY25569, BY25571, and BY25573 (Yeast Genetic Resource center, Japan), more preferably It may be one selected from the group consisting of KCTC 7155, KCTC 17631, BY25569, BY25571, and BY25573, and most preferably may be BY25571.

In one embodiment of the present invention, 15 kinds of Cluyveromyces maxianus strains were distributed in the Gene Bank of Daejeon Korea Research Institute of Bioscience and Biotechnology, and 3 kinds of Cluiberomyces maxianus were obtained from Yeast Genetic Resource center (Japan). Strains were distributed and examined for fibrinolytic activity, high temperature resistance (Examples 1-1 and 1-2). First, as shown in Table 1, cellulose (C1) decomposing ability of cellulose containing 2% Carboxymethyl cellulose (CMC) and cellobiose (CB), 1% Yeast extract, 2% Peptone As a result of growing in medium, it was confirmed that six strains of KCTC 7155, KCTC 17724, KCTC 17631, BY25569, BY25571, and BY25573 had high growth ability and high fibrinolytic substrate degradation ability.

In addition, as a result of confirming the high temperature resistance to six strains with high fibrinolytic substrate degradation ability, it was confirmed that the KCTC 7155, BY25569, BY25571, BY25573 strains have high heat resistance (Fig. 1). Accordingly, the four strains were confirmed to have desirable characteristics as parent strains. In the experiment as described above, strains having high fibrin substrate degrading ability and high temperature resistance were selected as parent strains, and thus, the following strains were prepared. Specifically, among the four strains having high fiber substrate degradation ability and high temperature resistance, the BY25571 strain was representatively selected as a parent strain to prepare a mutant strain.

On the other hand, it is an object of the present invention to provide a strain having a characteristic of low alcohol production and increased production of a specific organic acid. Therefore, the pyruvate decarboxylase activity can be removed to make a transformant having less alcohol production and increased production of a specific organic acid based on the natural strains.

As used herein, the term "pyruvate decarboxylase (PDC)" refers to an enzyme that catalyzes the reaction of pyruvate to produce carbonic acid and acetaldehyde (CH ₃ COCOOH-> CH ₃ CHO + CO ₂ ). . When it comes to alcohol production, it is an essential enzyme in one stage of alcohol fermentation. Therefore, in the present invention, it is possible to provide a strain in which alcohol production by fermentation is inhibited by inactivating pyruvate decarboxylase present in the strain. Preferably, the strain may be a strain in which the production of ethanol is reduced compared to the strain in which pyruvate decarboxylase is not inactivated when cultured in a sugar-containing medium. In addition, the inactivation of the pyruvate decarboxylase may be performed by substitution, deletion or addition to the gene of the enzyme.

In general, the inactivation of the gene in the strain can be produced in various forms. For example, gene expression can be reduced by alteration of the signal structure of gene expression or by antisense-RNA techniques. For example, the signal structure of gene expression can be, for example, a suppressor gene, an activator gene, an operator, a promoter, an attenuator, a ribosomal binding site, a start codon and Terminators are not limited thereto.

In addition, by introducing a double-stranded RNA (dsRNA) consisting of a sense strand having a sequence homologous to the mRNA of the target gene and an antisense strand having a sequence complementary thereto, the expression of the target gene is suppressed by inducing degradation of the target gene mRNA. RNA interference (RNAi) method can be used. Alternatively, a method may be employed in which a mutation through a transposon, which is a DNA sequence capable of moving to another position in the genome of a single cell, causes a function of a target gene to be blocked and inactivated. Mutations that induce changes or decreases in the catalytic activity of genetic proteins are well known in the art (Qiu and Goodman (Journal of Biological Chemistry 272: 8611 8617 (1997))).

Preferably, the inactivation of the gene comprises: mutation of a single or complex sequence, deletion of a single or complex gene, insertion of a foreign gene into the gene, deletion of the entire gene group, insertion of a suppression sequence of a promoter of the gene group, mutation of a promoter, Expression suppression control insertion of a gene group, RNAi introduction into a single or complex sequences of a gene group, transposon mediated mutations, or a combination of these variants can be performed.

The methods described above are generally understood by one of ordinary skill in the art and may be performed as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press 2001). .

By changing a single or complex sequence of active sites in the gene's base sequence, the activity of the protein expressed from the gene may be very low, and a single or complex gene may be missed, or antibiotic resistance genes or other genes that are foreign genes within the base sequence. It may be a method of inserting to prevent the expression of the complete protein. Preferably, a method may be used in which all of the nucleotide sequences of genes present in the chromosome are deleted. Or in combination with the variants by the above methods.

The pyruvate decarboxylase can be found in GenBank, etc. of NCBI, which is a known database, but is not limited thereto, and may be a gene including SEQ ID NO: 41.

The pyruvate decarboxylase inactivated strain is not limited thereto, but may be a strain having accession number KCTC12225BP.

Meanwhile, enzymes involved in alcohol production include alcohol dehydrogenase (ADH) in addition to pyruvate decarboxylase. The "alcohol dehydrogenase" of the present invention is an enzyme that is involved in alcohol fermentation and is a catalyst that desorbs hydrogen from alcohol to form aldehydes or ketones, and is a reversible catalyst, which is a catalyst that can advance the reaction in the direction of alcohol production. (CH ₃ CH ₂ OH + NAD ⁺ <-> CH ₃ CHO + NADH + H ⁺ ). Its broad substrate specificity also affects other alcohols.

The strain of the present invention is preferably in addition to inactivation of pyruvate decarboxylase, preferably further inactivation of alcohol dehydrogenase. The alcohol dehydrogenase may be identified in GenBank, etc. of NCBI, which is a known database, but is not limited thereto. The alcohol dehydrogenase may be a gene including one or more of the group consisting of SEQ ID NO: 37 to SEQ ID NO: 40.

In one embodiment of the present invention, genome analysis of the Kluyveromyces maximus strain confirmed that four ADH genes and one PDC gene were present. In order to prepare a strain that inhibits ethanol production, a strain (Accession Number KCTC12225BP) deleted from the alcohol dehydrogenase gene (ADH) or pyruvate decarboxylase gene (PDC1) of the Kluyveromyces maximus BY25571 strain was prepared (Accession No. KCTC12225BP). Example 2).

Cell growth was confirmed in YP-2% ethanol medium to determine if the five deletion strains use ethanol as a carbon source. As a result of comparing YP-2% glucose as a control, it was confirmed that all five strains use ethanol as a carbon source well (FIG. 7).

In addition, the ethanol production of each strain was compared through ethanol fermentation. 24 hours of each strain in a minimum nutrient medium seed culture and then 50g / ethanol fermentation medium (YE 0.5% glucose is added to the _{L, pepton 0.5%, KH 2} PO 4, Ammonia sulfate 0.2%, MgSO 4 · H 2 O 0.04% ) Were grown at 30 ° C. and 150 rpm, and cell growth and ethanol production were measured. As a result, the adh mutant strains produced ethanol without significant difference when compared with the natural strain, but the pdc1 mutant strain was confirmed that does not produce any ethanol (Fig. 7).

It is also an object of the present invention to provide a strain having the characteristics of producing a specific organic acid. To this end, various organic acids can be produced using a strain in which a variety of exogenous genes related to specific organic acid production are introduced into a Krebtri negative strain (Cluyberomyces maximans) blocked by the alcohol production pathway.

As used herein, the term “exogenous gene” refers to a gene of interest when a nucleic acid sequence that does not belong to a native strain is introduced from the outside for a certain purpose. In particular, in the present invention, it means a gene associated with the production of a specific organic acid.

Preferably, in the present invention, the specific organic acid targeted for the strain may be lactic acid. More preferably, it may be L-lactic acid or D-lactic acid which is an optical isomer.

The exogenous gene introduced into the strain to produce the lactic acid may preferably be a gene having lactate dehydrogenase (LDH) activity.

As used herein, the term "Lactate Dehydrogenase" is an enzyme that generally decomposes hydrogen from L-lactic acid or D-lactic acid using NAD ⁺ to pyruvic acid, and catalyzes the corresponding final step of reverse reaction. It is an enzyme that produces L-lactic acid or D-lactic acid by reducing pyruvic acid using NADH. That is, it may be L-lactate dehydrogenase or D-lactate dehydrogenase. The gene having lactate dehydrogenase activity may include at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of natural lactate dehydrogenase. Genes of enzymes having the same activity with homology are included. Mutation genes which induce mutations in the natural lactate dehydrogenase are also included in the lactate dehydrogenase of the present invention irrespective of the sequence, as long as they have lactate dehydrogenase activity.

The lactate dehydrogenase may be identified in GenBank, etc. of NCBI, which is a known database, but is not limited thereto. The lactate dehydrogenase may be a gene including a nucleotide sequence selected from the group consisting of SEQ ID NOs: 42 to 50. In particular, the lactate dehydrogenase producing L-lactic acid may be a gene including one nucleotide sequence selected from the group consisting of SEQ ID NOs: 42 to 46, and the lactate dehydrogenase producing D-lactic acid is SEQ ID NOs: 47 to It may be a gene comprising one nucleotide sequence selected from the group consisting of 50. The lactate dehydrogenase in the strains of the present invention can be expressed using the TEF1a promoter derived from Cluyveromyces macxanus.

The strain in which the lactate dehydrogenase is introduced into the Krebtri negative strain of which the alcohol production pathway is blocked is not limited thereto, but may be a strain having accession number KCTC12226BP.

As used herein, the term “mutation” refers to any action that changes the genetic function of a gene. Specifically, a mutation of a gene refers to a mutation caused by a quantitative or qualitative change of a gene among various variations of an organism. . That is, a change in the molecular level at which one or more of the DNA bases is substituted with another base, ie, a base sequence resulting from substitution, deletion, addition, inversion, duplication, or insertion or deletion of one or two bases in the DNA. Molecular changes, such as frame shift, mean that the enzymes or peptides produced according to the genetic information of the DNA are not made or the activity is lost or the original activity is increased or changed. Mutation methods for damaging or altering the genes of the present invention can be used without limitation mutation methods commonly used in the art.

In one embodiment of the present invention , genomic DNA was extracted from Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactococcus lactis in order to secure L-lactate or D-lactate dehydrogenase (LDH) gene. PCR was performed on the extracted samples, and the amplified genes were cloned into the T-easy vector to secure each gene. Eight vectors were constructed by inserting the L-LDH gene and the D-LDH gene into plasmid pTEFp-GAPt for expression in Kluyveromyces maximians. The constructed vector was treated with restriction enzyme NheI to make a linear DNA state, transformed to BY25571 strain URA3 auxotroph strain and BY25571Δpdc1 strain PDC1 mutant strain, and a single strain (Accession No. KCTC12226BP) was selected from the transformants. Strains producing L-lactic acid and D-lactic acid at the highest concentration were selected by comparing lactic acid and D-lactic acid production (Example 5).

The strain of the present invention may be a mutant strain further inactivated L-lactate cytochrome-c oxidoreductase (CYB2). That is, the skin bait decarboxylase may be inactivated and in addition, the L-lactate cytochrome-c oxidoreductase may be inactivated. Inactivation in the present invention is as described above.

In the present invention, "L-lactate cytochrome-c oxidoreductase (CYB2)" is an enzyme that promotes oxidation of L-lactate. In particular, the L-lactate cytochrome-c oxidoreductase can be identified in GenBank of NCBI, which is a well-known database, but is not limited thereto, and may include the nucleotide sequence of SEQ ID NO: 57.

 The strain of the present invention may be a mutant strain having enhanced plasma membrane ATPase (PMA1) activity.

In the present invention, "plasma membrane ATPase (PMA1)" is one of hydrogen ion pumps, which is an enzyme that gives various driving force for active transport through maintaining the quantum slope. In particular, the plasma membrane ATPase of the present invention can be identified from GenBank, etc. of NCBI, which is a known database, but is not limited thereto, and may be represented by the nucleotide sequence of SEQ ID NO: 58.

Method for enhancing the activity of the protein in the present invention can be applied to a variety of methods well known in the art. For example, the method may increase the number of copies by additionally inserting a polynucleotide including a nucleotide sequence encoding the protein and an expression control region introduced by itself or externally into a chromosome or by introducing into a vector system. Method of controlling the expression of the gene, including the substitution of the regulatory regulatory region with other regulatory sequences, modification of the entire or part of the nucleotide sequence of the expression regulatory region, and the method by strengthening the enzyme activity by introducing the mutation itself, etc. It may be, but is not limited thereto.

Therefore, the strain of the present invention may be a BY25571Δpdc1, cyb2-PMA1 // L-LpLDH strain of accession number KCTC12435BP.

In one embodiment of the present invention, in order to increase the lactic acid productivity at low pH, the strain (KCTC12225BP) prepared in Example 2 deleted KmCYB2 (L-lactate cytochrome-c oxidoreductase) gene and KmPMA1 (plasma membrane ATPase) gene A mutation was introduced that overexpressed. L-lactate dehydrogenase (L-LpLDH) was introduced. The strain thus prepared was named BY25571Δpdc1, cyb2-PMA1 // L-LpLDH, and was deposited on June 26, 2013 at the Korea Institute of Bioscience and Biotechnology (KCTC), and received accession number KCTC12435BP. As a result of culturing the strains and confirming the L-lactic acid production through HPLC, the production of 25571Δpdc1, cyb2-PMA1 / L-LpLDH strain was 20% higher than that of 25571Δpdc1 / L-LpLDH. It was confirmed that the degree increased (Table 6).

As another aspect, the present invention provides a method for producing lactic acid, comprising culturing the strain.

The method for producing lactic acid of the present invention may include culturing a transformed strain in which the ethanol production pathway is blocked in the above strains, and the growth ability of lactic acid is maintained while the growth ability is maintained.

Cultivation of the strain in the present invention can be carried out according to well-known methods, conditions such as culture temperature, incubation time and pH of the medium can be appropriately adjusted. Suitable culture methods include fed-batch culture, batch culture, and continuous culture (cintinuous culture) and the like, preferably batch culture, but is not limited thereto.

The culture medium used should suitably meet the requirements of a particular strain. Culture media for various microorganisms are known (see, for example, " Manual of Methods for General Bacteriology ", from the American Society for Bacteriology (Washington D.C., USA, 1981)). Carbon sources in the medium include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seed oil, peanut oil and coconut oil). ), Fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, organic acids such as acetic acid, and the like.

In the present invention, the strain may be cultured using a nutrient source of biomass pork potato powder that can be supplied at low cost in large quantities. The term "pig potato powder medium" of the present invention is a nutrition medium containing only pork potato powder without any pretreatment. Since it is a complete medium, it cannot be used as a medium for selection.

The materials constituting the culture medium can be used individually or as a mixture. Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, juice, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, Ammonium), and these materials may also be used individually or as a mixture. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium containing salts can be used as the phosphorus source. In addition, the culture medium may contain metal salts essential for growth (for example, magnesium sulfate or ferrous sulfate), and finally essential growth-promoting substances such as amino acids and vitamins may be used in addition to the above-mentioned substances. Additional suitable precursors may be added to the culture medium. The feed material may be added all at once to the culture, or may be supplied appropriately during the culture.

Preferably, the lactic acid produced may be L-lactic acid and D-lactic acid which are optical isomers. This may vary depending on the type of LDH introduced.

The mutant strain in which the pyruvate decarboxylase activity is inactivated does not produce ethanol, and a method for effectively producing organic acids from biomass through a strain capable of converting pyruvate to an organic acid by introducing an exogenous gene into the strain. It is about.

1 is a diagram confirming the high temperature resistance of strains Kluyveromyces maximians.
2 is a diagram confirming the ADH1 gene disruption process using the URA3 cassette and the ADH1 gene disruption using PCR.
3 is a diagram confirming the ADH2 gene disruption process using the URA3 cassette and the ADH2 gene disruption using PCR.
4 is a diagram confirming the ADH3 gene disruption process using the URA3 cassette and the ADH3 gene disruption using PCR.
5 is a diagram confirming the ADH4 gene disruption process using the URA3 cassette and the ADH4 gene disruption using PCR.
6 is a diagram confirming the PDC1 gene disruption process using the URA3 cassette and the PDC1 gene disruption using PCR.
7 is a graph measuring ethanol production according to cell growth in ethanol production medium.
Figure 8 is a schematic diagram showing the Kluyveromyces maximans vector for L-LDH or D-LDH expression.
Figure 9 is a graph measuring the L-lactic acid production, glucose consumption at 30 ℃ (A) and 35 ℃ (B).
10 is a graph measuring L-lactic acid production (A), D-lactic acid production (B), and glucose consumption from pork potato powder.
11 is a diagram confirming the CYB2 gene disruption process using the URA3 cassette and the CYB2 gene disruption using PCR.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example  One. Cluey Bromyces Maxianus  Strain selection

Unlike common yeast strains, Kluyveromyces marxianus (KM) has been reported with a large number of strains and accordingly has a wide variety of physiological characteristics, even the same strain has been reported different results in different groups. Therefore, screening strains suitable for the intended use is a very efficient way to utilize Kluyveromyces maximus as an industrial strain.

Fibrin substrate decomposing ability was obtained from 15 genes of Kluyveromyces maxianus strains from Gene Bank of Daejeon Korea Biotechnology Research Institute and 3 types of Kluyveromyces maxianus strains from Yeast Genetic Resource Center (Japan). And high temperature resistance were examined.

Example  1-1: Comparison of Fibrous Substrate Degradation Capacity

 KM strains are not originally known to have fibrinolytic function but are known to have weak fibrinolytic capacity in some strains. Therefore, in order to select strains with strong fibrinolytic effect, 18 strains of Kluyveromyces maximanus strains were treated with YP (1% Yeast extract, 2% Peptone) medium containing 2% Carboxymethyl cellulose (CMC) and cellobiose (CB). Incubated for 48 hours at 30 ℃ to analyze the cell growth capacity of the cellulase (cellulase) activity. It was confirmed that the growth of most strains in the medium requiring the fibrinolytic ability was weak, and in particular, the growth ability of strains KCTC 7155 and BY25569, BY25571, BY25573 was relatively high (Table 1).

Comparison of Cell Growth Capacity Using Fibrin Substrate of Kluyveromyces Maktians Strains strain Cell growth YP + CB YP + CMC KCTC 7001 ++ + KCTC 7118 - - KCTC 7149 ++ + KCTC 7150 ++ + KCTC  7155 +++ +++ KCTC 7524 ++ + KCTC 17212 ++ + KCTC 17544 ++ + KCTC 17555 ++ ++ KCTC  17631 +++ ++ KCTC 17694 ++ + KCTC 17724 ++ ++ KCTC 17725 ++ + KCTC 17759 ++ + BY25569 +++ +++ BY25571 +++ +++ BY25573 +++ +++

Example  1-2: high temperature resistance comparison

Since the optimum temperature of fibrinolytic enzyme is 40-50 ° C. when the fiber substrate is used as a bioethanol fermentation strain, it is necessary to develop a yeast resistant at such temperature. Six strains with good growth potential were selected from the fibrin substrate to compare their resistance to high temperatures. Cell cultures were dotting in YPD (1% Yeast extract, 2% Peptone and 2% glucose) medium and incubated at 42 ° C, 45 ° C, and 50 ° C for 24 hours.The KTCT 17724 strain had low heat resistance and KTCT 7155 , BY25569, BY25571, BY25573 strains were confirmed to have high heat resistance (Fig. 1). Accordingly, the four strains were confirmed to have desirable characteristics as parent strains. Among the four strains with high fibrinolytic ability and heat resistance, BY25571 strain was selected as a parent strain to prepare a mutant strain.

Example  2. ADH1 , ADH2 , ADH3 , ADH4 , PDC1  Gene deletion strain production

As a result of genome analysis, unlike Saccharomyces cerevisiae, the Kluyveromyces maxilianus strain has four alcohol dehydrogenase genes (ADH) and one pyruvate decarboxylase gene (PDC1). . In order to prepare a strain that inhibits ethanol production, a strain in which the alcohol dehydrogenase gene or the pyruvate decarboxylase gene of the strain Cluiberomyces maximus BY25571 was deleted was prepared.

Example  2-1: ADH1  Gene deletion strain production

Using genomic DNA as a template, DNA fragments of 300 bp in the upstream and downstream of the ADH1 ORF were used using HJ368 (SEQ ID NO: 1), HJ369 (SEQ ID NO: 2), HJ370 (SEQ ID NO: 3), and HJ371 (SEQ ID NO: 4) primers. After amplification, amplified URA3 DNA fragments and overlap extension PCR were performed using primers Tc-f (SEQ ID NO: 6), U200r (SEQ ID NO: 7), U200f (SEQ ID NO: 8), and Tc-r (SEQ ID NO: 9). . After mixing each of the gene fragments, the mixture was used as a template to link the two fragments.The PCR fragment was transformed into BY25571 strain, and the strain introduced into ADH1 locus was selected as a medium without uracil (0.67% yeast nitrogen base without amino acids, 0.077% Ura-DO supplement, 2% glucose, 2% agar). Transformation was confirmed by PCR using primers HJ368 and HJ372 (SEQ ID NO: 5), U200f and HJ372, and PCR bands were identified at expected positions. In order to confirm whether the remaining ADH1 gene was confirmed by PCR using HJ394 (SEQ ID NO: 10) and HJ371 primer, it was confirmed that the ADH1 gene (SEQ ID NO: 37) was knocked out by the URA3 cassette (Fig. 2).

Example  2-2: ADH2  Gene deletion strain production

Using genomic DNA as a template, DNA fragments of 300 bp in the upstream and downstream of the ADH2 ORF were prepared using HJ458 (SEQ ID NO: 11), HJ459 (SEQ ID NO: 12), HJ460 (SEQ ID NO: 13), and HJ461 (SEQ ID NO: 14) primers. After amplification, URA3 DNA fragments amplified using primers Tc-f and U200r, U200f and Tc-r, and overlap extension PCR were performed. After mixing each of the gene fragments, this mixture was used as a template to link the two fragments.The PCR fragment was transformed into 25571 strain, and the strain introduced into ADH2 locus was selected as a medium without uracil (0.67% yeast nitrogen base without amino acids, 0.077% Ura-DO supplement, 2% glucose, 2% agar). Transformation was confirmed by PCR using primers HJ458 and HJ462 (SEQ ID NO: 15), U200f and HJ462, and PCR bands were identified at expected locations. In order to confirm whether the remaining ADH2 gene was confirmed by PCR using HJ463 (SEQ ID NO: 16) and HJ461 primer, it was confirmed that the ADH2 gene (SEQ ID NO: 38) was knocked out by the URA3 cassette (Fig. 3).

Example  2-3: ADH3  Gene deletion strain production

Using genomic DNA as a template, DNA fragments of 300 bp in the upstream and downstream of the ADH3 ORF were used using HJ412 (SEQ ID NO: 17), HJ413 (SEQ ID NO: 18), HJ414 (SEQ ID NO: 19), and HJ415 (SEQ ID NO: 20) primers. After amplification, URA3 DNA fragments amplified using primers Tc-f and U200r, U200f and Tc-r, and overlap extension PCR were performed. After mixing the respective gene fragments, the mixture was used as a template to link the two fragments.The PCR fragment was transformed into 25571 strain, and the strain introduced into ADH3 locus was selected as a medium without uracil (0.67% yeast nitrogen base without amino acids, 0.077% Ura-DO supplement, 2% glucose, 2% agar). Transformation was confirmed by PCR using primers HJ412 and HJ416 (SEQ ID NO: 21), U200f and HJ416, and PCR bands were identified at expected positions. In order to confirm that the remaining ADH3 gene was confirmed by PCR using HJ434 (SEQ ID NO: 22) and HJ415 primer, it was confirmed that the ADH3 gene (SEQ ID NO: 39) was knocked out by the URA3 cassette (Fig. 4).

Example  2-4: ADH4  Gene deletion strain production

Using genomic DNA as a template, DNA fragments of 300 bp in the upstream and downstream of the ADH4 ORF were prepared using HJ482 (SEQ ID NO: 23), HJ483 (SEQ ID NO: 24), HJ484 (SEQ ID NO: 25), and HJ485 (SEQ ID NO: 26) primers. After amplification, URA3 DNA fragments amplified using primers Tc-f and U200r, U200f and Tc-r, and overlap extension PCR were performed. After mixing the respective gene fragments, the mixture was used as a template to connect the two fragments.The PCR fragment was transformed into 25571 strain and the strain introduced into ADH4 locus was selected as a medium without uracil (0.67% yeast nitrogen base without amino acids, 0.077% Ura-DO supplement, 2% glucose, 2% agar). Transformation was confirmed by PCR using primers HJ482 and HJ486 (SEQ ID NO: 27), U200f and HJ486, and PCR bands were identified at expected positions. In order to confirm that the remaining ADH4 gene was confirmed by PCR using HJ487 (SEQ ID NO: 28) and HJ485 primer, it was confirmed that the ADH4 gene (SEQ ID NO: 40) was knocked out by the URA3 cassette (Fig. 5).

Example  2-5: PDC1  Gene deletion strain production

 Using genomic DNA as a template, DNA fragments of 300 bp in the upstream and downstream of the PDC1 ORF were prepared using HJ464 (SEQ ID NO: 29), HJ465 (SEQ ID NO: 30), HJ466 (SEQ ID NO: 31), and HJ467 (SEQ ID NO: 32) primers. After amplification, URA3 DNA fragments amplified using primers Tc-f and U200r, U200f and Tc-r, and overlap extension PCR were performed. After mixing each of the gene fragments, this mixture was used as a template to link the two fragments.The PCR fragment was transformed into 25571 strain, and the strain introduced into PDC1 locus was selected as a medium without uracil (0.67% yeast nitrogen base without amino acids, 0.077% Ura-DO supplement, 2% glucose, 2% agar). Transformation was confirmed by PCR using primers HJ464 and HJ486 (SEQ ID NO: 33), U200f and HJ468, and PCR bands were identified at expected locations. In order to confirm whether the PDC1 gene remains, it was confirmed by PCR using HJ469 (SEQ ID NO: 34) and HJ467 primer, and it was confirmed that the PDC1 gene (SEQ ID NO: 41) was knocked out by the URA3 cassette (FIG. 6).

The strain prepared by the deletion of the pyruvate decarboxylase gene of the strain Kluyveromyces maxianus BY25571 was deposited on June 20, 2012 to KCTC of the Korea Research Institute of Bioscience and Biotechnology, and received the accession number KCTC12225BP. received.

Example  3. Ethanol Fermentation of Strains

The ethanol production of each of the mutant strains was compared through ethanol fermentation. 24 hours of each strain in a minimum nutrient medium seed culture and then 50g / ethanol fermentation medium (YE 0.5% glucose is added to the _{L, pepton 0.5%, KH 2} PO 4, Ammonia sulfate 0.2%, MgSO 4 · H 2 O 0.04% ) Was incubated at 30 ° C, 150rpm and cell growth and ethanol production were measured. As a result, adh mutant strains produced ethanol without significant difference compared to wild type strains, and as expected, pdc1 mutant strains reduced cell growth by 50% compared to wild type strains and produced no ethanol at all. 7). In order to convert pyruvate to ethanol, acetaldehyde is produced by pyruvate decarboxylase and then to ethanol by alcohol dehydrogenase. Since four ADH genes are present in the Kluyveromyces maxianus strain, the deletion of one or two ADH genes does not completely block the conversion of pyruvate to ethanol, whereas the pyruvate decarboxylase gene is 1 Since only dogs existed, the deletion of the pdc1 gene alone was a complete blockade of the ethanol production pathway. The reason for the pdc1 deletion strain growth is reduced compared to the wild-type strain in order to eliminate the NAD ⁺ / NADH imbalance is formed in the process requires the ethanol fermentation process, but in the pdc1 deletion strain because the ethanol fermentation route is blocked the NAD ⁺ / NADH imbalance This imbalance is caused by a decrease in growth because it acts as a barrier to strain growth. This phenomenon is expected to be able to solve the growth degradation problem caused by the pdc1 deletion strain because lactic acid fermentation can solve the NAD ⁺ / NADH imbalance in place of ethanol fermentation if the LDH gene expression was carried out.

Example  4. L- derived from microorganism Lactate  Or D- Lactate Decarbonate  enzyme ( LDH ) Expression

Lactobacillus, known for its excellent lactic acid dehydrogenase enzyme activity to secure L-lactate dehydrogenase and D-lactate dehydrogenase (LDH) genes rhamnosus , Lactobacillus plantarum , Lactobacillus acidophilus , Lactococcus Genomic DNA was extracted from lactis . L-LDH and D-LDH obtained by PCR with the extracted sample was cloned into a T-easy vector to secure each gene.

Eight vectors were constructed by inserting the L-LDH gene and the D-LDH gene into plasmid pTEF1 for expression in Kluyveromyces maximians. The pTEF1 vector contains a promoter of translation elongation factor 1a (TEF1a), a gene derived from Kluyveromyces maximans, for the powerful and constitutive expression of the LDH gene, and glyceraldehyde 3-phosphate to terminate the transcription of the LDH gene. It contains the transcription termination site of the dehydrogenase (GAP) gene. The constructed vector was treated with restriction enzyme NheI to make a linear DNA state, transformed to BY25571 strain URA3 auxotroph strain and BY25571Δpdc1 strain PDC1 mutant strain, and single strains were selected from the transformants. It was confirmed that it was introduced.

Among them, the strain which introduced L-LpLDH into the accession number KCTC12225BP strain prepared by deleting the pyruvate decarboxylase gene of the strain Kluyveromyces maxianus BY25571 was sent to the KCTC of the Korea Institute of Bioscience and Biotechnology (KCTC) in 2012. Deposited June 20, accession number KCTC12226BP was awarded.

Example  5. Lactic Acid Production According to Reaction Temperature

Flask culture was carried out at 30 ℃, 35 ℃ to determine the optimum reaction temperature for lactic acid production. Each strain was shaken in YPD medium to recover cells, washed twice with distilled water, and then inoculated in YPD10 liquid medium (1% yeast extract, 2% peptone, 10% glucose) containing calcium carbonate and incubated for 72 hours. It was. As a result of confirming the lactic acid production through HPLC, it was confirmed that the lactic acid production and yield is higher at 35 ℃ than 30 ℃. At 30 ° C, lactic acid production was highest in 25571pdc1 / L-LpLDH and 25571pdc1 / D-LrLDH strains, and at 35 ° C, lactic acid production was high in 25571pdc1 / L-LpLDH and 25571pdc1 / D-LpLDH strains. From the above results, it can be confirmed that the production amount of each gene is different depending on the temperature (Table 2). Finally, L-lactic acid was produced using 25571pdc1 / L-LpLDH strain and D-lactic acid was produced using 25571pdc1 / D-LpLDH strain.

Comparative Analysis of Lactic Acid Production at 30 ℃ and 35 ℃ Strain 30 ℃ fermentation 35 ℃ fermentation Lactic Acid Production (g / L) productivity(%) water Lactic Acid Production (g / L) productivity(%) water 25571pdc1 / L-LaLDH 25 82 99 42 73 99 25571pdc1 / L-LpLDH 31 79 99 46 79 99 25571pdc1 / L-LrLDH 25 73 99 46 78 99 25571pdc1 / L-LlLDH 18 53 99 22 61 99 25571pdc1 / D-LaLDH 0.5 2 99 10 58 99 25571pdc1 / D-LpLDH 39 63 99 40 78 99 25571pdc1 / D-LrLDH 49 76 99 31 70 99 25571pdc1 / D-LlLDH 30 45 99 30 45 99

Example  6. Glucose Production of high concentrations of lactic acid

In order to confirm the effect of lactic acid production according to temperature through fermentation and increase the productivity of lactic acid, fed-batch culture was performed with 25571pdc1 / L-LpLDH strain. Before entering the main culture, the culture was activated by incubating in 100 ml of YPD liquid medium, and then inoculated in the main culture solution and incubated at 30 ° C. for 24 hours. 40% glucose (final 10%) was fed to the high concentration fermentation broth to perform lactic acid conversion at 30 ° C and 35 ° C. As a result of confirming lactic acid production through HPLC, 97.3 g / l of lactic acid was produced from 140 g of glucose at 30 ° C, which showed 79.2% conversion, and 105.5 g / l of lactic acid was produced at 35 ° C, indicating 88.1% of conversion. 9). Therefore, as confirmed in the flask culture, it was confirmed that the production increased by about 10% when incubated at 35 ° C.

Example  7. Lactic Acid Production from Pork Potato Starch

The supernatant obtained by autoclave (20 minutes at 121 ° C) of pork potato starch was analyzed by HPLC and found to contain 60% of inulin. In order to compare lactic acid production according to the concentration of Inulin, porcine potato starch was added to each concentration and cultured in flask. Cells were harvested by shaking culture in YPD medium, washed twice with distilled water, and then inoculated in porcine potato starch added with 3% calcium carbonate and incubated for 84 hours to compare lactic acid conversion rate.

The results are summarized in Table 3 below.

Analysis of Lactic Acid Production According to Inulin Content Inulin Content (%) Sugar consumption
(g / L) Lactic Acid Production
(g / L) yield
(%) Lactate Optical Purity
(%) 2 20.0 19.7 98.5 99 5 50.0 47.5 95.0 99 10 50.1 42.2 86.2 99 14 44.3 23.0 51.9 99

As can be seen in Table 3 above, when the concentration of Inulin was 5% or more, the amount of lactic acid was decreased, and the lower the level of inulin, the better the lactic acid conversion. Although the initial strain inoculation was not high, it was confirmed that all of the Inulin used under the four conditions tested were degraded to fructose. Therefore, it was confirmed that optimizing the culture conditions through fermentation can produce high concentrations of lactic acid from pig potatoes.

Example  8. Nitrogen source Lactic acid production according to

In general, in order to produce lactic acid from swine potatoes, most microorganisms must supply insufficient nitrogen source, and thus, lactic acid production was analyzed by adding yeast extract to each concentration of pork starch. Cells were harvested by shaking culture in YPD medium, washed twice with distilled water, and then inoculated in porcine potato starch added with 3% calcium carbonate using a flask and incubated for 72 hours to compare lactic acid conversion. 4).

Analysis of Lactic Acid Production by Yeast Extract Addition Yeast extract
Addition amount (%) Sugar consumption
(g / L) Lactic Acid Production
(g / L) yield
(%) Lactate Optical Purity
%) 0 56.8 53.3 93.8 99 0.1 58.3 50.4 86.3 99 0.2 59.0 50.3 85.3 99 0.3 59.0 50.3 85.2 99 0.4 58.1 49.2 84.7 99 0.5 59.4 48.9 82.3 99 One 57.6 48.2 83.7 99

As can be seen in Table 4, the lactic acid production does not increase even if the addition amount of yeast extract is increased, so it was confirmed that no nitrogen source other than pork potato starch was needed.

Example  9. Production of high concentration lactic acid from pork potato starch

Batch culture was performed to produce high concentrations of lactic acid from pig potatoes using 25571pdc1 / L-LpLDH strain and 25571pdc1 / D-LpLDH. After activating by culturing in 100ml YPD liquid medium and inoculating the culture medium and incubated at 30 ℃ for 24 hours to recover the cells, washed twice with distilled water and then inoculated in pork potato starch and incubated for 66 hours while lactic acid Conversion rates were compared. As a result of confirming the amount of lactic acid produced by HPLC, L-lactic acid produced 130.1 g / l showed a conversion of 98.9%, D-lactic acid produced 122.3 g / l showed a conversion of 95.0% (Fig. 10).

Table 5 compares the results of the present invention with the results of recent studies conducted to produce lactic acid using pork potatoes.

Comparison of Lactic Acid Production Methods Using Pork Potato as a Nutrient Source Nutrition D / L type Used strain Fermentation method Optical purity Productivity (g / L / hr) Final concentration
(g / L) efficiency
(g / g) references Pork potato powder digest + corn steep powder L B. coagulans fed-
batch 99 2.5 134 96 Wang, L. et al. (2013) Bioreso urce technology , 130, 174-180. Pork Potato Powder Degradation + yeast extract L Immobilized L. lactis fed-
batch 2.85 142 92 Shi, Z. et al. (2012) Enzyme and microbial technology , 51, 263-268. Pork potato powder high temperature extract + yeast extract L L. paracasei batch 93.2 0.25 92.5 98 Choi, HY et al. (2012) Bioresource technology , 114, 745-747. Pork Potato Powder + Sodium Citrate L L. casei fed-
batch 4.7 141.5 93.6 Ge, XY et al. (2010) Journal of microbiology and biotechnology , 20, 101-109. Pork Potato Powder + soybean flour + sucrose L A. niger + Lactobacillus fed-
batch 3.6 120.5 94.5 Ge, XY et al. (2009) Bioresource technology , 100, 1872-1874 Pork potato powder L K. marxianus batch 99 2 130 98 Invention D K. marxianus batch 99 2 122 95

As can be seen in Table 5, the case of producing D-lactic acid at a concentration similar to the present invention has not been reported so far, and in the present invention using only pork potato powder that has not been treated in the case of L-lactic acid as a nutrient source No production of L-lactic acid at the indicated concentrations has been reported. By using the Cluiberomyces Maktianus strain of the present invention, lactic acid can be produced using only the pre-treated pork potato powder as a nutrient source, thereby reducing the cost of the medium, which takes up a large proportion in the lactic acid production process, and of high purity. Optical isomers can be produced selectively.

Example  10. Strain Preparation and Lactic Acid for Increasing Lactic Acid Productivity Production capacity  Measure

In the process of producing lactic acid, the growth environment of the strain is gradually changed to low pH. In order to increase lactic acid productivity at such low pH, the present inventors deleted the KmCYB2 (L-lactate cytochrome-c oxidoreductase) gene from the strain prepared in Example 2 (KCTC12225BP) and overexpressed the KmPMA1 (plasma membrane ATPase) gene. Mutations were introduced.

First, the same method as the deletion of the ADH gene described in Example 2 was used to inactivate the KmCYB2 gene. Specifically, DNA fragments of 300 bp in the upstream and downstream of the KmCYB2 ORF using the BY25571 genomic DNA as a template were identified by HJ628 (SEQ ID NO: 51), HJ629 (SEQ ID NO: 52), HJ630 (SEQ ID NO: 53), and HJ631 (SEQ ID NO: 54). After amplification using primers, overlap extension PCR with URA3 DNA fragments amplified using primers Tc-f and U200r, U200f and Tc-r was performed. After mixing each gene fragment, this mixture was used as a template to connect the two fragments, and the overlap extension PCR product was transformed into BY25571Δpdc1 strain (KCTC12225BP). Strains in which the introduced PCR fragments were introduced into the CYB2 locus were selected in a selection medium without uracil. Transformation was confirmed by PCR using primers HJ628 and HJ632 (SEQ ID NO: 55), U200f and HJ632, and PCR bands were identified at expected positions. In order to confirm whether the CYB2 gene remains, it was confirmed by PCR using the HJ633 (SEQ ID NO: 56) and the HJ631 primer, and it was confirmed that the CYB2 gene (SEQ ID NO: 57) was deleted by the URA3 cassette (FIG. 11). In order to remove the URA3 gene present in the URA3 cassette, 4-6 hours of incubation in YPD medium was selected in 5-FOA medium. As a result of PCR, the URA3 gene pop-out was confirmed and the strain BY25571Δpdc1, cyb2 from which the URA3 gene was removed was obtained.

Subsequently, in order to obtain a strain overexpressing the PMA1 gene (SEQ ID NO: 58), a gene was obtained from BY25571 genomic DNA, and the expression vector was prepared by cloning the URA3 gene of the pTEF1 vector with the LEU2 gene. The produced vector was linearized with restriction enzyme PmlI, transformed into the PDC1, CYB2 mutant strain BY25571Δpdc1, cyb2 prepared above, and a single strain was selected from the transformants. PTEF1-L-LpLDH used in Example 4 to introduce the L-LpLDH gene into the transformant obtained was treated with NheI and transformed to select a single strain.

The strain prepared above was named BY25571Δpdc1, cyb2-PMA1 // L-LpLDH, and was deposited on June 26, 2013 to the Bioresource Center (KCTC) of the Korea Research Institute of Bioscience and Biotechnology, and received accession number KCTC12435BP.

Then, in order to confirm the lactic acid production capacity of the prepared transformant, cells were recovered by shaking culture in YPD medium, washed twice with distilled water, and then inoculated in YPD10 liquid medium or YPD10 liquid medium containing calcium carbonate. Incubated for 72 hours. As a result of confirming the production of L-lactic acid through HPLC, the production of 25571Δpdc1, cyb2-PMA1 / L-LpLDH strain showed a 20% increase in lactic acid production than 25571Δpdc1 / L-LpLDH in both the conditions of pH control and the condition of no pH control. (Table 6). These results may be due to the inactivation of the KmCYB2 gene and overexpression of the KmPMA1 gene, which prevented the redox reaction of L-lactic acid and increased migration out of cells.

Analysis of Lactic Acid Production Capacity of pH Related Mutant Strains division Strain Sugar consumption
(g / L) Lactic Acid Production yield(%) Optical purity of lactic acid (%) pH
Unregulated 25571Δpdc1 / L-LpLDH 16.0 13.3 83.0 99 25571Δpdc1, cyb2-PMA1 / L-LpLDH 19.5 15.6 79.6 99 pH
control 25571Δpdc1 / L-LpLDH 51.3 40.9 79.6 99 25571Δpdc1, cyb2-PMA1 / L-LpLDH 61.9 48.6 78.4 99

Summarizing the above experimental results, it was confirmed that the lactic acid production was increased in the Krebtri negative strain (Cluyberomyces maximans) in which pyruvate decarboxylase was inactivated in the present invention, and the alcohol dehydrogenase was further inactivated. Or by introducing lactate dehydrogenase was confirmed to produce a strain capable of producing a high concentration of lactic acid. In addition, the strain of the present invention can selectively introduce the lactate dehydrogenase to be introduced as D or L-lactate dehydrogenase to selectively produce the lactic acid produced, and can use porcine potato powder as a nutrient source, in particular It was confirmed that the pork potato powder can be used as it is without any pretreatment.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed to cover all modifications and variations that come within the meaning and range of equivalents of the following claims and their equivalents, rather than the foregoing detailed description.

Korea Biotechnology Research Institute KCTC12225BP 20120620 Korea Biotechnology Research Institute KCTC12226BP 20120620 Korea Biotechnology Research Institute KCTC12435BP 20130626

<110> Korea Research Institute of Bioscience and Biotechnology <120> Kluyveromyces marxianus deficient in the ethanol fermentation          and use thereof <130> PA130599KR <150> 10-2012-0068809 <151> 2012-06-26 <160> 58 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> H223 368 primer <400> 1 gaaagtggaa tccagtccgg 20 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> H223 369 primer <400> 2 cgaccacacc cgtcctgttg tgttgtgtat atgatt 36 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HJ 370 primer <400> 3 gatgctgtcg gaatggacgc aaggaccctt ttgata 36 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> H223 371 primer <400> 4 ggcattaccg gtcggtccaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HJ 372 primer <400> 5 gcaagaatct ggcaaattgg 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Tc-f primer <400> 6 acaggacggg tgtggtcgcc atg 23 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> U200r primer <400> 7 gttgacccaa tgcgtctccc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> U200f primer <400> 8 gcaagaatct ggcaaattgg 20 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Tc-r primer <400> 9 gtccattccg acagcatcgc cag 23 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HJ394 primer <400> 10 atggctattc cagaaactca aaag 24 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HJ458 primer <400> 11 tggacaccaa gttgccattg 20 <210> 12 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> HJ459 primer <400> 12 cgaccacacc cgtcctgtgt cgtgggtgta accggac 37 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HJ460 primer <400> 13 gatgctgtcg gaatggacct tgccagaaat ttacga 36 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ461 primer <400> 14 ttaatctaaa ttgtatca 18 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ462 primer <400> 15 gatcgtcaat ttacagtc 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ463 primer <400> 16 caatacgcta ccgctgac 18 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ412 primer <400> 17 tggctaactg actgactg 18 <210> 18 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> HJ413 primer <400> 18 cgaccacacc cgtcctgttt ctagttgttg gttgttg 37 <210> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HJ414 primer <400> 19 gatgctgtcg gaatggacga ctgtaaattg acgatc 36 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HJ415 primer <400> 20 tcaagcttat accaatttc 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HJ416 primer <400> 21 tctataaaga aggatggggg 20 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ434 primer <400> 22 taagggctgg gaaatcgg 18 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ482 primer <400> 23 gacgacttgg gcttggtg 18 <210> 24 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HJ483 primer <400> 24 cgaccacacc cgtcctgtgt ataatgtggg tgtacg 36 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HJ484 primer <400> 25 gatgctgtcg gaatggacac gaagctcagt ccaact 36 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ485 primer <400> 26 ggtaaaatgg ataatcgc 18 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ486 primer <400> 27 gtagaacaaa gaataggc 18 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ487 primer <400> 28 atgttcagac tagcacgc 18 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HJ464 primer <400> 29 atgtctgaaa ttactctag 19 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HJ465 primer <400> 30 cgaccacacc cgtcctgtac caacaacgtg caaaac 36 <210> 31 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HJ466 primer <400> 31 gatgctgtcg gaatggactt gttcgtcttg aacaac 36 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HJ467 primer <400> 32 ctattcttgc ttggcgttg 19 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ468 primer <400> 33 tctctttatc ctctccct 18 <210> 34 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HJ469 primer <400> 34 gaatgtcttc caacatttc 19 <210> 35 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HJ403 primer <400> 35 agggccccag gcgcacagca cccagac 27 <210> 36 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> HJ390 primer <400> 36 aattgctcag caaagattac atatg 25 <210> 37 <211> 1047 <212> DNA <213> KM ADH1 <400> 37 atggctattc cagaaactca aaagggtgtt atcttctacg aaaacggtgg tgagttgcaa 60 tacaaggaca ttccagttcc aaagccaaag ccaaacgaac ttttgatcaa cgttaagtac 120 tctggtgtgt gtcacaccga tttgcacgca tggcaaggtg actggccatt ggacaccaag 180 ttgccattgg tgggtggtca cgaaggtgct ggtattgttg ttgccatggg tgagaacgtt 240 actggctggg aaatcggtga ctatgctggt atcaagtggt tgaacggttc ctgtatgtct 300 tgtgaggagt gtgagttgtc gaacgaacca aactgtccaa aggccgactt gtctggttac 360 acacacgacg gttctttcca acaatacgct accgctgacg ctgtccaggc tgccagaatt 420 ccaaagaacg tcgacttggc cgaggttgcc ccaatcttgt gtgccggtgt taccgtgtac 480 aaggctttga agtctgctca catcaaggct ggtgactggg tcgccatctc tggtgcatgt 540 ggtggtctag gttccttggc catccaatac gccaaggcta tgggttacag agtgctaggt 600 atcgatgctg gtgacgaaaa ggccaaattg ttcaaggaat tgggcggtga atactttatc 660 gactttacca agaccaagga tatggtagca gaagtcattg aggccaccaa cggtggtgcc 720 cacgccgtca ttaacgtgtc tgtgtccgaa gccgccatct ctacctctgt cttgtacacc 780 agatcaaacg gtaccgtcgt cttggtcggt ttgccaagag acgctcaatg taagtctgat 840 gtcttcaacc aagtcgtcaa gtccatctcc attgttggtt cttacgttgg taacagagca 900 gacaccagag aagccctaga cttcttctcc agaggtttgg tcaaggcccc aattaagatt 960 ctcggcttgt ccgaattggc aaccgtttac gacaagatgt ccaagggcca aatcattggt 1020 agaattgtcg ttgacacttc caaataa 1047 <210> 38 <211> 1047 <212> DNA <213> KM ADH2 <400> 38 atgtctattc caactactca aaagggtgtt atcttctacg aaaacggtgg tcaattgtac 60 tacaaggaca tcccagtccc aaagccaaag tctaacgaac ttttgatcaa cgttaagtac 120 tccggtgtct gccacaccga tttgcacgcc tggaagggtg actggccatt ggacaccaag 180 ttgccattgg tcggtggtca cgaaggtgcc ggtgtcgtcg tcgccatggg tgacaatgtc 240 aagggctgga agatcggtga ccttgccggt atcaaatggt tgaacggttc ttgtatgaac 300 tgtgaagaat gtgaattgtc caacgaatcc aactgtccag acgctgactt gtccggttac 360 acccacgacg gttctttcca acaatacgct actgctgacg ctgtccaagc cgctcacatc 420 ccagctggta ccgacttggc tcaagtcgcc ccaatcttgt gtgccggtgt taccgtctac 480 aaggctttga agaccgctga aatgaaggct ggtgactggg tcgccatctc cggtgctgct 540 ggtggtctag gttccttggc cgtccaatac gccaaggcca tgggtttcag agtcctaggt 600 atcgatggtg gtgaaggtaa ggaagaattg ttcaagagct tgggtggtga agtcttcatt 660 gacttcacca agtctaagga cattgtcggt gaagtcatca aggctaccaa cggtggtgct 720 cacggtgtca tcaacgtctc cgtctccgaa aaggccatcg aatcctccat cgaatactgt 780 agatccaacg gtaccgtcgt tctagtcggt ttgccaaagg atgctaagtg taagtccgat 840 gtcttcaacc aagtcgtcaa gtccatccac atcgttggtt cttacgtcgg taacagagct 900 gacaccagag aagctcttga cttcttctgc agaggtctag tcaacgcccc aatcaaggtt 960 gtcggtttgt ccaccttgcc agaaatttac gaaaagatgg aacaaggtaa ggttctaggt 1020 agatacgttg ttgacacttc caaataa 1047 <210> 39 <211> 1128 <212> DNA <213> KM ADH3 <400> 39 atgcttagat taactaacgc cagaacattt gtcaagccat tgcgcgctac tgcattcggt 60 tccttcagaa cgatggcctc tgttgctatt ccagaaaagc aaaagggtgt tattttctac 120 gaaaacggtg gtaaactaga atacaaggac attccagttc caaagccaaa gccaaacgaa 180 atcttgatca acgtcaagta ctctggtgtg tgtcacacag atttgcacgc ctggaagggt 240 gactggccat tggccaccaa gttgcctttg gtcggtggtc acgaaggtgc cggtgtcgtt 300 gtcgccatgg gtgaaaacgt taagggctgg gaaatcggtg actatgccgg tatcaagtgg 360 ttgaacggtt cttgtatgtc ctgtgaattc tgtgaattgt ccaacgaatc taactgtcca 420 gacgccgact tgtccggtta cacccacgac ggttccttcc aacaatacgc tactgcagat 480 gctgtgcaag ctgcaagaat tccaaagggt accgatttgg ctgaaatcgc cccaatcttg 540 tgtgctggtg ttaccgtcta caaggctttg aagaccgctg gcttgaaggc tggtgactgg 600 gtcgccatct ctggtgccgc tggtggtcta ggttccttgg ctgtccaata cgccaaggca 660 atgggttaca gagttgtcgg tattgatggt ggtgaagaaa agggtaagtt ggccaagcaa 720 ttgggtgccg aagccttcgt tgacttcacc aagaccaagg acatgattgg tgaaatccaa 780 gaaatcacca acggtggtcc acacggtgtc atcaacgtct ccgtctccga agccgccatg 840 aacgcttcca cccaatacgt cagaccaact ggtactgtcg tgttggttgg tttgccagcc 900 ggtgcagtca tcaagtctga ggtcttctcc cacgtcgtta agtccattgc catcaagggt 960 tcttacgtcg gtaacagagc tgacaccaga gaagccattg agttcttcgc tgctggtaag 1020 gttaagtctc caattaaggt tgttggcttg tctgaattgc caaaggttta cgaactaatg 1080 gaacaaggta agatcttggg tagatacgtc gtcgacactg aaaaataa 1128 <210> 40 <211> 1140 <212> DNA <213> KM ADH4 <400> 40 atgttcagac tagcacgcgc tcagaccagc attaccacca ccagcaaggc tctaggtggc 60 tccagaagac tattcgtcag actaaactcc tctttcgcca tcccagaatc ccaaaagggt 120 gtgattttct acgaaaacgg cggtaagttg gaatacaagg accttccagt tccaaagcca 180 aagccaaatg aaatcttgat caacgtcaag tactccggtg tgtgtcacac tgatttgcac 240 gcctggaagg gtgactggcc attgccagtt aagttgcctt tggtcggtgg tcacgaaggt 300 gccggtgtcg tcgttgccaa gggtgaaaac gttaccaact tcgagatcgg tgactacgca 360 ggtatcaagt ggttgaacgg ttcttgtatg tcttgtgaac tctgtgaaca aggttacgaa 420 tccaactgtt tgcaagctga cttgtctggt tacacccacg acggttcctt ccaacaatat 480 gccactgctg acgctgttca agctgctcaa attccaaagg gtaccgattt ggctgaaatc 540 gccccaatct tgtgtgccgg tgtcaccgtc tacaaggctc taaagaccgc tgacttgcaa 600 ccaggtcaat ggatcgctat ttccggtgct gctggtggtc ttggttccct agccgttcaa 660 tacgccaagg caatgggtct aagagttcta ggtatcgacg gtggtccagg taaggaagaa 720 ttgttcaaga gcttgggtgg tgaagtcttc attgacttca ccaagtccaa ggacatggtc 780 gcagacatcc aggaagccac caacggtggt cctcacggtg tgatcaacgt ctccgtctcc 840 gaggccgcta tctccatgtc caccgagtac gtcagaccaa ccggtgtggt cgttctagtc 900 ggtttgccag cccacgctta cgtcaagtcc gaagtcttct cccacgtcgt caagtctatc 960 tctattaagg gttcttacgt cggtaacaga gcagacacca gagaagctat tgacttcttc 1020 accagaggtt tggtcaagtc tccaatcaag gttgttggtt tgtctgaatt gccaaaggtt 1080 tatgaattga tggaagctgg taagatcttg ggtagatacg tcgttgacac ttccaaataa 1140                                                                         1140 <210> 41 <211> 1695 <212> DNA <213> KM PDC1 <400> 41 atgtctgaaa ttactctagg tcgttacttg ttcgaaagat taaagcaagt cgaagtccaa 60 accatcttcg gtttgccagg tgacttcaac ttgtccctat tggacaagat ctacgaagtc 120 ccaggtatga gatgggctgg taacgctaac gaattgaacg ctgcttacgc tgctgatggt 180 tacgccagat taaagggtat ggcctgtgtc atcaccacct tcggtgttgg tgaattgtct 240 gccttgaacg gtattgccgg ttcttacgct gaacacgttg gtgttttgca cgttgttggt 300 gttccatcca tctcttccca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360 gacttcactg ttttccacag aatgtcttcc aacatttctg aaaccactgc tatgatcact 420 gacatcaaca gtgctccatc tgaaatcgac agatgtatca gaactaccta catctctcaa 480 agaccagttt acttgggttt gccagctaac ttggttgacc tgaaggttcc agcttctcta 540 ttggaaaccc caattgactt gagcttgaag ccaaacgacc cagaagctga aaacgaagtt 600 ctagaaaccg ttttggaatt gatcaaggac gccaagaacc cagttatctt ggccgatgct 660 tgttgttcca gacacaacgt taaggctgaa accaagaagt tgattgacat cactcaattc 720 ccagccttcg ttaccccaat gggtaagggt tccattgacg aacaacaccc aagattcggt 780 ggtgtctacg tcggtacctt gtcttctcca gaagttaagg aagctgttga atccgctgac 840 ttggttctat ctgtcggtgc tctattgtcc gatttcaaca ccggttcttt ctcttactct 900 tacaagacca agaacattgt cgaattccac tccgactaca tcaaggtcag aaacgccact 960 ttcccaggtg tccaaatgaa gttcgtcttg caaaagttgt tgaccaaggt caaggatgct 1020 gctaagggtt acaagccagt tccagttcct cacgctccaa gagacaacaa gccagttgct 1080 gactctactc cattgaagca agaatgggtc tggactcaag tcggtaagtt cctacaagaa 1140 ggtgatgttg ttctaactga aaccggtacc tccgctttcg gtatcaacca aacccacttc 1200 ccaaatgaca cctacggtat ctcccaagtc ttgtggggtt ccattggttt caccggtggt 1260 gctactctag gtgctgcttt cgctgctgaa gaaatcgatc caaagaagag agttatcttg 1320 ttcattggtg acggttcttt gcaattgact gtccaagaaa tctccaccat gatcagatgg 1380 ggtttgaagc catacttgtt cgtcttgaac aacgatggtt acaccattga aagattgatt 1440 cacggtgaaa ccgctcaata caactgtatc caatcttgga agcacttgga cctattgcca 1500 accttcggtg ccaaggacta cgaagccgtc agagtcgcca ccactggtga atggaacaag 1560 ttgaccactg acaagaagtt ccaagaaaac tctaagatca gattgatcga agtcatgttg 1620 ccagtcatgg atgctccatc caacttggtc aagcaagctc aattgactgc ttccatcaac 1680 gccaagcaag aatag 1695 <210> 42 <211> 981 <212> DNA <213> L. casei LDH <400> 42 atggcaagta ttacggataa ggatcaccaa aaagttattc tcgttggtga tggcgccgtt 60 ggttcaagct atgcctatgc aatggttttg caaggtatcg ctcaagaaat cgggatcgtt 120 gacattttca aggacaagac gaaaggtgac gcaattgatt taagcaacgc gctcccattc 180 accagtccta agaagattta ttctgctgaa tacagcgatg ccaaggatgc tgatctggtt 240 gttatcactg ctggtgctcc tcaaaagcct ggcgaaactc gcttggatct ggttaacaag 300 aacttgaaga ttttgaagtc cattgttgat cctatcgtgg attctggctt taacggtatc 360 ttcttggttg ctgctaaccc agttgatatc ttgacttatg ctacctggaa gctttccgga 420 ttcccgaaga accgggttgt tggttcaggt acttctctgg acaccgctcg tttccgtcaa 480 tccattgctg aaatggttaa cgttgatgct cgttccgtcc acgcatatat catgggtgaa 540 cacggcgaca cagaattccc tgtatggtcc cacgctaaca ttggtggcgt taccatcgct 600 gaatgggtta aagcacatcc agaaatcaaa gaagacaaac ttgttaagat gtttgaagac 660 gttcgtgacg ctgcttacga aatcatcaaa ctcaagggtg caaccttcta cggtatcgca 720 actgctttgg cacggatttc caaagcaatt cttaacgatg aaaatgcggt actcccattg 780 tccgtttaca tggacggcca atatggcttg aacgacatct acattggtac acctgctgtg 840 atcaaccgca atggtatcca gaacattctg gaaatcccat tgaccgacca cgaagaagaa 900 tccatgcaga agtcggcttc acaattgaag aaagttctga ccgatgcgtt tgctaagaac 960 gacatcgaaa cacgtcagta a 981 <210> 43 <211> 963 <212> DNA <213> L-LpLDH <400> 43 atgtcaagca tgccaaatca tcaaaaagtt gtgttagtcg gcgacggcgc tgttggttct 60 agttacgctt ttgccatggc acaacaagga attgctgaag aatttgtaat tgtcgatgtt 120 gttaaagatc ggacaaaggg tgacgccctt gatcttgaag acgcccaagc attcaccgct 180 cccaagaaga tttactcagg cgaatattca gattgtaagg acgctgactt agttgttatt 240 acagccggtg cgcctcaaaa gcctggtgaa tcacgtttag acttagttaa caagaattta 300 aatatcctat catccattgt caaaccagtt gttgactccg gctttgacgg catcttctta 360 gttgctgcta accctgttga catcttaact tacgctactt ggaaattctc aggtttccca 420 aaggatcgtg tcattggttc agggacttcc ttagactctt cacgtttacg cgttgcgtta 480 ggcaaacaat tcaatgttga tcctcgttcc gttgatgctt acatcatggg tgaacacggt 540 gattctgaat ttgctgctta ctcaactgca accatcggga cacgtccagt tcgcgatgtt 600 gctaaggaac aaggcgtttc tgacgaagat ttagccaagt tagaagacgg cgttcgtaac 660 aaagcttacg acatcatcaa cttgaagggt gccacgttct acggtattgg gactgcttta 720 atgcggattt ccaaagccat tttacgtgat gaaaatgccg ttttaccagt aggtgcctac 780 atggacggcc aatacggctt aaacgacatt tatatcggga ctccggctgt gattggtgga 840 actggtttga aacaaatcat cgaatcacca ctttcagctg acgaactcaa gaagatgcaa 900 gattccgccg caactttgaa aaaagtgctt aacgacggtt tagctgaatt agaaaataaa 960 taa 963 <210> 44 <211> 972 <212> DNA <213> L-LaLDH <400> 44 atggcaagag ttgaaaaacc tcgtaaagtt attttagtcg gtgatggtgc tgtaggttct 60 acctttgcat tttcaatggt acaacaaggt atcgctgaag aattaggtat tatcgatatc 120 gctaaggaac acgttgaagg tgacgcaatc gatttagctg acgcaactcc ttggacttct 180 ccaaagaaca tttacgcagc tgactaccca gattgtaagg atgctgactt agttgttatt 240 actgctggtg ctccacaaaa gccaggcgaa actcgtcttg atcttgttaa caagaacttg 300 aagattttat catcaatcgt tgaaccagtt gttgaatcag gttttgaagg tattttctta 360 gtagttgcta acccagttga tatcttaact cacgcaactt ggagaatgtc aggcttccct 420 aaggatcgtg ttatcggttc aggtacttca cttgatactg gtcgtcttca aaaagttatt 480 ggtaaaatgg aaaacgttga cccaagttca gttaatgcat acatgcttgg tgaacacggt 540 gatactgaat tcccagcatg gagctacaac aatgtgggtg gcgtaaaggt tgctgactgg 600 gttaaggctc acaacatgcc tgaatctaag cttgaagaca tccaccaaga agttaaggac 660 atggcttacg acattattaa caagaaaggt gctaccttct acggtatcgg tactgcttca 720 gcaatgatcg ctaaggctat cttgaacgat gaacaccgtg tacttccact ttcagtacca 780 atggatggtg aatatggttt acacgatctt cacatcggta ctcctgcagt tgttggccgc 840 aagggtcttg aacaagttat cgaaatgcca ttaagcgata aggaacaaga attaatgact 900 gcttcagcag atcaattaaa gaaggttatg gacaaggcct tcaaagaaac tggcgttaag 960 gttcgtcaat aa 972 <210> 45 <211> 981 <212> DNA <213> L-LrLDH <400> 45 atggcaagta ttacggataa ggatcaccaa aaagttattc tcgttggtga tggcgccgtt 60 ggttcaagct atgcctatgc aatggttttg caaggtatcg ctcaagaaat cgggattgtt 120 gacattttca aggacaagac gaaaggtgac gcaattgatt taagcaacgc gctcccattc 180 accagtccta agaagattta ttctgctgaa tacagcgatg ccaaggatgc tgatctggtt 240 gttatcactg ctggtgctcc tcaaaagcct ggcgaaactc gcttggatct ggttaacaag 300 aacttgaaga ttttgaagtc cattgttgat ccgatcgtgg attctggctt taacggtatc 360 ttcttggttg ctgctaaccc agttgatatc ttgacttatg ctacttggaa gctttccgga 420 ttcccgaaga gccgggttgt tggttcaggt acttctctgg acaccgctcg tttccgtcaa 480 tccattgctg aaatggttaa cgttgatgct cgttccgtcc acgcatatat catgggtgaa 540 cacggcgaca cagaattccc tgtatggtcc cacgctaaca ttggtggcgt taccatcgct 600 gaatgggtta aagcacatcc agaaatcaaa gaagacaaac ttgttaagat gtttgaagac 660 gttcgtgacg ctgcttacga aatcatcaaa ctcaagggtg caaccttcta cggtatcgca 720 actgctttgg cacggatttc caaagcaatt cttaacgatg aaaatgcggt actcccattg 780 tccgtttaca tggacggcca atatggcttg aacgacatct acattggtac acctgctgtg 840 atcaccgca atggtattca gaacattctg gaaatcccat tgaccgacca cgaagaagaa 900 tccatgcaga agtcggcttc acaattgaag aaagttctga ccgatgcgtt tgctaagaac 960 gacatcgaaa cacgtcagta a 981 <210> 46 <211> 978 <212> DNA <213> L-LlLDH <400> 46 atggctgata aacaacgtaa aaaagttatc cttgtaggtg acggtgctgt aggttcatca 60 tacgcttttg ctcttgtaaa ccaagggatt gcacaagaat taggaattgt tgaccttttt 120 aaagaaaaaa ctcaaggaga tgcagaagac ctttctcatg ccttggcatt tacttcacct 180 aaaaagattt actctgcaga ctactctgat gcaagcgacg ctgacctcgt tgtcttgact 240 tctggtgctc cacaaaaacc aggtgaaact cgtcttgacc ttgttgaaaa aaatcttcgt 300 atcactaaag atgttgtaac taaaattgtt gcttcaggtt tcaaaggaat cttccttgtt 360 gctgctaacc cagttgatat cttgacatac gctacttgga aattctcagg tttccctaaa 420 aaccgcgttg taggttcagg tacttcactt gatactgcac gtttccgtca agcattggca 480 gaaaaagttg atgttgacgc tcgttcaatc cacgcataca tcatgggtga acacggtgac 540 tcagaatttg ccgtttggtc acacgctaac gttgctggtg ttaaattgga acaatggttc 600 caagaaaatg actaccttaa cgaagctgaa atcgttgaat tgtttgaatc tgtacgtgat 660 gctgcttact caatcatcgc taaaaaaggt gcaacattct acggtgtcgc tgtagctctt 720 gctcgtatta ctaaagcaat tcttgatgat gaacatgcag tacttcctgt atcagtattc 780 caagatggac aatatggcgt aagcgactgc taccttggtc aaccagctgt agttggtgct 840 gaaggtgttg ttaacccaat ccacattcca ttgaatgatg ctgaaatgca aaaaatggaa 900 gcttctggtg ctcaattgaa agcaatcatt gacgaagctt ttgctaaaga agaatttgct 960 tctgcagtta aaaactaa 978 <210> 47 <211> 999 <212> DNA <213> D-LpLDH <400> 47 atgaaaatta ttgcatatgc tgtacgtgat gacgaacgtc cattcttcga tacttggatg 60 aaagaaaacc cagatgttga agttaaatta gttccagaat tacttactga agacaacgtt 120 gacttagcta aaggcttcga cggtgccgat gtataccaac aaaaggacta tactgctgaa 180 gtattgaaca agttagccga cgaaggggtt aagaacatct ctcttcgtaa cgttggtgtt 240 gataacttgg acgttcctac tgttaaagca cgtggcttaa acatttctaa cgtacctgca 300 tactcaccaa atgcgattgc tgaattatca gtaacgcaat tgatgcaatt attacgtcaa 360 accccattgt tcaataagaa gttagctaag caagacttcc gttgggcacc agatattgcc 420 aaggaattaa acaccatgac tgttggtgtt atcggtactg gtcggattgg ccgtgctgcc 480 atcgatattt tcaaaggctt cggcgctaag gttatcggtt acgatgttta ccggaatgct 540 gaacttgaaa aggaaggcat gtacgttgac accttggacg aattatacgc ccaagctgat 600 gttatcacgt tacacgttcc tgcattgaag gataactacc acatgttgaa tgcggatgcc 660 ttcagcaaga tgaaagatgg cgcctacatc ttgaactttg ctcgtgggac actcatcgat 720 tcagaagact tgatcaaagc cttagacagt ggcaaagttg ccggtgccgc tcttgatacg 780 tatgaatacg aaactaagat cttcaacaaa gaccttgaag gtcaaacgat tgatgacaag 840 gtcttcatga acttgttcaa ccgcgacaat gttttgatta caccacatac ggctttctac 900 actgaaactg ccgttcacaa catggtgcac gtttcaatga acagtaacaa acaattcatc 960 gaaactggta aagctgatac gcaagttaag tttgactaa 999 <210> 48 <211> 1050 <212> DNA <213> D-LaLDH <400> 48 atggtcatac taataaattt tacggaggtt gaatttatga caaagatttt tgcttacgct 60 attcgaaaag acgaagaacc attcttgaat gaatggaagg aagctcacaa ggatatcgat 120 gttgattaca ctgataagct tttgactcct gaaactgcaa agcttgctaa gggtgcagat 180 ggtgttgttg tttaccaaca attagactac actgcagata ctcttcaagc tttagcagac 240 gctggcgtaa ctaagatgtc attacgtaac gttggtgtcg acaacattga catggacaag 300 gctaaagaac ttggctttga aattactaat gttccagttt actcaccaga cgctattgct 360 gaacatgctg ctattcaagc tgcacgtgta ttacgtcaag acaagcgcat ggacgaaaag 420 atggctaaac atgatttacg ttgggcacca actatcggcc gtgaagttcg tgaccaagtt 480 gtcggtgttg ttggtactgg tcacattggt caagtattta tgagaattat ggaaggtttc 540 ggcgcaaagg ttattgctta cgatatcttc aagaacccag aacttgaaaa gaagggttac 600 tacgttgact cacttgacga cttgtacaag caagctgatg taatttcact tcacgtacca 660 gatgttccag ctaacgttca catgatcaac gacaagtcaa tcgctgaaat gaaagacggc 720 gttgtaattg taaactgctc acgtggtcca cttgttgaca ctgatgctgt aattcgtggt 780 ttggactcag gcaagatctt cggcttcgtt atggatactt acgaagacga agttggtgta 840 tttaacaagg attgggaagg taaagaattc ccagacaagc gtttggcaga cttaattgat 900 cgtccaaacg tattggtaac tccacacacc gccttctaca ctactcacgc tgtacgtaac 960 atggttgtta aggcattcaa caacaacttg aagttaatca acggtgaaaa accagattcc 1020 ccagttgctt tggacaagaa caagttctaa 1050 <210> 49 <211> 1002 <212> DNA <213> D-LrLDH <400> 49 atgaagatta ttgcatatgg tgcacgcgtg gatgagatcc aatatttcaa gcagtgggct 60 aaggaaaccg gcaacacgct ggaatatcat acggaatttc ttgatgagca tactgttgaa 120 tgggcaaaag gctttgacgg cattaactca ctacaaacga cgccatacgc agctggtgtg 180 tttgaaaaaa tgcacgaata tggcatcaag tttctcacca tccgcaatgt cggaaccgac 240 aatatcgata tggcggcgat gaataaatat ggcattcgct taagtaatgt tcccgcatat 300 tcaccggctg ccattgctga atttgcgtta accgatacgt tatatctact tcgcaacatg 360 ggaaaggttc aagcacagct acaggcaggc gactacgaaa aagccggcac cttcatcggt 420 aaagaacttg gtcagcaaac agtcggcgtg atgggtaccg ggcacattgg tcgcgttgct 480 atcaagctct tcaaaggttt tggtgccaaa gtgattgctt acgatccata tccgatgaaa 540 ggcgatcatc cggactttga atatgtcagc ctggaagaac tattcaaaca aagtgacatc 600 attgatcttc acgttccggg cattaaacaa aatacccaca ttatcaacga ggcagcgttt 660 gatcttatga agccaggcgt gattgtggtt aacaccgcgc ggccgaatct gattgatacc 720 caggcgatgc tcagcaatct gaagtccggt aaactggctg gcgtcgggat cgatacgtac 780 gaatatgaaa ccgaagatct gttgaacctc gccaaacacg gtagcttcaa ggatccgtta 840 tgggatgaac tgctcgctat gccaaatgtt gtcctcagcc cgcatattgc gtactacaca 900 gaaaccgccg tgcacaacat ggtttacttc tcactgcaga atttagtcga ctttttgaca 960 aagggaaaga cgaatactga agtgacagca ccggcgaaat aa 1002 <210> 50 <211> 1680 <212> DNA <213> D-LlLDH <400> 50 atggacttag tcgaagaact agtcaatgtt gttggtaaaa aaaatgttat tactaacaaa 60 gcaaaatctt tacgttttag ggaagggtac cgctcaggta agggcgatgc agttgcagtc 120 gtatttccaa ccactttaat ggcaatgtgg cgtgtgctta atgttttgca tgataacaat 180 attatcatta ttatgcaagc agccaacact agtttgactg agggatccgt accagcccca 240 ggttatgacc gttctgcagt tgttgtgaac ggcatgaaaa ttaaagattt acagttaatc 300 aataatgggg aacaagtact agcattcccc ggtacgacac tgtttcactt agaaaatgaa 360 ttgcgaccac ttaaacgtga accacattca gtcattggct catctaattt gggtgcatcg 420 gtcattggtg gtattaacaa caattctggt ggtgctttga tcagaagagg tccagcttat 480 acagagctat cattgtatgt ttggattgat gaacatgacg aaatgcactt agttaaccat 540 ttaggcattg atttgggcaa aacgcccgaa gagattatta ctaatttgca gaaccgcaat 600 tttgatttga atcaagtacc agctactgaa catcatggtt caatgttcca tcatgaacag 660 gttgtgcgtg acgttgaatc tgataagcct attcgataca atgcaaatcc taaagaattg 720 tatgaagtat ctggatcgtc cggtcacgtt gcggcattgg ctgtccgctt agatacattc 780 ccaatggata aaaaaacaca aatgttttac attggaacga ataatcccga tgagttggaa 840 gatattcgtc gccatattat gacgacattc aaagaattgc ctgtttctgg tgaatatatg 900 cataaaaatg cttataaatt ggccaagaaa tatggtaaag actcactaat tgtgattgaa 960 aaaattggta cgggtcattt gccccaaatg tttgcgctaa aagcatgggg cgaacgcttt 1020 ttgaagcaca ttccgttttt caaaccttat ttcccagatc gtttgcttca aacattaagt 1080 aatttatttc ctaatcaaat gccaaagcgg ttagatgatt attacgaaaa gtacgaccat 1140 tacctacaat tgaaaatggc tggtaatggt attgaagaag cgcgagagta tctcaaatct 1200 tattttgaca aagccagtgg cgactacttt gaagccgatg ccaatgagac cagtaaagcg 1260 gcaactcacc gctatgtaac agctggggtt gcgattcgat atcaagaatt aaaacaggat 1320 agtattgaca ttttgccact agatatcgcg ttggcaagta atgattataa gtggttcgag 1380 catttgccta aagagattga agacaaaata gaacacaaaa tttattatgg tcacttactt 1440 gatcacgtta tgcaccagga ttatattcta aaacctggcg tcgatgcgca tgaacttaaa 1500 aaagaaatgc ttaaaatttt ggatgagcgt cacgctgttt atccagccga acacaatgtt 1560 gggcatctat atttggcagc accggcctta atcaagcact ataagaaaaa tgatccaact 1620 aatagtttca accctggcgt tggtaaacaa acaatgtcta aatattgggg cgaatactaa 1680                                                                         1680 <210> 51 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HJ628 primer <400> 51 aagatggaag ggaaggaag 19 <210> 52 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HJ629 primer <400> 52 cgaccacacc cgtcctgtga acttttaccc tgtccg 36 <210> 53 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HJ630 primer <400> 53 gatgctgtcg gaatggactg tatctatctc tcctcc 36 <210> 54 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ631 primer <400> 54 gacgttatgt aattaagg 18 <210> 55 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HJ632 primer <400> 55 gccattcagg ttaaatcg 18 <210> 56 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HJ633 primer <400> 56 atgttgtttt tggctgctg 19 <210> 57 <211> 1614 <212> DNA <213> CYB2 <400> 57 atgttgtttt tggctgctgg ggcttcagct gtagctggga tcgggctgct ctctcaattt 60 agcgacagct tgcaaaatgc tacgaaagaa gagctaaaca agccaaaggt ttccccgctc 120 gaggtcgcca agcactcgag tcccgatgac tgctgggtgg tgatcgatgg gtttgtctac 180 aatttgacgg aattcattag cgcccatcct ggtggaccag ctatcatcga gaacaacgcc 240 ggtaaggatg tgacggcgat ctttggccca attcatgcgc cagatgtcat tgagaagtac 300 attgctccgg agaaccggat cggtcctcta gacgggaaaa tgcccgacga cttgatctgt 360 gcgccattga cgcccggtga gactcctgag gatgttgcca gaaaggagga gttgcgtcaa 420 aatatgccag atctcgactc gttggtcaac atttacgatt tcgagttctt ggcgtcccag 480 attttgacca aacaggcatg gtcctactac tcttctgctg ccgatgacga agtcacccac 540 agagagaacc atgctgcata ccaccgtatc ttcttcaagc caagaatcct agtcaacgtc 600 aaggaggtgg acacttccac caccatgttg ggtgaaaaag tgggtgttcc attctatgtg 660 tctgctaccg ctctatgtaa attgggtaat ccaaaggaag gtgaaaagga tatcgctaga 720 ggttgtggcg agagcgacgt gaagccaatc cagatgattt ccactttggc ctcttgttct 780 ttgcaagaaa tcgtcgaagc agcaccttcc aaggaccaaa tccaatggtt ccaattgtat 840 gtcaacagtg accgtaagat taccgaggaa ttgatcaaaa acgtcgaaaa gctgggcttg 900 aaggctatct tcgttactgt ggatgcccca tctctcggta acagagaaaa ggatgctaag 960 gtcaaattca ccaacaagga cagttccgca aaggccatgg aaaagagcaa cgtcaaggaa 1020 tccaagggtg cttccagagc tctttccact ttcattgacc ctgctctatg ctgggatgat 1080 atcgttacct tgaaatcaaa gaccaagttg ccaattgtca ttaagggtgt tcaatgcgtc 1140 gaagacgtct tgaaagccgc agaaattggt gccgccggtg tcgtcttgtc caaccacggt 1200 ggtagacaac tagacttttc cagagcgcca atcgaagtct tggccgaaac aatgcctatt 1260 ctaaaagaaa agaaactaga cgacaagatc gaaatcttca tcgatggtgg tgtcagaaga 1320 ggtaccgata tattgaaggc cttgtgtctt ggtgccaagg gtgtcggttt aggtagacca 1380 ttcttgtacg caaacagttg ttacggtaag gaaggtgtca agaaggctat cgaattgcta 1440 aaggacgaac tagaaatgtc aatgagattg cttggtgtca ccagcatcga ccaattgtct 1500 gagaagtatc tggatctatc tactcttcac ggtagaactg ttagcgtacc tcgtgacaac 1560 ttgtacaacg gagtgtatgt tccacacgaa ccaactgact tcaaggaaaa ttga 1614 <210> 58 <211> 2700 <212> DNA <213> PMA1 <400> 58 atgtcagctg ccactgaacc aactaaggaa aagcctgtta aagcccagga ctccgatgat 60 gaagatgaag atatcgatca attgatcgaa gacttgcaat ctcatcacgg attagacgat 120 gacagtgatg acgatggtca tcacgccgct ggtactgcta gaccagttcc agaagaacta 180 ttacaaactg acccatctta cggtttgact tctgacgaag ttgccaagag aagaaagaag 240 tacggtttga accaaatgtc tgaagaagtt gaaaacttgc ttgtcaagtt cttgatgttc 300 ttcatcggtc caattcaatt cgttatggaa gccgctgctg ttttggctgc tggtttggaa 360 gattgggtcg atttcggtgt tatctgtggt ttattgtttt tgaacgctgg tgttggtttc 420 atccaagaat accaagccgg ttccattgtc gaagaattga agaagacttt ggccaacact 480 gctgttgtta tcagagacgg taacttggtt gaaatcccag ccaacgaagt tgtcccaggt 540 gatatcttgc aattggaaga tggtgttgtt atcccagccg atggtcgttt ggttaccgaa 600 gactgtttcg tccaaatcga tcaatctgct atcactggtg aatctttggc cgtcgacaag 660 agatacggtg actccacttt ctcttcttct actgttaaga gaggtgaagc tttcatgatt 720 gttactgcta ctggtgactc tactttcgtc ggtagagctg ctgctttggt taacaaggct 780 tccggtggta ctggtcactt cactgaagtt ttgaacggta tcggtactat tttgttgatt 840 ttggttattg tcactttgtt gttggtctgg gttgcttcct tctacagaac caacaagatt 900 gtcagaatct tgagatacac tttggccatc accattgttg gtgtcccagt tggtttgcca 960 gctgtcgtta ccaccaccat ggctgtcggt gctgcttact tggctaagaa gcaagctatt 1020 gttcaaaagc tatctgctat tgaatctttg gctggtgtcg aaatcttgtg ttccgacaag 1080 accggtactt tgaccaagaa caagttgtct ttgcacgaac catacactgt tgaaggtgtt 1140 gacccagatg acttgatgtt gactgcttgt ttggctgctt ctagaaagaa gaagggtttg 1200 gatgctattg acaaggcctt cttgaagtct ttgattaact acccaagagc taaggctgct 1260 ttgactaagt acaagttgtt ggaattccac ccattcgacc ctgtctctaa gaaggttact 1320 gctattgttg aatccccaga aggtgaaaga atcatctgtg ttaagggtgc tccattgttc 1380 gttttgaaga ctgttgaaga agaacatcca attccagaag atgtccgtga aaactacgaa 1440 aacaaggttg ctgaattggc ttccagaggt ttcagagctt tgggtgtcgc tagaaagaga 1500 ggtgaaggtc actgggaaat cttgggtgtt atgccatgta tggatcctcc aagagatgac 1560 actgctcaaa ctgttaacga agctaagcac ttgggtttga gagttaagat gttaactggt 1620 gacgctgtcg gtattgctaa ggaaacttgt agacaattgg gtttgggtac caacatttac 1680 aacgctgaaa gactaggtct aggtggtggt ggtgacatgc caggttctga attggctgac 1740 tttgttgaaa acgctgatgg tttcgctgaa gttttcccac aacacaagta caatgtcgtt 1800 gaaatcttgc aacaaagagg ttacttggtt gctatgactg gtgatggtgt taacgatgct 1860 ccttctttga agaaggctga tactggtatt gctgtcgaag gtgctactga tgctgctaga 1920 tctgctgctg atatcgtttt cttggctcca ggtttgtctg ctattattga tgctttgaag 1980 acctctagac aaattttcca cagaatgtac tcatacgtcg tttaccgtat cgctttgtcc 2040 ttgcatttgg aaattttctt gggtctatgg attgctatct tgaaccaatc tctaaacatt 2100 gacttggttg ttttcattgc tattttcgct gatgttgcta ccttggctat tgcttacgac 2160 aacgctccat actctccaaa gccagttaag tggaacttga gaagactatg gggtatgtcc 2220 gtcattttgg gtataatttt ggctatcggt acttggatta ccttgacctc tatgttcgtt 2280 ccaaagggtg gtatcatcca aaacttcggt tctattgacg gtgttttgtt cttgcaaatc 2340 tccttgaccg aaaactggtt gattttcatc actagagctg ctggtccatt ctggtcttcc 2400 atcccatctt ggcaattatc tggtgccgtt ctagctgttg acatcatcgc caccatgttc 2460 accttgttcg gctggtggtc tcaaaactgg aacgacatcg ttactgtcgt tagagtctgg 2520 attttctctt tcggtgtctt ctgtgtcatg ggtggtgctt actacatgat gtccgaatct 2580 gaagcctttg acagattcat gaacggtaag tcctctaagg aaagaccatc tggtagatct 2640 gttgaagatt tcctaatggc catgcaaaga gtctccactc aacacgaaaa ggaaaactaa 2700                                                                         2700

Claims (19)

In the parent strain Kluyveromyces marxianus , the pyruvate decarboxylase is inactivated, and the parent strain is Accession No. KCTC 7001, Group consisting of KCTC 7118, KCTC 7149, KCTC 7150, KCTC 7155, KCTC 7524, KCTC 17212, KCTC 17544, KCTC 17555, KCTC 17631, KCTC 17694, KCTC 17724, KCTC 17725, KCTC 17759, BY25569, BY25571, and BY25573 It is a variant strain.
The method of claim 1,
The parent strain is BY25571, mutant strain.
The method of claim 1,
The mutant strain is that the production of ethanol when cultured in a medium containing sugar is reduced compared to the strain in which pyruvate decarboxylase is not inactivated.
The method of claim 1,
The inactivation is that the pyruvate decarboxylase gene is missing, mutant strain.
5. The method of claim 4,
The variant strain is Accession No. KCTC12225BP, variant strain.
The method of claim 1,
Mutant strain further inactivated alcohol dehydrogenase.
The method of claim 1,
A mutant strain in which Lactate Dehydrogenase activity is introduced.
The method of claim 7, wherein
The gene having lactate dehydrogenase activity is derived from Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, or Lactococcus lactis. It is one, strain strain.
9. The method of claim 8,
The gene having lactate dehydrogenase activity is a nucleotide sequence selected from the group consisting of SEQ ID NOs: 42 to 50, variant strain.
The method of claim 7, wherein
The lactate dehydrogenase is a variant strain that is expressed using a TEF1a promoter derived from Cluyveromyces maximans.
The method of claim 7, wherein
The variant strain is Accession No. KCTC12226BP, Mutant strain.
The method of claim 1,
A variant strain wherein L-lactate cytochrome-c oxidreductase (CYB2) is further inactivated.
The method of claim 12,
Mutant strain with enhanced plasma membrane ATPase (PMA1) activity.
The method of claim 12,
The L-lactate cytochrome-c oxidoreductase is represented by the nucleotide sequence of SEQ ID NO: 57, variant strain.
13. The method of claim 13,
The plasma membrane ATPase is represented by the nucleotide sequence of SEQ ID NO: 58, variant strain.
The method of claim 13,
The variant strain is Accession No. KCTC12435BP, variant strain.
A method for producing lactic acid, comprising culturing the strain of claim 1.
18. The method of claim 17,
The step of culturing the strain is that the pig potato powder as a nutrient source.
18. The method of claim 17,
Wherein the lactic acid is L-lactic acid or D-lactic acid.

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