KR20150096270A - Lactate dehydrogenase mutant, polynucleotide coding the mutant, yeast cell having the polynucleotide, method of the mutant, and method of producing the lactate using the same - Google Patents

Abstract

A lactate dehydrogenase mutant, a polynucleotide encoding the mutant, a yeast cell containing the polynucleotide, a method for producing the mutant, and a method for producing lactate using the mutant.

Description

TECHNICAL FIELD [0001] The present invention relates to a lactate dehydrogenase mutant, a polynucleotide encoding the mutant, a yeast cell containing the polynucleotide, a method for producing the mutant, and a lactate dehydrogenase mutant, a polynucleotide coding mutant, having the polynucleotide, method of the mutant, and method of producing the lactate using the same}

Lactate dehydrogenase mutants, polynucleotides encoding the mutants, yeast cells containing the polynucleotides, a method for producing the mutant, and a lactate production method using the mutant.

Lactate is an organic acid widely used in various industries such as food, pharmaceutical, chemical, and electronics. Lactate is a colorless, odorless and low volatile substance that is well soluble in water. Lactate is not toxic to the human body and is used as a flavoring agent, an acidifier, a preservative, etc. It is also an environmentally friendly alternative polymer material and is a raw material of polylactic acid (PLA) which is a biodegradable plastic.

PLA is technically a ring-open polymerization polyester resin which is converted into dimer lactide for polymer polymerization and can be processed in various ways such as film, sheet, fiber, injection and the like. Therefore, PLA has been in great demand recently as a bioplastics that can widely replace conventional general petrochemical plastics such as polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), and polystyrene (PS).

In addition, since lactate has both a hydroxyl group and a carboxyl group at the same time, its reactivity is very high, and thus it is easy to convert it into an industrially important compound such as lactate ester, acetaldehyde, propylene glycol and the like, And is attracting attention as a raw material.

At present, lactate is produced industrially by petrochemical synthesis process and biotechnological fermentation process. The petrochemical synthesis process is produced by oxidizing ethylene derived from crude oil, making lactonitrile by hydrogenation of cyanide via acetaldehyde, purifying by distillation, and hydrolyzing using hydrochloric acid or sulfuric acid. In addition, the biotechnological fermentation process can produce lactate using a regenerable carbohydrate such as starch, sucrose, maltose, glucose, fructose, xylose as a substrate.

Therefore, even with such conventional techniques, there is a demand for a lactate dehydrogenase mutant and a method for efficiently producing lactate using the lactate dehydrogenase mutant.

One aspect provides a lactate dehydrogenase variant having activity catalyzing the conversion of pyruvate to lactate.

Another aspect provides a polynucleotide encoding said lactate dehydrogenase variant.

Another aspect provides a process for preparing a lactate dehydrogenase variant having activity catalyzing the conversion of pyruvate to lactate.

Another aspect provides a yeast cell comprising a gene encoding the lactate dehydrogenase variant. Another aspect provides a method for producing lactate using yeast cells comprising the lactate dehydrogenase mutant and / or the gene encoding the lactate dehydrogenase.

One aspect provides a lactate dehydrogenase variant having activity catalyzing the conversion of pyruvate to lactate. The lactate dehydrogenase may have the amino acid sequence of SEQ ID NO: 1, 25, 27, 29, 31 or 33.

The term "lactate" as used herein means "lactic acid" or a salt thereof.

The lactate dehydrogenase mutant may be one wherein at least one amino acid residue of the substrate binding site is mutated so as to shorten the distance between the substrate pyruvate and the catalytic site of the lactate dehydrogenase. For example, the catalytic site of the lactate dehydrogenase may be the 106th Arg, 169th Arg, and 193th His in SEQ ID NO: 1. As another example, the catalytic site of the lactate dehydrogenase may be selected from the group consisting of 181th His in SEQ ID NO: 25, 181th His in SEQ ID NO: 27, 181th His in SEQ ID NO: 29, 179th His in SEQ ID NO: 31, 33 to 179th His. The amino acid residue to be mutated may be the 108th Asn in SEQ ID NO: 1 to shorten the distance between the pyruvate and the catalytic site of the lactate dehydrogenase. The lactate dehydrogenase mutant may be one in which the 108th Asn in SEQ ID NO: 1 is substituted with a nonpolar amino acid. The amino acid substituting Asn may be selected from the group consisting of Arg, Gly, Leu and Met. Specifically, the amino acid replacing Asn may be GIy or Leu.

In addition, the lactate dehydrogenase variant may be one wherein one or more amino acid residues are mutated such that the pKa of the proton donor residue is increased. For example, the 193th His of SEQ ID NO: 1, the 181th His of SEQ ID NO: 25, the 181th His of SEQ ID NO: 27, the 181th His of SEQ ID NO: 29, the 179th His of SEQ ID NO: 31, One or more amino acid residues may be mutated to increase the pKa value of His. The amino acid residue to be mutated may be the 108th Asn in SEQ ID NO: 1 to increase the pKa value of the 193th His in SEQ ID NO: The lactate dehydrogenase mutant may be one in which the 108th Asn in SEQ ID NO: 1 is substituted with a nonpolar amino acid. The amino acid substituting Asn may be selected from the group consisting of Arg, Gly, Leu and Met. Specifically, the nonpolar amino acid may be Gly or Leu.

Lactate dehydrogenase (LDH) or its variants may be enzymes that catalyze the conversion of pyruvate to lactate. The lactate dehydrogenase or a variant thereof may be an NAD (P) -dependent enzyme, may also act on pyruvate, and may act on L-lactate or D-lactate. The NAD (P) -independent enzyme may be an enzyme classed as EC 1.1.1.27 which acts on L-lactate, or EC 1.1.1.28 which acts on D-lactate.

The lactate dehydrogenase or variant thereof may be obtained from Bos Taurus or Bos Indicus ). The GenBank Accession number of the lactate dehydrogenase may be AAI46211.1 and the GI may be 148878492. The lactate dehydrogenase may have the amino acid sequence of SEQ ID NO: 1. The polynucleotide encoding the lactate dehydrogenase is called Bos Taurus ). The gene may be one having the nucleotide sequence of SEQ ID NO: 2.

In addition, the dehydrogenase or a variant thereof as the lactate dehydrogenase is Kasei Lactobacillus (Lactobacillus casei ), the amino acid sequence of SEQ ID NO: 25, and the gene encoding the same may have the nucleotide sequence of SEQ ID NO: 26 (GenBank: D12591.1). In addition, the lactate dehydrogenase or a variant thereof may be Bifidobacterium longum) may be derived, have an amino acid sequence of SEQ ID NO: 27, the genes encoding the nucleotide sequence is (GenBank of SEQ ID NO: 28: may be one having a M33585.1). In addition, the lactate dehydrogenase or a variant thereof may be obtained from Bacillus megaterium megaterium ), the amino acid sequence of SEQ ID NO: 29, and the gene encoding the same may have the nucleotide sequence of SEQ ID NO: 30 (GenBank: M22305.1). The lactate dehydrogenase or a variant thereof may be derived from Lactobacillus helveticus , and the gene encoding the amino acid sequence of SEQ ID NO: 31, the nucleotide sequence of SEQ ID NO: 32 (GenBank: Z81318.1). In addition, the lactate dehydrogenase or a variant thereof may be derived from Pediococcus acidylactici , the amino acid sequence of SEQ ID NO: 33, and the gene encoding the same may have the nucleotide sequence of SEQ ID NO: 34 (GenBank: X70927.1).

The lactate dehydrogenase variant may be modified to have improved specific activity over the non-modified lactate dehydrogenase specific activity. The activity of the variant is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 50%, at least about 60% greater than the inactivity of the non-modified lactate dehydrogenase , At least about 70%, or at least about 100%. For example, the lactate dehydrogenase variant of SEQ ID NO: 1 has an increase of at least 0.020 U / mg, at least 0.025 U / mg, at least 0.030 U / mg, or at least about 0.033 U / mg over unmodified lactate dehydrogenase Lt; / RTI > The lactate dehydrogenase variant may be modified to have an improved Km or Kcat over the Km or Kcat of the unmodified lactate dehydrogenase. The Km of the mutant may have an increased value of, for example, about 1.5 times, about 2 times, about 3 times, or about 4 times greater than the Km of the non-modified lactate dehydrogenase. The Kcat of the mutant May have an increased value of, for example, about 1.5 times, about 2 times, or about 2.5 times greater than the Kcat of the non-modified lactate dehydrogenase.

Another aspect provides a polynucleotide encoding said lactate dehydrogenase variant.

The term "polynucleotide" encompasses DNA and RNA molecules, such as gDNA and cDNA, wherein the nucleotide, the basic building block in the polynucleotide, includes not only natural nucleotides but also analogues in which the sugar or base moieties are modified . The polynucleotide may be an isolated polynucleotide.

Another aspect provides a vector comprising a polynucleotide encoding said lactate dehydrogenase. The polynucleotide may be operably linked to a regulatory sequence. The regulatory sequence may comprise a promoter, terminator, or enhancer. The promoter may also be operatively associated with a sequence encoding the gene. The term "operably linked" may refer to a functional linkage between a nucleic acid expression control sequence and another nucleotide sequence. As such, the regulatory sequence may regulate transcription and / or translation of the nucleotide sequence encoding the gene.

Another aspect provides a process for preparing a lactate dehydrogenase variant having activity catalyzing the conversion of pyruvate to lactate.

The method includes the step of mutating one or more amino acid residues to shorten the distance between the pyruvate and the catalytic site of the lactate dehydrogenase. The method also includes mutating one or more amino acid residues such that the pKa of the proton donor residue is increased. The amino acid residues are as described above.

Another aspect provides a cell comprising a polynucleotide encoding a lactate dehydrogenase having activity catalyzing the conversion of pyruvate to lactate.

The cells may be yeast cells, for example, ascomycota. The scallop fungi may be saccharomycetaceae. Wherein the saccharide with a fine-shi include saccharide as MY access (Saccharomyces), A Cluj Vero My process (Kluyveromyces) genus Candida (Candida) in blood teeth (Pichia) in director Chen Escherichia (Issatchenkia), A debari Oh, my process (Debaryomyces ), Zygosaccharomyces , or Saccharomycopsis. ≪ / RTI > The genus Saccharomyces is, for example, S. cerevisiae , S. bayanus , S. boulardii , S. cerevisiae, S. bulderi , S. cariocanus , S. cariocus , S. chevalieri , and S. cerevisiae, In the case of S. dairenensis , S. ellipsoideus , S. eubayanus , S. exiguus , S. florentinus , S. kluyveri , S. martiniae , S. monacensis , and Sacharinia sp . romayi access Nord Ben systems (S. norbensis), reading in my process parameters Sakae's (S. paradoxus), four Romayi process Pas thoria Taunus (S. pastorianus), a saccharide as MY ROOM process Spencer (S. spencerorum), my process Turi sensor system as Saccharomyces (S. turicensis), my process as Saccharomyces Uni loose spokes (S. unisporus), My access to the Saccharomyces can be Uva Room (S. uvarum), or Saccharomyces access to My Bluetooth Jonas (S. zonatus). The genus Cluyveromyces is the cluyveromyces thermotolans thermotolerans . The genus Candida is Candida glabrata . The genus Zygosaccharomyces is the genus Zygosaccharomyces bailli ) or Zygosaccharomyces rouxii .

 The yeast cells may comprise non-native yeast cells. The term "non-native" means having one or more genetic alterations not normally found in the native strain of the reference species, including wild type strains of the reference species. Genetic alterations include, for example, introduction of nucleotides encoding polypeptides, addition of other nucleotides, deletion of nucleotides, and / or other functional disruption of the yeast cell genetic material. Such modifications include, for example, coding regions for polypeptides of both heterologous, homologous, or heterologous and homologous to the reference species and functional fragments thereof. Additional modifications include, for example, non-coding regulatory regions wherein said modifications alter the expression of the gene or operon. Exemplary metabolic polypeptides include enzymes or proteins in the biosynthetic pathway to lactate, including lactate. Thus, a non-native yeast cell may comprise a genetic modification to a nucleic acid encoding a metabolic polypeptide or a functional fragment thereof.

The yeast cell can express the lactate dehydrogenase variant described above. The yeast cell may have increased conversion activity from pyruvate to lactate. The increase of the activity means that the conversion activity from the pyruvate to the lactate is higher than that of the parent strain, and the parent strain may mean a strain which is a source of yeast cells. The yeast cell may have lactate production ability. The activity of catalyzing the conversion of pyruvate to lactate may be increased to an extent sufficient to produce lactate. The activity is at least about 5.0%, at least about 5.5%, at least about 6.0%, at least about 6.5%, at least about 7.0%, at least about 7.5%, at least about 8.0%, at least about 8.5% About 10%, about 10%, about 11.0%, about 11.5%, about 12.0%, about 12.5%, about 13.0%, about 13.5%, about 14.0% Greater than about 14.5%, greater than about 15.0%, greater than about 15.5%, greater than about 16.0%, or greater than about 16.5% lactate productivity.

The yeast cell may be one in which the activity of the polypeptide converting pyruvate to acetaldehyde is removed or reduced. The term "reduction" may be relative to activity in the yeast cell engineered relative to untreated yeast cells.

The yeast cell may be one in which a gene encoding a polypeptide that converts pyruvate to acetaldehyde is inactivated or reduced. The term " inactivation "may mean a gene that is not expressed at all, or a gene that is not active when expressed. The term " depression "may mean that the expression of the gene is expressed at a lower level than that of the untreated yeast cell, or even if it is expressed, its activity is low. The activity may be at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 80% , About 85%, about 90%, about 95%, or about 100%.

The inactivation or reduction may be achieved by transforming a vector containing a partial sequence of the gene into a cell, culturing the cell so that homologous recombination with the endogenous gene of the cell occurs, and then introducing the homologous recombination- . ≪ / RTI > The selectable marker may be a marker that confers a selectable phenotype, such as drug resistance, nutritional requirement, tolerance to cytotoxic agents, or expression of surface proteins.

Polypeptides that convert pyruvate to acetaldehyde may be enzymes classified as EC 4.1.1.1. The polypeptide converting the pyruvate to acetaldehyde may have the amino acid sequence of SEQ ID NO: 7. The gene encoding the polypeptide converting the pyruvate to acetaldehyde may have the nucleotide sequence of SEQ ID NO: The gene may be pdc1, pdc2, pdc5, or pdc6 encoding pyruvate decarboxylase (PDC).

Another aspect is a method for producing a yeast cell, comprising culturing the yeast cell described above; And recovering the lactate from the culture.

The term "cultivation "," fermentation ", or "fermentative production" is used interchangeably and refers to appropriate growth, including carbon sources, which are used for the growth of strains and / Means growing the strain on a culture medium. The appropriate medium is a medium suitable for culturing and growing the strain. Such media are well known in the field of fermentation of strains depending on the strain to be cultured. A suitable culture medium comprises a source of carbon, which means any carbon source that can be metabolized by the strain. The metabolism refers to the conversion of energy and matter to allow the strain to grow or at least to sustain life.

In this culture, the cells may be cultured in a batch, fed-batch or continuous manner. The culture may be carried out in a medium containing a carbon source, for example, glucose. The medium used for yeast cell culture may be any conventional medium suitable for growth of the host cell, such as a minimal or complex medium containing appropriate replenishment. Suitable media are available from commercial vendors or may be prepared according to known manufacturing methods.

The medium used for the above culture may be a medium that can satisfy the requirements of a specific yeast cell. The medium may be a medium selected from the group consisting of carbon sources, nitrogen sources, salts, trace elements, and combinations thereof.

Culturing conditions can be suitably adjusted to obtain lactate in the genetically engineered yeast cells. The cells are cultured under aerobic conditions for proliferation. The cells are then incubated in anaerobic conditions to produce lactate. The anaerobic condition may be a microaerobic condition such as a dissolved oxygen (DO) concentration of 0% to 10%, such as 0 to 8%, 0 to 6%, 0 to 4%, or 0 to 2% Conditions.

The term "culture conditions" means conditions for culturing yeast cells. Such a culture condition may be, for example, a carbon source, a nitrogen source, or an oxygen condition used by yeast cells. Carbon sources available to yeast include monosaccharides, disaccharides or polysaccharides. Specifically, glucose, fructose, mannose, or galactose may be used. The source of nitrogen available to the yeast cell may be an organic nitrogen compound or an inorganic nitrogen compound. Specifically, it may be an amino acid, amide, amine, nitrate, or ammonium salt. Oxygenation conditions for culturing yeast cells include aerobic conditions of normal oxygen partial pressure, hypoxic conditions containing 0.1% to 10% oxygen in the atmosphere, or anaerobic conditions without oxygen. The metabolic pathway can be modified to accommodate the carbon source and nitrogen source in which yeast cells are actually available.

The separation of lactate from the culture can be separated by conventional methods known in the art. Such a separation method may be a method such as centrifugation, filtration, ion exchange chromatography or crystallization. For example, the culture can be centrifuged at low speed to remove the biomass, and the resulting supernatant can be separated through ion exchange chromatography.

According to one aspect of the present invention, a lactate dehydrogenase mutant, a polynucleotide encoding the polynucleotide, a vector containing the polynucleotide, and a yeast cell containing the polynucleotide may have lactate production ability .

According to one aspect of the method for producing a lactate dehydrogenase mutant, a mutant having an efficient lactate-producing ability can be produced.

According to a method of producing lactate according to one aspect, lactate can be efficiently produced.

Figure 1 shows the pRS416 vector.
2 is a schematic diagram of the pGEM-PDCp-Gal10t vector.
3 is a schematic diagram of the pGEM-PDCp-NPT-Gal10t vector. The pGEM-PDCp-NPT-Gal10t vector is an NPT overexpression vector that serves as a template for producing cassettes for PDC1 deletion.
Fig. 4 shows a process for producing a mutant strain in which PDC1 has been deleted from the parent strain Saccharomyces cerevisiae CEN.PK2-1D.
Fig. 5 is a schematic diagram of a pathway for producing lactate in yeast cells. Fig.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One. homology modeling through Lactate Dehydrogenase Mutant  Selection

(1.1) Enzyme structure prediction

Bos Taurus ( Bos The structure of lactate dehydrogenase (BtLDH) derived from taurus was predicted by homology modeling using Discovery studio 3.5 software (Accelrys Inc.). homology modeling is a method of predicting the structure of a new protein based on the structure of a template protein already known in structure. The L-LDH derived from Squalus acanthias (PDB database ID: 3LDH, Mol.Biol. 102 (1976) : 759-779). The structure of wild type BtLDH was predicted using the structure as a template. As a result, the predicted structure of wild-type BtLDH predicted the wild-type structure of BtLDH more stably by energy minimization using Discovery Studio 3.5. Using the structure of wild-type BtLDH, we predicted the mutant structure of BtLDH with the "build mutant" option of Discovery Studio 3.5, and predicted a more stable structure by minimizing the energy of the BtLDH mutant structure as described above. The mutants transformed the residues 108 of wild-type BtLDH into different amino acids to produce 19 variants. Table 1 shows variants of BtLDH.

(1.2) Lactate Dehydrogenase Mutant  The catalytic site Pyruvate  Distance measurement

The distances between pyruvate, a ligand of lactate dehydrogenase, and residues 106, 193 and 169, which are catalytic sites of the mutant, in the predicted lactate dehydrogenase mutant structure were measured, respectively. Table 1 shows the distance between the catalytic site of the mutant BtLDH and pyruvate. As the distance between the pyruvate and the residue is closer to that of the wild-type lactate dehydrogenase, strong coupling between the pyruvate and the lactate dehydrogenase is possible, resulting in the formation of lactate from lactate dehydrogenase mutant pyruvate The conversion activity is expected to be advantageous. As shown in Table 1, the distance (d106) between the pyruvate and Arg, the 106th residue of the mutant in the case of the lactate dehydrogenase variants Asn108Gly, Asn108Leu, Asn108Arg, and Asn108Met, the His (193th residue of pyruvate and variant, His (D193) and the distance (d169) between pyruvate and the 169th residue of the mutant, Arg (d169), were both closer to the wild type lactate dehydrogenase distances (6.48, 5.04, and 4,49, respectively).

(1.3) Lactate Dehydrogenase Mutant His193 of pKa  prediction

Among the mutant structure and wild-type structure shown in Table 1, the pKa value of His193, which serves as a proton donor at the active site, was predicted using Discovery Studio 3.5 software (Accelrys Inc.). The activity of converting the lactate dehydrogenase mutant into pyruvate to lactate is expected to be advantageous because a higher pKa of the His193 can release protons better. As shown in Table 1, in the case of the lactate dehydrogenase variant Asn108Gly, Asn108Leu, Asn108Ala, Asn108Asp, Asn108Gln, Asn108Gln, Asn108His, Asn108Phe, Asn108Ser, Asn108Trp and Asn108Tyr, pKa of pyruvate and His193 in the mutant, Was higher than the pKa of His193 in dehydrogenase.

From the results shown in Examples 1.2 and 1.3, not only the distance between the catalytic site of the lactate dehydrogenase mutant and the pyruvate is close to that of the mutant, but also the mutant having a high pKa of His193 indicates that the 108th residue of the mutant is substituted with Gly or Leu .

Distance to Pyr
Asn108 variant d106
(A) d193
(A) d169
(A) PK of His193 Asn (W / T) 6.48 5.04 4.49 5.93 Ala 6.08 4.17 4.71 5.99 Arg 6.42 4.27 4.48 5.73 Asp 6.32 4.28 4.54 6.09 Cys 6.52 4.26 4.36 5.86 Gln 6.26 4.31 4.54 5.97 Glu 6.25 4.25 4.56 6.46 Gly 6.42 4.31 4.43 6.07 His 6.40 4.25 4.49 6.00 Ile 6.32 4.21 4.51 5.79 Leu 6.41 4.26 4.47 6.15 Lys 6.68 4.24 4.45 5.89 Met 6.41 4.22 4.41 5.81 Phe 6.11 4.22 4.72 5.96 Pro 6.11 4.22 4.70 5.49 Ser 6.57 4.22 4.45 6.04 Thr 6.01 4.22 4.93 5.92 Trp 6.37 4.21 4.52 6.10 Tyr 6.45 4.29 4.57 6.15 Val 6.63 4.24 4.34 5.86

Example  2. Lactate  For production Lactate Dehydrogenase (L- LDH ) Construction of Expression Vector

A cassette for introducing a wild-type lactate dehydrogenase or a lactate dehydrogenase (L-LDH) variant was prepared as follows. In order to clone the promoter portion (CCW12p) of the CCW12 gene known to induce gene expression in Saccharomyces cerevisiae, Saccharomyces cerevisiae (CEN.PK2-1D, genotype: MATα ura3-52; trp1-289 (98 ° C for 5 minutes, 30 times) using primers of SEQ ID NOS: 9 and 10, using the genomic DNA of the genomic DNA of the nucleotide sequences of SEQ ID NOS: 9 and 10, leu2-3,112; his3 ㅿ1; MAL2-8 ^C ; SUC2, EUROSCARF accession number: Due to perform a 98 ℃ 30 seconds, 55 ℃ 30 seconds, 1 minute, 72 ℃ performed after 72 ℃ 1 min), and then, cutting the resulting PCR fragment with SacI and XbaI and introduces it in the vector pRS416 (ATCC ^® 87521 ™ pRS416- CCW 12p was constructed. Fig. 1 is a diagram showing a pRS416 vector.

Bos Taurus ( Bos Taurus ) as a template and amplifying the wild type lactate dehydrogenase gene of SEQ ID NO: 2 by PCR using the primers of SEQ ID NOS: 11 and 12. The obtained gene encoding the lactate dehydrogenase wild type was introduced into pRS416-CCW12p using restriction enzyme EcoR1 / SalI to prepare pRS416-CCW12p-BtLDH _wt .

In addition, site-directed mutagenesis was performed using the obtained wild-type lactate dehydrogenase gene as a template, and the mutants of BtLDH (N108G) and BtLDH (N108L), which are variants of lactate dehydrogenase, Gene (Btldh _M ). The sequence numbers of the mutant genes are SEQ ID NOS: 5 or 6, respectively. Each of the genes encoding the mutants was introduced into pRS416-CCW12p using restriction enzyme EcoR1 / SalI to prepare pRS416-CCW12p-BtLDH (N108G) and pRS416-CCW12p-BtLDH (N108L).

Example  3. PDC1  Genetically modified cassette production

To delete the pyruvate decarboxylase 1 (PDC1) involved in the production of ethanol from pyruvate by homologous recombination, the gene deletion vector was constructed as follows.

To use the antibiotic marker, a Gal10 terminator (Gal10t) PCR fragment was prepared using the genomic DNA of S. cerevisiae ( CEN.PK2-1D) as a template and the primers of SEQ ID NOS: 13 and 14 And then digested with NotI and introduced into pGEM-5Zf (Promega USA) to prepare pGEM-Gal10t.

The PDC promoter (PDCp) was obtained by PCR using the genomic DNA of S. cerevisiae ( CEN.PK2-1D) as a template using SEQ ID NOs: 15 and 16, and the prepared fragment was digested with EcoRI And ligated to pGEM-Gal10t digested with the same EcoRI to prepare pGEM-PDCp-Gal10t. 2 is a schematic diagram of the pGEM-PDCp-Gal10t vector. A pGEM-PDCp-Gal10t vector was constructed for overexpression by inserting NPT, a geneticin antibiotic resistance gene.

Then, the neomycin phosphotransferase (NPT) gene, which is capable of resistance to geneticin (G418) antibiotics, was amplified with pcDNA3.3-TOPO (Invitrogen) using the primers of SEQ ID NOs: The obtained fragment was digested with XhoI and BamHI and ligated to pGEM-PDCp-Gal10t digested with the same restriction enzymes to prepare pGEM-PDCp-NPT-Gal10t. 3 is a schematic diagram of the pGEM-PDCp-NPT-Gal10t vector. The pGEM-PDCp-NPT-Gal10t vector is an NPT overexpression vector that serves as a template for producing cassettes for PDC1 deletion.

PDC1 gene deletion cassette was constructed by PCR using pGEM-PDCp-NPT-Gal10t as a template and the primers of SEQ ID NOs: 19 and 20 below.

Example  4. PDC1 This fruitful Sakaromayses Selivijia Production of strain

The mutant strains in which PDC1 was deleted in Saccharomyces cerevisiae were prepared as follows. Fig. 4 shows a process for producing a mutant strain in which PDC1 has been deleted from the parent strain Saccharomyces cerevisiae CEN.PK2-1D. Sakaromasse Serebijia CEN.PK2-1D was plated on solid medium of YPD (10 g yeast extract, 20 g peptone, 20 g glucose) and cultured at about 30 ° C for about 24 hours. The colonies were inoculated into about 10 ml of YPD liquid medium Lt; RTI ID = 0.0 > 30 C. < / RTI > The fully grown culture was inoculated 1% (v / v) in about 50 ml of YPD liquid medium in a 250 ml flask and cultured at about 230 rpm at about 30 캜 in an incubator. After about 4-5 hours, when the OD ₆₀₀ reached about 0.5, the cells were centrifuged at about 4,500 rpm for about 10 minutes to obtain saccharomyces cerevisiae cells. Then, the saccharomyces cerevisiae cells were obtained in about 100 mM of a lithium acetate solution Cells were resuspended. Thereafter, centrifugation was carried out at about 4,500 rpm for about 10 minutes to obtain resuspended cells. Then, cells obtained in a 1 M lithium acetate solution to which about 15% glycerol had been added were resuspended, Respectively.

For removal of PDC1, the PDC1 gene-deficient cassette cassette prepared in Example 3 was mixed with 50% polyethylene glycol, single stranded carrier DNA, and reacted for about 1 hour in a water bath at 42 캜. After that, was added to YPD containing ug / ml geneticin and cultured at about 30 캜 for about 24 hours or more. Eight colonies (mutation strains) formed on the plate were selected and transferred to a YPD solid medium containing about 100 ug / ml of genericin. At the same time, the selected colonies were cultured in a liquid medium of the same composition, and genomic DNA was isolated from the cultured colonies using a commercial kit (Gentra Puregene Cell kit, Qiagen, USA). PCR was carried out using the primers of SEQ ID NOS: 21 and 22 to confirm deletion of PDC1 using the genomic DNA of the isolated mutant strain as a template, and the obtained PCR products were subjected to electrophoresis to confirm deletion of PDC1. As a result, Saccharomyces cerevisiae CEN.PK2-1D (PDC1) was obtained.

Example  5. PDC1 This fruitful Sakaromayses To Servejiae  L- LDH  Production of strain into which an expression vector was introduced

Each of the pRS416-CCW12p-BtLDH (N108G) and pRS416-CCW12p-BtLDH (N108L) prepared in Example 2 was treated with Saccharomyces cerevisiae CEN.PK2-1D (PDC1) strain in which the PDC1 of Example 4 was deleted The following procedure was carried out.

The pRS416-CCW12p-BtLDH (N108G) prepared in Example 2 was mixed with 50% polyethylene glycol, single-stranded carrier DNA and reacted in a water bath at 42 ° C for 1 hour. The culture broth was added to a minimal solid medium (uracil, ura) L of amino acid dropout mix (-ura), and then cultured at about 30 ° C for about 24 hours or more.

Eight colonies (mutant strains) formed on the plate were selected, transferred to a YSD (-ura) solid medium, and cultured in a YSD (-ura) liquid medium. Yeast plasmid isolation kit (Clontech) was used from the strain And the plasmid DNA was isolated. In order to identify a plasmid containing BtLDH (N108G) using the separated plasmid DNA as a template, PCR was carried out using the primers of SEQ ID NOs: 23 and 24, and the obtained PCR product was subjected to electrophoresis to confirm that the inserted plasmid contained pRS416 -CCW12-BtLDH (N108G). As a result, Saccharomyces cerevisiae CEN.PK2-1D (? PDC1 + BtLDH (N108G)) was obtained.

The pRS416-CCW12p-BtLDH (N108L) prepared in Example 2 was mixed with 50% polyethylene glycol, single-stranded carrier DNA, and reacted in a water bath at 42 ° C for 1 hour. After that, the culture solution was added with minimal amount of uracil The cells were plated on medium (YSD, 6.4 g / L yeast nitrogen base without amino acids, 1.4 g / L Amino acid dropout mix (-ura)) and cultured at about 30 ° C for about 24 hours or more to obtain Saccharomyces cerevisiae CEN .PK2-1D (? PDC1 + BtLDH (N108L)) was obtained. The pRS416-CCW12p-BtLDHwt prepared in Example 2 was also transformed into Saccharomyces cerevisiae CEN.PK2-1D (PDC1) in the same manner as described above to produce Saccharomyces cerevisiae CEN.PK2 -1D (? PDC1 + BtLDHwt).

Example  6. L- LDH Mutant  Introduced Sakaromayses Selivijia L-Lac Tei Production

Saccharomyces cerevisiae CEN.PK2-1D strain containing the wild type, BtLDH (N108G) and BtLDH (N108L) mutants prepared in Example 5 was plated on YSD (-ura) solid medium, And incubated for 16 hours at 30 ° C in an aerobic condition with 50 ml YSD (-ura) containing 40 g / L glucose.

The fermentation was carried out by measuring the cell concentration in the culture medium of each 50 ml of the above by using a spectrophotometer to quantify an optical density (OD) of about 5.0 at about 600 nm, centrifuging the supernatant, Was resuspended and re-inoculated into a new approximately 50 ml YSD (-ura) containing approximately 40 g / L glucose. The cells were incubated at about 30 ° C for about 8 hours in a stirred incubator kept at about 90 rpm to ferment the cells. The initially provided sugar was 80 g and samples were taken periodically from the flask during fermentation approximately 17 hours after the cells had consumed the initially provided sugars. The collected samples were centrifuged at about 13,000 rpm for about 10 minutes, and the concentrations of lactate, glycerol, and ethanol were analyzed by liquid chromatography (HPLC). Yield was expressed as a percentage of the lactate (g) divided by the sugar (g) initially provided.

As shown in Table 2, the strain Saccharomyces cerevisiae CEN.PK2-1D (ㅿ PDC1 + BtLDH (N108G)) into which the BtLDH (N108G) expression vector was introduced showed a productivity of about 10.97 g / (BtLDH) was introduced into the strain, the lactate productivity and yield were improved. The strain also exhibited low productivity of glycerol and ethanol.

In addition, the strain Saccharomyces cerevisiae CEN.PK2-1D (ㅿ PDC1 + BtLDH (N108L)) into which the BtLDH (N108L) expression vector was introduced showed a productivity of about 10.10 g / L and a yield of 12.55% L-LDH (BtLDH) -induced strains showed higher lactate productivity and yield. The strain also exhibited low productivity of glycerol and ethanol.

Sakaromayses Selivijia CEN. PK2 -1D PDC1 ) Vector introduced into the strain Lactate
(g / L) Yield%
(g / g) Glycerol
(g / L) EtOH
(g / L) btLDH wild type 9.40 11.68 2.31 32.28 btLDH (N108G) 10.97 13.49 2.15 31.23 btLDH (N108L) 10.10 12.55 2.08 31.67

Example  7. Lactate Dehydrogenase Mutant  Activity evaluation

The wild-type lactate dehydrogenase and its variant expression vectors prepared in Example 2 were expressed in E. coli with a histidine tag and purified by TALON (R) metal affinity resin (Clontech, Inc.). The specific activity of the wild type lactate dehydrogenase and its variants was then measured and shown in Table 3. Km and Kcat of btLDH (N108G), which exhibited high lactate productivity in Example 6, were also measured and are shown in Table 4. Inactivity, Km, and Kcat were determined according to the method described in Microbiology 1994 140: 2077-2084.

As shown in Table 3, the specific activity of BtLDH (N108G) was about 0.167 U / mg at pH 7.5, which was about 24.6% higher than that of wild-type BtLDH as a control. In addition, the specific activity of BtLDH (N108L) was about 0.156 U / mg at pH 7.5, about 11.6% higher than that of wild type BtLDH as a control. In addition, the lactate dehydrogenase BtLDH (N108G) showed a higher specific activity at a small pH such as pH 5.2, and a change in the inactivity value smaller than the change in the inactivity value due to the pH change of the wild type lactate dehydrogenase Respectively.

enzyme Inactive (U / mg , pH  7.5) Wild type BtLDH 0.134 BtLDH (N108G) 0.167 BtLDH (N108L) 0.156

In addition, as shown in Table 4, Km and Kcat of BtLDH (N108G) were about 0.48 mM and 2279 / min, respectively, which were about 4.36 times and 2.76 times higher than Km and Kcat of wild type BtLDH as the control group, respectively.

enzyme Km (mM) Kcat (/ min) Wild type BtLDH 0.11 826 BtLDH (N108G) 0.48 2279

<110> Samsung Electronic Co. Ltd <120> Lactate dehydrogenase mutant, polynucleotide coding the mutant,          yeast cell having the polynucleotide, method of the mutant, and          method of producing the same lactate using the same <130> PN102303 <160> 34 <170> Kopatentin 2.0 <210> 1 <211> 332 <212> PRT <213> Bos Taurus <400> 1 Met Ala Thr Leu Lys Asp Gln Leu Ile Gln Asn Leu Leu Lys Glu Glu   1 5 10 15 His Val Pro Gln Asn Lys Ile Thr Ile Val Gly Val Gly Ala Val Gly              20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu Val          35 40 45 Ala Leu Val Asp Val Met Glu Asp Lys Leu Lys Gly Glu Met Met Asp      50 55 60 Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser Gly  65 70 75 80 Lys Asp Tyr Asn Val Thr Ala Asn Ser Arg Leu Val Ile Ile Thr Ala                  85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln Arg             100 105 110 Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Ile Val Lys Tyr Ser         115 120 125 Pro Asn Cys Lys Leu Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr     130 135 140 Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile Gly 145 150 155 160 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu                 165 170 175 Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Ile Leu Gly Glu             180 185 190 His Gly Asp Ser Ser Val Val Val Trp Ser Gly Val Asn Val Ala Gly         195 200 205 Val Ser Leu Lys Asn Leu His Pro Glu Leu Gly Thr Asp Ala Asp Lys     210 215 220 Glu Gln Trp Lys Ala Val His Lys Gln Val Val Asp Ser Ala Tyr Glu 225 230 235 240 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val                 245 250 255 Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His Pro             260 265 270 Ile Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Glu Asp Val Phe         275 280 285 Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp Val Val     290 295 300 Lys Val Thr Leu Thr His Glu Glu Glu Ala Cys Leu Lys Lys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe                 325 330 <210> 2 <211> 999 <212> DNA <213> Bos Taurus <400> 2 atggcaacat taaaagatca actaatccag aatttgttga aagaggagca tgttccacaa 60 aacaaaatca caatcgtcgg cgtaggtgca gtaggtatgg cttgtgccat atccatcttg 120 atgaaagact tagctgatga ggtcgcgctg gttgatgtaa tggaggacaa acttaaagga 180 gaaatgatgg atcttcaaca tggttcactc tttttgagaa ctcctaaaat tgtatccggg 240 aaagattata acgttaccgc caattctaga cttgttataa tcacggctgg tgcaagacaa 300 caggaaggcg aatcaagact taacttagtt cagagaaacg taaacatttt caagtttatc 360 atcccaaata ttgtaaaata ctccccaaat tgcaagttgc tggttgtttc aaatcctgtt 420 gacatattga cttacgttgc ttggaagatt tcaggtttcc caaagaatag agtaatcgga 480 tctggttgca atctcgattc tgctcgtttt aggtatctga tgggtgaaag attaggggtt 540 catccattga gttgtcacgg atggattcta ggtgaacatg gagatagttc tgtgcctgtt 600 tggtcaggtg tcaacgtagc aggtgtctct ttgaaaaatc tacacccaga actaggaaca 660 gatgccgaca aggaacaatg gaaggccgtc cacaaacaag tggtggattc tgcctacgaa 720 gtcatcaaat tgaagggcta cacatcttgg gcaattggct tatccgtcgc tgatctggct 780 gaatcaataa tgaaaaacct ccgtagagtg catcctataa gtactatgat taagggttta 840 tacgggatca aggaagatgt ttttctatct gtgccatgta ttttgggcca aaatggaatt 900 tctgacgttg ttaaagtgac acttactcat gaagaggaag cgtgtttgaa aaagagcgca 960 gacaccttat ggggcatcca aaaggaatta caattctaa 999 <210> 3 <211> 332 <212> PRT <213> Bos Taurus <400> 3 Met Ala Thr Leu Lys Asp Gln Leu Ile Gln Asn Leu Leu Lys Glu Glu   1 5 10 15 His Val Pro Gln Asn Lys Ile Thr Ile Val Gly Val Gly Ala Val Gly              20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu Val          35 40 45 Ala Leu Val Asp Val Met Glu Asp Lys Leu Lys Gly Glu Met Met Asp      50 55 60 Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser Gly  65 70 75 80 Lys Asp Tyr Asn Val Thr Ala Asn Ser Arg Leu Val Ile Ile Thr Ala                  85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Gly Leu Val Gln Arg             100 105 110 Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Ile Val Lys Tyr Ser         115 120 125 Pro Asn Cys Lys Leu Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr     130 135 140 Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile Gly 145 150 155 160 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu                 165 170 175 Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Ile Leu Gly Glu             180 185 190 His Gly Asp Ser Ser Val Val Val Trp Ser Gly Val Asn Val Ala Gly         195 200 205 Val Ser Leu Lys Asn Leu His Pro Glu Leu Gly Thr Asp Ala Asp Lys     210 215 220 Glu Gln Trp Lys Ala Val His Lys Gln Val Val Asp Ser Ala Tyr Glu 225 230 235 240 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val                 245 250 255 Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His Pro             260 265 270 Ile Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Glu Asp Val Phe         275 280 285 Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp Val Val     290 295 300 Lys Val Thr Leu Thr His Glu Glu Glu Ala Cys Leu Lys Lys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe                 325 330 <210> 4 <211> 332 <212> PRT <213> Bos Taurus <400> 4 Met Ala Thr Leu Lys Asp Gln Leu Ile Gln Asn Leu Leu Lys Glu Glu   1 5 10 15 His Val Pro Gln Asn Lys Ile Thr Ile Val Gly Val Gly Ala Val Gly              20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu Val          35 40 45 Ala Leu Val Asp Val Met Glu Asp Lys Leu Lys Gly Glu Met Met Asp      50 55 60 Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser Gly  65 70 75 80 Lys Asp Tyr Asn Val Thr Ala Asn Ser Arg Leu Val Ile Ile Thr Ala                  85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Leu Leu Val Gln Arg             100 105 110 Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Ile Val Lys Tyr Ser         115 120 125 Pro Asn Cys Lys Leu Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr     130 135 140 Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile Gly 145 150 155 160 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu                 165 170 175 Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Ile Leu Gly Glu             180 185 190 His Gly Asp Ser Ser Val Val Val Trp Ser Gly Val Asn Val Ala Gly         195 200 205 Val Ser Leu Lys Asn Leu His Pro Glu Leu Gly Thr Asp Ala Asp Lys     210 215 220 Glu Gln Trp Lys Ala Val His Lys Gln Val Val Asp Ser Ala Tyr Glu 225 230 235 240 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val                 245 250 255 Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His Pro             260 265 270 Ile Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Glu Asp Val Phe         275 280 285 Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp Val Val     290 295 300 Lys Val Thr Leu Thr His Glu Glu Glu Ala Cys Leu Lys Lys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe                 325 330 <210> 5 <211> 999 <212> DNA <213> Bos Taurus <400> 5 atggcaacat taaaagatca actaatccag aatttgttga aagaggagca tgttccacaa 60 aacaaaatca caatcgtcgg cgtaggtgca gtaggtatgg cttgtgccat atccatcttg 120 atgaaagact tagctgatga ggtcgcgctg gttgatgtaa tggaggacaa acttaaagga 180 gaaatgatgg atcttcaaca tggttcactc tttttgagaa ctcctaaaat tgtatccggg 240 aaagattata acgttaccgc caattctaga cttgttataa tcacggctgg tgcaagacaa 300 caggaaggcg aatcaagact tggcttagtt cagagaaacg taaacatttt caagtttatc 360 atcccaaata ttgtaaaata ctccccaaat tgcaagttgc tggttgtttc aaatcctgtt 420 gacatattga cttacgttgc ttggaagatt tcaggtttcc caaagaatag agtaatcgga 480 tctggttgca atctcgattc tgctcgtttt aggtatctga tgggtgaaag attaggggtt 540 catccattga gttgtcacgg atggattcta ggtgaacatg gagatagttc tgtgcctgtt 600 tggtcaggtg tcaacgtagc aggtgtctct ttgaaaaatc tacacccaga actaggaaca 660 gatgccgaca aggaacaatg gaaggccgtc cacaaacaag tggtggattc tgcctacgaa 720 gtcatcaaat tgaagggcta cacatcttgg gcaattggct tatccgtcgc tgatctggct 780 gaatcaataa tgaaaaacct ccgtagagtg catcctataa gtactatgat taagggttta 840 tacgggatca aggaagatgt ttttctatct gtgccatgta ttttgggcca aaatggaatt 900 tctgacgttg ttaaagtgac acttactcat gaagaggaag cgtgtttgaa aaagagcgca 960 gacaccttat ggggcatcca aaaggaatta caattctaa 999 <210> 6 <211> 999 <212> DNA <213> Bos Taurus <400> 6 atggcaacat taaaagatca actaatccag aatttgttga aagaggagca tgttccacaa 60 aacaaaatca caatcgtcgg cgtaggtgca gtaggtatgg cttgtgccat atccatcttg 120 atgaaagact tagctgatga ggtcgcgctg gttgatgtaa tggaggacaa acttaaagga 180 gaaatgatgg atcttcaaca tggttcactc tttttgagaa ctcctaaaat tgtatccggg 240 aaagattata acgttaccgc caattctaga cttgttataa tcacggctgg tgcaagacaa 300 caggaaggcg aatcaagact tttattagtt cagagaaacg taaacatttt caagtttatc 360 atcccaaata ttgtaaaata ctccccaaat tgcaagttgc tggttgtttc aaatcctgtt 420 gacatattga cttacgttgc ttggaagatt tcaggtttcc caaagaatag agtaatcgga 480 tctggttgca atctcgattc tgctcgtttt aggtatctga tgggtgaaag attaggggtt 540 catccattga gttgtcacgg atggattcta ggtgaacatg gagatagttc tgtgcctgtt 600 tggtcaggtg tcaacgtagc aggtgtctct ttgaaaaatc tacacccaga actaggaaca 660 gatgccgaca aggaacaatg gaaggccgtc cacaaacaag tggtggattc tgcctacgaa 720 gtcatcaaat tgaagggcta cacatcttgg gcaattggct tatccgtcgc tgatctggct 780 gaatcaataa tgaaaaacct ccgtagagtg catcctataa gtactatgat taagggttta 840 tacgggatca aggaagatgt ttttctatct gtgccatgta ttttgggcca aaatggaatt 900 tctgacgttg ttaaagtgac acttactcat gaagaggaag cgtgtttgaa aaagagcgca 960 gacaccttat ggggcatcca aaaggaatta caattctaa 999 <210> 7 <211> 563 <212> PRT <213> Saccharomyces cerevisiae <400> 7 Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln   1 5 10 15 Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser              20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn          35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile      50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser  65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu                  85 90 95 His Val Val Gly Val Ser Ser Ser Ser Gln Ala Lys Gln Leu Leu             100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met         115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Ala Thr     130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Val Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val                 165 170 175 Pro Ala Lys Leu Leu Gln Thr Pro Ile Asp Met Ser Leu Lys Pro Asn             180 185 190 Asp Ala Glu Ser Glu Lys Glu Val Ile Asp Thr Ile Leu Ala Leu Val         195 200 205 Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg     210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln His                 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu Val             260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu         275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys     290 295 300 Asn Ile Val Glu Phe His Ser Asp His Met Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Asn                 325 330 335 Ile Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Ala Arg             340 345 350 Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gln Glu         355 360 365 Trp Met Trp Asn Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp Val Val     370 375 380 Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Thr Phe 385 390 395 400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly                 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile             420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln         435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro     450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro Lys Ala Gln Tyr Asn Glu Ile Gln Gly Trp Asp His Leu                 485 490 495 Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His Arg Val             500 505 510 Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp Lys Ser Phe Asn         515 520 525 Asp Asn Ser Lys Ile Arg Met Ile Glu Val Met Leu Pro Val Phe Asp     530 535 540 Ala Pro Gln Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln             <210> 8 <211> 1692 <212> DNA <213> Saccharomyces cerevisiae <400> 8 atgtctgaaa ttactttggg taaatatttg ttcgaaagat taaagcaagt caacgttaac 60 accgttttcg gtttgccagg tgacttcaac ttgtccttgt tggacaagat ctacgaagtt 120 gaaggtatga gatgggctgg taacgccaac gaattgaacg ctgcttacgc cgctgatggt 180 tacgctcgta tcaagggtat gtcttgtatc atcaccacct tcggtgtcgg tgaattgtct 240 gctttgaacg gtattgccgg ttcttacgct gaacacgtcg gtgttttgca cgttgttggt 300 gtcccatcca tctcttctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360 gacttcactg ttttccacag aatgtctgcc aacatttctg aaaccactgc tatgatcact 420 gacattgcta ccgccccagc tgaaattgac agatgtatca gaaccactta cgtcacccaa 480 agaccagtct acttaggttt gccagctaac ttggtcgact tgaacgtccc agctaagttg 540 ttgcaaactc caattgacat gtctttgaag ccaaacgatg ctgaatccga aaaggaagtc 600 attgacacca tcttggcttt ggtcaaggat gctaagaacc cagttatctt ggctgatgct 660 tgttgttcca gacacgacgt caaggctgaa actaagaagt tgattgactt gactcaattc 720 ccagctttcg tcaccccaat gggtaagggt tccattgacg aacaacaccc aagatacggt 780 ggtgtttacg tcggtacctt gtccaagcca gaagttaagg aagccgttga atctgctgac 840 ttgattttgt ctgtcggtgc tttgttgtct gatttcaaca ccggttcttt ctcttactct 900 tacaagacca agaacattgt cgaattccac tccgaccaca tgaagatcag aaacgccact 960 ttcccaggtg tccaaatgaa attcgttttg caaaagttgt tgaccaatat tgctgacgcc 1020 gctaagggtt acaagccagt tgctgtccca gctagaactc cagctaacgc tgctgtccca 1080 gcttctaccc cattgaagca agaatggatg tggaaccaat tgggtaactt cttgcaagaa 1140 gt; ccaaacaaca cctacggtat ctctcaagtc ttatggggtt ccattggttt caccactggt 1260 gctaccttgg gtgctgcttt cgctgctgaa gaaattgatc caaagaagag agttatctta 1320 ttcattggtg acggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg 1380 ggcttgaagc catacttgtt cgtcttgaac aacgatggtt acaccattga aaagttgatt 1440 cacggtccaa aggctcaata caacgaaatt caaggttggg accacctatc cttgttgcca 1500 actttcggtg ctaaggacta cgaaacccac agagtcgcta ccaccggtga atgggacaag 1560 ttgacccaag acaagtcttt caacgacaac tctaagatca gaatgattga ggttatgttg 1620 ccagtcttcg atgctccaca aaacttggtt gaacaagcta agttgactgc tgctaccaac 1680 gctaagcaat aa 1692 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 9 cgagctcttc gcggccacct acgccgctat c 31 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 10 gctctagata ttgatatagt gtttaagcga at 32 <210> 11 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 11 aattgaattc atggcaacat taaaagatca acta 34 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 12 aattgtcgac ttagaattgt aattcctttt ggatg 35 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 13 gcggccgcga attcggatcc gtagatacat tgatgctatc 40 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 14 gcggccgctc cgcggctcgt gctatattc 29 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 15 gaattcaaca agctcatgca aag 23 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 16 gaattcctcg aggatttgac tgtgtta 27 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 17 ccgctcgaga tgattgaaca agatgg 26 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 18 cgcggatcct cagaagaact cgtcaag 27 <210> 19 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 19 aagatctacg aagttgaagg tatgagatgg gctggtaacg taatacgact cactataggg 60                                                                           60 <210> 20 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 20 gcttccttaa cttctggctt ggacaaggta ccgacgtaaa aacaagctca tgcaaagagg 60 t 61 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 21 gctcttctct accctgtcat tc 22 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 22 tagtgtacag ggtgtcgtat ct 22 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 23 actaaaggga acaaaagctg g 21 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 24 gacataacta attacatgac tcga 24 <210> 25 <211> 326 <212> PRT <213> Lactobacillus casei <400> 25 Met Ala Ser Ile Thr Asp Lys Asp His Gln Lys Val Ile Leu Val Gly   1 5 10 15 Asp Gly Ala Val Gly Ser Ser Tyr Ala Tyr Ala Met Val Leu Gln Gly              20 25 30 Ile Ala Gln Glu Ile Gly Ile Val Asp Ile Phe Lys Asp Lys Thr Lys          35 40 45 Gly Asp Ala Ile Asp Leu Ser Asn Ala Leu Pro Phe Thr Ser Pro Lys      50 55 60 Lys Ile Tyr Ser Ala Glu Tyr Ser Asp Ala Lys Asp Ala Asp Leu Val  65 70 75 80 Val Ile Thr Ala Gly Ala Pro Gln Lys Pro Gly Glu Thr Arg Leu Asp                  85 90 95 Leu Val Asn Lys Asn Leu Lys Ile Leu Lys Ser Ile Val Asp Pro Ile             100 105 110 Val Asp Ser Gly Phe Asn Gly Ile Phe Leu Val Ala Ala Asn Pro Val         115 120 125 Asp Ile Leu Thr Tyr Ala Thr Trp Lys Leu Ser Gly Phe Pro Lys Asn     130 135 140 Arg Val Val Gly Ser Gly Thr Ser Leu Asp Thr Ala Arg Phe Arg Gln 145 150 155 160 Ser Ile Ala Glu Met Val Asn Val Asp Ala Arg Ser Val His Ala Tyr                 165 170 175 Ile Met Gly Glu His Gly Asp Thr Glu Phe Pro Val Trp Ser His Ala             180 185 190 Asn Ile Gly Gly Val Thr Ile Ala Glu Trp Val Lys Ala His Pro Glu         195 200 205 Ile Lys Glu Asp Lys Leu Val Lys Met Phe Glu Asp Val Arg Asp Ala     210 215 220 Ala Tyr Glu Ile Ile Lys Leu Lys Gly Ala Thr Phe Tyr Gly Ile Ala 225 230 235 240 Thr Ala Leu Ala Arg Ile Ser Lys Ala Ile Leu Asn Asp Glu Asn Ala                 245 250 255 Val Leu Pro Leu Ser Val Tyr Met Asp Gly Gln Tyr Gly Leu Asn Asp             260 265 270 Ile Tyr Ile Gly Thr Pro Ala Val Ile Asn Arg Asn Gly Ile Gln Asn         275 280 285 Ile Leu Glu Ile Pro Leu Thr Asp His Glu Glu Glu Ser Met Gln Lys     290 295 300 Ser Ala Ser Gln Leu Lys Lys Val Leu Thr Asp Ala Phe Ala Lys Asn 305 310 315 320 Asp Ile Glu Thr Arg Gln                 325 <210> 26 <211> 1364 <212> DNA <213> Lactobacillus casei <400> 26 aagcttttag tcctcgtgaa aatcgctcat gcaagcgttt tcgttcctga aagcgagctt 60 gtcacaggat tcacaagtct tgctattgta aggctgagcg cgacttttta accatggcaa 120 aatttgaaa aagtctgtga attttgttcc ggcgaattga taatgtgtta tactcacaat 180 gaaatgcagt ttgcatgcac ataagaaagg atgatatcac cgtggcaagt attacggata 240 aggatcacca aaaagttatt ctcgttggtg acggcgccgt tggttcaagt tatgcctatg 300 caatggtatt gcaaggtatt gcacaagaaa tcgggatcgt tgacattttt aaggacaaga 360 cgaagggtga cgcgattgac ttatcgaacg cgctgccatt caccagccca aagaagattt 420 attcagctga atacagcgat gccaaggatg ctgatctggt tgttatcact gctggtgctc 480 ctcagaagcc aggcgaaacc cgcttggatc tggttaacaa gaacttgaag atcttgaagt 540 ccattgttga tccgattgtg gattctggct ttaacggtat cttcttggtt gctgccaacc 600 cagttgatat cttgacctat gcaacttgga aactttccgg cttcccgaag aaccgggttg 660 ttggttcagg tacttcattg gataccgcac gtttccgtca gtccattgct gaaatggtta 720 acgttgatgc acgttcggtc catgcttaca tcatgggtga acatggtgac actgaattcc 780 ctgtatggtc acacgctaac atcggtggcg ttactattgc cgaatgggtt aaagcacatc 840 cggaaatcaa ggaagacaag cttgttaaga tgtttgaaga cgttcgtgac gctgcttacg 900 aaatcatcaa actcaagggc gcaaccttct atggtatcgc aactgctttg gcacgtatct 960 ccaaggctat cctgaacgat gaaaatgctg ttctgccact gtccgtttac atggatggtc 1020 aatatggctt gaacgacatc tacatcggta ccccagctgt gatcaaccga aatggtatcc 1080 agaacattct ggaaattcca ttgaccgacc acgaagagga atccatgcag aaatctgctt 1140 cacaattgaa gaaggttctg actgatgcct tcgcgaagaa cgacatcgaa acccgtcagt 1200 aatcatcatc atgactgagt ctagaacagc cgggcaactg cccggttgtt ctttttttaa 1260 tctcgaaaat gattagggac tttagcgcat ttctctcgct tggcagcatc ctgaaagtat 1320 aatgtttttt taaagttgtc agatgccgtc cgctaatggt taac 1364 <210> 27 <211> 320 <212> PRT <213> Bifidobacterium longum <400> 27 Met Ala Glu Thr Thr Val Lys Pro Thr Lys Leu Ala Val Ile Gly Ala   1 5 10 15 Gly Ala Val Gly Ser Thr Leu Ala Phe Ala Ala Ala Gln Arg Gly Ile              20 25 30 Ala Arg Glu Ile Val Leu Glu Asp Ile Ala Lys Glu Arg Val Glu Ala          35 40 45 Glu Val Leu Asp Met Gln His Gly Ser Ser Phe Tyr Pro Thr Val Ser      50 55 60 Ile Asp Gly Ser Asp Asp Pro Glu Ile Cys Arg Asp Ala Asp Met Val  65 70 75 80 Val Ile Thr Ala Gly Pro Arg Gln Lys Pro Gly Gln Ser Arg Leu Glu                  85 90 95 Leu Val Gly Ala Thr Val Asn Ile Leu Lys Ala Ile Met Pro Asn Leu             100 105 110 Val Lys Val Ala Pro Asn Ale Ile Tyr Met Leu Ile Thr Asn Pro Val         115 120 125 Asp Ile Ala Thr His Val Ala Gln Lys Leu Thr Gly Leu Pro Glu Asn     130 135 140 Gln Ile Phe Gly Ser Gly Thr Asn Leu Asp Ser Ala Arg Leu Arg Phe 145 150 155 160 Leu Ile Ala Gln Gln Thr Gly Val Asn Val Lys Asn Val His Ala Tyr                 165 170 175 Ile Ala Gly Glu His Gly Asp Ser Glu Val Pro Leu Trp Glu Ser Ala             180 185 190 Thr Ile Gly Gly Val Pro Met Cys Asp Trp Thr Pro Leu Pro Gly His         195 200 205 Asp Pro Leu Asp Ala Asp Lys Arg Glu Glu Ile His Gln Glu Val Lys     210 215 220 Asn Ala Tyr Lys Ile Ile Asn Gly Lys Gly Ala Thr Asn Tyr Ala 225 230 235 240 Ile Gly Met Ser Gly Val Asp Ile Ile Glu Ala Val Leu His Asp Thr                 245 250 255 Asn Arg Ile Leu Pro Val Ser Ser Met Leu Lys Asp Phe His Gly Ile             260 265 270 Ser Asp Ile Cys Met Ser Val Pro Thr Leu Leu Asn Arg Gln Gly Val         275 280 285 Asn Asn Thr Ile Asn Thr Pro Val Ser Asp Lys Glu Leu Ala Ala Leu     290 295 300 Lys Arg Ser Ala Glu Thr Leu Lys Glu Thr Ala Ala Gln Phe Gly Phe 305 310 315 320 <210> 28 <211> 1767 <212> DNA <213> Bifidobacterium longum <400> 28 gtcgacgcgg tcaatgacgt gttggcggac atcgaaggca cggcctcgat tccgcgtatt 60 ctcgtattca acaaggccga tcaggcggac gaggcgactc gtgaacgact cgccgcgctg 120 cagccagatg cgttcatcgt ctccgcctat accggtgagg gattggacga gctgcgtacc 180 gcggtcgaaa gtctgctgcc ggtcccgcat gtgcatgtca acgctctgct gccgtatacc 240 gctggctccc tgatctctcg tgtacgcgaa tacggcaagg tagacaaggt ggagtaccgc 300 gatgatggca tacagcttga agcggacgtt gatgcccatc ttgcgctcag gtggtcgaac 360 agtccattga ctaacgtgat aaacatcaca gtatattcgt gagcgctaac aaccgttgaa 420 aacattacca tacggttgtc aaacagggtg gtgtgccggt agcaaaacgt cttagcgggt 480 ttatagagtg aagacgttag ttacaaggcc tgccattcat cagcagaccg cctttgaaga 540 gaggttcatc catcatggcg gaaactaccg ttaagcccac gaagcttgct gttattggtg 600 ccggtgccgt tggctccacc ctcgccttcg ccgctgccca gcgtggcatc gctcgcgaga 660 tcgtgcttga agacatcgcc aaggagcgcg tggaagccga agtgctcgac atgcagcatg 720 gctccagctt ctacccgacc gtgtccatcg acggttccga cgatcctgag atctgccgcg 780 acgccgacat ggtcgtcatc accgctggtc cgcgtcagaa gccgggtcag tctcgtcttg 840 agctcgttgg cgctaccgtc aacatcctca aggccatcat gccgaacctg gtcaaggtgg 900 ctccgaacgc catctacatg ctcatcacca acccggtcga catcgctacc cacgtggctc 960 agaagctcac cggtctgccc gagaaccaga tcttcggttc cggcaccaac ctggactccg 1020 ctcgtctgcg cttcctgatt gcccagcaga ccggcgtcaa cgtcaagaac gtgcacgcct 1080 acatcgccgg cgagcacggc gactccgaag tcccgctgtg ggagtccgcc accatcggtg 1140 gcgtccccat gtgcgactgg accccgctgc ccggccacga tccgctcgac gccgacaagc 1200 gcgaggagat ccaccaggaa gtcaagaacg ccgcttacaa gatcatcaac ggtaagggtg 1260 ccaccaacta cgccatcggc atgtccggcg tcgacatcat cgaagccgtc ctgcacgaca 1320 ccaaccgcat tctgcccgtg agctccatgc tcaaggactt ccacggcatc tccgacatct 1380 gcatgtccgt gccgaccctc ctcaaccgtc agggcgtcaa caacaccatc aacaccccgg 1440 tctccgacaa ggagctcgcc gctctgaagc gctctgccga gacgctgaag gaaaccgccg 1500 cccagttcgg cttctgataa aaaatcgctg tacggagcgc ctttcgcacc gcagcaagac 1560 tcgacctacc tttgtaggcc ttcaccttgt gcggcacgaa aatcgcacac gtacagcgat 1620 tttttatacc ctgctgaatg ctcccgttgg gagcattttt tattcgtgga gttcgttgga 1680 ggagggggag ctatagccct cgggctcgag ctggaaggtg gtgtggggga ccgagaccgg 1740 gaagtgctcg cgcaggcaat cctgcag 1767 <210> 29 <211> 318 <212> PRT <213> Bacillus megaterium <400> 29 Met Lys Thr Gln Phe Thr Pro Lys Thr Arg Lys Val Ala Val Ile Gly   1 5 10 15 Thr Gly Phe Val Gly Ser Ser Tyr Ala Phe Ser Met Val Asn Gln Gly              20 25 30 Ile Ala Asn Glu Leu Val Leu Ile Asp Met Asn Lys Glu Lys Ala Glu          35 40 45 Gly Glu Ala Arg Asp Ile Asn His Gly Met Pro Phe Ala Thr Pro Met      50 55 60 Lys Ile Trp Ala Gly Asp Tyr Lys Asp Cys Ala Asp Ala Asp Leu Ala  65 70 75 80 Val Ile Thr Ala Gly Ala Asn Gln Ala Pro Gly Glu Thr Arg Leu Asp                  85 90 95 Leu Val Glu Lys Asn Val Lys Ile Phe Glu Cys Ile Val Lys Asp Ile             100 105 110 Met Asn Ser Gly Phe Asp Gly Ile Ile Leu Val Ala Thr Asn Pro Val         115 120 125 Asp Ile Leu Ala His Val Thr Gln Lys Val Ser Gly Leu Pro Asn Gly     130 135 140 Arg Val Ile Gly Ser Gly Thr Ile Leu Asp Thr Ala Arg Phe Arg Tyr 145 150 155 160 Leu Leu Ser Asp Tyr Phe Glu Val Asp Ser Arg Asn Val His Ala Tyr                 165 170 175 Ile Met Gly Glu His Gly Asp Thr Glu Phe Pro Val Trp Ser His Ala             180 185 190 Gln Ile Gly Gly Val Lys Leu Glu His Phe Ile Asn Thr Ala Ala Ile         195 200 205 Glu Lys Glu Pro Asp Met Gln His Leu Phe Glu Gln Thr Arg Asp Ala     210 215 220 Ala Tyr His Ile Ile Asn Arg Lys Gly Ala Thr Tyr Tyr Gly Ile Ala 225 230 235 240 Met Gly Leu Val Arg Ile Thr Lys Ala Ile Leu Asp Asp Glu Asn Ser                 245 250 255 Ile Leu Thr Val Ser Ala Leu Leu Glu Gly Gln Tyr Gly Ile Ser Asp             260 265 270 Val Tyr Ile Gly Val Pro Ala Ile Ile Asn Lys Asn Gly Val Arg Gln         275 280 285 Ile Ile Glu Leu Asn Leu Thr Pro His Glu Gln Gln Gln Leu Glu His     290 295 300 Ser Ala Ser Ile Leu Lys Gln Thr Arg Asp Arg Ala Phe Val 305 310 315 <210> 30 <211> 1423 <212> DNA <213> Bacillus megaterium <400> 30 taacatacaa atagagaatg ttaaactctt tagctttctg caaaacaagt tgcctttact 60 caacacttta aacgtttaag atctttctaa atgtggcact tcatgtagaa ggacattttt 120 ttaaatcgcc ctgtaaaatt gtttactatt ttttaaaggg tgtgattttt atcacagctg 180 aagctattta ttcttgttac actaacattg tgaaaaacat cacaaaataa aattcaaagg 240 atgatacaaa tgaaaacaca atttacacca aaaacacgaa aagttgccgt tatcggaact 300 ggttttgttg gctcaagcta cgctttttca atggtgaatc aaggtattgc caatgaatta 360 gtgttaatcg atatgaacaa agaaaaagca gaaggtgaag cacgtgatat caatcatgga 420 atgccatttg ccacaccgat gaaaatctgg gctggagatt ataaagactg tgctgacgct 480 gatttagcag ttattacagc gggcgctaat caagctccag gggaaacacg cttagatcta 540 gttgaaaaaa acgttaaaat tttcgaatgc attgtaaaag atattatgaa cagcggattt 600 gacggcatca ttttagtggc aacaaatcca gttgatattc tcgcacacgt tacacaaaaa 660 gtatcaggat taccaaacgg acgggtaatt ggttcaggaa cgattcttga cacagctcgc 720 ttccgctact tgttaagcga ctatttcgaa gtagattctc gcaacgtcca cgcttatatt 780 atgggggaac atggagatac ggaatttcct gtttggagcc acgcgcaaat tggcggtgtg 840 aagctcgaac attttatcaa tactgccgct attgaaaaag aaccggatat gcagcatcta 900 ttcgaacaaa cccgcgatgc ggcttaccat attattaatc gaaaaggagc gacttattac 960 ggaattgcaa tggggcttgt acgcattacc aaggctattt tagatgatga aaattctatt 1020 ttaacagtat ctgctttatt agaaggacaa tacggtattt ctgatgtgta tatcggcgta 1080 ccagctatca ttaataaaaa cggcgtgcgt caaattattg aattgaattt aactcctcac 1140 gaacagcagc agctcgagca ctctgctagc attcttaagc aaactcgcga cagagctttt 1200 gtgtaacatc taaagatttt tgcgggggac tcccccgcac cttgttttaa atatgtagta 1260 gaggtgtatg aatatgacgt ggactcaggt atataatcct ttagataata tttggctttc 1320 tgcactaatt gcactcattc ccattatctt tttctttatt gctttaactc ttttgaaatt 1380 aaaaggacac attgcttgcc ggtattacgg tgcttctttc tat 1423 <210> 31 <211> 323 <212> PRT <213> Lactobacillus helveticus <400> 31 Met Ala Arg Glu Glu Lys Pro Arg Lys Val Ile Leu Val Gly Asp Gly   1 5 10 15 Ala Val Gly Ser Thr Phe Ala Phe Ser Met Val Gln Gln Gly Ile Ala              20 25 30 Glu Glu Leu Gly Ile Ile Asp Ile Ala Lys Glu His Val Glu Gly Asp          35 40 45 Ala Ile Asp Leu Ala Asp Ala Thr Pro Trp Thr Ser Pro Lys Asn Ile      50 55 60 Tyr Ala Asp Tyr Pro Asp Cys Lys Asp Ala Asp Leu Val Val Ile  65 70 75 80 Thr Ala Gly Ala Pro Gln Lys Pro Gly Glu Thr Arg Leu Asp Leu Val                  85 90 95 Asn Lys Asn Leu Lys Ile Leu Ser Ser Ile Val Glu Pro Val Val Glu             100 105 110 Ser Gly Phe Glu Gly Ile Phe Leu Val Val Ala Asn Pro Val Asp Ile         115 120 125 Leu Thr His Ala Thr Trp Arg Met Ser Gly Phe Pro Lys Asp Arg Val     130 135 140 Ile Gly Ser Gly Thr Ser Leu Asp Thr Gly Arg Leu Gln Lys Val Ile 145 150 155 160 Gly Lys Met Glu Asn Val Asp Pro Ser Ser Val Asn Ala Tyr Met Leu                 165 170 175 Gly Glu His Gly Asp Thr Glu Phe Pro Ala Trp Ser Tyr Asn Asn Val             180 185 190 Ala Gly Val Lys Val Ala Asp Trp Val Lys Ala His Asn Met Pro Glu         195 200 205 Ser Lys Leu Glu Asp Ile His Gln Glu Val Lys Asp Met Ala Tyr Asp     210 215 220 Ile Ile Asn Lys Lys Gly Ala Thr Phe Tyr Gly Ile Gly Thr Ala Ser 225 230 235 240 Ala Met Ile Ala Lys Ala Ile Leu Asn Asp Glu His Arg Val Leu Pro                 245 250 255 Leu Ser Val Pro Met Asp Gly Glu Tyr Gly Leu His Asp Leu His Ile             260 265 270 Gly Thr Pro Ala Val Val Gly Arg Lys Gly Leu Glu Gln Val Ile Glu         275 280 285 Met Pro Leu Ser Asp Lys Glu Glu Glu Leu Met Thr Ala Ser Ala Asp     290 295 300 Gln Leu Lys Lys Val Met Asp Lys Ala Phe Lys Glu Thr Gly Val Lys 305 310 315 320 Val Arg Gln             <210> 32 <211> 1350 <212> DNA <213> Lactobacillus helveticus <400> 32 cattaatttt ttgcttagtc caatgtcctt caaacttgtc tttgtctaat gacaaacctt 60 tttcattaag ataatggtcc aacgtcataa aaccagtgtt gtgcttggtt ttatcatact 120 tttgacctgg attacctaaa cctgcaatta ttttcattta tatatacccc tcattacatc 180 ataatctttc atataatagc acaatcagca aattaataat tttattaatg cctactatca 240 tggtataatt tttttgttaa aattatcact aataaaaagg agatttattg ttatggcaag 300 agaggaaaaa cctcgtaaag ttattttagt cggtgatggt gctgtaggtt ctacctttgc 360 attttcaatg gtacaacaag gtatcgctga agaattaggt attatcgata tcgctaagga 420 acacgttgaa ggtgacgcaa tcgatttagc tgacgcaact ccttggactt ctccaaagaa 480 catttacgca gctgactacc cagattgtaa ggatgctgac ttagttgtta ttactgctgg 540 tgctccacaa aagccaggcg aaactcgtct tgatcttgtt aacaagaact tgaagatttt 600 atcatcaatc gttgaaccag ttgttgaatc aggttttgaa ggtattttct tagtagttgc 660 taacccagtt gatatcttaa ctcacgcaac ttggagaatg tcaggcttcc ctaaggatcg 720 tgttatcggt tcaggtactt cacttgatac tggtcgtctt caaaaagtta ttggtaaaat 780 ggaaaacgtt gacccaagtt cagttaatgc atacatgctt ggtgaacacg gtgatactga 840 attcccagca tggagctaca acaatgttgc tggcgtaaag gttgctgact gggttaaggc 900 tcacaacatg cctgaatcta agcttgaaga catccaccaa gaagttaagg acatggctta 960 cgacattatt aacaagaaag gtgctacctt ctacggtatc ggtactgctt cagcaatgat 1020 cgctaaggct atcttgaacg atgaacaccg tgtacttcca ctttcagtac caatggatgg 1080 tgaatatggt ttacacgatc ttcacatcgg tactcctgca gttgttggcc gcaagggtct 1140 tgaacaagtt atcgaaatgc cattaagcga taaggaacaa gaattaatga ctgcttcagc 1200 agatcaatta aagaaggtta tggacaaggc cttcaaagaa actggcgtta aggttcgtca 1260 ataatcttta attttgttca ttaataaaga gcatctcttt tcttaggaag agatgctttt 1320 ttatttcact aactattgtt aaaatatata 1350 <210> 33 <211> 323 <212> PRT <213> Pediococcus acidilactici <400> 33 Met Ser Asn Ile Gln Asn His Gln Lys Val Val Leu Val Gly Asp Gly   1 5 10 15 Ala Val Gly Ser Ser Tyr Ala Phe Ala Met Ala Glu Glu Gly Ile Ala              20 25 30 Glu Glu Phe Val Ile Val Asp Val Val Lys Val Arg Thr Val Gly Asp          35 40 45 Ala Leu Asp Leu Glu Asp Ala Thr Pro Phe Thr Ala Pro Lys Asn Ile      50 55 60 Tyr Ser Gly Glu Tyr Ser Asp Cys Lys Asp Ala Asp Leu Val Val Ile  65 70 75 80 Thr Ala Gly Ala Pro Gln Lys Pro Gly Glu Thr Arg Leu Asp Leu Val                  85 90 95 Asn Lys Asn Leu Asn Ile Leu Ser Thr Ile Leu Lys Pro Val Val Asp             100 105 110 Ser Gly Phe Asp Gly Ile Phe Leu Val Ala Ala Asn Pro Val Asp Ile         115 120 125 Leu Thr Tyr Ala Thr Trp Lys Phe Ser Gly Phe Pro Lys Glu Lys Val     130 135 140 Ile Gly Ser Gly Ile Ser Leu Asp Thr Ala Arg Leu Arg Val Ala Leu 145 150 155 160 Gly Lys Lys Phe Asn Val Ser Pro Glu Ser Val Asp Ala Tyr Ile Leu                 165 170 175 Gly Glu His Gly Asp Ser Glu Phe Ala Ala Tyr Ser Ser Ala Thr Ile             180 185 190 Gly Thr Lys Pro Leu Leu Glu Ile Ala Lys Glu Glu Gly Val Ser Thr         195 200 205 Asp Glu Leu Ala Glu Ile Glu Asp Ser Val Arg Asn Lys Ala Tyr Glu     210 215 220 Ile Ile Asn Lys Lys Gly Ala Thr Phe Tyr Gly Val Gly Thr Ala Leu 225 230 235 240 Met Arg Ile Ser Lys Ala Ile Leu Arg Asp Glu Asn Ala Val Leu Pro                 245 250 255 Val Gly Ala Tyr Met Asp Gly Glu Tyr Gly Leu Asn Asp Ile Tyr Ile             260 265 270 Gly Thr Pro Ala Val Ile Asn Gly Gln Gly Leu Asn Arg Val Ile Glu         275 280 285 Ala Pro Leu Ser Asp Asp Glu Lys Lys Lys Met Thr Asp Ser Ala Thr     290 295 300 Thr Leu Lys Lys Val Leu Thr Asp Gly Leu Asn Ala Leu Ala Glu Lys 305 310 315 320 Gln Asp Lys             <210> 34 <211> 1628 <212> DNA <213> Pediococcus acidilactici <400> 34 gctagtgcgt ctaccaccat aaagccggtg ttgtgccgcg tttgatcata ttcttttcca 60 atgtttccta aaccaacaat cattttcatt atttatcatc ctcatacgca aaaatagctg 120 aaaccaatcc gccccttcag cttgagctcg ttttaaataa tcttatcata tatcgccaca 180 aaagcttaga tattacttct gttcgctcgg taattatttt tggcaaactt atttgttttc 240 tgaaaattag gtctcaactt tcccctgtta aaatgctata atactttggt ggaccaaata 300 tataatctaa taaatgaagg gaatggatat tatgtctaat attcaaaatc atcaaaaagt 360 tgtcctcgtc ggtgacggtg ccgtaggttc tagttacgca ttcgcgatgg cagaagaagg 420 aatcgctgaa gaattcgtca ttgtcgacgt tgttaaggta cgtacagttg gggacgcatt 480 ggaccttgaa gatgctactc cattcacagc tccaaagaac atctactctg gtgaatactc 540 agactgcaag gatgctgact tagttgttat cacagctggc gcaccacaaa agccaggtga 600 aacacgtctt gaccttgtta acaagaactt aaacatcctt tcaacgattc ttaaaccagt 660 tgttgattct ggttttgatg gtatcttcct tgttgctgct aacccagttg atatccttac 720 ttacgcaaca tggaaattct ctggcttccc taaggaaaaa gttatcggtt caggtatctc 780 acttgacaca gctcgtttgc gcgtagctct tggtaagaaa ttcaacgtta gcccagaatc 840 tgtagatgct tacatcttag gtgaacatgg tgacagtgaa tttgctgctt actcatcagc 900 tacaatcggt acaaagccat tgcttgaaat cgctaaagaa gaaggcgttt caactgacga 960 attggctgaa atcgaagaca gcgtacgtaa caaagcttac gaaatcatca acaagaaggg 1020 tgctacattc tacggtgttg gtactgcatt gatgcgcatt tctaaagcaa ttcttcgcga 1080 cgaaaacgcc gtattgcctg ttggtgcata catggatggc gaatatggtt tgaacgacat 1140 ttacattggt actcctgcag ttatcaatgg tcaaggtcta aaccgcgtta tcgaagcacc 1200 acttagcgat gacgaaaaga agaagatgac tgactcagca actactttga agaaggttct 1260 tactgacggt ctaaacgctc ttgctgaaaa acaagacaaa taatcattaa atttatcaac 1320 gaaagcaccg gttgcaaccg gtgctttttt tgtacctatt tttatgtccc ttccccaaac 1380 agcttgtact aaaatcagtt aattgtgcaa atattggtgt caaacccaga tttttttgtt 1440 aagattctca tagtagactt gatatattcg aaatattacg taatgttaca gtaatgttac 1500 agaagaaagt aaaagtaaaa gtaaagaatt aaatcatttg ggggaagtac tatgcaacaa 1560 cgcaaacatc agcaccagca gccacaccat tctaagatcg ccatcacaat cggcgcggtg 1620 gttatctt 1628

Claims (20)

Claims 1. A lactate dehydrogenase having activity to catalyze the conversion of pyruvate to lactate, wherein at least one amino acid residue is added to shorten the distance between the pyruvate and the catalytic site of the lactate dehydrogenase Wherein at least one of the at least one amino acid residue is mutated such that the pKa of the mutated or proton donor residue is increased. The lactate dehydrogenase according to claim 1, wherein the lactate dehydrogenase has the amino acid sequence of SEQ ID NO: 1, 25, 27, 29, 31 or 33. The method of claim 1, wherein the 193th His of SEQ ID NO: 1, the 181th His of SEQ ID NO: 25, the 181th His of SEQ ID NO: 27, the 181th His of SEQ ID NO: 29, the 179th His of SEQ ID NO: 31, Wherein one or more amino acid residues are mutated to increase the pKa value of the 179th His. The lactate dehydrogenase according to claim 1, wherein the catalytic site of the lactate dehydrogenase is the 106th Arg, the 169th Arg, and the 193th His in SEQ ID NO: 1. The lactate dehydrogenase of claim 1, wherein the amino acid residue mutated to shorten the distance between the pyruvate and the catalytic site of the lactate dehydrogenase is the 108th Asn in SEQ ID NO: 1. The lactate dehydrogenase according to claim 1, wherein the amino acid residue mutated to increase the pKa value of the pyruvate and the 193th His of SEQ ID NO: 1 is the 108th Asn in SEQ ID NO: 1. The lactate dehydrogenase according to claim 1, wherein the 108th Asn in SEQ ID NO: 1 is substituted with a non-polar amino acid. The lactate dehydrogenase according to claim 7, wherein the nonpolar amino acid is selected from the group consisting of Arg, Gly, Leu and Met. The lactate dehydrogenase according to claim 7, wherein the nonpolar amino acid is Gly or Leu. A polynucleotide encoding the lactate dehydrogenase of any one of claims 1 to 9. 12. A vector comprising the polynucleotide of claim 10 operably linked to a regulatory sequence. A process for producing a lactate dehydrogenase mutant having activity to catalyze the conversion of pyruvate to lactate,
Mutating one or more amino acid residues to shorten the distance between the pyruvate and the catalytic site of the lactate dehydrogenase or increasing the pKa of the proton donor residue to one or more Lt; RTI ID = 0.0 &gt; amino acid residues. &Lt; / RTI &gt; A yeast cell comprising the polynucleotide of claim 10. 14. The yeast cell according to claim 13, wherein the activity of the polypeptide converting pyruvate to acetaldehyde is removed or reduced. The yeast cell according to claim 14, wherein the polypeptide converting pyruvate to acetaldehyde has the amino acid sequence of SEQ ID NO: 7. 16. The yeast cell according to claim 15, wherein the gene encoding the polypeptide converting pyruvate to acetaldehyde has the nucleotide sequence of SEQ ID NO: 8. The yeast cell according to claim 13, which is ascomycota. The method according to claim 13, Mai to as My process (Saccharomyces), Cluj Vero My process (Kluyveromyces), Candida (Candida), blood teeth (Pichia), director Chen Escherichia (Issatchenkia), debari Oh, my process (Debaryomyces), Eisai Kosaka Saccharomyces Zygosaccharomyces , or Saccharomycopsis . 14. The yeast cell according to claim 13, wherein the yeast cell is Saccharomyces cerevisiae. Culturing the yeast cells of any one of claims 13 to 19; And
And recovering the lactate from the culture.

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