Microorganism producing lactic acid and method for producing lactic acid using same

The present application relates to a microorganism of the genus Saccharomyces producing lactic acid and a method for producing lactic acid using the same.

Lactic acid has a wide range of applications in the industry including foods, medicines, cosmetics, etc. Recently, due to the use of lactic acid as a monomer for polylactic acid, the demand has significantly increased. Methods for producing lactic acid include a traditional chemical synthesis and a biological fermentation process, which has carbohydrates as substrates, and the latter has been favored recently.

Generally, in yeast-based lactic acid-producing microorganisms, lactate dehydrogenase (LDH) and pyruvate decarboxylase (PDC) compete for the use of pyruvate as a substrate. In this regard, it is necessary to minimize the functions of PDC for higher production of lactic acid (LA) by LDH for maximizing the production yield of pyruvate. In Saccharomyces cerevisiae, PDC is present in three different forms of isozymes, i.e., PDC1, PDC5, and PDC6. Therefore, for maximizing lactic acid production, a method for preparing a strain with a simultaneous triple-deletion of PDC 1, PDC5, and PDC6 may be used. However, although the inactivation of the PDC activity may increase the yield of lactic acid, it also has disadvantages in that it reduces productivity and that strains cannot be grown smoothly (European Patent No. ΕΡ2041264), thus making it difficult to produce a desired amount of lactic acid. Additionally, yeasts, such as the genus Saccharomyces, cannot exactly predict whether a desired amount of lactic acid can be produced by a simple gene manipulation by various cell organelles and various organic systems, unlike in prokaryotes such as bacteria.

The present inventors have endeavored to develop a method for increasing both production yield and production amount of lactic acid while maintaining a smooth growth of lactic acid-producing microorganism, and as a result, have confirmed that enhancing the supply pathway of acetyl-CoA and the supply pathway from oxaloacetate (ΟΑΑ) to pyruvate improved the amount of lactic acid production, thereby completing the present application.

An object of the present application is to provide a microorganism of the genus Saccharomyces producing lactic acid.

Another object of the present application is to provide a method for producing lactic acid using the microorganism.

The modified lactic acid-producing strain of the present application, where PDC activity is inactivated, the foreign ACL activity is introduced, and the biosynthetic pathway for pyruvate is enhanced, has excellent lactic acid fermentation yield and productivity compared to the conventional strains, by minimizing the fermentation of producing the alcohol and blocking of the lactic acid decomposition pathway. Therefore, the growth of the lactic acid-producing strain has been increased, and the modified lactic acid-producing strain of the present application can be widely used for improving the productivity of various products prepared using lactic acid as a raw material. The thus-produced lactic acid can be provided as a raw material for various kinds of products.

FIG. 1 is a pathway for enhancing the biosynthesis of pyruvate via an overexpression of PEP carboxykinase (PCK 1, EC 4.1.1.49) and pyruvate kinase (ΡΥΚ 2, EC 2.7.1.40) present in a yeast microorganism, and shows a schematic diagram illustrating a strategy for improving lactic acid productivity by the introduction of foreign ACL and the enhancement of PCK and ΡΥΚ pathways of the present application.

FIG. 2 is a pathway for enhancing the biosynthesis of pyruvate by the enhancement of malate dehydrogenase 2 (MDH2, EC 1.1.1.37) and cytosolic malic enzyme 1 (ΜΑΕ1, EC 1.1.1.38) present in a yeast microorganism, and shows a schematic diagram illustrating a strategy for improving lactic acid productivity by the introduction of foreign ACL and through the MDH and cytosolic MAE pathways of the present application.

To achieve the above objects, in an aspect, the present application provides a microorganism of the genus Saccharomyces producing lactic acid, in which the microorganism is modified to inactivate the activity of pyruvate decarboxylase (PDC) compared to its endogenous activity, to introduce the activity of ATP-citrate lyase (ACL), and to enhance pyruvate biosynthetic pathway compared to its endogenous biosynthetic pathway.

The microorganism of the genus Saccharomyces producing lactic acid may be a microorganism, in which lactic acid fermentation yield is increased and/or growth of the body of a microorganism of the genus Saccharomyces is increased (the increase of the growth of the microorganism with productivity) and/or lactic acid productivity is increased, compared to the unmodified strains, wherein ACL activity is not introduced and/or the biosynthetic pathway of pyruvate is not enhanced relative to the endogenous biosynthetic pathway .

As used herein, the term “lactic acid (LA)” refers to an organic acid represented by C2H4OHCOOH. When such lactic acid is produced by a chemical synthesis, lactic acid is produced in the fonn of a racemic mixture in which D-type lactic acid and Z-type lactic acid are mixed in a 50/50 ratio, and it is impossible to control the composition ratio. Therefore, when polylactic acid is prepared, the lactic acid becomes an amorphous polymer with a low melting point, and thus there are many limitations in developing its use. In contrast, when lactic acid is produced by a biological fermentation method using a microorganism, D-type lactic acid and Z-type lactic acid can be selectively produced according to the bacteria being used or the lactate dehydrogenase (LDH) being introduced therein.

As used herein, the term “a microorganism producing lactic acid” refers to a microorganism strain, which produces lactic acid productivity, in the present application, can convert sugar into lactic acid, and for example, may include any yeast microorganism without any limitation as long as it includes lactic acid synthesis pathway and acetyl-CoA synthesis pathway of the present application.

According to their shapes, yeast microorganisms may be classified into the genus Saccharomyces, the genus Pichia, the genus Candida, and the genus Saccharomycopsis, and specifically, the microorganism of the genus Saccharomyces including various species may be used in the present application as long as the microorganism can produce lactic acid. Specifically, the Saccharomyces sp. microorganism may be one selected from the group consisting of Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguus, Saccharomyces florentinus, Saccharomyces kluyveri, Saccharomyces martiniae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, and Saccharomyces zonatus, and more specifically Saccharomyces cerevisiae.

The microorganism of the genus Saccharomyces producing lactic acid of the present application may be a microorganism, wherein the microorganism is modified to inactivate the activity of pyruvate decarboxylase (PDC) compared to its endogenous activity, to introduce the activity of ATP-citrate lyase, and to enhance pyruvate biosynthetic pathway compared to its endogenous biosynthetic pathway.

Specifically, the Saccharomyces sp. microorganism producing lactic acid may be a microorganism, wherein the microorganisms is modified (i) to inactivate the activity of pyruvate decarboxylase (PDC) compared to its endogenous activity, (ii) to introduce the activity of ATP-citrate lyase, and (iii) to enhance pyruvate biosynthetic pathway compared to its endogenous biosynthetic pathway, and is further modified (iv) to introduce the activity of lactate dehydrogenase (LDH), (ν) to weaken or inactivate the activity of alcohol dehydrogenase 1 compared to its endogenous activity, (vi) to weaken or inactivate the activity of pyruvate decarboxylase 1 compared to its endogenous activity, and/or (vii) to weaken or inactivate the activity of D-lactate dehydrogenase 1 compared to its endogenous activity.

Additionally, the microorganism of the present application may be further modified (i) to inactivate the activity of alcohol dehydrogenase 1 (ADH1) compared to its endogenous activity;

(ii) to inactivate the activity of pyruvate decarboxylase 1 (PDC1) compared to its endogenous activity; and (iii) to inactivate the activity of D-lactate dehydrogenase 1 (DLD1) compared to its endogenous activity.

As used herein, the term “pyruvate decarboxylase (PDC)”, which may be used interchangeably with an enzyme that catalyzes the decarboxylation of pyruvate, refers to an enzyme that converts pyruvate into acetaldehyde and carbon dioxide (CO2). Pyruvate decarboxylase is an enzyme involved in a fermentation process in an anaerobic condition occurring in yeasts, in particular in a Saccharomyces sp., and it is an enzyme that produces ethanol by fermentation. Generally, the PDC in a Saccharomyces sp. is present in three different forms of isozymes, i.e., PDC1, PDC5, and PDC6. The protein and gene sequences of the PDC may be obtained from a known database such as GenBank of NCBI, but is not limited thereto. Specifically, regarding the enzyme, PDC1 may be a protein represented by an amino acid sequence of SEQ ID NO: 39, PDC5 may be a protein represented by an amino acid sequence of SEQ ID NO: 41, and PDC 6 may be a protein represented by an amino acid sequence of SEQ ID NO: 43, but any amino acid sequence having the activity of PDC can be included without limitation. Additionally, the genes encoding PDC1, PDC5, and PDC6 may be specifically represented by the nucleotide sequences of, for example, SEQ ID NOS: 40, 42, and 44, respectively, but any nucleotide sequence that can encode the enzyme may be included without limitation.

As used herein, the term “ATP-citrate lyase (ACL, EC 2.3.3.8)” refers to an enzyme which converts citrate into oxaloacetate (ΟΑΑ) and acetyl-CoA and is known to be present in higher organisms and some yeasts (ATP-citrate lyase: A mini-review, Biochemical and Biophysical Research Communications, 422, (2012), 1 - 2).

The reaction scheme is shown below:

Citric acid + ATP + CoA + H₂0 -> ΟΑΑ + Acetyl-CoA + A + Pi

Acetyl-CoA is an essential enzyme for the growth of microorganisms and its importance has been highlighted in various references recently. As a representative example, there was a report on a study for improving productivity in a eukaryotic organism capable of producing 1, 3-butanediol (1,3-BDO) by providing cytosol acetyl-coA through a non-natural pathway (International Patent Publication No. WO 2013/036764).

In this regard, it is made possible to provide the acetyl-CoA, which is essential for the growth of a strain in which the activity of PDC is weakened or removed, by the introduction of exogenous ACL, thereby enabling the microorganism to grow in a manner independent of PDC activity. The protein- and gene sequences may be obtained from a known database, e.g., GenBank of NCBI, etc., but is not limited thereto. Specifically, ATP-citrate lyase may have an amino acid sequence of SEQ ID NO: 29, but any protein sequence having the enzyme activity may be included without limitation. Additionally, the gene encoding the ACL may be specifically represented by the nucleotide sequence of SEQ ID NO: 30, but any sequence encoding the enzyme may be included without limitation.

As used herein, the term “pyruvate biosynthesis pathway”, which refers to a biosynthetic pathway that can provide pyruvate in a microorganism of the genus Saccharomyces, may be a supply route from ΟΑΑ to pyruvate. Specific examples are shown in FIGS. 1 and 2.

In an exemplary embodiment, the pyruvate biosynthesis pathway may be performed by modifying the activities of phosphoenolpyruvate carboxykinase 1 (PCK1) or pyruvate kinase 2 (ΡΥΚ2) or both enzymes to enhance their activities compared to their endogenous activities.

Alternatively, the pyruvate biosynthesis pathway may be performed by modifying the activities of malate dehydrogenase 2 (MDH2) or cytosolic malic enzyme 1 (cytosolic ΜΑΕ1) or both enzymes to enhance their activities compared to their endogenous activities.

As used herein, the term “phosphoenolpyruvate carboxykinase 1 (PCK1)” refers to an enzyme that catalyzes the conversion of ΟΑΑ into posphoenolpyruvate (PEP). PCK1 is an enzyme necessary for gluconeogenesis to convert ΟΑΑ into PEP in a yeast microorganism and its expression is known to be inhibited in the presence of glucose (Differential post-transcriptional regulation of yeast mRNAs in response to high and low glucose concentrations. Mol Microbiol 35 (3): 553 - 65 (2000)).

As used herein, the term “pyruvate kinase 2 (PYK2)” refers to an enzyme, which catalyzes the production of pyruvate and ATP by delivering a phosphate group from PEP to ADP. ΡΥΚ2 is the enzyme in the final step of glycolysis in a yeast microorganism and its expression is also known to be inhibited in the presence of glucose (Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate, JBacteriol. 1997 May; 179 (9): 2987 - 93).

Each of the protein and gene sequences may be obtained from a known database, e.g., GenBank of NCBI, etc., but is not limited thereto. The PCK1 may have an amino acid sequence of SEQ ID NO: 31, but any protein sequence having the enzyme activity may be included without limitation. Additionally, the gene encoding the PCK1 may be specifically represented by the nucleotide sequence of SEQ ID NO: 32, but any sequence encoding the enzyme may be included without limitation. The ΡΥΚ2 may have an amino acid sequence of SEQ ID NO: 33, but any protein sequence having the enzyme activity may be included without limitation. Additionally, as a specific example, the gene encoding the ΡΥΚ2 may be represented by the nucleotide sequence of SEQ ID NO: 34, but any sequence that can encode the enzyme may be included without limitation.

As used herein, the term “malate dehydrogenase 2 (MDH2)” refers to a reversible enzyme which converts ΟΑΑ into malate. The MDH2 is an enzyme, which is originally located in the cytosol.

As used herein, the term “cytosolic malic enzyme 1 (MAE1)” refers to an enzyme which was modified to be located in the cytosol by removing the mitochondrial targeting sequence from the ΜΑΕ1, which is an enzyme to substitute malate with pyruvate. The ΜΑΕ1 enzyme is a protein originally located in the mitochondria, and it converts malate, which is an intermediate material in the tricarboxylic acid (TCA) cycle, into pyruvate in the mitochondria (Metabolic Engineering, 6 (2004), 352 - 363). Each of the protein and gene sequences may be obtained from a known database, e.g., GenBank of NCBI, etc., but is not limited thereto. The MDH2 may have an amino acid sequence of SEQ ID NO: 35, but any protein sequence having the enzyme activity may be included without limitation. Additionally, as a specific example, the gene encoding the MDH2 may be represented by the nucleotide sequence of SEQ ID NO: 36, but any sequence which can encode the enzyme may be included without limitation. The ΜΑΕ1 may have an amino acid sequence of SEQ ID NO: 37, but any protein sequence having the enzyme activity may be included without limitation. Additionally, for the ΜΑΕ1 to be present in the cytosol, the ΜΑΕ1 may have a sequence of the amino acid sequence of SEQ ID NO: 37 in which the amino acid residues at positions from the beginning to position 30 are removed (i.e., a sequence from which the amino acid sequence of SEQ ID NO: 51, a mitochondrial targeting sequence, is removed), and the sequence is represented by SEQ ID NO: 52. Additionally, in a specific example, the gene encoding the ΜΑΕ1 may be represented by the nucleotide sequence of SEQ ID NO: 38, but any sequence that can encode the enzyme may be included without limitation.

As used herein, the tenn “lactate dehydrogenase (LDH)” refers to an enzyme, which can catalyze the conversion of lactate to pyruvate and back, and the protein and gene sequences may be obtained from a known database, e.g., GenBank of NCBI, etc., but is not limited thereto. The LDH may have an amino acid sequence of SEQ ID NO: 49, but any protein sequence having the enzyme activity may be included without limitation. Additionally, the gene encoding the LDH may be represented by the nucleotide sequence of SEQ ID NO: 50, but any sequence which can encode the enzyme may be included without limitation.

Each of the enzymes described above may include without limitation, in addition to the amino acid sequences represented by SEQ ID NOS, any amino acid sequence, which has a homology of 70% or higher, specifically 80% or higher, more specifically 90% or higher, even more specifically 95% or higher, yet even more specifically 98% or higher, and yet even still more specifically 99% or higher, to each of the above-listed amino acid sequences, as long as the enzyme exhibits practically the same or corresponding effect to each of the enzymes. Additionally, it is obvious that any modified enzyme, which has the homology described above and has the effect corresponding to each enzyme, can belong to the scope of the present application, although the enzyme may have an amino acid sequence with a partial deletion, modification, substitution, or addition.

Additionally, the genes encoding each of the enzymes may also include without limitation, in addition to the nucleotide sequences represented by SEQ ID NOS, any gene sequence encoding the enzymes, which has a homology of 80% or higher, specifically 90% or higher, more specifically 95% or higher, even more specifically 98% or higher, and yet even more specifically 99% or higher, to each of the above-listed nucleotide sequences, as long as the sequence encodes an enzyme which has substantially the same or corresponding effect to each of the enzymes. Additionally, it is obvious that any nucleotide sequence, which has the above homology can belong to the scope of the present application, although the sequence may have a partial deletion, modification, substitution, or addition therein.

As used herein, the term “homology” refers to a percentage of identity between two polynucleotide or polypeptide moieties. Sequence correspondence from one moiety to another may be determined by a known technique in the art. For example, homology may be determined by directly aligning the sequence information (e.g., parameters such as score, identity, and similarity) on two polynucleotide molecules or two polypeptide molecules using a computer program (e.g., BLAST 2.0) that is readily available and capable of aligning sequence information. Additionally, homology may be determined by hybridizing the polynucleotides under the condition for forming a stable double-strand in the homologous regions and digesting the hybridized strand by a single-strand-specific nuclease to determine the size of digested fragments.

As used herein, the term “endogenous activity” refers to a condition, where a microorganism has a natural state of enzymes or an activation level of enzymes prior to the modification of the corresponding enzymes.

As used herein, the term “the activity of an enzyme is modified for inactivation compared to its endogenous activity” refers to that a gene encoding an enzyme is not expressed at all compared to that of the native strain or a strain before modification, or even when the gene is expressed, there is no activity or the activity is reduced.

The above reduction is a concept, which includes a case where the activity of an enzyme itself is reduced due to a modification of the gene encoding the enzyme, etc., compared to that of the endogenous activity originally possessed by a microorganism, a case where the overall level of enzyme activity within a cell is lower compared to that of the wild-type strain or the strain before modification, and also a combination thereof.

The inactivation of an enzyme may be achieved by various methods known in the art. Examples of the methods may include a method to substitute the gene encoding the enzyme on the chromosome with a gene mutated to reduce the enzymatic activity, including the case where the enzyme activity is removed; a method of introducing a modification in the expression regulatory sequence of the gene encoding the enzyme on the chromosome; a method of substituting the expression regulatory sequence of the gene encoding the enzyme with a sequence having weak or no activity; a method of deleting the entirety or a part of the gene encoding the enzyme on the chromosome; a method of introducing an antisense oligonucleotide (e.g., antisense RNA), which binds complementary to a transcript of the gene on the chromosome, thereby inhibiting the translation from the mRNA into the enzyme; a method of artificially incorporating a complementary sequence to the SD sequence into the upstream of the SD sequence of the gene encoding the enzyme, forming a secondary structure, thereby making the attachment of ribosome thereto impossible; a method of incorporating a promoter to the 3’ terminus of the open reading frame (ORF) to induce a reverse transcription (reverse transcription engineering (RTE)), etc., and also a combination thereof, but are not limited thereto.

Specifically, the method of deleting the entirety or a part of a gene encoding an enzyme may be performed by substituting the polynucleotide encoding the endogenous target protein within the chromosome with a polynucleotide or marker gene having a partial deletion in the nucleic acid sequence using a vector for chromosomal insertion within a strain. In an exemplary embodiment of the method of deleting a part or the entirety of a gene, a method for deleting a gene by homologous recombination may be used.

As used herein, the term “a part” may vary depending on the kinds of polynucleotides, and it may specifically refer to 1 to 300, more specifically 1 to 100, and even more specifically 1 to 50, but is not particularly limited thereto.

As used herein, the term “homologous recombination” refers to a genetic recombination that occurs via crossover at genetic chain loci having a mutual homology.

Specifically, the expression regulatory sequence may be modified by inducing a modification of the expression regulatory sequence by a deletion, an insertion, a non-conservative or conservative substitution, or a combination thereof in the nucleic acid sequence of the expression regulatory sequence; or by substituting with a weaker promoter, etc. The expression regulatory sequence may include a promoter, an operator sequence, a sequence encoding a ribosome-binding region, and sequences controlling the termination of transcription and translation.

Furthermore, the gene sequence on the chromosome may be modified by inducing a modification in the sequence by a deletion, an insertion, a non-conservative or conservative substitution, or a combination thereof in the gene sequence for reducing the enzyme activity; or by substituting with a gene sequence which was improved to have a weaker activity or a gene sequence which was improved to have no activity.

As used herein, the term “enhancement of activity compared to its endogenous activity” refers to increasing the intracellular activity of a protein (or enzyme) in a microorganism by modifying the protein to improve the intracellular activity compared to the activity of the protein possessed in its natural state. The “enhancement” may include the drawing of a higher effect than the original function due to the increase in the activity of the protein (or enzyme) itself, and it may be performed by at least one method selected from the group consisting of a method of increasing the copy number of a polynucleotide encoding the protein (or enzyme), a method of introducing a modification in the regulatory sequence of a gene encoding the protein (or enzyme), a method of substituting the regulatory sequence of a gene encoding the protein (or enzyme) on the chromosome with a sequence having strong activity, a method of substituting the gene encoding the protein (or enzyme) with a mutated gene to increase the activity of the protein (or enzyme), and a method of introducing a modification in the gene encoding the protein (or enzyme) on the chromosome to enhance the activity of the protein (or enzyme), but any known method which can enhance the activity of the protein (or enzyme) compared to its endogenous activity or enhance the introduced activity may be included without limitation.

As used herein, the term “introduction of the activity of a protein (or enzyme)” refers to providing an activity of a particular protein (or enzyme) to a microorganism, which does not have the activity of the particular protein (or enzyme); or increasing the intracellular activity of a particular protein (or enzyme) in a microorganism, which does not have the activity of the particular protein (or enzyme) by modifying the microorganism to further improve the intracellular activity of the protein (or enzyme) after providing the activity of the particular protein (or enzyme) to the microorganism.

The “introduction of the activity of a protein (or enzyme)” may be performed in various methods known in the art, for example: a method of inserting a polynucleotide including a nucleotide sequence encoding the protein (or enzyme) into the chromosome; a method of increasing the copy number of a polynucleotide by a method such as introducing the polynucleotide to a microorganism via an introduction into a vector system; a method of introducing a promoter capable of exhibiting improved activity or introducing the protein (or enzyme) with a modification in the promoter, into an upstream region of the nucleotide sequence encoding the protein (or enzyme); a method of introducing a nucleotide variant sequence encoding the protein (or enzyme); etc., but any known method that can introduce the activity of a protein (or enzyme) may be included without limitation.

In the above, the increase of copy number of a polynucleotide may be performed in a form in which the polynucleotide is operably linked to a vector, or by inserting the polynucleotide into the chromosome of a host cell, although the method is not particularly limited thereto. Specifically, the increase of copy number of a polynucleotide may be performed by introducing a vector, which can replicate and function regardless of a host cell and the polynucleotide encoding the protein of the present application is operably linked thereto; or may be performed by introducing a vector, which can insert the polynucleotide into the chromosome of a host cell and the polynucleotide is operably linked thereto, into a host cell.

The vector is a DNA construct including the sequence of a polynucleotide encoding a target peptide, which is operably linked to an appropriate regulatory sequence to enable the expression of the target peptide in a host cell. The regulatory sequence includes a promoter capable of initiating transcription, any operator sequence for the regulation of the transcription, a sequence encoding an appropriate mRNA ribosome-binding domain, and a sequence regulating the termination of transcription and translation. The vector, after being transformed into an appropriate host cell, may be replicated or function regardless of the host genome, or may be integrated into the host genome itself.

For the yeast expression vector, both an integrative yeast plasmid (Yip) and an extrachromosomal plasmid vector may be used. The extrachromosomal plasmid vector may include episomal yeast plasmid (ΥΕρ), replicative yeast plasmid (YRp), and yeast centromer plasmid (YCp). Additionally, artificial yeast chromosomes (YACs) may be also used as expression vectors according to the present application. For example, the vectors to be used in the present application may include pESC-HIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, ρΑ0815, pGAPZ A, pGAPZ Β, pGAPZ C, pGAPa A, pGAPa Β, pGAPa C, pPIC3.5K, pPIC6 A, pPIC6 Β, pPIC6 C, pPIC6a A, pPIC6a Β, pPIC6a C, pPIC9K, pYC2/CT, pYDl Yeast Display Vector, pYES2, pYES2/CT, pYES2/NT A, pYES2/NT Β, pYES2/NT C, pYES2/CT, pYES2.1, pYES-DEST52, pTEFl/Zeo, pFLDl, PichiaPink™, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, ρΒΥ011, pSGP47, pSGP46, pSGP36, pSGP40, ΖΜ552, ρΑG303GAL-ccdB,_PAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD Τ7, pGBK Τ7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A form, pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405, andpYJ406, but are not limited thereto.

More specifically, the yeast vector may be a yeast replication plasmid including replication origin (οή) and an antibiotic resistance cassette which can be proliferated and selected in Ε. coli. Generally, expression vectors may include an expression construct of promoter-gene-transcription termination sequence.

For example, when the host cells is a yeast, the promoters that can be used in the expression construct may include TEF1 promoter, TEF2 promoter, GAL 10 promoter, GA L / promoter, AD HI promoter, ADH2 promoter, ΡΗ05 promoter, GAL1-10 promoter, TDH3 promoter (GPD promoter), TDH2 promoter, TDH1 promoter, PGK1 promoter, ΡΥΚ2 promoter, ENOl promoter, ΕΝ02 promoter, and TPI1 promoter, but are not limited thereto.

The transcription tennination sequences that can be used in the expression construct may include ADH1 tenninator, CYC I terminator, GAL 10 terminator, PGK1 terminator, ΡΗ05 terminator, ENOl terminator, ΕΝ02 terminator, and TPI1 tenninator, but are not limited thereto.

Additionally, the polynucleotide encoding the endogenous target protein may be replaced with a modified polynucleotide within the chromosome by a vector for the insertion of chromosome within the host cell. Alternatively, the polynucleotide encoding a foreign target protein to be introduced into the chromosome may be replaced with a modified polynucleotide. The insertion of the polynucleotide into the chromosome may be performed using any known method in the art, for example, by homologous recombination. Since the vector of the present application can be inserted into the chromosome via homologous recombination, a selection marker for confirmation of the insertion into the chromosome may be further included. The selection marker is used for the selection of a transfonned cell, i.e., to confirm whether the target polynucleotide has been inserted, and markers capable of providing selectable phenotypes such as drug resistance, nutrient requirement, resistance to cytotoxic agents, and expression of surface proteins may be used. Under the circumstances treated with selective agents, only the cells capable of expressing the selection markers can survive or express other phenotypic traits, and thus the transformed cells can be selected.

As used herein, the term “transformation” refers to a process of introducing a vector including a polynucleotide encoding a target protein into a host cell, thereby enabling the expression of the polynucleotide encoded by the protein in the host cell. For the transfonned polynucleotide, it does not matter whether it is inserted into the chromosome of a host cell and located therein or located outside the chromosome, as long as it can be expressed in the host cell. Additionally, the polynucleotide includes DNA and RNA, which encode the target protein. The polynucleotide may be inserted in any form as long as it can be introduced into a host cell and expressed therein. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all essential elements required for self-expression. The expression cassette may conventionally include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome-binding domain, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide may be introduced into a host cell as it is and operably linked to a sequence essential for its expression in the host cell.

Additionally, as used herein, the term “operably linked” refers to a functional connection between a promoter sequence, which initiates and mediates the transcription of the polynucleotide encoding the target protein of the present application, and the above target gene sequence.

The method of transforming a vector of the present application may include any method which can introduce nucleic acids into a cell, and the transformation may be performed by selecting an appropriate technique as known in the art according to the host cell. For example, the method may include electroporation, calcium phosphate (CaPC>4) precipitation, calcium chloride (CaCF) precipitation, micro injection, a polyethylene glycol (PEG) method, a DEAE-dextran method, a cationic liposome calcium, and a lithium acetate/DMSO method, etc., but are not limited thereto.

Specifically, the host cell to be used should have high efficiency of DNA introduction and high expression efficiency of the introduced DNA, and for the purpose of the present application, the host cell may be a microorganism of the genus Saccharomyces.

Then, the introduction of a modification in the expression regulatory sequence for increasing the expression of a polynucleotide, although not particularly limited thereto, may be performed by inducing modification in the nucleic acid sequence via deletion, insertion, conservative substitution or non-conservative substitution, or a combination thereof in order to further enhance the activity of the expression regulatory sequence; or by replacing the polynucleotide sequence with a nucleic acid sequence with enhanced activity. The expression regulatory sequence, although not particularly limited thereto, may include a promoter, an operator sequence, a sequence encoding a ribosome-binding domain, and a sequence for regulating the termination of transcription and translation, etc.

A strong exogenous promoter, instead of the original promoter, may be linked to the upstream region of the expression unit of the polynucleotide.

Generally, the introduction or enhancement of the activity of a protein may increase the activity or concentration of the corresponding protein relative to the activity or concentration of a wild-type protein or in a microorganism strain from at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, or 500%, to a maximum of 1000% or 2000%.

In another aspect, the present application provides a method for producing lactic acid including a) culturing a novel microorganism of the genus Saccharomyces producing lactic acid , wherein the microorganism is modified to inactivate the activity of pyruvate decarboxylase (PDC) compared to its endogenous activity, to introduce the activity of ATP-citrate lyase (ACL), and to enhance pyruvate biosynthetic pathway compared to its endogenous biosynthetic pathway in a medium; and b) recovering lactic acid from the cultured microorganism and the culture.

The microorganism of the genus Saccharomyces producing lactic acid is the same as described above.

As used herein, the term “culturing” refers to growing a microorganism in an appropriately artificially adjusted environment. In the present application, the culturing using the microorganism of the genus Saccharomyces may be performed by an appropriate method well known in the art. Specifically, the culturing may be perfonned continuously in a batch process, a fed batch, or a repeated fed batch process, but is not limited thereto.

The media used for culturing the microorganism of the present application and other culture conditions are not particularly limited but any medium used for the conventional culturing of the microorganism of the genus Saccharomyces may be used. Specifically, the microorganism of the present application may be cultured in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorous sources, inorganic compounds, amino acids and/or vitamins, etc., in an aerobic condition while adjusting temperature, pH, etc.

As an example of the carbon sources, sucrose or glucose may be used, and molasses containing a large amount of sucrose may also be used as a carbon source, and an appropriate amount of other various kinds of carbon sources may be used.

Examples of the nitrogen sources may include organic nitrogen sources such as peptone, yeast extract, meat gravy, malt extract, corn steep liquor, and soybean flour; and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. These nitrogen sources may be used alone or in combination. In the above medium, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and corresponding sodium-containing salts may be contained as phosphorus sources. Additionally, metal salts, such as magnesium sulfate or iron sulfate, may be contained. Furthermore, amino acids, vitamins, and appropriate precursors may be contained. These media or precursors may be added in a batch culture process or a continuous culture process to the culture.

During the period of the culture, the pH of a culture may be adjusted by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in an appropriate manner. Additionally, during the period of the culture, an antifoaming agent, such as fatty acid polyglycol ester, may be added to prevent foam generation. Additionally, for maintaining the aerobic state of the culture, oxygen or an oxygen-containing gas may be injected into the culture, and for maintaining the anaerobic and microaerophillic states of the culture, nitrogen, hydrogen, or carbon dioxide gas may be injected without the injection of an air.

The culture temperature may normally be from 20°C to 40°C, specifically, from 25°C to 35°C, and more specifically 30°C, but may vary without limitation according to the desired purposes. Additionally, the culturing may be continued until desired amount of product can be obtained, and specifically for 10 hours to 100 hours, but is not limited thereto.

The method of producing lactic acid of the present application may include recovering lactic acid from the cultured microorganism or the culture. The method of recovering the lactic acid from the microorganism or the culture may be performed using the appropriate method known in the art, e.g., centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc., but is not limited thereto.

The recovering may include a purification process.

Hereinbelow, the present application will be described in detail with accompanying exemplary embodiments. However, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present application.

In order to prepare a representative lactic-acid producing strain to be used in the present application, Saccharomyces cerevisiae CEN. PK2-1D, which is a representative yeast strain among the wild-type yeast strains obtained from Euroscarf, were subjected to a series of genetic manipulation.

Specifically, alcohol dehydrogenase 1 (ADH1) and pyruvate decarboxylase 1 (PDC1) were deleted for minimizing the fermentation of producing the alcohol, whereas for blocking the pathway of lactic acid decomposition, the strain with a deletion in D-lactatc dehydrogenase 1 (DLD1) was used as the base strain of the present application.

DLD1 is not a factor that directly affects the improvement of growth but DLD1, being a dehydrogenase of D-typc lactic acid, is known as a major enzyme that converts lactic acid into pyruvate using NAD⁺. Accordingly, subsequent stains were prepared based on the strain with a deletion of the DLD1 gene, which is a lactic acid-consuming enzyme, and the lactic acid productivity was compared.

In the present application, the genetic manipulation was performed using a general molecular cloning method. First, the experiments on the deletion of ADH1 and PDC1 genes of the enzyme were performed usingpWALlOO andpWBRIOO plasmids based on the disclosure on the reference (Lee ΤΗ, et αℓ., J. Microbiol. Biotechnol. (2006), 16 (6), 979 - 982). Each insert incorporated into each plasmid was prepared by polymerase chain reaction (PCR) using primers corresponding to each insert (SEQ ID NO: 1 to SEQ ID NO: 8).

PCR was performed while using the genomic DNA of wild-type yeast strains as a template. For the deletion of ADH1, PCR was performed using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, and the resultant was cloned into pWALlOO using the restriction enzymes, BamHl and NcoI. Additional PCR was performed using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 and the resultant was cloned into pWBRIOO using the restriction enzymes, BamHl and Ncol. PCR was performed by denaturation at 95°C for 5 min, annealing at 53°C for 1 min, and polymerization at 72°C for 1 min 30 sec.

For the deletion of PDC1, PCR was performed using the primers of SEQ ID NO: 5 and SEQ ID NO: 6, and the resultant was cloned into pWALlOO using the restriction enzymes, BamHl and Ncol. Additional PCR was performed using the primers of SEQ ID NO: 7 and SEQ ID NO: 8 and the resultant was cloned into pWBRIOO using the restriction enzymes, BamHl and Ncol. PCR was performed by denaturation at 95°C for 5 min, annealing at 53°C for 1 min, and polymerization at 72°C for 1 min 30 sec.

Additionally, for the deletion of DLD1 gene, HIS3 marker gene was deleted by introduction via double crossover. The DNA fragments used therein were obtained by PCR perfonned using the genomic DNA of the wild-type yeast stain, along with primers of SEQ ID NO: 9 and SEQ ID NO: 10. PCR was performed by denaturation at 95°C for 5 min, annealing at 53°C for 1 min, and polymerization at 72°C for 1 min 30 sec.

The primers used in the gene manipulation are summarized in Table 1 below.

primer (SEQ ID NO: 10) Τ AC AT A AG A AC AC C TTTGG

Based on the strains having deletions of three genes (ADH1, PDC1, and DLD1), the D-lactate dehydrogenase (D-LDH) for lactic acid production was introduced. The 5’ terminus and 3’ terminus of IdhD gene derived from Lb. plantarum were respectively inserted to p413TEFl vector so that the IdhD gene can be included between the TEF1 promoter and the CYC1 terminator derived from S. cerevisiae, in which the insert was prepared by a double digestion with Sax \IPvu11. The vector was made blunt-ended using Mungbean nuclease in the DNA fragments double-digested with BamHl/Notl of the ρ-δ-η℮ο vector, and treated again with Sac I, thereby generating the vector portion having a Sac I sticky end and a BamHl blunt end.

The thus-obtained vector and the insert were ligated to complete the ρΤΕ573 vector and named as ρΤΕ573 vector. The ρΤΕ573 plasmid contains the IdhD gene derived from Lb. plantarum and was designed so that multiple copies can be randomly inserted into the 6-sequence, which is a part of the region of the retrotransposable element of S. cerevisiae CEN. PK2-1D pdcl adhl dldl strain. For the multiple insertion of the corresponding gene, the ρΤΕ573 plasmid was digested with SacI to prepare a DNA fragment that can induce a single crossover in the 8-scqucncc. The resultant was introduced into a parent strain by transfection and numerous colonies were obtained in YPD (1% yeast extract, 2% bacto-peptone, and 2% glucose) medium in a maximum concentration of 5 mg/mL G418. It was confirmed that the thus-obtained strain was finally inserted with a multiple number of Lb. plantarum-dQrrvQd D-LDH for providing D-type lactic acid productivity and the strain was named as CC02-0064.

A strain with a reduced titer or inactivation of PDC was prepared by preparing a strain, which had a deletion of PDC5, i.e., a PDC isozyme, in the CC02-0064 strain, which is the base strain prepared in Example 1, and a strain which had deletions of both PDC5 and PDC6 in the CC02-0064 strain.

Specifically, for the deletion of the gene of a yeast ,pWAL100 and pWBRIOO plasmids (J. Microbiol. Biotechnol., (2006) 16 (6), 979 - 982) were used.

For the deletion of PDC5, PCR was performed using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 and the resultant was cloned into pWALlOO using the restriction enzymes, BamHl and Notl. Additional PCR was performed using the primers of SEQ ID NO: 13 and SEQ ID NO: 14 and the resultant was cloned into pWBRIOO using the restriction enzymes, SpeI and Ncol. PCR was performed by denaturation at 95°C for 5 min, annealing at 53°C for 1 min, and polymerization at 72°C for 1 min 30 sec.

For the deletion of PDC6, PCR was performed using the primers of SEQ ID NO: 15 and SEQ ID NO: 16 and the resultant was cloned into pWALlOO using the restriction enzymes, BamHl and Notl. Additional PCR was performed using the primers of SEQ ID NO: 17 and SEQ ID NO: 18 and the resultant was cloned into pWBRIOO using the restriction enzymes, Spel and Ncol. PCR was performed by denaturation at 95°C for 5 min, annealing at 53°C for 1 min, and polymerization at 72°C for 1 min 30 sec.

The novel strains prepared above were named as CC02-0256 and CC02-0553, respectively, and the genetic traits of the novel strains are summarized in Table 3 below.

[Table 3] Strains with reduced or inactivated PDC activities

Strain Genetic Traits

CC02-0256 Saccharomyces cerevisiae δ:: IdhD adhlAdldlA pdclApdc5A

CC02-0553 Saccharomyces cerevisiae δ:: IdhD adhlAdldlA pdclApdc5Apdc6A

The media used for the evaluation of strains were synthetic complex media (SC). For the preparation of the media, an amino acid dropout mix (Sigma) was mixed to the 0.67% yeast nitrogen base without amino acid, which was used as the base, according to the manufacturer’s protocol, and the amino acids not included therein were added as necessary. Leucine was added to a concentration of 380 mg/L, and uracil, tryptophan, and histidine were added to a concentration of 76 mg/L, respectively, and glucose (8%) as a carbon source and 1% CaCCL as a neutralizing agent were added thereto. The thus-prepared media were used for the evaluation of lactic acid fermentation of yeast strains.

As the conditions for the evaluation of lactic acid fermentation ability of the strains, the media prepared for the evaluation of lactic acid fermentation were aliquoted in an amount of 25 mL per each flask and inoculated with each of the yeast strains, cultured at 30°C aerobically for 48 hours, and the amount of lactic acid present in the fermentation liquid was analyzed by HPLC.

The results of the experiments are summarized in Table 4 below.

[Table 4] Comparison of the results of fermentation of strains with reduced or inactivated PDC activities

A recombinant vector for the introduction of a foreign ACL enzyme and the simultaneous overexpression of PCK1 and ΡΥΚ2, one of the pathways of pyruvate biosynthesis, was prepared.

For the foreign ACL, a gene derived from Mus muscuius, a mammal, was used and the corresponding gene was confirmed by NCBI (Accession no. ΝΡ 001186225).

Specifically, the gene was synthesized using the amino acid sequence of SEQ ID NO: 29 (or an amino acid sequence of SEQ ID NO: 30), the vector was prepared using the GPD promoter based on pRS415, a gene expression vector for yeasts, and the vector inserted with the gene was prepared and named asp415GPDpro-ACL.

A recombinant vector for the simultaneous overexpression of PCK1 and ΡΥΚ2 for the enhancement of pyruvate biosynthesis pathway was prepared.

ΡΥΚ2 is a gene present in a yeast microorganism and may be represented by SEQ ID NO:

33. PCR was performed using the genomic DNA of S. cerevisiae as a template along with the primers of SEQ ID NOS: 19 and 20, and the fragments of ΡΥΚ2 gene were obtained therefrom. PCR was performed by denaturation at 95°C for 5 min, annealing at 53°C for 1 min, and polymerization at 72°C for 1 min 30 sec. The cloning was performed using the gene fragments and the restriction enzymes within the yeast expression vector derived from pRS416, i.e., SpeI, Xhol, and the overexpression was performed using the TEF1 promoter. The corresponding recombinant vector was named as pRS416-TEFlpro-PYK2.

ΡΥΚ1 is also a gene present in a yeast microorganism and may be represented by SEQ ID NO: 31. PCR was performed using the genomic DNAof S. cerevisiae as a template along with the primers of SEQ ID NOS: 23 and 24, and the fragment of ΡΥΚ1 gene were obtained therefrom. PCR was performed by denaturation at 95°C for 5 min, annealing at 53°C for 1 min, and polymerization at 72°C for 1 min 30 sec. PCR was perfonned under the same conditions for obtaining the PCK1 fragment using the genomic DNA of S. cerevisiae as a template along with the primers of SEQ ID NOS: 21 and 22 so that ΡΥΚ1 can be expressed using the TEF2 promoter, and the fragment of the TEF2 promoter were obtained. Then, for the simultaneous expression of PCK1 and ΡΥΚ2 in a single recombinant vector, the pRS416-TEFlpro-PYK2 recombinant vector prepared above was digested with Xhol, and at the time, the fragment of the TEF2 promoter and the PCK1 fragment were cloned using the In-Fusion cloning kit (Clontech). Finally, a single recombinant vector which can overexpress PCK1 and ΡΥΚ2 with the TEF2 promoter and the TEF1 promoter, respectively, and the vector was named as pRS416-TEFlpro-PYK2-TEF2pro-PCKl. The primers used in the preparation of the vector for the overexpression of PCK1 and ΡΥΚ2 are summarized in Table 5 below.

The foreign ACL prepared in Example 4-(1) based on the CC02-0553 strain prepared in Example 2 was introduced, and the vector for the simultaneous overexpression of PCK1/PYK2 prepared in Example 4-(2) was inserted by transfection.

The transfection was performed using a method, which includes treating the CC02-0553 strain cultured in YPD (1 % yeast extract, 2% bacto-peptone, and 2% glucose) medium for 18 hours with a solution containing 0.1 Μ Lithum Acetate, 0.01 Μ Tris-HCl, and 0.001 Μ EDTA (hereinafter, LiAc/TE buffer), and heat-treated along with the LiAc/TE buffer containing 40% PEG at 42°C for 15 minutes for the insertion of a recombinant vector. The thus-prepared strains were named as CC02-0652 and CC02-0765, respectively, and the genetic traits are summarized in Table 6 below.

[Table 6] Introduction of ACL based on strains with inactivated PDC potency, and strains with enhanced activities of PCK1 and ΡΥΚ2

For the evaluation of the ACL-PCKl-PYK2-enhanced strains, the lactic acid fermentation ability was evaluated in the strains with inactivated PDC potencyprepared in Example 4-(3) in the same manner as in Example 3. The results are summarized in Table 7 below.

[Table 7] Introduction of ACL based on strains with inactivated PDC titer and evaluation of strains with enhanced activities of PCK1 and ΡΥΚ2

Furthermore, by the result of the fermentation result of the CC02-0765 strain, it was confirmed that productivity of lactic acid fermentation can be further increased by the enhancement of pyruvate biosynthesis, and thus it was confirmed that the method for lactic acid production by the strategy of the present application employing acetyl-CoA production by a new pathway and enhancement of pyruvate biosynthesis is a method which can not only increase the lactic acid fermentation yield and enhance the growth of a given microorganism but also can increase the lactic acid fermentation productivity, unlike the existing technology.

Accordingly, the CC02-0765 strain was deposited in the Korean Culture Center of Microorganisms (KCCM) on November 28, 2014, with the accession number KCCM11616Ρ under the Budapest Treaty.

Based on the result of Example 5, it was confirmed that the strategy of producing acetyl-CoA by a new pathway and enhancing pyruvate biosynthesis is an effective method for increasing the yield of lactic acid fermentation, enhancing the growth of a microorganism with productivity, and increasing the productivity of lactic acid fermentation, and as such, the present inventors have attempted to confirm whether the enhancement of pyruvate biosynthesis using other genes may have similar effects.

(1) Preparation of a vector with enhanced activities of MDH2 and cytosolic ΜΑΕ1 Since the ΟΑΑ produced by the introduction of a foreign ACL can be biosynthesized into pyruvate by a different pathway, the present inventors attempted to overexpress MDH2, which is originally located in the cytosol, and overexpress ΜΑΕ1, which is an enzyme located in the mitochondria, by changing its location into the cytosol. To this end, a recombinant vector was prepared.

MDH2 is a gene present in yeast microorganisms and can be represented by an amino acid sequence of SEQ ID NO: 35. The fragment of the MDH2 gene was obtained by PCR which was performed using the genomic DNA of S. cerevisiae as a template along with the primers of SEQ ID NOS: 25 and 26. PCR was perfonned by denaturation at 95°C for 5 min, annealing at 53°C for 1 min, and polymerization at 72°C for 1 min. The thus-obtained fragment of the MDH2 gene was cloned based on the pRS414 vector after digesting with restriction enzymes, SpeI and Xhol, in which the MDH2 gene was set up to be overexpressed using the TEF1 promoter. The thus-obtained recombinant vector was named as pRS414-TEF1pro -MDH2.

ΜΑΕ1 is a gene originally present in the mitochondria of yeast microorganisms and can be represented by an amino acid sequence of SEQ ID NO: 37. For the expression of ΜΑΕ1 gene in the cytosol, the ΜΑΕ1 gene was cloned, excluding the mitochondrial target sequence (represented by the amino acid sequence of SEQ ID NO: 51), which consists of a sequence of 90 nucleotides from the start codon of the ΜΑΕ1 gene. PCR was performed using the genomic DNA of S. cerevisiae as a template along with the primers of SEQ ID NOS: 27 and 28. PCR was performed by denaturation at 95°C for 5 min, annealing at 53°C for 1 min, and polymerization at 72°C for 2 min. The thus-obtained PCR fragments were cloned based on the pRS416 vector after digesting with restriction enzymes, Spe I and Xma\. The primer of SEQ ID NO: 27 was prepared in such a manner to obtain the nucleotide sequences starting from the position 91, in order to remove the sequence of the 90 nucleotides starting from the ΜΑΕ1 ORF start codon. Additionally, it was attempted to overexpress the cytosolic ΜΑΕ1 using the TEF1 promoter. Additionally, the thus-prepared recombinant vector was named as pRS416-TEFlpro-cytosolic ΜΑΕ1. The primers used for the preparation of the recombinant vectors, i.℮., pRS4I4-TEFIpro-MDI 12 and pRS416-TEFlpro-cytosolic MAE 1, are summarized in Table 8 below.

[Table 8] Primers for the overexpression of MDH2 and cytosolic ΜΑΕ1

Primer Sequence (5’ -> 3’)

F-MDH2-SpeI CTAGAACTAGTATGCCTCACTCAGTTACACCATCCA

The vector introduced with a foreign ACL prepared in Example 4-(1), and the MDH2 overexpression vector and the cytosolic ΜΑΕ1 overexpression vector prepared in Example 6-(Τ) were cloned by transfection based on the CC02-0553 strain prepared in Example 2. The transfection was performed using the method explained in Example 4-(3). The thus-prepared strain was named as CC02-0821, and the genetic trait of the strain is summarized in Table 9 below.

[Table 9] Introduction of ACL in a strain with inactivated PDC, and a strain with enhanced activity of MDH2 and cytosolic ΜΑΕ1

For the evaluation on the ACL-MDH2-cytosolic MAE 1-enhanced strains, the lactic acid fermentation ability was evaluated in the strains with inactivated PDC prepared in Example 6 in the same manner as in Example 2. The results are summarized in Table 10 below.

[Table 10] Evaluation of fermentation ability of ACL-MDH2-cytosolic MAE 1-enhanced strains based on strains with inactivated PDC

As can be confirmed in Table 10 above, as a result of fermentation ability evaluation of the CC02-0821 strain, in which pyruvate biosynthesis was enhanced by overexpression of MDH2 and cytosolic MAE 1, also showed an increase in OD600 value, which represents the bacterial growth relative to the strain where the PDC activity was removed, by 110%; an increase in the amount of glucose consumption during the same period by 80%; and an improvement in the yield of lactic acid fermentation by 6%, as is the case with the CC02-0765 strain. Additionally, the strains finally showed an improvement of 90% in lactic acid productivity. Based on the results above, it was confirmed that not only the introduction of a foreign ACL for the improvement of lactic acid in a yeast microorganism but also the enhancement of pyruvate biosynthesis pathways via various routes is also effective for further improvement of lactic acid productivity.

Conclusively from the results in Examples, it was confirmed that the strains, in which PDC titer was removed, could increase the productivity of lactic acid fermentation by the introduction of a foreign ACL and the enhancement of pyruvate biosynthesis pathway. As such, the present inventors have attempted to confirm whether the same result could be obtained from the strains, in which the PDC titer was reduced, as well as in the strains, in which the PDC titer was removed.

The recombinant vectors, i.e., pRS415-GPDpro-ACL and pRS416-TEFlpro-PYK2-TEF2p-PCKl, prepared in Example 4 were inserted based on the CC02-0256 strain, which is a strain with reduced PDC activity prepared in Example 2, in the same manner as in Example 4-(3). The thus-prepared strains were named as CC02-0819 and CC02-0820, respectively. Furthermore, the recombinant vectors, i.e., pRS415-GPDpro-ACL prepared in Example 4 and pRS414-TEFlpro-MDH2 and pRS416-TEFlpro-cytosolic ΜΑΕ1, prepared in Example 6-(1) were inserted based on the CC02-0256 strain, which is a strain with reduced PDC activity prepared in Example 2, in the same manner as in Example 4-(3). The thus-prepared strain was named as CC02-0831, and the genetic trait of the strain is summarized in Table 11 below.

[Table 11] Introduction of ACL based on strains with reduced PDC activity and preparation of strains with enhanced pyruvate biosynthesis pathway

The lactic acid fermentation ability of CC02-0819 and CC02-0820 strains prepared in Example 8 was evaluated along with the CC02-0256 strain, which is a control group, in the same manner as in Example 3. The results of the experiments are summarized in Table 12 below.

[Table 12] Introduction of ACL based on strains with reduced PDC activity and evaluation of the fermentation of strains with enhanced activities of PCK1 and ΡΥΚ2

2 above, the CC02-0256 strain did not show a significant increase in effects by increasing the introduction of a foreign ACL and enhancing the pyruvate biosynthesis pathway, unlike in the CC02-0553 strain, but there were an improvement in lactic acid fermentation yield, an increase in bacterial growth of microorganisms with productivity, and an increase in productivity of lactic acid fermentation as well.

The CC02-0820 strain, in which a foreign ACL was introduced and PCK1 and ΡΥΚ2 activities were enhanced, showed an increase in Οϋ℮οο value, which represents the bacterial growth relative to the strain with reduced PDC titer, by 80%; an increase in the amount of glucose consumption during the same period by 40%; and an improvement in the yield of lactic acid fermentation by 2%. Additionally, the lactic acid productivity was finally improved by 40%. The CC02-0820 strain showed increases in all of the OD value, the amount of glucose consumption during the same period, and the yield of lactic acid fermentation, compared to the CC02-0819 strain, which was introduced with a foreign ACL only, and showed a 20% increase in productivity.

The CC02-0831 strain prepared in Example 8 was evaluated along with the CC02-0256 and CC02-0819 strains, which are control groups, in the same manner as in Example 3. The results of the experiments are summarized in Table 13 below.

[Table 13] Introduction of ACL based on strains with reduced PDC titer and evaluation of the fermentation of strains with enhanced activities of MDH2 and cytosolic MAE 1

The above results support that the method for lactic acid production by the strategy employing the acetyl-CoA production by a new pathway and enhanced pyruvate biosynthesis is a method, which can not only increase the yield of lactic acid fermentation and the enhancement of the growth of the microorganism with productivity, but also can increase the productivity of lactic acid fennentation, unlike the existing technology. In particular, the results suggest that the microorganism, which was prepared by the strategy of employing the acetyl-CoA production by a new pathway due to the introduction of a foreign ACL and enhanced pyruvate biosynthesis, can not only increase the yield of lactic acid fennentation and the enhancement of the growth of the microorganism with productivity, but also can increase the productivity of lactic acid fermentation, and thus can be provided as an excellent lactic acid-producing microorganism.

From the foregoing, a skilled person in the art to which the present application pertains will be able to understand that the present application may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present application. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present application. On the contrary, the present application is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents and other embodiments that may be included within the spirit and scope of the present application as defined by the appended claims.

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

r η

To. CJ Cheiljedang Corporation

CJ CHEILJEDANG CENTER,

330, DONGHO-RO,

JUNG-GU, SEOUL 100-400,

REPUBLIC OF KOREA

L J

RECEIPT IN THE CASE OF AN ORIGINAL

issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

Identification reference given by the

DEPOSITOR :

Saccharomyces cerevisiae CC02-0765

Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY:

KCCM11616P

The microorganism identified under I above was accompanied by:

□ a scientific description

□ a proposed taxonomic designation

(Mark with a cross where applicable)

This International Depositary Authority accepts the microorganism identified under I above, which was received by it on November. 28. 2014. (date of the original deposit)¹

Name : Korean Culture Center of Microorganisms

Address : Yurim B/D

45, Hongjenae-2ga-gil

Seodaemun-gu

SEOUL 120-861

Republic of Korea

Signature (s) of person (s) having the power

to represent the International Depositary

Authority or of authorized official (s) :

Date: November. 28. 2014.

Hjjliuc

Mi

||Dfr

¹ Where Rule 6.4(d) applies, such date is the date on which the status of intern

was acquired i where a deposit made outside the

international depositary authority is converted into

date on which the microorganism was received by

Form BP/4

γ authority Budapest Treaty after the acquisition of the status of a deposit under the Budapest Treaty, such date is the the international depositary authority.

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