MICROORGANISM HAVING ENHANCED PRODUCTIVITY OF LACTIC ACID AND A PROCESS FOR PRODUCING LACTIC ACID USING THE SAME

Abstract

The present invention relates to Saccharomyces sp. capable of producing lactic acid with a decreased activity of pyruvate decarboxylase (PDC) and increased activities of aldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS), and a method of producing lactic acid from the culture medium obtained by culturing the microorganism.

Claims

1. An isolated Saccharomyces cerevisiae microorganism having enhanced productivity of lactic acid, wherein the microorganism is modified so that: a) the activity of pyruvate decarboxylase (PDC) of the microorganism is decreased compared to that of a non-modified lactic acid-producing strain; and b) the activities of aldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS) of the microorganism are enhanced compared to that of the non-modified lactic acid-producing strain.

2. The microorganism according to claim 1, wherein the pyruvate decarboxylase is at least one selected from the group consisting of PDC1, PDC5, and PDC6.

3. The microorganism according to claim 2, wherein the microorganism is modified to: i) inactivate PDC1 activity and decrease PDC5 activity; or ii) decrease PDC1 activity and inactivate PDC5 activity.

4. The microorganism according to claim 1, wherein the aldehyde dehydrogenase is at least one selected from the group consisting of ALD2 and ALD3, and the acetyl-CoA synthetase is ACS1.

5. The microorganism according to claim 1, wherein alcohol dehydrogenase (ADH) is further inactivated.

6. The microorganism according to claim 1, wherein D-lactic acid dehydrogenase (DLD) is further inactivated.

7. A method for producing lactic acid comprising: a) culturing the microorganism according to claim 1 in the culture medium; and b) recovering lactic acid from the culture medium or the microorganism in step a).

8. A method for producing lactic acid comprising: a) culturing the microorganism according to claim 2 in the culture medium; and b) recovering lactic acid from the culture medium or the microorganism in step a).

9. A method for producing lactic acid comprising: a) culturing the microorganism according to claim 3 in the culture medium; and b) recovering lactic acid from the culture medium or the microorganism in step a).

10. A method for producing lactic acid comprising: a) culturing the microorganism according to claim 4 in the culture medium; and b) recovering lactic acid from the culture medium or the microorganism in step a).

11. A method for producing lactic acid comprising: a) culturing the microorganism according to claim 5 in the culture medium; and b) recovering lactic acid from the culture medium or the microorganism in step a).

12. A method for producing lactic acid comprising: a) culturing the microorganism according to claim 6 in the culture medium; and b) recovering lactic acid from the culture medium or the microorganism in step a).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0040] FIG. 1 is a schematic diagram illustrating the relationship between the lactic acid production pathway of a Saccharomyces sp microorganism, the alcohol fermentation pathway and the acetyl-CoA production pathway.

BEST MODE FOR CARRYING OUT INVENTION

[0041] Hereinbelow, the present invention 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 invention.

Example 1: Preparation of Lactic Acid-Producing Strain

[0042] To prepare lactic acid-producing strains, Saccharomyces cerevisiae CEN.PK2-1D, a representative wild type yeast obtained from EUROSCARF, was subject to genetic manipulation.

[0043] Specifically, a strain, where alcohol dehydrogenase 1 (ADH1) and pyruvate decarboxylase 1 (PDC1) were defective to minimize the loss of pyruvate to the alcohol synthesis pathway, and d-lactic acid dehydrogenase 1 (DLD1) was defective for blocking the D-type lactic acid decomposition pathway, was used as a base strain.

[0044] DLD1 is not a crucial factor that may have a direct impact on the growth improvement, but has been known as a major enzyme capable of converting D-lactic acid to pyruvate using NAD.sup.+ as D-lactic acid dehydrogenase. Accordingly, a subsequent strain was constructed based on the strain having gene defects in DLD1, an enzyme that consumes the prepared lactic acid thereof, to compare a complete fermentation productivity of D-type lactic acid-producing yeast, which is intended to be prepared in the present invention. As a result, the fermentation productivity was compared.

[0045] In the present invention, a general molecular cloning was employed for the gene manipulation.

[0046] First, in order to delete ADH1 and PDC1 genes of the yeast strains, an experiment was conducted with reference to the content disclosed in the Reference by Lee T H, et al. (J. Microbiol. Biotechnol. (2006), 16(6), 979-982), using plasmids pWAL100 and pWBR100. Each insert, which was introduced into the vector plasmids, was prepared using the suitable primers (corresponding to nucleotide sequences of SEQ ID NOS: 1 to 8) via PCR.

[0047] In addition, for the deletion of DLD1 gene, HIS3, which is a marker gene, was introduced by double crossover, and made it defective. The DNA fragments used therein were prepared using primers corresponding to nucleotide sequences of SEQ ID NOS: 9 and 10. The primers used in the gene manipulation are summarized in Table 1 below.

TABLE-US-00001 TABLE1 Primersusedforthe productionofthebaseyeaststrain Primer 5.fwdarw.3 sequence ADH1upstream CGGGATCCACTGTAGCCCTAGACTT forwardprimer GATAGCC (SEQIDNO:1) ADH1upstream ATAAGAATGCGGCCGCTGTATATGA reverseprimer GATAGTTGATTGTATGCTT (SEQIDNO:2) ADH1downstream GACTAGTGCGAATTTCTTATGATTT forwardprimer ATGATTTTTATT (SEQIDNO:3) ADH1downstream ACATGCCATGgAAGCATGCACGTAT reverseprimer ACACTTGAGTAA (SEQIDNO:4) PDC1upstream CGGGATCCATTATGTATGCTCTTCT forwardprimer GACTTTTCGT (SEQIDNO:5) PDC1upstream ATAAGAATGCGGCCGCTTTGATTGA reverseprimer TTTGACTGTGTTATTTTGC (SEQIDNO:6) PDC1downstream CGGGATCCGCGATTTAATCTCTAAT forwardprimer TATTAGTTAAAG (SEQIDNO:7) PDC1downstream ATAAGAATGCGGCCGCTTTCAATCA reverseprimer TTGGAGCAATCATTTTACA (SEQIDNO:8) DLD1-HIS3 GCGTAGTTGGCCCCAACTGGTGCAG upstream TAATACGTTTTAAGAGCTTGGTGAG linkingprimer (SEQIDNO:9) DLD1-HIS3 CGTGAAGGGTGAAAAAGGAAAATCA downstream GATACCTACATAAGAACACCTTTGG linkingprimer (SEQIDNO:10)

[0048] D-lactic acid dehydrogenase (D-LDH) specifically required for D-lactic acid production was introduced based on the strain having defects in the three genes such as ADH1, PDC1 and DLD1.

[0049] D-LDH was then cloned into a vector having restriction enzyme sites of XhoI and SpeI at 5 and 3 termini, respectively, in order for ldhD derived from lactobacillus plantarum (Lb. plantarum) to be included between TEF1 promoter derived from S. cerevisiae and CYC1 terminator. In particular, the insert was prepared by double-digestion of SacI/PvuII, and the vector was blunt ended by Mungbean nuclease from the DNA fragment, which was double-digested from p--neo into BamHI/NotI. Lastly, the vector was treated with Sac I to thereby obtain a vector having a SacI sticky end and BamHI derived blunt end.

[0050] The construction of pTL573 vector was completed by the ligation of the obtained vector with the insert. The plasmid pTL573 contains the ldhD gene derived from Lb. plantarum, and it was designed so that it may include a random insertion of multiple copies of genes into partial domain of -sequence among retrotransposable element of S. cerevisiae CEN.PK2-1D pdc1 adh1 dld1 strain. For multiple insertion of a corresponding gene, DNA fragments capable of inducing single crossover on the -sequence were constructed by digesting plasmid pTL573 with SalI. By introducing the DNA fragments into a parent strain via transformation, a multiple colonies were obtained from YPD plate (1% yeast extract, 2% bacto-peptone, and 2% glucose) at a maximum concentration of 5 mg/mL G418. Finally, it was confirmed that the thus-obtained strain, the Lb. plantarum derived D-LDH, was multiply inserted for the purpose of providing D-lactic acid-producing-ability, and was assigned CC02-0064 strain.

Example 2: Preparation of Mutant Strains Having Decreased PDC5 Activity

[0051] A mutant strain having substituted PDC5 promoter was prepared based on CC02-0064 strain prepared in Example 1. During the process, processes of cassette preparation and strain selection were conducted according to the method disclosed in Lee T. H. et al. (Development of reusable split URA3-marked knockout vectors for budding yeast, Saccharomyces cerevisiae. J Microbiol Biotechnol, 2006, 16:979-982).

[0052] Specifically, a total of five novel strains were prepared by substituting PDC5 promoter of the CC02-0064 strain with SCO1, SCO2, ACS1, IDP2, and FBA1 promoters, respectively, and subsequently, promoter-substituted cassettes were prepared using primers corresponding to nucleotide sequences of SEQ ID NOS: 11 to 36.

[0053] The primers used in the promoter substitution are summarized in Table 2 below.

TABLE-US-00002 TABLE2 Primersusedforthe preparationofpromoter-substitutedstrains Primers 5.fwdarw.3 sequence F_PDC5_UP_676 GTCAGCATTGACACGTTCGATT (SEQIDNO:11) R_KlURA3-PDC5_UP TCTACCCAGAATCACTTCTTTCGAGAGA (SEQIDNO:12) TTGTCATAATC F_PDC5_UP- CAATCTCTCGAAAGAAGTGATTCTGGGT AL_KlURA3 AGAAGATCGG (SEQIDNO:13) R_AL_KlURA3 GAGCAATGAACCCAATAACGAAATCTT (SEQIDNO:14) F_BR_KlURA3 CTTGACGTTCGTTCGACTGATGAG (SEQIDNO:15) R_PDC5_DOWN_522 CAAGTCAACCAAGTTAGCTGGC (SEQIDNO:16) R_SCO1p-BR_KlURA3 CTCTCCTAATAGACGTGGTGTCACCATG (SEQIDNO:17) AACGACAATTCTTAA F_SCO1p_500 CGTTCATGGTGACACCACGTCTATTAGG (SEQIDNO:18) AGAGCCATTC R_PDC5_DOWN_500- AAGGTTATTTCAGACATCTTTTCTACGT SCO1p TTGCTGTTTTTTC (SEQIDNO:19) F_SCO1p- CAGCAAACGTAGAAAAGATGTCTGAAAT PDC5_DOWN_500 AACCTTAGGTAAAT (SEQIDNO:20) R_SCO2p-BR_KlURA3 ATCGAATAAGTAACAAGCGTGTCACCAT (SEQIDNO:21) GAACGACAATTCTTAA F_SCO2p_500 CGTTCATGGTGACACGCTTGTTACTTAT (SEQIDNO:22) TCGATAACGC R_PDC5_DOWN_500- AAGGTTATTTCAGACATTTTACTCTCGC SCO2p TTCCCAAATTCC (SEQIDNO:23) F_SCO2p- GGAAGCGAGAGTAAAATGTCTGAAATAA PDC5_DOWN_500 CCTTAGGTAAAT (SEQIDNO:24) R_IDP2p-BR_KlURA3 TAAAAATAAATAGATAGACGTGTGTCAC (SEQIDNO:25) CATGAACGACAATTCTTAA F_IDP2p_500 CGTTCATGGTGACACACGTCTATCTATT (SEQIDNO:26) TATTTTTATAACTC R_PDC5_DOWN_500- AAGGTTATTTCAGACATTACGATTTTAT IDP2p ATATATACGTACGTTA (SEQIDNO:27) F_IDP2p- CGTATATATATAAAATCGTAATGTCTGA PDC5_DOWN_500 AATAACCTTAGGTAAAT (SEQIDNO:28) R_ACS1p-BR_KlURA3 CTGGACGTATGTGCACAGTGTCACCATG (SEQIDNO:29) AACGACAATTCTTAA F_ACS1p_500 CGTTCATGGTGACACTGTGCACATACGT (SEQIDNO:30) CCAGAATGAT R_PDC5_DOWN_500- AAGGTTATTTCAGACATAGCACAGTGGG ACS1p CAATGTCTTTC (SEQIDNO:31) F_ACS1p- CATTGCCCACTGTGCTATGTCTGAAATA PDC5_DOWN_500 ACCTTAGGTAAAT (SEQIDNO:32) R_FBA1p-BR_KlURA3 TTATTTACGTAATGACCCAGTGTCACCA (SEQIDNO:33) TGAACGACAATTCTTAA F_FBA1p_500 CGTTCATGGTGACACTGGGTCATTACGT (SEQIDNO:34) AAATAATGATAG R_PDC5_DOWN_500- AAGGTTATTTCAGACATTTTGAATATGT FBA1p ATTACTTGGTTATGGT (SEQIDNO:35) F_FBA1p- CCAAGTAATACATATTCAAAATGTCTGA PDC5_DOWN_500 AATAACCTTAGGTAAAT (SEQIDNO:36)

[0054] The thus-prepared novel strains were assigned CC02-0167, CC02-0168, CC02-0169, CC02-0170, and CC02-0174, respectively. The corresponding strains and their genetic traits are summarized in Table 3 below.

TABLE-US-00003 TABLE 3 PDC5 promoter-mutated strains Strains Genetic Traits CC02-0167 CC02-0064 PDC5 promoter::KlURA3-SCO1 promoter CC02-0168 CC02-0064 PDC5 promoter::KlURA3-SCO2 promoter CC02-0169 CC02-0064 PDC5 promoter::KlURA3-ACS1 promoter CC02-0170 CC02-0064 PDC5 promoter::KlURA3-IDP2 promoter CC02-0174 CC02-0064 PDC5 promoter::KlURA3-FBA1 promoter

Example 3: Evaluation of Lactic Acid Fermentation for Mutant Strains Having Decreased PDC5 Activity

[0055] An evaluation of lactic acid fermentation was conducted for the PDC5 promoter-mutated strains prepared in Example 2. In this regard, a specific medium was prepared for the evaluation of lactic acid fermentation.

[0056] Specifically, in order to prepare a synthetic complex media (SC media), a limiting medium for yeast, 0.67% yeast nitrogen base without amino acids serving as a base was mixed with amino acid dropout mix (Sigma) according to the protocol of the manufacturer, and added with the amino acids that were excluded in the base, as needed. In addition, 380 mg/L of leucine was added to the resultant, and uracil, tryptophan, and histidine were added at a concentration of 76 mg/L, respectively, 8% of glucose as a carbon source and 1% of CaCO.sub.3 as a neutralizing agent were also added. The thus-prepared medium was used for the evaluation of lactic acid fermentation of the yeast strains.

[0057] Among the PDC5 promoter-mutated strains prepared in Example 2, the mutant strains substituted with a weaker promoter than the original PDC5 promoter failed to grow, whereas the mutant strains substituted with a stronger promoter showed improved growth. Specifically, the mutant strains substituted with promoters of SCO1, SCO2, IDP2 or ACS1, which are weaker promoters than PDC5 promoter, failed to grow, leaving the strains whose promoter was substituted with FBA1 promoter the only strains to be evaluated. The evaluation result of the lactic acid fermentation for CC02-0064 and CC02-0174 strains, which were measurable, is summarized in Table 4 below.

TABLE-US-00004 TABLE 4 Evaluation of lactic acid fermentation for PDC5 promoter-mutated strains 24 hours 48 hours Glucose Lactic Glucose Lactic Yield Strain OD Consumed acid OD Consumed acid (%) CC02- 3.9 15.0 10.9 8.7 63.4 41.6 65.7 0064 CC02- 5.7 25.0 19.8 9.4 69.9 47.3 67.7 0174

[0058] As shown in the evaluation above, it was confirmed that, during the pathway promoting acetyl-CoA production, the strain where the wild-type PDC5 promoter was substituted with FBA1 promoter showed improved cell growth rate and lactic acid productivity thereof, compared to those of the original strain (CC02-0064). However, when the result of samples collected at 24 hours and 48 hours, respectively, were compared, it was confirmed that the improvements on the cell growth rate and the lactic acid productivity thereof according to the time were continued to reduce by a mere strengthening of a single PDC activity without strengthening ALD and ACS activities, which are involved in the subsequent acetyl-CoA producing pathway. In an example of the present invention, the improvement in the glucose consumption by the strengthening PDC activity was 10.3%, and the maximum lactic acid production concentration was 47.3 g/l. Accordingly, the overall improvement of the lactic acid productivity was 13.7%.

Example 4: Preparation of a Strain Having a PDC5 Gene Defect

[0059] In addition to the strain having a PDC1 gene defect and decreased PDC5 activity prepared in Example 2, a strain having a defect in PDC5 gene and decreased PDC1 activity was prepared to thereby confirm whether PDC pathway was attenuated in the corresponding strain.

[0060] Specifically, for the purpose of a PDC5 gene defect, the primers corresponding to nucleotide sequences of SEQ ID NOS: 37 to 40 were used to prepare PDC5 gene defect cassette based on the CC02-0064 strain. The defective strain was prepared by the same method described the literature of Example 1. The primers used in Example 4 are summarized in Table 5 below.

TABLE-US-00005 TABLE5 Primersusedforthe preparationofthestrainhavingPDC5defects Primers 5.fwdarw.3 sequence F-ALPDC5- GAGCTCGGATCCAAGGAAATAAAGCAAA BamHI TAACAATAACACC (SEQIDNO:37) R-ALPDC5- ACCATGGCGGCCGCTTTGTTCTTCTTGT NotI TATTGTATTGTGTTG (SEQIDNO:38) F-BRPDC5- GGATCCACTAGTGCTAATTAACATAAAA SpeI CTCATGATTCAACG (SEQIDNO:39) R-BRPDC5- CAGCTGCCATGGTATTCTAAATAAGATG NcoI TAAGGCCTTGTAAT (SEQIDNO:40)

[0061] The thus-prepared strain having a PDC5 gene defect was assigned CC02-0450 (CC02-0064, pdc5).

Example 5: Preparation of PDC1 Promoter-Mutated Strains Based on the Strain Having a PDC5 Defect

[0062] A strain having substituted PDC1 promoter was prepared based on the CC02-0450 strain prepared in Example 4. In this regard, a strain CC02-0451 (CC02-0450, PDC1p-PDC1), where the defect in PDC1 gene was recovered, was prepared to serve as a comparative group, and a strain CC2-0452 (CC02-0450, IDP1p-PDC1) having decreased PDC1 activity was prepared to serve as an experimental group.

[0063] Each strain was prepared in such a way that the vectors of PDC1p-PDC1-CYC1t and pRS406-IDP2p-PDC1-CYC1, which were constructed by cloning a target gene cassette into a pRS406 vector without a replication origin in the yeast, to be included in the strain.

[0064] Specifically. PCR was conducted using primers having nucleotide sequences of SEQ ID NOS: 41 and 42 with chromosomal DNA of the yeast serving as a template, to thereby obtain a product including PDC1 gene. Subsequently, a sequence of CYC1 terminator was obtained using primers having nucleotide sequences of SEQ ID NOS: 43 and 44. In addition, DNA fragments connecting PDC1 and CYC1 terminator were obtained via PCR using primers corresponding nucleotide sequences of SEQ ID NOS: 41 and 44 with the PDC1 and the CYC1 terminator sequences, respectively, serving as a template. A plasmid vector of pRS406-PDC1-CYC1t was obtained by treating DNA fragments of PDC1-CYC1 terminator and pRS406 vector with SpeI and XhoI restriction enzymes followed by ligation thereof. Meanwhile, for the introduction of the promoter domain into the thus-obtained plasmid vectors, plasmid vectors, into which promoters of PDC1 and IDP2 promoters were respectively incorporated, were obtained by a primer fusion of primers having nucleotide sequences of SEQ ID NOS: 45 and 46, and 47 and 48, respectively, via PCR using chromosomal DNA as a template. DNA fragments including each promoter and pRS406-PDC1-CYC1t plasmid were digested and ligated, to thereby prepare plasmid vectors of pRS406-PDC1p-PDC1-CYC1t and pRS406-IDP2p-PDC1-CYC1t, respectively, which are plasmid required for the yeast chromosomal insertion, designed such that the gene expression is controlled by PDC1 promoter and IDP2 promoter.

[0065] The primers used in Example 5 are summarized in Table 6.

TABLE-US-00006 TABLE6 Primersusedforpreparationofthestrains havingaPDC5defectanddecreasedPDC1activity Primers 5.fwdarw.3 sequence F_PDC1 ATAACTAGTATGTCTGAAATTACTTTG (SEQIDNO:41) GGTAAATATTT R_PDC1 CAAAGGAAAAGGGGCCTGTTTATTGCT (SEQIDNO:42) TAGCGTTGGTAGCAGCA F_CYC1t TACCAACGCTAAGCAATAAACAGGCCC (SEQIDNO:43) CTTTTCCTTTGTCGAT R_CYC1t ATACTCGAGGCAAATTAAAGCCTTCGA (SEQIDNO:44) GCGTCC F_PDC1p AAAGAGCTCCATGCGACTGGGTGAGCA (SEQIDNO:45) TATGTT R_PDC1p ATAACTAGTTTTGATTGATTTGACTGT (SEQIDNO:46) GTTATTTTGC F_IDP2p AAAGAGCTCACGTCTATCTATTTATTT (SEQIDNO:47) TTATAACTCC R_IDP2p ATAACTAGTTACGATTTTATATATATA (SEQIDNO:48) CGTACGTTAC

[0066] The two thus-prepared plasmid vectors were digested by StuI, respectively, and inserted into the strains immediately. The final strains were assigned CC02-0451(CC02-0450, PDC1p-PDC1) and CC02-0452(CC02-0450, IDP2p-PDC1), respectively. The thus-prepared strains and their genetic traits are summarized in Table 7.

TABLE-US-00007 TABLE 7 Strains having PDC5 defect and decreased PDC1 activity Strains Genetic Traits CC02-0450 CC02-0064 pdc5 CC02-0451 CC02-0450 PDC1p-PDC1-CYC1t CC02-0452 CC02-0450 IDP2p-PDC1-CYC1t

Example 6: Preparation of Strains Having Double or Triple Defects in PDC Genes

[0067] Strains having a single defect in PDC1 gene, a double defect in PDC1 and PDC5 genes, and a triple defect in PDC1, PDC5, and PDC6 genes were intended to be prepared from PDC family genes. CC02-0064 strain prepared in Example 1 was used as a strain having a single defect in PDC1 gene. A cassette for PDC5 defect was prepared using primers corresponding nucleotide sequences of SEQ ID NOS: 49 to 56, and inserted into CC02-0064 to prepare a strain having double defects in PDC1 and PDC5 genes. Subsequently, the thus-prepared strain was assigned CC02-0256. In addition, a strain having a triple defect in PDC1, PDC5, and PDC6 genes was prepared based on the strain having double defects in PDC1 and PDC5 genes using primers corresponding nucleotide sequence of SEQ ID NOS: 57 to 64 to, and was assigned CC02-0257.

[0068] The defect cassette preparation and strain selection process were conducted by the same method described in the literature disclosed in Example 1. The primers used in Example 6 are summarized in Table 8.

TABLE-US-00008 TABLE8 Primersusedforpreparationofthestrains havingdoubleortripledefectsinPDCgenes Primers 5.fwdarw.3 sequence F_BamHI- CGGGATCCAGGCCAAGGAAATAAAGCA PDC5_UP AATAACAA (SEQID.49) R_NotI-PDC5_UP ATAAGAATGCGGCCGCTTTGTTCTTCT (SEQIDNO:50) TGTTATTGTATTGTGTT F_BamHI- CGGGATCCGCTAATTAACATAAAACTC PDC5_DOWN ATGATTCAA (SEQIDNO:51) R_NotI- ATAAGAATGCGGCCGCTATTCTAAATA PDC5_DOWN AGATGTAAGGCCTTGTA (SEQIDNO:52) F_PDC5_UP AGGCCAAGGAAATAAAGCAAATAACAA (SEQIDNO:53) R_AL_KlURA3 GAGCAATGAACCCAATAACGAAATCTT (SEQIDNO:54) F_BR_KlURA3 CTTGACGTTCGTTCGACTGATGAG (SEQIDNO:55) R_PDC5_DOWN TATTCTAAATAAGATGTAAGGCCTTGT (SEQIDNO:56) A F_BamHI- CGGGATCCTGTTATAGAGTTCACACCT PDC6_UP TATTCACA (SEQIDNO:57) R_NotI-PDC6_UP ATAAGAATGCGGCCGCTTTGTTGGCAA (SEQIDNO:58) TATGTTTTTGCTATATTA F_BamHI- CGGGATCCGCCATTAGTAGTGTACTCA PDC6_DOWN AACGAAT (SEQIDNO:59) R_NotI- ATAAGAATGCGGCCGCGATGCAGAATG PDC6_DOWN AGCACTTGTTATTTAT (SEQIDNO:60) F_PDC6_UP TGTTATAGAGTTCACACCTTATTCACA (SEQIDNO:61) R_AL_KlURA3 GAGCAATGAACCCAATAACGAAATCTT (SEQIDNO:62) F_BR_KlURA3 CTTGACGTTCGTTCGACTGATGAG (SEQIDNO:63) R_PDC6_DOWN GATGCAGAATGAGCACTTGTTATTTAT (SEQIDNO:64)

[0069] The thus-prepared strains and their genetic traits are summarized in Table 9.

TABLE-US-00009 TABLE 9 Strains having double or triple defects in PDC genes Strains Genetic traits CC02-0256 CC02-0064 pdc5 CC02-0257 CC02-0256 pdc6

Example 7: Preparation of ALD and ACS1 Overexpressing Strains

[0070] For the preparation of ALD and ACS1 over expressing strains, ALD2, ALD3, and ACS1 over expressing plasmids were prepared.

[0071] Specifically, an open reading frame (ORF) of ALD2 was prepared using primers corresponding nucleotide sequences of SEQ ID NOS: 65 and 66, an ORF of ALD3 was prepared using primers corresponding nucleotide sequences of SEQ ID NOS: 67 and 68, and an ORF of ACS1 was prepared using primers corresponding nucleotide sequences of SEQ ID NOS: 69 and 70. In addition, p415ADH-ALD2, p415ADH-ALD3, p414ADH-ACS1 and p416ADH-ACS1, which are p414ADH, p415ADH and p416ADH plasmid-based recombinant vectors, were prepared by SpeI, XhoI or EcoRI restriction enzymes. The primers used in Example 7 are summarized in Table 10 below.

TABLE-US-00010 TABLE10 Primersusedforthepreparation ofALDandACS1overexpressingstrains Primers 5.fwdarw.3 sequence F_SpeI_ALD2 CAAGCTGGCCGCTCTAGAACTAGTATGC (SEQIDNO:65) CTACCTTGTATACTGATATCGA R_XhoI_ALD2 ACATAACTAATTACATGACTCGAGTTAG (SEQIDNO:66) TTGTCCAAAGAGAGATTTATGT F_SpeI_ALD3 CAAGCTGGCCGCTCTAGAACTAGTATGC (SEQIDNO:67) CTACCTTGTATACTGATATCGA R_XhoI_ALD3 ACATAACTAATTACATGACTCGAGTTAT (SEQIDNO:68) TTATCCAATGAAAGATCCACAT F_SpeI_ACS1 TCCAAGCTGGCCGCTCTAGAACTAGTAT (SEQIDNO:69) GTCGCCCTCTGCCGTACA R_EcoRI_ACS1 TATCGATAAGCTTGATATCGAATTCTTA (SEQIDNO:70) CAACTTGACCGAATCAATTAGA

[0072] The thus-prepared recombinant plasmids were introduced into the strains including CC02-0064, CC02-0168, CC02-0170, CC02-0256, CC2-0257, CC02-0451, and CC02-0452 via a yeast transformation by p415ADH-ALD2, p414ADH-ACS1 combination, p415ADH-ALD3, p414ADH-ACS1 combination, p415ADH-ALD2, p416ADH-ACS1 combination, or p415ADH-ALD3, p416ADH-ACS1 combination. However, no transformant was obtained in the CC02-0257 strain having triple defects in PDC genes where no PDC activity was exhibited.

[0073] The thus-prepared strains and their genetic traits and summarized in Table 11.

TABLE-US-00011 TABLE 11 ALD and ACS overexpressing strains Strains Genetic Traits CC02-0225 CC02-0064 p415ADH, p416ADH CC02-0226 CC02-0064 p415ADH-ALD2, p416ADH-ACS1 CC02-0227 CC02-0064 p415ADH-ALD3, p416ADH-ACS1 CC02-0356 CC02-0168 p414ADH, p415ADH CC02-0275 CC02-0168 p414ADH-ACS1, p415ADH-ALD2 CC02-0276 CC02-0168 p414ADH-ACS1, p415ADH-ALD3 CC02-0357 CC02-0170 p414ADH, p415ADH CC02-0277 CC02-0170 p414ADH-ACS1, p415ADH-ALD2 CC02-0278 CC02-0170 p414ADH-ACS1, p415ADH-ALD3 CC02-0444 CC02-0256 p415ADH, p416ADH CC02-0361 CC02-0256 p415ADH-ALD2, p416ADH-ACS1 CC02-0362 CC02-0256 p415ADH-ALD3, p416ADH-ACS1 CC02-0453 CC02-0451 p414ADH, p415ADH CC02-0454 CC02-0451 p414ADH-ACS1, p415ADH-ALD2 CC02-0455 CC02-0451 p414ADH-ACS1, p415ADH-ALD3 CC02-0456 CC02-0452 p414ADH, p415ADH CC02-0457 CC02-0452 p414ADH-ACS1, p415ADH-ALD2 CC02-0458 CC02-0452 p414ADH-ACS1, p415ADH-ALD3

Example 8: Evaluation of Lactic Acid Fermentation for the Yeast Strains

[0074] An evaluation of lactic acid fermentation-ability for the ALD and ACS1 overexpressing strains, prepared in Example 7, was conducted.

[0075] Specifically, the yeast was inoculated into each flask containing 25 ml of the medium, prepared in Example 3 for the purpose of lactic acid fermentation evaluation and was cultured under aerobic condition at 30 C. for 71 hours. The amount of D-type lactic acid present in the fermented broth was analyzed, and an enzymatic analysis (Acetic acid, R-Biopharm, Germany) was conducted to determine the amount of acetic acid present therein.

[0076] The above experiment results are summarized in Table 12 below.

TABLE-US-00012 TABLE 12 Evaluation of the growth rate, lactic acid fermentation, by-products, and production yield, etc., for the ALD and ACS overexpressing strains Initial Residual D-lactic Produc- Final glucose glucose Acetate acid Yield tivity Strains OD (g/L) (g/L) (g/L) (g/L) (g/g) (g/l .Math. h) CC02- 9.3 88 10 2.87 41.1 0.53 0.579 0225 CC02- 9.3 83 10.5 2.91 42.4 0.59 0.597 0226 CC02- 10.1 84 9.5 2.91 41.8 0.56 0.589 0227 CC02- 6.9 88 26 0.02 27.6 0.45 0.389 0356 CC02- 11.6 88 11.5 0.01 47.6 0.62 0.670 0275 CC02- 10.6 88 11 0.01 46.8 0.61 0.659 0276 CC02- 12.2 88 13 0.04 38.1 0.51 0.537 0357 CC02- 17.8 88 1 0.03 58.6 0.67 0.825 0277 CC02- 18.8 88 0 0.02 56.9 0.66 0.801 0278 CC02- 9.8 88 8.5 2.2 38.9 0.49 0.548 0453 CC02- 10.2 88 8.1 2.4 39.5 0.49 0.556 0454 CC02- 9.2 88 8.8 2.1 40 0.51 0.563 0455 CC02- 12 88 10.1 0.02 38.5 0.49 0.542 0456 CC02- 18.1 88 0 0.02 55.8 0.63 0.786 0457 CC02- 18.5 88 0 0.02 56.5 0.64 0.800 0458

[0077] As verified in Table 12, the strains having decreased PDC5 activity by IDP2 promoter or SCO2 promoter had a dramatic reduction in the accumulation of the acetate, a by-product, i.e., little detection of acetate was confirmed, compared to the strain with normal PDC5 activity. In such case, the final cell concentration of the strains, in which the activities of ALD and ACS were not increased, tended to decrease according to the PDC5 promoter substitutions. On the contrary, the strains, where the PDC5 expression was reduced and the activities of ALD and ACS were increased, showed an increase in the final cell concentration. Accordingly, the improvement in the cell growth was confirmed. Specifically, the strains of CC02-0277 and CC02-0278 having increased ALD and ACS activities prepared based on the CC02-0170 strain, where PDC5 promoter was substituted with IDP2, showed improved growth rate, D-lactic acid concentration of production, production yield thereof, and fermentation productivity as increasing in the ALD and ACS activities.

[0078] In summary, the strain where PDC5 was substituted with a weak expression of IDP2 had a reduction in acetate accumulation, and the final OD was 1.3 times higher compared to the strain exhibiting normal expression of PDC5. In addition, it was confirmed that, when ALD and ACS were co-expressed under the control of the ADH1 promoter, the glucose consumption and the rate thereof were increased, and finally, the percentage yield was increased from 56% or %59% to 66% or 67%, respectively, showing improved yield.

[0079] Specifically, by comparing the two kinds of promoters applied to the weak expression of PDC5, it was confirmed that, the lactic acid productivity was improved in both strains having SCO2 promoter and IDP2 promoter, respectively. However, the strain having IDP2 promoter may be considered as the most optimized form of a strain in terms of the overall cell concentration, the glucose consumption, and the rate thereof.

Example 9: Evaluation of Lactic Acid Fermentation for the Yeast Strain Having Double Defects in PDC1 and PDC5 Genes, and Increase in ALD and ACS Activities

[0080] Since the effects of the cell growth and the yield improvements resulted from the decreased PDC5 activity have been clearly confirmed, an evaluation was undertaken to determine the effects of additional PDC gene defect in lactic acid production. The evaluation method for each strain was identical to the method described in Example 8, and the culturing was conducted for 74 hours.

[0081] The thus-obtained experiment results are summarized in Table 13 below.

TABLE-US-00013 TABLE 13 The evaluation results of lactic acid fermentation for the strains having double defects in PDC1 and PDC5 genes Initial Residual D-lactic Produc- Final Glucose Glucose Acetate acid Yield tivity Strains OD (g/L) (g/L) (g/L) (g/L) (g/g) (g/l .Math. h) CC02- 3.2 78.5 52 0.10 20.8 78.5 0.281 0444 CC02- 3.9 78.5 49 0.05 25.4 86.3 0.343 0361 CC02- 3.8 78.5 51 0.08 22.8 82.8 0.308 0362

[0082] As confirmed in Table 13, the acetate concentration was clearly reduced in the strains having double defects in PDC1 and PDC5 genes, however, a reduction in the D-lactic acid concentration of production was also observed due to the reduction in the cell growth and the glucose consumption. In addition, the strain where the PDC pathway is almost inactivated, which was resulted from the double defects in PDC1 and PDC5 genes, did not have any improvement in the cell growth, the glucose consumption, and the productivity thereof, although the strain exhibited increased activities of ALD and ACS.

Example 10: Evaluation of Lactic Acid Fermentation for the Strains, where PDC Pathway is Attenuated, Using Sucrose

[0083] For the purpose of a fermentation evaluation using sucrose, the lactic acid-producing yeast strains, where PDC pathway is attenuated, the identical strains evaluated in Example 8 and 9 were used to confirm the effect of the lactic acid production. In this regard, sucrose was employed as a carbon source instead of glucose. The evaluation method was performed in the same manner as Example 8.

[0084] The thus-obtained experiment results are summarized in Table 14 below.

TABLE-US-00014 TABLE 14 The evaluation results of lactic acid fermentation for the strains having double defects in PDC1 and PDC5 genes or decreased PDC5 activity Initial Residual D-lactic Produc- Final Glucose Glucose Acetate acid Yield tivity Strains OD (g/L) (g/L) (g/L) (g/L) (g/g) (g/l .Math. h) CC02- 5.15 91.5 27.5 1.95 25.19 39.36 0.34 0225 CC02- 6.9 91.5 15 1.92 30.89 40.38 0.42 0226 CC02- 6.18 91.5 15 1.98 29.59 38.68 0.40 0227 CC02- 1.6 91.5 26.75 0.02 11.72 18.11 0.16 0356 CC02- 1.88 91.5 21.75 0.02 13.79 19.77 0.19 0275 CC02- 2.2 91.5 18.25 0.01 16.04 21.9 0.22 0276 CC02- 2.78 91.5 22 0.03 17.08 24.58 0.23 0357 CC02- 12.15 91.5 7.75 0.02 44.65 53.31 0.60 0277 CC02- 11.88 91.5 6.25 0.01 43.84 51.43 0.60 0278 CC02- 2.45 91.5 37 0.02 21.94 40.25 0.30 0444 CC02- 2.5 91.5 18.25 0.02 21.73 29.66 0.29 0361 CC02- 3.08 91.5 15.25 0.02 24.92 32.67 0.34 0362

[0085] The use of sucrose instead of glucose for the strains, used in the same manner as in Examples 8 and 9, allowed for improved effects of the growth and fermentation yield by increasing the activities of ALD and ACS in the strains where PDC pathway is attenuated, showing the same pattern of results as the strains in Example 8 where glucose was employed as a carbon source. Accordingly, the present invention confirms that the improved effects of fermentation yield and growth due to the decreased PDC activity and increased ALD and ACS activities, which were confirmed in the present invention, are not limited to the type of sugar used.

[0086] To summarize the above results, it was confirmed that, when the strains were mutated in such a way that PDC pathway was attenuated, and the activities of ALD and ACS were improved compared to that of the non-mutated strains, the lactic acid production was increased, and the growth rate thereof was maintained simultaneously.

[0087] From the foregoing, a skilled person in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present invention. 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 invention. On the contrary, the present invention 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 invention as defined by the appended claims.