TW201702372A - A microorganism having enhanced productivity of lactic acid and a process for producing lactic acid using the same - Google Patents

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.

Description

Microorganism having enhanced lactic acid productivity and method for producing lactic acid using the same

The present invention relates to a microorganism of the genus Saccharomyces sp. which produces lactic acid, and a method for producing lactic acid from a medium containing the microorganism by culturing the microorganism.

In general, lactic acid is an important organic acid having a wide range of applications including food additives (eg, food preservatives), perfumes, or acidulants; it has been widely used in industrial applications such as cosmetics, chemicals, metals, electronics, fabrics, Textile printing and dyeing, and the pharmaceutical industry. In addition, lactic acid is an essential component of polylactic acid, a biodegradable plastic, and thus the demand for lactic acid has been significantly increased. Lactic acid is also used as an important material for the production of many chemical compounds including polylactic acid, acetaldehyde, polypropylene glycol, acrylic acid, and 2,3-pentathione. Specifically, D-type lactic acid is an essential component for producing a stereoscopic complex PLA which is an optical isomer required for producing highly heat-resistant PLA.

Specifically, methods for producing lactic acid include tradition Chemical synthesis and biological fermentation. When lactic acid is produced by chemical synthesis, the lactic acid produced is in the form of a racemic mixture consisting of 50% D-type lactic acid and 50% L-type lactic acid, and the composition ratio is difficult to control, so that the resulting polylactic acid may become A low melting point amorphous polymer, thereby imposing limitations on the development of its use. On the other hand, depending on the strain to be used, the biological fermentation method can selectively produce D-type lactic acid or L-type lactic acid. Therefore, the latter is commercially preferred due to the possibility of producing a specific lactic acid isoform.

At the same time, by introducing an enzyme gene which is converted into D-type lactic acid, it has been attempted to increase the productivity of lactic acid by using a yeast microorganism having a D-lactic acid-producing ability through various genetic operations. Specifically, the activity of lactic dehydrogenase (LDH) has been utilized while reducing the activity of pyruvate decarboxylase (PDC), aldehyde dehydrogenase (ALD), and/or acetoin coenzyme A synthetase (ACS). Attempts to increase the productivity of lactic acid (US Patent Application Bulletin Nos. 2012-021421, 2010-0248233, and 2006-0148050). However, since the cells of the lactic acid producing strain grow slowly, the overall fermentation productivity is low.

The present inventors have made intensive efforts to obtain microorganisms having lactic acid productivity which have high efficiency of cell growth while reducing PDC activity. As a result, it has been confirmed that a strain controlling isotype PDC activity and increasing aldehyde dehydrogenase activity and acetamyl coenzyme A activity can increase the yield of lactic acid production and promote cell growth of the strain, thereby enhancing the overall lactic acid fermentation productivity, thus resulting in The completion of the invention.

It is an object of the present invention to provide a microorganism of the genus Yeast which has an improved productivity of lactic acid.

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

The present invention relates to the use of microorganisms for improving the productivity of lactic acid fermentation by controlling the activity of homologous PDC and increasing the activity of aldehyde dehydrogenase (ALD) and acetoin coenzyme A synthetase (ACS); therefore, it can be widely applied to lactic acid fermentation production. In the industry.

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In the first aspect of the present invention, in order to achieve the above object, there is provided a microorganism of the genus Saccharomyces having a lactic acid-promoting property, wherein the microorganism is subjected to a mutation such that (a) is compared with an unmutated lactic acid producing strain, acetone The activity of the acid decarboxylase is decreased; and (b) the activity of the aldehyde dehydrogenase and the acetoacetin A synthetase is enhanced compared to the lactic acid producing strain which has not been mutated.

In general, lactic acid producing yeast microorganisms use pyruvate as a substrate to produce lactic acid via lactate dehydrogenase (LDH). Using the pyruvic acid as a representative metabolic pathway of the common substrate, the ethanol fermentation pathway and the acetaminophen coenzyme A production pathway were blocked. Reducing PDC activity may help The production of lactic acid and its yield increase, however, when the degree of reduction reaches a certain level, an insufficient amount of cytosolic acetaminophen coenzyme A is generated, which in turn blocks the growth of the cells, and thus normal fermentation cannot be achieved. Therefore, the present inventors have developed a yeast microorganism having a lactic acid-promoting productivity by increasing the productivity of the overall lactic acid fermentation, wherein the growth of the lactic acid productivity is improved by controlling the acetaminophen A pathway to a minimum. rate.

As used herein, the term "pyruvate decarboxylase (PDC)" refers to a protein which is capable of transmitting an activity responsible for the reaction of carbonic acid with acetaldehyde from pyruvate, but is not limited to any derivative or isoform thereof having the same activity. . This protein is known to be involved in the steps of ethanol fermentation, mainly in yeast and plants. The pyruvate decarboxylase of the present invention may be present in the microorganism of the genus Saccharomyces in nature, or may be PDC1, PDC5, and/or PDC6, or specifically PDC1, PDC5, and/or Saccharomyces cerevisiae . Or PDC6, but not limited to this. The protein may include any variant or analog thereof as long as it is biologically identical to the protein and has activity corresponding to the protein. The amino acid sequence of the protein can be obtained from a known database or the like, for example, GenBank of NCBI, etc., but is not limited thereto. Specifically, PDC1 may consist of the amino acid sequence of SEQ ID NO: 71, PDC5 consists of the amino acid sequence of SEQ ID NO: 71, and PDC6 consists of the amino acid sequence of SEQ ID NO: 73. The protein may comprise an amine having greater than 70%, specifically greater than 80%, more specifically greater than 90%, and more specifically greater than 95% homology to each of the above listed amino acid sequences. Base acid sequence. Any variant of the above listed sequences encoding the same amino acid sequence produced by genetic code degeneracy can also be included in the present invention.

The term "homology" as used herein refers to the degree of similarity between a plurality of nucleotide sequences or amino acid sequences, and which is a sequence which has the same sequence as the amino acid sequence or nucleotide sequence of the present invention. Unit, the probability of which is equal to or greater than the above possibilities. This homology can be determined by visual comparison of two established sequences, but more appropriately, sequence comparison program measurements can be performed by easily arranging the sequences to be compared in parallel to indicate the degree of homology. Sequence comparison programs are known in the art to which the invention pertains, including FASTP, BLAST, BLAST2, PSIBLAST, and software containing CLUSTAL W.

There have been many reports on the use of lactic acid production which is defective in PDC1 which allows to exhibit the main activity in lactic acid production [Appl Microbiol Biotechnol. 2009, 82(5): 883-90]. In this case, since PDC6 is rarely expressed, the actual PDC activity occurs due to the expression of the PDC5 gene. According to reports, PDC1 deficiency alone does not prevent cell growth of wild-type strains, and maintains about 60 to 70% of PDC activity compared to wild type, so no significant phenotypic changes have been observed in this strain [J Bacteriol. 1990, 172(2): 678-685]. Meanwhile, in order to maximize lactic acid via the LDH pathway which competes with PDC for pyruvic acid, strains having a triple defect at the same time as PDC1, PDC5, and PDC6 can be prepared. In this case, the yield of lactic acid fermentation can be maximized, but inhibition of glycolysis induced by the presence of glucose may further inhibit the metabolism of ethanol and acetic acid, thereby reducing cell growth and ultimately reducing fermentation productivity [(Curr Genet) .2003, 43(3): 139-160)].

Since PDC6 is rarely expressed, the actual PDC activity is due to the expression of the PDC5 gene. According to reports, PDC1 deficiency alone does not prevent cell growth of wild-type strains, and maintains about 60 to 70% of PDC activity compared to wild type, so no significant phenotypic changes have been observed in this strain [J Bacteriol. 1990, 172(2): 678-685]. Alternatively, a strain having both defects in both of PDC1 and PDC5 (which is equivalent to exhibiting major PDC activity in yeast) can be prepared. In this case, lactic acid fermentation can be carried out using a sugar source such as hepatic sugar in the absence of a co-matrix such as acetic acid or ethanol. However, since the PDC activity is rapidly reduced, the growth rate of the yeast strain is lowered, thereby reducing the fermentation productivity of lactic acid [Biosci Biotechnol Biochem. 2006, 70(5): 1148-1153].

In particular, the reduced pyruvate decarboxylase (PDC) activity of the invention can i) deactivate PDC1 activity and reduce PDC5 activity; or ii) reduce PDC1 activity and deactivate PDC5 activity.

In an exemplary embodiment of the invention, four different strains are prepared based on a S. cerevisiae strain in which PDC1 activity is deactivated, including: a PDC5 activity-reduced strain via a substitution of the PDC5 gene promoter, and a PDC5-induced PDC1 activity. A strain defective in the gene t, a strain causing a double defect in the PDC1 and PDC5 genes, and a strain causing a triple defect in the PDC1, PDC5, and PDC6 genes. Among the strains thus prepared, the strain having the triple gene defect showed little cell growth.

The term "aldehyde dehydrogenase (ALD)" as used herein refers to A protein which mainly produces an acetic acid activity from acetaldehyde by a protein which produces an activity of a carboxylic acid or a thiol group by oxidation of an aldehyde, and is not limited to a derivative or an isoform thereof having the same activity in the present invention. The aldehyde dehydrogenase of the present invention may be derived from a microorganism of the genus Yeast, or may be ALD2 and/or ALD3. Specifically, the protein may be ALD2 and/or ALD3 of S. cerevisiae, but is not limited thereto, and may include any variant or analog thereof as long as it is biologically identical to the protein and has a corresponding The activity of the protein can be. The amino acid sequence of the protein can be obtained from a database known in the art to which the invention pertains, for example, GenBank of NCBI, etc., but is not limited thereto. In particular, ALD2 may consist of the amino acid sequence of SEQ ID NO: 74, and ALD3 may consist of the amino acid sequence of SEQ ID NO: 75. The protein may comprise an amino acid sequence having greater than 70%, specifically greater than 80%, more specifically greater than 90%, and more specifically greater than 95% homology to the amino acid sequences. Any sequence variant encoding a completely identical amino acid sequence produced by degeneracy of the genetic code can also be included in the present invention.

The term "acetamide coenzyme A synthetase (ACS)" as used herein refers to a protein having an activity of conjugation with ATP decomposition reaction, which catalyzes the esterification of acetic acid with coenzyme A, but is not limited thereto, and has the same in the present invention. Active derivative or isoform. It is generally known that the protein is present in microorganisms, plants, animals, and the like. The acetaminophen coenzyme A synthetase of the present invention may be derived from a microorganism of the genus Saccharomyces or may be ACS1. Specifically, the protein may be ACS1 of Saccharomyces cerevisiae, but is not limited thereto, and may include any variant or analog thereof as long as it is biologically identical to the protein and has activity corresponding to the protein. can. Amino acid sequence of the protein Columns are available from known databases, such as NCBI's GenBank, etc., but are not subject to this limitation. In particular, ACS1 may consist of the amino acid sequence of SEQ ID NO: 76 and may comprise greater than 70%, specifically greater than 80%, more specifically greater than 90%, and more specifically with the amino acid sequence. An amino acid sequence of greater than 95% homology. A protein mutant sequence encoding a completely identical amino acid sequence produced by degeneracy of the genetic code can also be included in the present invention.

In an exemplary embodiment of the invention, strains having ALD2 and ACS activity, or increased ALD3 and ACS activity are prepared to obtain strains having reduced PDC activity compared to unmutated microorganisms. Specifically, a strain having an increased ALD and ACS activity was prepared based on a strain having deactivated PDC1 via a PDC1 defect and a PDC5-reducing activity via a promoter having a low expression ability in place of the PDC5 gene promoter. More specifically, a yeast strain microorganism strain in which PDC1 activity deactivation, PDC5 activity is decreased, activity selected from at least one of a group consisting of ALD2 and ALD3 is increased, and ACS1 activity is increased is prepared. Therefore, it was confirmed that the growth rates of the strains, the D-lactic acid production rate, and the yield thereof were remarkably enhanced.

The term "deactivation" of the enzyme activity of the present invention refers to a method for deactivating the activity of an enzyme, including any method of inhibiting the expression of an enzyme, or allowing the expression of an enzyme that does not exhibit its original activity. The method may include partial gene deletion or total gene deletion caused by homologous recombination, inhibition of enzyme expression caused by insertion of a foreign gene into a related gene, inhibition of enzyme expression by substitution or modification of an enzyme gene promoter sequence, or The mutation caused by the substitution or modification of the enzyme becomes the deactivated leaven whose original function is missing. Prime, etc., but not limited to this.

The term "reduction" of the activity of an enzyme as used herein refers to a method for reducing the activity of an enzyme, including any method for reducing the amount of enzyme expression or reducing the activity of an enzyme being expressed. The method may include, without limitation, a substitution or modification of an enzyme gene promoter sequence resulting in a decrease in expression, or a mutation caused by substitution or modification of an enzyme, such as an enzyme having reduced activity.

The term "increased" in the activity of an enzyme as used herein refers to the insertion of a plastid containing an enzyme gene, the number of genes encoding an enzyme on a chromosome, or the increase in the activity of an enzyme caused by substitution, modification or mutation of an enzyme gene promoter sequence. However, this is not the limit.

The term "yeast microorganism" as used herein refers to a microorganism belonging to the genus Eumycetes , but as long as it involves any of a lactic acid production pathway, an ethanol production pathway, and/or an acetaminophen A production pathway. Not limited to this. Depending on the shape of the yeast, the yeast microorganism can be classified into the genus Saccharomyces, Pichia sp. , Candida sp. , and Saccharomycopsis sp .; In other words, a yeast comprising a plurality of species can be used in the present invention; in detail, the microorganism can be selected from the group consisting of Saccharomyces bayanus , Saccharomyces boulardii , and Iridium yeast. Saccharomyces bulderi ), Saccharomyces cariocanus , Saccharomyces cariocus , Saccharomyces cerevisiae, Saccharomyces chevalieri , Saccharomyces dairenensis , Saccharomyces ellipsoideus , Saccharomyces eubayanus , Saccharomyces exiguus , Fro Saccharomyces florentinus , Saccharomyces kluyveri , Saccharomyces martiniae , Saccharomyces monacensis , Saccharomyces norbensis , Saccharomyces paradoxus , Pasteurian ( Saccharomyces pastorianus) ), Sa Ccharomyces spencerorum , Saccharomyces turicensis , Saccharomyces unisporus , Saccharomyces uvarum , and Saccharomyces zonatus ; more specifically, it may be Saccharomyces cerevisiae.

The use of a representative example of the genus Saccharomyces cerevisiae to prepare microorganisms having reduced PDC activity and enhanced ALD and ACS activity confirmed a significant increase in the production of lactic acid.

The microorganism of the present invention may comprise an ethanol dehydrogenase (ADH) which is further deactivated.

As used herein, the term "ethanol dehydrogenase" refers to a protein having a catalytic activity responsible for the removal of hydrogen from ethanol to produce an aldehyde or ketone, but is not limited to derivatives or isoforms having the same activity. The alcohol dehydrogenase of the present invention may be derived from the genus Yeast, or may be ADH1. Specifically, the protein may be ADH1 of Saccharomyces cerevisiae, but is not limited thereto, and may include any variant or analog thereof as long as it is biologically identical to the protein and has activity corresponding to the protein. can. The amino acid sequence of the protein can be obtained from a known database or the like, for example, GenBank of NCBI, etc., but is not limited thereto. In particular, ADH1 may consist of the amino acid sequence of SEQ ID NO: 77 and may comprise greater than the amino acid sequence 70%, in particular greater than 80%, more specifically greater than 90%, and more specifically greater than 95% homologous amino acid sequence. A protein mutant sequence encoding a completely identical amino acid sequence produced by degeneracy of the genetic code can also be included in the present invention.

The microorganism of the present invention may comprise D-lactate dehydrogenase (DLD) which is further deactivated.

The term "D-lactate dehydrogenase" as used herein refers to a protein having an activity of producing pyruvic acid via anhydration of D-lactic acid, but is not limited to, an isoform having the same activity. The D-lactate dehydrogenase of the present invention can be derived from the genus Saccharomyces, specifically DLD1. In particular, the protein may be DLD1 of Saccharomyces cerevisiae, but is not limited thereto, and may include any variant or analog thereof as long as it is biologically identical to the protein and has activity corresponding to the protein. can. The amino acid sequence of the protein can be obtained from a known database or the like, for example, GenBank of NCBI, etc., but is not limited thereto. In particular, DLD1 may consist of the amino acid sequence of SEQ ID NO: 78 and may comprise greater than 70%, specifically greater than 80%, more specifically greater than 90%, and more specifically with the amino acid sequence. An amino acid sequence of greater than 95% homology. Any variant sequence encoding the exact same amino acid sequence produced by degeneracy of the genetic code can also be included in the present invention.

In the present invention, a strain having defects in ADH1 (an enzyme involved in an ethanol fermentation route using an aldehyde further produced from pyruvic acid as a substrate) and a defect in DLD1 (an enzyme derived from decomposition of lactic acid) are used for accurate measurement according to an acetic acid production route. Regulation of lactic acid fermentation cells Chemical. In an exemplary embodiment of the invention, wherein PDC, ALD, and strains mediated by ACS activity, showed significantly increased lactic acid fermentation productivity; the results are summarized in Table 12.

In another aspect, the invention provides a method of producing lactic acid using the microorganism of the invention.

Specifically, in an exemplary embodiment of the present invention, the present invention provides a method for producing lactic acid, which comprises culturing a microorganism of the present invention in a medium, and collecting lactic acid from the microorganism or a medium containing the microorganism.

The cultivation can be carried out using appropriate medium and culture conditions known in the art to which the invention pertains. The culture process can be easily adjusted by the person skilled in the art depending on the strain used. Examples of the culture method include batch type, continuous type, and feed batch type, but are not limited thereto. The medium used in the culture process should be appropriately adapted to the needs of the particular strain.

The medium used in the present invention contains sucrose or glucose as a main carbon source, and molasses containing a high concentration of sucrose may also be used as a carbon source. Other carbon sources can be used in various appropriate amounts. Organic nitrogen sources include peptone, yeast extract, meat extract, malt extract, corn extract, and soybean wheat, and inorganic nitrogen sources including elements, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Can be used as a nitrogen source. These nitrogen sources can be used singly or in combination. A phosphorus source such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a corresponding sodium salt may be added to the medium. Further, the medium may contain metal salts such as magnesium sulfate and iron sulfate. Furthermore, the medium may be supplemented with amino acids, vitamins, and appropriate precursors. These media or precursors can be used in batch or continuous mode Add to the culture.

During the cultivation, a compound such as ammonium hydroxide, potassium hydroxide, phosphoric acid, or sulfuric acid may be appropriately added to adjust the pH of the culture. Further, an antifoaming agent such as a fatty acid polyethylene glycol ester may be added to inhibit the formation of foam in the culture. In addition, in order to maintain the culture in an aerobic state, oxygen or an oxygen-containing gas may be injected into the culture; to maintain the culture in an anaerobic or microaerobic state, nitrogen, hydrogen, or carbon dioxide gas may be injected without injecting any gas. .

The culture temperature can be maintained at 20 to 40 ° C, specifically 25 to 35 ° C, more specifically 30 ° C. The cultivation is continued until the desired amount of the desired material is obtained, specifically 10 to 100 hours.

Depending on the culture method, for example, batch, continuous, or feed batch, the culture process of the present invention can be collected from microorganisms or medium containing/without microorganisms using suitable methods known in the art to which the invention pertains. Production of lactic acid.

Fig. 1 is a schematic view showing the relationship between the lactic acid production pathway of the microorganism of the genus Saccharomyces, the ethanol fermentation pathway, and the acetylcholine A production pathway.

Hereinafter, the present invention will be described in detail by way of the accompanying exemplified embodiments. However, the specific examples disclosed herein are for illustrative purposes only and therefore should not be construed as limiting the scope of the invention.

Example 1: Preparation of a lactic acid producing strain

In order to prepare a lactic acid producing strain, genetic manipulation was carried out from the representative wild type yeast of EUROSCARF, Saccharomyces cerevisiae CEN.PK2-1D.

In particular, the use of ethanol dehydrogenase 1 (ADH1) and pyruvate decarboxylase 1 (PDC1) is defective to minimize pyruvate consumption in the ethanol synthesis pathway, and d-lactate dehydrogenase 1 (DLD1) is defective A strain which blocks the decomposition pathway of D-type lactic acid is used as a basic strain.

DLD1 is not a key factor that has a direct impact on growth, but it is known to use NAD ⁺ as a D-lactate dehydrogenase to convert D-lactic acid into the main enzyme of pyruvate. Thus, based on the strain having the genetic defect DLD1 (the enzyme which consumes the lactic acid prepared therefrom), the strain which was later prepared in the present invention was constructed to be compared with the complete fermentation productivity of the D-type lactic acid producing yeast. As a result, the fermentation productivity was compared.

In the present invention, the genetic manipulation system uses a general molecular selection method.

First, in order to delete the ADH1 and PDC1 genes of the yeast strain, pWAL100 and pWBR100 plastids were used, see Lee TH, et al. [J. Microbiol. Biotechnol. (2006), 16(6), 979-982] The contents revealed in the experiment were carried out. Each insert introduced into the vector plastids was prepared via PCR using appropriate primers (corresponding to the nucleotide sequences of SEQ ID NOS: 1 to 8).

Furthermore, in order to delete the DLD1 gene, the marker gene HIS3 was introduced via a double crossover to make it defective. The DNA fragment used therein was prepared using primers corresponding to the nucleotide sequences of SEQ ID NOS: 9 and 10.

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

D-lactate dehydrogenase (D-LDH), which is particularly required for D-lactic acid production, is introduced on the basis of a strain having defects in the three genes of ADH1, PDC1 and DLD1. Then, D-LDH was cloned into a vector having XhoI and SpeI restriction enzyme sites at the 5' and 3' ends, respectively, and the 1dhD derived from Lactobacillus plantarum ( Lb. plantarum ) was included in the derivative. Between the TEF1 promoter of S. cerevisiae and the CYC1 terminator. In particular, the embedded system was prepared by double-digestion of SacI/PvuII , and the vector was blunt by digesting the DNA fragment of p-δ-neo into BamHI/NotI using mung bean nuclease. Blunt ended. Finally, the vector processing SacI to obtain a support having a blunt end-derived SacI and BamHI sticky end of.

The resulting vector and the insert are ligated to complete the construction of the pTL573 vector. The plastid pTL573 contains the 1dhD gene derived from Lactobacillus genus, which is designed to be localized between the reverse transposons of S. cerevisiae CEN.PK2-1D pdc1Δadh1Δdld1Δ strain, δ-sequence In the region, it contains random embedding of multiple gene sets. In order to correspond to the multiple insertion of genes, a DNA fragment capable of inducing single exchange on the δ-sequence was constructed by cleavage of the plastid pTL573 by SalI. The DNA fragment was introduced into the mother strain via transformation, and multiple colonies were obtained from a YPD disk (1% yeast extract, 2% bacterial peptone, and 2% glucose) at a maximum concentration of 5 mg/mL G418. Finally, it was confirmed that the thus obtained D-LDH-derived Lactobacillus plantarum strain was subjected to multiple embedding to achieve the purpose of providing D-lactic acid productivity, and was referred to as CC02-0064 strain.

Example 2: Preparation of mutant strains with reduced PDC5 activity

Based on the CC02-0064 strain prepared in Example 1, a mutant having a substituted PDC5 promoter was prepared. In this process, according to the method disclosed by Lee TH et al. (Development of reusable split URA3-marked knockout vectors for budding yeast, Saccharomyces cerevisiae. J Microbiol Biotechnol, 2006, 16: 979-982), the preparation of the cassette and the screening of the strain were carried out. program.

In detail, a total of 5 novel strains were prepared by substituting the PDC5 promoter of CC02-0064 strain with the promoters of SCO1, SCO2, ACS1, IDP2, and FBA1, respectively, and subsequently, corresponding to SEQ ID NOS: 11 to 36 The primer of the nucleotide sequence is prepared to prepare a promoter-substituted cassette.

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

The novel strains thus prepared are referred to as CC02-0167, CC02-0168, CC02-0169, CC02-0170, and CC02-0174. Corresponding strains and their genetic characteristics are summarized in Table 3 below.

Example 3: Evaluation of lactic acid fermentation with mutants with reduced PDC5 activity

The PDC5 promoter mutant prepared in Example 2 was subjected to lactic acid fermentation evaluation. Accordingly, a specific medium was prepared for evaluation by lactic acid fermentation.

In particular, in order to prepare a synthetic complex medium (SC medium), a restrictive medium for yeast is based on a 0.67% yeast nitrogen base without amino acid, according to the manufacturer's experimental procedure. This was mixed with an amino acid dropout mix (Sigma), and an amino acid excluded from the base was added as needed. In addition, 380 mg/L leucine was added to the product, and uridine, tryptophan, and histidine at a concentration of 76 mg/L were separately added; 8% glucose was also added as a carbon source and 1% CaCO _{3 was} used as a medium. And agent. The lactic acid fermentation of the yeast strain was evaluated using the medium thus prepared.

In the PDC5 promoter mutant prepared in Example 2, Mutants that were replaced with a promoter that was weaker than the original PDC5 promoter could not grow, while mutants that were replaced with a stronger promoter showed enhanced growth. In particular, a mutant strain substituted with a promoter such as SCO1, SCO2, IDP2 or ACS1 which is weaker than the PDC5 promoter cannot grow, leaving a strain in which the promoter was substituted with the FBA1 promoter, and was the only strain to be evaluated. The measurable evaluation results of lactic acid fermentation of CC02-0064 and CC02-0174 strains are summarized in Table 4 below.

As shown in the above evaluation, the strain that replaced the wild-type PDC5 promoter with the FBA1 promoter showed enhanced cell growth rate and lactic acid productivity during the promotion of the acetaminophen A production pathway compared to the original strain (CC02-0064). . However, when comparing the results of the samples collected at 24 hours and 48 hours, respectively, it was confirmed that the ALD and ACS activities involving the subsequent acetaminophen-CoA production pathway were not enhanced, and only the single PDC activity was enhanced, and the cell growth rate depending on time was determined. The increase in its lactic acid productivity continues to decrease. In the examples of the present invention, the increase in glucose consumption by the enhanced PDC activity was 10.3%, and the maximum lactic acid production concentration was 47.3 g/l. Therefore, the overall increase in lactic acid productivity was 13.7%.

Example 4: Preparation of a PDC5 gene-deficient strain

In addition to the strain having the PDC1 gene defect and the PDC5-reducing activity prepared in Example 2, a strain having a PDC5 gene defect and a PDC1-reducing activity was prepared, thereby confirming whether or not the PDC pathway was attenuated in the corresponding strain.

In particular, for the purpose of the PDC5 gene defect, a gene defect cassette based on the CC02-0064 strain was prepared using primers corresponding to the nucleotide sequences of SEQ ID NOS: 37 to 40. Defective strains were prepared in the same manner as described in the literature of Example 1. The primers used in Example 4 are summarized in Table 5 below.

The strain thus prepared having the defect of the PDC5 gene is referred to as CC02-0450 (CC02-0064, pdc5?).

Example 5: Preparation of a PDC1 promoter mutant based on a PDC5-deficient strain

A strain having a substituted PDC1 promoter was prepared based on the CC02-0450 strain prepared in Example 4. Accordingly, strain CC02-0451 (CC02-0450, PDC1p-PDC1) which restored the PDC1 gene defect was prepared as a comparison group, and strain CC02-0452 (CC02-0450, IDP1p-PDC1) having reduced PDC1 activity was prepared as an experimental group.

Each strain was prepared as follows. In the yeast, the target gene was cloned into the pRS406 vector without replication initiation, and the constructed PDC1p-PDC1-CYC1t and pRS406-IDP2p-PDC1-CYC1 vectors would contain In this strain.

Specifically, PCR was carried out using the primers having the nucleotide sequences of SEQ ID NOS: 41 and 42 using the chromosomal DNA of the yeast as a template to obtain a product containing the PDC1 gene. Subsequently, the sequences of the CYC1 terminator were obtained using primers having the nucleotide sequences of SEQ ID NOS: 43 and 44. Further, using the primers of the nucleotide sequences corresponding to SEQ ID NOS: 41 and 44, and the PDC1 and CYC1 terminator sequences as templates, respectively, a DNA fragment which ligated the PDC1 and CYC1 terminator was obtained by PCR. A DNA fragment of the PDC1-CYC1 terminator and the pRS406 vector was treated with SpeI and XhoI restriction enzymes, followed by ligation to obtain a plastid vector of pRS406-PDC1-CYC1t. Meanwhile, in order to introduce the promoter functional region into the plastid vector thus obtained, primers having SEQ ID NOS: 45 and 46, and 47 and 48 nucleotide sequences, respectively, using chromosomal DNA as a template, and primers via PCR were used. Fusion, plastid vectors incorporating the PDC1 promoter and the IDP2 promoter, respectively, were obtained. The DNA fragment containing each promoter and pRS406-PDC1-CYC1t plastid was digested and ligated to prepare plastid vectors of pRS406-PDC1p-PDC1-CYC1t and pRS406-IDP2p-PDC1-CYC1t, respectively, which are yeast chromosome embedding. The desired plastid is designed such that gene expression is under the control of the PDC1 promoter and the IDP2 promoter.

The primers used in Example 5 are summarized in Table 6.

The two plasmid vectors thus prepared were digested with StuI, respectively, and immediately embedded in the strain. The final strains are referred to as CC02-0451 (CC02-0450, PDC1p-PDC1) and CC02-0452 (CC02-0450, IDP2p-PDC1). The strain thus prepared and its genetic characteristics are summarized in Table 7.

Example 6: Preparation of a double or triple defective strain in the PDC gene

A strain with a single defect in the PDC1 gene, a double defect in the PDC1 and PDC5 genes, and a triple defect in the PDC1, PDC5, and PDC6 genes was prepared from the PDC family gene. The CC02-0064 strain prepared in Example 1 was used as a strain having a single defect in the PDC1 gene. A cassette for PDC5 deficiency was prepared using primers corresponding to the nucleotide sequences of SEQ ID NOS: 49 to 56, which were inserted into CC02-0064 to prepare a strain having a double defect in the PDC1 and PDC5 genes. Then, the strain thus prepared was referred to as CC02-0256. In addition, using a primer corresponding to the nucleotide sequence of SEQ ID NOS: 57 to 64, based on a double-defective strain in the PDC1 and PDC5 genes, a strain having a triple defect in the PDC1, PDC5, and PDC6 genes was prepared, alleged CC02-0257.

Defect cassette preparation and strain screening procedures were carried out using the same method as described in the literature disclosed in Example 1. The primers used in Example 6 are summarized in Table 8.

The strain thus prepared and its genetic characteristics are summarized in Table 9.

Example 7: Preparation of ALD and ACS1 overexpressing strains

In order to prepare ALD and ACS1 highly expressed strains, ALD2, ALD3, and ACS1 highly expressed plastids were prepared.

In particular, an open reading frame (ORF) of ALD2 was prepared using primers corresponding to the nucleotide sequences of SEQ ID NOS: 65 and 66; ORFs of ALD3 were prepared using primers corresponding to the nucleotide sequences of SEQ ID NOS: 67 and 68 And using the primers corresponding to the nucleotide sequences of SEQ ID NOS: 69 and 70 to prepare the ORF of ACS1. In addition, recombinant vectors p415ADH-ALD2, p415ADH-ALD3, p414ADH-ACS1 and p416ADH-ACS1 based on p414ADH, p415ADH and p416ADH plastids were prepared using Spe I, Xho I or EcoR I restriction enzymes. The primers used in Example 7 are summarized in Table 10 below.

The recombinant plastid thus prepared is combined with p414ADH-ALD2 in combination with p414ADH-ACS1, p415ADH-ALD3 in combination with p414ADH-ACS1, p415ADH-ALD2 in combination with p416ADH-ACS1, or p415ADH-ALD3 in combination with p416ADH-ACS1, via yeast transformation It was introduced into strains including CC02-0064, CC02-0168, CC02-0170, CC02-0256, CC02-0257, CC02-0451, and CC02-0452. However, the CC02-0257 strain which did not exhibit PDC activity and had a triple defect in the PDC gene did not obtain a transgenic strain.

The strain thus prepared and its genetic characteristics are summarized in Table 11.

Example 8: Evaluation of lactic acid fermentation of yeast strain

The lactic acid fermentation capacity evaluation of the ALD and ACS1 highly expressed strains prepared in Example 7 was carried out.

Specifically, the yeast was inoculated into a culture flask containing 25 ml of the medium prepared in the third embodiment for the purpose of lactic acid fermentation evaluation, and cultured under aerobic conditions at 30 ° C for 71 hours. The amount of D-type lactic acid present in the fermentation broth was analyzed and subjected to enzyme analysis (acetic acid, R-Biopharm, Germany) to determine the amount of acetic acid present therein.

The above experimental results are summarized in Table 12 below.

As demonstrated in Table 12, the accumulation of acetic acid by the strain having reduced PDC5 activity via the IDP2 promoter or the SCO2 promoter was significantly reduced, that is, a small amount of acetic acid was confirmed as compared with the strain having normal PDC5 activity. . In this case, the final cell concentration of the strain in which ALD and ACS activity were not increased, there was a tendency to decrease according to the substitution of the PDC5 promoter. Conversely, strains with reduced PDC5 expression and increased ALD and ACS activity showed an increase in final cell concentration. Therefore, it is confirmed that the growth in cell growth. In particular, CC02-0437 and CC02-0278 strains with increased ALD and ACS activity based on the CC02-0170 strain in which the PDC5 promoter was replaced with IDP2 showed growth as ALD and ACS activity increased. Rate, generated D-lactic acid concentration, productivity, and increase in fermentation productivity.

In summary, strains in which PDC5 was replaced by weakly expressed IDP2 showed a decrease in acetic acid accumulation and a final OD 1.3 fold compared to strains exhibiting normal PDC5 expression. Furthermore, it was confirmed that glucose consumption and its rate were increased when ALD and ACS were co-expressed under the control of the ADH1 promoter, and finally, the percentage yield increased from 56% or 59% to 66% or 67%, respectively, indicating an increased yield.

In particular, it was confirmed by comparing the two promoters applied to the weak expression of PDC5 that the lactic acid productivity was improved in the two strains having the SCO2 promoter and the IDP2 promoter, respectively. However, in terms of overall cell concentration, glucose consumption, and its rate, strains having the IDP2 promoter can be considered as strains that are optimized.

Therefore, its growth rate, D-lactic acid production concentration, yield, The CC02-0437 strain, which has been confirmed with the fermentation productivity, was deposited on November 22, 2013 in the International Depository Agency under the Budapest Treaty, the Korea Microbial Center (KCCM) (registration number KCCM 11489P); CC02-0278 strain, in 2015 On June 29th, it was deposited with KCCM (registration number KCCM 11715P) under the Budapest International Treaty.

Example 9: Evaluation of lactic acid fermentation of yeast strains with double defects and increased ALD and ACS activity in PDC1 and PDC5 genes

Since the effect of reduced PDC5 activity leading to cell growth and yield improvement has been clearly demonstrated, an assessment was made to determine the effect of more PDC gene defects in lactic acid production. The evaluation method of each strain was exactly the same as that described in Example 8, and the culture was carried out for 74 hours.

The experimental results thus obtained are summarized in Table 13 below.

As shown in Table 13, the acetic acid concentration of the double-defective strain in the PDC1 and PDC5 genes was significantly reduced, whereas the decrease in D-lactic acid concentration production was also observed due to a decrease in cell growth and glucose consumption. In addition, A double defect in which the PDC1 and PDC5 genes cause the PDC pathway to be almost deactivated, although exhibiting increased ALD and ACS activity, has no increase in cell growth, glucose consumption, and its productivity.

Example 10: Evaluation of lactic acid fermentation of PDC pathway-attenuating strains using sucrose

In order to achieve the purpose of evaluating the fermentation using sucrose, the identical strains evaluated in Examples 8 and 9 were used, in which the PDC-attenuated lactic acid-producing yeast was used to confirm the effect of lactic acid production. Accordingly, sucrose is used instead of glucose as a carbon source. The evaluation method was carried out in the same manner as in Example 8.

The experimental results thus obtained are summarized in Table 14 below.

For the same method as in Examples 8 and 9, the use of sucrose instead of glucose in the strains used allowed the increase in ALD and ACS activity in strains attenuated by the PDC pathway to provide an increase in growth and fermentation yield, as shown in Example 8. The same result pattern for strains using glucose as a carbon source. Therefore, the present invention confirmed that the effect of improving the fermentation yield and growth of PDC activity and increasing ALD and ACS activity confirmed in the present invention is not limited to the kind of sugar used.

Summary The above results demonstrate that when the strain is mutated in a manner that attenuates the PDC pathway and enhances ALD and ACS activity compared to the unmutated strain, lactic acid production can be increased while maintaining its growth rate.

From the above, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit and scope of the invention. Accordingly, the exemplification of the specific examples disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of the invention. Rather, the invention is not intended to be limited to the details of the details of the invention, and the various alternatives, modifications, equivalents, and other specific examples, which may be included in the spirit and scope as defined by the appended claims.

【Biomaterial Storage】

Foreign deposit information [please note according to the country, organization, date, number order]

1. Republic of Korea, Korea Microbiology Conservation Center, November 22, 2013, KCCM11489P

2. Republic of Korea, Korea Microbiology Conservation Center, June 29, 2015, KCCM11715P

<110> CJ First Sugar Co., Ltd.

<120> A microorganism having an increase in lactic acid productivity and a method for producing lactic acid using the same

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

<220>

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<400> 28

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<211> 43

<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<400> 30

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<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<211> 44

<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

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<223> F-ALPDC5-BamHI

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<212> DNA

<213> Artificial sequence

<220>

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<400> 38

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<212> DNA

<213> Artificial sequence

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<400> 39

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<212> DNA

<213> Artificial sequence

<220>

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<400> 40

<210> 41

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> F_PDC1

<400> 41

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<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> R_PDC1

<400> 42

<210> 43

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> F_CYC1t

<400> 43

<210> 44

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> R_CYC1t

<400> 44

<210> 45

<211> 33

<212> DNA

<213> Artificial sequence

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<211> 37

<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

<220>

<223> F_IDP2p

<400> 47

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<211> 37

<212> DNA

<213> Artificial sequence

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<223> R_IDP2p

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<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

<220>

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<211> 44

<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

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<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

<220>

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<400> 76

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<212> PRT

<213> S. cerevisiae DLD1

<400> 78

Claims (7)

An isolated Saccharomyces cerevisiae microorganism having enhanced lactic acid productivity, wherein the microorganism is modified to: a) pyruvate decarboxylase of the microorganism compared to an unmodified lactic acid producing strain ( The activity of PDC) is reduced; and b) the activity of the aldehyde dehydrogenase (ALD) and the acetoin coenzyme A synthetase (ACS) of the microorganism is enhanced compared to the unmodified lactic acid producing strain. The microorganism according to claim 1, wherein the pyruvate decarboxylase is at least one selected from the group consisting of PDC1, PDC5, and PDC6. The microorganism of claim 2, wherein the microorganism is modified to: i) deactivate PDC1 activity and reduce PDC5 activity; or ii) reduce PDC1 activity and deactivate PDC5 activity. The microorganism according to claim 1, wherein the aldehyde dehydrogenase is selected from at least one of the group consisting of ALD2 and ALD3, and the aceto-CoA synthetase system ACS1. The microorganism of claim 1, wherein the ethanol dehydrogenase (ADH) is further deactivated. The microorganism of claim 1, wherein the D-lactate dehydrogenase (DLD) is further deactivated. A method for producing lactic acid, the method comprising: a) cultivating in a medium according to the scope of claims 1 to 6 A microorganism according to any one of the invention; and b) recovering lactic acid from the medium or the microorganism in step a).