JP2008283917A - Method for producing lactic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method by which the amount and yield of lactic acid are improved efficiently in the method for producing the lactic acid, by using a yeast having lactic acid-synthesizing ability. <P>SOLUTION: The method for producing the lactic acid includes adding a hydroxide of calcium, magnesium, copper, vanadium, nickel, barium, strontium, cadmium, beryllium, zinc, cobalt, iron or the like to a fermentation medium, and using the yeast (Saccharomyces), to which a lactate dehydrogenase (L-lactate dehydrogenase) gene derived from a frog (Xenopus laevis) is introduced. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing lactic acid by fermentation using yeast.

  In recent years, production of industrial raw materials using materials derived from biological resources has been actively conducted for the construction of a resource recycling society. Among them, polylactic acid using lactic acid, which is a biological material, as a raw material has attracted attention as a polymer having excellent properties, and there is a problem of how to supply lactic acid as the raw material at low cost.

  Lactic acid production is mainly carried out by lactic acid bacteria represented by the genus Lactobacillus and Lactococcus, but in order to accumulate a large amount of lactic acid in the culture solution, Therefore, it is necessary to carry out culture with addition of a neutralizing agent such as calcium carbonate, ammonia, or sodium hydroxide, which requires a great deal of cost for the neutralization and purification steps.

  On the other hand, a plurality of methods for producing an organic acid using yeast that can grow under acidic conditions have been proposed (see Patent Documents 1 to 3 and Non-Patent Documents 1 and 2). According to these proposals, it is possible to reduce the neutralization operation and construct a low-cost lactic acid production process by introducing a lactic acid synthase gene into yeast that does not originally have lactic acid production ability to produce lactic acid. is there. Furthermore, it is possible to construct a cheaper method for producing an organic acid by improving the production ability of an organic acid such as lactic acid by yeast using a technique such as genetic recombination.

However, in the above-mentioned conventional production method, the substrate concentration is 10% even in the lactic acid production of lactic acid bacteria by alkali neutralization such as calcium carbonate, ammonia, sodium hydroxide and the like, and the lactic acid production under low acid conditions by genetically modified yeast. Therefore, there is a problem that lactic acid can be produced only up to 2 to 7% (see Non-Patent Documents 1 to 4).
JP 2001-204468 A Special table 2001-51658 pamphlet JP 2003-259878 A Applied and Environmental Microbiology, 71, 1964 (2005) Applied and Environmental Microbiology, 71, 2789 (2005), Applied and Environmental Microbiology (Applied and Environmental Microbiology), 71, 2789 (2005) Journal of Bioscience and Bioengineering (Journal of Bioscience and Bioengineering), 101, 172 (2006) Biotechnology Progress (Biotechnology Progress) 11, 294 (1995)

  In order to supply lactic acid at low cost, it is necessary to create microorganisms with high lactic acid production capacity and to examine culture conditions with high lactic acid production efficiency. Then, this invention aims at providing the manufacturing method of lactic acid which improves productivity and a yield at low cost.

  As a result of earnest research to solve the above problems, the present inventor is a method for producing lactic acid by fermentation using yeast, which is produced at low cost by a method for producing lactic acid using a hydroxide of divalent ions. The inventors have found that lactic acid can be produced with improved properties and yield, and have completed the present invention.

  According to the present invention, the amount of lactic acid produced and the production efficiency can be improved. That is, the present invention provides a method for producing lactic acid using yeast having an ability to synthesize lactic acid and an effective neutralization operation for producing lactic acid at a high level. Furthermore, in lactic acid fermentation performed so far, for example, lactic acid can be produced at low cost by using biomass-derived sugar.

  The present invention is a method for producing lactic acid by fermentation using yeast, and is a method for producing lactic acid using a hydroxide of divalent ions. In the lactic acid production method of the present invention, divalent ion hydroxide is usually used for the neutralization operation of the fermentation broth.

  In the method for producing lactic acid of the present invention, preferably, the hydroxide of divalent ions is calcium hydroxide, magnesium hydroxide, copper hydroxide (divalent), vanadium hydroxide, nickel hydroxide, barium hydroxide, water. At least one selected from strontium oxide, cadmium hydroxide, beryllium hydroxide, zinc hydroxide, cobalt hydroxide, and iron hydroxide.

  In the method for producing lactic acid of the present invention, the divalent ion hydroxide is more preferably calcium hydroxide or magnesium hydroxide, and the divalent ion hydroxide is calcium hydroxide. Is particularly preferred.

  In the method for producing lactic acid of the present invention, the concentration of calcium hydroxide is preferably 3.7 g or more and 370 g or less with respect to 1 kg of water, and more preferably 7.4 g or more and 296 g or less with respect to 1 kg of water. More preferably, it is 18.5 g or more and 74 g or less with respect to 1 kg of water. In the method for producing lactic acid according to the present invention, calcium hydroxide can be used in a slurry form.

  In the method for producing lactic acid according to the present invention, the concentration of magnesium hydroxide is preferably 2.9 g or more and 292 g or less with respect to 1 kg of water, and more preferably 5.8 g or more and 233 g or less with respect to 1 kg of water. Particularly preferably, it is 14.6 g or more and 58 g or less with respect to 1 kg of water. In the method for producing lactic acid according to the present invention, magnesium hydroxide can be used in a slurry form.

  In the method for producing lactic acid of the present invention, the divalent ion hydroxide may be contained in the lactic acid production medium, or may be added to the fermentation broth during the culture or continuously. Divalent ion hydroxide is contained in the lactic acid production medium, and can also be divided into fermentation broths during culture or in the form of a combination that is continuously added.

  In the present invention, the hydroxide of divalent ions can be divided during culture or added continuously. The number of additions may be one time or two or more times.

  The fermentation raw material in the lactic acid production medium used in the present invention contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the yeast, and can be a natural medium as long as the yeast can be cultured efficiently. Either a synthetic medium or a synthetic medium may be used.

  The carbon source contained in the fermentation raw material is saccharides such as sucrose, fructose, glucose, galactose, lactose, maltose, starch containing these saccharides, starch hydrolysate, sweet potato molasses, sugar beet molasses, high test molasses, Cane juice, cane juice extract or concentrate, raw sugar refined or crystallized from cane juice, refined sugar crystallized or crystallized from cane juice, organic acids such as acetic acid, alcohols such as ethanol , A raw material of sugar such as glycerin, or one containing at least one selected from these.

  The carbon source may be added all at once at the start of culture, or may be added in portions during culture or continuously.

  In the present invention, the concentration of the carbon source is preferably 10 g or more and 1000 g or less in 1 L culture solution, more preferably 50 g or more and 300 g or less in 1 L culture solution, and particularly preferably 50 g or more and 200 g or less in 1 L culture solution. It is. It is preferable that the concentration of the carbon source is 10 g or more and 1000 g or less in a 1 L culture solution because yeast having lactic acid synthesis ability grows normally and the lactic acid production of the yeast is efficient.

  In the present invention, the lactic acid production medium used in the method for producing lactic acid is more preferably a lactic acid production medium containing at least one selected from sucrose, fructose, glucose, galactose, lactose, and maltose. The lactic acid production medium used in the method for producing lactic acid is starch, starch hydrolyzate, sweet potato molasses, sugar beet molasses, high test molasses, cane juice, cane juice extract or concentrate, purified from cane juice or It is a lactic acid production medium containing at least one selected from crystallized raw sugar, refined sugar from cane juice or crystallized purified sugar, organic acids such as acetic acid, alcohols such as ethanol, and glycerin, preferable. In the present invention, these fermentation raw materials can be diluted as necessary.

  The nitrogen source contained in the fermentation raw material is ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein decomposition products, Other amino acids, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are preferably used.

  Moreover, phosphate, magnesium salt, calcium salt, iron salt, manganese salt etc. can be added suitably as inorganic salt contained in a fermentation raw material.

  An antifoaming agent can be used in the lactic acid production medium used in the present invention, if necessary.

  The lactic acid production medium used in the present invention is preferably added as a specific nutrient for a microorganism to grow or a natural product containing a specific nutrient.

  The present invention is a method for producing lactic acid by fermentation using yeast, and the lactic acid is preferably L-lactic acid.

  Examples of the yeast used in the present invention include yeasts belonging to the genus Saccharomyces, Schizosaccharomyces or Kluyveromyces, more preferably yeasts belonging to the genus Saccharomyces, Saccharomyces cerevisiae. Specifically, yeasts used in the present invention are particularly preferably NBRC10505 strain and NBRC10506 strain.

  In the lactic acid production method of the present invention, the yeast is preferably a yeast into which a lactic acid synthase gene has been introduced. In the present invention, the lactic acid synthase gene (ldh gene) means lactate dehydrogenation having an activity of converting reduced nicotinamide adenine dinucleotide (NADH) and pyruvate into lactic acid and oxidized nicotinamide adenine dinucleotide (NAD +). A gene encoding an enzyme. In the present invention, the lactate synthase gene (ldh gene) is a lactate dehydrogenase (ldh) gene.

  The lactate dehydrogenase gene has various homologues depending on the type of organism or in vivo. The lactate dehydrogenase gene in the present invention may be a lactate dehydrogenase gene artificially synthesized by chemical synthesis or genetic engineering techniques, in addition to a naturally occurring lactate synthase gene.

  In the present invention, the gene encoding lactic acid synthase to be introduced into yeast is preferably DNA encoding lactic acid synthase having strong enzyme activity.

  Examples of naturally occurring lactic acid synthase genes include those derived from prokaryotic organisms such as lactic acid bacteria or eukaryotic microorganisms such as molds, and lactate synthase genes derived from higher eukaryotes including mammals such as cattle and humans. it can.

  Examples of the lactate dehydrogenase gene that is preferably used in the present invention include, for example, frog-derived, Rhacophoridae, Ranaidae, Hyridae, Microhydridae, Bufondae, Examples thereof include lactic dehydrogenase genes belonging to the family of Hyperoliidae, Pelobatinae, Discoglossidae, and Pipidae. Among these, it is preferable to use a lactate dehydrogenase gene belonging to the family Pipidae, and among the frogs belonging to the family Comidae, it is preferably a lactate dehydrogenase gene derived from Xenopus laevis that is easily available. .

  The lactate dehydrogenase gene preferably used in the present invention is preferably a gene encoding frog-derived L-lactate dehydrogenase.

  The lactate dehydrogenase gene preferably used in the present invention is preferably a gene encoding L-lactate dehydrogenase derived from Xenopus laevis.

  The lactate dehydrogenase gene preferably used in the present invention is more preferably a lactate dehydrogenase gene having the nucleic acid sequence shown in SEQ ID NO: 1, more preferably encoding a frog-derived L-lactate dehydrogenase gene. A gene having the nucleic acid sequence shown in SEQ ID NO: 1.

  In the lactic acid production method of the present invention, the lactate dehydrogenase gene is preferably a gene encoding L-lactate dehydrogenase, and the gene encoding L-lactate dehydrogenase encodes L-lactate dehydrogenase. More preferably, it is introduced downstream of a promoter that enables gene expression. Furthermore, in the method for producing lactic acid of the present invention, it is more preferable that the gene encoding L-lactate dehydrogenase is introduced in a state in which it can be expressed downstream of the promoter of pyruvate decarboxylase 1 gene on the chromosome.

  The lactate dehydrogenase gene preferably used in the present invention includes genetic polymorphisms and mutant genes caused by mutagenesis. Here, the genetic polymorphism means that the base sequence of a gene is partially changed due to a natural mutation on the gene. Mutagenesis refers to artificially introducing a mutation into a gene. For example, a method using a site-specific mutagenesis kit (Mutant-K (manufactured by Takara Bio Inc.)) or a random mutagenesis kit. There is a method using (BD Diversity PCR Random Mutagenesis (manufactured by CLONTECH)).

  Examples of DNA encoding lactic acid synthase with enhanced enzyme activity include, for example, DNA encoding lactic acid synthase with strong enzyme activity obtained by a mutation technique using site-directed mutagenesis, and a strong promoter. Examples include DNA linked with DNA encoding lactic acid synthase under control.

  The yeast used in the method for producing lactic acid of the present invention can be preferably produced by introducing a gene encoding a lactic acid synthase (lactate dehydrogenase) using a technique such as genetic recombination. . In addition, when yeast already has a gene encoding a target lactic acid synthase, it is not always necessary to introduce the gene.

  The yeast used in the present invention may be haploid but may be polyploid. It is haploid that one set of chromosomes is a polyploid. For example, if there are two pairs of chromosomes, it is called a diploid.

  In the method for producing lactic acid according to the present invention, a method for introducing a gene encoding lactate dehydrogenase into yeast includes, for example, cloning the lactate dehydrogenase gene and transforming the expression vector incorporating the cloned gene into yeast. Examples include, but are not limited to, a method for conversion and a method for inserting the cloned gene into a target site on a chromosome by homologous recombination.

  There is no restriction | limiting in particular as a method of cloning the lactate dehydrogenase gene preferably used by this invention, A known method can be used. For example, based on known gene information, a method of amplifying and acquiring a necessary genetic region using PCR (Polymerase Chain Reaction) method, a method of cloning from a genomic library or cDNA library using homology or enzyme activity as an index, etc. Is mentioned. A method of chemical synthesis or genetic engineering based on known protein information is also possible.

  As an expression vector into which the cloned lactate dehydrogenase gene is incorporated, an expression vector generally used in yeast can be used. An expression vector widely used in yeast has a sequence necessary for autonomous replication in yeast cells, a sequence necessary for autonomous replication in E. coli cells, a yeast selection marker, and an E. coli selection marker. In addition, in order to express the incorporated lactate dehydrogenase gene, it is desirable to have a so-called regulatory sequence such as an operator, promoter, terminator or enhancer that regulates the expression.

  Here, the sequence necessary for autonomous replication in yeast cells is, for example, a yeast autonomous replication origin (ARS1) and a centromere sequence pair or the replication origin of a yeast 2 μm plasmid. The sequence necessary for autonomous replication is, for example, the ColE1 replication origin of Escherichia coli. Examples of yeast selection markers include auxotrophic complementary genes such as URA3 or TRP1, or drug resistance genes such as G418 resistance gene or neomycin resistance gene. Examples of selection markers for Escherichia coli include ampicillin resistance gene or kanamycin resistance gene. And antibiotic resistance genes such as Examples of regulatory sequences include GAPDH (glyceraldehyde-3-phosphate dehydrogenase) promoter, ADH (alcohol dehydrogenase) promoter, and GAPDH terminator. However, the expression vector is not limited to these.

  By introducing a lactate dehydrogenase gene downstream of the expression vector promoter, a vector capable of expressing the gene can be obtained. By transforming the obtained lactate dehydrogenase gene expression vector into yeast by the method described later, the lactate dehydrogenase gene can be introduced into yeast.

  As a method of inserting the lactate dehydrogenase gene into the target site on the chromosome, preferably downstream of the promoter of the pdc1 gene by homologous recombination, homologous recombination can be performed upstream and downstream of the lactate dehydrogenase gene. Examples include, but are not limited to, a method of performing PCR using a primer designed to add a portion and transforming the obtained PCR fragment into yeast by the method described later. In order to facilitate selection of transformants, the PCR fragment preferably contains a yeast selection marker.

  The method of adjusting the PCR fragment used here can be performed, for example, by the steps 1 to 3 of the following (1) to (3). The outline is shown in FIG.

  (1) Step 1: Using a plasmid having a terminator connected downstream of a lactate dehydrogenase gene as a template and primers 1 and 2 as a set, a fragment containing the lactate dehydrogenase gene and terminator is amplified by PCR. Primer 1 is designed so as to add a homologous sequence of 40 bp or more upstream of the target introduction site, and primer 2 is designed based on a sequence derived from a plasmid downstream from the terminator. Preferably, the sequence homologous to the upstream side of the introduction target site added to the primer 1 is a sequence homologous upstream of the pyruvate decarboxylase 1 gene (pdc1 gene).

  (2) Step 2: A fragment containing a yeast selection marker is amplified by PCR using a plasmid having a yeast selection marker, for example, pRS424, pRS426, etc. as a template and primers 3 and 4 as a set. Primer 3 is designed so that a sequence homologous to the sequence downstream from the terminator of the PCR fragment in Step 1 adds 30 bp or more, and primer 4 has a sequence of 40 bp or more homologous downstream of the target site of introduction. Design to add. Preferably, the sequence homologous to the downstream side of the target site to be added to the primer 4 is a sequence homologous downstream of the pdc1 gene.

  (3) Step 3: PCR is carried out using the mixture of the PCR fragments obtained in Steps 1 and 2 as a template and Primers 1 and 4 as a set, so that both ends are upstream and downstream of the introduction target site. A PCR fragment containing a lactate dehydrogenase gene, a terminator, and a yeast selection marker, to which a homologous sequence has been added, is obtained. Preferably, the PCR fragment is a PCR fragment containing an ldh gene, a terminator, and a marker gene with homologous sequences added upstream and downstream of the pdc1 gene at both ends.

  In order to introduce the lactate dehydrogenase gene expression vector or PCR fragment obtained above into yeast, a method such as transformation, transduction, transfection, cotransfection or electroporation can be used. Specific examples include a method using lithium acetate and a protoplast method.

  As a method for culturing the obtained transformant, for example, a known method described in “MD Rose et al.,“ Methods In Yeast Genetics ”, Cold Spring Harbor Laboratory Press (1990)” is used. Can be used. Yeast into which a lactate dehydrogenase gene expression vector or PCR fragment has been introduced can be selected by culturing in a nutrient-free medium or a drug-added medium according to the yeast selection marker possessed by the expression vector or PCR fragment.

  Lactic acid can be produced in the medium by culturing the yeast introduced with the lactate dehydrogenase gene preferably used in the present invention.

  If the expression vector to be introduced is to be retained in yeast, it is preferable to use a medium to which a selective pressure is applied by a selection marker. Examples of the medium include a synthetic medium in which an amino acid encoded by a selection marker possessed by a vector is removed. A particularly preferable medium is a synthetic medium containing 1 to 10% of glucose as a carbon source and 0.67% of yeast nitrogen base with amino acid (manufactured by DIFCO) as a nitrogen source, to which an appropriate amino acid is added.

  The culture can be performed by shaking culture or stirring culture. Furthermore, as a culture form, batch culture, semi-batch culture, continuous culture and the like can be performed. The oxygen supply conditions are not particularly limited, but can be performed under aerobic conditions or microaerobic conditions. The culture temperature is preferably 20 to 40 degrees Celsius, preferably 25 to 37 degrees, more preferably 30 to 34 degrees. The culture time is usually 24 hours to 5 days. The pH of the culture solution during the culture is desirably maintained at 2.0 to 10.0, preferably 2.5 to 8, and more preferably 5. This pH adjustment can be performed using an alkaline solution, ammonia, calcium carbonate, calcium hydroxide, or the like.

  In the production of lactic acid, first, the yeast of the present invention is pre-cultured, and the pre-culture solution is transferred to a new medium to perform main culture, whereby lactic acid can be produced in the culture solution. The culture temperature is not particularly limited as long as the growth of the strain is not substantially inhibited and lactic acid can be produced, but is preferably a temperature in the range of 20 to 40 degrees Celsius, more preferably 25 degrees Celsius. The temperature is in the range of -37 degrees, more preferably 30-34 degrees Celsius. Any method of standing, stirring or shaking can be employed for the culture.

  By culturing under the above conditions, 1-20% lactic acid can be obtained in the medium. The method for measuring the obtained lactic acid is not particularly limited, and examples include a method using HPLC and a method using F-kit (Roche).

  In the present invention, lactate dehydrogenase activity indicates the activity of converting pyruvate and NADH into lactic acid and NAD +. Although not limited, lactate dehydrogenase activity can be compared using specific activity as an index. That is, yeast having the same ldh gene introduction method and the same genetic background is cultured under the same conditions, and the change in absorbance at 340 nm due to the decrease in NADH is measured using a protein extracted from the cultured cells. At that time, the specific activity of lactate dehydrogenase is expressed by the formula (1) by defining the amount of enzyme that decreases 1 μmol of NADH per minute at room temperature as 1 unit (Unit). Here, Δ340 is the decrease in absorbance at 340 nm per minute, and 6.22 is the mmol molecular extinction coefficient of NADH.

  Lactic acid in the obtained culture broth can be purified by a conventionally known method. For example, a method in which a fermentation broth obtained by centrifuging microorganisms is adjusted to pH 1 or less and then extracted with diethyl ether, ethyl acetate or the like, a method in which it is adsorbed and washed on an ion exchange resin, and then eluted, or reacted with alcohol in the presence of an acid catalyst. Then, there are a method of distilling as an ester and a method of crystallization as a calcium salt or a lithium salt.

  Hereinafter, the present invention will be described based on examples.

  Hereinafter, the frog lactate dehydrogenase gene is selected as the lactate dehydrogenase gene, and Saccharomyces cerevisiae is selected as the yeast to show specific embodiments, and the present invention will be described in more detail.

Reference Example 1 (Creation of yeast capable of synthesizing lactic acid)
As the lactate dehydrogenase (ldh) gene, a lactate dehydrogenase gene derived from Xenopus laevis having the nucleic acid sequence shown in SEQ ID NO: 1 was used. The lactate dehydrogenase gene was cloned by the PCR method. For PCR, phagemid DNA prepared from a Xenopus laevis kidney-derived cDNA library (manufactured by STRATAGENE) according to the attached protocol was used as a template.

  For the PCR amplification reaction, KOD-Plus polymerase (manufactured by Toyobo Co., Ltd.) was used, and the attached reaction buffer, dNTPmix, etc. were used. A 50 μl reaction system was prepared so that the phagemid DNA prepared as described above was 50 ng / sample, the primer was 50 pmol / sample, and the KOD-Plus polymerase was 1 unit / sample. The reaction solution was thermally denatured at 94 ° C. for 5 minutes with a PCR amplification apparatus iCycler (manufactured by BIO-RAD), then 94 ° C. (thermal denature): 30 seconds, 55 ° C. (primer annealing): 30 seconds, 68 C. (Complementary strand extension): One cycle was performed for 30 cycles, and then cooled to a temperature of 4.degree. The gene amplification primer (SEQ ID NO: 2 and 3) was prepared such that a SalI recognition sequence was added to the 5 terminal side and a NotI recognition sequence was added to the 3 terminal side.

  The PCR amplified fragment was purified, and the end was phosphorylated with T4 polynucleotide Kinase (manufactured by Takara Bio Inc.), and then ligated to the pUC118 vector (which was cleaved with the restriction enzyme HincII and subjected to dephosphorylation treatment). Ligation was performed using DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). The ligation solution was transformed into competent cells of Escherichia coli DH5α (manufactured by Takara Bio Inc.) and plated on an LB plate containing 50 μg / mL of the antibiotic ampicillin and cultured overnight. For the grown colonies, plasmid DNA was collected with a miniprep, cut with restriction enzymes SalI and NotI, and a plasmid into which the ldh gene was inserted was selected. All of these series of operations were performed according to the attached protocol.

  The pUC118 vector in which the lactate dehydrogenase gene was inserted was cleaved with restriction enzymes SalI and NotI, the DNA fragments were separated by 1% agarose gel electrophoresis, and the fragment containing the lactate dehydrogenase gene was purified according to a conventional method. The obtained fragment containing the lactate dehydrogenase gene is ligated to the XhoI / NotI cleavage site of the expression vector pTRS11 shown in FIG. 2, the plasmid DNA is recovered by the same method as described above, and cleaved with the restriction enzymes XhoI and NotI. Thus, an expression vector into which the lactate dehydrogenase gene was inserted was selected. Hereinafter, an expression vector incorporating the lactate dehydrogenase gene prepared in this manner was designated as pTRS102.

  A 1.3 kb DNA fragment containing a lactate dehydrogenase gene and a GAPDH terminator sequence was amplified by PCR using the obtained expression vector pTS102 as an amplification template and oligonucleotides (SEQ ID NOs: 4 and 5) as primer sets (FIG. 1 corresponding to step 1). Here, SEQ ID NO: 4 was designed to add a sequence having homology to 65 bp upstream of the pdc1 gene.

  Next, a 1.2 kb DNA fragment containing the TRP1 gene as a yeast selection marker was obtained by PCR using plasmid pRS424 having a plasmid yeast selection marker as an amplification template and oligonucleotides (SEQ ID NOs: 6 and 7) as primer sets. Amplified. Here, SEQ ID NO: 7 was designed so that a sequence having homology was added to 65 bp downstream of the pdc1 gene.

  Each DNA fragment was separated by 1.5% agarose gel electrophoresis and purified according to a conventional method. A mixture of each 1.3 kb fragment and 1.2 kb fragment obtained here was used as an amplification template, and a PCR method using oligonucleotides (SEQ ID NOs: 4 and 7) as a primer set was used for the lactate dehydrogenase gene, GAPDH. A DNA fragment of about 2.5 kb to which the terminator and TRP1 gene were linked was amplified.

  The above DNA fragment is separated by 1.5% agarose gel electrophoresis, purified according to a conventional method, transformed into yeast Saccharomyces cerevisiae NBRC10505, and cultured in a medium without tryptophan, whereby the ldh gene is transformed into pdc1 on the chromosome. A transformant introduced downstream of the gene promoter was selected.

  Confirmation that the transformant obtained as described above was a yeast in which the lactate dehydrogenase gene was introduced downstream of the pdc1 gene promoter on the chromosome was performed as follows. First, the genomic DNA of the transformant was prepared using a genomic DNA extraction kit Gen Toru-kun (manufactured by Takara Bio Inc.), and this was used as an amplification template, and by PCR using oligonucleotides (SEQ ID NOs: 7 and 8) as primer sets, This was confirmed by obtaining an amplified DNA fragment of 2.8 kb. For non-transformed strains, an amplified DNA fragment of about 2.1 kb can be obtained by the PCR. Hereinafter, a transformant in which the lactate dehydrogenase gene is introduced downstream of the pdc1 gene promoter on the chromosome is referred to as B2 strain.

Example 1
Using the B2 strain prepared in Reference Example 1, a lactic acid fermentation test was performed.

  10 mL of a medium (hereinafter abbreviated as “natural medium”) containing cane juice shown in Table 1 was placed in a test tube, a small amount of B2 strain was inoculated therein, and cultured overnight at 30 ° C. (pre-culture). Next, 100 mL of a natural medium was put into a 500-mL Erlenmeyer flask, and the whole culture solution was inoculated in advance, and cultured with shaking at 30 ° C. for 24 hours (pre-culture). Subsequently, a pre-culture solution 24 hours after the start of pre-culture was inoculated in a mini jar fermenter (manufactured by Maruhishi Bio-Engineering Co., Ltd., volume 5 L) charged with 1 L of a natural medium, and 1N aqueous calcium hydroxide solution was used. The culture was performed while neutralizing. The culture was carried out at a constant stirring speed (120 rpm), aeration volume (0.1 L / min), temperature (30 ° C.), and pH (pH 5) (main culture). The culture solution was centrifuged every 10 hours after the start of the main culture, the obtained supernatant was subjected to membrane filtration, and the amount of L-lactic acid was measured by HPLC under the conditions shown below.

Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
Detection method: electrical conductivity Temperature: 45 ° C.

  The production amount of L-lactic acid calculated from the measurement results is shown in FIG. In lactic acid fermentation in a natural medium with 1N aqueous calcium hydroxide, the production yield of lactic acid was improved to 83.5%.

Example 2
A lactic acid fermentation test was conducted in the same manner as in Example 1 except that a 1N magnesium hydroxide aqueous solution was used instead of the 1N calcium hydroxide aqueous solution.

  In lactic acid fermentation in a natural medium with 1N magnesium hydroxide aqueous solution, the production yield of lactic acid was 74.9%.

Comparative Example 1
Using the B2 strain produced in Reference Example 1, a lactic acid fermentation test using sodium hydroxide was performed.

  10 mL of natural medium was taken in a test tube, a small amount of B2 strain was inoculated therein, and cultured overnight at 30 ° C. (pre-culture). Next, 100 mL of a natural medium was placed in a 500-mL Erlenmeyer flask, and the whole culture solution was inoculated in advance and cultured with shaking at 30 ° C. for 24 hours (pre-culture). Subsequently, a pre-culture solution 24 hours after the start of the pre-culture was inoculated in a mini jar fermenter (manufactured by Maruhishi Bio-Engineering Co., Ltd., volume 5 L) into which 1 L of natural medium had been added, and the medium was added with a 1N sodium hydroxide aqueous solution. The culture was performed while performing a Japanese procedure. Culturing was carried out at a constant stirring speed (120 rpm), aeration volume (0.1 L / min), temperature (30 ° C.), and pH (pH 5) (main culture). The culture broth was centrifuged every 10 hours after the start of the main culture, the obtained supernatant was subjected to membrane filtration, and the amount of L-lactic acid was measured by HPLC in the same manner as in Example 1.

  The production amount of L-lactic acid calculated from the measurement results is shown in FIG.

Comparative Example 2
A lactic acid fermentation test was conducted in the same manner as in Example 1 except that a 1N potassium hydroxide aqueous solution was used instead of the 1N sodium hydroxide aqueous solution. The production amount of L-lactic acid calculated from the measurement results is shown in FIG.

  As a result, lactic acid fermentation in a natural medium with 1N sodium hydroxide aqueous solution (Comparative Example 1) yielded 65.0% lactic acid, and lactic acid fermentation in a natural medium with 1N potassium hydroxide aqueous solution (comparison) In Example 2), the production yield of lactic acid is 62.1%, whereas in lactic acid fermentation in a natural medium with 1N aqueous magnesium hydroxide, the production yield of lactic acid is 74.9% (Example 2). ) In lactic acid fermentation in a natural medium with 1N calcium hydroxide aqueous solution, the production yield of lactic acid was improved to 83.5% (Example 1). Neutralization of divalent ions with hydroxide in natural medium was effective in the production yield of lactic acid.

Example 3
Using the B2 strain produced in Reference Example 1, a lactic acid fermentation test using a synthetic medium was performed.

  A medium (hereinafter abbreviated as “synthetic medium”) having a composition shown in Table 3 was placed in a test tube, a small amount of B2 strain was inoculated therein, and cultured overnight at 30 ° C. (pre-culture). Next, 100 mL of the synthetic medium was placed in a 500-mL Erlenmeyer flask, and the whole culture solution was inoculated in advance and cultured with shaking at 30 ° C. for 24 hours (pre-culture). Subsequently, a pre-culture solution 24 hours after the start of pre-culture was inoculated in a mini jar fermenter (manufactured by Maruhishi Bio-Engineering Co., Ltd., volume 5 L) into which 1 L of synthetic medium had been added, and the stirring speed (120 rpm) and aeration were performed. The culture was carried out with the amount (0.1 L / min), temperature (30 ° C.), and pH (pH 5) being constant (main culture). The cells were cultured while neutralizing with a 1N calcium hydroxide aqueous solution. The culture broth was centrifuged every 10 hours after the start of the main culture, the obtained supernatant was subjected to membrane filtration, and the amount of L-lactic acid was measured by HPLC in the same manner as in Example 1.

  The production amount of L-lactic acid calculated from the measurement results is shown in FIG. In lactic acid fermentation in a synthetic medium using 1N calcium hydroxide aqueous solution, the production yield of lactic acid was improved to 69.1%.

Comparative Example 3
Using the B2 strain produced in Reference Example 1, a lactic acid fermentation test using a synthetic medium was performed.

  10 mL of a synthetic medium was taken in a test tube, a small amount of B2 strain was inoculated therein, and cultured overnight at 30 ° C. (pre-culture). Next, 100 mL of the synthetic medium was placed in a 500-mL Erlenmeyer flask, and the whole culture solution was inoculated in advance and cultured with shaking at 30 ° C. for 24 hours (pre-culture). Subsequently, the entire preculture solution 24 hours after the start of preculture was inoculated into a mini-jar fermenter (manufactured by Maruhishi Bioengineer, 5 L) charged with 1 L of a synthetic medium, and neutralized with 1N sodium hydroxide. The culture was performed while operating. Culturing was carried out at a constant stirring speed (120 rpm), aeration volume (0.1 L / min), temperature (30 ° C.), and pH (pH 5) (main culture). The culture broth was centrifuged every 10 hours after the start of the main culture, the obtained supernatant was subjected to membrane filtration, and the amount of L-lactic acid was measured by HPLC in the same manner as in Example 1.

  The production amount of L-lactic acid calculated from the measurement results is shown in FIG.

Comparative Example 4
A lactic acid fermentation test was conducted in the same manner as in Example 1 except that a 1N potassium hydroxide aqueous solution was used instead of the 1N sodium hydroxide aqueous solution. The production amount of L-lactic acid calculated from the measurement results is shown in FIG.

  As a result, lactic acid fermentation in a synthetic medium using a 1N aqueous sodium hydroxide solution (Comparative Example 3) yielded a lactic acid production yield of 60.0%, and in a synthetic medium using a 1N aqueous potassium hydroxide solution. In lactic acid fermentation (Comparative Example 4), the production yield of lactic acid is 58.8%, whereas in lactic acid fermentation in a synthetic medium using 1N calcium hydroxide aqueous solution, the production yield of lactic acid is 69.1%. (Example 3).

FIG. 1 is a schematic diagram for explaining a method of introducing a lactate dehydrogenase gene (ldh gene) downstream of the pdc1 gene promoter. FIG. 2 is a diagram showing a physical map of the yeast expression vector pTRS11 used in the present invention. FIG. 3 is a graph of an increase in lactic acid production in the natural medium. FIG. 4 is a graph of an increase in lactic acid production in the synthetic medium.

Claims (11)

A method for producing lactic acid by fermentation using yeast, which uses a divalent hydroxide. Divalent ion hydroxide is calcium hydroxide, magnesium hydroxide, copper hydroxide (divalent), vanadium hydroxide, nickel hydroxide, barium hydroxide, strontium hydroxide, cadmium hydroxide, beryllium hydroxide, water The method for producing lactic acid according to claim 1, wherein the method is at least one selected from zinc oxide, cobalt hydroxide, and iron hydroxide. The production method according to claim 1 or 2, wherein the lactic acid is L-lactic acid. The method for producing lactic acid according to any one of claims 1 to 3, wherein the yeast is a yeast into which a lactic acid synthase gene has been introduced. The method for producing lactic acid according to claim 4, wherein the lactic acid synthase gene is a gene in which the gene encoding the frog-derived L-lactate dehydrogenase has the nucleic acid sequence shown in SEQ ID NO: 1. The method for producing lactic acid according to claim 4, wherein the lactic acid synthase gene is a gene encoding an L-lactic acid dehydrogenase derived from Xenopus laevis. The method for producing lactic acid according to claim 6, wherein a gene encoding L-lactate dehydrogenase is introduced downstream of a promoter that enables expression of the gene encoding L-lactate dehydrogenase. The method for producing lactic acid according to claim 6, wherein the gene encoding L-lactate dehydrogenase is introduced in a state that can be expressed downstream of the promoter of pyruvate decarboxylase 1 gene on the chromosome. The method for producing lactic acid according to any one of claims 1 to 8, wherein the yeast belongs to the genus Saccharomyces. The method for producing lactic acid according to claim 1, wherein the lactic acid production medium used in the method for producing lactic acid is a lactic acid production medium containing at least one selected from sucrose, fructose, glucose, galactose, lactose, and maltose. The lactic acid production medium used in the method for producing lactic acid is starch, starch hydrolyzate, sweet potato molasses, sugar beet molasses, high test molasses, cane juice, cane juice extract or concentrate, purification or crystallization from cane juice 11. A lactic acid production medium comprising at least one selected from raw material sugars, purified sugars purified from cane juice or crystallized sugars, organic acids such as acetic acid, alcohols such as ethanol, and glycerin. The manufacturing method of lactic acid as described.

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