JP2008301766A - Culture medium for producing lactic acid, and method for producing lactic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently improve the yield of lactic acid in a method for producing the lactic acid, using microorganisms having the ability of synthesizing lactic acid. <P>SOLUTION: The method for producing the lactic acid comprises culturing the microorganism having the ability to synthesize the lactic acid by using a culture medium comprising folic acid. The culture medium for producing the lactic acid comprises the folic acid. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a medium for producing lactic acid by fermentation for culturing microorganisms and a method for producing lactic acid by fermentation for culturing microorganisms.

  While there are concerns about the worsening of environmental problems, there is a need to build a sustainable resource recycling society, and in the materials field, production of industrial raw materials called green plastics using materials derived from biological resources is actively performed. Yes. Among them, polylactic acid using lactic acid as a raw material has attracted attention as a polymer having excellent properties, and is expected as a next-generation material.

  Lactic acid as a raw material has been produced by a fermentation method mainly using sucrose, glucose, starch or the like as a carbon source. However, at the present stage, many problems remain in the production amount and production yield, and the lactic acid as a polymer raw material cannot be stably supplied to the market.

  In fermentation production of lactic acid using yeast, a YPAD medium (Non-patent Document 1) generally known as a medium for culturing yeast, a yeast extract and peptone contained in YPAD are added in excess. It is well known that the amount of lactic acid produced and the production yield are improved by culturing. However, in the production of lactic acid in such a medium, the cost of the medium has been a problem.

  Therefore, studies are being made on effective medium components for lactic acid production using an inexpensive medium. So far, nicotinic acid (Non-Patent Document 2) and fatty acids (see Patent Document 1 and Non-Patent Documents 3 and 4) have been proposed as medium components for improving lactic acid productivity. These are that nicotinic acid is a precursor of NADH, a coenzyme required by lactate dehydrogenase (ldh), and that fatty acids have a reduced content of unsaturated fatty acids in yeast during lactic acid production. Is known, and the addition of these improves the lactic acid production yield per carbon source in the medium.

  However, even if these methods are used, the production yield of lactic acid required industrially has not been reached, and the medium price remains high. Moreover, many problems remain in practical use, such as the complexity of the production process after lactic acid production, by using yeast extract or peptone.

Therefore, a method for improving the production yield of lactic acid more effectively is desired.
JP 2000-37185 A "Methods in East Genetics", Cold Spring Harbor Laboratory Volume 194, 1991, pp. 12-18. Applied and Environmental Microbiology, 71, 2789 (2005) Journal of Bioscience and Bioengineering, 99, 512 (2005) Applied and Environmental Microbiology, 69, 1499 (2003)

  An object of the present invention is to produce lactic acid at low cost and high lactic acid production efficiency (for sugar yield) by fermentation for culturing microorganisms.

  As a result of intensive studies by the present inventors in order to solve the above problems, lactic acid can be produced at a high sugar yield by adding folic acid to the medium in lactic acid fermentation using yeast having lactic acid synthesis ability. As a result, the present invention has been completed.

  That is, the present invention is a medium for producing lactic acid by fermentation for culturing a microorganism having lactic acid synthesis ability, and is a medium for producing lactic acid containing folic acid or a folic acid precursor in an amount of 10 μg / L to 70 μg / L.

  The preferable aspect of the culture medium for lactic acid production of this invention contains sugarcane pressing juice.

  In a preferred embodiment of the lactic acid production medium of the present invention, the microorganism having the ability to synthesize lactic acid is yeast.

  Moreover, this invention is a manufacturing method of lactic acid using the culture medium for lactic acid manufacture of the said invention.

  Furthermore, the present invention is a method for producing lactic acid by fermentation of culturing a microorganism having lactic acid synthesis ability, and is a method for producing lactic acid using a medium containing folic acid or a folic acid precursor in an amount of 10 μg / L to 70 μg / L. .

  In a preferred embodiment of the method for producing lactic acid according to the present invention, the microorganism is yeast. In a more preferred embodiment, the yeast belongs to the genus Saccharomyces.

  In a preferred embodiment of the method for producing lactic acid according to the present invention, the yeast is a yeast into which a lactate dehydrogenase gene has been introduced. In a preferred embodiment of the method for producing lactic acid of the present invention, the lactate dehydrogenase gene is a gene encoding L-lactate dehydrogenase derived from Xenopus laevis. Moreover, the preferable aspect of the manufacturing method of lactic acid of this invention is a gene in which the gene which codes this L-lactic acid dehydrogenase has the base sequence shown to sequence number 1.

  In a preferred embodiment of the method for producing lactic acid according to the present invention, the gene encoding L-lactate dehydrogenase is introduced downstream of a promoter that enables expression of the gene encoding L-lactate dehydrogenase. . In a preferred embodiment of the method for producing lactic acid according to the present invention, a gene encoding L-lactate dehydrogenase is introduced in a state that can be expressed downstream of a promoter of pyruvate decarboxylase 1 gene on the chromosome. .

  Moreover, the preferable aspect of the manufacturing method of lactic acid of this invention culture | cultivates microorganisms in a 25-37 degreeC culture medium.

  According to the present invention, lactic acid can be produced at low cost and high lactic acid production efficiency by fermentation for culturing microorganisms.

  The present invention is a medium for producing lactic acid by fermentation of culturing microorganisms having lactic acid synthesis ability, and is a medium for producing lactic acid containing folic acid or a folic acid precursor in an amount of 10 μg / L to 70 μg / L.

  The concentration of folic acid or folic acid precursor contained in the lactic acid production medium of the present invention is 10 μg / L or more and 70 μg / L or less. Preferably, the medium contains 20 μg / L to 60 μg / L of folic acid or folic acid precursor, more preferably 30 μg / L to 50 μg / L.

  Folic acid contained in the lactic acid production medium of the present invention is also called vitamin M, vitamin B9, and pteroylglutamic acid, and is a physiologically active substance classified as a water-soluble vitamin. Folic acid undergoes various reductions in microorganisms and is converted into tetrahydrofolic acid via dihydrofolic acid, and then acts mainly as a coenzyme involved in C1 metabolism.

  In the lactic acid production medium of the present invention, folic acid may be included as a natural product containing folic acid. Examples of natural products containing folic acid include green and yellow vegetables, sugar cane, sugar beet, cassava, corn, and the like, among which sugar cane is preferably used.

  Folic acid may also be contained in the medium in the form of a precursor. The term “folic acid precursor” as used herein refers to a substance converted into folic acid in a microorganism or in a culture solution during culture. Examples of the precursor of folic acid include paraaminobenzoic acid and tetrahydrofolic acid. Among these, paraaminobenzoic acid can be preferably used.

  The folic acid may be added to the medium before or after the culture, and the number of additions may be once or twice or more.

  The medium for lactic acid production of the present invention may contain a carbon source, a nitrogen source and / or inorganic salts that can be assimilated by microorganisms as fermentation raw materials. The lactic acid production medium of the present invention is a natural medium that uses natural products such as animal and plant broth, soil and humic extracts in an almost intact composition, a synthetic medium prepared from commercially available chemicals, Any of a semi-synthetic medium obtained by adding a natural product to a synthetic medium may be used.

  Examples of the carbon source include sugars such as glucose, sucrose, fructose, galactose, and lactose, starch containing these sugars, starch hydrolyzate, sugar cane and corn press juice, sugar cane molasses, sugar beet molasses, high test molasses, Moreover, organic acids, such as acetic acid, alcohols, such as ethanol, glycerol, etc. can be used.

  Of these carbon sources, sugar cane juice is preferred. It is particularly preferable to use a permeate obtained by centrifuging the sugar cane juice at, for example, 8000 rpm and then filtering the supernatant with, for example, Vivaflow 50 (manufactured by Satorius AG Biotechnology). . Sugarcane can be sterilized by filtering or autoclaving, and the number of autoclaves may be one or more. In addition, folic acid may be contained in sugarcane, and it can be used as a natural product containing folic acid. Sugar cane press juice can be used as a preferred carbon source, particularly when yeast is used as a microorganism.

  Examples of the nitrogen source include ammonia gas, aqueous ammonia, ammonium salts, sulfates such as urea and ammonium sulfate, nitrates, and other organic nitrogen sources used as auxiliary substances such as oil cakes, soybean hydrolysate, casein Decomposed 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 used.

  Moreover, as said inorganic salt, a phosphate, magnesium salt, calcium salt, iron salt, manganese salt etc. can be added suitably.

  In the present invention, when the microorganism to be used is auxotrophic, that is, when a specific nutrient is required for the growth of the microorganism, the medium for producing lactic acid of the present invention is the nutrient or a substance containing the nutrient. Or it is preferable to contain the substance converted into this nutrient.

  Specific examples of nutrients required when the microorganism is auxotrophic include methionine, tyrosine, isoleucine, phenylalanine, glutamic acid, threonine, aspartic acid, valine, serine, arginine, uracil, adenine, lysine, tryptophan, leucine, Histidine and the like are known. For example, when the microorganism is yeast, the nutrients required by the yeast are histidine, leucine, lysine, tryptophan, uracil, and adenine.

  The genotypes of microorganisms exhibiting auxotrophy include methionine requirement: met1, met2, met3, met4, met5, met6, met7, met8, met10, met13, met14, met20, tyrosine requirement: tyr1, isoleucine, valine. Requirements: ilv1, ilv2, ilv3, ilv5, phenylalanine requirement: pha2, glutamate requirement: GLU3, threonine requirement thr1, thr4, aspartate requirement: asp1, asp5, serine requirement: ser1, ser2, arginine requirement : Arg1, arg3, arg4, arg5, arg8, arg9, arg80, arg81, arg82, arg84, uracil requirements: ura1, ura2, ura3, ura4, ura6, adenine required : Ade1, ade2, ade3, ade4, ade5, ade6, ade8, ade9, ade12, ADE15, lysine requirements: lys1, lys2, lys4, lys5, lys7, lys9, lys11, lys13, lys14, trp 1 Trp2, trp3, trp4, trp5, leucine requirement: leu1, leu2, leu3, leu4, leu5, histidine requirement: his1, his2, his3, his4, his5, his6, his7, his8, etc. are mainly used.

  Each component such as the carbon source, nitrogen source, inorganic salts, and nutrients contained in the lactic acid production medium of the present invention may be added all at once at the start of the culture, or divided or continuously added during the culture. You can also An antifoaming agent is also used as necessary.

  Preferred specific examples of the lactic acid production medium of the present invention include a medium having the composition shown in Table 1 (hereinafter sometimes abbreviated as MMF3) and a medium having the composition shown in Table 2 (hereinafter abbreviated as CMF4 medium). ) Etc.

  MMF3 medium can be prepared as follows. For example, among the nutrients shown in Table 1, vitamins and metal salts are sterilized by filtering, and the others are divided into sugar, amino acid, nucleic acid and inorganic salt groups, and each group is sterilized by autoclaving. And after cooling to room temperature or lower, the concentration shown in Table 1 is prepared. Folic acid can be sterilized by filtration and added in the required amount.

  CMF4 medium can be prepared as follows. For example, first, sugarcane press juice is centrifuged at 8000 rpm, and the supernatant is filtered using, for example, Vivaflow 50 (Satorius AG Biotechnology), and the sugar cane press filtrate is used. Prepare in advance. Next, the amino acids, nucleic acids and ammonium sulfate shown in Table 2 are each sterilized by autoclaving, cooled to room temperature or lower, and then combined with the sugar cane juice filtrate to prepare the concentrations shown in Table 2. Folic acid can be sterilized by filtration and added in the required amount.

  Examples of microorganisms used in the lactic acid production medium of the present invention include yeasts such as baker's yeast often used in the fermentation industry, bacteria such as E. coli, coryneform bacteria and lactic acid bacteria, filamentous fungi, actinomycetes, animal cells, and Examples include insect cells. The microorganism to be used may be isolated from the natural environment, or may be partially modified by mutation or genetic recombination.

  Of these microorganisms, yeast is preferred. Examples of the yeast include yeasts belonging to the genus Saccharomyces, and among these, yeasts belonging to (Genus Saccharomyces) and yeasts belonging to Saccharomyces cerevisiae.

  Examples of the microorganism having lactic acid synthesis ability used in the lactic acid production medium of the present invention include microorganisms having lactate dehydrogenase (lactic acid synthase), microorganisms into which lactate dehydrogenase has been introduced, and the like. For example, yeast, Escherichia coli, bacteria such as coryneform bacteria and lactic acid bacteria, filamentous fungi, actinomycetes, animal cells and insect cells into which a lactate dehydrogenase gene has been introduced may be mentioned. Lactic acid bacteria can be used as they are as microorganisms capable of synthesizing lactic acid without introducing a lactate dehydrogenase gene.

  In the present invention, a lactate dehydrogenase gene (ldh gene) is a lactic acid having an activity to convert reduced nicotinamide adenine dinucleotide (NADH) and pyruvate into lactic acid and oxidized nicotinamide adenine dinucleotide (NAD +). This gene encodes dehydrogenase.

  Lactate dehydrogenase has various homologues depending on the type of microorganism or in the microorganism. The lactate dehydrogenase possessed by the microorganism used in the present invention or introduced into the microorganism includes naturally occurring lactate dehydrogenase, as well as lactic acid artificially synthesized by chemical synthesis or genetic engineering techniques. Also included are dehydrogenases. First, naturally occurring lactate dehydrogenases include prokaryotic organisms such as lactic acid bacteria or those derived from eukaryotic microorganisms such as fungi, and lactate dehydrogenase genes derived from higher eukaryotes including mammals such as cattle, frogs and humans. Mention may be made of enzymes consisting of the encoded protein. Among these, the genes encoding frog-derived lactate dehydrogenase include Rhocophoridae, Ranaidae, Hyridae, Microhyridae, Bufondae, Examples include ldh genes belonging to the family Hyperideidae, Pelobatinae, Discoglossidae, and Pipidae, among which the ldh gene belonging to Pipidae is preferably used. Among the frogs belonging to the frog family, it is more preferable to use a gene encoding lactate dehydrogenase derived from Xenopus laevis, which is easily available, particularly a gene encoding L-lactate dehydrogenase. Specifically, a particularly preferred ldh gene is the ldh gene having the base sequence shown in SEQ ID NO: 1.

  In addition, the ldh gene introduced into the microorganism in the present invention includes genetic polymorphisms and mutated 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 (Mutan-K (manufactured by Takara Bio Inc.)) or a random mutagenesis kit. (BD Diversify PCR Random Mutagenesis (manufactured by CLONTECH)).

  Furthermore, it is preferable to use a microorganism into which DNA encoding lactate dehydrogenase with enhanced enzyme activity has been introduced. Examples of DNA encoding lactate dehydrogenase with enhanced enzyme activity include, for example, DNA encoding lactate dehydrogenase with strong enzyme activity obtained by a mutation technique using site-directed mutagenesis, and a strong promoter And the like can be exemplified by DNA linked with DNA encoding lactate dehydrogenase under the control of

  The method for producing lactic acid according to the present invention is a method for producing lactic acid by fermentation of cultivating a microorganism having a lactic acid synthesis ability, and is a production method using a medium containing 10 μg / L to 70 μg / L of folic acid or a folic acid precursor. is there.

  Moreover, the manufacturing method of the lactic acid of this invention is a manufacturing method using the culture medium for lactic acid manufacture of this invention which contains the above-mentioned folic acid or a folic acid precursor 10 microgram / L or more and 70 microgram / L or less.

  The folic acid or folic acid precursor used in the lactic acid production method of the present invention is the same as that used in the lactic acid production medium of the present invention. Folic acid may be folic acid itself, a natural product containing folic acid, or a precursor of folic acid.

  The method for producing lactic acid according to the present invention uses a medium containing folic acid or a folic acid precursor at a concentration of 10 μg / L to 70 μg / L. Similarly to the lactic acid production medium of the present invention, the concentration of folic acid or folic acid precursor is preferably 20 μg / L to 60 μg / L, more preferably 30 μg / L to 50 μg / L.

  The microorganism used in the method for producing lactic acid of the present invention is a microorganism having a lactic acid synthesis ability similar to that used in the lactic acid production medium of the present invention, and is a lactic acid dehydrogenase (lactic acid synthase). ), Microorganisms into which lactate dehydrogenase has been introduced, and the like. Specific examples of the microorganism include bacteria such as yeast, Escherichia coli, and coryneform bacteria, filamentous fungi, actinomycetes, animal cells, and insect cells. Among these, yeast is preferable.

  When yeast is used as the microorganism used in the method for producing lactic acid of the present invention, a yeast into which a gene encoding lactate dehydrogenase (lactic acid synthase) has been introduced using a technique such as genetic recombination should be used. However, when yeast already has a gene encoding the desired lactate dehydrogenase, it is not always necessary to introduce the gene.

  The yeast used in the method for producing lactic acid of the present invention is preferably a yeast into which a lactate dehydrogenase gene can be introduced, and yeast belonging to the genus Saccharomyces, Schizosaccharomyces, or Kluyveromyces is used. Can be mentioned. Saccharomyces cerevisiae is preferable, and specifically, NBRC10505 strain and NBRC10506 strain are preferable.

  As a gene encoding lactate dehydrogenase to be introduced into yeast, as in the lactic acid production medium of the present invention, a prokaryote such as lactic acid bacteria or a eukaryotic microorganism such as mold, bovine, Examples thereof include lactate dehydrogenase genes derived from higher eukaryotes including mammals such as frogs and humans. Among these, the frog-derived lactate dehydrogenase genes include Rhocophoridae, Ranadae, Hyridae, Microhydridae, Bufondae, and Hydraid H ), Ldh genes belonging to the family Pelobatinae, Discogrossidae, and Pipidae, among which the ldh gene belonging to Pipidae is preferably used. Among the frogs belonging to the frog family, it is more preferable to use a gene encoding lactate dehydrogenase derived from Xenopus laevis, which is easily available, particularly a gene encoding L-lactate dehydrogenase. Specifically, a particularly preferable gene encoding lactate dehydrogenase is a gene encoding L-lactate dehydrogenase having the base sequence shown in SEQ ID NO: 1.

  Introduction of a gene encoding lactate dehydrogenase into yeast can be performed using a technique such as gene recombination. Examples of a method for introducing a gene (ldh gene) encoding lactate dehydrogenase into yeast include, for example, a method of cloning the ldh gene, transforming an expression vector incorporating the cloned gene into yeast, and the cloned gene However, the method is not limited to these.

  The method for cloning the ldh gene used in the present invention is not particularly limited, and a known technique can be used. For example, based on known gene information, a method for amplifying and obtaining a necessary genetic region using PCR (Polymerase Chain Reaction) method, a method for 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 for incorporating the cloned ldh gene, an expression vector generally used in yeast can be used. The expression vector that is 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 ldh 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.

  The gene encoding the L-lactate dehydrogenase is preferably introduced downstream of a promoter that enables expression of the gene in an expression vector. The ldh gene can be introduced into yeast by transforming the obtained ldh gene expression vector into yeast by the method described later.

  The gene encoding L-lactate dehydrogenase is preferably introduced in a state that can be expressed downstream of the promoter of pyruvate decarboxylase 1 gene (pdc1 gene) on the chromosome. As a method of inserting the pyruvate decarboxylase 1 gene into the downstream of the promoter by homologous recombination, PCR is performed using primers designed to add a homologous portion to the target site upstream and downstream of the ldh gene. A method of transforming the obtained PCR fragment into yeast by the method described later is not limited thereto. 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: A plasmid containing a terminator connected to the downstream of the ldh gene is used as a template, and a fragment containing the ldh gene and the terminator is amplified by PCR using primers 1 and 2 as a set. 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 the ldh gene, terminator, and yeast selectable marker with homologous sequences 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 ldh gene expression vector or PCR fragment obtained above into yeast, methods such as transformation, transduction, transfection, co-transfection 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)” may be used. it can. Yeast into which the ldh 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.

  By culturing the yeast introduced with the ldh gene of the present invention, lactic acid can be produced in the medium.

  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 without amino acid (manufactured by DIFCO) as a nitrogen source and supplemented with an appropriate amino acid.

  In the method for producing lactic acid according to the present invention, microorganisms can be cultured 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 25 to 35 degrees Celsius, and the culture time is usually 24 hours to 5 days. It is desirable to maintain the pH of the culture medium during culture at 2.5 to 5.0, and this pH can be adjusted using an alkaline solution, ammonia, calcium carbonate, calcium hydroxide, or the like.

  In the method for producing lactic acid of the present invention, folic acid may be added to the medium before or after the start of culture, and the number of additions of folic acid may be once or twice or more. In addition, each component such as the carbon source, nitrogen source, inorganic salts, and nutrients may be added all at once at the start of the culture, or may be added in portions during the culture or continuously. Moreover, you may use an antifoamer as needed.

  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.

  Further, the folic acid concentration in the medium can be measured by HPLC under the following conditions, for example.

Column: TSK-GEL ODS-80TM 4.6 × 250mm
Mobile phase: A = 0.3% phosphoric acid aqueous solution, B = acetonitrile Detection method: electrical conductivity Temperature: 50 ° C

  Hereinafter, the L-ldh gene derived from a frog is selected as the ldh and Saccharomyces cerevisiae is selected as the yeast to show specific embodiments, and the present invention will be described in more detail. The technical scope of the present invention Is not limited to the following examples.

(Reference Example 1: Construction of yeast having lactic acid synthesis ability)
The ldh gene derived from Xenopus laevis having the base sequence shown in SEQ ID NO: 1 was used as the ldh gene. The ldh 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 primers (SEQ ID NOs: 2 and 3) were 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, the end was phosphorylated with T4 polynucleotide Kinase (manufactured by Takara Bio Inc.), and then ligated to the pUC118 vector (cut with the restriction enzyme HincII and the cut surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver.2 (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 ldh 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 ldh gene was purified according to a conventional method. The fragment containing the obtained ldh gene was ligated to the XhoI / NotI cleavage site of the expression vector pTRS11 shown in FIG. 2, the plasmid DNA was recovered by the same method as above, and cleaved with restriction enzymes XhoI and NotI. An expression vector into which the ldh gene was inserted was selected. Hereinafter, an expression vector incorporating the ldh gene prepared in this manner is referred to as pTRS102.

  A 1.3 kb DNA fragment containing the ldh gene and the 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 (step of FIG. 1). 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 primer sets was used for ldh gene, GAPDH terminator and TRP1. A DNA fragment of about 2.5 kb to which the gene was 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 ldh 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, the transformed strain in which the ldh gene is introduced downstream of the pdc1 gene promoter on the chromosome is referred to as B2 strain.

(Comparative Example 1: L-lactic acid productivity test using MMF3 medium without addition of folic acid)
Take 10 mL of the minimum synthetic medium (hereinafter abbreviated as MMF3) shown in Table 1 in a test tube, inoculate a small amount of the B2 strain prepared in Reference Example 1, and culture overnight at 30 ° C. (pre-culture) ). Next, 100 ml of MMF3 medium was placed in a 500 ml Erlenmeyer flask, and each pre-culture solution was inoculated in its entirety and cultured with shaking at 30 ° C. for 24 hours (pre-culture). Subsequently, a pre-culture solution 24 hours after the start of the preculture was inoculated in a total amount in a mini jar fermenter (manufactured by Maruhishi Bioengine, 5 L) charged with 1 L of MMF3 medium. 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 production amount of L-lactic acid was measured by HPLC under the following conditions. The L-lactic acid production amount calculated from the measurement results is shown in FIG. 3, and the lactic acid production yield (sugar yield: lactic acid production amount per carbon source introduced into the medium) is shown in Table 3.

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.

(Example 1: L-lactic acid productivity test 1 using MMF3 medium supplemented with folic acid)
A pre-culture solution was prepared in the same manner as in Comparative Example 1, and the entire amount of the pre-culture solution was inoculated into a mini jar fermenter (manufactured by Maruhishi Bio-Engineering Co., Ltd., volume 5 L) charged with 10 L of folic acid and 1 L of MMF3 medium. Then, it culture | cultivated by the method similar to the comparative example 1, and measured lactic acid. The production amount of L-lactic acid calculated from the measurement results is shown in FIG. 3, and the lactic acid production yield (sugar yield) is shown in Table 3.

(Example 2: L-lactic acid productivity test 2 using MMF3 medium supplemented with folic acid)
A pre-culture solution was prepared in the same manner as in Comparative Example 1, and the whole pre-culture solution was inoculated into a mini jar fermenter (manufactured by Maruhishi Bioengineering Co., Ltd., volume 5 L) charged with 30 μg of folic acid and 1 L of MMF3 medium. Then, it culture | cultivated by the method similar to the comparative example 1, and measured lactic acid. The production amount of L-lactic acid calculated from the measurement results is shown in FIG. 3, and the lactic acid production yield (sugar yield) is shown in Table 3.

(Example 3: L-lactic acid productivity test 3 using MMF3 medium supplemented with folic acid)
A pre-culture solution was prepared in the same manner as in Comparative Example 1, and the whole amount of the pre-culture solution was inoculated into a mini jar fermenter (manufactured by Maruhishi Bio-Engineering Co., Ltd., volume 5 L) charged with 40 L of folic acid and 1 L of MMF3 medium. Then, it culture | cultivated by the method similar to the comparative example 1, and measured lactic acid. The production amount of L-lactic acid calculated from the measurement results is shown in FIG.

(Example 4: L-lactic acid productivity test 4 using MMF3 medium supplemented with folic acid)
A pre-culture solution was prepared in the same manner as in Comparative Example 1, and the whole amount of the pre-culture solution was inoculated into a mini jar fermenter (manufactured by Maruhishi Bio-Engineering Co., Ltd., volume 5 L) charged with 50 L of folic acid and 1 L of MMF3 medium. Then, it culture | cultivated by the method similar to the comparative example 1, and measured lactic acid. The production amount of L-lactic acid calculated from the measurement results is shown in FIG. 3, and the lactic acid production yield (sugar yield) is shown in Table 3.

  As a result of Comparative Example 1 and Examples 1 to 4, the production yield of lactic acid was 40.2% under the conditions where folic acid was not added (Comparative Example 1), whereas folic acid was added (Examples 1 to 4). ) Improved the production yield of lactic acid (yield to sugar). In particular, the production yield of lactic acid was improved to 57.3% under the condition of adding 30 μg / L of folic acid (Example 3). Addition of folic acid was effective in improving the production yield of lactic acid.

(Comparative Example 2: L-lactic acid productivity test using CMF4 medium without folic acid added)
Using the B2 strain prepared in Reference Example 1, a lactic acid fermentation test using a medium to which folic acid was added was performed as follows.

  10 mL of a medium having the composition shown in Table 2 (hereinafter abbreviated as CMF4 medium) 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 CMF4 medium was placed in a 500 mL Erlenmeyer flask, and each pre-culture solution was inoculated in its entirety 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 CMF4 medium was added, and the stirring speed (120 rpm), Cultivation was performed with the aeration rate (0.1 L / min), temperature (30 ° C.), and pH (pH 5) being constant (main culture). The culture solution was centrifuged every 10 hours after the start of the main culture, and the obtained supernatant was subjected to membrane filtration. Then, the amount of L-lactic acid was measured by HPLC in the same manner as in Comparative Example 1. The L-lactic acid production amount calculated from the measurement results is shown in FIG. 4, and the lactic acid production yield (vs. sugar yield) is shown in Table 4.

(Example 5: L-lactic acid productivity test 1 using CMF4 medium supplemented with folic acid)
A pre-culture solution was prepared in the same manner as in Comparative Example 2, and the entire amount of the pre-culture solution was inoculated into a mini jar fermenter (manufactured by Maruhishi Bio-Engineering Co., Ltd., volume 5 L) charged with 10 L of folic acid and 1 L of CMF4 medium. Then, it culture | cultivated by the method similar to the comparative example 1, and measured lactic acid. The L-lactic acid production amount calculated from the measurement results is shown in FIG. 4, and the lactic acid production yield (vs. sugar yield) is shown in Table 4.

(Example 6: L-lactic acid productivity test 2 using CMF4 medium supplemented with folic acid)
A pre-culture solution was prepared in the same manner as in Comparative Example 2, and the entire pre-culture solution was inoculated into a mini jar fermenter (manufactured by Maruhishi Bioengineering Co., Ltd., volume 5 L) charged with 30 μg of folic acid and 1 L of CMF4 medium. Then, it culture | cultivated by the method similar to the comparative example 1, and measured lactic acid. The L-lactic acid production amount calculated from the measurement results is shown in FIG. 4, and the lactic acid production yield (vs. sugar yield) is shown in Table 4.

(Example 7: L-lactic acid productivity test 3 using CMF4 medium supplemented with folic acid)
A pre-culture solution was prepared in the same manner as in Comparative Example 2, and the entire pre-culture solution was inoculated into a mini jar fermenter (manufactured by Maruhishi Bio-Engineering Co., Ltd., volume 5 L) charged with 40 μg of folic acid and 1 L of CMF4 medium. Then, it culture | cultivated by the method similar to the comparative example 1, and measured lactic acid. The production amount of L-lactic acid calculated from the measurement results is shown in FIG.

(Example 8: L-lactic acid productivity test 4 using CMF4 medium supplemented with folic acid)
A pre-culture solution was prepared in the same manner as in Comparative Example 2, and the entire amount of the pre-culture solution was inoculated into a mini jar fermenter (manufactured by Maruhishi Bio-Engineering Co., Ltd., volume 5 L) charged with 50 L of folic acid and 1 L of CMF4 medium. Then, it culture | cultivated by the method similar to the comparative example 1, and measured lactic acid. The L-lactic acid production amount calculated from the measurement results is shown in FIG. 4, and the lactic acid production yield (vs. sugar yield) is shown in Table 4.

  As a result of Comparative Example 2 and Examples 5 to 8, the production yield of lactic acid was 65.3% under the condition where folic acid was not added (Comparative Example 2), whereas the condition where folic acid was added (Examples 5 to 8). ) Improved the production yield of lactic acid (yield to sugar). In particular, under the conditions where folic acid was added at 30 μg / L or 50 μg / L (Examples 5 and 7), the production yield of lactic acid was improved to 71%. Addition of folic acid was effective in improving the production yield of lactic acid.

  According to the present invention, in fermentation production of lactic acid using a microorganism capable of synthesizing lactic acid, the amount of lactic acid produced (yield to sugar) is improved, and lactic acid can be produced efficiently.

FIG. 1 is a schematic diagram illustrating a method for introducing an 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 showing the effect of increasing the amount of lactic acid produced by the addition of folic acid in the MMF3 medium. The horizontal axis represents the culture time, and the vertical axis represents the production amount of L-lactic acid. FIG. 4 is a graph showing the effect of increasing the amount of lactic acid produced by the addition of folic acid in the CMF4 medium. The horizontal axis represents the culture time, and the vertical axis represents the production amount of L-lactic acid.

SEQ ID NO: 1—Frog-derived L-lactate dehydrogenase: DNA
SEQ ID NO: 2—Description of Artificial DNA Sequence: Synthetic DNA
SEQ ID NO: 4—Description of Artificial DNA Sequence: Synthetic DNA
SEQ ID NO: 5—Description of Artificial DNA Sequence: Synthetic DNA
SEQ ID NO: 6—Description of Artificial DNA Sequence: Synthetic DNA
SEQ ID NO: 7--Description of Artificial DNA Sequence: Synthetic DNA
SEQ ID NO: 8--Description of Artificial DNA Sequence: Synthetic DNA

Claims (13)

  A medium for producing lactic acid by fermentation of culturing a microorganism having a lactic acid synthesis ability, the medium comprising folic acid or a folic acid precursor in an amount of 10 μg / L to 70 μg / L.   The medium for lactic acid production according to claim 1, comprising sugarcane press.   The medium for producing lactic acid according to claim 1 or 2, wherein the microorganism is yeast.   The manufacturing method of lactic acid using the culture medium for lactic acid manufacture in any one of Claims 1-3.   A method for producing lactic acid by fermentation of culturing a microorganism having lactic acid synthesis ability, wherein a medium containing folic acid or a folic acid precursor in an amount of 10 μg / L to 70 μg / L is used.   The method for producing lactic acid according to claim 5, wherein the microorganism is yeast.   The method for producing lactic acid according to claim 8, wherein the yeast belongs to the genus Saccharomyces.   The method for producing lactic acid according to claim 6 or 7, wherein the yeast is a yeast into which a lactate dehydrogenase gene has been introduced.   The method for producing lactic acid according to claim 8, wherein the lactate dehydrogenase gene is a gene encoding L-lactate dehydrogenase derived from Xenopus laevis.   The method for producing lactic acid according to claim 9, wherein the gene encoding L-lactate dehydrogenase is a gene having the base sequence shown in SEQ ID NO: 1.   The method for producing lactic acid according to claim 9 or 10, wherein a gene encoding L-lactate dehydrogenase is introduced downstream of a promoter that enables expression of the gene encoding L-lactate dehydrogenase.   The production of lactic acid according to any one of claims 9 to 11, wherein a gene encoding L-lactate dehydrogenase is introduced in a state capable of being expressed downstream of a promoter of pyruvate decarboxylase 1 gene on a chromosome. Method. The method for producing lactic acid according to any one of claims 5 to 12, wherein the microorganism is cultured in a medium at 25 to 37 ° C.

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