JP2012235714A - Method for producing useful substance by using candida utilis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for adjusting a pH of a culture solution in culture of Candida utilis.SOLUTION: The method for adjusting the pH of a culture solution upward during culturing Candida utilis, includes adding nitrate into the culture solution.

Description

Background of the Invention

TECHNICAL FIELD The present invention relates to a method for producing a useful substance using Candida utilis which is a club tree negative yeast as a host.

BACKGROUND ART In recent years, in view of the risk of depleting fossil resources in the future, attention has been focused on efforts to produce chemical products and their raw materials by fermentation using plant biomass as a raw material. Plastic is one of them, and organic acids that are metabolites of living organisms can be used as the material. An example of a plastic made from an organic acid is polylactic acid, which is a polymer of lactic acid.

  Lactic acid has L-form and D-form. In order to produce polylactic acid having a high melting point, it is necessary to polymerize L-form or D-form lactic acid having high optical purity. Moreover, it is possible to further increase the melting point by mixing equal amounts of poly L lactic acid and poly D lactic acid to form a stereo complex. For this reason, it is required to independently produce L-lactic acid and D-lactic acid having high optical purity. That is, the fermentation production system for D-lactic acid is the key to producing stereocomplex lactic acid.

  In the fermentation production of lactic acid, there is a problem that the activity of the bacterial cells is reduced or the bacteria are killed by the extreme bias of the pH to the acidic range as the product lactic acid accumulates. Therefore, in order to produce lactic acid at a high concentration, the bacterium is required not only to have a high fermentability but also to be resistant to a high concentration of lactic acid.

  In industrial fermentative production of lactic acid, neutralizers such as calcium carbonate are generally used in order to avoid an extreme decrease in pH. However, the use of a neutralizing agent synthesized through a chemical process does not necessarily meet the biomass plastic production concept of reducing environmental impact. This includes pyruvic acid [Non-patent document 1: Appl Environ Microbiol Vol. 70: p159-66 (2004)], malic acid and succinic acid [Non-patent document 2: Appl Environ Microbiol Vol. 74: p. This is a common problem when fermenting and producing various organic acids other than lactic acid.

Among the yeasts that have been most well studied to date and have accumulated genetic knowledge, there is a yeast of the genus Saccharomyces, which has been studied as a host for the production of various substances.
In recent years, methods for transforming several species such as Pichia yeast, Hansenula yeast, Kluyveromyces yeast, Candida yeast as yeast other than Saccharomyces yeast have been developed and are useful substances. It has been studied as a production host. Among these, Candida spp. Has characteristics that are not found in Saccharomyces spp. Such as a wide carbon utilization range.

Among the Candida genus yeasts, Candida utilis exhibits excellent assimilability to pentose including xylose. In addition, unlike Saccharomyces yeast, ethanol is not produced by culturing under aerobic conditions and growth inhibition is not caused thereby, so that efficient microbial cell production by continuous culture at high density is possible. Therefore, Candida utilis has once attracted attention as a protein source, and industrial production of microbial cells using a saccharified solution of broad-leaved trees containing a large amount of pentose and a sulfite pulp waste solution as a sugar source has been performed. Moreover, this yeast US FDA (Food and Drug Administration), Saccharomyces cerevisiae (Saccharomyces cerevisiae), with Saccharomyces fragilis (Saccharomyces fragilis), are recognized as safe yeast that can be used as a food additive. In fact, Candida utilis is manufactured in various countries around the world, including Germany, the United States, Taiwan, and Brazil, and is used as a diet. In addition to the use as a microbial protein, Candida utilis has been widely used in the industry as a production strain for pentose and xylose fermentation strains, ethyl acetate, L-glutamine, glutathione, invertase and the like. .

Appl Environ Microbiol Vol.70: p159-66 (2004) Appl Environ Microbiol Vol.74: p.2766-77 (2008)]

  The present inventors have found that when culturing Candida utilis, the pH of the culture solution increases with time by adding nitrate to the culture solution. Furthermore, when the present inventors produce an organic acid by culturing Candida utilis, a decrease in the pH of the culture solution accompanying the production of the organic acid is suppressed by adding nitrate to the culture solution. I found out. Furthermore, the present inventors have succeeded in developing a Candida utilis yeast strain that produces D-lactic acid by genetic manipulation. The present invention is based on these findings.

  Accordingly, an object of the present invention is to provide a method and a pH regulator for adjusting the pH of a culture solution, a method for producing an organic acid by culturing Candida utilis, and D-lactic acid in Candida utilis culture. The object is to provide a yeast strain that is produced with high efficiency.

  The pH adjustment method according to the present invention is a method for upwardly adjusting the pH of a culture solution during the cultivation of Candida utilis, and comprising adding nitrate to the culture solution.

  The method for producing an organic acid according to the present invention is a method for producing an organic acid by culturing Candida utilis, which comprises adding a nitrate to the culture solution.

  The pH adjusting agent according to the present invention is a pH adjusting agent for adjusting the pH of a culture solution upward during cultivation of Candida utilis, comprising nitrate.

  The yeast strain according to the present invention is transformed with at least one copy of the gene operably linked to a promoter sequence that enables expression of a gene encoding a polypeptide having D-lactate dehydrogenase activity. In addition, a yeast strain of Candida utilis, in which an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity is disrupted.

  According to the present invention, the pH of the culture solution can be adjusted upward in the cultivation of Candida utilis. Such an effect is advantageous in preventing adverse effects caused by a decrease in pH accompanying the production of organic acid, for example, killing of bacterial cells, for example, when organic acid is produced by culturing Candida utilis. It is. Another advantage of the present invention is that no neutralizing agent is required to prevent such a decrease in pH. Furthermore, the above effect is particularly advantageous in the cultivation of Candida utilis strains that produce D-lactic acid at high concentrations, such as the yeast strain according to the present invention.

Detailed description of the invention

  The pH adjustment method according to the present invention is a method for adjusting the pH of a culture solution upward during the cultivation of Candida utilis. The method comprises adding nitrate to the culture medium.

  In the present specification, the term “adjusting the pH upward” and derivatives thereof mean that the pH of the culture solution is adjusted to the alkali side as compared with the case where nitrate is not added to the culture solution. In other words, this term does not mean that the pH increases over time from the start of culture, but that the curve of the graph showing the change in pH over time is on the alkali side compared to the case where nitrate is not added. To do. Therefore, even if the pH decreases to the acidic side over time from the start of the culture, it corresponds to “adjusting the pH upward” if it is on the alkaline side as compared with the case where nitrate is not added.

  The yeast used in the present invention is Candida utilis, which is a club tree negative yeast. The strain of Candida utilis may be various strains known in the art, for example, NBRC0626 strain, NBRC0639 strain, NBRC0988 strain, NBRC1086 strain and the like.

  The nitrate used in the present invention is not particularly limited as long as it can be assimilated by Candida utilis. Such nitrates are well known in the art, but can preferably be sodium nitrate, potassium nitrate or calcium nitrate, or any combination thereof, more preferably sodium nitrate.

  The amount of nitrate added to the culture solution is not particularly limited as long as it is the amount of a normal nitrogen source used for Candida utilis culture. Thus, the amount of nitrate added can be appropriately determined by those skilled in the art, but preferably, the concentration of nitrate ions derived from nitrate is 1 to 80 mM, more preferably 20 to 80 mM, and most preferably about 50 mM. can do.

  The timing of adding nitrate to the culture solution is not particularly limited because it can be appropriately selected by those skilled in the art, such as when the pH of the culture solution is desired to be adjusted upward. For example, nitrate may be already added at the start of culture or may be added during the culture after the start of culture. In addition, nitrate may be added at the start of the culture and further added during the culture.

  The culture solution may contain a nitrogen source other than nitrate, and this does not impair the pH adjustment effect by nitrate. However, in order to obtain a high pH adjusting effect, it is preferable that the nitrogen source other than nitrate in the culture solution is as small as possible. For example, the molar concentration of a nitrogen source other than nitrate is preferably the same as or less than the molar concentration of nitrate ions derived from nitrate. According to a preferred embodiment of the invention, the added nitrate is present in the culture as the main nitrogen source assimilated by Candida utilis. Here, the term “major nitrogen source” means that 50% or more of the assimilated nitrogen is derived from nitrate. According to a particularly preferred embodiment of the invention, the added nitrate is present in the culture as the substantially only nitrogen source assimilated by Candida utilis. Here, the term “substantially” means that the amount of nitrogen source other than nitrate is less than the amount that can contribute to the growth of Candida utilis, that is, when nitrate is removed from the culture solution. This means that Candida utilis cannot grow.

  The composition of the culture solution other than the nitrogen source can be determined following a normal medium used for Candida utilis culture. For example, as a carbon source contained in the culture solution, for example, xylose or sucrose can be used in addition to glucose as long as it can be assimilated. Moreover, as a nutrient source contained in the culture solution, for example, yeast extract, peptone, whey and the like are used. Furthermore, the culture solution may contain potassium phosphate, magnesium sulfate, Fe (iron), Mn (manganese) compound, etc. as an inorganic nutrient source. The culture solution may be a completely synthetic medium such as SC medium, but may be a natural medium containing molasses and / or sulfite pulp waste liquid as a carbon source and peptones as a nitrogen source.

  As described above, it is an advantage of the present invention that a neutralizing agent for preventing a decrease in pH is not required. Therefore, according to a preferred embodiment of the present invention, the culture solution used does not contain a pH regulator such as a neutralizing agent.

  The culture temperature can be selected within the range in which the Candida utilis strain used can grow. The culture temperature can be, for example, about 15 to 45 ° C, more preferably 20 to 40 ° C, still more preferably 25 to 35 ° C, and most preferably about 30 ° C.

  The culture time is not particularly limited, and is carried out for any reaction time as long as the effect of the present invention is recognized. Those skilled in the art can easily optimize these conditions.

  In the present invention, Candida utilis can be cultured for the purpose of producing a substance by fermentation of Candida utilis. In particular, if the substance to be manufactured is an acidic substance such as an organic acid, the pH that should drop with the production of the substance is adjusted upward by the effect of nitrate. This is advantageous in maintaining a pH suitable for utilis culture.

  Therefore, according to another aspect of the present invention, there is provided a method for producing an organic acid by culturing Candida utilis, the method comprising adding nitrate to the culture solution.

  The organic acid produced in the present invention is not particularly limited as long as it is an organic acid that can be produced by culturing a wild strain or mutant strain of Candida utilis. Examples of such organic acids include, but are not limited to, lactic acid, pyruvic acid, malic acid, succinic acid, and the like. According to a preferred embodiment of the present invention, the organic acid produced according to the present invention is L-lactic acid or D-lactic acid, more preferably D-lactic acid.

  For the production of the organic acid, it is advantageous to use a mutant obtained by genetically modifying a wild strain of Candida utilis. Therefore, according to a preferred embodiment of the present invention, the Candida utilis is a genetically modified strain. The genetic manipulation of yeast is well known in the art, and thus a person skilled in the art can genetically modify Candida utilis to produce the desired organic acid. For example, a Candida utilis strain that produces L-lactic acid is described in International Publication No. 2010/095751.

  According to a preferred embodiment of the present invention, the Candida utilis strain used in the present invention is supposed to produce D-lactic acid, and such a strain is, for example, against the wild strain of Candida utilis. (i) transformed with at least one copy of the gene operably linked to a promoter sequence enabling expression of a gene encoding a polypeptide having D-lactate dehydrogenase activity; and (ii) It is assumed that the endogenous gene encoding a polypeptide having pyruvate decarboxylase activity has been disrupted. The Candida utilis strain subjected to the genetic modification of (i) and (ii) has a particularly excellent ability to produce D-lactic acid. Therefore, the Candida utilis strain is one embodiment of the present invention. Make. When such a Candida utilis strain is cultured, the pH drops sharply due to the highly efficient production of D-lactic acid. Therefore, it is preferable to adjust the pH upward by culturing in the presence of nitrate. .

The yeast strain according to the present invention retains a gene ( D-LDH gene) encoding a polypeptide having D-lactate dehydrogenase activity. Since yeast originally has no ability to produce lactic acid, the gene ( D-LDH ) encoding the polypeptide having the lactate dehydrogenase activity of the yeast strain according to the present invention is foreign. Examples of a gene encoding a polypeptide having the activity of D-lactate dehydrogenase used in the present invention include naturally-derived D-LDH, as well as D-LDH artificially synthesized by chemical synthesis or genetic engineering techniques. Is also included. Examples of organisms having D-LDH include prokaryotes such as lactic acid bacteria, eukaryotes such as fungi, and higher eukaryotes such as plants, animals and insects. D-LDH used in the present invention is preferably derived from lactic acid bacteria, and in particular, derived from Lactobacillus helveticus.

  According to a preferred embodiment of the present invention, the polypeptide having D-lactate dehydrogenase activity is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2. The polypeptide having D-lactate dehydrogenase activity includes an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 2, and It may be a polypeptide having the activity of D-lactate dehydrogenase.

  Here, amino acid deletion, substitution, addition, or insertion can be performed by modifying a gene encoding the above polypeptide by a technique known in the art. Mutation can be introduced into a gene by a known method such as the Kunkel method or Gapped duplex method or a method similar thereto, for example, a mutation introduction kit using site-directed mutagenesis, such as Mutant-K (Takara Bio Inc.), Mutant-G (Takara Bio Inc.), etc. or using Takara Bio Inc. LA PCR in vitro Mutagenesis series kit, KOD-Plus-Mutageness Kit (TOYOBO), etc. be able to. The activity of D-lactate dehydrogenase can be confirmed by a technique known in the art.

  Furthermore, the gene encoding the polypeptide having the activity of D-lactate dehydrogenase to be introduced into the host is a nucleotide sequence encoding the amino acid sequence of the enzyme derived from Lactobacillus helveticus described in SEQ ID NO: 2. Is preferably artificially synthesized in consideration of the codon usage frequency of Candida utilis. Such artificial synthesis can be appropriately performed by those skilled in the art, but a particularly preferred nucleotide sequence is the nucleotide sequence represented by SEQ ID NO: 1.

  According to a preferred embodiment of the present invention, the gene encoding a polypeptide having D-lactate dehydrogenase activity is a gene comprising the nucleotide sequence represented by SEQ ID NO: 1, or an equivalent thereof. . This equivalent means a gene having some nucleotide residues different on the condition that it has a function equivalent to that of the gene containing the nucleotide sequence represented by SEQ ID NO: 1. Such an equivalent includes 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, most preferably 95% or more homology with the nucleotide sequence represented by SEQ ID NO: 1. And a gene comprising a nucleotide sequence encoding a polypeptide having a D-lactate dehydrogenase activity. The equivalent further includes a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 1 or a complementary sequence thereof under stringent conditions and encodes a polypeptide having the activity of D-lactate dehydrogenase. Including genes. The equivalent further includes a sequence in which one or several nucleotide residues are deleted, substituted, added, or inserted in the nucleotide sequence represented by SEQ ID NO: 1, and the activity of D-lactate dehydrogenase And a gene containing a nucleotide sequence encoding a polypeptide having According to a particularly preferred embodiment of the present invention, the gene encoding a polypeptide having D-lactate dehydrogenase activity is a gene comprising the nucleotide sequence represented by SEQ ID NO: 1.

  Here, deletion, substitution, addition, or insertion of a nucleotide residue can be performed by modifying a gene containing the above sequence by a technique known in the art. Mutation can be introduced into a gene by a known method such as Kunkel method or Gapped duplex method or a method similar thereto, for example, a mutation introduction kit using site-directed mutagenesis may be used. For example, using Mutant-K (Takara Bio) or Mutant-G (Takara Bio) or using Takara Bio's LA PCR in vitro Mutagenesis series kit, KOD-Plus-Mutageness Kit (TOYOBO), etc. Mutations can be introduced. The activity of D-lactate dehydrogenase can be confirmed by a technique known in the art.

  The numerical value (%) indicating homology is calculated using default (initial setting) parameters using a base sequence comparison program: for example, GENETYX-WIN 7.0.0. That is, each gene on the yeast chromosome may be replaced by a gene encoding a polypeptide that is not identical but has an equivalent function, ie, each activity, through homologous recombination or the like. The activity of D-lactate dehydrogenase can be confirmed by a technique known in the art.

  The stringent conditions are, for example, rapid-hyb buffer (manufactured by GE Healthcare Bioscience), preferably at a temperature of 40 to 70 ° C., more preferably 60 ° C. Hybridization conditions. Then, for example using a general method of the person skilled in the art, washing for 5 minutes with a solution consisting of 2 × SSC and 0.1% (w / v) SDS, followed by 1 × SSC and 0.1% (w / v) v) Refers to washing for 10 minutes with a solution consisting of SDS, and further washing for 10 minutes with a solution consisting of 0.1 × SSC and 0.1% (w / v) SDS. However, by setting conditions such as the temperature conditions during hybridization and the salt concentration of the solution used for the subsequent membrane washing, it can be set to a certain level (70%, 80%, 85%, 90%, or 95%). ) DNA containing a base sequence having the above homology can be cloned. The gene thus obtained may be replaced by homologous recombination or the like with a gene that encodes a polypeptide that is not identical in sequence but has an equivalent function, that is, each activity. The activity of D-lactate dehydrogenase can be confirmed by a technique known in the art.

The gene encoding a polypeptide having D-lactate dehydrogenase activity is preferably provided so that it can be expressed under the control of a promoter having strong promoter activity. For example, in Candida utilis, a GAP gene promoter encoding a polypeptide having the activity of glyceraldehyde-3-phosphate dehydrogenase of Candida utilis encodes a polypeptide having the activity of phosphoglycerate kinase. Examples of the promoter of the PGK gene and the promoter of the PMA gene that encodes a polypeptide having plasma membrane proton ATPase activity (above, JP-A No. 2003-144185), and the like, more preferably pyruvate decarboxylase It is a promoter of gene 1 ( CuPDC1 gene) encoding a polypeptide having activity.

The gene encoding a polypeptide having D-lactate dehydrogenase activity is preferably provided so that it can be expressed under the control of the CuPDC1 gene promoter on the yeast chromosome. Candida utilis used as a host of the yeast strain according to the present invention is presumed to have at least one PDC gene ( CuPDC1 gene). By gene CuPDC1 gene controlled by the CuPDC1 gene promoter encoding a polypeptide having a disrupted by activity of lactate dehydrogenase is expressed in place, lowering the lactic acid effectively pyruvate decarboxylase activity The dehydrogenase activity can be expressed at the same time.

The yeast strain according to the present invention has no or reduced pyruvate decarboxylase (PDC) activity. This enzyme is an enzyme that converts pyruvate to acetaldehyde in the alcohol fermentation pathway, and yeast that performs alcohol fermentation inherently has a gene encoding a polypeptide having pyruvate decarboxylase activity on its chromosome. Yes. Saccharomyces cerevisiae has three types of genes ( ScPDC1 , ScPDC5, and ScPDC6 ) that encode polypeptides having pyruvate decarboxylase activity, and these function by a so-called autoregulation mechanism. Moreover, the homology at the nucleotide level of each gene is as high as 70% or more. The proteins encoded by these genes are composed of an N-terminal TPP binding region and a C-terminal PDC active region. The gene encoding PDC is also present in other yeasts. For example, the KlPDC1 gene of Kluyveromyces lactis has high homology with the ScPDC1 gene. On the other hand, Candida utilis has one type of gene ( CuPDC1 ) encoding a polypeptide having pyruvate decarboxylase activity, and there may be another similar gene, but at least CuPDC1 By destroying the gene, almost no alcoholic fermentation takes place.

  Here, “there is no or reduced PDC activity” means that there is no PDC activity, the enzyme having an activity lower than that of the wild type is produced, or the production amount of the enzyme is wild type. Means less than. The yeast strain having no or reduced PDC activity may be obtained by artificial manipulation or may be found by screening. Artificial manipulations for extinction or reduction of enzyme activity include RNAi, replacement with other genes such as all or part of the selectable marker, and insertion of meaningless sequences inside the gene. It can carry out by a method well-known in this technical field. Among these, it is preferable to destroy (knock out) the gene encoding the polypeptide having the enzyme activity. As such a method, among the above-mentioned methods, all or a part of the sequence of the selectable marker is used. And a method of exchanging the gene of PDC with a gene encoding PDC.

The gene encoding the polypeptide having the activity of pyruvate decarboxylase to be destroyed originally exists in Candida utilis. For example, one nucleotide sequence of the allele of CuPDC1 gene present in NBRC0988 strain is SEQ ID NO: The amino acid sequence represented and encoded by 7 is represented by SEQ ID NO: 8. When using other strains of Candida utilis, for example, NBRC0626 strain, NBRC0639 strain, NBRC1086 strain, etc., even if they are different from the sequences, if there are those having equivalent functions, ie, activities It can be targeted for destruction.

  According to a preferred embodiment of the present invention, the endogenous gene encoding a polypeptide having the activity of pyruvate decarboxylase to be destroyed is a gene encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 8 More preferably, the gene comprises the nucleotide sequence represented by SEQ ID NO: 7.

  Molecular breeding of the yeast strain according to the present invention can be performed by introducing a gene encoding a polypeptide having D-lactate dehydrogenase activity into a host yeast in a state where it can be expressed. In that case, it is preferable to involve destruction of the gene which codes PDC with respect to host yeast.

  The disruption of the PDC gene and the introduction of a gene encoding a polypeptide having the activity of L-lactate dehydrogenase are described in WO 2010/095751. Therefore, the yeast strain according to the present invention can be obtained by using the above-mentioned D-lactate dehydrogenase instead of L-lactate dehydrogenase as described in WO 2010/095751. In addition, in the NBRC0988 strain of Candida utilis, a strain that is completely deficient in the CuPDC1 gene encoding pyruvate decarboxylase: the Cupdc1delta4 strain has been constructed by Ikushima et al. [Biosci Biotechnol Biochem. Vol. 73: p1818- 24 (2009)]. Therefore, in breeding the yeast strain according to the present invention, it is advantageous to use this Cupdc1delta4 strain.

The position on the chromosome where the gene encoding the polypeptide having the activity of D-lactate dehydrogenase is integrated into the yeast genome is not particularly limited, but the polypeptide having the activity of pyruvate decarboxylase is not limited. Advantageously, the gene locus is encoded. As a result, a gene encoding a polypeptide having lactate dehydrogenase activity can be placed under the control of the full-length promoter of the PDC gene, and thus high expression efficiency can be obtained.

In addition to D-lactic acid and L-lactic acid, it is possible to construct a Candida utilis strain that produces organic acids such as pyruvic acid, malic acid, and succinic acid. For example, by continuously culturing S. cerevisiae that is completely deficient in the PDC gene, a strain that can grow even in the presence of a high concentration of glucose is selected. It has been reported that pyruvate can be produced at high concentration and high efficiency by aerobic culture of this strain under the condition of glucose as a sugar source [Appl. Environ. Microbiol. 70 (1), p159- 66 (2004)]. Furthermore, it has been reported that a malic acid high production strain can be constructed by adding the following modification to this glucose resistant strain: pyruvate carboxylase enzyme (PYC2) gene overexpression, recombined to function in the cytoplasm High expression of the malate dehydrogenase (MDH3) gene and functional expression of the malate transporter (SpMAE1) gene from the budding yeast Schizosaccharomyces pombe [Appl. Environ. Microbiol. 74 (9), p.2766- 77 (2008)]. It has also been confirmed that this high malic acid-producing strain produces succinic acid [Appl. Environ. Microbiol. 74 (9), p.2766-77 (2008)]. By applying these techniques to Candida utilis, it is possible to construct a yeast strain that produces pyruvic acid, malic acid, or succinic acid.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, these Examples do not restrict | limit the technical scope of this invention.

Example 1: Construction of Candida utilis strain into which D-LDH gene was introduced In NBRC0988 strain of Torula yeast Candida utilis, a strain completely lacking the CuPDC1 gene encoding pyruvate decarboxylase: The Cupdc1delta4 strain has already been constructed by Ikushima et al. [Biosci Biotechnol Biochem. Vol. 73: p1818-24 (2009)]. A D-lactate dehydrogenase (D-LDH) gene derived from the lactic acid bacterium Lactobacillus helveticus was introduced into this strain. The DNA fragment used here was artificially synthesized by adapting the frequency of codon usage to Candida utilis so that there was no amino acid substitution (SEQ ID NO: 1). The plasmid containing the fragment was designated as pDLDH. The procedure for constructing the plasmid used for the introduction is shown below.

  First, the following three types of polymerase chain reaction (PCR) were performed: (1) IM-341 (SEQ ID NO: 3: actcgcggccgctctagaCACCAACTTTGAAGATAGGG) and IM-678 (SEQ ID NO: 4) using the genomic DNA of C. utilis NBRC0988 strain as a template. : GtaggcgaagaccttggtcatGGTATCGATTGTTTTAGTTTTGTTTGTTTGTTGTGTATAACGGG) was used as a primer (elongation reaction 2 minutes), and the promoter region of CuPDC1 gene was amplified; 6: AtactcagatctTCATCAGAACTTGTTCTTGTTC) was used as a primer (extension reaction 1 minute) to amplify the D-LDH structural gene; (3) The DNA fragment amplified in (1) and (2) was used as a template, and IM- PCR was performed using 341 and IM-672 (extension reaction 3 minutes). Next, the DNA fragment amplified in (3) was digested with NotI and BglII, and the obtained DNA fragment was ligated to pCU675 (also called pPGtHPt) cleaved with NotI and BamHI. Here, pCU675 is a plasmid already constructed by Ikushima et al. [Biosci Biotechnol Biochem. Vol. 73: p1818-24 (2009)].

  The plasmid pCU871 obtained through the above-described steps is, in order, at the BssHII site of pBluescriptIISK (+), CuPDC1 gene promoter region, D-LDH structural gene, PGK (3-phosphoglycerokinase) gene terminator, loxP, PGK gene promoter. A DNA fragment consisting of a downstream region of the HPT gene (hygromycin B resistance gene), GAP (glyceraldehyde 3-phosphate dehydrogenase) gene terminator, loxP, and CuPDC1 gene is inserted. On the side of the inserted DNA fragment of the two BssHII recognition sequences, a BglII recognition sequence is provided immediately after the recognition sequence. Therefore, pCU871 was digested with BglII to obtain a DNA fragment consisting of the downstream region of the CuPDC1 gene from the above-described CuPDC1 gene promoter, and cupdc1delta4 was transformed by this using the electric pulse method. Here, the pulse conditions were according to the method of Ikushima et al. [Biosci Biotechnol Biochem. Vol. 73: p879-84 (2009)]. As a positive clone, a strain grown on YPD medium (1% yeast extract, 2% peptone, 2% glucose) containing 600 μg / ml hygromycin B was selected. This was named CDLA01 strain.

The CDLA01 strain thus obtained was cultured in a 100 ml YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) aerobically at 30 ° C. for 24 hours using a 500 ml scale Sakaguchi flask. . This strain was inoculated into 15 ml of eutrophic medium (1% yeast extract, 2% peptone, 90.7 g / l glucose) in a 100 ml Erlenmeyer flask so that the OD ₆₀₀ was 0.3. To this medium, 45 g / l calcium carbonate was added as a neutralizing agent. 24 hours after the start of the culture, all the sugar was consumed, and instead, D-lactic acid having an optical purity of 99.9% was produced at a concentration of 86.8 g / l. Assuming that the yield of D-lactic acid is theoretically maximum from the sugar contained in the medium, the yield is more than 95%.

Next, the CDLA01 strain was aerobically shake-cultured at 30 ° C. in a YPD2 liquid medium (1% yeast extract, 2% peptone, 2% glucose) in a 300 mL scale Sakaguchi flask. This strain was inoculated to 2.0 L of molasses medium (18% molasses, 2% peptone) at an initial OD ₆₀₀ of 0.1 in a 5 L scale jar fermenter and cultured at 35 ° C. with stirring. The aeration amount was 1.0 vvm, the stirring speed was 300 rpm, and the pH was maintained at 5.0 by using 10N NaOH and 10N HCl as neutralizing agents. When 72 hours passed from the start of culture, D-lactic acid with an optical purity exceeding 99.5% was produced at a concentration of 160 g / L (FIG. 1). This is a value that reaches 100% in terms of the conversion efficiency for the sugar consumed at that time.

Example 2: Candida utilis culture in medium with nitrate as the only nitrogen source
C. utilis NBRC 0988 wild type strain was aerobically shake cultured at 30 ° C. This strain was inoculated in a 5 L scale jar fermenter in 2.0 L of synthetic medium (5% glucose, 0.17% Yeast Nitrogen Base, 50 mM sodium nitrate) to an initial OD ₆₀₀ of 0.5, The culture was stirred at 30 ° C. The aeration rate was 2.0 L / min, and the stirring speed was 300 rpm. As a result, the pH of the culture broth increased with time and reached pH 8.6 after 27 hours from the start of the culture (FIG. 2).

  Next, when the same test was performed on the CDLA01 strain using the above-mentioned synthetic medium, the pH of the culture solution was stable at about 3 without adding a neutralizing agent, and even if lactic acid accumulated, There was no bias toward the acid range. Due to this effect, it was possible to avoid the death of the yeast due to the decrease in pH. As a result, 15.4 g / L of D-lactic acid was produced 27 hours after the start of the culture (FIG. 3).

Example 3: Candida utilis culture in a medium using nitrate and other nitrogen-containing substances as nitrogen sources In a synthetic medium containing a combination of 25 mM sodium nitrate and 25 mM ammonium chloride as a nitrogen source, C. The utilis NBRC 0988 wild strain was cultured. When ammonium chloride is used as a single nitrogen source, the viable cell count is usually drastically reduced after about 24 hours of cultivation, along with a rapid drop in pH. On the other hand, when sodium nitrate having the same molar concentration as ammonium chloride was mixed, it was clarified that the decrease in pH was suppressed and the culture could be continued for 40 hours or more (FIG. 4).

  This result shows that in the cultivation of Candida utilis in a medium containing nitrate as a nitrogen source, even if other nitrogen sources coexist, the pH increasing effect by nitrate is not inhibited.

Example 4: Cultivation of Candida utilis in medium with various nitrates as the only nitrogen source Instead of 50 mM sodium nitrate , medium supplemented with 50 mM potassium nitrate or 25 mM calcium nitrate as a single nitrogen source was used. C. utilis NBRC 0988 wild type strain was cultured in the same manner as in 2. As a result, in any case, the pH increase with time was the same as when sodium nitrate was used (FIG. 5).

  From this result, it is shown that in the cultivation of Candida utilis in a medium containing nitrate as a nitrogen source, the pH increase effect by nitrate is not affected by the type of nitrate cation.

Example 5: Cultivation of various strains of Candida utilis in medium with nitrate as the only nitrogen source Candida utilis strain ATCC 15239, NBRC 0619, NBRC 0626, and NBRC 10708 The pH raising effect of the liquid was tested. Specifically, in a 200 mL Erlenmeyer flask, each strain was inoculated into 50 mL of the nitric acid medium described in Example 2 so that the initial OD ₆₀₀ was 0.5 and cultured at 30 ° C. with shaking. As a result, in all the strains, the pH of the culture broth increased with time as in the case of NBRC 0988 strain (FIG. 6).

  From this result, it is shown that in the cultivation of Candida utilis in a medium containing nitrate as a nitrogen source, the pH increasing effect by nitrate is not affected by the difference in strains, but is generally observed in Candida utilis.

Example 6: Culture of yeast other than Candida utilis in a medium containing nitrate as the only nitrogen source As yeasts other than Candida utilis that can grow using nitrate as a single nitrogen source, Pichia angusta, paxolene A culture test similar to that of Example 5 was performed using three kinds of tannofilus (Pachysolen tannophilus) and Hansenula polymorpha. The results are shown in Table 1.

  As is clear from Table 1, when any of the three types of yeast was cultured, no rapid increase in pH as observed in Candida utilis was observed.

Fig. 1 shows that CDLA01 strain was inoculated in 2.0L molasses medium (18% molasses, 2% peptone) to an initial OD600 of 0.1 in a 5L scale jar fermenter and stirred at 35 ° C. The culture results are shown. In this culture, the aeration rate was 1.0 vvm, the stirring speed was 300 rpm, and the pH was maintained at 5.0 by using 10N NaOH and 10N HCl as neutralizing agents. The white circle and white triangle in the figure indicate the concentration of D-lactic acid in the culture mixture, and the black circle and black triangle indicate the concentration of glucose in the culture mixture. FIG. 2 shows changes in pH when C. utilis NBRC 0988 wild strain was cultured in a medium containing sodium nitrate as the sole nitrogen source. The solid line in the figure indicates the pH of the culture solution, and the white circle (dot) indicates the value of OD ₆₀₀ . FIG. 3 shows changes in pH and production of D-lactic acid when the CDLA01 strain is cultured in a medium containing sodium nitrate as the sole nitrogen source. The solid line in the figure indicates the pH of the culture solution, and the white circle (dot) indicates the concentration of D-lactic acid (g / l). FIG. 4 shows changes in pH when C. utilis NBRC 0988 wild strain was cultured in a medium containing a combination of sodium nitrate and ammonium chloride as a nitrogen source. The solid line in the figure indicates the pH of the culture solution, and the white circle (dot) indicates the value of OD ₆₀₀ . FIG. 5 shows the pH when C. utilis NBRC 0988 wild strain was cultured in a medium containing any one of sodium nitrate (white circle), potassium nitrate (white rhombus) and calcium nitrate (white triangle) as a sole nitrogen source. Shows changes. Fig. 6 shows Candida utilis ATCC 15239 (white circle), NBRC 0619 (white rhombus), NBRC 0626 (white triangle), and NBRC 10708 (white square). The change of pH at the time of culture | cultivating in the culture medium containing as is shown.

Claims (12)

  A method for adjusting the pH of a culture solution upward during cultivation of Candida utilis, comprising adding nitrate to the culture solution.   The method of claim 1, wherein the added nitrate is present in the culture as a primary nitrogen source assimilated by Candida utilis.   A method for producing an organic acid by culturing Candida utilis, comprising adding nitrate to a culture solution.   4. The method of claim 3, wherein the added nitrate is present in the culture as the primary nitrogen source assimilated by Candida utilis.   The method according to claim 3 or 4, wherein the organic acid is L-lactic acid or D-lactic acid.   The method according to any one of claims 3 to 5, wherein Candida utilis is a genetically modified strain. The strain is a wild strain of Candida utilis,
Transformed with at least one copy of the gene operably linked to a promoter sequence enabling expression of a gene encoding a polypeptide having D-lactate dehydrogenase activity; and
The method according to claim 6, wherein an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity is disrupted.   A pH adjuster for adjusting the pH of a culture solution upward during culture of Candida utilis, comprising nitrate.   D-lactate dehydrogenase is transformed with at least one copy of the gene operably linked to a promoter sequence enabling expression of a gene encoding a polypeptide having the activity of pyruvate dehydrogenase, and pyruvate decarboxylase A yeast strain of Candida utilis, wherein an endogenous gene encoding a polypeptide having the activity of is disrupted. A polypeptide having the activity of D-lactate dehydrogenase is
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, or (b) an amino acid wherein one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 2 The yeast strain according to claim 9, which is a polypeptide comprising a sequence and having a D-lactate dehydrogenase activity. A gene encoding a polypeptide having the activity of D-lactate dehydrogenase,
(A) the nucleotide sequence represented by SEQ ID NO: 1,
(B) a nucleotide sequence encoding a polypeptide having 85% or more homology with the nucleotide sequence represented by SEQ ID NO: 1 and having D-lactate dehydrogenase activity, or (c) represented by SEQ ID NO: 1. The yeast according to claim 9 or 10, comprising a nucleotide sequence that hybridizes with a nucleotide sequence or a complementary sequence thereof under stringent conditions and encodes a polypeptide having D-lactate dehydrogenase activity. Strain.   An endogenous gene encoding a polypeptide having pyruvate decarboxylase activity comprises a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 8, or a nucleotide sequence represented by SEQ ID NO: 7. The yeast strain according to any one of claims 9 to 11.

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