JP4700395B2 - Promoters for use under acidic conditions and use thereof - Google Patents

Description

  The present invention relates to a promoter for use under acidic conditions, a DNA construct for gene recombination, a transformant, and a substance production method.

  Advances in recombinant DNA technology have led to the development of techniques for obtaining target gene products by expressing foreign genes in hosts such as microorganisms, molds, animals and plants, and insects, and proliferating the transformants. For example, by culturing genetically modified yeast or the like, it is possible to produce a large amount of a target gene product by fermentation production. Regarding the production of useful organic acids such as L-lactic acid, many attempts have been reported to introduce L-lactic acid dehydrogenase gene into the genus Saccharomyces cerevisiae to produce L-lactic acid.

  In order to establish high productivity of a target product in a recombinant, it is necessary to construct a stable gene high expression system. The present inventors have already disrupted the PDC1 gene in the yeast chromosome at the same time as binding the intended useful gene (in this case, L-lactate dehydrogenase gene) downstream of the PDC1 gene promoter in the chromosome. Thus, a system has been developed that eliminates the expression of the PDC1 protein expressed by the original promoter while expressing the L-lactate dehydrogenase gene using the original PDC1 gene promoter function. It has been found that a stable gene high expression system, which has been difficult in the past, can be established by using this system (Patent Document 1).

  Even though such a stable gene high expression system has been established, there is still a demand for further enhancing the productivity of recombinant products in this system. For example, when producing lactic acid using genetically modified microorganisms, since the pH in the culture solution is lowered by the lactic acid produced, it is common to culture while neutralizing with alkali and collect it as lactate. is there. When collected as a lactate, a separate process for desalting it into free lactic acid is required. In view of the mass production of lactic acid, it is desirable to eliminate this process. It is also desirable to avoid charging the neutralizing agent itself.

In order to improve productivity, it is necessary to search for a new promoter. In yeast, glyceraldehyde triphosphate dehydrogenase 1 gene (TDH1 gene) promoter (Non-patent document 1), triose phosphate isomerase 1 gene (TPI1 gene) promoter (Non-patent document 2), cell wall-related protein 12 gene ( CCW12 gene) promoter (Non-patent document 3) and ribosomal protein S31 gene (RSP31 gene) promoter (Non-patent document 4) are known, but the characteristics of these promoter activities are not well known.
JP 2003-164295 A Applied and Environmental Microbiology, Jan. 2003, p.495-503 Applied and Environmental Microbiology, June 2002, p.3024-3030 Journal of Bacteriology, May 1999, p. 3076-3086 Journal of Biological Chemistry, vol.273, No.49, Issue of December 4, pp. 32870-32877 (1998)

  Although the above-mentioned PDC1 gene promoter is an effective promoter in organic acid production using a neutralizing agent, the present inventors have found that its expression ability is reduced under acidic conditions. In consideration of further simplification of the manufacturing process, a new promoter that is highly expressed specifically under acidic conditions has been required.

  Accordingly, an object of the present invention is to provide a promoter that is highly expressed under acidic conditions. Another object of the present invention is to provide a promoter, a recombinant DNA construct, a transformant, and a substance production method suitable for fermentation production capable of obtaining a free organic acid as a recombinant product from a medium. To do. Another object of the present invention is to provide a promoter, a recombinant DNA construct, a transformant and a method for producing a substance that can be subjected to organic acid fermentation without using a neutralizing agent. Furthermore, the present invention further provides a promoter, a recombinant DNA construct, a transformant, and a substance production method suitable for producing an organic acid in a state where expression of a pyruvate decarboxylase gene in yeast is suppressed. One other purpose.

  The present inventors have focused on obtaining a free organic acid from a medium by subjecting a recombinant organism to lactic acid fermentation under non-neutralizing conditions, and searched for a promoter capable of such lactic acid fermentation. That is, in non-neutralizing conditions, in the production of organic acids, genes that were highly expressed when yeast was made to produce lactic acid under acidic conditions were searched for. As a result, dozens of types of highly expressed genes were found, further selected, and finally when lactate dehydrogenase genes were bound downstream of the promoters of these highly expressed genes, We have found four types of promoters that produce lactic acid with high efficiency. None of these promoters are known as high expression promoters, but can be said to be promoters that are specifically resistant to acidic conditions. According to the present invention, the following means are provided.

According to one aspect of the present invention, there is provided a promoter selected from the following sequences (a) to (c), which has promoter activity under acidic conditions and is used under acidic conditions: Is done. In addition, the promoter of this aspect may be a promoter for use under acidic conditions having a part of the polynucleotide and having promoter activity for expression under acidic conditions.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1 (b) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1 or a base sequence complementary thereto, Nucleotide (c) a polynucleotide comprising a base sequence in which one or more bases have been deleted, substituted and / or added in the base sequence set forth in SEQ ID NO: 1

According to another embodiment of the present invention, a polynucleotide selected from the following sequences (a) to (c) for use under acidic conditions having promoter activity under acidic conditions: A promoter is provided. In addition, the promoter of this aspect may be a promoter for use under acidic conditions having a part of the polynucleotide and having promoter activity for expression under acidic conditions.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2 (b) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2 or a base sequence complementary to a part thereof. Nucleotide (c) a polynucleotide comprising a base sequence in which one or more bases are deleted, substituted and / or added in the base sequence set forth in SEQ ID NO:

According to another embodiment of the present invention, a polynucleotide selected from the following sequences (a) to (c) for use under acidic conditions having promoter activity under acidic conditions: A promoter is provided. In addition, the promoter of this aspect may be a promoter for use under acidic conditions having a part of the polynucleotide and having promoter activity for expression under acidic conditions.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 3 (b) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the base sequence set forth in SEQ ID NO: 3 or a base sequence complementary thereto, Nucleotide (c) a polynucleotide comprising a base sequence in which one or more bases are deleted, substituted and / or added in the base sequence set forth in SEQ ID NO:

According to another aspect of the present invention, a polynucleotide selected from the following sequences (a) to (c), which is used under acidic conditions having promoter activity under acidic conditions: A promoter is provided. In addition, the promoter of this aspect may be a promoter for use under acidic conditions having a part of the polynucleotide and having promoter activity for expression under acidic conditions.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 4 (b) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the base sequence set forth in SEQ ID NO: 4 or a base sequence complementary thereto. A polynucleotide comprising a nucleotide sequence obtained by deleting, substituting and / or adding one or more nucleotides in the nucleotide sequence of nucleotide (c) SEQ ID NO: 4

  Furthermore, according to another aspect of the present invention, there is provided any one of the above promoters which is a promoter for organic acid production in a microorganism. In particular, these promoters can be promoters for organic acid production by yeast. Moreover, it is good also as a promoter for organic acid production which produces organic acid on non-neutralization conditions, and is good also as a promoter for organic acid production which produces organic acid in pH 1.0-3.0. Furthermore, it is good also as a promoter for organic acid production for producing the organic acid derived from pyruvic acid in the state in which the expression of the pyruvate decarboxylase gene of the host is suppressed.

  Any of the above promoters can be used as an acidic condition-inducible promoter. This acidic condition can be a lactic acid acidic condition.

  According to another embodiment of the present invention, glyceraldehyde triphosphate dehydrogenase 1 gene (TDH1 gene), triose phosphate isomerase 1 gene (TPI1 gene), cell wall-related protein 12 gene (CCW12 gene) and A promoter for use under acidic conditions having the promoter activity of any gene selected from the ribosomal protein S31 gene (RSP31 gene) is provided.

  According to another aspect of the present invention, there is provided a DNA construct comprising DNA encoding a protein involved in organic acid production operably linked to any of the above promoters.

  In this embodiment, the protein can be a protein having lactate dehydrogenase activity. The protein may be bovine-derived lactate dehydrogenase. Furthermore, it can be set as the structure provided with the DNA for homologous recombination for the gene disruption of the yeast gene provided with an autoregulation mechanism. This yeast gene can be a pyruvate decarboxylase gene.

  According to another embodiment of the present invention, a transformant carrying any of these DNA constructs is provided.

  In this embodiment, the promoter and the DNA encoding the protein are preferably integrated on the host chromosome. The host is preferably yeast. Furthermore, the yeast preferably belongs to the genus Saccharomyces. In addition, expression of a yeast gene having an autoregulation mechanism on the host chromosome may be suppressed, and the yeast gene having the autoregulation mechanism is preferably a pyruvate decarboxylase gene. Furthermore, the protein preferably has lactate dehydrogenase activity.

  According to another aspect of the present invention, there is provided an organic acid production method comprising the steps of preparing any one of the above transformants and culturing the transformant in a medium containing an organic acid. Is done.

  In this aspect, in the culturing step, an organic acid may be supplied to the medium by organic acid production by the host cell. Further, in the culturing step, an organic acid may be added to the medium. The protein is a protein having lactate dehydrogenase activity, and the culturing step can be a step of producing lactic acid. Furthermore, the culture step can be a step of culturing the transformant without neutralizing the medium. Moreover, the said culture | cultivation process can also be set as the process of culturing the said transformant in the range of pH of a culture medium 1.0-3.0.

(promoter)
Whether the promoter of the present invention activates the expression of a DNA encoding a protein operably linked to the promoter for the first time in the presence of an organic acid, or promotes the expression compared to the absence of an organic acid, Alternatively, it has a promoter activity that is promoted when the concentration of the organic acid is increased (hereinafter, this activity is referred to as this promoter activity). As used herein, “functional binding” refers to binding that causes the expression of DNA encoding the bound protein to be under the influence or control of this promoter. The “organic acid” is an organic compound exhibiting acidity, but the acidic group provided in the organic acid is preferably a carboxylic acid group. Organic acids include organic acids in addition to free acids. Specific examples of such organic acids include lactic acid, butyric acid, acetic acid, pyruvic acid, succinic acid, formic acid, malic acid, citric acid, malonic acid, propionic acid, ascorbic acid, and adipic acid. Preferably, it is lactic acid. Lactic acid includes L-lactic acid, D-lactic acid, and DL-lactic acid, and includes any of these. “In the presence of an organic acid” means that the organic acid is present in the growth environment of the host retaining the promoter of the present invention, and the organic acid is produced by the host retaining the promoter of the present invention. Alternatively, it may be supplied from other than the host, such as added to the medium, or both of them. The concentration of the organic acid in the medium is not particularly limited as long as the DNA functionally bound to the promoter of the present invention is expressed or promoted.

  Examples of the promoter for use under acidic conditions of the present invention include a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 and having promoter activity under acidic conditions. Furthermore, the following promoters can be used as such promoters and corresponding promoters.

  One homologous promoter includes a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1. Such a polynucleotide is obtained by a general hybridization technique (Southern, EM., J Mol Biol, 1975, 98, 503.) using DNA consisting of the base sequence shown in SEQ ID NO: 1 or a part thereof as a probe. Obtainable. In addition, such DNA is prepared by PCR technology (Saiki, RK. Science, 1985, 230, 1350., et al., Saiki) using, as a primer, an oligonucleotide that specifically hybridizes to the DNA having the base sequence set forth in SEQ ID NO: 1. , RK. Et al., Science, 1988, 239, 487). The stringent conditions preferably used for the isolation of such DNA mean hybridization conditions in which the hybridization temperature is 37 ° C. in the presence of 50% formamide or hybridization conditions with the same stringency. According to conditions with higher stringency, DNA with higher homology can be isolated. Such hybridization conditions include, for example, hybridization conditions of about 42 ° C. in the presence of 50% formamide, and conditions of about 65 ° C. in the presence of 50% formamide. .

  Another homologous promoter is a polynucleotide comprising a nucleotide sequence in which one or more bases are deleted, substituted and / or added in the nucleotide sequence set forth in SEQ ID NO: 1, and the promoter under acidic conditions The thing which has activity is mentioned. Such DNA can be obtained by the hybridization technique and the PCR technique already described, and the Site-directedmutageness method (Kramer, W. & Fritz, HJ., Method Enzymol., 1987, 154, 350). Can also be obtained by artificially introducing a mutation into the base sequence described in SEQ ID NO: 1.

  The homology with the nucleotide sequence described in SEQ ID NO: 1 in these homologous promoters is preferably 70% or more, more preferably 80% or more, and still more preferably 90% in the isolated DNA. That's it. The homology of the DNA base sequence can be determined by a known gene analysis program BLAST or the like. The DNA base sequence homology is determined by the gene analysis program BLAST (http://blast.genome.ad.jp), FASTA (http://fasta.genome.ad.jp/SIT/FASTA.html), or the like. Can be determined.

  Examples of the promoter of the present invention include a polynucleotide having any one of the nucleotide sequences selected from the nucleotide sequences described in SEQ ID NOs: 2 to 4, and having the present promoter activity. For each of these SEQ ID NOs: 2 to 4, homologous promoters similar to those described in SEQ ID NO: 1 can be used.

  The promoters represented by the nucleotide sequences described in SEQ ID NOs: 1 to 4 of these promoters of the present invention are glyceraldehyde triphosphate dehydrogenase 1 gene (TDH1 gene) and triose phosphate isomerase of yeast Saccharomyces cerevisiae, respectively. 1 DNA (TPI1 gene), cell wall-related protein 12 gene (CCW12 gene) and ribosomal protein S31 gene (RSP31 gene) were obtained as DNA fragments having promoter activity. Therefore, the promoter of the present invention may be DNA having the promoter activity of these genes in yeast or Saccharomyces yeast, or DNA having homology to such a promoter region and having this promoter activity. Such DNA is obtained by performing a hybridization technique using a probe consisting of at least a part of the base sequence described in any one of SEQ ID NOs: 1 to 4 or a PCR technique using an oligonucleotide probe that hybridizes to the base sequence. It can be obtained from yeast. Furthermore, DNA that hybridizes under the stringent conditions described above can be selected.

  The promoter of the present invention may be a part of such various forms of DNA as long as it has this promoter activity.

  In addition, each DNA which consists of a base sequence of sequence number 1-4 does not add a neutralizing agent in the lactic acid production yeast which introduce | transduced the lactate dehydrogenase gene under control of the PDC1 gene promoter while the PDC1 gene is destroyed. It was selected through screening under lactic acid fermentation under conditions (non-neutralizing conditions; pH 1.0 to pH 3.0). In screening, lactic acid fermentation is performed using the lactic acid-producing yeast under the neutralization conditions, and a high expression level is obtained by quantifying cDNA obtained from mRNA collected after a certain period of time after the start of fermentation by a quantitative PCR technique or the like. The gene which showed is selected. By such screening, it is possible to search for a gene that is highly expressed during lactic acid production (in the presence of an organic acid) in the lactic acid-producing yeast as described above, and by isolating the promoter of such gene, A promoter having a high transcription activity can be obtained (in the presence of an organic acid).

  The promoter obtained by such screening is a promoter that is simultaneously resistant to acidic conditions including acidic conditions with organic acids such as lactic acid, and is an acidic condition-inducible promoter that activates gene expression under such acidic conditions. It can be said that there is. Therefore, by functionally binding a DNA encoding a protein having an enzyme activity involved in organic acid production such as lactate dehydrogenase to such a promoter, the production of organic acid is not suppressed due to an increase in the production amount of organic acid. Alternatively, conversely, it is highly expected to obtain a transformant such as a practical organic acid-producing yeast in which the production of organic acid is further promoted when the acidity is increased by increasing the production amount of organic acid. The PDC1 promoter is a constitutive promoter and has a certain ability to produce lactic acid even under acidic conditions. However, as lactic acid production proceeds and the acidity in the medium increases, the promoter activity may stagnate or decrease. However, according to the present promoter, such a tendency to decrease the promoter activity is suppressed or avoided.

  None of the activities of the promoter of the present invention is a known feature. In addition, the promoter obtained by such screening can be said to be a promoter that activates transcription in an abnormal state for yeast in which the expression of the pyruvate decarboxylase gene of the host is suppressed. These known promoters are presumed to be promoters having strong promoter activity at the time of such specific abnormalities.

  Therefore, this promoter can be used as a promoter for organic acid production in microorganisms. In particular, it can be used as a promoter for organic acid production by yeast. The yeast as the microorganism will be described in detail later. In addition, the promoter of the present invention can be used as a promoter for organic acid production that produces an organic acid under non-neutralizing conditions. Furthermore, the promoter of this invention can be used as a promoter for organic acid production which produces organic acid in pH 1.0-3.0. Furthermore, the promoter of the present invention is for producing an organic acid derived from pyruvate in a state in which the expression of the host pyruvate decarboxylase gene (typically pyruvate decarboxylase 1 gene) is suppressed. It can be used as a promoter for organic acid production.

  In addition, it can also be confirmed by the reporter assay using a reporter gene well-known in the art whether the acquired DNA has this promoter activity. As the reporter gene, a known gene can be used without particular limitation, and examples thereof include chloramphenicol acetyltransferase gene (CAT), β-galactosidase gene (LacZ), luciferase gene (LUC), β-glucuronidase and Examples thereof include green florescent protein gene (GFP). In addition, the presence or absence of this promoter activity is confirmed using a gene that can measure the amount of protein encoded by the gene and the activity of the protein (enzyme) (for example, the amount of metabolites). It is also possible. Furthermore, this promoter activity can also be confirmed by measuring the transcription level of a gene using Northern hybridization method or quantitative PCR method using mRNA obtained by gene expression. . Since this promoter activity activates or promotes expression in the presence of an organic acid, a medium containing the organic acid or a host that produces the organic acid should be used to confirm the promoter activity. preferable.

  The polynucleotide constituting the promoter of the present invention may be genomic DNA or DNA obtained artificially by a chemical technique or the like.

(DNA construct)
The present invention also provides a DNA construct comprising this promoter. The DNA construct of the present invention is not particularly limited, and a plasmid (DNA), virus (DNA), bacteriophage (DNA), retrotransposon (DNA), artificial chromosome (YAC, PAC, BAC, MAC, etc.) It can be selected according to the form of introduction (extrachromosomal or intrachromosomal) or the type of host cell and can take the form of a vector. Therefore, the present DNA construct can comprise the present promoter DNA, as well as DNA as a vector of any one of these aspects. The DNA construct preferably takes the form of a plasmid vector or a viral vector. Preferred prokaryotic vectors, eukaryotic cells, animal cells, and plant cells are well known in the art. This DNA construct may be retained outside the host chromosome in the cytoplasm or host cell, or may be incorporated and retained in the host chromosome.

  The present DNA construct includes a DNA encoding a desired protein operably linked to the present promoter (hereinafter also referred to as a coding DNA). The coding DNA may include not only cDNA but also a DNA sequence that is transcribed but not translated. Although the protein is not particularly limited, the coding DNA may be DNA for producing an organic acid such as lactic acid, that is, DNA coding for a protein having an enzyme activity related to organic acid production. It is expected that organic acid production is synergistically promoted by functionally binding DNA encoding such a protein to this promoter. Examples of such enzymes include L-lactate dehydrogenase and D-lactate dehydrogenase in the case of lactic acid production, pyruvate kinase and the like in the case of pyruvate production, and pyruvin in the case of acetic acid production. Examples of acid oxidase include succinyl-CoA synthetase in the case of succinic acid production, fumarate hydratase in the case of malic acid, and citrate synthetase in the case of citric acid production.

  As a lactate dehydrogenase (LDH), various homologues exist depending on the kind of organism or in vivo. The lactate dehydrogenase used in the present invention includes not only naturally derived LDH but also LDH synthesized chemically or genetically. LDH is preferably derived from prokaryotes such as lactic acid bacteria or eukaryotic microorganisms such as mold, more preferably derived from higher eukaryotes such as plants, animals and insects, and more preferably cows and the like. It is derived from higher eukaryotes including mammals. L-LDH is preferably bovine-derived LDH (L-LDH). For example, a protein having the amino acid sequence shown in SEQ ID NO: 6 can be mentioned as LDH derived from bovine. An example of such a DNA encoding LDH is a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 5. Furthermore, LDH in the present invention includes homologues of these LDHs. An LDH homolog is a protein having an LDH activity, which is an amino acid sequence having one or several amino acid substitutions, deletions, insertions and / or additions in the amino acid sequence of naturally occurring LDH, and a naturally occurring LDH It contains a protein having LDH and amino sequence homology of at least 70%, preferably 80% or more and having LDH activity.

  This DNA construct can be provided with a DNA sequence for homologous recombination for integrating the promoter and coding DNA into a host chromosome by homologous recombination. By providing such a DNA, the integration of these DNAs at a desired site of the host chromosome can be achieved, and the destruction of a desired gene can be achieved simultaneously. The DNA sequence for homologous recombination is a DNA sequence that is homologous to the DNA sequence at or near the target site into which the present DNA is to be introduced in the host chromosome. The DNA sequence for homologous recombination has one sequence that is homologous to at least one location in the vicinity of one target gene, or preferably, preferably a sequence that is homologous to at least two locations in the vicinity of the target gene. I have. For example, two DNA sequences for homologous recombination are made DNA sequences homologous to the upstream and downstream DNAs of the target site on the chromosome, and the promoter or It is preferable to link the coding DNA.

  The present DNA construct is a case where the present DNA is introduced into the host chromosome by homologous recombination on the host chromosome, and the promoter is selected at a desired site on the host chromosome by selecting an appropriate DNA for homologous recombination. And the coding DNA is incorporated, and the expression of the coding DNA can be controlled by the promoter DNA. The selection of DNA for homologous recombination for realizing such integration on a chromosome is well known to those skilled in the art, and those skilled in the art can select appropriate DNA for homologous recombination as needed and perform homologous recombination. Recombinant DNA constructs can be constructed.

  By selecting the DNA for homologous recombination, the desired gene on the host chromosome can be destroyed simultaneously with the chromosomal integration of the promoter DNA. The gene to be destroyed is preferably a gene having an autoregulation mechanism, which will be described later. For example, when an organic acid is produced using a DNA encoding a protein involved in organic acid production in yeast such as Saccharomyces yeast, the pyruvate decarboxylase gene (particularly, the pyruvate decarboxylase 1 gene) is destroyed. It is preferable to do. This is because the promoter of pyruvate decarboxylase 1 gene is strong and has an autoregulation mechanism as described later. Chromosomal integration that disrupts the gene can suppress the expression of pyruvate decarboxylase and simultaneously express the protein encoded by the foreign DNA. Furthermore, since the expression of the foreign coding DNA is activated under acidic conditions by this promoter, the expression of the foreign coding DNA is further promoted by promoting the production of organic acid by the expressed protein.

  In particular, when lactic acid is produced using DNA encoding lactate dehydrogenase, the disrupted form is more effective. This is because the pyruvate decarboxylase 1 gene acts competitively with lactate dehydrogenase using pyruvate, which is a substrate for lactate dehydrogenase, as a substrate. By disrupting the gene, the expression of pyruvate decarboxylase can be suppressed and lactate dehydrogenase can be expressed. At the same time, lactate dehydrogenase is activated in the presence of organic acid (lactic acid) by this promoter DNA. Therefore, lactic acid production is expected to be further promoted. In order to achieve such integration on the chromosome, the present DNA construct can be used, for example, a part of the structural gene of pyruvate decarboxylase or a sequence in the vicinity thereof (sequence in the vicinity of the start codon, upstream region of the start codon). Or a DNA sequence homologous to the upstream region and / or the downstream side of the gene on the chromosome. Preferably, a Saccharomyces genus (especially cerevisiae) is used as a host, and a DNA construct targeting the pyruvate decarboxylase 1 gene of this host is used.

  Since the pyruvate decarboxylase gene promoter is a strong promoter, it can be used in combination with this promoter DNA. That is, a protein having lactate dehydrogenase activity in place of a structural gene to be originally controlled by the gene promoter at the endogenous site of the pyruvate decarboxylase gene (preferably pyruvate decarboxylase 1 gene) on the yeast chromosome A DNA construct for homologous recombination that introduces a DNA encoding DNA, and a protein having lactate dehydrogenase activity in place of a structural gene that should be originally controlled by the DNA promoter at a site endogenous to the DNA promoter on the yeast chromosome DNA constructs for homologous recombination that introduce DNA to be used can be used. Expected to produce more effective lactic acid by promoting expression by both PDC (preferably PDC1) gene promoter and this promoter and suppressing expression of pyruvin decarboxylase gene by disrupting PDC (preferably PDC1) structural gene Is done. Even if the PDC1 gene is disrupted, pyruvate decarboxylase is biosynthesized by another PDC5 gene or the like, so that the growth performance of yeast is ensured. In such a DNA construct, DNA having PDC1 promoter activity can be retained as exhibiting the functions of both promoter DNA and DNA for homologous recombination. PDC1 promoter DNA includes DNA consisting of the base sequence set forth in SEQ ID NO: 9, DNA that hybridizes with the DNA under stringent conditions, substitution or deletion of one or more bases in the base sequence, DNA consisting of added and / or inserted sequences can be used. Such DNA can be isolated in the same manner as the promoter DNA.

  As already disclosed by the present inventors, the PDC1 gene is a gene having an autoregulation mechanism (Japanese Patent Laid-Open No. 2003-164295). The autoregulation mechanism is that multiple genes with the same function exist in the same organism, and at least one of them is normally expressed, but the rest is suppressed. It means a mechanism in which the remaining genes are expressed and continue their functions only when they stop functioning. Since such a mechanism exists, for example, even if the yeast PDC1 gene is disrupted, the PDC5 gene is activated, the yeast ethanol production function is maintained, and the physiological function is maintained. By destroying the gene in which such an autoregulation mechanism exists, it is possible to maintain the survival and proliferation function of the organism itself, and as a result, the target product while maintaining the growth of the transformant holding the foreign DNA. Can be produced effectively.

  In addition, when the host has this promoter on the host chromosome as the promoter of the endogenous gene, by selecting an appropriate DNA for homologous recombination, at the site where this promoter originally exists on the host chromosome. The promoter and coding DNA can also be incorporated. By doing so, the exogenous coding DNA can be controlled by this promoter in place of the endogenous coding DNA that should be controlled by the present promoter on the chromosome. As a result, the expression of the endogenous coding DNA that should be activated originally is suppressed, while the expression of the foreign coding DNA is activated by the promoter DNA at the site on the chromosome where the promoter DNA is originally present. It can be made. Such a DNA construct can be incorporated into such a host chromosome by providing the present promoter and at least a part of the endogenous structural gene controlled by the present promoter as DNA for homologous recombination.

  In addition, a terminator, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, and a ribosome binding sequence (SD sequence) can be linked to the DNA construct as necessary. The selectable marker is not particularly limited, and various known selectable marker genes such as drug resistance genes and auxotrophic genes can be used. For example, ampicillin resistance gene, kanamycin resistance G418 gene, hygromycin resistance gene, bleomycin resistance gene, neomycin resistance gene, dihydrofolate reductase gene, chloramphenicol resistance gene, cyclohexamide resistance gene and the like can be used.

(Transformant)
This DNA construct can be applied to an appropriate host cell by transformation method, transfection method, conjugation method, protoplast fusion, electroporation method, lipofection method, lithium acetate method, particle gun method, calcium phosphate precipitation method, Agrobacterium method This can be introduced by any suitable means such as PEG method, direct microinjection method and the like. Thereby, the transformant of the present invention can be obtained. After the introduction of the DNA construct, the host cell is cultured in a selective medium.

  Host cells include bacteria such as Eshrichia coli and Bacillus subtilis, yeasts such as Saccharomyces pombe such as Saccharomyces pombe, yeasts such as Pichia pastoris, and insect cells such as sf9 and sf21. Animal cells such as COS cells and Chinese hamster ovary cells (CHO cells), plant cells such as sweet potatoes and tobacco. Preferred are microorganisms that perform alcoholic fermentation such as yeast or acid-resistant microorganisms, for example, yeasts including Saccharomyces genus such as Saccharomyces cerevisiae. Specifically, Saccharomyces cerevisiae IFO2260 strain and YPH strain can be exemplified.

  Whether the present DNA construct has been introduced into the host or whether the present DNA construct has been introduced at a desired site on the chromosome can be confirmed by PCR or Southern hybridization. For example, it can be confirmed by preparing DNA from a transformant, performing PCR with an introduction site-specific primer, and detecting the expected band in electrophoresis for the PCR product. Alternatively, it can be confirmed by performing PCR with a primer labeled with a fluorescent dye or the like. These methods are well known to those skilled in the art.

  In a transformant transformed with the present DNA construct, the present promoter, coding DNA, and the like, which are components of the DNA construct, are present on a chromosome or on an extrachromosomal factor (including an artificial chromosome). When a DNA construct for homologous recombination that is capable of achieving homologous recombination is introduced, the coding DNA functionally linked to the promoter and the DNA at a desired position on the host chromosome when the DNA construct is introduced. Is obtained.

  One preferred form of a transformant obtained by homologous recombination with a host chromosome is a form comprising a coding DNA operably linked to the promoter at a site on the chromosome where the promoter is inherently present. According to such a transformant, since the expression of the coding DNA is activated by this promoter under acidic conditions, the target product can be effectively obtained under acidic conditions. In this form, preferably the coding DNA encodes a protein having lactate dehydrogenase activity. Such a transformant produces lactic acid by the lactic acid dehydrogenase activity, and further expression of the enzyme activity is promoted by the lactic acid production, thereby promoting lactic acid production.

  In another preferred form of the transformant of the present invention, expression of a gene having an autoregulation mechanism on a host (yeast, etc.) chromosome is suppressed, and the present promoter and This is a form comprising coding DNA operably linked to DNA. According to such a transformant, in particular, these DNAs can express the coding DNA without giving a fatal functional disorder to the host. When a DNA encoding a protein having lactate dehydrogenase activity is used as the encoding DNA, lactic acid can be produced while inhibiting the host's growth properties from being impaired. Note that the expression of a gene is suppressed when the gene is destroyed by introduction of a foreign gene, or when the activity of a transcript or expression product is reduced due to an artificial or natural mutation in the gene. However, it is not limited to these.

  Another preferred form of the transformant of the present invention is that the PDC gene (preferably PDC1 gene) on the host (yeast etc.) chromosome is disrupted and the promoter DNA is replaced with the promoter of the gene. In this mode, the coding DNA is functionally linked to the DNA. According to such a transformant, particularly when DNA encoding a protein having lactate dehydrogenase activity is used as the coding DNA, the expression of the competing pyruvate decarboxylase is suppressed, and lactate dehydrogenation is performed by this promoter DNA. An enzyme can be expressed, and a transformant suitable for lactic acid production can be obtained. As described above, even if the PDC1 gene is disrupted, the gene has an autoregulation mechanism, so that the host's growth ability and the like are maintained.

  Still another preferred form of the transformant comprises the present promoter DNA and a coding DNA operably linked to the DNA on the host chromosome (for example, any one of the above preferred three forms or a combination thereof). ), On the host chromosome or outside the host chromosome, the PDC1 promoter and a coding DNA operably linked to the DNA. According to this form, when all of the coding DNA encodes a protein having an enzyme activity related to biosynthesis of organic acid such as lactic acid, the expression of the organic acid biosynthetic enzyme is constitutively caused by the PDC1 promoter. On the other hand, since this promoter activates the expression of organic acid biosynthetic enzymes in an organic acid-inducible manner, high-efficiency organic acid production is expected. In particular, the PDC1 promoter is preferably in a form that utilizes an endogenous promoter on the host chromosome. In this case, stable expression by the PDC1 promoter is possible.

  In these transformants, the host is preferably yeast, and among them, yeast of the genus Saccharomyces is preferable, and Saccharomyces cerevisiae is more preferable. Furthermore, the coding DNA is preferably DNA encoding a protein having lactate dehydrogenase activity, more preferably DNA encoding bovine lactate dehydrogenase.

  In the present invention, it is important that the coding DNA is expressed by the promoter in the host chromosome. In particular, when trying to obtain a transformant that originally does not produce an organic acid or increases the production amount from the original organic acid production amount, a method using a YEP type plasmid vector using 2 μm DNA is often used. In this case, however, it has been reported that such coding DNA is difficult to be retained. However, in the present invention, a high expression activity that cannot be considered only due to such a high DNA retention rate has been found. Therefore, according to the present invention, the present promoter is utilized on the chromosome under acidic conditions, and the expression of the coding DNA operably linked to the DNA is activated, so that the coding DNA is stable and has a higher expression than expected. Can be constructed. Furthermore, when the coding DNA encodes a protein having an enzyme activity related to the biosynthesis of organic acid such as lactate dehydrogenase, it is the final product of the coding DNA whose expression is activated by this promoter. Further activation of the coding DNA by this promoter can be expected by the organic acid. That is, synergistic and continuous protein biosynthesis and final product biosynthesis can be promoted.

  In addition, in the case where DNA encoding a protein having lactate dehydrogenase activity is used as the coding DNA, this promoter and the coding DNA are introduced into the PDC1 gene of the host chromosome and destroyed, in addition to the above effects. Lactic acid production can be effectively promoted by the effect of suppressing the expression of pyruvate decarboxylase by gene disruption.

  Transformation in which a plurality of gene transfer fragments are introduced into the chromosome for the purpose of increasing the amount of lactic acid production when DNA encoding lactate dehydrogenase is linked downstream of the promoter of the present invention and introduced into the chromosome You can make a body. In this case, the present promoter can be used in a single type, or two or more of the present promoters can be used in combination. Here, the form in which two or more of the present promoters are used in combination refers to a form in which a coding DNA is bound to each promoter and lactate dehydrogenase is expressed by each promoter. For example, a transformant in which one or more gene transfer fragments prepared by binding a DNA encoding a protein having lactate dehydrogenase activity to each of two or more promoter DNAs of the present promoter is introduced. Can be made. More specifically, the CCW12 gene can be obtained by introducing into a host a gene introduction fragment in which a coding DNA is bound downstream of a TDH1 gene promoter and a gene introduction fragment in which a coding DNA is bound downstream of a TPI1 gene promoter. A transformant expressed by combining expression of a protein having lactate dehydrogenase activity by a promoter and expression of a protein having lactate dehydrogenase activity by an RPS31 gene promoter in the same transformant can be obtained.

  Although not restricting the present invention, the DNA encoding a protein having lactate dehydrogenase activity using a promoter (this promoter or PDC1 promoter) inherently present on the chromosome in the host chromosome as described above. By activating the expression, an unexpected increase in lactic acid production was obtained because of the structural genes that are supposed to be controlled by these promoters at the sites of these promoters on the chromosome. Instead, it is considered that there is a main reason for expressing DNA encoding a protein having lactate dehydrogenase activity. That is, other lactate dehydrogenases are alternatively expressed using these promoters at these promoter sites on the chromosome, or lactate dehydrogenase is substituted by other promoters in place of the promoter and structural gene. It is thought that it is important to make it express. Furthermore, in addition to these causes, regarding the PDC1 promoter, it is considered that the PDC1 promoter is a strong constitutive promoter and also a gene promoter having an autoregulation mechanism, which contributes to an increase in lactic acid production. Moreover, it is considered that this promoter contributes to being an inducible promoter that activates expression in the presence of an organic acid (lactic acid).

(Methods for producing organic acids and other substances)
The substance production method of the present invention comprises the steps of preparing one of these transformants and culturing the transformant in a medium containing an organic acid. By culturing a transformant obtained by introducing the present DNA construct, a protein that is an expression product of a foreign gene can be produced in a medium such as a culture solution. According to such a substance production method, since the expression of the coding DNA is promoted under acidic conditions, the production of useful proteins is promoted by adding the organic acid to the medium, for example, by adding the organic acid to the medium. be able to. When the coding DNA encodes a protein involved in organic acid production, the organic acid can be supplied to the medium by organic production by the transformant so that the organic acid is present in the medium. As a result, if the expression of the coding DNA is promoted and an organic acid is produced, the expression of the coding DNA is further promoted thereby. Even if the coding DNA encodes a protein involved in organic acid production, an organic acid can be added to the medium from the outside.

  In the method of the present invention, in the culture step, the transformant can be cultured without neutralizing the medium. By not neutralizing the medium, the neutralization operation can be omitted. Moreover, when manufacturing organic acids, such as lactic acid, by the method of this invention, the process of desalting the organic acid salt produced by neutralization again to make a free organic acid can be omitted. In such a culturing step, for example, in the case of a method for producing lactic acid as an organic acid, the pH is usually in the range of 1.0 to 3.0 when no neutralizing agent is used. An organic acid or an inorganic acid can be added so that This range is because the pH conditions allow the promoter of the present invention to be highly expressed specifically. Moreover, more preferable pH is 2.0 or more, More preferably, it is 2.5 or more. Moreover, it is preferable that it is 3.0 or less, More preferably, it is 2.8 or less.

  As other culture conditions, aeration conditions such as stationary culture, shaking culture, and aeration-agitation culture can be appropriately selected and performed. The temperature can be about 25 ° C. to 40 ° C., and the culture time can be about 24 to 120 hours. During the culture, antibiotics such as ampicillin and tetracycline can be added to the medium as needed.

  In addition, in the culture step, culture conditions can be selected according to the type of transformant. Such culture conditions are well known to those skilled in the art. In the production of an organic acid such as lactic acid, neutralization of the product lactic acid or the like can be performed as necessary, or treatment such as continuous removal of lactic acid can be performed. As a medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host, the medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms. As long as the medium can be used, either a natural medium or a synthetic medium can be used. As the carbon source, carbohydrates such as glucose, fructose, sucrose, starch and cellulose, and alcohols such as ethanol and propanol can be used. As the nitrogen source, ammonium salt of inorganic acid or organic acid such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, etc. may be used. it can. Examples of inorganic substances that can be used include potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. The organic acid is not necessarily added, but organic acids such as acetic acid, propionic acid, and lactic acid can be added.

  An organic acid can be obtained by carrying out the step of separating the organic acid from the medium. In the present invention, the culture medium includes cultured cells or microbial cells, cells or crushed cells, in addition to the culture supernatant. In this method, when an organic acid such as lactic acid is produced by carrying out the culturing step without using an alkali neutralizing agent, the organic acid is contained in the medium in a free state, so that it can be easily separated. The desalting process and the subsequent recovery process can be omitted.

  In order to separate the gene product from the culture after completion of the culture, ordinary lactic acid purification means can be used. For example, when produced in transformed cells, the cells can be disrupted by ultrasonic disruption treatment, grinding treatment, pressure disruption, etc., and the gene product can be separated from the cells by conventional methods. In this case, protease is added as necessary. When a gene product is produced in the culture supernatant, the solid content of this solution is removed by filtration, centrifugation, or the like.

  For these crudely extracted fractions, lactic acid can be purified using various conventionally known purification separation methods. Moreover, various lactic acid derivatives can be obtained by performing esterification etc. with respect to the said crude extraction fraction and its refined | purified material as needed.

Although the specific example of this invention is described below, it is not the meaning which limits this invention to the following specific example, and can be implemented with a various aspect in the range which does not deviate from the summary of this invention.
(Promoter expression analysis by quantitative PCR)
As a recombinant yeast having the ability to produce lactic acid, a recombinant yeast prepared in JP-A No. 2003-259878 (Japanese Patent Application No. 2002-65879) is used, under non-neutralizing conditions (pH 1.0 to pH 3.0) without using a neutralizing agent. ), Lactic acid fermentation was performed, and the bacterial cells were collected 6 hours and 30 hours after the start of the fermentation, and RNA was prepared therefrom. For RNA adjustment, an RneasyMini kit (Qiagen) was used. The obtained RNA was subjected to cDNA synthesis according to a first strand cDNA synthesis kit (Roche Diagnostics).

  The recombinant yeast used was obtained by culturing a tryptophan synthesis-deficient strain of yeast IFO2260 strain (registered by Issuance Laboratory) in 10 ml YPD medium at 30 ° C. until the logarithmic growth phase, and collecting the cells and washing with TE buffer. Next, 0.5 ml TE buffer and 0.5 mM lithium acetate were added, and after shaking culture at 30 ° C., the pBTrp-PDC1-LDHKCB vector described later was treated with restriction enzymes ApaI and SacI (both manufactured by Takara Shuzo). The obtained colonies are cultured in a tryptophan selective medium and obtained as a strain that has confirmed the stability of tryptophan synthesis and introduction of the DNA fragment at a predetermined chromosome position. can do.

  After mixing the above-mentioned sample, the primer for the target gene described later, and Light Cycler DNA Master SYBR Green I (Roche Diagnostics), the quantitative PCR analyzer Light Cycler (Roche Diagnostics) Expression analysis. Details were performed according to the attached protocol. Moreover, each gene fragment amount 4 fM, 20 fM, and 100 fM was set similarly, and the expression level was measured using this as a control.

  A total of 15 genes obtained by primary screening using a DNA microarray or the like were used as target genes, and their expression was analyzed. In addition, the PDC1 gene was also analyzed as a control as a highly expressed gene. As a result of quantitative PCR, a TDH1 gene promoter, a TPI1 gene promoter, a CCW12 gene promoter, and an RSP31 gene promoter were selected. These genes in lactic acid-producing yeast showed high expression compared to the PDC1 gene. Any of these genes can exhibit an expression level higher than that in the non-recombinant strain. From the above, it was found that the expression of these genes was activated by a promoter that was inducibly expressed under acidic conditions.

  Table 1 shows the primer sequences used and the Tm values when analyzed with a light cycler for the four highly expressed candidate genes that were finally confirmed to be effective. Similarly, the PDC1 gene used as a control also shows the primer sequence and Tm value.

(Acquisition of promoter and determination of nucleotide sequence)
As high expression candidate promoters that showed effective expression in Example 1, the DNA fragments of the promoters of the above four genes were cloned. As a promoter evaluation control, a PDC1 gene promoter known as a high expression promoter was also obtained.

  The gene resource for obtaining the promoter was isolated by a PCR amplification method using the genomic DNA of yeast IFO2260 strain (a strain registered with the Fermentation Research Institute) as a template. This strain was cultured overnight in 2 ml of YPD culture solution, and genomic DNA was prepared using a genomic DNA preparation kit, Gen Toru-kun (trademark) -for yeast (manufactured by Takara Bio Inc.). The prepared genomic DNA was measured for the DNA concentration with a spectrophotometer Ultra spec 3000 (manufactured by Amersham Pharmacia Biotech).

  For the PCR reaction, KOD plus DNA polymerase (manufactured by Toyobo Co., Ltd.), which is said to have high accuracy of the amplified fragment, was used as an amplification enzyme. 50 ng of total reaction of 50 ng of genomic DNA 50 ng of the prepared yeast IFO2260 strain, primer DNA 50 pmol × 2, 10-fold concentrated KOD enzyme reaction buffer 5 μl, 25 mM MgSO4 2 μl, 2 mM dNTPmix 5 μl, KOD plus DNA polymerase 1.0 unit The solution was amplified by a PCR amplification device Gene Amp PCR system 9700 (manufactured by PE Applied Biosystems). The reaction conditions of the PCR amplification apparatus are: heat treatment at 96 ° C. for 2 minutes, one cycle of three temperature changes of 96 ° C. for 30 seconds, 53 ° C. for 30 seconds, and 72 ° C. for 60 seconds. Repeat and finally 4 ° C. 5 μl of this reaction sample was electrophoresed on a 1% TBE agarose gel (containing 0.5 μg / ml ethidium bromide), and the gel band was detected by 254 nm ultraviolet irradiation (manufactured by Funakoshi) to detect gene amplification. Confirmed.

Primer sequences are shown in Table 2 below.

  The obtained PCR fragment was subcloned into pBluescript II SK + vector (manufactured by Toyobo). A series of reaction operations were performed according to a general DNA subcloning method. That is, each promoter fragment treated with the same restriction enzyme was ligated to the above-mentioned vector treated with the restriction enzymes NotI and SpeI (both manufactured by Takara Bio Inc.) with T4 DNA Ligase. For the T4 DNA ligase reaction, LigaFast Rapid DNA Ligation (manufactured by Promega) was used, and the details followed the attached protocol.

  Next, the ligation reaction solution was introduced into E. coli competent cells to transform E. coli. The Escherichia coli competent cells used were JM109 strain (manufactured by Toyobo Co., Ltd.), and detailed handling was in accordance with the attached protocol. Colonies were selected under an LB plate containing 100 μg / ml of the antibiotic ampicillin, plasmid DNA was prepared from each selected colony, and each of the colonies was confirmed by colony PCR using the primer DNAs listed in Table 2. The promoter sequence was subcloned. A detailed manual of a series of operations such as ethanol precipitation and restriction enzyme treatment was in accordance with Molecular Cloning-A Laboratory Manual second edition- (Maniatis et al., Cold Spring Harbor Laboratory Press. 1989).

  Each vector containing the isolated promoter sequence was prepared by an alkaline extraction method, and this was column purified using a GFX DNA Purification kit (Amersham Pharmacia Biotech). Next, the DNA concentration was measured with a spectrophotometer Ultra spec 3000 (manufactured by Amersham Pharmacia Biotech), and the DNA base sequence kit BigDye Terminator Cycle Sequential Ready Reaction Kit (manufactured by PE Applied Bioscience) was used. The reaction sample was set in a base sequence analyzer ABI PRISM 310 Genetic Analyzer (manufactured by PE Applied Biosystem), and the base sequences of six types of promoters were determined. The details of how to use the equipment were in accordance with the manual attached to this device. DNA sequences determined by sequence analysis are shown in SEQ ID NOs: 1-4.

(Construction of recombinant vector)
The newly constructed chromosome-introduced vectors were named pBTRP-TDH1P-LDH vector, pBTRP-TPI1P-LDH vector, pBTRP-CCW12P-LDH vector, and pBTRP-RSP31P-LDH vector, respectively. As a promoter comparison control, a vector capable of expressing the L-LDH gene under the PDC1 gene promoter was also constructed and named pBTRP-PDC1P-LDH vector. Details of the vector construction process in this example will be described below with reference to FIG. 1, but the vector construction procedure is not limited thereto. In addition, a series of reaction operations in the vector construction was performed according to a general DNA subcloning method, and a series of enzymes were manufactured by Takara Bio.

(Pre-vector construction)
The prevector pBTRP-LDH was constructed according to a general DNA subcloning method. PBluescript II SK + vector in which a fragment obtained by treating the pBTrp-PDC1-LDHKCB vector (described in JP-A-2003-259878) already constructed by the present inventors with the restriction enzymes ApaI and PstI is treated with the same restriction enzymes. (Toyobo Co., Ltd.) and constructed pBTRP-PDC1D vector. Subsequently, the TRX1 fragment (700 bp) obtained by PCR amplification and treated with restriction enzymes SacI and NotI and the LDHKCB fragment (2000 bp) obtained by PCR amplification and treated with restriction enzymes SpeI and BamHI are sequentially ligated to this vector. The prevector pBTRP-LDH was constructed.

  The pBTrp-PDC1P-LDHKCB vector is constructed by the following method. That is, it encodes the amino acid sequence of bovine-derived L-lactate dehydrogenase in order to efficiently produce L-lactate dehydrogenase, which is a protein derived from bovine, a higher eukaryote, in the genus Saccharomyces cerevisiae. A novel gene sequence (SEQ ID NO: 27) that is not naturally occurring was designed using the following items as design guidelines for DNA, and the method of Fujimoto et al. (Fujimoto Hideya, The DNA was synthesized using a synthetic gene production method, PCR experiment protocol for plant cell engineering series 7 plants, 1997, Shujunsha, p95-100) (the synthesized DNA was referred to as LDHKCB sequence). A yeast chromosome introduction type vector pBTRP-PDC1-LDHKCB was constructed using the fully synthesized LDHKCB sequence. The LDHKCB sequence was enzymatically treated with EcoRI, and similarly, ligated to pCR2.1TOPOVector (Invitrogen) enzymatically treated with EcoRI by a conventional method and called a pBTOPO-LDHKCB vector.

1. The PDC1P fragment for constructing pBTrp-PDC1-LDHKCB was isolated by PCR amplification using the genomic DNA of Saccharomyces cerevisiae YPH strain (Stratagene) as a template. Genomic DNA of Saccharomyces cerevisiae YPH strain was prepared using Fast DNA Kit (Bio 101), which is a genome preparation kit, according to the attached protocol for details. The DNA concentration was measured with a spectrophotometer Ultra spec 3000 (Amersham Pharmacia Biotech). For the PCR reaction, Pyrobest DNA Polymerase (Takara Shuzo), which is said to have high accuracy of the amplified fragment, was used as an amplification enzyme. Saccharomyces cerevisiae YPH strain genomic DNA 50 ng / sample, primer DNA 50 pmol / sample, and Pyrobest DNA polymerase 0.2 unit / sample prepared in the above manner were prepared in a total of 50 μl of reaction system. The reaction solution was subjected to DNA amplification with a PCR amplifier Gene Amp PCR system 9700 (PE Applied Biosystems). The reaction conditions of the PCR amplification apparatus were as follows: heat treatment at 96 ° C. for 2 minutes, 25 cycles of 96 ° C. for 30 seconds, 53 ° C. for 30 seconds, and 72 ° C. for 60 seconds, and then 4 ° C. It was. The amplified fragment of the PDCl primer was confirmed by 1% TBE agarose gel electrophoresis. The primer DNA used in the reaction was synthetic DNA (Sawaday Technology), and the DNA sequence of this primer was as follows.
・ PDCIP-LDH-U (31 mer, Tm value 58.3 ° C.) Add restriction enzyme BamH1 site to the end: ata tat ggatcc gcg tttatt tac ctatctc (SEQ ID NO: 28)
PDClP-LDH-D (31 mer, Tm value 54.4 ° C.) Add restriction enzyme EcoRI site to the end: atatgaga tctttgattg atttgactgt g (SEQ ID NO: 29)

2. The PDCl gene promoter fragment (PDCIP) 971 bp and the PDCl gene downstream region fragment (PDC1D) 518 bp are isolated by PCR amplification using the genomic DNA of Saccharomyces cerevisiae YPH as a template as described above. Went. The procedure for PCR amplification was as described above, and the following primers were used for amplification of the PDCl gene downstream region fragment.
・ PDC1D-LDH-U (34mer, Tm value 55.3 ° C.) Add restriction enzyme XhoI site at the end: atat ct ctc gag gcc agctaa ctt cttggtcga c (SEQ ID NO: 30)
PDCID-LDH-D (31 mer, Tm value 54.4 ° C.): Add restriction enzyme ApaI site at the end: ata tat gaa ttc tttgat tga ttt gac tgtg (SEQ ID NO: 31)

3. Each of the PDC1P and PDC1D gene amplified fragments obtained in the above reaction was purified by ethanol precipitation, and then the PDC1P amplified fragment was subjected to a restriction enzyme reaction treatment with restriction enzymes BamHI / EcoRI and PDC1D amplified fragment with restriction enzymes XhoI / ApaI. went. The enzymes used below were all manufactured by Takara Shuzo. Further, a detailed manual of a series of procedures for ethanol precipitation treatment and restriction enzyme treatment was according to Molecular Cloning A Laboratory Manual second edition (Maniatis et al., Cold Spring Harbor Laboratory Press. 1989). A pBluescript II SK + vector (Toyobo Co., Ltd.) subjected to restriction enzyme treatment with BamHI / EcoRI (Takara Shuzo Co., Ltd.) and dephosphorylating enzyme Alkaline Phosphatase (BAP, Takara Shuzo Co., Ltd.) Ligation was performed by T4 DNA Ligase reaction. For the T4 DNA ligase reaction, LigaFast Rapid DNA Ligation System (Promega) was used, and the details followed the attached protocol.

4). Next, transformation into competent cells was performed using the solution subjected to the ligation reaction. E. coli JM109 strain (Toyobo Co., Ltd.) was used as the competent cell, and details were performed according to the attached protocol. The obtained culture broth was spread on an LB plate containing 100 μg / ml of antibiotic ampicillin and cultured overnight. The grown colonies were confirmed by colony PCR using the insert fragment primer DNA and by restriction enzyme treatment of the plasmid DNA preparation solution by miniprep, and the vector pBPDC1P was isolated.

5. Subsequently, the LDHKCB gene fragment obtained by subjecting the previously constructed pBTOPO-LDHKCB vector to the restriction enzyme EcoRI treatment and the end modification enzyme T4 DNA polymerase treatment, the pBPDC1P vector also subjected to the restriction enzyme EcoRI treatment and the end modification enzyme T4DNA polymerase treatment. Inside, subcloning was performed in the same manner as described above to prepare a pBPDC1P-LDHKCB vector.

6). On the other hand, a fragment of the LDH gene (derived from Bifidobacterium longum) obtained by treating the already constructed pYLD1 vector with the restriction enzyme EcoRI / AatII treatment and the end modification enzyme T4 DNA polymerase, was also treated with the restriction enzyme EcoRI treatment and the end modification enzyme T4DNA. Subcloning was performed in the same manner as described above in the pBPDC1P vector that had been subjected to the polymerase treatment, to prepare a pBPDC1P-LDH1 vector. The above-mentioned pYLDl vector was introduced into Escherichia coli (name: “E.coll pYLD1”). -7423 is deposited internationally based on the Budabest Convention (original deposit date: October 26, 1999).

7). Subsequently, this vector was treated with XhoI / ApaI, and an amplified PDC1D fragment similarly treated with a restriction enzyme was ligated to prepare a pBPDC1P-LDH vector. Finally, the pBT4041P-LDHII vector treated with EcoRV was ligated with the Trp marker fragment obtained by treating the pRS404 vector (Stratagene) with AatII / SspI and T4 DNA polymerase to construct a pBTrp-PDCl-LDH vector.

8). Next, the pBPDC1P-LDHKCB vector was treated with a restriction enzyme with ApaI / EcoRI, while the pBTrp-PDC1-LDH vector was treated with a fragment containing a Trp marker treated with the restriction enzymes ApaI and StuI, and the amplified fragment was treated. Ligation was performed to construct a chromosome-introduced pBTrp-PDC1-LDHKCB vector as the final construct.

(Construction and confirmation of final vector)
The prevector pBTRP-LDH was treated with restriction enzymes NotI and SpeI, and the promoter sequence obtained in Example 3 was treated with the same restriction enzyme, and the final vectors were ligated to each other. Detailed maps of the pBTRP-TDH1P-LDH vector, the pBTRP-TPI1P-LDH vector, the pBTRP-CCW12P-LDH vector, the pBTRP-RSP31P-LDH vector, and the pBTRP-PDC1P-LDH vector prepared as a comparative control are shown in FIG. To FIG.

  The above-described series of DNA ligation reactions used LigaFast Rapid DNA Ligation (manufactured by Promega), and details followed the attached protocol. In addition, Escherichia coli JM109 strain (manufactured by Toyobo Co., Ltd.) was used for transformation of the Ligation reaction solution into competent cells. In either case, colony selection was performed under an LB plate containing 100 μg / ml of the antibiotic ampicillin, and colony PCR using each colony was performed to confirm whether it was the target vector. The detailed manual of the series of operations such as ethanol precipitation and restriction enzyme treatment was in accordance with Molecular Cloning “A Laboratory Manual second edition” (Maniatis et al., Cold Spring Harbor Press. 1989).

(Production of transformed yeast)
A strain lacking the ability to synthesize tryptophan of yeast IFO2260 strain (a strain registered in the Institute for Fermentation) is hosted in 10 ml of YPD culture solution at 30 ° C. until the logarithmic growth phase (OD600 nm = 0.8). Cultured. A competent cell was prepared using a Frozen-EZ Yeast Transformation II kit (manufactured by ZYMO RESEARCH). According to the protocol attached to the kit, this competent cell was treated with the restriction enzyme PvuII and the gene introduced into the chromosomal transfer vector constructed in Example 4 described above. Specific introduction vectors include pBTRP-TDH1P-LDH vector, pBTRP-TPI1P-LDH vector, pBTRP-CCW12P-LDH vector, pBTRP-RSP31P-LDH vector, and pBTRP-PDC1P-LDH vector prepared as a comparative control. It is a kind. These transformed samples were washed, dissolved in 100 μl of sterilized water, smeared on a tryptophan selection medium, and each was selected for transformants under static culture at 30 ° C.

  Each of the obtained colonies was again isolated with a new tryptophan selection medium, and a strain that stably maintained the growth ability was used as a transformation candidate strain. Next, these candidate strains were cultured overnight in 2 ml of YPD culture solution, and genomic DNA was prepared using a genomic DNA preparation kit, Gen Toru-kun (trademark) -for yeast (manufactured by Takara Bio Inc.). PCR analysis was carried out using each adjusted genomic DNA as a template, and a transformant was confirmed if the presence or absence of the transgene was confirmed.

(Verification of each promoter by fermentation test)
By measuring the amount of L-lactic acid produced in the produced transformant and comparing it with the amount of lactic acid produced using the PDC1 promoter known as a high expression promoter, the activity of the four types of promoters obtained under non-neutralizing conditions Verified. The obtained four kinds of transformed yeast were inoculated into 5 ml of YPD liquid medium, and cultured overnight at 30 ° C. and 130 rpm with shaking, and those having an OD of 600 nm = 1.2 were used as the initial cells. 2 ml of this was inoculated into 20 ml of YPD culture solution containing 10% glucose and 20 ml of cane juice culture solution containing 10% sucrose (total amount: 22 ml), and calcium carbonate (manufactured by Nacalai Tesque, Inc.) as a neutralizing agent ) 1 g added was statically cultured at 30 ° C. for 4 days. For the measurement of the amount of lactic acid produced, a multifunctional biosensor BF-4 apparatus (manufactured by Oji Scientific Instruments) was used, and the details of the specifications were in accordance with the attached manual. These results are shown in FIG.

  As shown in FIG. 6, for the TDH1 gene promoter, the TPI1 gene promoter, the RSP31 gene promoter, and the CCW12 gene promoter, the expression intensity of the control PDC1 promoter or higher was confirmed. In particular, a high expression intensity of 114% (105 to 117%) on average for the TDH1 gene promoter and 117% (113 to 126%) on average for the TPI1 gene promoter was confirmed.

  Each of these promoters has a activity equivalent to or higher than the PDC1 promoter, which is a strong constitutive promoter, and has a high transcription activity under acidic conditions. Therefore, an organic acid such as lactic acid is contained in the medium. It was found to be a high expression promoter suitable for organic acid production that can maintain high transcriptional activity even when accumulated. In addition, since these promoters all disrupt the PDC1 gene of the host yeast and express the LDH gene at the locus, an organic acid such as lactic acid can be used in a state where the expression of the gene in the yeast is suppressed. It was found to be a suitable promoter for production.

SEQ ID NO: 7 to 26 Synthetic primer SEQ ID NO: 27 DNA encoding modified bovine-derived L-lactate dehydrogenase
Sequence number: 28-31 Synthetic primer

It is a figure which shows a chromosome introduction | transduction type | mold vector construction process. It is a figure which shows the map of pBTRP-TDH1P-LDH vector. It is a figure which shows the map of pBTRP-TPI1P-LDH vector. It is a figure which shows the map of pBTRP-CCW12P-LDH vector. It is a figure which shows the map of pBTRP-RSP31P-LDH vector. It is a graph which shows the result (YPD culture solution) of the lactic acid fermentation test in various transformed yeast strains.

Claims (24)

A promoter for use under acidic conditions, which is a polynucleotide selected from the following sequences (a) to (c) and has promoter activity for organic acid production under acidic conditions.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1 (b) hybridizing under stringent conditions with a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1 or a base sequence complementary thereto, A polynucleotide having a homology of 90% or more with the base sequence described in SEQ ID NO: 1 (c) a base in which one or more bases are deleted, substituted and / or added in the base sequence described in SEQ ID NO: 1 A polynucleotide comprising the sequence and having a homology of 90% or more with the base sequence set forth in SEQ ID NO: 1
The promoter according to claim 1, which is a promoter for organic acid production in a microorganism. The promoter according to claim 2 , which is a promoter for organic acid production by yeast. The promoter according to claim 2 or 3 , wherein the promoter is an organic acid producing promoter that produces an organic acid under non-neutralizing conditions. The promoter according to any one of claims 2 to 4 , which is a promoter for producing an organic acid that produces an organic acid at a pH of 1.0 to 3.0. The promoter according to any one of claims 2 to 5 , which is a promoter for producing an organic acid for producing an organic acid derived from pyruvic acid in a state where expression of a pyruvate decarboxylase gene in a host is suppressed. The promoter according to any one of claims 1 to 6 , which is an acidic condition-inducible promoter. The promoter according to any one of claims 1 to 7 , wherein the acidic condition is a lactic acid acidic condition. Comprising a DNA encoding a protein involved in operably linked organic acid production promoter according to any one of claims. 1 to 8, DNA construct. The DNA construct according to claim 9 , wherein the protein is a protein having lactate dehydrogenase activity. The DNA construct according to claim 10 , wherein the protein is bovine-derived lactate dehydrogenase. The DNA construct according to any one of claims 9 to 11 , further comprising DNA for homologous recombination for gene disruption of a pyruvate decarboxylase gene having an autoregulation mechanism. A transformant carrying the DNA construct according to claim 9 . The transformant according to claim 13 , wherein the DNA encoding the promoter and the protein is integrated on a host chromosome. The transformant according to claim 13 or 14 , which is a yeast. The transformant according to claim 15 , wherein the yeast belongs to the genus Saccharomyces. The transformant according to claim 15 or 16 , wherein expression of a pyruvate decarboxylase gene having an autoregulation mechanism on a host chromosome is suppressed. The transformant according to any one of claims 13 to 17 , wherein the protein has lactate dehydrogenase activity. Preparing a transformant according to any of claims 13 to 17 ,
Culturing the transformant in a medium containing an organic acid;
An organic acid production method comprising: 20. The method for producing a substance according to claim 19 , wherein the culturing step is a step including supplying an organic acid to the medium by organic acid production by the host cell. The method according to claim 19 or 20 , wherein the culturing step includes adding an organic acid to the medium. The protein is a protein having lactate dehydrogenase activity,
The method according to any one of claims 19 to 21 , wherein the culturing step is a step of producing lactic acid. The method according to any one of claims 19 to 22 , wherein the culturing step is a step of culturing the transformant without neutralizing a medium. 24. The method according to any one of claims 19 to 23 , wherein the culturing step is a step of culturing the transformant in a medium having a pH of 1.0 to 3.0.

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