JP6671923B2 - Method for producing C4 dicarboxylic acid - Google Patents

Description

  The present invention relates to biological C4 dicarboxylic acid production.

  C4 dicarboxylic acid is a substance of high industrial value, such as being used for various purposes in the food industry as an acidulant, antibacterial agent and pH adjuster, and also used as a raw material for synthetic resins and biodegradable polymers. is there. C4 dicarboxylic acids are industrially produced either by chemical synthesis from petrified raw materials or by microbial fermentation. Previously, the chemical synthesis method was the mainstream because of its lower cost. However, in recent years, from the viewpoint of soaring raw materials and environmental burden, a production method by microbial fermentation using circulating recycled resources as raw materials has attracted attention.

  It is known that fumaric acid, which is one of the C4 dicarboxylic acids, can be produced by using a fermentative bacterium such as Rhizopus. Rhizopus bacteria produce fumaric acid using glucose as a carbon source and excrete them outside the cells. Heretofore, as for techniques for increasing the production of fumaric acid by Rhizopus genus, improvement of culture methods, production of high-producing strains by mutation breeding, and the like have been known. However, since the genetic background of Rhizopus has not been sufficiently studied, it has not been easy to develop technology for increasing the production of fumaric acid in Rhizopus by genetic recombination, and there are few reports. Slightly introducing a gene encoding pyruvate carboxylase derived from Saccharomyces cerevisiae into Rhizopus delemer (Patent Document 1), and introducing a gene encoding phosphoenolpyruvate carboxylase derived from Escherichia coli into Rhizopus oryzae. (Non-Patent Document 1) reports an improvement in fumaric acid productivity.

Chinese Patent Publication No. 103013843

Metabolic Engineering, 2012, 14: 512-520

  The present invention relates to a polypeptide having an effect of improving the ability to produce C4 dicarboxylic acid for a host cell, a gene encoding the polypeptide, a transformed cell containing the gene, and a method for producing C4 dicarboxylic acid using the transformed cell. About.

  As a result of intensive studies, the present inventors have found that cells in which the expression of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 has been enhanced improve its C4 dicarboxylic acid-producing ability.

  Accordingly, the present invention provides a transformed cell comprising a foreign polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or a polypeptide consisting of an amino acid sequence having at least 80% identity to the sequence.

  The present invention also provides a method for producing C4 dicarboxylic acid, which comprises culturing the above transformed cell.

  Furthermore, the present invention relates to a method for producing a transformed cell, comprising a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 80% identity to the sequence, A method comprising introducing the containing vector into a host cell is provided.

  Furthermore, the present invention relates to a method for improving the ability of a host cell to produce C4 dicarboxylic acid, which comprises a polypeptide encoding an amino acid sequence represented by SEQ ID NO: 2 or a polypeptide comprising an amino acid sequence having at least 80% identity to the sequence. A method comprising introducing a nucleotide, or a vector containing it, into a host cell is provided.

  The present invention provides a polypeptide having a function of enhancing the ability of a cell to produce C4 dicarboxylic acid, and a transformed cell in which expression of the polypeptide has been enhanced (for example, a gene encoding the polypeptide has been introduced). The transformed cells of the present invention can produce more C4 dicarboxylic acids. Therefore, the transformed cells of the present invention are useful for biological production of C4 dicarboxylic acid. The above and other features and advantages of the present invention will become more apparent from the description herein below.

(1. Definition)
In the present specification, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, the genetic information processing software GENETYCS Ver. It is calculated by performing analysis using Machos, -1 for Mismatches, 1 for Gaps, and * N + 2 using the amino acid sequence x amino acid sequence maximum matching or the nucleotide sequence x nucleotide sequence maximum matching of 12 homology analysis programs.

  As used herein, “at least 80% identity” with respect to an amino acid sequence or a nucleotide sequence refers to 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably Refers to identity of 96% or more, more preferably 97% or more, still more preferably 98% or more, and even more preferably 99% or more.

  In the present specification, the “corresponding region” on an amino acid sequence or a nucleotide sequence is an alignment (alignment) between a target sequence and a reference sequence (eg, the amino acid sequence represented by SEQ ID NO: 2) so as to give maximum homology. ) Can be determined. Alignment of amino acid or nucleotide sequences can be performed using known algorithms, the procedures of which are known to those skilled in the art. For example, the alignment can be performed manually based on the above-described Lipman-Pearson method or the like, but the Clustal W multiple alignment program (Thompson, JD et al, 1994, Nucleic Acids Res. 22: 4673-4680). ) With default settings. Clustal W is available, for example, from the European Bioinformatics Institute (European Bioinformatics Institute: EBI [www.ebi.ac.uk/index.html]) and the Japan National DNA Institute's DNA Data Bank (DDBJ [www. ddbj.nig.ac.jp/Welcome-j.html]). A region of the target sequence aligned corresponding to an arbitrary region of the reference sequence by the above-mentioned alignment is regarded as a “corresponding region” to the arbitrary region.

  As used herein, the term "amino acid sequence in which one or more amino acids have been deleted, substituted, added, or inserted" refers to one or more and ten or less, preferably one or more and eight or less, and more preferably one or more. This refers to an amino acid sequence in which at least 5 or less, more preferably 1 to 3 amino acids have been deleted, substituted, added, or inserted. The term “nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted” in the present specification refers to 1 to 30 nucleotides, preferably 1 to 24 nucleotides, more preferably 1 to 24 nucleotides. The nucleotide sequence refers to a nucleotide sequence in which at least 15 nucleotides, even more preferably at least 1 and 9 nucleotides have been deleted, substituted, added, or inserted. As used herein, the term "addition" of an amino acid or nucleotide includes addition of an amino acid or nucleotide to one end and both ends of the sequence.

  As used herein, “upstream” and “downstream” with respect to a gene refer to upstream and downstream in the transcription direction of the gene. For example, “a gene located downstream of the promoter” means that the gene is present 3 ′ to the promoter in the DNA sense strand, and “upstream of the gene” is 5 ′ to the gene in the DNA sense strand. Area.

  As used herein, the term "operably linked" between a regulatory region and a gene means that the gene and the regulatory region are linked so that the gene can be expressed under the control of the regulatory region. . Procedures for "operably linking" a gene and a control region are well known to those skilled in the art.

  In the present specification, the term “original” used for a function, property, or trait of a cell is used to indicate that the function, property, or trait exists in a wild type of the cell. In contrast, the term “foreign” is used to describe a function, property, or trait that is not originally present in the cell but is introduced from outside. For example, a “foreign” gene or polynucleotide is a gene or polynucleotide that has been introduced into a cell from outside. The foreign gene or polynucleotide may be from the same organism as the cell into which it has been introduced, or may be from a different organism (ie, a heterologous gene or polynucleotide).

  As used herein, the “C4 dicarboxylic acid-producing ability” of a cell is expressed as the yield (%) of C4 dicarboxylic acid in the culture medium of the cell, and more specifically, the consumption of the carbon source in the medium of the cell. And expressed as mass (%) of the amount of C4 dicarboxylic acid produced by the cells. The amount of C4 dicarboxylic acid produced by the cells can be calculated as the amount of C4 dicarboxylic acid in the culture supernatant obtained by removing the cells from the cell culture. The consumption of the carbon source in the medium can be calculated by subtracting the amount of the carbon source in the culture supernatant from the initial concentration of the carbon source in the medium. The amounts of C4 dicarboxylic acid and carbon source in the culture supernatant can be measured by high performance liquid chromatography (HPLC) or the like. A more specific measurement procedure is exemplified in Reference Example 1 described later.

As used herein, “improved C4 dicarboxylic acid-producing ability” in a transformed cell means that the transformed cell has an improved C4 dicarboxylic acid-producing ability as compared to a host cell or a control cell. The improvement rate of the C4 dicarboxylic acid producing ability in the transformed cells is calculated by the following formula.
Improvement rate (%)
= (C4 dicarboxylic acid-producing ability of transformed cells / C4 dicarboxylic acid-producing ability of host cells or control cells) × 100-100
Here, a transformed cell is a cell in which the expression of the polypeptide of the present invention is enhanced, for example, a cell into which a polynucleotide encoding the polypeptide of the present invention or a vector containing the same has been introduced, and a host cell is Refers to the host cell (parent cell) of the transformed cell. The control cell refers to a cell into which a vector that does not contain the polynucleotide encoding the polypeptide of the present invention has been introduced. Preferably, the improvement rate of the C4 dicarboxylic acid-producing ability is determined by the C4 dicarboxylic acid-producing ability of each cell at the time when the C4 dicarboxylic acid concentration in the culture supernatant of the transformed cells (not including the cells) is maximized. Calculated based on Therefore, as used herein, the term "transformed cell having an improved C4 dicarboxylic acid-producing ability of X% or more" refers to a transformed cell having a C4 dicarboxylic acid-producing ability improved by X% or more calculated by the above formula. In addition, "improvement of C4 dicarboxylic acid-producing ability by X% or more" in a cell means that the rate of improvement of C4 dicarboxylic acid-producing ability of the cell calculated by the above formula is X% or more.

  Examples of C4 dicarboxylic acids produced according to the present invention include fumaric acid, malic acid, and succinic acid, preferably fumaric acid.

  As used herein, the term “multiple transmembrane polypeptide” refers to a transmembrane helix structure predicted to have a plurality of transmembrane helix structures by analysis using a cell transmembrane region prediction program. Of the polypeptide. Examples of the transmembrane region prediction program include TMHMM Server, v. 2.0 (Journal of Molecular Biology, 2001, 305: 567-580), DAS-TMfilter (Protein Eng., 2002, Volume 15, Issue 9: 745-752), PRED-TMR2 (Protein Eng., 1999, Vol. : 631-634) and the like.

(2. Transformed cells with improved ability to produce C4 dicarboxylic acid)
The present inventors have found that a cell into which a polynucleotide encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 has been introduced, has improved C4 dicarboxylic acid-producing ability. Therefore, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a polypeptide having a function equivalent thereto (hereinafter, these may be collectively referred to as “polypeptide of the present invention”) , C4 dicarboxylic acid producing ability.

  Therefore, in one embodiment, the present invention provides a transformed cell having an improved ability to produce C4 dicarboxylic acid. The transformed cell of the present invention is a cell containing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a foreign polynucleotide encoding a polypeptide having a function equivalent thereto.

  The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is a protein of unknown function (accession number: RO3G — 06858) derived from Rhizopus delema RA 99-880 strain. TMHMM Server, v. A transmembrane region prediction program. As a result of analysis using 2.0, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 was found to have a region from position 13 to position 32, a region from position 42 to position 64, a region from position 71 to position 93, 97 Regions 119 to 153, 163 to 182, 189 to 206, and 212 to 234 each have a transmembrane helix structure. It was predicted to be a transmembrane polypeptide. Therefore, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is assumed to be a multiple transmembrane polypeptide. Also, generally, considering that a transporter polypeptide having an activity of transporting a substance into and out of a cell membrane has a multiple transmembrane structure, it is presumed that the polypeptide is a transporter-like polypeptide. You.

  The polypeptide having a function equivalent to that of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 includes a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO: 2. In a preferred embodiment, the polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence represented by SEQ ID NO: 2, and has a multiple transmembrane helix structure. It is. In a more preferred embodiment, the polypeptide consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 2, and a region from position 13 to position 32 of the amino acid sequence shown in SEQ ID NO: 2, 42-64 region, 71-93 region, 97-119 region, 131-153 region, 163-182 region, 189-206 region, and 212-234 region It is a multiple transmembrane polypeptide having a transmembrane helix structure in the corresponding regions.

  Examples of the amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO: 2 include deletion, substitution, addition, or deletion of one or more amino acids from the amino acid sequence represented by SEQ ID NO: 2. Inserted amino acid sequence.

Methods for introducing mutations such as amino acid deletion, substitution, addition, or insertion into the amino acid sequence include, for example, deletion, substitution, addition, or insertion of nucleotides into the nucleotide sequence encoding the amino acid sequence. And the like. As a method for introducing a mutation into a nucleotide sequence, for example, a chemical mutagen such as ethyl methanesulfonate, N-methyl-N-nitrosoguanidine, nitrous acid or a physical mutagen such as ultraviolet ray, X-ray, gamma ray, ion beam, etc. Mutagenesis, site-directed mutagenesis, and the method described in Dieffenbach et al. (Cold Spring Harbor Laboratory Press, New York, 581-621, 1995). Methods for site-directed mutagenesis include a method using Splitting overwrap extension (SOE) PCR (Horton et al., Gene 77, 61-68, 1989) and an ODA method (Hashimoto-Gotoh et al., Gene, 152). , 271-276, 1995) and the Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Alternatively, a Site-Directed Mutagenesis System Mutan-SuperExpress Km kit (Takara Bio Inc.), a Transformer ^™ Site-Directed Mutagenesis kit (Clonetech Inc.), and a KOD-Pusite-specific mutagenesis-based mutagenesis of Transformer ^™ Site-Directed Mutagenesis kit Kits can also be used.

  The foreign polynucleotide encoding the polypeptide of the present invention contained in the transformed cell of the present invention includes a nucleotide sequence represented by SEQ ID NO: 1 or a polynucleotide consisting of a nucleotide sequence having at least 80% identity to the sequence. Is mentioned. In a preferred embodiment, the polynucleotide has a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, or an eight transmembrane helix structure consisting of an amino acid sequence having at least 80% identity to the sequence. Encodes a multiple transmembrane polypeptide. In a more preferred embodiment, the polynucleotide comprises a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80% identity to the sequence, and the amino acid sequence shown in SEQ ID NO: 2. Regions 13 to 32, regions 42 to 64, regions 71 to 93, regions 97 to 119, regions 131 to 153, regions 163 to 182, regions 189 to 206, And a multiple transmembrane polypeptide having a transmembrane helix structure in regions corresponding to the regions 212 to 234.

  Examples of nucleotide sequences having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 1 include deletion, substitution, addition, or deletion of one or more nucleotides from the nucleotide sequence shown in SEQ ID NO: 1. Inserted nucleotide sequence. The method for introducing a mutation such as deletion, substitution, addition or insertion of a nucleotide into a nucleotide sequence is as described above. The polynucleotide contained in the transformed cell of the present invention may be in a single-stranded or double-stranded form, or may be DNA or RNA. The DNA may be an artificial DNA such as a cDNA or a chemically synthesized DNA.

  In the transformed cell of the present invention, the polynucleotide encoding the polypeptide of the present invention may be incorporated into a vector. Preferably, the vector containing the polynucleotide is an expression vector. Also preferably, the vector is an expression vector capable of introducing the polynucleotide into a host cell and expressing the polynucleotide in the host cell. Preferably, the vector comprises a polynucleotide encoding the polypeptide of the present invention and a control region operably linked thereto. The vector may be a vector such as a plasmid capable of autonomous propagation and replication outside a chromosome, or may be a vector integrated into a chromosome.

  Examples of specific vectors include, for example, pBluescript II SK (-) (Stratagene), pUC-based vectors such as pUC18 (Takara Bio), pET-based vectors (Takara Bio), pGEX-based vectors (GE Healthcare), pCold System vector (Takara Bio), pHY300PLK (Takara Bio), pUB110 (Mckenzie, T. et al., 1986, Plasmid 15 (2): 93-103), pBR322 (Takara Bio), pRS403 (Stratagene), pMW218 / 219 (Nippon Gene), pRI vectors (Takara Bio), pBI vectors (Clontech), IN3 vectors (Implanta Innovations) and the like.

  Alternatively, in the transformed cell of the present invention, the polynucleotide encoding the polypeptide of the present invention may be DNA integrated into genomic DNA. In this case, a DNA fragment containing the polynucleotide encoding the polypeptide of the present invention may be constructed and introduced into a host cell. Examples of the DNA fragment include a PCR amplified DNA fragment and a restriction enzyme digested DNA fragment. Preferably, the DNA fragment may be an expression cassette containing a polynucleotide encoding the polypeptide of the present invention and a control region operably linked thereto.

  The control region contained in the vector or the DNA fragment is a sequence for expressing a polynucleotide encoding the polypeptide of the present invention in a host cell into which the vector or the DNA fragment has been introduced, such as a promoter or a terminator. Examples include an expression control region and a replication origin. The type of the control region can be appropriately selected according to the type of the host cell into which the vector or the DNA fragment is introduced. If necessary, the vector or the DNA fragment may further have a selection marker such as an antibiotic resistance gene and a gene related to amino acid synthesis.

  The transformed cell of the present invention can be obtained by introducing a vector or a DNA fragment containing the polynucleotide encoding the polypeptide of the present invention into a host cell. In a transformed cell containing a vector or a foreign DNA fragment containing the polynucleotide encoding the polypeptide of the present invention, the gene encoding the polypeptide of the present invention is compared with its host cell (parent cell). The transfer amount has been improved. The amount of gene transcription can be measured by measuring the amount of mRNA by quantitative PCR, RNA-Seq analysis using a next-generation sequencer, DNA microarray analysis, or the like.

  Any of microbial cells, plant cells, and animal cells may be used as host cells for the transformed cells. From the viewpoint of the production efficiency of C4 dicarboxylic acid, the host cell is preferably a microbial cell. The microorganism may be either prokaryotic or eukaryotic. Among these, from the viewpoint of C4 dicarboxylic acid productivity, the microorganism is preferably a filamentous fungus or yeast, and more preferably a filamentous fungus. Examples of the filamentous fungi include all filamentous fungi belonging to the subdivision fungi (Eumycota) and the oomycetes (Oomycota) (Hawksworth et al., In, Ainsworth and Bisby's Dictionary of the Fungi, 8th Edition). , 1995, CAB International, bUniversity, Press, Cambridge, UK). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is by hyphal expansion and carbon metabolism is obligately aerobic.

  Preferred examples of the filamentous fungus used as the host cell of the transformed cell of the present invention include the genus Acremonium, the genus Aspergillus, the genus Aureobasidium, the genus Bjerkandera, the genus Ceriporiopsis, the genus Chrysosporium, the genus Copriolus, the genus Copriolus, and the genus Coriolius. Genus, Fusarium genus, Humicola genus, Magnaporthe genus, Mucor genus, Myceliophthora genus, Neocallimastix genus, Neurospora genus, Paecilomyces genus, Parasitella genus, Peacilium genus, Petaphilium, Phenicium genus us spp, Rhizopus spp, Schizophyllum sp, Talaromyces sp, Thermoascus sp., Thielavia genus Tolypocladium sp, Trametes sp, and include filamentous fungus Trichoderma sp. Of these, from the viewpoint of productivity of C4 dicarboxylic acids, Rhizopus delemar, Rhizopus arrhizus, Rhizopus chinensis, Rhizopus nigricans, Rhizopus tonkinensis, Rhizopus tritici, preferably Rhizopus genus, such as Rhizopus oryzae, Rhizopus delemar and Rhizopus oryzae more preferably , Rhizopus delemar are more preferred.

  For the introduction of the vector or DNA fragment into the above host cells, general transformation methods such as electroporation, transformation, transfection, conjugation, protoplast, particle gun, and Agrobacterium are used. Etc. can be used.

  Transformed cells into which the desired vector or DNA fragment has been introduced can be selected using a selection marker. For example, when the selectable marker is an antibiotic resistance gene, transformed cells into which the target vector or DNA fragment has been introduced can be selected by culturing the cells in the antibiotic-added medium. Further, for example, when the selectable marker is an amino acid synthesis-related gene, a transformed cell into which a target vector or a DNA fragment has been introduced using the presence or absence of the amino acid requirement as an index after introducing the gene into the host cell requiring the amino acid. Can be selected. Alternatively, the introduction of the target vector or DNA fragment can be confirmed by examining the DNA sequence of the transformed cell by PCR or the like.

  The above-mentioned transformed cell of the present invention has an improved transcription amount of a polynucleotide encoding the above-mentioned polypeptide of the present invention, and has enhanced expression of the polypeptide of the present invention. Thereby, the transformed cell has improved C4 dicarboxylic acid-producing ability. The transformed cells have improved C4 dicarboxylic acid-producing ability by preferably 10% or more, more preferably 15% or more, and still more preferably 20% or more, as compared with the host cells (parent cells).

(3. Production of C4 dicarboxylic acid)
The transformed cell of the present invention has an improved ability to produce C4 dicarboxylic acid. Therefore, the present invention also provides a method for producing C4 dicarboxylic acid, comprising culturing the above-described transformed cell of the present invention. The C4 dicarboxylic acid produced by the production method of the present invention includes fumaric acid, malic acid, and succinic acid, and is preferably fumaric acid.

  The culturing of the transformed cell in the production method of the present invention includes culturing a microorganism, a plant, an animal, or a cell or tissue containing the transformed cell. The medium and culture conditions for culturing the transformed cells can be appropriately selected depending on the type of host of the transformed cells. Generally, a medium and culture conditions usually used for the host of the transformed cell can be adopted.

  For example, when the transformed cells are filamentous fungi, the culture temperature may be, for example, 10 ° C. to 50 ° C., preferably 25 ° C. to 45 ° C., and the desired C4 dicarboxylic acid is sufficiently produced during the culture period. The period is not particularly limited, but may be, for example, 1 to 240 hours, preferably 12 to 120 hours, and preferably 24 to 72 hours. It is preferable to culture under stirring or aeration.

  As a medium for culturing filamentous fungi, a commonly used medium may be used. Preferably, the medium is a liquid medium, and may be any of a synthetic medium, a natural medium, and a semi-synthetic medium obtained by adding a natural component to a synthetic medium. Commercially available PDB medium (potato dextrose medium; manufactured by Becton, Dickinson and Company, etc.), PDA medium (manufactured by Becton, Dickinson and Company, etc.), LB medium (Luria-Bertani medium; manufactured by Nippon Pharmaceutical Co., Ltd. (trade name: Daigo), etc.) ), NB medium (Nutrient Broth; manufactured by Becton Dickinson and Company, etc.), SB medium (Sabouraud medium; manufactured by OXOID, etc.), SD medium (Synthetic Dropout Broth; for example, Clontech) and the like can also be used. The medium generally contains a carbon source, a nitrogen source, an inorganic salt and the like, but the composition of each component can be appropriately selected.

  Hereinafter, preferred medium compositions for culturing filamentous fungi will be described in detail. The concentration of each component in the medium described below indicates the initial concentration (at the time of medium preparation or at the start of culture).

  Examples of the carbon source in the medium include glucose, maltose, starch hydrolysate, fructose, xylose, and sucrose, among which glucose and fructose are preferred. These saccharides can be used alone or in combination of two or more. The concentration of the carbon source in the medium is preferably 1% (w / v) or more, more preferably 5% (w / v) or more, and preferably 40% (w / v) or less, more preferably Is not more than 30% (w / v). Alternatively, the concentration of the carbon source in the medium is preferably 1 to 40% (w / v), more preferably 5 to 30% (w / v).

  Examples of the nitrogen source in the medium include nitrogen-containing compounds such as ammonium sulfate, urea, ammonium nitrate, potassium nitrate, and sodium nitrate. The concentration of the nitrogen source in the medium is preferably 0.001 to 0.5% (w / v), more preferably 0.001 to 0.2% (w / v).

  The medium can contain sulfate, magnesium salt, zinc salt and the like. Examples of the sulfate include magnesium sulfate, zinc sulfate, potassium sulfate, sodium sulfate, ammonium sulfate and the like. Examples of magnesium salts include magnesium sulfate, magnesium nitrate, magnesium chloride and the like. Examples of zinc salts include zinc sulfate, zinc nitrate, zinc chloride and the like. The concentration of sulfate in the medium is preferably 0.01 to 0.5% (w / v), more preferably 0.02 to 0.2% (w / v). The concentration of the magnesium salt in the medium is preferably 0.001 to 0.5% (w / v), more preferably 0.01 to 0.1% (w / v). The concentration of the zinc salt in the medium is preferably 0.001 to 0.05% (w / v), more preferably 0.005 to 0.05% (w / v).

  The pH (25 ° C.) of the above medium is preferably 3 to 7, more preferably 3.5 to 6. The pH of the medium can be adjusted using a base such as calcium hydroxide, sodium hydroxide, calcium carbonate, ammonia or the like, or an acid such as sulfuric acid or hydrochloric acid.

  Preferred examples of the medium include 7.5 to 30% carbon source, 0.001 to 0.2% ammonium sulfate, 0.01 to 0.6% potassium dihydrogen phosphate, 0.01 to 0.1% sulfuric acid. A liquid medium containing magnesium heptahydrate, 0.005 to 0.05% zinc sulfate heptahydrate, and 3.75 to 20% calcium carbonate (all concentrations are% (w / v)) No.

  In order to more efficiently produce C4 dicarboxylic acid using a transformed cell using a filamentous fungus as a host, production may be carried out in the following steps. That is, a spore suspension of a transformed cell is prepared (Step A), and the resulting suspension is cultured in a culture solution to germinate spores to prepare a mycelium (Step B1). (Step B2) Then, the prepared mycelium is cultured to produce C4 dicarboxylic acid (Step C), whereby C4 dicarboxylic acid can be efficiently produced. However, the step of culturing transformed cells in the present invention is not limited to the following steps.

<Step A: Preparation of spore suspension>
The transformed spores of the filamentous fungus are added to, for example, an inorganic agar medium (composition example: 2% glucose, 0.1% ammonium sulfate, 0.06% potassium dihydrogen phosphate, 0.025% magnesium sulfate heptahydrate, 0.009% zinc sulfate heptahydrate, 1.5% agar, each having a concentration of% (w / v), PDA medium, etc., and inoculated at 10 to 40 ° C, preferably 27 to 30 ° C. A spore suspension can be prepared by performing static culture at 7 ° C. for 7 to 10 days to form spores, and then suspending the spores in physiological saline or the like. The spore suspension may or may not contain mycelium.

<Step B1: Preparation of mycelium>
The spore suspension obtained in the step A is inoculated into a culture solution and cultured, and spores are germinated to obtain mycelium. The number of spores of the filamentous fungus to be inoculated into the culture solution is 1 × 10 ² to 1 × 10 ⁸ -spore / mL-culture solution, preferably 1 × 10 ^{2 to} 5 × 10 ⁴ -spore / mL-culture solution, More preferably, it is 5 × 10 ² to 1 × 10 ⁴ -spore / mL-culture solution, and still more preferably, 1 × 10 ³ to 1 × 10 ⁴ -spore / mL-culture solution. As the culture solution, a commercially available medium such as a PDB medium, an LB medium, an NB medium, an SB medium, and an SD medium can be used. From the viewpoints of germination rate and cell growth, the culture solution contains, as a carbon source, monosaccharides such as glucose and xylose; oligosaccharides such as sucrose, lactose and maltose; and polysaccharides such as starch; glycerin and citric acid. Biological components; ammonium sulfate, urea, amino acids, and the like as a nitrogen source; and various salts such as sodium, potassium, magnesium, zinc, iron, and phosphoric acid as other inorganic substances can be appropriately added. Preferred concentrations of monosaccharide, oligosaccharide, polysaccharide and glycerin are 0.1-30% (w / v), preferred concentration of citric acid is 0.01-10% (w / v), preferred of ammonium sulfate, urea and amino acids. The concentration is 0.01 to 1% (w / v), and the preferable concentration of the inorganic substance is 0.0001 to 0.5% (w / v). The culture is inoculated with a spore suspension, and the mixture is preferably stirred at 80 to 250 rpm, more preferably 100 to 170 rpm, under a culture temperature control of 25 to 42.5 ° C., preferably for 24 to 120 hours. Culture is preferably performed for 48 to 72 hours. The amount of the culture solution to be used for the culture may be appropriately adjusted according to the culture container.For example, the volume is about 50 to 100 mL for a 200 mL baffled flask, and about 100 to 300 mL for a 500 mL baffled flask. I just need. By this culture, the inoculated spores germinate and grow into mycelium.

<Step B2: Propagation of mycelium>
From the viewpoint of improving the C4 dicarboxylic acid-producing ability, it is preferable to perform a step of further culturing and growing the mycelium obtained in the step B1 (step B2). The culture medium for growth used in the step B2 is not particularly limited, but may be any commonly used inorganic culture medium containing glucose, for example, 7.5 to 30% glucose, 0.05 to 2% ammonium sulfate, 0 to 2%. 0.03 to 0.6% potassium dihydrogen phosphate, 0.01 to 0.1% magnesium sulfate heptahydrate, 0.005 to 0.05% zinc sulfate heptahydrate, and 3.75 to A culture solution containing 20% calcium carbonate (all concentrations are% (w / v)) is exemplified. The amount of the culture solution may be appropriately adjusted according to the culture vessel. For example, in the case of a 500 mL Erlenmeyer flask, the volume may be 50 to 300 mL, preferably 100 to 200 mL. The culture cultivated in the step B1 is inoculated into the culture solution so as to have a wet weight of 1 to 6 g-cells / 100 mL-culture solution, preferably 3 to 4 g-cells / 100 mL-culture solution. The culture is performed for 12 to 120 hours, preferably for 24 to 72 hours under a culture temperature control of 25 to 42.5 ° C. while stirring at 300 rpm, preferably 170 to 230 rpm.

<Step C: C4 dicarboxylic acid production>
The mycelium of the filamentous fungus obtained in the above procedure (step B1 or B2) is cultured to cause the fungus to produce C4 dicarboxylic acid. The culturing conditions may be in accordance with the usual culturing conditions for filamentous fungi described above. The amount of the medium can be about 20 to 80 mL for a 200 mL Erlenmeyer flask, about 50 to 200 mL for a 500 mL Erlenmeyer flask, and about 10 L to 15 L for a 30 L jar fermenter. What is necessary is just to adjust suitably also. The inoculum amount of the cells obtained in step B1 or B2 to the medium may be preferably 5 g to 90 g-cells / 100 mL-medium, more preferably 5 g to 50 g-cells / 100 mL-medium as wet weight. Suitably, the culturing is carried out at a temperature of 25 to 45 ° C. with stirring at 100 to 300 rpm, preferably 150 to 230 rpm for 2 to 240 hours, preferably 12 to 120 hours. When a jar fermenter is used, ventilation is preferably performed at 0.05 to 2 vvm, more preferably 0.1 to 1.5 vvm.

  The transformed cells of the present invention are cultured by the above procedure to produce C4 dicarboxylic acid. After culturing, the C4 dicarboxylic acid is recovered from the culture. If necessary, the recovered C4 dicarboxylic acid may be further purified. The method for recovering or purifying C4 dicarboxylic acid from the culture is not particularly limited, and may be performed according to a known recovery or purification method. For example, a gradient method, filtration, removing cells and the like from the culture by centrifugation, etc., after concentrating the remaining culture as necessary, crystallization method, ion exchange method, methods such as solvent extraction method, or By applying these combinations, C4 dicarboxylic acid in the culture can be recovered or purified.

  The transformed cells of the present invention isolated from the culture can be reused for C4 dicarboxylic acid production. For example, newly adding the above-described medium to the transformed cells of the present invention separated from the culture, culturing again under the above conditions to produce C4 dicarboxylic acid, and then recovering the produced C4 dicarboxylic acid from the medium. Can be. This process can be further repeated. In the production method of the present invention, the culturing of the transformed cells and the recovery of C4 dicarboxylic acid may be performed by any of batch, semi-batch and continuous methods.

(4. Exemplary Embodiment)
The following materials, methods of manufacture, uses, or methods are further disclosed herein as exemplary embodiments of the invention. However, the present invention is not limited to these embodiments.

[1] A transformed cell comprising a foreign polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or a polypeptide consisting of an amino acid sequence having at least 80% identity to the sequence.

[2] Preferably, the amino acid sequence having at least 80% identity to the amino acid sequence represented by SEQ ID NO: 2 is
80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 96% or more, still more preferably 97% or more, still more preferably the amino acid sequence represented by SEQ ID NO: 2. Is an amino acid sequence having 98% or more, more preferably 99% or more identity; or 1 to 10 amino acids, preferably 1 to 8 amino acids to the amino acid sequence represented by SEQ ID NO: 2, More preferably, the amino acid sequence has 1 to 5 amino acids, more preferably 1 to 3 amino acids deleted, substituted, added, or inserted.
[1] The transformed cell according to [1].

[3] a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 80% identity to the sequence,
Preferably, a multiple transmembrane polypeptide having eight transmembrane helix structures,
More preferably, the amino acid sequence represented by SEQ ID NO: 2 is in the region from position 13 to position 32, from position 42 to position 64, from position 71 to position 93, from position 97 to position 119, from position 131 to position 153, and position 163. To 182 to 182, 189 to 206, and 212 to 234 corresponding to the transmembrane helix structure.
The transformed cell according to [1] or [2].

[4] Preferably, the polynucleotide is any one of [1] to [3], wherein the polynucleotide is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 80% identity to the sequence. The transformed cell according to claim 1.

[5] Preferably, a nucleotide sequence having at least 80% identity to the nucleotide sequence represented by SEQ ID NO: 1 is
80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 96% or more, still more preferably 97% or more, still more preferably 97% or more, of the nucleotide sequence represented by SEQ ID NO: 1. Is a nucleotide sequence having 98% or more, more preferably 99% or more identity; or 1 to 30 or less, preferably 1 or more to 24 or less with respect to the nucleotide sequence represented by SEQ ID NO: 1, More preferably, it is a nucleotide sequence in which 1 to 15 nucleotides, more preferably 1 to 9 nucleotides are deleted, substituted, added, or inserted.
[4] The transformed cell according to [1].

[6] The transformed cell according to any one of [1] to [5], which preferably comprises a vector containing the polynucleotide, or the polynucleotide is integrated into genomic DNA.

[7] Preferably, the transformed cell of [6], wherein the vector further comprises a control region operably linked to the polynucleotide.

[8] Preferably, the transformed cell according to [6], wherein the polynucleotide is integrated into genomic DNA together with a control region operably linked to the polynucleotide.

[9] The transformed cell according to any one of [1] to [8], wherein the cell is preferably a microbial cell.

[10] The transformed cell of [9], wherein the microorganism is preferably a filamentous fungus.

[11] The transformed cell according to [10], wherein preferably the filamentous fungus is of the genus Rhizopus.

[12] the Rhizopus genus,
Preferable is Rhizopus delemar or Rhizopus oryzae,
More preferably Rhizopus delemar,
[11] The transformed cell according to [1].

[13] The transformed cell according to any one of [1] to [12], which preferably has an improved ability to produce C4 dicarboxylic acid.

[14] The transformed cell according to [13], wherein the ability to produce C4 dicarboxylic acid is improved by preferably 10% or more, more preferably 15% or more, and still more preferably 20% or more.

[15] Preferably, the transformed cell according to [13] or [14], wherein the C4 dicarboxylic acid is fumaric acid, malic acid, or succinic acid.

[16] A method for producing C4 dicarboxylic acid, comprising culturing the transformed cell according to any one of [1] to [15].

[17] The method of [16], further comprising recovering C4 dicarboxylic acid from the culture.

[18] The method of [16] or [17], wherein the C4 dicarboxylic acid is fumaric acid, malic acid or succinic acid.

[19] A method for producing a transformed cell,
Introducing a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 80% identity to the sequence, or a vector containing the same into a host cell,
Method.

[20] A method for improving C4 dicarboxylic acid producing ability in a host cell,
Introducing a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 80% identity to the sequence, or a vector containing the same into a host cell,
Method.

[21] The method according to [20], wherein the C4 dicarboxylic acid-producing ability in the host cell is improved by preferably 10% or more, more preferably 15% or more, and still more preferably 20% or more.

[22] The method according to [20] or [21], wherein the C4 dicarboxylic acid is preferably fumaric acid, malic acid or succinic acid.

[23] Preferably, the amino acid sequence having at least 80% identity to the amino acid sequence represented by SEQ ID NO: 2 is
80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 96% or more, still more preferably 97% or more, still more preferably the amino acid sequence represented by SEQ ID NO: 2. Is an amino acid sequence having 98% or more, more preferably 99% or more identity; or 1 to 10 amino acids, preferably 1 to 8 amino acids to the amino acid sequence represented by SEQ ID NO: 2, More preferably, the amino acid sequence has 1 to 5 amino acids, more preferably 1 to 3 amino acids deleted, substituted, added, or inserted.
The method according to any one of [19] to [22].

[24] a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 80% identity to the sequence,
Preferably, a multiple transmembrane polypeptide having eight transmembrane helix structures,
More preferably, the amino acid sequence represented by SEQ ID NO: 2 is in the region from position 13 to position 32, from position 42 to position 64, from position 71 to position 93, from position 97 to position 119, from position 131 to position 153, and position 163. To 182 to 182, 189 to 206, and 212 to 234 corresponding to the transmembrane helix structure.
The method according to any one of [19] to [23].

[25] Preferably, any of [19] to [24], wherein the polynucleotide is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 80% identity to the sequence. The method of claim 1.

[26] Preferably, a nucleotide sequence having at least 80% identity to the nucleotide sequence represented by SEQ ID NO: 1 is
80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 96% or more, still more preferably 97% or more, still more preferably 97% or more, of the nucleotide sequence represented by SEQ ID NO: 1. Is a nucleotide sequence having 98% or more, more preferably 99% or more identity; or 1 to 30 or less, preferably 1 or more to 24 or less with respect to the nucleotide sequence represented by SEQ ID NO: 1, More preferably, it is a nucleotide sequence in which 1 to 15 nucleotides, more preferably 1 to 9 nucleotides are deleted, substituted, added, or inserted.
[25] The method according to the above.

[27] The method of any one of [19] to [26], wherein the host cell is preferably a microbial cell.

[28] The method of [27], wherein the microorganism is preferably a filamentous fungus.

[29] The method according to [28], wherein preferably the filamentous fungus is of the genus Rhizopus.

[30] the Rhizopus genus,
Preferable is Rhizopus delemar or Rhizopus oryzae,
More preferably Rhizopus delemar,
[29] The method according to the above.

  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Preparation of Transformed Cell (1) Genome Extraction Spores of Rhizopus delemar JCM (Japan Collection of Microorganisms / RIKEN) 5557 strain (hereinafter referred to as 5557 strain) were inoculated on a PDA medium and cultured at 30 ° C. for 5 days. Was done. After the culture, the cells were placed in a 3 mL crushing tube together with a 3 mL metal cone (Yasui Kikai), and immediately frozen in liquid nitrogen for 10 minutes or more. Thereafter, the cells were disrupted for 10 seconds at 1700 rpm using a multi-beads shocker (Yasui Kikai). 400 μL of TE Buffer (pH 8.0) (Nippon Gene) was added to the crushed vessel, mixed by inversion, and 250 μL was transferred to a 1.5 mL tube. Genome was extracted from the cell solution using "Gen Toru-kun (for yeast)" (Takara Bio) according to the protocol. 1 μL of RNase A (Roche) was added to 50 μL of the obtained genomic solution, and reacted at 37 ° C. for 1 hour. After the reaction, an equal amount of phenol-chloroform was added, mixed by tapping, and centrifuged at 4500C and 14,500 rpm for 5 minutes, and the supernatant was transferred to a new 1.5 mL tube. The phenol / chloroform treatment was repeated again, followed by ethanol precipitation to obtain a purified genomic solution of 5557 strain.

(2) Preparation of cDNA (i) Total RNA Extraction 6 g of the cells of 5557 strain-wet weight were added to 40 mL of liquid medium (0.1 g / L (NH ₄ ) ₂ SO ₄ , 0.6 g / L KH ₂ PO ₄ , 0.25 g / L MgSO ₄ .7H ₂ O, 0.09 g / L ZnSO ₄ .7H ₂ O, 50 g / L calcium carbonate, 100 g / L glucose) and cultured at 35 ° C. and 170 rpm for 8 hours. The cells were filtered and collected from the culture solution, and washed twice with 100 mL of 0.85% physiological saline. After washing, excess water was removed by suction filtration. Then, 0.3 g was weighed, placed in a 3 mL crushing tube together with a 3 mL metal cone (Yasui Kikai), immediately poured into liquid nitrogen, and frozen. The obtained frozen cells were disrupted for 10 seconds at 1700 rpm using a multi-beads shocker (Yasui Kikai). 500 μL of RLT buffer was added to the disrupted cells, and after inverting the mixture, 450 μL was subjected to RNeasy Plant Mini Kit (Qiagen) to extract total RNA. To 40 μL of the obtained RNA solution, 1 μL of DNaseI (TaKaRa) and 5 μL of 10 × DNaseI buffer (USB Corporation) were added, and after filling up to 50 μL with RNase free water, the reaction was allowed to proceed at 37 ° C. for 30 minutes or more to obtain a solution. Of residual DNA was removed. An additional 1 μL of DNase I was added, reacted at 37 ° C. for 30 minutes, extracted with phenol / chloroform, and then precipitated with ethanol. The precipitate was dissolved in 50 μL of sterilized water, and the concentration and purity of the RNA solution were measured using Qubit (Life Technologies). In addition, the RNA solution was appropriately diluted, and RNA extracted using an Agilent 2100 Bioanalyzer (Agilent) and RNA6000 Pico Kit (Agilent) was assayed. It was confirmed that “RNA Integrity Number (RIN value)”, which is an index of RNA degradation, was 6.0 or more, and the obtained RNA solution was obtained as total RNA.

(Ii) cDNA synthesis cDNA synthesis was performed using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). That is, 1 μg of the RNA solution obtained in (i) is filled up to 8 μL with DEPC water, and then 10 μL of 2 × RT Reaxion Mix and 2 μL of RT Enzyme Mix are added, gently mixed, and mixed at 25 ° C. for 10 minutes. At 50 ° C. for 30 minutes and at 85 ° C. for 5 minutes. 1 μL of RNase H was added to the solution after the reaction, and reacted at 37 ° C. for 20 minutes to obtain a cDNA solution.

(3) Preparation of plasmid vector (i) Introduction of trpC gene region into pUC18 Using the genomic DNA of the 5557 strain obtained in the above (1) as a template, a DNA fragment containing the trpC gene (SEQ ID NO: 3) was used as a primer oJK162. (SEQ ID NO: 4) and oJK163 (SEQ ID NO: 5). Next, a DNA fragment was amplified by PCR using the plasmid pUC18 as a template and primers oJK164 (SEQ ID NO: 6) and oJK165 (SEQ ID NO: 7). The above two fragments were ligated using an In-Fusion HD Cloning Kit (Clontech) to construct a plasmid pUC18-trpC.

(Ii) Cloning of ADH1 promoter and terminator Using the genomic DNA of 5557 strain obtained in the above (1) as a template, a DNA fragment containing the ADH1 promoter sequence (SEQ ID NO: 8) and a DNA containing the terminator sequence (SEQ ID NO: 9) The fragments were amplified by PCR using primers oJK202 (SEQ ID NO: 10) and oJK204 (SEQ ID NO: 11), and oJK205 (SEQ ID NO: 12) and oJK216 (SEQ ID NO: 13), respectively. Next, using the plasmid pUC18-trpC obtained in (i) as a template, a DNA fragment was amplified by PCR using primers oJK210 (SEQ ID NO: 14) and oJK211 (SEQ ID NO: 15). The above three fragments were ligated in the same manner as in (i) to construct a plasmid pUC18-trpC-Padh-Tadh. In the obtained plasmid, an ADH1 promoter and a terminator are sequentially arranged downstream of the trpC gene region. Further, a Not I restriction enzyme recognition sequence is arranged downstream of the ADH1 terminator.

(Iii) Preparation of plasmid vector Using the cDNA of the 5557 strain obtained in the above (2) as a template, a DNA fragment containing the gene represented by SEQ ID NO: 1 (hereinafter, referred to as rdt6) was synthesized with primers oJK515 (SEQ ID NO: 16) and Amplification was performed by PCR using oJK516 (SEQ ID NO: 17). Next, a DNA fragment was amplified by PCR using primers oJK204 (SEQ ID NO: 11) and oJK269-4 (SEQ ID NO: 18) using the plasmid pUC18-trpC-Padh-Tadh obtained in (ii) as a template. The above two fragments were ligated in the same manner as in (i) to construct a plasmid pUC18-trpC-Padh-rdt6-Tadh. In the obtained plasmid, the rdt6 gene represented by SEQ ID NO: 1 was inserted between the ADH promoter and the terminator.

  Table 1 shows the PCR primers used for construction of the plasmid vector pUC18-trpC-Padh-rdt6-Tadh.

(4) Gene transfer into host cells (i) Preparation of tryptophan auxotroph strain The tryptophan auxotroph strain used as the host cell for gene transfer was selected from among the strains mutated by ion beam irradiation into 5557 strain. I got it. The ion beam irradiation was performed at the ion irradiation facility (TIARA) of the Takasaki Quantum Application Research Institute, Japan Atomic Energy Agency. The irradiation was performed by accelerating ¹² C ⁵⁺ using an AVF cyclotron and irradiating 100 to 1250 Gray at an energy of 220 MeV. Spores were collected from the irradiated cells, and a Rhizopus delemar 02T6 strain exhibiting tryptophan auxotrophy (hereinafter referred to as 02T6 strain) was obtained from the spores. The 02T6 strain has a single nucleotide deletion at position 2093 in the total length of 2,298 bp of the trpC gene coding region (SEQ ID NO: 3).

(Ii) Amplification of plasmid vector Competent cell transformation of Escherichia coli DH5α strain (Nippon Gene) using the plasmid vectors pUC18-trpC-Padh-Tadh and pUC18-trpC-Padh-rdt6-Tadh prepared in (3) above. For transformation. The obtained transformed cells are allowed to stand at 37 ° C. overnight, and the obtained colonies are inoculated into 2 mL of LBamp liquid medium (Bacto Trypton 1%, Yeast Extract 0.5%, NaCl 1%, Ampicillin sodium 50 μg / mL). And cultured overnight at 37 ° C. Each plasmid vector was purified from this culture using a High Pure Plasmid Isolation Kit (Roche Life Science).

(Iii) Introduction of plasmid vector into host cells 10 μL of each DNA solution (1 μg / μL) of plasmid vector pUC18-trpC-Padh-Tadh and pUC18-trpC-Padh-rdt6-Tadh obtained in (ii) was used as gold particles. After 100 μL of the solution (60 mg / mL) was added and mixed, 40 μL of 0.1 M spermidine was added, and the mixture was vortexed well. Further, 100 μL of 2.5 M CaCl ₂ was added, the mixture was vortexed for 1 minute, and then centrifuged at 6000 rpm for 30 seconds to remove the supernatant. 200 μL of 70% EtOH was added to the obtained precipitate, and the mixture was vortexed for 30 seconds, and then centrifuged at 6000 rpm for 30 seconds to remove the supernatant. The resulting precipitate was resuspended in 100 μL of 100% EtOH.

Next, the spores of the 02T6 strain prepared in (i) were transfected with GDS-80 (Nepagene) using the DNA-gold particle solution described above. The spores after the gene transfer were collected on an inorganic agar medium (20 g / L glucose, 1 g / L ammonium sulfate, 0.6 g / L potassium dihydrogen phosphate, 0.25 g / L magnesium sulfate · heptahydrate, 0.09 g / L) (Zinc sulfate heptahydrate, 15 g / L agar) at 30 ° C. for about one week. A portion of the grown cells was scraped off with an inoculated ear and suspended in TE (pH 8.0) (Nippon Gene). The suspension was treated at 95 ° C. for 15 minutes to extract nucleic acids from the transformed strain. Using this nucleic acid as a template, a PCR reaction was performed using primers oJK438 (SEQ ID NO: 19) and oJK439 (SEQ ID NO: 20), and a strain in which the introduction of the target DNA fragment was confirmed was selected as a transformant. The PCR primers are shown in Table 2. The strain into which pUC18-trpC-Padh-rdt6-Tadh containing DNA with the rdt6 gene linked downstream of the ADH1 promoter was introduced was designated as RDT6 strain, while the plasmid vector pUC18-trpC-Padh containing DNA without rdt6 gene inserted thereinto. -The strain into which Tadh was introduced was obtained as a negative control strain (hereinafter, NC strain). The remaining cells were scraped off with an inoculation ear and mixed vigorously in a spore recovery solution (8.5 g / L sodium chloride, 0.5 g / L polyoxyethylene sorbitan monooleate). The spore suspension after mixing was filtered with a 3GP100 cylindrical funnel type glass filter (Shibata Chemical) to obtain a spore solution. The number of spores in the spore fluid was measured using a hemocytometer (hemocytometer, D = 1/50 mm / 1/400 mm ² ).

Example 2 C4 dicarboxylic acid-producing ability of RDT6 strain (1) Culture of transformed strain (i) Preparation of mycelium In a 500 mL Erlenmeyer flask with baffle (Asahi Glass), sorbitan monolaurate (Reodol SP-L10 (Kao)) Was added to a final concentration of 0.5% (v / v) to provide 200 mL of an SD / -Trp medium (Clontech), and the spore solutions of the RDT6 strain and the NC strain prepared in Example 1 were 1 × 10 ³ -spores. After inoculation so as to be / mL-medium, the mixture was cultured at 27 ° C. for 3 days with stirring at 170 rpm. The obtained culture was filtered using a 250-μm mesh stainless steel sieve (AS ONE) which had been sterilized in advance, and the cells were collected on the filter.

(Ii) Propagation of mycelium 100 mL of inorganic culture solution (0.1 g / L (NH ₄ ) ₂ SO ₄ , 0.6 g / L KH ₂ PO ₄ , 0.25 g / L MgSO _{4. 7H 2 O, 0.09g / L ZnSO} 4 · 7H 2 O, 50g / L of calcium carbonate, to 100 g / L glucose) were inoculated with wet cells 5.0~8.0g recovered in (i), 27 Culturing was performed at 220 ° C. for about 40 hours at 220 ° C. with stirring. The obtained culture was filtered using a stainless steel screen filter holder (MILLIPORE) previously sterilized, and the cells were collected on the filter. Further, the cells were washed on the filter holder with 200 mL of physiological saline. The physiological saline used for washing was removed by suction filtration.

(2) Evaluation of C4 dicarboxylic acid productivity of the transformed strain 6.0 g of wet cells of the RDT6 strain and the NC strain obtained in the above (1) were supplied to a 200 mL Erlenmeyer flask, and 40 mL of an inorganic culture solution for productivity evaluation was used. _{(0.0175g / L (NH 4)} 2 SO 4, 0.06g / L KH 2 PO 4, 0.375g / L MgSO 4 · 7H 2 O, 0.135g / L ZnSO 4 · 7H 2 O, 50g / L calcium carbonate, 100 g / L glucose), and cultured with stirring at 35 ° C. and 170 rpm. After 56 hours of culture, the culture supernatant containing no cells was collected, and fumaric acid, malic acid, succinic acid, and glucose were quantified by the procedure described in Reference Example 1 described below, and each C4 dicarboxylic acid from glucose was determined. Was calculated. Based on the obtained conversion rates of the RDT6 strain and the NC strain, the rate of improvement in the productivity of each C4 dicarboxylic acid in the RDT6 strain was calculated according to the following formula.
Improvement rate (%)
= (Conversion rate in 6 strains of RDT / conversion rate in NC strain) × 100-100
Table 3 shows the results. Compared with the NC strain into which the rdt6 gene was not introduced, in the RDT6 strain, malic acid was 49.5%, fumaric acid was 21.1%, and succinic acid was 28.6%, and the productivity was improved. Was.

Reference Example 1 Quantification of C4 dicarboxylic acid and glucose C4 dicarboxylic acid (fumaric acid, malic acid and succinic acid) and glucose in the culture supernatant were quantified by HPLC.
The culture supernatant to be subjected to HPLC analysis is appropriately diluted in advance with 37 mM sulfuric acid, and then, using DISMIC-13 cp (0.20 μm cellulose acetate membrane, ADVANTEC) or Acroprep 96 filter plate (0.2 μm GHP membrane, Nippon Pole). Insolubles were removed.
The HPLC apparatus used was LaChrom Elite (Hitachi High Technologies). The analysis column was connected to an IC Sep ICE-ION-300 Guard Column Cartridge (4.0 mm ID × 2.0 cm, TRANSGENOMIC), and a polymer column for organic acid analysis IC Sep ICE-ION-300 (7.8 mm ID. Elution was performed using 10 mM sulfuric acid, a flow rate of 0.5 mL / min, and a column temperature of 50 ° C. using × 30 cm (TRANSGENOMC). For the detection of each C4 dicarboxylic acid and glucose, a UV detector (detection wavelength 210 nm) and a suggestive refractive index detector (RI detector) were used. The concentration calibration curve was obtained from a standard sample [fumaric acid (sales source code: 063-00655, Wako Pure Chemical Industries), malic acid (sales source code: 135-00562, Wako Pure Chemical Industries), succinic acid (sales source code: 194-04335, (Wako Pure Chemical Industries, Ltd.) and glucose (Seller code 045-31162, Wako Pure Chemical Industries)]. Each component was quantified based on each concentration calibration curve.

  The value obtained by subtracting the determined amount of glucose in the medium from the initial amount of glucose in the medium was defined as glucose consumption. The ratio (%) of the amount of each C4 dicarboxylic acid quantified to the glucose consumption was calculated as the conversion (yield) of each C4 dicarboxylic acid.

Claims (10)

A method for producing a C4 dicarboxylic acid, comprising culturing a transformed cell comprising a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 90 % identity to the sequence. The method according to claim 1, wherein the polynucleotide is a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence having at least 90 % identity to the sequence.   The production method according to claim 1, wherein the transformed cell contains a vector containing the polynucleotide.   The method according to any one of claims 1 to 3, wherein the cells are microbial cells.   The method according to claim 4, wherein the microorganism is a filamentous fungus.   The method according to claim 5, wherein the filamentous fungus is of the genus Rhizopus.   The method according to claim 6, wherein the genus Rhizopus is Rhizopus delemar.   The method according to any one of claims 1 to 7, further comprising recovering C4 dicarboxylic acid from the culture.   The method according to any one of claims 1 to 8, wherein the C4 dicarboxylic acid is fumaric acid, malic acid or succinic acid. The method according to any one of claims 1 to 9, wherein the polypeptide has a function of improving C4 dicarboxylic acid producing ability of a cell.