JP2008182936A - cis-ACONITIC ACID DECARBOXYLATION ENZYME AND GENE ENCODING THE SAME - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cis-aconitic acid decarboxylation enzyme, and to provide a gene encoding the cis-aconitic acid decarboxylation enzyme catalyzing from cis-aconitic acid to itaconic acid. <P>SOLUTION: A protein comprises a specific amino acid sequence and having a cis-aconitic acid decarboxylation activity, a gene encodes the protein, a recombinant vector contains the gene, a microorganism transforms with the vector, and a method for produces itaconic acid with the microorganism or its preparation. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a gene encoding cis-aconitic acid decarboxylase (CAD) and cis-aconitic acid decarboxylase that catalyzes a decarboxylation reaction from cis-aconitic acid to itaconic acid. The present invention also relates to a recombinant vector containing the gene and a microorganism transformed with the vector. Furthermore, it is related with the manufacturing method of itaconic acid using this microorganism or its preparation.

  Itaconic acid is a dibasic acid compound having an ethylenically unsaturated double bond and a carboxyl group. Using these functionalities, synthetic resins, adhesives, detergents, latex, paints, printing inks and acrylic fibers It is a very useful compound that is widely used as a raw material for chemical industry applications such as modifiers.

  Itaconic acid is conventionally produced by fermentation using mono- and di-saccharides such as glucose, sucrose and fructose, and carbohydrates such as starch as raw materials. As microorganisms used for fermentation production, molds such as microorganisms belonging to the genus Aspergillus (patent document 1) and microorganisms belonging to the genus Rhizopus (patent document 2), and microorganisms belonging to the genus Candida (patent document 3). ) And other yeasts are known. Patent Document 4 discloses a technique for obtaining a high-temperature resistant strain by mutation treatment of Aspergillus terreus, which is an itaconic acid-producing bacterium.

  However, the above-mentioned various patent documents only disclose a culture technique and do not mention any itaconic acid-producing metabolic pathway that works in itaconic acid-producing cells.

  Further, in Non-Patent Document 1, among several possible metabolic pathways from glucose to itaconic acid production in Aspergillus terreus, itaconic acid is cis-aconitic acid produced by the TCA cycle. Although it has been specified that itaconic acid is produced by the action of decarboxylase (hereinafter also referred to as CAD), the CAD itself has not been provided with any genetic engineering knowledge and analysis.

  Further, in Non-Patent Document 2, CAD was isolated and purified from Aspergillus terreus strain, and the CAD catalytic activity regarding decarboxylation reaction (cell-free reaction) from cis-aconitic acid to itaconic acid in CAD solution was investigated. However, although knowledge about the separation / purification technology of CAD and its catalytic function is disclosed, no genetic engineering knowledge of CAD expressed in microbial cells is provided.

If CAD can be highly expressed by genetic engineering techniques based on genetic information, it is possible to provide a highly efficient production technology for itaconic acid. That is, it is possible to acquire a high production technology for CAD and establish itaconic acid production technology by various conditions and methods according to the suitability of the host by a wide range of host recombination technologies not limited to conventional itaconic acid-producing bacteria.
US Pat. No. 5,673,485 JP-A 61-209596 JP-A-55-34017 JP-A-6-38774 P. BONNARME and 5 others, Journal of Bacteriology, June issue, 1995, p. 3573-3578 IES DWIARTI, 4 others, Journal of Bioscience and Bioengineering, Vol. 94, No. 1, 29-33, 2002.

  The present invention provides cis-aconitic acid decarboxylase (CAD) that catalyzes the decarboxylation of cis-aconitic acid to itaconic acid and the gene that encodes it. The present invention also provides a recombinant vector containing the gene. Furthermore, a microorganism (transformant) obtained by transforming a host with the vector is provided. Furthermore, the present invention provides a method for producing itaconic acid using the microorganism or a preparation thereof.

  In order to obtain a gene encoding cis-aconitic acid decarboxylase that catalyzes a decarboxylation reaction from cis-aconitic acid to itaconic acid, for example, an Aspergillus terreus strain is produced. As a result of selecting the gene having CAD activity from the total soluble enzyme protein to be cloned, and cloning and expressing the gene sequence obtained from the amino acid sequence, it was found that it was the target gene sequence. The present invention has been completed.

That is, the present invention
[1] Gene shown in the following (a), (b), (c) or (d):
(A) a gene comprising DNA having the base sequence shown in SEQ ID NO: 1
(B) consisting of a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA of the base sequence shown in SEQ ID NO: 1 and encodes a protein having cis-aconitic acid decarboxylase activity gene:
(C) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2:
(D) From an amino acid sequence shown in SEQ ID NO: 2 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and encoding a protein having cis-aconitic acid decarboxylase activity The gene,
[2] Protein shown in the following (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2:
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having cis-aconitic acid decarboxylase activity;
[3] A recombinant vector containing the gene according to [1],
[4] A microorganism obtained by transforming a host with the recombinant vector according to [3],
[5] The microorganism according to [4] above, wherein the host is selected from Escherichia coli, Corynebacterium and yeast.
[6] The microorganism according to [5] above, wherein the microorganism is Escherichia coli JM109 / cad001 strain (Accession No. FERM P-21146),
[7] The microorganism described in [5] above, wherein the microorganism is Corynebacterium glutamicum R / cad002 strain (Accession No. FERM P-21147), and [8] any one of [4] to [7] A method for producing itaconic acid, which comprises contacting the microorganism or the preparation thereof with a liquid containing cis-aconitic acid, thereby producing and accumulating itaconic acid in the liquid, and collecting it.
About.

  According to the present invention, it is possible to provide a gene encoding cis-aconitic acid decarboxylase and cis-aconitic acid decarboxylase that catalyzes a decarboxylation reaction from cis-aconitic acid to itaconic acid. In addition, the recombinant vector containing the gene can be used for the production of the transformed microorganism of the present invention. Furthermore, the microorganism of the present invention can produce cis-aconitic acid decarboxylase, can be used for production of cis-aconitic acid decarboxylase, and a microorganism transformed with the vector or a preparation thereof can be used. By using itaconic acid, it can be produced with high efficiency.

Cis-aconitic acid decarboxylase (CAD) is a protein enzyme that catalyzes the decarboxylation reaction from cis-aconitic acid to itaconic acid. Therefore, if a significant amount of this CAD can be accumulated in the microbial cells, itaconic acid can be produced with high efficiency and high productivity from the CAD-producing microorganism or its preparation.
As the CAD of the present invention, for example, a protein comprising the amino acid sequence shown in SEQ ID NO: 2 is preferably mentioned. The CAD of the present invention includes an amino acid sequence in which one or more (about 2 to 20, preferably about 2 to 10) amino acids have been deleted, substituted or added in SEQ ID NO: 2, In addition, proteins having CAD activity are also included.

  Examples of the gene encoding CAD used in the present invention include Aspergillus genus, Candida genus, Rhizopus genus, Ustilago genus, Pseudozyma genus, Helicobacidium genus and Rhodotorula genus. A gene derived from a microorganism, more preferably a gene derived from Aspergillus terreus, particularly preferably a gene derived from Aspergillus terreus NBRC6123 strain (preservation strain of National Institute of Biotechnology, National Institute of Technology and Evaluation) It is done.

  As a gene containing DNA encoding CAD derived from Aspergillus terreus NBRC6123 strain, for example, a gene containing DNA having a base sequence represented by SEQ ID NO: 1 identified for the first time in the present invention can be mentioned.

  A gene comprising a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 and encodes a protein having CAD activity, It is a gene used in the present invention. The DNA of the present invention can also be prepared by chemical synthesis. In addition, the present invention encompasses DNAs encoding artificial mutant proteins having CAD activity from these DNAs of the present invention. That is, in order to obtain the artificial mutant DNA of the present invention relating to the corresponding base sequence or amino acid sequence, a known mutagenesis method may be used. For example, a mutation can be easily introduced using a commercially available mutagenesis kit. it can.

The “base sequence that hybridizes under stringent conditions” means, for example, DNA obtained by colony hybridization method or plaque hybridization method using the base sequence shown in SEQ ID NO: 1 as a probe.
“Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify this condition clearly, for example, DNAs having high homology, at least about 50%, preferably about 60% or more, more preferably at least the base sequence represented by SEQ ID NO: 1 A condition in which DNAs having a homology of about 80% or more hybridize and DNAs having lower homology do not hybridize, or an SSC solution having a concentration of about 0.1 to 2 times The SSC solution refers to a hybridization condition at a temperature of about 65 ° C., which consists of 150 mM sodium chloride and 15 mM sodium citrate). The homology can be calculated by base sequence analysis software such as EMBOSS (The European Molecular Biology Open Software Suite).
However, the gene in the present invention is not limited to the above-mentioned gene, and includes all genes encoding CAD including the amino acid sequence represented by SEQ ID NO: 2.

  In the present invention, the term “gene” or “DNA” includes not only DNA but also mRNA and cDNA thereof. Accordingly, the genes of the present invention include all of these DNAs, mRNAs and cDNAs.

  The “preparation” of the microorganism in the present invention refers to an enzyme that is produced by accumulating and extracting cis-aconitic acid decarboxylase (CAD) in a transformant, preferably an immobilized enzyme that is purified and immobilized on a resin carrier or the like. Means an enzyme. Furthermore, an immobilized microbial cell in which a microbial cell transformed with a gene encoding CAD or a vector containing the gene is immobilized on a carrier such as acrylamide or carrageenan is also included.

  A microorganism transformed with a gene encoding CAD of the present invention or a vector containing the gene can be mutated by an artificial mutagenesis method using ultraviolet rays, X-rays, drugs, etc., and the mutant strain thus obtained Even so, as long as it has the ability to produce a protein enzyme that catalyzes the decarboxylation reaction from cis-aconitic acid to itaconic acid, which is the object of the present invention, it is a transformed microorganism of the present invention.

  Isolation of CAD from Aspergillus tereus NBRC6123 strain and determination of its amino acid sequence. Although it is a different strain, a sequence highly homologous to the target gene of the present invention is selected from the approximately 10,000 gene group possessed by Aspergillus terreus NIH2624 strain that has completed the whole genome draft sequence (presumed gene) . Next, among these putative genes, a gene expressed in Aspergillus terreus NBRC6123 strain producing itaconic acid is searched. For the putative gene sequence finally selected, a cloning vector having the gene function is introduced into, for example, Escherichia coli to express the recombinant protein. Furthermore, by confirming that the enzyme protein thus induced for expression has CAD activity, the putative gene selected as described above can be determined to be a gene encoding the CAD of the present invention. A series of these embodiments is described in detail below.

i) Cultivation of Aspergillus terreus NBRC6123 strain and isolation of CAD Aspergillus terreus NBRC6123 strain was cultured aerobically at about 30 ° C. using, for example, the solid medium shown in Table 1, and then the liquid shown in Table 2 for example. Inoculate the medium and culture for about one week to collect the cells.

In addition to the above, any medium composition can be preferably used as long as the medium composition allows Aspergillus terreus to produce itaconic acid.
The cells are collected by filtration or centrifugation, and then lyophilized. For example, after the dried cells are ground in a mortar or the like, a crude enzyme solution can be obtained by dispersing in a buffer solution having a pH of 5 to 7, preferably about 6.5, and removing insoluble substances by centrifugation or the like. it can.
The method for purifying CAD protein from the crude enzyme solution is a known technique used for general protein purification as shown in Non-Patent Document 2, column chromatography methods such as precipitation fractionation, ion exchange, and hydrophobic interaction. It can be done by combining. The identification of CAD protein can be confirmed by performing an assay by a conversion reaction from cis-aconitic acid to itaconic acid by a purified enzyme.

ii) Amino acid sequence analysis of isolated CAD Internal amino acid sequence analysis of purified CAD is performed by degrading purified CAD protein using an appropriate proteolytic enzyme such as lysyl endopeptidase or trypsin, and Separation and collection can be performed by high performance liquid chromatography or electrophoresis, and the amino acid terminals can be determined by an amino acid sequence analyzer such as Procise 494HT Protein Sequencing System (Applied Biosystems).

iii) Cloning of a putative gene encoding CAD based on amino acid sequence analysis A homology search is performed on the determined amino acid sequence using a program such as Blast (Basic Local Alignment Search Tool). If a gene showing high homology exists through such analysis results, an oligonucleotide primer for amplifying the DNA sequence by a PCR (Polymerase chain reaction) method is prepared. Examples of such primers include those represented by the nucleotide sequences of SEQ ID NOS: 6 and 7. For the PCR method, a known PCR device such as a thermal cycler can be used. The PCR cycle may be performed according to a known technique. For example, denaturation, annealing, and extension are defined as one cycle, and usually about 10 to 100 cycles, preferably about 20 to 50 cycles. As a template for PCR reaction, RNA isolated from Aspergillus terreus producing itaconic acid or single strand cDNA (first strand cDNA) synthesized from RNA by reverse transcription reaction, RT (reverse transcription)- CAD cDNA can be synthesized by PCR or PCR. The gene obtained by the PCR method can be introduced into an appropriate cloning vector. As a cloning method, a commercially available PCR cloning system such as pGEM-T easy vector system (Promega), TOPO TA-cloning system (Invitrogen), Mighty Cloning Kit (Takara), etc. is preferable. Can be mentioned.

  Next, a cloning vector containing the gene obtained by PCR is introduced into a microorganism, for example, Escherichia coli JM109 strain, and the strain is transformed. The transformed strain is cultured in a medium containing an appropriate antibiotic (for example, ampicillin, chloramphenicol, etc.), and the cells are recovered from the culture. Plasmid DNA is extracted from the collected cells. Extraction of plasmid DNA can be performed by a known technique, or can be easily extracted using a commercially available plasmid DNA extraction kit. Examples of commercially available plasmid extraction kits include Qiaquick plasmid purification kit (trade name: Qiaquick plasmid purification kit, manufactured by Qiagen). By determining the base sequence of the extracted plasmid DNA, the gene sequence encoding the CAD of the present invention can be determined.

  Determination of the base sequence of DNA can be determined by a known method such as dideoxynucleotide enzyme method. In addition, using a capillary electrophoresis system, the base sequence can be determined using a multi-fluorescence technique for detection. It can also be determined using a DNA sequencer such as ABI PRISM 3730xl DNA Analyzer (Applied Biosystems).

  As described above, the gene sequence encoding CAD can be determined, and for example, the base sequence shown in SEQ ID NO: 1 can be determined. Then, the base sequence can be translated into an amino acid sequence, and the entire amino acid sequence of CAD shown in SEQ ID NO: 2 can be determined.

iv) Expression of CAD gene by host Next, a recombinant CAD expression vector can be prepared by inserting a gene encoding the enzyme whose entire amino acid sequence has been determined into an appropriate expression vector. Examples of expression vectors include pET, pMal, pCold, pGEX, pCRB1 and derivatives thereof, general bacterial plasmids, pUC18, 19, pBR322 and the like, and yeast plasmids (YEp type, YCp type, etc.), Examples of phage DNA and mammalian cell vectors include viral DNA such as baculovirus, vaccinia virus, adenovirus, SV40 and its derivatives, and any other vector can be used as long as it can replicate in the host.

  The vector includes, for example, an origin of replication, a selection marker, and a promoter. If necessary, an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a tag sequence for purification and detection such as His-tag or GST-tag, and polyadenylation A signal may be included. Desirably, the vector contains a polylinker with various restriction enzyme sites therein, or a single restriction enzyme site. Examples of restriction enzyme sites include PstI site, NdeI site, SalI site, EcoRI site, HindIII site and the like. These restriction enzyme sites can be cleaved with restriction enzymes PstI, NdeI, SalI, EcoRI or HindIII, respectively.

  Gene transfer into the vector can be performed by known means. Specifically, it is preferable that a specific restriction enzyme site in the vector is cleaved with a specific restriction enzyme, and the gene of the present invention is inserted into the cleavage site. In this way, a recombinant vector containing the gene of the present invention can be prepared.

  For example, a primer added with a recognition sequence of, for example, the restriction enzyme NdeI upstream of the 5 ′ end sequence of the enzyme gene shown in SEQ ID NO: 1, and a primer added with a recognition sequence of, for example, the restriction enzyme EcoRI downstream of the 3 ′ end sequence Is used to amplify DNA encoding CAD by PCR. This amplified fragment is treated with the restriction enzyme NdeI and the restriction enzyme EcoRI, and ligated with an expression vector similarly treated with both restriction enzymes, for example, pColdI (manufactured by Takara), that is, a recombinant vector containing the gene of the present invention, CAD expression vectors can be obtained.

  Next, this expression vector is inserted into a host, and the host (strain) is transformed. In the present invention, a host to be transformed is transformed with a recombinant vector containing a gene encoding a protein having cis-aconitic acid decarboxylase activity, and as a result, expresses cis-aconitic acid decarboxylase activity. There is no particular limitation as long as it is an organism that can be used. One skilled in the art can select an appropriate host and vector combination. Examples of the host include bacteria such as Escherichia coli (eg, Escherichia coli JM109 strain), Streptococcus, Staphylococcus, Enterococcus, Corynebacterium, and Bacillus. (Bacillus) (eg, Bacillus subtilis), Streptomyces, etc .; fungal cells, eg, Aspergillus, etc .; yeast cells, eg, baker's yeast, methanol-utilizing yeast, etc .; insect cells, eg, Drosophila S2 (Drosophila Schneider-2), Spodoptera Sf9, etc .; mammalian cells including human cultured cells, such as CHO, COS, BHK, 3T3, C127, etc .; or competent cells thereof, preferably E. coli competent Cell.

  Transformation can be performed by a known method such as calcium chloride / rubidium chloride method, calcium phosphate method, DEAE-dextran mediated transfection, or electroporation. Specifically, for example, a transformed microorganism can be obtained by bringing a recombinant vector containing the gene of the present invention into contact with competent cells of Escherichia coli JM109 (hereinafter also referred to as E. coli JM109).

The above method can be performed based on a conventional method of genetic engineering experiment. Information on vectors of various microorganisms such as E. coli and actinomycetes and methods for introducing and expressing foreign genes are described in many experimental documents (for example, Sambrook, J., Russel, DW, Molecular Cloning A Laboratory Manual). ^{, 3 rd Edition, CSHL Press,} 2001; Hopwood in DA, Bibb, MJ, Chater, KF, Bruton, CJ, Kieser, HM, Lydiate, DJ, Smith, CP, Ward, JM, Schrempf, H.Genetic manipulation of Streptomyces : A laboratory manual, The John lnnes institute, Norwich, UK, 1985, etc.), vector selection, gene introduction and expression can be performed according to them.

A microorganism transformed with a recombinant vector containing the gene encoding CAD (hereinafter simply referred to as a transformed microorganism or transformed microorganism) is a microorganism culture medium or the like described below, and is cultured at a culture temperature of about 15 to 37 ° C. The culture is preferably performed at a pH of about 5.0 to 8.0 for about 1 to 7 days.
In addition, the transformed microorganism can be mutated by an artificial mutagenesis method using ultraviolet rays, X-rays, chemicals, or the like, and any mutant strain obtained in this way is the CAD object of the present invention. As long as it has productivity, it can be used as the transformed microorganism of the present invention.

  The microorganism culture medium can be preferably used as long as it is a medium used for normal microorganism culture. For example, a natural medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts, and other nutrient substances is used. It is done.

  Examples of the carbon source include glucose and sugar alcohol such as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, galactose, starch, molasses, sorbitol or glycerin, acetic acid, citric fermentation, lactic acid, fumaric acid, maleic acid or Examples include organic acids such as gluconic acid, alcohols such as ethanol or propanol, and the like. Moreover, hydrocarbons, such as normal paraffin, etc. can be used if desired. A carbon source may be used individually by 1 type, and may mix and use 2 or more types. The concentration of these carbon sources in the medium is usually about 0.1 to 10% (wt).

  Nitrogen sources include, but are not limited to, nitrogen compounds such as inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate, urea, aqueous ammonia, sodium nitrate, or potassium nitrate. In addition, corn steep liquor, meat extract, bepton, NZ-amine, protein hydrolyzate, nitrogen-containing organic compounds such as amino acids, and the like can also be used. A nitrogen source may be used individually by 1 type, and may mix and use 2 or more types. The medium concentration of the nitrogen source varies depending on the nitrogen compound used, but is usually about 0.1 to 10% (wt).

  Examples of the inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate. These inorganic salts may be used alone or in a combination of two or more. The medium concentration of inorganic salts varies depending on the inorganic salt used, but is usually about 0.01 to 1.0% (wt).

Examples of the nutrient substance include meat extract, peptone, polypeptone, yeast extract, dry yeast, corn steep liquor, skim milk powder, defatted soy hydrochloride hydrolyzate, extracts of animals and plants or microbial cells, and degradation products thereof. The medium concentration of the nutrient substance varies depending on the nutrient substance used, but is usually about 0.1 to 10% (wt). Furthermore, vitamins can be added as necessary. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid and the like.
The pH of the medium is preferably about 5.0 to 8.0.
Preferred microorganism culture media include LB (Luria-Bertani) medium, NZYM medium, TB (Terrific Broth) medium, SOB medium, 2 × YT medium, AHC medium, χ1776 medium, M9 medium, YPD medium, SD medium, YPAD medium. Or a super broth culture medium etc. are mentioned.

v) Itaconic acid production reaction by expressed CAD The itaconic acid synthesis reaction of the present invention is carried out by contacting the isolated and purified CAD which is a preparation of the transformed microorganism of the present invention with cis-aconitic acid, or the transformation of the present invention. It can be carried out as a one-step synthesis reaction by contacting cis-aconitic acid as a metabolite in microbial cells with expressed CAD.
To prepare an enzyme solution containing CAD from the transformed microorganism, the collected supernatant of the centrifuged microorganism is disrupted with, for example, ultrasonic waves, glass beads, or commercially available E. coli lysate in an aqueous medium. Can be recovered. Further, CAD can be purified from the supernatant by a known technique.

  The reaction for producing itaconic acid from cis-aconitic acid by CAD is preferably carried out at a pH of about 5 to 7 and at a temperature of 4 to 50 ° C, particularly about 10 to 40 ° C. The reaction time is usually preferably 1 to 48 hours.

  The present invention will be specifically described below based on examples. However, the present invention is not limited to these examples.

[Example 1]
Isolation of cis-aconitic acid decarboxylase (CAD) from Aspergillus terreus NBRC6123 strain Aspergillus terreus NBRC6123 strain was cultured at 30 ° C. in the solid medium shown in Table 1. This was inoculated into a liquid medium shown in Table 2 and statically cultured at 30 ° C. for 7 days. The cultured cells were separated from the culture solution using a filter cloth (trade name: Miracloth, manufactured by Calbiochem) and washed carefully with pure water. The cells thus obtained were sufficiently drained, frozen at −80 ° C. and then lyophilized. About 2 g of quartz sand was added to about 2 g of the dried cells and pulverized with a mortar. To this, 25 ml of a buffer solution (0.2 M phosphate buffer pH 6.5, 1 mM DTT, 1 mM EDTA, 1 μM pepsin) was added, homogenized, and centrifuged (5000 g, 30 minutes) to obtain a total soluble enzyme solution. . A 30 to 60% ammonium sulfate precipitate fraction was recovered from this enzyme solution. The obtained precipitate was suspended in 5 ml of Buffer 1 (50 mM phosphate buffer pH 6.5, hereinafter referred to as Buffer 1), and this was desalted by equilibration with Buffer 1 (trade name). : 10DG column, manufactured by Bio-Rad). This was added to an anion exchange column (trade name: DEAE-HiPrep 16/10 column, manufactured by GE Healthcare Biosciences) that had been equilibrated with Buffer 1. Thereafter, the column was washed with 20 ml of buffer solution 1 and gradient elution (200 ml) with a sodium chloride concentration of 0 to 0.5 M was performed to collect fractions with a sodium chloride concentration of 0.18 to 0.35 M including CAD activity. In addition, the measurement of CAD activity was performed by quantifying the produced itaconic acid using Hartford's method (Analytical chemistry, 426-428, 34, 1962) using cis-aconitic acid as a substrate. A hydrophobic interaction column (ammonium sulfate was added to this fraction to a final concentration of 2M and equilibrated in advance with buffer 2 (50 mM phosphate buffer pH 6.5, 2M ammonium sulfate, hereinafter referred to as buffer 2) ( It was added to a Resource ISO column (manufactured by GE Healthcare Bioscience). Thereafter, after washing with 5 ml of buffer 2, gradient elution (45 ml) with an ammonium sulfate concentration of 2 to 0 M was performed to collect fractions containing CAD activity. The recovered fraction was desalted by the method shown above and then added to a chromatography column (trade name: Bio-Scale CHT2-I column, manufactured by Bio-Rad) that had been equilibrated with buffer 1 in advance. Thereafter, after washing with 5 ml of buffer 3 (5 mM phosphate buffer pH 6.5, hereinafter referred to as buffer 3), gradient elution (50 ml) with a phosphate concentration of 0 to 250 mM is performed, and 51 to 53 containing CAD activity. The 5 mM fraction was collected. The enzyme solution thus obtained was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and showed a unique band at a molecular weight of about 50 kDa. The purified enzyme was transferred to a PVDF membrane (manufactured by Bio-Rad) after SDS-PAGE and subjected to amino acid sequence analysis.

  The amino acid sequence analysis is performed by proteolytic enzyme (lysyl endopeptidase), amino terminal sequencing is performed on the resulting peptide fragment, protein sequencer (trade name: Procise 494HT Protein Sequencing System, manufactured by Applied Biosystems) It was performed using. As a result, the amino acid sequences of SEQ ID NOs: 3 to 5 were determined.

[Example 2]
Isolation of cis-aconitic acid decarboxylase gene Using the identified amino acid sequence, homology search was performed on the genome sequence of Aspergillus tereus NIH2624 strain. As a result, all of the three amino acid fragments (SEQ ID NOs: 3 to 5) identified in Example 1 were used for a putative gene with unknown function existing in the vicinity of lovF, which is one of the lovastatin biosynthesis genes of Aspergillus terreus NIH2624 strain. It was found that the homology was very high. Therefore, primers 1 and 2 (SEQ ID NOs: 6 and 7) were prepared based on the base sequence of this putative gene, and cis-aconitic acid decarboxylase gene cDNA was amplified by PCR (polymerase chain reaction). First, Aspergillus terreus was cultured in a liquid medium as described above, and the cells were collected after 6 days. Isolation of total RNA uses about 0.1 g (wet weight) of bacterial cells by the AGPC (acid guanidinium phenol-chloroform) method (Chonczynski P., Sacchi N., Analytical Biochemistry, 162, 156-159, 1987). I went. The isolated total RNA was recovered by fractionating polyA RNA using an RNA purification kit (trade name: oligotex-dT30 <Super> mRNA Purification Kit, manufactured by Takara). 60 ng of PolyA RNA was reverse-transcribed with Superscript II (manufactured by Invitrogen) using primer 3 (SEQ ID NO: 8) according to the instruction manual to obtain single-stranded cDNA (first strand cDNA). To 1 μl of the synthesized single-stranded cDNA solution, 0.2 μM of primers 1 and 2 (SEQ ID NOs: 6 and 7), 0.2 mM of dNTP (deoxyribonucleoside triphosphate), 2 U of PCR enzyme (Ex-Taq, manufactured by Takara) ) 3 μl of buffer (10 × Ex-Taq buffer, manufactured by Takara) was added to make the final volume 30 μl. For PCR, a thermal cycler (trade name: GeneAmp PCR System 9700, manufactured by Applied Biosystems) was used to perform reaction 1 (94 ° C. for 30 seconds, 58 ° C. for 30 seconds, 72 ° C. for 1.5 minutes, 30 minutes). Cycle: reaction solution, dNTP 0.2 mM, Ex-Taq 2U, 10 × Ex-Taq buffer 3 μl, primer 0.2 μM, final solution volume 30 μl, hereinafter referred to as reaction 1). The resulting reaction solution was subjected to agarose electrophoresis, and then a DNA fragment of about 1.5 kbp was recovered from the gel using a DNA gel extraction kit (trade name: Minelute Gel Extraction Kit, manufactured by Qiagen). The DNA fragment was ligated to a pGEM-T vector (Promega) according to the instruction manual, Escherichia coli JM109 strain was transformed using this vector, and the colony containing the target DNA fragment was sequenced to sequence the DNA sequence. Were determined. The DNA sequencer was ABI PRISM 3100 (Applied Biosystems), and the sequence reaction was ABI PRISM Cycle sequencing Kit (Applied Biosystems).
The full-length cDNA sequence was determined by 5'- and 3'-RACE (rapid amplification of cDNA ends) -PCR. In addition, 5'-RACE-PCR was performed according to the instruction manual using 5'-Full RACE Core Set (manufactured by Takara). First, primer 4 (SEQ ID NO: 9) was prepared based on the previously determined base sequence of partial CAD cDNA, and single-stranded cDNA was synthesized. After concatemerizing the single-stranded cDNA thus obtained, PCR was carried out using primers 5 and 6 (SEQ ID NOs: 10 and 11) using 1 μl of a 100-fold dilution as a template. PCR reaction conditions were 30 cycles of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds. PCR was performed using primers 1 and 8 (SEQ ID NOS: 12 and 13) using 1 μl of the reaction solution diluted 100-fold as a template. The PCR reaction conditions were 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 30 seconds, and 22 cycles. 3′-RACE-PCR was carried out under the conditions of reaction 1 using primers 1 and 9 (SEQ ID NOs: 6 and 14) using 1 μl of single-stranded cDNA used in 5′-RACE-PCR as a template. The reaction solution obtained as a result of the PCR shown above was subjected to agarose electrophoresis and then about 0.6 kbp (5 mg) using a DNA gel extraction kit (trade name: Minelute Gel Extraction Kit, manufactured by Qiagen). '-RACE-PCR) and 1.5 kbp (3'-RACE-PCR) DNA fragments were recovered from the gel. The DNA fragment was ligated to a pGEM-T vector (Promega) according to the instruction manual, Escherichia coli JM109 strain was transformed using this vector, and the colony containing the target DNA fragment was sequenced to sequence the DNA sequence. Were determined. The sequence obtained in this way has the same sequence over the base sequence of the obtained CAD gene fragment over about 300 bp, and the full-length CAD gene cDNA sequence (SEQ ID NO: 1) is determined by linking these sequences. I was able to. Further, the CAD amino acid sequence (SEQ ID NO: 2) deduced from the sequence after ligation had the N-terminal amino acid sequence determined by the purified enzyme (SEQ ID NO: 3 to 5, amino acid 61 in SEQ ID NO: 2). -68, 218-229, 392-399).

[Example 3]
Expression of cis-aconitic acid decarboxylase gene in E. coli DNA fragments having NdeI and HindIII recognition sequences added to the 5'- and 3'-ends of CAD cDNA were used as primers 10, 11 (SEQ ID NOs: 15 and 16). Used and amplified under the conditions of Reaction 1. The amplification product was ligated to the pGEM-T vector by the method described above. A DNA fragment containing CAD cDNA excised from Ndel and HindIII from the plasmid thus prepared was prepared, and a plasmid pCAD was prepared by inserting this into the NdeI-HindIII site of the expression vector pColdI (manufactured by Takara). The prepared plasmid is shown in FIG. This plasmid was introduced into Escherichia coli JM109 strain to obtain a transformant Escherichia coli JM109 / cad001 strain. Escherichia coli JM109 / cad001 strain was deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST), 1st-6th, 1st East, Tsukuba City, Ibaraki, Japan (zip code 305-8666). December 27, 2006, accession number: FERM P-21146). The transformed strain Escherichia coli JM109 / cad001 strain was grown at 37 ° C. in 100 ml of LB liquid medium until the turbidity reached 0.5, then the culture temperature was 15 ° C., and after 1 hour, IPTG (Isopropyl-β -D-thiogalactopyranoside) was added to a concentration of 0.1 mM to induce CAD protein expression. After 24 hours, the cells were collected (dry cell weight: 39 mg). This bacterial cell was suspended in 1.8 ml of 50 mM phosphate buffer pH 7.0, 200 μl of bacterial cell lysis buffer (trade name: FastBreak Cell Lysis Reagent, manufactured by Promega) was added and mixed by inversion. After 15 minutes, insoluble materials were separated by centrifugation (10,000 g, 5 minutes) to obtain a cell extract. The cell extract thus prepared contained 35700 units of CAD (1 Unit is the amount of enzyme that converts 1 μmol of aconitic acid to itaconic acid per minute). The separation and purification of the recombinant CAD enzyme was performed using a recombinant protein purification kit (trade name: Ni-NTA Fast Start Kit, manufactured by Qiagen). A final concentration of 0.3 M sodium chloride and 5 mM imidazole were added to the prepared cell extract. This solution is added to a Ni-NTA column and washed with 8 ml of washing solution (50 mM phosphate buffer pH 7, 0.3 M sodium chloride, 60 mM imidazole), and then 2 ml of eluent (50 mM phosphate buffer pH 7, 0). The recombinant CAD was eluted with 3M sodium chloride, 200 mM imidazole). The analysis results of purified CAD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis are shown in FIG. The CAD expressed by Escherichia coli JM109 / cad001 strain can obtain CAD with the same degree of purity by a much easier method than that obtained by separating and purifying CAD from Aspergillus terreus NBRC6123 strain (Example 1). all right. In FIG. 2, the CAD (lane 3) separated and purified in Example 3 has a molecular weight of about 1. 5 compared with the CAD (lane 2) separated and purified from Aspergillus terreus NBRC6123 strain by adding His-tag sequence or the like. It is 3 kDa larger.

[Example 4]
Effect of pH on the activity of recombinant CAD A recombinant CAD enzyme was prepared and purified in the same manner as in Example 3. The effect of pH on the activity of recombinant CAD was examined using an acetic acid seed buffer at pH 5-6 and a phosphate buffer at pH 6-8. The composition of the reaction was 130 μl 0.2 M acetic acid or phosphate buffer, 50 μl (6 μg) purified CAD, 20 μl 10 mM cis-aconitic acid. The reaction was performed at 30 ° C. for 1 hour. The reaction solution was analyzed by the aforementioned Hartford method, and itaconic acid was quantified. The result is shown in FIG. From this result, it became clear that pH 6.0 vicinity is reaction conditions which exhibit the highest CAD activity.

[Example 5]
Expression of cis-aconitic acid decarboxylase gene by Corynebacterium sp. A recombinant CAD expression system was produced using the gram-positive bacterium Corynebacterium glutamicum widely used in industry as a host. DNA fragments having EcoRI and BamHI recognition sequences added to the 5′- and 3′-ends of CAD cDNA were amplified under the conditions of reaction 1 using primers 12 and 13 (SEQ ID NOs: 17 and 18). The amplified product was ligated to the pGEM-T vector by the method described above, and the DNA sequence was confirmed. A DNA fragment containing CAD cDNA excised from EcoRI and BamHI was prepared from the plasmid thus prepared, and a plasmid pCAD2 was prepared by inserting it into the EcoRI-BamHI site of the corynebacterium expression vector pCRB1 (FIG. 4). This plasmid was introduced into Corynebacterium glutamicum R strain (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary FERM P-18976) to obtain a transformed strain Corynebacterium glutamicum R / cad002. Corynebacterium glutamicum R / cad002 strain was deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST), 1st, 1st, 1st-6, Tsukuba, Ibaraki, Japan (zip code 305-8666). Date: December 27, 2006, accession number: FERM P-21147). The transformed strain was cultured aerobically at 33 ° C. in 100 ml of the liquid A medium shown in Table 3. After about 24 hours, the cells were collected by centrifugation (5000 g, 5 minutes) (dry cell weight: 80 mg). The collected cells are suspended in 5 ml of 50 mM phosphate buffer pH 7.0, about 0.5 g of glass beads are added, and then 15 minutes by an ultrasonic crusher (trade name: Biorupter, manufactured by Cosmo Bio). It was crushed. Insoluble material was separated by centrifugation (10,000 g, 5 minutes) to obtain a cell extract. The cell extract thus obtained contained 19300 units of CAD.

[Comparative example]
The production rate of recombinant CAD using Escherichia coli JM109 / cad001 strain and Corynebacterium glutamicum R / cad002 strain obtained in Examples 3 and 5 as a host was compared with that of Aspergillus tereus NBRC6123 strain. . Aspergillus terreus was cultured according to the conditions shown in Example 1. That is, Aspergillus terreus NBRC6123 strain was inoculated into three 500 ml flasks containing 70 ml of itaconic acid production medium (described in Table 2 above) and cultured at 30 ° C. for 6 days. The cells were washed and collected using a filter cloth (trade name: Miracloth) and then freeze-dried (dry cell weight: 733 mg). From this microbial cell, 66200 unit of CAD was obtained from the crude enzyme solution (10 ml) by the method shown in Example 1. The recombinant CAD production rate by Escherichia coli JM109 / cad001 strain and Corynebacterium glutamicum R / cad002 strain was calculated from the results shown in Examples 3 and 5. As a result, the productivity of Escherichia coli JM109 / cad001 strain is about 61 times higher, and that of Corynebacterium glutamicum R / cad002 strain is about 16 times higher than that of Aspergillus terreus NBRC6123 strain. It became clear that. Table 4 shows a comparison result of the production rate of itaconic acid.

  According to the present invention, itaconic acid can be produced with high efficiency using a recombinant microorganism or a preparation thereof that has introduced and highly expressed a gene encoding cis-aconitic acid decarboxylase (CAD), a synthetic resin, an adhesive, It can be widely used as a raw material for chemical industry, such as detergent, latex, paint, printing ink and acrylic fiber modifier.

FIG. 1 shows the vector pCAD prepared in Example 3. FIG. 2 shows the results of polyacrylamide gel electrophoresis of CAD purified from Aspergillus terreus NBRC6123 strain (lane 2) described in Example 1 and Escherichia coli JM109 / cad001 strain (lane 3) described in Example 3 (lane 3). Lane 1 shows molecular weight markers). FIG. 3 shows the optimum pH for CAD activity. FIG. 4 shows the vector pCAD2 prepared in Example 5.

Claims (8)

Genes shown in the following (a), (b), (c) or (d):
(A) a gene comprising DNA having the base sequence shown in SEQ ID NO: 1
(B) consisting of a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA of the base sequence shown in SEQ ID NO: 1 and encodes a protein having cis-aconitic acid decarboxylase activity gene:
(C) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2:
(D) From an amino acid sequence shown in SEQ ID NO: 2 consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and encoding a protein having cis-aconitic acid decarboxylase activity The gene that becomes. Proteins shown in the following (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2:
(B) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2 and having cis-aconitic acid decarboxylase activity.   A recombinant vector containing the gene according to claim 1.   A microorganism obtained by transforming a host with the recombinant vector according to claim 3.   The microorganism according to claim 4, wherein the host is selected from Escherichia coli, Corynebacterium, and yeast.   The microorganism according to claim 5, which is Escherichia coli JM109 / cad001 strain (Accession No. FERM P-21146).   The microorganism according to claim 5, wherein the microorganism is Corynebacterium glutamicum R / cad002 strain (Accession No. FERM P-21147).   The microbe or preparation thereof according to any one of claims 4 to 7 is brought into contact with a liquid containing cis-aconitic acid, thereby producing and accumulating itaconic acid in the liquid, and collecting this. A method for producing itaconic acid.