JP2010057396A - New microorganism having putrescine synthesis ability, putrescine synthesis gene and production method of putrescine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism having high productivity of putrescine as compared to that of conventional microorganisms and to provide a putrescine synthesis gene having excellent putrescine synthesis ability. <P>SOLUTION: The microorganism has a 16S rDNA sequence having ≥90% homology to a base sequence made of a specific sequence and belongs to the genus Citrobacter having ability of producing putrescine. Examples of those microorganisms belonging to Citrobacter include Citrobacter sp. PR-144-5-1 strain (accession number:NITE P-620). <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a microorganism capable of producing putrescine (1,4-diaminobutane) as a raw material for nylon 46, a putrescine synthesis gene, and a method for producing putrescine using the microorganism or the gene.

  Nylon 46 is made of 1,4-diaminobutane (hereinafter referred to as putrescine) or a functional derivative thereof and adipic acid or a functional derivative thereof, and is characterized by higher heat resistance than a general-purpose polyamide centering on nylon 66. To do. In addition to excellent heat resistance, nylon 46 also has excellent mechanical strength such as tensile strength, bending strength, impact strength, sliding properties, and fatigue resistance. The use value is large, and demand is expanding as useful engineering plastics.

  Putrescine, which is a raw material of nylon 46, is chemically synthesized by reacting acrylonitrile with hydrogen cyanide to succinic nitrile, and then adding hydrogen (Non-patent Document 1). However, this chemical synthesis uses highly toxic hydrogen cyanide, which has a large environmental burden and high manufacturing costs.

  On the other hand, there have been reports on microbiological methods for producing putrescine, and Escherichia coli (Non-patent Document 2) and lactic acid bacteria (Non-patent Document 3) are used. However, conventional microorganisms have a low production capacity, which is about 0.3 gram per liter of the culture solution after 10 days of culture, and the produced putrescine is metabolized and accumulated in the bacteria or in the culture solution. I can't. Furthermore, microorganisms belonging to the genus Enterobacter that synthesize putrescine have also been reported (Patent Document 1). The microorganism disclosed in Patent Document 1 has a very excellent ability to synthesize putrescine compared to the microorganisms disclosed in Non-Patent Documents 1 to 3, but in order to perform putrescine production with higher efficiency, A microorganism having a better ability to synthesize putrescine and a novel gene involved in the synthesis of putrescine are desired.

Fine Chemical Market Data, Volume 1, 1999, CMC Corporation The Acetylation of Polyamines in Escherichia coli ’DONALD T. DUBIN AND SANFORD M. ROSENTHAL The Jounal of Biological Chemistry Vol.235, No.3, March 1960 M.E.Arena, et al., Journal of Applied Microbiology 2001, 90 (2), 158-162 JP 2006-191808 A

  Therefore, the present invention provides a microorganism having a higher ability to produce putrescine than conventional microorganisms, provides a gene involved in putrescine synthesis, and efficiently and inexpensively putrescine using the microorganism or the gene. The purpose is to provide a production method.

  The present inventor conducted extensive studies to solve the above problems, and screened microorganisms collected from various environments using the ability to synthesize putrescine. As a result, among the microorganisms belonging to the genus Citrobacter, the production of putrescine was dramatically high. The present inventors have found a microorganism exhibiting ability and completed the present invention.

  That is, the novel microorganism according to the present invention is a microorganism belonging to the genus Citrobacter having a 16S rDNA sequence that is 90% or more homologous to the base sequence shown in SEQ ID NO: 1 and having the ability to produce putrescine. Examples of the microorganism belonging to the genus Citrobacter according to the present invention include Citrobacter sp. PR-144-5-1 strain (deposit number: NITE P-620).

The putrescine synthetic gene according to the present invention encodes any one of the following proteins (a) to (c).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 3 (b) consisting of an amino acid sequence in which one or a plurality of amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and the ability to synthesize putrescine (C) a protein comprising an amino acid sequence having a homology of 95% or more with respect to the amino acid sequence shown in SEQ ID NO: 3, and having a putrescine synthesis ability

The putrescine synthetic gene according to the present invention includes, for example, any of the following polynucleotides (a) to (c).
(A) a polynucleotide comprising the base sequence shown in SEQ ID NO: 2 (b) a base sequence shown in SEQ ID NO: 2 consisting of a base sequence in which one or more bases are deleted, substituted, added or inserted, and putrescine synthesis A polynucleotide encoding a protein having the ability to synthesize putrescine, comprising a base sequence that hybridizes under stringent conditions to a complementary strand of the base sequence shown in SEQ ID NO: 2 nucleotide

  Furthermore, the method for producing putrescine according to the present invention includes the above-described microorganism according to the present invention or the above-described microorganism expressing the putrescine synthetic gene according to the present invention in a medium to which a substrate for producing putrescine and a nitrogen source are added. Culturing and collecting putrescine from the culture. Here, examples of the substrate for producing putrescine include arginine and ornithine.

  According to the present invention, a microorganism having high ability to produce putrescine is provided. Therefore, by culturing this microorganism with a substrate for producing putrescine, putrescine, which is a raw material of nylon 46 useful as an engineering plastic, can be produced at high cost at a low cost.

  In addition, according to the present invention, a novel putrescine synthesis gene excellent in putrescine synthesis ability is provided. Therefore, by using this novel putrescine synthesizing gene, putrescine can be produced with higher efficiency than in the case of using a conventionally known putrescine synthesizing gene.

Hereinafter, the present invention will be described in detail.
1. Microorganism having the ability to produce putrescine The microorganism of the present invention is a microorganism having a 16S rDNA sequence that is 90% or more homologous to the nucleotide sequence shown in SEQ ID NO: 1 and having the ability to produce putrescine. As an example of such a microorganism, PR-144-5-1 strain isolated from a soil sample collected by the present inventors in Okazaki City, Aichi Prefecture can be mentioned.

  The base sequence of 16S rDNA gene of PR-144-5-1 strain is shown in SEQ ID NO: 1. Based on the nucleotide sequence shown in SEQ ID NO: 1, identification was performed using NCBI program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). PR-144-5-1 The strain was found to belong to the genus Citrobacter. That is, the base sequence of 16S rDNA gene of PR-144-5-1 strain is included in the cluster formed by 16s rDNA of Citrobacter, and shows 99.0% homology with 16S rDNA gene of Citrobacter murliniae CDC2970-59 strain, Citrobacter braakii showed a 98.9% homology with the 16S rDNA gene of CDC080-58. Further, only Citrobacter-derived 16s rDNA showed a homology rate of PR-144-5-1 with the 16S rDNA gene of 97.0% or more. Therefore, PR-144-5-1 strain belongs to the genus Citrobacter but was not classified as a known species. Therefore, this strain was named Citrobacter sp. PR-144-5-1 strain. This strain was deposited under the accession number NITE P-620 on July 31, 2008 at the National Institute of Technology and Evaluation (NPMD) (2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture). ing.

  In addition to the PR-144-5-1 strain, the microorganisms according to the present invention include related microorganisms. As a related microorganism of PR-144-5-1 strain, for example, the nucleotide sequence of 16S rDNA gene is 90% or more, preferably 95% or more, more preferably 98% or more, most preferably, the nucleotide sequence of SEQ ID NO: 1. Refers to a microorganism that is 99% or more homologous and has a high ability to produce putrescine. Here, the high productivity of putrescine means, for example, the ability to produce 0.1 g / L or more, preferably 0.5 g / L or more of putrescine per day.

  A related microorganism of the PR-144-5-1 strain can be isolated using the above-mentioned putrescine production ability and the base sequence described in SEQ ID NO: 1 as indicators. For example, as shown in the Examples below, a soil sample is added to a medium containing arginine and cultured under anaerobic conditions, and the culture solution is subjected to GC / MS analysis to select a soil sample in which high production of putrescine is recognized. (Primary screening). Next, the culture solution of the selected soil sample is cultured on a plate, colonies are formed and microorganisms are isolated.The isolated microorganisms are cultured under anaerobic conditions, and GC / MS analysis is performed to increase the production of putrescine. Recognized microorganisms (secondary screening), and the nucleotide sequence database of NCBI program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) for the 16S rDNA nucleotide sequence of this microorganism strain A homology search is performed on.

  The microorganism of the present invention can be cultured and propagated in the same manner as known microorganisms belonging to the genus Citrobacter. As the medium, any of a natural medium and a synthetic medium may be used as long as the medium appropriately contains an assimitable carbon source, nitrogen source, inorganic salts, and necessary nutrient sources. Any carbon source may be used as long as the microorganism can assimilate, carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid, propionic acid, maleic acid, fumaric acid, and malic acid, ethanol, propanol, and the like Alcohols are used. Examples of nitrogen sources include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or nitrogen-containing compounds such as peptone, yeast extract, malt extract, meat extract, and corn steep liquor. Used. As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

  What is necessary is just to set the culture temperature to the range of the growth temperature of the microorganisms of this invention, Preferably the range of the optimal growth temperature, for example, the range of 25-40 degreeC is mentioned. The culture time is usually about 24 to 72 hours. Moreover, it is preferable to maintain pH at 5.0-7.0 during a culture | cultivation period. The pH of the medium is adjusted using an inorganic or organic acid, an alkaline solution, or the like.

2. The putrescine synthetic gene of the present invention can be identified based on the base sequence of the SpeF gene of E. coli in the genome of the microorganism of the present invention described above. The genome of the microorganism according to the present invention described above can be extracted and isolated according to a conventional method using a probe or primer set designed based on the base sequence of the SpeF gene of Escherichia coli. The SpeF gene of Escherichia coli encodes ornithine decarboxylase, and a paper by Kashiwagi, K. et al. (J. Biol. Chem. "Coexistence of the genes for putrescine transport protein and ornithine decarboxylase at 16 min on Escherichia coli chromosome. (1991), Volume 266, p20922-20927).

  Among the microorganisms according to the present invention, the base sequence of the putrescine synthesis gene isolated from Citrobacter sp. PR-144-5-1 strain and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 2 and 3, respectively. That is, as an example of the putrescine synthetic gene according to the present invention, a polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 3 can be mentioned. Similarly, as an example of the putrescine synthetic gene according to the present invention, a polynucleotide comprising the base sequence shown in SEQ ID NO: 2 can be mentioned.

  However, the putrescine synthetic gene according to the present invention is not limited to the one encoding the protein having the amino acid sequence shown in SEQ ID NO: 3. The putrescine synthetic gene according to the present invention encodes a protein having the ability to synthesize putrescine, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3. It may be a thing. Here, examples of the plurality of amino acids include 2 to 70 amino acids, preferably 2 to 50, more preferably 2 to 30, further preferably 2 to 15, most preferably Mention may be made of 2 to 10 amino acids.

  In addition, amino acid deletion, substitution, or addition can be performed by modifying the base sequence encoding the gene by a technique known in the art. Mutation can be introduced into a base sequence by a known method such as Kunkel method or Gapped duplex method or a method according thereto, for example, a mutation introduction kit (for example, Mutant-) using site-directed mutagenesis. Mutations are introduced using K, Mutant-G (both trade names, manufactured by TAKARA) or the like, or LA PCR in vitro Mutagenesis series kit (trade name, manufactured by TAKARA).

  Furthermore, examples of the putrescine synthesis gene according to the present invention include an amino acid sequence having 95% or more homology with the amino acid sequence shown in SEQ ID NO: 3 and encoding a protein having putrescine synthesis ability. . The amino acid sequence shown in SEQ ID NO: 3 has 91.7% homology to the amino acid sequence of the protein encoded by the putrescine synthesis gene derived from E. coli. Despite having such a high homology, the protein consisting of the amino acid sequence shown in SEQ ID NO: 3 is superior to the protein encoded by the putrescine synthesis gene derived from E. coli. Have the ability. Therefore, a protein comprising an amino acid sequence having 95% or more homology, preferably 98% or more homology, more preferably 99% or more homology based on the amino acid sequence shown in SEQ ID NO: 3 is very It is considered to have excellent ability to synthesize putrescine. The homology value means a value obtained by default setting using a computer program in which the blast algorithm is implemented and a database storing gene sequence information.

  Furthermore, the putrescine synthetic gene according to the present invention is not limited to the one containing the polynucleotide consisting of the base sequence shown in SEQ ID NO: 2, and for example, in the base sequence shown in SEQ ID NO: 2, one or more bases Examples include those comprising a nucleotide sequence consisting of a base sequence that is deleted, substituted, added or inserted and encoding a protein having the ability to synthesize putrescine. Here, examples of the plurality of bases include 2 to 200 bases, preferably 2 to 150, more preferably 2 to 100, still more preferably 2 to 50, and most preferably. 2-30 bases can be mentioned. The method for introducing a mutation into the base sequence is the same as that described above.

  Furthermore, the putrescine synthesizing gene according to the present invention comprises a base sequence that hybridizes under stringent conditions to the complementary strand of the base sequence shown in SEQ ID NO: 2, and encodes a protein having the ability to synthesize putrescine. It may contain a polynucleotide. Here, hybridizing under stringent conditions means maintaining binding at 60 ° C. under 2 × SSC washing conditions. Hybridization can be performed by a conventionally known method such as the method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

  The putrescine synthetic gene according to the present invention described above can be incorporated into an expression vector so that it can be expressed in an appropriate host according to a conventional method. Although it does not specifically limit as a host, Bacteria, such as colon_bacillus | E._coli and Bacillus subtilis, yeast, a plant cell, an insect cell, and an animal cell are mentioned. The expression vector can be appropriately selected depending on the host to be used, and examples thereof include plasmids, shuttle vectors, helper plasmids and the like.

  As plasmid DNA, plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YCp50, etc.) And λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.

  In order to insert the putrescine synthetic gene according to the present invention into a vector, first, a purified polynucleotide containing the putrescine synthetic gene is cleaved with an appropriate restriction enzyme and inserted into a restriction enzyme site or a multiple cloning site of an appropriate vector DNA. Then, a method of connecting them is adopted. The putrescine synthetic gene according to the present invention may be linked to a control region capable of controlling gene expression in an expression vector. Examples of the control region include conventionally known promoters and enhancers.

3. Production of putrescine using the microorganism of the present invention or putrescine synthetic gene The microorganism according to the present invention described above and the putrescine synthetic gene according to the present invention can be used for the production of putrescine. The microorganism strain obtained in the above 1 can be cultured in a medium to which a substrate for producing putrescine and a nitrogen source are added to produce putrescine. Alternatively, the host into which the putrescine synthetic gene obtained in 2 above has been introduced can be similarly cultured in a medium to which a substrate for producing putrescine and a nitrogen source have been added to produce putrescine. The substrate for producing putrescine is preferably arginine, but may be ornithine. Moreover, although it does not specifically limit as a nitrogen source, Natural nitrogen sources, such as a yeast extract and a meat extract, are preferable among the said nitrogen sources. The culture is performed at 25 to 40 ° C. for about 24 to 72 hours, for example. In the culture, it is preferable to use aerobic conditions.

  In addition, since the production of putrescine per unit culture solution and unit time is higher when a strain with higher growth ability is used, the cells are first activated by pre-culture, and this is used in the main culture for producing putrescine. It is preferable to inoculate the medium and culture.

  The medium only needs to contain at least a substrate and a nitrogen source, and the other carbon components, inorganic salts, and the like that are usually used in the technical field can be added to other components.

  The amount of the microbial strain used in the medium may be, for example, 0.1 to 2.0 mg, preferably 0.2 to 0.5 mg per 1 L of the medium when inoculated with bacterial cells, and may be appropriately increased or decreased depending on the substrate content. The total amount of the substrate may be added at the start of the culture, or may be added continuously or intermittently during the culture. Moreover, the produced putrescine may be taken out after completion of the culture, or may be taken out continuously or intermittently during the culture.

  The culture in the present invention can be terminated when the desired production amount of putrescine in the culture solution reaches the maximum, so that the putrescine in the culture solution can be gas chromatographed, high performance liquid chromatography, thin layer chromatography, etc. It is preferable to carry out the measurement while measuring by a known method.

  Putrescine accumulated in the culture solution is purified from the culture solution by a conventional method after completion of the culture. The purification may be performed by appropriately combining well-known means usually used in the technical field, for example, filtration, centrifugation, ion exchange or adsorption chromatography, solvent extraction, and the like as necessary. The form of the microorganism is not particularly limited, and may be either a microorganism cell or a treated cell of the microorganism. Examples of the processed microbial cells include crushed cells, enzymes extracted from cultures (bacteria and culture supernatant), and the like.

  The microbial cells or the processed microbial cells can be immobilized and used. Examples of the immobilization method include conventionally known methods such as a carrier binding method, a crosslinking method, and a comprehensive method. In the carrier binding method, cells or enzymes are immobilized on a carrier, but the immobilization may be any of physical adsorption, ionic bond, and covalent bond. As the carrier, polysaccharides (acetylcellulose, agarose), inorganic substances (porous glass, metal oxide), synthetic polymers (polyacrylamide, polystyrene) and the like are used. In the cross-linking method, the cells are immobilized by cross-linking and bonding the cells and enzymes using a bifunctional reagent such as glutaraldehyde. In the enveloping method, the enzyme and the fungus body are immobilized by wrapping them in a lattice or semipermeable membrane capsule of a polymer gel such as polysaccharide (alginic acid, carrageenan), polyacrylamide, collagen, polyurethane or the like.

  In the method for producing putrescine according to the present invention described above, since the microorganism according to the present invention described above or the putrescine synthetic gene according to the present invention is used, compared with the case where a conventionally known putrescine synthetic gene is used. Productivity is improved. In particular, when a recombinant using the putrescine synthetic gene according to the present invention, for example, recombinant Escherichia coli, 2.4 g / L or more of putrescine can be synthesized per day. This is a production difference of about 2.5 to 3.0 times or more when using recombinant Escherichia coli using a conventionally known putrescine synthesis gene (SpeF gene) derived from Escherichia coli.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, a novel microorganism having an excellent ability to synthesize putrescine was isolated. Soil samples collected in various parts of Japan are added to a liquid medium supplemented with a pH indicator having the composition shown in Table 1 below, and at 30 ° C. under a mixed gas (composition = nitrogen: carbon dioxide: hydrogen = 8: 1: 1). Cultured for 7 days. Since putrescine is alkaline, the culture solution in which the culture solution is changed from orange to red is mixed with a medium having the composition shown in Table 1 above with agar 1.5 g / L in a mixed gas (composition is the same as above). Cultured.

  Next, isolate the bacteria whose solid medium around the colony on the plate has changed from orange to red, and again culture the isolated bacteria in a liquid medium (Table 2) under a mixed gas (same composition as above), After the silylation treatment of the culture solution, it was analyzed with a gas chromatograph mass spectrometer (GC / MS) under the conditions shown in Table 3, and the putrescine production amount was measured.

  As a result, a strain capable of producing putrescine at high production (0.6 or more / L / 4 days) was obtained and named PR-144-5-1 strain. As of July 31, 2008, PR-144-5-1 shares were registered with NITE (National Institute of Technology and Evaluation) (NPMD) (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture) with the accession number NITE. Deposited as P-620.

Identification of PR-144-5-1 strain
The homology search of PR-144-5-1 strain by 16S rDNA base sequence was performed as follows. For the extraction of the genome, InstraGene Matrix (BIO RAD, CA, USA) was used, and the operation followed the protocol of BIO RAD. Using the extracted genomic DNA as a template, PCR (Ready-To-Go) using primer 9F (5′-GAGTTTGATCCTGGCTCAG-3 ′: SEQ ID NO: 4) and primer 1510R (5′-GGCTACCTTGTTACGA-3 ′: SEQ ID NO: 5) PCR Beads: Amersham Pharmacia Biotech, NJ, USA) was used to amplify a region of the entire base sequence 1500-1600 bp of the 16S ribosomal RNA gene (16S rDNA). Thereafter, the amplified 16S rDNA was sequenced (ABI PRISM 3100 DNA Sequencer: Applied Biosystems, CA, USA), and the base sequence of 16S rDNA was determined. The determined base sequence is shown in SEQ ID NO: 1.

  A homology search was performed using the base sequence of the obtained 16S rDNA. MicroSeq Microbial Identification System Software V.1.4.1 (Applied Biosystem, CA, USA) was used as a database for homology search. Furthermore, a homology search was performed on the GeneBank DNA base sequence database (GeneBank / DDBJ / EMBL) using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). As a result, the base sequence of 16S rDNA gene of PR-144-5-1 strain is included in the cluster formed by 16s rDNA of Citrobacter and shows 99.0% homology with 16S rDNA gene of Citrobacter murliniae CDC2970-59 strain The Citrobacter braakii CDC080-58 strain showed a 98.9% homology with the 16S rDNA gene. Further, only Citrobacter-derived 16s rDNA showed a homology rate of PR-144-5-1 with the 16S rDNA gene of 97.0% or more. Therefore, PR-144-5-1 strain belongs to the genus Citrobacter but was not classified as a known species. Therefore, this strain was named Citrobacter sp. PR-144-5-1 strain.

[Example 2]
In this example, the putrescine synthesis gene was cloned from the Citrobacter sp. PR-144-5-1 strain identified in Example 1. In this example, a medium (M11 medium) having the composition shown in Table 2 and a medium (LB medium and putrescine production medium) having the composition shown in Table 4 were used.

  Glcose was prepared at 20% concentration (autoclave sterilization) and added to the medium to 0.4%. Thiamine and Tryptophan were prepared in × 100 and sterilized with a 0.22 μl filter. Furthermore, Sol.1 and Sol.2 were prepared at x10 and sterilized by filter.

Although detailed description is omitted, a novel putrescine synthesis gene was identified from Citrobacter sp. PR-144-5-1 as follows. First, the first half of the new putrescine synthesis gene is Citrobacter sp. Amplified by PCR using genomic DNA extracted from PR-144-5-1 strain as a template, degenerate primers designed based on the SpeF gene base sequence in E. coli, ligated to the vector, and then base sequence determined did. As a result, about 1600 bp of the first half of the novel putrescine synthesis gene was identified.

  Next, in order to identify the latter half of the novel putrescine synthesis gene, a DNA fragment obtained by amplifying a part of the SpeF gene derived from E. coli was used as a probe for Southern hybridization. Southern hybridization was performed according to a standard method. That is, first, genomic DNA was extracted from Citrobacter sp. PR-144-5-1 strain cultured in M11 medium for 3-4 days using QIAGEN DNeasy Tissue Kit (manufactured by QIAGEN) according to the protocol. This genomic DNA was fragmented by a plurality of restriction enzyme treatments, subjected to agarose gel electrophoresis, and transferred to a membrane filter. Then, the fragment hybridized with the probe was excised, and then the excised fragment was used for self-ligation and impurse PCR to determine the base sequence of the putrescine synthetic gene.

Since the nucleotide sequence of the region containing the novel putrescine synthetic gene was determined from the above experiment, primers for amplifying the putrescine synthetic gene by PCR from the genomic DNA of Citrobacter sp. PR-144-5-1 were as follows: Designed.
-Primer 1 (SpeFF-B) 5'-CTGGATCCATGTCAGAATTAAAAATTGCCGTAAGCC-3 '(SEQ ID NO: 6)
-Primer 2 (SpeFR1-E) 5'-CTGAATTCCTCATAACATTTCCCCTTTCAACAGA-3 '(SEQ ID NO: 7)
Table 5 shows PCR using the genomic DNA of Citrobacter sp. PR-144-5-1 as a template and the above primer sets (Primer 1 (SpeFF-B) and Primer 2 (SpeFR1-E)). The reaction was carried out with the composition of the reaction solution (to a total of 100 μl with sterile water).

  PCR reaction conditions were 1 minute at 94 ° C, followed by 25 cycles (1 cycle at 98 ° C for 10 seconds, 68 ° C for 1 minute and 72 ° C for 3 minutes. As a result of PCR, about 2199 bp) The amplified DNA fragment was excised from the gel, treated with BamH I and EcoR I, cloned into pETBlue-2 (Novagen) treated with BamH I and EcoR I, and Escherichia coli JM109 strain was isolated. It was confirmed that the novel putrescine synthetic gene from Citrobacter sp.PR-144-5-1 was cloned and the nucleotide sequence of the novel proresin synthetic gene and the protein encoded by the gene The amino acid sequences of are shown in SEQ ID NOs: 2 and 3, respectively, and after treating the plasmid with Sma I and BamH I, the 5 ′ protruding ends were smoothed with Klenow Fragment and then ligated, thereby shifting the frame. Corrected and obtained The expression vector was pET-SF, and pET-SF was introduced into NovaBlue [DE3] (Novagen) to obtain a transformant (referred to as recombinant E. coli pET-SF) for producing a new putrescine. .

  For comparison, the SpeF gene in E. coli was amplified by PCR, an expression vector was constructed in the same manner, and a transformant expressing the putrescine synthesis gene derived from E. coli (referred to as recombinant E. coli pET-E-F) was also prepared.

  Recombinant E. coli pET-S-F and recombinant E. coli pET-E-F obtained as described above were pre-cultured at 37 ° C. using LB medium. Next, the preculture was inoculated into the putrescine production medium to 2%, and the main culture was started. After 4 hours, IPTG and ornithine were added to induce expression of the putrescine synthesis gene and to disclose putrescine synthesis. The culture was sampled every few hours and analyzed with a gas chromatograph mass spectrometer (GC / MS) to analyze putrescine production. GC / MS analysis conditions are as shown in Table 2 above.

  The above-described culture test was performed twice, the first result is shown in FIG. 1, and the second result is shown in FIG. As shown in FIGS. 1 and 2, recombinant E. coli pET-SF introduced with a putrescine synthesis gene derived from Citrobacter sp. PR-144-5-1 strain was compared with recombinant E. coli pET-EF and wild type E. coli. It was revealed that putrescine was synthesized with very good productivity. From the above results, the putrescine synthesizing gene derived from Citrobacter sp. PR-144-5-1 cloned in Example 2 has superior ability to synthesize putrescine as compared with the conventionally known E. coli-derived putrescine synthesizing gene. Turned out to be.

It is a characteristic figure which shows the result of the 1st culture test which examined the putrescine production ability in recombinant Escherichia coli pET-S-F and recombinant Escherichia coli pET-E-F. It is a characteristic figure which shows the result of the 2nd culture test which examined the putrescine production ability in recombinant Escherichia coli pET-S-F and recombinant Escherichia coli pET-E-F.

Claims (6)

  A microorganism belonging to the genus Citrobacter having a 16S rDNA sequence that is 90% or more homologous to the base sequence shown in SEQ ID NO: 1 and having the ability to produce putrescine.   The microorganism according to claim 1, wherein the microorganism belonging to the genus Citrobacter is Citrobacter sp. PR-144-5-1 strain (deposit number: NITE P-620). A putrescine synthetic gene encoding any one of the following proteins (a) to (c).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 3 (b) consisting of an amino acid sequence in which one or a plurality of amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and the ability to synthesize putrescine (C) a protein comprising an amino acid sequence having a homology of 95% or more with respect to the amino acid sequence shown in SEQ ID NO: 3, and having a putrescine synthesis ability The putrescine synthetic gene according to claim 3, comprising any one of the following polynucleotides (a) to (c):
(A) a polynucleotide comprising the base sequence shown in SEQ ID NO: 2 (b) a base sequence shown in SEQ ID NO: 2 consisting of a base sequence in which one or more bases are deleted, substituted, added or inserted, and putrescine synthesis A polynucleotide encoding a protein having the ability to synthesize putrescine, comprising a base sequence that hybridizes under stringent conditions to a complementary strand of the base sequence shown in SEQ ID NO: 2 nucleotide   The microorganism according to claim 1 or 2 or the microorganism expressing the putrescine synthesis gene according to claim 3 or 4 is cultured in a medium to which a substrate for producing putrescine and a nitrogen source are added, and the putrescine is collected from the culture. A method for producing putrescine, comprising:   The method for producing putrescine according to claim 5, wherein the substrate for producing putrescine is arginine or ornithine.

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