JP5553394B2 - Method for producing cadaverine - Google Patents

Description

  The present invention relates to a method for producing cadaverine. Cadaverine is expected to be a synthetic raw material such as a pharmaceutical intermediate and a polymer raw material, and its demand is increasing.

  Conventionally, it is known that cadaverine can be obtained by boiling lysine in cyclohexanol containing a trace amount of tetralin peroxide (Masaka Suyama, Kiyozo Kaneo; Decarboxylation of amino acids (Part 4) Pharmaceutical Journal, vol.85 (6), P.531-533 (1965)). However, large amounts of energy and organic solvents are required and production efficiency is very low (36%). Cadaverine is a biogenic amine that exists universally in the body, and its biosynthetic system is being elucidated (Celia white tabor and Herbert tabor; Microbiological Reviews, vol. 49, P. 81-99 (1985)). . It is known that introduction of a gene encoding lysine decarboxylase derived from microorganisms into plants increases the accumulation of cadaverine (Lothar F. Fecker, Christiane Rugenhagen and Jochen Berlin; Plant Molecular Biology, vol. 23, P.11-21 (1993)). In addition, lysine decarboxylase genes with different optimum pH derived from E. coli are known (Shi-yuanmeng and George N. Bennett; Journal of Bacteriology, vol. 174, P. 2659-2669 (1992)) (Yoshimi kikuchi Hiroyuki kojima, Takashi tanaka, Yumiko, Takatsuka and Yoshiyuki kamio; Journal of Bacteriology, vol. 179, P. 4486-4492 (1997)). Furthermore, it is known that lysine can be produced by fermentation with high efficiency (Schilling BM, Pfefferle W, Bachmann B, Leuchtenberger W and Deckwer W; Biotechnol Bioeng, vol 64, P. 599-606 (1999)). However, no practical production technique has been established for the production of cadaverine, and it is desired to develop a method for producing cadaverine efficiently and under milder conditions.

Problems to be solved by the invention

  An object of the present invention is to provide an industrial production method of cadaverine with high efficiency and high yield of cadaverine by microorganisms, which consumes less energy and does not require an organic solvent.

Means for solving the problem

In order to solve the above-mentioned problems, the present inventors have conducted intensive research on a method for producing cadaverine with higher productivity. As a result, 4 Escherichia coli having enhanced lysine decarboxylase activity and lysine-cadaverine antiporter enzyme activity were obtained. By culturing for 120 hours from the time, and isolating and purifying cadaverine from the culture solution, it was found that the accumulated concentration and production yield of cadaverine in the culture solution were remarkably improved, and the present invention was achieved .

That is, the present invention provides a feature to "lysine cultured 120 hours decarboxylase and lysine-cadaverine antiporter 4 hours E.coli with enhanced enzymatic activity, be isolated and purified cadaverine from the culture broth To produce cadaverine. "

In a preferred embodiment of the present invention, using the activity enhanced host in the cell of a lysine decarboxylase and lysine-cadaverine antiporter. Limitations on the method to enhance the activity in the cells of the host cell lysine decarboxylase within and lysine-cadaverine antiporter is not. Specifically, for example, be a method to increase the amount of enzyme lysine decarboxylase and lysine-cadaverine antiporter or by introducing a mutation into the structural gene itself of the enzyme, increases the specific activity of the enzyme itself Etc.

  Means for increasing the amount of enzyme in the cell include improving the transcriptional regulatory region of the gene, increasing the copy number of the gene, and improving the efficiency of translation into protein.

  Improving the transcriptional regulatory region means adding a modification that increases the amount of gene transcription. For example, the promoter can be strengthened by introducing a mutation into the promoter, and the transcription amount of the downstream gene can be increased. In addition to introducing mutations into the promoter, a promoter that is strongly expressed in the host may be introduced. For example, in E. coli, promoters such as lac, tac, and trp can be mentioned. In addition, the amount of gene transcription can be increased by newly introducing an enhancer. The introduction of a gene such as a chromosomal DNA promoter is described in, for example, JP-A-1-215280.

  Specifically, the increase in the copy number of a gene can be achieved by connecting the gene to a multi-copy vector to produce a recombinant DNA and allowing the host cell to hold the recombinant DNA. Here, vectors include those widely used such as plasmids and phages, but besides these, transposon (Berg, DE and Berg. CM, Bio / Technol., Vol. 1, P.417 (1983) ) And Mu phage (Japanese Patent Laid-Open No. 2-109985). It is also possible to increase the copy number by incorporating a gene into a chromosome by a method using a plasmid for homologous recombination.

  As a method for increasing the translation efficiency of proteins, for example, in prokaryotes, SD sequences (Shine, J. and Dalgarno, L., Proc. Natl. Acad. Sci. USA, 71, 1342-1346 (1974)), true In nuclear organisms, the introduction and modification of Kozak consensus sequences (Kozak, M., Nuc. Acids Res., Vol.15, p.8125-8148 (1987)) and optimization of codons used (JP-A-59) -125895).

As a means to increase the specific activity in cells of the lysine decarboxylase and lysine-cadaverine antiporter, method by introducing a mutation into the structural gene itself of the enzyme, selecting elevated enzyme more specific activity Is mentioned.

  In order to generate mutations in genes, site-specific mutagenesis (Kramer, W. and frita, HJ., Methods in Enzymology, vol.154, P.350 (1987)) Recombinant PCR (PCRTechnology, Stockton Press (1989) ), A method of chemically synthesizing a specific portion of DNA, a method of treating the gene with hydroxyamine, and a method of treating a strain having the gene with ultraviolet irradiation or a chemical agent such as nitrosoguanidine or nitrous acid. .

  When the host has a lysine metabolism system other than cadaverine, such as lysine monooxygenase, lysine oxidase, and lysine mutase, the lysine monooxygenase gene, lysine oxidase gene, and lysine mutase gene are destroyed in order to selectively produce cadaverine. May be.

  The method for obtaining the lysine decarboxylase gene and the lysine / cadaverine antiporter gene is not particularly limited, and a PCR method, a screening method from a genomic library or cDNA library, and the like are used. In the present invention, these genes include mutants due to genetic polymorphism and the like. Genetic polymorphism means that the base sequence of a gene is partially changed due to a natural mutation on the gene.

  The obtained gene is incorporated into an appropriate expression vector and introduced into the host.

  The type of expression vector used is not particularly limited as long as it is stably maintained in the host cell. For example, in E. coli, pBR322, pUC19, etc. that can replicate in E. coli can be used as vectors.

  There are no particular limitations on the expression vector to be produced. One kind of lysine decarboxylase gene may be incorporated and expressed in one type of expression vector that can replicate in the host. Alternatively, two or more lysine decarboxylase genes may be incorporated into one expression vector and expressed under the control of the same promoter or different promoters. Furthermore, two or more types of expression vectors having different origins of replication and different promoters may be used, and different lysine decarboxylase genes may be incorporated into the respective expression vectors, and each may be expressed under the control of different promoters.

  When two or more types of lysine decarboxylase genes are incorporated, it is preferable to incorporate lysine decarboxylase genes having different optimum pH values for the respective enzymes.

  More preferably, a lysine-cadaverine antiporter may be incorporated into the same expression vector as these lysine decarboxylase expression vectors and expressed under the control of the same promoter. In addition, the lysine-cadaverine antiporter gene is incorporated into an expression vector having a different origin of replication from these lysine decarboxylase expression vectors and a different promoter, and lysine decarboxylase and lysine-cadaverine antiporter are expressed under the control of different promoters You may let them.

  The introduction method is not particularly limited. For example, in the case of bacteria, a calcium chloride method, an electroporation method, or the like is used.

Host is E. coli is used.

  In addition, the host may have properties such as drug resistance and auxotrophy.

  The gene encoding lysine decarboxylase and the gene encoding lysine cadaverine antiporter to be incorporated into the host are particularly restricted if they are expressed by the host and retain their activity as lysine decarboxylase and lysine cadaverine antiporter. Although genes derived from microorganisms, animals, plants or insects can be used, genes encoding lysine decarboxylase derived from microorganisms and genes encoding lysine-cadaverine antiporter are preferred.

As a gene encoding lysine decarboxylase of a microorganism, those derived from bacteria are preferable, and Escherichia coli, Bacillus halodurans, Bacillus subtilis, Selenomonas ruminantium, Vibrio cholera. (Vibrio cholerae), Vibrio parahaemolyticus, Streptomyces coelicolor, Streptomyces pilosus, Eikenella corrodens, Iyubacterium asidamino film Eubacterium acidaminophilum, Salmonella typhimurium, Hafniaalvei, Neisseria meningitidis, Thermoplasma acidop (Thermoplasma acidop) Hilum) or Pyrococcus Abishi (Pyrococcus abyssi) that has any genes from are known, they are preferably used. More preferably, genes derived from E. coli (cadA, ldc) are used.

  Known genes encoding lysine / cadaverine antiporter include Escherichia coli, Thermoplasma acidophilum, Vibrio cholerae, and the like, which are preferably used. More preferably, a gene derived from E. coli (cadB) is used.

The culture method in the present invention will be described. Medium carbon source, nitrogen source, ordinary media containing other organic trace components as inorganic ions, and requires a usable capacity is not particularly limited as long as cadaverine is produced.

  Carbon sources include sugars such as glucose, fructose and molasses, organic acids such as fumaric acid, citric acid and succinic acid, alcohols such as methanol, ethanol and glycerol, etc., and organic substances such as ammonium acetate as a nitrogen source. Inorganic ammonium salts such as ammonium salt, ammonium sulfate, ammonium chloride, ammonium phosphate, and ammonium nitrate, ammonia gas, aqueous ammonia, urea, etc. are 0.1% to 4.0%. Organic trace components such as pyridoxal phosphate, biotin, etc. A medium containing 0.0000001% to 0.1% of the required substance, each in an appropriate amount, is used.

  In addition to these, calcium phosphate, magnesium sulfate, calcium chloride, sodium chloride, zinc sulfate, copper sulfate, ferrous sulfate and the like are added as necessary as trace substances. Furthermore, required vitamins such as thiamine and niacin, or yeast extract, corn steep liquor and other natural products containing these may be used.

  Cultivation is usually performed under aerobic conditions, but can be performed under anaerobic conditions depending on the production of cadaverine. During culture, the pH of the medium rises according to the production and accumulation of cadaverine. Therefore, it is effective to adjust to pH 4 to pH 8 with an acid such as hydrochloric acid or sulfuric acid. Preferably, an antifoaming agent or the like is also added to stabilize the culture conditions. The culture temperature is 15 ° C to 45 ° C, preferably 20 ° C to 37 ° C.

  Preferable results can be obtained by shaking or stirring for 4 to 120 hours under these conditions.

Cadaverine produced in the culture solution is isolated and purified by a conventional method after removing the cells by centrifugation or the like .

Hereinafter, the present invention will be described specifically by way of examples.
Example 1
Cloning of lysine decarboxylase gene and lysine cadaverine antiporter gene and preparation of expression vector under the same promoter control In order to produce cadaverine from lysine produced by host cells, Cloning was performed.

  PCR primers were designed based on the base sequences of lysine decarboxylase gene (cadA) and lysine cadaverine antiporter gene (cadB) (Accession No. M76411) registered in the database (GenBank). The nucleotide sequences are shown in Sequence Listing 1 and Sequence Listing 2. A HindIII cleavage site and an XbaI cleavage site are added to the ends of the PCR primers.

Using these primers, PCR was performed using E. coli K12 strain (ATCC10798) genomic DNA as a template to obtain an amplified fragment of 3,622 base pairs. This amplified fragment was cleaved with HindIII and XbaI and then introduced into the HindIII / XbaI cleavage site of pUC19 (Takara Shuzo) to prepare a lysine decarboxylase expression vector pCAD1 (FIG. 1). cadA and cadB cloned in pCAD1 are expressed under the control of the lac promoter of pUC19.
(Example 2)
Introduction of expression vector into host The expression vector pCAD1 prepared in Example 1 was introduced into E. coli strain JM109. After introduction, recombinant E. coli was selected using resistance to ampicillin, an antibiotic, as an index, and transformants were obtained. This transformed strain was named E. coli CAD1 strain.
(Example 3)
Expression of cadaverine by the transformed strain The parent strain Escherichia coli JM109 and this transformed strain were cultured as follows. The JM109 strain and this CAD1 strain were cultured. That is, each of these strains was inoculated with 1 platinum ear in 5 ml of LB medium, and precultured by shaking at 30 ° C. for 24 hours.

  Next, 45 ml of LB medium was placed in a 50 ml Erlenmeyer flask and preliminarily steam sterilized at 115 ° C. for 10 minutes. The above strain cultured in advance on this medium was inoculated, and 4 hours later, 1 mM IPTG (isopropyl-1-thio-β-D-galactoside) was added and cultured for 50 hours.

  After completion of the culture, the cadaverine concentration in the culture supernatant from which the cells were removed was measured according to the method described in (Phan, APH, TTNgo and HMLenhoff; Anal. Biochem., Vol120, P.193-197 (1982)) did. The results are shown below.

Cadaverine concentration E. coli JM109 strain 0.01 g / L
E. coli CAD1 strain 1.22g / L
The amount of cadaverine accumulated was increased in the recombinant E. coli CAD1 strain compared to the control E. coli strain JM109.

Effect of the invention

  According to the present invention, when cadaverine is produced in a culture solution by fermentation, cadaverine can be produced more economically than existing methods.

Sequence listing

  FIG. 3 is a diagram showing a physical map of lysine decarboxylase and lysine-cadaverine antiporter expression vector pCAD1.

Claims (8)

Lysine was decarboxylase activity and 120 hours lysine-cadaverine antiporter activity enhanced E. coli from 4 hours incubation, the production method of cadaverine, characterized in that the isolated and purified cadaverine from the culture broth.
The method for producing cadaverine according to claim 1, wherein the Escherichia coli is a microorganism into which a gene encoding lysine decarboxylase and / or lysine cadaverine antiporter is incorporated.
Genes encoding lysine decarboxylase, 1 or 2 or more in the production method according to claim 2 Symbol placement of cadaverine is.
The method for producing cadaverine according to claim 2 or 3 , wherein the gene encoding lysine decarboxylase is derived from a microorganism.
The method for producing cadaverine according to any one of claims 2 to 4 , wherein the gene encoding lysine decarboxylase is derived from Escherichia coli.
The method for producing cadaverine according to any one of claims 2 to 5 , wherein the gene encoding lysine cadaverine antiporter is derived from a microorganism.
The method for producing cadaverine according to any one of claims 2 to 6 , wherein the gene encoding lysine cadaverine antiporter is derived from Escherichia coli.
The method for producing cadaverine according to any one of claims 2 to 7 , wherein the lysine decarboxylase gene and the lysine cadaverine antiporter gene are expressed under the control of the same promoter.

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