CN102154393A

CN102154393A - Production method and strain of gamma-aminobutyric acid (GABA)

Info

Publication number: CN102154393A
Application number: CN2011100206068A
Authority: CN
Inventors: 史锋; 李佑新; 李永富; 王小元
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2011-01-18
Filing date: 2011-01-18
Publication date: 2011-08-17

Abstract

The invention discloses a production method and strain of gamma-aminobutyric acid (GABA), belonging to the technical field of gene engineering. The method comprises the following steps of: introducing a glutamate decarboxylase gene into a glutamic acid production bacterium to construct a gene engineering bacterium; and removing a carboxyl from a self-accumulated glutamic acid by using glutamate decarboxylase secreted by the gene engineering bacterium to synthesize the GABA. In the invention, fermentation of glutamic acid is combined with transformation of the GABA, so that the production steps of the GABA are simplified; moreover, glucose, urea and the like are taken as culturing raw materials, so that the production cost of the method disclosed by the invention is remarkably lowered compared with a biological transformation method in which L-glutamic acid or L-sodium glutamate serving as a precursor needs to be exogenously added.

Description

A kind of production method of γ-An Jidingsuan and production bacterial strain thereof

Technical field

The present invention relates to the production method of γ-An Jidingsuan and produce bacterial strain, belong to gene engineering technology field.

Background technology

γ-An Jidingsuan (γ-Aminobutyric acid is called for short GABA), (Glutamic acid is Glu) through L-Glutamic decarboxylase (Glutamate decarboxylase is called for short GAD) catalytic decarboxylation by L-glutamic acid.γ-An Jidingsuan is the important inhibitory nerve mediator in the mammalian central nervous system.Have the important physical function, the physiologically active of having reported has blood pressure regulation, impels ataraxy, promotes the brain blood flow, promotes the brain vigor, the trophic nerve cell, increase growth hormone secretion, strong liver sharp kidney, prevention of obesity, promotion alcohol metabolism (sobering up), improve multiple efficacies such as climacteric syndrome.

Utilize genetic engineering technique overexpression glutamic acid decarboxylase gene at present, further improve the existing relevant report of research of the output of GABA.In this research field, mainly be that these glutamic acid decarboxylase genes that derive from GABA accumulation bacterial strain are imported in certain wild type strain of intestinal bacteria or subtilis, and L-Glutamic decarboxylase is efficiently expressed in the host bacterium, thereby strengthen the ability that the host bacterium produces GABA, but adopt this method to produce GABA, need to add a large amount of L-L-glutamic acid or L-Sodium Glutamate as precursor substance, and L-L-glutamic acid or L-Sodium Glutamate are produced by the Corynebacterium glutamicum fermentation usually.Therefore, prior art is produced process complexity and the production cost height of GABA.

Summary of the invention

The invention provides a kind of production method of γ-An Jidingsuan, be glutamic acid decarboxylase gene to be imported to make up genetic engineering bacterium in the glutamate producing bacterium, utilize genetic engineering bacterium excretory L-Glutamic decarboxylase that the L-glutamic acid that himself accumulates is sloughed a carboxyl and synthesize γ-An Jidingsuan.

Concrete grammar is as follows: with Corynebacterium glutamicum ATCC13032 is starting strain, it has the ability of accumulation L-glutamic acid, glutamic acid decarboxylase gene gadB1 imported to made up the reorganization Corynebacterium glutamicum among the Corynebacterium glutamicum ATCC13032, make L-Glutamic decarboxylase gadB1 that the reorganization bacterium utilizes heterogenous expression that the L-glutamic acid of himself accumulation is sloughed a carboxyl and synthesize γ-An Jidingsuan.

Described glutamic acid decarboxylase gene gadB1 comes the short lactobacillus of self-produced γ-An Jidingsuan, this bacterial strain is on December 27th, 2010, be preserved in Chinese typical culture collection center, address: Chinese Wuhan, Wuhan University, deposit number: CCTCC NO:M 2010367, the taxonomy name: short lactobacillus Lb85 ( Lactobacillus brevisLb85).

This bacterial strain is the milk-acid bacteria that γ-An Jidingsuan is produced in the strain that utilizes the screening of ply of paper analysis method to obtain from the series milk-acid bacteria of this laboratory, identify by microscopy and 16SrDNA, the homology of the 16SrDNA of the various short lactobacillus of announcing among confirmation and the GenBank is 99%, thereby determines that this bacterial strain is a short lactobacillus.

Described glutamic acid decarboxylase gene gadB1 is that the glutamic acid decarboxylase gene of the short lactobacillus ATCC 367 that announced according to genom sequence is for reference to the design primer, the genome that produces the short lactobacillus of GABA with this strain that filters out is a template, utilize round pcr that the gene gadB1 of coding L-Glutamic decarboxylase in its genome is increased out, and carry out gene sequencing, its nucleotide sequence such as Seq ID NO .1.

The present invention also provides the production bacterial strain of a kind of GABA, intestinal bacteria-coryneform bacteria fabric shuttle-type inducible expression carrier pDXW-8(the number of patent application 200910233618.1 that adopts this laboratory to make up), the goal gene gadB1 that amplifies is connected on the carrier, construct L-Glutamic decarboxylase recombinant expression plasmid pDXW8-gadB1, recombinant plasmid is imported among the Corynebacterium glutamicum ATCC13032 by the method that electricity transforms, and Corynebacterium glutamicum pDXW8-gadB1/ATCC13032 obtains recombinating.

The accurate quantification of GABA in the fermented liquid: automatic analyzer for amino acids method.With static 30min after 25 times of the fermented liquid dilutions, the centrifugal 10min protein precipitation of 12000rpm is got supernatant liquor, detects with the L835-50 of Hitachi type automatic analyzer for amino acids with 5% trichoroacetic acid(TCA).Automatic analyzer for amino acids employing 2619# resin (cationic exchange coloum f2.6 * 150mm), 53 ℃ of analyses, GABA is quantitative with external standard method.

The present invention utilizes the reorganization glutamate producing bacterium, and the L-L-glutamic acid that fermentation is produced directly is transformed into γ-An Jidingsuan, has following advantage:

1) fermentation of L-glutamic acid and the conversion of GABA are united two into one, simplified the GABA production stage;

2) substratum with glucose as inorganic nitrogens such as carbon source, urea as nitrogenous source, with respect to needing external source to add L-L-glutamic acid or the L-Sodium Glutamate is the bio-transformation of precursor substance, production cost obviously reduces.

Description of drawings

The structure synoptic diagram of Fig. 1 recombinant plasmid pDXW8-gadB1.

Embodiment

Come further to illustrate the present invention by the following examples, the following example is used for illustration purpose but not is used to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is all operated according to the common described condition of molecular cloning handbook basically.

Material and reagent:

Used restriction enzyme, T4 dna ligase, PCR reagent etc. are all purchased the precious biotech firm in TaKaRa; E. coli jm109 is available from sky root biotech firm; Primer, plasmid extraction kit, PCR product purification test kit are all purchased in Shanghai life worker bio-engineering corporation; Electroporation is available from Bio-Rad company; Other reagent are the analytical reagent that home or overseas is bought.

Embodiment one: the acquisition of glutamic acid decarboxylase gene gadB1

From this laboratory series milk-acid bacteria, utilize the ply of paper analysis method to filter out the milk-acid bacteria that γ-An Jidingsuan is produced in a strain, identify by microscopy and 16SrDNA, the homology of the 16SrDNA of the various short lactobacillus of announcing among confirmation and the GenBank is 99%, thereby determines that this bacterial strain is a short lactobacillus.This bacterial strain is preserved in Chinese typical culture collection center, the address on December 27th, 2010: Chinese Wuhan, Wuhan University, and deposit number: CCTCC NO:M 2010367, the taxonomy name: short lactobacillus Lb85 ( Lactobacillus brevisLb85).

The glutamic acid decarboxylase gene LVIS_1847 of the short lactobacillus ATCC 367 that has announced according to genom sequence is for reference to the design primer, the genome that produces the short lactobacillus of GABA with this strain that filters out is a template, utilize round pcr that the gene gadB1 of coding L-Glutamic decarboxylase in its genome is increased out, and carry out gene sequencing.

Upstream primer B1F:

5’-CTAGCTAGCAGAAGGAGATATACC ATGGCTATGTTGTATGGAAAACAC-3’

Downstream primer B1R:

5’-CCGAAGCTT TTAGTGCGTGAACCCGTATTTTTTA-3’

Embodiment two: the structure of reorganization Corynebacterium glutamicum pDXW8-gadB1/ATCC13032:

1. gadB1 and pDXW-8 are carried out double digestion with same two kinds of restriction enzyme NheI, HindIII, reclaim 1.43kb and 9.48kb endonuclease bamhi respectively, then these two fragments are connected with T4 dna ligases, and be transformed into and finish pDXW8-gadB1 construction of recombinant plasmid (Fig. 1) in the e. coli jm109;

2. recombinant plasmid pDXW8-gadB1 is imported among the Corynebacterium glutamicum ATCC13032 through the method that electricity transforms, Corynebacterium glutamicum pDXW8-gadB1/ATCC13032 obtains recombinating.

The competent preparation of Corynebacterium glutamicum ATCC13032.With the single bacterium colony of Corynebacterium glutamicum ATCC13032 in liquid LBG substratum (10g/L peptone, the 5g/L yeast powder, 10g/L NaCl, 5g/L glucose) 30 ℃ of overnight incubation in, transfer in 30mL competence substratum (10g/L peptone with 10% inoculum size subsequently, the 5g/L yeast powder, 10g/L NaCl, the 25g/L glycine, 0.1% Tween-80) in, cultivates 3-5h for 30 ℃, afterwards bacterium liquid is placed ice-water bath cooling 15min, the centrifugal supernatant of abandoning, in ice-cold 10% glycerine of 30mL, recentrifuge is abandoned supernatant with cell suspension, and with 10% glycerine repeated washing 2 times, use the cold glycerine re-suspended cell of 400 μ L 10% at last, preserve or be directly used in electricity in-70 ℃ after the packing and transform.

Embodiment three: reorganization Corynebacterium glutamicum fermentative production GABA

Substratum: seed culture medium (glucose 2.5%, urea 0.6%, corn steep liquor 3.0%, K ₂HPO ₄3H ₂O 0.15%, MgSO ₄7H ₂O 0.05%, and pH 7.0～7.5); Fermention medium (glucose 16%, urea 0.4%, corn steep liquor 0.3%, K ₂HPO ₄3H ₂O 0.2%, MgSO ₄7H ₂O 0.08%, MnSO ₄2mg/L, FeSO ₄2mg/L, pH 7.0～7.5)

Culture condition: the reorganization Corynebacterium glutamicum is in seed culture medium behind 30 ℃ of shaking culture 12h, inoculum size with 10% is transferred in the fermention medium ferments, adding 1mM IPTG when fermentation 12h induces, and the method by intermittence interpolation urea makes the medium pH value be controlled at neutral range within 24h subsequently, continue afterwards to ferment, measure alpha-aminobutyric acid content in the fermented liquid to 60h.

Recombinating after testing, the content of γ-An Jidingsuan is 5.6mM in the fermented liquid, and does not detect γ-An Jidingsuan in the wild bacterium ATCC13032 fermented liquid.

Sequence table

＜110〉Southern Yangtze University

＜120〉a kind of production method of γ-An Jidingsuan and production bacterial strain thereof

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ccccgtgaag ccgatcggtt ggttcgcgat caactgctag atgaaggaaa ctcgcggctg 180

aatctcgcca cgttctgtca gacttacatg gaaccggaag ctgttgaact catgaaggat 240

acactggaga aaaacgccat cgataaatcc gagtatcctc ggaccgctga aattgaaaat 300

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gtggatgact acaccattgg tatcgttggc attatgggca tcacttatac tggacaatac 660

gacgatttag cccgattaga tgccgttgta gagcggtata atcggacgac taagttcccg 720

gtatatatcc atgtcgatgc cgcttccggc ggattttaca cgccgtttat tgaacccgag 780

ctcaagtggg acttccgttt aaacaacgtg atttccatca atgcctccgg ccacaaatat 840

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Claims

1. the production method of a γ-An Jidingsuan, be glutamic acid decarboxylase gene to be imported to make up genetic engineering bacterium in the glutamate producing bacterium, utilize genetic engineering bacterium excretory L-Glutamic decarboxylase that the L-glutamic acid that himself accumulates is sloughed the synthetic γ-An Jidingsuan of a carboxyl.

2. according to the described method of claim 1, it is characterized in that described glutamic acid decarboxylase gene gadB1 is shown in Seq ID NO .1.

3. according to the described method of claim 1, it is characterized in that described glutamate producing bacterium is Corynebacterium glutamicum ATCC13032.

4. according to the described method of claim 1, it is characterized in that described plasmid adopts intestinal bacteria-coryneform bacteria fabric shuttle-type inducible expression carrier pDXW-8.

5. according to the described method of claim 1, it is characterized in that, glutamic acid decarboxylase gene gadB1 imported to obtain genetic engineering bacterium pDXW8-gadB1/ATCC13032 among the Corynebacterium glutamicum ATCC13032, utilize genetic engineering bacterium fermentative production γ-An Jidingsuan.

6. a genetic engineering bacterium that adopts claim 1 method to make up is that glutamic acid decarboxylase gene is imported to the genetic engineering bacterium that makes up in the glutamate producing bacterium.

7. according to the described genetic engineering bacterium of claim 6, it is characterized in that this bacterial strain is pDXW8-gadB1/ATCC13032.

8. the application of the described genetic engineering bacterium of claim 6 in the γ-An Jidingsuan fermentative production.