CN101381698A - Recombinant corynebacterium crenatum for over expression of N-acetylglutamate kinase and application thereof - Google Patents

Recombinant corynebacterium crenatum for over expression of N-acetylglutamate kinase and application thereof Download PDF

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CN101381698A
CN101381698A CNA2008101551330A CN200810155133A CN101381698A CN 101381698 A CN101381698 A CN 101381698A CN A2008101551330 A CNA2008101551330 A CN A2008101551330A CN 200810155133 A CN200810155133 A CN 200810155133A CN 101381698 A CN101381698 A CN 101381698A
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tac
argb
pjc1
corynebacterium
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饶志明
许正宏
徐美娟
张晓梅
许泓瑜
窦文芳
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Jiangnan University
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Abstract

The invention relates to a recombinant corynebacterium crenatum to enhance the expression of N-acetyl glutamic acid kinase and the application thereof, which belongs to the technical field of gene engineering. The classification and nomenclature of the recombinant corynebacterium crenatum to enhance the expression of N-acetyl glutamic acid kinase is corynebacterium crenatum SYPA/pJC1-tac-argB with an access number of CCTCC NO: M 208133; The application is as follows: using a key enzyme N-acetyl glutamic acid kinase from corynebacterium crenatum pathway arginine to construct a corynebacterium crenatum expression vector pJC1-tac-argB; introducing the corynebacterium crenatum expression vector pJC1-tac-argB into corynebacterium crenatum SYPA by electrotransformation to obtain the recombinant corynebacterium crenatum SYPA/pJC1-tac-argB; and excessively expressing N-acetyl glutamic acid kinase, which greatly enhances the utilization rate of a precursor glutamic acid and allows the metabolic flow to flow to the arginine synthesis pathway, weakens the synthesis of proline, achieves the purpose of improving the output of arginine and the output of arginine is increased by 23.4 percent than C. crenatum SYPA.

Description

Add the application of the kinase whose recombinant corynebacterium crematum of strongly expressed N-acetylglutamat and this bacterium
Technical field
Add the application of the kinase whose recombinant corynebacterium crematum of strongly expressed N-acetylglutamat and this bacterium, the present invention relates to utilize key gene in the L-arginine circulation route of synthesis in Corynebacterium crenatum, to add strongly expressed, to improve the L-arginine yield, belong to gene engineering technology field.
Background technology
L-arginine (L-arginine) is human body and animal intravital half an essential basic aminoacids, is the important source material of synthetic protein creatine, also is a kind of important mesostate of organism ornithine cycle, has the physiology and the pharmacological action of multiple uniqueness.The L-arginine is of many uses in clinical medicine, food, makeup and relevant biological study field.Fermentation method is that present L-arginine is commercially produced more effective and economic method.
In prokaryotic micro-organisms, the synthetic of L-Arg is from L-glutamic acid, experienced 8 kinds of enzyme catalysiss and formed.At first, the acetylize under of the amino group of L-glutamic acid by the effect of the acetylglutamate synthetase of argA genes encoding, acetylizing has not only stoped spontaneous cyclisation but also stoped the L-glutamic acid carboxylic group to be modified the formation proline(Pro).The 5th step of this approach is that acetylornithice deacetylate group under the effect of enzyme forms ornithine, difference according to deacetylation group mode, the metabolic pathway of synthesizing of L-Arg is divided into 2 kinds, a kind ofly be referred to as linear approach, promptly acetylornithice forms L-Arg precursor---ornithine under the hydrolytic action by the acetyl-ornithinase of argE genes encoding; Another kind is referred to as the circulation approach; when to be acetylornithice form ornithine under the effect by the ornithine acetyltransferase of argJ genes encoding; acetyl group is transferred to L-glutamic acid and forms acetylglutamate; in this approach; ornithine acetyltransferase shows double function---not only have the function of acetyl-ornithinase but also have the function of acetylglutamate synthetase; so approach is called economic circulation approach again; at enterobacteria (Escherichiacoli); sulfolobus (Sulfolobussolf ataricus) in false unit cell pseudomonas bacteria (Pseudomonas aeruginosa) and the archeobacteria is produced L-Arg with linear approach; and the every other prokaryotic micro-organisms of studying so far; comprise and have a liking for the warm nature archeobacteria; shaft-like fatty thermophile bacteria (Bacillusstearothermophilus); Neisseria gonorrheae (Neisseria gonorrboeae) and Bacillaceae, and eukaryotic microorganisms all adopts the circulation approach.Have report to show, in enterobacteria, synthetic L-Arg the 1st enzyme---acetylglutamate synthetase is the target of end product L-Arg feedback inhibition; And the microorganism of adopting the circulation approach, the key enzyme that is suppressed by L-Arg---acetylglutamate kinase or the 5th enzyme---ornithine acetyltransferase that is the 2nd enzyme of route of synthesis.The key enzyme that is suppressed by L-Arg in the Corynebacterium glutamicum is an acetylglutamate kinase, this enzyme not only by the L-Arg feedback inhibition also by its feedback repression.
Raising along with China's medicine trophic level, growing to arginic demand, but the superior strain of traditional mode of production L-Arg adopts the method for mutagenesis screening to obtain mostly, but this method has blindness height, workload big, and has mutant strain disorder, easy limitation such as degeneration; After the DNA recombinant technology occurred, people began exploration and utilize the synthetic Arg pathways metabolism of DNA recombinant technology regulation and control, to reach the purpose that improves L-Arg output.
Corynebacterium crenatum (Corynebacterium crenatum) is a kind of cognate shape, the nonspore-bearing gram-positive microorganism that China investigator is separated to, its mutant strain is widely used in the amino acid production at home, but the research of its genetic background also is in space state.Corynebacterium crenatum C.crenatum SYPA is the high yield arginine mutant strain that this laboratory screening obtains.C.crenatum utilizes the circulation approach to synthesize arginine, and acetylglutamate kinase (NAGK) etc. is the key enzyme in the route of synthesis, and some can be subjected to arginic feedback inhibition of product and feedback repression.Utilizing metabolic engineering to improve one of most important strategy of L-arginine yield is exactly to improve key enzyme enzyme amount, by means of the gene cloning and expression technology, key enzyme encoding gene in the L-Arg biosynthetic pathway is imported in the production bacterium, by increasing gene dosage and changing expression regulation element, strengthen and express, thereby reach the purpose that improves arginine yield.The key gene that imports both can be a native gene of producing bacterium self, also from the foreign gene of nonproductive bacterium.
Corynebacterium crenatum (Corynebacterium crenatum) SYPA is the industrial bacterial strain of Gram-positive, does not see the research report of the genetic expression system of Corynebacterium crenatum as yet.
Summary of the invention
The purpose of this invention is to provide the application that a strain adds the kinase whose recombinant corynebacterium crematum of strongly expressed N-acetylglutamat and this bacterium, the clone is from the key gene argB in the arginine route of synthesis of high yield arginine mutant strain C.crenatum SYPA, strengthen the expression of enzyme in its route of synthesis again by Corynebacterium crenatum expression vector establishment recombinant corynebacterium crematum, thereby improve output and the yield of purpose product L-Arg in the fermenting process.
Technical scheme of the present invention: a strain adds the kinase whose recombinant corynebacterium crematum of strongly expressed N-acetylglutamat, its classification called after Corynebacterium crenatum (Corynebacterium crenatum) SYPA/pJC1-tac-argB, be preserved in Chinese typical culture collection center, deposit number is CCTCC NO:M208133.
Described recombinant corynebacterium crematum, it makes up used expression vector pJC1-tac-argB, adopts the tac promotor.
Described recombinant corynebacterium crematum is the arginine route of synthesis key enzyme N-acetylglutamat kinases encoding gene argB that has increased self-produced arginic Corynebacterium crenatum first.
Described recombinant corynebacterium crematum reaches the kinase whose strongly expressed that adds of synthetic arginine key enzyme N-acetylglutamat of Corynebacterium crenatum self, improves the arginic output of fermentative Production L-.
The construction process of described recombinant corynebacterium crematum will come from the synthetic arginic key enzyme N-acetylglutamat kinases NAGK encoding gene argB of Corynebacterium crenatum circulation approach, make up Corynebacterium crenatum expression vector pJC1-tac-argB; This expression vector pJC1-tac-argB electricity is changed among the Corynebacterium crenatum SYPA, obtained recombinant corynebacterium crematum (Corynebacterium crenatum) SYPA/pJC1-tac-argB;
(1) obtain the tac promotor:
According to the chloramphenicol acetyl transferasegene cat sequence that GenBank announces, the design primer amplifies the cat sequence by round pcr from plasmid pAC, and design of primers is as follows:
pCm1100F?EcoRI：5’-CGC GAATTCTTCGAATTTCTGCCATTCATC-3’
pCm1100R?Hind?III：5’-CGC AAGCTTGCGGTGCTTTTGCCGTTACG-3’
With pAC is template, with pCm1100F EcoRI and pCm1100R Hind III is primer, PCR obtains to contain the cat fragment of EcoRI and two restriction enzyme sites of HindIII, be connected with pMD18-T, make up cloning vector pMD 18-T-cat, cloning vector pMD18-T-cat is converted into E.coli JM109 recipient bacterium, coat on the LB agar plate that contains penbritin Amp, isopropyl-IPTG, semi-lactosi analogue X-gal, after 37 ℃ of overnight incubation, grow many bluenesss, white colony on the flat board; Picking white is cloned at random, enzyme is cut and is identified the back order-checking, obtain containing the cat fragment of EcoRI and two restriction enzyme sites of Hind III with EcoRI and HindIII double digestion, the pEtac that cuts with same enzyme is connected, obtain transformant at kantlex and chlorampenicol resistant LB plate screening behind the transformed into escherichia coli JM109, obtain plasmid pEtac-cat;
According to the sequence of complete sequence and the cat of pEtac, design primer amplification-tac-cat-gene; Design of primers:
Ptac?F?Pst?I：5＇-AAAA CTGCAGGGAGCTTATCGACTGCACG-3＇
PT7?R?Pst?I：5＇-AAAA CTGCAGATCCGGATATAGTTCCTCCTTTC-3＇
With pEtac-cat is template, Ptac F Pst I, PT7 R Pst I is a primer, high-fidelity PCR obtains gene fragment-tac-promoter-cat-T7 terminator-, be connected conversion with the pJC1 that same enzyme is cut, coating kalamycin resistance flat board, picking transformant, enzyme is cut checking, promptly obtains recombinant plasmid pJC1-tac-cat;
(2) make up Corynebacterium crenatum expression vector pJC1-tac-argB:
As follows according to the Corynebacterium glutamicum coding acetylglutamate kinase gene argB nucleotide sequence design primer of announcing among the GenBank:
Upstream primer P1:5 ＇-CGC GTCGACGGTCATCAGTGACGGTTGC-3 ＇
Downstream primer P2:5 ＇-CGC GAATTCATGAATGACTTGATCAAAG-3 ＇
With the C.crenatum genomic dna is template, and reaction system is for getting dna profiling 1 μ L, 10 * ExPCR damping fluid, 5 μ L, and each 1 μ L of upstream and downstream primer, dNTPs 4 μ L, ExTaq archaeal dna polymerase 0.5 μ L, total reaction volume is 50 μ L; The pcr amplification parameter is 94 ℃ of sex change 1.5min, 55 ℃ of renaturation 1min, and 72 ℃ are extended 1.5min, 35 circulations; Gained fragment glue reclaims the back and is connected Transformed E .coli JM109, picking positive transformant with cloning vector pMD18-T; The evaluation of extraction plasmid enzyme restriction, and with recombinant plasmid called after pMD18-T-argB, and order-checking;
With the pMD18-T-argB double digestion, glue reclaims the argB fragment, with its with through the expression vector pJC1-tac of identical linearization for enzyme restriction connection, obtain recombinant expression plasmid pJC1-tac-argB;
(3) obtain recombinant corynebacterium crematum SYPA/pJC1-tac-argB
Extract plasmid pJC1-tac-argB, electricity consumption commentaries on classics method transforms Corynebacterium crenatum SYPA, coat contain 10 μ g/mL kantlex contain glucose LB flat board, grow transformant behind the 36h, promptly obtain recombinant corynebacterium crematum SYPA/pJC1-tac-argB.
With the arginic method of described recombinant corynebacterium crematum fermentative production L-:
Starting strain is recombinant corynebacterium crematum SYPA/pJC1-tac-argB;
Substratum is formed: the shake-flask seed substratum is in g/L: glucose 30, corn steep liquor 20, (NH 4) 2 SO 420, KH 2PO 41, MgSO 47H 2O 0.5, urea 1.5, and pH 7.0～7.2,121 ℃ of sterilization 20min, liquid amount 20mL/250mL;
The shake flask fermentation substratum is in g/L: glucose 150, corn steep liquor 4, (NH 4) 2 SO 420, KH 2PO 41.5, MgSO 47H 2O 0.5, FeSO 47H 2O 0.02, MnSO 4H 2O 0.02, vitamin H 8 * 10 -5, L-Histidine 5 * 10 -4, CaCO 330; PH 7.0～7.2,121 ℃ of sterilization 10min, 20mL/250mL triangular flask;
Jar top fermentation substratum is in g/L: glucose 150, corn steep liquor 4, (NH 4) 2 SO 420, KH 2PO 41.5, MgSO 47H 2O 0.5, FeSO 47H 2O 0.02, MnSO 4H 2O 0.02, vitamin H 8 * 10 -5, L-Histidine 5 * 10 -4, 7.0～7.2,121 ℃ of sterilizations of ammoniacal liquor control pH 10min, liquid amount 3L/5L fermentor tank;
Culture condition: seed culture: the single colony inoculation of picking Corynebacterium crenatum is in seed culture medium in the solid medium flat board, and 150r/min cultivates 15h for 30 ℃;
Shake flask fermentation is cultivated: after the seed culture, be transferred to the mid-reciprocating type shaking table of fermention medium by 5% inoculum size, 110r/min cultivates 96h for 30 ℃;
The 5L fermentation tank culture medium: liquid amount 3L, inoculum size 5%～10%, 31 ℃, rotating speed 600r/min, air flow 3L/min, adding control pH with the ammoniacal liquor Continuous Flow is 7.0～7.2, cultivates 96h.
The present invention with chloramphenicol acetyl transferasegene (cat) as reporter gene, successfully made up the Corynebacterium crenatum expression system that contains the tac promotor, and its promoter activity in intestinal bacteria and Corynebacterium crenatum is studied the back find, the tac promotor all has promoter function in intestinal bacteria and Corynebacterium crenatum, and does not also exist certain background to express under the inductive condition.
Wherein the replaceable encoding gene for other key enzymes in the circulation approach of argB comprises argC, argD, argE, argF, argG, argJ, argH etc.
Beneficial effect of the present invention: successfully made up and carried the expression plasmid pJC1-tac-argB that derives from the synthetic key gene argB of Corynebacterium crenatum SYPA arginine etc., and electricity changes Corynebacterium crenatum SYPA over to.Picking 8 Corynebacterium crenatum transformant C.c (pJC1-tac-argB) and pick out the highest transformant C.c2 of arginine yield by shake flask fermentation, N-acetylglutamat kinases (NAGK) has been carried out enzyme is lived and fermentation character has been done initial analysis.The enzyme of NAGK is lived and is 44.5U/g among the C.c2, compares with the enzyme of NAGK among starting strain C.crenatumSYPA 31.2U/g alive and has improved about 42.6%; The L-arginine yield reaches 35.8g/L and compares with the 29.8g/L of starting strain C.crenatum SYPA and improved about 23.4%.By automatic analyzer for amino acids to amino acid whose quantitative analysis in the fermented liquid, the overexpression that has proved NAGK has promoted the utilization ratio of precursor substance L-glutamic acid greatly and has impelled the metabolism stream of L-glutamic acid to flow to the approach that arginine generates in a large number, improves arginic output.
Description of drawings
The structure of Fig. 1 recombinant plasmid pJC1-tac-cat.
The enzyme of Fig. 2 plasmid pEtac-cat is cut proof diagram; 1: λ DNA/Hind III marker; 2:pEtac-cat/Eco RI+Hind III double digestion; 3:pEtac-28a/Eco RI+Hind III double digestion; 4:cat PCR product; 5:DL2000Marker.
The enzyme of Fig. 3 plasmid pJC1-tac-cat is cut proof diagram; 1: λ DNA/Hind III marker; The 2:pJC1/PstI single endonuclease digestion; 3:-tac-promoter-cat-T7 terminator-PCR product; 4:pJC1-tac-cat/Pst I single endonuclease digestion; 5:DL2000 Marker.
The preliminary of Fig. 4 promotor intensity determined; Ctac (b): induce preceding C.C. (pJC1-tac-cat); Ctac (a): induce back C.C. (pJC1-tac-cat).
The structure of Fig. 5 recombinant plasmid pJC1-tac-argB.
The enzyme of Fig. 6 plasmid pJC1-tac-argB is cut proof diagram; 1, DNA Marker DL2000; 2, argBgene; 3 pJC1-tac/BamHI+SalI; 4, pJC1-tac-argB/BamHI; 5, pJC1-tac-argB/BamHI+SalI; 6, λ DNA/HindIII.
Fig. 7 ferment essence propylhomoserin process parameter over time; (A) thalli growth curve; (B) ferment essence propylhomoserin summation curve; (C) Tang consumption curve.
The biological material specimens preservation
One strain adds the kinase whose recombinant corynebacterium crematum of strongly expressed N-acetylglutamat, its classification called after Corynebacterium crenatum (Corynebacterium crenatum) SYPA/pJC1-tac-argB, be preserved in Chinese typical culture collection center, preservation date: on September 22nd, 2008, deposit number is CCTCC NO:M 208133.
Embodiment
Embodiment 1: with the structure of chloramphenicol acetyl transferasegene cat as the Corynebacterium crenatum genetic expression system of reporter gene:
According to the chloramphenicol acetyl transferasegene cat sequence that GenBank announces, the design primer amplifies the cat sequence by round pcr from plasmid pAC.Design of primers is as follows:
pCm1100F?Eco?RI：5’-CGC GAATTCTTCGAATTTCTGCCATTCATC-3’
pCm1100R?Hind?III：5’-CGC AAGCTTGCGGTGCTTTTGCCGTTACG-3’
With plasmid pAC is template, is primer with pCm1100F EcoRI and pCm1100R Hind III, and reaction system is for getting dna profiling 1 μ L, 10 * ExPCR damping fluid, 5 μ L, each 1 μ L of upstream and downstream primer, dNTPs 4 μ L, ExTaq archaeal dna polymerase 0.5 μ L, total reaction volume is 50 μ L.The pcr amplification parameter is 94 ℃ of sex change 1.5min, 55 ℃ of renaturation 1min, and 72 ℃ are extended 1min, 30 circulations; PCR obtains to contain the cat fragment of EcoRI and two restriction enzyme sites of Hind III, is connected with pMD 18-T, makes up cloning vector pMD18-T-cat (Eco RI+Hind III).Cloning vector pMD18-T-cat (Eco RI+Hind III) is converted into E.coli JM109 recipient bacterium, coat on the LB agar plate that contains penbritin Amp, isopropyl-IPTG, semi-lactosi analogue X-gal, after 37 ℃ of overnight incubation, grow many bluenesss, white colony on the flat board.Picking white is cloned at random, enzyme is served the order-checking of sea living worker's biotechnology company limited after cutting evaluation, obtain containing the cat fragment of EcoRI and two restriction enzyme sites of HindIII with EcoRI and Hind III double digestion, the pEtac that cuts with same enzyme is connected, obtain transformant at kantlex and chlorampenicol resistant LB plate screening behind the transformed into escherichia coli JM109, receive plasmid pEtac-cat.Figure 2 shows that recombinant plasmid pEtac-cat enzyme cuts the checking result.
According to the sequence of complete sequence and the cat of pEtac, design primer amplification-tac-cat-gene.Design of primers is as follows:
Ptac?F?Pst?I：5＇-AAAA CTGCAGGGAGCTTATCGACTGCACG-3＇
PT7R?Pst?I：5＇-AAAA CTGCAGATCCGGATATAGTTCCTCCTTTC-3＇
With pEtac-cat is template, Ptac F Pst I, and PT7R Pst I is a primer, reaction system is for getting dna profiling 1 μ L, 10 * ExPCR damping fluid, 5 μ L, each 1 μ L of upstream and downstream primer, dNTPs 4 μ L, ExTaqDNA polysaccharase 0.5 μ L, total reaction volume is 50 μ L; The pcr amplification parameter is 94 ℃ of sex change 1.5min, 57 ℃ of renaturation 1min, and 72 ℃ are extended 1.5min, 35 circulations; PCR obtains gene fragment-tac-promoter-cat-T7 terminator-, is connected conversion with the pJC1 that same enzyme is cut, coating kalamycin resistance flat board, and the picking transformant, enzyme is cut checking, promptly obtains recombinant plasmid pJC1-tac-cat (Fig. 1).
Or check order after gained gene fragment-tac-promoter-cat-T7 terminator-connected pMD 18-T carrier in the same way.After using PstI single endonuclease digestion-tac-promoter-cat-T7 terminator-then, standby, make and needn't carry out pcr amplification at every turn, can simplify the operation.Figure 3 shows that recombinant plasmid pJC1-tac-cat enzyme cuts the checking result.
Recombinant plasmid pEtac-cat obtains the dna fragmentation of about 650bp and 5622bp behind EcoRI and HindIII double digestion, respectively big or small consistent with cat gene fragment and plasmid pEtac.Recombinant plasmid pJC1-tac-cat obtains the dna fragmentation of about 1098b after Pst I enzyme is cut, size and PCR acquisition-the tac-promoter-cat-T7terminator-fragment is consistent.
It is correct that above result proves that recombinant plasmid pJC1-tac-cat makes up.
Extract plasmid pJC1-tac-cat, electricity consumption commentaries on classics method transforms Corynebacterium crenatum SYPA, coat contain 20 μ g/mL kantlex contain glucose LB flat board, grow transformant behind about 36h.After the some bacterium colonies of picking are cultivated at random, extract plasmid in the LB substratum.The enzyme of plasmid cuts the result and the linearizing size is all consistent with the purpose plasmid.
Embodiment 2: with the preliminary assessment of chloramphenicol acetyl transferasegene cat as the Corynebacterium crenatum genetic expression system of reporter gene:
Picking is cultured to OD at the Corynebacterium crenatum colony inoculation LB substratum of the flat board growth that contains 20 μ g/mL kantlex in 30 ℃ of shaking tables 600After reaching 0.6, add IPTG to final concentration be 20 μ mol/L, induce 4h.Thalline after inducing is inoculated on the chlorampenicol resistant flat board of different concns incubation growth, the intensity of tentatively definite promotor.
Found that the recon pJC1-tac-cat before and after inducing can grow on the chlorampenicol resistant flat board, can not on the chlorampenicol resistant flat board, grow and contrast (not containing recombinant plasmid pJC1-tac-cat).Illustrate that the tac promotor has the function of promotor in Corynebacterium crenatum.And the tac promotor has background to express in Corynebacterium crenatum.
Next, the C.C. (pJC1-tac-cat) before and after inducing is inoculated on the chlorampenicol resistant flat board of different concns the intensity of each promotor of rough determination respectively.Shown in Figure 4, be the expression amount of C.C. (pJC1-tac-cat).
In order to determine the power of promotor more accurately, further the enzyme work of the expressed E.C. 2.3.1.28 of C.C. (pJC1-tac-cat) is measured.The result is as shown in table 1, and it is consistent testing with flat board than the general trend of vigor, and the tac promotor has background to express before inducing, and the expression amount after inducing is higher.
The CAT of table 1 different strains compares vigor
Figure A200810155133D00101
Embodiment 3:C.crenatum synthesizes the cloning and expression of arginic key enzyme N-acetylglutamat kinases (NAGK) in Corynebacterium crenatum:
As follows according to the Corynebacterium glutamicum coding acetylglutamate kinase gene argB nucleotide sequence design primer of announcing among the GenBank:
Upstream primer P1:5 ＇-CGC GTCGACGGTCATCAGTGACGGTTGC-3 ＇ (SalI) downstream primer P2:5 ＇-CGC GAATTCATGAATGACTTGATCAAAG-3 ＇ (EcoRI)
With the C.crenatum genomic dna is template, and reaction system is for getting dna profiling 1 μ L, 10 * ExPCR damping fluid, 5 μ L, and each 1 μ L of upstream and downstream primer, dNTPs 4 μ L, ExTaq archaeal dna polymerase 0.5 μ L, total reaction volume is 50 μ L.The pcr amplification parameter is 94 ℃ of sex change 1.5min, 55 ℃ of renaturation 1min, and 72 ℃ are extended 1.5min, 35 circulations; Gained fragment glue reclaims the back and is connected Transformed E .coli JM109, picking positive transformant with cloning vector pMD 18-T carrier.The evaluation of extraction plasmid enzyme restriction, and with recombinant plasmid called after pMD18-T-argB, and order-checking.
With the pMD18-T-argB double digestion, glue reclaims the argB fragment, with its with connect through the expression vector pJC1-tac of identical linearization for enzyme restriction, obtain recombinant expression plasmid pJC1-tac-argB, and with its transformed into escherichia coli, coating kalamycin resistance flat board, picking transformant, enzyme is cut checking, promptly obtains recombinant plasmid pJC1-tac-argB (Fig. 5).
As shown in Figure 6, the pJC1-tac-argB enzyme is cut the checking result.Recombinant plasmid pJC1-tac-argB obtains the dna fragmentation of about 1000bp after enzyme is cut, size is consistent with the fragment that PCR obtains.It is correct that above result proves that recombinant plasmid pJC1-tac-argB makes up.
Extract plasmid pJC1-tac-argB, electricity consumption commentaries on classics method transforms Corynebacterium crenatum, coat contain 10 μ g/mL kantlex contain glucose LB flat board, grow transformant behind about 36h.After the some bacterium colonies of picking are cultivated at random, extract plasmid in the LB substratum.The enzyme of plasmid cuts the result and the linearizing size is all consistent with the purpose plasmid, proves that Corynebacterium crenatum reorganization bacterium C.C. (pJC1-tac-argB) successfully constructs.
After obtaining recombinant corynebacterium crematum C.C. (pJC1-tac-argB), it is carried out the expression study of NAGK.
Selecting a C.C (pJC1-tac-argB) transformant and starting strain C.crenatum SYPA is inoculated in after LB adds the culture medium culturing 24h of 0.5% glucose, inoculum size with 10% is transferred and collect thalline after LB is added 0.5% dextrose culture-medium cultivation 9h, record the reorganization NAGK enzyme 44.5U/g of being alive, starting strain NAGK enzyme is lived and is 31.2U/g, improve approximately 42.6%, the result is as shown in table 2.Illustrate that plasmid pJC1-tac-argB is in C.crenatum SYPA successful expression.
Table 2 recombinase vigor is analyzed
Figure A200810155133D00111
Embodiment 4: the reorganization bacterium produces the arginine characteristic relatively with the fermentation of the strain C.crenatum SYPA that sets out:
Fermentation condition technical scheme as above of the present invention is described.Picking 8 recombinant corynebacterium crematum transformant C.c (pJC1-tac-argB) and starting strain C.crenatum SYPA shake flask fermentation simultaneously, with 8 transformants difference called after C.c (1-8), it is parallel that each transformant is done 3 fermentations, behind the shaking culture 96h, measure its arginine yield, wherein transformant C.c2 output is the highest, has improved approximately 21.4% than starting strain C.crenatum SYPA acid yield, and concrete outcome is as shown in table 3.
The arginine yield of table 3 recombinant corynebacterium crematum transformant
Figure A200810155133D00121
Select recombinant corynebacterium crematum C.c2 and starting strain C.crenatum SYPA top fermentation simultaneously jar to cultivate 96h, different time respectively sampling analysis contrast both fermentation character.The fermentation result as shown in Figure 7, by Fig. 7 (A) as can be seen under identical culture condition, the growth curve basically identical of C.c2 and starting strain C.crenatum SYPA, from the 8h that begins to measure is the logarithmic phase of thalline to 40h, also be the critical period that arginine is produced the stationary phase that is thalli growth from 40h to 96h, and thalline falls into a decline after the 96h, has entered the growth decline phase, this moment, the viscosity of fermented liquid obviously increased, and a large amount of autolysis of thalline occurred.The growing state of C.c2 is poor slightly than starting strain but by Fig. 7 (A) as can be seen.
Fig. 7 (B) is arginic production curve, and C.c2 there is no clear superiority at 40h with interior arginic output and starting strain, may be owing to be not arginic a large amount of generation period at the logarithmic phase of thalline, and the slower reason of the reorganization bacterium C.c2 speed of growth.Arginine is synthetic in a large number as seen from the figure after entering stationary phase of thalli growth, and reorganization bacterium C.c2 produces arginic advantage and also displays gradually, and output reaches the highest when 88h, improves about 23.4% than the production peak of starting strain C.crenatum SYPA.
By Fig. 7 (C) as can be known the consumption sugar situation of C.c2 and starting strain C.crenatum SYPA do not have difference substantially, so the metabolism of carbon source to flow to may be the major reason that causes the growth differences of reorganization bacterium and produce arginine difference.
Embodiment 5: recombinant corynebacterium crematum C.c2 and starting strain C.crenatum SYPA fermented liquid amino acid chromatographic instrument are analyzed:
According to L-arginine synthetic metabolism network, influencing L-arginine synthetic main node is pyruvic acid and L-glutamic acid node.L-glutamic acid is arginine synthetic precursor substance, it is first reactant of synthetic 9 step of L-arginine straight chain reaction, its accumulation volume and assignment of traffic are directly connected to the arginic amount of accumulation L-, but L-glutamic acid also is the precursor of the synthetic branch road of proline(Pro) simultaneously, so the accumulation volume of L-glutamic acid and proline(Pro) is that we study the important reference of the overexpression of NAGK to the arginine metabolic effect.
With above-mentioned C.c2 and C.crenatum SYPA fermentor cultivation 88h secondary fermentation liquid in the centrifugal 10min of 12000r/min, get after 50 times of the supernatant liquor dilutions with automatic analyzer for amino acids amino acid quantitative assay in the fermented liquid, table 4 is L-glutamic acid, proline(Pro) and arginic quantitative assay result.
Table 4 L-glutamic acid, proline(Pro) and arginic quantitative assay
The content of L-glutamic acid and proline(Pro) has reduced by 80.3% and 57.8% than the L-glutamic acid in the original strain C.crenatum SYPA fermented liquid and the content of proline(Pro) respectively in the C.c2 fermented liquid as shown in Table 4, and arginic output has improved 23.4% than C.crenatum SYPA.Therefore as can be seen according to above data, overexpression produces the utilization ratio that arginine key enzyme NAGK has strengthened precursor L-glutamic acid greatly in C.c2, and allow the metabolism stream of L-glutamic acid flow to arginic route of synthesis in a large number and weakened the synthetic of proline(Pro) simultaneously, thereby reach the purpose that improves arginine yield.
Embodiment 6: the research of recombinant corynebacterium crematum plasmid stability:
Recombinant corynebacterium crematum C.c2 is carried out plasmid stability measure, up to fermentation ends, test-results shows that the fermentation ends plasmid stability is 95%, the plasmid kept stable every the 24h sampling.As shown in table 5.
The research of table 5 recombinant corynebacterium crematum C.c2 plasmid stability
Figure A200810155133D00132

Claims (6)

1, a strain adds the kinase whose recombinant corynebacterium crematum of strongly expressed N-acetylglutamat, its classification called after Corynebacterium crenatum (Corynebacterium crenatum) SYPA/pJC1-tac-argB, be preserved in Chinese typical culture collection center, deposit number is CCTCC NO:M208133.
2, recombinant corynebacterium crematum according to claim 1 is characterized in that making up used expression vector pJC1-tac-argB, adopts the tac promotor.
3, recombinant corynebacterium crematum according to claim 1, the arginine route of synthesis key enzyme N-acetylglutamat kinases encoding gene argB of the next self-produced arginic Corynebacterium crenatum that it is characterized in that increasing first.
4, recombinant corynebacterium crematum according to claim 1 is characterized in that reaching the kinase whose strongly expressed that adds of synthetic arginine key enzyme N-acetylglutamat of Corynebacterium crenatum self, improves the arginic output of fermentative Production L-.
5, the construction process of the described recombinant corynebacterium crematum of claim 1, it is characterized in that to come from the synthetic arginic key enzyme N-acetylglutamat kinases NAGK encoding gene argB of Corynebacterium crenatum circulation approach, make up Corynebacterium crenatum expression vector pJC1-tac-argB; This expression vector pJC1-tac-argB electricity is changed among the Corynebacterium crenatum SYPA, obtained recombinant corynebacterium crematum (Corynebacterium crenatum) SYPA/pJC1-tac-argB;
(1) obtain the tac promotor:
According to the chloramphenicol acetyl transferasegene cat sequence that GenBank announces, the design primer amplifies the cat sequence by round pcr from plasmid pAC, and design of primers is as follows:
pCm?1100F?EcoR?I：5’-CGC GAATTCTTCGAATTTCTGCCATTCATC-3’
pCm?1100R?Hind?III：5’-CGC AAGCTTGCGGTGCTTTTGCCGTTACG-3’
With pAC is template, with pCm1100F EcoRI and pCml100R Hind III is primer, PCR obtains to contain the cat fragment of EcoRI and two restriction enzyme sites of HindIII, be connected with pMD18-T, make up cloning vector pMD18-T-cat, cloning vector pMD18-T-cat is converted into E.coli JM109 recipient bacterium, coat on the LB agar plate that contains penbritin Amp, isopropyl-IPTG, semi-lactosi analogue X-gal, after 37 ℃ of overnight incubation, grow many bluenesss, white colony on the flat board; Picking white is cloned at random, enzyme is cut and is identified the back order-checking, obtain containing the cat fragment of EcoRI and two restriction enzyme sites of Hind III with EcoRI and HindIII double digestion, the pEtac that cuts with same enzyme is connected, obtain transformant at kantlex and chlorampenicol resistant LB plate screening behind the transformed into escherichia coli JM109, obtain plasmid pEtac-cat;
According to the sequence of complete sequence and the cat of pEtac, design primer amplification-tac-cat-gene; Design of primers:
Ptac?F?Pst?I:5′-AAAA CTGCAGGGAGCTTATCGACTGCACG-3′
PT7R?Pst?I:5′-AAAA CTGCAGATCCGGATATAGTTCCTCCTTTC-3′
With pEtac-cat is template, Ptac F Pst I, PT7R Pst I is a primer, high-fidelity PCR obtains gene fragment-tac-promoter-cat-T7 terminator-, be connected conversion with the pJC1 that same enzyme is cut, coating kalamycin resistance flat board, picking transformant, enzyme is cut checking, promptly obtains recombinant plasmid pJC1-tac-cat;
(2) make up Corynebacterium crenatum expression vector pJC1-tac-argB:
As follows according to the Corynebacterium glutamicum coding acetylglutamate kinase gene argB nucleotide sequence design primer of announcing among the GenBank:
Upstream primer P1:5 '-CGC GTCGACGGTCATCAGTGACGGTTGC-3 '
Downstream primer P2:5 '-CGC GAATTCATGAATGACTTGATCAAAG-3 '
With the C.crenatum genomic dna is template, and reaction system is for getting dna profiling 1 μ L, 10 * ExPCR damping fluid, 5 μ L, and each 1 μ L of upstream and downstream primer, dNTPs 4 μ L, ExTaq archaeal dna polymerase 0.5 μ L, total reaction volume is 50 μ L; The pcr amplification parameter is 94 ℃ of sex change 1.5min, 55 ℃ of renaturation 1min, and 72 ℃ are extended 1.5min, 35 circulations; Gained fragment glue reclaims the back and is connected Transformed E .coli JM109, picking positive transformant with cloning vector pMD18-T; The evaluation of extraction plasmid enzyme restriction, and with recombinant plasmid called after pMD18-T-argB, and order-checking;
With the pMD18-T-argB double digestion, glue reclaims the argB fragment, with its with through the expression vector pJC1-tac of identical linearization for enzyme restriction connection, obtain recombinant expression plasmid pJC1-tac-argB;
(3) obtain recombinant corynebacterium crematum SYPA/pJC1-tac-argB:
Extract plasmid pJC1-tac-argB, electricity consumption commentaries on classics method transforms Corynebacterium crenatum SYPA, coat contain 10 μ g/mL kantlex contain glucose LB flat board, grow transformant behind the 36h, promptly obtain recombinant corynebacterium crematum SYPA/pJC1-tac-argB.
6, with the arginic method of claim 1 described recombinant corynebacterium crematum fermentative production L-, it is characterized in that:
Starting strain is recombinant corynebacterium crematum SYPA/pJC1-tac-argB;
Substratum is formed: the shake-flask seed substratum is in g/L: glucose 30, corn steep liquor 20, (NH 4) 2SO 420, KH 2PO 41, MgSO 47H 2O 0.5, urea 1.5, pH7.0～7.2,121 ℃ sterilization 20min, liquid amount 20mL/250mL;
The shake flask fermentation substratum is in g/L: glucose 150, corn steep liquor 4, (NH 4) 2SO 420, KH 2PO 41.5, MgSO 47H 2O 0.5, FeSO 47H 2O 0.02, MnSO 4H 2O 0.02, vitamin H 8 * 10 -5, L-Histidine 5 * 10 -4, CaCO 330; PH7.0～7.2,121 ℃ sterilization 10min, the 20mL/250mL triangular flask;
Jar top fermentation substratum is in g/L: glucose 150, corn steep liquor 4, (NH 4) 2SO 420, KH 2PO 41.5, MgSO 47H 2O 0.5, FeSO 47H 2O 0.02, MnSO 4H 2O 0.02, vitamin H 8 * 10 -5, L-Histidine 5 * 10 -4, ammoniacal liquor control pH7.0～7.2,121 ℃ sterilization 10min, liquid amount 3L/5L fermentor tank;
Culture condition: seed culture: the single colony inoculation of picking Corynebacterium crenatum is in seed culture medium in the solid medium flat board, and 150r/min cultivates 15h for 30 ℃;
Shake flask fermentation is cultivated: after the seed culture, be transferred to the mid-reciprocating type shaking table of fermention medium by 5% inoculum size, 110r/min cultivates 96h for 30 ℃;
The 5L fermentation tank culture medium: liquid amount 3L, inoculum size 5%～10%, 31 ℃, rotating speed 600r/min, air flow 3L/min, adding control pH with the ammoniacal liquor Continuous Flow is 7.0～7.2, cultivates 96h.
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