CN102021154B - Method for improving yield of arginine by mutation of Corynebacterium crenatum N-acetyl glutamic acid kinase - Google Patents
Method for improving yield of arginine by mutation of Corynebacterium crenatum N-acetyl glutamic acid kinase Download PDFInfo
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- CN102021154B CN102021154B CN201010166997XA CN201010166997A CN102021154B CN 102021154 B CN102021154 B CN 102021154B CN 201010166997X A CN201010166997X A CN 201010166997XA CN 201010166997 A CN201010166997 A CN 201010166997A CN 102021154 B CN102021154 B CN 102021154B
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Abstract
The invention relates to a method for improving the yield of arginine by the mutation of Corynebacterium crenatum N-acetyl glutamic acid kinase. L-arginine is one of semi-essential basic amino acids for a human body, and has various particular physiology and pharmacology functions. High-yield arginine mutant strain C. crenatum SYPA5-5 is compounded into the arginine through a circulating way, and N-acetyl glutamic acid kinase (NAGK) is a key enzyme in the compounding way and is subject to the feedback inhibition of the product arginine. By using an overlapping PCR (Polymerase Chain Reaction) technique, a GAT (glycerol phosphate acyl transferase) codon used for coding aspartic acid is used for substituting a GGA (General Gonadotropic activity) codon used for coding glycine at the site 287 in the coding NAGK albumen, and the NAGK which has high activity and arginine with obviously reduced by the feedback inhibition can be obtained after the mutation. An argBSD gene is brought into the Corynebacterium crenatum of the high-yield arginine through pJCl-tac, and the expression volume of the key enzyme is further improved. The final acid yield is increased to 36.3g/L from original 28g/L, and the yield of the L-arginine is increased by 29.7 percent.
Description
Technical field
The present invention relates to industrial microorganism production, be specifically related to produce arginic method.More specifically; The present invention relates to l-arginine and produce key enzyme---the kinase whose purposes of N-acetylglutamat; It is transformed, and is that a kind of new sudden change N-acetylglutamat kinases and a kind of genetically engineered recombinant corynebacterium crematum that contains this enzyme are produced arginic method.
Background technology
The L-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-l-arginine is of many uses in clinical medicine, food, makeup and relevant biological study field.Fermentation method is that present L-l-arginine is commercially produced more effective and economic method.
In prokaryotic micro-organisms, synthetic arginic approach has two kinds of linear path and circulation approach.Arginic synthesizing of L-in the linear path is to begin 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, and promptly acetylornithice forms l-arginine precursor---ornithine under the hydrolytic action by the acetyl-ornithinase of argE genes encoding.Therefore roll into a ball the difference of mode according to deacetylation; The arginic another kind of route of synthesis of L-is 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 the sulfolobus (Sulfolobussolf ataricus) that approach is called again in economic circulation approach enterobacteria (Escherichia coli), false unit cell pseudomonas bacteria (Pseudomonas aeruginosa) and the archeobacteria is produced l-arginine with linear approach, and the every other prokaryotic micro-organisms of studying so far; Comprise and have a liking for warm nature archeobacteria, shaft-like fatty thermophile bacteria (Bacillus stearothermophilus), Neisseria gonorrheae (Neisseriagonorrboeae) and Bacillaceae, and eukaryotic microorganisms all adopts the circulation approach.Have report to show, in enterobacteria, synthetic arginic the 1st enzyme---acetylglutamate synthetase is the target of end product l-arginine feedback inhibition; And the mikrobe of adopting the circulation approach, the key enzyme that is suppressed by l-arginine---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-arginine in the Corynebacterium glutamicum is an acetylglutamate kinase, this enzyme not only by the l-arginine feedback inhibition also by its feedback repression.
Raising along with China's medicine trophic level; Arginic increasing demand is increased; But the arginic superior strain of traditional mode of production L-adopts the method for mutagenesis screening to obtain mostly, and blindness is high, workload is big but this method has, and has limitation such as mutant strain disorder, easy degeneration; After the DNA recombinant technology occurred, people began exploration and utilize the synthetic l-arginine pathways metabolism of DNA recombinant technology regulation and control, to reach the purpose that improves arginine yield.Corynebacterium crenatum (Corynebacterium crenatum) is the blue formula positive bacteria of a kind of cognate shape, nonspore-bearing leather that Research in China person 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.C.crenatum SYPA5-5 is the high yield l-arginine mutant strain that this laboratory screening obtains.C.crenatum utilizes the circulation approach to synthesize l-arginine, and acetylglutamate kinase (NAGK) etc. is the key enzyme in the route of synthesis, and verified this enzyme receives 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; Through technology such as rational protein design and rite-directed mutagenesises key enzyme N-acetylglutamat kinases in the l-arginine production approach is transformed; Thereby obtain to remove the N-acetylglutamat kinases of product l-arginine feedback inhibition, its mutator gene is imported produce in the bacterium, thereby the intensity of enzymatic reaction in the enhancing thalline; Strengthen and express, reach the purpose that improves arginine yield.
Summary of the invention
Corynebacterium crenatum (Corynebacterium crenatum) SYPA5-5 is that Gram-positive is produced the arginic industrial bacterial strain of L-, the present invention relates in intestinal bacteria and Corynebacterium crenatum, be structured in sudden change and the N-acetylglutamat kinases (NAGK) high reactivity and anti-l-arginine feedback inhibition that plays a crucial role in the arginic biosynthesizing.
In the present invention, put forward the GGA codon that GAT codon with coding asparagicacid residue in the argB gene replaces in the coding NAGK albumen 287 glycocoll.The replacement of amino-acid residue has caused having the expression of the mutain of natural radioactivity level in the aminoacid sequence.Discovery said mutation NAGK becomes arginic feedback inhibition is obviously reduced.The inventor finds with the argB with sudden change then
SDThe Corynebacterium crenatum that the DNA of gene transforms produces the l-arginine ability improves, and has therefore accomplished the present invention.
The invention provides following content:
1, the N-acetylglutamat kinases from Corynebacterium crenatum is transformed, made it receive arginic feedback inhibition downward modulation.One or several amino acid whose disappearance, replacement, insertion or the interpolation of the one or more positions beyond sudden change NAGK can be included in 287, prerequisite are the active not destruction of NAGK." NAGK is active " expression catalysis generates the reactive activity of N-acetylglutamat phosphoric acid from L-glutamic acid with ATP.
(1) contain N-acetylglutamat kinases just like the aminoacid sequence shown in the SEQ ID NO:1 in the sequence table, wherein 287 glycine residue corresponding to SEQ ID NO:1 is replaced by asparagicacid residue.
(2) according to any one N-acetylglutamat kinases of (1), wherein this enzyme clone is from Corynebacterium crenatum SYPA5-5.
(3) the N-acetylglutamat kinases of basis (1) and (2); The one or more positions that comprise (as 281,268 etc.) beyond 287 of the aminoacid sequence shown in a kind of SEQ IDNO:1 in sequence table have the aminoacid sequence of one or several amino acid whose disappearance, replacement, insertion or interpolation.
(4) according to the N-acetylglutamat kinases of (1) or (3), wherein corresponding to 287 the glycine residue of SEQ ID NO:1 in the sequence table by the aminoacid replacement beyond the asparagicacid residue.
2, the present invention introduces the sudden change of NAGK enzyme in intestinal bacteria, and with being included in recombinant DNA transformed into escherichia coli and the Corynebacterium crenatum that belongs to the argB gene of acting carrier and sudden change in the intestinal bacteria, wherein used production bacterial strain is a Corynebacterium crenatum.
Be used for introducing sudden change argB
SDThe example of the carrier of gene is a plasmid vector, pBR322, and pUC18, pET serial carrier etc., the plasmid vector that uses among the present invention is pET28a.Be used for intestinal bacteria---the Corynebacterium crenatum shuttle expression carrier pJC1-tac that the clonal expression carrier of Corynebacterium crenatum makes up for the inventor voluntarily.ArgB with reduction that obtains or releasing feedback inhibition
SDGene is introduced through pJC1-tac and is produced in the arginic production bacterial strain Corynebacterium crenatum, thereby further increases the arginic output of L-.
Description of drawings
Fig. 1 representes primer P1, P2 and P3, the relative position of P4 and overlapping PCR synoptic diagram.
Fig. 2 representes overlapping PCR nucleic acid electrophoresis figure
1:DL2000Marker; 2,3: first round PCR product; Take turns the PCR product at 4: the second;
Fig. 3 SDS-PAGE electrophoretic analysis behind bacterium whole-cell protein and the NAGK purifying of recombinating
1.Protein markers (kDa); 2. E.coli BL21/pET-28a (+)-argB recombinates
SD3. NAGK behind the purifying
The practical implementation method
Increase with the PCR program, obtain wild-type argB gene, and be cloned into carrier pET28a, produce plasmid pET28a-argB.As template, the oligonucleotide P1 that describes among the SEQ ID NO:3 and 4 and P2 are as primer with the genomic dna of Corynebacterium crenatum, and the pcr amplification parameter is 94 ℃ of sex change 90s, 55 ℃ of renaturation 1min, 72 ℃ of extension 1.5min, 35 circulations.Gained glue reclaims product and is connected with the pMD18-T carrier, order-checking.Sequencing result is the nucleotide sequence that shows among the SEQ ID NO:2.Utilize the argB gene of the method for overlapping PCR through two-wheeled PCR acquisition sudden change.Two-wheeled pcr amplification parameter is 94C sex change 30s, 52 ℃ of renaturation 30s, and 72 ℃ are extended 30s, 25 circulations.Extract plasmid pMD18-T-argB as the template of carrying out the reaction of the rite-directed mutagenesis first round; Respectively with SEQ ID NO:3 and 6 and SEQ ID NO:5 and 4 carry out pcr amplification; The product that obtains is the two ends product of sudden change argB gene; Be template with these two sections products again, carry out second with primer SEQ IDNO:3 and 4 and take turns the PCR reaction, thereby obtained the argB of sudden change
SDGene.(Fig. 2)
With the argB that obtains through two-wheeled PCR among the embodiment 1
SDMutator gene is through EcoRI and SalI double digestion, and glue reclaims argB
SDFragment is connected it with the plasmid pET-28a that cuts through same enzyme, among the transformed into escherichia coli BL21 (DE3), the screening positive transformant obtains positive transformant BL21/pET-28a-argB
SDSelecting positive transformant is inoculated in the LB liquid nutrient medium that contains 50 μ g/mL kantlex; 37 ℃ of shaking table overnight cultures; Transfer by 1% inoculum size next day and in the triangular flask of the 250mL that the 50mL substratum is housed, continue to cultivate 4h to logarithmic phase; Add IPTG (final concentration is 1mmol/L), 16 ℃ of abduction deliverings that spend the night.Utilize and contain the 6HisTag encoding sequence among the coli expression carrier pET-28a, the method purifying acetylglutamate kinase NAGK of available Ni-NTA column purification target protein.The NAGK that purifying is obtained carries out the mensuration of protein-active, and adds l-arginine and measure its inhibition concentration.With compare without the NAGK enzyme work of sudden change, the 287th is the reduction of living of the NAGK enzyme after the amino-acid residue sudden change, but its inhibition concentration I
0.5 ArgBe increased to 0.2M from 0.4mM, inhibition concentration has improved 500 times.Continuation is carried out other site mutation to NAGK, finally obtains the N-acetylglutamat kinases of anti-l-arginine feedback inhibition.Embodiment 3. makes up the recombinant corynebacterium crematum mutant strain with argB mutator gene
Utilize construction recombination plasmid pET-28a-argB among the embodiment 2
SDConstruction process construction recombination plasmid pJC1-tac-argB
SDExtract plasmid pJC1-tac-argB
SD, electricity consumption commentaries on classics method transforms Corynebacterium crenatum SYPA, coat contain 10 μ g/mLKan to contain glucose LB dull and stereotyped, 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 Corynebacterium crenatum reorganization bacterium C.C. (pJC1-tac-argB
SD) make up successfully.
Success obtains recombinant corynebacterium crematum C.C. (pJC1-tac-argB
SD) after, transformant of picking is cultivated thalline to logarithmic phase in the l-arginine fermention medium immediately, utilizes the ultrasonic disruption method to the Corynebacterium crenatum bacterial cell disruption, measures parameter such as work of NAGK enzyme and l-arginine inhibition concentration in its born of the same parents.The result shows that the NAGK activity ratio wild-type Corynebacterium crenatum enzyme work in its born of the same parents has improved 40.8%, and it receives arginic inhibition concentration I
0.5 ArgBe increased to 0.13M.And Corynebacterium crenatum fermentation product arginine yield reaches 0.17M, therefore can confirm on the basis of this output, has tentatively removed it and has received the arginic product inhibition of product.
Immediately picking 6 recombinant corynebacterium crematum transformant C.C (pJC1-tac-argB
SD) and starting strain C.crenatumSYPA while shake flask fermentation, it is parallel that each transformant is done 3 fermentations, behind the shaking culture 96h, measures its arginine yield, wherein transformant C.C (pJC1-tac-argB
SD) output is the highest, improved approximately 29.7% than starting strain C.crenatum SYPA acid yield, concrete outcome is as shown in table 1.
The arginine yield of table 1 recombinant corynebacterium crematum transformant
Sequence table
< 110>Southern Yangtze University
< 120>Corynebacterium crenatum N-acetylglutamat kinase mutant improves the method for arginine yield
<140>201010166997.X
<141>2010-05-10
<160>6
<210>1
<211>316
<212>PRT
< 213>artificial sequence
<220>
<22?1>VARIANT
<222>(287)
< 223>aspartic acid substituted glycinic acid
<400>1
M?N?D?L?I?K?D?L?G?S?E?V?R?A?N
1 6 11
V?L?A?E?A?L?P?W?L?Q?H?F?R?D?K
16 21 26
I?V?V?V?K?Y?G?G?N?A?M?V?D?D?D
31 36 41
L?K?A?A?F?A?A?D?M?V?F?L?R?T?V
46 51 56
G?A?K?P?V?V?V?H?G?G?G?P?Q?I?S
61 66 71
E?M?L?N?R?V?G?L?Q?G?E?F?K?G?G
76 81 86
F?R?V?T?T?P?E?V?M?D?I?V?R?M?V
91 96 101
L?F?G?Q?V?G?R?D?L?V?G?L?I?N?S
106 111 116
H?G?P?Y?A?V?G?T?S?G?E?D?A?G?L
121 126 131
F?T?A?Q?K?R?M?V?N?I?D?G?V?P?T
136 141 146
D?I?G?L?V?G?D?I?I?N?V?D?A?S?S
151 156 161
L?M?D?I?I?E?A?G?R?I?P?V?V?S?T
166 171 176
I?A?P?G?E?D?G?Q?I?Y?N?I?N?A?D
181 186 191
T?A?A?G?A?L?A?A?A?I?G?A?E?R?L
196 201 206
L?V?L?T?N?V?E?G?L?Y?T?D?W?P?D
211 216 221
K?S?S?L?V?S?K?I?K?A?T?E?L?E?A
226 231 236
I?L?P?G?L?D?S?G?M?I?P?K?M?E?S
241 246 251
C?L?N?A?V?R?G?G?V?S?A?A?H?V?I
256 261 266
D?G?R?I?A?H?S?V?L?L?E?L?L?T?M
271 276 281
G?GIG?T?M?V?L?P?D?V?F?D?R?E
286 291 296
N?Y?P?E?G?T?V?F?R?K?D?D?K?D?G
301 306 311
E?L
316
<210>2
<211>954
<212>DNA
< 2 13>artificial sequence
<220>
<221>gene
<222>(859)...(861)
< 223>sudden change N-acetylglutamat kinases coding gene sequence sports GAT by GGA
<400>2
atgaatgact?tgatcaaaga?tttaggctct?gaggtgcgcg?caaatgtcct?cgctgaggcg 60
ttgccatggt?tgcagcactt?ccgcgacaag?attgttgtcg?tgaaatatgg?cggaaacgcc 120
atggtggatg?atgatctcaa?ggctgctttt?gctgccgaca?tggtcttctt?gcgcaccgtg 180
ggcgcaaaac?cagtggtggt?gcacggtggt?ggacctcaga?tttctgagat?gctaaaccgt 240
gtgggtctcc?agggcgagtt?caagggtggt?ttccgtgtga?ccactcctga?ggtcatggac 300
attgtgcgca?tggtgctctt?tggtcaggtc?ggtcgcgatt?tagttggttt?gatcaactct 360
catggccctt?acgctgtggg?aacctccggt?gaggatgccg?gcctgtttac?cgcgcagaag 420
cgcatggtca?acatcgatgg?cgtacccact?gatattggtt?tggtcggaga?catcattaat 480
gtcgatgcct?cttccttgat?ggatatcatc?gaggccggtc?gcattcctgt?ggtctctacg 540
attgctccag?gcgaagacgg?ccagatttac?aacattaacg?ccgataccgc?agcaggtgct 600
ttggctgcag?cgattggtgc?agaacgcctg?ctggttctca?ccaatgtgga?aggtctgtac 660
accgattggc?ctgataagag?ctcactggtg?tccaagatca?aggccaccga?gctggaggcc 720
attcttccgg?gacttgattc?cggcatgatt?ccaaagatgg?agtcttgctt?gaacgcggtg 780
cgtgggggag?taagtgctgc?tcatgtcatt?gacggccgca?tcgcgcactc?ggtgttgctg 840
gagcttttga?ccatgggtgg?aattggcacg?atggtgctgc?cggatgtttt?tgatcgggag 900
aattatcctg?aaggcaccgt?ttttagaaaa?gacgacaagg?atggggaact?gtaa 954
<210>3
<211>28
<212>DNA
< 213>Corynebacterium crenatum (C.crenatum) argB upstream region of gene primer P1 sequence
<400>3
cgcgaattca?tgaatgactt?gatcaaag 28
<210>4
<211>28
<212>DNA
< 213>Corynebacterium crenatum (C.crenatum) argB gene downstream primer P2 sequence
<400>4
cgcgtcgact?tacagttccc?catccttg 28
<210>5
<211>28
<212>DNA
< 213>artificial sequence
<220>
<221>gene
<222>(8)...(10)
< 223>sport GAT by GGA
<400>5
catgggtgat?attggcacga?tggtgctg 28
<210>6
<211>28
<212>DNA
< 213>artificial sequence
<220>
<221>gene
<222>(18)...(20)
< 223>sport ATC by TCC
<400>6
agcaccatcg?tgccaatatc?acccatgg 28
Claims (2)
1. a mutant N-acetylglutamat kinases is characterized in that be aspartic acid with kinase whose the 287th of sequence N-acetylglutamat shown in SEQ NO.1 by glycine mutation.
2. one kind is utilized the said mutant N-acetylglutamat of claim 1 kinases to produce arginic method; It is characterized in that adopting electrotransformation that the said mutant N-acetylglutamat of claim 1 kinase gene is changed in the Corynebacterium crenatum; Obtain to produce l-arginine and use bacterial strain, said bacterial strain is used for l-arginine production.
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| CN103074343B (en) * | 2013-02-04 | 2015-10-14 | 江南大学 | A kind of method utilizing the endogenous High-expression promoter of 2-DE technology screening Corynebacterium crenatum |
| CN103320438B (en) * | 2013-05-24 | 2015-06-24 | 江南大学 | Screening method for corynebacterium crenatum dissolved oxygen inducible promoter |
| CN104059863A (en) * | 2014-05-06 | 2014-09-24 | 江南大学 | Metabolic transformation method for efficiently improving production capacity of corynebacterium crenatum SYPA5-5 L-arginine |
| CN104031933A (en) * | 2014-05-08 | 2014-09-10 | 江南大学 | Construction and application method for L-ornithine synthesis bacteria |
| KR101835935B1 (en) | 2014-10-13 | 2018-03-12 | 씨제이제일제당 (주) | A microorganism of genus corynebacterium having an ability to produce L-arginine and a method for producing L-arginine using the same |
| CN105543303A (en) * | 2016-01-19 | 2016-05-04 | 江南大学 | Biosynthesis method for increasing yield of trans-4-hydroxyproline by knocking out other metabolic pathway |
| CN105838690B (en) * | 2016-05-03 | 2019-05-17 | 江南大学 | A kind of N-acetylglutamat kinase mutants that thermal stability significantly improves |
| CN105907735B (en) * | 2016-05-03 | 2019-07-30 | 江南大学 | A kind of N-acetylglutamat kinase mutants of catalytic efficiency and thermal stability raising |
| CN105838689B (en) * | 2016-05-03 | 2019-05-21 | 江南大学 | A kind of N-acetylglutamat kinase mutants that catalytic efficiency improves |
| CN106434594B (en) * | 2016-11-08 | 2019-09-10 | 江南大学 | A kind of N-acetylglutamat kinase mutants and its application |
| CN112877269B (en) * | 2020-01-15 | 2021-12-24 | 中国科学院天津工业生物技术研究所 | Microorganism producing lysine and method for producing lysine |
| CN111718883A (en) * | 2020-06-28 | 2020-09-29 | 江南大学 | Recombinant corynebacterium crenatum capable of producing agmatine and application thereof |
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| CN101381698A (en) * | 2008-10-23 | 2009-03-11 | 江南大学 | Recombinant corynebacterium crenatum for over expression of N-acetylglutamate kinase and application thereof |
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| Title |
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| HongXu et al.A two-stage oxygen supply strategy for enhanced L-arginine production by Corynebacterium crenatum based on metabolic fluxes analysis.《Biochemical Engineering Journal》.2009,第43卷41-51. * |
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