CN1884501A - Glutamine synthetase and its dedicated expression engineered bacteria and uses - Google Patents
Glutamine synthetase and its dedicated expression engineered bacteria and uses Download PDFInfo
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention discloses a glutamine synthetase and its special-purpose expression project bacterium and its allocation, which is to provide a glutamine synthetase and its special-purpose expression project bacterium and its allocation. The said glutamine synthetase is prepared by abrupt change of the 405th amino acid residue of amino end to phenylalanine corynebacterium glutamicum. The engineering bacteria prepare the recombination bacteria by importing the recombination expression carrier containing the said glutamine synthetase gene into host bacteria. The glutamine synthetase produced by the engineering bacteria can specially act on NH4+ and aminoglutaric acid, the durability to the high concentration ammonium ion has been distinctively improved because of the dissolution of adenosine acylation modification, the major bottleneck of glutamine with enzymatical production has been solved, and the concentration of the obtained glutamine can reach 47.6g/L. The invention has higher practicability and flackery and has wide application foreground in glutamine commercial manufacture.
Description
Technical field
The present invention relates to glutamine synthetase and dedicated expression engineered bacteria thereof and application, particularly relate to the application in producing glutamine of a kind of glutamine synthetase and dedicated expression engineered bacteria thereof and this project bacterium.
Background technology
Glutamine is a kind of important foodstuff additive and medicine material, has higher nutritive value and pharmaceutical use.The market requirement of glutamine is growing, at present, the glutamine market value is about ten thousand yuans of 10-15 per ton, the demand of domestic glutamine is about 5000 tons/year, market value is above 500,000,000 yuan, if can create bigger economic benefit after being processed into medicament or healthcare products, market outlook are wide.The major country of production of glutamine is Japan, Korea S and China in the world, and the fermentation concentration of Japan is up to 60g/L, and Korea S has also reached 50g/L.At present, in the domestic optimization that mainly rests on selection by mutation and zymotechnique in the research aspect the glutamine ferment.Though obtained the production bacterial strain of some glutamine by selection by mutation, final fermentation concentration and productive rate be low than Japan and Korea S all, thereby be difficult to provide support for the scale operation of glutamine.
The Production by Enzymes glutamine has great application prospect, its basic ideas be with monosodium glutamate and ammonium salt as raw material, glutamine synthetase (glutamine synthetase, GS) and the synthetic glutamine of catalysis under the condition of energy ATP is provided.Compare with microbe fermentation method, the Production by Enzymes glutamine has advantages such as cost is low, reactions steps is simple, side reaction is few, easily separated.At present, the existing report that successfully synthesizes glutamine on a small quantity with enzyme process, Yang Chunyu etc. utilize Corynebacterium glutamicum (C.glutamicum) fermentation to obtain the GS enzyme, through obtaining containing the crude enzyme liquid of GS enzyme after the cytoclasis, join again and contain L-glutamic acid, in the phosphate buffered saline buffer of ammonium salt and ATP, the transformation efficiency of L-glutamic acid has reached 94.8% (Yang Chunyu after testing, Ma Cuiqing, permitted equality. the enzymatic conversion method glutamine. the process engineering journal, 2002,2 (6): 529 ~ 533), but this method adopts the mode that adds ATP that energy is provided, ATP costs an arm and a leg, thereby can't be used for suitability for industrialized production; Chen Qunying etc. utilize engineered method, realized the high expression level of GS enzyme in intestinal bacteria, after extracting the GS enzyme of expressing in the reorganization mycetocyte it is joined in the fermentation system of fresh yeast saccharomyces cerevisiae, utilize the ATP that glycolysis glucose produces in the yeast fermentation process to be GS enzyme catalysis L-glutamic acid and NH
4 +Synthetic glutamine provides energy, in the method, the GS enzyme of escherichia coli expression accounts for 80% of total mycoprotein, and utilize yeast fermentation to produce the energy derive problem that ATP has solved GS enzyme catalysis process well, but the fermentative preparation of GS enzyme and enzyme catalysis process are finished in two living things systems, need carry out ultrasonic broken wall to recombination bacillus coli and extract the GS enzyme, in addition, for make ATP and ADP can be between yeast cell and catalyst system cyclic regeneration, also to change its permeability with the O for toluene yeast cells wall, thereby complicated operation, the suitability for industrialized production difficulty big (Chen Qunying, Chen Guoan, Xue Bin etc. the research of the efficient synthetic L-glutaminate of genetically engineered enzyme process combining yeast energy coupling connection. the biotechnology journal, 2004,20 (3): 456-460).Above-mentioned result of study shows that adopting yeast fermentation to produce ATP and enzyme process produces glutamine the heavy industrialization application difficulty of coupling connection is bigger mutually.
The Production by Enzymes glutamine is to use NH
4 +Make raw material with L-glutamic acid, through glutamine synthetase catalytic production glutamine.There are two kinds of forms in the GS enzyme in bacterial body: a kind of is unmodified, and another kind is through the adenosine covalent modification.To be exactly AMP combine the process that produces GS (AMP) with covalent and the tyrosine residues on the peptide chain to the adenylylation of GS enzyme, the adenylylation of GS enzyme and go adenylylation all by adenylyl transferase (adenylyltransferase, ATase) catalysis.Adenylylation can make the activity of GS enzyme reduce or forfeiture.In addition, the adenylylation of GS enzyme modification inactivation and inverse process thereof also are subjected to the regulation and control of ammonium salt concentration, grow into the intracellular GS enzyme of stationary phase and do not modified by adenosine under the culture condition of restriction ammonium salt; Under the culture condition of excessive ammonium salt, the adenylylation degree strengthens, and the GS enzyme activity descends even inactivation.But in the process of Production by Enzymes glutamine, ammonium salt is the catalytic substrate of GS enzyme, and desire improves glutamine output, must strengthen the supply of ammonium salt, to realize the conversion to glutamine of L-glutamic acid and ammonium salt.Therefore, press for the method for a kind of releasing, to keep the activity of GS enzyme in the catalytic production glutamine process to the modification of GS enzyme adenylylation.
Summary of the invention
The purpose of this invention is to provide a kind of glutamine synthetase of removing the adenylylation modification.
Glutamine synthetase provided by the present invention is the glutamine synthetase that sports the Corynebacterium glutamicum (C.glutamicum) of phenylalanine from aminoterminal the 405th amino acids residue.
The gene of above-mentioned glutamine synthetase of encoding also belongs to protection scope of the present invention.
The 3rd purpose of the present invention provides a kind of dedicated expression engineered bacteria of glutamine synthetase.
The dedicated expression engineered bacteria of glutamine synthetase provided by the present invention is that the recombinant expression vector that will contain described glutamine synthetase gene imports the reorganization bacterium that obtains in the host bacterium.
The carrier that sets out that is used to make up described recombinant expression vector can be at expression in escherichia coli expression of exogenous gene carrier, as pET-3a, pET-30a, pET-28a, pET-28b or pET-28c etc.
Be the carrier that sets out with pET-3a, the recombinant expression vector that contains described glutamine synthetase gene of structure is pET-3a/GSIM.
Described host can be intestinal bacteria, excellent bacillus, yeast or Bacillus subtilus etc., is preferably intestinal bacteria.
Colibacillary concrete bacterial strain is not had particular requirement, and selection is that non-constant width is general, all can such as E.coli BL21 (DE3), E.coli BL21 (DE3) plys, BLR (DE3) or B834 etc.
With E.coli BL21 (DE3) is starting strain, and pET-3a/GSIM is imported reorganization bacterium called after BL21 (DE3) GSIM that E.coli BL21 (DE3) obtains.
Above-mentioned recombinant expression vector and reorganization bacterium all can make up according to ordinary method.
Another object of the present invention provides a kind of preparation method of glutamine.
The preparation method of glutamine provided by the present invention, be the dedicated expression engineered bacteria of the above-mentioned glutamine synthetase of fermentation, obtain glutamine synthetase, add substrate ammonium salt and Sodium Glutamate again, continue fermentation 24-48 hour down at 20-37 ℃, obtain glutamine; NH in the described fermented liquid
4 +Concentration be 10-100g/L, the concentration of Sodium Glutamate is 10-150g/L.
Can adopt the conventional culture condition that makes up the used starting strain of engineering bacteria that engineering bacteria is cultivated, when described engineering bacteria is recombination bacillus coli, need to add the IPTG inductor, add IPTG concentration be 0.01-1.2mmol/L, inducing temperature is 20-39 ℃, and induction time is 2-8 hour.
The selection of described ammonium salt is diversified, as ammonium chloride, ammonium sulfate, ammonium nitrate or ammonium acetate etc.; Leavening temperature behind the adding substrate is preferably 30 ℃.
The invention provides a kind of glutamine synthetase.This enzyme is on the basis of the glutamine synthetase of original Corynebacterium glutamicum, and the tyrosine residues in adenylylation site is sported phenylalanine.And utilize prokaryotic expression carrier will be transformed in the host bacterium encoding gene of this glutamine synthetase, obtained the expression engineering bacteria of glutamine synthetase.This project bacterium can produce enzyme higher glutamine synthetase alive after fermentation, enzyme activity can reach 1200U/L, be significantly higher than the enzyme activity of the glutamine synthetase of producing with existing bacterial strain, and this project bacterium has the expression amount height, and production cost is low, is easy to the advantage of industrialization.The glutamine synthetase that engineering bacteria of the present invention produces can change the NH that acts on of a property
4 +And L-glutamic acid, and because of having removed the adenylylation modification tolerance of high density ammonium ion is significantly improved, having solved the important bottleneck of Production by Enzymes glutamine, the concentration of the glutamine of generation is up to 47.6g/L.But the present invention has advantages of high practicability and generalization, has broad application prospects in the suitability for industrialized production of glutamine.
Below in conjunction with specific embodiment the present invention is described in further detail.
Description of drawings
Fig. 1 is the structure schema of pET-3a/GSIM
Fig. 2 is the growing state comparative result of control strain and recombinant bacterial strain in the fermenting process
Fig. 3 prepares the comparative result of glutamine for the glutamine synthetase that produces with control strain and recombinant bacterial strain fermentation
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.The primer sequence is synthetic by match Parkson company.
The acquisition of embodiment 1, glutamine synthetase rite-directed mutagenesis gene
Adopt overlapping PCR method to the glutamine synthetase gene of Corynebacterium glutamicum (C.glutamicum) (NCBIGenBank number: Y13221) carry out rite-directed mutagenesis; the codon mutation that is about to from 5 ' end adenylylation site, 1213-1215 position tyrosine is the phenylalanine codon; and introduce the recognition site of restriction enzyme Nde I and Hind III respectively at the sequence two ends, concrete grammar may further comprise the steps:
1, first round amplification
Genomic dna with Corynebacterium glutamicum (C.glutamicum) is a template, by primer 1:5 '-ATAAGGGAGGAGTG
CATATG1) and primer 3:5 '-GGTAG (band underscore base is a restriction enzyme Nde I recognition site to GCGTTTGAAA-3 ', SEQ ID № in the sequence table:
(base is the sudden change codon to GAAGAGGTCCTTGTCCACTGGAG-3 ' in the square frame, SEQ ID № in the sequence table: 3) under the right guiding of the primer of Zu Chenging, the upstream segment of pcr amplification glutamine synthetase gene is also introduced mutating alkali yl, and the PCR reaction conditions is: 94 ℃ of sex change of elder generation 5 minutes; 94 ℃ of sex change are 30 seconds then, 58 ℃ of annealing 45 seconds, and 72 ℃ were extended totally 30 circulations 1.5 minutes; Last 72 ℃ were extended 10 minutes, took out after being cooled to 4 ℃.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, pcr amplification has gone out the single band of big or small about 1240bp as a result, conforms to expected results.
2, second take turns amplification
Genomic dna with Corynebacterium glutamicum (C.glutamicum) is a template, by primer 2: 5 '-CAAGGACCTCTTC
(base is the sudden change codon to CTACCACCAGAGGAA-3 ' in the square frame, SEQ ID № in the sequence table: 2) and primer 4:5 '-AATCAAGCTTACCACACGGAACCGTCTCACT-3 ' (band underscore base is a restriction enzyme Hind III recognition site, SEQ ID № in the sequence table: 4) under the right guiding of the primer of Zu Chenging, the downstream segment of pcr amplification glutamine synthetase gene is also introduced mutating alkali yl, and the PCR reaction conditions is: 94 ℃ of sex change of elder generation 5 minutes; 94 ℃ of sex change are 30 seconds then, 54 ℃ of annealing 45 seconds, and 72 ℃ were extended totally 30 circulations 30 seconds; Last 72 ℃ were extended 10 minutes, took out after being cooled to 4 ℃.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, pcr amplification has gone out the single band of big or small about 440bp as a result, conforms to expected results.
3, third round amplification
Upstream and downstream segment with the glutamine synthetase gene of step 1 and step 2 amplification is a template, under the right guiding of the primer of being made up of primer 1 and primer 4, carries out overlapping pcr amplification, and the PCR reaction conditions is: first 94 ℃ of sex change 5 minutes; 94 ℃ of sex change are 30 seconds then, 62 ℃ of annealing 45 seconds, and 72 ℃ were extended totally 30 circulations 1.5 minutes; Last 72 ℃ were extended 10 minutes, took out after being cooled to 4 ℃.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, pcr amplification has gone out the single band of big or small about 1660bp as a result, conforms to expected results.Reclaim and this purpose segment of purifying, it is checked order, sequencing result shows that the glutamine synthetase mutator gene of the overlapping PCR method amplification of this usefulness has SEQ ID № in the sequence table: 5 nucleotide sequence, from 5 ' end 1213-1215 bit base is the mutational site, codon mutation by tyrosine is the phenylalanine codon, and is consistent with expected results.
The structure of embodiment 2, glutamine synthetase dedicated expression engineered bacteria
One, the structure of glutamine synthetase gene expression of recombinant e. coli carrier
The glutamine synthetase mutator gene of embodiment 1 amplification is carried out after enzyme cuts with restriction enzyme Nde I and HindIII, be connected with the plasmid pET-3a (Novagen) that contains penbritin (Amp) resistance marker through the same enzyme double digestion, to connect product Transformed E .coli BL21 (DE3) competent cell, the transformant that screening has the Amp resistance, obtain containing glutamine synthetase mutator gene recombinant expression vector, called after pET-3a/GSIM, it makes up schema and sees Fig. 1.
Two, with recombinant expression vector pET-3a/GSIM transformed into escherichia coli
Recombinant expression vector pET-3a/GSIM CaCl with the step 1 structure
2Method Transformed E .coli BL21 (DE3) competent cell is coated transformant on the LB resistant panel that contains 100 μ g/mL Amp, cultivates 12-16h down at 37 ℃.After growing single bacterium colony, picking list bacterium colony is as template, carrying out bacterium colony PCR under the guiding of primer 1 and primer 4 identifies, can obtain the segmental positive clone of 1660bp through pcr amplification, the upgrading grain, carrying out single endonuclease digestion with restriction enzyme Nde I and Hind III respectively earlier identifies, cut the segmental positive plasmid of acquisition 5780bp through Nde I enzyme, cut the segmental positive plasmid of acquisition 5780bp through Hind III enzyme, carrying out double digestion with Nde I and Hind III again identifies, cut acquisition 4120bp and the segmental positive plasmid of 1660bp through enzyme, qualification result shows and has obtained the positive recombinant that correct conversion has goal gene, can be used as the engineering bacteria of expressing glutamine synthetase, with this project bacterium called after BL21 (DE3) GSIM.
The preparation of embodiment 2, glutamine
Reorganization bacterium BL21 (DE3) GSIM that embodiment 1 is made up is inoculated in the 50mL LB liquid nutrient medium, be contrast with E.coli BL21 (DE3), 2-3 hour OD to bacterium liquid of cultivation in 37 ℃, 170rpm shaking table
600Value is 0.6-1.0, the basic identical (see figure 2) of the growing state of control strain and recombinant bacterial strain, add the IPTG that final concentration is 0.1mmol/L then, continue cultivation and made the GS expression of enzymes in 23 hours, GS enzyme work in the recombinant bacterial strain can reach 122.84U/L after testing, comparison has improved 6 times according to bacterium E.coli BL21 (DE3), adds substrate NH then
4Cl (10-100g/L fermented liquid) and Sodium Glutamate (10-150g/L fermented liquid) are adjusted shaking table temperature to 30 ℃, continue fermentation 24-48h, make the GS enzyme catalysis L-glutamic acid and the synthetic glutamine of ammonium of fermentation expression.After the fermentation ends; detect the glutamine concentration in the fermented liquid; the concentration that the result produces the glutamine of GS enzyme preparation with the fermentation of reorganization bacterium is up to 47.6g/L; be 51.8 times of (see figure 3)s that contrast bacterium E.coliBL21 (DE3) produces glutamine concentration; show that glutamine synthetase adenylylation site mutation has significantly improved the ability of glutamine synthetase tolerance high density ammonium ion; solved the important bottleneck of Production by Enzymes glutamine, engineering strain of the present invention can be the large-scale industrial production glutamine and provides support.
Sequence table
<160>5
<210>1
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
ataagggagg?agtgcatatg?gcgtttgaaa 30
<210>2
<211>31
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
caaggacctc?ttcgaactac?caccagagga?a 31
<210>3
<211>31
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
ggtagttcga?agaggtcctt?gtccactgga?g 31
<210>4
<211>31
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
aatcaagctt?accacacgga?accgtctcac?t 31
<210>5
<211>1434
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
atggcgtttg?aaaccccgga?agaaattgtc?aagttcatca?aggatgaaaa?cgtcgagttc 60
gttgacgttc?gattcaccga?ccttcccggc?accgagcagc?acttcagcat?cccagctgcc 120
agcttcgatg?cagatacaat?cgaagaaggt?ctcgcattcg?acggatcctc?gatccgtggc 180
ttcaccacga?tcgacgaatc?tgacatgaat?ctcctgccag?acctcggaac?ggccaccctt 240
gatccattcc?gcaaggcaaa?gaccctgaac?gttaagttct?tcgttcacga?tcctttcacc 300
cgcgaggcat?tctcccgcga?cccacgcaac?gtggcacgca?aggcagagca?gtacctggca 360
tccaccggca?ttgcagacac?ctgcaacttc?ggcgccgagg?ctgagttcta?cctcttcgac 420
tccgttcgct?actccaccga?gatgaactcc?ggcttctacg?aagtagatac?cgaagaaggc 480
tggtggaacc?gtggcaagga?aaccaacctc?gacggcaccc?caaacctggg?cgcaaagaac 540
cgcgtcaagg?gtggctactt?cccagtagca?ccatacgacc?aaaccgttga?cgtgcgcgat 600
gacatggttc?gcaacctcgc?agcttccggc?ttcgctcttg?agcgtttcca?ccacgaagtc 660
ggtggcggac?agcaggaaat?caactaccgc?ttcaacacca?tgctccacgc?ggcagatgat 720
atccagacct?tcaagtacat?catcaagaac?accgctcgcc?tccacggcaa?ggctgcaacc 780
ttcatgccta?agccactggc?tggcgacaac?ggttccggca?tgcacgctca?ccagtccctc 840
tggaaggacg?gcaagccact?cttccacgat?gagtccggct?acgcaggcct?gtccgacatc 900
gcccgctact?acatcggcgg?catcctgcac?cacgcaggcg?ctgttctggc?gttcaccaac 960
gcaaccctga?actcctacca?ccgtctggtt?ccaggcttcg?aggctccaat?caacctggtg 1020
tactcacagc?gcaaccgttc?cgctgctgtc?cgtatcccaa?tcaccggatc?caacccgaag 1080
gcaaagcgca?tcgaattccg?cgctccagac?ccatcaggca?acccatacct?gggctttgca 1140
gcgatgatga?tggccggcct?cgacggcatc?aagaaccgca?tcgagccaca?cgctccagtg 1200
gacaaggacc?tcttcgaact?accaccagag?gaagctgcat?ccattccaca?ggcaccaacc 1260
tccctggaag?catccctgaa?ggcactgcag?gaagacaccg?acttcctcac?cgagtctgac 1320
gtcttcaccg?aggatctcat?cgaggcgtac?atccagtaca?agtacgacaa?cgagatctcc 1380
ccagttcgcc?tgcgcccaac?cccgcaggaa?ttcgaattgt?acttcgactg?ctaa 1434
Claims (9)
1, glutamine synthetase is the glutamine synthetase that sports the Corynebacterium glutamicum of phenylalanine from aminoterminal the 405th amino acids residue.
2, the gene of the described glutamine synthetase of coding claim 1.
3, expressing the engineering bacteria of glutamine synthetase, is that the recombinant expression vector that will contain the described glutamine synthetase gene of claim 2 imports the reorganization bacterium that obtains in the host bacterium.
4, engineering bacteria according to claim 3 is characterized in that: the carrier that sets out that is used to make up described recombinant expression vector is a coli expression carrier.
5, engineering bacteria according to claim 4 is characterized in that: described recombinant expression vector is pET-3a/GSIM.
6, according to claim 3 or 4 or 5 described engineering bacterias, it is characterized in that: described host is intestinal bacteria.
7, a kind of preparation method of glutamine, be the engineering bacteria of the described expression glutamine synthetase of fermentation claim 3, obtain glutamine synthetase, add substrate ammonium salt and Sodium Glutamate again, continue fermentation 24-48 hour down at 20-37 ℃, obtain glutamine; NH in the described fermented liquid
4 +Concentration be 10-100g/L, the concentration of Sodium Glutamate is 10-150g/L.
8, method according to claim 7 is characterized in that: when described engineering bacteria is recombination bacillus coli, add the IPTG inductor, add IPTG concentration be 0.01-1.2mmol/L, inducing temperature is 20-39 ℃, induction time is 2-8 hour.
9, according to claim 7 or 8 described methods, it is characterized in that: described ammonium salt is ammonium chloride, ammonium sulfate, ammonium nitrate or ammonium acetate; Leavening temperature behind the adding substrate is 30 ℃.
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| CN107603938A (en) * | 2017-10-30 | 2018-01-19 | 天津科技大学 | It is overexpressed the genetic engineering bacterium and its construction method of heterologous glutamyl amine synzyme |
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|---|---|---|---|---|
| RU2230114C2 (en) * | 2001-11-30 | 2004-06-10 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" | Mutant glutamine synthetase, dna fragment, strain of escherichia coli as p roducer of l-glutamine and method for preparing l-amino acids |
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| JP2022529297A (en) * | 2020-03-04 | 2022-06-20 | シージェイ チェイルジェダン コーポレーション | Glutamine synthetase mutant polypeptide and L-glutamine production method using it |
| JP7371119B2 (en) | 2020-03-04 | 2023-10-30 | シージェイ チェイルジェダン コーポレーション | Glutamine synthetase mutant polypeptide and L-glutamine production method using the same |
| CN114375332B (en) * | 2020-03-04 | 2024-01-12 | Cj第一制糖株式会社 | Modified polypeptide of glutamine synthetase and method for producing L-glutamine using the same |
| WO2023180398A1 (en) | 2022-03-23 | 2023-09-28 | Boehringer Ingelheim International Gmbh | Bacterial glutamine synthetase as selection marker in mammalian cells |
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