CN105695383A - Recombinant strain and application thereof - Google Patents
Recombinant strain and application thereof Download PDFInfo
- Publication number
- CN105695383A CN105695383A CN201610119402.2A CN201610119402A CN105695383A CN 105695383 A CN105695383 A CN 105695383A CN 201610119402 A CN201610119402 A CN 201610119402A CN 105695383 A CN105695383 A CN 105695383A
- Authority
- CN
- China
- Prior art keywords
- gene
- glutamic acid
- bacterial strain
- gdh
- mhz
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- 229920001214 Polysorbate 60 Polymers 0.000 description 2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/14—Glutamic acid; Glutamine
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Abstract
The invention relates to the field of microorganisms, in particular to a recombinant strain and application thereof. Experimental results show that before modification, wild corynebacterium glutanmicum ATCC 13869 almost generates no glutamic acid, and the yield of strain MHZ-0112-1 glutamic acid is 4.7 g/L; the yield of MHZ-0112-2 glutamic acid is 18.5 g/L; the yield of strain MHZ-0112-4 glutamic acid and the yield of MHZ-0112-5 glutamic acid are 23.4 g/L and 26.4 g/L respectively. When a strong promoter Psod is independently utilized for expressing a gdh gene, the yield of glutamic acid is increased about 1.8 times; when a promoter Ptuf is independently utilized for expressing a gltA gene, the enzyme activity of CS is improved 4 times, and the yield of glutamic acid is increased to 30.8 g/L; after the two genes are expressed at the same time, the yield of glutamic acid continues to be increased to 35.5 g/L, and the conversion rate reaches about 59%.
Description
Technical field
The present invention relates to microorganism field, particularly to recombinant bacterial strain and application thereof。
Background technology
Glutamic acid (Glutamicacid), its formal name used at school is 2-amino-5-shuttle base valeric acid, is one of 20 kinds of common amino acids constituting protein。Glutamic acid is the aminoacid kind that yield is maximum in the world at present, and the annual demand to glutamic acid in the whole world reaches 25,000,000 tons。Pidolidone is the levo form of glutamic acid, it has, and increasing is fresh, reduce blood ammonia, improve central nervous system, improve children ' s intelligence development, expand blood vessel, strengthen blood circulation, promote the functions such as Inflorescence development, and many important downstream product can be produced, such as L-sodium, L-threonine, polyglutamic acid etc., it is widely used in the industries such as food, medicine, artificial process hides, cosmetics, agricultural。
The preparation of glutamic acid originated in for 18th century。Germanization scholar in 1866 extracts from the hydrolysate of wheat gluten and obtains glutamic acid, nineteen fifty-seven, Japan took the lead in separating from nature obtaining Corynebacterium glutamicum (Corynebacteriumglutamicum), open the research utilizing sugar and ammoniacal fermentation to produce glutamic acid, because fermentation method has the advantages such as cost of material is low, reaction condition is gentle, can be mass-produced, it is still the main method that glutamic acid produces at present。
Corynebacterium glutamicum be a kind of aerobic, do not produce embrace son a gram-positive bacterium, Genome Size is 3282kb, it can not only utilize glucose to grow for sole carbon source, and other organic acid and carbohydrate of including acetic acid and bluff bat acid can be utilized to grow for sole carbon source or mixed carbon source。Corynebacterium glutamicum is biotin deficiency, it is necessary to supplementing biotin could grow。When the biotin in culture medium is excessive, thalline does not produce glutamic acid to cell exocrine；When biotin limited the quantity interpolation time, thalline then in a large number secretion produce glutamic acid, thus the Corynebacterium glutamicum of wild type can by biotin limit the quantity interpolation come secretion inducing produce glutamic acid。Additionally, the excessive synthesis glutamic acid of Corynebacterium glutamicum also can be induced when biotin is excessive by interpolation certain surfactant such as Twee40 or Tween60。
In recent years, completing and Corynebacterium glutamicum gene operating technology constantly perfect along with Corynebacterium glutamicum type strain ATCC13032 gene order-checking so that carry out Corynebacterium glutamicum relevant being transformed into reality by molecular biology method。Glutamic acid metabolic pathway of synthesizing in vivo is also reported in succession, sees Fig. 1。Glutamate producing bacterium kind has been done some genetic modifications according to the metabolic pathway of glutamic acid synthesis by Ajincomoto Co., Inc of Japan, and the citric acid synthesized enzyme gene as introduced bar shaped bacteria makes the ability that enterobacteria produces glutamic acid strengthen；Increase the copy number of pyruvate dehydrogenase gene and strengthen pyruvate dehydrogenase activity, and make the glutamic acid-producing ability of bar shaped bacteria improve；Process LAN YHFK gene makes Pidolidone output increased etc.。
Prior art generally requires adds excessive biotin and surfactant such as Twee40 or Tween60 in the medium, adds production cost, and productivity is often relatively low。
Summary of the invention
In view of this, the present invention provides a kind of recombinant bacterial strain and application thereof。Compared with wild strain, bacterial strain provided by the invention notable (P < 0.05) or extremely notable (P < 0.01) improve the fermentation yield of glutamic acid。
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides a kind of recombinant bacterial strain, it comprises the mutant-yggB gene of encoding mutant yggB protein。
In some specific embodiments of the present invention, the yggB protein that suddenlys change described in described recombinant bacterial strain is that A106 aminoacid place has sudden change。
In some specific embodiments of the present invention, described in described recombinant bacterial strain suddenly change yggB protein have A106V sudden change, it is thus achieved that Strain Designation be MHZ-0112-1。
In some specific embodiments of the present invention, described recombinant bacterial strain also includes coding and reduces the sudden change odhA gene of ODHC activity；Described sudden change odhA gene for carrying out inactivation transformation to odhA gene。Concrete, the Strain Designation containing sudden change odhA gene provided by the invention is MHZ-0112-2。Preparation method is as follows:
From glutamic acid metabolic pathway of synthesizing (Fig. 1), tricarboxylic acid cycle is the main competition approach of glutamic acid anabolism by the α-ketoglutaric acid of ketoglurate dehydrogenase complex (ODHC) catalysis to the conversion of S-(3-carboxy-propionyl)-coenzyme-A, in order to reduce the activity of ODHC, the encoding gene odhA of its Elo subunit need to be carried out inactivation transformation。The means adopted are introduced into base deletion, make to occur in protein synthesis frameshift mutation, thus reaching the purpose of inactivation。
NCBIGenBank data base obtains the nucleotide sequence (SEQIDNo.9) of Corynebacterium glutamicum (CorynebacteriumglutamicumATCC13869) odhA gene (GeneBankNO.NCgl1084), thus nucleotide sequence design introduces base deletion so that odhA gene inactivation at ad-hoc location, has synthesized four primers (as shown in SEQIDNO.10-13) based on base sequence and selected deletion sites。Utilize the super proofreading polymerase of Phusion (NewEnglandBioLabs), with B1/B2, B3/B4 as primer, prepare the upstream and downstream fragment in odhA base deletion site using the genomic DNA of Corynebacterium glutamicum ATCC13869 as template。PCR program is, 98 DEG C of degeneration 10s, 50 DEG C of renaturation 20s, and 72 DEG C extend 20s, after circulating 30 times。Two fragments of gained reclaim after test kit (sky root) purification through agarose gel, utilize primer sets B1/B4 to carry out overlapPCR amplification and obtain product, reclaiming through agarose gel utilizes EcoRI/BamHI to digest after test kit (sky root) purification, utilize EcoRI/BamHI to digest pK18-mobsacB simultaneously, and with T4DNA ligase (TransGenBiotech), fragment and carrier are attached, convert Trans1T1 competent cell (TransGenBiotech), that resistance clone of picking card, EcoRI/BamHI enzyme action identifies that obtaining overlapPCR fragment inserts the positive colony of pK18mobsacB, identify that the fragment of insertion treats the upstream and downstream fragment of deletion segment really for odhA gene by check order (Invitrogen company) further。
By obtained plasmid called after pK18-odhAda1。PK18-odhAda1 is proceeded in MHZ-0112-1 bacterial strain。Selective agar medium containing the kanamycin of 15mg/L selects single-swap transformant, wherein with the sequence of odhA base deletion by being incorporated in genome together with recombinant vector with source odhA homologous recombination。Utilize FastTaqDNA polymerase (TransGenBiotech), carry out bacterium colony PCR with B1/T2, T1/B4 primer pair and identify KanRClone, PCR parameter is: 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 40s, totally 26 circulations, and it is positive colony that two primer pairs all amplify the clone of purpose fragment。The positive colony that will be singled out inoculates cultivation 12～14h in nonreactive BHI culture medium, bacterium solution is diluted cultivation 36h in the solid BHI culture medium being coated on after 100-1000 times containing 10% sucrose, the bacterial strain filtered out is carried out further the checking of that resistant phenotype of card, selects KanSRecon utilize B1/B4 primer pair amplifies purpose fragment, the purpose fragment of positive colony is little compared with the fragment that wild type expands, and positive colony is carried out sequence verification, obtains the bacterial strain of odhA base deletion, called after MHZ-0112-2。
In some specific embodiments of the present invention, described recombinant bacterial strain also includes sudden change gdh gene；Described sudden change gdh gene is process LAN gdh gene。
In some specific embodiments of the present invention, described recombinant bacterial strain also includes sudden change gltA gene；Described sudden change gltA gene is process LAN gltA gene。
In some specific embodiments of the present invention, process LAN described in described recombinant bacterial strain strengthens the expression of described gene by building the mode of over-express vector or insertion promoter。
In some specific embodiments of the present invention, over-express vector described in described recombinant bacterial strain is pEC-gdh or pEC-gltA；Described promoter is sod or tuf。
Primer is designed respectively, as shown in SEQIDNO.16～19 according to the sequence (SEQNO.14) of gdh gene (GeneBankNO:NCgl1084) in NCBIGenBank data base and the sequence (SEQNO.15) of gltA gene (GeneBankNO:NCgl0795)。
The structure of integrated plasmid pK18-Psodgdh, pK18-tufgltA:
Using Corynebacterium glutamicum ATCC13869 genome as template, respectively with C1/C2, the full length fragment of D1/D2 two genes of primer pair amplifies gdh and gltA, after agarose gel electrophoresis and glue reclaim purification, gained fragment is utilized respectively EcoRI/SalI double digestion, plasmid pEC-XK99E (GI:29164935) is also carried out identical double digestion, T4DNA ligase is utilized to be attached, screening positive clone after conversion Trans1T1 competent cell, extract plasmid and carry out EcoRI/SalI double digestion qualification respectively, finally carry out order-checking to confirm, gained plasmid is respectively designated as pEC-gdh, pEC-gltA。Prepare the competent cell of MHZ-0112-2 and control plasmid pEC-XK99E and above-mentioned constructed pEC-gdh, pEC-gltA are proceeded in MHZ-0112-2 bacterial strain。Utilizing Kan15BHI flat screen to select positive colony, and extract plasmid checking, three obtained bacterial strains with replicability plasmid are respectively designated as MHZ-0112-3, MHZ-0112-4, MHZ-0112-5。
With the genome of Corynebacterium glutamicum ATCC13869 for template, respectively with C3/C4, C5/C6, C7/C8 primer pair (as Suo Shi SEQIDNO.20～31) pcr amplification under the effect of the super proofreading polymerase of Phusion, obtaining the downstream homology arm of the upstream homology arm of gdh start codon, sod promoter and start codon, PCR parameter is 98 DEG C of 10s, 50 DEG C of 20s, 72 DEG C of 20s, totally 30 circulations。PCR primer agarose gel is reclaimed test kit and carries out rubber tapping recovery。With above-mentioned obtained sheet section mixture for template, overlapPCR amplification is carried out with C3/C8 primer pair, and recovery of tapping rubber, product SalI and HindIII double digestion, pK18mobsacB cuts with same enzyme, T4DNA ligase is utilized to connect 1-2h at 22 DEG C, convert Trans1T1 competent cell, that resistance clone of picking card, SalI/HindIII enzyme action identifies that obtaining overlapPCR fragment inserts the positive colony of pK18mobsacB, and the fragment identifying insertion by checking order further is really for the addition of the gdh gene upstream and downstream homology arm of Psod promoter。Obtained plasmid called after pK18-Psodgdh。
The building process of pK18-tufgltA is similar to the above, the primer upstream and downstream homology arm to expanding gltA start codon for D3/D4, D7/D8, expands promoter Ptuf, OverlapPCR the primer to for D3/D8 with D5/D6。
The preparation of high expressed bacterial strain:
Prepare the competent cell of MHZ-0112-2 bacterial strain, respectively plasmid pK18-Psodgdh, pK18-tufgltA are converted。
Selective agar medium containing the kanamycin of 15mg/L selects single-swap transformant, wherein with the sequence of sod (tuf) promoter by being incorporated in genome together with recombinant vector with source gdh (gltA) homologous recombination。Utilize FastTaqDNA polymerase, carry out bacterium colony PCR with C3/T2, T1/C6 primer pair (or D3/T2, T1/D6 primer pair) and identify KanRClone, ibid, it is positive colony that two primer pairs all amplify the clone of purpose fragment to PCR parameter。The positive colony that will be singled out inoculates cultivation 12-14h in nonreactive BHI culture medium, bacterium solution is diluted cultivation 36h in the solid BHI culture medium being coated on after 100-1000 times containing 10% sucrose, the bacterial strain filtered out is carried out further the checking of that resistant phenotype of card, selects KanSRecon utilize C3/C6 primer pair (or D3/D6 primer pair) to expand purpose fragment, the purpose fragment of positive colony is bigger than the fragment that wild type expands, positive colony is carried out sequence verification, obtain sod (tuf) promoter and be successfully integrated into the bacterial strain before gdh (gltA) Gene A TG, called after MHZ-0112-6 (MHZ-0112-7)。
In like manner, prepare MHZ-0112-6 competent cell, pK18-tufgltA is converted, through two-wheeled restructuring screening, obtain the mutant MHZ-0112-8 of gdh and gltA superposition。
In some specific embodiments of the present invention, the deposit number of described recombinant bacterial strain MHZ-0112-8 is CGMCCNo.11941。
Present invention also offers the application in described recombinant bacterial strain fermenting and producing glutamic acid。
Present invention also offers the construction method of described recombinant bacterial strain, comprise the steps:
With wild type glutamic acid bar bacterium ATCC13869 for starting strain；
Step 1, in yggB introduce point mutation A106V；
Step 2, odhA gene is carried out inactivation transformation；
Step 3, process LAN gdh gene；
Step 4, process LAN gltA gene。
In some specific embodiments of the present invention, process LAN described in the step 3 of described construction method or step 4 strengthens the expression of described gene by building the mode of over-express vector or insertion promoter；Described over-express vector is pEC-gdh or pEC-gltA；Described promoter is sod or tuf。
A kind of method that present invention also offers fermenting and producing glutamic acid, with described recombinant bacterial strain for fermentation strain。
The invention provides recombinant bacterial strain and the purposes for fermenting and producing glutamic acid thereof。Test result indicate that, wild type glutamic acid bar bacterium ATCC13869 does not substantially produce glutamic acid before not engineered, after by the yggB genetic modification of coding pass albumen (MHZ-0112-1), culture fluid has a small amount of glutamic acid accumulation 4.7g/L, compared with wild strain, this bacterial strain notable (P < 0.05) improves the fermentation yield of glutamic acid。After the competition approach of glutamic acid route of synthesis is blocked (MHZ-0112-2), the yield of glutamic acid is 18.5g/L, compare MHZ-0112-1 and improve about 4 times, compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。By pEC-gdh, pEC-gltA proceeds in MHZ-0112-2 bacterial strain, prepare bacterial strain MHZ-0112-4 and MHZ-0112-5, the yield of glutamic acid respectively 23.4g/L and 26.4g/L, compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。Gdh gene is being expressed individually with strong promoter Psod, GDH enzyme relatively wild type alive improves about 2.5 times, glutamic acid yield improves about 1.8 times, and compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。Adding about 4 times individually with Ptuf promoter expression gltA gene by alive for CS enzyme, glutamic acid yield is increased to 30.8g/L, and compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid；Two genes are carried out process LAN simultaneously after, glutamic acid yield continues to be increased to 35.5g/L, and conversion ratio reaches about 59%, and compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。
Biological deposits explanation
Biomaterial MHZ-0112-8, Classification And Nomenclature: Corynebacterium glutamicum, Corynebacteriumglutamicum is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 25th in December in 2015, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, and deposit number is CGMCCNo.11941。
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, the accompanying drawing used required in embodiment or description of the prior art will be briefly described below。
Fig. 1 shows glutamic acid metabolic pathway of synthesizing in vivo；
Fig. 2 shows sacB screening system schematic diagram。
Detailed description of the invention
The invention discloses a kind of bacterial strain and application thereof, those skilled in the art can use for reference present disclosure, is suitably modified technological parameter and realizes。Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are considered as including in the present invention。Method and the application of the present invention are described already by preferred embodiment, method described herein and application substantially can be modified or suitably change and combination by related personnel in without departing from present invention, spirit and scope, realize and apply the technology of the present invention。
The competent preparation of Corynebacterium glutamicum: in-80 DEG C of refrigerators, line is transferred out on Corynebacterium glutamicum kind ATCC13869 to BHI flat board, 30 DEG C of cultivations；Picking list bacterium colony, is transferred in 5mlBHI fluid medium (3.7% brain heart leaching powder) test tube, and 200rpm cultivates 12h for 30 DEG C；Being seeded in 100mlBHIS fluid medium (3.7% brain heart leaching powder, 9.1% sorbitol) by 1% inoculum concentration, 200rpm, 30 DEG C are cultured to OD600 and reach 1.5；4 DEG C, 6000rpm is centrifuged 20min, collects thalline, abandons supernatant；In 4 DEG C after suspending with TGBuffer (1mMTris, 10% glycerol (v/v), pH7.5), 6000rpm is centrifuged 20min, abandons supernatant, and comes again；With 10% glycerol suspend after in 4 DEG C, 6000rpm is centrifuged 20min, abandons supernatant, and comes again；Subpackage after addition 1ml10% glycerol suspension thalline。Competent cell is put in-70 DEG C of Refrigerator stores or is directly used in electroporated。
Electricity converts: is taken out from refrigerator by competent cell and melts in frozen water, add 10-15 μ l recombinant plasmid dna and enter in the competent cell dissolved, transfer them to 1mm after pressure-vaccum mixing and shock by electricity in cup (BioRad Products), in 1.8kv, in the upper electric shock of electric shock instrument (BioRad Products) when 5ms, add the BHI broth of 46 DEG C of preheatings immediately after, mix gently, mixed liquor is proceeded in 15ml centrifuge tube, 46 DEG C of water-baths 6 minutes, 30 DEG C of activation culture 2h, centrifugal collection thalline, thalline is coated on the BHI solid medium containing 25mg/L, 36h is cultivated for 30 DEG C in calorstat。
Bacterial strain screening: for yggB genetic modification, first with blocking that resistance screening, as it has been described above, the thalline after being turned by electricity is coated in the solid BHI culture medium containing that resistance of card, selects resistance clone。Sequential design checking primer pK18T1/pK18T2 (sequence is in Table 1) according to pK18mobsacB plasmid MCS two ends, utilize FastTaqDNA polymerase (Quan Shi King Company), with A1/T2, T1/A4 primer pair carries out bacterium colony PCR and identifies KanR clone, PCR parameter is: 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 40s, totally 26 circulations, it is positive colony that two primer pairs all amplify the clone of 1000bp fragment。The positive colony that will be singled out inoculates cultivation 12-14h in nonreactive BHI culture medium, allows thalline that homologous recombination occurs in the process of breeding。Bacterium solution is diluted cultivation 36h in the solid BHI culture medium being coated on after 100-1000 times containing 10% sucrose, deposits in case at sucrose, do not occur the bacterial strain of second time homologous recombination because of with sacB gene can not survive (schematic diagram is shown in Fig. 2)。Owing to sucrose screening exists the false positive of about 20%, the bacterial strain filtered out need to be carried out that resistant phenotype of card checking further。Designing MAMAPCR (MismatchAmplificationMutationAssyPCR) according to point mutation and identify primer 106T (sequence is in Table 1), the recon selecting KanS utilizes 106T/A4 to carry out point mutation checking, and parameter is ibid。To verify that correct recon utilizes A1/A4 to carry out sequence verification after expanding purpose fragment。
The method of fermentation checking glutamic acid yield: frozen wild type 13869 bacterial strain in-80 DEG C of glycerol pipes, MHZ-0112-1 bacterial strain and MHZ-0112-2 are inoculated in above-mentioned slant medium and activate, grows lawn after cultivating 24h at 31.5 DEG C；From the inclined-plane of fresh activation, the lawn of the above-mentioned three strain bacterium of picking, is inoculated in above-mentioned seed culture medium, and in 31.5 DEG C, under 220rpm/min, shaken cultivation is to logarithmic growth middle and late stage (about 12h), prepares seed liquor；Above-mentioned seed liquor is seeded to equipped with in the 500ml shaking flask of 20ml fermentation medium by the inoculum concentration with 10%, at 31.5 DEG C, and shaken cultivation。After being totally consumed at sugar, measure the concentration of the Pidolidone of accumulation in culture medium。Result is (OD600 is that the culture fluid diluting 100 times at the turbidity of 600nm and represents cell concentration, and Glu (g/L) represents the amount of the Pidolidone of accumulation) as shown in table 4。
GDH enzyme activity determination method: the antibacterial after cultivating is centrifuged 5min with 10000rpm/min at 4 DEG C, collects thalline, with 0.01mol/L phosphate buffer (PBS, pH7.4) wash once, then be resuspended in the PBS of 1/10 original bacteria liquid volume, ice bath, (350W, continues 5s, interval 10s to carrying out ultrasonic bacteria breaking, totally 20 times) after, 10000rpm/min is centrifuged 10min, goes precipitation, obtains thick enzymatic solution, being measured with NADP+ for coenzyme, reaction system is: 0.5mmol/LNADP+, 15mmol/L glutamic acid, 25mmol/LTris-HCl, pH8.0。Take sample enzyme liquid and measure GDH catalysis coenzyme NAD H+The change at 340nm wavelength place of the absorbance value caused by reduction, 1 μm of olNADP of catalysis per minute+Enzyme amount needed for reduction is defined as an enzyme activity unit (U)。Enzyme work=Δ OD340× 0.2 × 2/0.0486, protein concentration=1.45 × OD280-0.74×OD260, unit enzyme work=enzyme work/protein concentration。All mensuration all complete on BECKMANDU800 spectrophotometer。Measuring the protein concentration in crude enzyme liquid with ProtienAssay (Bio-Rad), use bovine serum albumin is standard protein。
CS enzyme activity determination method: centrifugation medium is with separating thallus。Clean cell with the 50mmol/LTris buffer (pH7.5) containing 200mmol/L sodium glutamate, then suspend with same solution again。Ice bath, after carrying out ultrasonic bacteria breaking (350W, continues 5s, interval 10s, totally 20 times), 10000rpm/min is centrifuged 10min, goes precipitation, obtains thick enzymatic solution, measures the activity of citrate synthetase according to MethodsEnzymol.13,3-11 (1969)。Specifically, this thick enzymatic solution is added containing 100mmo/LTris-HCl (pH8), 0.1mmol/LDTNB, 200mmol/L sodium glutamate, in the reactant liquor of 0.3mmol/L second phthalidyl coenzyme A, then with spectrophotometer at 30 DEG C, 412nm measures its absorbance to be increased, with this background value the most, add oxalacetic acid further and make its ultimate density be 0.5mmol/L。Measure the increase at 412nm place light absorption value, calculate that from background value the enzyme obtaining citrate synthetase is lived。Measuring the protein concentration in crude enzyme liquid with ProtienAssay (Bio-Rad), use bovine serum albumin is standard protein。
The invention provides recombinant bacterial strain and the purposes for fermenting and producing glutamic acid thereof。Test result indicate that, wild type glutamic acid bar bacterium ATCC13869 does not substantially produce glutamic acid before not engineered, after by the yggB genetic modification of coding pass albumen (MHZ-0112-1), culture fluid has a small amount of glutamic acid accumulation 4.7g/L, compared with wild strain, this bacterial strain notable (P < 0.05) improves the fermentation yield of glutamic acid。After the competition approach of glutamic acid route of synthesis is blocked (MHZ-0112-2), the yield of glutamic acid is 18.5g/L, compare MHZ-0112-1 and improve about 4 times, compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。By pEC-gdh, pEC-gltA proceeds in MHZ-0112-2 bacterial strain, prepare bacterial strain MHZ-0112-4 and MHZ-0112-5, the yield of glutamic acid respectively 23.4g/L and 26.4g/L, compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。Gdh gene is being expressed individually with strong promoter Psod, GDH enzyme relatively wild type alive improves about 2.5 times, glutamic acid yield improves about 1.8 times, and compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。Adding about 4 times individually with Ptuf promoter expression gltA gene by alive for CS enzyme, glutamic acid yield is increased to 30.8g/L, and compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid；Two genes are carried out process LAN simultaneously after, glutamic acid yield continues to be increased to 35.5g/L, and conversion ratio reaches about 59%, and compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。
Recombinant bacterial strain provided by the invention and all can being buied by market for bacterial strain involved in the purposes of fermenting and producing glutamic acid, carrier, promoter, preparation。
Terminological interpretation:
PCR: polymerase chain reaction
KanR: kalamycin resistance
KanS: kanamycin is sensitive。
Below in conjunction with embodiment, the present invention is expanded on further:
The introducing of embodiment 1yggB Gene A 106V point mutation
(1) for the vector construction of yggBA106V point mutation
DNA sequence (SEQNO.1) according to Corynebacterium glutamicum (CorynebacteriumglutamicumATCC13869) yggB gene (GeneBankNO.:NCgl1221) designs primer and carries out pcr amplification, and primer sequence is as shown in table 1。
Table 1 primer sequence
Extract test kit (Tian Gen company) with genome DAN and extract wild type glutamic acid bar bacterium C.glutamicumATCC13869 whole genome sequence。With the genome of extracting for template, respectively with A1/A2, A3/A4 primer pair pcr amplification under the effect of the super proofreading polymerase of Phusion (NewEnglandBioLabs) obtains the homology arm of yggB gene 106 amino acids upstream and downstream, PCR parameter is 98 DEG C of 10s, 50 DEG C of 20s, 72 DEG C of 20s, totally 30 circulations。PCR primer agarose gel is reclaimed test kit (Tian Gen company) and carries out rubber tapping recovery。With above-mentioned gained upstream and downstream homology arm 1:1 mixture for template, overlapPCR amplification is carried out with A1/A4 primer pair, and recovery of tapping rubber, product SalI and HindIII double digestion, pK18mobsacB cuts with same enzyme, utilize connection 1-2h at T4DNA ligase (Quan Shi King Company) 22 DEG C, convert Trans1T1 competent cell (Quan Shi King Company), that resistance clone of picking card, SalI/HindIII enzyme action identifies that obtaining overlapPCR fragment inserts the positive colony of pK18mobsacB, identify that the fragment of insertion is really for the upstream and downstream fragment of yggB gene 106 amino acids by check order (Invitrogen company) further。Obtained plasmid called after pK18-yggBA106V。
Sequencing result is such as shown in SEQIDNo.14。
(2) preparation of the Corynebacterium glutamicum MHZ-0112-1 of yggBA106V point mutation
Bacterial strain screening with yggBA106V point mutation: first with blocking that resistance screening, as it has been described above, the thalline after being turned by electricity is coated in the solid BHI culture medium containing that resistance of card, select resistance clone。Sequential design checking primer pK18T1/pK18T2 (sequence is in Table 1) according to pK18mobsacB plasmid MCS two ends, utilizes FastTaqDNA polymerase (Quan Shi King Company), carries out bacterium colony PCR with A1/T2, T1/A4 primer pair and identifies KanRClone, PCR parameter is: 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 40s, totally 26 circulations, and it is positive colony that two primer pairs all amplify the clone of 1000bp fragment。The positive colony that will be singled out inoculates cultivation 12-14h in nonreactive BHI culture medium, allows thalline that homologous recombination occurs in the process of breeding。Bacterium solution is diluted cultivation 36h in the solid BHI culture medium being coated on after 100-1000 times containing 10% sucrose, deposits in case at sucrose, do not occur the bacterial strain of second time homologous recombination because of with sacB gene can not survive (schematic diagram is shown in Fig. 2)。Owing to sucrose screening exists the false positive of about 20%, the bacterial strain filtered out need to be carried out that resistant phenotype of card checking further。Design MAMAPCR (MismatchAmplificationMutationAssyPCR) according to point mutation and identify primer 106T (sequence is in Table 1), select KanSRecon utilize 106T/A4 to carry out point mutation checking, parameter is ibid。To verify that correct recon utilizes A1/A4 to carry out sequence verification after expanding purpose fragment。The Strain Designation obtained is MHZ-0112-1。Sequencing result is such as shown in SEQIDNo.15。
The inactivation of odhA gene in embodiment 213869-yggBA106V bacterial strain
From glutamic acid metabolic pathway of synthesizing (Fig. 1), tricarboxylic acid cycle is the main competition approach of glutamic acid anabolism by the α-ketoglutaric acid of ketoglurate dehydrogenase complex (ODHC) catalysis to the conversion of S-(3-carboxy-propionyl)-coenzyme-A, in order to reduce the activity of ODHC, the encoding gene odhA of its Elo subunit need to be carried out inactivation transformation。The means adopted are introduced into base deletion, make to occur in protein synthesis frameshift mutation, thus reaching the purpose of inactivation。
(1) for odhA base deletion vector construction
First, in NCBIGenBank data base, obtain the nucleotide sequence (SEQIDNo.9) of Corynebacterium glutamicum (CorynebacteriumglutamicumATCC13869) odhA gene (GeneBankNO.NCgl1084), thus nucleotide sequence design introduces base deletion so that odhA gene inactivation at ad-hoc location, four primers (SEQIDNO.10-13, table 2) have been synthesized based on base sequence and selected deletion sites。
Table 2 primer sequence
Utilize the super proofreading polymerase of Phusion (NewEnglandBioLabs), with B1/B2, B3/B4 as primer, prepare the upstream and downstream fragment in odhA base deletion site using the genomic DNA of Corynebacterium glutamicum ATCC13869 as template。PCR program is, 98 DEG C of degeneration 10s, 50 DEG C of renaturation 20s, and 72 DEG C extend 20s, after circulating 30 times。Two fragments of gained reclaim after test kit (sky root) purification through agarose gel, utilize primer sets B1/B4 to carry out overlapPCR amplification and obtain product, reclaiming through agarose gel utilizes EcoRI/BamHI to digest after test kit (sky root) purification, utilize EcoRI/BamHI to digest pK18-mobsacB simultaneously, and with T4DNA ligase (TransGenBiotech), fragment and carrier are attached, convert Trans1T1 competent cell (TransGenBiotech), that resistance clone of picking card, EcoRI/BamHI enzyme action identifies that obtaining overlapPCR fragment inserts the positive colony of pK18mobsacB, identify that the fragment of insertion treats the upstream and downstream fragment of deletion segment really for odhA gene by check order (Invitrogen company) further。Obtained plasmid called after pK18-odhAda1。Sequencing result is such as shown in SEQIDNo.33。
(2) odhA base deletion carrier introducing in MHZ-0112-1
Utilize and with the preparation of competence described in embodiment 1 and method for transformation, pK18-odhAda1 is proceeded in MHZ-0112-1 bacterial strain。
Selective agar medium containing the kanamycin of 15mg/L selects single-swap transformant, wherein with the sequence of odhA base deletion by being incorporated in genome together with recombinant vector with source odhA homologous recombination。Utilize FastTaqDNA polymerase (TransGenBiotech), carry out bacterium colony PCR with B1/T2, T1/B4 primer pair and identify KanRClone, PCR parameter is: 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 40s, totally 26 circulations, and it is positive colony that two primer pairs all amplify the clone of purpose fragment。The positive colony that will be singled out inoculates cultivation 12-14h in nonreactive BHI culture medium, bacterium solution is diluted cultivation 36h in the solid BHI culture medium being coated on after 100-1000 times containing 10% sucrose, the bacterial strain filtered out is carried out further the checking of that resistant phenotype of card, selects KanSRecon utilize B1/B4 primer pair amplifies purpose fragment, the purpose fragment of positive colony is little compared with the fragment that wild type expands, and positive colony is carried out sequence verification, obtains the bacterial strain of odhA base deletion, called after MHZ-0112-2。Sequencing result is such as shown in SEQIDNo.34。
The partial nucleotide sequence of table 3odhA gene
(3) MHZ-0112-2 glutamic acid produces performance detection
Culture medium prescription is as follows:
Slant medium: yeast powder 5g/L, Carnis Bovis seu Bubali cream 10g/L, peptone 10g/L, sodium chloride 10g/L, agar powder 2.5g/L, pH7.0-7.2,121 DEG C of 0.1MPa sterilizing 30min；
Seed culture medium: glucose 25g/L, carbamide 3g/L, K2HPO4·3H2O2.2g/L, MgSO4.7H2O.0.9g/L, Semen Maydis pulp 33mL/L, soybean cake hydrolyzed solution 22mL/L, pH7.0-7.2,121 DEG C of 0.1MPa sterilizing 15min；
Fermentation medium: glucose 60g/L, ammonium sulfate 15g/L, KH2PO41g/L, MgSO4·7H2O0.4g/L, FeSO4·7H2O1mg/L, MnSO4·4-5H2O1mg/L, VB1200 μ g/L, biotin 300 μ g/L, 0.48g/L Soy hydrolysate, regulate to pH7.2-7.5 with NaOH, 121 DEG C of 0.1MPa sterilizing 15min, the rear interpolation heat-killed calcium carbonate of 1.0g。
Frozen wild type 13869 bacterial strain in-80 DEG C of glycerol pipes, MHZ-0112-1 bacterial strain and MHZ-0112-2 are inoculated in above-mentioned slant medium and activate, after cultivating 24h at 31.5 DEG C, grows lawn；From the inclined-plane of fresh activation, the lawn of the above-mentioned three strain bacterium of picking, is inoculated in above-mentioned seed culture medium, and in 31.5 DEG C, under 220rpm/min, shaken cultivation is to logarithmic growth middle and late stage (about 12h), prepares seed liquor；Above-mentioned seed liquor is seeded to equipped with in the 500ml shaking flask of 20ml fermentation medium by the inoculum concentration with 10%, at 31.5 DEG C, and shaken cultivation。After being totally consumed at sugar, measure the concentration of the Pidolidone of accumulation in culture medium。Result is (OD600 is that the culture fluid diluting 100 times at the turbidity of 600nm and represents cell concentration, and Glu (g/L) represents the amount of the Pidolidone of accumulation) as shown in table 4。
Find that 13869 bacterial strains do not accumulate Pidolidone, being carried out by odhA after base deletion makes in thalline ketoglurate dehydrogenase inactivation, culture medium has the accumulation of glutamic acid。
The yield of table 4 glutamic acid
| OD600(×100) | Glu(g/L) | |
| ATCC 13869 | 0.625 | 0.2 |
| MHZ-0112-1 | 0.597 | 4.7 |
| MHZ-0112-2 | 0.453 | 18.5 |
Result shows that wild type glutamic acid bar bacterium ATCC13869 does not substantially produce glutamic acid before not engineered, after by the yggB genetic modification of coding pass albumen, culture fluid has a small amount of glutamic acid accumulation, compared with wild strain, this bacterial strain notable (P < 0.05) improves the fermentation yield of glutamic acid；After being blocked by the competition approach of glutamic acid route of synthesis, the output increased of glutamic acid about 4 times, compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。
The enhancing of embodiment 3gdh, gltA gene expression
(1) structure of over-express vector pEC-gdh, pEC-gltA
Primers according to the sequence of gdh gene (GeneBankNO:NCgl1084) in NCBIGenBank data base and gltA gene (GeneBankNO:NCgl0795) respectively, as shown in table 5。
Table 5 primer sequence
Using Corynebacterium glutamicum ATCC13869 genome as template, respectively with C1/C2, the full length fragment of D1/D2 two genes of primer pair amplifies gdh and gltA, after agarose gel electrophoresis and glue reclaim purification, gained fragment is utilized respectively EcoRI/SalI double digestion, plasmid pEC-XK99E (GI:29164935) is also carried out identical double digestion, T4DNA ligase is utilized to be attached, screening positive clone after conversion Trans1T1 competent cell, extract plasmid and carry out EcoRI/SalI double digestion qualification respectively, finally carry out order-checking to confirm, gained plasmid is respectively designated as pEC-gdh, and (sequencing result is such as shown in SEQIDNo.35。), (sequencing result is such as shown in SEQIDNo.36 for pEC-gltA。)。
(2) with the detection of the bacterial strain glutamic acid-producing ability of gdh, gltA gene high expression plasmid and enzyme activity determination
Utilize and prepare the competent cell of MHZ-0112-2 with competence preparation method described in embodiment 1 and control plasmid pEC-XK99E and above-mentioned constructed pEC-gdh, pEC-gltA are proceeded in MHZ-0112-2 bacterial strain。Utilizing Kan15BHI flat screen to select positive colony, and extract plasmid checking, three obtained bacterial strains with replicability plasmid are respectively designated as MHZ-0112-3, MHZ-0112-4, MHZ-0112-5。
Two obtained bacterial strains and 13869-yggBA106V-odhAda1 bacterial strain are inoculated in the culture medium containing composition shown in table 6 respectively, the later stage of exponential phase it is cultured at 31 DEG C, mensuration to glutamte dehydrogenase in thalline (GDH) and the expression of citrate synthase (CS) respectively, the assay method of two enzyme work is as follows:
GDH enzyme activity determination method: the antibacterial after cultivating is centrifuged 5min with 10000rpm/min at 4 DEG C, collects thalline, with 0.01mol/L phosphate buffer (PBS, pH7.4) wash once, then be resuspended in the PBS of 1/10 original bacteria liquid volume, ice bath, (350W, continues 5s, interval 10s to carrying out ultrasonic bacteria breaking, totally 20 times) after, 10000rpm/min is centrifuged 10min, goes precipitation, obtains thick enzymatic solution, being measured with NADP+ for coenzyme, reaction system is: 0.5mmol/LNADP+, 15mmol/L glutamic acid, 25mmol/LTris-HCl, pH8.0。Take sample enzyme liquid and measure GDH catalysis coenzyme NAD H+The change at 340nm wavelength place of the absorbance value caused by reduction, 1 μm of olNADP of catalysis per minute+Enzyme amount needed for reduction is defined as an enzyme activity unit (U)。Enzyme work=Δ OD340× 0.2 × 2/0.0486, protein concentration=1.45 × OD280-0.74×OD260, unit enzyme work=enzyme work/protein concentration。All mensuration all complete on BECKMANDU800 spectrophotometer。Measuring the protein concentration in crude enzyme liquid with ProtienAssay (Bio-Rad), use bovine serum albumin is standard protein。
CS enzyme activity determination method: centrifugation medium is with separating thallus。Clean cell with the 50mmol/LTris buffer (pH7.5) containing 200mmol/L sodium glutamate, then suspend with same solution again。Ice bath, after carrying out ultrasonic bacteria breaking (350W, continues 5s, interval 10s, totally 20 times), 10000rpm/min is centrifuged 10min, goes precipitation, obtains thick enzymatic solution, measures the activity of citrate synthetase according to MethodsEnzymol.13,3-11 (1969)。Specifically, this thick enzymatic solution is added containing 100mmo/LTris-HCl (pH8), 0.1mmol/LDTNB, 200mmol/L sodium glutamate, in the reactant liquor of 0.3mmol/L second phthalidyl coenzyme A, then with spectrophotometer at 30 DEG C, 412nm measures its absorbance to be increased, with this background value the most, add oxalacetic acid further and make its ultimate density be 0.5mmol/L。Measure the increase at 412nm place light absorption value, calculate that from background value the enzyme obtaining citrate synthetase is lived。Measuring the protein concentration in crude enzyme liquid with ProtienAssay (Bio-Rad), use bovine serum albumin is standard protein。
The detection of table 6 glutamic acid-producing ability and enzyme activity determination
By pEC-gdh, pEC-gltA proceeds in MHZ-0112-2 bacterial strain, prepare bacterial strain MHZ-0112-4 and MHZ-0112-5, the yield of glutamic acid respectively 23.4g/L and 26.4g/L, compared with wild strain, bacterial strain MHZ-0112-4 and MHZ-0112-5 extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。Compared with MHZ-0112-3, MHZ-0112-4 improves 4 times than MHZ-0112-3GDH expression, and CS expression remains basically stable, and glutamic acid improves 41.8%；MHZ-0112-5 remains basically stable than MHZ-0112-3GDH expression, and CS expression improves 7.4 times, and glutamic acid improves 60%。MHZ-0112-4 and MHZ-0112-5 extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。
(3) structure of integrated plasmid pK18-Psodgdh, pK18-tufgltA
With the genome of Corynebacterium glutamicum ATCC13869 for template, respectively with C3/C4, C5/C6, C7/C8 primer pair (sequence is in Table 7) pcr amplification under the effect of the super proofreading polymerase of Phusion, obtaining the downstream homology arm of the upstream homology arm of gdh start codon, sod promoter and start codon, PCR parameter is 98 DEG C of 10s, 50 DEG C of 20s, 72 DEG C of 20s, totally 30 circulations。PCR primer agarose gel is reclaimed test kit and carries out rubber tapping recovery。With above-mentioned obtained sheet section mixture for template, overlapPCR amplification is carried out with C3/C8 primer pair, and recovery of tapping rubber, product SalI and HindIII double digestion, pK18mobsacB cuts with same enzyme, T4DNA ligase is utilized to connect 1-2h at 22 DEG C, convert Trans1T1 competent cell, that resistance clone of picking card, SalI/HindIII enzyme action identifies that obtaining overlapPCR fragment inserts the positive colony of pK18mobsacB, and the fragment identifying insertion by checking order further is really for the addition of the gdh gene upstream and downstream homology arm of Psod promoter。(sequencing result is such as shown in SEQIDNo.37 for obtained plasmid called after pK18-Psodgdh。)。
(sequencing result is such as shown in SEQIDNo.38 for pK18-tufgltA。) building process similar to the above, the primer to for D3/D4, D7/D8 expand gltA start codon upstream and downstream homology arm, with D5/D6 expand promoter Ptuf, OverlapPCR the primer to for D3/D8。
Table 7 primer sequence
(4) preparation of high expressed bacterial strain
Described in embodiment 1, prepare the competent cell of MHZ-0112-2 bacterial strain, respectively plasmid pK18-Psodgdh, pK18-tufgltA are converted。
Selective agar medium containing the kanamycin of 15mg/L selects single-swap transformant, wherein with the sequence of sod (tuf) promoter by being incorporated in genome together with recombinant vector with source gdh (gltA) homologous recombination。Utilize FastTaqDNA polymerase, carry out bacterium colony PCR with C3/T2, T1/C6 primer pair (or D3/T2, T1/D6 primer pair) and identify KanRClone, ibid, it is positive colony that two primer pairs all amplify the clone of purpose fragment to PCR parameter。The positive colony that will be singled out inoculates cultivation 12-14h in nonreactive BHI culture medium, bacterium solution is diluted cultivation 36h in the solid BHI culture medium being coated on after 100-1000 times containing 10% sucrose, the bacterial strain filtered out is carried out further the checking of that resistant phenotype of card, selects KanSRecon utilize C3/C6 primer pair (or D3/D6 primer pair) to expand purpose fragment, the purpose fragment of positive colony is bigger than the fragment that wild type expands, positive colony is carried out sequence verification, obtain sod promoter and be successfully integrated into the bacterial strain before gdh Gene A TG, called after MHZ-0112-6；Sequencing result is such as shown in SEQIDNo.39。
Obtain tuf promoter and be successfully integrated into the bacterial strain before gltA Gene A TG, called after MHZ-0112-7。Sequencing result is such as shown in SEQIDNo.40。
In like manner, preparing MHZ-0112-6 competent cell, converted by pK18-tufgltA, through two-wheeled restructuring screening, obtain the mutant MHZ-0112-8 of gdh and gltA superposition, sequencing result is such as shown in SEQIDNo.41。
(5) the corresponding enzyme activity determination of high expressed bacterial strain and glutamic acid yield are determined
Obtained bacterial strain MHZ-0112-6, MHZ-0112-7 and MHZ-0112-8 are accessed in BHI culture medium, at 31 DEG C, is cultured to the later stage of exponential phase, measure GDH and CS expression of enzymes activity；Be connected in the fermentation medium described in embodiment 2 to be cultured to sugar consumption completely after, measure yield and the OD of glutamic acid, respectively the results are shown in Table 8。
Table 8 enzyme activity determination and glutamic acid yield result
As shown in table 8, expressing gdh gene individually with strong promoter Psod, GDH enzyme relatively wild type alive improves about 2.5 times, and glutamic acid yield improves about 1.8 times, compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。
Adding about 4 times individually with Ptuf promoter expression gltA gene by alive for CS enzyme, glutamic acid yield is increased to 30.8g/L, and compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。
MHZ-0112-8 and MHZ-0112-6, MHZ-0112-7 compare, and glutamic acid yield improves 19.2% and 14.6%。
Two genes are carried out process LAN simultaneously after, glutamic acid yield continues to be increased to 35.5g/L, and conversion ratio reaches about 59%, and compared with wild strain, this bacterial strain extremely notable (P < 0.01) improves the fermentation yield of glutamic acid。
The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the principles of the invention; can also making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention。
Claims (10)
1. recombinant bacterial strain, it is characterised in that it comprises the mutant-yggB gene of encoding mutant yggB protein。
2. recombinant bacterial strain according to claim 1, it is characterised in that described sudden change yggB protein is that A106 aminoacid place has sudden change；
Described sudden change yggB protein has A106V sudden change；
Also include coding and reduce the sudden change odhA gene of ODHC activity；Described sudden change odhA gene for carrying out inactivation transformation to odhA gene。
3. recombinant bacterial strain according to claim 1 and 2, it is characterised in that also include sudden change gdh gene；Described sudden change gdh gene is process LAN gdh gene；
Also include sudden change gltA gene；Described sudden change gltA gene is process LAN gltA gene。
4. recombinant bacterial strain according to claim 3, it is characterised in that the described process LAN in described process LAN gdh gene or process LAN gltA gene strengthens the expression of described gene by building the mode of over-express vector or insertion promoter。
5. recombinant bacterial strain according to claim 4, it is characterised in that described over-express vector is pEC-gdh or pEC-gltA；Described promoter is sod or tuf。
6. the recombinant bacterial strain according to any one of claim 1 to 5, it is characterised in that its deposit number is CGMCCNo.11941。
7. the application in the recombinant bacterial strain fermenting and producing glutamic acid according to any one of claim 1 to 6。
8. the construction method of the recombinant bacterial strain according to any one of claim 1 to 6, it is characterised in that comprise the steps:
With wild type glutamic acid bar bacterium ATCC13869 for starting strain；
Step 1, in yggB introduce point mutation A106V；
Step 2, odhA gene is carried out inactivation transformation；
Step 3, process LAN gdh gene；
Step 4, process LAN gltA gene。
9. construction method according to claim 8, it is characterised in that process LAN described in step 3 or step 4 strengthens the expression of described gene by building the mode of over-express vector or insertion promoter；Described over-express vector is pEC-gdh or pEC-gltA；Described promoter is sod or tuf。
10. the method for a fermenting and producing glutamic acid, it is characterised in that with the recombinant bacterial strain as described in any one of claim 1 to 6 for fermentation strain。
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| EP4183880A4 (en) * | 2020-07-20 | 2024-08-14 | Tianjin Inst Ind Biotechnology Cas | Mutant of glutamate dehydrogenase gene promoter and application thereof |
| EP4067484A4 (en) * | 2021-01-27 | 2024-01-24 | CJ CheilJedang Corporation | Novel galactoside o-acetyltransferase variant, and method for producing l-glutamic acid using same |
| CN115851688A (en) * | 2021-09-23 | 2023-03-28 | 胡丹 | TrpCF mutant and application thereof |
| CN115927325A (en) * | 2022-09-20 | 2023-04-07 | 中国科学院天津工业生物技术研究所 | Citrate synthase promoter mutant and application thereof |
| CN115927325B (en) * | 2022-09-20 | 2024-07-09 | 中国科学院天津工业生物技术研究所 | Citrate synthase promoter mutant and application thereof |
| CN116004501A (en) * | 2023-01-10 | 2023-04-25 | 呼伦贝尔东北阜丰生物科技有限公司 | NADP-ferredoxin reductase mutant and application thereof in production of glutamic acid |
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