CN105602881A - Temperature-sensitive recombinant corynebacterium glutamicum producing glutamic acid and application thereof - Google Patents
Temperature-sensitive recombinant corynebacterium glutamicum producing glutamic acid and application thereof Download PDFInfo
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- CN105602881A CN105602881A CN201610139302.6A CN201610139302A CN105602881A CN 105602881 A CN105602881 A CN 105602881A CN 201610139302 A CN201610139302 A CN 201610139302A CN 105602881 A CN105602881 A CN 105602881A
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- 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.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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- 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.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01158—UDP-N-acetylmuramate dehydrogenase (1.1.1.158), i.e. UDP-N-acetylenolpyruvoylglucosamine reductase
Abstract
The invention provides a temperature-sensitive recombinant corynebacterium glutamicum producing glutamic acid. The temperature-sensitive recombinant corynebacterium glutamicum is obtained by transforming wild type corynebacterium glutamicum ( C. glutamicum ATCC 13032) and knocking out two key genes in a genome of the corynebacterium glutamicum in a peptidoglycan synthesis route, namely UDP-N-enol form acetyl glucosamine transferase gene (murA) and UDP-N-acetyl cell wall acidohydrogenase gene (murB), the glutamic acid yield of the recombinant corynebacterium glutamicum is improved obviously, and the glutamic acid yield is 4-7 g/L under the condition that the fermentation temperature rises to 38.5 DEG C.
Description
Technical field
The invention belongs to genetic engineering and field of fermentation engineering, particularly utilize technique for gene engineering to Corynebacterium glutamicumCorynebacteriumglutamicumATCC13032 carries out molecular modification, obtains the temperature sensitive type recombinant bacterium that produces glutamic acid.
Background technology
Glutamic acid is one of primary amino acid of nitrogen metabolism in living organism, significant in metabolism. At food, medicineHave been widely used with industrial aspect. In order to improve glutamic acid production rate, what in the research work of glutamate producing bacterium, give prominence to the most isThe seed selection work of high yield glutamic acid bacterium. Separate the glutamate producing bacterium wild strain obtaining from nature, aspect production performance oftenCan not meet the requirement of fermenting and producing development. Glutamate producing bacterium strain has larger natural variation ability, conventionally minus variant frequencyHigher than plus variant frequency, result causes the aging of bacterial classification and bacterial strain vigor to go down, and causes domestic glutamic acid production industry ubiquityThe bottleneck problems such as acid production rate level is low, glucose acid invert ratio is low, cost is high, energy consumption is high, the therefore seed selection of good production bacterial classification,It is one of industrial Main Topics of glutamic acid fermentation always.
In China's glutamic acid industrial production, the bacterial strain that most of enterprise is used is glutamic acid temperature sensitive mutant, this bacterium at presentStrain has temperature sensitive characteristic, improves cultivation temperature in the time that thalline enters exponential phase of growth, forces cell to be made the transition by normal cellFor the cell of Cell wall synthesis defect, promote the secretion of glutamic acid, thereby reach the object of synthesizing and accumulate in a large number glutamic acid. ShouldThe feature of technique is: (I) has resistance to biotin, temperature sensitive feature, be at higher temperature could normal fermentation and acid(being generally 37 DEG C-40 DEG C). (II) temperature transition is that sour key is produced in impact, and temperature has productive to glutamic acid fermentationImpact shows, after temperature transition, must carry out the growth of appropriateness residue, completes from the non-accumulation type cell of glutamic acid to accumulation type cellTransformation, to ensure high yield glutamic acid in the culture medium that is rich in biotin. (III) raw materials for production are easy to get, and not only can utilize lifeThe high raw material of thing cellulose content is as dry in molasses, potato etc. directly carries out fermenting and producing, and can utilize starchy material. (IV) fermentationTechnique is tending towards that simple sweat is easily controlled, glutamic acid yield is high, fermentation period is short, it is stable and save energy consumption to produce acid, fallsLow cost etc.
As far back as 20 century 70s, just there is the research report of responsive to temperature type glutamic acid bacterium in Japan. Domestic report is the earliest 1985Year Shanghai City industrial microorganism Sun Zhifeng etc. to responsive to temperature type variant D0The seed selection of bacterium and the report of effect thereof, they withThis bacterium is starting strain, and through DES and the processing of NTG continually mutagenize, seed selection obtains the good responsive to temperature type NI bacterium of a strain, this bacteriumBiotin accommodation wider (25 μ g/l-200 μ g/l), simultaneously will higher temperature (37 DEG C-40 DEG C) is lower could be normalFermentation and acid. NI bacterium is used for to rice hydrolysis sugar at 6.5m3On fermentation tank, produce glutamic acid, average acid production rate reaches 5.89%, flatAll glucose acid invert ratio is 47.25%, and the average fermentation cycle is 26.5h. Chen Ning in 2002 etc. report that directive breeding has gone out to have widowThe temperature-sensitive mutation strain TMGO106 of mycin resistance, glutamic acid oxygen oximate resistance is then, high with this mutant strain and acid production rateTianjin brevibacterium TG961 be parental plant, by Protoplast Fusion Technique, successfully selected the high fusant of acid production rateCN1021, at 6m3On fermentation tank, its glutamic acid yield reaches 14.6%, and glucose acid invert ratio reaches 62.8%, and this bacterial strain is that temperature is quickSense type bacterial strain, can be used for glutamic acid forced fermentation.
As can be seen here, for the seed selection of responsive to temperature type corynebacterium glutamicum strain, China is also in by traditional breeding technique (being mainlyWild strain glutamate producing bacterium is carried out to autotelic directional transformation, or adopt the physical mutagenesis factor or the nitrosoguanidines etc. such as ultraviolet rayThe mutagenesis factor is carried out conventional mutation breeding) to the transition process of DNA recombinant technique, adopt technique for gene engineering to carry out temperatureThe transformation of quick type corynebacterium glutamicum strain need further with deep.
Summary of the invention
The object of this invention is to provide a kind of temperature sensitive type restructuring corynebacterium glutamicum that produces glutamic acid, is by transformation wild type paddyPropylhomoserin rod bacillus (C.glutamicumATCC13032), knocks out two crucial bases in peptide glycan route of synthesis in its genomeBecause of UDP-N-enol form acetylglucosamine transferase gene (murA) and UDP-N-acetylmuramic acid dehydrogenase gene (murB),Obtain temperature sensitive type recombinant bacterium, the glutamic acid yield of recombinant bacterium is obviously promoted.
The present invention also provides a kind of method of utilizing restructuring corynebacterium glutamicum fermenting and producing glutamic acid, specifically comprises the steps:
(1) thalline is inoculated in slant medium and is activated, cultivate 24-36h for 32 DEG C;
(2) by the bacterial classification access seed culture medium having activated, 32 DEG C, 200rpm, cultivates 6-8h;
(3) cultured seed is accessed in fermentation medium by 10% inoculum concentration, 32 DEG C, 200rpm fermented and cultured;
(4) treat that fermented and cultured is to thalline OD600When=12-16, fermentation temperature is increased to 38.5 DEG C, 200rpm is cultured to alwaysFermentation 28-32h.
The composition of described slant medium: peptone 1%, dusty yeast 0.5%, beef extract 1%, NaCl0.25%, MgSO40.05％，KH2PO40.1%, corn steep liquor 2%, agar 2%.
The composition of described seed culture medium: glucose 30g/L, K2HPO4·3H2O0.2g/L，MgSO4·7H2O0.8g/L，MnSO42mg/L，FeSO42mg/L，VB10.3mg/L，VH0.2mg/L, dusty yeast 10g/L, peptone
5g/L, urea 0.5g/L, Met1g/L.
The composition of described fermentation medium: glucose 8g/L, K2HPO4·3H2O0.4g/L，VH0.35mg/L，VB1
0.5mg/L, betaine 5g/L, MnSO430mg/L，FeSO430mg/L，MgSO4·7H2O2g/L, dusty yeast 15G/L, peptone 5g/L.
The method inactivation Corynebacterium glutamicum wild-type strain C.glutamicumATCC13032 of the present invention by genetic recombinationTwo gene murA in peptide glycan route of synthesis and murB, disturb the normal synthetic of this strain cell wall, causes bacterial strain to occurTemperature sensitive proterties, is conducive to strengthen the transhipment effect of part intracellular metabolite concentration outside born of the same parents, promotes as metabolins such as glutamic acidSynthetic. Utilize method provided by the invention, other Corynebacterium glutamicums are transformed, can make more bacterial strains obtain temperatureResponsive proterties. In actual applications, improve cultivation temperature and can make the cell wall structure of temperature sensitive type recombinant bacterium change, fromIn cellular morphology, show as cell rounding, become large, the cell membrane permeability of these cells strengthens, and is beneficial to intracellular product and is secreted into trainingSupport in base, remove feedback repression and inhibition in born of the same parents, thereby promote the synthetic of object product.
Brief description of the drawings
Fig. 1: the growth song of original bacterium ATCC13032 and recombinant bacterium C.glutamicumATCC13032 △ murA △ murBLine.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these examples are only exemplary, scope of the present invention are not formed to any restriction. Those skilled in the art should understand thatBe, can the details of technical solution of the present invention and form modified or be replaced lower without departing from the spirit and scope of the present invention,But these amendments and replacement all fall within the scope of protection of the present invention. The method, composition and the consumption that in following embodiment, relate to, asWithout specified otherwise, all conventional methods known to those skilled in the art.
Research foundation of the present invention is that the existing glutamic acid temperature sensitive mutant obtaining by mutagenesis screening is carried out to full geneThe order-checking of group, is studied and is found that this bacterial strain has lacked murA and these two genes of murB by comparative genomics, and by strikingExcept-covering experiment, the function of these two genes is verified, whether the existence that has confirmed these two genes has determined whether tool of bacterial strainThere is responsive to temperature characteristic;
The existing glutamic acid temperature sensitive mutant C.glutamicumCN1021 obtaining by mutagenesis screening is carried out to full geneGroup order-checking, studies discovery by comparative genomics, and this bacterial strain has lacked on murA and two peptide glycan route of synthesis of murBKey gene.
By the method for PCR, from Corynebacterium glutamicum C.glutamicumATCC13032, clone obtains murA and murBTwo genes. The method of cutting connection by enzyme is connected to over-express vector pXMJ19 above by two genetic fragments, builds expressionPlasmid. Then the method transforming by chemical conversion and electricity, it is C.glutamicumCN that mistake expression plasmid is imported to object hostIn 1021. By shake flask fermentation, determine the covering of murA and murB gene, can remove to a certain extent temperature-sensitive mutationThe temperature-sensing property of strain C.glutamicumCN1021.
For further checking murA and these two genes of murB are for the impact of glutamic acid production secretion, the present invention is with C.GlutamicumATCC13032 is starting strain, is increased and is obtained murA upstream homology arm and murB downstream by PCR methodHomology arm, and by overlapping PCR by overlapping upstream and downstream homology arm, obtain overlapping fragments. Cut the method for connection by enzyme, connectTo pK18mobsacB shuttle vector, build murA and murB gene knockout plasmid with this, the method that adopts electricity to transform will be struckExcept plasmid electricity goes in C.glutamicumATCC13032, filter out the recon of twice homologous recombination, realizeKnocking out of murA, murB gene, thus the responsive to temperature type recombinant bacterium of C.glutamicumATCC13032 obtained.
Specifically comprise the steps:
(1) acquisition of genes of interest: according to GenBank accession number NCgl0345 (murA gene order), NCgl0346 (murBGene order) design primer, adopt PCR method amplification to obtain object fragment murA upstream homology arm, murB downstream homologyArm, and pass through overlapping PCR by overlapping upstream and downstream homology arm, obtain overlapping fragments;
PCR system is as follows:
Overlapping PCR system is as follows:
PCR reaction condition: then the first step is carried out under the following conditions 28 at 95 DEG C of denaturation 5min and taken turns circulation: 95 DEG CSex change 30s, (55 DEG C) annealing 30s under suitable annealing temperature, 72 DEG C are extended 1min, after last circulation completes,72 DEG C are continued to extend 10min, then stop at 20 DEG C.
(2) by EcoRI and HindIII double digestion for the overlapping fragments obtaining, with the expression vector of same double digestionPK18mobsacB connects, construction recombination plasmid;
(3) the recombinant plasmid electricity building is converted in bacterial strain C.glutamicumATCC13032;
(4) cultivation of going down to posterity, filters out double crossing over recombinant bacterial strain C.glutamicumATCC13032 △ murA △ murB.
Test by shaking flask temperature-variable fermentation, contrasted and knocked out murA and murB recombinant bacterium C.glutamicumATCC afterwards13032 △ murA △ murB and original bacterium C.glutamicumATCC13032 are at thalli growth and produce the difference between acid.
(1) thalline is inoculated in slant medium and is activated, cultivate 24-36h for 32 DEG C;
(2) by the bacterial classification access seed culture medium having activated, 32 DEG C, 200rpm, cultivates 6-8h;
(3) cultured seed is accessed in fermentation medium by 10% inoculum concentration, 32 DEG C, 200rpm fermented and cultured;
(4) treat that fermented and cultured is to thalline OD600When=12-16, fermentation temperature is increased to 38.5 DEG C, 200rpm is cultured to alwaysFermentation 28-32h.
The composition of described slant medium: peptone 1%, dusty yeast 0.5%, beef extract 1%, NaCl0.25%, MgSO40.05％，KH2PO40.1%, corn steep liquor 2%, agar 2%.
The composition of described seed culture medium: glucose 30g/L, K2HPO4·3H2O0.2g/L，MgSO4·7H2O0.8g/L，MnSO42mg/L，FeSO42mg/L，VB10.3mg/L，VH0.2mg/L, dusty yeast 10g/L, peptone 5g/L, urineElement 0.5g/L, Met1g/L.
The composition of described fermentation medium: glucose 8g/L, K2HPO4·3H2O0.4g/L，VH0.35mg/L，VB1
0.5mg/L, betaine 5g/L, MnSO430mg/L，FeSO430mg/L，MgSO4·7H2O2g/L, dusty yeast 15G/L, peptone 5g/L.
Can find out from experimental result (Fig. 1), C.glutamicumATCC13032 △ murA △ murB is in thalli growth sideFace is lower slightly compared with former bacterium; And producing in acid, former bacterium C.glutamicumATCC13032 produces glutamic acid hardly, C.GlutamicumATCC13032 △ murA △ murB glutamic acid yield can reach 4-7g/L.
Claims (7)
1. a temperature sensitive type restructuring corynebacterium glutamicum that produces glutamic acid, is characterized in that knocking out wild type Corynebacterium glutamicum C.The UDP-N-enol form acetylglucosamine transferase gene of glutamicumATCC13032 and UDP-N-acetyl cell wallAcidohydrogenase gene, obtains temperature sensitive type recombinant bacterium.
2. a kind of temperature sensitive type restructuring corynebacterium glutamicum that produces glutamic acid as claimed in claim 1, is characterized in that, with C.GlutamicumATCC13032 is starting strain, by PCR method increase obtain murA upstream region of gene homology arm andMurB gene downstream homology arm, and by overlapping PCR by overlapping upstream and downstream homology arm, obtain overlapping fragments; By superimposed sheetsEcoRI and HindIII double digestion for section, connect with the expression vector pK18mobsacB of same double digestion, builds restructuringPlasmid; Recombinant plasmid electricity is converted in bacterial strain C.glutamicumATCC13032; Filter out homology twiceThe recon of restructuring, realizes knocking out of murA, murB gene, obtains temperature sensitive type recombinant bacterium.
3. the purposes of temperature sensitive type restructuring corynebacterium glutamicum described in claim 1 or 2, is characterized in that, for fermenting and producing paddyPropylhomoserin.
4. a method of utilizing the corynebacterium glutamicum fermenting and producing glutamic acid of recombinating described in claim 1 or 2, is characterized in that,Comprise the steps:
(1) thalline is inoculated in slant medium and is activated, cultivate 24-36h for 32 DEG C;
(2) by the bacterial classification access seed culture medium having activated, 32 DEG C, 200rpm, cultivates 6-8h;
(3) cultured seed is accessed in fermentation medium by 10% inoculum concentration, 32 DEG C, 200rpm fermented and cultured;
(4) treat that fermented and cultured is to thalline OD600When=12-16, fermentation temperature is increased to 38.5 DEG C, 200rpm is cultured to total sending outFerment 28-32h.
5. method as claimed in claim 4, is characterized in that, the consisting of of described slant medium: peptone 1%, dusty yeast0.5%, beef extract 1%, NaCl0.25%, MgSO40.05％，KH2PO40.1%, corn steep liquor 2%, agar 2%.
6. method as claimed in claim 4, is characterized in that, the consisting of of described seed culture medium: glucose 30g/L,K2HPO4·3H2O0.2g/L，MgSO4·7H2O0.8g/L，MnSO42mg/L，FeSO42mg/L，VB10.3mg/L，VH0.2mg/L, dusty yeast 10g/L, peptone 5g/L, urea 0.5g/L, Met1g/L.
7. method as claimed in claim 4, is characterized in that, the consisting of of described fermentation medium: glucose 8g/L,K2HPO4·3H2O0.4g/L，VH0.35mg/L，VB10.5mg/L, betaine 5g/L, MnSO430mg/L，FeSO430mg/L，MgSO4·7H2O2g/L, dusty yeast 15g/L, peptone 5g/L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106191153A (en) * | 2016-08-31 | 2016-12-07 | 菱花集团有限公司 | The method that glutamic acid fermentation produces |
| CN109337854A (en) * | 2018-11-19 | 2019-02-15 | 南京工业大学 | One plant of Corynebacterium glutamicum for knocking out extracellular nuclease ExeR and its construction method and application |
| CN113444655A (en) * | 2020-03-26 | 2021-09-28 | 吉林中粮生化有限公司 | Corynebacterium glutamicum, temperature-sensitive strain with high glutamic acid yield, acquisition method and application of temperature-sensitive strain and glutamic acid fermentation method |
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| CN103243131A (en) * | 2013-05-28 | 2013-08-14 | 山东祥维斯生物科技有限公司 | Method for preparing L-glutamic acid by fermentation |
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| CN103243131A (en) * | 2013-05-28 | 2013-08-14 | 山东祥维斯生物科技有限公司 | Method for preparing L-glutamic acid by fermentation |
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| TAKASHI HIRASAWA ET AL.: "A Mutation in the Corynebacterium glutamicum ltsA Gene Causes Susceptibility to Lysozyme, Temperature-Sensitive Growth, and L-Glutamate Production", 《JOURNAL OF BACTERIOLOGY》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106191153A (en) * | 2016-08-31 | 2016-12-07 | 菱花集团有限公司 | The method that glutamic acid fermentation produces |
| CN109337854A (en) * | 2018-11-19 | 2019-02-15 | 南京工业大学 | One plant of Corynebacterium glutamicum for knocking out extracellular nuclease ExeR and its construction method and application |
| CN113444655A (en) * | 2020-03-26 | 2021-09-28 | 吉林中粮生化有限公司 | Corynebacterium glutamicum, temperature-sensitive strain with high glutamic acid yield, acquisition method and application of temperature-sensitive strain and glutamic acid fermentation method |
| CN113444655B (en) * | 2020-03-26 | 2023-05-16 | 吉林中粮生化有限公司 | Corynebacterium glutamicum, temperature-sensitive strain with high glutamic acid yield, obtaining method and application thereof, and glutamic acid fermentation method |
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