CN105602881B - A kind of temperature sensitive type recombination corynebacterium glutamicum and its application producing glutamic acid - Google Patents

A kind of temperature sensitive type recombination corynebacterium glutamicum and its application producing glutamic acid Download PDF

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CN105602881B
CN105602881B CN201610139302.6A CN201610139302A CN105602881B CN 105602881 B CN105602881 B CN 105602881B CN 201610139302 A CN201610139302 A CN 201610139302A CN 105602881 B CN105602881 B CN 105602881B
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glutamic acid
temperature sensitive
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corynebacterium glutamicum
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谢希贤
陈宁
石拓
马跃超
徐庆阳
张成林
李燕军
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Tianjin University of Science and Technology
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
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    • 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

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Abstract

The present invention provides a kind of temperature sensitive type recombination corynebacterium glutamicum for producing glutamic acid, it is by the way that wild type glutamic acid bar bacterium (C.glutamicum ATCC 13032) is transformed, knock out two key gene UDP-N- enol form acetylglucosamine transferase genes (murA) in its genome in peptide glycan route of synthesis and UDP-N- acetylmuramic acid dehydrogenase gene (murB), obtain temperature sensitive type recombinant bacterium, the glutamic acid yield of recombinant bacterium is obviously improved, and when fermentation temperature is improved to 38.5 DEG C, glutamic acid yield is 4-7g/L.

Description

A kind of temperature sensitive type recombination corynebacterium glutamicum and its application producing glutamic acid
Technical field
The invention belongs to genetic engineering and field of fermentation engineering, in particular to using technique for gene engineering to glutamic acid rod Bacterium Corynebacterium glutamicum ATCC 13032 carries out molecular modification, obtains the temperature sensitive type recombination for producing glutamic acid Bacterium.
Background technique
Glutamic acid is one of the primary amino acid of nitrogen metabolism in living organism, is of great significance in metabolism.Food, Medicine has been widely used with industrial aspect.In order to improve glutamic acid production rate, dash forward the most in the research work of glutamate producing bacterium Out be high yield glutamic acid producer breeding work.The glutamate producing bacterium wild strain isolated from nature, in production performance Aspect tends not to the requirement for meeting fermenting and producing development.Glutamate producing bacterium strain has biggish natural variation ability, usually Minus variant frequency is higher than plus variant frequency, as a result causes the aging and the decline of bacterial strain vigor of strain, domestic glutamic acid is caused to produce The bottleneck problems such as industry is low in the prevalence of acid production rate level, saccharic acid conversion ratio is low, at high cost, energy consumption is high, therefore excellent production The breeding of strain is always industrial one of the Main Topics of glutamic acid fermentation.
At present in China's glutamic acid industrial production, the bacterial strain that most of enterprise uses is glutamic acid temperature sensitive mutation Strain, the bacterial strain have temperature sensitive characteristic, and cultivation temperature is improved when thallus enters exponential phase of growth, force cell by normal Cell transition is the cell of Cell wall synthesis defect, promotes the secretion of glutamic acid, largely synthesizes to reach and accumulate glutamic acid Purpose.The characteristics of technique is: (I) has the characteristics that resistance to biotin, temperature sensitive, at a higher temperature could be normal Fermentation and acid (generally 37 DEG C -40 DEG C).(II) temperature transition is the key that influence to produce acid, and temperature has glutamic acid fermentation After productive influence shows temperature transition, it is necessary to carry out the remaining growth of appropriateness, complete from the non-accumulation type cell vector product of glutamic acid The transformation of tired type cell, to guarantee the high yield glutamic acid in the culture medium rich in biotin.(III) raw materials for production are easy to get, and not only may be used With using biological cellulose content high raw material such as molasses, the potato directly progress fermenting and producing such as dry, and it can use starchy material. (IV) zymotechnique tends to that simple fermentation process, which is easy to control, glutamic acid yield is high, fermentation period is short, produces acid stablizes and save Energy consumption reduces cost etc..
Early in the 1970s, originally there is the research of responsive to temperature type glutamic acid producer to report day.Domestic earliest report Be Shanghai City industrial microorganism in 1985 the temperature sensitive form variation strain D such as Sun Zhifeng0The breeding of bacterium and its report of effect Road, they are using the bacterium as starting strain, and through the continuous mutagenic treatment of DES and NTG, breeding obtains one plant of excellent responsive to temperature type NI Bacterium, the biotin adaptation range of this bacterium is wider (25 μ g/l-200 μ g/l), while will be under higher temperature (37 DEG C -40 DEG C) It can normal fermentation production acid.NI bacterium is used for rice hydrolysis sugar in 6.5m3Glutamic acid is produced on fermentor, average acid production rate reaches 5.89%, average saccharic acid conversion ratio is 47.25%, and the average fermentation period is 26.5h.Chen Ning in 2002 etc. reports directive breeding The temperature-sensitive mutation strain TMGO106 with oligomycin resistance, glutamic acid oxygen oximate resistance is gone out, then, with the mutant strain The Tianjin brevibacterium TG961 high with acid production rate is parental plant, by Protoplast Fusion Technique, has successfully selected acid production rate height Fusant CN1021, in 6m3Its glutamic acid yield is up to 14.6% on fermentor, and saccharic acid conversion ratio is up to 62.8%, and the bacterium Strain responsive to temperature type bacterial strain, can be used for glutamic acid forced fermentation.
It can be seen that the breeding for responsive to temperature type corynebacterium glutamicum strain, China is also in (main by traditional breeding technique If wild strain glutamate producing bacterium is carried out purposive directional transformation, or using the physical mutagenesis factor or nitrous such as ultraviolet lights The chemical mutagenesis factor such as base guanidine carries out conventional mutation breeding) to the transition process of DNA recombinant technique, using technique for gene engineering The transformation for carrying out temperature sensitive type corynebacterium glutamicum strain need further and gos deep into.
Summary of the invention
The object of the present invention is to provide a kind of temperature sensitive types for producing glutamic acid to recombinate corynebacterium glutamicum, is wild by transformation Raw type Corynebacterium glutamicum (C.glutamicum ATCC 13032), knocks out two in its genome in peptide glycan route of synthesis A key gene UDP-N- enol form acetylglucosamine transferase gene (murA) and UDP-N- acetylmuramic acid dehydrogenase base Because of (murB), temperature sensitive type recombinant bacterium is obtained, the glutamic acid yield of recombinant bacterium is obviously improved.
The present invention also provides it is a kind of using recombination corynebacterium glutamicum fermenting and producing glutamic acid method, specifically include as Lower step:
(1) thallus is inoculated into slant medium and is activated, 32 DEG C of culture 24-36h；
(2) activated strain is accessed in seed culture medium, 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) to fermented and cultured to thallus OD600When=12-16, fermentation temperature is improved to 38.5 DEG C, 200rpm cultivate to Total fermentation 28-32h.
The composition of the slant medium: peptone 1%, yeast powder 0.5%, beef extract 1%, NaCl 0.25%, MgSO40.05%, KH2PO40.1%, corn pulp 2%, agar 2%.
The composition of the seed culture medium: glucose 30g/L, K2HPO4·3H2O 0.2g/L, MgSO4·7H2O 0.8g/ L, MnSO42mg/L, FeSO42mg/L, VB10.3mg/L, VH0.2mg/L, yeast powder 10g/L, peptone
5g/L, urea 0.5g/L, Met1g/L.
The composition of the fermentation medium: glucose 8g/L, K2HPO4·3H2O 0.4g/L, VH0.35mg/L, VB1
0.5mg/L, glycine betaine 5g/L, MnSO430mg/L, FeSO430mg/L, MgSO4·7H2O 2g/L, yeast powder 15g/L, peptone 5g/L.
The present invention inactivates Corynebacterium glutamicum wild-type strain C.glutamicum ATCC by the method for genetic recombination Two genes murA and murB in 13032 peptide glycan route of synthesis, interfere the normal synthesis of the strain cell wall, lead to bacterium There is temperature sensitive character in strain, is conducive to strengthening part intracellular metabolite concentration and acts on to extracellular transhipment, promotes such as glutamic acid The synthesis of equal metabolins.Using method provided by the invention, other Corynebacterium glutamicums are transformed, more bacterial strains can be made Obtain temperature sensitivity shape.In practical applications, improving cultivation temperature enables to the cell wall structure of temperature sensitive type recombinant bacterium to send out Changing shows as cell rounding from cellular morphology, becomes larger, and the cell wall permeability enhancing of these cells is conducive to production intracellular Object is secreted into culture medium, releases feedback repression and inhibition intracellular, to promote the synthesis of purpose product.
Detailed description of the invention
The growth of 13032 △ murA △ murB of Fig. 1: original bacteria ATCC 13032 and recombinant bacterium C.glutamicum ATCC Curve.
Specific embodiment
Further describe the present invention below in conjunction with specific embodiment, the advantages and features of the present invention will be with description and more It is clear.But these examples be only it is exemplary, it is not intended to limit the scope of the present invention in any way.Those skilled in the art answer It should be appreciated that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form repair Change or replace, but these modifications and replacement are fallen within the protection scope of the present invention.Method involved in following embodiments, ingredient And dosage, unless otherwise specified, conventional method known to those skilled in the art.
Research foundation of the invention is carried out to the existing glutamic acid temperature sensitive mutant obtained by mutagenesis screening The sequencing of full-length genome finds that the bacterial strain has lacked the two genes of murA and murB by comparing genomics research, and The function of the two genes is verified by knockout-covering experiment, it was confirmed that the presence or absence of the two genes determines Whether bacterial strain has temperature sensitive properties；
To the existing glutamic acid temperature sensitive mutant C.glutamicum CN 1021 obtained by mutagenesis screening into Row genome sequencing, by comparing genomics the study found that the bacterial strain has lacked two peptide glycan synthesis of murA and murB Key gene in approach.
By the method for PCR, from Corynebacterium glutamicum C.glutamicum ATCC 13032 clone obtain murA and Two genes of murB.Two genetic fragments are connected on over-express vector pXMJ19 by the method that digestion connects, were constructed Expression plasmid.Then the method for passing through chemical conversion and electrotransformation will be overexpressed plasmid and imported into purpose host i.e. In C.glutamicum CN1021.Pass through shake flask fermentation, it is determined that the covering of murA and murB gene, it can be to a certain extent Release the temperature-sensing property of temperature sensitive mutant C.glutamicum CN 1021.
In order to further verify the two genes of murA and murB for glutamic acid production secretion influence, the present invention with C.glutamicum ATCC 13032 is starting strain, expands to obtain the upstream murA homology arm and the downstream murB by PCR method Homology arm, and be overlapped upstream and downstream homology arm by over-lap PCR, obtain overlapping fragments.The method connected by digestion, is connected to PK18mobsacB shuttle vector constructs murA with this and murB gene knocks out plasmid, will knock out plasmid using the method for electrotransformation Electricity is gone in C.glutamicum ATCC 13032, filters out the recon for completing homologous recombination twice, realize murA, The knockout of murB gene, to obtain the responsive to temperature type recombinant bacterium of C.glutamicum ATCC 13032.
Specifically comprise the following steps:
(1) acquisition of target gene: according to GenBank accession number NCgl0345 (murA gene order), NCgl0346 It is homologous to expand to obtain the upstream target fragment murA homology arm, the downstream murB using PCR method for (murB gene sequence) design primer Arm, and be overlapped upstream and downstream homology arm by over-lap PCR, obtain overlapping fragments；
PCR system is as follows:
Over-lap PCR system is as follows:
PCR reaction condition: then the first step carries out 28 wheel circulations: 95 DEG C of changes in 95 DEG C of initial denaturation 5min under the following conditions Property 30s, (55 DEG C) annealing 30s, 72 DEG C of extension 1min under suitable annealing temperature, after the completion of the last one circulation, 72 DEG C of continuation Extend 10min, then stops at 20 DEG C.
(2) expression vector by obtained overlapping fragments EcoR I and Hind III double digestion, with same double digestion PK18mobsacB connection, construction recombination plasmid；
(3) by the recombinant plasmid electrotransformation built into bacterial strain C.glutamicum ATCC 13032；
(4) secondary culture filters out 13032 △ murA △ murB of double exchange reorganization bacterial strain C.glutamicum ATCC.
It is tested by shaking flask temperature-variable fermentation, compared knocking out the recombinant bacterium C.glutamicum after murA and murB ATCC13032 △ murA △ murB and original bacteria C.glutamicum ATCC 13032 is in thalli growth and produces the difference between acid It is different.
(1) thallus is inoculated into slant medium and is activated, 32 DEG C of culture 24-36h；
(2) activated strain is accessed in seed culture medium, 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) to fermented and cultured to thallus OD600When=12-16, fermentation temperature is improved to 38.5 DEG C, 200rpm cultivate to Total fermentation 28-32h.
The composition of the slant medium: peptone 1%, yeast powder 0.5%, beef extract 1%, NaCl 0.25%, MgSO40.05%, KH2PO40.1%, corn pulp 2%, agar 2%.
The composition of the seed culture medium: glucose 30g/L, K2HPO4·3H2O 0.2g/L, MgSO4·7H2O 0.8g/ L, MnSO42mg/L, FeSO42mg/L, VB10.3mg/L, VH0.2mg/L, yeast powder 10g/L, peptone 5g/L, urea 0.5g/L, Met1g/L.
The composition of the fermentation medium: glucose 8g/L, K2HPO4·3H2O 0.4g/L, VH0.35mg/L, VB1
0.5mg/L, glycine betaine 5g/L, MnSO430mg/L, FeSO430mg/L, MgSO4·7H2O 2g/L, yeast powder 15g/L, peptone 5g/L.
From experimental result (Fig. 1) as can be seen that 13032 △ murA △ murB of C.glutamicum ATCC is in thalli growth Aspect is slightly lower compared with opportunistic pathogen；And on producing acid, opportunistic pathogen C.glutamicum ATCC 13032 hardly produces glutamic acid, 13032 △ murA △ murB glutamic acid yield of C.glutamicum ATCC can reach 4-7g/L.

Claims (4)

1. a kind of temperature sensitive type for producing glutamic acid recombinates corynebacterium glutamicum, it is characterised in that knock out wild type glutamic acid bar bacterium The UDP-N- enol form acetylglucosamine transferase gene and UDP-N- acetylmuramic acid of C.glutamicum ATCC 13032 Dehydrogenase gene obtains temperature sensitive type recombinant bacterium.
2. a kind of temperature sensitive type for producing glutamic acid as described in claim 1 recombinates corynebacterium glutamicum, which is characterized in that with C.glutamicum ATCC 13032 is starting strain, expands to obtain murA upstream region of gene homology arm and murB by PCR method Downstream of gene homology arm, and be overlapped upstream and downstream homology arm by over-lap PCR, obtain overlapping fragments；By overlapping fragments EcoR I and Hind III double digestion is connect, construction recombination plasmid with the expression vector pK18mobsacB of same double digestion；Matter will be recombinated Grain electrotransformation is into bacterial strain C.glutamicum ATCC 13032；The recon for completing homologous recombination twice is filtered out, is realized The knockout of murA, murB gene obtain temperature sensitive type recombinant bacterium.
3. the purposes of temperature sensitive type recombination corynebacterium glutamicum as claimed in claim 1 or 2, which is characterized in that be used for fermenting and producing Glutamic acid.
4. a kind of method using the as claimed in claim 1 or 22 recombination corynebacterium glutamicum fermenting and producing glutamic acid, feature exist In including the following steps:
(1) thallus is inoculated into slant medium and is activated, 32 DEG C of culture 24-36h；
(2) activated strain is accessed in seed culture medium, 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) to fermented and cultured to thallus OD600When=12-16, fermentation temperature is improved to 38.5 DEG C, 200rpm is cultivated to total hair Ferment 28-32h；
The composition of the slant medium are as follows: peptone 1%, yeast powder 0.5%, beef extract 1%, NaCl 0.25%, MgSO4 0.05%, KH2PO40.1%, corn pulp 2%, agar 2%；
The composition of the seed culture medium are as follows: glucose 30g/L, K2HPO4·3H2O 0.2g/L, MgSO4·7H2O 0.8g/L, MnSO42mg/L, FeSO42mg/L, VB10.3mg/L, VH0.2mg/L, yeast powder 10g/L, peptone 5g/L, urea 0.5g/ L, Met1g/L；
The composition of the fermentation medium are as follows: glucose 8g/L, K2HPO4·3H2O 0.4g/L, VH0.35mg/L, VB10.5mg/ L, glycine betaine 5g/L, MnSO430mg/L, FeSO430mg/L, MgSO4·7H2O 2g/L, yeast powder 15g/L, peptone 5g/ L。
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A Mutation in the Corynebacterium glutamicum ltsA Gene Causes Susceptibility to Lysozyme, Temperature-Sensitive Growth, and L-Glutamate Production;TAKASHI HIRASAWA et al.;《JOURNAL OF BACTERIOLOGY》;20000530;第182卷(第10期);摘要、第2697页左栏倒数第4段、第2699页左栏最后一段、第2700页右栏第2段、第2697页左栏倒数第3段
General principles for the formation and proliferation of a wall-free (L-form) state in bacteria;Romain Mercier et al.;《eLIFE》;20141030;第3卷;第1页倒数第2段、第4页第2段、第8页第3段、图1
甜菜碱对谷氨酸温度敏感突变株发酵的影响;常立群 等;《发酵科技通讯》;20130430;第42卷(第2期);第1-3页

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