CN102533626A - Genetic engineering strain for producing succinic acid by utilizing glucose and acidogenic fermentation method thereof - Google Patents
Genetic engineering strain for producing succinic acid by utilizing glucose and acidogenic fermentation method thereof Download PDFInfo
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- CN102533626A CN102533626A CN2011104131376A CN201110413137A CN102533626A CN 102533626 A CN102533626 A CN 102533626A CN 2011104131376 A CN2011104131376 A CN 2011104131376A CN 201110413137 A CN201110413137 A CN 201110413137A CN 102533626 A CN102533626 A CN 102533626A
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- succinic acid
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- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 41
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- 238000000855 fermentation Methods 0.000 title claims abstract description 31
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- 239000008103 glucose Substances 0.000 title claims abstract description 25
- 238000010353 genetic engineering Methods 0.000 title claims abstract description 8
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
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- HQABUPZFAYXKJW-UHFFFAOYSA-N N-butylamine Natural products CCCCN HQABUPZFAYXKJW-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- OVYQSRKFHNKIBM-UHFFFAOYSA-N butanedioic acid Chemical compound OC(=O)CCC(O)=O.OC(=O)CCC(O)=O OVYQSRKFHNKIBM-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention belongs to the field of biology engineering technology, and relates to a genetic engineering strain for producing succinic acid by utilizing glucose and an acidogenic fermentation method of the genetic engineering strain. The genetic engineering strain for producing succinic acid by utilizing glucose is named as Escherichia coli BA205 and the preservation number is registered as CCTCC No.M2011447. In the construction process, Escherichia coli which is short of lactic dehydrogenase (LDH) gene and Pyruvate formate-lyase (PFL) gene activity is mainly used as an original strain; phosphoenolpyruvate carboxylase (PPC) gene is removed by utilizing a homologous recombination technology; and phosphoenolpyruvate carboxylase and nicotinic acid phosphoribosyl transferase are excessively co-expressed; therefore the synthesis efficiency of succinic acid is greatly increased. In the fermentation method, a two-stage fermentation manner is adopted, the biomass is improved in an aerobic stage and the acidogenic fermentation is carried out in an anaerobic stage.
Description
Technical field
The invention belongs to technical field of bioengineering; Relating to engineering strain and the fermentation and acid method thereof of utilizing the glucose succinic acid-producing, specifically is that a plant height is imitated the method for utilizing glucose growth and succinic acid-producing recombinant bacterial strain and utilizing this strain fermentation production Succinic Acid.
Background technology
Succinic Acid (succinic acid) claim succsinic acid again; Be widely used in industries such as medicine, agricultural chemicals, dyestuff, spices, paint, food and plastics; As C4 hardware and software platform compound; Can be used for synthesizing 1, organic chemicals and poly butylene succinate (PBS) type Biodegradable materials such as 4-butyleneglycol, THF, gamma-butyrolactone are thought one of biorefinery product of following 12 kinds of most worthies by USDOE.
The working method of Succinic Acid mainly comprises chemical synthesis and microbe fermentation method; Utilize microbe fermentation method to transform renewable resources (glucose, wood sugar etc.),, pollute little because raw material sources are extensive and cheap; Environmental friendliness, and can absorb fixation of C O during the fermentation
2, can effectively alleviate Greenhouse effect, opened up the new way that the greenhouse gases carbonic acid gas utilizes, become the focus of research this year.The production bacterial strain of Succinic Acid mainly concentrates on
Anaerobiospirillum succiniciproducens,
Actinobacillus succinogenes,
Mannheimia succiniciproducens, reorganization Corynebacterium glutamicum and recombination bacillus coli.Obtained higher production concentration though utilize wild strain to produce Succinic Acid, the culturing process culture medium cost is higher, and byproducts build-up such as formic acid, acetate are more, hindered its process of industrialization.
E.coliBecause clear, easy to operate, the easy-regulating of genetic background, substratum require simple and the advantage such as rapid of growing, and are widely used in research in recent years to obtain the outstanding bacterial strain of succinic acid-producing.
The structure thinking of existing succinic acid-producing recombination bacillus coli comprises that mainly the inactivation by product generates the key enzyme (like pyruvate formate-lyase and serum lactic dehydrogenase) of approach, the activity that strengthens enzyme (like phosphoric acid enol pyruvic acid carboxylase) in the Succinic Acid route of synthesis and external source importing and can guide the enzyme (like pyruvate carboxylase) of synthesizing succinic acid to improve its utilization ratio and throughput rate to glucose.Wherein,
E. coliNZN111 since simultaneously inactivation pyruvate formate-lyase and serum lactic dehydrogenase, NADH can not in time be regenerated as NAD
+, cause the imbalance (NADH/NAD of coenzyme NAD (H) in the born of the same parents
+Ratio surpasses 2), cause finally that bacterial strain can not utilize glucose under the anaerobic condition.Its spontaneous mutation strain
E. coliAFP111 is owing to suddenlyd change in the glucose obligate movement system
PtsGGene; Reduce the generation speed of NADH in EMP Embden Meyerbof Parnas pathway, recovered NAD (H) balance, made bacterial strain under anaerobic can utilize glucose; And product is mainly Succinic Acid; Cultivate in the AFP111 process at aerobic anaerobism two stage fermentations, the Succinic Acid mass yield reaches 96%, and production intensity is 1.21 gL
-1H
-1Therefore, in high succinic acid-producing coli strain building process, guarantee that the balance of coenzyme NAD (H) in the born of the same parents is one of key factor of the high succinic acid-producing of recombination bacillus coli.
Biosynthesizing and the decomposition approach of NAD in the intestinal bacteria (H) are as shown in Figure 1, relate to its synthetic gene mainly contain three (
PncB,
NadD,
NadE), relate to catabolic gene mainly contain two (
YjaD,
YrfE), and NAD
+Then reach more than 300 with NADH conversion reaction each other.Correlative study shows that utilizing the DNA recombinant technology to transform NAD (H) biosynthetic pathway is the effective means that improves NAD (H) total amount.People such as San (Metab Eng, 2002,4:238-247; Metab Eng, 2002,4:182-192) in the influence process of research cofactor regulation and control, make interior NAD (H) total amount of born of the same parents improve 41.7% through overexpression nicotinic acid phosphoribosyl transferase (NAPRTase) to intestinal bacteria metabolism distributions; People such as Heuser (Eng Life Sci; 2007,7:343-353), perhaps express this two enzymes simultaneously through overexpression nicotinic acid phosphoribosyl transferase and NAD synthetic enzyme; Interior NAD (H) total amount of bacterial strain born of the same parents has been improved more than 2 times; And apply it in synthetic (the R)-methyl of enzymatic conversion-3-hydroxyl butylamine process, make the amount of NAD (H) no longer become limiting factor, thereby improved the efficient of enzymatic conversion.Numerous scientific practices also prove and utilize the fermentation control means can effectively regulate NAD (H) total amount and NADH/NAD
+Ratio, and then effectively improve the utilization ratio and the product production level of substrate.Utilizing
Saccharomyces cerevisiaeTMB3001 (Biotechnol Bioeng, 2002,78:172-178) with
Fusarium oxysporum(J Biosci Bioeng, 2004,97:299-304. Enzyme Micro Technol, 2005,36:100-106) xylose-fermenting is produced and is added acetoin in the alcoholic acid process as the external source electron acceptor(EA), has increased NAD in the born of the same parents effectively
+Content has improved ethanol yield; People such as San (Metab Eng, 2002,4:182-192) utilizing intestinal bacteria to produce 1, in the 2-Ucar 35 process, find in thinning ratio to be 0.1 h
-1In the perseveranceization anaerobism culture systems, along with the increase of carbon source reductibility, NADH/NAD in the born of the same parents
+Ratio is increased to 0.75 (glucose) and 0.94 (sorbyl alcohol) from 0.51 (glucono-), and causes center metabolism apoblema ethanol (consuming 2 mol NADH) that the ratio of acetate (not consuming NADH) is respectively 0.29,1 and 3.62.The nicotinic acid phosphoribosyltransferase is rate-limiting step enzyme in NAD (H) building-up process and the participation (see figure 1) that needs ATP.
PEP generates oxaloacetic acid through phosphoric acid enol pyruvic acid carboxylase in intestinal bacteria, in this process, does not have the generation of ATP, but
Bacillus subtilisIn, PEP generates oxaloacetic acid through PEP carboxylation kinases, and the generation of ATP is arranged in this process, and overexpression in intestinal bacteria such as Millard
E. coli ppcWith
Pck, discover overexpression
PpcCan make the primary product of succsinic acid, and output improves 3.5 times than starting strain as mixed acid fermentation, and overexpression
PckTo not influence of fermentation result, but
PpcIn the defective bacterial strain,
PckOverexpression can improve the output of succsinic acid.The patented claim formerly of the application contriver team relates to the engineering strain that a strain can utilize the wood-sugar fermentation succinic acid-producing; And carried out the preservation of bacterial strain patent; Number of patent application 201110380396.3, November 25 2011 applying date, biomaterial deposit number CCTCC NO:M2011207.This bacterial strain can efficiently utilize the wood-sugar fermentation succinic acid-producing, but can not utilize glucose fermentation to produce Succinic Acid.
If to lack lactate dehydrogenase gene; Pyruvate formate-lyase gene and phosphoric acid enol pyruvic acid carboxylase gene are active; And the kinase whose bacterial strain intestinal bacteria of overexpression PEP carboxylation BA204 is a starting strain; Behind the overexpression nicotinic acid phosphoribosyltransferase, obtain efficiently to utilize glucose growth and succinic acid-producing genetic engineering bacterium again.
Summary of the invention
The object of the present invention is to provide a kind of engineering strain and construction process thereof that can efficiently utilize glucose growth and succinic acid-producing; And utilizing this bacterial strain anaerobically fermenting to produce Succinic Acid, the construction process that reaches bacterial strain is simple and convenient, makes up the strain fermentation method simple possible that obtains; Be easy to industriallization; The purpose that acid producing ability is strong, thus reduce production costs greatly, increase economic efficiency.
For realizing the object of the invention, the present invention adopts following technical scheme.
One, the present invention provides a strain succinic acid-producing genetically engineered bacteria strain, its classification called after ETEC BA205 (
Escherichia coliBA205), its deposit number is CCTCC NO:M 2011447.
Two, the construction process of ETEC BA205 of the present invention; It is characterized in that to lack serum lactic dehydrogenase (LDH) gene; The bacterial strain intestinal bacteria of pyruvate formate-lyase (PFL) gene activity are starting strain; Utilize homologous recombination technique to knock out phosphoric acid enol pyruvic acid carboxylase (PPC) gene; And behind excessive coexpression PEP carboxylation kinases and the nicotinic acid phosphoribosyltransferase, obtain efficiently to utilize glucose growth and succinic acid-producing ETEC BA205.
Further, described concrete construction step is following:
(1) with lack lactate dehydrogenase gene (
LdhA), the pyruvate formate-lyase gene (
PflB) active
E.coliThe NZN111 bacterial strain is a starting strain, knocks out wherein phosphoric acid enol pyruvic acid carboxylase (PPC) gene, is lacked simultaneously
LdhA,
PflBWith
PpcThe competence bacterial strain; (2) synthetic a pair of 5 ' end has the primer of restriction enzyme site, with
Bacillus subtilisGenomic dna is a template, and purifying amplifies
PckBehind the gene, expression plasmid pTrc99a uses consistent enzyme double digestion, the connection of restriction enzyme site that is designed with primer to obtain recombinant plasmid pTrc99a-
Pck
(3) synthetic a pair of 5 ' end has the primer of identical restriction enzyme site, is template with the e. coli k12 genomic dna, and purifying amplifies
PncBBehind the gene, the recombinant plasmid pTrc99a-that has made up
PckUse consistent enzyme single endonuclease digestion, the connection of restriction enzyme site that is designed with primer to obtain recombinant plasmid pTrc99a-
Pck-
PncB
(4) with recombinant plasmid pTrc99a-
Pck-
PncBImport the competence bacterial strain that step (1) obtains, obtain positive transformant;
(5) utilize the excessive coexpression PEP of the positive transformant carboxylation kinases and the nicotinic acid phosphoribosyltransferase of step (4); Recover its ability of metabolizable glucose under anaerobic, efficiently utilized glucose metabolism succinic acid-producing genetic engineering bacterium ETEC BA205.
Three, utilize the method for ETEC BA205 fermentation production of succinic acid of the present invention, it is characterized in that adopting two stage fermentation modes, the aerobic stage is improved living weight, anaerobic stages fermentation and acid.
Further, concrete steps are following.
ETEC BA205 is inoculated aerobic cultivation in the aerobic stage fermentation substratum by 1% (v/v) inoculum size, as aerobic culture bacteria body OD
600IPTG to 0.4～0.6 usefulness, 0.3 mM is induced to OD
600, be forwarded to anaerobically fermenting in the anaerobic stages fermention medium at=3 o'clock by inoculum size 10%.
Wherein said aerobic stage fermentation substratum is that aerobic is cultivated the colibacillary conventional substratum of succinic acid-producing in the prior art; Described anaerobic stages fermention medium is to be that the succinic acid-producing intestinal bacteria of carbon source are used fermention medium with glucose.
Beneficial effect of the present invention is: to lack lactate dehydrogenase gene; Pyruvate formate-lyase gene and phosphoric acid enol pyruvic acid carboxylase gene are active; And the kinase whose bacterial strain intestinal bacteria of overexpression PEP carboxylation BA204 is a starting strain; Again behind the overexpression nicotinic acid phosphoribosyltransferase; Obtain efficiently to utilize glucose growth and succinic acid-producing genetic engineering bacterium, overcome the defective that former BA204 bacterial strain can not utilize glucose, strengthened the accommodation of this bacterial strain.
Description of drawings
The biosynthesizing of NAD in Fig. 1 intestinal bacteria (H) and decomposition approach.
Fig. 2 recombinant plasmid pTrc99a-
PckThe structure collection of illustrative plates.
Fig. 3 recombinant plasmid pTrc99a-
Pck-pncBThe structure collection of illustrative plates.
Fig. 4 PCR product
PckAgarose gel electrophoresis identify figure.
Fig. 5 PCR product
PncBAgarose gel electrophoresis identify figure.
Fig. 6 recombinant plasmid pTrc99a-
PckDouble digestion identify figure.
Fig. 7 recombinant plasmid pTrc99a-
Pck-pncBDouble digestion identify figure.
Microorganism classification called after ETEC BA205 of the present invention (
Escherichia coliBA205), preservation date is on December 7th, 2011, and depositary institution's full name is Chinese typical culture collection center, abbreviates CCTCC as, depositary institution address: China. Wuhan. and Wuhan University; Deposit number: CCTCC NO:M 2011447.
Embodiment
Following embodiment elaborates to the present invention, but to not restriction of the present invention.
The source of apramycin resistant gene of the present invention is: pIJ773, and available from the azure professor of Shao of Nanjing Normal University place.
The source of plasmid that can abduction delivering λ recombinase of the present invention is: pKD46, and available from Introvegen company.
The source of the plasmid that produces the FLP recombinase of can inducing of the present invention is: pCP20, and available from Introvegen company.
Of the present invention
Bacillus subtilisGenomic source is: available from ATCC 23857.
Expression plasmid of the present invention with the source of pTrc99a is: available from Introvegen company.
Starting strain of the present invention
E.coliThe source of NZN111 (CGSC#:7726) is: Biotechnol Bioeng, 2001,74:89～95.
The present embodiment explanation utilizes homologous recombination technique to knock out phosphoric acid enol pyruvic acid carboxylase among the starting strain NZN111
PpcGene, the process of the apramycin resistant strain that is eliminated.
1, utilizes the LB substratum, in 37 ℃, the following intestinal bacteria NZN111 to OD that cultivates of aerobic conditions
600=0.4～0.6, being prepared into electricity changes competence.
2, change plasmid pKD46 electricity over to competent intestinal bacteria NZN111.The electric shock condition is: 200 Ω, 25 μ F, electric shock voltage 2.3 kV, electric shock times 4～5 ms.The SOC substratum that rapidly thalline is added precooling 1 mL after shocking by electricity, 150 r/min, 30 ℃ of cultivation 1 h coat and be with the LB culture medium flat plate of penbritin (amp) to filter out positive transformant intestinal bacteria NZN111 (pKD46) afterwards.
3, in the LB substratum, add the L-arabinose of 10 mM, under 30 ℃, induce plasmid pKD46 to give expression to the λ recombinase, process electricity and change competence.
4, the apramycin resistant gene that has a FRT site with both sides is a template, utilizes high-fidelity pcr amplification system, is template with plasmid pIJ773, and the design two ends have the amplimer of PPC homologous fragment, amplifies the linear DNA homologous fragment, and primer sequence is following:
Upper reaches band homology arm primer H1-P1, underscore is a homologous fragment:
5’-
ATGAACGAACAATATTCCGCATTGCGTAGTAATGTCAGTATGCTCGGCATTCCGGGGATCCGTCGACC-3’。
Downstream band homology arm primer H2-P2, underscore is a homologous fragment:
5’-
AGCACGAGGGTTTGCAGAAGAGGAAGATTAGCCGGTATTACGCATACCTGTAGGCTGGAGCTGCTTC-3’。
Reaction system: each 0.5 μ L of upstream and downstream primer (100 pmol/ μ L) of band homology arm; Template DNA (100 ng/ μ L) 0.5 μ L; 10 * buffer, 5 μ L; Each 1 μ L of dNTPs (10 mM); DMSO (100%) 2.5 μ L; Pyrobest archaeal dna polymerase (2.5 U/ μ L) 1 μ L; DdH
2O 36/35.5 μ L; TV 50 μ L.
Reaction conditions: 94 ℃, 2 min; (94 ℃ of 45 sec; 50 ℃ of 45 sec; 72 ℃ of 90 sec; 10 circulations); (94 ℃ of 45 sec; 50 ℃ of 45 sec; 72 ℃ of 90 sec; 15 circulations); 72 ℃, 5 min.
The evaluation of linear DNA fragment such as Fig. 2.
5, electricity changes linear DNA fragment intestinal bacteria NZN111 (pKD46) competence of abduction delivering λ recombinase extremely, and coats and be with the LB flat screen of apramycin to select positive recombinant, and has carried out the PCR evaluation, and electrophorogram is as shown in Figure 3.
6, positive recombinant process pour into after the competence can abduction delivering FLP recombinase plasmid pCP20, after the FLP recombinase is expressed in 42 ℃ of heat shocks, can eliminate the apramycin resistance.Utilize pair of plates, carry out parallel point sample, can on the non-resistant flat board, grow, but the bacterial strain that has all very knocked out resistance that can not on resistant panel, grow.
The present embodiment explanation makes up the expression plasmid of excessive coexpression PEP carboxylation kinases and nicotinic acid phosphoribosyltransferase, recovers the recombinant bacterial strain ability of metabolizable glucose under anaerobic, obtains bacterial strain
Escherichia coliThe method of BA205.
1, makes up pTrc99a-
PckPlasmid, its process comprises:
(1) synthetic having
SacI with
XbaThe primer of I restriction enzyme site,
Upstream primer: 5 '-CGAGCTCATGAACTCAGTTGATTTGACCG-3 '.
Downstream primer: 5 '-GCTCTAGAGCATTCCGTCAATTAAAACAAG-3 '.
(2) with
Bacillus subtilisGenomic dna is a template, the pcr amplification target gene fragment, and reaction conditions is: 94 ℃, 5 min; (94 ℃ of 45 s, 53 ℃ of 45 s, 72 ℃ of 100 s, 35 circulations); 72 ℃, 10 min.Purifying amplifies
PckBehind the gene, expression plasmid is used respectively with pTrc99a
SacI with
XbaI double digestion, connection obtain recombinant plasmid pTrc99a-
Pck
2, the expression plasmid of excessive coexpression PEP carboxylation kinases and nicotinic acid phosphoribosyltransferase, its process comprises:
(1) synthetic upstream and downstream primer all has
HindThe primer of III restriction enzyme site,
Upstream primer: 5 '-CCCAAGCTTATGACACAATTCGCTTCTCCTG-3 '.
Downstream primer: 5 '-CCCAAGCTTCACTTGTCCACCCGTAAATGG-3 '.
(2) be template with e. coli k12 series, the pcr amplification target gene fragment, reaction conditions is: 94 ℃, 5 min; (94 ℃ of 45 s, 55 ℃ of 45 s, 72 ℃ of 1 min, 35 circulations); 72 ℃, 10 min.Purifying amplifies
PncBBehind the gene, plasmid pTrc99a-
PckUse
HindIII single endonuclease digestion, connection obtain recombinant plasmid pTrc99a-
Pck-
PncB
3, with plasmid pTrc99a-
Pck-
PncBLack when importing embodiment 1
LdhA,
PflBWith
PpcThe competence bacterial strain, the positive transformant of acquisition is new structure bacterial strain of the present invention
Escherichia coliBA205.
Embodiment 3
Present embodiment is explained the new reorganization large intestine bacterial strain that makes up of excessive coexpression
Escherichia coliNAD (H) total amount and the NADH/NAD of the elimination apramycin resistant strain that BA205 and embodiment 1 obtain
+The comparison of ratio, and the contrast that consumes sugar and acid producing ability in both fermenting processs.
When importing plasmid pTrc99a-
Pck-
PncBAfter; Eliminate apramycin resistant strain excessive coexpression PEP carboxylation kinases and nicotinic acid phosphoribosyltransferase and recovered the anaerobic condition redox equilibrium of reorganization bacterium down; The total amount of NAD (H) is significantly improved; Also recovered simultaneously the ability of metabolizable glucose under the anaerobic condition, main product is a Succinic Acid simultaneously, the accumulation of no formic acid and lactic acid.
ETEC BA205 is inoculated aerobic cultivation in the aerobic stage fermentation substratum by 1% (v/v) inoculum size, as aerobic culture bacteria body OD
600IPTG to 0.4～0.6 usefulness, 0.3 mM is induced to OD
600, be forwarded to anaerobically fermenting in the anaerobic stages fermention medium at=3 o'clock by inoculum size 10%.
Wherein said aerobic stage fermentation substratum is that aerobic is cultivated the colibacillary conventional substratum of succinic acid-producing in the prior art; Substratum in the present embodiment is: the LB substratum.
Described anaerobic stages fermention medium is to be that the succinic acid-producing intestinal bacteria of carbon source are used fermention medium with glucose.Substratum in the present embodiment is following.
The fermentation of anaerobism serum bottle uses substratum to be: LB+ glucose (20g/L)+magnesium basic carbonate 0.48g++Amp (penbritin 50 μ g/mL)+0.3mM IPTG+0.5mM NA (nicotinic acid).
The mensuration result that the anaerobism serum bottle is cultivated the various parameters in back sees table 1.
Table 1 anaerobism serum bottle is cultivated the mensuration result of the various parameters in back
Claims (6)
1. a strain succinic acid-producing genetically engineered bacteria strain, its classification called after ETEC BA205 (
Escherichia coliBA205), its preservation registration number is CCTCC M 2011447.
2. the construction process of the described ETEC BA205 of claim 1; It is characterized in that to lack lactate dehydrogenase gene; The bacterial strain intestinal bacteria of pyruvate formate-lyase gene activity are starting strain; Utilize homologous recombination technique to knock out phosphoric acid enol pyruvic acid carboxylase gene, and behind excessive coexpression PEP carboxylation kinases and the nicotinic acid phosphoribosyltransferase, obtain efficiently to utilize glucose growth and succinic acid-producing ETEC BA205.
3. the construction process of ETEC BA205 according to claim 1 is characterized in that concrete construction step is following:
(1) to lack lactate dehydrogenase gene, the pyruvate formate-lyase gene activity
E.coliThe NZN111 bacterial strain is a starting strain, knocks out wherein phosphoric acid enol pyruvic acid carboxylase gene, is lacked simultaneously
LdhA,
PflBCompetence bacterial strain with PPC;
(2) synthetic a pair of 5 ' end has the primer of restriction enzyme site, with
Bacillus subtilisGenomic dna is a template, and purifying amplifies
PckBehind the gene, expression plasmid pTrc99a uses consistent enzyme double digestion, the connection of restriction enzyme site that is designed with primer to obtain recombinant plasmid pTrc99a-
Pck
(3) synthetic a pair of 5 ' end has the primer of identical restriction enzyme site, is template with the e. coli k12 genomic dna, and purifying amplifies
PncBBehind the gene, the recombinant plasmid pTrc99a-that has made up
PckUse consistent enzyme single endonuclease digestion, the connection of restriction enzyme site that is designed with primer to obtain recombinant plasmid pTrc99a-
Pck-pncB
(4) with recombinant plasmid pTrc99a-
Pck-pncBImport the competence bacterial strain that step (1) obtains, obtain positive transformant;
(5) utilize the excessive coexpression PEP of the positive transformant carboxylation kinases and the nicotinic acid phosphoribosyltransferase of step (4); Recover its ability of metabolizable glucose under anaerobic, efficiently utilized glucose metabolism succinic acid-producing genetic engineering bacterium ETEC BA205.
4. utilize the method for the described ETEC BA205 of claim 1 fermentation production of succinic acid, it is characterized in that adopting two stage fermentation modes, the aerobic stage is improved living weight, anaerobic stages fermentation and acid.
5. method according to claim 4 is characterized in that ETEC BA205 is inoculated aerobic cultivation in the aerobic stage fermentation substratum by 1% (v/v) inoculum size, as aerobic culture bacteria body OD
600IPTG to 0.4～0.6 usefulness, 0.3 mM is induced to OD
600, be forwarded to anaerobically fermenting in the anaerobic stages fermention medium at=3 o'clock by inoculum size 10%.
6. method according to claim 4 is characterized in that described anaerobic stages fermention medium is is that the succinic acid-producing intestinal bacteria of carbon source are used fermention medium with glucose.
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| CN102864116A (en) * | 2012-10-16 | 2013-01-09 | 南京工业大学 | Genetic engineering bacterium for producing succinic acid, and construction and application thereof |
| WO2013086907A1 (en) * | 2011-12-13 | 2013-06-20 | 南京工业大学 | Genetic engineering strain for producing succinic acid by using glucose and method for producing acid by fermenting the strain |
| CN103937733A (en) * | 2014-03-18 | 2014-07-23 | 南京工业大学 | Genetic engineering strain utilize sucrose to produce succinic acid from and method for production of succinic acid by fermenting the same |
| CN105543214A (en) * | 2014-10-30 | 2016-05-04 | 华东理工大学 | Construction method and applications of metabolic engineering escherichia coli strain for producing succinic acid by using acetic acid |
| CN112280725A (en) * | 2020-10-29 | 2021-01-29 | 江南大学 | Recombinant escherichia coli for efficiently producing succinic acid and construction method thereof |
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| WO2013086907A1 (en) * | 2011-12-13 | 2013-06-20 | 南京工业大学 | Genetic engineering strain for producing succinic acid by using glucose and method for producing acid by fermenting the strain |
| CN102864116A (en) * | 2012-10-16 | 2013-01-09 | 南京工业大学 | Genetic engineering bacterium for producing succinic acid, and construction and application thereof |
| CN103937733A (en) * | 2014-03-18 | 2014-07-23 | 南京工业大学 | Genetic engineering strain utilize sucrose to produce succinic acid from and method for production of succinic acid by fermenting the same |
| CN105543214A (en) * | 2014-10-30 | 2016-05-04 | 华东理工大学 | Construction method and applications of metabolic engineering escherichia coli strain for producing succinic acid by using acetic acid |
| CN105543214B (en) * | 2014-10-30 | 2019-01-08 | 华东理工大学 | Utilize the metabolic engineering coli strain construction method of acetic acid production succinic acid and application |
| CN112280725A (en) * | 2020-10-29 | 2021-01-29 | 江南大学 | Recombinant escherichia coli for efficiently producing succinic acid and construction method thereof |
| CN115895989A (en) * | 2022-08-05 | 2023-04-04 | 湖北工业大学 | Escherichia coli with high succinic acid yield as well as preparation method and application thereof |
| CN115895989B (en) * | 2022-08-05 | 2024-05-10 | 江苏寒武纪生物细胞科学有限公司 | Escherichia coli for high yield of succinic acid and preparation method and application thereof |
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Application publication date: 20120704 |