CN104232553A - Engineering strain capable of producing succinic acid at low pH value and method for producing succinic acid by fermentation - Google Patents
Engineering strain capable of producing succinic acid at low pH value and method for producing succinic acid by fermentation Download PDFInfo
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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 103
- 239000001384 succinic acid Substances 0.000 title claims abstract description 50
- 238000000855 fermentation Methods 0.000 title claims abstract description 30
- 230000004151 fermentation Effects 0.000 title claims abstract description 30
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 37
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- 230000000694 effects Effects 0.000 claims abstract description 8
- 230000035772 mutation Effects 0.000 claims abstract description 8
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- 108091000080 Phosphotransferase Proteins 0.000 claims abstract description 7
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- 241000193830 Bacillus <bacterium> Species 0.000 claims description 32
- 210000001072 colon Anatomy 0.000 claims description 32
- 230000001580 bacterial effect Effects 0.000 claims description 18
- 241000894006 Bacteria Species 0.000 claims description 17
- 239000002054 inoculum Substances 0.000 claims description 15
- 230000008569 process Effects 0.000 claims description 14
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a genetic engineering strain capable of producing succinic acid at a low pH value. The classification name of the genetic engineering strain is Escherichia coli BA601, and the collection number is CCTCCNO: M2014210. The invention further discloses a method for producing succinic acid by using the strain. According to the strain disclosed by the invention, an Escherichia coli AFP111 which is lack of lactate dehydrogenase (LDH) genes and has activity of pyruvate formate lyase (PFL) genes and ptsG spontaneous mutation of a phosphotransferase system is taken as a starting strain for over-expression of acid-resistant genes gadBC. The method of enabling the Escherichia coli AFP111 which can not grow under low-pH (pH 5.6) conditions originally and can not produce acid by fermentation to efficiently utilize glucose to produce succinic acid through a biological and microbial acclimation means is not disclosed before, and the application can greatly promote the progress and development of the succinic acid industry.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to the method for a strain succinic acid-producing genetic engineering bacterium and fermentation production of succinic acid thereof, specifically strain succinic acid-producing recombinant bacterial strain and utilize this strain fermentation to produce the method for succinic acid at low ph values.
background technology
Succinic acid (succinic acid) is also known as succsinic acid, be widely used in the industries such as medicine, agricultural chemicals, dyestuff, spices, paint, food and plastics, as C4 platform chemicals, can be used for synthesis 1, organic chemicals and poly butylene succinate (PBS) the class Biodegradable materials such as 4-butyleneglycol, tetrahydrofuran (THF), gamma-butyrolactone, thought one of biorefinery product of following 12 kinds of most worthies by USDOE.In addition, succinic acid is also beneficial to human health intermediate for the production of medicine, microbiotic, amino acid and VITAMIN etc.
The production method of succinic acid mainly comprises chemical synthesis and microbe fermentation method, utilizes microbe fermentation method to transform renewable resources, because raw material sources are extensive and cheap, pollutes little, environmental friendliness, and can absorb fixation of C O during the fermentation
2, effectively can alleviate Greenhouse effect, open the new way that GHG carbon dioxide utilizes, become the focus of research this year.The production bacterial strain of succinic acid mainly concentrates on
anaerobiospirillum succiniciproducens,
actinobacillus succinogenes,
mannheimia succiniciproducens, restructuring Corynebacterium glutamicum and recombination bacillus coli.Although wherein utilize wild strain to produce succinic acid obtain higher production concentration, culturing process culture medium cost is higher, and the byproducts build-up such as formic acid, acetic acid is more, hinders its process of industrialization.And recombination bacillus coli is because genetic background is clear, easy to operate, easy-regulating, substratum require simple and grow the advantages such as rapid, be widely used in research in recent years to obtain the outstanding bacterial strain of succinic acid-producing.
The Constructed wetlands of existing succinic acid-producing recombination bacillus coli mainly comprises the key enzyme (as pyruvate formate-lyase and serum lactic dehydrogenase) of inactivation by product constructive ways, the activity strengthening enzyme (as phosphoric acid enol pyruvic acid carboxylase) in succinic acid route of synthesis and external source and imports the enzyme of synthesizing succinic acid (as pyruvate carboxylase) can be guided to improve it to the utilization ratio of glucose and throughput rate.Wherein,
e. colinZN111 due to while inactivation pyruvate formate-lyase and serum lactic dehydrogenase, NADH can not be regenerated as NAD in time
+, cause the imbalance (NADH/NAD of coenzyme NAD in born of the same parents (H)
+ratio is more than 2), under finally causing anaerobic condition, bacterial strain can not utilize glucose.Its spontaneous mutation strain
e. coliaFP111 is owing to having suddenlyd change in glucose obligate movement system
ptsGgene, reduce the generation speed of NADH in EMP Embden Meyerbof Parnas pathway, recover NAD (H) balance, make bacterial strain under anaerobic can utilize glucose, and product is mainly succinic acid, in aerobic anaerobism two benches fermentation culture AFP111 process, succinic acid mass yield reaches 96%, and production intensity is 1.21 g L
-1h
-1.
The cost utilizing biotechnology to produce 60-70% in succinic acid process is used in the succinate produced finally fermenting to be separated the purification phase obtaining free succinic acid, and this is also the stage that in whole process, Expenses Cost is maximum.In order to reduce production cost, the most feasible method need not or make free succinic acid become primary product with the neutralization of a small amount of alkalimetric titration in fermenting process.But the bacterium produced for succinic acid up to now all effectively can not grow and succinic acid-producing in low ph value.But the prokaryotic micro-organisms that effectively can maintain pH in born of the same parents is then used in low ph value bottom fermentation production succinic acid.
Intestinal bacteria are widely used in industry, but current most of intestinal bacteria are due to can not in tolerance low ph value, thus can not fermentation production of succinic acid at low ph values.
Summary of the invention
The object of the present invention is to provide strain succinic acid-producing coli strain under pH value, and utilize this bacterial strain anaerobically fermenting to produce succinic acid, the method reaching bacterial strain is simple and convenient, the strain fermentation method simple possible obtained, be easy to industrialization, the object that acid producing ability is strong, thus greatly reduce production cost, increase economic efficiency.
For realizing the object of the invention, the present invention by the following technical solutions:
One, the invention provides a strain succinic acid-producing genetically engineered bacteria strain, its Classification And Nomenclature is colon bacillus BA601(
escherichia colibA601), its preserving number is numbered CCTCC NO:M2014210.
Two, colon bacillus of the present invention (
escherichia coli) construction process of BA601, it is characterized in that lacking serum lactic dehydrogenase (LDH) gene, pyruvate formate-lyase (PFL) gene activity and phosphotransferase system
ptsGthe intestinal bacteria of karyomit(e) generation spontaneous mutation are starting strain, after utilizing process LAN L-Glutamic decarboxylase, γ-aminobutyric acid antiporter gene, cultivate through continuous domestication, and obtaining can high-yield succinic colon bacillus BA601 at low ph values;
Further, described concrete steps are as follows:
(1) CaCl is utilized
2legal system is standby lacks lactate dehydrogenase gene, pyruvate formate-lyase gene activity and phosphotransferase system
ptsGkaryomit(e) generation spontaneous mutation
e.colicompetence bacterial strain;
(2) purifying amplifies and comprises its acidproof original paper
gadl-glutamic acid (Glumate) decarboxylase (GadB) of system, L-glutamic acid-γ-aminobutyric acid (GABA) antiporter protein (GadC) gene
gadBCplasmid;
(3) by the competence bacterial strain that the plasmid described in step (2) utilizes thermal shock method steps for importing (1) to obtain, positive transformant is obtained;
(4) positive transformant of step (3) is utilized to express resistance to acid gene
gadBC, obtain a strain and to ferment at low ph values the colon bacillus BA600(of succinic acid-producing
escherichia colibA600).
(5) colon bacillus BA600 is after continuous domestication is cultivated, utilize the solid medium of low ph value dull and stereotyped, screening obtains the mutant strain that can grow fast, then to obtain through the screening of anaerobism shake flask fermentation the bacterial strain of efficient succinic acid-producing to be at the low ph aimed strain colon bacillus BA601(
escherichia colibA601).
Three, utilize the method for colon bacillus BA601 fermentation production of succinic acid of the present invention, it is characterized in that adopting two benches fermentation mode, two-stage improves biomass, anaerobic stages fermentation and acid.
Further, concrete steps are as follows:
Colon bacillus BA601 is inoculated aerobic in two-stage fermention medium by 1% (v/v) inoculum size cultivate, aerobic cultivates thalline to OD
600when=3, be forwarded in the serum bottle containing anaerobic stages fermention medium by inoculum size 10%, fill CO
22 min, and at 37 DEG C, 200 r/min anaerobically fermenting 48 h.
Or by colon bacillus BA601 by 10% inoculum size by seed liquor access be equipped with in 3 L fermentor tanks of 1.5 L fermention mediums, 37 DEG C of aerobics are cultured to thalli growth and reach stationary phase, 37 DEG C, 200 r/min continue to pass into CO with the speed of 0.5 L/min
2, carry out anaerobically fermenting, preferably, wherein two-stage fermention medium maintains pH 5.6.
Further, described anaerobic stages fermention medium maintains pH 5.6.
Beneficial effect of the present invention is: the present invention to lack serum lactic dehydrogenase (LDH) gene, pyruvate formate-lyase (PFL) gene activity and phosphotransferase system
ptsGthe intestinal bacteria AFP111 of spontaneous mutation is starting strain, its resistance to acid gene of overexpression
gadBC.This by molecular biology and microbial acclimation means, the method enabling originally to grow under low pH (pH5.6) condition the intestinal bacteria AFP111 efficiency utilization glucose succinic acid-producing of also fermentation and acid has no open, and this application will advance progress and the development of succinic acid industry greatly.
Accompanying drawing explanation
Resistance to acid gene in Fig. 1 intestinal bacteria
gadBCacidproof approach.
Fig. 2 PCR primer
gadBCgel electrophoresis qualification figure.
Fig. 3 plasmid pMD19-T-
gadBCsingle endonuclease digestion, double digestion electroresis appraisal figure.
Fig. 4 colon bacillus (
escherichia coli) BA601 succinic acid-producing result when pH 5.6.
Microorganism classification called after colon bacillus BA601(of the present invention
escherichia colibA601), its preservation date is on May 18th, 2014, and depositary institution's full name is China typical culture collection center, and referred to as CCTCC, preservation address is China. Wuhan. and Wuhan University, its preserving number is numbered CCTCC NO:M2014210.
Embodiment
The following examples elaborate to the present invention, but do not limit the present invention.
The source of plasmid pMD19-T of the present invention is: purchased from Takara company
Starting strain of the present invention:
e.colithere are two places in the source of the competence bacterial strain of AFP111:
(1) Applied and environmental microbiology, 2001,67 (1): 148-154. applicants first by finding the above-mentioned document source of this biomaterial, and contacted by Univ Chicago USA of utterer system
david P. Clarkprofessor, and its this biomaterial of gifting of mail requests, and freely obtain this biomaterial; And applicant ensured to provide this biomaterial to the public in Two decades years from the application's day.
(2) this biomaterial also discloses and obtains the authorization in the patent documentation of Chinese patent (application number CN 96198547, applying date 1996.10.31, authorize day to be on January 1st, 2003, Authorization Notice No. is CN1097632 C).
Primer Source of the present invention is: designed, designed also outer Si Rui biotech company covered with gold leaf synthesis.
embodiment 1
The present embodiment illustrates the method building colon bacillus BA600.
(1) utilize LB substratum, in 37 DEG C, cultivate intestinal bacteria AFP111 to OD under aerobic conditions
600=0.5 ~ 0.6, utilize CaCl
2legal system standby shortage lactate dehydrogenase gene (
ldhA), pyruvate formate-lyase gene (
pflB) active and phosphotransferase system
ptsGthe intestinal bacteria AFP111 competence bacterial strain of gene karyomit(e) spontaneous mutation;
The formula of LB substratum is: peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L.(2) primer of synthesis not with restriction enzyme site,
Upstream primer: 5 '-ACACGAGTCCTTTGCACTTGCTTACTTT-3 ';
Downstream primer: 5 '-CGCTCCCTTGTCTTATAACCATTCAGAC-3 '.
Extract
e. colithis laboratory of JM109(is preserved) genome, with
e. colijM109 genome is template, pcr amplification goal gene fragment, and concrete reaction conditions is: 94 DEG C, 5 min; (94 DEG C of 45 s, 60 DEG C of 45 s, 72 DEG C of 4 min, 36 circulations); 72 DEG C, 10 min.Purifying amplifies and comprises resistance to acid gene
gadBCand its own promoter fragment.PCR primer
gadBCqualification as shown in Figure 2.
Reclaim product for template with PCR, add a certain amount of enzyme, damping fluid, dNTP, 72 DEG C of 20 min reaction, final fragment smooth end adds " A " base respectively.Through adding the reacted fragment of A, carry out TA with pMD19-T carrier and be connected and obtain recombinant plasmid pMD19-T-
gadBC.Structure obtains utilizing fragment its own promoter to express resistance to acid gene
gadBCexpression plasmid.Plasmid pMD19-T-
gadBCsingle endonuclease digestion qualification as shown in Figure 3.
(3) by the competence bacterial strain that the plasmid described in step (2) utilizes thermal shock method steps for importing (1) to obtain, positive transformant is obtained;
(4) positive transformant of step (3) is utilized to express resistance to acid gene
gadBC, obtain a strain can when pH 5.6 efficiency utilization glucose fermentation succinic acid-producing colon bacillus BA600.
embodiment 2
The present embodiment illustrate by colon bacillus (
escherichia coli) BA600 carries out the method for cultured continuously domestication.
The colon bacillus BA600 that obtains will be built as starting strain in embodiment 1.1% (v/v) inoculum size from cryopreservation tube access test tube, 37 DEG C, 200 rpm overnight incubation, then with in 1% (v/v) inoculum size access triangular flask, 37 DEG C, 200 rpm cultivate the bacterium liquid that 6-8 h obtain logarithmic phase; By the bacterium liquid of logarithmic phase with 10%(v/v) inoculum size be inoculated into and be equipped with in 500 mL bactogens of 300mL fermention medium, 37 DEG C of heating in water bath, pass into the CO of filtration sterilization
2maintain anaerobic environment, and add fresh fermention medium with the speed of 1.5 mL/h to stream in culture apparatus.Timing sampling detects the density of thalline in culture apparatus, when in reactor, cell density reaches OD
600=2 ~ 3, and keep 48 h without larger change, illustrate that representing that thalline grows under this flow acceleration stablizes, obtain mutant strain, process of now taming completes a circulation.The flow acceleration of fresh fermention medium is doubled, carries out the cultured continuously of next circulation.Until the flow acceleration of fermention medium reaches 12 mL/h, mutant strain growth performance remains stable under this condition.
Wherein, the formula of described seed culture medium is: peptone 10g/L, yeast powder 5g/L, NaCl 5g/L.
The formula of described fermention medium is: citric acid 3 gL
-1, Na
2hPO
412H
2o 4 gL
-1, KH
2pO
48 gL
-1, (NH
4)
2hPO
48 gL
-1, NH
4cl 0.2 gL
-1, (NH
4)
2sO
40.75 gL
-1, MgSO
47H
2o 1 gL
-1, CaCl
22H
2o 10.0 mgL
-1, ZnSO
47H
2o 0.5 mgL
-1, CuCl
22H
2o 0.25 mgL
-1, MnSO
4h
2o 2.5 mgL
-1, CoCl
26H
2o 1.75 mgL
-1, H
3bO
30.12 mgL
-1, Al
2(SO4)
31.77 mgL
-1, Na
2moO
42H
2o 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, 20.0 mg L
-1vB
1, 2.0 mg L
-1vitamin H, glucose 30-40 g/L.
embodiment 3
The present embodiment illustrates that fermention medium can be maintained the carbonate of lower ph by screening.
Add the different carbonate of different volumetric molar concentration in the fermentation medium, before investigating its sterilizing and after sterilizing, lead to 2 min CO
2after pH value, filter out and pH value can be made to be adjusted to carbonate near 5.6.
The formula of described fermention medium is: citric acid 3 gL
-1, Na
2hPO
412H
2o 4 gL
-1, KH
2pO
48 gL
-1, (NH
4)
2hPO
48 gL
-1, NH
4cl 0.2 gL
-1, (NH
4)
2sO
40.75 gL
-1, MgSO
47H
2o 1 gL
-1, CaCl
22H
2o 10.0 mgL
-1, ZnSO
47H
2o 0.5 mgL
-1, CuCl
22H
2o 0.25 mgL
-1, MnSO
4h
2o 2.5 mgL
-1, CoCl
26H
2o 1.75 mgL
-1, H
3bO
30.12 mgL
-1, Al
2(SO4)
31.77 mgL
-1, Na
2moO
42H
2o 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, 20.0 mg L
-1vB
1, 2.0 mg L
-1vitamin H, glucose 30-40 g/L.
The different carbonate that table 1 screening obtains regulate pH to compare
?
the pH regulation and control of each carbonate to fermention medium are as shown in table 1.Can 5 × 10 be found out from this table
-4mol/L CaCO
3the pH of substratum can be made to reach 5.79, closest to pH 5.6.
embodiment 4
The present embodiment illustrate screening obtain excellent colon bacillus (
escherichia coli) method of BA601.
Screening step:
1, solid plate primary dcreening operation
Under aseptic technique, from bactogen, take out 2-4 mL bacterium liquid, with sterilized water dilution 1 × 10
4doubly, get 100 μ L and be coated on the solid plate of pH 5.6, cultivate 12 h, pick out and select fast growth, comparatively full mutant strain list bacterium colony for 37 DEG C.
2, solid plate sieves again
By the mutant strain turning point cultivation repeatedly on flat board screened, finally obtain bacterial strain BA601, BA613, BA621 shows stronger growth velocity and growth stability.
3, shake flask fermentation screening
Mutant strain BA600, BA601, BA616 and BA628 are accessed in seed culture medium and cultivates, 37 DEG C, 200 r/min, cultivate 12 h, contain in the triangular flask of 50 mL substratum with the access of 1% (v/v) inoculum size again, 37 DEG C, 200 rpm growth 6-8 h.Then be inoculated in fermention medium, 100 mL anaerobism serum bottle liquid amount 30 mL, inoculum size 10%(v/v), carbonating 2 min, 37 DEG C, 200 r/min, cultivate 48 h.
Wherein, the culture medium prescription used is as follows:
Solid plate substratum: citric acid 3 gL
-1, Na
2hPO
412H
2o 4 gL
-1, KH
2pO
48 gL
-1, (NH
4)
2hPO
48 gL
-1, NH
4cl 0.2 gL
-1, (NH
4)
2sO
40.75 gL
-1, MgSO
47H
2o 1 gL
-1, CaCl
22H
2o 10.0 mgL
-1, ZnSO
47H
2o 0.5 mgL
-1, CuCl
22H
2o 0.25 mgL
-1, MnSO
4h
2o 2.5 mgL
-1, CoCl
26H
2o 1.75 mgL
-1, H
3bO
30.12 mgL
-1, Al
2(SO
4)
31.77 mgL
-1, Na
2moO
42H
2o 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, 20.0 mg L
-1vB
1, 2.0 mg L
-1vitamin H, agar 15-20 g/L, glucose 20 g/L.
Seed culture medium: peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L.
Fermention medium: citric acid 3 gL
-1, Na
2hPO
412H
2o 4 gL
-1, KH
2pO
48 gL
-1, (NH
4)
2hPO
48 gL
-1, NH
4cl 0.2 gL
-1, (NH
4)
2sO
40.75 gL
-1, MgSO
47H
2o 1 gL
-1, CaCl
22H
2o 10.0 mgL
-1, ZnSO
47H
2o 0.5 mgL
-1, CuCl
22H
2o 0.25 mgL
-1, MnSO
4h
2o 2.5 mgL
-1, CoCl
26H
2o 1.75 mgL
-1, H
3bO
30.12 mgL
-1, Al
2(SO4)
31.77 mgL
-1, Na
2moO
42H
2o 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, 20.0 mg L
-1vB
1, 2.0 mg L
-1vitamin H, calcium carbonate 0.5 g/L, glucose 30-40g/L.
Table 2 screen strain excellent and the starting strain obtained growth and produce acid compare
Fermentation results is as shown in table 2.Can find out that starting strain AFP111 cannot grow the fermention medium of pH 5.6 from this table, BA600 after molecular modification can grow in the fermention medium of pH 5.6, but its poor growth and the output of succinic acid is lower, but the bacterial strain colon bacillus obtained after bactogen domestication (
escherichia coli) BA601, the speed of growth is in the fermentation medium very fast, and the Yield compari@of succinic acid is high, reaches 28 g/L.
embodiment 5
The present embodiment explanation colon bacillus (
escherichia coli) BA601 to ferment the ability of succinic acid-producing at pH5.6.
Colon bacillus BA601 accesses test tube by 1% (v/v) inoculum size from cryopreservation tube, 37 DEG C, 200rpm overnight incubation, then with the access of 1% (v/v) inoculum size containing in the triangular flask of LB substratum, 37 DEG C, 200rpm growth.When aerobic cultivates thalline OD
600to about 3 time, anaerobically fermenting in the serum bottle being forwarded to containing anaerobically fermenting substratum by inoculum size 10%, logical CO
2two minutes, and at 37 DEG C, 200 r/min anaerobically fermentings 48 hours.
Two-stage substratum is: LB(peptone 10g/L, yeast powder 5g/L, NaCl 5 g/L)+Amp (penbritin 100 μ g/mL)+Chl (paraxin 25 μ g/mL)+Kar (kantlex 30 μ g/mL)
Anaerobic stages substratum is: citric acid 3 gL
-1, Na
2hPO
412H
2o 4 gL
-1, KH
2pO
48 gL
-1, (NH
4)
2hPO
48 gL
-1, NH
4cl 0.2 gL
-1, (NH
4)
2sO
40.75 gL
-1, MgSO
47H
2o 1 gL
-1, CaCl
22H
2o 10.0 mgL
-1, ZnSO
47H
2o 0.5 mgL
-1, CuCl
22H
2o 0.25 mgL
-1, MnSO
4h
2o 2.5 mgL
-1, CoCl
26H
2o 1.75 mgL
-1, H
3bO
30.12 mgL
-1, Al
2(SO4)
31.77 mgL
-1, Na
2moO
42H
2o 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, 20.0 mg L
-1vB
1, 2.0 mg L
-1vitamin H+glucose (30 g/L)+calcium carbonate 0.5 g/L+Amp (penbritin 100 μ g/mL)+Chl (paraxin 25 μ g/mL)+Kar (kantlex 30 μ g/mL)
Shown in fermentation results table 2, detect after fermentation culture 48 h that the output of succinic acid is 28 g/L, succinic acid transformation efficiency is 88 %, and fermenting process proves that BA601 has higher succinic acid-producing ability in pH5.6 fermention medium.
Table 3 colon bacillus (
escherichia coli) BA601 utilizes sucrose to ferment the ability of succinic acid-producing
?
embodiment 5
The present embodiment explanation colon bacillus (
escherichia coli) BA601 ferments the ability of succinic acid-producing in pH 5.6 fermention medium.
Colon bacillus (
escherichia coli) seed liquor access is equipped with in 3 L fermentor tanks of 1.5 L fermention mediums, containing the glucose of 30 g/L in substratum by the inoculum size of 10% by BA501.37 DEG C of aerobic residual sugars be cultured in substratum are less than 0.5 g/L, 37 DEG C, 200 r/min continue to pass into CO with the speed of 0.5 L/min
2, carry out anaerobically fermenting.
Seed culture medium: LB(peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L)+Amp (penbritin 100 μ g/mL)+Chl (paraxin 25 μ g/mL)+Kar (kantlex 30 μ g/mL)
Fermention medium: citric acid 3 gL
-1, Na
2hPO
412H
2o 4 gL
-1, KH
2pO
48 gL
-1, (NH
4)
2hPO
48 gL
-1, NH
4cl 0.2 gL
-1, (NH
4)
2sO
40.75 gL
-1, MgSO
47H
2o 1 gL
-1, CaCl
22H
2o 10.0 mgL
-1, ZnSO
47H
2o 0.5 mgL
-1, CuCl
22H
2o 0.25 mgL
-1, MnSO
4h
2o 2.5 mgL
-1, CoCl
26H
2o 1.75 mgL
-1, H
3bO
30.12 mgL
-1, Al
2(SO
4)
31.77 mgL
-1, Na
2moO
42H
2o 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, 20.0 mg L
-1vB, 2.0 mgL
-1vitamin H+glucose (65 g/L)+Amp (penbritin 100 μ g/mL)+Chl (paraxin 25 μ g/mL)+Kar (kantlex 30 μ g/mL)
As shown in Figure 4, detect after fermentation culture 48 h that the output of succinic acid is 41 g/L, succinic acid transformation efficiency is 96% to fermentation results, and fermenting process proves that BA601 can grow and the efficient succinic acid-producing that ferments in the fermention medium of pH 5.6.
Claims (8)
1. a strain succinic acid-producing genetically engineered bacteria strain, its Classification And Nomenclature is colon bacillus BA601(
escherichia colibA601), its preserving number is numbered CCTCC NO:M2014210.
2. colon bacillus BA601(according to claim 1
escherichia colibA601) construction process, is characterized in that: to lack lactate dehydrogenase gene, pyruvate formate-lyase gene activity and phosphotransferase system
ptsGthe intestinal bacteria of karyomit(e) generation spontaneous mutation are starting strain, after utilizing process LAN L-Glutamic decarboxylase, γ-aminobutyric acid antiporter gene, cultivate through continuous domestication, and obtaining can high-yield succinic colon bacillus BA601 at low ph values.
3. colon bacillus BA601 construction process according to claim 2, is characterized in that concrete steps are as follows:
Utilize CaCl
2legal system is standby lacks lactate dehydrogenase gene, pyruvate formate-lyase gene activity and phosphotransferase system
ptsGkaryomit(e) generation spontaneous mutation
e.colicompetence bacterial strain;
Purifying amplifies and comprises its acidproof original paper
gadthe L-Glutamic decarboxylase of system, L-glutamic acid-γ-aminobutyric acid antiporter gene
gadBCplasmid;
By the competence bacterial strain that the plasmid described in step (2) utilizes thermal shock method steps for importing (1) to obtain, obtain positive transformant;
(4) positive transformant of step (3) is utilized to express resistance to acid gene
gadBC, obtain a strain and to ferment at low ph values the colon bacillus BA600(of succinic acid-producing
escherichia colibA600);
(5) colon bacillus BA600 is after continuous domestication is cultivated, utilize the solid medium of low ph value dull and stereotyped, screening obtains the mutant strain that can grow fast, then to obtain through the screening of anaerobism shake flask fermentation the bacterial strain of efficient succinic acid-producing to be at the low ph aimed strain colon bacillus BA601(
escherichia colibA601).
4. utilize the method for the colon bacillus BA601 fermentation production of succinic acid described in claim 2, it is characterized in that adopting two benches fermentation mode, two-stage improves biomass, anaerobic stages fermentation and acid.
5. method according to claim 4, it is characterized in that colon bacillus BA601 is inoculated aerobic in two-stage fermention medium by 1% (v/v) inoculum size to be cultivated, aerobic cultivates thalline to OD
600when=3, be forwarded in the serum bottle containing anaerobic stages fermention medium by inoculum size 10%, fill CO
2two minutes, and at 37 DEG C, 200 r/min anaerobically fermenting 48 h.
6. method according to claim 4, it is characterized in that by colon bacillus BA601 by 10% inoculum size by seed liquor access be equipped with in 3 L fermentor tanks of 1.5 L fermention mediums, 37 DEG C of aerobics are cultured to thalli growth and reach stationary phase, 37 DEG C, 200 r/min continue to pass into CO with the speed of 0.5 L/min
2, carry out anaerobically fermenting.
7. the method according to claim 5 or 6, is characterized in that described anaerobic stages fermention medium maintains pH 5.6.
8. method according to claim 6, is characterized in that two-stage fermention medium maintains pH5.6.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676052A (en) * | 2017-03-21 | 2017-05-17 | 南京工业大学 | Method for constructing succinic-acid-producing Escherichia coli and application of Escherichia coli |
| CN111411119A (en) * | 2020-03-13 | 2020-07-14 | 南京凯诺生物科技有限公司 | Construction and application of recombinant escherichia coli for coupling production of pentanediamine and succinic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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2014
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
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| 丁秋露: "大肠杆菌抗酸机制研究进展", 《淮南师范学院学报》, vol. 10, no. 5, 30 September 2008 (2008-09-30), pages 23 - 25 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676052A (en) * | 2017-03-21 | 2017-05-17 | 南京工业大学 | Method for constructing succinic-acid-producing Escherichia coli and application of Escherichia coli |
| CN111411119A (en) * | 2020-03-13 | 2020-07-14 | 南京凯诺生物科技有限公司 | Construction and application of recombinant escherichia coli for coupling production of pentanediamine and succinic acid |
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