CN105543214B - Utilize the metabolic engineering coli strain construction method of acetic acid production succinic acid and application - Google Patents
Utilize the metabolic engineering coli strain construction method of acetic acid production succinic acid and application Download PDFInfo
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Abstract
The present invention discloses construction method and the application of a kind of metabolic engineering coli strain using acetic acid production succinic acid, the Escherichia coli of metabolic engineering are that fermenting raw materials produce succinic acid using acetic acid, its rebuilding approach is to block TCA circulation, and and/or block succinic acid utilization ways, and and/or enhancing acetic acid intake and oxaloacetic acid supply, strengthen glyoxalic acid circulation, and/or missing by-product constructive ways, and/or it reduces from pyruvate decarboxylation and produces inefficient cycle caused by acetic acid, and/or dredge acetyl-CoA node metabolic fluxes.The present invention is transformed Escherichia coli by the analysis to metabolic pathway and regulation, using genetic engineering means, and the bacterial strain of acquisition can generate succinic acid in the culture medium using acetic acid as carbon source, and generate without by-product.
Description
Technical field
The invention belongs to technical field of bioengineering, more particularly, are related to the weight that building utilizes acetic acid production succinic acid
Group coli strain, and the metabolic engineering Escherichia coli fermentation synthesizing succinic acid is utilized for primary raw material by acetic acid.
Background technique
Succinic acid also known as succinic acid are a kind of four carbon dicarboxylic acids, are widely used in agricultural, food and pharmaceuticals industry.Fourth two
Acid is the precursor of many essential industry chemicals, including hexanedioic acid, 1,4-butanediol, tetrahydrofuran, N-Methyl pyrrolidone, 2-
Pyrrolidones, succinate and gamma-butyrolacton.It can also be used in the polymer poly succinic acid-butanediol ester of synthesizing biological degradable
(PBS) and polyamide polymer (x,4).Currently, business has been passed through via liquefied petroleum gas or mineral oil with succinic acid
Chemical synthesis production is changed into biofermentation production (Catal.Today (2014), http://dx.doi.org/10.1016/
J.cattod.2014.05.035), succinic acid is produced by biological fermentation process using renewable resource more to pass through than petroleum refining
Ji and social benefit, therefore receive significant attention (Enzyme and microbial technology, 2006,39 (3): 352-
361)。
It is mainly succinic acid-producing anaerobism spirillum Anaerobiospirillum that succinic acid, which produces bacterial strain,
Succiniciproducens, succinic acid-producing Actinobacillus Actinobacillus succinogenes, Mannheim succinic acid-producing
Bacterium Mannheimia succiniciproducens, recombination bacillus coli Escherichia coli, saccharomyces cerevisiae
Saccharomyce scerevisiae (Metab Eng, 2010,12 (6): 518-525), Corynebacterium glutamicum
Corynebacterium glutamicum (PLoS One, 2013,8 (4): e60659) etc..Wherein Escherichia coli are due to gene
Genetic background understands that genetic manipulation is simple, while fermentation substrate is mostly renewable resource, such as glucose, xylose, glycerol, wheat straw,
The lignocellulosics such as corn stover, the starchy materials such as cassava, jerusalem artichoke, and have more industrial value.
Using the production of recombination bacillus coli fermentation succinic acid, extensive work is transformed for the metabolic pathway of glucose, packet
The metabolic fluxes for including glucose to succinic acid under aerobic and anaerobic conditions guide.San etc. (Biotechnol Bioeng, 2005,
90 (6): 775-779) building Escherichia coli HL27658k (pKK313), block TCA circulation, cut off by-product forming feature,
It releases glyoxylate bypass to check, realizes that the aerobic conversion of glucose accumulates succinic acid 58.3g/L, yield is 0.94 ± 0.07mol/
Mol glucose, production intensity are 1.08 ± 0.06g/ (L.h).(the Journal of Industrial such as Zhao
Microbiology&Biotechnology, 2013,40 (12): 1461-1475) building Escherichia coli E2- Δ sdh-ppc-
SucAB blocks TCA circulation and is overexpressed under the transformation of phosphoric acid enol pyruvic acid carboxylase, succinic thiokinase,
In the basic salt culture medium of glycerol, the maximum volume throughput rate of succinic acid and average volume production rate are respectively 19.2 Hes
6.55mM h-1。
Acetic acid is mainly prepared by the carbonylation of methanol, i.e. methanol and carbon monoxide preparation, is a kind of a large amount of and cheap carbon
Source, while being also one of the Main By product formed in ligno-cellulosic materials sour water solution.Acetic acid is many microbiological anaerobic generations
The incomplete oxidation product of the final product thanked and aerobic metabolism, Escherichia coli can aerobic metabolism acetic acid grown, and with
When acetic acid is sole carbon source aerobic growth, the key enzyme activity of glyoxylate bypass and gluconeogenesis approach can be significantly improved, and have
Conducive to it is subsequent carry out glucose convert to the anaerobism of succinic acid (Applied and Environmental Microbiology,
2007,73(24):7837-7843.).But there has been no use acetic acid as the direct biosynthesis succinic acid of carbon source so far
Research report.The present invention from enhancing substrate uptake, block downstream catabolism and the various aspects such as dredge metabolic fluxes to large intestine bar
Bacterium is transformed, and can be directly that carbon source produces succinic acid using acetic acid.
Summary of the invention
The first purpose of this invention is to provide a kind of metabolic engineering Escherichia coli bacterium using acetic acid production succinic acid
The construction method of strain.
Second object of the present invention is to provide a kind of metabolic engineering coli strain.
Third object of the present invention is to provide a kind of metabolic engineering coli strain answering in production succinic acid
With.
Fourth object of the present invention is to provide a kind of side using metabolic engineering coli strain production succinic acid
Method.
In order to achieve the above object, the present invention discloses following technical scheme: a kind of metabolism work using acetic acid production succinic acid
The construction method of journey coli strain, which is characterized in that the Escherichia coli of metabolic engineering are fermenting raw materials using acetic acid
Succinic acid is produced, rebuilding approach is missing succinate dehydrogenase Complex Gene to block TCA to recycle, and and/or missing amber
Amber acyl CoA constructive ways key gene and/or is overexpressed acetic acid intake gene, and/or lacks to block succinic acid utilization ways
It loses the adjusting gene of glyoxalic acid circulation and TCA circulation, and/or is overexpressed glyoxalic acid circulation and TCA circulation key gene to increase
Strength acetic acid intake and oxaloacetic acid supply, strengthen glyoxalic acid circulation, and/or reduce the decarboxylic reaction of malic acid to lack by-product
Constructive ways, and/or reduce from pyruvate decarboxylation and produce inefficient cycle caused by acetic acid, and/or missing lactic acid and ethyl alcohol production
Key gene is in approach to dredge acetyl-CoA node metabolic fluxes.
As a preferred embodiment, rebuilding approach are as follows: include at least
(1) sdhAB is lacked
And one or more of following approach:
(2) sucABCD and/or fumC is lacked；
(3) it is overexpressed acs and/or ackA；
(4) iclR, fadR and/or arcA are lacked；
(5) it is overexpressed gltA and/or aceA and/or acnB；
(6) sfcA and/or maeB is lacked；
(7) poxB is lacked；
(8) adhE and/or ldhA is lacked.
As a preferred embodiment, rebuilding approach are as follows: missing sdhAB, missing iclR and overexpression gltA.
As a preferred embodiment, rebuilding approach are as follows: missing sdhAB lacks iclR, lacks maeB, is overexpressed gltA.
As a preferred embodiment, rebuilding approach are as follows: missing sdhAB lacks iclR, lacks maeB, is overexpressed aceA
With overexpression gltA.
As a preferred embodiment, rebuilding approach are as follows: missing sdhAB, lack iclR, lack maeB, be overexpressed acs and
It is overexpressed gltA.
As a preferred embodiment, rebuilding approach are as follows: missing sdhAB lacks iclR, lacks maeB, is overexpressed aceA,
It is overexpressed acs and is overexpressed gltA.
Second purpose, the present invention disclose following technical scheme to realize the present invention: being obtained using above-mentioned construction method
Metabolic engineering coli strain.
Third purpose to realize the present invention, the present invention disclose following technical scheme: being obtained using above-mentioned construction method
Application of the metabolic engineering coli strain in production succinic acid.
The 4th purpose, the present invention disclose following technical scheme to realize the present invention: a kind of big using above-mentioned metabolic engineering
The method of enterobacteria bacterial strain production succinic acid, which is characterized in that utilize using acetic acid as the common culture of the Escherichia coli of primary carbon source
Base, fermentating metabolism engineering colon bacillus, obtains succinic acid.
Present invention building efficiently utilizes the metabolic engineering Escherichia coli of acetic acid production succinic acid, i.e., according to existing information to big
Enterobacteria approach is analyzed, and is transformed using genetic engineering means to bacillus coli gene, and is carried out to its metabolic condition
Analysis is obtained the metabolic engineering Escherichia coli that can be produced succinic acid under aerobic condition using acetic acid as carbon source, and utilizes transformation
Metabolic engineering bacterial strain afterwards produces succinic acid by raw material of acetic acid.
Method of the invention: (1) using molecular biology method, absorbs approach to acetic acid and glyoxalic acid circulation adds
By force, it is taken the photograph respectively using carrier pTrc99a and pBAD33 expression glyoxalic acid circulation key gene gltA, aceA, acnB and acetic acid
Take gene acs and ackA；(2) using red gene recombination technology obtain gene sdhAB, iclR, arcA, ldhA, sfcA, maeB,
The host e. coli that singly missing or combination lack such as sucABCD, poxB, adhE, fadR and fumC；Combine (1) and (2) building
Succinic acid produces Escherichia coli.
The present invention is that using wild-type e. coli or have the bacterial strains of other transformations (embodiment is wild as starting strain
Raw type bacterial strain is starting strain), succinate dehydrogenase Complex Gene sdhAB is on the one hand lacked, blocks TCA circulation to reach long-pending
The purpose of tired succinic acid.Acetic acid is converted into acetyl coenzyme A after entering cell, and acetyl coenzyme A is followed by TCA circulation and glyoxalic acid
Two kinds of approach of ring are metabolized.So can choose further missing glyoxalic acid circulation and TCA circulation adjusting gene iclR,
ArcA, and/or it is overexpressed aceA, gltA, acnB.Missing sfcA and maeB can reduce the decarboxylic reaction of malic acid, maintain oxalyl
The supply of acetic acid.Missing poxB produces inefficient cycle caused by acetic acid to reduce from pyruvate decarboxylation.Meanwhile lacking lactic acid and second
The by-product the way of production of alcohol.Another aspect of the present invention is the flux for enhancing acetic acid utilization ways.Acetic acid passes through acetyl coenzyme A
Two kinds of approach of synzyme and ack-pta are utilized, and research is overexpressed influence of the acs and ackA to Metabolism of E. coli acetic acid.
The present invention has the advantages that the present invention utilizes genetic engineering means pair by the analysis to metabolic pathway and regulation
Escherichia coli are transformed, and the bacterial strain of acquisition can generate succinic acid in the culture medium using acetic acid as carbon source, and without pair
Product generates.
Specific embodiment
Present invention will be further explained below with reference to specific examples.Experimental method used in following embodiments for example without
Specified otherwise is conventional method.The materials, reagents and the like used in the following examples unless otherwise specified can be from business way
Diameter obtains.It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.In the following example
Test method without specific conditions, usually according to normal condition, such as J. Pehanorm Brooker (Joseph Sambrook) etc.
Condition described in " Molecular Cloning:A Laboratory guide " write, or tested according to the condition that manufacturer is recommended.
The present invention is analyzed by metabolic pathway, in order to reinforce the utilization of acetic acid, needs to be transformed acetic acid intake approach.
Acs and ackA is overexpressed as carrier using pBAD33, is attached using restriction enzyme site.
Since acetic acid is mainly metabolized by glyoxalic acid circulation, the metabolic flux for reinforcing flowing to glyoxalic acid circulation is needed.
On the one hand, the adjusting gene for needing to knock out glyoxalic acid circulation and TCA circulation, releases the inhibition for adjusting gene pairs metabolic fluxes, that is, strikes
Except iclR, fadR, arcA.On the other hand, be overexpressed the key gene that glyoxalic acid recycles and TCA is recycled, i.e. overexpression gltA,
AceA and acnB is attached using pTrc99a as carrier using restriction enzyme site.
The efficient accumulation of succinic acid needs that TCA is blocked to recycle, i.e. knockout gene sdhAB, fumC.In addition, also needing to block amber
The aerobic degradation approach of amber acid, i.e. succinyl CoA constructive ways, therefore knock out sucABCD.
For the decarboxylic reaction for reducing malic acid, the supply of oxaloacetic acid is maintained, lacks sfcA and maeB.
It avoids generating acetic acid by pyruvate decarboxylation, forms inefficient cycle, lack poxB.
Possible metabolic by-product approach is knocked out, metabolic fluxes is made to flow to target product, while avoiding the life of metabolic by-product
At, downstream separation cost is saved, so knock out the key gene ldhA and adhE in lactic acid and ethyl alcohol the way of production.
Direction of each single item as transformation in 1-8 above-mentioned, but be not all of and be transformed in each single item.Multiple combinations
Merely to improving production efficiency and yield.
Using Red genetic recombination method building gene delection recombination bacillus coli (Folia Microbiologica,
2011,56(3):253-263).Using the primer for having target gene 5 0bp homology arm, with pKD4 or single missing of corresponding gene
Bacterium is the kan resistance gene fragment that template amplification two sides have the site FRT.Helper plasmid pKD46 is transferred in host strain, at 30 DEG C
Exo, bet, gam gene of homologous recombination are assisted with L-arabinose inducing expression, cultivate a period of time plasmid loss at 42 DEG C.
Electrotransformation promotes gene that homologous recombination occurs, and target gene is replaced with kan resistant gene.High temperature induction expression is finally transferred to turn over
Turn the plasmid pCP20 of enzyme recombinase (FLP), eliminates the resistant gene of homologous recombination on chromosome, which is responsive to temperature type
Ampicillin and chlorampenicol resistant plasmid induce the generation of FLP enzyme, while plasmid loss at 42 DEG C.
Embodiment 1. knocks out gene to obtain succinic acid production bacterial strain
Using red recombinant technique knock out gene sdhAB, iclR, arcA, ldhA, sfcA, maeB, sucABCD, poxB,
adhE,fumC,fadR.Concrete operations are as follows:
For the knockout of gene sdhAB, it is with the primer 1 (F-sdhAB) and sequence that sequence is SEQ ID NO:1 first
The primer 2 (R-sdhAB) of SEQ ID NO:2 clones the DNA fragmentation of about 1700bp using pKD4 as template PCR.Calcium turns in host strain
Change and import plasmid pKD46, recon is screened with ampicillin.30 DEG C of recombinant bacterium for importing pKD46 are cultivated to OD600About 0.3
When, L-arabinose is added and induces 1 hour, then prepares electricity using 10% glycerol and turns competence.Electrotransformation uses bacterium mould
Formula 1 (1.8KV, 5ms) carries out.The transformant of homologous recombination occurs using kanamycin screening.It the use of sequence is then SEQ ID
The primer 4 (R-sdhAB-check) that the primer 3 (F-sdhAB-check) and sequence of NO:3 is SEQ ID NO:4 carries out bacterium colony
PCR, it is variant to knock out the target fragment size that successful transformant and wild mushroom PCR are obtained, can preliminary judgement whether recombinate
Success.PCR product send sequencing, and wild mushroom pcr gene band is identical as sdhAB gene order, and in recombinant bacterium pcr gene band
Between part it is identical as kan gene order, then prove that gene knocks out success really.
SdhAB knocks out successful recombinant bacterium, 37 DEG C after culture 5-6 hours, is transferred to 42 DEG C of overnight incubations, separation single colonie,
Then resistance is verified.Only kalamycin resistance is bacterium colony that pKD46 has been eliminated without amicillin resistance.Then in the bacterium
In be transferred to plasmid pCP20, after 30 DEG C of culture a period of times, be transferred to 42 DEG C of overnight incubations, separate single colonie, then verify resistance.
Picking is only grown on non-resistant plate, and non-growing bacterium on kalamycin resistance plate and amicillin resistance plate
Drop into capable identification.It the use of the primer 3 (F-sdhAB-check) and sequence that sequence is SEQ ID NO:3 is drawing for SEQ ID NO:4
Object 4 (R-sdhAB-check) carries out bacterium colony PCR, and it is big that resistance eliminates the target fragment that successful transformant and wild mushroom PCR are obtained
It is small to have notable difference, it can determine whether resistant gene is eliminated and recombinate successfully.
It for iclR, arcA gene, successively knocks out according to the method described above, the primer is shown in Table 3.It is obtained after gene knockout
Bacterial strain is shown in Table 1.
For the knockout of gene maeB, from CGSC (http://cgsc.biology.yale.edu/index.php) strain
The single-gene that phase is searched in library lacks bacterium, obtains bacterial strain JW2447-5.Then the primer 13 for the use of sequence being SEQ ID NO:13
(F-maeB-check) and sequence is the primer 14 (F-maeB-check) of SEQ ID NO:14, is with JW2447-5 full-length genome
Template, PCR clone kalamycin resistance gene.The preparation of other competence, electrotransformation and the homogenic sdhAB of bacterial strain screening method strike
Except method.
The knockout of gene ldhA, adhE, fumC, fadR, sfcA, sucABCD, poxB, the knockout technique with gene maeB
Identical, the primer is shown in Table 3.The bacterial strain obtained after gene knockout is shown in Table 1.
Embodiment 2. is overexpressed glyoxalic acid and recycles key gene gltA, aceA and acnB.
The citric acid synthesized enzyme gene gltA of Escherichia coli uses plasmid pTrc99a overexpression.Sequence is used first
The primer 36 that the primer 35 (pTrc99a-gltA-F (BamH1)) and sequence for being classified as SEQ ID NO:29 are SEQ ID NO:30
(pTrc99a-gltA-R-Hind3), the gltA gene of restriction enzyme site is had using Escherichia coli MG1655 as template PCR clone.So
The target fragment double digestion recycled afterwards using BamH I and Hind III to pTrc99a and PCR is connected after digestion products recycling using T4
Connect 16 DEG C of enzyme connections.Then calcium conversion enters in purpose bacterial strain.Successful recombinant bacterium is constructed using amicillin resistance screening.
Protein electrophoresis determines whether albumen expresses.It expresses correct bacterial strain and takes out plasmid, send sequencing, be denoted as pTrc99a-gltA.
The overexpression of aceA and acnB equally uses plasmid pTrc99a.Method is same as above, and is denoted as pTrc99a- respectively
aceA、pTrc99a-acnB。
GltA respectively with aceA, acnB expressing in series when, be attached with restriction enzyme site, obtained plasmid is denoted as respectively
pTrc99a-aceA-gltA、pTrc99a-acnB-gltA。
It is overexpressed acs and ackA and uses carrier pBAD33, which can exist simultaneously in Escherichia coli with pTrc99a.
The construction method of recombinant plasmid is same as above.It constructs successful plasmid and is shown in Table 2.
1 strain of table
2 plasmid of table
3 list of primers of table
3. succinic acid-producing bacterial strain complex medium shake flask fermentation of embodiment
Using Escherichia coli MG1655 as starting strain, gene sdhAB is knocked out, blocks TCA circulation, obtains producing succinic acid basis
Bacterial strain MG01.
Singly lack bacterium MG01 on the basis of, respectively knock out gene iclR, fadR, arcA and fumC, obtain bacterial strain MG02,
MG022,MG023,MG024.Through it was found that, MG02 effect is optimal, therefore next using MG02 as starting strain.
On the basis of double missing bacterium MG02, verifying blocks anaplerotic sequence and TCA circulation is activated to produce succinic acid to fermentation
It influences, therefore knocks out gene arcA, maeB, sfcA respectively again, obtain bacterial strain MG03, MG032, MG033.Fermentation results show
MG032 effect is optimal, therefore subsequent adaptation is carried out based on MG032.
To understand influence of the missing by-product constructive ways to succinate fermentative, further knocked out gene poxB,
AdhE, ldhA obtain bacterial strain MG042, MG043, MG044.Since gene arcA significantly affects succinic acid to the yield of acetic acid, therefore
ArcA has been knocked out in bacterial strain MG032, has constructed bacterial strain MG04.
Gene arcA is knocked out on the basis of MG042, obtains bacterial strain MG05, is knocked out gene adh E and ldhA and is obtained bacterial strain
MG06。
After the completion of above-mentioned bacterial strains building, (25%v/v) is stored in glycerol.And the various plasmids conversion that application build is completed
Each deletion mycopremna obtains the recombinant bacterium for being overexpressed each gene.Shake flask fermentation verifies the fermenting property of each bacterial strain.
The performance of recombinant bacterium is evaluated based on culture in 250ml shaking flask using the LB culture of addition 10g/l sodium acetate.Culture
37 DEG C of temperature, revolving speed 200rpm.Liquid amount 50ml.Kanamycins or ampicillin are added as needed.With recombination matter
The bacterial strain of grain cultivates 2 hours (OD600About 0.4-0.6) after, 1mM IPTG induction is added.
It is sampled after culture, 12000rpm is centrifuged 10 minutes separating thallus and supernatant.After fermented liquid supernatant suitably dilutes,
Through 0.22um micro-pore-film filtration, using the organic acid in Japan shimadzu high performance liquid chromatograph monitoring fermented liquid supernatant.Chromatographic column
For BioRadAminex HPX-87 ion chromatographic column (300mm*7.8mm), it be furnished with UV detector and differential refraction detector.Stream
Dynamic is mutually the H of 5mM2SO4, flow velocity 0.6ml/min, 65 DEG C of column temperature.
The measurement of biomass is using the light absorption value under spectrophotometer method measurement 600nm.
Wild mushroom MG1655 consumes 7.11g/l acetic acid under the condition of culture, generates without succinic acid.
Bacterial strain MG1655pTrc99a-gltA under the condition of culture is generated without succinic acid.
Bacterial strain MG02 consumes 0.76g/l acetic acid under the condition of culture, generates 0.01g/l succinic acid, yield 0.01mol/
Mol acetic acid.
And bacterial strain MG02/pTrc99a-gltA consumes 3.31g/l acetic acid under the condition of culture, generates 0.85g/l succinic acid,
Yield is 0.131mol/mol acetic acid.
4 different strain complex medium shake flask fermentation result of table
ND: product is not detected.
4. acetic acid bioconversion of embodiment produces succinic acid
In order to improve the throughput rate of succinic acid, each fermenting performance is verified using biotransformation method shaking flask culture.Tool
Gymnastics is made as follows:
The seed inoculation being stored in glycerol tube and overnight incubation in the test tube equipped with 3ml LB, are then transferred to and are equipped with
In the conical flask of 50ml LB culture medium, inoculum concentration 2%, the addition 1mM IPTG induction in about 2 hours of the strain culturing containing plasmid
(or switching in the M9 culture medium of the glucose containing 10g/l, 1mM IPTG induction is added in inoculum concentration 4% after culture 5 hours).It lures
After leading 5 hours, 8000rpm is centrifuged 10min and collects thallus.Basic salt culture medium washed once.Thallus is resuspended in without nitrogen source
In M9 culture medium (not adding ammonium chloride in culture medium), carbon source is the sodium acetate of 5-10g/l.It is added as needed in culture medium
Antibiotic and IPTG appropriate.Condition of culture is 37 DEG C, 220rpm.
M9 culture medium group becomes (every liter): Na2HPO4·12H2O 15.12g, KH2PO43g, NaCl 0.5g, MgSO4·
7H2O 0.5g, CaCl20.011g, ammonium chloride 1g, 1% vitamin B10.2mL and microelement (TE) mixed liquor 0.2mL.It is micro-
Secondary element (TE) mixed liquor group becomes (every liter): Na2MoO4·2H2O 2.0g, FeSO4·7H2O 80g, MnSO4·H2O 10g,
ZnSO4·7H2O 2.0g, CoCl24.0g, CuCl2·2H2O1.0g, H3BO40.5g, AlCl3·6H2O 10g。
The measuring method of organic acid and the measurement of biomass are the same as embodiment 3.
Shake flask fermentation result is as shown in Table 5,6.
By table 5 as it can be seen that wild-type e. coli MG1655 is only generated when carrying out transformation experiment with LB culture thallus
0.024g/l succinic acid, yield are only 0.003mol/mol acetic acid.
By transformation, single bacterial strain MG01 for knocking out gene sdhAB and obtaining, succinic acid production rises to 0.16g/l, in yield
Rise to 0.047mol/mol acetic acid.Double deletion mycopremna MG02 that gene sdhAB and iclR are obtained are knocked out, the yield of succinic acid has gently
Micro- rising, is 0.19g/l, and yield is not substantially change.
And three deletion mycopremna MG032, i.e., the bacterial strain of gene maeB acquisition is further knocked out on the basis of MG02, consumption
1.98g/l acetic acid generates 0.75g/l succinic acid, and 30 times of output increased, yield reaches 0.194mol/mol acetic acid.
On the basis of MG032, further knocks out gene poxB and obtain bacterial strain MG042, compare wild mushroom MG1655, amber
The yield and yield of acid have obvious rising, respectively 0.67g/l and 0.169mol/mol, but there is no be better than bacterial strain
MG032。
The growth of five deletion mycopremna MG05 is significantly inhibited, and the yield of succinic acid is 0.42g/l, and yield it is obvious on
It rises, is 0.421mol/mol acetic acid, close to theoretical yield 0.5mol/mol.
On the basis of bacterial strain MG02, citric acid synthesized enzyme gene gltA is expressed, obtains bacterial strain MG02pTrc99a-gltA
(being shown in Table 6), the bacterial strain consume 1.9g/l acetic acid, 0.80g/l succinic acid are generated, relative to wild mushroom MG1655, output increased
33.3 times, yield is further increased to 0.214mol/mol acetic acid.
Optimum organization MG032pTrc99a-gltA consumes 2.7g/l acetic acid, generates 1.50g/l succinic acid, and yield is
0.283mol/mol acetic acid.
Optimum organization MG032pTrc99a-aceA-gltA consumes 3.5g/l acetic acid, generates 2.2g/l succinic acid, and yield is
0.320mol/mol acetic acid.
Optimum organization MG032pTrc99a-aceA-gltA pBAD-acs consumes 3.6g/l acetic acid, generates 2.6g/l amber
Acid, yield are 0.367mol/mol acetic acid.
Other strain fermentations the results are shown in Table 5,6.
In addition to use LB be culture medium culture thallus other than, also can be used using glucose as the basic salt culture medium culture of carbon source
Thallus.Fermentation results are as shown in table 7.
Under this condition of culture, bacterial strain MG02pTrc99a-gltA consumes acetic acid 2.34g/l, generates succinic acid 0.84g/l, obtains
Rate is 0.184mol/mol.Bacterial strain MG02pTrc99a-gltA consumes acetic acid 1.53g/l, generates succinic acid 0.97g/l, and yield is
0.322mol/mol。
In conclusion the recombination bacillus coli obtained can produce succinic acid by carbon source of acetic acid.
Table 5 carries out transformation experiment shake flask results 1 with LB culture thallus
Table 6 carries out transformation experiment shake flask results 2 with LB culture thallus
Table 7 carries out transformation experiment shake flask results with glucose culture thallus
Fermentation of the 5. succinic acid-producing bacterial strain of embodiment in fed-batch fermentation tank
Using the mode of fed-batch culture, cultivated in 3L fermentor to evaluate the Fermented of above-mentioned succinic acid-producing bacterium
Energy.
Fermentation medium are as follows: 10g/l peptone, 5g/l yeast extract, 10g/l sodium chloride, 10g/l sodium acetate.
When bacterial strain preculture, using the small test tube that 3ml LB culture medium is housed, 37 DEG C are cultivated 9 hours or so, gained culture
Object is as first order seed.Then first order seed is transferred in the triangular flask equipped with 30ml LB, inoculum concentration 2% is cultivated 2 hours
Left and right is added 1mM IPTG and induces 7 hours to be used as secondary seed.Start to ferment in secondary seed access 3L fermentor.Fermentation temperature
37 DEG C, inoculum concentration 2% adjusts stirring and ventilates to keep dissolved oxygen to be higher than 30% saturation oxygen concentration.Using 4M sodium hydroxide and
2M sulphur acid for adjusting pH is 7.0.Fermentation is initial, and appropriate antibiotic is added as needed.Culture culture to OD600 be 2 or so when,
1mM IPTG induction is added.Acetic acid is added in fermentation process, controls residual acetic acid concentration not higher than 5g/l.
Bacterial strain MG032pTrc99a-gltA ferments 56 hours, and thalli growth to OD600 is 10.2, generates succinic acid 5g/l.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (8)
1. a kind of construction method of the metabolic engineering coli strain using acetic acid production succinic acid, which is characterized in that it changes
Make approach are as follows: lack sdhAB, missing iclR and overexpression gltA including (1)；
And one or more of following approach:
(2) sucABCD and/or fumC is lacked；
(3) it is overexpressed acs and/or ackA；
(4) fadR and/or arcA is lacked；
(5) it is overexpressed aceA and/or acnB；
(6) sfcA and/or maeB is lacked；
(7) poxB is lacked；
(8) adhE and/or ldhA is lacked.
2. a kind of building side of metabolic engineering coli strain using acetic acid production succinic acid according to claim 1
Method, which is characterized in that its rebuilding approach are as follows: missing sdhAB lacks iclR, missing maeB and overexpression gltA.
3. a kind of building side of metabolic engineering coli strain using acetic acid production succinic acid according to claim 1
Method, which is characterized in that its rebuilding approach are as follows: missing sdhAB lacks iclR, lacks maeB, is overexpressed aceA and overexpression
gltA。
4. a kind of building side of metabolic engineering coli strain using acetic acid production succinic acid according to claim 1
Method, which is characterized in that its rebuilding approach are as follows: missing sdhAB lacks iclR, lacks maeB, is overexpressed acs and is overexpressed gltA.
5. a kind of building side of metabolic engineering coli strain using acetic acid production succinic acid according to claim 1
Method, which is characterized in that its rebuilding approach are as follows: missing sdhAB, lack iclR, lack maeB, be overexpressed aceA, be overexpressed acs and
It is overexpressed gltA.
6. the metabolic engineering coli strain obtained using any construction method of claim 1-5.
7. the metabolic engineering coli strain obtained using any construction method of claim 1-5 is in production fourth two
Application in acid.
8. a kind of method using the production succinic acid of metabolic engineering coli strain described in claim 6, which is characterized in that benefit
Culture medium is often used to the Escherichia coli that acetic acid is primary carbon source, metabolic engineering Escherichia coli described in claim 6 of fermenting obtain
Succinic acid.
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| CN107881186B (en) * | 2017-10-18 | 2021-02-02 | 华东理工大学 | Construction method and application of metabolic engineering escherichia coli strain for producing hydroxypropionic acid by using acetic acid |
| CN107841515B (en) * | 2017-11-17 | 2021-06-22 | 华东理工大学 | Method for biologically synthesizing succinic acid by using papermaking waste liquid |
| CN110656075B (en) * | 2018-06-28 | 2022-11-11 | 中国科学院青岛生物能源与过程研究所 | Universal underpan cell for synthesizing acetyl coenzyme A derivative product and construction method and application thereof |
| CN110964680B (en) * | 2018-09-29 | 2022-06-28 | 中国石油化工股份有限公司 | Engineering strain and method for preparing farnesene by using cellulose |
| CN109468257A (en) * | 2018-11-29 | 2019-03-15 | 江苏师范大学 | A kind of metabolic engineering Pseuomonas denitrifican producing succinic acid, its construction method and its application |
| CN115960808A (en) * | 2022-11-24 | 2023-04-14 | 苏州聚维元创生物科技有限公司 | Gene-knocked-out recombinant escherichia coli and construction method thereof |
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| CN101363032B (en) * | 2008-08-08 | 2011-03-30 | 山东大学 | Method for coproducing succinic acid and poly beta-hydroxybutyrate using recombination of escherichia coli |
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