CN105543214A - Construction method and applications of metabolic engineering escherichia coli strain for producing succinic acid by using acetic acid - Google Patents
Construction method and applications of metabolic engineering escherichia coli strain for producing succinic acid by using acetic acid Download PDFInfo
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Abstract
The present invention discloses a construction method and applications of a metabolic engineering escherichia coli strain for producing succinic acid by using acetic acid, wherein escherichia coli transformed through metabolic engineering uses acetic acid as a raw material to perform fermented production of succinic acid, and the transformation pathway comprises: blocking TCA cycle, and/or blocking succinic acid utilization pathway, and/or enhancing acetic acid uptake and oxaloacetic acid supply, strengthening glyoxylic acid cycle, and/or deleting by-product generation pathway, and/or reducing futile cycle caused by acetic acid production using pyruvic acid decarboxylation, and/or dredging acetyl CoA node metabolic flow. According to the present invention, through the analysis on the metabolic pathway and the regulation, the escherichia coli is transformed by using the genetic engineering way, the obtained strain can produce succinic acid in the culture medium adopting acetic acid as the carbon source, and no by-product is generated.
Description
Technical field
The invention belongs to technical field of bioengineering, more particularly, relate to the recombinant escherichia coli strain building and utilize acetic acid production succinic acid, and be that main raw material utilizes this metabolic engineering Escherichia coli fermentation synthesizing succinic acid by acetic acid.
Background technology
Succinic acid, has another name called succsinic acid, is a kind of four carbon dicarboxylic acid, is widely used in agricultural, food and pharmaceutical industries.Succinic acid is the precursor of many essential industry chemical, comprises adipic acid, BDO, tetrahydrofuran (THF), N-Methyl pyrrolidone, 2-Pyrrolidone, succinate and gamma-butyrolactone.Also can be used for the polymer poly succinic acid-butanediol ester (PBS) of synthesizing biological degradable and polyamide polymer (
x, 4).At present, business succinic acid is produced through chemosynthesis by liquefied petroleum gas (LPG) or mineral oil, change biological fermentation into and produce (Catal.Today (2014), http://dx.doi.org/10.1016/j.cattod.2014.05.035), utilize renewable resources to produce succinic acid by biological fermentation process and have more economic and social benefit than refining of petroleum, therefore extensive concern (Enzymeandmicrobialtechnology is subject to, 2006,39 (3): 352-361).
Succinic acid is produced bacterial strain and is mainly succinic acid-producing anaerobism spirillum Anaerobiospirillumsucciniciproducens, succinic acid-producing actinobacillus Actinobacillussuccinogenes, mannheim succinic acid-producing bacterium Mannheimiasucciniciproducens, recombination bacillus coli Escherichiacoli, Saccharomyces Cerevisiae in S accharomycescerevisiae (MetabEng, 2010,12 (6): 518-525), Corynebacterium glutamicum Corynebacteriumglutamicum (PLoSOne, 2013,8 (4): e60659) etc.Wherein intestinal bacteria due to gene genetic background clear, genetic manipulation is simple, and fermentation substrate mostly is renewable resources simultaneously, as glucose, wood sugar, glycerine, the lignocellulose such as wheat straw, maize straw, the starchy material such as cassava, jerusalem artichoke, and have more industrial value.
Utilize recombination bacillus coli to ferment the production of succinic acid, extensive work is transformed for the pathways metabolism of glucose, is included in glucose under aerobic and anaerobic conditions and guides to the metabolic fluxes of succinic acid.(the BiotechnolBioeng such as San, 2005,90 (6): 775-779) the intestinal bacteria HL27658k (pKK313) built, block TCA circulation, excision by product forming feature, removes glyoxylate cycle and checks, and realizes glucose aerobic conversion accumulation succinic acid 58.3g/L, yield is 0.94 ± 0.07mol/mol glucose, and production intensity is 1.08 ± 0.06g/ (L.h).(the JournalofIndustrialMicrobiology & Biotechnology such as Zhao, 2013,40 (12): 1461-1475) the intestinal bacteria E2-Δ sdh-ppc-sucAB built, block TCA circulation and process LAN phosphoric acid enol pyruvic acid carboxylase, succinic thiokinase transformation under, in the basic salt culture medium of glycerine, maximum volume throughput rate and the average volume production speed of succinic acid are respectively 19.2 and 6.55mMh
-1.
Acetic acid is prepared primarily of the carbonylation of methyl alcohol, i.e. methyl alcohol and carbon monoxide preparation, and be a kind of carbon source of cheapness in a large number, be also one of Main By product formed in ligno-cellulosic materials acid hydrolysis simultaneously.Acetic acid is the end product of many microbiological anaerobic metabolism and the incomplete oxidation product of aerobic metabolism, intestinal bacteria can grow by aerobic metabolise acetic acid, and when taking acetic acid as sole carbon source aerobic growth, the key enzyme activity of glyoxylate cycle and glyconeogenesis approach can be significantly improved, be conducive to carrying out subsequently glucose and transform (AppliedandEnvironmentalMicrobiology to the anaerobism of succinic acid, 2007,73 (24): 7837-7843.).But not yet have so far and adopt acetic acid to report as the research of the direct biosynthesizing succinic acid of carbon source.The present invention from enhancing substrate uptake, block downstream katabolism and dredge that metabolic fluxes etc. is many-sided to be transformed intestinal bacteria, can directly utilize acetic acid to produce succinic acid for carbon source.
Summary of the invention
First object of the present invention is to provide a kind of construction process utilizing the metabolic engineering coli strain of acetic acid production succinic acid.
Second object of the present invention is to provide a kind of metabolic engineering coli strain.
3rd object of the present invention is to provide a kind of metabolic engineering coli strain producing the application in succinic acid.
4th object of the present invention is to provide a kind of method utilizing metabolic engineering coli strain to produce succinic acid.
For realizing above object, the present invention discloses following technical scheme: a kind of construction process utilizing the metabolic engineering coli strain of acetic acid production succinic acid, it is characterized in that, the intestinal bacteria of metabolic engineering use acetic acid to produce succinic acid for fermenting raw materials, its rebuilding approach is for disappearance succinate dehydrogenase Complex Gene is to block TCA circulation, and and/or disappearance succinyl CoA constructive ways key gene to block succinic acid utilization ways, and and/or process LAN acetic acid picked-up gene, and/or the regulatory gene of disappearance glyoxylate cycle and TCA circulation, and/or process LAN glyoxylate cycle and TCA circulation key gene are to strengthen acetic acid picked-up and oxaloacetic acid supplies, strengthening glyoxylate cycle, and/or the decarboxylic reaction of minimizing oxysuccinic acid is to lack by product constructive ways, and/or reduce the inefficient cycle caused from pyruvate decarboxylation production acetic acid, and/or disappearance lactic acid and alcohol production approach in key gene to dredge acetyl-CoA node metabolic fluxes.
As a preferred version, its rebuilding approach is: at least comprise
(1) sdhAB is lacked
And one or more in following approach:
(2) sucABCD and/or fumC is lacked;
(3) process LAN acs and/or ackA;
(4) iclR, fadR and/or arcA is lacked;
(5) process LAN 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 version, its rebuilding approach is: disappearance sdhAB, disappearance iclR and process LAN gltA.
As a preferred version, its rebuilding approach is: disappearance sdhAB, disappearance iclR, disappearance maeB, process LAN gltA.
As a preferred version, its rebuilding approach is: disappearance sdhAB, disappearance iclR, disappearance maeB, process LAN aceA and process LAN gltA.
As a preferred version, its rebuilding approach is: disappearance sdhAB, disappearance iclR, disappearance maeB, process LAN acs and process LAN gltA.
As a preferred version, its rebuilding approach is: disappearance sdhAB, disappearance iclR, disappearance maeB, process LAN aceA, process LAN acs and process LAN gltA.
For realizing the present invention's second object, the present invention discloses following technical scheme: utilize the metabolic engineering coli strain that above-mentioned construction process obtains.
For realizing the present invention's the 3rd object, the present invention discloses following technical scheme: the metabolic engineering coli strain utilizing above-mentioned construction process to obtain is producing the application in succinic acid.
For realizing the present invention's the 4th object, the present invention discloses following technical scheme: a kind of method utilizing above-mentioned metabolic engineering coli strain to produce succinic acid, it is characterized in that, utilization is that the intestinal bacteria of primary carbon source commonly use substratum with acetic acid, metabolic engineering intestinal bacteria described in fermentation claim 3, obtain succinic acid.
The present invention builds the metabolic engineering intestinal bacteria of efficiency utilization acetic acid production succinic acid, namely according to existing information, E. coli pathway is analyzed, genetic engineering means is utilized to transform bacillus coli gene, and its metabolic condition is analyzed, the metabolic engineering intestinal bacteria of succinic acid can be produced under obtaining aerobic condition using acetic acid as carbon source, and utilize improved metabolic engineering bacterial strain to take acetic acid as raw material production succinic acid.
Method of the present invention: (1) adopts molecular biology method, acetic acid picked-up approach and glyoxylate cycle are strengthened, uses carrier pTrc99a and pBAD33 to express glyoxylate cycle key gene gltA, aceA, acnB and acetic acid picked-up gene acs and ackA respectively; (2) red gene recombination technology is utilized to obtain the host e. coli of single disappearance such as gene sdhAB, iclR, arcA, ldhA, sfcA, maeB, sucABCD, poxB, adhE, fadR and fumC or combination disappearance; Combination (1) and (2) builds succinic acid and produces intestinal bacteria.
The present invention is for starting strain (embodiment be that wild type strain is for starting strain) with bacterial strains of wild-type e. coli or other transformations existing, disappearance succinate dehydrogenase Complex Gene sdhAB on the one hand, blocks TCA circulation to reach the object of accumulation succinic acid.Acetic acid is converted into acetyl-CoA after entering cell, acetyl-CoA by TCA circulation and glyoxylate cycle two kinds of approach by metabolism.So the regulatory gene iclR, the arcA that lack glyoxylate cycle and TCA circulation further can be selected, and or process LAN aceA, gltA, acnB.Disappearance sfcA and maeB can reduce the decarboxylic reaction of oxysuccinic acid, maintains the supply of oxaloacetic acid.Disappearance poxB reduces the inefficient cycle caused from pyruvate decarboxylation production acetic acid.Meanwhile, the by product the way of production of lactic acid and ethanol is lacked.Another aspect of the present invention is the flux strengthening acetic acid utilization ways.Acetic acid is utilized by acetyl-CoA-synthetase and ack-pta two kinds of approach, and research process LAN acs and ackA is on the impact of Metabolism of E. coli acetic acid.
The invention has the advantages that: the present invention is by the analysis to pathways metabolism and regulation and control, and utilize genetic engineering means to transform intestinal bacteria, the bacterial strain of acquisition, in the substratum taking acetic acid as carbon source, can produce succinic acid, and not have by product to produce.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.The experimental technique used in following embodiment if no special instructions, is ordinary method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the condition described in " Molecular Cloning: A Laboratory guide " that such as J. Pehanorm Brooker (JosephSambrook) etc. are write, or test according to the condition that manufacturer is recommended.
The present invention is analyzed by pathways metabolism, in order to add the utilization of strength acetic acid, needs to transform acetic acid picked-up approach.Adopt pBAD33 as carrier process LAN acs and ackA, use restriction enzyme site to connect.
Because acetic acid carries out metabolism mainly through glyoxylate cycle, need the metabolic flux strengthening flowing to glyoxylate cycle.On the one hand, need the regulatory gene knocking out glyoxylate cycle and TCA circulation, remove regulatory gene to the suppression of metabolic fluxes, namely knock out iclR, fadR, arcA.On the other hand, the key gene of process LAN glyoxylate cycle and TCA circulation, i.e. process LAN gltA, aceA and acnB, adopts pTrc99a as carrier, uses restriction enzyme site to connect.
The efficient accumulation of succsinic acid, needs to block TCA circulation, namely knocks out gene sdhAB, fumC.In addition, also need the aerobic degradation approach blocking succsinic acid, i.e. succinyl CoA constructive ways, therefore knock out sucABCD.
For reducing the decarboxylic reaction of oxysuccinic acid, maintain the supply of oxaloacetic acid, disappearance sfcA and maeB.
Avoid generating acetic acid by pyruvate decarboxylation, form inefficient cycle, disappearance poxB.
Knock out possible metabolic by-prods approach, make metabolic fluxes flow to target product, avoid the generation of metabolic by-prods simultaneously, save downstream separation cost, so knock out key gene ldhA and adhE in lactic acid and ethanol the way of production.
In above-mentioned 1-8 item, each is all as the direction of transformation, but is not all transform in each.Multiple combination is only to improve production efficiency and yield.
The method of Red gene recombination is adopted to build genetically deficient recombination bacillus coli (FoliaMicrobiologica, 2011,56 (3): 253-263).Use the primer with target gene 5 0bp homology arm, lack bacterium for template amplification both sides are with the kan resistance gene fragment in FRT site with the list of pKD4 or corresponding gene.Proceed to helper plasmid pKD46 in Host Strains, assist exo, bet, gam gene of homologous recombination at 30 DEG C with L-arabinose abduction delivering, at 42 DEG C, cultivate for some time plasmid loss.Electricity transforms and impels gene generation homologous recombination, goal gene is replaced with kan resistant gene.Finally proceed to the plasmid pCP20 that high temperature induction expresses Flippases recombinase (FLP), eliminate the resistant gene of homologous recombination on karyomit(e), this plasmid is responsive to temperature type penbritin and chlorampenicol resistant plasmid, induces the generation of FLP enzyme when 42 DEG C, simultaneously plasmid loss.
Embodiment 1. knocks out gene and produces bacterial strain to obtain succinic acid
Red recombinant technology is adopted to knock out gene sdhAB, iclR, arcA, ldhA, sfcA, maeB, sucABCD, poxB, adhE, fumC, fadR.Concrete operations are as follows:
For knocking out of gene sdhAB, first with the primer 2 (R-sdhAB) of sequence to be the primer 1 (F-sdhAB) of SEQIDNO:1 and sequence be SEQIDNO:2, take pKD4 as the DNA fragmentation that template PCR clones about 1700bp.In Host Strains, calcium transforms and imports plasmid pKD46, with penbritin screening recon.The recombinant bacterium 30 DEG C importing pKD46 is cultured to OD
600when being about 0.3, adding L-arabinose and induce 1 hour, then use the glycerine of 10% to prepare electricity and turn competence.Electricity transforms and adopts bacterium pattern 1 (1.8KV, 5ms) to carry out.Use kantlex screening that the transformant of homologous recombination occurs.The primer 3 (F-sdhAB-check) that uses sequence to be SEQIDNO:3 subsequently and sequence are that the primer 4 (R-sdhAB-check) of SEQIDNO:4 carries out bacterium colony PCR, knock out the object clip size that successful transformant and wild mushroom PCR obtain variant, can whether recombinate successfully by preliminary judgement.PCR primer send order-checking, and wild mushroom pcr gene band is identical with sdhAB gene order, and recombinant bacterium pcr gene band middle portion is identical with kan gene order, then prove that gene knocks out successfully really.
SdhAB knocks out successful recombinant bacterium, cultivates after 5-6 hour, proceeds to 42 DEG C of overnight incubation, be separated single bacterium colony, then verify resistance for 37 DEG C.Only has kalamycin resistance without the bacterium colony eliminated for pKD46 of amicillin resistance.Then in this bacterium, proceed to plasmid pCP20, after 30 DEG C of cultivation for some time, proceed to 42 DEG C of overnight incubation, be separated single bacterium colony, then verify resistance.Picking is only at non-resistant grow on plates, and kalamycin resistance is dull and stereotyped and bacterium colony that amicillin resistance flat board does not all grow is identified.The primer 3 (F-sdhAB-check) that use sequence is SEQIDNO:3 and sequence are that the primer 4 (R-sdhAB-check) of SEQIDNO:4 carries out bacterium colony PCR, resistance eliminates the object clip size that successful transformant and wild mushroom PCR obtain notable difference, can judge whether resistant gene is eliminated and recombinate successfully.
For iclR, arcA gene, knock out successively according to the method described above, the primer is in table 3.The bacterial strain obtained after gene knockout is in table 1.
For knocking out of gene maeB, from CGSC (http://cgsc.biology.yale.edu/index.php) strain library, search the single-gene disappearance bacterium of phase, obtain bacterial strain JW2447-5.Then the primer 13 (F-maeB-check) that uses sequence to be SEQIDNO:13 and sequence are the primer 14 (F-maeB-check) of SEQIDNO:14, and with JW2447-5 full-length genome for template, PCR clones kalamycin resistance gene.The preparation of other competence, electricity transform and the homogenic sdhAB knockout technique of bacterial strain screening method.
Gene ldhA, adhE, fumC, fadR, sfcA, sucABCD, poxB knock out, and identical with the knockout technique of gene maeB, the primer is in table 3.The bacterial strain obtained after gene knockout is in table 1.
Embodiment 2. process LAN glyoxylate cycle key gene gltA, aceA and acnB.
The citric acid synthesized enzyme gene gltA of Escherichia coli uses plasmid pTrc99a overexpression.First the primer 35 (pTrc99a-gltA-F (BamH1)) that uses sequence to be SEQIDNO:29 and sequence are the primer 36 (pTrc99a-gltA-R-Hind3) of SEQIDNO:30, with intestinal bacteria MG1655 for template PCR clones gltA gene with restriction enzyme site.Then the object fragment double digestion using BamH I and Hind III couple of pTrc99a and PCR to reclaim, after digestion products reclaims, uses the 16 DEG C of connections of T4 ligase enzyme.Then calcium transforms and enters in object bacterial strain.Amicillin resistance is used to screen the recombinant bacterium successfully constructed.Whether protein electrophoresis determination albumen expresses.Express correct bacterial strain and take out plasmid, send order-checking, be designated as pTrc99a-gltA.
The overexpression of aceA and acnB uses plasmid pTrc99a equally.Method is the same, is designated as pTrc99a-aceA, pTrc99a-acnB respectively.
When gltA is respectively with aceA, acnB tandem expression, connect with restriction enzyme site, the plasmid obtained is designated as pTrc99a-aceA-gltA, pTrc99a-acnB-gltA respectively.
Process LAN acs and ackA adopts carrier pBAD33, and this plasmid can be present in intestinal bacteria with pTrc99a simultaneously.The construction process of recombinant plasmid is the same.The plasmid successfully constructed is in table 2.
Table 1 bacterial classification
Table 2 plasmid
Table 3 list of primers
Embodiment 3. succinic acid-producing bacterial strain complex medium shake flask fermentation
With intestinal bacteria MG1655 for starting strain, knock out gene sdhAB, block TCA circulation, obtain producing succsinic acid basis bacterial strain MG01.
On the basis of single disappearance bacterium MG01, knock out gene iclR, fadR, arcA and fumC respectively, obtain bacterial strain MG02, MG022, MG023, MG024.Through comparing discovery, MG02 effect is optimum, therefore is next starting strain with MG02.
On the basis of two disappearance bacterium MG02, checking blocks anaplerotic sequence and activates succsinic acid is produced in TCA circulation impact on fermentation, therefore knocks out gene arcA, maeB, sfcA respectively again, obtains bacterial strain MG03, MG032, MG033.Fermentation results shows, MG032 effect is optimum, therefore carries out subsequent adaptation based on MG032.
For understanding disappearance by product constructive ways to the impact of succinate fermentative, knocking out gene poxB, adhE, ldhA further again, having obtained bacterial strain MG042, MG043, MG044.Because gene arcA remarkably influenced succsinic acid is to the yield of acetic acid, therefore knock out arcA in bacterial strain MG032, construct bacterial strain MG04.
The basis of MG042 knocks out gene arcA, obtains bacterial strain MG05, knock out gene adh E and ldhA and obtain bacterial strain MG06.
After above-mentioned bacterial strains has built, be stored in (25%v/v) in glycerine.And each deletion mycopremna of various Plastid transformation that application build completes, obtain the recombinant bacterium of each gene of process LAN.Shake flask fermentation verifies the leavening property of each bacterial strain.
Use the LB adding 10g/l sodium acetate to cultivate and cultivate based in 250ml shaking flask the performance evaluating recombinant bacterium.Culture temperature 37 DEG C, rotating speed 200rpm.Liquid amount 50ml.Add kantlex or penbritin as required.With the bacterial strain of recombinant plasmid, cultivate 2 hours (OD
600be about 0.4-0.6) after, add 1mMIPTG induction.
Cultivation terminates rear sampling, 12000rpm centrifugal 10 minutes separating thallus and supernatant.After fermented liquid supernatant is suitably diluted, through 0.22um micro-pore-film filtration, adopt the organic acid in Japanese Shimadzu high performance liquid chromatograph monitoring fermented liquid supernatant.Chromatographic column is BioRadAminexHPX-87 ion chromatographic column (300mm*7.8mm), is furnished with UV detector and differential refraction detector.Moving phase is the H of 5mM
2sO
4, flow velocity 0.6ml/min, column temperature 65 DEG C.
Light absorption value under the mensuration employing spectrophotometer method mensuration 600nm of biomass.
Under this culture condition, wild mushroom MG1655 consumes 7.11g/l acetic acid, does not have succsinic acid to produce.
Bacterial strain MG1655pTrc99a-gltA under this culture condition, does not have succsinic acid to produce.
Under this culture condition, bacterial strain MG02 consumes 0.76g/l acetic acid, and generate 0.01g/l succinic acid, yield is 0.01mol/mol acetic acid.
And bacterial strain MG02/pTrc99a-gltA consumes 3.31g/l acetic acid under this culture condition, generate 0.85g/l succinic acid, yield is 0.131mol/mol acetic acid.
Table 4 different strain complex medium shake flask fermentation result
ND: product do not detected.
Succinic acid is produced in the bio-transformation of embodiment 4. acetic acid
In order to improve the throughput rate of succsinic acid, biotransformation method shake-flask culture is adopted to verify each fermenting performance.Concrete operations are as follows:
The seed be kept in glycerine pipe is inoculated and overnight incubation in the test tube of 3mlLB is housed, then be transferred in the Erlenmeyer flask that 50mlLB substratum is housed, inoculum size 2%, strain culturing containing plasmid within about 2 hours, add 1mMIPTG induction (or transfer in containing 10g/l glucose M9 substratum in, inoculum size 4%, cultivates after 5 hours and adds 1mMIPTG induction).Induce after 5 hours, the centrifugal 10min of 8000rpm collects thalline.Basic salt culture medium washing once.Thalline is resuspended in the M9 substratum (namely not adding ammonium chloride in substratum) in nonnitrogenous source, and carbon source is the sodium acetate of 5-10g/l.Suitable microbiotic and IPTG is added as required in substratum.Culture condition is 37 DEG C, 220rpm.
M9 substratum consists of (often liter): Na
2hPO
412H
2o15.12g, KH
2pO
43g, NaCl0.5g, MgSO
47H
2o0.5g, CaCl
20.011g, ammonium chloride 1g, 1% vitamins B
10.2mL and trace element (TE) mixed solution 0.2mL.Trace element (TE) mixed solution consists of (often liter): Na
2moO
42H
2o2.0g, FeSO
47H
2o80g, MnSO
4h
2o10g, ZnSO
47H
2o2.0g, CoCl
24.0g, CuCl
22H
2o1.0g, H
3bO
40.5g, AlCl
36H
2o10g.
The mensuration of organic acid measuring method and biomass is with embodiment 3.
Shake flask fermentation result as shown in Table 5,6.
From table 5, when carrying out transformation experiment with LB cultivation thalline, wild-type e. coli MG1655 only produces 0.024g/l succsinic acid, and yield is only 0.003mol/mol acetic acid.
Through transformation, singly knock out the bacterial strain MG01 that gene sdhAB obtains, succinic acid production rises to 0.16g/l, and yield rises to 0.047mol/mol acetic acid.Knock out two deletion mycopremna MG02 that gene sdhAB and iclR obtains, the output of succsinic acid has slight rising, and be 0.19g/l, yield does not obviously change.
And three deletion mycopremna MG032, on MG02 basis, namely knock out the bacterial strain that gene maeB obtains further, consume 1.98g/l acetic acid, generate 0.75g/l succsinic acid, output increased 30 times, yield reaches 0.194mol/mol acetic acid.
On the basis of MG032, knock out gene poxB further and obtain bacterial strain MG042, compare wild mushroom MG1655, the output of succsinic acid and yield all have obvious rising, are respectively 0.67g/l and 0.169mol/mol, but are not better than bacterial strain MG032.
The growth of five deletion mycopremna MG05 is subject to obvious suppression, and the output of succsinic acid is 0.42g/l, and yield obviously rises, and is 0.421mol/mol acetic acid, close to theoretical yield 0.5mol/mol.
On the basis of bacterial strain MG02, express citric acid synthesized enzyme gene gltA, obtain bacterial strain MG02pTrc99a-gltA (see table 6), this bacterial strain consumes 1.9g/l acetic acid, generate 0.80g/l succsinic acid, relative to wild mushroom MG1655, output increased 33.3 times, yield brings up to 0.214mol/mol acetic acid further.
Optimum combination MG032pTrc99a-gltA, consumes 2.7g/l acetic acid, and generate 1.50g/l succsinic acid, yield is 0.283mol/mol acetic acid.
Optimum combination MG032pTrc99a-aceA-gltA, consumes 3.5g/l acetic acid, and generate 2.2g/l succsinic acid, yield is 0.320mol/mol acetic acid.
Optimum combination MG032pTrc99a-aceA-gltApBAD-acs, consumes 3.6g/l acetic acid, and generate 2.6g/l succsinic acid, yield is 0.367mol/mol acetic acid.
Other strain fermentations the results are shown in Table 5,6.
Except using LB for except culture medium culturing thalline, also can use with glucose is that the basic salt culture medium of carbon source cultivates thalline.Fermentation results is as shown in table 7.
Under this culture condition, bacterial strain MG02pTrc99a-gltA consumes acetic acid 2.34g/l, and produce succsinic acid 0.84g/l, yield is 0.184mol/mol.Bacterial strain MG02pTrc99a-gltA consumes acetic acid 1.53g/l, and produce succsinic acid 0.97g/l, yield is 0.322mol/mol.
In sum, the recombination bacillus coli of acquisition can be that carbon source produces succinic acid with acetic acid.
Table 5 is cultivated thalline with LB and is carried out transformation experiment shake flask results 1
Table 6 is cultivated thalline with LB and is carried out transformation experiment shake flask results 2
Table 7 is cultivated thalline with glucose and is carried out transformation experiment shake flask results
The fermentation of embodiment 5. succinic acid-producing bacterial strain in fed-batch fermentation tank
Use the mode of fed batch cultivation, in 3L fermentor tank, cultivate the leavening property evaluating above-mentioned succinic acid-producing bacterium.
Fermention medium is: 10g/l peptone, 5g/l yeast extractive substance, 10g/l sodium-chlor, 10g/l sodium acetate.
During bacterial strain preculture, use the small test tube that 3mlLB substratum is housed, cultivate 9 hours for 37 DEG C, gained culture is as first order seed.Then first order seed is transferred in the triangular flask that 30mlLB is housed, inoculum size 2%, cultivate 2 hours and add 1mMIPTG and induce 7 hours as secondary seed.Fermentation is started in secondary seed access 3L fermentor tank.Leavening temperature 37 DEG C, inoculum size 2%, regulates and stirs and ventilate to keep dissolved oxygen higher than 30% saturated oxygen concentration.4M sodium hydroxide and 2M sulphur acid for adjusting pH is used to be 7.0.Fermentation is initial, adds suitable microbiotic as required.Culture is cultured to OD600 when being about 2, adds 1mMIPTG induction.Add acetic acid in fermenting process, control residual acetic acid concentration not higher than 5g/l.
Bacterial strain MG032pTrc99a-gltA ferments 56 hours, and thalli growth is 10.2 to OD600, produces succsinic acid 5g/l.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. one kind utilizes the construction process of the metabolic engineering coli strain of acetic acid production succinic acid, it is characterized in that, the intestinal bacteria of metabolic engineering use acetic acid to produce succinic acid for fermenting raw materials, its rebuilding approach is for disappearance succinate dehydrogenase Complex Gene is to block TCA circulation, and and/or disappearance succinyl CoA constructive ways key gene to block succinic acid utilization ways, and and/or process LAN acetic acid picked-up gene, and/or the regulatory gene of disappearance glyoxylate cycle and TCA circulation, and/or process LAN glyoxylate cycle and TCA circulation key gene are to strengthen acetic acid picked-up and oxaloacetic acid supplies, strengthening glyoxylate cycle, and/or the decarboxylic reaction of minimizing oxysuccinic acid is to lack by product constructive ways, and/or reduce the inefficient cycle caused from pyruvate decarboxylation production acetic acid, and/or disappearance lactic acid and alcohol production approach in key gene to dredge acetyl-CoA node metabolic fluxes.
2. a kind of construction process utilizing the metabolic engineering coli strain of acetic acid production succinic acid according to claim 1, it is characterized in that, its rebuilding approach is: at least comprise
(1) sdhAB is lacked
And one or more in following approach:
(2) sucABCD and/or fumC is lacked;
(3) process LAN acs and/or ackA;
(4) iclR, fadR and/or arcA is lacked;
(5) process LAN 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.
3. a kind of construction process utilizing the metabolic engineering coli strain of acetic acid production succinic acid according to claim 2, it is characterized in that, its rebuilding approach is:
Disappearance sdhAB, disappearance iclR and process LAN gltA.
4. a kind of construction process utilizing the metabolic engineering coli strain of acetic acid production succinic acid according to claim 2, it is characterized in that, its rebuilding approach is:
Disappearance sdhAB, disappearance iclR, disappearance maeB and process LAN gltA.
5. a kind of construction process utilizing the metabolic engineering coli strain of acetic acid production succinic acid according to claim 2, it is characterized in that, its rebuilding approach is:
Disappearance sdhAB, disappearance iclR, disappearance maeB, process LAN aceA and process LAN gltA.
6. a kind of construction process utilizing the metabolic engineering coli strain of acetic acid production succinic acid according to claim 2, it is characterized in that, its rebuilding approach is:
Disappearance sdhAB, disappearance iclR, disappearance maeB, process LAN acs and process LAN gltA.
7. a kind of construction process utilizing the metabolic engineering coli strain of acetic acid production succinic acid according to claim 2, it is characterized in that, its rebuilding approach is:
Disappearance sdhAB, disappearance iclR, disappearance maeB, process LAN aceA, process LAN acs and process LAN gltA.
8. utilize the metabolic engineering coli strain that the construction process described in claim 1 or 2 obtains.
9. the metabolic engineering coli strain utilizing the construction process described in claim 1 or 2 to obtain is producing the application in succinic acid.
10. utilize metabolic engineering coli strain described in claim 5 to produce a method for succinic acid, it is characterized in that, utilizing with acetic acid is that the intestinal bacteria of primary carbon source commonly use substratum, and metabolic engineering intestinal bacteria described in fermentation claim 3, obtain succinic acid.
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| PCT/CN2014/094215 WO2016065709A1 (en) | 2014-10-30 | 2014-12-18 | Construction method and use of metabolically engineered e. coli strains for producing succinic acid by using acetic acid |
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| CN107881186A (en) * | 2017-10-18 | 2018-04-06 | 华东理工大学 | Construction method and application using the metabolic engineering coli strain of acetic acid production hydracrylic acid |
| CN107868795B (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 acetone or isopropanol by using acetic acid |
| 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 |
| CN107841515A (en) * | 2017-11-17 | 2018-03-27 | 华东理工大学 | A kind of method using papermaking wastewater biosynthesis succinic 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 |
| CN110656075A (en) * | 2018-06-28 | 2020-01-07 | 中国科学院青岛生物能源与过程研究所 | Universal chassis cell for synthesizing acetyl coenzyme A derived product and construction method and application thereof |
| CN110964680A (en) * | 2018-09-29 | 2020-04-07 | 中国石油化工股份有限公司 | Engineering strain and method for preparing farnesene by using cellulose |
| 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 |
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| CN105543214B (en) | 2019-01-08 |
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