CN102559736B - Gene engineering bacterial strain capable of improving yield of fumaric acid and establishing method and application thereof - Google Patents
Gene engineering bacterial strain capable of improving yield of fumaric acid and establishing method and application thereof Download PDFInfo
- Publication number
- CN102559736B CN102559736B CN201210044717.7A CN201210044717A CN102559736B CN 102559736 B CN102559736 B CN 102559736B CN 201210044717 A CN201210044717 A CN 201210044717A CN 102559736 B CN102559736 B CN 102559736B
- Authority
- CN
- China
- Prior art keywords
- ldhl
- gene
- fumaric acid
- substratum
- fragment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention discloses a gene engineering bacterial strain capable of improving yield of fumaric acid and an establishing method and application thereof. The establishing method comprises the following steps of: (1) cloning a lactate dehydrogenase gene ldhL; (2) establishing a recombinant fragment which contains a fungal promoter sequence, a reverse ldhL gene sequence and terminator sequence; (3) cloning a hygromycin-resistant gene; (4) establishing a recombinant gene fragment containing a resistant gene; (5) establishing a recombinant plasmid; (6) obtaining a recombinant Agrobacterium pus bacillus; and (7) obtaining a recombinant Rhizopus oryzae strain. The gene engineering bacterial strain capable of improving the yield of the fumaric acid can accumulate high-concentration fumaric acid through glucose fermentation under fully aerobic conditions; compared with the wild Rhizopus oryzae strain, the lactic acid content of a byproduct is low, the final fermentation concentration and the production intensity for producing the fumaric acid are significantly improved, and the production time is shortened, thereby reducing the separation difficulty and the separation cost of the fumaric acid in a fermentation liquid and laying a good foundation for industrial production of the bio-based fumaric acid by a microbial fermentation method.
Description
Technical field
The invention belongs to technical field of biochemical industry, relate to a kind of engineering strain and construction process and application thereof that improves fumaric acid output.
Background technology
Fumaric acid is a kind of important basic chemical raw materials and fine chemical product, is widely used in the fields such as coating, resin, medicine, softening agent.Industrially can be used for producing unsaturated polyester and Synolac, and as the raw material of producing electrocoating paint.With cinnamic multipolymer be the raw material of producing glass reinforced plastic, with the multipolymer of vinyl acetate be good tackiness agent.The food grade fumaric acid is the acidic flavoring agent of pure taste, as the acidic flavoring agent of beverage, drinks, cake and salted vegetables etc., and plays a part food preservatives, antioxidant and pH adjusting agent.The sodium fumarate that fumaric acid is reacted to preparation with sodium carbonate is a kind of flavour enhancer of foodstuff additive; Feed grade fumaric acid and acids thereof can improve specific absorption and the biology utilization ratio of organic substance in feed, reduce human body energy consumption, so also have the weightening finish of promotion, wait effect.Simultaneously, take fumaric acid can further synthesize multiple high value derivative as raw material.Fumaric acid and alcohols carry out esterification under the effect of sulfuric acid catalyst can generate fumarate, developed a series of fumaric acid esters anti-mould and moth-proof agent modern age, as dimethyl fumarate (DMF), diethyl ester (DEF), dipropyl (DPF), dibutylester (DBF), mono-methyl (MMF) etc., the class low toxicity that its mainstream product dimethyl fumarate is external the eighties exploitation, efficiently, inexpensive, the novel mildew resistant sanitas of wide spectrum, than phenylformic acid, Sorbic Acid, dehydro-acetic acid, it is few that propionic acid and corresponding salt have a consumption, cost is low, the advantage such as effective, be widely used in already the antimildew agent for food industry abroad.Using fumaric acid as raw material, can further synthesize multiple medicine, the substitution product Ferrous Fumarate of sodium fumarate and ferrous sulfate has been widely used in medically treating the microcytic cell anemia of human body, other are such as fumaric acid Kui Horizon, ketotifen fumarate, the clemastine fumarate sheet, bisoprolol tablets etc. all listing are at home and abroad used.In addition, fumaric acid is as a kind of four important carbon hardware and software platform compounds, can also be by C4 compounds such as explained hereafter L-Aspartic acid, oxysuccinic acid, succsinic acid, toxilic acid, BDO, gamma-butyrolactone and tetrahydrofuran (THF) such as enzyme catalysis conversion, esterification, hydrogenation.The bacterial classification commonly used that microbe fermentation method prepares fumaric acid is Rhizopus oryzae, Roryzae, unrooted rhizopus and bread mould, professors Tsao etc. have reported and have utilized fermentation with Rhizopus oryzae fumaric acid (Appl Environ Microbiol, 1996,62:2926～2931.), by the synthetic fumaric acid metabolisming way of Rhizopus oryzae is analyzed, glucose generates pyruvic acid by glycolytic cycle, pyruvic acid is in TCA and anti-TCA approach generation fumaric acid, also can under the effect of serum lactic dehydrogenase (LDH), generate lactic acid, the existence of lactic acid approach has weakened the accumulation ability of fumaric acid greatly.The researchist attempts by bacterial strain mutagenesis means screening lactic acid deficient strain, but is limited to low screening efficiency and the restriction of the limiting factor such as screening method is incorrect always, fails to obtain the low lactic acid producing Rhizopus oryzae strain of inheritance stability.Rhizopus oryzae is after the cellar culture that has experienced certain hour, and ldhL gradually plays leading role in the product accumulation, thereby makes the ability of Rhizopus oryzae accumulation lactic acid progressively improve, and has had a strong impact on the synthetic of fumaric acid.Therefore will change the distribution of carbon metabolism flow by certain technique means blocking-up ldhL gene, and weaken or reject the lactic acid metabolism branch road fully, thereby reaching the purpose that improves the fumaric acid metabolic flux.And how effectively to suppress the ldhL activity and, on the basis of metabolism migration, carry out the regulation and control of metabolism stream, redistribute metabolic flux, will be to realize utilizing gene Knockout to transform the key of R.oryzae high-throughput accumulation fumaric acid.
Summary of the invention:
The purpose of this invention is to provide a kind of engineering strain that improves fumaric acid output.
Another object of the present invention is to provide a kind of construction process that improves the engineering strain of fumaric acid output.
The 3rd purpose of the present invention is to provide a kind of purposes that improves the engineering strain of fumaric acid output.
Technical scheme of the present invention is summarized as follows:
A kind of construction process that improves the engineering strain of fumaric acid output, comprise the steps:
(1) clone of lactate dehydrogenase gene ldhL:
The Rhizopus oryzae cell genomic dna extracted of take is template, take respectively sequence table SEQ ID No.1 and SEQ ID No.2 is the upstream and downstream primer, carry out the PCR reaction, amplify the serum lactic dehydrogenase ldhL gene order shown in SEQ ID No.5, purify, serum lactic dehydrogenase ldhL gene order after purifying is connected with the pMD18-simple-T carrier, the pMD18-simple-T carrier obtains recombinating, carry out sequencing, enzyme is cut described restructuring pMD18-simple-T carrier, obtains the ldhL gene fragment that comprises restriction enzyme site;
(2) recombinant fragment that comprises fungal promoters sequence, reverse ldhL gene order and terminator sequence builds:
Utilize Not I, the fragment that Xho I obtains step (1) oppositely is inserted into the downstream of the plasmid pGAPZB promotor shown in SEQ ID No.7, obtain recombinant plasmid pGAPZB-ldhL, use Bgl II, BamH I enzyme is cut the described pGAPZB-ldhL of recombinant plasmid, obtains the recombinant fragment pGAP-ldhL-AOX1 that comprises fungal promoters sequence, reverse ldhL gene order and terminator sequence A OX1;
(3) clone of hygromycin gene:
The plasmid pDx-Los shown in SEQ ID No.8 of take is template, take sequence table SEQ ID No.3 and SEQ ID No.4 is the upstream and downstream primer, the pcr amplification hygromycin gene, pcr amplification product is purified, with the pMD18-simple-T carrier, be connected, the pMD18-simple-T carrier that obtains recombinating, enzyme is cut described restructuring pMD18-simple-T carrier, obtains the hygromycin gene fragment that comprises restriction enzyme site shown in SEQ ID No.6;
(4) structure that contains resistant gene recombination fragment:
The recombinant fragment that step (2) is obtained utilizes BamH I site to be connected with the gene fragment that step (3) obtains, by the restriction enzyme EcoR I checking fragment order of connection, screening hygromycin gene fragment is positioned at the connexon in pGAP-ldhL-AOX1 fragment downstream, obtains recombinant fragment pGAP-ldhL-AOX1-chao
r;
(5) structure of recombinant plasmid:
Utilize restriction endonuclease sites BamH I, Hind III is by described recombinant fragment pGAP-ldhL-AOX1-chao
rinsert plasmid pCAMB3300, obtain recombinant plasmid p CAMB3300-pGAP-ldhL-AOX1-chao
r;
(6) acquisition of restructuring crown gall purulence bacillus:
The recombinant plasmid p CAMB3300pGAP-ldhL-AOX1-chao that step (5) is obtained
rdirectly transform crown gall purulence bacillus LBA4404 by shocking by electricity, coating contains the kantlex LB plate culture medium of 50 μ g/mL, the picking positive transformant, and carry out the Molecular Identification positive colony, obtain containing recombinant plasmid p CAMB3300pGAP-ldhL-AOX1-chao
rcrown gall purulence oxydans genetic engineering bacterial strain, called after Agrobacterium tumefaciens LBA4404-pCAMB3300-pGAP-ldhL-AOX1-chao
r
(7) acquisition of restructuring Rhizopus oryzae bacterial strain:
1. inoculation step (6) obtained is in containing in the LB substratum of kantlex, rifomycin and Streptomycin sulphate, 28-32 ℃, and the 180-220r/min shaking table is cultivated 30-42h, centrifugal collection thalline, with the resuspended thalline of isopyknic IM substratum, cultivate 5-8h, make the mycetocyte final concentration reach 10
11-10
12individual/mL; The concentration of described kantlex in the LB substratum is 40-60 μ g/mL, and the concentration of described rifomycin in the LB substratum is 40-60 μ g/mL, and the concentration of described Streptomycin sulphate in the LB substratum is 20-40 μ g/mL;
2. the Rhizopus oryzae spore liquid is inoculated in the YPD solid medium, cultivates 6-8 days for 33-35 ℃; Collect fresh spore with the sterilized water wash-out, be resuspended in the aseptic CaCl with 20g/l
2in the aqueous solution, make the Rhizopus oryzae spore concentration reach 10
8-10
9individual/mL;
Each the 50 μ L of resuspended bacterium liquid that get step and 1. and 2. obtain add in the IM substratum that contains Syringylethanone, kantlex and Totomycin, and at 28-32 ℃, 180-220r/min cultivates 12-16h; Dilution 10
-6doubly, get 200 μ L diluent coatings and be covered with the IM+AS culture medium flat plate of glassine paper, and be placed in 33-35 ℃ of cultivation 40-50h; Glassine paper is transferred on the YPD substratum that contains Totomycin and cultivates 90-100h, screen positive Rhizopus oryzae engineering strain, and carry out the Molecular Identification on gene level, called after Rhizopus oryzae CM 08/LDH
-, the concentration of described Syringylethanone in the IM substratum is 200 μ mol/L, and the concentration of kantlex in the IM substratum is 50 μ g/mL, and the concentration of Totomycin in the IM substratum is 50 μ g/mL, and the concentration of described Totomycin in the YPD substratum is 50 μ g/mL.
A kind of engineering strain Rhizopus oryzae CM 08/LDH that improves fumaric acid output that aforesaid method builds
-.
Above-mentioned a kind of engineering strain that improves fumaric acid output take the purposes of glucose as fermenting substrate production fumaric acid.
Beneficial effect of the present invention:
Bacterial strain of the present invention, fully under aerobic condition, utilizing glucose fermentation high density accumulation fumaric acid, has solved the higher problem of traditional fermentation with Rhizopus oryzae fumaric acid by product lactic acid content.Fed-batch fermentation is complete: can utilize the glucose of 150g/L, obtain fumaric acid 120g/L, and molar yield 80%, production intensity reaches 1.2g/L/h; Under the same conditions, original strain utilizes the glucose of 150g/L, obtains the fumaric acid of 80g/L, and production intensity is only 0.8g/L/h, with the wild rice rhizopus strains, compare, final fermentation concentration and the production intensity of the production fumaric acid of recombinant bacterial strain of the present invention are significantly improved.Report that with current document utilizing the fermentation of wild rice rhizopus strains to produce the fumaric acid process compares, improved production intensity, shortened the production time, the concentration of by product lactic acid significantly reduces, target product concentration promotes obviously, thereby reduced separating difficulty and the separation costs of fumaric acid in the fermented liquid, for the fumaric acid in microbe fermentation method suitability for industrialized production bio-based source is had laid a good foundation.
Embodiment
Below by specific embodiment, the present invention is further illustrated, and embodiments of the invention are in order to enable those skilled in the art to understand better the present invention, but the present invention are not done to any restriction.
Embodiment 1
A kind of construction process that improves the engineering strain of fumaric acid output, comprise the steps:
(1) clone of lactate dehydrogenase gene ldhL:
The Rhizopus oryzae cell genomic dna extracted of take is template, take respectively sequence table SEQ ID No.1 and SEQ ID No.2 is the upstream and downstream primer, carry out the PCR reaction, amplify the serum lactic dehydrogenase ldhL gene order shown in SEQ ID No.5, purify, serum lactic dehydrogenase ldhL gene order after purifying is connected with the pMD18-simple-T carrier, obtains
restructuring the pMD18-simple-T carrier, carrying out sequencing, enzyme is cut described
restructuring pMD18-simple-T carrier, obtain the ldhL gene fragment that comprises restriction enzyme site;
(2) recombinant fragment that comprises fungal promoters sequence, reverse ldhL gene order and terminator sequence builds:
Utilize Not I, the fragment that Xho I obtains step (1) oppositely is inserted into the downstream of the plasmid pGAPZB promotor shown in SEQ ID No.7, obtain recombinant plasmid pGAPZB-ldhL, use Bgl II, BamH I enzyme is cut the described pGAPZB-ldhL of recombinant plasmid, obtains the recombinant fragment pGAP-ldhL-AOX 1 that comprises fungal promoters sequence, reverse ldhL gene order and terminator sequence A OX1;
(3) clone of hygromycin gene:
The plasmid pDx-Los shown in SEQ ID No.8 of take is template, take sequence table SEQ ID No.3 and SEQ ID No.4 is the upstream and downstream primer, the pcr amplification hygromycin gene, pcr amplification product is purified, with the pMD18-simple-T carrier, be connected, the pMD18-simple-T carrier that obtains recombinating, enzyme is cut described restructuring pMD18-simple-T carrier, obtains the hygromycin gene fragment that comprises restriction enzyme site shown in SEQ ID No.6;
(4) structure that contains resistant gene recombination fragment:
The recombinant fragment that step (2) is obtained utilizes BamH I site to be connected with the gene fragment that step (3) obtains, by the restriction enzyme EcoR I checking fragment order of connection, screening hygromycin gene fragment is positioned at the connexon in pGAP-ldhL-AOX1 fragment downstream, obtains recombinant fragment pGAP-ldhL-AOX1-chao
r;
(5) structure of recombinant plasmid:
Utilize restriction endonuclease sites BamH I, Hind III is by described recombinant fragment pGAP-ldhL-AOX1-chao
rinsert plasmid pCAMB3300, obtain recombinant plasmid p CAMB3300-pGAP-ldhL-AOX1-chao
r;
(6) acquisition of restructuring crown gall purulence bacillus:
The recombinant plasmid p CAMB3300pGAP-ldhL-AOX1-chao that step (5) is obtained
rdirectly transform crown gall purulence bacillus LBA4404 by shocking by electricity, coating contains the kantlex LB plate culture medium of 50 μ g/mL, the picking positive transformant, and carry out the Molecular Identification positive colony, obtain containing recombinant plasmid p CAMB3300pGAP-ldhL-AOX1-chao
rcrown gall purulence oxydans genetic engineering bacterial strain, called after Agrobacterium tumefaciens LBA4404-pCAMB3300-pGAP-ldhL-AOX1-chao
r
(7) acquisition of restructuring Rhizopus oryzae bacterial strain:
1. inoculation step (6) obtained is in containing the LB substratum of kantlex, rifomycin and Streptomycin sulphate, and 30 ℃, the 200r/min shaking table is cultivated 36h, centrifugal collection thalline, with the resuspended thalline of isopyknic IM substratum, cultivate 6h, make the mycetocyte final concentration reach 10
11individual/mL; The concentration of described kantlex in the LB substratum is 50 μ g/mL, and the concentration of described rifomycin in the LB substratum is 50 μ g/mL, and the concentration of described Streptomycin sulphate in the LB substratum is 30 μ g/mL;
2. the Rhizopus oryzae spore liquid is inoculated in the YPD solid medium, cultivates 7 days for 34 ℃; Collect fresh spore with the sterilized water wash-out, be resuspended in the aseptic CaCl with 20g/l
2in the aqueous solution, make the Rhizopus oryzae spore concentration reach 10
9individual/mL;
Each the 50 μ L of resuspended bacterium liquid that get step and 1. and 2. obtain add in the IM substratum that contains Syringylethanone, kantlex and Totomycin, and at 30 ℃, 200r/min cultivates 14h; Dilution 10
-6doubly, get 200 μ L diluent coatings and be covered with the IM+AS culture medium flat plate of glassine paper, and be placed in 34 ℃ of cultivation 40-50h; Glassine paper is transferred on the YPD substratum that contains Totomycin and cultivates 95h, screen positive Rhizopus oryzae engineering strain, and carry out the Molecular Identification on gene level, called after Rhizopusoryzae CM 08/LDH
-, the concentration of described Syringylethanone in the IM substratum is 200 μ mol/L, and the concentration of kantlex in the IM substratum is 50 μ g/mL, and the concentration of Totomycin in the IM substratum is 50 μ g/mL, and the concentration of described Totomycin in the YPD substratum is 50 μ g/mL.
Embodiment 2
A kind of construction process that improves the engineering strain of fumaric acid output, comprise the steps:
(1) clone of lactate dehydrogenase gene ldhL:
With embodiment 1 step (1)
(2) recombinant fragment that comprises fungal promoters sequence, reverse ldhL gene order and terminator sequence builds:
With embodiment 1 step (2)
(3) clone of hygromycin gene:
With embodiment 1 step (3)
(4) structure that contains resistant gene recombination fragment:
With embodiment 1 step (4)
(5) structure of recombinant plasmid:
With embodiment 1 step (5)
(6) acquisition of restructuring crown gall purulence bacillus:
With embodiment 1 step (6)
(7) acquisition of restructuring Rhizopus oryzae bacterial strain:
1. inoculation step (6) obtained is in containing the LB substratum of kantlex, rifomycin and Streptomycin sulphate, and 28 ℃, the 220r/min shaking table is cultivated 42h, centrifugal collection thalline, resuspended with isopyknic IM substratum, cultivate 8h, make the mycetocyte final concentration reach 10
12individual/mL; The concentration of described kantlex in the LB substratum is 60 μ g/mL, and the concentration of described rifomycin in the LB substratum is 60 μ g/mL, and the concentration of described Streptomycin sulphate in the LB substratum is 40 μ g/mL;
2. the Rhizopus oryzae spore liquid is inoculated in the YPD solid medium, cultivates 8 days for 33 ℃; Collect fresh spore with the sterilized water wash-out, be resuspended in the aseptic CaCl with 20g/l
2in the aqueous solution, make the Rhizopus oryzae spore concentration reach 10
8individual/mL;
Each the 50 μ L of bacterium liquid that get step and 1. and 2. obtain add in the IM substratum that contains Syringylethanone, kantlex and Totomycin, and at 28 ℃, 220r/min cultivates 16h; Dilution 10
-6doubly, get 200 μ L diluent coatings and be covered with the IM+AS culture medium flat plate of glassine paper, and be placed in 35 ℃ of cultivation 40h; Glassine paper is transferred on the YPD substratum that contains Totomycin and cultivates 90h, screen positive Rhizopus oryzae engineering strain, and carry out the Molecular Identification on gene level, called after Rhizopus oryzae CM08/LDH
-, the concentration of described Syringylethanone in the IM substratum is 200 μ mol/L, and the concentration of kantlex in the IM substratum is 50 μ g/mL, and the concentration of Totomycin in the IM substratum is 50 μ g/mL, and the concentration of described Totomycin in the YPD substratum is 50 μ g/mL.
Embodiment 3
A kind of construction process that improves the engineering strain of fumaric acid output, comprise the steps:
(1) clone of lactate dehydrogenase gene ldhL:
With embodiment 1 step (1)
(2) recombinant fragment that comprises fungal promoters sequence, reverse ldhL gene order and terminator sequence builds:
With embodiment 1 step (2)
(3) clone of hygromycin gene:
With embodiment 1 step (3)
(4) structure that contains resistant gene recombination fragment:
With embodiment 1 step (4)
(5) structure of recombinant plasmid:
With embodiment 1 step (5)
(6) acquisition of restructuring crown gall purulence bacillus:
With embodiment 1 step (6)
(7) acquisition of restructuring Rhizopus oryzae bacterial strain:
1. inoculation step (6) obtained is in containing the LB substratum of kantlex, rifomycin and Streptomycin sulphate, and 32 ℃, the 180r/min shaking table is cultivated 30h, centrifugal collection thalline, resuspended with isopyknic IM substratum, cultivate 5h, make the mycetocyte final concentration reach 10
11individual/mL; The concentration of described kantlex in the LB substratum is 40 μ g/mL, and the concentration of described rifomycin in the LB substratum is 40 μ g/mL, and the concentration of described Streptomycin sulphate in the LB substratum is 20 μ g/mL;
2. the Rhizopus oryzae spore liquid is inoculated in the YPD solid medium, cultivates 6 days for 35 ℃; Collect fresh spore with the sterilized water wash-out, be resuspended in the aseptic CaCl with 20g/l
2in the aqueous solution, make the Rhizopus oryzae spore concentration reach 10
9individual/mL;
Each the 50 μ L of bacterium liquid that get step and 1. and 2. obtain add in the IM substratum that contains Syringylethanone, kantlex and Totomycin, and at 32 ℃, 180r/min cultivates 12h; Dilution 10
-6doubly, get 200 μ L diluent coatings and be covered with the IM+AS culture medium flat plate of glassine paper, and be placed in 33 ℃ of cultivation 50h; Glassine paper is transferred on the YPD substratum that contains Totomycin and cultivates 100h, screen positive Rhizopus oryzae engineering strain, and carry out the Molecular Identification on gene level, called after Rhizopus oryzae CM 08/LDH
-, the concentration of described Syringylethanone in the IM substratum is 200 μ mol/L, and the concentration of kantlex in the IM substratum is 50 μ g/mL, and the concentration of Totomycin in the IM substratum is 50 μ g/mL, and the concentration of described Totomycin in the YPD substratum is 50 μ g/mL.
Embodiment 4
To recombinant bacterium and wild rice rhizopus strains fermentation culture, step is as follows:
By original Rhizopus oryzae and the new Rhizopus oryzae CM 08/LDH built
-under substratum below of bacterium and culture condition, fermented:
(1) original Rhizopus oryzae bacterial strain, its called after Rhizopus oryzae CICC 40506;
(2) seed culture medium: glucose 20g/l, potassium primary phosphate 0.8g/l, ammonium sulfate 2g/l, sal epsom 0.6g/l, zinc sulfate 0.13g/l, ferrous sulfate 0.01g/l, the aseptic dilute sulphuric acid of the complete use of sterilizing is regulated initial pH to 3.0 and is cultivated for seed growth.
Seed culture: 500ml triangular flask, liquid amount 100ml, 33 ℃ of culture temperature, spore inoculating amount 5.0 * 10
7/ 100ml seed liquor, shaking speed 200r/min, cultivate 14h, and this is primary seed solution.Primary seed solution is seeded to cultivation 14h in new sterile culture based component with 10% inoculum size and is secondary seed solution; Secondary seed solution is seeded to cultivation 14h in new sterile culture based component with 10% inoculum size and is three grades of seed liquor; Two, three grades of seed culture medium components and all the other each parameters are all with the one-level seed liquor.
(3) fermention medium: glucose 80g/l, potassium primary phosphate 0.8g/l, urea 0.2g/l, sal epsom 0.6g/l, zinc sulfate 0.13g/l, ferrous sulfate: 0.01g/l, calcium carbonate 50g/l.
Culture condition: the 7L fermentor tank, liquid amount 4L, inoculate three grades of seed liquor, inoculum size 10%, 35 ℃ of leavening temperatures, rotating speed 300rpm/min, blowing air strategy: fermentation 0-24h 1vvm, 24-36h 0.6vvm, 36-48h 0vvm, 48-60h 0.6vvm, 60-72h 1vvm, maintain air flow 1vvm after 72h starts, approximately to 72h, fermentation ends is in Table 1.
Table 1:
Data results illustrates by experiment: final fermentation concentration and the production intensity of the production fumaric acid of recombinant bacterial strain of the present invention are significantly improved; Shortened the production time; The concentration of by product lactic acid significantly reduces.
Claims (3)
1. improve the construction process of the engineering strain of fumaric acid output, it is characterized in that comprising the steps:
(1) clone of lactate dehydrogenase gene ldhL:
Rhizopus oryzae (Rhizopus oryzae) cell genomic dna extracted of take is template, take respectively sequence table SEQ ID No.1 and SEQ ID No.2 is the upstream and downstream primer, carry out the PCR reaction, amplify the serum lactic dehydrogenase ldhL gene order shown in SEQ ID No.5, purify, serum lactic dehydrogenase ldhL gene order after purifying is connected with the pMD18-simple-T carrier, the pMD18-simple-T carrier obtains recombinating, carry out sequencing, enzyme is cut described restructuring pMD18-simple-T carrier, obtains the ldhL gene fragment that comprises restriction enzyme site;
(2) recombinant fragment that comprises fungal promoters sequence, reverse ldhL gene order and terminator sequence builds:
Utilize Not I, the fragment that Xho I obtains step (1) oppositely is inserted into the downstream of the plasmid pGAPZB promotor shown in SEQ ID No.7, obtain recombinant plasmid pGAPZB-ldhL, use the Bgl II, BamH I enzyme is cut recombinant plasmid pGAPZB-ldhL, obtains the recombinant fragment pGAP-ldhL-AOX1 that comprises fungal promoters sequence, reverse ldhL gene order and terminator sequence A OX1;
(3) clone of hygromycin gene:
The plasmid pDx-Los shown in SEQ ID No.8 of take is template, take sequence table SEQ ID No.3 and SEQ ID No.4 is the upstream and downstream primer, the pcr amplification hygromycin gene, pcr amplification product is purified, with the pMD18-simple-T carrier, be connected, the pMD18-simple-T carrier that obtains recombinating, enzyme is cut described restructuring pMD18-simple-T carrier, obtains the hygromycin gene fragment that comprises restriction enzyme site shown in SEQ ID No.6;
(4) structure that contains resistant gene recombination fragment:
The recombinant fragment that step (2) is obtained utilizes BamH I site to be connected with the gene fragment that step (3) obtains, by the restriction enzyme EcoR I checking fragment order of connection, screening hygromycin gene fragment is positioned at the connexon in pGAP-ldhL-AOX1 fragment downstream, obtains recombinant fragment pGAP-ldhL-AOX1-chao
r;
(5) structure of recombinant plasmid:
Utilize restriction endonuclease sites BamH I, Hind III is by described recombinant fragment pGAP-ldhL-AOX1-chao
rinsert plasmid pCAMB3300, obtain recombinant plasmid p CAMB3300-pGAP-ldhL-AOX1-chao
r;
(6) acquisition of restructuring crown gall purulence bacillus:
The recombinant plasmid p CAMB3300pGAP-ldhL-AOX1-chao that step (5) is obtained
rdirectly transform crown gall purulence bacillus (Agrobacterium tumefaciens) LBA4404 by shocking by electricity, coating is containing the kantlex LB plate culture medium of 50 μ g/mL, the picking positive transformant, and carry out the Molecular Identification positive colony, obtain containing recombinant plasmid p CAMB3300pGAP-ldhL-AOX1-chao
rcrown gall purulence oxydans genetic engineering bacterial strain, called after Agrobacterium tumefaciens LBA4404-pCAMB3300-pGAP-ldhL-AOX1-chao
r;
(7) acquisition of restructuring Rhizopus oryzae bacterial strain:
1. inoculation step (6) obtained is in containing the LB substratum of kantlex, rifomycin and Streptomycin sulphate, 28-32 ℃, the 180-220r/min shaking table is cultivated 30-42h, centrifugal collection thalline, with the resuspended thalline after centrifugal of isopyknic IM substratum, cultivate 5-8h, make the mycetocyte final concentration reach 10
11-10
12individual/mL; The concentration of described kantlex in the LB substratum is 40-60 μ g/mL, and the concentration of described rifomycin in the LB substratum is 40-60 μ g/mL, and the concentration of described Streptomycin sulphate in the LB substratum is 20-40 μ g/mL;
2. the Rhizopus oryzae spore liquid is inoculated in the YPD solid medium, cultivates 6-8 days for 33-35 ℃; Collect fresh spore with the sterilized water wash-out, be resuspended in the aseptic CaCl with 20g/l
2in the aqueous solution, make the Rhizopus oryzae spore concentration reach 10
8-10
9individual/mL;
Each the 50 μ L of resuspended bacterium liquid that get step and 1. and 2. obtain add in the IM substratum that contains Syringylethanone, kantlex and Totomycin, and at 28-32 ℃, 180-220r/min cultivates 12-16h; Dilution 10
-6doubly, get 200 μ L diluent coatings and be covered with the IM+AS culture medium flat plate of glassine paper, and be placed in 33-35 ℃ of cultivation 40-50h; Glassine paper is transferred on the YPD substratum that contains Totomycin and cultivates 90-100h, screen positive Rhizopus oryzae engineering strain, and carry out the Molecular Identification on gene level, called after Rhizopus oryzae CM08/LDH
-, the concentration of described Syringylethanone in the IM substratum is 200 μ mol/L, and the concentration of kantlex in the IM substratum is 50 μ g/mL, and the concentration of Totomycin in the IM substratum is 50 μ g/mL, and the concentration of described Totomycin in the YPD substratum is 50 μ g/mL.
Claim 1 a kind of engineering strain Rhizopus oryzae CM08/LDH that improves fumaric acid output of building of method
-.
3. a kind of engineering strain that improves fumaric acid output of claim 2 take the purposes of glucose as fermenting substrate production fumaric acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210044717.7A CN102559736B (en) | 2012-02-24 | 2012-02-24 | Gene engineering bacterial strain capable of improving yield of fumaric acid and establishing method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210044717.7A CN102559736B (en) | 2012-02-24 | 2012-02-24 | Gene engineering bacterial strain capable of improving yield of fumaric acid and establishing method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102559736A CN102559736A (en) | 2012-07-11 |
| CN102559736B true CN102559736B (en) | 2014-01-08 |
Family
ID=46406342
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210044717.7A Active CN102559736B (en) | 2012-02-24 | 2012-02-24 | Gene engineering bacterial strain capable of improving yield of fumaric acid and establishing method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102559736B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115093977B (en) * | 2022-07-21 | 2023-09-01 | 中国海洋大学 | Brevibacterium pullulans strain EP01 for producing fumaric acid and use method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011041402A1 (en) * | 2009-09-29 | 2011-04-07 | Butamax(Tm) Advanced Biofuels Llc | Improved flux to acetolactate-derived products in lactic acid bacteria |
| CN102174419A (en) * | 2011-03-24 | 2011-09-07 | 南京工业大学 | Rhizopus oryzae strain, mutagenic screening method thereof and method for producing fumaric acid through fermentation |
-
2012
- 2012-02-24 CN CN201210044717.7A patent/CN102559736B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011041402A1 (en) * | 2009-09-29 | 2011-04-07 | Butamax(Tm) Advanced Biofuels Llc | Improved flux to acetolactate-derived products in lactic acid bacteria |
| CN102174419A (en) * | 2011-03-24 | 2011-09-07 | 南京工业大学 | Rhizopus oryzae strain, mutagenic screening method thereof and method for producing fumaric acid through fermentation |
Non-Patent Citations (7)
| Title |
|---|
| Ashraf S. Ibrahim.Native and modiWed lactate dehydrogenase expression in a fumaric acid producing isolate Rhizopus oryzae 99-880.《Curr Genet》.2007,第52卷1-3. * |
| Ayumi Abe et al..Rhizopus delemar is the proper name for Rhizopus oryzae fumaric-malic acid producers.《MYCOLOGIA》.2007,第99卷(第5期),全文. |
| Christopher D. Skory & Ashraf S. Ibrahim.Native and modiWed lactate dehydrogenase expression in a fumaric acid producing isolate Rhizopus oryzae 99-880.《Curr Genet》.2007,第52卷1-3. |
| Christopher D. Skory & * |
| Genetic diversity in Rhizopus oryzae strains as revealed by the sequence of lactate dehydrogenase genes;Katsuichi Saito et al.;《Arch Microbiol》;20041231;全文 * |
| Katsuichi Saito et al..Genetic diversity in Rhizopus oryzae strains as revealed by the sequence of lactate dehydrogenase genes.《Arch Microbiol》.2004,1-3. |
| Rhizopus delemar is the proper name for Rhizopus oryzae fumaric-malic acid producers;Ayumi Abe et al.;《MYCOLOGIA》;20071231;第99卷(第5期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102559736A (en) | 2012-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103881954B (en) | Gamma-polyglutamic acid-genetic engineering bacterium and high yield gamma-polyglutamic acid-method thereof are produced in one strain | |
| CN103243064B (en) | Escherichia coli engineered strain and application in succinic acid production through aerobic-microaerobic-anaerobic full-stage fermentation of Escherichia coli engineered strain | |
| CN107586789B (en) | High-yield acidic protease aspergillus niger recombinant expression strain and construction method and application thereof | |
| CN107142225B (en) | A kind of pichia yeast recombinant bacterium of the source overexpression Streptomyces sp.FA1 zytase | |
| CN103740629B (en) | The genetically engineered acetic bacteria of overexpression coenzyme PQQ synthetic proteins and application thereof | |
| CN102286415B (en) | Bacterial strain for high yield of succinic acid and application thereof | |
| CN106148209A (en) | New binary organic acid produces bacterial strain and preparation thereof and application | |
| CN101531988B (en) | Alkaline pectinase genetic engineering bacteria and construction method thereof | |
| CN102199570B (en) | Method for constructing gene engineering bacterium for improving microbial fermentation for1,3-propanediol production from glycerol | |
| CN102816729A (en) | Construction and application of polygene knockout strain of Halomonas sp. TD01 | |
| CN105400796A (en) | Gene for adjusting and controlling production of long-chain diacid and application of gene | |
| CN110272858B (en) | Bacillus coagulans for high yield of L-lactic acid and application thereof | |
| CN104232722A (en) | Method for producing 9-alpha-hydroxyandrostenedione by microbial fermentation | |
| CN105400711B (en) | The structure of the saccharomyces cerevisiae engineered yeast of one plant of production L MALIC ACID and application | |
| CN105543214A (en) | Construction method and applications of metabolic engineering escherichia coli strain for producing succinic acid by using acetic acid | |
| CN103571772A (en) | Novel strain for producing butanol and method for producing butanol | |
| CN101260379B (en) | Gene engineering bacterium for producing 1,3-propanediol and its preparation method and application | |
| CN105400831B (en) | Utilize the method for recombination Corynebacterium glutamicum coproduction 1,3-PD and glutamic acid | |
| CN102559736B (en) | Gene engineering bacterial strain capable of improving yield of fumaric acid and establishing method and application thereof | |
| CN105420265A (en) | Genetic engineering acetic bacteria for over-expressing ATP (Adenosine Triphosphate) enzyme as well as construction method and application thereof | |
| CN101205541B (en) | Recombinant expression carrier and method for high-yield of 1,3-propanediol by fermenting glycerin using host cell converted thereby | |
| CN102286387A (en) | Construction method and use of fumaric acid producing candida glabrata engineering strain | |
| CN106148255B (en) | Engineering bacteria lacking organic acid production way and application thereof in co-production of 1, 3-propylene glycol, 2, 3-butanediol and ethanol | |
| CN104293817B (en) | Construction methods and uses of recombinant plasmid for producing succinic acid, and genetic engineering bacterium | |
| CN102827800B (en) | Escherichia coli engineering strain and application thereof in production of succinic acid by low-oxygen fermentation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |