CN104830851A - Recombinant bacterium of formate dehydrogenase and application of recombinant bacterium - Google Patents
Recombinant bacterium of formate dehydrogenase and application of recombinant bacterium Download PDFInfo
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Abstract
The invention discloses a primer pair used for formate dehydrogenase amplification, further discloses a recombinant bacterium of heterogenous expression formate dehydrogenase, further discloses a construction method of the recombinant bacterium of heterogenous expression formate dehydrogenase, and further discloses an application of the recombinant bacterium of heterogenous expression formate dehydrogenase in preparation of R,R-2,3-butanediol. A result of Paenibacillus polymyxa ZJ-9 fermented in an NBS 7.5L fermentation tank shows that the yield of DCW is increased by 3.9% and reaches 23.1g/L; the yield of R,R-2,3 butanediol is increased by 10.2% and reaches 36.8g/L; and the content of by-product formic acid is reduced from 3.3g/L to 0.9g/L. Reduction of the content of by-product formic acid facilitates separation and purification of fermentation liquid in a later stage, and a foundation is laid for R,R-2,3-BD industrialization.
Description
Technical field
The invention belongs to technical field of biochemical industry, be specifically related to a kind of recombinant bacterium and application thereof of heterogenous expression hydrogenlyase.
Background technology
2,3-butanediol is a kind of important industrial chemicals and liquid fuel, is widely used in the fields such as chemical industry, food, fuel and aerospace, has biological degradability.2,3-butanediol contains two chiral carbon atoms, therefore has three kinds of optically active isomers (being respectively R, R-2,3-butyleneglycol, S, S-2,3-butyleneglycol and meso-2,3-butyleneglycol).R, R-2,3-butyleneglycol is except having such use, or excellent antifreezing agent, is also the important intermediate of synthesis of chiral reagent, chiral ligand, in the synthesis of chiral drug and natural product, also have potential important application.At present, study discovery and metabolism can synthesize R, R-2, the microorganism of 3-butyleneglycol is very few, and Paenibacillus polymyxa (Paenibacillus polymyxa, front title Bacillus polymyxa) wherein uniquely possesses suitability for industrialized production R, the microorganism of R-2,3-butyleneglycol potentiality.Only a few bacterial strain in P.polymyxa can produce inulinase, can directly utilize energy-source plant jerusalem artichoke inulin (synanthrin) one-step fermentation to prepare the R of high added value, R-2,3-butyleneglycol.Wherein coenzyme NAD H plays a part key as reducing equivalent, and the height of its content directly has influence on the output of 2,3-butanediol, and these NADH are mainly synthetic regenerated by inulin oxidative pathway.Therefore, suitably improve NADH concentration and NADH/NAD ratio in born of the same parents, be expected to improve 2,3-butanediol synthesis concentration.
Due to shortcomings such as coenzyme NAD H structure are complicated, unstable and expensive, it is obviously infeasible for constantly adding coenzyme NAD H in the industrial production, and thus the regeneration of coenzyme NAD H just becomes the important step reducing production cost.In recent years, hydrogenlyase (FDH) regenerating coenzyme system causes the extensive concern of people, and is successfully applied to the industrial production of S-Leucine.Hydrogenlyase belongs to D-2-alcohol acid dehydrogenase type, in the bacterium being extensively present in methanol using type and yeast.It can by NAD while catalysis formic acid is converted into carbonic acid gas
+be reduced into NADH.The present invention selects Candida boidinii as the starting strain of molecular biology manipulations, obtained the encoding gene (fdh gene) of hydrogenlyase from the genome amplification of this bacterial strain by round pcr, Paenibacillus polymyxa ZJ-9 is as host, successfully constructing can the recombinant bacterium of high expression hydrogenlyase, improve R, the output of R-2,3-butyleneglycol, decrease the content of byproduct formic acid, have good prospects for commercial application.
Summary of the invention
Goal of the invention: first object of the present invention is to provide a kind of primer pair for the hydrogenlyase that increases.
Second object of the present invention is to provide a kind of recombinant bacterium of heterogenous expression hydrogenlyase.
3rd object of the present invention is to provide a kind of construction process of recombinant bacterium of heterogenous expression hydrogenlyase.
4th object of the present invention is to provide the application of recombinant bacterium at preparation R, R-2,3-butyleneglycol aspect expressing a kind of heterogenous expression hydrogenlyase.5th object of the present invention is to provide a kind of production method of R, R-2,3-butyleneglycol.
Technical scheme: in order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of primer pair for the hydrogenlyase that increases, and it is as follows that described primer pair comprises two groups: first group of primer pair:
F1：5′-AGGCCGGGGCATATGGGAAACA-3′
R1：5′-TAAGACTAAAACGATCTTCATAAGCTTCTGTTATTAATTCTT-3′
Second group of primer pair is as follows:
F2：5′-AAGAATTAATAACAGAAGCTTATGAAGATCGTTTTAGTCTTA-3′
R2：5′-CCGGGATCCTTATTTCTTATCGTGTTTACC-3′。
A kind of recombinant bacterium of heterogenous expression hydrogenlyase, it is for template carries out with first group of primer pair the P43 promoter fragment that pcr amplification obtains acquisition with B.subtilis 168 genome, with the genome of candiyeast Candida boidinii for template obtains formate dehydrogenase gene fragment fdh with second group of primer pair pcr amplification, again the P43 promoter fragment of acquisition and formate dehydrogenase gene fragment are carried out over-lap PCR, the product of over-lap PCR acquisition is connected with plasmid pMA5 under the effect of T4 ligase enzyme and obtains recombinant plasmid pMA5-P43-fdh, finally recombinant plasmid pMA5-P43-fdh electricity is proceeded in P.polymyxa ZJ-9 and obtain recombinant bacterium P.polymyxa GX-1.
A construction process for the recombinant bacterium of heterogenous expression hydrogenlyase, comprises the following steps:
1) with B.subtilis 168 genome for stencil design first group of primer pair, as shown in above-mentioned sequence, pcr amplification obtains P43 strong promoter fragment, cut glue reclaim for subsequent use;
2) with the genome of candiyeast Candida boidinii for template, design second group of primer pair, as shown in above-mentioned sequence, pcr amplification obtains formate dehydrogenase gene fragment fdh;
3) the P43 strong promoter fragment of acquisition and formate dehydrogenase gene fdh are carried out over-lap PCR;
4) through restriction enzyme BamH I and Nde I double digestion, under the effect of T4 ligase enzyme, carry out connection with the plasmid pMA5 through same double digestion and obtain recombinant plasmid pMA5-P43-fdh;
5) recombinant plasmid pMA5-P43-fdh electricity is proceeded to P.polymyxa ZJ-9, obtain recombinant bacterium P.polymyxa GX-1.
The application of recombinant bacterium at preparation a R, R-2,3-butyleneglycol aspect of heterogenous expression hydrogenlyase.
A kind of production method of R, R-2,3-butyleneglycol, obtains in fermentation cylinder for fermentation with above-mentioned recombinant bacterium P.polymyxa GX-1.
Wherein, above-mentioned fermentation culture conditions is: temperature 30 DEG C, air flow 1vvm, liquid amount 4L, inoculum size 8%, front 22h rotating speed is 300r/min, rear 22h is adjusted to 200r/min, pH regulating strategy is: the fermentation initial stage does not control fermented liquid pH, in fermenting process along with pH value drop to below 6.0 time, with 4M NaOH adjust pH be 6.0.
Wherein, activation medium in above-mentioned fermenting process: peptone 10g/L, extractum carnis 3g/L, NaCl 5g/L, agar 10 ~ 20g/L, pH 7.0; Seed culture medium: inulin 20g/L, peptone 10g/L, yeast extract paste 3g/L, NaCl5g/L, pH 7.0; Fermention medium: inulin 75g/L, yeast extract paste 6g/L, peptone 2g/L, MgSO
47H
2o0.5g/L, K
2hPO
43.09g/L, MnSO
4h
2o 0.001g/L, NH
4cl 0.93g/L, FeSO
47H
2o 0.04g/L, ZnSO
47H
2o 0.001g/L, pH 6.0.
Beneficial effect: compared with prior art, advantage of the present invention is: the present invention is by importing in P.polymyxa ZJ-9 by external source hydrogenlyase, realize the regenerating coenzyme of this bacterial strain, finally improve R, R-2, the output of 3-butyleneglycol, reduce the content of byproduct formic acid, be conducive to the extraction and isolation of product, the method is simple to operate, environmental friendliness, recombinant bacterium is applicable to large-scale industrial production.Paenibacillus polymyxa ZJ-9 of the present invention, by the display of NBS 7.5L ferment tank result, DCW improves 3.9%, reach 23.1g/L, R, R-2,3-butyleneglycol output increased 10.2%, reach 36.8g/L, byproduct formic acid content is reduced to 0.9g/L by 3.3g/L.The reduction of byproduct formic acid content, is conducive to the separation and purification of later stage fermentation liquid, for R, R-2,3-BD industrialization lays the first stone.
Accompanying drawing explanation
Fig. 1 P43, fdh gene fragment glue reclaims rear electrophoresis figure;
The P43-fdh fragment electrophoretic figure of Fig. 2 over-lap PCR amplification;
Fig. 3 construction of recombinant plasmid schema;
The checking electrophorogram of Fig. 4 recombinant bacterium P.polymyxa GX-1.
Embodiment
Below by specific embodiment, the present invention is further described; it should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention; can also make some modification and improvement, these also should be considered as belonging to protection scope of the present invention.
Embodiment 1: the extraction of genomic dna
With Genomic DNA Purification Kit (Takara, Dalian) extract respectively and be in the Candidaboidinii (purchase of Beijing North Na Kaichuan Bioisystech Co., Ltd) of logarithmic phase and the genomic dna of B.subtilis 168 (purchase of Beijing North Na Kaichuan Bioisystech Co., Ltd), and with the agarose gel electrophoresis of 1% (10g/L), obtained genomic dna to be detected.
Embodiment 2: strong promoter (P43 gene), the clone of hydrogenlyase (fdh gene) and the structure of carrier
2.1PCR amplifying target genes
According to strong promoter (P43 gene) sequence that the B.subtilis 168 that Genbank has reported originates, use following two primers of Vector NTI software design:
F1:5 '-AGGCCGGGG
cATATGgGAAACA-3 ' (underscore place is Nde I restriction enzyme site)
R1：5′-TAAGACTAAAACGATCTTCATAAGCTTCTGTTATTAATTCTT-3′
With the genome of candiyeast Candida boidinii for template, design primer is as follows:
F2：5′-AAGAATTAATAACAGAAGCTTATGAAGATCGTTTTAGTCTTA-3′
R2:5 '-CCG
gGATCCtTATTTCTTATCGTGTTTACC-3 ' (underscore place is BamH I restriction enzyme site)
According to Standard PCR reaction system and condition, pcr amplification obtains P43 promoter fragment and fdh gene fragment, 1% (10g/L) agarose gel electrophoresis checking PCR primer, reach a conclusion: P43 promoter fragment size is about 500bp, fdh gene fragment size is about 1100bp, all consistent with expected results, so reclaim with DNA purification kit.Result is as Fig. 1.
Over-lap PCR reaction is increased in two steps, and the first step system is as follows: 10 × PCR buffer 2.5 μ L, Mg
2+2 μ L, dNTP 2 μ L, each 2 μ L of fragment template to be overlapped, Ex Taq DNA polymerase 0.5 μ L, adds ddH
2o is 25 μ L to reaction cumulative volume.Amplification program is as follows: 95 DEG C of annealing 4min; 94 DEG C of 55s, 65 DEG C of 40s, 72 DEG C of 1min, circulate 5 times; 72 DEG C extend 2min.
Second step adds 25 μ L reactive material again on the first step basis, and system is as follows: 10 × PCR buffer 5 μ L, Mg
2+2 μ L, dNTP 4 μ L, fragment template P43 upstream primer to be overlapped and each 2 μ L of template fdh downstream primer, ExTaqDNA polysaccharase 1 μ L, adds ddH
2o is 50 μ L to reaction cumulative volume.Amplification program is as follows: 95 DEG C of annealing 4min; 94 DEG C of 50s, 55 ~ 60 DEG C of 40s, 72 DEG C of 1min30s, circulate 30 times; 72 DEG C extend 10min.As shown in Figure 2, stripe size is about 1600bp to the electrophorogram of overlapping fragments after cutting glue and reclaiming, and shows P43 promotor to be connected with fdh gene.
Utilize pMA5 plasmid construction expression vector, schema is shown in Fig. 3.
2.2 restriction endonuclease reaction, purifying and ligation
The PCR primer that embodiment 2.1 obtains is connected with pMD18-T carrier, product conversion competent cell E.coli JM109 will be connected, will the bacterium liquid of positive colony be accredited as (containing Amp
+lB substratum) send Nanjing Genscript Biotechnology Co., Ltd. to check order.
Double digestion reaction is carried out with the enzyme of correspondence.In this experiment, restriction enzyme used is Nde I and BamH I.The enzyme system of cutting is: carrier T 50 μ L, BamH I 2.5 μ L, Nde I 2.5 μ L, 10 × damping fluid 10 μ L, ddH
2o35 μ L, cumulative volume 100 μ L.PCR primer after DNA Purification Kit enzyme is cut.
Same is that BamH I and Nde I carries out enzyme to pMA5 plasmid and cuts with restriction enzyme, because BamH I and Nde I close proximity in the multiple clone site of pMA5 plasmid (general as the purchase of spit of fland biotechnology (Beijing) company limited), about 130bp, so the plasmid after linearizing only needs through DNA Purification Kit.
Be connected through the PCR primer after purifying and pMA5 linearization plasmid carries out.Linked system is: enzyme cuts the PCR primer 3 μ L of purifying, and enzyme cuts the pMA5 plasmid 2 μ L of purifying, Solution I 5 μ L.Spend the night to connect at 37 DEG C and obtain recombinant plasmid pMA5-P43-fdh.
The conversion of 2.3 recombinant plasmid pMA5-P43-fdh
Calcium Chloride Method prepares competence Bacillus coli cells.
(1) 10 μ L recombinant plasmid pMA5-P43-fdh are got in 200 μ L intestinal bacteria Escherichia coli JM109 (purchase of Beijing North Na Kaichuan Bioisystech Co., Ltd) competent cells, ice bath 30min.
(2) 42 DEG C of water-bath heat shock 90s, are placed in 2 ~ 3min on ice fast.
(3) fresh LB liquid nutrient medium 800 μ L is added, in 37 DEG C of shaking culture 45 ~ 60min.
(4) get 150 μ L thalline and coat LB solid culture primary surface containing 20 μ g/mL ammonia benzyl resistances.Cultivate 12 ~ 16h for 37 DEG C to occur to single bacterium colony.
After correct for 37 DEG C of cultivation 12h, the bacterium liquid pcr checkings in the LB liquid nutrient medium containing ammonia benzyl (20 μ g/mL) of single colony inoculation, extract the sub-plasmid of positive colony with plasmid extraction kit.
The acquisition of 2.4 recombinant bacterium P.polymyxa GX-1
Take out the P.polymyxa ZJ-9 of slant preservation, Paenibacillus polymyxa paenibacillus polymyxa CGMCC 3044 in the patent (application number is 201410018001.9) that this bacterial strain is applied for before being exactly us, in 30 DEG C of LB liquid nutrient medium activation about 12h, prepare the triangular flask of 20 250mL, the bottled 50mL of each triangle adds the LB substratum of 0.5mol/L sorbyl alcohol, regulates pH to be 7.0, the bacterial classification of the activation of access 6%, 30 DEG C, 200r/min cultivates.Take out a triangular flask every 1h and measure its OD
660the nonvaccinated LB substratum containing 0.5mol/L sorbyl alcohol is as blank, dilute with blank solution when concentration is excessive, keep OD at 0.2-0.8, actual OD value is multiplied by extension rate by surveying OD, take time as X-coordinate, actual OD value draws out the growth curve of p.polymyxa ZJ-9 for ordinate zou, chooses the bacterium liquid of OD value when 0.7-0.9 as competence.
Getting P.polymyxa ZJ-9 is inoculated in LB liquid nutrient medium, cultivates 12h for 30 DEG C; Be transferred to 50mL containing in the LB liquid nutrient medium of 0.5mol/L sorbyl alcohol with the inoculum size of 6% (V/V), 30 DEG C, 200r/min is cultured to 8h, now OD
660be about 0.8, stop cultivating, place 20min on ice, the centrifugal 10min of 6000 × g, collect thalline; With containing 0.5mol/L sorbyl alcohol, the N.F,USP MANNITOL of 0.5mol/L, the liquid scrubbing thalline of the glycerine of 10% 2-3 time; Finally use the cleaning mixture re-suspended cell of 500 μ L, be distributed into 80 μ L/ and manage ,-70 DEG C of preservations.
The recombinant plasmid pMA5-P43-fdh successfully constructed getting 1 μ L adds in the p.polymyxa ZJ-9 competent cell of 80 μ L, mixed solution is transferred in the electric shock cup of 0.1cm, places 5min on ice, electric shock, parameter is 2.2kV, 4ms, then adds the LB liquid nutrient medium that 1mL contains the sorbyl alcohol of 0.5mol/L and the N.F,USP MANNITOL of 0.38mol/L, 28 DEG C, 4h cultivated by 200r/min shaking table, then coat on the LB substratum containing kantlex, 30 DEG C of incubated overnight, picking positive transformant.
At the dull and stereotyped enterprising row filter of kalamycin resistance, obtain 10 left/right rotation beggars altogether.By whole for these recombinant bacteriums picking to containing in the liquid nutrient medium of kalamycin resistance, extract plasmid checking.As shown in Figure 4, swimming lane 1 is recombinant bacterium extraction plasmid result to one of them positive strain result, and result shows have plasmid to proceed in this recombinant bacterium.For verifying whether plasmid is recombinant vectors pMA5-P43-fdh further, we carry out single double digestion checking with BamH I single endonuclease digestion and with Nde I and BamHI to these three plasmids respectively, result is as shown in the swimming lane 2,3 of Fig. 4, there is the band of about 9000bp in swimming lane 2, there is about 7500bp and about 1600bp band in swimming lane 3, size is identical with expection, illustrates that pMA5-P43-fdh successfully proceeds to P.polymyxa ZJ-9, by recombinant bacterium called after P.polymyxa GX-1.
Embodiment 3: utilize genetic engineering bacterium to produce R, R-2,3-butyleneglycol
Activation medium (g/L): peptone 10, extractum carnis 3, NaCl 5, agar 10 ~ 20, pH 7.0.Getting Storage in refrigerator bacterial strain is scoring on activation medium, to be placed in incubator 30 DEG C of constant temperature culture 1 day.
Seed culture medium (g/L): inulin 20, peptone 10, yeast extract paste 3, NaCl 5, pH 7.0, the seed liquor prepared 115 DEG C of sterilizing 30min, are placed in 30 DEG C, cultivate 22h to the logarithmic growth middle and later periods in the shaking table of 200r/min.
Fermention medium (g/L): inulin 75, yeast extract paste 6, peptone 2, MgSO
47H
2o 0.5, K
2hPO
43.09, MnSO
4h
2o 0.001, NH
4cl 0.93, FeSO
47H
2o 0.04, ZnSO
47H
2o 0.001, pH 6.0.Add 5 μ g/mL kantlex in the medium during recombinant bacterium fermentation, prevent the loss of plasmid.
7.5L fermentor cultivation (BioFlo110, NBS company of the U.S.): temperature 30 DEG C, air flow 1vvm, liquid amount 4L, inoculum size 8%, front 22h rotating speed is 300r/min, rear 22h is adjusted to 200r/min, pH regulating strategy is: the fermentation initial stage does not control fermented liquid pH, in fermenting process along with pH value drop to below 6.0 time, with 4M NaOH adjust pH be 6.0.
Recombinant bacterium and wild mushroom ferment 44h, and get 1mL fermented liquid 1000 × g, centrifugal 5min obtains supernatant, carry out liquid phase analysis, chromatographic column: AminexHPX-87H after crossing the film of 0.22 μm; The H of moving phase: 4mM
2sO
4; Flow velocity: 0.6mL/min; Sample size: 20 μ L; Column temperature: 60 DEG C, Composition distribution detects.Fermentation results shows, during wild mushroom fermentation 44h, and R, the output of R-2,3-butyleneglycol reaches 33.4g/L, and byproduct formic acid content is 3.3g/L, and the output of recombinant bacterium R, R-2,3-butyleneglycol reaches 36.8g/L, compared with wild mushroom, output increased 10.2%, byproduct formic acid content is reduced to 0.9g/L, illustrate that cell produces formate dehydrogenase enzymes metabolism in the reorganized bacterium of a part of by product and falls, be converted into metabolizable energy and facilitate R, the generation of R-2,3-butyleneglycol.
Above are only the preferred embodiment of the invention, be not restricted to the present invention.To those of ordinary skill in the art, other multi-form change or variations can also be made on the basis of the above description.Here without the need to also illustrating all embodiments.And thus scheme the apparent change of extending out or variation be still within protection scope of the present invention.
Claims (7)
1. for a primer pair for the hydrogenlyase that increases, it is characterized in that, it is as follows that described primer pair comprises two groups: first group of primer pair:
F1：5′-AGGCCGGGGCATATGGGAAACA-3′
R15′-TAAGACTAAAACGATCTTCATAAGCTTCTGTTATTAATTCTT-3′
Second group of primer pair is as follows:
F2：5′-AAGAATTAATAACAGAAGCTTATGAAGATCGTTTTAGTCTTA-3′
R2：5′-CCGGGATCCTTATTTCTTATCGTGTTTACC-3′。
2. the recombinant bacterium of a heterogenous expression hydrogenlyase, it is characterized in that, it is for template carries out with first of claim 1 group of primer pair the P43 promoter fragment that pcr amplification obtains acquisition with B.subtilis 168 genome, with the genome of candiyeast Candida boidinii for template obtains formate dehydrogenase gene fragment fdh with second of claim 1 group of primer pair pcr amplification, again the P43 promoter fragment of acquisition and formate dehydrogenase gene fragment are carried out over-lap PCR, the product of over-lap PCR acquisition is connected with plasmid pMA5 under the effect of T4 ligase enzyme and obtains recombinant plasmid pMA5-P43-fdh, finally recombinant plasmid pMA5-P43-fdh electricity is proceeded in P.polymyxa ZJ-9 and obtain recombinant bacterium P.polymyxa GX-1.
3. the construction process of the recombinant bacterium of a kind of heterogenous expression hydrogenlyase according to claim 1, is characterized in that, comprise the following steps:
1) with B.subtilis 168 genome for stencil design first group of primer pair, as shown in claim 1 sequence, pcr amplification obtains P43 strong promoter fragment, cut glue reclaim for subsequent use;
2) with the genome of candiyeast Candida boidinii for template, design second group of primer pair, as shown in claim 1 sequence, pcr amplification obtains formate dehydrogenase gene fragment fdh;
3) the P43 strong promoter fragment of acquisition and formate dehydrogenase gene fdh are carried out over-lap PCR;
4) through restriction enzyme BamH I and Nde I double digestion, under the effect of T4 ligase enzyme, carry out connection with the plasmid pMA5 through same double digestion and obtain recombinant plasmid pMA5-P43-fdh;
5) recombinant plasmid pMA5-P43-fdh electricity is proceeded to P.polymyxa ZJ-9, obtain recombinant bacterium P.polymyxa GX-1.
4. the application of recombinant bacterium at preparation R, R-2,3-butyleneglycol aspect of a kind of heterogenous expression hydrogenlyase according to claim 2.
5. the production method of a R, R-2,3-butyleneglycol, is characterized in that, obtains in fermentation cylinder for fermentation with recombinant bacterium P.polymyxa GX-1 according to claim 2.
6. the production method of a kind of R, R-2,3-butyleneglycol according to claim 5, it is characterized in that, described fermentation culture conditions is: temperature 30 DEG C, air flow 1vvm, liquid amount 4L, inoculum size 8%, front 22h rotating speed is 300r/min, and rear 22h is adjusted to 200r/min, and pH regulating strategy is: the fermentation initial stage does not control fermented liquid pH, in fermenting process along with pH value drop to below 6.0 time, with 4M NaOH adjust pH be 6.0.
7. the production method of a kind of R, R-2,3-butyleneglycol according to claim 5, is characterized in that, activation medium in described fermenting process: peptone 10g/L, extractum carnis 3g/L, NaCl 5g/L, agar 10 ~ 20g/L, pH 7.0; Seed culture medium: inulin 20g/L, peptone 10g/L, yeast extract paste 3g/L, NaCl 5g/L, pH 7.0; Fermention medium: inulin 75g/L, yeast extract paste 6g/L, peptone 2g/L, MgSO
47H
2o 0.5g/L, K
2hPO
43.09g/L, MnSO
4h
2o 0.001g/L, NH
4cl 0.93g/L, FeSO
47H
2o 0.04g/L, ZnSO
47H
2o 0.001g/L, pH 6.0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107299074A (en) * | 2017-08-30 | 2017-10-27 | 山东省科学院生态研究所 | The construction method of hydrogenlyase engineered strain and application |
| CN107354178A (en) * | 2017-07-20 | 2017-11-17 | 盐城工学院 | A kind of method added amino acid and promote the butanediol of Microbe synthesis 2,3 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565683A (en) * | 2009-06-01 | 2009-10-28 | 盐城工学院 | Bacillus polymyxa and method for preparing optically-pure R, R type 2, 3-butanedio by utilizing same |
| CN103060255A (en) * | 2013-01-24 | 2013-04-24 | 盐城工学院 | Genetically engineering bacterium capable of producing S-3-hydroxy-butanone as well as construction method and application thereof |
| CN103451141A (en) * | 2013-08-09 | 2013-12-18 | 天津大学 | Bacillus subtilis strain for producing high-purity chiral meso-2,3-butanediol as well as construction method and application of strain |
| CN103756949A (en) * | 2014-01-15 | 2014-04-30 | 盐城工学院 | Gene engineering bacteria for producing ultrahigh-optical purity R,R-2,3-butanediol as well as construction method and application thereof |
-
2015
- 2015-05-11 CN CN201510236085.8A patent/CN104830851A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565683A (en) * | 2009-06-01 | 2009-10-28 | 盐城工学院 | Bacillus polymyxa and method for preparing optically-pure R, R type 2, 3-butanedio by utilizing same |
| CN103060255A (en) * | 2013-01-24 | 2013-04-24 | 盐城工学院 | Genetically engineering bacterium capable of producing S-3-hydroxy-butanone as well as construction method and application thereof |
| CN103451141A (en) * | 2013-08-09 | 2013-12-18 | 天津大学 | Bacillus subtilis strain for producing high-purity chiral meso-2,3-butanediol as well as construction method and application of strain |
| CN103756949A (en) * | 2014-01-15 | 2014-04-30 | 盐城工学院 | Gene engineering bacteria for producing ultrahigh-optical purity R,R-2,3-butanediol as well as construction method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| YU WANG ET AL.: "Engineering of cofactor regeneration enhances (2S,3S)-2,3-butanediol production from diacetyl", 《SCIENTIFIC REPORTS》 * |
| YU WANG ET AL.: "Engineering of cofactor regeneration enhances (2S,3S)-2,3-butanediol production from diacetyl.", 《SCIENTIFIC REPORTS》 * |
| 王青艳 等: "微生物法合成(R,R)-2,3-丁二醇的研究进展与展望", 《基因组学与应用生物学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107354178A (en) * | 2017-07-20 | 2017-11-17 | 盐城工学院 | A kind of method added amino acid and promote the butanediol of Microbe synthesis 2,3 |
| CN107299074A (en) * | 2017-08-30 | 2017-10-27 | 山东省科学院生态研究所 | The construction method of hydrogenlyase engineered strain and application |
| CN107299074B (en) * | 2017-08-30 | 2020-06-16 | 山东省科学院生态研究所 | Construction method and application of formate dehydrogenase engineering strain |
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