CN104560856A - Escherichia coli for aerobically synthesizing vitamin B12 as well as construction and application of escherichia coli - Google Patents
Escherichia coli for aerobically synthesizing vitamin B12 as well as construction and application of escherichia coli Download PDFInfo
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Abstract
The invention discloses an escherichia coli for aerobically synthesizing vitamin B12 as well as construction and application of the escherichia coli, and belongs to the field of metabolic engineering and microorganism fermentations. The Escherichia coli is a host cell, the genes deficient in the escherichia coli genome from rhodobacter sphaeroides and pseudomonas putida vitamin B12 synthesis path are assembled in a modular mode and expressed by using a compatible expression vector, and a recombinant escherichia coli engineering strain is constructed; the vitamin B12 is synthesized by the escherichia coli through the fermenting verification.
Description
Technical field
The present invention relates to a kind of aerobic synthesise vitamins B
12intestinal bacteria and study on its developing, belong to metabolic engineering and field of microbial fermentation.
Background technology
Vitamins B
12(Vitamin B
12, VB
12), be also called cobalami, molecular formula is C
63h
88coN
14o
14p, molecular weight is 1355.38.It is the general name that a class contains the corrin compounds of cobalt, is maximum, the most complicated vitamin molecules found at present, is also the VITAMIN uniquely containing metal ion; Its crystallization is red, therefore also known as red VITAMIN.
1956, Hodgkin etc. demonstrated its crystalline structure by x-ray method, i.e. center corrin ring, the axial Co β aglucon part of center ring and 1 Co α aglucon containing Nucleotide ring, and structure is very complicated.Vitamins B
12as a kind of important VITAMIN, its main Physiological Function participates in manufacturing erythrocyte, prevents pernicious anemia; Cerebral nerve is prevented to be damaged.At present, the fields such as feed, food, medical and health and makeup have been widely used in.
1972, American scientist Woodward, R.B. led 100 multidigit co-workers to last 11 years, jointly complete vitamins B
12full chemosynthesis.But because its chemosynthesis step is many, yield is extremely low, and therefore chemosynthesis is very expensive.In early days, people are from the tissue extraction such as animal livers, kidney vitamins B
12, but yield and benefit are also very low.Afterwards, extract from streptomycin fermentation waste liquid, yield is still very low.At present, vitamins B in industrial production
12production mainly adopt microbe fermentation method.
Occurring in nature, vitamins B
12de novo synthesis is divided into two approach, article one, be anaerobism route of synthesis, be present in the bacteriums such as propionibacterium freudenreichii (Propionibacteriumfreudenreichi), Xie Shi propionibacterium (Propionibacterium shermanii), salmonella (Salmonella sp.) and bacillus megaterium (Bacillus megaterium); Another is aerobic route of synthesis, is mainly present in Pseuomonas denitrifican (Pseudomonas denitrificans) (aerobic bacterial strain) and hydrogenlike silicon ion (Rhodobacter sphaeroides) (facultative aerobe strain).At present, for industrial vitamins B
12bacterial strain mainly P.shermanii and P.denitrificans of fermentation.Substratum take glucose as primary carbon source, and corn steep liquor is major nitrogen source, adds cobalt ion and precursor 5,6-dimethyl benzene imipramine (DMBI).In recent years, because aerobic fermentation bacterial strain has plurality of advantages, as grow fast, culture condition is comparatively simple and it is easy to control etc. to ferment.Aerobic fermentation process is the main technique of current suitability for industrialized production vitamin B12, the output in the whole world more than 80% is from this technique, be produce bacterial strain with P.denitrificans, substratum adopts beet sirup or maltose to be primary carbon source, corn steep liquor or yeast extract paste are nitrogenous source, add inorganic salt, cobalt ion and DMBI.At present, attention is placed on the zymotechnique by optimizing P.denitrificans by relevant scholar both at home and abroad, to improving vitamins B
12output.
The present invention derives from hydrogenlike silicon ion and pseudomonas putida vitamins B at expression in escherichia coli
12the gene of route of synthesis, adopts the expression vector of different copy number to carry out modularization assembling to gene, builds vitamins B
12the strain of from the beginning aerobic route of synthesis colibacillus engineering.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of aerobic synthesise vitamins B
12colibacillus engineering strain, be the vitamins B adopting the carrier module assembling expression of different copy number to derive from hydrogenlike silicon ion (Rhodobactersphaeroides) and pseudomonas putida (Pseudomonasputida) in intestinal bacteria
12the gene of route of synthesis, building with glucose is substrate from the beginning aerobic synthesise vitamins B
12approach.
Described vitamins B
12the gene of route of synthesis comprises cobA, cobI, cobG, cobJ, cobM, cobF, cobK, cobL, cobH, cobB, cobN, cobS, cobT, cobR, cobO, cobQ, cobC and cobD.
In one embodiment of the invention, described vitamins B
12the gene cobA of route of synthesis, cobI, cobJ, cobM, cobF, cobK, cobL, cobH, cobB, cobN, cobS, cobT, cobO, cobQ, cobC and cobD derive from hydrogenlike silicon ion, cobG and cobR derives from pseudomonas putida.
In one embodiment of the invention, the nucleotide sequence of described cobA is as shown in SEQ ID NO.1, the nucleotide sequence of cobI is as shown in SEQ ID NO.2, the nucleotide sequence of cobG is as shown in SEQ ID NO.3, the nucleotide sequence of cobJ is as shown in SEQ ID NO.4, the nucleotide sequence of cobM is as shown in SEQ ID NO.5, the nucleotide sequence of cobF is as shown in SEQID NO.6, the nucleotide sequence of cobK is as shown in SEQ ID NO.7, the nucleotide sequence of cobL is as shown in SEQ ID NO.8, the nucleotide sequence of cobH is as shown in SEQ ID NO.9, the nucleotide sequence of cobB is as shown in SEQ ID NO.10, the nucleotide sequence of cobN is as shown in SEQ ID NO.11, the nucleotide sequence of cobS is as shown in SEQ ID NO.12, the nucleotide sequence of cobT is as shown in SEQ ID NO.13, the nucleotide sequence of cobR is as shown in SEQ ID NO.14, the nucleotide sequence of cobO is as shown in SEQ ID NO.15, the nucleotide sequence of cobQ is as shown in SEQ ID NO.16, the nucleotide sequence of cobC is as shown in SEQ ID NO.17, the nucleotide sequence of cobD is as shown in SEQ ID NO.18.
In one embodiment of the invention, described escherichia coli host comprises DH5 α, JM109, W3110, BL21 (DE3), MG1655.
In one embodiment of the invention, described intestinal bacteria are BL21 (DE3).
Described different copy number expression vector is respectively pACYCDuet-1, pCDFDuet-1, pETDuet-1 and pRSFDuet-1.
In one embodiment of the invention, cobA, cobI, cobG, cobJ and cobM is expressed with pACYCDuet-1, pCDFDuet-1 expresses cobF, cobK, cobL, cobH and cobB, pETDuet-1 expresses cobN, cobS and cobT, and pRSFDuet-1 expresses cobR, cobO, cobQ, cobC and cobD.
In one embodiment of the invention, before the netic module of each independent assembling, add PgapA promotor and ribosome bind site.
In one embodiment of the invention, use that pMD19 is carrier, isocaudarner assembles gene.
In one embodiment of the invention, the construction process of described colibacillus engineering strain mainly comprises the following steps:
(1) respectively by PgapA and cobM, cobJ and cobG, cobI with cobA is connected by fusion DNA vaccine method, is assembled into PgapA-cobM, cobJG, cobIA.Then be carrier with pMD19, utilize isocaudarner SpeI with XbaI and BamHI with BglII 3 fragments to be connected, build plasmid pMD19-PgapA-cobMJGIA.Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pACYCDuet-1 cut through same enzyme, build plasmid pACYCDuet-1-PgapA-cobMJGIA.
(2) respectively by PgapA and cobB, cobH and cobL, cobK with cobF is connected by fusion DNA vaccine method, is assembled into PgapA-cobB, cobHL, cobKF.Then be carrier with pMD19, utilize isocaudarner NsiI with PstI and SpeI with XbaI 3 fragments to be connected, build plasmid pMD19-PgapA-cobBHLKF.Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pCDFDuet-1 cut through same enzyme, build plasmid pCDFDuet-1-PgapA-cobBHLKF.
(3) respectively PgapA and cobN, cobS and cobT are connected by fusion DNA vaccine method, are assembled into PgapA-cobN, cobST.Then be carrier with pMD19, utilize isocaudarner SpeI with XbaI 2 fragments to be connected, build plasmid pMD19-PgapA-cobNST.Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pETDuet-1 cut through same enzyme, build plasmid pETDuet-1-PgapA-cobNST.
(4) respectively by PgapA and cobD, cobC and cobQ, cobO with cobR is connected by fusion DNA vaccine method, is assembled into PgapA-cobD, cobCQ, cobOR.Then be carrier with pMD19, utilize isocaudarner SpeI with XbaI and BamHI with BglII 3 fragments to be connected, build plasmid pMD19-PgapA-cobDCQOR.Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pRSFDuet-1 cut through same enzyme, build plasmid pRSFDuet-1-PgapA-cobDCQOR.
(5) recombinant plasmid pACYCDuet-1-PgapA-cobMJGIA, pCDFDuet-1-PgapA-cobBHLKF, pETDuet-1-PgapA-cobNST and pRSFDuet-1-PgapA-cobDCQOR cotransformation E.coli BL21 (DE3) will built, obtains recombinant bacterial strain VB12-MBND (E.coli BL21 (DE3)/pACYCDuet-1-cobMJGIA pCDFDuet-1-cobBHLKF pETDuet-1-cobNSTpRSFDuet-1-cobDCQOR).
The present invention also provides a kind of application described colibacillus engineering fermentative production vitamins B
12method, be will recombinant bacterium activate after be transferred in fermention medium with the inoculum size of 2-5%, add 0.1-0.5mM IPTG inducible gene expression during 0h, add paraxin, Streptomycin sulphate, penbritin, kantlex as required, 30-37 DEG C, 200r/min cultivate, cycle 60-72h.Fermention medium (g/L): glucose 60-80, yeast extract paste 25-30, (NH
4)
2hPO
42.5-3.0, MgSO
47H
2o 1.5-2.0, CoCl6H
2o 0.05-0.1,5,6-dimethylbenzimidazole (DMBI) 0.01-0.05,5-ALA 0.05-0.1, ZnSO
47H
2o 0.05-0.1, pH 7.0 ~ 7.2.
In one embodiment of the invention, with 2% inoculum size switching after recombinant bacterium is activated, 0.1-0.5mMIPTG inducible gene expression is added during 0h, add paraxin (34 μ g/mL), Streptomycin sulphate (100 μ g/mL) as required, penbritin (100 μ g/mL), kantlex (50 μ g/mL), 30-37 DEG C, 200r/min cultivates, cycle 60-72h.Fermention medium (g/L): glucose 80, yeast extract paste 30, (NH
4)
2hPO
43.0, MgSO
47H
2o 2.0, CoCl6H
2o 0.1,5,6-dimethylbenzimidazole (DMBI) 0.05,5-ALA 0.1, ZnSO
47H
2o 0.1, pH 7.0 ~ 7.2.
The present invention adopts the carrier module assembling expression of different copy number to derive from hydrogenlike silicon ion and pseudomonas putida vitamins B in intestinal bacteria
12the gene cobA of route of synthesis, cobI, cobG, cobJ, cobM, cobF, cobK, cobL, cobH, cobB, cobN, cobS, cobT, cobR, cobO, cobQ, cobC and cobD, build vitamins B
12from the beginning aerobic route of synthesis, realize microbe fermentation method direct aerobic fermentation glucose synthesise vitamins B
12.
Accompanying drawing explanation
Fig. 1: gene assembling and plasmid construction figure
Fig. 2: vitamins B is produced in colibacillus engineering strain fermentation
12mass Spectrometric Identification
A: cyanocobalamin standard specimen (100mg/L)
B: control strain: E.coli BL21 (DE3)
C: recombinant bacterial strain: E.coli BL21 (DE3) VB12-MBND
Embodiment
Vitamins B
12analytical procedure (high performance liquid chromatography):
Sample preparation: add 8%NaNO in the 10mL fermented liquid got ready
3solution and each 2.5mL of Glacial acetic acid, shake up, put after heating 30min in 95 ~ 100 DEG C of water-baths and take out, be cooled to room temperature, add deionized water after illumination 30min under 50W incandescent light and be settled to 50mL, filter, gained filtrate filters 1mL to sample bottle by 0.22 μm of millipore filtration syringe filters, draws sodium cyanide solution 20 μ L and puts in human sample's bottle, put by sample bottle in people's 35 ~ 40 DEG C of water-baths and react 1h with microsyringe, take out, carry out liquid-phase chromatographic analysis under prescribed conditions.
High-efficient liquid phase chromatogram condition: moving phase: 250mmol/L phosphate aqueous solution-acetonitrile (30:70, v/v), chromatographic column: BackmanC18 post (4.6mm × 250mm, 5 μm); Determined wavelength: 361nm; Sample size: 20 μ L; Flow velocity: 1.0mL/min; Column temperature: 25 DEG C.
Substratum:
Slant medium (g/L): peptone 10, sodium-chlor 10, yeast powder 5.0, agar 20, pH 7.0;
Seed culture medium (g/L): glucose 40, yeast extract paste 10, NH
4cl 1.0, MgSO
47H
2o 0.5, ZnSO
47H
2o0.1, pH 7.2 ~ 7.4;
Fermention medium (g/L): glucose 80, yeast extract paste 30, (NH
4)
2hPO
43.0, MgSO
47H
2o 2.0, CoCl6H
2o0.1,5,6-dimethylbenzimidazole (DMBI) 0.05,5-ALA 0.1, ZnSO
47H
2o 0.1, pH 7.0 ~ 7.2.
Culture condition:
Spawn culture: glycerine pipe is rule, and then picking list bacterium colony streak plate 37 DEG C cultivation, as seed source;
Seed culture: dull and stereotyped picking thalline, 37 DEG C, 200r/min, add paraxin 34 μ g/mL as requested, Streptomycin sulphate 100 μ g/mL, penbritin 100 μ g/mL, kantlex 50 μ g/mL, cultivates about 12h, switching fermention medium;
Fermentation culture: with 2% inoculum size switching, 0.1-0.5mM IPTG inducible gene expression is added during 0h, add paraxin (34 μ g/mL), Streptomycin sulphate (100 μ g/mL) as required, penbritin (100 μ g/mL), kantlex (50 μ g/mL), 30-37 DEG C, 200r/min cultivate, cycle 60-72h.
The structure of embodiment 1 recombinant plasmid and engineering strain
(1) gene assembling and plasmid construction
Gene is carried out modularization assembling, before the netic module of each independent assembling, adds PgapA promotor and ribosome bind site.Because assembled gene is more and fragment is longer, restriction enzyme site available in connection procedure is less, and the present embodiment uses pMD19 to be carrier, uses isocaudarner and carries out gene assembling (Fig. 1).Construction recombination plasmid pACYCDuet-1-PgapA-cobMJGIA, pCDFDuet-1-PgapA-cobBHLKF, pETDuet-1-PgapA-cobNST and pRSFDuet-1-PgapA-cobDCQOR:
(1) respectively by PgapA and cobM, cobJ and cobG, cobI with cobA is connected by fusion DNA vaccine method, is assembled into PgapA-cobM, cobJG, cobIA.Then be carrier with pMD19, utilize isocaudarner SpeI with XbaI and BamHI with BglII 3 fragments to be connected, build plasmid pMD19-PgapA-cobMJGIA.Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pACYCDuet-1 cut through same enzyme, build plasmid pACYCDuet-1-PgapA-cobMJGIA.
(2) respectively by PgapA and cobB, cobH and cobL, cobK with cobF is connected by fusion DNA vaccine method, is assembled into PgapA-cobB, cobHL, cobKF.Then be carrier with pMD19, utilize isocaudarner NsiI with PstI and SpeI with XbaI 3 fragments to be connected, build plasmid pMD19-PgapA-cobBHLKF.Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pCDFDuet-1 cut through same enzyme, build plasmid pCDFDuet-1-PgapA-cobBHLKF.
(3) respectively PgapA and cobN, cobS and cobT are connected by fusion DNA vaccine method, are assembled into PgapA-cobN, cobST.Then be carrier with pMD19, utilize isocaudarner SpeI with XbaI 2 fragments to be connected, build plasmid pMD19-PgapA-cobNST.Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pETDuet-1 cut through same enzyme, build plasmid pETDuet-1-PgapA-cobNST.
(4) respectively by PgapA and cobD, cobC and cobQ, cobO with cobR is connected by fusion DNA vaccine method, is assembled into PgapA-cobD, cobCQ, cobOR.Then be carrier with pMD19, utilize isocaudarner SpeI with XbaI and BamHI with BglII 3 fragments to be connected, build plasmid pMD19-PgapA-cobDCQOR.Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pRSFDuet-1 cut through same enzyme, build plasmid pRSFDuet-1-PgapA-cobDCQOR.
(2) vitamins B
12the structure of recombinant escherichia coli strain
By recombinant plasmid pACYCDuet-1-PgapA-cobMJGIA, pCDFDuet-1-PgapA-cobBHLKF, pETDuet-1-PgapA-cobNST and pRSFDuet-1-PgapA-cobDCQOR cotransformation E.coli BL21 (DE3) built, obtain recombinant bacterial strain VB
12-MBND.
Embodiment 2 recombination bacillus coli engineering strain shake flask fermentation is verified
Recombination bacillus coli VB
12-MBND ferments checking in 250mL triangular flask, inoculum size 2%, and initial glucose concentration is that 80g/L, 0h add 0.1-0.5mM IPTG induction and corresponding microbiotic, after fermentation ends, processes somatic cells, analyzes vitamins B
12synthesis, measure through liquid phase and mass spectroscopy, vitamins B
12output be about 100 μ g/L (Fig. 2).
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. an aerobic synthesise vitamins B
12colibacillus engineering strain, it is characterized in that, be in intestinal bacteria, adopt the carrier moduleization of different copy number to assemble express to derive from the vitamins B of hydrogenlike silicon ion and pseudomonas putida
12the gene of route of synthesis, building with glucose is the aerobic synthesise vitamins B of substrate
12approach.
2. colibacillus engineering strain according to claim 1, is characterized in that, described vitamins B
12the gene of route of synthesis comprises cobA, cobI, cobG, cobJ, cobM, cobF, cobK, cobL, cobH, cobB, cobN, cobS, cobT, cobR, cobO, cobQ, cobC and cobD.
3. colibacillus engineering strain according to claim 2, is characterized in that, described vitamins B
12gene cobA, cobI, cobJ, cobM, cobF, cobK, cobL, cobH, cobB, cobN, cobS, cobT, cobO, cobQ, cobC and cobD of route of synthesis derive from hydrogenlike silicon ion, cobG and cobR derives from pseudomonas putida.
4. the colibacillus engineering strain according to Claims 2 or 3, it is characterized in that, the nucleotide sequence of described cobA is as shown in SEQ ID NO.1, the nucleotide sequence of cobI is as shown in SEQ ID NO.2, the nucleotide sequence of cobG is as shown in SEQID NO.3, the nucleotide sequence of cobJ is as shown in SEQ ID NO.4, the nucleotide sequence of cobM is as shown in SEQ ID NO.5, the nucleotide sequence of cobF is as shown in SEQ ID NO.6, the nucleotide sequence of cobK is as shown in SEQ ID NO.7, the nucleotide sequence of cobL is as shown in SEQ ID NO.8, the nucleotide sequence of cobH is as shown in SEQ ID NO.9, the nucleotide sequence of cobB is as shown in SEQ ID NO.10, the nucleotide sequence of cobN is as shown in SEQ ID NO.11, the nucleotide sequence of cobS is as shown in SEQ ID NO.12, the nucleotide sequence of cobT is as shown in SEQ ID NO.13, the nucleotide sequence of cobR is as shown in SEQ ID NO.14, the nucleotide sequence of cobO is as shown in SEQ ID NO.15, the nucleotide sequence of cobQ is as shown in SEQ ID NO.16, the nucleotide sequence of cobC is as shown in SEQ ID NO.17, the nucleotide sequence of cobD is as shown in SEQ IDNO.18.
5. colibacillus engineering strain according to claim 1, is characterized in that, described intestinal bacteria comprise DH5 α, JM109, W3110, BL21 (DE3), MG1655.
6. colibacillus engineering strain according to claim 1, is characterized in that, described different copy number expression vector is respectively pACYCDuet-1, pCDFDuet-1, pETDuet-1 and pRSFDuet-1.
7. colibacillus engineering strain according to claim 1, it is characterized in that, cobA, cobI, cobG, cobJ and cobM is expressed with pACYCDuet-1, pCDFDuet-1 expresses cobF, cobK, cobL, cobH and cobB, pETDuet-1 expresses cobN, cobS and cobT, and pRSFDuet-1 expresses cobR, cobO, cobQ, cobC and cobD.
8. colibacillus engineering strain according to claim 7, is characterized in that, by PgapA and cobM, cobJ and cobG, cobI and cobA are connected by fusion DNA vaccine method, are assembled into PgapA-cobM, cobJG, cobIA, be connected with plasmid pACYCDuet-1 after connection again; By PgapA and cobB, cobH and cobL, cobK with cobF is connected by fusion DNA vaccine method, is assembled into PgapA-cobB, and cobHL, cobKF are connected with pCDFDuet-1 after connection again; PgapA and cobN, cobS and cobT are connected by fusion DNA vaccine method, are assembled into PgapA-cobN, cobST, be connected with pETDuet-1 again after connection, build plasmid pETDuet-1-PgapA-cobNST; By PgapA and cobD, cobC and cobQ, cobO with cobR is connected by fusion DNA vaccine method, is assembled into PgapA-cobD, cobCQ, cobOR, connects and is connected with pRSFDuet-1.
9. build a method for colibacillus engineering strain described in claim 1, it is characterized in that, mainly comprise the following steps:
(1) respectively by PgapA and cobM, cobJ and cobG, cobI with cobA is connected by fusion DNA vaccine method, is assembled into PgapA-cobM, cobJG, cobIA; Then be carrier with pMD19, utilize isocaudarner SpeI with XbaI and BamHI with BglII 3 fragments to be connected, build plasmid pMD19-PgapA-cobMJGIA; Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pACYCDuet-1 cut through same enzyme, build plasmid pACYCDuet-1-PgapA-cobMJGIA;
(2) respectively by PgapA and cobB, cobH and cobL, cobK with cobF is connected by fusion DNA vaccine method, is assembled into PgapA-cobB, cobHL, cobKF.Then be carrier with pMD19, utilize isocaudarner NsiI with PstI and SpeI with XbaI 3 fragments to be connected, build plasmid pMD19-PgapA-cobBHLKF; Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pCDFDuet-1 cut through same enzyme, build plasmid pCDFDuet-1-PgapA-cobBHLKF;
(3) respectively PgapA and cobN, cobS and cobT are connected by fusion DNA vaccine method, are assembled into PgapA-cobN, cobST; Then be carrier with pMD19, utilize isocaudarner SpeI with XbaI 2 fragments to be connected, build plasmid pMD19-PgapA-cobNST; Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pETDuet-1 cut through same enzyme, build plasmid pETDuet-1-PgapA-cobNST;
(4) respectively by PgapA and cobD, cobC and cobQ, cobO with cobR is connected by fusion DNA vaccine method, is assembled into PgapA-cobD, cobCQ, cobOR.Then be carrier with pMD19, utilize isocaudarner SpeI with XbaI and BamHI with BglII 3 fragments to be connected, build plasmid pMD19-PgapA-cobDCQOR; Finally utilize restriction enzyme site AvrII with KpnI to be connected with the plasmid pRSFDuet-1 cut through same enzyme, build plasmid pRSFDuet-1-PgapA-cobDCQOR;
(5) recombinant plasmid pACYCDuet-1-PgapA-cobMJGIA, pCDFDuet-1-PgapA-cobBHLKF, pETDuet-1-PgapA-cobNST and pRSFDuet-1-PgapA-cobDCQOR cotransformation E.coli BL21 (DE3) will built, obtains recombinant bacterial strain VB12-MBND (E.coli BL21 (DE3)/pACYCDuet-1-cobMJGIA pCDFDuet-1-cobBHLKF pETDuet-1-cobNSTpRSFDuet-1-cobDCQOR).
10. an application rights requires colibacillus engineering strain fermentative production vitamins B described in 1
12method, it is characterized in that, be transferred in fermention medium with the inoculum size of 2-5% after recombinant bacterium is activated, 0.1-0.5mM IPTG inducible gene expression is added during 0h, add paraxin, Streptomycin sulphate, penbritin, kantlex as required, 30-37 DEG C, 200r/min cultivate, cycle 60-72h; Fermention medium (g/L): glucose 60-80, yeast extract paste 25-30, (NH
4)
2hPO
42.5-3.0, MgSO
47H
2o 1.5-2.0, CoCl6H
2o 0.05-0.1,5,6-dimethylbenzimidazole (DMBI) 0.01-0.05,5-ALA 0.05-0.1, ZnSO
47H
2o 0.05-0.1, pH 7.0 ~ 7.2.
Priority Applications (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105112436A (en) * | 2015-06-29 | 2015-12-02 | 江南大学 | Complete-biological synthesis method of adipic acid |
| CN109825543A (en) * | 2018-11-24 | 2019-05-31 | 浙江华康药业股份有限公司 | The method of xylose mother liquid microbial fermentation vitamin B12 based on illumination regulation |
| WO2019109975A1 (en) * | 2017-12-08 | 2019-06-13 | 中国科学院天津工业生物技术研究所 | Recombinant strain of escherichia coli for de novo synthesis of vitamin b12, construction method therefor and application thereof |
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| WO2008131359A1 (en) * | 2007-04-20 | 2008-10-30 | Waltham Technologies Inc. | Genetically modified biological cells |
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| WO2008131359A1 (en) * | 2007-04-20 | 2008-10-30 | Waltham Technologies Inc. | Genetically modified biological cells |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112436A (en) * | 2015-06-29 | 2015-12-02 | 江南大学 | Complete-biological synthesis method of adipic acid |
| CN105112436B (en) * | 2015-06-29 | 2018-08-28 | 江南大学 | A kind of full biological synthesis method of adipic acid |
| WO2019109975A1 (en) * | 2017-12-08 | 2019-06-13 | 中国科学院天津工业生物技术研究所 | Recombinant strain of escherichia coli for de novo synthesis of vitamin b12, construction method therefor and application thereof |
| CN109825543A (en) * | 2018-11-24 | 2019-05-31 | 浙江华康药业股份有限公司 | The method of xylose mother liquid microbial fermentation vitamin B12 based on illumination regulation |
| CN109825543B (en) * | 2018-11-24 | 2021-04-30 | 浙江华康药业股份有限公司 | Method for fermenting vitamin B12 by xylose mother liquor microorganism based on illumination regulation |
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