CN104560856B - A kind of aerobic synthetic vitamin B12Escherichia coli and its build with application - Google Patents

A kind of aerobic synthetic vitamin B12Escherichia coli and its build with application Download PDF

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CN104560856B
CN104560856B CN201510017545.8A CN201510017545A CN104560856B CN 104560856 B CN104560856 B CN 104560856B CN 201510017545 A CN201510017545 A CN 201510017545A CN 104560856 B CN104560856 B CN 104560856B
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康振
陈坚
堵国成
张俊丽
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Jiangnan University
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Abstract

The invention discloses a kind of aerobic synthetic vitamin B12Escherichia coli and its build with application, belong to metabolic engineering and field of microbial fermentation.Escherichia coli of the present invention are host cell, using compatible expression vector, will derive from hydrogenlike silicon ion and pseudomonas putida vitamin B12The gene that genome of E.coli is lacked in route of synthesis carries out modularization assembling and expressed, and builds recombination bacillus coli engineered strain, is verified by fermenting, realizes Escherichia coli synthetic vitamin B12。

Description

A kind of aerobic synthetic vitamin B12Escherichia coli and its build with application
Technical field
The present invention relates to a kind of aerobic synthetic vitamin B12Escherichia coli and its build with application, belong to metabolic engineering and Field of microbial fermentation.
Background technology
Vitamin B12(Vitamin B12,VB12), also known as cobalamin, molecular formula is C63H88CoN14O14P, molecular weight is 1355.38.It is the general name for the corrin class compound that a class contains cobalt, is maximum, the most complicated vitamin having now been found that Molecule, is also unique vitamin containing metal ion；It is red that it, which is crystallized, therefore also known as red vitamin.
1956, Hodgkin etc. demonstrated its crystal structure, i.e. center corrin ring, center ring axial direction by x-ray method Co β match somebody with somebody base section and 1 Co α aglucon containing nucleotides ring, and structure is sufficiently complex.Vitamin B12It is used as a kind of important dimension life Element, its main Physiological Function is to participate in manufacture erythrocyte, prevents pernicious anaemia；Prevent cerebral nerve from being destroyed.At present, It is widely used to the fields such as feed, food, medical and health and cosmetics.
1972, American scientist Woodward, R.B. 100 multidigit partners of leader lasted 11 years, and dimension is completed jointly Raw element B12Full chemistry synthesis.But because its chemical synthesis step is more, yield is extremely low, therefore chemical synthesis is sufficiently expensive.In early days, People are from the tissue extraction vitamin B such as animal's liver, kidney12, but yield and benefit are also very low.Later, sent out from streptomysin Extracted in ferment waste liquid, yield is still very low.At present, vitamin B in industrial production12Production mainly use microbe fermentation method.
In nature, vitamin B12De novo synthesis is divided into two approach, and one is anaerobism route of synthesis, is present in expense Family name's Propionibacterium (Propionibacteriumfreudenreichi), Xie Shi Propionibacteriums (Propionibacterium Shermanii), Salmonella (Salmonella sp.) and bacillus megaterium (Bacillus megaterium) etc. are thin In bacterium；Another is aerobic route of synthesis, is primarily present in Pseuomonas denitrifican (Pseudomonas denitrificans) In (aerobic bacterial strain) and hydrogenlike silicon ion (Rhodobacter sphaeroides) (facultative aerobe strain).It is presently used for industry Vitamin B12The bacterial strain of fermentation is mainly P.shermanii and P.denitrificans.Culture medium is using glucose as main carbon Source, corn steep liquor is main nitrogen, adds cobalt ions and precursor 5,6- dimethyl benzenes imipramine (DMBI).In recent years, due to aerobic Fermentation strain has many advantages, such as that such as growth is fast, condition of culture is relatively simple and ferments easy to control.Aerobic fermentation process is The main technique of current industrialized production vitamin B12, the yield in the whole world more than 80% comes from the technique, with P.denitrificans is production bacterial strain, and culture medium uses beet molasses or maltose for primary carbon source, corn steep liquor or yeast extract For nitrogen source, inorganic salts, cobalt ions and DMBI are added.At present, notice is placed on by optimization by related scholar both at home and abroad P.denitrificans zymotechnique, to improve vitamin B12Yield.
The present invention derives from hydrogenlike silicon ion and pseudomonas putida vitamin B in expression in escherichia coli12Route of synthesis Gene, using different copy numbers expression vector to gene carry out modularization assembling, build vitamin B12It is from the beginning aerobic conjunction Into the strain of approach colibacillus engineering.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of aerobic synthetic vitamin B12Colibacillus engineering strain, be Hydrogenlike silicon ion (Rhodobacter is derived from using the carrier moduleization assembling expression of different copy numbers in Escherichia coli Sphaeroides) and pseudomonas putida (Pseudomonasputida) vitamin B12The gene of route of synthesis, build with Glucose is substrate from the beginning aerobic synthetic vitamin B12Approach.
The vitamin B12The gene of route of synthesis includes 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, the vitamin B12Gene cobA, cobI, the cobJ of route of synthesis, CobM, cobF, cobK, cobL, cobH, cobB, cobN, cobS, cobT, cobO, cobQ, cobC and cobD are red from class ball Bacterium, cobG and cobR derive from pseudomonas putida.
In one embodiment of the invention, the nucleotide sequence of the cobA is as shown in SEQ ID NO.1, cobI's Nucleotide sequence is as shown in SEQ ID NO.2, and cobG nucleotide sequence is as shown in SEQ ID NO.3, cobJ nucleotides sequence Row are as shown in SEQ ID NO.4, and cobM nucleotide sequence is as shown in SEQ ID NO.5, cobF nucleotide sequence such as SEQ Shown in ID NO.6, cobK nucleotide sequence is as shown in SEQ ID NO.7, cobL nucleotide sequence such as SEQ ID NO.8 institutes Show, cobH nucleotide sequence is as shown in SEQ ID NO.9, and cobB nucleotide sequence is as shown in SEQ ID NO.10, cobN Nucleotide sequence as shown in SEQ ID NO.11, cobS nucleotide sequence is as shown in SEQ ID NO.12, cobT nucleosides Acid sequence is as shown in SEQ ID NO.13, and cobR nucleotide sequence is as shown in SEQ ID NO.14, cobO nucleotide sequence As shown in SEQ ID NO.15, cobQ nucleotide sequence is as shown in SEQ ID NO.16, cobC nucleotide sequence such as SEQ Shown in ID NO.17, cobD nucleotide sequence is as shown in SEQ ID NO.18.
In one embodiment of the invention, the escherichia coli host includes DH5 α, JM109, W3110, BL21 (DE3)、MG1655。
In one embodiment of the invention, the Escherichia coli are BL21 (DE3).
The different copy number expression vectors be respectively pACYCDuet-1, pCDFDuet-1, pETDuet-1 and pRSFDuet-1。
In one embodiment of the invention, cobA, cobI, cobG, cobJ and cobM are expressed with pACYCDuet-1, PCDFDuet-1 expresses cobF, cobK, cobL, cobH and cobB, and pETDuet-1 expresses cobN, cobS and cobT, PRSFDuet-1 expresses cobR, cobO, cobQ, cobC and cobD.
In one embodiment of the invention, before each netic module individually assembled, PgapA promoters are added And ribosome bind site.
In one embodiment of the invention, the use of pMD19 is that carrier, isocaudarner are assembled to gene.
In one embodiment of the invention, the construction method of the colibacillus engineering strain mainly includes following step Suddenly：
(1) respectively by PgapA and cobM, cobJ and cobG, cobI and cobA are connected by fusion DNA vaccine method, are assembled into PgapA-cobM, cobJG, cobIA.Then using pMD19 as carrier, isocaudarner SpeI and XbaI and BamHI and BglII is utilized 3 fragments are connected, plasmid pMD19-PgapA-cobMJGIA is built.Finally using restriction enzyme site AvrII and KpnI with through phase With the plasmid pACYCDuet-1 connections of digestion, plasmid pACYCDuet-1-PgapA-cobMJGIA is built.
(2) respectively by PgapA and cobB, cobH and cobL, cobK and cobF are connected by fusion DNA vaccine method, are assembled into PgapA-cobB, cobHL, cobKF.Then, will using isocaudarner NsiI and PstI and SpeI and XbaI using pMD19 as carrier 3 fragment connections, build plasmid pMD19-PgapA-cobBHLKF.Finally using restriction enzyme site AvrII and KpnI with through identical The plasmid pCDFDuet-1 connections of digestion, build plasmid pCDFDuet-1-PgapA-cobBHLKF.
(3) PgapA and cobN, cobS and cobT are connected by fusion DNA vaccine method respectively, are assembled into PgapA-cobN, cobST.Then using pMD19 as carrier, 2 fragments are connected using isocaudarner SpeI and XbaI, plasmid pMD19- is built PgapA-cobNST.Restriction enzyme site AvrII and KpnI and the plasmid pETDuet-1 connections through identical digestion are finally utilized, is built Plasmid pETDuet-1-PgapA-cobNST.
(4) respectively by PgapA and cobD, cobC and cobQ, cobO and cobR are connected by fusion DNA vaccine method, are assembled into PgapA-cobD, cobCQ, cobOR.Then, will using isocaudarner SpeI and XbaI and BamHI and BglII using pMD19 as carrier 3 fragment connections, build plasmid pMD19-PgapA-cobDCQOR.Finally using restriction enzyme site AvrII and KpnI with through identical The plasmid pRSFDuet-1 connections of digestion, build plasmid pRSFDuet-1-PgapA-cobDCQOR.
(5) by recombinant plasmid pACYCDuet-1-PgapA-cobMJGIA, pCDFDuet-1-PgapA- of structure CobBHLKF, pETDuet-1-PgapA-cobNST and pRSFDuet-1-PgapA-cobDCQOR cotransformation E.coli BL21 (DE3) recombinant bacterial strain VB12-MBND (E.coli BL21 (DE3)/pACYCDuet-1-cobMJGIA pCDFDuet-1-, are obtained cobBHLKF pETDuet-1-cobNST pRSFDuet-1-cobDCQOR)。
The present invention also provides a kind of application colibacillus engineering fermenting and producing vitamin B12Method, be to recombinate It is transferred to after bacterium activation with 2-5% inoculum concentration in fermentation medium, 0.1-0.5mM IPTG inducible gene expressions is added during 0h, It is added as needed on chloramphenicol, streptomysin, ampicillin, kanamycins, 30-37 DEG C, 200r/min cultures, cycle 60- 72h.Fermentation medium (g/L)：Glucose 60-80, yeast extract 25-30, (NH4)2HPO42.5-3.0, MgSO4·7H2O 1.5- 2.0, CoCl6H2O 0.05-0.1,5,6- dimethylbenzimidazoles (DMBI) 0.01-0.05,5-ALA 0.05- 0.1, ZnSO4·7H2O 0.05-0.1, pH 7.0~7.2.
In one embodiment of the invention, it is, with the switching of 2% inoculum concentration, 0.1- to be added during 0h after recombinant bacterium is activated 0.5mM IPTG inducible gene expressions, are added as needed on chloramphenicol (34 μ g/mL), streptomysin (100 μ g/mL), ammonia benzyl mould Plain (100 μ g/mL), kanamycins (50 μ g/mL), 30-37 DEG C, 200r/min cultures, cycle 60-72h.Fermentation medium (g/ L)：Glucose 80, yeast extract 30, (NH4)2HPO43.0, MgSO4·7H2O 2.0, CoCl6H2O 0.1,5,6- dimethyl benzene And imidazoles (DMBI) 0.05,5-ALA 0.1, ZnSO4·7H2O 0.1, pH 7.0~7.2.
The present invention derives from hydrogenlike silicon ion in Escherichia coli using the carrier moduleization assembling expression of different copy numbers With pseudomonas putida vitamin B12Gene cobA, cobI, cobG, cobJ, cobM, cobF, cobK, the cobL of route of synthesis, CobH, cobB, cobN, cobS, cobT, cobR, cobO, cobQ, cobC and cobD, build vitamin B12From the beginning aerobic synthesis Approach, realizes the direct aerobic fermentation glucose synthetic vitamin B of microbe fermentation method12。
Brief description of the drawings
Fig. 1：Gene is assembled and plasmid construction figure
Fig. 2：Colibacillus engineering strain fermentation production vitamin B12Mass 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
Vitamin B12Analysis method (high performance liquid chromatography)：
Sample preparation：8%NaNO is added in the 10mL zymotic fluids got ready3Solution and each 2.5mL of glacial acetic acid, shake up, put Heat after 30min and take out in 95~100 DEG C of water-baths, be cooled under room temperature, 50W incandescent lamps after illumination 30min plus deionized water is determined Hold to 50mL, filtering, gained filtrate filters 1mL to sample bottle with 0.22 μm of miillpore filter syringe filters, uses microsyringe Draw the μ L of sodium cyanide solution 20 to put in human sample's bottle, sample bottle is put in 35~40 DEG C of water-baths of people and reacts 1h, is taken out, in given bar Liquid-phase chromatographic analysis is carried out under part.
High-efficient liquid phase chromatogram condition：Mobile phase：250mmol/L phosphate aqueous solutions-acetonitrile (30：70, v/v), chromatographic column： BackmanC18 posts (4.6mm × 250mm, 5 μm)；Detection wavelength：361nm；Sample size：20μL；Flow velocity：1.0mL/min；Post Temperature：25℃.
Culture medium：
Slant medium (g/L)：Peptone 10, sodium chloride 10, dusty yeast 5.0, agar 20, pH 7.0；
Seed culture medium (g/L)：Glucose 40, yeast extract 10, NH4Cl 1.0, MgSO4·7H2O 0.5, ZnSO4· 7H2O0.1, pH 7.2~7.4；
Fermentation medium (g/L)：Glucose 80, yeast extract 30, (NH4)2HPO43.0, MgSO4·7H2O 2.0, CoCl 6H2O0.1,5,6- dimethylbenzimidazoles (DMBI) 0.05,5-ALA 0.1, ZnSO4·7H2O 0.1, pH 7.0 ~7.2.
Condition of culture：
Spawn incubation：Glycerol tube is rule, and then the 37 DEG C of cultures of picking single bacterium colony streak plate, are used as seed source；
Seed culture：Flat board picking thalline, 37 DEG C, 200r/min adds the μ g/mL of chloramphenicol 34, streptomysin as requested 100 μ g/mL, the μ g/mL of ampicillin 100, the μ g/mL of kanamycins 50, cultivate about 12h, fermentation medium of transferring；
Fermented and cultured：Transferred with 2% inoculum concentration, 0.1-0.5mM IPTG inducible gene expressions are added during 0h, as needed Addition chloramphenicol (34 μ g/mL), streptomysin (100 μ g/mL), ampicillin (100 μ g/mL), kanamycins (50 μ g/mL), 30-37 DEG C, 200r/min cultures, cycle 60-72h.
The structure of the recombinant plasmid of embodiment 1 and engineered strain
(1) gene assembling and plasmid construction
Gene is subjected to modularization assembling, before each netic module individually assembled, addition PgapA promoters and core Sugared body binding site.By the gene assembled is more and fragment is longer, available restriction enzyme site is less in connection procedure, this Embodiment is carrier using pMD19, borrows 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 and cobA are connected by fusion DNA vaccine method, are assembled into PgapA-cobM, cobJG, cobIA.Then using pMD19 as carrier, isocaudarner SpeI and XbaI and BamHI and BglII is utilized 3 fragments are connected, plasmid pMD19-PgapA-cobMJGIA is built.Finally using restriction enzyme site AvrII and KpnI with through phase With the plasmid pACYCDuet-1 connections of digestion, plasmid pACYCDuet-1-PgapA-cobMJGIA is built.
(2) respectively by PgapA and cobB, cobH and cobL, cobK and cobF are connected by fusion DNA vaccine method, are assembled into PgapA-cobB, cobHL, cobKF.Then, will using isocaudarner NsiI and PstI and SpeI and XbaI using pMD19 as carrier 3 fragment connections, build plasmid pMD19-PgapA-cobBHLKF.Finally using restriction enzyme site AvrII and KpnI with through identical The plasmid pCDFDuet-1 connections of digestion, build plasmid pCDFDuet-1-PgapA-cobBHLKF.
(3) PgapA and cobN, cobS and cobT are connected by fusion DNA vaccine method respectively, are assembled into PgapA-cobN, cobST.Then using pMD19 as carrier, 2 fragments are connected using isocaudarner SpeI and XbaI, plasmid pMD19- is built PgapA-cobNST.Restriction enzyme site AvrII and KpnI and the plasmid pETDuet-1 connections through identical digestion are finally utilized, is built Plasmid pETDuet-1-PgapA-cobNST.
(4) respectively by PgapA and cobD, cobC and cobQ, cobO and cobR are connected by fusion DNA vaccine method, are assembled into PgapA-cobD, cobCQ, cobOR.Then, will using isocaudarner SpeI and XbaI and BamHI and BglII using pMD19 as carrier 3 fragment connections, build plasmid pMD19-PgapA-cobDCQOR.Finally using restriction enzyme site AvrII and KpnI with through identical The plasmid pRSFDuet-1 connections of digestion, build plasmid pRSFDuet-1-PgapA-cobDCQOR.
(2) vitamin B12The structure of recombinant escherichia coli strain
By the recombinant plasmid pACYCDuet-1-PgapA-cobMJGIA of structure, pCDFDuet-1-PgapA-cobBHLKF, PETDuet-1-PgapA-cobNST and pRSFDuet-1-PgapA-cobDCQOR cotransformation E.coli BL21 (DE3), are obtained Recombinant bacterial strain VB12-MBND。
The recombination bacillus coli engineered strain shake flask fermentation of embodiment 2 is verified
Recombination bacillus coli VB12- MBND ferments checking, inoculum concentration 2%, initial glucose concentration in 250mL triangular flasks Induced and corresponding antibiotic for 80g/L, 0h addition 0.1-0.5mM IPTG, after fermentation ends, at somatic cells Reason, analyzes vitamin B12Synthesis, determined through liquid phase and mass spectral analysis, vitamin B12Yield be about 100 μ g/L (Fig. 2).
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention Enclose being defined of being defined by claims.

Claims (5)

1. a kind of aerobic synthetic vitamin B12Colibacillus engineering strain, it is characterised in that be using not in Escherichia coli Carrier moduleization assembling expression with copy number is from hydrogenlike silicon ion and the vitamin B of pseudomonas putida12Route of synthesis Gene, build using glucose as the aerobic synthetic vitamin B of substrate12Approach；The vitamin B12The gene bag of route of synthesis Include cobA, cobI, cobG, cobJ, cobM, cobF, cobK, cobL, cobH, cobB, cobN, cobS, cobT, cobR, cobO, CobQ, cobC and cobD；The nucleotide sequence of the cobA is as shown in SEQ ID NO.1, cobI nucleotide sequence such as SEQ Shown in ID NO.2, cobG nucleotide sequence is as shown in SEQ ID NO.3, cobJ nucleotide sequence such as SEQ ID NO.4 institutes Show, cobM nucleotide sequence as shown in SEQ ID NO.5, cobF nucleotide sequence as shown in SEQ ID NO.6, cobK's Nucleotide sequence is as shown in SEQ ID NO.7, and cobL nucleotide sequence is as shown in SEQ ID NO.8, cobH nucleotides sequence Row are as shown in SEQ ID NO.9, and cobB nucleotide sequence is as shown in SEQ ID NO.10, cobN nucleotide sequence such as SEQ Shown in ID NO.11, cobS nucleotide sequence is as shown in SEQ ID NO.12, cobT nucleotide sequence such as SEQ ID Shown in NO.13, cobR nucleotide sequence is as shown in SEQ ID NO.14, cobO nucleotide sequence such as SEQ ID NO.15 institutes Show, cobQ nucleotide sequence is as shown in SEQ ID NO.16, and cobC nucleotide sequence is as shown in SEQ ID NO.17, cobD Nucleotide sequence as shown in SEQ ID NO.18；CobA, cobI, cobG, cobJ and cobM are expressed with pACYCDuet-1, PCDFDuet-1 expresses cobF, cobK, cobL, cobH and cobB, and pETDuet-1 expresses cobN, cobS and cobT, PRSFDuet-1 expresses cobR, cobO, cobQ, cobC and cobD.
2. colibacillus engineering according to claim 1 strain, it is characterised in that the Escherichia coli include DH5 α, JM109、W3110、BL21(DE3)、MG1655。
3. colibacillus engineering according to claim 1 strain, it is characterised 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, after connection again with matter Grain pACYCDuet-1 connections；By PgapA and cobB, cobH and cobL, cobK and cobF are connected by fusion DNA vaccine method, assembling For PgapA-cobB, cobHL, cobKF, it is connected again with pCDFDuet-1 after connection；By PgapA and cobN, cobS and cobT are logical The connection of fusion DNA vaccine method is crossed, PgapA-cobN, cobST is assembled into, is connected again with pETDuet-1 after connection, plasmid is built pETDuet-1-PgapA-cobNST；By PgapA and cobD, cobC and cobQ, cobO and cobR are connected by fusion DNA vaccine method Connect, be assembled into PgapA-cobD, cobCQ, cobOR, connection is connected with pRSFDuet-1 again.
4. a kind of method for building colibacillus engineering strain described in claim 1, it is characterised in that mainly including following step Suddenly：
(1) respectively by PgapA and cobM, cobJ and cobG, cobI and cobA are connected by fusion DNA vaccine method, are assembled into PgapA-cobM, cobJG, cobIA；Then using pMD19 as carrier, isocaudarner SpeI and XbaI and BamHI and BglII is utilized 3 fragments are connected, plasmid pMD19-PgapA-cobMJGIA is built；Finally using restriction enzyme site AvrII and KpnI with through phase With the plasmid pACYCDuet-1 connections of digestion, plasmid pACYCDuet-1-PgapA-cobMJGIA is built；
(2) respectively by PgapA and cobB, cobH and cobL, cobK and cobF are connected by fusion DNA vaccine method, are assembled into PgapA-cobB, cobHL, cobKF；Then, will using isocaudarner NsiI and PstI and SpeI and XbaI using pMD19 as carrier 3 fragment connections, build plasmid pMD19-PgapA-cobBHLKF；Finally using restriction enzyme site AvrII and KpnI with through identical The plasmid pCDFDuet-1 connections of digestion, build plasmid pCDFDuet-1-PgapA-cobBHLKF；
(3) PgapA and cobN, cobS and cobT are connected by fusion DNA vaccine method respectively, are assembled into PgapA-cobN, cobST；Then using pMD19 as carrier, 2 fragments are connected using isocaudarner SpeI and XbaI, plasmid pMD19- is built PgapA-cobNST；Restriction enzyme site AvrII and KpnI and the plasmid pETDuet-1 connections through identical digestion are finally utilized, is built Plasmid pETDuet-1-PgapA-cobNST；
(4) respectively by PgapA and cobD, cobC and cobQ, cobO and cobR are connected by fusion DNA vaccine method, are assembled into PgapA-cobD, cobCQ, cobOR；Then, will using isocaudarner SpeI and XbaI and BamHI and BglII using pMD19 as carrier 3 fragment connections, build plasmid pMD19-PgapA-cobDCQOR；Finally using restriction enzyme site AvrII and KpnI with through identical The plasmid pRSFDuet-1 connections of digestion, build plasmid pRSFDuet-1-PgapA-cobDCQOR；
(5) by the recombinant plasmid pACYCDuet-1-PgapA-cobMJGIA of structure, pCDFDuet-1-PgapA-cobBHLKF, PETDuet-1-PgapA-cobNST and pRSFDuet-1-PgapA-cobDCQOR cotransformation E.coli BL21 (DE3), are obtained Recombinant bacterial strain VB12-MBND (E.coli BL21 (DE3)/pACYCDuet-1-cobMJGIA pCDFDuet-1-cobBHLKF pETDuet-1-cobNST pRSFDuet-1-cobDCQOR)。
5. colibacillus engineering strain fermenting and producing vitamin B described in one kind application claim 112Method, it is characterised in that It is to be transferred to after recombinant bacterium is activated in fermentation medium with 2-5% inoculum concentration, 0.1-0.5mM IPTG inductions is added during 0h Gene expression, is added as needed on chloramphenicol, streptomysin, ampicillin, kanamycins, 30-37 DEG C, 200r/min is cultivated, Cycle 60-72h；Fermentation medium (g/L)：Glucose 60-80, yeast extract 25-30, (NH4)2HPO42.5-3.0, MgSO4· 7H2O 1.5-2.0, CoCl2·6H2O 0.05-0.1,5,6- dimethylbenzimidazole 0.01-0.05,5-ALA 0.05-0.1, ZnSO4·7H2O 0.05-0.1, pH 7.0~7.2.
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