CN104988173B - It improves Lincomycin A component and reduces recombination engineering and the application of Lincomycin B component - Google Patents
It improves Lincomycin A component and reduces recombination engineering and the application of Lincomycin B component Download PDFInfo
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- OJMMVQQUTAEWLP-KIDUDLJLSA-N lincomycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@@H](C)O)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 OJMMVQQUTAEWLP-KIDUDLJLSA-N 0.000 title claims abstract description 52
- 229960005287 lincomycin Drugs 0.000 title claims abstract description 51
- 238000005215 recombination Methods 0.000 title claims abstract description 41
- 230000006798 recombination Effects 0.000 title claims abstract description 41
- DZSDDKNXMARQMJ-AVXYAQEDSA-N (2S,4R)-4-ethyl-N-[(1R,2R)-2-hydroxy-1-[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-methylsulfanyloxan-2-yl]propyl]-1-methylpyrrolidine-2-carboxamide Chemical compound CN1C[C@H](CC)C[C@H]1C(=O)N[C@H]([C@@H](C)O)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 DZSDDKNXMARQMJ-AVXYAQEDSA-N 0.000 title claims abstract description 28
- CWPMAVJRTHPUNS-UHFFFAOYSA-N Lincomycin B Natural products CCC1CC(NC(=O)C(C(C)O)C2OC(SC)C(O)C(O)C2O)N(C)C1 CWPMAVJRTHPUNS-UHFFFAOYSA-N 0.000 title claims abstract description 26
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- 239000000969 carrier Substances 0.000 claims abstract description 57
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 32
- 230000029087 digestion Effects 0.000 claims abstract description 24
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- 230000004151 fermentation Effects 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 13
- 241000894006 Bacteria Species 0.000 claims abstract description 4
- 101100023016 Methanothermobacter marburgensis (strain ATCC BAA-927 / DSM 2133 / JCM 14651 / NBRC 100331 / OCM 82 / Marburg) mat gene Proteins 0.000 claims description 14
- 101150095438 metK gene Proteins 0.000 claims description 14
- 238000007857 nested PCR Methods 0.000 claims description 3
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- 238000004519 manufacturing process Methods 0.000 abstract description 4
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 4
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- XZNUGFQTQHRASN-XQENGBIVSA-N apramycin Chemical compound O([C@H]1O[C@@H]2[C@H](O)[C@@H]([C@H](O[C@H]2C[C@H]1N)O[C@@H]1[C@@H]([C@@H](O)[C@H](N)[C@@H](CO)O1)O)NC)[C@@H]1[C@@H](N)C[C@@H](N)[C@H](O)[C@H]1O XZNUGFQTQHRASN-XQENGBIVSA-N 0.000 description 3
- 229950006334 apramycin Drugs 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
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- 108090000364 Ligases Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical class N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
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- 235000005822 corn Nutrition 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- 229940041028 lincosamides Drugs 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses the recombination engineerings and application, recombination engineering that improve Lincomycin A component and reduction Lincomycin B component to be built with following methods：(1) using Str. lincolnensis genome as template, using sequence shown in SEQ ID No.1 as sense primer, using sequence shown in SEQ ID No.2 as downstream primer, lmbW genes are cloned；(2) lmbW genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP04 carriers；(3) pAP04 carriers are transformed into Str. lincolnensis, screened, obtain the recombination engineering for improving Lincomycin A component and reducing Lincomycin B component.Recombinant bacterium of the present invention not only makes Lincomycin A component fermentation titer high, but also Lincomycin B component declines.Be conducive to simplify downstream separation purification process, reduce production cost, energy saving, emission reduction, increase economic efficiency.
Description
Technical field
The invention belongs to biotechnology, more particularly to a kind of raising Lincomycin A component and reduction Lincomycin B group
The recombination engineering of part and application.
Background technology
Lincomycin is the lincosamides generated by Str. lincolnensis, is mainly used for treating Gram-positive
Microbial infectious diseases.In Str. lincolnensis fermentation process, two kinds of components of Lincomycin A and B are generated.Lincomycin A group
Part is drug activity component, and Lincomycin B component is by-product, and activity only has the 25% of component A, and is more toxic.Fermentation
The excessive Lincomycin B needs of middle generation remove in downstream separation purifying process, could meet the requirement of drug.Therefore it improves
Lincomycin A component reduces Lincomycin B component, is very important to lincomycin fermentation industrial process.
Invention content
The purpose of the present invention is being directed to current lincomycin production status, provides and improve Lincomycin A component and reduction woods
Can mycin B component recombination engineering.
Second object of the present invention is to provide above-mentioned raising Lincomycin A component and reduces the weight of Lincomycin B component
Group engineering bacteria improves Lincomycin A component in fermentation and reduces the application of B component.
Lincomycin A component is improved third object of the present invention is to provide second and reduces Lincomycin B component
Recombination engineering.
Fourth object of the present invention is to provide second of raising Lincomycin A component and reduces Lincomycin B component
Recombination engineering improves Lincomycin A component in fermentation and reduces the application of B component.
Lincomycin A component is improved fifth object of the present invention is to provide the third and reduces Lincomycin B component
Recombination engineering.
Lincomycin A component is improved sixth object of the present invention is to provide the third and reduces Lincomycin B component
Recombination engineering improves Lincomycin A component in fermentation and reduces the application of B component.
The 7th purpose of the present invention is to provide the 4th kind and improves Lincomycin A component and reduce Lincomycin B component
Recombination engineering.
The 8th purpose of the present invention is to provide the 4th kind and improves Lincomycin A component and reduce Lincomycin B component
Recombination engineering improves Lincomycin A component in fermentation and reduces the application of B component.
Technical scheme of the present invention is summarized as follows：
A kind of recombination engineering for improving Lincomycin A component and reducing Lincomycin B component, is built with following methods：
(1) using Str. lincolnensis genome as template, using sequence is sense primer shown in SEQ ID No.1, with SEQ ID
Sequence shown in No.2 is downstream primer, clones lmbW genes；
(2) lmbW genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP04 carriers；
(3) pAP04 carriers are transformed into Str. lincolnensis, screened, obtained raising Lincomycin A component and reduction woods can be mould
The recombination engineering of plain B component.
A kind of above-mentioned recombination engineering improves Lincomycin A component in fermentation and reduces the application of B component.
Second of recombination engineering for improving Lincomycin A component and reduction Lincomycin B component, with following methods structure
It builds：
(1) using Str. lincolnensis genome as template, using sequence is sense primer shown in SEQ ID No.3, with SEQ ID
Sequence shown in No.4 is downstream primer, clones metK genes；
(2) metK genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP05 carriers；
(3) pAP05 carriers are transformed into Str. lincolnensis, screened, obtained raising Lincomycin A component and reduction woods can be mould
The recombination engineering of plain B component.
Above-mentioned second of recombination engineering improves Lincomycin A component in fermentation and reduces the application of B component.
The third recombination engineering for improving Lincomycin A component and reducing Lincomycin B component, with following methods structure
It builds：
(1) using Str. lincolnensis genome as template, using sequence is sense primer shown in SEQ ID No.1, with SEQ ID
Sequence shown in No.2 is downstream primer, clones lmbW genes；
(2) lmbW genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP04 carriers；
(3) using pAP04 carriers as template, using sequence shown in SEQ ID No.5 as sense primer, with SEQ ID No.2 institutes
Show that sequence is downstream primer, clones lmbW genes；
(4) using Str. lincolnensis genome as template, using sequence is sense primer shown in SEQ ID No.3, with SEQ ID
Sequence shown in No.4 is downstream primer, clones metK genes；
(5) metK genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP05 carriers；It will
The lmbW genes that step (3) obtains are connected in such a way that digestion connects on pAP05 carriers, build pAP06 carriers；
(6) pAP06 carriers are transformed into Str. lincolnensis, screened, obtained and improve Lincomycin A component and reduction B component
Recombination engineering.
The third above-mentioned recombination engineering improves Lincomycin A component in fermentation and reduces the application of B component.
4th kind is improved Lincomycin A component and reduces the recombination engineering of Lincomycin B component, with following methods structure
It builds：
(1) using Str. lincolnensis genome as template, using sequence is sense primer shown in SEQ ID No.1, with SEQ ID
Sequence shown in No.2 is downstream primer, clones lmbW genes；
(2) lmbW genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP04 carriers；
(3) using pAP04 carriers as template, using sequence shown in SEQ ID No.5 as sense primer, with SEQ ID No.2 institutes
Show that sequence is downstream primer, clones lmbW genes；
(4) using Str. lincolnensis genome as template, using sequence is sense primer shown in SEQ ID No.3, with SEQ ID
Sequence shown in No.4 is downstream primer, clones metK genes；
(5) metK genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP05 carriers；It will
The lmbW genes that step (3) obtains are connected in such a way that digestion connects on pAP05 carriers, build pAP06 carriers；
(6) using Str. lincolnensis genome as template, using sequence is sense primer shown in SEQ ID No.6, with SEQ ID
Sequence shown in No.7 is downstream primer, clones lmbJ genes；Using sequence is sense primer shown in SEQ ID No.8, with SEQ ID
Sequence shown in No.9 is downstream primer, clones lmbG genes；
(7) using lmbJ genes and lmbG genes as template, using SEQ ID No.6 as sense primer, it is with SEQ ID No.9
Downstream primer carries out Overlap extension PCR, obtains lmbJ-lmbG series connection segments；
(8) lmbJ-lmbG series connection segment is connected on pAP06 carriers, constructs pAP07 carriers；
(9) pAP07 carriers are transformed into Str. lincolnensis, screened, obtained raising Lincomycin A component and reduction woods can be mould
The recombination engineering of plain B component.
Above-mentioned 4th kind of recombination engineering improves Lincomycin A component in fermentation and reduces the application of B component.
Recombinant bacterium of the present invention not only makes Lincomycin A component fermentation titer high, but also Lincomycin B component declines.Favorably
In simplifying downstream separation purification process, production cost, energy saving, emission reduction are reduced, is increased economic efficiency.
Specific implementation mode
Str. lincolnensis used in various embodiments of the present invention derives from ATCC 25466, and competent E.coli is impression
State DH5 α.
With reference to specific embodiment, the present invention is further illustrated.
Embodiment 1
A kind of recombination engineering for improving Lincomycin A component and reducing B component, is built with following methods：
(1) using Str. lincolnensis genome as template, following primer is designed：Using sequence shown in SEQ ID No.1 as upstream
Primer clones lmbW genes (GenBank using sequence shown in SEQ ID No.2 as downstream primer:EU124663.1)；
5’-GCCCAAGCTTCTTCAGACGGACCGAGGTGCC-3 ' (being HindIII restriction enzyme sites at underscore)；SEQ
ID No.1
5’-GCCGGGATCCTCCGCGTGGTTCAGGGCACA-3 ' (being BamHI restriction enzyme sites at underscore)；SEQ ID
No.2
50 μ L PCR reaction systems are：5 × buffer of 10 μ L, 5 μ L dNTPs, 5 μ L DMSO, 1 μ L sense primers, 1 μ L
Downstream primer, 1 μ L genomic templates, 1 μ L polymerases, surplus pure water polishing.
PCR parameters are as follows：95℃5min；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 30 cycles；72℃10min；4℃+
∞。
Amplification obtains lmbW genetic fragments.With DNA purification kits, PCR fragment is recycled.
(2) by lmbW genes by digestion connect in a manner of, be connected to pUWL201apr carriers (Du, L., Liu, R-H.,
Ying,L.,Zhao,G-R.(2012)An efficient intergeneric conjugation of DNA from
Escherichia coli to mycelia of the lincomycin-producer Streptomyces
Lincolnensis.Int J Mol Sci13,4797-4806.) on, construct pAP04 carriers；
50 μ L digestion systems are：5 10 × buffer of μ L, 30 μ L plasmids or genetic fragment, 5 μ L restriction enzymes, surplus
With pure water polishing.37 DEG C of reaction 30min.After reaction, with DNA purification kits, digestion products are recycled.
Genetic fragment and carrier segments are attached with T4 ligases, 22 DEG C of reaction 30min.
Apramycin antibiotic, mixing, paving is added in LB solid mediums in connection product transformed competence colibacillus Escherichia coli
Tablet.37 DEG C of cultures, overnight incubation screen positive transformant.The carrier built is sequenced, it is ensured that the correctness of sequence.
(3) pAP04 carriers are transformed into Str. lincolnensis, screened, obtained raising Lincomycin A component and reduction woods can be mould
The recombination engineering of plain B component.
Screening step is：According to the conventional method of streptomycete genetic transformation, including protoplast transformation or Escherichia coli-chain
Mould Conjugative tiansfer method, pAP04 carriers are transformed into Str. lincolnensis.With apramycin antibiotic, screening is recombinated
Bacterial strain.Propagative spore is preserved with 20% glycerine at -80 DEG C.
Embodiment 2
Second of recombination engineering for improving Lincomycin A component and reduction Lincomycin B component, with following methods structure
It builds：
(1) using Str. lincolnensis genome as template, following primer is designed：Using sequence shown in SEQ ID No.3 as upstream
Primer clones metK genes (GenBank using sequence shown in SEQ ID No.4 as downstream primer：KP225159)；
5’-ACGCAAGCTTGTGTCCCGTCGTCTGTTCAC-3 ' (being HindIII restriction enzyme sites at underscore)；SEQ
ID No.3
5’-GCCGGAATTCGGGGCACCGCGTAGTCGAAGTA-3 ' (being EcoRI restriction enzyme sites at underscore)；SEQ
ID No.4
PCR reaction systems and parameter setting are the same as embodiment 1 (wherein primer is different).
Amplification obtains metK genetic fragments.With DNA purification kits, PCR fragment is recycled.
(2) metK genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP05 carriers.
The operation of digestion connection, conversion, screening positive transformant and verification operation with 1 step of embodiment (2).
(3) pAP05 carriers are transformed into Str. lincolnensis, screened, obtained raising Lincomycin A component and reduction woods can be mould
The recombination engineering of plain B component.
Screening of the screening step with 1 step of embodiment (3).
Embodiment 3
The third recombination engineering for improving Lincomycin A component and reducing Lincomycin B component, with following methods structure
It builds：
(1) the pAP04 carriers obtained using embodiment 1 is templates, using sequence shown in SEQ ID No.5 as sense primer, with
Sequence shown in SEQ ID No.2 is downstream primer, clones lmbW genes；
5’-GCCGGAATTCGCCCGATGCTAGTCGCGGTTG-3 ' (being EcoRI restriction enzyme sites at underscore)；SEQ ID
No.5 PCR reaction systems and parameter setting are the same as embodiment 1 (wherein primer and template are different).
Amplification obtains lmbW genetic fragments, with DNA purification kits, recycles PCR fragment.
(2) it by the lmbW genes of acquisition in such a way that digestion connects, is connected on the pAP05 carriers of the acquisition of embodiment 2, structure
The concatenated pAP06 expression vectors of metK and lmbW are built.
The operation of digestion connection, conversion, screening positive transformant and verification operation with 1 step of embodiment (2).
(3) pAP06 carriers are transformed into Str. lincolnensis, screened, obtained raising Lincomycin A component and reduction woods can be mould
The recombination engineering of plain B component.
Screening of the screening step with 1 step of embodiment (3).
Embodiment 4
4th kind is improved Lincomycin A component and reduces the recombination engineering of Lincomycin B component, with following methods structure
It builds：
(1) using Str. lincolnensis genome as template, using sequence is sense primer shown in SEQ ID No.6, with SEQ ID
Sequence shown in No.7 is downstream primer, PCR amplification lmbJ genetic fragments；Using sequence shown in SEQ ID No.8 as sense primer, with
Sequence shown in SEQ ID No.9 is downstream primer, clones lmbG genetic fragments；
5’-GAAGATCTGACTGTCCCTCGCGTCCCA-3 ' (being BglII restriction enzyme sites at underscore)；SEQ ID
No.6
5’-GTTGTGGGGTGGTCAGCCAGTCTCCTCGGCGGTGGTGTC-3’；SEQ ID No.7
5’-GACACCACCGCCGAGGAGACTGGCTGACCACCCCACAAC-3’；SEQ ID No.8
5’-GGACTAGTCAGGTACAGCGGCAGACGG-3 ' (being SpeI restriction enzyme sites at underscore)；SEQ ID No.9
PCR reaction systems are the same as embodiment 1 (wherein primer is different).
PCR parameters are as follows：95℃5min；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 45s, 30 cycles；72℃10min；4℃+
∞。
Amplification obtains lmbJ and lmbG genetic fragments, with DNA purification kits, recycles PCR fragment.
(2) using segment lmbJ and lmbG as template, using SEQ ID No.6 as sense primer, using SEQ ID No.9 as downstream
Primer carries out Overlap extension PCR, obtains lmbJ-lmbG series connection segments.With DNA purification kits, PCR fragment is recycled.
(3) lmbJ-lmbG series connection segment is connected on the pAP06 that embodiment 3 obtains, constructs carrier pAP07.
Digestion is carried out with SpeI and BamHI carriers pAP06, digestion is carried out to segment lmbJ-lmbG with SpeI and BglII
60min, respectively purifying recycling.
Since BglII and BamHI are isocaudarners, by after purification carrier segments and segment lmbJ-lmbG, with T4 ligases
It is attached.
Apramycin antibiotic, mixing, paving is added in LB solid mediums in connection product transformed competence colibacillus Escherichia coli
Tablet.37 DEG C of cultures, overnight incubation screen positive transformant.The carrier built is sequenced, it is ensured that the correctness of sequence.
(4) pAP07 carriers are transformed into Str. lincolnensis, screened, obtained raising Lincomycin A component and reduction woods can be mould
The recombination engineering of plain B component.
Screening of the screening step with 1 step of embodiment (3).
Embodiment 5
The fermentation for the recombination engineering that embodiment 1, embodiment 2, embodiment 3, embodiment 4 obtain and HPLC are measured
According to the zymotechnique of conventional streptomycete, tablet spore, liquid seeds are prepared, are transferred in fermentation medium.
30 DEG C, 250r/min is cultivated 7 days, terminates fermentation.
The 7th day recombined engineering fermented liquid is taken, centrifuges, takes supernatant.Methanol, concussion mixing is added.Centrifugation, takes supernatant,
With 0.45 μm of F membrane filtration, analyzed for HPLC.
Lincomycin HPLC testing conditions：It is filler with octadecylsilane chemically bonded silica；0.005mol/L ammonium acetates
Solution (adjusting pH value to 9.0 with ammonia spirit)-methanol (5:5) it is mobile phase；Detection wavelength is 214nm；25 DEG C of column temperature；Flow velocity
1.0mL/min。
Fermentative medium formula (mass percent)：10% glucose, 2% analysis for soybean powder, 0.15% corn steep liquor, 0.8% nitre
Sour sodium, 0.5% sodium chloride, 0.03% ammonium sulfate, 0.03% dipotassium hydrogen phosphate, 0.8% calcium carbonate adjust pH to 7.1, and surplus is
Pure water.
The fermentation results of each recombination engineering are shown in Table 1.
The fermentation titer of 1 recombination engineering of table
Claims (4)
1. a kind of recombination engineering for improving Lincomycin A component and reducing Lincomycin B component, it is characterised in that with following sides
Method is built：
(1) using Str. lincolnensis genome as template, using sequence shown in SEQ ID No.1 as sense primer, with SEQ ID No.2
Shown sequence is downstream primer, clones lmbW genes；
(2) lmbW genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP04 carriers；
(3) using pAP04 carriers as template, using sequence shown in SEQ ID No.5 as sense primer, with sequence shown in SEQ ID No.2
It is classified as downstream primer, clones lmbW genes；
(4) using Str. lincolnensis genome as template, using sequence shown in SEQ ID No.3 as sense primer, with SEQ ID No.4
Shown sequence is downstream primer, clones metK genes；
(5) metK genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP05 carriers；By step
(3) the lmbW genes obtained are connected in such a way that digestion connects on pAP05 carriers, build pAP06 carriers；
(6) pAP06 carriers are transformed into Str. lincolnensis, screened, obtain the weight for improving Lincomycin A component and reducing B component
Group engineering bacteria.
2. a kind of recombination engineering of claim 1 improves Lincomycin A component in fermentation and reduces the application of B component.
3. a kind of recombination engineering for improving Lincomycin A component and reducing Lincomycin B component, it is characterised in that with following sides
Method is built：
(1) using Str. lincolnensis genome as template, using sequence shown in SEQ ID No.1 as sense primer, with SEQ ID No.2
Shown sequence is downstream primer, clones lmbW genes；
(2) lmbW genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP04 carriers；
(3) using pAP04 carriers as template, using sequence shown in SEQ ID No.5 as sense primer, with sequence shown in SEQ ID No.2
It is classified as downstream primer, clones lmbW genes；
(4) using Str. lincolnensis genome as template, using sequence shown in SEQ ID No.3 as sense primer, with SEQ ID No.4
Shown sequence is downstream primer, clones metK genes；
(5) metK genes are connected in such a way that digestion connects on pUWL201apr carriers, build pAP05 carriers；By step
(3) the lmbW genes obtained are connected in such a way that digestion connects on pAP05 carriers, build pAP06 carriers；
(6) using Str. lincolnensis genome as template, using sequence shown in SEQ ID No.6 as sense primer, with SEQ ID No.7
Shown sequence is downstream primer, clones lmbJ genes；Using sequence shown in SEQ ID No.8 as sense primer, with SEQ ID No.9
Shown sequence is downstream primer, clones lmbG genes；
(7) using lmbJ genes and lmbG genes as template, using SEQ ID No.6 as sense primer, using SEQ ID No.9 as downstream
Primer carries out Overlap extension PCR, obtains lmbJ-lmbG series connection segments；
(8) lmbJ-lmbG series connection segment is connected on pAP06 carriers, constructs pAP07 carriers；
(9) pAP07 carriers are transformed into Str. lincolnensis, screened, obtained and improve Lincomycin A component and reduction Lincomycin B
The recombination engineering of component.
4. a kind of recombination engineering of claim 3 improves Lincomycin A component in fermentation and reduces the application of B component.
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Citations (2)
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| CN102719388A (en) * | 2011-03-08 | 2012-10-10 | 上海交通大学 | Method for improving yield of streptomyces antibiotics and plasmids thereof |
| CN104164462A (en) * | 2014-08-12 | 2014-11-26 | 天方药业有限公司 | Method for reducing lincomycin B components in fermentation process |
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| CN102719388A (en) * | 2011-03-08 | 2012-10-10 | 上海交通大学 | Method for improving yield of streptomyces antibiotics and plasmids thereof |
| CN104164462A (en) * | 2014-08-12 | 2014-11-26 | 天方药业有限公司 | Method for reducing lincomycin B components in fermentation process |
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