CN104988173A - Recombinant bacterium for improving lincomycin component A and reducing lincomycin component B and application thereof - Google Patents
Recombinant bacterium for improving lincomycin component A and reducing lincomycin component B and application thereof 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 abstract description 50
- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 title abstract description 12
- 229960005287 lincomycin Drugs 0.000 title abstract description 12
- 241000894006 Bacteria Species 0.000 title abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 41
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 30
- 238000012216 screening Methods 0.000 claims abstract description 25
- 238000000855 fermentation Methods 0.000 claims abstract description 20
- 230000004151 fermentation Effects 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 18
- 230000001580 bacterial effect Effects 0.000 claims description 46
- 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 claims description 35
- 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 claims description 35
- DZSDDKNXMARQMJ-UHFFFAOYSA-N N-Desmethyllincomycin Natural products CN1CC(CC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 DZSDDKNXMARQMJ-UHFFFAOYSA-N 0.000 claims description 35
- 108090000790 Enzymes Proteins 0.000 claims description 24
- 102000004190 Enzymes Human genes 0.000 claims description 24
- 239000012634 fragment Substances 0.000 claims description 22
- 101150095438 metK gene Proteins 0.000 claims description 16
- 239000013598 vector Substances 0.000 claims description 13
- 238000007857 nested PCR Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 abstract description 7
- 241000187399 Streptomyces lincolnensis Species 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 238000004134 energy conservation Methods 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 11
- 108091008146 restriction endonucleases Proteins 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000000968 intestinal effect Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 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
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 241001655322 Streptomycetales Species 0.000 description 2
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- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000588724 Escherichia coli 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
- 235000010469 Glycine max Nutrition 0.000 description 1
- 101100023016 Methanothermobacter marburgensis (strain ATCC BAA-927 / DSM 2133 / JCM 14651 / NBRC 100331 / OCM 82 / Marburg) mat gene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 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
- 238000007599 discharging Methods 0.000 description 1
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- 229940023064 escherichia coli Drugs 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
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- 235000011187 glycerol Nutrition 0.000 description 1
- 229940041028 lincosamides Drugs 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a recombinant bacterium for improving the lincomycin component A and reducing the lincomycin component B and the application thereof. The building method of the recombinant bacterium comprises the steps that 1, streptomyces lincolnensis genome is used as a template, the sequence shown in the SEQ ID No.1 is used as an upstream primer, the sequence shown in the SEQ ID No.2 is used as a downstream primer, and a lmbW gene is cloned; 2, the lmbW gene is connected to a pUML201apr carrier in an enzyme-cut and link up mode, and a pAP04 carrier is built; 3, the pAP04 carrier is converted to streptomyces lincolnensis, screening is carried out, and therefore the recombinant bacterium for improving the lincomycin component A and reducing the lincomycin component B is obtained. According to the recombinant bacterium, the fermentation potency of the lincomycin component A is high, the lincomycin component B is reduced, the downstream separation and purification process can be simplified, the production cost is reduced, energy conservation and emission reduction are achieved, and economic benefits are improved.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of recombinant bacterial strain and application improving LCM component and reduce Lincomycin B component.
Background technology
Lincomycin be by Str. lincolnensis produce lincosamides, be mainly used in treating the infectious diseases that causes of gram-positive microorganism.In Str. lincolnensis fermenting process, produce LCM and B two kinds of components.LCM component is drug activity component, and Lincomycin B component is by product, and its activity only has 25% of component A, and toxicity is larger.The excessive Lincomycin B produced in fermentation needs to remove in downstream separation purifying process, could meet the requirement of medicine.Therefore improve LCM component, reducing Lincomycin B component, is very important to lincomycin fermentation commercial run.
Summary of the invention
The object of the invention is for current lincomycin production status, the recombinant bacterial strain improving LCM component and reduce Lincomycin B component is provided.
The recombinant bacterial strain that second object of the present invention is to provide above-mentioned raising LCM component and minimizing Lincomycin B component improves LCM component in fermentation and reduces the application of B component.
3rd object of the present invention is to provide the recombinant bacterial strain that the second improves LCM component and reduces Lincomycin B component.
4th object of the present invention is to provide the second and improves the recombinant bacterial strain of LCM component and minimizing Lincomycin B component in fermentation raising LCM component and the application reducing B component.
5th object of the present invention is to provide the third recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
6th object of the present invention is to provide the application that the third recombinant bacterial strain improving LCM component and reduce Lincomycin B component improves LCM component in fermentation and reduces B component.
7th object of the present invention is to provide the 4th kind of recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
8th object of the present invention is to provide the 4th kind of recombinant bacterial strain improving LCM component and minimizing Lincomycin B component and improves LCM component in fermentation and reduce the application of B component.
Technical scheme of the present invention is summarized as follows:
Improve a recombinant bacterial strain for LCM component and minimizing Lincomycin B component, build by following method:
(1) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.1 for upstream primer, with sequence shown in SEQ ID No.2 for downstream primer, clone's lmbW gene;
(2) lmbW gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP04 carrier;
(3) by pAP04 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
Above-mentioned a kind of recombinant bacterial strain improves LCM component in fermentation and reduces the application of B component.
The second improves LCM component and reduces the recombinant bacterial strain of Lincomycin B component, builds by following method:
(1) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.3 for upstream primer, with sequence shown in SEQ ID No.4 for downstream primer, clone's metK gene;
(2) metK gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP05 carrier;
(3) by pAP05 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
Above-mentioned the second recombinant bacterial strain improves LCM component in fermentation and reduces the application of B component.
The third recombinant bacterial strain improving LCM component and reduce Lincomycin B component, builds by following method:
(1) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.1 for upstream primer, with sequence shown in SEQ ID No.2 for downstream primer, clone's lmbW gene;
(2) lmbW gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP04 carrier;
(3) with pAP04 carrier for template, with sequence shown in SEQ ID No.5 for upstream primer, with sequence shown in SEQ ID No.2 for downstream primer, clone's lmbW gene;
(4) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.3 for upstream primer, with sequence shown in SEQ ID No.4 for downstream primer, clone's metK gene;
(5) metK gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP05 carrier; LmbW gene step (3) obtained cuts the mode of connection with enzyme, be connected on pAP05 carrier, builds pAP06 carrier;
(6) by pAP06 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce B component.
The third recombinant bacterial strain above-mentioned improves LCM component in fermentation and reduces the application of B component.
The 4th kind of recombinant bacterial strain improving LCM component and reduce Lincomycin B component, builds by following method:
(1) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.1 for upstream primer, with sequence shown in SEQ ID No.2 for downstream primer, clone's lmbW gene;
(2) lmbW gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP04 carrier;
(3) with pAP04 carrier for template, with sequence shown in SEQ ID No.5 for upstream primer, with sequence shown in SEQ ID No.2 for downstream primer, clone's lmbW gene;
(4) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.3 for upstream primer, with sequence shown in SEQ ID No.4 for downstream primer, clone's metK gene;
(5) metK gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP05 carrier; LmbW gene step (3) obtained cuts the mode of connection with enzyme, be connected on pAP05 carrier, builds pAP06 carrier;
(6) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.6 for upstream primer, with sequence shown in SEQ ID No.7 for downstream primer, clone's lmbJ gene; With sequence shown in SEQ ID No.8 for upstream primer, with sequence shown in SEQ ID No.9 for downstream primer, clone lmbG gene;
(7) with lmbJ gene and lmbG gene for template, with SEQ ID No.6 for upstream primer, with SEQ ID No.9 for downstream primer, carry out Overlap extension PCR, obtain lmbJ-lmbG and to connect fragment;
(8) fragment of being connected by lmbJ-lmbG is connected on pAP06 carrier, constructs pAP07 carrier;
(9) by pAP07 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
Above-mentioned 4th kind of recombinant bacterial strain improves LCM component in fermentation and reduces the application of B component.
Recombinant bacterium of the present invention not only makes LCM component fermentation titer high, and Lincomycin B component declines.Be conducive to simplifying downstream separation purge process, reduce production cost, energy-conservation, reduction of discharging, increase economic efficiency.
Embodiment
The Str. lincolnensis that various embodiments of the present invention use derives from ATCC 25466, and competence intestinal bacteria are competence DH5 α.
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment 1
Improve a recombinant bacterial strain for LCM component and minimizing B component, build by following method:
(1) with Str. lincolnensis genome for template, design following primer: with sequence shown in SEQ ID No.1 for upstream primer, with sequence shown in SEQ ID No.2 for downstream primer, clone lmbW gene (GenBank:EU124663.1);
5 '-GCCC
aAGCTTcTTCAGACGGACCGAGGTGCC-3 ' (underscore place is HindIII restriction enzyme site); SEQ ID No.1
5 '-GCCG
gGATCCtCCGCGTGGTTCAGGGCACA-3 ' (underscore place is BamHI restriction enzyme site); SEQ ID No.2
50 μ L PCR reaction systems are: the 5 × buffer of 10 μ L, 5 μ L dNTPs, 5 μ L DMSO, 1 μ L upstream primer, 1 μ L downstream primer, 1 μ L genomic templates, 1 μ L polysaccharase, surplus pure water polishing.
PCR parameter is as follows: 95 DEG C of 5min; 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 30 circulations; 72 DEG C of 10min; 4 DEG C of+∞.
Amplification obtains lmbW gene fragment.Use DNA purification kit, reclaim PCR fragment.
(2) lmbW gene is cut the mode of connection with enzyme, be connected to pUWL201apr carrier (Du, L., Liu, R-H., Ying, L., Zhao, G-R. (2012) An efficient intergeneric conjugation of DNA from Escherichiacoli to mycelia of the lincomycin-producer Streptomyces lincolnensis.Int J Mol Sci13,4797-4806.) on, construct pAP04 carrier;
The 50 μ L enzyme systems of cutting are: 5 μ L 10 × buffer, 30 μ L plasmid or gene fragments, 5 μ L restriction enzymes, surplus pure water polishing.37 DEG C of reaction 30min.After reaction terminates, use DNA purification kit, reclaim digestion products.
With T4 ligase enzyme, gene fragment is connected with carrier segments, 22 DEG C of reaction 30min.
Connect product conversion competence intestinal bacteria, at LB solid medium, add apramycin microbiotic, mixing, paves plate.37 DEG C of cultivations, overnight incubation, screening positive transformant.By the carrier order-checking built, guarantee the exactness of sequence.
(3) by pAP04 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
Screening step is: according to the ordinary method of streptomycete genetic transformation, comprise protoplast transformation or intestinal bacteria-streptomycete Conjugative tiansfer method, by pAP04 vector in Str. lincolnensis.Use apramycin microbiotic, screening obtains recombinant bacterial strain.Propagative spore, uses 20% glycerine ,-80 DEG C of preservations.
Embodiment 2
The second improves LCM component and reduces the recombinant bacterial strain of Lincomycin B component, builds by following method:
(1) with Str. lincolnensis genome for template, design following primer: with sequence shown in SEQ ID No.3 for upstream primer, with sequence shown in SEQ ID No.4 for downstream primer, clone metK gene (GenBank:KP225159);
5 '-ACGC
aAGCTTgTGTCCCGTCGTCTGTTCAC-3 ' (underscore place is HindIII restriction enzyme site); SEQ ID No.3
5 '-GCCG
gAATTCgGGGCACCGCGTAGTCGAAGTA-3 ' (underscore place is EcoRI restriction enzyme site); SEQ ID No.4
PCR reaction system and optimum configurations are with embodiment 1 (wherein primer is different).
Amplification obtains metK gene fragment.Use DNA purification kit, reclaim PCR fragment.
(2) metK gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP05 carrier.
Enzyme cuts the operation with embodiment 1 step (2) of connection, conversion, screening positive transformant and verification operation.
(3) by pAP05 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
Screening step is with the screening of embodiment 1 step (3).
Embodiment 3
The third recombinant bacterial strain improving LCM component and reduce Lincomycin B component, builds by following method:
(1) the pAP04 carrier obtained with embodiment 1 for template, with sequence shown in SEQ ID No.5 for upstream primer, with sequence shown in SEQ IDNo.2 for downstream primer, clone's lmbW gene;
5 '-GCCG
gAATTCgCCCGATGCTAGTCGCGGTTG-3 ' (underscore place is EcoRI restriction enzyme site); SEQ ID No.5PCR reaction system and optimum configurations are with embodiment 1 (wherein primer is different with template).
Amplification obtains lmbW gene fragment, uses DNA purification kit, reclaims PCR fragment.
(2) the lmbW gene of acquisition is cut the mode of connection with enzyme, be connected on the pAP05 carrier of embodiment 2 acquisition, construct the pAP06 expression vector of metK and lmbW series connection.
Enzyme cuts the operation with embodiment 1 step (2) of connection, conversion, screening positive transformant and verification operation.
(3) by pAP06 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
Screening step is with the screening of embodiment 1 step (3).
Embodiment 4
The 4th kind of recombinant bacterial strain improving LCM component and reduce Lincomycin B component, builds by following method:
(1) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.6 for upstream primer, with sequence shown in SEQ ID No.7 for downstream primer, pcr amplification lmbJ gene fragment; With sequence shown in SEQ ID No.8 for upstream primer, with sequence shown in SEQ IDNo.9 for downstream primer, clone lmbG gene fragment;
5 '-GA
aGATCTgACTGTCCCTCGCGTCCCA-3 ' (underscore place is BglII restriction enzyme site); SEQ ID No.6
5’-GTTGTGGGGTGGTCAGCCAGTCTCCTCGGCGGTGGTGTC-3’；SEQ ID No.7
5’-GACACCACCGCCGAGGAGACTGGCTGACCACCCCACAAC-3’；SEQ ID No.8
5 '-GG
aCTAGTcAGGTACAGCGGCAGACGG-3 ' (underscore place is SpeI restriction enzyme site); SEQ ID No.9
PCR reaction system is with embodiment 1 (wherein primer is different).
PCR parameter is as follows: 95 DEG C of 5min; 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 45s, 30 circulations; 72 DEG C of 10min; 4 DEG C of+∞.
Amplification obtains lmbJ and lmbG gene fragment, uses DNA purification kit, reclaims PCR fragment.
(2) with fragment lmbJ and lmbG for template, with SEQ ID No.6 for upstream primer, with SEQ ID No.9 for downstream primer, carry out Overlap extension PCR, obtain lmbJ-lmbG connect fragment.Use DNA purification kit, reclaim PCR fragment.
(3) fragment of being connected by lmbJ-lmbG is connected on the pAP06 that embodiment 3 obtains, and constructs carrier pAP07.
Carry out enzyme with SpeI and BamHI carrier pAP06 to cut, carry out enzyme cut 60min with SpeI and BglII to fragment lmbJ-lmbG, purifying reclaims respectively.
Because BglII and BamHI is isocaudarner, by the carrier segments after purifying and fragment lmbJ-lmbG, connect with T4 ligase enzyme.
Connect product conversion competence intestinal bacteria, at LB solid medium, add apramycin microbiotic, mixing, paves plate.37 DEG C of cultivations, overnight incubation, screening positive transformant.By the carrier order-checking built, guarantee the exactness of sequence.
(4) by pAP07 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
Screening step is with the screening of embodiment 1 step (3).
Embodiment 5
The fermentation of the recombinant bacterial strain that embodiment 1, embodiment 2, embodiment 3, embodiment 4 obtain and HPLC measure
The conveniently zymotechnique of streptomycete, prepares dull and stereotyped spore, liquid seeds, is transferred in fermention medium.At 30 DEG C, 250r/min cultivates 7 days, terminates fermentation.
Get the 7th day recombined engineering fermented liquid, centrifugal, get supernatant liquor.Add methyl alcohol, concussion mixing.Centrifugal, get supernatant, with 0.45 μm of F membrane filtration, analyze for HPLC.
Lincomycin HPLC testing conditions: be weighting agent with octadecylsilane chemically bonded silica; 0.005mol/L ammonium acetate solution (by ammonia soln adjust ph to 9.0)-methyl alcohol (5:5) is moving phase; Determined wavelength is 214nm; Column temperature 25 DEG C; Flow velocity 1.0mL/min.
Fermentative medium formula (mass percent): 10% glucose, 2% analysis for soybean powder, 0.15% corn steep liquor, 0.8% SODIUMNITRATE, 0.5% sodium-chlor, 0.03% ammonium sulfate, 0.03% dipotassium hydrogen phosphate, 0.8% calcium carbonate, regulate pH to 7.1, surplus is pure water.
The fermentation results of each recombinant bacterial strain is in table 1.
The fermentation titer of table 1 recombinant bacterial strain
Claims (8)
1. improve a recombinant bacterial strain for LCM component and minimizing Lincomycin B component, it is characterized in that building by following method:
(1) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.1 for upstream primer, with sequence shown in SEQ ID No.2 for downstream primer, clone's lmbW gene;
(2) lmbW gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP04 carrier;
(3) by pAP04 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
2. a kind of recombinant bacterial strain of claim 1 improves LCM component in fermentation and reduces the application of B component.
3. improve a recombinant bacterial strain for LCM component and minimizing Lincomycin B component, it is characterized in that building by following method:
(1) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.3 for upstream primer, with sequence shown in SEQ ID No.4 for downstream primer, clone's metK gene;
(2) metK gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP05 carrier;
(3) by pAP05 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
4. a kind of recombinant bacterial strain of claim 3 improves LCM component in fermentation and reduces the application of B component.
5. improve a recombinant bacterial strain for LCM component and minimizing Lincomycin B component, it is characterized in that building by following method:
(1) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.1 for upstream primer, with sequence shown in SEQ ID No.2 for downstream primer, clone's lmbW gene;
(2) lmbW gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP04 carrier;
(3) with pAP04 carrier for template, with sequence shown in SEQ ID No.5 for upstream primer, with sequence shown in SEQ ID No.2 for downstream primer, clone's lmbW gene;
(4) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.3 for upstream primer, with sequence shown in SEQ ID No.4 for downstream primer, clone's metK gene;
(5) metK gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP05 carrier; LmbW gene step (3) obtained cuts the mode of connection with enzyme, be connected on pAP05 carrier, builds pAP06 carrier;
(6) by pAP06 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce B component.
6. a kind of recombinant bacterial strain of claim 5 improves LCM component in fermentation and reduces the application of B component.
7. improve a recombinant bacterial strain for LCM component and minimizing Lincomycin B component, it is characterized in that building by following method:
(1) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.1 for upstream primer, with sequence shown in SEQ ID No.2 for downstream primer, clone's lmbW gene;
(2) lmbW gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP04 carrier;
(3) with pAP04 carrier for template, with sequence shown in SEQ ID No.5 for upstream primer, with sequence shown in SEQ ID No.2 for downstream primer, clone's lmbW gene;
(4) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.3 for upstream primer, with sequence shown in SEQ ID No.4 for downstream primer, clone's metK gene;
(5) metK gene is cut the mode of connection with enzyme, be connected on pUWL201apr carrier, build pAP05 carrier; LmbW gene step (3) obtained cuts the mode of connection with enzyme, be connected on pAP05 carrier, builds pAP06 carrier;
(6) with Str. lincolnensis genome for template, with sequence shown in SEQ ID No.6 for upstream primer, with sequence shown in SEQ ID No.7 for downstream primer, clone's lmbJ gene; With sequence shown in SEQ ID No.8 for upstream primer, with sequence shown in SEQ ID No.9 for downstream primer, clone lmbG gene;
(7) with lmbJ gene and lmbG gene for template, with SEQ ID No.6 for upstream primer, with SEQ ID No.9 for downstream primer, carry out Overlap extension PCR, obtain lmbJ-lmbG and to connect fragment;
(8) fragment of being connected by lmbJ-lmbG is connected on pAP06 carrier, constructs pAP07 carrier;
(9) by pAP07 vector to Str. lincolnensis, screening, obtains the recombinant bacterial strain improving LCM component and reduce Lincomycin B component.
8. a kind of recombinant bacterial strain of claim 7 improves LCM component in fermentation and reduces the application of B component.
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| CN108004194A (en) * | 2017-12-07 | 2018-05-08 | 天津大学前沿技术研究院有限公司 | A kind of construction method of recombination engineering for improving coban yield and application |
| CN111117942A (en) * | 2020-01-16 | 2020-05-08 | 华东理工大学 | Genetic engineering bacterium for producing lincomycin and construction method and application thereof |
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Cited By (2)
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| CN108004194A (en) * | 2017-12-07 | 2018-05-08 | 天津大学前沿技术研究院有限公司 | A kind of construction method of recombination engineering for improving coban yield and application |
| CN111117942A (en) * | 2020-01-16 | 2020-05-08 | 华东理工大学 | Genetic engineering bacterium for producing lincomycin and construction method and application thereof |
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