CN111979136B - Carbon-phosphorus compound derived from streptomyces, and preparation method and application thereof - Google Patents
Carbon-phosphorus compound derived from streptomyces, and preparation method and application thereof Download PDFInfo
- Publication number
- CN111979136B CN111979136B CN201910440511.8A CN201910440511A CN111979136B CN 111979136 B CN111979136 B CN 111979136B CN 201910440511 A CN201910440511 A CN 201910440511A CN 111979136 B CN111979136 B CN 111979136B
- Authority
- CN
- China
- Prior art keywords
- streptomyces
- compound
- carbon
- culture medium
- albus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- -1 Carbon-phosphorus compound Chemical class 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims description 14
- 241000187747 Streptomyces Species 0.000 title abstract description 14
- 241000187180 Streptomyces sp. Species 0.000 claims abstract description 44
- 229940125904 compound 1 Drugs 0.000 claims abstract description 38
- 241000828256 Streptomyces albus J1074 Species 0.000 claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 26
- 230000004151 fermentation Effects 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 22
- 239000002609 medium Substances 0.000 claims description 38
- 239000001963 growth medium Substances 0.000 claims description 34
- 241000588724 Escherichia coli Species 0.000 claims description 32
- 239000007788 liquid Substances 0.000 claims description 29
- 108091008053 gene clusters Proteins 0.000 claims description 22
- 239000013612 plasmid Substances 0.000 claims description 19
- 238000011218 seed culture Methods 0.000 claims description 12
- 238000012546 transfer Methods 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 8
- 238000010367 cloning Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 238000009629 microbiological culture Methods 0.000 claims description 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 4
- 235000010469 Glycine max Nutrition 0.000 claims description 4
- 244000068988 Glycine max Species 0.000 claims description 4
- 229930195725 Mannitol Natural products 0.000 claims description 4
- 239000000594 mannitol Substances 0.000 claims description 4
- 235000010355 mannitol Nutrition 0.000 claims description 4
- 238000005215 recombination Methods 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 238000003306 harvesting Methods 0.000 claims description 3
- 101100226347 Escherichia phage lambda exo gene Proteins 0.000 claims description 2
- 210000000349 chromosome Anatomy 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 abstract description 8
- 238000005481 NMR spectroscopy Methods 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 5
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 238000004949 mass spectrometry Methods 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 49
- 239000000047 product Substances 0.000 description 37
- 239000006228 supernatant Substances 0.000 description 24
- 239000000203 mixture Substances 0.000 description 22
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 19
- 239000000499 gel Substances 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- 239000011347 resin Substances 0.000 description 15
- 229920005989 resin Polymers 0.000 description 15
- 239000008223 sterile water Substances 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 230000001580 bacterial effect Effects 0.000 description 13
- 239000003480 eluent Substances 0.000 description 13
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 13
- 238000001228 spectrum Methods 0.000 description 13
- 238000004090 dissolution Methods 0.000 description 12
- 239000000725 suspension Substances 0.000 description 11
- 230000003321 amplification Effects 0.000 description 10
- 150000001875 compounds Chemical class 0.000 description 10
- 239000011521 glass Substances 0.000 description 10
- 238000003199 nucleic acid amplification method Methods 0.000 description 10
- 108020004465 16S ribosomal RNA Proteins 0.000 description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 238000012258 culturing Methods 0.000 description 9
- 239000003814 drug Substances 0.000 description 9
- 238000012795 verification Methods 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 8
- 239000000287 crude extract Substances 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 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 7
- 229940041514 candida albicans extract Drugs 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 229960005091 chloramphenicol Drugs 0.000 description 7
- 239000008103 glucose Substances 0.000 description 7
- 235000011187 glycerol Nutrition 0.000 description 7
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 7
- 239000002244 precipitate Substances 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 239000002689 soil Substances 0.000 description 7
- 239000012138 yeast extract Substances 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 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 6
- 229950006334 apramycin Drugs 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 229930014626 natural product Natural products 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 238000004440 column chromatography Methods 0.000 description 5
- 238000004925 denaturation Methods 0.000 description 5
- 230000036425 denaturation Effects 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 241001655322 Streptomycetales Species 0.000 description 4
- 238000000137 annealing Methods 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 230000001851 biosynthetic effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000005100 correlation spectroscopy Methods 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000012160 loading buffer Substances 0.000 description 3
- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 description 3
- 238000001394 phosphorus-31 nuclear magnetic resonance spectrum Methods 0.000 description 3
- 238000012257 pre-denaturation Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 239000012137 tryptone Substances 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- JOCBASBOOFNAJA-UHFFFAOYSA-N N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Chemical compound OCC(CO)(CO)NCCS(O)(=O)=O JOCBASBOOFNAJA-UHFFFAOYSA-N 0.000 description 2
- 101710118509 Phosphoenolpyruvate phosphomutase Proteins 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 101100397226 Schizosaccharomyces pombe (strain 972 / ATCC 24843) isp4 gene Proteins 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 241000187759 Streptomyces albus Species 0.000 description 2
- 239000007994 TES buffer Substances 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- JXBAVRIYDKLCOE-UHFFFAOYSA-N [C].[P] Chemical compound [C].[P] JXBAVRIYDKLCOE-UHFFFAOYSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000001351 cycling effect Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 210000003128 head Anatomy 0.000 description 2
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229960000210 nalidixic acid Drugs 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000012807 shake-flask culturing Methods 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- WGLYHYWDYPSNPF-RQFIXDHTSA-N Apramycin sulfate Chemical compound OS(O)(=O)=O.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 WGLYHYWDYPSNPF-RQFIXDHTSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- SRBFZHDQGSBBOR-OWMBCFKOSA-N L-ribopyranose Chemical compound O[C@H]1COC(O)[C@@H](O)[C@H]1O SRBFZHDQGSBBOR-OWMBCFKOSA-N 0.000 description 1
- 244000245214 Mentha canadensis Species 0.000 description 1
- 235000016278 Mentha canadensis Nutrition 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- PNNNRSAQSRJVSB-BXKVDMCESA-N aldehydo-L-rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000078 anti-malarial effect Effects 0.000 description 1
- 229940059720 apra Drugs 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- BKHZIBWEHPHYAI-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol Chemical compound ClC(Cl)Cl.CC(C)CCO BKHZIBWEHPHYAI-UHFFFAOYSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/28—Phosphorus compounds with one or more P—C bonds
- C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a carbon-phosphorus compound, which has a structure shown in a formula 1:
the compound 1 is derived from a Streptomyces sp.LS131440 heterologous expression recombinant S.albus J1074 (NC 30-M), the carbon-phosphorus compound derived from Streptomyces belongs to a novel carbon-phosphorus compound, the used microbial fermentation method selects the Streptomyces sp.LS131440 heterologous expression recombinant S.albus J1074 (NC 30-M), the extraction method is mature, the process is simple and convenient, the yield of the obtained product is high, and the structure is correct through nuclear magnetic resonance and mass spectrometry detection.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a special carbon-phosphorus compound separated from a Streptomyces sp.LS131440 heterologous expression recombinant S.albus J1074 (NC 30-M) of a Streptomyces terrestris strain, and a preparation method and application thereof.
Background
Streptomyces (Streptomyces) is an important strain for producing antibiotics, and a large amount of antibiotics are separated and identified from the Streptomyces, such as penicillin, vancomycin, platemycin and the like. At present, a novel carbon-phosphorus compound is separated from Streptomyces sp.LS131440 heterologous expression recombinant S.albus J1074 (NC 30-M), and the compound is a micromolecular natural product containing carbon-phosphorus bonds. The carbon-phosphorus natural product has wide application, has important application in the fields of antibiotics, herbicides, pesticides, inhibitors of bacteria and parasites and the like, has the drug rate of 15 percent, and has great development potential and social application value compared with the drug rate of 0.1 percent of other natural products. Therefore, intensive research on natural products of activated carbon and phosphorus is carried out, and the method has important significance for finding compounds with new structures and new activities.
Disclosure of Invention
The first purpose of the invention is to provide a carbon-phosphorus compound derived from streptomyces.
The second purpose of the invention is to provide a preparation method of the carbon-phosphorus compound derived from streptomyces.
The third purpose of the invention is to provide the application of the carbon-phosphorus compound derived from streptomyces.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a Streptomyces sp.LS131440 strain, which is preserved in China general microbiological culture Collection center (CGMCC for short, the address: beijing university No.1 of North Chen West Lu, institute of microbiology, china academy of sciences, postal code 100101) at 29.04.2019 in China general microbiological culture Collection management Committee, and the preservation number is CGMCC No.17649.
The culture method of the Streptomyces sp.LS131440 strain comprises the following steps: sterilizing the plate culture medium, preparing into plate, inoculating Streptomyces sp.LS131440 strain spores into the plate culture medium, and culturing at 28 deg.C for 7 days until the produced spores are rich and there is no contamination of infectious microbes, to obtain plate strain.
The plate culture medium consists of the following components: yeast extract, malt extract, glucose, agar and water; the concentrations of the components in the plate culture medium are respectively (g/L): yeast extract 4g/L, malt extract 10g/L, glucose 4g/L and agar 20g/L, the pH value of the plate culture medium is 7.2.
The method for separating the Streptomyces sp.LS131440 strain comprises the following steps:
taking 1.0g of soil sample (longitude and latitude are E116 degrees 30.36 degrees, N27 degrees 12.37 degrees, and soil sample sampling depth is 20 cm.), placing the soil sample into a 50mL centrifuge tube filled with 9.0mL sterile water, shaking at 180rpm for 2h, and carrying out ultrasonic treatment at 20KHz and 100W power for 2min; taking 1.0mL of the suspension, putting the suspension into a 50mL centrifuge tube filled with 9.0mL of sterile water, and uniformly mixing; taking 1.0mL of the suspension, putting the suspension into a 50mL centrifuge tube filled with 9.0mL of sterile water, and uniformly mixing; dilution series 10 -3 And 10 -4 (ii) a Covering a pipe cover tightly, keeping the temperature at 100 ℃ for 1h, taking 0.2mL of coated plates, placing the plates at 28 ℃ for culturing for about 7 days, selecting the plates with clear bacterial colonies and no overlapping, roughly selecting streptomycete basically meeting the requirements according to the introduction of the bacterial colony appearance of streptomycete in Bergey bacillus species identification manual, carrying out bacterial colony shake flask culture on the selected single bacterial colony, adopting a bacterial strain separation culture medium for culturing until a small amount of hyphae is generated at the edge of an experimental group tissue block, and timely transferring the cultured hyphae into another culture plate by adopting a hyphae tip picking method for culturing.
The preparation method of the strain isolation medium comprises the following steps: 20g of oatmeal and 1000mL of artificial seawater, filtering the mixture after 20min with soft fire, taking the filtrate, metering the volume to 1000mL, and adding 20g of agar powder.
The second aspect of the present invention provides a carbon-phosphorus compound, which has a structure represented by formula 1:
the compound 1 is derived from Streptomyces sp.LS131440 heterologous expression recombinant S.albus J1074 (NC 30-M), and the compound shown in the formula 1 belongs to a novel compound containing a carbon-phosphorus bond and is not reported in patents and literatures.
The third aspect of the present invention provides a method for preparing the carbon-phosphorus compound, comprising the steps of:
sterilizing a fermentation culture medium (sterilizing for 30 minutes at 115 ℃), inoculating the primary seed solution to the fermentation culture medium according to the inoculation amount of 5 percent by volume, performing rotary culture for 6 days at 28 ℃, harvesting a fermentation liquid which is all substances in a container, and separating and purifying the fermentation liquid to obtain the compound 1.
The fermentation medium comprises the following components: 20g/L of mannitol, 20g/L of soybean cake powder and 7.8 of pH value of the fermentation medium.
The preparation method of the first-stage seed liquid comprises the following steps: sterilizing the seed culture medium (sterilizing at 115 deg.C for 30 min), inoculating the plate culture medium containing heterologous expression recombinant S.albus J1074 (NC 30-M) obtained by digging block (0.5 cm × 0.5 cm) of plate strain, and rotary culturing at 28 deg.C in rotary shaker (rotating speed of 220 rpm) for 24 hr to obtain first-stage seed solution.
The seed culture medium consists of the following components: tryptone Soy Broth (TSB); the concentration of the components in the seed culture medium is as follows: TSB 30g/L; the pH value of the seed culture medium is 7.2.
The method for obtaining the compound 1 by separating and purifying the fermentation liquor comprises the following steps:
centrifuging the obtained fermentation liquor at 4 ℃, collecting supernatant, evaporating the supernatant to dryness to recover crude extract, dissolving the crude extract in an ammonia water solution, adding pretreated DOWEX 21K XLT resin, oscillating and mixing uniformly, collecting supernatant, placing the supernatant in a refrigerator at 4 ℃ for storage, adding the adsorbed DOWEX 21K XLT resin into hydrochloric acid for elution and filtration, concentrating and freezing eluent (placed in a refrigerator at-80 ℃) to obtain crude extract, marking as S-NC30, and sequentially carrying out Sephadex LH-20 column chromatography and HPLC separation on the crude extract for three times to obtain the compound 1.
The third Sephadex LH-20 column chromatography comprises the following steps:
dissolving the prepared S-NC30 component in water, separating and purifying by using 1.4L Sephadex LH-20 chromatographic column, using water as eluent, wherein the flow rate of the eluent is 8 seconds per drop, and obtaining 5 parts, wherein the carbon-phosphorus compound mainly exists in the 3 rd part and is marked as S-NC30-3;
dividing the prepared S-NC30-3 into a 3A component and a 3B component, dissolving the 3A component in water, separating and purifying by using a 400mL Sephadex LH-20 chromatographic column, using water as an eluent, wherein the flow rate of the eluent is 7 seconds per drop to obtain 7 fractions, and the carbon-phosphorus compound mainly exists in the 5 th fraction and is marked as 3A-5; dissolving the 3B in water, and separating and purifying by using 1.4mL Sephadex LH-20 chromatographic column, wherein water is used as eluent, the flow rate of the eluent is 7 seconds per drop, 6 fractions are obtained, and the carbon-phosphorus compound mainly exists in the 4 th fraction and the 5 th fraction and is marked as 3B-4C;
dissolving the prepared 3B-4C in water, separating and purifying by using a 400mL Sephadex LH-20 chromatographic column, using water as an eluent, wherein the flow rate of the eluent is 7 seconds per drop to obtain 60 fractions, and the carbon-phosphorus compound mainly exists in the 21 st to 23 rd fractions; the 21 st-23 st fraction of 3B-4C was combined with the 3A-5 fraction and recorded as NC30-D.
The HPLC separation comprises the following steps:
prepared NC30-D was dissolved in water to a final concentration of 102.7mg/mL and compound 1 was prepared using reverse phase HPLC under the conditions: thermo Hypercarb column, 4.6 x 150mm, detection wavelength 270nm, mobile phase 0.1% ammonia (a) and acetonitrile, mobile phase gradient (B): 0.00min,1%;3.00min,1%;3.50min,5%;4.00min,20%;4.01min,99%;9.00min,99%;9.01min,1%;13.00min,1 percent; collecting the effluent with retention time of 2min to obtain compound 1.
The heterologous expression recombinant S.albus J1074 (NC 30-M) is obtained by cloning Streptomyces sp.LS131440-NC30 gene cluster and conjugal transfer of three parents.
The cloning method of the Streptomyces sp.LS131440-NC30 gene cluster is as follows: when gene cluster cloning is carried out, taking a genome of a strain Streptomyces sp.LS131440 as an enzyme cutting template, selecting enzyme cutting sites at the outer sides of two ends of a target gene cluster for enzyme cutting to obtain a Streptomyces sp.LS131440-NC30 gene cluster, grabbing a carbon-phosphorus compound biosynthesis gene cluster Streptomyces sp.LS131440-NC30 by a RecET recombination technology, integrating a conjugative transfer element by a Red alpha beta recombination technology, and finally constructing a recombinant plasmid p15A-NC30-M and an Escherichia coli E.coli DH10B (NC 30-M); or when carrying out three-parent combined transfer, stably combining the Streptomyces sp.LS131440-NC30 gene cluster on the chromosome of a heterologous expression host Streptomyces albus J1074 with the aid of a plasmid pUB307 to obtain a recombinant S.albus J1074 (NC 30-M).
The cleavage sites are AvrII and PspXI.
Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:
the carbon-phosphorus compound derived from Streptomyces provided by the invention belongs to a novel carbon-phosphorus compound, the used microbial fermentation method selects Streptomyces sp.LS131440 heterologous expression recombinant S.albus J1074 (NC 30-M), the extraction method is mature, the process is simple and convenient, the yield of the obtained product is high, and the structure is correct through nuclear magnetic resonance and mass spectrometry detection.
The advantages of the compound are: the commercialization probability of the carbon phosphorus compound is as high as 15%.
The carbon-phosphorus compound is derived from a recombinant S.albus J1074 (NC 30-M), and the carbon-phosphorus compound biosynthesis gene cluster Streptomyces sp.LS131440-NC30 in the heterologous expression recombinant is derived from a Streptomyces sp.LS131440 strain.
Preservation information of biological material sample:
the preservation unit: china general microbiological culture Collection center (CGMCC for short)
Address: microbial research institute of western road 1 institute No. 3 of China academy of sciences, beijing, chaoyang
The preservation date is as follows: 29/04/2019
The preservation number is as follows: CGMCC No.17649
And (3) classification and naming: streptomyces sp.
Drawings
FIG. 1 is an open reading frame analysis of the Streptomyces sp.LS131440-NC30 biosynthetic gene cluster prepared in the examples of the present invention.
FIG. 2 is a restriction enzyme digestion verification spectrum of p15A-NC30-M plasmid prepared in the embodiment of the present invention.
FIG. 3 shows compound 1 prepared in the examples of the present invention dissolved in D 2 ESI-MS plot under negative ion source in O.
FIG. 4 shows compound 1 prepared in the examples of the present invention dissolved in D 2 In O 1 H-NMR (700 MHz) spectrum.
FIG. 5 shows compound 1 prepared in the examples of the present invention dissolved in D 2 In O 13 C-NMR (150 MHz) spectrum.
FIG. 6 shows the dissolution of Compound 1 in D prepared in the examples of the present invention 2 In O 31 P-NMR spectrum.
FIG. 7 shows the dissolution of Compound 1 in D prepared in the examples of the present invention 2 DEPT spectra in O.
FIG. 8 shows the dissolution of Compound 1 in D prepared in the examples of the present invention 2 In O of ( 1 H- 1 H) COSY spectrum.
FIG. 9 shows the dissolution of Compound 1 in D prepared in the examples of the present invention 2 HSQC spectrum in O.
FIG. 10 shows the dissolution of Compound 1 in D prepared in the examples of the present invention 2 In O of ( 1 H- 13 C) HMBC spectrum.
FIG. 11 shows Compound 1 prepared in accordance with the present invention in D 2 In O of ( 1 H- 31 P) HMBC spectrum.
FIG. 12 is a phylogenetic tree of the Streptomyces sp.LS131440 strain of Streptomyces based on the 16S rRNA gene sequence.
Fig. 13 is a PCR verification spectrum of e.coli DH10B (NC 30-M) prepared in the example of the present invention.
Fig. 14 is a PCR verification spectrum of s.albus J1074 (NC 30-M) prepared according to an example of the present invention.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention are commercially available unless otherwise specified.
Chloroform isoamyl alcohol (25: 24) is purchased from Beijing Solebao scientific Co., ltd, and the product catalog number is P1012; ethanol was purchased from Shanghai Tantake Technique, inc. under the product catalog number 01470949; acetone was purchased from Shanghai Tantake Technique, inc. under the product catalog number 01693227; methanol was purchased from Shanghai Tantake Technique, inc. under the product catalog number 01226745; agar was purchased from shanghai hadamard ltd under the product catalog number 88930B; the yeast extract is obtained from Shanghai Jinpan Biotech, inc., and has a catalog number of 01284983; the malt extract powder is purchased from Shanghai Hadamard GmbH, with a product catalog number of 124980A; tryptone was purchased from Shanghai Jinpan Biotech, inc.; the product catalog number is LP0042; glycine was purchased from shanghai hadamard ltd with product catalog number B65953C; the D-arabinose is purchased from Shanghai Hadamard GmbH, with the product catalog number of 59668C; l-rhamnose was purchased from Haemax corporation, shanghai under the catalog number 70440C; EDTA available from Hainan Inc. of Shanghai under the catalog number 68915A; DMSO is available from Hainan Inc. of Shanghai under the catalog number 38114B; the tryptone soy peptone liquid medium is purchased from Qingdao Haibo biotechnology limited, and the product catalog number is HBKP4114-19; potassium acetate was purchased from shanghai hadamard ltd under the product catalog number 18899a; acetic acid was purchased from Haohai Hadamard, inc. under the product catalog number 73562B; mannitol was purchased from shanghai hadamard ltd under product catalog number 77529C; ammonia water was purchased from Hakka limited, product catalog number 20877R; tris-hydrochloric acid is purchased from pharmaceutical industry GmbH of national drug group with the product catalog number of 1.08128e +010; glucose purchased from Beijing YinuoKai technologyCompany Limited, product catalog number 01185932; the sodium chloride is purchased from pharmaceutical industry of the national drug group, and the product catalog number is 10019369; the sodium hydroxide is purchased from pharmaceutical industry of the national drug group, and the product catalog number is 10019718; the acetonitrile is purchased from pharmaceutical industry GmbH of the national medicine group, and the product catalog number is 01270576; taKaRa Ex
Purchased from TaKaRa Bio-engineering Co., ltd, and having a product catalog number of RR001A;
GXLDNA Polymerase is purchased from TaKaRa Bio-engineering Co., ltd, and the product catalog number is R050Q; the restriction endonuclease AvrII was purchased from british biotechnology limited, under the product catalog number R0174L; restriction endonuclease PspXI was purchased from British Biotechnology, inc., N.Y., under product catalog number R0656L; the plasmid small-amount extraction kit is purchased from Aisijin biotechnology limited company, and the product catalog number is AP-MN-P-250; the DNA gel recovery kit is purchased from Aisijin biotechnology limited company, and the product catalog number is AP-GX-50; SDS was purchased from Aisijin Biotechnology Co., ltd, and the catalog number of the product is L5750; the agarose gel was purchased from Aisijin Biotechnology Ltd, and the product catalog number is 01375654; the Marker is purchased from Biotechnology engineering Co., ltd, and the product catalog number is B500345-0050; kanamycin was purchased from Biotechnology, inc., product catalog number MFCD00070253; the apramycin is purchased from Biotechnology engineering, inc., and has a product catalog number of MFCD06200257; the chloramphenicol is purchased from Biotechnology engineering, inc., and has a product catalog number of MFCD00078159; naphthatonic acid was purchased from Biotechnology, inc., under the product catalog number MFCD00006884; the soybean cake powder is purchased from Tianjin pre-crown chemical industry Jingzhe, and the product catalog number is THHD200223; ISP4 medium was purchased from Bidi medical devices, inc. under the product catalog number 277210.
The strain used in the invention is Streptomyces terreus strain sp.LS131440 which has been preserved in China general microbiological culture Collection center (CGMCC for short, address: beijing city Shangyang district Beichen Xilu No. 3, china academy of sciences microorganism, postal code 100101) in 29.04.2019, and the preservation number is CGMCC No.17649.
Example 1
1. Construction of heterologous expression recombinants for preparation of Compound 1
1. Seed plate culture of wild type strain
(1) Sterilizing the plate culture medium at 115 deg.C for 30min, making into plate, inoculating Streptomyces sp.LS131440 strain spore to the plate culture medium, and culturing at 28 deg.C for 7 days until the produced spore is rich and has no contamination of infectious microbes, to obtain plate strain.
The plate culture medium consists of the following components: yeast extract, malt extract, glucose, agar and water; the concentrations of the components in the plate culture medium are respectively (g/L): 4g/L of yeast extract, 10g/L of malt extract, 4g/L of glucose and 20g/L of agar. The pH of the plate medium was 7.2.
The method for separating and identifying the Streptomyces sp.LS131440 is as follows:
strain-derived samples: collected from a soil sample of China. The latitude and longitude are E116 degrees 30.36 degrees, N27 degrees 12.37 degrees, and the sampling depth of the soil sample is 20cm.
The strain separation method comprises the following steps:
the wet-heat dilution coating method comprises the following specific operation methods:
taking 1.0g of soil sample, putting the soil sample into a 50mL centrifuge tube filled with 9.0mL of sterile water, shaking at 180rpm for 2h, and carrying out ultrasonic treatment at 20KHz and 100W power for 2min; taking 1.0mL of the suspension, putting the suspension into a 50mL centrifuge tube filled with 9.0mL of sterile water, and uniformly mixing; taking 1.0mL of the suspension, putting the suspension into a 50mL centrifuge tube filled with 9.0mL of sterile water, and uniformly mixing; dilution series 10 -3 And 10 -4 (ii) a The tube cover is tightly covered, the temperature is kept at 100 ℃ for 1h, 0.2mL of coated plate is taken, and the plate is placed at 28 ℃ for culture for about 7 days. Selecting a flat plate with clear bacterial colony and no overlapping, roughly selecting streptomycete basically meeting the requirements according to the introduction of the bacterial colony appearance of streptomycete in Bergey's bacillus species identification handbook, and carrying out separation on the selected single bacterial colonyAnd (3) carrying out colony shake flask culture, adopting a strain isolation culture medium for culture, culturing until a small amount of hyphae are generated at the edge of the experimental group tissue block, and transferring the experimental group tissue block into another culture flat plate for culture by adopting a hyphae tip picking method in time. And performing physical and chemical index detection according to Bergey 'S manual, extracting total genome DNA of the strains meeting the physical and chemical indexes of the Bergey' S manual, amplifying 16S rRNA sequences by PCR and sequencing.
The preparation method of the strain isolation medium comprises the following steps: 20g of oatmeal and 1000mL of artificial seawater, filtering the mixture after 20min with soft fire, taking the filtrate, metering the volume to 1000mL, and adding 20g of agar powder.
The strain preservation method comprises the following steps: storing in 25% glycerol freezing tube at-80 deg.C.
The sequence determination and phylogenetic analysis method of the 16S rRNA of the strain are as follows:
genomic DNA from Streptomyces sp.M10731 was extracted using the TINAamp Bacteria DNA Kit according to the Kit instructions and subjected to 16S rDNA amplification using the universal primers (27f. PCR amplification reaction of 16S rDNA was performed on a TaKaRa PCR Thermal Cycler [ 25. Mu.L amplification system: mu.L of 20. Mu.M primer, 2.5. Mu.L of 10 Xbuffer (TaKaRa, chinese Dalian), 2.5. Mu.L of 2.5nM dNTP (TaKaRa, dalian, china), 2U of rTap polymerase (TaKaRa, chinese Dalian), 1. Mu.L of DNA template ], primary denaturation at 94 ℃ for 5 minutes, then denaturation at 94 ℃ for 1 minute, cycling 30 times, annealing at 55 ℃ for 1 minute, extension at 72 ℃ for 1 minute and 15 seconds, and finally extension at 72 ℃ for 10 minutes, and sending the PCR amplification product to the sequencing company. Performing multi-sequence alignment analysis on the obtained 16S rRNA sequences by using CLSSTAL W sequence analysis software, and generating a phylogenetic tree by using an adjacent method in MEGA6.0 software, as shown in FIG. 12, wherein FIG. 12 is a phylogenetic tree of Streptomyces sp.LS131440 based on 16S rRNA gene sequences, and the step gap values are set as follows: 1000.
as a result: according to the colony morphology characteristics: the culture medium is orange yellow, white spore is produced, and the result of the 16S rRNA gene sequence shows that Streptomyces sp.LS131440 belongs to Streptomyces, and the similarity of the Streptomyces sp.LS131440 to the model strain S.nogalatter JCM 4799 reaches 100 percent in the 16S rRNA gene sequence.
2. Cloning of Streptomyces sp.LS131440-NC30 Gene Cluster
(1) Streptomyces sp.LS131440 mycelium Collection
The agar block containing the spore-forming colonies was scooped up to 0.5 cm. Times.0.5 cm in a clean bench by using an inoculating scoop, added to a liquid medium, and cultured in a constant temperature shaker at 28 ℃ and 220rpm for 48 hours. Putting the mycelium into a 50mL centrifuge tube, balancing, and centrifuging at 9000rpm for 5min to collect mycelium from the supernatant.
The liquid medium consists of the following components: yeast extract, malt extract, glucose and water; the concentrations of the components in the liquid culture medium are respectively (g/L): 4g/L of yeast extract, 10g/L of malt extract and 4g/L of glucose. The pH of the liquid medium was 7.2.
(2) Streptomyces sp.LS131440 genome extraction
Adding 8mL of SET solution into the 50mL centrifuge tube to resuspend the thallus, adding 10mg of lysozyme, mixing uniformly, placing in a constant-temperature water bath kettle at 37 ℃ for 1h, and destroying cell walls. Add 500. Mu.L proteinase K and mix gently, add 1mL 10% SDS and mix gently. When the genome DNA is released, the genome must be gently prevented from being broken when mixed. Keeping the temperature in a constant temperature water bath kettle at 50 ℃ for 2h. Add 3.5mL of 5M NaCl and mix gently. 15mL of a mixture of phenol, chloroform, isoamyl alcohol (25. The mixture was then trimmed and centrifuged in a bench top centrifuge at 9000rpm for 30min. mu.L of the upper aqueous phase was taken with a pipette tip into a 2ml sterile EP tube, 35. Mu.L of 3M sodium acetate pH 7.5 was added and mixed gently. 1.2mL of absolute ethanol was added and mixed gently. White flocculent precipitate was visible at this time. A2 ml sterile EP tube was prepared, 1.5mL of 70% ethanol was added, and the white flocculent precipitate was scooped in with a 200. Mu.L sterile tip. After washing, the mixture was centrifuged at 9000rpm for 1min by a desk centrifuge, the supernatant was discarded, and the EP tube was inverted for 30min to evaporate water in the tube. This step should be done to prevent the flocculent DNA precipitate from slipping off the tube wall, and the flocculent DNA precipitate will change from white to colorless and transparent during the evaporation of water. The genomic DNA was dissolved by adding 500. Mu.L of 10mM Tris-Cl at pH 8.0. The genomic DNA concentration was 500 ng/. Mu.L as determined using a Nanodrop2000 nucleic acid concentration detector.
(3) Streptomyces sp.LS131440 genomic cleavage
A400-. Mu.L system was prepared from 250. Mu.L of the above genomic DNA (125. Mu.g), 2. Mu.L of RnaseA (10. Mu.g/. Mu.L), 29. Mu.L of 500units/mLAvrII, 59. Mu.L of 1000units/mL PspXI (59. Mu.L), and 40. Mu.L of 10 Xdigestion Buffer, and digested in a 37 ℃ thermostatic water bath for 1 hour. After completion of the digestion, 400. Mu.L of phenol, chloroform and isoamyl alcohol (25. The supernatant after centrifugation of 300. Mu.L was put into a clean 1.5mL centrifuge tube, 20. Mu.L of 3M sodium acetate was added and mixed gently, 800. Mu.L of absolute ethanol was added and mixed gently, and centrifuged at 9000rpm for 2min in a centrifuge. After discarding the supernatant, 1mL of 70% ethanol was added and centrifuged at 9000rpm for 1min in a centrifuge. Discarding the supernatant, drying the centrifuge tube in a 45 deg.C oven for 10min, adding 12 μ LddH 2 O。
FIG. 1 shows the open reading frame analysis of Streptomyces sp.LS131440-NC30 biosynthetic gene cluster prepared in the example of the present invention, orf 67 is a PEP mutase coding gene, and it can be shown that the biosynthetic gene cluster contains a coding gene for a PEP mutase key enzyme.
Table 1 shows the function of the open reading frame presumed for the Streptomyces sp.LS131440-NC30 biosynthetic gene cluster prepared in the examples of the present invention.
TABLE 1
(4) preparation of p15A Linear vector
The amplification of the p15A vector was carried out using the primers (p 15A-f:5'-acttgcggcgcacccggagcatgtcgtccaggaattcctcgtcctggcgcatgagtccgaaattggagtcggcgagcatcaggatagatccgaaaaccccaagttacg-3', p 15A-R5'-cgctgctggaccaggtcgccgcggacaacccgtcggtgaccaagcgctgggtggacgccgggttcaagaacgcggtcattagatcctttctcctctttagatc-3') and the p15A-cm-tetR-tet0-hyg-ccdB plasmid as a template. PCR amplification reaction of p15A vector was performed on TaKaRa PCR Thermal Cycler [50. Mu.L amplification system: 2. Mu.L 10. Mu.M primer, 5. Mu.L 10 Xbuffer (TaKaRa, chinese Dalian), 5. Mu.L2.5nM dNTP (TaKaRa), 2GXL polymerase (TaKaRa, chinese Dalian), 1. Mu.L DNA template, 24. Mu.L ddH 2 O]Pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 68 ℃ for 3min, circulating for 30 times, and finally extension at 72 ℃ for 10min. PCR product purification was performed according to Axygen PCR clean kit AP-PCR-50 instructions.
(5) Preparation of E.coli electroporation competent cells
mu.L of E.coli GB05RedTrfA (pSC 101-BAD-ETgA-tet) was removed from the stock using 200. Mu.L of sterile Huang Qiangtou. And (3) coating the mixture on an LB solid plate containing 500ng of tetracycline by a four-area scribing method, sealing the edge of the plate by a sealing film, wrapping the plate by tinfoil paper, and performing inverted light-proof culture in an incubator at 30 ℃ for 12 hours to obtain the Escherichia coli monoclonal antibody. Coli single clones in the center of the plate were picked with 10. Mu.L of sterilized Bai Qiangtou and cultured in 30mL brown screw glass vials containing LB liquid medium. The liquid loading of a 30mL screw glass vial was 6mL of liquid LB medium containing 300ng of the corresponding antibiotic. The inoculated 30mL screw glass vial was placed in an incubator at 30 ℃ and 220rpm for 12 hours. Aspirate 160. Mu.L of E.coli culture into another 30mL brown screw glass vial. The liquid content of the 30mL screw glass vial was 6mL. Liquid LB medium contained 300ng tetracycline. The inoculated 30mL screw glass bottle is put into an incubator at 30 ℃ and 220rpm for culture for 2h, and the OD600 reaches 0.35-0.40. 150 μ L of 10% arabinose was added to the above 30mL screw glass vial for induction. The induced 30mL screw glass vial was placed in an incubator at 37 ℃ and 220rpm for about 1h, at which time the OD600 should reach 0.7-0.8. 2mL of the above-induced E.coli culture was pipetted into a 2mL centrifuge tube using a 1mL tipper sterile blue-gun tip, centrifuged at 12000rpm for 30s in a desktop centrifuge, and the supernatant was removed rapidly, all following this step on ice. 2mL of pre-cooled sterile water was added to the centrifuge tube and centrifuged in a tabletop centrifuge at 12000rpm for 30s and the supernatant was removed quickly. After washing with sterile water for 3 times, 20. Mu.L of sterile water was added, and the competence was placed on ice for electroporation. Competence needs to be prepared as it is.
(6) The p15A linear vector is connected with a Streptomyces sp.LS131440-NC30 gene cluster
Mu.g of the target fragment of the Streptomyces sp.LS131440-NC30 gene cluster described above and the 1. Mu. g p15A linear vector described above were added to 20. Mu.L of E.coli competent cell solution. 200. Mu.L of sterilized Huang Qiangtou was gently mixed and added to a 1mm electric rotor pre-cooled at-20 ℃ for electric rotation at 1350V, 10. Mu.F and 600. Omega. After the electrotransfer, 1mL of an antibiotic-free LB liquid medium was added, the mixture was transferred to a 1.5mL sterilized EP tube, and the tube was precultured for 1 hour in an incubator at 37 ℃ and 220 rpm. After pre-culture for 1h, the pre-culture is centrifuged for 1min at 8000rpm in a desktop centrifuge, 900 μ L of supernatant is discarded, the centrifuged and precipitated cells are mixed uniformly, and the mixture is spread on LB solid culture medium containing 600 μ g of chloramphenicol resistance in four areas, and cultured in an incubator at 37 ℃ for 12h until single clones grow out. The single colonies on the plate were individually picked up with a sterilized white tip into a sterile 2mL EP tube containing 1mL LB liquid medium. LB liquid medium contained 20. Mu.g of chloramphenicol. The EP tube was cultured in a shaker at 37 ℃ for 12 hours at 220rpm, and E.coli was designated E.coli DH10B (NC 30).
(7) Splice transfer element modification
50 μ L of 1 μ g R K-phiC31-oriT-apra plasmid, 6 μ L of 20U/. Mu.L Asel, 124.0ddH 2 O and 20. Mu.L of 10 Xdigestion Buffer were combined to prepare a 200. Mu.L system, and the system was digested in a 37 ℃ thermostatic water bath for 1 hour. The above digestion mixture was purified using PCR purification kit and finally dissolved in 50. Mu.L ddH 2 In O, the resulting oriT-attP-phiC 31-conjugated transfer element had a DNA concentration of 100 ng/. Mu.L as determined by Nanodrop 2000.
Coli E.coli DH10B (NC 30) competent cells were prepared as described in (5) above, with resistance to chloramphenicol. 500ng of oriT-attP-phiC31 binding transfer element was added to 20. Mu.L of E.coli competent cell solution. Gently mixed with 200. Mu.L of sterilized Huang Qiangtou and added into a 1mm electric rotary cup precooled at-20 ℃. The electrotransfer was carried out under the conditions of 1350V, 10. Mu.F, and 600. Omega. After the electro-transformation, 1mL of LB liquid medium without antibiotics was added, and the mixture was pre-cultured in an incubator at 37 ℃ and 220rpm for 1 hour. The preculture was centrifuged at 8000rpm for 1min in a desk centrifuge, 900. Mu.L of the supernatant was discarded, the centrifuged and precipitated cells were mixed well and spread on LB solid medium containing 600. Mu.g of apramycin resistance in four zones, and cultured in an incubator at 37 ℃ for 12 hours until single colonies grew out. Single colonies on the plates were individually picked with a sterile white tip into sterile 2mL EP tubes containing 1mL LB broth. LB liquid medium contained 10. Mu.g of apramycin. The EP tubes were incubated for 12h at 220rpm in a shaker at 37 ℃. In which 500. Mu.L of the culture was mixed with 500. Mu.L of 30% sterile glycerol and stored in a freezer at-80 ℃ in the form of a frozen tube of 15% glycerol at the final concentration. The remaining culture was used for enzyme digestion validation. Coli after enzyme digestion verification is named as E.coli DH10B (NC 30-M)
(8) Extraction of large plasmids from E.coli
The culture of 20mL E.coli DH10B (NC 30) is taken, centrifuged at 12000rpm for 2min to discard the supernatant, the bacteria are suspended by 4mL of solution I for plasmid extraction, 8mL of solution II for plasmid extraction is added, the mixture is repeatedly inverted and uniformly mixed, then 6mL of solution III for plasmid extraction is added and uniformly mixed, finally 6mL of chloroform is added, the mixture is centrifuged at 12000rpm for 2min to take 14mL of supernatant clear liquid after uniform mixing, 9.8mL of isopropanol is added, the mixture is centrifuged at 12000rpm for 20min to discard the supernatant, white flaky precipitates can be seen, the precipitates are rinsed for 2 times by using 75% ethanol, and the precipitates are centrifuged and dried at 60 ℃ to obtain the plasmid p15A-NC30. To an EP tube, 50. Mu.L of sterile water was added, and the concentration was 100 ng/. Mu.L as measured by Nanodrop 2000.
(9) Verification E.coli DH10B (NC 30-M)
mu.L of the above 10. Mu.L total 1. Mu. g p15A-NC30-M plasmid, 6. Mu.L 10000units/mL KpnI or PvuII, and 9. Mu.L ddH 2 O and 3. Mu.L of 10 Xdigestion Buffer were combined to prepare a 30. Mu.L system, and the mixture was digested in a 37 ℃ thermostatic water bath for 1 hour. Add 2. Mu.L of 6 × loading buffer to the above digestion system, mix gently. And sequentially adding the sample and 2 mu L of Marker into the gel hole, wherein the electrophoresis condition is 200V and 30min. And taking out the gel after the electrophoresis is finished, putting the gel into an imaging analyzer to shoot a gel picture and storing the gel picture. As shown in FIG. 2, FIG. 2 is a graph showing the restriction enzyme digestion verification of the p15A-NC30-M plasmid prepared in the example of the present invention.
The size of a band of a p15A-NC30-M plasmid digestion map in the figure 2 accords with the size of a p15A-NC30-M plasmid digestion simulation band in the table 2, and the p15A-NC30-M plasmid is a positive plasmid.
(10) PCR validation E.coli DH10B (NC 30-M)
The amplification of the above-mentioned PCR template was carried out using primers (NC 30-top-yz-1:5'-tcgtcctgccgttggagctgg-3', NC30-top-yz-2:5'-accgaaggagcgaacgaccccttc-3', NC30-mid-yz-1:5'-tcccttgtgcgtgttctgc-3', NC30-mid-yz-2:5'-tgcgggtaagcctgtagtcc-3', NC30-end-yz-1:5 '-4984 zxft 84-3', NC30-end-yz-2:5'-atcgacccgatcggtttcgaccaag-3', yz-phic31-1-F:5'-aggatccgatatccagatcgc-3', yz-phic31-1-R: 3424 zxft 24, yz-phic-3431-3535-phi 3535-3531, NC 30-M-z-3584 (NC 30-3). Sterile water was used as a template as a blank control. PCR amplification reactions were performed on a TaKaRa PCR Thermal Cycler [ 50.0. Mu.L amplification system: 1. Mu.L of 10.0. Mu.M primer, 5.0. Mu.L of 10 Xbuffer (TaKaRa, chinese Dalian), 8.0. Mu.L of 2.5nM dNTP (TaKaRa), 0.5. Mu.L of rTaq enzyme (TaKaRa, chinese Dalian), 1. Mu.L of E.coli DH10B (NC 30-M) bacterial liquid template, 5.0. Mu.L of DMSO, 24.0. Mu.L of ddH2O ], pre-denaturation at 95 ℃ for 2min, denaturation at 98 ℃ for 1s, annealing at 60 ℃ for 15s, extension at 68 ℃ for 1min, cycling for 30 times, and extension at 72 ℃ for 10min. Add 2. Mu.L of 6 × loading buffer to the 10. Mu.L amplification system and mix gently. And sequentially adding the sample and 2 mu L of Marker into the gel hole, wherein the electrophoresis condition is 200V and 30min. And taking out the gel after the electrophoresis is finished, putting the gel into an imaging analyzer to shoot a gel picture and storing the gel picture. Fig. 13 is a PCR verification spectrum of e.coli DH10B (NC 30-M) prepared in the example of the present invention, as shown in fig. 13. All experimental groups in FIG. 13 obtained target bands, blank control did not obtain bands, and E.coli DH10B (NC 30-M) was a positive clone.
(11) Bond transfer
mu.L of E.coli DH10B (NC 30-M) was pipetted from a glycerin tube using a 200. Mu.L sterilized yellow tip, streaked onto LB plates containing 500. Mu.g of apramycin, and cultured in a 37 ℃ incubator for 12 hours. A single E.coli DH10B (NC 30-M) clone was picked up and inoculated into 6mL of LB liquid medium containing 300. Mu.g of apramycin. The culture was carried out in a 37 ℃ constant temperature shaker at 220rpm for 12h. Coli DH10B (NC 30-M) cultured for 12h, the OD600 was 0.898 after four-fold dilution. A2% inoculum size of a 12-hour culture broth of E.coli DH10B (NC 30-M) was inoculated into 6mL of LB broth containing 300. Mu.g of apramycin, and the OD600 of E.coli DH10B (NC 30-M) cultured for 2 hours after the transfer was 0.511.
mu.L of E.coli ET12567 (pUB 307) was pipetted from a glycerin tube using a 200. Mu.L sterilized yellow tip, streaked onto LB plates containing 500. Mu.g of chloramphenicol and 500. Mu.g of kanamycin, and cultured in a 37 ℃ incubator for 12 hours. Coli ET12567 (pUB 307) was picked up as a single clone and inoculated into 6mL of LB liquid medium containing 300. Mu.g of chloramphenicol and 300. Mu.g of kanamycin. The culture was carried out in a 37 ℃ constant temperature shaker at 220rpm for 12h. Coli ET12567 (pUB 307), the OD600 was 0.929 after 12h of culture medium dilution. The bacterial suspension cultured for 12 hours in E.coli ET12567 (pUB 307) was inoculated at 2% inoculum size into 6mL LB liquid medium containing 300. Mu.g chloramphenicol and 300. Mu.g kanamycin, and 2 hours after the transfer culture, E.coli ET12567 (pUB 307) OD600 was 0.323.
Taking out the S.albus J1074 glycerol freezing tube from a refrigerator at minus 80 ℃, sucking 15 mu L of the bacterial liquid of the S.albus J074 from the glycerol tube by using a 200 mu L sterilizing yellow gun head, streaking the bacterial liquid on a MS plate culture medium without an antibody, culturing the bacterial liquid in a constant temperature incubator at 30 ℃ for 4d, and growing white spore-producing monoclonals by naked eyes. Spore-producing monoclonals were picked from the center of the plate with 200. Mu.L of sterilized Huang Qiangtou, spread evenly on MS solid plate medium, and cultured in a constant temperature incubator at 28 ℃ for 4 days with white spores visible to the naked eye. After wetting with sterilized cotton swabs in TES buffer, S.albus J074 spores on MS solid medium were collected with the wetted cotton swabs and collected in 10mL of non-resistant LB liquid medium. 10mL of spore harvest was centrifuged at 8000rpm for 5min in a centrifuge and the supernatant discarded. The spores were suspended by adding 10mL of TES buffer, and the spore suspension was heat shocked in a thermostatic water bath pan at 50 ℃ for 10min. After heat shock, the mixture was taken out and added with 10mL of 2 Xpre-germination solution, and pre-germination was carried out in a constant temperature shaker at 37 ℃ for 2h at a rotation speed of 220 rpm. The germinated spores were centrifuged at 8000rpm for 5min and the supernatant was discarded. Spores were suspended by adding 1mL of non-resistant LB liquid medium. At this point the spore suspension was diluted 3-fold and had an OD600 of 0.483.
50 μ L of E.coli DH10B (NC 30-M), 70 μ L E.coli ET12567 (pUB 307) and 100 μ L S.albus J1074 were gently mixed and spread on ISP4 solid medium, and blown dry on a clean bench for 1h to prevent the growth of the water-excess E.coli from fast growth and adversely affecting the growth of the recombinants. The plate coated and dried is placed upright and cultured in a constant temperature incubator at 30 ℃ for 11h. The plate was taken out and placed in a clean bench, and 1mL of sterile water was added to cover the entire plate, and then the sterile water was aspirated off with a sterile blue-gun head. 1mL of sterile water is sucked up again, and 500. Mu.g of nalidixic acid and 300. Mu.g of aprazalide are added, at which time a turbidity visible to the naked eye is generated in the solution. The aqueous solution is evenly covered on a flat plate and dried for 3 hours on an ultra-clean workbench. Collecting the dried flat plate, and placing the flat plate upside down in a constant temperature incubator at 28 ℃ for culturing for 4-5d until the recombinant grows out.
The recombinants were picked onto MS solid medium plates containing 500ng nalidixic acid and 300ng aprazasin using 200. Mu.L sterile Huang Qiangtou to purify the recombinants from contaminating E.coli. And (4) inversely placing the plate of the transmissible recombinant into a constant-temperature incubator at 28 ℃ for 3d until the purified recombinant grows out. The purified recombinants were plated on MS solid plate medium containing 300ng of aprazasin. The cells were inverted and cultured in a 28 ℃ incubator for 3 days, and the recombinant was named S.albus J1074 (NC 30-M).
(12) PCR verification S.albus J1074 (NC 30-M)
The amplification of the above-mentioned PCR template was carried out using primers (NC 30-top-yz-1:5'-tcgtcctgccgttggagctgg-3', NC30-top-yz-2:5'-accgaaggagcgaacgaccccttc-3', NC30-mid-yz-1:5'-tcccttgtgcgtgttctgc-3', NC30-mid-yz-2:5'-tgcgggtaagcctgtagtcc-3', NC30-end-yz-1:5 '-4984 zxft 84-3', NC30-end-yz-2:5'-atcgacccgatcggtttcgaccaag-3', yz-phic31-1-F:5'-aggatccgatatccagatcgc-3', yz-phic31-1-R: 3424 zxft 24, yz-phic-3431-3535 zpic 31-3531, 3531-3531, NC 30-J-3531, NC 3-3, and Z-3, and S30-Z-3, and S3, and M3, and M.3. Streptomyces sp.ls131440 genome was used as positive control template, sterile water as blank control template, and s.albus J1074 as negative control template. PCR amplification reactions were performed on a TaKaRa PCR Thermal Cycler [ 50.0. Mu.L amplification System: 1. Mu.L of 10.0. Mu.M primer, 5.0. Mu.L of 10 Xbuffer (TaKaRa, conn.), 8.0. Mu.L of 2.5nM dNTP (TaKaRa), 0.5. Mu.L of rTaq enzyme (TaKaRa, conn.), S.albus J1074 (NC 30-M) monoclonal template, 5.0. Mu.L of DMSO, 24.0. Mu.L of ddH 2 O]Pre-denaturation at 95 ℃ for 2min, then denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 15s, and extension at 68 DEG CStretch for 1min, cycle for 30 times, and finally stretch for 10min at 72 ℃. Add 2. Mu.L of 6 × loading buffer to the 10. Mu.L amplification system and mix gently. And sequentially adding the sample and 2 mu L of Marker into the gel hole, wherein the electrophoresis condition is 200V and 30min. And taking out the gel after the electrophoresis is finished, putting the gel into an imaging analyzer to shoot a gel picture and storing the gel picture. As shown in fig. 14, fig. 14 is a PCR verification spectrum of s.albus J1074 (NC 30-M) prepared according to an embodiment of the present invention, in which the same band is obtained for the experimental group and the positive control, and the same band is obtained for both the blank control and the negative control in fig. 14. The S.albus J1074 (NC 30-M) is a positive clone.
2. Fermentative preparation of Compound 1
1. Seed culture
Seed culture medium: 40mL of seed medium was placed in each of 4 250mL triangular glass flasks, sterilized at 115 ℃ for 30 minutes, and the plate medium containing the heterologously expressed recombinant S.albus J1074 (NC 30-M) obtained in the above step (11) was inoculated with a plate seed cut (0.5 cm. Times.0.5 cm), and cultured by rotation (220 rpm) on a rotary shaker at 28 ℃ for 24 hours to obtain a primary seed solution.
The seed culture medium consists of the following components: TSB;
the concentrations of the components in the seed culture medium are respectively (g/L): TSB 30g/L; the pH value of the seed culture medium is 7.2, a spring is added into a triangular glass bottle, and 8 layers of gauze are sealed.
2. Fermentation culture
Preparing a fermentation medium (the components of the fermentation medium comprise 20g/L of mannitol and 20g/L of soybean cake powder, and the pH value of the fermentation medium is 7.8). 300mL of fermentation medium was filled in a 1000mL Erlenmeyer flask, the first-stage seed solution obtained in step 1 was inoculated to the fermentation medium in an inoculum size of 5% (volume percentage) after sterilization (30 minutes at 115 ℃), and the fermentation broth, which was all the substances in the vessel, was harvested after 6 days of rotary culture (220 rpm) at 28 ℃.
2. Isolation of Compound 1
1. Pretreatment of DOWEX 21K XLT resin
And (3) soaking 1L DOWEX 21K XLT resin into 500mL of water to fully expand the resin, discarding the upper layer liquid, adding 500mL of 2-4% dilute sodium hydroxide solution for mild disturbance, and fully washing to remove organic impurities. The above operation was repeated 3 times. Then 500mL of distilled water was added to wash the resin until the resin was neutral. And adding 500mL of 4-5% diluted hydrochloric acid solution, gently disturbing, fully washing and taking out inorganic impurities. Then 500mL of distilled water was added to wash the resin until the resin became neutral.
2. Supernatant of DOWEX 21K XLT resin enriched fermentation liquor
The fermentation broth obtained above was centrifuged at 8000 rpm/min at 4 ℃ for 8 min, and the supernatant was collected. The supernatant was evaporated to dryness by a rotary evaporator to recover the crude extract. Weighing 20g of the crude extract obtained by decompressing and recovering the fermented supernatant, dissolving the crude extract in 100mL of ammonia water solution with the pH value of 10, adding 200mL of pretreated DOWEX 21K XLT resin, and uniformly mixing the mixture at 28 ℃ and 200rpm for 20min in a shaking way, so that the resin can absorb the carbon-phosphorus natural product in the supernatant under the alkaline condition. Collecting supernatant, storing in a refrigerator at 4 deg.C, adding 200mL 1mol/L hydrochloric acid into the adsorbed DOWEX 21K XLT resin, eluting at 220rpm for 30min, filtering with 0.45 μm filter membrane, and removing residual resin and other insoluble impurities from the eluate. The eluate was placed in a suspension-evaporation flask, and the eluate was concentrated to about 2mL with a rotary evaporator and then transferred to a 4mL sample vial. Sealing the bottle mouth with sealing film, and pricking with needle to keep the pressure inside and outside the bottle consistent. And (3) freezing the sample in the sample bottle in a refrigerator at the temperature of-80 ℃, and then putting the sample in a freeze dryer for freeze drying overnight. Finally, the crude extract containing the compound 1 after resin enrichment is obtained, and the dry weight is 3721.0mg which is recorded as S-NC30.
3. Sephadex LH-20 column chromatography of S-NC30 Sephadex
The 3721.0mg S-NC30 fraction prepared in step 1 was dissolved in 5mL of water, and 1.4L Sephadex LH-20 column (brand GE healthcare) was used to separate and purify the phosphorocarbon compound, using water as eluent at a flow rate of 8 seconds per drop, to give 5 fractions (S-NC 30-1, S-NC30-2, S-NC30-3, S-NC30-4, S-NC 30-5) in which the phosphorocarbon compound was mainly present in fraction 3 (1321.7 mg), denoted S-NC30-3.
4. Sephadex LH-20 column chromatography separation of S-NC30-3
And (3) dividing the 1321.7mg S-NC30-3 prepared in the step 2 into a 200mg 3A component and a 1000mg 3B component. The 200mg of 3A was dissolved in 1mL of water, separated and purified using 400mL of Sephadex LH-20 column (brand: GE healthcare), and 7 fractions (3A-1, 3A-2, 3A-3, 3A-4, 3A-5, 3A-6, 3A-7) were obtained using water as eluent at a flow rate of 7 sec/drop, and the carbon phosphorus compound was mainly present in the 5 th fraction (48.8 mg in total), and was recorded as 3A-5. The 1000mg of 3B was dissolved in 4mL of water, and separated and purified by using 1.4mL of Sephadex LH-20 column (brand: GE healthcare) using water as an eluent at a flow rate of 7 seconds/drop to give 6 fractions (3B-1, 3B-2, 3B-3, 3B-4, 3B-5, 3B-6) in total, and the carbon phosphorus compound was mainly present in the 4 th and 5 th fractions (total 513.7 mg) and was designated as 3B-4C.
5. Sephadex LH-20 column chromatography separation of 3B-4C
The 513.7mg of 3B-4C prepared in step 3 was dissolved in 3mL of water and separated and purified using 400mL of Sephadex LH-20 column (brand: GE healthcare) using water as eluent at a flow rate of 7 sec/drop to give 60 fractions and the carbon phosphorus compound was mainly present in 21 st to 23 th fractions (156.5 mg total). The 156.5mg 3B-4C 21-23 fractions were combined with the 3A-5 fractions (205.3 mg total) and noted NC30-D.
6. Preparation of Compound 1
The NC30-D prepared in step 4 was dissolved in 2mL of water to a final concentration of 102.7mg/mL and compound 1 was prepared using reverse phase HPLC under the conditions: thermo Hypercarb column, 4.6 x 150mm, detection wavelength 270nm, mobile phase 0.1% ammonia (a) and acetonitrile. The mobile phase gradient was (B): 0.00min,1%;3.00min,1 percent; 3.50min,5%;4.00min,20%;4.01min,99%;9.00min,99%;9.01min,1%;13.00min,1 percent. Collecting the effluent with retention time of 2min to obtain compound 1.
3. Identification of Compound 1
1. mass spectrum: as shown in FIG. 3, FIG. 3 shows the dissolution of Compound 1 in D prepared in the example of the present invention 2 ESI-MS graph under a negative ion source in O, HRESIMS showing that the peak of the excimer ion is m/z 224.9799[ M-H ])] - And the mass spectrogram acquisition is performed by using a Saimer fly Q-active plus.
2. Nuclear magnetic resonance spectroscopy: as shown in FIGS. 4 to 11, FIG. 4 shows the solubility of Compound 1 in D prepared in the examples of the present invention 2 In O is 1 H-NMR (700 MHz) spectrum, FIG. 5 shows the dissolution of Compound 1 prepared in the example of the present invention in D 2 In O 13 C-NMR (150 MHz) spectrum, FIG. 6 shows the dissolution of Compound 1 prepared in the example of the present invention in D 2 In O is 31 P-NMR spectrum, FIG. 7 shows the dissolution of Compound 1 in D prepared in example of the present invention 2 DEPT spectra in O, FIG. 8 shows the dissolution of Compound 1 in D prepared in the examples of the present invention 2 In O of ( 1 H- 1 H) COSY spectrum, FIG. 9 shows that compound 1 prepared in the example of the invention is dissolved in D 2 HSQC spectrum in O, FIG. 10 shows the dissolution of Compound 1 in D prepared according to the example of the present invention 2 In O of ( 1 H- 13 C) HMBC chromatogram, FIG. 11 shows the dissolution of Compound 1 in D prepared in the example of the invention 2 In O of ( 1 H- 31 P) HMBC spectrum.
According to the compound 1 H NMR、 13 C NMR and HSQC spectra show 2 carboxyl carbons (. Delta.) C 173.9,173.4, positions 5, 1) in the following structural formula, 1 sp 2 Quaternary carbon (. Delta.) C 143.9, position 2) in the following formula, 2 sp 3 Methine radical (. Delta.) C 126.7,71.0, positions 3, 4 in the following structural formula). 31 P NMR spectrum showed 1 phosphorus signal (. Delta.) P 15.4)。 1 H- 1 H COSY shows H-3 (. Delta.) H 6.05 Is with H-4 (. Delta.) H 4.47 Coupled). 1 H- 31 P HMBC spectra show the presence of H-3, H-4 and 4-P (. Delta.) ( P 15.4 ) of the received signal is detected, and, 1 H- 13 c HMBC shows H-4 and C-5 (. Delta.) C 173.9)、C-2(δ C 143.9)、C-3(δ C 126.7 And H-3 and C-1 (. Delta.) C 173.4)、C-4(δ C 71.0 C-5, C-2, in combination with the exact molecular formula shown by the high resolution mass spectrometry, the NMR of Compound 1 was studied and examined 13 C signals were assigned and, as shown in table 2, the planar structure of compound 1 was finally determined as follows:
TABLE 2 NMR peak assignment of Compound 1
The compound 1 provided by the invention belongs to a novel carbon-phosphorus compound and is not reported in patents and literatures.
Use of compound 1: the compound shown in the formula 1 is a carbon-phosphorus compound, the medicine rate of a natural product of carbon-phosphorus small molecules is 15%, and the compound shown in the formula 1 is expected to become a novel antibiotic or be predicted to become an anti-cancer medicine and an anti-malaria medicine.
Although the present invention has been described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the present invention.
Claims (1)
1. A method for preparing a carbon-phosphorus compound, wherein the carbon-phosphorus compound is a compound 1,
compound 1 is derived from Streptomyces sp.LS131440 heterologous expression recombinant S.albus J1074 (NC 30-M); the Streptomyces sp.LS131440 is preserved in China general microbiological culture Collection center at 29.04.2019 with the preservation number of CGMCCNo.17649;
the preparation method comprises the following steps:
sterilizing a fermentation culture medium, inoculating a primary seed solution to the fermentation culture medium according to an inoculation amount of 5% by volume, performing rotary culture at 28 ℃ for 6 days, and then harvesting a fermentation broth, wherein the fermentation broth is all substances in a container, and separating and purifying the fermentation broth to obtain the compound 1;
the fermentation medium comprises the following components: 20g/L of mannitol, 20g/L of soybean cake powder and 7.8 of pH value of the fermentation medium;
the first-stage seed liquid is prepared by the following preparation method:
sterilizing a seed culture medium, digging and inoculating a flat strain to obtain a flat culture medium containing a heterologous expression recombinant S.albus J1074 (NC 30-M), and performing rotary culture on a rotary table at 28 ℃ for 24 hours to obtain a primary seed solution;
wherein the seed culture medium is a culture medium of TSB 30g/L; the pH value of the seed culture medium is 7.2;
the heterologous expression recombinant S.albus J1074 (NC 30-M) is obtained by cloning Streptomyces sp.LS131440-NC30 gene cluster and conjoint transfer of three parents;
the cloning method of the Streptomyces sp.LS131440-NC30 gene cluster is as follows: when gene cluster cloning is carried out, taking a genome of a strain Streptomyces sp.LS131440 as an enzyme cutting template, selecting enzyme cutting sites at the outer sides of two ends of a target gene cluster for enzyme cutting to obtain a Streptomyces sp.LS131440-NC30 gene cluster, grabbing a carbon-phosphorus compound biosynthesis gene cluster Streptomyces sp.LS131440-NC30 by a RecET recombination technology, integrating a conjugative transfer element by a Red alpha beta recombination technology, and finally constructing a recombinant plasmid p15A-NC30-M and an Escherichia coli E.coli DH10B (NC 30-M); when carrying out three-parent combination transfer, the Streptomyces sp.LS131440-NC30 gene cluster is stably combined to the chromosome of a heterologous expression host Streptomyces albus J1074 with the aid of a plasmid pUB307, and a recombinant S.albus J1074 (NC 30-M) is obtained.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910440511.8A CN111979136B (en) | 2019-05-24 | 2019-05-24 | Carbon-phosphorus compound derived from streptomyces, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910440511.8A CN111979136B (en) | 2019-05-24 | 2019-05-24 | Carbon-phosphorus compound derived from streptomyces, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111979136A CN111979136A (en) | 2020-11-24 |
| CN111979136B true CN111979136B (en) | 2023-03-10 |
Family
ID=73436724
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910440511.8A Active CN111979136B (en) | 2019-05-24 | 2019-05-24 | Carbon-phosphorus compound derived from streptomyces, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111979136B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3658987A (en) * | 1964-11-18 | 1972-04-25 | Rhone Poulenc Sa | Antibiotic 8036 r.p. and process for the production thereof |
| JPS58216689A (en) * | 1982-06-10 | 1983-12-16 | Meiji Seika Kaisha Ltd | Novel phosphorus-containing compound and its preparation |
| CN101213205A (en) * | 2004-12-22 | 2008-07-02 | 希龙公司 | Group B streptococcus |
| CN105925637A (en) * | 2016-04-27 | 2016-09-07 | 中国科学院南海海洋研究所 | Method for preparing antibiotic dihydrotetrodecamycin through Streptomyces sp. SCSIO 5604 |
-
2019
- 2019-05-24 CN CN201910440511.8A patent/CN111979136B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3658987A (en) * | 1964-11-18 | 1972-04-25 | Rhone Poulenc Sa | Antibiotic 8036 r.p. and process for the production thereof |
| JPS58216689A (en) * | 1982-06-10 | 1983-12-16 | Meiji Seika Kaisha Ltd | Novel phosphorus-containing compound and its preparation |
| CN101213205A (en) * | 2004-12-22 | 2008-07-02 | 希龙公司 | Group B streptococcus |
| CN105925637A (en) * | 2016-04-27 | 2016-09-07 | 中国科学院南海海洋研究所 | Method for preparing antibiotic dihydrotetrodecamycin through Streptomyces sp. SCSIO 5604 |
Non-Patent Citations (2)
| Title |
|---|
| Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide;Blodgett 等;《NATURE CHEMICAL BIOLOGY》;20070831;第3卷(第8期);第480-485页 * |
| 磷霉素类新抗生素磷酰胺霉素;刘诗通;《国外医药.抗生素分册》;19831231(第03期);第240-246、187页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111979136A (en) | 2020-11-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107501086B (en) | Sixteen-carbon-chain fatty acid antagonistic substance generated by Bacillus amyloliquefaciens SQR9 and application thereof | |
| CN114317317A (en) | Salt-tolerant Bacillus belgii with high lipopeptide yield and application thereof | |
| WO2016004848A1 (en) | Fidaxomicin purification method | |
| CN112608965B (en) | Culture medium for producing bleomycin E by using deep sea streptomycete and preparation method thereof | |
| CN116286560B (en) | Raoult bacterium HC6 and application thereof in low-temperature production of 2, 3-butanediol | |
| CN111979136B (en) | Carbon-phosphorus compound derived from streptomyces, and preparation method and application thereof | |
| CN111778172A (en) | Streptomyces for producing antibacterial active compound and separation method and application thereof | |
| CN108101778B (en) | Fourteen-carbon-chain fatty acid antagonistic substance generated by Bacillus amyloliquefaciens SQR9 and application thereof | |
| CN109280034B (en) | Benzoxazepine compound with antibacterial activity and preparation method and application thereof | |
| CN106978457B (en) | Preparation method of antibiotic fusaricidin A | |
| CN110564718A (en) | Method and strain for screening high-yield amphotericin B-tubercle streptomycete by high-throughput mutagenesis | |
| CN102453708B (en) | Method for increasing yield of streptomyces coeruleorubidus daunorubicin | |
| CN115109023A (en) | Macrolide compound FWYZ52-A, and fermentation strain, fermentation method and application thereof | |
| CN110092758B (en) | Novel alkaloid compound and wart spore strain for preparing compound by fermentation | |
| CN108949610B (en) | Streptomyces and angucycline compound generated by streptomyces as well as preparation and application of angucycline compound | |
| CN111979137B (en) | Carbon-phosphorus compound derived from marine streptomyces and preparation method and application thereof | |
| CN112239740B (en) | Streptomyces strain for producing piericidin A1 and inhibiting growth of plant pathogenic xanthomonas | |
| CN107699532B (en) | 3, 7-dihydroxy tropolone high-yield strain and fermentation culture method thereof | |
| CN106434785B (en) | The preparation method of 2-n- heptyl -4- oxyquinoline | |
| CN114606135B (en) | Sponge coanda fungus and application thereof in preparation of sterols | |
| CN111269867B (en) | Gene engineering strain for directionally producing antiviral and antibacterial active compound and application thereof | |
| CN114292193B (en) | Depsipeptide cyclic ether compound, bacterial strain, preparation method and application | |
| CN113912658B (en) | 4' -N-methyl-vicencarptin, and preparation method and application thereof | |
| CN111285828B (en) | Compound proximicin and preparation method and application thereof | |
| CN116676353A (en) | Streptomyces coelicolor M145-chry and application thereof in fermentation production of aureomycin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |