CN104164399B - The superior strain and its construction method of cyclic ester peptide antibiotics Hei Motading - Google Patents
The superior strain and its construction method of cyclic ester peptide antibiotics Hei Motading Download PDFInfo
- Publication number
- CN104164399B CN104164399B CN201410372728.7A CN201410372728A CN104164399B CN 104164399 B CN104164399 B CN 104164399B CN 201410372728 A CN201410372728 A CN 201410372728A CN 104164399 B CN104164399 B CN 104164399B
- Authority
- CN
- China
- Prior art keywords
- gene
- nov
- himastatinicus
- himastatin
- hmta
- 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.)
- Expired - Fee Related
Links
- 238000010276 construction Methods 0.000 title claims abstract description 6
- -1 cyclic ester Chemical class 0.000 title abstract description 4
- 239000003910 polypeptide antibiotic agent Substances 0.000 title abstract description 4
- 241000855330 Streptomyces himastatinicus Species 0.000 claims abstract description 70
- 108700024144 himastatin Proteins 0.000 claims abstract description 54
- OZPNLJQULOIOGU-UPABGHPRSA-N himastatin Chemical compound O=C([C@@H]1C[C@@]2(O)C3=C4)N[C@H](C(C)C)C(=O)O[C@@H](C(C)C)C(=O)N5NC[C@H](O)C[C@@H]5C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]([C@H](C)O)C(=O)N1[C@@H]2NC3=CC=C4C(C=C12)=CC=C2N[C@@H]2[C@@]1(O)C[C@H]1C(=O)N[C@H](C(C)C)C(=O)O[C@@H](C(C)C)C(=O)N3NC[C@H](O)C[C@@H]3C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]([C@H](C)O)C(=O)N12 OZPNLJQULOIOGU-UPABGHPRSA-N 0.000 claims abstract description 54
- 101100071243 Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H) hmtA1 gene Proteins 0.000 claims abstract description 47
- 239000002773 nucleotide Substances 0.000 claims abstract description 23
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 23
- 101150013565 hmtB gene Proteins 0.000 claims abstract description 17
- 230000002779 inactivation Effects 0.000 claims abstract description 11
- 230000033228 biological regulation Effects 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 24
- 101100178222 Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H) hmtB gene Proteins 0.000 abstract description 19
- 230000003115 biocidal effect Effects 0.000 abstract description 18
- 238000000034 method Methods 0.000 abstract description 16
- 108091008053 gene clusters Proteins 0.000 abstract description 11
- 125000004122 cyclic group Chemical group 0.000 abstract description 8
- 230000008569 process Effects 0.000 abstract description 8
- 238000003786 synthesis reaction Methods 0.000 abstract description 7
- 108010028921 Lipopeptides Proteins 0.000 abstract description 6
- 230000002103 transcriptional effect Effects 0.000 abstract description 3
- 238000011144 upstream manufacturing Methods 0.000 abstract description 3
- 101800001241 Acetylglutamate kinase Proteins 0.000 abstract description 2
- 108091077303 mer family Proteins 0.000 abstract description 2
- 230000000415 inactivating effect Effects 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 23
- 150000001875 compounds Chemical class 0.000 description 16
- 238000004128 high performance liquid chromatography Methods 0.000 description 14
- 238000000855 fermentation Methods 0.000 description 12
- 230000004151 fermentation Effects 0.000 description 12
- 238000003752 polymerase chain reaction Methods 0.000 description 12
- 241000588724 Escherichia coli Species 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 239000012071 phase Substances 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 229940125904 compound 1 Drugs 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000187747 Streptomyces Species 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000005215 recombination Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 229930000044 secondary metabolite Natural products 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 101100071248 Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H) hmtA2 gene Proteins 0.000 description 3
- 101100397226 Schizosaccharomyces pombe (strain 972 / ATCC 24843) isp4 gene Proteins 0.000 description 3
- 241001655322 Streptomycetales Species 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 235000002639 sodium chloride Nutrition 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108010069514 Cyclic Peptides Proteins 0.000 description 2
- 102000001189 Cyclic Peptides Human genes 0.000 description 2
- 229930105110 Cyclosporin A Natural products 0.000 description 2
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 2
- 108010036949 Cyclosporine Proteins 0.000 description 2
- 108010013198 Daptomycin Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108700023479 Glutamate 5-kinases Proteins 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 108700005078 Synthetic Genes Proteins 0.000 description 2
- 108010059993 Vancomycin Proteins 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 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 2
- 229950006334 apramycin Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000007622 bioinformatic analysis Methods 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 229960001265 ciclosporin Drugs 0.000 description 2
- 239000004927 clay Substances 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- DOAKLVKFURWEDJ-QCMAZARJSA-N daptomycin Chemical compound C([C@H]1C(=O)O[C@H](C)[C@@H](C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)[C@H](C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-QCMAZARJSA-N 0.000 description 2
- 229960005484 daptomycin Drugs 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class 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 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000012269 metabolic engineering Methods 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 229960003165 vancomycin Drugs 0.000 description 2
- 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 2
- 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 2
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 1
- 241000186046 Actinomyces Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000251557 Ascidiacea Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 230000003682 DNA packaging effect Effects 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000194033 Enterococcus Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 235000019733 Fish meal Nutrition 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000005133 Glutamate 5-kinase Human genes 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- YJPIGAIKUZMOQA-UHFFFAOYSA-N Melatonin Natural products COC1=CC=C2N(C(C)=O)C=C(CCN)C2=C1 YJPIGAIKUZMOQA-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000187708 Micromonospora Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 241001575310 Polyzoa Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 244000191761 Sida cordifolia Species 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000187391 Streptomyces hygroscopicus Species 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- NGXUUAFYUCOICP-UHFFFAOYSA-N aminometradine Chemical compound CCN1C(=O)C=C(N)N(CC=C)C1=O NGXUUAFYUCOICP-UHFFFAOYSA-N 0.000 description 1
- 229960001887 aminometradine Drugs 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000001716 anti-fugal effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000078 anti-malarial effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003430 antimalarial agent Substances 0.000 description 1
- 239000003972 antineoplastic antibiotic Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 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 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000013601 cosmid vector Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 150000001923 cyclic compounds Chemical class 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- DRLFMBDRBRZALE-UHFFFAOYSA-N melatonin Chemical compound COC1=CC=C2NC=C(CCNC(C)=O)C2=C1 DRLFMBDRBRZALE-UHFFFAOYSA-N 0.000 description 1
- 229960003987 melatonin Drugs 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 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 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 108010091748 peptide A Proteins 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000004763 spore germination Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010809 targeting technique Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 1
- 229960001082 trimethoprim Drugs 0.000 description 1
- GSXRBRIWJGAPDU-BBVRJQLQSA-N tyrocidine A Chemical compound C([C@H]1C(=O)N[C@H](C(=O)N[C@@H](CCCN)C(=O)N[C@H](C(N[C@H](CC=2C=CC=CC=2)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N1)=O)CC(C)C)C(C)C)C1=CC=C(O)C=C1 GSXRBRIWJGAPDU-BBVRJQLQSA-N 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 238000005292 vacuum distillation Methods 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses the superior strains and its construction method of cyclic ester peptide antibiotics Hei Motading.The superior strain of Hei Motading is to inactivate the hmtA gene of S.himastatinicus sp.Nov or hmtB gene, thus to obtain himastatin high productive mutant S.himastatinicus sp.Nov, the nucleotide sequence of the hmtA gene is as shown in SEQ ID NO.1, and the nucleotide sequence of the hmtB gene is as shown in SEQ ID NO.3.The mutant strain S.himastatinicus sp.Nov Ju2015 that the present invention respectively obtains mutant strain S.himastatinicus sp.Nov Ju2014 and the hmtB inactivation of hmtA inactivation by inactivating the Mer family transcriptional modulatory gene hmtA and acetylglutamate kinase gene hmtB of the biological synthesis gene cluster upstream of the Cyclic lipopeptide antibiotic himastatin of S.himastatinicus sp.Nov.The yield of the himastatin of mutant strain S.himastatinicus sp.Nov Ju2014 and Ju2015 are 10 times or more of wild type S.himastatinicus sp.Nov.Therefore, the success of two high productive mutants of Cyclic lipopeptide antibiotic himastatin is configured to accelerate himastatin industrialization process that bring may be with hope.
Description
Technical field:
The invention belongs to microbiological genetic engineerings and metabolic engineering field, and in particular to cyclic ester peptide antibiotics Hei Motading
(himastatin) superior strain and its construction method.
Background technique:
Cyclic peptide compound is a kind of cyclic compound formed with amido bond, mainly by amino acid, glycopeptide, lipopeptid and
A series of complicated molecule that nucleosides peptide is composed after modifications.Such compound be widely present in invertebrate (sponge,
Ascidian, ectoproctous polyzoa etc.), bacterium, cyanobacteria, in fungi and actinomyces, majority has complicated novel structure feature, most prominent
Out the characteristics of is cyclic structure and various amino acid derivativges structural units.Cyclic structure assigns such compound certain object
Reason rigidity and the tolerance to proteolysis, and different derivative structures more makes such compound have various physiology
Biochemical speciality.Because its majority has outstanding biology performance, (antibacterial, antiviral, immunosuppressive activity, resists anti-inflammatory, anticancer
Worm, antimalarial and Physiology and biochemistry regulating and controlling effect etc.) so that such compound has become synthesis chemistry, drug screening and exploitation
Pay close attention to object.Some of which compound has been used successfully to the treatment of clinical disease.Such as antifungic action quarter butt
Bacterium junket peptide A (tyrocidine A), for the cyclosporin A (cyclosporin A) of the anti-repulsive interaction of organ transplant, to methoxy
The drug resistant S. aureus L-forms in XiLin have the vancomycin (Vancomycin), to Mrsa In Rabbits and through the ages of excellent activity
Mycin drug resistance enterococcus all has Daptomycin (daptomycin) of good antibacterial activity etc., is that such compound is used for
The successful model of clinical treatment.
But some of which cyclic peptide compound is since its water solubility is poor, side effect is larger or the yield of compound
Low reason and exited clinical test, but this also side light carries out structure of modification and optimization to these compounds or improves
The necessity and urgency of its yield.And since these compound majorities have labyrinth, numerous chiral centres and close
The selection of ring position seriously restricts the process of efficiency or industrialization that structure of modification is carried out to this kind of compound.For traditional
The limitation of natural products screening and research and development means is developed one kind in nineteen nineties and is ground with genetic engineering and zymetology
Biosynthesis technology based on studying carefully --- Combinatorial biosynthesis (combinatorial biosynthesis), not only compensates for
The deficiency of tradition research method, and new Research Thinking and research side are provided for further expand of biomedicine field
To.And with the rapid development of genomic sequencing technique and gene recombination technology etc., Combinatorial biosynthesis is led to biology
Domain brings revolutionary variation, at the same also to chemistry subject create new chemical structure and building compound library bring it is new
Method and provide beneficial complement.In addition, the biosynthesis gene of antibiotic is usually at one section of chromosome or huge plasmid
The feature of continuum cluster arrangement, (including structural gene, ego resistance gene, transporter gene and controlling gene) is further
Exploitation and transformation antibiotic provide new opportunity.By 2006, has the biology of nearly 300 kinds of microbial secondary metabolites
Synthetic gene cluster is cloned and Function Identification, to utilize Combinatorial biosynthesis technology, metabolic engineering technology and external zymetology skill
Art is created or is transformed to obtain new compound and lay a good foundation.There are many researchs to pass through biosynthesis and combination biology
Synthetic technology has successfully completed the orientation product of the structural modification of more complicated natural products, transformation or certain target components
It is tired, a series of suitable activity or the stronger derivative of activity or high productive mutant are constructed, is that the genetic engineering of streptomycete is transformed
The reference sample of beneficial methodology is provided, for the resistance problems and tumor disease for solving increasingly intractable pathogenic microorganism
Treatment brings new hope.
Cyclic lipopeptide antibiotic Hei Motading (himastatin), structure as shown in Figure 1, external activity analysis shows that its
There is preferable bacteriostatic activity to gram-positive bacteria, has significant cell to P388 cell line and melatonin tumor B16 cell line
Cytotoxic activity.
Summary of the invention:
The object of the present invention is to provide Hei Motading (himastatin) high productive mutant Streptomyces
himastatinicus sp.Nov。
Himastatin high productive mutant Streptomyces himastatinicus sp.Nov of the invention is
The Streptomyces himastatinicus sp.Nov of hmtA gene or hmtB gene inactivation, the hmtA gene
Nucleotide sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of the hmtB gene is as shown in SEQ ID NO.3.
The present invention also provides himastatin high productive mutant Streptomyces himastatinicus sp.Nov
Construction method, which is characterized in that be by the hmtA gene of Streptomyces himastatinicus sp.Nov or
HmtB gene inactivation, thus to obtain himastatin high productive mutant Streptomyces himastatinicus sp.Nov,
The nucleotide sequence of the hmtA gene is as shown in SEQ ID NO.1, the nucleotide sequence such as SEQ of the hmtB gene
Shown in ID NO.3.
It is described the present invention also provides the application of hmtA gene or hmtB gene in regulation himastatin yield
The nucleotide sequence of hmtA gene is as shown in SEQ ID NO.1, the nucleotide sequence of the hmtB gene such as SEQ ID NO.3
It is shown.
Original strain Streptomyces himastatinicus sp.Nov of the invention is one plant of Lu Yuan streptomycete, point
Do not pass through 1 MerR family transcriptional modulatory gene, the 1 acetyl paddy ammonia in the biological synthesis gene cluster to its secondary metabolite
The genetic modification of kinase gene has obtained the mutant strain Streptomyces of 2 plants of energy high yield himastatin
Himastatinicus sp.Nov Ju2014 and Ju2015.With the culture medium fermentation strain of himastatin, ethyl acetate is used
Extractive fermentation liquid, by its secondary metabolite of the analysis of HPLC-DAD-UV.Program analysis is carried out with HPLC program, HPLC divides
Analysis map shows that elution time is at 17.45min and has feature ultraviolet absorption value (λmax210,285) flow point, the flow point are answered
For himastatin.In order to ensure at 17.45 min and having feature ultraviolet absorption value (λmax210,285) flow point is
Himastatin, therefore the fermentation process of 6L scale is used, then analyze to obtain the sterling of compound 1 by HPLC-UV；Through nuclear-magnetism
Resonance analyzing, 1D (1H,13C) NMR spectroscopy data and the map of himastatin reported in the literature are completely the same, that is, determine
Compound 1 at 17.45 min of retention time is antibiotic himastatin (its structural formula is as shown in Figure 1).
The invention further relates to the genome-wide screenings and antibiosis of Streptomyces himastatinicus sp.Nov
The confirmation of the biological synthesis gene cluster of plain himastatin.In order to find the gene cluster of antibiotic himastatin biosynthesis,
The method that we use genome-wide screening, utilizes Illumina ' s Solexa sequencing technologies, to Streptomyces
The complete genome DNA of himastatinicus sp.Nov bacterial strain carries out sequencing, a size as the result is shown for gene annotation
Gene information necessary to himastatin biosynthesis, further biological information may be contained for the continuous fragment of 60 Kb
The DNA sequence dna that credit analysis discloses 20 about 45 Kb sizes of open reading frame contains in antibiotic himastatin biosynthesis
Regulation, structure, ego resistance, transhipment and after modify isogenic information (see Fig. 2).The sequence of gene cluster is by EMBL number
It is included according to library, sequence number FR823394.
The present invention relates to the gene hmtA (cores for being responsible for transcriptional control effect in himastatin biological synthesis gene cluster
Nucleotide sequence is as shown in SEQ ID NO.1, and the amino acid sequence of the albumen HmtA of coding is as shown in SEQ ID NO.2), acetyl
(for nucleotide sequence as shown in SEQ ID NO.3, the amino acid sequence of the albumen HmtB of coding is such as by gamma-Glutamate kinase gene hmtB
Shown in SEQ ID NO.4), the engineered strain of high yield antibiotic himastatin will can be obtained after its individually progress replacement mutation.
In the upstream of himastatin biological synthesis gene cluster, there is the gene for being named as hmtA, it, which encodes one, there are 247 amino acid
Polypeptide, identify that the peptide chain belongs to MerR family transcription regulator by bioinformatic analysis.In order to verify hmtA and hmtB
Specific effect of the protein HmtA and HmtB of gene coding in the biosynthesis of antibiotic himastatin, has with one
The DNA fragmentation of apramycin resistance carries out replacing in reading frame respectively, so that hmtA and hmtB inactivation.Mutation clay is transferred to
Into the E.coli ET12567/pUZ8002 bacterial strain of Methylation deficient, with wild type S.himastatinicus sp.Nov bacterium
Strain carries out inter-species engagement transfer, obtains some joint elements, each respectively to choose 3 resistant phenotypes (A Pula tolerance, kanamycins are quick
Sense) and the correct mutant strain S.himastatinicus sp.Nov Ju2014 of genotype, mutant strain
S.himastatinicus sp.Nov Ju2015, with wild type S.himastatinicus sp.Nov bacterial strain same condition simultaneously
Fermentation, wild-type strain is as positive control.The fermentation liquid ethyl acetate extraction of mutant strain Δ hmtA, knot are analyzed by HPLC
Fruit shows the consistent high yield himastatin of two mutant strain clones, and the yield of the target product is higher by compared with wild-type strain
10 times or more.These data demonstrate HmtA and HmtB albumen has negative regulatory factor in the biosynthesis of himastatin
Effect.
Nucleotide sequence provided by the present invention or partial nucleotide sequence, using polymerase chain reaction (PCR)
Method or the 1st~744 DNA comprising sequence shown in SEQ ID NO.1 of the present invention are as probe with Southern hybridization etc.
Method obtains gene similar with hmtA from other organisms.
Can be modified in vitro in vivo comprising nucleotide sequence provided by the present invention or at least partly nucleotide sequence or
It is mutated, including insertion, displacement or missing, polymerase chain reaction, mistake mediated polymerization enzyme chain reaction, locus specificity
Mutation, not homotactic reconnect or is oriented evolutions with the homologous sequence in other sources at the different piece of sequence, or leads to
Cross ultraviolet light or chemical reagent mutagenesis etc..
Clone gene comprising nucleotide sequence provided by the present invention or at least partly nucleotide sequence can pass through conjunction
Suitable expression system is expressed in foreign host to obtain corresponding enzyme or other higher bioactive substances or yield.These
Foreign host includes Escherichia coli, streptomycete, micromonospora, pseudomonas, bacillus, yeast, plant and animal etc..
Albumen required for amino acid sequence provided by the present invention can be used to separate and the preparation that can be used for antibody.
Polypeptide comprising amino acid sequence provided by the present invention or at least partly sequence may remove or substitute it is certain
Still there is bioactivity even to have new biological activity after amino acid, or improves yield or optimize protein dynamics spy
Sign or other properties being dedicated to.
The mutant strain for the yield for improving himastatin or derivatives thereof can be used to construct comprising DNA fragmentation or gene,
The present invention provides the approach that yield is improved in genetically engineered microorganism.
The biology that the present invention passes through the Cyclic lipopeptide antibiotic himastatin of inactivation S.himastatinicus sp.Nov
The Mer family transcriptional modulatory gene hmtA and acetylglutamate kinase gene hmtB of synthetic gene cluster upstream and respectively obtain hmtA
The mutant strain S.himastatinicus of mutant strain S.himastatinicus sp.Nov Ju2014 and the hmtB inactivation of inactivation
sp.Nov Ju2015.The yield of the himastatin of mutant strain S.himastatinicus sp.Nov Ju2014 and Ju2015
It is 10 times or more of wild type S.himastatinicus sp.Nov.Therefore, Cyclic lipopeptide antibiotic himastatin two
The success of high productive mutant is configured to accelerate himastatin industrialization process that bring may be with hope.
Black not its fourth streptomycete Streptomyces himastatinicus sp.Nov of the invention is purchased from U.S.'s mould
Formula culture collection warehousing (American type culture collection), number are as follows: ATCC53653, the bacterial strain are external
Sale, anyone can buy from the preservation library.
Detailed description of the invention:
Fig. 1 is the chemical structural formula of black not its fourth (himastatin).
Fig. 2 is himastatin biosynthesis gene in streptomycete Streptomyces himastatinicus sp.Nov
Cluster organization chart.
Fig. 3 is the building of hmtA double crossing over mutant strain Streptomyces himastatinicus sp.Nov Ju2014
Schematic diagram, wherein Mutant Ju2014 indicates mutant strain Streptomyces himastatinicus sp.Nov Ju2014；
Fig. 4 is that the PCR of hmtA double crossing over mutant strain Streptomyces himastatinicus sp.Nov Ju2014 is tested
Card figure, wt indicate wild type Streptomyces himastatinicus sp.Nov, Δ hmtA1, Δ hmtA2, Δ hmtA3 table
Show three clones of mutant strain Streptomyces himastatinicus sp.Nov Ju2014；
Fig. 5 is the building of hmtB double crossing over mutant strain Streptomyces himastatinicus sp.Nov Ju2015
Schematic diagram, wherein Mutant Ju2015 indicates mutant strain Streptomyces himastatinicus sp.Nov Ju2015；
Fig. 6 is that the PCR of hmtB double crossing over mutant strain Streptomyces himastatinicus sp.Nov Ju2015 is tested
Card figure, wherein wt indicates wild type Streptomyces himastatinicus sp.Nov, Δ hmtB1, Δ hmtB2, Δ
Three clones of hmtB3 expression mutant strain Streptomyces himastatinicus sp.Nov Ju2015；
Fig. 7 be hmtA double crossing over mutant strain Streptomyces himastatinicus sp.Nov Ju2014 with it is wild
HPLC analysis after fermenting respectively under type equal conditions is as a result, wherein wt indicates wild type Streptomyces
Himastatinicus sp.Nov, Δ hmtA1, Δ hmtA2, Δ hmtA3 indicate mutant strain Streptomyces
Three clones of himastatinicus sp.Nov Ju2014, Δ hmtA1, Δ hmtA2, Δ hmtA3 are after diluting 10 times
HPLC result after sample introduction.
Fig. 8 be hmtB double crossing over mutant strain Streptomyces himastatinicus sp.Nov Ju2015 with it is wild
HPLC analysis after fermenting respectively under type equal conditions is as a result, wherein wt indicates wild type Streptomyces
Himastatinicus sp.Nov, Δ hmtB1, Δ hmtB2, Δ hmtB3 indicate mutant strain Streptomyces
Three clones of himastatinicus sp.Nov Ju2015, Δ hmtB1, Δ hmtB2, Δ hmtB3 are after diluting 10 times
HPLC result after sample introduction.
Fig. 9 is the H spectrum of reactive compound 1.
Specific embodiment:
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1:
The culture of 1.S.himastatinicus sp.Nov
The culture used in S.himastatinicus sp.Nov wild-type strain of the invention and mutant strain growth
Base, every liter is prepared in this way: fish meal 10g, glycerol 20g, calcium carbonate 5g and yeast extract 5g being added in 1L water, pH is
Between 7.2-7.4, conventional sterilant is spare.The temperature of its most suitable growth is at 28 DEG C -30 DEG C.
2. the secondary metabolite himastatin's of streptomycete S.himastatinicus sp.Nov isolating and purifying and tying
Structure identification
Firstly, 250 mL triangular flasks of the S.himastatinicus sp.Nov bacterial strain in the culture medium equipped with 50mL step 1
Middle fermentation 7 days, 28~30 DEG C of temperature, 200 rpm of revolving speed；Then, the ethyl acetate of 2 times of volumes of fermentation liquid extracts, and upper layer has
50 DEG C of machine phase vacuum distillations, are dissolved with 1mL methanol, obtain crude extract, carry out HPLC analysis, carry out mesh according to characteristic ultraviolet absorption
Mark the calibration (210nm, 285nm) of compound.Followed by same fermentation condition, to S.himastatinicus sp.Nov
Carry out 4L scale fermentation.The separation of progress target compound after HPLC analysis is carried out to the runic object of tunning.HPLC analysis
The composition of mobile phase used is as follows: A phase ,+0.1% glacial acetic acid of+85% water of 15% acetonitrile；B phase ,+80% acetonitrile of 20% water+
0.1% glacial acetic acid.HPLC program are as follows: 0%~80% gradient elution of 0~20 min, B phase；21~23min, B phase 80%~
100% gradient elution；24~30min, 100%B phase elute.(210 nm, 285nm, flow velocity are 1 min/mL to UV Detection wavelength.
In this program, the retention time of target compound 1 about 17.45min.HR-ESI-MS, 1D are carried out to reactive compound 1
(1H,13C) NMR (Fig. 9) is tested, comprehensive analysis spectral data, and document: JOHN E.LEET, DANIEL R.SCHROEDER,
BALA S.KRISHNAN and JAMES A.MATSON,HIMASTATIN,A NEW ANTITUMOR ANTIBIOTIC FROM
STREPTOMYCES HYGROSCOPICUS
Ⅱ.ISOLATION AND CHARACTERIZATION,THE JOURNAL OF ANTIBIOTICS,VOL.XLⅢ
The nuclear magnetic data of NO.8, the himastatin in 961~966,1990,8. compare, and carry out the confirmation of target compound 1,
Identify that target compound 1 is himastatin, structural formula is as shown in Figure 1.
3. the screening of the Cosmid 12H8 containing gene hmtA and hmtB
The building of S.himastatinicus sp.Nov genomic library: using SuperCos I as carrier, detailed process ginseng
According to the operation manual of 1 Cosmid Vector Kit of SuperCos.Operating process can be substantially summarized as follows: from
Genomic DNA is extracted in S.himastatinicus sp.Nov mycelium, through Sau3AI Partial digestion, with calf intestinal phosphatase
Dephosphorylation is then attached to by 1 carrier of SuperCos of XbaI and BamHI enzymolysis processing, uses Packagene
Lambda DNA packaging system carries out in vitro package, Escherichia coli LE392 is infected, in LB+kanamycin (Kan, 50 μ of final concentration
G/mL it) is screened on plate.About 1920 clones are chosen to select into 20 piece of 96 orifice plate, after cultivating 12h, the bodies such as addition in every hole
The glycerol of product 50%.- 80 DEG C of preservations.
The gene order-checking of S.himastatinicus sp.Nov transfers to Hua Da company to complete.It is provided according to Hua Da company
Sequence.In order to find the gene cluster of antibiotic himastatin biosynthesis, the method that we use genome-wide screening,
Illumina ' s Solexa sequencing technologies are utilized, to the full base of Streptomyces himastatinicus sp.Nov bacterial strain
Because group DNA carries out sequencing, the continuous fragment that a size is 60 Kb as the result is shown of gene annotation may contain
Gene information necessary to himastatin biosynthesis, further bioinformatic analysis disclose 20 open reading frame about
The DNA sequence dna of 45 Kb sizes contain regulation in antibiotic himastatin biosynthesis, structure, ego resistance, transhipment and
After modify isogenic information, thus it is speculated that the institutional framework of himastatin biological synthesis gene cluster is as shown in Figure 2.himastatin
The sequence of gene cluster is included by EMBL database, sequence number FR823394.Wherein hmtA gene (its nucleosides in gene cluster
Acid sequence is as shown in SEQ ID NO.1) coding MerR family transcription regulatory factor；HmtB gene (its nucleotide sequence such as SEQ
Shown in ID NO.3) encoding acetyl gamma-Glutamate kinase.In order to study hmtA and hmtB gene in himastatin biosynthetic process
In function, we carry out the knockout of gene to it respectively.It is operated and is required according to PCR-targeting, it in order to obtain can be right
The clay that hmtA and hmtB are knocked out, we devise two pairs of primers, SAF (5 '-near hmtA sequence
CGCAGATCCACGCCGCACTC-3 '), SAR (5 '-AGT TCCGCGTGACGGAGGTG-3 ') and SBF (5 '-
CGCCACCTGCTCGTCACCG-3 '), SBR (5 '-TCG GCG TAC AGC CAC AGC AG-3 ').Obtained by two pairs of primer PCRs
Purpose band is respectively 622 bp and 1679bp.It is utilized respectively these two pair primer, round pcr is utilized from the genomic library of building
Screening positive clone.PCR program is as follows: 95 DEG C of 5min；95 DEG C of 45s, 58 DEG C of 45s, 72 DEG C of 90s, 30 circulations；72℃10min.
Wherein screen multiple positive colonies.The positive colony of our selected corresponding positions (H, 8) of 12nd piece of 96 orifice plates, is named as
Cosmid 12H8 carries out the knockout of the hmtA and hmtB gene of next step containing complete hmtA and hmtB gene.
The building of 4.hmtA gene deletion mutants Streptomyces himastatinicus sp.Nov Ju2014
Inactivation is the PCR-Targeting technology mediated using λ-RED in the reading frame of hmtA gene.It include hmtA
The Cosmid 12H8 of gene is transformed into E.coli BW25113/pIJ790, is named as E.coli BW25113/pIJ790/
12H8.It is same with 39bp is respectively provided with hmtA gene using the pIJ773 plasmid digested by EcoRI and HindIII as template
Knockout primer (the hmtAdelF:5 '-of source armacgctcggcgcgacatacgcccggacccgacgacccgatATTCCGGGGA
TCCGT CGAC-3’,hmtAdelR:5’-tagccggtacccgacgtcgatggcctcgagcccgaggagTGTAGGCTGGA
TC-3 ' the underscore part GCTGCT represents and hmtA DNA homolog) carry out aac (3) the IV-oriT resistance piece with Apr resistance
The pcr amplification reaction of section.The aac expanded (3) IV-oriT resistance fragments electrotransformation is entered into E.coli BW25113/pIJ790/
In 12H8 competent cell, improved recombination cosmid is obtained by the homologous recombination that λ-RED is mediated, is named as recombination
cosmid pJu2014.With the PCR of hmtA gene verifying primer (IDhmtAF:5 '-GAACCGATTCCGCCTGACC-3 ',
IDhmtAR:3 '-ACATCGCCCTCGCCCTCAC-3 ') carry out amplification reaction the correctness for determining recombination cosmid pJu2014.
Then, recombination cosmid pJu2014 is transferred into E.coli ET12567/pUZ8002, is named as E.coli ET12567/
PUZ8002/p Ju2014 carries out engagement transfer with wild-type strain S.himastatinicus sp.Nov.
The process for engaging transfer is as follows: by the spore of wild type S.himastatinicus sp.Nov in TSB culture solution
In, 28 DEG C shake culture 12-18 hours, make its spore germination；E.coli ET12567/pUZ8002/pJu2014 is added with card
(Cml, end are dense for that mycin (Kan, final concentration of 50 μ g/mL), ampicillin (Amp, final concentration of 100 μ g/mL), chloramphenicol
Degree is culture in 25 μ g/mL) and the LB culture solution of A Baila mycin (Apm, final concentration of 50 μ g/mL) to OD value OD=
0.6, cell is collected by centrifugation, is washed twice with sterile LB liquid medium, is then mixed with the wild type spore sprouted, it will
Mixed liquor is laid in added with 10mM MgSO4M-ISP4On solid medium；After 28 DEG C grow 23 hours, each solid culture
800 μ L are coated on base plate added with the sterile water of Trimethoprim and the antibiotic medicine of apramycin, wherein containing first
Oxygen benzyl Aminometradine (Tmp, 50 mg/mL) 30 μ L, A Baila mycin (Apm, 50 mg/mL) 30 μ L；Allow it under 30 DEG C of incubators
Growth 4-5 days occurs being double crossing over mutant strain until joint element, is named as Streptomyces himastatinicus
sp.Nov Ju2014.The double crossing over mutant strain passes through its resistant phenotype (Kan to antibioticS&ApmR) and genotype and by
Screening and identification come out, and wherein the PCR verifying of hmtA gene delection utilizes the carry out PCR expansion of primer (IDhmtAF, IDhmtAR)
Increase reaction to be confirmed.Thus the mutant strain Streptomyces himastatinicus sp.Nov that hmtA is knocked is obtained
Ju2014。
The building process of hmtB deletion mutant bacterial strain is with hmtA gene knockout, but the deletion-primers of hmtB are as follows
hmtBdelF:5’-cttggcgtccggtacgaccgtggtgccggaccgctcgtcATTCCGGGGATCCGTCGAC-3 ',
hmtBdelR:5’-gaggcgtcgttcgccgacgacgtcgtacggatgcaccggLower stroke of TGTAGGCTGGAGCTGCTTC-3 '
Line part represents and hmtB DNA homolog.The PCR of hmtB gene verifies primer are as follows: IDhmtBF:5 '-
aggctcgtgtcggaggtagt-3',IDhmtBR:5'-cggcgggacagaatcttg-3'.Thus obtain what hmtB was knocked
Mutant strain Streptomyces himastatinicus sp.Nov Ju2015.
The building schematic diagram of specific mutant strain Streptomyces himastatinicus sp.Nov Ju2014 is as schemed
Shown in 3, the PCR proof diagram of mutant strain Streptomyces himastatinicus sp.Nov Ju2014 is as shown in Figure 4.
The building schematic diagram of specific mutant strain Streptomyces himastatinicus sp.Nov Ju2015 is as schemed
Shown in 5, the PCR proof diagram of mutant strain Streptomyces himastatinicus sp.Nov Ju2015 is as shown in Figure 6.
5. the mutant strain Streptomyces that wild-type strain and double crossing over mutant strain hmtA are knocked
The mutant strain Streptomyces himastatinicus that himastatinicus sp.Nov Ju2014 and hmtB are knocked
The fermentation of sp.Nov Ju2015 and its HPLC analysis
Wild-type strain and double crossing over mutant strain are respectively 30 in the ISP4 of improvement (M-ISP4) solid medium tablets
DEG C culture 7 days, the appropriate spore inoculating of picking in equipped with 50mL himastatin step 1 culture medium triangular flask in, at 28 DEG C
It is cultivated 7 days on the shaking table of 200rpm revolving speed.The ethyl acetate of 2 times of volumes of fermentation liquid extracts, and the extract liquor of organic phase is through revolving
It steams after instrument is evaporated and is dissolved in 1mL methanol, after 12000rpm is centrifuged 5min, supernatant is taken to carry out HPLC analysis.HPLC analyzes result
See Fig. 7 and Fig. 8.Pass through the integral radiometer of the HPLC analysis detection and HPLC map of mutant strain and wild-type strain tunning
The multiple of himastatin output increased in mutant strain is calculated.As can be seen from Figures 7 and 8, wild type
Streptomyces himastatinicus sp.Nov, mutant strain Streptomyces himastatinicus sp.Nov
The mutant strain Streptomyces himastatinicus sp.Nov Ju2015 that Ju2014 and hmtB are knocked is in equal conditions
Lower fermentation, compared with wild type, mutant strain Streptomyces himastatinicus sp.Nov Ju2014 and hmtB is struck
The yield of the target product himastatin of the mutant strain Streptomyces himastatinicus sp.Nov Ju2015 removed
10 times or more have been higher by compared with wild-type strain.Therefore, two kinds of high productive mutants of Cyclic lipopeptide antibiotic himastatin at
The industrialization process that function is configured to acceleration antibiotic himastatin is brought may be with hope.
M-ISP4 culture medium (the ISP4 fluid nutrient medium of improvement): be on the basis of ISP4 culture medium be added peptone,
Yeast extract and sea salt make whole mass fraction be respectively as follows: 0.1%, 0.05%, 3%.That is the formula of M-ISP4 culture medium are as follows:
Every liter of M-ISP4 culture medium contains: soluble starch 10g, K2HPO41g, MgSO4.7H2O 1g, NaCl 1g, (NH4)2SO42g,
CaCO32g, FeSO4.7H2O 0.001g, MnCl2.4H2O 0.001g, ZnSO4.7H2O 0.001g, peptone 1g, yeast extract
Object 0.5g, coarse sea salt 30g, surplus are water, pH7.0~7.4.Solid medium is added on the basis of liquid medium within
1.5% agar.
Claims (3)
1.himastatin high productive mutant Streptomyces himastatinicus sp.Nov, which is characterized in that it is
The Streptomyces himastatinicus sp.Nov of hmtA gene or hmtB gene inactivation, the hmtA gene
Nucleotide sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of the hmtB gene is as shown in SEQ ID NO.3.
2. a kind of himastatin high productive mutant Streptomyces himastatinicus described in claim 1
The construction method of sp.Nov, which is characterized in that be by the hmtA gene of Streptomyces himastatinicus sp.Nov
Or hmtB gene inactivation, thus to obtain himastatin high productive mutant Streptomyces himastatinicus
Sp.Nov, the nucleotide sequence of the hmtA gene is as shown in SEQ ID NO.1, the nucleotide sequence of the hmtB gene
As shown in SEQ ID NO.3.
The application of 3.hmtA gene or hmtB gene in regulation himastatin yield, the nucleotide of the hmtA gene
Sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of the hmtB gene is as shown in SEQ ID NO.3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410372728.7A CN104164399B (en) | 2014-07-31 | 2014-07-31 | The superior strain and its construction method of cyclic ester peptide antibiotics Hei Motading |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410372728.7A CN104164399B (en) | 2014-07-31 | 2014-07-31 | The superior strain and its construction method of cyclic ester peptide antibiotics Hei Motading |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104164399A CN104164399A (en) | 2014-11-26 |
| CN104164399B true CN104164399B (en) | 2019-06-11 |
Family
ID=51908381
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410372728.7A Expired - Fee Related CN104164399B (en) | 2014-07-31 | 2014-07-31 | The superior strain and its construction method of cyclic ester peptide antibiotics Hei Motading |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104164399B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106497827B (en) * | 2016-10-09 | 2019-08-02 | 中国科学院南海海洋研究所 | A kind of engineering strain and its application of orientation production anti-tubercular and active compound for anti tumor |
-
2014
- 2014-07-31 CN CN201410372728.7A patent/CN104164399B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| "Biosynthesis of himastatin：Assembly Line and Characterization of Three Cytochrome P450";Junying Ma 等;《Angewandte Chemie》;20111231;第123卷(第34期);第7797-77798页，表1 * |
| "CBZ42135.1";Ma，J.Y.等;《GenBank》;20110731;第1页 * |
| "CBZ42136.1";Ma，J.Y.等;《GenBank》;20110731;第1页 * |
| "FR823394.1";Ma，J.Y.等;《GenBank》;20110731;第1-2页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104164399A (en) | 2014-11-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cacho et al. | Identification and characterization of the echinocandin B biosynthetic gene cluster from Emericella rugulosa NRRL 11440 | |
| JP6421115B2 (en) | Symbiotic selection by screening multiple host-symbiotic associations | |
| Zhou et al. | Endophytism or saprophytism: decoding the lifestyle transition of the generalist fungus Phomopsis liquidambari | |
| CN106497827B (en) | A kind of engineering strain and its application of orientation production anti-tubercular and active compound for anti tumor | |
| Liu et al. | Rational construction of genome-reduced Burkholderiales chassis facilitates efficient heterologous production of natural products from proteobacteria | |
| Benson et al. | The biology of Frankia sp. strains in the post-genome era | |
| Yang et al. | Streptomyces alleviate abiotic stress in plant by producing pteridic acids | |
| CN111876514B (en) | Rapid detection method for gibberellin miniascape generated in bakanae disease germ of rice | |
| CN104164399B (en) | The superior strain and its construction method of cyclic ester peptide antibiotics Hei Motading | |
| US20220272935A1 (en) | Novel Xanthomonas Strains and Related Methods | |
| CN108611361A (en) | A kind of genetic screening method of streptomycete biological synthesis gene cluster negative regulatory factor | |
| WO2014092345A1 (en) | Fusaricidin-producing strain, and method for mass producing fusaricidin using same | |
| CN110484481A (en) | A method of lincomycin yield is improved by transformation Str. lincolnensis SLCG_3128 gene | |
| CN109321618A (en) | A method of yield of erythrocin is improved by the red mould SACE_5717 gene of the more spores of sugar | |
| US9872502B2 (en) | Brachiaria-urochloa endophytes | |
| CN109136253A (en) | Method for improving yield of erythromycin through saccharopolyspora erythraea SACE _5754 gene approach | |
| CN102373171A (en) | Nucleoside antibiotic A201A superior strain and construction method thereof | |
| CN105985967A (en) | Biosynthetic gene cluster of oosporein and application of biosynthetic gene cluster | |
| CN111139207A (en) | Brevibacillus brevis gene recombinant strain and preparation method and application thereof | |
| CN110305881A (en) | The biological synthesis gene cluster of polyketides neoenterocins a kind of and its application | |
| CN113755517B (en) | Construction method and application of SLCG _5407 gene modified streptomyces lincolnensis | |
| Feng et al. | Siderophore Synthesis Ability of the Nitrogen-Fixing Bacterium (NFB) GXGL-4A is Regulated at the Transcriptional Level by a Transcriptional Factor (tr X) and an Aminomethyltransferase-Encoding Gene (amt) | |
| CN114561405B (en) | Application of synthetic gene cluster of trichoderma Harzianolide in preparation of preparation for improving plant gray mold resistance | |
| CN108929860A (en) | A kind of genetic engineering bacterium and its preparation method and application producing chloroeremomycin | |
| CN116144637A (en) | Rumex olivaceus histone deacetylase and encoding gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190611 |