WO2017114034A1 - 可利霉素生物合成基因簇 - Google Patents

可利霉素生物合成基因簇 Download PDF

Info

Publication number
WO2017114034A1
WO2017114034A1 PCT/CN2016/107039 CN2016107039W WO2017114034A1 WO 2017114034 A1 WO2017114034 A1 WO 2017114034A1 CN 2016107039 W CN2016107039 W CN 2016107039W WO 2017114034 A1 WO2017114034 A1 WO 2017114034A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
genes
amino acid
sequence
seq
Prior art date
Application number
PCT/CN2016/107039
Other languages
English (en)
French (fr)
Inventor
王以光
姜洋
赵小峰
赫卫清
戴剑漉
Original Assignee
沈阳同联集团有限公司
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority to UAA201808040A priority Critical patent/UA124058C2/uk
Priority to US16/067,392 priority patent/US10858659B2/en
Application filed by 沈阳同联集团有限公司 filed Critical 沈阳同联集团有限公司
Priority to AU2016383270A priority patent/AU2016383270A1/en
Priority to PE2018001213A priority patent/PE20190221A1/es
Priority to ES16880824T priority patent/ES2812256T7/es
Priority to JP2018553280A priority patent/JP6882330B2/ja
Priority to PL16880824T priority patent/PL3385386T6/pl
Priority to CA3009706A priority patent/CA3009706A1/en
Priority to EP16880824.4A priority patent/EP3385386B3/en
Priority to DK16880824.4T priority patent/DK3385386T6/da
Priority to RU2018125487A priority patent/RU2719189C2/ru
Priority to BR112018013309-4A priority patent/BR112018013309A2/zh
Priority to MYPI2018001122A priority patent/MY193028A/en
Priority to KR1020187022082A priority patent/KR20180093083A/ko
Publication of WO2017114034A1 publication Critical patent/WO2017114034A1/zh
Priority to PH12018501393A priority patent/PH12018501393A1/en
Priority to ZA2018/04397A priority patent/ZA201804397B/en
Priority to CONC2018/0007130A priority patent/CO2018007130A2/es

Links

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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01064—Testosterone 17-beta-dehydrogenase (NADP+) (1.1.1.64)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01188—Prostaglandin-F synthase (1.1.1.188)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01008—Acyl-CoA dehydrogenase (NADP+) (1.3.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01013—Glycine N-acyltransferase (2.3.1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/02—Thioester hydrolases (3.1.2)
    • C12Y301/02007—Glutathione thiolesterase (3.1.2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/02—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
    • C12Y305/02003—Dihydroorotase (3.5.2.3)

Definitions

  • the invention belongs to the field of microbial gene resources and genetic engineering, and specifically relates to cloning, analysis, functional research and application of genetic engineering antibiotic biosynthesis gene cluster.
  • Colimycin has used the famous biotechnologymycin, Bite spiramycin, and is a 16-membered ring macrolide antibiotic developed by synthetic biology technology [Patent No.: ZL971044406, ZL 021487715], which is 4"- Isoamyl spiromycin III, II, I as the main component of the acyl group in the 4"-position hydroxyl group, wherein the III component accounts for more than 30%, and the II component is about 25%. Group I The score is no more than 10%.
  • Colimycin has strong activity against Gram-positive bacteria, resistant to erythromycin and ⁇ -lactam antibiotics, influenza bacillus, gonococcus, Legionella, Bacteroides fragilis, Clostridium perfringens It has antibacterial activity. In particular, it has strong activity against Mycoplasma pneumoniae, Chlamydia trachomatis and Chlamydia pneumoniae [Yu Lanxiang et al., Sichuan Journal of Physiological Sciences; 1998, 20(3), Patent No.: 2003101224209], with good post-antibiotic effect and antibiotic sub-inhibitory concentration effect. It is not completely cross-resistant with similar drugs.
  • Colimycin is a direct fermentation product of genetic engineering bacteria obtained by genetic recombination technology, and the preparation process is simple, and can effectively avoid chemical pollution and save energy. Its oral preparation is convenient to take, and it only needs to be taken once a day, which is beneficial to improve the patient's medication compliance and easy access to the basic medical insurance series.
  • Colimycin is a gene-engineered strain (Streptomyces spiramyceticus WSJ-), which uses the gene recombination technology to clone and express the carbomycin producing strain 4"-isovalyl transferase gene in Streptomyces spiramyceticus F21. 1) Fermentation product.
  • the spiromycin producing strain (Streptomyces spiramyceticus F21) was isolated from the soil of Yongchang County, Gansu province, China in 1982. The morphological culture characteristics, physiological and biochemical characteristics, and cell wall chemistry of the strain were obtained.
  • the macrolide antibiotic biosynthesis gene cluster is about 50-80 kb in length, and its common feature is that the polyketide synthase (PKS) and polyketone synthesis extended unit are encoded by a 16-member macrolide ring biosynthesis modular structure.
  • PKS polyketide synthase
  • Related enzymes enzymes responsible for the modification of different groups of lactone rings, genes for glycosyl synthesis and transfer-related enzymes, and genes involved in resistance and regulatory functions.
  • Macrolides are catalyzed by a PKS consisting of a modular structure that catalyzes the formation of simple carboxylic acid molecules by a continuous condensation reaction in a manner similar to fatty acid biosynthesis.
  • Each module is responsible for only one condensation reaction during the formation of the polyketone chain. It contains at least one ⁇ -keto ester synthase (KS) domain, an acyltransferase (AT) domain and an acyl carrier protein (ACP). Domain.
  • KS ⁇ -keto ester synthase
  • AT acyltransferase
  • ACP acyl carrier protein
  • KR beta-ketoacyl reductase
  • DH dehydratase
  • ER ester acyl reductase
  • a modification step such as hydroxylation, methylation, methoxylation, and acylation is carried out to form a variety of macrolide antibiotic structures.
  • macrolide is linked to a different number of glycosyl groups (or sugar amine groups), such as colimycin containing three sugar groups, respectively, floroxamine, carbamelamine and carbofuran. They are responsible for glycosylation and transfer related enzymes.
  • glycosyl groups or sugar amine groups
  • colimycin containing three sugar groups, respectively, floroxamine, carbamelamine and carbofuran. They are responsible for glycosylation and transfer related enzymes.
  • the resistance gene confers the ability of the producing strain to antagonize its own production of antibiotics, usually associated with ABC transporters. Regulatory function-related genes are involved in the regulation of self-biosynthesis antibiotics.
  • the genetically produced bacteria can be further manipulated to obtain new and more effective antibiotics, such as genetically modified to change the modular structure of PKS synthesis, post-esterification modification of lactone ring, sugar Gene replacement or modification to create new macrolide antibiotics; antibiotic production can also be increased by genetic manipulation of resistance genes or regulatory genes.
  • antibiotics such as genetically modified to change the modular structure of PKS synthesis, post-esterification modification of lactone ring, sugar Gene replacement or modification to create new macrolide antibiotics
  • antibiotic production can also be increased by genetic manipulation of resistance genes or regulatory genes.
  • the invention provides a gene cluster of colimycin biosynthesis, which has a total open reading frame (orf) of 44 genes, and the nucleotide sequence has a total length of 89,315 bp (Seq. 1), and contains five codes encoding polyketide synthase (orf10-). 14), including 8 modules, 37 domains, and 9 orches (1, 4-6, 15, 36-39) associated with polyketide extension units and modifications, orf associated with glycosylation Sixteen (9, 16-22, 24, 26, 28, 29, 33-35, 41), there are six orfs associated with glycosyl transfer (7, 8, 30-32, 40). In addition, there are two orfs related to resistance (3 and 25), and there are four orfs related to regulation (2, 23, 27, 42).
  • nucleotide sequences are respectively selected from orf1 (1-645), orf2 (1810-1208), orf3 (3133-2285), orf4 (3614-4840), orf5 (4846-5511), orf6 in Seq. 7150-5801), orf7 (8444-7179), orf8 (9729-8482), orf9 (10543-9830), orf10 (16215-10543), orf11 (21076-16328), orf12 (32511-21124), orf13 (38599 -32585), orf14 (52259-38643), orf15 (53099-54310), orf16 (54495-54845), orf17 (54842-56041), orf18 (56038-56946), orf19 (56930-57967), orf20 (57937- 60174), orf21 (60836-61984), orf22 (62796-62077), orf23 (63633-65645), orf24 (67379-66318), orf25 (69004-67352), or
  • the present invention also provides an amino acid sequence of a 4' phosphopantetheinyl transferase (PPT) consisting of 214 amino acids in the Seq.3 sequence, designated as IA-W1, and the nucleotide sequence of the coding gene is selected. From 1-645 bases in Seq.1.
  • PPT 4' phosphopantetheinyl transferase
  • the present invention also provides an amino acid sequence of a TetR family transcriptional regulator consisting of 200 amino acids in the Seq.4 sequence, designated as IA-W2, and the nucleotide sequence encoding the gene is selected from 1810-1208 in Seq. Base.
  • the present invention also provides an amino acid sequence of a 23S rRNA methyltransferase consisting of 282 amino acids in the Seq.5 sequence, designated as IA-W3, and the nucleotide sequence encoding the gene is selected from 3133- in Seq. 2285 bases.
  • the present invention also provides an amino acid sequence of a 3-O-acyltransferase consisting of 408 amino acids in the Seq.6 sequence, designated IA-W4, and the nucleotide sequence encoding the gene is selected from 3614 in Seq. -4840 bases.
  • the present invention also provides an amino acid sequence of an O-methyltransferase consisting of 221 amino acids in the Seq.7 sequence, designated IA-W5, and the nucleotide sequence encoding the gene is selected from 4846- in Seq. 5511 bases.
  • the present invention also provides an amino acid sequence of a crotonyl-CoA reductase consisting of 449 amino acids in the Seq.8 sequence, designated as IA-W6, and the nucleotide sequence encoding the gene is selected from 7150-5801 in Seq. Base.
  • the present invention also provides an amino acid sequence of a glycosyltransferase consisting of 421 amino acids in the Seq.9 sequence, designated as IA-W7, and the nucleotide sequence encoding the gene is selected from 8444-7179 bases in Seq. .
  • the present invention also provides an amino acid sequence of a glycosyltransferase accessory protein consisting of 415 amino acids in the sequence of Seq. 10, designated as IA-W8, and the nucleotide sequence encoding the gene is selected from 9729-8482 in Seq. Base.
  • the present invention also provides an amino acid sequence of NDP-aminohexose N-dimethyltransferase consisting of 237 amino acids in the sequence of Seq.11, designated as IA-W9, and the nucleotide sequence encoding the gene is selected from Seq 10543-9830 bases in .1.
  • the present invention also provides a keto synthase (KS) 8 -acyltransferase (AT) 8 -ketoreductase (KR) 8 -acyl carrier protein domain (ACP) 8 -chain-releasing thioesterase (TE)
  • KS keto synthase
  • AT 8 -acyltransferase
  • KR ketoreductase
  • ACP acyl carrier protein domain
  • TE chain-releasing thioesterase
  • the polyketide synthase domain amino acid sequence consists of 1890 amino acids in the Seq. 12 sequence, designated IA-W10, and the nucleotide sequence encoding the gene is selected from 16215-10543 bases in Seq.
  • the present invention also provides a core domain amino acid sequence comprising polyketide synthase structure 7 -AT 7 -KR 7 -ACP 7 is KS, the sequence Seq.13 1582 amino acids, designated as IA-W11, the gene encoding
  • the nucleotide sequence is selected from the 21076-16328 bases in Seq.1.
  • the present invention also provides a comprising KS 5 -AT 5 -KR 5 -ACP 5 -KS 6 -AT 6 -DH 6 ( dehydratase) -ER 6 (enoyl reductase) -KR polyketone engagement of 6 -ACP 6
  • the amino acid sequence of the enzyme domain consists of 3795 amino acids in the Seq.14 sequence, designated IA-W12, and the nucleotide sequence encoding the gene is selected from 32511-21124 bases in Seq.
  • the present invention also provides a domain amino acid sequence comprising a polyketide synthase structure 4 -AT 4 -DH 4 -KR 4 -ACP 4 is KS, the sequence Seq.15 2004 amino acids, designated as IA-W13, encoding
  • the nucleotide sequence of the gene is selected from the base of 38599-32585 in Seq.
  • the present invention further provides a package KS 1 -AT 1 -ACP 1 -KS 2 -AT 2 -KR 2 -ACP 2 -KS 3 -AT 3 -DH 3 -KR 3 -ACP polyketide synthase domain 3 amino acid
  • the sequence consists of 4538 amino acids in the Seq.16 sequence, designated IA-W14, and the nucleotide sequence encoding the gene is selected from 52259-38643 bases in Seq.
  • the present invention also provides an amino acid sequence of a cytochrome P-450 oxidase consisting of 403 amino acids in the Seq.17 sequence, designated as IA-W15, and the nucleotide sequence encoding the gene is selected from 53093 in Seq. -54310 bases.
  • the present invention also provides an amino acid sequence of NDP-hexose isomerase consisting of 116 amino acids in the sequence of Seq. 18, designated as IA-W16, and the nucleotide sequence encoding the gene is selected from 54495 in Seq. -54845 bases.
  • the present invention also provides an amino acid sequence of an NDP-hexose aminotransferase consisting of 399 amino acids in the sequence of Seq.19, Named IA-W17, the nucleotide sequence encoding the gene was selected from 54482-56041 bases in Seq.
  • the present invention also provides an amino acid sequence of NDP-glucose synthase consisting of 302 amino acids in the Seq.20 sequence, designated as IA-W18, and the nucleotide sequence encoding the gene is selected from 56038-56946 in Seq. Base.
  • the present invention also provides an amino acid sequence of NDP-glucose-4,6 dehydratase consisting of 345 amino acids in the sequence of Seq.21, designated as IA-W19, and the nucleotide sequence encoding the gene is selected from Seq. 56930-57967 bases.
  • the present invention also provides an NDP-hexose 2,3 dehydratase/thioesterase amino acid sequence consisting of 745 amino acids in the Seq.22 sequence, designated IA-W20, and the nucleotide sequence encoding the gene is selected from the group consisting of 57937-60174 bases in Seq.1.
  • the present invention also provides an amino acid sequence of an NDP-hexose aminotransferase consisting of 382 amino acids in the Seq.23 sequence, designated as IA-W21, and the nucleotide sequence encoding the gene is selected from 60836 in Seq. -61984 bases.
  • the present invention also provides an amino acid sequence of NDP-aminohexose N-dimethyltransferase consisting of 239 amino acids in the Seq.24 sequence, designated IA-W22, and the nucleotide sequence encoding the gene is selected from Seq. 62796-62077 bases in 1.
  • the present invention also provides an amino acid sequence of a transcriptional regulatory factor consisting of 670 amino acids in the Seq.25 sequence, designated IA-W23, and the nucleotide sequence encoding the gene is selected from 63633-65645 bases in Seq. .
  • the present invention also provides an amino acid sequence of NDP-aminohexose isomerase consisting of 354 amino acids in the sequence of Seq. 26, designated as IA-W24, and the nucleotide sequence encoding the gene is selected from the group consisting of Seq. 67379-66318 base.
  • the present invention also provides an amino acid sequence of an ABC transporter consisting of 550 amino acids in the Seq.27 sequence, designated IA-W25, and the nucleotide sequence encoding the gene is selected from 69004-67352 bases in Seq. .
  • the present invention also provides an amino acid sequence of an NDP-hexose dehydratase consisting of 433 amino acids in the sequence of Seq. 28, designated as IA-W26, and the nucleotide sequence encoding the gene is selected from 69349 in Seq. 70,650 bases.
  • the present invention also provides an amino acid sequence similar to GTPase, consisting of 482 amino acids in the Seq.29 sequence, designated IA-W27, and the nucleotide sequence encoding the gene is selected from 72156-70708 base in Seq. base.
  • the present invention also provides an amino acid sequence of an NDP-sugar isomerase consisting of 346 amino acids in the Seq.30 sequence, designated IA-W28, and the nucleotide sequence encoding the gene is selected from 72422 in Seq. 73,462 bases.
  • the present invention also provides an amino acid sequence of an NDP-hexoketosyl reductase consisting of 346 amino acids in the sequence of Seq. 31, designated as IA-W29, and the nucleotide sequence encoding the gene is selected from the group consisting of Seq. 74601-73561 bases.
  • the present invention also provides an amino acid sequence of a glycosyltransferase accessory protein consisting of 415 amino acids in the Seq.32 sequence, designated as IA-W30, and the nucleotide sequence encoding the gene is selected from 74913-76160 in Seq. Base.
  • the present invention also provides an amino acid sequence of a glycosyltransferase consisting of 422 amino acids in the sequence of Seq.33, designated as IA-W31, and the nucleotide sequence encoding the gene is selected from 76218-77486 base in Seq. base.
  • the present invention also provides an amino acid sequence of a glycosyltransferase consisting of 391 amino acids in the sequence of Seq. 34, designated as IA-W32, and the nucleotide sequence encoding the gene is selected from 77606-78781 base in Seq. base.
  • the present invention also provides an amino acid sequence of an NDP-hexoketosyl reductase consisting of 330 amino acids in the Seq.35 sequence, designated as IA-W33, and the nucleotide sequence encoding the gene is selected from the group consisting of Seq. 78783-79775 bases.
  • the present invention also provides an amino acid sequence of an NDP-hexose reductase, Seq.36 consisting of 335 amino acids in the sequence, designated IA-W34, and the nucleotide sequence encoding the gene is selected from 79972- in Seq. 80779 bases.
  • the present invention also provides an amino acid sequence of an NDP-hexose methyltransferase consisting of 410 amino acids in the sequence of Seq.37, designated as IA-W35, and the nucleotide sequence encoding the gene is selected from the group consisting of Seq. 82055-80823 base.
  • the present invention also provides an amino acid sequence of a methoxymalonyl synthase consisting of 370 amino acids in the sequence of Seq. 38, designated as IA-W36, and the nucleotide sequence encoding the gene is selected from the group consisting of Seq. 83164-82052 base.
  • the invention also provides an amino acid sequence of a dehydrogenase consisting of 373 amino acids in the sequence of Seq. 39, designated as IA-W37,
  • the nucleotide sequence encoding the gene is selected from the 87400-83279 bases in Seq.
  • the present invention also provides an amino acid sequence of an acyl carrier protein consisting of 106 amino acids in the Seq.40 sequence, designated as IA-W38, and the nucleotide sequence encoding the gene is selected from 84713-84393 bases in Seq. .
  • the present invention also provides an amino acid sequence of a methoxymalonyl dehydrogenase consisting of 288 amino acids in the sequence of Seq.41, designated as IA-W39, and the nucleotide sequence encoding the gene is selected from Seq. 85576-84710 bases.
  • the present invention also provides an amino acid sequence of a glycosyltransferase consisting of 405 amino acids in the Seq.42 sequence, designated as IA-W40, and the nucleotide sequence encoding the gene is selected from the 85825-87042 base in Seq. base.
  • the present invention also provides an amino acid sequence of NDP-hexose isomerase consisting of 202 amino acids in the sequence of Seq.43, designated as IA-W41, and the nucleotide sequence encoding the gene is selected from 87094 in Seq. -87702 bases.
  • the present invention also provides an amino acid sequence of a transcriptional regulator protein consisting of 390 amino acids in the Seq.44 sequence, designated IA-W42, and the nucleotide sequence encoding the gene is selected from the 89315-88143 base in Seq. base.
  • the present invention also provides an amino acid sequence of an exogenously inserted 23S rRNA methylase (thiostrepton thiostrepton, tsr resistance marker-related), which is composed of 269 amino acids in the Seq.45 sequence and is named IA-W43.
  • the nucleotide sequence encoding the gene is selected from 866-57 bases in Seq.
  • the present invention also provides an exogenously inserted 4" carotenose glycosyltransferase amino acid sequence consisting of 388 amino acids in the Seq.46 sequence, designated IA-W44, and the nucleotide sequence encoding the gene is selected from Seq 2337-1171 bases in .2.
  • polyketide synthase gene orf10-14 a total of 5 genes
  • Exogenously introduced genetically engineered bacterial marker gene orf43 (thiostrepton-thiostrepton, tsr resistance gene) and its linked carbonaceous 4"-O-isovaleryltransferase gene orf44, total 2 Genes.
  • Orf14 contains 3 modules: loading domain 1 and modules 2 and 3; in the loading module domain, KS 1 , AT 1 and ACP 1 are responsible for the initial synthesis of the lactone ring, catalyzing an acetic acid as the starting unit and module 2 containing the KS 2 , AT 2 , KR 2 and ACP 2 domains; Module 3 contains KS 3 , AT 3 , DH 3 , KR 3 and ACP 3 domains, responsible for the introduction of two other acetic acid extension units, which ultimately form colimycin C11- 15 carbon chain skeleton.
  • Orf13 comprises module 4, which contains KS 4 , AT 4 , DH 4 , KR 4 , ACP 4, is responsible for the extension of the third acetic acid unit, and finally forms the calicheamicin C9-10 carbon chain backbone;
  • Orf12 comprises modules 5 and 6, 5 module containing KS 5 -AT 5 -KR 5 -ACP 5 domain extension unit responsible for introducing acid; module 6 containing KS 6 -AT 6 -DH 6 -KR 6 -ER 6 -KR 6 -ACP 6 domains, Responsible for the introduction of butyric acid extension units, and finally the formation of the Coriomycin C5-C8 carbon chain skeleton.
  • Orf11 7 includes a module containing KS 7 -AT 7 -KR 7 -ACP 7 domain, responsible for introducing extending glycolic units eventually form C3-C4 Currie rapamycin backbone carbon chain.
  • Orf10 8 includes a module containing KS 8 -AT 8 -KR 8 -ACP 8 -TE domain, responsible for introducing an acetic acid unit extends, and cyclization and release of carbon chains with the participation of a thioesterase (TE).
  • TE thioesterase
  • IA-W1 encoding a PPT-modified polyketone body Synthetic acyl carrier protein (ACP) to make it active Protein
  • IA-W4 encodes a 3-O-acyltransferase responsible for the hydroxylation of coordinin at position 3
  • IA-W5 and IA-W6 encode O-methylase and crotonyl-CoA reductase, respectively, responsible for aggregation
  • IA-W15 encodes P450 cytochrome monooxygenase, which is responsible for the oxidation of polyketone carbon chains
  • IA-W36-39 encodes methoxymalonyl synthase, dehydrogenase, acyl carrier protein, respectively.
  • methoxymalonyl dehydrogenase which are involved in the synthesis and modification
  • orf7 encodes a carbonase aminoglycosylase
  • orf8 encodes a glycosylation accessory protein
  • orf31 and 32 Encoding a forrosamine glycosylation enzyme
  • orf30 encodes a glycosylation accessory protein
  • orf40 encodes a carbonase glycosylation enzyme.
  • orf2 encodes a transcriptional regulator of TetR family, which may be involved in the negative regulation of corimycin biosynthesis
  • Orf23, 42 respectively Encoding two positive regulatory transcription factors, the latter is a pathway-specific positive regulator, which directly regulates the biosynthesis of colimycin
  • orf27 encodes a GTPase, which may regulate the biosynthesis of colimycin by regulating cell function.
  • orf43 and orf44 are introduced exogenously, and orf43 encodes a 23S rRNA methylase gene associated with thiostrepton resistance, which is different from the 4"-O-hydroxyl of carbonaceous sugar.
  • the pentamyl transferase gene orf44 is linked, and its resistance expression can provide a differential marker for the gluconmycin genetically engineered bacteria.
  • the complementary sequences of Seq.1 and 2 of the present invention can be obtained at any time according to the principle of DNA base complementation.
  • the nucleotide sequence or partial nucleotide sequence of Seq. 1, 2 can be obtained by polymerase chain reaction (PCR) or by restriction endonuclease digestion with the corresponding DNA or using other suitable techniques.
  • the nucleotide sequence or partial nucleotide sequence provided by the present invention can be obtained from other organisms by a method of polymerase chain reaction (PCR) or a DNA comprising the sequence of the present invention as a probe for Southern hybridization. A gene similar to the colimycin biosynthesis gene.
  • the present invention also provides a method of constructing a recombinant DNA vector by at least obtaining a DNA sequence in a portion of Seq.1, 2.
  • the present invention also provides a pathway for blocking a gene encoding a colimycin biosynthesis, at least one of which comprises a nucleotide sequence in Seq.
  • a cloned gene or DNA fragment of the nucleotide sequence or at least a partial sequence provided by the present invention can be obtained by blocking one or several steps of colimycin biosynthesis.
  • the inclusion of DNA fragments or genes can be used to increase the yield of colimycin or its derivatives.
  • the nucleotide sequence or at least a partial sequence of the cloned DNA provided by the present invention can be used to locate more library quality from the genomic library. grain.
  • These library plasmids contain at least a partial sequence of the present invention, and also contain DNA adjacent to the region of the genome of the colimycin producing bacteria.
  • nucleotide sequences provided by the present invention may be modified or mutated. Routes include insertion or substitution, polymerase chain reaction, error-mediated polymerase chain reaction, site-specific mutation, re-ligation of different sequences, or mutation by UV or chemical reagents.
  • the nucleotide sequence provided by the present invention can be directly shuffling by homologous sequences of different parts of the sequence or other sources.
  • a nucleotide sequence or a fragment or domain or module or gene of at least a partial sequence of the invention can be used to construct a polyketide synthase library or a polyketide synthase derivatization library or combinatorial library. Generating new ones by deleting or inactivating one or more polyketide synthase domains, modules or genes from the same or different polyketide synthase systems, or by adding one or more polyketide synthase domains, modules or genes Polyketone compound.
  • the nucleotide sequence of the biosynthetic modification gene, glycosylation synthesis and glycosyltransferase gene of the present invention provides a pathway for obtaining a colimycin derivative by deleting, replacing or modifying these glycosyl synthesis, transfer and modification genes .
  • the nucleotide sequence or fragment or domain or module or gene of at least a partial sequence provided by the present invention can increase the yield of colimycin or a derivative thereof by doubling the amount.
  • the nucleotide sequence of the present invention or at least a partial sequence of the cloned gene can be expressed in a foreign host by a suitable expression system to obtain a modified or higher biological activity or a higher yield of colimycin.
  • exogenous hosts include Streptomyces, Escherichia coli, Bacillus, yeast, plants and animals, and the like.
  • Nucleotide sequences or at least partial sequences of genes or gene clusters of the invention can be expressed in heterologous hosts and their function in the host metabolic chain can be understood by DNA chip technology.
  • the amino acid sequence or at least a partial sequence of the polypeptide of the present invention may still have biological activity or even new biological activity after removing or replacing one or some amino acids, or increase yield or optimize protein dynamics or other commitments.
  • the properties obtained. By the deletion of a suitable technique, by ligating the amino acid sequence of the present invention, a new protein or enzyme can be obtained, thereby producing a new or associated product.
  • amino acid sequences provided by the present invention can be used to isolate desired proteins and can be used in antibody preparation.
  • amino acid sequence described in the present invention provides the possibility of predicting the three-dimensional structure of polyketide synthase.
  • genes, proteins, and antibodies provided by the present invention can also be used to screen and develop compounds or proteins for use in medicine, industry, and agriculture.
  • FIG. 4 Schematic diagram of the construction of IA-W42 transcriptional regulation and other gene blocking recombinant plasmid
  • III-DNA marker III C-original; M-gene blocking variant
  • I, II, III- are the absorption peaks of three components of the main component of colimycin, isovalerylspiramycin I, II and III, respectively.
  • the present invention obtains a mutant strain by a gene blocking experiment, and experimentally proves that the gene blockade causes the mutant strain to produce a change in the colimycin component, or no longer produces colimycin, thereby suggesting that the obtained gene cluster information is Associated with corimycin biosynthesis.
  • Example 1 Total DNA extraction of S. spiramyceticus
  • S.spiramyceticus strain was inoculated in 25ml R 2 YE medium, shaken at 28°C for 48h, transferred to 100ml R 2 YE medium, shaken at 28°C for 24h, centrifuged at 5000rpm for 10-15min, and collected the bacteria (about 10g).
  • the bacteria are mainly operated according to the product specifications of UPTECHTMlife science.
  • Example 2 verifies the function of Seq.1 gene information by gene blocking
  • the IA-W1 and IA-W42 and IA-W4, 17, 21, 23 and 27 genes were ligated at both ends of the gene cluster to obtain the mutant strains, and it was proved by experiments that these blocking strains produced colimycin ability. Change, or no longer produce colimycin. It is thus suggested that the obtained gene cluster information is necessary for the production of colimycin.
  • Primers were designed based on the above-described coding gene and its upstream and downstream sequences, and inserted into appropriate restriction sites. The primer sequences are shown in Table 2.
  • the corresponding homologous gene fragments were obtained by PCR amplification, and the corresponding restriction enzyme sites were inserted and inserted into the screening marker resistance gene (Apmycin-Am) to the temperature-sensitive vector pKC1139 [Bierman M. et al Gene1992] ;116(1):43-9] or Escherichia coli/streptavidin vector pGH112 [Youbao Biotechnology Co., Ltd.], obtained a recombinant plasmid containing a homologous gene, transformed by protoplasts, and transferred into a colimycin producing strain. After the cultivation, single colony isolation was carried out to obtain a homologous fragment double-exchange gene blocking strain.
  • the screening marker resistance gene Apmycin-Am
  • the IA-W4 3-O-acyltransferase gene blocking recombinant plasmid construction and gene homologous fragment double exchange are shown in Figure 3.
  • the construction of the IA-W42 transcriptional regulatory gene and other blocking recombinant plasmids is shown in Figure 4.
  • PCR was performed on the total DNA of the blocking strain and the original strain using the corresponding primers, as shown in Figs. 5A and B.
  • the results in Fig. 5A indicate that the coding gene orf4 of the IA-W4 gene-blocking mutant lacks 613 bp.
  • the results of PCR verification are shown by way of example in Figure 5B, indicating that the length of the PCR product is increased due to the insertion of the screening marker resistance gene compared to the original strain.
  • Seq.1 gene cluster provided by the present invention is involved in the biosynthesis of colimycin.
  • Example 3 gene transfer of colimycin producing bacteria and screening of blocking strains
  • Plasmid DNA transformation into protoplasts Take 100 ⁇ l of protoplasts, add 10 ⁇ l of plasmid DNA solution, mix the light bulb wall, and quickly add 400 ⁇ l of P-buffer containing 25% PEG-1000 (Koch-light, UK). After standing at room temperature for 5 min, 200 ⁇ l was applied to a dehydrated R 2 YE plate, cultured at 28 ° C for 20 h, covered with 50 ⁇ g/ml tsr of sterile water, cultured at 28 ° C for 5-7 days, and the transformants were picked.
  • the transformants were picked up in Tsr 50 ⁇ g/ml medium.
  • Example 4 fermentation and product activity detection and identification of colimycin producing strain and gene blocking strain
  • the strain is in slant medium (g/L):
  • the strain was inoculated and inoculated into a 100 mL flask containing 30 ml of fermentation medium, and cultured at 28 ° C for 96-120 h.
  • the fermentation broth is diluted with the supernatant and centrifuged, and Bacillus subtilis is used as the test bacteria.
  • Bacillus subtilis is used as the test bacteria.
  • the cup-and-disk method was used to measure by the standard curve method.
  • the fermentation broth was centrifuged at 3000 rpm for 15 min at room temperature.
  • the supernatant was adjusted to pH 8.5 with 1 M NaOH, extracted with 1/2 volume of ethyl acetate, and the ethyl acetate phase was taken out and dried in a dish. After being evaporated, it was dissolved in purified methanol and filtered. After injection, 10-20 ⁇ l.

Abstract

提供一种可利霉素生物合成基因簇，共有44个基因开放阅读框(orf)，其中含有5个编码聚酮合酶的orf(orf10-14)、9个聚酮合成延长单位及修饰相关的orf (orf1、4-6、15和36-39)、16个与糖基合成相关的orf (orf9、16-22、24、26、28、29、33-35和41)、6个与糖基转移相关的orf (orf7、8、30-32和40)、2个与抗性相关的orf (orf3和25)、4个与调控可能相关的orf (orf2、23、27和42)、tsr抗性标记基因orf（orf43）和4"碳霉糖苷异戊酰转移酶基因orf（orf44）。

Description

可利霉素生物合成基因簇 技术领域：
本发明属于微生物基因资源和基因工程领域，具体涉及基因工程抗生素生物合成基因簇的克隆、分析、功能研究及其应用。
技术背景：
可利霉素曾用名生技霉素、必特螺旋霉素，是利用合成生物学技术研制的16元环大环内酯类抗生素[专利号：ZL971044406、ZL 021487715]，是以4”-异戊酰螺旋霉素Ⅲ、Ⅱ、Ⅰ为主组分的在4”-位羟基多种酰基化的螺旋霉素，其中Ⅲ组分约占30％以上、Ⅱ组分25％左右，Ⅰ组分不超过10％。
Figure PCTCN2016107039-appb-000001
可利霉素结构式
异戊酰螺旋霉素Ⅲ：R＝COCH2CH3     R′＝COCH2CH(CH3)2
异戊酰螺旋霉素Ⅱ：R＝COCH3        R′＝COCH2CH(CH3)2
异戊酰螺旋霉素Ⅰ：R＝H              R′＝COCH2CH(CH3)2
可利霉素对革兰氏阳性菌有较强的活性，对红霉素和β-内酰胺类抗生素耐药菌、流感杆菌、淋球菌、军团菌、脆弱拟杆菌、产气荚膜梭菌有抗菌活性。尤其对肺炎支原体、沙眼衣原体和肺炎衣原体具有较强的活性[余兰香等，四川生理科学杂志；1998,20(3),专利号：2003101224209]，有较好的抗生素后效应和抗生素亚抑菌浓度效应。其与同类药没有完全交叉耐药性。药代动力学研究表明，可利霉素有较高的亲脂性，在胞内抗菌活性强，口服吸收快，绝对生物利用度高，组织渗透性强，其组织浓度高于血浆浓度，组织分布广，消除半衰期长，在体内维持时间长[孙丽文等，中国药理学通报2000，16(6)：694-8；钟大放等，J chromatography B.2003，791：45；史向国等，Asian Journal of Drug Metabolism and Pharmacokinetics.2003，3(2)：134；史向国等，Chinese Chemical letter 2004,15:431；史向国等，Acta Pharmacologica Sinica,2004,25:1396]。药理、毒理及已完成的临床三期研究结果表明，可利霉素用于治疗呼吸道感染其疗效确切，不良反应率低，尤其对肝脏损害小，安全性好[林赴田等，第八次全国抗生素学术会议论文汇编1997,p.167；赵春燕等，中国抗生素杂志1998,23(4)：306；孙涛等，中国抗生素杂志2001，26(1)：49-51]。可利霉素是利用基因重组技术获得的基因工程菌发酵直接产物，制备工艺简便、可以有效地避免化学污染及节省能源。其口服制剂服用方便，每日只需服用一次，有利于提高患者的用药依从性，也便于进入基本医保药物系列。
可利霉素是利用基因重组技术，将碳霉素产生菌4”-异戊酰基转移酶基因在螺旋霉素产生菌(Streptomyces spiramyceticus F21)中进行克隆表达，获得基因工程菌(Streptomyces spiramyceticus WSJ-1)的发酵产物。所述螺旋霉素产生菌(Streptomyces spiramyceticus F21)，是本实验室于1982年从中国甘肃永昌县土壤中分离得到的，该菌的形态培养特征、生理生化特征、细胞壁化学组成以及16S rRNA基因序列和5个看家基因蛋白水平在系统发育树分析中的地位，与国外报道的螺旋霉素产生菌 Streptomyces ambofaciens ATCC23877和已报道的链霉菌均无共同之处，故极可能为一株链霉菌新种[戴剑漉等，微生物学通报2012，39(4)：503-514]。
螺旋霉素产生菌Streptomyces ambofaciens ATCC23877中与螺旋霉素生物合成相关基因簇测序已经完成[Karray F.Microbiology 2007,153:4111-4122]，其他大环内酯类抗生素如阿维菌素、刀豆霉素、红霉素、查耳霉素、泰洛菌素和麦迪霉素生物合成基因簇序列也已有报道[Ikeda H.et al Nat.Biotechnol.2003,21(5):526-531、Haydock et al Microbiology 2005,151,3161-3169；Oliynyk M.et al Nat.Biotechnol.2007,25(4):447-453；Wards L.et al Antimicrob.Agents&Chemotherapy 2004,48(12):4703-4712；Cundiffe E.et al Antonie Van Leeuwenhoek 2001,79(3-4):229-234；Midoh Naoki et al US patent 7070980]。大环内酯类抗生素生物合成基因簇全长约50-80kb，其共同特征是，由编码16元大环内酯环生物合成模块式结构的聚酮合酶(PKS)、聚酮合成延长单位相关酶、负责内酯环不同基团修饰的酶、糖基合成和转移相关酶的基因以及与抗性和调控功能相关基因等组成。大环内酯是由模块结构形式组成的PKS催化，以类似脂肪酸生物合成的方式，通过连续缩合反应将一些简单的羧酸分子催化形成的。每一个模块在聚酮链形成过程中只负责一步缩合反应，它至少包含一个β-酮酯酰合成酶(KS)结构域，一个酰基转移酶(AT)结构域和一个酰基载体蛋白(ACP)结构域。此外，它还可能包含一个β-酮酯酰还原酶(KR)结构域，一个脱水酶(DH)结构域和一个酯酰还原酶(ER)结构域，它们决定了加入延伸单位的还原步骤。同时，还需要硫酯酶(TE)结构域的作用，以催化聚酮链的环化与释放。最后，还要经过羟基化、甲基化、甲氧基化和酰基化等修饰步骤，形成多种多样的大环内酯类抗生素的结构。通常大环内酯均与不同数量的糖基(或糖胺基)相连，如可利霉素含有三个糖基，分别是福洛糖胺、碳霉糖胺和碳霉糖。它们是由糖基合成和转移相关酶负责。抗性基因赋予产生菌拮抗自身产生抗生素的能力，通常与ABC转运蛋白相关。调控功能相关的基因参与自身生物合成抗生素的调控。
通过基因簇序列信息和结构分析，可以进一步对其产生菌进行遗传操作，获得新型、更有效的抗生素，如通过基因操作来改变其PKS合成模块式结构、进行内酯环后修饰的改变、糖基因的置换或修饰，创造新的大环内酯类抗生素；也可以通过对抗性基因或调节基因的遗传操作，提高抗生素的产量。[Wilkinson B.et al Chem Biol 2000,7(2):111-117；Kalz L.et al Med Res Rev 1999,19(6):543-58；Goodman CD et al Antimicrobial Agents and Chemotherapy,2013,57(2):907–913；Wang W et al Proc Natl Acad Sci U S A 2014,111(15)：5688-93；Stratigopoulos G et al Mol Microbiol.2004,54(5)：1326-34；Novakova R et al Folia Microbiol.2011,56(3):276-82]。
发明内容：
本发明提供了可利霉素生物合成连锁基因簇，共有44个基因开放阅读框(orf)，核苷酸序列全长89315bp(Seq.1)，其中含有5个编码聚酮合酶(orf10-14)，包括8个模块，37个结构域，以及与聚酮合成延长单位及修饰相关的orf有9个(1、4-6、15、36-39)，与糖基合成相关的orf有16个(9、16-22、24、26、28、29、33-35、41)，与糖基转移相关的orf有6个(7、8、30-32、40)。此外，还有与抗性相关的orf有2个(3和25)，与调控可能相关的orf有4个(2、23、27、42)。这些核苷酸序列分别选自Seq.1中的orf1(1-645)、orf2(1810-1208)、orf3(3133-2285)、orf4(3614-4840)、orf5(4846-5511)、orf6(7150-5801)、orf7(8444-7179)、orf8(9729-8482)、orf9(10543-9830)、orf10(16215-10543)、orf11(21076-16328)、orf12(32511-21124)、orf13(38599-32585)、orf14(52259-38643)、orf15(53099-54310)、orf16(54495-54845)、orf17(54842-56041)、orf18(56038-56946)、orf19(56930-57967)、orf20(57937-60174)、orf21(60836-61984)、orf22(62796-62077)、orf23(63633-65645)、orf24(67379-66318)、orf25(69004-67352)、orf26(69349-70650)、orf27(72156-70708)、orf28(72422-73462)、orf29(74601-73561)、orf30(74913-76160)、orf31(76218-77486)、orf32(77606-78781)、orf33 (78783-79775)、orf34(79772-80779)、orf35(82055-80823)、orf36(83164-82052)、orf37(84400-83279)、orf38(84713-84393)、orf39(85576-84710)、orf40(85825-87042)、orf41(87094-87702)、orf42(89315-88143)。此外，还含有与Seq1.不连锁的外源基因Seq.2全长2337bp中的orf43(866-60)和orf44(2337-1174)。
本发明还提供了一个4’磷酸泛酰巯基乙胺基转移酶(PPT)的氨基酸序列，由Seq.3序列中的214个氨基酸组成，命名为IA-W1，编码基因的核苷酸序列选自Seq.1中的1-645碱基。
本发明还提供了一个TetR家族转录调控因子的氨基酸序列，由Seq.4序列中的200个氨基酸组成，命名为IA-W2，编码基因的核苷酸序列选自Seq.1中的1810-1208碱基。
本发明还提供了一个23S rRNA甲基转移酶的氨基酸序列，由Seq.5序列中的282个氨基酸组成，命名为IA-W3，编码基因的核苷酸序列选自Seq.1中的3133-2285碱基。
本发明还提供了一个3-O-酰基转移酶的氨基酸序列，由Seq.6序列中的408个氨基酸组成，命名为IA-W4，编码基因的核苷酸序列选自Seq.1中的3614-4840碱基。
本发明还提供了一个O-甲基转移酶的氨基酸序列，由Seq.7序列中的221个氨基酸组成，命名为IA-W5，编码基因的核苷酸序列选自Seq.1中的4846-5511碱基。
本发明还提供了一个巴豆酰基辅酶A还原酶的氨基酸序列，由Seq.8序列中449个氨基酸组成，命名为IA-W6，编码基因的核苷酸序列选自Seq.1中的7150-5801碱基。
本发明还提供了一个糖苷转移酶的氨基酸序列，由Seq.9序列中的421个氨基酸组成，命名为IA-W7，编码基因的核苷酸序列选自Seq.1中的8444-7179碱基。
本发明还提供了一个糖苷转移酶辅助蛋白的氨基酸序列，由Seq.10序列中的415个氨基酸组成，命名为IA-W8，编码基因的核苷酸序列选自Seq.1中的9729-8482碱基。
本发明还提供了一个NDP-氨基己糖N-二甲基转移酶的氨基酸序列，由Seq.11序列中的237个氨基酸组成，命名为IA-W9，编码基因的核苷酸序列选自Seq.1中的10543-9830碱基。
本发明还提供了一个包括酮基合成酶(KS)8-酰基转移酶(AT)8-酮基还原酶(KR)8-酰基载体蛋白结构域(ACP)8-链释放硫酯酶(TE)的聚酮合酶结构域氨基酸序列，由Seq.12序列中1890个氨基酸组成，命名为IA-W10，编码基因的核苷酸序列选自Seq.1中的16215-10543碱基。
本发明还提供了一个包括KS7-AT7-KR7-ACP7的聚酮合酶结构域氨基酸序列，由Seq.13序列中的1582个氨基酸组成，命名为IA-W11，编码基因的核苷酸序列选自Seq.1中的21076-16328碱基。
本发明还提供了一个包括KS5-AT5-KR5-ACP5-KS6-AT6-DH6(脱水酶)-ER6(烯酰基还原酶)-KR6-ACP6的聚酮合酶结构域氨基酸序列，由Seq.14序列中的3795个氨基酸组成，命名为IA-W12，编码基因的核苷酸序列选自Seq.1中的32511-21124碱基。
本发明还提供了一个包括KS4-AT4-DH4-KR4-ACP4的聚酮合酶结构域氨基酸序列，由Seq.15序列中的2004个氨基酸组成，命名为IA-W13，编码基因的核苷酸序列选自Seq.1中的38599-32585碱基。
本发明还提供了一个包KS1-AT1-ACP1-KS2-AT2-KR2-ACP2-KS3-AT3-DH3-KR3-ACP3的聚酮合酶结构域氨基酸序列，由Seq.16序列中的4538个氨基酸组成，命名为IA-W14，编码基因的核苷酸序列选自Seq.1中的52259-38643碱基。
本发明还提供了一个细胞色素P-450氧化酶的氨基酸序列，由Seq.17序列中的403个氨基酸组成，命名为IA-W15，编码基因的核苷酸序列选自Seq.1中的53099-54310碱基。
本发明还提供了一个NDP-己糖异构酶的氨基酸序列，由Seq.18序列中的116个氨基酸组成，命名为IA-W16，编码基因的核苷酸序列选自Seq.1中的54495-54845碱基。
本发明还提供了一个NDP-己糖氨基转移酶的氨基酸序列，由Seq.19序列中的399个氨基酸组成， 命名为IA-W17，编码基因的核苷酸序列选自Seq.1中的54842-56041碱基。
本发明还提供了一个NDP-葡萄糖合酶的氨基酸序列，由Seq.20序列中的302个氨基酸组成，命名为IA-W18，编码基因的核苷酸序列选自Seq.1中的56038-56946碱基。
本发明还提供了一个NDP-葡萄糖-4,6脱水酶的氨基酸序列，由Seq.21序列中的345个氨基酸组成，命名为IA-W19，编码基因的核苷酸序列选自Seq.1中的56930-57967碱基。
本发明还提供了一个NDP-己糖2,3脱水酶/硫酯酶的氨基酸序列，由Seq.22序列中的745个氨基酸组成，命名为IA-W20，编码基因的核苷酸序列选自Seq.1中的57937-60174碱基。
本发明还提供了一个NDP-己糖氨基转移酶的氨基酸序列，由Seq.23序列中的382个氨基酸组成，命名为IA-W21，编码基因的核苷酸序列选自Seq.1中的60836-61984碱基。
本发明还提供了一个NDP-氨基己糖N二甲基转移酶的氨基酸序列，由Seq.24序列中的239个氨基酸组成，命名为IA-W22，编码基因的核苷酸序列选自Seq.1中的62796-62077碱基。
本发明还提供了一个转录调控因子的氨基酸序列，由Seq.25序列中的670个氨基酸组成，命名为IA-W23，编码基因的核苷酸序列选自Seq.1中的63633-65645碱基。
本发明还提供了一个NDP-氨基己糖异构酶的氨基酸序列，由Seq.26序列中的354个氨基酸组成，命名为IA-W24，编码基因的核苷酸序列选自Seq.1中的67379-66318碱基。
本发明还提供了一个ABC转运蛋白的氨基酸序列，由Seq.27序列中的550个氨基酸组成，命名为IA-W25，编码基因的核苷酸序列选自Seq.1中的69004-67352碱基。
本发明还提供了一个NDP-己糖脱水酶的氨基酸序列，由Seq.28序列中的433个氨基酸组成，命名为IA-W26，编码基因的核苷酸序列选自Seq.1中的69349-70650碱基。
本发明还提供了一个类似于GTP酶的氨基酸序列，由Seq.29序列中的482个氨基酸组成，命名为IA-W27，编码基因的核苷酸序列选自Seq.1中的72156-70708碱基。
本发明还提供了一个NDP-糖异构酶的氨基酸序列，由Seq.30序列中的346个氨基酸组成，命名为IA-W28，编码基因的核苷酸序列选自Seq.1中的72422-73462碱基。
本发明还提供了一个NDP-己糖酮基还原酶的氨基酸序列，由Seq.31序列中的346个氨基酸组成，命名为IA-W29，编码基因的核苷酸序列选自Seq.1中的74601-73561碱基。
本发明还提供了一个糖基转移酶辅助蛋白的氨基酸序列，由Seq.32序列中415个氨基酸组成，命名为IA-W30，编码基因的核苷酸序列选自Seq.1中的74913-76160碱基。
本发明还提供了一个糖基转移酶的氨基酸序列，由Seq.33序列中的422个氨基酸组成，命名为IA-W31，编码基因的核苷酸序列选自Seq.1中的76218-77486碱基。
本发明还提供了一个糖基转移酶的氨基酸序列，由Seq.34序列中的391个氨基酸组成，命名为IA-W32，编码基因的核苷酸序列选自Seq.1中的77606-78781碱基。
本发明还提供了一个NDP-己糖酮基还原酶的氨基酸序列，由Seq.35序列中的330个氨基酸组成，命名为IA-W33，编码基因的核苷酸序列选自Seq.1中的78783-79775碱基。
本发明还提供了一个NDP-己糖还原酶的氨基酸序列，Seq.36由序列中的335个氨基酸组成，命名为IA-W34，编码基因的核苷酸序列选自Seq.1中的79772-80779碱基。
本发明还提供了一个NDP-己糖甲基转移酶的氨基酸序列，由Seq.37序列中的410个氨基酸组成，命名为IA-W35，编码基因的核苷酸序列选自Seq.1中的82055-80823碱基。
本发明还提供了一个甲氧基丙二酰合成酶的氨基酸序列，由Seq.38序列中的370个氨基酸组成，命名为IA-W36，编码基因的核苷酸序列选自Seq.1中的83164-82052碱基。
本发明还提供了一个脱氢酶的氨基酸序列，由Seq.39序列中的373个氨基酸组成，命名为IA-W37， 编码基因的核苷酸序列选自Seq.1中的84400-83279碱基。
本发明还提供了一个酰基携带蛋白的氨基酸序列，由Seq.40序列中的106个氨基酸组成，命名为IA-W38，编码基因的核苷酸序列选自Seq.1中的84713-84393碱基。
本发明还提供了一个甲氧基丙二酰脱氢酶的氨基酸序列，由Seq.41序列中的288个氨基酸组成，命名为IA-W39，编码基因的核苷酸序列选自Seq.1中的85576-84710碱基。
本发明还提供了一个糖基转移酶的氨基酸序列，由Seq.42序列中的405个氨基酸组成，命名为IA-W40，编码基因的核苷酸序列选自Seq.1中的85825-87042碱基。
本发明还提供了一个NDP-己糖异构酶的氨基酸序列，由Seq.43序列中的202个氨基酸组成，命名为IA-W41，编码基因的核苷酸序列选自Seq.1中的87094-87702碱基。
本发明还提供了一个转录调控因子蛋白的氨基酸序列，由Seq.44序列中的390个氨基酸组成，命名为IA-W42，编码基因的核苷酸序列选自Seq.1中的89315-88143碱基。
本发明还提供了外源插入的23S rRNA甲基化酶(硫链丝菌素thiostrepton、tsr抗性标记相关)氨基酸序列，由Seq.45序列中的269个氨基酸组成，命名为IA-W43，编码基因的核苷酸序列选自Seq.2中的866-57碱基。
本发明还提供了外源插入的4”碳霉糖苷异戊酰转移酶氨基酸序列，由Seq.46序列中的388个氨基酸组成，命名为IA-W44，编码基因的核苷酸序列选自Seq.2中的2337-1171碱基。
在获得可利霉素生物合成基因簇信息、通过基因阻断及同源性比较分析各基因编码蛋白可能功能基础上，进一步描述本发明整个可利霉素生物合成基因簇共44个基因，其基因簇结构如图1所示，具体为：
(1)聚酮合酶基因orf10-14，共5个基因；
(2)与聚酮合成延长单位及修饰相关的基因orf1、orf4-6、15、36-39，共9个基因；
(3)与糖基合成相关基因orf9、16-22、24、26、28、29、33-35、41，共16个基因；
(4)与糖基转移相关基因orf7、8、30-32、40，共6个基因；
(5)与抗性相关基因orf3和25，共2个基因；
(6)与生物合成调控相关基因orf2、23、27、42，共4个基因；
(7)外源引入的基因工程菌标记基因orf43(硫链丝菌素-thiostrepton、tsr抗性基因)和与之连锁的碳霉糖4”-O-异戊酰转移酶基因orf44，共2个基因。
Seq.1中有5个聚酮合酶基因(orf10-14)，核苷酸互补序列及其氨基酸序列，是可利霉素内酯环合成所必须的，其中，包含8个模块，37个结构域如图2所示。Orf14包含3个模块：加载结构域1以及模块2和3；加载模块域中，KS1、AT1和ACP1负责内酯环的起始合成，催化一个乙酸作为起始单位，模块2含有KS2、AT2、KR2和ACP2结构域；模块3含有KS3、AT3、DH3、KR3和ACP3结构域，负责引入另2个乙酸延伸单位，最终形成可利霉素C11-15碳链骨架。Orf13包括模块4，含有KS4、AT4、DH4、KR4、ACP4，负责第3个乙酸单位的延伸，最终形成可利霉素C9-10碳链骨架；Orf12包括模块5和6，模块5含有KS5-AT5-KR5-ACP5结构域，负责引入丙酸延伸单位；模块6含有KS6-AT6-DH6-KR6-ER6-KR6-ACP6结构域，负责引入丁酸延伸单位，最终形成可利霉素C5-C8碳链骨架。Orf11包括模块7，含有KS7-AT7-KR7-ACP7结构域，负责引入乙醇酸延伸单位，最终形成可利霉素C3-C4碳链骨架。Orf10包括模块8，含有KS8-AT8-KR8-ACP8-TE结构域，负责引入一个乙酸延伸单位，并在硫酯酶(TE)参与下完成碳链的环化及释放。可利霉素聚酮合酶基因结构示意图见图2。聚酮合酶基因各结构域及其氨基酸的位置如表1所示。
可利霉素聚酮合成延长单位及修饰相关基因orf1、orf4-6、15、36-39的核苷酸序列或互补序列及其相应的氨基酸序列：即IA-W1，编码PPT修饰聚酮体合成的酰基载体蛋白(ACP)，使之成为有活性的 蛋白；IA-W4编码3-O-酰基转移酶,负责可利霉素3位羟基酰基化；IA-W5和IA-W6分别编码O-甲基化酶和巴豆酰辅酶A还原酶，负责聚酮体延长单位的供应；IA-W15编码P450细胞色素单氧化酶，负责聚酮体碳链的氧化；IA-W36-39分别编码甲氧基丙二酰合成酶、脱氢酶、酰基携带蛋白和甲氧基丙二酰脱氢酶，它们均参与聚酮体延长单位的合成与修饰。
表1聚酮合酶基因各结构域及其氨基酸的位置：
表1.1聚酮合酶基因IA-W14各结构域及其氨基酸的位置
Figure PCTCN2016107039-appb-000002
表1.2聚酮合酶基因IA-W13各结构域及其氨基酸的位置
Figure PCTCN2016107039-appb-000003
表1.3聚酮合酶基因IA-W12各结构域及其氨基酸的位置
Figure PCTCN2016107039-appb-000004
表1.4聚酮合酶基因IA-W11各结构域及其氨基酸的位置
Figure PCTCN2016107039-appb-000005
Figure PCTCN2016107039-appb-000006
表1.5聚酮合酶基因IA-W10各结构域及其氨基酸的位置
Figure PCTCN2016107039-appb-000007
与可利霉素糖基合成相关基因orf9、16-22、24、26、28、29、33-35和41，共12个基因，其中：orf18,19和28编码参与可利霉素基本糖基单元合成、脱水和异构化酶；orf9、20、21、24、26和29编码参与福洛糖胺合成中NDP-氨基己糖N-二甲基化、2,3脱水、氨基化、异构化、脱水和酮基还原酶；orf16、17、22编码参与碳霉糖胺NDP-氨基己糖异构化、氨基化和N-二甲基化酶；orf33、34、35和41编码碳霉糖NDP-氨基己糖酮基还原、甲基化和异构化酶。
与可利霉素糖基转移相关基因orf7、8、30-32、40，共6个基因，其中：orf7编码碳霉糖胺糖基化酶；orf8编码其糖基化辅助蛋白；orf31和32编码福洛糖胺糖基化酶；orf30编码其糖基化辅助蛋白；orf40编码碳霉糖糖基化酶。
与可利霉素抗性相关基因orf3和25，共2个基因，其中：orf3编码23S rRNA甲基化酶；orf25编码ABC转运蛋白，它们通过对核糖体RNA甲基化以及泵出机制，赋予可利霉素产生菌对自身产生抗生素的抗性。
与可利霉素生物合成调控相关基因orf 2、23、27、42，共4个基因，其中：orf2编码TetR家族转录调控抑制因子，可能参与可利霉素生物合成负调控；Orf23、42分别编码两个正调控转录因子，后者为途径特异正调控因子，对可利霉素的生物合成直接进行调控；orf27编码GTP酶，可能通过调控细胞的功能对可利霉素的生物合成进行调控。
与可利霉素生物合成相关，外源引入orf43和orf44，其中orf43编码与硫链丝菌素抗性相关的23S rRNA甲基化酶基因，该基因与碳霉糖4”-O-羟基异戊酰转移酶基因orf44连锁，其抗性表达可以为可利霉素基因工程菌提供鉴别性标记。
本发明Seq.1、2的互补序列可依据DNA碱基互补原则随时得到。Seq.1、2的核苷酸序列或部分核苷酸序列，可以通过聚合酶链式反应(PCR)或用合适的限制性内切酶，酶切相应的DNA或使用其它合适的技术得到。通过本发明所提供的核苷酸序列或部分核苷酸序列，可利用聚合酶链式反应(PCR)的方法或包含本发明序列的DNA作为探针进行Southern杂交的方法，从其它生物体得到与可利霉素生物合成基因相似的基因。
本发明还提供了至少获得部分Seq.1、2中DNA序列构建重组DNA载体的途径。
本发明还提供了阻断可利霉素生物合成基因的途径，至少其中之一的基因包含有Seq.1中的核苷酸序列。
本发明所提供核苷酸序列或至少部分序列的克隆基因或DNA片段，可以通过阻断可利霉素生物合成的一个或几个步骤而得到新的可利霉素衍生物。包含DNA片段或基因，可以用来提高可利霉素或其衍生物的产量。
本发明所提供核苷酸序列或至少部分序列的克隆DNA，可用来从基因组文库中定位更多的文库质 粒。这些文库质粒至少包含有本发明中的部分序列，也包含有可利霉素产生菌基因组中与之邻近区域的DNA。
本发明所提供的核苷酸序列可以被修饰或突变。其途径包括插入或置换、聚合酶链式反应、错误介导聚合酶链式反应、位点特异性突变、不同序列的重新连接或通过紫外线或化学试剂的突变。
本发明所提供的核苷酸序列，可以通过序列的不同部分或其它来源的同源序列进行直接进化(DNA shuffling)。
本发明的核苷酸序列或至少部分序列的片段或结构域或模块或基因，可以用来构建聚酮合酶库或聚酮合酶衍生库或组合库。通过缺失或失活来自相同或不同聚酮合酶系统的一个或多个聚酮合酶结构域、模块或基因，或增加一个或多个聚酮合酶结构域、模块或基因，产生新的聚酮化合物。
本发明的生物合成修饰基因、糖基合成及糖基转移酶基因的核苷酸序列，提供了通过缺失、置换或改造这些糖基合成、转移和修饰基因而得到可利霉素衍生物的途径。
本发明提供的核苷酸序列或至少部分序列的片段或结构域或模块或基因，可以通过加倍量来提高可利霉素或其衍生物的产量。
本发明的核苷酸序列或至少部分序列的克隆基因，可以通过合适的表达系统在外源宿主中表达，以得到修饰的或更高生物活性或更高产量的可利霉素。这些外源宿主包括链霉菌、大肠杆菌、芽孢杆菌、酵母、植物和动物等。
本发明的核苷酸序列或至少部分序列的基因或基因簇，可以在异源宿主中表达并通过DNA芯片技术，了解它们在宿主代谢链中的功能。
本发明的氨基酸序列或至少部分序列的多肽，可能在去除或替代某个或某些氨基酸之后仍有生物活性甚至有新的生物学活性，或者提高了产量或优化蛋白动力学特征或其它致力于得到的性质。通过合适的技术缺失，连接本发明中的氨基酸序列，可以得到新的蛋白或酶，进而产生新的或相关联的产物。
本发明所提供的氨基酸序列，可以用来分离需要的蛋白质并可用于抗体制备。
本发明所所述的氨基酸序列，提供了预测聚酮合酶三维结构的可能。
本发明所提供的基因及其蛋白质、抗体，也可用以筛选和发展用于医药、工业、农业的化合物或蛋白。
附图说明：
图1-可利霉素生物合成基因簇结构
图2-可利霉素聚酮合酶基因结构
图3-IA-W4 3-O-酰基转移酶基因阻断重组质粒的构建及双交换示意图
图4-IA-W42转录调控等基因阻断重组质粒的构建示意图
图5A-IA-W4 3-O-酰基转移酶基因阻断PCR验证
其中：1-原株；2、3、4-基因阻断变株；5-DNA markerIII
图5B-其他基因阻断的PCR验证
其中：III-DNA markerIII；C-原株；M-基因阻断变株
图6-IA-W4 3-O-酰基转移酶基因阻断变株发酵产物HPLC分析
其中：a-可利霉素对照品b-基因阻断变株发酵提取物
Ⅰ、Ⅱ、Ⅲ-分别为可利霉素主组分异戊酰螺旋霉素Ⅰ、Ⅱ、Ⅲ三个组分吸收峰
本发明通过基因阻断实验获得了突变菌株，并通过实验证明，基因阻断导致突变菌株产生可利霉素组分变化，或者不再产生可利霉素，从而提示，所获基因簇信息是与可利霉素生物合成相关。本发明外源引入的硫链丝菌素抗性标记基因(orf43)和与之连锁的碳霉糖4”O-羟基异戊酰转移酶基因(orf44)， 是通过基因同源重组整合在染色体上的，本实验室已经研究证明其为可利霉素生物合成所必须的(1999年生物工程学报15卷2期171-176)。
实施方案：
以下所提供实施例，仅为帮助本领域技术人员更好地理解本发明，但不以任何方式限制本发明。
《实施例1》可利霉素产生菌(S.spiramyceticus)总DNA提取
R2YE培养基配方(g/100ml)：
Figure PCTCN2016107039-appb-000008
加入微量元素溶液0.2ml，定容至100ml蒸馏水，pH6.5
微量元素溶液(g/100ml)：
Figure PCTCN2016107039-appb-000009
S.spiramyceticus菌种接种于25ml R2YE培养基，28℃摇床培养48h，转种于100ml R2YE培养基，28℃摇床培养24h，离心5000rpm 10-15min，收集菌体(约10g菌体)，主要按UPTECHTMlife science公司产品说明书操作。加入50ml的25mM的EDTA溶液震荡洗涤，离心，弃上清；用25ml溶菌酶溶液(10mg/ml，用10mM的pH8.0的Tris-HCl、2mM EDTA、1.2％TritonX-100配制，加入0.5ml 100mg/ml RNase)悬浮菌丝体，37℃培养约1-2h，培养细胞至半透明状；加入2.5ml蛋白酶K溶液，55℃30min；加入20ml 10％SDS溶液70℃10min；加入等体积无水乙醇，充分震荡；将溶液转移到DNA纯化柱，12000rpm离心1min；加入50ml含蛋白酶溶液洗柱，室温离心12000rpm 1min；再用50ml漂洗液洗柱二次，每次离心12000rpm 1min；加入5-10ml TE洗脱液，室温放置2-5min，12000rpm离心1min；收集溶液，-20℃保存总DNA。
《实施例2》通过基因阻断验证Seq.1基因信息的功能
选取基因簇两端IA-W1和IA-W42以及IA-W4、17、21、23和27等基因进行阻断，获得突变菌株，并通过实验证明，这些阻断菌株产生可利霉素能力发生变化，或者不再产生可利霉素。从而提示，所获得的基因簇信息是产生可利霉素所必须的。根据上述编码基因及其上下游序列设计引物，并插入合适的酶切位点，引物序列见表2。
表2基因阻断实验所设计使用的引物序列
Figure PCTCN2016107039-appb-000010
Figure PCTCN2016107039-appb-000011
经PCR扩增分别获得相应的同源基因片段，采用相应的酶切位点，并插入筛选标记抗性基因(阿普霉素-Am)连接到温敏型载体pKC1139[Bierman M.et al Gene1992；116(1):43-9]或大肠杆菌/链霉菌载体pGH112[优宝生物公司]，获得含有同源基因的重组质粒，经原生质体转化，转入可利霉素产生菌中，经培养后，进行单菌落分离，获得同源片段双交换基因阻断株。IA-W4 3-O-酰基转移酶基因阻断重组质粒构建及基因同源片段双交换示意见图3。IA-W42转录调控基因等阻断重组质粒构建示意见图4。
分别采用相应引物对阻断株及原株的总DNA进行PCR验证，如图5A和B所示。图5A结果表明，IA-W4基因阻断变株的编码基因orf4中缺失613bp。图5B中举例显示PCR验证结果，表明与原株相比，阻断变株相关编码基因由于插入筛选标记抗性基因，PCR产物长度呈现增加。
发酵实验及对产物的HPLC检测证明，IA-W4阻断变株不再产生4”-异戊酰螺旋霉素Ⅲ和Ⅱ，而是以4”-异戊酰螺旋霉素Ⅰ为主组分(图6)。证明本发明所提供Seq.1基因信息中的IA-W4 3-O-酰基转移酶基因，参与了可利霉素的生物合成，该基因的阻断，使变株丧失了可利霉素内酯环3位羟基被酰基化的功能。
其他基因阻断株经发酵实验及对产物抗菌活性和HPLC检测，证明阻断株已不再产生有活性的可利霉素。说明本发明所提供Seq.1基因簇参与可利霉素的生物合成。
《实施例3》可利霉素产生菌的基因转移及阻断株的筛选
3.1原生质体的制备：取新鲜的可利霉素产生菌斜面孢子接种于R2YE液体培养基中，28℃,220rpm摇瓶振荡培养48h，培养液以10％的转种量，转种于新鲜的补加了0.5％甘氨酸的R2YE液体培养基中，28℃振荡培养20h。取10ml菌液于离心管中，3000rpm离心收集菌丝体，沉淀用P-buffer
Figure PCTCN2016107039-appb-000012
Figure PCTCN2016107039-appb-000013
洗涤2次后用适量P-buffer悬浮，加入溶菌酶的P-buffer溶液(终浓度为2mg/ml)，混合均匀，于37℃水浴中保温30-45min，每隔10-15min振荡一次。用10×40的相差显微镜观察原生质体的形成情况，当镜检显示绝大部分菌丝已形成原生质体时，停止酶解。经脱脂棉过滤，滤液用P-buffer离心洗涤2次。最后用1ml P-buffer悬浮原生质体，分装EP管、100μl/管，-70℃保存备用。
3.2质粒DNA转化原生质体：取100μl原生质体，加入10μl质粒DNA溶液，轻弹管壁混匀，迅速加入400μl含25％PEG-1000(英国Koch-light公司产品)的P-buffer，吹吸混匀，室温放置5min，取200μl涂布于失水的R2YE平板上，28℃培养20h，以50μg/ml tsr的无菌水覆盖，28℃培养5～7天，挑取转化子。
3.3基因阻断变株的筛选
将转化子挑取在加有Tsr 50μg/ml培养基
Figure PCTCN2016107039-appb-000014
去离子水配制，自然pH值，15磅灭菌，121℃，30min。
28℃培养5～7天，在不加药的培养基传四-五代，分离单孢子，将单孢子分别对应地在加阿普霉素(Am 50μg/ml)的培养基上进行筛选，筛选出在Am生长和不在Tsr生长的基因阻断株。挑取抗性标记表达稳定的阻断株，提取基因组DNA，采用实施例2中相应引物PCR扩增，根据产物大小及DNA测序判断基因阻断的正确性。
《实施例4》可利霉素产生菌及基因阻断株发酵、产物活性检测及鉴定
4.1发酵
菌株在斜面培养基(g/L)：
Figure PCTCN2016107039-appb-000015
去离子水配制，自然pH值，15磅灭菌，121℃，30min。
28℃培养10-12d，菌株长好后挖块接种到装量30ml发酵培养基的100mL三角瓶，28℃振荡培养96-120h。
发酵培养基(g/L)：
Figure PCTCN2016107039-appb-000016
去离子水配制，15磅灭菌，121℃，30min。
4.2发酵产物活性检测：
发酵液经离心取上清液稀释后，以枯草芽孢杆菌为检定菌，参考《中华人民共和国药典》2005年版(二部)乙酰螺旋霉素微生物检定法。采用杯碟法，用标准曲线法进行测定。
4.3发酵产物提取及鉴定：
发酵液室温3000rpm离心15min，上清液用1M NaOH调至pH 8.5后，用1/2体积乙酸乙酯萃取， 取出乙酸乙酯相于平皿中吹干，挥干后溶于色谱纯甲醇，过滤后进样10-20μl。色谱议：岛津LC-10ATvp液相色谱议，二级管阵列检测器，色谱柱：Kromasil C18(4.5mm×150mm，5μm)，流动相：CH3OH/1％NaH2PO4(55:45)，检测波长：231nm，流速：1ml/min柱温：25℃。以可利霉素标准品作为对照(购自中国药品生物制品检定所)，对变株发酵产物进行鉴定。
本发明所述基因和蛋白见序列表。

Claims (10)

  1. 一种可利霉素生物合成基因簇，其特征是，所述生物合成基因簇共有44个基因，具体为：
    1)聚酮合酶基因orf10-14，共5个基因；
    2)与聚酮合成延长单位及修饰相关的基因orf1、orf4-6、15、36-39，共有9个基因；
    3)与糖基合成相关基因orf9、16-22、24、26、28、29、33-35、41，共16个基因；
    4)与糖基转移相关基因orf7、8、30-32、40，共6个基因；
    5)与抗性相关基因orf3和25，共2个基因；
    6)与生物合成调控相关基因orf2、23、27、42，共4个基因；
    7)外源引入的基因工程菌标记基因orf43和与之连锁的碳霉糖4”O-羟基异戊酰转移酶基因orf44，共2个基因。
  2. 如权利要求1所述生物合成基因簇，其特征是，所述的5个聚酮合酶基因编码聚酮体生物合成酶，所述聚酮体生物合成酶催化可利霉素16元内酯环的合成，所述5个聚酮合酶基因orf10-14的核苷酸序列或互补序列所对应的氨基酸序列为IA-W10、IA-W11、IA-W12、IA-W13和IA-W14。
  3. 如权利要求1或2所述的生物合成基因簇，其特征是，5个聚酮合酶包括酮基合成酶(KS)、酰基转移酶(AT)、酮基还原酶(KR)、脱水酶(DH)、烯酰基还原酶(ER)、酰基载体蛋白(ACP)、硫酯酶结构域的核苷酸序列或互补序列所对应的氨基酸序列。
  4. 如权利要求3所述的生物合成基因簇，其特征是，5个聚酮合酶包含模块或结构域。
  5. 如权利要求1所述的生物合成基因簇，其特征是，所述聚酮合成延长单位及修饰相关基因orf1、orf4-6、15、36-39的核苷酸序列或互补序列所对应的氨基酸序列为IA-W1、IA-W4、IA-W5、IA-W6、IA-W15、IA-W36、IA-W37、IA-W38和IA-W39。
  6. 如权利要求1所述的生物合成基因簇，其特征是，所述糖基合成相关基因orf9、16-22、24、26、28、29、33-35、41的核苷酸序列或互补序列所对应的氨基酸序列为IA-W9、IA-W16、IA-W17、IA-W18、IA-W19、IA-W20、IA-W21、IA-W22、IA-W24、IA-W26、IA-W28、IA-W29、IA-W33、IA-W34、IA-W35、IA-W41。
  7. 如权利要求1所述的生物合成基因簇，其特征是，所述糖基转移相关基因orf7、 8、30-32、40的核苷酸序列或互补序列所对应的氨基酸序列为IA-W7、IA-W8、IA-W30、IA-W31、IA-W32、和IA-W40。
  8. 如权利要求1所述的生物合成基因簇，其特征是，所述抗性相关基因orf3和25的核苷酸序列或互补序列所对应的氨基酸序列为IA-W3及IA-W25。
  9. 如权利要求1所述的生物合成基因簇，其特征是，所述生物合成调控相关基因orf2、23、27、42的核苷酸序列或互补序列所对应的氨基酸序列为IA-W2、IA-W23、IA-W27、IA-W42。
  10. 如权利要求1所述的生物合成基因簇，其特征是，外源引入的基因工程菌标记基因orf43和与之连锁的orf44的核苷酸序列或互补序列所对应的氨基酸序列为IA-W43、IA-W44。
PCT/CN2016/107039 2015-12-31 2016-11-24 可利霉素生物合成基因簇 WO2017114034A1 (zh)

Priority Applications (17)

Application Number Priority Date Filing Date Title
MYPI2018001122A MY193028A (en) 2015-12-31 2016-11-24 Biosynthetic gene cluster of carrimycin
CA3009706A CA3009706A1 (en) 2015-12-31 2016-11-24 Biosynthetic gene cluster of carrimycin
AU2016383270A AU2016383270A1 (en) 2015-12-31 2016-11-24 Carrimycin biosynthetic gene cluster
PE2018001213A PE20190221A1 (es) 2015-12-31 2016-11-24 Agrupacion de genes biosinteticos de carrimicina
ES16880824T ES2812256T7 (es) 2015-12-31 2016-11-24 Agrupación de genes de biosíntesis de carrimicina
JP2018553280A JP6882330B2 (ja) 2015-12-31 2016-11-24 カリマイシン（Ｃａｒｒｉｍｙｃｉｎ）生合成遺伝子クラスタ
PL16880824T PL3385386T6 (pl) 2015-12-31 2016-11-24 Biosyntetyczny klaster genów karymycyny
UAA201808040A UA124058C2 (uk) 2015-12-31 2016-11-24 Кластер генів біосинтезу кариміцину
EP16880824.4A EP3385386B3 (en) 2015-12-31 2016-11-24 Carrimycin biosynthetic gene cluster
RU2018125487A RU2719189C2 (ru) 2015-12-31 2016-11-24 Биосинтетический генный кластер карримицина
DK16880824.4T DK3385386T6 (da) 2015-12-31 2016-11-24 Biosyntetisk carrimycin-gencluster
BR112018013309-4A BR112018013309A2 (zh) 2015-12-31 2016-11-24 Canlomycin biosynthetic gene cluster
US16/067,392 US10858659B2 (en) 2015-12-31 2016-11-24 Biosynthetic gene cluster of carrimycin
KR1020187022082A KR20180093083A (ko) 2015-12-31 2016-11-24 켈리마이신 생합성 유전자 클러스터
PH12018501393A PH12018501393A1 (en) 2015-12-31 2018-06-28 Biosyhthetic gene cluster of carrimycin
ZA2018/04397A ZA201804397B (en) 2015-12-31 2018-06-29 Biosynthetic gene cluster of carrimycin
CONC2018/0007130A CO2018007130A2 (es) 2015-12-31 2018-07-09 Agrupación de genes biosintéticos de carrimicina

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201511028754.9 2015-12-31
CN201511028754.9A CN105505954B (zh) 2015-12-31 2015-12-31 可利霉素生物合成基因簇

Publications (1)

Publication Number Publication Date
WO2017114034A1 true WO2017114034A1 (zh) 2017-07-06

Family

ID=55714247

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/107039 WO2017114034A1 (zh) 2015-12-31 2016-11-24 可利霉素生物合成基因簇

Country Status (20)

Country Link
US (1) US10858659B2 (zh)
EP (1) EP3385386B3 (zh)
JP (1) JP6882330B2 (zh)
KR (1) KR20180093083A (zh)
CN (2) CN107868789B (zh)
AU (1) AU2016383270A1 (zh)
BR (1) BR112018013309A2 (zh)
CA (1) CA3009706A1 (zh)
CL (1) CL2018001796A1 (zh)
CO (1) CO2018007130A2 (zh)
DK (1) DK3385386T6 (zh)
ES (1) ES2812256T7 (zh)
MY (1) MY193028A (zh)
PE (1) PE20190221A1 (zh)
PH (1) PH12018501393A1 (zh)
PL (1) PL3385386T6 (zh)
RU (1) RU2719189C2 (zh)
UA (1) UA124058C2 (zh)
WO (1) WO2017114034A1 (zh)
ZA (1) ZA201804397B (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020125531A1 (zh) * 2018-12-20 2020-06-25 沈阳福洋医药科技有限公司 一种螺旋霉素产生菌、可利霉素产生菌、构建方法、应用及提高产物产量的方法

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106916835B (zh) * 2015-12-24 2022-08-12 武汉合生科技有限公司 化合物的生物合成基因簇及其应用
CN107868789B (zh) 2015-12-31 2020-11-17 沈阳福洋医药科技有限公司 可利霉素生物合成基因簇
AU2018247555B2 (en) 2017-04-06 2024-04-18 Shenyang Fuyang Pharmaceutical Technology Co., Ltd. Use of carrimycin and pharmaceutically acceptable salt thereof for manufacture of medicament for treatment and/or prevention of tumors
CN111197019B (zh) * 2020-01-10 2022-04-15 安徽大学 一种通过糖多孢红霉菌sace_1906基因途径提高红霉素产量的方法
CN114517175B (zh) * 2020-11-20 2024-04-09 上海医药工业研究院 基因工程菌及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1174238A (zh) * 1997-06-03 1998-02-25 中国医学科学院医药生物技术研究所 一种利用基因工程技术制造生技霉素的方法
US7070980B2 (en) 2002-07-19 2006-07-04 Meiji Seika Kaisha, Ltd. Midecamycin biosynthetic genes
CN105505954A (zh) * 2015-12-31 2016-04-20 沈阳同联集团有限公司 可利霉素生物合成基因簇

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5068189A (en) * 1988-05-13 1991-11-26 Eli Lilly And Company Recombinant dna vectors encoding a 4"-o-isovaleryl acylase derived from a carbomycin biosynthetic gene, designated care, for use in streptomyces and other organisms
CA2197160C (en) * 1996-02-22 2007-05-01 Stanley Gene Burgett Platenolide synthase gene
US7109019B2 (en) * 1999-01-06 2006-09-19 The Regents Of The University Of California Gene cluster for production of the enediyne antitumor antibiotic C-1027
CA2394849A1 (en) 2002-07-24 2004-01-24 Ian Kerr Improved prefabricated door frame and door
CN1169947C (zh) 2002-11-19 2004-10-06 中国医学科学院医药生物技术研究所 必特螺旋霉素的基因工程菌株螺旋霉素链霉菌wsj-195
CN1237976C (zh) 2003-12-23 2006-01-25 沈阳同联集团有限公司 必特螺旋霉素及其在抗感染性疾病中的应用
CN101054553A (zh) * 2007-04-09 2007-10-17 中国医学科学院医药生物技术研究所 异戊酰螺旋霉素i基因工程菌株的构建
CN101649325B (zh) * 2009-07-03 2011-09-07 中国医学科学院医药生物技术研究所 一种提高基因工程异戊酰螺旋霉素主组分含量的基因串连技术
CN101914482B (zh) * 2010-07-23 2012-07-04 中国医学科学院医药生物技术研究所 一株异戊酰螺旋霉素i组分高含量、高产量基因工程菌
CN102115757B (zh) * 2010-12-14 2015-10-28 中国科学院南海海洋研究所 台勾霉素的生物合成基因簇及其应用
CN103820474A (zh) * 2012-11-16 2014-05-28 中国医药集团总公司四川抗菌素工业研究所 一种多烯多醇大环内脂类化合物的生物合成基因簇

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1174238A (zh) * 1997-06-03 1998-02-25 中国医学科学院医药生物技术研究所 一种利用基因工程技术制造生技霉素的方法
US7070980B2 (en) 2002-07-19 2006-07-04 Meiji Seika Kaisha, Ltd. Midecamycin biosynthetic genes
CN105505954A (zh) * 2015-12-31 2016-04-20 沈阳同联集团有限公司 可利霉素生物合成基因簇

Non-Patent Citations (26)

* Cited by examiner, † Cited by third party
Title
BIERMAN M. ET AL., GENE, vol. 116, no. 1, 1992, pages 43 - 9
CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 15, no. 2, 1999, pages 171 - 176
CUNDIFFE E. ET AL., ANTONIE VAN LEEUWENHOEK, vol. 79, no. 3-4, 2001, pages 229 - 234
DAI, JIANLU ET AL., JOURNAL OF MICROBIOLOGY, vol. 39, no. 4, 2012, pages 503 - 514
GOODMAN CD ET AL., ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 57, no. 2, 2013, pages 907 - 913
HAYDOCK ET AL., MICROBIOLOGY, vol. 151, 2005, pages 3161 - 3169
IKEDA H. ET AL., NAT.BIOTECHNOL., vol. 21, no. 5, 2003, pages 526 - 531
KALZ L. ET AL., MED RES REV., vol. 19, no. 6, 1999, pages 543 - 58
KARRAY F., MICROBIOLOGY, vol. 153, 2007, pages 4111 - 4122
LIN, FUTIAN ET AL., EIGHTH NATIONWIDE ANTIBIOTIC ACADEMIC CONFERENCE PAPER COMPILATION, 1997, pages 167
MA, CHUNYAN ET AL.: "Construction of 4''-Isovalerylspiramycin-I-Producing Strain by In-Frame Partial Deletion of 3-0-Acyltransferase Gene in Streptomyces spiramyceticus WSJ-1, the Bitespiramycin Producer", CURRENT MICROBIOLOGY, vol. 62, 31 January 2011 (2011-01-31), pages 16 - 20, XP019869333, ISSN: 0343-8651 *
NOVAKOVA R ET AL., FOLIA MICROBIOL., vol. 56, no. 3, 2011, pages 276 - 82
OLIYNYK M. ET AL., NAT. BIOTECHNOL., vol. 25, no. 4, 2007, pages 447 - 453
SHANG, GUANGDONG ET AL.: "Construction of a Stable Bioengineered Strain of Biotechmycin", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 15, no. 2, 23 April 1999 (1999-04-23), pages 171 - 175, ISSN: 1000-3061 *
SHI, XIANGGUO ET AL., ACTAPHARMACOLOGICASINICA, vol. 25, 2004, pages 1396
SHI, XIANGGUO ET AL., ASIAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS, vol. 3, no. 2, 2003, pages 134
SHI, XIANGGUO ET AL., CHINESE CHEMICAL LETTER, vol. 15, 2004, pages 431
STRATIGOPOULOS G ET AL., MOLMICROBIOL, vol. 54, no. 5, 2004, pages 1326 - 34
SUN, LIWEN ET AL., CHINESE PHARMACOLOGICAL BULLETIN, vol. 16, no. 6, 2000, pages 694 - 8
SUN, TAO ET AL., CHINESE JOURNAL OF ANTIBIOTICS, vol. 26, no. 1, 2001, pages 49 - 51
WANG W ET AL., PROCNATLACADSCI USA, vol. 111, no. 15, 2014, pages 5688 - 93
WARDS L. ET AL., ANTIMICROB. AGENTS & CHEMOTHERAPY, vol. 48, no. 12, 2004, pages 4703 - 4712
WILKINSON B. ET AL., CHEM BIOL., vol. 7, no. 2, 2000, pages 111 - 117
YU, LANXIANG ET AL., SICHUAN JOURNAL OF PHYSIOLOGICAL SCIENCES, vol. 20, no. 3, 1998
ZHAO, CHUNYAN ET AL., CHINESE JOURNAL OF ANTIBIOTICS, vol. 23, no. 4, 1998, pages 306
ZHONG, DAFANG ET AL., J CHROMATOGRAPHY B., vol. 791, 2003, pages 45

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020125531A1 (zh) * 2018-12-20 2020-06-25 沈阳福洋医药科技有限公司 一种螺旋霉素产生菌、可利霉素产生菌、构建方法、应用及提高产物产量的方法

Also Published As

Publication number Publication date
PE20190221A1 (es) 2019-02-13
PL3385386T3 (pl) 2020-11-30
BR112018013309A2 (zh) 2018-12-11
EP3385386A1 (en) 2018-10-10
AU2016383270A1 (en) 2018-07-26
US20190093112A1 (en) 2019-03-28
JP2019500903A (ja) 2019-01-17
KR20180093083A (ko) 2018-08-20
EP3385386B3 (en) 2020-11-18
RU2018125487A (ru) 2020-01-31
DK3385386T3 (da) 2020-08-31
CO2018007130A2 (es) 2018-07-19
CN105505954A (zh) 2016-04-20
RU2719189C2 (ru) 2020-04-17
CN107868789B (zh) 2020-11-17
ES2812256T3 (es) 2021-03-16
ZA201804397B (en) 2019-09-25
EP3385386B1 (en) 2020-05-27
MY193028A (en) 2022-09-23
CN107868789A (zh) 2018-04-03
US10858659B2 (en) 2020-12-08
PL3385386T6 (pl) 2021-04-19
ES2812256T7 (es) 2021-09-22
UA124058C2 (uk) 2021-07-14
JP6882330B2 (ja) 2021-06-02
PH12018501393A1 (en) 2019-02-27
CL2018001796A1 (es) 2018-09-28
EP3385386A4 (en) 2018-10-10
DK3385386T6 (da) 2021-02-08
RU2018125487A3 (zh) 2020-02-14
CN105505954B (zh) 2019-01-22
CA3009706A1 (en) 2017-07-06

Similar Documents

Publication Publication Date Title
WO2017114034A1 (zh) 可利霉素生物合成基因簇
Marcone et al. Old and new glycopeptide antibiotics: From product to gene and back in the post-genomic era
August et al. Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699
Mendes et al. The two-component phoR-phoP system of Streptomyces natalensis: inactivation or deletion of phoP reduces the negative phosphate regulation of pimaricin biosynthesis
US10047363B2 (en) NRPS-PKS gene cluster and its manipulation and utility
CN102224242B (zh) 新基因簇
CN103131716A (zh) 盐霉素的生物合成基因簇及其应用
RU2394906C2 (ru) Генетически модифицированный микроорганизм и способ получения макролидного соединения с гидроксильной группой в 16-положении с использованием таких микроорганизмов
CN106754608A (zh) 生产米尔贝霉素的重组链霉菌及其制备方法和应用
CN108753674A (zh) 一种调控米尔贝霉素合成的基因簇、重组链霉菌及其制备方法和应用
JP7086984B2 (ja) Ｓｔｒｅｐｔｏｍｙｃｅｓ ｆｕｎｇｉｃｉｄｉｃｕｓの遺伝子組換え株におけるエンデュラシジンの産生を増強するための組成物及び方法
CN101892186B (zh) 产表柔红霉素的天蓝淡红链霉菌的基因工程菌及其制备方法
CN106754986A (zh) 创新霉素生物合成基因簇及其应用
CN102373171A (zh) 一种核苷类抗生素a201a高产菌株及其构建方法
US8207321B2 (en) Method of obtaining idolocarbazoles using biosynthetic rebeccamycin genes
KR101349436B1 (ko) 해양 미생물 하헬라 제주엔시스의 제주엔올라이드 생합성 유전자 클러스터
KR101327798B1 (ko) 아글루코반코마이신을 생산하는 미생물
CA2354030A1 (en) Micromonospora echinospora genes encoding for biosynthesis of calicheamicin and self-resistance thereto
Pageni et al. Characterization of a chalcosyltransferase (gerGTII) in dihydrochalcomycin biosynthesis
EP2247740B1 (en) Genes encoding the biosynthetic pathway for etnangien production
CN107881138A (zh) 中断缺失的高产安丝菌素菌株及其制备方法
JP2004173537A (ja) カナマイシン生合成遺伝子
Arakawa Characterization of the Biosynthetic Gene Clusters Located on the Streptomyces Linear Plasmid
Pelzer et al. Procaryotic and eucaryotic cells in biotech production
JP2004534502A (ja) 糖部分を変更するための方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16880824

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3009706

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 12018501393

Country of ref document: PH

Ref document number: 001213-2018

Country of ref document: PE

Ref document number: MX/A/2018/008080

Country of ref document: MX

ENP Entry into the national phase

Ref document number: 2018553280

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2016880824

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112018013309

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 2016880824

Country of ref document: EP

Effective date: 20180702

ENP Entry into the national phase

Ref document number: 2016383270

Country of ref document: AU

Date of ref document: 20161124

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20187022082

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2018125487

Country of ref document: RU

ENP Entry into the national phase

Ref document number: 112018013309

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20180628