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Bitespiramycin biosynthetic gene cluster

A technology of biosynthesis and carrimycin, applied in the field of microbial genetic resources and genetic engineering, can solve the problem of no complete cross-resistance

Active Publication Date: 2016-04-20
SHANGHAI TONGLIAN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has no complete cross-resistance with similar drugs

Method used

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  • Bitespiramycin biosynthetic gene cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] 《Example 1》Crimycin producing bacteria (S.spiramyceticus) total DNA extraction

[0115] R 2 YE medium formula (g / 100ml):

[0116]

[0117] Add 0.2ml of trace element solution, dilute to 100ml distilled water, pH6.5

[0118] Trace element solution (g / 100ml):

[0119]

[0120] 15 pounds of sterilization, 121 ℃, 20min

[0121] S.spiramyceticus strains were inoculated in 25mlR 2 YE medium, cultivated on a shaker at 28°C for 48 hours, and transferred to 100ml R 2 YE culture medium, cultured on a shaker at 28°C for 24h, centrifuged at 5000rpm for 10-15min, collected bacteria (about 10g of bacteria), and mainly operated according to the product instructions of UPTECHTM lifescience company. Add 50ml of 25mM EDTA solution to shake and wash, centrifuge, discard supernatant; use 25ml lysozyme solution (10mg / ml, prepare with Tris-HCl, 2mM EDTA, 1.2% TritonX-100 of 10mM pH8.0, add 0.5ml100mg / ml mlRNase) suspended mycelium, cultured at 37°C for about 1-2h, and cultured ce...

Embodiment 2

[0122] 《Example 2》Verify the function of Seq.1 gene information by gene blocking

[0123] IA-W1 and IA-W42 as well as IA-W4, 17, 21, 23 and 27 genes at both ends of the gene cluster were selected for blocking to obtain mutant strains, and experiments proved that the ability of these blocking strains to produce corimycin occurred changes, or no longer produces corimycin. Thus it is suggested that the obtained gene cluster information is necessary for the production of carrimycin. Primers were designed according to the above coding genes and their upstream and downstream sequences, and inserted into appropriate restriction sites. The primer sequences are shown in Table 2.

[0124] Table 2 Primer sequences designed and used in gene blocking experiments

[0125]

[0126]

[0127] The corresponding homologous gene fragments were respectively obtained by PCR amplification, and the corresponding restriction sites were used, and the selection marker resistance gene (Apramycin-...

Embodiment 3

[0131] "Example 3" Gene transfer of corimycin-producing bacteria and screening of blocking strains

[0132] 3.1 Preparation of protoplasts: inoculate the slant spores of fresh carrimycin-producing bacteria in R 2 In YE liquid medium, 28°C, 220rpm shaking flask cultured for 48h, the culture solution was transferred to fresh R with 0.5% glycine supplemented with 10% transfer amount. 2 In YE liquid medium, shake culture at 28°C for 20h. Take 10ml of bacterial liquid in a centrifuge tube, centrifuge at 3000rpm to collect mycelia, and use P-buffer for precipitation

[0133]

[0134] Sterilize at pH7.615 lbs, 121°C, 30min.

[0135] After washing twice, suspend with an appropriate amount of P-buffer, add the P-buffer solution of lysozyme (final concentration is 2mg / ml), mix well, keep warm in a 37°C water bath for 30-45min, and shake once every 10-15min. Use a 10×40 phase-contrast microscope to observe the formation of protoplasts. When the microscopic examination shows that mo...

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Abstract

The invention provides a bitespiramycin biosynthetic gene cluster. The bitespiramycin biosynthetic gene cluster totally has 44 gene open reading frames, a total length of a nucleotide sequence is 89315 bp, and the bitespiramycin biosynthetic gene cluster contains 5 encoded polyketide synthases and comprises 8 modules, 37 structural domains, 9 orf related to the polyketone synthesis extension unit and modification, 16 orf related to glycosyl synthesis and 6 orf related to glycosyl transfer. By virtue of the analysis on the sequence information and structure of the gene cluster, the genetic manipulation can be further performed on a producing strain to obtain a novel and more effective antibiotic, for example, a novel macrolides antibiotic can be created by adopting the genetic manipulation to change the PKS synthetic modular structure, performing the alteration of lactonic ring post-translational modification and replacing or modifying the glycosyl, and the yield of the antibiotic can be increased by virtue of the genetic manipulation on a resistance gene or a regulation gene. The amino acid sequence provided by the invention can be used for separating needed proteins and can be used for preparing an antibody.

Description

Technical field: [0001] The invention belongs to the field of microbial gene resources and genetic engineering, in particular to the cloning, analysis, function research and application of genetic engineering antibiotic biosynthesis gene clusters. technical background: [0002] Klimycin, formerly known as Shengjimycin and Bitespiramycin, is a 16-membered ring macrolide antibiotic developed by synthetic biology technology [Patent No.: ZL971044406, ZL021487715], based on the 4”-iso Valeroylspiramycin III, II, and I are the main components of spiramycins acylated at the 4”-position hydroxyl group, of which the III component accounts for more than 30%, the II component about 25%, and the I component No more than 10%. [0003] [0004] Carrimycin structural formula [0005] Isovalerylspiramycin III: R=COCH 2 CH 3 R'=COCH 2 CH(CH 3 ) 2 [0006] Isovalerylspiramycin II: R=COCH 3 R'=COCH 2 CH(CH 3 ) 2 [0007] Isovalerylspiramycin I: R=HR'=COCH 2 CH(CH 3 ) 2 [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52
CPCC12N15/52C12N9/00C12P19/62C12N9/0006C12N9/001C12N9/1029C12N9/16C12N9/86C12Y101/01064C12Y101/01188C12Y103/01008C12Y203/01013C12Y301/02007C12Y305/02003
Inventor 王以光姜洋赵小峰赫卫清戴剑漉
Owner SHANGHAI TONGLIAN PHARMA CO LTD
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