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6-methy salicylic acid synthetase transformed by genetic engineering and combinatorial biosynthesis of spirocyclic acetoacetic acid lactone antiboitic

A technology of spirocyclic acetoacetic acid and antibiotics, applied in the field of biotechnology engineering, can solve problems such as limited production prospects

Active Publication Date: 2012-08-29
国科新研国际技术转移有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its complex chemical structure has brought great challenges to chemical synthesis and structure derivation. Due to the excessive chiral centers and concentrated functional groups, chemical synthesis has very limited prospects for the production of CHL and its analogues. An important supplement, the development of combinatorial biosynthesis technology provides a new method for the acquisition of complex natural products and their analogs

Method used

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  • 6-methy salicylic acid synthetase transformed by genetic engineering and combinatorial biosynthesis of spirocyclic acetoacetic acid lactone antiboitic
  • 6-methy salicylic acid synthetase transformed by genetic engineering and combinatorial biosynthesis of spirocyclic acetoacetic acid lactone antiboitic
  • 6-methy salicylic acid synthetase transformed by genetic engineering and combinatorial biosynthesis of spirocyclic acetoacetic acid lactone antiboitic

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Point mutation and heterologous expression of DH and KR domains of ChlB1

[0044] 1. Acquisition of ChlB1 with point mutations in DH and KR domains

[0045] 1) Mutation of the DH domain

[0046] Inactivate the DH domain of ChlB1, choose to mutate its 947 histidine residue, and mutate it into glycine or phenylalanine. First, take two pairs of designed primers, and the primer sequences are as follows:

[0047] 947 position H is mutated to A

[0048] Upstream: 5’-C TAC CCC GGC AGC GCC ACC ATC AAC GGC ACG-3’

[0049] Downstream: 5’-CGT GCC GTT GAT GGT GGC GCT GCC GGG GTA G-3’

[0050] 947 position H is mutated to F

[0051] Upstream: 5’-C TAC CCC GGC AGC TTC ACC ATC AAC GGC ACG-3’

[0052] Downstream: 5’-CGT GCC GTT GAT GGT GAA GCT GCC GGG GTA G-3’

[0053] Using pAL1084 (a fragment of the wild-type DH domain cloned into pSP72) as a template template, dNTP, DMSO, enzyme-free water, high-fidelity primestar enzyme and its buffer constitute a PCR reaction syst...

Embodiment 2

[0087] Example 2, KR mutated ChlB1 complementary ChlB1 mutants produce new analogs of CHL

[0088] 1. Intergeneric conjunctive transfer between E.coli ET12567 and Streptomyces antibioticus DSM40725(ΔchlB1)

[0089] Pick a single colony from the transformed E.coli ET12567 culture plate and transfer it to a test tube for overnight culture, pipette 0.5ml of the bacterial solution into 25ml LB, and culture it in a shaker at 37°C until OD 600 It is 0.3-0.4, or 0.4-0.6. The bacteria were collected by centrifugation, washed twice with an equal volume of LB medium, and the bacteria were collected by centrifugation and suspended in 1ml of LB medium. as a DNA donor.

[0090] Take out the spore suspension (3 × 10 9 cells / mL), centrifuge at 8,000rpm for 3 minutes to remove the supernatant, then wash twice with 0.5ml TES buffer (0.05M, pH 8.0), resuspend in 500μl TES buffer (0.05M, pH 8.0), heat shock at 50°C for 10 minutes To stimulate spore germination, then add 500μl TSB, mix well, ...

Embodiment 3

[0096] Example 3, fermentation isolation and purification of new compounds of mutant strains, structural identification and determination of biological activity

[0097] When the mutant strain was fermented in a small amount, two possible new compounds 7 and 8 were detected by HPLC and LC-MS. In order to identify the structures of these two compounds, we carried out a large amount of fermentation on the mutant strain. See Example 2 for the fermentation method. After the culture medium is freeze-dried, mash it and add an equal volume of anhydrous methanol, sonicate for 10 minutes (ultrasound for 10 seconds, interval of 50 seconds), stir with a stirrer for 1 hour, filter (or centrifuge) to collect methanol, and rotate to remove methanol , dispersed with 1 / 5 volume of water, adjusted to pH 2.0-3.0, extracted three times with an equal volume of ethyl acetate, and dried under reduced pressure to obtain a dark brown paste. The first step of the paste is coarse separation, mix the pa...

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Abstract

The invention discloses a novel method for synthesizing spirocyclic acetoacetic acid lactone antibiotic with higher activity by using combinatorial biosynthesis based on a genetic engineering method. The method is characterized by carrying out specific locus mutation on the keto-reduction (KR) conservation activity locus of a 6-methy salicylic acid (6-MSA) synthetase and genetically transforming the producing strain Streptomyces antibioticus DSM40725 of chlorothrichin (CHL) and fermenting to generate new chlorothrichin analogues 7 and 8 with improved biological activity. The favorable biological activity of the new chlorothrichin analogues 7 and 8 has values to be developed into medicaments.

Description

technical field [0001] The invention belongs to the field of biotechnological engineering, and specifically relates to the function of genetic engineering modification of 6-MSA synthetase, the fermentation, separation, structure identification and activity determination of new compound biosynthesis in recombinant bacterial strain combination. Background technique [0002] Natural compounds are a major source of drug discovery and development, such as penicillin and erythromycin for anti-infection, bleomycin and etomycin for anti-tumor, avermectin for anti-parasite and immunosuppressant cyclosporine etc., have been the main drugs for human treatment of diseases in the past few decades. Therefore, the research and development of new drugs based on natural compounds has always been a key area of ​​concern in the chemical and medical circles. Among them, the natural products derived from microorganisms account for an important part. There are thousands of microbial natural prod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H17/08C12N9/00C12N15/52C12N15/76C12P19/62A61K31/7048A61P31/04A61P35/00
Inventor 刘文丁伟
Owner 国科新研国际技术转移有限公司
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