A mutant strain of Streptomyces flavum, its construction method and its use

A technology of Streptomyces flavum and mutant strains, which is applied in the field of microbial genetic resources and genetic engineering, and can solve the problems of complicated process and high cost of separation and purification of SFA

Active Publication Date: 2011-11-30
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Since S.flaveolus produces a large number of other impurity compounds while producing SFA, in the actual

Method used

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  • A mutant strain of Streptomyces flavum, its construction method and its use
  • A mutant strain of Streptomyces flavum, its construction method and its use
  • A mutant strain of Streptomyces flavum, its construction method and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Example 1: Cloning of a large number of actively transcribed polyketide synthase genes under fermentation conditions.

[0130] Inoculate 500 microliters of S.flaveolus spores into 50 milliliters of sanglifellin-fermented seed culture medium, and cultivate them for 24 hours at 27 degrees and 250 rpm; transfer 3 milliliters of the culture medium to 50 milliliters of the main fermentation medium And cultivated for 4 days under the conditions of 25 degrees and 250 rotations. Centrifuge the fermentation broth at 4 degrees and 5000 rpm, pour off the supernatant, and gently scrape a little bacteria (about 100 microliters) attached to the surface of the sediment with a steel spoon. After quick freezing and grinding in liquid nitrogen, RNA was extracted with Trizol (purchased from Shanghai Yingjun Company) (see Practical Streptomyces Genetics for the operation steps), and finally dissolved in 100 microliters of water. Take out 43 microliters of RNA, add 5 microliters of 10×DNas...

Embodiment example 4

[0142] Example 4: Interruption of PKS and NRPS genes can completely block the biosynthesis of aminosatriene, confirming that the gene cluster is the biosynthetic gene cluster of aminosatriene

[0143] The plasmid containing five sets of PKS genes and one set of NRPS gene fragments was digested with EcoRI and HindIII, and then cloned into the EcoRI-HindIII site of pKC1139. These plasmids were transformed into sangliferin-producing bacteria through the method of indirect conjugative transfer of E. coli-Streptomyces developed by the present inventors (Mol. BioSyst. 2011, 7, 852-861). The resulting zygote was streaked on the ISP-4 medium containing 50 micrograms / ml of apramycin (Apramycin) and cultured at 37 degrees to induce gene disruption, to obtain the gene disruption mutant strain TL3016 (interrupted mycD1 gene ), TL3017 (disrupted mycD2 gene), TL3018 (disrupted mycD3 gene), TL3019 (disrupted mycD4 gene), TL3020 (disrupted mycD5 gene), TL3021 (disrupted mycC gene). genotype ...

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Abstract

The invention discloses a mutant strain of Streptomyces flaveolus, a construction method and application thereof. The mutant strain is a mutant strain obtained by gene substitution or a mutant strain obtained by gene disruption. The collection number of the mutant strain obtained by gene substitution is CGMCC 4948, and the construction of the mutant strain is realized by knocking out a part of Ansatrienin biosynthetic genes of the Streptomyces flaveolus. The mutant strain can be used for fermentation production of Sanglifehrin A, Ansatrienin impurities are avoided, and the Sanglifehrin A can be separated and purified conveniently.

Description

technical field [0001] The invention belongs to the field of microbial genetic resources and genetic engineering, and specifically relates to a mutant strain of Streptomyces flavum, a construction method and its application, and a light yellow strain that does not produce aminosatriene impurities is obtained by means of gene knockout. Streptomyces mutant strain CGMCC 4948 and its application to the fermentation of sangliferin. technical background [0002] Sanglifehrin A (Sanglifehrin A, abbreviated as SFA, with the following structural formula, is a polyketide-polypeptide hybrid natural product produced by Streptomyces flavolus DSM 9954 (J. Antibiot. (Tokyo) 1999, 52, 466 -473). Among the more than 20 SFA structural analogues (J.Am.Chem.Soc.2002, 124, 4257-4270) that have been isolated, SFA has the highest immunosuppressive activity. In addition to having a strong immunosuppressive In addition to activity (J.Immunol.2001, 166, 7165-7171; J.Immunol.2003, 171, 542-546), SFA ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/76C12P17/18C12R1/465
Inventor 刘文瞿旭东雷春
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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