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Method for improving fermentation level of salinomycin by increasing supply of precursors

A technology of fermentation level and salinomycin, which is applied in the field of bioengineering, can solve the problems of affecting polyketide chain extension, unclear antibiotic fermentation level, and affecting the final yield of polyketides, so as to reduce fermentation costs and improve salinomycin Effect of fermentation and improvement of fermentation yield

Active Publication Date: 2015-02-18
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

[0004] Through literature research, it is found that sufficient supply of precursors will affect the extension of polyketide chains, thereby affecting the final yield of polyketides
In Streptomyces coelicolor, doubling the acetyl-CoA carboxylase gene can increase actin rhodopsin production by 6 times; 06 yield increased by 2 times; however, for Streptomyces albicans DSMZ41398, it is not clear whether the bacteria can produce more precursors by doubling the rate-limiting genes in the precursor synthesis pathway, thereby increasing the fermentation level of antibiotics

Method used

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  • Method for improving fermentation level of salinomycin by increasing supply of precursors
  • Method for improving fermentation level of salinomycin by increasing supply of precursors
  • Method for improving fermentation level of salinomycin by increasing supply of precursors

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Embodiment

[0028] Step 1: Construction of plasmids pLQ59, pLQ60 and pLQ61

[0029] Using Streptomyces coelicolor genomic DNA as a template, two subunits accA2 (1802bp) and accBE (1839bp) of acetyl-CoA carboxylase were amplified by PCR using two sets of primers accA2-F / R and accBE-F / R respectively ), the correctness of the sequence was confirmed by gene sequencing. AccA2 (NdeI / HindIII) and accBE (HindIII / EcoRI) were inserted into the NdeI / EcoRI site of plasmid pIB139, and the plasmid pLQ59 for acetyl-CoA carboxylase doubling was obtained by the above method. Under the condition of water bath at 37 degrees Celsius, two target bands of 1802bp and 1839bp can be observed by using NdeI, HindIII and EcoRI three restriction endonucleases for enzyme digestion, indicating that the plasmid is constructed correctly.

[0030]Using Streptomyces albicans DSMZ41398 genomic DNA as a template, the crotonyl-CoA reductase gene ccr (1338bp) was amplified by PCR using primers ccr-F / R, and the correctness of ...

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Abstract

The invention discloses a method for improving the fermentation level of salinomycin by increasing supply of precursors. The method comprises the following steps: doubling acetyl coA carboxylase genes from streptomyces coelicolor, methyl malonyl coA mutase genes from streptomyces albus DSMZ41398 and crotonoyl coA reductase genes from the streptomyces albus DSMZ41398 on the chromosomes of the streptomyces albus DSMZ41398 through an integrating vector pSET152 respectively to increase supply of three precursors required for synthesizing salinomycin so as to increase the salinomycin yield. According to the method, the final fermentation yield of engineering strain salinomycin is increased by 260% and the shake flask level of a lab reaches 2 g / L.

Description

technical field [0001] The invention relates to bioengineering technology, in particular to a method for increasing the supply of precursors to improve the fermentation level of salinomycin. technical background [0002] Actinomycetes are a class of Gram-positive bacteria with high GC content, and more than 60% of known antibiotics are synthesized by actinomycetes. Streptomyces is a class of higher actinomycetes, which has strong antibiotic synthesis ability and complex morphological differentiation, so it has always been concerned by people. Polyketides are a class of important secondary metabolites produced by actinomycetes, which have extremely important applications in the fields of anti-tumor, anti-parasite, and antibiotics. Previous experiments have proved that the biosynthesis of polyketides mainly depends on type I, type II and type III polyketide biosynthesis gene clusters, and the main precursors used are acetyl-CoA, malonyl-CoA, methyl Malonyl-CoA, ethylmalonyl-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/18C12N1/21C12R1/47
Inventor 白林泉芦晨阳蒋明康前进
Owner SHANGHAI JIAO TONG UNIV
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