Method for efficiently synthesizing mequinomycin A in streptomyces albus

A technology of Streptomyces albicans and moronomycin, which is applied in the directions of microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc. Create job problems, etc.

Active Publication Date: 2021-02-26
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

However, the yield of moenomycin in the actinomycete Streptomyces garnerii ATCC14672 is extremely low, and it is a mixture of multiple compounds, which is extremely unfavorable to the biosynthesis of moenomycin and the creation of new structural clinical drugs, which severely limits sugar phosphate Research progress of lipid compound clinical drug

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  • Method for efficiently synthesizing mequinomycin A in streptomyces albus
  • Method for efficiently synthesizing mequinomycin A in streptomyces albus
  • Method for efficiently synthesizing mequinomycin A in streptomyces albus

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Embodiment

[0058] Construction of Streptomyces albus LX03 High-Producing Strain of Moenomyces A

[0059] Step 1: Construction of the fosmid plasmid pJQK455 containing the moenomycin biosynthesis gene cluster cluster 1; through antiSMASH online analysis, an incomplete moenomycin biosynthesis gene was found in the whole genome of Streptomyces lincoeum (GenBank: CP016438.1) Cluster cluster 1 (GenBank assembly accession: GCA_003344445.1RefSeq assembly accession: GCF_003344445.1). The designed primers (Moe-L-F / Moe-L-R and Moe-R-F / Moe-R-R) were screened by polymerase chain reaction shrinkage genome library method to obtain fosmid plasmids 5B1, 8F3, 9A8 containing moenomycin biosynthesis gene cluster and 16G7 were confirmed to be correct by restriction enzyme digestion with BamHI and KpnⅠ, and 5B1 was selected for sequencing and confirmed to be the correct plasmid, named pJQK455 ( Figure 5 ).

[0060] The primers used in the above step 1 are:

[0061] Primer name base sequence ...

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Abstract

The invention discloses a method for efficiently synthesizing mequinomycin A in streptomyces albus. The invention is based on the method of introducing site-directed mutant A penicillin binding protein gene into the heterologous expression host Streptomyces.albus of the mequinomycin A biosynthesis gene cluster to optimize the heterologous host, and the mequinomycin A acts on peptidoglycan transferase structural domain active sites of bacterial cell walls aPBP to effectively inhibit the synthesis of the cell walls of Gram-positive bacteria. Large-fragment assembly and promoter optimization arecarried out on the mequinomycin biosynthesis gene cluster by means of genetic engineering, and the mequinomycin A resistance gene MaPBP is introduced into the optimized strain II to further improve the mequinomycin resistance of the strain II. Finally, the heterologous expression yield of the mequinomycin A in the strain LX03 is increased by 110% compared with that of the original strain I. According to the invention, not only the genetic engineering modification method of large-segment gene clusters is described, but also a precedent that a Streptomyces. Albus host is optimized by a resistance gene to improve the yield of a target compound is started.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for efficiently synthesizing moenomycin A in Streptomyces albicans, in particular to a method based on heterologous expression of moenomycin A biosynthesis gene cluster and host strain moenomycin A method for optimizing the A resistance gene MaPBP, specifically a heterologous expression host Streptomyces.albus J1074 based on the moenomycin A biosynthetic gene cluster to introduce site-directed mutation type A penicillin-binding proteins (Mutant class Apenicillin-binding proteins, MaPBP) A method for genetically optimizing its heterologous host. Background technique [0002] Moenomycin compounds have a good killing or inhibitory effect on many Gram-positive bacteria by binding to the active site of bacterial cell wall peptidoglycantransferase (PGT). The minimum inhibitory concentration (MIC) of moenomycin is only 1-100ng / mL, and its biological activity is about 10-1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/76C12N15/65C12N15/31C12P19/26C12P19/44C12R1/47
CPCC12N9/00C12N15/52C12N15/76C12N15/65C07K14/36C12P19/26C12P19/44C12N2800/22C12N2800/101
Inventor 康前进李兴白林泉欧一新
Owner SHANGHAI JIAO TONG UNIV
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