Herbicidin biosynthetic gene cluster and application thereof

A gene and analog technology, applied in the field of Herbicidin biosynthesis gene cluster, can solve the problem that the biosynthesis gene cluster has not been reported.

Active Publication Date: 2019-08-23
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The structure of Herbicidin F has been determined [6] , but before the start of this work, its biosynthetic gene cluster has not been reported

Method used

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  • Herbicidin biosynthetic gene cluster and application thereof
  • Herbicidin biosynthetic gene cluster and application thereof
  • Herbicidin biosynthetic gene cluster and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1. Bioinformatics analysis and identification of herbicidin F biosynthetic gene cluster (hcd) in S. mobaraensis US-43

[0113] S. mobaraensis US-43 is a relatively mature strain (CPCC 204095) studied in our laboratory. Over the years, research on the metabolites of this strain has found that it can produce a large number of other types of compounds in addition to glycopeptide antibiotics. In order to systematically explore other types of metabolites, we sequenced the whole genome of S. mobaraensis US-43, and predicted that its genome contains at least 47 secondary metabolite biosynthesis gene clusters by using antiSMASH 4.0.0 software [20] . Through LC-MS analysis of the fermentation broth of the strain, compound 1 was detected as a trace component, and compound 1 was presumed to be herbicidin F by UV and MS / MS data analysis.

[0114] Although the structure of Herbicidin F has been determined, its biosynthetic pathway has not been reported yet. Compound aureon...

Embodiment 2

[0118] Example 2, Confirmation of pathway-specific regulatory proteins in hcd

[0119] The predicted hcd gene cluster contains three transcriptional regulatory genes hcdR1, hcdR2, and hcdR3. We constructed overexpressed strains of each transcriptional regulatory gene, and detected the changes in the yield of herbicidin F in the fermentation broth of the overexpressed strains by HPLC to judge the prediction Whether the gene cluster hcd is responsible for herbicidin F biosynthesis, and further determine whether hcdR1, hcdR2 and hcdR3 are pathway-specific regulatory genes in herbicidin F biosynthesis. We cloned each transcriptional regulator gene into pSET152 [21] The derived expression plasmid vector pL646 [22] Above, it contains the φC31attP site, which can be integrated into the attB site in the Streptomyces genome, and contains a strong promoter ermE*p and the SD sequence of the tuf1 gene upstream of the multiple cloning site. The constructed overexpression plasmids pL-hcdR...

Embodiment 3

[0125] Example 3, HcdR2 is a relatively conserved pathway-specific regulatory protein in herbicidin biosynthesis

[0126] The pathway-specific regulatory protein HcdR2 belongs to the LuxR family. According to BLASTp analysis, there is a typical helix-turn-helix (HTH) DNA-binding domain (DBD) at the C-terminus of the regulatory protein. We searched for similar sequences in GenBank with hcdB / C / D / E as the target, and found that at least 15 actinomycetes contained hcd / hbc / her / anm similar gene clusters ( image 3 ), and 11 gene clusters contained transcriptional regulatory proteins belonging to the LuxR protein family. Among these LuxR family regulatory proteins, except for the LuxR protein in the gene cluster of strain S. mobaraensis NBRC 13819 (comparison analysis with HcdR2, 99.78% amino acid similarity), the rest of the LuxR regulatory proteins were compared with HcdR2, and the comparison results They were shown to have 30-43% amino acid similarity over the full length. HHpre...

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Abstract

The invention belongs to the technical field of medical biology, and particularly relates to a method for improving the yield of fermented herbicidin F and analogues thereof. The method comprises thestep that HcdR2 genes are over-expressed in a host strain for fermentation production of the herbicidin F and the analogues thereof, wherein the host strain is streptomycete and is preferably an S.mobaraensis US-43 strain, HcdR2 is a transcription regulation protein of the LuxR protein family, and the amino acid sequence of the HcdR2 is shown in SEQ ID NO.1. The invention further relates to a group of novel structural analogue compounds of the herbicidin F.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and specifically relates to a herbicidin biosynthetic gene cluster and its application. Background technique [0002] Streptomyces mobaraensis US-43 (formerly named S. verticillus var. pingyangensis n. var) is a strain of Streptomyces isolated from the soil in Pingyang, Zhejiang, China. It can produce a series of glycopeptide antibiotics with the same properties as bleomycin [1-3] , among them, pingyangmycin and boanmycin have been approved by SFDA as anti-tumor drugs. As a laboratory-preserved strain, we studied the secondary metabolites of this strain under different fermentation conditions. Piericidin A1 and a series of isocoumarins compounds have been found in its fermentation broth. In addition, a trace component in the fermentation broth was detected by LC-MS method, which was identified by UV and MS / MS data and presumed to be herbicidin F. [0003] Herbicidins ( figure 1 ) ...

Claims

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Application Information

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IPC IPC(8): C12N15/76C12P17/18C07D519/00A61K31/52A61P31/10A61P35/00
CPCA61P35/00A61P31/10C12P17/188C07K14/4702
Inventor 洪斌解云英侍媛媛谷仁杰李怡宏王新位任卫聪李星星王丽非张丛
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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