Abstract

The polyene macrolide rimocidin, produced by Streptomyces rimosus M527, was found to be highly effective against a broad range of fungal plant pathogens. Current understanding of the regulatory mechanism of rimocidin biosynthesis and morphological differentiation in S. rimosus M527 is limited. NsdA is considered a negative regulator involved in morphological differentiation and biosynthesis of secondary metabolites in some Streptomyces species. In this study, nsdA_sr was cloned from S. rimosus M527. The role of nsdA_sr in rimocidin biosynthesis and morphological differentiation was investigated by gene deletion, complementation, and over-expression. A ΔnsdA_sr mutant was obtained using CRISPR/Cas9. The mutant produced more rimocidin (46%) and accelerated morphological differentiation than the wild-type strain. Over-expression of nsdA_sr led to a decrease in rimocidin production and impairment of morphological differentiation. Quantitative RT-PCR analysis revealed that transcription of rim genes responsible for rimocidin biosynthesis was upregulated in the ΔnsdA_sr mutant but downregulated in the nsdA_sr over-expression strain. Similar effects have been described for Streptomyces coelicolor M145 and the industrial toyocamycin-producing strain Streptomyces diastatochromogenes 1628.

Key points

• A negative regulator for sporulation and rimocidin production was identified.

• The CRISPR/Cas9 system was used for gene deletion in S. rimosus M527.

中文翻译：

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鉴定来自链霉菌M527的基因，该基因对rimocidin的生物合成和形态分化产生负面影响

摘要

发现由链霉菌M527生产的多烯大环内酯rimocidin对多种真菌植物病原体具有很高的功效。rimocidin在生物合成和形态分化的调节机制的当前理解S. rimosus M527是有限的。NsdA被认为是负调节剂，参与某些链霉菌属物种的次级代谢物的形态分化和生物合成。在这项研究中，nsdA _sr克隆自rimosus M527。的作用nsdA基因_SR在rimocidin生物合成和形态分化通过基因缺失，互补，和过表达的影响。ΔnsdA使用CRISPR / Cas9获得_sr突变体。该突变体比野生型菌株产生更多的rimocidin（46％）并加速了形态分化。nsdA _sr的过表达导致rimocidin的产生减少和形态分化受损。定量RT-PCR分析表明的转录轮缘负责rimocidin生物合成基因在ΔnsdA上调_SR突变体，但在下调nsdA基因_SR过表达菌株。对于腔链链霉菌M145和生产Toytocamycin的工业链霉菌产色链霉菌1628，已经描述了类似的效果。

关键点

•确定了孢子形成和rimocidin产生的负调节剂。

• CRISPR / Cas9系统用于S. rimosus M527中的基因缺失。