龟裂霉素高效合成的分子机制解析及其理性设计改造

Rimocidin is a member of the polyketide antibiotic family. Recently the rimocidin was found to be highly efficient against a broad range of plant pathogenic fungi. Therefore, rimocidin has been recognized as an ideal fungicide that can be utilized to control the occurrence of plant diseases in the agriculture field. So far, Streptomyces rimosus M527 as only a rimocidin producer has the important research value, however, the rimocidin production is limited in this strain, it can’t meet the requirement of industrial production and limited its application. Therefore, analysis of crucial metabolic nodes and regulatory mechanism of efficient rimocidin biosynthesis in S. rimosus M527 is of great significance. Recently, the mutants M527-G6 and M527-G11 which showed the highest (2.7 times higher) and lowest (4.5 times lower) rimocidin production comparing to original strain M527, respectively, were isolated by using ribosome engineering technology. In this study, in order to explore the key factors involved in metabolism and regulatory of rimocidin overproduction, the combined transcriptomic (RNA-Seq) and proteomic (iTRAQ) strategy was employed to analyze and identify the distinct differences of gene expression among the mutants and wild-type strain, and to reveal the relationship between identified genes and rimocidin high-production. Subsequently, the strain was engineered and construction of higher-producing rimodicin strain was carried out by rational design. It shall be beneficial for the industrial production of rimocidin.

聚酮类抗生素龟裂霉素(Rimocidin)可有效防治多种植物真菌病害，作为目前唯一生物合成龟裂霉素的菌株S. rimosus M527具有重要的研究价值，但其产量较低，限制了其广泛应用。因此，从分子水平解析龟裂霉素高效合成的代谢节点与调控机制具有重要的研究意义。近期，利用核糖体工程技术筛选获得了突变株M527-G6和M527-G11，其龟裂霉素产量较原始菌株分别提高和降低了2.7倍和4.5倍。本研究以原始菌株S. rimosus M527，突变株M527-G6和M527-G11为研究对象，联合利用转录组学RNA-seq技术和蛋白质组学iTRAQ技术，筛选相关的显著差异表达基因，并验证相关表达基因与龟裂霉素高效合成的相关性，从而阐明全局代谢调控网络中影响龟裂霉素高效合成的关键节点；在此基础上，理性设计改造显著提高菌株龟裂霉素的合成水平，构建工程菌株，为实现龟裂霉素的生物创制奠定基础。

聚酮类抗生素龟裂霉素(Rimocidin)可有效防治多种植物真菌病害，作为生物合成龟裂霉素的菌株S. rimosus M527具有重要的研究价值，但其产量较低，限制了其广泛应用。. 首先，在合成龟裂霉素基因簇内发现了至今没有发现的一个调控基因rimR2。经比对，发现其与常见的LuxR家族调节子具有较高的同源性，为了验证该基因的功能，通过敲除、回补以及过量表达等实验显示，rimR2是一个参与龟裂霉素合成的正向调节子。通过荧光定量PCR和EMSA显示，rimR2调控簇内rim基因的转录而影响龟裂霉素的合成。. 利用核糖体工程技术筛选一株双抗（庆大霉素和利福平）Genr-Rifr突变株M527-GR7，其5L发酵罐水平上的龟裂霉素产量达到673.1 mg/L，较原始菌株M527提高了62.89%。以原始菌株S. rimosus M527以及突变株M527-GR7为研究对象，利用比较蛋白质组学(iTRAQ)技术开展高产龟裂霉素分子机理与代谢工程改造方面的研究。鉴定得到差异表达蛋白NsdAsr。克隆了1476 bp的基因nsdAsr（GenBank accession No. MN395042），通过敲除、回补以及过量表达等实验显示，nsdAsr是一个参与调控龟裂霉素合成和菌株形态分化的负调控因子。. 另考察了不同生物诱导子对S. rimosus M527合成龟裂霉素的影响，结果显示：2%（v/v）的酿酒酵母滤液和2%（v/v）的黄瓜枯萎病原菌活细胞，对提高龟裂霉素产量的效果最显著。