PKS/PKS-NRPS基因簇对武夷菌素生物合成的竞争机制研究

Wuyiencin is a kind biological pesticide，which has good control effect for variety fungal disease on vegetables. However, the difficulty of further expand application was the high cost of production of industrialization. Therefore, we have the aim of improving the industrial production level and market competitiveness. This project used the high-throughput technology of third generation for sequencing combined with analyzing of the secondary metabolites bioinformatics，it will determine the secondary metabolites gene clusters type of wuyiencin and clear the competitive pathway of genetic variety PKS/PKS-NRPS type for biosynthesis metabolic, screening important candidate gene clusters between wuyiencin and competition relations clusters. We have used the effectively method of Gibson assembly to build the vector of gene clusters knockout, alliance with functional recovering and active testing methods to identify the function of PKS/PKS-NRPS candidate gene clusters. The target is to determine its competitive role in the wuyiencin biosynthesis to realize the successful implementation metabolic engineering on wuyiencin biosynthesis with a directional high-yield strain of wuyiencin. This project is expected to establishing a method of raising the yield of antibiotics though competitive gene clusters knockout and to breeding with independent intellectual property rights of wuyiencin high-yield strains based on metabolic engineering technology, which will provide theoretical and technical support on improved the level of industrialization and reduced cost of production for wuyiencin.

武夷菌素是一种对蔬菜真菌病害具有良好防治效果的生物农药。但是，生产成本高严重制约其产业化发展。因此，以提高生物农药武夷菌素工业化生产水平和市场竞争力为总体目标，本项目利用三代测序技术结合次级代谢产物信息学分析方法，确定武夷菌素产生菌次级代谢产物基因簇类型，明确生物合成竞争代谢途径PKS/PKS-NRPS基因簇种类，筛选与武夷菌素生物合成存在重要竞争关系的候选基因簇。采用Gibson组装的方法高效构建敲除载体和基因簇快速敲除、功能回补、结合活性物质检测等方法鉴定PKS/PKS-NRPS候选基因簇功能，从而确定其在武夷菌素生物合成中的竞争作用，采用代谢工程技术对武夷菌素生物合成进行定向改造，获得武夷菌素高产菌株。本课题有望通过敲除竞争途径，建立一种以代谢工程技术为基础的提高抗生素产量的育种方法，选育具有自主知识产权的武夷菌素高产新菌株，为提高武夷菌素工业化水平、降低产品成本提供理论和技术支持。

本项目以提高生物农药武夷菌素工业化生产水平和市场竞争力为总体目标，利用三代测序技术结合次级代谢产物信息学分析方法，确定武夷菌素产生菌次级代谢产物基因簇类型，明确生物合成竞争代谢途径PKS/PKS-NRPS基因簇种类，筛选与武夷菌素生物合成存在重要竞争关系的候选基因簇。采用Gibson组装的方法高效构建敲除载体和基因簇快速敲除、功能回补、结合活性物质检测等方法鉴定PKS/PKS-NRPS候选基因簇功能，从而确定其在武夷菌素生物合成中的竞争作用，采用代谢工程技术对武夷菌素生物合成进行定向改造，获得武夷菌素高产菌株。研究表明，不吸水链霉菌武夷变种（Streptomyces ahygroscopicus var. wuyiensis）CK-15基因组中含有39个次级代谢基因簇，包括聚酮（PKS）和非核糖体肽（NRPS）基因簇有12个，核苷类基因簇有2个，丁内脂类有4个及其他类型基因簇21个。构建属于PKS/PKS-NRPS候选基因簇的制霉菌素生物合成基因簇中nysA-nysR各基因的缺失突变株、过表达菌株及回补菌株，发现制霉菌素生物合成基因簇与武夷菌素生物合成基因簇不存在显著的竞争关系。高效构建2个核苷类基因簇丰加霉素基因簇和谷氏菌素基因簇中关键基因的缺失突变株、过表达菌株及回补菌株，通过功能验证发现丰加霉素基因簇中toyF和toyG能够影响武夷菌素的生物合成。toyF基因缺失突变株和toyG基因缺失突变株武夷菌产量较野生菌株分别提高了23.06%和19.45%，证实武夷菌素与丰加霉素存在明确的竞争作用, 初步解析toyF和toyG为竞争作用的中的关键因子。同时，本研究还获得了4株丰加霉素产量显著提高的过表达菌株S.albulus ootoyA，ootoyF，ootoyG和ootoyH，其中toyA基因过表达菌株丰加霉素产量达到217.46mg/L为野生菌株的2.74倍。本课题成功建立一种以代谢工程技术为基础的提高抗生素产量的育种方法，选育具有自主知识产权的武夷菌素高产新菌株，为提高武夷菌素工业化水平、降低产品成本提供高产基因工程菌株。