武夷菌素生物合成调控基因的功能鉴定

Wuyiencin is a new agricultural antibiotics in safety and efficiency with independent intellectual property rights, which has been applied to industrialization production in large area, especially for vegetables fungal disease. In order to reduce the cost of manufacture of Wuyiencin and overcome the shortcomings of mutation breeding to select the high-yield producing strains, the aim of the project is to study and establish the site-directed breeding based on understanding biosynthesis gene clusters. This project includes many works as following. The first is to constructure of genome Fosmid bank of Wuyiencin producing strain, optimize the specific primers and then clone the complete biosynthesis gene culsters of Wuyiencin. The second is to establish a new method using Wuyiencin biosynthesis gene deletion and over-expression in producing strain. At same time, it need to observe the changes of producing strain on the biological and culture characteristis, the inhibiting spectrum to pathogenc fungi and the compositin of Wuyiencin in fermentation liquid checked by HLPC. It is also important to make sure the key genes among the gene cluster which decides the Wuyiencin biosynthesis of producing strain, as well as the gateway of Wuyiencin biosynthesis and make sure the mechanism of biosynthesis regulation. The expected outcomes will lead to establishing a new metheod of site-directed breeding to improve the yield of Wuyiencin and to get Wuyiencin's derivatives from the producing strain. This study has important significance in enhancing the level of industrial production of Wuyiencin and the ability of independent innovation of biopesticide.

武夷菌素是具有我国自主知识产权的安全、高效新型生物农药，对多种蔬菜真菌病害具有良好的防治效果，并已产业化生产和大面积应用。为了进一步降低武夷菌素的生产成本，突破采用传统诱变方法选育高产菌株面临的效率低、目标产物不明确的限制，进而建立以明确武夷菌素生物合成基因簇及其代谢调控机理为前提的定向育种方法。本项目拟在已构建武夷菌素产生菌基因组Fosmid文库的基础上，优化特异性引物，克隆完整的武夷菌素生物合成基因簇。通过目的基因缺失和过表达，结合产生菌的生物学特性、抑菌谱和发酵液中武夷菌素有效成分的高压液相色谱测定，确定基因簇中与武夷菌素合成相关的关键调控基因。为利用定向育种和代谢工程技术提高武夷菌素产量或获得更高活性的武夷菌素衍生物提供理论、技术支持。项目研究成果对提高武夷菌素产业化水平和增强我国生物农药创新能力具有重要意义。

武夷菌素是一种具有自主知识产权的高效、广谱、低毒生物农药，对粮食、蔬菜和果树及经济作物的真菌病害具有良好的防治效果。为了进一步提高武夷菌素产量、降低成本，本研究通过全基因组测序技术，挖掘出39种武夷菌素产生菌次级代谢基因簇。结合基因克隆方法，首次完成并解析了武夷菌素生物合成基因簇的序列特征、功能及化学结构。采用同源重组双交换方法，分别构建调控基因wysR、wysRⅢ、wysPⅡ、wysPⅢ的工程菌株，通过比较缺失突变株、回复突变株、过表达菌株以及有野生菌株的生物学性状以及发酵产物差异，发现wysR基因在武夷菌素生物合成途径中正调控武夷菌素产量，wysRⅢ、wysPⅡ、wysPⅢ基因不影响武夷菌素产量，对菌株的生长表型有影响。在此基础上，构建了武夷菌素高产菌株W-273，平均效价达到7215μg/mL，较原始菌株效价提高26.5%，且最佳发酵时间缩短4-8h。该研究为探明武夷菌素生物合成机制和获得结构独特的新兴化合物提供了理论依据，同时为武夷菌素工业化生产及大面积应用奠定了基础。