冰城链霉菌前体代谢对杀虫抗生素米尔贝霉素生物合成的影响研究

Milbemycins are possessed of extremely high activity against various insects, such as mites and aphis. Streptomyces bingchenggensis is a milbemycin producing strain with self-owned intellectual property rights. And its complete genome has been sequenced. Two mutant strains with high yield of milbemycins were obtained by physical and chemical mutagenesis technologies. A remarkable difference in constituent proportion of milbemycins A3 and A4 was observed between the two mutants. Then the genomes of the two mutants were re-sequenced and compared with that of the wild-type strain. Based on the speculative diagram of primary metabolic pathway, we found that eight genes with mutation were related to primary metabolism which was associated with the precursors of milbemycins. The mutation of these eight genes may influence the yield of milbemycins and the constituent proportion of milbemycins A3 and A4. We will study the eight genes thoroughly by site-directed mutagenesis, gene disruption and complementation, and over express two important primary metabolic enzymes, acetyl-coA carboxylase and propionyl coA synthetase in vivo to nail down the key primary metabolic genes which can affect the yield and constituent proportion of milbemycins A3 and A4. This study is aimed to pave a profound way for the change of metabolic fluxes and the construction of engineered strains with high-yield and unicomponent milbemycins, and to provide data for constructing the actual diagram of primary metabolic pathway.

以代谢高效防治螨、蚜虫等的米尔贝霉素的冰城链霉菌为研究对象。前期完成、发表了冰城链霉菌全基因组测序，并诱变选育获得两株高产米尔贝霉素、且米尔贝霉素A3、A4两个组分比例差异大的突变菌株。对这两株高产突变菌株进行了全基因组重测序，比较两株突变株与原始菌株，两株突变菌株之间全基因组序列的位点差异，并根据构建的冰城链霉菌初级代谢通路图，发现8个突变基因与米尔贝霉素生物合成所需前体的初级代谢相关，它们可能影响米尔贝霉素发酵单位产量和米尔贝霉素A3、A4生物合成的比例。本项目拟对这8个突变基因进行基因点突变，基因替换与回补，以及体内过表达重要影响初级代谢的乙酰辅酶A羧化酶和丙酰辅酶A合成酶。明确能提高米尔贝霉素发酵单位产量，和影响米尔贝霉素A3、A4生物合成比例的初级代谢关键酶基因。为冰城链霉菌代谢流的改造，及高产、单组分米尔霉素优良工程菌的构建奠定基础，为实际构建冰城链霉菌初级代谢通路图积累数据。

米尔贝霉素是一种广泛应用于农业害虫防治的杀螨剂，具有高效、低毒、低抗性、环境友好、对人畜安全等优点，被认为是最有前途的生物农药之一。其中，米尔贝霉素A3/A4具有较高的杀菌活性。米尔贝霉素 A3/A4 聚酮骨架生物合成的延伸单元为7个丙二酰辅酶A (M-CoAs) 和5个甲基丙二酰辅酶A (MM-CoAs)，是典型的Ⅰ型PKS。米尔贝霉素A3/A4生物合成的起始单元分别为乙酰辅酶A和丙酰辅酶A。在农业上广泛使用的弥拜霉素是一种由米尔贝霉素A3/A4以3:7的比例混合而成的物质。因此，在增加米尔贝霉素产量的同时，保持米尔贝霉素A3与米尔贝霉素A4的比例是十分必要的。我们利用时序启动子控制冰城链霉菌中米尔贝霉素生物合成的不同前体供给途径，达到增加并平衡前体供给的目标，实现了菌株米尔贝霉素产量的显著提高。过表达乙酰辅酶A羧化酶（ACC）提高了胞内M-CoA的浓度从而提高了米尔贝霉素A3的生物合成。进而，通过过表达丙酰辅酶A合酶（prpE）提高丙酰辅酶A的方法提高了米尔贝霉素A3/A4产量。最终，利用自身时序启动子控制丙酰辅酶A羧化酶（PCC）优化适配起始单元丙酰辅酶A与延伸单元M-CoA 和 MM-CoA的比例，使菌株的米尔贝霉素产量提高39.5%，达到了3.42g/L。本研究首次报道了一种利用自身时序启动子动态调节前体供应途径来增强前体水平的策略，为米尔贝霉素的合成提供了一种高效的方法。同时，时序启动子作为一种有效的代谢工程工具，对米尔贝霉素其他途径的代谢工程改造提供理论基础。