林可霉素正丙基脯氨酸结构单元生物合成机制研究及工业生产菌株的代谢工程改造

Lincomycin and its chemical derivative clindamycin are potent antibacterial agents that have long been used in clinical. The biosynthetic gene cluster of lincomycin was reported in 1995; however, the detailed mechanisms regarding its biosynthesis remain elusive. In fact, only a few genes have been functionally assigned. Benefited from the dramatical increasing of understanding the biosynthesis of various natural products, the forming pathways of certain antibiotics can be speculated according to the established logic of the "biosynthetic gene-enzyme-corrresponding structure". In this study, starting with comparative analysis of lincomycin and its structurally related natural products at the level of the genetic basis, we found the genes involved in propyl-L-proline(PLP) biosyntheis, and plan to perform the in vivo and in vitro invesitgations into the biochemistry of the unusual PLP building block in lincomycin biosynthesis. Elucidation of this may facilitate the application of the rational metabolic engineering into the pathway, aiming at the elimination of the associated by-products of lincomycin and improvement of lincomycin production at the fermentation stage.

林可霉素及其下游产品克林霉素都是临床广泛使用多年疗效明显的药物。早在1995年，林可霉素生物合成基因簇就已经被报道，然而对林可霉素生物合成机制方面的了解却非常少，迄今为止仍然只有少数几个基因的功能被证实。随着越来越多的抗生素的生物合成基因簇被克隆报道，我们可以根据"生物合成基因-酶-分子结构"的一一对应关系，来推测其可能的生物合成途径。因此本项目从林可霉素与多个含有相似结构单元分子的生物合成基因簇同源性比较出发，找出可能负责林可霉素PPL结构单元的基因，体内基因敲除/前体喂养实验结合体外酶学测活的方法，阐明林可霉素PPL结构单元的生物合成途径，揭示其蕴含的新型酶学催化机制；从而将生物合成机制研究的结果应用于代谢工程改造林可霉素工业生产菌株，优化林可霉素发酵产物的组份，提高林可霉素的产量。

林可霉素及其下游产品克林霉素都是临床广泛使用多年疗效明显的药物。然而对其生物合成机制方面的了解却非常少。我们阐明了林可霉素PPL结构单元的生物合成机制，尤其是阐明了一个γ-谷氨酰基转移酶LmbA具有N-端亲和水解酶的活性催化PPL生物合成过程中独特的C-C键断裂反应。我们还揭示了两个小分子硫醇MSH和EGT介导的林可酰胺类抗生素的生物合成机制。以及两个具有较高同源性的PLP依赖的蛋白CcbF和LmbF催化相似的中间产物发生不同的半胱氨酸基团剪切方式，再在不同的后修饰酶的协同作用下，最终生成了林可霉素和天青菌素中不同的硫修饰结构。