解淀粉芽孢杆菌Sfp乙酰化修饰调控表面活性素合成的机制研究

Lipopeptide surfactin, a secondary metabolite, presents various biological activities including anti-microbial, anti-virus and anti-tumor, but low fermentation yield limits its application. We found that the phosphopantethein transferase Sfp in Bacillus amyloliquefaciens can regulate the synthesis of surfactin by Lys38 acetylation, but how acetylation modification alters the Sfp activity, and what enzymes are responsible for the acetylation/deacetylation of Sfp, these key issues are not yet clear. For this project, the site-directed mutation of Lys38 was designed to mimic the acetylation/deacetylation modification. To clarify the molecular mechanism of acetylation modification regulating Sfp activity, the stability of Sfp protein and the interaction between Sfp and its substrate peptidyl carrier protein before and after mutation will be analyzed; according to the increased/decreased Sfp acetylation levels in acetyltransferase/deacetylase knockout mutants, acetyltransferase/deacetylase of Sfp can be identified; through the relationship between the production and activity of surfactin and Sfp acetylation level, Sfp activity in bacterial physiological state, reveal the molecular mechanism of lysine acetylation of Sfp in regulating surfactin synthesis. This study will not only help reveal the regulation mechanism of acetylation in the synthesis of secondary metabolites, but also provide important basis for breeding of high-producing lipopeptides strains.

脂肽类次生代谢产物表面活性素具有抗菌、抗病毒、抗肿瘤等活性，但菌株发酵产量低限制了它的应用。前期我们发现解淀粉芽孢杆菌合成表面活性素的关键酶磷酸泛酰巯基乙胺转移酶Sfp通过乙酰化修饰其38位赖氨酸调控表面活性素的合成，但乙酰化修饰如何改变Sfp的催化活性，以及何种酶负责Sfp的乙酰化/去乙酰化, 这些关键问题尚不清楚。本项目拟将Lys38定点突变模拟乙酰化/去乙酰化修饰，通过突变前后Sfp蛋白稳定性、Sfp与底物肽酰载体蛋白互作强弱，解析乙酰化对Sfp酶活的调控；通过分析乙酰基转移酶/去乙酰化酶基因敲除突变体中Sfp乙酰化水平升降，鉴定乙酰基转移酶/去乙酰化酶；基于此，分析菌体生理状态下Sfp乙酰化水平与Sfp活性、表面活性素合成的相互关系，揭示Sfp乙酰化修饰调控表面活性素合成的分子机制。本研究结果将有助于揭示乙酰化修饰在次生代谢产物合成中的调控机制，并为脂肽类高产菌株选育提供重要依据。

脂肽类次生代谢产物表面活性素具有抗菌、抗病毒、抗肿瘤等活性，但菌株发酵产量低限制了它的应用。磷酸泛酰巯基乙胺转移酶（Sfp）是解淀粉芽孢杆菌中表面活性素合成的关键酶，其38位赖氨酸的乙酰化修饰可以调控表面活性素的合成。将Sfp的Lys38突变为谷氨酰胺（Q）/精氨酸（R）模拟乙酰化/去乙酰化修饰，与野生型相比，突变菌株Sfp（K38Q）表面活性素产量大幅降低，而菌株Sfp（K38R）产量显著提高。原核表达并纯化蛋白，构建体外反应体系，分析乙酰化修饰对酶活的影响。与野生型Sfp相比，蛋白Sfp（K38Q）酶活显著降低，蛋白Sfp（K38R）酶活显著提高。等温滴定微量热技术分析结果显示与野生型Sfp相比，蛋白Sfp（K38Q）Km值增加，蛋白Sfp（K38R）Km值减小。这表明Sfp的乙酰化修饰能够改变Sfp与底物的亲和性，从而改变酶的催化活性，调控表面活性素的合成。采用柠檬酸和葡萄糖为碳源，发酵合成表面活性素，分析生理状态下，Sfp酶活、表面活性素合成以及Sfp乙酰化水平的动态变化。Sfp酶活与表面活性素的合成量呈正相关，酶活高，表面活性素产量高。不同碳源条件下，Sfp乙酰化修饰水平的动态变化趋势基本一致，发酵初期乙酰化修饰水平高，在指数期迅速下降，进入稳定期乙酰化水平趋于平稳。生理状态下，较低的Sfp乙酰化修饰水平对应较高的Sfp酶活，同时具有较高的表面活性素产量。项目揭示了乙酰化修饰在脂肽类次生代谢产物合成中的调控作用，拓展了乙酰化修饰的生物学意义，同时为微生物次生代谢产物的合成调控提供新的研究思路，为通过分子手段选育脂肽类高产菌株提供重要依据。