基于枯草芽孢杆菌fengycin合成酶结构修饰的新型抗菌脂肽探索

During our previous studies, toxicological assessment, antimicrobial mechanism and application in food for lipopeptide fengicin had been accomplished, the research results show that fengycin, a kind of antimicrobial lipopeptide produced by Bacillus subtilis fmbJ have the property of high security, broad antimicrobial spectrum and high stability. Thus, there is a potential possibility to develope fengycin as natural food preservative. Fengycin was synthesized on non-ribosomal peptide synthetase (NRPS) which is very complicated, to date there are still many mechanism and rule remained unknown for the NRPS. On this account, thorough investigation will be proceeded subsequently to explore the relevance between fengycin synthetase structure and lipopeptide diversity. The combinatorial biosynthesis methods will be applied to make module or domain substitution and induce the formation of heterozyme, modify linker sequence as well as induce module skipping in fengycin synthetase to produce novel lipopeptide with different length and composition. The research aim is to explore the regularity between structural modification of fengycin synthetase and novel lipopeptide biosynthesis, to reveal the relation between lipopeptide structure and its antimicrobial activity, which can provide a theory foundation to the biosynthesis of lipopeptide food preservative with high antimicrobial activity and broad spectrum for food industry.

课题组前期研究已经通过毒理学评估、抑菌机制和食品应用等确定Bacillus subtilis fmbJ 产生的抗菌脂肽fengycin具有安全性高、抑菌谱广、性能稳定等优势，因此有潜力开发成为新一代天然食品防腐剂和保鲜剂。脂肽fengycin是由非核糖体肽合成酶-fengycin合成酶催化合成，该酶结构复杂目前还存在许多未知规律。基于此，本研究将在原有工作基础上将探索fengycin合成酶结构与脂肽多样性的关系。通过组合生物合成的方法对fengycin 合成酶内部模块或结构域进行替代形成杂合酶；修饰fengycin 合成酶内部linker 的结构、诱导fengycin 合成酶产生模块跳跃，从而产生肽链组成和长度不同的脂肽，探索由于fengycin合成酶结构改变而引起新型抗菌脂肽形成的规律，揭示新型脂肽与抗菌生物活性之间的联系，为广谱高效脂肽型食品防腐剂的生物合成奠定理论基础。

本研究通过探索fengycin （plipastatin）合成酶结构与脂肽多样性的关系，通过NRPS合成酶修饰对plipastatin合成酶内部模块或结构域进行替代形成杂合酶；通过plipastatin 合成酶内部linker序列的功能与改造、诱导plipastatin 合成酶产生模块跳跃，形成肽链组成和长度多样的脂肽，取得的重要结果：.1 通过同源重组方法，获得了一株生产脂肽plipastatin和surfactin的重组菌株，命名为B. subtilis pB2-L，经检测对金黄色葡萄球菌和尖孢镰刀菌具有显著的抑菌活性。.2 Plipastatin合成酶中TE结构域的前移来设计新的脂肽， 采用原始的第10位T结构域与TE之间的linker来替换上述位移中的linker序列，通过高分辨LC-ESI-MS/MS检测鉴定到了相应预测的plipastatin衍生脂肽：线性七肽、八肽和九肽，环形七肽和八肽。.3 通过模块敲除和模块置换方法进行Plipastatin合成酶linker序列的功能与改造，敲除ppsC的COMD序列鉴定获得新型线性的R-ABC，当ppsD的供体替换为ppsC的供体时，获得新型线性6肽β-OHFA-ABCD；将 ppsB 的受体替换为ppsC的受体，得到新型线性8肽β-OHFA-ABCD。当将ppsC供体替换为 ppsD供体，获得新型线性7肽β-OHFA-ABCE。当将ppsC供体敲除和 ppsC 受体替换为 ppsB受体， 指导合成了新型β-OHFA-ABDE环8肽。线性六肽、线性七肽和线性九肽抗菌脂肽对供试真菌和细菌均有抑制作用。 .4利用组合生物合成策略构建ppsC-A2结构域、ppsC-Glu模块、ppsC亚基敲除菌株，分析发现ppsC-A2结构域敲除菌株Plipastatin生物合成途径中第六模块被完整跳跃。构建ppsC-C2结构域和ppsC-PCP2结构域敲除菌株，发现ppsC-PCP2结构域敲除菌株出现模块跳跃现象。.5 利用同源重组操作，获得缺失一个氨基酸的三种新型Surfactin抗菌脂肽：[△Leu3]Surfactin、[△Asp5]Surfactin和[△Leu6]Surfactin。.6 本项目科研成果申报专利4项，已经发表SCI论文8篇，中文核心期刊6篇。.研究成果揭示了NRPS合成酶结构修饰与新型脂肽合成之间的规律