基于烷基脂酰结构定向合成脂肽surfactin新化合物的组合生物合成策略

The cyclic lipopeptides, assembled by nonribosomal peptide synthetases (NRPSs) as secondary metabolites in microorganisms, are a new class of antibiotics. They are also characterized by having a medium to long fatty acyl chain attached to the macrocyclic peptide core. Structural activity relationship (SAR) studies have shown that variations in the lipid side chains of lipopeptide have significant affects on their bioactivity and properties. Surfactin, a cyclic lipoheptapeptide lactone produced by Bacillus subtilis, contains seven amino acid residues and one 3-hydroxy fatty acid. Besides its excellent properties as a biosurfactant, surfactin also exhibits antibacterial, -viral, -tumoral, and antimycoplasmatic activities, which make it an interesting target for therapeutic applications. Only its cytotoxicity against erythrocytes, which is greatly affected by the fatty acid moieties, prevents surfactin from clinical applications. The fatty acyl chains of surfactin are also the first substructure incorporated into the peptidyl backbone via a process known as lipoinitiation. Therefore, understanding the pathways of fatty acyl precursor for surfactin synthesis is of special interest as it might facilitate the design of new products with improved properties. In this proposal, we want to investigate and modify the lipid chains of surfactins, the detailed work including: (1) investigating the dissected initiation module (C-AGlu-PCP) of surfactin synthetase SrfAA in vitro to gain its substrate specificity for medium to long chain of CoA-activated 3-hydroxy fatty acids; (2) identifying the enzymes for 3-hydroxy-fatty acids(or CoA-activated 3-hydroxy fatty acids) from fatty acid de nove biosynthesis pathway or beta-oxidation pathway; (3) constructing pathways for directed biosynthesis of CoA-activated 3-hydroxy fatty acids by feeding fatty acids as precursor and subsequently incorporated into the peptidyl backbone via lipoinitiation process to form designed surfactin new products.

表面活性素（surfactin）是由枯草芽孢杆菌非核糖体肽合成酶（NRPS）合成的典型脂肽（lipopeptides）化合物，除具有优异的表面活性外，还具有抗菌、抑癌和抗病毒等生物学活性，但surfactin的溶血活性阻碍了进一步应用。surfactin烷基脂酰结构不但对其理化性质和生物学活性有显著的影响，还是surfactin合成的起始底物。本项目研究枯草芽孢杆菌surfactin合成酶srfAA的合成起始模块（C-AGlu-PCP）对脂酰结构前体（中长链R-3羟基脂酰CoA）的底物选择性，基于组合生物合成技术提升srfAA起始模块的合成能力，解析脂肪酸代谢途径对脂酰结构前体的合成和调控机制，重构脂肪酸β-氧化途径定向合成脂酰基结构前体，获得特定脂酰结构的surfactin新分子。定向合成surfactin烷基脂酰结构的设计理念和技术平台，对于开发脂肽类新结构化合物具有重要的示范意义。

脂肽surfactin是一类重要的生物表面活性剂，其分子结构中的亲水基和亲油基分别由多肽与脂肪酸构成，脂肪酸链以C14和C15组分为主，其结构差异会对物理化学性质和生物活性产生显著的影响。本项目重点关注了surfactin脂酰基结构合成途径及强化策略，通过对野生高产菌株BS-37的基因组和转录组分析以及对枯草芽孢杆菌168菌株的代谢改造等研究，获得了有效的surfactin合成强化策略以及脂酰基结构改造策略，为促进surfactin进入微生物采油（MEOR）应用提供了参考价值。.具体结果包括：1.解析了野生高产菌株Bacillus velezensis BS-37的基因组及转录组，获得了surfactin合成模块中的重要功能元件基因：如4’-磷酸泛酰巯基转移酶编码基因sfp、脂肪酰CoA连接酶编码基因yhfl、乙酰乳酸合酶编码基因ilvB等，为构建高产surfactin的基因工程菌提供了基础；2. 高产基因工程菌构建：基于脂酰基合成模块的强化策略，在枯草芽孢杆菌168菌株中构建了高效的surfactin合成途径，surfactin产量达4000 mg/L；3. 新脂酰基结构surfactin的开发：针对surfactin合成模块中NRPS的初始模块（C-AGlu-PCP），将合成大侧柏素(plipatatin)初始结构域中的7-187aa与surafctin的NRPS进行模块替换，获得了新型结构surfactin，表面张力30.6mN/m，产量50 mg/L左右；4. 以木糖/廉价木质纤维素资源发酵产surfactin的发酵技术：工程菌168菌株利用木糖时surfactin产量达2203mg/L,发现了利用木糖可以减少有机酸副产物合成的现象，进一步开发了无磷酸缓冲的发酵体系，产量可达2032mg/L，是目前利用木糖产surfactin的最高产量; 5. MEOR用途surfactin的发酵体系：考察了初级代谢产物有机酸及多元醇对surfactin物化性质及洗油效果的影响，结果表明乙偶姻会影响surfactin的胶束形成及组装构象，破坏surfactin对柴油的乳化活性，降低对原油的表面浸润性，表明降低发酵产物中的乙偶姻对MEOR应用具有重要性。.已发表研究论文7篇，做学术交流报告2次，申请专利4项，培养硕士/博士6人，在投论文2-3篇。