新抗霉素生物合成及其α-酮基酸加载元件的功能研究

Neoantimycins (nat) are depsipeptide natural products with outstanding anti-tumor activities, whereas its biosynthesis process is remained obscure. We recently identified two nat compounds produced from S. conglobatus. Bioinformatical analysis suggests the nat biosynthesis mainly consists of a start unit biosynthesis and the NRPS -PKS , of which an unusual adenylation domain, putatively activating α-keto-phenylpropanoic acid (α-KPP), should be present. And the hydroxylation modification on neoantimycin skeleton and the N-formylation modification on the start unit are to be explained for further understanding of nat biosynthesis. In the proposal, nat gene cluster will be extensively elucidated by high efficient heterologous expression, gene knock-out and characterization of the resulted new compounds. Exchanging of the α-keto-acid activating domains within the nat NRPS modules will be carried out to generate the compounds with improved pharmacological activities. The unusual α-KPP activating A domain will be explored by site mutations and in vitro assays to understand the substrate conferring residues. Furthermore, the α-KPP activating A domain is designed to replace the native A domain of the NRPS modules in the biosynthesis of either antimycins with potent anti-tumor activity or antimycobacterial pyridomycin to introduce aromatic moiety in each molecule respectively and therefore to verify its potential to produce new depsipeptedes with improved pharmaceutical properties by the way of combinatorial biology.

新抗霉素（neoantimycin）是具有显著抗肿瘤活性的缩酚肽类天然产物，其生物合成机制仍不清楚。申请人从链霉菌S. conglobatus中发现了两个新抗霉素化合物，生物信息学推断其生物合成主要由NRPS-PKS和起始单位合成元件构成，含有新颖的α-酮基苯丙酸 (α-KPP) 加载腺苷酰化（A）功能域，其分子骨架的羟基化和起始单位的氮-甲酰化修饰元件仍待确认。本项目拟对新抗霉素基因簇进行异源高效表达、基因敲除和新化合物结构鉴定，深入系统解析新抗霉素生物合成途径；通过置换α-酮基酸加载A功能域优化新抗霉素的结构；通过点突变探索α-KPP加载A功能域的底物选择决定位点；应用α-KPP加载A功能域分别在抗肿瘤抗霉素和抗结核吡啶霉素分子结构中引入芳香侧链，优化其药理活性，探索该功能域元件对组合生物改造缩酚肽类分子结构的重要价值。

新抗霉素（neoantimycin, NAT）是具有显著抗肿瘤活性的缩酚肽类天然产物，探明NAT生物合成机制将使得通过合成生物学改造NAT分子结构和建立其高效生物合成体系成为可能，为开发新型抗肿瘤候选药物创造条件。本项目从链霉菌S. conglobatus发酵产物中发现了10个NAT结构类似物，考察了其抗肿瘤活性构效关系，并确认了其生物合成基因簇为非核糖体多肽合成酶和聚酮合成酶（NRPS-PKS）的杂合类型，随后通过克隆并异源表达NAT基因簇、遗传突变关键基因、分离鉴定突变株积累的中间产物、构建体外酶催化过程的方法，系统解析了NAT生物合成途径。在此基础上，通过体内遗传和体外生化实验探明了选择性抗肿瘤活性NAT新结构衍生物UAT（unantimycin）的生物合成机制，进而构建了可定向积累UAT的基因工程菌株，为后续该抗肿瘤候选药物研发的药源供应创造了条件。此外，作为课题的延伸，本项目从细菌来源NRPS生物合成途径中发现了新家族多肽环化硫酯酶PBP-like TE，确认了其合成药用活性环肽的应用潜力。

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