"AT-less" Type I Polyketide Synthases

性状（由申请方提供）：来那霉素（LNM）、偏头痛抑制素（MGS）、多瑞戈辛（DGN）和乳酰氨霉素（LTM）具有广谱生物活性。本申请提出（1）研究LNM、MGS/DGN和LTM生产的生物化学和遗传学，作为“无AT”I型聚酮合酶（PKS）催化的聚酮生物合成的模型，（2）开发用于工程化聚酮结构多样性的新的组合生物合成策略，和（3）操纵LNM、MGS/DGN和LTM生物合成以生产生物活性化合物。假设是：（1）“AT-less”I型PKS代表了一种前所未有的PKS结构，对它的研究将为PKS催化的分子机理提供新的认识：（2）“AT-less”I型PKS为PKS工程、方法和策略提供了新的机遇，将进一步扩大通过组合生物合成可获得的聚酮产物的数量和多样性;（3）LNM、MGS/DGN和LTM生物合成是前所未有的，其表征将揭示用于聚酮生物合成的新化学;和（4）LNM、MGS/DGN和LTM是用于组合生物合成的优异起始分子支架，其衍生可导致发现治疗上有用的药剂。所提出的研究的结果将（1）扩展用于组合生物合成的PKS基因库;（2）提供用于采用“无AT”I型PKS的组合生物合成的新策略和方法;和（3）潜在地导致LNM、MGS/DGN和LTM类似物作为新型治疗剂的产生。本项目的具体目标是：（1）通过体内靶向基因失活，对S.（2）通过体内靶向基因失活，对黑乳链霉菌mgs/dgn生物合成基因簇进行功能分析。（3）通过体内靶向基因失活分析S. platensis中Itm生物合成基因簇的功能。（4）开发基于LNM、MGS/DGN、LTM生物合成机制的组合生物合成策略和方法用于新型聚酮化合物的生产。

DESCRIPTION (provided by applicant): Leinamycin (LNM), migrastatin (MGS), dorrigocin (DGN), and lactimidomycin (LTM) encompass a broad spectrum of biological activities. This application proposes (1) to study the biochemistry and genetics of LNM, MGS/DGN, and LTM production as models for "AT-less" type I polyketide synthase (PKS)- catalyzed polyketide biosynthesis, (2) to develop new combinatorial biosynthesis strategies for engineering polyketide structural diversity, and (3) to manipulate LNM, MGS/DGN, and LTM biosyntheses for the production of biologically active compounds. The hypotheses are: (1) "AT-less" type I PKSs represent an unprecedented PKS structure, the studies of which will reveal new insights into the molecular mechanism of PKS catalysis; (2) "AT-less" type I PKS provides new opportunities for PKS engineering, methods and strategies based on which will further expand the number and diversity of polyketide products accessibly by combinatorial biosynthesis; (3) LNM, MGS/DGN, and LTM biosyntheses are unprecedented, the characterization of which will uncover new chemistry for polyketide biosynthesis; and (4) LNM, MGS/DGN, and LTM are excellent starting molecular scaffolds for combinatorial biosynthesis, derivation of which could lead to the discovery of therapeutically useful agents. The outcome of the proposed studies will (1) expand the repertoire of PKS genes for combinatorial biosynthesis; (2) provide new strategies and methods for combinatorial biosynthesis employing "AT-less" type I PKS; and (3) potentially lead to the production of LNM, MGS/DGN, and LTM analogs as novel therapeutic agents. The specific aims for this grant period are: (1) functional analysis of the Inm biosynthetic gene cluster by targeted gene inactivation in vivo in S. atroolivaceus and by biochemical characterization of the LNM hybrid nonribosomal peptide synthetase (NRPS)-"AT-less" type I PKS megasynthetase in vitro; (2) functional analysis of the mgs/dgn biosynthetic gene cluster by targeted gene inactivation in vivo in S. platensis and by biochemical characterization of the MGS/DGN "AT-less" type I PKS in vitro; (3) functional analysis of the Itm biosynthetic gene cluster by targeted gene inactivation in vivo in S. amphibiosporus and by biochemical characterization of the LTM "AT-less" type I PKS in vitro; (4) development of combinatorial biosynthesis strategies and methods based on the LNM, MGS/DGN, LTM biosynthetic machinery for novel polyketide production.