"AT-less" Type I Polyketide Synthases

说明(申请人提供)：来那霉素(LNM)、米曲他汀(MGS)、多利高霉素(DGN)和乳糖霉素(LTM)包括广泛的生物活性。本申请建议(1)研究LNM、MGS/DGN和LTM产物的生物化学和遗传学，作为“无AT”I型聚酮合成酶(PKS)催化的聚酮生物合成的模型；(2)开发新的组合生物合成策略，用于工程聚酮结构多样性；(3)操纵LNM、MGS/DGN和LTM生物合成，以生产具有生物活性的化合物。提出的假设是：(1)“无AT”的I型PKS代表了一种前所未有的PKS结构，其研究将揭示PKS催化的分子机制；(2)“无AT”的I型PKS为PKS工程、方法和策略提供了新的机遇，基于此将进一步扩大可通过组合生物合成获得的聚酮产品的数量和多样性；(3)LNM、MGS/DGN和LTM生物合成是前所未有的，其表征将为聚酮的生物合成开辟新的化学途径；(4)LNM、MGS/DGN和LTM是组合生物合成的良好起始分子支架，其衍生可能导致发现治疗上有用的药物。这些研究的结果将：(1)扩大用于组合生物合成的PKS基因谱系；(2)为利用“无AT”的I型PKS进行组合生物合成提供新的策略和方法；以及(3)潜在地导致LNM、MGS/DGN和LTM类似物作为新的治疗药物的产生。这一授权期的具体目标是：(1)通过体内靶向基因失活和体外LNM杂合非核糖体多肽合成酶(NRPS)-“AT-less”I型PKS巨合酶的生化特征分析INM生物合成基因簇的功能；(2)通过体内靶向基因失活和体外MGS/DGN“无AT”型PKS的生化特征分析MGS/DGN生物合成基因簇的功能；(3)通过体内靶向基因失活和体外LTM“AT-less”I型PKS的生化特性分析ITM生物合成基因簇的功能；(4)基于LNM、MGS/DGN、LTM生物合成机制的组合生物合成策略和方法的发展，用于新型聚酮的生产。

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.