"AT-less" Type I Polyketide Synthases

“无 AT”I 型聚酮化合物合成酶

• 批准号: 7053378
• 负责人: Ben Shen
• 金额: $ 28.95万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7053378
• 关键词: Streptomyces; acyltransferase; antineoplastics; bacterial genetics; biochemistry; combinatorial chemistry; enzyme activity; high performance liquid chromatography; liquid chromatography mass spectrometry; piperidine; polyketide synthase; protein biosynthesis

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.

描述（由申请人提供）：Leinamycin (LNM)、migrastatin (MGS)、dorrigocin (DGN) 和lactimidomycin (LTM) 涵盖广泛的生物活性。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)开发用于工程聚酮结构多样性的新组合生物合成策略，以及(3)操纵LNM， MGS/DGN 和 LTM 生物合成用于生产生物活性化合物。假设是：（1）“无AT”I型PKS代表了一种前所未有的PKS结构，其研究将为PKS催化的分子机制提供新的见解； （2）“AT-less”I型PKS为PKS工程、方法和策略提供了新的机遇，在此基础上将进一步扩大通过组合生物合成获得的聚酮化合物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，其表征将揭示聚酮化合物生物合成的新化学； (4) LNM、MGS/DGN 和 LTM 是用于组合生物合成的优异起始分子支架，其衍生可能导致治疗有用药物的发现。拟议研究的结果将（1）扩展用于组合生物合成的 PKS 基因库； （2）为采用“AT-less”I型PKS的组合生物合成提供新的策略和方法； (3) 可能导致 LNM、MGS/DGN 和 LTM 类似物作为新型治疗剂的生产。本资助期的具体目标是：（1）通过 S. atroolivaceus 体内靶向基因失活和体外 LNM 杂合非核糖体肽合成酶（NRPS）-“AT-less”I 型 PKS 大合成酶的生化表征，对 Inm 生物合成基因簇进行功能分析； (2) 通过钝顶链球菌体内靶向基因失活和体外 MGS/DGN“AT-less”I 型 PKS 的生化表征，对 mgs/dgn 生物合成基因簇进行功能分析； (3) 通过两栖链球菌体内靶向基因失活和体外 LTM“AT-less”I 型 PKS 的生化表征，对 Itm 生物合成基因簇进行功能分析； (4)开发基于LNM、MGS/DGN、LTM生物合成机械的组合生物合成策略和方法，用于新型聚酮化合物的生产。