Biosynthesis of Fungal Natural Products on Collaborating Iterative Polyketide Synthases

聚酮化合物是天然产物中最大的家族之一，具有惊人的结构多样性和显著的生物活性。聚酮化合物在真菌中通过聚酮化合物合成酶（PKS）多酶生物合成，所述多酶的单个酶结构域被重复使用。 虽然只有一个单一的PKS多酶是足以产生大多数真菌聚酮化合物，这些天然产物中的一些是由两个合作的真菌PKS组装。这种合作代表了真菌聚酮生物合成的典型生物合成逻辑的重大偏离。 目前的项目的首要目标是澄清目前未知的分子机制的细节，组装聚酮化合物对这种合作的PKS，使用的生物合成的间苯二酚内酯（RAL）和相关的二羟基苯乙酸内酯（DAL）作为模型。具体而言，该项目旨在：1：表征DAL型天然产物脱氢弯孢菌素的生物合成; 2：在替代微生物宿主中表达RAL和DAL聚酮化合物合成酶，其适当的突变体和杂合酶，并使用这些酶和合成底物评估聚酮化合物的生产; 3：通过结合相关的生物合成途径展示组合生物合成。 这些实验将阐明从简单代谢物组装聚酮化合物的编程规则，并控制这些天然产物的最终形状（环化模式）。 这些经验教训将促进我们对迭代酶催化的理解，并有助于开发生产“设计师”聚酮化合物的组合生物合成方法，这些聚酮化合物可能在未来用作新型农用化学品或高价值特种化学品。将为研究生和本科生提供广泛的研究培训机会，包括来自少数民族和代表性不足群体的学生。 该项目还将培训社区大学的本科生实习生，并可能在亚利桑那大学一个成功的外联方案的主持下接待高中高年级学生作为实习生。 所有学生将学习微生物代谢物生物合成的跨学科科学和组合生物合成的实践，从而为他们在生物技术行业和学术研究的职业生涯做好准备。 此外，还将为K-12教育工作者提供专业发展机会，以了解天然产物及其微生物生物合成：这些知识以后可能会融入他们的科学课程。该项目由分子和细胞生物科学部门的代谢生物化学计划和化学部门的生命过程化学计划共同支持。

Polyketides are one of the largest families of natural products with an astonishing structural variety and a remarkable range of biological activities. Polyketides are biosynthesized in fungi by polyketide synthase (PKS) multienzymes whose individual enzymatic domains are used repeatedly. While only a single PKS multienzyme is sufficient to produce most fungal polyketides, some of these natural products are assembled by two collaborating fungal PKSs. Such collaborations represent significant departures from the canonical biosynthetic logic of fungal polyketide biosynthesis. The overarching goal of the current project is to clarify the presently unknown molecular mechanistic details of the assembly of polyketides on such collaborating PKSs, using the biosynthesis of resorcylic acid lactones (RAL) and the related dihydroxybenzeneacetic acid lactones (DAL) as models. Specifically, the project aims to: 1: Characterize the biosynthesis of dehydrocurvularin, a DAL-type natural product; 2: Express RAL and DAL polyketide synthetases, their appropriate mutants, and hybrid enzymes in alternative microbial hosts, and evaluate the production of polyketides using these enzymes and synthetic substrates; and 3: Demonstrate combinatorial biosynthesis by combining related biosynthetic pathways. These experiments will clarify the programming rules that dictate the assembly of polyketides from simpler metabolites, and control the final shape (cyclization patterns) of such natural products. The lessons learned will advance our understanding of iterative enzymatic catalysis, and contribute to the development of combinatorial biosynthetic methods for the production of "designer" polyketides that may be used as novel agrochemicals or high value specialty chemicals in the future.Broader ImpactsEqually important, the project will incorporate training, education and outreach activities. Extensive research-based training opportunities will be provided to graduate and undergraduate students, including those from minority and underrepresented groups. The project will also train community college undergraduate student interns, and may host high school seniors as interns under the auspices of a successful outreach program of the University of Arizona. All students will learn the interdisciplinary science of microbial metabolite biosynthesis and the practice of combinatorial biosynthesis, thus preparing them for careers in the biotechnology industry and in academic research. Further, professional development opportunities will also be provided to K-12 educators to learn about natural products and their biosynthesis by microorganisms: this knowledge may later be integrated into their science curricula.This project is jointly supported by the Metabolic Biochemistry Program of the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes Program in the Chemistry Division.