Biosynthesis of Fungal Natural Products on Collaborating Iterative Polyketide Synthases

聚酮类化合物是最大的天然产物家族之一，具有惊人的结构多样性和显著的生物活性范围。聚酮类化合物是由真菌中的聚酮合成酶（PKS）多酶合成的，其单个酶结构域被重复使用。虽然只有一个单一的PKS多酶足以产生大多数真菌聚酮，但其中一些天然产物是由两个合作的真菌PKS组装而成的。这样的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。当前项目的总体目标是利用间环酸内酯（RAL）和相关的二羟基苯乙酸内酯（DAL）的生物合成作为模型，澄清目前未知的聚酮在这些合作PKSs上组装的分子机制细节。具体而言，该项目旨在：1 .表征dal型天然产物脱氢曲vularin的生物合成；2：在替代微生物宿主中表达RAL和DAL聚酮合成酶及其相应的突变体和杂交酶，并评估使用这些酶和合成底物生产聚酮；3：通过结合相关的生物合成途径来展示组合生物合成。这些实验将阐明编程规则，这些规则决定了从更简单的代谢物中组装聚酮，并控制了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，并有助于开发组合生物合成方法来生产“设计”聚酮，这些聚酮可能在未来被用作新型农用化学品或高价值的特种化学品。更广泛的影响同样重要的是，该项目将包括培训、教育和外联活动。将为研究生和本科生提供广泛的以研究为基础的培训机会，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成功的外展计划的支持下，接待高中毕业生作为实习生。所有学生都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中的职业生涯做好准备。此外，还将向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的生物合成：这些知识以后可能会纳入他们的科学课程。本项目由分子与细胞生物科学部代谢生物化学项目和化学部生命过程化学项目联合支持。

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.