CAREER: Investigation and Redesign of Nonribosomal Peptide Synthetases

知识价值：该项目涉及已知天然产物生物分子的生物合成途径的调查和重新设计。这种途径的工程化是目前最有希望开发和生产新型结构多样的生物活性分子的途径之一。有效的组合生物合成主要依赖于参与天然产物形成的酶的机制信息。在双插层天然产物硫代珊瑚碱的基因簇中发现了不完全了解的，有趣的，甚至是独特的酶。该项目主要通过诱变操作、单个酶的机制和结构表征，在体外重建硫代可拉啉生物合成途径，以实现以下目标：(1)获得操纵氨基酸l -色氨酸衍生化的能力，用于未来生产新型双插层剂和硫肽抗生素；（ii）了解mbth样蛋白刺激腺苷化结构域（非核糖体肽合成酶装配线的组成部分）活性的机制以及这些酶在组合生物合成中的潜在作用；（iii）深入了解中断的腺苷化结构域的功能；（iv）开发具有广泛或改变底物特异性的新型腺苷化结构域，以扩展操纵非核糖体肽合成酶装配线进行组合合成的能力。该研究有可能促进对独特酶转化机制的理解，并有望为自然生物合成途径工程的新工具的开发做出贡献。更广泛的影响：通过该项目的拟议活动，来自密歇根大学周围当地高中的学生，以及本科生和研究生，将获得生物化学和结构生物学领域现代研究的实践经验。通过开发一个免费访问的非核糖体多肽、聚酮及其杂合体数据库，本科生和药学博士学生将被引入研究，并有机会提高他们的整体科学素养。最后，通过高中外展计划，高中生不仅将被介绍到现代和跨学科的科学，而且将充分了解和指导规划一个成功的和有回报的科学生涯。目标是建立一个综合的教育和研究计划，以培养对化学和生物学界面工作感兴趣的未来科学家，并激发和培养学生在不同教育阶段对科学的兴趣。

Intellectual merit: The project involves an investigation and redesign of biosynthetic pathways of known natural product biomolecules. The engineering of such pathways is currently one of the most promising routes toward the development and production of novel structurally diverse biologically active molecules. Efficient combinatorial biosynthesis critically depends on mechanistic information on enzymes involved in natural product formation. Incompletely understood, intriguing, and even unique enzymes are found in the gene cluster of the bisintercalator natural product thiocoraline. The project uses in vitro reconstitution of the thiocoraline biosynthetic pathway, predominantly through mutagenetic manipulation, mechanistic, and structural characterization of individual enzymes, in working towards (i) gaining the ability to manipulate the derivatization of the amino acid L-tryptophan for future production of novel bisintercalators and thiopeptide antibiotics, (ii) understanding the mechanism of stimulation of activity of adenylation domains (integral components of nonribosomal peptide synthetase assembly-lines) by MbtH-like proteins and the potential role of these enzymes in combinatorial biosynthesis, (iii) gaining insight into the function of interrupted adenylation domains, and (iv) developing novel adenylation domains with broad or altered substrate specificity to extend the ability to manipulate nonribosomal peptide synthetase assembly-lines for combinatorial synthesis. The research has the potential to advance the understanding of the mechanism of unique enzymatic transformations and is expected to contribute to the development of new tools for engineering of natural biosynthetic pathways.Broader impacts: Through the proposed activities of the project, students from local high schools surrounding the University of Michigan, as well as undergraduate and graduate students, will receive the hands-on experience of modern research in the fields of biochemistry and structural biology. Through the development of a freely accessible database on nonribosomal peptides, polyketides, and their hybrids, undergraduate and PharmD students will be introduced to research and will have the opportunity to raise their overall scientific literacy. Finally, through a high school outreach program, high school students will not only be introduced to modern and interdisciplinary science, but also will be well informed on and provided with guidance for planning a successful and rewarding scientific career. The goal is to establish an integrated educational and research program to develop future scientists interested in working at the interface of chemistry and biology and to spark and foster the interest of students in science at different stages of their education.