Cytochrome P450 Enzymes from Streptomyces: Diverse Biocatalysts in Natural Products Biosynthesis

来自链霉菌的细胞色素 P450 酶：天然产物生物合成中的多种生物催化剂

• 批准号: 9371636
• 负责人: Jeffrey Daniel Rudolf
• 金额: $ 9万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-18 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9371636
• 关键词: Actinomyces Infections; Actinomycetales; Advisory Committees; Anabolism; Biochemical; Bioinformatics; Biological; Biological Process; Biology; Biomedical Engineering; Biotechnology; Breathing; Catalysis; Chemicals; Chemistry; Collection; Cytochrome P450; Data; Databases; Development; Development Plans; Diterpenes; Drug Metabolic Detoxication; Engineering; Ensure; Environment; Enzymatic Biochemistry; Enzymes; Foundations; Future; Generations; Genetic; Genome; Genomics; Goals; Gram-Positive Bacteria; Hemeproteins; Homologous Gene; Hormones; Human; Hydroxylation; In Vitro; Life; Mammals; Mentors; Metabolism; Mining; Modeling; Molecular; Natural Product Drug; Natural Products; Nature; Organic Synthesis; Outcome; Pathway Analysis; Pathway interactions; Phase; Physiological; Positioning Attribute; Proteins; Research; Research Institute; Research Project Grants; Research Training; Roentgen Rays; Role; Science; Site-Directed Mutagenesis; Skeleton; Streptomyces; Structure; Structure-Activity Relationship; Terpenes; Testing; Time; Training; Universities; Utah; Variant; X-Ray Crystallography; Xenobiotics; analog; biosynthetic product; career; career development; chemical reaction; design; drug development; drug metabolism; experience; genetic manipulation; hormone biosynthesis; human disease; in vivo; innovation; interdisciplinary approach; microbial; molecular array; novel; post-doctoral training; preference; programs; scaffold; small molecule; structural biology; tenure track

Project Summary/Abstract Research. The goal of this K99/R00 project is to test the hypothesis that cytochromes P450 from Actinomycetales, a diverse subfamily of remarkable biocatalysts, may be used as a means to discover natural products with novel functionalities. We propose to (Aim 1) investigate the sequence–structure–function relationships of novel P450s from selected natural product biosynthetic pathways, (Aim 2) use a P450-focused genome mining approach to discover natural products that have undergone unique P450-catalyzed transformations, and (Aim 3) employ the newly identified P450s, and their homologues, as models for P450 enzymology and bioengineering. The multidisciplinary approach involves bioinformatics analysis, in vitro enzymology, protein X-ray crystallography, in vivo pathway engineering, (un)natural product isolation and structural determination. Superb preliminary data supports both the feasibility of the proposed mentored phase in a two-year time span, and a successful transition to an independent research program. The innovation of this application lies in leveraging the diversity of natural P450 variants from a proprietary Actinomycetales strain collection for discovering unprecedented chemistries that imbue natural products with new functionalities. The significance of this project is the advancement of P450 catalysis and enzymology and the discovery of novel natural products, laying the foundation for future treatments of human diseases. Candidate and Environment. Dr. Rudolf obtained graduate training in mechanistic enzymology and organic synthesis from Dr. C. Dale Poulter, a preeminent terpenoid biochemist, at the University of Utah. He then joined Dr. Ben Shen, a recognized leader in natural products discovery and biosynthesis, at The Scripps Research Institute (TSRI), and received postdoctoral training in actinomycetes genetics and natural products biosynthesis. His long-term career goals include establishing an independent, competitive research program that emphasizes using natural products as inspirations for the discovery and study of novel chemistries, enzymologies, and biologies. His immediate career goals include obtaining training in P450 enzymology and structural biology, and in utilizing genomics for the discovery of novel enzymes and natural products. Career development plan. This proposal was designed to ensure the realization of his career goals by providing intellectual, technical, and professional training during the mentored phase that will lead to an effective transition to an independent, tenure-track position. The primary mentor, Dr. Shen, and co-mentor, Dr. George Phillips, an expert in X-ray crystallography and heme proteins, were chosen for their scientific expertise and mentoring experience. An advisory committee, including Drs. Bradley Moore, Wilfred van der Donk, Sean Brady, and Michael Smanski, all experts in natural products, was explicitly assembled to oversee both the science and career development aspects of this proposal. The research training and excellent mentoring will be supplemented with numerous opportunities for scientific and professional development available at TSRI.

项目概要/摘要 该 K99/R00 项目的研究目标是检验细胞色素 P450 来自的假设。 放线菌是一个具有卓越生物催化剂的多样化亚科，可作为发现天然催化剂的一种手段。 我们建议（目标1）研究序列-结构-功能。 来自所选天然产物生物合成途径的新型 P450 之间的关系，（目标 2）使用以 P450 为重点的 基因组挖掘方法发现经过独特 P450 催化的天然产物 转化，并且（目标 3）采用新鉴定的 P450 及其同源物作为 P450 的模型 酶学和生物工程涉及体外生物信息学分析。 酶学、蛋白质 X 射线晶体学、体内途径工程、（非）天然产物分离和 出色的初步数据支持了拟议导师的可行性。 两年的时间跨度阶段，并成功过渡到独立研究计划。 该应用的创新在于利用专有技术中天然 P450 变体的多样性 放线菌菌株系列，用于发现前所未有的化学物质，使天然产品具有 该项目的意义在于P450催化和酶学的进步。 新型天然产物的发现，为未来治疗人类疾病奠定了基础。 鲁道夫博士获得了机械酶学和有机学的研究生培训。 由犹他大学杰出萜类生物化学家 C. Dale Poulter 博士合成。 加入斯克里普斯自然产品发现和生物合成领域公认的领导者 Ben Shen 博士 研究院（TSRI），并接受放线菌遗传学和天然产物博士后培训 他的长期职业目标包括建立一个独立的、有竞争力的研究项目。 强调使用天然产品作为发现​​和研究新型化学物质的灵感， 他的近期职业目标包括获得 P450 酶学和生物学方面的培训。 结构生物学，以及利用基因组学发现新型酶和天然产物。 该提案旨在通过提供确保实现他的职业目标。 在指导阶段进行智力、技术和专业培训，这将导致有效的 过渡到独立的终身教授职位。主要导师沉博士和共同导师乔治博士。 菲利普斯是 X 射线晶体学和血红素蛋白方面的专家，因其科学专业知识和 顾问委员会，包括 Bradley Moore 博士、Wilfred van der Donk 博士、Sean 博士。 布雷迪和迈克尔·斯曼斯基都是天然产品领域的专家，他们被明确召集来监督这两个项目 该提案的科学和职业发展方面将提供研究培训和出色的指导。 TSRI 提供大量科学和专业发展机会作为补充。

项目成果

The Natural Products Atlas 2.0: a database of microbially-derived natural products.

• DOI: 10.1093/nar/gkab941
• 发表时间: 2022-01-07
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: van Santen JA;Poynton EF;Iskakova D;McMann E;Alsup TA;Clark TN;Fergusson CH;Fewer DP;Hughes AH;McCadden CA;Parra J;Soldatou S;Rudolf JD;Janssen EM;Duncan KR;Linington RG
• 通讯作者: Linington RG