Biosynthesis of Antifungal Lipopeptides from Filamentous Fungi

丝状真菌抗真菌脂肽的生物合成

• 批准号: 8664531
• 负责人: NEIL K GARG
• 金额: $ 34.02万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8664531
• 关键词: Address; Amino Acids; Anabolism; Anidulafungin; Antifungal Agents; Aspergillus nidulans; Bacterial Infections; Biochemical; Biochemistry; Biological Assay; Biological Factors; Biology; Chemistry; Complex; Constitution; Cyclosporine; Cyclosporins; Cytochrome P450; Daptomycin; Engineering; Enzymes; Family; Future; Gene Cluster; Genes; Genetic; Genome; Glean; Goals; Health; Human; Hydroxyl Radical; In Vitro; Individual; Iron; Kinetics; Knock-in Mouse; Knock-out; Knowledge; Laboratory culture; Ligase; Methods; Mining; Mixed Function Oxygenases; Modeling; Molds; Mycoses; Organism; Ornithine; Oxygenases; Pathway interactions; Penicillins; Peptides; Process; Proline; Role; Saccharomyces cerevisiae; Source; Specificity; Structure; Substrate Specificity; System; Therapeutic; Time; Transcriptional Activation; Vancomycin; Work; base; chemical synthesis; combat; drug discovery; enzyme pathway; experience; fungal genetics; fungus; genome sequencing; innovation; oxidation; peptide synthase; polypeptide; reconstitution; tool; weapons

DESCRIPTION (provided by applicant): Nonribsomal peptide (NRP) natural products biosynthesized by nonribosomal peptide synthetases (NRPSs) are among the most important therapeutics known to mankind, including penicillin, vancomycin and cyclosporin. Newly approved lipo-NRPs such as daptomycin and anidulafungin (a semisynthetic derivative of echinocandin B, the target of this proposal) are important weapons in combating bacterial and fungal infections, respectively. Filamentous fungi are prolific producers of NRPs and represent an important source for future natural-product based drug discovery. Recent genome sequencing of >30 fungi species have revealed each genome encodes far more NRPS than the identified NRPs, with a majority of the NRPS genes being silent during laboratory culturing conditions. Therefore, having the abilities to predict NRP product structure based on sequence, to activate otherwise cryptic pathways in both native and heterologous hosts, and to manipulate the NRPSs towards the biosynthesis of targeted derivatives are important goals towards realizing the fungal biosynthetic potential. Our proposed work here represents the first comprehensive study of a fungal NRPS to date. We will focus on the biosynthesis of echinocandin B, which is a fungal lipopeptide that contains six highly hydroxylated amino acids. We have recently identified the gene cluster from the producing organism Aspergillus nidulans sp. This collaborative proposal between biosynthetic (Tang) and a synthetic (Garg) labs aims to address the unique chemistry and biology associated with fungal NRPSs. Our proposed work is based on extensive preliminary results that have generated deep knowledge with the fungal systems, as well genetic and biochemical tools. Using a combination of genetic knockout/knock-in, heterologous expression in Saccharomyces cerevisiae and in vitro biochemical interrogation, we will dissect the echinocandin NRPS (EcdA) and associated enzymes using the following four aims: 1) Examine the activation and biosynthesis of unusual amino acids; 2) Characterization of the hydroxylases in the echinocandin pathway; 3) Heterologous reconstitution of fungal NRPS; and 4) Genome mining of cryptic fungal NRPSs.

描述（由申请人提供）：由非核糖体肽合成酶（NRPS）生物合成的非核糖体肽（NRP）天然产物是人类已知的最重要的治疗药物之一，包括青霉素、万古霉素和环孢菌素。新批准的脂质体-NRP如达托霉素和阿尼芬净（棘白菌素B的半合成衍生物，该提案的目标）分别是对抗细菌和真菌感染的重要武器。丝状真菌是NRP的多产者，代表了未来基于天然产物的药物发现的重要来源。最近对>30种真菌物种的基因组测序已经揭示了每个基因组编码的NRPS远多于所鉴定的NRPS，其中大多数NRPS基因在实验室培养条件下是沉默的。因此，具有基于序列预测NRP产物结构的能力，激活天然和异源宿主中的其他隐蔽途径，以及操纵NRPS朝向靶向衍生物的生物合成的能力是实现真菌生物合成潜力的重要目标。我们提出的工作是迄今为止对真菌NRPS的第一次全面研究。我们将重点研究棘白菌素B的生物合成，棘白菌素B是一种含有六个高度羟基化氨基酸的真菌脂肽。我们最近已经确定了生产生物构巢曲霉属物种的基因簇。生物合成（唐）和合成（加格）实验室之间的合作建议旨在解决与真菌NRPS相关的独特化学和生物学。我们提出的工作是基于广泛的初步结果，这些结果已经产生了对真菌系统以及遗传和生物化学工具的深入了解。本论文采用基因敲除/敲入、酵母异源表达和体外生物化学研究相结合的方法，对棘白菌素NRPS（EcdA）及其相关酶进行了研究，主要目的如下：1）研究棘白菌素NRPS的激活和生物合成，2）棘白菌素途径中的羟化酶的特性，3）真菌NRPS的异源重组，4）棘白菌素NRPS的生物合成。（4）隐生真菌NRPS的基因组挖掘。