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）：非ribsomal肽（NRP）天然产物由非核糖体肽合成酶（NRPS）（NRPSS）是人类已知的最重要的治疗方法之一，包括青霉素，宽霉素和环孢菌素。新批准的脂肪NRP，例如Daptomycin和Anidulafungin（Echinocandin B的半合成衍生物，该提案的靶标）是对细菌和真菌感染的分别进行对抗的重要武器。丝状真菌是NRP的产生者，代表了未来基于自然产品的药物发现的重要来源。最近的30种真菌种类的基因组测序揭示了每个基因组的编码远比已鉴定的NRP的NRP要多得多，而在实验室培养条件下，大多数NRP基因都保持沉默。因此，具有根据序列预测NRP产品结构的能力，激活天然和异源宿主中的其他神秘途径，并操纵NRPS朝着靶向衍生物的生物合成的生物合成是实现真菌生物合成潜力的重要目标。我们在这里提出的工作代表了迄今为止真菌NRP的首次全面研究。我们将重点关注棘皮素B的生物合成，它是一种真菌脂肽，其中含有六种高度羟化氨基酸。我们最近从生产生物曲霉的nidulans sp中确定了基因簇。生物合成（Tang）与合成（GARG）实验室之间的这种协作提议旨在解决与真菌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，这些结果与真菌系统以及遗传和生化工具产生了深厚的知识。使用遗传基因敲除/敲除，酿酒酵母中的异源表达和体外生化询问，我们将使用以下四个目的剖析echinocandin nrps（ECDA）和相关的酶，以下四个目的：1）检查激活和生物合成的非唯一氨基酸的激活和生物合成； 2）在棘齿素途径中的羟化酶的表征； 3）真菌NRP的异源重构； 4）神秘真菌NRPS的基因组开采。