Biosynthesis of Antifungal Lipopeptides from Filamentous Fungi

描述(申请人提供)：由非核糖体多肽合成酶(NRPS)生物合成的天然产物非核糖体多肽(NRP)是人类已知的最重要的疗法之一，包括青霉素、万古霉素和环孢素。新批准的脂氧核糖核酸，如达托霉素和阿尼杜拉芬净(本提案的靶标棘球菌素B的半合成衍生物)分别是对抗细菌和真菌感染的重要武器。丝状真菌是NRPs的高产生产者，是未来基于天然产物的药物发现的重要来源。最近对30种真菌的基因组测序显示，每个基因组编码的NRPS远远多于已鉴定的NRPS，大多数NRPS基因在实验室培养条件下是沉默的。因此，根据序列预测NRP产物的结构，激活本源和异源宿主中原本难以理解的途径，并操纵NRPS以合成目标化合物，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对真菌NRPS的第一次全面研究。我们将重点介绍棘球菌素B的生物合成，它是一种真菌脂肽，含有六个高度羟化的氨基酸。我们最近从产生菌Aspergillusnidulans sp.中鉴定了该基因簇。生物合成(Tang)和合成(GAG)实验室之间的这项合作提案旨在解决与真菌NRPS相关的独特化学和生物学问题。我们提出的工作是基于广泛的初步结果，这些结果产生了对真菌系统的深入知识，以及遗传和生化工具。利用基因敲除/敲入、异源表达和体外生化检测相结合的方法，我们将从以下四个方面剖析棘球蛋白NRPS(Echocandin NRPS)及其相关的酶：1)检测异常氨基酸的激活和生物合成；2)表征Echinocandin途径中的羟基酶；3)真菌NRPS的异源重组；以及4)隐匿真菌NRPS的基因组挖掘。

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.