Biosynthesis of Antifungal Lipopeptides from Filamentous Fungi

描述（由申请人提供）：由非核糖体肽合成酶（NRPSs）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、万古霉素和环孢素。新近批准的脂质体nrps，如达托霉素和anidulafungin（棘白菌素B的半合成衍生物，本提案的目标）分别是对抗细菌和真菌感染的重要武器。丝状真菌是NRPs的多产生产者，是未来基于天然产物的药物发现的重要来源。最近对bbbb30种真菌的基因组测序显示，每个基因组编码的NRPS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，基于序列预测NRP产物结构、激活原生和异源宿主中其他隐秘的途径、操纵NRP进行目标衍生物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NRPS的第一个全面研究。我们将重点介绍棘白菌素B的生物合成，这是一种真菌脂肽，含有六个高度羟基化的氨基酸。我们最近从生产真菌的芽曲霉（Aspergillus nidulans sp.）中鉴定出了基因簇。这是生物合成实验室（Tang）和合成实验室（Garg）之间的合作提议，旨在解决与真菌NRPSs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，这些结果已经对真菌系统以及遗传和生化工具产生了深入的了解。通过基因敲除/敲入、酿酒酵母菌的异源表达和体外生化分析，我们将对棘白菌素NRPS （EcdA）及其相关酶进行分析，目的如下：1)研究异常氨基酸的激活和生物合成；2)棘白菌素途径中羟基化酶的表征；3)真菌NRPS的异源重组；4)隐真菌NRPSs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.