Development of an efficient cyanobacterial platform for heterologous expression and biosynthetic interrogations of natural products.

项目摘要 该项目将克服阻碍充分开发海洋天然产品的主要障碍 健康应用程序。目前，还没有有效的遗传方法来询问和修饰内源NP 海洋丝状蓝藻的生物合成途径，以及没有强大的平台支持异源生物 蓝藻NP途径的表达与基因工程。该项目将开发方法和工具 用于工程蓝藻对NP基因簇的异源表达和操纵鉴定 在不适用于遗传方法的生物体中，或在 环境DNA序列。该项目将使用合成生物学菌株细长聚球藻。 PCC7942和海洋丝状蓝藻Leptolyngbya(ISB-3N94-8PLP)提供了两种截然不同的 而是为NP途径的表达和工程提供互补的遗传平台。长期的 该项目的目标是提供高效的平台，以生产适合于 研究它们的生物活性和化学修饰以增强这些对健康的活性 申请。尽管一些相对简单的海洋蓝藻NPs已经在异源的 宿主如大肠杆菌，某些酶和大的复杂途径的表达仍然困难 在系统发育上相距遥远的宿主。我们假设这些蓝藻基因和基因簇的表达 在蓝藻中，宿主将克服这一障碍。本研究的三个具体目标如下。(1) 将Leptolynbya建立为一个广泛适用的识别、表达和询问的遗传平台 NP通路。(2)开发改进的遗传工具，使转移、重构和过度表达成为可能 蓝藻平台菌株的大型生物合成途径。(3)快线两条孤儿径 长链藻和细长链霉属海洋蓝藻Okeania。新的遗传工具和方法将是 将包括TAR克隆载体、穿梭质粒、染色体 整合位点、构成和调控启动子、报告基因和抗生素耐药性标记 促进内源性NP途径的操纵以及来自其他来源的途径的异源表达 有机体。该项目将结合基因组学、转录组学、生物信息学等最先进的方法 次生代谢物途径、蓝藻基因工程和NP化学 海洋蓝藻中与医学上重要的NP生物合成相关的缺口。该项目将得到发展 Leptolyngbya和Septolyngbya长链霉菌成为蓝藻次生代谢产物广泛适用的表达宿主 酶和整个NP生物合成途径，这些宿主和遗传工具将提供给 NP研究社区。

Project Summary This project will overcome a main obstacle impeding progress to fully exploit marine natural products (NPs) for health applications. Currently, there are no efficient genetic methods to interrogate and modify endogenous NP biosynthetic pathways from marine filamentous cyanobacteria, and no robust platforms for heterologous expression and genetic engineering of cyanobacterial NP pathways. This project will develop methods and tools for engineering cyanobacteria for the heterologous expression and manipulation of NP gene clusters identified in organisms that are not amenable to genetic methods, or orphan NP gene clusters that are identified in environmental DNA sequences. The project will use the synthetic-biology strain Synechococcus elongatus PCC7942 and the marine filamentous cyanobacterium Leptolyngbya (ISB-3N94-8PLP) to provide two distinct but complementary genetic platforms for the expression and engineering of NP pathways. The long-term objectives of this project are to provide efficient platforms for the production of NPs in quantities suitable for studying their biological activities and for chemical modifications to enhance those activities for health applications. Although a few relatively simple marine cyanobacterial NPs have been produced in heterologous hosts such as E. coli, difficulties remain for the expression certain enzymes and of large complex pathways in phylogenetically distant hosts. We hypothesize that expression of these cyanobacterial genes and gene clusters in cyanobacterial hosts will overcome this obstacle. The 3 specific aims of this research are as follows. (1) Establish Leptolyngbya as a broadly applicable genetic platform for identification, expression, and interrogation of NP pathways. (2) Develop improved genetic tools to enable the transfer, refactoring, and overexpression of large biosynthetic pathways in platform strains of cyanobacteria. (3) Express two orphan pathways from the marine cyanobacterial genus Okeania in S. elongatus and Leptolyngbya. New genetic tools and methods will be created for S. elongatus and Leptolyngbya that will include TAR cloning vectors, shuttle plasmids, chromosomal integration sites, constitutive and regulated promoters, reporter genes, and antibiotic-resistance markers to facilitate manipulation of endogenous NP pathways and for the heterologous expression of pathways from other organisms. This project will combine state-of-the-art approaches in genomics, transcriptomics, bioinformatics of secondary metabolite pathways, cyanobacterial genetic engineering, and NP chemistry to address knowledge gaps related to medically important NP biosynthesis in marine cyanobacteria. The project will develop Leptolyngbya and S. elongatus into broadly useful expression hosts for cyanobacterial secondary-metabolite enzymes and entire NP biosynthetic pathways, and these hosts and genetic tools will be made available to the NP research community.