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

开发用于天然产物异源表达和生物合成研究的高效蓝藻平台。

• 批准号: 9238314
• 负责人: LENA GERWICK
• 金额: $ 29.34万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-12 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9238314
• 关键词: Address; Anabolism; Antibiotic Resistance; Antineoplastic Agents; Area; Biochemical Pathway; Bioinformatics; Biological; Biology; Chemicals; Cloning; Cloning Vectors; Communities; Complex; Cyanobacterium; DNA Sequence; Data; Development; Devices; Distant; Engineering; Enzymes; Escherichia coli; Gene Cluster; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genetic study; Genomics; Goals; Health; Human; Investigation; Knock-out; Knowledge; Marines; Medical; Methods; Modeling; Modification; Molecular; Molecular Biology; Natural Products; Natural Products Chemistry; Organism; Orphan; Outcome; Pathway interactions; Pharmaceutical Preparations; Phylogenetic Analysis; Planets; Plasmids; Production; Regulation; Regulator Genes; Regulatory Element; Reporter Genes; Research; Research Personnel; Synechococcus; Yeasts; antineoplastic antibiotics; base; biosynthetic product; health application; improved; integration site; interest; marine natural product; metabolic profile; novel therapeutics; overexpression; promoter; synthetic biology; tool; transcriptome sequencing; transcriptomics

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.

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。目前，还没有有效的遗传学方法来询问和修饰内源NP 来自海洋丝状蓝细菌的生物合成途径，并且没有强大的异源平台 蓝藻NP途径的表达和基因工程。该项目将开发方法和工具 用于异源表达和操纵鉴定的NP基因簇的工程化蓝细菌 在不适合遗传方法的生物体中，或者在 环境DNA序列该项目将使用合成生物菌株细长聚球藻 PCC 7942和海洋丝状蓝细菌细鳞藻（ISB-3 N94 - 8 PLP）的杂交，以提供两种不同的 而是用于NP途径的表达和工程化的互补遗传平台。长期 该项目的目标是提供有效的平台，用于生产适合于 研究其生物活性，并进行化学修饰，以增强这些活性， 应用.虽然一些相对简单的海洋蓝藻纳米颗粒已经产生了异源 宿主如E.在大肠杆菌中，某些酶的表达和大型复杂途径的表达仍然存在困难。 遗传学上远距离的宿主。我们假设这些蓝藻基因和基因簇的表达 在蓝藻宿主将克服这一障碍。本研究的三个具体目标如下。（一） 将细舌丝藻作为一个广泛适用的遗传平台，用于鉴定、表达和询问 NP路径(2)开发改进的遗传工具，使转移，重构和过度表达 蓝藻平台菌株中的大生物合成途径。(3)从 海洋蓝藻属Okeania在S. elongatus和Leptolyngbya。新的基因工具和方法将被 为S而建。细长体和细叶藓属的克隆载体，包括TAR克隆载体、穿梭质粒、染色体 整合位点、组成型和调控型启动子、报告基因和抗肿瘤标记物， 促进内源性NP途径的操作和来自其它NP途径的途径的异源表达， 有机体该项目将联合收割机结合基因组学、转录组学、生物信息学和基因组学的最新方法， 次级代谢途径，蓝藻基因工程和NP化学，以解决知识 与海洋蓝细菌中医学上重要的NP生物合成有关的空白。该项目将开发 Leptolyngbya和S. elongatus转化为蓝藻次级代谢产物的广泛有用的表达宿主 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