Genomic and synthetic biology tools for expressing natural product gene clusters

用于表达天然产物基因簇的基因组和合成生物学工具

基本信息

批准号：
9316665
负责人：
Ronald Wayne Davis
金额：
$ 214.6万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9316665
关键词：
Address Anabolism Animal Model Anti-Bacterial Agents Antineoplastic Agents Biological Biosensor Bypass Chemicals Collection Communities Coupled Couples DNA DNA biosynthesis Data Directed Molecular Evolution Distant Engineering Ensure Evolution Explosion Fungal Genes Fungal Genome Gene Cluster Gene Library Genes Genetic Genetic Transcription Genome Genomic Library Genomics Growth Health Human Impairment Introns Laboratories Libraries Location Measures Methods Molds Molecular Chaperones Mutagenesis Mutation Natural Product Drug Natural Products Nature Oligonucleotides Organism Pathway interactions Peptide Hydrolases Pharmacology Problem Solving Process Production Productivity Proteins RNA Splicing Saccharomyces cerevisiae Series Spliceosomes Structure Technology Therapeutic Translating Translations Yeasts base cost design functional genomics fungus gene product genetic selection genome sequencing genome-wide genome-wide analysis genomic data improved interest next generation novel overexpression programs promoter protein degradation protein expression public health relevance response scaffold screening success synthetic biology tool whole genome

项目摘要

DESCRIPTION (provided by applicant): The untapped chemical diversity in nature holds tremendous promise of biological and pharmacological relevance. Indeed, as highlighted in this RFA, roughly 75% of antibacterial and anticancer drugs are natural products or inspired by natural products (NPs). However, discovery of secondary metabolite NPs has historically been a laborious and costly process involving producer species that are often difficult to impossible to cultivate. The recent explosion in genome sequence data has additionally revealed that only a fraction of the secondary metabolites from even well-studied species have actually been discovered, due to the clusters being transcriptionally silent under laboratory conditions. Efforts to activate these cryptic or silent gene clusters have been laborious and not scalable to high-throughput discovery. We propose to address this bottleneck in NP discovery by altogether bypassing native, uncultivable hosts and instead developing yeast as a "super-host" capable of expressing a large variety of NP gene clusters. We propose to achieve this objective through four specific aims. Aim 1. Tools for NP DNA design and synthesis. We will use de novo DNA synthesis and develop new synthetic biology tools and genomic technologies to allow heterologous expression of diverse NP pathways in yeast. We will develop this host and its tools while performing heterologous expression of ~600 natural product gene clusters mined from 10 filamentous fungi of diverse ecological origin. Aim 2. Tools to improve yeast as a host for heterologous transcription of fungal NP gene clusters. We will characterize promoter libraries and engineer a fungal spliceosome in S. cerevisiae to allow proper intron splicing. Aim 3. Tools to improve yeast as a host for heterologous translation of fungal NP gene clusters. We will identify yeast strains that serve as improved hosts for heterologous protein expression by screening genome-wide yeast and fungal gene libraries and performing directed in-lab evolution, coupled with a high-throughput readout for protein expression. Aim 4. Tools to improve yeast as a host for heterologous NP production. We will use the library and in-lab evolution screening developed in Aim 3, here applied to a screen for improved NP production. We will also develop tools that users can use to improve expression their own NP pathways of interest. Our proposal addresses all aspects of the genome to NP process, starting with genomic sequence and ending with NPs. The strategy outlined here will allow discovery of an unprecedented number of new NPs.

描述（由申请人提供）：本质上未开发的化学多样性具有生物学和药理相关性的巨大希望。确实，正如该RFA中突出的那样，大约75％的抗菌和抗癌药是天然产物或受天然产物（NP）启发的。但是，从历史上看，二级代谢物NP的发现一直是一个费力且昂贵的过程，涉及生产者物种，这些物种通常难以耕种。基因组序列数据中最近的爆炸还表明，由于在实验室条件下的转录群集在转录中静音，实际上只发现了来自甚至良好的物种的次生代谢产物的一部分。努力激活这些神秘或无声的基因簇很费力，无法扩展到高通量发现。我们建议通过完全绕过天然，无法养活的宿主，而是将酵母作为一种能够表达大量NP基因簇的“超级主持人”来解决NP发现中的这种瓶颈。我们建议通过四个特定目标来实现这一目标。目标1。NP DNA设计和合成的工具。我们将使用从头DNA的合成，并开发新的合成生物学工具和基因组技术，以允许酵母中各种NP途径的异源表达。我们将开发该宿主及其工具，同时进行〜600个自然产物基因簇的异源表达，这些基因簇从10种生态起源的10种丝状真菌开采。目标2。改善酵母作为真菌NP基因簇异源转录的宿主的工具。我们将表征启动子库和工程师在酿酒酵母中的真菌剪接体，以允许适当的内含子剪接。目标3。改善酵母作为真菌NP基因簇异源翻译的宿主的工具。我们将通过筛选全基因组酵母和真菌基因库并进行定向在LAB进化中，再加上蛋白质表达的高通量读数，从而确定酵母菌菌株可作为改进的异源蛋白表达的宿主。目标4。改善酵母作为异源NP生产的宿主的工具。我们将使用AIM 3中开发的图书馆和LAB内进化筛选，此处应用于屏幕以改善NP的生产。我们还将开发用户可以用来改善表达自己感兴趣的NP途径的工具。我们的建议从基因组序列开始，并以NP结束，解决了基因组到NP过程的各个方面。此处概述的策略将允许发现前所未有的新NP。