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）：自然界中未开发的化学多样性具有生物和药理学相关性的巨大前景。事实上，正如本 RFA 所强调的，大约 75% 的抗菌和抗癌药物是天然产品或受天然产品 (NP) 启发。然而，次级代谢产物纳米颗粒的发现历来是一个费力且成本高昂的过程，涉及的生产物种往往很难甚至不可能培养。最近基因组序列数据的爆炸式增长还表明，即使是经过充分研究的物种，实际上也只发现了一小部分次生代谢物，因为这些簇在实验室条件下转录沉默。努力激活这些神秘或沉默的基因簇非常费力，而且无法扩展到高通量发现。我们建议通过完全绕过天然的、不可培养的宿主，而是开发酵母作为能够表达多种 NP 基因簇的“超级宿主”，来解决 NP 发现中的这一瓶颈。我们建议通过四个具体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 DNA 从头合成并开发新的合成生物学工具和基因组技术，以允许在酵母中异源表达不同的 NP 途径。我们将开发该宿主及其工具，同时对从不同生态起源的 10 种丝状真菌中开采的约 600 个天然产物基因簇进行异源表达。目标 2. 改进酵母作为真菌 NP 基因簇异源转录宿主的工具。我们将表征启动子文库并在酿酒酵母中设计真菌剪接体以允许正确的内含子剪接。目标 3. 改进酵母作为真菌 NP 基因簇异源翻译宿主的工具。我们将通过筛选全基因组酵母和真菌基因库并进行定向实验室进化，并结合蛋白质表达的高通量读数，来鉴定作为异源蛋白质表达改良宿主的酵母菌株。目标 4. 改进酵母作为异源 NP 生产宿主的工具。我们将使用目标 3 中开发的文库和实验室进化筛选，此处应用于筛选以改进 NP 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。我们的提案涉及从基因组到 NP 过程的所有方面，从基因组序列开始到 NP 结束。这里概述的策略将允许发现数量空前的新纳米颗粒。