Genomic and synthetic biology tools for expressing natural product gene clusters

描述(由申请人提供)：自然界中尚未开发的化学多样性具有巨大的生物学和药理学意义。事实上，正如本RFA中强调的那样，大约75%的抗菌和抗癌药物是天然产品或受到天然产品(NP)的启发。然而，从历史上看，发现次生代谢物NPs是一个费力且昂贵的过程，涉及生产者物种，而这些物种往往难以培养。最近基因组序列数据的爆炸性增长还表明，实际上只发现了一小部分来自研究得很好的物种的次生代谢物，因为在实验室条件下，这些簇在转录上是沉默的。努力 激活这些隐秘的或沉默的基因簇一直是费力的，而且不能扩展到高通量发现。我们建议通过完全绕过天然的、不可培养的宿主来解决NP发现中的这个瓶颈，而是将酵母发展为能够表达大量NP基因簇的“超级宿主”。我们建议通过四个具体目标来实现这一目标。目的1.设计和合成NP DNA的工具。我们将使用从头合成DNA，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。我们将开发这种宿主及其工具，同时对从10个不同生态来源的丝状真菌中挖掘的约600个天然产物基因簇进行异源表达。目的2.提高酵母作为真菌NP基因簇异源转录宿主的工具。我们将确定启动子文库的特征，并在酿酒酵母中设计一个真菌剪接体，以实现正确的内含子剪接。目的3.提高酵母作为真菌NP基因簇异源翻译宿主的方法。我们将通过筛选全基因组酵母和真菌基因库，并进行实验室内的定向进化，以及高通量的蛋白质表达读数，来确定作为异源蛋白表达的改进宿主的酵母菌株。目的4.提高酵母作为异源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内进化筛选，这里应用于改进NP生产的筛选。我们还将开发工具，用户可以使用这些工具来改善他们自己感兴趣的NP途径的表达。我们的建议涉及基因组到NP过程的方方面面，从基因组序列开始，以NP结束。这里概述的战略将允许发现前所未有的数量的新核动力源。

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.