Genomic and synthetic biology tools for expressing natural product gene clusters

描述（由申请人提供）：自然界中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所强调的，大约75%的抗菌和抗癌药物是天然产物或由天然产物（NPs）启发的。然而，从历史上看，次级代谢物NPs的发现是一个费力且昂贵的过程，涉及通常难以甚至不可能培养的生产者物种。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，也只有一小部分次生代谢物被发现，因为这些簇在实验室条件下是转录沉默的。努力

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.