Engineered Actinomycete Production Hosts for Heterologous Natural Product Discovery

 描述（由申请人提供）：现在众所周知，大多数天然产物化学多样性尚未使用传统的，依赖于培养的策略进行探索。此外，来自培养的微生物来源的基因簇的显著部分在标准发酵条件下保持沉默。理论上，天然产物基因簇的异源表达可以提供来自未培养生物和沉默途径的化学多样性。然而，最近的研究表明，这种方法的主要限制是在未工程化的异源底盘中缺乏朝向次级代谢途径的足够的代谢通量，这限制了产量，使得去复制和鉴定异源产生的天然产物具有挑战性。为了解决这些限制，我们提出开发一组稳健的异源生产宿主，以实现大规模、系统性的去复制和天然产物的发现。该系统还将允许快速， 直接优化现有天然产品的生产和产品纯化，为商业化发挥重要作用。与更广泛使用的生物制造宿主（例如大肠杆菌和酿酒酵母）相比，本申请中的工程化放线菌天然产生复杂的天然产物，并且含有必需的次级代谢前体、用于大型生物合成蛋白质折叠的伴侣机制、转录激活机制和用于产生复杂天然产物的内源性抗性机制。该I期申请旨在开发一组用于异源生产天然产物的优化放线菌宿主，这些宿主一起提供了一种用于一致生产广谱天然产物类别的工具，而无论其天然表达水平如何。这些放线菌菌株因其能够以高滴度生产不同类别的天然产物而被选择，首先将B合理的工程改造，以去除竞争性天然产物途径，以增加重要前体的可用性，并将添加针对核糖体的靶向突变。这些策略已被证明是一个成功的发展链霉菌coelicolor到一个改进的异源宿主。通过生成一组这些“超级宿主”，我们将扩大可以利用这项技术的天然产品群的范围。此外，我们将使用新的微流体菌株改良技术在这一宿主群体的基础上建立。这种微流体液滴培养和筛选平台允许菌落筛选速度提高>10，000倍，并将用于鉴定进一步提高这些优化宿主中天然产物产量的因果突变。总的来说，本申请旨在开发一组优化的生产菌株，其将允许更系统地、去复制和发现来自未栽培和遗传沉默来源的天然产物。在这样做时，他们将提供一个 来自天然产物的生物活性化学多样性的可再生来源。

 DESCRIPTION (provided by applicant): It is now well-known that the majority of natural product chemical diversity has not been explored using traditional, cultivation-dependent strategies. Furthermore, a significant fraction of gene clusters from cultivated microbial sources remain silent under standard fermentation conditions. In theory, the heterologous expression of natural product gene clusters can provide access to chemical diversity from both uncultivated organisms and silent pathways. Recent studies have shown, however, that a major limitation of this approach is a lack of sufficient metabolic flux toward secondary metabolic pathways in an unengineered heterologous chassis, which limits production yield, making it challenging to dereplicate and identify heterologously produced natural products. To address these limitations we propose to develop a panel of robust heterologous production hosts to enable large- scale, systematic de-replication and discovery of natural products. This system will also allow for rapid, drop-in optimization of manufacturing and product purification for existing natural products, playing an important role for commercialization. In contrast to more widely-used biomanufacturing hosts, such as Escherichia coli and Saccharomyces cerevisiae, the engineered actinomycetes in this application produce complex natural products natively and contain the necessary secondary metabolic precursors, chaperone mechanisms for large biosynthetic protein folding, transcriptional activation machinery, and endogenous resistance mechanisms for the production of complex natural products. This Phase I application aims to develop a panel of optimized actinomycete hosts for heterologous production of natural products that, together, provide a tool for the consistent production of a broad spectrum of natural product classes regardless of their native expression levels. These actinomycete strains, chosen for their ability to produce diverse classes of natural products at high titer, will first b rationally engineered to remove competing natural product pathways to increase the availability of important precursors and targeted mutations to the ribosome will be added. These strategies have been shown to be a successful in developing Streptomyces coelicolor into an improved heterologous host. By generating a panel of these "superhosts", we will broaden the spectrum of natural product clusters that can utilize this technology. In addition, we will build upon this panl of hosts using a novel microfluidic strain improvement technology. This microfluidic droplet cultivation and screening platform allows for a >10,000x improvement in the speed in colony screening and will be used to identify causal mutations that further increase the natural product production yield in these optimized hosts. Overall, this application aims to develop a panel of optimized production strains that will allow for more systematic, de-replication and discovery of natural products from uncultivated and genetically silent sources. In doing so, they will provide a renewable source of biologically-active chemical diversity from natural products.