Mechanistic Studies of the Functional Consequences of Heterologous Expression of Actinomycetal Megasynthases

项目总结/摘要 来自放线菌目的细菌，特别是来自链霉菌属的那些， 生物活性天然产品的一些最多产的生产商，包括超过75%的 市售抗生素。尽管其他细菌的生物合成 放线菌的代谢天赋是制药的黄金标准， 的发现特别是，它们含有许多模块化的途径，如I型聚酮化合物 核糖体肽合成酶（PKS）和I型非核糖体肽合成酶（NRPS），它们已经被 以其在工程生物合成方面的巨大潜力而闻名，已有30多年的历史。然而，在这方面， 放线菌的生长特征和遗传系统带来了许多挑战， 作为异源宿主不太理想，特别是对于工程化的 系统或用于酶学研究的蛋白质生产。此外，它们的高GC基因组 （~75% GC）使常规分子生物学、遗传操作和转录和 翻译效率这对于大合成酶如PKS尤其如此 和NRPS，其尺寸大，并且通常表达差，作为截短产物，或者不表达。 不能正确折叠。虽然这些问题在天然产物酶学中进行了讨论， 社区，很少有系统的研究存在于文献中，以了解功能 异源宿主选择和重构的后果，特别是在它 影响蛋白质功能。此外，许多理想的系统， 大合成酶（如体外转录-翻译; TXTL）在细胞内产生不规则性， 到截断和折叠，这也是很差的特点。我们将研究功能 宿主和重构依赖性表达对这类放线菌的影响 megasynthases在蛋白质水平。本提案中描述的特征将导致 更系统地了解不同制度的利弊， 用于不同应用的异源表达，包括表征和分析 工程化的大合成酶并最终产生小分子代谢物。这项工作将 提供实现蛋白质的全部潜力从放线菌作为一个丰富的来源， 合成生物学是工程代谢的一部分。

PROJECT SUMMARY/ABSTRACT Bacteria from the order Actinomycetales, especially those from the genus Streptomyces, are some of the most prolific producers of bioactive natural products, including over 75% of commercially available antibiotics. Although other orders of bacteria harbor biosynthetic pathways, the metabolic giftedness of actinomycetes is the gold standard for pharmaceutical discovery. In particular, they harbor many pathways that are modular such as type I polyketide synthases (PKSs) and type I non-ribosomal peptide synthetases (NRPSs), which have been noted for their considerable potential for engineered biosynthesis for well over 30 years. However, actinomycetes present a number of challenges with their growth profiles and genetic systems that serve as less than ideal as heterologous hosts, especially for rapid profiling of engineered systems or for protein production for enzymology studies. Furthermore, their high GC genomes (~75% GC) complicate routine molecular biology, genetic manipulations, and transcriptional and translational efficiency. This is particularly the case with megasynthase enzymes such as PKSs and NRPSs, which are large in size and frequently express poorly, as truncated products, or do not fold correctly. While such issues are discussed in the natural products enzymology community, few systematic studies exist in the literature to understand the functional consequences of heterologous host choice and refactoring, especially at the level at which it affects protein function. Furthermore, many of the systems that would be ideal to profile megasynthases (such as in vitro transcription-translation; TXTL) create irregularities with regard to truncation and folding, which are also poorly characterized. We will examine the functional consequences of host and refactoring-dependent expression on such actinomycetal megasynthases at the protein level. The characterization described in this proposal will lead to a more systematic understanding of the benefits and drawbacks of different systems of heterologous expression for different applications which include characterizing and profiling engineered megasynthases and ultimately producing small molecule metabolites. This work will afford the realization of the full potential of proteins from actinomycetes as a rich source for synthetic biology parts for engineered metabolism.