A Robust Platform for Reconstituting and Engineering Iterative Megasynthases

描述(申请人提供)：大自然使用一系列令人惊叹的酶来制造小分子天然产品。在最有趣但最不被了解的酶中，这些化合物是在丝状真菌中发现的迭代聚酮合成酶(IPKS)。与以流水线方式工作的细菌I型PKS不同，IPKS是一种超级合成酶，它通过在不同的组合中重复使用一组催化结构域来产生结构不同的真菌代谢物来迭代地发挥作用。对最近测序的真菌物种基因组的生物信息学分析表明，每个基因组都包含大量编码IPKS的基因。IPKS的总数明显多于从这些物种中分离到的已知多酮和聚酮非核糖体肽，这表明在培养条件下这些真菌中的大多数生物合成基因是沉默的。这反过来又表明，真菌物种可能具有合成大量天然产品的未开发潜力。此外，由于无法从天然真菌宿主或异源曲霉宿主中获得足够数量的功能性纯化的巨合酶，IPKs的分析和工程一直受到阻碍。因此，对IPKS代谢产物组装的程序设计还不是很清楚。有待阐明的关键方面包括：1)每个结构域在巨合酶中的催化和结构作用；2)催化结构域的底物特异性及其对巨合酶功能扰动的耐受性；以及3)在每次催化迭代中控制不同催化结构域选择的因素。这项计划的目的是开发遗传优势的酿酒酵母作为异源宿主，用于IPKs的重组、分析和工程，特别是神秘的高度还原的IPKs，如与洛伐他汀生物合成相关的LOVB。我们已经积累了大量的初步数据来证明酿酒酵母是以功能形式表达这些巨合酶的高度强健的宿主，并且可以利用纯化的酶在体内和体外促进聚酮产物的产生。具体目标如下：1)设计和优化酿酒酵母以产生真菌代谢物和巨合酶；2)在酿酒酵母中重组真菌巨合酶；3)利用酿酒酵母对真菌PKS进行生化分析；以及4)以酿酒酵母为宿主进行丝状真菌基因组挖掘。 与公共卫生相关：丝状真菌是天然产品的丰富来源。其中，多酮类化合物是一类结构多样的重要天然产物。聚酮是由聚酮合成酶(PKSS)产生的。许多真菌物种的基因组测序显示，每个生物体都含有大量的PKS，但其中许多PKS具有未知的功能或产生未知的代谢物。因此，丝状真菌可以被认为是天然产品生产商的“后进者”。我们建议使用酿酒酵母作为表达宿主来异源生产真菌PKS。我们将利用这种遗传优势宿主来重建已知和未知功能的真菌PKS，对已测序的真菌物种进行基因组挖掘，并对新的真菌衍生天然产物进行工程生物合成。

DESCRIPTION (provided by applicant): Nature uses an amazing array of enzymes to make small molecule natural products. Among the most interesting but least understood enzymes making these compounds are the iterative polyketide synthases (IPKSs) found in filamentous fungi. In contrast to the well-studied bacterial type I PKSs that operate in an assembly-line fashion, IPKSs are megasynthases that function iteratively by using a single set of catalytic domains repeatedly in different combinations to produce structurally diverse fungal metabolites. Bioinformatics analysis of the genomes of recently sequenced fungal species revealed that each genome contains a large number of genes encoding IPKSs. The total numbers of IPKSs significantly outnumber the known polyketides and polyketide-nonribosomal peptides isolated from these species, suggesting that a majority of biosynthetic genes are silent in these fungi under cultivating conditions. This in turn suggests that the fungal species may have untapped potential to synthesize a much large number of natural products. Furthermore, analysis and engineering of IPKSs have been hampered by inability to obtain sufficient amounts of the functional purified megasynthase from either the native fungal host or heterologous Aspergillus hosts. As a result, the programming that governs metabolite assembly by IPKSs is not understood. Key aspects that remain to be elucidated include: 1) the catalytic and structural roles of each domain in the megasynthase; 2) substrate specificities of the catalytic domains and their tolerance to perturbation in megasynthase functions; and 3) factors governing the choice of different combinations of catalytic domains during each iteration of catalysis. The objective of this proposal is to develop the genetically superior Saccharomyces cerevisiae as a heterologous host for reconstitution, analysis and engineering of IPKSs, especially the enigmatic highly-reducing IPKS, such as LovB associated with Lovastatin biosynthesis. We have accumulated a significant body of preliminary data to demonstrate that S. cerevisiae is a highly robust host for expressing these megasynthases in functional forms, and can facilitate the production of polyketide products both in vivo and in vitro with purified enzymes. The following specific aims will be pursued: 1) Engineer and optimize S. cerevisiae towards producing fungal metabolites and megasynthases; 2) Reconstitution of fungal megasynthases in S. cerevisiae; 3) Biochemical analysis of fungal PKS using S. cerevisiae; and 4) Genome mining of filamentous fungi using S. cerevisiae as a host. PUBLIC HEALTH RELEVANCE: Filamentous fungi are a rich source of natural products. Among them, polyketides represent an important family of structurally diverse natural products. Polyketides are produced by polyketide synthase (PKSs). Genome sequencing of many fungal species has revealed each organism contains a large number of PKSs, yet many of these PKSs have unknown functions or produce unknown metabolites. Therefore, filamentous fungi can be considered "underachievers" of natural product producers. We propose here to use Saccharomyces cerevisiae as an expression host to heterologously produce fungal PKSs. We will use this genetically superior host to reconstitute fungal PKSs of both known and unknown functions, perform genome mining of sequenced fungal species and engineered biosynthesis of new fungal-derived natural products.