A Robust Platform for Reconstituting and Engineering Iterative Megasynthases

描述（由申请人提供）：大自然使用一系列惊人的酶来制造小分子天然产物。在制造这些化合物的酶中，最有趣但最不为人所知的是在丝状真菌中发现的迭代聚酮合成酶（IPKSs）。与被充分研究过的以流水线方式运作的细菌I型pks不同，ipks是一种大型合成酶，它通过以不同的组合重复使用一组催化结构域来迭代地起作用，从而产生结构多样的真菌代谢物。对最近测序的真菌物种基因组的生物信息学分析表明，每个基因组包含大量编码ipks的基因。ipks的总数明显超过从这些真菌中分离到的已知聚酮和聚酮-非核糖体肽，这表明在培养条件下，这些真菌中大多数生物合成基因是沉默的。这反过来表明，真菌物种可能有未开发的潜力来合成大量的天然产物。此外，由于无法从原生真菌宿主或异源曲霉宿主中获得足够数量的功能性纯化巨合成酶，ipks的分析和工程一直受到阻碍。因此，控制ipks代谢物组装的编程尚不清楚。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成酶功能扰动的耐受性；3)每次催化过程中控制不同催化结构域组合选择的因素。本研究的目的是开发具有遗传优势的酿酒酵母作为IPKS重组、分析和工程的异源宿主，特别是与洛伐他汀生物合成相关的高还原IPKS，如LovB。我们已经积累了大量的初步数据，证明酿酒葡萄球菌是一个高度健壮的宿主，可以以功能形式表达这些巨合酶，并且可以在体内和体外用纯化的酶促进聚酮产物的生产。具体目标如下：1)设计和优化酿酒酵母生产真菌代谢物和巨合成酶；2)酿酒葡萄球菌真菌巨合成酶的重组；3)利用酿酒酵母对真菌PKS进行生化分析；4)以酿酒葡萄球菌为寄主的丝状真菌基因组挖掘。

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.