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品。其中最有趣的，但最不了解的酶，使这些化合物是迭代聚酮酶（IPKS）发现在丝状真菌。与以装配线方式操作的充分研究的细菌I型PKS相反，IPKS是通过以不同组合重复使用单组催化结构域以产生结构多样的真菌代谢物而迭代地起作用的大合成酶。对最近测序的真菌物种的基因组的生物信息学分析显示，每个基因组包含大量编码IPKS的基因。IPKS的总数显着超过了已知的聚酮和聚酮非核糖体肽从这些物种中分离，这表明大多数的生物合成基因是沉默的，在这些真菌培养条件下。这反过来表明，真菌物种可能具有尚未开发的潜力，可以合成大量的天然产物。此外，IPKS的分析和工程改造由于不能从天然真菌宿主或异源曲霉属宿主获得足够量的功能性纯化的大合成酶而受到阻碍。因此，通过IPKS控制代谢物组装的编程尚不清楚。仍有待阐明的关键方面包括：1）大合成酶中每个结构域的催化和结构作用; 2）催化结构域的底物特异性及其对大合成酶功能扰动的耐受性;以及3）在每次催化迭代期间控制催化结构域的不同组合的选择的因素。 本研究的目的是开发遗传上上级的酿酒酵母作为异源宿主，用于重组、分析和工程化IPKS，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S。酿酒酵母是以功能形式表达这些大合成酶的高度稳健的宿主，并且可以促进用纯化的酶在体内和体外产生聚酮化合物产物。具体目标如下：1）设计和优化S。2）在S. cerevisiae中重建真菌大合成酶; 3）利用S.酿酒酵母;和4）使用S.酿酒酵母作为宿主。 公共卫生相关性：丝状真菌是天然产品的丰富来源。其中，聚酮化合物代表了结构多样的天然产物的重要家族。聚酮化合物由聚酮化合物合成酶（PKS）产生。许多真菌的基因组测序显示，每种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产物。因此，丝状真菌可以被认为是天然产物生产者的“后进生”。我们在这里建议使用酿酒酵母作为表达宿主异源产生真菌PKSs。我们将使用这种遗传上优越的上级宿主来重建已知和未知功能的真菌PKS，进行测序真菌物种的基因组挖掘和新的真菌衍生天然产物的工程生物合成。

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.