Characterization and Engineering of Fungal Megasynthases in Escherichia coli

描述(申请人提供)：丝状真菌是农业和药学上重要的天然产品的丰富来源。例如，真菌多酮他汀类药物，如洛伐他汀，是通过抑制胆固醇生物合成来预防和治疗动脉粥样硬化的最广泛的处方药之一。真菌多酮类化合物属于间苯二酸内酯家族，具有很强的抗增殖活性，是一种选择性的激酶抑制剂。迭代真菌聚酮合成酶(PKS)在合成复杂多酮时使用一套独特的生化规则。这些规则规定了聚酮起始剂单元的选择、链长度控制和PKS后处理。虽然细菌多酮的生物合成起源已被广泛研究，并导致了具有重要药用价值的非天然天然产物的组合生物合成，但真菌PKS的生物合成机制尚不清楚，其组合生物合成的潜力尚未实现。这在很大程度上是由于在它们的天然或相关真菌宿主中操纵这些巨合酶，以及获得用于生化分析的完整酶的困难。这项建议的目的是弥合这些重要的知识和技术差距，并提供使用主要生物大肠杆菌的真菌聚酮生物合成的多角度图像。我们利用大肠杆菌作为异源宿主，获得了大量的表达、重组和工程化赤霉菌PKS4(GfPKS4)和玉米赤霉菌Pks 13(GzPKS13)的生化数据。这项建议将评估以下假设：1)真菌PKS巨合酶可以在细菌宿主(如大肠杆菌)中进行功能重组；2)真菌PKS含有可用于组合生物合成的起始和环化结构域；3)真菌和细菌的催化成分可以催化整合，以合成新的聚酮。为了在五年的时间里解决这些假设，我们定义了以下三个具体目标：1)gfPKS4和gzPKS13启动域的生化特征；2)gfPKS4和gzPKS13环化区域的生物化学特征；3)真菌和细菌PKS的催化整合。项目简介我们已经提出了生化和代谢工程研究，以研究真菌多酮合成酶。真菌聚酮合成酶是一种重复的巨型合成酶，催化许多生物活性化合物的生物合成，包括那些抗癌和抗高胆固醇血症的化合物。我们将使用强大的异源宿主大肠杆菌来研究完整合成酶的起始、延伸、终止和环化步骤。从这些研究中获得的知识将对这些酶的工程设计以及体内和体外合成新化合物具有重要意义。

DESCRIPTION (provided by applicant): Filamentous fungi are a rich source of agriculturally and pharmaceutically important natural products. For example, the fungal polyketide statins, such as lovastatin, are among the most widely-prescribed drugs for the prevention and treatment of atherosclerosis by inhibiting cholesterol biosynthesis. Fungal polyketides belonging to the resorcylic acid lactone family exhibit potent antiproliferative activities as selective kinase inhibitors. Iterative fungal polyketide synthases (PKSs) use a unique set of biochemical rules in the synthesis of complex polyketides. These rules dictate polyketide starter unit selection, chain length control, and post-PKS processing. While the biosynthetic origins of bacterial polyketides have been studied extensively and have led to the combinatorial biosynthesis of pharmaceutically important unnatural natural products, the biosynthetic mechanisms of fungal PKSs are not well understood and their potential for combinatorial biosynthesis has not yet been realized. This is largely due to difficulties associated with manipulating these megasynthases in their native or related fungal hosts, and with obtaining intact enzymes for biochemical analysis. The objective of this proposal is to bridge these important knowledge and technical gaps and provide a multi- angled picture of the fungal polyketide biosynthesis employing the workhorse organism Escherichia coli. We have obtained extensive preliminary biochemical data on the expression, reconstitution and engineering of PKS4 from Gibberella fujikuroi (gfPKS4) and PKS13 from Gibberella zeae (gzPKS13) using E. coli as the heterologous host. This proposal will evaluate the following hypotheses: 1) Fungal PKS megasynthases can be functionally reconstituted in a bacterial host, such as E. coli; 2) Fungal PKS contains initiation and cyclization domains that can be exploited for combinatorial biosynthesis; 3) Fungal and bacterial catalytic components can be catalytically integrated towards the synthesis of novel polyketides. To address these hypotheses in a five-year period, we have defined the following three SPECIFIC AIMS: 1) Biochemical Characterization of gfPKS4 and gzPKS13 Initiation Domains; 2) Biochemical Characterization of gfPKS4 and gzPKS13 Cyclization Domains and 3) Catalytic Integration of fungal and bacterial PKSs. Project Narrative We have proposed biochemical and metabolic engineering studies to investigate fungal polyketide synthases. Fungal polyketide synthases are iterative megasynthases that catalyze the biosynthesis of a number of biological active compounds, including those that are anticancer and antihypercholesterolemia. We will use the robust heterologous host Escherichia coli to study the initiation, elongation, termination and cyclization steps of the intact synthases. Knowledge gained from these studies will be valuable in the engineering of these enzymes towards synthesis of novel compounds both in vivo and in vitro.