Characterization and Engineering of Fungal Megasynthases in Escherichia coli

描述（由申请人提供）：丝状真菌是农业和药学上重要的天然产物的丰富来源。例如，真菌聚酮他汀类药物，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。属于间苯二酚内酯家族的真菌聚酮化合物作为选择性激酶抑制剂表现出有效的抗增殖活性。迭代真菌聚酮酶（PKS）在复杂聚酮化合物的合成中使用一套独特的生化规则。这些规则规定了聚酮化合物起始单元的选择、链长控制和PKS后处理。虽然细菌聚酮化合物的生物合成起源已被广泛研究，并已导致药学上重要的非天然天然产物的组合生物合成，但真菌PKS的生物合成机制尚未得到很好的理解，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其天然或相关真菌宿主中操作这些大合成酶以及获得完整的酶用于生化分析的困难。本建议的目的是弥合这些重要的知识和技术差距，并提供了一个多角度的图片真菌聚酮生物合成采用主力生物大肠杆菌。我们已经获得了大量的初步生化数据的表达，重建和工程的PKS 4从赤霉菌藤仓（gfPKS 4）和PKS 13从玉米赤霉菌（gzPKS 13）使用E。coli作为异源宿主。本研究将对以下假设进行评估：1）真菌PKS大合成酶可以在细菌宿主中功能性重构，如E。大肠杆菌; 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）真菌和细菌催化组分可催化整合以合成新型聚酮化合物。为了在五年内解决这些假设，我们定义了以下三个具体目标：1）gfPKS 4和gzPKS 13起始结构域的生化表征; 2）gfPKS 4和gzPKS 13环化结构域的生化表征; 3）真菌和细菌PKS的催化整合。我们已经提出了生物化学和代谢工程研究，以调查真菌聚酮酶。真菌聚酮脱氢酶是催化许多生物活性化合物（包括抗癌和抗高胆固醇血症的那些）的生物合成的迭代大合成酶。我们将使用强大的异源宿主大肠杆菌来研究完整的酶的起始、延伸、终止和环化步骤。从这些研究中获得的知识将是有价值的，在这些酶在体内和体外合成新的化合物的工程。

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.