Metabolic Engineering of Polyketide Production in E.coli

描述（由申请人提供）：聚酮合成酶（pks）是一个多酶组合的家族，催化合成许多结构复杂和生物学上重要的天然产物。模块化PKSs，如6-脱氧红素内酯B合成酶（DEBS），是一个特别有趣的PKSs亚类，可以合成复杂的多酮，如大环内酯类。在过去的十年中，人们对研究这些巨合酶，并利用它们的模块化和广泛的底物特异性来进行“非自然”天然产物的工程生物合成产生了相当大的兴趣。模块化PKSs的大多数产品是由相对未表征的细菌产生的。因此，每当发现一种具有良好生物学特性的新天然产物时，必须花费大量的时间和费用才能从天然来源获得可靠数量的化合物，而在生物合成途径适合合理的工程设计之前，则需要更大的投资。另一种选择是开发强大且普遍适用的技术，用于在特征良好的微生物宿主中异种表达多酮。在过去的提议期间，模型细菌大肠杆菌的代谢被改造为产生6-脱氧红素内酯B (6dEB)，这是抗生素红霉素的大环核心。该工程菌株携带了5个内源性基因的修饰；它还包含来自三个不同异种来源的七个新基因。所得的细胞催化剂在5天的过程中将外源性丙酸转化为6dEB，量接近200 mg/L。

DESCRIPTION (provided by applicant): Polyketide synthases (PKSs) are a family of multi-enzyme assemblies that catalyze the synthesis of numerous structurally complex and biologically important natural products. Modular PKSs, such as the 6-deoxyerythronolide B synthase (DEBS), are a particularly interesting sub-class of PKSs that synthesize complex polyketides such as macrolides. Over the past decade, there has been considerable interest in studying these megasynthases, and in exploiting their modularity and broad substrate specificity for the engineered biosynthesis of "unnatural" natural products. Most products of modular PKSs are produced by relatively uncharacterized bacteria. As a result, every time a new natural product with promising biological properties is discovered, a considerable amount of time and expense must be incurred to obtain reliable quantities of the compound from natural sources, and an even greater investment is demanded before the biosynthetic pathway becomes amenable to rational engineering. An alternative is to develop robust and generally applicable technologies for the heterologous expression of polyketides in well-characterized microbial hosts. During the past proposal period, the metabolism of the model bacterium Escherichia coli was engineered to produce 6-deoxyerythronolide B (6dEB), the macrocyclic core of the antibiotic erythromycin. This engineered strain of E. coli harbors modifications in five endogenous genes; it also contains seven new genes from three different heterologous sources. The resulting cellular catalyst converts exogenous propionate into 6dEB in quantities approaching 200 mg/L over a 5-day process. During the next 3-year proposal period, we will focus on improving and extending the properties of E. coli as a host of choice for the biosynthesis of natural and unnatural polyketides. This will be accomplished through a combination of molecular biological tools, metabolic engineering strategies and fermentation technology development. The Specific Aims are: I] Engineering new pathways for precursor and product biosynthesis in E. coli; II] Improved fermentation protocols for enhancing polyketide productivity in E. coli; III] Further improvements in polyketide productivity of E. coli using functional genornic and metabolic engineering approaches; & IV] Heterologous production of two new complex natural products in E. coli. The implications of this research are 3-fold. First, given the availability of scalable protocols for fermenting E. coli to overproduce bioproducts, the ability to synthesize complex polyketides in this heterologous host will bode well for the practical production of these expensive bioactive natural products as well as their engineered derivatives. Second, the use of E. coli as a host for polyketide production opens the door for harnessing E. coli to engineer modular PKSs using directed and random approaches. Finally, the project is a good opportunity to train students at the interface of metabolic engineering & natural product biosynthesis.