Metabolic Engineering of Polketide Production in E.coli

描述(申请人提供)：聚酮合成酶(PKS)是一个多酶组合家族，催化合成许多结构复杂和具有生物重要性的天然产物。模块化PKS，如6-脱氧赤藓内酯B合成酶(Debs)，是一类特别有趣的合成大环内酯类等复杂多酮的PKS的子类。在过去的十年里，人们对这些超级合成酶的研究以及利用它们的模块化和广泛的底物专一性来工程合成“非天然”天然产物产生了相当大的兴趣。大多数模块化PKS的产物是由相对未经鉴定的细菌产生的。因此，每次发现具有良好生物学特性的新天然产物，都必须花费相当多的时间和费用从自然来源获得可靠数量的化合物，甚至需要更大的投资，才能使生物合成途径适合于合理的工程。另一种选择是开发健壮且普遍适用的技术，用于在特征良好的微生物宿主中异源表达聚酮。在过去的建议期内，模式菌大肠杆菌的代谢被改造为产生6-脱氧赤藓内酯B(6dEB)，这是抗生素红霉素的大环核心。这个工程化的大肠杆菌菌株有五个内源基因的修改；它还包含来自三个不同异源来源的七个新基因。由此产生的细胞催化剂在5天的过程中将外源丙酸转化为6dEB，数量接近200毫克/L。 在接下来的三年建议书期间，我们将重点改善和扩大大肠杆菌的性质，使其成为生物合成天然和非天然聚酮的选择。这将通过结合分子生物学工具、代谢工程策略和发酵技术开发来实现。具体目标是： I]在大肠杆菌中设计前体和产物生物合成的新途径； 2]改进发酵方案，以提高大肠杆菌中聚酮的产量； 利用功能基因组和代谢工程方法进一步提高大肠杆菌的聚酮产量； 在大肠杆菌中异源生产两种新的复杂天然产物。 这项研究的影响有三个方面。首先，鉴于可扩展的方案可用于发酵大肠杆菌以大量生产生物制品，在这种异种宿主中合成复杂聚酮的能力将预示着这些昂贵的生物活性天然产物及其工程衍生物的实际生产将是一个好兆头。其次，利用大肠杆菌作为聚酮生产的宿主，为利用大肠杆菌利用定向和随机方法设计模块化PKS打开了大门。最后，该项目是一个很好的机会，培养学生在代谢工程和天然产物生物合成的界面。

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.