Metabolic Engineering of Polketide Production in E.coli

描述（由申请人提供）：聚酮化合物脱氢酶（PKS）是一个多酶组装体家族，催化合成许多结构复杂且具有生物学重要性的天然产物。模块化PKS，如6-脱氧腺苷酸B合酶（DEBS），是合成复杂聚酮化合物如大环内酯的PKS的特别有趣的亚类。在过去的十年中，有相当大的兴趣在研究这些megasynthases，并在利用其模块性和广泛的底物特异性的工程生物合成的“非天然”的天然产物。模块化PKS的大多数产物由相对未表征的细菌产生。因此，每次发现具有有希望的生物学特性的新天然产物时，必须花费相当多的时间和费用来从天然来源获得可靠量的化合物，并且在生物合成途径变得适合于合理工程之前需要甚至更大的投资。另一种方法是开发用于在充分表征的微生物宿主中异源表达聚酮化合物的稳健且普遍适用的技术。在过去的提案期间，模型细菌大肠杆菌的代谢被改造为产生6-脱氧红霉素B（6 dEB），抗生素红霉素的大环核心。该工程菌是E.大肠杆菌在五个内源基因中进行了修饰，它还包含来自三个不同异源来源的七个新基因。所得的细胞催化剂在5天的过程中将外源性丙酸盐以接近200 mg/L的量转化为6dEB。 在未来3年的建议期内，我们将专注于改善和扩展E。大肠杆菌作为生物合成天然和非天然聚酮化合物的首选宿主。这将通过分子生物学工具、代谢工程策略和发酵技术开发的组合来实现。具体目标是： 在大肠杆菌中设计前体和产物生物合成的新途径。大肠杆菌; 用于提高E.大肠杆菌; 进一步提高E.大肠杆菌中使用功能基因和代谢工程方法; 在E.杆菌 这项研究的意义有三个方面。 首先，考虑到发酵E.由于大肠杆菌能够过量生产生物产品，因此在这种异源宿主中合成复杂聚酮化合物的能力对于这些昂贵的生物活性天然产品及其工程衍生物的实际生产将是一个好兆头。第二，利用E.大肠杆菌作为聚酮化合物生产的宿主为利用大肠杆菌生产聚酮化合物打开了大门。大肠杆菌工程模块PKS使用定向和随机的方法。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接口。

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.