ME: Combinatorial, in Vitro Manipulation of a Polyketide Synthase Pathway on a Microscale

拟议的研究的目的是使用组合生物学和微尺度处理相结合的体外代谢工程，以评估一个复杂的代谢网络，即聚酮合成的功能多样性。 研究人员将开发一种微型微流体装置，用于复杂聚酮化合物的体外组合生物合成。 此外，由于聚酮化合物合成的微尺度组分和模块性，研究人员期望使用多步酶网络的原理（例如，代谢途径）以改变途径的进程并产生独特的化合物。 本研究中使用的聚酮化合物类由少量多肽组成，每个多肽含有多个模块。 每个模块负责一轮链延伸和缩合后修饰。 模块将被固定到微制造装置的通道中，突变硫酯酶结构域将被工程化到每个模块中（释放和运输到下一个模块所必需的），并且产物将从一个模块运输到下一个模块。 这项研究可以合成具有新的和不寻常功能的分子。 此外，这项研究可以增加对代谢途径和微流体的基本理解。

The objective of the proposed research is to use in vitro metabolic engineering combined with combinatorial biology and microscale processing to evaluate the functional diversity of a complex metabolic network, namely, polyketide synthesis. The investigators will develop a microscale, microfluidic device for the in vitro combinatorial biosynthesis of complex polyketides. Also, because of the microscale component and the modularity of the polyketide synthesis, the investigators expect to use the principles of multi-step enzymatic networks (e.g., a metabolic pathway) to alter the progress of the pathway and generate unique compounds. The class of polyketides used in this research consists of a small number of polypeptides each containing multiple modules. Each module is responsible for one round of chain extension and post-condensation modifications. The modules will be immobilized into the channels of the microfabricated device, a mutant thioesterase domain will be engineered into each module (necessary for release and transport to the next module), and the products will be transported from one module to the next. This research could enable the synthesis of molecules with new and unusual functions. Also, this research could add to the fundamental understanding of metabolic pathways and microfluidics.