3-羟基丙酸生物合成关键酶—丙二酸单酰辅酶A还原酶的催化机制研究和功能改造

Malonyl-coenzyme A (MCR) of Chloroflexus aurantiacus, a thermophilic and phototrophic bacterium, can convert the basic metabolic intermediate malonyl-CoA to an important platform chemical 3-hydroxypropionate (3HP), which is listed in top 12 value added biomass-based chemicals by US Department of Energy.MCR has a great application potential in biotechnology field, however the application of MCR was limited by its low activity due to that the high temperature and autotrophic conditions can not be achieved in a recombinant strain. MCR does not show significant sequence identity with any known protein, and its protein structure and catalytic mechanism remain unknown. So it is an urgent request for elucidating MCR catalytic mechanism and improving its enzyme activity. It was proved that dissection of MCR into two functional fragments can improve its enzyme activity. In this study, the crystal structures of MCR protein and its two fragments will be determined by X-ray crystallography method, and the MCR fragments will be engineered by directed evolution and rational design to improve their enzymatic acitivity. The kinetic processes of MCR and its fragments binding substrate and catalyzing the reaction will be simulated and analyzed to elucidate the MCR catalytic mechanism and to understand how dissection of MCR and other mutations enhance its activity. MCR derivatives with higher enzyme activity will be used to construct a recombinant strain producing 3HP, and this strain will be used in fermentation to test the 3HP yield. This study will not only enrich our knowledge about the reductase catalytic mechanism, improve the 3HP biological synthesis technology, but also provide scientific information and reference for improving enzyme performance using both structural biology and molecular biology methods.

丙二酸单酰辅酶A还原酶(MCR)来源于嗜热光合细菌，可将生物体内的基础代谢中间体丙二酸单酰辅酶A转化为重要的平台化合物3-羟基丙酸(3HP)，在生物工程领域具有非常重要的应用价值，但由于其催化活性较低而受到很大限制。MCR与已知蛋白不具序列同源性，蛋白三维结构和催化机制未知，所以阐明MCR反应机理，提高其催化活性成为了亟待解决的问题。前期研究表明将MCR蛋白分为两个功能独立的片段可增强其活性，本研究将利用X射线晶体学方法解析MCR蛋白及其两个功能片段的三维结构，阐明MCR蛋白及其片段的催化机理；通过定向进化和理性设计方法获得高活性的MCR功能片段突变体，解析MCR结构拆分和突变提高酶活性的机制；将得到的MCR片段突变体应用于3HP工程菌株的构建，提高3HP产量。本研究将丰富人们对于还原酶催化机理的认识，为微生物法合成3HP的技术提升奠定理论基础。

3-羟基丙酸是重要的平台化合物，可用于合成丙烯酸、丙烯酰胺等多种化工产品，被美国能源部列入最具开发潜力的12种生物基化学品之一。丙二酸单酰辅酶A还原酶（MCR）可以将微生物体内的代谢中间体—丙二酸单酰辅酶A转化为3-羟基丙酸，但因其催化活性较低而受到很大限制。在前期研究中，我们已将双功能酶MCR拆分为两个具有独立功能的片段：MCR-C负责催化丙二酸单酰辅酶A到丙二酸半醛，MCR-N负责催化丙二酸半醛到3-羟基丙酸。为提高MCR的催化能力，我们建立了针对限速步骤MCR-C的迭代定向进化系统，通过多轮筛选获得了高活性的MCR-C突变体；成功表达纯化了MCR-N和MCR-C蛋白，分别解析了MCR-N和MCR-C两个蛋白的三维结构。结果发现二者均由多结构域组成，综合其结构和活性中心特征可知，MCR-N和MCR-C不同于已知短链氧化还原酶（short-chain dehydrogenase/reductase，SDR）超家族成员的特点，属该超家族蛋白的新类型；发现了底物结合可以改变限速酶MCR-C的构象，明确了MCR-C处于催化与非催化构象的结构特点；MCR-N结构与MCR-C存在明显差异，与其较短的氨基酸序列和较小的底物分子相匹配，且其与底物结合前后不发生构象变化。针对在定向进化中发现的可提高MCR-C酶活性但远离活性中心的氨基酸突变位点，我们解析了突变体MCR-C蛋白的三维结构，发现这些位点突变可以稳定蛋白的活性构像，进而增加MCR-C的催化活性。综上所述，我们获得了高活性的NCR-C突变体，解析了MCR-C和MCR-N蛋白的三维结构和催化机制，为进一步理性改造MCR和提升3-羟基丙酸生物合成技术奠定了理论基础。

内容获取失败，请点击重试