通过定向进化策略改善蓝细菌脂肪醛脱甲酰加氧酶活性的研究

A cyanobacterial pathway consisting of an acyl–acyl carrier protein reductase (AAR) and an aldehyde-deformylating oxygenase (ADO), which converts acyl–acyl carrier proteins into corresponding n-1 hydrocarbons, has been revealed and hotly discussed since 2010. As one of the key catalysts in the hydrocarbon biosynthetic pathway that has important application prospect in the production of advanced biofuels using photosynthetic microorganisms, ADO has gained great attention in the fields of metabolic engineering and enzyme engineering. However, the poor catalytic activity and stability of the enzyme have posed a serious barrier to its further study and industrial application. In this project, directed evolution of the cyanobacterial aldehyde-deformylating oxygenase will be performed to improve its catalytic performance by error-prone PCR and DNA shuffling. A high-throughput screening (HTS) approach will also be developed to facilitate the implementation of the evolution strategy, basing on a hydrocarbon bioreporter biobrick as well as the green fluorescent protein (GFP)-labeled cell sorting method. The advantageous ADO mutants will be separated and characterized. The role of the mutations will be investigated by combinatorial mutagenesis and site-saturation mutagenesis. This project will promote the understanding of the relationship between the primary sequences with the catalytic function, and provide biocatalysts with higher activity and stability for the construction of highly efficient photosynthetic microbial cell factories for the industrial production of advanced hydrocarbon biofuels.

由脂酰ACP还原酶和脂肪醛脱甲酰加氧酶（ADO）组成的蓝细菌脂肪烃生物合成途径于2010年被发现。脂肪烃生物合成在利用光合微生物生产生物质燃料方面重要具有应用前景。作为该途径的一个关键催化剂，ADO受到相关领域研究者的广泛关注。然而，由于ADO活性较低，稳定性较差，极大制约了对该酶的深入研究与大规模工业应用。本项目将采用定向进化策略对蓝细菌脂肪醛脱甲酰加氧酶进行改造，利用易错PCR、DNA改组等手段，获得具有优良催化性能的ADO突变体；基于脂肪烃生物传感器元件和GFP荧光标记快速细胞分选技术，构建高效产烃大肠杆菌高通量筛选平台；通过组合突变、定点饱和突变等手段揭示关键氨基酸位点和序列对ADO催化特性的影响。本项目将有助于增进对ADO蛋白质结构与功能之间关系的理解，促进对ADO等关键催化剂的分子改造，也为高效光合微生物细胞工厂的构建与优质脂肪烃燃料的工业生产提供更加高效和稳定的催化剂。

由脂酰ACP还原酶（AAR）和脂肪醛脱甲酰加氧酶（ADO）组成的蓝细菌脂肪烃生物合成途径发现于2010年。由于蓝细菌脂肪烃合成途径在生物质燃料的光合生物合成方面的重要应用前景，作为这一途径的关键催化剂之一，ADO受到相关领域研究者的广泛关注。然而，由于ADO活性低，稳定性差，极大制约了对该酶的深入研究与应用。本项目致力于采用定向进化策略对蓝细菌脂肪醛脱甲酰加氧酶进行改造，利用易错PCR、定点饱和突变等手段，改造获得在大肠杆菌中产烃效率提高的ADO突变体。围绕这一目的，本研究主要开展了以下两部分工作。.（1）实现快速检测细胞内的脂肪烃合成水平是实施ADO改造乃至高产烃细胞工厂构建的必要技术前提。针对脂肪烃检测手段缺乏的问题，受某些天然微生物脂肪烃响应和降解机制的启发，我们在大肠杆菌中整合来源于不动杆菌和假单胞菌的基因元件AlkR-PalkM，利用绿色荧光蛋白（GFP）作为报告蛋白，成功实现将细胞内的脂肪烃浓度信号转变为能够直观反映烃浓度高低的荧光信号，为进一步依据荧光实现高通量筛选提供了技术基础。.（2）在成功开发脂肪烃检测元件的基础上，我们以来源于聚球藻Synechococcus elongates PCC7942的ADO作为定向进化的对象，以大肠杆菌为筛选宿主，采用易错PCR引入随机突变的方法实施定向进化，并通过荧光活化流式细胞分选技术（FACS）从表达文库中筛选优势突变株，经过两轮筛选，获得产烃能力为出发菌株3倍的优势突变株；通过定点突变和组合突变，获得产烃能力为出发菌株4.7倍的优势突变株。这一工作首次尝试将定向进化和高通量筛选技术应用于脂肪烃脱甲酰加氧酶的改造，并取得了较好的进展，为进一步通过基因工程改造提高微生物脂肪烃合成效率提供了有益的启示。