辅因子自平衡的2-苯乙醇合成途径的胞内空间组装研究

Using synthetic biomolecular scaffolds to co-localize key enzymes of synthetic pathways can improve the local concentration of enzymes and metabolites, reduce the loss of intermediates, and avoid the accumulation of toxic metabolites. In cofactor regeneration studies，spatial organization of pathway and cofactor regeneration module will form a NAD(P)H circulation channel and greatly improve the productivity, which has attracted much attention. In our previous study, we established a self-sufficient cofactor regeneration biocatalyst producing 2-phenylthanol (2-PE) from L-phenylalanine with no need for amino receptor and redox addition. The 2-PE production was further enhanced by spatial assembly of the key enzymes and cofactors regeneration module. In this study, we attempt to use plasmid DNA as a stable, robust, configurable scaffold for arranging biosynthetic enzymes and cofactors regeneration module in a designable manner. Optimize the 2-PE productivity by altering multi-factor such as the spacers between the DNA binding site, the ratio of each enzyme, protein expression level and the number of binding units in a single cell, then establish a new way to arrange the synthetic pathway and cofactors regeneration module by a ring organization. Compare the differences of intermediate accumulation and production efficiency between traditional linear assemble and ring assemble, we will able to reveal the key factors that influence efficiency of 2-PE biosynthesis in spatial organization, and lay the foundation for self-sufficient cofactors regeneration amine-alcohol bioconversion system spatial assembling.

对代谢途径的空间组装能够提高局部酶和代谢物浓度，减少中间代谢物流失，避免有毒中间代谢物积累。在辅因子再生研究中，对合成酶与辅因子再生酶的空间组装有利于形成NAD(P)H的物质循环通道，提高产物合成速率，因而受到了广泛的关注。在前期研究中，申请人建立了一个辅因子自平衡的2-苯乙醇的生物合成系统，对该系统的关键酶和辅因子再生模块的空间组装初步结果显示，空间组装能够有效提高产物的合成效率。本研究拟使用DNA作为骨架实现2-苯乙醇合成途径与辅因子平衡模块的胞内空间组装，优化空间组装中酶的空间距离、各酶比例、蛋白表达水平和锚定单元数量多个因素，并建立一种全新的胞内辅因子平衡模块与合成途径的环形组装方法，进一步比较和分析传统线性组装与环形组装状态下的中间代谢物积累、产物合成效率，揭示影响2-苯乙醇生物合成系统空间组装的关键因素，为胞内辅因子自平衡的胺-醇转化体系的空间组装奠定基础。

2-苯乙醇是一种具有玫瑰花香气的芳香族化合物，广泛的应用于香水、医药、食品等领域。天然2-苯乙醇主要从玫瑰精油分离获得，由于其含量低、获取困难因此具有极高的价格。近年来，利用生物方法生物转化合成2-苯乙醇由于其具有反应条件温和、合成效率高、副产物少受到广泛关注。本项目在大肠杆菌中搭建了一个以L-苯丙氨酸为底物合成2-苯乙醇的合成途径，将锌指结构域与途径酶融合赋予酶DNA结合能力，通过对DNA锚定位点间距和比例的调节实现高效的2-苯乙醇合成途径空间组装，揭示影响合成途径空间组装的关键因素，获得一种通用的、可编程的合成途径空间组装方法。取得的研究结果如下：1.在大肠杆菌中构建了芳香族化合物2-苯乙醇的合成途径，获得了具有不同DNA序列锚定能力的锌指-酶；2.以DNA为支架在大肠杆菌胞内实现了2-苯乙醇合成途径的空间组装，经过筛选当间距为10bp，三种催化酶的比例为1:3:1时2-苯乙醇产量最高，达到20.5 mM；3.对限速酶苯丙酮酸脱羧酶进行更加深入的优化，获得了1个具有良好催化能力的苯丙酮酸脱羧酶。4.合成途径与辅因子再生模块空间组装后，2-苯乙醇合成量达49.60 mM，摩尔转化率达到99.2%。研究结果建立了一个以DNA为支架的辅因子自平衡的芳香胺到芳香醇的全细胞生物工厂。在本项目的资助下，共发表论文7篇，主持人为第一作者或通讯作者5篇，其中SCI论文4篇，国内核心期刊3篇，培养研究生3名。