基于二醇脱水酶分子改造全细胞催化L-阿拉伯糖合成2-脱氧L-核糖研究

The compound 2-deoxy-L-ribose constitutes the central building block for antitumor drug and shows high application potential in pharmaceutical industry; however, chemical synthesis method needs lots of steps and suffers from low yield. This project presents a new whole cell transformation strategy based on directed evolution of diol dehydratase (DDH) to synthesize 2-deoxy-L-ribose directly from L-arabinose. We have made a molecular docking between DDH and L-arabitol and got some candidate sites which influenced the substrate binding. We have also construed a high throughput selection method for DDH and got some mutants which showed the dehydration ability to L-arabitol. In this study, we will employ structure-guided triple code saturation mutagenesis and iterative combinatorial mutagenesis strategy based on semi-rational design to further improve DDH activity. The catalytic mechanism of DDH mutant to L-arabitol will be clearly revealed. Then, the metabolic engineering strategy will be applied to construct 2-deoxy-L-ribose synthesis pathway in Escherichia coli based on aldose reductase (AR), DDH mutant, and glucose dehydrogenase (GDHs). The endogenous L-arabinose isomerase will be deleted to decrease L-arabinose metabolic consumption, and the expression level of heterologous genes will be modulated to achieve higher 2-deoxy-L-ribose production ability. This project will pave the way to green synthesis of 2-deoxy-L-ribose.

2-脱氧L-核糖作为抗肿瘤药物中间体，具有重要市场前景和开发价值。但由于化学法合成脱氧糖过程繁琐，本研究提出了基于二醇脱水酶（DDH）分子改造全细胞催化L-阿拉伯糖合成2-脱氧L-核糖的新思路，前期利用DDH蛋白结构模拟及半理性设计了相关突变位点，建立了高通量筛选方法，经过筛选初步验证若干DDH突变体对L-阿拉伯糖醇具有脱水催化活性。本课题拟首先运用三重密码饱和突变和迭代饱和突变策略进一步改造DDH，筛选对L-阿拉伯糖醇具有更高催化活性的突变体，解析突变体针对糖醇脱水作用的催化机理；进一步以大肠杆菌为底盘微生物，运用代谢工程技术，构建由醛糖还原酶(AR)、DDH突变体和葡萄糖脱氢酶（GDH）组成的酶级联反应合成2-脱氧L-核糖途径；通过敲除L-阿拉伯糖异构酶基因降低底物代谢消耗，调控关键基因表达水平优化合成途径代谢流，提高全细胞转化合成效率，为2-脱氧L-核糖的绿色合成奠定基础。

2-脱氧L-核糖是合成L-核苷类药物的重要中间体，化学法合成脱氧糖需要多步保护和脱保护步骤导致收率低、合成过程繁锁，因此，开发绿色、环保、高效的生物合成方法，对于制备2-脱氧L-核糖至关重要。本项目提出基于多元醇脱水反应和羟醛缩合反应生物合成2-脱氧L-核糖的新思路，重点开展了二醇脱水酶分子改造提高针对长链糖醇的催化活性研究，建立了高通量筛选方法，获得了二醇脱水酶突变体GDHtS302AQ337A，实现针对L-阿拉伯糖醇等多种多元醇具有脱水活性；通过筛选获得了6种氧化还原酶，实现转化L-阿拉伯糖合成L-阿拉伯糖醇，并建立了针对NAD/NADH/NADPH的辅酶再生体系；基于乙醛依赖的醛缩酶（DERA）建立催化乙醛和L-甘油醛缩合反应合成2-脱氧L-核糖反应，分子改造醛缩酶DERA获得突变体，提高催化活性近3倍；建立全细胞催化反应体系合成2-脱氧L-核糖；提出基于小分子合成2-脱氧L-核糖的新思路，建立由甲醛连接酶（FLS），果糖6-磷酸醛缩酶(FSA)和DERA组成的多酶级联体系，采用模块化组装策略，实现以廉价甲醛和乙醛为原料合成了2-脱氧L-核糖等C3，C4，C5和C6功能糖化合物。本项目提供了生物合成2-脱氧L-核糖的新方法，对于其他种类2-脱氧糖的高效制备具有借鉴意义。

内容获取失败，请点击重试