基于酶催化羟醛反应立体选择性构建谷氨酸棒杆菌模式细胞生物合成稀少糖

Rare sugars refer to monosaccharides and their derivatives that rarely exist in nature. The sugars have various known biological functions and have been used as food additives, cancer cell suppressors and building blocks for anticancer and antiviral drugs. Biotransformations have been developed for synthesis of rare sugars; however, those methods were only carried out in laboratory scale because the limitation of substrate and conversion rate. We have constructed a rare sugar biosynthesis pathway based on aldol reaction in C. glutamicum strain and obtained two ketohexoses. In this study, we would screen different type of DHA/DHAP-dependent aldolase using sequence alignments, enzyme structure analysis, molecular docking between enzyme and substrate, and directed evolution. The aldolases were characterized to uncover the mechanism of aldol reaction to specific substrate and product formation. The “one-pot” reaction system containing the preferable aldolases would be applied to synthesize many kinds of rare sugars. Furthermore, the donor DHA/DHAP would be accumulated from glucose through glycolytic pathway or from glycerol though glycerol aerobic/aerobiotic metabolic pathway. Then, the accepter would be obtained from alcohol or carboxylic acid though corresponding oxidordeuctases. The rare sugar formation pathways based on aldolase and phosphorylase were constructed and optimized. On this basis, a RBS library with different expression strengths would be isolated and applied to regulate the expression level of those three modules. Finally, a microbial model cell to synthesize C5 or C6 D/L- isomers sugars and deoxy sugars from glucose and glycerol would be constructed in the study.

稀少糖是自然界中稀少存在的一类单糖及其衍生物，具有降血糖、抑制癌细胞生长与脂肪积累等多重功能。目前稀少糖主要以单糖生物转化方法合成，其受底物及转化效率局限，探索新的合成方法具有重要意义。前期已构建了由RhaD醛缩酶利用非糖前体合成己酮糖途径，在此基础上，本研究将利用基因序列比对、酶立体结构解析、分子对接模拟等方法，挖掘或改造不同类型的DHA/DHAP依赖型醛缩酶，对其进行功能表征，并揭示这些酶在羟醛反应中对特定底物立体选择性的催化机制，从而合成不同R, S构型稀少糖。进一步以谷氨酸棒杆菌为底盘微生物，优化糖酵解途径与重构甘油代谢途径积累酮供体，以小分子醇或酸为底物转化形成乙醛、甘油醛等受体，模拟糖异生进行C-C键缩合羟醛反应构建稀少糖合成新途径；通过生物学元件设计，建立一系列RBS文库，对宿主细胞外源基因表达及其所催化代谢途径进行调控，建立以可再生甘油、葡萄糖为碳源全细胞合成稀少糖体系。

稀少糖是自然界中存在极为罕见的单糖及其衍生物，在食品、保健品、医药与化妆品等领域具有十分重要的应用价值，传统生物合成方法依赖于单糖异构反应，受原料和热力学平衡限制，导致产物转化率低、产物构型少。本项目提出基于酶羟醛缩合反应构建生物合成途径，转化小分子合成稀少糖的新策略，开展了醛缩酶筛选和表征研究、解析酶羟醛缩合催化机制、阐明产物构型与结构关系以及构建体外多酶催化体系和微生物细胞工厂合成稀少糖研究。筛选和表征了不同物种来源的二羟丙酮/磷酸二羟丙酮(DHA/DHAP)依赖的醛缩酶，发现醛缩酶具有催化酮基和酮基之间羟醛缩合反应的新功能，并结合分子动力学模拟解析催化机制；基于醛缩酶和果糖1-磷酸酶建立“一锅”双酶级联反应，实现催化DHAP和多种醛合成C4, C5, C6和C7等10余种稀少糖，基于模块化组装策略，采用分步级联催化方法，实现转化甲醛合成L-赤藓酮糖、L-山梨糖等多种稀少糖; 以谷氨酸棒杆菌为出发菌株，构建了羟醛缩合途径，实现发酵葡萄糖和受体醛合成多种稀少糖，为了降低醛受体对细胞毒性影响，筛选获得了催化小分子醇和酸转化为醛的醇脱氢酶，并构建了受体醛合成途径，通过模块化途径组装，实现发酵葡萄糖和甘油合成3种5-脱氧糖；在谷氨酸棒杆菌中构建甘油利用途径，使得甘油利用能力显著提升，通过组合甘油利用途径，L-甘油醛合成途径，羟醛缩合途径，实现工程菌株发酵单一碳源甘油合成4种L-己酮糖；基于醛缩酶催化DHAP和DHA之间羟醛缩合反应的新功能，通过增强催化DHAP转化为DHA的磷酸酶表达水平和引入羟醛缩合途径，实现发酵葡萄糖合成支链酮糖。本项目实现基于C-C连接反应合成稀少糖的目标，与传统单糖异构转化反应相比，具有转化率高、产物构型广泛等优势，为更多种类稀少糖的生物合成提供借鉴意义。