植物乳杆菌UDP-GlcNAc代谢调控机制及在N-乙酰神经氨酸合成中的应用

UDP-GlcNAc is the key intermediates that involved the biosynthesis of both the cell wall and extracellular polysaccharides. It is also the precursor of lipopolysaccharides, proteoglycan, etc. In this study, we will analyze the regulation of the key intermediates GlcN-6P and UDP-GlcNAc that involved in cell wall formation and amino sugar biosynthesis in Lactobacillus plantarum. The potential riboswitch structure and regulator binding site in this pathway will be determined, the regulation mechanism will be proposed. Then, the metabolic engineering approach shall be employed to improve the accumulation of UDP-GlcNAc in Lb. plantarum. Based on these results, we will construct the N-acetylneuraminic acid (NeuAc) biosynthesis pathway in Lb. plantarum. A series of riboswitch which sense different concentration of NeuAc should be designed to construct the NeuAc evolution and screening system. Through this study, we will set up new technologies in evolution and screening of lactic acid bacteria, and will benefit to the rational design and modification of other microorganisms.

UDP-GlcNAc（尿嘧啶二核苷酸-N-乙酰氨基葡萄糖）是合成胞外多糖、氨基糖类和细胞壁成分的重要前体。作为许多糖基化反应的糖基供体，它还参与了多种具有重要价值的糖胺多糖、蛋白多糖、脂多糖等的合成。本研究拟以植物乳杆菌（Lactobacillus plantarum）为对象，分析细菌细胞壁产生及氨基糖类合成中的关键中间代谢产物GlcN-6P和UDP-GlcNAc的合成调控，阐明其代谢调控的分子机制和调控模型，并通过代谢工程手段提高UDP-GlcNAc的积累。在此基础上，构建N-乙酰神经氨酸（NeuAc）生物合成途径，验证UDP-GlcNAc积累对生物合成的影响。构建了以核糖开关（riboswitch）为基础的NeuAc动态感应系统，筛选通过单链核苷酸重组技术（ssDNA recombineering）进化的高产NeuAc的植物乳杆菌菌株，为理性设计和改造其它氨基糖类的代谢提供了依据。

N-乙酰神经氨酸（NeuAc）是细胞保护分子，主要位于细胞膜的表面，具有多种细胞生物学功能，同时也是重要的药物前体和保健品。本研究以植物乳杆菌（Lactobacillus plantarum）为对象，通过基于单链核苷酸重组技术（ssDNA recombineering）双链核苷酸重组技术（dsDNA recombineering）的基因组工程对植物乳杆菌进行基因组改造，解除N-乙酰葡萄糖胺合成途径中的反馈抑制及调控，通过代谢工程手段提高植物乳杆菌NeuAc的前体N-乙酰葡萄糖胺的产量；构建了NeuAc生物传感器，并利用传感器对NeuAc代谢途径进行优化并对途径关键酶N-乙酰神经氨酸合酶进行定向进化提高NeuAc产量；建立了体内进化核糖开关方法，提高了NeuAc核糖开关的阈值，利用NeuAc核糖开关定向进化提高NeuAc产量；理性改造NeuAc工程菌株平衡代谢流量，获得了报道以来葡萄糖生产NeuAc的最高产量。