谷氨酸棒杆菌中N-乙酰神经氨酸合成的调控

N-acetylneuraminic acid serves as important food supplement and precursor for pharmaceuticals synthesis since it can help facilitate the development and maintain the function of the brain. However, its wide application is restricted by its high price due to the high production cost in raw material extraction and whole-cell catalysis. In this project, we assume to investigate N-acetylneuraminic acid synthesis from glucose in Corynebacterium glutamicum. First, we will engineer the N-acetylneuraminic acid lyase pathway in an N-acetylneuraminic acid producing Corynebacterium glutamicum, and thus use N-acetylglucosamine and the readily available pyruvate in the cell as key precursors for N-acetylneuraminic acid biosynthesis. The expression of key genes in the lyase pathway will be subsequently optimized by modulating their transcription. Next, we aim to establish a metabolic regulation method based on the availability of the two cofactors D-pantothenate and biotin in the medium for dynamically tuning the carbon flux distribution at the pyruvate node.The molecular mechanism of the regulation method will be revealed by comparative analysis of the transcriptome, metabolome and key enzymes activity. Finally, we will utilize this dynamic regulation system to balance N-acetylglucosamine and pyruvate supply for N-acetylneuraminic acid biosynthesis, and thus reveal the regulation mechanism of its efficient synthesis. Results obtained in this project will shed light on the microbial production of N-acetylneuraminic acid from glucose. Methods and strategies applied in this project will be applicable for investigating the metabolic regulation of other chemicals biosynthesis.

N-乙酰神经氨酸具有促进大脑发育和维持脑功能健康的生理功效，是重要的食品添加剂和新型药物前体。化学提取和全细胞转化法生产N-乙酰神经氨酸需使用昂贵的原料或前体，高昂的生产成本使其在食品中的广泛应用受到限制。本课题拟以一株可利用葡萄糖合成N-乙酰神经氨酸的重组谷氨酸棒杆菌为基础，首先通过构建N-乙酰神经氨酸裂合酶途径，实现利用N-乙酰氨基葡萄糖和胞内较易积累的丙酮酸作为关键合成前体；通过优化关键基因的转录，获得最优途径基因表达策略。再通过赋予菌株D-泛酸和生物素双重营养缺陷型表型，建立基于辅因子供给水平的丙酮酸节点代谢流的动态调控方法，并通过组学分析阐明调控的分子机制。最后，利用该动态调控方法，考察N-乙酰氨基葡萄糖和丙酮酸平衡供给对N-乙酰神经氨酸合成的影响，解析N-乙酰神经氨酸合成的调控机制。本项目中采用的方法策略和阐明的调控机制将为其他相关化学品的代谢调控机理研究提供理论和方法参考。

N-乙酰神经氨酸具有促进大脑发育和维持脑功能健康的生理功效，是重要的食品添加剂和新型药物前体。化学提取和全细胞转化法生产N-乙酰神经氨酸需使用昂贵的原料或前体，高昂的生产成本使其在食品中的广泛应用受到限制。本课题以一株可利用葡萄糖合成N-乙酰神经氨酸的重组谷氨酸棒杆菌为基础，首先构建了N-乙酰神经氨酸裂合酶途径，实现了利用N-乙酰氨基葡萄糖和胞内较易积累的丙酮酸作为关键前体合成N-乙酰神经氨酸；其次优化了合成途径基因的转录，获得了一株能利用N-乙酰神经氨酸裂合酶途径合成N-乙酰神经氨酸的重组谷氨酸棒杆菌；接着重构了中心代谢途径以提高前体丙酮酸的胞内积累浓度，但由于N-乙酰神经氨酸裂合酶对底物丙酮酸的亲和性太差，致使丙酮酸在胞内外过量积累影响了细胞正常生长，使得N-乙酰神经氨酸的合成反而受阻；随后采用了N-乙酰神经氨酸合酶途径进行进一步的研究，通过赋予菌株D-泛酸和生物素双重营养缺陷型表型，建立了基于辅因子供给水平的丙酮酸节点代谢流动态调控方法，并以阐明调控的分子机制为目的，进行了转录组学分析。本研究最终构建得到的谷氨酸棒杆菌N-乙酰神经氨酸生产菌可胞外积累约1g/L N-乙酰神经氨酸,具有潜在应用前景，而本研究所获得的数据及建立的方法对代谢工程改造谷氨酸棒杆菌生产其他营养品具有指导意义。