生物转化合成N-乙酰神经氨酸的关键因素

Key Limiting Factors for the Whole-Cell Synthesis of N-Acetyl Neuraminic Acid by RecombinantEscherichia coli

DOI：10.3969/j.issn.1673-1689.2019.04.010

中文关键词: 唾液酸 N-乙酰神经氨酸 磷酸烯醇式丙酮酸 N-乙酰氨基葡萄糖 全细胞生物转化

英文关键词: sialic acid,Neu5Ac,phosphoenolpyruvate,GlcNAc,whole-cell catalysis

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中文摘要:

在前期工作中已构建了一株能以外源N-乙酰氨基葡萄糖（GlcNAc）和胞内磷酸烯醇式丙酮酸（PEP）为底物，生物转化合成N-乙酰神经氨酸（Neu5Ac）的重组大肠杆菌，但其合成Neu5Ac的效率有待进一步提高。通过调整生物转化阶段培养基中氮源和碳源的组成，探寻Neu5Ac合成过程中的关键限制性因素。结果表明，增加唾液酸合成酶NeuB活性的措施没有提高Neu5Ac的产率，而底物之一胞内PEP的供给不足是限制Neu5Ac合成的关键性因素。通过限制转化培养基中的氮源，抑制重组大肠杆菌的生长，避免了PEP被用于细胞自身生长而更多地流向Neu5Ac合成方向；通过改用不依赖PTS跨膜转运系统的甘油作为碳源，在甘油/无氮转化培养基中Neu5Ac合成量最终达到（4.42±0.08） g/L，比初始合成条件高出10.3倍。

英文摘要:

We constructed a recombinantEscherichia coliwhich could synthesize Neu5Ac with exogenous GlcNAc as sole substrate in previous work. Various nitrogen and carbon sources were used to investigate their effects on the production of Neu5Ac，and the key limiting factors in fluencing the whole process were revealed. It was shown that the increase of NeuB activity could not improve the Neu5Ac yield and the NeuB activity was not the limiting factor. The intracellular PEP was supposed to be the limiting factor for Neu5Ac production. The intracellular PEP was saved and shifted to the synthesis of Neu5Ac by adjusting the nitrogen source and restraining the cell growth. In addition，by using glycerol as sole carbon source which was PTS-independent，Neu5Ac synthesis in the glycerol/nitrogen-free medium reached （4.42±0.08） g/L，10.3 times higher than the initial synthesis conditions.

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