N-acetylneuraminate (NeuAc) biosynthesis has drawn much attention owing to its wide applications in many aspects. Previously, we engineered for the first time an artificial NeuAc biosynthetic pathway in Escherichia coli using glucose as sole substrate. However, rigorous requirements for the flux and cofactor balance make subsequent strain improvement rather difficult. In this study, an in vivo NeuAc biosensor was designed and applied for genetic screening the mutant library of NeuAc producer. Its NeuAc responsive manner was demonstrated using sfgfp as a reporter and a Ni2+-based selection system was developed to couple the cell growth with in vivo NeuAc concentration. Employing this selection system, the NeuAc biosynthesis pathway was optimized and the key enzyme NeuAc synthase was evolved, which improved the titer by 34% and 23%, respectively. The final strain produced up to 8.31g/L NeuAc in minimal medium using glucose as sole carbon source. This work demonstrated the effectiveness of NeuAc biosensor in genetic screening and great potential in metabolic engineering of other organisms.

中文翻译：

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使用基于体内aptazyme的生物传感器的N-乙酰神经氨酸生物合成的途径优化和关键酶进化。

N-乙酰神经氨酸（NeuAc）生物合成因其在许多方面的广泛应用而备受关注。以前，我们首次使用葡萄糖作为唯一底物在大肠杆菌中设计了一条人工的NeuAc生物合成途径。然而，对通量和辅助因子平衡的严格要求使得随后的应变改善相当困难。在这项研究中，设计了一种体内NeuAc生物传感器，并将其用于遗传筛选NeuAc生产者的突变体文库。使用sfgfp作为报告基因证明了其对NeuAc的响应方式，并开发了基于Ni2 +的选择系统以将细胞生长与体内NeuAc浓度耦合在一起。使用该选择系统，可以优化NeuAc的生物合成途径，并进化出关键的酶NeuAc合酶，其效价分别提高了34％和23％。使用葡萄糖作为唯一碳源，最终菌株在基本培养基中可产生高达8.31g / L NeuAc。这项工作证明了NeuAc生物传感器在基因筛选中的有效性以及在其他生物的代谢工程中的巨大潜力。