Abstract:Cytidine 5?-monophosphate (5?-CMP), a fundamental component of RNA and a key intermediate for nucleotide derivatives, has broad applications in the medical, food, and agricultural industries. However, the biosynthesis of 5?-CMP faces challenges such as low enzyme catalytic efficiency, low substrate conversion rates, and high production costs. To address these limitations, we first screened and identified a cytidine kinase (MmUCK) with high product tolerance and strong stability. After that, an AMP/ATP regeneration system was introduced to reduce ATP consumption. With cytidine, sodium hexametaphosphate, and adenosine monophosphate (AMP) as substrates, 5?-CMP was efficiently synthesized via a one-pot, dual-enzyme biocatalytic system. That is, (76.94±3.26) mmol/L 5?-CMP was produced when 100 mmol/L cytidine was used as the substrate. Furthermore, the cytidine deaminase gene (cdd) and the pyrimidine-specific ribonucleoside hydrolase gene (rihC) in the cytidine branch were knocked out, which increased the molar conversion rate to 98.1%. Finally, in a 10 L bioreactor, (563.93±8.84) mmol/L 5?-CMP was synthesized after 7 h of enzymatic reaction with 600 mmol/L cytidine, 150 mmol/L sodium hexametaphosphate, and 5 mmol/L AMP as substrates, and a molar conversion rate of 94.2% was achieved. Our study significantly improves catalytic efficiency and conversion rates by using a highly tolerant MmUCK enzyme, constructing an AMP/ATP regeneration system, and knocking out genes related to branch metabolic pathways, providing an economically efficient and feasible route for the industrial biomanufacturing of 5?-CMP.
