分别在大肠杆菌BL21(DE3)中构建胞苷激酶(CDK)与乙酸激酶(ACK)的共表达及融合表达体系和胞苷酸激酶(CMK)与乙酸激酶(ACK)的融合表达体系,其中融合菌株SUMO-CDK-L2-ACK与CMK-L2-ACK构成的双融合催化体系具有较好的催化活性。搭建了双融合菌全细胞一锅法催化合成胞苷三磷酸(CTP)反应体系,并对其温度、pH、金属离子、乙酰磷酸投加量及菌粉投加量等进行优化。结果表明,在优化反应体系中,反应75 min时CTP产量达到最高为108 mmol/L,总收率达86.4%,实现了CTP的一锅法高效合成。

Cytidine triphosphate (CTP) is synthesized through a one-pot whole-cell catalysis pathway with cytidine as substrate.The co-expression and fusion expression systems of cytidine kinase (CDK) and acetate kinase (ACK),and the fusion expression system of cytidylate kinase (CMK) and ACK are respectively constructed in Escherichia coli BL21 (DE3),in which the dual fusion catalytic system composed of SUMO-CDK-L2-ACK fusion strain and CMK-L2-ACK fusion strain is found to have better catalytic activity.A dual fusion strains whole-cell one-pot catalysis reaction system is established for the synthesis of CTP.The system is optimized in terms of temperature,pH,metal ions,acetyl phosphate dosage and bacterial powder dosage.It is indicated that in the optimized reaction system,the highest CTP yield reaches 108 mmol·L^-1 in 75 min,and the total yield reaches 86.4%,which realizes the efficient one-pot synthesis of CTP.

沈来伟(1996-),男,硕士生,研究方向为生物催化,787156353@qq.com;任宇红(1967-),男,博士,研究员,研究方向为生物催化与生物过程工程,通讯联系人,yhren@ecust.edu.cn。