Metabolic engineering of Escherichia coli to produce cytidine 5 \' -monophosphate

• 1、School of biotechnology, Jiangnan University, Wuxi 214122

Abstract：Cytidine 5'-monophosphate (5'-CMP) is a key intermediate for the preparation of various nucleotide derivatives and is widely used in food and pharmaceutical industries. In this study, E. coli MG1655 was used as the starting strain to construct a strain efficiently producing 5'-CMP by metabolic engineering. By knocking out the gene ppnN to block the degradation pathway of 5'-CMP to cytosine, the titer of 5\'-CMP in the recombinant strain E. coli CM001 reached 20.58 mg/L. The engineering strain E. coli CM006 was constructed by knocking out the genes ushA, yrfG, yjjG, umpH and umpG to block the degradation pathway of 5'-CMP, the titer of which reached 133.4 mg/L. The 5'-CMP titer of recombinant strain E. coli CM007 was increased by 17.1% compared with the control strain after overexpression of 5'-CTP diphosphate hydrolase gene nudG. In order to reduce the accumulation of intermediate metabolite orotic acid, the orotic acid phosphoribosyltransferase gene pyrE was expressed to promot the synthesis of 5'-CMP. The uridine kinase mutant gene PyrH (D93A) was integrated and expressed to weak the rate-limiting step in the 5'-CMP synthesis pathway, and the 5'-CMP titer of the recombinant strain E. coli CM009 was significantly improved. By knocking out the cytidine deaminase gene cdd and overexpressing the uridine-cytidine kinase gene udk, the accumulation level of 5'-CMP was significantly increased. The integrated expression genes prs, zwf and gnd enhanced the supply of precursor PRPP, and the 5'-CMP yield of the recombinant strain E.coli CM012 was increased to 417.9 mg/L.

Keywords： Escherichia coli cytidine 5 \' -monophosphate de novo synthesis pathway metabolic engineering.