Metabolic Engineering Of Escherichia Coli To Produce Cytidine 5’-monophosphate

Cytidine 5’-monophosphate（5’-CMP）is a key intermediate in the production of variousnucleotide derivatives and is widely used in the food and pharmaceutical industries.As the development of antiviral and anti-tumour drugs and the functionalisation of pyrimidine nucleosides continues to expand,the market demand for pyrimidine nucleosides continues to grow and there is an urgent need to develop low-cost production processes for pyrimidine nucleosides that can be applied on a large scale.Traditional methods for producing 5’-CMP include nucleic acid hydrolysis and chemical synthesis.However,nucleic acid hydrolysis and chemical synthesis have the disadvantages of a long reaction time and a complicated separation process.The synthesis of 5’-CMP through synthetic biology by engineering microorganisms has attracted much attention.In this study,an engineered strain for 5’-CMP production was constructed for the first time in Escherichia coli using metabolic engineering,and its yield was improved by a systematic pathway optimisation strategies.Main research contents include:（1）E.coli MG1655 was used to construct an efficient cytidine 5’-CMP production strain by metabolic engineering.The 5’-CMP degradation pathway was knocked down using CRISPR-Cas9 gene editing technology.The engineered strain E.coli CM006 was constructed by knocking out the gene ppn N to block the degradation pathway of 5’-CMP to cytosine,and knocking out the genes ush A,yrf G,yjj G,ump H and ump G to block the degradation pathway of5’-CMP to cytosine.It could efficiently accumulate 5’-CMP,the yield of which reached 133.4mg·L^-1.（2）Enhancement of 5’-CMP accumulation by increasing the expression levels of key enzymes in the 5’-CMP synthesis pathway.The recombinant strain E.coli CM007 was constructed by overexpressing the 5’-CTP diphosphate hydrolase gene nud G from CTP to 5’-CMP,the 5’-CMP yield of which reached 183.0 mg·L^-1.The orotic acid phosphoribosyl transferase gene pyr E was overexpressed to reduce the accumulation of the intermediate metabolite orotic acid to promote the synthesis of 5’-CMP.The yield of recombinant strain E.coli CM009 constructed reached 253.0 mg·L^-1 by integrating the expression of the uridylate kinase mutant gene pyr H（D93A）,which weakened the rate-limiting step in the 5’-CMP synthesis pathway.（3）The accumulation of 5’-CMP was increased by reducing cytidine accumulation.The yield of 5’-CMP was significantly increased by knocking out the cytidine deaminase gene cdd and overexpressing the uridine cytidine kinase gene udk.The supply of the precursor PRPP was increased by integranted expression of the genes prs,zwf and gnd of PPP pathway to construct the recombinant strain E.coli CM012,the 5’-CMP yield of which reached 417.9 mg·L^-1.（4）The recombinant strain E.coli CM016 was constructed by introducing the pyrimidine manipulator gene cluster from Bacillus subtilis in E.coli CM012.The UMP synthesis pathway was reconstructed in E.coli to improve the supply of precursors for the synthesis of 5’-CMP,and its yield of 5’-CMP reached 468.0 mg·L^-1.