Pyrimidine nucleosides, including uridine and cytidine, are important intermediates in the production of antiviral and antitumor drugs. To obtain a high-yield cytidine-producing strain, this research took Escherichia coli UR6, a high-yield uridine-producing strain, as the starting strain and carried out a serious of metabolic engineering transformations using CRISPR/Cas9 mediated genome editing technology. The results showed that the knockout of cdd, cmk and ygdH genes blocked the main degradation pathways of cytidine and its precursors and the production of cytosine reached 2.8 g/L, while the uridine production remained basically unchanged. The introduction of mutants PyrHecj(D93A) and PyrGcgl(D160E, E162, E168K) combined with the double copy of nudG gene effectively enhanced the cytidine synthetic pathway, resulting in the increase of cytidine production to 5.6 g/L, and the uridine production decrease to 9.3 g/L. The introduction of 5'-nucleotidase PHM8 from Saccharomyces cerevisiae increased the uridine production to 11.5 g/L and had little effect on the accumulation of cytidine. The fusion expression of orotidine-5'-phosphate decarboxylase and uridine kinase promoted UMP flow to cytidine synthesis, which further increased the cytidine production to 7.2 g/L and reduced the uridine production to 8.2 g/L. The final strain can be used for co-production of cytidine and uridine and has a good prospect for industrial application.
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