Developing the E. coli platform for efficient production of UMP-derived chemicals

5-Methyluridine (5-MU) is a prominent intermediate for industrial synthesis of several antiviral-drugs, however, its availability over the past decades has overwhelmingly relied on chemical and enzymatic strategies. Here, we have realized efficient production of 5-MU in E. coli, for the first time, via a designer artificial pathway consisting of a two-enzyme cascade (UMP 5-methylase and phosphatase). More importantly, we have engineered the E. coli cell factory to boost 5-MU production by systematic evaluation of multiple strategies, and as a proof of concept, we have further developed an antibiotic-free fermentation strategy to realize 5-MU production (10.71 g/L) in E. coli MB229 (a ΔthyA strain). Remarkably, we have also established a versatile and robust platform with exploitation of the engineered E. coli for efficient production of diversified UMP-derived chemicals. This study paves the way for future engineering of E. coli as a synthetic biology platform for acceleratively accessing UMP-derived chemical diversities. •A designer artificial pathway comprising a two-enzyme cascade has been constructed for 5-MU biosynthesis.•5-MU production has reached to 9.94 g/L in engineered E. coli with employing multiple synthetic biology strategies.•An antibiotic-free strategy has been developed for robust production of 5-MU (10.71 g/L) in the strain E. coli MB229.•A versatile E. coli platform has been further established for efficient production of diverse UMP-derived chemicals.