Submitted to: Metabolic Engineering
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/21/2024
Publication Date: 3/29/2024
Citation: Yu, L., Gao, Y., He, Y., Liu, Y., Shen, J., Liang, H., Gong, R., Duan, H., Price, N.P.J., Song, X., Deng, Z., Chen, W. 2024. Developing the E. coli platform for efficient production of UMP-derived chemicals. Metabolic Engineering. 83:61-74. https://doi.org/10.1016/j.ymben.2024.03.004.
DOI: https://doi.org/10.1016/j.ymben.2024.03.004

Interpretive Summary: 5-Methyluridine (5-MU) is an important chemical intermediate that is used as a starting material to make several antiviral drugs, such as zidovudine (AZT), stavudine and alovudine. Currently, 5-MU is manufactured using a chemical synthesis process that is inefficient and requires expensive starting materials. In this study, we have developed an economical environmentally friendly approach for producing 5-MU by efficient bioengineering of a microbial strain commonly used for fermentation. The best of these strains produces 5-MU in high yields and has the potential to make other similar intermediate chemicals. The approach described in this study is expected to improve production of antiviral drugs and have many potential uses for making antimicrobial agents for veterinary medicine and agriculture.

Technical Abstract: 5-Methyluridine (5-MU) is a prominent intermediate for industrial synthesis of several antiviral-drugs, such as zidovudine, stavudine and alovudine, however, the availability of this chemical over the past decades has overwhelmingly relied on chemical and enzymatic strategies. In this study, we have realized efficient production of 5-MU in E. coli, for the first time, via a designer artificial biosynthetic pathway consisting of a two-enzyme cascade of UMP 5-methylase (PolB and its homologs) and phosphatase (Pbs1). More specifically, we have engineered the E. coli cell factory to boost 5-MU production by systematic evaluation of several biosynthetic strategies, including 5-MU degradation pathway disruption, transcriptional modulation, UMP metabolic flux-limitation, phosphatase-screening, indirect precursor-supply enhancement, an extrinsic pyrimidine operon introduction, and by PolB-homolog screening and application. The resulting 5-MU titer has achieved 4.36 mg/mL by shaking flask fermentation (72 h) and 9.94 mg/mL by fed batch fermentation in 5 L bioreactor (120 h). More importantly, as a proof of concept we have developed a novel antibiotic-free strategy to realize 5-MU production (10.65 mg/mL) in E. coli MB229 (A thyA strain complemented by a plasmid). This also establishes a versatile and robust platform with exploitation of the engineered E. coli strain for efficient production of multiple UMP-derived chemicals. This study paves the way for future engineering of E. coli as a synthetic biology platform for access to greater diversification of UMP-derived chemicals.