Orotic acid, an essential intermediate in pyrimidine nucleotide biosynthesis, has broad applications in the food, pharmaceutical, and cosmetic industries. It serves as a carrier for essential metal ions including magnesium, zinc, and lithium, and exhibits physiological functions supporting growth, cellular repair, and liver health. While microbial production offers advantages over traditional chemical synthesis—including milder reaction conditions and reduced environmental impact—previous production levels have been insufficient for large-scale industrial implementation.
The research team employed rational modular metabolic engineering strategies to construct a high-yielding orotic acid producer. The entire metabolic pathway was divided into three functional modules: downstream degradation, midstream synthesis, and upstream precursor supply. The strategy involved blocking downstream consumption by deleting the pyrE gene, enhancing midstream pathway expression through optimized plasmid combinations, and boosting precursor availability by relieving transcriptional repression and redirecting metabolic flux.
A key innovation of this study is the implementation of a toxin-antitoxin (TA) system for plasmid stabilization. Traditional industrial fermentation requires antibiotics to maintain plasmid stability, which increases production costs and complicates downstream purification. The TA system selectively eliminates plasmid-free cells through post-segregational killing, achieving over 90% plasmid retention after six days of cultivation without antibiotics. This antibiotic-free approach not only reduces production costs but also meets safety requirements for food and pharmaceutical applications.
The final engineered strain represents the highest orotic acid titer reported in E. coli to date, establishing a competitive microbial platform for industrial production. The modular engineering framework developed in this study provides a blueprint for biosynthesis of other pyrimidine nucleotide derivatives.
The work entitled “Construction of an antibiotic-free orotic acid producer in Escherichia coli via modular molecular engineering combined with a plasmid stabilization system” was published on Systems Microbiology and Biomanufacturing (published on Mar. 19, 2026).
DOI:10.1007/s43393-026-00451-x