High-level and -yield orotic acid production in Escherichia coli through systematic modular engineering and "Chaos to Order Cycles" fermentation.

[Display omitted] • Escherichia coli was engineered to produce industrial-yields of orotic acid. • A plasmid-free, uninduced and nonauxotrophic orotic acid strain was constructed. • Metabolic flux for orotic acid biosynthesis was optimized by systematic engineering. • A novel "Chaos to Order Cycles" fermentation method was developed. • The highest production titer, productivity and yield of orotic acid was achieved. Orotic acid is widely used in healthcare and cosmetic industries. However, orotic acid-producing microorganisms are auxotrophic, which results in inefficient microbial production. Herein, a plasmid-free, uninduced, non-auxotrophic orotic acid hyperproducer was constructed from Escherichia coli W3110. Initially, the orotic acid degradation pathway was blocked and the carbamoyl phosphate supply was enriched. Subsequently, pyr operon from Bacillus subtilis F126 was heterologously expressed and precursors' supply was optimized. Thereafter, pyrE was dynamically regulated to reconstruct the non-auxotrophic pathway. Employing fed-batch cultivation, orotic acid titer, yield, and productivity of strain Ora21 reached 182.5 g/L, 0.58 g/g, and 3.80 g/L/h, respectively, the highest levels reported so far. Finally, a novel "Chaos to Order Cycles (COC)" fermentation was developed, which effectively increased the yield to 0.63 g/g. This research is a remarkable achievement in orotic acid production by microbial fermentation and has vast potential for industrial applications. [ABSTRACT FROM AUTHOR]