De novo Biosynthesis of Caffeic Acid and Chlorogenic Acid in Escherichia coli via Enzyme Engineering and Pathway Engineering.

Caffeic acid (CA) and chlorogenic acid (CGA) have diverse health benefits, including hemostatic, antioxidant, and antiinflammatory, highlighting their potential for medical applications. However, the absence of high-performance production strains increases production costs, limiting their wider application. In this study, we engineered Escherichia coli for the de novo production of CA and CGA. To improve production, a highly efficient mutant tyrosine ammonia-lyase from Rhodotorula taiwanensis (RtTAL^T415M/Y458F) was identified using genome mining and protein engineering. By engineering the tyrosine biosynthetic pathway through the deletion of pheA and tyrR, along with the overexpression of aroG^fbr and tyrA^fbr, we developed an engineered E. coli strain, CA11, which produced 6.36 g/L of CA with a yield of 0.06 g/g glucose and a productivity of 0.18 g/L/h. This represents the highest titer reported for microbial synthesis of CA using glucose as the sole carbon source in E. coli. Based on strain CA11, we further developed strain CGA13, with optimized replicons, promoters, and ribosome-binding sites, which produced 1.53 g/L of CGA in fed-batch fermentation, highlighting its potential for industrial-scale production.