Optimization of Culture Conditions for the Efficient Biosynthesis of Trilobatin from Phloretin by Engineered Escherichia coli Harboring the Apple Phloretin-4′-O-glycosyltransferase

Trilobatin, a dihydrochalcone glucoside and natural sweetener, has diverse biological and therapeutic properties. In the present study, we developed a microbial system to produce trilobatin from phloretin using Escherichia coli (E. coli) overexpressing the phloretin-4'-O-glycosyltransferase from Malus x domestica Borkh. Various optimization strategies were employed for the efficient production of trilobatin using a one-factor-at-a-time method. The effect of UDP-glucose supplementation, substrate, and inducer concentrations, time of substrate feeding as well as protein induction, and different culture media combinations were evaluated and optimized to enhance the production of trilobatin. As a result, the highest trilobatin production, 246.83 μM (107.64 mg L^-1), was obtained with an LB-TB medium combination, 22 h of induction with 0.1 mM IPTG followed by 4 h of feeding with 250 μM phloretin and without extracellular UDP-glucose supplementation. These results demonstrate the efficient production of trilobatin and constitute a promising foundation for large-scale production of the dihydrochalcone glycosides in engineered E. coli.