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

Abstract

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.

Identification and Characterization of CtUGT3 as the Key Player of Astragalin Biosynthesis in Carthamus tinctorius L.

“…UDP-glucose was used as a sugar donor. Naringenin chalcone 2′-O-glucoside was elucidated by its 1 H NMR spectrum (Figure S10) and 13 C NMR spectrum (Figure S11), and the other products were matched with commercial standards. CtUGT3 catalyzed all of the substrates and exhibited stronger glycosylation activity than the other two candidates.…”

“…The basic metabolic pathway of flavonoid biosynthesis is well known, particularly in model plants . In safflower flowers, many functional genes involved in flavonoid biosynthesis have been cloned and analyzed, including CHS , CHI , FLS , and F3H . − Several studies have been published on flavonoid glycosyltransferases (GTs) in different plants, including O -GTs, − C -GTs, and di- C -GTs. − However, only two GTs have been identified in safflower, and their functions have only been confirmed in vitro. , It is necessary to screen and identify new glycosyltransferases in C. tinctorius.…”