Engineering a Robust <scp> <i>Escherichia coli</i> </scp> W Platform for Scalable Production of Flavonoid‐O‐Glucosides

Flavonoids are valuable for pharmaceutical, cosmetic and food applications. However, poor solubility and bioavailability limit their widespread use. Biotechnological glycosylation of flavonoids helps address these limitations, but such bioprocesses remain constrained by the cost and availability of uridine diphosphate glucose (UDPG) and the inherent toxicity of flavonoids. In this study we demonstrate that Escherichia coli W is an optimal microbial host for glycosylation bioprocesses using sucrose as a carbon and UDPG source. Escherichia coli W outperforms the model E. coli K12 strain in terms of flavonoid tolerance and glycosylation capabilities. Optimization of sucrose metabolism through adaptive laboratory evolution (ALE) and targeted metabolic engineering to reroute glucose metabolism to UDPG further enhances E. coli W's glycosylation abilities. We validated our glycosylation platform for bench-scale production of chrysin-7-O-glucoside (C7O), a valuable flavonoid glucoside, overcoming key challenges related to the low solubility and bioavailability of its precursor, chrysin. To address bioavailability limitations, we implemented a fed-batch bioprocess in a 3 L bioreactor which returned 1844 mg/L (3.3 mM) C7O, a specific production rate of 0.17 mmol C7O/g DCW·h and a 25.24 mg/g Yp/s after 76 h. An 82.1% yield (1515 mg/L C7O) post extraction and purification demonstrates the efficiency and scalability of the process for industrial bioproduction.