Efficient Production of Cyanidin 3-O-Glucoside and Cyanidin 3-O-Rutinoside in Escherichia coli via Metabolic Engineering and Process Optimization.

Cyanidin-3-O-rhamnoside (C3R) and cyanidin-3-O-glucoside (C3G) show strong anti-inflammatory and antioxidant activities with great potential for chronic disease prevention. However, C3R biosynthesis from (+)-catechin has not been reported, and C3G production from this substrate is low for industrial applications. Here, we constructed a microbial platform for the efficient C3R and C3G production via genetic engineering and metabolic optimization. We screened glucosyltransferases and rhamnosyltransferases from various plants to identify the optimal combinations for C3G and C3R biosynthesis. Tuning UDP-glucose and UDP-rhamnose balance by plasmid copy number increased the C3R concentration by 120%. Overexpression of MdtH and yadH improved extracellular C3R and C3G accumulation by 16% and 31%, respectively. After optimizing substrate and culture conditions, the engineered strains produced 529.01 mg/L of C3R and 926.47 mg/L of C3G, with C3R synthesis reported for the first time. This study underscores the key roles of glycosyltransferases and UDP-sugar modulation, providing a versatile strategy for producing other disaccharide- or rhamnose-containing natural products.