Cascade biocatalysis systems for bioactive naringenin glucosides and quercetin rhamnoside production from sucrose

Two sustainable and cost-effective cascade enzymatic systems were developed to regenerate uridine diphosphate (UDP)-α-D-glucose and UDP-β-L-rhamnose from sucrose. The systems were coupled with the UDP generating glycosylation reactions of UDP sugar-dependent glycosyltransferase (UGT) enzymes mediated reactions. As a result, the UDP generated as a by-product of the GT-mediated reactions was recycled. In the first system, YjiC, a UGT from Bacillus licheniformis DSM 13, was used for transferring glucose from UDP-α-D-glucose to naringenin, in which AtSUS1 from Arabidopsis thaliana was used to synthesize UDP-α-D-glucose and fructose as a by-product from sucrose. In the second system, flavonol 7-O-rhamnosyltransferase (AtUGT89C1) from A. thaliana was used to transfer rhamnose from UDP-β-L-rhamnose to quercetin, in which AtSUS1 along with UDP-β-L-rhamnose synthase (AtRHM1), also from A. thaliana, were used to produce UDP-β-L-rhamnose from the same starter sucrose. The established UDP recycling system for the production of naringenin glucosides was engineered and optimized for several reaction parameters that included temperature, metal ions, NDPs, pH, substrate ratio, and enzymes ratio, to develop a highly feasible system for large-scale production of different derivatives of naringenin and other natural products glucosides, using inexpensive starting materials. The developed system showed the conversion of about 37 mM of naringenin into three different glucosides, namely naringenin, 7-O-β-D-glucoside, naringenin, 4'-O-β-D-glucoside, and naringenin, 4',7-O-β-D-diglucoside. The UDP recycling (RC_max) was 20.10 for naringenin glucosides. Similarly, the conversion of quercetin to quercetin 7-O-α-L-rhamnoside reached a RC_max value of 10.0.