Evaluation of double expression system for co-expression and co-immobilization of flavonoid glucosylation cascade.

Glucosylation cascade consisting of Leloir glycosyltransferase and sucrose synthase with in situ regeneration system of expensive and low available nucleotide sugars is a game-changing strategy for enzyme-based production of glycoconjugates of relevant natural products. We designed a stepwise approach including co-expression and one-step purification and co-immobilization on glass-based EziG resins of sucrose synthase from Glycine max (GmSuSy) with promiscuous glucosyltransferase YjiC from Bacillus licheniformis to produce efficient, robust, and versatile biocatalyst suited for preparative scale flavonoid glucosylation. The undertaken investigations identified optimal reaction conditions (30 °C, pH 7.5, and 10 mM Mg 2+ ) and the best-suited carrier (EziG Opal). The prepared catalyst exhibited excellent reusability, retaining up to 96% of initial activity after 12 cycles of reactions. The semi-preparative glucosylation of poorly soluble isoflavone Biochanin A resulted in the production of 73 mg Sissotrin (Biochanin A 7-O-glucoside). Additionally, the evaluation of the designed double-controlled, monocistronic expression system with two independently induced promoters (rhaBAD and trc) brought beneficial information for dual-expression plasmid design. KEY POINTS: • Simultaneous and titratable expression from two independent promoters is possible, although full control over the expression is limited. • Designed catalyst managed to glucosylate poorly soluble isoflavone. • The STY of Sissotrin using the designed catalyst reached 0.26 g/L∙h∙g of the resin.