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 ( Gm SuSy) 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 . Graphical Abstract