Glycosyltransferase Co‐Immobilization for Natural Product Glycosylation: Cascade Biosynthesis of theC‐Glucoside Nothofagin with Efficient Reuse of Enzymes

Abstract Sugar nucleotide‐dependent (Leloir) glycosyltransferases are synthetically important for oligosaccharides and small molecule glycosides. Their practical use involves one‐pot cascade reactions to regenerate the sugar nucleotide substrate. Glycosyltransferase co‐immobilization is vital to advance multi‐enzyme glycosylation systems on solid support. Here, we show glycosyltransferase chimeras with the cationic binding module Z basic2 for efficient and well‐controllable two‐enzyme co‐immobilization on anionic (ReliSorb SP400) carrier material. We use the C ‐glycosyltransferase from rice ( Oryza sativa ; Os CGT) and the sucrose synthase from soybean ( Glycine max ; Gm SuSy) to synthesize nothofagin, the natural 3’‐ C ‐β‐ d ‐glucoside of the dihydrochalcone phloretin, with regeneration of uridine 5’‐diphosphate (UDP) glucose from sucrose and UDP. Exploiting enzyme surface tethering via Z basic2 , we achieve programmable loading of the glycosyltransferases (∼18 mg/g carrier; 60%–70% yield; ∼80% effectiveness) in an activity ratio ( Os CGT: Gm SuSy=∼1.2) optimal for the overall reaction rate (∼0.2 mmol h −1 g −1 catalyst; 30 °C, pH 7.5). Using phloretin solubilized at 120 mM as inclusion complex with 2‐hydroxypropyl‐β‐cyclodextrin, we demonstrate complete substrate conversion into nothofagin (∼52 g/L; 21.8 mg product h −1 g −1 catalyst) at 4% mass loading of the catalyst. The UDP‐glucose was recycled 240 times. The solid catalyst showed excellent reusability, retaining ∼40% of initial activity after 15 cycles of phloretin conversion (60 mM) with a catalyst turnover number of ∼273 g nothofagin/g protein used. Our study presents important progress towards applied bio‐catalysis with immobilized glycosyltransferase cascades. magnified image