Glycosyltransferase Co-Immobilization for Natural Product Glycosylation: Cascade Biosynthesis of the C-Glucoside Nothofagin with Efficient Reuse of Enzymes

Sugar nucleotide-dependent (Leloir) glycosyltransferases are synthetically important for oligosac-charides 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; OsCGT) and the sucrose synthase from soybean (Glycine max; GmSuSy) to synthesize nothofagin, the natural 3'-C-beta-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 (similar to 18 mg/g carrier; 60%-70% yield; similar to 80% effectiveness) in an activity ratio (OsCGT:GmSuSy = similar to 1.2) optimal for the overall reaction rate (similar to 0.2 mmolh(-1) g(-1) catalyst; 30 degrees C, pH 7.5). Using phloretin solubilized at 120 mM as inclusion complex with 2-hydroxypropyl-beta-cyclodextrin, we demonstrate complete substrate conversion into nothofagin (similar to 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 similar to 273 g nothofaginlg protein used. Our study presents important progress towards applied bio-catalysis with immobilized glycosyltransferase cascades.

Automated summary

In this study, we demonstrate the efficient and controllable co-immobilization of two enzymes using glycosyltransferase chimeras with the cationic binding module Z(basic2) for the synthesis of nothofagin. The enzyme surface tethering via Z(basic2) allowed for programmable loading of glycosyltransferases, achieving a high yield of 60%-70% and an effectiveness of approximately 80%. Under optimal conditions, the reaction rate reached approximately 0.2 mmolh(-1) g(-1) catalyst, with successful regeneration of UDP glucose and complete conversion of the substrate.