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.