Glycosyltransferase Co‐Immobilization for Natural Product Glycosylation: Cascade Biosynthesis of the C‐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 Zbasic2 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‐β‐d‐glucoside of the dihydrochalcone phloretin, with regeneration of uridine 5’‐diphosphate (UDP) glucose from sucrose and UDP. Exploiting enzyme surface tethering via Zbasic2, we achieve programmable loading of the glycosyltransferases (∼18 mg/g carrier; 60%–70% yield; ∼80% effectiveness) in an activity ratio (OsCGT:GmSuSy=∼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.

Leloir glycosyltransferases enabled to flow synthesis: Continuous production of the natural C‐glycoside nothofagin

“…The product solution therefore contained 0.5 g unreacted phloretin. The catalyst half-life of approximately 200 h under in operando conditions of the flow reactor (Figure 5a) was consistent with evidence from an earlier study (Liu et al, 2021) that used recycling of co-immobilized Z-OsCGT and Z-GmSuSy in repeated batch reaction.…”

“…BSA, bovine serum albumin; GmSuSy, sucrose synthase from soybean (Glycine max); HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; OsCGT, C-glycosyltransferase from rice (Oryza sativa); UDP, uridine 5ʹ-diphosphate that could use multivalency from four Z basic2 modules for binding, the immobilization of Z-GmSuSy is not much stronger than that of Z-OsCGT. Liu et al (2021) discuss the results in relation to structural features of the sucrose synthase. Here, total protein loading was varied between 122 mg/g and 240 mg/g and the immobilization results are summarized in Table S3.…”

“…Based on Liu et al (2021), who showed that a Z-OsCGT/Z-GmSuSy activity ratio of approximately 1.2 in the loaded mixture of cell lysates was optimal for the overall synthetic activity of the co-immobilized enzyme preparation, we here aimed at enhancing the immobilized nothofagin activity (U/g carrier) while keeping the loaded enzyme activity ratio constant. Individual immobilization of Z-OsCGT and Z-GmSuSy on Relisorb SP400 was studied in detail before (Liu et al, 2021) S2). (b) Reaction of Z-GmSuSy at varied τ res and UDP concentration (in mM; ▽, 1.0; ○, 2.0; △, 5.0; ■, 8.0; •, 10).…”

Structure–function relationship of terpenoid glycosyltransferases from plants

“…To date, only a few C -UGTs have been characterized although they show great biotechnological potential as biocatalysts. 156 In the future, research on these enzymes is likely to be further intensified. Similarly, more attention will be paid to N - and S -UGTs, since numerous physiologically active natures are N - and S -glycosides.…”

Exploring the in Vitro Operating Window of Glycosyltransferase PtUGT1 from Polygonum Tinctorium for a Biocatalytic Route to Indigo Dye

“…Successful examples thereof were recently published by Trobo-Maseda et al 24 and Liu et al 25 who co-immobilized a sucrose synthase with their glycosyltransferase. We understand that while this measure shifts the problem away from the expensive cosubstrate, it still requires hyperstoichiometric amounts of sucrose.…”