Biocatalysis for rare ginsenoside rh2 production in high level with co-immobilized udp-glycosyltransferase bs-yjic mutant and sucrose synthase atsusy

Rare ginsenoside Rh2 exhibits diverse pharmacological effects. UDP-glycosyltransferase (UGT) catalyzed glycosylation of protopanaxadiol (PPD) has been of growing interest in recent years. UDP-glycosyltransferase Bs-YjiC coupling sucrose synthase in one-pot reaction was successfully applied to ginsenoside biosynthesis with UDP-glucose regeneration from sucrose and UDP, which formed a green and sustainable approach. In this study, the his-tagged UDP-glycosyltransferase Bs-YjiC mutant M315F and sucrose synthase AtSuSy were co-immobilized on heterofunctional supports. The affinity adsorption significantly improved the capacity of specific binding of the two recombinant enzymes, and the dual enzyme covalently cross-linked by the acetaldehyde groups significantly promoted the binding stability of the immobilized bienzyme, allowing higher substrate concentration by easing substrate inhibition for the coupled reaction. The dual enzyme amount used for ginsenoside Rh2 biosynthesis is Bs-YjiC-M315F: AtSuSy = 18 mU/mL: 25.2 mU/mL, a yield of 79.2% was achieved. The coimmobilized M315F/AtSuSy had good operational stability of repetitive usage for 10 cycles, and the yield of ginsenoside Rh2 was kept between 77.6% and 81.3%. The high titer of the ginsenoside Rh2 cumulatively reached up to 16.6 mM (10.3 g/L) using fedbatch technology, and the final yield was 83.2%. This study has established a green and sustainable approach for the production of ginsenoside Rh2 in a high level of titer, which provides promising candidates for natural drug research and development.