Protein Engineering to Change the Sugar Donor and Improve the Activity of PnUGT94Q39 for Efficient Ginsenoside Rf Biosynthesis.

Ginsenoside Rf, a rare protopanaxatriol-type ginsenoside derived from Panax ginseng, exhibits notable neuroprotective, anti-inflammatory, and antitumor properties. However, its low natural abundance has impeded large-scale production. Here, we reprogrammed the sugar donor specificity of PnUGT94Q39 from UDP-xylose to UDP-glucose, thereby enabling efficient ginsenoside Rf biosynthesis. A single-point mutation (I142T) within a flexible loop not only switched the sugar donor preference to UDP-glucose but also established efficient catalysis (kcat/Km, 1.18 μM-1·min-1) toward this donor. Coupling the I142T mutant with sucrose synthase GmSUS in Escherichia coli enabled the production of ginsenoside Rf (Rf) from ginsenoside Rh1 (Rh1) without exogenous UDP-glucose addition. Under optimized whole-cell biocatalysis conditions, a ginsenoside Rf titer of 1219.49 mg·L-1 was achieved, corresponding to a 97.26% conversion rate from Rh1 to Rf. Collectively, this work generated an efficient PnUGT94Q39-I142T mutant and established a sustainable, high-efficiency route for ginsenoside Rf production.