Engineering of the UGT86C11 Glycosyltransferase for Improving Catalytic Efficiency and Efficient Biosynthesis of Neoandrographolide

Neoandrographolide is a bioactive diterpenoid biosynthesized in the medicinal plant Andrographis paniculata by the C₁₉-O-glycosyltransferase UGT86C11. Neoandrographolide supply typically suffers owing to its inadequate amounts in A. paniculata, highlighting the need for an efficient bioprocess to produce this compound. We combined computational modeling of UGT86C11 structure and diterpenoid binding dynamics with site-directed mutagenesis and developed UGT86C11 variants (E163A, T403A, and F404A) exhibiting ∼2-fold increased catalytic efficiency. E163A, T403A, and F404A displayed increased turnover of the C₁₉-O-glycosylation reaction. Moreover, T403A and F404A showed higher affinity for the UDP-glucose sugar donor. Escherichia coli expression of UGT86C11 variants, coupled with an improved UDP-glucose regeneration system, enabled a remarkable biotransformation rate of neoandrographolide, reaching up to 91% conversion as compared to ∼45% using native UGT86C11. Besides, UGT86C11 variants demonstrated ∼6-fold higher neoandrographolide biosynthesis in Nicotiana benthamiana. Thus, the engineered UGT86C11 presents a promising avenue for the efficient production of neoandrographolide using both plant and microbial hosts.