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

Abstract

Neoandrographolide is a bioactive diterpenoid biosynthesized in the medicinal plant <i>Andrographis paniculata</i> by the C₁₉-<i>O</i>-glycosyltransferase UGT86C11. Neoandrographolide supply typically suffers owing to its inadequate amounts in <i>A. paniculata</i>, 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-<i>O</i>-glycosylation reaction. Moreover, T403A and F404A showed higher affinity for the UDP-glucose sugar donor. <i>Escherichia coli</i> 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 <i>Nicotiana benthamiana</i>. Thus, the engineered UGT86C11 presents a promising avenue for the efficient production of neoandrographolide using both plant and microbial hosts.

Affiliated Institutions

• Council of Scientific and Industrial Research IN
• Academy of Scientific and Innovative Research IN
• Central Institute of Medicinal and Aromatic Plants IN
• Brno University of Technology CZ

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