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.