Rational engineering of a nucleotide-promiscuous glycosyltransferase for enhanced gastrodin biosynthesis

The stringent nucleotide specificity of native glycosyltransferases blocks access to alternative nucleoside diphosphate glucose (NDPG) donors, a limitation that persists even in microbial gastrodin synthesis where exogenous uridine diphosphate-glucose (UDPG) is dispensed with. To address this limitation, the GT1-family glycosyltransferase itUGT2 from Indigofera tinctoria was developed into a nucleotide-promiscuous biocatalyst. Combining phylogenetic conservation analysis with molecular docking, key residues governing nucleotide recognition were mapped, and rational mutagenesis of their flanking regions produced the superior variant M25. Against the wild-type, M25 showed dramatically improved affinity and catalytic efficiency ( kcat/Km ​) toward the non-natural donor thymidine diphosphate glucose (TDPG), 4.98-fold for TDPG and 6.01-fold for p-hydroxybenzyl alcohol (pHBA), and with TDPG as the sole donor, replicated the gastrodin synthesis rate of the wild-type enzyme using UDPG. confirmed that these gains are underpinned by a more compact and conformationally stable protein architecture. Expressed in a whole-cell fermentation host, the M25 strain produced 5.82 g/L gastrodin, an 81.31% increase over the wild-type benchmark. This study validates rational nucleotide-binding pocket engineering as a robust strategy for unlocking GT1 donor promiscuity and establishes a replicable framework for exploiting endogenous NDPG diversity in microbial glycoside synthesis.