High-Level De Novo Biosynthesis of Gastrodin in Escherichia coli by Protein and Metabolic Engineering

Gastrodin is a naturally occurring phenolic glycoside abundant in the agricultural and medicinal plant Gastrodia elata and has attracted increasing interest as a high-value-added compound due to its antioxidant and cytoprotective properties. However, conventional production through plant extraction is constrained by low natural abundance, long cultivation cycles, and increasing pressure on agricultural resources, while chemical synthesis suffers from low efficiency and environmental concerns. Here, we constructed an efficient and sustainable microbial fermentation platform for the de novo biosynthesis of gastrodin in Escherichia coli through integrated protein and metabolic engineering. Through rational and combinatorial mutagenesis of the glycosyltransferase AtUGT, the catalytic efficiency was markedly enhanced, and the resulting triple mutant AtUGT T282S/A294E/V463L exhibited a 74.84% increase in the level of gastrodin formation compared with the wild type. To strengthen aromatic precursor supply, key genes of the shikimate pathway and associated precursor-providing modules were assembled, enabling a gastrodin titer of 1.54 g/L. Furthermore, reconstruction of an independent UDP-glucose regeneration pathway further boosted production to 2.18 g/L without compromising cell growth. Ultimately, fed-batch fermentation under oxygen-controlled conditions yielded 13.73 g/L gastrodin in a 1.1-L bioreactor, representing the highest reported titer in E. coli . This study establishes a scalable and environmentally friendly strategy for the production of a phenolic glycoside, providing a sustainable alternative to plant extraction for agricultural purposes and a broadly applicable framework for microbial production of glycosylated aromatics.