Engineering of 4-Hydroxyphenylacetate-3-hydroxylase from Escherichia Coli for Efficient Biosynthesis of Piceatannol

Piceatannol, a catechol compound, exhibits various health benefits, with great application value in food, medicine, and cosmetics. However, the low natural abundance of piceatannol limits its cost-effective isolation, and chemical synthesis is difficult. Here, we report an efficient biocatalytic system for the production of piceatannol from resveratrol. Structure-based semi-rational engineering was used to generate HpaBCG209F, a novel HpaBC mutant of 4-hydroxyphenylacetate-3-hydroxylase from Escherichia coli. Biocatalytic activity assays revealed that HpaBCG209F exhibits 3.12 times higher activity toward resveratrol than wild-type HpaBC. We also introduced formic acid dehydrogenase (FDH) as an NADH-regeneration system for HpaBCG209F to support piceatannol production. Co-expression of HpaBCG209F and FDH in E. coli BL21(DE3) enabled the generation of 8.23 mM piceatannol from 10.2 mM resveratrol within 9.5 h under optimized conditions, representing an 80.6% molar conversion ratio. This study therefore successfully developed a highly active HpaBC variant for hydroxylating resveratrol to piceatannol, as well as demonstrated a promising biopathway for piceatannol production.