Hyperoside is used as the standard to assess the quality ofHyperin perforatumL and exhibits many biological properties. To improve the yield of hyperoside inEscherichia coli,four UDP-galactose synthesis pathways were screened and a recombinant strain was reconstructed by using an efficient UDP-galactose generation system coupled withPetunia hybridaglycosyltransferase to biosynthesis hyperoside from quercetin. By adopting the resting cell transformation, maximal hyperoside production of 4574 mg/L and average productivity of 63.5 mg/L/h were achieved after 72 h of biotranformation. Subsequently through recycling of resting cells, average productivity within 72 h reached 126 mg/L/h, which was 100% higher than that in the batch fermentation. Hyperoside production reached equivalent to 18,000 mg/L by recycling seven times of resting cell in 168 h, which was 393% of that in the batch fermentation and the first to reach 10 g per liter scale inE. coli. Therefore, this study provides an efficient method for construction of UDP-galactose synthesis pathway and hyperoside production inE. coli. [GRAPHICS] .