Escherichia coli BL21 (DE3) was engineered by blocking glucose-1-phosphate utilizing glucose phosphate isomerase (pgi), glucose-6-phosphate dehydrogenase (zwf) and uridylyltransferase (galU) genes to produce pool of four different rare dTDP-sugars. The cytosolic pool of dTDP-L-rhamnose, dTDP-D-viosamine, dTDP-4-amino 4,6-dideoxy-D-galactose, and dTDP-3-amino 3,6-dideoxy-D-galactose was generated by overexpressing respective dTDP-sugars biosynthesis genes from various microbial sources. A flexible glycosyltransferase YjiC, from Bacillus licheniformis DSM 13 was also overexpressed to transfer sugar moieties to 3-hydroxyl group of 3-hydroxyflavone, a core unit of flavonoids. Among four rare dTDP-sugars generated in cytosol of engineered strains, YjiC solely transferred L-rhamnose from dTDP-L-rhamnose and tuned to rhamnosyltransferase. (C) 2016 Published by Elsevier Inc.