A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d - and l -sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α- d -glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3- O -β- d -glucoside, resveratrol 4′- O -β- d -glucoside, resveratrol 3,5- O -β- d -diglucoside, and resveratrol 3,5,4′- O -β- d -triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3- O -β- d -2-deoxyglucoside and resveratrol 3,5- O -β- d -di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4′- O -β- d -galactoside, resveratrol 4′- O -β- d -viosaminoside, resveratrol 3- O -β- l -rhamnoside, and resveratrol 3- O -β- l -fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.

Ramesh Prasad Pandey, Prakash Parajuli, Niranjan Koirala, Joo Ho Lee, Yong Il Park, and Jae Kyung Sohng. Mol. Cells 2014;37:172-7. https://doi.org/10.14348/molcells.2014.2348

Multi-monocistronic and multi-variate vectors were designed, built, and tested for the improved production of cyanidin 3-O-glucoside (C3G) in Escherichia coli BL21 (DE3). The synthetic bio-parts were designed in such a way that multiple genes can be assembled using the bio-brick system, and expressed under different promoters in a single vector. The vectors harbor compatible cloning sites, so that the genes can be shuffled from one vector to another in a single step, and assembled into a single vector. The two required genes: anthocyanidin synthase (PhANS) from Petunia hybrida, and cyanidin 3-O-glucosyltransferase (At3GT) from Arabidopsis thaliana, were individually cloned under PT7, Ptrc, and PlacUV5 promoters. Both PhANS and At3GT were shuffled back and forth, so as to generate a combinatorial system for C3G production. The constructed systems were further coupled with the genes for UDP-d-glucose synthesis, all cloned in a multi-monocistronic fashion under PT7. Finally, the production of C3G was checked and confirmed using the modified M9 media, and analyzed through various chromatography and spectrometric analyses. The engineered strains endowed with newly generated vectors and the genes for C3G biosynthesis and UDP-d-glucose synthesis were fed with 2 mM (+)-catechin and d-glucose for the production of cyanidin, and its subsequent conversion to C3G. One of the engineered strains harboring At3GT and PhANS under Ptrc promoter and UDP-d-glucose biosynthesis genes under PT7 promoter led to the production of ~ 439 mg/L of C3G within 36 h of incubation, when the system was exogenously fed with 5% (w/v) d-glucose. This system did not require exogenous supplementation of UDP-d-glucose. A synthetic vector system using different promoters has been developed and used for the synthesis of C3G in E. coli BL21 (DE3) by directing the metabolic flux towards the UDP-d-glucose. This system has the potential of generating better strains for the synthesis of valuable natural products.