An engineeredE.colistrain was developed to produce the noncaloric sweetener rebaudioside D (RD) from rebaudioside A (RA) through whole cell biocatalysis. In order to increase endogenous production of the essential active sugar donor of the catalytic reaction; uridine diphosphate glucose (UDPG); host strainE.coliBL21(DE3) was subjected to chromosomal modifications and the resultant genetically modified host strains were named as SG1; SG2; SG3 and SG4; respectively. These modified hosts were transformed with plasmid pYF09 harboring the glycosyltransferase EUGT11 that catalyze the biosynthesis of RD from RA; and the resultant engineered cells were then used to catalyze the biosynthesis of RD. The strain pYF09-SG4 was selected for further studies on whole cell catalysis conditions. The results showed that using fresh cells collected from 50 mL liquid culture and under conditions 100 mmol/L pH 8.0 sodium phosphate buffer 5 mmol/L ZnCl2; 80 mmol/L sodium citrate; 0.1% volumetric percent of Triton X100; 500 g/L sucrose; temperature 42 ℃; and 1 d incubation time; 98.5% of 1 mmol/L RA was catalyzed to RD with a yield of 1 112.21 mg/L.