Steviol glycosides from Stevia rebaudiana leaves are used in stevia-based sweeteners for their intense sweetness and low calories. Rebaudioside D is present in leaves in minute quantities (∼0.4-0.5% w/w total dry weight)， but it is ∼350 times sweeter than sucrose， and sweeter than the more abundant rebaudioside A and stevioside. In the present study， pathways for rebaudioside D synthesis and UDP-glucose recycling were developed by coupling recombinant UDP-glucosyltransferase UGTSL2 from Solanum lycopersicum and sucrose synthase StSUS1 from Solanum tuberosum. Reaction parameters， including substrate ratio， sucrose concentration， temperature， crude extract concentration， and reaction time， were evaluated， and 17.4 g/l of rebaudioside D (yield = 74.6%) was obtained from 20 g/l of rebaudioside A after 20 h， using UDP or UDP-glucose in recombinant cell crude extracts. Extending the reaction time generated rebaudioside M2 from further glycosylation of rebaudioside D. K values for UGTSL2 indicated a higher affinity for rebaudioside D than for rebaudioside A.