Nucleotide sugar-dependent (Leloir) glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine-5-diphosphate glucose (UDP-glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at approximate to 100g scale synthesis. Constitutive expression in Escherichia coli shifted the recombinant protein production mainly to the stationary phase and enhanced the specific enzyme activity to a level (approximate to 480U/g(cell dry weight)) suitable for whole-cell biotransformation. The UDP-glc production had excellent performance metrics of approximate to 100g(product)/L, 86% yield (based on UDP), and a total turnover number of 103g(UDP-glc)/g(cell dry weight) at a space-time yield of 10g/L/h. Using efficient chromatography-free DSP, the UDP-glc was isolated in a single batch with 90% purity and in 73% isolated yield. Overall, the process would allow production of approximate to 0.7kg of isolated product/L E. coli bioreactor culture, thus demonstrating how integrated process design promotes the practical use of a GT conversion. Biotechnol. Bioeng. 2017;114: 924-928. (c) 2016 Wiley Periodicals, Inc.