Glycosyl transferases (GT) are extremely efficient biocatalysts that can be used for the synthesis of special carbohydrates and glycoconjugates. Famous examples of such products include human milk oligosaccharides that serve as prebiotics, and glycosides of flavonoids with improved stability and solubility. Unfortunately, the large-scale application of GT has been hampered by their low operational stability and by the high cost of their glycosyl donor. This project aims to solve both problems by a combination of enzyme and process engineering. On the one hand, the production of stable biocatalysts will be facilitated by the development of a suitable expression system, the design of optimized variants and the development of immobilized formulations. On the other hand, the production and recycling of nucleotide-activated sugars will be accomplished by exploiting the reaction of sucrose synthase (SuSy).With the help of SuSy, UDP-glucose can be produced from sucrose as cheap and abundant substrate. Furthermore, other nucleotide sugars can be obtained when sucrose analogues are employed. These alternative substrates will be produced here with fructansucrase (FS), an enzyme that can couple fructose to various monosaccharides. However, the activity of both FS and SuSy towards sucrose analogues will have to be improved to become economically viable. In that way, the proposed concept can be developed into a generic procedure, in which the nucleotide moiety can be recycled to establish a constant supply of glycosyl donor. The three enzymatic steps will be optimized and integrated into an efficient process for the production of glycosylated compounds. The economical potential of this technology will be demonstrated by the scale-up of selected reactions at the industrial facilities available in our consortium. The participants in this project comprise 3 universities, 1 research institute and 4 SME with complementary skills and expertise.