This PhD project focuses on the development of a cell-free protein synthesis workflow which allows in-depth characterization of small, multidomain enzymes responsible for specialty carbohydrates biosynthesis. These specialty carbohydrates have been known for many decades, yet their bioactivity is poorly investigated and their application potential far from fully exploited, mainly due to their restricted availability. To enable a microbial production process, a sustainable, efficient and scalable production method, these enzymes need to be characterized. Hence, in this project we will develop an in vitro protein synthesis workflow for specialty carbohydrates genes, circumventing the hurdles associated with arduous in vivo characterization. Additionally, chemical structure elucidation of these specialty carbohydrates and their bioactive properties will be investigated. Besides the natural sugars produced by these multidomain enzymes, a portfolio of new (-to-nature) sugars with specific bioactivites will be developed.