The quest for attractive alternative carbohydrates is hampered by the natural scarcity of many of these compounds, complicating evaluation of their properties and application potential. Enzymes, especially glycoside phosphorylases, are promising biocatalysts for the production of these so-called rare sugars. Unfortunately, the limited availability of the glycosyl donors these enzymes require, like β-glucose 1-phosphate (βGlc1P), complicates their commercial exploitation. It is currently impossible to produce βGlc1P in a practical and cost-efficient manner. This PhD project will address this issue by creating an inverting sucrose phosphorylase that allows the production of βGlc1P from the cheap, abundant and highly reactive substrate sucrose. Directed evolution and rational enzyme engineering techniques will be combined to finally obtain an industrially relevant biocatalyst. The feasibility of this project will be demonstrated by two proof-of-concept setups. First, the target compound βGlc1P will be produced on 100 g scale and purified. Secondly, the novel enzyme will be applied for the production of interesting rare sugars, namely trehalose analogues. The successful execution of this project holds great potential for applications in food, pharmaceutical, chemical and biotechnological industries.