Bio-based surface-active agents (biosurfactants) are renewable and environmentally friendly alternatives for conventional fossil- and oleochemical based surfactants. In this project, the biosurfactants of interest are branched sophorolipids (BSL) produced by the basidiomycete yeast Pseudohyphozyma bogoriensis. These microbial biosurfactants have potential applications in amongst others the pharma, food and cosmetic sectors. However, BSL biosynthesis by this non-conventional microorganism is not at all efficient, which hampers industrial production of these interesting compounds. Moreover, the BSL biosynthetic pathway remains unknown. To deal with these hurdles, a molecular toolbox will be developed for P. bogoriensis focusing on the development of a CRISPR-based genome editing toolbox. The latter will be useful to apply a knockout strategy aiming to elucidate the BSL biosynthetic pathway. In vitro enzymatic assays will be performed in parallel to achieve the same goal. A pathway tuning tool will be developed and applied once the biosynthetic pathway is elucidated, aiming to boost BSL biosynthesis in P. bogoriensis. Furthermore, the production of BSLs will be evaluated in S. bombicola, an efficient producer of (non-branched) SLs. Together, this will translate into the efficient microbial production of these compounds with promising industrial applications, and on the long term make BSLs economically attractive as alternatives to conventional surfactants.