In this chapter, a step-by-step approach on how to transform non-conventional yeasts or fungi into platform organisms is described. The non-conventional glycolipid producing yeast Starmerella bombicola (and in some cases also Pseudohyphozyma bogoriensis) is used as a case study. And more specifically how to engineer it toward production of new-to-nature glycolipids like bola sophorolipids. When starting genetic engineering efforts for non-lab strains, one should start at the very basis: identifying selection markers and possibly developing auxotrophic strains. Once this is done, the quest for the development of an optimal transformation method can be started. After optimization thereof, knock-out and knock-in strains can be generated based upon the specific strategy/aim. Sometimes this can lead to unexpected, but yet very interesting findings. To fully and efficiently expand the potential as a production platform of these yeast strains, a range of additional molecular tools are required. A well-equipped molecular toolbox should contain a set of characterized promotors, terminators, and defined genomic landing paths. The availability of an episomal system greatly facilitates engineering and screening efforts, but also offers the possibility of developing more advanced engineering techniques such as Crispr-Cas. InBio.be is a world leading pioneer to do this for the yeast S. bombicola and combined, these efforts will result in the commercialization of new types of glycolipids in the next few years.
Keywords: Biosurfactants; Genetic engineering; Glycolipids; Metabolic engineering; Pseudohyphozyma bogoriensis; Sophorolipids; Starmerella bombicola.