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The production at industrial scale of recombinant proteins (rProt) and metabolites is of increasing economic importance. Among the different microbial chassis that have been developed for that purpose, yeasts are regarded as the preferred option for the production of recombinant enzymes, therapeutic proteins and metabolites. The main advantage of yeast over bacterial system such as Escherichia coli relies on the possibility to obtain post-translational modified proteins in the culture supernatant at a gram per liter scale. Historically, Saccharomyces cerevisiae has been used as the reference eukaryotic chassis, however it suffers several drawbacks such as low protein productivity, overflow metabolism and hyperglycosylation phenomenon. Moreover, it is less metabolically adapted to catabolize raw carbon and nitrogen sources, which are nowadays increasingly considered as feedstock in bioprocesses with the intention to reduce the process cost. Non-conventional yeasts, such as Pichia pastoris and Yarrowia lipolytica, are considered today as realistic alternatives to S. cerevisiae for these bioprocesses. They both combine advantages of growing at high cell density, to produce and secrete rProt and metabolite at high yield and to have low nutritional requirements, allowing thus to grow them on raw materials or industrial byproducts. Here we will present the peculiar physiological traits of both P. pastoris and Y. lipolytica that enabled the development metabolic engineering tools and efficient bioreactor production processes. Several examples will be detailed such as the sweetener erythritol biosynthesis in Y. lipolytica and the extracellular synthesis of lipase CalB in P. pastoris.