The use of synthetic biology on yeasts has enhanced the production of commercially relevant chemicals, from biofuels to recombinant therapeutic proteins, to name just a few. Despite most of these advances had already been studied and described in Saccharomyces cerevisiae, during the last years the attention has turned to the use of alternative expression systems with a higher yield and quality such as non-conventional yeasts. Recently, there has been an increase in studies about non-conventional yeasts due to advantages based on their natural capacity to tolerate harsh conditions or the wide range of carbon sources they need during the generation of specific products. This chapter, therefore, aims to describe the current status of the most used non-conventional yeasts in metabolite production as well as the engineering behind them in order to optimize or regulate protein expression: Pichia pastoris, Kluyveromyces marxianus, Kluyveromyces lactis and Yarrowia lipolytica.