Mannosylerythritol lipids (MELs) are natural glycolipids that possess biosurfactant properties and are abundantly produced by Pseudozyma sp. Due to their specific characteristics, such as biodegradability and low toxicity, MELs have attracted significant interest as an alternative to petroleum-based surfactants in medical and cosmetic fields. The present study describes a novel expression system and optimal transformation conditions of Pseudozyma sp. SY16 for producing MELs. The hygromycin resistance gene under the control of Deinococcus radiodurans-derived Kat1 promoter was used as selection marker and the superfolder green fluorescent protein under the control of the yeast glyceraldehyde- 3-phosphate dehydrogenase promoter was used for confirming successful expression. Using this expression system, several transformants overexpressing genes related to MEL production, including emt1, mmf1, and mat1, were generated. Among them, MMF1-2 strain exhibited an MEL yield of 27.9 g/L, which was 31.6% higher than that of the wild-type strain. Altogether, this study demonstrates that engineered yeast strains hold potential for large-scale production of MELs.