General assessment of genetically-encoded reporters in Yarrowia lipolytica

The development of genetically-encoded reporters is important to characterize gene expression dynamics and investigate cellular-level events. Unlike Bakers yeast, the thick cell wall and the high oil content in oleaginous yeast have restricted our ability to establish efficient fluorescence or enzyme-based reporters. In this book chapter, we detailed the protocol for how to clone and construct fluorescence reporters including hrGFP, TurboGFP, and mScarlet-I3, and the Nanoluc luciferase reporters. We quantified the fluorescence/luciferase reporter efficiency with 96-well microplate readers, flow cytometry, and confocal microscopy. Our results indicate that TurboGFP and hrGFP have a relatively low signal/noise ratio, and mScarlet-I3 yields a very high signal/noise ratio. Compared to fluorescence, luciferase Nanoluc exhibits the highest signal/noise ratio. The downside of using luciferase is the relatively laborious procedure and the related high cost. This chapter may guide us to establish an efficient and reliable reporter system to study gene expression or protein-labeling in nonconventional yeast.