Isolation of Mutants Defective in Cytosolic β-galactosidase Degradation in the Methylotrophic Yeast Komagataella phaffii

The certain cytosolic enzymes and recombinant proteins in methylotrophic yeast Komagataella phaffii expressed under methanol-induced promoters undergo selective degradation and proteolysis when transferred from methanol to glucose medium via autophagic pathway. However, the mechanism underlying the specific degradation cytosolic proteins in yeast, remain unclear. To this end, we developed a simple method for isolating mutants with impaired degradation of cytosolic proteins in our previous study. This method utilized the K. phaffii strain expressing beta-galactosidase under the control of the methanol-induced FLD1 promoter, fused with GFP. The beta-galactosidase of methanol-grown K. phaffii strain can be directly assayed on plates through X-Gal staining, therefore, offering an opportunity to isolate the mutants defective in inactivation of cytosolic proteins in K. phaffii. In this study, chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was applied to select autophagy-defective mutants in this system. Mutants showing bule color on YPD plates with X-Gal after methanol induction were obtained. Selected mutans display higher specific activity of beta-galactosidase shift from methanol to glucose compared to the wild-type strain. It was also observed that, after shift from methanol to glucose, the mutants exhibited stronger fluorescence compared to the parental strain. The specific activity of alcohol oxidase, which is involved in methanol metabolism, varies among the individual mutants. Additionally, the analyzed mutants exhibited growth defects under nitrogen starvation conditions and showed increased phloxine B staining, indicating impairments in general autophagy.