ZUO M. Regulation of β-galactosidase degradation and metabolic engineering of xylose fermentation in the methylotrophic yeasts Komagataella phaffii and Ogataea polymorpha

This dissertation primarily focuses on three topics: the first involves isolating mutants with defects in cytosolic β-galactosidase degradation in the methylotrophic yeast Komagataella phaffii through the use of the chemical mutagen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG); The second is to develop new dominant selectable markers for future applications in metabolic engineering of the yeast O. polymorpha; the third addresses the development and implementation of metabolic engineering strategies to produce Ogataea polymorpha strains with enhanced efficiency of ethanol production from xylose. Komagataella phaffii (previously known as Pichia pastoris) is an obligate aerobic, methylotrophic yeast capable of using methanol as sole carbon and energy source (Mastropietro et al., 2021). In this dissertation, the chemical mutagen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) was utilized to select new mutant strains exhibiting impaired degradation of β-galactosidase. The impairment of this enzyme’s degradation in the selected mutants was assessed based on their blue color on YPD with X-gal plates following the shift from methanol to glucose. Four mutants displaying elevated β-galactosidase activity on glucose compared to the parental strain were identified. Viability assays and phloxine B assays conducted under nitrogen starvation conditions revealed growth defects in these mutants. Furthermore, the biomass of the mutant strains was significantly diminished relative to that of the parental strain, indicating an impairment in autophagy. These findings suggest that the selected mutants possess defects in autophagy. Additionally, to investigate pexophagy, residual activity of the key peroxisomal matrix protein alcohol oxidase (AOX) was measured in the selected mutants after shifting cells from methanol to glucose. The results demonstrated that mutants MNNG-1 and MNNG-3 exhibited defects in both selective autophagy and pexophagy, while mutants MNNG-2 and MNNG-4 were characterized exclusively by defects in selective autophagy of cytosolic β-galactosidase. This study thus establishes a foundational basis for further investigations into autophagy and protein degradation, paving the way for advances in biotechnology and pharmaceutical applications where precise control of autophagic processes is essential. In another topic of this dissertation, novel dominant selectable markers were developed for the nonconventional yeast Ogataea polymorpha. Therefore, in this study, we evaluated two potential dominant selective markers for O. polymorpha: the mutated AUR1 gene and the native IMH3 gene, which confer resistance to aureobasidin and mycophenolic acid, respectively. These markers were tested for their potential application in metabolic engineering of O. polymorpha. Two mutant versions of the AUR1 gene were created to develop aureobasidin-resistant O. polymorpha strains. In the third topic in this dissertation, we primarily focus on elevated bioethanol production from xylose in O. polymorpha. Therefore, this dissertation focuses on investigating the roles of the hexose sensor gene HXS1 and the AZF1 gene, which encodes the O. polymorpha homolog of the S. cerevisiae transcription factor with sensing properties, in the alcoholic fermentation of xylose and glucose by O. polymorpha. The data suggest that overexpressing the AZF1 and HXS1 genes has a positive impact on ethanol production from xylose in O. polymorpha strains. Given that efficient xylose fermentation is essential for optimizing bioethanol production from lignocellulosic biomass, understanding these mechanisms is necessary for advancing industrial applications and enhancing the economic viability of biofuels. Besides, the strains developed in this study can be further utilized to establish stable ethanol superproducers.