The Komagatella phaffii ACG1 gene, encoding β-1,6-N-acetylglucosaminyltransferase, is involved in the autophagy of cytosolic and peroxisomal proteins.

Abstract

The methylotrophic yeast Komagataella phaffii is considered one of the most effective producers of recombinant proteins of industrial importance. Effective producers should be characterized by the maximal reduction of degradation of the cytosolic recombinant proteins. The mechanisms of degradation of cytosolic proteins in K. phaffii have not been elucidated; however, data suggest that they are partially degraded in the autophagic pathway. To identify factors that influence this process, a developed system for the selection of recombinant strains of K. phaffii with impaired autophagic degradation of the heterologous model cytosolic protein (yeast β-galactosidase) was used for insertional tagging of the genes involved in cytosolic proteins degradation. In one of the obtained strains, the insertion cassette disrupted the open reading frame of the gene encoding β-1,6-N-acetylglucosaminyltransferase. A recombinant strain with deletion of this gene was also obtained. The rate of degradation of the β-galactosidase enzyme was two times slower in the insertion mutant and 1.5 times slower in the deletion strain as compared to the parental strain with native β-1,6-N-acetylglucosaminyltransferase. The rate of degradation of native K. phaffii cytosolic and peroxisomal enzymes, formaldehyde dehydrogenase, formate dehydrogenase, and alcohol oxidase, respectively, showed similar trends to that of β-galactosidase-slower degradation in the deletion and insertional mutants as compared to the wild-type strain, but faster protein degradation relative to the strain completely defective in autophagy. We conclude that K. phaffii gene designated ACG1, encoding β-1,6-N-acetylglucosaminyltransferase, is involved in autophagy of the cytosolic and peroxisomal proteins.