Degradation of Methanol Catabolism Enzymes of Formaldehyde Dehydrogenase and Formate Dehydrogenase in Methylotrophic Yeast Komagataella phaffii

Dmytruk, Olena V.; Bulbotka, N. V.; Sibirny, Andriy А.

doi:10.3103/s0095452720050047

Cited by 2 publications

(2 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To test whether the observed deficiency in cytosolic protein degradation under conditions of impaired β‐1,6‐N‐acetylglucosaminyltransferase activity is specific to certain proteins (like recombinant ones) or a more general phenomenon, we decided to estimate the rate of degradation of the native K. phaffii cytosolic enzymes formaldehyde dehydrogenase and formate dehydrogenase, which are involved in methanol metabolism and are degraded by autophagy (Dmytruk et al, 2020).…”

Section: Resultsmentioning

confidence: 99%

“…The activity of formaldehyde dehydrogenase and formate dehydrogenase was determined according to the method described in Dmytruk et al (2020). Formaldehyde dehydrogenase activity was determined in a reaction mixture of the following composition (per 1 mL): 1 M potassium phosphate buffer, pH 8–100 μL; 1 M formaldehyde—0.3 μL; 50 mM NAD + (Sigma‐Aldrich; 43410)—20 μL; 100 mM glutathione (Sigma‐Aldrich; G4251)—20 μL.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Zazulya

Semkiv

Stec

et al. 2023

Yeast

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%