Degradation of Methanol Catabolism Enzymes of Formaldehyde Dehydrogenase and Formate Dehydrogenase in Methylotrophic Yea
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adation of Methanol Catabolism Enzymes of Formaldehyde Dehydrogenase and Formate Dehydrogenase in Methylotrophic Yeast Komagataella phaffii O. V. Dmytruka, *, N. V. Bulbotkaa, and A. A. Sibirnya, b aDepartment
of Molecular Genetics and Biotechnology, Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, 79005 Ukraine b Department of Biotechnology and Microbiology, University of Rzeszow, Rzeszow, 35-601 Poland *e-mail: [email protected] Received March 10, 2020; revised March 24, 2020; accepted September 18, 2020
Abstract—The investigation of the mechanisms of cytosolic protein degradation is of great fundamental and applied importance. The decrease in the specific activity of formaldehyde dehydrogenase (Fldh1) and formate dehydrogenase (Fdh1) in the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene, and the strain that is defective in autophagy pathway SMD1163 of K. phaffii during short-term and long-term induction with methanol, with or without the addition of the MG132 (proteasome degradation inhibitor), was investigated. It was shown that the duration of cell incubation on methanol had no particular effect on the inactivation of enzymes. The effect of the proteasome inhibitor MG132 was insignificant. Catabolic inactivation of cytosolic and peroxisomal enzymes was damaged in the gss1Δ mutant since glucose signaling was impaired. Fldh1 and Fdh1 are probably degraded via the vacuolar pathway regardless of the duration of methanol induction. Keywords: formaldehyde dehydrogenase, formate dehydrogenase, specific activity, degradation, vacuoles, yeast DOI: 10.3103/S0095452720050047
INTRODUCTION One of the most striking achievements of modern biotechnology is the rapid development of the production of recombinant proteins used in various sectors of the economy and medicine. Methylotrophic yeast (organisms capable of using methanol as a growth substrate) is considered one of the most effective producers of its own and recombinant proteins of industrial importance, in particular, insulin, hepatitis B surface antigen, interferons, and such enzymes as alcohol oxidase, and nitrilase [1–3]. Methylotrophic yeast Komagataella phaffii (formerly Pichia pastoris) [4] is widely used for the production of recombinant proteins for basic and applied research. Promoters of genes encoding enzymes of methanol metabolism are among the strongest and most strictly regulated. Methods of genetic manipulation are well developed for K. phaffii, in particular, highly efficient transformation, integration of DNA into target loci, and cloning of genes by means of functional complementation. These properties, as well as such characteristics of K. phaffii as high biomass production and efficient secretion and yield of target proteins, provide this organism significant advantage over other systems for the production of heterologous proteins. One of the
unsolved problems is the stability of heterologous proteins during their over-synthesis. Increased expression of recombinant proteins in K. pha
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