Yap1-Regulated Glutathione Redox System Curtails Accumulation of Formaldehyde and Reactive Oxygen Species in Methanol Metabolism of Pichia pastoris

Abstract

The glutathione redox system, including the glutathione biosynthesis and glutathione regeneration reaction, has been found to play a critical role in the yeast Pichia pastoris during growth on methanol, and this regulation was at least partly executed by the transcription factor PpYap1. During adaptation to methanol medium, PpYap1 transiently localized to the nucleus and activated the expression of the glutathione redox system and upregulated glutathione reductase 1 (Glr1). Glr1 activates the regeneration of the reduced form of glutathione (GSH). Depletion of Glr1 caused a severe growth defect on methanol and hypersensitivity to formaldehyde (HCHO), which could be complemented by addition of GSH to the medium. Disruption of the genes for the HCHO-oxidizing enzymes PpFld1 and PpFgh1 caused a comparable phenotype, but disruption of the downstream gene PpFDH1 did not, demonstrating the importance of maintaining intracellular GSH levels. Absence of the peroxisomal glutathione peroxidase Pmp20 also triggered nuclear localization of PpYap1, and although cells were not sensitive to HCHO, growth on methanol was again severely impaired due to oxidative stress. Thus, the PpYap1-regulated glutathione redox system has two important roles, i.e., HCHO metabolism and detoxification of reactive oxygen species.

Expression of the Laccase Gene from a White Rot Fungus in Pichia pastoris Can Enhance the Resistance of This Yeast to H 2 O 2 -Mediated Oxidative Stress by Stimulating the Glutathione-Based Antioxidative System

“…PpYAP1 can act as a regulator of the redox system in Pichia pastoris. It plays an important role in the defense against oxidative stress by activating the expression of other antioxidative genes involved in the glutathione-based antioxidant system (55,56). Based on our results and previous research, we propose the following hypothesis to address the issue of how laccase expression is linked to defense mechanisms like enhanced glutathione production.…”

“…Previous research has revealed that ScYAP1 is an important transcription factor in Saccharomyces cerevisiae, in which it can stimulate the expression of several genes of the glutathionedependent antioxidative system, such as GLR1, GPX2, and GSH1 (28, 38-40, 53, 55). Recent studies have shown that the PpYAP1-regulated glutathione redox system plays an important role in the detoxification of reactive oxygen species in the methanol metabolism of Pichia pastoris (55). PpYAP1 can act as a regulator of the redox system in Pichia pastoris.…”

“…This causes strong oxidative stress. The glutathione redox system, which includes synthesis and recycling, has been found to play an important role in scavenging hydrogen peroxide, detoxifying reactive oxygen species, and protecting against oxidative stress in Pichia pastoris (55,56,58). We have previously cloned and characterized a laccase gene, lac5930-1, and its corresponding full-length cDNA from white rot fungus Trametes sp.…”

Msn5p Is Involved in Formaldehyde Resistance but Not in Oxidative Stress Response in the Methylotrophic YeastCandida boidinii

“…In addition, a transcription factor termed Yap1p plays an important role in the upregulation of GSH-related enzymes in P. pastoris. 15,16) In that the PpYap1p-depleted strain proved sensitive to H 2 O 2 but not to HCHO, methylotrophic yeasts must have an alternative transcriptional regulatory system to controll the expression of the genes for HCHO resistance.To date, in the budding yeast Saccharomyces cerevisiae, stress-inducible transcription factors Msn2p/ Msn4p and heat shock protein Ssa4p, and their carrier Msn5p, have been reported to play important roles in protecting cells from stress-induced cell death. [17][18][19][20][21] Among these factors, we focused on Msn5p, which belongs to the importin-family and participates in the nuclear export of several proteins.…”

The Peroxisomal Catalase Gene in the Methylotrophic YeastPichia methanolica

“…Recently, the mechanisms by which methylotrophic yeasts respond to oxidative stress derived from H 2 O 2 or other reactive oxygen species (ROS) were elucidated in P. pastoris. 18,19) This knowledge should improve the utility of the P. methanolica gene expression system, as it is necessary to understand not only the scavenging system for ROS but also the mechanism of response to ROS.…”