Yeast Methylotrophy: Metabolism, Gene Regulation and Peroxisome Homeostasis

Abstract

Eukaryotic methylotrophs, which are able to obtain all the carbon and energy needed for growth from methanol, are restricted to a limited number of yeast species. When these yeasts are grown on methanol as the sole carbon and energy source, the enzymes involved in methanol metabolism are strongly induced, and the membrane-bound organelles, peroxisomes, which contain key enzymes of methanol metabolism, proliferate massively. These features have made methylotrophic yeasts attractive hosts for the production of heterologous proteins and useful model organisms for the study of peroxisome biogenesis and degradation. In this paper, we describe recent insights into the molecular basis of yeast methylotrophy.

Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

“…Among the tested promoters, the activities of P AOD1 in all disruptant strains remained at 16 to 28% of the levels of the wild-type strain, whereas the activities of P DAS1 and P FDH1 decreased to levels below 10% of those of the wild-type strain. Because P AOD1 is partly activated by glucose derepression, which is independent of methanol-specific induction (6,8), CbHap2p, CbHap3p, and CbHap5p are assumed to be more involved in methanol-specific induction.…”

“…A chromatin immunoprecipitation (ChIP) assay was performed as described previously (6,8), and primers used for this assay are listed in Table S1 in the supplemental material. CbHAP2-YFP/Cbhap2⌬, CbHAP3-YFP/Cbhap3⌬, and CbHAP5-YFP/ Cbhap5⌬ cells grown to mid-log phase in YND or YNM medium were crosslinked with 1% formaldehyde for 10 min.…”

“…Efficient production has been achieved with their strong and tightly regulated methanolinducible gene promoters in combination with high-cell-density cultures in methanol-containing medium. The genes encoding methanol-metabolic enzymes are highly induced in the presence of methanol, i.e., alcohol oxidase (AOD), dihydroxyacetone synthase (DAS), glutathione-dependent formaldehyde dehydrogenase (FLD), and formate dehydrogenase (FDH), and these promoter regions have been used commercially for heterologous gene expression (5)(6)(7).…”

“…Methanol-inducible gene expression is assumed to be controlled under three distinct modes of regulation: glucose repression, glucose derepression, and methanol-specific induction (6,8). In C. boidinii, the promoter of the AOD-encoding gene (P AOD1 ) is completely repressed in the presence of glucose and derepressed upon exhaustion of glucose or in the presence of a nonfermentable carbon source without methanol, exhibiting ϳ10% of the maximum AOD expression level.…”

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

“…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.…”