Since methylotrophic yeasts such as Ogataea methanolica can use methanol as a sole carbon feedstock, they could be applied to produce valuable products from methanol, a next-generation energy source synthesized from natural gases, using genetic engineering tools. In this study, metabolite profiling of O. methanolica was conducted under glucose (Glc) and low and high methanol (L-and H-MeOH) conditions to show the adaptation mechanism to a H-MeOH environment. The yeast strain responded not only to the presence of methanol but also to its concentration based on the growth condition. Under H-MeOH conditions, O. methanolica downregulated the methanol utilization, glycolytic pathway and alcohol oxidase (AOD) isozymes and dihydroxyacetone synthase (DAS) expression compared with L-MeOH-grown cells. However, levels of energy carriers, such as ATP, were maintained to support cell survival. In H-MeOH-grown cells, reactive oxygen species (ROS) levels were significantly elevated. Along with increasing ROS levels, ROS scavenging system expression was significantly increased in H-MeOH-grown cells. Thus, we concluded that formaldehyde and H 2 O 2 , which are products of methanol oxidation by AOD isozymes in the peroxisome, are overproduced in H-MeOH-grown cells, and excessive ROS derived from these cells is generated in the cytosol, resulting in upregulation of the antioxidant system and downregulation of the methanol-utilizing pathway to suppress overproduction of toxic intermediates.

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