Abstract
BackgroundSome yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date.ResultsIn this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation.ConclusionsTaken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0186-5) contains supplementary material, which is available to authorized users.
Highlights
Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source
The higher oxygen uptake rate of methanol/glycerol-grown P. pastoris can be explained by the higher degree of reduction of methanol and glycerol compared to glucose
The biomass yield was slightly higher for cells grown on methanol/glycerol compared to glucose, which is in good agreement with data from the literature [28, 30]
Summary
Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Hansenula polymorpha, another methylotrophic yeast, harbors only a single, small peroxisome which can serve as a source for proliferation by fission when induction is triggered by shifting the cells to methanol [10, 11]. The first steps of methanol assimilation involve an alcohol oxidase (AOX) to convert methanol to formaldehyde, and a special transketolase named dihydroxyacetone synthase (DAS) to form a C-C bond with the C1 molecule formaldehyde. The reactions of these two enzymes and their localization to peroxisomes are well described [12, 13]. The further reaction cycle of methanol assimilation is supposed to involve pentose phosphate reactions, but the details are not fully clarified to date
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