Systems-level organization of yeast methylotrophic lifestyle

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.

Reorganization of budding yeast cytoplasm upon energy depletion

“…The observed changes in lipid droplet (LDs) morphology might be indicative of the dramatic effects that the scarcity of nutrients/energy has on the regulation of lipid metabolism (Rußmayer et al, 2015). In yeast cells, LDs are particularly dynamic throughout the growth phase and depending on the nutritional status of the cell (Kurat et al, 2006;Bozaquel-Morais et al, 2010).…”

The effect of hypoxia on the lipidome of recombinant Pichia pastoris

“…The lipid composition of this yeast has also been studied with regard to a carbon source effect [38]. However, such fundamental studies were performed exclusively using wild type strains.…”

Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter

“…In addition, as our previous transcriptomic study revealed a high impact of the overexpression of ROL on the peroxisomal pentose phosphate regeneration pathway (PPP) and peroxisome biogenesis in ROL multicopy strains (Cámara et al, 2017), the mRNA levels of fructose 1,6-bisphosphate aldolase (FBA1-2) and peroxine 6 (PEX6) were selected as markers of these pathways. In the case of FBA1-2, an increase of 40% and 36% was detected for 4C 6 -2MXR1* and 4C-MIT1 strains, respectively, supporting the role of the peroxisomal PPP in methanol assimilation (Rußmayer et al, 2015).…”

“…In the case of FBA1-2, an increase of 40% and 36% was detected for 4C 6 -2MXR1* and 4C-MIT1 strains, respectively, supporting the role of the peroxisomal PPP in methanol assimilation (Rußmayer et al, 2015). In the case of FBA1-2, an increase of 40% and 36% was detected for 4C 6 -2MXR1* and 4C-MIT1 strains, respectively, supporting the role of the peroxisomal PPP in methanol assimilation (Rußmayer et al, 2015).…”