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. 2015 Sep 23:13:80.
doi: 10.1186/s12915-015-0186-5.

Systems-level organization of yeast methylotrophic lifestyle

Hannes Rußmayer  1   2 , Markus Buchetics  1   2 , Clemens Gruber  2   3 , Minoska Valli  1   2 , Karlheinz Grillitsch  4   5 , Gerda Modarres  2   6 , Raffaele Guerrasio  2   3   7 , Kristaps Klavins  2   3   8 , Stefan Neubauer  2   3   9 , Hedda Drexler  2   3 , Matthias Steiger  1   2 , Christina Troyer  2   3 , Ali Al Chalabi  10 , Guido Krebiehl  10 , Denise Sonntag  10 , Günther Zellnig  11 , Günther Daum  4   5 , Alexandra B Graf  2   5 , Friedrich Altmann  2   3 , Gunda Koellensperger  12 , Stephan Hann  2   3 , Michael Sauer  1   2 , Diethard Mattanovich  13   14 , Brigitte Gasser  1   2
Affiliations

Systems-level organization of yeast methylotrophic lifestyle

Hannes Rußmayer et al. BMC Biol. .

Abstract

Background: Some 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.

Results: In 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.

Conclusions: Taken 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.

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Figures

Fig. 1
Fig. 1
Co-regulation of transcripts and proteins of P. pastoris grown on methanol/glycerol compared to glucose in chemostats at μ = 0.1 h−1. Log2 fold changes of transcripts versus log2 fold changes of proteins for the 575 data pairs are shown which have been grouped based on their regulation patterns. Significantly enriched GO terms in these groups are indicated. Criteria for up/down-regulation of transcript and protein levels are described in the Methods section
Fig. 2
Fig. 2
Extracted ion chromatogram (m/z = 368.9993 ± 10 ppm) of a sample grown on methanol (red) or glucose (blue) showing the sedoheptulose-1,7-bisphosphate peak at a retention time of 12 min. In the upper left corner the mass spectrum (m/z) of the peak of the methanol-grown sample after background subtraction is shown. The difference between the measured accurate mass and the calculated exact mass is 0.53 ppm (0.2 mDa)
Fig. 3
Fig. 3
Regeneration of pentose phosphates. Left: Methanol assimilation through the xylulose-monophosphate cycle: proposed rearrangements employing an alternative XYL5P forming pathway via S1,7BP. The net reaction of methanol assimilation is the formation of one GAP molecule from three methanol molecules. Right: Rearrangement reactions of the Calvin cycle. Regeneration of ribulose-1,5-bisphosphate (Rul-1,5-BP) needed for CO2 fixation in chloroplasts of plants via S1,7BP. For simplicity, the initial reaction steps after carbon fixation are condensed. The enzyme RuBisCO catalyzes the fixation of CO2 to Rul-1,5-BP, which yields two 3-phosphoglycerate molecules, which are phosphorylated to 1,3-bisphosphoglycerate by phosphoglycerate kinase, and then reduced to GAP by glyceraldehyde 3-phosphate dehydrogenase. Involved metabolites are in oval signs, genes/proteins are shown in rectangular signs. The colors of the individual metabolites serve for better readability of the figure, that is, chemically related compounds share the same color. The regulation pattern and the cellular localization of the proteins is given in Table 2
Fig. 4
Fig. 4
Differential regulation of central carbon metabolism comparing methanol/glycerol- and glucose-grown cells. Visualization of changes in transcript (square, upper symbol), protein (square, lower symbol), and metabolite (oval) levels. Red: up-regulation on methanol/glycerol; blue: down-regulation on methanol/glycerol; gray: not differentially regulated; white/no symbol: not measured. Criteria for up-/down-regulation of transcript, protein, and metabolite levels are described in the Methods section
Fig. 5
Fig. 5
Intracellular metabolic flux distributions of methanol/glycerol- and glucose-grown cells. The flux values are normalized to glycerol or glucose uptake, respectively, and presented in [% Cmol]. The upper value in the rectangular boxes represents the flux distribution on glucose and the lower value the flux distribution on methanol/glycerol. For reversible reactions only the net fluxes are presented
Fig. 6
Fig. 6
Differential regulation of amino acid synthesis pathways comparing methanol/glycerol- and glucose-grown cells. Visualization of changes in transcript, protein and metabolite levels. For an explanation of the symbols see legend to Fig. 4
Fig. 7
Fig. 7
Differential regulation of riboflavin, thiamine, and heme synthesis pathways comparing methanol/glycerol- and glucose-grown cells. Visualization of changes in transcript, protein, and metabolite levels. For an explanation of the symbols see legend to Fig. 4
Fig. 8
Fig. 8
Electron microscopy of P. pastoris grown on glucose or methanol. Cells were cultivated in complex media containing either glucose or methanol as the sole carbon source until they reached the late exponential growth phase. N, Nucleus; M, Mitochondria; LD, Lipid droplet; Px, Peroxisome. Scale bar: 1 μm
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