Systems-level organization of yeast methylotrophic lifestyle

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-015-0186-5) contains supplementary material, which is available to authorized users

Melatonin Minimizes the Impact of Oxidative Stress Induced by Hydrogen Peroxide in Saccharomyces and Non-conventional Yeast

Melatonin (N-acetyl-5-methoxytryptamine) is synthesized from tryptophan by Saccharomyces cerevisiae and non-conventional yeast species. Antioxidant properties have been suggested as a possible role of melatonin in a S. cerevisiae wine strain. However, the possible antioxidant melatonin effect on non-Saccharomyces species and other strains of S. cerevisiae must be evaluated. The aim of this study was to determine the antioxidant capacity of melatonin in eight S. cerevisiae strains and four non-conventional yeasts (Torulaspora delbrueckii, Metschnikowia pulcherrima, Starmerella bacillaris, and Hanseniaspora uvarum). Therefore, the ROS formation, lipid peroxidation, catalase activity, fatty acid composition, and peroxisome proliferation were investigated. The results showed that the presence of melatonin increases peroxisome accumulation and slightly increases the catalase activity. When cells grown in the presence of melatonin were exposed to oxidative stress induced by H2O2, lower ROS accumulation and lipid peroxidation were observed in all tested strains. Therefore, the increased catalase activity that was a consequence of oxidative stress was lower in the presence of melatonin. Moreover, the presence of MEL modulates cell FA composition, increasing oleic and palmitoleic acids and leading to higher UFA/SFA ratios, which have been previously related to a higher tolerance to H2O2. These findings demonstrate that melatonin can act as an antioxidant compound in both S. cerevisiae and non-Saccharomyces yeasts

The effect of hypoxia on the lipidome of recombinant Pichia pastoris

Cultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. In addition, transcriptomic analyses of hypoxic P. pastoris pointed out important regulation of lipid metabolism and unfolded protein response (UPR). Notably, UPR that plays a role in the regulation of lipid metabolism, amino acid metabolism and protein secretion, was found to be upregulated under hypoxia. To improve our understanding of the interplay between lipid metabolism, UPR and protein secretion, the lipidome of a P. pastoris strain producing an antibody fragment was studied under hypoxic conditions. Furthermore, lipid composition analyses were combined with previously available transcriptomic datasets to further understand the impact of hypoxia on lipid metabolism. Chemostat cultures operated under glucose-limiting conditions under normoxic and hypoxic conditions were analyzed in terms of intra/extracellular product distribution and lipid composition. Integrated analysis of lipidome and transcriptome datasets allowed us to demonstrate an important remodeling of the lipid metabolism under limited oxygen availability. Additionally, cells with reduced amounts of ergosterol through fluconazole treatment were also included in the study to observe the impact on protein secretion and its lipid composition. Our results show that cells adjust their membrane composition in response to oxygen limitation mainly by changing their sterol and sphingolipid composition. Although fluconazole treatment results a different lipidome profile than hypoxia, both conditions result in higher recombinant protein secretion levels. The online version of this article (doi:10.1186/s12934-017-0699-4) contains supplementary material, which is available to authorized users

Identification of triacylglycerol and steryl ester synthases of the methylotrophic yeast Pichia pastoris

AbstractIn yeast like in many other eukaryotes, fatty acids are stored in the biologically inert form of triacylglycerols (TG) and steryl esters (SE) as energy reserve and/or as membrane building blocks. In the present study, we identified gene products catalyzing formation of TG and SE in the methylotrophic yeast Pichia pastoris. Based on sequence homologies to Saccharomyces cerevisiae, the two diacylglycerol acyltransferases Dga1p and Lro1p and one acyl CoA:sterol acyltransferase Are2p from P. pastoris were identified. Mutants bearing single and multiple deletions of the respective genes were analyzed for their growth phenotype, lipid composition and the ability to form lipid droplets. Our results indicate that the above mentioned gene products are most likely responsible for the entire TG and SE synthesis in P. pastoris. Lro1p which has low fatty acid substrate specificity in vivo is the major TG synthase in this yeast, whereas Dga1p contributes less to TG synthesis although with some preference to utilize polyunsaturated fatty acids as substrates. In contrast to S. cerevisiae, Are2p is the only SE synthase in P. pastoris. Also this enzyme exhibits some preference for certain fatty acids as judged from the fatty acid profile of SE compared to bulk lipids. Most interestingly, TG formation in P. pastoris is indispensable for lipid droplet biogenesis. The small amount of SE synthesized by Are2p in a dga1∆lro1∆ double deletion mutant is insufficient to initiate the formation of the storage organelle. In summary, our data provide a first insight into the molecular machinery of non-polar lipid synthesis and storage in P. pastoris and demonstrate specific features of this machinery in comparison to other eukaryotic cells, especially S. cerevisiae

Channeling of fatty acids from depots to biomembranes in the yeast

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