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. 2010 Jan 4:11:1.
doi: 10.1186/1471-2164-11-1.

Adaptation of Hansenula polymorpha to methanol: a transcriptome analysis

Tim van Zutphen  1 , Richard J S Baerends, Kim A Susanna, Anne de Jong, Oscar P Kuipers, Marten Veenhuis, Ida J van der Klei
Affiliations

Adaptation of Hansenula polymorpha to methanol: a transcriptome analysis

Tim van Zutphen et al. BMC Genomics. .

Abstract

Background: Methylotrophic yeast species (e.g. Hansenula polymorpha, Pichia pastoris) can grow on methanol as sole source of carbon and energy. These organisms are important cell factories for the production of recombinant proteins, but are also used in fundamental research as model organisms to study peroxisome biology. During exponential growth on glucose, cells of H. polymorpha typically contain a single, small peroxisome that is redundant for growth while on methanol multiple, enlarged peroxisomes are present. These organelles are crucial to support growth on methanol, as they contain key enzymes of methanol metabolism.In this study, changes in the transcriptional profiles during adaptation of H. polymorpha cells from glucose- to methanol-containing media were investigated using DNA-microarray analyses.

Results: Two hours after the shift of cells from glucose to methanol nearly 20% (1184 genes) of the approximately 6000 annotated H. polymorpha genes were significantly upregulated with at least a two-fold differential expression. Highest upregulation (> 300-fold) was observed for the genes encoding the transcription factor Mpp1 and formate dehydrogenase, an enzyme of the methanol dissimilation pathway. Upregulated genes also included genes encoding other enzymes of methanol metabolism as well as of peroxisomal beta-oxidation.A moderate increase in transcriptional levels (up to 4-fold) was observed for several PEX genes, which are involved in peroxisome biogenesis. Only PEX11 and PEX32 were higher upregulated. In addition, an increase was observed in expression of the several ATG genes, which encode proteins involved in autophagy and autophagy processes. The strongest upregulation was observed for ATG8 and ATG11.Approximately 20% (1246 genes) of the genes were downregulated. These included glycolytic genes as well as genes involved in transcription and translation.

Conclusion: Transcriptional profiling of H. polymorpha cells shifted from glucose to methanol showed the expected downregulation of glycolytic genes together with upregulation of the methanol utilisation pathway. This serves as a confirmation and validation of the array data obtained. Consistent with this, also various PEX genes were upregulated. The strong upregulation of ATG genes is possibly due to induction of autophagy processes related to remodeling of the cell architecture required to support growth on methanol. These processes may also be responsible for the enhanced peroxisomal beta-oxidation, as autophagy leads to recycling of membrane lipids. The prominent downregulation of transcription and translation may be explained by the reduced growth rate on methanol (td glucose 1 h vs td methanol 4.5 h).

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Figures

Figure 1
Figure 1
Transcript level of AOX and DHAS. RT-PCR was performed on RNA samples using AOX and DHAS primers from glucose containing precultures (OD 2,3), and cultures shifted for 1 or 2 hrs to methanol medium. As loading control transcript levels of actin were analysed.
Figure 2
Figure 2
FUNCATS. Representation of functional groups among up- and downregulated genes is shown in a diagram. For comparison, a similar diagram is made for the total number of genes in Hansenula polymorpha (up-, down- and non-regulated genes). Genes are classified in one or multiple groups based on the Functional Catalogue.
Figure 3
Figure 3
Methanol metabolism. Overview of the methanol metabolism in H. polymorpha. The fold upregulation of the indicated genes is shown in red. GAP: glyceraldehyde-3-phosphate; DHA: dihydroxyacetone; DHAP: dihydroxyacetone phosphate; FBP: fructose bisphosphate; F6P: fructose-6-phosphate; XU5P: xylulose-5-phosphate; GSH: reduced glutathione; GS-CH2OH: S-hydroxymethylglutathione; GS-CHO: S-formylglutathione. AOX: alcohol oxidase; CAT: catalase; DHAS: dihydroxyacetone synthase; DAK: dihydroxyacetone kinase; FLD: formaldehyde dehydrogenase; FGH: S-formyl glutathione hydrolase and FMD: formate dehydrogenase.
Figure 4
Figure 4
Ultrastructural analysis of the adaptation of cells to methanol. Glucose-grown H. polymorpha WT cells were extensively analysed at different time-points after the shift to methanol by electron microscopy of KMnO4-fixed samples. (A) Control glucose-grown cell and (B) after 2 h of incubation in the presence of methanol. A clear increase in peroxisome size was observed, cross-sections of representative cells are depicted. Note the association of the organelles with strands of ER (indicated by arrow). (C) 2 hours after the shift a clear increase was observed in large vacuolar autophagic bodies, indicative of induction of autophagy. N - Nucleus, M - Mitochondrion, V - Vacuole, AV - Autophagosome. The bar represents 0.5 μm.
Figure 5
Figure 5
Fluorescence microscopy of mitochondria. Fluorescence microscopy images demonstrating the increase in mitochondrial volume fractions in cells after 2 hours of incubation on methanol (B), relative to those of the glucose inoculum cells (A). Mitochondria are marked by MitoTracker Orange.
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