BACKGROUND
hansenula polymorpha is an important cell factory for the production of pharmaceutical proteins  <cit> .
moreover, it is extensively used in fundamental research aiming at understanding the molecular principles of peroxisome biology  <cit> .
as cell factory, h. polymorpha has several important advantages.
first, it contains very strong and inducible promoters derived from the methanol metabolism pathway.
also, the organism is thermotolerant  and tolerates various environmental stresses.
h. polymorpha does not hyperglycosylate secreted proteins, which often is a problem in heterologous protein production in s. cerevisiae.
in h. polymorpha peroxisomes massively develop during growth on methanol as sole source of carbon and energy.
methanol is oxidized by the enzyme alcohol oxidase , which is localized in peroxisomes together with catalase and dihydroxyacetone synthase , the first enzyme of the assimilation pathway.
peroxisomes are not required for primary metabolism when cells are grown on glucose.
moreover, glucose represses the key enzymes of methanol metabolism aox and dhas.
therefore, during growth on glucose h. polymorpha cells contain only a single, small peroxisome.
upon a shift to methanol media, the enzymes of methanol metabolism are induced paralleled by an increase in peroxisome size and abundance.
the initial small peroxisome serves as the target organelle for the enzymes of methanol metabolism and proliferates by fission  <cit> .
ultimately, in exponentially growing cells, each cell contains several enlarged peroxisomes  <cit> .
a wealth of information is now available of individual genes encoding enzymes of the methanol metabolism as well as on genes involved in peroxisome formation .
however, genomics approaches in h. polymorpha are still rare.
we used genome-wide transcriptional profiling to dissect the initial events accompanying the adaptation of glucose grown h. polymorpha cells to methanol metabolism.
this will gain information on the induction and repression of metabolic genes as well as on non-metabolic genes, including pex genes.
RESULTS
all experiments described in this paper were performed in batch cultures.
we chose not to grow the cells in carbon-limited chemostats, as glucose-limitation results in derepression of genes involved in methanol metabolism  <cit> .
h. polymorpha cells were extensively pre-cultivated in batch cultures on mineral media supplemented with glucose as sole carbon source in order to fully repress the enzymes of methanol metabolism.
glucose cultures in the late exponential growth phase were transferred to fresh mineral medium containing methanol as sole carbon and energy source.
as shown in figure  <dig>  rt-pcr indicated that the inoculum cells  did not contain transcript of alcohol oxidase  or dihydroxyacetone synthase , key enzymes of methanol metabolism.
however, two hours after the shift to medium containing methanol, mrnas of both genes were readily detected, a time-point which has also been identified as threshold for the detection of first aox enzyme activity  <cit> .
therefore,  <dig> hours incubation on methanol was selected as sampling point of cells for transcriptome analysis.
replicates were obtained by growing  <dig> independent cultures on glucose that were independently transferred to fresh media containing methanol.
of each culture, mrna isolated from the glucose and the methanol sample was labeled  and hybridized on two arrays per culture.
in addition, as a control aox transcript levels of these samples were determined by rt-pcr, confirming the absence of transcript in the glucose-grown pre-cultures and the presence of aox transcript after  <dig> hours incubation .
overview of the dna microarray data
the dna microarray analyses data were analyzed to generate the ratio between the transcripts on methanol and glucose for each gene to identify any differential expression and to determine the p-value to assess the significance and the a-value to check the intensity of the signals.
presents an overview of the array results.
of the nearly  <dig> annotated h. polymorpha genes that are listed, approximately 20%  are upregulated, while another 20%  are downregulated with at least a two-fold differential expression, meeting the significance and signal intensity criteria.
of the upregulated genes,  <dig> are more than  <dig> times upregulated,  <dig> genes show a 10-100-fold upregulation,  <dig> genes increase between  <dig> and 10-fold and the remaining  <dig> genes are less than 5-fold upregulated.
highest upregulated are the central methanol metabolism regulator mpp <dig>  and the gene encoding formate dehydrogenase , required in methanol catabolism.
also the other components of the methanol metabolic pathway are highly upregulated.
moreover, crc <dig> is highly induced, encoding a mitochondrial inner membrane carnitine transporter, which is required for the transport of acetyl-coa from peroxisomes to mitochondria during fatty acid beta-oxidation .
in line with crc <dig>  also the genes involved in the beta-oxidation of fatty acids are overrepresented among the highly upregulated genes .
furthermore, approximately 13% of the more than 10-fold upregulated genes is involved in transport.
the upregulation of hexose transporters may be important for the uptake of the residual glucose that was present in the inoculum.
of the downregulated genes, the highest fold downregulation  is observed for hp24g <dig>  encoding a protein with strong similarity to sik1p of s. cerevisiae, which is involved in pre-rrna processing.
this predicted function is consistent with the general trend among the downregulated genes, since of the  <dig> genes that are over 10-fold downregulated, nearly 50% code for products that function in either transcription or translation processes.
of the other downregulated genes,  <dig> show a 5- to 10-fold downregulation.
of these, nearly 40% encode proteins involved in transcription and translation.
the other  <dig> genes are less than 5-fold reduced in transcript levels.
functional overview dna microarray data - funcats
to obtain an overview of the functions of the differentially expressed genes, these were categorized according to the functional catalogue, funcat  <cit> .
in this system, each gene is classified in one or more groups, depending on its function.
the number of genes in each category is shown as the percentage of the total number of up- or downregulated genes in the diagrams shown in figure  <dig>  for comparison, a diagram showing the contribution of each functional category to the total number of genes in h. polymorpha is included.
to construct this diagram, all known h. polymorpha genes are used; both up- and downregulated genes as well as non-regulated genes.
as expected, genes involved in metabolic pathways strongly contribute to both the up- and downregulated genes , reflecting the large-scale adaptations accompanying the shift from glucose to methanol.
however, metabolism is a large group also in the total genome and the contribution in percentages does not reflect the nature of the metabolic pathways that are regulated .
in contrast to metabolism, some other functional categories display a difference in contribution to the upregulated compared to the downregulated genes.
one such functional category is the group of protein synthesis genes, which is almost absent among upregulated genes , while it composes a large portion of the downregulated genes .
of the total genome of h. polymorpha only approximately 4% is involved in protein synthesis, reflecting the considerable effect of a shift to methanol on protein synthesis.
in addition, also the group of genes involved in transcription is more predominant among downregulated genes .
in concurrence with the trend of genes related to transcription and translation, also genes related to nucleotide biosynthesis are mostly downregulated , yet genes involved in amino acid biosynthesis show a less clear trend .
the observed downregulation of major anabolic processes most likely is related to the reduction in doubling time  and may reflect the accompanying decrease in dna replication, rna transcription and translation.
stress response genes form only relatively small categories among both the upregulated genes relative to the downregulated genes .
based on the functional catalogue, only  <dig> of the nearly  <dig> annotated h. polymorpha genes are indicated as stress response genes.
however, based on several studies by gasch in saccharomyces cerevisiae  <cit> , many more genes could contribute to the cellular stress response.
hence, most likely also genes classified in other groups play a role in coping with stress accompanying a shift in cultivation conditions.
thus, the contribution of the stress response to the total differential expression in h. polymorpha upon transfer to methanol medium is probably larger than the observed  <dig> % upregulation and  <dig> % downregulation.
a last remarkable group in the functional catalogue diagram is the category of unclassified proteins, showing that 33% of the upregulated genes and  <dig> % of the downregulated genes are thus far not experimentally characterized, relative to 25% of the genes of the total genome of h. polymorpha.
this observation suggests that our current knowledge on adaptation to methanol is far from complete.
metabolic pathways - upregulation of methanol metabolism
as expected, genes involved in methanol metabolism are highly upregulated.
in figure  <dig> an overview of the methanol metabolism, including the microarray data, is presented  <cit> .
in peroxisomes, methanol is oxidized to formaldehyde and hydrogen peroxide by alcohol oxidase , which is  <dig>  times upregulated at the transcriptional level.
the hydrogen peroxide is detoxified by catalase  to water and oxygen .
formaldehyde can be further metabolized via two different routes: 1) dissimilation via formaldehyde dehydrogenase , s-formyl glutathione hydrolase  and formate dehydrogenase  to co <dig>  generating nadh and co <dig> or 2) assimilation via the peroxisome-borne enzyme dihydroxyacetone synthase  to generate cell constituents.
dhas is part of the xylulose-5-phosphate cycle and catalyzes the formation of two c3-molecules  from one c <dig>  and one c <dig> compound   <cit> .
promoter studies in candida boidinii using phosphatase as a reporter enzyme revealed that upon a shift to methanol medium fmd was induced first, followed by dhas and even later aox  <cit> .
the early induction of fmd  relative to aox and dhas  suggests that a similar induction pattern may exist in h. polymorpha.
the differences in induction of the genes  <dig> hours after the shift to methanol medium does not reflect the ultimate protein levels in the cells, as aox and dhas are the major protein bands in extracts prepared from methanol grown h. polymorpha cells  <cit> .
pex genes
pex genes control the development and function of a specialized class of organelles, the peroxisomes.
most of the pex genes showed a moderate induction upon the shift to methanol .
this is in line with earlier studies of s. cerevisiae cells that were shifted from glucose to the peroxisome-inducing carbon source oleate  <cit> .
of the pex genes involved in import of peroxisomal matrix enzymes , the highest upregulation was observed for pex <dig>  pex <dig>  pex <dig>  pex <dig>  pex <dig> and pex <dig>  which all encode key components pts <dig> protein import machinery  <cit> .
highest upregulation was observed for pex <dig>  and pex <dig> .
pex11p, together with pex25p and pex11cp, are the three members of the pex11p protein family in h. polymorpha  <cit> .
these proteins are implicated in regulating peroxisome proliferation.
also in bakers' yeast cells shifted from glucose to oleic acid medium pex <dig> increased by far the most  <cit> .
all genes shown have a p-value below  <dig> .
negative values indicate downregulation on methanol, positive values indicate upregulation on methanol
h. polymorpha pex <dig> was upregulated  <dig> -fold, whereas pex11c, whose function is still unknown, showed a  <dig> -fold downregulation  <cit> .
pex32p is a member of the pex <dig> protein family, a group of membrane proteins with unknown function  <cit> .
y. lipolytica pex <dig> mutants cannot grow on oleate and partially mislocalize peroxisomal proteins to the cytosol, suggesting a role in matrix protein import.
in contrast however, s. cerevisiae pex23p, pex31p and pex32p are not required for protein import but play a role in peroxisome proliferation.
where scpex23p appears to be a positive regulator of peroxisome size, scpex31p and scpex32p have been suggested to negatively regulate this process.
the function of h. polymorpha pex32p is not yet known.
based on our current study this protein may be, together with pex11p, responsible for the initial increase in size of the peroxisomes, as was observed by detailed ultrastructural analysis  and in concurrence with earlier findings  <cit> .
the relatively high induction of this peroxin makes it an interesting candidate for further study in h. polymorpha.
metabolic pathways - downregulation of glucose utilisation
as depicted in table  <dig>  the majority of the genes involved in glycolysis are downregulated .
since the genes are listed in order of appearance in the pathway, it is evident that the strongest downregulation is observed in the steps before fructose  <dig>  bisphosphate aldolase.
this corresponds with the fact that the products of methanol metabolism, dihydroxyacetone and glyceraldehyde  <dig> phosphate, enter the glycolytic pathway directly after this step, so the subsequent enzymes are still required for progression with the pathway towards the tca cycle.
the observed mild decrease in expression of their encoding genes can be attributed to the reduction in the substrate flow, when switching from glucose to methanol utilisation.
however, it should be noted that the enzymes of the final part of glycolysis in majority also function in the direction of gluconeogenesis, by catalyzing the reverse reactions.
finally, the upregulation of the gene encoding fructose  <dig>  bisphosphate aldolase which, on methanol, catalyzes the formation of fructose  <dig>  bisphosphate from dihydroxyacetone and glyceraldehyde-3-phosphate, reflects an increase of this reaction, which has been shown to be important in the rearrangement reactions to replenish the cell with xylulose-5-phosphate to the downstream reactions in methanol metabolism  <cit> .
* with the exception of the one marked with an asterisk, all genes shown have a p-value below  <dig> .
regulatory networks
accompanying the changes in expression of many metabolic genes, also changes in the underlying regulatory networks are expected.
in addition to global changes, the dna microarray data reflect the initial adaptation to methanol and thus enable the investigation of more specific changes resulting in activation of methanol-dependent genes or in repression of glucose-dependent genes.
among the upregulated genes,  <dig> are involved in regulation of transcription.
most of these encode general transcription factors or transcription factors which have not been linked to a specific process.
regulators involved in stress response , glucose sensing/derepression , and peroxisome-related pathways  are overrepresented, as is expected due to the change in carbon source.
the expression of genes coding for peroxisome assembly and function is controlled by a transcriptional regulatory network, which has been studied extensively in s. cerevisiae in response to oleate  <cit> .
the oaf1-pip <dig> complex plays a prominent role, although the putative h. polymorpha homolog of pip <dig> is only moderately upregulated during adaptation to methanol.
however the homolog of adr <dig> , a second activator of oleate-responsive genes is strongly upregulated in h. polymorpha and is also involved in regulation of the response to both oleate and methanol in p. pastoris , thus suggesting it is a master regulator of methanol-responsive genes  <cit> .
swi <dig> and snf <dig> also belong to a regulatory complex involved in gene expression of methanol-related genes as well as peroxisomal assembly, however their encoding genes were not induced in the early adaptation phase to methanol  <cit> .
among the downregulated genes, the decreased transcription of rap <dig>  is remarkable.
this transcriptional regulator is known to activate transcription of genes encoding ribosomal proteins  <cit>  and its downregulation is consistent with the observed massive decrease in transcripts for ribosomal proteins.
interestingly, this gene is also shown to be repressed during the environmental stress response in s. cerevisiae as described by gasch et al.,  <cit> .
autophagy
adaptation of h. polymorpha cells to methanol requires a major rearrangement of the cellular architecture.
the finding that most atg genes, which are involved in autophagy and autophagy related processes  <cit> , as well as several proteasomal genes are upregulated , suggests that cellular reorganisation requires massive degradation of cellular components.
interestingly, the highest upregulation is observed for atg <dig> and atg <dig> .
atg <dig> has a prominent role in various selective and non-selective macroautophagic processes, whereas atg <dig> is specifically involved in selective macroautophagy  <cit> .
the function of hpatg19-like, of which the encoding gene is also upregulated, is not known.
however, hpatg19-like is not involved in selective degradation of peroxisomes .
remarkably only atg <dig> is significantly downregulated.
atg <dig> is involved in selective peroxisome degradation by macropexophagy, but not in microautophagy  <cit> .
*with the exception of the one marked with an asterisk, all genes shown have a p-value below  <dig> .
ultrastructural analysis of cells at different time-points after the shift indeed showed strong morphological evidence for increased autophagy, reflected in the massive uptake of cytoplasmic components in the vacuole .
recent findings showed the importance of autophagy during methanol adaptation of p. pastoris, not only for cell remodeling, but also to provide amino acids  <cit> .
consistent with these findings, we also observed that h. polymorpha atg mutants showed a slight delay in methanol adaptation .
β-oxidation of fatty acids
a significant upregulation of genes encoding proteins related to β-oxidation of fatty acids was observed  <cit> .
this unexpected cluster is listed in table  <dig>  the regulation of these genes could be merely due to derepression as a result of decreasing glucose levels.
however we consider this less likely since the observed ratio's and signals are quite substantial.
another explanation could be co-regulation of multiple peroxisomal pathways by common regulators.
a third option is the increase in cellular fatty acid levels, the substrate for peroxisomal β-oxidation.
this might originate from the observed autophagy, leading to recycling of intracellular membrane lipids.
all genes shown have a p-value below  <dig> .
mitochondria
remarkably, the shift of cells from glucose to methanol is associated with a significant increase in mitochondrial volume fractions .
several functional links exist between peroxisomes and mitochondria, both for metabolic pathways and for biogenesis of both organelles  <cit> .
of the genes involved in mitochondrial function or assembly  <dig> are down- and  <dig> are upregulated.
genes coding for components of the dnm1-dependent fission machinery of both organelles are not differentially expressed  <cit> .
similar to all downregulated genes, of the mitochondrial downregulated genes almost 50% is involved in transcription and translation processes.
in addition the genes coding for tom and tim protein import complexes are also mostly down-regulated.
genes involved in fes cluster, heme biosynthesis and cytochrome c assembly are overrepresented among upregulated mitochondrial genes , in agreement with the prominent role for mitochondria as the sole site of atp generation during methanol-metabolism  <cit> .
heme is also the co-factor of peroxisomal catalase which is highly induced.
fes cluster formation is also coupled to the glutathione-based redox regulation system via grx <dig>  <cit> .
reactive oxygen species
although mitochondria were long considered as the main source of reactive oxygen species , also peroxisomes actually defined as organelles that harbour h2o2-producing enzymes as well as catalase are now recognized as a significant contributor to ros production  <cit> .
besides catalase , peroxisomes also contain the peroxiredoxin pmp <dig>  that is 11-fold upregulated, both indicative of an increase in peroxisomal ros production  <cit> .
we also observed an increase of several pivotal genes involved in cytosolic and mitochondrial ros detoxification; like the superoxide dismutase sod <dig> , the glutathione transferase gto <dig> , the glutathione reductase gpx <dig> , the glutaredoxin grx <dig>  and the thioredoxin peroxidase tsa <dig> .
the remaining members of the glutathione- and trx-based system are not differentially expressed , except for the thioredoxin reductase trr <dig> .
induction of these key enzymes involved in sustaining the redox balance of the cytosol, suggests that methanol-metabolism also results in enhanced cytosolic ros levels, which may originate from peroxisomal metabolism.
comparison with other dna microarray analyses
back in  <dig>  the proof of principle for the use of dna microarray technology to investigate transcriptional changes was shown for s. cerevisiae  <cit> .
since then, dna microarray analysis has become a regular, well-established tool, facilitated by the now commercial available array slides.
for many other yeast species however, thusfar the usage of dna microarray analysis is still rather limited.
only recently, species-specific dna microarray studies have been presented for e.g.
debaryomyces hansenii  <cit>  and p. pastoris  <cit> .
for h. polymorpha, oh et al.,  <cit>  presented a partial, dna microarray study, followed by a full dna microarray analysis describing the transcriptional response of h. polymorpha to exposure to cadmium  <cit> .
however, this study was not related to the metabolic and architectural alterations associated with a change in cellular metabolism.
smith et al.
<cit>  published two studies in which bakers' yeast was shifted to oleate.
the first study focused mainly on transcriptional changes of genes encoding peroxisomal proteins and peroxins, the second one on the regulatory network coordinating the response to oleate.
the current study is the first in which arrays were used to study the shift from glucose to methanol in the yeast h. polymorpha.
sauer et al.
<cit>  was the first to publish transcriptional profiling of the methylotrophic yeast p. pastoris, also upon a shift to methanol.
however, in that study, yeast cells were transferred from glycerol to methanol and heterologous hybridisation onto s. cerevisiae dna-microarrays was performed.
still, the same trend in regulated functional groups was observed.
also, similar effects were reported on carbohydrate metabolism and the regulation of ribosomal genes.
although some of the data obtained for h. polymorpha are comparable with those obtained for p. pastoris, a significant drawback of the latter study is the absence of p. pastoris specific or methylotrophic yeast specific genes on the dna-microarray slides.
there is indeed a substantial amount of methanol-responsive h. polymorpha genes found in our study which were not observed in p. pastoris using s. cerevisiae microarrays  .
CONCLUSIONS
the current dna microarray study revealed the expected repression of genes involved in glucose metabolism concomitant with the induction of the genes of methanol metabolism in response of a shift of h. polymorpha cells from glucose to methanol.
also genes involved in peroxisome biogenesis  are upregulated, with pex <dig> being the most strongly upregulated pex gene.
a surprising finding was the upregulation of autophagy- and of beta-oxidation-genes.
the first can likely be explained by the need for cellular reorganisations and is confirmed by electron microscopy studies showing active uptake of cytoplasmic components in the vacuoles of the cells.
the induction of beta-oxidation is thought to be a consequence of the cellular reorganisations and thereby the high turnover of lipids, serving as substrates for beta-oxidation.
a final interesting but not yet uncovered group consists of the 33% of the upregulated genes that have no known function.
these genes reflect a large potential of h. polymorpha or methylotrophic yeast specific genes with a specialized role in adaptations to growth on methanol as the sole source of carbon and can form interesting targets for future research.

 hansenula polymorpha is an important cell factory for the production of pharmaceutical proteins. moreover, it is extensively used in fundamental research aiming at understanding the molecular principles of peroxisome biology. as cell factory, h. polymorpha has several important advantages. first, it contains very strong and inducible promoters derived from the methanol metabolism pathway. also, the organism is thermotolerant and tolerates various environmental stresses. h. polymorpha does not hyperglycosylate secreted proteins, which often is a problem in heterologous protein production in s. cerevisiae. in h. polymorpha peroxisomes massively develop during growth on methanol as sole source of carbon and energy. methanol is oxidized by the enzyme alcohol oxidase, which is localized in peroxisomes together with catalase and dihydroxyacetone synthase, the first enzyme of the assimilation pathway. peroxisomes are not required for primary metabolism when cells are grown on glucose. moreover, glucose represses the key enzymes of methanol metabolism aox and dhas. therefore, during growth on glucose h. polymorpha cells contain only a single, small peroxisome. upon a shift to methanol media, the enzymes of methanol metabolism are induced paralleled by an increase in peroxisome size and abundance. the initial small peroxisome serves as the target organelle for the enzymes of methanol metabolism and proliferates by fission. ultimately, in exponentially growing cells, each cell contains several enlarged peroxisomes. a wealth of information is now available of individual genes encoding enzymes of the methanol metabolism as well as on genes involved in peroxisome formation. however, genomics approaches in h. polymorpha are still rare. we used genome-wide transcriptional profiling to dissect the initial events accompanying the adaptation of glucose grown h. polymorpha cells to methanol metabolism. this will gain information on the induction and repression of metabolic genes as well as on non-metabolic genes, including pex genes.  all experiments described in this paper were performed in batch cultures. we chose not to grow the cells in carbon-limited chemostats, as glucose-limitation results in derepression of genes involved in methanol metabolism. h. polymorpha cells were extensively pre-cultivated in batch cultures on mineral media supplemented with glucose as sole carbon source in order to fully repress the enzymes of methanol metabolism. glucose cultures in the late exponential growth phase were transferred to fresh mineral medium containing methanol as sole carbon and energy source. as shown in figure rt-pcr indicated that the inoculum cells did not contain transcript of alcohol oxidase or dihydroxyacetone synthase, key enzymes of methanol metabolism. however, two hours after the shift to medium containing methanol, mrnas of both genes were readily detected, a time-point which has also been identified as threshold for the detection of first aox enzyme activity. therefore, hours incubation on methanol was selected as sampling point of cells for transcriptome analysis. replicates were obtained by growing independent cultures on glucose that were independently transferred to fresh media containing methanol. of each culture, mrna isolated from the glucose and the methanol sample was labeled and hybridized on two arrays per culture. in addition, as a control aox transcript levels of these samples were determined by rt-pcr, confirming the absence of transcript in the glucose-grown pre-cultures and the presence of aox transcript after hours incubation. overview of the dna microarray data the dna microarray analyses data were analyzed to generate the ratio between the transcripts on methanol and glucose for each gene to identify any differential expression and to determine the p-value to assess the significance and the a-value to check the intensity of the signals. presents an overview of the array results. of the nearly annotated h. polymorpha genes that are listed, approximately 20% are upregulated, while another 20% are downregulated with at least a two-fold differential expression, meeting the significance and signal intensity criteria. of the upregulated genes, are more than times upregulated, genes show a 10-100-fold upregulation, genes increase between and 10-fold and the remaining genes are less than 5-fold upregulated. highest upregulated are the central methanol metabolism regulator mpp and the gene encoding formate dehydrogenase, required in methanol catabolism. also the other components of the methanol metabolic pathway are highly upregulated. moreover, crc is highly induced, encoding a mitochondrial inner membrane carnitine transporter, which is required for the transport of acetyl-coa from peroxisomes to mitochondria during fatty acid beta-oxidation. in line with crc also the genes involved in the beta-oxidation of fatty acids are overrepresented among the highly upregulated genes. furthermore, approximately 13% of the more than 10-fold upregulated genes is involved in transport. the upregulation of hexose transporters may be important for the uptake of the residual glucose that was present in the inoculum. of the downregulated genes, the highest fold downregulation is observed for hp24g encoding a protein with strong similarity to sik1p of s. cerevisiae, which is involved in pre-rrna processing. this predicted function is consistent with the general trend among the downregulated genes, since of the genes that are over 10-fold downregulated, nearly 50% code for products that function in either transcription or translation processes. of the other downregulated genes, show a 5- to 10-fold downregulation. of these, nearly 40% encode proteins involved in transcription and translation. the other genes are less than 5-fold reduced in transcript levels. functional overview dna microarray data - funcats to obtain an overview of the functions of the differentially expressed genes, these were categorized according to the functional catalogue, funcat. in this system, each gene is classified in one or more groups, depending on its function. the number of genes in each category is shown as the percentage of the total number of up- or downregulated genes in the diagrams shown in figure for comparison, a diagram showing the contribution of each functional category to the total number of genes in h. polymorpha is included. to construct this diagram, all known h. polymorpha genes are used; both up- and downregulated genes as well as non-regulated genes. as expected, genes involved in metabolic pathways strongly contribute to both the up- and downregulated genes, reflecting the large-scale adaptations accompanying the shift from glucose to methanol. however, metabolism is a large group also in the total genome and the contribution in percentages does not reflect the nature of the metabolic pathways that are regulated. in contrast to metabolism, some other functional categories display a difference in contribution to the upregulated compared to the downregulated genes. one such functional category is the group of protein synthesis genes, which is almost absent among upregulated genes, while it composes a large portion of the downregulated genes. of the total genome of h. polymorpha only approximately 4% is involved in protein synthesis, reflecting the considerable effect of a shift to methanol on protein synthesis. in addition, also the group of genes involved in transcription is more predominant among downregulated genes. in concurrence with the trend of genes related to transcription and translation, also genes related to nucleotide biosynthesis are mostly downregulated, yet genes involved in amino acid biosynthesis show a less clear trend. the observed downregulation of major anabolic processes most likely is related to the reduction in doubling time and may reflect the accompanying decrease in dna replication, rna transcription and translation. stress response genes form only relatively small categories among both the upregulated genes relative to the downregulated genes. based on the functional catalogue, only of the nearly annotated h. polymorpha genes are indicated as stress response genes. however, based on several studies by gasch in saccharomyces cerevisiae, many more genes could contribute to the cellular stress response. hence, most likely also genes classified in other groups play a role in coping with stress accompanying a shift in cultivation conditions. thus, the contribution of the stress response to the total differential expression in h. polymorpha upon transfer to methanol medium is probably larger than the observed % upregulation and % downregulation. a last remarkable group in the functional catalogue diagram is the category of unclassified proteins, showing that 33% of the upregulated genes and % of the downregulated genes are thus far not experimentally characterized, relative to 25% of the genes of the total genome of h. polymorpha. this observation suggests that our current knowledge on adaptation to methanol is far from complete. metabolic pathways - upregulation of methanol metabolism as expected, genes involved in methanol metabolism are highly upregulated. in figure an overview of the methanol metabolism, including the microarray data, is presented. in peroxisomes, methanol is oxidized to formaldehyde and hydrogen peroxide by alcohol oxidase, which is times upregulated at the transcriptional level. the hydrogen peroxide is detoxified by catalase to water and oxygen. formaldehyde can be further metabolized via two different routes: 1) dissimilation via formaldehyde dehydrogenase, s-formyl glutathione hydrolase and formate dehydrogenase to co generating nadh and co or 2) assimilation via the peroxisome-borne enzyme dihydroxyacetone synthase to generate cell constituents. dhas is part of the xylulose-5-phosphate cycle and catalyzes the formation of two c3-molecules from one c and one c compound. promoter studies in candida boidinii using phosphatase as a reporter enzyme revealed that upon a shift to methanol medium fmd was induced first, followed by dhas and even later aox. the early induction of fmd relative to aox and dhas suggests that a similar induction pattern may exist in h. polymorpha. the differences in induction of the genes hours after the shift to methanol medium does not reflect the ultimate protein levels in the cells, as aox and dhas are the major protein bands in extracts prepared from methanol grown h. polymorpha cells. pex genes pex genes control the development and function of a specialized class of organelles, the peroxisomes. most of the pex genes showed a moderate induction upon the shift to methanol. this is in line with earlier studies of s. cerevisiae cells that were shifted from glucose to the peroxisome-inducing carbon source oleate. of the pex genes involved in import of peroxisomal matrix enzymes, the highest upregulation was observed for pex pex pex pex pex and pex which all encode key components pts protein import machinery. highest upregulation was observed for pex and pex. pex11p, together with pex25p and pex11cp, are the three members of the pex11p protein family in h. polymorpha. these proteins are implicated in regulating peroxisome proliferation. also in bakers' yeast cells shifted from glucose to oleic acid medium pex increased by far the most. all genes shown have a p-value below. negative values indicate downregulation on methanol, positive values indicate upregulation on methanol h. polymorpha pex was upregulated -fold, whereas pex11c, whose function is still unknown, showed a -fold downregulation. pex32p is a member of the pex protein family, a group of membrane proteins with unknown function. y. lipolytica pex mutants cannot grow on oleate and partially mislocalize peroxisomal proteins to the cytosol, suggesting a role in matrix protein import. in contrast however, s. cerevisiae pex23p, pex31p and pex32p are not required for protein import but play a role in peroxisome proliferation. where scpex23p appears to be a positive regulator of peroxisome size, scpex31p and scpex32p have been suggested to negatively regulate this process. the function of h. polymorpha pex32p is not yet known. based on our current study this protein may be, together with pex11p, responsible for the initial increase in size of the peroxisomes, as was observed by detailed ultrastructural analysis and in concurrence with earlier findings. the relatively high induction of this peroxin makes it an interesting candidate for further study in h. polymorpha. metabolic pathways - downregulation of glucose utilisation as depicted in table the majority of the genes involved in glycolysis are downregulated. since the genes are listed in order of appearance in the pathway, it is evident that the strongest downregulation is observed in the steps before fructose bisphosphate aldolase. this corresponds with the fact that the products of methanol metabolism, dihydroxyacetone and glyceraldehyde phosphate, enter the glycolytic pathway directly after this step, so the subsequent enzymes are still required for progression with the pathway towards the tca cycle. the observed mild decrease in expression of their encoding genes can be attributed to the reduction in the substrate flow, when switching from glucose to methanol utilisation. however, it should be noted that the enzymes of the final part of glycolysis in majority also function in the direction of gluconeogenesis, by catalyzing the reverse reactions. finally, the upregulation of the gene encoding fructose bisphosphate aldolase which, on methanol, catalyzes the formation of fructose bisphosphate from dihydroxyacetone and glyceraldehyde-3-phosphate, reflects an increase of this reaction, which has been shown to be important in the rearrangement reactions to replenish the cell with xylulose-5-phosphate to the downstream reactions in methanol metabolism. * with the exception of the one marked with an asterisk, all genes shown have a p-value below. regulatory networks accompanying the changes in expression of many metabolic genes, also changes in the underlying regulatory networks are expected. in addition to global changes, the dna microarray data reflect the initial adaptation to methanol and thus enable the investigation of more specific changes resulting in activation of methanol-dependent genes or in repression of glucose-dependent genes. among the upregulated genes, are involved in regulation of transcription. most of these encode general transcription factors or transcription factors which have not been linked to a specific process. regulators involved in stress response, glucose sensing/derepression, and peroxisome-related pathways are overrepresented, as is expected due to the change in carbon source. the expression of genes coding for peroxisome assembly and function is controlled by a transcriptional regulatory network, which has been studied extensively in s. cerevisiae in response to oleate. the oaf1-pip complex plays a prominent role, although the putative h. polymorpha homolog of pip is only moderately upregulated during adaptation to methanol. however the homolog of adr, a second activator of oleate-responsive genes is strongly upregulated in h. polymorpha and is also involved in regulation of the response to both oleate and methanol in p. pastoris, thus suggesting it is a master regulator of methanol-responsive genes. swi and snf also belong to a regulatory complex involved in gene expression of methanol-related genes as well as peroxisomal assembly, however their encoding genes were not induced in the early adaptation phase to methanol. among the downregulated genes, the decreased transcription of rap is remarkable. this transcriptional regulator is known to activate transcription of genes encoding ribosomal proteins and its downregulation is consistent with the observed massive decrease in transcripts for ribosomal proteins. interestingly, this gene is also shown to be repressed during the environmental stress response in s. cerevisiae as described by gasch et al.,. autophagy adaptation of h. polymorpha cells to methanol requires a major rearrangement of the cellular architecture. the finding that most atg genes, which are involved in autophagy and autophagy related processes, as well as several proteasomal genes are upregulated, suggests that cellular reorganisation requires massive degradation of cellular components. interestingly, the highest upregulation is observed for atg and atg. atg has a prominent role in various selective and non-selective macroautophagic processes, whereas atg is specifically involved in selective macroautophagy. the function of hpatg19-like, of which the encoding gene is also upregulated, is not known. however, hpatg19-like is not involved in selective degradation of peroxisomes. remarkably only atg is significantly downregulated. atg is involved in selective peroxisome degradation by macropexophagy, but not in microautophagy. *with the exception of the one marked with an asterisk, all genes shown have a p-value below. ultrastructural analysis of cells at different time-points after the shift indeed showed strong morphological evidence for increased autophagy, reflected in the massive uptake of cytoplasmic components in the vacuole. recent findings showed the importance of autophagy during methanol adaptation of p. pastoris, not only for cell remodeling, but also to provide amino acids. consistent with these findings, we also observed that h. polymorpha atg mutants showed a slight delay in methanol adaptation. β-oxidation of fatty acids a significant upregulation of genes encoding proteins related to β-oxidation of fatty acids was observed. this unexpected cluster is listed in table the regulation of these genes could be merely due to derepression as a result of decreasing glucose levels. however we consider this less likely since the observed ratio's and signals are quite substantial. another explanation could be co-regulation of multiple peroxisomal pathways by common regulators. a third option is the increase in cellular fatty acid levels, the substrate for peroxisomal β-oxidation. this might originate from the observed autophagy, leading to recycling of intracellular membrane lipids. all genes shown have a p-value below. mitochondria remarkably, the shift of cells from glucose to methanol is associated with a significant increase in mitochondrial volume fractions. several functional links exist between peroxisomes and mitochondria, both for metabolic pathways and for biogenesis of both organelles. of the genes involved in mitochondrial function or assembly are down- and are upregulated. genes coding for components of the dnm1-dependent fission machinery of both organelles are not differentially expressed. similar to all downregulated genes, of the mitochondrial downregulated genes almost 50% is involved in transcription and translation processes. in addition the genes coding for tom and tim protein import complexes are also mostly down-regulated. genes involved in fes cluster, heme biosynthesis and cytochrome c assembly are overrepresented among upregulated mitochondrial genes, in agreement with the prominent role for mitochondria as the sole site of atp generation during methanol-metabolism. heme is also the co-factor of peroxisomal catalase which is highly induced. fes cluster formation is also coupled to the glutathione-based redox regulation system via grx. reactive oxygen species although mitochondria were long considered as the main source of reactive oxygen species, also peroxisomes actually defined as organelles that harbour h2o2-producing enzymes as well as catalase are now recognized as a significant contributor to ros production. besides catalase, peroxisomes also contain the peroxiredoxin pmp that is 11-fold upregulated, both indicative of an increase in peroxisomal ros production. we also observed an increase of several pivotal genes involved in cytosolic and mitochondrial ros detoxification; like the superoxide dismutase sod, the glutathione transferase gto, the glutathione reductase gpx, the glutaredoxin grx and the thioredoxin peroxidase tsa. the remaining members of the glutathione- and trx-based system are not differentially expressed, except for the thioredoxin reductase trr. induction of these key enzymes involved in sustaining the redox balance of the cytosol, suggests that methanol-metabolism also results in enhanced cytosolic ros levels, which may originate from peroxisomal metabolism. comparison with other dna microarray analyses back in the proof of principle for the use of dna microarray technology to investigate transcriptional changes was shown for s. cerevisiae. since then, dna microarray analysis has become a regular, well-established tool, facilitated by the now commercial available array slides. for many other yeast species however, thusfar the usage of dna microarray analysis is still rather limited. only recently, species-specific dna microarray studies have been presented for e.g. debaryomyces hansenii and p. pastoris. for h. polymorpha, oh et al., presented a partial, dna microarray study, followed by a full dna microarray analysis describing the transcriptional response of h. polymorpha to exposure to cadmium. however, this study was not related to the metabolic and architectural alterations associated with a change in cellular metabolism. smith et al. published two studies in which bakers' yeast was shifted to oleate. the first study focused mainly on transcriptional changes of genes encoding peroxisomal proteins and peroxins, the second one on the regulatory network coordinating the response to oleate. the current study is the first in which arrays were used to study the shift from glucose to methanol in the yeast h. polymorpha. sauer et al. was the first to publish transcriptional profiling of the methylotrophic yeast p. pastoris, also upon a shift to methanol. however, in that study, yeast cells were transferred from glycerol to methanol and heterologous hybridisation onto s. cerevisiae dna-microarrays was performed. still, the same trend in regulated functional groups was observed. also, similar effects were reported on carbohydrate metabolism and the regulation of ribosomal genes. although some of the data obtained for h. polymorpha are comparable with those obtained for p. pastoris, a significant drawback of the latter study is the absence of p. pastoris specific or methylotrophic yeast specific genes on the dna-microarray slides. there is indeed a substantial amount of methanol-responsive h. polymorpha genes found in our study which were not observed in p. pastoris using s. cerevisiae microarrays.  the current dna microarray study revealed the expected repression of genes involved in glucose metabolism concomitant with the induction of the genes of methanol metabolism in response of a shift of h. polymorpha cells from glucose to methanol. also genes involved in peroxisome biogenesis are upregulated, with pex being the most strongly upregulated pex gene. a surprising finding was the upregulation of autophagy- and of beta-oxidation-genes. the first can likely be explained by the need for cellular reorganisations and is confirmed by electron microscopy studies showing active uptake of cytoplasmic components in the vacuoles of the cells. the induction of beta-oxidation is thought to be a consequence of the cellular reorganisations and thereby the high turnover of lipids, serving as substrates for beta-oxidation. a final interesting but not yet uncovered group consists of the 33% of the upregulated genes that have no known function. these genes reflect a large potential of h. polymorpha or methylotrophic yeast specific genes with a specialized role in adaptations to growth on methanol as the sole source of carbon and can form interesting targets for future research. 
a moderate increase in transcriptional levels was observed for several pex genes, which are involved in peroxisome biogenesis. consistent with this, also various pex genes were upregulated. these organelles are crucial to support growth on methanol, as they contain key enzymes of methanol metabolism. upregulated genes also included genes encoding other enzymes of methanol metabolism as well as of peroxisomal β-oxidation. 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. 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. two hours after the shift of cells from glucose to methanol nearly 20% of the approximately annotated h. polymorpha genes were significantly upregulated with at least a two-fold differential expression. 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. methylotrophic yeast species 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. in addition, an increase was observed in expression of the several atg genes, which encode proteins involved in autophagy and autophagy processes. 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. highest upregulation was observed for the genes encoding the transcription factor mpp and formate dehydrogenase, an enzyme of the methanol dissimilation pathway. these processes may also be responsible for the enhanced peroxisomal β-oxidation, as autophagy leads to recycling of membrane lipids.
 methylotrophic yeast species 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.  two hours after the shift of cells from glucose to methanol nearly 20% of the approximately annotated h. polymorpha genes were significantly upregulated with at least a two-fold differential expression. highest upregulation was observed for the genes encoding the transcription factor mpp 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 β-oxidation. a moderate increase in transcriptional levels was observed for several pex genes, which are involved in peroxisome biogenesis. only pex and pex 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 atg and atg approximately 20% of the genes were downregulated. these included glycolytic genes as well as genes involved in transcription and translation.  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 β-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. 