BACKGROUND
the response of cells to high osmotic pressure and increased salinity has been a subject of close investigation in many different organisms  <cit> .
depending on the intensity of the osmotic shock the immediate response to high osmolarity usually includes the activation of the environmental stress response  and of the high osmolarity glycerol  pathway to induce changes that are necessary to cope with this stressful environmental condition in saccharomyces cerevisiae  <cit> .
in batch culture, osmotic shock usually implies a temporary growth arrest to adapt the cellular metabolism  <cit> .
major adjustments of gene transcription in saccharomyces cerevisiae and other yeasts include the induction of glycerol-3-phosphate dehydrogenase gpd <dig> transcription  <cit> , transcriptional repression of the plasma membrane glycerol efflux channel fps <dig>  <cit> , but also the adjustment of ribosome biogenesis and the translation and protein folding machinery  <cit> .
glycerol production, but also the production of other small organic molecules, is induced in different yeast species to compensate variations of osmotic conditions  <cit> .
polyols, such as glycerol, pertain to a class of small molecules known as compatible solutes, which, in contrast to inorganic ions, can be safely accumulated and degraded in the cell without impairing cellular function or having detrimental effects on protein and nucleic acid stability  <cit> .
furthermore, biomass yield is reduced upon exposure to high osmolarity because of higher maintenance energy in both, batch and chemostat cultures  <cit> .
however, it is known that after the immediate shock response, transcript levels of many stress responsive genes return to near basal levels after cells have adapted to the new environmental conditions  <cit> .
the effect of osmolarity on cellular physiology is not only of particular interest for the basic research community.
as biotechnological production processes aim at high cell and product concentrations, cultivation media usually employ high concentrations of nutrient salts and carbon source resulting in high osmolarities.
additionally, there is some evidence that exposure to osmotic stress can have a beneficial effect on recombinant protein production in bacterial, yeast and mammalian host organisms  <cit> .
unfortunately, the positive effect of increased osmolarity on heterologous protein production is, at least in mammalian cells, often cell line specific  <cit>  and e.g.
in case of the yeast pichia pastoris it remains anecdotal.
the genome sequence of p. pastoris has been recently published  <cit>  and with a publicly available sequence at hand thorough physiological investigations and characterization of this biotechnologically relevant organism becomes feasible.
in this context the effect of osmolarity on the physiology of p. pastoris was analyzed in both a non-expressing wild type  strain and a recombinant protein secreting strain.
the protein secretion strain expressed the antibody fab fragment 3h <dig>  <cit>  under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase  promoter.
the effect of osmolarity was monitored in steady state by applying chemostat cultivation in both strains.
although chemostat cultivation differs from batch and fed batch systems, which are usually applied for large scale production of recombinant proteins, long term suboptimal growth conditions as they occur during batch and fed batch cultivation can also be applied in steady state chemostat conditions  <cit> .
furthermore, chemostat cultivation offers the advantage that growth rate related effects, which otherwise would interfere with high throughput protein and mrna analytics, can be avoided  <cit> .
to analyze the effect of increased osmolarity on host cell physiology, 2d fluorescence difference gel electrophoresis  and dna microarray analyses were applied.
these techniques have already been successfully applied to monitor the effect of environmental factors, such as temperature and osmolarity in yeasts  <cit> .
furthermore, hplc analysis was applied to analyze to intracellular polyol and trehalose contents.
the obtained data indicated an unfolded protein response  like response upon growth at increased osmolarity in the non-expressing wt strain of p. pastoris.
in the recombinant protein secreting strain, the upr was obviously already induced due to protein overexpression.
the observed overlap of the response to increased osmolarity and the response to recombinant protein production, lead to less adaptations/changes upon high osmolarity on both, the transcriptome and proteome scale, in the fab secreting strain than in the non-expressing wt strain.
RESULTS
general characteristics of cultures at different osmotic conditions
chemostat cultivations of p. pastoris were performed at three different osmotic conditions, which were achieved by different concentrations of kcl in the growth medium.
this resulted in supernatant osmolarities of approximately  <dig>   <dig> and  <dig> mosmol kg- <dig>  which will be named low, medium and high osmolarity thereafter.
samples were taken at steady state, which means fully adapted cells were analyzed.
the characteristics of the chemostat cultures did not dramatically change upon cultivation at different osmotic conditions .
biomass yield decreased with increasing osmolarity in the wt strain and fab 3h <dig> producing strain.
however, the decrease in biomass yield was only statistically significant  between cultivations at low and high osmolarity in both strains.
the amount of total protein secreted into the culture supernatant did not change upon higher osmotic pressure, but was generally higher in the fab 3h <dig> production strain.
osmolarity of the growth medium did not significantly influence specific productivity  of the fab 3h <dig>  generally, increased osmotic pressure poses a severe stress condition to cells  <cit> .
although the osmolarity was increased approximately 6-fold and ten-fold in the current study, no decrease of cell viability was observed.
more than 97% of the cells in chemostat culture represented viable cells throughout all cultivations .
according to flow cytometry data, increased osmolarity resulted in a decrease of the mean cell size as indicated by a decrease of the mean forward scatter of the cells .
wt represents the non-expressing wild type strain.
fab represents the recombinant protein producing strain.
osmolarity values represent actual measured values of the culture supernatant.
+/- represents the standard error of the mean.
- not applicable.
production of compatible solutes and trehalose in p. pastoris upon growth at different osmolarities
in yeasts, glycerol is a very common solute but other polyols such as arabitol, mannitol and erythritol are also produced in some yeast species  <cit> .
to analyze whether p. pastoris produces any of these substances, cell extracts were analyzed by hplc.
it turned out that very low levels of mannitol and nearly no detectable amounts of erythritol were present in p. pastoris cells .
intracellular glycerol levels were higher than mannitol and erythritol levels and a slight significant increase in the wt strain from low to high and medium to high osmolarity occurred , whereas no significant changes of glycerol content were observed in the fab 3h <dig> expressing strain .
surprisingly, arabitol was the most abundant compound of the analyzed polyols in p. pastoris cells  and showed statistically significant increased levels  when shifting growth conditions towards high osmolarity .
a 3-fold increase of intracellular arabitol levels was observed when comparing low and medium osmolarity conditions and a 4-fold increase when comparing cells grown at low and high osmolarity.
furthermore, intracellular levels of trehalose were analyzed as trehalose is thought to be involved in relieving or impeding protein folding stress  <cit> , which may also occur during salt stress  <cit> .
intracellular trehalose levels were in the same range as glycerol levels but showed a significant trend  towards decreased concentrations at medium and high osmolarity growth conditions in the wt strain but slightly missed the threshold p-value in the 3h <dig> fab expressing strain
the effect of osmolarity on the p. pastoris intracellular proteome
as 2d-dige has already been successfully applied to monitor changes in the p. pastoris proteome upon growth at different temperatures  <cit> , this method was also applied to track changes upon growth at elevated osmolarity.
in the wt strain of p. pastoris approximately  <dig> proteins passed the criteria , whereas about  <dig> proteins passed these criteria in the recombinant protein producing strain.
most of the protein spots represented low abundant proteins with too small quantities of proteins on the 2d gels to be confidently identified, resulting in  <dig> successfully identified proteins .
a list of all identified protein spots with corresponding peptides, obtained from ms/ms analysis, is available in additional file  <dig>  as already reported in previous studies  <cit> , additionally to most likely full length proteins, protein fragments were identified according to the spot position on the gel .
furthermore, protein levels showed the largest changes when low and medium and low and high osmolarity cultivations were compared, whereas only minor changes occurred when comparing medium and high osmolarity setpoints .
analysis was performed by 2d-dige and subsequent lc esi-ms/ms identification.
protein standard name , protein functional description, theoretical molecular weight  and theoretical isoelectric point , average expression values and 1-anova values are shown.
significant anova values are indicated in bold letters.
the average protein abundance fold change of the comparison of low and high osmolarity setpoints  are shown.
additional protein fold change data are available through additional file  <dig>
as can be seen in figure  <dig>  the major impact of osmolarity on the p. pastoris proteome was on proteins involved in energy metabolism and protein folding.
whereas protein levels of a major spot of aconitate hydratase  were increased at medium and high osmolarity in the wt strain, they were not significantly affected by osmolarity in the fab 3h <dig> producing strain.
in contrast, in the fab 3h <dig> expressing strain three minor isoforms or degradation products were significantly down-regulated at higher osmolarity, but showed no altered abundance in the wt strain.
furthermore, citrate synthase  protein levels were decreased at high salt concentrations in the wt strain but showed no significant change in the fab producing strain.
formate dehydrogenase , glycerol kinase , and isocitrate lyase  showed similar trends towards lower protein levels during medium and high osmolarity cultivations in both strains, whereas pyruvate kinase  and phosphoglycerate kinase  were generally up-regulated at higher osmolarity .
phosphoglucose isomerase  showed lower levels at medium osmolarity in the production strain and a protein identified as atp3p  was massively down-regulated at medium and high salt concentrations in the wt strain.
furthermore, alcohol oxidase , a key enzyme in methanol utilization, was down-regulated at medium and high osmolarity in the wt strain but did not show a significant change in the fab 3h <dig> expressing strain.
it should be pointed out that aox <dig> transcription is thought to be repressed during growth on glucose and that the current study was performed with glucose as carbon source to constitutively express the heterologous protein under the control of the gap-promoter.
however, it was shown previously that basal levels of aox1p were actually present during glucose limited growth of p. pastoris  <cit> .
similar discrepancies between the wt and the recombinant protein expressing strain were observed for proteins involved in protein folding and secretion and folding stress response.
whereas the major er chaperone and unfolded protein response  sensor kar2p/bip and the protein disulfide isomerase pdi1p were up-regulated at medium and high osmolarity in the wt strain, no changes of these two proteins were observed in the fab producing strain.
more prominently increased levels of cytosolic and mitochondrial chaperones ssc1p, sse1p, ssz1p and hsp60p were observed at medium and high osmolarity in the wt strain than in the fab 3h <dig> producing strain.
the stress induced chaperone ssa4p showed increased levels at medium salt concentrations in both strains but returned to below basal levels at high salt conditions in the fab producing strain.
ino1p, a protein involved in synthesis of inositol phosphates and inositol-containing phospholipids and which is linked to the upr  <cit> , was also up-regulated at high salt concentrations in both strains analyzed.
other protein spots that changed their abundance upon cultivation at increased salt concentrations were agx1p and gdh1p .
both of them showed higher protein levels during growth at high osmolarity.
the effect of osmolarity on the p. pastoris transcriptome
to analyze the effect of different osmolarities on the p. pastoris transcriptome, p. pastoris specific microarrays were applied .
to support microarray analysis, real-time pcr was performed.
real-time pcr data proved to be consistent with microarray results .
more significant changes on the transcriptome level were observed in the wt strain than in the recombinant protein expressing strain .
low p-values can result from a high technical variation within the replicates or reflect the biological truth within the samples.
to determine if the lower amount of significantly regulated genes in the fab expressing strain is a technical artefact, the correlation, standard deviation and the coefficient of variation for the replicates of the wt and expressing strain were compared.
correlation of intensity values was generally high between all microarrays of one group  with r <dig> values between  <dig>  and  <dig> .
the values for standard deviation and coefficient of variation  indicated that the variance in replicates of the expressing strain was slightly but consistently higher than for the wt strain .
based on these results additional microarray experiments were performed to exclude any bias in the data.
these additional data did not change the result or number of regulated genes, suggesting a true biological difference.
to eliminate the possibility that the samples of the expressing strain vary more than the ones of the wt strain, hierarchical cluster analysis  and gene set analysis  were performed on the fold change data and indicated that regulation was indeed different in the two strains analyzed .
wt - non-expressing wild type strain.
fab - fab 3h <dig> expressing strain.
the number of genes that are similarly regulated in both strains are listed as common genes.
because most of the genes that were regulated when comparing low to medium osmolarity were also regulated when comparing low to high osmolarity, the following data presentation and discussion will focus on the effects that were observed when low and high osmotic conditions were compared.
to get an overview of the general adaptations during steady-state cultivation, fisher's exact test was performed to identify cellular processes, which were affected by different osmolarities on the transcript level.
a total of  <dig> go categories were either affected in both or at least in one of the analyzed strains .
concordant with the mere number of regulated genes, there appeared more significantly affected cellular processes in the wt strain than in the heterologous protein expressing strain.
only  <dig> go categories occurred to be affected in both strains, namely go: <dig> , go: <dig>  and go: <dig> .
additionally, in the wt strain the go terms go: <dig> , go: <dig> , go: <dig>  and go: <dig>  were affected by increased extracellular osmolarity.
regarding ion transport, uptake and metabolism, high osmolarity resulted in increased expression of the iron transporters ftr <dig>  sit <dig> and the vacuolar iron reductase fre <dig> in both strains.
calcium ion homeostasis and calcium dependent signal transduction were obviously affected by high osmolarity in the wt strain as the ca2+ transporter pmc <dig> and calcineurin a  were down-regulated at high osmolarity.
a major effect was apparent for genes involved in cell wall organization and its biogenesis.
whereas 21% of the genes belonging to this go group were down-regulated at high osmolarity in the wt strain, a similar effect, albeit with fewer significant genes, was observed in the 3h <dig> fab secreting strain .
additionally, a putative extracellular or cell wall associated protein with homology to the s. cerevisiae pry <dig> gene was up-regulated in both strains at high osmolarity.
increased levels of a gene with homology to s. cerevisiae pry <dig> upon increased salinity have also been reported for the halotolerant yeast hortaea werneckii previously  <cit> .
however, no changes in the protein pattern, indicating higher protein levels, were observed by sds-page of the culture supernatant .
a signaling cascade for sensing and adaptation to osmotic stress in s. cerevisiae has already been established based on the available data  <cit>  and genes with homology to the corresponding genes in s. cerevisiae were also identified in p. pastoris.
none of the genes involved in osmotic stress sensing upstream of the mitogen activated protein kinase  hog <dig> showed significant regulation in the fab 3h <dig> expressing strain, whereas sho <dig>  ssk <dig> and ptp <dig> were up-regulated and ste <dig> was down-regulated at increased osmolarity in the wt strain .
several genes involved in energy metabolism and storage carbohydrate metabolism were affected by increased osmolarity.
fba <dig>  a key enzyme in glycolysis and gluconeogenesis, was up-regulated in the heterologous protein expressing strain at high osmolarity.
the acetyl-coa synthetase acs <dig> was up-regulated in the wt strain at high osmolarity.
transcript levels of several genes involved in the tricarboxylic acid  cycle and the glyoxylate cycle, namely aco <dig>  fum <dig>  mdh <dig>  sfc <dig> and icl <dig> were reduced and subunits of the atp synthase  were up-regulated during growth at high osmolarity in the p. pastoris wt strain.
in the fab producing strain, tkl <dig>  involved in the pentose phosphate  pathway was significantly up-regulated.
glycogen synthesis was also affected by high osmolarity as glg <dig>  gsy <dig> and glc <dig> showed decreased transcript levels during growth at high osmolarity.
decreased levels of dga <dig>  gut <dig> and gtp <dig> indicated changes in glycerol and lipid metabolism.
additionally, a homologue to the s. cerevisiae putative passive glycerol channel yfl054c was down-regulated and the active glycerol importer stl <dig> was up-regulated during growth at high osmotic conditions in the wt strain.
furthermore, significant down-regulation of the p. pastoris alcohol oxidase aox <dig> was observed in the wt strain at high osmolarity, whereas no significant regulation was observed in the fab 3h <dig> expressing strain.
these data are concordant with data on the proteome level .
in the wt strain approximately 7% of the genes involved in ribosome biogenesis and assembly were up-regulated during steady state cultivation at high osmolarity.
the effect of recombinant protein production on the transcriptome of p. pastoris at low osmolarity
as the experiment was performed with a non-expressing wt and a recombinant protein expressing strain of p. pastoris the effect of recombinant protein expression itself on the cellular transcriptome at low osmolarity could also be analyzed.
the most prominent effect of recombinant protein production on host cell physiology is the induction of the unfolded protein response   <cit> .
therefore, we analyzed whether this effect could be observed at low osmolarity in glucose limited chemostat cultures of p. pastoris.
it turned out that the transcriptional response to recombinant protein production during chemostat cultivation at low osmolarity was low.
only  <dig> mrnas were significantly regulated when comparing the two strains using a strict cut-off .
using a less strict cut-off   <dig> genes were significantly regulated .
hac <dig> was up-regulated in the fab expressing strain and indicated the induction of the upr in the fab expressing strain.
in contrast to hac <dig> other members of the core upr and targets of the hac <dig> transcription factor showed no significant response by applying a p-value cut-off.
however, gene set analysis  applied on the microarray data revealed increased expression of genes related to the go term go: <dig>  .
the upr  is a specific response to unfolded protein in the er and is a subcategory of go: <dig>
the up-regulation of the hac <dig> transcript as well as the significant upregulation of the response to unfolded protein indicated the induction of the upr in the fab 3h <dig> expressing strain.
the effect was lower than in previous studies, which in fact were performed in non-carbon limited batch cultures.
it is known that during carbon-limited chemostat cultivation of s. cerevisiae metabolic control can be more important than gene regulation  <cit> .
furthermore, it is known that part of the regulation of the upr can be performed on a post-transcriptional or even post-translational level in s. cerevisiae and aspergillus niger  <cit> .
less information about control mechanisms are available for p. pastoris.
we conclude that similar to these previous studies, a substantial part of regulation, including the regulation of the upr, is achieved on a post-transcriptional level during glucose-limited chemostat cultivation of p. pastoris.
regarding the response to unfolded protein, gsa indicated significantly increased transcription of genes related to this go category  in the wt strain at high osmolarity.
in contrast, higher growth medium osmolarity did not result in an induction of the response to unfolded protein in fab 3h <dig> expressing strain .
salt tolerance of pichia pastoris
as no data on the salt tolerance of p. pastoris compared to s. cerevisiae were found in literature, a growth test on ypd agar plates, containing different amounts of nacl or kcl, was performed.
this growth test indicated higher tolerance to growth on nacl and a less pronounced higher tolerance to growth on kcl of p. pastoris compared to s. cerevisiae .
discussion
production of compatible solutes
to counterbalance the osmotic pressure by high or low salt or solute concentrations in the growth medium, microorganisms produce various compatible solutes.
in s. cerevisiae and many other organisms glycerol is the main osmolyte accumulated during osmotic stress.
however, we found that intracellular glycerol levels were low at all osmotic conditions in p. pastoris.
on the other hand, arabitol was more abundant than glycerol, even at low osmolarity, and it was accumulated in p. pastoris during growth at elevated osmotic pressure .
glycerol production in s. cerevisiae depends on the increased expression of glycerol-3-phosphate dehydrogenase gpd <dig> and glycerol-3-phosphatase gpp <dig>  <cit> .
nevertheless, we could not find neither of the two genes involved in glycerol metabolism, gpd <dig> and gpp <dig>  to be up-regulated on the transcript level and did not identify protein spots with altered expression which would match these two genes in p. pastoris.
furthermore, it was shown in debaryomyces hansenii that nacl stress lead to increased levels of proteins involved in the upper part of glycolysis and down-regulation of proteins involved in the tca-cycle  <cit> .
it was concluded that these changes may favor the accumulation of dihydroxyacetonephosphate and consequently the production of glycerol  <cit> .
no changes related to the upper part of glycolysis were observed in the current study, although it is clear that the need for compatible solutes leads to a redirection of a part of the carbon source to alleviate the stress induced by increased osmolarity.
this would make sense as arabitol obviously plays a more important role as compatible solute than glycerol in p. pastoris.
however, the regulation of genes involved in glycerol uptake and efflux, such as the up-regulation of stl <dig> and the down-regulation of yfl054c may be beneficial for p. pastoris as well.
the loss of minor osmolytes may result in detrimental effects on cellular integrity at high kcl concentrations.
no significant regulation was observed for other putative glycerol transporters of p. pastoris recently described by mattanovich and co-workers  <cit> .
arabitol synthesis is linked to the pentose phosphate  pathway.
however, no changes possibly linked to the pp pathway and arabitol synthesis were observed on the transcript or the proteome level.
the regulation of arabitol synthesis might be mainly achieved on a post-transcriptional level by increased translation or by protein modification and changes of enzyme activity during chemostat cultivation of p. pastoris.
nevertheless, concordant with other studies, arabitol obviously is of particular importance for the metabolism of p. pastoris as it is also secreted into the supernatant at certain growth conditions, such as low oxygenation  <cit> .
trehalose has been previously shown to play an important role in heat shock induced refolding of proteins in baker's yeast  <cit>  and in vitro  <cit> .
furthermore, trehalose may also be involved in the response to temperature induced stress in p. pastoris as intracellular levels increased at elevated temperature .
however, as trehalose levels were lower during growth at high osmolarity and no changes of the stress induced cytosolic trehalase nth <dig>  <cit>  were observed on the protein or mrna level, trehalose may not be directly involved in the protection of proteins against osmotic induced protein denaturation or damage.
it is more likely that, similar to s. cerevisiae, trehalose degradation may play a role during growth at elevated osmolarity  <cit> , or that trehalose levels may be simply lower due to a redirection of carbon source to the production of arabitol rather then to the production of trehalose.
effect on energy metabolism
the differential response of aco1p and the differences of transcript levels of genes involved the tca cycle to different osmotic conditions between the wt and the fab 3h <dig> expressing strain lead to the conclusion that recombinant protein production influenced the osmo-dependent adaptation of the energy metabolism.
previous data already indicated a metabolic burden and influence of recombinant protein production on energy metabolism in p. pastoris  <cit> .
furthermore, the key enzyme of methanol utilization, aox <dig>  was differently regulated in the two strains and indicated significant differences in the regulation of energy metabolism.
protein and transcript levels of the alcohol oxidase  were significantly negatively affected by growth at high osmolarity in the wt strain but not in the fab 3h <dig> secreting strain.
p. pastoris aox <dig> seems to be tightly regulated upon exposure to various stresses and might represent an ideal candidate as a marker gene/protein to monitor diverse external and internal stresses in p. pastoris.
apart from these additional data supporting the idea of a metabolic burden during recombinant protein production in p. pastoris, no clear interpretation about the changes of energy metabolism upon growth at different osmolarities in chemostat cultures emerged.
further investigations using a different approach to the one used in the current study will be necessary to elucidate the effect of osmolarity on the energy metabolism of p. pastoris.
activation of translation, ribosome biogenesis and the response to unfolded protein at high osmolarity
another major effect was the massive increase of chaperones and upr related proteins at high osmolarity.
the upr, including heat shock proteins and cellular chaperones, plays an essential role in the response to various stresses  <cit> .
apart from its role in the esr of unicellular organisms, the upr is also of great importance in human disease as highlighted by its involvement in the development of several human maladies such as diabetes, neurodegenerative disorders and cancer  <cit> .
the observation of increased levels of molecular chaperones during growth at high osmolarity is concordant with previous results for aspergillus nidulans  <cit>  and similar to results obtained for d. hansenii  <cit>  and s. cerevisiae  <cit>  in batch culture.
furthermore, high osmotic pressure resulted in increased levels of pdi1p and kar2p, indicating endoplasmic reticulum related protein folding stress.
unlike s. cerevisiae, upr induction has been reported to be a main event upon exposure to salt stress in the halotolerant yeast rhodotorula mucilaginosa  <cit> .
generally, the induction of the upr may not only be a result of high concentrations of ionic solutes such as salts but the response to unfolded proteins is also triggered by high osmotic pressure induced by other substances such as sugar compounds.
it has been reported for mammalian cells that low as well as high hexose concentrations can lead to upr induction  <cit> .
the upr has been described to be mainly a transcriptional response, but recently post-transcriptional and post-translational regulation has been described for fungal organisms  <cit> .
in this context, mrna levels of the upr transcription factor hac <dig> are increased in the fab expressing strain as a reaction to recombinant protein production.
on the other hand, gsa of the microarray data also showed induction of "responses to unfolded protein" at high osmolarity in the wt strain.
these results are supported by the effects observed at the proteome level.
a conventional induction of the upr by increased hac <dig> levels was not observed at increased osmolarity in neither of the strains.
however, increased osmolarity resulted in increased kar2p and pdi1p on the proteome level in the wt strain.
as this clearly demonstrated er associated protein folding stress we refer to a upr-like response of p. pastoris wild type cells at high osmolarity.
although a direct comparison between the wt and recombinant protein producing strain was not possible on the proteome level, this comparison was possible on the transcript level and strongly indicated the upregulation of processes involved in response to unfolded protein in the recombinant fab producing strain.
thus, we hypothesize that the up-regulation of these er resident proteins as well as other chaperones was obviously not necessary in a fab 3h <dig> producing strain at high osmolarity, as these changes had already been triggered by the upr that was induced by the recombinant protein.
additionally to this upr-like response, the induction of ribosome biogenesis and translation were apparent on the transcript level in the wt strain.
the up-regulation of genes involved in protein synthesis during osmotic stress has been reported for the salt-tolerant yeasts h. werneckii and d. hansenii  <cit> .
furthermore, studies on brewing strains of s. cerevisiae concluded that the faster adaption to higher salt concentration compared to a laboratory strain was achieved by higher expression levels of genes involved in protein synthesis  <cit> .
similar to other environmental factors, such as temperature  <cit> , translation might become a rate-limiting factor during growth at high osmolarities because of stress related to decreased intracellular water availability.
boosting the protein synthesis machinery might be necessary for growth of p. pastoris at elevated osmolarity.
the fab 3h <dig> expressing strain of p. pastoris did not show this increase of the protein synthesis machinery.
we have shown previously that over-expression of the transcription factor hac <dig> in p. pastoris batch cultures resulted in increased expression of genes involved in mrna translation and to a massive increase of genes involved in ribosome biogenesis and assembly  <cit> .
obviously the up-regulation of ribosome biogenesis, translation and other co-regulated processes at high osmolarity was not necessary in the fab 3h <dig> producing strain as these changes had already been induced by recombinant protein production itself.
this upr-like response at high osmolarity also points to the fact that, similar to halotolerant yeast species like r. mucilaginosa, p. pastoris might use different mechanisms for gaining osmotic stress resistance than s. cerevisiae.
this hypothesis was supported by the growth tests for salt tolerance, which were performed with p. pastoris and s. cerevisiae.
p. pastoris showed indeed higher resistance to increased salt concentrations in the growth medium than s. cerevisiae .
many changes observed in the wt upon a change from low to high osmolarity were not observed in the recombinant protein expressing strain.
although high osmolarity triggered the response to unfolded protein, ribosome biogenesis and translation in the wt strain, the activation of these apparently co-regulated processes was compensated in the fab 3h <dig> strain by recombinant protein induced upr.
other cellular responses
increased osmolarity also influenced other cellular mechanisms, such as some parts of the oxidative damage response, which apparently were not co-regulated with the protein synthesis and folding machinery.
therefore, these processes which seem to be an essential part of the response to increased osmolarity were monitored in both strains of p. pastoris.
the interrelation of salt and oxidative stress is already established in plants  <cit>  and the interrelation and cross-talk of the hog pathway and other pathways such as protein kinase c  and calcineurin dependent signaling are also established in yeasts  <cit> .
changes in cell wall integrity signaling, which were evident by altered expression levels of cell wall components in both strains, may be directly related to the changes of the cna <dig> and pmc <dig> transcripts, as some of these cell wall synthesis related genes are dependent on calcineurin signaling  <cit> .
for example, decreased transcript levels of crh <dig> and gas <dig> at high osmolarity may indicate a change of cell wall rigidity at high osmolarity.
high osmolarity results in decreased turgor pressure when compared with low or hypo-osmotic conditions.
thus, increased osmolarity results in cell shrinkage and smaller cells, which in fact was obvious by a decreased mean forward scatter of the cells in the present study .
in this context the down-regulation of cell wall components, which would result in lower cell wall rigidity, at high osmolarity makes sense.
it has been shown in aspergillus nidulans that salt addition to the growth medium resulted in decreased cell wall rigidity  <cit> .
a further indication that high osmolarity is compatible with lower cell wall rigidity is the fact that the swollen cellular phenotype of gas <dig> mutant cells, a gene which is also downregulated at high osmolarity in the current study, in s. cerevisiae is compensated by growth at high osmolarity  <cit> .
although this effect was very evident on the transcript level we were not able to monitor it on the proteome level.
this may be simply due to the preparation of protein samples and the resulting absence of cell wall and membrane proteins, which are rather difficult to extract by standard protein preparation methods.
additionally to these events, the induction of iron transporters at high osmolarity also occurred in both strains.
a proteomic study of bacillus subtilis recently highlighted that salt stress had an impact on iron homeostasis  <cit> .
as already concluded for b. subtilis, also in p. pastoris the induction of iron uptake mechanisms might be of importance for growth in natural environments, where iron availability is generally scarce and may become even more limiting during growth implying high osmotic stress.
CONCLUSIONS
although the central esr pathways are well conserved among fungi, the up- and downstream elements can be significantly different among species to satisfy niche-specific requirements  <cit> .
most notably, the presented data demonstrate a very high similarity and/or cross-talk of the stress induced by recombinant protein production and the reaction to elevated osmolarity in p. pastoris.
growth at high osmolarity resulted in the induction of the response to unfolded proteins.
additionally ribosome biogenesis and translation processes were upregulated, whereas genes involved in cell wall synthesis were downregulated at high osmolarity.
osmotic stress is a common condition for biotechnological production processes, due to high nutrient concentrations.
in this light it is interesting to observe that p. pastoris is more osmo-tolerant than s. cerevisiae, and employs another main osmolyte, namely arabitol instead of glycerol, to compensate for osmotic stress.
the recombinant fab 3h <dig> secreting p. pastoris strain was less prone to osmotic induced stress.
distinct differences, especially in the central carbon metabolism and processes linked to the upr existed between the wt and the 3h <dig> fab producing strain, which can be at least partially explained as response to unfolded protein is significantly induced in the fab producing strain even at low osmolarity.
although in the current study elevated osmolarity did not result in increased productivity of recombinant fab 3h <dig>  the obtained data might be useful to explain the results of other research groups.
it has been reported previously that osmotic stress applied prior to induction of protein secretion resulted in higher levels of scfv antibody in p. pastoris in batch culture  <cit> .
because osmotic stress obviously results in a upr-like response in p. pastoris, it seems plausible that cells may be preconditioned for recombinant protein production as folding competence of the host cells may be increased compared to untreated cells.
in this respect, the data obtained in the present study might be exploited not only for improved bioprocesses, but also for novel routes of strain engineering.
according to the data presented in this study, post-translational control mechanisms play an essential role in p. pastoris, especially during chemostat cultivation.
other proteomic methods such as the analysis of the phosphoproteome  <cit>  might be very useful to gain detailed insight into these yet non-established mechanisms.
however, the current data represent a first step towards a systems wide approach to assess the response to environmental stresses, as well as their overlap with recombinant protein induced stress, in p. pastoris.

 the response of cells to high osmotic pressure and increased salinity has been a subject of close investigation in many different organisms. depending on the intensity of the osmotic shock the immediate response to high osmolarity usually includes the activation of the environmental stress response and of the high osmolarity glycerol pathway to induce changes that are necessary to cope with this stressful environmental condition in saccharomyces cerevisiae. in batch culture, osmotic shock usually implies a temporary growth arrest to adapt the cellular metabolism. major adjustments of gene transcription in saccharomyces cerevisiae and other yeasts include the induction of glycerol-3-phosphate dehydrogenase gpd transcription, transcriptional repression of the plasma membrane glycerol efflux channel fps, but also the adjustment of ribosome biogenesis and the translation and protein folding machinery. glycerol production, but also the production of other small organic molecules, is induced in different yeast species to compensate variations of osmotic conditions. polyols, such as glycerol, pertain to a class of small molecules known as compatible solutes, which, in contrast to inorganic ions, can be safely accumulated and degraded in the cell without impairing cellular function or having detrimental effects on protein and nucleic acid stability. furthermore, biomass yield is reduced upon exposure to high osmolarity because of higher maintenance energy in both, batch and chemostat cultures. however, it is known that after the immediate shock response, transcript levels of many stress responsive genes return to near basal levels after cells have adapted to the new environmental conditions. the effect of osmolarity on cellular physiology is not only of particular interest for the basic research community. as biotechnological production processes aim at high cell and product concentrations, cultivation media usually employ high concentrations of nutrient salts and carbon source resulting in high osmolarities. additionally, there is some evidence that exposure to osmotic stress can have a beneficial effect on recombinant protein production in bacterial, yeast and mammalian host organisms. unfortunately, the positive effect of increased osmolarity on heterologous protein production is, at least in mammalian cells, often cell line specific and e.g. in case of the yeast pichia pastoris it remains anecdotal. the genome sequence of p. pastoris has been recently published and with a publicly available sequence at hand thorough physiological investigations and characterization of this biotechnologically relevant organism becomes feasible. in this context the effect of osmolarity on the physiology of p. pastoris was analyzed in both a non-expressing wild type strain and a recombinant protein secreting strain. the protein secretion strain expressed the antibody fab fragment 3h under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. the effect of osmolarity was monitored in steady state by applying chemostat cultivation in both strains. although chemostat cultivation differs from batch and fed batch systems, which are usually applied for large scale production of recombinant proteins, long term suboptimal growth conditions as they occur during batch and fed batch cultivation can also be applied in steady state chemostat conditions. furthermore, chemostat cultivation offers the advantage that growth rate related effects, which otherwise would interfere with high throughput protein and mrna analytics, can be avoided. to analyze the effect of increased osmolarity on host cell physiology, 2d fluorescence difference gel electrophoresis and dna microarray analyses were applied. these techniques have already been successfully applied to monitor the effect of environmental factors, such as temperature and osmolarity in yeasts. furthermore, hplc analysis was applied to analyze to intracellular polyol and trehalose contents. the obtained data indicated an unfolded protein response like response upon growth at increased osmolarity in the non-expressing wt strain of p. pastoris. in the recombinant protein secreting strain, the upr was obviously already induced due to protein overexpression. the observed overlap of the response to increased osmolarity and the response to recombinant protein production, lead to less adaptations/changes upon high osmolarity on both, the transcriptome and proteome scale, in the fab secreting strain than in the non-expressing wt strain.  general characteristics of cultures at different osmotic conditions chemostat cultivations of p. pastoris were performed at three different osmotic conditions, which were achieved by different concentrations of kcl in the growth medium. this resulted in supernatant osmolarities of approximately and mosmol kg- which will be named low, medium and high osmolarity thereafter. samples were taken at steady state, which means fully adapted cells were analyzed. the characteristics of the chemostat cultures did not dramatically change upon cultivation at different osmotic conditions. biomass yield decreased with increasing osmolarity in the wt strain and fab 3h producing strain. however, the decrease in biomass yield was only statistically significant between cultivations at low and high osmolarity in both strains. the amount of total protein secreted into the culture supernatant did not change upon higher osmotic pressure, but was generally higher in the fab 3h production strain. osmolarity of the growth medium did not significantly influence specific productivity of the fab 3h generally, increased osmotic pressure poses a severe stress condition to cells. although the osmolarity was increased approximately 6-fold and ten-fold in the current study, no decrease of cell viability was observed. more than 97% of the cells in chemostat culture represented viable cells throughout all cultivations. according to flow cytometry data, increased osmolarity resulted in a decrease of the mean cell size as indicated by a decrease of the mean forward scatter of the cells. wt represents the non-expressing wild type strain. fab represents the recombinant protein producing strain. osmolarity values represent actual measured values of the culture supernatant. +/- represents the standard error of the mean. - not applicable. production of compatible solutes and trehalose in p. pastoris upon growth at different osmolarities in yeasts, glycerol is a very common solute but other polyols such as arabitol, mannitol and erythritol are also produced in some yeast species. to analyze whether p. pastoris produces any of these substances, cell extracts were analyzed by hplc. it turned out that very low levels of mannitol and nearly no detectable amounts of erythritol were present in p. pastoris cells. intracellular glycerol levels were higher than mannitol and erythritol levels and a slight significant increase in the wt strain from low to high and medium to high osmolarity occurred, whereas no significant changes of glycerol content were observed in the fab 3h expressing strain. surprisingly, arabitol was the most abundant compound of the analyzed polyols in p. pastoris cells and showed statistically significant increased levels when shifting growth conditions towards high osmolarity. a 3-fold increase of intracellular arabitol levels was observed when comparing low and medium osmolarity conditions and a 4-fold increase when comparing cells grown at low and high osmolarity. furthermore, intracellular levels of trehalose were analyzed as trehalose is thought to be involved in relieving or impeding protein folding stress, which may also occur during salt stress. intracellular trehalose levels were in the same range as glycerol levels but showed a significant trend towards decreased concentrations at medium and high osmolarity growth conditions in the wt strain but slightly missed the threshold p-value in the 3h fab expressing strain the effect of osmolarity on the p. pastoris intracellular proteome as 2d-dige has already been successfully applied to monitor changes in the p. pastoris proteome upon growth at different temperatures, this method was also applied to track changes upon growth at elevated osmolarity. in the wt strain of p. pastoris approximately proteins passed the criteria, whereas about proteins passed these criteria in the recombinant protein producing strain. most of the protein spots represented low abundant proteins with too small quantities of proteins on the 2d gels to be confidently identified, resulting in successfully identified proteins. a list of all identified protein spots with corresponding peptides, obtained from ms/ms analysis, is available in additional file as already reported in previous studies, additionally to most likely full length proteins, protein fragments were identified according to the spot position on the gel. furthermore, protein levels showed the largest changes when low and medium and low and high osmolarity cultivations were compared, whereas only minor changes occurred when comparing medium and high osmolarity setpoints. analysis was performed by 2d-dige and subsequent lc esi-ms/ms identification. protein standard name, protein functional description, theoretical molecular weight and theoretical isoelectric point, average expression values and 1-anova values are shown. significant anova values are indicated in bold letters. the average protein abundance fold change of the comparison of low and high osmolarity setpoints are shown. additional protein fold change data are available through additional file as can be seen in figure the major impact of osmolarity on the p. pastoris proteome was on proteins involved in energy metabolism and protein folding. whereas protein levels of a major spot of aconitate hydratase were increased at medium and high osmolarity in the wt strain, they were not significantly affected by osmolarity in the fab 3h producing strain. in contrast, in the fab 3h expressing strain three minor isoforms or degradation products were significantly down-regulated at higher osmolarity, but showed no altered abundance in the wt strain. furthermore, citrate synthase protein levels were decreased at high salt concentrations in the wt strain but showed no significant change in the fab producing strain. formate dehydrogenase, glycerol kinase, and isocitrate lyase showed similar trends towards lower protein levels during medium and high osmolarity cultivations in both strains, whereas pyruvate kinase and phosphoglycerate kinase were generally up-regulated at higher osmolarity. phosphoglucose isomerase showed lower levels at medium osmolarity in the production strain and a protein identified as atp3p was massively down-regulated at medium and high salt concentrations in the wt strain. furthermore, alcohol oxidase, a key enzyme in methanol utilization, was down-regulated at medium and high osmolarity in the wt strain but did not show a significant change in the fab 3h expressing strain. it should be pointed out that aox transcription is thought to be repressed during growth on glucose and that the current study was performed with glucose as carbon source to constitutively express the heterologous protein under the control of the gap-promoter. however, it was shown previously that basal levels of aox1p were actually present during glucose limited growth of p. pastoris. similar discrepancies between the wt and the recombinant protein expressing strain were observed for proteins involved in protein folding and secretion and folding stress response. whereas the major er chaperone and unfolded protein response sensor kar2p/bip and the protein disulfide isomerase pdi1p were up-regulated at medium and high osmolarity in the wt strain, no changes of these two proteins were observed in the fab producing strain. more prominently increased levels of cytosolic and mitochondrial chaperones ssc1p, sse1p, ssz1p and hsp60p were observed at medium and high osmolarity in the wt strain than in the fab 3h producing strain. the stress induced chaperone ssa4p showed increased levels at medium salt concentrations in both strains but returned to below basal levels at high salt conditions in the fab producing strain. ino1p, a protein involved in synthesis of inositol phosphates and inositol-containing phospholipids and which is linked to the upr, was also up-regulated at high salt concentrations in both strains analyzed. other protein spots that changed their abundance upon cultivation at increased salt concentrations were agx1p and gdh1p. both of them showed higher protein levels during growth at high osmolarity. the effect of osmolarity on the p. pastoris transcriptome to analyze the effect of different osmolarities on the p. pastoris transcriptome, p. pastoris specific microarrays were applied. to support microarray analysis, real-time pcr was performed. real-time pcr data proved to be consistent with microarray results. more significant changes on the transcriptome level were observed in the wt strain than in the recombinant protein expressing strain. low p-values can result from a high technical variation within the replicates or reflect the biological truth within the samples. to determine if the lower amount of significantly regulated genes in the fab expressing strain is a technical artefact, the correlation, standard deviation and the coefficient of variation for the replicates of the wt and expressing strain were compared. correlation of intensity values was generally high between all microarrays of one group with r values between and. the values for standard deviation and coefficient of variation indicated that the variance in replicates of the expressing strain was slightly but consistently higher than for the wt strain. based on these results additional microarray experiments were performed to exclude any bias in the data. these additional data did not change the result or number of regulated genes, suggesting a true biological difference. to eliminate the possibility that the samples of the expressing strain vary more than the ones of the wt strain, hierarchical cluster analysis and gene set analysis were performed on the fold change data and indicated that regulation was indeed different in the two strains analyzed. wt - non-expressing wild type strain. fab - fab 3h expressing strain. the number of genes that are similarly regulated in both strains are listed as common genes. because most of the genes that were regulated when comparing low to medium osmolarity were also regulated when comparing low to high osmolarity, the following data presentation and discussion will focus on the effects that were observed when low and high osmotic conditions were compared. to get an overview of the general adaptations during steady-state cultivation, fisher's exact test was performed to identify cellular processes, which were affected by different osmolarities on the transcript level. a total of go categories were either affected in both or at least in one of the analyzed strains. concordant with the mere number of regulated genes, there appeared more significantly affected cellular processes in the wt strain than in the heterologous protein expressing strain. only go categories occurred to be affected in both strains, namely go:, go: and go:. additionally, in the wt strain the go terms go:, go:, go: and go: were affected by increased extracellular osmolarity. regarding ion transport, uptake and metabolism, high osmolarity resulted in increased expression of the iron transporters ftr sit and the vacuolar iron reductase fre in both strains. calcium ion homeostasis and calcium dependent signal transduction were obviously affected by high osmolarity in the wt strain as the ca2+ transporter pmc and calcineurin a were down-regulated at high osmolarity. a major effect was apparent for genes involved in cell wall organization and its biogenesis. whereas 21% of the genes belonging to this go group were down-regulated at high osmolarity in the wt strain, a similar effect, albeit with fewer significant genes, was observed in the 3h fab secreting strain. additionally, a putative extracellular or cell wall associated protein with homology to the s. cerevisiae pry gene was up-regulated in both strains at high osmolarity. increased levels of a gene with homology to s. cerevisiae pry upon increased salinity have also been reported for the halotolerant yeast hortaea werneckii previously. however, no changes in the protein pattern, indicating higher protein levels, were observed by sds-page of the culture supernatant. a signaling cascade for sensing and adaptation to osmotic stress in s. cerevisiae has already been established based on the available data and genes with homology to the corresponding genes in s. cerevisiae were also identified in p. pastoris. none of the genes involved in osmotic stress sensing upstream of the mitogen activated protein kinase hog showed significant regulation in the fab 3h expressing strain, whereas sho ssk and ptp were up-regulated and ste was down-regulated at increased osmolarity in the wt strain. several genes involved in energy metabolism and storage carbohydrate metabolism were affected by increased osmolarity. fba a key enzyme in glycolysis and gluconeogenesis, was up-regulated in the heterologous protein expressing strain at high osmolarity. the acetyl-coa synthetase acs was up-regulated in the wt strain at high osmolarity. transcript levels of several genes involved in the tricarboxylic acid cycle and the glyoxylate cycle, namely aco fum mdh sfc and icl were reduced and subunits of the atp synthase were up-regulated during growth at high osmolarity in the p. pastoris wt strain. in the fab producing strain, tkl involved in the pentose phosphate pathway was significantly up-regulated. glycogen synthesis was also affected by high osmolarity as glg gsy and glc showed decreased transcript levels during growth at high osmolarity. decreased levels of dga gut and gtp indicated changes in glycerol and lipid metabolism. additionally, a homologue to the s. cerevisiae putative passive glycerol channel yfl054c was down-regulated and the active glycerol importer stl was up-regulated during growth at high osmotic conditions in the wt strain. furthermore, significant down-regulation of the p. pastoris alcohol oxidase aox was observed in the wt strain at high osmolarity, whereas no significant regulation was observed in the fab 3h expressing strain. these data are concordant with data on the proteome level. in the wt strain approximately 7% of the genes involved in ribosome biogenesis and assembly were up-regulated during steady state cultivation at high osmolarity. the effect of recombinant protein production on the transcriptome of p. pastoris at low osmolarity as the experiment was performed with a non-expressing wt and a recombinant protein expressing strain of p. pastoris the effect of recombinant protein expression itself on the cellular transcriptome at low osmolarity could also be analyzed. the most prominent effect of recombinant protein production on host cell physiology is the induction of the unfolded protein response. therefore, we analyzed whether this effect could be observed at low osmolarity in glucose limited chemostat cultures of p. pastoris. it turned out that the transcriptional response to recombinant protein production during chemostat cultivation at low osmolarity was low. only mrnas were significantly regulated when comparing the two strains using a strict cut-off. using a less strict cut-off genes were significantly regulated. hac was up-regulated in the fab expressing strain and indicated the induction of the upr in the fab expressing strain. in contrast to hac other members of the core upr and targets of the hac transcription factor showed no significant response by applying a p-value cut-off. however, gene set analysis applied on the microarray data revealed increased expression of genes related to the go term go:. the upr is a specific response to unfolded protein in the er and is a subcategory of go: the up-regulation of the hac transcript as well as the significant upregulation of the response to unfolded protein indicated the induction of the upr in the fab 3h expressing strain. the effect was lower than in previous studies, which in fact were performed in non-carbon limited batch cultures. it is known that during carbon-limited chemostat cultivation of s. cerevisiae metabolic control can be more important than gene regulation. furthermore, it is known that part of the regulation of the upr can be performed on a post-transcriptional or even post-translational level in s. cerevisiae and aspergillus niger. less information about control mechanisms are available for p. pastoris. we conclude that similar to these previous studies, a substantial part of regulation, including the regulation of the upr, is achieved on a post-transcriptional level during glucose-limited chemostat cultivation of p. pastoris. regarding the response to unfolded protein, gsa indicated significantly increased transcription of genes related to this go category in the wt strain at high osmolarity. in contrast, higher growth medium osmolarity did not result in an induction of the response to unfolded protein in fab 3h expressing strain. salt tolerance of pichia pastoris as no data on the salt tolerance of p. pastoris compared to s. cerevisiae were found in literature, a growth test on ypd agar plates, containing different amounts of nacl or kcl, was performed. this growth test indicated higher tolerance to growth on nacl and a less pronounced higher tolerance to growth on kcl of p. pastoris compared to s. cerevisiae. discussion production of compatible solutes to counterbalance the osmotic pressure by high or low salt or solute concentrations in the growth medium, microorganisms produce various compatible solutes. in s. cerevisiae and many other organisms glycerol is the main osmolyte accumulated during osmotic stress. however, we found that intracellular glycerol levels were low at all osmotic conditions in p. pastoris. on the other hand, arabitol was more abundant than glycerol, even at low osmolarity, and it was accumulated in p. pastoris during growth at elevated osmotic pressure. glycerol production in s. cerevisiae depends on the increased expression of glycerol-3-phosphate dehydrogenase gpd and glycerol-3-phosphatase gpp. nevertheless, we could not find neither of the two genes involved in glycerol metabolism, gpd and gpp to be up-regulated on the transcript level and did not identify protein spots with altered expression which would match these two genes in p. pastoris. furthermore, it was shown in debaryomyces hansenii that nacl stress lead to increased levels of proteins involved in the upper part of glycolysis and down-regulation of proteins involved in the tca-cycle. it was concluded that these changes may favor the accumulation of dihydroxyacetonephosphate and consequently the production of glycerol. no changes related to the upper part of glycolysis were observed in the current study, although it is clear that the need for compatible solutes leads to a redirection of a part of the carbon source to alleviate the stress induced by increased osmolarity. this would make sense as arabitol obviously plays a more important role as compatible solute than glycerol in p. pastoris. however, the regulation of genes involved in glycerol uptake and efflux, such as the up-regulation of stl and the down-regulation of yfl054c may be beneficial for p. pastoris as well. the loss of minor osmolytes may result in detrimental effects on cellular integrity at high kcl concentrations. no significant regulation was observed for other putative glycerol transporters of p. pastoris recently described by mattanovich and co-workers. arabitol synthesis is linked to the pentose phosphate pathway. however, no changes possibly linked to the pp pathway and arabitol synthesis were observed on the transcript or the proteome level. the regulation of arabitol synthesis might be mainly achieved on a post-transcriptional level by increased translation or by protein modification and changes of enzyme activity during chemostat cultivation of p. pastoris. nevertheless, concordant with other studies, arabitol obviously is of particular importance for the metabolism of p. pastoris as it is also secreted into the supernatant at certain growth conditions, such as low oxygenation. trehalose has been previously shown to play an important role in heat shock induced refolding of proteins in baker's yeast and in vitro. furthermore, trehalose may also be involved in the response to temperature induced stress in p. pastoris as intracellular levels increased at elevated temperature. however, as trehalose levels were lower during growth at high osmolarity and no changes of the stress induced cytosolic trehalase nth were observed on the protein or mrna level, trehalose may not be directly involved in the protection of proteins against osmotic induced protein denaturation or damage. it is more likely that, similar to s. cerevisiae, trehalose degradation may play a role during growth at elevated osmolarity, or that trehalose levels may be simply lower due to a redirection of carbon source to the production of arabitol rather then to the production of trehalose. effect on energy metabolism the differential response of aco1p and the differences of transcript levels of genes involved the tca cycle to different osmotic conditions between the wt and the fab 3h expressing strain lead to the conclusion that recombinant protein production influenced the osmo-dependent adaptation of the energy metabolism. previous data already indicated a metabolic burden and influence of recombinant protein production on energy metabolism in p. pastoris. furthermore, the key enzyme of methanol utilization, aox was differently regulated in the two strains and indicated significant differences in the regulation of energy metabolism. protein and transcript levels of the alcohol oxidase were significantly negatively affected by growth at high osmolarity in the wt strain but not in the fab 3h secreting strain. p. pastoris aox seems to be tightly regulated upon exposure to various stresses and might represent an ideal candidate as a marker gene/protein to monitor diverse external and internal stresses in p. pastoris. apart from these additional data supporting the idea of a metabolic burden during recombinant protein production in p. pastoris, no clear interpretation about the changes of energy metabolism upon growth at different osmolarities in chemostat cultures emerged. further investigations using a different approach to the one used in the current study will be necessary to elucidate the effect of osmolarity on the energy metabolism of p. pastoris. activation of translation, ribosome biogenesis and the response to unfolded protein at high osmolarity another major effect was the massive increase of chaperones and upr related proteins at high osmolarity. the upr, including heat shock proteins and cellular chaperones, plays an essential role in the response to various stresses. apart from its role in the esr of unicellular organisms, the upr is also of great importance in human disease as highlighted by its involvement in the development of several human maladies such as diabetes, neurodegenerative disorders and cancer. the observation of increased levels of molecular chaperones during growth at high osmolarity is concordant with previous results for aspergillus nidulans and similar to results obtained for d. hansenii and s. cerevisiae in batch culture. furthermore, high osmotic pressure resulted in increased levels of pdi1p and kar2p, indicating endoplasmic reticulum related protein folding stress. unlike s. cerevisiae, upr induction has been reported to be a main event upon exposure to salt stress in the halotolerant yeast rhodotorula mucilaginosa. generally, the induction of the upr may not only be a result of high concentrations of ionic solutes such as salts but the response to unfolded proteins is also triggered by high osmotic pressure induced by other substances such as sugar compounds. it has been reported for mammalian cells that low as well as high hexose concentrations can lead to upr induction. the upr has been described to be mainly a transcriptional response, but recently post-transcriptional and post-translational regulation has been described for fungal organisms. in this context, mrna levels of the upr transcription factor hac are increased in the fab expressing strain as a reaction to recombinant protein production. on the other hand, gsa of the microarray data also showed induction of "responses to unfolded protein" at high osmolarity in the wt strain. these results are supported by the effects observed at the proteome level. a conventional induction of the upr by increased hac levels was not observed at increased osmolarity in neither of the strains. however, increased osmolarity resulted in increased kar2p and pdi1p on the proteome level in the wt strain. as this clearly demonstrated er associated protein folding stress we refer to a upr-like response of p. pastoris wild type cells at high osmolarity. although a direct comparison between the wt and recombinant protein producing strain was not possible on the proteome level, this comparison was possible on the transcript level and strongly indicated the upregulation of processes involved in response to unfolded protein in the recombinant fab producing strain. thus, we hypothesize that the up-regulation of these er resident proteins as well as other chaperones was obviously not necessary in a fab 3h producing strain at high osmolarity, as these changes had already been triggered by the upr that was induced by the recombinant protein. additionally to this upr-like response, the induction of ribosome biogenesis and translation were apparent on the transcript level in the wt strain. the up-regulation of genes involved in protein synthesis during osmotic stress has been reported for the salt-tolerant yeasts h. werneckii and d. hansenii. furthermore, studies on brewing strains of s. cerevisiae concluded that the faster adaption to higher salt concentration compared to a laboratory strain was achieved by higher expression levels of genes involved in protein synthesis. similar to other environmental factors, such as temperature, translation might become a rate-limiting factor during growth at high osmolarities because of stress related to decreased intracellular water availability. boosting the protein synthesis machinery might be necessary for growth of p. pastoris at elevated osmolarity. the fab 3h expressing strain of p. pastoris did not show this increase of the protein synthesis machinery. we have shown previously that over-expression of the transcription factor hac in p. pastoris batch cultures resulted in increased expression of genes involved in mrna translation and to a massive increase of genes involved in ribosome biogenesis and assembly. obviously the up-regulation of ribosome biogenesis, translation and other co-regulated processes at high osmolarity was not necessary in the fab 3h producing strain as these changes had already been induced by recombinant protein production itself. this upr-like response at high osmolarity also points to the fact that, similar to halotolerant yeast species like r. mucilaginosa, p. pastoris might use different mechanisms for gaining osmotic stress resistance than s. cerevisiae. this hypothesis was supported by the growth tests for salt tolerance, which were performed with p. pastoris and s. cerevisiae. p. pastoris showed indeed higher resistance to increased salt concentrations in the growth medium than s. cerevisiae. many changes observed in the wt upon a change from low to high osmolarity were not observed in the recombinant protein expressing strain. although high osmolarity triggered the response to unfolded protein, ribosome biogenesis and translation in the wt strain, the activation of these apparently co-regulated processes was compensated in the fab 3h strain by recombinant protein induced upr. other cellular responses increased osmolarity also influenced other cellular mechanisms, such as some parts of the oxidative damage response, which apparently were not co-regulated with the protein synthesis and folding machinery. therefore, these processes which seem to be an essential part of the response to increased osmolarity were monitored in both strains of p. pastoris. the interrelation of salt and oxidative stress is already established in plants and the interrelation and cross-talk of the hog pathway and other pathways such as protein kinase c and calcineurin dependent signaling are also established in yeasts. changes in cell wall integrity signaling, which were evident by altered expression levels of cell wall components in both strains, may be directly related to the changes of the cna and pmc transcripts, as some of these cell wall synthesis related genes are dependent on calcineurin signaling. for example, decreased transcript levels of crh and gas at high osmolarity may indicate a change of cell wall rigidity at high osmolarity. high osmolarity results in decreased turgor pressure when compared with low or hypo-osmotic conditions. thus, increased osmolarity results in cell shrinkage and smaller cells, which in fact was obvious by a decreased mean forward scatter of the cells in the present study. in this context the down-regulation of cell wall components, which would result in lower cell wall rigidity, at high osmolarity makes sense. it has been shown in aspergillus nidulans that salt addition to the growth medium resulted in decreased cell wall rigidity. a further indication that high osmolarity is compatible with lower cell wall rigidity is the fact that the swollen cellular phenotype of gas mutant cells, a gene which is also downregulated at high osmolarity in the current study, in s. cerevisiae is compensated by growth at high osmolarity. although this effect was very evident on the transcript level we were not able to monitor it on the proteome level. this may be simply due to the preparation of protein samples and the resulting absence of cell wall and membrane proteins, which are rather difficult to extract by standard protein preparation methods. additionally to these events, the induction of iron transporters at high osmolarity also occurred in both strains. a proteomic study of bacillus subtilis recently highlighted that salt stress had an impact on iron homeostasis. as already concluded for b. subtilis, also in p. pastoris the induction of iron uptake mechanisms might be of importance for growth in natural environments, where iron availability is generally scarce and may become even more limiting during growth implying high osmotic stress.  although the central esr pathways are well conserved among fungi, the up- and downstream elements can be significantly different among species to satisfy niche-specific requirements. most notably, the presented data demonstrate a very high similarity and/or cross-talk of the stress induced by recombinant protein production and the reaction to elevated osmolarity in p. pastoris. growth at high osmolarity resulted in the induction of the response to unfolded proteins. additionally ribosome biogenesis and translation processes were upregulated, whereas genes involved in cell wall synthesis were downregulated at high osmolarity. osmotic stress is a common condition for biotechnological production processes, due to high nutrient concentrations. in this light it is interesting to observe that p. pastoris is more osmo-tolerant than s. cerevisiae, and employs another main osmolyte, namely arabitol instead of glycerol, to compensate for osmotic stress. the recombinant fab 3h secreting p. pastoris strain was less prone to osmotic induced stress. distinct differences, especially in the central carbon metabolism and processes linked to the upr existed between the wt and the 3h fab producing strain, which can be at least partially explained as response to unfolded protein is significantly induced in the fab producing strain even at low osmolarity. although in the current study elevated osmolarity did not result in increased productivity of recombinant fab 3h the obtained data might be useful to explain the results of other research groups. it has been reported previously that osmotic stress applied prior to induction of protein secretion resulted in higher levels of scfv antibody in p. pastoris in batch culture. because osmotic stress obviously results in a upr-like response in p. pastoris, it seems plausible that cells may be preconditioned for recombinant protein production as folding competence of the host cells may be increased compared to untreated cells. in this respect, the data obtained in the present study might be exploited not only for improved bioprocesses, but also for novel routes of strain engineering. according to the data presented in this study, post-translational control mechanisms play an essential role in p. pastoris, especially during chemostat cultivation. other proteomic methods such as the analysis of the phosphoproteome might be very useful to gain detailed insight into these yet non-established mechanisms. however, the current data represent a first step towards a systems wide approach to assess the response to environmental stresses, as well as their overlap with recombinant protein induced stress, in p. pastoris. 
therefore, these results might be used in future strain and bioprocess engineering of this biotechnologically relevant yeast. several studies indicated that increased osmolarity of the growth medium can have a beneficial effect on recombinant protein production in different host organisms. thus, the effect of osmolarity on the cellular physiology of pichia pastoris, a prominent host for recombinant protein production, was studied in carbon limited chemostat cultures at different osmolarities. while our main intention was to analyze the effect of different osmolarities on p. pastoris in general, this was complemented by studying it in context with recombinant protein production. the current data demonstrate a strong similarity of environmental stress response mechanisms and recombinant protein related stresses. increased osmolarity resulted in an unfolded protein response like response in p. pastoris and lead to pre-conditioning of the recombinant fab producing strain of p. pastoris to growth at high osmolarity. these data indicated that upon increased osmolarity less adaptations on both the transcript and protein level occurred in a p. pastoris strain, secreting the fab fragment, compared with the wild type strain. in contrast to the model yeast saccharomyces cerevisiae, the main osmolyte in p. pastoris was arabitol rather than glycerol, demonstrating differences in osmotic stress response as well as energy metabolism. the effect of osmolarity on cellular physiology has been subject of investigation in many different species. high osmolarity is of importance for biotechnological production processes, where high cell densities and product titers are aspired. no transcriptional activation of the high osmolarity glycerol pathway was observed at steady state conditions. 2d fluorescence difference gel electrophoresis and microarray analysis were applied and demonstrated that processes such as protein folding, ribosome biogenesis and cell wall organization were affected by increased osmolarity. transcriptome and proteome analyses were applied to assess differences upon growth at different osmolarities in both, a wild type strain and an antibody fragment expressing strain. furthermore, no change of the specific productivity of recombinant fab was observed at increased osmolarity. these data point out that the physiological response to increased osmolarity is different to s. cerevisiae.
 the effect of osmolarity on cellular physiology has been subject of investigation in many different species. high osmolarity is of importance for biotechnological production processes, where high cell densities and product titers are aspired. several studies indicated that increased osmolarity of the growth medium can have a beneficial effect on recombinant protein production in different host organisms. thus, the effect of osmolarity on the cellular physiology of pichia pastoris, a prominent host for recombinant protein production, was studied in carbon limited chemostat cultures at different osmolarities. transcriptome and proteome analyses were applied to assess differences upon growth at different osmolarities in both, a wild type strain and an antibody fragment expressing strain. while our main intention was to analyze the effect of different osmolarities on p. pastoris in general, this was complemented by studying it in context with recombinant protein production.  in contrast to the model yeast saccharomyces cerevisiae, the main osmolyte in p. pastoris was arabitol rather than glycerol, demonstrating differences in osmotic stress response as well as energy metabolism. 2d fluorescence difference gel electrophoresis and microarray analysis were applied and demonstrated that processes such as protein folding, ribosome biogenesis and cell wall organization were affected by increased osmolarity. these data indicated that upon increased osmolarity less adaptations on both the transcript and protein level occurred in a p. pastoris strain, secreting the fab fragment, compared with the wild type strain. no transcriptional activation of the high osmolarity glycerol pathway was observed at steady state conditions. furthermore, no change of the specific productivity of recombinant fab was observed at increased osmolarity.  these data point out that the physiological response to increased osmolarity is different to s. cerevisiae. increased osmolarity resulted in an unfolded protein response like response in p. pastoris and lead to pre-conditioning of the recombinant fab producing strain of p. pastoris to growth at high osmolarity. the current data demonstrate a strong similarity of environmental stress response mechanisms and recombinant protein related stresses. therefore, these results might be used in future strain and bioprocess engineering of this biotechnologically relevant yeast. 