BACKGROUND
bioethanol production from lignocellulosic biomass including agricultural and forestry residues has attracted increased attention worldwide  <cit> .
lignocellulosic biomass needs to be depolymerized into simple sugars in order to be utilized for microbial fermentation.
the commonly applied dilute acid pretreatment generates numerous chemical byproducts that inhibit cell growth and interfere with subsequent microbial fermentation  <cit> .
among numerous inhibitory compounds, furfural and 5-hydroxymethylfurfural  are commonly encountered inhibitors  <cit> .
furfural and hmf are formed by dehydration of pentoses and hexoses released from hemicellulose and cellulose, respectively  <cit> .
these inhibitors can damage cell structures, inhibit cell growth, reduce enzymatic activities, generate cellular reactive oxygen species , break down dna, and inhibit protein and rna synthesis  <cit> .
the presence of fermentation inhibitors represents a bottle neck in cellulosic ethanol conversion technology and overcoming the inhibitor effect is one of the fundamental challenges to the industrial production of bioethanol from lignocellulosic biomass.
furfural and its conversion product have been widely studied while knowledge of hmf conversion is limited due to a lack of commercial source of its conversion product  <cit> .
unlike evaporative furfural, hmf is more stable and difficult to degrade in cell culture.
recently, an hmf metabolic conversion product was isolated and identified as  <dig>  5-bis-hydroxymethylfuran   <cit> .
a dose-dependent response of yeast to hmf was demonstrated and a lag phase was used to measure levels of strain tolerance  <cit> .
the yeast saccharomyces cerevisiae is able to in situ detoxify hmf into the less toxic compound fdm through nadph-dependent reductions  <cit> .
typically, yeast strains show a lag of delayed cell growth after inhibitor challenge such as with furfural and hmf, under sublethal doses.
once hmf and furfural inhibitor levels were chemically reduced to a certain lower concentration, cell growth recovered and the glucose-to-ethanol conversion accelerated at a faster rate than would normally occur  <cit> .
it was suggested that genomic adaptation occurred during the lag phase  <cit> .
in fact, inhibitor-tolerant yeast strains showed significant shorter lag phases under the inhibitor challenges compared with a wild type strain  <cit> .
gene expressions of selected pathways of the tolerant yeast are distinct from the wild type control  <cit> .
sequence mutations are common and a large number of single nucleotide polymorphism  mutations were observed throughout all  <dig> chromosomes for a tolerant yeast strain .
adaptations appear to occur at the genome level.
however, little is known about gene expression response and regulatory events for yeast during the adaptation lag phase.
the objective of this study was to characterize transcriptome response of yeast during the lag phase after the hmf challenge.
using a comparative time course study, we investigated the dynamics of transcriptome profiling during this critical stage applying dna microarray assays and regulatory analysis.
important genes, together with transcription factors  involved in the hmf stress response, were identified.
the functions of selective candidate genes were verified by corresponding gene deletion mutation strains.
significant regulatory interaction networks were uncovered during the genome adaptation in yeast.
results of this study provide insight into mechanisms of yeast adaptation and tolerance to lignocellulose derived inhibitors.
this will directly aid engineering efforts for more tolerant strain development.
RESULTS
cell growth response and metabolic conversion profiles
compared to a non-treated control, yeast challenged by hmf displayed a significant drop in cell growth as measured by od <dig> absorbance  <dig> h after the treatment .
although the cell growth was recovered at a later time, cell density of the hmf-treated yeast was relatively low throughout the course of the study.
similarly, glucose consumption for the hmf-treated culture was slower and glucose was depleted at  <dig> h, approximately  <dig> h later than the non-treated control .
as expected, hmf was undetectable and fdm was detected as hmf conversion product  <cit>  in hmf-treated cultures less than  <dig> h after incubation .
no hmf or fdm was detected from the control culture.
transcription expression dynamics during the lag phase
clustering analysis distinguished significant differences for expression responses by hmf between the treated and untreated conditions over time .
among the more than  <dig>  genes of the yeast genome,  <dig> genes were identified as differentially expressed by anova for at least 2-fold changes during the lag phase of  <dig> to  <dig> min by the hmf challenge .
among these,  <dig> genes were induced constantly throughout the lag phase while  <dig> genes were repressed at various stages of the lag phase .
many of the induced genes showed immediate enhanced expressions within  <dig> min after the hmf challenge.
these genes mainly fall with functional categories of reductase, pleiotropic drug resistance , proteasome and ubiquitin, amino acids metabolism, stress response functions, and others .
for example, adh <dig>  encoding nadph-dependent medium chain alcohol dehydrogenase displayed the highest induction of more than 30-fold increase in mrna abundance at  <dig> min after the hmf treatment.
other significantly induced genes including ari <dig>  gre <dig>  pdr <dig>  rsb <dig>  put <dig>  cha <dig>  hsp <dig>  ssa <dig>  and oye <dig>  which showed more than 10-fold mrna increase at various times during the lag phase.
* genes in bold indicate their encoding proteins or enzymes are involved in more than one function
the repressed genes are mainly involved in the functional categories of ribosome biogenesis, amino acid and derivative metabolic process, rna metabolic process, transport, and others .
most of the genes encoding enzymes for arginine biosynthesis were severely repressed, such as arg <dig>  arg <dig>  arg <dig>  arg <dig> , arg <dig>  and arg <dig> .
for the repressed genes, three types of dynamic responses were observed.
a small group of two dozen genes showed transient inductions at  <dig> min but quickly turned into repressed after  <dig> min, such as pcl <dig> and pcl <dig> for glycogen metabolism, mal <dig>  mal <dig>  and mph <dig> for maltose utilization.
another group of about  <dig> genes were constantly repressed, and these were mainly in the functional categories of amino acid metabolism, such as arg <dig>  arg <dig>  arg <dig>  arg <dig> , arg <dig> for arginine metabolism, his <dig>  his <dig>  and his <dig> for histidine metabolism, aro <dig>  aro <dig>  hom <dig>  and hom <dig> for aromatic amino acid metabolism.
the third group of the repressed genes were initially repressed at  <dig> or  <dig> min but recovered at later time points.
this group of repressed genes fall within the categories of rrna processing, trna export, and ribosomal biogenesis such as nob <dig>  pus <dig>  rrp <dig>  nop <dig>  and cbf5; mitochondrial mrna maturase such as bi <dig> and bi3; vitamin b <dig> biosynthesis gene snz1; and telomere length maintenance gene yku <dig> .
relevant transcription factors
under the hmf challenge, we found that seven transcription factor genes, pdr <dig>  pdr <dig>  yap <dig>  yap <dig>  yap <dig>  rpn <dig>  and hsf <dig>  displayed significant greater expression during the lag phase in response to the hmf challenge .
except for hsf <dig>  most transcription factor genes displayed greater than 2-fold increase after the hmf treatment.
by the aid of t-profiler  <cit> , yeastract database  <cit>  and interactive pathway analysis using genespring gx  <dig> , we identified these genes as the most important transcription factor genes positively regulating gene expression response in adaptation to the hmf stress during the lag phase in yeast.
we further analyzed protein binding motifs for these genes and found each transcription factor gene harbored protein binding motifs for pdr1p, pdr3p, yap1p, yap5p, yap6p, rpn4p, and hsf1p.
dna binding motifs of pdr1/3p were found in promoter regions of pdr <dig>  yap <dig>  pdr <dig>  and rpn4; yap1p binding sites in all six transcription factor genes except for pdr1; and hsf1p sites in all six genes except for pdr <dig> .
except for pdr <dig> which had a single yap1p binding site, each of the other six transcription factor genes displayed multiple binding sites for multiple transcription factors.
for example, rpn <dig> had  <dig> binding sites of  <dig> transcription factors, and pdr <dig> had  <dig> sites for  <dig>  interactions involving multiple transcription factors apparently exist.
for example, highly expressed rpn <dig> in this study was found to be regulated by yap1p, pdr1p, pdr3p, and hsf1p that supported by chip-chip data and microarray assay of transcription factor mutations  <cit> .
on the other hand, it also demonstrated positive feedback to its regulators of yap1p and pdr1p  <cit> .
the presence of dna binding motifs of a transcription factors' own in its promoter region, such as pdr <dig>  yap <dig>  and hsf <dig> , suggested a self-regulated expression.
the highly induced expression of the seven transcription factor genes in response to the hmf challenge and multiple protein binding motifs across the transcription factors suggested co-regulation and interactions of multiple transcription factors under the stress.
as for many repressed expression responses to hmf, we identified five transcription factor genes arg <dig>  arg <dig>  gcn <dig>  fhl <dig>  and rap <dig> that displayed down-regulated expressions .
yap <dig> regulated gene expression networks
among the seven transcription factor genes, yap <dig> displayed consistently higher inductions, a 2- to 3-fold increase during the lag phase .
yap1p acts as a sensor for oxidative molecules, and activates the transcription response of anti-oxidant genes by recognizing yap1p response elements , 5'-tkactma-3', in the promoter region  <cit> .
a total of  <dig> hmf-induced genes were found to have the yre sequence in their promoter region .
many genes were confirmed to be regulated directly by yap <dig> or indirectly through yap <dig> and yap <dig> .
most yap1-regulated genes were classified in the functional categories of redox metabolism, amino acid metabolism, stress response, dna repair, and others .
for example, the highly induced oxidoreductase genes adh <dig>  gre <dig>  and oye <dig> were found as regulons of yap <dig>   <cit> .
adh <dig> and gre <dig> were also co-regulated by yap5p and yap6p  <cit> .
these two genes were among those confirmed as reductases actively involved in the hmf detoxification  <cit> .
ari <dig>  a recently characterized aldehyde reductase contributing to detoxification of furfural and hmf  <cit> , was found to be regulated by yap6p  <cit>  which is a regulon of yap <dig>  in addition, yap <dig> and other yap gene family members were shown to co-regulate numerous genes in a wide range of functional categories such as pdr, heat shock protein, chaperones, amino acid metabolism, as well as other regulators.
the significance of the role of the yap gene family in adaptation and tolerance to hmf is confirmed by growth responses of the deletion mutations.
single yap gene deletion mutations were able to grow normally without hmf treatment .
however, in the presence of  <dig> mm hmf, mutations Δyap <dig>  Δyap <dig>  Δyap <dig>  and Δyap <dig> showed delayed growth compared with their parental strain .
among these, Δyap <dig> displayed a 4-day long lag phase, indicating a profound functional defect affected by the yap <dig> gene.
pdr family and pdr1/ <dig> involved regulatory interactions
among the significantly induced genes by hmf, at least  <dig> genes were categorized into the pdr family .
many genes displayed consistent induced expressions ranging from 3- to 30-fold increases during the lag phase .
gene products of these increased transcripts were in the protein categories of drug/toxin transport for tpo <dig> and tpo <dig>  transport atpase for rsb <dig>  and abc transporters for pdr <dig> .
snq <dig>  yor <dig>  pdr <dig>  and pdr <dig> encoding proteins shared functions of all these three categories.
in addition, many pdr proteins have functions such as atp binding and chemical agent resistance .
most of these genes have the pleiotropic drug response element  in their promoter regions .
* proteins in bold indicate functions involved in more than one category
hmf-induced transcription factor genes pdr <dig> and pdr <dig> regulate gene expression under a large variety of unrelated chemical stress conditions by binding to the pdre of target genes  <cit> .
both pdr1p and pdr3p recognize cgg triplets oriented in opposite directions  to form an inverted repeat  <cit> , and able to form homodimers or heterodimers to activate target gene expression  <cit> .
many induced genes regulated by pdr1p and/or pdr3p in this group are involved in export of both xenobiotic compounds and endogenous toxic metabolites using atp-binding cassette  transporters , lipid composition of the plasma membrane , export of polyamines by polyamine transporters , dna repairing , and other functions   <cit> .
at least eight genes induced by hmf in this study were regulated by both pdr1p and pdr3p.
pdr1p and pdr3p also recognize and activate other subsets of genes.
pdr3p participates in certain processes that do not involve pdr1p, such as regulating dna damage-inducible genes mag <dig> and ddi <dig>  <cit> .
similarly, some genes are only regulated by pdr1p, such as rsb <dig>  <cit> , adh <dig>  and pre <dig>  <cit> .
we also found that the pdr <dig> promoter contains two pdres that can be autoregulated by itself in addition to being a regulon of pdr1p  <cit> .
pdr <dig> and pdr <dig> also demonstrated regulatory connections with a broad range of functional category genes as well as most active regulatory genes.
pdr  <dig> and pdr <dig> gene deletion mutations were assayed to confirm their influence on the expression of the potential regulons.
when examined by qrt-pcr, mutant Δpdr <dig> displayed reduced transcriptional abundance for many genes, such as pdr <dig>  pdr <dig>  pdr <dig>  yor <dig>  snq <dig>  ict <dig>  gre <dig>  tpo <dig>  ymr102c, and ygr035c compared with its parental strain by <dig>  <dig> h after exposure to furfural and hmf .
the mutation Δpdr <dig> appeared to have a similar regulatory effect but to a lesser degree and to fewer genes .
however, it was clear that expression of pga <dig> was affected by Δpdr <dig> but not Δpdr <dig>
regulatory interactions of rpn <dig> and hsf1
among the genes induced by hmf, at least  <dig> ubiquitin-related and proteasome genes for protein degradation were identified .
these genes, by encoding enzymes involving in the degradation of damaged proteins, maintain cell viability and functions under the inhibitor stress.
the induction of these genes was predicted to be under the control of the transcription factor rpn4p by binding to the proteasome-associated control element   <cit> , and the pace was found in the promoter of most ubiquitin-related and proteasome genes induced by hmf .
in this study, rpn <dig> was continuously enhanced over time during the lag phase .
rpn4p levels are regulated by the  <dig> s proteasome via a negative feedback control mechanism  <cit> .
it is also required for regulation of genes involved in dna repair and other cellular processes, such as dna damage-inducible genes mag <dig> and ddi <dig>  <cit> .
interestingly, rpn4p is a feedback regulator of yap <dig> and pdr <dig>  <cit> .
the consistent expression of rpn <dig> and its known complex functions including regulatory functions indicated a significant role of this transcription factor gene in regulating genomic adaptation networks during the lag phase.
this was further demonstrated by the comparative performance of the deletion mutation response to hmf.
while it was able to grow and establish a culture normally without hmf challenge, the strain harboring Δrpn <dig> failed to recover in the presence of  <dig> mm hmf  <dig> days after incubation .
although the levels of induction of hsf <dig> were not as great as rpn <dig>  we found its constantly enhanced expression response to hmf was statistically significant.
up-regulated genes hsp <dig> and ssa <dig> for protein folding and refolding in this study have been reported to be regulated by hsf1p  <cit> .
it was also a positive regulator of other transcription factor genes rpn <dig>  pdr <dig>  yap <dig>  and yap <dig>  <cit> .
hsf <dig> is likely involved in the complex co-regulation networks to the hmf stress.
regulatory interactions of repressed genes
for  <dig> significantly repressed genes, we found at least  <dig> important regulatory genes were involved in the down-regulated expression.
for example, arg <dig>  arg <dig>  arg <dig>  arg <dig> , arg <dig>  and arg <dig> involved in arginine biosynthesis repressed by hmf were regulated by the transcription factor genes arg <dig> and arg <dig>  as well as gcn <dig> .
these transcription factor genes were reported to regulate arginine metabolism  <cit> .
all of these genes were found to be down-regulated under the hmf stress in this study.
in addition to regulation of arginine biosynthesis, gcn <dig> regulates expression of many other genes related to amino acid biosynthesis , identified by natarajan et al  <cit> .
numerous genes involved in biosynthesis of histidine, leucine, and lysine were repressed under the control of gcn <dig>  among the genes repressed by hmf, a large number of genes are involved in ribosome biogenesis and protein translation processes, which were predicted to be regulated by transcription factor genes rap <dig> and fhl <dig> .
at the same time, rap <dig> and fhl <dig> also showed repressed expression response.
deletion mutation response to hmf
all selective single gene deletion mutations displayed normal growth similar to their parental strain in the absence of hmf treatment on sc medium .
in the presence of hmf, the parental strain by <dig> showed a delayed growth response on sc medium.
in contrast, all tested deletion mutations for genes yap <dig>  rpn <dig>  pdr <dig>  pdr <dig>  yap <dig>  yap <dig>  yap <dig>  adh <dig>  adh <dig>  ald <dig>  snq <dig>  ict <dig>  shp <dig>  otu <dig>  met <dig>  met <dig>  cha <dig>  alt <dig>  ssa <dig>  oye <dig>  npl <dig>  mag <dig>  gre <dig>  gre <dig>  ari <dig>  ybr062c, and yer137c, displayed varied lengths of lag phase .
these represent growth defects at different levels in the absence of the individual genes.
among which, the most profound effect was observed by Δrpn <dig> and Δyap <dig> for transcription factor genes rpn <dig> and yap <dig> as mentioned above.
metabolic conversion profiles were highly consistent with the growth response.
as assayed by hplc, no glucose consumption was observed for all tested strains during the lag phase .
discussion
yeast adaptation to lignocellulose derived inhibitor stress is manifest at genome level and likely during the lag phase  <cit> .
variation in the length of the lag phase has been widely used to measure the tolerance of strains to a specific inhibitor.
using dna 70-mer long oligo microarray and qrt-pcr assays, we investigated comparative transcriptome profilings of s. cerevisiae during the lag phase under hmf challenge in a time-course study.
our comprehensive analyses uncovered important transcription factor genes, including yap <dig>  yap <dig>  yap <dig>  pdr <dig>  pdr <dig>  rpn <dig>  and hsf <dig>  as key regulators for the yeast adaptation, as well as their co-regulation and complicated regulatory networks with numerous multiple function genes.
we identified more than  <dig> genes showing statistically significant differential expression responses that potentially affect yeast adaptation to the inhibitor challenge.
among which, more than  <dig> genes were consistently induced and more than  <dig> genes were repressed at varied stages during the lag phase.
this is the first report of systematic analysis on genomic expression to inhibitor stress during the lag phase in the context of yeast adaptation.
knowledge obtained from this study provides insight into global adaptive responses of the yeast to inhibitor stress and aids the dissection of tolerance mechanisms of the yeast.
our studies uncovered at least three significant elements for yeast adaptation to inhibitor stress and mechanisms of tolerance.
the first component involves the functional enzymes and related regulatory networks directly involved in biotransformation and inhibitor detoxification.
at a sublethal dose, yeasts are able to convert hmf into less toxic compound fdm.
the in situ detoxification of hmf has been identified as a primary mechanism of the tolerance for yeast strains  <cit> .
this is mainly accomplished via the activity of functional reductase and numerous enzymes possessing nadh-dependent aldehyde reduction activities, such as enzyme encoding genes adh <dig>  adh <dig>  ald <dig>  ari <dig>  ari <dig>  ari <dig>  oye <dig>  gre <dig>  and gre <dig> .
in this study, we found adh <dig>  ari <dig>  gre <dig>  and ald <dig> were immediately induced by the addition of hmf, especially for adh <dig> which displayed a greater than 30-fold increase in transcription abundance  <dig> min after the hmf addition and 80-fold increase at  <dig> h. the expression of adh <dig> was regulated by yap1p, yap5p, yap6p, and pdr1p .
multiple layers of up-regulated expressions of adh <dig> provide strong support for its extremely high levels of induction.
on the other hand, it indicated the significant roles of adh <dig> in adaptation to the aldehyde inhibitor challenge and tolerance to the inhibitor.
most reductase genes are regulated by yap1p and related regulons yap5p and yap6p.
a few enzyme encoding genes for example, ald <dig> and gre <dig> were co-regulated by pdr1p.
it should be pointed out that multiple functions of a gene are common and the co-regulation can be a reflection of the multi-functions.
as mentioned above, conversion of aldehyde inhibitors including hmf, consumes cofactor nadh and redox imbalance often causes damage in cell metabolism.
we have previously demonstrated that tolerant yeast cells utilize reprogrammed pathways to detoxify aldehyde inhibitors and favored pentose phosphate pathway in regeneration of cofactors keeping a well maintained redox balance  <cit> .
in this study, we found the yeast, during the lag phase, appeared to facilitate a short path to the tca cycle from which energy and nadh regeneration can be achieved.
this involved genes in the amino acids metabolism pathways closely related to the tca cycle, both induced genes such as cha <dig>  alt <dig>  put <dig>  put <dig>  and car <dig>  and repressed genes such as arg <dig>  arg <dig>  arg <dig>  arg <dig> , arg <dig>  arg <dig>  lys <dig>  lys <dig>  and lys <dig> .
the accelerated catabolism of proline, serine, and alanine, together with the reduced biosynthesis of arginine likely provided a shortcut for atp regeneration via the tca cycle.
thus, efficient energy metabolism can be maintained under the hmf stress.
these findings suggest the altered pathway is an adaptation response that allows sufficient production of intermediate substrates for energy and nadh regeneration through the tca cycle under the hmf challenge.
many of these genes, for example, put <dig> and alt <dig> are regulated by yap <dig> and its related yap gene family.
yap1p has been reported as involved in the regulation of numerous other anti-oxidant genes  <cit> .
it also plays a significant role for dna damage repairing  <cit> .
the preferred yap1p binding site is ttactaa  <cit> .
we found many reductase genes that contribute to the biotransformation of the inhibitors have the yap1p binding site in their promoter regions and are likely regulons of yap1p.
the second element we found to be significant for yeast survival and adaptation under the hmf challenge is the pdr gene family-centered functions that are regulated by pdr1/3p and as well as other regulator genes such as yap <dig> and hsf <dig>  some pdr genes function as transporters of atp-binding cassette proteins and encode plasma membrane proteins.
these genes mediate membrane translocation of ions and a wide range of substrates and often exhibit multiple functions in response to a large variety of unrelated chemical stresses  <cit> .
in this study, we found at least  <dig> members of the pdr gene family were significantly induced by hmf.
the membrane and transporter activity related functions are mainly documented for these genes.
for example, tpo <dig> and tpo <dig> encode proteins to function as drug/toxin transport and multidrug efflux pumps  <cit> , rsb <dig> for transport atpase, and pdr <dig> for abc transporters, specifically.
other genes encode proteins that have multiple functions covering all of these categories, such as snq <dig>  yor <dig>  pdr <dig>  and pdr <dig> .
in addition, proteins encoded by these genes also perform functions of atp binding and other cytoplasmic and molecular functions.
confirmed by deletion mutation assays of cell growth and qrt-pcr, we reasonably speculate that abc transporters play a key role to export excessive hmf and endogenous toxic metabolites from intracellular environment brought about by hmf damage.
as mentioned above, the shortcut of the tca cycle could provide energy for the pumping of hmf and toxic metabolites by abc transporters.
in this group, we observed induced transcriptional response of rsb <dig> and ict <dig>  these two genes are involved in phospholipid synthesis and transportation for membrane structure and functions, and are responsible for tolerance to organic solvents in s. cerevisiae  <cit> .
it is possible that the induction of these pdr genes prevents the fast influx of hmf into cytoplasm and important organelles by membrane remodeling, thus, increasing the cell's tolerance to hmf.
mag <dig> encodes a 3-methyladenine  dna glycosylase  <cit> , which acts in the first step of a multistage base excision repair pathway for the removal of lethal lesions such as 3mea and protects yeast cells from killing by dna-alkylating agents  <cit> .
ddi <dig>  located immediately upstream of mag <dig> and transcribed in an opposite direction, encodes an ubiquitin-related protein and is involved in a dna-damage cell-cycle checkpoint  <cit> .
another dna damage related gene rad <dig> was also induced by hmf  <cit> .
the induction of mag <dig>  ddi <dig>  and rad <dig> in this study are consistent with the potential dna damage by hmf and yeast defense response to the hmf challenge.
regulatory interactions of pdr gene family are complex and many genes appeared to be regulated by multiple transcription factor genes involving pdr <dig>  pdr <dig>  yap <dig>  and hsf <dig>  regulatory roles of pdr <dig> and pdr <dig> to hmf challenge were suggested by computational modeling  <cit> .
our deletion mutation assays using qrt-pcr suggest pdr <dig> may have direct interactive effects with more induced genes than pdr <dig>  but pga <dig> appeared to be regulated by pdr <dig>  however, detailed interactions of the multiple functions of most pdr genes remain largely unknown, which can be related to the multiple regulated interactions by yap <dig>  pdr <dig>  pdr <dig>  and hsf <dig> as outlined by this study.
the third component of the yeast adaptation response to hmf involves degradation of damaged proteins and protein modifications mainly regulated by transcription factor genes rpn <dig> and hsf <dig>  chemical stress causes damage to protein conformation leading to protein unfolding and aggregation  <cit> .
small heat shock proteins, acting as chaperones, assist in folding or refolding nascent or denatured proteins and enzymes to maintain a functional conformation  <cit> .
in this study, we found hsp <dig> and ssa <dig> encoding chaperones were significantly induced to counteract hmf stress damage to proteins.
the deletion mutation of ssa <dig> displayed a significant longer lag phase under the hmf challenge, indicating its important role in adaptation and tolerance to hmf.
while the presence of chaperones provides positive contribution to protein protection, severe or prolonged stress condition can result in irreversible protein damage.
misfolded or damaged proteins, especially aggregated proteins are highly toxic to cells  <cit> .
degradation of misfolded and damaged proteins by the ubiquitin-mediated proteasome pathway plays an important role in maintaining normal cell function and viability  <cit> .
denatured proteins are targeted via the covalent attachment of ubiquitin to a lysine side chain, and polyubiquitinated proteins are finally delivered to proteasome to be degraded.
we observed that at least  <dig> ubiquitin-related and proteasome genes were induced by hmf , indicating their important functions in adaptation to the hmf stress.
strains with deletion mutations in these genes were sensitive to hmf with an extended lag phase, for example, genes otu <dig> and shp <dig>  it was suggested that the degradation of proteins by the ubiquitin-mediated proteasome pathway has regulatory roles on cell cycle, metabolic adaptations, gene regulation, development, and differentiation  <cit> .
as indicated by our study, many genes involved in the degradation of damaged protein and protein modifications are regulated by transcription factor gene rpn <dig>  our deletion mutation assays of rpn <dig> showed normal growth in the absence of hmf but no growth with the hmf treatment.
these results confirmed the vital role of rpn <dig> involvement in adaptation to survival and coping with the hmf challenge.
since hsf <dig> is an essential gene, no deletion mutant test was performed.
CONCLUSIONS
among  <dig> genes identified as differentially expressed under hmf challenges, both induced and repressed genes of multiple functional categories are associated with the yeast adaptation to the inhibitor hmf during the lag phase.
transcription factor genes yap <dig>  pdr <dig>  pdr <dig>  rpn <dig>  and hsf <dig> were identified as key regulatory genes for yeast global adaptation.
functional enzyme coding genes, for example ari <dig>  adh <dig>  adh <dig>  and oye <dig>  as well as gene interactions involved in the biotransformation and regulated by yap <dig>  are directly involved in the conversion of hmf into the less toxic compound fdm.
pdr genes encode plasma membrane proteins and function as transporter of atp-binding cassette proteins.
the large number of induced pdr genes observed by our study suggests a hypothesis of the important pdr function of pumping hmf and endogenous toxic metabolites to maintain cell viability.
important pdr gene functions include specific transporter atpase gene rsb <dig>  toxin transporter genes tpo <dig> and tpo <dig>  and multiple cellular transport facilitator genes pdr <dig>  pdr <dig>  pdr <dig>  yor <dig>  and snq <dig>  in addition, highly expressed genes involving degradation of damaged proteins and protein modifications regulated by rpn <dig>  hsf <dig>  and other co-regulators appear to be necessary for yeast survival and adaption to the hmf stress.
mutant strain Δrpn <dig> was unable to recover growth in the presence of hmf suggesting a significant regulatory role of rpn <dig> for many regulons.
complex gene interactions and regulatory networks as well as co-regulation events exist in response to the lignocellulose derived inhibitor hmf .
results from this study provide insight into mechanisms of adaptation and tolerance by the yeast saccharomyces cerevisiae that will directly aid continued engineering efforts for more tolerant yeast development.

 bioethanol production from lignocellulosic biomass including agricultural and forestry residues has attracted increased attention worldwide. lignocellulosic biomass needs to be depolymerized into simple sugars in order to be utilized for microbial fermentation. the commonly applied dilute acid pretreatment generates numerous chemical byproducts that inhibit cell growth and interfere with subsequent microbial fermentation. among numerous inhibitory compounds, furfural and 5-hydroxymethylfurfural are commonly encountered inhibitors. furfural and hmf are formed by dehydration of pentoses and hexoses released from hemicellulose and cellulose, respectively. these inhibitors can damage cell structures, inhibit cell growth, reduce enzymatic activities, generate cellular reactive oxygen species, break down dna, and inhibit protein and rna synthesis. the presence of fermentation inhibitors represents a bottle neck in cellulosic ethanol conversion technology and overcoming the inhibitor effect is one of the fundamental challenges to the industrial production of bioethanol from lignocellulosic biomass. furfural and its conversion product have been widely studied while knowledge of hmf conversion is limited due to a lack of commercial source of its conversion product. unlike evaporative furfural, hmf is more stable and difficult to degrade in cell culture. recently, an hmf metabolic conversion product was isolated and identified as 5-bis-hydroxymethylfuran. a dose-dependent response of yeast to hmf was demonstrated and a lag phase was used to measure levels of strain tolerance. the yeast saccharomyces cerevisiae is able to in situ detoxify hmf into the less toxic compound fdm through nadph-dependent reductions. typically, yeast strains show a lag of delayed cell growth after inhibitor challenge such as with furfural and hmf, under sublethal doses. once hmf and furfural inhibitor levels were chemically reduced to a certain lower concentration, cell growth recovered and the glucose-to-ethanol conversion accelerated at a faster rate than would normally occur. it was suggested that genomic adaptation occurred during the lag phase. in fact, inhibitor-tolerant yeast strains showed significant shorter lag phases under the inhibitor challenges compared with a wild type strain. gene expressions of selected pathways of the tolerant yeast are distinct from the wild type control. sequence mutations are common and a large number of single nucleotide polymorphism mutations were observed throughout all chromosomes for a tolerant yeast strain. adaptations appear to occur at the genome level. however, little is known about gene expression response and regulatory events for yeast during the adaptation lag phase. the objective of this study was to characterize transcriptome response of yeast during the lag phase after the hmf challenge. using a comparative time course study, we investigated the dynamics of transcriptome profiling during this critical stage applying dna microarray assays and regulatory analysis. important genes, together with transcription factors involved in the hmf stress response, were identified. the functions of selective candidate genes were verified by corresponding gene deletion mutation strains. significant regulatory interaction networks were uncovered during the genome adaptation in yeast. results of this study provide insight into mechanisms of yeast adaptation and tolerance to lignocellulose derived inhibitors. this will directly aid engineering efforts for more tolerant strain development.  cell growth response and metabolic conversion profiles compared to a non-treated control, yeast challenged by hmf displayed a significant drop in cell growth as measured by od absorbance h after the treatment. although the cell growth was recovered at a later time, cell density of the hmf-treated yeast was relatively low throughout the course of the study. similarly, glucose consumption for the hmf-treated culture was slower and glucose was depleted at h, approximately h later than the non-treated control. as expected, hmf was undetectable and fdm was detected as hmf conversion product in hmf-treated cultures less than h after incubation. no hmf or fdm was detected from the control culture. transcription expression dynamics during the lag phase clustering analysis distinguished significant differences for expression responses by hmf between the treated and untreated conditions over time. among the more than genes of the yeast genome, genes were identified as differentially expressed by anova for at least 2-fold changes during the lag phase of to min by the hmf challenge. among these, genes were induced constantly throughout the lag phase while genes were repressed at various stages of the lag phase. many of the induced genes showed immediate enhanced expressions within min after the hmf challenge. these genes mainly fall with functional categories of reductase, pleiotropic drug resistance, proteasome and ubiquitin, amino acids metabolism, stress response functions, and others. for example, adh encoding nadph-dependent medium chain alcohol dehydrogenase displayed the highest induction of more than 30-fold increase in mrna abundance at min after the hmf treatment. other significantly induced genes including ari gre pdr rsb put cha hsp ssa and oye which showed more than 10-fold mrna increase at various times during the lag phase. * genes in bold indicate their encoding proteins or enzymes are involved in more than one function the repressed genes are mainly involved in the functional categories of ribosome biogenesis, amino acid and derivative metabolic process, rna metabolic process, transport, and others. most of the genes encoding enzymes for arginine biosynthesis were severely repressed, such as arg arg arg arg, arg and arg. for the repressed genes, three types of dynamic responses were observed. a small group of two dozen genes showed transient inductions at min but quickly turned into repressed after min, such as pcl and pcl for glycogen metabolism, mal mal and mph for maltose utilization. another group of about genes were constantly repressed, and these were mainly in the functional categories of amino acid metabolism, such as arg arg arg arg, arg for arginine metabolism, his his and his for histidine metabolism, aro aro hom and hom for aromatic amino acid metabolism. the third group of the repressed genes were initially repressed at or min but recovered at later time points. this group of repressed genes fall within the categories of rrna processing, trna export, and ribosomal biogenesis such as nob pus rrp nop and cbf5; mitochondrial mrna maturase such as bi and bi3; vitamin b biosynthesis gene snz1; and telomere length maintenance gene yku. relevant transcription factors under the hmf challenge, we found that seven transcription factor genes, pdr pdr yap yap yap rpn and hsf displayed significant greater expression during the lag phase in response to the hmf challenge. except for hsf most transcription factor genes displayed greater than 2-fold increase after the hmf treatment. by the aid of t-profiler, yeastract database and interactive pathway analysis using genespring gx, we identified these genes as the most important transcription factor genes positively regulating gene expression response in adaptation to the hmf stress during the lag phase in yeast. we further analyzed protein binding motifs for these genes and found each transcription factor gene harbored protein binding motifs for pdr1p, pdr3p, yap1p, yap5p, yap6p, rpn4p, and hsf1p. dna binding motifs of pdr1/3p were found in promoter regions of pdr yap pdr and rpn4; yap1p binding sites in all six transcription factor genes except for pdr1; and hsf1p sites in all six genes except for pdr. except for pdr which had a single yap1p binding site, each of the other six transcription factor genes displayed multiple binding sites for multiple transcription factors. for example, rpn had binding sites of transcription factors, and pdr had sites for interactions involving multiple transcription factors apparently exist. for example, highly expressed rpn in this study was found to be regulated by yap1p, pdr1p, pdr3p, and hsf1p that supported by chip-chip data and microarray assay of transcription factor mutations. on the other hand, it also demonstrated positive feedback to its regulators of yap1p and pdr1p. the presence of dna binding motifs of a transcription factors' own in its promoter region, such as pdr yap and hsf, suggested a self-regulated expression. the highly induced expression of the seven transcription factor genes in response to the hmf challenge and multiple protein binding motifs across the transcription factors suggested co-regulation and interactions of multiple transcription factors under the stress. as for many repressed expression responses to hmf, we identified five transcription factor genes arg arg gcn fhl and rap that displayed down-regulated expressions. yap regulated gene expression networks among the seven transcription factor genes, yap displayed consistently higher inductions, a 2- to 3-fold increase during the lag phase. yap1p acts as a sensor for oxidative molecules, and activates the transcription response of anti-oxidant genes by recognizing yap1p response elements, 5'-tkactma-3', in the promoter region. a total of hmf-induced genes were found to have the yre sequence in their promoter region. many genes were confirmed to be regulated directly by yap or indirectly through yap and yap. most yap1-regulated genes were classified in the functional categories of redox metabolism, amino acid metabolism, stress response, dna repair, and others. for example, the highly induced oxidoreductase genes adh gre and oye were found as regulons of yap. adh and gre were also co-regulated by yap5p and yap6p. these two genes were among those confirmed as reductases actively involved in the hmf detoxification. ari a recently characterized aldehyde reductase contributing to detoxification of furfural and hmf, was found to be regulated by yap6p which is a regulon of yap in addition, yap and other yap gene family members were shown to co-regulate numerous genes in a wide range of functional categories such as pdr, heat shock protein, chaperones, amino acid metabolism, as well as other regulators. the significance of the role of the yap gene family in adaptation and tolerance to hmf is confirmed by growth responses of the deletion mutations. single yap gene deletion mutations were able to grow normally without hmf treatment. however, in the presence of mm hmf, mutations Δyap Δyap Δyap and Δyap showed delayed growth compared with their parental strain. among these, Δyap displayed a 4-day long lag phase, indicating a profound functional defect affected by the yap gene. pdr family and pdr1/ involved regulatory interactions among the significantly induced genes by hmf, at least genes were categorized into the pdr family. many genes displayed consistent induced expressions ranging from 3- to 30-fold increases during the lag phase. gene products of these increased transcripts were in the protein categories of drug/toxin transport for tpo and tpo transport atpase for rsb and abc transporters for pdr. snq yor pdr and pdr encoding proteins shared functions of all these three categories. in addition, many pdr proteins have functions such as atp binding and chemical agent resistance. most of these genes have the pleiotropic drug response element in their promoter regions. * proteins in bold indicate functions involved in more than one category hmf-induced transcription factor genes pdr and pdr regulate gene expression under a large variety of unrelated chemical stress conditions by binding to the pdre of target genes. both pdr1p and pdr3p recognize cgg triplets oriented in opposite directions to form an inverted repeat, and able to form homodimers or heterodimers to activate target gene expression. many induced genes regulated by pdr1p and/or pdr3p in this group are involved in export of both xenobiotic compounds and endogenous toxic metabolites using atp-binding cassette transporters, lipid composition of the plasma membrane, export of polyamines by polyamine transporters, dna repairing, and other functions. at least eight genes induced by hmf in this study were regulated by both pdr1p and pdr3p. pdr1p and pdr3p also recognize and activate other subsets of genes. pdr3p participates in certain processes that do not involve pdr1p, such as regulating dna damage-inducible genes mag and ddi. similarly, some genes are only regulated by pdr1p, such as rsb, adh and pre. we also found that the pdr promoter contains two pdres that can be autoregulated by itself in addition to being a regulon of pdr1p. pdr and pdr also demonstrated regulatory connections with a broad range of functional category genes as well as most active regulatory genes. pdr and pdr gene deletion mutations were assayed to confirm their influence on the expression of the potential regulons. when examined by qrt-pcr, mutant Δpdr displayed reduced transcriptional abundance for many genes, such as pdr pdr pdr yor snq ict gre tpo ymr102c, and ygr035c compared with its parental strain by h after exposure to furfural and hmf. the mutation Δpdr appeared to have a similar regulatory effect but to a lesser degree and to fewer genes. however, it was clear that expression of pga was affected by Δpdr but not Δpdr regulatory interactions of rpn and hsf1 among the genes induced by hmf, at least ubiquitin-related and proteasome genes for protein degradation were identified. these genes, by encoding enzymes involving in the degradation of damaged proteins, maintain cell viability and functions under the inhibitor stress. the induction of these genes was predicted to be under the control of the transcription factor rpn4p by binding to the proteasome-associated control element, and the pace was found in the promoter of most ubiquitin-related and proteasome genes induced by hmf. in this study, rpn was continuously enhanced over time during the lag phase. rpn4p levels are regulated by the s proteasome via a negative feedback control mechanism. it is also required for regulation of genes involved in dna repair and other cellular processes, such as dna damage-inducible genes mag and ddi. interestingly, rpn4p is a feedback regulator of yap and pdr. the consistent expression of rpn and its known complex functions including regulatory functions indicated a significant role of this transcription factor gene in regulating genomic adaptation networks during the lag phase. this was further demonstrated by the comparative performance of the deletion mutation response to hmf. while it was able to grow and establish a culture normally without hmf challenge, the strain harboring Δrpn failed to recover in the presence of mm hmf days after incubation. although the levels of induction of hsf were not as great as rpn we found its constantly enhanced expression response to hmf was statistically significant. up-regulated genes hsp and ssa for protein folding and refolding in this study have been reported to be regulated by hsf1p. it was also a positive regulator of other transcription factor genes rpn pdr yap and yap. hsf is likely involved in the complex co-regulation networks to the hmf stress. regulatory interactions of repressed genes for significantly repressed genes, we found at least important regulatory genes were involved in the down-regulated expression. for example, arg arg arg arg, arg and arg involved in arginine biosynthesis repressed by hmf were regulated by the transcription factor genes arg and arg as well as gcn. these transcription factor genes were reported to regulate arginine metabolism. all of these genes were found to be down-regulated under the hmf stress in this study. in addition to regulation of arginine biosynthesis, gcn regulates expression of many other genes related to amino acid biosynthesis, identified by natarajan et al. numerous genes involved in biosynthesis of histidine, leucine, and lysine were repressed under the control of gcn among the genes repressed by hmf, a large number of genes are involved in ribosome biogenesis and protein translation processes, which were predicted to be regulated by transcription factor genes rap and fhl. at the same time, rap and fhl also showed repressed expression response. deletion mutation response to hmf all selective single gene deletion mutations displayed normal growth similar to their parental strain in the absence of hmf treatment on sc medium. in the presence of hmf, the parental strain by showed a delayed growth response on sc medium. in contrast, all tested deletion mutations for genes yap rpn pdr pdr yap yap yap adh adh ald snq ict shp otu met met cha alt ssa oye npl mag gre gre ari ybr062c, and yer137c, displayed varied lengths of lag phase. these represent growth defects at different levels in the absence of the individual genes. among which, the most profound effect was observed by Δrpn and Δyap for transcription factor genes rpn and yap as mentioned above. metabolic conversion profiles were highly consistent with the growth response. as assayed by hplc, no glucose consumption was observed for all tested strains during the lag phase. discussion yeast adaptation to lignocellulose derived inhibitor stress is manifest at genome level and likely during the lag phase. variation in the length of the lag phase has been widely used to measure the tolerance of strains to a specific inhibitor. using dna 70-mer long oligo microarray and qrt-pcr assays, we investigated comparative transcriptome profilings of s. cerevisiae during the lag phase under hmf challenge in a time-course study. our comprehensive analyses uncovered important transcription factor genes, including yap yap yap pdr pdr rpn and hsf as key regulators for the yeast adaptation, as well as their co-regulation and complicated regulatory networks with numerous multiple function genes. we identified more than genes showing statistically significant differential expression responses that potentially affect yeast adaptation to the inhibitor challenge. among which, more than genes were consistently induced and more than genes were repressed at varied stages during the lag phase. this is the first report of systematic analysis on genomic expression to inhibitor stress during the lag phase in the context of yeast adaptation. knowledge obtained from this study provides insight into global adaptive responses of the yeast to inhibitor stress and aids the dissection of tolerance mechanisms of the yeast. our studies uncovered at least three significant elements for yeast adaptation to inhibitor stress and mechanisms of tolerance. the first component involves the functional enzymes and related regulatory networks directly involved in biotransformation and inhibitor detoxification. at a sublethal dose, yeasts are able to convert hmf into less toxic compound fdm. the in situ detoxification of hmf has been identified as a primary mechanism of the tolerance for yeast strains. this is mainly accomplished via the activity of functional reductase and numerous enzymes possessing nadh-dependent aldehyde reduction activities, such as enzyme encoding genes adh adh ald ari ari ari oye gre and gre. in this study, we found adh ari gre and ald were immediately induced by the addition of hmf, especially for adh which displayed a greater than 30-fold increase in transcription abundance min after the hmf addition and 80-fold increase at h. the expression of adh was regulated by yap1p, yap5p, yap6p, and pdr1p. multiple layers of up-regulated expressions of adh provide strong support for its extremely high levels of induction. on the other hand, it indicated the significant roles of adh in adaptation to the aldehyde inhibitor challenge and tolerance to the inhibitor. most reductase genes are regulated by yap1p and related regulons yap5p and yap6p. a few enzyme encoding genes for example, ald and gre were co-regulated by pdr1p. it should be pointed out that multiple functions of a gene are common and the co-regulation can be a reflection of the multi-functions. as mentioned above, conversion of aldehyde inhibitors including hmf, consumes cofactor nadh and redox imbalance often causes damage in cell metabolism. we have previously demonstrated that tolerant yeast cells utilize reprogrammed pathways to detoxify aldehyde inhibitors and favored pentose phosphate pathway in regeneration of cofactors keeping a well maintained redox balance. in this study, we found the yeast, during the lag phase, appeared to facilitate a short path to the tca cycle from which energy and nadh regeneration can be achieved. this involved genes in the amino acids metabolism pathways closely related to the tca cycle, both induced genes such as cha alt put put and car and repressed genes such as arg arg arg arg, arg arg lys lys and lys. the accelerated catabolism of proline, serine, and alanine, together with the reduced biosynthesis of arginine likely provided a shortcut for atp regeneration via the tca cycle. thus, efficient energy metabolism can be maintained under the hmf stress. these findings suggest the altered pathway is an adaptation response that allows sufficient production of intermediate substrates for energy and nadh regeneration through the tca cycle under the hmf challenge. many of these genes, for example, put and alt are regulated by yap and its related yap gene family. yap1p has been reported as involved in the regulation of numerous other anti-oxidant genes. it also plays a significant role for dna damage repairing. the preferred yap1p binding site is ttactaa. we found many reductase genes that contribute to the biotransformation of the inhibitors have the yap1p binding site in their promoter regions and are likely regulons of yap1p. the second element we found to be significant for yeast survival and adaptation under the hmf challenge is the pdr gene family-centered functions that are regulated by pdr1/3p and as well as other regulator genes such as yap and hsf some pdr genes function as transporters of atp-binding cassette proteins and encode plasma membrane proteins. these genes mediate membrane translocation of ions and a wide range of substrates and often exhibit multiple functions in response to a large variety of unrelated chemical stresses. in this study, we found at least members of the pdr gene family were significantly induced by hmf. the membrane and transporter activity related functions are mainly documented for these genes. for example, tpo and tpo encode proteins to function as drug/toxin transport and multidrug efflux pumps, rsb for transport atpase, and pdr for abc transporters, specifically. other genes encode proteins that have multiple functions covering all of these categories, such as snq yor pdr and pdr. in addition, proteins encoded by these genes also perform functions of atp binding and other cytoplasmic and molecular functions. confirmed by deletion mutation assays of cell growth and qrt-pcr, we reasonably speculate that abc transporters play a key role to export excessive hmf and endogenous toxic metabolites from intracellular environment brought about by hmf damage. as mentioned above, the shortcut of the tca cycle could provide energy for the pumping of hmf and toxic metabolites by abc transporters. in this group, we observed induced transcriptional response of rsb and ict these two genes are involved in phospholipid synthesis and transportation for membrane structure and functions, and are responsible for tolerance to organic solvents in s. cerevisiae. it is possible that the induction of these pdr genes prevents the fast influx of hmf into cytoplasm and important organelles by membrane remodeling, thus, increasing the cell's tolerance to hmf. mag encodes a 3-methyladenine dna glycosylase, which acts in the first step of a multistage base excision repair pathway for the removal of lethal lesions such as 3mea and protects yeast cells from killing by dna-alkylating agents. ddi located immediately upstream of mag and transcribed in an opposite direction, encodes an ubiquitin-related protein and is involved in a dna-damage cell-cycle checkpoint. another dna damage related gene rad was also induced by hmf. the induction of mag ddi and rad in this study are consistent with the potential dna damage by hmf and yeast defense response to the hmf challenge. regulatory interactions of pdr gene family are complex and many genes appeared to be regulated by multiple transcription factor genes involving pdr pdr yap and hsf regulatory roles of pdr and pdr to hmf challenge were suggested by computational modeling. our deletion mutation assays using qrt-pcr suggest pdr may have direct interactive effects with more induced genes than pdr but pga appeared to be regulated by pdr however, detailed interactions of the multiple functions of most pdr genes remain largely unknown, which can be related to the multiple regulated interactions by yap pdr pdr and hsf as outlined by this study. the third component of the yeast adaptation response to hmf involves degradation of damaged proteins and protein modifications mainly regulated by transcription factor genes rpn and hsf chemical stress causes damage to protein conformation leading to protein unfolding and aggregation. small heat shock proteins, acting as chaperones, assist in folding or refolding nascent or denatured proteins and enzymes to maintain a functional conformation. in this study, we found hsp and ssa encoding chaperones were significantly induced to counteract hmf stress damage to proteins. the deletion mutation of ssa displayed a significant longer lag phase under the hmf challenge, indicating its important role in adaptation and tolerance to hmf. while the presence of chaperones provides positive contribution to protein protection, severe or prolonged stress condition can result in irreversible protein damage. misfolded or damaged proteins, especially aggregated proteins are highly toxic to cells. degradation of misfolded and damaged proteins by the ubiquitin-mediated proteasome pathway plays an important role in maintaining normal cell function and viability. denatured proteins are targeted via the covalent attachment of ubiquitin to a lysine side chain, and polyubiquitinated proteins are finally delivered to proteasome to be degraded. we observed that at least ubiquitin-related and proteasome genes were induced by hmf, indicating their important functions in adaptation to the hmf stress. strains with deletion mutations in these genes were sensitive to hmf with an extended lag phase, for example, genes otu and shp it was suggested that the degradation of proteins by the ubiquitin-mediated proteasome pathway has regulatory roles on cell cycle, metabolic adaptations, gene regulation, development, and differentiation. as indicated by our study, many genes involved in the degradation of damaged protein and protein modifications are regulated by transcription factor gene rpn our deletion mutation assays of rpn showed normal growth in the absence of hmf but no growth with the hmf treatment. these results confirmed the vital role of rpn involvement in adaptation to survival and coping with the hmf challenge. since hsf is an essential gene, no deletion mutant test was performed.  among genes identified as differentially expressed under hmf challenges, both induced and repressed genes of multiple functional categories are associated with the yeast adaptation to the inhibitor hmf during the lag phase. transcription factor genes yap pdr pdr rpn and hsf were identified as key regulatory genes for yeast global adaptation. functional enzyme coding genes, for example ari adh adh and oye as well as gene interactions involved in the biotransformation and regulated by yap are directly involved in the conversion of hmf into the less toxic compound fdm. pdr genes encode plasma membrane proteins and function as transporter of atp-binding cassette proteins. the large number of induced pdr genes observed by our study suggests a hypothesis of the important pdr function of pumping hmf and endogenous toxic metabolites to maintain cell viability. important pdr gene functions include specific transporter atpase gene rsb toxin transporter genes tpo and tpo and multiple cellular transport facilitator genes pdr pdr pdr yor and snq in addition, highly expressed genes involving degradation of damaged proteins and protein modifications regulated by rpn hsf and other co-regulators appear to be necessary for yeast survival and adaption to the hmf stress. mutant strain Δrpn was unable to recover growth in the presence of hmf suggesting a significant regulatory role of rpn for many regulons. complex gene interactions and regulatory networks as well as co-regulation events exist in response to the lignocellulose derived inhibitor hmf. results from this study provide insight into mechanisms of adaptation and tolerance by the yeast saccharomyces cerevisiae that will directly aid continued engineering efforts for more tolerant yeast development. 
complex gene interactions and regulatory networks as well as co-regulations exist in yeast adaptation and tolerance to the lignocellulose derived inhibitor hmf. the length of lag phase for cell growth in response to the inhibitor challenge has been used to measure tolerance of strain performance. using systems biology approach, this study investigated comparative transcriptome profiling, metabolic profiling, cell growth response, and gene regulatory interactions of yeast strains and selective gene deletion mutations in response to hmf challenges during the lag phase of growth. second, a large number of pdr genes, mainly regulated by pdr and pdr were induced during the lag phase and the pdr gene family-centered functions, including specific and multiple functions involving cellular transport such as tpo tpo rsb pdr pdr yor and snq promoted cellular adaptation and survival in order to cope with the inhibitor stress. first, functional enzyme coding genes such as ari adh adh and oye as well as gene interactions involved in the biotransformation and inhibitor detoxification were the direct driving force to reduce hmf damages in cells. third, expressed genes involving degradation of damaged proteins and protein modifications such as shp and ssa regulated by rpn hsf and other co-regulators, were necessary for yeast cells to survive and adapt the hmf stress. the yeast saccharomyces cerevisiae is able to adapt and in situ detoxify lignocellulose derived inhibitors such as furfural and hmf. transcription factor genes yap pdr pdr rpn and hsf appeared to play key regulatory rules for global adaptation in the yeast s. cerevisiae. we identified candidate genes and found at least significant components involving some of these genes that enable yeast adaptation and tolerance to hmf in yeast. both induced and repressed genes involving diversified functional categories are accountable for adaptation and energy rebalancing in yeast to survive and adapt the hmf stress during the lag phase of growth. mechanisms of yeast tolerance at the genome level remain unknown.
 the yeast saccharomyces cerevisiae is able to adapt and in situ detoxify lignocellulose derived inhibitors such as furfural and hmf. the length of lag phase for cell growth in response to the inhibitor challenge has been used to measure tolerance of strain performance. mechanisms of yeast tolerance at the genome level remain unknown. using systems biology approach, this study investigated comparative transcriptome profiling, metabolic profiling, cell growth response, and gene regulatory interactions of yeast strains and selective gene deletion mutations in response to hmf challenges during the lag phase of growth.  we identified candidate genes and found at least significant components involving some of these genes that enable yeast adaptation and tolerance to hmf in yeast. first, functional enzyme coding genes such as ari adh adh and oye as well as gene interactions involved in the biotransformation and inhibitor detoxification were the direct driving force to reduce hmf damages in cells. expressions of these genes were regulated by yap and its closely related regulons. second, a large number of pdr genes, mainly regulated by pdr and pdr were induced during the lag phase and the pdr gene family-centered functions, including specific and multiple functions involving cellular transport such as tpo tpo rsb pdr pdr yor and snq promoted cellular adaptation and survival in order to cope with the inhibitor stress. third, expressed genes involving degradation of damaged proteins and protein modifications such as shp and ssa regulated by rpn hsf and other co-regulators, were necessary for yeast cells to survive and adapt the hmf stress. a deletion mutation strain Δrpn was unable to recover the growth in the presence of hmf.  complex gene interactions and regulatory networks as well as co-regulations exist in yeast adaptation and tolerance to the lignocellulose derived inhibitor hmf. both induced and repressed genes involving diversified functional categories are accountable for adaptation and energy rebalancing in yeast to survive and adapt the hmf stress during the lag phase of growth. transcription factor genes yap pdr pdr rpn and hsf appeared to play key regulatory rules for global adaptation in the yeast s. cerevisiae. 