Uncovering transcriptional regulation of glycerol metabolism in Aspergilli through genome-wide gene expression data analysis

  • Margarita Salazar
  • Wanwipa Vongsangnak
  • Gianni Panagiotou
  • Mikael R. Andersen
  • Jens NielsenEmail author
Original Paper


Glycerol is catabolized by a wide range of microorganisms including Aspergillus species. To identify the transcriptional regulation of glycerol metabolism in Aspergillus, we analyzed data from triplicate batch fermentations of three different Aspergilli (Aspergillus nidulans, Aspergillus oryzae and Aspergillus niger) with glucose and glycerol as carbon sources. Protein comparisons and cross-analysis with gene expression data of all three species resulted in the identification of 88 genes having a conserved response across the three Aspergilli. A promoter analysis of the up-regulated genes led to the identification of a conserved binding site for a putative regulator to be 5′-TGCGGGGA-3′, a binding site that is similar to the binding site for Adr1 in yeast and humans. We show that this Adr1 consensus binding sequence was over-represented on promoter regions of several genes in A. nidulans, A. oryzae and A. niger. Our transcriptome analysis indicated that genes involved in ethanol, glycerol, fatty acid, amino acids and formate utilization are putatively regulated by Adr1 in Aspergilli as in Saccharomyces cerevisiae and this transcription factor therefore is likely to be cross-species conserved among Saccharomyces and distant Ascomycetes. Transcriptome data were further used to evaluate the high osmolarity glycerol pathway. All the components of this pathway present in yeast have orthologues in the three Aspergilli studied and its gene expression response suggested that this pathway functions as in S. cerevisiae. Our study clearly demonstrates that cross-species evolutionary comparisons among filamentous fungi, using comparative genomics and transcriptomics, are a powerful tool for uncovering regulatory systems.


Aspergillus species Glycerol metabolism Transcriptional regulation 



The authors would like to thank Tina Johansen, Pia Friis and Lene Christiansen at Technical University of Denmark for assistance with the experimental work and Dr. Kim Hansen for supervising the fermentations with A. oryzae, Lone Vuholm and Anne Kejser Jensen at Novozymes for technical assistance. We would like to thank National Council of Research Conacyt-Mexico and Chalmers University of Technology for financial support to MS; Novozymes Bioprocess Academy and Chalmers University of Technology for financial support to WV; Danish Research Council for Technology and Production Sciences and Lundbeck Foundation for financial support to GP and Danish Research Agency for Technology and Production for financial support to MRA. We thank Dr. Gerald Hofmann for revision of the manuscript and good scientific discussion.

Supplementary material

438_2009_486_MOESM1_ESM.pdf (3.5 mb)
A. nidulans differentially expressed genes mapped to metabolic map of A. oryzae resulting from glucose versus glycerol t-test analysis (PDF 3.54 MB)
438_2009_486_MOESM2_ESM.pdf (3.5 mb)
A. oryzae differentially expressed genes mapped to metabolic map of A. oryzae resulting from glucose versus glycerol t-test analysis (PDF 3.54 MB)
438_2009_486_MOESM3_ESM.pdf (3.5 mb)
A. niger differentially expressed genes mapped to metabolic map of A. oryzae resulting from glucose versus glycerol t-test analysis (PDF 3.54 MB)
438_2009_486_MOESM4_ESM.pdf (90 kb)
Significant genes differentially expressed and mapped to the metabolic maps of A. niger and A. oryzae. Selected pathways included: central carbon metabolism, TCA cycle, C2 and C3 carbon metabolism and fatty acid metabolism. Complete metabolic maps of A. nidulans, A. oryzae and A. niger, using A. oryzae as a template are included in Supplementary Figs 1, 2 and 3, respectively. The abbreviation of metabolites is described as follows. C2 metabolism: ETH, ethanol; AC, acetate, ACAL, acetaldehyde; ACCOA, acetyl-CoA. C3 metabolism: GL, glycerol; GLYAL, D-glyceraldehyde; GLYN, glycerone; GL3P, sn-glycerol 3-phosphate; T3P2, glycerone phosphate. Pyruvate metabolism: F6P, Beta-D-fructose 6-phosphate; FDP, Beta-D-fructose 1,6-bisphosphate; T3P1, D-glyceraldehyde 3-phosphate; 13PDG, 1,3-Bisphospho-D-glycerate; 3PG, 3-Phospho-D-glycerate 2PG, 2-Phospho-D-glycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; MTHGXL, methylglyoxal; RGT, glutathione; LACAL, D-lactaldehyde; LAC, D-lactate; LGT, (R)-S-lactoylglutathione; LLAC, L-lactate. TCA cycle: OA, oxaloacetate; CIT, citrate; ACO, Cis-aconitate; ICIT, isocitrate AKG, 2-oxoglutarate; SUCCOA, succinyl coenzyme A; SUCC, succinate; FUM, fumarate; MAL, (S)-malate; GABAL, 4-aminobutyraldehyde; GABA, 4-aminobutanoate; GLU, L-glutamate; SUCCSAL, succinate semialdehyde. Fatty acid catabolism: C120COA, dodecanoyl-Coenzyme A; C120CAR, dodecanoyl-carnitine; C12DCOA, dodecanoyl-dehydro-Coenzyme A; C12HCOA, dodecanoyl-Hydroxy-Coenzyme A; C12OCOA, dodecanoyl-oxo-Coenzyme A; C140COA, myristoyl-Coenzyme A; C140CAR, myristoyl-carnitine; C14DCOA, myristoyl-dehydro-Coenzyme A; C14HCOA, myristoyl-Hydroxy-Coenzyme A; C14OCOA, myristoyl-oxo-Coenzyme A; C160COA, hexadecanoyl-Coenzyme A; C160CAR, hexadecanoyl-carnitine; C16DCOA, hexadecanoyl-dehydro-Coenzyme A; C16HCOA, hexadecanoyl-Hydroxy-Coenzyme A; C160COA, hexadecanoyl-Coenzyme A; C180COA, stearoyl-Coenzyme A; C180CAR, octadecanoyl-carnitine; C18DCOA, stearoyl-dehydro-Coenzyme A; C18HCOA, stearoyl-Hydroxy-Coenzyme A; C180COA, Stearoyl-oxo-Coenzyme A. Extracellular metabolites are designated by subscript ‘e’; mitochondrial metabolites by subscript ‘m’ (PDF 90.2 KB)
438_2009_486_MOESM5_ESM.pdf (204 kb)
Supplementary Table 1. (PDF 204 KB)
438_2009_486_MOESM6_ESM.xls (50 kb)
Supplementary Table 2. (XLS 50 KB)
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Supplementary Table 3. (XLS 457 KB)
438_2009_486_MOESM8_ESM.xls (44 kb)
Supplementary Table 4. (XLS 43.5 KB)
438_2009_486_MOESM9_ESM.pdf (25 kb)
Supplementary Table 5. (PDF 24.6 KB)
438_2009_486_MOESM10_ESM.xls (34 kb)
Supplementary Table 6. (XLS 34.0 KB)
438_2009_486_MOESM11_ESM.xls (274 kb)
Supplementary Table 7. (XLS 274 KB)


  1. Affymetrix: GeneChip expression analysis technical manual, with specific protocols for using the GeneChip hybridization, wash, and stain kit (2007) P/N 702232, Affymetrix, Santa Clara, CA, Revision 2Google Scholar
  2. Albertyn J, Hohmann S, Thevelein JM et al (1994) GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic-stress in Saccharomyces cerevisiae, and its expression is regulated by the high osmolarity glycerol response pathway. Mol Cell Biol 14:4135–4144PubMedGoogle Scholar
  3. Alexa A, Rahnenfuhrer J, Lengauer T (2006) Improved scoring of functional groups from gene expression data by decorrelating GO graph structure. Bioinformatics 22:1600–1607CrossRefPubMedGoogle Scholar
  4. Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  5. Andersen MR, Vongsangnak W, Panagiotou G et al (2008) A trispecies Aspergillus microarray: comparative transcriptomics of three Aspergillus species. Proc Natl Acad Sci USA 105:4387–4392CrossRefPubMedGoogle Scholar
  6. Appleyard M, McPheat WL, Stark MJR (2000) A novel ‘two-component’ protein containing histidine kinase and response regulator domains required for sporulation in Aspergillus nidulans. Curr Genet 37:364–372CrossRefPubMedGoogle Scholar
  7. Beever RE, Laracy EP (1986) Osmotic adjustment in the filamentous fungus Aspergillus nidulans. J Bacteriol 168:1358–1365PubMedGoogle Scholar
  8. Bembom O, Keles S, van der Laan MJ (2007) Supervised detection of conserved motifs in DNA sequences with cosmo. Stat Appl Genet Mol Biol 6:Article 8Google Scholar
  9. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate—a practical and powerful approach to multiple testing. J R Stat Soc B Methodol 57:289–300Google Scholar
  10. Blomberg A, Adler L (1992) Physiology of osmotolerance in fungi. Adv Microb Physiol 33:145–212CrossRefPubMedGoogle Scholar
  11. Blumenstein A, Vienken K, Tasler R et al (2005) The Aspergillus nidulans phytochrome FphA represses sexual development in red light. Curr Biol 15:1833–1838CrossRefPubMedGoogle Scholar
  12. Carlsen M, Nielsen J (2001) Influence of carbon source on alpha-amylase production by Aspergillus oryzae. Appl Microbiol Biotechnol 57:346–349PubMedGoogle Scholar
  13. Cheng C, Kacherovsky N, Dombek KM et al (1994) Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1. Mol Cell Biol 14:3842–3852PubMedGoogle Scholar
  14. Das HK, Baez ML (2008) ADR1 interacts with a down-stream positive element to activate PS1 transcription. Front Biosci 13:3439–3447CrossRefPubMedGoogle Scholar
  15. David H, Hofmann G, Oliveira A et al (2006) Metabolic network driven analysis of genome-wide transcription data from Aspergillus nidulans. Genome Biol 7(11):R108Google Scholar
  16. DeRisi JL, Iyer VR, Brown PO (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278:680–686CrossRefPubMedGoogle Scholar
  17. Dudoit S, Gendeman RC, Quackenbush J (2003) Open source software for the analysis of microarray data. Biotechniques (Suppl):45–51Google Scholar
  18. Felenbok B, Kelly JM (1996) Regulation of carbon metabolism in mycelia fungi. In: Marzluf G, Brambl R (eds) The Mycota: III: Biochemistry and molecular biology. Springer, Berlin, pp 369–380Google Scholar
  19. Fujimura M, Ochiai N, Oshima M et al (2003) Putative homologs of SSK22 MAPKK kinase and PBS2 MAPK kinase of Saccharomyces cerevisiae encoded by os-4 and os-5 genes for osmotic sensitivity and fungicide resistance in Neurospora crassa. Biosci Biotechnol Biochem 67:186–191CrossRefPubMedGoogle Scholar
  20. Furukawa K, Hoshi Y, Maeda T et al (2005) Aspergillus nidulans HOG pathway is activated only by two-component signalling pathway in response to osmotic stress. Mol Microbiol 56:1246–1261CrossRefPubMedGoogle Scholar
  21. Gentleman RC, Carey VJ, Bates DM et al (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5(10):R80Google Scholar
  22. Han KH, Prade RA (2002) Osmotic stress-coupled maintenance of polar growth in Aspergillus nidulans. Mol Microbiol 43:1065–1078CrossRefPubMedGoogle Scholar
  23. Hohmann S (2002) Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66(2):300–372CrossRefPubMedGoogle Scholar
  24. Hondmann DHA, Busink R, Witteveen CFB et al (1991) Glycerol catabolism in Aspergillus nidulans. J Gen Microbiol 137:629–636PubMedGoogle Scholar
  25. Hynes MJ, Murray SL, Duncan A et al (2006) Regulatory genes controlling fatty acid catabolism and peroxisomal functions in the filamentous fungus Aspergillus nidulans. Eukaryotic Cell 5:794–805CrossRefPubMedGoogle Scholar
  26. Hynes MJ, Murray SL, Khew GS et al (2008) Genetic analysis of the role of peroxisomes in the utilization of acetate and fatty acids in Aspergillus nidulans. Genetics 178:1355–1369CrossRefPubMedGoogle Scholar
  27. Irizarry RA, Hobbs B, Collin F et al (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4:249–264CrossRefPubMedGoogle Scholar
  28. Kobayashi T, Abe K, Asai K et al (2007) Genomics of Aspergillus oryzae. Biosci Biotechnol Biochem 71:646–670CrossRefPubMedGoogle Scholar
  29. Maeda T, Takekawa M, Saito H (1995) Activation of yeast Pbs2 MAPKK by MAPKKKs of by binding of an SH3-containing osmosensor. Science 269:554–558CrossRefPubMedGoogle Scholar
  30. Maggio-Hall LA, Keller NP (2004) Mitochondrial beta-oxidation in Aspergillus nidulans. Mol Microbiol 54:1173–1185CrossRefPubMedGoogle Scholar
  31. Norbeck J, Pahlman AK, Akhtar N et al (1996) Purification and characterization of two isoenzymes of DL-glycerol-3-phosphatase from Saccharomyces cerevisiae—identification of the corresponding GPP1 and GPP2 genes and evidence for osmotic regulation of Gpp2p expression by the osmosensing mitogen-activated protein kinase signal transduction pathway. J Biol Chem 271:13875–13881CrossRefPubMedGoogle Scholar
  32. Panagiotou G, Andersen MR, Grotkjaer T et al (2008) Systems analysis unfolds the relationship between the phosphoketolase pathway and growth in Aspergillus nidulans. PLoS ONE 3:3847CrossRefGoogle Scholar
  33. Pedersen H, Beyer M, Nielsen J (2000) Glucoamylase production in batch, chemostat and fed-batch cultivations by an industrial strain of Aspergillus niger. Appl Microbiol Biotechnol 53:272–277CrossRefPubMedGoogle Scholar
  34. Posas F, WurglerMurphy SM, Maeda T et al (1996) Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 ‘‘two-component’’ osmosensor. Cell 86:865–875CrossRefPubMedGoogle Scholar
  35. R Development Core Team (2007) A Language and Environment for Statistical ComputingGoogle Scholar
  36. Roberts GG, Hudson AP (2006) Transcriptome profiling of Saccharomyces cerevisiae during a transition from fermentative to glycerol-based respiratory growth reveals extensive metabolic and structural remodeling. Mol Genet Genomics 276:170–186CrossRefPubMedGoogle Scholar
  37. Ronnow B, Kielland-Brandt MC (1993) GUT2, a gene for mitochondrial glycerol 3-phosphate dehydrogenase of Saccharomyces cerevisiae. Yeast 9:1121–1130CrossRefPubMedGoogle Scholar
  38. Ruijter GJG, Visser J (1997) Carbon repression in Aspergilli. FEMS Microbiol Lett 151:103–114CrossRefPubMedGoogle Scholar
  39. Schneider TD, Stephens RM (1990) Sequence logos—a new way to display consensus sequences. Nucleic Acids Res 18:6097–6100CrossRefPubMedGoogle Scholar
  40. Shani N, Valle D (1996) A Saccharomyces cerevisiae homolog of the human adrenoleukodystrophy transporter is a heterodimer of two half ATP-binding cassette transporters. Proc Natl Acad Sci USA 93:11901–11906CrossRefPubMedGoogle Scholar
  41. Smyth G (2004) Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3:Article 3Google Scholar
  42. Strauss J, Mach RL, Zeilinger S et al (1995) Cre1, the carbon catabolite repressor protein from Trichoderma reesei. FEBS Lett 376:103–107CrossRefPubMedGoogle Scholar
  43. Suzuki A, Kanamaru K, Azuma N et al (2008) GFP-Tagged expression analysis revealed that some histidine kinases of Aspergillus nidulans show temporally and spatially different expression during the life cycle. Biosci Biotechnol Biochem 72:428–434CrossRefPubMedGoogle Scholar
  44. van Helden J, Andre B, Collado-Vides J (1998) Extracting regulatory sites from the upstream region of yeast genes by computational analysis of oligonucleotide frequencies. J Mol Biol 281:827–842CrossRefPubMedGoogle Scholar
  45. Visser J, Vanrooijen R, Dijkema C et al (1988) Glycerol uptake mutants of the hyphal fungus Aspergillus nidulans. J Gen Microbiol 134:655–659PubMedGoogle Scholar
  46. Vongsangnak W, Olsen P, Hansen K et al (2008) Improved annotation through genome-scale metabolic modeling of Aspergillus oryzae. BMC Genomics 9:14CrossRefGoogle Scholar
  47. Vongsangnak W, Salazar M, Hansen K et al (2009) Genome-wide analysis of maltose utilization and regulation in aspergilli. Microbiology (in press)Google Scholar
  48. Wei HJ, Requena N, Fischer R (2003) The MAPKK kinase SteC regulates conidiophore morphology and is essential for heterokaryon formation and sexual development in the homothallic fungus Aspergillus nidulans. Mol Microbiol 47:1577–1588CrossRefPubMedGoogle Scholar
  49. Witteveen CFB, Visser J (1995) Polyols pools in Aspergillus niger. FEMS Microbiol Lett 134:57–62CrossRefPubMedGoogle Scholar
  50. Witteveen CFB, Vandevondervoort P, Dijkema C et al (1990) Characterization of a glycerol kinase mutant of Aspergillus niger. J Gen Microbiol 136:1299–1305PubMedGoogle Scholar
  51. Workman C, Jensen LJ, Jarmer H et al (2002) A new non-linear normalization method for reducing variability in DNA microarray experiments. Genome Biol 3(9)Google Scholar
  52. Young ET, Kacherovsky N, Van Riper K (2002) Snf1 protein kinase regulates Adr1 binding to chromatin but not transcription activation. J Biol Chem 277:38095–38103CrossRefPubMedGoogle Scholar
  53. Young ET, Dombek KM, Tachibana C et al (2003) Multiple pathways are co-regulated by the protein kinase Snf1 and the transcription factors Adr1 and Cat8. J Biol Chem 278:26146–26158CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Margarita Salazar
    • 1
  • Wanwipa Vongsangnak
    • 1
  • Gianni Panagiotou
    • 2
  • Mikael R. Andersen
    • 2
  • Jens Nielsen
    • 1
    Email author
  1. 1.Department of Chemical and Biological EngineeringChalmers University of TechnologyGöteborgSweden
  2. 2.Department of Systems Biology, Center for Microbial BiotechnologyTechnical University of DenmarkKongens LyngbyDenmark

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