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Applied Microbiology and Biotechnology

, Volume 99, Issue 3, pp 1375–1388 | Cite as

The transcriptomic profile of Pseudozyma aphidis during production of mannosylerythritol lipids

  • Michael Günther
  • Christian Grumaz
  • Stefan Lorenz
  • Philip Stevens
  • Elena Lindemann
  • Thomas Hirth
  • Kai Sohn
  • Susanne Zibek
  • Steffen Rupp
Genomics, transcriptomics, proteomics

Abstract

The basidiomycetous fungus Pseudozyma aphidis is able to convert vegetable oils to abundant amounts of the biosurfactant mannosylerythritol lipid (MEL) with a unique product pattern of MEL-A, MEL-B, MEL-C, and MEL-D. To investigate the metabolism of MEL production, we analyzed the transcriptome of P. aphidis DSM 70725 under MEL-inducing and non-inducing conditions using deep sequencing. Following manual curation of the previously described in silico gene models based on RNA-Seq data, we were able to generate an experimentally verified gene annotation containing 6347 genes. Using this database, our expression analysis revealed that only four of the five cluster genes required for MEL synthesis were clearly induced by the presence of soybean oil. The acetyltransferase encoding gene PaGMAT1 was expressed on a much lower level, which may explain the secretion of MEL with different degrees of acetylation in P. aphidis. In parallel to MEL synthesis, microscopic observations showed morphological changes accompanied by expression of genes responsible for cell development, indicative of a coregulation between MEL synthesis and cell morphology. In addition a set of transcription factors was identified which may be responsible for regulation of MEL synthesis and cell development. The upregulation of genes required for nitrogen metabolism and other assimilation processes indicate additional metabolic pathways required under the MEL-inducing conditions used. We also searched for a conserved gene cluster for cellobiose lipids (CL) but only found seven genes with limited homology distributed over the genome. However, we detected characteristic TLC spots in fermentations using P. aphidis DSM 70725, indicative of CL secretion.

Keywords

Transcriptome Pseudozyma aphidis Mannosylerythritol lipids MEL gene cluster Cellobiose lipids Morphologic switch 

Notes

Acknowledgments

This work was funded by an ERA-NET grant (no. 0315928A, ERA-IB10.039, “BioSurf—Novel Production Strategies for Biosurfactants”). It was further supported by a PhD scholarship of the “Deutsche Bundestiftung Umwelt” (DBU).

Supplementary material

253_2014_6359_MOESM1_ESM.pdf (451 kb)
ESM 1 (PDF 450 kb)

References

  1. Anderson MD, Che P, Song J, Nikolau BJ, Wurtele ES (1998) 3-Methylcrotonyl-coenzyme A carboxylase is a component of the mitochondrial leucine catabolic pathway in plants. Plant Physiol 118:1127–1138. doi: 10.1104/pp. 118.4.1127 PubMedCentralPubMedCrossRefGoogle Scholar
  2. Arutchelvi JI, Bhaduri S, Uppara PV, Doble M (2008) Mannosylerythritol lipids: a review. J Ind Microbiol Biotechnol 35:1559–1570. doi: 10.1007/s10295-008-0460-4 PubMedCrossRefGoogle Scholar
  3. Bölker M, Basse CW, Schirawski J (2008) Ustilago maydis secondary metabolism—from genomics to biochemistry. Fungal Genet Biol 45(Suppl 1):88–93. doi: 10.1016/j.fgb.2008.05.007 CrossRefGoogle Scholar
  4. Boothroyd B, Thorn JA, Haskins RH (1955) Biochemistry of the Ustilaginales X. The biosynthesis of ustilagic acid. Can J Biochem Physiol 33:289–296. doi: 10.1139/o55-039 PubMedCrossRefGoogle Scholar
  5. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676. doi: 10.1093/bioinformatics/bti610 PubMedCrossRefGoogle Scholar
  6. Cosgrove DJ (1996) Plant cell enlargement and the action of expansins. Bioessays 18:533–540. doi: 10.1002/bies.950180704 PubMedCrossRefGoogle Scholar
  7. Elìas-Villalobos A, Fernández-Álvarez A, Ibeas JI (2011) The general transcriptional repressor Tup1 is required for dimorphism and virulence in a fungal plant pathogen. PLOS Pathog. doi: 10.1371/journal.ppat.1002235
  8. Freitag J, Ast J, Linne U, Stehlik T, Martorana D, Bölker M, Sandrock B (2014) Peroxisomes contribute to biosynthesis of extracellular glycolipids in fungi. Mol Microbiol 93:24–36. doi: 10.1111/mmi.12642 PubMedCrossRefGoogle Scholar
  9. Grumaz C, Lorenz S, Stevens P, Lindemann E, Schock U, Retey J, Rupp S, Sohn K (2013) Species and condition specific adaptation of the transcriptional landscapes in Candida albicans and Candida dubliniensis. BMC Genomics 14:212. doi: 10.1186/1471-2164-14-212 PubMedCentralPubMedCrossRefGoogle Scholar
  10. Guida A, Lindstadt C, Maguire SL, Ding C, Higgins DG, Corton NJ, Berriman M, Butler G (2011) Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis. BMC Genomics 12:628. doi: 10.1186/1471-2164-12-628 PubMedCentralPubMedCrossRefGoogle Scholar
  11. Hewald S, Linne U, Scherer M, Marahiel MA, Kämper J, Bölker M (2006) Identification of a gene cluster for biosynthesis of mannosylerythritol lipids in the basidiomycetous fungus Ustilago maydis. Appl Environ Microbiol 72:5469–5477. doi: 10.1128/AEM. 00506-06 PubMedCentralPubMedCrossRefGoogle Scholar
  12. Horst RJ, Zeh C, Saur A, Sonnewald S, Sonnewald U, Voll LM (2012) The Ustilago maydis Nit2 homolog regulates nitrogen utilization and is required for efficient induction of filamentous growth. Eukaryot Cell 11:368–380. doi: 10.1128/EC.05191-11 PubMedCentralPubMedCrossRefGoogle Scholar
  13. Im JH, Yanagishita H, Ikegami T, Takeyama Y, Idemoto Y, Koura N, Kitamoto D (2003) Mannosylerythritol lipids, yeast glycolipid biosurfactants, are potential affinity ligand materials for human immunoglobulin G. J Biomed Mater Res A 65:379–385. doi: 10.1002/jbm.a.10491 PubMedCrossRefGoogle Scholar
  14. Imura T, Ohta N, Inoue K, Yagi N, Negishi H, Yanagishita H, Kitamoto D (2006) Naturally engineered glycolipid biosurfactants leading to distinctive self-assembled structures. Chemistry 12:2434–2440. doi: 10.1002/chem.200501199 PubMedCrossRefGoogle Scholar
  15. Imura T, Masuda Y, Ito S, Worakitkanchanakul W, Morita T, Fukuoka T, Sakai H, Abe M, Kitamoto D (2008) Packing density of glycolipid biosurfactant monolayers give a significant effect on their binding affinity toward immunoglobulin G. J Oleo Sci 57:415–422. doi: 10.5650/jos.57.415 PubMedCrossRefGoogle Scholar
  16. Inoh Y, Furuno T, Hirashima N, Kitamoto D, Nakanishi M (2010) The ratio of unsaturated fatty acids in biosurfactants affects the efficiency of gene transfection. Int J Pharm 398:225–230. doi: 10.1016/j.ijpharm.2010.07.042 PubMedCrossRefGoogle Scholar
  17. Inoh Y, Furuno T, Hirashima N, Kitamoto D, Nakanishi M (2011) Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes. Biochem Biophys Res Commun 414:635–640. doi: 10.1016/j.bbrc.2011.09.147 PubMedCrossRefGoogle Scholar
  18. Kämper J, Kahmann R, Bölker M, Ma LJ, Brefort T, Saville BJ, Banuett F, Kronstad JW, Gold SE, Müller O, Perlin MH, Wösten HA, de Vries R, Ruiz-Herrera J, Reynaga-Pena CG, Snetselaar K, McCann M, Perez-Martín J, Feldbrügge M, Basse CW, Steinberg G, Ibeas JI, Holloman W, Guzman P, Farman M, Stajich JE, Sentandreu R, González-Prieto JM, Kennell JC, Molina L, Schirawski J, Mendoza-Mendoza A, Greilinger D, Münch K, Rössel N, Scherer M, Vranes M, Ladendorf O, Vincon V, Fuchs U, Sandrock B, Meng S, Ho EC, Cahill MJ, Boyce KJ, Klose J, Klosterman SJ, Deelstra HJ, Ortiz-Castellanos L, Li W, Sanchez-Alonso P, Schreier PH, Häuser-Hahn I, Vaupel M, Koopmann E, Friedrich G, Voss H, Schlüter T, Margolis J, Platt D, Swimmer C, Gnirke A, Chen F, Vysotskaia V, Mannhaupt G, Güldener U, Münsterkötter M, Haase D, Oesterheld M, Mewes HW, Mauceli EW, DeCaprio D, Wade CM, Butler J, Young S, Jaffe DB, Calvo S, Nusbaum C, Galagan J, Birren BW (2006) Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444:97–101. doi: 10.1038/nature05248 PubMedCrossRefGoogle Scholar
  19. Kitamoto D, Nemoto T, Yanagishita H, Nakane T, Kitamoto H, Nakahara T (1993) Fatty-acid metabolism of mannosylerythritol lipids as biosurfactants produced by Candida antarctica. J Jpn Oil Chem Soc 42:346–358CrossRefGoogle Scholar
  20. Kitamoto D, Ikegami T, Suzuki GT, Sasaki A, Takeyama Y, Idemoto Y, Koura N, Yanagishita H (2001) Microbial conversion of n-alkanes into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma (Candida antarctica). Biotechnol Lett 23:1709–1714. doi: 10.1023/A:1012464717259 CrossRefGoogle Scholar
  21. Klose J, de Sa MM, Kronstad JW (2004) Lipid-induced filamentous growth in Ustilago maydis. Mol Microbiol 52:823–835. doi: 10.1111/j.1365-2958.2004.04019.x PubMedCrossRefGoogle Scholar
  22. Konishi M, Hatada Y, Horiuchi J (2013) Draft genome sequence of the basidiomycetous yeast-like fungus Pseudozyma hubeiensis SY62, which produces an abundant amount of the biosurfactant mannosylerythritol lipids. Genome Announc 1:e00409–00413. doi: 10.1128/genomeA. 00409-13 PubMedCentralPubMedCrossRefGoogle Scholar
  23. Lorenz S, Günther M, Grumaz C, Rupp S, Zibek S, Sohn K (2014) Genome sequence of the basidiomycetous fungus Pseudozyma aphidis DSM 70725, an efficient producer of biosurfactant mannosylerythritol lipids. Genome Announc 2:e00053–00014. doi: 10.1128/genomeA. 00053-14 PubMedCentralPubMedCrossRefGoogle Scholar
  24. Morita T, Konishi M, Fukuoka T, Imura T, Kitamoto D (2007a) Physiological differences in the formation of the glycolipid biosurfactants, mannosylerythritol lipids, between Pseudozyma antarctica and Pseudozyma aphidis. Appl Microbiol Biotechnol 74:307–315. doi: 10.1007/s00253-006-0672-3 PubMedCrossRefGoogle Scholar
  25. Morita T, Konishi M, Fukuoka T, Imura T, Kitamoto HK, Kitamoto D (2007b) Characterization of the genus Pseudozyma by the formation of glycolipid biosurfactants, mannosylerythritol lipids. FEMS Yeast Res 7:286–292. doi: 10.1111/j.1567-1364.2006.00154.x PubMedCrossRefGoogle Scholar
  26. Morita T, Kitagawa M, Suzuki M, Yamamoto S, Sogabe A, Yanagidani S, Imura T, Fukuoka T, Kitamoto D (2009) A yeast glycolipid biosurfactant, mannosylerythritol lipid, shows potential moisturizing activity toward cultured human skin cells: the recovery effect of MEL-A on the SDS-damaged human skin cells. J Oleo Sci 58:639–642. doi: 10.5650/jos.58.639 PubMedCrossRefGoogle Scholar
  27. Morita T, Ito E, Kitamoto HK, Takegawa K, Fukuoka T, Imura T, Kitamoto D (2010) Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica. Yeast 27:905–917. doi: 10.1002/yea.1794 PubMedCrossRefGoogle Scholar
  28. Morita T, Ishibashi Y, Hirose N, Wada K, Takahashi M, Fukuoka T, Imura T, Sakai H, Abe M, Kitamoto D (2011) Production and characterization of a glycolipid biosurfactant, mannosylerythritol lipid B, from sugarcane juice by Ustilago scitaminea NBRC 32730. Biosci Biotechnol Biochem 75:1371–1376. doi: 10.1271/bbb.110221 PubMedCrossRefGoogle Scholar
  29. Morita T, Fukuoka T, Imura T, Kitamoto D (2013a) Accumulation of cellobiose lipids under nitrogen-limiting conditions by two ustilaginomycetous yeasts, Pseudozyma aphidis and Pseudozyma hubeiensis. FEMS Yeast Res 13:44–49. doi: 10.1111/1567-1364.12005 PubMedCrossRefGoogle Scholar
  30. Morita T, Koike H, Koyama Y, Hagiwara H, Ito E, Fukuoka T, Imura T, Machida M, Kitamoto D (2013b) Genome sequence of the basidiomycetous yeast Pseudozyma antarctica T-34, a producer of the glycolipid biosurfactants mannosylerythritol lipids. Genome Announc 1:e0006413. doi: 10.1128/genomeA. 00064-13 PubMedCrossRefGoogle Scholar
  31. Morita T, Fukuoka T, Imura T, Kitamoto D (2013c) Production of mannosylerythritol lipids and their application in cosmetics. Appl Microbiol Biotechnol 97:4691–4700. doi: 10.1007/s00253-013-4858-1 PubMedCrossRefGoogle Scholar
  32. Morita T, Koike H, Hagiwara H, Ito E, Machida M, Sato S, Habe H, Kitamoto D (2014) Genome and transcriptome analysis of the basidiomycetous yeast Pseudozyma antarctica producing extracellular glycolipids, mannosylerythritol lipids. PLoS ONE 9:e86490. doi: 10.1371/journal.pone.0086490 PubMedCentralPubMedCrossRefGoogle Scholar
  33. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628. doi: 10.1038/nmeth.1226 PubMedCrossRefGoogle Scholar
  34. Ness SA (1999) Myb binding proteins: regulators and cohorts in transformation. Oncogene 18:3039–3046. doi: 10.1038/sj.onc.1202726 PubMedCrossRefGoogle Scholar
  35. Nikolaidis N, Doran N, Cosgrove DJ (2014) Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion. Mol Biol Evol 31:376–386. doi: 10.1093/molbev/mst206 PubMedCrossRefGoogle Scholar
  36. Rau U, Nguyen LA, Roeper H, Koch H, Lang S (2005a) Fed-batch bioreactor production of mannosylerythritol lipids secreted by Pseudozyma aphidis. Appl Microbiol Biotechnol 68:607–613. doi: 10.1007/s00253-005-1906-5 PubMedCrossRefGoogle Scholar
  37. Rau U, Nguyen LA, Roeper H, Koch H, Lang S (2005b) Downstream processing of mannosylerythritol lipids produced by Pseudozyma aphidis. Eur J Lipid Sci Technol 107:373–380. doi: 10.1002/ejlt.200401122 CrossRefGoogle Scholar
  38. Smyth GK (2005) Limma: linear models for microarray data. In: Gentleman R, Carey VJ, Huber W, Irizarry RA, Dudoit S (eds) Bioinformatics and computational biology solutions using R and Bioconductor. Springer, New York, NY, pp 397–420CrossRefGoogle Scholar
  39. Storey JD, Tibshirani R (2003) Statistical significance for genomewide studies. Proc Natl Acad Sci U S A 100:9440–9445. doi: 10.1073/pnas.1530509100 PubMedCentralPubMedCrossRefGoogle Scholar
  40. Teichmann B, Linne U, Hewald S, Marahiel MA, Bölker M (2007) A biosynthetic gene cluster for a secreted cellobiose lipid with antifungal activity from Ustilago maydis. Mol Microbiol 66:525–533. doi: 10.1111/j.1365-2958.2007.05941.x PubMedCrossRefGoogle Scholar
  41. Teichmann B, Liu L, Schink KO, Bölker M (2010) Activation of the ustilagic acid biosynthesis gene cluster in Ustilago maydis by the C2H2 zinc finger transcription factor Rua1. Appl Environ Microbiol 76:2633–2640. doi: 10.1128/AEM. 02211-09 PubMedCentralPubMedCrossRefGoogle Scholar
  42. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105–1111. doi: 10.1093/bioinformatics/btp120 PubMedCentralPubMedCrossRefGoogle Scholar
  43. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562–578. doi: 10.1038/nprot.2012.016 PubMedCentralPubMedCrossRefGoogle Scholar
  44. Wang L, Feng Z, Wang X, Wang X, Zhang X (2010) DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 26:136–138. doi: 10.1093/bioinformatics/btp612 PubMedCrossRefGoogle Scholar
  45. Yamamoto S, Morita T, Fukuoka T, Imura T, Yanagidani S, Sogabe A, Kitamoto D, Kitagawa M (2012) The moisturizing effects of glycolipid biosurfactants, mannosylerythritol lipids, on human skin. J Oleo Sci 61:407–412. doi: 10.5650/jos.61.407 PubMedCrossRefGoogle Scholar
  46. Yamamoto S, Fukuoka T, Imura T, Morita T, Yanagidani S, Kitamoto D, Kitagawa M (2013) Production of a novel mannosylerythritol lipid containing a hydroxy fatty acid from castor oil by Pseudozyma tsukubaensis. J Oleo Sci 62:381–389. doi: 10.5650/jos.62.381 PubMedCrossRefGoogle Scholar
  47. Yoshida S, Morita T, Shinozaki Y, Watanabe T, Sameshima-Yamashita Y, Koitabashi M, Kitamoto D, Kitamoto H (2014) Mannosylerythritol lipids secreted by phyllosphere yeast Pseudozyma antarctica is associated with its filamentous growth and propagation on plant surfaces. Appl Microbiol Biotechnol 98:6419–6429. doi: 10.1007/s00253-014-5675-x PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Michael Günther
    • 1
  • Christian Grumaz
    • 1
  • Stefan Lorenz
    • 1
  • Philip Stevens
    • 2
    • 3
  • Elena Lindemann
    • 1
  • Thomas Hirth
    • 1
    • 2
  • Kai Sohn
    • 1
  • Susanne Zibek
    • 1
  • Steffen Rupp
    • 1
    • 2
  1. 1.Department of Molecular BiotechnologyFraunhofer Institute for Interfacial Engineering and BiotechnologyStuttgartGermany
  2. 2.Institute of Interfacial Process Engineering and Plasma Technology, University of StuttgartStuttgartGermany
  3. 3.Max F. Perutz Laboratories, Center for Integrative BioinformaticsUniversity of ViennaViennaAustria

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