Current Microbiology

, Volume 64, Issue 5, pp 477–485 | Cite as

In Silico Cloning and Characterization of the Glycerol-3-Phosphate Dehydrogenase (GPDH) Gene Family in the Green Microalga Chlamydomonas reinhardtii

  • Virginia A. Herrera-Valencia
  • Laura A. Macario-González
  • Melissa L. Casais-Molina
  • Anayeli G. Beltran-Aguilar
  • Santy Peraza-Echeverría


Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the conversion of dihydroxyacetone phosphate (DHAP) and NADH to glycerol-3-phosphate (G3P) and NAD+. G3P is important as a precursor for glycerol and glycerolipid synthesis in microalgae. A GPDH enzyme has been previously purified from the green microalga Chlamydomonas reinhardtii, however, no genes coding for GPDH have been characterized before. In this study, we report the in silico characterization of three putative GPDH genes from C. reinhardtii: CrGPDH1, CrGPDH2, and CrGPDH3. These sequences showed a significant similarity to characterized GPDH genes from the microalgae Dunaliella salina and Dunaliella viridis. The prediction of the three-dimensional structure of the proteins showed the characteristic fold topology of GPDH enzymes. Furthermore, the phylogenetic analysis showed that the three CrGPDHs share the same clade with characterized GPDHs from Dunaliella suggesting a common evolutionary origin and a similar catalytic function. In addition, the K a/K s ratios of these sequences suggested that they are under purifying selection. Moreover, the expression analysis showed a constitutive expression of CrGPDH1, while CrGPDH2 and CrGPDH3 were induced in response to osmotic stress, suggesting a possible role for these two sequences in the synthesis of glycerol as a compatible solute in osmoregulation, and perhaps also in lipid synthesis in C. reinhardtii. This study has provided a foundation for further biochemical and genetic studies of the GPDH family in this model microalga, and also opportunities to assess the potential of these genes to enhance the synthesis of TAGs for biodiesel production.


Microalgae Osmotic Stress Chlamydomonas Dunaliella Chloroplast Transit Peptide 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This research was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT, México) Project No. CB-169217 and Centro de Investigación Científica de Yucatán (CICY, Mexico) Project FB0054. Laura Anahi Macario-González, Melissa Lessen Casais-Molina and Anayeli Beltrán Aguilar are grateful to Consejo Nacional de Ciencia y Tecnología (CONACYT, Mexico) for the Scholarship Nos. 235997, 228280 and 264772, respectively. Authors are grateful to Ileana C. Borges Argáez for technical support and Miguel Ángel Vallejo Reyna for assistance on the protein modeling.

Supplementary material

284_2012_95_MOESM1_ESM.docx (12 kb)
Supplementary material 1 (DOCX 12 kb)
284_2012_95_MOESM2_ESM.rtf (293 kb)
Supplementary material 2 (RTF 292 kb)


  1. 1.
    Albertyn J, Hohmann S, Thevelein JM, Prior BA (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
  2. 2.
    Altschul SF, Madden TL, Schäffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  3. 3.
    Ansell R, Granath K, Hohmann S, Thevelein JM, Adler L (1997) The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation. EMBO J 16:2179–2187PubMedCrossRefGoogle Scholar
  4. 4.
    Bordoli L, Kiefer F, Arnold K et al (2009) Protein structure homology modeling using SWISS-MODEL workspace. Nature Protoc 4:1–13CrossRefGoogle Scholar
  5. 5.
    Chen H, Lao Y-M, Jiang J-G (2011) Effects of salinities on the gene expression of a (NAD+)-dependent glycerol-3-phosphate dehydrogenase in Dunaliella salina. Sci Total Environ 409:1291–1297PubMedCrossRefGoogle Scholar
  6. 6.
    Chen X, Fang H, Rao Z et al (2008) Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer. FEMS Yeast Res 8:725–734PubMedCrossRefGoogle Scholar
  7. 7.
    Fan J, Andre C, Xu C (2011) A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii. FEBS Lett 585:1985–1991PubMedCrossRefGoogle Scholar
  8. 8.
    Gee R, Goyal A, Byerrum RU, Tolbert NE (1993) Two isoforms of dihydroxyacetone phosphate reductase from the chloroplast of Dunaliella tertiolecta. Plant Physiol 103:243–249PubMedGoogle Scholar
  9. 9.
    Goshal D, Mach D, Agarwal M et al (2002) Osmoregulatory isoform of dihydroxyacetone phosphate reductase from Dunaliella tertiolecta: purification and characterization. Protein Expr Purif 24:404–411CrossRefGoogle Scholar
  10. 10.
    Harris EH (1989) The Chlamydomonas sourcebook, a comprehensive guide to biology and laboratory use. Academic Press, Inc, San DiegoGoogle Scholar
  11. 11.
    He Q, Qiao D, Bai L et al (2007) Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina. J Plant Physiol 164:214–220PubMedCrossRefGoogle Scholar
  12. 12.
    He Y, Meng X, Fan Q et al (2009) Cloning and characterization of two novel chloroplastic glycerol-3-phosphate dehydrogenases from Dunaliella viridis. Plant Mol Biol 71:193–205PubMedCrossRefGoogle Scholar
  13. 13.
    Heckman DS, Geiser DM, Eidell BR et al (2001) Molecular evidence for the early colonization of land by fungi and plants. Science 293:1129–1133PubMedCrossRefGoogle Scholar
  14. 14.
    Herrera-Valencia VA, Contreras-Pool PY, López-Adrián SJ et al (2011) The green microalga Chlorella saccharophila as a suitable source of oil for biodiesel production. Curr Microbiol 63:151–157PubMedCrossRefGoogle Scholar
  15. 15.
    Hu Q, Sommerfeld M, Jarvis E et al (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54:621–639PubMedCrossRefGoogle Scholar
  16. 16.
    Husic HD, Tolbert NE (1986) Effect of osmotic stress on carbon metabolism in Chlamydomonas reinhardtii. Plant Physiol 82:594–596PubMedCrossRefGoogle Scholar
  17. 17.
    Klöck G, Kreuzberg K (1989) Kinetic properties of a sn-glycerol-3-phosphate dehydrogenase purified from the unicellular alga Chlamydomonas reinhardtii. Biochim Biophys Acta 991:347–352PubMedCrossRefGoogle Scholar
  18. 18.
    Larkin M, Blackshields N, Brown R et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948PubMedCrossRefGoogle Scholar
  19. 19.
    Larsson C, Pahlman I, Ansell R et al (1998) The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae. Yeast 14:347–357PubMedCrossRefGoogle Scholar
  20. 20.
    Lee D, Kim M, Ryu Y, Seo J (2008) Cloning and characterization of CmGPD1, the Candida magnoliae homologue of glycerol-3-phosphate dehydrogenase. FEMS Yeast Res 8:1324–1333PubMedCrossRefGoogle Scholar
  21. 21.
    León R, Galván F (1994) Halotolerance studies on Chlamydomonas reinhardtii: glycerol excretion by free and immobilized cells. J Appl Phycol 6:13–20CrossRefGoogle Scholar
  22. 22.
    Letunic I, Doerks T, Bork P (2009) SMART 6: recent updates and new developments. Nucleic Acids Res 37:D229–D232PubMedCrossRefGoogle Scholar
  23. 23.
    Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452PubMedCrossRefGoogle Scholar
  24. 24.
    Miller R, Wu G, Deshpande RR et al (2010) Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism. Plant Physiol 154:1737–1752PubMedCrossRefGoogle Scholar
  25. 25.
    Ou X, Ji C, Han X et al (2006) Crystal structures of human glycerol-3-phosphate dehydrogenase 1 (GPD1). J Mol Biol 357:858–869PubMedCrossRefGoogle Scholar
  26. 26.
    Peng F, Li G, Wang X et al (2010) Cloning and characterization of a glycerol-3-phosphate dehydrogenase (NAD+) gene from halotolerant yeast Pichia farinosa. Yeast 27:115–121PubMedGoogle Scholar
  27. 27.
    Rao AR, Dayananda C, Sarada R et al (2007) Effect of salinity on growth of green alga Botryococcus braunii and its constituents. Bioresour Technol 98:560–564PubMedCrossRefGoogle Scholar
  28. 28.
    Riekhof WR, Sears BB, Benning C (2005) Annotation of genes involved in glycerolipid biosynthesis in Chlamydomonas reinhardtii: discovery of betaine lipid synthase BTA1Cr. Eukaryot Cell 4:242–252PubMedCrossRefGoogle Scholar
  29. 29.
    Ronnow B, Kielland-Brandt MC (1993) GUT2, a gene for mitochondrial glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Yeast 9:1121–1130PubMedCrossRefGoogle Scholar
  30. 30.
    Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  31. 31.
    Shen W, Wei Y, Dauk M et al (2006) Involvement of a glycerol-3-phosphate dehydrogenase in modulating the NADH/NAD+ ratio provides evidence of a mitochondrial glycerol-3-phosphate shuttle in Arabidopsis. Plant Cell 18:422–441PubMedCrossRefGoogle Scholar
  32. 32.
    Siaut M, Cuiné S, Cagnon C et al (2011) Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves. BMC Biotechnol 11:1–15CrossRefGoogle Scholar
  33. 33.
    Sugase Y, Hirono M, Kindle K, Kamiya R (1996) Cloning and characterization of the actin-encoding gene of Chlamydomonas reinhardtii. Gene 168:117–121PubMedCrossRefGoogle Scholar
  34. 34.
    Takagi M, Karseno, Yoshida T (2006) Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunalliela cells. J Biosci Bioeng 101:223–226PubMedCrossRefGoogle Scholar
  35. 35.
    Tamura K, Peterson D, Peterson N et al. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. doi: 10.1093/molbev/msr121
  36. 36.
    Thomé P (2004) Isolation of a GPD gene from Debaryomyces hansenii encoding a glycerol-3-phosphate dehydrogenase (NAD+). Yeast 21:119–126PubMedCrossRefGoogle Scholar
  37. 37.
    Vigeolas H, Geigenberger P (2004) Increased levels of glycerol-3-phosphate lead to a stimulation of flux into tryacylglycerol synthesis after supplying glycerol to developing seeds of Brassica napus L. in planta. Planta 219:827–835PubMedCrossRefGoogle Scholar
  38. 38.
    Vigeolas H, Waldeck P, Zank T, Geigenberger P (2007) Increasing seed oil content in oil-seed rape (Brassica napus L.) by over-expression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed-specific promoter. Plant Biotechnol J 5:431–441PubMedCrossRefGoogle Scholar
  39. 39.
    Watanabe Y, Tsuchimoto S, Tamai Y (2004) Heterologous expression of Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) and glycerol dehydrogenase gene (ZrGCY1) in Saccharomyces cerevisiae. FEMS Yeast Res 4:505–510PubMedCrossRefGoogle Scholar
  40. 40.
    Yang ZH, Bielawski JP (2000) Statistical methods for detecting molecular adaptation. Trends Ecol Evol 15:496–503PubMedCrossRefGoogle Scholar
  41. 41.
    Yang W, Cao Y, Sun X et al (2007) Isolation of a FAD-GPDH gene encoding a mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase from Dunaliella salina. J Basic Microbiol 47:266–274PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Virginia A. Herrera-Valencia
    • 1
  • Laura A. Macario-González
    • 1
  • Melissa L. Casais-Molina
    • 1
  • Anayeli G. Beltran-Aguilar
    • 1
  • Santy Peraza-Echeverría
    • 1
  1. 1.Unidad de BiotecnologíaCentro de Investigación Científica de Yucatán (CICY)MéridaMexico

Personalised recommendations