Molecular Biotechnology

, Volume 45, Issue 2, pp 121–128 | Cite as

Molecular Cloning and Characterization of a Malic Enzyme Gene from the Oleaginous Yeast Lipomyces starkeyi

  • Wei Tang
  • Sufang Zhang
  • Haidong Tan
  • Zongbao K. Zhao
Research

Abstract

The malic enzyme-encoding cDNA (GQ372891) from the oleaginous yeast Lipomyces starkeyi AS 2.1560 was isolated, which has an 1719-bp open reading frame flanked by a 290-bp 5′ untranslated sequence and a 92-bp 3′ untranslated sequence. The proposed gene, LsME1, encoded a protein with 572 amino acid residues. The protein presented 58% sequence identity with the malic enzymes from Yarrowia lipolytica CLIB122 and Aspergillus fumigatus Af293. The LsME1 gene was cloned into the vector pMAL-p4x to express a fusion protein (MBP-LsME1) in Escherichia coli TB1. The fusion protein was purified and then cleaved by Factor Xa to give the recombinant LsME1. This purified enzyme took either NAD+ or NADP+ as the coenzyme but preferred NAD+. The Km values for malic acid, NAD+ and NADP+ were 0.85 ± 0.05 mM, 0.34 ± 0.08 mM, and 7.4 ± 0.32 mM, respectively, at pH 7.3.

Keywords

Lipomyces starkeyi Malic enzyme RACE Protein expression Enzyme kinetics 

References

  1. 1.
    Fall, R., Phelps, P., & Spindler, D. (1984). Bioconversion of xylan to triglycerides by oil-rich yeasts. Applied and Environmental Microbiology, 47, 1130–1134.Google Scholar
  2. 2.
    Jakobsen, A. N., Aasen, I. M., Josefsen, K. D., & Strom, A. R. (2008). Accumulation of docosahexaenoic acid-rich lipid in thraustochytrid Aurantiochytrium sp. strain T66: effects of N and P starvation and O2 limitation. Applied Microbiology Biotechnology, 80, 297–306.CrossRefGoogle Scholar
  3. 3.
    Zhao, Z. B. (2005). Toward cheaper microbial oil for biodiesel. China Biotechnology, 25, 8–11.Google Scholar
  4. 4.
    Li, Y., Horsman, M., Wang, B., Wu, N., & Lan, C. (2008). Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Applied Microbiology and Biotechnology, 81, 629–636.CrossRefGoogle Scholar
  5. 5.
    Li, Y. H., Zhao, Z. B., & Bai, F. W. (2007). High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzyme and Microbial Technology, 41, 312–317.CrossRefGoogle Scholar
  6. 6.
    Roesler, K., Shintani, D., Savage, L., Boddupalli, S., & Ohlrogge, J. (1997). Targeting of the arabidopsis homomeric acetyl-coenzyme A carboxylase to plastids of rapeseeds. Plant Physiology, 113, 75–81.CrossRefGoogle Scholar
  7. 7.
    Dehesh, K., Tai, H., Edwards, P., Byrne, J., & Jaworski, J. G. (2001). Overexpression of 3-ketoacyl-acyl-carrier protein synthase IIIs in plants reduces the rate of lipid synthesis. Plant Physiology, 125, 1103–1114.CrossRefGoogle Scholar
  8. 8.
    Mysyakina, I. S., & Funtikova, N. S. (2008). Activity of NAD-dependent isocitrate dehydrogenase, isocitrate lyase, and malate dehydrogenase in Mucor circinelloides var. lusitanicus INMI under different modes of nitrogen supply. Microbiology, 77, 400–406.CrossRefGoogle Scholar
  9. 9.
    Sourdioux, M., Brevelet, C., Delabrosse, Y., & Douaire, M. (1999). Association of fatty acid synthase gene and malic enzyme gene polymorphisms with fatness in turkeys. Poultry Science, 78, 1651–1657.Google Scholar
  10. 10.
    Zhang, Y., Adams, I. P., & Ratledge, C. (2007). Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation. Microbiology, 153, 2013–2025.CrossRefGoogle Scholar
  11. 11.
    Chang, G. G., & Tong, L. (2003). Structure and function of malic enzymes, a new class of oxidative decarboxylases. Biochemistry, 42, 12721–12733.CrossRefGoogle Scholar
  12. 12.
    Frenkel, R. (1975). Regulation and physiological functions of malic enzymes. Current Topics in Cellular Regulation, 9, 157–181.Google Scholar
  13. 13.
    Fukuda, W., Ismail, Y. S., Fukui, T., Atomi, H., & Imanaka, T. (2005). Characterization of an archaeal malic enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Archaea, 1, 293–301.CrossRefGoogle Scholar
  14. 14.
    Viljoen, M., Subden, R. E., Krizus, A., & Van Vuuren, H. J. (1994). Molecular analysis of the malic enzyme gene (mae2) of Schizosaccharomyces pombe. Yeast, 10, 613–624.CrossRefGoogle Scholar
  15. 15.
    Schomburg, D., & Stephan, D. (1995). Enzyme handbook 9. Class 1.1 oxidoreductases EC 1.1.1.1−EC 1.1.1.149. Berlin: Springer Verlag Press.Google Scholar
  16. 16.
    Moreadith, R. W., & Lehninger, A. L. (1984). The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)+-dependent malic enzyme. The Journal of Biological Chemistry, 259, 6215–6221.Google Scholar
  17. 17.
    Hill, S., Winning, B., Jenner, H., Knorpp, C., & Leaver, C. (1996). Role of NAD+-dependent ‘malic’ enzyme and pyruvate dehydrogenase complex in leaf metabolism. Biochemical Society Transactions, 24, 743–746.Google Scholar
  18. 18.
    Goodridge, A. G., Klautky, S. A., Fantozzi, D. A., Baillie, R. A., Hodnett, D. W., Chen, W., et al. (1996). Nutritional and hormonal regulation of expression of the gene for malic enzyme. Progress in Nucleic Acid Research and Molecular Biology, 52, 89–122.CrossRefGoogle Scholar
  19. 19.
    Saayman, M., van Zyl, W. H., & Viljoen-Bloom, M. (2006). Cloning, characterisation, and heterologous expression of the Candida utilis malic enzyme gene. Current Genetics, 49, 248–258.CrossRefGoogle Scholar
  20. 20.
    Sinsuwongwat, S., Kodera, A., Kaneko, T., Tabata, S., Nomura, M., & Tajima, S. (2002). Cloning and characterization of a NADP+-malic enzyme gene from Bradyrhizobium japonicum USDA110. Soil Science and Plant Nutrition, 48, 711–717.Google Scholar
  21. 21.
    Gourdon, P., Baucher, M. F., Lindley, N. D., & Guyonvarch, A. (2000). Cloning of the malic enzyme gene from Corynebacterium glutamicum and role of the enzyme in lactate metabolism. Applied and Environmental Microbiology, 66, 2981–2987.CrossRefGoogle Scholar
  22. 22.
    Tang, W., Zhang, S. F., Wang, Q., Tan, H. D., & Zhao, Z. B. (2009). The isocitrate dehydrogenase gene of oleaginous yeast Lipomyces starkeyi is linked to lipid accumulation. Canadian Journal of Microbiology, 55, 1062–1069.CrossRefGoogle Scholar
  23. 23.
    Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual. New York: CSHL Press.Google Scholar
  24. 24.
    Ausubel, F. M., Brent, R., Kingston, R. E., & Moore, D. D. (2005). Short protocols in molecular biology. New Jersey: Wiley.Google Scholar
  25. 25.
    Tamura, K., Dudley, J., Nei, M., & Kumar, S. (2007). MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24, 1596–1599.CrossRefGoogle Scholar
  26. 26.
    Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 72, 248–254.CrossRefGoogle Scholar
  27. 27.
    Kuo, C. W., Hung, H. C., Tong, L., & Chang, G. G. (2004). Metal-induced reversible structural interconversion of human mitochondrial NAD(P)+-dependent malic enzyme. Proteins, 54, 404–411.CrossRefGoogle Scholar
  28. 28.
    Yilmaz, E. (2002). Kinetic studies with crude tomato alcohol dehydrogenase. Turkish Journal of Agriculture and Forestry, 26, 141–145.Google Scholar
  29. 29.
    Rothermel, B. A., & Nelson, T. (1989). Primary structure of the maize NADP-dependent malic enzyme. Journal of Biological Chemistry, 264, 19587–19592.Google Scholar
  30. 30.
    Wierenga, R. K., Terpstra, P., & Hol, W. G. J. (1986). Prediction of the occurrence of the ADP-binding βαβ-fold in proteins, using an amino acid sequence fingerprint. Journal of Molecular Biology, 187, 101–107.CrossRefGoogle Scholar
  31. 31.
    Hsu, R. Y., Mildvan, A. S., Chang, G. G., & Fung, C. H. (1976). Mechanism of malic enzyme from pigeon liver—Magnetic resonance and kinetic studies of role of Mn2+. The Journal of Biological Chemistry, 251, 6574–6583.Google Scholar
  32. 32.
    Kulkarni, G., Cook, P. F., & Harris, B. G. (1993). Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum malic enzyme. Archives of Biochemistry and Biophysics, 300, 231–237.CrossRefGoogle Scholar
  33. 33.
    Hsu, R. Y. (1982). Pigeon liver malic enzyme. Molecular and Cellular Biochemistry, 43, 3–26.CrossRefGoogle Scholar
  34. 34.
    Boles, E., de Jong-Gubbels, P., & Pronk, J. T. (1998). Identification and characterization of MAE1, the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme. The Journal of Bacteriology, 180, 2875–2882.Google Scholar
  35. 35.
    Smyth, D. R., Mrozkiewicz, M. K., McGrath, W. J., Listwan, P., & Kobe, B. (2003). Crystal structures of fusion proteins with large-affinity tags. Protein Science, 12, 1313–1322.CrossRefGoogle Scholar
  36. 36.
    Biolabs. (2001). pMAL™ protein fusion and purification system. MA: New England, Inc. Press.Google Scholar
  37. 37.
    Sanwal, B. D. (1970). Regulatory characteristics of the diphosphopyridine nucleotide-specific malic enzyme of Escherichia coli. The Journal of Biological Chemistry, 245, 1212–1216.Google Scholar
  38. 38.
    Wang, J. X., Tan, H. D., & Zhao, Z. B. (2007). Overexpression, purification, and characterization of recombinant NAD-malic enzyme from Escherichia coli K12. Protein Expression and Purification, 53, 97–103.CrossRefGoogle Scholar
  39. 39.
    Kawai, S., Suzuki, H., Yamamoto, K., Inui, M., Yukawa, H., & Kumagai, H. (1996). Purification and characterization of a malic enzyme from the ruminal bacterium Streptococcus bovis ATCC 15352 and cloning and sequencing of its gene. Applied and Environmental Microbiology, 62, 2692–2700.Google Scholar
  40. 40.
    Chen, F., Okabe, Y., Osano, K., & Tajima, S. (1998). Purification and characterization of an NAD-malic enzyme from Bradyrhizobium japonicum A1017. Applied and Environmental Microbiology, 64, 4073–4075.Google Scholar
  41. 41.
    Kobayashi, K., Doi, S., Negoro, S., Urabe, I., & Okada, H. (1989). Structure and properties of malic enzyme from Bacillus stearothermophilus. The Journal of Biological Chemistry, 264, 3200–3205.Google Scholar
  42. 42.
    Fuck, E., & Radler, F. (1972). Malic acid metabolism of Saccharomyces. 1. Anaerobic decomposition of malic acid by Saccharomyces cerevisiae. Archiv Fur Mikrobiologe, 87, 149–164.CrossRefGoogle Scholar
  43. 43.
    Colombo, S. L., Andreo, C. S., & Podesta, F. E. (1997). Carbon metabolism in germinating Ricinus communis cotyledons. Purification, characterization and developmental profile of NADP-dependent malic enzyme. Physiologia Plantarum, 101, 821–826.CrossRefGoogle Scholar
  44. 44.
    Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G. G., & Chou, W. Y. (2000). Lysine residues 162 and 340 are involved in the catalysis and coenzyme binding of NADP+-dependent malic enzyme from pigeon. Biochemical and Biophysical Research Communications, 270, 821–825.CrossRefGoogle Scholar
  45. 45.
    Hsieh, J. Y., Liu, G. Y., Chang, G. G., & Hung, H. C. (2006). Determinants of the dual cofactor specificity and substrate cooperativity of the human mitochondrial NAD(P)+-dependent malic enzyme—Functional roles of glutamine 362. The Journal of Biological Chemistry, 281, 23237–23245.CrossRefGoogle Scholar
  46. 46.
    Coleman, R., & Bell, R. M. (1978). Evidence that biosynthesis of phosphatidylethanolamine, phosphatidylcholine, and triacylglycerol occurs on cytoplasmic side of microsomal vesicles. The Journal of Cell Biology, 76, 245–253.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Wei Tang
    • 1
    • 2
  • Sufang Zhang
    • 1
  • Haidong Tan
    • 1
  • Zongbao K. Zhao
    • 1
  1. 1.Dalian Institute of Chemical Physics, CASDalianPeople’s Republic of China
  2. 2.Graduate School of the Chinese Academy of SciencesBeijingPeople’s Republic of China

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