1887

Abstract

Malic enzyme (ME; NADP-dependent; EC 1 . 1 . 1 . 40) has been postulated to be the rate-limiting step for fatty acid biosynthesis in oleaginous fungi in which the extent of lipid accumulation is below the maximum possible. The genes encoding the isoform of ME involved in fatty acid synthesis were identified in and , two commercially useful oil-producing fungi, using degenerate primers. Both showed high similarity with ME genes from other micro-organisms. The whole-length ME gene from each source was cloned into a leucine auxotroph of and placed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene () promoter. After confirming correct expression of the ME genes, the two recombinant strains were grown in fully controlled, submerged-culture bioreactors using a high C : N ratio medium for lipid accumulation. Activities of ME were increased by two- to threefold and the lipid contents of the cells, in both recombinants, were increased from 12 % of the biomass to 30 %. Simultaneously, the degree of fatty acid desaturation increased slightly. Thus, increased expression of the ME gene leads to both increased biosynthesis of fatty acids and formation of unsaturated fatty acids, including -linolenic acid (18 : 3 n-6). At the end of lipid accumulation (96 h), ME activity in the recombinant strains had ceased, as it had done in the parent wild-type cells, indicating that additional, but unknown, controls over its activity must be in place to account for this loss of activity: this may be due to the presence of a specific ME-cleaving enzyme. The hypothesis that the rate-limiting step of fatty acid biosynthesis is therefore the supply of NADPH, as generated specifically and solely by ME, is therefore considerably strengthened by these results.

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2007-07-01
2024-03-29
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References

  1. Appel K. F., Wolff A. M., Arnau J. 2004; A multicopy vector sysem for genetic studies in Mucor circinelloides and other zygomycetes. Mol Genet Genomics 271:595–602 [CrossRef]
    [Google Scholar]
  2. Borsch D., Westhoff P. 1990; Primary structure of NADP+-dependent malic enzyme in the dicotyledonous C4 plant Flaveria trinervia. FEBS Lett 273:111–115 [CrossRef]
    [Google Scholar]
  3. Botham P. A., Ratledge C. 1979; A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous microorganisms. J Gen Microbiol 114:361–375 [CrossRef]
    [Google Scholar]
  4. Boulton C. A., Ratledge C. 1981; Correlation of lipid accumulation in yeasts with possession of ATP : citrate lyase. J Gen Microbiol 127:169–176
    [Google Scholar]
  5. Broun P., Gettner S., Somerville C. 1999; Genetic engineering of plant lipids. Annu Rev Nutr 19:197–216 [CrossRef]
    [Google Scholar]
  6. Castelein H., Gulick T., Declercq P. E., Mannaerts G. P., Moore D. D., Baes M. I. 1994; The peroxisome proliferator activated receptor regulates malic enzyme gene expression. J Biol Chem 269:26754–26758
    [Google Scholar]
  7. Ceddia R. B., William W. N., Lima F. B., Flandin P., Curi R., Giacobino J. P. 2000; Leptin stimulates uncoupling protein-2 mRNA expression and Krebs cycle activity and inhibits lipid synthesis in isolated rat white adipocytes. Eur J Biochem 267:5952–5958 [CrossRef]
    [Google Scholar]
  8. Chang G. G., Tong L. 2003; Structure and function of malic enzymes, a new class of oxidative decarboxylases. Biochemistry 42:12721–12733 [CrossRef]
    [Google Scholar]
  9. Chang G. G., Wang J. K., Huang T. M., Lee H. J., Chou W. Y., Meng C. L. 1991; Purification and characterisation of the NADP+-dependent cytosolic malic enzyme from human cancer cell line. Eur J Biochem 202:681–688 [CrossRef]
    [Google Scholar]
  10. Cohen Z., Ratledge C. 2005 Single Cell Oils Champaign, IL: AOCS Press;
    [Google Scholar]
  11. Dean R. A., Talbot N. J., Ebbole D. J., Farman M. L., Mitchell T. K., Orbach M. J., Thon M., Kulkarni R., Xu J. R. other authors 2005; The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980–986 [CrossRef]
    [Google Scholar]
  12. Drincovich M. F., Casati P., Andreo C. S. 2001; NADP-malic enzyme from plants: a ubiquitous enzyme involved in different metabolic pathways. FEBS Lett 490:1–6 [CrossRef]
    [Google Scholar]
  13. Eichinger L., Pachebat J. A., Glockner G., Rajandream M. A., Sucgang R., Berriman M., Song J., Olsen R., Szafranski K. other authors 2005; The genome of the social amoeba Dictyostelium discoideum. Nature 435:43–57 [CrossRef]
    [Google Scholar]
  14. Evans C. T., Ratledge C. 1983; Biochemical activities during lipid accumulation in Candida curvata. Lipids 18:630–635 [CrossRef]
    [Google Scholar]
  15. Evans C. T., Ratledge C. 1984; Phosphofructokinase and the regulation of the flux of carbon from glucose to lipid in the oleaginous yeast Rhodosporidium toruloides. J Gen Microbiol 130:3251–3264
    [Google Scholar]
  16. Evans C. T., Ratledge C. 1985; Possible regulatory roles of ATP : citrate lyase, malic enzyme and AMP deaminase in lipid accumulation by the oleaginous yeast Rhodosporidium toruloides CBS 14. Can J Microbiol 31:1000–1005 [CrossRef]
    [Google Scholar]
  17. Galagan J. E., Calvo S. E., Borkovich K. A. 2003; The genome sequence of the filamentous fungus Neurospora crassa. Nature 422:859–868 [CrossRef]
    [Google Scholar]
  18. Galagan J. E., Calvo S. E., Cuomo C., Ma L. J., Wortman J. R., Batzoglou S., Lee S. I., Basturkmen M., Spevak C. C. other authors 2005; Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438:1105–1115 [CrossRef]
    [Google Scholar]
  19. Hillgartner F. B., Charron T. 1998; Glucose stimulates transcription of fatty acid synthase and malic enxyme in avian hepatocytes. Am J Physiol 274:E493–E501
    [Google Scholar]
  20. Kendrick A., Ratledge C. 1992; Desaturation of polyunsaturated fatty acids in Mucor circinelloides and the involvement of a novel membrane-bound malic enzyme. Eur J Biochem 209:667–673 [CrossRef]
    [Google Scholar]
  21. Kulkarni G., Cook P. R., Harris B. G. 1993; Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum malic enzyme. Arch Biochem Biophys 300:231–237 [CrossRef]
    [Google Scholar]
  22. Li Y., Adams I. P., Wynn J. P., Ratledge C. 2005; Cloning and characterization of a gene encoding a malic enzyme involved in anaerobic growth in Mucor circinelloides. Mycol Res 109:461–468 [CrossRef]
    [Google Scholar]
  23. Mackenzie D. A., Wongwathanarat P., Carter A. T., Archer D. B. 2000; Isolation and use of a homologous histone H4 promoter and a ribosomal DNA region in a transformation vector for the oil-producing fungus Mortierella alpina. Appl Environ Microbiol 66:4655–4661 [CrossRef]
    [Google Scholar]
  24. Michaelson L. V., Lazarus C. M., Griffiths G., Napier J. A., Stobart K. A. 1998; Isolation of a Δ5-fatty acid desaturase gene from Mortierella alpina. J Biol Chem 273:19055–19059 [CrossRef]
    [Google Scholar]
  25. Ohlrogge J. B., Jaworski J. G. 1997; Regulation of fatty acid synthesis. Annu Rev Plant Physiol Plant Mol Biol 48:109–136 [CrossRef]
    [Google Scholar]
  26. Ramli U. S., Salas J. J., Quant P. A., Harwood J. L. 2005; Metabolic control analysis reveals an important role for diacylglycerol acyltransferase in olive but not in oil palm lipid accumulation. FEBS J 272:5764–5770 [CrossRef]
    [Google Scholar]
  27. Rangasamy D., Ratledge C. 2000; Genetic enhancement of fatty acid synthesis by targeting rat liver ATP : citrate lyase into plastids of tobacco. Plant Physiol 122:1231–1238 [CrossRef]
    [Google Scholar]
  28. Ratledge C. 1997; Microbial lipids. In Biotechnology , 2nd edn. vol. 7 pp 135–197 Edited by Kleinkauf H., Dohren H. Weinheim, Germany: VCH;
    [Google Scholar]
  29. Ratledge C. 2004; Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production. Biochimie 86:807–815 [CrossRef]
    [Google Scholar]
  30. Ratledge C. 2005; Single Cell Oils for the 21st Century. In Single Cell Oils, pp 1–20 Edited by Cohen Z., Ratledge C. Champaign, IL: AOCS Press;
    [Google Scholar]
  31. Ratledge C., Gilbert S. C. 1985; Carnitine acetyltransferase activity in oleaginous yeasts. FEMS Microbiol Lett 27:273–275 [CrossRef]
    [Google Scholar]
  32. Ratledge C., Hopkins S. 2006a; Lipids from microbial sources. In Modifying Lipids for Use in Foods, pp 80–113 Edited by Gunstone F. Abington, UK: Woodhead Publishing;
    [Google Scholar]
  33. Ratledge C., Hopkins S. 2006b; Applications and safety of microbial oils. In Modifying Lipids for Use in Foods pp 567–585 Edited by Gunstone F. Abington, UK: Woodhead Publishing;
    [Google Scholar]
  34. Ratledge C., Wynn J. P. 2002; The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 51:1–51
    [Google Scholar]
  35. Rognstad R., Katz J. 1979; Effects of 2,4-dihydroxybutyrate on lipogenesis in rat hepatocytes. J Biol Chem 254:11969–11972
    [Google Scholar]
  36. Rothermel B. A., Nelson T. 1989; Primary structure of the maize NADP+-dependent malic enzyme. J Biol Chem 264:19587–19592
    [Google Scholar]
  37. Schipper M. A. A. 1976; On Mucor circinelloides, Mucor racemosus and related species. Stud Mycol 12:1–40
    [Google Scholar]
  38. Shimomura I., Shimano H., Korn B. S., Bashmakov Y., Horton J. D. 1998; Nuclear sterol regulatory element-binding proteins activate genes responsible for the entire program of unsaturated fatty acid biosynthesis in transgenic mouse liver. J Biol Chem 273:35299–35306 [CrossRef]
    [Google Scholar]
  39. Song Y., Wynn J. P., Li Y., Grantham D., Ratledge C. 2001; A pregenetic study of the isoforms of malic enzyme associated with lipid accumulation in Mucor circinelloides. Microbiology 147:1507–1515
    [Google Scholar]
  40. Sourdioux M., Brevelet C., Delabrosse Y., Douaire M. 1999; Association of fatty acid synthase gene and malic enzyme gene polymorphisms with fatness in turkeys. Poult Sci 78:1651–1657 [CrossRef]
    [Google Scholar]
  41. Thelen J. J., Ohlrogge J. B. 2002; Metabolic engineering of fatty acid biosynthesis in plants. Metab Eng 4:12–21 [CrossRef]
    [Google Scholar]
  42. van Heeswijck R., Roncero M. I. 1984; High frequency transformation of Mucor with recombinant plasmid DNA. Carlsberg Res Commun 49:691–701 [CrossRef]
    [Google Scholar]
  43. Velayos A., Blasco J. L., Alvarez M. I., Iturriaga E. A., Eslava A. P. 2000; Blue-light regulation of phytoene dehydrogenase ( carB ) gene expression in Mucor circinelloides. Planta 210:938–946 [CrossRef]
    [Google Scholar]
  44. Wierenga R. K., Terpstra P., Hol W. G. J. 1986; Prediction of the occurrence of the ADP-binding β α β -fold in proteins, using an amino acid sequence fingerprint. J Mol Biol 187:101–107 [CrossRef]
    [Google Scholar]
  45. Wolff A. M., Appel K. F., Petersen J. B., Poulsen U., Arnau J. 2002; Identification and analysis of genes involved in the control of dimorphism in Mucor circinelloides (syn. racemosus. FEMS Yeast Res 2:203–213
    [Google Scholar]
  46. Wongwathanarat P., Michaelson L. V., Carter A. T., Lazarus C. M., Griffiths G., Stobart A. K., Archer D. B., MacKenzie D. A. 1999; Two fatty acid delta 9-desaturase genes, ole1 and ole2 , from Mortierella alpina complement the yeast ole1 mutation. Microbiology 145:2939–2946
    [Google Scholar]
  47. Wynn J. P., Ratledge C. 1997; Malic enzyme is a major source of NADPH for lipid accumulation by Aspergillus nidulans. Microbiology 143:253–257 [CrossRef]
    [Google Scholar]
  48. Wynn J. P., Ratledge C. 2000; Evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of the 18 : 3 to 20 : 3 elongase. Microbiology 146:2325–2331
    [Google Scholar]
  49. Wynn J. P., Kendrick A., Ratledge C. 1997; Sesamol as an inhibitor of growth and lipid metabolism in Mucor circinelloides via its action on malic enzyme. Lipids 32:605–610 [CrossRef]
    [Google Scholar]
  50. Wynn J. P., Hamid A. A., Midgley M., Ratledge C. 1998; Widespread occurrence of ATP : citrate lyase and carnitine acetyltransferase in filamentous fungi. World J Microbiol Biotechnol 14:145–147
    [Google Scholar]
  51. Wynn J. P., Hamid A. A., Ratledge C. 1999; The role of malic enzyme in the regulation of lipid accumulation in filamentous fungi. Microbiology 145:1911–1917 [CrossRef]
    [Google Scholar]
  52. Wynn J. P., Hamid A. A., Li Y., Ratledge C. 2001; Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina. Microbiology 147:2857–2864
    [Google Scholar]
  53. Yang Z., Floyd D. L., Loeber G., Tong L. 2000; Structure of a closed form of human malic enzyme and implications for catalytic mechanism. Nat Struct Biol 7:251–257 [CrossRef]
    [Google Scholar]
  54. Yang Z., Zhang H., Hung H. C., Kuo C. C., Tsai L. C., Yuan H. S., Chou W. Y., Chang G. G., Tong L. 2002; Structural studies of the pigeon cytosolic NADP+-dependent malic enzyme. Protein Sci 11:332–341
    [Google Scholar]
  55. Zhang Y. 2005; A biochemical and molecular study of the roles of malic enzyme in lipid accumulation in Mortierella alpina and Mucor circinelloides . PhD thesis University of Hull;
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