Skip to main content
SpringerLink
Log in

Fatty Acyl-CoA Reductase and Wax Synthase from Euglena gracilis in the Biosynthesis of Medium-Chain Wax Esters

  • Original Article
  • Published:
Lipids

Abstract

Euglena gracilis, a unicellular phytoflagellate, can accumulate a large amount of medium-chain wax esters under anaerobic growth conditions. Here we report the identification and characterization of two genes involved in the biosynthesis of wax esters in E. gracilis. The first gene encodes a fatty acyl-CoA reductase (EgFAR) involved in the conversion of fatty acyl-CoAs to fatty alcohols and the second gene codes for a wax synthase (EgWS) catalyzing esterification of fatty acyl-CoAs and fatty alcohols, yielding wax esters. When expressed in yeast (Saccharomyces cerevisiae), EgFAR converted myristic acid (14:0) and palmitic acid (16:0) to their corresponding alcohols (14:0Alc and 16:0Alc) with myristic acid as the preferred substrate. EgWS utilized a broad range of fatty acyl-CoAs and fatty alcohols as substrates with the preference towards myristic acid and palmitoleyl alcohol. The wax biosynthetic pathway was reconstituted by co-expressing EgFAR and EgWS in yeast. When myristic acid was fed to the yeast, myristyl myristate (14:0–14:0), myristyl palmitoleate (14:0–16:1), myristyl palmitate (14:0–16:0) and palmityl myristate (16:0–14:0) were produced. These results indicate EgFAR and EgWS are likely the two enzymes involved in the biosynthesis of medium-chain wax esters in E. gracilis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

CoA:

Coenzyme A

FAMEs:

Fatty acid methyl esters

FAR:

Fatty acyl-CoA reductase

RACE:

Rapid amplification of cDNA ends

WS:

Wax synthase

References

  1. Inui H, Miyatake K, Nakano Y, Kitaoka S (1982) Wax ester fermentation in Euglena gracilis. FEBS Lett 150:89–93

    Article  CAS  Google Scholar 

  2. Rosenberg A (1963) A comparison of lipid patterns in photosynthesizing and non-photosynthesizing cells of Euglena gracilis. Biochemistry 2:1148–1154

    Article  CAS  PubMed  Google Scholar 

  3. Tucci S, Vacula R, Krajcovic J, Proksch P, Martin W (2009) Variability of wax ester fermentation in natural and bleached Euglena gracilis strains in response to oxygen and the elongase Inhibitor Flufenacet. J Eukaryot Microbiol 57:63–69

    Google Scholar 

  4. Koritala S (1989) Microbiological synthesis of wax esters by Euglena gracilis. J Am Oil Chem Soc 66:133–134

    Article  CAS  Google Scholar 

  5. Schneider T, Betz A (1985) Waxmonoester fermentation in Euglena gracilis T. Factors favouring the synthesis of odd-numbered fatty acids and alcohols. Planta 166:67–73

    Article  CAS  Google Scholar 

  6. Tani Y, Okumura M, Li S (1987) Liquid wax ester production by Euglena gracilis. Agric Biol Chem 51:225–230

    CAS  Google Scholar 

  7. Regnault A, Chervin D, Chammai A, Pitona F, Calvayraca R, Mazliakb P (1995) Lipid composition of Euglena gracilis in relation to carbon–nitrogen balance. Phytochemistry 40:725–733

    Article  CAS  Google Scholar 

  8. Kawabata A, Inui H, Miyatake K, Nakano Y, Kitaoka S (1990) Production and composition of Euglena wax esters at high temperature. Agric Biol Chem 54:811–812

    CAS  Google Scholar 

  9. Samuels L, Kunst L, Jetter R (2008) Sealing plant surfaces: cuticular wax formation by epidermal cells. Annu Rev Plant Biol 59:683–707

    Article  CAS  PubMed  Google Scholar 

  10. Cheng JB, Russell DW (2004) Mammalian wax biosynthesis. I. Identification of two fatty acyl-coenzyme A reductases with different substrate specificities and tissue distributions. J Biol Chem 279:37789–37797

    Article  CAS  PubMed  Google Scholar 

  11. Moto K, Yoshiga T, Yamamoto M, Takahashi S, Okano K, Ando T, Nakata T, Matsumoto S (2003) Pheromone gland-specific fatty-acyl reductase of the silkmoth, Bombyx mori. Proc Natl Acad Sci USA 100:9156–9161

    Article  CAS  PubMed  Google Scholar 

  12. Antony B, Fujii T, Moto K, Matsumoto S, Fukuzawa M, Nakano R, Tatsuki S, Ishikawa Y (2009) Pheromone-gland-specific fatty-acyl reductase in the adzuki bean borer, Ostrinia scapulalis (Lepidoptera: Crambidae). Insect Biochem Mol Biol 39:90–95

    Article  CAS  PubMed  Google Scholar 

  13. Miwa T (1971) Jojoba oil wax esters and derived fatty acids and alcohols: gas chromatographic analyses. J Am Oil Chem Soc 48:259–264

    Article  CAS  Google Scholar 

  14. Doan TT, Carlsson AS, Hamberg M, Bulow L, Stymne S, Olsson P (2008) Functional expression of five Arabidopsis fatty acyl-CoA reductase genes in Escherichia coli. J Plant Physiol 166:787–796

  15. Rowland O, Zheng H, Hepworth SR, Lam P, Jetter R, Kunst L (2006) CER4 encodes an alcohol-forming fatty acyl-coenzyme A reductase involved in cuticular wax production in Arabidopsis. Plant Physiol 142:866–877

    Article  CAS  PubMed  Google Scholar 

  16. Wang A, Xia Q, Xie W, Dumonceaux T, Zou J, Datla R, Selvaraj G (2002) Male gametophyte development in bread wheat (Triticum aestivum L.): molecular, cellular, and biochemical analyses of a sporophytic contribution to pollen wall ontogeny. Plant J 30:613–623

    Article  CAS  PubMed  Google Scholar 

  17. Cheng JB, Russell DW (2004) Mammalian wax biosynthesis. II. Expression cloning of wax synthase cDNAs encoding a member of the acyltransferase enzyme family. J Biol Chem 279:37798–37807

    Article  CAS  PubMed  Google Scholar 

  18. Lardizabal KD, Metz JG, Sakamoto T, Hutton WC, Pollard MR, Lassner MW (2000) Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic Arabidopsis. Plant Physiol 122:645–655

    Article  CAS  PubMed  Google Scholar 

  19. King A, Nam JW, Han J, Hilliard J, Jaworski JG (2007) Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes wax-esters. Planta 226:381–394

    Article  CAS  PubMed  Google Scholar 

  20. Kalscheuer R, Steinbuchel A (2003) A novel bifunctional wax ester synthase/acyl-CoA: diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278:8075–8082

    Article  CAS  PubMed  Google Scholar 

  21. Li F, Wu X, Lam P, Bird D, Zheng H, Samuels L, Jetter R, Kunst L (2008) Identification of the wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase WSD1 required for stem wax ester biosynthesis in Arabidopsis. Plant Physiol 148:97–107

    Article  CAS  PubMed  Google Scholar 

  22. Khan AA, Kolattukudy PE (1973) Control of synthesis and distribution of acyl moieties in etiolated Euglena gracilis. Biochemistry 12:1939–1948

    Article  CAS  PubMed  Google Scholar 

  23. Kolattukudy PE (1970) Reduction of fatty acids to alcohols by cell-free preparations of Euglena gracilis. Biochemistry 9:1095–1102

    Article  CAS  PubMed  Google Scholar 

  24. Schlösser UG (1994) SAG—Sammlung von Algenkulturen at the University of Göttingen, catalogue of strains. Bot Acta 107:113–186

    Google Scholar 

  25. Meesapyodsuk D, Reed DW, Covello PS, Qiu X (2007) Primary structure, regioselectivity, and evolution of the membrane-bound fatty acid desaturases of Claviceps purpurea. J Biol Chem 282:20191–20199

    Article  CAS  PubMed  Google Scholar 

  26. Sandager L, Gustavsson MH, Stahl U, Dahlqvist A, Wiberg E, Banas A, Lenman M, Ronne H, Stymne S (2002) Storage lipid synthesis is non-essential in yeast. J Biol Chem 277:6478–6482

    Article  CAS  PubMed  Google Scholar 

  27. Katavic V, Mietkiewska E, Barton DL, Giblin EM, Reed DW, Taylor DC (2002) Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution. Eur J Biochem 269:5625–5631

    Article  CAS  PubMed  Google Scholar 

  28. Li R, Reed DW, Liu E, Nowak J, Pelcher LE, Page JE, Covello PS (2006) Functional genomic analysis of alkaloid biosynthesis in Hyoscyamus niger reveals a cytochrome P450 involved in littorine rearrangement. Chem Biol 13:513–520

    Article  CAS  PubMed  Google Scholar 

  29. Rossmann MG, Moras D, Olsen KW (1974) Chemical and biological evolution of nucleotide-binding protein. Nature 250:194–199

    Article  CAS  PubMed  Google Scholar 

  30. Aarts MG, Hodge R, Kalantidis K, Florack D, Wilson ZA, Mulligan BJ, Stiekema WJ, Scott R, Pereira A (1997) The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes. Plant J 12:615–623

    Article  CAS  PubMed  Google Scholar 

  31. Metz JG, Pollard MR, Anderson L, Hayes TR, Lassner MW (2000) Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed. Plant Physiol 122:635–644

    Article  CAS  PubMed  Google Scholar 

  32. Kalscheuer R, Luftmann H, Steinbuchel A (2004) Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase. Appl Environ Microbiol 70:7119–7125

    Article  CAS  PubMed  Google Scholar 

  33. Kolattukudy PE (1971) Enzymatic synthesis of fatty alcohols in Brassica oleracea. Arch Biochem Biophys 142:701–709

    Article  CAS  PubMed  Google Scholar 

  34. Reiser S, Somerville C (1997) Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl coenzyme A reductase. J Bacteriol 179:2969–2975

    CAS  PubMed  Google Scholar 

  35. Vioque J, Kolattukudy PE (1997) Resolution and purification of an aldehyde-generating and an alcohol-generating fatty acyl-CoA reductase from pea leaves (Pisum sativum L.). Arch Biochem Biophys 340:64–72

    Article  CAS  PubMed  Google Scholar 

  36. Pollard MR, McKeon T, Gupta LM, Stumpf PK (1979) Studies on biosynthesis of waxes by developing jojoba seed. II. The demonstration of wax biosynthesis by cell-free homogenates. Lipids 14:651–662

    Article  CAS  Google Scholar 

  37. Moore C, Snyder F (1982) Properties of microsomal acyl coenzyme A reductase in mouse preputial glands. Arch Biochem Biophys 214:489–499

    Article  CAS  PubMed  Google Scholar 

  38. Wang X, Kolattukudy PE (1995) Solubilization, purification and characterization of fatty acyl-CoA reductase from duck uropygial gland. Biochem Biophys Res Commun 208:210–215

    Article  CAS  PubMed  Google Scholar 

  39. Johnson RC, Gilbertson JR (1972) Isolation, characterization, and partial purification of a fatty acyl coenzyme A reductase from bovine cardiac muscle. J Biol Chem 247:6991–6998

    CAS  PubMed  Google Scholar 

  40. Falk-Petersen S, Hagen W, Kattner G, Clarke A, Sargent JR (2000) Lipids, trophic relationships, and biodiversity in Arctic and Antarctic krill. Can J Fish Aquat Sci 57:178–191

    Article  CAS  Google Scholar 

  41. Kattner G, Hagen W (1998) Lipid metabolism of the Antarctic euphausid Euphausia crystallorophias and its ecological implications. Mar Ecol Prog Ser 170:203–213

    Article  CAS  Google Scholar 

  42. Kolattukudy PE, Bohnet S, Rogers L (1987) Diesters of 3-hydroxy fatty acids produced by the uropygial glands of female mallards uniquely during the mating season. J Lipid Res 28:582–588

    CAS  PubMed  Google Scholar 

  43. Rijpstra WI, Reneerkens J, Piersma T, Damste JS (2007) Structural identification of the β-hydroxy fatty acid-based diester preen gland waxes of shorebirds. J Nat Prod 70:1804–1807

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are very grateful to Patricia Vrinten for technical assistance in gene isolation. We thank Devin Polichuk, Darwin Reed and Valerie Catinot for technical help and discussions. This work is part of ICON, a European Community Seventh Framework Programme.

Author information

Authors and Affiliations

  1. Department of Food and Bioproduct Sciences, College of Agriculture and Bioresources, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, S7N 5A8, Canada

    Prapapan Teerawanichpan & Xiao Qiu

Authors
  1. Prapapan Teerawanichpan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiao Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiao Qiu.

About this article

Cite this article

Teerawanichpan, P., Qiu, X. Fatty Acyl-CoA Reductase and Wax Synthase from Euglena gracilis in the Biosynthesis of Medium-Chain Wax Esters. Lipids 45, 263–273 (2010). https://doi.org/10.1007/s11745-010-3395-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11745-010-3395-2

Keywords

Search

Navigation