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Alteration of Wax Ester Content and Composition in Euglena gracilis with Gene Silencing of 3-ketoacyl-CoA Thiolase Isozymes


Euglena gracilis produces wax ester under hypoxic and anaerobic culture conditions with a net synthesis of ATP. In wax ester fermentation, fatty acids are synthesized by reversing beta-oxidation in mitochondria. A major species of wax ester produced by E. gracilis is myristyl myristate (14:0-14:0Alc). Because of its shorter carbon chain length with saturated compounds, biodiesel produced from E. gracilis wax ester may have good cold flow properties with high oxidative stability. We reasoned that a slight metabolic modification would enable E. gracilis to produce a biofuel of ideal composition. In order to produce wax ester with shorter acyl chain length, we focused on isozymes of the enzyme 3-ketoacyl-CoA thiolase (KAT), a condensing enzyme of the mitochondrial fatty acid synthesis pathway in E. gracilis. We performed a gene silencing study of KAT isozymes in E. gracilis. Six KAT isozymes were identified in the E. gracilis EST database, and silencing any three of them (EgKAT1-3) altered the wax ester amount and composition. In particular, silencing EgKAT1 induced a significant compositional shift to shorter carbon chain lengths in wax ester. A model fuel mixture inferred from the composition of wax ester in EgKAT1-silenced cells showed a significant decrease in melting point compared to that of the control cells.

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Coenzyme A


Differential scanning calorimetry


Expressed sequence tag


Fatty acid


Fatty alcohol


Fatty acid methyl ester


Type-2 fatty acid synthase


3-Ketoacyl-CoA thiolase


Reverse transcription




  1. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54:621–639

    Article  CAS  PubMed  Google Scholar 

  2. Sirevåg R, Levine RP (1972) Fatty acid synthetase from Chlamydomonas reinhardi. J Biol Chem 247:2586–2591

    PubMed  Google Scholar 

  3. Fan J, Andre C, Xu C (2011) A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii. FEBS Lett 585:1985–1991

    Article  CAS  PubMed  Google Scholar 

  4. Fan J, Yan C, Andre C, Shanklin J, Schwender J, Xu C (2012) Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii. Plant Cell Physiol 53:1380–1390

    Article  CAS  PubMed  Google Scholar 

  5. Knothe G (2009) Improving biodiesel fuel properties by modifying fatty ester composition. Energy Environ Sci 2:759–766

    Article  CAS  Google Scholar 

  6. Stansell GR, Gray VM, Sym SD (2011) Microalgal fatty acid composition: implications for biodiesel quality. J Appl Phycol 24:791–801

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Inui H, Miyatake K, Nakano Y, Kitaoka S (1983) Production and composition of wax esters by fermentation of Euglena gracilis. Agric Biol Chem 47:2669–2671

    Article  CAS  Google Scholar 

  9. Inui H, Miyatake K, Nakano Y, Kitaoka S (1984) Fatty acid synthesis in mitochondria of Euglena gracilis. Eur J Biochem 142:121–126

    Article  CAS  PubMed  Google Scholar 

  10. Teerawanichpan P, Qiu X (2010) Fatty acyl-CoA reductase and wax synthase from Euglena gracilis in the biosynthesis of medium-chain wax esters. Lipids 45:263–273

    Article  CAS  PubMed  Google Scholar 

  11. Fox AR, Soto G, Mozzicafreddo M, Garcia AN, Cuccioloni M, Angeletti M, Salerno JC, Ayub ND (2014) Understanding the function of bacterial and eukaryotic thiolases II by integrating evolutionary and functional approaches. Gene 533:5–10

    Article  CAS  PubMed  Google Scholar 

  12. Oda Y, Nakano Y, Kitaoka S (1982) Utilization and toxicity of exogenous amino acids in Euglena gracilis. J Gen Microbiol 128:853–858

    CAS  Google Scholar 

  13. Koren LE, Hutner SH (1967) High-yield media for photosynthesizing Euglena gracilis z. J Protozool 14(Supplement):17

    Google Scholar 

  14. Sauch JF, Flanigan D, Galvin ML, Berman D, Jakubowski W (1991) Propidium iodide as an indicator of Giardia cyst viability. Appl Environ Microbiol 57:3243–3247

    PubMed Central  CAS  PubMed  Google Scholar 

  15. O’Brien EA, Koski LB, Zhang Y, Yang L, Wang E, Gray MW, Burger G, Lang BF (2007) TBestDB: a taxonomically broad database of expressed sequence tags (ESTs). Nucleic Acids Res 35:D445–D451

    Article  PubMed Central  PubMed  Google Scholar 

  16. Nakazawa M, Minami T, Teramura K, Kumamoto S, Hanato S, Takenaka S, Ueda M, Inui H, Nakano Y, Miyatake K (2005) Molecular characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitratelyase, in Euglena gracilis. Comp Biochem Physiol B Biochem Mol Biol 141:445–452

    Article  PubMed  Google Scholar 

  17. Tessier L, Keller M, Chan RL, Fournier R, Weil JH, Imbault P (1991) RNAs to pre-mature mRNAs by trans-splicing in Euglena. EMBO J 10:2621–2625

    PubMed Central  CAS  PubMed  Google Scholar 

  18. Iseki M, Matsunaga S, Murakami A, Ohno K, Shiga K, Yoshida K, Sugai M, Takahashi T, Hori T, Watanabe M (2002) A blue-light-activated adenylyl cyclase mediates photo avoidance in Euglena gracilis. Nature 415:1047–1051

    Article  CAS  PubMed  Google Scholar 

  19. Ishikawa T, Tajima N, Nishikawa H, Gao Y, Rapolu M, Shibata H, Sawa Y, Shigeoka S (2010) Euglena gracilisas corbate peroxidase forms an intramolecular dimeric structure: its unique molecular characterization. Biochem J 426:125–134

    Article  CAS  PubMed  Google Scholar 

  20. Takenaka S, Kondo T, Nazeri S, Tamura Y, Tokunaga M, Tsuyama S, Miyatake K, Nakano Y (1997) Accumulation of trehalose as a compatible solute under osmotic stress in Euglena gracilis Z. J Eukaryot Microbiol 44:609–613

    Article  CAS  Google Scholar 

  21. Hodge JE, Hofreiter BT (1962) Determination of reducing sugars and carbohydrates. In: Whistler RL, Wolfrom ML (eds) Methods in carbohydrate chemistry, vol 1. Academic press, New York, pp 380–394

    Google Scholar 

  22. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917

    Article  CAS  PubMed  Google Scholar 

  23. Christie WW (1993) Preparation of ester derivatives of fattyacids for chromatographic analysis. In: Christie WW (ed) Advances in lipid methodology—two. The Oily Press, Dundee, UK, pp 69–111

    Google Scholar 

  24. Dunn RO (1999) Thermal analysis of alternative diesel fuels from vegetable oils. J Am Oil Chem Soc 76:109–115

    Article  CAS  Google Scholar 

  25. Meriläinen G, Poikela V, Kursula P, Wierenga RK (2009) The thiolase reaction mechanism: the importance of Asn316 and His348 for stabilizing the enolate intermediate of the Claisen condensation. Biochemistry 48:11011–11025

    Article  PubMed  Google Scholar 

  26. Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971

    Article  CAS  PubMed  Google Scholar 

  27. Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–36

    Article  CAS  PubMed  Google Scholar 

  28. Inui H, Ochi H, Nogami K, Miyatake K, Nakano Y, Kitaoka S (1988) Effect of thiamin deficiency on wax ester fermentation in Euglena gracilis. Agric Biol Chem 52:49–54

    Article  CAS  Google Scholar 

  29. Thelen JJ, Ohlrogge JB (2002) Metabolic engineering of fatty acid biosynthesis in plants. Metab Eng 4:12–21

    Article  CAS  PubMed  Google Scholar 

  30. Eccleston V, Ohlrogge J (1998) Expression of lauroyl-acyl carrier protein thioesterase in Brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and implies a set point for triacylglycerol accumulation. Plant Cell 10:613–622

    PubMed Central  CAS  PubMed  Google Scholar 

  31. Radakovits R, Eduafo P, Posewitz MC (2010) Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum. Metab Eng 13:89–95

    Article  PubMed  Google Scholar 

  32. Dellomonaco C, Clomburg JM, Miller EN, Gonzalez R (2011) Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature 476:355–359

    Article  CAS  PubMed  Google Scholar 

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This work was supported by the Japan Science and Technology Agency (JST), PRESTO program. The authors thank Dr. Joseph Rodrigue for critical reading of the manuscript.

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The authors declare that they have no competing interests.

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Correspondence to Masami Nakazawa.

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Nakazawa, M., Andoh, H., Koyama, K. et al. Alteration of Wax Ester Content and Composition in Euglena gracilis with Gene Silencing of 3-ketoacyl-CoA Thiolase Isozymes. Lipids 50, 483–492 (2015).

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  • Euglena gracilis
  • Wax ester fermentation
  • 3-Ketoacyl-CoA thiolase
  • Mitochondrial fatty acid synthesis
  • Acyl chain length modification
  • Algal lipids