Comparative proteomic analysis of mitochondria isolated from Euglena gracilis under aerobic and hypoxic conditions

Autoři: Shun Tamaki aff001;  Kohei Nishino aff001;  Takahisa Ogawa aff001;  Takanori Maruta aff001;  Yoshihiro Sawa aff001;  Kazuharu Arakawa aff003;  Takahiro Ishikawa aff001
Působiště autorů: Institute of Agricultural and Life Sciences, Academic Assembly, Shimane University, Matsue, Shimane, Japan aff001;  Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo, Japan aff002;  Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan aff003;  Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa, Japan aff004
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


The unicellular microalga Euglena gracilis produces wax esters for ATP acquisition under low-oxygen conditions. The regulatory mechanism of wax ester production is not yet understood. Indeed, our previous transcriptomic analysis showed that transcript levels of genes involved in the wax ester synthesis hardly changed under hypoxic conditions, suggesting contribution of post-transcriptional regulation. In this study, we conducted a proteome analysis of E. gracilis mitochondria, as this organelle employs the fatty-acid synthesis pathway under hypoxic conditions. Mitochondria were isolated from E. gracilis SM-ZK strain treated with both aerobic and hypoxic conditions and used for shotgun proteomic analysis. Three independent proteomic analyses succeeded in identifying a total of 714 non-redundant proteins. Of these, 229 were detected in common to all experiments, and 116 were significantly recognized as differentially expressed proteins. GO enrichment analysis suggested dynamic changes in mitochondrial metabolic pathways and redox reactions under aerobic and hypoxic conditions. Protein levels of bifunctional enzymes isocitrate lyase and malate synthase in glyoxylate cycle were 1.35-fold higher under hypoxic conditions. Abundances of the propionyl-CoA synthetic enzymes, succinyl-CoA synthetase and propionyl-CoA carboxylase, were also 1.35- and 1.47-fold higher, respectively, under hypoxic conditions. Protein levels of pyruvate:NADP+ oxidoreductase, a key enzyme for anaerobic synthesis of acetyl-CoA, which serves as a C2 donor for fatty acids, showed a 1.68-fold increase under hypoxic conditions, whereas those of pyruvate dehydrogenase subunits showed a 0.77–0.81-fold decrease. Protein levels of the fatty-acid synthesis enzymes, 3-ketoacyl-CoA thiolase isoforms (KAT1 and KAT2), 3-hydroxyacyl-CoA dehydrogenases, and acyl-CoA dehydrogenase were up-regulated by 1.20- to 1.42-fold in response to hypoxic treatment. Overall, our proteomic analysis revealed that wax ester synthesis-related enzymes are up-regulated at the protein level post-transcriptionally to promote wax ester production in E. gracilis under low-oxygen conditions.

Klíčová slova:

Dehydrogenases – Enzymes – Esters – Fatty acids – Hypoxia – Mitochondria – Proteomics – Waxes


1. Krajčovič J, Vesteg M, Schwartzbach SD. Euglenoid flagellates: a multifaceted biotechnology platform. J. Biotechnol. 2015; 202: 135–45. doi: 10.1016/j.jbiotec.2014.11.035 25527385

2. Barras DR, Stone BA (1968) In: Buetow DE, editor. Biology of Euglena, vol II. Academic Press New York; 1968; 141–91.

3. Inui H, Miyatake K, Nakano Y, Kitaoka S. Wax ester fermentation in Euglena gracils. FEBS Lett. 1982; 150: 89–93.

4. Inui H, Ishikawa T, Tamoi M. Wax ester fermentation and its application for biofuel production. Adv. Exp. Med. Biol. 2017; 979: 269–83. doi: 10.1007/978-3-319-54910-1_13 28429326

5. Yanowitz J, Ratcliff MA, McCormick RL, Taylor JD, Murphy MJ. Compendium of experimental cetane numbers. In: Technical Report. National Renewable Energy Laboratory. 2014;

6. Inui H, Miyatake K, Nakano Y, Kitaoka S. Fatty acid synthesis in mitochondria of Euglena gracilis. Eur J Biochem. 1984; 142: 121–6. doi: 10.1111/j.1432-1033.1984.tb08258.x 6146525

7. Inui H, Miyatake K, Nakano Y, Kitaoka S. Pyruvate:NADP+ oxidoreductase from Euglena gracilis: mechanism of O2-inactivation of the enzyme and its stability in the aerobe. Arch Biochem Biophys. 1990; 280: 292–8. doi: 10.1016/0003-9861(90)90332-s 2114824

8. Nakazawa M, Hayashi R, Takenaka S, Inui H, Ishikawa T, Ueda M, et al. Physiological functions of pyruvate:NADP+ oxidoreductase and 2-oxoglutarate decarboxylase in Euglena gracilis under aerobic and anaerobic conditions. Biosci. Biotechnol. Biochem. 2017; 81: 1386–93. doi: 10.1080/09168451.2017.1318696 28463550

9. Nakazawa M, Andoh H, Koyama K, Watanabe Y, Nakai T, Ueda M, et al. Alteration of wax ester content and composition in Euglena gracilis with gene silencing of 3-ketoacyl-CoA thiolase isozymes. Lipids 2015; 50: 483–92. doi: 10.1007/s11745-015-4010-3 25860691

10. Hoffmeister M, Piotrowski M, Nowitzki U, Martin W. Mitochondrial trans-2-enoyl-CoA reductase of wax ester fermentation from Euglena gracilis defines a new family of enzymes involved in lipid synthesis. J. Biol. Chem. 2005; 280: 4329–38. doi: 10.1074/jbc.M411010200 15569691

11. Tomiyama T, Goto K, Tanaka Y, Maruta T, Ogawa T, Sawa Y, et al. A major isoform of mitochondrial trans-2-enoyl-CoA reductase is dispensable for wax ester production in Euglena gracilis under anaerobic conditions. PLoS One. 2019; e0210755 doi: 10.1371/journal.pone.0210755 30650145

12. Teerawanichpan P, Qiu X. Fatty acyl-CoA reductase and wax synthase from Euglena gracilis in the biosynthesis of medium-chain wax esters. Lipids. 2010; 45: 263–73. doi: 10.1007/s11745-010-3395-2 20195781

13. Tomiyama T, Kurihara K, Ogawa T, Maruta T, Ogawa T, Ohta D, et al. Wax ester synthase/diacylglycerol acyltransferase isoenzymes play a pivotal role in wax ester biosynthesis in Euglena gracilis. Sci. Rep. 2016; 7: 13504.

14. Tomita Y, Yoshioka K, Iijima H, Nakashima A, Iwata O, Suzuki K, et al. Succinate and lactate production from Euglena gracilis during dark, anaerobic conditions. Front. Microbiol. 2016; 7: 2050. doi: 10.3389/fmicb.2016.02050 28066371

15. Yoshida Y, Tomiyama T, Maruta T, Tomita M, Ishikawa T, Arakawa K. De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions. BMC Genomics. 2016; 17: 182. doi: 10.1186/s12864-016-2540-6 26939900

16. Koren LE, Hutner SH. High-yield media for photosynthesizing Euglena gracilis Z. J. Protozool. 1967; 14: 17.

17. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976; 72: 248–254. doi: 10.1006/abio.1976.9999 942051

18. Hoffmeister M, van der Klei A, Rotte C, van Grinsven KW, van Hellemond JJ, Henze K, et al. Euglena gracilis rhodoquinone:ubiquinone ratio and mitochondrial proteome differ under aerobic and anaerobic conditions. J. Biol. Chem. 2004; 279: 22422–9. doi: 10.1074/jbc.M400913200 15014069

19. Shingaki-Wells R, Millar AH, Whelan J, Narsai R. What happens to plant mitochondria under low oxygen? An omics review of the responses to low oxygen and reoxygenation. Plant Cell Environ. 2014; 37: 2260–77. doi: 10.1111/pce.12312 24575773

20. Shigeoka S, Onishi T, Maeda K, Nakano Y, Kitaoka S. Occurrence of thiamin pyrophosphate-dependent 2-oxoglutarate decarboxylase in mitochondria of Euglena gracilis. FEBS Lett. 1986; 195: 43–7.

21. Nakazawa M. C2 metabolism in Euglena. Adv. Exp. Med. Biol. 2017; 979: 39–45. doi: 10.1007/978-3-319-54910-1_3 28429316

22. Santiago-Martínez MG, Lira-Silva E, Encalada R, Pineda E, Gallardo-Pérez JC, Zepeda-Rodriguez A, et al. Cadmium removal by Euglena gracilis is enhanced under anaerobic growth conditions. J. Hazard. Mater. 2015; 288: 104–12. doi: 10.1016/j.jhazmat.2015.02.027 25698571

23. Inui H, Miyatake K, Nakano Y, Kitaoka S. Synthesis of reserved polysaccharide from wax esters accumulated as the result of anaerobic energy generation in Euglena gracilis returned from anaerobic to aerobic conditions. Int. J. Biochem. 1992; 24: 799–803

24. Nakazawa M, Nishimura M, Inoue K, Ueda M, Inui H, Nakano Y, et al. Characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, of Euglena gracilis. J. Eukaryot. Microbiol. 2011; 58: 128–33. doi: 10.1111/j.1550-7408.2011.00534.x 21332878

25. Matsuda F, Hayashi M, Kondo A. Comparative profiling analysis of central metabolites in Euglena gracilis under various cultivation conditions. Biosci. Biotechnol. Biochem. 2011; 75: 2253–6. doi: 10.1271/bbb.110482 22056447

26. Keller M, Chan RL, Tessier LH, Weil JH, Imbault P. Post-transcriptional regulation by light of the biosynthesis of Euglena ribulose-1,5-bisphosphate carboxylase/ oxygenase small subunit. Plant Mol. Biol. 1991; 17: 73–82. doi: 10.1007/bf00036807 1907872

27. Kishore R, Schwartzbach SD. (1992a) Photo and nutritional regulation of the light-harvesting chlorophyll a/b-binding protein of photosystem II mRNA levels in Euglena. Plant Physiol. 1992a; 98: 808–12.

28. Kishore R, Schwartzbach SD. Translational control of the synthesis of the Euglena light harvesting chlorophyll a/b binding protein of photosystem II. Plant Sci. 1992b; 85: 79–89.

29. Levasseur PJ, Meng Q, Bouck GB. Tubulin genes in the algal protist Euglena gracilis. J. Eukaryot. Microbiol. 1994; 41: 468–77. doi: 10.1111/j.1550-7408.1994.tb06044.x 7804247

30. Madhusudhan R, Ishikawa T, Sawa Y, Shigeoka S, Shibata H, Post-transcriptional regulation of ascorbate peroxidase during light adaptation of Euglena gracilis, Plant Sci. 2003; 165: 233–8.

31. Saint-Guily A, Schantz ML, Schantz R. Structure and expression of a cDNA encoding a histone H2A from Euglena gracilis. Plant Mol. Biol. 1994; 24: 941–948. doi: 10.1007/bf00014447 8204830

32. Vacula R, Steiner JM, Krajcovic J, Ebringer L, Löffelhardt W. Nucleus-encoded precursors to thylakoid lumen proteins of Euglena gracilis possess tripartite presequences. DNA Res. 1999; 6: 45–9. doi: 10.1093/dnares/6.1.45 10231029

33. Vacula R, Steiner JM, Krajcovic J, Ebringer L, Löffelhardt W. Plastid state- and light-dependent regulation of the expression of nucleus-encoded genes for chloroplast proteins in the flagellate Euglena gracilis. Folia Microbiol. 2001; 46: 433–41.

34. Vesteg M, Vacula R, Burey S, Loffelhardt W, Drahovska H, Martin W, et al. Expression of nucleus-encoded genes for chloroplast proteins in the flagellate Euglena gracilis. J. Eukaryot. Microbiol. 2009; 56: 159–66. doi: 10.1111/j.1550-7408.2008.00383.x 19457056

35. Vesteg M, Vacula R, Steiner JM, Mateásiková B, Löffelhardt W, Brejová B, et al. A possible role for short introns in the acquisition of stroma-targeting peptides in the flagellate Euglena gracilis. 2010; 17: 223–31.

36. Tamaki S, Kato S, Shinomura T, Ishikawa T, Imaishi H. Physiological role of β-carotene monohydroxylase (CYP97H1) in carotenoid biosynthesis in Euglena gracilis. Plant Sci. 2019; 278: 80–7. doi: 10.1016/j.plantsci.2018.10.017 30471732

37. Nakazawa M, Ando H, Nishimoto A, Ohta T, Sakamoto K, Ishikawa T, et al. Anaerobic respiration coupled with mitochondrial fatty acid synthesis in wax ester fermentation by Euglena gracilis. FEBS Lett. 2018; 592: 4020–7. doi: 10.1002/1873-3468.13276 30328102

38. Steverding D, Sexton DW, Wang X, Gehrke SS, Wagner GK, Caffrey CR. Trypanosoma brucei: chemical evidence that cathepsin L is essential for survival and a relevant drug target. Int. J. Parasitol. 2012; 42: 481–8. doi: 10.1016/j.ijpara.2012.03.009 22549023

39. Mus F, Dubini A, Seibert M, Posewitz MC, Grossman AR. Anaerobic acclimation in Chlamydomonas reinhardtii: anoxic gene expression, hydrogenase induction, and metabolic pathways. J. Biol. Chem. 2007; 282: 25475–86. doi: 10.1074/jbc.M701415200 17565990

40. Fricker R, Brogli R, Luidalepp H, Wyss L, Fasnacht M, Joss O, et al. A tRNA half modulates translation as stress response in Trypanosoma brucei. Nat. Commun. 2019;10:118. doi: 10.1038/s41467-018-07949-6 30631057

41. Kimura M, Ishikawa T. Suppression of DYRK ortholog expression affects wax ester fermentation in Euglena gracilis. J. Appl. Phycol. 2018; 30: 367–73.

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