Advertisement

Metabolic Engineering of Cyanidioschyzon merolae

  • Nobuko Sumiya
  • Shinya MiyagishimaEmail author
Chapter

Abstract

Algae are expected to be promising alternative sources of biofuels, foods, and cosmetics. The unicellular red alga Cyanidioschyzon merolae is potentially useful for producing high concentrations of desirable biomaterials by metabolic engineering. C. merolae is genetically traceable and can thrive at low pH (1–5) and high temperatures (25–50 °C), which are harmful to many other organisms. Thus, this alga can be suitable for outdoor cultivation without the risk for contamination from other undesirable organisms. Recent studies regarding C. merolae have reported enhanced triacylglycerol (TAG) production, which can be used for biodiesel production, by genetic modification. Introducing cyanobacterial acyl-acyl carrier protein (ACP) reductase in C. merolae led to temporary TAG accumulation via an artificial metabolic pathway. The omics analyses showed that acyl-ACP reductase expression resulted in upregulating endogenous aldehyde dehydrogenase and the endogenous fatty acid synthetic pathway in chloroplasts. Another study expressed the 12-kDa FK506-binding protein of Saccharomyces cerevisiae in C. merolae and succeeded in increasing TAG levels by adding rapamycin. The omics analyses suggested that the target of rapamycin (TOR) regulated the expression of TAG-synthesizing enzymes, glycerol-3-phosphate acyltransferase, and acyl-CoA:diacylglycerol acyltransferase. Therefore, the combination of metabolic engineering and the evaluation of the effects in C. merolae by omics analyses will help in understanding the regulatory mechanism of metabolism. In addition, recent studies have started to find culture conditions that increase TAG accumulation while maintaining the cellular growth. Combinations of these cultivation techniques and genetic manipulations will leads to production of desirable biomolecules on a large scale in the future.

Keywords

Triacylglycerol Lipid droplets Acyl-ACP reductase Aldehyde dehydrogenase TOR Rapamycin Cyanidiales Cyanidioschyzon merolae 

Notes

Acknowledgments

Our study was partly supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research 25251039 (to S.M.) and by the Core Research for Evolutional Science and Technology Program of the Japan Science and Technology Agency (to S.M.).

References

  1. Beck T, Hall MN (1999) The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402:689–692. https://doi.org/10.1038/45287 CrossRefPubMedGoogle Scholar
  2. Cooper TG (2002) Transmitting the signal of excess nitrogen in Saccharomyces cerevisiae from the Tor proteins to the GATA factors: connecting the dots. FEMS Microbiol Rev 26:223–238. https://doi.org/10.1111/j.1574-6976.2002.tb00612.x CrossRefPubMedPubMedCentralGoogle Scholar
  3. De Virgilio C, Loewith R (2006) The TOR signalling network from yeast to man. Int J Biochem Cell Biol 38:1476–1481. https://doi.org/10.1016/j.biocel.2006.02.013 CrossRefPubMedGoogle Scholar
  4. Fujiwara T, Ohnuma M, Yoshida M, Kuroiwa T, Hirano T (2013) Gene targeting in the red alga Cyanidioschyzon merolae: single- and multi-copy insertion using authentic and chimeric selection markers. PLoS One 8:e73608. https://doi.org/10.1371/journal.pone.0073608 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Ge F, Huang W, Chen Z, Zhang C, Xiong Q, Bowler C, Yang J, Xu J, Hu H (2014) Methylcrotonyl-CoA carboxylase regulates triacylglycerol accumulation in the model diatom Phaeodactylum tricornutum. Plant Cell 26:1681–1697. https://doi.org/10.1105/tpc.114.124982 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Heitman J, Movva N, Hall M (1991) Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253:905–909. https://doi.org/10.1126/science.1715094 CrossRefPubMedGoogle Scholar
  7. Hlavová M, Turóczy Z, Bišová K (2015) Improving microalgae for biotechnology – from genetics to synthetic biology. Biotechnol Adv 33:1194–1203. https://doi.org/10.1016/j.biotechadv.2015.01.009 CrossRefPubMedGoogle Scholar
  8. Ho S-H, Ye X, Hasunuma T, Chang J-S, Kondo A (2014) Perspectives on engineering strategies for improving biofuel production from microalgae – a critical review. Biotechnol Adv 32:1448–1459. https://doi.org/10.1016/j.biotechadv.2014.09.002 CrossRefPubMedGoogle Scholar
  9. Holme IB, Wendt T, Holm PB (2013) Intragenesis and cisgenesis as alternatives to transgenic crop development. Plant Biotechnol J 11:395–407. https://doi.org/10.1111/pbi.12055 CrossRefPubMedGoogle Scholar
  10. Imamura S, Ishiwata A, Watanabe S, Yoshikawa H, Tanaka K (2013) Expression of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga Cyanidioschyzon merolae. Biochem Biophys Res Commun 439:264–269. https://doi.org/10.1016/j.bbrc.2013.08.045 CrossRefPubMedGoogle Scholar
  11. Imamura S, Kawase Y, Kobayashi I, Sone T, Era A, Miyagishima SY, Shimojima M, Ohta H, Tanaka K (2015) Target of rapamycin (TOR) plays a critical role in triacylglycerol accumulation in microalgae. Plant Mol Biol 89:309–318. https://doi.org/10.1007/s11103-015-0370-6 CrossRefPubMedGoogle Scholar
  12. Imamura S, Kawase Y, Kobayashi I, Shimojima M, Ohta H, Tanaka K (2016) TOR (target of rapamycin) is a key regulator of triacylglycerol accumulation in microalgae. Plant Signal Behav 11:e1149285. https://doi.org/10.1080/15592324.2016.1149285 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Kaiser BK, Carleton M, Hickman JW, Miller C, Lawson D, Budde M, Warrener P, Paredes A, Mullapudi S, Navarro P, Cross F, Roberts JM (2013) Fatty aldehydes in cyanobacteria are a metabolically flexible precursor for a diversity of biofuel products. PLoS One 8:e58307. https://doi.org/10.1371/journal.pone.0058307 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Koyama Y, Takimoto K, Kojima A, Asai K, Matsuoka S, Mitsui T, Matsumoto K, Hara H, Ohta N (2011) Characterization of the nuclear- and plastid-encoded secA-homologous genes in the unicellular red alga Cyanidioschyzon merolae. Biosci Biotechnol Biochem 75:2073–2078. https://doi.org/10.1271/bbb.110338 CrossRefPubMedGoogle Scholar
  15. Laplante M, Sabatini David M (2012) mTOR signaling in growth control and disease. Cell 149:274–293. https://doi.org/10.1016/j.cell.2012.03.017 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Li Y, Horsman M, Wu N, Lan CQ, Dubois-Calero N (2008) Biofuels from microalgae. Biotechnol Prog 24:815–820. https://doi.org/10.1021/bp070371k PubMedGoogle Scholar
  17. Matsuzaki M, Misumi O, Shin IT, Maruyama S, Takahara M, Miyagishima SY, Mori T, Nishida K, Yagisawa F, Yoshida Y, Nishimura Y, Nakao S, Kobayashi T, Momoyama Y, Higashiyama T, Minoda A, Sano M, Nomoto H, Oishi K, Hayashi H, Ohta F, Nishizaka S, Haga S, Miura S, Morishita T, Kabeya Y, Terasawa K, Suzuki Y, Ishii Y, Asakawa S, Takano H, Ohta N, Kuroiwa H, Tanaka K, Shimizu N, Sugano S, Sato N, Nozaki H, Ogasawara N, Kohara Y, Kuroiwa T (2004) Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428:653–657. https://doi.org/10.1038/nature02398 CrossRefPubMedGoogle Scholar
  18. Merchant SS, Kropat J, Liu B, Shaw J, Warakanont J (2012) TAG, You’re it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation. Curr Opin Biotechnol 23:352–363. https://doi.org/10.1016/j.copbio.2011.12.001 CrossRefPubMedGoogle Scholar
  19. Minoda A, Sakagami R, Yagisawa F, Kuroiwa T, Tanaka K (2004) Improvement of culture conditions and evidence for nuclear transformation by homologous recombination in a red alga, Cyanidioschyzon merolae 10D. Plant Cell Physiol 45:667–671. https://doi.org/10.1093/pcp/pch087 CrossRefPubMedGoogle Scholar
  20. Misumi O, Sakajiri T, Hirooka S, Kuroiwa H, Kuroiwa T (2008) Cytological studies of metal ion tolerance in the red algae Cyanidioschyzon merolae. Cytologia (Tokyo) 73:437–443. https://doi.org/10.1508/cytologia.73.437 CrossRefGoogle Scholar
  21. Miyagishima SY, Fujiwara T, Sumiya N, Hirooka S, Nakano A, Kabeya Y, Nakamura M (2014) Translation-independent circadian control of the cell cycle in a unicellular photosynthetic eukaryote. Nat Commun 5:3807. https://doi.org/10.1038/ncomms4807 CrossRefPubMedGoogle Scholar
  22. Mori N, Moriyama T, Toyoshima M, Sato N (2016) Construction of global Acyl lipid metabolic map by comparative genomics and subcellular localization analysis in the red alga Cyanidioschyzon merolae. Front Plant Sci 7:958. https://doi.org/10.3389/fpls.2016.00958 PubMedPubMedCentralGoogle Scholar
  23. Nozaki H, Takano H, Misumi O, Terasawa K, Matsuzaki M, Maruyama S, Nishida K, Yagisawa F, Yoshida Y, Fujiwara T, Takio S, Tamura K, Chung S, Nakamura S, Kuroiwa H, Tanaka K, Sato N, Kuroiwa T (2007) A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae. BMC Biol 5:1–8. https://doi.org/10.1186/1741-7007-5-28 CrossRefGoogle Scholar
  24. Ohnuma M, Yokoyama T, Inouye T, Sekine Y, Tanaka K (2008) Polyethylene glycol (PEG)-mediated transient gene expression in a red alga, Cyanidioschyzon merolae 10D. Plant Cell Physiol 49:117–120. https://doi.org/10.1093/pcp/pcm157 CrossRefPubMedGoogle Scholar
  25. Ohnuma M, Misumi O, Fujiwara T, Watanabe S, Tanaka K, Kuroiwa T (2009) Transient gene suppression in a red alga, Cyanidioschyzon merolae 10D. Protoplasma 236:107–112. https://doi.org/10.1007/s00709-009-0056-5 CrossRefPubMedGoogle Scholar
  26. Ohta N, Sato N, Kuroiwa T (1998) Structure and organization of the mitochondrial genome of the unicellular red alga Cyanidioschyzon merolae deduced from the complete nucleotide sequence. Nucleic Acids Res 26:5190–5198. https://doi.org/10.1093/nar/26.22.5190 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Ohta N, Matsuzaki M, Misumi O, Miyagishima SY, Nozaki H, Tanaka K, Shin IT, Kohara Y, Kuroiwa T (2003) Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae. DNA Res 10:67–77. https://doi.org/10.1093/dnares/10.2.67 CrossRefPubMedGoogle Scholar
  28. Sheehan J, Dunahay T, Benemann J, Roessler P (1998) A look back at the US Department of energy’s aquatic species program: biodiesel from algae, vol 328. National Renewable Energy Laboratory, GoldenCrossRefGoogle Scholar
  29. Sumiya N, Fujiwara T, Kobayashi Y, Misumi O, Miyagishima SY (2014) Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae. PLoS One 9:e111261. https://doi.org/10.1371/journal.pone.0111261 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Sumiya N, Kawase Y, Hayakawa J, Matsuda M, Nakamura M, Era A, Tanaka K, Kondo A, Hasunuma T, Imamura S, Miyagishima SY (2015) Expression of cyanobacterial acyl-ACP reductase elevates the triacylglycerol level in the red alga Cyanidioschyzon merolae. Plant Cell Physiol 56:1962–1980. https://doi.org/10.1093/pcp/pcv120 CrossRefPubMedGoogle Scholar
  31. Sumiya N, Fujiwara T, Era A, Miyagishima SY (2016) Chloroplast division checkpoint in eukaryotic algae. Proc Natl Acad Sci U S A 113:E7629–E7638. https://doi.org/10.1073/pnas.1612872113 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Taki K, Sone T, Kobayashi Y, Watanabe S, Imamura S, Tanaka K (2015) Construction of a URA5.3 deletion strain of the unicellular red alga Cyanidioschyzon merolae: a backgroundless host strain for transformation experiments. J Gen Appl Microbiol 61:211–214. https://doi.org/10.2323/jgam.61.211 CrossRefPubMedGoogle Scholar
  33. Watanabe S, Ohnuma M, Sato J, Yoshikawa H, Tanaka K (2011) Utility of a GFP reporter system in the red alga Cyanidioschyzon merolae. J Gen Appl Microbiol 57:69–72. https://doi.org/10.2323/jgam.57.69 CrossRefPubMedGoogle Scholar
  34. Watanabe S, Sato J, Imamura S, Ohnuma M, Ohoba Y, Chibazakura T, Tanaka K, Yoshikawa H (2014) Stable expression of a GFP-reporter gene in the red alga Cyanidioschyzon merolae. Biosci Biotechnol Biochem 78:175–177. https://doi.org/10.1080/09168451.2014.877823 CrossRefPubMedGoogle Scholar
  35. Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell 124:471–484. https://doi.org/10.1016/j.cell.2006.01.016 CrossRefPubMedGoogle Scholar
  36. Yan N, Fan C, Chen Y, Hu Z (2016) The potential for microalgae as bioreactors to produce pharmaceuticals. Int J Mol Sci 17:962. https://doi.org/10.3390/ijms17060962 CrossRefPubMedCentralGoogle Scholar
  37. Zienkiewicz M, Krupnik T, Drożak A, Golke A, Romanowska E (2017a) Chloramphenicol acetyltransferase—a new selectable marker in stable nuclear transformation of the red alga Cyanidioschyzon merolae. Protoplasma 254:587–596. https://doi.org/10.1007/s00709-015-0936-9 CrossRefPubMedGoogle Scholar
  38. Zienkiewicz M, Krupnik T, Drożak A, Golke A, Romanowska E (2017b) Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments. Plant Mol Biol 93:171–183. https://doi.org/10.1007/s11103-016-0554-8 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2017

Authors and Affiliations

  1. 1.Department of BiologyKeio UniversityYokohamaJapan
  2. 2.Department of Cell GeneticsNational Institute of GeneticsShizuokaJapan

Personalised recommendations