Improving the co-production of triacylglycerol and isoprenoids in Chlamydomonas

Document Type : Research Paper

Authors

1 Microalgal Molecular Genetics and Functional genomics Special Research Unit, Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.

2 Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University (CASTNAR, NRU-KU), Kasetsart University, Bangkok 10900, Thailand.

3 Department of Botany, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.

Abstract

Biodiesel and natural products derived from microalgae require a smaller land area and have higher production rates compared to plants and animals and has recently attracted considerable interest. However, biodiesel production from microalgal triacylglycerol is still far from commercial realization due to its high production cost. One way to overcome this obstacle is to improve the triacylglycerol accumulation and couple its production with other high-value compounds. Of particular interest is the sterol biosynthetic pathway with squalene as an intermediate due to its close relationship with triacylglycerol and carotenoid biosynthetic pathways. Besides, both squalene and carotenoids are isoprenoid lipids that have health benefits. Perturbation of one pathway has been suggested to affect other pathways. Three terbinafine-sensitive mutants of the green microalga Chlamydomonas reinhardtii were isolated using terbinafine, a drug that inhibits squalene epoxidase, leading to squalene accumulation.One of the mutants, tfs2, accumulated twice the amount of wild-type triacylglycerol. As well as squalene accumulation, the presence of terbinafine further increased the triacylglycerol content. The level of prenyl lipid carotenoid and chlorophyll was also more significant than that of the wild type. Growth and photosynthesis were not compromised in this mutant. This is the first study that has demonstrated amutant screening method to improve the co-production of TAG and isoprenoid lipids in a green microalga.

Graphical Abstract

Improving the co-production of triacylglycerol and isoprenoids in Chlamydomonas

Highlights

  • A mutant screening method for improving triacylglycerol (TAG) and isoprenoid content.
  • Terbinafine-sensitive mutants were isolated in Chlamydomonas.
  • One mutant exhibited 2X higher level of triacylglycerol and 1.5X higher level of photosynthetic pigments.
  • The presence of terbinafine further increased the triacylglycerol level along with the accumulation of squalene.
  • The mutant exhibited normal growth and photosynthesis.

Keywords

References

[1] Aburai, N., Abe, K., 2015. Metabolic switching: synergistic induction of carotenogenesis in the aerial microalga Vischeria helvetica under environmental stress conditions by inhibitors of fatty acid biosynthesis. Biotechnol. Lett. 37(5), 1073-1080. 
[2] Adams, C., Godfrey, V., Wahlen, B., Seefeldt, L., Bugbee, B., 2013. Understanding precision nitrogen stress to optimize the growth and lipid content trade of in oleaginous green microalgae. Bioresour. Technol. 131, 188-194
[3] Berges, J.A., Charlebois, D.O., Mauzerall, D.C., Falkowski, P.G., 1996. Differential effects of nitrogen limitation on photosynthetic efficiency of photosystems I and II in microalgae. Plant. Physiol. 110, 689-696.
[4] Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917.
[5] Brumfield, K.M., Laborde, S.M., Moroney, J.V., 2017. A model for the ergosterol biosynthetic pathway in Chlamydomonas reinhardtii. Eur. J. Phycol. 52(1), 64-74.
[6] Cooksey, K., Guckert, J., Williams, S., Callis, P., 1987. Fluorometric determination of the neutral lipid content of microalgal cells using Nile red. J. Microbiol. Meth. 6(6), 333-345.
[7] Du, Z.-Y., Benning, C., 2016. Triacylglycerol accumulation in photosynthetic cells in plants and algae, in: Nakamura, Y., Li-Beisson, Y. (Eds.), Lipids in plant and algae development. Springer, Cham, pp. 179-205.
[8] Delrue, F., Setier, P.A., Sahut, C., Cournac, L., Roubaud, A., Peltier, G., Froment, A.K., 2012. An economic sustainability and energetic model of biodiesel production from microalgae. Bioresour. Technol. 111, 191-200
[9] Fang, Y., Hu, L., Zhou, X., Jaiseng, W., Zhang, B., Takami, T., Kuno, T., 2012. A genomewide screen in Schizosaccharomyces pombe for genes affecting the sensitivity of antifungal drugs that target ergosterol biosynthesis. Antimicrob. Agents Chemother. 56(4), 1949-1959.
[10] Fox, C., 2009. Squalene emulsions for parenteral vaccine and drug delivery. Molecules. 14, 3286-3312
[11] Francois, I., 2007. Investigating the role of reactive oxygen species in the mechanism of action of miconazole and effects on the treatment of diaper dermatitis. J. Am. Acad. Dermatol. 56, AB77.
[12] Garaiová, M., Zambojová, V., Šimová, Z., Griač, P., Hapala, I., 2014. Squalene epoxidase as a target for manipulation of squalene levels in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. 14(2), 310-323.
[13] Halim, R., Gladma, B., Danquah, M.K., Webley, P.A., 2011. Oil extraction from microalgae for biodiesel production. Bioresour. Technol. 102, 178-185.
[14] Heredia-Martínez, L.G., Andrés-Garrido, A., Martínez-Force, E., Pérez-Pérez, M.E., Crespo, J.L., 2018. Chloroplast damage induced by the inhibition of fatty acid synthesis triggers autophagy in chlamydomonas. Plant. Physiol. 178, 1112-1129.
[15] 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.
[16] Huang, Z.R, Lin, Y.K, Fang, J.Y, 2009. Biological and pharmacological activities of squalene and related compounds: potential uses in cosmetic dermatology. Molecules. 14(1), 540-554.
[17] Kajikawa, M., Kinohira, S., Ando, A., Shimoyama, M., Kato, M., Fukuzawa, H., 2015. Accumulation of squalene in a microalga Chlamydomonas reinhardtii by genetic modification of squalene synthase and squalene epoxidase genes. PLoS One. 10(3), e0120446.
[18] Kim, H., Jang, S., Kim, S., Yamaoka, Y., Hong, D., Song, W.Y., Nishida, I., Li-Beisson, Y., Lee, Y., 2015. The small molecule fenpropimorph rapidlyconverts chloroplast membrane lipids to triacylglycerols in Chlamydomonas reinhardtii. Front. Microbiol. 6, 54.
[19] Kou, Z., Bei, S., Sun, J., Pan, J., 2013.  Fluorescent measurement of lipid content in the model organism Chlamydomonas reinhardtti. J. Appl. Phycol. (25), 1633-1641.
[20] Lee, S.J., Yoon, B.D., Oh, H.M., 1998. Rapid method for the determination of lipid from the green algae Botryococcus braunii. Biotechnol. Tech. 553, 556.
[21] Leu, S., Boussiba, S., 2014. Advances in the production of high-value products by microalgae. Ind. Biotechnol. 10, 169-183.
[22] Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods. Enzymol. 148, 350-382.
[23] Liefer, J.D., Garg, A., Campbell, D.A., Irwin, A.J., Finkel, Z.V., 2018. Nitrogen starvation induces distinct photosynthetic responses and recovery dynamics in diatoms and prasinophytes. PLoS One. 13, e0195705.
[24] Lu, H.T., Jiang, Y., Chen, F., 2004. Determination of squalene using high-performance liquid chromatography with diode array detection. Chromatographia. 59, 367-71.
[25] Lu, Y., Zhou, W., Wei, L., Li, J., Jia, J., Li, F., Smith, S.M., Xu, J., 2014. Regulation of the cholesterol biosynthetic pathway and its integration with fatty acid biosynthesis in the oleaginous microalga Nannochloropsis oceanica. Biotechnol. Biofuels. 7(1), 81.
[26] Panis, G., Carreon, J.R., 2016. Commercial astaxanthin production derived by green alga Haematococcus pluvialis: a microalgae process model and a techno-economic assessment all through production line. Algal. Res. 18, 175-190.
[27] Patel, A., Pravez, M., Deeba, F., Pruthi, V., Singh, R.P., Pruthi, P.A., 2014. Boosting accumulation of neutral lipids in Rhodosporidium kratochvilovae HIMPA1 grown on hemp (Cannabis sativa Linn) seed aqueous extract as feedstock for biodiesel production. Bioresour. Technol. 165, 214-222.
[28] Shekhova, E., Kniemeyer, O., Brakhage, A.A., 2017. Induction of mitochondrial reactive oxygen species production by itraconazole, terbinafine, and amphotericin B as a mode of action against Aspergillus fumigatus. Antimicrob. Agents Chemother. 61 (11), e00978-17.
[29] Solymosi, K., Mysliwa-Kurdziel, B., 2017. Chlorophylls and their derivatives used in food industry and medicine. Mini. Rev. Med. Chem. 17, 1194-222.
[30] Thevissen, K., Ayscough, K.R., Aerts, A.M., Du, W., De Brucker, K., Meert, E.M.K., Ausma, J., Borgers, M., Cammue, B.P.A., François, I.E.J.A., 2007. Miconazole induces changes in actin cytoskeleton prior to reactive oxygen species induction in yeast. J. Biol. Chem. 282 (30), 21592-21597.
[31] Tossavainen, M., Ilyass, U., Ollilainen, V., Valkonen, K., Ojala, A., Romantschuk, M., 2019. Influence of long termnitrogen limitation on lipid, protein and pigment production of Euglena gracilisin photoheterotrophic cultures. Peer J. 7, e6624
[32] Wentzinger, L.F., Bach, T.J., Hartmann, M.A., 2002. Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Plant. Physiol. 130(1), 334-346.

Files

Cited by

Share

How to cite

Statistics