Skip to main content
SpringerLink
Log in

Enhanced Lutein Production in Chlamydomonas reinhardtii by Overexpression of the Lycopene Epsilon Cyclase Gene

  • Original Article
  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Chlamydomonas reinhardtii is a well-established microalgal model species with a shorter doubling time, which is a promising natural source for the efficient production of high-value carotenoids. In the microalgal carotenoid biosynthetic pathway, lycopene is converted either into β-carotene by lycopene β-cyclase or into α-carotene by lycopene ε-cyclase (LCYE) and lycopene β-cyclase. In this study, we overexpressed the LCYE gene in C. reinhardtii to estimate its effect on lycopene metabolism and lutein production. Chlamydomonas transformants (CrLCYE#L1, #L5, and #L6) produced significantly increased amounts of lutein per culture (up to 2.6-fold) without a decrease in cell yields. Likewise, the expression levels of LCYE gene in transformants showed a significant increase compared with that of the wild-type strain. These results suggest that LCYE overexpression enhances the conversion of lycopene to α-carotene, which in turn improves lutein productivity. Interestingly, their β-carotene productivity appeared to increase slightly rather than decrease. Considering that the inhibition of the lycopene cyclization steps often induces higher expression in genes upstream of metabolic branches, this result implies that the redirection from β-carotene to α-carotene by LCYE overexpression might also enhance upstream gene expression, thereby leading to auxiliary β-carotene production.

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

Data Availability

Not applicable

Code Availability

Not applicable

References

  1. Saini, R. K., Nile, S. H., & Park, S. W. (2015). Carotenoids from fruits and vegetables: Chemistry, analysis, occurrence, bioavailability and biological activities. Food Research International, 76(Pt 3), 735–750. https://doi.org/10.1016/j.foodres.2015.07.047.

    Article  CAS  PubMed  Google Scholar 

  2. De Jesus Raposo, M. F., De Morais, A. M. M. B., & De Morais, R. M. S. C. (2015). Carotenoids from marine microalgae: A valuable natural source for the prevention of chronic diseases. Marine Drugs, 13(8), 5128–5155. https://doi.org/10.3390/md13085128.

    Article  CAS  Google Scholar 

  3. Koller, M., Muhr, A., & Braunegg, G. (2014). Microalgae as versatile cellular factories for valued products. Algal Research, 6(PA), 52–63. https://doi.org/10.1016/j.algal.2014.09.002.

    Article  Google Scholar 

  4. Cezare-Gomes, E. A., Mejia-da-Silva, L. d. C., Pérez-Mora, L. S., Matsudo, M. C., Ferreira-Camargo, L. S., Singh, A. K., & de Carvalho, J. C. M. (2019). Potential of microalgae carotenoids for industrial application. Applied Biochemistry and Biotechnology, 188(3), 602–634. https://doi.org/10.1007/s12010-018-02945-4.

    Article  CAS  PubMed  Google Scholar 

  5. Lohr, M., Im, C. S., & Grossman, A. R. (2005). Genome-based examination of chlorophyll and carotenoid biosynthesis in Chlamydomonas reinhardtii. Plant Physiology, 138(1), 490–515. https://doi.org/10.1104/pp.104.056069.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lohr, M., Schwender, J., & Polle, J. E. W. (2012). Isoprenoid biosynthesis in eukaryotic phototrophs: A spotlight on algae. Plant Science, 185–186, 9–22. https://doi.org/10.1016/j.plantsci.2011.07.018.

    Article  CAS  PubMed  Google Scholar 

  7. Christaki, E., Bonos, E., Giannenas, I., & Florou-Paneri, P. (2013). Functional properties of carotenoids originating from algae. Journal of the Science of Food and Agriculture, 93(1), 5–11. https://doi.org/10.1002/jsfa.5902.

    Article  CAS  PubMed  Google Scholar 

  8. Scaife, M. A., Nguyen, G. T. D. T., Rico, J., Lambert, D., Helliwell, K. E., & Smith, A. G. (2015). Establishing Chlamydomonas reinhardtii as an industrial biotechnology host. The Plant Journal, 82(3), 532–546. https://doi.org/10.1111/tpj.12781.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Cordero, B. F., Couso, I., León, R., Rodríguez, H., & Vargas, M. Á. (2011). Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis. Applied Microbiology and Biotechnology, 91(2), 341–351. https://doi.org/10.1007/s00253-011-3262-y.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Couso, I., Vila, M., Rodriguez, H., Vargas, M. A., & León, R. (2011). Overexpression of an exogenous phytoene synthase gene in the unicellular alga Chlamydomonas reinhardtii leads to an increase in the content of carotenoids. Biotechnology Progress, 27(1), 54–60. https://doi.org/10.1002/btpr.527.

    Article  CAS  PubMed  Google Scholar 

  11. Liu, J., Gerken, H., Huang, J., & Chen, F. (2013). Engineering of an endogenous phytoene desaturase gene as a dominant selectable marker for Chlamydomonas reinhardtii transformation and enhanced biosynthesis of carotenoids. Process Biochemistry, 48(5), 788–795. https://doi.org/10.1016/j.procbio.2013.04.020.

    Article  CAS  Google Scholar 

  12. Morikawa, T., Uraguchi, Y., Sanda, S., Nakagawa, S., & Sawayama, S. (2018). Overexpression of DnaJ-like chaperone enhances carotenoid synthesis in Chlamydomonas reinhardtii. Applied Biochemistry and Biotechnology, 184(1), 80–91. https://doi.org/10.1007/s12010-017-2521-5.

    Article  CAS  PubMed  Google Scholar 

  13. Kumari, S., Vira, C., Lali, A. M., & Prakash, G. (2020). Heterologous expression of a mutant Orange gene from Brassica oleracea increases carotenoids and induces phenotypic changes in the microalga Chlamydomonas reinhardtii. Algal Research, 47, 101871. https://doi.org/10.1016/j.algal.2020.101871.

    Article  Google Scholar 

  14. Perozeni, F., Cazzaniga, S., Baier, T., Zanoni, F., Zoccatelli, G., Lauersen, K. J., Wobbe, L., & Ballottari, M. (2020). Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii. Plant Biotechnology Journal, 18(10), 2053–2067. https://doi.org/10.1111/pbi.13364.

    Article  CAS  PubMed Central  Google Scholar 

  15. Tan, C.-P., Zhao, F.-Q., Su, Z.-L., Liang, C.-W., & Qin, S. (2007). Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii. Journal of Applied Phycology, 19(4), 347–355. https://doi.org/10.1007/s10811-006-9141-8.

    Article  CAS  Google Scholar 

  16. Kindle, K. L. (1990). High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences of the United States of America, 87(3), 1228–1232. https://doi.org/10.1073/pnas.87.3.1228.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Packeiser, H., Lim, C., Balagurunathan, B., Wu, J., & Zhao, H. (2013). An extremely simple and effective colony PCR procedure for bacteria, yeasts, and microalgae. Applied Biochemistry and Biotechnology, 169(2), 695–700. https://doi.org/10.1007/s12010-012-0043-8.

    Article  CAS  PubMed  Google Scholar 

  18. Kubo, Y., Shiroi, M., Higashine, T., Mori, Y., Morimoto, D., Nakagawa, S., & Sawayama, S. (2020). Enhanced production of astaxanthin without decrease of DHA content in Aurantiochytrium limacinum by overexpressing multifunctional carotenoid synthase gene. Applied Biochemistry and Biotechnology, 193(1), 52–64. https://doi.org/10.1007/s12010-020-03403-w.

    Article  CAS  PubMed  Google Scholar 

  19. Moellering, E. R., & Benning, C. (2010). RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii. Eukaryotic Cell, 9(1), 97–106. https://doi.org/10.1128/EC.00203-09.

    Article  CAS  PubMed  Google Scholar 

  20. Pogson, B., McDonald, K. A., Truong, M., Britton, G., & DellaPenna, D. (1996). Arabidopsis carotenoid mutants demonstrate that lutein is not essential for photosynthesis in higher plants. The Plant Cell, 8(9), 1627–1639. https://doi.org/10.1105/tpc.8.9.1627.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yu, B., Lydiate, D. J., Young, L. W., Schäfer, U. A., & Hannoufa, A. (2008). Enhancing the carotenoid content of Brassica napus seeds by downregulating lycopene epsilon cyclase. Transgenic Research, 17(4), 573–585. https://doi.org/10.1007/s11248-007-9131-x.

    Article  CAS  PubMed  Google Scholar 

  22. Diretto, G., Tavazza, R., Welsch, R., Pizzichini, D., Mourgues, F., Papacchioli, V., Beyer, P., & Giuliano, G. (2006). Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase. BMC Plant Biology, 6(1), 13. https://doi.org/10.1186/1471-2229-6-13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Harjes, C. E., Rocheford, T. R., Bai, L., Brutnell, T. P., Kandianis, C. B., Sowinski, S. G., et al. (2008). Natural genetic variation in lycopene epsilon cyclase tapped for Maize biofortification. Science, 319(5861), 330–333. https://doi.org/10.1126/science.1150255.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Farré, G., Sanahuja, G., Naqvi, S., Bai, C., Capell, T., Zhu, C., & Christou, P. (2010). Travel advice on the road to carotenoids in plants. Plant Science, 179(1–2), 28–48. https://doi.org/10.1016/j.plantsci.2010.03.009.

    Article  CAS  Google Scholar 

  25. Osmond, C. B., Foyer, C. H., Bock, G., Pogson, B. J., & Rissler, H. M. (2000). Genetic manipulation of carotenoid biosynthesis and photoprotection. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 355(1402), 1395–1403. https://doi.org/10.1098/rstb.2000.0701.

    Article  Google Scholar 

  26. Liang, M. H., Hao, Y. F., Li, Y. M., Liang, Y. J., & Jiang, J. G. (2016). Inhibiting lycopene cyclases to accumulate lycopene in high β-carotene-accumulating Dunaliella bardawil. Food and Bioprocess Technology, 9(6), 1002–1009. https://doi.org/10.1007/s11947-016-1681-6.

    Article  CAS  Google Scholar 

  27. Fazeli, M. R., Tofighi, H., Madadkar-Sobhani, A., Shahverdi, A. R., Nejad-Sattari, T., Sako, M., & Jamalifar, H. (2009). Nicotine inhibition of lycopene cyclase enhances accumulation of carotenoid intermediates by Dunaliella salina CCAP 19/18. European Journal of Phycology, 44(2), 215–220. https://doi.org/10.1080/09670260802578526.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Prof. Tatsuya Sugawara and Assistant Prof. Yuki Manabe for the constructive comments and discussion.

Funding

This work was partly supported by the Sun Chlorella Corp. (Kyoto, Japan).

Author information

Authors and Affiliations

  1. Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan

    Saki Tokunaga, Daichi Morimoto, Takahisa Koyama, Yuki Kubo, Mai Shiroi, Kanta Ohara, Tokuhiro Higashine, Yuki Mori, Satoshi Nakagawa & Shigeki Sawayama

Authors
  1. Saki Tokunaga
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daichi Morimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takahisa Koyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuki Kubo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mai Shiroi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kanta Ohara
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tokuhiro Higashine
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yuki Mori
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Satoshi Nakagawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Shigeki Sawayama
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

ST conducted experiments. DM analyzed data and wrote the manuscript. KO partly contributed figure preparation. TK, TH, and YM partly contributed establishment of experimental methods. SN and SS conceived and designed research. All authors read and approved the manuscript.

Corresponding author

Correspondence to Daichi Morimoto.

Ethics declarations

Ethics Approval

This article does not contain any studies with human participants by any of the authors.

Consent to Participate

Not applicable

Consent for Publication

All authors consent to the submission and publication of this manuscript in Applied Microbiology and Biotechnology.

Competing Interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

ESM 1

(DOCX 959 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tokunaga, S., Morimoto, D., Koyama, T. et al. Enhanced Lutein Production in Chlamydomonas reinhardtii by Overexpression of the Lycopene Epsilon Cyclase Gene. Appl Biochem Biotechnol 193, 1967–1978 (2021). https://doi.org/10.1007/s12010-021-03524-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-021-03524-w

Keywords

Search

Navigation