Skip to main content
Log in

Metabolic Engineering of Chlamydomonas reinhardtii for Enhanced β-Carotene and Lutein Production

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

Abstract

The metabolic engineering of Chlamydomonas reinhardtii, one of the fastest-growing microalgae, is a potential alternative for enhanced carotenoid productivity. CrtYB (phytoene-β-carotene synthasePBS) gene from red yeast Xanthophyllomyces dendrorhous encodes for a bifunctional enzyme that harbours both phytoene synthase (psy) and lycopene cyclization (lcyb) activities. Heterologous expression of this bifunctional PBS gene led to 38% enhancement in β-carotene along with 60% increase in the lutein yields under low light conditions of 75 μmol photons m−2 s−1. Short Duration-High Light induction strategy led to overall 72% and 83% increase in β-carotene and lutein yield reaching up to 22.8 mg g−1 and 8.9 mg g−1, respectively. This is the first report of expression of heterologous bifunctional PBS gene resulting in simultaneous enhancement in β-carotene and lutein content in phototrophic engineered cells.

Graphical Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Koller, M., Muhr, A., & Braunegg, G. (2014). Microalgae as versatile cellular factories for valued products. Algal Research, 6, 52–63.

    Article  Google Scholar 

  2. Cezare-Gomes, E. A., del Carmen Mejia-da-Silva, L., 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 & Biotechnology, 188(3), 602–634.

  3. Scaife, M. A., Nguyen, G. 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.

    Article  CAS  PubMed Central  Google Scholar 

  4. 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.

    Article  CAS  PubMed Central  Google Scholar 

  5. Couso, I., Vila, M., Rodriguez, H., Vargas, M. A., & Leon, 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.

    Article  CAS  PubMed Central  Google Scholar 

  6. 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(5-6), 788–795.

    Article  CAS  Google Scholar 

  7. Vira, C., Prakash, G., Rathod, J. P., & Lali, A. M. (2016). Cloning, expression, and purification of Chlamydomonas reinhardtii CC-503 sedoheptulose 1, 7-bisphosphatase in Escherichia coli. Preparative Biochemistry & Biotechnology, 46(8), 810–814.

    Article  CAS  Google Scholar 

  8. Rasala, B. A., Lee, P. A., Shen, Z., Briggs, S. P., Mendez, M., & Mayfield, S. P. (2012). Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide. PLoS One, 7(8), e43349.

    Article  CAS  PubMed Central  Google Scholar 

  9. Leon, R., Couso, I., & Fernández, E. (2007). Metabolic engineering of ketocarotenoids biosynthesis in the unicelullar microalga Chlamydomonas reinhardtii. Journal of Biotechnology, 130(2), 143–152.

    Article  CAS  PubMed Central  Google Scholar 

  10. Rathod, J. P., Prakash, G., Vira, C., & Lali, A. M. (2016). Trehalose phosphate synthase overexpression in Parachlorella kessleri improves growth and photosynthetic performance under high light conditions. Preparative Biochemistry & Biotechnology, 46(8), 803–809.

    Article  CAS  Google Scholar 

  11. Garcia-Plazaola, J. I., & Becerril, J. M. (1999). A rapid high-performance liquid chromatography method to measure lipophilic antioxidants in stressed plants: simultaneous determination of carotenoids and tocopherols. Phytochemical Analysis, 10(6), 307–313.

    Article  CAS  Google Scholar 

  12. Visser, H., van Ooyen, A. J., & Verdoes, J. C. (2003). Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous. FEMS Yeast Research, 4(3), 221–231.

    Article  CAS  Google Scholar 

  13. Verdoes, J. C., Krubasik, P., Sandmann, G., & van Ooyen, A. J. J. (1999). Isolation and functional characterisation of a novel type of carotenoid biosynthetic gene from Xanthophyllomyces dendrorhous. Molecular and General Genetics MGG, 262(3), 453–461.

    Article  CAS  Google Scholar 

  14. Verwaal, R., Wang, J., Meijnen, J. P., Visser, H., Sandmann, G., van den Berg, J. A., & van Ooyen, A. J. (2007). High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Applied and Environmental Microbiology, 73(13), 4342–4350.

    Article  CAS  PubMed Central  Google Scholar 

  15. Koh, H. G., Kang, N. K., Kim, E. K., Jeon, S., Shin, S. E., Lee, B., & Chang, Y. K. (2018). Advanced multigene expression system for Nannochloropsis salina using 2A self-cleaving peptides. Journal of Biotechnology, 278, 39–47.

    Article  CAS  Google Scholar 

  16. Molino, J. V. D., de Carvalho, J. C. M., & Mayfield, S. P. (2018). Evaluation of secretion reporters to microalgae biotechnology: blue to red fluorescent proteins. Algal Research, 31, 252–261.

    Article  Google Scholar 

  17. Scaife, M. A., & Smith, A. G. (2016). Towards developing algal synthetic biology. Biochemical Society Transactions, 44(3), 716–722.

    Article  CAS  Google Scholar 

  18. Verdoes, J. C., Sandmann, G., Visser, H., Diaz, M., van Mossel, M., & van Ooyen, A. J. (2003). Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Applied and Environmental Microbiology, 69(7), 3728–3738.

    Article  CAS  PubMed Central  Google Scholar 

  19. Paliwal, C., Mitra, M., Bhayani, K., Bharadwaj, S. V., Ghosh, T., Dubey, S., & Mishra, S. (2017). Abiotic stresses as tools for metabolites in microalgae. Bioresource Technology, 244, 1216–1226.

    Article  CAS  Google Scholar 

  20. Couso, I., Vila, M., Vigara, J., Cordero, B. F., Vargas, M. Á., Rodríguez, H., & León, R. (2012). Synthesis of carotenoids and regulation of the carotenoid biosynthesis pathway in response to high light stress in the unicellular microalga Chlamydomonas reinhardtii. European Journal of Phycology, 47(3), 223–232.

    Article  CAS  Google Scholar 

  21. Ma, R., Zhao, X., Xie, Y., Ho, S. H., & Chen, J. (2018). Enhancing lutein productivity of Chlamydomonas sp. via high-intensity light exposure with corresponding carotenogenic genes expression profiles. Bioresource Technology, 275, 416–420.

    Article  PubMed Central  Google Scholar 

  22. Schroda, M., Blöcker, D., & Beck, C. F. (2000). The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. The Plant Journal, 21(2), 121–131.

    Article  CAS  PubMed Central  Google Scholar 

  23. Gimpel, J. A., Henríquez, V., & Mayfield, S. P. (2015). In metabolic engineering of eukaryotic microalgae: potential and challenges come with great diversity. Frontiers in Microbiology, 6, 1376.

    Article  PubMed Central  Google Scholar 

  24. 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.

    Article  CAS  PubMed Central  Google Scholar 

  25. Harjes, C. E., Rocheford, T. R., Bai, L., Brutnell, T. P., Kandianis, C. B., Sowinski, S. G., Stapleton, A. E., Vallabhaneni, R., Williams, M., Wurtzel, E. T., Yan, J., & Buckler, E. S. (2008). Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification. Science, 319(5861), 330–333.

    Article  CAS  PubMed Central  Google Scholar 

  26. Cordero, B. F., Couso, I., Leon, R., Rodriguez, H., & Vargas, M. A. (2012). Isolation and characterization of a lycopene ε-cyclase gene of Chlorella (Chromochloris) zofingiensis. Regulation of the carotenogenic pathway by nitrogen and light. Marine Drugs, 10(9), 2069–2088.

    Article  CAS  PubMed Central  Google Scholar 

  27. Ben-Amotz, A., Katz, A., & Avron, M. (1982). Accumulation of β-carotene in halotolerant alge: purification and characterization of β-carotene-rich globules from Dunaliella bardawil (Chlorophyceae). Journal of Phycology, 18(4), 529–537.

    Article  CAS  Google Scholar 

  28. Guedes, A. C., Amaro, H. M., & Malcata, F. X. (2011). Microalgae as sources of carotenoids. Marine Drugs, 9(4), 625–644.

    Article  CAS  PubMed Central  Google Scholar 

  29. Lin, J. H., Lee, D. J., & Chang, J. S. (2015). Lutein production from biomass: marigold flowers versus microalgae. Bioresource Technology, 184, 421–428.

    Article  CAS  PubMed Central  Google Scholar 

  30. Sun, H., Mao, X., Wu, T., Ren, Y., Chen, F., & Liu, B. (2018). Novel insight of carotenoid and lipid biosynthesis and their roles in storage carbon metabolism in Chlamydomonas reinhardtii. Bioresource Technology, 263, 450–457.

    Article  CAS  PubMed Central  Google Scholar 

  31. Fields, F. J., Ostrand, J. T., & Mayfield, S. P. (2018). Fed-batch mixotrophic cultivation of Chlamydomonas reinhardtii for high-density cultures. Algal Research, 33, 109–117.

    Article  Google Scholar 

  32. Kadono T., Kira N., Suzuki K., Iwata O., Ohama T., Okada S., Nishimura Y., Akakabe M., Tsuda M.,  & Adachi M. (2015) Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum. Marine Drugs 13(8):5334–5357

  33. Ismaiel M. M., El-Ayouty Y. M., Said A. A.,  & Fathey H. A. (2018) Transformation of Dunaliella parva with PSY gene: Carotenoids show enhanced antioxidant activity under polyethylene glycol and calcium treatments. Biocatalysis and Agricultural Biotechnology 16:378–384

  34. Lohr M., Im C. S., & Grossman A. R. (2005) Genome-Based Examination of Chlorophyll and Carotenoid Biosynthesis in Chlamydomonas reinharditii. Plant Physiology 138(1):490–515

  35. Contreras G., Barahona S., Rojas M. C., Baeza M., Cifuentes V., & Alcaíno J. (2013) Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous. BMC Biotechnology 13(1):84

Download references

Funding

This study was supported by the Department of Biotechnology, Ministry of Science & Technology, Government of India (No. BT/PR13796/PBD/ 26/139/2010). Jayant Pralhad Rathod is thankful to the Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi, for providing the fellowship during the above research work.

Author information

Authors and Affiliations

Authors

Contributions

Experiments were planned by JPR, CV, AML and GP and conducted by JPR. Data was analyzed by JPR, CV and GP. Manuscript is written by GP and CV. Funding for the work was facilitated by AML.

Corresponding author

Correspondence to Gunjan Prakash.

Additional information

Publisher’s Note

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

Electronic supplementary material

ESM 1

(DOCX 80 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rathod, J.P., Vira, C., Lali, A.M. et al. Metabolic Engineering of Chlamydomonas reinhardtii for Enhanced β-Carotene and Lutein Production. Appl Biochem Biotechnol 190, 1457–1469 (2020). https://doi.org/10.1007/s12010-019-03194-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-019-03194-9

Keywords

Navigation