Skip to main content
SpringerLink
Log in

Carotenoid Pathway Engineering in Tobacco Chloroplast Using a Synthetic Operon

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

The carotenoid pathway in plants has been altered through metabolic engineering to enhance their nutritional value and generate keto-carotenoids, which are widely sought after in the food, feed, and human health industries. In this study, the aim was to produce keto-carotenoids by manipulating the native carotenoid pathway in tobacco plants through chloroplast engineering. Transplastomic tobacco plants were generated that express a synthetic multigene operon composed of three heterologous genes, with Intercistronic Expression Elements (IEEs) for effective mRNA splicing. The metabolic changes observed in the transplastomic plants showed a significant shift towards the xanthophyll cycle, with only a minor production of keto-lutein. The use of a ketolase gene in combination with the lycopene cyclase and hydroxylase genes was a novel approach and demonstrated a successful redirection of the carotenoid pathway towards the xanthophyll cycle and the production of keto-lutein. This study presents a scalable molecular genetic platform for the development of novel keto-carotenoids in tobacco using the Design–Build–Test–Learn (DBTL) approach.

Graphical Abstract

This study corroborates chloroplast metabolic engineering using a synthetic biology approach for producing novel metabolites belonging to carotenoid class in industrially important tobacco plant. The synthetic multigene construct resulted in producing a novel metabolite, keto-lutein with high accumulation of xanthophyll metabolites. This figure was drawn using BioRender (https://www.biorender.com).

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

Similar content being viewed by others

Data availability

Datasets generated from this study are provided in the main article and as supplemental files.

Abbreviations

IEE:

Intercistronic expression element

aadA:

Spectinomycin

CrtW/bkt:

β-Carotene ketolase

CrtZ/bhy:

β-Carotene hydroxylase

lcy:

β-Lycopene cyclase

CrtE:

GGPP synthase

CrtB:

Phytoene synthase

CrtI:

Phytoene synthase

CrtY:

Lycopene cyclase

LB:

Luria–Bertani media

SBMSN:

Super broth with ammonium and sucrose medium

2YT:

Yeast extract and tryptone media

TB:

Tryptone broth

MS:

Murashige and Skoog medium

References

  1. Ruiz-Sola, M. Á., & Rodríguez-Concepción, M. (2012). Carotenoid biosynthesis in Arabidopsis: A colorful pathway. Arabidopsis Book, 10, e0158.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Mann, V., Harker, M., Pecker, I., & Hirschberg, J. (2000). Metabolic engineering of astaxanthin production in tobacco flowers. Nature Biotechnology, 18(8), 888–892.

    Article  CAS  PubMed  Google Scholar 

  3. Kim, S. H., Ahn, Y. O., Ahn, M.-J., Lee, H.-S., & Kwak, S.-S. (2012). Down-regulation of β-carotene hydroxylase increases β-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweet potato. Phytochemistry, 74, 69–78.

    Article  CAS  PubMed  Google Scholar 

  4. Swapnil, P., Meena, M., Singh, S. K., Dhuldhaj, U. P., Harish, & Marwal, A. (2021). Vital roles of carotenoids in plants and humans to deteriorate stress with its structure, biosynthesis, metabolic engineering and functional aspects. Current Plant Biology, 26, 100203.

    Article  CAS  Google Scholar 

  5. Aziz, E., Batool, R., Akhtar, W., Rehman, S., Shahzad, T., Malik, A., Shariati, M. A., Laishevtcev, A., Plygun, S., Heydari, M., Rauf, A., & Ahmed Arif, S. (2020). Xanthophyll: Health benefits and therapeutic insights. Life Sciences, 240, 117104.

    Article  CAS  PubMed  Google Scholar 

  6. Naguib, Y. M. (2000). Antioxidant activities of astaxanthin and related carotenoids. Journal of Agricultural and Food Chemistry, 48(4), 1150–1154.

    Article  CAS  PubMed  Google Scholar 

  7. Misawa, N. (2009). Pathway engineering of plants toward astaxanthin production. Plant Biotechnology, 26(1), 93–99.

    Article  CAS  Google Scholar 

  8. Jin, S., & Daniell, H. (2015). The engineered chloroplast genome just got smarter. Trends in Plant Science, 20(10), 622–640.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Llorente, B., Torres-Montilla, S., Morelli, L., Florez-Sarasa, I., Matus, J. T., Ezquerro, M., D’andrea, L., Houhou, F., Majer, E., & Picó, B. (2020). Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development. Proceedings of the National Academy of Sciences of USA, 117(35), 21796–21803.

    Article  CAS  Google Scholar 

  10. Hasunuma, T., Miyazawa, S. I., Yoshimura, S., Shinzaki, Y., Tomizawa, K. I., Shindo, K., Choi, S. K., Misawa, N., & Miyake, C. (2008). Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering. The Plant Journal, 55(5), 857–868.

    Article  CAS  PubMed  Google Scholar 

  11. Harada, H., Maoka, T., Osawa, A., Hattan, J.-I., Kanamoto, H., Shindo, K., Otomatsu, T., & Misawa, N. (2014). Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids. Transgenic Research, 23(2), 303–315.

    Article  CAS  PubMed  Google Scholar 

  12. Jayaraj, J., Devlin, R., & Punja, Z. (2008). Metabolic engineering of novel ketocarotenoid production in carrot plants. Transgenic Research, 17(4), 489–501.

    Article  CAS  PubMed  Google Scholar 

  13. Lu, Y., Rijzaani, H., Karcher, D., Ruf, S., & Bock, R. (2013). Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons. Proceedings of the National Academy of Sciences of USA, 110(8), E623–E632.

    Article  Google Scholar 

  14. Zhou, F., Karcher, D., & Bock, R. (2007). Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons. The Plant Journal, 52(5), 961–972.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Apel, W., & Bock, R. (2009). Enhancement of carotenoid biosynthesis in transplastomic tomatoes by induced lycopene-to-provitamin A conversion. Plant Physiology, 151(1), 59–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhou, P., Ye, L., Xie, W., Lv, X., & Yu, H. (2015). Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt. Applied Microbiology and Biotechnology, 99(20), 8419–8428.

    Article  CAS  PubMed  Google Scholar 

  17. Maliga, P., Tungsuchat-Huang, T., & Lutz, K. A. (2021). Transformation of the plastid genome in tobacco: The model system for chloroplast genome engineering. Methods in Molecular Biology, 2317, 135–153.

    Article  CAS  PubMed  Google Scholar 

  18. Ruf, S., Hermann, M., Berger, I. J., Carrer, H., & Bock, R. (2001). Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nature Biotechnology, 19(9), 870.

    Article  CAS  PubMed  Google Scholar 

  19. McBride, K. E., Svab, Z., Schaaf, D. J., Hogan, P. S., Stalker, D. M., & Maliga, P. (1995). Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Bio/Technology, 13(4), 362–365.

    CAS  PubMed  Google Scholar 

  20. Daniell, H., Jin, S., Zhu, X. G., Gitzendanner, M. A., Soltis, D. E., & Soltis, P. S. (2021). Green giant—A tiny chloroplast genome with mighty power to produce high-value proteins: History and phylogeny. Plant Biotechnology Journal, 19(3), 430–447.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ruf, S., & Bock, R. (2014). Plastid transformation in tomato. In Chloroplast biotechnology (pp. 265–276). Springer.

  22. Verma, D., Samson, N. P., Koya, V., & Daniell, H. (2008). A protocol for expression of foreign genes in chloroplasts. Nature Protocols, 3(4), 739–758.

    Article  CAS  PubMed  Google Scholar 

  23. Day, A., & Goldschmidt-Clermont, M. (2011). The chloroplast transformation toolbox: Selectable markers and marker removal. Plant Biotechnology Journal, 9(5), 540–553.

    Article  CAS  PubMed  Google Scholar 

  24. Doyle, J. (1991). DNA protocols for plants. In Molecular techniques in taxonomy (pp. 283–293). Springer.

  25. Lichtenthaler, H. K., & Buschmann, C. (2001). Chlorophylls and carotenoids: Measurement and characterization by UV–Vis spectroscopy. Current Protocols in Food Analytical Chemistry, 1(1), F4.3.1-F4.3.8.

    Article  Google Scholar 

  26. Zielewicz, W., Wróbel, B., & Niedbała, G. (2020). Quantification of chlorophyll and carotene pigments content in Mountain Melick (Melica nutans L.) in relation to edaphic variables. Forests, 11(11), 1197.

    Article  Google Scholar 

  27. Huang, J.-C., Zhong, Y.-J., Liu, J., Sandmann, G., & Chen, F. (2013). Metabolic engineering of tomato for high-yield production of astaxanthin. Metabolic Engineering, 17, 59–67.

    Article  CAS  PubMed  Google Scholar 

  28. Campbell, R., Morris, W. L., Mortimer, C. L., Misawa, N., Ducreux, L. J., Morris, J. A., Hedley, P. E., Fraser, P. D., & Taylor, M. A. (2015). Optimising ketocarotenoid production in potato tubers: Effect of genetic background, transgene combinations and environment. Plant Science, 234, 27–37.

    Article  CAS  PubMed  Google Scholar 

  29. Zhu, C., Naqvi, S., Breitenbach, J., Sandmann, G., Christou, P., & Capell, T. (2008). Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize. Proceedings of the National Academy of Sciences of USA, 105(47), 18232–18237.

    Article  CAS  Google Scholar 

  30. Pierce, E. C., LaFayette, P. R., Ortega, M. A., Joyce, B. L., Kopsell, D. A., & Parrott, W. A. (2015). Ketocarotenoid production in soybean seeds through metabolic engineering. PLoS ONE, 10(9), e0138196.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Fujisawa, M., Takita, E., Harada, H., Sakurai, N., Suzuki, H., Ohyama, K., Shibata, D., & Misawa, N. (2009). Pathway engineering of Brassica napus seeds using multiple key enzyme genes involved in ketocarotenoid formation. Journal of Experimental Botany, 60(4), 1319–1332.

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  33. Stålberg, K., Lindgren, O., Ek, B., & Höglund, A. S. (2003). Synthesis of ketocarotenoids in the seed of Arabidopsis thaliana. The Plant Journal, 36(6), 771–779.

    Article  PubMed  Google Scholar 

  34. Ralley, L., Enfissi, E. M., Misawa, N., Schuch, W., Bramley, P. M., & Fraser, P. D. (2004). Metabolic engineering of ketocarotenoid formation in higher plants. The Plant Journal, 39(4), 477–486.

    Article  CAS  PubMed  Google Scholar 

  35. Zhang, Y., & Fernie, A. R. (2021). Metabolons, enzyme–enzyme assemblies that mediate substrate channeling, and their roles in plant metabolism. Plant Communications, 2(1), 100081.

    Article  CAS  PubMed  Google Scholar 

  36. Sun, T., Rao, S., Zhou, X., & Li, L. (2022). Plant carotenoids: Recent advances and future perspectives. Molecular Horticulture, 2(1), 3.

    Article  CAS  Google Scholar 

  37. Grossman, A. R., Harris, E. E., Hauser, C., Lefebvre, P. A., Martinez, D., Rokhsar, D., Shrager, J., Silflow, C. D., Stern, D., & Vallon, O. (2003). Chlamydomonas reinhardtii at the crossroads of genomics. Eukaryotic Cell, 2(6), 1137–1150.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Allen, Q. M., Febres, V. J., Rathinasabapathi, B., & Chaparro, J. X. (2022). Engineering a plant-derived astaxanthin synthetic pathway into Nicotiana benthamiana. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2021.831785

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors are grateful to Professor Dr. Ralph Bock and Dr. Stephanie Ruf from Max Planck Institute for Molecular Plant Physiology, Germany for providing us the chloroplast-specific vector pRB96 and resolving few queries for chloroplast transformation. We thank Professor K. C. Bansal for his guidance for the project and Professor Dr. Damodhar Reddy from ICAR-Central Tobacco Research Institute, India for sending us tobacco seeds for the study. We would also like to extend our gratitude to Dr. P. Maheswara Reddy and his research students for helping us to carry out this work in lab.

Funding

The authors thank Deakin University, Australia for partially funding the research and providing Post-Graduate Research Scholarship DUPRS to NT. We also thank TERI-Nanobiotechnology Centre, India to provide research infrastructure and internal funding support for the study.

Author information

Authors and Affiliations

  1. TERI-Deakin Nano-Biotechnology Centre, The Energy Resources Institute (TERI), New Delhi, 110003, India

    Neha Tanwar & Sangram K. Lenka

  2. School of Life and Environmental Sciences, Deakin University, Waurn Ponds Campus, Geelong, VIC, 3216, Australia

    Neha Tanwar, James E. Rookes & David M. Cahill

  3. Department of Plant Biotechnology, Gujarat Biotechnology University, Gandhinagar, 382355, India

    Sangram K. Lenka

Authors
  1. Neha Tanwar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. James E. Rookes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David M. Cahill
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sangram K. Lenka
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

NT conducted experiments, analysed data and drafted the MS. JER and DMC participated in drafting and data analysis and contributed reagents/chemicals. SKL participated in drafting and data analysis, contributed reagents/chemicals and designed experiments. All the authors read and approved the final MS.

Corresponding author

Correspondence to Sangram K. Lenka.

Ethics declarations

Conflict of interest

The authors declare no competing financial interest or any conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors. Further, the authors have considered Committee on Publication Ethics (COPE) guidelines for preparing this article.

Additional information

Publisher's Note

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

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 214 kb)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tanwar, N., Rookes, J.E., Cahill, D.M. et al. Carotenoid Pathway Engineering in Tobacco Chloroplast Using a Synthetic Operon. Mol Biotechnol 65, 1923–1934 (2023). https://doi.org/10.1007/s12033-023-00693-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-023-00693-3

Keywords

Search

Navigation