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Engineering of ketocarotenoid biosynthetic pathway in Chlamydomonas reinhardtii through exogenous gene expression

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Abstract

Microalgal biorefineries have emerged as significant reservoirs of therapeutic compounds, including pigments and proteins. Facilitating a robust circular bioeconomy necessitates the augmentation of pigment synthesis alongside algae biofuel production. Nevertheless, inherent constraints in ketocarotenoid synthesis exist in naturally fast-growing microalgae strains, such as Chlamydomonas reinhardtii. To address this limitation, we overexpressed two pivotal enzymes in the carotenoid biosynthetic pathway, namely β-carotene hydroxylase (crt) and β-carotene ketolase (bkt), in C. reinhardtii utilizing strong promoters to amplify carotenoid production. The genetically modified (GM) microalgae were validated through PCR, Southern hybridization, and Western blot assays, confirming the presence and expression of both genes in the C. reinhardtii strains. These GM lines exhibited a substantial enhancement over wild-type (WT) algae, showcasing a remarkable 5.39-fold increase in β-carotene concentration and twofold increase in total carotenoids compared to the WT microalgae. Notably, the GM microalgae achieved astaxanthin production up to 1.47 ± 0.063 mg/g DCW, a compound absent in WT C. reinhardtii. These findings indicate the successful functionalization of Hematococcus pluvialis genes through nuclear expression in C. reinhardtii, facilitating ketocarotenoid production. This study presents a valuable strategy to boost carotenoid production in microalgae by stable overexpression of two heterologous genes within the nuclear genome of C. reinhardtii.

Graphical abstract

Graphical abstract for the study carried out which represents the in silico plasmid vector designing, algae transformation by electroporation, selection on antibiotic plates, PCR amplification for GM confirmation, Southern hybridization to confirm gene integration, Western blotting to check protein expression, pigment quantification, and algae growth determination.

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All data generated or analyzed during this study are included in this published article [and its supplementary information files].

References

  1. Fal S, Smouni A, Arroussi EH. Integrated microalgae-based biorefinery for wastewater treatment, industrial CO2 sequestration and microalgal biomass valorization: a circular bioeconomy approach. Environ Adv. 2023;12: 100365. https://doi.org/10.1016/j.envadv.2023.100365.

    Article  CAS  Google Scholar 

  2. Sharma A, Sarkar P, Chhabra M, Kumar A, Kumar A, Kothadia H, Mallick A. Carbon capture from petrol-engine flue gas: Reviving algae-based sequestration with integrated microbial fuel cells. Chem Eng J. 2023. https://doi.org/10.1016/j.cej.2023.146578.

    Article  Google Scholar 

  3. Sreenikethanam A, Raj S, Gugulothu P, Bajhaiya AK. Genetic engineering of microalgae for secondary metabolite production: Recent developments, challenges, and future prospects. Front Bioeng Biotechnol. 2022. https://doi.org/10.3389/fbioe.2022.836056.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Ganesan, A, Nawkarkar, P, Ali, S, Kajlaand, S, Kumar, S. Genetic engineering of microalgae for enhanced photosynthetic efficiency, CO2 fixation, and fuel-based products. In: Microalgae for sustainable products: the green synthetic biology platform. The Royal Society of Chemistry. 2022;134–154. https://doi.org/10.1039/9781839167508-00134

  5. Yadav K, Vasistha S, Nawkarkar P, Kumar S, Rai MP. Algal biorefinery culminating multiple value-added products: recent advances, emerging trends, opportunities, and challenges. 3 Biotech. 2022;12(10):244. https://doi.org/10.1007/s13205-022-03288-y.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Song I, Kim J, Baek K, Choi Y, Shin B, Jin E. The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii. Microb Cell Factories. 2020;19:1–9. https://doi.org/10.1186/s12934-020-01480-4.

    Article  CAS  Google Scholar 

  7. Velmurugan A, Muthukaliannan GK. Genetic manipulation for carotenoid production in microalgae an overview. Curr Res Biotechnol. 2022;4:221–8. https://doi.org/10.1016/j.crbiot.2022.03.005.

    Article  CAS  Google Scholar 

  8. Cordero BF, Couso I, León R, Rodríguez H, Vargas MÁ. Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis. Appl Microbiol Biotechnol. 2011;91:341–51. https://doi.org/10.1007/s00253-011-3262-y.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lin B, Cui Y, Yan M, Wang Y, Gao Z, Meng C, Qin S. Construction of astaxanthin metabolic pathway in the green microalga Dunaliella viridis. Algal Res. 2019;44: 101697. https://doi.org/10.1016/j.algal.2019.101697.

    Article  Google Scholar 

  10. Cao K, Cui Y, Sun F, Zhang H, Fan J, Ge B, Gao Z. Metabolic engineering and synthetic biology strategies for producing high-value natural pigments in Microalgae. Biotechnol Adv. 2023. https://doi.org/10.1016/j.biotechadv.2023.108236.

    Article  PubMed  Google Scholar 

  11. Kang NK, Baek K, Koh HG, Atkinson CA, Ort DR, Jin YS. Microalgal metabolic engineering strategies for the production of fuels and chemicals. Bioresour Technol. 2022;345: 126529. https://doi.org/10.1016/j.biortech.2021.126529.

    Article  CAS  PubMed  Google Scholar 

  12. Tsai TH, Lin JY, Ng IS. Cooperation of phytoene synthase, pyridoxal kinase and carbonic anhydrase for enhancing carotenoids biosynthesis in genetic Chlamydomonas reinhardtii. J Taiwan Inst Chem Eng. 2022;137: 104184. https://doi.org/10.1016/j.jtice.2021.104184.

    Article  CAS  Google Scholar 

  13. Tokunaga S, Morimoto D, Koyama T, Kubo Y, Shiroi M, Ohara K, Sawayama S. Enhanced lutein production in Chlamydomonas reinhardtii by overexpression of the lycopene epsilon cyclase gene. Appl Biochem Biotechnol. 2021;193:1967–78. https://doi.org/10.1007/s12010-021-03524-w.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  15. Chen Y, Du H, Liang H, Hong T, Li T. Enhanced carotenoid production in Chlamydomonas reinhardtii via overexpression of endogenous and exogenous beta-carotene ketolase (bKT) genes. Int J Mol Sci. 2023;24(14):11382. https://doi.org/10.3390/ijms241411382.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Huang D, Liu C, Su M, Zeng Z, Wang C, Hu Z, Li H. Enhancement of β-carotene content in Chlamydomonas reinhardtii by expressing bacterium-driven lycopene β-cyclase. Biotechnol Biofuels Bioprod. 2023;16(1):1–8. https://doi.org/10.1186/s13068-023-02377-1.

    Article  CAS  Google Scholar 

  17. Perozeni F, Cazzaniga S, Baier T, Zanoni F, Zoccatelli G, Lauersen KJ, Ballottari M. Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii. Plant Biotechnol J. 2020;18(10):2053–67. https://doi.org/10.1111/pbi.13364.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Rathod JP, Vira C, Lali AM, Prakash G. Metabolic engineering of Chlamydomonas reinhardtii for enhanced β-carotene and lutein production. Appl Biochem Biotechnol. 2020;190:1457–69. https://doi.org/10.1007/s12010-019-03194-9.

    Article  CAS  PubMed  Google Scholar 

  19. Tran NT, Kaldenhoff R. Metabolic engineering of ketocarotenoids biosynthetic pathway in Chlamydomonas reinhardtii strain CC-4102. Sci Rep. 2020;10(1):1–11. https://doi.org/10.1038/s41598-020-67756-2.

    Article  CAS  Google Scholar 

  20. Zheng K, Wang C, Xiao M, Chen J, Li J, Hu Z. Expression of bkt and bch genes from Haematococcus pluvialis in transgenic Chlamydomonas. Sci China Life Sci. 2014;57:1028–33. https://doi.org/10.1007/s11427-014-4729-8.

    Article  CAS  PubMed  Google Scholar 

  21. Jeong BR, Jang J, Jin E. Genome engineering via gene editing technologies in microalgae. Bioresour Technol. 2023. https://doi.org/10.1016/j.biortech.2023.128701.

    Article  PubMed  Google Scholar 

  22. Perozeni F, Baier T. Current nuclear engineering strategies in the green microalga Chlamydomonas reinhardtii. Life. 2023;13(7):1566. https://doi.org/10.3390/life13071566.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sharma A, Chhabra M. Performance evaluation of a photosynthetic microbial fuel cell (PMFC) using Chlamydomonas reinhardtii at cathode. Bioresour Technol. 2021;338: 125499. https://doi.org/10.1016/j.biortech.2021.125499.

    Article  CAS  PubMed  Google Scholar 

  24. Ahmad I, Sharma AK, Daniell H, Kumar S. Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2. Plant Biotechnol J. 2015;13(4):540–50. https://doi.org/10.1111/pbi.12278.

    Article  CAS  PubMed  Google Scholar 

  25. Zohir WF, Kapase VU, Kumar S. Identification and characterization of a new microalga Dysmorphococcus globosus-HI from the Himalayan Region as a potential source of natural astaxanthin. Biology. 2022;11(6):884. https://doi.org/10.3390/biology11060884.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kindle KL. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci. 1990;87(3):1228–32. https://doi.org/10.1073/pnas.87.3.1228.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Nawkarkar P, Kapase VU, Chaudhary S, Kajla S, Kumar S. Heterogeneous diacylglycerol acyltransferase expression enhances lipids and PUFA in Chlorella species. GCB Bioenergy. 2023. https://doi.org/10.1111/gcbb.13089.

    Article  Google Scholar 

  28. Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull. 1987;19:11-–5.

    Google Scholar 

  29. Hurkman WJ, Tanaka CK. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 1986;81(3):802–6. https://doi.org/10.1104/pp.81.3.802.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lichtenthaler HK, Buschmann C. Chlorophylls and carotenoids: measurement and characterization by UV-VIS spectroscopy. Curr Protoc Food Anal Chem. 2001;1(1):F4–F3. https://doi.org/10.1002/0471142913.faf0403s01.

    Article  Google Scholar 

  31. Tan CP, Zhao FQ, Su ZL, Liang CW, Qin S. Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii. J Appl Phycol. 2007;19:347–55. https://doi.org/10.1007/s10811-006-9141-8.

    Article  CAS  Google Scholar 

  32. Sun JP, Wei XH, Cong XM, Zhang WH, Qiu LX, Zang XN. Expression of fatty acid related gene promotes astaxanthin heterologous production in Chlamydomonas reinhardtii. Front Nutr. 2023;10:1130065. https://doi.org/10.3389/fnut.2023.1130065.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Gong M, Bassi A. Carotenoids from microalgae: a review of recent developments. Biotechnol Adv. 2016;34(8):1396–412. https://doi.org/10.1016/j.biotechadv.2016.10.005.

    Article  CAS  PubMed  Google Scholar 

  34. Kim U, Cho DH, Heo J, Yun JH, Choi DY, Cho K, Kim HS. Two-stage cultivation strategy for the improvement of pigment productivity from high-density heterotrophic algal cultures. Bioresour Technol. 2020;302: 122840. https://doi.org/10.1016/j.biortech.2020.122840.

    Article  CAS  PubMed  Google Scholar 

  35. Vila M, Galván A, Fernández E, León R. Ketocarotenoid biosynthesis in transgenic microalgae expressing a foreign β-C-4-carotene oxygenase gene. Microbial carotenoids from bacteria and microalgae. Methods Protoc. 2012;10:283–95. https://doi.org/10.1007/978-1-61779-879-5_17.

    Article  CAS  Google Scholar 

  36. Huang K, Su Z, He M, Wu Y, Wang M. Simultaneous accumulation of astaxanthin and β-carotene in Chlamydomonas reinhardtii by the introduction of foreign β-carotene hydroxylase gene in response to high light stress. Biotechnol Lett. 2022;44(2):321–31. https://doi.org/10.1007/s10529-022-03230-5.

    Article  CAS  PubMed  Google Scholar 

  37. Chen JH, Nagarajan D, Huang Y, Zhu X, Liao Q, Chang JS. A novel and effective two-stage cultivation strategy for enhanced lutein production with Chlorella sorokiniana. Biochem Eng J. 2022;188: 108688. https://doi.org/10.1016/j.bej.2022.108688.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to gratefully acknowledge Dr. Sarika Chaudhary, Associate Professor, Bennett University, for helping with the Western blot protocol, and providing her valuable suggestions. AS thanks ICGEB New Delhi and IIT Jodhpur for providing research facilities, and DST-INSPIRE for Ph.D. fellowship (Grant no. IF190164). Ms. Apoorva Malik is gratefully acknowledged for helping in HPLC.

Funding

This work was partially supported by DST INSPIRE Ph.D. Fellowship (Grant no. IF190164).

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Authors and Affiliations

  1. Environmental Biotechnology Laboratory, Indian Institute of Technology Jodhpur (IITJ), Jodhpur, Rajasthan, 342030, India

    Arti Sharma & Meenu Chhabra

  2. Metabolic Engineering Laboratory, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India

    Arti Sharma, Prachi Nawkarkar, Vikas U. Kapase & Shashi Kumar

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Contributions

Arti Sharma: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, review & editing. Prachi Nawkarkar: Supervision, Methodology, Formal analysis, Investigation, Writing – review & editing. Vikas U. Kapase: Formal analysis, Writing – review & editing. Meenu Chhabra: Conceptualization, Methodology, Formal analysis, Writing – review & editing. Shashi Kumar: Project administration, Supervision, Conceptualization, Methodology, Investigation, Writing – review & editing, Funding acquisition.

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Correspondence to Meenu Chhabra or Shashi Kumar.

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This article does not contain any studies with human participants or animals. The algae work complies the relevant institutional, national, and international guidelines and legislation.

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Sharma, A., Nawkarkar, P., Kapase, V.U. et al. Engineering of ketocarotenoid biosynthetic pathway in Chlamydomonas reinhardtii through exogenous gene expression. Syst Microbiol and Biomanuf (2024). https://doi.org/10.1007/s43393-024-00240-4

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