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
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull. 1987;19:11-–5.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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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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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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DOI: https://doi.org/10.1007/s43393-024-00240-4