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Enhanced production of triacylglycerols and polyunsaturated fatty acids in novel acid-tolerant mutants of the green microalga Chlorella saccharophila

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Abstract

In this study, the microalga Chlorella saccharophila was subjected to ultraviolet (UV) mutagenesis, and mutant screening was conducted based on acidity tolerance to generate mutants with increased triacylglycerol (TAG) and polyunsaturated fatty acid (PUFA) contents. Two improved mutant strains (M1 and M5) were generated. M1 and M5 accumulated 27.2% and 27.4% more TAG, respectively, and showed stronger fluorescence intensity than the wild-type (WT) strain when the cells of these mutants were stained with the lipophilic Nile Red stain. In the M1 mutant, 50.5% of the fatty acid methyl esters (FAMEs) were saturated (C16:0 and C18:0) and 25.27% were monounsaturated (C18:1) fatty acids which are suitable for biofuels production. In the M5 mutant, 65.19% of the total FAMEs were nutritional PUFAs (C16:2, C18:2, and C18:3), while these FAMEs were not detected in the WT. These results demonstrated that UV mutagenesis coupled to an acid pH screening strategy represents a valuable and fast platform to generate mutants of C. saccharophila with improved TAG and PUFA contents for biofuels and nutraceutical applications, respectively.

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Acknowledgements

This study was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT México) Project no. 169217. Jalsen Iván Teco Bravo received support from CONACYT (scholarship no. 404430). The authors thank Ileana C. Borges Argáez, Tanit Toledano Thompson and Fray Martin Baas Espinola for technical support.

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Authors and Affiliations

  1. Unidad de Biotecnología, Centro de Investigación Científica de Yucatán (CICY), Calle 43 No. 130 x 32 y 34, Col. Chuburná de Hidalgo, C.P. 97205, Mérida, Yucatán, México

    Jalsen Iván Teco-Bravo, Virginia Aurora Herrera-Valencia & Santy Peraza-Echeverria

  2. Unidad de Energía Renovable, Centro de Investigación Científica de Yucatán (CICY), Carretera Sierra Papacal-Chuburná Puerto, km 5. Sierra Papacal, C.P. 97302, Mérida, Yucatán, México

    Luis Felipe Barahona-Pérez

  3. Departamento de Ingeniería Química y Bioquímica, Tecnológico Nacional de México, Instituto Tecnológico de Mérida, Avenida Tecnológico S/N, C.P. 97118, Mérida, Yucatán, México

    Carlos Francisco Reyes-Sosa

  4. Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán (CICY), Calle 43 No. 130 x 32 y 34, Col. Chuburná de Hidalgo, C.P. 97205, Mérida, Yucatán, México

    Ángela Francisca Ku-González

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  1. Jalsen Iván Teco-Bravo
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Correspondence to Virginia Aurora Herrera-Valencia or Santy Peraza-Echeverria.

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Supplementary Fig.

 1 Comparison of the tolerance to acidity between the WT and mutant strains on a pH gradient plate. A) Control plate; only the WT strain was inoculated. B-F) The WT and mutant strains were streaked on the same pH gradient plate. Approximately 1x105 WT and mutagenized cells were streaked on the gradient in the direction from the acid to alkaline region (TIFF 5889 kb)

Supplementary Fig.

 2 Growth patterns observed with the WT and mutant strains of C. saccharophila on pH gradient agar plates. Approximately 4x107 cells were spread over the surface (TIFF 8373 kb)

Supplementary Fig.

 3 Lipid content of the WT and mutant strains of C. saccharophila at pH 6 and 7. A) Lipid extract in mg L-1 culture; B) Lipid content as a percentage of dry biomass weight (% DBW). Cells were incubated in 50 mL of TAP medium adjusted to the indicated pH for 10 days. Values are the mean ± standard deviation (n = 3). Means with a common letter are not significantly different (Fisher´s least significant difference, p ˂ 0.05) (TIFF 3264 kb)

Supplementary Fig.

 4 Productivity of the WT and mutant strains of C. saccharophila cultured at pH 6 and 7. A) Biomass productivity; B) Lipid productivity; C) TAG productivity. Cells were incubated in 50 mL of TAP medium adjusted to the pH indicated for 10 days. Values are the mean ± standard deviation (n = 3). Means with a common letter are not significantly different (Fisher´s least significant difference, p ˂ 0.05) (TIFF 2595 kb)

Supplementary Table

 1 Fatty acid methyl ester (FAME) composition of the WT and mutant strains (M1 and M5) of C. saccharophila (percentage of total FAMEs) (TIFF 795 kb)

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Teco-Bravo, J.I., Barahona-Pérez, L.F., Reyes-Sosa, C.F. et al. Enhanced production of triacylglycerols and polyunsaturated fatty acids in novel acid-tolerant mutants of the green microalga Chlorella saccharophila. Bioprocess Biosyst Eng 42, 1561–1571 (2019). https://doi.org/10.1007/s00449-019-02153-2

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