Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing
The development of effective tools for the sustainable supply of phyto-ingredients and natural substances with reduced environmental footprints can help mitigate the dramatic scenario of climate change. Plant cell cultures-based biorefineries can be a technological advancement to face this challenge...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-06-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2022.913374/full |
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| author | Elisa Cappetta Monica De Palma Rosa D’Alessandro Alessandra Aiello Raffaele Romano Giulia Graziani Alberto Ritieni Dario Paolo Franca Locatelli Francesca Sparvoli Teresa Docimo Marina Tucci |
| author_facet | Elisa Cappetta Monica De Palma Rosa D’Alessandro Alessandra Aiello Raffaele Romano Giulia Graziani Alberto Ritieni Dario Paolo Franca Locatelli Francesca Sparvoli Teresa Docimo Marina Tucci |
| author_sort | Elisa Cappetta |
| collection | DOAJ |
| description | The development of effective tools for the sustainable supply of phyto-ingredients and natural substances with reduced environmental footprints can help mitigate the dramatic scenario of climate change. Plant cell cultures-based biorefineries can be a technological advancement to face this challenge and offer a potentially unlimited availability of natural substances, in a standardized composition and devoid of the seasonal variability of cultivated plants. Monounsaturated (MUFA) fatty acids are attracting considerable attention as supplements for biodegradable plastics, bio-additives for the cosmetic industry, and bio-lubricants. Cardoon (Cynara cardunculus L. var. altilis) callus cultures accumulate fatty acids and polyphenols and are therefore suitable for large-scale production of biochemicals and valuable compounds, as well as biofuel precursors. With the aim of boosting their potential uses, we designed a biotechnological approach to increase oleic acid content through Agrobacterium tumefaciens-mediated metabolic engineering. Bioinformatic data mining in the C. cardunculus transcriptome allowed the selection and molecular characterization of SAD (stearic acid desaturase) and FAD2.2 (fatty acid desaturase) genes, coding for key enzymes in oleic and linoleic acid formation, as targets for metabolic engineering. A total of 22 and 27 fast-growing independent CcSAD overexpressing (OE) and CcFAD2.2 RNAi knocked out (KO) transgenic lines were obtained. Further characterization of five independent transgenic lines for each construct demonstrated that, successfully, SAD overexpression increased linoleic acid content, e.g., to 42.5%, of the relative fatty acid content, in the CcSADOE6 line compared with 30.4% in the wild type (WT), whereas FAD2.2 silencing reduced linoleic acid in favor of the accumulation of its precursor, oleic acid, e.g., to almost 57% of the relative fatty acid content in the CcFAD2.2KO2 line with respect to 17.7% in the WT. Moreover, CcSADOE6 and CcFAD2.2KO2 were also characterized by a significant increase in total polyphenolic content up to about 4.7 and 4.1 mg/g DW as compared with 2.7 mg/g DW in the WT, mainly due to the accumulation of dicaffeoyl quinic and feruloyl quinic acids. These results pose the basis for the effective creation of an engineered cardoon cells-based biorefinery accumulating high levels of valuable compounds from primary and specialized metabolism to meet the industrial demand for renewable and sustainable sources of innovative bioproducts. |
| first_indexed | 2024-04-13T14:47:01Z |
| format | Article |
| id | doaj.art-6e47c3da9aae4b7c922e60c66ef31485 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-04-13T14:47:01Z |
| publishDate | 2022-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-6e47c3da9aae4b7c922e60c66ef314852022-12-22T02:42:43ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2022-06-011310.3389/fpls.2022.913374913374Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 SilencingElisa Cappetta0Monica De Palma1Rosa D’Alessandro2Alessandra Aiello3Raffaele Romano4Giulia Graziani5Alberto Ritieni6Dario Paolo7Franca Locatelli8Francesca Sparvoli9Teresa Docimo10Marina Tucci11National Research Council, Institute of Bioscience and Bioresources, Portici, ItalyNational Research Council, Institute of Bioscience and Bioresources, Portici, ItalyNational Research Council, Institute of Bioscience and Bioresources, Portici, ItalyDepartment of Agricultural Sciences, University of Naples Federico II, Portici, ItalyDepartment of Agricultural Sciences, University of Naples Federico II, Portici, ItalyDepartment of Pharmacy, University of Naples Federico II, Naples, ItalyDepartment of Pharmacy, University of Naples Federico II, Naples, ItalyNational Research Council, Institute of Agricultural Biology and Biotechnology, Milan, ItalyNational Research Council, Institute of Agricultural Biology and Biotechnology, Milan, ItalyNational Research Council, Institute of Agricultural Biology and Biotechnology, Milan, ItalyNational Research Council, Institute of Bioscience and Bioresources, Portici, ItalyNational Research Council, Institute of Bioscience and Bioresources, Portici, ItalyThe development of effective tools for the sustainable supply of phyto-ingredients and natural substances with reduced environmental footprints can help mitigate the dramatic scenario of climate change. Plant cell cultures-based biorefineries can be a technological advancement to face this challenge and offer a potentially unlimited availability of natural substances, in a standardized composition and devoid of the seasonal variability of cultivated plants. Monounsaturated (MUFA) fatty acids are attracting considerable attention as supplements for biodegradable plastics, bio-additives for the cosmetic industry, and bio-lubricants. Cardoon (Cynara cardunculus L. var. altilis) callus cultures accumulate fatty acids and polyphenols and are therefore suitable for large-scale production of biochemicals and valuable compounds, as well as biofuel precursors. With the aim of boosting their potential uses, we designed a biotechnological approach to increase oleic acid content through Agrobacterium tumefaciens-mediated metabolic engineering. Bioinformatic data mining in the C. cardunculus transcriptome allowed the selection and molecular characterization of SAD (stearic acid desaturase) and FAD2.2 (fatty acid desaturase) genes, coding for key enzymes in oleic and linoleic acid formation, as targets for metabolic engineering. A total of 22 and 27 fast-growing independent CcSAD overexpressing (OE) and CcFAD2.2 RNAi knocked out (KO) transgenic lines were obtained. Further characterization of five independent transgenic lines for each construct demonstrated that, successfully, SAD overexpression increased linoleic acid content, e.g., to 42.5%, of the relative fatty acid content, in the CcSADOE6 line compared with 30.4% in the wild type (WT), whereas FAD2.2 silencing reduced linoleic acid in favor of the accumulation of its precursor, oleic acid, e.g., to almost 57% of the relative fatty acid content in the CcFAD2.2KO2 line with respect to 17.7% in the WT. Moreover, CcSADOE6 and CcFAD2.2KO2 were also characterized by a significant increase in total polyphenolic content up to about 4.7 and 4.1 mg/g DW as compared with 2.7 mg/g DW in the WT, mainly due to the accumulation of dicaffeoyl quinic and feruloyl quinic acids. These results pose the basis for the effective creation of an engineered cardoon cells-based biorefinery accumulating high levels of valuable compounds from primary and specialized metabolism to meet the industrial demand for renewable and sustainable sources of innovative bioproducts.https://www.frontiersin.org/articles/10.3389/fpls.2022.913374/fullMUFAcardoon callimetabolic engineeringdesaturasesoil cropsbiorefinery |
| spellingShingle | Elisa Cappetta Monica De Palma Rosa D’Alessandro Alessandra Aiello Raffaele Romano Giulia Graziani Alberto Ritieni Dario Paolo Franca Locatelli Francesca Sparvoli Teresa Docimo Marina Tucci Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing Frontiers in Plant Science MUFA cardoon calli metabolic engineering desaturases oil crops biorefinery |
| title | Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing |
| title_full | Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing |
| title_fullStr | Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing |
| title_full_unstemmed | Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing |
| title_short | Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing |
| title_sort | development of a high oleic cardoon cell culture platform by sad overexpression and rnai mediated fad2 2 silencing |
| topic | MUFA cardoon calli metabolic engineering desaturases oil crops biorefinery |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2022.913374/full |
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