Towards a Cardoon (Cynara cardunculus var. altilis)-Based Biorefinery: A Case Study of Improved Cell Cultures via Genetic Modulation of the Phenylpropanoid Pathway

Paolo, Dario; Locatelli, Franca; Cominelli, Eleonora; Pirona, Raul; Pozzo, Sara; Graziani, Giulia; Ritieni, Alberto; Palma, Monica De; Docimo, Teresa; Tucci, Marina; Sparvoli, Francesca

doi:10.3390/ijms222111978

Cited by 4 publications

(6 citation statements)

References 112 publications

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“…industries [ 36 , 37 , 38 , 39 , 40 , 41 , 42 ]. Provided that plant cell bioreactors are designed to reduce inputs, especially energy, water, and waste, they can be an efficient and sustainable technique to save land and reduce agricultural footprints [ 25 , 42 , 43 ]. Moreover, cell cultures do not suffer from seasonal variation in yield and quality, typical of field production.…”

Section: Discussionmentioning

confidence: 99%

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Abiotic Stresses Elicitation Potentiates the Productiveness of Cardoon Calli as Bio-Factories for Specialized Metabolites Production

et al. 2022

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Cultivated cardoon (Cynara cardunculus L. var altilis) is a Mediterranean traditional food crop. It is adapted to xerothermic conditions and also grows in marginal lands, producing a large biomass rich in phenolic bioactive metabolites and has therefore received attention for pharmaceutical, cosmetic and innovative materials applications. Cardoon cell cultures can be used for the biotechnological production of valuable molecules in accordance with the principles of cellular agriculture. In the current study, we developed an elicitation strategy on leaf-derived cardoon calli for boosting the production of bioactive extracts for cosmetics. We tested elicitation conditions that trigger hyper-accumulation of bioactive phenolic metabolites without compromising calli growth through the application of chilling and salt stresses. We monitored changes in growth, polyphenol accumulation, and antioxidant capability, along with transcriptional variations of key chlorogenic acid and flavonoids biosynthetic genes. At moderate stress intensity and duration (14 days at 50–100 mM NaCl) salt exerted the best eliciting effect by stimulating total phenols and antioxidant power without impairing growth. Hydroalcoholic extracts from elicited cardoon calli with optimal growth and bioactive metabolite accumulation were demonstrated to lack cytotoxicity by MTT assay and were able to stimulate pro-collagen and aquaporin production in dermal cells. In conclusion, we propose a “natural” elicitation system that can be easily and safely employed to boost bioactive metabolite accumulation in cardoon cell cultures and also in pilot-scale cell culture production.

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Section: Discussionmentioning

confidence: 99%

“…Callus cultures were established according to [ 25 ] and maintained in continuous dark conditions at 24 °C in a growth chamber. Once established, callus cultures were subcultured every 30 days on agarized Gamborg B5 medium with the same composition as for induction.…”

Section: Methodsmentioning

confidence: 99%

Abiotic Stresses Elicitation Potentiates the Productiveness of Cardoon Calli as Bio-Factories for Specialized Metabolites Production

et al. 2022

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“…The oil from the samples was extracted using solvent extraction as reported in Paolo et al [ 40 ]. Briefly, lipids were extracted from 10 g of freeze-dried sample using 100 mL of hexane shaking the mixture for 24 h in a conical flask.…”

Section: Methodsmentioning

confidence: 99%

Use of Different Organic Carbon Sources in Cynara cardunculus Cells: Effects on Biomass Productivity and Secondary Metabolites

Oliviero

Langellotti

Russo

et al. 2022

Plants

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Cynara cardunculus (Asteraceae family) is a perennial plant native to Mediterranean regions. This plant represents a source of high-value compounds, such as polyphenols and fatty acids that have several industrial applications. However, in vitro plant cell cultures can represent a valid alternative to in-field cultivation and facilitate the extraction of metabolites of commercial interest. Generally, sucrose is the main sugar used for plant cell cultures, but other carbon sources can be considered. Here, we investigated the potential use of alternative organic carbon sources, such as galactose, maltose, glucose, glycerol, fructose, lactose, and starch, for the cultivation of C. cardunculus cells. Moreover, cardoon cells were collected, and an extraction of polyphenols and oils was performed to study the effects of different carbon sources on the production of bioactive molecules. This study provided evidence that cardoon cell growth can be supported by carbon sources other than sucrose. However, the carbon source inducing optimum growth, did not necessarily induce the highest production of high-value compounds.

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“…Friable callus was induced from cardoon leaves of the cv "Spagnolo" as described (Paolo et al, 2021) and maintained in the dark at 25 • C on Gamborg B5 (GB5) growth medium including vitamins (Duchefa Biochemie, Haarlem, Netherlands), supplied with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg/L adenine, 0.1 mg/L kinetin, 3% (w/v) sucrose, and 8% (w/v) agar, adjusted to pH 5.8.…”

Section: Plant Materials and Growth Conditionsmentioning

confidence: 99%

“…For these reasons, the development of cardoon cell cultures for biorefinery applications could be a valuable alternative strategy. A significant step toward the industrial use of cardoon cell cultures was recently obtained by Paolo et al (2021), who obtained AtMYB4 overexpressing callus lines characterized by the faster growth rate, reduction in lignin content, and improved accessibility of the biomass to enzymatic degradation.…”

Section: Introductionmentioning

confidence: 99%

Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing

et al. 2022

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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.

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Towards a Cardoon (Cynara cardunculus var. altilis)-Based Biorefinery: A Case Study of Improved Cell Cultures via Genetic Modulation of the Phenylpropanoid Pathway

Cited by 4 publications

References 112 publications

Abiotic Stresses Elicitation Potentiates the Productiveness of Cardoon Calli as Bio-Factories for Specialized Metabolites Production

Abiotic Stresses Elicitation Potentiates the Productiveness of Cardoon Calli as Bio-Factories for Specialized Metabolites Production

Use of Different Organic Carbon Sources in Cynara cardunculus Cells: Effects on Biomass Productivity and Secondary Metabolites

Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing

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