ABSTRACT
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.
ABSTRACT
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.
