Crocin production in engineered Nicotiana tabacum L. cell cultures: metabolic characterization, bioreactor cultivation and neuroprotective potential.
TL;DR
Results demonstrate that engineered N. tabacum callus cultures are a promising platform for scalable crocin production and might represent a potential source of bioactive apocarotenoids for pharmaceutical applications and demonstrates the potential of engineered tobacco cell cultures for sustainable large-scale crocin production.
Abstract
Crocins are water-soluble apocarotenoids from saffron (Crocus sativus L.) with potent antioxidant and neuroprotective properties, yet their supply is limited by low yields and labor-intensive cultivation. We previously engineered Nicotiana tabacum L. to express carotenoid cleavage dioxygenase 2 (CCD2) to establish an alternative production platform. Transgenic lines accumulated crocins at variable levels, including a subset of high-producing genotypes. However, these lines displayed chlorosis, impaired growth, and reduced viability in soil, highlighting a trade-off between crocin biosynthesis and photosynthetic function. To overcome this limitation, selected best performer lines were subsequently used to establish callus cultures, which were grown under light and dark conditions, elicited using nanoparticles and finally analyzed for crocin accumulation, carotenoid precursor profiles, and expression of key biosynthetic genes. Integration of metabolite and transcript data revealed complex relationships between carotenoid metabolism and crocin production, indicating that crocin accumulation might likely depend not only on gene expression but also on precursor availability, post-translational and traditional effects and metabolic flux. To develop a scalable production platform, the highest-producing line was transferred to suspension cultures and, for the first time, successfully cultivated in a bioreactor system. This approach yielded crocin-enriched extracts dominated by glycosylated crocin species and demonstrates the potential of engineered tobacco cell cultures for sustainable large-scale crocin production. Finally, hydroalcoholic extracts from the bioreactor culture showed no cytotoxicity in the human SK-N-BE neuroblastoma cell line and significantly protected cells from hydrogen peroxide-induced oxidative stress. Overall, these results demonstrate that engineered N. tabacum callus cultures are a promising platform for scalable crocin production and might represent a potential source of bioactive apocarotenoids for pharmaceutical applications.