Abstract
In the Medicago genus, triterpene saponins are a group of bioactive compounds extensively studied for their different biological and pharmaceutical properties. In this work, the CRISPR/Cas9-based approach with two single-site guide RNAs was used in Medicago truncatula (barrel medic) to knock-out the CYP93E2 and CYP72A61 genes, which are responsible for the biosynthesis of soyasapogenol B, the most abundant soyasapogenol in Medicago spp. No transgenic plants carrying mutations in the target CYP72A61 gene were recovered while fifty-two putative CYP93E2 mutant plant lines were obtained following Agrobacterium tumefaciens-mediated transformation. Among these, the fifty-one sequenced plant lines give an editing efficiency of 84%. Sequencing revealed that these lines had various mutation patterns at the target sites. Four T0 mutant plant lines were further selected and examined for their sapogenin content and plant growth performance under greenhouse conditions. The results showed that all tested CYP93E2 knock-out mutants did not produce soyasapogenols in the leaves, stems and roots, and diverted the metabolic flux toward the production of valuable hemolytic sapogenins. No adverse influence was observed on the plant morphological features of CYP93E2 mutants under greenhouse conditions. In addition, differential expression of saponin pathway genes was observed in CYP93E2 mutants in comparison to the control. Our results provide new and interesting insights into the application of CRISPR/Cas9 for metabolic engineering of high-value compounds of plant origin and will be useful to investigate the physiological functions of saponins in planta.
Highlights
Saponins are a large group of triterpene or steroid glycosides common to many plant species (Haralampidisis et al, 2002) exhibiting a wide spectrum of biological and pharmacological activities (Sparg et al, 2004; Tava and Avato, 2006; Augustin et al, 2011; Moses et al, 2014a)
The major requirement for the successful engineering of specific saponins is the knowledge of the genes/enzymes involved in their biosynthesis
The economic relevance of these valuable natural compounds led to a substantial understanding of the biosynthetic routes of triterpene saponins, in particular in M. truncatula, a rich source of different saponins and a model plant species for legumes (Pateraki et al, 2015; Carelli et al, 2020)
Summary
Saponins are a large group of triterpene or steroid glycosides common to many plant species (Haralampidisis et al, 2002) exhibiting a wide spectrum of biological and pharmacological activities (Sparg et al, 2004; Tava and Avato, 2006; Augustin et al, 2011; Moses et al, 2014a). These properties have, in the last decades, stimulated research on triterpene saponin biosynthetic. Hemolytic saponins from Medicago spp. are valuable specialized metabolites to be employed in the agro-industry and for pharmaceutical applications (Ellen et al, 2007; Bora and Sharma, 2011; Rafinska et al, 2017)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
