The genetic basis of biosynthesis and anti-inflammatory activity of calendula officinalis triterpene fatty acid esters Daria Golubova , Honghao Su , Melissa Salmon, Connor Tansley, Maria O’Connell, Nicola Patron Earlham Institute, University of East Anglia, UK Plants produce a wide range of specialised metabolites that play important roles in interactions with their environments. Many of these natural products are also useful in food, industry, and medicine due to their biological activities. Until recently, extraction and chemical synthesis have been the primary means to obtain these molecules, but many natural products are difficult to purify, only accumulate in small quantities, or have complex structures making chemical synthesis challenging or uneconomical. In the past decade, the biosynthetic pathways of useful molecules have been elucidated and rebuilt in so-called ‘chassis’ organisms using synthetic biology approaches. However, the genetic basis of most plant natural products remains unknown. Our work focuses on identifying the genetic basis and investigating the bioactivity of C:16 hydroxylated triterpene fatty acid esters (TFAE) found in flowers of Calendula officinalis (pot marigold). Floral extracts enriched in these molecules have previously been associated with anti-inflammatory activity. By combining metabolomics, genomics, and transcriptomics with rapid, transient expression in Nicotiana benthamiana, we have identified and characterized an oxidosqualene cyclase (OSC) that produces the ψ-taraxasterol and taraxasterol triterpene scaffolds found in this species and across Asteraceae lineages in varying ratios. Mutagenesis of this enzyme has identified amino acids residues that contribute to differences in product ratios. We have also identified two cytochrome P450s (CYPS) that catalyse C:16 hydroxylation, identifying two amino residues important for substrate specificity, and an acyltransferase (ACT), enabling us to make progress towards pathway reconstruction. Finally, we have conducted preliminary cytotoxicity and anti-inflammatory bioassays with human leukaemia monocytic cell lines, which suggest anti-inflammation is activated through the IL-6 pathway. Our work provides insights into how plants evolve novel chemodiversity and provides a route for accessing new molecules via biosynthesis. It additionally demonstrates the value of data emerging from the Darwin Tree of Life in accessing the enormous potential of UK floral diversity.
P24F
© The Author(s), 2022
Made with FlippingBook Learn more on our blog