The P450 multigene family of Fontainea and insights into diterpenoid synthesis

Abstract

Background Cytochrome P450s (P450s) are enzymes that play critical roles in the biosynthesis of physiologically important compounds across all organisms. Although they have been characterised in a large number of plant species, no information relating to these enzymes are available from the genus Fontainea (family Euphorbiaceae). Fontainea is significant as the genus includes species that produce medicinally significant epoxy-tigliane natural products, one of which has been approved as an anti-cancer therapeutic. Results A comparative species leaf metabolome analysis showed that Fontainea species possess a chemical profile different from various other plant species. The diversity and expression profiles of Fontainea P450s were investigated from leaf and root tissue. A total of 103 and 123 full-length P450 genes in Fontainea picrosperma and Fontainea venosa, respectively (and a further 127/125 partial-length) that were phylogenetically classified into clans, families and subfamilies. The majority of P450 identified are most active within root tissue (66.2% F. picrosperma, 65.0% F. venosa). Representatives within the CYP71D and CYP726A were identified in Fontainea that are excellent candidates for diterpenoid synthesis, of which CYP726A1, CYP726A2 and CYP71D1 appear to be exclusive to Fontainea species and were significantly more highly expressed in root tissue compared to leaf tissue. Conclusion This study presents a comprehensive overview of the P450 gene family in Fontainea that may provide important insights into the biosynthesis of the medicinally significant epoxy-tigliane diterpenes found within the genus.

Identification of Gene Biomarkers for Tigilanol Tiglate Content in Fontainea picrosperma

“…Roots are reported as the likely site of biosynthesis for macrocyclic diterpenes (e.g., jatrophane and ingenane) in other taxa of Euphorbiaceae [5], and recent studies suggest a similar situation with F. picrosperma with core enzymes encoding for the diterpenoid biosynthetic pathways found to be most highly expressed in roots [17]. A large repertoire of cytochrome P450 genes (P450s) was also reported that showed varied gene expression in both leaf and root tissue, which may correlate with different levels of EBC-46 [11].…”

“…Based on the EBC-46 quantitative analysis, three high and three low EBC-46 producers (H EBC-46 and L EBC-46 ) were used for RNA sequencing of leaf and root samples (raw reads were deposited into the NCBI, Sequence Read Archive (SRA) database under accession number PRJNA592624). Reads were mapped to the reference F. picrosperma transcriptome assembly, previously reported in Mitu et al [11], derived from leaf and root RNAs. The number of mapped clean reads for each gene was counted and normalized into transcripts per million (TPM) to calculate the expression level of the gene.…”

“…Approximately 100 mg of leaf and root tissue from 3 H EBC-46 and 3 L EBC-46 F. picrosperma was collected between 9 and 10 a.m. once a month over a year. RNA extraction followed a procedure previously described by Mitu et al [11]. The RNA integrity was assessed by visualization in 2.0% agarose gels, as well as Nanodrop spectrophotometer 2000c (Thermo Scientific, Waltham, MA, USA) at 260 and 280 nm.…”

“…Molecules 2022, 27, 3980 2 of 11 EBC-46 is a novel epoxy-tigliane diterpene ester extracted from the fruit of F. picrosperma [7,10]. TT is likely present in all tissues of the plant, and biosynthesis occurs in the roots [11]. It is of particular interest due to its effectiveness as a local treatment for a range of cancers in humans and animals [7,12,13].…”

“…EBC-46 was approved for use as a local intratumoural therapy for canine mast cell tumours by major regulatory authorities, including the European Medicines Agency and the United States Food and Drug Administration in 2020 [10,14] and is currently in clinical development for a range of cancers in humans and in companion animals [12,15]. EBC-46 is not amenable to synthesis on a commercial scale and is currently produced by isolation and purification from the fruit of F. picrosperma [7,11,16]. Consequently, there is considerable interest in the potential for selective breeding of F. picrosperma to improve yields of EBC-46.…”