Promiscuous CYP87A enzyme activity initiates cardenolide biosynthesis in plants.

Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families1,2. Cardenolides play protective roles in plants, but these molecules, including digoxin from foxglove (Digitalis spp.), are better known for treatment of congenital heart failure, atrial arrhythmia, various cancers and other chronic diseases3-9. However, it is still unknown how plants synthesize 'high-value', complex cardenolide structures from, presumably, a sterol precursor. Here we identify two cytochrome P450, family 87, subfamily A (CYP87A) enzymes that act on both cholesterol and phytosterols (campesterol and ß-sitosterol) to form pregnenolone, the first committed step in cardenolide biosynthesis in the two phylogenetically distant plants Digitalis purpurea and Calotropis procera. Arabidopsis plants overexpressing these CYP87A enzymes ectopically accumulated pregnenolone, whereas silencing of CYP87A in D. purpurea leaves by RNA interference resulted in substantial reduction of pregnenolone and cardenolides. Our work uncovers the key entry point to the cardenolide pathway, and expands the toolbox for sustainable production of high-value plant steroids via synthetic biology.

Single-cell multi-omics in the medicinal plant Catharanthus roseus.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Inhibition of miR31 and miR92a as Oncological Biomarkers in RKO Colon Cancer Cells Treated with Kaempferol-3-O-Glycoside Isolated from Black Bean.

MicroRNAs (miRNAs) are small molecules of 19-23 nucleotides of RNA that act as regulators of the expression of proteins in eukaryotic cells. Currently, the participation of miRNAs in the development of different types of cancer has been observed. To evaluate the inhibitory effect of kaempferol-3-O-glycoside on the expression of oncological biomarkers, miR31 and miR92a in a colon cancer cell line (RKO) were analyzed. Cells were cultured and treated with 1 mM kaempferol-3-O-glycoside isolated from black bean. Expression levels of miR31 and miR92a were evaluated by real-time PCR using TaqMan probes; in addition, two oncogenes (KRAS and c-MYC) and two tumor suppressors (AMP-activated protein kinase [AMPK] and adenomatous tumors of polyposis coli [APC]) were quantified to validate the biological effects; normalization of expression levels were carried out by 2-ΔΔCt. Results were analyzed by one-way ANOVA. The expression levels of miR31, miR92a, KRAS oncogene, and the c-MYC transcription factor were subexpressed upon 72 h post-treatment with kaempferol-3-O-glycoside compared with the control without treatment (P < .05); in contrast, the tumor suppressor genes AMPK (∼4.85, P = .005) and APC (∼2.71, P = .066) tumor suppressors genes were overexpressed. Our results showed the inhibitory effect of isolated black bean flavonoid kaempferol-3-O-glycoside on cancer biomarkers: miR31 and miR92a; based on our results, this flavonoid may have interesting nutritional, therapeutic, and/or prophylactic applications to combat colon cancer.