A metabolic gene cluster in Lotus japonicus discloses novel enzyme functions and products in triterpene biosynthesis

Abstract

SummaryGenes for triterpene biosynthetic pathways exist as metabolic gene clusters in oat and Arabidopsis thaliana plants. We characterized the presence of an analogous gene cluster in the model legume Lotus japonicus.In the genomic regions flanking the oxidosqualene cyclase AMY2 gene, genes for two different classes of cytochrome P450 and a gene predicted to encode a reductase were identified. Functional characterization of the cluster genes was pursued by heterologous expression in Nicotiana benthamiana. The gene expression pattern was studied under different developmental and environmental conditions. The physiological role of the gene cluster in nodulation and plant development was studied in knockdown experiments.A novel triterpene structure, dihydrolupeol, was produced by AMY2. A new plant cytochrome P450, CYP71D353, which catalyses the formation of 20-hydroxybetulinic acid in a sequential three-step oxidation of 20-hydroxylupeol was characterized. The genes within the cluster are highly co-expressed during root and nodule development, in hormone-treated plants and under various environmental stresses. A transcriptional gene silencing mechanism that appears to be involved in the regulation of the cluster genes was also revealed.A tightly co-regulated cluster of functionally related genes is involved in legume triterpene biosynthesis, with a possible role in plant development.

Something old, something new: Conserved enzymes and the evolution of novelty in plant specialized metabolism

“…After the duplication event, loss of one duplicate gene is the most likely fate of newly duplicated genes (Lynch and Conery, 2000;Krokida et al, 2013), and the examples noted above represent the rare surviving cases of successful retention and functional diversification. Although multiple factors were noted previously to influence the retention of duplicate genes (Birchler and Veitia, 2012;, two questions regarding the duplicated enzyme may be quite relevant with regard to recruitment into specialized metabolism.…”

CYP72A67 Catalyzes a Key Oxidative Step in Medicago truncatula Hemolytic Saponin Biosynthesis

“…Despite their physical proximity on chromosome 2 (distance between the two genes of 14 Kb), CYP72A68-430 and CYP72A68-470 did not show similar expression patterns in leaves suggesting they did not act as a paralog gene cluster. On the contrary, a consistent expression in different tissues and developmental stages has been found for genes -an oxido-squalene cyclase and two CYP450s -belonging to a putative metabolic cluster involved in triterpene biosynthesis in Lotus japonicus (Krokida et al, 2013).…”

Biosynthesis and biotechnological production of ginsenosides

“…In Arabidopsis, two gene clusters for the synthesis of novel triterpenes, thalianol and marneral, were identified (Field and Osbourn, 2008;Field et al, 2011). Gene cluster for the biosynthesis of a novel triterpene, dihydroluepol was also recently discovered in the model legume plant Lotus japonicus (Krokida et al, 2013). Most clusters have shown that the pair of terpene synthase and CYP are clustered and coordinately expressed across monocots and eudicots (Boutanaev et al, 2015).…”