Functional characterization of an oxidosqualene cyclase (PdFRS) encoding a monofunctional friedelin synthase in Populus davidiana

Abstract

An oxidosqualene cyclase (PdFRS) from Populus davidiana was characterized as a monofunctional friedelin synthase by its heterologous expression in yeast and overexpression in plants. Triterpenes are one of the largest classes of plant chemical compounds composed of three terpene units, which form the basic skeleton of all sterols and saponins. Friedelin (friedelan-3-one), a pentacyclic triterpene, occurs in many plant families and is particularly present in rich amounts in cork tissues from trees. The biosynthesis of friedelin occurs through the oxidosqualene cyclase (OSC) enzyme that generates friedelin from 2,3-oxidosqualene after the maximum rearrangement of a triterpene skeleton. Populus davidiana is called Korean aspen and grows in northern East Asia. From 57,322 unique sequences generated from the P. davidiana transcriptome database, one complete coding sequence (PdFRS) was obtained from a contig, which showed 74% identity to Betula platyphylla β-amyrin synthase and 73% identity with friedelin synthase from Maytenus ilicifolia. The open reading frame (ORF) region of the PdFRS sequence was 2280 bp long and composed a 759 amino acid protein with a predicted molecular mass of 87.81 kDa. qPCR analysis revealed that methyl jasmonate treatments strongly upregulated PdFRS gene expression and resulted in enhanced friedelin accumulation in leaves. Heterologous expression of the PdFRS gene in yeast resulted in the production of friedelin triterpene as a single product, which was confirmed by comparison with the mass fragmentation pattern from an authentic friedelin standard by GC/MS analysis. Transgenic P. davidiana overexpressing the PdFRS gene was constructed via Agrobacterium-mediated transformation. Overexpression of PdFRS in transgenic P. davidiana lines resulted in enhanced friedelin production.

Friedelane‐type triterpene cyclase in celastrol biosynthesis from Tripterygium wilfordii and its application for triterpenes biosynthesis in yeast

“…By contrast, Tw OSC1 and Tw OSC3 clustered with Mi FRSs and Pd FRS into a clade. As previously described (Souzamoreira et al ., ; Han et al ., ), both Mi FRSs and Pd FRS were monofunctional friedelin synthases, that can produce friedelin only. Interestingly, there was also a multiproduct friedelin synthase, Kd FRS (Wang et al ., ), which could produce friedelin as a major product, taraxerol, and β‐amyrin as a minor product.…”

Oxidosqualene cyclases involved in the biosynthesis of triterpenoids in Quercus suber cork

“…4C), including residues in positions 749 and 426 (associated with lupeol synthesis), 742 (associated with multifunctional amyrin synthase activity), and 496 of our alignment (associated with friedelin synthesis). Previous sequence comparisons 37 and point mutation studies 38,39 demonstrated the residue in the latter position (Leu-491 in QsOSC3, position 496 in the alignment) as critical for friedelin synthesis. These results, combined with ours, underscore the power of association analyses to identify residues associated with specific catalytic activity and the extensive impact single amino acid substitutions can have on the catalytic activity of specialized metabolic enzymes.…”

Overexpression of TwSQS, TwSE and TwOSC Regulates Celastrol Accumulation in Cambial Meristematic Cells and Dedifferentiated Cells

“…After SE overexpression, the content of ganoderic acid in Ganoderma lingzhi was 2 times higher than that of the wild type (Zhang et al 2017a). In Populus davidiana, overexpression of OSC increased friedelin accumulation by 233-445% compared with the non-transgenic plants (Han et al 2019). However, the effects of overexpression of TwSQS, TwSE and TwOSC on celastrol biosynthesis in T. wilfordii CMCs and DDCs still remain unknown.…”