Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha

Abstract

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono-and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg 2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases.

Extending a Single Residue Switch for Abbreviating Catalysis in Plant ent-Kaurene Synthases

“…Intriguingly, a recently identified monofunctional KS from the liverwort Marchantia polymorpha (MpKS) (Kumar et al, 2016) was found to contain an Ile at the key position previously identified in spermatophyta KSs ( Figure 1 ). Moreover, this Ile is further conserved in the only characterized KS from a lycophyte (i.e., Selaginella moellendorffii , SmKS) (Shimane et al, 2014), as well as the bifunctional CPS/KSs identified from other bryophytes (Hayashi et al, 2006; Kawaide et al, 2011).…”

“…The enzymes investigated here are pseudomature constructs suitable for recombinant expression in Escherichia coli , as previously described for MpKS (Kumar et al, 2016), PpCPS/KS (Hayashi et al, 2006), OsKSL5i (Xu et al, 2007a), and AtKS (Yamaguchi et al, 1998). Site-directed mutants were constructed by whole-plasmid PCR amplification of the relevant pENTR/SD/d-TOPO constructs using the primers described in Supplementary Table S1, and AccuPrime TM Pfx DNA Polymerase.…”

“…While the reactions catalyzed by CPSs and KSs are mechanistically distinct and carried out in distinct active sites, in plants these enzymes seem to be derived from fusion of the genes for each enzyme – i.e., CPS and KS – potentially from bacteria (Morrone et al, 2009; Gao et al, 2012). However, the presumably bifunctional nature of the initially resulting enzyme appears to have been retained only in certain bryophytes (Hayashi et al, 2006; Kawaide et al, 2011; Kumar et al, 2016). The production of 1 in most plants appears to depend on separate CPS and KS that arose from duplication and sub-functionalization of the ancestral fused bifunctional enzyme, with retention of the original multi-domain structure (Zi et al, 2014).…”

“…However, such conservation of function versus parallel evolution from related but functionally divergent ancestors is difficult to discern from phylogenetic analyses given the extended timescale over which land plants have evolved (Kumar et al, 2016). For example, while KSs contain highly conserved DDxxD and NDxx(G/S/T)xxxE amino acid sequence motifs, these are found in class I terpene synthases more generally.…”

Sesquiterpenoids from Streptomyces anulatus isolated from Giraffa camelopardalis feces

“…Owing to low yields and difficulties in detection and isolation, the presence of terpenoids in the metabolites of bacteria, particularly actinomycetes, is likely to be overlooked. However, an in‐depth understanding of genome sequence of terpenes and the progress in fermentation and isolation provided the stimulus for the isolation of a large number of bacterial terpene metabolites …”