Discovery of bifunctional diterpene cyclases/synthases in bacteria supports a bacterial origin for the plant terpene synthase gene family

Abstract

Land plants are well-known producers of terpenoids that play diverse roles in plant-environment interactions. The vast chemical diversity of terpenoids is initiated by terpene synthases. Plants contain a distinct mid-sized terpene synthase gene family termed TPS, which appears to have an ancient origin in a fused bacterial Class I (di)terpene synthase (TS) and Class II diterpene cyclase (DTC), corresponding to the catalytically relevant α-domain and βγ-didomains, respectively. However, while such fused tridomain bifunctional (Class I/II) diterpene cyclases/synthases (DCSs) have been found in plants (and fungi), no examples have been reported from bacteria, leaving the origin of the fusion event initiating the TPS gene family opaque. Here, the discovery of such tridomain bifunctional DCSs in bacteria is reported. Extensive genome mining unearthed five putative bacterial DCSs, with biochemical characterization revealing the expected bifunctional activity for three. The most intriguing was CseDCS from Candidatus sericytochromatia bacterium, which produces ent-kaurene, an intermediate in plant hormone biosynthesis, as this is the hypothesized activity for the ancestral TPS. Unlike the extant functionally equivalent TPSs, it was possible to split CseDCS into separate, independently acting DTC and TS, with the first producing the expected ent-copalyl diphosphate (CPP), serving as a CPP synthase (CPS), while the second converts this to ent-kaurene, serving as a kaurene synthase (KS). Nevertheless, sequence alignment and mutation analysis revealed intriguing similarities between this cyanobacterial fused CPS-KS and functionally equivalent TPSs. Regardless of the exact relationship, the discovery of fused bifunctional DCSs in bacteria supports the hypothesized origin of the plant TPS family from such a bacterial gene.

Acknowledgements

This work was supported by an ASAP-SPRINT award from the University of Tennessee, AgResearch (to F.C.) and a grant from the NIH (GM131885 to R.J.P.).

Author contributions

F.C. and R.J.P. designed the research. X.C., M.X., J.H., and M.S-D. performed research. X.C., M.X., J.H., M.S-D., R.J.P., and F.C. analyzed data. F.C. and R.J.P. wrote the paper.

Data availability

The sequences for the biochemically characterized terpene synthases reported in this paper have been deposited in the GenBank database (accession numbers OR920195-OR920199).

Conflict of interest statement

None declared.

Supplementary Data

Supplementary data are available at Horticulture Research online.

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