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

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 , 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 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 bacterium, which produces -kaurene, an intermediate in plant hormone biosynthesis, as this is the hypothesized activity for the ancestral . Unlike the extant functionally equivalent , it was possible to split CseDCS into separate, independently acting DTC and TS, with the first producing the expected -copalyl diphosphate (CPP), serving as a CPP synthase (CPS), while the second converts this to -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 . Regardless of the exact relationship, the discovery of fused bifunctional DCSs in bacteria supports the hypothesized origin of the plant family from such a bacterial gene.