Functional identification of specialized diterpene synthases from Chamaecyparis obtusa and C. obtusa var. formosana to illustrate the putative evolution of diterpene synthases in Cupressaceae.

Chamaecyparis obtusa and C. obtusa var. formosana of the Cupressaceae family are well known for their fragrance and excellent physical properties. To investigate the biosynthesis of unique diterpenoid compounds, diterpene synthase genes for specialized metabolite synthesis were cloned from C. obtusa and C. obtusa var. formosana. Using an Escherichia coli co-expression system, eight diterpene synthases (diTPSs) were characterized. CoCPS and CovfCPS are class II monofunctional (+)-copalyl diphosphate synthases [(+)-CPSs]. Class I monofunctional CoLS and CovfLS convert (+)-copalyl diphosphate [(+)-CPP] to levopimaradiene, CoBRS, CovfBRS1, and CovfBRS3 convert (+)-CPP to (-)-beyerene, and CovfSDS converts (+)-CPP to (-)-sandaracopimaradiene. These enzymes are all monofunctional diterpene syntheses in Cupressaceae family of gymnosperm, and differ from those in Pinaceae. The discovery of the enzyme responsible for the biosynthesis of tetracyclic diterpene (-)-beyerene was characterized for the first time. Diterpene synthases with different catalytic functions exist in closely related species within the Cupressaceae family, indicating that this group of monofunctional diterpene synthases is particularly prone to the evolution of new functions and development of species-specific specialized diterpenoid constituents.

Discovery and characterization of diterpene synthases in Chamaecyparis formosensis Matsum. which participated in an unprecedented diterpenoid biosynthesis route in conifer.

Chamaecyparis formosensis Matsum. is an endemic and precious coniferous species of Taiwan, and is known for a high abundance of specialized metabolites, which contributes to the excellent timber durability. Several terpenoids were identified and isolated from C. formosensis wood and needles, and exhibit anti-fungal and anti-bacterial bioactivities, which may participate in plant defense against pathogens. In various identified compounds, not only cadinene and ferruginol, were identified in C. formosensis extracts but also unique diterpenoids, which include pisferal, totarol, and derivates of isoabienol. To understand the biosynthesis of these specific diterpenoids, we conducted a series of functional characterization of the C. formosensis diterpene synthases (CfdiTPSs), which participate in skeleton formation and differentiation of diterpenes. In this study, we identified eight diTPSs from C. formosensis transcriptome, and they all contain either class I or class II motif, which indicates they are all monofunctional enzymes. These candidates consist of three class II diTPSs and five class I diTPSs, and after conducting in vivo and in vitro assays, class II diTPS CfCPS1 was characterized as a (+)-copalyl diphosphate synthase ((+)-CPS), and class I diTPSs CfKSL1 could further convert (+)-copalyl diphosphate ((+)-CPP) to levopimaradiene. Meanwhile, CfKSL1 also accepted labda-13-en-8-ol diphosphate (LPP) as substrate and formed monoyl oxide. Another class I diTPS, CfKSL4, exhibits a strong enzymatic ability of isoabienol synthase, which is firstly reported in conifer. This finding provides potential participants in the biosynthesis of unique diterpenoids, and with this knowledge, we can further expand our understanding of diterpenoid metabolism in Cupressaceae and their potential role in plant defense.