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.

Biochemical characterization of diterpene synthases of Taiwania cryptomerioides expands the known functional space of specialized diterpene metabolism in gymnosperms.

Taiwania cryptomerioides is a monotypic gymnosperm species, valued for the high decay resistance of its wood. This durability has been attributed to the abundance of terpenoids, especially the major diterpenoid metabolite ferruginol, with antifungal and antitermite activity. Specialized diterpenoid metabolism in gymnosperms primarily recruits bifunctional class-I/II diterpene synthases (diTPSs), whereas monofunctional class-II and class-I enzymes operate in angiosperms. In this study, we identified a previously unrecognized group of monofunctional diTPSs in T. cryptomerioides, which suggests a distinct evolutionary divergence of the diTPS family in this species. Specifically, five monofunctional diTPS functions not previously observed in gymnosperms were characterized, including monofunctional class-II enzymes forming labda-13-en-8-ol diphosphate (LPP, TcCPS2) and (+)-copalyl diphosphate (CPP, TcCPS4), and three class-I diTPSs producing biformene (TcKSL1), levopimaradiene (TcKSL3) and phyllocladanol (TcKSL5), respectively. Methyl jasmonate (MeJA) elicited the accumulation of levopimaradiene and the corresponding biosynthetic diTPS genes, TcCPS4 and TcKSL3, is consistent with a possible role in plant defense. Furthermore, TcCPS4 and TcKSL3 are likely to contribute to abietatriene biosynthesis via levopimaradiene as an intermediate in ferruginol biosynthesis in Taiwania. In conclusion, this study provides deeper insight into the functional landscape and molecular evolution of specialized diterpenoid metabolism in gymnosperms as a basis to better understand the role of these metabolites in tree chemical defense.

Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis).

(+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis nootkatensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was detected at 144±10µg/L yeast culture. CnVO belongs to a new subfamily of the CYP706 family of cytochrome P450 oxidases.

Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.

Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential.

Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica).

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.