Figure 1.
Schematic biosynthetic pathway for santalols and bergamotol in sandalwood.
Compounds identified with numbers are: α-santalene (1), α-exo-bergamotene (2), epi-β-santalene (3), β-santalene (4), (Z)-α-santalol (5), (E)-α-santalol (7), (Z)-α-exo-bergamotol (6), (E)-α-exo-bergamotol (8), (Z)-epi-β-santalol (9), (E)-epi-β-santalol (11), (Z)-β-santalol (10), (E)-β-santalol (12). Numbers match the numbers in Table 1. DMADP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; SaSSy, S. album santalene synthase.
Figure 2.
Phylogenetic tree of S. album CYP76F proteins and related terpene-modifying P450s.
The neighbor-joining tree was constructed with members of the CYP71 clan, using Picea sitchensis PsCYP720B4 (ADR78276) as an outgroup. S. album CYP76F proteins fell into two clades, clade I santalene/bergamotene oxidases and clade II bergamotene oxidases. CaCYP76B4, Camptotheca acuminata putative geraniol-10-hydroxylase (AES93118); CrCYP76B6, Catharanthus roseus geraniol 10-hydroxylase (Q8VWZ7); SmCYP76B4, Swertia mussotii geraniol 10-hydroxylase (D1MI46); OsCYP76M7 Oryza sativa ent-cassadiene C11a-hydroxylase (NP_001047185); MpCYP71A32, Mentha x piperita menthofuran synthase (Q947B7); PaCYP71A1, Persea americana (P24465); CiCYP71AV8, Cichorium intybus valencene oxidase (ADM86719); MpCYP71D13, Mentha x piperita; (−)-limonene-3-hydroxylase (AY281027); NtCYP71D20, Nicotiana tabacum, 5-epi-aristolochene-1,3-dihydroxylase (AF368376); GaCYP706B1, Gossypium arboreum (+)-delta-cadinene-8-hydroxylase (AAK60517). This work: SaCYP76F37v1 (KC533717); SaCYP76F37v2 (KC698966); SaCYP76F38v1 (KC533715); SaCYP76F38v2 (KC533718); SaCYP76F39v1 (KC533716); SaCYP76F39v2 (KC698967); SaCYP76F40 (KC698968); SaCYP76F41 (KC698969); SaCYP76F42 (KC698965); SaCYP76F43 (KC533719).
Figure 3.
GCMS analysis of products formed in vitro with SaCYP76F39v1.
A sesquiterpene mixture of α-, β- and epi-β-santalene and α-exo-bergamotene (Figure S3) was incubated with microsomes containing SaCYP76F39v1 and SaCPR produced in yeast. (A) Product profile (extracted ion chromatogram, EIC) of assays with SaCYP76F39v1. (B) Authentic S. album oil. (C) Control assays were performed with microsomes isolated from yeast cells transformed with the empty vector. Mass spectra of compounds corresponding to peaks 5–12 identified in assays with SaCYP76F39v1 (left panel) and S. album oil (right panel) are shown in Figure S4. Peak numbers match the numbers in Table 1 and Figure 1.
Table 1.
Retention indices of sesquiterpenes and sesquiterpenols identified in the enzyme assays with cytochromes P450 of the S. album CYP76F subfamily and of sesquiterpene alcohols of S. album oil.
Figure 4.
GCMS analysis of products formed in vitro with clade I SaCYP76Fs.
GCMS analysis (extracted ion chromatograms) of products formed in vitro with (A) SaCYP76F39v2; (B) SaCYP76F40; (C) SaCYP76F41; (D) SaCYP76F42. Assays were performed with a sesquiterpene mixture of α-, β- and epi-β-santalene and α-exo-bergamotene (Figure S3) as substrate and microsomes prepared from yeast transformed with SaCPR, individual clade I candidate SaCYP76F cDNAs, or (E) empty vector as control. Peak numbers match the numbers in Table 1 and Figure 1.
Figure 5.
GCMS analysis of products formed in vitro with clade II SaCYP76Fs.
GCMS analysis (extracted ion chromatograms) of products formed in vitro with (A) SaCYP76F38v1; (B) SaCYP76F38v2; (C) SaCYP76F37v1; (D) SaCYP76F37v2. Assays were performed with a sesquiterpene mixture of α-, β- and epi-β-santalene and α-exo-bergamotene (Figure S3) as substrate and microsomes prepared from yeast transformed with SaCPR, individual clade II candidate SaCYP76F cDNAs, or (E) empty vector as control. Peak numbers match the numbers in Table 1 and Figure 1.
Figure 6.
Relative activities of SaCYP76F39v1 and SaCYP76F37v1 with different sesquiterpenes.
Relative activities represent rate of product formation relative to product formation by SaCYP76F39v1 with β-santalene.
Figure 7.
GCMS analysis of products formed in vivo with SaCYP76F39v1.
GCMS analysis (extracted ion chromatograms) of compounds formed in vivo in yeast cells expressing SaSSY, SaCPR2 and (A) SaCYP76F39v1 or (B) an empty vector. (C) Mass spectra of compounds corresponding to peaks 5–12 identified in (A). Peak numbers match the numbers in Table 1 and Figure 1. Peaks in (A) and (B) marked with symbol (*) correspond to farnesol also produced in yeast cells without SaCYP76F. Peaks in (A) marked with symbol (#) represent yeast in vivo modifications of santalols (see Figure S6).