Biosynthesis of Sandalwood Oil: <i>Santalum album</i> CYP76F Cytochromes P450 Produce Santalols and Bergamotol

<div><p>Abstract</p><p>Sandalwood oil is one of the world’s most highly prized essential oils, appearing in many high-end perfumes and fragrances. Extracted from the mature heartwood of several <i>Santalum</i> species, sandalwood oil is comprised mainly of sesquiterpene olefins and alcohols. Four sesquiterpenols, α-, β-, and <i>epi</i>-β-santalol and α-<i>exo-</i>bergamotol, make up approximately 90% of the oil of <i>Santalum album.</i> These compounds are the hydroxylated analogues of α-, β-, and <i>epi</i>-β-santalene and α-<i>exo</i>-bergamotene. By mining a transcriptome database of <i>S. album</i> for candidate cytochrome P450 genes, we cloned and characterized cDNAs encoding a small family of ten cytochrome P450-dependent monooxygenases annotated as <i>Sa</i>CYP76F37v1, <i>Sa</i>CYP76F37v2, <i>Sa</i>CYP76F38v1, <i>Sa</i>CYP76F38v2, <i>Sa</i>CYP76F39v1, <i>Sa</i>CYP76F39v2, <i>Sa</i>CYP76F40, <i>Sa</i>CYP76F41, <i>Sa</i>CYP76F42, and <i>Sa</i>CYP76F43. Nine of these genes were functionally characterized using <i>in vitro</i> assays and yeast <i>in vivo</i> assays to encode santalene/bergamotene oxidases and bergamotene oxidases. These results provide a foundation for production of sandalwood oil for the fragrance industry by means of metabolic engineering, as demonstrated with proof-of-concept formation of santalols and bergamotol in engineered yeast cells, simultaneously addressing conservation challenges by reducing pressure on supply of sandalwood from native forests.</p></div

Retention indices of sesquiterpenes and sesquiterpenols identified in the enzyme assays with cytochromes P450 of the <i>S. album</i> CYP76F subfamily and of sesquiterpene alcohols of <i>S. album</i> oil.

1<p>These numbers are used as identifiers for compounds and corresponding peaks in.</p><p>GC traces throughout the paper and figures.</p>2<p>Linear retention indices (LRI) measured on a DBwax column.</p>3<p>Linear retention indices (LRI) measured on an HP5 column.</p

GCMS analysis of products formed <i>in vitro</i> with clade I <i>Sa</i>CYP76Fs.

<p>GCMS analysis (extracted ion chromatograms) of products formed <i>in vitro</i> with (<b>A</b>) <i>Sa</i>CYP76F39v2; (<b>B</b>) <i>Sa</i>CYP76F40; (<b>C</b>) <i>Sa</i>CYP76F41; (<b>D</b>) <i>Sa</i>CYP76F42. Assays were performed with a sesquiterpene mixture of α-, β- and <i>epi</i>-β-santalene and α-<i>exo</i>-bergamotene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone.0075053.s003" target="_blank">Figure S3</a>) as substrate and microsomes prepared from yeast transformed with <i>Sa</i>CPR, individual clade I candidate <i>Sa</i>CYP76F cDNAs, or (<b>E</b>) empty vector as control. Peak numbers match the numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone-0075053-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone-0075053-g001" target="_blank">Figure 1</a>.</p

Phylogenetic tree of <i>S. album</i> CYP76F proteins and related terpene-modifying P450s.

<p>The neighbor-joining tree was constructed with members of the CYP71 clan, using <i>Picea sitchensis Ps</i>CYP720B4 (ADR78276) as an outgroup. <i>S. album</i> CYP76F proteins fell into two clades, clade I santalene/bergamotene oxidases and clade II bergamotene oxidases. <i>Ca</i>CYP76B4, <i>Camptotheca acuminata</i> putative geraniol-10-hydroxylase (AES93118); <i>Cr</i>CYP76B6, <i>Catharanthus roseus</i> geraniol 10-hydroxylase (Q8VWZ7); <i>Sm</i>CYP76B4, <i>Swertia mussotii</i> geraniol 10-hydroxylase (D1MI46); <i>Os</i>CYP76M7 <i>Oryza sativa ent</i>-cassadiene C11a-hydroxylase (NP_001047185); <i>Mp</i>CYP71A32, <i>Mentha x piperita</i> menthofuran synthase (Q947B7); <i>Pa</i>CYP71A1, <i>Persea americana</i> (P24465); <i>Ci</i>CYP71AV8, <i>Cichorium intybus</i> valencene oxidase (ADM86719); <i>Mp</i>CYP71D13, <i>Mentha x piperita</i>; (−)-limonene-3-hydroxylase (AY281027); <i>Nt</i>CYP71D20, <i>Nicotiana tabacum</i>, 5-<i>epi</i>-aristolochene-1,3-dihydroxylase (AF368376); <i>Ga</i>CYP706B1, <i>Gossypium arboreum</i> (+)-delta-cadinene-8-hydroxylase (AAK60517). This work: <i>Sa</i>CYP76F37v1 (KC533717); <i>Sa</i>CYP76F37v2 (KC698966); <i>Sa</i>CYP76F38v1 (KC533715); <i>Sa</i>CYP76F38v2 (KC533718); <i>Sa</i>CYP76F39v1 (KC533716); <i>Sa</i>CYP76F39v2 (KC698967); <i>Sa</i>CYP76F40 (KC698968); <i>Sa</i>CYP76F41 (KC698969); <i>Sa</i>CYP76F42 (KC698965); <i>Sa</i>CYP76F43 (KC533719).</p

Schematic biosynthetic pathway for santalols and bergamotol in sandalwood.

<p>Compounds identified with numbers are: α-santalene (1), α-<i>exo</i>-bergamotene (2), <i>epi</i>-β-santalene (3), β-santalene (4), (<i>Z</i>)-α-santalol (5), (<i>E</i>)-α-santalol (7), (<i>Z</i>)-α-<i>exo</i>-bergamotol (6), (<i>E</i>)-α-<i>exo</i>-bergamotol (8), (<i>Z</i>)-<i>epi</i>-β-santalol (9), (<i>E</i>)-<i>epi</i>-β-santalol (11), (<i>Z</i>)-β-santalol (10), (<i>E</i>)-β-santalol (12). Numbers match the numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone-0075053-t001" target="_blank">Table 1</a>. DMADP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; <i>Sa</i>SSy, <i>S. album</i> santalene synthase.</p

GCMS analysis of products formed <i>in vivo</i> with <i>Sa</i>CYP76F39v1.

<p>GCMS analysis (extracted ion chromatograms) of compounds formed <i>in vivo</i> in yeast cells expressing <i>Sa</i>SSY, <i>Sa</i>CPR2 and (<b>A</b>) <i>Sa</i>CYP76F39v1 or (<b>B</b>) an empty vector. (<b>C</b>) Mass spectra of compounds corresponding to peaks 5–12 identified in (<b>A</b>). Peak numbers match the numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone-0075053-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone-0075053-g001" target="_blank">Figure 1</a>. Peaks in (<b>A</b>) and (<b>B</b>) marked with symbol (*) correspond to farnesol also produced in yeast cells without <i>Sa</i>CYP76F. Peaks in (<b>A</b>) marked with symbol (#) represent yeast <i>in vivo</i> modifications of santalols (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone.0075053.s006" target="_blank">Figure S6</a>).</p

GCMS analysis of products formed <i>in vitro</i> with <i>Sa</i>CYP76F39v1.

<p>A sesquiterpene mixture of α-, β- and <i>epi</i>-β-santalene and α-<i>exo</i>-bergamotene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone.0075053.s003" target="_blank">Figure S3</a>) was incubated with microsomes containing <i>Sa</i>CYP76F39v1 and <i>Sa</i>CPR produced in yeast. (<b>A</b>) Product profile (extracted ion chromatogram, EIC) of assays with <i>Sa</i>CYP76F39v1. (<b>B</b>) Authentic <i>S. album</i> oil. (<b>C</b>) Control assays were performed with microsomes isolated from yeast cells transformed with the empty vector. Mass spectra of compounds corresponding to peaks 5<b>–</b>12 identified in assays with <i>Sa</i>CYP76F39v1 (left panel) and <i>S. album</i> oil (right panel) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone.0075053.s004" target="_blank">Figure S4</a>. Peak numbers match the numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone-0075053-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone-0075053-g001" target="_blank">Figure 1</a>.</p

GCMS analysis of products formed <i>in vitro</i> with clade II <i>Sa</i>CYP76Fs.

<p>GCMS analysis (extracted ion chromatograms) of products formed <i>in vitro</i> with (<b>A</b>) <i>Sa</i>CYP76F38v1; (<b>B</b>) <i>Sa</i>CYP76F38v2; (<b>C</b>) <i>Sa</i>CYP76F37v1; (<b>D</b>) <i>Sa</i>CYP76F37v2. Assays were performed with a sesquiterpene mixture of α-, β- and <i>epi</i>-β-santalene and α-<i>exo</i>-bergamotene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone.0075053.s003" target="_blank">Figure S3</a>) as substrate and microsomes prepared from yeast transformed with <i>Sa</i>CPR, individual clade II candidate <i>Sa</i>CYP76F cDNAs, or (<b>E</b>) empty vector as control. Peak numbers match the numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone-0075053-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075053#pone-0075053-g001" target="_blank">Figure 1</a>.</p

Relative activities of <i>Sa</i>CYP76F39v1 and <i>Sa</i>CYP76F37v1 with different sesquiterpenes.

<p>Relative activities represent rate of product formation relative to product formation by <i>Sa</i>CYP76F39v1 with β-santalene.</p