The Yml081Wp effect on acetate production requires <i>ALD6</i>.

<p><i>ALD4</i>-null and <i>ALD6</i>-null M2 yeast strains with and without YML081W were grown to mid-log phase, and then assayed for acetic acid production by HPLC. The dashed line indicates the initial acetic acid level of the media. As expected, with the full complement of <i>ALD</i> genes, eliminating YML081W produced a significant reduction in acetic acid levels (compare the two leftmost columns). Eliminating <i>ALD6</i> produced a lower acetic acid level compared to wild-type (compare the first and fifth columns). However, the further elimination of YML081W had no effect (compare the fifth and sixth columns). Therefore, YML081W requires <i>ALD6</i> to mediate its effects on acetic acid levels.</p

Yml081Wp regulates acetaldehyde dehydrogenase activity.

<p>M2 yeast cells were grown to mid-log phase, then harvested and lysed. ACDH specific activity was assayed as described in the Materials and Methods section. The numbers represent (nmol NAD(P)H formed/min) per mg protein. Cells without Yml081Wp contained lower ACDH activity, while cells overexpressing YML081W contained higher ACDH activity. These differences were statistically significant (p<0.05).</p

Yml081Wp regulates <i>ALD</i> gene expression and protein levels.

<p>(<b>A</b>) Acetate biosynthesis pathway, adapted from Saint-Prix <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051551#pone.0051551-SaintPrix1" target="_blank">[6]</a>. (<b>B</b>) <i>ALD</i> gene expression was measured in mid-log phase wild-type and YML081W-null cells by quantitative PCR. Removal of YML081W resulted in a significant reduction in <i>ALD4</i> and <i>ALD6</i> mRNA levels. (<b>C</b>) <i>ALD</i> gene expression was measured in mid-log phase yeast cells overexpressing YML081W under the control of the <i>PGK1</i> promoter, and compared to wild-type cells. YML081W-overexpressing cells produced significantly higher levels of ALD4 and ALD6 mRNA compared to wild-type cells. (<b>D</b>) <i>ALD4</i> and <i>ALD6</i> genes were tagged with the FLAG epitope at the C-terminus in wild-type, YML081W-null and YML081Woverexpressing strains. The cells were grown to mid-log phase, then harvested and lysed. The lysates were immunoblotted with an α-FLAG antibody to detect the levels of Ald4p-FLAG and Ald6p-FLAG protein. Cells lacking YML081W produced lower levels of FLAG-tagged Ald4p and Ald6p, compared to wild-type cells. Cells overexpressing YML081W produced significantly higher levels of Ald4p-FLAG protein compared to wild-type cells. ALD6p-FLAG levels did not appear to change significantly. Membrane staining shown below the immunoblots indicates the equivalance of total protein loading between lanes.</p

The acetic acid effect is much stronger with YML081W than with its ohnolog <i>RSF2</i>.

<p>Wild-type, YML081W-null and <i>RSF2</i>-null strains were grown in triplicate to mid-log phase in YPD media, and the resulting media was assayed for acetic acid. The dashed line indicates the initial acetic acid level of the media. Elimination of YML081W has a much more dramatic effect on acetic acid levels than elimination of <i>RSF2</i>.</p

Yml081Wp regulates the <i>ALD6</i> promoter.

<p>(<b>A</b>) A schematic diagram of the <i>ALD6</i> promoter sequence. Numbers on the left indicate the position of the first nucleotide in the promoter construct relative to the start codon. The positions and sequences of sites matching the previously published Yml081Wp binding sites are highlighted above the full-length promoter. (<b>B</b>) LacZ reporter activity from the <i>ALD6</i> promoter. Reporter plasmids carrying <i>ALD6</i> promoter fragments of the indicated lengths were transformed into wild-type M2 and YML081W-null strains. Three independent transformants for each reporter were grown to log phase, and assayed for β-galactosidase activity. Truncation of the reporter resulted in progressively lower activity levels. Importantly, cells lacking Yml081Wp produced significantly lower β-galactosidase activity than their wild-type counterparts (except for the shortest reporter fragment), suggesting that this transcription factor plays a positive role in stimulating <i>ALD6</i> transcription. (<b>C</b>) A consensus binding site at −590 plays an important role in mediating Yml081Wp transcriptional activity on the <i>ALD6</i> promoter. A reporter plasmid carrying a mutation of the YML081W consensus binding site was constructed for comparison to its wild-type counterpart. In cells producing normal levels of Yml081Wp, the mutation resulted in a 54% reduction in β-galactosidase activity (* indicates p<0.05 for a two-tailed Student t-test, compared to wild-type). However, in cells lacking Yml081Wp, the mutation had no significant effect on β-galactosidase activity. This result suggests that the consensus binding site at position −590 mediates Yml081Wp transcriptional activity on the <i>ALD6</i> promoter.</p

The Fermentation Stress Response Protein Aaf1p/Yml081Wp Regulates Acetate Production in <em>Saccharomyces cerevisiae</em>

<div><p>The production of acetic acid during wine fermentation is a critical issue for wineries since the sensory quality of a wine can be affected by the amount of acetic acid it contains. We found that the C2H2-type zinc-finger transcription factor YML081Wp regulated the mRNA levels of <em>ALD4 and ALD6</em>, which encode a cytosolic acetaldehyde dehydrogenase (ACDH) and a mitochondrial ACDH, respectively. These enzymes produce acetate from acetaldehyde as part of the pyruvate dehydrogenase bypass. This regulation was also reflected in the protein levels of Ald4p and Ald6p, as well as total ACDH activity. In the absence of <em>ALD6</em>, YML081W had no effect on acetic acid levels, suggesting that this transcription factor’s effects are mediated primarily through this gene. <em>lacZ</em> reporter assays revealed that Yml081wp stimulates <em>ALD6</em> transcription, in large part from a GAGGGG element 590 base pairs upstream of the translation start site. The non-annotated ORF YML081W therefore encodes a transcription factor that regulates acetate production in <em>Saccharomyces cerevisiae.</em> We propose <em>AAF1</em> as a gene name for the YML081W ORF.</p> </div

Yml081Wp regulates acetic acid levels.

<p>(<b>A</b>) Wild-type, YML081W-null and YML081W-overexpressing M2 yeast cells were used in a wine fermentation with sterile Chardonnay grape juice. Once fermentation was complete, the wine was assayed for acetic acid by HPLC. YML081W-null cells produced significantly less acetic acid, while YML081W-overexpressing cells produced significantly more acetic acid. In this figure, and all subsequent figures, * indicates p<0.05 for a two-tailed Student t-test, compared to wild-type. (<b>B</b>) These same strains were grown in triplicate in YPD media. At 4 hour intervals, media samples were removed, and assayed for acetic acid as above. YML081-null cells (▪, dotted line) produced significantly less acetic acid in the media than wild-type cells (•, solid line), while YML081W-overexpressing cells (⧫, dashed line) produced significantly more acetic acid.</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

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

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