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Decarboxylation of sorbic acid by spoilage yeasts is associated with the PAD1 gene - PubMed

Decarboxylation of sorbic acid by spoilage yeasts is associated with the PAD1 gene

Malcolm Stratford et al. Appl Environ Microbiol. 2007 Oct.

Abstract

The spoilage yeast Saccharomyces cerevisiae degraded the food preservative sorbic acid (2,4-hexadienoic acid) to a volatile hydrocarbon, identified by gas chromatography mass spectrometry as 1,3-pentadiene. The gene responsible was identified as PAD1, previously associated with the decarboxylation of the aromatic carboxylic acids cinnamic acid, ferulic acid, and coumaric acid to styrene, 4-vinylguaiacol, and 4-vinylphenol, respectively. The loss of PAD1 resulted in the simultaneous loss of decarboxylation activity against both sorbic and cinnamic acids. Pad1p is therefore an unusual decarboxylase capable of accepting both aromatic and aliphatic carboxylic acids as substrates. All members of the Saccharomyces genus (sensu stricto) were found to decarboxylate both sorbic and cinnamic acids. PAD1 homologues and decarboxylation activity were found also in Candida albicans, Candida dubliniensis, Debaryomyces hansenii, and Pichia anomala. The decarboxylation of sorbic acid was assessed as a possible mechanism of resistance in spoilage yeasts. The decarboxylation of either sorbic or cinnamic acid was not detected for Zygosaccharomyces, Kazachstania (Saccharomyces sensu lato), Zygotorulaspora, or Torulaspora, the genera containing the most notorious spoilage yeasts. Scatter plots showed no correlation between the extent of sorbic acid decarboxylation and resistance to sorbic acid in spoilage yeasts. Inhibitory concentrations of sorbic acid were almost identical for S. cerevisiae wild-type and Deltapad1 strains. We concluded that Pad1p-mediated sorbic acid decarboxylation did not constitute a significant mechanism of resistance to weak-acid preservatives by spoilage yeasts, even if the decarboxylation contributed to spoilage through the generation of unpleasant odors.

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Figures

FIG. 1.
FIG. 1.

Chemical structures of (A) sorbic acid (2,4-hexadienoic acid) and (B) 1,3-pentadiene. Sorbic acid could be converted into 1,3-pentadiene by removal of the carboxylic acid group.

FIG. 2.
FIG. 2.

Time course of growth and 1,3-pentadiene formation by S. cerevisiae BY4741 (wild type [WT]) and S. cerevisiae Y05833 (pad1Δ) in multiple replicates of 30-ml bottles containing 10 ml YEPD, pH 4.0, 1 mM sorbic acid incubated and shaken at 120 rpm and 28°C. Each point indicates the mean and standard deviation of two replicate samples at each time point. Following pressure equalization, 10% of the headspace was sampled, and 1,3-pentadiene was detected by GCMS. Yeast cell density was determined by the optical density at 600 nm and converted to dry weight by calibration curve.

FIG. 3.
FIG. 3.

Percentages of decarboxylation of sorbic acid to 1,3-pentadiene (dark histograms) and cinnamic acid to styrene (light histograms) in S. cerevisiae strains containing or lacking PAD1. Strains were S. cerevisiae BY4741 (wild type), Y05833 (pad1Δ), AP001 (Y05833 transformed with pPad1), AP002 (Y05833 transformed with pESC). Tests were carried out over 10 h at 28°C. Data are based on two independent determinations. Error bars indicate standard deviations.

FIG. 4.
FIG. 4.

Northern blot analysis of PAD1 mRNA levels measured at 0, 10, 24, 33, 48, 72, and 96 h during growth in the presence of 1 mM sorbic acid. Growth of cultures during this experiment is shown in Fig. 2. Maximum PAD1 expression was found to coincide with exponential growth. ACT1 expression and 26S and 18S rRNA are used as loading controls.

FIG. 5.
FIG. 5.

Long-term growth (bottom) and rate of formation of styrene (top) by S. cerevisiae BY4741 in closed 30-ml bottles (multiple replicates) containing 10 ml YEPD, pH 4.0, 0.5 mM cinnamic acid incubated, with shaking at 120 rpm and 28°C. Following pressure equalization, styrene formation was detected by GCMS and expressed in milligrams against styrene standards. Yeast cell density was determined by optical density at 600 nm and converted to dry weight by calibration curve. Each point represents the mean of two cultures sampled at that time point. Error bars indicate standard deviations.

FIG. 6.
FIG. 6.

Growth and inhibition of S. cerevisiae BY4741 (closed symbols) and S. cerevisiae Y05833 (open symbols) by cinnamic acid or sorbic acid in YEPD, pH 4.0, after 14 days of incubation at 28°C. Data are based on the means of three independent determinations, and error bars indicate standard deviations. OD600, optical density at 600 nm.

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