Biodegradation Mechanisms of Patulin in Candida guilliermondii: An iTRAQ-Based Proteomic Analysis

Abstract

Patulin, a potent mycotoxin, contaminates fruits and derived products worldwide, and is a serious health concern. Several yeast strains have shown the ability to effectively degrade patulin. However, the mechanisms of its biodegradation still remain unclear at this time. In the present study, biodegradation and involved mechanisms of patulin by an antagonistic yeast Candida guilliermondii were investigated. The results indicated that C. guilliermondii was capable of not only multiplying to a high population in medium containing patulin, but also effectively reducing patulin content in culture medium. Degradation of patulin by C. guilliermondii was dependent on the yeast cell viability, and mainly occurred inside cells. E-ascladiol was the main degradation product of patulin. An iTRAQ-based proteomic analysis revealed that the responses of C. guilliermondii to patulin were complex. A total of 30 differential proteins involved in 10 biological processes were identified, and more than two-thirds of the differential proteins were down-accumulated. Notably, a short-chain dehydrogenase (gi|190348612) was markedly induced by patulin at both the protein and mRNA levels. Our findings will provide a foundation to help enable the commercial development of an enzyme formulation for the detoxification of patulin in fruit-derived products.

Keywords: Penicillium; contamination; detoxification; mycotoxins; patulin; yeast.

Figure 1

Biodegradation of patulin by C. guilliermondii. (A) Effects of patulin on the growth of C. guilliermondii; (B) Biodegradation assay of patulin by co-incubating with living and dead yeast cells; (C) Biodegradation assay of patulin by co-incubating with supernatant of the yeast culture and intracellular protein extracts. Error bars indicate standard deviations of the means from three replicates. Values followed by different letters are significantly different according to a Duncan’s multiple range test (p < 0.05).

Figure 2

Biodegradation product of patulin by C. guilliermondii. (A) HPLC profiles of filtered mixtures before and after co-incubation of C. guilliermondii and patulin; (B) Changes in the peak area of peak 1 and peak 2 during the co-incubation; Error bars indicate standard deviations of the means from three replicates; (C) Conversion from patulin to E-ascladiol.

Figure 3

LC-TOF-MS and MS/MS analysis on the biodegradation product of patulin. (A,B) Extracted ion chromatograms of the 48 h sample (at m/z 155.035) and E-ascladiol standard (at m/z 155.0360); (C,D) Corresponding MS/MS profiles of the 48 h sample and E-ascladiol standard.

Figure 4

Differential proteins and their functional categories in iTRAQ-based quantitative proteomic analysis. (A,C) Numbers of up- and down- accumulated proteins at 24 and 48 h, respectively; (B,D), functional categories of up- and down-accumulated proteins according to Blast2GO analysis.

Figure 5

Changes in the relative expression patterns of genes encoding differential proteins. Bolded gi numbers represent up-accumulated proteins, as based upon proteomic analysis. The non-bolded gi numbers represent proteins that were down-accumulated in the proteomic analysis. Error bars indicate the standard deviations of the means from three replicates.