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Dekkera/Brettanomyces bruxellensis , the main spoilage yeast in barrel-aged wine, metabolize hydroxycinnamic acids into off-flavors, namely ethylphenols. Recently, both the enzymes involved in this transformation, the cinnamate decarboxylase ( DbCD ) and the vinylphenol reductase ( DbVPR ), have been identified. To counteract microbial proliferation in wine, sulfur dioxide (SO 2 ) is used commonly to stabilize the final product, but limiting its use is advised to preserve human health and boost sustainability in winemaking. In the present study, the influence of SO 2  was investigated in relation with pH and ethanol factors on the expression of  DbCD  and  DbVPR  genes and volatile phenol production in  D. bruxellensis  CBS2499 strain under different model wines throughout a response surface methodology (RSM). In order to ensure an exact quantification of  DbCD  and  DbVPR  expression, an appropriate housekeeping gene was sought among  DbPDC ,  DbALD ,  DbEF ,  DbACT , and  DbTUB  genes by GeNorm and Normfinder algorithms. The latter gene showed the highest expression stability and it was chosen as the reference housekeeping gene in qPCR assays. Even though SO 2  could not be commented as main factor because of its statistical irrelevance on the response of  DbCD  gene, linear interactions with pH and ethanol concurred to define a significant effect ( p  < 0.05) on its expression. The  DbCD  gene was generally downregulated respect to a permissive growth condition (0 mg/L mol. SO 2 , pH 4.5 and 5% v/v ethanol); the combination of the factor levels that maximizes its expression (0.83-fold change) was calculated at 0.25 mg/L mol. SO 2 , pH 4.5 and 12.5% (v/v) ethanol. On the contrary,  DbVPR  expression was not influenced by main factors or by their interactions; however, its expression is maximized (1.80-fold change) at the same conditions calculated for  DbCD  gene. While no linear interaction between factors influenced the off-flavor synthesis, ethanol and pH produced a significant effect as individual factors. The obtained results can be useful to improve the SO 2  management at the grape harvesting and during winemaking in order to minimize the  D./B. bruxellensis  spoilage.

frontiers in microbiology

A Response Surface Methodology Approach to Investigate the Effect of Sulfur Dioxide, pH, and Ethanol on DbCD and DbVPR Gene Expression and on the Volatile Phenol Production in Dekkera/Brettanomyces bruxellensis CBS2499