Molecular techniques for the detection of Dekkera bruxellensis in wine and the genetic basis of relevant physiological characteristics

: a review

Abstract

Brettanomyces/Dekkera is a wine contaminating yeast responsible for serious economic losses due to the production of volatile phenols and the consequent negative impact on wine quality. Despite its importance to the wine industry it has remained poorly understood at the genetic level for a long period of time. In recent years, a genomic survey sequencing was implemented on Dekkera bruxellensis by some research groups providing resources for further investigation on this species. This thesis aims to make a review on how the acquired genetic knowledge has been applied for the development of molecular techniques for the detection, identification and enumeration of Brettanomyces/Dekkera in wine. Furthermore it is evaluated whether the accessibility to the whole genome sequence of D. bruxellensis allows a better understanding of the adaptation and survival mechanisms of this yeast in wine and the genetic bases of some relevant phenotypic traits. It is extremely important to find rapid and effective ways to detect Brettanomyces/Dekkera in wine. Conventional methods are based on culturing the yeast on selective media, but these are laborious and time consuming, delaying corrective actions on the wine processing. Various studies based on molecular methods have been developed as alternatives to the traditional ones. They offer faster results and increased sensibility and specificity. However some limitations can be pointed out such as high detection limits and the quantification of non viable cells as is the case of QPCR. Beyond this, high cost equipment and skilled technicians are needed to execute the methods and for interpretation of the results. Various genes and proteins were identified and related with the capacity of Brettanomyces/Dekkera to survive in the wine environment. These genes are responsible for glucose transport, carbon metabolism, anaerobic growth, fermenting characteristics, nitrate assimilation and osmotic stress response. Proteins involved in cell wall budding, adhesion and pseudohyphal growth were also identified and seem to be related with the capacity of cells to survive on the surface of grapes and barrels. The analysis of the genome sequence has also revealed one ORF that encodes a protein related to sulphite tolerance of Brettanonomyces/Dekkera. However, further studies on the expression and regulation of these genes are still necessary. The production of volatile phenols is the major spoilage activity of Brettanonomyces/Dekkera. This requires the activity of two enzymes, the phenolic acid decarboxylase (PAD) and the vinylphenol reductase (VPR). The sequencing of Brettanomyces/Dekkera genome revealed the presence of the DbPAD gene. Two paralogous genes that encode PAD were identified and they differ in the gene expression and type of metabolism, which may explain the differences found between different isolates of Brettanomyces/Dekkera. The VPR protein has been isolated and purified but the gene sequence has yet to be found. Although the sequencing of the genome of Brettanomyces/Dekkera has brought new opportunities to better understand the behaviour of this yeast in wine, the molecular mechanisms are still poorly understood and further investigation is necessary.

Date of Award	31 Jul 2015
Original language	English
Awarding Institution	Universidade Católica Portuguesa
Supervisor	José António Couto (Supervisor)