Project 3.4.1

Understanding Brettanomyces and its adaptation to control measures

Project summary

Brettanomyces yeast cause wine spoilage by producing 4-ethylphenol and 4-ethylguiacol which are responsible for ‘phenolic’, ‘leather’, ‘sweaty’ and ‘medicinal’ aromas (collectively known as ‘Brett’ character). Previous AWRI research has shown that it is possible for sulfite-resistant Brettanomyces strains to evolve and develop even greater levels of sulfite tolerance (when subjected to directed evolution under laboratory conditions), although the genetic basis for this adaptive response remains to be determined.

New molecular tools, including genetic transformation and gene knockout technology have recently been developed, and these now provide a powerful means to assist in the understanding of the evolution of Brettanomyces both in the laboratory and in the field.

This project will therefore extend the results of previous work by combining a new field survey of Brettanomyces (using both high-throughput phenotyping and whole genome sequencing to determine if further adaptive responses are occurring in the winery environment), with detailed molecular analysis of the genes responsible for resistance to sulfite and the production of the key sensory compounds responsible for Brettanomyces spoilage character (4-ethyl phenol (4-EP) and 4-ethyl guaiacol (4-EG)).

Latest information

Development of sulfur dioxide tolerance
A key question for the Australian wine industry is whether Brettanomyces may be developing tolerance to SO2, as this would severely constrain current control strategies. Studies conducted using industry isolates sourced from 2004 to 2019 identified many highly tolerant isolates, especially from more recent years, suggesting that tolerance to SO2 may be increasing over time. Whole-genome sequencing of these tolerant isolates was initiated to determine if the tolerant strains represented a novel genetic group or evolved members of an existing genotype. Genetic comparisons showed that the tolerant strains were all members of the existing AWRI 1499 clade, a group of strains already known to be tolerant to SO2, albeit to levels lower than those observed in many of these newer isolates (Figure 1). Detailed genomic analysis is now being used in an effort to uncover the precise genomic changes that may be responsible for the increased levels of SO2 tolerance observed in these strains compared to older members of the AWRI 1499 clade.

Figure 1. Genetic analysis of SO2-tolerant Brettanomyces strains. A whole-genome phylogeny was assembled from a collection of reference strains (coloured circles) and SO2-tolerant industry isolates (black circles). The phylogeny could be sub-divided into six clear genetic groups as indicated by the color-coding. All industry isolates were shown to cluster within the AWRI 1499-like clade (shown in green). Phylogeny is scaled by substitutions per site.

Molecular engineering of Brettanomyces bruxellensis
The ability to genetically manipulate microorganisms is essential for understanding their biology and metabolism. CRISPR technology was used to develop a way to manipulate the genome of Brettanomyces bruxellensis for the first time. This enabled the creation of defined mutant strains through deletion of specific genes and allowed for the role of these genes in the growth of B. bruxellensis in wine to be elucidated. Using this methodology, the SO2-transporter gene SSU1 was deleted from the genome of B. bruxellensis, confirming its role as a major facilitator of SO2 tolerance in this spoilage species