Understanding Brettanomyces and its adaptation to control measures

Abstract

Brettanomyces bruxellensis represents a major source of microbiological spoilage in winemaking. Current control measures rely heavily on the use of SO2 following primary fermentation; however, recent research suggests that Brettanomyces can evolve tolerance to this antimicrobial. This project applied a multifaceted approach to understand Brettanomyces spoilage. A survey of industrial isolates was conducted, which showed increasing tolerance to SO2, while elucidating the associated genetic alterations. A framework for rapid and sensitive detection of Brettanomyces was established, which also predicted SO2 tolerance levels, while additional control options, including a potential biocontrol strategy, were assessed for their efficacy in controlling tolerant Brettanomyces strains.

Summary

The propensity of Brettanomyces yeast to spoil wine remains one of the foremost microbiological problems faced by winemakers. Brettanomyces spoilage is currently managed through the application of a multi-faceted strategy previously developed by AWRI in collaboration with Australian winemakers. While the initiation of these efforts in the early 2000s facilitated industry-wide decreases in spoilage of finished wines, this strategy does not eliminate Brettanomyces yeasts from wineries and is heavily reliant upon use of the preservative SO2 to inhibit the growth of Brettanomyces. To ensure that Australian winemakers can continue to manage Brettanomyces in a cost-effective manner, this project examined interactions between Brettanomyces genetics and SO2 tolerance, and options for rapid detection and control.

More than 300 industry isolates were phenotyped, with assessments including SO2 tolerance and the production of volatile phenols. For the volatile phenol screening, a new high-throughput method was developed that used coumaric acid consumption as a proxy for the production of ethyl phenols. A range of SO2 tolerance levels was observed, from highly sensitive to levels that well exceed those there were considered to be the highest expected for Brettanomyces. Far less variation was observed in the consumption of coumaric acid, with all strains consuming amounts of coumaric acid that would be predicted to produce significant wine spoilage.

Twenty-four of the most SO2-tolerant isolates were fully genotyped using whole-genome sequencing. All of these isolates were shown to represent members of the previously identified AWRI 1499 clade, indicating that continued adaptation of this group is occurring, as opposed to the appearance of a new genetic clade.

B. bruxellensis has been shown to have a highly variable genetic make-up. The most prevalent (and most SO2-tolerant) genetic group that had previously been isolated from Australian wineries has a hybrid genome, which is predicted to have arisen through a ‘rare-mating’ event. The high level of SO2 tolerance that was produced through this mating is predicted to have allowed for a ‘selective-sweep’, which allowed this new variant to largely replace other B. bruxellensis strains in the winemaking environment.

The discovery of contemporary strains with higher levels of SO2 tolerance than observed previously, posed the question as to whether these new strains represented further adaptation by members of the original hybrid group, or the appearance of a completely new type of strain.
Genomic comparison of these new tolerant isolates showed that they were not a new type of B. bruxellensis, but represented adapted versions of the original hybrid group. More detailed analysis highlighted an amplification of a specific gene, SSU1, as being important for this additional adaptation to SO2, providing a potential molecular marker for use in genetic testing for SO2 tolerance.

While genetic transformation of B. bruxellensis had recently been established, which enabled the introduction of new genetic material, no methodology existed for creating specific alterations, such as gene deletions, to the existing genome. As CRISPR-based gene editing tools had been shown to provide an efficient means to perform genetic alterations in other yeast species, a CRISPR-based platform was developed for genetic engineering in Brettanomyces. The method that was established was shown to be able to efficiently and accurately create specific deletions in the Brettanomyces genome and was used to investigate genes responsible for SO2 tolerance and the formation of volatile phenols. The production of volatile phenols represents the major detrimental impact of Brettanomyces on wine quality. Harnessing tools such as CRISPR-gene deletion that were developed in this project, it was possible to investigate the metabolic pathways that impacted the formation of volatile phenols by Brettanomyces.

Both random mutagenesis and candidate gene approaches were used to identify components of the volatile phenol pathway. While the identity of the gene that encodes the final step of the pathway (vinyl-phenol reductase) remains to be identified, it was possible to formally characterise the Brettanomyces gene PAD1 as encoding the phenylacrylic acid decarboxylase enzyme that is responsible for the first step in the pathway, which converts hydroxycinnamic acids to their vinyl derivative.

Phenol-negative Brettanomyces strains were developed through CRISPR-based deletion of the PAD1 gene that was identified as encoding the phenylacrylic acid decarboxylase enzyme during this project.

These phenol-negative strains were shown to be fully capable of growth in wine but did not produce detectable spoilage compounds. This key characteristic was then investigated for its potential use as a bioprotective strategy, with inoculation of the phenol-negative strain shown to inhibit both the growth and volatile phenol production of other strains of Brettanomyces. Phenol-negative Brettanomyces therefore represents an innovative means of biological control of Brettanomyces spoilage.

This project has developed foundational datasets on the current state-of-play in Brettanomyces population characteristics and genomics and combined this with a suite of potential tools to aid in addressing wine spoilage by this yeast.