Understanding Brettanomyces and its adaptation to control measures
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)).
Understanding the development of sulfur dioxide tolerance
Whole-genome sequencing of SO2-tolerant Brettanomyces strains isolated from industry between 2004 and 2019 found that these isolates displayed structural genomics changes that included amplification of the SO2 transporter gene SSU1. Laboratory-based directed evolution was also used to assess the ability of Brettanomyces strains to evolve higher tolerance to SO2. Three B. bruxellensis strains, representing the known genetic variation within the species, were subjected to increasing sub-lethal sulfur dioxide concentrations. Individual isolates from the evolved populations displayed between 1.6 and 2.5 times higher SO2 tolerance than the original parental strains. Whole-genome sequencing revealed many structural changes to the genomes of the evolved isolates; however, as seen in the industry isolates, the SO2 transporter gene, SSU1, was amplified in all tolerant clones. This work, combined with the industry isolate testing, clearly demonstrates that as for Saccharomyces cerevisiae, alterations in the gene SSU1 is a key mechanism driving the development of SO2 tolerance in Brettanomyces. This discovery will help guide future work.
Anthony Borneman, Cristian Varela