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)).
Sulfur dioxide tolerance of new industry isolates
The results of laboratory-scale directed evolution provided the first direct evidence that Brettanomyces bruxellensis strains have the capacity to adapt to the use of SO2 as a control agent by increasing their level of SO2 tolerance. However, a key question for the Australian wine industry is whether this may be happening in the field. Previous industry-based population surveys in the early 2000s had already shown that the strains of Brettanomyces with the highest levels of SO2 resistance were most frequently isolated from Australian wineries (Curtin et al. 2008, Curtin et al. 2012). Since this original study was performed, Australian winemakers have become increasingly aware of the importance of SO2 management for Brettanomyces spoilage control. However, these changing practices could potentially provide conditions that could promote the evolution of SO2 tolerance.
Historical industry isolates were therefore sourced from the AWRI Wine Microorganism Culture Collection (including those isolated during the original Curtin et al. 2008 study), in addition to new industry isolates sourced from a commercial partner in 2016 and 2017. The strains were tested to determine if average levels of SO2 resistance had changed over time (Figure 14). Strains isolated from 2000 to 2004 displayed levels of SO2 resistance that broadly represented the range of tolerances observed in the original study. Strains isolated from 2010 to 2014 did not show a significant difference in their median SO2 resistance, although there were a small number that displayed higher levels of SO2 resistance than those seen in the 2000-2004 cohort. Interestingly, the 2016-2017 isolates, sourced from 16 different tank and barrel samples, displayed greater tolerance to SO2, growing at concentrations significantly higher than those observed from the two previous cohorts. It should be noted that the 2016-2017 isolates were sourced from only two wine companies (although one is a multi-site producer). As such, they represent a small part of the overall industry; however, these preliminary data suggest that strains with significantly higher levels of SO2 resistance are present in the field. While this situation will need to be monitored by further sampling, results from a recent AWRI survey of Australian bottled wine suggest that the existence of these SO2-tolerant strains is not yet translating to elevated 4-ethylphenol levels in wines in the market.
Genetic transformation of Brettanomyces
Genetic transformation is a foundational technology that enables the comprehensive study of a species by applying a multitude of molecular biology tools, such as gene overexpression, gene deletion, incorporation of marker genes for competition experiments and tagging for visualisation or purification. Transformation has been available in Saccharomyces cerevisiae for more than 30 years, with much of the knowledge that has been generated for this species due to the early development of this technique. While, genetic transformation for Brettanomyces bruxellensis has been developed very recently (Miklenic et al. 2015, Schifferdecker et al. 2016), it has suffered from a very low efficiency that limited the scope of tools that could be developed. To address these shortcomings, a new set of gene transformation cassettes was developed that were specifically tailored for B. bruxellensis. While these new cassettes provide multiple drug markers, they also provide for increased transformation efficiencies. In addition to the standard drug-selections cassettes, constructs were created that enable Brettanomyces cells to be labelled with either green- or blue-fluorescent proteins. Cells that express these proteins glow when exposed to certain wavelengths of light and this enables the rapid identification and enumeration of these cells during fermentation (Figure 15).
Curtin, C., Bramley, B., Cowey, G., Holdstock, M., Kennedy, E., Lattey, K., Coulter, A., Henschke, P., Francis, L., Godden, P. 2008. Sensory perceptions of ‘Brett’ and relationship to consumer preference. Blair, R.J., Williams, P.J., Pretorius, I.S. (eds.) Proceedings of the thirteenth Australian wine industry technical conference, Adelaide, SA, 29 July–2 August 2007. Adelaide, SA: Australian Wine Industry Technical Conference Inc.: 207–211.
Curtin, C., Kennedy, E., Henschke, P.A. 2012. Genotype-dependent sulphite tolerance of Australian Dekkera (Brettanomyces) bruxellensis wine isolates. Lett. Appl. Microbiol. 55(1): 56-61.
Miklenic, M., Zunar, B., Stafa, A, Svetec, I.K. 2015 Improved electroporation procedure for genetic transformation of Dekkera/Brettanomyces bruxellensis. FEMS Yeast Res. 15(8): fov096.
Schifferdecker, A.J., Siurkus, J., Andersen, M.R., Joerck-Ramberg, D., Ling, Z., Zhou, N., Blevins, J.E., Sibirny, A.A., Piškur, J., Ishchuk, O.P. 2016. Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast. Appl. Microbiol. Biotech. 100(7): 3219-3231.