LAUSR

&NA; Brettanomyces bruxellensis is usually considered a spoilage microorganism, responsible for significant economic losses during the production of fermented beverages such as wine, beer and cider, though for some styles of beer its influence is essential. In recent years, the competitiveness of this yeast in bioethanol production processes has brought to attention its broader biotechnological potential. Furthermore, the species has evolved key fermentation traits in parallel with Saccharomyces cerevisiae. Attempts to better understand B. bruxellensis physiology through genomics‐driven research have been hampered by a lack of functional genomics tools. Genetic transformation for B. bruxellensis has only been developed recently and with limited efficiency. Here we describe gene transformation cassettes tailored for B. bruxellensis, which provide multiple drug‐resistant markers and the ability to tag B. bruxellensis with different fluorescent proteins. All marker cassettes resulted in increased transformation efficiency compared to the maximum reported in literature, with one cassette, TDH1p natMX, showing five times greater efficiency. Transformation cassettes encoding fluorescent proteins enabled discrimination between subpopulations of transformed B. bruxellensis cells by flow cytometry and fluorescent microscopy. Thus, the genetic transformation toolkit described here unlocks several molecular applications such as strain tagging, insertional mutagenesis and potentially targeted gene deletion.