Ethanol production from sugarcane is a key renewable fuel industry in Brazil. Major drivers of this alcoholic fermentation are Saccharomyces cerevisiae strains that originally were contaminants to the system and yet prevail in the industrial process. Here we present newly sequenced genomes (using Illumina short-read and PacBio long-read data) of two monosporic isolates (H3 and H4) of the S. cerevisiae PE-2, a predominant bioethanol strain in Brazil. The assembled genomes of H3 and H4, together with 42 draft genomes of sugarcane-fermenting (fuel ethanol plus cachaça) strains, were compared against those of the reference S288C and diverse S. cerevisiae. All genomes of bioethanol yeasts have amplified SNO2(3)/SNZ2(3) gene clusters for vitamin B1/B6 biosynthesis, and display ubiquitous presence of a particular family of SAM-dependent methyl transferases, rare in S. cerevisiae. Widespread amplifications of quinone oxidoreductases YCR102C/YLR460C/YNL134C, and the structural or punctual variations among aquaporins and components of the iron homeostasis system, likely represent adaptations to industrial fermentation. Interesting is the pervasive presence among the bioethanol/cachaça strains of a five-gene cluster (Region B) that is a known phylogenetic signature of European wine yeasts. Combining genomes of H3, H4, and 195 yeast strains, we comprehensively assessed whole-genome phylogeny of these taxa using an alignment-free approach. The 197-genome phylogeny substantiates that bioethanol yeasts are monophyletic and closely related to the cachaça and wine strains. Our results support the hypothesis that biofuel-producing yeasts in Brazil may have been co-opted from a pool of yeasts that were pre-adapted to alcoholic fermentation of sugarcane for the distillation of cachaça spirit, which historically is a much older industry than the large-scale fuel ethanol production.

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