Isolation, genetic identification of Amazonian yeasts and analysis of thermotolerance and alcohol tolerance of Saccharomyces cerevisiae from Theobroma grandiflorum and Eugenia stipitata

Although yeasts of the Saccharomyces cerevisiae species are industrially significant, few studies have investigated their presence in environmental samples from the Amazon rainforest. This study aimed to isolate S. cerevisiae yeasts associated with trees of the Amazon Forest and investigate their thermotolerance, alcohol tolerance, and single nucleotide polymorphism (SNP) characteristics, along with those of regional strains from previous research and reference strains from the industry. We collected fruits, bark and decaying plant material from Theobroma grandiflorum, Spondias mombin L., Mangifera indica L., and Eugenia stipitata, and isolated yeasts using the culture media. To identify the yeasts, we conducted morphological and biochemical analyses, including sugar assimilation and fermentation, and sequencing analyses of the rDNA (ITS and LSU (D1 and D2)). We also performed fermentation tests to determine the optimum temperature, thermotolerance and ethanol tolerance. Finally, we subjected the selected strains to SNP analysis to study the reported genes that are important for alcohol tolerance in S. cerevisiae: FPS1 (farnesyl diphosphate synthase1) and ASR1/YPR093 (alcohol sensitive RING/PHD finger1) genes. As a result, we isolated 53 yeasts, and 10 of which exhibited a sugar assimilation and fermentation profile that was similar to that of S. cerevisiae. These ten isolates were identified using sequencing of the ITS and LSU regions, which revealed the species to be Wickerhamomyces anomalus (n = 4), Torulaspora pretoriensis (n = 3), Debaryomyces hansenni (n = 1), and Saccharomyces cerevisiae (n = 2). Through the analysis of the ASR1 and FPS1 regions, we found an SNP at nucleotide 1552 A > G (FPS1), which was associated with ethanol tolerance under our experimental conditions. This work is significant because it is one of the first studies to focus specifically on the isolation of S. cerevisiae from samples in the Amazon region. Furthermore, the SNP analysis allowed us to differentiate isolates that showed greater tolerance to ethanol.