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Flor strains of Saccharomyces cerevisiae are principal microbial agents responsible for biological wine aging used for production of sherry-like wines. The flor yeast velum formed on the surface of fortified fermented must is a major adaptive and technological characteristic of flor yeasts that helps them to withstanding stressful winemaking conditions and ensures specific biochemical and sensory oxidative alterations typical for sherry wines. We have applied RNAseq technology for transcriptome analysis of an industrial flor yeast strain at different steps of velum development over 71 days under experimental winemaking conditions. Velum growth and maturation was accompanied by accumulation of aldehydes and acetales. We have identified 1490 differentially expressed genes including 816 genes upregulated and 674 downregulated more than 2-fold at mature biofilm stage as compared to the early biofilm. Distinct expression patterns of genes involved in carbon and nitrogen metabolism, respiration, cell cycle, DNA repair, cell adhesion, response to various stresses were observed. Many genes involved in response to different stresses, oxidative carbon metabolism, high affinity transport of sugars, glycerol utilization, sulfur metabolism, protein quality control and recycling, cell wall biogenesis, apoptosis were induced at the mature biofilm stage. Strong upregulation was observed for FLO11 flocculin while expression of other flocculins remained unaltered or moderately downregulated. Downregulated genes included those for proteins involved in glycolysis, transportation of ions, metals, aminoacids, sugars, indicating repression of some major transport and metabolic process at the mature biofilm stage. Presented results are important for in-depth understanding of cell response elicited by velum formation and sherry wine manufacturing conditions, and for the comprehension of relevant regulatory mechanisms. Such knowledge may help to better understand the molecular mechanisms that flor yeasts use to adapt to winemaking environments, establish the functions of previously uncharacterized genes, improve the technology of sherry- wine production, and find target genes for strain improvement., (Copyright © 2020 Mardanov, Eldarov, Beletsky, Tanashchuk, Kishkovskaya and Ravin.)

Flor yeast strains represent a specialized group of Saccharomyces cerevisiae yeasts used for biological wine aging. We have sequenced the genomes of three flor strains originated from different geographic regions and used for production of sherry-like wines in Russia. According to the obtained phylogeny of 118 yeast strains, flor strains form very tight cluster adjacent to the main wine clade. SNP analysis versus available genomes of wine and flor strains revealed 2,270 genetic variants in 1,337 loci specific to flor strains. Gene ontology analysis in combination with gene content evaluation revealed a complex landscape of possibly adaptive genetic changes in flor yeast, related to genes associated with cell morphology, mitotic cell cycle, ion homeostasis, DNA repair, carbohydrate metabolism, lipid metabolism, and cell wall biogenesis. Pangenomic analysis discovered the presence of several well-known "non-reference" loci of potential industrial importance. Events of gene loss included deletions of asparaginase genes, maltose utilization locus, and FRE-FIT locus involved in iron transport. The latter in combination with a flor-yeast-specific mutation in the Aft1 transcription factor gene is likely to be responsible for the discovered phenotype of increased iron sensitivity and improved iron uptake of analyzed strains. Expansion of the coding region of the FLO11 flocullin gene and alteration of the balance between members of the FLO gene family are likely to positively affect the well-known propensity of flor strains for velum formation. Our study provides new insights in the nature of genetic variation in flor yeast strains and demonstrates that different adaptive properties of flor yeast strains could have evolved through different mechanisms of genetic variation.