SummaryMycolic acids are major components of the cell envelope of mycobacteria, such as Mycobacterium tuberculosis, and play an important role in its architecture, impermeability and interaction with the environment. Synthesis of mycolic acids is carried out by two types of fatty acid synthases (FAS) working in concert: type I FAS, a multifunctional enzyme capable of de novo synthesis of medium-chain fatty acids, and type II FAS, responsible for their elongation. In this article we report the identification and characterization of a transcriptional regulator (MabR), whose binding to the FAS-II promoter region was demonstrated in vitro and in vivo. Overexpression and knock-down studies in Mycobacterium smegmatis revealed the repressor nature of MabR, with reduced amounts of FAS-II transcripts and fatty acids in the overproducing strain. Under these conditions, downregulation of fas transcription was also observed, thereby suggesting the existence of cross-talk between the two FAS, mediated by MabR. Finally, the finding that a mabR knockout mutant could only be obtained in a merodiploid strain of M. smegmatis, confirmed the predicted essentiality, thus implying an essential role for MabR in mycobacterial fatty acid metabolism.

Hanseniaspora is the main genus of the apiculate yeast group that represents approximately 70% of the grape-associated microflora. Hanseniaspora vineae is emerging as a promising species for quality wine production compared to other non-Saccharomyces species. Wines produced by H. vineae with Saccharomyces cerevisiae consistently exhibit more intense fruity flavors and complexity than wines produced by S. cerevisiae alone. In this work, genome sequencing, assembling, and phylogenetic analysis of two strains of H. vineae showed that it is a member of the Saccharomyces complex and it diverged before the whole-genome duplication (WGD) event from this clade. Specific flavor gene duplications and absences were identified in the H. vineae genome compared to 14 fully sequenced industrial S. cerevisiae genomes. The increased formation of 2-phenylethyl acetate and phenylpropanoids such as 2-phenylethyl and benzyl alcohols might be explained by gene duplications of H. vineae aromatic amino acid aminotransferases (ARO8 and ARO9) and phenylpyruvate decarboxylases (ARO10). Transcriptome and aroma profiles under fermentation conditions confirmed these genes were highly expressed at the beginning of stationary phase coupled to the production of their related compounds. The extremely high level of acetate esters produced by H. vineae compared to that by S. cerevisiae is consistent with the identification of six novel proteins with alcohol acetyltransferase (AATase) domains. The absence of the branched-chain amino acid transaminases (BAT2) and acyl coenzyme A (acyl-CoA)/ethanol O-acyltransferases (EEB1) genes correlates with H. vineae's reduced production of branched-chain higher alcohols, fatty acids, and ethyl esters, respectively. Our study provides sustenance for understanding and potentially utilizing genes that determine fermentation aromas.IMPORTANCE The huge diversity of non-Saccharomyces yeasts in grapes is dominated by the apiculate genus Hanseniaspora. Two native strains of Hanseniaspora vineae applied to winemaking because of their high oenological potential in aroma and fermentation performance were selected to obtain high-quality genomes. Here, we present a phylogenetic analysis and the complete transcriptome and aroma metabolome of H. vineae during three fermentation steps. This species produced significantly richer flavor compound diversity than Saccharomyces, including benzenoids, phenylpropanoids, and acetate-derived compounds. The identification of six Downloaded from proteins, different from S. cerevisiae ATF, with diverse acetyltransferase domains in H. vineae offers a relevant source of native genetic variants for this enzymatic activity. The discovery of benzenoid synthesis capacity in H. vineae provides a new eukaryotic model to dilucidate an alternative pathway to that catalyzed by plants' phenylalanine lyases.

Benzyl alcohol and other benzenoid-derived metabolites of particular importance in plants confer floral and fruity flavors to wines. Among the volatile aroma components in Vitis vinifera grape varieties, benzyl alcohol is present in its free and glycosylated forms. These compounds are considered to originate from grapes only and not from fermentative processes. We have found increased levels of benzyl alcohol in red Tannat wine compared to that in grape juice, suggesting de novo formation of this metabolite during vinification. In this work, we show that benzyl alcohol, benzaldehyde, p-hydroxybenzaldehyde, and p-hydroxybenzyl alcohol are synthesized de novo in the absence of grape-derived precursors by Hanseniaspora vineae. Levels of benzyl alcohol produced by 11 different H. vineae strains were 20-200 times higher than those measured in fermentations with Saccharomyces cerevisiae strains. These results show that H. vineae contributes to flavor diversity by increasing grape variety aroma concentration in a chemically defined medium. Feeding experiments with phenylalanine, tryptophan, tyrosine, p-aminobenzoic acid, and ammonium in an artificial medium were tested to evaluate the effect of these compounds either as precursors or as potential pathway regulators for the formation of benzenoid-derived aromas. Genomic analysis shows that the phenylalanine ammonia-lyase (PAL) and tyrosine ammonia lyase (TAL) pathways, used by plants to generate benzyl alcohols from aromatic amino acids, are absent in the H. vineae genome. Consequently, alternative pathways derived from chorismate with mandelate as an intermediate are discussed.