Identification of novel alleles conferring superior production of rose flavor phenylethyl acetate using polygenic analysis in yeast.

Flavor compound metabolism is one of the last areas in metabolism where multiple genes encoding biosynthetic enzymes are still unknown. A major challenge is the involvement of side activities of enzymes having their main function in other areas of metabolism. We have applied pooled-segregant whole-genome sequence analysis to identify novel Saccharomyces cerevisiae genes affecting production of phenylethyl acetate (2-PEAc). This is a desirable flavor compound of major importance in alcoholic beverages imparting rose- and honey-like aromas, with production of high 2-PEAc levels considered a superior trait. Four quantitative trait loci (QTLs) responsible for high 2-PEAc production were identified, with two loci each showing linkage to the genomes of the BTC.1D and ER18 parents. The first two loci were investigated further. The causative genes were identified by reciprocal allele swapping into both parents using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. The superior allele of the first major causative gene, FAS2, was dominant and contained two unique single nucleotide polymorphisms (SNPs) responsible for high 2-PEAc production that were not present in other sequenced yeast strains. FAS2 encodes the alpha subunit of the fatty acid synthetase complex. Surprisingly, the second causative gene was a mutant allele of TOR1, a gene involved in nitrogen regulation. Exchange of both superior alleles in the ER18 parent strain increased 2-PEAc production 70%, nearly to the same level as in the best superior segregant. Our results show that polygenic analysis combined with CRISPR/ Cas9-mediated allele exchange is a powerful tool for identification of genes encoding missing metabolic enzymes and for development of industrial yeast strains generating novel flavor profiles in alcoholic beverages

Variable flocculation profiles of yeast strains isolated from cacha?a distilleries.

In cacha?a production, the use of yeast cells as starters with predictable flocculation behavior facilitates the cell recovery at the end of each fermentation cycle. Therefore, the aim of this work was to explain the behavior of cacha?a yeast strains in fermentation vats containing sugarcane through the determination of biochemical and molecular parameters associated with flocculation phenotypes. By analyzing thirteen cacha?a yeast strains isolated from different distilleries, our results demonstrated that neither classic biochemical measurements (e.g., percentage of flocculation, EDTA sensitivity, cell surface hydrophobicity, and sugar residues on the cell wall) nor modern molecular approaches, such as polymerase chain reaction (PCR) and real-time PCR (q-PCR), were sufficient to distinctly classify the cacha?a yeast strains according to their flocculation behavior. It seems that flocculation is indeed a strain-specific phenomenon that is difficult to explain and/or categorize by the available methodologie

Regulation of the N-acetyl-beta-D-glucosaminidase produced by Trichoderma harzianum : evidence that cAMP controls the expression.

Trichoderma harzianum is a filamentous fungus reported to be a producer of extracellular N-acetyl-?-D-glucosaminidase (NAGase) when grown in chitin-containing medium. An approximately 64-kDa protein with NAGase activity was purified by gel filtration and ion exchange chromatography. The involvement of cyclic AMP (cAMP) in the synthesis of NAGase from T. harzianum in chitin-containing medium was also investigated. Molecules that increase the intracellular levels of cAMP, including caffeine, aluminium tetrafluoride and dinitrophenol, were used. Western blot analysis showed that NAGase synthesis was repressed by increasing the levels of intracellular cAMP. Using specific nag primers in a reverse transcription-polymerase chain reaction-based approach, NAGase synthesis was shown to be regulated at the level of gene transcription

New aspects of the glucose activation of the H-ATPase in the yeast Saccharomyces cerevisae.

The glucose-induced activation of plasma membrane ATPase from Saccharomyces cerevisiae was first described by Serrano in 1983. Many aspects of this signal transduction pathway are still obscure. In this paper, evidence is presented for the involvement of Snf3p as the glucose sensor related to this activation process. It is shown that, in addition to glucose detection by Snf3p, sugar transport is also necessary for activation of the ATPase. The participation of the G protein, Gpa2p, in transducing the internal signal (phosphorylated sugars) is also demonstrated. Moreover, the involvement of protein kinase C in the regulation of ATPase activity is confirmed. Finally, a model pathway is presented for sensing and transmission of the glucose activation signal of the yeast HM-ATPase

Fractionation of sugarcane bagasse using hydrothermal and advanced oxidative pretreatments for bioethanol and biogas production in lignocellulose biorefineries.

The fractionation of sugarcane bagasse (SB) by hydrothermal pretreatment (HP, autohydrolysis) followed by alkaline extraction (AE) and advanced oxidative pretreatment (AOP) for production of second-generation ethanol and biogas was investigated. The AOP of SB was optimized using a Doehlert design, varying the applied H2O2 load, liquid-to-solid ratio (LSR), and time. The responses evaluated were yield (Y), residual cellulose (RC), delignification (DE), and enzymatic conversion (EC). The AE of SB pretreated by HP led to 61.8% DE (using 0.2?mol?L?1 NaOH). This high lignin removal enabled substantial savings of H2O2 in the AOP. The optimized AOP conditions led to 78% Y, 82.2% RC, 42.7% DE, and 88.9% EC (overall glucose yield of 60.9%). Fermentation of the enzymatic hydrolysate with Saccharomyces cerevisiae yielded 190.8?Lethanol?tonSB?1. Biogas production by anaerobic digestion of residual liquid streams of the pretreatment steps yielded 27.46?NLCH4?kgSB?1. An energy balance was estimated for the SB fractionation

The PLC1 encoded phospholipase C in the yeast Saccharomyces cerevisiae is essential for glucose-induced phosphatidylinositol turnover and activation of plasma membrane H -ATPase.

Addition of glucose to glucose-deprived cells of the yeast Saccharomyces cerevisiae triggers rapid turnover of phosphatidylinositol, phosphatidylinositol-phosphate and phosphatidylinositol 4,5-bisphosphate. Glucose stimulation of PI turnover was measured both as an increase in the specific ratio of 32P-labeling and as an increase in the level of diacylglycerol after addition of glucose. Glucose also causes rapid activation of plasma membrane H.-ATPase. We show that in a mutant lacking the PLC1 encoded phospholipase C, both processes were strongly reduced. Compound 48/80, a known inhibitor of mammalian phospholipase C, inhibits both processes. However, activation of the plasma membrane H.- ATPase is only inhibited by concentrations of compound 48/80 that strongly inhibit phospholipid turnover. Growth was inhibited by even lower concentrations. Our data suggest that in yeast cells, glucose triggers through activation of the PLC1 gene product a signaling pathway initiated by phosphatidylinositol turnover and involved in activation of the plasma membrane H.-ATPase

Isolation of Saccharomyces cerevisiae strains producing higher levels of flavoring compounds for production of ??cacha?a?? the Brazilian sugarcane spirit.

In Brazil, spontaneous fermentation and open vessels are still used to produce cachac?a (the Brazilian sugarcane spirit) and this fermentation is characterized by mixed cultures with continuous succession of yeast species. This work shows the development of a methodology for isolation of yeasts, particularly Saccharomyces cerevisiae, used in the production of cachac?a. According to the proposed strategy, the strains were selected for their ability to adapt to stress conditions encountered during fermentation of the sugarcane juice such as high sucrose concentration; high temperatures and high alcohol concentration; for their capacity to flocculate; and for their higher fermentative ability. For strains with such characteristics, specific procedures were employed to select for 5,5,5-trifluoro-dl-leucine (TFL) and cerulenin-resistant strains, since these characteristics are related to a higher capacity of production of the flavoring compounds isoamyl alcohol and caproic acid, respectively. The effectiveness of such a selection strategy was documented. Taken together, the results obtained present the development of a new strategy to isolate yeast strains with appropriated characteristics to be used in the cachac?a industry. Moreover, the results obtained offer an explanation for the great variability in terms of chemical composition found in products obtained even in a single distillery

Lpx1p links glucose-induced calcium signaling and plasma membrane H+-ATPase activation in Saccharomyces cerevisiae cells.

In yeast, as in other eukaryotes, calcium plays an essential role in signaling transduction to regulate different processes. Many pieces of evidence suggest that glucose-induced activation of plasma membrane H+-ATPase, essential for yeast physiology, is related to calcium signaling. Until now, it was not identified any protein that could be regulated by calcium in this context. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase activation, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, and by using different approaches, we showed many pieces of evidence suggesting that the requirement of calcium signaling for activation of the plasma membrane H+-ATPase is due to its requirement for activation of Lpx1p. According to the current model, activation of Lpx1p would cause hydrolysis of an acetylated tubulin that keeps the plasma membrane H+-ATPase in an inactive state. Therefore, after its activation, Lpx1p would hydrolyze the acetylated tubulin making the plasma membrane H+-ATPase phosphorylation accessible for at least one protein kinase

Adhesion on yeast cell surface as a trapping mechanism of pathogenic bacteria by Saccharomyces probiotics.

Recently, much attention has been given to the use of probiotics as an adjuvant for the prevention or treatment of gastrointestinal pathology. The great advantage of therapy with probiotics is that they have few side effects such as selection of resistant bacteria or disturbance of the intestinal microbiota, which occur when antibiotics are used. Adhesion of pathogenic bacteria onto the surface of probiotics instead of onto intestinal receptors could explain part of the probiotic effect. Thus, this study evaluated the adhesion of pathogenic bacteria onto the cell wall of Saccharomyces boulardii and Saccharomyces cerevisiae strains UFMG 905, W303 and BY4741. To understand the mechanism of adhesion of pathogens to yeast, cell-wall mutants of the parental strain of Saccharomyces cerevisiae BY4741 were used because of the difficulty of mutating polyploid yeast, as is the case for Saccharomyces cerevisiae and Saccharomyces boulardii. The tests of adhesion showed that, among 11 enteropathogenic bacteria tested, only Escherichia coli, Salmonella Typhimurium and Salmonella Typhi adhered to the surface of Saccharomyces boulardii, Saccharomyces cerevisiae UFMG 905 and Saccharomyces cerevisiae BY4741. The presence of mannose, and to some extent bile salts, inhibited this adhesion, which was not dependent on yeast viability. Among 44 cell-wall mutants of Saccharomyces cerevisiae BY4741, five lost the ability to fix the bacteria. Electron microscopy showed that the phenomenon of yeast?bacteria adhesion occurred both in vitro and in vivo (in the digestive tract of dixenic mice). In conclusion, some pathogenic bacteria were captured on the surface of Saccharomyces boulardii, Saccharomyces cerevisiae UFMG 905 and Saccharomyces cerevisiae BY4741, thus preventing their adhesion to specific receptors on the intestinal epithelium and their subsequent invasion of the host

High?affinity transport, cyanide?resistant respiration, and ethanol production under aerobiosis underlying efficient high glycerol consumption by Wickerhamomyces anomalus.

Wickerhamomyces anomalus strain LBCM1105 was originally isolated from the wort of cacha?a (the Brazilian fermented sugarcane juice-derived Brazilian spirit) and has been shown to grow exceptionally well at high amounts of glycerol. This paramount residue from the biodiesel industry is a promising cheap carbon source for yeast biotechnology. The assessment of the physiological traits underlying the W. anomalus glycerol consumption ability in opposition to Saccharomyces cerevisiae is presented. A new WaStl1 concentrative glycerol-H+ symporter with twice the affinity of S. cerevisiae was identified. As in this yeast, WaSTL1 is repressed by glucose and derepressed/induced by glycerol but much more highly expressed. Moreover, LBCM1105 aerobically growing on glycerol was found to produce ethanol, providing a redox escape to compensate the redox imbalance at the level of cyanide-resistant respiration (CRR) and glycerol 3P shuttle. This work is critical for understanding the utilization of glycerol by non-Saccharomyces yeasts being indispensable to consider their industrial application feeding on biodiesel residue