Enhancing probiotic impact: engineering Saccharomyces boulardii for optimal acetic acid production and gastric passage tolerance.

Saccharomyces boulardii has been a subject of growing interest due to its potential as a probiotic microorganism with applications in gastrointestinal health, but the molecular cause for its probiotic potency has remained elusive. The recent discovery that S. boulardii contains unique mutations causing high acetic acid accumulation and inhibition of bacterial growth provides a possible clue. The natural S. boulardii isolates Sb.P and Sb.A are homozygous for the recessive mutation whi2S270* and accumulate unusually high amounts of acetic acid, which strongly inhibit bacterial growth. However, the homozygous whi2S270* mutation also leads to acetic acid sensitivity and acid sensitivity in general. In the present study, we have constructed a new S. boulardii strain, derived from the widely therapeutically used CMCN I-745 strain (isolated from the pharmaceutical product Enterol), producing even higher levels of acetic acid while keeping the same tolerance toward low pH as the parent Enterol (ENT) strain. This newly engineered strain, named ENT3, has a homozygous deletion of ACH1 and strong overexpression of ALD4. It is also able to accumulate much higher acetic acid concentrations when growing on low glucose levels, in contrast to the ENT wild-type and Sb.P strains. Moreover, we show the antimicrobial capacity of ENT3 against gut pathogens in vitro and observed that higher acetic acid production might correlate with better persistence in the gut in healthy mice. These findings underscore the possible role of the unique acetic acid production and its potential for improvement of the probiotic action of S. boulardii.IMPORTANCESuperior variants of the probiotic yeast Saccharomyces boulardii produce high levels of acetic acid, which inhibit the growth of bacterial pathogens. However, these strains also show increased acid sensitivity, which can compromise the viability of the cells during their passage through the stomach. In this work, we have developed by genetic engineering a variant of Saccharomyces boulardii that produces even higher levels of acetic acid and does not show enhanced acid sensitivity. We also show that the S. boulardii yeasts with higher acetic acid production persist longer in the gut, in agreement with a previous work indicating competition between probiotic yeast and bacteria for residence in the gut.

Genomic approachesidentifySTT4 as a new component in glucose-induced activation of yeast plasma membrane H+-ATPase.

Many studies have focused on identifying the signaling pathway by which addition of glucose triggers post-translational activation of the plasma membrane H+-ATPase in yeast. They have revealed that calcium signaling is involved in the regulatory pathway, supported for instance by the phenotype of mutants inARG82 that encodes an inositol kinase that phosphorylates inositol triphosphate (IP3). Strong glucose-induced calcium signaling, and high glucose-induced plasma membrane H+-ATPase activation have been observed in a specific yeast strain with the PJ genetic background. In this study, we have applied pooled-segregant whole genome sequencing, QTL analysis and a new bioinformatics methodology for determining SNP frequencies to identify the cause of this discrepancy and possibly new components of the signaling pathway. This has led to the identification of an STT4 allele with 6 missense mutations as a major causative allele, further supported by the observation that deletion of STT4 in the inferior parent caused a similar increase in glucose-induced plasma membrane H+-ATPase activation. However, the effect on calcium signaling was different indicating the presence of additional relevant genetic differences between the superior and reference strains. Our results suggest that phosphatidylinositol-4-phosphate might play a role in the glucose-induced activation of plasma membrane H+-ATPase by controlling intracellular calcium release through the modulation of the activity of phospholipase C.

The molecular biology of fruity and floral aromas in beer and other alcoholic beverages.

Aroma compounds provide attractiveness and variety to alcoholic beverages. We discuss the molecular biology of a major subset of beer aroma volatiles, fruity and floral compounds, originating from raw materials (malt and hops), or formed by yeast during fermentation. We introduce aroma perception, describe the most aroma-active, fruity and floral compounds in fruits and their presence and origin in beer. They are classified into categories based on their functional groups and biosynthesis pathways: (1) higher alcohols and esters, (2) polyfunctional thiols, (3) lactones and furanones, and (4) terpenoids. Yeast and hops are the main sources of fruity and flowery aroma compounds in beer. For yeast, the focus is on higher alcohols and esters, and particularly the complex regulation of the alcohol acetyl transferase ATF1 gene. We discuss the release of polyfunctional thiols and monoterpenoids from cysteine- and glutathione-S-conjugated compounds and glucosides, respectively, the primary biological functions of the yeast enzymes involved, their mode of action and mechanisms of regulation that control aroma compound production. Furthermore, we discuss biochemistry and genetics of terpenoid production and formation of non-volatile precursors in Humulus lupulus (hops). Insight in these pathways provides a toolbox for creating innovative products with a diversity of pleasant aromas.