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

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 contains unique mutations causing high acetic acid accumulation and inhibition of bacterial growth provides a possible clue. The natural isolates Sb.P and Sb.A are homozygous for the recessive mutation and accumulate unusually high amounts of acetic acid, which strongly inhibit bacterial growth. However, the homozygous mutation also leads to acetic acid sensitivity and acid sensitivity in general. In the present study, we have constructed a new 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 and strong overexpression of . 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 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 .IMPORTANCESuperior variants of the probiotic yeast 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 that produces even higher levels of acetic acid and does not show enhanced acid sensitivity. We also show that the 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.