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.

Saccharomyces boulardii promoters for control of gene expression in vivo.

BACKGROUND: Interest in the use of engineered microbes to deliver therapeutic activities has increased in recent years. The probiotic yeast Saccharomyces boulardii has been investigated for production of therapeutics in the gastrointestinal tract. Well-characterised promoters are a prerequisite for robust therapeutic expression in the gut; however, S. boulardii promoters have not yet been thoroughly characterised in vitro and in vivo. RESULTS: We present a thorough characterisation of the expression activities of 12 S. boulardii promoters in vitro in glucose, fructose, sucrose, inulin and acetate, under both aerobic and anaerobic conditions, as well as in the murine gastrointestinal tract. Green fluorescent protein was used to report on promoter activity. Promoter expression was found to be carbon-source dependent, with inulin emerging as a favourable carbon source. Furthermore, relative promoter expression in vivo was highly correlated with expression in sucrose (R = 0.99). CONCLUSIONS: These findings provide insights into S. boulardii promoter activity and aid in promoter selection in future studies utilising S. boulardii to produce therapeutics in the gut.

New biomarkers underlying acetic acid tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii.

Evolutionary engineering experiments, in combination with omics technologies, revealed genetic markers underpinning the molecular mechanisms behind acetic acid stress tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii. Here, compared to the ancestral Ent strain, evolved yeast strains could quickly adapt to high acetic acid levels (7 g/L) and displayed a shorter lag phase of growth. Bioinformatic-aided whole-genome sequencing identified genetic changes associated with enhanced strain robustness to acetic acid: a duplicated sequence in the essential endocytotic PAN1 gene, mutations in a cell wall mannoprotein (dan4Thr192del), a lipid and fatty acid transcription factor (oaf1Ser57Pro) and a thiamine biosynthetic enzyme (thi13Thr332Ala). Induction of PAN1 and its associated endocytic complex SLA1 and END3 genes was observed following acetic acid treatment in the evolved-resistant strain when compared to the ancestral strain. Genome-wide transcriptomic analysis of the evolved Ent acid-resistant strain (Ent ev16) also revealed a dramatic rewiring of gene expression among genes associated with cellular transport, metabolism, oxidative stress response, biosynthesis/organization of the cell wall, and cell membrane. Some evolved strains also displayed better growth at high acetic acid concentrations and exhibited adaptive metabolic profiles with altered levels of secreted ethanol (4.0-6.4% decrease), glycerol (31.4-78.5% increase), and acetic acid (53.0-60.3% increase) when compared to the ancestral strain. Overall, duplication/mutations and transcriptional alterations are key mechanisms driving improved acetic acid tolerance in probiotic strains. We successfully used adaptive evolutionary engineering to rapidly and effectively elucidate the molecular mechanisms behind important industrial traits to obtain robust probiotic yeast strains for myriad biotechnological applications. KEY POINTS: •Acetic acid adaptation of evolutionary engineered robust probiotic yeast S. boulardii •Enterol ev16 with altered genetic and transcriptomic profiles survives in up to 7 g/L acetic acid •Improved acetic acid tolerance of S. boulardii ev16 with mutated PAN1, DAN4, OAF1, and THI13 genes.

Evaluation of the impact of polypeptide-p on diabetic rats upon its cloning, expression, and secretion in Saccharomyces boulardii.

This study was designed to evaluate the effectiveness of recombinant polypeptide-p derived from Momordica charantia on diabetic rats. In this research, the optimized sequence of polypeptide-p gene fused to a secretion signal tag was cloned into the expression vector and transformed into probiotic Saccharomyces boulardii. The production of recombinant secretion protein was verified by western blotting, HPLC, and mass spectrometry. To assay recombinant yeast bioactivity in the gut, diabetic rats were orally fed wild-type and recombinant S. boulardii, in short SB and rSB, respectively, at two low and high doses as well as glibenclamide as a reference drug. In untreated diabetic and treated diabetic + SB rats (low and high doses), the blood glucose increased from 461, 481, and 455 (mg/dl), respectively, to higher than 600 mg/dl on the 21st day. Whereas glibenclamide and rSB treatments showed a significant reduction in the blood glucose level. The result of this study promised a safe plant-source supplement for diabetes through probiotic orchestration.

Programming Probiotics: Diet-Responsive Gene Expression and Colonization Control in Engineered S. boulardii.

Saccharomyces boulardii (Sb) is an emerging probiotic chassis for delivering biomolecules to the mammalian gut, offering unique advantages as the only eukaryotic probiotic. However, precise control over gene expression and gut residence time in Sb have remained challenging. To address this, we developed five ligand-responsive gene expression systems and repaired galactose metabolism in Sb, enabling inducible gene expression in this strain. Engineering these systems allowed us to construct AND logic gates, control the surface display of proteins, and turn on protein production in the mouse gut in response to dietary sugar. Additionally, repairing galactose metabolism expanded Sb's habitat within the intestines and resulted in galactose-responsive control over gut residence time. This work opens new avenues for precise dosing of therapeutics by Sb via control over its in vivo gene expression levels and localization within the gastrointestinal tract.