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.

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.

Development of a Novel ATP Bioluminescence Assay Based on Engineered Probiotic Saccharomyces boulardii Expressing Firefly Luciferase.

Quantitative analysis of adenosine triphosphate (ATP) has been widely used as a diagnostic tool in the food and medical industries. Particularly, the pathogenesis of a few diseases including inflammatory bowel disease (IBD) is closely related to high ATP concentrations. A bioluminescent D-luciferin/luciferase system, which includes a luciferase (FLuc) from the firefly Photinus pyralis as a key component, is the most commonly used method for the detection and quantification of ATP. Here, instead of isolating FLuc produced in recombinant Escherichia coli, we aimed to develop a whole-cell biocatalyst system that does not require extraction and purification of FLuc. To this end, the gene coding for FLuc was introduced into the genome of probiotic Saccharomyces boulardii using the CRISPR/Cas9-based genome editing system. The linear relationship (r2 = 0.9561) between ATP levels and bioluminescence generated from the engineered S. boulardii expressing FLuc was observed in vitro. To explore the feasibility of using the engineered S. boulardii expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (i.e., ATP) in the gut, a colitis mouse model was established using dextran sodium sulfate as a colitogenic compound. Our findings demonstrated that the whole-cell biosensor can detect elevated ATP levels during gut inflammation in mice. Therefore, the simple and powerful method developed herein could be applied for non-invasive IBD diagnosis.

Microbiome Engineering Using Probiotic Yeast: Saccharomyces boulardii and the Secreted Human Lysozyme Lead to Changes in the Gut Microbiome and Metabolome of Mice.

The probiotic yeast Saccharomyces boulardii has great potential for use as a chassis for microbiome engineering because of its high resistance to environmental stress, well-developed genetic tools, and the ability to secrete recombinant proteins in the intestine. As oral feeding of lysozyme has been reported to change the gut microbiome and fecal metabolites, we engineered S. boulardii to secrete human lysozyme, and investigated the changes in the microbiome and fecal metabolites in response to the administration of the engineered probiotic yeast into mice. Administration of S. boulardii changed the structure of the gut microbiome by promoting the growth of clostridia and increasing the diversity of strains. The human lysozyme secreted by S. boulardii in the intestine resulted in a unique gut microbiome structure through selective growth. In addition, the administration of probiotic yeast S. boulardii affected host energy metabolism and decreased blood urea and fructose levels, suggesting a mechanism of health benefits in mice. IMPORTANCE Our study identified changes in the microbiome by administering wild-type S. boulardii in mice to healthy mice based on long-read sequencing and demonstrated that a recombinant protein secreted by engineered S. boulardii in the intestine could change the microbiome. Our results provide valuable information for the development of therapeutics using engineered S. boulardii that changes the gut microbiome and host physiology.

Improving therapeutic protein secretion in the probiotic yeast Saccharomyces boulardii using a multifactorial engineering approach.

The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb's ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.

Anticancer Properties of Probiotic Saccharomyces boulardii Supernatant on Human Breast Cancer Cells.

Saccharomyces boulardii, a variety of S. cerevisiae, is used as a probiotic yeast in food and drug industries. However, S. boulardii is an opportunistic pathogen, and the supernatant of this organism has recently been recommended for its health-promoting benefits. Breast cancer is the most frequent cancer disease in women worldwide. The objective of this study was to investigate the effects of S. boulardii supernatant (SBS) on cell viability, inducing apoptosis and suppression of survivin gene expression in MCF-7 and MCF-7/MX as human non-drug-resistant and multidrug-resistant breast cancer cells respectively. The IC50 value of SBS against MCF-7 was calculated 1037, 542, and 543 µg/mL for 24, 48, and 72 h treatments, respectively. Also, this value against MCF-7/MX cells were measured 1242, 616, and 444 µg/mL after 24, 48, and 72 h respectively. We found that suppression of survivin gene expression should be one of the main molecular antitumor mechanisms which is contributed to apoptosis in breast cancer cells. However, anticancer activity of SBS was observed more efficient against MCF-7 than that against MCF-7/MX cells. SBS is suggested to be considered as one of the prospective anticancer drugs to treat human breast carcinoma. More investigations especially in vivo studies are strongly recommended to be implemented to characterize other antitumor mechanisms of SBS against breast carcinoma.

A Tunable and Expandable Transactivation System in Probiotic Yeast Saccharomyces boulardii.

Precise transcriptional modulation is a key requirement for developing synthetic probiotics with predictably tunable functionalities. In this study, an expandable and tunable transactivation system was constructed and validated in probiotic yeast Saccharomyces boulardii. The use of nuclease-null Cas9 and scaffold RNA (scRNA) directed regulation enabled transactivation under the control of a synthetic promoter in S. boulardii. A synthetic promoter consisting of the scRNA target sequence and the core GAL7 promoter region restricted interference from the native galactose regulon. The system was readily expanded by introducing new target sequences to the promoter and scRNA. Complementarity between the promoter and scRNA, and binding specificity between scRNA and transcriptional activator, served as two layers of orthogonality of the transactivation. In addition, activator expression under the control of an inducible promoter enabled control of the transactivation via chemical inducer. The described system has the potential to enable engineering of probiotic yeast to more precisely perform therapeutic functions.

Production of neoagarooligosaccharides by probiotic yeast Saccharomyces cerevisiae var. boulardii engineered as a microbial cell factory.

BACKGROUND: Saccharomyces cerevisiae var. boulardii is a representative probiotic yeast that has been widely used in the food and pharmaceutical industries. However, S. boulardii has not been studied as a microbial cell factory for producing useful substances. Agarose, a major component of red macroalgae, can be depolymerized into neoagarooligosaccharides (NAOSs) by an endo-type ß-agarase. NAOSs, including neoagarotetraose (NeoDP4), are known to be health-benefiting substances owing to their prebiotic effect. Thus, NAOS production in the gut is required. In this study, the probiotic yeast S. boulardii was engineered to produce NAOSs by expressing an endo-type ß-agarase, BpGH16A, derived from a human gut bacterium Bacteroides plebeius. RESULTS: In total, four different signal peptides were compared in S. boulardii for protein (BpGH16A) secretion for the first time. The SED1 signal peptide derived from Saccharomyces cerevisiae was selected as optimal for extracellular production of NeoDP4 from agarose. Expression of BpGH16A was performed in two ways using the plasmid vector system and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system. The production of NeoDP4 by engineered S. boulardii was verified and quantified. NeoDP4 was produced by S. boulardii engineered using the plasmid vector system and CRISPR-Cas9 at 1.86 and 0.80 g/L in a 72-h fermentation, respectively. CONCLUSIONS: This is the first report on NAOS production using the probiotic yeast S. boulardii. Our results suggest that S. boulardii can be considered a microbial cell factory to produce health-beneficial substances in the human gut.

In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii.

Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (ß-carotene) and drug (violacein) titers. We found that S. boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled ß-carotene synthesis (194 µg total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional ß-carotene recovered in feces was 56-fold higher than the ß-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.

Capric acid secreted by Saccharomyces boulardii influences the susceptibility of Candida albicans to fluconazole and amphotericin B.

The effect of capric acid, secreted by the probiotic yeasts Saccharomyces boulardii, was evaluated on the activities of fluconazole (FLC) and amphotericin B (AMB) against pathogenic Candida albicans fungus. The findings indicated that capric acid may be a promising additive for use in combination with FLC. A FLC-capric acid combination led to reduced efflux activity of multidrug resistance (MDR) transporter Cdr1p by causing it to relocalize from the plasma membrane (PM) to the interior of the cell. The above effect occurred due to inhibitory effect of FLC-capric acid combination of ergosterol biosynthesis. However, capric acid alone stimulated ergosterol production in C. albicans, which in turn generated cross resistance towards AMB and inhibited its action (PM permeabilization and cytoplasm leakage) against C. albicans cells. This concluded that AMB should not be administered among dietary supplements containing capric acid or S. boulardii cells.

Heterologous Expression of the Leuconostoc Bacteriocin Leucocin C in Probiotic Yeast Saccharomyces boulardii.

The yeast Saccharomyces boulardii is well known for its probiotic effects such as treating or preventing gastrointestinal diseases. Due to its ability to survive in stomach and intestine, S. boulardii could be applied as a vehicle for producing and delivering bioactive substances of interest to human gut. In this study, we cloned the gene lecC encoding the antilisterial peptide leucocin C from lactic acid bacterium Leuconostoc carnosum in S. boulardii. The constructed S. boulardii strain secreted a peptide, which had molecular weight corresponding to leucocin C in SDS-PAGE. The peptide band inhibited Listeria monocytogenes in gel overlay assay. Likewise, concentrated S. boulardii culture supernatant inhibited the growth of L. monocytogenes. The growth profile and acid tolerance of the leucocin C secreting S. boulardii were similar as those of the strain carrying the empty vector. We further demonstrated that the cells of the leucocin C producing S. boulardii efficiently killed L. monocytogenes, also without antibiotic selection pressure. These results showed that antilisterial activity could be added to the arsenal of probiotic activities of S. boulardii, demonstrating its potential as a carrier for therapeutics delivery.

Yeasts as probiotics: Mechanisms, outcomes, and future potential.

The presence of commensal fungal species in the human gut indicates that organisms from this kingdom have the potential to benefit the host as well. Saccharomyces boulardii, a yeast strain isolated about a hundred years ago, is the most well-characterized probiotic yeast. Though for the most part it genetically resembles Saccharomyces cerevisiae, specific phenotypic differences make it better suited for the gut microenvironment such as better acid and heat tolerance. Several studies using animal hosts suggest that S. boulardii can be used as a biotherapeutic in humans. Clinical trials indicate that it can alleviate symptoms from gastrointestinal (GI) tract infections to some extent, but further trials are needed to understand the full therapeutic potential of S. boulardii. Improvement on probiotic function using engineered yeast is an attractive future direction, though genome modification tools for use in S. boulardii have been limited until recently. However, some tools available for S. cerevisiae should be applicable for S. boulardii as well. In this review, we summarize the observed probiotic effect of this yeast and the state of the art for genome engineering tools that could help enhance its probiotic properties.

Unique genetic basis of the distinct antibiotic potency of high acetic acid production in the probiotic yeast Saccharomyces cerevisiae var. boulardii.

The yeast Saccharomyces boulardii has been used worldwide as a popular, commercial probiotic, but the basis of its probiotic action remains obscure. It is considered conspecific with budding yeast Saccharomyces cerevisiae, which is generally used in classical food applications. They have an almost identical genome sequence, making the genetic basis of probiotic potency in S. boulardii puzzling. We now show that S. boulardii produces at 37°C unusually high levels of acetic acid, which is strongly inhibitory to bacterial growth in agar-well diffusion assays and could be vital for its unique application as a probiotic among yeasts. Using pooled-segregant whole-genome sequence analysis with S. boulardii and S. cerevisiae parent strains, we succeeded in mapping the underlying QTLs and identified mutant alleles of SDH1 and WHI2 as the causative alleles. Both genes contain a SNP unique to S. boulardii (sdh1 F317Y and whi2 S287*) and are fully responsible for its high acetic acid production. S. boulardii strains show different levels of acetic acid production, depending on the copy number of the whi2 S287* allele. Our results offer the first molecular explanation as to why S. boulardii could exert probiotic action as opposed to S. cerevisiae They reveal for the first time the molecular-genetic basis of a probiotic action-related trait in S. boulardii and show that antibacterial potency of a probiotic microorganism can be due to strain-specific mutations within the same species. We suggest that acquisition of antibacterial activity through medium acidification offered a selective advantage to S. boulardii in its ecological niche and for its application as a probiotic.

Enterobacteriaceae are essential for the modulation of colitis severity by fungi.

BACKGROUND: Host-microbe balance maintains intestinal homeostasis and strongly influences inflammatory conditions such as inflammatory bowel diseases (IBD). Here we focused on bacteria-fungi interactions and their implications on intestinal inflammation, a poorly understood area. METHODS: Dextran sodium sulfate (DSS)-induced colitis was assessed in mice treated with vancomycin (targeting gram-positive bacteria) or colistin (targeting Enterobacteriaceae) and supplemented with either Saccharomyces boulardii CNCM I-745 or Candida albicans. Inflammation severity as well as bacterial and fungal microbiota compositions was monitored. RESULTS: While S. boulardii improved DSS-induced colitis and C. albicans worsened it in untreated settings, antibiotic treatment strongly modified DSS susceptibility and effects of fungi on colitis. Vancomycin-treated mice were fully protected from colitis, while colistin-treated mice retained colitis phenotype but were not affected anymore by administration of fungi. Antibacterial treatments not only influenced bacterial populations but also had indirect effects on fungal microbiota. Correlations between bacterial and fungal relative abundance were dramatically decreased in colistin-treated mice compared to vancomycin-treated and control mice, suggesting that colistin-sensitive bacteria are involved in interactions with fungi. Restoration of the Enterobacteriaceae population by administrating colistin-resistant Escherichia coli reestablished both beneficial effects of S. boulardii and pathogenic effects of C. albicans on colitis severity. This effect was at least partly mediated by an improved gut colonization by fungi. CONCLUSIONS: Fungal colonization of the gut is affected by the Enterobacteriaceae population, indirectly modifying effects of mycobiome on the host. This finding provides new insights into the role of inter-kingdom functional interactions in intestinal physiopathology and potentially in IBD.

Application of Saccharomyces cerevisiae var. boulardii in food processing: a review.

Probiotics are increasingly being added to food in order to develop products with health-promoting properties. Particularly, Saccharomyces cereviceae var. boulardii yeast is recently being investigated like a starting-culture for development of functional and probiotic foods. Although the literature is abundant on the beneficial effects of S. boulardii on health, slight information is available on the effects of supplementing this probiotic to food systems. The aim of this paper is to examine the applications of S. boulardii to different food matrices and its implication in food processing (stability, sensorial properties and other technological implications) and the concomitant effects on nutrition and health.

A Mutation in PGM2 Causing Inefficient Galactose Metabolism in the Probiotic Yeast Saccharomyces boulardii.

The probiotic yeast Saccharomyces boulardii has been extensively studied for the prevention and treatment of diarrheal diseases, and it is now commercially available in some countries. S. boulardii displays notable phenotypic characteristics, such as a high optimal growth temperature, high tolerance against acidic conditions, and the inability to form ascospores, which differentiate S. boulardii from Saccharomyces cerevisiae The majority of prior studies stated that S. boulardii exhibits sluggish or halted galactose utilization. Nonetheless, the molecular mechanisms underlying inefficient galactose uptake have yet to be elucidated. When the galactose utilization of a widely used S. boulardii strain, ATCC MYA-796, was examined under various culture conditions, the S. boulardii strain could consume galactose, but at a much lower rate than that of S. cerevisiae While all GAL genes were present in the S. boulardii genome, according to analysis of genomic sequencing data in a previous study, a point mutation (G1278A) in PGM2, which codes for phosphoglucomutase, was identified in the genome of the S. boulardii strain. As the point mutation resulted in the truncation of the Pgm2 protein, which is known to play a pivotal role in galactose utilization, we hypothesized that the truncated Pgm2 might be associated with inefficient galactose metabolism. Indeed, complementation of S. cerevisiaePGM2 in S. boulardii restored galactose utilization. After reverting the point mutation to a full-length PGM2 in S. boulardii by Cas9-based genome editing, the growth rates of wild-type (with a truncated PGM2 gene) and mutant (with a full-length PGM2) strains with glucose or galactose as the carbon source were examined. As expected, the mutant (with a full-length PGM2) was able to ferment galactose faster than the wild-type strain. Interestingly, the mutant showed a lower growth rate than that of the wild-type strain on glucose at 37°C. Also, the wild-type strain was enriched in the mixed culture of wild-type and mutant strains on glucose at 37°C, suggesting that the truncated PGM2 might offer better growth on glucose at a higher temperature in return for inefficient galactose utilization. Our results suggest that the point mutation in PGM2 might be involved in multiple phenotypes with different effects.IMPORTANCESaccharomyces boulardii is a probiotic yeast strain capable of preventing and treating diarrheal diseases. However, the genetics and metabolism of this yeast are largely unexplored. In particular, molecular mechanisms underlying the inefficient galactose metabolism of S. boulardii remain unknown. Our study reports that a point mutation in PGM2, which codes for phosphoglucomutase, is responsible for inferior galactose utilization by S. boulardii After correction of the mutated PGM2 via genome editing, the resulting strain was able to use galactose faster than a parental strain. While the PGM2 mutation made the yeast use galactose slowly, investigation of the genomic sequencing data of other S. boulardii strains revealed that the PGM2 mutation is evolutionarily conserved. Interestingly, the PGM2 mutation was beneficial for growth at a higher temperature on glucose. We speculate that the PGM2 mutation was enriched due to selection of S. boulardii in the natural habitat (sugar-rich fruits in tropical areas).

Complete genome sequence and comparative genomics of the probiotic yeast Saccharomyces boulardii.

The probiotic yeast, Saccharomyces boulardii (Sb) is known to be effective against many gastrointestinal disorders and antibiotic-associated diarrhea. To understand molecular basis of probiotic-properties ascribed to Sb we determined the complete genomes of two strains of Sb i.e. Biocodex and unique28 and the draft genomes for three other Sb strains that are marketed as probiotics in India. We compared these genomes with 145 strains of S. cerevisiae (Sc) to understand genome-level similarities and differences between these yeasts. A distinctive feature of Sb from other Sc is absence of Ty elements Ty1, Ty3, Ty4 and associated LTR. However, we could identify complete Ty2 and Ty5 elements in Sb. The genes for hexose transporters HXT11 and HXT9, and asparagine-utilization are absent in all Sb strains. We find differences in repeat periods and copy numbers of repeats in flocculin genes that are likely related to the differential adhesion of Sb as compared to Sc. Core-proteome based taxonomy places Sb strains along with wine strains of Sc. We find the introgression of five genes from Z. bailii into the chromosome IV of Sb and wine strains of Sc. Intriguingly, genes involved in conferring known probiotic properties to Sb are conserved in most Sc strains.