Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts

G protein-coupled receptors (GPCRs) enable cells to sense environmental cues and are indispensable for coordinating vital processes including quorum sensing, proliferation, and sexual reproduction. GPCRs comprise the largest class of cell surface receptors in eukaryotes, and for more than three decades the pheromone-induced mating pathway in baker's yeast Saccharomyces cerevisiae has served as a model for studying heterologous GPCRs (hGPCRs). Here we report transcriptome profiles following mating pathway activation in native and hGPCR-signaling yeast, and use a model-guided approach to correlate gene expression to morphological changes. From this we demonstrate mating between haploid cells armed with hGPCRs and endogenous biosynthesis of their cognate ligands. Furthermore, we devise a ligand-free screening strategy for hGPCR compatibility with the yeast mating pathway and enable hGPCR-signaling in the probiotic yeast Saccharomyces boulardii. Combined, our findings enable new means to study mating, hGPCR-signaling, and cell-cell communication in a model eukaryote and yeast probiotics.

Tryptophol acetate and tyrosol acetate, metabolites secreted by a probiotic yeast, halt cytokine storm

Probiotic fermented foods are perceived as contributing to human health and capable of protecting against inflammation, however solid mechanistic evidence for the presumptive therapeutic benefits is lacking. Here we report that tryptophol acetate and tyrosol acetate, small molecule metabolites secreted by the probiotic milk-fermented yeast Kluyveromyces marxianus exhibit remarkable anti-inflammatory properties. Comprehensive in vivo, ex vivo and in vitro experiments, employing LPS-induced 'cytokine storm' models, reveal dramatic effects of the two molecules, added in tandem, on mice morbidity, laboratory parameters and mortality. In parallel, significant attenuation of pro-inflammatory cytokines including IL-6, IL-1α, IL-1β and TNF-κB, and reduction of reactive oxygen species were recorded. Importantly, tryptophol acetate and tyrosol acetate did not completely suppress cytokine generation, but rather brought their concentrations back to baseline levels, further maintaining core immune functions, including phagocytosis. The anti-inflammatory effects of tryptophol acetate and tyrosol acetate were mediated through downregulation of TLR4, IL-1R, and TNFR signaling pathways and increased A20 expression, attenuating NF-kB level. In addition, the two molecules had a significant impact on mice microbiome, increasing the abundance of the genus Bactericides, known to exhibit anti-inflammatory properties. Overall, this work illuminates pronounced and broad-based immune modulation properties of probiotic yeast-secreted metabolites, uncovering their mechanism of action and underscoring potential new therapeutic avenues for severe inflammation.

Investigation of the Effects of Probiotics on Sub-Chronic Neonicotinoid Toxicity in Rats

Probiotics have been shown to have positive effects when it comes to combating various health issues when consumed, preventing even the absorption of environmental toxins. One of the main environmental toxins encountered today is pesticide residues. Neonicotinoids, widely applied today in countries that have approved of them, are a known class of insecticides with an excellent and effective potency. Neonicotinoids have been shown to cause various toxic effects, either acutely or chronically, on human health and on beneficial insects when exposed. To clarify the assumption that probiotics could counteract these toxic effects, especially on vital organs, the probiotic yeast “Saccharomyces boulardii” (S. boulardii) was tested against the neonicotinoids, acetamiprid (ACE) and imidacloprid (IMI), as it has outstanding physiological and metabolic properties. The results obtained from the studies indicated that although ACE and IMI induced liver, kidney, brain and bowel damage, there was a considerable level of protection by the dietary supplementation of S. boulardii, as it reduced the absorption of these insecticides.

Virulence Factors and in-Host Selection on Phenotypes in Infectious Probiotic Yeast Isolates (Saccharomyces ‘boulardii’)

Saccharomyces yeast probiotics (S. ’boulardii’) have long been applied in the treatment of several gastrointestinal conditions. Despite their widespread use, they are rare opportunistic pathogens responsible for a high proportion of Saccharomyces mycosis cases. The potential virulence attributes of S. ’boulardii’ as well as its interactions with the human immune system have been studied, however, no information is available on how these yeasts may change due to in-host evolution. To fill this gap, we compared the general phenotypic characteristics, cell morphology, virulence factors, epithelial and immunological interactions, and pathogenicity of four probiotic product samples, two mycosis, and eight non-mycosis samples of S. ’boulardii’. We assessed the characteristics related to major steps of yeast infections. Mycosis and non-mycosis isolates both displayed novel characters when compared to the product isolates, but in the case of most virulence factors and in pathogenicity, differences were negligible or, surprisingly, the yeasts from products showed elevated levels. No isolates inflicted considerable damage to the epithelial model or bore the hallmarks of immune evasion. Our results show that strains in probiotic products possess characteristics that enable them to act as pathogens upon permissive conditions, and their entry into the bloodstream is not due to active mechanisms but depends on the host. Survival in the host is dependent on yeast phenotypic characteristics which may change in many ways once they start evolving in the host. These facts call attention to the shortcomings of virulence phenotyping in yeast research, and the need for a more thorough assessment of probiotic use.

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 β-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 β-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. Graphical abstract