Cellulosic plant biomass is a promising sustainable resource for generating alternative biofuels and biochemicals with microbial factories. But a remaining bottleneck is engineering microbes that are tolerant of toxins generated during biomass processing, because mechanisms of toxin defense are only beginning to emerge. Here, we exploited natural diversity in 165 Saccharomyces cerevisiae strains isolated from diverse geographical and ecological niches, to identify mechanisms of hydrolysate-toxin tolerance. We performed genome-wide association (GWA) analysis to identify genetic variants underlying toxin tolerance, and gene knockouts and allele-swap experiments to validate the involvement of implicated genes. In the process of this work, we uncovered a surprising difference in genetic architecture depending on strain background: in all but one case, knockout of implicated genes had a significant effect on toxin tolerance in one strain, but no significant effect in another strain. In fact, whether or not the gene was involved in tolerance in each strain background had a bigger contribution to strain-specific variation than allelic differences. Our results suggest a major difference in the underlying network of causal genes in different strains, suggesting that mechanisms of hydrolysate tolerance are very dependent on the genetic background. These results could have significant implications for interpreting GWA results and raise important considerations for engineering strategies for industrial strain improvement. Understanding the genetic architecture of complex traits is important for elucidating the genotype-phenotype relationship. Many studies have sought genetic variants that underlie phenotypic variation across individuals, both to implicate causal variants and to inform on architecture. Here we used genome-wide association analysis to identify genes and processes involved in tolerance of toxins found in plant-biomass hydrolysate, an important substrate for sustainable biofuel production. We found substantial variation in whether or not individual genes were important for tolerance across genetic backgrounds. Whether or not a gene was important in a given strain background explained more variation than the alleleic differences in the gene. These results suggest substantial variation in gene contributions, and perhaps underlying mechanisms, of toxin tolerance.

Bovine respiratory disease causes morbidity and mortality in cattle of all ages. Supplementing with postbiotic products from Saccharomyces cerevisiae fermentation (SCFP) has been reported to improve growth and provide metabolic support required for immune activation in calves. The objective of this study was to determine effects of SCFP supplementation on the transcriptional response to coinfection with bovine respiratory syncytial virus (BRSV) and Pasteurella multocida in the lung using RNA sequencing. Twenty-three calves were enrolled and assigned to 2 treatment groups: control (n = 12) or SCFP-treated (n = 11, fed 1 g/d SmartCare in milk and 5 g/d NutriTek on starter grain; both from Diamond V Mills Inc.). Calves were infected with ∼10⁴ median tissue culture infectious dose per milliliter of BRSV, followed 6 d later by intratracheal inoculation with ∼10¹⁰ cfu of Pasteurella multocida (strain P1062). Calves were euthanized on d 10 after viral infection. Blood cells were collected and assayed on d 0 and 10 after viral infection. Bronchoalveolar lavage (BAL) cells were collected and assayed on d 14 of the feeding period (preinfection) and d 10 after viral infection. Blood and BAL cells were assayed for proinflammatory cytokine production in response to stimulation with lipopolysaccharide (LPS) or a combination of polyinosinic:polycytidylic acid and imiquimod, and BAL cells were evaluated for phagocytic and reactive oxygen species production capacity. Antemortem and postmortem BAL and lesioned and nonlesioned lung tissue samples collected at necropsy were subjected to RNA extraction and sequencing. Sequencing reads were aligned to the bovine reference genome (UMD3.1) and edgeR version 3.32.1 used for differential gene expression analysis. Supplementation with SCFP did not affect the respiratory burst activity or phagocytic activity of either lung or blood immune cells. Immune cells from the peripheral blood of SCFP-supplemented calves produced increased quantities of IL-6 in response to toll-like receptor stimulation, whereas cells from the BAL of SCFP-treated calves secreted fewer proinflammatory cytokines and less tumor necrosis factor-α (TNF-α) and IL-6 in response to the same stimuli. Transcriptional responses in lung tissues and BAL samples from SCFP-fed calves differed from the control group. The top enriched pathways in SCFP-treated lungs were associated with decreased expression of inflammatory responses and increased expression of plasminogen and genes involved in glutathione metabolism, supporting effective lung repair. Our results indicate that supplementing with SCFP postbiotics modulates both systemic and mucosal immune responses, leading to increased resistance to bovine respiratory disease.

This study evaluated the effects of adding a Saccharomyces cerevisiae fermentation product (SCFP) to adult cat diets on palatability, intestinal health, nutrient digestibility, immune parameters, and the fecal microbiome over 42 days. Sixty-three healthy Domestic Short-hair cats were randomized to three diets: a control diet (CD) without SCFP, or the same diet containing 1.0% or 2.0% SCFP, targeting daily intakes of 150 or 300 mg/kg body weight, respectively. Body weight and blood parameters remained within reference ranges across all groups. Stool quality was largely not affected, although compared with controls, SCFP-supplemented cats had slightly firmer stools at day 21, and increased fecal pH. Shotgun metagenomic sequencing revealed that microbiome diversity remained steady in SCFP-fed cats, whereas diversity in the control group declined over time. Fecal immunoglobulin A concentrations were lower in cats supplemented with SCFP at 150 mg/kg BW by the end of the study, and positive shifts in the circulatory leukocyte profile were observed at both inclusion levels. Apparent total tract macronutrient digestibility did not differ among groups. Palatability tests showed diets with SCFP were generally preferred, indicating a potential benefit for commercial feline feeds, particularly at the 150 mg/kg BW level, which was preferred over 300 mg/kg BW. Overall, these findings suggest that SCFP can act as a functional ingredient in feline nutrition to maintain microbial diversity and enhance diet acceptance without compromising digestibility.

Hose J, Yong CM, Sardi M, Wang Z, Newton MA, Gasch AP. 2015. Dosage compensation can buffer copy-number variation in wild yeast. eLife 4:e05462. doi: 10.7554/eLife.05462.

Controlled, evidence-based research studies are essential to ensure functional pet food, treat and supplement ingredients are efficacious. However, understanding how nutritional technologies influence pet health and well-being in the pet's home environment is an additional way to derive further insight on performance and effectiveness. Therefore, the objective of this study was to assess the efficacy of supplemental Saccharomyces cerevisiae fermentation product (SCFP) using subjective pet owner insights coupled with an analysis of fecal metagenomic characteristics in dogs using an in-home testing approach. Healthy adult dogs (n = 348; 170 males and 178 females; mean age = 6.0 y; mean BW = 18.2 kg) were recruited from across the U.S. and randomly allotted to one of two treatment groups blocked by breed, gender, age and BW. Owners were asked to feed their dogs their usual food, treats and supplements throughout the 60-d study. The SCFP was delivered in a daily chew. The control was a placebo chew void of SCFP (CON), and the treatment chew (EPI) was formulated to deliver 7 mg· kg BW^-1· d^-1 SCFP (EpiCor postbiotic, Ankeny, IA). Surveys were fielded on d 0, 15, 30, 45 and 60 to obtain pet owner subjective ratings of supplement performance across key health attributes. Fecal sample collection kits were provided to respondents on d 0, 30 and 60 for shotgun metagenomic analysis. All statistical analyses were conducted in R v.4.1.2. Ordinal logistic regression using the package ordinal in R was conducted on the survey data to understand relationships between the predictor variables and the ranked outcomes. Metagenomic data were analyzed using phyloseq, vegan, lme4, LinDA and emmeans packages in R. Differences P < 0.05 were considered significant and P ≤ 0.10 as trends after FDR correction. Dogs receiving the EPI chew received numerically higher immunity ratings throughout the study and overall tended to be 95% more likely (P = 0.10) to receive a higher immune health rating versus CON. By d 60, EPI dogs were more likely (211% greater odds; P = 0.007) to exhibit less breath odor and 165% more likely to be less anxious (P = 0.02) than CON dogs. An increased relative abundance in butyrate producing taxa Anaerostipes hadrus (P = 0.002) and Megamonas (P = 0.001) and Fusobacteria (P = 0.004) were observed in EPI dogs emerging at d 30. Under the conditions of this study, daily supplementation of a SCFP supplement provided a spectrum of positive health outcomes in healthy adult dogs. Moreover, there was consistency in physiological benefits noted in previous controlled studies in healthy dogs fed SCFP to the owner-perceived changes in several health attributes in this study. Our data also suggest that SCFP may beneficially affect key indicators of breath odor and anxiety in dogs.

Provisoy, produced by hydro, thermal, and mechanical process using soybean meal (SBM) has been shown to improve both post-weaning pig growth and gut health compared with SBM. It was hypothesized that Provisoy could improve the utilization of both protein and fiber fractions along the gastrointestinal tract of a nursery pig resulting in a more balanced nutrient environment and robust microbiome in the hindgut compared with SBM. A total of 268 weaned pigs (body weight, 6.82 ± 0.85 kg) were allotted to either a regular nursery condition (5 pigs/pen, 10-11 pens/treatment) or subjected to ETEC challenge (3 pigs/pen, 12 pens/treatment, 1 pig per pen was orally gavaged with O149:K91:K88 strain on d 5 post-weaning). Pigs received one of the following diets: SBM, Provisoy or HP300 (soy protein from Hamlet Protein, used as a benchmark). A standard wheat-barley-SBM based diet was used, and test soy products were added to replace SBM in the treatment diets during the first three weeks of nursery program. All diets were isocaloric with the same standardized ileal digestible lysine level in a three-phase (38 days) nursery program. Fecal samples were collected on d 13 or d 14 post-weaning and subjected to DNA extraction and Nanopore shotgun sequencing. Data were center-log-ratio transformed and subjected to statistical analysis. Microbiome composition was affected by dietary treatment (P < 0.01) and challenge (P < 0.05), but their interaction was not significant. In regular nursery condition, both Provisoy and HP300 tended (P < 0.10) to have greater microbiome alpha-diversity compared with SBM, but no difference was observed between these two treatments. Only SBM fed pigs subjected to ETEC challenge had greater (P = 0.02) alpha-diversity compared with their non-challenged counterparts. Compared with the SBM treatment, pigs that received Provisoy in the regular condition had greater abundance of Coprococcus and Dialister, which were positively correlated with greater average daily gain (ADG; r = 0.53, P < 0.01, and r = 0.45, P = 0.01, respectively). Under ETEC challenge, an increase in abundance of Dialister was observed in pigs that received Provisoy treatment with a positive correlation with ADG (r = 0.42, P = 0.01). In conclusion, dietary inclusion of Provisoy or HP300 improved microbiome alpha-diversity compared with SBM. Provisoy helped promote the growth of beneficial bacteria in the hindgut of nursery pigs which were positively correlated with gain.