Saccharomyces cerevisiae derived postbiotic alters gut microbiome metabolism in the human distal colon resulting in immunomodulatory potential in vitro.

The yeast-based postbiotic EpiCor is a well-studied formulation, consisting of a complex mixture of bioactive molecules. In clinical studies, EpiCor postbiotic has been shown to reduce intestinal symptoms in a constipated population and support mucosal defense in healthy subjects. Anti-inflammatory potential and butyrogenic properties have been reported in vitro, suggesting a possible link between EpiCor's gut modulatory activity and immunomodulation. The current study used a standardized in vitro gut model, the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®), to obtain a deeper understanding on host-microbiome interactions and potential microbiome modulation following repeated EpiCor administration. It was observed that EpiCor induced a functional shift in carbohydrate fermentation patterns in the proximal colon environment. Epicor promoted an increased abundance of Bifidobacterium in both the proximal and distal colon, affecting overall microbial community structure. Co-occurrence network analysis at the phylum level provided additional evidence of changes in the functional properties of microbial community promoted by EpiCor, increasing positive associations between Actinobacteria with microbes belonging to the Firmicutes phylum. These results, together with a significant increase in butyrate production provide additional support of EpiCor benefits to gut health. Investigation of host-microbiome interactions confirmed the immunomodulatory potential of the applied test product. Specific microbial alterations were observed in the distal colon, with metabotyping indicating that specific metabolic pathways, such as bile acid and tryptophan metabolism, were affected following EpiCor supplementation. These results, especially considering many effects were seen distally, further strengthen the position of EpiCor as a postbiotic with health promoting functionality in the gut, which could be further assessed in vivo.

A Randomized Controlled Trial to Evaluate the Impact of a Novel Probiotic and Nutraceutical Supplement on Pruritic Dermatitis and the Gut Microbiota in Privately Owned Dogs.

Pruritic dermatitis (PD) is a common presentation of canine allergic skin diseases, with diversity in severity and treatment response due to complex etiopathogenesis. Evidence suggests the gut microbiota (GM) may contribute to the development of canine allergies. A 10-week double-blind randomized controlled trial evaluated a novel probiotic and nutraceutical blend (PNB) on clinical signs of skin allergy, health measures, and the GM of privately owned self-reported pruritic dogs. A total of 105 dogs were enrolled, with 62 included in pruritus and health analysis and 50 in microbiome analysis. The PNB supported greater improvement of owner-assessed clinical signs of PD at week 2 than the placebo (PBO). More dogs that received the PNB shifted to normal pruritus (digital PVAS10-N: <2) by week 4, compared to week 7 for the PBO. While a placebo effect was identified, clinical differences were supported by changes in the GM. The PNB enriched three probiotic bacteria and reduced abundances of species associated with negative effects. The PBO group demonstrated increased abundances of pathogenic species and reduced abundances of several beneficial species. This trial supports the potential of the PNB as a supplemental intervention in the treatment of PD; however, further investigation is warranted, with stricter diagnostic criteria, disease biomarkers and direct veterinary examination.

Comparison of a Daily Steviol Glycoside Beverage compared with a Sucrose Beverage for Four Weeks on Gut Microbiome in Healthy Adults.

BACKGROUND: Recent studies suggest that some nonnutritive sweeteners (NNS) have deleterious effects on the human gut microbiome (HGM). The effect of steviol glycosides on the HGM has not been well studied. OBJECTIVE: We aimed to evaluate the effects of stevia- compared with sucrose-sweetened beverages on the HGM and fecal short-chain fatty acid (SCFA) profiles. METHODS: Using a randomized, double-blinded, parallel-design study, n = 59 healthy adults [female/male, n = 36/23, aged 31±9 y, body mass index (BMI): 22.6±1.7 kg/m2] consumed 16 oz of a beverage containing either 25% of the acceptable daily intake (ADI) of stevia or 30 g of sucrose daily for 4 weeks followed by a 4-week washout. At weeks 0 (baseline), 4, and 8, the HGM was characterized via shotgun sequencing, fecal SCFA concentrations were measured using ultra-high performance liquid chromatography-tandem mass spectrometry and anthropometric measurements, fasting serum glucose, insulin and lipids, blood pressure, pulse, and 3-d diet records were obtained. RESULTS: There were no significant differences in the HGM or fecal SCFA between the stevia and sucrose groups at baseline (P > 0.05). At week 4 (after intervention), there were no significant differences in the HGM at the phylum, family, genus, or species level between the stevia and sucrose groups and no significant differences in fecal SCFA. At week 4, BMI had increased by 0.3 kg/m2 (P = 0.013) in sucrose compared with stevia, but all other anthropometric and cardiometabolic measures and food intake did not differ significantly (P > 0.05). At week 8 (after washout), there were no significant differences in the HGM, fecal SFCA, or any anthropometric or cardiometabolic measure between the stevia and sucrose groups (P > 0.05). CONCLUSIONS: Daily consumption of a beverage sweetened with 25% of the ADI of stevia for 4 weeks had no significant effects on the HGM, fecal SCFA, or fasting cardiometabolic measures, compared with daily consumption of a beverage sweetened with 30 g of sucrose. TRIAL REGISTRATION: clinicaltrials.gov as NCT05264636.

Saccharomyces cerevisiae fermentation product improves robustness of equine gut microbiome upon stress.

Introduction: Nutritional and environmental stressors can disturb the gut microbiome of horses which may ultimately decrease their health and performance. We hypothesized that supplementation with a yeast-derived postbiotic (Saccharomyces cerevisiae fermentation product-SCFP) would benefit horses undergoing an established model of stress due to prolonged transportation. Methods: Quarter horses (n = 20) were blocked based on sex, age (22 ± 3 mo) and body weight (439 ± 3 kg) and randomized to receive either a basal diet of 60% hay and 40% concentrate (CON) or the basal diet supplemented with 21 g/d Diamond V TruEquine C (SCFP; Diamond V, Cedar Rapids, IA) for 60 days. On day 57, horses were tethered with their heads elevated 35cm above wither height for 12 h to induce mild upper respiratory tract inflammation. Fecal samples were collected at days 0, 28, and 56 before induction of stress, and at 0, 12, 24, and 72 h post-stress and subjected to DNA extraction and Nanopore shotgun metagenomics. Within sample (alpha) diversity was evaluated by fitting a linear model and between sample (beta) diversity was tested with permutational ANOVA. Results: The SCFP stabilized alpha diversity across all time points, whereas CON horses had more fluctuation (P < 0.05) at 12, 24, and 72 h post-challenge compared to d 56. A significant difference between CON and SCFP was observed at 0 and 12 h. There was no difference in beta-diversity between SCFP and CON on d 56. Discussion: Taken together, these observations led us to conclude that treatment with SCFP resulted in more robust and stable microbial profiles in horses after stress challenge.

Perspective: Leveraging the Gut Microbiota to Predict Personalized Responses to Dietary, Prebiotic, and Probiotic Interventions.

Humans often show variable responses to dietary, prebiotic, and probiotic interventions. Emerging evidence indicates that the gut microbiota is a key determinant for this population heterogeneity. Here, we provide an overview of some of the major computational and experimental tools being applied to critical questions of microbiota-mediated personalized nutrition and health. First, we discuss the latest advances in in silico modeling of the microbiota-nutrition-health axis, including the application of statistical, mechanistic, and hybrid artificial intelligence models. Second, we address high-throughput in vitro techniques for assessing interindividual heterogeneity, from ex vivo batch culturing of stool and continuous culturing in anaerobic bioreactors, to more sophisticated organ-on-a-chip models that integrate both host and microbial compartments. Third, we explore in vivo approaches for better understanding of personalized, microbiota-mediated responses to diet, prebiotics, and probiotics, from nonhuman animal models and human observational studies, to human feeding trials and crossover interventions. We highlight examples of existing, consumer-facing precision nutrition platforms that are currently leveraging the gut microbiota. Furthermore, we discuss how the integration of a broader set of the tools and techniques described in this piece can generate the data necessary to support a greater diversity of precision nutrition strategies. Finally, we present a vision of a precision nutrition and healthcare future, which leverages the gut microbiota to design effective, individual-specific interventions.

The microbiota-gut-brain axis: pathways to better brain health. Perspectives on what we know, what we need to investigate and how to put knowledge into practice.

The gut and brain link via various metabolic and signalling pathways, each with the potential to influence mental, brain and cognitive health. Over the past decade, the involvement of the gut microbiota in gut-brain communication has become the focus of increased scientific interest, establishing the microbiota-gut-brain axis as a field of research. There is a growing number of association studies exploring the gut microbiota's possible role in memory, learning, anxiety, stress, neurodevelopmental and neurodegenerative disorders. Consequently, attention is now turning to how the microbiota can become the target of nutritional and therapeutic strategies for improved brain health and well-being. However, while such strategies that target the gut microbiota to influence brain health and function are currently under development with varying levels of success, still very little is yet known about the triggers and mechanisms underlying the gut microbiota's apparent influence on cognitive or brain function and most evidence comes from pre-clinical studies rather than well controlled clinical trials/investigations. Filling the knowledge gaps requires establishing a standardised methodology for human studies, including strong guidance for specific focus areas of the microbiota-gut-brain axis, the need for more extensive biological sample analyses, and identification of relevant biomarkers. Other urgent requirements are new advanced models for in vitro and in vivo studies of relevant mechanisms, and a greater focus on omics technologies with supporting bioinformatics resources (training, tools) to efficiently translate study findings, as well as the identification of relevant targets in study populations. The key to building a validated evidence base rely on increasing knowledge sharing and multi-disciplinary collaborations, along with continued public-private funding support. This will allow microbiota-gut-brain axis research to move to its next phase so we can identify realistic opportunities to modulate the microbiota for better brain health.

Effect of erythritol and xylitol on Streptococcus pyogenes causing peritonsillar abscesses.

Polyols are effective against caries-causing streptococci but the effect on oropharynx-derived pyogenic streptococci is not well characterised. We aimed to study the effect of erythritol (ERY) and xylitol (XYL) against Streptococcus pyogenes isolated from peritonsillar abscesses (PTA). We used 31 clinical isolates and 5 throat culture collection strains. Inhibition of bacterial growth by polyols at 2.5%, 5% and 10% concentrations was studied and the results were scored. Amylase levels in PTA pus were compared to polyol effectivity scores (PES). Growth curves of four S. pyogenes isolates were analysed. Our study showed that XYL was more effective than ERY inhibiting 71-97% and 48-84% of isolates, respectively, depending of concentrations. 48% of clinical and all throat strains were inhibited by polyols in all concentrations (PES 3). PES was negative or zero in 26% of the isolates in the presence of ERY and in 19% of XYL. ERY enhanced the growth of S. pyogenes isolated from pus with high amylase levels. Polyols in all concentrations inhibited the growth in exponential phase. In conclusion, ERY and XYL are potent growth inhibitors of S. pyogenes isolated from PTA. Therefore, ERY and XYL may have potential in preventing PTA in the patients with frequent tonsillitis episodes.

Exploring Valine Metabolism in Astrocytic and Liver Cells: Lesson from Clinical Observation in TBI Patients for Nutritional Intervention.

The utilization of alternative energy substrates to glucose could be beneficial in traumatic brain injury (TBI). Recent clinical data obtained in TBI patients reported valine, ß-hydroxyisobutyrate (ibHB) and 2-ketoisovaleric acid (2-KIV) as three of the main predictors of TBI outcome. In particular, higher levels of ibHB, 2-KIV, and valine in cerebral microdialysis (CMD) were associated with better clinical outcome. In this study, we investigate the correlations between circulating and CMD levels of these metabolites. We hypothesized that the liver can metabolize valine and provide a significant amount of intermediate metabolites, which can be further metabolized in the brain. We aimed to assess the metabolism of valine in human-induced pluripotent stem cell (iPSC)-derived astrocytes and HepG2 cells using 13C-labeled substrate to investigate potential avenues for increasing the levels of downstream metabolites of valine via valine supplementation. We observed that 94 ± 12% and 84 ± 16% of ibHB, and 94 ± 12% and 87 ± 15% of 2-KIV, in the medium of HepG2 cells and in iPSC-derived astrocytes, respectively, came directly from valine. Overall, these findings suggest that both ibHB and 2-KIV are produced from valine to a large extent in both cell types, which could be of interest in the design of optimal nutritional interventions aiming at stimulating valine metabolism.

Integrative phenotyping of glycemic responders upon clinical weight loss using multi-omics.

Weight loss aims to improve glycemic control in obese but strong variability is observed. Using a multi-omics approach, we investigated differences between 174 responders and 201 non-responders, that had lost >8% body weight following a low-caloric diet (LCD, 800 kcal/d for 8 weeks). The two groups were comparable at baseline for body composition, glycemic control, adipose tissue transcriptomics and plasma ketone bodies. But they differed significantly in their response to LCD, including improvements in visceral fat, overall insulin resistance (IR) and tissue-specific IR. Transcriptomics analyses found down-regulation in key lipogenic genes (e.g. SCD, ELOVL5) in responders relative to non-responders; metabolomics showed increase in ketone bodies; while proteomics revealed differences in lipoproteins. Findings were consistent between genders; with women displaying smaller improvements owing to a better baseline metabolic condition. Integrative analyses identified a plasma omics model that was able to predict non-responders with strong performance (on a testing dataset, the Receiving Operating Curve Area Under the Curve (ROC AUC) was 75% with 95% Confidence Intervals (CI) [67%, 83%]). This model was based on baseline parameters without the need for intrusive measurements and outperformed clinical models (p = 0.00075, with a +14% difference on the ROC AUCs). Our approach document differences between responders and non-responders, with strong contributions from liver and adipose tissues. Differences may be due to de novo lipogenesis, keto-metabolism and lipoprotein metabolism. These findings are useful for clinical practice to better characterize non-responders both prior and during weight loss.

Exploration of singular and synergistic effect of xylitol and erythritol on causative agents of dental caries.

Non-cariogenic sweet substances, like sugar alcohols, are used to decrease the risk of caries by reducing the growth of dental plaque. The aim of our study was to reveal the impact of xylitol and erythritol on the growth and biofilm formation of cariogenic bacteria including as a novelty, set of clinical mutans streptococci and Scardovia wiggsiae and to assess the possible synergistic influence of these polyols. We found both xylitol and erythritol to express high growth inhibition effect on cariogenic bacteria. In synergistic effect experiments, 10% polyol combination with excess of erythritol was found to be more effective against growth of Streptococcus mutans and the combination with excess of xylitol more effective against growth of Streptococcus sobrinus and S. wiggsiae. In biofilm inhibition experiments, solutions of 10% polyols in different combinations and 15% single polyols were equally effective against mutans streptococci. At the same time, higher biofilm formation of S. wiggsiae compared to experiments without polyols was detected in different polyol concentrations for up to 34%. In conclusion, both erythritol and xylitol as well as their combinations inhibit the growth of different cariogenic bacteria. Biofilm formation of mutans streptococci is also strongly inhibited. When applying polyols in caries prophylaxis, it is relevant to consider that the profile of pathogens in a particular patient may influence the effect of polyols used.

Impact of multi-micronutrient supplementation on lipidemia of children and adolescents.

BACKGROUND: Micronutrient supplementation has been extensively explored as a strategy to improve health and reduce risk of chronic diseases. Fat-soluble vitamins like A and E with their antioxidant properties and mechanistic interactions with lipoproteins, have potentially a key impact on lipid metabolism and lipidemia. OBJECTIVE: The impact of micronutrients on lipid metabolism requires further investigation including characterization of plasma lipidome following supplementation and any cause-effect on circulating lipids. DESIGN: In this study, we elucidate the effect and associations of a multi-micronutrient intervention in Brazilian children and teens with lipoprotein alterations and lipid metabolism. RESULTS: Our analysis suggests a combination of short and long-term impact of supplementation on lipid metabolism, potentially mediated primarily by α-tocopherol (vitamin E) and retinol (vitamin A). Among the lipid classes, levels of phospholipids, lysophospholipids, and cholesterol esters were impacted the most along with differential incorporation of stearic, palmitic, oleic and arachidonic acids. Integrated analysis with proteomic data suggested potential links to supplementation-mediated alterations in protein levels of phospholipases and pyruvate dehydrogenase kinase 1 (PDK1). CONCLUSIONS: Associations between the observed differences in lipidemia, total triglyceride, and VLDL-cholesterol levels suggest that micronutrients may play a role in reducing these risk factors for cardiovascular disease in children. This would require further investigation.

Differential Metabolism of Medium-Chain Fatty Acids in Differentiated Human-Induced Pluripotent Stem Cell-Derived Astrocytes.

Medium-chain triglyceride (MCT) ketogenic diets increase ketone bodies, which are believed to act as alternative energy substrates in the injured brain. Octanoic (C8:0) and decanoic (C10:0) acids, which produce ketone bodies through ß-oxidation, are used as part of MCT ketogenic diets. Although the ketogenic role of MCT is well-established, it remains unclear how the network metabolism underlying ß-oxidation of these medium-chain fatty acids (MCFA) differ. We aim to elucidate basal ß-oxidation of these commonly used MCFA at the cellular level. Human-induced pluripotent stem cell-derived (iPSC) astrocytes were incubated with [U-13C]-C8:0 or [U-13C]-C10:0, and the fractional enrichments (FE) of the derivatives were used for metabolic flux analysis. Data indicate higher extracellular concentrations and faster secretion rates of ß-hydroxybutyrate (ßHB) and acetoacetate (AcAc) with C8:0 than C10:0, and an important contribution from unlabeled substrates. Flux analysis indicates opposite direction of metabolic flux between the MCFA intermediates C6:0 and C8:0, with an important contribution of unlabeled sources to the elongation in the C10:0 condition, suggesting different ß-oxidation pathways. Finally, larger intracellular glutathione concentrations and secretions of 3-OH-C10:0 and C6:0 were measured in C10:0-treated astrocytes. These findings reveal MCFA-specific ketogenic properties. Our results provide insights into designing different MCT-based ketogenic diets to target specific health benefits.

Discovery and validation of temporal patterns involved in human brain ketometabolism in cerebral microdialysis fluids of traumatic brain injury patients.

BACKGROUND: Traumatic brain injury (TBI) is recognized as a metabolic disease, characterized by acute cerebral glucose hypo-metabolism. Adaptive metabolic responses to TBI involve the utilization of alternative energy substrates, such as ketone bodies. Cerebral microdialysis (CMD) has evolved as an accurate technique allowing continuous sampling of brain extracellular fluid and assessment of regional cerebral metabolism. We present the successful application of a combined hypothesis- and data-driven metabolomics approach using repeated CMD sampling obtained routinely at patient bedside. Investigating two patient cohorts (n = 26 and n = 12), we identified clinically relevant metabolic patterns at the acute post-TBI critical care phase. METHODS: Clinical and CMD metabolomics data were integrated and analysed using in silico and data modelling approaches. We used both unsupervised and supervised multivariate analysis techniques to investigate structures within the time series and associations with patient outcome. FINDINGS: The multivariate metabolite time series exhibited two characteristic brain metabolic states that were attributed to changes in key metabolites: valine, 4-methyl-2-oxovaleric acid (4-MOV), isobeta-hydroxybutyrate (iso-bHB), tyrosyine, and 2-ketoisovaleric acid (2-KIV). These identified cerebral metabolic states differed significantly with respect to standard clinical values. We validated our findings in a second cohort using a classification model trained on the cerebral metabolic states. We demonstrated that short-term (therapeutic intensity level (TIL)) and mid-term patient outcome (6-month Glasgow Outcome Score (GOS)) can be predicted from the time series characteristics. INTERPRETATION: We identified two specific cerebral metabolic patterns that are closely linked to ketometabolism and were associated with both TIL and GOS. Our findings support the view that advanced metabolomics approaches combined with CMD may be applied in real-time to predict short-term treatment intensity and long-term patient outcome.

Transcriptomics-driven lipidomics (TDL) identifies the microbiome-regulated targets of ileal lipid metabolism.

The gut microbiome and lipid metabolism are both recognized as essential components in the maintenance of metabolic health. The mechanisms involved are multifactorial and (especially for microbiome) poorly defined. A strategic approach to investigate the complexity of the microbial influence on lipid metabolism would facilitate determination of relevant molecular mechanisms for microbiome-targeted therapeutics. E. coli is associated with obesity and metabolic syndrome and we used this association in conjunction with gnotobiotic models to investigate the impact of E. coli on lipid metabolism. To address the complexities of the integration of the microbiome and lipid metabolism, we developed transcriptomics-driven lipidomics (TDL) to predict the impact of E. coli colonization on lipid metabolism and established mediators of inflammation and insulin resistance including arachidonic acid metabolism, alterations in bile acids and dietary lipid absorption. A microbiome-related therapeutic approach targeting these mechanisms may therefore provide a therapeutic avenue supporting maintenance of metabolic health.

Aging and sarcopenia associate with specific interactions between gut microbes, serum biomarkers and host physiology in rats.

The microbiome has been demonstrated to play an integral role in the maintenance of many aspects of health that are also associated with aging. In order to identify areas of potential exploration and intervention, we simultaneously characterized age-related alterations in gut microbiome, muscle physiology and serum proteomic and lipidomic profiles in aged rats to define an integrated signature of the aging phenotype. We demonstrate that aging skews the composition of the gut microbiome, in particular by altering the Sutterella to Barneseilla ratio, and alters the metabolic potential of intestinal bacteria. Age-related changes of the gut microbiome were associated with the physiological decline of musculoskeletal function, and with molecular markers of nutrient processing/availability, and inflammatory/immune status in aged versus adult rats. Altogether, our study highlights that aging leads to a complex interplay between the microbiome and host physiology, and provides candidate microbial species to target physical and metabolic decline during aging by modulating gut microbial ecology.

Complete Genome Sequence of Escherichia coli Strain M8, Isolated from ob/ob Mice.

Escherichia coli is one of the common inhabitants of the mammalian gastrointestinal track. We isolated a strain from an ob/ob mouse and performed whole-genome sequencing, which yielded a chromosome of ~5.1 Mb and three plasmids of ~160 kb, ~6 kb, and ~4 kb.

Population Heterogeneity in the Epithelial to Mesenchymal Transition Is Controlled by NFAT and Phosphorylated Sp1.

Epithelial to mesenchymal transition (EMT) is an essential differentiation program during tissue morphogenesis and remodeling. EMT is induced by soluble transforming growth factor ß (TGF-ß) family members, and restricted by vascular endothelial growth factor family members. While many downstream molecular regulators of EMT have been identified, these have been largely evaluated individually without considering potential crosstalk. In this study, we created an ensemble of dynamic mathematical models describing TGF-ß induced EMT to better understand the operational hierarchy of this complex molecular program. We used ordinary differential equations (ODEs) to describe the transcriptional and post-translational regulatory events driving EMT. Model parameters were estimated from multiple data sets using multiobjective optimization, in combination with cross-validation. TGF-ß exposure drove the model population toward a mesenchymal phenotype, while an epithelial phenotype was enhanced following vascular endothelial growth factor A (VEGF-A) exposure. Simulations predicted that the transcription factors phosphorylated SP1 and NFAT were master regulators promoting or inhibiting EMT, respectively. Surprisingly, simulations also predicted that a cellular population could exhibit phenotypic heterogeneity (characterized by a significant fraction of the population with both high epithelial and mesenchymal marker expression) if treated simultaneously with TGF-ß and VEGF-A. We tested this prediction experimentally in both MCF10A and DLD1 cells and found that upwards of 45% of the cellular population acquired this hybrid state in the presence of both TGF-ß and VEGF-A. We experimentally validated the predicted NFAT/Sp1 signaling axis for each phenotype response. Lastly, we found that cells in the hybrid state had significantly different functional behavior when compared to VEGF-A or TGF-ß treatment alone. Together, these results establish a predictive mechanistic model of EMT susceptibility, and potentially reveal a novel signaling axis which regulates carcinoma progression through an EMT versus tubulogenesis response.

Resolving microbial membership using Abundance and Variability In Taxonomy ('AVIT ).

Development of NGS has revolutionized the analysis in microbial ecology contributing to our deeper understanding of microbiota in health and disease. However, the quality, quantity and confidence of summarized taxonomic abundances are in need of further scrutiny due to sample dependent and independent effects. In this article we introduce 'AVIT (Abundance and Variability In Taxonomy), an unbiased method to enrich for assigned members of microbial communities. As opposed to using a priori thresholds, 'AVIT uses inherent abundance and variability of taxa in a dataset to determine the inclusion or rejection of each taxa for further downstream analysis. Using in-vitro and in-vivo studies, we benchmarked performance and parameterized 'AVIT to establish a framework for investigating the dynamic range of microbial community membership in clinically relevant scenarios.

Identification of metabolic engineering targets for the enhancement of 1,4-butanediol production in recombinant E. coli using large-scale kinetic models.

Rational metabolic engineering methods are increasingly employed in designing the commercially viable processes for the production of chemicals relevant to pharmaceutical, biotechnology, and food and beverage industries. With the growing availability of omics data and of methodologies capable to integrate the available data into models, mathematical modeling and computational analysis are becoming important in designing recombinant cellular organisms and optimizing cell performance with respect to desired criteria. In this contribution, we used the computational framework ORACLE (Optimization and Risk Analysis of Complex Living Entities) to analyze the physiology of recombinant Escherichia coli producing 1,4-butanediol (BDO) and to identify potential strategies for improved production of BDO. The framework allowed us to integrate data across multiple levels and to construct a population of large-scale kinetic models despite the lack of available information about kinetic properties of every enzyme in the metabolic pathways. We analyzed these models and we found that the enzymes that primarily control the fluxes leading to BDO production are part of central glycolysis, the lower branch of tricarboxylic acid (TCA) cycle and the novel BDO production route. Interestingly, among the enzymes between the glucose uptake and the BDO pathway, the enzymes belonging to the lower branch of TCA cycle have been identified as the most important for improving BDO production and yield. We also quantified the effects of changes of the target enzymes on other intracellular states like energy charge, cofactor levels, redox state, cellular growth, and byproduct formation. Independent earlier experiments on this strain confirmed that the computationally obtained conclusions are consistent with the experimentally tested designs, and the findings of the present studies can provide guidance for future work on strain improvement. Overall, these studies demonstrate the potential and effectiveness of ORACLE for the accelerated design of microbial cell factories.

Towards kinetic modeling of genome-scale metabolic networks without sacrificing stoichiometric, thermodynamic and physiological constraints.

Mathematical modeling is an essential tool for the comprehensive understanding of cell metabolism and its interactions with the environmental and process conditions. Recent developments in the construction and analysis of stoichiometric models made it possible to define limits on steady-state metabolic behavior using flux balance analysis. However, detailed information on enzyme kinetics and enzyme regulation is needed to formulate kinetic models that can accurately capture the dynamic metabolic responses. The use of mechanistic enzyme kinetics is a difficult task due to uncertainty in the kinetic properties of enzymes. Therefore, the majority of recent works considered only mass action kinetics for reactions in metabolic networks. Herein, we applied the optimization and risk analysis of complex living entities (ORACLE) framework and constructed a large-scale mechanistic kinetic model of optimally grown Escherichia coli. We investigated the complex interplay between stoichiometry, thermodynamics, and kinetics in determining the flexibility and capabilities of metabolism. Our results indicate that enzyme saturation is a necessary consideration in modeling metabolic networks and it extends the feasible ranges of metabolic fluxes and metabolite concentrations. Our results further suggest that enzymes in metabolic networks have evolved to function at different saturation states to ensure greater flexibility and robustness of cellular metabolism.