Metabolic modeling of Halomonas campaniensis improves polyhydroxybutyrate production under nitrogen limitation.

Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform. KEY POINTS: A comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed. Experiments and simulations predict N limitation in minimal media under aerobiosis. In silico media design increased experimental biomass yield and PHB titer.

Genome-scale metabolic modeling of the human milk oligosaccharide utilization by Bifidobacterium longum subsp. infantis.

Bifidobacterium longum subsp. infantis is a representative and dominant species in the infant gut and is considered a beneficial microbe. This organism displays multiple adaptations to thrive in the infant gut, regarded as a model for human milk oligosaccharides (HMOs) utilization. These carbohydrates are abundant in breast milk and include different molecules based on lactose. They contain fucose, sialic acid, and N-acetylglucosamine. Bifidobacterium metabolism is complex, and a systems view of relevant metabolic pathways and exchange metabolites during HMO consumption is missing. To address this limitation, a refined genome-scale network reconstruction of this bacterium is presented using a previous reconstruction of B. infantis ATCC 15967 as a template. The latter was expanded based on an extensive revision of genome annotations, current literature, and transcriptomic data integration. The metabolic reconstruction (iLR578) accounted for 578 genes, 1,047 reactions, and 924 metabolites. Starting from this reconstruction, we built context-specific genome-scale metabolic models using RNA-seq data from cultures growing in lactose and three HMOs. The models revealed notable differences in HMO metabolism depending on the functional characteristics of the substrates. Particularly, fucosyl-lactose showed a divergent metabolism due to a fucose moiety. High yields of lactate and acetate were predicted under growth rate maximization in all conditions, whereas formate, ethanol, and 1,2-propanediol were substantially lower. Similar results were also obtained under near-optimal growth on each substrate when varying the empirically observed acetate-to-lactate production ratio. Model predictions displayed reasonable agreement between central carbon metabolism fluxes and expression data across all conditions. Flux coupling analysis revealed additional connections between succinate exchange and arginine and sulfate metabolism and a strong coupling between central carbon reactions and adenine metabolism. More importantly, specific networks of coupled reactions under each carbon source were derived and analyzed. Overall, the presented network reconstruction constitutes a valuable platform for probing the metabolism of this prominent infant gut bifidobacteria.IMPORTANCEThis work presents a detailed reconstruction of the metabolism of Bifidobacterium longum subsp. infantis, a prominent member of the infant gut microbiome, providing a systems view of its metabolism of human milk oligosaccharides.

LooplessFluxSampler: an efficient toolbox for sampling the loopless flux solution space of metabolic models.

BACKGROUND: Uniform random sampling of mass-balanced flux solutions offers an unbiased appraisal of the capabilities of metabolic networks. Unfortunately, it is impossible to avoid thermodynamically infeasible loops in flux samples when using convex samplers on large metabolic models. Current strategies for randomly sampling the non-convex loopless flux space display limited efficiency and lack theoretical guarantees. RESULTS: Here, we present LooplessFluxSampler, an efficient algorithm for exploring the loopless mass-balanced flux solution space of metabolic models, based on an Adaptive Directions Sampling on a Box (ADSB) algorithm. ADSB is rooted in the general Adaptive Direction Sampling (ADS) framework, specifically the Parallel ADS, for which theoretical convergence and irreducibility results are available for sampling from arbitrary distributions. By sampling directions that adapt to the target distribution, ADSB traverses more efficiently the sample space achieving faster mixing than other methods. Importantly, the presented algorithm is guaranteed to target the uniform distribution over convex regions, and it provably converges on the latter distribution over more general (non-convex) regions provided the sample can have full support. CONCLUSIONS: LooplessFluxSampler enables scalable statistical inference of the loopless mass-balanced solution space of large metabolic models. Grounded in a theoretically sound framework, this toolbox provides not only efficient but also reliable results for exploring the properties of the almost surely non-convex loopless flux space. Finally, LooplessFluxSampler includes a Markov Chain diagnostics suite for assessing the quality of the final sample and the performance of the algorithm.

Microbial interactions and the homeostasis of the gut microbiome: the role of Bifidobacterium.

The human gut is home to trillions of microorganisms that influence several aspects of our health. This dense microbial community targets almost all dietary polysaccharides and releases multiple metabolites, some of which have physiological effects on the host. A healthy equilibrium between members of the gut microbiota, its microbial diversity, and their metabolites is required for intestinal health, promoting regulatory or anti-inflammatory immune responses. In contrast, the loss of this equilibrium due to antibiotics, low fiber intake, or other conditions results in alterations in gut microbiota composition, a term known as gut dysbiosis. This dysbiosis can be characterized by a reduction in health-associated microorganisms, such as butyrate-producing bacteria, enrichment of a small number of opportunistic pathogens, or a reduction in microbial diversity. Bifidobacterium species are key species in the gut microbiome, serving as primary degraders and contributing to a balanced gut environment in various ways. Colonization resistance is a fundamental property of gut microbiota for the prevention and control of infections. This community competes strongly with foreign microorganisms, such as gastrointestinal pathogens, antibiotic-resistant bacteria, or even probiotics. Resistance to colonization is based on microbial interactions such as metabolic cross-feeding, competition for nutrients, or antimicrobial-based inhibition. These interactions are mediated by metabolites and metabolic pathways, representing the inner workings of the gut microbiota, and play a protective role through colonization resistance. This review presents a rationale for how microbial interactions provide resistance to colonization and gut dysbiosis, highlighting the protective role of Bifidobacterium species.

Metabolic Modeling and Bidirectional Culturing of Two Gut Microbes Reveal Cross-Feeding Interactions and Protective Effects on Intestinal Cells.

The gut microbiota is constituted by thousands of microbial interactions, some of which correspond to the exchange of metabolic by-products or cross-feeding. Inulin and xylan are two major dietary polysaccharides that are fermented by members of the human gut microbiota, resulting in different metabolic profiles. Here, we integrated community modeling and bidirectional culturing assays to study the metabolic interactions between two gut microbes, Phocaeicola dorei and Lachnoclostridium symbiosum, growing in inulin or xylan, and how they provide a protective effect in cultured cells. P. dorei (previously belonging to the Bacteroides genus) was able to consume inulin and xylan, while L. symposium only used certain inulin fractions to produce butyrate as a major end product. Constrained-based flux simulations of refined genome-scale metabolic models of both microbes predicted high lactate and succinate cross-feeding fluxes between P. dorei and L. symbiosum when growing in each fiber. Bidirectional culture assays in both substrates revealed that L. symbiosum growth increased in the presence of P. dorei. Carbohydrate consumption analyses showed a faster carbohydrate consumption in cocultures compared to monocultures. Lactate and succinate concentrations in bidirectional cocultures were lower than in monocultures, pointing to cross-feeding as initially suggested by the model. Butyrate concentrations were similar across all conditions. Finally, supernatants from both bacteria cultured in xylan in bioreactors significantly reduced tumor necrosis factor-α-induced inflammation in HT-29 cells and exerted a protective effect against the TcdB toxin in Caco-2 epithelial cells. Surprisingly, this effect was not observed in inulin cocultures. Overall, these results highlight the predictive value of metabolic models integrated with microbial culture assays for probing microbial interactions in the gut microbiota. They also provide an example of how metabolic exchange could lead to potential beneficial effects in the host. IMPORTANCE Microbial interactions represent the inner connections in the gut microbiome. By integrating mathematical modeling tools and microbial bidirectional culturing, we determined how two gut commensals engage in the exchange of cross-feeding metabolites, lactate and succinate, for increased growth in two fibers. These interactions underpinned butyrate production in cocultures, resulting in a significant reduction in cellular inflammation and protection against microbial toxins when applied to cellular models.

The topology of genome-scale metabolic reconstructions unravels independent modules and high network flexibility.

The topology of metabolic networks is recognisably modular with modules weakly connected apart from sharing a pool of currency metabolites. Here, we defined modules as sets of reversible reactions isolated from the rest of metabolism by irreversible reactions except for the exchange of currency metabolites. Our approach identifies topologically independent modules under specific conditions associated with different metabolic functions. As case studies, the E.coli iJO1366 and Human Recon 2.2 genome-scale metabolic models were split in 103 and 321 modules respectively, displaying significant correlation patterns in expression data. Finally, we addressed a fundamental question about the metabolic flexibility conferred by reversible reactions: "Of all Directed Topologies (DTs) defined by fixing directions to all reversible reactions, how many are capable of carrying flux through all reactions?". Enumeration of the DTs for iJO1366 model was performed using an efficient depth-first search algorithm, rejecting infeasible DTs based on mass-imbalanced and loopy flux patterns. We found the direction of 79% of reversible reactions must be defined before all directions in the network can be fixed, granting a high degree of flexibility.

Metabolic Modeling of Wine Fermentation at Genome Scale.

Wine fermentation is an ancient biotechnological process mediated by different microorganisms such as yeast and bacteria. Understanding of the metabolic and physiological phenomena taking place during this process can be now attained at a genome scale with the help of metabolic models. In this chapter, we present a detailed protocol for modeling wine fermentation using genome-scale metabolic models. In particular, we illustrate how metabolic fluxes can be computed, optimized and interpreted, for both yeast and bacteria under winemaking conditions. We also show how nutritional requirements can be determined and simulated using these models in relevant test cases. This chapter introduces fundamental concepts and practical steps for applying flux balance analysis in wine fermentation, and as such, it is intended for a broad microbiology audience as well as for practitioners in the metabolic modeling field.

Modeling approaches for probing cross-feeding interactions in the human gut microbiome.

Microbial communities perform emergent activities that are essentially different from those carried by their individual members. The gut microbiome and its metabolites have a significant impact on the host, contributing to homeostasis or disease. Food molecules shape this community, being fermented through cross-feeding interactions of metabolites such as lactate, acetate, and amino acids, or products derived from macromolecule degradation. Mathematical and experimental approaches have been applied to understand and predict the interactions between microorganisms in complex communities such as the gut microbiota. Rational and mechanistic understanding of microbial interactions is essential to exploit their metabolic activities and identify keystone taxa and metabolites. The latter could be used in turn to modulate or replicate the metabolic behavior of the community in different contexts. This review aims to highlight recent experimental and modeling approaches for studying cross-feeding interactions within the gut microbiome. We focus on short-chain fatty acid production and fiber fermentation, which are fundamental processes in human health and disease. Special attention is paid to modeling approaches, particularly kinetic and genome-scale stoichiometric models of metabolism, to integrate experimental data under different diet and health conditions. Finally, we discuss limitations and challenges for the broad application of these modeling approaches and their experimental verification for improving our understanding of the mechanisms of microbial interactions.

GRASP: a computational platform for building kinetic models of cellular metabolism.

Summary: Kinetic models of metabolism are crucial to understand the inner workings of cell metabolism. By taking into account enzyme regulation, detailed kinetic models can provide accurate predictions of metabolic fluxes. Comprehensive consideration of kinetic regulation requires highly parameterized non-linear models, which are challenging to build and fit using available modelling tools. Here, we present a computational package implementing the GRASP framework for building detailed kinetic models of cellular metabolism. By defining the mechanisms of enzyme regulation and a reference state described by reaction fluxes and their corresponding Gibbs free energy ranges, GRASP can efficiently sample an arbitrarily large population of thermodynamically feasible kinetic models. If additional experimental data are available (fluxes, enzyme and metabolite concentrations), these can be integrated to generate models that closely reproduce these observations using an approximate Bayesian computation fitting framework. Within the same framework, model selection tasks can be readily performed. Availability and implementation: GRASP is implemented as an open-source package in the MATLAB environment. The software runs in Windows, macOS and Linux, is documented (graspk.readthedocs.io) and unit-tested. GRASP is freely available at github.com/biosustain/GRASP. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Screening of COVID-19 cases through a Bayesian network symptoms model and psychophysical olfactory test.

The sudden loss of smell is among the earliest and most prevalent symptoms of COVID-19 when measured with a clinical psychophysical test. Research has shown the potential impact of frequent screening for olfactory dysfunction, but existing tests are expensive and time consuming. We developed a low-cost ($0.50/test) rapid psychophysical olfactory test (KOR) for frequent testing and a model-based COVID-19 screening framework using a Bayes Network symptoms model. We trained and validated the model on two samples: suspected COVID-19 cases in five healthcare centers (n = 926; 33% prevalence, 309 RT-PCR confirmed) and healthy miners (n = 1,365; 1.1% prevalence, 15 RT-PCR confirmed). The model predicted COVID-19 status with 76% and 96% accuracy in the healthcare and miners samples, respectively (healthcare: AUC = 0.79 [0.75-0.82], sensitivity: 59%, specificity: 87%; miners: AUC = 0.71 [0.63-0.79], sensitivity: 40%, specificity: 97%, at 0.50 infection probability threshold). Our results highlight the potential for low-cost, frequent, accessible, routine COVID-19 testing to support society's reopening.

Inferring composition and function of the human gut microbiome in time and space: A review of genome-scale metabolic modelling tools.

The human gut hosts a complex community of microorganisms that directly influences gastrointestinal physiology, playing a central role in human health. Because of its importance, the metabolic interplay between the gut microbiome and host metabolism has gained special interest. While there has been great progress in the field driven by metagenomics and experimental studies, the mechanisms underpinning microbial composition and interactions in the microbiome remain poorly understood. Genome-scale metabolic models are mathematical structures capable of describing the metabolic potential of microbial cells. They are thus suitable tools for probing the metabolic properties of microbial communities. In this review, we discuss the most recent and relevant genome-scale metabolic modelling tools for inferring the composition, interactions, and ultimately, biological function of the constituent species of a microbial community with special emphasis in the gut microbiota. Particular attention is given to constraint-based metabolic modelling methods as well as hybrid agent-based methods for capturing the interactions and behavior of the community in time and space. Finally, we discuss the challenges hindering comprehensive modelling of complex microbial communities and its application for the in-silico design of microbial consortia with therapeutic functions.

Estrategias de evaluación funcional en deportistas / Functional evaluation strategies in athletes

El deporte se ha asociado a las posibles apariciones de lesiones ya sea durante entrenamientos como en competencia; pero, en términos de intensidad en los deportes de alto rendimiento se hace más probable la aparición de las mismas. Teniendo en cuenta lo anterior, una lesión deportiva, es el daño causado por el ejercicio físico, a la estructura, ya sea del músculo, hueso o tejido blando provocando molestia, sensación de dolor y como consecuencia un deceso del rendimiento deportivo para evitar que esto suceda es importante la valoración y monitoreo constante de sus cualidades físicas por medio de los test o baterías de específicas en este caso de evaluación funcional, las cuales evalúan la calidad de los movimientos específicos de cada deportista y disciplina, el reingreso al deporte y como alguno autores lo refieren la predicción de lesiones musculares. Objetivo: identificar los test o escalas usadas actualmente para la evaluación funcional en los deportistas. Materiales y métodos: se realizó una revisión documental de la literatura existente en el período 2010-2020 de acuerdo con los criterios de inclusión y exclusión propuestos. Resultados y conclusiones: los resultados nos indica que en el ámbito de la evaluación funcional al deportista existe poca evidencia científica en ciertos test por lo cual se necesita incursionar y plantear la posibilidad de crear nuevas baterías de evaluación específicas para cada disciplina deportiva, con el fin de complementar las funciones del fisioterapeuta desde aspectos de prevención de lesiones deportivas, potenciación y rehabilitación deportiva.

Engineering Saccharomyces cerevisiae for the Overproduction of ß-Ionone and Its Precursor ß-Carotene.

ß-ionone is a commercially attractive industrial fragrance produced naturally from the cleavage of the pigment ß-carotene in plants. While the production of this ionone is typically performed using chemical synthesis, environmentally friendly and consumer-oriented biotechnological production is gaining increasing attention. A convenient cell factory to address this demand is the yeast Saccharomyces cerevisiae. However, current ß-ionone titers and yields are insufficient for commercial bioproduction. In this work, we optimized S. cerevisiae for the accumulation of high amounts of ß-carotene and its subsequent conversion to ß-ionone. For this task, we integrated systematically the heterologous carotenogenic genes (CrtE, CrtYB and CrtI) from Xanthophyllomyces dendrorhous using markerless genome editing CRISPR/Cas9 technology; and evaluated the transcriptional unit architecture (bidirectional or tandem), integration site, and impact of gene dosage, first on ß-carotene accumulation, and later, on ß-ionone production. A single-copy insertion of the carotenogenic genes in high expression loci of the wild-type yeast CEN.Pk2 strain yielded 4 mg/gDCW of total carotenoids, regardless of the transcriptional unit architecture employed. Subsequent fine-tuning of the carotenogenic gene expression enabled reaching 16 mg/gDCW of total carotenoids, which was further increased to 32 mg/gDCW by alleviating the known pathway bottleneck catalyzed by the hydroxymethylglutaryl-CoA reductase (HMGR1). The latter yield represents the highest total carotenoid concentration reported to date in S. cerevisiae for a constitutive expression system. For ß-ionone synthesis, single and multiple copies of the carotene cleavage dioxygenase 1 (CCD1) gene from Petunia hybrida (PhCCD1) fused with a membrane destination peptide were expressed in the highest ß-carotene-producing strains, reaching up to 33 mg/L of ß-ionone in the culture medium after 72-h cultivation in shake flasks. Finally, interrogation of a contextualized genome-scale metabolic model of the producer strains pointed to PhCCD1 unspecific cleavage activity as a potentially limiting factor reducing ß-ionone production. Overall, the results of this work constitute a step toward the industrial production of this ionone and, more broadly, they demonstrate that biotechnological production of apocarotenoids is technically feasible.

Factors Associated with Cardiovascular Risk in High School Students of a Public School in the City of Santiago de Cali, Colombia / Factores asociados a riesgo cardiovascular en estudiantes de bachillerato de un colégio público de la ciudad de Santiago de Cali, Colombia / Fatores associados a risco cardiovascular em estudantes de educação secundária de uma escola pública da cidade de Santiago de Cali, Colômbia

Abstract Introduction: Cardiovascular diseases are the leading cause of death worldwide, and they have become a problem of interest of public health, both for developed and developing countries. In this regard, the decrease in time devoted to physical activity and the increase of sedentary activities stand out, thus making physical inactivity one of the many risk factors for Cardiovascular diseases. The objective was to determine the factors associated with cardiovascular risk in students of an educational institution in the city of Cali, Colombia. Materials and Methods: It is an observational and analytical epidemiological study with a sample of 227 randomly selected students, with 95 % reliability. To determine the level of physical activity in schoolchildren, researchers applied the international IPAQ physical activity questionnaire, and took anthropometric measures such as weight, height, and waist and hip circumference. Results: 96 % of the participants presented low cardiovascular risk. Likewise, the variables that showed a statistically significant association with cardiovascular risk were alcohol consumption and body mass index, which predict 81 % of the possibility of being at risk. Conclusions: The low cardiovascular risk and the high level of physical activity prevailed in the evaluated schoolchildren, significant differences regarding gender were observed, finding that men have higher values in weight, height, and waist/hip ratio compared to females. Finally, it was established that the factors associated with cardiovascular risk are body mass index and alcohol consumption.

Resumen Introducción: las enfermedades cardiovasculares son la principal causa de muerte en todo el mundo, lo que las convierte en un problema de interés en salud pública, tanto para países desarrollados, como en vías de desarrollo. La disminución del tiempo dedicado a la actividad física y el aumento de las actividades sedentarias son parte de los muchos factores de riesgo para las enfermedades cardiovasculares. El objetivo fue determinar los factores asociados al riesgo cardiovascular en los estudiantes de una institución educativa de la ciudad de Cali, Colombia. Materialesy métodos: estudio epidemiológico observacional y analítico, con una muestra de 227 estudiantes seleccionados de forma aleatoria, con una confiabilidad del 95 %. Para determinar el nivel de actividad física en los escolares, se aplicó el cuestionario internacional de actividad física IPAQ; igualmente, se tomaron medidas antropométricas como peso, talla, perímetro de cintura y cadera. Resultados: el 96 % de los participantes presentaron riesgo cardiovascular bajo. Así mismo, las variables que mostraron una asociación estadísticamente significativa con el riesgo cardiovascular fueron el consumo de alcohol y el índice de masa corporal, los cuales predicen en un 81 % la posibilidad de tener riesgo. Conclusiones: prevaleció el bajo riesgo cardiovascular y el alto nivel de actividad física en los escolares evaluados, con diferencias significativas respecto al sexo. Los hombres presentan valores más altos en peso, talla y relación cintura/cadera en comparación con las mujeres. Se estableció que los factores asociados con el riesgo cardiovascular son el índice de masa corporal y el consumo de alcohol.

Resumo Introdução: as doenças cardiovasculares são a principal causa de morte no mundo, convertendo-se em um problema de interesse em saúde pública, tanto para países desenvolvidos quanto em vias de desenvolvimento. Neste sentido, ressalta-se a diminuição de tempo dedicado à atividade física e o aumento das atividades sedentárias, convertendo a inatividade física em um dos muitos fatores de risco para as doenças cardiovasculares. O objetivo consistiu em determinar os fatores associados a risco cardiovascular nos estudantes de uma instituição educativa da cidade de Cali, Colômbia. Materiais e métodos: estudo epidemiológico observacional e analítico com uma amostra de 227 estudantes selecionados de maneira aleatória com uma confiabilidade do 95 %. Para determinar o nível de atividade física nos escolares, aplicou-se o questionário internacional de atividade física IPAQ; igualmente tomaram-se medidas antropométricas como peso, estatura, perímetro de cintura e quadril. Resultados: o 96 % dos participantes apresentaram risco cardiovascular baixo. Igualmente, as variáveis que mostraram associação estatisticamente significativa com o risco cardiovascular foram o consumo de álcool e o índice de massa corporal, os quais predizem em um 81 % a possibilidade de ter risco. Conclusões: prevaleceu o baixo risco cardiovascular e o alto nível de atividade física nos escolares avaliados, observando diferenças significativas com respeito ao sexo, encontrando que os homens apresentam valores maiores em peso, estatura e relação cintura/quadril em comparação com as mulheres, estabelecendo finalmente que os fatores associados com o risco cardiovascular são índice de massa corporal e o consumo de álcool.

Genomic integration of unclonable gene expression cassettes in Saccharomyces cerevisiae using rapid cloning-free workflows.

Most DNA assembly methods require bacterial amplification steps, which restrict its application to genes that can be cloned in the bacterial host without significant toxic effects. However, genes that cannot be cloned in bacteria do not necessarily exert toxic effects on the final host. In order to tackle this issue, we adapted two DNA assembly workflows for rapid, cloning-free construction and genomic integration of expression cassettes in Saccharomyces cerevisiae. One method is based on a modified Gibson assembly, while the other relies on a direct assembly and integration of linear PCR products by yeast homologous recombination. The methods require few simple experimental steps, and their performance was evaluated for the assembly and integration of unclonable zeaxanthin epoxidase expression cassettes in yeast. Results showed that up to 95% integration efficiency can be reached with minimal experimental effort. The presented workflows can be employed as rapid gene integration tools for yeast, especially tailored for integrating unclonable genes.

Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris.

Pichia pastoris is recognized as a biotechnological workhorse for recombinant protein expression. The metabolic performance of this microorganism depends on genetic makeup and culture conditions, amongst which the specific growth rate and oxygenation level are critical. Despite their importance, only their individual effects have been assessed so far, and thus their combined effects and metabolic consequences still remain to be elucidated. In this work, we present a comprehensive framework for revealing high-order (i.e., individual and combined) metabolic effects of the above parameters in glucose-limited continuous cultures of P. pastoris, using thaumatin production as a case study. Specifically, we employed a rational experimental design to calculate statistically significant metabolic effects from multiple chemostat data, which were later contextualized using a refined and highly predictive genome-scale metabolic model of this yeast under the simulated conditions. Our results revealed a negative effect of the oxygenation on the specific product formation rate (thaumatin), and a positive effect on the biomass yield. Notably, we identified a novel positive combined effect of both the specific growth rate and oxygenation level on the specific product formation rate. Finally, model predictions indicated an opposite relationship between the oxygenation level and the growth-associated maintenance energy (GAME) requirement, suggesting a linear GAME decrease of 0.56 mmol ATP/gDCW per each 1% increase in oxygenation level, which translated into a 44% higher metabolic cost under low oxygenation compared to high oxygenation. Overall, this work provides a systematic framework for mapping high-order metabolic effects of different culture parameters on the performance of a microbial cell factory. Particularly in this case, it provided valuable insights about optimal operational conditions for protein production in P. pastoris.

Build Your Bioprocess on a Solid Strain-ß-Carotene Production in Recombinant Saccharomyces cerevisiae.

Robust fermentation performance of microbial cell factories is critical for successful scaling of a biotechnological process. From shake flask cultivations to industrial-scale bioreactors, consistent strain behavior is fundamental to achieve the production targets. To assert the importance of this feature, we evaluated the impact of the yeast strain design and construction method on process scalability -from shake flasks to bench-scale fed-batch fermentations- using two recombinant Saccharomyces cerevisiae strains capable of producing ß-carotene; SM14 and ßcar1.2 strains. SM14 strain, obtained previously from adaptive evolution experiments, was capable to accumulate up to 21 mg/gDCW of ß-carotene in 72 h shake flask cultures; while the ßcar1.2, constructed by overexpression of carotenogenic genes, only accumulated 5.8 mg/gDCW of carotene. Surprisingly, fed-batch cultivation of these strains in 1L bioreactors resulted in opposite performances. ßcar1.2 strain reached much higher biomass and ß-carotene productivities (1.57 g/L/h and 10.9 mg/L/h, respectively) than SM14 strain (0.48 g/L/h and 3.1 mg/L/h, respectively). Final ß-carotene titers were 210 and 750 mg/L after 80 h cultivation for SM14 and ßcar1.2 strains, respectively. Our results indicate that these substantial differences in fermentation parameters are mainly a consequence of the exacerbated Crabtree effect of the SM14 strain. We also found that the strategy used to integrate the carotenogenic genes into the chromosomes affected the genetic stability of strains, although the impact was significantly minor. Overall, our results indicate that shake flasks fermentation parameters are poor predictors of the fermentation performance under industrial-like conditions, and that appropriate construction designs and performance tests must be conducted to properly assess the scalability of the strain and the bioprocess.

Síndrome metabólico en conductores de transporte intermunicipal de Tunja, Boyacá / Metabolic Syndrome in Intermunicipal Transportation Drivers Tunja, Boyacá / Síndrome metabólico em condutores de transporte intermunicipal Tunja, Boyacá

Resumen Introducción: el Síndrome Metabólico (SM) se considera una epidemia mundial, el aumento global de su prevalencia se ha extendido en los países tanto industrializados como en vía de desarrollo, es el resultado de una mayor proporción de obesidad y sedentarismo en la población. El objetivo de esta investigación fue determinar la prevalencia de SM en conductores de transporte intermunicipal de Tunja en el año 2017. Materiales y métodos: se realizó un estudio analítico de corte transversal a una muestra de 115 conductores de transporte intermunicipal en la ciudad de Tunja, se evaluaron índices antropométricos, glicemia, triglicéridos, HDL y se aplicó el cuestionario internacional de actividad física IPAQ. La presencia de SM se identificó de acuerdo con los criterios de NCEP/ATP III, se calculó la razón de prevalências y se determinó significancia mediante la prueba exacta de Fisher y U de Mann- Whitney. Resultados: se encontró una prevalencia de SM del 10.4 % (12/115; IC 95 % 4.6-15.7) y se hallaron diferencias significativas entre los pacientes con y sin SM en relación con la hipertensión (p = 0.03), niveles de glucosa (p = 0.0004), triglicéridos (p = <0.001), HDL (p = 0.00004), perímetro abdominal (p = 0.008) e índice de masa corporal (p = 0.001). Conclusión: en esta población de conductores, la hipertrigliceridemia fue el criterio más frecuente entre los que tenían síndrome metabólico, y se observó una tendencia hacia el sobrepeso y la obesidad, que determina la importancia de la generación de programas de información, educación y comunicación dirigidos a estas poblaciones que promuevan la alimentación saludable y la actividad física.

Abstract Introduction: Metabolic Syndrome (SM) is considered a global epidemic. The increase of its prevalence is widely extended in both industrialized and developing countries and is the outcome of a rise in the proportion of obesity and sedentary lifestyle in the population. The objective of this research was to determine the prevalence of SM and associated factors in transport drivers of Tunja in the year 2017. Materials and Methods: An analytical cross-sectional study was carried out on a sample of 115 inter-municipal transportation drivers in the city of Tunja, evaluating anthropometric indexes, glycemia, triglycerides, HDL and the IPAQ international physical activity questionnaire was applied. The presence of SM evaluatio followed the NCEP/ATP III criteria, the prevalence ratio was found, and Fisher's exact test and Mann-Whitney U test were used to determine the significance. Results: A prevalence of 10.4% (12/115; IC 95% 4.6-15.7) was found with significant differences between the patients with and without SM in relation with hypertension (p = 0.03), glucose levels (p = 0.0004), triglycerides (p < 0.001), HDL (p = 0.00004), abdominal perimeter (p = 0.008) and body mass index (p = 0.001). Conclusion: In this population of drivers, hypertriglyceridemia was the altogether criteria among those with metabolic syndrome as well as an observed trend towards overweight and obesity, which determines the importance of generating information, education and communication programs aimed at these populations to promote healthy eating and physical activity.

Resumo Introdução: a Síndrome Metabólica (SM) considera-se uma epidemia mundial, o aumento global de sua prevalência se tem estendido nos países tanto industrializados quanto em via de desenvolvimento, é o resultado de uma maior proporção de obesidade e sedentarismo na população. O objetivo desta pesquisa foi determinar a prevalências da SM em motoristas de transporte intermunicipal de Tunja no ano 2017. Materiais e métodos: se realizou um estudo analítico de corte transversal a uma amostra de 115 motoristas de transporte intermunicipal da cidade de Tunja; avaliaram-se índices antropométricos, glicemia, triglicérides, HDL e aplicou-se o questionário internacional de atividade física IPAO. A presença de SM se identificou de acordo aos critérios de NCEP/ATP III, calculara-se a razão de prevalências e determinou-se significância mediante a prova exata de Fisher e U de Mann - Whitney. Resultados: encontrou-se uma prevalência de SM do 10.4% (12/115; IC 95 % 4.6-15.7), achando diferenças significativas entre os pacientes com e sem SM em relação com a hipertensão (p = 0.03), níveis de glucose (p= 0.0004), triglicérides (p = <0.001), HDL (p = 0.00004), perímetro abdominal (p = 0.008) e índice de massa corporal (p = 0.001). Conclusão: nesta população de condutores de hipertrigliceridemia foi o critério mais frequente entre os que tinham síndrome metabólica, e observou-se uma tendência ao sobrepeso e a obesidade, que determina a importância da geração de programas de informação, educação e comunicação dirigidos a estas populações que promovam a alimentação saudável e a atividade física.

Expanding Metabolic Capabilities Using Novel Pathway Designs: Computational Tools and Case Studies.

Design and selection of efficient metabolic pathways is critical for the success of metabolic engineering endeavors. Convenient pathways should not only produce the target metabolite in high yields but also are required to be thermodynamically feasible under production conditions, and to prefer efficient enzymes. To support the design and selection of such pathways, different computational approaches have been proposed for exploring the feasible pathway space under many of the above constraints. In this review, an overview of recent constraint-based optimization frameworks for metabolic pathway prediction, as well as relevant pathway engineering case studies that highlight the importance of rational metabolic designs is presented. Despite the availability and suitability of in silico design tools for metabolic pathway engineering, scarce-although increasing-application of computational outcomes is found. Finally, challenges and limitations hindering the broad adoption and successful application of these tools in metabolic engineering projects are discussed.

From reconstruction to C4 metabolic engineering: A case study for overproduction of polyhydroxybutyrate in bioenergy grasses.

The compartmentalization of C4 plants increases photosynthetic efficiency, while constraining how material and energy must flow in leaf tissues. To capture this metabolic phenomenon, a generic plant metabolic reconstruction was replicated into four connected spatiotemporal compartments, namely bundle sheath (B) and mesophyll (M) across the day and night cycle. The C4 leaf model was used to explore how amenable polyhydroxybutyrate (PHB) production is with these four compartments working cooperatively. A strategic pattern of metabolite conversion and exchange emerged from a systems-level network that has very few constraints imposed; mainly the sequential two-step carbon capture in mesophyll, then bundle sheath and photosynthesis during the day only. The building of starch reserves during the day and their mobilization during the night connects day and night metabolism. Flux simulations revealed that PHB production did not require rerouting of metabolic pathways beyond what is already utilised for growth. PHB yield was sensitive to photoassimilation capacity, availability of carbon reserves, ATP maintenance, relative photosynthetic activity of B and M, and type of metabolites exchanged in the plasmodesmata, but not sensitive towards compartmentalization. Hence, the compartmentalization issues currently encountered are likely to be kinetic or thermodynamic limitations rather than stoichiometric.