DetSpace: a web server for engineering detectable pathways for bio-based chemical production.

Tackling climate change challenges requires replacing current chemical industrial processes through the rational and sustainable use of biodiversity resources. To that end, production routes to key bio-based chemicals for the bioeconomy have been identified. However, their production still remains inefficient in terms of titers, rates, and yields; because of the hurdles found when scaling up. In order to make production more efficient, strategies like automated screening and dynamic pathway regulation through biosensors have been applied as part of strain optimization. However, to date, no systematic way exists to design a genetic circuit that is responsive to concentrations of a given target compound. Here, the DetSpace web server provides a set of integrated tools that allows a user to select and design a biological circuit that performs the sensing of a molecule of interest by its enzymatic conversion to a detectable molecule through a transcription factor. In that way, the DetSpace web server allows synthetic biologists to easily design biosensing routes for the dynamic regulation of metabolic pathways in applications ranging from genetic circuits design, screening, production, and bioremediation of bio-based chemicals, to diagnostics and drug delivery.

Computer-aided design and implementation of efficient biosynthetic pathways to produce high added-value products derived from tyrosine in Escherichia coli.

Developing efficient bioprocesses requires selecting the best biosynthetic pathways, which can be challenging and time-consuming due to the vast amount of data available in databases and literature. The extension of the shikimate pathway for the biosynthesis of commercially attractive molecules often involves promiscuous enzymes or lacks well-established routes. To address these challenges, we developed a computational workflow integrating enumeration/retrosynthesis algorithms, a toolbox for pathway analysis, enzyme selection tools, and a gene discovery pipeline, supported by manual curation and literature review. Our focus has been on implementing biosynthetic pathways for tyrosine-derived compounds, specifically L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine, with significant applications in health and nutrition. We selected one pathway to produce L-DOPA and two different pathways for dopamine-one already described in the literature and a novel pathway. Our goal was either to identify the most suitable gene candidates for expression in Escherichia coli for the known pathways or to discover innovative pathways. Although not all implemented pathways resulted in the accumulation of target compounds, in our shake-flask experiments we achieved a maximum L-DOPA titer of 0.71 g/L and dopamine titers of 0.29 and 0.21 g/L for known and novel pathways, respectively. In the case of L-DOPA, we utilized, for the first time, a mutant version of tyrosinase from Ralstonia solanacearum. Production of dopamine via the known biosynthesis route was accomplished by coupling the L-DOPA pathway with the expression of DOPA decarboxylase from Pseudomonas putida, resulting in a unique biosynthetic pathway never reported in literature before. In the context of the novel pathway, dopamine was produced using tyramine as the intermediate compound. To achieve this, tyrosine was initially converted into tyramine by expressing TDC from Levilactobacillus brevis, which, in turn, was converted into dopamine through the action of the enzyme encoded by ppoMP from Mucuna pruriens. This marks the first time that an alternative biosynthetic pathway for dopamine has been validated in microbes. These findings underscore the effectiveness of our computational workflow in facilitating pathway enumeration and selection, offering the potential to uncover novel biosynthetic routes, thus paving the way for other target compounds of biotechnological interest.

Reconstrucción biológica de grandes defectos óseos con autoinjerto de peroné vascularizado en huesos largos / Biological reconstruction of large bone defects with vascularized fibular autograft

Introducción: El autoinjerto vascular de peroné se presenta como una muy buena opción en la reconstrucción de grandes defectos óseos en huesos largos gracias a sus características estructurales y propiedades biológicas. Materiales y Métodos: Se realizó un estudio observacional descriptivo y retrospectivo que incluyó a todos los pacientes operados con un injerto vascular de peroné aislado o asociado a injerto estructural (técnica de Capanna) desde el 1 de enero de 2014 hasta el 1 de enero de 2021 en nuestro hospital. Resultados: Se realizaron 26 cirugías mediante un injerto vascular de peroné; en 8 de ellas, se utilizó el colgajo vascularizado de peroné para la reconstrucción del defecto óseo en hueso largo. El tamaño medio del defecto era de 7,7 cm. El origen del defecto era postraumático en 5 casos y tumoral en el resto. Se consiguió la consolidación completa en todos los pacientes. Los resultados clínicos y funcionales en las escalas de valoración fueron mejores en pacientes operados en el miembro inferior. Conclusiones: El uso de un colgajo vascularizado de peroné asociado o no a aloinjerto estructural es una estrategia útil en la reconstrucción de grandes defectos óseos (≥5 cm), independientemente de la causa de la lesión; la supervivencia del injerto y la función son buenas, con una tasa de complicaciones aceptable. Nivel de Evidencia: IV

Background: Given its biological and structural qualities, vascular fibular autograft is a good option for the reconstruction of large defects in long bones. Materials and Methods: A descriptive and retrospective observational study was conducted. We included all cases of patients who underwent surgery in our hospital between January 1, 2014, and January 1, 2021, and who had a vascular fibula autograft either standalone or in combination with a structural graft (Capanna technique). Results:There were 26 documented vascular fibula autograft procedures. Eight of the procedures involved the reconstruction of a long bone defect. The bone defect was an average of 7.7 cm in length. In five of the cases, the origin of the bone defect was post-traumatic, and in the remaining cases, it was tumoral. In all cases, complete consolidation was achieved. Surgical procedures performed on the lower extremities yielded better clinical and functional outcomes. Conclusions:Vascular fibula autograft either on its own or in combination with a structural graft, as described in the Capanna technique, is an excellent alternative for the reconstruction of bone defects ≥ 5 cm. Radiological, clinical and functional outcomes are good, with an acceptable rate of complications. Level of Evidence: IV

Nueva mutación fundadora en MYBPC3: comparación fenotípica con la mutación de MYBPC3 más frecuente en España / A novel founder mutation in MYBPC3: Phenotypic comparison with the most prevalent MYBPC3 mutation in Spain

Introducción y objetivos: Las mutaciones en MYBPC3 son causa de miocardiopatía hipertrófica (MCH). A pesar de que la mayoría de ellas producen una proteína truncada, la gravedad del fenotipo es diversa. Se describe el fenotipo clínico de una nueva mutación en MYBPC3, p.Pro108Alafs*9, presente en 13 familias del sur de España, y se compara con la mutación de MYBPC3 con mayor prevalencia en dicha región (c.2308 + 1 G > A). Métodos: Se estudió a 107 familiares de 13 casos índice que tenían diagnóstico de MCH y portaban la mutación p.Pro108Alafs*9. Se realizó un análisis del árbol genealógico, junto con una evaluación clínica y determinación del genotipo. Resultados: Se identificó en total a 54 portadores de la mutación p.Pro108Alafs*9, de los que 39 tenían MCH. Hubo 5 casos de muerte súbita en las 13 familias. La penetrancia de la enfermedad aumentaba a medida que se incrementaba la edad, y los pacientes con MCH fueron con más frecuencia varones, y estos contrajeron la enfermedad más precozmente que las mujeres. El fenotipo fue similar en la p.Pro108Alafs*9 y la c.2308 + 1 G > A, pero se observaron diferencias en varios factores de riesgo y en la supervivencia. Hubo tendencia a mayor masa ventricular izquierda en la p.Pro108Alafs*9 que en la c.2308 + 1G > A. La resonancia magnética cardiaca reveló una extensión y un patrón de fibrosis similares en ambas. Conclusiones: La mutación p.Pro108Alafs*9 se asoció a MCH, alta penetrancia y aparición de la enfermedad a mediana edad (AU)

Introduction and objectives: Mutations in MYBPC3 are the cause of hypertrophic cardiomyopathy (HCM). Although most lead to a truncating protein, the severity of the phenotype differs. We describe the clinical phenotype of a novel MYBPC3 mutation, p.Pro108Alafs*9, present in 13 families from southern Spain and compare it with the most prevalent MYBPC3 mutation in this region (c.2308 + 1 G > A). Methods: We studied 107 relatives of 13 index cases diagnosed as HCM carriers of the p.Pro108Alafs*9 mutation. Pedigree analysis, clinical evaluation, and genotyping were performed. Results: A total of 54 carriers of p.Pro108Alafs*9 were identified, of whom 39 had HCM. There were 5 cases of sudden death in the 13 families. Disease penetrance was greater as age increased and HCM patients were more frequently male and developed disease earlier than female patients. The phenotype was similar in p.Pro108Alafs*9 and in c.2308 + 1 G > A, but differences were found in several risk factors and in survival. There was a trend toward a higher left ventricular mass in p.Pro108Alafs*9 vs c.2308 + 1G > A. Cardiac magnetic resonance revealed a similar extent and pattern of fibrosis. Conclusions: The p.Pro108Alafs*9 mutation is associated with HCM, high penetrance, and disease onset in middle age (AU)