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Debido a la abundancia y toxicidad del arsénico, muchos organismos han desarrollado diferentes estrategias de interacción con este metaloide, i.e., mecanismos de resistencia/detoxificación y mecanismos metabólicos de estado rédox que utilizan el arsénico como aceptor final o fuente de electrones1. La beta-proteobacteria Aromatoleum sp. CIB es un bacilo anaerobio facultativo capaz de interaccionar con la raíz de ciertas plantas (e.g., arroz), y de resistir concentraciones moderadas de arsénico. En este trabajo presentamos los resultados de un análisis genómico, transcriptómico y funcional de la bacteria CIB cultivada en presencia de arsénico, lo que nos ha permitido identificar el arsenoma de este microorganismo. Se ha caracterizado el cluster arsRCDAB relacionado con la resistencia a arsenito/arsenato y el cluster arx, responsable de la oxidación anaerobia de arsenito2. Además, el estudio genómico ha identificado los genes arsP, que codifica un presunto transportador de especies metiladas de arsénico, arsS, que codifica una enzima SAM-dependiente relacionada con la síntesis de arsenoazúcares3, y arsM, el cual se ha sobreexpresado y caracterizado como una SAM-metiltransferasa de arsenito que produce ácido dimetilarsínico (DMA(V)). Por otro lado, se ha caracterizado el regulador transcripcional ArsR que responde a arsenito como inductor y controla la expresión del operón arsRCDAB, si bien no parece estar implicado en la regulación de otros genes del arsenoma de la cepa CIB. Todo este nuevo conocimiento del arsenoma de CIB nos ha permitido definir el mapa de la respuesta molecular de Aromatoleum sp. CIB a la presencia de arsenito, explorar el uso de nuevas proteínas Ars para la construcción de biosensores específicos de arsenito, y plantear el uso de la cepa CIB como agente bioprotector de plantas de arroz en suelos contaminados con arsénico

Los ésteres de ftalatos (PAEs) son derivados esterificados del ácido o-ftálico (PA) y los plastificantes más utilizados en la actualidad. Dado que se unen de manera no covalente a los plásticos, difunden fácilmente al medio ambiente y representan uno de los contaminantes orgánicos emergentes más frecuentes debido a sus distintos efectos tóxicos para la vida. La degradación bacteriana de PAEs se presenta como una de las estrategias más prometedoras para su eliminación. Las rutas catabólicas de los PAEs comprenden un metabolismo periférico que transforma el PAE en PA (y el correspondiente alcohol) y metabolismo central para la mineralización del PA (1). En trabajos previos se caracterizó el metabolismo central (transporte y degradación del PA vía benzoil-CoA), y se demostró la posibilidad de reconducir el flujo de carbono hacia la producción de bioplásticos (polihidroxibutirato, PHB) (2). En este trabajo se aborda el metabolismo periférico de un PAE altamente utilizado, di-n-butilftalato (DBP), mediante el diseño de una ruta sintética que expresa esterasas específicas de DBP y su implantación en biocatalizadores optimizados para la conversión de PA en PHB. Con este fin, se han desarrollado cassettes recombinantes que expresan distintas esterasas con elevada actividad hidrolítica frente a distintos PAEs (3,4) y otras todavía sin caracterizar como EstB de Halomonas sp.ATBC28 (5). Ensayos bioquímicos de la enzima EstB revelan que se trata de una diesterasa muy activa capaz de generar mono-n-butil-ftalato (MBP) como producto final. La utilización de enzimas con doble actividad (di y mono-esterasa) o la combinación de EstB con monoesterasas que convierten MBP en PA son dos estrategias alternativas que se están analizando con el objetivo de diseñar la ruta periférica más eficiente y versátil (amplio rango de sustratos PAEs) posible para el desarrollo de biocatalizadores recombinantes que faciliten la conversión de PAEs en PHB.

The environmental challenge associated with conventional plastics has created a need for alternative solutions, placing bio-based polymers like polyhydroxyalkanoates (PHAs) in a promising position.These polyesters are produced under nutritional stress conditions by several microorganisms including the model bacterium Pseudomonas putida. Carbon sources for PHA synthesis can be classified into two categories: i) PHA related substrates, primarily fatty acids, which provide high production yields, and ii) non-PHA related substrates, such as sugars, whose conversion into PHA precursors through de novo fatty acid synthesis route has been associated with low production yields [1]. Significant efforts have been devoted for the use of carbohydrates as carbon sources within a circular economy framework. To address this challenge, systems biology approaches, like genomescale metabolic models, offer valuable insights into the metabolic features of PHA cycle in P. putida KT2440 [2]. These approaches enable the prediction of the key reaction to be deleted in order to redirect the metabolic routes of interest, and represent powerful tools for optimizing and enhancing the utilization of non-PHA related carbon sources in a sustainable manner. In order to construct an optimized PHA producer P. putida KT2440 strain when using sugars as feedstock, we employed the iJN1411 genome-scale metabolic model to predict those genes to be deleted for eliminating potential competing pathways and redirecting carbon flux from acetyl-CoA towards PHA biosynthesis [3], and those genes to be added for using sucrose as carbon source. We obtained the engineered MT9 strain which was tested to produce PHA from sucrose. Additionally, the synthesis of the antimicrobial biopolymer PHACOS was explored by co-feeding the chemical precursor 6-acetylthiohexanoic acid (6-ATH) and glucose or sucrose [4]. The results revealed that MT9 cultures grown with sucrose as carbon sources achieved a PHA accumulation of 35% per

An efficient and cheap energization of microbial biocatalysts is essential in current biotechnological processes. A promising alternative to the use of common organic or inorganic electron donors are the semiconductor nanoparticles (SNs) that absorb light and transfer electrons (photoelectrons) behaving as artificial photosynthetic systems (biohybrid systems). Excited photoelectrons generated by illuminated SNs are highly reductive and readily accepted by membrane-bound proteins and electron shuttles to drive specific cell reduction processes and energy generation in microbes. However, the operational mechanisms of these hybrid systems are still poorly understood, specially at the material-microbe interface, and therefore the design and production of efficient biohybrids is challenging. Some major limitations/challenges and future prospects of SNs as microbial energization systems are discussed.

We have identified and characterized the AccS multidomain sensor kinase that mediates the activation of the AccR master regulator involved in carbon catabolite repression (CCR) of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB. A truncated AccS protein that contains only the soluble C-terminal autokinase module (AccS′) accounts for the succinate-dependent CCR control. In vitro assays with purified AccS′ revealed its autophosphorylation, phosphotransfer from AccS′∼P to the Asp60 residue of AccR, and the phosphatase activity toward its phosphorylated response regulator, indicating that the equilibrium between the kinase and phosphatase activities of AccS′ may control the phosphorylation state of the AccR transcriptional regulator. Oxidized quinones, e.g., ubiquinone 0 and menadione, switched the AccS′ autokinase activity off, and three conserved Cys residues, which are not essential for catalysis, are involved in such inhibition. Thiol oxidation by quinones caused a change in the oligomeric state of the AccS′ dimer resulting in the formation of an inactive monomer. This thiol-based redox switch is tuned by the cellular energy state, which can change depending on the carbon source that the cells are using. This work expands the functional diversity of redox-sensitive sensor kinases, showing that they can control new bacterial processes such as CCR of the anaerobic catabolism of aromatic compounds. The AccSR two-component system is conserved in the genomes of some betaproteobacteria, where it might play a more general role in controlling the global metabolic state according to carbon availability.

Most of the current methods for controlling the formation rate of a key protein or enzyme in cell factories rely on the manipulation of target genes within the pathway. In this article, we present a novel synthetic system for post-translational regulation of protein levels, FENIX, which provides both independent control of the steady-state protein level and inducible accumulation of target proteins. The FENIX device is based on the constitutive, proteasome-dependent degradation of the target polypeptide by tagging with a short synthetic, hybrid NIa/SsrA amino acid sequence in the C-terminal domain. Protein production is triggered via addition of an orthogonal inducer ( i.e., 3-methylbenzoate) to the culture medium. The system was benchmarked in Escherichia coli by tagging two fluorescent proteins (GFP and mCherry), and further exploited to completely uncouple poly(3-hydroxybutyrate) (PHB) accumulation from bacterial growth. By tagging PhaA (3-ketoacyl-CoA thiolase, first step of the route), a dynamic metabolic switch at the acetyl-coenzyme A node was established in such a way that this metabolic precursor could be effectively redirected into PHB formation upon activation of the system. The engineered E. coli strain reached a very high specific rate of PHB accumulation (0.4 h-1) with a polymer content of ca. 72% (w/w) in glucose cultures in a growth-independent mode. Thus, FENIX enables dynamic control of metabolic fluxes in bacterial cell factories by establishing post-translational synthetic switches in the pathway of interest.