Highlights First test in which whey and E. coli are used to obtain L-valine. Kinetic properties and kinetic models of L-valine production were studied. Highest L-valine production was obtained when whey was used as substrate. Highest specific growth rate was achieved when using deproteinized whey.

Replacing traditional substrates in industrial bioprocesses to advance the sustainable production of chemicals is an urgent need in the context of the circular economy. However, since the limited degradability of non-conventional carbon sources often returns lower yields, effective exploitation of such substrates requires a multi-layer optimization which includes not only the provision of a suitable feedstock but the use of highly robust and metabolically versatile microbial biocatalysts. We tackled this challenge by means of systems metabolic engineering and validated Escherichia coli W as a promising cell factory for the production of the key building block chemical 2-ketoisovalerate (2-KIV) using whey as carbon source, a widely available and low-cost agro-industrial waste. First, we assessed the growth performance of Escherichia coli W on mono and disaccharides and demonstrated that using whey as carbon source enhances it significantly. Second, we searched the available literature and used metabolic modeling approaches to scrutinize the metabolic space of E. coli and explore its potential for overproduction of 2-KIV identifying as basic strategies the block of pyruvate depletion and the modulation of NAD/NADP ratio. We then used our model predictions to construct a suitable microbial chassis capable of overproducing 2-KIV with minimal genetic perturbations, i.e., deleting the pyruvate dehydrogenase and malate dehydrogenase. Finally, we used modular cloning to construct a synthetic 2-KIV pathway that was not sensitive to negative feedback, which effectively resulted in a rerouting of pyruvate towards 2-KIV. The resulting strain shows titers of up to 3.22 ± 0.07 g/L of 2-KIV and 1.40 ± 0.04 g/L of L-valine in 24 h using whey in batch cultures. Additionally, we obtained yields of up to 0.81 g 2-KIV/g substrate. The optimal microbial chassis we present here has minimal genetic modifications and is free of nutritional autotrophies to deliver high 2-KIV production rates using whey as a non-conventional substrate.

El metabolismo representa el nivel biológico que más se relaciona con los fenotipos de la célula y, las alteraciones o reprogramaciones de éste pueden, entre otras, (i) afectar la producción de metabolitos primarios o secundarios en microorganismos de interés biotecnológico, (ii) favorecer o no la inhibición del crecimiento en organismos patógenos y (iii) desarrollar desórdenes metabólicos como la obesidad o la diabetes. Es por ello, que el estudio del metabolismo, el rediseño, y el redireccionamiento de fluxes metabólicos se ha convertido en un área importante de investigación (también conocida como Ingeniería Metabólica), ya que ha permitido el desarrollo y diseño de procesos biológicos mejorados, la identificación de blancos terapéuticos, el diseño de estrategias terapéuticas para curar desordenes metabólicos y la identificación de biomarcadores en cáncer, entre otros. Actualmente, se han desarrollado metodologías computacionales que permiten estudiar el metabolismo celular a diferentes condiciones medioambientales, dirigiendo la experimentación con las predicciones del modelo. El propósito de esta revisión es resaltar la importancia del análisis de fluxes metabólicos como una metodología general para estudiar la reprogramación metabólica en distintos organismos de interés biotecnológico, médico, y terapéutico. Este trabajo condensa las bases teóricas y los conceptos claves para entender el análisis de fluxes metabólicos, lo cual será un insumo fundamental para aquellos que se están adentrando al mundo de la biología de sistemas o áreas afines.