From fossil fuel-based plastics to bioplastics through model-driven and synthetic biology-assisted engineering of Pseudomonas putida KT2440

Abstract

1 p.Pseudomonas putida KT2440 is a model environmental bacterium in the production of medium-chain-length polyhydroxyalkanoates (PHAs). Its metabolism has been extensively characterized and a high-quality metabolic model is available (iJN1441), allowing for the systems evaluation of its metabolic capabilities. The use of plastic wastes, like polyethylene terephthalate (PET) and polyurethane (PU), has been proposed as a novel second-generation carbon source for biotechnology. In this work, by using the iJN1411 as a computational platform, we present a set of in silico and in vivo validated growth-coupled PHA overproducer P. putida strains, using monomers derived from the hydrolysis of these polymers, as carbon sources. Cutting edge synthetic biology approaches were used to generate the high number of gene knockouts and knockins required to systematically remove all potential competing pathways and to reroute the carbon flux towards PHA biosynthesis. The effect on PHA production of the iterative rounds of model-guided genetic modifications was subsequently evaluated by means of high-throughput physiological experiments.Peer reviewe