À la carte synthesis of polyhydroxyalkanoates using Pseudomonas putida: exploiting the possibilities within the synthetic biology

Abstract

Bio-based materials are a sustainable alternative for the substitution of oil derived plastics. Within this group we can highlight the polyhydroxyalkanoates (PHAs), biodegradable polymers with similar properties to conventional plastics. The physicochemical properties of the PHA can be tuneable and strongly depends on its hydroxyacids’ monomeric composition1. The synthesis of these PHAs is triggered in many bacteria by a complex regulatory network driven by many factors such as the nutritional imbalance, where the cells experience an excess of carbon coupled with a limitation of other nutrients such as nitrogen. In this study we focus on the model bacterium Pseudomonas putida KT2440 for the production of à la carte PHAs due to its versatile metabolism, resistance to stress, and that naturally produces medium-chain-length-PHA. In order to avoid the complexity of the native regulatory system in this chassis, the entire pha operon was deleted generating the P. putida ∆pha strain, named as PP05_01. Using the Golden Gate technology, a battery of synthetic operons was constructed bearing the minimum set of genes needed for tuneable PHA production in PP05_01. For this, we explored a range of enzymatic set scenarios from different microorganisms (i.e. Rhodospirillum rubrum, Cupriavidus necator, P. putida) producing a panel of PHA with different monomer composition. The most promising candidates were expressed in a multicopy system in PP05_01 strain chassis and the amount and composition of PHA synthesized from various substrates was determined. This study provides P. putida optimized cell factories for the production of tailored PHAs.