Improved genetic tools for rapid engineering of Pseudomonas putida

Pseudomonas putida strains are highly robust bacteria known for their ability to efficiently utilize a variety of carbon sources, including aliphatic and aromatic compounds. Recently, P. putida has been engineered to valorize the lignin stream of a lignocellulosic biomass pretreatment process. Compared to platform organisms such as Escherichia coli, the toolkit for engineering P. putida is underdeveloped, hindering metabolic engineering and synthetic biology applications. Replicative plasmids are the state of the art for rapid prototyping in P. putida, but problems with plasmid instability and copy number variance provide additional challenges. Integration of synthetic constructs into the genome would address these problems. Therefore, we are developing systems to more rapidly integrate constructs into the genome of P. putida. Most engineering efforts in P. putida to date combine gene deletions with constitutive overexpression of exogenous pathways. Work in other organisms such as E. coli has demonstrated the importance of tuning the expression of synthetic pathways to balance flux, decrease metabolic burden and increase host fitness. To improve the P. putida toolkit, we utilized an integration system with a fluorescent reporter platform, mNeonGreen, to rapidly assay the strength of a library of synthetic promoters. This practical test of the integration system resulted in the identification of promoters with a wide variety of expression levels, which can be used to fine tune expression of exogenous pathways. Furthermore, the genome integration and reporter systems are extensible to assay for terminator strength or rapidly integrate exogenous pathways for testing under a variety of expression levels.