Development of SynBio Tools for Pseudomonas chlororaphis: A Versatile Non-Pathogenic Bacterium Host

Abstract

Pseudomonas chlororaphis ATCC 9446 is a non-pathogenic bacterium associated with the rhizosphere. It is commonly used as a biocontrol agent against agricultural pests. This organism can grow on a variety of carbon sources, has a robust secondary metabolism, and produces secondary metabolites with antimicrobial properties. This makes it an alternative host organism for synthetic biology applications. However, as a novel host there is a need for well-characterized molecular tools that allow fine control of gene expression and exploration of its metabolic potential. In this work we developed and characterized expression vectors for P. chlororaphis. We used two different promoters: the exogenously induced lac-IPTG promoter, and LuxR-C6-AHL, which we evaluated for its auto-inducible capacities, as well as using an external addition of C6-AHL. The expression response of these vectors to the inducer concentration was characterized by detecting a reporter fluorescent protein (YFP: yellow fluorescent protein). Furthermore, the violacein production operon was evaluated as a model heterologous pathway. We tested violacein production in shake flasks and a 3 L fermenter, showing that P. chlororaphis possesses a vigorous aromatic amino acid metabolism and was able to produce 1 g/L of violacein in a simple batch reactor experiment with minimal medium using only glucose as the carbon source. We compared the experimental results with the predictions of a modified genome scale model. The presented results show the potential of P. chlororaphis as a novel host organism for synthetic biology applications.