(450d) Development of a Synthetic Biology Toolbox for Soil Bacteria

Soil microbes have the potential to greatly increase food yield and improve disease resistance in relevant crops; however, the ability to manipulate these non-model organisms is currently limited. In this study, we have developed tools for genetic manipulation and heterologous gene expression in two species of interest: Pseudomonas putida and Azotobacter vinelandii. P. putida is a common soil microorganism previously shown to associate with crops such as maize, whereas A. vinelandii is a robust nitrogen-fixing organism that has previously been engineered to supply additional ammonium to the soil. We characterized and optimized several plasmids, constitutive promoters, and different induction systems in Azotobacter vinelandii DJ and Pseudomonas putida KT2440. These characterized tools and induction systems were subsequently used to develop high-efficiency, λRed/CRISPR and I-SceI recombineering strategies for genetic modification of both microbes in order to create scarless knockouts and gene insertions.

We were able to identify stable plasmids for both organisms, characterizing their copy numbers using qPCR, as well as relative expression strength using a GFP reporter. Additionally, we identified reliable constitutive promoters for both P. putida and A. vinelandii, including induction systems that demonstrated approximately 100-fold induction for both organisms. We show that optimization of repressor systems from E. coli can greatly increase induction range. Additionally, the recombineering approaches highlighted here resulted in high efficiency knockouts in Pseudomonas (>90% efficiency of desired knockouts with optimized protocol), and genetic integrations were used to produce several heterologous natural products. Finally, we developed nitrogen-secretion strains of A. vinelandii using an I-SceI recombineering strategy. This study demonstrates synthetic biology tools for reliable gene expression in P. putida and A. vinelandii, two soil microbes which may have a role in developing robust synthetic communities for various crops in the future.