Cyclical Genome-streamlining of the Cell Factory Pseudomonas putida

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Brendan Ryback, Audrey Leprince, Mark van Passel, Vítor Martins dos Santos

France

Top-down approaches to functional genomics traditionally rely on the targeted deletion of individual genes. However, the necessity to tailor the recombination sequences of knock-out constructs to each target poses an obstacle for automation and high-throughput experimentation. In order to rapidly streamline the genome of the biotechnologically important gram-negative bacterium Pseudomonas putida, we developed a recyclable three-step deletion method capable of excising large genomic segments.

The method combines random insertions of selectable mini-Tn5 transposons with the site-specific Flp-FRT recombination system to generate successive random deletions in a single strain in which parts of the genome are excised via the action of the cognate flippase.  These mini-Tn5 transposons carry different selectable markers and each has an FRT (Flippase Recognition Target) site. Upon induction, both FRT sites are recognized by the flippase, resulting in the deletion of the intervening genomic segment along with the transposon backbones without the inheritance of any marker genes.

A cyclical application of the method generated four double-deletion mutants of which a maximum of ~ 7.4% of the chromosome (~ 6.9% of the gene count) was excised. Comparative Phenotype Microarray analysis between the deletion mutants and their ancestors analysis revealed an unexpected increase in respiratory capacity when utilizing a number of sugars and organic acids as carbon sources, and a decreased capacity to metabolize certain amino acids.

This procedure demonstrates a new approach to streamlining bacterial genomes and generating large libraries of deletion mutants. Combined with high-throughput ~omics analyses, the application of this method will lead to a better understanding of the metabolic and regulatory interactions relevant to design and optimization of heterologous metabolic pathways – and eventually result in a streamlined bacterial chassis for the sustainable production of chemicals.