Development of genetic tools for large-scale deletions and streamlining of a Pseudomonas putida strain

In the last decades, the white biotechnology was highly developed and many bacteria were used for diverse purposes (e.g. production of chemical compounds). Among them the Pseudomonas putida species provided good natural candidates, for example for the bioremediation of polluted soils. Despite the fully sequenced genomes, several genes remain of uncharacterized function (code for hypothetical and conserved hypothetical proteins as well as for proteins of unknown functions) and create instability during the manipulation of the bacteria. Furthermore some genes with characterized functions are considered (experimentally proved or predicted in silico) as being non essential under specific laboratory conditions (e.g. certain redundant genes). In order to reduce the complexity and to render the genome more stable, the idea was developed to reduce the genome size by removing random fragments from the chromosome. The PhD work was initiated on the reduction of the 6.18 Mbp genome from P. putida strain TEC1 (uracil auxotroph from strain KT2440) under specific laboratory conditions. For this purpose a combination of random insertions, using two modified mini-Tn5 mini-transposons, and the Flp-FRT site specific recombination system (from the yeast Saccharomyces cerevisiae) was applied. After a first round of insertion and deletion/recombination events the genome of P. putida TEC1 was reduced by 4.1% of its size. The repetition of these events increased the first deletion and generated a double-deleted P. putida Δ2 mutant that lost 4.8% of its genome. The first genetic and metabolic analyses of the mutant confirmed the presence of in silico predicted essential genes but revealed also potential new branches of certain metabolic pathways. This study allowed the establishment of a combinatorial method for streamlining a P. putida strain, which offers new insights into the function of the genes and the metabolic pathways.