1 1 2 A second chromosomal copy of the catA gene endows Pseudomonas putida mt - 2 3 with an enzymatic safety valve for excess of catechol 4 5 by 6 7

1 2 Pseudomonas putida mt-2 harbors two different routes for catabolism of catechol, namely one meta 3 pathway encoded by the xyl genes of the TOL plasmid pWW0 and one ortho pathway determined by the 4 chromosomal ben and cat genes. P. putida mt-2 has a second chromosomal copy of the catA gene 5 (named catA2) located downstream of the ben operon that encodes an additional catechol-1,26 dioxygenase. The metabolic and regulatory phenotypes of strains lacking one enzyme, the other and both 7 of them in cells with and without the TOL plasmid were evaluated. The data consistently indicated that 8 induction of the ortho pathway by benzoate plasmid-less strain P. putida KT2440 led to catechol surplus, 9 the toxicity of which at high concentrations being counteracted by CatA2. Cells carrying pWW0 but 10 lacking catA2 experienced both a rapid loss of the plasmid when grown on benzoate (a substrate of the 11 lower pathway) and a slowdown of their growth rate when cultured with benzylalcohol (a substrate 12 converted to benzoate by the upper pathway). These data reveal the role of CatA2 as a type of metabolic 13 safety valve for excess catechol that alleviates the metabolic conflict generated by simultaneous 14 expression of the meta and ortho pathways, thereby facilitating their co-existence. 15 ____________________________________________________________________________________ 16