Generation of a catR deficient mutant of P. putida KT2440 that produces cis, cis-muconate from benzoate at high rate and yield.

Pseudomonas putida KT2440-JD1 was derived from P. putida KT2440 after N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-mutagenesis and exposure to 3-fluorobenzoate (3-FB). The mutant was no longer able to grow using benzoate as a sole carbon source, but co-metabolized benzoate to cis, cis-muconate during growth on glucose, which accumulated in the growth medium. The specific production rate (q(pm)) was 0.18±0.03 g cis, cis-muconate/(g(DCW) h) in continuous cultures, and increased to 1.4 g cis, cis-muconate/(g(DCW) h) during wash-out cultivation. Transcriptome analysis showed that the cat operon was not induced in P. putida KT2440-JD1 in the presence of 5mM benzoate, due to a point mutation in the highly conserved DNA binding domain of the transcriptional regulator (catR) of the cat operon. The ben operon was highly expressed in the presence of benzoate in the mutant and its parental strain. This operon contains PP_3166 (catA2), which was shown to be a second catechol 1,2-dioxygenase besides catA. P. putida KT2440-JD1 is the first cis, cis-muconate-accumulating mutant that was characterized at the genetic level. The specific production rate achieved is at least eight times higher than those reported for other cis, cis-muconate-producing strains.