Production of cis, cis-muconate from benzoate at high rate and yield by a mutant of P. putida KT2440

P. putida KT2440-JD1 was derived from P. putida KT2440 after NTG-mutagenesis and exposure to 3-fluorobenzoate. The strain was no longer able to grow with benzoate as a single source of carbon and energy. Instead, benzoate was co-metabolized to cis, cis-muconate, which accumulated in the culture medium while grown on glucose. During continuous cultivations with glucose as a growth substrate, benzoate was co-metabolized to cis, cis-muconate with a molar product yield (Y(P/S)) of 0.88 ±0.01 mol cis, cis-muconate/ mol benzoate and a specific production rate (qpm) of 0.18 ±0.03 g cis, cis-muconate/ (gDCW* h). During wash-out cultivation a maximum growth rate of 0.6 h-1 and a maximum qpm of 1.4 g cis, cis-muconate/ (gDCW* h) were obtained. Analysis of the transcriptome revealed that the cat operon was no longer induced in P. putida KT2440-JD1 in the presence of 5 mM benzoate. This was also confirmed on the proteomic level for CatA and CatB. A point mutation in catR in P. putida KT2440-JD1 changed the highly conserved Arg50 in the DNA binding domain to Cys50, resulting in the inactivation of the transcriptional regulator of the cat operon. A catechol 1,2-dioxygenase that is encoded by gene PP_3166 (catA2) on the ben operon is likely to convert catechol to cis, cis-muconate in P. putida KT2440-JD1. wt, JD1 both twice on 10 mM glucose and 10 mM glucose + 5 mM benzoate in total 8 chips