Cloning and Expression in E. coli of the Genes Responsible for Degradation of 4-Chlorobenzoate and 4-Chlorocatechol from Pseudomonas sp. Strain S-47

Pseudomonas sp. strain S.47 can grow on ii-chlorobenzoate (4CBA) and transform 4CBA to 4-chlorocatechol (4CC) under aerobic conditions, which is subsequently degraded to produce 2-hydroxypent-2,4-dienoate (2H-2,4DA). The upper steps for conversion of 4CBA to 4CC are recognized to be conducted by the benzoate-1,2-dioxygenase (B12O) system encoded by benABC and ben. The ensving meta-cleavage reaction of 4CC is catalyzed by catechol 2,3-dioxygenase (C23O) encoded by the xylE gene. The benABCD and the xylE genes were cloned from the chromosome of Pseudomonas sp. S-47 into pCS1 (48.7 kb), pCS101 (24.4 kb), pCS201 (17.7 kb), and pCS202 (6.7 kb) recombinant plasmids, and were well expressed in E. coli XL1-Blue host cells. The PstI-insert (4.0 kb) of pCS202 was found to contain the benABCD and xylE genes and to have 2 EcoRV, 1 SphI, and 3 SacII restriction sites.