MOLECULAR CHARACTERIZATION OF THE 4-HYDROXYPHENYLACETATE CATABOLIC PATHWAY OF ESCHERICHIA-COLI W - ENGINEERING A MOBILE AROMATIC DEGRADATIVE CLUSTER

We have determined and analyzed the nucleic acid sequence of a 14,855- bp region that contains the complete gene cluster encoding the 4-hydro xyphenylacetic acid (4-HPA) degradative pathway of Escherichia coli W (ATCC 11105). This catabolic pathway is composed by 11 genes, i.e., 8 enzyme-encoding genes distributed in two putative operons, hpaBC (4-HP A hydroxylase operon) and hpaGEDFHI (meta-cleavage operon); 2 regulato ry genes, hpaR and hpaA; and the gene, hpaX, that encodes a protein re lated to the superfamily of transmembrane facilitators and appears to be cotranscribed with hpaA. Although comparisons with other aromatic c atabolic pathways revealed interesting similarities, some of the genes did not present any similarity to their corresponding counterparts in other pathways, suggesting different evolutionary origins. The cluste r is flanked by two genes homologous to the cstA (carbon starvation pr otein) and tsr (serine chemoreceptor) genes off. coli K-12. A detailed genetic analysis of this region has provided a singular example of ho w E. colt becomes adapted to novel nutritional sources by the recruitm ent of a catabolic cassette, Furthermore, the presence of the pac gene in the proximity of the 4-HPA cluster suggests that the penicillin G acylase was a recent acquisition to improve the ability of E. colt W t o metabolize a wider range of substrates, enhancing its catabolic vers atility, Five repetitive extragenic palindromic sequences that might b e involved in transcriptional regulation were found within the cluster , The complete 4-HPA cluster was cloned in plasmid and transposon clon ing vectors that were used to engineer E. coli K-12 strains able to gr ow on 4-HPA, We report here also the in vitro design of new biodegrada tive capabilities through the construction of a transposable cassette containing the nide substrate range 4-HPA hydroxylase, in order to exp and the ol tho-cleavage pathway of Pseudomonas putida KT2442 and allow the new recombinant strain to use phenol as the only carbon source,