Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster

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Abstract
We have determined and analyzed the nucleic acid sequence of a 14,855-bp region that contains the complete gene cluster encoding the 4-hydroxyphenylacetic 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-HPA hydroxylase operon) and hpaGEDFHI (meta-cleavage operon); 2 regulatory genes, hpaR and hpaA; and the gene, hpaX, that encodes a protein related to the superfamily of transmembrane facilitators and appears to be cotranscribed with hpaA. Although comparisons with other aromatic catabolic 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 cluster is flanked by two genes homologous to the estA (carbon starvation protein) and tsr (serine chemoreceptor) genes of E. coli K-12. A detailed genetic analysis of this region has provided a singular example of how E. coli becomes adapted to novel nutritional sources by the recruitment 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. coli W to metabolize a wider range of substrates, enhancing its catabolic versatility. Five repetitive extragenic palindromic sequences that might be involved in transcriptional regulation were found within the cluster. The complete 4-HPA cluster was cloned in plasmid and transposon cloning vectors that were used to engineer E. coli K-12 strains able to grow on 4-HPA. We report here also the in vitro design of new biodegradative capabilities through the construction of a transposable cassette containing the wide substrate range 4-HPA hydroxylase, in order to expand the ortho-cleavage pathway of Pseudomonas putida KT2442 and allow the new recombinant strain to use phenol as the only carbon source.

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