Genetic characterization and expression in heterologous hosts of the 3-(3-hydroxyphenyl)propionate catabolic pathway of Escherichia coli K-12

Abstract

We report the complete nucleotide sequence of the gene cluster encoding the 3-(3-hydroxyphenyl)propionate (3-HPP) catabolic pathway of Escherichia coli K-12. Sequence analysis revealed the existence of eight genes that map at min 8 of the chromosome, between the lac and hemB regions. Six enzyme-encoding genes account for a flavin-type monooxygenase (mhpA), the extradiol dioxygenase (mhpB), and the meta-cleavage pathway (mhpCDFE). The order of these catabolic genes, with the sole exception of mhpF, parallels that of the enzymatic steps of the pathway. The mhpF gene may encode the terminal acetaldehyde dehydrogenase (acylating) not reported previously in the proposed pathway. Enzymes that catalyze the early reactions of the pathway, MhpA and MhpB, showed the lowest level of sequence similarity to analogous enzymes of other aromatic catabolic pathways. However, the genes mhpCDFE present the same organization and appear to be homologous to the Pseudomonas xyl, dmp, and nah meta-pathway genes, supporting the hypothesis of the modular evolution of catabolic pathways and becoming the first example of this type of catabolic module outside the genus Pseudomonas. Two bacterial interspersed mosaic elements were found downstream of the mhpABCDFE locus and flank a gene, orfT, which encodes a protein related to the superfamily of transmembrane facilitators that might be associated with transport. All of the genes of the 3-HPP cluster are transcribed in the same direction, with the sole exception of mhpR. Inducible expression of the mhp catabolic genes depends upon the presence, in the cis or trans position, of a functional mhpR gene, which suggests that the mhpR gene product is the activator of the 3-HPP biodegradative pathway. The primary structure of MhpR revealed significant similarities to that of members of the IclR subfamily of transcriptional regulators. A 3-HPP catabolic DNA cassette was engineered and shown to be functional not only in enteric bacteria (E. coli and Salmonella typhimurium) but also in Pseudomonas putida and Rhizobium meliloti, thus facilitating its potential application to improve the catabolic abilities of bacterial strains for degradation of aromatic compounds.

Regulation of the mhp Cluster Responsible for 3-(3-Hydroxyphenyl)propionic Acid Degradation in Escherichia coli

“…Therefore, the intergenic mhpR-mhpA region should contain the Pr and Pa promoters driving expression of the mhp regulatory and catabolic genes, respectively. Whereas the ATG translational start codon of the first catabolic 3HPP Degradation in E. coli 27578 gene (mhpA) has been identified, two potential start codons of the regulatory mhpR gene were proposed (14). To define precisely the intergenic mhpR-mhpA region, we have determined the N-terminal amino acid sequence of MhpR as indicated under "Experimental Procedures."…”

“…On the other hand, aerobic degradation of phenylpropionic acid, 3-(3-hydroxyphenyl)propionic acid (3HPP) 1 and 3-hydroxycinnamic acid usually involves an initial oxygenation step with formation of 2,3-dihydroxyphenylpropionate (2,3-dihydroxyphenylcinnamate) that is further degraded via a metacleavage hydrolytic pathway (5). These pathways have been described in different bacterial genera (Pseudomonas, Arthrobacter, Ralstonia, Acinetobacter, Comamonas, Escherichia, Rhodococcus) (5-13), but only a few reports about their genetic characterization have been published (11)(12)(13)(14)(15).…”

“…1) (14). Amino acid sequence comparison analyses revealed that the MhpR protein from E. coli belongs to the IclR family of regulatory proteins (14,17 (18,19), PcaR of the protocatechuate degradation in Pseudomonas putida (20) and Agrobacterium tumefaciens (21), and CatR and PcaR of the catechol and protocatechuate degradation in R. opacus 1CP (22,23), are controlling ortho-cleavage pathways, the MhpR protein of E. coli becomes the first IclR-type regulator that is controlling the expression of a meta-cleavage pathway for degradation of aromatic compounds. On the other hand, there are no reports on the promoters driving expression of the genes involved in catabolism of HPP.…”

“…The 3HPP (and 3-hydroxycinnamic acid) degradation pathway from Escherichia coli is encoded by the mhp cluster located at min 8.0 of the genome, and it was the first HPP degradation pathway that was described both at the biochemical and genetic levels (5,14,16,17). The 3HPP pathway is initiated by the MhpA monooxygenase that transforms 3HPP into 2,3-dihydroxyphenylpropionate, which is then converted to succinate, pyruvate, and acetyl-CoA through the action of a metacleavage hydrolytic route that involves a dioxygenase (MhpB), hydrolase (MhpC), hydratase (MhpD), aldolase (MhpE), and acetaldehyde dehydrogenase (MhpF) activity (5,17) (Fig.…”

“…The mhp cluster is arranged as follows: (i) six catabolic genes encoding the initial monooxygenase (mhpA), the extradiol dioxygenase (mhpB), and the hydrolytic meta-cleavage enzymes (mhpCDFE); (ii) a gene (mhpT) that encodes a potential transporter; and (iii) a regulatory gene (mhpR) that is adjacent to the catabolic genes but transcribed in the opposite direction ( Fig. 1) (14). A similar gene arrangement has been observed in Klebsiella pneumoniae (17).…”