p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate

Abstract

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.

Expansion of growth substrate range in Pseudomonas putida F1 by mutations in both cymR and todS, which recruit a ring-fission hydrolase CmtE and induce the tod catabolic operon, respectively

“…It was concluded that P. putida CE2010 first degrades biphenyl to 6-phenyl-HOHD using the tod pathway enzymes, and degraded further by CmtE (Fig. 1), which is constitutively expressed by the less effective binding of the repressor CymR to the cmt operator region (Eaton, 1997) due to the changed nucleotide. No base difference was found in cymR in P. putida strains F1 and CE2010.…”

“…These operons encode enzymes that catalyse p-cymene to p-cumate, and p-cumate to TCA cycle intermediates, respectively ( Fig. 1) (Eaton, 1996(Eaton, , 1997. The cmt degradation pathway is similar to the tod pathway, except that the former incorporates one more reaction step of decarboxylation after ring cleavage.…”

“…However, the mechanisms for the control of gene expression exploited are completely different in the two pathways. In the cym and cmt degradation pathways, CymR of a TetR protein family (Hillen & Berens, 1994) acts as a repressor in both catabolic operons with p-cumate as an effector (Eaton, 1997). The locations of the cymR, cym and cmt gene clusters with respect to the tod operons are shown in Fig.…”

“…1). The cmt operon is induced in the presence of p-cumate and regulated by a repressor CymR (Eaton, 1997). For the adapted strains to express cmtE in the absence of an effector, the CymR protein has to be incapable of functioning.…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…This family of oxygenases is able to introduce one atom of oxygen into hydrophobic substrate using one atom of oxygen originating from molecular oxygen. The other oxygen atom is reduced to water using two electrons from NAD(P)H. The electrons are transferred from the NAD(P)H to the monooxygenase by either ferredoxin and ferredoxin reductase or cytochrome b5 in the case of fatty acid desaturases [156] rubredoxin and rubredoxin reductase in the case of alkane hydroxylases (AlkB) [157,158] or a ferredoxin-ferredoxin reductase fusion protein for xylene monooxygenases [22,[159][160][161]. Three, four or six membranespanning elements can be identified in all primary amino acid sequences of proteins belonging to this super-family, with varying numbers of amino acids between each transmembrane helix [162,163].…”