RESUMO
o-Xylene is one of the most difficult-to-degrade environmental pollutants. We report here Rhodococcus genes mediating oxygenation in the first step of o-xylene degradation. Rhodococcus opacus TKN14, isolated from soil contaminated with o-xylene, was able to utilize o-xylene as the sole carbon source and to metabolize it to o-methylbenzoic acid. A cosmid library from the genome of this strain was constructed in Escherichia coli. A bioconversion analysis revealed that a cosmid clone incorporating a 15-kb NotI fragment had the ability to convert o-xylene into o-methylbenzyl alcohol. The sequence analysis of this 15-kb region indicated the presence of a gene cluster significantly homologous to the naphthalene-inducible dioxygenase gene clusters (nidABCD) that had been isolated from Rhodococcus sp. strain I24. Complementation studies, using E. coli expressing various combinations of individual open reading frames, revealed that a gene (named nidE) for rubredoxin (Rd) and a novel gene (named nidF) encoding an auxiliary protein, which had no overall homology with any other proteins, were indispensable for the methyl oxidation reaction of o-xylene, in addition to the dioxygenase iron-sulfur protein genes (nidAB). Regardless of the presence of NidF, the enzyme composed of NidABE was found to function as a typical naphthalene dioxygenase for converting naphthalene and various (di)methylnaphthalenes into their corresponding cis-dihydrodiols. All the nidABEF genes were transcriptionally induced in R. opacus TKN14 by the addition of o-xylene to a mineral salt medium. It is very likely that these genes are involved in the degradation pathways of a wide range of aromatic hydrocarbons by Rhodococcus species as the first key enzyme.
Assuntos
Oxigenases de Função Mista/genética , Rhodococcus/genética , Sequência de Bases , Cosmídeos , Primers do DNA , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Dioxigenases/genética , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Família Multigênica , Naftalenos/metabolismo , Fases de Leitura Aberta , Plasmídeos/genética , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Rhodococcus/enzimologia , Transcrição GênicaRESUMO
An aldehyde dehydrogenase gene, designated phnN, was isolated from a genome library of the 1,4-dimethylnaphthalene-utilizing soil bacterium, Sphingomonas sp. 14DN61. Escherichia coli expressing the phnN gene converted 1,4-dihydroxymethylnaphthalene to 1-hydroxymethyl-4-naphthoic acid. The putative amino acid sequence of the phnN gene product had 31-42% identity with those of NAD(+)-dependent short-chain aliphatic aldehyde dehydrogenases and a secondary alcohol dehydrogenase. The NAD(P)(+)-binding site and two consensus sequences involved in the active site for aldehyde dehydrogenase are conserved among these proteins. The PhnN enzyme purified from recombinant E. coli showed broad substrate specificity towards various aromatic aldehydes, i.e., 1- and 2-naphaldehydes, cinnamaldehyde, vanillin, syringaldehyde, benzaldehyde and benzaldehydes substituted with a hydroxyl, methyl, methoxy, chloro, fluoro, or nitro group were converted to their corresponding carboxylic acids. Interestingly, E. coli expressing phnN was able to biotransform a variety of not only aromatic aldehydes, but also aromatic alcohols to carboxylic acids.
Assuntos
Aldeído Desidrogenase/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Ácidos Carboxílicos/metabolismo , Sphingomonas/enzimologia , Álcoois/metabolismo , Aldeído Desidrogenase/química , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Ácidos Carboxílicos/química , Catálise , DNA Bacteriano/química , DNA Ribossômico/química , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Sphingomonas/classificação , Sphingomonas/genética , Especificidade por SubstratoRESUMO
A gene coding for lycopene beta-monocyclase, which metabolizes lycopene (psi,psi-carotene) to gamma-carotene (beta,psi-carotene), was isolated for the first time from a unique marine bacterium strain P99-3 that produces myxol (a gamma-carotene derivative). This lycopene beta-monocyclase gene (designated crtYm) was included in the gene cluster which contained carotenoid biosynthetic gene (crtI, crtB, crtZ, crtY, and crtA) homologs. CrtYm, the CrtY homolog, metabolized lycopene to gamma-carotene, which was confirmed by deletion/expression analysis of the crtYm and by subsequent analysis of the metabolites from lycopene based on the retention times on high-performance liquid chromatography, UV-visible absorption spectra, and mass spectrometry.
