Degradation of Alkyl Methyl Ketones by Pseudomonas veronii MEK700

Abstract

Pseudomonas veronii MEK700 was isolated from a biotrickling filter cleaning 2-butanone-loaded waste air. The strain is able to grow on 2-butanone and 2-hexanol. The genes for degradation of short chain alkyl methyl ketones were identified by transposon mutagenesis using a newly designed transposon, mini-Tn5495, and cloned in Escherichia coli. DNA sequence analysis of a 15-kb fragment revealed three genes involved in methyl ketone degradation. The deduced amino acid sequence of the first gene, mekA, had high similarity to BaeyerVilliger monooxygenases; the protein of the second gene, mekB, had similarity to homoserine acetyltransferases; the third gene, mekR, encoded a putative transcriptional activator of the AraC/XylS family. The three genes were located between two gene groups: one comprising a putative phosphoenolpyruvate synthase and glycogen synthase, and the other eight genes for the subunits of an ATPase. Inactivation of mekA and mekB by insertion of the mini-transposon abolished growth of P. veronii MEK700 on 2-butanone and 2-hexanol. The involvement of mekR in methyl ketone degradation was observed by heterologous expression of mekA and mekB in Pseudomonas putida. A fragment containing mekA and mekB on a plasmid was not sufficient to allow P. putida KT2440 to grow on 2-butanone. Not until all three genes were assembled in the recombinant P. putida was it able to use 2-butanone as carbon source. The Baeyer-Villiger monooxygenase activity of MekA was clearly demonstrated by incubating a mekB transposon insertion mutant of P. veronii with 2-butanone. Hereby, ethyl acetate was accumulated. To our knowledge, this is the first time that ethyl acetate by gas chromatographic analysis has been definitely demonstrated to be an intermediate of MEK degradation. The mekB-encoded protein was heterologously expressed in E. coli and purified by immobilized metal affinity chromatography. The protein exhibited high esterase activity towards short chain esters like ethyl acetate and 4-nitrophenyl acetate.

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters

“…embers of the Pseudomonas genus are highly versatile and known to colonize diverse habitats such as humans, animals, plants, water, and soil (1), and Pseudomonas veronii strains have been implicated in human pathogenicity (2), heavy metal biosorption (3), and dibenzo-p-dioxin, chloroaromatic, and alkyl methyl ketone degradation (4,5). P. veronii strains were also pointed out as key catabolic players in a BTEX (benzene, toluene, ethylbenzene, xylenes)-contaminated area where benzene had been the major contaminant (6).…”

Genome Sequence of Pseudomonas aeruginosa DQ8, an Efficient Degrader of n -Alkanes and Polycyclic Aromatic Hydrocarbons

“…Some members of the genus Pseudomonas are able to metabolize chemical pollutants in the environment, such as heterocyclic compounds and aliphatic or aromatic hydrocarbons, many of which are mutagenic and carcinogenic (2,3,4,5,9).…”

A Novel Subfamily Esterase with a Homoserine Transacetylase-like Fold but No Transferase Activity

“…However, these two members of the HTA family were reported to function as esterases rather than transacetylases (19,20). Both MekB and CgHle preferred to hydrolyze acetyl esters (19,20).…”