Degradation of alkyl methyl ketones by Pseudomonas veronii MEK700

Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart; Institut für Siedlungswasserbau, Wassergüte- und Abfallwirtschaft, Universität Stuttgart, Abteilung Biologische Abluftreinigung, Bandtäle 2, D-70569 Stuttgart, Germany

^* To whom correspondence should be addressed. Email: khe{at}iswa.uni-stuttgart.de.

	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 miniTn5495 and cloned in E. 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 Baeyer-Villiger monooxygenases, the protein of the second gene mekB to homoserine acetyltransferases and the third gene mekR encoded a putative transcriptional activator of the AraC/XylS family. The three genes were located between two gene groups: the one comprising a putative phosphoenolpyruvate synthase and glycogen synthase, the other eight genes for the subunits of an ATPase. Inactivation of mekA and mekB by insertion of the minitransposon 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 P. 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, it was able to use 2-butanone as carbon source. The Baeyer-Villiger monooxygenase (BVMO) 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 was 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.