Involvement of a Novel Enzyme, MdpA, in Methyl tert-Butyl Ether Degradation in Methylibium petroleiphilum PM1

ABSTRACT Methylibium petroleiphilum PM1 is a well-characterized environmental strain capable of complete metabolism of the fuel oxygenate methyl tert-butyl ether (MTBE). Using a molecular genetic system which we established to study MTBE metabolism by PM1, we demonstrated that the enzyme MdpA is involved in MTBE removal, based on insertional inactivation and complementation studies. MdpA is constitutively expressed at low levels but is strongly induced by MTBE. MdpA is also involved in the regulation of tert-butyl alcohol (TBA) removal under certain conditions but is not directly responsible for TBA degradation. Phylogenetic comparison of MdpA to related enzymes indicates close homology to the short-chain hydrolyzing alkane hydroxylases (AH1), a group that appears to be a distinct subfamily of the AHs. The unique, substrate-size-determining residue Thr59 distinguishes MdpA from the AH1 subfamily as well as from AlkB enzymes linked to MTBE degradation in Mycobacterium austroafricanum.

Biodegradation of 2-Ethylhexyl Nitrate by Mycobacterium austroafricanum IFP 2173

ABSTRACT 2-Ethyhexyl nitrate (2-EHN) is a major additive of fuel that is used to increase the cetane number of diesel. Because of its wide use and possible accidental release, 2-EHN is a potential pollutant of the environment. In this study, Mycobacterium austroafricanum IFP 2173 was selected from among several strains as the best 2-EHN degrader. The 2-EHN biodegradation rate was increased in biphasic cultures where the hydrocarbon was dissolved in an inert non-aqueous-phase liquid, suggesting that the transfer of the hydrophobic substrate to the cells was a growth-limiting factor. Carbon balance calculation, as well as organic-carbon measurement, indicated a release of metabolites in the culture medium. Further analysis by gas chromatography revealed that a single metabolite accumulated during growth. This metabolite had a molecular mass of 114 Da as determined by gas chromatography/mass spectrometry and was provisionally identified as 4-ethyldihydrofuran-2(3H)-one by liquid chromatography-tandem mass spectrometry analysis. Identification was confirmed by analysis of the chemically synthesized lactone. Based on these results, a plausible catabolic pathway is proposed whereby 2-EHN is converted to 4-ethyldihydrofuran-2(3H)-one, which cannot be metabolized further by strain IFP 2173. This putative pathway provides an explanation for the low energetic efficiency of 2-EHN degradation and its poor biodegradability.

Genes involved in the methyl tert-butyl ether (MTBE) metabolic pathway of Mycobacterium austroafricanum IFP 2012

Methyl tert-butyl ether (MTBE) is a persistent pollutant of surface and groundwater, and the reasons for its low biodegradability are poorly documented. Using one of the rare bacterial strains able to grow in the presence of MTBE, Mycobacterium austroafricanum IFP 2012, the protein profiles of crude extracts after growth in the presence of MTBE and glucose were compared by SDS-PAGE. Ten proteins with molecular masses of 67, 64, 63, 55, 50, 27, 24, 17, 14 and 11 kDa were induced after growth in the presence of MTBE. Partial amino acid sequences of N-terminal and internal peptide fragments of the 64 kDa protein were used to design degenerate oligonucleotide primers to amplify total DNA by PCR, yielding a DNA fragment that was used as a probe for cloning. A two-step cloning procedure was performed to obtain a 10 327 bp genomic DNA fragment containing seven ORFs, including a putative regulator, mpdR, and four genes, mpdC, orf1, mpdB and orf2, in the same cluster. The MpdB protein (64 kDa) was related to a flavoprotein of the glucose–methanol–choline oxidoreductase family, and the MpdC protein (55 kDa) showed a high similarity with NAD(P) aldehyde dehydrogenases. Heterologous expression of these gene products was performed in Mycobacterium smegmatis mc2 155. The recombinant strain was able to degrade an intermediate of MTBE biodegradation, 2-methyl 1,2-propanediol, to hydroxyisobutyric acid. This is believed to be the first report of the cloning and characterization of a cluster of genes specifically involved in the MTBE biodegradation pathway of M. austroafricanum IFP 2012.