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The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen.

Petasch J, Disch EM, Markert S, Becher D, Schweder T, Hüttel B, Reinhardt R, Harder J - BMC Microbiol. (2014)

Bottom Line: An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes.CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase.Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Max Planck Institute for Marine Microbiology, Celsiusstraße 1, Bremen D-28359, Germany. jharder@mpi-bremen.de.

ABSTRACT

Background: The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic β-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway.

Results: Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58EuT, than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate.

Conclusions: The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen.

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GC chromatogram of metabolite accumulation. Extracts of nitrate-limited cultures were obtained from the primary stationary phase of cultures grown with 20 mM acetate as co-substrate and 3 mM (R)-(+)-limonene.
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Figure 2: GC chromatogram of metabolite accumulation. Extracts of nitrate-limited cultures were obtained from the primary stationary phase of cultures grown with 20 mM acetate as co-substrate and 3 mM (R)-(+)-limonene.

Mentions: To demonstrate the in vivo formation of perillyl alcohol from limonene, an in-frame deletion mutant of geoB was generated. The deletion of the alcohol dehydrogenase gene geoA caused a decreased growth rate and biomass formation with several monoterpenes as growth substance. We attribute this residual growth with the expression of a second alcohol dehydrogenase in cells of the in-frame ΔgeoA mutant [27]. The deletion mutant C. defragrans 65Phen ΔgeoB grew on acetate and perillic acid, but lacked the ability to grow on limonene, β-myrcene, perillyl alcohol and perillyl aldehyde (data not shown). To analyze the metabolite formation, the mutant and for comparison the wild type were anaerobically cultured with 3 mM (R)-(+)-limonene and 20 mM acetate as co-substrates in the presence of 20 mM nitrate. To increase the mass transfer, the organic carrier phase 2,2,4,4,6,8,8-heptamethylnonane (HMN) - and with it the two-phase system - was replaced by 0.5% v/v Tween 20 in a homogeneous phase. In the early stationary phase after seven days, hydrophobic compounds were salted out and extracted with isopropanol. The metabolites in the isopropanol phase were analyzed stereospecifically by GC and GC-MS (Figure 2). (R)-(+)-perillyl alcohol as well as (R)-(+)-perillyl aldehyde were not detected in an abiotic control without cells and in the culture of the wild type, but in the deletion mutant ΔgeoB. This in vivo formation of (R)-(+)-perillyl alcohol from (R)-(+)-limonene suggested an oxidation of limonene at the methyl group as initial reaction of the degradation pathway.


The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen.

Petasch J, Disch EM, Markert S, Becher D, Schweder T, Hüttel B, Reinhardt R, Harder J - BMC Microbiol. (2014)

GC chromatogram of metabolite accumulation. Extracts of nitrate-limited cultures were obtained from the primary stationary phase of cultures grown with 20 mM acetate as co-substrate and 3 mM (R)-(+)-limonene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4109377&req=5

Figure 2: GC chromatogram of metabolite accumulation. Extracts of nitrate-limited cultures were obtained from the primary stationary phase of cultures grown with 20 mM acetate as co-substrate and 3 mM (R)-(+)-limonene.
Mentions: To demonstrate the in vivo formation of perillyl alcohol from limonene, an in-frame deletion mutant of geoB was generated. The deletion of the alcohol dehydrogenase gene geoA caused a decreased growth rate and biomass formation with several monoterpenes as growth substance. We attribute this residual growth with the expression of a second alcohol dehydrogenase in cells of the in-frame ΔgeoA mutant [27]. The deletion mutant C. defragrans 65Phen ΔgeoB grew on acetate and perillic acid, but lacked the ability to grow on limonene, β-myrcene, perillyl alcohol and perillyl aldehyde (data not shown). To analyze the metabolite formation, the mutant and for comparison the wild type were anaerobically cultured with 3 mM (R)-(+)-limonene and 20 mM acetate as co-substrates in the presence of 20 mM nitrate. To increase the mass transfer, the organic carrier phase 2,2,4,4,6,8,8-heptamethylnonane (HMN) - and with it the two-phase system - was replaced by 0.5% v/v Tween 20 in a homogeneous phase. In the early stationary phase after seven days, hydrophobic compounds were salted out and extracted with isopropanol. The metabolites in the isopropanol phase were analyzed stereospecifically by GC and GC-MS (Figure 2). (R)-(+)-perillyl alcohol as well as (R)-(+)-perillyl aldehyde were not detected in an abiotic control without cells and in the culture of the wild type, but in the deletion mutant ΔgeoB. This in vivo formation of (R)-(+)-perillyl alcohol from (R)-(+)-limonene suggested an oxidation of limonene at the methyl group as initial reaction of the degradation pathway.

Bottom Line: An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes.CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase.Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Max Planck Institute for Marine Microbiology, Celsiusstraße 1, Bremen D-28359, Germany. jharder@mpi-bremen.de.

ABSTRACT

Background: The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic β-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway.

Results: Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58EuT, than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate.

Conclusions: The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen.

Show MeSH
Related in: MedlinePlus