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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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Proposed partial degradation pathway of monocyclic limonene by C. defragrans 65Phen. Enzymes of C. defragrans 65Phen predicted to catalyze reactions of the limonene metabolism: CtmAB, limonene dehydrogenase; GeoA, geraniol dehydrogenase; GeoB, geranial dehydrogenase; GeoC perillate-CoA ligase; MrcF, perillyl-CoA hydratase; MrcD, 2-hydroxy-4-isopropenylcyclohexane-1-carboxyl-CoA dehydrogenase; MrcE, 4-isopropenyl-2-oxocyclohexane-1-carboxyl-CoA hydrolase (Ctm cyclic terpene metabolism, Mrc monoterpene ring cleavage).
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Figure 3: Proposed partial degradation pathway of monocyclic limonene by C. defragrans 65Phen. Enzymes of C. defragrans 65Phen predicted to catalyze reactions of the limonene metabolism: CtmAB, limonene dehydrogenase; GeoA, geraniol dehydrogenase; GeoB, geranial dehydrogenase; GeoC perillate-CoA ligase; MrcF, perillyl-CoA hydratase; MrcD, 2-hydroxy-4-isopropenylcyclohexane-1-carboxyl-CoA dehydrogenase; MrcE, 4-isopropenyl-2-oxocyclohexane-1-carboxyl-CoA hydrolase (Ctm cyclic terpene metabolism, Mrc monoterpene ring cleavage).

Mentions: The ring cleavage of the cyclic perillyl-CoA resembles the cyclohex-1-ene-1-carboxyl-CoA degradation in anaerobic benzoate degraders or the described monocyclic monoterpene degradation pathways of Pseudomonas putida[34] or Geobacillus (ex. Bacillus) stearothermophilus[35] (Figure 3). We named the genes monoterpene ring cleavage-associated genes (mrc). Perillyl-CoA may be hydrated by MrcF to 2-hydroxy-4-isopropenylcyclohexane-1-carboxyl-CoA. Oxidation by the dehydrogenase MrcD may yield 4-isopropenyl-2-oxocyclohexane-1-carboxyl-CoA that may be hydrolysed by MrcE to 4-isopropenylpimelyl-CoA. Related enzymes of the anaerobic benzoate catabolism, BadK, BadH and BadI, catalyze the β-oxidation-like oxidation of cyclohexenecarboxyl-CoA, forming pimelyl-CoA. Among the strains of Azoarcus and Thauera, the enzymes of Azoarcus sp. KH32C are most closely related to MrcDEF. BadK (YP_007598293) catalyzes the hydration of cyclohex-1-ene-1-carboxyl-CoA and has an amino acid identity of 53% to MrcF, affiliating to the enoyl-CoA hydratase/isomerase superfamily. The dehydrogenase BadH (YP_007598295) has a high identity of 85% to MrcD. The formation of pimelyl-CoA is catalzyed by BadI (YP_007598294) which is highly similar to MrcE (88% identity). All three proteins MrcDEF were expressed in cells grown on α-phellandrene. In C. defragrans 65Phen, the genes required for ring cleavage are located in a cluster (mrcABCDEFGH). An insertion mutant of mrcF did not grow on monoterpenes like limonene or α-phellandrene. A second mutant strain with an insertion in the gene mrcC also lacked the capability to grow on limonene. MrcC was annotated as a 2,4-dienoyl reductase.


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)

Proposed partial degradation pathway of monocyclic limonene by C. defragrans 65Phen. Enzymes of C. defragrans 65Phen predicted to catalyze reactions of the limonene metabolism: CtmAB, limonene dehydrogenase; GeoA, geraniol dehydrogenase; GeoB, geranial dehydrogenase; GeoC perillate-CoA ligase; MrcF, perillyl-CoA hydratase; MrcD, 2-hydroxy-4-isopropenylcyclohexane-1-carboxyl-CoA dehydrogenase; MrcE, 4-isopropenyl-2-oxocyclohexane-1-carboxyl-CoA hydrolase (Ctm cyclic terpene metabolism, Mrc monoterpene ring cleavage).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Proposed partial degradation pathway of monocyclic limonene by C. defragrans 65Phen. Enzymes of C. defragrans 65Phen predicted to catalyze reactions of the limonene metabolism: CtmAB, limonene dehydrogenase; GeoA, geraniol dehydrogenase; GeoB, geranial dehydrogenase; GeoC perillate-CoA ligase; MrcF, perillyl-CoA hydratase; MrcD, 2-hydroxy-4-isopropenylcyclohexane-1-carboxyl-CoA dehydrogenase; MrcE, 4-isopropenyl-2-oxocyclohexane-1-carboxyl-CoA hydrolase (Ctm cyclic terpene metabolism, Mrc monoterpene ring cleavage).
Mentions: The ring cleavage of the cyclic perillyl-CoA resembles the cyclohex-1-ene-1-carboxyl-CoA degradation in anaerobic benzoate degraders or the described monocyclic monoterpene degradation pathways of Pseudomonas putida[34] or Geobacillus (ex. Bacillus) stearothermophilus[35] (Figure 3). We named the genes monoterpene ring cleavage-associated genes (mrc). Perillyl-CoA may be hydrated by MrcF to 2-hydroxy-4-isopropenylcyclohexane-1-carboxyl-CoA. Oxidation by the dehydrogenase MrcD may yield 4-isopropenyl-2-oxocyclohexane-1-carboxyl-CoA that may be hydrolysed by MrcE to 4-isopropenylpimelyl-CoA. Related enzymes of the anaerobic benzoate catabolism, BadK, BadH and BadI, catalyze the β-oxidation-like oxidation of cyclohexenecarboxyl-CoA, forming pimelyl-CoA. Among the strains of Azoarcus and Thauera, the enzymes of Azoarcus sp. KH32C are most closely related to MrcDEF. BadK (YP_007598293) catalyzes the hydration of cyclohex-1-ene-1-carboxyl-CoA and has an amino acid identity of 53% to MrcF, affiliating to the enoyl-CoA hydratase/isomerase superfamily. The dehydrogenase BadH (YP_007598295) has a high identity of 85% to MrcD. The formation of pimelyl-CoA is catalzyed by BadI (YP_007598294) which is highly similar to MrcE (88% identity). All three proteins MrcDEF were expressed in cells grown on α-phellandrene. In C. defragrans 65Phen, the genes required for ring cleavage are located in a cluster (mrcABCDEFGH). An insertion mutant of mrcF did not grow on monoterpenes like limonene or α-phellandrene. A second mutant strain with an insertion in the gene mrcC also lacked the capability to grow on limonene. MrcC was annotated as a 2,4-dienoyl reductase.

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