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

Abstract

BackgroundThe 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.ResultsCastellaniella 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.ConclusionsThe 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.

The anaerobic linalool metabolism in Thauera linaloolentis 47 Lol

“…M1 changed the expression of nearly 30 % of the genome in response to a change from lactate to myrcene, including genes involved in the Atu-pathway, citric acid cycle and β-oxidation, genes for a restructuring of the LPS layer and membranes and an up-regulation of nitrate-respiration gene clusters [ 28 ]. The toxicity of monoterpenes was also reflected in the proteomes of C. defragrans 65Phen cells grown on acetate and α-phellandrene: among the 107 identified induced proteins were several transporters and membrane biosynthesis proteins [ 7 ]. For this organism, a similar transposon mutagenesis identified the central degradation pathway for monoterpenes [ 7 ], but these alkenes are less toxic than monoterpene alcohols [ 6 ].…”

“…The toxicity of monoterpenes was also reflected in the proteomes of C. defragrans 65Phen cells grown on acetate and α-phellandrene: among the 107 identified induced proteins were several transporters and membrane biosynthesis proteins [ 7 ]. For this organism, a similar transposon mutagenesis identified the central degradation pathway for monoterpenes [ 7 ], but these alkenes are less toxic than monoterpene alcohols [ 6 ].…”

“…We did not detect isoenzymes of the acyclic terpene utilization (Atu) and leucin isovalerate utilization (Liu) pathway (see Table 1 ). Candidate genes for a second pathway were also not found: monoterpene synthases or the ctm operon that was recently identified for limonene degradation [ 7 ]. The cytochrome P450 enzyme linalool 8-monooxygenase was also not present, albeit the genome contains mono- and dioxygenases.…”

“…The further degradation pathway to geranic acid is catalyzed by a NAD + -dependent geraniol and a geranial dehydrogenase (GeoA, GeoB) [ 6 ]. However, a pathway for geranic acid degradation was not found in the genome of C. defragrans 65Phen [ 7 ]. Expected were genes and operons as described for the degradation of the monoterpene alcohols geraniol and citronellol in Pseudomonas aeruginosa PAO1.…”

Planimetric and Biomechanical Study of Local Effect of Pulegone on Full Thickness Wound Healing in Rat

“…These compounds are inexpensive and have been widely used in flavourings and fragrances since the beginning of the 19th century. More recently, in the pharmaceutical industry, they play a great role because of their therapeutic potential (19). Pulegone, a naturally occurring monoterpene ketone, has been reported to be able to reduce the number of writhings induced by acetic acid with the same intensity shown by carvone (20).…”