Rhodococcus erythropolis DCL14 contains a novel degradation pathway for limonene

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism w as identified as a strain of Rhadococcus erythropolis by chemotaxonomic and genetic studies. R erythropolis DCL14 also assimilated the terpenes limone ne-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (-)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption ra tes with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 cont ained the following four novel enzymatic activities involved in the limonen e degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADN-dependent limonene 1,a-monooxygenase activity, a cofactor-indepen dent limonene-1,2-epoxide hydrolase activity, a dichlorophenol-indophenol-d ependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1 -hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation stu dies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolinuo nene, and (3R)3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)l imonene degradation pathway. The opposite enantiomers [(LR,2R,4S)-limonene- 1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxohept anoate] mere found in the (4S)-limonene degradation pathway,while accumulat ion of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. T hese results show that R, erythropolis DCL14 metabolizes both enantiomers o f limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequen tly converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy- 4-isopropenyl-7-methyl-2-oxo-oxepanone, This lactone spontaneously rearrang es to form 3-isopropenyl-6-oxobeptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the hi gh levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the beta-oxidation pathw ay.