van der Werf M J, Swarts H J, de Bont J A
Division of Industrial Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands.
Appl Environ Microbiol. 1999 May;65(5):2092-102. doi: 10.1128/AEM.65.5.2092-2102.1999.
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 was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-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 rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1, 2-monooxygenase activity, a cofactor-independent limonene-1, 2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S, 4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R, 4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the beta-oxidation pathway.
从淡水沉积物样本中分离出能够以柠檬烯作为唯一碳源和能源生长的菌株DCL14。通过化学分类学和遗传学研究，该微生物被鉴定为红平红球菌菌株。红平红球菌DCL14还能同化萜类化合物柠檬烯-1,2-环氧化物、柠檬烯-1,2-二醇、香芹醇、香芹酮和（-）-薄荷醇，而紫苏醇未被用作碳源和能源。对在柠檬烯上生长的细胞进行的诱导试验表明，柠檬烯-1,2-环氧化物、柠檬烯-1,2-二醇、1-羟基-2-氧代柠檬烯和香芹醇的耗氧率较高。柠檬烯诱导的红平红球菌DCL14细胞含有参与该微生物柠檬烯降解途径的以下四种新的酶活性：黄素腺嘌呤二核苷酸和NADH依赖性柠檬烯1,2-单加氧酶活性、不依赖辅因子的柠檬烯-1,2-环氧化物水解酶活性、二氯酚靛酚依赖性柠檬烯-1,2-二醇脱氢酶活性和NADPH依赖性1-羟基-2-氧代柠檬烯1,2-单加氧酶活性。产物积累研究表明，（1S,2S,4R）-柠檬烯-1,2-二醇、（1S,4R）-1-羟基-2-氧代柠檬烯和（3R）-3-异丙烯基-六氢-6-氧代庚酸酯是（4R）-柠檬烯降解途径中的中间体。在（4S）-柠檬烯降解途径中发现了对映体（1R,2R,4S）-柠檬烯-1,2-二醇、（1R,4S）-1-羟基-2-氧代柠檬烯和（3S）-3-异丙烯基-六氢-6-氧代庚酸酯，同时也观察到（4S）-柠檬烯积累了（1R,2S,4S）-柠檬烯-1,2-二醇。这些结果表明，红平红球菌DCL14通过一种新的降解途径代谢柠檬烯的两种对映体，该途径始于在1,2双键处环氧化形成柠檬烯-1,2-环氧化物。该环氧化物随后转化为柠檬烯-1,2-二醇、1-羟基-2-氧代柠檬烯和7-羟基-4-异丙烯基-7-甲基-2-氧代-氧杂环庚酮。该内酯自发重排形成3-异丙烯基-六氢-6-氧代庚酸酯。在辅酶A和ATP存在下，该酸进一步转化，这一发现以及柠檬烯生长细胞提取物中高水平的异柠檬酸裂合酶活性表明，进一步的降解通过β-氧化途径进行。
