Cloning and characterization of a gene cluster for cyclododecanone oxidation in Rhodococcus ruber SC1

Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cel l. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone, as the sole carbon sou rce. A reverse genetic approach, was used to isolate a 10-kb gene cluster c ontaining all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxi dation were only marginally similar to the analogous genes for cyclohexanon e oxidation. The biochemical function of the enzymes encoded on the 10-kb g ene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol d ehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. coli strains grown in the presence of cyclododecanone accumulated lauryl lactone , 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyc lododecanone monooxygenase is, a type 1 Baeyer-Villiger flavin monooxygenas e (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C-11 to C-15), which is different from the specificity of c yclohexanone monooxygenase favoring short-chain cyclic compounds (C-5 to C- 7).