Identification of two gene clusters involved in cyclohexanone oxidation in Brevibacterium epidermidis strain HCU.

Brevibacterium epidermidis HCU can grow on cyclic ketones and alcohols as a sole carbon source. We have previously reported the identification of two cyclohexanone-induced Bayer-Villiger monooxygenase genes by mRNA differential display. Using the related technique of Out-PCR, we have amplified large DNA fragments flanking the two monooxygenase genes. Two large gene clusters were sequenced. Several ORFs in each gene cluster encoded proteins homologous to cyclohexanol and cyclohexanone oxidation enzymes from Acinetobacter. However, the structure of these two gene clusters differs significantly from that of Acinetobacter, where the complete pathway has been described. To assess activity of these genes, they were cloned and expressed in Escherichia coli. In vivo and in vitro assays enabled us to assign functions to the expressed ORFs. These ORFs included a cyclohexanol dehydrogenase, two different epsilon-caprolactone hydrolases and two 6-hydroxyhexanoate dehydrogenases belonging to different enzyme families. Because this environmental isolate is difficult to manipulate, we cannot determine at this time which cluster is involved in the degradation of cyclohexanone under physiological conditions. However, the original differential display experiments and some of the experiments reported here suggest the involvement of both gene clusters in the oxidation of cyclic ketones.

Simultaneous identification of two cyclohexanone oxidation genes from an environmental Brevibacterium isolate using mRNA differential display.

The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacterium environmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.