Cloning of Four Genes Involved in Limonene Hydroxylation from Enterobacter cowanii 6L

Genes encoding proteins responsible for limonene catabolism were cloned from a limonene-degrading microorganism, Enterobacter cowanii 6L, which was isolated from citron (Citrus junos) peel. The 8.6, 4.7, and 7.7 kb fragments (CD3, CD4, and CD6) of E. cowanii 6L chromosomal DNA that confer to E. coli the ability to grow on limonene have been cloned and their corresponding DNA sequences were determined. Nine open reading frames (ORFs) were identified, and the four ORFs (921 bp of CD3-2; 1,515 bp of CD4-1; 1,776 bp of CD6-1; and 1,356 bp of CD6-2) that encode limonene hydroxylase were confirmed by independently expressing these genes in E. coli. FAD and NADH were found to stimulate the hydroxylation reaction if added to cell extracts from E. coli recombinants, and multiple compounds (linalool, dihydrolinalool, perillyl alcohol, (${\alpha}-terpineol$, and ${\gamma}-terpineol$) were the principal products observed. Our results suggest that the isolate E. cowanii 6L has a broad metabolic capability including utilization of limonene. This broad metabolic ability was confirmed by identifying four novel limonene hydroxylase functional ORFs in E. cowanii 6L.