Isolation and characterization of alkane hydroxylases from a metagenomic library of Pacific deep-sea sediment.

Two clones 9E7 and 21G8 in a metagenomic library of the east Pacific deep-sea sediment were found to contain alkane hydroxylase genes (alkB). The whole insert sequences of the two cosmid clones were determined. The insert sequences of 9E7 and 21G8 are 40 and 35 kb, respectively. Besides alkB, several alcohol/aldehyde dehydrogenase genes were also determined. A homolog of rubredoxin 2 of Pseudomonas putida was identified on 9E7 immediately downstream the alkB gene, but was lacking on 21G8. Unlike previous reports, the alkB genes on 9E7 and 21G8 have opposite transcription directions to those of linked alcohol/aldehyde dehydrogenase genes. Phylogenetic analysis put these two deep-sea AlkBs into a unique branch of integral membrane hydroxylases. The two alkB genes (9E7-alkB and 21G8-alkB) were cloned into pCom8 and introduced into two alkB expression host systems P. fluorescens KOB2 Delta 1 and P. putida GPo12 (pGEc47 Delta B). The transformed strains can grow on the n-alkanes from C5 to C16, indicating that both 9E7-AlkB and 21G8-AlkB have a wide substrate range. The data further indicate that the deep sea would be a rich resource for exploring novel alkane-degrading strains and genes.