Functional Analysis of Novel alkB Genes Encoding Long-Chain n-Alkane Hydroxylases in Rhodococcus sp. Strain CH91

Rhodococcus sp. strain CH91 is capable of utilizing long-chain n-alkanes as the sole carbon source. Two new genes (alkB1 and alkB2) encoding AlkB-type alkane hydroxylase were predicted by its whole-genome sequence analysis. The purpose of this study was to elucidate the functional role of alkB1 and alkB2 genes in the n-alkane degradation of strain CH91. RT-qPCR analyses revealed that the two genes were induced by n-alkanes ranging from C16 to C36 and the expression of the alkB2 gene was up-regulated much higher than that of alkB1. The knockout of the alkB1 or alkB2 gene in strain CH91 resulted in the obvious reduction of growth and degradation rates on C16-C36 n-alkanes and the alkB2 knockout mutant exhibited lower growth and degradation rate than the alkB1 knockout mutant. When gene alkB1 or alkB2 was heterologously expressed in Pseudomonas fluorescens KOB2Δ1, the two genes could restore its alkane degradation activity. These results demonstrated that both alkB1 and alkB2 genes were responsible for C16-C36 n-alkanes’ degradation of strain CH91, and alkB2 plays a more important role than alkB1. The functional characteristics of the two alkB genes in the degradation of a broad range of n-alkanes make them potential gene candidates for engineering the bacteria used for bioremediation of petroleum hydrocarbon contaminations.