Functional characterization of two alkane hydroxylases in a versatile Pseudomonas aeruginosa strain NY3

Abstract

Pseudomonas aeruginosa strain NY3 has an extraordinary capacity to utilize a wide range of substrates, including n–alkanes of lengths C5 to C34, aromatic compounds, phenols, diesel and crude oil, and it can produce a variety of small bioactive molecules, including rhamnolipids, which can enhance its metabolic capacity for hydrophobic organic pollutants. This capacity makes NY3 a good candidate for use in environmental pollution remediation. Alkane hydroxylases catalyze both the initial and rate-limiting step of the terminal oxidation of n–alkanes. To better understand the genetic mechanisms by which P. aeruginosa NY3 degrades such a wide range of n–alkanes, two putative coding genes of alkane hydroxylases were functionally characterized using a gene-knockout approach with three different degradation systems. The single n–alkane test indicated that the hydroxylase AlkB2 acted in the early growth phase and played a major role in the utilization of C12–C18. However, a double mutant showed a trend towards recovery when C20–C24 were used as sole carbon source. This suggests that there are other enzymes capable of utilizing n–alkanes longer than C20. Tests of both artificial n–alkanes mixture and crude oil-containing waste water showed similar results, suggesting that both AlkB1 and AlkB2 are involved in n–alkane degradation, and, moreover, that AlkB2 plays a major role. Finally, given the wider functional range of both AlkBs in the mixture of n–alkanes compared to that of single n–alkanes, these results hint at co-metabolism.