Gene cloning and functional analysis of triple alkane monooxygenases from Geobacillus thermoleovorans B23

An extremely thermophilic bacterium, Geobacillus thermoleovorans B23 which was isolated from a deep subterranean oil reservoir at Niigata, Japan, is capable of degrading broad range alkanes (C11-C32) at 70℃ by terminal oxidation pathway, followed by β-oxidation pathway. Whole genome sequence analysis revealed that B23 did not have alkB-type alkane monooxygenases genes like most alkane degrading bacteria but it carried three gene homologs namely ladAαB23, ladAβB23 and ladBB23 on its chromosome in a unique region, namely “ladAB gene island”. The amino acid sequences deduced from these three gene homologs were significantly similar to FMN- dependent alkane monooxygenase, LadA, encoded in a plasmid of Geobacillus thermodetrificans NG 80-2 with 50%, 33% and 21% identity, respectively. I also found a gene encoding FMN reductase in the upstream of ladAB gene island, which provides reduced flavin for the monooxygenase enzyme to activate oxygen molecule in alkane oxidation reaction.In order to confirm that the three gene products are active alkane monooxygenases, heterologous expression of ladAαB23, ladAβB23 and ladBB23 have been performed in Pseudomonas fluorescens KOB2Δ1. The strain KOB2Δ1 is a mutant of alkane degrading P. fluorescens CHA0 with alkB1 deletion which no longer grows in medium containing C12 - C16 of n-alkanes as sole carbon sources. It was found that all these gene homologs functioned in P. fluorescens KOB2Δ1 and partially restored alkane degradation activity. Alkane degradation test at 35℃ for 8 days revealed that all the recombinants harboring either ladAαB23, ladAβB23 or ladAγB23 degraded more C12-C23 alkanes than vector only. Alkane degradation activity of the three recombinant strains was ladAαB23< ladAβB23 < ladBB23. It was also found that the ladAB gene structure is widely shared in Geobacillus strains. Here, I report that a group of G. thermoleovorans utilizes multiple LadA-type alkane monooxygenases for degradation of wide range alkanes.