Unravelling the role of the group 6 soluble di‐iron monooxygenase (SDIMO) SmoABCD in alkane metabolism and chlorinated alkane degradation

Soluble di‐iron monooxygenases (SDIMOs) are multi‐component enzymes catalysing the oxidation of various substrates. These enzymes are characterized by high sequence and functional diversity that is still not well understood despite their key role in biotechnological processes including contaminant biodegradation. In this study, we analysed a mutant of Rhodoccocus aetherivorans BCP1 (BCP1‐2.10) characterized by a transposon insertion in the gene smoA encoding the alpha subunit of the plasmid‐located SDIMO SmoABCD. The mutant BCP1‐2.10 showed a reduced capacity to grow on propane, lost the ability to grow on butane, pentane and n‐hexane and was heavily impaired in the capacity to degrade chloroform and trichloroethane. The expression of the additional SDIMO prmABCD in BCP1‐2.10 probably allowed the mutant to partially grow on propane and to degrade it, to some extent, together with the other short‐chain n‐alkanes. The complementation of the mutant, conducted by introducing smoABCD in the genome as a single copy under a constitutive promoter or within a plasmid under a thiostreptone‐inducible promoter, allowed the recovery of the alkanotrophic phenotype as well as the capacity to degrade chlorinated n‐alkanes. The heterologous expression of smoABCD allowed a non‐alkanotrophic Rhodococcus strain to grow on pentane and n‐hexane when the gene cluster was introduced together with the downstream genes encoding alcohol and aldehyde dehydrogenases and a GroEL chaperon. BCP1 smoA gene was shown to belong to the group 6 SDIMOs, which is a rare group of monooxygenases mostly present in Mycobacterium genus and in a few Rhodococcus strains. SmoABCD originally evolved in Mycobacterium and was then acquired by Rhodococcus through horizontal gene transfer events. This work extends the knowledge of the biotechnologically relevant SDIMOs by providing functional and evolutionary insights into a group 6 SDIMO in Rhodococcus and demonstrating its key role in the metabolism of short‐chain alkanes and degradation of chlorinated n‐alkanes. In this study, we analysed a mutant of Rhodoccocus aetherivorans BCP1 characterized by a transposon insertion in the gene operon smoABCD encoding a soluble di‐iron monooxygenase of the rare SDIMO group 6. The smo mutant showed a reduced capacity to grow on propane, lost the ability to grow on butane, pentane and n‐hexane and was heavily impaired in the capacity to degrade chloroform and trichloroethane. Phylogenetic analyses showed that SmoABCD o