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

Abstract

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.

FIGURE 1

. Growth assays of R. aetherivorans BCP1 on short‐chain n‐alkanes. Growth was measured as OD₆₀₀ values at two different time points (72 and 168 h) after the inoculation on minimal medium (MSM) supplied with C₃ (A), C₄ (B), C₅ (C) or C₆ (D) as only carbon and energy source. Results shown are means ± standard deviation with n = 3. Asterisks indicate statistically different groups respect BCP1 wild type (BCP1 WT) according to one‐way ANOVA analysis. *p < 0.5, **p < 0.01.

FIGURE 2

The soluble di‐iron monooxygenase (SDIMO) Smo from Rhodococcus aetherivorans BCP1. (A) Schematic organization of the gene cluster smoABCD encoding the soluble di‐iron monooxygenase (SDIMO) Smo and the flanking regions that are co‐localized on the megaplasmid pBMC2 in R. aetherivorans BCP1 genome. Gene are displayed as arrows. The single genes of the smoABCD operon code for smoA, the monooxygenase alpha subunit, smoB, the monooxygenase beta subunit, smoC the coupling protein, smoD, the reductase. The flanking genes code for ald DH, the aldehyde dehydrogenase (DH), alc DH the alcohol DH, groEL GroEL the chaperonin, hisK the histidine kinase, luxR‐like the cognate response regulator. The transposable element of pTNR‐TA that interrupt smoA gene in the mutant 2.10 is shown together with the 1070 nt‐long thiostrepton resistance gene (thio ^R ) and the insertion site (at the nucleotide 789 of smoA). The locus tags of the genes go from N505_0128845 (for hisK) to N505_0128885 (for groEL) B. (B) Cofactor composition of the Smo components and predicted enzymatic reactions catalysed by Smo, Alc DH and Ald DH. The metabolic pathway and reaction intermediates for the alkanes are typed in black, the reaction involved in the degradation of chlorinated alkanes are in blue. Dashed lines correspond to more reactions.

FIGURE 3

Heterologous expression of smoABCD in R. erythropolis MTF. Growth of MTF wild type (MTF) and MTF transformed with pTipQT1‐smoABCD/dhs/groEL (MTF pTipQT1‐smoABCD/dhs/groEL) or the empty vector (MTF pTipQT1) is shown as OD₆₀₀ values measured after 72 h of culture in minimal medium (MSM) supplied with C₃, C₄, C₅ and C₆ (0.1% v/v) as only carbon and energy source. Results shown are means ± SD. Statistically significant differences (using one‐way ANOVA analysis) respect to the MTF wild type are shown with asterisks *p < 0.05, **p < 0.01.

FIGURE 4

Role of the monooxygenase Smo in the degradation of short‐chain n‐alkanes and chlorinated alkanes in Rhodococcus aetherivorans BCP1. The capability of the wild‐type strain BCP1 (BCP1), the mutant 2.10 strain (2.10) and the complemented mutant strain (2.10 pNitsmoABCD) to degrade short chain alkanes (each supplied in MSM at 0.1% v/v) (A) and chlorinated n‐alkanes (chloroform, CF, and 1,1,2‐trichloroethane, TCA, each supplied at the concentrations 2 and 10 μM) (B, C) are shown in terms of specific degradation rates (i.e. concentration of the compound degraded per mg of cellular proteins per day). Asterisks indicate statistical significance (one‐way ANOVA analysis compared to BCP1). *p < 0.05, **p < 0.01.

FIGURE 5

Maximum likelihood phylogenetic tree and distribution of the SDIMO groups (from 1 to 6) of Mycobacteriaceae family with an enlargement on the SDIMO group 6 sub‐clusters (from 6‐1 to 6‐5). (A) Phylogenetic distances in the phylogenetic tree were calculated using the Q.yeast+F + I + G4 substitution model. Ultrafast bootstrap support values >80 (1000 bootstrap replicates) are indicated as black dots. R. aetherivorans BCP1 SmoA is framed by a red box. Besides each strain name, NCBI protein accession is indicated between brackets together with genomic location, if known, corresponding to P, for plasmid or C, for chromosome. (B) The distribution of each SDIMO group among different Mycobacteriaceae genera is shown by framing each bar plot in boxes that are coloured in the same way as the phylogenetic tree.

FIGURE 6

Predicted GeneRax horizontal gene transfer (HGT) events of group 6 SDIMOs between members of Rhodococcus and Mycobacterium (only these bacterial genera possess group 6 SDIMOs). HGT events occurring between Rhodococcus spp. strains are shown as pink arrows. Phylogenetic distances were calculated using the WAG substitution model. Ultrafast bootstrap support values >80 (1000 bootstrap replicates) are indicated as yellow dots.