Mycobacterium strain NBB4 was isolated on ethene as part of a bioprospecting study searching for novel monooxygenase (MO) enzymes of interest to biocatalysis and bioremediation. Previous work indicated that strain NBB4 contained an unprecedented diversity of MO genes, and we hypothesized that each MO type would support growth on a distinct hydrocarbon substrate. Here, we attempted to untangle the relationships between MO types and hydrocarbon substrates. Strain NBB4 was shown to grow on C2 -C4 alkenes and C2 -C16 alkanes. Complete gene clusters encoding six different monooxygenases were recovered from a fosmid library, including homologues of ethene MO (etnABCD), propene MO (pmoABCD), propane MO (smoABCD), butane MO (smoXYB1C1Z), cytochrome P450 (CYP153; fdx-cyp-fdr) and alkB (alkB-rubA1-rubA2). Catabolic enzymes involved in ethene assimilation (EtnA, EtnC, EtnD, EtnE) and alkane assimilation (alcohol and aldehyde dehydrogenases) were identified by proteomics, and we showed for the first time that stress response proteins (catalase/peroxidase, chaperonins) were induced by growth on C2 -C5 alkanes and ethene. Surprisingly, none of the identified MO genes could be specifically associated with oxidation of small alkanes, and thus the nature of the gaseous alkane MO in NBB4 remains mysterious.

Alkene monooxygenases (MOs) are soluble di-iron-containing enzymes found in bacteria that grow on alkenes. Here, we report improved heterologous expression systems for the propene MO (PmoABCD) and ethene MO (EtnABCD) from strain NBB4. Strong functional expression of PmoABCD and EtnABCD was achieved in mc155, yielding epoxidation activities (62 and 27 nmol/min/mg protein, respectively) higher than any reported to date for heterologous expression of a di-iron MO system. Both PmoABCD and EtnABCD were specialized for the oxidation of gaseous alkenes (C to C), and their activity was much lower on liquid alkenes (C to C). Despite intensive efforts to express the complete EtnABCD enzyme in , this was not achieved, although recombinant EtnB and EtnD proteins could be purified individually in soluble form. The biochemical function of EtnD as an oxidoreductase was confirmed (1.36 μmol cytochrome reduced/min/mg protein). Cloning the EtnABCD gene cluster into KT2440 yielded detectable epoxidation of ethene (0.5 nmol/min/mg protein), and this could be stimulated (up to 1.1 nmol/min/mg protein) by the coexpression of chaperonins from either spp. or Successful expression of the ethene MO in a Gram-negative host was validated by both whole-cell activity assays and peptide mass spectrometry of induced proteins seen on SDS-PAGE gels. Alkene MOs are of interest for their potential roles in industrial biocatalysis, most notably for the stereoselective synthesis of epoxides. Wild-type bacteria that grow on alkenes have high activities for alkene oxidation but are problematic for biocatalysis, since they tend to consume the epoxide products. Using recombinant biocatalysts is the obvious alternative, but a major bottleneck is the low activities of recombinant alkene MOs. Here, we provide new high-activity recombinant biocatalysts for alkene oxidation, and we provide insights into how to further improve these systems.

ABSTRACT1,2-Dichloroethane (DCA) is a problematic xenobiotic groundwater pollutant. Bacteria are capable of biodegrading DCA, but the evolution of such bacteria is not well understood. In particular, the mechanisms by which bacteria acquire the key dehalogenase genes dhlA and dhlB have not been well defined. In this study, the genomic context of dhlA and dhlB was determined in three aerobic DCA-degrading bacteria (Starkeya novella strain EL1, Xanthobacter autotrophicus strain EL4, and Xanthobacter flavus strain EL8) isolated from a groundwater treatment plant (GTP). A haloalkane dehalogenase gene (dhlA) identical to the canonical dhlA gene from Xanthobacter sp. strain GJ10 was present in all three isolates, and, in each case, the dhlA gene was carried on a variant of a 37-kb circular plasmid, which was named pDCA. Sequence analysis of the repA replication initiator gene indicated that pDCA was a member of the pTAR plasmid family, related to catabolic plasmids from the Alphaproteobacteria, which enable growth on aromatics, dimethylformamide, and tartrate. Genes for plasmid replication, mobilization, and stabilization were identified, along with two insertion sequences (ISXa1 and ISPme1) which were likely to have mobilized dhlA and dhlB and played a role in the evolution of aerobic DCA-degrading bacteria. Two haloacid dehalogenase genes (dhlB1 and dhlB2) were detected in the GTP isolates; dhlB1 was most likely chromosomal and was similar to the canonical dhlB gene from strain GJ10, while dhlB2 was carried on pDCA and was not closely related to dhlB1. Heterologous expression of the DhlB2 protein confirmed that this plasmid-borne dehalogenase was capable of chloroacetate dechlorination. IMPORTANCEEarlier studies on the DCA-degrading Xanthobacter sp. strain GJ10 indicated that the key dehalogenases dhlA and dhlB were carried on a 225-kb linear plasmid and on the chromosome, respectively. The present study has found a dramatically different gene organization in more recently isolated DCA-degrading Xanthobacter strains from Australia, in which a relatively small circular plasmid (pDCA) carries both dhlA and dhlB homologs. pDCA represents a true organochlorine-catabolic plasmid, first because its only obvious metabolic phenotype is dehalogenation of organochlorines, and second because acquisition of this plasmid provides both key enzymes required for carbon-chlorine bond cleavage. The discovery of the alternative haloacid dehalogenase dhlB2 in pDCA increases the known genetic diversity of bacterial chloroacetate-hydrolyzing enzymes. O rganochlorine chemicals have been widely used as pesticides, solvents, and for plastic manufacture. Many of these compounds are xenobiotic, and, as a result, their environmental breakdown is often slow (1). The environmental persistence of organochlorines is a problem, since many of these chemicals are damaging to human and environmental health (2). One organochlorine of concern is 1,2-dichloroethane (DCA), which is often present as a groundwater contaminant (3, 4). DCA is not known to exi...

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