Heterologous Expression of Mycobacterium Alkene Monooxygenases in Gram-Positive and Gram-Negative Bacterial Hosts

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 Mycobacterium chubuense strain NBB4. Strong functional expression of PmoABCD and EtnABCD was achieved in Mycobacterium smegmatis mc²155, 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 (C₂ to C₄), and their activity was much lower on liquid alkenes (C₅ to C₈). Despite intensive efforts to express the complete EtnABCD enzyme in Escherichia coli, 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 c reduced/min/mg protein). Cloning the EtnABCD gene cluster into Pseudomonas putida 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 cpn60 chaperonins from either Mycobacterium spp. or E. coli 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.IMPORTANCE 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.