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

Abstract

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.

A novel soluble di‐iron monooxygenase from the soil bacterium Solimonas soli

“…Since the plasmid construct used in both hosts was identical, there must be trans‐acting factors in the KT2440 genome that enable this MO to function. Candidates for this role include chaperonins and oxidative stress defence enzymes (Coleman et al, 2011; Furuya et al, 2012; McCarl et al, 2018), but it is notable that unlike the case for some other SDIMOs, no such accessory genes were seen near the Group 7 SDIMOs in their host genomes, and co‐expression of an E. coli chaperonin (GroEL) did not stimulate activity (Supporting Information Figure S5). P. putida was used previously to express the ethene MO EtnABCD (McCarl et al, 2018), although that enzyme gave much higher activity in a Mycobacterium smegmatis host.…”

“…The SDIMOs are best categorized based on amino acid sequence data, but simpler schemes based on the number of enzyme subunits (ranging from four to six), the arrangement of the gene clusters, or the nature of their canonical substrate are possible (Leahy et al, 2003). While the last of these methods is unreliable due to the broad substrate range of these enzymes, this system has nevertheless been widely used and organizes the SDIMOs into six groups (Coleman et al, 2006;Notomista et al, 2003): toluene MOs (Group 1) (Bertoni et al, 1996(Bertoni et al, , 1998Cafaro et al, 2004); phenol MOs (Group 2) (Arai et al, 1998;Hino et al, 1998;Zhou et al, 2016); soluble methane MOs (sMMO; Group 3) (Cardy et al, 1991;Sayavedra-Soto et al, 2001;Stainthorpe et al, 1990); alkene MOs (Group 4) (Mattes et al, 2005;McCarl et al, 2018;Suzuki et al, 2019); propane-2-MOs (Group 5) (Furuya et al, 2011(Furuya et al, , 2015Kotani et al, 2003); and propane-1-MOs (Group 6) (Deng et al, 2018;Kotani et al, 2006;Masuda et al, 2012b). The known sequence diversity of SDIMO genes has greatly expanded in recent years with genomic and metagenomic sequencing, but in many cases, the biochemistry of the enzymes and their role in the physiology of the hosts remain unknown.…”

Synthetic Biology Approaches to Hydrocarbon Biosensors: A Review

“…There are fewer examples of alkene-oxidising enzymes ( Table 1 ), with the two best-studied systems being the propene monooxygenases of Rhodococcus rhodochrous ( Gallagher et al, 1997 ; Gallagher et al, 1998 ; Smith et al, 1999 ) and Xanthobacter Py2 ( Ginkel, 1987 ; Ginkel et al, 1987 ; Small and Ensign, 1997 ; Champreda et al, 2004 ). Despite having the same primary substrate, these two enzymes are diverse in both sequence and structure; the former is encoded by four genes encoding three enzyme subunits ( Smith et al, 1999 ) while the latter is a six gene, four component system ( Small and Ensign, 1997 ; McCarl et al, 2018 ). Major advances since the alkene monooxygenases were last reviewed ( Ensign, 2001 ; Shennan, 2006 ) include the identification, characterisation, and heterologous expression of the genes encoding the ethene monooxygenases (EtnABCD) found in Nocardioides and Mycobacterium spp.…”

“…Major advances since the alkene monooxygenases were last reviewed ( Ensign, 2001 ; Shennan, 2006 ) include the identification, characterisation, and heterologous expression of the genes encoding the ethene monooxygenases (EtnABCD) found in Nocardioides and Mycobacterium spp. ( Coleman and Spain, 2003b ; Mattes et al, 2005 ; Coleman et al, 2011a ; McCarl et al, 2018 ) and the investigation of these enzymes as biocatalysts for epoxide synthesis ( Owens et al, 2009 ; Cheung et al, 2013 ).…”

“…The substrate ranges of the NBB4 ethene and propene monooxygenases (EtnABCD and PmoABCD, respectively) are similar; both enzymes show activity on C 2 -C 8 alkenes, with stronger activity on gaseous alkenes (C 2 -C 4 ) ( McCarl et al, 2018 ). The fact that strain NBB4 possesses two distinct SDIMO enzyme systems with very close overlap in substrate ranges is unusual and poses questions about why this genotype has evolved and how these genes are regulated.…”