The mycobacterial binuclear iron monooxygenases require a specific chaperonin‐like protein for functional expression in a heterologous host

Abstract

The mimABCD gene clusters in Mycobacterium smegmatis strain mc2155 and Mycobacterium goodii strain 12523 encode binuclear iron monooxygenases that oxidize propane and phenol. In this study, we attempted to express each mimABCD gene cluster in a heterologous host. The actinomycetous strain Rhodococcus opacus B‐4, which is phylogenetically close to Mycobacterium, was selected as the host. Each mimABCD gene cluster was cloned into the Rhodococcus–Escherichia coli shuttle vector, pTip‐QC2, and then introduced into R. opacus cells. Although whole‐cell assays were performed with phenol as a substrate, the transformed R. opacus cells did not oxidize this substrate. SDS/PAGE analysis revealed that the oxygenase large subunit MimA was expressed in the insoluble fraction of R. opacus cells. We found that a gene designated mimG, which lies downstream of mimABCD, exhibits similarity in the amino acid sequence of its product with the products of genes encoding the chaperonin GroEL. When the mimG gene was cloned and coexpressed with each mimABCD gene cluster in R. opacus strain B‐4, this host successfully acquired oxidation activity towards phenol. SDS/PAGE and western blotting analyses demonstrated that MimA was clearly soluble when in the presence of MimG. These results indicated that MimG played essential roles in the productive folding of MimA, and that the resulting soluble MimA protein led to the active expression of MimABCD.

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

“…We have already found that MimABCD requires the specific chaperonin-like protein MimG, which is encoded downstream from the mimABCD gene cluster (Fig. 1), for functional expression in the Rhodococcus host; when the mimG gene was coexpressed with the mimABCD gene cluster in R. opacus strain B-4, this host successfully acquired oxidation activity toward phenol (20). Furthermore, we demonstrated that MimG was involved in the productive folding of the oxygenase large subunit MimA (20).…”

“…We have previously attempted to express the mimABCD gene cluster in E. coli cells using pETmimABCD, in which the tandem mimABCD gene cluster was located downstream from the T7 promoter and the ribosome-binding site (RBS) on the pET21a vector (20). In this plasmid, the mimA gene utilizes the vector-derived RBS, whereas the mimB, mimC, and mimD genes have their natural RBSs.…”

“…1), for functional expression in the Rhodococcus host; when the mimG gene was coexpressed with the mimABCD gene cluster in R. opacus strain B-4, this host successfully acquired oxidation activity toward phenol (20). Furthermore, we demonstrated that MimG was involved in the productive folding of the oxygenase large subunit MimA (20). We speculated that this chaperonin-like protein might also be an important factor in active expression in E. coli.…”

“…In whole-cell assays using transformed E. coli cells, the reaction mixture (250 l) contained cells of the E. coli strain (2 g dry cell weight per liter), the substrate phenol (10 mM), ethanol (1%, vol/vol), and aqueous basal medium (20,26) containing glucose (5 g/liter).…”

“…More recently, we succeeded in functionally expressing the mimABCD gene clusters from Mycobacterium smegmatis strain mc 2 155 and Mycobacterium goodii strain 12523 in the actinomycetous strain Rhodococcus opacus B-4 (20). The four genes mimA, mimB, mimC, and mimD encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively (Fig.…”