Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetoneinduced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc 2 155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc 2 155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc 2 155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc 2 155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.Mycobacterium goodii strain 12523 is a unique actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone (20). This microorganism was discovered for application in biocatalysis: chemical synthesis of hydroquinone is accompanied by side reactions leading to undesired by-products such as catechol, whereas M. goodii strain 12523 enabled gram-scale production of hydroquinone without by-products (21). Although M. goodii strain 12523 cannot utilize phenol, this microorganism can use acetone and methylethylketone as sources of carbon and energy. Interestingly, the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone and methylethylketone (20). To date, there have been only a few reports concerning monooxygenases that convert phenol to hydroquinone. P450BM-3, a cytochrome P450 monooxygenase from Bacillus megaterium, exhibits such an activity, although this activity is very low (23). A P450BM-3 mutant was then created, which exhibited 16.5 times higher activity than the wild type (23). A strain with a mutation of toluene-o-xylene monooxygenase, a binuclear iron monooxygenase from Pseudomonas stutzeri, also converts phenol to hydroquinone and catechol in a proportion of 80:20, although the wild type produces only catechol (27).In this study, we identified a monooxygenase gene clu...
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.
Overexpression and production of the high concentration of hydroxynitrile lyase from cassava (Manihot esculenta (MeHNL, EC 4.1.2.39)) were investigated. Hydroxynitrile lyase is a useful enzyme for the production of optically active cyanohydrin compounds. The production of MeHNL was increased by changing the rare codons of the original sequence of cassava MeHNL. However, most of the produced MeHNL was in the insoluble form. In order to increase the solubility of MeHNL, the effects of the cultivation temperature were investigated. When the cultivation temperature was reduced, the cell yield and the ratio of soluble MeHNL increased significantly. The enzyme activity and yield at low-temperature cultures (17 degrees C) were 850 times higher than those obtained at the optimum growth temperature of 37 degrees C. The rate of MeHNL production in the present study was calculated as 3,000 unit/h. Low-temperature cultivation was very effective in improving the productivity of the active form of MeHNL. Unlike the temperature-shift method, low-temperature cultivation has more potential for the large-scale production of MeHNL for the optically active cyanohydrin production.
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