Mining Genomes for Novel BVMOs

A direct method to obtain novel Baeyer-Villiger monooxygenases is to search available sequenced genomes. As indicated above, a large number of BVMO genes can be identified. The first genome mining efforts focusing on BVMO discovery have just been reported. PAMO was the first BVMO identified by this strategy [35].

Recently, all six BVMO genes identified in the M. tuberculosis genome have been cloned and explored for their biocatalytic potential [72]. One of these genes had already been studied in some detail as it is involved in multidrug resistance, as discussed above. It was found that four out of six genes are readily expressed in E. coli. Using a test set of ketones and a sulfide, catalytic activities of cells expressing these monooxygenases were tested. While only one clone displayed activity in oxidizing a sulfide, the other three clones also performed Baeyer-Villiger oxidations. The type I BVMO corresponding to gene Rv3049c ("Mtub5" in Fig. 3.4) was studied in more detail since it catalyzed a kinetic resolution of racemic bicyclo[3.2.0]hept-2-en-6-one. Conversion of this benchmark substrate for BVMOs in a 4 L fermentation reaction resulted in production of (1R,5S)-(+)-bicyclo[3.2.0]hept-2-en-6-one with >95% enantiomeric excess and with a modest yield of 19%. In a recent follow-up study it was demonstrated, using a series of cyclic aliphatic ketones, that this BVMO exhibits exquisite enantio-selective behavior, enabling preparative kinetic resolution of cyclic ketones and production of enantio pure lactones [73]. These resolutions cannot be achieved using one of the other available BVMOs. Therefore, this novel BVMO represents a useful biocatalyst as it extends the biocatalytic repertoire in the BVMO toolbox.

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