Enabling the P450 complement of Beauveria bassiana and Rhodococcus jostii for biocatalysis

Enzyme discovery today is proceeding at an enormous pace due to ever-growing technology development. As a result of this, more than 21000 cytochrome P450s have been identified in all kingdoms of life to date, making a wide range of enzyme resources with outstanding potential for biocatalytical implementation available. P450s from filamentous fungi, such as Beauveria bassiana, represent particularly compelling targets for the discovery of novel enzymes as these organisms have a long history of application in industrial hydroxylation reactions, many of which are believed to be P450-dependent. In addition, the genome sequence of B. bassiana has recently been completed revealing 83 putative P450s.

In order to uncover new cytochrome P450-based biocatalysts from the fungus Beauveria bassiana extensive bioinformatics analysis of the Beauveria CYPome were performed. As a result 7 genes encoding for heme domains with possible alkane hydroxylase function and one encoding a naturally fused P450 with homology to P450foxy from Fusarium oxysporum could be identified for subsequent cloning, heterologous expression and characterization. Different expression hosts as well as various expression conditions have been investigated. Despite our efforts, delivery of active biocatalysts could not be realized. However, empirical data acquired in this project will be of value for future studies of fungal P450s.

In addition, 23 cytochrome P450 heme domains from Rhodococcus jostii fused to the P450 reductase domain (RhfRED) of cytochrome P450Rhf from Rhodococcus sp. NCIMB 9784 have been investigated in a further strand of this work and provided a screening platform that could be applied for industrial purposes.