LICRED: A Versatile Drop‐In Vector for Rapid Generation of Redox‐Self‐Sufficient Cytochrome P450s

Abstract

Cytochromes P450 (P450s) are a family of haem-containing oxidases with considerable potential as tools for industrial biocatalysis. Organismal genomes are revealing thousands of gene sequences that encode P450s of as yet unknown function, the exploitation of which will require high-throughput tools for their isolation and characterisation. In this report, a ligationindependent cloning vector "LICRED" is described that enables the high-throughput generation of libraries of redox-self-sufficient P450s by fusing a range of P450 haem domains to the reductase of P450RhF (RhF-Red) in a robust and generically applicable way. Cloning and expression of fusions of RhF-Red with the haem domains of P450cam and P450-XplA resulted in soluble, active, redox-self-sufficient, chimeric enzymes. In vitro studies also revealed that electron transfer from NADPH to haem was primarily intramolecular. The general applicability of the LICRED platform was then demonstrated through the creation of a library of RhF-Red fusion constructs by using the diverse complement of P450 haem domains identified in the genome of Nocardia farcinica. The resultant fusion-protein library was then screened against a panel of substrates; this revealed chimeric enzymes competent for the hydroxylation of testosterone and methyltestosterone, and the dealkylation of 7-ethoxycoumarin.

Molecular identification and functional characterization of cytochrome P450 monooxygenases from the brown-rot basidiomycete Postia placenta

“…In addition to the biological importance of P450s, it will be of great interest to utilize their catalytic potentials in biotechnology, because regio-and stereo-speciWc oxidations by P450s promise practical advantages (Guengerich 2002;Ro et al 2006;Urlacher and Eiben 2006;Chang et al 2007;Gillam 2008;Grogan 2011). Hence, increase in both molecular and functional information relating to P450s would facilitate advanced research into fungal biology and applied biotechnology (Lamb et al 2002;Yadav et al 2006;Sabbadin et al 2010;Nazir et al 2011;Hirosue et al 2011).…”

Construction and Application of a Functional Library of Cytochrome P450 Monooxygenases from the Filamentous Fungus Aspergillus oryzae

“…Biocatalytic regiospecific and stereospecific oxidations by CYPs attract much attention in industry because (i) use of biocatalysts allows reduction in the number of tedious steps, such as blocking and deblocking, that are common in conventional chemical synthesis, (ii) a single product without by-product formation reduces the cost of downstream purification steps, and (iii) enantiomeric mixtures (rather than racemic mixtures) are strictly required in some fields, such as the pharmaceutical industry (12,33,39,42). Therefore, a rational comprehensive approach toward elucidating catalytic potentials and utilities of CYPs is required to develop bioindustrial applications (8,19,34).…”

“…For catalytic activity, CYPs must be associated with electron transfer systems providing reducing equivalents. Thus, systematic production of both CYPs and their redox partner(s) are important to facilitate comprehensive functional screening (19,34). In this study, we employed S. cerevisiae as a host strain for heterologous expression because (i) a yeast expression system seemed appropriate to obtain recombinant eukaryotic CYP without genetic engineering of cDNA, (ii) S. cerevisiae has only three CYP genes, and their catalytic functions are well characterized (26), and (iii) S. cerevisiae is potentially useful for industrial applications as well as basic research.…”

Strategies for the construction of insect P450 fusion enzymes

“…LICRED is a high-throughput method for the development of P450 fusion proteins in which a vector containing a ligation-independent cloning (LIC) site adjacent to the CYP116B2 reductase domain (RED) allows the rapid insertion of P450 genes [59,60]. PUPPET is a platform in which three proliferating cell nuclear antigen (PCNA) fusion proteins are used to form a heterotrimer that recruits P450 into a complex with its electron donors, hence PUPPET refers to "proliferating cell nuclear antigen-utilized protein complex of P450 and its two electron transfer-related proteins" [61,62].…”