LICRED: A versatile drop-in vector for rapid generation of redox-self-sufficient cytochromes P450

Abstract

Cytochromes P450 (P450s) are a family of heme-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 characterization. Here, we describe a new ligation-independent cloning vector (LICRED) that enables the high-throughput generation of libraries of redox-self-suf ficient P450s, by fusing a range of P450 heme domains to the reductase of P450RhF (RhF-Red) in a robust and generically applicable way. © Springer Science+Business Media New York 2013.