RESUMEN
Bacterial cytochromes P450 (P450s) have been recognized as attractive targets for biocatalysis and protein engineering. They are soluble cytosolic enzymes that demonstrate higher stability and activity than their membrane-associated eukaryotic counterparts. Many bacterial P450s possess broad substrate spectra and can be produced in well-known expression hosts like Escherichia coli at high levels, which enables quick and convenient mutant libraries construction. However, the majority of bacterial P450s interacts with two auxiliary redox partner proteins, which significantly increase screening efforts. We have established recombinant E. coli cells for screening of P450 variants that rely on two separate redox partners. In this chapter, a case study on construction of a selective P450 to synthesize a precursor of several chemotherapeutics, (-)-podophyllotoxin, is described. The procedure includes co-expression of P450 and redox partner genes in E. coli with subsequent whole-cell conversion of the substrate (-)-deoxypodophyllotoxin in 96-deep-well plates. By omitting the chromatographic separation while measuring mass-to-charge ratios specific for the substrate and product via MS in so-called multiple injections in a single experimental run (MISER) LC/MS, the analysis time could be drastically reduced to roughly 1 min per sample. Screening results were verified by using isolated P450 variants and purified redox partners.
