Bacterial cytochrome P450 enzymes: Semi-rational design and screening of mutant libraries in recombinant Escherichia coli cells.

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.

Effect of chromosomal integration on catalytic performance of a multi-component P450 system in Escherichia coli.

Cytochromes P450 are useful biocatalysts in synthetic chemistry and important bio-bricks in synthetic biology. Almost all bacterial P450s require separate redox partners for their activity, which are often expressed in recombinant Escherichia coli using multiple plasmids. However, the application of CRISPR/Cas recombineering facilitated chromosomal integration of heterologous genes which enables more stable and tunable expression of multi-component P450 systems for whole-cell biotransformations. Herein, we compared three E. coli strains W3110, JM109, and BL21(DE3) harboring three heterologous genes encoding a P450 and two redox partners either on plasmids or after chromosomal integration in two genomic loci. Both loci proved to be reliable and comparable for the model regio- and stereoselective two-step oxidation of (S)-ketamine. Furthermore, the CRISPR/Cas-assisted integration of the T7 RNA polymerase gene enabled an easy extension of T7 expression strains. Higher titers of soluble active P450 were achieved in E. coli harboring a single chromosomal copy of the P450 gene compared to E. coli carrying a medium copy pET plasmid. In addition, improved expression of both redox partners after chromosomal integration resulted in up to 80% higher (S)-ketamine conversion and more than fourfold increase in total turnover numbers.