Cofactor-free light-driven whole-cell cytochrome P450 catalysis.

Area of Science:

• Biocatalysis
• Photochemistry
• Molecular Biology

Background:

• Cytochromes P450 (P450s) are crucial enzymes for catalyzing oxidation reactions.
• Traditional P450 biocatalysis often requires expensive cofactors and redox partners.
• Developing cofactor-free systems is essential for sustainable biocatalysis.

Purpose of the Study:

• To design a novel light-driven platform for cofactor-free, whole-cell P450 photo-biocatalysis.
• To utilize eosin Y (EY) as a photosensitizer for P450 activation.
• To demonstrate the broad applicability of the system with various P450s and substrates.

Main Methods:

• Engineered Escherichia coli for whole-cell P450 expression.
• Incorporated eosin Y (EY) as a photosensitizer that enters the bacterial cytoplasm.
• Utilized visible light illumination to activate the P450 catalytic cycle via photoinduced electron transfer from EY to the P450 heme domain.
• Tested the system with bacterial P450 variants and human P450s (CYPs).

Main Results:

• Successfully achieved cofactor-free, light-driven P450 photo-biocatalysis in whole cells.
• Demonstrated direct electron transfer from photosensitized EY to the P450 heme domain.
• Showcased the system's efficacy with diverse P450s, including P450 BM3 variants and human CYPs (1A1, 1A2, 1B1, 2A6, 2E1, 3A4).
• Successfully converted various substrates, including drugs (simvastatin, lovastatin, omeprazole) and a steroid (17β-estradiol).

Conclusions:

• Developed a generalizable, light-driven, cofactor-free platform for P450 biocatalysis.
• The system enables efficient P450 activation using visible light and a photosensitizer.
• This approach offers a sustainable and versatile alternative for P450-mediated biotransformations.