Optimisation of Cytochrome P450 BM3 Assisted by Consensus-Guided Evolution.

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Industrial Chemistry

Background:

• Cytochrome P450 enzymes, particularly bacterial BM3 (CYP102A1), are valuable for oxygenating C-H bonds in chemical synthesis.
• BM3's solubility and fused redox partner facilitate its use, but instability and reliance on expensive NADPH limit industrial application.

Purpose of the Study:

• To enhance the performance of the BM3 enzyme using inexpensive cofactors NADH and NBAH.
• To engineer mutations in the reductase domain of BM3 to improve catalytic efficiency and product yield.

Main Methods:

• Site-directed mutagenesis was employed to introduce specific mutations (A769S, S847G, S850R, E852P, V978L) into a pre-existing BM3 mutant (R966D/W1046S).
• The performance of the engineered mutants (NTD5 and NTD6) was evaluated using NADH and NBAH as cofactors, measuring total product output.
• Comparison of mutant performance against the parent BM3 mutant and wild-type BM3 with NADPH.

Main Results:

• The NTD5 mutant, using NBAH, achieved a 5.24-fold increase in total product output compared to the R966D/W1046S mutant.
• The NTD6 mutant, using NADH, demonstrated a 2.3-fold increase in total product output compared to the R966D/W1046S mutant.
• The NTD6 mutant using NADH surpassed wild-type BM3's output with NADPH and retained high activity with NADPH.

Conclusions:

• Engineered mutations in the reductase domain of BM3 significantly enhance its utility with inexpensive cofactors.
• These advancements address key limitations of BM3, paving the way for more cost-effective industrial biocatalysis.
• The developed mutants offer a promising alternative for sustainable production of pharmaceuticals and fine chemicals.