Use of Whole Cells and Cell-Free Extracts of Catalase-Deficient E. coli for Peroxygenase-Catalyzed Reactions

Unspecific peroxygenases (UPOs) and cytochrome P450 monooxygenases (CYPs) are increasingly used as biocatalysts for oxyfunctionalization reactions. A group of researchers created a catalase-deficient E. coli strain using CRISPR/Cas to prevent hydrogen peroxide degradation, allowing for more efficient peroxygenase reactions. The study results indicate that using this strain for whole-cell or cell-free extracts (CFEs) improved hydrogen peroxide stability, enabling better performance in sulfoxidation, epoxidation, and benzylic hydroxylation reactions compared to wild-type strains, and allowed for high-throughput screening and preparative-scale reactions. The publication recently appeared in Biotechnology and Bioengineering. Below you can read the abstract. The link to the whole publication can be found in the reference at the bottom.

"Unspecific peroxygenases (UPOs) and cytochrome P450 monooxygenases (CYPs) with peroxygenase activity are becoming the preferred biocatalysts for oxyfunctionalization reactions. While whole cells (WCs) or cell-free extracts (CFEs) of Escherichia coli are often preferred for cofactor-dependent monooxygenase reactions, hydrogen peroxide (H₂O₂) driven peroxygenase reactions are generally performed with purified enzymes, because the catalases produced by E. coli are expected to quickly degrade H₂O₂. We used the CRISPR/Cas system to delete the catalase encoding chromosomal genes, katG, and katE, from E. coli BL21-Gold(DE3) to obtain a catalase-deficient strain. A short UPO, DcaUPO, and two CYP peroxygenases, SscaCYP_E284A and CYP102A1_21B3, were used to compare the strains for peroxygenase expression and subsequent sulfoxidation, epoxidation, and benzylic hydroxylation activity. While 10 mM H₂O₂ was depleted within 10 min after addition to WCs and CFEs of the wild-type strain, at least 60% remained after 24 h in WCs and CFEs of the catalase-deficient strain. CYP peroxygenase reactions, with generally lower turnover frequencies, benefited the most from the use of the catalase-deficient strain. Comparison of purified peroxygenases in buffer versus CFEs of the catalase-deficient strain revealed that the peroxygenases in CFEs generally performed as well as the purified proteins. We also used WCs from catalase-deficient E. coli to screen three CYP peroxygenases, wild-type SscaCYP, SscaCYP_E284A, and SscaCYP_E284I for activity against 10 substrates comparing H₂O₂ consumption with substrate consumption and product formation. Finally, the enzyme-substrate pair with highest activity, SscaCYP_E284I, and trans-β-methylstyrene, were used in a preparative scale reaction with catalase-deficient WCs. Use of WCs or CFEs from catalase-deficient E. coli instead of purified enzymes can greatly benefit the high-throughput screening of enzyme or substrate libraries for peroxygenase activity, while they can also be used for preparative scale reactions."

Ebrecht AC, Luelf UJ, Govender K, Opperman DJ, Urlacher VB, Smit MS, 2025, Use of Whole Cells and Cell-Free Extracts of Catalase-Deficient E. coli for Peroxygenase-Catalyzed Reactions, Biotechnology and Bioengineering, online access, https://doi.org/10.1002/bit.28959

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