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Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

preprint
submitted on 10.09.2019 and posted on 11.09.2019 by Huifang Xu, Weinan Liang, Linlin Ning, Yuanyuan Jiang, Wenxia Yang, Cong Wang, Feifei Qi, Li Ma, Lei Du, Laurent Fourage, Yongjin J. Zhou, Shengying Li
P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient Escherichia coli host strain and report an HTS approach based on colorimetric detection of H2O2-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H2O2 as co-substrate.

History

Email Address of Submitting Author

xu_hf@qibebt.ac.cn

Institution

Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences

Country

China

ORCID For Submitting Author

0000-0002-1963-1780

Declaration of Conflict of Interest

The authors declare no conflict of interest.

Version Notes

An earlier version of this manuscript was submitted to Angewandte Chemie International Edition on May 02, 2019. The current revised version has been submitted to ChemCatChem on July 26, 2019 following editor’s transfer suggestion.

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