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High-Throughput Screening and Directed Evolution of Methionine Adenosyltransferase from Escherichia coli

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Abstract

S-adenosyl-L-methionine (SAM) is the active form of methionine, which participates in various metabolic reactions and plays a vital role. It is mainly used as a precursor by three key metabolic pathways: trans-methylation, trans-sulfuration, and trans-aminopropylation. Methionine adenosyltransferase (MAT) is the only enzyme to produce SAM from methionine and ATP. However, there is no efficient and accurate method for high-throughput detection of SAM, which is the major obstacles of directed evolution campaigns for MAT. Herein, we established a colorimetric method for directed evolution of MAT based on detecting SAM by using glycine oxidase and glycine/sarcosine N-methyltransferase enzyme. Screening of MAT libraries revealed variant I303V/Q22R with 2.13-fold improved activity towards SAM in comparison to the wild type. Molecular dynamic simulation indicates that the loops more flexible and more conducive to SAM release.

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Data Availability

The data presented in this study are available on request from the corresponding author.

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Funding

This study was funded by the National Key Research and Development Program of China (grant number 2021YFC2101000). This study was also funded by the National Natural Science Foundation of China (grant numbers 52073022, 21978017).

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Authors and Affiliations

  1. Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing, 100029, People’s Republic of China

    Chenqi Cao, Kaili Nie, Haijun Xu & Luo Liu

Authors
  1. Chenqi Cao
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  2. Kaili Nie
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  3. Haijun Xu
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  4. Luo Liu
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Chennqi Cao and Kaili Nie. The first draft of the manuscript was written by Chennqi Cao, Haijun Xu, and Luo Liu, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Luo Liu.

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Cao, C., Nie, K., Xu, H. et al. High-Throughput Screening and Directed Evolution of Methionine Adenosyltransferase from Escherichia coli. Appl Biochem Biotechnol 195, 4053–4066 (2023). https://doi.org/10.1007/s12010-023-04314-2

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  • DOI: https://doi.org/10.1007/s12010-023-04314-2

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