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Development of high-performance nitrile hydratase whole-cell catalyst by automated structure- and sequence-based design and mechanism insights

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Abstract

Nitrile hydratase (NHase) is a metalloenzyme that catalyzes the conversion of nitrile to amide and is widely used in the biocatalysis of bulk chemicals such as acrylamide and nicotinamide. Improving the thermostability, activity, and soluble expression of natural NHase is crucial for its industrial application. However, conventional engineering strategies are often based on the design and evaluation of single-point mutations, followed by multiple rounds of iterative combinations, which are inefficient and difficult to predict the evolutionary direction of the combinatorial mutations due to epistatic effects. In this study, we used PROSS, an automated design tool based on structural and sequence information, to design a thermophilic NHase from Pseudonocardia thermophila JCM3095 (PtNHase). By sequentially applying subunit-independent mutations, subunit-synergistic mutations, and single-point revertant mutations, we obtained the superior mutant A2B1–β221. This mutant exhibited 1.4-fold and 2.3-fold higher activity towards acrylonitrile and 3-cyanopyridine, respectively, compared to the wild type. Additionally, A2B1–β221 showed a significant enhancement in thermostability. Moreover, benefiting from the enhanced soluble expression, a high-performance whole-cell catalyst for NHase was obtained. Furthermore, conventional molecular dynamics simulations and metadynamics simulations were employed to resolve the molecular mechanisms underlying the high activity and thermostability of A2B1–β221. This study not only provided highly efficient whole-cell catalyst for NHase, but also demonstrated the efficacy of utilizing automated design tools and molecular dynamics simulations in the engineering of heterologous multimeric proteins, offering valuable insights into their applicability.

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Data availability

All data included in this study are available upon request by contact with the corresponding author.

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Acknowledgements

This work is financially supported by the National Natural Science Foundation of China (32201034, 32271301), China Innovation Challenge (NingBo) Major Project (2023T020), the Natural Science Foundation of Jiangsu (BK20210470). Many thanks to Professor Lukasz Peplowski of the Nicolaus Copernicus University in Toruń, Poland, for generously sharing the topology and force field parameter files of the NHase active site, which supported the MD simulations in this study. The MD simulation and structural analysis in this study were supported by the high-performance computing cluster platform of the School of Biotechnology, Jiangnan University.

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  1. Key Laboratory of Industrial Biotechnology (Ministry of Education), School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China

    Meng Li, Dong Ma, Zhongyi Cheng, Qiong Wang, Zhemin Zhou & Laichuang Han

  2. Jiangnan University (Rugao) Food Biotechnology Research Institute, Rugao, Jiangsu, China

    Zhemin Zhou

  3. Ningbo Institute of Marine Medicine, Peking University, Ningbo, Zhejiang, China

    Jun Qiao

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LH and ZZ conceived the project and wrote the paper. ML, DM, JQ and ZC designed and performed all the experiments. LH and QW performed structural analysis and computational simulation. ML, ZZ and LH analyzed the results.

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Li, M., Ma, D., Qiao, J. et al. Development of high-performance nitrile hydratase whole-cell catalyst by automated structure- and sequence-based design and mechanism insights. Syst Microbiol and Biomanuf (2024). https://doi.org/10.1007/s43393-024-00239-x

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