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Improving D-carbamoylase thermostability through salt bridge engineering for efficient D-p-hydroxyphenylglycine production

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Abstract

D-p-hydroxyphenylglycine (D-HPG) is an important intermediate in the pharmaceutical industry, and it is commonly synthesized by cascading D-hydantoinase (DHase) and D-carbamoylase (DCase). In this study, the stability of DCase was identified as the main problem that limits its application. Therefore, the complexed structure of AkDCase (DCase from the Agrobacterium sp. strain KNK712) with the substrate N-carbamoyl-D-p-hydroxyphenylglycine (CpHPG) (with 2.52 Å resolution) and catalytic mechanism were resolved. Based on the catalytic mechanism and electrostatic stabilization, salt bridge engineering was adopted to improve AkDCase thermostability. The best variant, AkDCaseD30A, increased the Tm by 2.91 °C and half-life (t1/2) at 40 and 60 °C by 18.43 h and 23.21 min, respectively. After AkDCaseD30A was assembled with GsDHase (DHase from Geobacillus stearothermophilus SD-1) in a single Escherichia coli cell, the recombinant strain could produce 29.53 g/L D-HPG within 12 h, with a 97% conversion and a 2.46 g/(L·h) space–time yield (STY). The titer of D-HPG increased by 40.55% compared to the E. coli cell harboring pETduet-1-AkDCase- GsDHase. The recombinant strain could be used for two cycles. Our research provides a basis for the industrial production of D-HPG.

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All data generated or analyzed during this study are included in this published article and its supplementary information files.

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Funding

This work was financially supported by the National Key R&D Program of China (Grant No. 2021YFC2100100), the General Program of National Natural Science Foundation of China (22178146), the Program for Young Talents in China, and the Fundamental Research Funds for the Central Universities (JUSRP622011).

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Authors and Affiliations

  1. School of Life Science and Health Engineering, Jiangnan University, Wuxi, 214122, China

    Landong Zhang, Wei Song & Jing Wu

  2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China

    Wei Song, Wanqing Wei, Cong Gao, Xiulai Chen, Jia Liu & Liming Liu

  3. Department of Cardiology, Affiliated Hospital of Jiangnan University, Wuxi, 214122, China

    Changzheng Gao

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  1. Landong Zhang
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Contributions

LDZ and CZG conceived the study. LDZ and WS made contributions to the design of the experiments, the acquisition of data, the analysis and interpretation of data and contributed to manuscript writing. WQW, CG, XLC, JL, LML and JW conceived and organized the study, helped to draft the manuscript and revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Jing Wu.

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Zhang, L., Gao, C., Song, W. et al. Improving D-carbamoylase thermostability through salt bridge engineering for efficient D-p-hydroxyphenylglycine production. Syst Microbiol and Biomanuf 4, 250–262 (2024). https://doi.org/10.1007/s43393-023-00176-1

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