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Computer-aided engineering of adipyl-CoA synthetase for enhancing adipic acid synthesis

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Abstract

Objective

To enhance adipic acid production, a computer-aided approach was employed to engineer the adipyl-CoA synthetase from Thermobifida fusca by combining sequence analysis, protein structure modeling, in silico site-directed mutagenesis, and molecular dynamics simulation.

Results

Two single mutants of T. fusca adipyl-CoA synthetase, E210βN and E210βQ, achieved a specific enzyme activity of 1.95 and 1.84 U/mg, respectively, which compared favorably with the 1.48 U/mg for the wild-type. The laboratory-level fermentation experiments showed that E210βN and E210βQ achieved a maximum adipic acid titer of 0.32 and 0.3 g/L. In contrast, the wild-type enzyme yielded a titer of 0.15 g/L under the same conditions. Molecular dynamics (MD) simulations revealed that the mutants (E210βN and E210βQ) could accelerate the dephosphorylation process in catalysis and enhance enzyme activity.

Conclusions

The combined computational-experimental approach provides an effective strategy for enhancing enzymatic characteristics, and the mutants may find a useful application for producing adipic acid.

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Acknowledgements

We thank the National Key R&D Program of China (2019YFA0905502), the National Natural Science Foundation of China (21877053 and 31900066), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-24), and the Fundamental Research Funds for the Central Universities (JUSRP51705A, JUSRP11964) for financial support. We also acknowledge the Top-Notch Academic Programs Project of Jiangsu Higher Education Institutions (TAPP), the Open Project Program of China-Canada Joint Lab of Food Nutrition and Health, Beijing Technology and Business University (BTBU), and the Jiangsu Province Scientific Research Innovation Project for Postgraduates (KYLX16_0805) for supporting this project.

Supporting information

Supplementary Table S1—Strains and plasmids used in this work.

Supplementary Table S2—Primers used in this work.

Supplementary Table S3—Sequence identity between ACS and SCSs.

Supplementary Fig. S1—Multiple sequence alignment by Clustal Omega.

Author information

Authors and Affiliations

  1. National Engineering Laboratory for Cereal Fermentation Technology (NELCF), School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, People’s Republic of China

    Ju Yang, Yafeng Wei, Guohui Li, Shenghu Zhou & Yu Deng

  2. Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, People’s Republic of China

    Ju Yang, Yafeng Wei, Guohui Li, Shenghu Zhou & Yu Deng

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  1. Ju Yang
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  2. Yafeng Wei
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  3. Guohui Li
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  5. Yu Deng
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Correspondence to Yu Deng.

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Yang, J., Wei, Y., Li, G. et al. Computer-aided engineering of adipyl-CoA synthetase for enhancing adipic acid synthesis. Biotechnol Lett 42, 2693–2701 (2020). https://doi.org/10.1007/s10529-020-02978-y

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  • DOI: https://doi.org/10.1007/s10529-020-02978-y

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