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Enhancing the substrate tolerance of DszC by a combination of alanine scanning and site-directed saturation mutagenesis

  • Metabolic Engineering and Synthetic Biology - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

The biodesulfurization 4S pathway can specifically desulfurize an aromatic S heterocyclic compound (which is difficult to desulfurize by hydrodesulfurization) and maintain the integrity of its combustion value. The four Dsz enzymes in the pathway convert the model compound dibenzothiophene (DBT) into the sulfur-free compound 2-hydroxybiphenyl (HBP). DszC is the first enzyme in the 4S pathway and is subject to feedback inhibition and substrate inhibition. This study is the first attempt to further modify the DszC mutant AKWC to improve its tolerance to DBT. Alanine scanning was performed on the dimeric surface of the DszC mutant AKWC, and the HBP yield of the BAD (AKWCP413A) strain was increased compared to the BAD (AKWC) strain. Site-directed saturation mutagenesis was performed on the 413th amino acid of AKWC, and the substrate inhibition parameter KI value of the mutant AKWCPI was 5.6 times higher than that of AKWC. When the DBT concentration was 0.25 mM, the HBP production of the recombinant strain overexpressing AKWCPI was increased by approximately 1.4-fold compared to the BL21(DE3)/BADC*+C* strain. The protein engineering of DszC further improved the substrate tolerance after overcoming the feedback inhibition, which provided a reference for the analysis of the inhibition mechanism of DszC substrate. Overexpression of DszC-beneficial mutants also greatly improved the efficiency of desulfurization.

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Abbreviations

HDS:

Hydrodesulfurization

BDS:

Biodesulfurization

DBT:

Dibenzothiophene

HBP:

2-Hydroxybiphenyl

DBTO2 :

Dibenzothiophene sulfone

HBPS:

2-(2-Hydroxybiphenyl)-2-sulfonic acid salt

HPLC:

High-performance liquid chromatography

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Acknowledgements

This research was financially supported by the National Natural Science Foundation of China (Grant No. 31470159) and the National Science Foundation for Young Scientists of China (Grant No. 31400062).

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Author notes

  1. Lu Li and Lei Ye have contributed equally.

Authors and Affiliations

  1. Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China

    Lu Li, Ying Lin, Wei Zhang, Xihao Liao & Shuli Liang

  2. Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China

    Lu Li, Ying Lin, Wei Zhang, Xihao Liao & Shuli Liang

  3. Institute of Food Safety and Nutrition, Jinan University, Guangzhou, 510006, China

    Lei Ye

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  1. Lu Li
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Correspondence to Shuli Liang.

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Li, L., Ye, L., Lin, Y. et al. Enhancing the substrate tolerance of DszC by a combination of alanine scanning and site-directed saturation mutagenesis. J Ind Microbiol Biotechnol 47, 395–402 (2020). https://doi.org/10.1007/s10295-020-02274-8

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  • DOI: https://doi.org/10.1007/s10295-020-02274-8

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