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Cloning, Expression, Mutagenesis Library Construction of Glycerol Dehydratase, and Binding Mode Simulation of Its Reactivase with Ligands

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Abstract

The production of 1, 3-propanediol (1, 3-PD) and 3-hydroxypropionaldehyde (3-HPA) by enzyme reaction has been a hot field, and glycerol dehydratase (GDHt) is the key and rate-limiting enzyme involved in their biosynthesis. The gldABC gene encoding GDHt was cloned from Klebsiella pneumoniae, and the activity of the corresponding proteins expressed extracellularly and intracellularly was 6.8 and 3.2 U/mg, respectively, about six and three times higher than that of the wild strain. The change of amino acids for the β subunit can adjust the length of the Co–N bond and affect the homolysis rate of the Co–C bond to change GDHt activity. The expression plasmid, pET-32a-gldAC (containing no gldB which encodes the β subunit of GDHt), was constructed to build the mutagenesis library to improve the GDHt activity. The binding models of glycerol dehydratase reactivation factor (GDHtR) with ATP, CTP, or GTP were simulated by semi-flexible docking, respectively, and there was almost no difference between them. This research provided the basis for studying the quantitative structure-activity relationships between GDHtR and its ligands, as well as searching inexpensive ligands to replace ATP. These results and methods are of great use in economical and highly efficient production of 3-HPA and 1, 3-PD by the enzyme method.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 41176111 and No. 41306124), the State Key Program of the National Natural Science Foundation of China (No. 21336009), the Fundamental Research Funds for the Central Universities (No. 2013121029), the Foundation of South Oceanographic Research Center of China in Xiamen (No. 14GYY011NF11), and the Public Science and Technology Research Funds Projects of Ocean (No. 201505032-6).

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Authors and Affiliations

  1. Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China

    Wei Jiang, Shizhen Wang & Baishan Fang

  2. The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China

    Wei Jiang, Shizhen Wang & Baishan Fang

  3. Hangzhou DAC Biotech Co., Ltd, Hangzhou, 310000, China

    Wenjun Li

  4. Jingdezhen Ceramic Institute of Materials Science and Engineering, Jingde Zhen, 333000, China

    Yan Hong

  5. The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian, 361005, China

    Baishan Fang

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  1. Wei Jiang
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Correspondence to Baishan Fang.

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Conflict of Interest

The authors state that they have no competing interests.

Authors’ Contributions

WJ designed and supervised the experiments and drafted this manuscript. WJL performed the binding modes and revised the manuscript. YH performed gene cloning and expression in E. coli, and revised the manuscript. SZW constructed a mutagenesis library and revised the manuscript. BSF conceived the study, designed and supervised the experiments, and is the corresponding author. All authors have read and approved the manuscript.

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Highlights

• The specific activities of pET-32a-gldABC and pET-22b-gldABC were six and three times those of the wild type.

• An expression plasmid without gldB was built to construct a mutagenesis library.

• The binding modes of the GDHtR with ATP, CTP, or GTP, respectively, were simulated.

• The binding modes were almost no different basis for finding out inexpensive ligands to replace ATP.

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Jiang, W., Li, W., Hong, Y. et al. Cloning, Expression, Mutagenesis Library Construction of Glycerol Dehydratase, and Binding Mode Simulation of Its Reactivase with Ligands. Appl Biochem Biotechnol 178, 739–752 (2016). https://doi.org/10.1007/s12010-015-1906-6

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  • DOI: https://doi.org/10.1007/s12010-015-1906-6

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