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High-Throughput Screening of T7 Promoter Mutants for Soluble Expression of Cephalosporin C Acylase in E. coli

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Abstract

Cephalosporin C acylase (CCA) is the key enzyme in the production of 7-aminocephalosporanic acid (7-ACA) via a one-step enzymatic process. To improve the soluble expression level of CCA in recombinant Escherichia coli at elevated temperatures, a library of T7 promoter mutants was created by site-saturation mutagenesis, and a series of mutated promoters were subsequently screened. Green fluorescent protein (GFP) was fused to the C-terminus of CCA to facilitate library screening, and the expression of the CCA and GFP fusion proteins was investigated under the control of the T7 promoter. Twenty-four mutants were selected by detecting the fluorescence intensity of colonies on agar plates to form a library with different expression levels. The enzyme activities of the mutants were positively correlated with their fluorescence intensities. The highest enzyme activity among these mutant promoters was 1.3-fold higher than the enzyme activity resulting from the wild-type promoter when the cells were cultured at 32 °C for 16 h. In addition, the transcription and expression levels of several typical promoters were discussed, and the effects of GFP fusion on the enzyme activity of CCA were investigated.

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Acknowledgments

The authors would like to thank Shandong Lukang Pharmaceutical Co., Ltd. for the generous supply of CPC and 7-ACA. The authors thank Prof. Stephanopoulos for the gift of the plasmid pZE-GFP.

Funding

This work was supported by the National Natural Science Foundation of China (21276023; 21476025).

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Authors and Affiliations

  1. Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing, 100083, China

    Zihao Nie, Hui Luo, Jiufeng Li, Hongxu Sun, Ying Xiao, Ruiqi Jia & Tianjiao Liu

  2. Department of Environmental Engineering, University of Science and Technology Beijing, Beijing, 100083, China

    Yanhong Chang

  3. Beijing Key Laboratory of Resource-Oriented Treatment of Industrial Pollutants, Beijing, 100083, China

    Yanhong Chang

  4. Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China

    Huimin Yu & Zhongyao Shen

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  1. Zihao Nie
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Correspondence to Hui Luo.

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Nie, Z., Luo, H., Li, J. et al. High-Throughput Screening of T7 Promoter Mutants for Soluble Expression of Cephalosporin C Acylase in E. coli. Appl Biochem Biotechnol 190, 293–304 (2020). https://doi.org/10.1007/s12010-019-03113-y

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