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

• Published in: Applied biochemistry and biotechnology 2020-01, Vol.190 (1), p.293-304
• Main Authors: Nie, Zihao, Luo, Hui, Li, Jiufeng, Sun, Hongxu, Xiao, Ying, Jia, Ruiqi, Liu, Tianjiao, Chang, Yanhong, Yu, Huimin, Shen, Zhongyao
• Format: Article
• Language: English
• Subjects: Amidohydrolases - genetics; Amidohydrolases - metabolism; Aminocephalosporanic acid; Antibiotics; Bacteriophage T7 - genetics; Biochemistry; Biotechnology; C-Terminus; Cephalosporin C; Cephalosporin C acylase; Cephalosporins - metabolism; Chemistry; Chemistry and Materials Science; E coli; Enzymatic activity; Enzyme activity; Enzymes; Escherichia coli - genetics; Fluorescence; Genes, Viral; Green fluorescent protein; Green Fluorescent Proteins - genetics; High temperature; High-throughput screening; High-Throughput Screening Assays; Levels; Libraries; Mutants; Mutation; Promoter Regions, Genetic; Promoters; Proteins; Saturation mutagenesis; Screening; Transcription; Transcription, Genetic
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-019-03113-y

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.