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Regulatory mode of the pac gene expression
1Shanghai Research Center of Biotechnology, Chinese Academy of Sciences, Shanghai.
Chinese Journal of Biotechnology
|January 1, 1992
Summary
Titrating the pac operator in E. coli D816 cells enhances penicillin acylase production. This occurs because increased operator copies on a plasmid reduce regulatory protein binding, boosting pac gene expression at the transcriptional level.
Area of Science:
- Molecular Biology
- Microbial Genetics
Background:
- Penicillin acylase (pac) gene expression is crucial for industrial applications.
- Understanding gene regulation mechanisms is key to optimizing enzyme production.
Purpose of the Study:
- To investigate the effect of operator titration on pac gene expression in E. coli.
- To elucidate the regulatory mechanism controlling pac gene transcription.
Main Methods:
- Cloning a DNA fragment containing the pac operator into a high-copy-number plasmid (pPA41).
- Transformation of E. coli D816 with the constructed plasmid.
- Quantification of penicillin acylase activity and pac mRNA levels via RNA-DNA hybridization.
Main Results:
- E. coli cells harboring the pPA41 plasmid showed significantly higher penicillin acylase activity.
- Pac mRNA concentration was elevated in cells with the pPA41 plasmid, correlating with enzyme activity.
- Operator titration on the plasmid competed with chromosomal operators, reducing free regulatory proteins.
Conclusions:
- Pac gene expression is negatively regulated at the transcriptional level.
- Operator titration is a viable strategy to enhance pac gene expression and penicillin acylase production.