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Improvement of Sec-dependent secretion of a heterologous model protein in Bacillus subtilis by saturation mutagenesis of the N-domain of the AmyE signal peptide

By M. Caspers, U. Brockmeier, C. Degering, T. Eggert and R. Freudl


Due to the lack of an outer membrane, Gram-positive bacteria (e.g., Bacillus species) are considered as promising host organisms for the secretory production of biotechnologically relevant heterologous proteins. However, the yields of the desired target proteins were often reported to be disappointingly low. Here, we used saturation mutagenesis of the positively charged N-domain (positions 2-7) of the signal peptide of the Bacillus subtilis alpha-amylase (AmyE) as a novel approach for the improvement of the secretion of a heterologous model protein, cutinase from Fusarium solani pisi, by the general secretory pathway of B. subtilis. Automated high-throughput screening of the resulting signal peptide libraries allowed for the identification of four single point mutations that resulted in significantly increased cutinase amounts, three of which surprisingly reduced the net charge of the N-domain from +3 to +2. Characterization of the effects of the identified mutations on protein synthesis and export kinetics by pulse-chase analyses indicates that an optimal balance between biosynthesis and the flow of the target protein through all stages of the B. subtilis secretion pathway is of crucial importance with respect to yield and quality of secreted heterologous proteins

Topics: info:eu-repo/classification/ddc/570, Bacillus subtilis: genetics, Bacillus subtilis: metabolism, Bacterial Proteins: chemistry, Bacterial Proteins: genetics, Bacterial Proteins: metabolism, Biotechnology, Carboxylic Ester Hydrolases: genetics, Carboxylic Ester Hydrolases: metabolism, Fungal Proteins: genetics, Fungal Proteins: metabolism, Fusarium: enzymology, Fusarium: genetics, Mutagenesis, Mutant Proteins: metabolism, Peptide Library, Point Mutation, Protein Sorting Signals: genetics, Protein Structure, Tertiary, Recombinant Fusion Proteins: genetics, Recombinant Fusion Proteins: metabolism, alpha-Amylases: chemistry, alpha-Amylases: genetics, alpha-Amylases: metabolism, Bacterial Proteins, Fungal Proteins, Mutant Proteins, Peptide Library, Protein Sorting Signals, Recombinant Fusion Proteins, Carboxylic Ester Hydrolases, cutinase, alpha-Amylases, J, Heterologous protein secretion, Signal peptide, Saturation mutagenesis, Bacillus subtilis
Publisher: Springer
Year: 2010
DOI identifier: 10.1007/s00253-009-2405-x
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