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

• Published in: Applied microbiology and biotechnology Vol. 86; no. 6; pp. 1877 - 1885
• Main Authors: Caspers, Michael, Brockmeier, Ulf, Degering, Christian, Eggert, Thorsten, Freudl, Roland
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.05.2010; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: alpha-Amylases - chemistry; alpha-Amylases - genetics; alpha-Amylases - metabolism; Amino acids; Applied Genetics and Molecular Biotechnology; Automation; Bacillus; Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Carboxylic Ester Hydrolases - genetics; Carboxylic Ester Hydrolases - metabolism; Cloning; E coli; Enzymes; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Fungal Proteins - metabolism; Fusarium - enzymology; Fusarium - genetics; Fusarium solani pisi; Gram-positive bacteria; Life Sciences; Microbial Genetics and Genomics; Microbiology; Mutagenesis; Mutant Proteins - metabolism; Mutation; Peptide Library; Peptides; Plasmids; Point Mutation; Protein Sorting Signals - genetics; Protein Structure, Tertiary; Protein synthesis; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Studies; Saturation mutagenesis; Signal peptide; Heterologous protein secretion; Leader peptide; Bacillus subtilis; Secretion; Bacillaceae; Optimization; Bacillales; Mutagenesis; Bacteria; Models; Recombinant protein
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-009-2405-x

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 α-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.