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.
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.
Caspers
Michael
M
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany.
Brockmeier
Ulf
U
Degering
Christian
C
Eggert
Thorsten
T
Freudl
Roland
R
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001709:Biotechnology; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology / Q000235:genetics; D016296:Mutagenesis; D050505:Mutant Proteins / Q000378:metabolism; D019151:Peptide Library; D017354:Point Mutation; D021382:Protein Sorting Signals / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D005656:Fungal Proteins; D050505:Mutant Proteins; D019151:Peptide Library; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D000516:alpha-Amylases