We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
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.
- Authors
Caspers, Michael; Brockmeier, Ulf; Degering, Christian; Eggert, Thorsten; Freudl, Roland
- Abstract
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.
- Subjects
SIGNAL peptidases; MUTAGENESIS; GENETIC mutation; BACILLUS subtilis biotechnology; MICROBIAL biotechnology; GRAM-positive bacteria; BACILLUS genetics; BIOCHEMICAL engineering; MICROBIOLOGY
- Publication
Applied Microbiology & Biotechnology, 2010, Vol 86, Issue 6, p1877
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-009-2405-x