Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis.
Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis alpha-amylase (AmyL), Escherichia coli TEM beta-lactamase (Bla), human pancreatic alpha-amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of B. subtilis cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.
Bolhuis
A
A
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands.
Tjalsma
H
H
Smith
H E
HE
de Jong
A
A
Meima
R
R
Venema
G
G
Bron
S
S
van Dijl
J M
JM
D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009113:Muramidase / Q000276:immunology; D010179:Pancreas / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D011994:Recombinant Proteins; D000516:alpha-Amylases; D009113:Muramidase; D001618:beta-Lactamases