Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation.
OBJECTIVE
To explore the potential to enhance secretion of heterologous proteins in Bacillus subtilis by engineering cell factors affecting extracytoplasmic protein folding and degradation.


RESULTS
Bottleneck components affecting the extracytoplasmic phase of protein secretion were genetically engineered and their effects on the secretion of 11 industrially interesting heterologous proteins were studied by Western blotting and enzymatic assays. Overproduction of PrsA lipoprotein enhanced the secretion of alpha-amylase of Bacillus stearothermophilus (fourfold) and pneumolysin (1.5-fold). Increasing the net negative charge of the cell wall because of lack of the d-alanine substitution of anionic cell wall polymers enhanced the secretion of pneumolysin c. 1.5-fold. Decreasing the level of HtrA-type quality control proteases caused harmful effects on growth and did not enhance secretion. Pertussis toxin subunit, S1 was found to be a substrate for HtrA-type proteases and its secretion was dependent on these proteases.


CONCLUSIONS
Secretion of heterologous proteins can be enhanced by engineering components involved in late stages of secretion in a protein-dependent manner.


CONCLUSIONS
The study revealed both possibilities and limitations of modulating the post-translocational phase of secretion as a means to improve the yield of heterologous proteins.
Vitikainen
M
M
Vaccine Development Laboratory, National Public Health Institute, Helsinki, Finland.
Hyyryläinen
H-L
HL
Kivimäki
A
A
Kontinen
V P
VP
Sarvas
M
M
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D002473:Cell Wall / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D001411:Geobacillus stearothermophilus / Q000378:metabolism; D008074:Lipoproteins / Q000096:biosynthesis; D008565:Membrane Proteins / Q000096:biosynthesis; D010447:Peptide Hydrolases / Q000378:metabolism; D037342:Pertussis Toxin / Q000378:metabolism; D011096:Polygalacturonase / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D012697:Serine Endopeptidases / Q000378:metabolism; D013301:Streptolysins / Q000378:metabolism; D014178:Translocation, Genetic; D000516:alpha-Amylases / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism
D001426:Bacterial Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D013301:Streptolysins; C083388:pertussis toxin, S1 subunit; C083389:pertussis toxin, S4 subunit; C032290:plY protein, Streptococcus pneumoniae; C082160:prsA protein, bacteria; D037342:Pertussis Toxin; D000516:alpha-Amylases; D011096:Polygalacturonase; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C419211:YkdA protein, Bacillus subtilis; C419213:YvtA protein, Bacillus subtilis; D001618:beta-Lactamases