Coexpression of the type I signal peptidase gene sipM increases recombinant protein production and export in Bacillus megaterium MS941.
The removal of the signal peptide from a precursor protein is a crucial step of protein secretion. In order to improve Bacillus megaterium as protein production and secretion host, the influence of homologous type I signal peptidase SipM overproduction on recombinant Leuconostoc mesenteroides dextransucrase DsrS synthesis and export was investigated. The dsrS gene was integrated as a single copy into the chromosomal bgaM locus encoding beta-galactosidase. Desired clones were identified by blue-white selection. In this strain, the expression of sipM from a multicopy plasmid using its own promoter increased the amount of secreted DsrS 3.7-fold. This increase in protein secretion by SipM overproduction was next transferred to a high level DsrS production strain using a multicopy plasmid encoding sipM with its natural promoter and dsrS under control of a strong xylose-inducible promoter. No further increase in DsrS export were observed when this vector was carrying two sipM copies. Similarly, bicistronic sipM and dsrS high level expression did not enhance DsrS secretion, indicating the natural limitation of the approach. Interestingly, SipM-enhanced DsrS secretion also resulted in an overall increase of DsrS production.
Malten
Marco
M
Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
Nahrstedt
Hannes
H
Meinhardt
Friedhelm
F
Jahn
Dieter
D
D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D002876:Chromosomes, Bacterial; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007934:Leuconostoc / Q000201:enzymology / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000378:metabolism
D008565:Membrane Proteins; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C020025:dextransucrase; D001616:beta-Galactosidase; D012697:Serine Endopeptidases; C026515:type I signal peptidase