Expression of the pgsB encoding the poly-gamma-DL-glutamate synthetase of Bacillus subtilis (natto).
An industrial strain of Bacillus subtilis (natto) was used to produce poly-gamma-DL-glutamate (gammaPGA), a polymer of DL-glutamate linked by a gamma-peptide bond. In spite of efforts to improve gammaPGA production by modifying the medium, little attention has been paid to the expression of the gammaPGA synthetase gene. In this study, we investigated the expression of the gammaPGA synthetic gene and the gammaPGA product under various conditions with the LacZ-fusion of the synthetic gene (pgsB-lacZ). The 5' upstream regulatory region of the pgsB gene was also investigated by constructing deletion mutations of lacZ-fusion. The pgsB-lacZ was clearly expressed in the early stationary phase and was abolished by degU gene disruption. The results showed that pgsB-lacZ expression was repressed in rich media, and that gammaPGA production was limited by the substrate supply rather than by the amount of synthetase. Adding D-glutamate to the medium reduced gammaPGA production and synthetic gene expression. The transcription start point was determined by primer extension, and it was found that up to -721 bp (translation start point = +1) of the 5' untranslated region (UTR) was required for optimal pgsB-lacZ fusion gene expression.
Division of Applied Microbiology, National Food Research Institute, Tsukuba, Ibaraki, Japan. keitarou@affrc.go.jp
Keitarou
K
Kimura
Lam-Son Phan
LS
Tran
Thi-Huyen
TH
Do
Yoshifumi
Y
Itoh
D020121:5' Untranslated Regions / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D018628:Gene Dosage; D015870:Gene Expression / Q000187:drug effects; D005970:Glutamate Synthase / Q000235:genetics* / Q000378:metabolism; D009876:Operon / Q000235:genetics; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D017384:Sequence Deletion; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D003470:Culture Media; D011993:Recombinant Fusion Proteins; C511775:poly(gamma-glutamic acid); D011099:Polyglutamic Acid; D005970:Glutamate Synthase