TY - JOUR
T1 - High external pH enables more efficient secretion of alkaline α-amylase AmyK38 by Bacillus subtilis
AU - Manabe, Kenji
AU - Kageyama, Yasushi
AU - Tohata, Masatoshi
AU - Ara, Katsutoshi
AU - Ozaki, Katsuya
AU - Ogasawara, Naotake
N1 - Funding Information:
We thank Junichi Sekiguchi (Shinshu University), Kouji Nakamura (University of Tsukuba), Shigehiko Kanaya, Shu Ishikawa (Nara Institute of Science and Technology), Fujio Kawamura (University of Rikkyo), and Yasutaro Fujita (Fukuyama University) for valuable discussions. We also thank Yoshiharu Kimura and Yoshinori Takema (Kao Corp.) for valuable advice. This study was performed in collaboration with Takuya Morimoto (Kao Corp.), Shengao Liu (Kao Corp.), Takeko Kodama (Kao Corp.), and Hiroshi Kakeshita (Kao Corp.). This research was conducted as part of the Project for the Development of a Technological Infrastructure for Industrial Bioprocesses through R&D on New Industrial Science and Technology Frontiers of the Ministry of Economy, Trade, and Industry (METI), Japan, and was supported by the New Energy and Industrial Technology Development Organization (NEDO), Japan.
PY - 2012/6/8
Y1 - 2012/6/8
N2 - Background: Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor) system.Results: Compared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH 3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l -1) of AmyK38 was obtained using strain MGB874.Conclusions: We demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38.
AB - Background: Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor) system.Results: Compared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH 3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l -1) of AmyK38 was obtained using strain MGB874.Conclusions: We demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38.
KW - AmyK38
KW - Bacillus subtilis
KW - Cssrs
KW - Genome reduction
KW - HtrA
KW - HtrB
KW - MGB874
KW - Secretion stress response
KW - α-amylase
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U2 - 10.1186/1475-2859-11-74
DO - 10.1186/1475-2859-11-74
M3 - Article
C2 - 22681752
AN - SCOPUS:84861936885
SN - 1475-2859
VL - 11
JO - Microbial Cell Factories
JF - Microbial Cell Factories
M1 - 74
ER -