Improved production of secreted heterologous enzyme in Bacillus subtilisstrain MGB874 via modification of glutamate metabolism and growth conditions

Microbial Cell Factories

Research

Eri Shimizu¹  Katsuya Ozaki²  Takuya Morimoto²  Yasushi Kageyama²  Kenji Manabe³  Katsutoshi Ara⁴  Naotake Ogasawara⁵  Shigehiko Kanaya⁶  Hiroki Takahashi⁷ 

[1] Analytical Science Laboratories, Kao Corporation, 2606 Akabane, 321-3497, Ichikai, Haga, Tochigi, Japan;Biological Science Laboratories, Kao Corporation, 2606 Akabane, 321-3497, Ichikai, Haga, Tochigi, Japan;Biological Science Laboratories, Kao Corporation, 2606 Akabane, 321-3497, Ichikai, Haga, Tochigi, Japan;Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, 630-0101, Ikoma, Nara, Japan;Fundamental Technology Research Laboratories, Kao Corporation, 623 Zi Ri Rd, 200241, Minhang, Shanghai, China;Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, 630-0101, Ikoma, Nara, Japan;Graduate School of Information Science, Nara Institute of Science and Technology, 8916-5 Takayama, 630-0101, Ikoma, Nara, Japan;Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, 260-8673, Chuo-ku, Chiba, Japan;

关键词: Bacillus subtilis;    Protein secretion;    Genome reduction;    Glutamate metabolism;   

DOI  :  10.1186/1475-2859-12-18

 received in 2012-12-09, accepted in 2013-02-06,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundThe Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular enzymes under batch fermentation conditions. We predicted that deletion of the gene for RocG, a bi-functional protein that acts as a glutamate dehydrogenase and an indirect repressor of glutamate synthesis, would improve glutamate metabolism, leading to further increased enzyme production. However, deletion of rocG dramatically decreased production of the alkaline cellulase Egl-237 in strain MGB874 (strain 874∆rocG).ResultsTranscriptome analysis and cultivation profiles suggest that this phenomenon is attributable to impaired secretion of alkaline cellulase Egl-237 and nitrogen starvation, caused by decreased external pH and ammonium depletion, respectively. With NH3-pH auxostat fermentation, production of alkaline cellulase Egl-237 in strain 874∆rocG was increased, exceeding that in the wild-type-background strain 168∆rocG. Notably, in strain 874∆rocG, high enzyme productivity was observed throughout cultivation, possibly due to enhancement of metabolic flux from 2-oxoglutarate to glutamate and generation of metabolic energy through activation of the tricarboxylic acid (TCA) cycle. The level of alkaline cellulase Egl-237 obtained corresponded to about 5.5 g l-1, the highest level reported so far.ConclusionsWe found the highest levels of production of alkaline cellulase Egl-237 with the reduced-genome strain 874∆rocG and using the NH3-pH auxostat. Deletion of the glutamate dehydrogenase gene rocG enhanced enzyme production via a prolonged auxostat fermentation, possibly due to improved glutamate synthesis and enhanced generation of metabolism energy.

【 授权许可】

Unknown   
© Manabe et al; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.