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

Kenji Manabe, Yasushi Kageyama, Takuya Morimoto, Eri Shimizu, Hiroki Takahashi, Shigehiko Kanaya, Katsutoshi Ara, Katsuya Ozaki, Naotake Ogasawara

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Background: The 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{increment}rocG).Results: Transcriptome 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 NH 3 -pH auxostat fermentation, production of alkaline cellulase Egl-237 in strain 874{increment}rocG was increased, exceeding that in the wild-type-background strain 168{increment}rocG. Notably, in strain 874{increment}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.Conclusions: We found the highest levels of production of alkaline cellulase Egl-237 with the reduced-genome strain 874{increment}rocG and using the NH 3 -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.

Original languageEnglish
Article number18
JournalMicrobial Cell Factories
Volume12
Issue number1
DOIs
Publication statusPublished - 2013 Feb 18
Externally publishedYes

Fingerprint

Cellulase
Bacilli
Bacillus subtilis
Metabolism
Glutamic Acid
Enzymes
Fermentation
Growth
Glutamate Dehydrogenase
Genes
Genome
Citric Acid Cycle
Gene Deletion
Gene Expression Profiling
Starvation
Ammonium Compounds
Energy Metabolism
Nitrogen
Productivity
Chemical activation

Keywords

  • Bacillus subtilis
  • Genome reduction
  • Glutamate metabolism
  • Protein secretion

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Improved production of secreted heterologous enzyme in Bacillus subtilis strain MGB874 via modification of glutamate metabolism and growth conditions. / Manabe, Kenji; Kageyama, Yasushi; Morimoto, Takuya; Shimizu, Eri; Takahashi, Hiroki; Kanaya, Shigehiko; Ara, Katsutoshi; Ozaki, Katsuya; Ogasawara, Naotake.

In: Microbial Cell Factories, Vol. 12, No. 1, 18, 18.02.2013.

Research output: Contribution to journalArticle

Manabe, Kenji ; Kageyama, Yasushi ; Morimoto, Takuya ; Shimizu, Eri ; Takahashi, Hiroki ; Kanaya, Shigehiko ; Ara, Katsutoshi ; Ozaki, Katsuya ; Ogasawara, Naotake. / Improved production of secreted heterologous enzyme in Bacillus subtilis strain MGB874 via modification of glutamate metabolism and growth conditions. In: Microbial Cell Factories. 2013 ; Vol. 12, No. 1.
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abstract = "Background: The 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{increment}rocG).Results: Transcriptome 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 NH 3 -pH auxostat fermentation, production of alkaline cellulase Egl-237 in strain 874{increment}rocG was increased, exceeding that in the wild-type-background strain 168{increment}rocG. Notably, in strain 874{increment}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.Conclusions: We found the highest levels of production of alkaline cellulase Egl-237 with the reduced-genome strain 874{increment}rocG and using the NH 3 -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.",
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T1 - Improved production of secreted heterologous enzyme in Bacillus subtilis strain MGB874 via modification of glutamate metabolism and growth conditions

AU - Manabe, Kenji

AU - Kageyama, Yasushi

AU - Morimoto, Takuya

AU - Shimizu, Eri

AU - Takahashi, Hiroki

AU - Kanaya, Shigehiko

AU - Ara, Katsutoshi

AU - Ozaki, Katsuya

AU - Ogasawara, Naotake

PY - 2013/2/18

Y1 - 2013/2/18

N2 - Background: The 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{increment}rocG).Results: Transcriptome 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 NH 3 -pH auxostat fermentation, production of alkaline cellulase Egl-237 in strain 874{increment}rocG was increased, exceeding that in the wild-type-background strain 168{increment}rocG. Notably, in strain 874{increment}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.Conclusions: We found the highest levels of production of alkaline cellulase Egl-237 with the reduced-genome strain 874{increment}rocG and using the NH 3 -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.

AB - Background: The 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{increment}rocG).Results: Transcriptome 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 NH 3 -pH auxostat fermentation, production of alkaline cellulase Egl-237 in strain 874{increment}rocG was increased, exceeding that in the wild-type-background strain 168{increment}rocG. Notably, in strain 874{increment}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.Conclusions: We found the highest levels of production of alkaline cellulase Egl-237 with the reduced-genome strain 874{increment}rocG and using the NH 3 -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.

KW - Bacillus subtilis

KW - Genome reduction

KW - Glutamate metabolism

KW - Protein secretion

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DO - 10.1186/1475-2859-12-18

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JO - Microbial Cell Factories

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