title: 13 C-metabolic flux analysis in heterologous cellulase production by Bacillus subtilis genome-reduced strain.
abstract: The great potential of Bacillus subtilis to produce biomaterials would be further enhanced by the development of strains with deletions of non-essential genomic regions. Here, using stationary (13)C-metabolic flux analysis ((13)C-MFA), we investigated the metabolism during cellulase production by the genome-reduced B. subtilis strain MGB874. We transformed MGB874 and wild-type strains with the heterologous cellulase gene, and cultured these on a synthetic medium containing glucose as carbon source. The addition of glutamate and the genome reduction enhanced cellulase production, which led us to use (13)C-MFA to assess the effects of glutamate addition and gene deletions on metabolism. We found that there was a significant increase in the flux in the pentose phosphate (PP) pathway, whereas the fluxes of reactions from acetyl-CoA to α-ketoglutarate were repressed in the presence of glutamate. We hypothesize that the increase in the PP pathway flux was caused by the decrease of citrate synthase flux through the accumulation of glycolytic intermediates. Excess NADPH produced by the PP pathway may affect the increase in cellulase production. Furthermore, the fluxes on glycolysis and the acetate formation of the cellulase-producing wild-type strain were significantly larger than that of the cellulase-producing MGB874 strain when the strains were cultured with glucose and glutamate.
authors.affiliation: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan; Advanced Low Carbon Technology Research and Development Program, Japan Science and Technology Agency (JST, ALCA), Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Toya
authors.affiliation: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan; Advanced Low Carbon Technology Research and Development Program, Japan Science and Technology Agency (JST, ALCA), Japan; Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori, Yokohama, Kanagawa 226-8501, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Hirasawa
authors.affiliation: Advanced Low Carbon Technology Research and Development Program, Japan Science and Technology Agency (JST, ALCA), Japan; Biological Science Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Morimoto
authors.affiliation: Advanced Low Carbon Technology Research and Development Program, Japan Science and Technology Agency (JST, ALCA), Japan; Biological Science Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Masuda
authors.affiliation: Advanced Low Carbon Technology Research and Development Program, Japan Science and Technology Agency (JST, ALCA), Japan; Biological Science Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kageyama
authors.affiliation: Advanced Low Carbon Technology Research and Development Program, Japan Science and Technology Agency (JST, ALCA), Japan; Biological Science Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Ozaki
authors.affiliation: Advanced Low Carbon Technology Research and Development Program, Japan Science and Technology Agency (JST, ALCA), Japan; Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
authors.forename: Naotake
authors.initials: N
authors.lastname: Ogasawara
authors.affiliation: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan; Advanced Low Carbon Technology Research and Development Program, Japan Science and Technology Agency (JST, ALCA), Japan. Electronic address: shimizu@ist.osaka-u.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shimizu
mesh_terms: D001412:Bacillus subtilis / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D002247:Carbon Isotopes / Q000032:analysis*; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics; D002950:Citrate (si)-Synthase / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D020043:Genes, Essential; D016680:Genome, Bacterial; D005947:Glucose / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D006019:Glycolysis; D010427:Pentose Phosphate Pathway
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D003470:Culture Media; D018698:Glutamic Acid; D002950:Citrate (si)-Synthase; D002480:Cellulase; C421179:Egl-237 protein, Bacillus; D005947:Glucose
keywords: (13)C-metabolic flux analysis; Bacillus subtilis; Cellulase production; Genome-reduced strain