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Accumulation of gene-targeted Bacillus subtilis mutations that enhance fermentative inosine production

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Abstract

In order to test a possible approach to enhance fermentative inosine production by Bacillus subtilis, seven gene-targeted mutations were introduced in the laboratory standard strain168 in a stepwise fashion. The mutations were employed in order to prevent inosine 5′-monophosphate (IMP) from being consumed for AMP and GMP synthesis, to minimize inosine degradation, and to expand the intracellular IMP pool. First, the genes for adenylosuccinate synthase (purA) and IMP dehydrogenase (guaB) were inactivated. Second, two genes for purine nucleoside phosphorylase, punA and deoD, were inactivated. Third, to enhance purine nucleotide biosynthesis, the pur operon repressor PurR and the 5′-UTR of the operon, containing the guanine riboswitch, were disrupted. Finally, the -10 sequence of the pur promoter was optimized to elevate its transcription level. The resulting mutant was capable of producing 6 g/L inosine from 30 g/L glucose in culture broth without the detectable by-production of hypoxanthine. This indicates the validity of this approach for the breeding of the next generation of B. subtilis strains for industrial nucleoside production.

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Acknowledgements

We are grateful to S. Kondo for technical assistance, including construction of plasmids and strains, and A. L. Sonenshein for careful reading of the manuscript and valuable comments.

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Authors and Affiliations

  1. Fermentation and Biotechnology Laboratories, Ajinomoto Co. Inc, 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-8681, Japan

    Takayuki Asahara & Yukiko Mori

  2. Ajinomoto-Genetika Research Institute, 1st Dorozhny Proezd, 1-1, Moscow, 117545, Russia

    Natalia P. Zakataeva, Vitaliy A. Livshits & Kiyoshi Matsuno

  3. Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Kobe, 657-8501, Japan

    Ken-ichi Yoshida

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  1. Takayuki Asahara
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  2. Yukiko Mori
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  3. Natalia P. Zakataeva
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  4. Vitaliy A. Livshits
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  5. Ken-ichi Yoshida
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Correspondence to Takayuki Asahara or Kiyoshi Matsuno.

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Asahara, T., Mori, Y., Zakataeva, N.P. et al. Accumulation of gene-targeted Bacillus subtilis mutations that enhance fermentative inosine production. Appl Microbiol Biotechnol 87, 2195–2207 (2010). https://doi.org/10.1007/s00253-010-2646-8

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