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Glutamyl- and Glutaminyl-tRNA Synthetases Are a Promising Target for the Design of an L-Threonine–Producing Strain

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Abstract

The present work describes an approach that uses a reduction in biomass accumulation during fermentation to improve the properties of a strain producing L-threonine. Glutamyl- and glutaminyl-tRNA synthetases were chosen as targets. Mutants carrying temperature-sensitive alleles of the mentioned enzymes were obtained. It was shown with this system that suppression of the function of tRNA synthetases led to the rapid arrest of culture growth and an increase in the productivity and conversion of L-threonine synthesis. One of the temperature-sensitive strains was used to obtain mutants with the ts phenotype under nonpermissive conditions. Some of these mutants accumulated less biomass and produced 10–12% more threonine than the original strain.

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REFERENCES

  1. Yuzbashev, T.V., Vybornaya, T.V., Larina, A.S., et al., Directed modification of Escherichia coli metabolism for the design of threonine-producing strains, Appl. Biochem. Microbiol., 2013, vol. 49, no. 9, pp. 723–742. https://doi.org/10.1134/S0003683813090056

    Article  CAS  Google Scholar 

  2. Lee, K.H., Park, J.H., Kim, T.Y., et al., Systems metabolic engineering of Escherichia coli for L-threonine production, Mol. Syst. Biol., 2007, vol. 3, no. 149. https://doi.org/10.1038/msb4100196

  3. Okamoto, K., Kino, K., and Ikeda, M., Hyperproduction of L-threonine by an Escherichia coli mutant with impaired L-threonine uptake, Biosci. Biotechnol. Biochem., 1997, vol. 61, no. 11, pp. 1877–1882. https://doi.org/10.1271/bbb.61.1877

    Article  CAS  PubMed  Google Scholar 

  4. Haseltine, W.A. and Block, R., Synthesis of guanosine tetra-and pentaphosphate requires the presence of a codon-specific, uncharged transfer ribonucleic acid in the acceptor site of ribosomes, Proc. Natl. Acad. Sci. U. S. A., 1973, vol. 70, no. 5, pp. 1564–1568. https://doi.org/10.1073/pnas.70.5.1564

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Potrykus, K. and Cashel, M., (p)ppGpp: still magical?, Ann. Rev. Microbiol., 2008, vol. 62, pp. 35–51. https://doi.org/10.1146/annurev.micro.62.081307.162903

    Article  CAS  Google Scholar 

  6. Paul, B.J., Berkmen, M.B., and Gourse, R.L., DksA potentiates direct activation of amino acid promoters by ppGpp, Proc. Natl. Acad. Sci. U. S. A., 2005, vol. 102, no. 22, pp. 7823–7828. https://doi.org/10.1073/pnas.0501170102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Taguchi, M., Izui, K., and Katsuki, H., Activation of Escherichia coli phosphoenolpyruvate carboxylase by guanosine-5′-diphosphate-3′-diphosphate, FEBS Lett., 1977, vol. 77, no. 2, pp. 270–272. https://doi.org/10.1016/0014-5793(77)80249-4

  8. Pao, C.C. and Dyess, B.T., Effect of unusual guanosine nucleotides on the activities of some Escherichia coli cellular enzymes, Biochim. Biophys. Acta, 1981, vol. 677, nos. 3–4, pp. 358–362. https://doi.org/10.1016/0304-4165(81)90247-6

  9. Bubnov, D.M., Yuzbashev, T.V., Vybornaya, T.V., et al., Development of new versatile plasmid-based systems for λRed-mediated Escherichia coli genome engineering, J. Microbiol. Methods, 2018, vol. 151, pp. 48–56. https://doi.org/10.1016/j.mimet.2018.06.001

    Article  CAS  PubMed  Google Scholar 

  10. Lutz, R. and Bujard, H., Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements, Nucleic Acids Res., 1997, vol. 25, no. 6, pp. 1203–1210. https://doi.org/10.1093/nar/25.6.1203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Thomason, L.C., Costantino, N., and Court, D.L., E. coli genome manipulation by P1 transduction, Curr. Protoc. Mol. Biol., 2007, vol. 1, pp. 1–17. https://doi.org/10.1002/0471142727.mb0117s79

    Article  PubMed  Google Scholar 

  12. Vybornaya, T.V., Yuzbashev, T.V., Fedorov, A.S., et al., The use of an alternative pathway for isoleucine synthesis in Escherichia coli strains–threonine producers, Biotekhnologiya, 2019, vol. 35, no. 4, pp. 42–54. https://doi.org/10.21519/0234-2758-2019-35-4-42-54

    Article  Google Scholar 

  13. Korner, A., Magee, B.B., Liska, B., et al., Isolation and partial characterization of a temperature-sensitive Escherichia coli mutant with altered glutaminyl-transfer ribonucleic acid synthetase, J. Bacteriol., 1974, vol. 120, no. 1, pp. 154–158.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Isaksson, L.A., Skold, S.E., Skjoldebrand, J., et al., A procedure for isolation of spontaneous mutants with temperature sensitive of RNA and/or protein, Mol. Gen. Genet., 1977, vol. 156, no. 3, pp. 233–237. https://doi.org/10.1007/bf00267177

    Article  CAS  PubMed  Google Scholar 

  15. Englisch-Peters, S., Conley, J., Plumbridge, J., et al., Mutant enzymes and tRNAs as probes of the glutaminyl-tRNA synthetase: tRNA (Gln) interaction, Biochimie, 1991, vol. 73, no. 12, pp. 1501–1508. https://doi.org/10.1016/0300-9084

  16. Kaspy, I., Rotem, E., Weiss, N., et al., HipA-mediated antibiotic persistence via phosphorylation of the glutamyl-tRNA-synthetase, Nat. Commun., 2013, vol. 4, no. 3001. https://doi.org/10.1038/ncomms4001

  17. Das, A. and Wolska, K., Transcription antitermination in vitro by lambda N gene product: requirement for a phage nut site and the products of host nusA, nusB, and nusE genes, Cell, 1984, vol. 38, no. 1, pp. 165–173. https://doi.org/10.1016/0092-8674

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ACKNOWLEDGMENTS

The work was carried out on the equipment of the Multipurpose Scientific Installation of All-Russia Collection of Industrial Microorganisms of the Kurchatov Institute National Resource Center, GOSNIIgenetika.

Funding

This work was supported by the Ministry of Science and Higher Education of the Russian Federation (Project no. RFMEFI61017X0011).

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Authors and Affiliations

  1. State Research Institute for Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center (Kurchatov Institute NRC, GOSNIIgenetika), 117545, Moscow, Russia

    D. M. Bubnov, T. V. Yuzbashev, A. S. Fedorov, F. V. Bondarenko, T. V. Vybornaya, S. S. Filippova & S. P. Sineoky

  2. Department of Bioengineering, Imperial College of London, SW72AZ, London, Great Britain

    T. V. Yuzbashev

  3. Department of Integrated Biosciences, University of Tokyo, Tokyo, Japan

    A. S. Savchenko

Authors
  1. D. M. Bubnov
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  2. T. V. Yuzbashev
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  3. A. S. Fedorov
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  4. F. V. Bondarenko
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  5. A. S. Savchenko
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  6. T. V. Vybornaya
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  7. S. S. Filippova
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  8. S. P. Sineoky
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Corresponding author

Correspondence to D. M. Bubnov.

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The authors declare that they have no conflicts of interest.

This article does not contain any studies involving animals performed by any of the authors.

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Translated by I. Gordon

Abbreviations: CL—culture liquid; fbr—resistant to feedback inhibition; LB medium—lysogeny-broth medium; OD600—optical density at a wavelength of 600 nm; ppGpp—guanosine tetraphosphate; pppGpp—guanosine pentaphosphate; SpR—spectinomycin resistance; ts—temperature-sensitive.

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Bubnov, D.M., Yuzbashev, T.V., Fedorov, A.S. et al. Glutamyl- and Glutaminyl-tRNA Synthetases Are a Promising Target for the Design of an L-Threonine–Producing Strain. Appl Biochem Microbiol 56, 837–846 (2020). https://doi.org/10.1134/S0003683820080037

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