Microbial synthesis of 2H-labelled L-phenylalanine with different levels of isotopic enrichment by a facultative methylotrophic bacterium Brevibacterium methylicum with RuMP assimilation of carbon

  • O. V. Mosin
  • V. I. Shvets
  • D. A. Skladnev
  • I. Ignatov
Article

Abstract

Using the L-phenylalanine secreting strain of Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via the ribulose-5-monophosphate (RuMP) cycle of carbon assimilation, as an example, we have continued studies on the use of methylotrophic bacteria for the preparative microbial synthesis of amino acids labeled with stable isotopes, including deuterium (2H), suitable for biomedical applications and clinical diagnostics. Here we demonstrate the data on adaptation of the methylotrophic bacterium B. methylicum to the maximal concentration of deuterium in the growth medium with 98% (v/v) 2H2O and 2% (v/v) [2H]MeOH, and biosynthesis of deuterium labeled L-phenylalanine with different levels of isotopic enrichment. The strain was adapted to 2H2O by means of plating of initial cells on solid (2% agarose) minimal growth media M9 with an increasing gradient of 2H2O concentration from 0, 24.5, 49.0, 73.5 up to 98% (v/v) 2H2O and subsequent selection of individual colonies stable to the action of 2H2O, which were capable to produce L-phenylalanine. L-phenylalanine was extracted from the growth medium with isopropanol followed by subsequent crystallization in ethanol (output 0.65 g/L). Using the developed method of microbial synthesis it is possible to obtain deuterated L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2H2O in growth media, from 17% (the growth medium with 24.5% (v/v) 2H2O) right up to 75% (the growth medium with 98% (v/v) 2H2O) of deuterium as evidenced by results of the electron impact (EI) mass-spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfonyl chloride (dansyl) phenylalanine isolated from growth media under different experimental conditions.

Keywords

Brevibacterium methylicum L-phenylalanine biosynthesis heavy water electron impact mass spectrometry 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Rerefences

  1. 1.
    LeMaster, D.M., Annu. Rev. Biophys. Chem., 1990, vol. 19, pp. 243–266.CrossRefGoogle Scholar
  2. 2.
    Vertes, A., in Elements and Isotopes: Formation, Transformation, Distribution, Vertes A., Ed., Dordrecht: Kluwer Acad. Publ., 2003, pp. 101–112.Google Scholar
  3. 3.
    Crespi, H.L., in Proceedings of the Second Inter. Symp. on Synthesis and Applications of Isotopically Labeled Compounds, Muccino, R.R., Ed., New York: Elsevier, 1986, pp. 111–112.Google Scholar
  4. 4.
    MacCarthy, P., J. Chem. Educ., 1985, vol. 62, pp. 633–638.CrossRefGoogle Scholar
  5. 5.
    Mosin, O.V., Skladnev, D.A., Egorova, T.A., and Shvets, V.I., Bioorgan. Khim., 1996, vol. 22, pp. 856–869.Google Scholar
  6. 6.
    Kushner, D.J., Baker, A., and Dunstall, T.G., Can. J. Physiol. Pharmacol., 1999, vol. 77, pp. 79–88.CrossRefGoogle Scholar
  7. 7.
    Skladnev, D.A., Mosin, O.V., Egorova, T.A., Eremin, S.V., and Shvets, V.I., Biotechnologiya, 1996, no. 5, pp. 25–34.Google Scholar
  8. 8.
    Mosin, O.V., Issledovanie metodov biotekhnologicheskogo polucheniya aminokislot, belkov i nukleozidov mechennykh stabil’nymi izotopami 2 H i 13 C s vysokimi urovnyami izotopnogo obogashcheniya (The study of methods of biotechnological preparation of amino acids, protein and nucleosides, labeled with stable isotopes 2H and 13C with high levels of isotopical enrichment), Extended abstract of candidate’s dissertation, Moscow Lomonosov State Academy of Fine Chemical Technology, 1996.Google Scholar
  9. 9.
    Dewick, P.M., in Medicinal Natural Products: A Biosynthetic Approach, Derwik, P.M., Ed., 3rd ed., Chichester John Wiley and Sons Ltd, 2009, pp. 137–186.Google Scholar
  10. 10.
    Karnaukhova, E.N., Mosin, O.V., and Reshetova, O.S., Amino Acids, 1993, vol. 5, pp. 125–126.Google Scholar
  11. 11.
    Mosin, O.V., Skladnev, D.A., Egorova, T.A., Yurkevich, A.M., and Shvets, V.I., Biotechnologiya, 1996, no. 3, pp. 3–12.Google Scholar
  12. 12.
    Mosin, O.V., Karnaukhova, E.N., Pshenichnikova, A.B., Skladnev, D.A., and Akimova, O.L., Biotechnologiya, 1993, no. 9, pp. 16–20.Google Scholar
  13. 13.
    de Boer, L., Harder, W, and Dijkhuizen, L., Arch Microbiol., 1988, vol.149, pp. 459–465.CrossRefGoogle Scholar
  14. 14.
    Bohinski, R., Modern Concepts in Biochemistry 4th ed., Boston: Allyn and Bacon Inc., 1987.Google Scholar
  15. 15.
    Herrmann, K.M., Weaver, L.M., Annu. Rev. Plant Physiol. Plant Mol. Biol., 1999, vol. 50, pp. 473–503.CrossRefGoogle Scholar
  16. 16.
    Rouse, B., Azen, B., Koch, R., Matalon, R., Hanley, W., de la Cruz, F., Trefz, F., Friedman, E., and Shifrin, H., Am. J. Med. Genet., 1997, vol. 69, pp. 89–95.CrossRefGoogle Scholar
  17. 17.
    Mosin, O.V., Skladnev, D.A., and Shvets, V.I., Biosci., Biotechnol. Biochem., 1998, vol. 62, pp. 225–229.CrossRefGoogle Scholar
  18. 18.
    Mosin, O.V., Skladnev, D.A., and Shvets, V.I., Biotekhnologiya, 2000, no. 10, pp. 16–23.Google Scholar
  19. 19.
    Trotzenko, Yu.A., Biokhimiya i fiziologiya metolotrofnykh organizmov (Biochemistry and physiology of methylotrophic microorganisms), A collection of articles, USSR Acad. Sci., Institute of Biochemistry and Physiology of Microorganisms, Pushchino, 1987, p. 15.Google Scholar

Copyright information

© Pleiades Publishing, Ltd. 2013

Authors and Affiliations

  • O. V. Mosin
    • 1
  • V. I. Shvets
    • 1
  • D. A. Skladnev
    • 2
  • I. Ignatov
    • 3
  1. 1.Lomonosov Moscow University of Fine Chemical TechnologyMoscowRussia
  2. 2.Institute for Genetics and Selection of Industrial MicroorganismsMoscowRussia
  3. 3.Scientific Research Center of Medical BiophysicsSofiaBulgaria

Personalised recommendations