Microbiological Synthesis of 2H-Labeled Phenylalanine, Alanine, Valine, and Leucine/Isoleucine with Different Degrees of Deuterium Enrichment by the Gram-Positive Facultative Methylotrophic Bacterium Вrevibacterium Methylicum

Oleg V. Mosin, PhD¹*, Ignat Ignatov, ScD²

¹Moscow State University of Applied Biotechnology, Moscow, Russian Federation

²Scientific Research Center of Medical Biophysics (SRCMB), Sofia, Bulgaria

* Corresponding author: Dr. Oleg V. Mosin, PhD. Scientist employee Moscow State University of Applied Biotechnology. 7-25 Mira st, Krasnogorsk, 143444, Moscovskaya oblast (mkr. Opalikha), Russian Federation. Tel8 (915) 054 79 73. E-mail: mosin-oleg@yandex.ru

Published: June 25, 2013


The microbiological synthesis of [2H]amino acids was performed by the conversion of low molecular weight substrates ([U-2H]MeOH and 2H2O)  using the Gram-positive aerobic facultative methylotrophic bacterium Brevibacterium methylicum, an L-phenylalanine producer, realizing the NAD+ dependent methanol dehydrogenase (EC variant of the ribulose-5-monophosphate (RuMP) cycle of carbon assimilation. In this process, the adapted cells of the methylotroph with enhanced growth characteristics were used on a minimal salt medium M9, supplemented with 2% (v/v) [U-2H]MeOH and an increasing gradient of 2Н2O concentration from 0; 24.5, 49.0; 73.5 up to 98% (v/v) 2Н2O. Alanine, valine, and leucine/isoleucine were produced and accumulated exogeneously in quantities of 5–6 mmol, in addition to the main product of biosynthesis. This method enables the production of [2Н]amino acids with different degrees of deuterium enrichment, depending on the 2Н2O concentration in the growth medium, from 17 at.% 2Н (on the growth medium with 24.5 % (v/v) 2Н2О) up to 75 at.% 2Н (on the growth medium with 98 % (v/v) 2Н2О). This has been confirmed with the data from the electron impact (EI) mass spectrometry analysis of the methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride [2H]amino acids under these experimental conditions.

[2H]amino acids; biosynthesis; Brevibacterium methylicum; EI mass spectrometry; heavy water; [U-2H]methanol.

1. LeMaster DM. Deuterium labeling in NMR structural analysis of larger proteins. Q Rev Biophys 1990; 23(2):133–174.

2. Vertes A. Physiological effects of heavy water. Elements and isotopes: formation, transformation, distribution. Dordrecht: Kluwer Acad Publ; 2004.

3. Blomquist AT, Cedergren RJ, Hiscock BF, Tripp SL, Harp DN. Synthesis of highly deuterated amino acids. Proc Natl Acad Sci USA 1966; 55(3):453–6.

4. Walker TE, Matheny C. An Efficient Chemomicrobiological Synthesis of Stable Isotope-Labeled L-Tyrosine and L-Phenylalanine. J Org Chem 1986; 51:1175–1179.

5. Faleev NG, Ruvinov SB, Saporovskaya MB, Belikov VM, Zakomyrdina LN. Preparation of a-Deuterated Amino Acids by E. coli Cells Containing Tryptophanase. Izv Akad Nauk USSR Ser Khim 1989; 10:2341–2343. [Article in Russian].

6. Cox J, Kyli D. Stable Isotope Labeled Biochemicals from Microalgae. Trends Biotechnol 1988; 6:279–282.

7. Crespi HL. Biosynthesis and uses of per-deuterated proteins. In: Muccino RR, editor. Synthesis and Applications of Isotopically labeled Compounds, Proceedings of the 2nd Intern Symp, Kansas City, Missouri, USA, 3–6 September 1985. Elsevier: Amsterdam-Oxford-New York; 1986:111-112.

8. Kushner DJ, Baker A, Dunstall TG. Pharmacological uses and perspectives of heavy water and deuterated compounds. Can J Physiol Pharmacol 1999; 77(2):79–88.

9. Mosin OV, Ignatov I. Isotope effects of deuterium in bacterial and microalgae cells at growth on heavy water (D2O). Water: Chemistry and Ecology 2012; 3:83–94. [Article in Russian].

10. Antony C. Bacterial oxidation of methane and methanol. Adv Microb Physiol 1986; 27:113-210.

11. Karnaukhova EN, Mosin OV, Reshetova OS. Biosynthetic production of stable isotope labeled amino acids using methylotroph Methylobacillus flagellatum. Amino Acids 1993; 5(1):125–126.

12. Skladnev DA, Mosin OV, Egorova TA, Eremin SV, Shvets VI. Methylotrophic bacteria are sourses of isotopically labelled 2Н- and 13С-amino acids. Biotechnologija 1996; 5:25–34. [Article in Russian].

13. Mosin OV, Skladnev DA, Shvets VI. Biosynthesis of 2H-labeled phenylalanine by a new methylotrophic mutant Brevibacterium methylicum. Biosci Biotechnol Biochem 1998; 62(2):225–229.

14. Conn EE. Recent Advances in Phytochemistry. In: Conn EE, editor. The Shikimic Acid Pathway, 2nd ed. New York: Plenum Press; 1986:20–22.

15. Herrmann KM, Weaver LM. The Shikimate Pathway. Ann Rev Plant Physiol Plant Mol Biol 1999; 50:473–503.

16. Antony C, Williams PW. The structure and function of methanol dehydrogenase. Biochim Biophys Acta 2003; 1467:18–23.

17. Lindstom ME, Stirling DI. Methylotrophs: genetics and commercial applications. Annu Rev Microbiol 1990; 44:27–58.

18. Wendisch VF. Amino Acid Biosynthesis – Pathways, Regulation and Metabolic Engineering. Springer –Verlag Berlin Heidelberg, 2007.

19. Boer L de, Harder W, Dijkhuizen L. Phenylalanine and Tyrosine Metabolism in the Facultative Methylotroph Nocardia sp. 239. Arch Microbiol 1988; 149:459–465.

20. Abou-Zeid A, Euverink G, Hessels GI, Jensen RA, Dijkhuizen L. Biosynthesis of L-Phenylalanine and L-Tyrosine in the Actinomycete Amycolatopsis methanolica. Appl Environ Microbiol 1995; 61(4):1298–302.

21. Maksimova NP, Dobrozhinetskaia EV, Fomichev IuK. Regulation of phenylalanine biosynthesis in the obligate methylotroph Methylobacillus M75. Mol Gen  Microbiol  Virusol 1990; 10:28–30. [Article in Russian].

22. Mosin OV, Skladnev DA, Egorova TA, Shvets VI. Mass-spectrometric determination of levels of enrichment of 2Н and 13С in molecules of amino acids of various bacterial objects. Bioorganic Chemistry 1996; 22(10–11):856–869. [Article in Russian].

The fully formatted PDF version is available.

Download Article

Int J Biomed. 2013; 3(2):132-138. © 2013 International Medical Research and Development Corporation. All rights reserved.