Microbic synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium brevibacterium methylicum with RMP assimilation of carbon

Mosin O.V.1 , Shvets V.I.1, Skladnev D.A.2, Ignatov I.3

1. Lomonosov Moscow University of Fine Chemical Technology, Federation
2. Research Institute for Genetics and Selection of Industrial Microorganisms
3. Scientific Research Center of Medical Biophysics
Section: Experimental Study
DOI: 10.18097/pbmc20146004448      UDK: 579.871.08+577.385.4.08      PubMed Id: 25249528
Year: 2014  Volume: 60  Issue: 4  Pages: 448-461
The preparative microbial synthesis of amino acids labelled with stable isotopes, including deuterium ( 2 H), suitable for biomedical applications by methylotrophic bacteria was studied using L-phenylalanine as example. This amino acid is secreted by Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of assimilation of carbon, The data on adaptation of L-phenylalanine secreted by methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in the growth medium with 98% 2 Н 2 O and 2% [ 2 Н]methanol, and biosynthesis of deuterium labelled L-phenylalanine With different levels of enrichment are presented. The strain was adapted by means of plating initial cells on firm (2% agarose) minimal growth media with an increasing gradient of 2 Н 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% 2 Н 2 O followed by subsequent selection of separate colonies stable to the action of 2 Н 2 O. These colonies were capable to produce L-phenylalanine. L-phenylalanine was extracted from growth medium by extraction with isopropanol with the subsequent cristallization in ethanol (output 0.65 g/l). The developed method of microbial synthesis allows to obtain deuterium labelled L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2 Н 2 O in growth media, from 17% (on growth medium with 24,5% 2 Н 2 O) up to 75% (on growth medium with 98% 2 Н 2 O) of deuterium in the molecule that is confirmed with the data of the electron impact (EI) mass- spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) phenylalanine in these experimental conditions.
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Mosin O.V., Shvets V.I., Skladnev D.A., Ignatov I. (2014) Biomeditsinskaya khimiya, 60(4), 448-461.
This paper is also available as the English translation:10.1134/S1990750813030098
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