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

• Main Authors: Oleg V. Mosin, PhD¹, Ignat Ignatov, ScD²
• Format: Article
• Language: English
• Published: International Medical Research and Development Corporation 2013-06-01
• Series: International Journal of Biomedicine
• Subjects: [2H]amino acids; biosynthesis; Brevibacterium methylicum; EI mass spectrometry; heavy water; [U-2H]methanol
• Online Access: http://www.ijbm.org/articles/3_2_Biotech1.pdf
• ISSN: 2158-0510; 2158-0529

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 1.6.99.3) 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 mol, 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.