The Strain of Facultative Methylotrophic Bacterium ?revibacterium Methylicum 5662 with RuMP Cycle of Carbon Assimilation for Biosynthesis of Deuterium Labeled Phenylalanine

Abstract

We used Gram-positive aerobic facultative methylotrophic bacterium, Brevibacterium methylicum, L-phenylalanine producer with ribulose-5-monophosphate (RuMP) cycle for carbon assimilation for microbiological preparation of [2H]phenylalanine via conversion of low molecular weight substrates ([U-2H]MeOH and 2H2O). For this purpose, the cells of the methylotroph with improved growth characteristics were used on minimal salt media M9 supplemented with 2% (v/v) [U-2H]MeOH and increasing gradient of 2?2O concentration from 0; 24.5; 49.0; 73.5 up to 98% (v/v) 2?2O. L-phenylalanine was isolated from the growth medium after adding 5 M 2HCl (in 2?2?), pH = 2.0 by extraction with isopropanol and subsequent crystallization in ethanol (output 0.65 g/l). Alanine, valine, and leucine/isoleucine were produced and accumulated exogenously in amounts of 5–6 ?mol in addition to the main product of biosynthesis. The method allows to obtain [2?]amino acids with different levels of deuterium enrichment, depending on 2?2O concentration in growth media, from 17% 2? (2 deuterium atoms) (on the growth medium with 24.5% (v/v) 2?2?) up to 75% 2? (6 deuterium atoms) (on the growth medium with 98% (v/v) 2?2?) with introduction of deuterium to benzyl ?6?5??2-fragment of molecule that is confirmed with the data of electron impact (EI) mass spectrometry analysis of methyl ethers of N-5-dimethylamino(naphthalene)-1-sulfochloride [2H]amino acids after the separation by reverse-phase HPLC. Keywords: Brevibacterium methylicum, [U-2H]MeOH, heavy water, biosynthesis, [2H]amino acids, EI mass spectrometry, HPLC.