The production of 2H-labeled amino acids by a new mutant of RuMP fucultative methylotroph Вrevibacterium methylicum
Oleg V. Mosin1
1 Department of Biotechnology, M. V. Lomonosov State Academy of Fine Chemical Technology, Vernadskogo Prospekt 86, 117571, Moscow, Russia
Summary
The biosynthesis of 2H-labeled phenylalanine was done by converse of low molecular weight substrates ([U- 2H]methanol and 2H2O) in a new RuMP facultative methylotrophic mutant Brevibacterium methylicum. To make the process work, adapted cells with improved growth characteristics were used on minimal medium M9 with the maximum content of 2H-labeled substrates. Alanine, valine, and leucine/isoleucine were produced and accumulated exogeneously in addition to the main product of biosynthesis. Electron impact mass spectrometry of methyl esters of the N-Dns-amino acid mixture obtained after the chemical derivatization of growth medium with dansyl chloride and diazomethane, was done to calculate the deuterium enrichment of the amino acids synthesized. The experimental data testified to the character of labeling of amino acid molecules as heterogeneous; however, high levels of deuterium enrichment were detected in all presented molecules - for phenylalanine the enrichment was six, leucine/isoleucine - 5.1, valine - 4.7, and alanine - 3.1 deuterium atoms.
Keywords: Brevibacterium methylicum - Heavy water - Biosynthesis - 2H-Labeled amino acids - Phenylalanine - EIMS
Abbreviations: EI MS: electron impact mass spectrometry; TLC: thin layer chromatography; DNSCl: dansylchloride; DZM: diazomethane; N-NMU: N-nitroso-methylurea; RuMP: rybolose monophosphate; PenP: pentose phosphate; PEP: phosphoenolpyruvate; ERP: erythrose-4-phosphate.
Introduction
Labeling of amino acid molecules with deuterium is becoming an essential part for various biochemical studies with 2H-labeled molecules and investigation of certain aspects of their biosynthesis(LeMaster, 1990).
For introduction of deuterium into amino acid molecules either chemical or biosynthetical methods may be used. Chemical synthesis of these compounds has one significant limitation; it is a very laborious and costly multistep process resulting in a mixture of dl-racemates. This major disadvantage, however essentially delaying its development is a difficulty in preparing the appropriate 2H-labeled amino acids. Chemical synthesis usually results in obtaining a mixture of d,l-racemates (Daub, 1979). Although chemomicrobiological synthesis overcomes this problem (Walker, 1986), the amount of purified enzymes required is prohibitive (Faleev, 1989). By growing algae on media with 96% (v/v) 2H2O, the desired 2H-labeled biochemicals can be produced both at high yields and enrichments (Cox, 1988), but the process involves algae is limited by the expense of a mixture of 2H-labeled amino acids isolated from hydrolysates of biomass (Daboll, 1962). The using for this purpose a certain methylotrophs which assimilates MetOH as a source of carbon and energy via RuMP cycle has a great practical advantage because their ability to produce and acumulate a gram quantities of 2H-labeled amino acids during the growth on media with 2H2O and [U -2H]MetOH and the comparatively low price of [U -2H]MetOH (Karnaukhova, 1994).
The biosynthesis of 2H-labeled amino acids usually involves growth of an organism on selective media containing the labeled substrates: e.g., growth of algae autotrophically on media with content of 2H2O 90% and more, is a well established method for biosynthesis of numerous highly deuterated molecules. But this method, while being generally applicable, is limited by the low resistance of plant cells to 2H2O and expense of 2H-labeled amino acids isolated from algae hydrolysates. Alternative and relatively inexpensive objects for biosynthesis of 2H-labeled amino acids seem certain auxotrophic mutants of methylotrophic bacteria using methanol as a main source of carbon and energy via the ribulose-5-monophosphate (RuMP) and the serine cycle of carbon assimilation. These bacteria have a big advantage because of their ability to produce and accumulate gram quantities of highly enriched, 2H-labeled amino acids during growth on minimal salt media with [U- 2H]methanol and 2H2O and the comparatively low price of [U- 2H]methanol. It is only in recent years that some progress was made in the isolation of a number of versatile the RuMP cycle methylotrophic bacteria, suitable for such studies, though the research that has been done with methylotrophs was limited and suffered from low growth characteristics on 2H2O-containing media. Although the production of 2H-labeled amino acids by obligate methylotroph Methylobacillus flagellatum described by Karnaukhova involves the growth on media with approximately 75% (v/v) 2H2O. We have recently selected a new mutant of facultative methylotroph Brevibacterium methylicum, realizing the NAD+ dependent methanol gehydrogenase (EC 1.6.99.3) variant of RuMP cycle of carbon assimilation, which seems more convinient for the preparation of 2H-labeled amino acids than M. flagellatum because its ability to grow on liquid M9 with 98% (v/v) 2H2O (Mosin, 1995).
Thus, we have previously studied the applicability of the RuMP cycle obligate methylotrophic bacterium Methylobacillus flagellatum for biosynthesis of 2H-labeled leucine 8). This approach is not yet practical for the biosynthesis of 2H-labeled phenylalanine, mainly because of the absence of suitable methylotrophic producer of this amino acid. ............
