Pharmaceutical Biotechnology (Track)


Oleg V. Mosin , and Ignat Ignatov

V. Lomonosov State University for Fine Chemical Technology, Moscow 119571, Russia


Deuterated cells of various industrial microorganisms adapted to maximum concentration of 2H2O in growth media (95–98 at.% 2H) are convenient sourses for preparation of highly deuterated biological active compounds as amino acids and proteins. Both these natural [2H]compounds may find their further application in biomedical diagnostics, methabolic and structural-functional 1H-NNR-studies, because it is preferable, that used compounds possess maximum levels of isotopic enrichment.

We have investigated the prospects of using two various taxonomic groups of industrial methylotrophic microorganisms – L-leucine synthesising (0.75 g/l) L-isoleucine auxotroph of Gram-nagative obligate methylotrophic bacterium Methylobacillus flagellatum assimilating methanol as a sole source of carbon and energy via 2-keto 3-deoxy 6-phospho-6-gluconate aldolase/transaldolase (KDPGA/TA) variant of ribulose monophosphate (RMP) cycle and L-phenylalanine synthesising (1 g/l) L-leucine auxotroph of Gram-negative facultative methylotrophic bacterium Brevibacterium methylicum, realizing the NAD+ dependent methanol dehydrogenase (EC variant of RMP cycle of carbon assimilation for preparation of [2H]amino acids with high levels of isotopic enrichment up to 75 at.% 2Н. For fermentation which was carried out under batch conditions was used minimal salt medium M9 (g/l) KH2PO4 – 3; Na2HPO4 – 6; NaCl – 0.5; NH4Cl – 1, supplemented with L-Ileu and L-Leu (100 mg/l) with 0.5–2% (v/v) [2Н]methanol (depending on physiological requirement of bacteria) and an increasing gradient of 2Н2O concentration from 0, 24.5, 49.0, 73.5 up to 98% (v/v) 2Н2O. Clones resistent to deuterium were selected on solid 2% (m/m) agarose media M9 with gradually increasing concentrations of 2H2O in the presence of 2% (v/v) [2H]metahnol (degree of cell survive on maximum deuterated salt medium was about 40%). Deuterated cells were able to growth on maximum deuterated liquid media M9 and produce exogeniously amino acids. The transfer of fully deuterated cells to ordinary protonated medium results eventually in normal growth. М. flagellatum exerts a hypersensevity ibility to 2H2O: the inhibition of bacterial growth was observed at 74.5% (v/v) 2H2O while [2H]methanol did not render essential influence on bacterial growth. On the contrary, B. methylicum was adapted to 98% (v/v) 2H2O in growth medium.

The production of L-phenylalanine by B. methylicum was linear with respect to the time up to exponentaly grown cells. During the fermentation the formation rate of L-phenylalanine was about  5 mmol/day. The level of phenylalanine production of non-adapted bacteria on maximally deuterated medium M9 was 0.39 g/liter after 80 hours of growth. The level of phenylalanine production for adapted bacteria under those growth conditions was 0.9 g/liter. Thus, the use of adapted bacteria, enabled us to improve the level of [2H]phenylalanine production on maximally deuterated medium M9 by 2.3 times. However, phenylalanine was not the only product of biosynthesis; other metabolically related amino acids (alanine, valine, andleucine/isoleucine) were also produced and accumulated exoginiously  into the growth medium in amounts of 5–6 mmol in addition to phenylalanine. This fact requires isolation of [2H]phenylalanine from growth medium, which was carried out by extraction with iso-PrOH and the subsequent cristallization in EtOH (output 0.65 g/l). Analytical separation of [2H]phenylalanine and metabolically related [2H]amino acids was performed using a reversed-phase HPLC method developed for methyl esters of N-DNS-[2H]amino acids with chromatographic purity of 96–98% and yield of 67–89%.

For studies of deuterium enrichment of [2H]amino acid molecules 1H-NNR and EI mass-spectrometry technic was applied on using methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) [2H]amino acids after the chemical derivatization of lyophilized growth media M9 with dansyl chloride and diazomethan. With diazomethan treatment it was occured the derivatization on aNH2-group in the molecule, so that its N-methylated derivative was formed to the addition of methyl ester of N-Dns-Phe. The experimental data testified to the character of labeling of amino acid molecules as heterogeneous; however, high levels of deuterium were detected in all presented amino acid molecules. With increasing of 2H2O content in growth media M9, the levels of deuterium enrichment were varried propotionaly. Thus, in experiment where used maximum deuterated medium M9 with 98% (v/v) 2H2O and 2% (v/v) [2H]methanol the enrichment of Phe was 6 (75 at.% 2H), leucine/isoleucine – 5 (50 at.% 2H), valine –5 (62.5 at.% 2H), and alanine – 3 (55 at.% 2H) deuterium atoms. The developed method allows to obtain [2Н]phenylalanine with different levels of isotopic enrichment, depending on concentration of 2Н2O in growth media, from 17 at.% 2Н (2 atoms 2Н) (on growth medium with 24.5 vol.% 2Н2О) up to 75 at.% 2Н (6 atoms 2Н) (on growth medium with 98% (v/v) 2Н2О) with introduction of deuterium into benzyl С6Н5СН2-fragment of Phe molecule that was confirmed with the data of EI mass- spectrometry and 1H-NNR. The enrichment level was nevertheless less than we estimated theoretically, because L-Leu was added into growth medium in protonated form. This fact should be seriously scrutinised before the applying of this methylotrophic bacterium for large-scale preparation of [2H]phenylalanine.