Brevibacterium Methylicum Research Articles

Evaluation of Biosynthetic Pathways of 2Н- and 13С-Labeled Amino Acids by an Obligate Methylotrophic Bacterium Methylobacillus Flagellatum and a Facultative Methylotrophic Bacterium Brevibacterium Methylicum

Evaluation of Biosynthetic Pathways of 2Н- and 13С-Labeled Amino Acids by an Obligate Methylotrophic Bacterium Methylobacillus Flagellatum and a Facultative Methylotrophic Bacterium Brevibacterium Methylicum

English

By the method of microbiological synthesis were obtained and analyzed by electron impact mass-spectrometry 2H, 13C-labeled amino acids of a facultative methylotrophic bacterium Brevibacterium methylicum and an obligate methylotrophic bacterium Methylobacillus flagellatum and 2H, 13C-labeled amino acids of the total protein of biomass obtained on media containing as a source of stable isotopes [2H]methanol, [13C]methanol and 2H2O. It was also performed the incorporation of L-[2,3,4,5,6-2Н]phenylalanine, L-[3,5-2Н]tyrosine and L-[2,4,5,6,7-2Н]tryptopan into the membrane integral protein bacteriorhodopsin synthesised in purple membranes of photoorganotrophic halobacterium Halobacterium halobium ET 1001. For mass-spectrometric analysis the multicomponential mixtures of 2H- and 13C-labeled amino acids, derived from cultural media and protein hydrolysates after hydrolysis in 6 M 2HСl (3% phenol) and 2 M Ва(OH)2 were modified into N-benzyloxycarbonyl-derivatives of amino acids as well as into methyl esters of N-5(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl) derivatives of [2H, 13С]amino acids,

English

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 [ 2 H]phenylalanine via conversion of low molecular weight substrates ([U- 2 H]MeOH and 2 H2O). 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- 2 H]MeOH and increasing gradient of 2 Н2O concentration from 0; 24,5; 49,0; 73,5 up to 98 % (v/v) 2 Н2O. L-phenylalanine was isolated from the growth medium after adding 5 M 2 HCl (in 2 Н2О), 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 Н]amino acids with different levels of deuterium enrichment, depending on 2 Н2O concentration

Russian

With using of a strain of of L-phenylalanine secreted Gram-positive aerobic facultative methylotrophic bacteria Brevibacterium methylicum В-5652, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of carbon assimilation it was carried out the preparative microbiological synthesis of phenylalanine and metabolically related amino acids (alanine, valine, leucine/isoleucine in the amount of 5–6 mmol/l), labelled with deuterium (2H). The data on adaptation of L-phenylalanine secreted methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in minimal growth medium M9 with 98 % 2Н2O and 2 % [2Н]methanol, and data on biosynthesis of deuterium labelled L-phenylalanine with different levels of enrichment are submitted. The developed method for biosynthesis allows to obtain [2Н]phenylalanine with different levels of isotopic enrichment, depending on 2Н2O concentration in growth media M9, from 17 % (2 deuterium atoms) (on growth medium with 24,5 % 2Н2О) right up to 75 % (6 deuterium atoms) (on growth medium with 98 % 2Н2О) with introduction of deuterium to benzyl С6Н5СН2-fragment of molecule that is confirmed with the data of the electron impact (EI) mass-spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5- 25 European Journal of Molecular Biotechnology, 2014, Vol.(3), № 1 sulfochloride (dansyl) derivatives of [2Н]phenylalanine, isolated from the liquid culture after its separation by RP HPLC

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

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 crystallization 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.

Use of Gram-positive Chemoheterotrophic Bacterium Basillus subtilis В-3157 with HMP-cycle of Carbon Assimilation for Microbiological Synthesis of [2H]riboxine with High Level of Deuterium Enrichment

We studied the growth and biosynthetic properties of a strain of Gram-positive chemoheterotriphic bacterium Bacillus subtilis В-3157 producer of 2 H-labeled purine ribonucleoside riboxine (outcome is 3,9 g/l) in heavy hydrogen (HH) medium with high level of deuterium enrichment (99,8 at.% 2 H2O) with 2 % hydrolysate of deuterated biomass of methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of 2 H-labeled growth substrates, obtained in the minimal М9 growth medium of 98 % 2 Н2О and 2 % ( 2 H)methanol. Isolation of riboxine from liquid culture of riboxine producer strain was carried out by adsorption/resorption on a surface of activated carbon coal, extraction by 0.3 M NH4-formiate buffer (рН = 8.9) with the subsequent crystallization in 80 % of ethanol and column ion exchange chromatography on cation exchanger AG50WX 4, counterbalanced with 0.3 M NH4-formiate with 0.045 M NH4Cl (outcome of riboxine is 3.1 g/l (80 %)). The level of deuterium enrichment of biosynthetically prepared riboxine, analyzed by a method of fast atom bombardment (FAB) mass- spectrometry makes up 5 deuterium atoms with incorporation of 3 deuterium atoms into ribose and 2 deuterium atoms into hypoxantine fragments of the molecule.

Microbial synthesis of 2H-labelled L-phenylalanine with different levels of isotopic enrichment by a facultative methylotrophic bacterium Brevibacterium methylicum with RuMP assimilation of carbon

Microbial synthesis of 2H-labelled L-phenylalanine with different levels of isotopic enrichment by a facultative methylotrophic bacterium Brevibacterium methylicum with RuMP assimilation of carbon

Микробиологический синтез [2H]инозина высокого уровня дейтерированности грамположительной хемогетеротрофной бактериейBacillus subtilis

A 2H-labeled purine ribonucleoside inosine was microbiologically synthesized (yield, 3.9 g/L of culture liquid) using a deuterium-adapted strain ofthe gram-positive chemoheterotrophic bacterium Bacillus subtilis, cultivated in a heavy water medium with a high degree of deuteration (99.8 at % 2H) containing 2% hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum as a source of 2H-labeled growth substrate produced in an M9 minimal medium with 98% 2H20 and 2% [2H]-methanol. The inosine extracted from the culture liquid of the producer strain was fractionated by adsorption (desorption) on an activated carbon surface, extraction with 0.3 M ammonium-formate buffer (pH 8.9), subsequent crystallization in 80% ethanol, and ion exchange chromatography on a column with AG50WX 4 cation exchange resin equilibrated with 0.3 M ammonium-formate buffer containing 0.045 M NH4Cl. Fast atom bombardment (FAB) mass spectrometry demonstrated incorporation of five deuterium atoms in the inosine molecule (62.5% 2H), three of which were contained in the ribose moiety and two in the hypoxanthine moiety.

Biosynthesis of (2)H-Labeled Phenylalanine by a New Methylotrophic Mutant Brevibacterium methylicum.

The biosynthesis of (2)H-labeled phenylalanine was done by converse of low molecular weight substrates ([U-(2)H]methanol and (2)H2O) 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 (2)H-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-five, valine-five, and alanine-three deuterium atoms.

Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the Gram-positive methylotroph Brevibacterium methylicum by electrotransformation

Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells. In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B. methylicum transformants. With the exception of pZ6-1, all these plasmids were stably maintained in B. methylicum cells grown under non-selective conditions. When plasmid DNAs isolated from B. methylicum were used, the highest efficiency of transformation (10(5) transformants/micrograms DNA) was achieved.

Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the Gram-positive methylotroph Brevibacterium methylicum by electrotransformationy

Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the Gram-positive methylotroph Brevibacterium methylicum by electrotransformationy

Transformation of a new Gram-positive methylotroph, Brevibacterium methylicum, by plasmid DNA

Brevibacterium methylicum is a newly isolated Gram-positive facultatively methylotrophic bacterium that uses the NAD+-dependent methanol dehydrogenase for methanol oxidation and assimilates its carbon via the ribulose monophosphate cycle. Protoplasts prepared by lysozyme treatment of B. methylicum cells grown in the presence of glycine were transformed by plasmid shuttle vectors pCEM500 (16.5 kb; Smr/Spr, Kmr/Gmr) and pEC71 (7.1 kb; Kmr/Nmr) constructed on the basis of B. lactofermentum plasmid pAM330 and replicating in Escherichia coli and in amino-acid-producing coryneform bacteria. The resistance markers were found to be expressed in B. methylicum and autonomous plasmid DNAs of various size were isolated from the transformants. The presence of the pAM330 replicon in these plasmids was demonstrated by DNA-DNA hybridization experiments.