The production of 2H-labeled amino acids by a new mutant of RuMP fucultative methylotroph Вrevibacterium methylicum

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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. After selecting a new the RuMP cycle methylotrophic producer of phenylalanine leucine auxotroph Brevibacterium methylicum we have used this strain for this research.

Material and methods

2H2O (99.9 at.% 2H) was purchased from Russian Scientific Enterprises Sankt Petersburg. [U -2H]MetOH (97.5 at.% 2H) was from Biophysic Center Pushino. DNSCl of sequential grade was from Sigma Chemicals Corp. USA. DZM was prepared from N-NMU Pierce Chemicals Corp. USA. A gram-positive parental strain of RuMP facultative methylotroph Brevibacterium methylicum # 5662 was obtained from Russian State Scientific Center for Genetics and Selection of Industrial Microorganisms GNIIGENETIKA (Nesvera 1991).

            Basal salt medium M9 (Miller 1976) with MetOH as a carbon and energy source (2% v/v) and supplemented with Leu (100 mg/l) was used for bacterial growth. For isotopic experiments M9 was enriched with [U -2H]MetOH and 2H2O of various content (see Table below). The bacterial growth was carried out under batch conditions (Karnaukhova 1994). The exponentially growing cells (cell density 2.0 at absorbance 540 nm) were pelleted by centrifugation (1200 g for 15 min) the supernatant was lyophilized and used for chemical derivatization.

            The amount of Phe was determined for 10 ml aliquotes of liquid M9 by TLC with solvent of iso-PrOH-ammonia (7:3 v/v) using pure commercial available Phe as a standard. The spots were detected by 0.1% ninhydrine solution in acetone eluted by 0.5% CdCl2 solution in 50% EtOH (2 ml). The absorbance of the eluates was measured at 540 nm the concentration was calculated using a standard curve.

            The samples of lyophilized M9 were dansylated in 1 M sodium hydrohycarbonate-acetone (1:2 v/v) solution (pH 10-11) with tenfold excess of DNSCl and treated according to Devenyi (1976). The derivatization to methyl esters of N-DNS-amino acids was performed in a standard procedure with DZM (Greenstein 1976).

            EI MS was performed on Hitachi MB 80 spectrometer at ionizing energy 70 eV and an ion source temperature of 180oC.

 

Results and discussion

Phe is synthesized in most bacteria via shikimic acid pathway (Conn 1986). The precursors for the biosynthesis of Phe are PEP and ERP. The latter compound is an intermediate in the PenP pathway and in some methylotrophs the RuMP cycle of carbon assimilation (Antony 1982; Kletsova 1988). It is widely accepted that the native bacterial strains can not to be a strong producers of Phe owing to the effective mechanisms of its metabolic regulation although certain bacterial mutants with mutations of prephenate dehydrogenase (EC 1.3.1.12) prephenate hydratase (EC 4.2.1.51) chorismate mutase (EC 5.4.99.5) and a number of other several enzymes are proved to be an active producers of this amino acid (Umbarger 1978). That is why the best Phe producing strains once selected were the mutants partially or completely dependent on Tyr or Trp for growth. The reports about the other regulative mechanisms of Phe biosynthesis in bacterial cell are quite uncommon though today it is known a large number of RuMP cycle auxotrophic mutants of methylotrophs covering numerious steps in aromatic amino acid biosynthesis  (Dijkhuizen 1996). The selection of new producers of Phe has a big importance for studies of their regulating pathways and possible production of 2H-labeled Phe.

            For our studies we have used a new non-traditional producer of phenylalanine: a leucine auxotroph of the facultative methylotrophic bacterium Brevibacterium methylicum obtaining the NAD+ dependent methanol dehydrogenase (EC 1.6.99.3) variant of the RuMP cycle of carbon assimilation with maximum productivity of phenylalanine on protonated medium M9 - 0.95 gram per liter of growth medium. According to experiments various compositions of  [U- 2H]methanol and 2H2O were added to the growth media as hydrogen (deuterium) atoms could be assimilated both from methanol and H2O. The growth characteristics of the non-adapted bacteria and production of phenylalanine in the presence of increasing content of 2H2O are given in Table (Expts. 3-10) relative to the control (1) on protonated medium and to the adapted bacteria (Expt. 10’). The odd numbers of experiment were chosen to investigate whether the replacement of [U -2H]methanol of its protonated analogue has a positive effect on growth characteristics in the presence of 2H2O. The maximum deuterium content was reached under conditions (10) and (10’) in which we used 98% (v/v) 2H2O and 2% (v/v) [U -2H]methanol. In the control the duration of a lag-phase did not exeed twenty hours the yield of microbial biomass (wet weight) and production of phenylalanine were 150 and 0.95 gram per 1 liter of growth medium (see relative values in Table Expt. 1). The results suggested that below 49% (v/v) of 2H2O (Table Expts. 2-4) there was a small inhibition of growth indicators compared with the control (1) above 49% (v/v) of 2H2O (Table Expts. 5-8) however growth was markedly reduced while at the upper content of 2H2O (Table Expts. 9-10) growth was extremely small. With increasing content of 2H2O in the media there was a simultaneous increase both of the lag-phase and generation time. Thus under experimental conditions (10) where we used 98% (v/v) 2H2O and 2% (v/v) [U -2H]methanol the lag-phase was more than three and the generation time - 2.2 times that on ordinary protonated medium (1). The production of phenylalanine and yield of biomass were decreased on medium with 98% (v/v) 2H2O and 2% (v/v) [U -2H]methanol by 2.7 and 3.3 times respectively; in contrast to the adapted bacteria (10’) the growth characteristics of non-adapted bacteria on maximally deuterated medium were strongly inhibited (Table Expt. 10). The replacement of protonated methanol by [U- 2H]methanol caused small alterations in growth characteristics (Table Expts. 2 4 6 8 10) relative to experiments where we used protonated methanol (Table Expts. 3 5 7 9).


Table. Isotope components of growth media and characteristics of bacterial growth

 

Media components, % (v/v)

          

H2O          2H2O           MetOH      [U -2H]

                                                       MetOH

Lag-phase (h)

Yield of

biomass (%)

Generation time (h) Production of phenylalanine (%)
(a) 98 0 2 0 20 100.0 2.2 100.0
(b) 73.5 24.5 0 2 34 85.9 2.6 97.1
(c) 49.0 49.0 0 2 44 60.5 3.2 98.8
(d) 24.5 73.5 0 2 49 47.2 3.8 87.6
(e) 0 98.0 0 2 60 30.1 4.9 37.0

The production of L-phenylalanine was linear with respect to the time up to exponentaly growth cells (see Fig.1). During the fermentation the formation rate of L-phenylalanine was about  5 mmol/day. As shown in Fig. 1 the substitution by deuterium atoms pronons of water and methanol caused  the decreasing both the production of L-phenylalanine and the yieald of biomass. Hawever the decreasing of L-phenylalanine production (up to 0 5 gL) was observed in those experiments (10) (Fig.1) when using non adapted cells on media with 98 % (v/v) 2H2O. The growth rate and generation time for adapted cells were found to be the same as in control in ordinary water despite of small increasing of lag-phase. In contrast to adapted cells the growth of non-adapted species on maximal deuterated media was strongly inhibited by deuterium. These data are shown in Fig. 2.

A smart attempt was made to intensificate the growth and biosynthetic parameteres of cells to grow on media containing highly concentration of deuterated substrates. We employed a "step by step" adaptation method combined with the selection of clones resistent to deuterium using agaric media supplemented with C2H3O2H 2% (v/v) and with increasing 2H2O content starting from pure water up to 98 % (v/v) 2H2O. The degree of cell survive on maximum deuterated medium (10) containing 98 v/v.% 2H2O and 2 v/v.% C2H3O2H was about 40%. Figure 1 shows characteristic growth and biosynthesis curves for adapted to 2H2O (10’) and non-adapted  (10) cells in conditions compared with the control (1) in H2O. The transfer of fully deuterated cells to ordinary protonated medium results eventually in normal growth.

The results on adaptation testified that the generation time for adapted bacteria was approximately the same as in the control (1) despite the two-fold increase of the lag-phase (Table Expt. 10’). The yield of microbial biomass and level of phenylalanine production for adapted bacteria on maximally deuterated medium (Table Expt. 10’) were decreased relative to the control (1) by 13 and 5.3% respectively. Figure 1 shows growth (Expts. 1a 2 a 3 a) and production of phenylalanine (Expts. 1 b 2 b 3 b) for non-adapted (2) and adapted (3) bacteria on maximally deuterated medium under conditions like the control (1) on protonated medium. As shown from Fig. 1 the curves of phenylalanine production were close to a linear extrapolation with respect to the exponential phase of growth dynamics. The level of phenylalanine production of non-adapted bacteria on maximally deuterated medium was 0.39 g/liter after 80 hours of growth (Fig. 1 Expt. 2 b). The level of phenylalanine production for adapted bacteria under those growth conditions was 0.9 g/liter (Fig. 1 Expt. 3 b). Thus the use of adapted bacteria in growth conditions to be the same as in the control (1) enabled us to improve the level of phenylalanine production on maximally deuterated medium by 2.3 times. However phenylalanine is not the only product of biosynthesis; other metabolically related amino acids (alanine valine and leucine/isoleucine) were also produced and accumulated in the growth medium in amounts of 5-6 mmol in addition to phenylalanine. This fact required for the future prospects of the production of labeling molecules of amino acids with deuterium an efficient separation of 2H-labeled phenylalanine from other amino acids of growth medium. Recently such separation was done using a reversed-phase HPLC method developed for methyl esters of N-Dns- and Bzl-amino acids with chromatographic purity of 96-98 and yield of 67-89%.                                                                                                                                                                                               

            For evaluation of deuterium enrichment methyl esters of N-DNS-amino acids were applied because the peaks of molecular ions (M+) allow to monitor the enrichment of multicomponential mixtures of amino acids being in composition with growth media metabolites therefore EI MS allows to detect samples with amino acids of 10-9-10-12 mol (Karnaukhova 1994). N-DNS-amino acids were obtained through the derivatization of lyophilized M9 with DNSCl. To increase the volatality of N-DNS-amino acids the methylation with DZM was made to prevent the possible isotopic (1H-2H) exchange in molecule of Phe. With DZM treatment it 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.

            Mass spectra EI MS of methyl esters of N-DNS-amino acid mixtures obtained from M9 where used 0; 73.5

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