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

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

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 lagphase
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

Lagphase
(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

(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 and 98% (v/v) of 2H2O (Table, Expts.
(a), (d), (e)) are shown in consecutive order in Figs. 1-3. The fragmentation
pathways of methyl esters of N-Dns-amino acids by EI MS leads to the
formation of the molecular ions (M+) from whom the fragments with
smaller m/z ratio further are formed. Since the value of (M+)
for Leu is as the same as for Ile, these two amino acids could not be clearly
estimated by EI MS. A right region of mass spectra EI MS contains four peaks of
molecular ions (M+) of methyl esters of N-DNS-amino acids: Phe with m/z
412; Leu/Ile with m/z 378.5; Val with m/z 364.5; Ala with m/z
336.4 (see Fig. 1 as an example). A high continuous left background region at
m/z
80 — 311 is associated with the numerious peaks of concominant
metabolites and fragments of further decay of methyl esters of N-DNS-amino
acids.

The results, firmly established the labeling of amino acids
as heterogenious, juging by the presence of clasters of adduct peaks at their
molecular ions (M+); the species of molecules with different numbers
of deuterium atoms were visualised. The most aboundant peak (M+)
in each claster was registered by mass spectrometer as a peak with
average m/z ratio, from whom the enrichment of each individual amino
acid was calculated. Thus, in experiment (d) shown in Fig. 2 where used 73.5%
(v/v) 2H2O the enrichment of Phe was 4.1, calculated at
(M+) with m/z 416.1 (instead of m/z 412 (M+)
for non-labeled compound); Leu/Ile — 4.6 (M+) with m/z 383.1
instead of m/z with 378.5 (M+)); Val — 3.5 (M+ with
m/z 368 instead of m/z (M+) with 364.5); Ala — 2.5
deuterium atoms ((M+)  with m/z 338.9 instead of
m/z
with 336.4 (M+)).

            With
increasing of 2H2O content in liquid M9, the levels of
amino acid enrichment varried propotionaly. As seen in Fig. 3 in experiment (e)
where used 98% (v/v) 2H2O the enrichment of Phe was six
((M+) with m/z 418 instead of m/z 412 (M+));
Leu/Ile — 5.1 ((M+) with m/z 383.6 instead of m/z with
378.5 (M+));  Val — 4.7 ((M+) with m/z 369.2
instead of m/z (M+) with 364.5); Ala — 3.1 deuterium atoms 
(M+) with m/z 339.5 instead of m/z with 336.4 (M+)).
The label was distributed uniformely among the amino acid molecules, in
experiment (e) the enrichment of 2H-labeled amino acids was
nevertheless less than we estimated theoretically, because Leu was added in
growth medium in protonated form. This effect should be seriously scrutinised
before the applying this mutant for the preparation of 2H-labeled
amino acids.

 

 

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