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

The production of 2H-labeledamino acids by a new mutant of RuMP fucultative methylotroph Вrevibacterium methylicum
 
Oleg V. Mosin1
 
1 Department ofBiotechnology, M. V. Lomonosov State Academy of Fine Chemical Technology,Vernadskogo Prospekt 86, 117571, Moscow, Russia
 
Summary
The biosynthesis of 2H-labeledphenylalanine was done by converse of low molecular weight substrates ([U- 2H]methanoland 2H2O) in a new RuMP facultative methylotrophic mutantBrevibacterium methylicum. To make the process work, adapted cells withimproved growth characteristics were used on minimal medium M9 with the maximumcontent of 2H-labeled substrates. Alanine, valine, andleucine/isoleucine were produced and accumulated exogeneously in additionto the mainproduct of biosynthesis. Electron impact mass spectrometry of methyl esters ofthe N-Dns-amino acid mixture obtained after the chemical derivatizationof growth medium with dansyl chloride and diazomethane, was done to calculatethe deuterium enrichment of the amino acids synthesized. The experimental datatestified to the character of labeling of amino acid molecules asheterogeneous; however, high levels of deuterium enrichment were detected inall 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 impactmass 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 acidmolecules with deuterium is becoming an essential part for various biochemicalstudies with 2H-labeled molecules and investigation of certainaspects of their biosynthesis(LeMaster, 1990).
Forintroduction of deuterium into amino acid molecules either chemical or biosyntheticalmethods may be used. Chemical synthesis of these compounds has one significantlimitation; it is a very laborious and costly multistep process resulting in amixture of dl-racemates. This major disadvantage, however essentiallydelaying its development is a difficulty in preparing the appropriate 2H-labeledamino 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, thedesired 2H-labeled biochemicals can be produced both at high yieldsand enrichments  (Cox, 1988), but the process involves algae is limited by theexpense of a mixture of 2H-labeled amino acids isolated fromhydrolysates of biomass (Daboll, 1962). The using for this purpose a certainmethylotrophs which assimilates MetOH as a source of carbon and energy via RuMPcycle has a great practical advantage because their ability to produce andacumulate a gram quantities of 2H-labeled amino acids during thegrowth on media with 2H2O and [U -2H]MetOH andthe comparatively low price of [U -2H]MetOH (Karnaukhova, 1994).
Thebiosynthesis of 2H-labeled amino acids usually involves growth of anorganism on selective media containing the labeled substrates:e.g.,growth of algae autotrophically on media with content of 2H2O90% and more, is a well established method for biosynthesis of numerous highlydeuterated molecules. But this method, while being generally applicable, islimited by the low resistance of plant cells to 2H2O andexpense of 2H-labeled amino acids isolated from algae hydrolysates.Alternative and relatively inexpensive objects for biosynthesis of 2H-labeledamino acids seem certain auxotrophic mutants of methylotrophic bacteria usingmethanol as a main source of carbon and energy via theribulose-5-monophosphate (RuMP) and the serine cycle of carbon assimilation.These bacteria have a big advantage because of their ability to produce andaccumulate gram quantities of highly enriched, 2H-labeled aminoacids during growth on minimal salt media with [U- 2H]methanol and 2H2Oand the comparatively low price of [U- 2H]methanol. It is only inrecent years that some progress was made in the isolation of a number ofversatile the RuMP cycle methylotrophic bacteria, suitable for such studies,though the research that has been done with methylotrophs was limited andsuffered from low growth characteristics on 2H2O-containingmedia. Although the production of 2H-labeled amino acids by obligatemethylotroph Methylobacillus flagellatum described by Karnaukhovainvolves the growth on media with approximately 75% (v/v) 2H2O.We have recently selected a new mutant of facultative methylotroph Brevibacteriummethylicum, realizing the NAD+ dependent methanol gehydrogenase(EC 1.6.99.3) variant of RuMP cycle of carbon assimilation, which seems moreconvinient 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 havepreviously studied the applicability of the RuMP cycle obligate methylotrophicbacterium Methylobacillus flagellatum for biosynthesis of 2H-labeledleucine 8). This approach is not yet practical for the biosynthesisof 2H-labeled phenylalanine, mainly because of the absence ofsuitable methylotrophic producer of this amino acid. After selecting a new theRuMP cycle methylotrophic producer of phenylalanine, leucine auxotroph Brevibacteriummethylicum, 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 ofsequential grade was from Sigma Chemicals Corp., USA. DZM was prepared fromN-NMU, Pierce Chemicals, Corp., USA. A gram-positive parental strain of RuMPfacultative methylotrophBrevibacterium methylicum # 5662 was obtainedfrom Russian State Scientific Center for Genetics and Selection of IndustrialMicroorganisms GNIIGENETIKA (Nesvera, 1991).
            Basal salt medium M9(Miller, 1976) with MetOH as a carbon and energy source (2%, v/v) andsupplemented with Leu (100 mg/l) was used for bacterial growth. For isotopicexperiments M9 was enriched with [U -2H]MetOH and 2H2Oof various content (see Table below). The bacterial growth was carried outunder batch conditions (Karnaukhova, 1994). The exponentially growing cells(cell density 2.0 at absorbance 540 nm) were pelleted by centrifugation (1200 gfor 15 min), the supernatant was lyophilized and used for chemicalderivatization.
            The amount of Phe wasdetermined for 10 ml aliquotes of liquid M9 by TLC with solvent ofiso-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 wasmeasured at 540 nm, the concentration was calculated using a standard curve.
            The samples oflyophilized 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 performedin a standard procedure with DZM (Greenstein, 1976).
            EI MS was performed onHitachi MB 80 spectrometer at ionizing energy 70 eV and an ion sourcetemperature of 180oC.
 Results and discussion
Phe issynthesized in most bacteria via shikimic acid pathway (Conn, 1986). Theprecursors for the biosynthesis of Phe are PEP and ERP. The latter compound isan intermediate in the PenP pathway and, in some methylotrophs, the RuMP cycleof carbon assimilation (Antony, 1982; Kletsova, 1988). It is widely accepted,that the native bacterial strains can not to be a strong producers of Phe owingto the effective mechanisms of its metabolic regulation, although certainbacterial 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 anumber of other several enzymes are proved to be an active producers of thisamino acid (Umbarger, 1978). That is why the best Phe producing strains onceselected were the mutants partially or completely dependent on Tyr or Trp forgrowth. The reports about the other regulative mechanisms of Phe biosynthesisin bacterial cell are quite uncommon, though today it is known a large numberof RuMP cycle auxotrophic mutants of methylotrophs, covering numerious steps inaromatic amino acid biosynthesis  (Dijkhuizen, 1996). The selection of newproducers of Phe has a big importance for studies of their regulating pathwaysand possible production of 2H-labeled Phe.
            For our studies we haveused a new non-traditional producer of phenylalanine: a leucine auxotroph ofthe facultative methylotrophic bacterium Brevibacterium methylicum obtainingthe NAD+ dependent methanol dehydrogenase (EC 1.6.99.3) variant ofthe RuMP cycle of carbon assimilation, with maximum productivity ofphenylalanine on protonated medium M9 — 0.95 gram per liter of growth medium.According to experiments, various compositions of  [U- 2H]methanoland 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 ofphenylalaninein the presence of increasing content of 2H2O are givenin Table (Expts. 3-10) relative to the control (1) on protonated medium and tothe adapted bacteria (Expt. 10’). The odd numbers of experiment were chosen toinvestigate whether the replacement of [U -2H]methanol of itsprotonated analogue has a positive effect on growth characteristics in thepresence of 2H2O. The maximum deuterium content wasreached under conditions (10) and (10’) in which we used 98% (v/v) 2H2Oand 2% (v/v) [U -2H]methanol. In the control, the duration of a lag-phasedid not exeed twenty hours, the yield of microbial biomass (wet weight) andproduction 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 smallinhibition of growth indicators compared with the control (1), above 49% (v/v)of 2H2O (Table, Expts. 5-8), however growth was markedlyreduced, while at the upper content of 2H2O (Table,Expts. 9-10) growth was extremely small. With increasing content of 2H2Oin the media there was a simultaneous increase both of the lag-phase andgeneration time. Thus, under experimental conditions (10) where we used 98%(v/v) 2H2O and 2% (v/v) [U -2H]methanol, thelag-phase was more than three and the generation time — 2.2 times that onordinary protonated medium (1). The production of phenylalanine and yield ofbiomass were decreased on medium with 98% (v/v) 2H2O and2% (v/v) [U -2H]methanol by 2.7 and 3.3 times respectively; incontrast to the adapted bacteria (10’), the growth characteristics ofnon-adapted bacteria on maximally deuterated medium were strongly inhibited(Table, Expt. 10). The replacement of protonated methanol by [U- 2H]methanolcaused 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 mediaand characteristics of bacterial growth
 
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Media components, % (v/v)
          
H2O          2H2O           MetOH      [U -2H]
                                                       MetOH
Lag-phase (h)
Yield of
biomass (%) Generation time (h) Production of phenylalanine (%) (a) 98 2 20 100.0 2.2 100.0 (b) 73.5 24.5 2 34 85.9 2.6 97.1 (c) 49.0 49.0 2 44 60.5 3.2 98.8 (d) 24.5 73.5 2 49 47.2 3.8 87.6 (e) 98.0 2 60 30.1 4.9 37.0
The productionof L-phenylalanine was linear with respect to the time up to exponentaly growthcells (see Fig.1). During the fermentation the formation rate ofL-phenylalanine was about  5 mmol/day. As shown in Fig. 1, the substitution bydeuterium atoms pronons of water and methanol caused  the decreasing both theproduction of L-phenylalanine and the yieald of biomass. Hawever, thedecreasing of L-phenylalanine production (up to 0,5 g\L) was observed in thoseexperiments (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 sameas in control in ordinary water despite of small increasing of lag-phase. Incontrast to adapted cells, the growth of non-adapted species on maximaldeuterated media was strongly inhibited by deuterium. These data are shown inFig. 2.
A smartattempt was made to intensificate the growth and biosynthetic parameteres ofcells to grow on media containing highly concentration of deuterated substrates.We employed a «step bystep» adaptation method,combined with the selection of clones resistent to deuterium using agaric mediasupplemented with C2H3O2H 2% (v/v) and withincreasing 2H2O content starting from pure water up to 98% (v/v) 2H2O. The degree of cell survive on maximumdeuterated medium (10), containing 98 v/v.% 2H2O and 2v/v.% C2H3O2H was about 40%. Figure 1 showscharacteristic growth and biosynthesis curves for adapted to 2H2O(10’’) and non-adapted  (10) cells in conditions compared with thecontrol (1) in H2O. The transfer of fully deuterated cells toordinary protonated medium results eventually in normal growth.
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The results onadaptation testified, that the generation time for adapted bacteria wasapproximately the same as in the control (1) despite the two-fold increase ofthe lag-phase (Table, Expt. 10’). The yield of microbial biomass and level ofphenylalanine 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 ofphenylalanine (Expts. 1 b, 2 b, 3 b) for non-adapted (2) and adapted (3) bacteriaon maximally deuterated medium under conditions like the control (1) onprotonated medium. As shown from Fig. 1, the curves of phenylalanine productionwere close to a linear extrapolation with respect to the exponential phase ofgrowth dynamics. The level of phenylalanine production of non-adapted bacteriaon 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 underthose growth conditions was 0.9 g/liter (Fig. 1, Expt. 3 b). Thus, the use ofadapted bacteria in growth conditions to be the same as in the control (1),enabled us to improve the level of phenylalanine production on maximallydeuterated medium by 2.3 times. However, phenylalanine is not the only productof biosynthesis; other metabolically related amino acids (alanine, valine, andleucine/isoleucine) were also produced and accumulated in the growth mediumin amounts of 5-6 mmol in addition to phenylalanine. This fact required, for thefuture prospects of the production of labeling molecules of amino acids withdeuterium, an efficient separation of 2H-labeled phenylalanine fromother amino acids of growth medium. Recently such separation was done using areversed-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 ofdeuterium enrichment methyl esters of N-DNS-amino acids were applied becausethe peaks of molecular ions (M+) allow to monitor the enrichment ofmulticomponential mixtures of amino acids being in composition with growthmedia metabolites, therefore EI MS allows to detect samples with amino acids of10-9-10-12 mol (Karnaukhova, 1994). N-DNS-amino acidswere obtained through the derivatization of lyophilized M9 with DNSCl. To increasethe volatality of N-DNS-amino acids, the methylation with DZM was made toprevent the possible isotopic (1H-2H) exchange inmolecule of Phe. With DZM treatment it occured the derivatization on aNH2 group inthe molecule, so that its N-methylated derivative was formed to the addition ofmethyl ester of N-DNS-Phe.
            Mass spectra EI MS ofmethyl esters of N-DNS-amino acid mixtures, obtained from M9 where used 0; 73.5and 98% (v/v) of 2H2O (Table, Expts. (a), (d), (e)) areshown in consecutive order in Figs. 1-3. The fragmentation pathways of methylesters ofN-Dns-amino acids by EI MS leads to the formation of themolecular ions (M+) from whom the fragments with smaller m/zratio further are formed. Since the value of (M+) for Leu is as thesame 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/z378.5; Val with m/z 364.5; Ala with m/z 336.4 (see Fig. 1 as anexample). A high continuous left background region atm/z 80 — 311 isassociated with the numerious peaks of concominant metabolites and fragments offurther decay of methyl esters of N-DNS-amino acids.
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The results,firmly established the labeling of amino acids as heterogenious, juging by thepresence of clasters of adduct peaks at their molecular ions (M+);the species of molecules with different numbers of deuterium atoms werevisualised. The most aboundant peak (M+)in each clasterwas registered by mass spectrometer as a peak with average m/z ratio,from whom the enrichment of each individual amino acid was calculated. Thus, inexperiment (d) shown in Fig. 2 where used 73.5% (v/v) 2H2Othe enrichment of Phe was 4.1, calculated at (M+) with m/z416.1 (instead of m/z 412 (M+) for non-labeled compound);Leu/Ile — 4.6 (M+) with m/z 383.1 instead of m/z with378.5 (M+)); Val — 3.5 (M+ with m/z 368 instead ofm/z (M+) with 364.5); Ala — 2.5 deuterium atoms ((M+) with m/z 338.9 instead ofm/z with 336.4 (M+)).
            With increasing of 2H2Ocontent in liquid M9, the levels of amino acid enrichment varried propotionaly.As seen in Fig. 3 in experiment (e) where used 98% (v/v) 2H2Othe enrichment of Phe was six ((M+) with m/z 418 instead of m/z412 (M+)); Leu/Ile — 5.1 ((M+) with m/z 383.6instead 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 ofm/zwith 336.4 (M+)). The label was distributed uniformely among theamino acid molecules, in experiment (e) the enrichment of 2H-labeledamino acids was nevertheless less than we estimated theoretically, because Leuwas added in growth medium in protonated form. This effect should be seriouslyscrutinised before the applying this mutant for the preparation of 2H-labeledamino acids.
 
 
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