Methylotrophic biomass as 2H-labeledsubstrate for biosynthesis of inosine
Oleg V. Mosin1
1 M. V. Lomonosov State Academy of Fine Chemical Technology,Vernadskogo Prospect 86, Moscow, 117571
Abstract
It was proposed to use the2H-labeled hydrolysate of RuMP facultative methylotroph Brevibacteriummethylicum, obtained from deuterated salt medium dM9 as a substrate for thegrowth of inosine producing bacterium Bacillus subtilis. The growth ofthe bacterim was performed via glucose convertion on specially developed mediumdHM with 78.5% (m/m) 2H2O and supplimented with 2.5%(m/m) of 2H-labeled methylotrophic hydrolysate. To evaluate thelevel of deuterium enrichment FAB MS technique was used after the isolation of 2H-labeledinosine. 2H-labeled inosine obtained from dHM medium represented amixture of molecular species containing various number of included deuteriumatoms with different contribution to the enrichment. The level of enrichmetcalculated by the presence of most abandant peak of the molecular ion incluster ((M+H)+ at m/z 274) was estimated as five deuterium atoms,from which three are attributed to ribose and two to hypoxantine.
Keywords:2H-labeled growthsubstrates — Bacillus subtilis — Biosynthesis — 2H-labeled inosine
Introduction
Nucleosideslabeled with deuterium (2H) and other stable isotopes are becomingan indispensable tool for biomedical diagnostic and the investigation ofvarious aspects of the metabolism [1, 2]. Thus inosine which is known as animportant intermediate in the synthesis of inosine monophosphate (IMP) is inthe focal point of clinical interest in medical diagnostic of heart deceasesand in certain medical cases [3, 4].
Thereare several approaches reported for the preparation of 2H-nucleosides.Chemical synthesis are usually tedious and inefficient. Only by employingmutant forms of bacteria, which can produce a large quantities of thenucleosides when growing of an organism on media containing deuteratedsubstrates, the desired biochemicals can be obtained both with high yields andenrichments. On the microbial production of inosine, there have been manystudies so far [5-7]. .
For instance,a certain adenine, histidine and tyrosine auxotrophic mutants derived from Bacillussubtilis have been found to have a remarkable ability to produce a largeamount of inosine in the growth medium, and at the present it may be producedon an industrial scale.
Themajor disadvantage of production of 2H-nuclesides is difficulty inobtaining the appropriate deuterated growth substrates. One approach to solvethis problem is to use the extracts obtained from microorganisms growing onminimal media with 99,9 at.% 2H2O far [8]. Thus, werecently described a facultative methylotrophic bacteriumBrevibacteriummethylicum, which seems to be an an ideal source for the preparation ofuniformelly labeled growth substrates on the basis of its 2H-biomassprepared from 2H2O and [U -2H]MetOH [9, 10].In this article, we demonstrate the possibility of using the hydrolysates of 2H-labeledbiomass of this bacterium as substrates for growing the inosine producingmutant B. subtillis.
Materials and methods
Chemicals
2H2O(99.9 at.% 2H[1]) was obtained fromRussian Scientific Enterprises, Sanct Petersburg and purified by distillationfrom alkaline permanganate. [U -2H]methanol (95.7 at.% 2H)was from Biophysic Center, Pushino. All other chemicals were of reagent grade.
To create a high isotopic content in growth medium, 2H2Owith trade marked isotopic purity 99.9 at.% 2H, was used. However,the deuterium content of used 2H2O verified by NMR wasfound to be 97 at.% 2H. The water containing salts were severaltimes preliminarily crystallyzed in pure 2H2O and driedin vacuum before using (the true content of deuterium in growth media after theautoclaving was less smaller on 8-10% then isotopic purity of an initial 2H2O.
The bacterial strain
Adenine, tyrosine andhystidine auxotroph mutant B. subtilis B -3157 capable to produce andaccumulate 17 g/liter of inosine during the growth on protonated medium withglucose and yeast extract was employed. The strain was obtained from RussianState Scientific Center for Genetics and Selection of Industrial MicroorganismsGNIIGENETIKA.
Preparation of 2H-labeledgrowth substrates
The methylotrophicbacterium B. methylicum # 5662 was grown on salt medium dM9 with 93.5%(m/m) 2H2O and 2% (m/m) [U -2H]MetOH in massculture [11]. Cells were pelleted by centrifugation (2000 g, 10 min), washedonce with 2H2O and stored at -14 0C.Periodically, 10 g (wet weight) portions are thawed, suspended in 0.5 N 2HClsolution (in 2H2O) and autoclaved at 1200C for30 min. After adjusting pH till 7.0-7.2 with potassium hydroxide, thehydrolysate was used as a mixure of 2H-labeled growth substrates forthe growth ofinosine producing strain.
Media and growthconditions
The bacterial growth wascarried out on FM medium (m/m.%): glucose 12; yeast extract 2.5; ammoniumnitrate 3; magnium sulphate 2; chalk 2. The composition of dHM was as the sameas FM except dHM was prepared from 2H2O and thehydrolysate of 2H-labeled methylotrophic biomass was added. Themedia were sterilized by autoclaving at 1200C for 30 min and cooled.Glucose was sterilized separetely in 2H2O solution, andafter that added in growth medium. рН was adjusted till 6.5-6.7 with potassium hydroxide. The bacterium wasgrown in 250 ml Erlenmeyer flasks containing 20 ml of the medium at 32-34 0С and vigorously aerated on an orbitalshaker. After 7 days the cells were pelleted by centrifugation (2000 g, 10min). The supernatant was separated, lyophilized and used for the isolation of 2H-labeled inosine.
Isolation of inosine
MetOH solution in H2O(50 v/v %, 20 ml) was added to a lyophilized growth medium. The mixture wasallowed to — 4 0C and after 10 h the total protein was precipitatedand removed by centrifugation (1200 g, 10 min). MetOH was evaporated underreduced pressure. The resulting mixture was dissolved in 2H2O(30 ml) and 5 g of activated carbon was added. After keeping for 24 h at -4 0C,the inosine, eluting with ammonia, was concentrated and twice recrystallizedfrom MetOH (nd20 = 1.33). The purity of the product wasjudged by using controls of normal nucleosides, and running mixed TLC withgraded amounts of the neighboring nucleosides.
Quantitative determination
During the growth inosinewas separated by TLC on Silufol UV-254 plates with mobile phases: n -ButOH — AcOH — water (2:1:1, v/v) using pure commercial available inosine as astandard. The amount of inosine was determined for 10 ml aliquots of liquidgrowth medium by TLC. The sports were eluted by 0.1 N solution of HCl (10 ml).The absorbance of the eluates was measured at 249 nm and the content of inosinewas determined using a standard curve.
The convertion of glucosewas estimated enzymatically with glucoseoxydenase method [].
Equipment
Absorbance was measuredwith a spectrophotometer Beckman DU-6 (USA).
The analysis of proteinhydrolisates was carried out using a Biotronic LC 50001 chromatograph(Germany), 230 x 3.2 mm, working pressure 50-60 atm, flow-rate 18.5 ml/h.
The levels of deuteriumenrichment of amino acids were investigated with the aid of EI MS afterderivatization to methyl esters of N-Dns-amino acids [].
FAB MS was performed onHitachi MBA spectrometer (Japan) on glyserol template at potential 5 кV and an ion current of 0.6-0.8 мА.
RESULTS AND DISCUSSION
Production of 2H-labeledinosine
For biosynthesis of 2H-labeled inosine we employed bacterium Bacillus subtillis, whichcould produce and accumulate a conciderable amount of inosine exogeniously dueto an altered nucleoside metabolism. This strain displayed the maximumproductivity on FM medium, containing as a source of carbon and energy glucose(12 m/m.%), and as a source of growth factors and additional source of nitrogenthe yeast extract. Since the small availability of commercial available 2H-labeledbiomass prepared from yeast, it was necessary to find the more suitablemicrobial source, from which the 2H-labeled growth substrates couldbe obtained. For this purpose we employed the available RuMP facultativemethylotroph Brevibacterium methylicum [5] with the content of the totalprotein and polycarbohydrates in biomass 53 and 10% respectively [6].
The content ofamino acids in biomass ofB. methylicum and the deuterium enrichment areshown in Table.
Table:
The content of amino acids in biomass ofB.methylicum and the deuterium enrichment.Amino Acids The content in biomass, % Deuterium enrichment, % Glycine 9,69 90,0 Alanine 13,98 97,5 Valine 3,74 50,0 Leucine/Isoleucine 7,33/3,64 49,0 Phenylalanine 3,94 95,0 Tyrosine 1,82 92,8 Serine 4,90 90,0 Threonine 5,51 not determined Methionine 2,25 not determined Aspartic Acid 9,59 66,6 Glytamic Acid 10,38 70,0 Lysine 3,98 58,9 Arginine 5,27 not determined Histidine 3,72 not determined
The hydrolysis of 2H-labeledbiomass was performed in mild conditions via its autoclaving (30 min, 08 atm)in 0.5 N solution of 2HCl (in 2H2O). The dataon the amino acid composition of hydrolysate and levels of the enrichment areshown in Fig. 2. The contents of tyrosine and histidine in hydrolysate were1.82 and 3.72% and can ensure the polyauxotrophy of the inosine producingstrain. Another important parameter is a high level of amino acid enrichment.
Bacterial growth andproduction of inosine
Two following media wereused for the bacterial growth:
1). FM medium, prepared from ordinaryprotonated water and yeast extract.
2). dHM medium, prepared from 87.5%(v/v) 2H2O and 2.5% (m/m) of 2H-labeledmethylotrophic hydrolisate, obtained accordingly from medium dМ9.
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Fig.1
Curves, reflecting thegrowth dynamics (a), convercion of glucose (b) and production of inosine (c)are given in Fig. 1. A maximal level of inosine production on ordinaryprotonated medium was 17 g\liter. When growing on dHM medium the strainproduced only 3.9 g/liter of inosine throughout the whole course of the growth.The low level of inosine production was correlated with a degree of glucoseconversion in those conditions. 4m/m.% of non-assimilated glucose was detectedin medium dHM after the growth, that proved that glucose is metabolized lesseffectivelly on medium dHM, that is probably a result of non-equvalentreplacement of yeast extract by methylotrophic hydrolysate.
The absorptionspectra of inosine isolated from medium dHM (a) are shown in Fig. 2comparatively to the growth medium (b) and commertially available inosine (c).TLC of isolated inosine showed the presence of main spot with Rf=0.5(inosine) and additional spot with Rf=0.75 (hypoxantine). The outputof 2H-labeled inosine was 1 gram from 1 liter of growth medium.
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Fig.2
The evaluation of inosineenrichment
The method of FAB MS wasemployed for the evaluation of inosine enrichment. The fragmentation pathwaysof inosine by FAB MS are shown in Fig. 3. Two main decomposition processesarised from the molecule: sugar (m/z 133) and hypoxantine (m/z 136) formation.The compounds with a smaller m/z ratio may further to be formed as a result ofelitination of HCN and CO from hypoxantine. The level of deuterium enrichmentcould be evaluated from the FAB mass spectrum of 2H-labeled inosineshown in Fig. 3, b compared with the non-labeled inosine (a). The results,firmely established the labeling of inosine as heterogenious, juging by thepresence of clasters of adduct peaks at the molecular ion MH+; thespecies of molecules with different numbers of deuterium atoms were visualised.The most abundant peak with (M+H)+ at m/z 274 (instead of m/z 269for non-labeled compound) in the claster was registered by mass spectrometer asa peak with average m/z ratio, from whom the enrichment of inosine wascalculated as five deuterium atoms. The presence of peak corresponding to thehypoxantine fragment [C5H4ON4]+ at m/z 138(instead of m/z 136) and the peak of sugar fragment [C5H9O4]+at m /z 136 (instead of m/z 133) proved that two deuterium atoms are located inhypoxantine, however, three of them are attributed to the ribose pattern.
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Fig.3
Mainly two aspects ofthe enrichment of inosine were taken into account (scheme). First, becauseprotons in С’1-С’5 positions of ribose pattern ininosine could be originated from glucose, we assumed, that the character ofbiosynthetic enrichment of deuterium in sugar pattern of inosine is determinedmainly to the functioning of a number of processes of hexose monophosphateshunt of glucose assimilation. But since protonated glucose was added in growthmedium, its contribution in the inosine enrichment was minimal. Nevertheless,the results suggested, that ribose contained three deuterium atoms that couldnot stemp from glucose. Three deuterium atoms probably stemp via some minorreactions of glucose biosynthesis. Secondly, the numerous exchange processesand intermolecular regrouping reactions, occurring with participation of 2H2Ocould also be resulted in specific labelling of inosine. Such accessiblepositions are occupied by the easily exchangeable hydrogen (deuterium) atomsboth of hydroxylic- and imino groups of inosine. Two protons at C-H positionsin inosine could be replaced by deuterium via assimilation of 2H-labeledhydrolysate. The enrichment of inosine was approximately the same as 2H2Ocontent in growth medium (65.5-67.5%).
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