Production of Guanosine-5′-Monophosphate and Inosine-5′-Monophosphate by Fermentation

A biotin-requiring coryneform bacterium which produces glutamic acid was mutated to adenine dependency. The adenine-requiring strain, which excreted insoine-5′-monophosphate (IMP), was further mutated to xanthine dependency. As expected, IMP was also excreted by this mutant. The mutant strain was reverted to xanthine independence in an attempt to obtain a culture with an altered IMP dehydrogenase which would be less sensitive to feedback inhibition by guanosine-5′-monophosphate (GMP). A revertant was obtained which produced GMP and IMP, each at 0.5 g per liter. The reversion to xanthine independence had resulted in a concomitant requirement for isoleucine, leucine, and valine. Further mutation to increased nutritional requirements led to culture MB-1802, which accumulated 1 g per liter each of GMP and IMP. Both nucleotides were isolated in pure form. The concentrations of GMP and IMP produced by MB-1802 were four times that of cytidylate, uridylate, or adenylate, indicating that the mechanism of GMP and IMP production was direct and not via ribonucleic acid breakdown.     

Production of Xanthosine-5′-Monophosphate and Inosine-5′-Monophosphate by Auxotrophic Mutants of a Coryneform Bacterium

Although most microorganisms with genetic blocks in the purine nucleotide sequence excrete breakdown products, a coryneform bacterium was found to accumulate intact 5′-nucleotides in the extracellular medium. Adenineless mutants accumulated 0.4 to 0.6 g of inosine-5′-monophosphate per liter of broth. The yield of this nucleotide was increased to 0.8 to 0.9 g per liter when such mutants were mutated to xanthine dependence. Induction of a specific guanine requirement in adenineless auxotrophs resulted in cultures capable of producing high yields of xanthosine-5′-monophosphate (3 to 4 g per liter). Pure xanthosine-5′-monophosphate was isolated from broth by a procedure involving ion-exchange chromatography, charcoal adsorption, and barium precipitation.     

Regulatory Role of Adenine Nucleotides in the Biosynthesis of Guanosine 5′-Monophosphate

Derepression of the synthesis of inosine 5′-monophosphate (IMP) dehydrogenase and of xanthosine 5′-monophosphate (XMP) aminase in pur mutants of Escherichia coli which are blocked in the biosynthesis of adenine nucleotides and guanine nucleotides differs in two ways from derepression in pur mutants blocked exclusively in the biosynthesis of guanine nucleotides. (i) The maximal derepression is lower, and (ii) a sharp decrease in the specific activities of AMP dehydrogenase and XMP aminase occurs, following maximal derepression. From the in vivo and in vitro experiments described, it is shown that the lack of adenine nucleotides in derepressed pur mutants blocked in the biosynthesis of adenine and guanine nucleotides is responsible for these two phenomena. The adenine nucleotides are shown to play an important regulatory role in the biosynthesis of guanosine 5′-monophosphate (GMP). (i) They induce the syntheses of IMP dehydrogenase and XMP aminase. (The mechanism of induction may involve the expression of the gua operon.) (ii) They appear to have an activating function in IMP dehydrogenase and XMP aminase activity. The physiological importance of these regulatory characteristics of adenine nucleotides in the biosynthesis of GMP is discussed.     

5′-Nucleotidase Activity in Improved Inosine-producing Mutants of Bacillus subtilis

An inosine- and guanosine-producing strain, AJ11100, of Bacillus subtilis could not grow in the minimum medium supplemented with 50 µg of sulfaguanidine per ml. When sulfaguanidine resistant mutants were derived from AJ11100, the sulfaguanidine resistance was frequently accompanied by xanthine requirement. All the xanthine auxotrophic mutants required a large amount of xanthine for cell growth and inosine accumulation. Revertants were then derived from one of the xanthine auxotrophic mutants, AJ11101, and improved inosine producers were obtained. The best mutant, AJ11102, accumulated 20.6 g of inosine per liter.

Furthermore, enzyme activities of inosine 5′-monophosphate (IMP) dehydrogenase, 5′-nucleotidase and phosphoribosyl pyrophosphate (PRPP) amidotransferase were assayed to investigate why AJ11102 accumulated an increased amount of inosine. The results showed that the increase of specific activity of 5′-nucleotidase contributed much to the increased accumulation of inosine.     

Production of Nucleic Acid-related Substances by Fermentative Processes: XIX. Accumulation of 5′-Inosinic Acid by a Mutant of Brevibacterium ammoniagenes

The accumulation of 5′-inosinic acid (IMP) by a mutant, KY 13102, induced from Brevibacterium ammoniagenes ATCC 6872 by ultraviolet light irradiation, was examined. Although growth was stimulated by adenine or adenosine, the microorganism showed fair growth in the medium containing amino acids but no adenine. Among six kinds of natural nutrients tested, meat extract and Casamino Acids were suitable for the accumulation of IMP. Manganese ion strongly affected growth, the accumulation of IMP and hypoxanthine, and cell morphology. Among amino acids tested, L-methionine, L-proline, and L-valine stimulated IMP accumulation. In the medium containing 1.0 g of L-proline per liter, 12.8 mg of IMP per ml was accumulated. The mechanism of IMP accumulation by the mutant is discussed.     

Accumulation of cyclic guanosine 3':5'-monophosphate in the culture medium of growing cells of Escherichia coli.

In an exponentially growing culture of E. coli, the concentration of cyclic guanosine 3′:5′-monophosphate (cyclic GMP) was found to increase in parallel with the bacterial growth. As the cells approach the stationary phase of growth, the increment of cyclic GMP also ceases progressively to reach to a plateau. When cells are separated from the medium by centrifugation, almost all of the cyclic GMP is recovered in the culture supernatant. The amount of cyclic GMP accumulated is proportional to the number of cells present in the culture. These results suggest that a constant number of cyclic GMP molecules is synthesized each generation of E. coli, and is excreted from the cells to accumulate into the medium.     

Novel Characteristics of Trypanosoma brucei Guanosine 5'-monophosphate Reductase Distinct from Host Animals

The metabolic pathway of purine nucleotides in parasitic protozoa is a potent drug target for treatment of parasitemia. Guanosine 5’-monophosphate reductase (GMPR), which catalyzes the deamination of guanosine 5’-monophosphate (GMP) to inosine 5’-monophosphate (IMP), plays an important role in the interconversion of purine nucleotides to maintain the intracellular balance of their concentration. However, only a few studies on protozoan GMPR have been reported at present. Herein, we identified the GMPR in Trypanosoma brucei, a causative protozoan parasite of African trypanosomiasis, and found that the GMPR proteins were consistently localized to glycosomes in T. brucei bloodstream forms. We characterized its recombinant protein to investigate the enzymatic differences between GMPRs of T. brucei and its host animals. T. brucei GMPR was distinct in having an insertion of a tandem repeat of the cystathionine β-synthase (CBS) domain, which was absent in mammalian and bacterial GMPRs. The recombinant protein of T. brucei GMPR catalyzed the conversion of GMP to IMP in the presence of NADPH, and showed apparent affinities for both GMP and NADPH different from those of its mammalian counterparts. Interestingly, the addition of monovalent cations such as K⁺ and NH₄⁺ to the enzymatic reaction increased the GMPR activity of T. brucei, whereas none of the mammalian GMPR’s was affected by these cations. The monophosphate form of the purine nucleoside analog ribavirin inhibited T. brucei GMPR activity, though mammalian GMPRs showed no or only a little inhibition by it. These results suggest that the mechanism of the GMPR reaction in T. brucei is distinct from that in the host organisms. Finally, we demonstrated the inhibitory effect of ribavirin on the proliferation of trypanosomes in a dose-dependent manner, suggesting the availability of ribavirin to develop a new therapeutic agent against African trypanosomiasis.     

Studies on the presence of adenosine cyclic 3':5'-monophosphate in oat coleoptiles

The incorporation of adenosine-8-¹⁴C into adenosine cyclic 3′:5′-monophosphate in coleoptile-first leaf segments of Avena sativa L. was investigated. Homogenates of segments incubated in adenosine-8-¹⁴C for either 4 or 10 hours were partially purified by thin layer chromatography followed by paper electrophoresis. A radioactive fraction, less than 0.06% of the ¹⁴C present in the original homogenate, migrated as adenosine cyclic 3′:5′-monophosphate during electrophoresis. Upon treatment with cyclic nucleotide phosphodiesterase, however, less than 10% of this radioactive fraction appeared as 5′-AMP. Deamination with NaNO₂ as well as further chromatographical purification also suggested that only a small fraction of the ¹⁴C in the partially purified samples could be in adenosine cyclic 3′:5′-monophosphate. The data suggest that levels of this nucleotide can probably be no greater than 7 to 11 picomoles per gram of fresh weight in oat coleoptiles. Treatment of such coleoptiles with physiologically active concentrations of indoleacetic acid, furthermore, had no significant effect on the ¹⁴C radioactivity in marker adenosine cyclic 3′:5′-monophosphate-containing fractions at any stage of purification during several experiments.     

Production of Guanosine by Psicofuranine and Decoyinine Resistant Mutants of Bacillus subtilis

Growth of Bacillus subtilis AG169 that produced large amounts of xanthosine and guanosine was inhibited by psicofuranine. When AG169 was mutated to resistance against psicofuranine, a mutant, GP–1, which yielded more guanosine was obtained. Psicofuranine did not inhibit growth of GP–1 any more. The guanosine 5′-monophosphate (GMP) synthetase activities were then assayed. In GP–1, the specific activity decreased about half, the complete loss of repression by GMP was found, and the inhibition by GMP was slightly loosed, when compared with those of AG169.

Furthermore, as growth of GP–1 was strongly inhibited by decoyinine, decoyinine resistant mutants were derived from GP–1. Of these mutants, two strains, MG–1 and MG–4, were resistant to decoyinine completely and showed the exclusive accumulation of guanosine in high yields, i.e. 16.0 and 15.5 g of guanosine per liter with weight yields of 20.0 and 19.4% of consumed sugar, respectively. GMP synthetase activity of MG–1 increased remarkably in comparison with that of GP–1 or AG169, and the inhibitions by GMP, psicofuranine and decoyinine were completely released in MG–1. Namely, the psicofuranine and decoyinine resistances seemed to cause mainly variations of GMP synthetase, and as results, the conversion of xanthosine 5′-monophosphate (XMP) to GMP proceeded more smoothly, and a larger amount of guanosine was accumulated.     

Production of Nucleic Acid-related Substances by Fermentative Processes: XIX. Accumulation of 5′-Inosinic Acid by a Mutant of Brevibacterium ammoniagenes

The accumulation of 5′-inosinic acid (IMP) by a mutant, KY 13102, induced from Brevibacterium ammoniagenes ATCC 6872 by ultraviolet light irradiation, was examined. Although growth was stimulated by adenine or adenosine, the microorganism showed fair growth in the medium containing amino acids but no adenine. Among six kinds of natural nutrients tested, meat extract and Casamino Acids were suitable for the accumulation of IMP. Manganese ion strongly affected growth, the accumulation of IMP and hypoxanthine, and cell morphology. Among amino acids tested, L-methionine, L-proline, and L-valine stimulated IMP accumulation. In the medium containing 1.0 g of L-proline per liter, 12.8 mg of IMP per ml was accumulated. The mechanism of IMP accumulation by the mutant is discussed.     

Phosphohydrolases and the production of 5′-nucleotides by direct fermentation

A major problem involved in the direct fermentation of nucleotides is their breakdown by phosphohydrolases. Thus, adenine auxotrophs of most microorganisms produce hypoxanthine and/or inosine rather than inosine 5′-monophosphate (IMP) while guanine auxotrophs excrete xanthosine rather than xanthosine 5′-monophosphate (XMP). Examination of a Bacillus subtilis mutant producing hypoxanthine plus inosine revealed at least four phosphohydrolases, three of which could attack nucleotides. Even when the extracellular nucleotide phosphohydrolase was inhibited by Cu⁺² and its surface-bound alkaline phosphohydrolase was repressed and inhibited by inorganic phosphate, or removed by mutation, the breakdown products were still the only products of fermentation. Under these conditions, the third enzyme, a surface-bound non-repressible nucleotide phosphohydrolase was still active. It appears, at least in B. subtilis, that excretion is dependent upon breakdown by this enzyme and if hydrolysis does not occur, excretion of purine nucleotides is feedback inhibited by the resultant high intracellular IMP concentration. Corynebacterium glutamicum mutants, on the other hand, can excrete intact nucleotides, and direct fermentations for IMP, XMP, and GMP have been described. An examination of phosphohydrolases in a GMP-producing culture revealed no extracellular or surface enzymes. Disruption of the cells resulted in liberation of cellular phosphohydrolase activity with a substrate specificity remarkably similar to the flavorenhancing properties of the 5′-nucleotides. The order of decreasing susceptibility was GMP, IMP, XMP; AMP was not attacked.     

Linear velocity of cyclic adenosine 3',5'-monophosphate transport in corn coleoptiles

The transport of cyclic adenosine 3′, 5′-monophosphate in corn coleoptile segments is very rapid. The linear velocity of basipetal transport is 183 millimeters per hour, while the velocity of acropetal transport is 79 millimeters per hour. Transport velocity as well as intensity thus appear to be polar in the corn coleoptile. Application of metabolic inhibitors such as cyanide, ouabain, and 2,4-dinitrophenol increase rather than decrease the velocity and intensity of transport. The mechanism of transport in light of these data is discussed.     

Fermentative production of 5′-purine ribonucleotide

Micrococcus sodonensis KY 3765 and Arthrobacter citreus KY 3155 were found capable of accumulating IMP in media supplemented with hypoxanthine as a precursor. High concentrations of phosphate and magnesium salts were required for high yields of IMP. Manganese deficiency in the media was also essential. Excessive Mn²⁺ effects were also seen in the IMP fermentation carried out with an adenineless mutant, of Cornynebacterium glutamicum. In M. sodonensis, R5P-like substances, 5-phosphoribose pyrophosphokinase and IMP pyrophosphorylase, were leaked out, of the cells grown in suboptimal Mn²⁺ levels. This excretion was inhibited by high levels of Mn²⁺. Such a phenomenon was not noted in A. citreus. An adenineless mutant (KY 7208) of Brevibacterium ammoniagenes was found to accumulate an appreciable amount of IMP. The chemical changes in this fermentation showed that, hypoxanthine was first produced de novo, excreted, and then reconverted into IMP by a salvage pathway. When hypoxanthine was added to 7208 culture, IMP yield was increased appreciably. In fact exogenous ¹⁴C-hypoxanthine was incorporated into ¹⁴C-IMP. Subsequent experiments showed that indeed Br. ammoniagenes ATCC 6872, a parent culture of KY 7208, was able to produce IMP, GMP, and AMP, in good yield from hypoxanthine, guanine, and adenine, respectively.     

Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum

Uridine 5′-monophosphate (UMP) synthase mutants of tobacco have been produced from haploid cell-suspension cultures of a transgenic Nicotiana tabacum line, Tr25. The mutants were induced by incubating the suspension-cultured cells with 1 mm N-nitroso-N-methylurea for either 5 or 12 hours. Twenty mutant calli were isolated on selection medium containing 20 milligrams per liter of 5-fluoroorotic acid. Of those tested, most had reduced regeneration capacity. Characterization of UMP synthase activities in the isolated calli showed that UMP synthase activity varied from 8 to nearly 100% of the wild-type activity. The growth of the calli on the media containing different levels of 5-fluoroorotic acid correlated with decreasing UMP synthase activity. Because the UMP synthase enzyme has two separate enzymic activities (orotate phosphoribosyl transferase and orotidine-5′-monophosphate decarboxylase), several mutants were further characterized to determine how the mutations affected each of the two enzymic activities. In each case, the enzymic activity affected was the orotate phosphoribosyl transferase and not the orotidine-5′-monophosphate decarboxylase. The wound-inducible phenotype of the Tr25 plants as measured by the activation of the pin2-CAT gene remained unchanged by introduction of the UMP synthase mutations.     

Cyclic adenosine 3':5'-monophosphate in axenic rye grass endosperm cell cultures

Cyclic adenosine 3′:5′-monophosphate (cAMP) was extensively purified from rye grass (Lolium multiflorum) endosperm cells grown in axenic suspension culture. The cAMP was purified by neutral alumina and anion and cation exchange chromatography. The cAMP was quantitated by means of a radiochemical saturation assay using a beef heart cAMP-binding protein and also by an assay involving activation of beef heart protein kinase. The cAMP levels found (corrected for recovery of tracer cyclic 3′,5′-[8-³H]AMP included from the point of sample extraction) ranged from 2 to 12 pmol/g fresh weight. The material purified from rye grass cultures was indistinguishable from authentic cAMP with respect to chromatography in two cellulose thin layer systems, behavior on dilution in both the saturation and protein kinase activation assays, and rates of degradation by a mammalian cAMP phosphodiesterase. The cAMP from rye grass cultures was completely degraded by a mammalian cAMP phosphodiesterase, and 1-methyl-3-isobutylxanthine inhibited such degradation. The protein kinase activation and saturation assays gave essentially the same values for the cAMP content of axenic rye grass culture extracts. Material satisfying the above criteria for identity with cAMP was also isolated from the culture medium. The increase observed in medium cAMP levels during culture growth provides evidence for the synthesis and secretion of cAMP by rye grass endosperm cells in suspension culture.     

Mutation of an inosine-producing strain of Bacillus subtilis to DL-methionine sulfoxide resistance for guanosine production.

An inosine-producing strain of Bacillus subtilis was mutated to resistance against the antagonist of glutamine, DL-methionine sulfoxide. Among the mutants derived, guanosine producers were observed frequently. The best strain, 14119, produced 9.6 g of guanosine per liter at a weight yield of 12% from consumed sugar. Inosine production decreased concomitantly. When resistance was increased further by exposure to higher doses of DL-methionine sulfoxide, another strain, AG169, was obtained that did not excrete inosine but produced increased amounts of xanthosine. In these strains, the specific activity of 5'-nucleotidase was lower and that of inosine 5'-monophosphate (IMP) dehydrogenase was higher than the parent strain. It is speculated that the metabolic flow from IMP to xanthosine 5'-monophosphate proceeds more smoothly than that from IMP to inosine and yields more xanthosine and guanosine.     

Mutation of an inosine-producing strain of Bacillus subtilis to DL-methionine sulfoxide resistance for guanosine production.

An inosine-producing strain of Bacillus subtilis was mutated to resistance against the antagonist of glutamine, DL-methionine sulfoxide. Among the mutants derived, guanosine producers were observed frequently. The best strain, 14119, produced 9.6 g of guanosine per liter at a weight yield of 12% from consumed sugar. Inosine production decreased concomitantly. When resistance was increased further by exposure to higher doses of DL-methionine sulfoxide, another strain, AG169, was obtained that did not excrete inosine but produced increased amounts of xanthosine. In these strains, the specific activity of 5'-nucleotidase was lower and that of inosine 5'-monophosphate (IMP) dehydrogenase was higher than the parent strain. It is speculated that the metabolic flow from IMP to xanthosine 5'-monophosphate proceeds more smoothly than that from IMP to inosine and yields more xanthosine and guanosine.     

Biosynthesis of Caffeine in Leaves of Coffee

The levels of endogenous caffeine and theobromine were much higher in buds and young leaves of Coffea arabica L. cv Kent than in fully developed leaves. Biosynthesis of caffeine from 14C-labeled adenine, guanine, xanthosine, and theobromine was observed, whereas other studies (H. Ashihara, A.M. Monteiro, T. Moritz, F.M. Gillies, A. Crozier [1996] Planta 198: 334-339) have indicated that there is no detectable incorporation of label into caffeine when theophylline and xanthine are used as substrates for in vivo feeds with leaves of C. arabica. The capacity for caffeine biosynthesis, especially from guanine and xanthosine, was reduced markedly in both fully developed mature and aged leaves. Data obtained in pulse-chase experiments with young leaves indicate the operation of an AMP -> IMP -> xanthosine 5[prime]-monophosphate (or GMP -> guanosine) -> xanthosine -> 7-methylxanthosine -> 7-methylxanthine -> theobromine -> caffeine pathway. The data obtained provide strong evidence against proposals by G.M. Nazario and C.J. Lovatt ([1993] Plant Physiol 103: 1203-1210) concerning the independence of caffeine and theobromine biosynthesis pathways and the role of xanthine as a key intermediate in caffeine biosynthesis.     

Direct assay method for guanosine 5'-monophosphate reductase activity.

A sensitive and simple micromethod for the accurate measurement of GMP reductase (EC 1.6.6.8) activity in crude extracts is described. The reaction product of [8-14C]IMP was separated from the substrate [8-14C]GMP by descending chromatography on Whatman DE81 ion-exchange paper. This separation method provides an analysis of the possible interfering reactions, such as the metabolic conversion of the substrate GMP to GDP, GTP, and/or guanosine, and guanine and the loss of the product IMP to inosine, hypoxanthine, and other metabolites. Low blank values (70-90 cpm) were obtained consistently with this assay because the IMP spot moves faster than the GMP spot. The major advantages of this method are direct measurement of GMP reductase activity in crude extracts, high sensitivity (with a limit of detection of < 10 pmol of IMP production), high reproducibility (< +/- 5%), and capability to measure activity in small samples (9 micrograms protein).     

Production of Nucleic Acid-Related Substances by Fermentative Processes. XXVIII. Accumulation of 5′ Inosinic Acid by a Manganese-Insensitive Mutant of Brevibacterium ammoniagenes

A manganese-insensitive mutant, KY 13105, of Brevibacterium ammoniagenes which accumulates considerable amounts of 5' inosinic acid (IMP) in the presence of 100 to 1,000 mug of Mn(2+) per liter was obtained from an IMP-producing mutant of a manganese-sensitive strain, KY 13102. The effects of Mn(2+) at 0 to 30 mug/liter on IMP accumulation by KY 13105 were similar to those by KY 13102. However, the accumulation of IMP by KY 13105 was not affected by 100 to 1,000 mug of Mn(2+) per liter, showing a clear difference from KY 13102. The accumulation of IMP by KY 13105 was always accompanied by cellular morphological changes irrespective of Mn(2+) concentration. In the presence of Mn(2+), factors which affect IMP accumulation by KY 13105 were examined. Most of the nutrients tested stimulated IMP accumulation at a relatively low concentration (2 g/liter). Iron, calcium, and zinc were found to be essential for IMP accumulation and were independent of Mn(2+). Biotin regulated the growth but not the accumulation of IMP. Under limited or surplus amounts of Mn(2+), the dynamics of IMP fermentation were followed. Under both conditions, the fermentations proceeded in a similar way. The morphological changes were found to be closely related to IMP accumulation.