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.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

Brevibacterium insectiphilium KY 3446 (Steinhous, Breed AHU 1401) was found to accumulate IMP from hypoxanthine and UMP from uracil, respectively. This strain is thus considered to present the fourth example in salvage-type fermentation, in addition to Micrococcus sodonensis, Arthrobacter citreus and Brevibacterium ammoniagenes reported previously.

IMP from adenine and UMP from cytosine were also produced by KY 3446, respectively. Further, the addition of inosine and adenosine instead of the bases also caused IMP accumulation.

This strain grew well on sucrose medium, and produced IMP and UMP in higher yields on sucrose than on glucose medium.

Excessive amounts of Mn²⁺ stimulated growth, but markedly inhibited IMP production. The optimal concentration of Mn²⁺ for IMP accumulation induced morphogenetic alterations from normal and small to abnormal and large cells.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

An adenine-requiring mutant (KY7208) of Brevibacterium ammoniagenes ATTC 6872 was found to accumulate an appreciable quantity of IMP and hypoxanthine in the culture liquid.

Crystalline IMP was isolated from culture broth of KY7208 by the use of ion-exchange columns. The preparation obtained was definitely identified as 5′-IMP, based on the results on paperchromatography, UV and IR absorption spectra, and analyses of its hydrolysates.

Growth responses of this mutant were demonstrated to adenine and adenosine, but not to 5′-AMP, 3′-AMP and 5′-AMP.

Over 5 mg of IMP per ml of broth were produced by the organism in natural medium consisting of glucose, yeast extract, urea, high concentrations of phosphate and magnesim salts, and others. The chemical changes showed that hypoxanthine first accumulated in the earlier stage of fermentation, and IMP synthesis then took place with the disappearance of hypoxanthine in the later stage of fermentation.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

A seed medium and a fermentation medium for nucleotide fermentations such as 5′ IMP, 5′GMP (plus GDP and GTP) and 5′AMP (plus ADP and ATP) with Brevibacterirm ammoniagenes ATCC 6872 were entirely chemically defined, with the use of a mixture of five amino acids.

As a result, the presence of Zn²⁺, Fe²⁺ and Ca²+ in addition to Mn²⁺ was found to be essential for the nucleotide fermentations. In particular, Zn²⁺ levels as well as Mn²⁺ affected nucleotide productions remarkably. Various fermentations proceeded favorably only when suboptimum levels of manganese (20~30 μg/liter) and zinc (100~200 μg/liter) were simultaneously present. This effect of trace metals was attributed to the fact that the excretion of R5P, a precursor of nucleotides, and those enzymes catalyzing reactions synthesizing nucleotides from R5P, ATP and purine bases were greatly stimulated by trace metals in cooperation with two vitamins, Ca-pantothenate and thiamine, and presumably high concentrations of phosphate and magnesium.

Furthermore, it was revealed that some metals were able to control the amounts of nucleotides accumulated when they were added to the broth during fermentation. For example, Hg²⁺ and Ag⁺ could increase the amounts of 5′GMP or 5′AMP, and decrease those of GTP and ATP.

Growth responses of Brevibacterium ammoniagenes ATCC 6872, capable of accumulating purine nucleotides, were investigated by the use of completely defined media.

1. Casamino acids required for its growth could be replaced by a mixture of l-histidine, l-homoserine, glycine, d, l-alanine and l-lysine. A completely defined medium for nucleotide productions was thus established by the use of this mixture.

2. High levels of phosphate inhibited growth markedly, and this inhibition was overcome by the simultaneous addition 1) of hign levels of Mg²⁺ and 2) of Mn²⁺, 3) pantothenate and 4) thiamine. Ca²⁺ had also a stimulatory effect on the growth. Therefore, a clear growth response to Mn²⁺ levels and the requirement of the two vitamins for growth emerged only under the conditions of high phosphate and magnesium salts. These 4 factors were found entirely the same as factors essential for nucleotide accumulations by Br. ammoniagenes.

     

Production of Nucleic Acid-Related Substances by Fermentative Processes

Enzymatic studies with Brevibacterium ammoniagenes ATCC 6872 demonstrated that 5-phosphoribose pyrophosphokinase and purinenucleotide pyrophosphorylase were involved in the nucleotide synthesis from purine base by ATCC 6872 and that its actual accumulation from base seemed to take place extracellularly through the action of the salvage enzymes leaked out of cells. Mn²⁺ deficiency and the simultaneous presence of pantothenate and thiamine, essential for efficient nucleotide accumulation, caused the extracellular leakage of the two enzymes with the simultaneous excretion of R5P. In the direct IMP fermentation with the adenine auxotroph, it was verified that hypoxanthine first produced de novo was reconverted into IMP extracellularly by the salvage enzymes as speculated previously.

A guanine-requiring mutant of Brevibacterium ammoniagenes ATCC 6872 accumulated a large amonnt of 5′-xanthosine-monophosphate (abbreviated as XMP).

The quantity of XMP accumulated by the strain was affected significantly by guanine levels in the medium. The suppression of XMP accumulation by an excessive addition of guanine compounds was recovered by the supply of casamino acids in the medium.

An enzyme in the pathway of de novo XMP synthesis, IMP dehydrogenase (IMP: NAD oxidoreductase, EC 1.2.1.14), was repressed and inhibited by guanine compounds.

The facts that an exogenous xanthine was not converted to XMP by the growing cells and that the activity of XMP-pyrophosphorylase was very low or deficient suggest that XMP accumulation by the strain would be probably due to the direct excretion of the nucleotide from the cells.     

Pentose Metabolism in Candida utilis

In attempts to obtain GMP producing strains, Brevibacterium ammoniagenes was treated with UV, N.T.G. or D.E.S. as a mutagen. Adenine-guanine requiring mutants were obtained from an adenine-requiring mutant of Brev. ammoniagenes, KY 3482–9 and two of them, presumably adenine-xanthine requiring mutants, were then reverted to mutants which required only adenine for their growth.

Although these revertants were not able to accumulate a copious amount of GMP, most of them and of adenine-guanine requiring mutants produced larger amounts of IMP than the parent adenine-requiring strain.

Effects of Mn²⁺ and purine bases in the medium on IMP production by these mutants were examined and IMP productivities of these mutants were compared with the parent strain under optimal conditions.

These mutagenic treatments were thus proved to be effective for the increase of de novo IMP production by Brev. ammoniagenes mutants.

Brevibacterium ammoniagenes ATCC 6872 accumulates 5′-GDP and -GTP, or 5′-ADP and -ATP together with GMP or AMP in nucleotide fermentation by salvage synthesis.

With cell free extract of this strain, transphosphorylating reactions of AMP or GMP were investigated.

ATP-AMP transphosphorylating enzyme(s) was partially purified to 21.7 fold with acid treatment, salting-out and column chromatography.

In ATP-AMP and ATP-GMP transphosphorylating reactins, optimal conditions were decided such as for concentrations of enzyme, of MgCl₂ and of phosphate donor, pH and cell age as the enzyme sources.

Specificities of phosphate donors and acceptors were examined with both the partially purified enzymes or the sonicate. AMP and GMP were phosphorylated by ATP rapidly, but IMP and XMP were not, therefore supporting our previous finding that Brev. ammoniagenes could not accumulated IDP, ITP, XDP and XTP in IMP and XMP fermentation, respectively.

Although ATP was the best donor for both AMP and GMP phosphorylations, other nucleoside triphosphates and PRPP were used as phosphate donors.

Furthermore, phosphorylation of ADP to ATP was investigated and possible mechanisms of nucleoside di- or triphosphates synthesis in the nucleotide fermentation were discussed.

From these results, it is suggested as a possible mechanism for nucleoside di- and triphosphate accumulation by Brev. Ammoniagenes, that a nucleoside monophosphate formed is phosphorylated to a nucleoside di-phosphate with ATP or other phosphate donors and then the nucleoside diphosphate is converted to a triphosphate with these phosphate donors.

Both AMP and GMP were transphosphorylated rapidly to the corresponding nucleoside-diphosphates and triphosphates by ATP and by other high energy phosphate compounds with cell free extracts of Brevibacterium ammoniagenes.

Some enzyme inhibitors, such as metals and PCMB were shown to inhibit the phosphorylations of AMP and GMP. Higher levels of ATP, ADP, GTP and GDP also inhibited the activity of the partially purified ATP-AMP transphosphorylating enzyme(s).

In guanine nucleotides fermentation by salvage synthesis with this strain, addition of these inhibitors to the medium increased the amounts of GMP and total guanine nucleotides accumulated.

On the contrary, supplement of xylene or of other organic solvents to the medium stimulated the accumulation of both GTP and total guanine compouuds in this fermentation. From enzymatic studies, these solvents are presumed to have the ability to change cell permeability.

Such findings give an effective method for controlling the amounts of nucleotides accumulated in these fermentations.     

Conversion of 5' xanthylic acid to guanine and guanine nucleotides by a mutant of Brevibacterium ammoniagenes

A decoyinine resistant, KY 13501, isolated after nitrosoguanidine treatment from Brevibacterium ammoniagenes ATCC 6872 converted 5′XMP added in fermentation media to guanine derivatives and accumulated them in the media. The converted substances were identified as guanine, 5′GMP, 5′GDP, and 5′GTP. The conditions for the conversion were examined and the following points were clarified. (1) Very low concentration of manganese ion (Mn²⁺) showed profound effects on the conversion and the excessive amounts of the ion severely repressed the conversion. (2) Under limitation of Mn²⁺, 5′XMP was converted most efficiently when added at inoculation time. (3) The inhibition of the conversion by excessive amount of Mn²⁺ was completely released by addition of a surface activating agent, polyoxyethylene stearylamine. (4) For the conversion, it was essential to maintain pH of the media at 7.5 to 8.0 and supply ammonium ion.     

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.     

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.     

Production of Nucleic Acid-Related Substances by Fermentation Processes

The effects of manganese ion (Mn²⁺) and adenine on the accumulation of 5′ inosinic acid (IMP) by Brevibacterium ammoniagenes KY 13102, were examined. Adenine regulated the accumulation of IMP in the presence of limiting amounts of Mn²⁺ and the accumulation of hypoxanthine (Hx) in the presence of excessive amounts of the ion. Manganese ion markedly affected IMP accumulations, cell growth and cellular morphology. These biological changes caused by Mn²⁺ are related to changes in the syntheses of macromolecules. The cells cultivated under limitation of Mn²⁺ showed abnormally elongated and irregular forms irrespective of adenine levels and had smaller nucleotide pools than those of the cells in the presence of excessive Mn²⁺. The Mn²⁺ limited cells showed ability to accumulate IMP directly in the cell suspension but the Mn²⁺ excessive cells did not accumulated IMP but Hx. These results indicated that adenine and Mn²⁺ affected the IMP accumulation independently each other and adenine acted as a feedback regulator on de novo synthesis of purine nucleotide and limitation of Mn²⁺ caused morphological changes, resulting in changes of permeability of the cells. The fatty acid contents of the Mn²⁺ limited cells were higher than those of the Mn²⁺ excessive cells and the ratio of unsaturated fatty acid to saturated one was higher in the former cells.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

1. Suitable agar plate media were selected for isolation of nucleotide producing strains, by salvage synthesis, from natural sources. Since this agar medium contains a high concentration of phosphates, manganese and glucose, it is specific for these bacteria.

2. With this plate medium, 113 bacterial strains accumulating 5′inosinic acid (IMP) or IMP-like substances were isolated effectively from feces of a variety of birds and mammals and from soils.

Some of the strains isolated were recognized to accumulate other nucleotides, purine bases and sugars, such as guanine nucleotides, XMP, xanthine, ribulose or xylnlose, with or without hypoxanthine in the media.

3. Five strains of IMP accumulating bacteria were identified; two were classified as Brevibacteriurm, two as Corynebacterium and one as Arthrobacterium species by taxonomical studies. But their characteristics did not completely coincide with those of bacteria described in Bergey’s manual.

4. One of the IMP producing bacteria isolated, culture No. 21–26, actually consisted of two separate strains, namely No. 21–26–101 and No. 21–26–102. The highest production of IMP or guanine nucleotides was obtained, when each strain was inoculated together to the fermentation medium from each seed culture in the same inoculum size.

5. The nucleotide productions by No. 21–26–101 or No. 21–26–102 with authentic strains were examined by the mixed culture technique. It was found that production of IMP or guanine nucleotides by Brevibacterium ammoniagenes ATCC 6871 was stimulated remarkably in the presence of No. 21–26–102.     

Production of N-Succinyl-l-diaminopimelic Acid by Auxotrophic Mutants of Aerobacter aerogenes and Serratia marcescens

α,ε-Diaminopimelic acid (DAP)-requiring mutants isolated from Aerobacter aerogenes ATCC 8308 and Serratia marcescens ATCC 19180 were found to accumulate N-succinyl-l-diaminopimelic acid (SDAP) which was an intermediate in the biosynthesis of lysine in Escherichia coli. SDAP was isolated from the culture broth and identified by the behavior in paper chromatography, melting point, elementary analysis, infrared spectrum, and optical rotation.

The culture conditions for SDAP production by A. aerogenes KY 7049 (DAP^?) and S. marcescens KY 8921 (DAP^?/Lys^?) were investigated. A. aerogenes KY 7049 has an absolute requirement for DAP together with a relative requirement for l-lysine. High levels of DAP (2000~4000 μg/ml) were proved to be favorable for SDAP accumulation, while if lysine along with DAP was added to the fermentation medium, optimal level of DAP for SDAP production was relatively low (about 200 μg/ml at 200 μg/ml of lysine). A variety of compounds which may conceivably affect the course of a fermentation process, i.e., carbon source, inorganic nitrogen source, amino acids, vitamines, precursors, were screened at optimal levels of lysine and DAP. Thus, the amount of SDAP accumulation reached a level of 19.9 mg/ml with the medium containing 10% glucose and 2000 μg/ml of DAP. S. marcescens KY 8921 requires either DAP or lysine for growth. Optimal level of DAP and lysine for SDAP accumulation was 50~100μg/ml.     

Arrest of cell cycle by inhibition of ribonucleotide reductase induces accumulation of NAD+ by Mn2+-supplemented growth of Corynebacterium ammoniagenes

Cell division of the wild type strain Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 which requires 1 M Mn²⁺ for balanced growth was inhibited by addition of 20 mM hydroxyurea (HU) or 10 mM p-methoxyphenol (MP) to a Mn²⁺-supplemented fermentation medium at an appropriate time. Scanning electron microscopy (SEM) showed a restricted elongation characteristic of arrest of the cell cycle in coryneform bacteria. The cultures treated with HU or MP had, respectively, a fourfold or sixfold enhanced accumulation of NAD⁺ by a salvage biosynthetic pathway. An assay of nucleotide-permeable cells for ribonucleotide reductase activity using [³H-CDP] as substrate revealed a pre-early and complete decline of DNA precursor biosynthesis not found in the untreated control. Overproduction of NAD⁺ is an alternative to the conventional fermentation process using Mn²⁺ deficiency. A simple model is presented to discuss the metabolic regulation of the new process based on the presence of a manganese ribonucleotide reductase (Mn-RNR) in the producing strain.     

Growth of Yeasts on n-Alkanes: Inhibition by a Lactonic Sophorolipid Produced by

The effects of amino acids on IMP production were examined with a mutant strain, KY10895, derived from Corynebacterium ammoniagenes KY13374. l-Proline improved the productivity of IMP more than any other amino acid. The optimum concentration of l-proline for IMP production was 1–2% and the IMP productivity was about 70% more than that in the control medium. The effects of l-proline analogs on IMP production were also examined with the mutant KY10895. DL-3,4-Dehydroproline inhibited IMP production. Mutants resistant to growth inhibition by dl-3,4-dehydroproline were derived from strain KY10895. Among mutants thus obtained, strain H-7335 had the highest productivity. The intracellular concentrations of l-proline in strain H-7335 were higher than those of the parental strain, KY10895. These findings indicated that an increase in intracellular l-proline was linked with an increase of IMP productivity and strengthening the l-proline synthesis of a strain was an effective method for obtaining a hyper-producer of IMP.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

During the course of the investigation on the production of nucleotide by fermentative processes, it was found that a large amount of ATP and ADP or GTP and GDP, in addition to a smaller amount of AMP or GMP, accumulated in the culture broth when Brevibacterium ammoniagenes ATCC 6872 was incubated in a medium containing adenine or guanine.

After treatment of the culture filtrate with charcoal, the nucleotides were isolated by ion-exchange chromatography on Dowex-1 × 2 (Cl^?-form). They were identified by paper-chromatography, ultraviolet absorption spectra and analyses of base, ribose and phosphate. The ATP preparation from the broth had the same activity with that of authentic sample in the β-aspartokinase system from Corynebacterium glutamicum.     

Conversion of Purine Bases and Their Ribosides to 5′-Inosinic Acid by Bacillus subtilis

Among various nutritional mutants with weak 5′-nucleotidase derived from Bacillus subtilis IAM 1145, the adenine-requiring mutants could convert exogenously added hypo- xanthine, guanine, xanthine and their ribosides to 5′-inosinic acid (IMP) and accumulate it in the medium. Synthesis of IMP from purine derivatives was observed predominantly in an early stage of the cultivation. The conversion was stimulated by Fe²⁺ or Mn²⁺, and markedly depressed by an excess amount of adenine in the production-medium.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

The presence of psicofuranine in the fermentation medium caused the accumulation of a copious amount of 5′–XMP by Brevibacterium ammoniagenes. The accumulation of 5′–XMP in the medium was considered to be due to the inhibition of converting 5′–XMP to 5′–GMP by psicofuranine, which is known as a specific inhibitor of XMP aminase.

It was previously reported that in 5′–IMP fermentation with Br. ammoniagenes pantothenate and thiamine, in addition to biotin which was required for the growth of the microorganism, were exclusively required. This requirement for both vitamins was also observed in 5′–XMP production induced by the antibiotic.

The addition of manganese in excess to the fermentation medium promoted the bacterial growth greatly and inhibited IMP production, whereas XMP production induced by piscofuranine was not affected by the addition of excess manganese.

The accumulation of XMP induced by the antibiotic was completely suppressed by the presence of purine derivatives such as guanine, and xanthine derivatives, and partially by hypoxanthine.

5′–XMP was identified by chemical and enzymatic analyses and by UV absorption spectrum.     

Parameters of unbalanced growth and reversible inhibition of deoxyribonucleic acid synthesis in Brevibacterium ammoniagenes ATCC 6872 induced by depletion of Mn2+. Inhibitor studies on the reversibility of deoxyribonucleic acid synthesis

Unbalanced growth induced by depletion of manganese ions was a prerequisite for production of ribonucleotides in a high salt mineral medium with the wildtype strain Brevibacterium ammoniagenes ATCC 6872. The concentration of manganese strictly controlled the overall deoxyribonucleic acid (DNA) synthesis, whereas ribonucleic acid (RNA), protein and cell wall synthesis remained essentially unimpaired in the manganese-lacking cells.The reversibility of inhibition of overall DNA synthesis was shown by enhanced incorporation (up to threefold compared to the cultures supplied with sufficient manganese) of [8-¹⁴C] adenine into alkali-stable, trichloroacetic acid-insoluble material after subsequent addition of 10 M MnCl₂ to 15 h-old depleted cultures.The results of inhibitor studies on the restoration of overall DNA synthesis due to subsequent addition of manganese ions to depleted cultures suggest that ribonucleotide reduction is the primary target of the manganese starvation during nucleotide fermentation with Brevibacterium ammoniagenes ATCC 6872.     

Production of l-Leucine by a Mutant of Brevibacterium lactofermentum 2256

A potent l-leucine producer was screened among mutants of glutamic acid producing bacteria. This strain, No. 218, is one of 2-thiazolealanine resistant mutants derived from a methionine isoleucine double auxotroph of Brevibacterium lactofermentum 2256 by nitroso-guanidine.

Strain No. 218 produced 19 mg/ml of l-leucine after 72 hr cultivation when 8 % glucose and 4 % ammonium sulfate were supplied as a carbon and a nitrogen source, respectively, thus giving the yield of 23.1 % from glucose.

The addition of Fe²⁺ and Mn²⁺ in combination gave much more productivity than that of Fe²⁺ or Mn²⁺ alone.

Effects of amino acids, nucleic acids, vitamins, and the other nutrients on l-leucine production were investigated.

The fermentation product was isolated and purified from the culture, and identified as l-leucine.     

Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria

We previously reported on the secretion of Streptomyces mobaraensis transglutaminase by Corynebacterium glutamicum ATCC13869 (formerly classified as Brevibacterium lactofermentum). In the present work, we investigated whether any other coryneform bacteria showed higher productivity than C. glutamicum ATCC13869. We found that most coryneform species secreted pro-transglutaminase efficiently. Moreover, we confirmed that Corynebacterium ammoniagenes ATCC6872 produced about 2.5 g/l pro-transglutaminase over a 71-h period in a jar fermentor. Our findings suggest that some other coryneform bacteria, especially C. ammoniagenes ATCC6872, are potential hosts for industrial scale protein production.