Production of Nucleic Acid-Related Substances by Fermentation Processes: XXXIII. Accumulation of Inosine by a Mutant of Brevibacterium ammoniagenes

Inosine-producing cultures were found among mutants resistant to 6-mercaptoguanine (6MG) derived from a 5'-inosinic acid (IMP)-producing strain, KY 13102, of Brevibacterium ammoniagenes. Inosine-producing ability was very frequent among the mutants resistant to a low concentration (10 to 50 mug/ml) of 6MG. The accumulation of inosine by strain KY 13714 was stimulated by a low concentration of adenine (25 mg/liter) but was depressed by high levels of adenine. The accumulation by strain KY 13714 was not inhibited by manganese ion but instead was stimulated by its excess, in contrast to IMP accumulation by KY 13102. Addition of hypoxanthine at an early stage of cultivation accelerated inosine accumulation. Furthermore, on addition of hypoxanthine and of a surface-activating agent after 48 hr of cultivation, the simultaneous accumulation of IMP and inosine was observed. A 9.3-mg amount of inosine per ml accumulated after 4 days of cultivation at 30 C. The inosine-producing mutant did not differ from the IMP-producing strain either in 5' purine nucleotide degradation or in IMP formation from hypoxanthine. However, it was found to be completely devoid of purine nucleoside-degrading activity. The conversion of IMP accumulation to inosine can be explained by the lack of nucleosidedegrading activity. The relationship between deficiency of nucleoside-degrading activity and resistance to low levels of 6MG is discussed, and a new mechanism for 6MG resistance is presented.     

产氨短杆菌GMA-1112利用味精母液发酵生产肌苷

通过亚硝基胍或60 Coγ辐照等选育一株产氨短杆菌GMA 1 1 1 2 (Ade +Gua +VB1 +8 AGr+SGr+6 MPr) ,能以味精母液代替传统肌苷发酵添加酵母粉作为有机营养物 ,进行肌苷发酵 ,平均产肌苷率 2 5.4 0 g/L。具有降低发酵成本、提高产肌苷率等优点。     

Production of l-Lysine by Leucine Auxotrophs Derived from AEC Resistant Mutant of Brevibacterium lactofermentum

The effect of amino acids was examined on the production of l-lysine by AEC resistant mutant of B. lactofermentum. Among amino acids tested, only leucine showed strong specific inhibition. In order to release the production of l-lysine from this negative effect of leucine, leucine auxotrophs were derived from AEC resistant strain of B. lactofermentum. Most of these leucine auxotrophs produced larger amount of l-lysine (maximally 41 mg/ml) than the parental strain which produced about 18 mg/ml of l-lysine. It was confirmed that leucine auxotrophs derived from AEC resistant mutant of other glutamate producing bacteria, B. saccharolyticum and Corynebacterium glutamicum. These results suggested that leucine might directly or indirectly affect the biosynthesis of lysine.

However, this increase in lysine productivity of leucine auxotrophs could not be explained by the alteration of aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3). These enzymes are key enzymes in lysine and threonine biosynthesis, respectively.     

Fermentative Production of Imidazoleglycerol with a Histidine Auxotroph of Brevibacterium ammoniagenes

A histidine auxotioph of Brevibacterium ammoniagenes was found to accumulate imidazoleglycerol in the culture medium. The accumulation of it reached a level of 13 mg/ml as its monohydrochloride salt with a medium containing 12% glucose, 2% (NH₄)₂SO₄ and 2.5% meat extract. By-production of other imidazoles was little.     

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.     

短杆菌变异株FMP92814产苯丙氨酸的研究

经过多次的人工诱变筛选,获得一株来自乳糖发酵短杆菌ＡＴＣＣ１３８６９的变异株ＦＭＰ９２８１４。它对多种苯丙氨酸结构类似物具有抗性,并兼有酪氨酸营养缺陷型特性。我们对ＦＭＰ９２８１４菌株的发酵生产苯丙氨酸条件进行了研究。在５Ｌ台式小罐的发酵试验中,该菌株的苯丙氨酸产量可达２５．２ｇ／Ｌ,糖酸转化率１６．８％．。     

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.     

Fermentative Accumulation of Guanosine Polyphosphate by Brevibacterium ammoniagenes

Guanosine-3'-diphosphate-5'-monophosphate (3.35 mg/ml), guanosine-3'-diphosphate-5'-diphosphate (MSI) (5.21 mg/ml), and guanosine-3'-diphosphate-5'-triphosphate (MSII) (0.82 mg/ml), in addition to guanosine 5'-monophosphate, guanosine 5'-diphosphate, and guanosine 5'-triphosphate, were accumulated by microbial conversion of 5'-xanthylic acid with a mutant of Brevibacterium ammoniagenes.     

Accumulation of 6-Azauridine by Mutants of Brevibacterium ammoniagenes

From the inosine producing mutants of Brevibacterium ammoniagenes, KY 13761 was selected as a strain which produced 6-azauridine from 6-azauracil.

The conditions for the conversion were examined and the intermitent feeding of 6-azauracil was found effective for the accumulation.

In order to increase the accumulation, prototrophic revertants were induced from KY 13761 and KY 13021 was selected. By intermitent feeding of 6-azauracil of a final concentration of 6 mg per ml, a maximal accumulation, 12.4 mg/ml, of 6-azauridine was obtained with KY 13021.     

Production of L-Isoleucine by AHV Resistant Mutants of Brevibacterium flavum

The growth of Brevibacterium flavum No. 2247A was inhibited by α-amino-β-hydroxy-valeric acid (AHV), and the inhibition was partially reversed by L-isoleucine.

AHV resistant strain ARI-129, which was isolated on a medium supplemented with 2 mg/ml of AHV, produced 11 g/liter of L-isoleucine.

No difference was observed in threonine dehydratase between No. 2247A and ARI–129. Homoserine dehydrogenase from ARI–129 was insensitive to the feedback inhibition by L-isoleucine and L-threonine.

O-Methyl-L-threonine resistant mutant, strain AORI–126, which was derived from ARI–129, produced 14.5 g/liter of L-isoleucine. Specific activity of threonine dehydratase from AORI–126 increased about two-fold higher than those from No. 2247A and ARI–129, whereas degree of inhibition of the enzyme by L-isoleucine was the same among three strains.

Among auxotrophic mutants derived from ARI–129, adenine and lysine auxotrophs produced more L-isoleucine than the parent did.

In the adenine auxotroph, L-isoleucine production was markedly reduced by the addition of excess adenine.     

Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme

Ribonucleotide reduction and not DNA replication is the site for the specific manganese requirement of DNA synthesis and cell growth in the coryneform bacterium Brevibacterium ammoniagenes. To characterize the metal effect we have isolated and purified ribonucleoside-diphosphate reductase from overproducing bacteria that were first deprived of and then reactivated by manganese ions. Purification on columns of Sephacryl S400, DEAE-cellulose and hydroxyapatite provided an apparently homogeneous enzyme consisting of two protein subunits. These were characterized by affinity chromatography on 2',5'-ADP-Sepharose as nucleotide-binding protein B1 (Mr = 80,000) and catalytic protein B2 (Mr = 100,000, composed of two Mr = 50,000 polypeptides), which were both necessary for activity. In vitro the purified enzyme does not require added metal ions except for an unspecific, twofold activity increase observed in the presence of Mg2+ and other divalent cations. Enzyme activity is inhibited by hydroxyurea (I50 = 2.5 mM). The electronic spectrum with maxima around 455 nm and 485 nm closely resembles that of manganese(III)-containing pseudocatalase and of oxo-bridged binuclear Mn(III) model complexes. Denaturation of the enzyme in trichloroacetic acid liberated an equimolar amount of Mn(II) which was detected by EPR spectroscopy. It was not possible to remove and reintroduce metal ions without loss of enzyme activity. Manganese-deficient cell cultures were also grown in the presence of 54MnCl2. Ribonucleotide reductase activity and radioactivity cochromatographed in several systems. Non-denaturing polyacrylamide gel electrophoresis showed that protein subunit B2 was specifically 54Mn-labeled. All these properties suggest that the ribonucleotide reductase of B. ammoniagenes is a manganese-containing analog of the non-heme-iron-containing reductases of Escherichia coli and eukaryotes.     

A Metaphosphate-dependent Nicotinamide Adenine Dinucleotide Kinase from Brevibacterium ammoniagenes

The enzyme utilizing metaphosphate for nicotinamide adenine dinucleotide phosphorylation was purified 500-fold from B. ammoniagenes and its properties were studied. The isolated enzyme appeared homogeneous on disc gel electrophoresis; its molecular weight was determined to be 9.0 × 10⁴ by gel filtration. This enzyme specifically phosphorylated nicotinamide adenine dinucleotide at the optimum pH at 6.0. Of phosphoryl donors tested, metaphosphate was most effective for the reaction, and adenosine-5′-triphosphate was less effective. The activity was inhibited by adenosine-5′-monophosphate, adenosine-5′-diphosphate or reduced pyridine nucleotides. The enzyme did not exhibit catalytic activity in the absence of a divalent cation. We concluded that the enzyme phosphorylating nicotinamide adenine dinucleotide in the presence of metaphosphate is distinct from adenosine-5′-triphosphate-dependent nicotinamide adenine dinucleotide kinase, and tentatively designated it metaphosphate-dependent nicotinamide adenine dinucleotide kinase.     

Continuous production of NADP by immobilized Brevibacterium ammoniagenes cells.

Whole cells of Brevibacterium ammoniagenes IAM 1645 having the polyphosphate NAD-kinase were successfully immobilized in a polyacrylamide gel lattice. The immobilized cells were activated by treatment with organic solvents or detergents. The pH optimum of the immobilized cells for the production of NADP was 7.0, and divalent metal ions were required to maintain the elevated activity of polyphosphate NAD-kinase. Highly pure NADP was continuously produced in high yield by the immobilized cell column. The half-life of this column was about eight days.     

Effect of antibiotics on 5'-inosinic acid biosynthesis by a Brevibacterium ammoniagenes mutant

The effect of streptomycin, erythromycin, kanamycin and penicillin on the biosynthesis of 5'-inosinic acid (IMP) by the mutant strain Brevibacterium ammoniagenes was studied. It has been found that the efficiency of antibiotic action depends not only in its concentration but on the age of the culture. When the antibiotics were introduced into the culture broth at the beginning of fermentation, they inhibited the culture growth and accumulation of IMP in the cultural medium. Only after 36-72 hours of cultivation the addition of the antibiotics stimulated the biosynthesis. All the antibiotics tested when adding at the definite for each of them period of fermentation and at the definite concentration stimulated the accumulation of IMP. The stimulating effect appears to be connected with an increase in permeability. A considerable increase in the number of anormalous elongated and swollen cells and, as a rule, in the protein content of the cultural supernatant indicates the fact. Streptomycin and kanamycin were the most efficient antibiotics, as they increased the IMP yield from 10.4 to 17.5 g/l.     

Specific effect of endonucleases from Brevibacterium ammoniagenes on DNA

The previously described deoxyribonucleases from Brevibacterium ammoniagenes have been characterized. It was shown that they are endonucleases with molecular weights of 60 000 (I), 10 000 (II) and 20 000 (III). The rate of endonuclease I effect on native DNA exceeded that on the denatured DNA 2-fold. The mechanism of its action is of a single hit type. The enzyme hydrolyzes two chains of DNA simultaneously in two symmetrical sites and splits the bond 5'-P to form fragments with terminal 5'-OH and 3'-P. Endonuclease I was characterized as deoxyribonucleate-3'-oligonucleotide hydrolase (EC 3.1.4.6).     

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.     

A Euglena Mutant Resistant to N-Methyl-N-Nitroso-p-Toluenesulfonamide

SYNOPSIS. Treatment of Euglena gracilis strain Z with N-methyl-N-nitroso-p-toluenesulfonamide (MNTS) produced a mutant (designated MNTS-40A) showing 2.5-fold greater resistance to the killing effects of MNTS and 40-fold greater resistance to “bleaching” by MNTS. The mutant destroyed MNTS 2–3 times faster than strain Z. As a consequence the nucleic acids of strain 40A became methylated to a smaller extent than those of Z. Strain MNTS-40A was net cross-resistant to either N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light.     

Production of Ribotides of 4-Hydroxy- and 4-Aminopyrazolo[3, 4-d]pyrimidine by Brevibacterium ammoniagenes

Brevibacterium ammoniagenes ATCC 6872 was previously reported to accumulate large amounts of IMP, AMP, ADP, ATP, GMP, GDP and GTP from the corresponding purine bases. The organism was also reported to convert various derivatives of purine and 8-azapurine to the corresponding ribotides.

Using the similar process, ribotidation of pyrazolo[3, 4-d]pyrimidines was attempted, and it was found that the same organism was able to produce remarkable amounts of 4-hydroxy-1-β-d-ribofuranosylpyrazolo[3, 4-d]pyrimidine 5′-monophosphate (HPP-RP) from 4-hydroxypyrazolo[3, 4-d]pyrimidine (HPP, allopurinol) and 4-amino-1-β-d-ribofuranosylpyrazolo[3, 4-d]pyrimidine 5′-monophosphate and 5′-diphosphate from 4-amino-pyrazolo[3, 4-d]pyrimidine.

The crystals of HPP-RP (Na-salt) were isolated from the cultured broth of Br. ammoniagenes incubated with HPP, and characterized based on UV-spectra, IR-spectrum, NMR and others.

It was also found that HPP-RP was converted to the corresponding riboside by hydrolysis in aqueous solution (pH 4.0 ~ 9.0) for 6 hr at 140°C. The hydrolysis of HPP-RP was also accomplished with various organisms.