Production of Nucleic Acid-Related Substances by Fermentative Processes

Attempts were made with success to develop a chemically defined medium for 5′-purine ribonucleotide production by Brevibacterium ammoniagenes ATCC 6872 and its adenine auxotroph KY 7208.

The results demonstrated that the presence of pantothenate and thiamine and a limiting level of manganese in the medium are essential for IMP production from hypoxanthine. These conditions were likewise indispensable for GMP, GDP and GTP productions and AMP, ADP and ATP productions from corresponding bases by ATCC 6872 and for direct IMP fermentation with KY 7208 strain.

It was further shown that R5P accumulation by ATCG 6872 culture, in the absence of bases, was affected by the two vitamins and Mn²⁺ exactly in the same way as the nucleotide synthesis. Morphogenetic alterations were induced under such conditions as two vitamins added and Mn²⁺ kept deficient.     

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.     

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 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.     

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.     

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 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 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.     

Fermentative Production of l-Glutamine by Sulfaguanidine Resistant Mutants Derived from l-Glutamate Producing Bacteria

Excellent l-glutamine producers were screened for among sulfaguanidine resistant mutants derived from the wild type l-glutamic acid-producing bacteria, Brevibacterium flavum, Brevibacterium lac to fermentum, Corynebacterium glutamicum and Microbacterium ammoniaphilum.

The best strain, No. 1~60, was a sulfaguanidine resistant mutant derived from B. flavum 2247 by mutation. Strain No. 1~60 accumulated 41.0 mg/ml of l-glutamine after 48 hr of cultivation from 10% glucose as a carbon source. This yield was the highest among those so far reported.

The addition of Mn^{2 +} (2 ppm) to the standard medium for B. flavum 2247 decreased the l- glutamine production and increased the l-glutamic acid excretion markedly. On the contrary, strain 1 —60 was not affected the Mn²⁺ (2 ppm) addition at all.

Glutamate kinase activity and the intracellular content of ATP in sulfaguanidine resistant mutant No. 1~60 were higher than those in the parent strain, B. flavum 2247.

It was confirmed that the increase in glutamate kinase and the increase in internal ATP, which were important for the l-glutamine synthesis, were very effective for the improvement of l-glutamine-producing mutants.     

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: 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.     

Study on the Optical Rotatory Dispersion of Di-Peptides and its Application to the Recognition of Di-Peptides from Higher Peptides and Amino Acids

During the cource of the investigation of ribotidation of purine and pyrimidine bases by Brevibacterium ammoniagenes ATCC 6872, it was found that a large amount of uridine 5′-monophosphate (UMP) was accumulated in the culture broth when the organism was incubated in a medium containing uracil or orotic acid. The yields of UMP were 83% (4.8 mg/ml) from uracil and 100% (4.3 mg/ml) from orotic acid when each substrate was added at the concentration of 2 mg/ml.

Addition of 6-azauracil or 5-hydroxyuracil to the culture of the organism during cultivation led to the accumulation of both orotidine 5′-monophosphate (OMP) and UMP. The accumulation of OMP seemed to be due to the inhibition of OMP decarboxylase (E. C. 4.1.1.23) by the ribotide formed from each base. The OMP accumulation was enhanced by the addition of orotic acid in addition to 6-azauracil. When 6-azauracil was added to the medium before inoculation, UMP was predominantly accumulated, and when it was added after one day incubation, OMP was predominantly accumulated. A largest accumulation (3.6 mg/ml) of OMP was obtained when 6-azauracil was added on the 1st day and orotic acid was added on the 3rd day.

UMP and OMP accumulated in the medium were isolated from the cultured broth and identified by usual methods.     

Identification and biochemical characterization of a unique Mn2+-dependent UMP kinase from Helicobacter pylori

Uridine monophosphate (UMP) kinase converts UMP to the corresponding UDP in the presence of metal ions and ATP and is allosterically regulated by nucleotides such as UTP and GTP. Although the UMP kinase reported to date is Mg²⁺-dependent, we found in this study that the UMP kinase of Helicobacter pylori had a preference for Mn²⁺ over Mg²⁺, which may be related to a conformational difference between the Mn²⁺-bound and Mg²⁺-bound UMP kinase. Similar to previous findings, the UMP kinase activity of H. pylori UMP kinase was inhibited by UTP and activated by GTP. However, a relatively low GTP concentration (0.125 mM) was required to activate H. pylori UMP kinase to a level similar to other bacterial UMP kinases using a higher GTP concentration (0.5 mM). In addition, depending on the presence of either Mg²⁺ or Mn²⁺, a significant difference in the level of GTP activation was observed. It is therefore hypothesized that the Mg²⁺-bound and Mn²⁺-bound H. pylori UMP kinase may be activated by GTP through different mechanisms.     

Formation of Valine in the Presence of Sodium Acrylate Monomer by Glutamic Acid Producing Bacteria

Glutamic acid producing bacteria accumulated a large amount of valine in the presence of the excess biotin, when sodium acrylate monomer (Na-AM) was added at the earlier phase of culture. Brevibacterium roseum ATCC 13825, particularly, accumulated the large amount of valine among bacteria tested and the conditions of valine accumulation by this strain were investigated.

The most effective addition time of Na-AM was at the earlier phase of logarithmic phase. The optimal concentration of Na-AM for the accumulation of valine was 1.0 per cent (v/v). Most effective nitrogen sources were the combination of 1.0 per cent urea and 0.2 per cent ammonium sulphate. The additions of Mn²⁺ and Fe²⁺ increased valine accumulation. By the excess concentration of biotin for growth, 20 μg/liter or more, did not affected valine accumulation, while the presence of the suboptimal condition of biotin for growth was not good for the formation of valine even in the presence of Na-AM. The accumulation of valine reached 9.0 mg/ml from 75.0 mg/ml of glucose in the presence of 50 μg/liter of biotin and 1.0 per cent (v/v) of Na-AM.

This strain possessed considerable activity of valine formation regardless of the addition of Na-AM and promoted the accumulation of valine by the addition of Na-AM.     

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.     

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

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.     

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.     

Enhancing the production of uridine 5′-monophosphate by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using a whole cell biocatalytic process

Attempts were made with success to produce uridine 5′-monophosphate (UMP) from orotic acid by a recombinant Saccharomyces cerevisiae strain pYX212-URA5/BJX12, using the whole cell biocatalytic process. URA5 and URA3 genes, encoding orotate phosphoribosytransferase (OPRTase) and orotidine monophosphate decarboxylase (ODCase), respectively were successfully overexpressed in the industrial yeast strain. As a result, S .cerevisae pYX212-URA5/BJX12 exhibited the highest biocatalytic ability, in contrast with the original industrial yeast strain and S. cerevisae pYX212/BJX12 that overexpressed ODCase only. It indicated that the first step of UMP production from orotic acid is a rate-limiting step. Effects of cultivation for the recombinant strain and biocatalytic reaction conditions on UMP production were also investigated. Cultivating the cells in malt extract medium for 36 h in the exponential phase of growth is in favor of converting orotic acid to UMP. To acquire a higher UMP yield, the conditions of the whole cell biocatalytic reaction were optimized and up to 3.8 g l⁻¹ UMP was produced in 24 h consequently. The yield was fivefold higher than the original UMP yield from the industrial yeast. In addition, the accumulation of 2.6 g l⁻¹ UDP (uridne 5′-diphosphate) in the process demonstrated the possibility for further genetic manipulation: deleting the UMPK (Uridylate Kinase, catalyzing UMP–UDP).