UMP pyrophosphorylase of Tetrahymena pyriformis. Partial purification and properties.

UMP pyrophosphorylase (EC 2.4.2.9, UMP:pyrophosphate phosphoribosyltransferase) was purified approximately 85-fold from exponentially growing cells of Tetrahymena pyriformis GL-7. It was found to have a molecular weight of 36,000, and was active over a broad pH range, with an optimum at 7.5. The enzyme exhibited a temperature optimum at 40 °C, above which irreversible inactivation began to occur. The apparent K_m values for uracil and phosphoribosyl pyrophosphate (PRPP) were 0.4 and 6.9 m, respectively. The pyrophosphorylase exhibited a pyrimidine base specificity for uracil, although 5-fluorouracil was utilized by the enzyme. Neither cytosine, orotic acid, nor 6-azauracil competed with uracil for the enzyme or inhibited the production of UMP from uracil and PRPP. Although most triphosphates had little effect on pyrophosphorylase activity, UTP and dUTP, each at a concentration of 1 mm, depressed UMP formation by 86 and 59%, respectively. Thus, UMP pyrophosphorylase may be sensitive to feedback inhibition by the product of the pathway it initiates. UMP pyrophosphorylase specific activity in extracts of Tetrahymena grown in a medium containing uracil as the sole pyrimidine source was threefold higher than that in extracts of cells grown on uridine or UMP.     

The mechanisms of citrate on regulating the distribution of carbon flux in the biosynthesis of uridine 5′-monophosphate by Saccharomyces cerevisiae

A whole cell biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. The concentration of UMP was increased by 23% when 1 g l⁻¹ sodium citrate was fed into the broth. Effects of citrate addition on UMP production were investigated. Glucose-6-phosphate pool was elevated by onefold, while FBP and pyruvate were decreased by 42% and 40%, respectively. Organic acid pools such as acetate and succinate were averagely decreased by 30% and 49%. The results demonstrated that manipulation of citrate levels could be used as a novel tool to regulate the metabolic fluxes distribution among glycolysis, pentose phosphate pathway, and TCA cycle.     

Production of uridine 5′-monophosphate by Corynebacterium ammoniagenes ATCC 6872 using a statistically improved biocatalytic process

Attempts were made with success to develop a two-step biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Corynebacterium ammoniagenes ATCC 6872: the strain was first cultivated in a high salt mineral medium, and then cells were harvested and used as the catalyst in the UMP production reaction. Effects of cultivation and reaction conditions on UMP production were investigated. The cells exhibited the highest biocatalytic ability when cultivated in a medium containing corn steep liquor at pH 7.0 for 15 h in the exponential phase of growth. To optimize the reaction, both “one-factor-at-a-time” method and statistical method were performed. By “one-factor-at-a-time” optimization, orotic acid, glucose, phosphate ion (equimolar KH₂PO₄ and K₂HPO₄), MgCl₂, Triton X-100 were shown to be the optimum components for the biocatalytic reaction. Phosphate ion and C. ammoniagenes cell were furthermore demonstrated as the most important main effects on UMP production by Plackett–Burman design, indicating that 5-phosphoribosyl-1-pyrophosphate (PRPP) synthesis was the rate-limiting step for pyrimidine nucleotides production. Optimization by a central composition design (CCD) was then performed, and up to 32 mM (10.4 g l⁻¹) UMP was accumulated in 24 h from 38.5 mM (6 g l⁻¹) orotic acid. The yield was threefold higher than the original UMP yield before optimization.     

Studies on the Accumulation of Orotic Acid by Escherichia coli K12

More than 300 mg/liter of orotic acid was found to accumulate in the supernatants of the cultures of wild type strains of E. coli K12. The pyrimidine precursor was accumulated in a synthetic medium such as glucose-ammonium sulfate medium. The substance was isolated from the culture, crystallized, and identified as orotic acid. Orotic acid was excreted mainly during logarithmic phase of the bacterial growth. Yeast extract or nutrient broth stimulated bacterial growth, but suppressed orotic acid accumulation. E. coli strains other than K12 failed to accumulate orotic acid.

The results suggest that the accumulation of orotic acid is specific to E. coli K12.     

酿酒酵母中过表达URA5及URA3基因催化合成UMP的初步研究

为提高乳清酸到尿嘧啶核苷酸(UMP)的转化效率,利用PCR方法扩增酿酒酵母乳清酸磷酸核糖转移酶基因URA5, 并将其连接到携带乳清苷酸脱羧酶基因URA3的表达载体pYX212中,构建了重组质粒pYX212-URA5,然后转化到酿酒酵母BJX12中进行表达,并进行转化乳清酸到UMP的初步研究。试验结果表明: pYX212-URA5/ BJX12发酵培养40h后以32 mM乳清酸为底物催化产生UMP的量约为7 mM。明显高于同等条件下pYX212/ BJX12的UMP产量2.7 mM和对照组野生型BJX12的UMP产量2.4 mM。     

Mechanism of uridine production by Bacillus subtilis mutants

Summary Pyrimidine analogue-resistant mutants of Bacillus subtilis were found to produce a large amount of uridine. One of them accumulated 55 mg/ml of uridine in culture medium. The changes in enzymes involved in the metabolism of uridine 5-monophosphate (UMP) were examined with this mutant. All six enzymes of de novo UMP biosynthesis were completely free from regulation by uridine compounds, and the activities of these enzymes were 16- to 30-fold higher than those of the enzymes of the parental strain. In the mutant strain, the level of uridine phosphorylase, responsible for converting uridine to uracil, was extremely low, compared with that of the parental strain. No apparent change was observed between the strains in the activity of UMP dephosphorylation or uracil phosphoribosyltransferase. The implication of these findings is discussed in relation to the overproduction of uridine by the mutant.Microbial production of uridine. Part III     

6-azauracil-resistant variants of cultured plant cells lack uracil phosphoribosyltransferase activity

6-Azauracil-resistant variants of Haplopappus gracilis (Nutt.) Gray and Datura innoxia Mill. lack activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme that catalyzes the conversion of uracil and 6-azauracil to uridine-5′-monophosphate and 6-azauridine-5′-monophosphate, respectively. Resistant cells are competent to take up uracil from their growth medium but do not convert it into a form that can be used for macromolecular synthesis. In extracts from resistant cells, orotate monophosphate decarboxylase, a target enzyme of 6-azauridine monophosphate, is fully sensitive to the phosphorylated analog. These results strongly suggest that uracil phosphoribosyltransferase is the major pathway of pyrimidine salvage in cells of these species and that loss of this enzyme activity confers on the variants resistance to 6-azauracil.     

A method for assaying orotate phosphoribosyltransferase and measuring phosphoribosylpyrophosphate

An orotate phosphoribosyltransferase, OPRTase, assay method which relies upon binding reactant [3H]orotic acid and product [3H]orotidine-5'-monophosphate to polyethyleneimine-impregnated-cellulose resin and collecting on a GFC glass fiber filter is presented. Elution with 2 X 5 ml of 0.1 M sodium chloride in 5 mM ammonium acetate removes all of the orotate and leaves all of the product orotidine monophosphate (OMP) bound so that it may be measured in a scintillation counter. It was found that the addition of 10 microM barbituric acid riboside monophosphate to the reaction mixture prevented the conversion of OMP to UMP and products of UMP. The assay is suitable for measurement of OPRTase activity with purified enzyme or in crude homogenates. A modification of this scheme using commercially available yeast OPRTase and 10 microM of unlabeled OMP provides an assay for phosphoribosylpyrophosphate with a sensitivity such that 10 pmol of PRPP may be measured.     

Biosynthesis of pyrimidine nucleotides in cultured root callus ofCucurbita pepo

Summary Callus cultures derived from roots of summer squash (Cucurbita pepo L. c.v. Early Prolific Straightneck) grown in the dark at 27° C on Murashige and Skoog medium supplemented per liter with 30 g sucrose, 100 mg myo-inositol, 10 mg indole-butyric acid, 2 mg glycine, 1 mg thiamin, 0.5 mg nicotinic acid, 0.5 mg pyridoxine, and 2 g Gelrite were capable of synthesizing pyrimidine nucleotides both de novo and through salvage of existing pyrimidine nucleotides and bases. Evidence that the de novo biosynthesis of pyrimidine nucleotides proceeded via the orotate pathway in this tissue included: (a) demonstration of the incorporation of NaH¹⁴CO₃ and [¹⁴C₆]orotic acid into uridine nucleotides (ΣUMP), and (b) demonstration that the addition of 6-azauridine blocked the incorporation of these two precursors into ΣUMP. The synthesis of pyrimidine nucleotides through the salvage of existing pyrimidine bases and ribosides was demonstrated by measuring the incorporation of [¹⁴C₂]uracil and [¹⁴C₂]uridine into ΣUMP. Salvage of both [¹⁴C₂]uracil and [¹⁴C₂]uridine was sensitive to inhibition by 6-azauridine or one of its metabolites. The orotic acid pathway for the de novo biosynthesis of pyrimidine nucleotides was demonstrated to be sensitive to end-product inhibition. Uridine, or one of its metabolites, inhibited the incorporation of NaH¹⁴CO₃, but not [¹⁴C₆]orotic acid, into ΣUMP. Evidence is presented suggesting that Aspartate carbomoyltransferase is the site of feedback control. This work was supported by the Citrus Research Center and Agricultural Experiment Station of the University of California, Riverside, CA. Submitted in partial fulfillment of the requirements of the University of California for the Master of Science degree in botany (F-F.L.)     

Radioassay of orotic acid phosphoribosyltransferase and orotidylate decarboxylase utilizing a high-voltage paper electrophoresis technique or an improved 14CO2- release method.

Orotic acid phosphoribosyltransferase (EC 2.4.2.10) and orotidylate decarboxylase (EC 4.1.1.23) can be assayed independently of one another by the high voltage paper electrophoresis method described here, which separates orotic acid, OMP, and UMP, the substrates and/or products of these enzymes, from each other. The relative migration of other compounds, mainly other nucleotides, their bases, or other intermediates of the UMP biosynthetic pathway, has also been recorded. The method has allowed us to observe that OMP is not released to any significant degree from the enzyme complex of these two enzymes that occurs in Ehrlich ascites cells; rather orotic acid is converted stoichiometrically by the enzyme complex to UMP. For purification of the enzyme complex, we have found the release of ¹⁴CO₂ from [¹⁴C]carboxyl-labeled orotic acid (when phosphoribosyl pyrophosphate and Mg²⁺ are present) preferable to the HVPE method as a routine assay procedure. The most economical CO₂-absorbant for the assay of the enzyme complex or for orotidylate decarboxylase (and possibly for other enzymes which release CO₂) is an NaOH-soaked paper strip. As detailed here, its use allows one to repeatedly reuse the scintillation vials and fluid.     

Controlling selectivity and enhancing yield of flavonoid glycosides in recombinant yeast

Flavonoid glycosides are known for their medicinal properties and potential use as natural sweeteners. In this study, Saccharomyces cerevisiae expressing a flavonoid glucosyltransferase from Dianthus caryophyllus was used as a whole-cell biocatalyst. The yeast system’s performance was characterized using the flavanone naringenin as a model substrate for the production of naringenin glycosides. It was found that final naringenin glycoside yields increased in a dose-dependent manner with increasing initial naringenin substrate concentrations. However, naringenin concentrations >0.5 mM did not give further enhancements in glycoside yield. In addition, a method for controlling overall selectivity was discovered where the glucose content in the culture medium could be altered to control the selectivity, making either naringenin-7-O-glucoside (N7O) or naringenin-4′-O-glucoside (N4O) the major products. The highest yields achieved were 87 mg/L of N7O and 82 mg/L of N4O using 40MSGI and 2xMSGI media, respectively. The effects of two intermediates involved in UDP-glucose biosynthesis, uridine 5′-monophosphate (UMP) and orotic acid, on glycoside yields were also determined. Addition of UMP to the culture medium significantly decreased glycoside yield. In contrast, addition of orotic acid to the culture medium significantly enhanced the glycoside yield and shifted the selectivity toward N7O. The highest naringenin glycoside yield achieved using 10 mM orotic acid in the 40MSGI media was 155 mg/L, a 71% conversion of substrate to product.     

Fermentative Production of Nδ-Acetyl-L-ornithine with an Arginine and Pyrimidine Auxotroph of Paracolobactrum coliforme

In the course of selecting a useful mutant strain for a fermentative production of L-valine, it was found that an arginine-pyrimidine auxotroph of Paracolobactrum coliforme accumulated N^δ-acetyl-L-ornithine (δ-AO) in the culture medium. The accumulation of it reached a level of 16 mg/ml with medium containing 12.5 % glucose, 2.2% (NH₄)₂SO₄, 0.5% peptone and 300 μg/ml of uracil. The wild strain 775 also accumulated 1.4 mg/ml of δ-AO in the medium supplemented with a high level (300μg/ml) of uracil when L-ornithine (10 mg/ml) was added in the middle phase of fermentation. The mutant cells elongated under the condition with limited supply of uracil.

The mechanism of the accumulation of δ-AO was discussed from the information of relevant biosynthetic regulation in other organisms.     

Spores of microorganisms

6-Azauracil at a concentration of 1 μmole/ml inhibits by 50% the outgrowth of germinated spores of a strain ofBacillus cereus, concentration of 1.5 μmole/ml resulting in 100% inhibition. Two distinct maxima of sensitivity to 6-azauracil are observed during postgerminative development of spores. The first occurs during early stages of development (immediately after depolymerization period) and the second after about 60 min of cultivation (late stage of swelling). Uracil reverses the inhibition of the outgrowth of spores caused by 6-azauracil when added during 0–30 min of the spore development. The addition of uracil after 30 min of the germination does not bring about the reversion of the effect of 6-azauracil. An important role of pyrimidine pathway via orotidine 5′-phosphate in germinating spores was proved, suggesting a possible use of 6-azauracil in synchronization of the postgerminative development of spores.     

The capacity of orotic acid production in pyrimidinedeficient mutants ofBrevibacterium ammoniagenes

Eight uracil-dependent mutants ofBrevibacterium ammoniagenes CCEB 364 and three mutants ofCorynebacterium sp. 9366 were checked for the production of precursors of nucleic acids. Four of the strains liberated into the medium a substantial amount of orotic acid. The production of orotic acid by a mutant ofBrevibacterium ammoniagenes (1043) was examined on mineral media containing varying amounts of glucose in the presence of uracil. The optimum concentration of glucose for the production of orotic acid was found to be 5–8%. On media to which natural substrates were added the orotic acid production increased substantially. The maximum production (6.5 g orotic acid/liter) was reached in a medium containing 0.5% yeast extract and 5% glucose; addition of uracil to this medium had no effect on the production. The maximum rate of production occurred between 24 and 72 h of fermentation. After this period the concentration of orotic acid in the medium decreases.     

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).     

Growth Inhibition by Purine Derivatives and Its Reversal by Pyrimidine Derivatives in a Mutant of Escherichia coli K12

An adenosine-sensitive mutant was isolated from Escherichia coli K12 derivative strain C600. This mutant (designated as PS100) grew slower than parental strain C600in a minimal medium, and its growth was completely inhibited by addition of all kinds of purine bases, nucleosides and nucleotides tested. On the other hand, this growth inhibitory effect of purine derivatives was reversed by co-addition of uridine to the medium. Other pyrimidine derivatives such as uracil, UMP,cytosine, cytidine, CMP and thymidine were also effective for this reversal. The mutant strain, PS100, showed a lower level (7%) of activity for orotate phosphoribosyltransferase than strain C600 did, and accumulated orotic acid in the growth medium. Lysogenization of strain PS100 with λ transducing phage containing the gene for orotate phosphoribosyltransferase (pyrE) resulted in restoration of the activity for orotate phosphoribosyltransferase and removal of growth inhibition by purine derivatives.     

Biosynthesis of willardiine and isowillardiine in germinating pea seeds and seedlings

The synthesis of the pyrimidinyl amino acids willardiine and isowillardiine was studied in vivo and in vitro. Uracil derivatives stimulate the biosynthesis of both compounds; the free base is the most effective. Significant incorporation of [2-(14)C]uracil and [6-(14)C]orotate into willardiine and isowillardiine was found. Incorporation of [6-(14)C]orotate was substantially decreased in the presence of uracil, and to a lesser extent by uridine and UMP. [3-(14)C]Serine was incorporated into the alanine side chain of the two uracilylalanines but not into the ring. The effect of a number of uracil analogues and inhibitors of pyrimidine metabolism was examined. Some were shown to stimulate the biosynthesis; the most noticeable effects were obtained with 6-azauracil and 2-thiouracil. Attempts to obtain extracts capable of synthesizing the uracilylalanines from uracil and serine were unsuccessful, but weak activity was observed when serine was replaced by O-acetylserine.     

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.     

The conversion of orotic acid into uridine 5′-monophosphate by isolated perfused normal and regenerating rat livers

The release of (14)CO(2) from [7-(14)C]orotic acid was measured in isolated perfused normal and regenerating rat livers. With some limitations, the release of (14)CO(2) from [7-(14)C]orotic acid can be used to estimate UMP synthesis in perfused livers. Isolated perfused livers rapidly pick up labelled orotic acid added to perfusate and convert most of it into UMP. Perfused regenerating livers produce approx. 2.5 times as much UMP/g of liver as do perfused normal livers. However, the absolute amount of orotic acid converted into UMP is higher in perfused normal livers than in perfused regenerating livers. Perfused regenerating livers do not differ in their orotic acid uptake and UMP synthesis from livers of comparable size in which regeneration is not taking place. The total amount of orotic acid taken up by the liver (rather than the rate of uptake) and the size of the liver appear to be the determining factors in UMP production. The results suggest that the decrease in liver size caused by partial hepatectomy may be in itself sufficient to account for an increase in the flow of metabolites in the pyrimidine pathway at the early stages of liver regeneration.     

Amylose-like polysaccharide accumulation and hyphal cell-surface structure in relation to citric acid production by Aspergillus niger in shake culture

When 120 mg glucose/ml was used as a carbon source, in shake culture Aspergillus niger Yang no. 2 maximally produced only 15.4 mg citric acid/ml but accumulated 3.0 mg extracellular polysaccharide/ml. The polysaccharide secreted by mycelia of Yang no. 2 in shake culture was confirmed to be an amylose-like alpha-1,4-glucan by hydrolysis analysis with acid, amylase and glucoamylase. However, in static cultures, such as semisolid and surface cultures free from physical stresses caused by shaking damage, Yang no. 2 produced more citric acid but did not accumulate the polysaccharide. With cultivation time in shake culture, the amount of extracellular polysaccharide and the viscosity of the culture broth increased. The increase of shaking speed caused a remarkable increase in the accumulation of extracellular polysaccharide, e.g. 11.2 mg extracellular polysaccharide/ml was accumulated in the medium at a shaking speed of 200 rpm. The addition of 2.0 mg carboxymethylcellulose (CMC)/ml as a viscous additive to the medium reduced drastically the amount of extracellular polysaccharide accumulated to 1.5 mg/ml, but increased the citric acid produced to 52.0 mg/ml. However, intracellular polysaccharide accumulation kept up a steady rate of 0.26 microgram/mg dried mycelium through the entire period of cultivation. The addition of 3.0 mg polysaccharide/ml purified from the culture broth to the medium at the start of a culture resulted in a decrease of extracellular polysaccharide accumulation but an increase of citric acid accumulation. From electronmicroscopic observation, cell surfaces of hyphae cultivated with CMC were smooth, while hyphae cultivated without CMC had fibrous and granular polysaccharide on the cell surface. These results suggested that Yang no. 2 secreted the polysaccharide on the cell surface as a viscous substance and/or a shock absorber to protect itself from physical stresses caused by shaking damage in shake culture.