[Effects of overexpression of key enzyme genes on guanosine accumulation in Bacillus amyloliquefaciens].

OBJECTIVE: To study the effects of overexpression of key enzyme genes (prs, purF and guaB) on guanosine production in Bacillus amyloliquefaciens TA208. METHODS: The prs, purF, guaB and prs-purF genes were inserted into constructed expression plasmid PBE43. All these constructed plasmids were electroporated into B. amyloliquefaciens TA208. The transcriptional level of various genes in the resulting strains was tested by real-time quantitative PCR. The activity of inosine 5'-monophosphate dehydrogenase in the resulting strains was detected. Finally, cell growth, glucose consumption and guanosine production of 4 engineering strains along with control strain were examined. RESULTS: The transcriptional analysis showed that overexpression of prs, purF and guaB gene accompanied by their own transcription level up-regulated. Overexpression of prs or purF genes alone slightly down-regulated the transcriptional level of purine operon, but overexpression of guaB gene independently did not disturb the transcription of prs gene and purine operon. Enzyme activity analysis showed that overexpression of prs or purF gene did not change the activity of inosine 5'-monophosphate dehydrogenase and its activity increased by 126% through overexpression of guaB gene. Finally, by fermentation flask test, we found that overexpression of prs and purF gene alone could not promote guanosine accumulation. However, overexpression of guaB gene resulted in an increase in the production of guanosine, which was 20.7% higher than the control strain. The guanosine concentration and the conversion ratio from glucose to guanosine in the host strain containing co-expression plasmid were 14.4% and 6.8% higher than the control strain. CONCLUSION: Overexpression of guaB gene could enhance the guanosine yield in the culture broth. However, for prs and purF gene, only co-expression of them could lead to a significant improvement of guanosine production in B. amyloliquefaciens. It should provide a valuable insight into the construction of industrially important strains for guanosine production by metabolic engineering.

Phosphoribosylamidotransferase, the first enzyme for purine de novo synthesis, is required for conidiation in the sclerotial mycoparasite Coniothyrium minitans.

Coniothyrium minitans is an important sclerotial parasite of the fungal phytopathogen, Sclerotinia sclerotiorum. Previously, we constructed a T-DNA insertional library, and screened for many conidiation-deficient mutants from this library. Here, we report a T-DNA insertional mutant ZS-1T21882 that completely lost conidiation. In mutant ZS-1T21882, the T-DNA was integrated into a gene (CmPrat-1) which encodes phosphoribosylamidotransferase (PRAT, EC 2.4.2.14), an enzyme catalyzing the first committed step in de novo purine nucleotide synthesis. Gene replacement and complementation experiments confirmed that phosphoribosylamidotransferase is essential for conidiation of C. minitans. Mutant ZS-1T21882 did not grow on modified Czapek-Dox broth (MCD), but it grew well on MCD amended with IMP or AMP. The conidial production of this mutant was dependent on the dosage of IMP amended. At low concentrations, such as 0.1 mM and 0.25 mM, the mutant produced very few pycnidia, while up to 0.75 mM or higher, the conidiation of this mutant was restored completely. cAMP could not restore the conidiation of mutant ZS-1T21882 when amended into MCD, but could when amended into PDA. Neither GMP nor cGMP could restore the conidiation in MCD or in PDA. Our findings suggest that phosphoribosylamidotransferase is essential for conidiation of C. minitans via adenosine related molecules. Furthermore, when dual cultured with its host, this mutant produced conidia in the host mycelium and on the sclerotia of S. sclerotiorum, but not in dead mycelium or on dead sclerotia, suggesting that C. minitans is likely to able to obtain adenosine or related components from its host during parasitization.

Carrier proteins for fusion expression of antimicrobial peptides in Escherichia coli.

Antimicrobial peptides are an essential component of innate immunity and play an important role in host defence against microbial pathogens. They have received increasing attention recently as potential novel pharmaceutical agents. To meet the requirement for necessary basic science studies and clinical trials, large quantities of these peptides are needed. In general, isolation from natural sources and chemical synthesis are not cost-effective. The relatively low cost and easy scale-up of the recombinant approach renders it the most attractive means for large-scale production of antimicrobial peptides. Among the many systems available for protein expression, Escherichia coli remains the most widely used host. Antimicrobial peptides produced in E. coli are often expressed as fusion proteins, which effectively masks these peptides' potential lethal effect towards the bacterial host and protects the peptides from proteolytic degradation. Although some carriers confer peptide solubility, others promote the formation of inclusion bodies. The present minireview considers the most commonly used carrier proteins for fusion expression of antimicrobial peptides in E. coli. The favourable properties of SUMO (small ubiquitin-related modifier) as a novel fusion partner are also discussed.

The purine biosynthesis enzyme PRAT detected in proenzyme and mature forms during development of Drosophila melanogaster.

Glutamine phosphoribosylpyrophosphate amidotransferase (PRAT; EC 2.4. 2.14) is the first and rate-limiting enzyme of de novo purine biosynthesis. PRAT expression in Drosophila development was examined to determine if it is correlated with cell proliferation and/or nutritional isolation. An antiserum, raised against the 16 carboxyl-terminal amino acids of PRAT, detects two proteins corresponding to a 60 kDa proenzyme and 55 kDa mature enzyme, consistent with a 53 amino acid propeptide predicted from the gene sequence. Mature enzyme is maternally expressed, and proenzyme appears in embryos at 2-8 h, corresponding to the interval during which zygotic transcription is initiated. Upon hatching of first instar larvae, proenzyme levels are reduced and remain low relative to mature enzyme. Adult females have higher levels of both proteins relative to males, consistent with maternal expression. Maternal expression reflects a requirement for the enzyme during embryogenesis, while reduction in expression following hatching reflects a switch to an exogenous source of purines. Prat mRNA levels follow a similar overall pattern in the same developmental stages examined for the protein. Discovery of a second gene encoding PRAT with 78% amino acid identity leads to the possibility that the antiserum raised against the carboxyl-terminus detects two enzymes.

Biosynthesis of the pyrimidine moiety of thiamine independent of the PurF enzyme (Phosphoribosylpyrophosphate amidotransferase) in Salmonella typhimurium: incorporation of stable isotope-labeled glycine and formate.

Genetic analyses have suggested that the pyrimidine moiety of thiamine can be synthesized independently of the first enzyme of de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in Salmonella typhimurium. To obtain biochemical evidence for and to further define this proposed synthesis, stable isotope labeling experiments were performed with two compounds, [2-13C]glycine and [13C]formate. These compounds are normally incorporated into thiamine pyrophosphate (TPP) via steps in the purine pathway subsequent to PurF. Gas chromatography-mass spectrometry analyses indicated that both of these compounds were incorporated into the pyrimidine moiety of TPP in a purF mutant. This result clearly demonstrated that the pyrimidine moiety of thiamine was being synthesized in the absence of the PurF enzyme and strongly suggested that this synthesis utilized subsequent enzymes of the purine pathway. These results were consistent with an alternative route to TPP that bypassed only the first enzyme in the purine pathway. Experiments quantitating cellular thiamine monophosphate (TMP) and TPP levels suggested that the alternative route to TPP did not function at the same capacity as the characterized pathway and determined that levels of TMP and TPP in the wild-type strain were significantly altered by the presence of purines in the medium.

Isolation of a human cDNA encoding amidophosphoribosyltransferase and functional complementation of a CHO Ade-A mutant deficient in this activity.

We report here the isolation of a human cDNA encoding the first step in de novo purine biosynthesis, amidophosphoribosyltransferase (PRAT). The human PRAT cDNA was isolated by complementation of a Saccharomyces cerevisiae ade4 mutant deficient in PRAT enzymatic activity. The identity of the isolated cDNA, designated pAdeA-3, was confirmed by several independent methods. Genomic DNA sequences homologous to pAdeA-3 show coordinate segregation with the hypoxanthine nutritional requirement in Chinese hamster ovary (CHO) cell Ade-A-human hybrids, segregants of these hybrids, and irradiation reduction hybrids. The PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT enzyme deficiency in CHO Ade-A mutants. This correction was monitored by both cell-free PRAT assays and in vivo phosphoribosylformylglycinamide (FGAR) accumulation studies. FGAR accumulation is a classic method for assessment of the early steps of purine nucleotide biosynthesis. Two of the isolated transformants, designated PRAT-1 and PRAT-2, exhibited 22% and 53%, respectively, of wild-type CHO K1 PRAT enzymatic activity using a cell-free enzyme assay. These same two transformants plus an additional transformant, designated PRAT-13, showed FGAR accumulations of 150%, 260%, and 140%, respectively, compared to the levels of accumulation seen in CHO K1. Transformants PRAT-1 and PRAT-2 both contained a mRNA species recognized by the PRAT cDNA of identical size to a mRNA species in human fibroblasts homologous to the PRAT cDNA. This observation, along with the functionality of the cDNA in both yeast and CHO cells deficient in PRAT activity, suggests the isolated cDNA is full length.

Increased de novo purine synthesis by insulin through selective enzyme induction in primary cultured rat hepatocytes.

The proliferative effect of insulin on de novo purine synthesis and on the expression of various enzymes of purine metabolism were studied in primary cultured rat hepatocytes. Insulin greater than 1.5 x 10(-8) M increased DNA and de novo purine synthesis to 260-390 and 270-420%, respectively, 24 and 8 h after the administration. Insulin at 1.5 x 10(-7) M increased the specific activity of amidophosphoribosyltransferase (ATase) to 154-180%, hypoxanthine-guanine phosphoribosyltransferase to 129%, and adenine phosphoribosyltransferase (APRT) to 205%, in contrast to unchanged xanthine dehydrogenase at 80%. Enzyme induction was supported by the results of kinetic analysis and the inhibition of the insulin-induced increase in enzyme activities by protein synthesis inhibitors. Insulin increased ATP to 127% and decreased AMP, ADP, 5'-guanylic acid (GMP), and guanosine 5'-diphosphate (GDP), respectively, to 73, 69, 73, and 69%. Insulin increased adenylate energy charge from 0.83 to 0.90 without changing total feedback inhibitory potential on ATase. No obvious increase of 5-phosphoribosyl-1-pyrophosphate supply was suggested, although its apparent availability for purine ribonucleotide synthesis was increased to 208-245%, reflecting mainly induced APRT activity to 205%. It is concluded that hepatocyte proliferation by insulin, as evidenced by purine metabolism, is mediated by the selective gene activation of anabolic enzymes and increased ATP as the basis to activate multiple metabolic pathways without remarkable changes of substrate availability or feedback inhibition.

Purine nucleotide synthesis in normal and leukemic blood cells.

The present study was undertaken to obtain more precise information about the purine biosynthetic pathway in human blood cells. 5'phosphoribosyl-l-pyrophosphate (PP-ribose-P) amidotransferase was found in cell-free extracts from all leukemic cells and normal lymphocytes and therefore these cells could synthesize the first intermediate of the purine-de-novo-synthesis. Normal leucocytes, erythrocytes and bone marrow cells lack this enzyme system and have an absolute requirement for externally supplied purines via salvage pathway. Leukemic blast cells show different enzyme activities independent of their cell count. Kinetic studies with the crude enzymes showed sigmoidal substrate velocity curves for PP-ribose-P, whereas glutamine shows hyperbolic kinetics. The leukemic cell enzymes from all four donor types (ALL, CLL, AML and CML) are rapidly saturated with low concentrations of PP-ribose-P and less inhibited by the physiological feedback inhibitor, adenosine 5'monophosphate. The crude enzymes of normal spleen lymphocytes and leukemic cells were further purified (10 to 15-fold) and substrate velocity curves for PP-ribose-P and glutamine show now hyperbolic kinetics and double reciprocal plots were linear with and apparent Km for PP-ribose-P of 0.14 mM and for glutamine 2.0 mM. In the presence of different concentrations of AMP, the PP-ribose-P substrate velocity plot changed from a hyperbolic to a sigmoidal curve; no difference in the degree of the inhibition between both partially purified enzymes (normal spleen lymphocytes and leukemic cells from all four donor types) could now be observed.