Pur Operon Research Articles

Development of an Inosine Hyperproducer from Bacillus licheniformis by Systems Metabolic Engineering.

Inosine is widely used in food, chemical, and medicine. This study developed Bacillus licheniformis into an inosine hyperproducer through systems metabolic engineering. First, purine metabolism was activated by deleting inhibitors PurR and YabJ and overexpressing the pur operon. Then, the 5-phosphoribosyl-1-pyrophosphate (PRPP) supply was increased by optimizing the glucose transport system and pentose phosphate pathway, increasing the inosine titer by 97% and decreasing the titers of byproducts by 36%. Next, to prevent the degradation of inosine, genes deoD and pupG coding purine nucleoside phosphorylase were deleted, accumulating 0.91 g/L inosine in the culture medium. Additionally, the downregulation of adenosine 5'-monophosphate (AMP) synthesis pathway increased the inosine titer by 409%. Importantly, enhancing the glycine and aspartate supply increased the inosine titer by 298%. Finally, the guanosine synthesis pathway was blocked, leading to strain IR-8-2 producing 27.41 g/L inosine with a 0.46 g inosine/g glucose yield and a 0.38 g/(L·h) productivity in a shake flask.

Regulatory Functions of PurR in Yersinia pestis: Orchestrating Diverse Biological Activities.

The bacterium Yersinia pestis has developed various strategies to sense and respond to the complex stresses encountered during its transmission and pathogenic processes. PurR is a common transcriptional regulator of purine biosynthesis among microorganisms, and it modulates the transcription level of the pur operon to suppress the production of hypoxanthine nucleotide (IMP). This study aims to understand the functions and regulatory mechanisms of purR in Y. pestis. Firstly, we constructed a purR knockout mutant of Y. pestis strain 201 and compared certain phenotypes of the null mutant (201-ΔpurR) and the wild-type strain (201-WT). The results show that deleting purR has no significant impact on the biofilm formation, growth rate, or viability of Y. pestis under different stress conditions (heat and cold shock, high salinity, and hyperosmotic pressure). Although the cytotoxicity of the purR knockout mutant on HeLa and 293 cells is reduced, the animal-challenging test found no difference of the virulence in mice between 201-ΔpurR and 201-WT. Furthermore, RNA-seq and EMSA analyses demonstrate that PurR binds to the promoter regions of at least 15 genes in Y. pestis strain 201, primarily involved in purine biosynthesis, along with others not previously observed in other bacteria. Additionally, RNA-seq results suggest the presence of 11 potential operons, including a newly identified co-transcriptional T6SS cluster. Thus, aside from its role as a regulator of purine biosynthesis, purR in Y. pestis may have additional regulatory functions.

Improve uridine production by modifying related metabolic pathways in Bacillus subtilis.

The metabolic pathway related to uridine production was modified in Bacillus subtilis in order to increase the production of uridine. Decreasing the relative transcriptional level of pur operon in Bacillus subtilis TD300 to 80%, and the production of the derived strain TD312 was increased to 11.81g uridine/l and the yield was increased to 270mg uridine/g glucose. The expression of pucR gene in situ by PccpA resulting in a 194.01-fold increase in the relative transcriptional level of pucR gene and 349.71-fold increase in the relative transcriptional level of ure operon, respectively. Furthermore, the production of TD314 reached 13.06g uridine/l, while the yield reached 250mg uridine/g glucose. This is the first report that more than 13g uridine/l with a yield of 250mg uridine/g glucose is produced in shake flask fermentation of genetically engineered Bacillus subtilis.

Strategy for enhancing adenosine production under the guidance of transcriptional and metabolite pool analysis.

To rationally identify targets for enhancing adenosine production, transcription level of genes involved in adenosine synthesis of Bacillus subtilis XGL was detected during the fermentation process, complemented with metabolite pool analysis. PurR-regulated genes (pur operon and purA) and prs were down-regulated and 5-phosphoribosyl 1-pyrophosphate (PRPP) decreased considerably after 24 h when adenosine significantly accumulated. Since PRPP could strongly antagonize the binding of PurR to its targets, it was inferred that down-regulation of pur operon and purA might be due to a low PRPP pool, which was confirmed by metabolite analysis. So desensitized prs responsible for PRPP synthesis was overexpressed, resulting in increased PRPP concentration and pur operon transcription. To further enhance the adenosine production, desensitized purF and prs were co-overexpressed with integrating additional copy of purA to B. subtilis XGL genome, resulting in 24.3 % (1.29 g/g DCW) higher adenosine production than that by B. subtilis XG. Overexpression of prs, purF and purA under the guidance of transcriptional and metabolite pool analysis significantly increased adenosine production. Strategies used in this study have potential applications for rational modification of industrial microorganisms.

Deregulation of purine pathway in Bacillus subtilis and its use in riboflavin biosynthesis.

BackgroundPurine nucleotides are essential metabolites for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and biosynthesis of several amino acids and riboflavin. Owing to the pivotal roles of purines in cell physiology, the pool of intracellular purine nucleotides must be maintained under strict control, and hence the de novo purine biosynthetic pathway is tightly regulated by transcription repression and inhibition mechanism. Deregulation of purine pathway is essential for this pathway engineering in Bacillus subtilis.ResultsDeregulation of purine pathway was attempted to improve purine nucleotides supply, based on a riboflavin producer B. subtilis strain with modification of its rib operon. To eliminate transcription repression, the pur operon repressor PurR and the 5’-UTR of pur operon containing a guanine-sensing riboswitch were disrupted. Quantitative RT-PCR analysis revealed that the relative transcription levels of purine genes were up-regulated about 380 times. Furthermore, site-directed mutagenesis was successfully introduced into PRPP amidotransferase (encoded by purF) to remove feedback inhibition by homologous alignment and analysis. Overexpression of the novel mutant PurF (D293V, K316Q and S400W) significantly increased PRPP amidotransferase activity and triggered a strong refractory effect on purine nucleotides mediated inhibition. Intracellular metabolite target analysis indicated that the purine nucleotides supply in engineered strains was facilitated by a stepwise gene-targeted deregulation. With these genetic manipulations, we managed to enhance the metabolic flow through purine pathway and consequently increased riboflavin production 3-fold (826.52 mg/L) in the purF-VQW mutant strain.ConclusionsA sequential optimization strategy was applied to deregulate the rib operon and purine pathway of B. subtilis to create genetic diversities and to improve riboflavin production. Based on the deregulation of purine pathway at transcription and metabolic levels, an extended application is recommended for the yield of products, like inosine, guanosine, adenosine and folate which are directly stemming from purine pathway in B. subtilis.

Accumulation of gene-targeted Bacillus subtilis mutations that enhance fermentative inosine production

In order to test a possible approach to enhance fermentative inosine production by Bacillus subtilis, seven gene-targeted mutations were introduced in the laboratory standard strain168 in a stepwise fashion. The mutations were employed in order to prevent inosine 5'-monophosphate (IMP) from being consumed for AMP and GMP synthesis, to minimize inosine degradation, and to expand the intracellular IMP pool. First, the genes for adenylosuccinate synthase (purA) and IMP dehydrogenase (guaB) were inactivated. Second, two genes for purine nucleoside phosphorylase, punA and deoD, were inactivated. Third, to enhance purine nucleotide biosynthesis, the pur operon repressor PurR and the 5'-UTR of the operon, containing the guanine riboswitch, were disrupted. Finally, the -10 sequence of the pur promoter was optimized to elevate its transcription level. The resulting mutant was capable of producing 6 g/L inosine from 30 g/L glucose in culture broth without the detectable by-production of hypoxanthine. This indicates the validity of this approach for the breeding of the next generation of B. subtilis strains for industrial nucleoside production.

Transcriptome analysis guided metabolic engineering of Bacillus subtilis for riboflavin production

A comparative transcriptome profiling between a riboflavin-producing Bacillus subtilis strain RH33 and the wild-type strain B. subtilis 168 was performed, complemented with metabolite pool and nucleotide sequence analysis, to rationally identify new targets for improving riboflavin production. The pur operon ( purEKBCSQLFMNHD) together with other PurR-regulated genes ( glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO) was all down-regulated in RH33, which consequently limited the supply of the riboflavin precursors. As 5-phospho-ribosyl-1( a)-pyrophosphate (PRPP) strongly inhibits the binding of PurR to its targets, it was inferred that the reduced expression of PurR-regulated genes might be caused by a low PRPP pool, which was subsequently confirmed by metabolite analysis. Thus, we selected and co-overexpressed prs and ywlF genes in RH33, which are involved in the biosynthetic pathway of PRPP from ribulose-5-phosphate. This co-amplification led to an elevated PRPP pool and thus the increased transcript abundances of PurR-regulated genes participated in riboflavin precursor biosynthesis. The riboflavin titer was increased by 25% (up to 15 g l −1) in fed-batch fermentation.

Nucleotide mutations in purA gene and pur operon promoter discovered in guanosine- and inosine-producing Bacillus subtilis strains

The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5' untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains.

Mutational Analysis of the Bacillus subtilis purA Operator Site

The Bacillus subtilis purine repressor, PurR, regulates many genes involved in purine metabolism. These genes contain a conserved 14-nucleotide inverted repeat (PurBox). Both pur operon and purA, which are regulated by PurR, have this inverted repeat with a 16- or 17-nucleotide spacer, respectively. Mutational studies have earlier shown that PurR binding is dependent on the PurBox of pur operon. In contrast, these studies failed to establish the importance of purA PurBox to PurR binding. To examine this inconsistency, we studied the effects of PurBox mutations both in vivo and in vitro. The data presented here indicate that purA PurBox has a similar role as pur operon PurBox in PurR binding. In addition, our data suggest that the previously proposed classification of the two halves of the Purbox into weak and strong may need to be revised.

Mutations in PurBox1 of theBacillus subtilis pur operon control site affect adenine-regulated expressionin vivo

Transcription of the Bacillus subtilis pur operon is regulated by a purine repressor (PurR)-DNA control site interaction. The pur operon control site has two PurBoxes that are required for high-affinity PurR binding. An upstream, strong-binding PurBox1 is at position -81 to -68 relative to the transcription start site and a downstream weak-binding PurBox2 is at position -49 to -36. We constructed three PurBox1 mutations and the effects on binding of PurR to the control region in vitro and on regulation of pur operon expression in vivo were investigated. The mutations significantly reduced the binding of PurR to control region DNA. In strains with G-75A, G-75T and a five bp deletion (delta5) pur operon repression was defective in vivo. In addition in vivo PurR titration was used to confirm that sequences flanking PurBox1 and PurBox2 are required for PurR binding to the puroperon control site.

Mutations in PurBox1 of the Bacillus subtilis pur operon control site affect adenine-regulated expression in vivo

Mutations in PurBox1 of the Bacillus subtilis pur operon control site affect adenine-regulated expression in vivo

Mutations in the Bacillus subtilis purine repressor that perturb PRPP effector function in vitro and in vivo.

The Bacillus subtilis pur operon repressor (PurR) has a PRPP (5-phosphoribosyl 1-pyrophosphate) binding motif at residues 199-211. Two PurR PRPP binding region mutations (D203A and D204A) were constructed, and the effects on binding of repressor to the pur operon control site in vitro and on regulation of pur operon expression in vivo were investigated. PRPP significantly inhibited the binding of wild-type but not mutant PurR to pur operon control site DNA. In strains with the D203A and D204A mutations, pur operon expression in vivo was super-repressed by addition of adenine to the growth medium. These results support the role of PRPP in modulating the regulatory function of PurR in vivo. YabJ, the product of the distal gene in the bicistronic purR operon, is also required for PurR function in vivo.

Interaction of Bacillus subtilis purine repressor with DNA.

A purine repressor (PurR) mediates adenine nucleotide-dependent regulation of transcription initiation of the Bacillus subtilis pur operon. This repressor has been purified for the first time, and binding to control site DNA was characterized. PurR binds in vitro to four operons. Apparent Kd values for binding were 7 nM for the pur operon, 8 nM for purA, 13 nM for purR, and 44 nM for the pyr operon. In each case, DNase I footprints exhibited a pattern of protected and hypersensitive sites that extended over more than 60 bp. A GAAC-N24-GTTC sequence in the pur operon was necessary but not sufficient for the PurR-DNA interaction. However, this motif, which is conserved in the four binding sites, was not required for binding of PurR to purA. Thus, the common DNA recognition element for binding of PurR to the four operons is not known. Multiple PurR-pur operon DNA complexes having a binding stoichiometry that was either approximately two or six repressor molecules per DNA fragment were detected. The results of a torsional constraint experiment suggest that control site DNA forms one right-handed turn around PurR.

The 52 degrees-55 degrees segment of the Bacillus subtilis chromosome: a region devoted to purine uptake and metabolism, and containing the genes cotA, gabP and guaA and the pur gene cluster within a 34960 bp nucleotide sequence.

Within the framework of the international project for sequencing the entire Bacillus subtilis genome, we have determined the complete sequence of the segment flanking the purE-D gene cluster (55 degrees) as far as cotA (52 degrees). This segment (34960 bp) contains, as well as 12 genes already identified as part of the pur operon, 17 putative ORFs and one partial one. Two of them (gabP and guaA) are known B. subtilis genes. The gene product of cotA (formerly pig) shows significant similarity to oxidoreductases (phenoxazine synthase and bilirubin oxidase). The putative products of ORFs yeaB (Czd protein), yeaC (MoxR), yebA (CNG-channel and cGMP-channel proteins from eukaryotes), yebB (hypothetical 32.9 kDa protein of Escherichia coli), yecA (amino acid permease) and yecB (adenine deaminase) were similar to proteins in data banks.

Identification of the Bacillus subtilis pur operon repressor.

Transcription of the Bacillus subtilis pur operon is repressed in response to a signal of excess adenine. We have purified the repressor protein and have identified, cloned, and overexpressed the purR regulatory gene that controls transcription initiation of the operon. B. subtilis purR encodes a 62-kDa homodimer that binds to the pur operon control region. The PurR binding site which overlaps the promoter encompasses approximately 110 bp. The protein-DNA interaction is inhibited by 5-phosphoribosyl 1-pyrophosphate. A mutation that deletes the repressor binding site or one that disrupts purR abolishes binding activity in vitro and repression of transcription in vivo in response to the excess adenine signal. These results lead to a model in which an excess-adenine signal is transmitted to PurR via the 5-phosphoribosyl 1-pyrophosphate pool. In addition, purR is autoregulated. There is no structural or mechanistic similarity between the B. subtilis and Escherichia coli purine repressors.

Cloning and sequence of Bacillus subtilis purA and guaA, involved in the conversion of IMP to AMP and GMP.

Bacillus subtilis genes purA, encoding adenylosuccinate synthetase, and guaA, coding for GMP synthetase, appear to be lethal when cloned in multicopy plasmids in Escherichia coli. The nucleotide sequences of purA and guaA were determined from a series of gene fragments isolated by polymerase chain reaction amplification, library screening, and plasmid rescue techniques. Identifications were based on amino acid sequence alignments with enzymes from other organisms. Comparison of the 5'-flanking regions of purA and guaA with the pur operon suggests similarities in mechanisms for gene regulation. Nucleotide sequences are now available for all genes involved in the 14-step pathway for de novo purine nucleotide synthesis in B. subtilis.

Regulation of levels of purine biosynthetic enzymes in Bacillus subtilis: effects of changing purine nucleotide pools

The genes encoding the enzymes of IMP biosynthesis in Bacillus subtilis constitute the pur operon, whereas the genes encoding GMP biosynthetic enzymes, guaA (GMP synthetase) and guaB (IMP dehydrogenase), and the purA gene encoding adenylosuccinate (sAMP) synthetase all occur as single units. The purB gene encodes an enzyme involved in both IMP and AMP biosynthesis and is located in the pur operon. The levels of purine biosynthetic enzymes (except for GMP synthetase) were repressed in cells grown in the presence of purine compounds. Transcription of the pur operon is regulated negatively by adenine and guanine compounds. Our results suggest that ATP and guanine (or hypoxanthine) act as low molecular mass repressors. The level of IMP dehydrogenase was repressed by guanosine, but not in the presence of adenine, and was negatively correlated with the GTP/ATP pools ratio. The level of sAMP synthetase was repressed by adenine and increased by guanosine, and was positively correlated with the GTP/ATP pools ratio. It appears that the mode of regulating purine biosynthetic enzyme levels coincides with the cellular need for the individual enzymes.

Bacillus subtilis pur operon expression and regulation

The Bacillus subtilis pur operon is a 12-gene cluster, purEKB-purC(orf)QLF-purMNH(J)-purD, organized in groups of overlapping coding units separated by intercistronic gaps. Translational fusions of Escherichia coli lacZ were constructed to purE, purC, and purM, the first gene of each group. Analyses of gene fusions integrated into the chromosomal pur operon exclude the possibility of internal promoters in intercistronic regions and support the view that transcription is from the single sigma 43 promoter at the 5' end of the operon. Enzyme and mRNA measurements indicate that transcriptional regulation occurs solely at the 5' end of the operon. The relative levels of beta-galactosidase from purE-lacZ, purC-lacZ, and purM-lacZ were determined under repressing and nonrepressing conditions. These results indicate that expression of purC-lacZ was 3.0- to 6.8-fold higher than purE-lacZ because of enhanced translational efficiency. The enhanced translational efficiency of purC-lacZ was accompanied by a partial escape from regulation by purines. This anomalous effect on purC-lacZ was the only suggestion for posttranscriptional regulation.

Interaction of a Putative Repressor Protein with an Extended Control Region of the Bacillus subtilis pur Operon

Transcription of the Bacillus subtilis pur operon is regulated independently by adenine and guanine levels in the growth medium. Transcription initiation is regulated by adenine, transcription attenuation by guanine (Ebbole, D. J., and Zalkin, H. (1987) J. Biol. Chem. 262, 8274-8287). A purC-lacZ fusion was constructed to monitor gene expression. Expression was reduced 11- and 18-fold by excess adenine and guanine, respectively. We have characterized the mechanism for the adenine-mediated control of transcription initiation. Deletions of DNA between nucleotides -193 and -37, relative to the start of transcription, resulted in high level constitutive expression and thus identified a control site for negative regulation. A candidate for a pur operon repressor protein was identified in a cell extract and was partially purified. This protein interacts specifically with an extended 5' flanking region of the pur operon that includes the control site identified by deletion analysis. DNase I footprinting defined the region of DNA-protein interaction between nucleotides -145 and -29. The protein-DNA interaction was not dependent on adenine, adenosine, or adenine nucleotides in vitro. These results suggest that a complex interaction of protein with an extended control region may repress transcription by blocking the -35 promoter site. This protein-DNA interaction is unlike that which regulates pur gene expression in Escherichia coli.

Detection of pur operon-attenuated mRNA and accumulated degradation intermediates in Bacillus subtilis.

Transcription of the Bacillus subtilis pur operon is regulated independently by adenine and guanine nucleotides (Ebbole, D. J., and Zalkin, H. (1987) J. Biol. Chem. 262, 8274-8287). Guanine nucleotides regulate transcription by a termination-antitermination mechanism in a 242-nucleotide, 5'-untranslated mRNA leader region. We have identified an apparently intact, terminated transcript of approximately 200 nucleotides in length, having a half-life of about 0.7 min. The terminated transcript is degraded in a series of discrete steps resulting in the accumulation of stable intermediates in vivo. We have used Northern blot analysis, primer extension, and nuclease S1 mapping to align the degradation intermediates with the nucleotide sequence and assign secondary structures that may contribute to the stability of the intermediates. Degradation is initiated by endonucleolytic cleavage of the approximately 200-nucleotide terminated transcript generating approximately 93- and approximately 97-nucleotide 5' and 3' moieties, respectively. The approximately 93-nucleotide 5' and approximately 97-nucleotide 3' intermediates are further degraded to approximately 88 and approximately 58 nucleotides, respectively. The 5'-end of pur operon mRNA and the attenuated transcript are degraded by different pathways.