Dataset Information

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

ABSTRACT:

Background

Purine 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.

Results

Deregulation 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.

Conclusions

A 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.

SUBMITTER: Shi T

PROVIDER: S-EPMC4223553 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Publications

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

Shi Ting T Wang Yongcheng Y Wang Zhiwen Z Wang Guanglu G Liu Dingyu D Fu Jing J Chen Tao T Zhao Xueming X

Microbial cell factories 20140715

<h4>Background</h4>Purine 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 ...[more]

PMID: 25023436

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Project description:UnlabelledThe 2-thiouridine (s(2)U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s(2)U in Escherichia coli requires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). The E. coli MnmA adenylates and subsequently thiolates tRNA to form the s(2)U modification. Bacillus subtilis lacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene, yrvO, directly adjacent to mnmA. The genomic synteny of yrvO and mnmA combined with the absence of the Tus proteins indicated a potential functionality of these proteins in s(2)U formation. Here, we provide evidence that the B. subtilis YrvO and MnmA are sufficient for s(2)U biosynthesis. A conditional B. subtilis knockout strain showed that s(2)U abundance correlates with MnmA expression, and in vivo complementation studies in E. coli IscS- or MnmA-deficient strains revealed the competency of these proteins in s(2)U biosynthesis. In vitro experiments demonstrated s(2)U formation by YrvO and MnmA, and kinetic analysis established a partnership between the B. subtilis proteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype of E. coli ΔiscS and ΔmnmA strains associated with s(2)U depletion is recovered by B. subtilis yrvO and mnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s(2)U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.ImportanceThe 2-thiouridine (s(2)U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s(2)U in Escherichia coli requires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE). Bacillus subtilis and most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, the mnmA coding sequence is located adjacent to yrvO, encoding a cysteine desulfurase. In this work, we provide evidence that the B. subtilis YrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s(2)U biosynthesis in a pathway independent of the one used in E. coli.

Project description:BackgroundRiboflavin, an intermediate of primary metabolism, is one kind of important food additive with high economic value. The microbial cell factory Bacillus subtilis has already been proven to possess significant importance for the food industry and have become one of the most widely used riboflavin-producing strains. In the practical fermentation processes, a sharp decrease in riboflavin production is encountered along with a decrease in the dissolved oxygen (DO) tension. Influence of this oxygen availability on riboflavin biosynthesis through carbon central metabolic pathways in B. subtilis is unknown so far. Therefore the unveiled effective metabolic pathways were still an unaccomplished task till present research work.ResultsIn this paper, the microscopic regulation mechanisms of B. subtilis grown under different dissolved oxygen tensions were studied by integrating 13C metabolic flux analysis, metabolomics and transcriptomics. It was revealed that the glucose metabolic flux through pentose phosphate (PP) pathway was lower as being confirmed by smaller pool sizes of metabolites in PP pathway and lower expression amount of ykgB at transcriptional level. The latter encodes 6-phosphogluconolactonase (6-PGL) under low DO tension. In response to low DO tension in broth, the glucose metabolic flux through Embden-Meyerhof-Parnas (EMP) pathway was higher and the gene, alsS, encoding for acetolactate synthase was significantly activated that may result due to lower ATP concentration and higher NADH/NAD+ ratio. Moreover, ResE, a membrane-anchored protein that is capable of oxygen regulated phosphorylase activity, and ResD, a regulatory protein that can be phosphorylated and dephosphorylated by ResE, were considered as DO tension sensor and transcriptional regulator respectively.ConclusionsThis study shows that integration of transcriptomics, 13C metabolic flux analysis and metabolomics analysis provides a comprehensive understanding of biosynthesized riboflavin's regulatory mechanisms in B. subtilis grown under different dissolved oxygen tension conditions. The two-component system, ResD-ResE, was considered as the signal receiver of DO tension and gene regulator that led to differences between biomass and riboflavin production after triggering the shifts in gene expression, metabolic flux distributions and metabolite pool sizes.

Dataset Information

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

Background

Results

Conclusions

Publications

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

Similar Datasets

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