Effects ofeddandpgiDisruptions on Inosine Accumulation inEscherichia coli

Shimaoka, Megumi; Kawasaki, Hisashi; Takenaka, Yasuhiro; Kurahashi, Osamu; Matsui, Hiroshi

doi:10.1271/bbb.69.1248

Cited by 15 publications

(8 citation statements)

References 29 publications

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“…These are maintained by a balanced system with two compensatory operating parts: the de novo biosynthesis pathway, generating the purine intermediates from central carbon metabolism, and the salvage pathway , which can replenish corresponding pools (Figure 1) [6,7,10,15]. …”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine

et al. 2012

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BackgroundPurine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of Corynebacterium glutamicum.ResultsBlocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol gCDW-1. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol gCDW-1). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol gCDW-1) derived from IMP degradation.ConclusionsThe purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization.

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Section: Introductionmentioning

confidence: 99%

Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine

et al. 2012

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“…In addition to being central building blocks for DNA and RNA [1], they also act as co-substrates in enzymatic reactions, play crucial roles in activating metabolites, and serve as signal mediators [2]. Furthermore, several intermediates of the purine biosynthesis pathway exhibit additional characteristics-ranging from flavor-enhancing qualities (inosine monophosphate (IMP) and guanosine monophosphate (GMP) [3,4]) to drug-assisted therapy (e.g., inosine and purine analogues [5,6]). These properties make them attractive targets for the pharmaceutical and biotechnological industries.…”

Section: Introductionmentioning

confidence: 99%

Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS

et al. 2012

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Intermediates of the purine biosynthesis pathway play key roles in cellular metabolism including nucleic acid synthesis and signal mediation. In addition, they are also of major interest to the biotechnological industry as several intermediates either possess flavor-enhancing characteristics or are applied in medical therapy. In this study, we have developed an analytical method for quantitation of 12 intermediates from the purine biosynthesis pathway including important nucleotides and their corresponding nucleosides and nucleobases. The approach comprised a single-step acidic extraction/quenching procedure, followed by quantitative electrospray LC-MS/MS analysis. The assay was validated in terms of accuracy, precision, reproducibility, and applicability for complex biological matrices. The method was subsequently applied for determination of free intracellular pool sizes of purine biosynthetic pathway intermediates in the two Gram-positive bacteria Corynebacterium glutamicum and Corynebacterium ammoniagenes. Importantly, no ion pair reagents were applied in this approach as usually required for liquid chromatography analysis of large classes of diverse metabolites.

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“…Recently, the wild-type Escherichia coli strain W3110 has been subjected to rational engineering for inosine production. Inactivation of edd, pgi, xpa, and guaA (Shimaoka et al 2005;Shimaoka et al 2006) and overexpression of desensitized purF and prs (Shimaoka et al 2007) have been reported to enhance inosine accumulation in E. coli.…”

Section: Introductionmentioning

confidence: 99%

De novo engineering and metabolic flux analysis of inosine biosynthesis in Bacillus subtilis

Zhang

Deng

et al. 2011

Biotechnol Lett

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Wild-type B. subtilis strain W168 was de novo engineered for inosine biosynthesis. Inactivation of deoD and purA led to 0.15 ± 0.04 and 6.44 ± 0.39 g inosine/l yields, respectively. The deoD purA double mutant accumulated 7.6 ± 0.34 g inosine/l, with a 4.7% (w/w) conversion ratio from glucose to inosine. Comparative metabolic flux analysis revealed that the fluxes from inosine to hypoxanthine and from inosine monophosphate to adenosine monophosphate in the double mutant decreased to 14.0 and 0.61% of those in the wild-type strain. The major role of purA was demonstrated when inactivation of deoD and purA were found to contribute additively to inosine accumulation. This work is expected to contribute to the improvement of the fermentative production of purine nucleosides in the microbial industry.

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Effects ofeddandpgiDisruptions on Inosine Accumulation inEscherichia coli

Cited by 15 publications

References 29 publications

Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine

Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine

Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS

De novo engineering and metabolic flux analysis of inosine biosynthesis in Bacillus subtilis

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