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

Li, Haojian; Zhang, Guoqiang; Deng, Aihua; Chen, Ning; Wen, Tingyi

doi:10.1007/s10529-011-0597-5

Cited by 23 publications

(33 citation statements)

References 14 publications

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“…1). 2), a value that lies within the same range of the rationally engineered E. coli and B. subtilis strains reported so far (6-7 g/L; Asahara et al, 2010;Li et al, 2011;Shimaoka et al, 2007). 2).…”

Section: Introductionsupporting

confidence: 74%

“…Therefore, during the last years, considerable effort has been made to obtain super-producer strains by rational gene manipulation of B. subtilis (Asahara et al, 2010;Li et al, 2011;Sheremet et al, 2011) and E. coli (Shimaoka et al, 2007). Combinations of these modifications have achieved strains able to excrete $6-7 g/L to the culture medium (Asahara et al, 2010;Li et al, 2011;Shimaoka et al, 2007). Combinations of these modifications have achieved strains able to excrete $6-7 g/L to the culture medium (Asahara et al, 2010;Li et al, 2011;Shimaoka et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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The filamentous fungus Ashbya gossypii as a competitive industrial inosine producer

Ledesma-Amaro

Buey

Revuelta

2016

Biotech & Bioengineering

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Inosine is a nucleoside with growing biotechnological interest due to its recently attributed beneficial health effects and as a convenient precursor of the umami flavor. At present, most of the industrial inosine production relies on bacterial fermentations. In this work, we have metabolically engineered the filamentous fungus Ashbya gossypii to obtain strains able to excrete high amounts of inosine to the culture medium. We report that the disruption of only two key genes of the purine biosynthetic pathway efficiently redirect the metabolic flux, increasing 200-fold the excretion of inosine with respect to the wild type, up to 2.2 g/L. These results allow us to propose A. gossypii as a convenient candidate for large-scale nucleoside production, especially in view of the several advantages that Ashbya has with respect to the bacterial systems used at present for the industrial production of this food additive. Biotechnol. Bioeng. 2016;113: 2060-2063. © 2016 Wiley Periodicals, Inc.

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Section: Introductionsupporting

confidence: 74%

Section: Introductionmentioning

confidence: 99%

The filamentous fungus Ashbya gossypii as a competitive industrial inosine producer

Ledesma-Amaro

Buey

Revuelta

2016

Biotech & Bioengineering

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“…IMP and GMP [5-7], and act as potential drugs in medical therapy, e.g. inosine and purine analogs [8-10]. Due to their immense pharmaceutical and biotechnological potential, these compounds have been successfully synthesized over the past decades via fermentative production employing Corynebacteria , especially Corynebacterium ammoniagenes [11-13].…”

Section: Introductionmentioning

confidence: 99%

“…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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“…B. subtilis has a long tradition as safe and stable producer for purine nucleotides [[13],[20],[21]]. It should be noted that B. subtilis has constituted a paradigm of environmentally friendly “white” biotechnology with regard to industrial riboflavin overproduction [[22],[23]].…”

Section: Introductionmentioning

confidence: 99%

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

Shi

Wang

et al. 2014

Microb Cell Fact

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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.

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

Cited by 23 publications

References 14 publications

The filamentous fungus Ashbya gossypii as a competitive industrial inosine producer

The filamentous fungus Ashbya gossypii as a competitive industrial inosine producer

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

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

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