Abstract
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.
Highlights
Purine nucleotides exhibit various functions in cellular metabolism
De novo nucleotide synthesis begins with the activation of the pentose phosphate pathway (PPP) intermediate and direct precursor ribose 5-phosphate [16], which is further converted to IMP in a twelve-step biosynthetic process with concomitant consumption of 10 mol ATP
Metabolic snapshots of the purine pathway in wild type C. glutamicum Intracellular metabolite concentrations in C. glutamicum were quantified after whole culture extraction using LCESI-MS/MS [28]
Summary
Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. The diverse class of purine intermediates comprises phosphorylated nucleotides, nucleosides and nucleobases, exhibiting multiple functions in the cellular system [1]: for example, they serve as transmitters of the genetic information [2], as phosphate group donors, are involved in signal mediation [3] and ensure the energy supply of the living cell [4]. These intermediates are involved in almost every aspect of cellular metabolism. The salvage pathway, converts extracellular nucleobases or degraded purine compounds into the corresponding nucleosides and nucleotides [11,18], thereby preventing energetically expensive de novo synthesis of these compounds
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