In-depth analysis of the purine biosynthetic pathway of Corynebacterium glutamicum : from local pathway analysis to global phenotype profiling

This thesis focused on the purine biosynthetic pathway of C. glutamicum in order to increase the general understanding of this essential pathway. In a first step, a suitable approach for the reliable quantitation of intracellular purine pools was established and validated, thus allowing targeted metabolite profiling of genetically engineered C. glutamicum strains. Based on a metabolic engineering strategy, site-directed mutants were generated and comprised modifications of the purine precursor supply (pgi), deletions of degrading reactions (purA and guaB2) and a point mutation (purFK348Q) targeting at a deregulation of the feedback inhibitory control. The individual modifications were combined in C. glutamicum ΔpurA ΔguaB2 purFK348Q Δpgi. Conducting a systems-level approach, a metabolic shift of the purine intermediate distribution was revealed, promoting a tremendous increase of the intracellular concentration of the degradation product hypoxanthine at the expense of IMP. Furthermore, a global phenotypic adaptation, expressed in transient growth stagnation, was observed. These adverse effects were attributed to a decline in the ATP generating capacity and imbalances of the NADPH metabolism caused by the deletion of the first glycolytic enzyme, glucose 6-phosphate isomerase (pgi). Cultivations applied on complex substrates showed a release of these adverse effects, temporarily delaying the growth stagnation phenomenon.Im Mittelpunkt der Arbeit stand die Untersuchung des Purinbiosynthesewegs in C. glutamicum, welche auf eine Erweiterung des vorliegenden Kenntnisstandes über diesen essentiellen Stoffwechselweg abzielte. Zunächst wurde ein geeignetes Verfahren zur Quantifizierung intrazellulärer Purinpools etabliert und validiert. Dies bildete die Grundlage zur Erstellung gezielter metabolischer Profile genetisch veränderter C. glutamicum Stämme. Die eingebrachten genetischen Veränderungen umfassten die Modifizierung der Purin-Vorläuferbereitstellung (pgi), die Deletion beteiligter Abbauwege (purA und guaB2), sowie die Einführung einer Punktmutation (purFK348Q). Letztere hatte die Deregulation der Feedback-Inhibierung zum Ziel. Eine system-orientierte Analyse der Mutante C. glutamicum ΔpurA ΔguaB2 purFK348Q Δpgi wies eine ausgeprägte Verschiebung der intrazellulären Purinpools auf, die zu einem drastischen Konzentrationsanstieg des Abbauproduktes Hypoxanthin führte. Desweiteren resultierten die genetischen Veränderungen in einer phänotypisch-globalen Adaption, die sich durch einen vorübergehenden Wachstumsstillstand auszeichnete. Die nachteiligen Effekte - die sich sowohl lokal im Hinblick auf den Purinweg, aber auch global mit Auswirkungen auf den gesamten Phänotyp - zeigten, wurden auf ein verringertes Potential zur Energiegewinnung, sowie auf ein Ungleichgewicht im NADPH-Metabolismus zurückgeführt. Diese Effekte wurden durch den Einsatz komplexer Medienbestandteile teilweise eliminiert

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

Background: 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. 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. Results: Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 mumol 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 mumol gCDW-1). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 mumol gCDW-1) derived from IMP degradation. Conclusions: The 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

Physiological modeling of isoprene dynamics in exhaled breath

Human breath contains a myriad of endogenous volatile organic compounds (VOCs) which are reflective of ongoing metabolic or physiological processes. While research into the diagnostic potential and general medical relevance of these trace gases is conducted on a considerable scale, little focus has been given so far to a sound analysis of the quantitative relationships between breath levels and the underlying systemic concentrations. This paper is devoted to a thorough modeling study of the end-tidal breath dynamics associated with isoprene, which serves as a paradigmatic example for the class of low-soluble, blood-borne VOCs. Real-time measurements of exhaled breath under an ergometer challenge reveal characteristic changes of isoprene output in response to variations in ventilation and perfusion. Here, a valid compartmental description of these profiles is developed. By comparison with experimental data it is inferred that the major part of breath isoprene variability during exercise conditions can be attributed to an increased fractional perfusion of potential storage and production sites, leading to higher levels of mixed venous blood concentrations at the onset of physical activity. In this context, various lines of supportive evidence for an extrahepatic tissue source of isoprene are presented. Our model is a first step towards new guidelines for the breath gas analysis of isoprene and is expected to aid further investigations regarding the exhalation, storage, transport and biotransformation processes associated with this important compound.Comment: 14 page

A mathematical model for breath gas analysis of volatile organic compounds with special emphasis on acetone

Recommended standardized procedures for determining exhaled lower respiratory nitric oxide and nasal nitric oxide have been developed by task forces of the European Respiratory Society and the American Thoracic Society. These recommendations have paved the way for the measurement of nitric oxide to become a diagnostic tool for specific clinical applications. It would be desirable to develop similar guidelines for the sampling of other trace gases in exhaled breath, especially volatile organic compounds (VOCs) which reflect ongoing metabolism. The concentrations of water-soluble, blood-borne substances in exhaled breath are influenced by: (i) breathing patterns affecting gas exchange in the conducting airways; (ii) the concentrations in the tracheo-bronchial lining fluid; (iii) the alveolar and systemic concentrations of the compound. The classical Farhi equation takes only the alveolar concentrations into account. Real-time measurements of acetone in end-tidal breath under an ergometer challenge show characteristics which cannot be explained within the Farhi setting. Here we develop a compartment model that reliably captures these profiles and is capable of relating breath to the systemic concentrations of acetone. By comparison with experimental data it is inferred that the major part of variability in breath acetone concentrations (e.g., in response to moderate exercise or altered breathing patterns) can be attributed to airway gas exchange, with minimal changes of the underlying blood and tissue concentrations. Moreover, it is deduced that measured end-tidal breath concentrations of acetone determined during resting conditions and free breathing will be rather poor indicators for endogenous levels. Particularly, the current formulation includes the classical Farhi and the Scheid series inhomogeneity model as special limiting cases.Comment: 38 page

Identification of novel fusion genes in lung cancer using breakpoint assembly of transcriptome sequencing data

Genomic translocation events frequently underlie cancer development through generation of gene fusions with oncogenic properties. Identification of such fusion transcripts by transcriptome sequencing might help to discover new potential therapeutic targets. We developed TRUP (Tumor-specimen suited RNA-seq Unified Pipeline) (https://github.com/ruping/TRUP), a computational approach that combines split-read and read-pair analysis with de novo assembly for the identification of chimeric transcripts in cancer specimens. We apply TRUP to RNA-seq data of different tumor types, and find it to be more sensitive than alternative tools in detecting chimeric transcripts, such as secondary rearrangements in EML4-ALK-positive lung tumors, or recurrent inactivating rearrangements affecting RASSF8

Detaillierte Analyse des Purinbiosynthesewegs von Corynebacterium glutamicum : von lokaler Stoffwechselweganalyse zu globalem Phänotyp-Profiling

This thesis focused on the purine biosynthetic pathway of C. glutamicum in order to increase the general understanding of this essential pathway. In a first step, a suitable approach for the reliable quantitation of intracellular purine pools was established and validated, thus allowing targeted metabolite profiling of genetically engineered C. glutamicum strains. Based on a metabolic engineering strategy, site-directed mutants were generated and comprised modifications of the purine precursor supply (pgi), deletions of degrading reactions (purA and guaB2) and a point mutation (purFK348Q) targeting at a deregulation of the feedback inhibitory control. The individual modifications were combined in C. glutamicum ΔpurA ΔguaB2 purFK348Q Δpgi. Conducting a systems-level approach, a metabolic shift of the purine intermediate distribution was revealed, promoting a tremendous increase of the intracellular concentration of the degradation product hypoxanthine at the expense of IMP. Furthermore, a global phenotypic adaptation, expressed in transient growth stagnation, was observed. These adverse effects were attributed to a decline in the ATP generating capacity and imbalances of the NADPH metabolism caused by the deletion of the first glycolytic enzyme, glucose 6-phosphate isomerase (pgi). Cultivations applied on complex substrates showed a release of these adverse effects, temporarily delaying the growth stagnation phenomenon.Im Mittelpunkt der Arbeit stand die Untersuchung des Purinbiosynthesewegs in C. glutamicum, welche auf eine Erweiterung des vorliegenden Kenntnisstandes über diesen essentiellen Stoffwechselweg abzielte. Zunächst wurde ein geeignetes Verfahren zur Quantifizierung intrazellulärer Purinpools etabliert und validiert. Dies bildete die Grundlage zur Erstellung gezielter metabolischer Profile genetisch veränderter C. glutamicum Stämme. Die eingebrachten genetischen Veränderungen umfassten die Modifizierung der Purin-Vorläuferbereitstellung (pgi), die Deletion beteiligter Abbauwege (purA und guaB2), sowie die Einführung einer Punktmutation (purFK348Q). Letztere hatte die Deregulation der Feedback-Inhibierung zum Ziel. Eine system-orientierte Analyse der Mutante C. glutamicum ΔpurA ΔguaB2 purFK348Q Δpgi wies eine ausgeprägte Verschiebung der intrazellulären Purinpools auf, die zu einem drastischen Konzentrationsanstieg des Abbauproduktes Hypoxanthin führte. Desweiteren resultierten die genetischen Veränderungen in einer phänotypisch-globalen Adaption, die sich durch einen vorübergehenden Wachstumsstillstand auszeichnete. Die nachteiligen Effekte - die sich sowohl lokal im Hinblick auf den Purinweg, aber auch global mit Auswirkungen auf den gesamten Phänotyp - zeigten, wurden auf ein verringertes Potential zur Energiegewinnung, sowie auf ein Ungleichgewicht im NADPH-Metabolismus zurückgeführt. Diese Effekte wurden durch den Einsatz komplexer Medienbestandteile teilweise eliminiert

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

Abstract Background 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. 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. Results Blocking 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. Conclusions The 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.</p