Biology subjects

Kohlstedt, M.

Publications and source records attributed to Kohlstedt, M..

4 recordsLinked to original sources

bioRxiv2026

Rewiring central carbon metabolism reveals a trade-off between yield and production rate in poly-γ-glutamic acid synthesis by Bacillus subtilis

Improving carbon yield and process time is a key requirement for the industrially viable production of biopolymers. During poly-{gamma}-glutamic acid production with Bacillus subtilis, a considerable fraction of the carbon source is diverted into by-products. These originate from accumulating pyruvate, constraining carbon flux towards glutamate synthesis. Disrupting by-product pathways reduces their secretion, but the underlying imbalance at the pyruvate node persists. Here, we rewired the central carbon metabolism to redirect glycolytic flux from pyruvate formation towards oxaloacetate and 2-oxoglutarate in an improved medium. Deletion of pyruvate kinase combined with heterologous expression of phosphoenolpyruvate carboxylase established an alternative anaplerotic entry route into the TCA cycle. Subsequent disruption of gluconeogenetic reactions (OF5) reduced by-product formation by 81% and increased {gamma}-PGA yield to 0.41 g g-1 glucose, but lowered the glucose uptake rate. Alternatively, disruption of the TCA cycle downstream of 2-oxoglutarate combined with enhanced glutamate synthesis (OF4.2) increased substrate uptake and improved the {gamma}-PGA production rate by 39% compared to the wild type, while product and by-product titers remained comparable. Both strategies thus revealed a trade-off between improved yield or production rate. Subsequent 13C metabolic flux analysis and intracellular metabolite profiling revealed distinct carbon routing phenotypes. While strain OF5 maintained carbon within the TCA cycle, OF4.2 cycled carbon between oxaloacetate, malate and pyruvate, thereby altering redox and energy balances. These results highlight the need for coordinated control of carbon flux, redox metabolism and energy supply for improving {gamma}-PGA synthesis, and identify the oxaloacetate-malate-pyruvate node as a promising strain engineering target.

microbiology↗

bioRxiv2022

Systems-wide dissection of organic acid assimilation in Pseudomonas aeruginosa reveals a novel path to underground metabolism

The human pathogen Pseudomonas aeruginosa (Pa) is one of the most frequent and severe causes of nosocomial infection. This organism is also a major cause of airway infections in people with cystic fibrosis (CF). Pa is known to have a remarkable metabolic plasticity, allowing it to thrive in diverse environmental conditions and ecological niches, yet little is known about the central metabolic pathways which sustain its growth during infection, or precisely how these pathways operate. In this work, we used a combination of omics approaches (transcriptomics, proteomics, metabolomics and 13C-fluxomics) and reverse genetics to provide a systems-level insight into how the infection-relevant organic acids, succinate and propionate, are metabolized by Pa. Moreover, through structural and kinetic analysis of the 2-methylcitrate synthase (PrpC) and its paralogue, citrate synthase (GltA), we show how these two crucial enzymatic steps are interconnected in Pa organic acid assimilation. We found that Pa can rapidly adapt to the loss of GltA function by acquiring mutations in a transcriptional repressor, which then de-represses prpC expression. Our findings provide a clear example of how underground metabolism, facilitated by enzyme substrate promiscuity, "rewires" Pa metabolism, allowing it to overcome the loss of a crucial enzyme. This pathogen-specific knowledge is critical for the advancement of a model-driven framework to target bacterial central metabolism.

microbiology↗

bioRxiv2021

IsoSolve: an integrative framework to improve isotopic coverage and consolidate isotopic measurements by MS and/or NMR

Stable-isotope labeling experiments are widely used to investigate the topology and functioning of metabolic networks. Label incorporation into metabolites can be quantified using a broad range of mass spectrometry (MS)and nuclear magnetic resonance (NMR)spectroscopy methods, but in general, no single approach can completely cover isotopic space, even for small metabolites. The number of quantifiable isotopic species could be increased, and the coverage of isotopic space improved, by integrating measurements obtained by different methods; however, this approach has remained largely unexplored because no framework able to deal with partial, heterogeneous isotopic measurements has yet been developed. Here, we present a generic computational framework based on symbolic calculus that can integrate any isotopic dataset by connecting measurements to the chemical structure of the molecules. As a test case, we apply this framework to isotopic analyses of amino acids, which are ubiquitous to life, central to many biological questions, and can be analyzed by a broad range of MS and NMR methods. We demonstrate how this integrative framework helps to i) clarify and improve the coverage of isotopic space, ii) evaluate the complementarity and redundancy of different techniques, iii) consolidate isotopic datasets, iv) design experiments, and v) guide future analytical developments. This framework, which can be applied to any labeled element, isotopic tracer, metabolite, and analytical platform, has been implemented in IsoSolve (available at https://github.com/MetaSysLISBP/IsoSolve and https://pypi.org/project/IsoSolve), an open source software that can be readily integrated into data analysis pipelines.

systems biology↗

bioRxiv2019

Contextual flexibility in Pseudomonas aeruginosa central carbon metabolism during growth in single carbon sources

Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge towards a common metabolic program as the organism adapts to the CF airway environment. However, at a systems level, we still have only a limited understanding of how these transcriptional changes impact on metabolic flux. To address this, we analysed the transcriptome, proteome and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in the CF airways. We show that the flux of carbon through central metabolism is radically different on each carbon source. For example, the newly-recognised EDEMP cycle plays an important role in supplying NADPH during growth on glycerol. By contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the isocitrate dehydrogenase(s)-catalyzed reaction. Perhaps more importantly, our proteomic and transcriptomic analyses reveal a global remodelling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; a plasticity which allows the organism to seamlessly segue between different carbon sources, maximising the energetic yield from each.\n\nImportancePseudomonas aeruginosa is an opportunistic human pathogen, well-known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well-adapted to life in the CF lung, little is currently known about how the organism metabolises the nutrients available in the airways. In this work, we use a combination of gene expression and isotope tracer (\"fluxomic\") analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We find that carbon is routed (\"fluxed\") through very different pathways during growth on these substrates, and that this is accompanied by an unexpected remodelling of the cells electron transfer pathways. Having access to this \"blueprint\" is important because the metabolism of P. aeruginosa is increasingly being recognised as a target for the development of much-needed antimicrobial agents.

microbiology↗