Abstract
Disruption of specific metabolic pathways constitutes the mode of action of many known toxicants and it is responsible for the adverse phenotypes associated to human genetic defects. Conversely, many industrial applications rely on metabolic alterations of diverse microorganisms, whereas many therapeutic drugs aim to selectively disrupt pathogens’ metabolism. In this work we analyzed metabolic changes induced by auxotrophic starvation conditions in yeast in a non-targeted approach, using one-dimensional proton Nuclear Magnetic Resonance spectroscopy (1H NMR) and chemometric analyses. Analysis of the raw spectral datasets showed specific changes linked to the different stages during unrestricted yeast growth, as well as specific changes linked to each of the four tested starvation conditions (L-methionine, L-histidine, L-leucine and uracil). Analysis of changes in concentrations of more than 40 metabolites by Multivariate Curve Resolution – Alternating Least Squares (MCR-ALS) showed the normal progression of key metabolites during lag, exponential and stationary unrestricted growth phases, while reflecting the metabolic blockage induced by the starvation conditions. In this case, different metabolic intermediates accumulated over time, allowing identification of the different metabolic pathways specifically affected by each gene disruption. This synergy between NMR metabolomics and molecular biology may have clear implications for both genetic diagnostics and drug development.
Highlights
Metabolomics aims to identify the specific cellular processes undergoing in biological organisms by the identification and quantitation of dozens to thousands metabolites with high-throughput techniques, by using a non-aprioristic approach[1]
The study of different auxotrophic starvations allows a more detailed and specific analysis of the metabolic changes induced by characterized disruptions of the endogenous metabolic pathways
Metabolic changes during the 24 h-incubation periods in all five conditions were first analyzed by a global overview of the 1H Nuclear Magnetic Resonance (NMR) spectral dataset (Fig. 1b)
Summary
Metabolomics aims to identify the specific cellular processes undergoing in biological organisms by the identification and quantitation of dozens to thousands metabolites with high-throughput techniques, by using a non-aprioristic approach[1]. We present here an NMR analysis of the metabolome variations induced by auxotrophic starvation in yeast, which occurs when a strain lacking specific genes (in this case, HIS3, LEU2, MET15 and URA3, called genetic markers) is confronted with a medium devoid of one or more of the essential metabolites it can no longer synthesize (L-histidine, L-leucine, L-methionine and/or uracil, respectively). The study of different auxotrophic starvations allows a more detailed and specific analysis of the metabolic changes induced by characterized disruptions of the endogenous metabolic pathways. In this article we applied 1H NMR to the study of local disruptions of metabolic pathways, an approach that can be extrapolated to determine the specific triggers for other locally disrupted processes
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