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Adduct formation, fragmentation events and matrix effects impose special challenges to the identification and quantitation of metabolites in LC-ESI-MS datasets. An important step in compound identification is the deconvolution of mass signals. During this processing step, peaks representing adducts, fragments, and isotopologues of the same analyte are allocated to a distinct group, in order to separate peaks from coeluting compounds. From these peak groups, neutral masses and pseudo spectra are derived and used for metabolite identification via mass decomposition and database matching. Quantitation of metabolites is hampered by matrix effects and nonlinear responses in LC-ESI-MS measurements. A common approach to correct for these effects is the addition of a U-13C-labeled internal standard and the calculation of mass isotopomer ratios for each metabolite. Here we present a new web-platform for the analysis of LC-ESI-MS experiments. ALLocator covers the workflow from raw data processing to metabolite identification and mass isotopomer ratio analysis. The integrated processing pipeline for spectra deconvolution “ALLocatorSD” generates pseudo spectra and automatically identifies peaks emerging from the U-13C-labeled internal standard. Information from the latter improves mass decomposition and annotation of neutral losses. ALLocator provides an interactive and dynamic interface to explore and enhance the results in depth. Pseudo spectra of identified metabolites can be stored in user- and method-specific reference lists that can be applied on succeeding datasets. The potential of the software is exemplified in an experiment, in which abundance fold-changes of metabolites of the l-arginine biosynthesis in C. glutamicum type strain ATCC 13032 and l-arginine producing strain ATCC 21831 are compared. Furthermore, the capability for detection and annotation of uncommon large neutral losses is shown by the identification of (γ-)glutamyl dipeptides in the same strains. ALLocator is available online at: https://allocator.cebitec.uni-bielefeld.de. A login is required, but freely available.

Background: 3-Hydroxypropionic acid (3-HP) is a promising platform chemical with various industrial applications. Several metabolic routes to produce 3-HP from organic substrates such as sugars or glycerol have been implemented in yeast, enterobacterial species and other microorganisms. In this study, the native 3-HP metabolism of Cupriavidus necator was investigated and manipulated as it represents a promising chassis for the production of 3-HP and other fatty acid derivatives from CO 2 and H 2. Results: When testing C. necator for its tolerance towards 3-HP, it was noted that it could utilise the compound as the sole source of carbon and energy, a highly undesirable trait in the context of biological 3-HP production which required elimination. Inactivation of the methylcitrate pathway needed for propionate utilisation did not affect the organism's ability to grow on 3-HP. Putative genes involved in 3-HP degradation were identified by bioinformatics means and confirmed by transcriptomic analyses, the latter revealing considerably increased expression in the presence of 3-HP. Genes identified in this manner encoded three putative (methyl)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) and two putative dehydrogenases (hpdH and hbdH). These genes, which are part of three separate mmsA operons, were inactivated through deletion of the entire coding region, either singly or in various combinations, to engineer strains unable to grow on 3-HP. Whilst inactivation of single genes or double deletions could only delay but not abolish growth, a triple ∆mmsA1∆mmsA2∆mmsA3 knockout strain was unable utilise 3-HP as the sole source of carbon and energy. Under the used conditions this strain was also unable to co-metabolise 3-HP alongside other carbon and energy sources such as fructose and CO 2 /H 2. Further analysis suggested primary roles for the different mmsA operons in the utilisation of β-alanine generating substrates (mmsA1), degradation of 3-HP (mmsA2), and breakdown of valine (mmsA3). Conclusions: Three different (methyl)malonate semialdehyde dehydrogenases contribute to 3-HP breakdown in C. necator H16. The created triple ∆mmsA1∆mmsA2∆mmsA3 knockout strain represents an ideal chassis for autotrophic 3-HP production.