Time-Optimized Isotope Ratio LC–MS/MS for High-Throughput Quantification of Primary Metabolites

Cellular metabolite concentrations hold information on the function and regulation of metabolic networks. However, current methods to measure metabolites are either low-throughput or not quantitative. Here we optimized conditions for liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) for quantitative measurements of primary metabolites in 2 min runs. In addition, we tested hundreds of multiple reaction monitoring (MRM) assays for isotope ratio mass spectrometry of most metabolites in amino acid, nucleotide, cofactor, and central metabolism. To systematically score the quality of LC–MS/MS data, we used the correlation between signals in the ¹²C and ¹³C channel of a metabolite. Applying two optimized LC methods to bacterial cell extracts detected more than 200 metabolites with less than 20% variation between replicates. An exhaustive spike-in experiment with 79 metabolite standards demonstrated the high selectivity of the methods and revealed a few confounding effects such as in-source fragments. Generally, the methods are suited for samples that contain metabolites at final concentrations between 1 nM and 10 μM, and they are sufficiently robust to analyze samples with a high salt content

Systematic Identification of Protein–Metabolite Interactions in Complex Metabolite Mixtures by Ligand-Detected Nuclear Magnetic Resonance Spectroscopy

Protein–metabolite interactions play a vital role in the regulation of numerous cellular processes. Consequently, identifying such interactions is a key prerequisite for understanding cellular regulation. However, the noncovalent nature of the binding between proteins and metabolites has so far hampered the development of methods for systematically mapping protein–metabolite interactions. The few available, largely mass spectrometry-based, approaches are restricted to specific metabolite classes, such as lipids. In this study, we address this issue and show the potential of ligand-detected nuclear magnetic resonance (NMR) spectroscopy, which is routinely used in drug development, to systematically identify protein–metabolite interactions. As a proof of concept, we selected four well-characterized bacterial and mammalian proteins (AroG, Eno, PfkA, and bovine serum albumin) and identified metabolite binders in complex mixes of up to 33 metabolites. Ligand-detected NMR captured all of the reported protein–metabolite interactions, spanning a full range of physiologically relevant <i>K</i>_d values (low micromolar to low millimolar). We also detected a number of novel interactions, such as promiscuous binding of the negatively charged metabolites citrate, AMP, and ATP, as well as binding of aromatic amino acids to AroG protein. Using <i>in vitro</i> enzyme activity assays, we assessed the functional relevance of these novel interactions in the case of AroG and show that l-tryptophan, l-tyrosine, and l-histidine act as novel inhibitors of AroG activity. Thus, we conclude that ligand-detected NMR is suitable for the systematic identification of functionally relevant protein–metabolite interactions

Engineered Production of Short-Chain Acyl-Coenzyme A Esters in <i>Saccharomyces cerevisiae</i>

Short-chain acyl-coenzyme A esters serve as intermediate compounds in fatty acid biosynthesis, and the production of polyketides, biopolymers and other value-added chemicals. <i>S. cerevisiae</i> is a model organism that has been utilized for the biosynthesis of such biologically and economically valuable compounds. However, its limited repertoire of short-chain acyl-CoAs effectively prevents its application as a production host for a plethora of natural products. Therefore, we introduced biosynthetic metabolic pathways to five different acyl-CoA esters into <i>S. cerevisiae</i>. Our engineered strains provide the following acyl-CoAs: propionyl-CoA, methylmalonyl-CoA, <i>n</i>-butyryl-CoA, isovaleryl-CoA and <i>n</i>-hexanoyl-CoA. We established a yeast-specific metabolite extraction protocol to determine the intracellular acyl-CoA concentrations in the engineered strains. Propionyl-CoA was produced at 4–9 μM; methylmalonyl-CoA at 0.5 μM; and isovaleryl-CoA, <i>n</i>-butyryl-CoA, and <i>n</i>-hexanoyl-CoA at 6 μM each. The acyl-CoAs produced in this study are common building blocks of secondary metabolites and will enable the engineered production of a variety of natural products in <i>S. cerevisiae</i>. By providing this toolbox of acyl-CoA producing strains, we have laid the foundation to explore <i>S. cerevisiae</i> as a heterologous production host for novel secondary metabolites