Hydrophobicity and Charge Shape Cellular Metabolite Concentrations

What governs the concentrations of metabolites within living cells? Beyond specific metabolic and enzymatic considerations, are there global trends that affect their values? We hypothesize that the physico-chemical properties of metabolites considerably affect their in-vivo concentrations. The recently achieved experimental capability to measure the concentrations of many metabolites simultaneously has made the testing of this hypothesis possible. Here, we analyze such recently available data sets of metabolite concentrations within E. coli, S. cerevisiae, B. subtilis and human. Overall, these data sets encompass more than twenty conditions, each containing dozens (28-108) of simultaneously measured metabolites. We test for correlations with various physico-chemical properties and find that the number of charged atoms, non-polar surface area, lipophilicity and solubility consistently correlate with concentration. In most data sets, a change in one of these properties elicits a ∼100 fold increase in metabolite concentrations. We find that the non-polar surface area and number of charged atoms account for almost half of the variation in concentrations in the most reliable and comprehensive data set. Analyzing specific groups of metabolites, such as amino-acids or phosphorylated nucleotides, reveals even a higher dependence of concentration on hydrophobicity. We suggest that these findings can be explained by evolutionary constraints imposed on metabolite concentrations and discuss possible selective pressures that can account for them. These include the reduction of solute leakage through the lipid membrane, avoidance of deleterious aggregates and reduction of non-specific hydrophobic binding. By highlighting the global constraints imposed on metabolic pathways, future research could shed light onto aspects of biochemical evolution and the chemical constraints that bound metabolic engineering efforts

Metabolomics: Going Deeper, Going Broader, Going Further

Metabolomics is a continuously dynamic field of research that is driven by demanding research questions and technological advances alike. In this review we highlight selected recent and ongoing developments in the area of mass spectrometry-based metabolomics. The field of view that can be seen through the metabolomics lens can be broadened by adoption of separation techniques such as hydrophilic interaction chromatography and ion mobility mass spectrometry (going broader). For a given biospecimen, deeper metabolomic analysis can be achieved by resolving smaller entities such as rare cell populations or even single cells using nano-LC and spatially resolved metabolomics or by extracting more useful information through improved metabolite identification in untargeted metabolomic experiments (going deeper). Integration of metabolomics with other (omics) data allows researchers to further advance in the understanding of the complex metabolic and regulatory networks in cells and model organisms (going further). Taken together, diverse fields of research from mechanistic studies to clinics to biotechnology applications profit from these technological developments

Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production

Ustilago trichophora RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain U. trichophora TZ1. Medium optimization and investigation of process parameters enabled titers and rates that are able to compete with those of organisms overexpressing major parts of the underlying metabolic pathways. Metabolic engineering can likely further increase the efficiency of malate production by this organism, provided that basic genetic tools and methods can be established for this rarely used and relatively obscure species. Here we investigate and adapt existing molecular tools from U. maydis for use in U. trichophora. Selection markers from U. maydis that confer carboxin, hygromycin, nourseothricin, and phleomycin resistance are applicable in U. trichophora. A plasmid was constructed containing the ip-locus of U. trichophora RK089, resulting in site-specific integration into the genome. Using this plasmid, overexpression of pyruvate carboxylase, two malate dehydrogenases (mdh1, mdh2), and two malate transporters (ssu1, ssu2) was possible in U. trichophora TZ1 under control of the strong P etef promoter. Overexpression of mdh1, mdh2, ssu1, and ssu2 increased the product (malate) to substrate (glycerol) yield by up to 54% in shake flasks reaching a titer of up to 120 g L-1. In bioreactor cultivations of U. trichophora TZ1Petefssu2 and U. trichophora TZ1 Petefmdh2 a drastically lowered biomass formation and glycerol uptake rate resulted in 29% (Ssu1) and 38% (Mdh2) higher specific production rates and 38% (Ssu1) and 46% (Mdh2) increased yields compared to the reference strain U. trichophora TZ1. Investigation of the product spectrum resulted in an 87% closed carbon balance with 134 g L-1 malate and biomass (73 g L-1), succinate (20 g L-1), CO2 (17 g L-1), and α-ketoglutarate (8 g L-1) as main by-products. These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation

Efficient itaconic acid production from glycerol with Ustilago vetiveriae TZ1

Abstract Background The family of Ustilaginaceae is known for their capability to naturally produce industrially valuable chemicals from different carbon sources. Recently, several Ustilaginaceae were reported to produce organic acids from glycerol, which is the main side stream in biodiesel production. Results In this study, we present Ustilago vetiveriae as new production organism for itaconate synthesis from glycerol. In a screening of 126 Ustilaginaceae, this organism reached one of the highest titers for itaconate combined with a high-glycerol uptake rate. By adaptive laboratory evolution, the production characteristics of this strain could be improved. Further medium optimization with the best single colony, U. vetiveriae TZ1, in 24-deep well plates resulted in a maximal itaconate titer of 34.7 ± 2.5 g L−1 produced at a rate of 0.09 ± 0.01 g L−1 h−1 from 196 g L−1 glycerol. Simultaneously, this strain produced 46.2 ± 1.4 g L−1 malate at a rate of 0.12 ± 0.00 g L−1 h−1. Due to product inhibition, the itaconate titer in NaOH-titrated bioreactor cultivations was lower (24 g L−1). Notably, an acidic pH value of 5.5 resulted in decreased itaconate production, however, completely abolishing malate production. Overexpression of ria1 or mtt1, encoding a transcriptional regulator and mitochondrial transporter, respectively, from the itaconate cluster of U. maydis resulted in a 2.0-fold (ria1) and 1.5-fold (mtt1) higher itaconate titer in comparison to the wild-type strain, simultaneously reducing malate production by 75 and 41%, respectively. Conclusions The observed production properties of U. vetiveriae TZ1 make this strain a promising candidate for microbial itaconate production. The outcome of the overexpression experiments, which resulted in reduced malate production in favor of an increased itaconate titer, clearly strengthens its potential for industrial itaconate production from glycerol as major side stream of biodiesel production

Polyamine metabolism is a central determinant of helper T cell lineage fidelity

Polyamine synthesis represents one of the most profound metabolic changes during T cell activation, but the biological implications of this are scarcely known. Here, we show that polyamine metabolism is a fundamental process governing the ability of CD4+ helper T cells (TH) to polarize into different functional fates. Deficiency in ornithine decarboxylase, a crucial enzyme for polyamine synthesis, results in a severe failure of CD4+ T cells to adopt correct subset specification, underscored by ectopic expression of multiple cytokines and lineage-defining transcription factors across TH cell subsets. Polyamines control TH differentiation by providing substrates for deoxyhypusine synthase, which synthesizes the amino acid hypusine, and mice in which T cells are deficient for hypusine develop severe intestinal inflammatory disease. Polyamine-hypusine deficiency caused widespread epigenetic remodeling driven by alterations in histone acetylation and a re-wired tricarboxylic acid (TCA) cycle. Thus, polyamine metabolism is critical for maintaining the epigenome to focus TH cell subset fidelity