Streamlining the Analysis of Dynamic 13C-Labeling Patterns for the Metabolic Engineering of Corynebacterium glutamicum as l-Histidine Production Host

Today’s possibilities of genome editing easily create plentitudes of strain mutants that need to be experimentally qualified for configuring the next steps of strain engineering. The application of design-build-test-learn cycles requires the identification of distinct metabolic engineering targets as design inputs for subsequent optimization rounds. Here, we present the pool influx kinetics (PIK) approach that identifies promising metabolic engineering targets by pairwise comparison of up- and downstream 13C labeling dynamics with respect to a metabolite of interest. Showcasing the complex l-histidine production with engineered Corynebacterium glutamicuml-histidine-on-glucose yields could be improved to 8.6 ± 0.1 mol% by PIK analysis, starting from a base strain. Amplification of purA, purB, purH, and formyl recycling was identified as key targets only analyzing the signal transduction kinetics mirrored in the PIK values.

Calcium Influx Kinetics and the Characteristics of Potassium Channels in Peripheral T Lymphocytes in Systemic Sclerosis

<b><i>Background:</i></b> Systemic sclerosis (SSc) is a chronic, immune-mediated, connective tissue disease causing microvascular abnormalities and fibrosis. The cytoplasmic calcium influx kinetics in T lymphocytes governs lymphocyte activation in this inflammatory process. The inhibition of Kv1.3 and IKCa1 potassium channels reduces calcium influx. <b><i>Methods:</i></b> This study aimed to analyze cytoplasmic calcium influx kinetics following activation in Th1, Th2, and CD8 cells in peripheral blood of 12 healthy individuals and 16 patients with systemic sclerosis using flow cytometry. We also evaluated the effect of the specific inhibition of the Kv1.3 and IKCa1 potassium channels. <b><i>Results:</i></b> We observed higher levels of activation in CD8 compared with Th1 cells in SSc. However, the activation of CD8 cells was lower in SSc compared to healthy controls. Moreover, activation of Th1 lymphocytes was slower in SSc than in healthy controls. The inhibition of IKCa1 channels decreased the activation of Th1 cells, while the inhibition of Kv1.3 channels modified the dynamics of activation of Th1 and Th2 lymphocytes in SSc. <b><i>Conclusion:</i></b> Th1 and CD8 cells demonstrate specific activation dynamics and sensitivity to potassium channel inhibition in SSc, distinguishing this condition both from healthy controls and other autoimmune diseases.

Regulation of Gut Microbiota and Metabolic Endotoxemia with Dietary Factors

Metabolic endotoxemia is a condition in which blood lipopolysaccharide (LPS) levels are elevated, regardless of the presence of obvious infection. It has been suggested to lead to chronic inflammation-related diseases such as obesity, type 2 diabetes mellitus, non-alcoholic fatty liver disease (NAFLD), pancreatitis, amyotrophic lateral sclerosis, and Alzheimer’s disease. In addition, it has attracted attention as a target for the prevention and treatment of these chronic diseases. As metabolic endotoxemia was first reported in mice that were fed a high-fat diet, research regarding its relationship with diets has been actively conducted in humans and animals. In this review, we summarize the relationship between fat intake and induction of metabolic endotoxemia, focusing on gut dysbiosis and the influx, kinetics, and metabolism of LPS. We also summarize the recent findings about dietary factors that attenuate metabolic endotoxemia, focusing on the regulation of gut microbiota. We hope that in the future, control of metabolic endotoxemia using dietary factors will help maintain human health.

Functional roles of the A335 and G338 residues of the proton-coupled folate transporter (PCFT-SLC46A1) mutated in hereditary folate malabsorption

The proton-coupled folate transporter (PCFT-SLC46A1) mediates intestinal folate absorption and folate transport across the choroid plexus, processes defective in hereditary folate malabsorption (HFM). This paper characterizes the functional defect, and the roles of two mutated PCFT residues, associated with HFM (G338R and A335D). The A335D-PCFT and other mutations at this residue result in an unstable protein; when expression of a mutant protein was preserved, function was always retained. The G338R and other charged mutants resulted in an unstable protein; substitutions with small neutral and polar amino acids preserved protein but with impaired function. Pemetrexed and methotrexate (MTX) influx kinetics mediated by the G338C mutant PCFT revealed marked (15- to 20-fold) decreases in Kt and Vmax compared with wild-type PCFT. In contrast, there was only a small (∼2-fold) decrease in the MTX influx Ki and an increase (∼3-fold) in the pemetrexed influx Ki for the G338C-PCFT mutant. Neither a decrease in pH to 4.5, nor an increase to 7.4, restored function of the G338C mutant relative to wild-type PCFT excluding a role for this residue in proton binding or proton coupling. Homology modeling localized the A335 and G338 residues embedded in the 9th transmembrane, consistent with the inaccessibility of the A335C and G338C proteins to MTS reagents. Hence, the loss of intrinsic G338C-PCFT function was due solely to impaired oscillation of the carrier between its conformational states. The data illustrate how alterations in carrier cycling can impact influx Kt without comparable alterations in substrate binding to the carrier.

Nitrate and Ammonium Uptake in Vaccinium Species Differing in Tolerance to High Soil pH

Cultivated Vaccinium species (e.g. highbush blueberry, Vaccinium corymbosum, or cranberry, V. macrocarpon) commonly require acidic soil (pH 4.5 to 5.5) for optimum growth. Under these conditions, ammonium (NH4+) is the dominant form of inorganic N. In contrast, V. arboreum, the sparkleberry can tolerate higher-pH mineral soils, where nitrate (NO3-) is typically the predominant inorganic N form. This tolerance may be related to increased ability to acquire and utilize NO3—N. Measurements of 15NO3- and 15NH4+ influx kinetics in excised roots of V. arboreum, V. corymbosum, and V. macrocarpon did not support this hypothesis. NO3- influx kinetics measured from 10 micromolar to 200 micromolar NO3- were similar among all three species. NO3- influx was consistently lower than NH4+ influx at all concentrations for all three species.