Evolving a New Efficient Mode of Fructose Utilization for Improved Bioproduction in Corynebacterium glutamicum

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (∼10%) compared to glucose (∼60%). Consequently, the biosynthesis of NADPH demanding products, e.g., L-lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose-specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and, consequently, NADPH availability. Here, we generated deletion strains lacking either the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose-specific PTS as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose-specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, and P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased ODs, reduced side-product accumulation, and increased L-lysine production by 50%.

Evolving a new efficient mode of fructose utilization for improved bioproduction in Corynebacterium glutamicum

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (~10 %) compared to glucose (~60 %). Consequently, the biosynthesis of NADPH demanding products, e.g. L-lysine, by C. glutamicum is largely decreased, when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and consequently NADPH availability. Here, we generated deletion strains either lacking in the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose specific PTS, as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased biomass formation, reduced side-product accumulation, and increased L-lysine production by 50 %.

A Cyclic Metabolic Network inPseudomonas protegensPf-5 Prioritizes the Entner-Doudoroff Pathway and Exhibits Substrate Hierarchy during Carbohydrate Co-Utilization

ABSTRACTThe genetic characterization ofPseudomonas protegensPf-5 was recently completed. However, the inferred metabolic network structure has not yet been evaluated experimentally. Here, we employed13C-tracers and quantitative flux analysis to investigate the intracellular network for carbohydrate metabolism. In lieu of the direct phosphorylation of glucose by glucose kinase, glucose catabolism was characterized primarily by the oxidation of glucose to gluconate and 2-ketogluconate before the phosphorylation of these metabolites to feed the Entner-Doudoroff (ED) pathway. In the absence of phosphofructokinase activity, a cyclic flux from the ED pathway to the upper Embden-Meyerhof-Parnas (EMP) pathway was responsible for routing glucose-derived carbons to the non-oxidative pentose phosphate (PP) pathway. Consistent with the lack of annotated genes inP. protegensPf-5 for the transport or initial catabolism of pentoses and galactose, only glucose was assimilated into intracellular metabolites in the presence of xylose, arabinose, or galactose. However, when glucose was fed simultaneously with fructose or mannose, co-uptake of these hexoses was evident, but glucose was preferred over fructose (3 to 1) and over mannose (4 to 1). Despite gene annotation of mannose catabolism to fructose-6-phosphate, metabolite labeling patterns revealed that mannose was assimilated into fructose-1,6-bisphosphate, similarly to fructose catabolism. Remarkably, carbons from mannose and fructose were also found to cycle backward through the upper EMP pathway toward the ED pathway. Therefore, the operational metabolic network for processing carbohydrates inP. protegensPf-5 prioritizes flux through the ED pathway to channel carbons to EMP, PP, and downstream pathways.IMPORTANCESpecies of thePseudomonasgenus thrive in various nutritional environments and have strong biocatalytic potential due to their diverse metabolic capabilities. Carbohydrate substrates are ubiquitous both in environmental matrices and in feedstocks for engineered bioconversion. Here, we investigated the metabolic network for carbohydrate metabolism inPseudomonas protegensPf-5. Metabolic flux quantitation revealed the relative involvement of different catabolic routes in channeling carbohydrate carbons through a cyclic metabolic network. We also uncovered that mannose catabolism was similar to fructose catabolism, despite the annotation of a different pathway in the genome. Elucidation of the constitutive metabolic network inP. protegensis important for understanding its innate carbohydrate processing, thus laying the foundation for targeting metabolic engineering of this untappedPseudomonasspecies.

Alterations of Hippocampal Glucose Metabolism by Even versus Uneven Medium Chain Triglycerides

Medium chain triglycerides (MCTs) are used to treat neurologic disorders with metabolic impairments, including childhood epilepsy and early Alzheimer's disease. However, the metabolic effects of MCTs in the brain are still unclear. Here, we studied the effects of feeding even and uneven MCTs on brain glucose metabolism in the mouse. Adult mice were fed 35% (calories) of trioctanoin or triheptanoin (the triglycerides of octanoate or heptanoate, respectively) or a matching control diet for 3 weeks. Enzymatic assays and targeted metabolomics by liquid chromatography tandem mass spectrometry were used to quantify metabolites in extracts from the hippocampal formations (HFs). Both oils increased the levels of β-hydroxybutyrate, but no other significant metabolic alterations were observed after triheptanoin feeding. The levels of glucose 6-phosphate and fructose 6-phosphate were increased in the HF of mice fed trioctanoin, whereas levels of metabolites further downstream in the glycolytic pathway and the pentose phosphate pathway were reduced. This indicates that trioctanoin reduces glucose utilization because of a decrease in phosphofructokinase activity. Trioctanoin and triheptanoin showed similar anticonvulsant effects in the 6 Hz seizure model, but it remains unknown to what extent the anticonvulsant mechanism(s) are shared. In conclusion, triheptanoin unlike trioctanoin appears to not alter glucose metabolism in the healthy brain.

Inhibition of Rat Muscle and Liver Phosphofructokinases by High Doses of Ethanol

Activities of both rat muscle and liver phosphofructokinases are significantly inhibited after a single ethanol intake in the dose of 2.5 g per kg of body weight. This inhibitory effect is indirect, since ethanol in concentration (50 mM) close to that established after 2.5 g per kg of body weight intake cannot decrease their activitiesin vitro. Inhibition of liver phosphofructokinase activity after the 5.0 g per kg ethanol intake may be direct, since liver phosphofructokinase activity decreasesin vitrowhen ethanol is added to supernatants of rat liver tissue in 100 mM concentration. According to the results of molecular docking, ethanol at high concentrations can be bound by adenine-binding pocket of the allosteric ADP-binding site of liver phosphofructokinase (Asp543, Phe308, Phe538, and Phe671) and its activation by ADP can be blocked by C2H5OH molecule. Direct inhibition of muscle phosphofructokinase activity, probably due to the binding of ethanol to the similar ADP-binding site, is possible when the concentration of ethanol (500 mM) is much higher than the level which can be established in living cells. So, inhibition of muscle phosphofructokinase activity after a single 5.0 g per kg intake is indirect and probably linked with the inhibition of the enzyme by elevated citrate and phosphoenolpyruvate levels.