Project description:Previous findings have demonstrated that the NADH/NAD+ ratio has a strong impact on the glycolytic flux in C. glutamicum under anaerobic conditions in the absence of external electron acceptors. During an attempt to rewire anaerobic metabolism to achieve high yield formation of ethanol, we inactivated the malate dehydrogenase and lactate dehydrogenase in a C. glutamicum strain expressing pyruvate decarboxylase and alcohol dehydrogenase, to eliminate formation of the by-products succinate and lactate, respectively. This modification increased the yield of ethanol but had a negative effect on glycolysis, which we found to correlate with an elevated NADH/NAD+ ratio. The pyruvate dehydrogenase (PDH) of C. glutamicum is active under anaerobic conditions, and can potentially exacerbate the negative effect on glycolysis, due to NADH formation. To reduce PDH activity under anaerobic conditions, we decided to replace the gene encoding the E3 subunit of PDH with its Escherichia coli counterpart, as E. coli PDH has been reported to be functional under aerobic conditions only. The resultant strain JS133 produced far less acetate with a further increased ethanol production, however, the glycolytic flux was still low. After observing differences in glycolytic flux for JS133 on glucose and fructose, we speculated that the pentose phosphate pathway (PPP) might be involved in the reduced flux on glucose. To prove this, we deleted the zwf gene, encoding glucose-6-phosphate dehydrogenase, which is the entry point into PPP, and immediately observed a stimulating effect on glycolysis. Subsequent characterization revealed a direct correlation between the intracellular NADH/NAD+ and NADPH/NADP+ ratios under anoxic conditions. Based on these findings we managed to re-channel the metabolism of C. glutamicum successfully towards either to ethanol or D-lactate with 92% and 98% of the theoretical yield respectively, which is the highest yields for D-lactate production thus far reported in the literature.

Project description:The escalating prevalence of metabolic syndrome (MetS) poses significant risks to type 2 diabetes mellitus, cardiovascular diseases, and non-alcoholic fatty liver disease. High fructose intake has emerged as an environmental risk for MetS and the associated metabolic diseases. To examine inter-individual variability in MetS susceptibility in response to fructose consumption, here we fed three inbred mouse strains, namely C57BL/6J (B6), DBA (DBA) and FVB/NJ (FVB) with 8% fructose in drinking water for 12 weeks. We found that fructose-fed DBA mice had significantly higher amount of body weight, adiposity, and glucose intolerance starting from the 4th week of fructose feeding compared to the control group, while B6 and FVB showed no differences in these phenotypes over the course of fructose feeding. In addition, elevated insulin levels were found in fructose-fed DBA and FVB mice, and cholesterol levels were uniquely elevated in B6 mice. To explore the molecular underpinnings of the observed distinct phenotypic responses among strains, we applied RNA sequencing to investigate the effect of fructose on the transcriptional profiles of liver and hypothalamus tissues, revealing strain- and tissue-specific patterns of transcriptional and pathway perturbations. Strain-specific liver pathways altered by fructose include fatty acid and cholesterol metabolic pathways for B6 and PPAR signaling for DBA. In hypothalamus tissue, only B6 showed significantly enriched pathways such as protein folding, pancreatic secretion, and fatty acid beta-oxidation. Using network modeling, we predicted potential strain-specific key regulators of fructose response such as Fgf21 (DBA) and Lss (B6) in liver, and Fmod (B6) in hypothalamus. We validated strain-biased responses of Fgf21 and Lss to fructose in primary hepatocytes. Our findings support that fructose perturbs different tissue networks and pathways in genetically diverse mice and associates with distinct features of metabolic dysfunctions. These results highlight individualized molecular and metabolic responses to fructose consumption and may help guide the development of personalized strategies against fructose-induced MetS.

Project description:Cell proliferation is achieved by numerous enzyme reactions. Temperature governs the activity of each enzyme, which overall determines the optimal growth temperature. Synthesizing useful chemicals and fuels utilizes only part of the metabolic pathways, especially the central metabolic pathways such as glycolysis and TCA cycle to metabolize glucose. However, the optimal temperature for the activity of the central metabolic pathways is inconclusive whether it is correlated with the optimal temperature for cell proliferation. Here, we found that Corynebacterium glutamicum wild type increased the metabolic activity to consume glucose under anaerobic (oxygen deprived) conditions at 42.5ºC, in which the cell hardly grows under aerobic conditions. Glucose consumption rate was increased by 24% at 42.5ºC compared to that at the optimal growth temperature of 30.0ºC. Transcriptional analysis showed that gapX gene encoding glycelaldehydre-3-phosphate dehydrogenase, glucokinase gene, and a gene involved in glucose uptake were upregulated at 42.5ºC. Production of fermentative lactate was increased by 69% than that at 30.0ºC, whereas succinate production was decreased by 13%. In addition to several glycolytic enzymes, the activity of pyruvate kinase was increased with increasing the temperature, whereas the activity of phosphoenolpyruvate carboxylase in anapleotic pathway leading to succinate synthesis was decreased. However, a metabolically engineered succinate over-producing strain, in which lactate production was shut off and pyruvate carboxylase encoding gene in another anapleotic pathway was overexpressed, produced 34% higher succinate at 42.5ºC than that at 30.0ºC with increased glucose consumption. This study provides the evidence that the optimal reaction temperature for production of fermentative products can be set beyond upper limit of growth temperature in C. glutamicum, and hence it could be applicable for producing various chemicals and fuels in C. glutamicum under anaerobic conditions and non-growing cell reactions in other microorganisms.

Dataset Information

Transcriptome analysis of the pfkB1 deletion mutant cultivated with glucose or fructose

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