Investigation Of Metabolic Characteristics Of Microorganisms Based On Metabolic Flux Analysis

Gene expression is regulated through different transcriptional regulators, which can either enhance or inhibit gene transcription. The transcriptional regulator isocitrate lyase regulator (IclR) of E. coli represses the expression of the aceBAK operon, which codes for the glyoxylate pathway enzymes. In this study, physiological and metabolic responses of iclR deletions on E. coli BW25113 were investigated based on metabolic flux analysis.The deletion of the iclR gene resulted in a decrease in the growth rate, glucose uptake rate and the acetate secretion rate, but an increase in biomass yield. The increase in biomass yield can be explained by the lower metabolic fluxes through several CO2 producing pathways in E. coli BW25113ΔiclR knockout strain. Due to the deletion of the iclR gene, the glyoxylate pathway was activated, resulting in the redirection of 38% of isocitrate molecules directly to succinate and malate without CO2 production, and the flux through the PP pathway was reduced. Furthermore, activation of the glyoxylate pathway of E. coli BW25113 AiclR might be explained by the change of isocitrate lyase activity. In the E. coli BW25113ΔiclR knockout strain, the activation of the glyoxylate pathway is linked to only a minor increase in the flux from oxaloacetate to PEP, implying that the PEP-glyoxylate cycle was not active.Microbial fermentation for producing biodiesel from lignocellulosic hydrolysates is receiving increasing attention. An oleaginous yeast Trichosporon cutaneum with high lipid content and a strong tolerance to lignocellulose hydrolysates was screened previously. In order to systematically identify metabolic pathways and understand the mechanisms of lipid accumulation in this strain, metabolic analysis based on flux quantification was performed for cells grown on high nitrogen condition and/or low nitrogen condition.Compared with the results under high nitrogen condition, the biomass yield of cells was increased and the oil content reached approximately 31%(g/gDCW) when the nitrogen concention was low. Besed on the growth and metabolic characteristics of T. cutaneum, a metabolic model was established. In the case of low nitrogen, both the PP pathway flux and the activity of cytosolic NADP+ dependent malic enzyme were found higher than those under high nitrogen condition. In addition, the activity of cytosolic NADP+ dependent malic enzyme was increased in lipid accumulation phase compared to that in cell growth phase. All these metabolic changes suggested the excessive NADPH requirement for lipid biosynthesis. In addition, the reaction via cytosolic NADP+ dependent malic enzyme was shown to be the major source of reducing equivalent for lipid accumulation.The flux ratio-based analysis also indicated that the citrate pyruvate cycle played an essential role in generating cytosolic acetyl-CoA for the lipid biosynthesis.