Year: 2005
Type: dissertation
Abstract: In this study, understanding of the central metabolism was improved by quantification of metabolite concentrations, enzyme activities, protein abundances, and gene transcript concentrations. Intracellular fluxes were estimated by applying stoichiometric models of metabolism. The methods were applied in the study of yeast Saccharomyces cerevisiae in two separate projects. A xylose project aimed at improved utilization of D-xylose as a substrate for, e.g., producing biomaterial-based fuel ethanol. A mannose project studied the production of GDP-mannose from D-mannose in a strain lacking the gene for phosphomannose isomerase (PMI40 deletion). Hexose, D-glucose is the only sugar more abundant than pentose D-xylose. D-xylose is common in hardwoods (e.g. birch) and crop residues (ca. 25% of dry weight). However, S. cerevisiae is unable to utilize D-xylose without a recombinant pathway where D-xylose is converted to D-xylulose. In this study D-xylose was converted in two steps via xylitol: by D-xylose reductase and xylitol dehydrogenase encoded by XYL1 and XYL2 from Pichia stipitis, respectively. Additionally, endogenous xylulokinase (XKS1) was overexpressed in order to increase the consumption of D-xylose by enhancing the phosphorylation of D-xylulose. Despite of the functional recombinant pathway the utilization rates of D-xylose still remained low. This study proposes a set of limitations that are responsible for the low utilization rates of D-xylose under microaerobic conditions. Cells compensated for the cofactor imbalance, caused by the conversion of D-xylose to D-xylulose, by increasing the flux through the oxidative pentose phosphate pathway and by shuttling NADH redox potential to mitochondrion to be oxidized in oxidative phosphorylation. However, mitochondrial NADH inhibits citrate synthase in citric acid cycle, and consequently lower flux through citric acid cycle limits oxidative phosphorylation. Further, limitations in the uptake of D-xylose, in the pentose phosphate pathway, and in the citric acid cycle were alleviated in xylose chemostat isolates with three-fold improved xylose utilization rates. Uptake rate of D-xylose, assayed in vitro with radioactive D-xylose, was improved by 60% in the chemostat isolates grown under aerobic conditions on D-xylose. In the pentose phosphate pathway activities of transketolase and transaldolase were increased two-fold, and consequently concentrations of their substrates were decreased two-fold in the chemostat isolates. Finally, less pyruvate and citrate, but more malate accumulated in the chemostat isolates than in the original strain grown on D-xylose under aerobic conditions. In a S. cerevisiae strain with PMI40 deletion, growth on media without D-mannose and D-glucose is disabled. Phosphomannose isomerase encoded by PMI40 connects D-mannose to glycolysis, which is the main pathway for D-glucose utilization. Hypothetically, a PMI40 deletion strain would direct all its D-mannose into the biosynthesis of GDP-mannose. However, in the PMI40 deletion strain increased initial D-mannose concentrations led to increased intracellular mannose 6-phosphate concentrations. Mannose 6-phosphate inhibited activity of phosphoglucose isomerase (encoded by PGI1) in glycolysis, which in essence is equivalent to suppressed expression of PGI1. Subsequently, reduced availability of glycolysis intermediates, due to inhibition of phosphoglucose isomerase, led to a decrease in the glycolytic flux. Eventually, increased initial D-mannose concentrations resulted in a starvation response, which was accompanied by slower cell cycle and slower growth rate.
Source: Aaltodoc (Aalto University)
Author: Juha‐Pekka Pitkänen
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: Helsingin Yliopisto, Tekes
OpenAlex ID: W2370189545