A wide variety of microorganisms are increasingly being employed for the production of a broad diversity of compounds, instead of using fossil fuel. The production of such compounds faces different challenges for an optimized production. Identifying the bottlenecks in the synthesis of such products offers the possibility to reduce these bottlenecks and increase the production efficiency. This could lead to economically feasible production of these compounds without utilizing fossil fuels. This thesis focuses specifically on the production of PHB using E. coli, by studying the redox modified metabolism. Different metabolic pathways that might favour the flux towards PHB production were evaluated. More specifically, the bottlenecks in the synthesis and the conditions that might favor or limit its production were carefully analyzed in this thesis. The main bottlenecks in PHB production that have been identified and discussed in literature are the precursor acetyl-CoA and the co-factor NADPH. However, their role has not been entirely clarified in the field of metabolic engineering. Most studies use flux balance analysis to investigate the roles of acetyl-CoA and NADPH. However, these analyses only provide information about the stoichiometry of a pathway with the flux distribution, while analyzing them through thermodynamics gives the specific reaction that is furthest from equilibrium and therefore a bottleneck for the synthesis of a product. In this thesis we combine both flux balance analysis and thermodynamics for understanding the pathways EMP (Embden-Meyerhof-Parnas pathway), Entner–Doudoroff pathway (EDP), and modified Embden-Meyerhof-Parnas pathway (mEMP).@en

Biotechnological industry strives to develop anaerobic bioprocesses fueled by abundant and cheap carbon sources, like sucrose. However, oxygen-limiting conditions often lead to by-product formation and reduced ATP yields. While by-product formation is typically decreased by gene deletion, the breakdown of oligosaccharides with inorganic phosphate instead of water could increment the ATP yield. To observe the effect of oxygen limitation during sucrose consumption, a non-fermentative Escherichia coli K-12 strain was transformed with genes enabling sucrose assimilation. It was observed that the combined deletion of the genes adhE, adhP, mhpF, ldhA, and pta abolished the anaerobic growth using sucrose. Therefore, the biomass-specific conversion rates were obtained using oxygen-limited continuous cultures. Strains performing the breakdown of the sucrose by hydrolysis (SUC-HYD) or phosphorolysis (SUC-PHOSP) were studied in such conditions. An experimentally validated in silico model, modified to account for plasmid and protein burdens, was employed to calculate carbon and electron consistent conversion rates. In both strains, the biomass yields were lower than expected and, strikingly, SUC-PHOSP showed a yield lower than SUC-HYD. Flux balance analyses indicated a significant increase in the non-growth-associated ATP expenses by comparison with the growth on glucose. The observed fructose-1,6-biphosphatase and phosphoglucomutase activities, as well as the concentrations of glycogen, suggest the operation of ATP futile cycles triggered by a combination of the oxygen limitation and the metabolites released during the sucrose breakdown.

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Poly(3-hydroxybutyrate) (PHB) is an interesting biopolymer for replacing petroleum-based plastics, its biological production is performed in natural and engineered microorganisms. Current metabolic engineering approaches rely on high-throughput strain construction and screening. Analytical procedures have to be compatible with the small scale and speed of these approaches. Here, we present a method based on isotope dilution mass spectrometry (IDMS) and propanolysis extraction of poly(3-hydroxybutyrate) from an Escherichia coli strain engineered for PHB production. As internal standard (IS), we applied an uniformly labeled 13C-cell suspension, of an E. coli PHB producing strain, grown on U-13C-glucose as C-source. This internal 13C-PHB standard enables to quantify low concentrations of PHB (LOD of 0.01 µg/gCDW) from several micrograms of biomass. With this method, a technical reproducibility of about 1.8% relative standard deviation is achieved. Furthermore, the internal standard is robust towards different sample backgrounds and dilutions. The early addition of the internal standard also enables higher reproducibility and increases sensitivity and throughput by simplified sample preparation steps@en