Metabolic engineering and introduction of heterologous pathways are nowadays standard-procedure and high carbon yields can be obtained. To take the next step, the free energy conservation of the pathways from the extracellular substrate to extracellular product (and biomass) has to be improved, especially to apply cost-efficient anaerobic fermentation to a broad variety of commodity chemicals. One group of chemicals for which this energetic limitation occurs is the production of organic acids at low pH. Yeasts, such as S. cerevisiae, are being applied for the production of lactic acid or succinic acid.
Approach and Techniques
In the project, we aim at optimizing the energy conservation based on two major improvements:
The project aims at obtaining an improved quantitative understanding of energy dissipation in metabolic networks and emphasizes on substrate cycles in the cell, emphasizing on weak-acid cycling of lactate, its implications on overall ATP yield and interactions with central carbon metabolism. Especially at low pH several intracellular metabolites are found at high concentrations in the extracellular space. It is assumed that there are active export and import processes operating in parallel, leading to a ATP dissipation in case of H+ or ATP dependent transport mechanisms.
Substrate uptake and product export
Oligosaccharides open the possibility for increased free energy conservation (ATP) per monosaccharide unit. The first step to achieve this higher ATP gain has been demonstrated by introducing a heterologous maltose phosphorylase (see references). Another prerequisite is the proton neutral import of the oligosaccharide: For maltose this currently occurs through H+ symport, which requires transport engineering (maltose uniporter) and develop of methods to asses transport mechanisms in order to increase the net ATP gain, moreover implications of introducing those new pathways on energy state and metabolic regulation will be characterized.
Quantitative metabolomics on extra- and intracellular metabolites in vivo allow to quantify the cellular state in presence of stimuli. Coupling concentration information with 13C labeling information allows flux determination from dynamic and stationary experimental conditions. The project will also involve application of modeling approaches to deduct hypothesis from observables and to identify targets for metabolic engineering of cells towards increased free energy conservation.
- de Kok et al., Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase DOI
- van Maris et al., Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered Saccharomyces cerevisiae: Possible Consequence of Energy-Dependent Lactate Export DOI
- Abbott et al., Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids: current status and challenges.DOI
- Abate et al., Piecewise affine approximations of fluxes and enzyme kinetics from in vivo 13C labelling experiments DOI
- George E. P. Box, Science and Statistics DOI