Extension of the substrate range of the cell factory Saccharomyces cerevisiae to use C1-derived feedstocks: succinic acid as a model target product

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As a result of the preceding project, we have recently demonstrated the co-utilization of glycerol (C3) and CO2 (C1) for the production of the platform chemical succinic acid (C4) by after metabolic engineering of the yeast Saccharomyces cerevisiae. The product yield (per glycerol consumed) corresponded to 47% of the theoretical maximum. Further possibilities for improvements of the process have been explored in our current project. In the project proposed here, we plan to replace the carbon source glycerol by feedstocks that do not originate from edible plant biomass, and whose abundance is independent from the future of biodiesel production. Both methanol (C1) and dihydroxyacetone (DHA, C3) have the potential to become future carbon sources for biotechnological processes since they can be generated from synthesis gas or from CO2. An attractive approach is to equip our succinic acid producing strain with a linear pathway for conversion of methanol to SA (methanol dehydrogenase and formolase) and afterwards use adaptive laboratory evolution (ALE). We consider this a high-risk approach due to both thermodynamic and kinetic constraints. We still consider it promising. In fact, the proposed ALE strategy has the potential to optimize the kinetics of the enzyme formolase. In addition, our succinic acid pathway is considered a strong sink for NADH and can therefore attenuate the thermodynamic barrier of the NAD+-dependent methanol oxidation. As an alternative to methanol as the carbon source, we propose a second approach based on DHA (C3) as a carbon source. In the future, DHA could be generated from CO2 in a chemoenzymatic process and the channeling of DHA into the central metabolism of S. cerevisiae is straightforward. Our previously constructed S. cerevisiae strains with modified glycerol catabolism (in particular those with the DHA pathway) form an excellent basis for this goal. The improvement of tolerance towards higher DHA concentrations will be addressed by ALE. In order to provide sufficient cytosolic NADH for SA production, an auxiliary substrate (such as formate generated from CO2) will be fed.