Background: The microbial production of succinic acid (SA) from renewable carbon sources via the reverse TCA (rTCA) pathway is a process potentially accompanied by net-fixation of carbon dioxide (CO₂). Among reduced carbon sources, glycerol is particularly attractive since it allows a nearly twofold higher CO₂-fixation yield compared to sugars. Recently, we described an engineered Saccharomyces cerevisiae strain which allowed SA production in synthetic glycerol medium with a maximum yield of 0.23 Cmol Cmol⁻¹. The results of that previous study suggested that the glyoxylate cycle considerably contributed to SA accumulation in the respective strain. The current study aimed at improving the flux into the rTCA pathway accompanied by a higher CO₂-fixation and SA yield. Results: By changing the design of the expression cassettes for the rTCA pathway, overexpressing PYC2, and adding CaCO₃ to the batch fermentations, an SA yield on glycerol of 0.63 Cmol Cmol⁻¹ was achieved (i.e. 47.1% of the theoretical maximum). The modifications in this 2nd-generation SA producer improved the maximum biomass-specific glycerol consumption rate by a factor of nearly four compared to the isogenic baseline strain solely equipped with the dihydroxyacetone (DHA) pathway for glycerol catabolism. The data also suggest that the glyoxylate cycle did not contribute to the SA production in the new strain. Cultivation conditions which directly or indirectly increased the concentration of bicarbonate, led to an accumulation of malate in addition to the predominant product SA (ca. 0.1 Cmol Cmol⁻¹ at the time point when SA yield was highest). Off-gas analysis in controlled bioreactors with CO₂-enriched gas-phase indicated that CO₂ was fixed during the SA production phase. Conclusions: The data strongly suggest that a major part of dicarboxylic acids in our 2nd-generation SA-producer was formed via the rTCA pathway enabling a net fixation of CO₂. The greatly increased capacity of the rTCA pathway obviously allowed successful competition with other pathways for the common precursor pyruvate. The overexpression of PYC2 and the increased availability of bicarbonate, the co-substrate for the PYC reaction, further strengthened this capacity. The achievements are encouraging to invest in future efforts establishing a process for SA production from (crude) glycerol and CO₂.

Previously, our lab replaced the endogenous FAD-dependent pathway for glycerol catabolism in S. cerevisiae by the synthetic NAD-dependent dihydroxyacetone (DHA) pathway. The respective modifications allow the full exploitation of glycerol’s higher reducing power (compared to sugars) for the production of the platform chemical succinic acid (SA) via a reductive, carbon dioxide fixing and redox-neutral pathway in a production host robust for organic acid production. Expression cassettes for three enzymes converting oxaloacetate to SA in the cytosol (“SA module”) were integrated into the genome of UBR2_CBS-DHA, an optimized CEN.PK derivative. Together with the additional expression of the heterologous dicarboxylic acid transporter DCT-02 from Aspergillus niger, a maximum SA titer of 10.7 g/L and a yield of 0.22 ± 0.01 g/g glycerol was achieved in shake flask (batch) cultures. Characterization of the constructed strain under controlled conditions in a bioreactor supplying additional carbon dioxide revealed that the carbon balance was closed to 96%. Interestingly, the results of the current study indicate that the artificial “SA module” and endogenous pathways contribute to the SA production in a highly synergistic manner.