Carbon dioxide fixation via production of succinic acid from glycerol in engineered Saccharomyces cerevisiae.

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.

Novel Evolutionary Engineering Approach to Alter Substrate Specificity of Disaccharide Transporter Mal11 in Saccharomyces cerevisiae.

A major challenge in the research of transport proteins is to understand how single amino acid residues contribute to their structure and biological function. Amino acid substitutions that result in a selective advantage in adaptive laboratory evolution experiments can provide valuable hints at their role in transport proteins. In this study, we applied an evolutionary engineering strategy to alter the substrate specificity of the proton-coupled disaccharide transporter Mal11 in Saccharomyces cerevisiae, which has affinity for sucrose, maltose and glucose. The introduction of MAL11 in a strain devoid of all other sugar transporters and disaccharide hydrolases restored growth on glucose but rendered the strain highly sensitive to the presence of sucrose or maltose. Evolution in glucose-limited continuous cultures with pulse-wise addition of a concentrated sucrose solution at increasing frequency resulted in the enrichment of spontaneous mutant cells that were less sensitive to the presence of sucrose and maltose. Sequence analysis showed that in each of the two independent experiments, three mutations occurred in MAL11, which were found responsible for the disaccharide-insensitive phenotype via reverse engineering. Our work demonstrates how laboratory evolution with proton-motive force-driven uptake of a non-metabolizable substrate can be a powerful tool to provide novel insights into the role of specific amino acid residues in the transport function of Mal11.

Evolutionary engineering reveals amino acid substitutions in Ato2 and Ato3 that allow improved growth of Saccharomyces cerevisiae on lactic acid.

In Saccharomyces cerevisiae, the complete set of proteins involved in transport of lactic acid across the cell membrane has not been determined. In this study, we aimed to identify transport proteins not previously described to be involved in lactic acid transport via a combination of directed evolution, whole-genome resequencing and reverse engineering. Evolution of a strain lacking all known lactic acid transporters on lactate led to the discovery of mutated Ato2 and Ato3 as two novel lactic acid transport proteins. When compared to previously identified S. cerevisiae genes involved in lactic acid transport, expression of ATO3T284C was able to facilitate the highest growth rate (0.15 ± 0.01 h-1) on this carbon source. A comparison between (evolved) sequences and 3D models of the transport proteins showed that most of the identified mutations resulted in a widening of the narrowest hydrophobic constriction of the anion channel. We hypothesize that this observation, sometimes in combination with an increased binding affinity of lactic acid to the sites adjacent to this constriction, are responsible for the improved lactic acid transport in the evolved proteins.