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.

Non-domesticated, wild Saccharomyces yeasts have promising characteristics for beer diversification, particularly when used in the generation of de novo interspecific hybrids. A major motivation for the current work was the question whether attractive novel Saccharomyces interspecific hybrids can be created for the production of exotic lager beers without using the genomic resources of the ale yeast Saccharomyces cerevisiae. Importantly, maltotriose utilization is an essential characteristic typically associated with domesticated ale/lager brewing strains. A high-throughput screening on nearly 200 strains representing all eight species of the Saccharomyces genus was conducted. Three Saccharomyces mikatae strains were able to aerobically grow on maltotriose as the sole carbon source, a trait until recently unidentified for this species. Our screening also confirmed the recently reported maltotriose utilization of the S. jurei strain D5095^T. Remarkably, de novo hybrids between a maltotriose-utilizing S. mikatae or S. jurei strain and the maltotriose-negative Saccharomyces eubayanus strain CBS 12357^T displayed heterosis and outperformed both parents with regard to aerobically utilizing maltotriose as the sole source of carbon. Indeed, the maximum specific growth rates on this sugar were comparable to the well-known industrial strain, Saccharomyces pastorianus CBS 1513. In lager brewing settings (oxygen-limited), the new hybrids were able to ferment maltose, while maltotriose was not metabolized. Favorable fruity esters were produced, demonstrating that the novel hybrids have the potential to add to the diversity of lager brewing.

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.

Acetic acid tolerance of the yeast Saccharomyces cerevisiae is manifested in several quantifiable parameters, of which the duration of the latency phase is one of the most studied. It has been shown recently that the latter parameter is mostly determined by a fraction of cells within the population that resumes proliferation upon exposure to acetic acid. The aim of the current study was to identify genetic determinants of the difference in this parameter between the highly tolerant strain MUCL 11987-9 and the laboratory strain CEN.PK113-7D. To this end, a combination of genetic mapping and pooled-segregant RNA sequencing was applied as a new approach. The genetic mapping data revealed four loci with a strong linkage to strain MUCL 11987-9, each containing still a large number of genes making the identification of the causal ones by traditional methods a laborious task. The genes were therefore prioritized by pooled-segregant RNA sequencing, which resulted in the identification of six genes within the identified loci showing differential expression. The relevance of the prioritized genes for the phenotype was verified by reciprocal hemizygosity analysis. Our data revealed the genes ESP1 and MET22 as two, so far unknown, genetic determinants of the size of the fraction of cells resuming proliferation upon exposure to acetic acid.

Background: Acetic acid, released during hydrolysis of lignocellulosic feedstocks for second generation bioethanol production, inhibits yeast growth and alcoholic fermentation. Yeast biomass generated in a propagation step that precedes ethanol production should therefore express a high and constitutive level of acetic acid tolerance before introduction into lignocellulosic hydrolysates. However, earlier laboratory evolution strategies for increasing acetic acid tolerance of Saccharomyces cerevisiae, based on prolonged cultivation in the presence of acetic acid, selected for inducible rather than constitutive tolerance to this inhibitor. Results: Preadaptation in the presence of acetic acid was shown to strongly increase the fraction of yeast cells that could initiate growth in the presence of this inhibitor. Serial microaerobic batch cultivation, with alternating transfers to fresh medium with and without acetic acid, yielded evolved S. cerevisiae cultures with constitutive acetic acid tolerance. Single-cell lines isolated from five such evolution experiments after 50-55 transfers were selected for further study. An additional constitutively acetic acid tolerant mutant was selected after UV-mutagenesis. All six mutants showed an increased fraction of growing cells upon a transfer from a non-stressed condition to a medium containing acetic acid. Whole-genome sequencing identified six genes that contained (different) mutations in multiple acetic acid-tolerant mutants. Haploid segregation studies and expression of the mutant alleles in the unevolved ancestor strain identified causal mutations for the acquired acetic acid tolerance in four genes (ASG1, ADH3, SKS1 and GIS4). Effects of the mutations in ASG1, ADH3 and SKS1 on acetic acid tolerance were additive. Conclusions: A novel laboratory evolution strategy based on alternating cultivation cycles in the presence and absence of acetic acid conferred a selective advantage to constitutively acetic acid-tolerant mutants and may be applicable for selection of constitutive tolerance to other stressors. Mutations in four genes (ASG1, ADH3, SKS1 and GIS4) were identified as causative for acetic acid tolerance. The laboratory evolution strategy as well as the identified mutations can contribute to improving acetic acid tolerance in industrial yeast strains.