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

Abstract

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.

The Dicarboxylate Transporters from the AceTr Family and Dct-02 Oppositely Affect Succinic Acid Production in S. cerevisiae

“…For this purpose, we constructed the Saccharomyces cerevisiae strain engineered for succinic acid overproduction from glycerol as described in Materials and Methods and named it ‘strain SA’ throughout this study ( Table 1 ). In summary, the strain SA ( Figure 1 ) was equipped with the L-G3P and DHA pathways for efficient glycerol utilization [ 23 ], the UBR2 allele from the strain CBS 6412-13A, which is required for CEN.PK strains to grow in synthetic glycerol medium [ 32 ], and the reductive TCA (rTCA) pathway [ 12 ] for efficient cytosolic succinic acid production.…”

“…After glycerol exhaustion, nearly all ethanol was re-consumed by the strain SA in both conditions ( Figure 2 ). Notably, the strain SA did not produce any malic acid ( Figure 2 ), which is an intermediate of the rTCA pathway ( Figure 1 ), and a common by-product of yeast cell factories engineered for succinic acid production [ 12 , 33 , 34 ].…”

“…Dct-02 from A. niger was already employed in the past as a heterologous succinic acid exporter in yeast cell factories which achieved high succinic acid titers and yields [ 8 , 11 , 12 ]. However, there was little information available about the individual contribution of Dct-02 towards the reported achievements.…”

“…The strain carries an expression cassette for the Dct-02 transporter and the results obtained with an isogenic strain lacking the transporter confirmed the importance of Dct-02 for achieving significant succinic acid production in S. cerevisiae (unpublished data). An improved succinic acid producer (UBR2 CBS -DHA-SA-AnDCT-02 (2)-PYC2oe) has recently been published by Malubhoy, et al [ 12 ]. This strain produced 35 g/L of succinic acid from glycerol after four days of cultivation in CaCO 3 -buffered shake flasks and 20 g/L of succinic acid after three days of cultivation in unbuffered shake flasks, reaching yields of 0.60 and 0.53 g/g glycerol , respectively.…”

“…First, we constructed a S. cerevisiae SA strain with a very efficient succinic acid production pathway but without a heterologous exporter. The strain construction was based on our previous experience regarding succinic acid production from glycerol [ 11 , 12 , 23 ]. The resulting SA strain was used as a platform to express and characterize the above transporter variants.…”