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Growth parameters and sample analysis were performed as described under example 2C.4 with the following modifications: pre-culturing was performed using 2% glucose (w/v) as carbon source. In the production medium 10% glucose (w/v) was used as carbon source.
As depicted in Figure 23 strain SUC-230, overexpressing MDH3, FUMR and FRDg, produced up to 3.0 g/L succinic acid. Additional overexpression of PCKa increased succinic acid(cas:110-15-6) production up to 3.4 g/L (strain SUC-226), and additional overexpression of PYC2 increased succinic acid(cas:110-15-6) production up to 3.7 g/L (strain SUC-228). Surprisingly, overexpression of both PCKa and PYC2 (SUC-227) resulted in 1.5 increase of succinic acid production levels up to 5.0 g/L, as compared to the effect of PCK and PYC alone. These results show a synergistic effect of combined overexpression of both PEP carboxykinase from A. succinogenes (PCKa) and pyruvate carboxylase from S. cerevisiae(PYC2) on succinic acid production levels in S. cerevisiae.
Transformants were inoculated in 20 ml pre-culture medium consisting of Verduyn medium (Verduyn et al., 1992, Yeast. Jul;8(7):501-17) comprising 2% galactose (w/v) and grown under aerobic conditions in 100 ml shake flasks in a shaking incubator at 30°C at 250 rpm. After 72 hours, the culture was centrifuged for 5 minutes at 4750 rpm. 1 ml supernatant was used to measure succinic acid levels by HPLC as described in section 1.4. The remaining supernatant was decanted and the pellet (cells) was resuspended in 1 ml production medium. The production medium consisted of Verduyn medium with 10 % galactose (w/v) and 1% CaCO3 (w/v). The resuspended cells were inoculated in 50 ml production medium in 100 ml shake flasks and grown in a shaking incubator at 30°C at 100 rpm.
Strains transformed with empty vectors (control strain) produced up to 0.3 g/L succinic acid(cas:110-15-6). Overexpression of PEP carboxykinase from M. succiniciproducens (PCKm), peroxisomal malate dehydrogenase (MDH3) from S. cerevisiae and fumarase from R. oryzae (FUMR) resulted in production of 0.9 g/L succinic acid production. Overexpression of PEP carboxykinase from A. succinogenes (PCKa), MDH3 and FUMR resulted in a slight increase in succinic acid production to 1.0 g/L.
These results show that in S. cerevisiae as described increased succinic acid(cas:110-15-6) production about 3 times. Additional overexpression of mitochondrial fumarate reductase (FRDm1) from T. brucei further increased succinic acid(cas:110-15-6) production levels; overexpression of PCKa, MDH3, FUMR, FRDm1 resulted in production of 2.6 g/L succinic acid(cas:110-15-6), and overexpression of PCKm, MDH3, FUMR and FRDm1 resulted in production of 2.7 g/L succinic acid(cas:110-15-6). Overexpression of delta12NMDH2 in combination with PCKm, FUMR and FRDm1 resulted in production of 2.7 g/L succinic acid, indicating that similar levels of succinic acid were produced using either truncated MDH2 or MDH3. Additional overexpression of glycosomal fumarate reductase (FRDg) from T. brucei resulted in an even higher increase in succinic acid production levels; overexpression of PCKa, MDH3, FUMR and FRDg resulted in production of 3.9 g/L succinic acid, whereas overexexpression of PCKm, MDH3, FUMR and FRDg resulted in slightly lower production of 3.6 g/L succinic acid.
The results show addition of NAD(H) dependent fumarate reductase as disclosed herein in a S. cerevisiae comprising a genetic modification of PCKa/m, MDH3 and FUMR significantly increased succinic acid production levels. Overexpression of FRDg had a more positive effect on succinic acid(cas:110-15-6) production levels in S. cerevisiae compared to overexpression of FRDm1 in S. cerevisiae.
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