Abstract:
Saccharomyces cerevisiae has been regarded as one of the cell factories in biomanufacturing because of numerous advantages towards industrial fermentations, including robust growth in low pH, lower temperatures, high tolerance to shear stress, lack of phage contamination, and ease of separation. However, the Crabtree effect of
Saccharomyces cerevisiae leads to accumulation of ethanol and glycerol due to carbon overflow. For the production of intermediate derivatives of the TCA (Tricarboxylic acid cycle) cycle, such as itaconic acid, the Crabtree effect must be eliminated by using a suitable strategy. In this paper, the role of NOX and AOX1 on the Crabtree effect in batch fermentation of
Saccharomyces cerevisiae was investigated by expressing the NADH (Nicotinamide adenine dinucleotide) oxidase NOX and the alternative oxidase AOX1 with plasmids having different copy numbers. It was revealed that both strains expressing NOX and AOX1 in high copy vectors caused significant metabolic changes. The high copy expressing
nox strains were able to oxidize cytoplasmic NADH, glycerol secretion in the medium was reduced by 43.94%, and IA concentration was not changed. In contrast, strains with high copy expression of
aox1 had cytoplasmic residual AOX1, which oxidized cytoplasmic NADH and reduced glycerol accumulation. Further location of AOX1 to the mitochondria of
Saccharomyces cerevisiae with the mitochondrial location signals AAC2 and BCS1p weakened the effect of AOX1 on glycerol synthesis, and IA production was enhanced to 116.98 mg/L. However, none of the strains expressing AOX1, AAC2-AOX1 and BCS1p-AOX1 significantly alleviated the accumulation of ethanol in batch fermentation. This study helps to improve the production of TCA cycle derivatives from glucose by engineered
Saccharomyces cerevisiae, providing a reference for the production of TCA cycle derivatives from
Saccharomyces cerevisiae in the batch culture at high original glucose concentrations.