Saccharomyces cerevisiae MET5DeltaSIZ1Delta enhancing organic acid tolerance with XYL1 and XYL2 integration for ethanol yield improvement in the presence of xylose and low pH value

Organic acids are known to be stress inhibitors in Saccharomyces cerevisiae in industrial processes of ethanol production due to energy depletion, growth arrest, inhibition of enzymes, and ethanol productivity losses. To improve the tolerance of organic acids and increase the utilization efficiency of xylose, S. cerevisiae MET5 and SIZ1 were knocked out by XYL1 and XYL2 expression cassettes using the CRISPR-Cas9 approach. The results indicated that S. cerevisiae MET5ΔSIZ1Δ, after MET5 and SIZ1 co-deletion, possessed strong tolerance of organic acids such as acetic acid and lactic acid. Transcriptomics analysis revealed that the co-deletion of MET5 and SIZ1 mainly resulted in the up-regulation of carbon metabolism and the down-regulation of ribosome biogenesis. The ethanol concentration of S. cerevisiae MET5ΔSIZ1Δ (55 g/L) was 1.28-fold higher than that of the wild-type strain (43 g/L) when using kiwifruit as a fermentation substrate. In addition, S. cerevisiae MET5ΔSIZ1Δ produced an ethanol concentration of 34.1 g/L, which was 1.8 times that of the wild-type strain (19 g/L) using the enzymatic hydrolysate of corn stalk. Thus, S. cerevisiae MET5ΔSIZ1Δ, with its two remarkable advantages of organic acid tolerance and xylose consumption, had great potential for ethanol production from cellulosic hydrolysates and other substrates rich in organic acids.