The same genetic regulation strategy produces inconsistent effects in different Saccharomyces cerevisiae strains for 2-phenylethanol production.

A CRISPR/Cas9 system with gene editing efficiency of 100% in the industrial diploid Saccharomyces cerevisiae CWY-132 strain for 2-phenylethanol (2-PE) production was constructed. The effect of deletion of acetyltransferase gene ATF1 in the Ehrlich pathway on 2-PE synthesis was studied for the first time in S. cerevisiae. Laboratory and industrial strains were compared for the deletion effect of ATF1 and acetaldehyde dehydrogenase genes ALD2 and ALD3 involved in competing branches of the Ehrlich pathway on the 2-PE titer. The results showed that in 2-PE low-yielding haploid strain PK-2C, the ATF1∆ mutant produced 2-PE of 0.45 g/L, an increase of 114%, whereas in CWY-132, the 2-PE yield of ATF1∆ decreased significantly from 3.50 to 0.83 g/L. In PK-2C, the 2-PE yield of ALD2∆ increased from 0.21 to 1.20 g/L, whereas in CWY-132, it decreased from 3.50 to 3.02 and 2.93 g/L in ALD2∆ and ALD3∆ mutants, respectively, and to 1.65 g/L in ALD2∆ALD3∆. These results indicate that the same genetic manipulation strategy used for strains with different 2-PE yield backgrounds produces significantly different or even opposite effects. Moreover, we found that a supply of NADH or GSH increased the 2-PE production in S. cerevisiae. The correlation between the synthesis of 2-PE and ethanol was also revealed, and the tolerance of cells to 2-PE and ethanol was suggested to be a key limiting factor for further increase of 2-PE production in high-yielding strains. KEY POINTS: • Deletion of genes competing for 2-PE synthesis produces different effects in S. cerevisiae strains. • The ATF1∆, ALD2∆, or ALD3∆ increased 2-PE production in laboratory strains but not industrial strains. • The supply of NADH or GSH increased the titer of 2-PE in S. cerevisiae.