Adaptive evolution of engineered yeast for squalene production improvement and its genome-wide analysis.

In the present study, the adaptive evolution of a metabolically engineered Saccharomyces
cerevisiae strain in the presence of an enzyme inhibitor terbinafine for
enhanced squalene accumulation via serial transfer leads to the development of
robust strains. After adaptation for nearly 1500 h, a strain with higher squalene production
efficiency was identified at a specific growth rate of 0.28 h−1 with a final
squalene titer of 193 mg/L, which is 16.5-fold higher than the BY4741 and 3-fold
higher over the metabolically engineered SK22 strain. Whole-genome sequencing
comparison between the reference strain and the evolved variant SK23 has led to
the identification of 462 single-nucleotide variants (SNVs) between both strains, with
102 SNVs affecting metabolism-related genes. It was also established that F420I
mutation of ERG1 in S. cerevisiae improves squalene synthesis. Further, the effect of
increased squalene on lipid droplet and neutral lipid pattern in the evolved mutant
strains was investigated by fluorescent techniques proving that the neutral lipid content
and clustering of lipid droplets increase with an increase in squalene.