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

Abstract

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.

Advances in Metabolic Engineering Paving the Way for the Efficient Biosynthesis of Terpenes in Yeasts

“…When the mutant α-factor STE6 T1025N responsible for β-caryophyllene transport was overexpressed, the enhanced recombinant S. cerevisiae produced up to 13.8 mg/g DCW of β-caryophyllene in tube culture, which was four times higher than in the parental strain . Additionally, ALE provided key technology for increasing triterpene accumulation in S. cerevisiae to obtain robust strains with high squalene production …”

From Sharks to Yeasts: Squalene in the Development of Vaccine Adjuvants

“…In contrast, Saccharomyces cerevisiae ( S. cerevisiae ) and Escherichia coli ( E. coli ), are preferred experimental models for genetic manipulation, molecular and synthetic biology. Genome editing and metabolic optimization studies have generated strains that are increasingly efficient in producing commercially valuable bioactive molecules, including terpenoids such as squalene [ 29 , 30 , 31 , 32 , 80 , 81 ].…”

“…Importantly, the engineered strain showed steady β-farnesene production at industrial scale, in 200,000-L bioreactors, thus becoming a viable, cost-effective approach for large-scale production of acetyl-CoA-derived biomolecules [ 29 ]. Adaptive evolution of S. cerevisiae , in the presence of the isoprenoid pathway inhibitor terbinafine, has recently generated squalene-hyperproducing strains suiTable for industrial scale [ 31 ]. The evolved strains presented more than 100 single nucleotide variations (SNVs) in metabolism-related genes, including a point mutation in the ERG1 gene that encodes squalene epoxidase, responsible for the conversion of squalene into squalene epoxide [ 31 , 82 ].…”

“…Adaptive evolution of S. cerevisiae , in the presence of the isoprenoid pathway inhibitor terbinafine, has recently generated squalene-hyperproducing strains suiTable for industrial scale [ 31 ]. The evolved strains presented more than 100 single nucleotide variations (SNVs) in metabolism-related genes, including a point mutation in the ERG1 gene that encodes squalene epoxidase, responsible for the conversion of squalene into squalene epoxide [ 31 , 82 ]. The approach resulted in a maximum squalene production of 193 mg/L, higher than that of S. cerevisiae BY4741 which is commonly used as a source of bioactive molecules in the industrial setting ( Table 2 ; [ 7 , 80 ]).…”

“…When compared to sharks and plants, these microorganisms produce minimal quantities of squalene (reviewed in [ 3 ]). However, their easy maintenance, rapid life cycle and the possibility to genetically engineer and optimize their metabolism, make them appealing sources of metabolites with commercial value, such as squalene [ 7 , 21 , 29 , 30 , 31 ]. Here, the yeast Saccharomyces cerevisiae ( S. cerevisiae ) emerges as the standard model organism for the optimization of the isoprenoid synthetic pathway, via genetic engineering and metabolic manipulation [ 29 , 31 , 32 ].…”