Studies on Squalene Biosynthesis and the Standardization of Its Extraction Methodology from Saccharomyces cerevisiae

Abstract

In this study, a homogenization-based extraction method was developed and was compared to five conventional methods of squalene extraction. Squalene recovered from this novel procedure gave 3.5-fold, 10-fold, 16-fold, and 8.1-fold higher yield than standard procedures, viz., saponification with 60% KOH, acidic saponification, saponification with 18% KOH, and glass beads method, respectively. Furthermore, this procedure has been evaluated on laboratory Saccharomyces cerevisiae strains such as BY4742 and CEN.PK2-1C (native), deletion strains (ERG6 and ERG11), and tHMG1 overexpressed S. cerevisiae strains. When sonication method of cell lysis was replaced with homogenization, it was found that the yields were significantly higher and reached a value of 9 mg/g DCW in case of BY4742. In addition, squalene yield in ergosterol mutant strains has been analyzed and was found to be 1.8-fold and 3.4-fold higher in ERG6 and ERG11 deletion strains, respectively, than in BY4742. Squalene was also found to be higher at the optimized temperature of 30 °C and pH 6.0. Furthermore, tolerance of S. cerevisiae to external squalene at various concentrations has been carried and found that the organism was tolerant up to 25 g/L of squalene.

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

“…The extraction and analysis of squalene is according to Paramasivan et al (2018). In brief, cells were harvested by centrifugation to obtain cell pellets, and the cell pellets were then subjected to lyophilization at −50°C under vacuum for 4 h. The lyophilized cells were dispersed in chloroform/methanol (2:1, v/v) mixture followed by sonication.…”

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

“…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 ]). Still, newly introduced triterpenoid-related modifications must compete with the endogenous ergosterol pathway, as yeast uses most of its products in its endogenous metabolism.…”

“…Humans also synthesize squalene in the liver, which peaks around the second decade of life [ 5 ]. Skin and adipose tissues are the major sites for squalene storage in humans, where it accounts for up to 12% of all skin surface lipids [ 5 , 6 , 7 ].…”

“…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 ].…”

Recent advances in the microbial production of squalene

“…Knockout of the ERG6 gene coding for delta (24)-sterol C-methyltransferase along with a site-specific mutation in HMG2 (K6R) has enhanced squalene to 20-fold (Mantzouridou and Tsimidou 2010). Likewise, knockout of ERG6 gene along with knockdown of ERG11 gene involved in ergosterol biosynthesis has enhanced squalene up to 43 mg/g DCW in S. cerevisiae (Paramasivan et al 2018). In another study, knockdown of downstream genes involved in ergosterol synthesis and the alcohol dehydrogenase genes involved in ethanol biosynthesis combined with several other strategies has enhanced squalene to 304.49 mg/l (Rasool et al 2016a).…”