Mutation of ERG1 for improved squalene synthesis in an engineered saccharomyces cerevisiae

Squalene is a valuable triterpenoid compound, which is widely used in various applications such as cosmetics, pharmaceuticals, and nutraceutical industries. The core source is under limited conditions, and its production needs to be considered, and alternative strategies should have been developed. Nowadays, studies are trying to improve the production by using different methods, one of the methods is a diploid engineered strain of Saccharomyces cerevisiae through targeted mutation of the ERG1 gene using the CRISPR Cas9 method. The ERG1 gene encodes squalene epoxidase, which catalyses squalene into 2,3-oxidosqualene, and the partial inhibition of that gene is expected to increase intracellular squalene accumulation. The study involves the construction of guide RNA (gRNA) and the integration fragment amplification for the ERG1 mutation. The recombinant gRNA plasmid was introduced into a diploid yeast strain carrying Cas9, and site-directed mutagenesis was done to introduce the F420I mutation in the ERG1 gene. Successful mutation was confirmed by sequencing analysis. A comparative analysis between the mutant strain and the parental strains was carried out. Squalene production was analysed through growth studies, cell lysis, extraction, and analytical techniques like Thin Layer Chromatography (TLC) and Gas Chromatography (GC). The mutant strain showed slightly higher growth compared to the parental strain and maintained sufficient viability. Qualitative and quantitative analysis showed increased accumulation of squalene in the parental strain, it is observed as higher band intensity in TLC and increased peak area in GC. Bioreactor fermentation using a fed-batch process was carried out for further improvement in biomass and squalene production over 120 hours, with efficient glucose utilization confirmed by a GOD-POD assay. Overall the results shows that CRISPR-Cas9 mediated mutation of the ERG1 gene effectively increases squalene production but the parental strain is slightly higher than the mutant. This study provides a sustainable, scalable approach to improving squalene production and underscores the importance of combining metabolic engineering with bioprocess optimization for industrial applications.