Comparison between CRISPR/Cas9 and episomal plasmid methods in heterologous expression of cellulases in Saccharomyces cerevisiae industrial strains

Consolidated ethanol production is the best strategy to counter the emerging fossil fuel crisis. However, no native microorganism has all the characteristics necessary for this purpose. Advances in genetic engineering, however, allow us to build a microorganism that combines the ideal traits observed in other species. The genetic editing method, the number of copies inserted and stability over the generations are essential variables in the success of the transformation. Thus, the objective of this work was to evaluate the influence of the transformation method on the expression of endoglucanase (AsENDO) and β-glycosidase (AsGLI) in Saccharomyces cerevisiae industrial strains, aiming at saccharification and fermentation of biomass. The thermotolerant and parental industry strains of PE-2 and SA-1 (diploids) were transformed by the genomic integration method via CRISPR / Cas9 and multiple copy episomal plasmid in the expression of AsENDO and AsGLI genes isolated from Acremonium strictum. The CRISPR/Cas9 method presented higher activity than the episomal pathway with maximum AsENDO and AsGLI activity of 8.05 U.mL-1 and 0.177 U.mL-1, respectively. The CRISPR/Cas9 pathway was 3-fold higher in expression of AsGLI activity than the multiple copy plasmid method. This result shows that CRISPR/Cas9 genomic integration, even in single copy, is more relevant than insertion of multiple plasmid copies. Specific culture medium for maintaining the plasmid in the cell is essential. However, during cell division, inefficient plasmid partitioning may occur between the mother and daughter cells, resulting in plasmid-free cells, which explains the lower enzyme production of the culture. While genomic integration perpetuates heterologous genes across generations without the need for a specific and more expensive medium. Therefore, the CRISPR/Cas9 method is most recommended for yeast transformation, generating strains with high protein expression and simpler maintenance bias, lower cost and greater ease in large scale applications.