A single-vector CRISPR/Cas9 system for genome editing and heterologous enzyme secretion in Saccharomyces cerevisiae: a case study on pectate lyase for coffee mucilage removal.

The CRISPR/Cas9 system facilitates precise genome editing in various organisms. In this study, a single-vector CRISPR/Cas9 system was developed for Saccharomyces cerevisiae, employing a type II Cas9 enzyme from Streptococcus pyogenes and a single-guide RNA cassette targeting CAN1.Y locus on chromosome V. This system is broadly applicable across yeast strains, as it utilizes G418 selection, eliminating the need for auxotrophic markers. The efficiency of the CRISPR/Cas9 system was demonstrated, with editing efficiencies ranging from 70 to 100%. This system was utilized to integrate a cassette encoding secretory pectate lyase (PL) from Bacillus subtilis 168 into the yeast genome. The engineered S. cerevisiae strain secreted active PL, which exhibited pectin-degrading activity characterized by significant reductions in residual pectin and increased production of reducing sugars. Since pectin constitutes a major component of coffee mucilage, the secreted PL was applied to coffee beans for mucilage removal. The treated beans presented noticeably reduced residual mucilage, a purer green color, and decreased viscosity. These findings suggest the potential of the engineered S. cerevisiae strain for applications in coffee processing, particularly in efficient mucilage removal.