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 DOI
Linda Lam,

Nguyen Huynh Ha Nhi,

Vo Thi Hoang Lan

et al.

Published: April 7, 2025

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

Language: Английский

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 DOI
Linda Lam,

Nguyen Huynh Ha Nhi,

Vo Thi Hoang Lan

et al.

Published: April 7, 2025

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

Language: Английский

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