eGFP Gene Integration in HO: A Metabolomic Impact?

Area of Science:

• Yeast molecular biology
• Metabolomics
• Fermentation science

Background:

• Integrating fluorescent genes like enhanced green fluorescent protein (EGFP) into the yeast genome is common for cell visualization and monitoring.
• Such genomic integrations are often assumed to be phenotypically neutral.

Purpose of the Study:

• To investigate the impact of integrating an EGFP gene cassette into the *HO* gene of *Saccharomyces cerevisiae* on its exometabolome during alcoholic fermentation.
• To determine if CRISPR-Cas9 mediated integration affects cellular phenotype beyond basic growth and fermentation parameters.

Main Methods:

• CRISPR-Cas9 technology was used to integrate an EGFP gene cassette into the *HO* gene of a commercial *S. cerevisiae* strain.
• High-resolution mass spectrometry was employed to analyze the exometabolome, specifically focusing on peptide profiles.
• Phenotypic growth and fermentation parameters were monitored for wild-type and modified strains.

Main Results:

• The integration of the EGFP cassette resulted in significant changes to the exometabolome, with 41 and 82 unique biomarkers identified for the S3 and S3GFP strains, respectively.
• 28 biomarkers showed significant concentration variations between wild-type and modified strains, primarily corresponding to peptides.
• Despite similar growth and fermentation kinetics, the peptidome was demonstrably altered in the modified yeast strain.

Conclusions:

• Genomic integration of an EGFP cassette, even via CRISPR-Cas9 into the *HO* gene, is not phenotypically neutral at the exometabolome level during alcoholic fermentation.
• The study provides the first evidence that such integrations modify the yeast peptidome, impacting metabolic outputs.
• These findings highlight the importance of considering metabolic consequences when engineering yeast strains for biotechnological applications.