Desenvolvimento de linhagens de Saccharomyces cerevisiae com fenótipo POF (Phenolic Off-Flavor) negativo por edição do gene FDC1 via sistema CRISPR/Cas9

Abstract

Beer production relies on alcoholic fermentation carried out by Saccharomyces cerevisiae yeasts, which synthesize primary metabolites and secondary compounds responsible for the beverage’s aroma and flavor. Some of these compounds are volatile phenolics, known as Phenolic Off-Flavor (POF), formed through the decarboxylation of phenolic acids, a process mediated by the genes PAD1 (YDR538W) and FDC1 (YDR539W). The PAD1 gene activates FDC1, which encodes the enzyme feruloyl-CoA decarboxylase (FDC), responsible for catalyzing the conversion of phenolic acids into volatile derivatives, such as 4-vinylguaiacol (4-VG), whose phenolic aroma can be undesirable in certain beer styles. The aim of the present study was to develop POF-negative strains through genetic editing of the FDC1 gene, disrupting the 4-VG metabolic pathway. This goal was achieved by editing two POF-positive strains: WB06, widely used in the brewing industry, and LBGA 287, isolated from ethanol production processes and with potential application in brewing. The strategy employed involved the CRISPR/Cas9 system with a synthetic donor containing nucleotides that introduced an XbaI restriction site into this gene. After transformation, colonies from each strain were selected through molecular tests (PCR and Sanger sequencing) and olfactory sensory analysis, confirming the desired edition and the expected new POF-negative phenotype. Following plasmid removal, beers were produced at a laboratory scale, confirming the sensory characteristics during alcoholic fermentation. These results demonstrated the feasibility of yeast genetic editing to eliminate undesirable phenolic compounds, contributing to the production of beer with controlled aroma and improved sensory consistency. Thus, it was possible to generate POF-negative yeasts through FDC1 gene editing.