Development of a GMO-free de novo lager yeast with reduced phenolic off-flavor production through interspecific hybridization and UV mutagenesis.

Lager beers are traditionally brewed using Saccharomyces pastorianus, a natural hybrid between S. cerevisiae and S. eubayanus that combines robust fermentation with cold tolerance. However, commercial lager yeasts originate from a narrow genetic pool, limiting strain diversity and aromatic complexity. To meet growing demand for new lager beer profiles, the development of novel non-GMO strains is increasingly important. Many wild yeasts from non-brewing environments can ferment wort but produce phenolic off-flavor (POF+) by converting ferulic acid (FA) into 4-vinyl guaiacol (4-VG), restricting their industrial applicability. In this study, a de novo POF+ lager-like hybrid (FM1) generated by spore-to-spore mating between a monosporic culture of the sourdough strain S. cerevisiae Y15 and S. eubayanus x S. uvarum NBRC1948, was submitted to UV mutagenesis to reduce POF formation. Screening on FA-supplemented medium identified a POF-reduced mutant, FM1.45, showing lower ferulic acid conversion and higher cinnamic acid sensitivity than FM1. Wort microfermentation trials demonstrated that FM1 and FM1.45 both outperformed the parental strains in fermentation kinetics and produced similar aroma profiles, with FM1.45 generating substantially less 4-VG than FM1. Sanger sequencing results were consistent with a loss of the S. eubayanus PAD1-FDC1 gene cluster and a frameshift mutation in the S. cerevisiae FDC1 gene, leading to a non-functional ferulic acid decarboxylase. Flow cytometry supported a ploidy increase from 3n to 4n in FM1.45, which likely accounts for the restored spore viability observed in this mutant compared with the sterile FM1. Overall, this work demonstrates that combining interspecific hybridization with UV mutagenesis represents an effective GMO-free strategy for generating novel lager yeast strains with robust fermentation performance and reduced POF production.