Denzel G L Ignacia

Flocculation is a key technological trait in lager brewing, governing fermentation performance, yeast recovery, and beer quality. In the allo-aneuploid hybrid yeast Saccharomyces pastorianus , the genetic basis of flocculation remains poorly resolved due to its complex dual sub-genome architecture. Here, we systematically re-annotated and functionally characterized the complete FLO gene repertoire of the Group II strain CBS 1483. Thirteen FLO genes were identified, including allelic variants and a previously uncharacterized adhesin, Flo12, containing a Hyphal_reg_CWP domain instead of the canonical PA14 lectin-binding domain. Structural modeling revealed strong conservation of Ca² ⁺ -binding residues in PA14 domains, alongside repeat-region diversification likely contributing to functional variability. Using optogenetic expression in a FLO -null background, we demonstrated that Spc I- FLO9 -1 and Spc I- FLO9-2 _1 are the strongest drivers of flocculation, exhibiting NewFlo-like sugar sensitivity. Transcriptomic analysis during 17°P wort fermentation showed dynamic induction of these genes coinciding with flocculation onset. Surprisingly, deletion of both loci in CBS 1483 did not abolish but only delayed sedimentation in wort, accompanied by improved maltose utilization and attenuation. These findings reveal functional redundancy and compensatory mechanisms within the FLO network of lager yeast, highlighting the genetic complexity underlying flocculation, and providing a molecular framework to inform yeast selection, strain development, and optimization of the lager fermentation processes. IMPORTANCE: Flocculation, the process by which yeast cells aggregate and settle, is essential for producing clear, high-quality lager beer, and for efficient yeast recovery during brewing. However, the genetic basis of this trait in lager yeast has remained poorly understood because these strains possess unusually complex hybrid genomes. In this study, we systematically identified and characterized the complete set of flocculation genes in the industrial lager yeast Saccharomyces pastorianus CBS 1483. We demonstrated that lager yeast flocculation is not controlled by a single dominant gene, but instead emerges from the combined action of several moderately active adhesion proteins that are expressed at low levels during fermentation. Surprisingly, deleting the two strongest candidate genes only delayed, rather than eliminated, sedimentation, revealing a robust compensatory network that preserves brewing performance. These findings refine the current understanding of yeast flocculation and provide a molecular framework for developing brewing strains with improved fermentation efficiency, product consistency, and flavor quality.

Flocculation in Saccharomyces cerevisiae is a critical phenotype with ecological and industrial significance. This study aimed to functionally dissect the contributions of individual FLO genes (FLO1, FLO5, FLO9, FLO10, FLO11) to flocculation by employing an optogenetic circuit (OptoQ-AMP5) for precise, light-inducible control of gene expression. A FLO-null platform yeast strain was engineered allowing the expression of individual FLO genes without native background interference. Each FLO gene was reintroduced into the FLO-null background under the control of OptoQ-AMP5. Upon light induction, strains expressing FLO1, FLO5, or FLO10 demonstrated strong flocculation, with FLO1 and FLO5 forming large and structurally distinct aggregates. FLO9 induced a weaker phenotype. Sugar inhibition assays revealed distinct sensitivities among flocculins, notably FLO9’s novel sensitivity to fructose and maltotriose. Additionally, FLO-induced changes in cell surface hydrophobicity were quantified, revealing that FLO10 and FLO1 conferred the greatest hydrophobicity, correlating with their aggregation strength. This work establishes a robust platform for investigating flocculation mechanisms in yeast with temporal precision. It highlights the phenotypic diversity encoded within the FLO gene family and their differential responses to environmental cues. The optogenetic system provides a valuable tool for both fundamental studies and the rational engineering of yeast strains for industrial fermentation processes requiring controlled flocculation.