Identification and Characterization of a Novel Target for Diastatic Activity in Saccharomyces cerevisiae

Diastatic strains of Saccharomyces cerevisiae are major contaminants in industrial brewing fermentations.  They pose serious risks throughout the brewing process and especially post-packaging where super-attenuation and CO₂ production can lead to packaging failures.

Diastatic activity has been ascribed to the STA gene family, and Saccharomyces cerevisiae var diastaticus, is largely defined by the presence of the STA1 gene.  For this reason, many commercial diagnostic assays target STA1.  Unfortunately, STA1 genotype does not always accurately predict the diastatic spoilage potential (phenotype) of the yeast. As observed in this study, STA1 negative strains can potentially exhibit diastatic activity, and conversely, some STA1 positive strains lack a diastatic phenotype. 

In this study, we utilized two parallel sequencing strategies, Illumina short reads and Oxford Nanopore Technologies (ONT) long reads to investigate whether a novel genetic marker could be discovered that more accurately reflects the diastatic spoilage phenotype of yeast. 60 strains of S. cerevisiae encompassing both diastatic and non-diastatic strains were sequenced in this manner. Through comparative genomic analysis, we identified a novel genetic marker positively correlated with the diastatic phenotype, as defined by a bromocresol green maltodextrin (BGM) broth assay.

We developed a polymerase chain reaction (PCR) assay targeting the novel genetic region and assessed its functionality by testing 83 brewing yeast isolates. PCR results were compared with those obtained through the BGM phenotypic assay.  The parity of the results from the two methods in tandem with STA 1 genetic detection, demonstrates the reliability of the PCR assay to detect true diastatic potential, particularly in strains where STA genotype was not predictive of diastatic activity.

To demonstrate the utility of this PCR system as a viable diagnostic tool, we analyzed a yeast strain, isolated from a spoilage event in a German brewery.  This strain lacked the traditional STA1 gene target despite being implicated in a super-attenuation spoilage event.  The strain was tested in the laboratories of bioMérieux, and it was determined to be positive for both the novel target and demonstrated diastatic activity in the BGM phenotypic assay.

This research unveils a previously unrecognized marker for diastatic activity in S. cerevisiae that reliably predicts spoilage phenotype when tested in parallel with STA family targets. The development of a diagnostic PCR assay employing this novel target enhances the efficiency and accuracy of diastatic activity detection, facilitating improved quality control measures in brewing and fermentation industries. These findings contribute to our understanding of S. cerevisiae biology and its implications for industrial applications.