A deletion in theSTA1promoter determines maltotriose and starch utilization inSTA1+Saccharomyces cerevisiaestrains

Abstract

12Diastatic strains of Saccharomyces cerevisiae are common contaminants in beer fermentations and 13 are capable of producing an extracellular STA1-encoded glucoamylase. Recent studies have 14 revealed variable diastatic ability in strains tested positive for STA1, and here we elucidate genetic 15 determinants behind this variation. We show that poorly diastatic strains have a 1162 bp deletion in 16 the promoter of STA1. With CRISPR/Cas9-aided reverse engineering, we show that this deletion 17 greatly decreases the ability to grow in beer and consume dextrin, and the expression of STA1. New 18 PCR primers were designed for differentiation of highly and poorly diastatic strains based on the 19 presence of the deletion in the STA1 promoter. In addition, using publically available whole genome 20 sequence data, we show that the STA1 gene is prevalent in among the 'Beer 2'/'Mosaic Beer' 21 brewing strains. These strains utilize maltotriose efficiently, but the mechanisms for this have been 22 unknown. By deleting STA1 from a number of highly diastatic strains, we show here that 23 extracellular hydrolysis of maltotriose through STA1 appears to be the dominant mechanism 24 enabling maltotriose use during wort fermentation in STA1+ strains. The formation and retention of 25 STA1 seems to be an alternative evolutionary strategy for efficient utilization of sugars present in 26 brewer's wort. The results of this study allow for the improved reliability of molecular detection 27 methods for diastatic contaminants in beer, and can be exploited for strain development where 28 maltotriose use is desired. 29 the SGA1-derived peptide, while the FLO11-derived peptide allows for extracellular secretion of the 49 protein (Adam et al., 2004). The upstream sequences of STA1 and FLO11 are also nearly identical, 50 meaning that these genes are largely co-regulated (Gagiano et al., 1999). 51Current detection methods for diastatic S. cerevisiae rely mainly on either the microbiological 52 detection through culturing on specialized selective growth media, or the molecular detection of the 53 4 STA1 gene through conventional or quantitative PCR (Brandl, 2006;Meier-Dörnberg et al., 2018; 54 van der Aa Kühle, 1998;Yamauchi et al., 1998). The main weakness of the microbiological 55 methods is that they are time-consuming. While the molecular methods are rapid, a recent study 56 (Meier-Dörnberg et al., 2018) has revealed that there is considerable variability in diastatic ability 57 and beer-spoilage potential in strains carrying the STA1 gene. In fact, some strains that carry the 58 STA1 gene do not show spoilage potential, and these benign strains would be erroneously flagged as 59 diastatic with the current molecular methods. 60In this study, we examined the diastatic ability of a range of STA1+ S. cerevisiae strains. We also 61 sequenced the open reading frame and upstream sequence of STA1 in these same strains to search 62 for polymorphisms that might explain the variable diastatic ability. Sequencing revealed that the 63 poorly dias...