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Abstract
The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion (“attenuation”) of maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for maltotriose. Evolved populations exhibited an up to 5-fold lower residual maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labeled sugars revealed an up to 4.75-fold higher transport capacity for maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains.
Highlights
Over the last decades, the global beer industry has grown to reach a volume of 193 billion liters
To investigate whether the incomplete utilization of maltose and maltotriose was caused by a limiting amount of available nitrogen in the wort, additional experiments with S. pastorianus CBS1483 were performed in stirred bioreactors on synthetic medium with excess nitrogen and lower overall sugar concentration (20 g.L−1 of a sugar mixture containing 2.5% glucose, 28.0% maltose, 42.0% maltotriose and 26.4% higher dextrins)
Mutagenesis increases the likelihood of secondary mutations, which may negatively affect industrially relevant traits that are difficult to screen in high-throughput set-ups, such as the complex balance of flavor and aroma compounds in the final product
Summary
The global beer industry has grown to reach a volume of 193 billion liters (data for 2015, https://www.statista.com). In Saaz strains, a sizeable fraction of the S. cerevisiae subgenome (e.g., chromosomes (CHR) VI and VIII as well as parts of IV, XIII, and XV) is absent, whereas modern industrial Frohberg yeasts retain a near-complete set of chromosomes from both parents (Hewitt et al, 2014; Walther et al, 2014; van den Broek et al, 2015) Origins of these two lineages is still a matter of debate, some studies have advocated for two different hybridization histories (Dunn and Sherlock, 2008; Nguyen et al, 2011; Okuno et al, 2016) whereas others privileged a common primary hybridization event followed by divergent evolutionary paths (Okuno et al, 2016)
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