Diacetyl control during brewery fermentation via adaptive laboratory engineering of the lager yeast Saccharomyces pastorianus

Abstract

Diacetyl contributes to the flavor profile of many fermented products. Its typical buttery flavor is considered as an off flavor in lager-style beers, and its removal has a major impact on time and energy expenditure in breweries. Here, we investigated the possibility of lowering beer diacetyl levels through evolutionary engineering of lager yeast for altered synthesis of α-acetolactate, the precursor of diacetyl. Cells were exposed repeatedly to a sub-lethal level of chlorsulfuron, which inhibits the acetohydroxy acid synthase responsible for α-acetolactate production. Initial screening of 7 adapted isolates showed a lower level of diacetyl during wort fermentation and no apparent negative influence on fermentation rate or alcohol yield. Pilot-scale fermentation was carried out with one isolate and results confirmed the positive effect of chlorsulfuron adaptation. Diacetyl levels were over 60% lower at the end of primary fermentation relative to the non-adapted lager yeast and no significant change in fermentation performance or volatile flavor profile was observed due to the adaptation. Whole-genome sequencing revealed a non-synonymous SNP in the ILV2 gene of the adapted isolate. This mutation is known to confer general tolerance to sulfonylurea compounds, and is the most likely cause of the improved tolerance. Adaptive laboratory evolution appears to be a natural, simple and cost-effective strategy for diacetyl control in brewing.

Keywords: Beer; Chlorsulfuron; Diacetyl; Saccharomyces pastorianus; α-Acetolactate.

Fig. 1

Emergence of visible colonies from control and chlorsulfuron-exposed populations of the A15 lager yeast after spreading cells on agar plates supplemented with 500 mg l⁻¹ chlorsulfuron. Populations included a control population after EMS treatment, but not previously exposed to chlorsulfuron (T0), and populations exposed to 15 or 30 serial transfers to liquid media containing 100 mg l⁻¹ chlorsulfuron, T15 and T30, respectively. The number of new colonies emerging each day is expressed as a percentage of the total number of visible colonies after 10 days. Results are averages from three replicate agar plates each producing at least 120 visible colonies on day 10. Asterisk denotes significant difference (p < 0.01) at this time point as determined by unpaired, two-tailed Student’s t test

Fig. 2

Preliminary assessment of alcohol evolution and VDK level during fermentation of all-malt 15°P wort with the lager yeast A15 and chlorsulfuron-adapted isolates of A15. Concentration of the vicinal diketones diacetyl and pentanedione were measured in samples taken 72 h following inoculation, as indicated in the upper panel. Values are from single fermentations. VDK data are means of three technical replicates and error bars indicate standard deviation

Fig. 3

Alcohol content, total diacetyl and total 2,3 pentanedione concentrations during fermentation of 15°P all-malt wort at 30-l scale with the lager strain A15 (open symbols) and an A15-derived, chlorsulfuron-adapted variant (Isolate 8; closed symbols). Values are means of two replicates and error bars where visible indicate the range about the mean

Fig. 4

Genome coverage of the A15 lager strain (above) and A15 variant Isolate 8 (below) showing the contribution of individual chromosomes to the genome and the changes occurring after adaptation for lowered diacetyl production