Towards diacetyl‐less brewers' yeast. Influence of ilv2 and ilv5 mutations

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Abstract
During alcoholic fermentations, the off‐flavour compound diacetyl is formed non‐enzymatically from acetolactate leaking out from the cells. Acetolactate is an intermediate in the biosynthesis of valine. In beer fermentation, the amount of diacetyl is reduced to acceptable levels during maturation. A reduction of the time needed for maturation may be achieved by the use of a brewing yeast that produces less diacetyl.Saccharomyces cerevisiae laboratory strains with an inactive ilv2 gene can not form acetolactate, while ilv5 strains, blocked in the subsequent step, leak acetolactate in high amounts.Induction of recessive mutations in production strains of Saccharomyces carlsbergensis has not yet been achieved, as the yeast is polyploid and possibly a hybrid between S. cerevisiae and another Saccharomyces species. Thus, all chromosomes investigated so far are present in at least two genetically different versions. Genetic and molecular analysis has shown that the brewing yeast is structurally heterozygous for ILV2 and ILV5.Genetic modification of brewers' yeast to reduce diacetyl formation is being carried out by mutation of ILV2. Deletion mutations in both ILV2 alleles have been constructed in vitro to be used for gene replacement in the brewing strain. In addition, partial inactivation of the ILV2 function is carried out by selecting spontaneous dominant mutations resistant to the herbicide sulfometuron methyl. Among these mutants some produce only half the amount of diacetyl compared to the parental strain.An alternative way to reduce diacetyl production might be to increase the activity of the ILV5 gene product. Model experiments in S. cerevisiae show that the presence of the ILV5 gene on a 2‐micron based multi‐copy vector can reduce the diacetyl production by half.