I’ve been researching diacetyl formation and removal by brewing yeasts the past year(s), and we recently did some screening of various lager yeast (Saccharomyces pastorianus) strains for diacetyl production and subsequent transcriptional analysis of key genes in the isoleucine-valine catabolic pathway (diacetyl is formed as an indirect by-product of valine biosynthesis in yeast; more information available here). We did this in order to identify what genes contribute most to the differences in wort total diacetyl concentration we observed between the different strains. We observed that particularly the ILV6 gene, encoding for a regulatory subunit of the acetohydroxy acid synthase enzyme (responsible for converting pyruvate into the diacetyl precursor α-acetolactate), showed greater expression early during fermentation in a strain producing more diacetyl. To confirm the role of this ILV6 gene in controlling α-acetolactate/diacetyl formation, we overexpressed both the S. cerevisiae- and S. eubayanus-form of ILV6 in our standard production lager strain A15, and found that overexpression of either form of ILV6 resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. These results suggest that both forms of the gene influence diacetyl formation, and different levels of transcription (this could be a result of a gene-dosage effect, since different lager yeast hybrids have inherited different amounts of the parental genomes; for a recent review on the subject click here) may contribute to differences in diacetyl production in various lager yeast strains.
Link to the publication: http://onlinelibrary.wiley.com/doi/10.1002/yea.3026/abstract
E-mail me if you are interested in the article, but don’t have access to the full-text.
A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer’s wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation. Transcriptional analysis of key genes involved in valine biosynthesis showed differences between the two strains that were consistent with the differences in wort diacetyl concentration. In particular, the ILV6 gene, encoding a regulatory subunit of acetohydroxy acid synthase, showed early transcription (only 6 h after inoculation) and up to five-fold greater expression in W34 compared to A153. This earlier transcription was observed for both orthologues of ILV6 in the S. pastorianus hybrid (S. cerevisiae × S. eubayanus), although the S. cerevisiae form of ILV6 in W34 also showed a consistently higher transcript level throughout fermentation relative to the same gene in A153. Overexpression of either form of ILV6 (by placing it under the control of the PGK1 promoter) resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. The results confirm the role of the Ilv6 subunit in controlling α-acetolactate/diacetyl concentration and indicate no functional divergence between the two forms of Ilv6. The greater contribution of the S. cerevisiae ILV6 to acetolactate production in natural brewing yeast hybrids appears rather to be due to higher levels of transcription relative to the S. eubayanus form.