Abstract
Saccharomyces pastorianus is an interspecies hybrid between S. cerevisiae and S. eubayanus. The identification of the parental species of S. pastorianus enabled the de novo reconstruction of hybrids that could potentially combine a wide array of phenotypic traits. Lager yeasts are characterized by their inability to decarboxylate ferulic acid present in wort, a phenotype also known as Pof- (phenolic off-flavor). However, all known S. eubayanus strains characterized so far produce clove-like aroma specific of 4-vinyl guaiacol, a decarboxylated form of ferulic acid. This study explored a non-GMO approach to construct Pof- S. eubayanus variants derived from the parental strain S. eubayanus CBS 12357. To rapidly screen a population of UV-mutagenized cells two complementary assays were developed. The first assay was based on the difference of light absorption spectra of ferulic acid and 4-vinyl guaiacol, while the second was based on the difference of sensitivity of Pof- and Pof+ strains to cinnamic acid. The S. eubayanus variant HTSE042 was selected and was confirmed not to produce 4-vinyl guaiacol. Whole genome sequencing revealed that this variant lost the subtelomeric region of the CHRXIII right arm that carried the two clustered genes SePAD1- SeFDC1 whose deletion in a naïve S. eubayanus strain (CBS 12357/FM1318) resulted in an identical phenotype. Subsequently, the Pof- variant was crossed with a Pof- S. cerevisiae partner. The resulting hybrid was not able to convert ferulic acid demonstrating the undisputable value of the mutagenized variant HTSE042 to eventually construct S. cerevisiae × S. eubayanus hybrids with phenotypic characteristics of S. pastorianus.
Highlights
Since its discovery in Patagonia seven years ago (Libkind et al, 2011), Saccharomyces eubayanus received significant attention which to a large extent was related to its role in the reconstruction of the interspecies hybrid lager brewing yeast, S. pastorianus (Nakao et al, 2009; van den Broek et al, 2015; Okuno et al, 2016)
Yeast strain design has been enhanced by the emergence of genomeediting tools such as CRISPR-Cas9 (Jinek et al, 2012; Doudna and Charpentier, 2014), techniques that enable highly accurate genetic alterations such as gene deletion, in vivo site directed mutagenesis and novel genes chromosomal integration (DiCarlo et al, 2013; Mans et al, 2015; Jakociunas et al, 2016; de Vries et al, 2017) are available, but, consumer concerns about use of genetically modified (GM) organisms for food and beverage production preclude the use of these approaches in industry (Varzakas et al, 2007)
The single knockout strain IMK747 showed a ferulic acid consumption that was approximately half of CBS 12357, while in the double PAD1FDC1 knockout IMK749 the ferulic acid concentration dropped by only 13% (Figure 2A)
Summary
Since its discovery in Patagonia seven years ago (Libkind et al, 2011), Saccharomyces eubayanus received significant attention which to a large extent was related to its role in the reconstruction of the interspecies hybrid lager brewing yeast, S. pastorianus (Nakao et al, 2009; van den Broek et al, 2015; Okuno et al, 2016). S. eubayanus CBS 12357 is not able to consume all wort sugars, in particular maltotriose (Hebly et al, 2015; Krogerus et al, 2015), it exhibits several brewing traits suitable for lager brewing including growth at a temperature as low as 4◦C (Hebly et al, 2015), proficient maltose utilization and formation of desired aroma molecules (Mertens et al, 2015) These characteristics might be transferred to hybrids holding significant value for the brewing industry (Krogerus et al, 2017a). Yeast strain design has been enhanced by the emergence of genomeediting tools such as CRISPR-Cas (Jinek et al, 2012; Doudna and Charpentier, 2014), techniques that enable highly accurate genetic alterations such as gene deletion, in vivo site directed mutagenesis and novel genes chromosomal integration (DiCarlo et al, 2013; Mans et al, 2015; Jakociunas et al, 2016; de Vries et al, 2017) are available, but, consumer concerns about use of genetically modified (GM) organisms for food and beverage production preclude the use of these approaches in industry (Varzakas et al, 2007)
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