Abstract
Terpene flavor compounds are an important target for food improvement in a number of areas. One such area is the brewing of beer, particularly newer “craft” beers that feature large quantities of newer American varieties of hops bred for their aroma and flavor characteristics rather than their bittering properties. Beer fermentation by brewer’s yeast (Saccharomyces cerevisiae) results in changes to the profile of terpene compounds extracted from hops in a number of ways including degradation of flavor-active compounds, transformation between different classes, and hydrolysis of glycosidically bound precursors to make them flavoractive. All of these processes present targets for modification of yeast to change the profile of flavor compounds in finished beer. Genetically modifying yeast for a food product is problematic due to EU regulations on such products but by using socalled “selfcloning” techniques to transfer sequences from one region of the yeast genome to another (i.e. cisgenesis), we can make useful modifications without creating an organism that is considered a “GMO” by current regulation. Our project will be composed of a laboratory component involving molecular biology for the creation of new yeast strains and gas chromatography combined with mass spectrometry (GCMS) to analyze terpene profiles, a modeling and data analysis component in which we try to understand how terpene profiles influence flavor perception, and a legal component in which we attempt to create a legal pathway for bringing selfcloning yeast to the home brewing community.
Outcomes and progress:
Sequencing and cloning of exg1 and regulatory regions from homebrew strains
- We purchased homebrew yeast strains WLP001 (California Ale Yeast), WLP002 (English Ale Yeast), and WLP500 (Monastery Ale Yeast). We performed DNA extractions from these strains and sequenced the WLP500 strain at the exg1 locus. We found the sequence to be nearly identical to the published S. cerevisiae lab strain genome so primers designed against published sequence were likely to be valid in homebrew strains. We PCR-amplified the regulatory regions (~1kb upstream of the start codon of exg1) from each of the homebrew strains and cloned them upstream of mCherry fluorescent protein to create reporter constructs. We were hoping to find a diversity of regulatory sequence from which to choose variants for differing levels of Exg1 expression but sequences from WLP001 and WLP002 were identical (and identical to the published sequence) while WLP500 contained a total of 5 single nucleotide polymorphisms with respect to the other sequence within the 1kb cloned. These regulatory regions were therefore not likely to be useful as reporter constructs as their expression levels were likely to be very similar.
- Development of a fluorescence assay for Exg1 activity. Exg1 is an exo-beta-1,3-glucanase enzyme and as such should be secreted into growth media where its activity can be measured during growth of cultures. We developed a platereader-based assay using 4-methylumbelliferyl-beta-d-glucoside (4-MUG) as a fluorescent substrate to monitor the activity of extracellular glucanase activity in growing cultures of the three homebrew yeast strains. Consistent with the sequencing data, we did not observe measurable differences between the levels of fluorescence produced by the different strains.
Future work
We have performed the groundwork for analysing exg1 expression in homebrew strains of yeast but did not move forward into the engineering phase as planned as there was no evidence of differences in expression in the strains we analysed. Obviously this was a very small-scale pilot and future work would require casting a wider net for source material from which to perform ‘self-cloning’ transfers of genetic material. Another possibility is to expand into other genera such as Brettanomyces, which is reported to have higher glucanase activity. Utilizing CRISPR-mediated homologous recombination would provide a way of inserting genetic material without having to use selection constructs.
Outputs
Reporter constructs containing exg1 regulatory regions fused to mCherry and all sequencing data can be made available on request from Paul Grant (pg384@cam.ac.uk).