In hops and malt, thiols often exist in a bound form, typically conjugated with molecules e.g. glutathione or cysteine. The term conjugate in the context of thiol metabolism refers to the bound form of the thiol, where the thiol is chemically attached to another molecule. The thiol itself is a sulfur-containing compound, and when it binds to these other molecules, it forms a thiol conjugate. In their bound form, these thiols are non-volatile and odourless, meaning they don’t contribute to beer's aroma until they are released or separated from their conjugates. When we refer to precursors in the context of thiol metabolism, we are generally talking about the conjugates, because they are the preliminary forms that will release active thiols after enzymatic cleavage. Table 1 summarizes the most common thiol precursors.
The process of releasing these molecules from their bound forms is an intracellular process. Once internalized, intracellular enzymes convert these thiol precursors into their active, odorant forms. This means yeast cells must first take up these precursors, and if the process of internalization is inefficient, these molecules won’t be available for metabolism
This process is mainly governed by membrane transport proteins that allow specific molecules, such as cysteine- or glutathione-bound thiols (non-odorant precursors), to cross the yeast cell membrane. Several factors influence this transport:
1. Transporter Proteins: Yeast cells use specific amino acid transporters (like permeases and peptide transporters) to internalize these precursors. The availability and activity of these transporters depend on the yeast strain and its growth conditions.
2.pH of the Medium: External pH affects the charge and solubility of non-odorant precursors, impacting how they are recognized and transported by specific membrane transporters.
3. Yeast Metabolic State: Yeast cells express different levels of transport proteins depending on their growth phase (e.g., exponential growth vs. stationary phase). For example, during active fermentation, yeast cells upregulate transporter proteins to meet the high demand for amino acids and precursors
The IRC7 gene in Saccharomyces cerevisiae (brewer's yeast) has two common allele variants: IRC7S (short variant) and IRC7L (long variant). These variants differ in their sequence length, enzymatic activity, and their ability to release volatile thiols during fermentation, which affects the aroma profile of beer and wine.
Gene Length:
IRC7S (Short Variant): This is a truncated form of the IRC7 gene with a deletion in part of the coding sequence. This deletion results in a shorter protein lacking functional regions needed for full enzymatic activity.
IRC7L (Long Variant): The full-length version of the gene encodes the complete β-lyase enzyme, containing all necessary domains for full enzymatic function, including the cleavage of non-odorant thiol precursors.
The sequences of the two variants (IRC7s and IRC7L) were amplified through PCR, this technique is used to make millions to billions of copies of a specific DNA segment, enabling the gene to be studied and manipulated.
Expected Sizes:
IRC7L: The expected PCR product for the long allele is around 1,498 bp to 1,928 bp.
IRC7S: The expected PCR product for the short allele is around 1,462 bp to 1,896 bp, but shorter than IRC7L due to the truncation.
Genetic characterization was done on AEB yeast strains to correlate the presence of the IRC7 gene and β-lyase activity (CSL).
The technique used to separate molecules (e.g. DNA, RNA, or proteins) based on their size and charge. In our case PCR products (DNA fragments) based on their size, allowing the identification of different IRC7 gene alleles (long and short variants).
Lanes: Each lane on the gel represents a different yeast strain or control (e.g., molecular size ladder and negative control).
M (Marker): The molecular size ladder, which contains fragments of known lengths, is used as a reference to estimate the size of PCR products.
The PCR products are run in parallel lanes, with the size of the resulting DNA fragments showing whether the yeast strain carries the long or short variant of the IRC7 gene.
Visibility: DNA samples (in this case, PCR products) are mixed with a loading dye (a coloured solution that makes it easier to track the movement of the DNA) and are loaded into small wells at one end of the gel.
An electric field is applied across the gel by connecting the chamber to a power supply. The negatively charged DNA fragments move towards the positive electrode (anode) because DNA carries a negative charge due to its phosphate backbone.
The rate at which the DNA fragments move through the gel depends on their size. Smaller fragments move faster and migrate farther through the gel, while larger fragments move more slowly and do not travel as far.
Long Variant (IRC7L): Yeast strains carrying the long allele will show a PCR product of a larger size (approximately 1,500 base pairs (bp) or more), indicating the full-length IRC7 gene is present. The full-length allele produces a functional enzyme with high β-lyase activity, resulting in significant thiol release and the production of desirable fruity and tropical aromas in beer and wine.
Short Variant (IRC7S): Yeast strains carrying the short allele will show a PCR product of smaller size (around 1,000 bp or less), indicating that the IRC7 gene is truncated. This truncated allele produces an enzyme with little to no β-lyase activity, leading to low thiol release and a more muted aroma in fermentation products.
Ana Victoria Vasquez de la Peña
ana@neumaker.com.au
7 September 2024