A flavor compound called phenylethyl acetate, or 2-PEAc, imparts a hint of rose or honey to wherever it’s found – a dab of perfume, a sip of wine, a slug of beer. It has long been known to be produced in greater or lesser measure by different yeast strains, but exactly why some yeast strains generated more phenylethyl acetate, and produced superior alcoholic beverages, has been a mystery.
The specific yeast genes that produce higher levels of the prized flavor molecule have been identified by scientists at the Center for Microbiology at VIB, in Belgium. These scientists used “applied pooled-segregant whole-genome sequence analysis” to find four quantitative trait loci (QTLs) responsible for high production of phenylethyl acetate.
QTLs are swaths of DNA that contain multiple genes but only one causative gene. To go deeper, to identify the causative alleles, the scientists made use of CRISPR/Cas9 gene-editing technology. Details of this work appeared in the journal mBio, in an article entitled “Identification of Novel Alleles Conferring Superior Production of Rose Flavor Phenylethyl Acetate Using Polygenic Analysis in Yeast.”
Two QTLs each showed linkage to the genomes of the BTC.1D and ER18 parents. The first two loci were investigated further.
“The causative genes were identified by reciprocal allele swapping into both parents using clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9,” the article’s authors explained. “The superior allele of the first major causative gene, FAS2, was dominant and contained two unique single nucleotide polymorphisms (SNPs) responsible for high 2-PEAc production that were not present in other sequenced yeast strains. FAS2 encodes the alpha subunit of the fatty acid synthetase complex. Surprisingly, the second causative gene was a mutant allele of TOR1, a gene involved in nitrogen regulation.”
When CRISPR/Cas9 was used to swap the alleles between the parent strains, the production of phenylethyl acetate increased significantly.
“In some wines, you can smell the rose flavor above all the others,” said microbiologist Johan Thevelein, Ph.D. “But why certain yeast strains make more of this compound than other strains, there was no knowledge at all.” Thevelein led the study with Maria R. Foulquié-Moreno, Ph.D., also at VIB.
Yeast plays a critical role in shaping the flavor of a beer. During fermentation, it adds flavors and carbonation. In wine, most of the flavor comes from the grapes themselves; the metabolism of the yeast can alter those flavors, adding secondary flavors. Yeast also contributes its own flavors.
Enhancing industrial yeast strains for desirable flavors has been a challenge in the past, says Thevelein. Biologists can cross-breed strains to select for certain genes – so certain flavors – but the process is time-consuming, expensive, and may cause other unwanted changes in the yeast.
“You have to do two things,” noted Thevelein. “One is to improve the yeast trait that you want to improve. Second is to change nothing else in the yeast. In practice, the latter turns out to be much more difficult than the former.” A cross-bred yeast may work in the lab, but not in the brewery. “If the fermentation is bad, you have to throw away all the beer,” Thevelein added.
CRISPR/Cas9 offers a more efficient way to precisely engineer desirable traits in yeasts without affecting other traits, he says. In recent years, microbiologists have connected specific genes to an array of flavors, including nerolidol (a woody scent), ethyl acetate (a sweet smell, as in nail polish), and sulfur flavors. In their work at VIB, Thevelein and Foulquié-Moreno have also identified genes responsible for banana- and butter-like flavors.
CRISPR/Cas9 can add desirable genes and swap out the undesirables, usually without the time, expense, and unwelcome side effects of cross-breeding strains. “With CRISPR, we never leave a scar,” asserted Foulquié-Moreno. “Because the engineered yeast strains have alleles that have been swapped from other yeasts, they should be indistinguishable from strains produced by breeding or by mutagenesis and selection,” she said.
The researchers have partnered with a Belgian brewery to evaluate their experimental strains with several batches of beer, with hopes of clearing the highest hurdle of all: The taste test.