Novel Food Information: Saccharomyces cerevisiae (brewer's yeast) strain BY-989

Background

Health Canada has notified Berkeley Fermentation Science Inc. that it has no objection to the food use of genetically modified Saccharomyces cerevisiae (brewer's yeast) strain BY-989 to impart tropical fruit flavours and aromas in beer by converting flavourless conjugated thiols naturally present in barley and hops into free thiols. Liquid and dried commercial preparations of this strain will be sold as Tropics London and Dry Tropics London, respectively, and are intended to replace yeast from other available commercial sources in the fermentation of beer.

Health Canada conducted a comprehensive assessment of these products according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel characteristics.

The following provides a summary of the notification from Berkeley Fermentation Science Inc. and the evaluation conducted by Health Canada. This document contains no confidential business information.

Introduction

Strain BY-989 was genetically modified to express a protein engineered carbon-sulfur lyase (CSL) enzyme from Citrobacter freundii. In beer produced using strain BY-989, the fruity flavours come from the presence of 3-mercaptohexanol (3MH) and 3-mercaptohexyl acetate (3MHA). The precursor glutathione-3-mercaptohexan-1-ol (Glut-3MH) is converted into S-3-(hexan-1-ol)-L-cysteine (cys-3MH) by native transpeptidase enzymes, which is then converted into 3-mercaptohexanol (3MH) by native and recombinant CSL enzymes. The resulting 3MH is finally converted into 3MHA by native alcohol-O-acyltransferase enzymes.

Although brewer's yeast endogenously expresses two CSL enzymes, these are inefficient at converting precursors into 3MHA. In contrast, the recombinant CSL expressed by strain BY-989 significantly increases 3MHA concentration in beer compared to the commercially available brewing yeast strain used for its development.

The Food and Nutrition Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from S. cerevisiae strain BY-989 preparations are considered novel foods under the following part of the definition of novel foods:

"c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that

(iii) one or more characteristics of the plant, animal or microorganism no longer fall within the anticipated range for that plant, animal or microorganism."

Development of the Modified Microorganism

The bacterial species used as donor organism, C. freundii, is commonly found in the gastrointestinal tract of healthy humans. This species belongs to the C. freundii complex, which includes members identified as emerging and rare opportunistic nosocomial pathogens. The clinical importance of members of the C. freundii complex comes less from causing unique diseases and more from their moderate potential to develop resistance during treatment. However, the C. freundii CSL encoding sequence was synthetic, and no other DNA from the donor organism was used in the development of strain BY-989. This approach ruled out the possibility that genes of concern from C. freundii could be introduced into strain BY-989.

The wildtype CSL enzyme from C. freundii possesses both carbon-sulfur lyase and tryptophanase activities. The latter enzymatic activity results in trace levels of indole, which is considered safe for human consumption. For example, wine typically contains 10 μg/L of indole. The CSL coding sequence integrated in strain BY-989 has five amino acid changes compared to its wildtype counterpart sequence, and their collective effect is to reduce the tryptophanase activities while increasing specificity toward cys-3MH. The company demonstrated that beer produced using strain BY-989 contains levels of indole similar to beer made using the unmodified host strain, London Ale Yeast (LAY), which is a commercially available brewer's yeast strain widely used in the manufacturing of London ale.

S. cerevisiae strain BY-989 was developed using a single genomic integration using gene editing technology. The host strain LAY was co-transformed with the linear CSL expression cassette fragment and a co-transformation plasmid containing an endonuclease and a guide RNA to mediate targeted genomic integration of the CSL cassette at the desired locus. The co-transformation plasmid also contained geneticin and kanamycin resistance genes. The kanamycin resistance gene was used as part of the co-transformation plasmid construction while the geneticin resistance gene was used in the selection of successful transformants. The co-transformation plasmid was designed so that it would not integrate into the transformed cell genome.

Following transformation, cells were plated on a geneticin-containing medium to select for transformants and PCR analysis served to screen for transformants containing the intact insert at the desired locus. Passage of chosen transformants on non-selective media led to the loss of the co-transformation plasmid. The chosen isolate confirmed to contain the CSL expression cassette at the targeted locus and lack the co-transformation plasmid, demonstrated by sensitivity to geneticin, was designated as strain BY-989.

Characterization of the Modified Microorganism

The petitioner characterized strain BY-989 using a combination of PCR, amplicon sequencing, as well as whole genome sequencing (WGS), BLAST searches, and open reading frames (ORF) analysis.

Together, these analyses demonstrated that the intact CSL expression cassette was integrated into the genome at the intended location. A single nucleotide change was observed at the 5' end of the promoter sequence; however, this was not expected to pose a safety concern. As strain LAY is tetraploid (4n), the company also confirmed that the insert integrated as expected across all four homologous chromosomes. The insertion site was in an intergenic region such that native genes were not disrupted. The ORF analysis confirmed the absence of unintended putative proteins resulting from the insert's integration. No off-target edits were identified through means of WGS analysis and subsequent BLAST search.

The stability of the integrated genetic elements was determined by serially passaging the strain BY-989 on yeast peptone dextrose (YPD) medium to achieve over 100 doubling events, following which the location, orientation, and arrangement of the integration sites were confirmed to be unchanged. These results confirmed the genetic stability of the cassette insert contained in strain BY-989.

The growth characteristics of strains LAY and BY-989 were compared in terms of cell density and metabolic activity (input consumption and ethanol production) during a typical production run. Both strains exhibited similar growth performance. As part of organoleptic analysis, beer brewed using strain BY-989 was reportedly associated with tropical and fruity aromas and flavours compared to LAY, suggesting that the intended phenotype was produced as expected. Taken together, these data demonstrate the phenotypic stability of the characteristics developed in strain BY-989.

Based on the information provided, the Bureau of Microbial and Toxicological Food Safety does not have safety concerns regarding S. cerevisiae strain BY-989 from a molecular standpoint.

Product Information

The liquid and dried commercial preparations are produced through submerged fermentation of a pure culture of strain BY-989 under controlled conditions. Following the fermentation, the biomass is separated from the growth medium resulting in the liquid preparation. For the dried preparation, dewatering and drying steps are applied. The petitioner indicated that a good manufacturing process and a hazard analysis and critical control points (HACCP) plan are employed during the entire manufacturing process.

Dietary exposure

During brewing, yeast naturally settles following fermentation and is removed through standard processes such as racking. Consequently, only trace amounts remain in the final consumed beer product. Overall, exposure to strain BY-989 is expected to be minimal and comparable to that associated with conventional brewer's yeast.

Chemistry

The petitioner provided specifications for lead in strain BY-989 brewer's yeast indicating that the ingredient will meet the lead specification of no more than 1 ppm for dried yeast (brewer's yeast) established in the Food Chemicals Codex (FCC). Additionally, the estimated concentrations of lead in beer resulting from the use of this yeast product under the proposed conditions would comply with Health Canada's maximum level of 0.2 ppm lead in beverages, as consumed, as set out in the List of Contaminants and Other Adulterating Substances.

Using the provided specification values, the Food Contaminants Section (FCS) determined that the proposed use of strain BY-989 is not expected to result in increased dietary exposure to lead. On this basis, the FCS is of the opinion that strain BY-989 is not expected to pose a food safety concern from a chemical contaminants perspective.

A list of substances used during the manufacturing of S. cerevisiae strain BY-989 was provided by the petitioner. All substances used in the manufacture of S. cerevisiae strain BY-989 were stated by the petitioner to be of food-grade quality and meet their respective specifications set out in the most recent edition of either the FCC and/or the Combined Compendium of Food Additive Specifications established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA).

Ingredients of the fermentation media are not typically regulated as food additives and usually there is no requirement under the Food and Drug Regulations for their pre-market review. Based on information provided as part of the submission, substances from the growth and fermentation media are not expected to remain in the final product.

According to the petitioner, sorbitan monostearate is added to the yeast at the level of 0.8-1.2% during drying where it functions as an emulsifier and contributes to maintaining yeast viability during drying and rehydration. Sorbitan monostearate is a permitted emulsifying, gelling, stabilizing or thickening agent for use in dried yeast at a maximum level of use of 1.5%.

The petitioner provided the levels of 3MH and 3MHA in beer made with S. cerevisiae strain BY-989 in comparison to their levels in commercial beer and wine, produced using conventional yeast strains. The data provided show that the levels of 3MH and 3MHA imparted to beer through the use of S. cerevisiae strain BY-989 are within the range of levels in beer and wine produced by conventional strains and are within the ranges known for these substances when used as flavourings in food. The FAS does not have any concerns with the levels of 3MH and 3MHA produced in beer from the use of S. cerevisiae strain BY-989, as provided in the submission. In Canada, flavouring substances are generally not subject to premarket approval. Nevertheless, the use of food flavouring substances, as with any ingredient used in food manufacture, must not result in a violation of Section 4 of the Food and Drugs Act.

The manufacturer and distributor are responsible for ensuring that any substance used in the manufacture of this yeast complies with all regulatory requirements. This includes ensuring that food additives used in the manufacture of the yeast are in compliance with provisions set out in the Food and Drug Regulations and in the Lists of Permitted Food Additives; ensuring that all materials used in the manufacture of the yeast are of food-grade quality, or equivalent, and meet appropriate food-grade specifications. As with any food or food ingredient sold in Canada, it is the responsibility of the petitioner to ensure that its use does not result in a violation of Section 4 of the Food and Drugs Act.

The Bureau of Chemicals Safety has not identified any chemical food safety concerns with the proposed uses of S. cerevisiae strain BY-989 brewer's yeast.

Microbiology

The petitioner established different microbiological specifications for the liquid and powdered formats of BY-989. The liquid preparation must contain > 95 % viable cells and contain no detectable bacteria or wild yeast per 10⁶ cells of the preparation. For the powdered preparation, viability is > 5x10⁹ CFU/g, Escherichia coli, Staphylococcus aureus, Salmonella spp., and Listeria monocytogenes must all be absent in 25 g samples, and wild yeasts, lactic acid bacteria, and acetic acid bacteria must be < 10⁴ CFU/g. Internationally recognized methods were used to verify compliance with established microbiological specifications.

BY-989 is currently manufactured in a liquid format in the U.S. The petitioner indicated that they have recently expanded their product line to include a dried format with only one batch being produced so far. As a result, the company provided certificates of analysis for three non-consecutive batches of the liquid preparation and one batch of the powdered preparation.

Based on the test results provided, the microbiological specifications set for the liquid format were met across multiple non-consecutive batches, supporting the consistent production of a microbially safe product. Results for a single batch of the powder format were also compliant with the established specifications. Testing a single batch of the dried preparation is not a safety concern considering that the manufacturing steps differentiating the liquid preparation from its dried equivalent are not expected to introduce microbiological hazards and food grade ingredients are used. Further, the petitioner confirmed only batches that meet established specifications will be released for sale.

Based on the information provided, the Bureau of Microbial and Toxicological Food Safety does not have safety concerns regarding S. cerevisiae strain BY-989 from a microbiological standpoint.

Nutrition

Strain BY-989 can be used in the same way as any commercial brewing yeast and may substitute for any S. cerevisiae strain currently used in beer fermentation.

Beer is generally not consumed for its nutritional value. Therefore, from a nutritional standpoint, it was not deemed necessary to require compositional data that compare beer made with BY- 989 with beer made with traditional brewer's yeast.

The substrate (barley and hops), the yeast and enzyme (naturally occurring in the gastrointestinal system of most humans), and the reaction products are all commonly consumed as part of the diet, and thus raise no nutritional concerns.

The Bureau of Nutritional Sciences did not identify any nutritional concerns with the proposed use of S. cerevisiae strain BY-989 to increase thiol concentrations in finished beer.

Toxicology

No toxicity or clinical studies of the novel food S. cerevisiae strain BY-989 were submitted. However, a weight of evidence approach was considered based on the following evidence.

The host strain (London Ale Yeast) has a history of safe food use from decades of use in commercial beer production. With regards to the donor microorganism, the Pre-market Toxicology Assessment Section (PTAS) considered that the C. freundii CSL encoding sequence was synthetically synthesized, and no other DNA from the donor organism was used in the development of strain BY-989, hence ruling out the risk of genes of concerns from C. freundii being carried over into the genome of strain BY-989.

Additionally, the CSL protein does not have amino acid homology with known toxins based on an acceptable in silico analysis. Also, the CSL protein is not expected to be present at a significant level in the finished beer because the protein is produced intracellularly and most yeast will be removed from beer as part of the standard brewing process.

For all those reasons, the absence of toxicity studies in this case was deemed acceptable.

CSL reaction products (3MH and 3MHA) are recognized food flavouring ingredients by the Food Additives Section (FAS), and under the Food and Drug Regulations there are no numerical limits on the use of flavouring substances, provided their use is consistent with Good Manufacturing Practices (GMP). The FAS confirmed that the concentrations of 3MH and 3MHA reported in beer produced with S. cerevisiae strain BY-989 fall within levels typically associated with flavouring use. Based on these findings, the PTAS determined that it was not necessary to conduct a toxicological safety assessment for 3MH or 3MHA. Additionally, JECFA has determined that both compounds present no safety concern at current intake levels when used as flavouring agents.

Based on the information provided, the PTAS did not identify any toxicological food safety concerns with the use of S. cerevisiae BY-989 compared to the use of other strains of S. cerevisiae already used in brewing.

Allergenicity

The petitioner conducted a sequence homology search using the AllergenOnline database to identify potential cross-reactivity between the CSL enzyme and known allergens. The database was searched and no alignment met or exceeded the threshold of > 35% over a sliding window of 80-amino acid sequences, as well as that of 100% over 8-amino acids, both criteria being consistent with guidance from WHO/FAO.

No significant homology between the CSL enzyme and known or suspected food allergens was identified.

The PTAS did not identify any allergenic food safety concerns with the use of S. cerevisiae BY-989.

Conclusion

Health Canada's review of the information presented in support of the use of Saccharomyces cerevisiae strain BY-989 to replace yeast from other available commercial sources in the fermentation of beer does not raise concerns related to food safety.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food and Nutrition Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

For further information, please contact:

Novel Foods Section
Food and Nutrition Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
bmh-bdm@hc-sc.gc.ca

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2026-09-29