WO2026003169A1

WO2026003169A1 - Yeast strains producing high levels of hydrogen sulphide

Info

Publication number: WO2026003169A1
Application number: PCT/EP2025/068064
Authority: WO
Inventors: Klaus LENGELER; Carl Gustav Anton MALINA; Rikke Uhre VILANDT; Michael Katz; Jesper Harholt; Manuel Ernesto Arreola SEVILLA; Iben Moeller-Hansen; Zoran Gojkovic; Helena da Silva ATHANASOPOULOS
Original assignee: Carlsberg AS
Current assignee: Carlsberg AS
Priority date: 2024-06-26
Filing date: 2025-06-26
Publication date: 2026-01-02
Anticipated expiration: 2026-12-26

Abstract

The present invention relates to the field of fermented beverage, such as beer, production. More specifically, the invention provides yeast strains of the genus Starmera with one or more mutations in the gene MET30, which are particularly useful in the production of beverages with enhanced levels of hydrogen sulphide (H₂S).

Description

Yeast strains producing high levels of hydrogen sulphide

Technical field

The present invention relates to the field of beverage production. More specifically, the invention provides yeast strains of the genus Starmera with one or more mutations in the gene MET30. These strains are particularly useful in the production of beverages with enhanced levels of hydrogen sulphide (H₂S).

Background

Yeast strains are integral to the brewing industry, facilitating the fermentation of sugars derived from grains, such as barley, into alcohol and carbon dioxide, and contributing to the production of various flavour and aroma compounds. While traditional brewing yeast strains, like Saccharomyces cerevisiae, are widely used, there is increasing demand for yeast strains capable of producing low or non-alcoholic beverages while offering distinct sensory profiles, such as increased hydrogen sulphide (H₂S) content. At low levels, H₂S contributes to the characters of some types of beer, such as lagers. Even at higher concentrations, H₂S is considered acceptable in certain traditional beer styles such as English pale ales from Burton-on-Trent.

Starmera is a genus of yeast often associated with necrotic lesions in cacti. One notable species, Starmera caribaea - formerly known as Pichia caribaea - is a naturally occurring auxotroph requiring an organic sulphur source like L-methionine or L-cysteine for optimal growth, but that grows poorly on substrates containing other sulphur sources such as glutathione or thiosulfate. The species lacks homologs of several key sulphur genes found in Saccharomyces cerevisiae, such as MET3, MET14 and MET16, indicating that its sulphur metabolic pathway is likely substantially different. Importantly, the species produces very low amounts of alcohol during fermentation, making it a candidate for specialized brewing applications.

Summary

The present invention provides yeast strains of the genus Starmera with mutations in the gene encoding Met30. These strains produce significantly higher levels of hydrogen sulphide compared to wild type strains and this is particularly useful in production of beverages, such as beers, including lagers and ales, as well as ciders, wines and juices.

The MET30 gene in Saccharomyces cerevisiae encodes the Met30 protein, a crucial component of the SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. This complex is important in targeting specific proteins for ubiquitination and subsequent proteasomal degradation, regulating sulphur amino acid metabolism in S. cerevisiae, specifically methionine and cysteine biosynthesis, and plays a role in cell cycle progression.

Starmera caribaea comprises a gene with the sequence as set forth in SEQ ID NO: 2, encoding a protein having 54.2% sequence identity to the Met30 Saccharomyces cerevisiae protein. Despite the low sequence identity, this protein is also referred to as Met30. However, Starmera caribaea has a significantly altered sulphur metabolism compared to Saccharomyces cerevisiae as detailed in Example 3, and the function of Starmera caribaea Met30 can therefore not be predicted based on Saccharomyces cerevisiae sulphur metabolism. Surprisingly, the present invention shows that Starmera yeast carrying mutations in Starmera Met30 can lead to increased production of hydrogen sulphide.

In some aspects of the present disclosure is therefore provided a yeast strain of the genus Starmera, wherein said yeast strain comprises a mutant MET30 gene encoding a mutant Met30 polypeptide, wherein the wild type MET30 gene encodes i. wild type Met30 as set forth in SEQ ID NO: 1 , or ii. a functional homologue of SEQ ID NO: 1 with at least 90%, such as at least 95%, such as at least 98% sequence identity to SEQ ID NO: 1.

In some aspects of the present disclosure is also provided a method of producing a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, said method comprising the steps of: i) providing a beverage base, such as an aqueous extract of malt and/or cereal grains; ii) providing a Starmera yeast strain, wherein said yeast strain is as described elsewhere herein; and iii) fermenting the beverage base, such as the aqueous extract of malt and/or cereal grains, provided in step i) with said yeast strain of step ii), thereby obtaining a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains.

In some aspects of the present disclosure is also provided a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, prepared by the method as described elsewhere herein.

In some aspects of the present disclosure is provided a method for producing a beverage, such as a malt and/or cereal based beverage, said method comprising the steps of: a) preparing a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, by the method as described elsewhere herein, and b) processing said fermented beverage base into a beverage.

In some aspects of the present disclosure is also provided a beverage, such as a malt and/or cereal based beverage, prepared by the method as described elsewhere herein.

Description of Drawings

Figure 1 shows a visual representation of the breeding scheme performed to generate the yeast strains described in Example 1. “UV” denotes inducing mutagenesis using ultraviolet irradiation. “Cross” denotes the mating (crossing) of two haploid strains.

Figures 2-3 show the levels of H2S in different strains as measured by flow cytometry. The X-axis value corresponds to the fluorescence intensities detected for a population of yeast cells using a fluorescent H2S probe. The results are further described in Example 1.

Figure 4 shows the levels of H2S in different strains as measured by plating on lead nitrate containing plates. Darker colour corresponds to higher levels of produced H2S. The results are further described in Example 1.

Figure 5 shows growth of various strains described in Example 1 on plates with different sources of sulphur. The results are further described in Example 3. Abbreviations: GSS - reduced glutathione; TS - sodium thiosulfate; SO3 - sodium sulphite.

Detailed description

Definitions

As used herein, unless indicated otherwise, the gene “MET30" and the protein “Met30” refer to the Starmera caribaea gene and protein according to SEQ ID NO: 2 and SEQ ID NO: 1 , respectively, as well homologs thereof. As such, the term for the gene “MET30" may be used interchangeably with MET30, also known by the systematic name YIL046W, in Saccharomyces cerevisiae.

The term “hydrogen sulphide” refers to the chemical compound H2S.

The term “yeast(s)” is herein used interchangeably with the term “yeast cell(s)”

The term “beer” as used herein refers to a beverage prepared by fermentation of wort. Preferably, said fermentation is done by yeast.

The terms “non-alcoholic beverage” and “alcohol-free beverage” are used interchangeably herein to denote a beverage with an alcohol content of less than 0.5% vol. In preferred embodiments, the alcohol content of an alcohol-free beverage or beer is 0.05% vol. or less, such as 0.049% or less, for example 0.047% or less, for example 0.0%. The beverage may in particular be beer, and thus the terms “non-alcoholic beer” or “alcohol-free beer” denotes beer having aforementioned alcohol content.

As used herein, the term “0.0%” ethanol content refers to an ethanol content below 0.05% ABV.

The term “low-alcohol beverage”, such as “low-alcohol beer”, denotes a beverage, such as a beer, with an alcohol content from 0.5% vol. to 2.8% vol. In some embodiments, the alcohol content of a low-alcohol beverage or beer is from 0.5% vol. to 1.2% vol. The term “debrewing” as used herein refers to dilution of a beverage or beverage base, e.g. beer with water. Water. The water may e.g. be tap water, demineralised water or a mixture of both.

The term “cereal” as used herein refers to any plant of the grass family yielding an edible grain, such as wheat, millet, rice, barley, oats, rye, triticale, sorghum, and maize.

The term "grain" as used herein refers to seeds of a cereal comprising the cereal caryopsis, also denoted internal seed. In addition, the grain may comprise the lemma and palea. In most barley varieties, the lemma and palea adhere to the caryopsis and are a part of the grain following threshing. However, naked barley varieties also occur. In these, the caryopsis is free of the lemma and palea and threshes out free as in wheat. The terms "grain" and "kernel" are used interchangeably herein.

By the term "wort" is meant a liquid extract of malt and/or cereal grains and optionally additional adjuncts. Wort is in general obtained by mashing, optionally followed by "sparging" The mashing process typically involves mixing milled malt and/or grains with water and heating the mixture to predetermined temperatures to activate hydrolytic enzymes. Hydrolytic enzymes include amylases and glucanases, that break down the starches in the malt/grains into sugars. The temperatures and duration of the process are controlled to optimize enzyme activity and sugar extraction. Sparging is a process of extracting residual sugars and other compounds from spent grains after mashing with hot water. Sparging is typically conducted in a lauter tun, a mash filter, or another apparatus to allow separation of the extracted water from spent grains. The wort obtained after mashing is generally referred to as "first wort", while the wort obtained after sparging is generally referred to as the "second wort". If not specified, the term wort may be first wort, second wort, or a combination of both. During conventional beer production, wort is boiled together with hops. Wort without hops, may also be referred to as "sweet wort", whereas wort boiled with hops may be referred to as "boiled wort" or simply as wort.

The term ’’beverage base” as used herein refers to any beverage base, such as an aqueous extract of malt and/or cereal kernels, non-limiting examples hereof which can be wort with a given amount of fermentable sugars, or any other beverage base, such as fruits, such as apples, pears or grapes, or juice, e.g. grape, apple, pear or orange juice. The beverage base may also be made from steamed cereals, e.g rice, which optionally have been incubated with fungi, such as Aspergillus oryzae. For example, the beverage base may be shubo or moto.

The term “fermented beverage base” as used herein refers to any beverage base, such as any aqueous extract, incubated with a microorganism, such as a yeast strain. A fermented beverage base, may for example be a fermented malt and/or cereal based extract. Preferably, a fermented beverage base has been fermented for at least 2 days, such as at least 3 days, for example for 3 to 7 days, such as for 5 to 7 days.

The term “Alcohol by volume (ABV)” as used herein refers to the amount of alcohol (ethanol) in a given volume of an alcoholic beverage (expressed as a volume percent). It is defined as the number of milliliter of pure ethanol present in 100ml of solution at 20°C. ABV can be measured e.g. by gas chromatography or with an Alcolyzer.

The term “fermenting” as used herein refers to incubating a beverage base, such as an aqueous extract of malt and/or cereal grains, or another solution with a microorganism, such as a yeast strain.

The term "malt" as used herein refers to cereal kernels, which have been malted. The term “green malt” refers to germinated cereal kernels, which have not been subjected to a step of kiln drying. In some embodiments the green malt is milled green malt. The term "kiln dried malt" as used herein refers germinated cereal kernels, which have been dried by kiln drying. In some embodiments the kiln dried malt is milled kiln dried malt. In general, said cereal kernels have been germinated under controlled environmental conditions.

Amino acids may be named herein using the IIIPAC one-letter and three-letter codes. If not otherwise indicated the term “amino acid” refers to the standard amino acids.

The term ’’functional homologue” as used herein denotes a polypeptide sharing at least one biological function with a reference polypeptide. In general said functional homologue also shares a significant sequence identity with the reference polypeptide. Preferably a functional homologue of a reference polypeptide is a polypeptide, which has the same biological function as the reference protein and which shares a high level of sequence identity with the reference polypeptide.

In the context of the present disclosure, if a yeast carries a gene encoding a functional homologue of Met30 of SEQ ID NO: 1 , said yeast will have levels of sulfur compounds, such as H2S, comparable to wild type Starmera caribaea, preferably levels of H2S +/- 10% of the level in wild type Starmera caribaea.

The term “sequence identity” as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e. a candidate sequence (e.g. a mutant sequence) and a reference sequence (such as a wild type sequence) based on their pairwise alignment. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277,), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss_needle/). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment). The Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence corresponds to a given position of the reference sequence. For purposes of the present invention, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment). Sequence identity is always measured compared to the full-length reference sequence, i.e. truncated proteins with no gaps or mismatches are not considered 100% sequence identical to the reference sequence. In some embodiments of the present invention, the sequence identity between two amino acid sequences is determined using the BioPython implementation of BLAST found at https://biopython.Org/docs/1.76/api/Bio.Blast.Applications.html.

The term "mutations" as used herein include insertions, deletions, substitutions, transversions, and point mutations in the coding and noncoding regions of a gene. Point mutations may concern changes of one base pair, and may result in premature stop codons, frameshift mutations, mutation of a splice site or amino acid substitutions. A gene comprising a mutation may be referred to as a “mutant gene”. If said mutant gene encodes a polypeptide with a sequence different to the wild type, said polypeptide may be referred to as a “mutant polypeptide” and/or “mutant protein”. A mutant polypeptide may be described as carrying a mutation, when it comprises an amino acid sequence differing from the wild type sequence.

Yeast strain

The present disclosure relates to a yeast strain of the genus Starmera, that surprisingly produces high levels of hydrogens sulphide (H2S) during fermentation.

In some aspects of the present disclosure is provided a yeast strain of the genus Starmera, wherein said yeast strain comprises a mutant MET30 gene encoding a mutant Met30 polypeptide, wherein the wild type MET30 gene encodes i. wild type Met30 as set forth in SEQ ID NO: 1, or ii. a functional homologue of SEQ ID NO: 1 with at least 90%, such as at least 95%, such as at least 98% sequence identity to SEQ ID NO: 1.

In some embodiments of the present disclosure is therefore provided a yeast strain of the genus Starmera, wherein said yeast strain comprises a mutant MET30 gene encoding a mutant Met30 polypeptide, wherein the wild type MET30 gene encodes wild type Met30 as set forth in SEQ ID NO: 1.

In some embodiments of the present disclosure is provided a yeast strain of the genus Starmera, wherein said yeast strain comprises a mutant MET30 gene encoding a mutant Met30 polypeptide, wherein the wild type MET30 gene encodes a functional homologue of SEQ ID NO: 1 with at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, sequence identity to SEQ ID NO: 1.

In some embodiments, the mutant Met30 polypeptide has an amino acid substitution at position 101, 111 , 244 or 581 of SEQ ID NO: 1 , or an amino acid substitution in a corresponding position of a functional homologue thereof.

The term “corresponding amino acid” or “corresponding position”, as is generally understood in the art, refers to a residue on a second amino acid sequence which occupies the same (i.e. , equivalent) position as a residue on a first amino acid sequence, when the first and second sequences are optimally aligned for comparison purposes. The first amino acid sequence may be Met30 as set forth in SEQ ID NO: 1, while the second amino acid sequence may be a variant of sequence. Thus, a residue at a first position in a first peptide sequence does not necessarily correspond to a residue in said same first position in a second peptide sequence, but may instead correspond to a residue at a second position in the second peptide sequence that optimally aligns with the residue in said first position of said first peptide sequence, when the first and second peptide sequences are optimally aligned. Said alignment may be performed by any method known in the art, such as by using the Needleman- Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss_needle/). The parameters used may be a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. In some embodiments, said alignment is performed using the BioPython implementation of BLAST found at https://biopython.Org/docs/1.76/api/Bio.Blast.Applications.html.

Thus, corresponding amino acids may in some embodiments be identical amino acids. In other embodiments, corresponding amino acids are be non-identical amino acids with appropriately similar structural and/or functional characteristics. As is well known by those of ordinary skill in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, “charged” (either positive or negative) or “uncharged”, and/or as having "polar" or "non-polar" side chains.

Substitution of one amino acid for another of the same type may often be considered a homologous substitution. Typical amino acid categorizations are summarized in Table A, below.

Table A - Amino acid properties

Side Side chain

Amino Acid _Letter Letter ^c^^a‘ⁿ acidity or Charge Hydrophobic polarity basicity

Alanine Ala A nonpolar neutral Uncharged Yes

Arginine Arg R polar basic Positive No

Asparagine Asn N polar neutral Uncharged No

Aspartic acid Asp D polar acidic Negative No

Cysteine Cys C polar neutral Uncharged No

Glutamic acid Glu E polar acidic Negative No

Glutamine Gin Q polar neutral Uncharged No

Glycine Gly G nonpolar neutral Uncharged Yes

Histidine His H polar basic Positive No

Isoleucine lie I nonpolar neutral Uncharged Yes

Leucine Leu L nonpolar neutral Uncharged Yes

Lysine Lys K polar basic Positive No

Methionine Met M nonpolar neutral Uncharged Yes

Phenylalanine Phe F nonpolar neutral Uncharged Yes

Proline Pro P nonpolar neutral Uncharged Yes

Serine Ser S polar neutral Uncharged No

Threonine Thr T polar neutral Uncharged No

Tryptophan Trp W polar neutral Uncharged Yes

Tyrosine Tyr Y polar neutral Uncharged No

Valine Vai V nonpolar neutral Uncharged Yes

In some embodiments, the mutant Met30 polypeptide has an amino acid substitution at position 101 of SEQ ID NO: 1 or an amino acid substitution in a corresponding position of said functional homologue thereof. In some embodiments of the present disclosure, the substitution in SEQ ID NO: 1 or in said functional homologue thereof is a substitution of a hydrophobic amino acid to a polar, uncharged amino acid. In some embodiments, the substitution is substitution of a leucine (L) to a serine (S). In some embodiments, the substitution is substitution of the leucine (L) at position 101 of SEQ ID NO: 1, or of a corresponding amino acid, such as a leucine, in said functional homologue thereof, to a serine (S).

In some embodiments, the mutant Met30 polypeptide has an amino acid substitution at position 111 of SEQ ID NO: 1 or an amino acid substitution in a corresponding position of said functional homologue thereof. In some embodiments of the present disclosure, the substitution is substitution of a cysteine (C) to a hydrophobic amino acid. In some embodiments of the present disclosure, the substitution is substitution of a cysteine (C) to an amino acid comprising a side chain comprising an aromatic group. In some embodiments, the substitution is substitution of a cysteine (C) to a tryptophan (W). In some embodiments, the substitution is substitution of the cysteine (C) at position 111 of SEQ ID NO: 1, or of a corresponding amino acid, such as a cysteine, in said functional homologue thereof, to a tryptophan (W).

In some embodiments, the mutant Met30 polypeptide has an amino acid substitution at position 244 of SEQ ID NO: 1 or an amino acid substitution in a corresponding position of said functional homologue thereof. In some embodiments of the present disclosure, the substitution is a substitution of a hydrophobic amino acid to a charged amino acid. In some embodiments, the substitution is a substitution of a hydrophobic amino acid to a positively charged amino acid. In some embodiments, the substitution is a substitution of a tyrosine (Y) to a histidine (H). In some embodiments, the substitution is a substitution of the tyrosine (Y) at position 244 of SEQ I D NO: 1 , or of a corresponding tyrosine in said functional homologue thereof, to a histidine (H).

In some embodiments, the mutant Met30 polypeptide has an amino acid substitution at position 581 of SEQ ID NO: 1 or an amino acid substitution in a corresponding position of said functional homologue thereof. In some embodiments of the present disclosure, the substitution is a substitution of a glycine to a charged amino acid. In some embodiments, the substitution is a substitution of a glycine to a negatively charged amino acid. In some embodiments, the substitution is a substitution of a glycine (G) to an aspartic acid (D). In some embodiments, the substitution is a substitution of the glycine (G) at position 581 of SEQ ID NO: 1 , or of a corresponding glycine in said functional homologue thereof, to an aspartic acid (D). In some embodiments, the wild type MET30 gene comprises or consists of the sequence as set forth in SEQ ID NO: 2.

In some embodiments, the wild type MET30 gene comprises or consists of a homologous sequence of SEQ ID NO: 2, wherein said homologous sequence encodes a polypeptide with at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or such as 100% sequence identity to the polypeptide encoded by SEQ ID NO: 1.

In some embodiments of the present disclosure, the mutant MET30 gene has a T to C mutation in position 302 of SEQ ID NO: 2 or has a corresponding mutation, such as a T to C mutation, in a corresponding position in said homologous sequence of SEQ ID NO: 2.

In some embodiments of the present disclosure, the mutant MET30 gene has a T to G mutation in position 333 of SEQ ID NO: 2 or has a corresponding mutation, such as a T to G mutation, in a corresponding position in said homologous sequence of SEQ ID NO: 2.

In some embodiments of the present disclosure, the mutant MET30 gene has a T to C mutation in position 730 of SEQ ID NO: 2 or has a corresponding mutation, such as a T to C mutation, in a corresponding position in said homologous sequence of SEQ ID NO: 2.

In some embodiments of the present disclosure, the mutant MET30 gene has a G to A mutation in position 1742 of SEQ ID NO: 2 or has a corresponding mutation, such as a G to A mutation, in a corresponding position in said homologous sequence of SEQ ID NO: 2.

The Starmera yeast strains according to the present disclosure produce significantly higher levels of hydrogen sulphide compared to wild type Starmera yeast strains. In some embodiments, said production of sulfur compounds, such as H2S, is determined after fermentation is performed essentially as described in Example 2 or 4 (either Ankom fermentation or 50L fermentation). The skilled person will be able to adjust the fermentation conditions according to needs, e.g. by upscaling. The level of hydrogen sulphide may be measured after fermentation, e.g., essentially as described in Example 2 (section “Quantification of hydrogen sulphide by flow cytometry”).

In some embodiments of the present disclosure, the yeasts as described elsewhere herein comprise at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as at least 145%, such as at least 150%, such as at least 155%, such as at least 160%, such as at least 165%, such as at least 170%, such as at least 175%, such as at least 180%, such as at least 185%, such as at least 190%, such as at least 195%, such as at least 200%, such as at least 205%, such as at least 210%, such as at least 215%, such as at least 220%, such as at least 225%, such as at least 230%, such as at least 235%, such as at least 240%, such as at least 245%, such as at least 250%, such as at least 255%, such as at least 260%, such as at least 265%, such as at least 270%, such as at least 275%, such as at least 280%, such as at least 285%, such as at least 290%, such as at least 295%, such as at least 300%, such as at least 305%, such as at least 310%, such as at least 315%, such as at least 320%, such as at least 325%, such as at least 330%, such as at least 335%, such as at least 340%, such as at least 345%, such as at least 350%, such as at least 355%, such as at least 360%, such as at least 365%, such as at least 370%, such as at least 375%, such as at least 380%, such as at least 385%, such as at least 390%, such as at least 395%, such as at least 400% the level of hydrogen sulphide (H2S) compared to yeast of a reference strain of the genus Starmera.

In some embodiments, the yeasts as described herein comprise at least 125%, such as at least 130%, such as at least 135%, such as at least 140%, such as at least 145%, such as at least 150%, such as at least 155%, such as at least 160%, such as at least 165%, such as at least 170%, such as at least 175%, such as at least 180%, such as at least 185%, such as at least 190%, such as at least 195%, such as at least 200%, such as at least 205%, such as at least 210%, such as at least 215%, such as at least 220%, such as at least 225%, such as at least 230%, such as at least 235%, such as at least 240%, such as at least 245%, such as at least 250%, such as at least 255%, such as at least 260%, such as at least 265%, such as at least 270%, such as at least 275%, such as at least 280%, such as at least 285%, such as at least 290%, such as at least 295%, such as at least 300%, such as at least 305%, such as at least 310%, such as at least 315%, such as at least 320%, such as at least 325%, such as at least 330%, such as at least 335%, such as at least 340%, such as at least 345%, such as at least 350%, such as at least 355%, such as at least 360%, such as at least 365%, such as at least 370%, such as at least 375%, such as at least 380%, such as at least 385%, such as at least 390%, such as at least 395%, such as at least 400% the level of hydrogen sulphide (H2S) compared to yeast of a reference strain of the genus Starmera, wherein said reference strain has an identical genotype to said yeast strain except i. not comprising said mutant MET30 gene; and ii. comprising said wild type MET30 gene.

In some embodiments of the present disclosure, the yeasts as described herein comprise at least 4 pg/L H2S, such as at least 6 pg/L H2S, such as at least 8 pg/L H2S, such as at least 10 pg/L H2S, such as at least 15 pg/L H2S, such as at least 20 pg/L H2S, such as at least 25 pg/L H2S, such as at least 30 pg/L H2S, such as at least 35 pg/L H2S, such as at least 40 pg/L H2S, such as at least 50 pg/L H2S, such as at least 60 pg/L H2S, such as at least 70 pg/L H2S, such as at least 80 pg/L H2S, such as at least 90 pg/L H2S, such as at least 100 pg/L H2S, such as at least 110 pg/L H2S, such as at least 120 pg/L H2S, such as at least 130 pg/L H2S, such as at least 140 pg/L H2S, or such as at least 150 pg/L H2S.

In some embodiments, the yeasts as described herein comprise from 4 to 150 pg/L H2S, such as from 10 to 150 pg/L H2S, such as from 20 to 150 pg/L H2S, such as from 30 to 150 pg/L H2S, such as from 40 to 150 pg/L H2S, such as from 50 to 150 pg/L H2S, or such as from 60 to 150 pg/L H2S.

In some embodiments, the yeasts as described herein comprise from 4 to 100 pg/L H2S, such as from 10 to 100 pg/L H2S, such as from 20 to 100 pg/L H2S, such as from 30 to 100 pg/L H2S, such as from 40 to 100 pg/L H2S, such as from 50 to 100 pg/L H2S, or such as from 60 to 100 pg/L H2S. In some embodiments, the yeasts as described herein comprise from 10 to 140 pg/L H2S, such as from 20 to 130 pg/L H2S, such as from 30 to 120 pg/L H2S, such as from 40 to 110 pg/L H2S, such as from 60 to 90 pg/L H2S.

In some embodiments, said H2S concentration is determined after overnight cultivation of said yeast cells.

The H2S content of a yeast cell may be measured by any method known to the skilled person in the art suitable for measuring said H2S content.

In some embodiments, yeast content of H2S is measured by a method comprising the steps of: i. contacting a hLS-specific probe with said yeast; and ii. measuring the amount of said probe bound to said H2S within said yeast, such as by flow cytometry.

The yeast cells of the present disclosure may also produce higher levels of other sulphur-containing compounds in addition to higher levels of H2S. These compounds are in particular mercaptans, thiols and sulphides.

Thus in some embodiments, the yeasts as described herein comprise higher levels of one or more sulphur-containing compounds selected from the group consisting of sulfite, sulfur dioxide, carbon disulfide, methanethiol, ethylene sulfide, ethanethiol, propanethiol, dimelthyl sulfide, dimethyl disulfide, diethyl disulfide, dimethyl trisulfide, methyl thioacetate, ethyl thioacetate, methionol, methional, 3-methyl-2-butene-1 -thiol compared to a wild type Starmera yeast strain.

The yeast strains as disclosed herein are particularly suitable for producing beverages with low concentrations of alcohol, such as non-alcoholic beverages, due to their low production of ethanol during fermentation.

In some embodiments of the present disclosure, the yeast strain produces at the most 0.5%, such as at the most 0.4%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1%, such as at the most 0.09%, such as at the most 0.08%, such as at the most 0.07%, such as at the most 0.06%, such as at the most 0.05%, such as at the most 0.047%, such as at the most 0.04%, such as at the most 0.03%, such as at the most 0.02%, such as at the most 0.01%, such as at the most 0.0% alcohol by volume (ABV) at the end of fermentation.

The end of fermentation may in some embodiments be after fermentation for at least 2 days, such as for at least 3 days, for example for in the range of 3 to 7 days, such as for in the range of 5 to 7 days. The end of fermentation may also be when the level of Strecker aldehydes, such as 2-methylpropanal (2-MePr), 2-methylbutanal (2-Me-Bu), 3-methylbutanal (3-MeBu), fufural, methional and phenylacetaldehyde (PheAcel) are below the taste perception threshold in the fermented beverage base, such as the taste perception thresholds defined in Gernat et al., 2019 and/or Piornos et al., 2020. The end of fermentation may in particular be the point in time when fermentation is complete as described below in the section “Fermented beverage base and methods of production thereof”.

In some embodiments of the present disclosure, the yeast strain produces at the most 0.5%, such as at the most 0.4%, for example at the most 0.47%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1%, such as at the most 0.09%, such as at the most 0.08%, such as at the most 0.07%, such as at the most 0.06%, such as at the most 0.05%, such as at the most 0.047%, such as at the most 0.04%, such as at the most 0.03%, such as at the most 0.02%, such as at the most 0.01%, such as at the most 0.0% alcohol by volume (ABV) after fermentation in wort of 9 “Plato for 5 days at 16°C.

The methods of producing a beverage may in some embodiments comprise a step of debrewing said fermented beverage base. The aforementioned levels alcohol and other compounds are preferably the levels obtained before debrewing.

The yeast strain of the invention is in general a yeast of the genus Starmera. In preferred embodiments, the yeast strain is of the species Starmera caribaea, also known as Pichia caribaea.

As is known to the skilled person in the art, a yeast species may also be taxonomically defined according to its internal transcribed spacer (ITS) region, see e.g. Vu et al., 2016. ITS regions in yeast are non-coding sequences located between the small subunit rRNA and large subunit rRNA genes within the rRNA gene cluster. These regions are transcribed along with the rRNA genes but are removed during rRNA maturation. ITS regions are highly variable in sequence among different yeast species, making them useful for phylogenetic studies and species identification.

In some embodiments, a yeast of the genus Starmera is defined as a yeast comprising an internal transcribed spacer (ITS) region comprising or consisting of a sequence with at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90% sequence identity to SEQ ID NO: 6. In preferred embodiments, a yeast of the genus Starmera is defined as a yeast comprising an ITS region consisting of a sequence with at least 85% sequence identity to SEQ ID NO: 6.

In some embodiments, a yeast of the species Starmera caribaea is defined as a yeast comprising an internal transcribed spacer (ITS) region comprising or consisting of a sequence with at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 100% sequence identity to SEQ ID NO: 6. In preferred embodiments, a yeast of the species Starmera caribaea is defined as a yeast comprising an ITS region consisting of a sequence with at least 99% sequence identity to SEQ ID NO: 6.

In some embodiments, the yeast strain is haploid. In some embodiments, the yeast strain is diploid.

In some embodiments, the yeast strain is diploid and is homozygous for the mutation(s) in the MET30 gene or homolog thereof as described elsewhere herein.

In some embodiments, the yeast strain is auxotrophic for L-methionine and/or L- cysteine. In some embodiments, the yeast strain is auxotrophic for L-methionine. In some embodiments, the yeast strain is auxotrophic for L-cysteine. In some embodiments, the yeast strain is auxotrophic for L-methionine and L-cysteine.

In some embodiments, the yeast strain does not comprise a functional homolog of

Met3 as set forth in SEQ ID NO: 3, Met14 as set forth in SEQ ID NO: 4 and/or Met16 as set forth in SEQ ID NO: 5. In some embodiments, the yeast strain does not comprise a functional homolog of Met3 as set forth in SEQ ID NO: 3. In some embodiments, the yeast strain does not comprise a functional homolog of Met14 as set forth in SEQ ID NO: 4. In some embodiments, the yeast strain does not comprise a functional homolog of Met16 as set forth in SEQ ID NO: 5. In some embodiments, the yeast strain does not comprise a functional homolog of Met3 as set forth in SEQ ID NO: 3, Met14 as set forth in SEQ ID NO: 4 and Met16 as set forth in SEQ ID NO: 5.

Fermented beverage base and methods of production thereof

The present disclosure also provides methods of preparing fermented aqueous extract, using the yeast strains as described elsewhere herein.

In one aspect of the present disclosure is therefore provided a method of producing a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, said method comprising the steps of: i) Providing a beverage base, such as an aqueous extract of malt and/or cereal grains; ii) providing a Starmera yeast strain, wherein said yeast strain is as described elsewhere herein; and iii) fermenting the beverage base, such as the aqueous extract of malt and/or cereal grains, provided in step i) with said yeast strain of step ii), thereby obtaining a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains.

The beverage base may be any aqueous extract of kernels or any type of juice, such as apple, orange or grape juice, or any type of fruits, such as apples, pears or grapes, optionally in combination.

The juice may be any juice. In some embodiments, the juice is a pure fruit juice. The juice may also be provided in the form of a concentrate or as RTD (ready- to-d rink) juice.

The juice may be the juice of any fruit, such as berries, orange, apple, banana, lemon, lime, passion fruit, mango, pineapple, pears, kumquats, pomelo, pomegranate, rhubarb and/or grape. Non-limiting examples of useful juice includes apple juice and orange juice. The juice may be the juice of any vegetable, such as carrot juice.

In some embodiments it may be preferred that the juice is free of solid particles, e.g. that the juice is a fruit juice essentially clear of solid materials, such as pulp.

The gravity of the juice may for example be between 5 and 15° Plato, such as in the range of 8 to 12° Plato. Another measure for sugar content of a beverage is the BRIX value. RTD juice to be used with the present invention typically has an RTD in the range of 60 to 80 BRIX, such as in the range of 65 to 71 BRIX.

In some embodiments, the beverage base is an aqueous extract of malt and/or cereal grains. Thus, a non-limiting example hereof is wort. The aqueous extract may for example be prepared by preparing an extract of malt by mashing and optionally sparging as described herein in this section below.

Malt is cereal kernels, such as barley kernels, that have been malted. By the term "malting" is to be understood a process involving steeping and germination of kernels in a process taking place under controlled environmental conditions, optionally followed by a drying step. Said drying step may preferably be kiln drying of the germinated kernels at elevated temperatures. Green malt, which has not been subject to kilning may also be used, in particular malt obtained by the process described in WO 2018/001882 or WO 2019/129731.

Malting is important for the synthesis of numerous enzymes that cause kernel modification, processes that principally depolymerize starch and cell walls of the dead endosperm to mobilize the kernel nutrients and activate other depolymerases. In the subsequent drying process, flavour and colour are generated at least partly due to chemical browning reactions.

Steeping may be performed by any conventional method known to the skilled person. One non-limiting example involves steeping at a temperature in the range of 10°C to 25°C with alternating dry and wet conditions. Germination may be performed by any conventional method known to the skilled person. One non-limiting example involves germination at a temperature in the range of 10 to 25°C, optionally with changing temperature, in the range of 1 to 4 h. Steeping and germination may also be performed in a combined method, e.g. as described in international patent application WO 2018/001882 or WO 2019/129731 .

The kiln drying may be performed at conventional temperatures, such as at least 75°C, for example in the range of 80 to 90°C, such as in the range of 80 to 85°C. Thus, the malt may, for example be produced by any of the methods described by Briggs et al. (1981) and by Hough et al. (1982). However, any other suitable method for producing malt may also be used with the present invention, such as methods for production of specialty malts, including, but not limited to, methods of roasting the malt.

Malt may be further processed, for example by milling. Milling can be performed in a dry state, i.e. the malt is milled while dry or in a wet state if green malt is used.

The malt, e.g. the milled malt may be mashed to prepare an aqueous extract of said malt. The starting liquid for preparing the beverage may be an aqueous extract of malt, e.g. an aqueous extract of malt prepared by mashing.

Thus, the method for preparing a malt and/or cereal based fermented aqueous extract according to the invention may comprise a step of producing an aqueous extract, such as wort, by mashing malt and optionally additional adjuncts. Said mashing step may also optionally comprise sparging, and accordingly said mashing step may be a mashing step including a sparging step or a mashing step excluding a sparging step.

In general, the production of the aqueous extract is initiated by the milling of malt and/or kernels. If additional adjuncts are added, these may also be milled depending on their nature. If the adjunct is a cereal, it may for example be milled, whereas syrups, sugars and the like will generally not be milled. Milling will facilitate water access to kernel particles in the mashing phase. During mashing enzymatic depolymerization of substrates initiated during malting may be continued.

In general, the aqueous extract is prepared by combining and incubating milled malt and water, i.e. in a mashing process. During mashing, the malt/liquid composition may be supplemented with additional carbohydrate-rich adjunct compositions, for example milled barley, maize, or rice adjuncts. Unmalted cereal adjuncts usually contain little or no active enzymes, making it important to supplement with malt or exogenous enzymes to provide enzymes necessary for polysaccharide depolymerization etc.

During mashing, milled malt and/or milled grains - and optionally additional adjuncts are incubated with a liquid fraction, such as water. The incubation temperature is in general either kept constant (isothermal mashing), or gradually increased, for example increased in a sequential manner. In either case, soluble substances in the malt/kernel/adjuncts are liberated into said liquid fraction. A subsequent filtration confers separation of the aqueous extract and residual solid particles, the latter also denoted "spent kernel". The aqueous extract thus obtained may also be denoted "first wort". Additional liquid, such as water may be added to the spent kernels during a process also denoted sparging. After sparging and filtration, a "second wort" may be obtained. Further worts may be prepared by repeating the procedure. Non-limiting examples of suitable procedures for preparation of wort is described by Briggs et al. (1981) and Hough et al. (1982).

As mentioned above, the aqueous extract may also be prepared by mashing only unmalted kernels. Unmalted kernels lack or contain only a limited amount of enzymes beneficial for wort production, such as enzymes capable of degrading cell walls or enzymes capable of depolymerising starch into sugars. Thus, in embodiments of the invention where up to 80%, such as 90% or such as 100% of unmalted kernels, such as barley kernels, are used for mashing, it is preferred that one or more suitable, external brewing enzymes are added to the mash Suitable enzymes may be lipases, starch degrading enzymes (e.g. amylases), glucanases [preferably (1-4)- and/or (1-3,1- 4)-p-glucanase], and/or xylanases (such as arabinoxylanase), and/or proteases, or enzyme mixtures comprising one or more of the aforementioned enzymes, e.g. Cereflo, Ultraflo, or Ondea Pro (Novozymes). However, even if a lower amount of unmalted grains or no unmalted grains are used, enzymes may be added to the mash.

The aqueous extract may also be prepared by using a mixture of malted and unmalted kernels, in which case one or more suitable enzymes may be added during preparation. Even in embodiments, where malt is used enzymes may also be added. More specifically, kernels can be used together with malt in any combination for mashing - with or without external brewing enzymes - such as, but not limited to, the proportions of kernel: malt = approximately 100 : 0, or approximately 75 : 25, or approximately 50 : 50, or approximately 25 : 75.

The aqueous extract obtained after mashing may also be referred to as “sweet wort”. In conventional methods, the sweet wort is boiled with or without hops where after it may be referred to as boiled wort.

The beverage base, such as the aqueous extract of malt and/or cereal grains, may be heated or boiled before it is subjected to fermentation with the yeast of the invention. In one aspect of the invention, second and further worts may be combined, and thereafter subjected to heating or boiling. The beverage base, such as the aqueous extract of malt and/or cereal grains may be heated or boiled for any suitable amount of time, e.g. in the range of 60min to 120min. Said heating or boiling may preferably be performed in the presence of hops.

The outcome of the fermented beverage base, such as the malt and/or cereal based fermented aqueous extract, is highly dependent on the amount and type of fermentable sugars present in the beverage base, such as in the aqueous extract of malt and/or cereal kernels, as well as the characteristics of the yeast strain used during fermentation.

Thus, the beverage base, such as the aqueous extract of malt and/or cereal grains, e.g. wort, may be prepared as described above. The fermented beverage base, such as the malt and/or cereal based fermented aqueous extract, may be prepared by fermentation of said beverage base, such as said aqueous extract of malt and/or cereal grains, with the yeast strain as described elsewhere herein.

In preferred embodiments, the fermented beverage base, such as the fermented aqueous extract of malt and/or cereal grains, is a green beer, more preferably a green non-alcoholic beer or low-alcohol beer.

In general terms, alcoholic or non-alcoholic fermented beverage bases, such as fermented aqueous extracts - such as beer - may be manufactured from malted and/or unmalted kernels. Malt, in addition to hops and yeast, contributes to flavor and color of the beverage, such as beer. Furthermore, malt functions as a source of fermentable sugar and enzymes. Non-limited descriptions of examples of suitable methods for malting and brewing can be found, for example, in publications by Briggs et al. (1981) and Hough et al. (1982). Numerous, regularly updated methods for analyses of kernel, malt and beer products are available, for example, but not limited to, American Association of Cereal Chemists (1995), American Society of Brewing Chemists (1992), European Brewery Convention (1998), and Institute of Brewing (1997). It is recognized that many specific procedures are employed for a given brewery, with the most significant variations relating to local consumer preferences. Any such method of producing beer may be used with the present invention.

The first step of producing beer from wort preferably involves heating said wort as described herein above, followed by a subsequent phase of wort cooling and optionally whirlpool rest.

The method of the invention comprises a step of fermenting a beverage base, such as an aqueous extract of malt and/or cereal kernels, such as malt and/or cereal kernels, with the yeast strain according to the invention. Said fermentation may be a fermentation of an unfermented beverage base, such as an unfermented aqueous extract, or a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, still containing fermentable sugars for the yeast. Thus, in some embodiments said fermentation may be performed essentially immediately after completion of mashing or after heating of wort.

Fermentation may be performed in fermentation tanks containing yeast according to the invention. During the several-day-long fermentation process, flavour substances are developed. If the yeast strain is not capable of converting specific compounds, these will still be present after the fermentation step iii).

In some embodiments, the pitching rate is about 3x10⁶ yeast cells/mL. In some embodiments, the pitching rate is about 6x10⁶ yeast cells/mL.

In some embodiments, fermentation is performed essentially as described in Example

2 or 4 (either Ankom fermentation or 50L fermentation). The skilled person will be able to adjust the fermentation conditions according to needs, e.g. by upscaling. Fermentation with the yeast strains according to the present invention have the additional beneficial property of the fermented beverage base, such as the fermented aqueous extract of malt and/or cereal grains, being very low in alcohol already immediately at the end of fermentation. This enables further processing into a low- or non-alcoholic beverage without any additional steps to lower the alcohol content. Optionally, a step of debrewing may be included at the end of fermentation.

In some embodiments, fermentation is complete once the Strecker aldehydes, such as 2-methylpropanal (2-MePr), 2-methylbutanal (2-Me-Bu), 3-methylbutanal (3-MeBu), fufural, methional and phenylacetaldehyde (PheAcel) are below the taste perception threshold in the fermented beverage base, such as the taste perception thresholds defined in Gernat et al., 2019 and/or Piornos et al., 2020.

Strecker aldehydes in the beverage base may be measured by any method known to the skilled person in the art suitable for measuring said content. In some embodiments, said Strecker aldehyde levels are measured as described in Gernat et al., 2019 and/or Piornos et al., 2020. In preferred embodiments, said Strecker aldehydes are measured in fresh beer.

In some embodiments, fermentation is complete once the Strecker aldehydes are at or below the concentrations listed in Table B, below.

Table B. Threshold values for Strecker aldehydes for fermentation to be considered complete.

In some embodiments, fermentation is complete once the fermented beverage base, such as the fermented malt and/or cereal based aqueous extract, comprises

• 0-100 pg/L of 2-methylpropanal;

• 0-50 pg/L of 2-methylbutanal;

• 0-60 pg/L of 3-methylbutanal;

• 0-20 pg/L of methional;

• optionally, 0-60 pg/L of furfural; and optionally, 0-25 pg/L of phenyl acetaldehyde.

• 0-100 pg/L of 2-methylpropanal;

• 0-50 pg/L of 2-methylbutanal;

• 0-60 pg/L of 3-methylbutanal;

• 0-20 pg/L of methional;

• 0-60 pg/L of furfural; and

• 0-25 pg/L of phenyl acetaldehyde.

• 0-20 pg/L of 2-methylpropanal;

• 0-10 pg/L of 2-methylbutanal;

• 0-20 pg/L of 3-methylbutanal;

• 0-50 pg/L of furfural.

• 0-20 pg/L of methional; and

• 0-20 pg/L of phenyl acetaldehyde.

• 0-6 pg/L of 2-methylbutanal;

• 0-14 pg/L of 3-methylbutanal;

• 0-12 pg/L of 2-methylpropanal;

• 0-6 pg/L of methional;

• 8-100 pg/L of phenyl acetaldehyde; and

• 0-500 pg/L of furfural, such as 30-500 pg/L of furfural.

• 0-6 pg/L of 2-methylbutanal;

• 0-14 pg/L of 3-methylbutanal;

• 0-12 pg/L of 2-methylpropanal;

• 0-6 pg/L of methional; • 8-100 pg/L of phenyl acetaldehyde; and

• 0-500 pg/L of furfural, such as 30-500 pg/L of furfural, wherein

• the combination of 2-methylbutanal, 3-methylbutanal, 2-methylpropanal and methional is present in a combined concentration of X pg/L;

• phenyl acetaldehyde is present in a concentration of Y pg/L; and

• furfural is present in a concentration of Z pg/L; and wherein:

X:Y < 1 :2; and/or

X:Z < 1:20.

In some embodiments, fermentation is complete after fermentation for at least 2 days, such as for at least 3 days, for example for in the range of 3 to 7 days, such as for in the range of 5 to 7 days. In some embodiments, fermentation is complete after a maximum of 7 days, such as a maximum of 6 days, such as a maximum of 5 days, such as a maximum of 4 days, such as a maximum of 3 days. In preferred embodiments, fermentation is complete after a maximum or 5 days, such as after a maximum of 3 days, such as after a maximum of from 3 to 5 days.

In some embodiments, fermentation is complete after a maximum of 5 days, wherein the fermentation is performed as described in Example 2 (Ankom fermentation), i.e. with a 9° Plato Holsten Wort at 16°C.

In some embodiments, fermentation is complete after a maximum of 5 days wherein the fermentation is performed as described in Example 4 (Ankom fermentation or 50L fermentation). In some embodiments, fermentation is complete after a maximum of 4 days wherein the fermentation is performed as described in Example 4 (Ankom fermentation or 50L fermentation).

In some embodiments, the fermented beverage base, such as the fermented aqueous extract of malt and/or cereal grains, comprises at the most 0.5%, such as at the most 0.4%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1%, such as at the most 0.0% alcohol by volume (ABV). Preferably, the fermented beverage base, such as the fermented aqueous extract of malt and/or cereal grains, comprises at the most 0.5%, such as at the most 0.4%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1 %, such as at the most 0.09%, such as at the most 0.08%, such as at the most 0.07%, such as at the most 0.06%, such as at the most 0.05%, such as at the most 0.047%, such as at the most 0.04%, such as at the most 0.03%, such as at the most 0.02%, such as at the most 0.01%, such as at the most 0.0% alcohol by volume (ABV) immediately at the end of fermentation, i.e. immediately when fermentation is complete.

In some embodiments, the fermented beverage base, such as the fermented aqueous extract of malt and/or cereal grains, comprises from 0.5 to 0.0%, such as from 0.4 to 0.0%, such as from 0.3 to 0.0%, such as from 0.2 to 0.0%, such as from 0.1 to 0.0 alcohol by volume (ABV). Preferably, the fermented beverage base, such as the fermented aqueous extract of malt and/or cereal grains, comprises from 0.5 to 0.0%, such as from 0.4 to 0.0%, such as from 0.3 to 0.0%, such as from 0.2 to 0.0%, such as from 0.1 to 0.0 alcohol by volume (ABV) immediately at the end of fermentation, i.e. immediately when fermentation is complete.

In some aspects of the present disclosure is also provided a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, prepared by the method as described herein above.

Beverage and methods of production thereof

The fermented beverage base, such as the fermented aqueous extract, such as the malt and/or cereal based fermented aqueous extract, described herein above may be further processed into a beverage.

It is therefore a further aspect of the present disclosure to provide a method of producing a beverage, such as a malt and/or cereal based beverage, said method comprising the steps of: i. preparing a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, by the method as described elsewhere herein, and ii. processing said fermented beverage base into a beverage.

In some embodiment of the present invention, the beverage, such as the malt and/or cereal based beverage, is diluted with a liquid, such as water. Optionally, water can be used to dilute the beverage. In one embodiment of the present invention the proportions of water: beverage may be in the range of 0.1 to 5 parts water to 1 part beverage.

The further process may for example also include lagering, chilling and/or filtering of the beverage, such as the malt and/or cereal based beverage. Also additives may be added. Furthermore, CO2 may be added (carbonation). Finally, the beverage, such as the malt and/or cereal based beverage, such as a beer, may be pasteurized and/or filtered, before it is packaged (e.g. bottled or canned).

In some embodiments, the beverage has an ethanol content of 0.0%.

In some embodiments, the beverage comprises less than 2.00% ethanol. In some embodiments, the beverage comprises less than 1.75% ethanol. In some embodiments, the beverage comprises less than 1.50% ethanol. In some embodiments, the beverage comprises less than 1.25% ethanol. In some embodiments, the beverage comprises less than 1.00% ethanol. In some embodiments, the beverage comprises less than 0.75% ethanol. In some embodiments, the beverage comprises less than 0.50% ethanol. In some embodiments, the beverage comprises less than 0.47% ethanol. In some embodiments, the beverage comprises less than 0.40% ethanol. In some embodiments, the beverage comprises less than 0.30% ethanol. In some embodiments, the beverage comprises less than 0.20% ethanol. In some embodiments, the beverage comprises less than 0.10% ethanol. In some embodiments, the beverage comprises less than 0.09% ethanol. In some embodiments, the beverage comprises less than 0.08% ethanol. In some embodiments, the beverage comprises less than 0.07% ethanol. In some embodiments, the beverage comprises less than 0.06% ethanol. In some embodiments, the beverage comprises less than 0.05% ethanol. In some embodiments, the beverage comprises less than 0.04% ethanol. In some embodiments, the beverage comprises less than 0.03% ethanol. In some embodiments, the beverage comprises less than 0.02% ethanol. In some embodiments, the beverage comprises less than 0.01% ethanol. In some embodiments, the beverage comprises less than 0.0% ethanol. In preferred embodiments, the beverage is a beer. In some embodiments, the beer is a low-alcoholic beer. In some embodiments, the beer is a non-alcoholic or alcohol-free beer. Said beer may be any kind of beer, for example an alcohol-free beer of the lager type or ale type.

It is an aspect of the present disclosure is provided, that the beverage, such as the malt and/or cereal based beverage, produced by fermenting the beverage base, such as the aqueous extract of malt and/or cereal grains, with the yeast strain according to the present disclosure has a pleasant taste.

The taste of the beverage, such as the malt and/or cereal based beverage, produced by fermentation with the yeasts according to the invention may be analyzed, for example, by a specialist beer taste panel. Preferably, said panel is trained in tasting and describing beer flavors, with special focus on aldehydes, diacetyl, esters, higher alcohols, fatty acids and sulphury components.

In general, the taste panel will consist of in the range of 3 to 30 members, for example in the range of 5 to 15 members, preferably in the range of 8 to 12 members. The taste panel may evaluate the presence of various flavours, such as papery, oxidized, aged, and bready off-flavours as well as flavours of esters, higher alcohols, sulphur components and body of beer. The overall taste of the beer will generally be rated by the taste panel on several different characteristics on a scale from 1 to 9, where an average rating of over 5 signifies that the beer has an acceptable taste.

The present invention also provides beverages, such as malt and/or cereal based beverages, prepared by the methods described above.

In some embodiments, the beverage is a wine, such as a red wine or a white wine. In some embodiments, the beverage is a cider. In some embodiments, the beverage is a juice. In some embodiments, the beverage is a sake.

In preferred embodiments, the beverage is a beer. In some embodiments, the beer is a low-alcoholic beer. In some embodiments, the beer is a non-alcoholic or alcohol-free beer. Items

1. A yeast strain of the genus Starmera, wherein said yeast strain comprises a mutant MET30 gene encoding a mutant Met30 polypeptide, wherein the wild type MET30 gene encodes i. wild type Met30 as set forth in SEQ ID NO: 1, or ii. a functional homologue of SEQ ID NO: 1 with at least 90%, such as at least 95%, such as at least 98% sequence identity to SEQ ID NO: 1.

2. The yeast strain according to item 1 , wherein said mutant Met30 polypeptide has an amino acid substitution at position 101 , 111 , 244 or 581 of SEQ ID NO: 1 , or an amino acid substitution in a corresponding position of said functional homologue thereof.

3. The yeast strain according to item 2, wherein said substitution is substitution of a hydrophobic amino acid to a polar, uncharged amino acid.

4. The yeast strain according to item 3, wherein said substitution is substitution of a leucine (L) to a serine (S).

5. The yeast strain according to item 4, wherein said substitution is substitution of the leucine (L) at position 101 of SEQ ID NO: 1, or of a corresponding leucine in said functional homologue thereof, to a serine (S).

6. The yeast strain according to any one of items 2 to 5, wherein said substitution is substitution of a cysteine (C) to a hydrophobic amino acid.

7. The yeast strain according to item 6, wherein said substitution is substitution of a cysteine (C) to an amino acid comprising a side chain comprising an aromatic group.

8. The yeast strain according to item 6, wherein said substitution is substitution of a cysteine (C) to a tryptophan (W). 9. The yeast strain according to item 8, wherein said substitution is substitution of the cysteine (C) at position 111 of SEQ ID NO: 1 , or of a corresponding cysteine in said functional homologue thereof, to a tryptophan (W).

10. The yeast strain according to any one of items 2 to 8, wherein said substitution is substitution of a hydrophobic amino acid to a charged amino acid, such as a positively charged amino acid.

11. The yeast strain according to item 10, wherein said substitution is substitution of a tyrosine (Y) to a histidine (H).

12. The yeast strain according to item 11 , wherein said substitution is substitution of the tyrosine (Y) at position 244 of SEQ ID NO: 1, or of a corresponding tyrosine in said functional homologue thereof, to a histidine (H).

13. The yeast strain according to any one of items 2 to 12, wherein said substitution is substitution of a glycine to a charged amino acid, such as a negatively charged amino acid.

14. The yeast strain according to item 13, wherein said substitution is substitution of a glycine (G) to an aspartic acid (D).

15. The yeast strain according to item 14, wherein said substitution is substitution of the glycine (G) at position 581 of SEQ ID NO: 1, or of a corresponding glycine in said functional homologue thereof, to an aspartic acid (D).

16. The yeast strain according to any one of the preceding items, wherein said wild type MET30 gene comprises the sequence as set forth in SEQ ID NO: 2 or a homologous sequence thereof, wherein said homologous sequence encodes a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as 100% sequence identity to the polypeptide encoded by SEQ ID NO: 1.

17. The yeast strain according to item 16, wherein said mutant MET30 gene has a T to C mutation in position 302 of SEQ ID NO: 2 or in a corresponding position in said homologous sequence of SEQ ID NO: 2.

18. The yeast strain according to item 16, wherein said mutant MET30 gene has a T to G mutation in position 333 of SEQ ID NO: 2 or in a corresponding position in said homologous sequence of SEQ ID NO: 2.

19. The yeast strain according to item 16, wherein said mutant MET30 gene has a T to C mutation in position 730 of SEQ ID NO: 2, or in a corresponding position in said homologous sequence of SEQ ID NO: 2.

20. The yeast strain according to item 16, wherein said mutant MET30 gene has a G to A mutation in position 1742 of SEQ ID NO: 2 or in a corresponding position in said homologous sequence of SEQ ID NO: 2.

21 . The yeast strain according to any one of the preceding items, wherein yeast of said yeast strain comprises at least 125%, such as at least 150%, such as at least 175%, such as at least 200%, such as at least 225%, such as at least 250%, such as at least 275%, such as at least 300%, such as at least 325%, such as at least 350%, such as at least 375%, such as at least 400% the level of hydrogen sulphide (H2S) compared to yeast of a reference strain of the genus Starmera, optionally wherein said reference strain has an identical genotype to said yeast strain except i. not comprising said mutant MET30 gene; and ii. comprising said wild type MET30 gene.

22. The yeast strain according to any one of the preceding items, wherein yeast of said yeast strain comprises at least 4 pg/L H2S, such as at least 6 pg/L H2S, such as at least 8 pg/L H2S, such as at least 10 pg/L H2S, such as at least 15 pg/L H2S, such as at least 20 pg/L H2S, such as at least 25 pg/L H2S, such as at least 30 pg/L H2S, such as at least 35 pg/L H2S, such as at least 40 pg/L H2S, such as at least 50 pg/L H2S, such as at least 60 pg/L H2S, such as at least 70 pg/L H2S, such as at least 80 pg/L H2S, such as at least 90 pg/L H2S, such as at least 100 pg/L H2S, such as at least 110 pg/L H2S, such as at least 120 pg/L H2S, such as at least 130 pg/L H2S, such as at least 140 pg/L H2S, or such as at least 150 pg/L H2S.

23. The yeast strain according to any one of items 21 to 22, wherein yeast content of H2S is measured by a method comprising the steps of: i. contacting a H2S-specific probe with said yeast; and ii. measuring the amount of said probe bound to said H2S within said yeast, such as by flow cytometry.

24. The yeast strain according to any one of the preceding items, wherein said yeast strain produces at the most 0.5%, such as at the most 0.4%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1 %, such as at the most 0.0% alcohol by volume (ABV) after fermentation in wort of 9° Plato for 5 days at 16°C.

25. The yeast strain according to any one of the preceding items, wherein said yeast strain is of the species Starmera caribaea.

26. The yeast strain according to any one of the preceding items, wherein said yeast strain is auxotrophic for L-methionine and/or L-cysteine.

27. The yeast strain according to any one of the preceding items, wherein said yeast strain is haploid or diploid.

28. The yeast strain according to any one of the preceding items, wherein said yeast strain is diploid and is homozygous for said mutant MET30 gene.

29. The yeast strain according to any one of the preceding items, wherein said functional homologue of Met30 of SEQ ID NO: 1 catalyzes production of sulfur compounds when expressed in said yeast strain, comparable to the level produced when wild type Starmera caribaea Met30 of SEQ ID NO: 1 is expressed in a wild type Starmera caribaea strain, wherein said sulfur compounds for example is H2S.

30. The yeast strain according to item 29, wherein said functional homologue catalyzes production of levels of H2S when expressed in said strain that are +/- 10% of the level of H2S produced in said wild type Starmera caribaea strain comprising said wild type Starmera caribaea Met30 of SEQ ID NO: 1.

31. A method of producing a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, said method comprising the steps of: i) Providing a beverage base, such as an aqueous extract of malt and/or cereal grains; ii) providing a Starmera yeast strain, wherein said yeast strain is according to any one of items 1 to 30; and iii) fermenting the beverage base, such as the aqueous extract of malt and/or cereal grains, provided in step i) with said yeast strain of step ii), thereby obtaining a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains.

32. The method according to item 31 , wherein the beverage base, such as the aqueous extract of malt and/or cereal grains, is wort.

33. The method according to any one of items 31 to 32, wherein said fermented beverage base, such as said fermented aqueous extract, comprises at the most 0.5%, such as at the most 0.4%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1%, such as at the most 0.0% alcohol by volume (ABV).

34. A fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, prepared by the method according to any one of items 31 to 33.

35. A method for producing a beverage, such as a malt and/or cereal based beverage, said method comprising the steps of: i) preparing a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, by the method according to any one of items 31 to 33, and ii) processing said fermented beverage base into a beverage. 36. The method according to item 35, wherein the steps of processing comprise one or more of the following: a) Filtration, b) Carbonation, c) Maturation, or d) Bottling

37. A beverage prepared by the method according to any one of items 35 to 36.

38. The method according to any one of items 35 to 36 or the beverage according to item 37, wherein said beverage comprises less than 2% ethanol, such as less than 1.5% ethanol, such as less than 1.0% ethanol, such as less than 0.5% ethanol, such as less than 0.3% ethanol, such as less than 0.1 % ethanol.

39. The method or beverage according to item 38, wherein said beverage is an alcohol-free beverage, such as wherein said beverage has an ethanol content of 0.5% or less, such as wherein said beverage has an ethanol content of 0.0%.

40. The method or beverage according to any one of items 38 to 39, wherein said beverage is a beer, such as wherein said alcohol-free beverage is an alcohol- free beer.

Examples

Example 1 - Analysis of hydrogen sulphide levels

Materials and methods

Microorganisms and Media

Starmera caribaea strain NRRL Y-17468 (IB0015) was obtained from the agricultural Research Service Culture Collection. The yeast was stored in a -80°C freezer in 20% (V/V) glycerol stock until used. Prior to fermentation the yeast cells were prepared by streaking the content of a 1 l inoculation needle of the yeast glycerol cryo mixture onto a YPD agar plate (Yeast extract 10g, Peptone 20g, Dextrose 20g) and incubating the plate for 48-72h at 30°C.

Fermentation Media

In the experiments of these examples, the fermentation medium was 9° Plato Holsten Wort. The Holsten wort was prepared using standard 12° Plato wort prepared from a mixture of 87% Pilsner Malt and 13% Munich Malt using the mashing profile listed in table 1. After mashing wort was boiled. At the start of boiling bitter hops was added and boiling was achieved at 105°C for 50 minutes. The resulting 12° Plato wort was subsequently adjusted to 9° Plato by diluting with brewing water and adjusted to pH 4,4 using phosphoric acid to obtain the so-called AFB Holsten Wort with the following specifications: Original gravity: 9, 0-9, 5; pH: 4, 3-4,4; Color: 8,0-11,0; Bitterness Units: 33-39.

Table 1. Mashing profile for the preparation of 12° Plato wort before diluted to 9° Plato. Pre-Cultures, Inoculation & Propagation

Precultures for propagation and fermentations was prepared by dosing the content of 1 p inoculation needle of the cells from several colonies of the YPD plate into 50m L of 9° Plato Holsten wort. The pre-cultures were cultivated for 24h at 30°C in a 100mL Erlenmeyer flask on a shaking table (120 rpm).

Strain Construction

Creating haploid derivatives of Starmera caribaea NRRL Y17468 by sporulation: Starmera caribaea is a heterothallic yeast having two opposite mating types herein called MATa and MATalpha. Most natural isolates are diploids and to be able to breed in Starmera, haploid strains needed to be created. To that extent, the diploid S. caribaea strain NRRL Y-17468 (IB0015) was streaked onto a fresh YPD agar plate, and the plate was incubated overnight at 30°C. Using an inoculation loop, some of the growth was transferred onto a P-SPOR plate (see recipe below; Chen et al., 2012) the next day, and the plate was incubated for 5-7 days at RT. Periodically, the growth was microscopically checked for asci formation. Once enough asci have formed, a small amount of growth was transferred into 180pl 100mM phosphate buffer pH7 in an Eppendorf tube, 20pl of Zymolyase (10 mg/ml) were added and the cell suspension was incubated at 30°C for approx. 30min. Subsequently, 200pl 50mM EDTA and 200pl 0.9% saline containing 0.03% Triton X-100 were added, the tube was briefly vortexed, and the cells were spun down for 10min in an Eppendorf tabletop centrifuge at full speed. After the supernatant was removed, the cells/spores were suspended in 200- 400pl sterile water. Approx. 10pl of the cell/spore suspension were used to pick spores on a Singer MSM 400 dissection microscope on a YPD plate according to the manufacturers Saccharomyces cerevisiae tetrade dissection protocol. The YPD plate was incubated at 30°C for 2 to 3 days until colonies showed up. The spore clones were finally analyzed for mating type using the sexual agglutination technique (see below) and one well growing spore clone of each mating type (IB0040 MATalpha/IB0041 MATa) was chosen for further breeding efforts with Starmera caribaea.

Sexual agglutination test: Starmera caribaea and its close relatives were found to show a strong sexual agglutination phenotype, where cells of opposite mating type clump together when mixed, but strains of the same mating type, or diploid strains mixed with a haploid strain of either mating type, stay in suspension. The sexual agglutination analysis was performed essentially as described in Mendonga-Previato et al., 1981. More specifically, to perform a sexual agglutination analysis, a strain of interest and the two haploid tester strains of opposite mating type IB0040 (MATalpha) and IB0041 (MATa) were grown up in liquid YPD at 30°C overnight. On the next day, 50pl of cells from the strain of interest were mixed separately with 50pl of cells of either of the two tester strains at an approximate 1 :1 cell ratio in a 250pl PCR tube. In case the cell agglutination was not immediately visible after mixing, the PCR tubes were briefly spun (10-15sec) in a small tabletop centrifuge for PCR tubes. While cells that do NOT agglutinate would be collected at the bottom of the tube, agglutinated cells tend to stick to the wall of the tube. To countercheck, tubes can be vortexed after spinning, and cells that did NOT agglutinate will go back into suspension, while agglutinated cells will stick together. We have used this sexual agglutination technique to determine the mating type of haploid cells, but it is also a fast and easy way to confirm the diploid state of strains derived from mating experiments.

Mating reactions in S. caribaea and diploid strain identification: To mate two haploid S. caribaea strains of opposite mating type we made use of the sexual agglutination phenotype (see before). In principle, a sexual agglutination test reaction was set up and some of the cell clumps were transferred with an inoculation loop to a fresh YPD agar plate. The agar plate was subsequently incubated at 30°C at least overnight. With an inoculation loop a little bit of growth from the mating plate was transferred into saline containing 0.03% Triton X-100 and mixed well. The ODeoo of this cell suspension was determined, the cells were diluted to an ODeoo of -0.0001 and 10OpI were plated onto ploidy dye plates (PDP; see recipe below; Takagi et al., 1983;

Yamazaki et al., 1979). The ploidy dye plates were incubated for 5-7 days at 30°C in the dark (Trypan Blue is light sensitive!). Diploid cells were found to be usually more “pale-looking” on PSPs and don’t show as bright a color as haploid cells do. The diploid nature of picked “pale-looking” colonies was initially confirmed running a standard propidium iodide stain-based ploidy analysis on an Agilent NovoCyte flow cytometer using the original diploid S. caribaea strain Y-17468 and the two haploid spore clones IB0040 and IB0041 as controls. In later breeding efforts the ploidy of picked colonies was analyzed out of convenience performing a sexual agglutination test with known haploid tester strains IB0040 and IB0041.

Mutagenesis of Starmera caribaea-. S. caribaea was mutagenized by either UV irradiation or DNA methylation with methylnitronitrosoguanidine (MNNG). For either technique, the strain of interest was first grown on YPD plates at 30°C for 2-3 days. Several uniform looking colonies were harvested with an inoculation loop and the cells were washed with sterile water containing 0.3% Triton X-100. A cell suspension was made with a cell titer of ODeoo of ~1. To perform an UV mutagenesis 5ml of this cell suspension was pipetted into a sterile petri dish, and the cells were exposed to UV light in a Stratagene UV Stratalinker 1800. The mutagenized cells were harvested by centrifugation and recovered in liquid YPD medium at 30°C for 1-2 h. To run a MNNG mutagenesis 5pl MNNG (3mg/ml water) was added to 1ml of cells in a 2ml safe-lock Eppendorf tube, and the cells were incubated at 30°C and 1000 rpm on an Eppendorf shaker. After 30 min the reaction was stopped by spin/washing the cells three times with sterile water containing 0.3% Triton X-100. This procedure would result in a killing rate of approx. 50% for most S. caribaea strains used. After the final washing step, cells were recovered in YPD medium in analogy to the UV mutagenesis.

Identifying a H2S overproducing S. caribaea strain: To isolate strains of S. caribaea that produce a higher amount of H2S in comparison to wild type, UV mutagenized cells of strain IB0040 were plated for single colonies on lead nitrate containing plates (ESL plates; recipe see below; Espinoza-Simon et al., 2022). While wild type colonies usually stay white on these plates, strains that overproduce H2S would turn brownish due to a chemical reaction between H2S and the lead nitrate in the plates. Potential candidates were picked, grown up in liquid YPD medium and then re-spotted onto ESL plates to confirm the color change of the colonies. The H2S overproducing phenotype was subsequently confirmed and analyzed on YPD-Biggy agar plates, which were found to promote better and faster growth of S. caribaea strains than the ESL plates. Specifically, 5pl of cells ODeoo of ~10 were spotted onto Biggy/YPD or Biggy plates and incubated for between 24-72 hours at 30 °C.

Analytical Methods

Quantification of Sulphur by flow cytometry: Quantification of sulphur was carried out on a Novocyte Flowcytometer running an assay using a H2S specific probe purchased from Sigma Aldrich (WSP-5 - CAS number: 1593024-78-2). Cells to be examined were inoculated in YPD and grown O/N at 30 °C. The OD of the cells were the following day adjusted to 1 and cells were subsequently washed in 10mM PBS (pH 7,4) twice. Washed cells were resuspended in 10mM PBS buffer (pH 7,4) and CTAB in addition to WSP-5 probe was added to obtain a final concentration of 1mM CTAB and 1 OpM WSP-5 probe respectively. The culture was afterwards subject to thorough mixing and incubated for 30 minutes at RT. The reaction mix was afterwards transferred to a recording chamber and run at wavelength 488/530nm ex/em on a flow cytometer. Analysis on the flowcytometry data was done using the mean and median value of the fluorescence emitted by the selected cells in detection channel 530/30.

Sequencing methods

DNA Extraction and Library Preparation: For DNA extraction, cells were grown overnight in 100ml shake flasks containing 25ml YPD medium. Biomass was collected through centrifugation for 10 minutes at 4000xg and 4°C, washed with sterile water and stored at -20°C until analysis. Genomic DNA was extracted using the DNeasy PowerSoil Pro kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Purity of the DNA was checked using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) was used to check for purity of the DNA and quantification was performed using a Qubit 4 Fluorometer (Thermo Fisher Scientific) with the dsDNA Quantification Broad Range Assay kit (Thermo Fisher Scientific). For Nanopore sequencing, the library was prepared using the SQK-NBD114-24 kit (Oxford Nanopore Technologies, Oxford, United Kingdom). The sequencing was performed on a Minion Mk1 B device (Oxford Nanopore Technologies) using a FLO-MIN114 flow cell (Oxford Nanopore Technologies) FAST5 file from the Nanopore sequencing were basecalled using Guppy version 6.5.7. For Illumina sequencing of the reference strain, the Illumina DNA PCR-free kit (Illumina, San Diego, CA, United States) was used using the IDT for Illumina DNA/RNA UD indexes Set A (Integrated DNA Technologies, Coralville, IA, United States) and the sequencing was performed on a MiniSeq system (Illumina) using the 300-cycle high output kit. FASTQ read files were generated using the Local Run Manager GenerateFastQ module version 2.5.56.9. For the remaining strains, cells were harvested as described above, and genomic DNA extraction, PCR free short insert library preparation (<800bp), 150bp paired-end sequencing on a DNBSEQ instrument was performed by BGI Genomics Global (Hong Kong).

RNA Sequencing: For RNA sequencing, cells were harvested by centrifugation at 3000xg for 5 minutes at 4°C, washed in ice-cold water, flash frozen in liquid nitrogen and stored at -80°C until analysis. mRNA extraction and strand specific, 100bp paired-end mRNA library preparation and sequencing on DNBSEQ was performed by BGI Genomics Global (Hong Kong). Genome Assembly & Annotation: For the genome assembly adapters were removed from the raw basecalled Nanopore reads using porechop version 0.2.4, and the reads were filtered using filtlong version 0.2.1 , using a length cut-off of 1 kb. Genome was performed using Flye version 2.9 with parameters --keep-haplotypes and -nano-raw. After assembly, the genome was polished using Medaka version 1.6.0 using long reads, using pilon version 1.24 using short reads. The short reads were trimmed using trimmomatic version 0.39, using parameters SLIDINGWINDOW:4:20, and mapped to the assembly using bwa-mem version 0.7.17 and pilon was run with parameters -fix bases. Finally, Nanopore reads were mapped to the assembly using minimap2 version 2.24 and purge_dups version 1.2.5 was used to remove duplicate and artefactual contigs.

The genome assembly was annotated using funannotate version 1.8.15. To prepare the assembly for gene prediction and annotation, the genome assembly was filtered to retain only scaffolds above 1 kb in length using funannotate clean, sorted by length using funannotate sort and repeats were masked using RepeatMasker version 4.1.5. The resulting assembly was used for training a gene model using funannotate train, including stranded paired-end transcriptomics data as evidence. Next, gene prediction was performed using funannotate predict, using the assembled transcriptome from the previous step as transcript evidence, and all Saccharomycetes proteins available in the OrthoDb database version 11 (accessed 2023-01-17) and the UniProt-SwissProt database as protein evidence. Prior to functional annotation, a gene prediction was performed using InterProScan version 5.62-94.0 and secondary metabolite gene clusters were predicted using antiSMASH version 7.0.0 using parameters -taxon fungi, -fullhmmer, -clusterhmmer, -tigrfam, -asf, -cc-mbig, -cb-general, -cb-subclusters, - cb-known-clusters, -pfam2go and -smcog-trees. Finally, gene annotation was performed using funannotate, incorporating the results from InterProScan and antiSMASH.

Variant Calling & Annotation: The variant calling was performed according to the GATK best practices workflow (https://gatk.broadinstitute.org/hc/en- us/sections/360007226651-Best-Practices-Workflows) using gatk version 4.4.0.0. The trimmed reads were mapped to the genome assembly using bwa-mem version 0.7.17, and samtools sort version 1.15 was used to sort and generate a bam output file. The reference was indexed using samtools faidx version 1.15 and a sequence dictionary for the reference was generated using gatk CreateSequenceDictionary. Read group information for the mapping file was added using gatk AddOrReplaceReadGroups, followed by creating a bam index file using gatk MarkDuplicates, and removing duplicate reads using gatk MarkDuplicates. Variants were called using gatk HaplotypeCaller with parameters --emit-ref-confidence GVCF --min-base-quality-score 20, -standard-min- confidence-threshold-for-calling 50. The resulting gvcf files were combined using gatk CombineGVCFs and genotyped using gatk GenotypeGVCFs. Single nucleotide polymorphisms (SNPs) and insertions and deletions (indels) were filtered using gatk SelectVariants using parameters --select “QD >= 2.0 && FS >= 60.0 && MQ >= 40.0 && MQRankSum >= 12.5 && ReadPosRankSum >= -8.0” and --select-genotype-expression “DP >= 30 && GQ >= 30” for SNPs, and -select “QD >= 2.0 && FS >= 60.0 && ReadPosRankSum >= -20.0” and -select-genotype-expressions “DP >= 30.0 && GQ >= 30” for indels. The resulting files were merged using gatk MergeVcfs. Variants were annotated using snpEff version 5.2a using a custom database for Starmera caribaea.

Sequence alignment: Protein sequences in fasta format were aligned using clustalo version 1.2.4 with arguments -full and -outfmt clu to generate an output file in clustal format.

Identifying the causative mutation for H2S overproducing strains

To identify the causative mutation for the H2S overproduction phenotype, strain IB0042 was sporulated, spores were picked and plated on YPD-Biggy agar plates, as described above. 25 of the strains were selected for sequencing, of which 14 displayed the H2S overproduction phenotype and 11 did not display the phenotype. Variants were called and annotated, and mutations were filtered based on the expected genotype where the causative mutation of the H2S overproduction should be present only in strains displaying the phenotype.

Media recipes

Ploidy Dye Plates (PDP; based on Takagi et al., 1983 and Yamazaki et al., 1979)

1 g/l yeast extract

1 g/l peptone

1 ,5g/l KH2PO4

1 ,5g/l MgSO₄

20g/l glucose pH was adjusted to pH5,6. 15g/l agar-agar

After autoclaving, 15mg/l Trypan Blue and 10mg/l Phloxin B were added. Plates need to be stored in the dark since Trypan Blue is light sensitive.

Pichia pastoris sporulation plates (P-SPOR; Chen et al, 2012)

0,5% Na-acetate

1% KCI

1% glucoase

2% agar-agar

Lead Nitrate Plates (ESL, Espinoza-Simon et al., 2022)

2,4g/l peptone

4g/l yeast extract

0,16g/l (NH₄)₂SO₄

20g/l agar-agar

After autoclaving add to 32g/l glucose and 0,8g/l PbNCh.

YPD-Biggy Agar Plates (based on Nickerson et al, 1953)

To generate YPD-Biggy agar plates commercially available Biggy Agar (Fluka) was adjusted to YPD medium compositions by adding the following components before boiling the agar medium:

0.9g/l yeast extract

2g/l peptone

10g/I glucose

H2S Probe: As described in Peng, B. et al., 2013.

Results

Strains constructed

The strains as shown below in Table 2 were created as described in the section “Strain Construction” herein above and according to the breeding scheme outlined in Figure 1.

Table 2. Yeast strains constructed in this example

Analysis of levels of hydrogen sulphide by flow cytometry

As proof of concept of the method for measuring H2S levels in yeast strains by flow cytometry, the S. cerevisiae haploid strain BY4742 was measured together with an identical strain, except comprising the known Amet17 mutation, known to result in increased H2S levels (Linderholm et al., 2008). As can be seen in Figure 2, the right shift of the peak on the X-axis for the BY4742 Amet17 strain compared to the wild type strain clearly indicates this increased level of H2S in the BY4742 Amet17 strain. The mean fluorescent intensity (Green-H) for the BY4742 strain was 1,095, while it was 1 ,569 for BY4742 Amet17. As can be seen in Figure 3, the Starmera caribaea diploid wild type (IB0015) and heterozygous met30 Y244H mutant (IB0042) strains have very similar levels of H2S as measured by flow cytometry. In contrast, the homozygous met30 Y244H mutant (IB0045) shows a marked increase in H2S levels, as seen by the peak shift on the X- axis in Figure 3.

The results of this experiment is additionally shown in Table 3, below, and it is clear that the homozygous met30 Y244H mutant (IB0045) shows a marked increase in H2S levels.

Table 3. Levels of H2S as measured by flow cytometry for strains IB0015, IB0042 and IB0045.

Several additional Starmera caribaea strains with mutations in Met30 were generated in this example (e.g. L101S, C111W, and G581 D), and it is expected that one or more of these mutants have comparable phenotypes to the Y244H mutant.

Analysis of levels of hydrogen sulphide by plating on lead nitrate containing plates Several additional mutants were analysed for their level of H2S by plating on lead nitrate containing plates as shown in Figure 4. A darker colour denotes a higher level of H₂S.

As is clear from Figure 4, strains IB0027 and IB0043 (haploid strains each comprising the met30 Y244H mutation) produce an intermediately increased level of H2S, while strain IB0045 (diploid strain homozygous for the met30 Y244H mutation) produced a significantly higher level of this compound.

The H2S overproducing phenotype was subsequently confirmed and analyzed on YPD- Biggy agar plates by measuring the mean black intensity of various met30 mutant strains, as shown in Table 4, below. A higher mean black intensity denotes a higher level of H2S.

Table 4. Mean black intensity of various strains on Biggy agar plates

These results clearly demonstrate the effects of met30 mutations Y244H, C111 W, L101S and G581 D in increasing H2S levels.

Example 2 - Fermentation of wort with Starmera caribaea strains according to the invention

Materials and methods

Pre-Cultures, Inoculation & Propagation

Precultures for propagation and fermentations was prepared by dosing the content of 1 inoculation needle of the cells from several colonies of the YPD plate into 50ml of 9° Plato Holsten wort. The pre-cultures were cultivated for 24h at 30°C in a 100ml Erlenmeyer flask on a shaking table (120rpm).

For ANKOM fermentation trials 10ml of the preculture was dosed into 150ml of 9° Plato Holsten wort in a 250ml DURAN® bottle.

Propagation for 50L fermentation trials were carried out in SB Carlsberg Flasks (AlphaLaval Nordics A/S) containing 10L 9° Plato Holsten Wort sterilized by autoclavation at 100°C for 30 minutes and cooled down to RT before use. Aeration with sterile air were connected through a membrane sample valve with continuous moderate air flow of 5l/min measured using a flowmeter (Brooks Instruments). 50ml of pre-cultured yeast were pitched to the Carlsberg flask. A content of 50ml yeast was pitched aseptically into the Carlsberg flask using a syringe through a membrane fitting. Propagation was carried out for 3 days at RT and transferred under aseptic conditions to the 50L fermentation vessels for brewing.

Fermentation Conditions

Ankom Fermentations: Initial fermentation trials were carried out in the ANKOM RF Gas Production System (ANKOM Technology). The working volume in each fermentation vessel was 150ml. The wort was prior to fermentation pasteurized in a 5L blue cap bottle at 80°C for 40 minutes before it was homogenized by gentle shaking and transferred into the autoclaved ANKOM fermenters. The fermentations were carried out at 16°C and continuously agitation utilizing at magnet stirrer at 130rpm. Monitoring and measuring of ambient pressure as a proxy of gas production was carried out using the ANKOM system. ANKOM fermentations were all run for a length of 5 days in biological triplicates and samples were harvested at the end of the fermentation. Pitching rate was 3x10⁶ yeast cells/mL. Sample preparation was carried out by centrifuging the harvested fermentation liquid at 6000x g for 10 minutes and stored at -21 °C until analysis.

50L fermentations: Fermentations were performed in 50L scale as described in patent application WO 2022/002960, section Materials and Methods.

Analysis of ethanol content

Gas Chromatograph Mass Spectrometry SIM mode (GC-MS SIM) was used to detect and quantify ethanol levels by comparison to a standard curve. The system used was Agilent technologies 7890B GC (Agilent Technologies Denmark ApS, Glostrup, Denmark) fitted with an Agilent J&W GC DB-wax column, coupled with a computer with the analytical software MassHunter (version B.08.00). The analytical conditions were as follows: constant helium flow of 1.5ml/min, transfer line temp. 250°C, ion source temperature 230°C, MS quad temp. 150°C, injection temp. 250°C. Samples were incubated for 10min at 60°C with agitation of 500rpm. The injection was performed with static headspace with a gas-tight syringe. The injection volume was 10Opl. The column temperature program was: 7 min at 50°C, from 50°C to 240°C at 30 °C min"¹. The program was finished upon reaching 240°C. Quantification of hydrogen sulphide by flow cytometry

Quantification of hydrogen sulphide was carried out on a Novocyte Flowcytometer running an assay using a H2S specific probe purchased from Sigma Aldrich (WSP-5 - CAS number: 1593024-78-2). Cells to be examined were inoculated in YPD and grown O/N at 30°C. The OD of the cells were the following day adjusted to 1 and cells were subsequently washed in 10mM PBS (pH7,4) twice. Washed cells were resuspended in 10mM PBS buffer (pH7,4) and CTAB in addition to WSP-5 probe was added to obtain a final concentration of 1mM CTAB and 10pM WSP-5 probe respectively. The culture was afterwards subject to thorough mixing and incubated for 30 minutes at RT. The reaction mix was afterwards transferred to a recording chamber and run at wavelength 488/530nm es/em on a flow cytometer. Analysis on the flowcytometry data was done using the mean and median value of the fluorescence emitted by the selected cells in detection channel 530/30.

Results

Analysis of levels of ethanol at end of fermentation

Ankom trials

Results from Ankom fermentations with strains IB0015 and IB0045 (further described in Example 1), and reference S. pastorianus strain 1 comprising a wild type MET30 gene are shown in Table 5, below:

Table 5. Alcohol levels et end of Ankom fermentations (day 5)

As is clear from table 5, Starmera caribaea strains IB0015 (expressing wild type Met30) and IB0045 (expressing mutant Met30 with the Y244H mutation) show significantly reduced alcohol levels at the end of fermentation compared to the reference S. pastorianus strain.

50L trials

Results from fermentations in 50L scale with strains IB0015 and IB0045 (further described in Example 1), and reference S. pastorianus strain 1 comprising a wild type MET30 gene are shown in Table 6, below:

Table 6. Alcohol levels et end of 50L fermentations (day 7)

As is clear from Table 6, Starmera caribaea strains IB0015 (expressing wild type Met30) and IB0045 (expressing mutant Met30 with the Y244H mutation) show significantly reduced alcohol levels at the end of fermentation compared to the reference S. pastorianus strain.

Analysis of levels of hydrogen sulphide at end of fermentation

Analysis of H2S levels of various strains as measured by flow cytometry are shown in table 7, below. The genotypes of these strains are further described in Example 1.

Table 7. Analysis of H2S levels of various strains

As is clear from the above table, all met30 mutant strains (IB0027, IB0049, IB0050, IB0051, and IB0052) produce significantly higher levels of H2S compared to the reference strain (IB0040).

Example 3 - Analysis of Starmera caribaea sulphur metabolism compared to Saccharomyces cerevisiae Materials and methods

Determining homology to sulphur metabolism in Saccharomyces cerevisiae

The homology of sulphur metabolism in S. caribaea and S. cerevisiae was determined by performing a homology search for proteins involved in sulphur metabolism in S. cerevisiae (Huang et al., 2017) against the proteome of S. caribaea. The protein sequences from S. cerevisiae S288C were downloaded from the UniProt protein database and each of the sequences was compared to the S. caribaea proteome using BlastP, executed through the NcbiblastpCommandline module of BioPython (v1.79). For the proteins where no homolog was found, the conclusion that the proteins are missing in S. caribaea was drawn.

Protein sequences in fasta format were aligned using clustalo version 1.2.4 with arguments -full and -outfmt clu to generate an output file in clustal format.

Growth on plates with various sulphur sources

Strains were pre-grown overnight in liquid YPD medium into stationary phase (30°C). On the next day, the ODeoo of each culture was determined, and cell density was adjusted to ODeoo of ~1. Subsequently, five tenfold dilutions were done, and 5pl of each dilution was spotted from left to right onto agar plates, which were then incubated at 30°C for three or seven days respectively.

The SD medium used in this example contains 4g/L ammonium chloride as nitrogen source instead of ammonium sulfate.

Results

Table 8, below, shows the sequence identities between the most relevant proteins known to be involved in sulphur metabolism in S. cerevisiae with their identified corresponding homologs in S. caribaea.

Table 8. Sequence identities between S. cerevisiae and S. caribaea sulphur metabolism proteins

As is clear from the above table, several important sulphur metabolism homologs are completely absent in Starmera caribaea (Met3, Met4, and Met16). Additionally, a substantial number of other homologs show low sequence identities of under 60%, with the S. caribaea Met30 homolog showing only 54,2% sequence identity to the S. cerevisiae protein.

Growth of Starmera caribaea on various sulphur sources.

As shown in Figure 5, growth of all Starmera caribaea strains is sustained on methionine, and cysteine within 3 days, similarly to as what has been previously reported (see e.g. Phaff et al., 1992). Growth on reduced glutathione and thiosulfate only appears after 7 days of incubation indicating impaired or absent ability to grow on media with only these sulphur sources. This is in contrast to Saccharomyces cerevisiae, which is able to also grow efficiently using glutathione or thiosulfate as sulphur sources.

Taken together, these results indicate a significantly altered sulphur metabolism pathway in Starmera caribaea compared to Saccharomyces cerevisiae.

Example 4 - Alternative fermentation process in wort with Starmera caribaea strains

Materials and methods

Pre-Cultures, Inoculation & Propagation

Precultures, inoculation and propagation carried out as described in Example 2.

Fermentation Conditions

Ankom Fermentations: Initial fermentation trials were carried out in the ANKOM RF Gas Production System (ANKOM Technology). The working volume in each fermentation vessel was 150ml. The wort was prior to fermentation pasteurized in a 5L blue cap bottle at 80°C for 40 minutes before it was homogenized by gentle shaking and transferred into the autoclaved ANKOM fermenters. The fermentations were carried out at 14°C and continuously agitation utilizing at magnet stirrer at 130rpm. The pitching rate was 6x10⁶ yeast cells/mL. Monitoring and measuring of ambient pressure as a proxy of gas production was carried out using the ANKOM system. ANKOM fermentations were all run for a length of 4 days in biological triplicates and samples were harvested at the end of the fermentation. Sample preparation was carried out by centrifuging the harvested fermentation liquid at 6000x g for 10 minutes and stored at - 21°C until analysis.

50 L fermentations: Fermentations were performed in 50L scale as described in patent application WO 2022/002960, section Materials and Methods. However, the pitching rate was 6x10⁶ yeast cells/mL and the fermentations were carried out at 14 °C and run for a length of 4 days.

Sequence overview

References

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Claims

1. A yeast strain of the genus Starmera, wherein said yeast strain comprises a mutant MET30 gene encoding a mutant Met30 polypeptide, wherein the wild type MET30 gene encodes i. wild type Met30 as set forth in SEQ ID NO: 1, or ii. a functional homologue of SEQ ID NO: 1 with at least 90%, such as at least 95%, such as at least 98% sequence identity to SEQ ID NO: 1 , wherein a) yeast of said yeast strain comprises at least 125%, such as at least

150%, such as at least 175%, such as at least 200%, such as at least

225%, such as at least 250%, such as at least 275%, such as at least

300%, such as at least 325%, such as at least 350%, such as at least

375%, such as at least 400% of the level of hydrogen sulphide (H2S) compared to yeast of a reference strain of the genus Starmera, optionally wherein said reference strain has an identical genotype to said yeast strain except i. not comprising said mutant MET30 gene; and ii. comprising said wild type MET30 gene; b) said functional homologue of Met30 of SEQ ID NO: 1 catalyzes production of sulfur compounds when expressed in said yeast strain, comparable to the level produced when wild type Starmera caribaea Met30 of SEQ ID NO: 1 is expressed in a wild type Starmera caribaea strain, wherein said sulfur compounds for example is H2S, preferably wherein said functional homologue catalyzes production of levels of H2S when expressed in said strain that are +/- 10% of the level of H2S produced in said wild type Starmera caribaea strain comprising said wild type Starmera caribaea Met30 of SEQ ID NO: 1.

2. The yeast strain according to claim 1 , wherein said mutant Met30 polypeptide has an amino acid substitution at position 101 , 111 , 244 or 581 of SEQ ID NO: 1 , or an amino acid substitution in a corresponding position of said functional homologue thereof.

3. The yeast strain according to any one of the preceding claims, wherein said mutant Met30 polypeptide a. has an amino acid substitution of the leucine (L) at position 101 of SEQ ID NO: 1, or of a corresponding leucine in said functional homologue thereof, to a serine (S); and/or b. has an amino acid substitution of the cysteine (C) at position 111 of SEQ ID NO: 1, or of a corresponding cysteine in said functional homologue thereof, to a tryptophan (W); and/or c. has an amino acid substitution of the tyrosine (Y) at position 244 of SEQ ID NO: 1, or of a corresponding tyrosine in said functional homologue thereof, to a histidine (H); and/or d. has an amino acid substitution of the glycine (G) at position 581 of SEQ ID NO: 1, or of a corresponding glycine in said functional homologue thereof, to an aspartic acid (D).

4. The yeast strain according to any one of the preceding claims, wherein said wild type MET30 gene comprises the sequence as set forth in SEQ ID NO: 2 or a homologous sequence thereof, wherein said homologous sequence encodes a polypeptide with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as 100% sequence identity to the polypeptide encoded by SEQ ID NO: 1.

5. The yeast strain according to any one of the preceding claims, wherein yeast of said yeast strain comprises at least 4 pg/L H2S, such as at least 6 pg/L H2S, such as at least 8 pg/L H2S, such as at least 10 pg/L H2S, such as at least 15 pg/L H2S, such as at least 20 pg/L H2S, such as at least 25 pg/L H2S, such as at least 30 pg/L H2S, such as at least 35 pg/L H2S, such as at least 40 pg/L H2S, such as at least 50 pg/L H2S, such as at least 60 pg/L H2S, such as at least 70 pg/L H2S, such as at least 80 pg/L H2S, such as at least 90 pg/L H2S, such as at least 100 pg/L H2S, such as at least 110 pg/L H2S, such as at least 120 pg/L H2S, such as at least 130 pg/L H2S, such as at least 140 pg/L H2S, or such as at least 150 pg/L H2S.

6. The yeast strain according to any one of the preceding claims, wherein said yeast strain produces at the most 0.5%, such as at the most 0.4%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1%, such as at the most 0.0% alcohol by volume (ABV) after fermentation in wort of 9° Plato for 5 days at 16°C.

7. The yeast strain according to any one of the preceding claims, wherein said yeast strain is of the species Starmera caribaea.

8. A method of producing a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, said method comprising the steps of: i) Providing a beverage base, such as an aqueous extract of malt and/or cereal grains; ii) providing a Starmera yeast strain, wherein said yeast strain is according to any one of claims 1 to 7; and iii) fermenting the beverage base, such as the aqueous extract of malt and/or cereal grains, provided in step i) with said yeast strain of step ii), thereby obtaining a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains.

9. The method according to claim 8, wherein said fermented beverage base, such as said fermented aqueous extract, comprises at the most 0.5%, such as at the most 0.4%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1%, such as at the most 0.0% alcohol by volume (ABV).

10. A fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, prepared by the method according to any one of claims 8 to 9.

11. A method for producing a beverage, such as a malt and/or cereal based beverage, said method comprising the steps of: i) preparing a fermented beverage base, such as a fermented aqueous extract of malt and/or cereal grains, by the method according to any one of claims 8 to 9, and ii) processing said fermented beverage base into a beverage.

12. The method according to claim 11 , wherein the steps of processing comprise one or more of the following: a) Filtration, b) Carbonation, c) Maturation, or d) Bottling

13. A beverage, such as a beer, prepared by the method according to any one of claims 11 to 12.

14. The method according to any one of claims 11 to 12 or the beverage according to claim 13, wherein said beverage comprises less than 2% ethanol, such as less than 1.5% ethanol, such as less than 1.0% ethanol, such as less than 0.5% ethanol, such as less than 0.3% ethanol, such as less than 0.1 % ethanol, such as wherein said beverage has an ethanol content of 0.0%.