Recombinant yeast that has a positive phenotype for opacity.

Abstract

Provided herein is a recombinant organism comprising (a) a heterologous condition specific secretion 1 (CSS1) gene; or (b) a heterologous FLO5-CSS1 gene fusion operably linked to a promoter in the genome of the organism.

Description

1 / 35 “RECOMBINANT YEAST THAT HAS A POSITIVE PHENOTYPE FOR OPACITY” Incorporation by reference of the sequence listing.

[001] This application contains, as a separate part of the description, a Sequence Listing in machine-readable format (file name: 58543SeqListing.xml; Size: 27,530 bytes; Created: March 11, 2024), which is incorporated by reference into this document in its entirety. BACKGROUND

[002] Opacity is a well-known phenomenon in the beverage industry. Creating opacity in beer for modern pale ales is a desired visual attribute for many craft beer drinkers. Some yeast strains are better at promoting opacity than others. For example, the research described in this paper classified strains as opacity-positive or opacity-neutral based on the ease of producing opacity in dry-hopped beers. Yeast strains from the “London Ale 3” family of brewer's yeast were classified as opacity-positive. In contrast, the “Vermont” or “Conan” strains were classified as opacity-neutral. It is important to note that the opacity locus in the yeast genome has not yet been identified. Thus, the need remains in the art for methods of identifying a yeast with an opacity-positive phenotype at the genomic level. SUMMARY

[003] In one aspect, a recombinant organism comprising a condition-specific secretion heterologous gene 1 (CSS1) operably linked to a promoter is provided in the present document. Petition 870250083056, dated 09 / 15 / 2025, p. 7 / 88 2 / 35

[004] In another aspect, a recombinant organism comprising a fusion of the heterologous FLO5CSS1 gene operably linked to a promoter is described in this document.

[005] In another aspect, a method for identifying a yeast with a positive phenotype for opacity is described in this document, the method comprising (a) detecting the presence of a CSS1 gene in the yeast genome and (b) determining the length of the protein encoded by the CSS1 gene, wherein the presence of a Css1 protein comprising an amino acid sequence having a serine-rich region at the N-terminal that is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminal of SEQ ID NO: 1, and which is composed of at least 45% serine, identifies the yeast as having a positive phenotype for opacity. In some embodiments, the Css1 protein further comprises an amino acid sequence having a serine / threonine-rich region at the C-terminal that is expanded by at least 36 amino acids compared to the serine / threonine-rich region at the C-terminal of SEQ ID NO: 1.

[006] In another aspect, a method for identifying a yeast that has an opacity-neutral phenotype is described in this document, wherein the method comprises (a) detecting the presence of a CSS1 gene in the yeast genome and (b) determining the length of the protein encoded by the CSS1 gene, wherein the presence of a Css1 protein comprising an amino acid sequence lacking an N-terminal serine-rich region that is expanded by at least 14 amino acids in length compared to the N-terminal serine-rich region of SEQ ID NO: 1, and which is composed of at least 45% serine, identifies the yeast as having an opacity-neutral phenotype.

[007] In another aspect, a document is described Petition 870250083056, dated 09 / 15 / 2025, page 8 / 88 3 / 35 method for promoting a positive phenotype for opacity in a yeast, wherein the method comprises introducing a heterologous CSS1 gene operably linked to a promoter in the yeast genome.In some embodiments, the method comprises (a) identifying the presence of a short form of the CSS1 gene in the yeast genome, wherein the short form of the CSS1 gene encodes a Css1 protein comprising an amino acid sequence lacking an N-terminal serine-rich region that is expanded by at least 14 amino acids in length compared to the amino acid sequence established in SEQ ID NO: 1, and is composed of at least 45% serine; and (b) introducing an operably linked heterologous CSS1 gene to a promoter, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with an N-terminal serine-rich region that is expanded by at least 14 amino acids in length compared to the amino acid sequence established in SEQ ID NO: 1, and is composed of at least 45% serine.In some embodiments, the long form of the Css1 protein further comprises an amino acid sequence that has a serine / threonine-rich region at the C-terminal that is expanded by at least 36 amino acids compared to the serine / threonine-rich region at the C-terminal of SEQ ID NO: 1.

[008] In another aspect, a method for promoting a positive phenotype for opacity in yeast is described in this document, wherein the method comprises introducing an operably linked FLO5-CSS1 gene fusion into a promoter in the yeast genome.

[009] In another aspect, a method for promoting a neutral phenotype to opacity in yeast is described in this document, wherein the method comprises modifying a gene Petition 870250083056, dated 09 / 15 / 2025, p. 9 / 88 4 / 35 CSS1 in the yeast genome encodes a long form of a Css1 protein, where the modification step results in the inactivation of the CSS1 gene or its replacement with a short form of CSS1. BRIEF DESCRIPTION OF THE FIGURES

[0010] Figure 1 shows the results of the OYL-011 opacity-positive phenotype backcrossed with the Maxithiol wine strain (homozygous diploid) and the resulting opacity-positive isolates.

[0011] Figure 2 shows that the 0-100 kb region of chromosome IX exhibited the greatest sequence-specific variation for opacity-positive strains 7A and 7B. This region of chromosome IX contains the candidate opacity-positive locus.

[0012] Figure 3 shows that the positive phenotype for opacity correlates with the long CSS1 allele.

[0013] Figure 4 shows that the opacity-positive strain OYL077 was identified by the long CSS1 allele.

[0014] Figure 5 shows that CSS1 disruption in opacity-positive strains results in dry hop-dependent opacity loss.

[0015] Figure 6 shows that the long CSS1 allele is confirmed in additional opacity-positive strains.

[0016] Figure 7A is a schematic of the Css1 protein from S288C.

[0017] Figure 7B is a schematic of the short form of the protein. Css1.

[0018] Figure 7C is a schematic of a long form of the Css1 protein.

[0019] Figures 8A-8C. Characterization of the opacity phenotype. Figure 8A: Image and opacity measurements documenting the opacity-positive phenotype of OYL-011. Figure 8B: Image and opacity measurements documenting the opacity-neutral phenotype of OYL-004. Figure 8C: Opacity measurements with the addition of hops. Petition 870250083056, dated 09 / 15 / 2025, page 10 / 88 5 / 35 dry on the seventh day in a collection of brewing strains. The average of a minimum of three experimental replicates is represented for each strain with error bars representing the standard deviation. Dashed line at 200 NTUs indicates cutoff point to define the positive and neutral phenotype for opacity.

[0020] Figures 9A-9D. OYL-011 backcross and identification of a candidate opacity locus in the left telomeric region of Chr IX. Figure 9A: Schematic representation of the OYL-011 backcross and wine strain. Figure 9B: Opacity measurements of opacity-positive isolates from each backcross. Figure 9C: Opacity measurements of the parental strains and the two opacity-positive and two opacity-neutral BC7 isolates. Figure 9D: Variants specific to the two opacity-positive BC7 isolates map to a candidate opacity locus on the left arm of Chr IX. The proportion of variants found in the BC7 isolates relative to the original wine strain (geometric y-axis) is plotted with a 50 bp sliding window along the coordinates of chromosome IX of the S288C reference genome (x-axis).

[0021] Figures 10A-10D. Large expansions of intergenic repeats in the OYL-011 CSS1 allele are associated with opacity. Figure 10A: Coverage plot using short reads from Illumina whole-genome sequencing of the original wine strain (top row) and the original OYL-011 strain (bottom row) mapped to the S288C reference genome. Regions within the N-terminal and C-terminal show higher coverage in the OYL-011 strain, indicating possible repeat expansions in the CSS1 regions. Figure 10B: Coverage plot using short reads from Illumina whole-genome sequencing for isolates BC7-A and BC7-B and BC7-C and BC7D. Isolates BC7-A and BC7-B also exhibit higher coverage in the N-terminal and C-terminal regions. Figure 10C: Genotyping of Petition 870250083056, dated 09 / 15 / 2025, page 11 / 88 6 / 35 BC7-A spores for N-terminal expansion CSS1, short allele (~580 bp) and long allele (~2415 bp) and the correlation of the long allele with the positive phenotype for opacity. Figure 10D: Alignment of alleles S288C, wine strain and OYL-011 CSS1. Figure 10E: Schematic representation of intragenic repeats in CSS1 for alleles S288C, wine strain and OYL-011. The legend indicates identified repeat motifs along with secretory sequences and candidate GPI anchors.

[0022] Figures 11A-11D. N-terminal and C-terminal expansions in CSS1 in a collection of brewing strains. Figure 11A: Scheme of sequence markers used to extract reads from long-read sequencing datasets and determine N-terminal (tag1 and tag2) and C-terminal (tag2 and tag3) lengths in CSS1 alleles. Figure 11B: Violin plots indicating the size and distribution of the N-terminal (region between tag1 and tag2) in long reads obtained from various brewing strains. Those positive for opacity are indicated as green and those neutral for opacity as red. Strains not distinguished for the opacity phenotype are white. Figure 11C: Violin plots indicating the size and distribution of the C-terminal (region between tag2 and tag3) in long reads obtained from various brewing strains. Those positive for opacity are indicated as green and those neutral for opacity as red. Strains not distinguished for the opacity phenotype are white.Figure 11D: Opacity phenotype of strains identified as having an expanded N-terminal CSS1.

[0023] Figures 12A-12C. CSS1 is required for dry hop formation depending on opacity. Figure 12A: PCR confirmation of complete CRISPR / Cas9 disruption of the CSS1 gene in all css1 Δ strains. Figure 12B: The resulting opacity phenotype of css1 Δ strains. Figure 12C: Typical IPA recipe fermented with OYL-011 (left) and OYL-011 css1 Δ (right). Petition 870250083056, dated 09 / 15 / 2025, p. 12 / 88 7 / 35

[0024] Figure 13. Interruption of the fusion of the CSS1 gene and the gene FLO5-CSS1 results in a reduction in opacity.

[0025] Figure 14. The long CSS1 allele is sufficient for the formation of dry hop-dependent opacity. The resulting opacity in the opacity-neutral lager strain of the parent (OYL-106) and the modified strain where the native (short) CSS1 allele was replaced by the long CSS1 allele (OYL-106 + long CSS1 allele). DETAILED DESCRIPTION

[0026] The present description is based, in part, on the finding that yeasts with a long allele of the condition-specific secretion gene 1 (CSS1) (or a FLO5-CSS1 gene fusion) exhibit a positive phenotype for opacity. The expression “positive phenotype for opacity” refers to the ability of a yeast to produce beer that has a hazy appearance or turbidity after the addition of hop material during or after fermentation (dried hops). A yeast strain that results in a turbidity measurement above 200 NTU in a sample of beer with dried hops is considered “positive for opacity”. Turbidity is measured by different methods of turbid media photometry, such as nephelometry, opacimetry, and turbidimetry. In general, it is expressed in NTU (Nephelometric Turbidity Unit).In the brewing field, the units of measurement for the disorder are the EBC (European Brewing Convention), the ASBC (American Society of Brewing Chemists), the Helm, and the FTU (Formazine Nephelometric Unit). The relationship between these different units is as follows: 1 EBC = 69.2 ASBC = 40 Helm = 4 FTU (Analytica EBC - method 9.30).

[0027] Turbidity measurements are performed using a device such as a turbidimeter or nephelometer. It is generally a photoelectric receptor that measures the light scattered by the liquid. More specifically, it is the scattering of light by the suspensions that allows for the evaluation of... Petition 870250083056, dated 09 / 15 / 2025, p. 13 / 88 8 / 35 concentration of substances suspended in a liquid. This apparatus usually consists of a white light source or infrared light. In nephelometry, scattered light is measured at a 90° angle and at a 25° angle relative to the incident light. In turbidimetry, scattered light is measured by a detector placed on the axis of the incident light.

[0028] In one aspect, a recombinant organism comprising a heterologous condition-specific secretion gene 1 (CSS1) operably linked to a promoter is described in the present document. The CSS1 gene is located near the left telomere on chromosome IX of the S. cerevisiae genome and encodes an amino acid sequence 995 amino acids long (Uniprot Accession No. P40442) in a type strain of S. cerevisiae. Little or nothing is known about the function of the Css1 protein.In contrast to the surprising results demonstrated in this paper, previous studies indicated that CSS1 overexpression in wine yeast reduced wine opacity (termed HPF1' by Brown et al.). According to the consensus sequence for CSS1 in the Saccharomyces cerevisiae S288C reference genome (SEQ ID NO: 24), the Css1 protein contains a serine-rich region near the N-terminal (residues 27 to 303 of SEQ ID NO: 24), a central domain (residues 304-570 of SEQ ID NO: 24), and a serine / threonine-rich region near the C-terminal (residues 571 to 995 of SEQ ID NO: 24). It has a putative secretion signal at the N-terminal (residues 1 to 26 of SEQ ID NO: 24). Of the additional S. cerevisiae sequences provided in this document (short CSS1 allele SEQ ID NO: 1 and long CSS1 allele SEQ ID NO: 23), there is an additional hydrophobic region with a supposed GPI anchor attachment site at the C-terminal.This supposed GPI anchor is not present in the CSS1 consensus sequence provided by the S288C reference genome (SEQ ID NO. 24). As described in this document for the first time, there is a tremendous... Petition 870250083056, dated 09 / 15 / 2025, page 14 / 88 9 / 35 Diversity in the size of CSS1 proteins in industrial brewing strains. Expansion of the serine-rich region at the N-terminal and the serine / threonine-rich region at the C-terminal was correlated with increased stability of dry hop-dependent opacity. Deletion of CSS1 in opacity-positive brewing yeasts resulted in loss of dry hop-dependent opacity stability, demonstrating that expanded CSS1 alleles were necessary for dry hop-dependent opacity in beer.

[0029] In some embodiments, the CSS1 heterologous gene encodes a long form of a Css1 protein comprising an amino acid sequence with an N-terminal serine-rich region that is expanded by at least 14 amino acids in length compared to the N-terminal serine-rich region of SEQ ID NO: 1. In some embodiments, the CSS1 heterologous gene encodes a long form of a CSS1 protein comprising an amino acid sequence with an N-terminal serine-rich region that is expanded by at least 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 675, 700, 750, 77, 800 or more amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1.In some embodiments, the heterologous gene CSS1 encodes a long form of a CSS1 protein comprising an amino acid sequence with an N-terminal serine-rich region that is expanded by at least 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 675, 700, 750, 77, 800 or more amino acids in length compared to the N-terminal serine-rich region of SEQ ID NO: 1. In some embodiments, the long form of the Css1 protein comprises an amino acid sequence longer than 995 amino acids. In some forms, the long form of the Css1 protein with... Petition 870250083056, dated 09 / 15 / 2025, p. 15 / 88 10 / 35 comprises an amino acid sequence that is at least 1,200 amino acids long. In some embodiments, the long form of the Css1 protein comprises an amino acid sequence of at least 1,220; 1,250; 1,300; 1,350; 1,400; 1,450; 1,500; 1,550; or 2,000 amino acids in length.

[0030] In some embodiments, the serine-rich region at the N-terminal comprises the amino acid sequence set at SEQ ID NO: 2 (XXXXSSXSXXSSSX). In some embodiments, the serine-rich region at the N-terminal end comprises one or more amino acid sequences set at SEQ ID NOs: 3-19. In some embodiments, the long form of the Css1 protein comprises a serine-rich region at the N-terminal comprising at least 10 repeats (e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more repeats) of one or more SEQ ID NOs: 2-19.

[0031] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with an N-terminal serine-rich region that is composed of at least 45% serine. In some embodiments, the N-terminal serine-rich region is composed of at least 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65% or more serine. In some embodiments, the heterologous CSS1 gene encodes a long form of a CSS1 protein comprising an amino acid sequence with an N-terminal serine-rich region that is expanded by at least 644 amino acids and is composed of at least 50% serine.

[0032] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with a serine / threonine-rich region at the C-terminal that is expanded by at least 36 amino acids. Petition 870250083056, 15 / 09 / 2025, p. 16 / 88 11 / 35 mentum (for example, at least 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 576, 600, 650, 700 or more amino acids in length) compared to the C-terminus serine-rich region of SEQ ID NO: 1. In some embodiments, the C-terminus serine / threonine-rich region comprises the amino acid sequence established in one or more of SEQ ID NOs: 20-22. In some embodiments, the long form of the Css1 protein comprises a serine / threonine-rich C-terminus comprising at least 1 repeat (e.g., at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more repeats) of one or more of the SEQ ID NOs: 20-22.

[0033] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with a C-terminal serine / threonine-rich region that is composed of at least 40% serine and threonine. In some embodiments, the C-terminal serine / threonine-rich region is composed of at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65% or more serine and threonine.

[0034] In some embodiments, the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0035] In another aspect, a recombinant organism comprising a FLO5CSS1 gene fusion operably linked to a promoter is described in this document. In some embodiments, the FLO5-CSS1 gene fusion encodes an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95% or more) identical to the amino acid sequence established in SEQ ID NO: 25. In some embodiments, the FLO5-CSS1 gene fusion encodes an amino acid sequence established in Petition 870250083056, dated 09 / 15 / 2025, p. 17 / 88 12 / 35 SEQ ID NO: 25.

[0036] In some embodiments, the recombinant organism described in this document has a positive phenotype for opacity, as determined by a dry hop-induced opacity assay (as described in Example 1).

[0037] The terms “operably linked” or “functionally linked” refer to the association of nucleic acid sequences in a single nucleic acid fragment, such that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence encoding an RNA or a polypeptide if the two sequences are situated in such a way that the regulatory DNA sequence affects the expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in either sense or antisense orientation.

[0038] The term “promoter” refers to a nucleotide sequence, usually upstream (5') of its coding sequence, that controls the expression of the coding sequence by providing the recognition site for RNA polymerase and other factors necessary for proper transcription. “Promoter” includes a minimal promoter, which is a short DNA sequence composed, in some cases, of a TATA box and other sequences that serve to specify the transcription start site, to which regulatory elements are added to increase expression. “Promoter” also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements and that has the capacity to control the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and distal upstream elements. Petition 870250083056, dated 09 / 15 / 2025, page 18 / 88 13 / 35 such, the latter elements often called enhancers. Thus, an “enhancer” is a DNA sequence that can stimulate promoter activity and can be an innate promoter element or a heterologous element inserted to increase the level or tissue specificity of a promoter. It has the ability to operate in both orientations (normal or inverted) and has the ability to function even when moved upstream or downstream of the promoter. Both enhancers and other upstream promoter elements bind to sequence-specific DNA-binding proteins that mediate their effects. Promoters can be derived entirely from a native gene, or be composed of different elements derived from different promoters found in nature, or even be composed of synthetic DNA segments.

[0039] A promoter may also contain DNA sequences that are involved in binding protein factors, which control the effectiveness of transcription initiation in response to physiological or developmental conditions. The “initiation site” is the position around the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site, all other sequences of the gene and its control regions are numbered. Downstream sequences (i.e., other protein-coding sequences in the 3' direction) are termed positive, while upstream sequences (mainly control regions in the 5' direction) are termed negative.

[0040] In some embodiments, the promoter is a heterologous promoter (e.g., a promoter that is not native to the CSS1 gene). In some embodiments, the promoter is a CSS1 promoter. In some embodiments, the promoter is a TDH3 promoter, a TDH2 promoter, a CCW12 promoter, a PGK1 promoter, an ADH1 promoter, an ADH2 promoter, a CYC1 promoter, an HHF1 promoter, a pro Petition 870250083056, dated 09 / 15 / 2025, page 19 / 88 14 / 35 HHF2 motor, an aTEF1 promoter, a TEF2 promoter, an HTB2 promoter, a PAB1 promoter, an ALD6 promoter, an RNR1 promoter, an RNR2 promoter, a POP6 promoter, a RAD27 promoter, a PSP2 promoter, a REV1 promoter, an MFA1 promoter, an MFa2 promoter, a GAL1 promoter, a CUP1 promoter, a MET25 promoter, an ICL1 promoter, an ICL2 promoter, a GAL3 promoter, an HXT1 promoter, an HXT2 promoter, a MAL11 promoter, a MAL31 promoter, a MAL32 promoter, a MAL33 promoter, an MRK1 promoter or a SUC2 promoter.

[0041] In some embodiments, the recombinant organism is a yeast. In some embodiments, the yeast is of the genus Saccharomyces sp. In some embodiments, the yeast is Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces eubayanus, Saccharomyces paradoxus, Saccharomyces mikitae, Saccharomyces arboricolus, Saccharomyces kudriavzevii, Saccharomyces jurei, Saccharomyces pastorianus, Torulaspora delbrueckii, Wickerhamomyces anomolus, Pichia kluyveri, Metschnikowia reukaufii, Hanseniaspora uvarum, or Lachancea thermotolerans.

[0042] Techniques for recombinant expression of a heterologous gene in a cell and genetic modification of a recombinant yeast cell are well known to those skilled in the art. Typically, these techniques involve transforming a yeast cell with the nucleic acid construct comprising the relevant sequence (e.g., CSS1 gene). Such methods are, for example, known from standard manuals such as Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al., eds., Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York (1987). Methods for transformation and genetic modification of fungal host cells Petition 870250083056, dated 09 / 15 / 2025, page 20 / 88 15 / 35 cases are described, for example, in Application No. EP-A-0635574, International Patent Publication No. WO 98 / 46772, International Patent Publication No. WO 99 / 60102, International Patent Publication No. WO 00 / 37671, International Patent Publication No. WO 90 / 14423, Application No. EP-A-0481008, Application No. EP-A-0635574 and Pat. No. US 6,265,186, the descriptions of which are incorporated herein by reference in their entirety.

[0043] Recombinant host cells (e.g., yeast cells) can be cultured using procedures known in the art. For each combination of a promoter and a host cell, culture conditions are available that lead to the expression of the DNA sequence encoding the polypeptide. After reaching the desired cell density or polypeptide titer, the culture is stopped and the polypeptide is recovered using known procedures.

[0044] The fermentation medium may include a known culture medium containing a carbon source (e.g., glucose, maltose, molasses, etc.), a nitrogen source (e.g., ammonium sulfate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g., yeast extract, malt extract, peptone, etc.), and inorganic nutrient sources (e.g., phosphate, magnesium, potassium, zinc, iron, etc.). Optionally, an inducer (dependent on the expression construct used) may be included or subsequently added.

[0045] The selection of the appropriate medium may be based on the choice of expression host and / or on the regulatory requirements of the expression construct. Suitable media are well known to those skilled in the art. The medium may, if desired, contain additional components that favor the transformed expression hosts over other potentially infectious microorganisms. Petition 870250083056, dated 09 / 15 / 2025, p. 21 / 88 16 / 35 contaminants.

[0046] Fermentation can be carried out over a period of 0.5 to 30 days. Fermentation can be a batch, continuous, or fed-batch process, at a suitable temperature in the range of 0 °C to 45 °C and, for example, at a pH of 2 to 10. Preferred fermentation conditions include a temperature between 9 °C and 37 °C and / or a pH between 3 and 7. The appropriate conditions are generally selected based on the choice of fermenting organism and the beverage being fermented. Promoting a Positive Phenotype for Opacity

[0047] In another aspect, a method for promoting an opacity-positive phenotype in yeast is described in this document, comprising the introduction of a heterologous CSS1 gene operably linked to a promoter in the yeast genome. In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with an N-terminal serine-rich region that is expanded by at least 14 amino acids in length compared to the N-terminal serine-rich region of SEQ ID NO: 1.

[0048] In another aspect, a method for promoting a positive phenotype for opacity in yeast is described in this document, wherein the method comprises (a) identifying the presence of a short form of the CSS1 gene in the yeast genome, wherein the short form of the CSS1 gene encodes a Css1 protein comprising an amino acid sequence lacking an N-terminal serine-rich region that is expanded by at least 14 amino acids in length compared to the N-terminal serine-rich region of SEQ ID NO: 1; and (b) introducing an operably linked heterologous CSS1 gene into a promoter, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein comprising Petition 870250083056, dated 09 / 15 / 2025, page 22 / 88 17 / 35 of an amino acid sequence with an N-terminus serine-rich region that is expanded by at least 14 amino acids in length compared to the N-terminus serine-rich region of SEQ ID NO: 1.

[0049] In some embodiments, the Css1 protein encoded by the short form of the CSS1 gene lacks a C-terminal serine / threonine-rich region that is extended by at least 36 amino acids in length (e.g., at least 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 576, 600, 650, 700 or more amino acids in length) compared to the C-terminal serine / threonine-rich region of SEQ ID NO: 1.

[0050] In some embodiments, the heterologous CSS1 gene encodes a long form of a CSS1 protein comprising an amino acid sequence with an N-terminus serine-rich region that is expanded by at least 14 amino acids in length (e.g., at least 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 675, 700, 750, 77, 800 or more amino acids in length) compared to the N-terminus serine-rich region of SEQ ID NO: 1. In some embodiments, the heterologous CSS1 gene encodes a long form of a protein Css1 comprising an amino acid sequence with an N-terminus serine-rich region that is composed of at least 45% serine. In some embodiments, the N-terminus serine-rich region is composed of at least 45%, 46%, 47%, 48%, 49%, or 50% serine.In some embodiments, the N-terminal serine-rich region is expanded by at least 14 amino acids in length and is composed of at least 45% serine compared to the N-terminal serine-rich region of SEQ ID NO: 1.

[0051] In some forms, the serine-rich region in the N Petition 870250083056, dated 09 / 15 / 2025, page 23 / 88 The 18 / 35 terminal comprises the amino acid sequence established in SEQ ID NO: 2 (XXXXSSXSXXSSSX). In some embodiments, the serine-rich region at the N-terminal comprises one or more amino acid sequences established in SEQ ID NOs: 3-19. In some embodiments, the long form of the Css1 protein comprises a serine-rich region at the N-terminal comprising at least 10 repeats (e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more repeats) of one or more SEQ ID NOs: 219.

[0052] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with an N-terminus serine-rich region that is composed of at least 45% serine. In some embodiments, the N-terminus serine-rich region is composed of at least 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65% or more serine.

[0053] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with a C-terminal serine / threonine-rich region that is expanded by at least 36 amino acids in length (e.g., at least 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 576, 600, 650, 700 or more amino acids in length) to the C-terminal serine / threonine-rich region of SEQ ID NO: 1.In some embodiments, the serine / threonine-rich region at the C-terminal comprises the amino acid sequence established in one or more of the SEQ ID NOs: 20-22. In some embodiments, the long form of the Css1 protein comprises a serine / threonine-rich region at the C-terminal comprising at least 1 repeat (e.g., at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more repeats) of one or more of the SEQ ID NOs:. Petition 870250083056, dated 09 / 15 / 2025, p. 24 / 88 19 / 35 20-22.

[0054] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with a serine / threonine-rich region at the C-terminal that is composed of at least 40% serine. In some embodiments, the serine / threonine-rich region at the C-terminal is composed of at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65% or more serine and threonine.

[0055] In another aspect, a method for promoting an opacity-positive phenotype in yeast is described in this document, wherein the method comprises introducing an operably linked FLO5-CSS1 gene fusion into a promoter in the yeast genome. In some embodiments, the FLO5CSS1 gene fusion encodes an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95% or more) identical to the amino acid sequence established in SEQ ID NO: 25. In some embodiments, the FLO5-CSS1 gene fusion encodes an amino acid sequence established in SEQ ID NO: 25.

[0056] In some embodiments, the recombinant organism (e.g., yeast) described herein is transformed or transfected with a vector comprising the heterologous CSS1 gene. The term “vector” preferably encompasses phage, plasmid, viral or retroviral vectors, such as artificial chromosomes, such as bacterial or yeast artificial chromosomes. The vector may include selectable markers for propagation and / or selection in a host. The vector may be incorporated into a host cell by several sets of procedures well known in the art. If introduced into a host cell, the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it should be understood that the vector may also include nucleic acid sequences. Petition 870250083056, dated 09 / 15 / 2025, page 25 / 88 20 / 35 cos that allow homologous recombination or heterologous insertion. Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” can include one or more of a multitude of processes for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, carbon-based clusters, chemically mediated transfer, protoplast transformation, electroporation, or particle bombardment (e.g., “genetic weapon”). Suitable methods for the transformation or transfection of host cells, including yeast cells, can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed.(Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium Protocols, Ed.: Gartland and Davey, Humana Press, Totowa, New Jersey. Alternatively, a plasmid vector can be introduced by heat shock or electroporation techniques.

[0057] Preferably, the vector referred to in this document is suitable as a cloning vector, i.e., replicable in microbial systems. Such vectors ensure efficient cloning in bacteria and, preferably, yeasts or fungi. These vector systems, preferably, also comprise other cis-regulatory regions, such as promoters and terminators and / or selection markers with which suitable host cells or transformed organisms can be identified.

[0058] Examples of vectors and processes for the vector construct that are suitable for use in recombinant organisms in Petition 870250083056, dated 09 / 15 / 2025, p. 26 / 88 21 / 35 of the present document described include those described in detail in: van den Hondel, CAMJJ, & Punt, PJ (1991) Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, JF Peberdy et al., Ed., pages 128, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi (JW Bennett & LL Lasure, Ed., pages 396-428: Academic Press: San Diego).

[0059] The expression of the CSS1 heterologous gene can be determined by various techniques, for example, by Western Blot, Northern Blot or in situ hybridization techniques, as described, for example, in document WO 02 / 102970, the description of which is incorporated herein by reference in its entirety.

[0060] In some embodiments, the method also involves the deletion or inactivation of a gene for the opacity protection factor (HPF1) (Gene ID, 85410, www.ncbi.nlm.nih.gov / gene / 854010) from the yeast genome. Promoting the Neutral Phenotype for Opacity

[0061] In another aspect, a method for promoting a neutral phenotype to opacity in yeast is described in this document, wherein the method comprises modifying a CSS1 gene in the yeast genome that encodes a long form of a Css1 protein, wherein the modification step results in the inactivation of the CSS1 gene or replacement by a short form of CSS1.

[0062] In some embodiments, the recombinant organism (e.g., yeast) described herein is transformed or transfected with a vector comprising the short form of the CSS1 gene. The expression of the short form of the CSS1 gene can be determined by various techniques, for example, by Western blot, Northern blot, or in situ hybridization techniques, as described, for example, in WO 02 / 102970, the description of which is incorporated herein. Petition 870250083056, dated 09 / 15 / 2025, page 27 / 88 22 / 35 document for reference purposes in its entirety.

[0063] In another aspect, a method for promoting a neutral phenotype for opacity in yeast is described in this document, wherein the method comprises inactivating the FLO5-CSS1 gene fusion from the yeast genome. In some embodiments, the method comprises deleting the FLO5-CSS1 gene fusion from the yeast genome. Screening Methods

[0064] In another aspect, a method for identifying a yeast with a positive phenotype for opacity is described in this document, wherein the method comprises (a) detecting the presence of a CSS1 gene in the yeast genome and (b) determining the length of the protein encoded by the CSS1 gene, wherein the presence of a Css1 protein comprising an amino acid sequence with an N-terminal serine-rich region that is expanded by at least 14 amino acids in length (e.g., at least 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 175, 200, 300 or more amino acids in length) compared with the N-terminal serine-rich region of SEQ ID NO: 1, identifies the yeast as having a positive phenotype for opacity.

[0065] In some embodiments, the CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with an N-terminal serine-rich region that is expanded by at least 14 amino acids in length (e.g., at least 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 675, 700, 750, 77, 800 or more amino acids in length) compared to the N-terminal serine-rich region of SEQ ID NO: 1. In some embodiments, the CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with a region rich in Petition 870250083056, dated 09 / 15 / 2025, page 28 / 88 23 / 35 serine at the N-terminal that is composed of at least 45% serine. In some embodiments, the serine-rich region at the N-terminal is composed of at least 45%, 46%, 47%, 48%, 49%, or 50% serine. In some embodiments, the serine-rich region at the N-terminal is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminal of SEQ ID NO: 1 and is composed of at least 45% serine.

[0066] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with a C-terminal serine / threonine-rich region that is expanded by at least 36 amino acids in length (e.g., at least 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 576, 600, 650, 700 or more amino acids in length) compared to the C-terminal serine / threonine-rich region of SEQ ID NO: 1. In some embodiments, the The C-terminus serine / threonine-rich region comprises the amino acid sequence established in one or more of the SEQ ID NOs: 20-22. In some embodiments, the long form of the Css1 protein comprises a C-terminus serine-rich region comprising at least 1 repeat (e.g., at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more repeats) of one or more of the SEQ ID NOs: 20-22.

[0067] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with a serine / threonine-rich region at the C-terminal that is composed of at least 40% serine. In some embodiments, the serine / threonine-rich region at the C-terminal is composed of at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65% or more serine and threonine. Petition 870250083056, dated 09 / 15 / 2025, p. 29 / 88 24 / 35

[0068] In some embodiments, the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0069] In another aspect, a method for identifying a yeast with a neutral phenotype for opacity is described in this document, wherein the method comprises (a) detecting the presence of a CSS1 gene in the yeast genome and (b) determining the length of the protein encoded by the CSS1 gene, wherein the presence of a Css1 protein comprising an amino acid sequence lacking an N-terminal serine-rich region that is expanded by at least 14 amino acids in length compared to the N-terminal serine-rich region of SEQ ID NO: 1, identifies the yeast as having a neutral phenotype for opacity. The term “neutral phenotype for opacity” refers to the ability of a yeast to produce beer with a clear or unturbid appearance after the addition of hop material during fermentation (dried hops).A yeast strain that results in a turbidity measurement below 200 NTU in a sample of dry-hopped beer is considered "neutral for opacity".

[0070] Exemplary methods for determining whether a yeast has a positive or neutral phenotype for opacity are described in the Examples provided in this document.

[0071] All patent documents and non-patent literature referenced herein are incorporated herein by reference. EXAMPLES Example 1 - Identification of the Opacity Locus in Yeast

[0072] Yeast-Dependent Opacity Assay: To better understand the mechanisms of opacity formation in beer, an assay was developed to screen yeast strains that contain Petition 870250083056, dated 09 / 15 / 2025, page 30 / 88 25 / 35 contribute to the formation of opacity in beer with dry hops. The assay involved small-scale fermentations of beer wort prepared from barley malt that is inoculated with each candidate yeast strain at 10 million cells / ml. A collection of yeast strains from industrial brewing was tested with a minimum of three replicate fermentations. The fermentations were dry-hopped with 8 g / l of T90 hop pellets each day after inoculation until day 7. After a total of 14 days of fermentation, the beer samples were centrifuged at 5000 rpm to remove all yeast cells and particles. The resulting clarified beer samples were measured for opacity using an Anton Paar HazeQC turbidity meter. Opacity measurements were reported as NTU. The dried hops from day 7 exhibited the highest opacity measurements and were used to establish criteria for the positive and neutral opacity phenotype.Yeast strains with an average opacity measurement of 200 NTU or more with day 7 dried hops were classified as opacity positive. The remaining strains with less than 200 NTU opacity measurements with day 7 dried hops were classified as opacity neutral. Late fermentation dried hop additions between the fourth and seventh day showed the greatest difference in opacity measurements in strains OYL-011 and OYL-004 (Figures 6A and 6B, respectively). Day 7 dried hops were used to phenotype a collection of brewing strains (Figure 6C). A variety of opacity phenotypes were observed, and an arbitrary cutoff of 200 NTUs was used to define opacity positive strains (> 200 NTUs) and opacity neutral strains (< 200 NTUs). Traditional English and American ale strains were among the most positive for opacity, while Belgian ale and German lager strains were the most neutral for opacity. Petition 870250083056, dated 09 / 15 / 2025, p. 31 / 88 26 / 35

[0073] Genetic Backcrossing of Yeast: Genetic manipulations, including crosses, sporulation, and tetrad analysis, were performed using standard procedures (Guthrie et al. 2002). Briefly, a tetraploid heterozygous industrial brewing strain positive for opacity (OYL-011) was backcrossed with a homozygous diploid wine strain neutral for opacity (Maxithiol) for 7 generations. Figure 1. In each generation, only the resulting strains with the opacity-positive phenotype were used for subsequent backcrosses (Figure 1). After 7 backcrosses, two opacity-positive and two opacity-neutral isolates were obtained that were approximately 99.3% identical to the Maxithiol parent, allowing segregation and identification of candidate opacity loci. Illumina sequencing was performed to obtain > 1 Gbp of whole genome sequencing data for the parental strains along with the BC7-A, BC7-B, BC7-C, and BC7-D isolates.Variant calling was performed for BC7 isolates and the wine strain against the S288C reference genome. Variant frequencies were plotted by the corresponding genome coordinates as a ratio between BC7 isolates and the wine strain. A region on the left arm of chromosome IX (0-30,000 bp) contained the candidate opacity locus, as it was the only region that contained variants unique to the BC7-A and BC7-B opacity-positive isolates and not the parent wine strain or BC7-C and BC7-D opacity-neutral isolates (Figure 9D).

[0074] Variant Call and Distribution Mapping Variants: Complete genomic sequences with > 1 Gbp of sequencing data were obtained for the parental strains and four isolates from the backcrossing experiment. Quality trimming and adapter clipping were performed using Trimmomatic with standard parameters. The trimmed raw reads were mapped to Petition 870250083056, dated 09 / 15 / 2025, page 32 / 88 27 / 35 the S. cerevisiae reference genome (S288C) was aligned using minimap2. From the resulting alignments, variants were named using freebayes. A custom Python script was used to generate variant distribution plots for each strain to the corresponding S288C genome coordinates. These variant distribution plots allow visualization of the chromosomal position in Chr IX with the highest degree of variance for the S288C reference genome that co-segregates with the opacity-positive phenotype (Figure 2). This region corresponding to the opacity-positive locus contains the CSS1 gene.

[0075] The region between 23,000-26,000 bp showed a multi-fold increase in coverage in the parental OYL-011 and in isolates BC7-A and BC7-B, suggesting two potential repeat expansions within YIL169C at 25,400 bp and 23,800 bp corresponding to the N-terminal and C-terminal (Figures 10A-10B). As isolates BC7-A and BC7-B were heterozygous for YIL169C, BC7-A was sporulated to obtain meiotic segregants that were homozygous diploid and positive or neutral for opacity. The meiotic segregants were then phenotyped for opacity. Primers were designed to amplify the N-terminal of YIL169C. Two products of distinct sizes were identified, with the “long” allele corresponding to the positive phenotype for turbidity (Figure 10C).Furthermore, the PCR products of the “long” and “short” OYL-011 alleles of the YIL169C wine strain were directly sequenced using nanopore sequencing and aligned to the S288C YIL169C allele, confirming that the opacity-positive OYL011 allele contains expansions in the N-terminal and C-terminal regions (Figure 10D). From the amino acid sequence, we note that the N-terminal is strongly enriched in serine (OYL-011 “long” > 55%) and the C-terminal in serine and threonine (OYL-011 “long” > 45%). Upon closer examination of the YIL169C alleles, two mo. Petition 870250083056, dated 09 / 15 / 2025, p. 33 / 88 28 / 35 repeated motifs were identified: (1) a 14 aa motif at the N-terminal and (2) a 36 aa motif at the C-terminal (Figure 10E). The N-terminal repeat was expanded 53 times in the OYL-011 strain, while it was found only 15 times in the laboratory strain S288C and 7 times in the wine strain. The C-terminal repeat was expanded 17 times in the OYL-011 strain, while it was identified only once in the laboratory strain S288C and in the wine strain. Another unexpected finding was a predicted GPI anchor present in the parental strain of wine and OYL011, suggesting that YIL169C in the laboratory strain S288C lacked the GPI anchor.

[0076] The CSS1 long allele is correlated with the opacity phenotype. PCR primers were designed to determine the length of the CSS1 gene in meiotic spores from a resulting 7th generation backcrossed opacity-positive strain. The 7th generation backcrossed strain was heterozygous for CSS1 with one opacity-positive allele from OYL-011 and one opacity-neutral allele from the Maxithiol parent. The primers were designed to amplify the N-terminal of CSS1 from each spore. Two distinct-sized products were identified, with the long CSS1 allele corresponding to the opacity-positive phenotype (Figure 3). Furthermore, a PCR product obtained using primers flanking the CSS1 long allele sequence was directly sequenced using nanopore sequencing to provide the amino acid sequence established at SEQ ID NO: 2. A novel OYL-077 opacity-positive strain was identified by the CSS1 long allele. Figure 4.

[0077] Next, long-read nanopore sequencing data were obtained for a collection of strains and three highly conserved upstream (tag1 ChrIX:25,878-26,392), central domain (tag2 ChrIX:24,727-25,335) and downstream (tag3 ChrIX:22,324-22,442) sequences of the CSS1 gene were used to extract reads Petition 870250083056, dated 09 / 15 / 2025, page 34 / 88 29 / 35 containing the complete N-terminal and C-terminal sequences (Figure 11A). Because many beer strains have heterozygous tetraploid genomes and frequently aneuploidies, the sequence lengths of tag1-tag2 (N-term) and tag2-tag3 (C-term) were collected to capture all potential CSS1 alleles in each strain. The sequence lengths were then plotted using violin plots to observe both the size and distribution of the N-term (Figure 10B) and C-term (Figure 10C) in the various beer strains. Several strains with long N-term regions were previously identified as positive for opacity (OYL-011, OYL-017, OYL-032, OYL-045) in our initial screening. Three opacity-neutral strains (OYL-004, OYL-024, OYL-052) were identified as having low-frequency alleles with N-terminal expansions (Figure 11B). Furthermore, several opacity-positive strains lacked long CSS1 alleles (OYL-061, OYL-043, OYL-015, OYL-021), and therefore it is possible that additional genes are promoting opacity in these strains (Figure 11B). The C-terminal expansion exhibited less variation, with most expansions within the 500 bp distribution (Figure 11C). Interestingly, OYL-001, OYL-009, and OYL-077 were identified as having N-terminal expansions in CSS1 and, when tested for opacity, were among the most opacity-positive strains, suggesting that the length of the CSS1 N-terminal is partially predictive of an opacity-positive phenotype (Figure 11D).

[0078] CSS1 fiddle charts N-terminal lengths and C-terminal: Complete long-read genome sequences were obtained for the Omega Yeast Collection using Oxford Nanopore sequencing. Three sequence markers were designed in the most conserved regions around and within the CSS1 gene. All reads were mapped to the tag1 sequence with Petition 870250083056, dated 09 / 15 / 2025, page 35 / 88 Using minimap2, the resulting PAF file was converted into a BED file using a custom Python script. The readings were then clipped within this Python script and finally extracted using bedtools. All readings containing tag1 were then mapped to the tag2 sequence using the same methods, resulting in a final FASTA file with all readings mapped to tag1 and tag2 clipped at the markers. The length of all clipped readings for each sample was obtained using the bash sequence-stats package. Fiddle plots were generated to show the distribution of N-terminal lengths using the ggplot2 package in R. C-terminal plots were made following the same methodology listed above, except that the readings were mapped first to tag2 and then to the tag3 sequence. Example 2 - CSS1 Interruption in Opacity-Positive Strains Results in Dry Hop-Dependent Opacity Loss.

[0079] With a strong correlation between repeated expansions in CSS1 for the opacity phenotype was then disrupted using CRISPR / Cas9 in several of the most opacity-positive strains (OYL-011, OYL-009, OYL-077), as well as in one of the opacity-neutral strains (OYL-004).

[0080] Plasmids, DNA manipulations, and transformation methods for CSS1 / HPF1 disruption: CSS1 and HPF1 were disrupted in OYL-004, OYL-011, OYL-009, and OYL-077 using CRISPR / Cas9 gene editing. Plasmids pOY092 (CRISPR / Cas9, sgRNA-targeted CSS1, G418 drug-resistant cassette), pOY093 (CRISPR / Cas9, sgRNA-targeted HPF1, G418 drug-resistant cassette), and pOY035 (hygromycin B resistance cassette) were used to disrupt endogenous CSS1 and HPF1 genes. Briefly, primers with 70 bp homology were used. Petition 870250083056, dated 09 / 15 / 2025, page 36 / 88 31 / 35 for the upstream and downstream regions of the CSS1 and HPF1 genes were engineered for amplification of the hygromycin B drug resistance cassette. The resulting PCR products were transformed using the standard Li / Ac transformation protocol (Gietz et al. 2007) in yeast cells with the corresponding CRISPR / Cas9 and sgRNA targeting the CSS1 or HPF1 genes. Selection with G418 was used to screen for pOY092 or pOY093 transformants. Subsequently, the G418+ transformants were screened for hygromycin B resistance. The corresponding G418+ / HYGB+ transformants were confirmed as disrupted for CSS1 or HPF1 with PCR primers specific for HYGB insertion into the CSS1 or HPF1 locus.

[0081] The following strains were tested for the opacity-positive phenotype in flask fermentations with dry hop additions. Briefly, beer wort was inoculated with each yeast isolate at a standard addition rate of 10 million cells / ml. Fermentations proceeded for 7 days, after which a dry hop addition (8 g / l) was performed. The resulting fermentations were terminated on day 14 and opacity measurements are shown. The opacity-positive control strains (OYL-009, OYL-011, OYL077) with intact CSS1 and HPF1 strains exhibited the opacity-positive phenotype. Deletion of CSS1 (css^) in all opacity-positive strains resulted in a neutral opacity phenotype, while deletion of HPF1 (IιρHΔ) maintained the opacity-positive phenotype. The neutral opacity strain OYL-004 remained unchanged after the deletion of CSS1 and HPF1. See Figure 5.

[0082] The disruption of each allele was confirmed by PCR with complete loss of product for N-terminal CSS1 and gain of product indicating disruption of CSS1 (Figure 12A). Each of the resulting css^ strains showed a substantial decrease in opacity. Petition 870250083056, dated 09 / 15 / 2025, page 37 / 88 32 / 35 (Figure 12B). Even the opacity-neutral strain OYL-004 showed reduced opacity with CSS1 interruption. This confirmed that CSS1 was necessary for opacity formation in these strains. Example 3 - Identification of the CSS1 long allele in strains positive for additional opacity.

[0083] Using complete de novo genome assemblies from long and short read sequencing data, CSS1 alleles were determined for each of the industrial brewing strains analyzed for the opacity phenotype. From this list, several additional long CSS1 alleles were identified in opacity-positive strains. The expansion of the N-terminal serine-rich repeat domain (the expansion comprising at least 14 amino acids and composed of at least 45% serine compared to SEQ ID NO: 1) is noted as a shared characteristic of these long CSS1 alleles. See Figure 6. Example 4 - Cloning of CSS1 alleles

[0084] The BC7 opacity-positive isolate (OYR-329) was dissected, and tetrads were confirmed by PCR for the OYL-011 and CSS1 wine strain alleles. Each allele was amplified by PCR and subcloned into a transport vector with AMP and HYG-B drug-resistant cassettes (pOY064). The resulting vectors were sequenced using Oxford Nanopore long-read sequencing. The OYL-011 CSS1 allele was unstable and exhibited frequent loss of N-terminal and C-terminal repeat motifs; therefore, the PCR product was also sequenced using Oxford Nanopore long-read sequencing. In an attempt to alter the sequence containing DNA repeats and obtain a stable cloned OYL-011 CSS1 allele, a codon-optimized sequence was assembled using HiFi assembly. A clone resulting from the OYL-011 CSS1 allele was obtained with a slightly modified sequence containing 52 of the 52 N-terminal repeats and 14 Petition 870250083056, dated 09 / 15 / 2025, p. 38 / 88 33 / 35 of the 17 C-terminal repeats (pOY112). Example 5 - Beer Brewing Test and Tetrad Test

[0085] An IPA wort was brewed with 85% 2-row base malt and 15% Munich malt to 16.9 Plato. Hot-side hop additions included 1 g / l of Mosaic with 10 minutes remaining in the boil, and 2 g / l of Citra were added at the start of a 15-minute swirl. The wort was cooled to 20 °C (68 °F), aerated with oxygen, and transferred to two fermentation vessels. Yeast strains OYL-011 and OYL-011 css1 Δ were inoculated at 10 million cells / ml, and fermentations were maintained at 21 °C (70 °F). On day 7 of fermentation, the beers were dry-hopped with 16 g / l of Citra. Fermentation was completed on day 14 and the beverages were cooled to 0 °C (32 °F) for 4 days before being transferred to serving vessels for carbonation.

[0086] Opacity in the resulting beers from the two fermentation vessels. The yeasts OYL-011 and OYL-011 css1 Δ measured 428 NTUs and 40 NTUs, respectively. This difference was very visually striking and would present itself very differently to the beer consumer. A tetrad sensory test was performed when the beer samples were kept covered and in opaque glasses to prevent panel members from determining which was opaque or not. Only one of the eleven panel members was able to identify the correct combination, indicating that the aroma, mouthfeel, and flavor were not statistically different between opaque and non-opaque beers.

[0087] Discussion: Employing a classical genetic backcrossing approach, YIL169C was identified in CSS1 as a novel opacity gene. Through CSS1 knockout experiments and the correlation between the expansion of CSS1 intragenic repeats and the opacity-positive phenotype among brewing strains, it was concluded Petition 870250083056, dated 09 / 15 / 2025, page 39 / 88 34 / 35 was intended to provide further evidence that CSS1 promotes the opacity phenotype. Together, the results presented in this paper demonstrate the first evidence that the S. cerevisiae gene, CSS1, plays a critical role in promoting opacity in dry-hopped beer styles. Example 6 - Interruption of Css1 and Flo5-Css1 gene fusion results in reduced opacity.

[0088] Plasmids, DNA Manipulations, and Transformation Methods for FLO5-CSS1 Interruption: The FLO5-CSS1 fusion was interrupted in OYL-011 containing the CSS1 deletion (css^) using CRISPR / Cas9 gene editing. pOY092 (CRISPR / Cas9, CSS1 targeting sgRNA, G418 drug-resistant cassette) was transformed with a duplex oligonucleotide containing 45 bp homology with the FLO5 promoter and CSS1 terminator sequences. The sgRNA break introduced in the FLO5-CSS1 fusion is repaired with the duplex oligonucleotide, resulting in a complete deletion of the FLO5-CSS1 fusion. The yeast OYL-011 was transformed with pOY092 and the duplex oligonucleotide using the standard Li / Ac transformation protocol (Gietz et al. 2007). Selection with G418 was used for screening pOY092 transformants. Subsequently, G418+ transformants were screened by PCR to confirm successful deletion of the FLO5-CSS1 fusion.

[0089] The following OYL-011, OYL-011 cssM and OYL-011 flo5css^ were tested for the opacity-positive phenotype in flask fermentations with dry hop additions. Briefly, beer wort was inoculated with each yeast isolate at a standard addition rate of 10 million cells / ml. Fermentations proceeded for 7 days, during which a dry hop addition (8 g / l) was performed. The resulting fermentations were terminated on day 14 and the opacity measurements are shown in Figure 13. Petition 870250083056, dated 09 / 15 / 2025, page 40 / 88 35 / 35

[0090] As shown in Figure 13, a reduction in opacity from 496 NTUs to 194 NTUs after deletion of the CSS1 gene (reducing opacity by > 50%), with a further reduction to 37 NTUs with deletion of both the CSS1 gene and the fusion of the FLO5CSS1 gene (reducing opacity by > 90%). Example 7 - The Long CSS1 Allele is Sufficient for Opacity Formation

[0091] Using the CRISPR / Cas9 plasmid (pOY092), the locus Native CSS1 (short CSS1 allele) was targeted in the opacity-neutral lager strain. A repair template including the long CSS1 allele from OYL-011 was used to repair the CRISPR / Cas9-targeted CSS1 locus, resulting in a switch from the short CSS1 allele to the long CSS1 allele (encoding the amino acid sequence established in SEQ ID NO: 23). The corresponding strain (OYL-106 + long CSS1 allele) was verified using PCR primers to amplify the long CSS1 allele and followed by nanopore sequencing. The original opacity-neutral lager strain and the modified strain were tested for opacity phenotype in dry-hopped flask fermentations. The modified strain (OYL-106 + long CSS1 allele) resulted in substantially more opacity than the original strain (OYL-106), confirming that the long CSS1 allele is sufficient for opacity formation. References: Guthrie, Christine, and Gerald R. Fink. A Guide to Methods in Enzymology for Yeast Genetics and Molecular Biology: Part B. Burlington: Elsevier Science, 2002. Gietz, et al., Nat Protoc 2, 31-34 (2007). Petition 870250083056, dated 09 / 15 / 2025, p. 41 / 88

Claims

1 / 10 CLAIMS 1. Recombinant organism, characterized in that it comprises a heterologous condition-specific secretion 1 (CSS1) gene operably linked to a promoter.

2. Recombinant organism, according to claim 1, characterized in that the promoter is a native promoter of the CSS1 gene.

3. Recombinant organism, according to claim 1, characterized in that the promoter is a heterologous promoter.

4. Recombinant organism, according to claim 3, characterized in that the heterologous promoter is a TDH3 promoter, a TDH2 promoter, a CCW12 promoter, a PGK1 promoter, an ADH1 promoter, an ADH2 promoter, a CYC1 promoter, an HHF1 promoter, an HHF2 promoter, an aTEF1 promoter, a TEF2 promoter, an HTB2 promoter, a PAB1 promoter, an ALD6 promoter, an RNR1 promoter, an RNR2 promoter, a POP6 promoter, a RAD27 promoter, a PSP2 promoter, a REV1 promoter, an MFA1 promoter, an MFa2 promoter, a GAL1 promoter, a CUP1 promoter, a MET25 promoter, an ICL1 promoter, an ICL2 promoter, a GAL3 promoter, an HXT1 promoter, an HXT2 promoter, a A promoter of MAL11, a promoter of MAL31, a promoter of MAL32, a promoter of MAL33, a promoter of MRK1, or a promoter of SUC2.

5. Recombinant organism according to claim 1, characterized in that it is yeast.

6. Recombinant organism, according to claim 2, characterized by the fact that the yeast is Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces eubayanus, Saccharomyces paradoxus, Saccharomyces mikitae, Saccharomyces arbori Petition 870250083056, of 15 / 09 / 2025, p. 42 / 88 2 / 10 colus Saccharomyces kudriavzevii, Saccharomyces jurei, Saccharomyces pastorianus, Torulaspora delbrueckii, Wickerhamomyces anomolus, Pichia kluyveri, Metschnikowia reukaufii, Hanseniaspora uvarum or Lachancea thermotolerans.

7. Recombinant organism, according to any one of claims 1 to 6, characterized in that the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with an N-terminus serine-rich region that is expanded by at least 14 amino acids compared to the amino acid sequence established in SEQ ID NO:

1.

8. Recombinant organism, according to any one of claims 1 to 7, characterized in that the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with a C-terminal serine / threonine-rich region that is expanded by at least 36 amino acids compared to the C-terminal serine / threonine-rich region of SEQ ID NO:

1.

9. Recombinant organism, according to any one of claims 1 to 8, characterized in that the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with a serine-rich region at the N-terminus that is composed of at least 45% serine.

10. Recombinant organism, according to any one of claims 1 to 9, characterized in that the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with a serine / threonine-rich region at the C-terminus that is composed of at least 40% serine / threonine.

11. Recombinant organism, according to any one of claims 7 to 10, characterized in that the serine-rich region at the N-terminal end comprises the amino acid sequence set forth in SEQ ID NO: 2 (XXXXSSXSXXSSSX).

12. Recombinant organism, according to any one of claims 7 to 11, characterized in that the serine-rich region at the N-terminus comprises one or more amino acid sequences set forth in SEQ ID NO: 3-19.

13. Recombinant organism, according to any one of claims 10 to 12, characterized in that the serine / threonine rich region at the C-terminus comprises the amino acid sequence set forth in SEQ ID NO:

20.

14. Recombinant organism, according to any one of claims 10 to 13, characterized in that the serine / threonine rich region at the C-terminus comprises the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO:

22.

15. Recombinant organism, according to any one of claims 10 to 14, characterized in that the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO:

23.

16. Recombinant organism, according to any one of claims 5 to 15, characterized in that it comprises the detection of the presence of a FLO5-CSS1 gene fusion in the yeast genome.

17. Recombinant organism, according to claim 16, characterized in that the FLO5-CSS1 gene fusion comprises a nucleotide sequence encoding an amino acid sequence at least 70% identical to the amino acid sequence established in SEQ ID NO:

25.

18. Recombinant organism, according to claim 16 or 17, characterized in that the fusion of the FLO5 gene Petition 870250083056, dated 09 / 15 / 2025, p. 44 / 88 4 / 10 CSS1 encodes the amino acid sequence set forth in SEQ ID NO:

25.

19. Recombinant organism, according to any one of claims 10 to 18, characterized in that it has a positive phenotype for opacity, as determined by a dry hop-induced opacity assay.

20. Recombinant organism, according to any one of claims 1 to 18, characterized in that the heterologous CSS1 gene encodes a long form of a Css1 protein comprising (a) an amino acid sequence with an N-terminus serine-rich region that is expanded by at least 14 amino acids compared with the amino acid sequence established in SEQ ID NO: 1 and (b) an amino acid sequence with a C-terminus serine / threonine-rich region that is expanded by at least 36 amino acids compared with the C-terminus serine / threonine-rich region of SEQ ID NO:

1.

21. Method for identifying a yeast with an opacity phenotype, characterized in that it comprises (a) detecting the presence of a CSS1 gene in the yeast genome, and (b) determining the length of the protein encoded by the CSS1 gene, wherein the presence of a Css1 protein comprising an amino acid sequence with an N-terminus serine-rich region that is expanded by at least 14 amino acids in length compared to the amino acid sequence established in SEQ ID NO: 1 and that is composed of at least 45% serine identifies the yeast as having an opacity phenotype.

22. Method, according to claim 21, characterized by the fact that the serine-rich region at the N-terminal comprises an amino acid sequence that is expanded by at least 644 amino acids in length compared to the serine-rich region at the N-terminal of SEQ ID NO: 1 and that is composed of at least 50% serine.

23. Method, according to claim 21 or 22, characterized in that the heterologous CSS1 gene encodes a long form of a Css1 protein that further comprises an amino acid sequence with a serine / threonine-rich region at the C-terminal that is expanded by at least 36 amino acids compared to the C-terminal serine / threonine-rich region of SEQ ID NO:

1.

24. Method, according to any one of claims 21 to 23, characterized in that the serine-rich region at the N-terminal end comprises the amino acid sequence set forth in SEQ ID NO: 2 (XXXXSSXSXXSSSX).

25. Method, according to any one of claims 21 to 24, characterized in that the serine-rich region at the N-terminus comprises one or more of the amino acid sequences set forth in SEQ ID NO: 3-19.

26. Method, according to any one of claims 21 to 25, characterized in that the serine / threonine rich region at the C-terminus comprises the amino acid sequence set forth in SEQ ID NO:

20.

27. Method, according to any one of claims 21 to 26, characterized in that the serine / threonine-rich region at the C-terminus comprises one or more of the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO:

22.

28. Method, according to any one of claims 21 to 27, characterized in that the long form of the protein Petition 870250083056, dated 09 / 15 / 2025, p. 46 / 88 6 / 10 in Css1 comprises the amino acid sequence set forth in SEQ ID NO:

23.

29. A method, according to any one of claims 21 to 28, characterized in that it comprises detecting the presence of a FLO5-CSS1 gene fusion in the yeast genome.

30. Method according to claim 29, characterized in that the FLO5-CSS1 gene fusion comprises a nucleotide sequence encoding an amino acid sequence at least 70% identical to the amino acid sequence established in SEQ ID NO:

25.

31. Method, according to claim 29 or 30, characterized in that the fusion of the FLO5-CSS1 gene encodes the amino acid sequence established in SEQ ID NO:

25.

32. Method for identifying a yeast with an opacity-neutral phenotype, characterized in that it comprises (a) detecting the presence of a CSS1 gene in the yeast genome, and (b) determining the length of the protein encoded by the CSS1 gene, wherein the presence of a Css1 protein comprising an amino acid sequence lacking an N-terminal serine-rich region that is expanded by at least 14 amino acids in length compared to the N-terminal serine-rich region of SEQ ID NO: 1 and that is composed of at least 45% serine identifies the yeast as having an opacity-neutral phenotype.

33. Method for promoting a positive phenotype for opacity in a yeast, characterized in that it comprises the introduction of a heterologous CSS1 gene operably linked to a promoter in the yeast genome, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with a serine-rich region at the N-terminal that is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminal of SEQ ID NO: 1, and which is composed of at least 45% serine.

34. Method according to claim 33, characterized in that the heterologous CSS1 gene encodes a long form of a Css1 protein that further comprises an amino acid sequence with a C-terminus serine / threonine-rich region that is expanded by at least 36 amino acids compared to the C-terminus serine / threonine-rich region of SEQ ID NO:

1.

35. Method according to claim 32 or 33, characterized in that the serine-rich region at the N-terminus comprises the amino acid sequence set forth in SEQ ID NO: 2 (XXXXSSXSXXSSSX).

36. Method, according to any one of claims 32 to 35, characterized in that the serine-rich region at the N-terminus comprises one or more of the amino acid sequences set forth in SEQ ID NO: 3-19.

37. Method, according to any one of claims 32 to 35, characterized in that the serine / threonine rich region at the C-terminus comprises the amino acid sequence set forth in SEQ ID NO:

20.

38. Method, according to any one of claims 32 to 35, characterized in that the serine / threonine-rich region at the C-terminus comprises one or more of the amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO:

22.

39. Method, according to any one of claims 32 to 38, characterized in that the long form of the Css1 protein comprises the amino acid sequence set forth in Petition 870250083056, dated 09 / 15 / 2025, page 48 / 88 8 / 10 SEQ ID NO:

23.

40. A method according to any one of claims 32 to 39, characterized in that it further comprises the deletion of a gene for the anti-opacity factor (HPF1) from the yeast genome.

41. A method for promoting a positive phenotype for opacity in a yeast, characterized in that it comprises (a) identifying the presence of a short form of the CSS1 gene in the yeast genome, wherein the short form of the CSS1 gene encodes a Css1 protein comprising an amino acid sequence lacking an N-terminal serine-rich region that is expanded by at least 14 amino acids in length compared to the N-terminal serine-rich region of SEQ ID NO: 1, and which is composed of at least 45% serine; and (b) introducing an operably linked heterologous CSS1 gene to a promoter, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence with an N-terminal serine-rich region that is expanded by at least 14 amino acids in length compared to the N-terminal serine-rich region of SEQ ID NO: 1, and which is composed of at least 45% serine.

42. Method according to claim 41, characterized in that the heterologous CSS1 gene encodes a long form of a Css1 protein that further comprises an amino acid sequence with a C-terminal serine / threonine-rich region that is expanded by at least 36 amino acids compared to the C-terminal serine / threonine-rich region of SEQ ID NO:

1.

43. Method, according to claim 41 or 42, characterized in that the serine-rich region at the N-terminus comprises the amino acid sequence set forth in SEQ ID NO: Petition 870250083056, dated 09 / 15 / 2025, page 49 / 88 9 / 10 2 (XXXXSSXSXXSSSX).

44. Method, according to any one of claims 41 to 43, characterized in that the serine-rich region at the N-terminus comprises one or more of the amino acid sequences set forth in SEQ ID NO: 3-19.

45. Method, according to any one of claims 41 to 43, characterized in that the serine / threonine rich region at the C-terminus comprises the amino acid sequence set forth in SEQ ID NO:

20.

46. ​​Method, according to any one of claims 41 to 43, characterized in that the serine / threonine rich region at the C-terminus comprises one or more amino acid sequences set out in SEQ ID NO: 21 and SEQ ID NO:

22.

47. Method, according to any one of claims 41 to 43, characterized in that the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO:

23.

48. A method for promoting a positive phenotype for opacity in yeast, characterized in that it comprises the introduction of a heterologous FLO5-CSS1 gene fusion operably linked to a promoter in the yeast genome.

49. Method according to claim 36, characterized in that the FLO5-CSS1 gene fusion comprises a nucleotide sequence encoding an amino acid sequence at least 70% identical to the amino acid sequence established in SEQ ID NO:

25.

50. Method, according to claim 48 or 49, characterized in that the fusion of the FLO5-CSS1 gene encodes the amino acid sequence established in SEQ ID NO:

25.

51. Method for promoting a neutral phenotype for opacity in yeast, characterized in that it comprises modifying a CSS1 gene in the yeast genome that encodes a long form of a Css1 protein, wherein the modification step results in the inactivation of the CSS1 gene or its replacement by a short form of CSS1.

52. Method according to claim 51, characterized in that the long form of the Css1 protein comprises an amino acid sequence with a serine-rich region at the N-terminal that is expanded by at least 14 amino acids in length compared to the amino acid sequence established in SEQ ID NO: 1 and is composed of at least 45% serine.

53. A method for promoting a neutral phenotype for opacity in yeast, characterized by the fact that it comprises inactivating the FLO5-CSS1 gene fusion of the yeast genome.

54. Method according to claim 53, characterized in that the inactivation comprises deleting the FLO5-CSS1 gene fusion from the yeast genome.

55. Method, according to any one of claims 21 to 54, characterized in that the yeast is Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces eubayanus, Saccharomyces paradoxus, Saccharomyces mikitae, Saccharomyces arboricolus, Saccharomyces kudriavzevii, Saccharomyces jurei, Saccharomyces pastorianus, Torulaspora delbrueckii, Wickerhamomyces anomolus, Pichia kluyveri, Metschnikowia reukaufii, Hanseniaspora uvarum or Lachancea thermotolerans. Petition 870250083056, dated 15 / 09 / 2025, p. 51 / 88

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