WO2024192063A1 - Recombinant yeast having haze positive phenotype - Google Patents

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

RECOMBINANT YEAST HAVING HAZE POSITIVE PHENOTYPE

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

[0001] This application contains, as a separate part of disclosure, a Sequence Listing in computer-readable form (filename: 58543SeqListing.xml; Size: 27,530 bytes: Created: March 11 , 2024), which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] Haze is a well-known phenomenon in the beverage industry. Creating haze in beer for modern pale ales is a desired visual attribute for many craft beer drinkers. Some yeast strains are better at promoting haze than others. For example, research disclosed herein has classified strains as either haze positive or haze neutral based on the ease of haze production in dry hopped beers. Yeast strains in the “London Ale 3” family of brewing yeast were classified as haze positive. In contrast, “Vermont” or “Conan” strains were classified as haze neutral. Importantly, the haze locus in the yeast genome has not yet been identified. Thus, there remains a need in the art for methods of identifying a yeast having a haze positive phenotype at the genomic level.

SUMMARY

[0003] In one aspect, provided herein is a recombinant organism comprising a heterologous condition specific secretion 1 (CSS1) gene operably linked to a promoter.

[0004] In another aspect, described herein is a recombinant organism comprising a heterologous FLO5-CSS1 gene fusion operably linked to a promoter.

[0005] In another aspect, described herein is a method of identifying a yeast having a haze positive phenotype, the method comprising (a) detecting the presence of a CSS1 gene in the genome of the yeast, 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-terminus that is expanded by at least 14 amino acids in length compared to serine-rich region at the N-terminus of SEQ ID NO: 1 , and that is composed of at least 45% serine identifies the yeast as having a haze positive phenotype. In some embodiments, the Css1 protein further comprises an amino acid sequence having a serine/threonine-rich region at the C-terminus that is expanded by at least 36 amino acids compared to the serine/threonine-rich region at the C-terminus of SEQ ID NO: 1

[0006] In another aspect, described herein is a method of identifying a yeast having a haze neutral phenotype, the method comprising (a) detecting the presence of a CSS1 gene in the genome of the yeast, 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 that lacks a serine-rich region at the N-terminus that is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 , and that is composed of at least 45% serine identifies the yeast as having a haze neutral phenotype.

[0007] In another aspect, described herein is a method of promoting a haze positive phenotype in a yeast, the method comprising introducing a heterologous CSS1 gene operably linked to a promoter into the genome of the yeast. In some embodiments, the method comprises (a) identifying the presence of a short form of the CSS1 gene in the genome of the yeast, wherein the short form of the CSS1 gene encodes a Css1 protein comprising an amino acid sequence that lacks a serine-rich region at the N-terminus that is expanded by at least 14 amino acids in length compared to the amino acid sequence set forth in SEQ ID NO: 1 , and is composed of at least 45% serine; and (b) introducing a heterologous CSS1 gene operably linked to a promoter, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine-rich region at the N-terminus that is expanded by at least 14 amino acids in length compared to the amino acid sequence set forth 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 having a serine/threonine-rich region at the C-terminus that is expanded by at least 36 amino acids compared to the serine/threonine-rich region at the C- terminus of SEQ ID NO: 1 .

[0008] In another aspect, described herein is a method of promoting a haze positive phenotype in a yeast, the method comprising introducing a FLO5-CSS1 gene fusion operably linked to a promoter into the genome of the yeast.

[0009] In another aspect, described herein is a method of promoting a haze neutral phenotype in a yeast, the method comprising modifying a CSS1 gene in the genome of the yeast that encodes a long form of a Css1 protein, wherein the modifying step results in inactivation of the CSS1 gene or substitution with a short form of CSS1.

BRIEF DESCRIPTION OF THE FIGURES

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

[0011] Figure 2 depicts the region from 0-100 kb of chromosome IX exhibited the greatest sequence variation specific to the 7 A and 7B haze positive strains. This region of Chromosome IX contains the candidate haze positive locus.

[0012] Figure 3 shows that haze positive phenotype correlates with the long CSS1 allele. [0013] Figure 4 shows that haze positive strain OYL-077 was identified by long CSS1 allele.

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

[0015] Figure 6 shows that the long CSS1 allele is confirmed in additional haze 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 Css1 protein.

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

[0019] Figures 8A-8C. Characterization of the haze phenotype. Figure 8A: Image and haze measurements documenting the haze positive phenotype of OYL-011 . Figure 8B: Image and haze measurements documenting the haze neutral phenotype of OYL-004. Figure 8C: Measurements of haze with day seven dry hop addition in a collection of brewing strains. The average of a minimum of three experimental replicates are plotted for each strain with error bars representing standard deviation. Dashed line at 200 NTUs indicates cutoff to define haze positive and haze neutral phenotype.

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

[0021] Figures 10A-10D. Large intergenic repeat expansions in the OYL-011 CSS1 allele are associated with haze. Figure 10A: Coverage plot using short reads from Illumina whole genome sequencing from the parent wine strain (top line) and the parent OYL-011 strain (bottom line) mapped to S288C reference genome. Regions within the N-term and C-term show increased coverage in the OYL-011 strain indicating potential repeat expansions in regions of CSS1 . Figure 10B: Coverage plot using short reads from Illumina whole genome sequencing for the BC7-A and BC7-B isolates and BC7-C and BC7-D. BC7-A and BC7-B isolates also exhibit increased coverage in the N-term and C-term regions. Figure 10C: Genotyping of the BC7-A spores for CSS1 N-terminal expansion, short allele (-580 bp) and long allele (-2415 bp) and the correlation of long allele to the haze positive phenotype. Figure 10D: Alignment of S288C, wine strain and OYL-011 CSS1 alleles. Figure 10E: Schematic representation of the intragenic repeats in CSS1 for the S288C, wine strain and OYL-011 alleles. Legend indicates identified repeat motifs along with candidate secretory and GPI anchor sequences.

[0022] Figures 11 A-11 D. N-terminal and C-terminal expansions in CSS1 in a collection of brewing strains. Figure 11 A: Schematic of sequence tags used to extract reads from long read sequencing datasets and determine lengths of N-terminus (tag1 and tag2) and C- terminus (tag2 and tag3) in CSS1 alleles. Figure 11 B: Violin plots indicating the size and distribution the N-terminus (region between tag1 and tag2) in long reads obtained from various brewing strains. Haze positive are indicated as green and haze neutral as red.

Strains uncharacterized for haze phenotype are white. Figure 11 C: Violin plots indicating the size and distribution the C-terminus (region between tag2 and tag3) in long reads obtained from various brewing strains. Haze positive are indicated as green and haze neutral as red. Strains uncharacterized for haze phenotype are white. Figure 11 D: Haze phenotype of strains identified to have expanded CSS1 N-terminus.

[0023] Figures 12A-12C. CSS1 is necessary for the formation of dry hop depending haze. Figure 12A: PCR confirmation of the full CRISPR/Cas9 disruption of CSS1 gene in all css1A strains. Figure 12B: The resulting haze phenotype of css1A strains. Figure 12C: Typical IPA recipe fermented with OYL-011 (left) and OYL-011 css1A (right).

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

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

DETAILED DESCRIPTION

[0026] The present disclosure is based, in part, on the discovery that yeast having a long allele of the condition specific secretion 1 (CSS1) gene (or a FLO5-CSS1 gene fusion) presents with a haze positive phenotype. The phrase “haze positive” phenotype refers to the ability of a yeast to produce beer having a cloudy appearance, or turbidity following the addition of hop material during or after fermentation (dry hop). A yeast strain that results in a turbidity measurement above 200 NTU in a dry hopped beer sample is considered “haze positive.” Turbidity is measured by different photometry methods of turbid media such as nephelometry, opacimetry and turbidimetry. In general, it is expressed in NTU (Nephelometric Turbidity Unit). In the brewing field, the measurement units of 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 made using a device such as turbidimeter or nephelometer. It is usually a photoelectric receiver measuring the light scattered by the liquid. More particularly, it is the diffusion of light by the suspensions which makes it possible to evaluate the concentration of substances suspended in a liquid. This apparatus generally consists of a source of white light or infrared light. In nephelometry, the scattered light is measured at 90° angle and at 25° angle to the incident light. In turbidimetry, the scattered light is measured by a detector placed in the axis of the incident light.

[0028] In one aspect, described herein is a recombinant organism comprising a heterologous condition specific secretion 1 (CSS1) gene operably linked to a promoter. The CSS1 gene is located near the left telomere on Chromosome IX of the S. cerevisiae genome and encodes an amino acid sequence that is 995 amino acids in length (Uniprot Accession No. P40442) in a type strain of S. cerevisiae. Little to nothing is known about the function of the Css1 protein. In contrast to surprising results demonstrated herein, previous studies indicated that over-expression of CSS1 in wine yeast reduced wine haze (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-terminus (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- terminus (residues 571 to 995 of SEQ ID NO: 24). It has a putative secretion signal at the N- terminus (residues 1 to 26 of SEQ ID NO: 24). From the additional S. cerevisiae sequences provided herein (short CSS1 allele SEQ ID NO: 1 and long CSS1 allele SEQ ID NO: 23) there is an additional hydrophobic region with a putative GPI anchor attachment site at the C-terminus. This putative GPI anchor is not present in the CSS1 consensus sequence provided by the S288C reference genome (SEQ ID NO. 24). As disclosed herein for the first time, there is tremendous diversity in the size of Css1 proteins in industrial brewing strains. Expansion of the serine-rich region at the N-terminus and the serine/threonine-rich region at the C-terminus was correlated to increased dry hop-dependent haze stability. Deletion of CSS1 in haze positive brewing yeasts resulted in loss of dry hop-dependent haze stability, demonstrating that the expanded alleles of CSS1 were necessary for dry hop-dependent haze in beer. [0029] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine-rich region at the N- terminus that is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 . In some embodiments, the heterologous CSS1 gene encodes a long form of a CSS1 protein comprising an amino acid sequence having a serine-rich region at the N-terminus 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 CSS1 gene encodes a long form of a CSS1 protein comprising an amino acid sequence having a serine-rich region at the N-terminus 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: 11n some embodiments, the long form of the Css1 protein comprises an amino acid sequence that is more than 995 amino acids in length. In some embodiments, the long form of the Css1 protein comprises an amino acid sequence that is at least 1 ,200 amino acids in length. In some embodiments, the long form of the Css1 protein comprises an amino acid sequence that is 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-terminus comprises the amino acid sequence set forth in in SEQ ID NO: 2 (XXXXSSXSXXSSSX). In some embodiments, the serine-rich region at the N-terminus comprises one or more amino acid sequences set forth in SEQ ID NOs: 3-19. In some embodiments, the long form of the Css1 protein comprises a serine-rich region at the N-terminus that comprises 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 of 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 having a serine-rich region at the N- terminus that is composed of at least 45% serine. In some embodiments, the serine-rich region at the N-terminus 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 having a serine-rich region at the N-terminus that is expanded by at least 644 amino acids and that 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 having a serine/threonine-rich region at the C-terminus 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 serine- rich region at the C-terminus of SEQ ID NO: 1. In some embodiments, the serine/threonine- rich region at the C-terminus comprises the amino acid sequence set forth 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 region at the C-terminus that comprises 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 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 having a serine/threonine-rich region at the C-terminus that is composed of at least 40% serine and threonine. In some embodiments, the serine/threonine-rich region at the C-terminus 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, described herein is a recombinant organism comprising a FLO5- CSS1 gene fusion operably linked to a promoter. 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 set forth in SEQ ID NO: 25. In some embodiments, the FLO5-CSS1 gene fusion encodes an amino acid sequence set forth in SEQ ID NO: 25.

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

[0037] The terms “operably linked” or “functionally linked” refer to the association of nucleic acid sequences on single nucleic acid fragment so 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 that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects 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 sense or antisense orientation. [0038] The term “promoter” refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition site for RNA polymerase and other factors required for proper transcription. “Promoter” includes a minimal promoter that is a short DNA sequence comprised, in some cases, of a TATA box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for enhancement of expression.

“Promoter” also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements and that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence, which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements, derived from different promoters found in nature, or even be comprised of synthetic DNA segments.

[0039] A promoter may also contain DNA sequences that are involved in the binding of protein factors, which control the effectiveness of transcription initiation in response to physiological or developmental conditions. The "initiation site" is the position surrounding 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 controlling regions are numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated 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, a ADH1 promoter, a ADH2 promoter, a CYC1 promoter, a HHF1 promoter, a HHF2 promoter, aTEF1 promoter, a TEF2 promoter, a HTB2 promoter, a PAB1 promoter, a ALD6 promoter, a RNR1 promoter, a RNR2 promoter, a POP6 promoter, a RAD27 promoter, a PSP2 promoter, a REV1 promoter, a MFA1 promoter, a MFa2 promoter, a GAL1 promoter, a CUP1 promoter, a MET25 promoter, a ICL1 promoter, a ICL2 promoter, a GAL3 promoter, a HXT 1 promoter, a HXT2 promoter, a MAL11 promoter, a MAL31 promoter, a MAL32 promoter, a MAL33 promoter, a MRK1 promoter, or a SUC2 promoter.

[0041] In some embodiments, the recombinant organism is a yeast. In some embodiments, the yeast is from 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 the 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 such techniques involve transformation of a yeast cell with nucleic acid construct comprising the relevant sequence (e.g., CSS1 gene). Such methods are, for example, known from standard handbooks, 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 are described in, e.g., European 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, European Application No. EP-A-0481008, European Application No. EP-A-0635574 and U.S. Pat. No. 6,265,186, the disclosures of which are incorporated herein by reference in their entireties.

[0043] The recombinant host cells (e.g., yeast cells) may be cultured using procedures known in the art. For each combination of a promoter and a host cell, culture conditions are available which are conducive to the expression the DNA sequence encoding the polypeptide. After reaching the desired cell density or titre of the polypeptide the culturing is ceased and the polypeptide is recovered using known procedures.

[0044] Fermentation medium can comprise a known culture medium containing a carbon source (e.g., glucose, maltose, molasses, etc.), a nitrogen source (e.g., ammonium sulphate, 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 be added. [0045] The selection of the appropriate medium may be based on the choice of expression host and/or based 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 favoring the transformed expression hosts over other potentially contaminating microorganisms.

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

[0047] Promoting Haze Positive Phenotype

[0048] In another aspect, described herein is a method of promoting a haze positive phenotype in a yeast comprising introducing a heterologous CSS1 gene operably linked to a promoter into the genome of the yeast. In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine- rich region at the N-terminus that is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 .

[0049] In another aspect, described herein is a method of promoting a haze positive phenotype in a yeast, the method comprising (a) identifying the presence of a short form of the CSS1 gene in the genome of the yeast, wherein the short form of the CSS1 gene encodes a Css1 protein comprising an amino acid sequence that lacks a serine-rich region at the N-terminus that is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 ; and (b) introducing a heterologous CSS1 gene operably linked to a promoter, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine-rich region at the N-terminus that is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 .

[0050] In some embodiments, the Css1 protein encoded by the short form of the CSS1 gene lacks a serine/threonine-rich region at the C-terminus 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 serine/threonine-rich region at the C-terminus of SEQ ID NO: 1. [0051] In some embodiments, the heterologous CSS1 gene encodes a long form of a CSS1 protein comprising an amino acid sequence having a serine-rich region at the N- terminus 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 serine-rich region at the N-terminus of SEQ ID NO: 1. In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine- rich region at the N-terminus that is composed of at least 45% serine. In some embodiments, the serine-rich region at the N-terminus is composed of at least 45%, 46%, 47%, 48%, 49%, or 50% serine. In some embodiments, the serine-rich region at the N-terminus is expanded by at least 14 amino acids in length and is composed of at least 45% serine compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 .

[0052] In some embodiments, the serine-rich region at the N-terminus comprises the amino acid sequence set forth in in SEQ ID NO: 2 (XXXXSSXSXXSSSX). In some embodiments, the serine-rich region at the N-terminus comprises one or more amino acid sequences set forth SEQ ID NOs: 3-19. In some embodiments, the long form of the Css1 protein comprises a serine-rich region at the N-terminus that comprises 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 of SEQ ID NOs: 2-19.

[0053] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine-rich region at the N- terminus that is composed of at least 45% serine. In some embodiments, the serine-rich region at the N-terminus is composed of at least 45%, 46%, 47%, 48%, 49%, 50% , 55%, 60%, 65%, or more serine.

[0054] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine/threonine-rich region at the C-terminus 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 serine/threonine-rich region at the C-terminus of SEQ ID NO: 1 . In some embodiments, the serine/threonine-rich region at the C-terminus comprises the amino acid sequence set forth 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 region at the C-terminus that comprises 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 SEQ ID NOs: 20-22. [0055] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine/threonine-rich region at the C-terminus that is composed of at least 40% serine. In some embodiments, the serine/threonine-rich region at the C-terminus is composed of at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, or more serine and threonine.

[0056] In another aspect, described herein is a method of promoting a haze positive phenotype in a yeast, the method comprising introducing a FLO5-CSS1 gene fusion operably linked to a promoter into the genome of the yeast. 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 set forth in SEQ ID NO: 25. In some embodiments, the FLO5-CSS1 gene fusion encodes an amino acid sequence set forth in SEQ ID NO: 25.

[0057] 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 as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. The vector may comprise selectable markers for propagation and/or selection in a host. The vector may be incorporated into a host cell by various techniques 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 is to be understood that the vector may further comprise nucleic acid sequences which allow for 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” may include any one or more of a multiplicity of processes for introducing foreign nucleic acid (for example DNA) into a host cell, including calcium phosphate, rubidium chloride or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, carbonbased clusters, chemically mediated transfer, protoplast transformation, electroporation or particle bombardment (e.g., “gene-gun”). 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 may be introduced by heat shock or electroporation techniques.

[0058] Preferably, the vector referred to herein 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 further cis- regulatory regions such as promoters and terminators and/or selection markers with which suitable transformed host cells or organisms can be identified.

[0059] Examples of vectors and processes for the construction of vectors which are suitable for use in the recombinant organisms described herein comprise those which are described in detail in: van den Hondel, C.A.M.J.J., & Punt, P.J. (1991) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J.F. Peberdy et al., Ed., pp. 1-28, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi (J.W. Bennett & L.L. Lasure, Ed., pp. 396-428: Academic Press: San Diego).

[0060] The expression of the heterologous CSS1 gene can be determined by various techniques, e.g., by Western Blot, Northern Blot or in situ hybridization techniques as described in, for instance, WO 02/102970, the disclosure of which is incorporated herein by reference in its entirety.

[0061] In some embodiments, the method further comprises deleting or inactivating a haze protecting factor (HPF1) gene (Gene ID, 85410, www.ncbi.nlm.nih.gov/gene/854010) from the genome of the yeast.

[0062] Promoting Haze Neutral Phenotype

[0063] In another aspect, described herein is a method of promoting a haze neutral phenotype in a yeast, the method comprising modifying a CSS1 gene in the genome of the yeast that encodes a long form of a Css1 protein, wherein the modifying step results in inactivation of the CSS1 gene or substitution with a short form of CSS1.

[0064] 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, e.g., by Western Blot, Northern Blot or in situ hybridization techniques as described in, for instance, WO 02/102970, the disclosure of which is incorporated herein by reference in its entirety.

[0065] In another aspect, described herein is a method of promoting a haze neutral phenotype in a yeast, the method comprising inactivating the FLO5-CSS1 gene fusion from the genome of the yeast. In some embodiments, the method comprises deleting the FLO5- CSS1 gene fusion from the genome of the yeast.

[0066] Screening Methods [0067] In another aspect, described herein is a method of identifying a yeast having a haze positive phenotype, the method comprising (a) detecting the presence of a CSS1 gene in the genome of the yeast, 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-terminus 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 to the serine-rich region at the N-terminus of SEQ ID NO: 1 , identifies the yeast as having a haze positive phenotype.

[0068] In some embodiments, the CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having serine-rich region at the N-terminus 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 serine-rich region at the N-terminus of SEQ ID NO: 1 . In some embodiments, the CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine-rich region at the N-terminus that is composed of at least 45% serine. In some embodiments, the serine-rich region at the N-terminus is composed of at least 45%, 46%, 47%, 48%, 49%, or 50% serine. In some embodiments, the serine-rich region at the N-terminus is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 and is composed of at least 45% serine.

[0069] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine/threonine-rich region at the C-terminus 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 serine/threonine-rich region at the C-terminus of SEQ ID NO: 1. In some embodiments, the serine/threonine-rich region at the C-terminus comprises the amino acid sequence set forth in one or more of SEQ ID NOs: 20-22. In some embodiments, the long form of the Css1 protein comprises a serine-rich region at the C-terminus that comprises 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 SEQ ID NOs: 20-22.

[0070] In some embodiments, the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine/threonine-rich region at the C-terminus that is composed of at least 40% serine. In some embodiments, the serine/threonine-rich region at the C-terminus is composed of at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, or more serine and threonine. [0071] In some embodiments, the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0072] In another aspect, described herein is a method of identifying a yeast having a haze neutral phenotype, the method comprising (a) detecting the presence of a CSS1 gene in the genome of the yeast, 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 that lacks a serine-rich region at the N-terminus that is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 , identifies the yeast as having a haze neutral phenotype. The term “haze neutral” phenotype refers to the ability of a yeast to produce beer having a clear appearance, or lacks turbidity following the addition of hop material during fermentation (dry hop). A yeast strain that results in a turbidity measurement below 200 NTU in a dry hopped beer sample is considered “haze neutral”.

[0073] Exemplary methods of determining whether a yeast has a haze positive or haze neutral phenotype are described in the Examples provided herein.

[0074] All patent documents and non-patent literature referenced herein is incorporated herein by reference.

EXAMPLES

Example 1 - Identification of haze locus in yeast

[0075] Assay for Yeast-Dependent Haze: To better understand the mechanisms for haze formation in beer, an assay was developed to screen for yeast strains that contribute to the formation of haze in dry hopped beer. The assay involved small scale fermentations of brewer’s wort prepared from barley malt that is inoculated with each candidate yeast strain to 10 million cells/ml. A collection of industrial brewing yeast strains were assayed with a minimum of three replicate fermentations. The fermentations were dry hopped with 8 g/L of T90 pellet hops on each day following 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 particulate. The resulting clarified beer samples were measured for haze in an Anton Paar HazeQC turbidity meter. Haze measurements were reported as NTU. The day 7 dry hop exhibited the highest haze measurements and was further used to establish criteria for the haze positive and haze neutral phenotype. Yeast strains with an average haze measurement of 200 NTU or greater with the day 7 dry hop were classified as haze positive. The remaining strains with less than 200 NTU haze measurements with a day 7 dry hop were classified as haze neutral. The late fermentation dry hop additions between day four and day seven showed the greatest difference in haze measurements in the OYL-011 and OYL-004 strains (Figure 6A and 6B, respectively). The day seven dry hop was used to phenotype a collection of brewing strains (Figure 6C). A range of haze phenotypes was observed and an arbitrary cut off of 200 NTUs was used to define haze positive (>200 NTUs) and haze neutral (<200 NTLIs) strains. Traditional English and American ale strains were among the most haze positive strains, whereas Belgian ale strains and German lager strains were the most haze neutral.

[0076] Yeast Genetic Backcrossinq: Genetic manipulations including crosses, sporulation and tetrad analysis were carried out using standard procedures (Guthrie et al. 2002). Briefly, a haze positive tetrapioid heterozygous industrial brewing strain (OYL-011) was backcrossed to a haze neutral homozygous diploid wine strain (Maxithiol) for 7 generations. Figure 1 . At each generation, only the resulting strains with the haze positive phenotype were used for subsequent backcrosses (Figure 1 ). After 7 backcrosses, two haze positive and two haze neutral isolates were obtained that were approximately 99.3% identical to the Maxithiol parent, allowing for the segregation and identification of candidate haze loci. Illumina sequencing was performed to obtain >1 Gbp of whole genome sequencing data for the parent strains along with the BC7-A, BC7-B, BC7-C, and BC7-D isolates. Variant calling was performed for the BC7 isolates and the wine strain against the reference genome S288C. Variant frequencies were plotted by the corresponding genome coordinates as a ratio between the BC7 isolates to the wine strain. One region on the left arm of chromosome IX (0-30,000 bp) contained the candidate haze locus, as it was the only region that contained variants unique to the BC7-A and BC7-B haze positive isolates and not the wine strain parent or BC7-C and BC7-D haze neutral isolates (Figure 9D).

[0077] Variant Calling and Variant Distribution Mapping: Full genome sequences with >1 Gbp of sequencing data were obtained for the parent strains and four isolates from the backcrossing experiment. Quality trimming and adapter clipping was performed using Trimmomatic with default parameters. The trimmed raw reads were mapped to the S. cerevisiae reference genome (S288C) using minimap2. From the resulting alignments, variants were called using freebayes. A custom python script was used generate the variant distribution plots for each strain to the corresponding S288C genome coordinates. These variant distribution plots allow visualization of the chromosomal position on Chr IX with the highest degree of variance to the S288C reference genome that co-segregates with the haze positive phenotype (Figure 2). This region corresponding to the haze positive locus contains the CSS1 gene.

[0078] The region between 23,000-26,000 bp showed a several fold increase in coverage in the OYL-011 parent and BC7-A and BC7-B isolates suggesting two potential repeat expansions within YIL169C at 25,400 bp and 23,800 bp corresponding to the N-terminus and C-terminus (Figures 10A-10B). Since the BC7-A and BC7-B isolates were heterozygous for YIL169C, BC7-A was sporulated to obtain meiotic segregants that were homozygous diploid and either haze positive or haze neutral. The meiotic segregants were then phenotyped for haze. Primers were designed to amplify the N-terminus of YIL169C. Two products of distinct size were identified, with the “long” allele corresponding to the haze positive phenotype (Figurel OC). Furthermore, PCR products of the full length OYL-011 “long” and wine strain “short” alleles of YIL169C were directly sequenced using nanopore sequencing and aligned to the YIL169C allele of S288C confirming the haze positive OYL- 011 allele contains expansions in both the N-terminal and C-terminal regions (Figure 10D). From the amino acid sequence, we noted that the N-terminus is heavily enriched in serine (OYL-011 “long” >55%) and the C-terminus in serine and threonine (OYL-011 “long” >45%). When further examining the YIL169C alleles, two repeat motifs were identified: (1) a 14 aa motif in the N-terminus and (2) a 36 aa motif in the C-terminus (Figure 10E). The N-terminal repeat was expanded 53 times in the OYL-011 strain, while only found 15 times in the S288C lab strain and 7 times in the wine strain. The C-terminal repeat was expanded 17 times in the OYL-011 strain, while only identified once in both the S288C lab strain and wine strain. Another unanticipated finding was a predicted GPI anchor present in the wine parent strain and OYL-011 , suggesting that YIL169C in the S288C lab strain has lost the GPI anchor.

[0079] CSS1 long allele is correlated to haze phenotype. PCR Primers were designed to determine the length of the CSS1 gene in meiotic spores of a resulting generation 7 backcrossed haze positive strain. The backcrossed generation 7 strain was heterozygous for CSS1 with a haze positive allele from OYL-011 and haze neutral allele from the Maxithiol parent. Primers were designed to amplify the N-terminus of CSS1 from each spore. Two products of distinct size were identified, with the CSS1 long allele corresponding to the haze 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 set forth in SEQ ID NO: 2. A new haze positive strain OYL-077 was identified by long CSS1 allele. Figure 4.

[0080] Next, long read nanopore sequencing data was obtained for a collection of strains and three highly conserved sequences upstream (tag 1 ChrlX:25, 878-26, 392), in the central domain (tag2 ChrlX:24, 727-25, 335) and downstream (tag3 ChrlX:22, 324-22, 442) of the CSS1 gene was used to extract reads containing the full N-term and C-term sequences (Figure 11 A). Due to many brewing strains having heterozygous tetrapioid genomes and often aneuploidies, we collected the sequence lengths from tag1-tag2 (N-term) and tag2- tag3 (C-term) to capture all potential CSS1 alleles in each strain. The sequence lengths were then plotted using violin plots to look at both the size and distribution of N-term (Figure 10B) and C-term (Figure 10C) in the various brewing strains. Several strains with long N- term regions had previously been identified as haze positive (OYL-01 1 , OYL-017, OYL-032, OYL-045) in our initial screening. Three haze neutral strains (OYL-004, OYL-024, OYL-052) were identified to have low frequency alleles with N-term expansions (Figure 11 B). Also, several haze positive strains did not have long CSS1 alleles (OYL-061 , OYL-043, OYL-015, OYL-021 ) and therefore it is possible that additional genes are promoting haze in these strains (Figure 11 B). The C-term expansion exhibited smaller variation with the majority of expansions within 500 bp distribution (Figure 1 1 C). Interestingly, OYL-001 , OYL-009, OYL- 077 were identified to have N-term expansions in CSS1 and when assayed for haze were found to be among the most haze positive strains, suggesting the N-term length of CSS1 is partially predictive of a haze positive phenotype (Figure 11 D).

[0081] Violin Plots of CSS1 N-term and C-term Lengths: Full long read genome sequences were obtained for the Omega Yeast Collection using Oxford Nanopore sequencing. Three sequence tags were designed in the highest conserved regions surrounding and within the CSS1 gene. All reads were mapped to the tag 1 sequence using minimap2, the resulting paf file was converted to a bed file using a custom python script, the reads were trimmed within that python script and finally extracted using bedtools. All the tag 1 containing reads were then mapped to the tag2 sequence using the same methods which resulted in a final FASTA file with all the reads mapped to tag 1 and tag2 trimmed at the tags. The length of all the trimmed reads for each sample were obtained using the sequence-stats bash package. Violin plots were generated to show the distribution of N-term lengths using the ggplot2 package in R. The C-term plots were made following the same methodology as listed above with the exception that the reads were first mapped to tag2 and then the tag3 sequence.

Example 2 - Disruption of CSS1 in haze positive strains results in loss of dry hopdependent haze.

[0082] With a strong correlation between repeat expansions in CSS1 to the haze phenotype, CSS1 was then disrupted using CRISPR/Cas9 in several of the most haze positive strains (OYL-011 , OYL-009, OYL-077) as well as one of the haze neutral strains (OYL-004).

[0083] 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, CSS1 targeting sgRNA, G418 drug resistant cassette), pOY093 (CRISPR/Cas9, HPF1 targeting sgRNA, G418 drug resistant cassette) and pOY035 (hygromycin B drug resistance cassette) were used in the disruption of the endogenous CSS1 and HPF1 genes. Briefly, primers with 70bp homology to the upstream and downstream regions of the CSS1 and HPF1 genes were designed for the amplification of the Hygromycin B drug resistance cassette. The resulting PCR products were transformed using standard Li/Ac transformation protocol (Gietz et al.2007) into the yeast cells with the corresponding CRISPR/Cas9 and sgRNA targeting the either CSS1 or HPF1 genes. Selection with G418 was used to screen for pOY092 or pOY093 transformants. Subsequently, G418+ transformants were screened for hygromycin B resistance. The corresponding G418+/HYGB+ transformants were confirmed to be disrupted for CSS1 or HPF1 with PCR primers specific to the HYGB insertion at CSS1 or HPF1 locus.

[0084] The following strains were assayed for the haze positive phenotype in flask fermentations with dry hop additions. Briefly, brewers wort was inoculated with each yeast isolate at a standard addition rate of 10 million cells/ml. The fermentations proceeded for 7 days at upon which a dry hop addition (8 g/L) was performed. The resulting fermentations were terminated at day 14 and measurements for haze are displayed. The control haze positive strains (GYL-009, OYL-011 , OYL-077) with intact CSS1 an HPF1 strains exhibited haze positive phenotype. The CSS1 deletion (css/zf) in all haze positive strains resulted in a haze neutral phenotype, whereas the HPF1 deletion (hpfl ) retained the haze positive phenotype. The GYL-004 haze neutral strain remained unaffected upon deletion of CSS1 and HPF1. See Figure 5.

[0085] The disruption of each allele was confirmed by PCR with complete loss product for CSS1 N-terminus and gain of product indicating CSS1 disruption (Figure 12A). Each of the resulting css1A strains showed a substantial decrease in haze (Figure 12B). Even the OYL- 004 haze neutral strain showed reduced haze with CSS1 disruption. This confirmed CSS1 was necessary for haze formation in these strains.

Example 3 - Identification of long CSS1 allele in additional haze positive strains

[0086] Using de novo full genome assemblies from long and short read sequencing data, the CSS1 alleles were determined for each of the industrial brewing strains assayed for the haze phenotype. From this list, several additional long alleles of CSS1 were identified in haze positive strains. The expansion of the serine-rich N-terminal repeat domain (the expansion comprising at least 14 amino acids and that is composed of at least 45% serine compared to SEQ ID NO: 1) is noted as a shared feature of these long CSS1 alleles. See Figure 6. Example 4 - Cloning of CSS1 alleles

[0087] The haze positive isolate from BC7 (OYR-329) was dissected and tetrads were PCR confirmed for the OYL-011 and wine strain alleles of CSS1. Each allele was PCR amplified and subcloned into a shuttling vector with AMP and HYG-B drug resistant cassettes (pOY064). The resulting vectors were sequenced with oxford nanopore long read sequencing. The OYL-011 CSS1 allele was unstable and exhibited frequent loss of the N- terminal and C-terminal repeat motifs and thus the PCR product was also sequenced with oxford nanopore long read sequencing. In attempts to alter the sequence containing DNA repeats and obtain a stable cloned OYL-011 CSS1 allele, a codon optimized sequence was assembled with HiFi assembly. A resulting clone of the OYL-011 CSS1 allele was obtained with a slightly modified sequence containing 52 of the 52 N-terminal repeats and 14 of the 17 C-terminal repeats (pOY112).

Example 5 - Brewing Trial and Tetrad Test

[0088] An IPA wort was prepared with 85% 2-row base malt and 15% Munich malt to 16.9 plato. Hotside hop additions includedl g/L Mosaic at 10 minutes remaining in the boil, and 2 g/L of Citra was added at the beginning of a 15 minute whirlpool. The wort was chilled to 68°F, aerated with oxygen and transferred to two fermentation vessels. OYL-011 and OYL- 011 cssl yeast strains were pitched at 10 million cells/ml and fermentations were maintained at 70°F. On day 7 of fermentation, the beers were dry hopped with 16 g/L of Citra. The fermentations were complete on day 14 and were cooled to 32°F for 4 days before transferring to serving vessels to carbonate.

[0089] The haze in the resulting beers from the two fermentation vessels. OYL-011 and OYL-011 cssl . yeast measured 428 NTUs and 40 NTUs, respectively. This difference was very visually striking and would present very differently to the beer consumer. A tetrad sensory test was performed where the beer samples were kept covered and in opaque cups to prevent panelists from determining which was hazy or not hazy. Only one out of eleven panelists was able to identify the correct pairing, indicating that the aroma, mouthfeel and taste were not statistically different between the hazy and non-hazy beers.

[0090] Discussion: Employing a classic genetic backcrossing approach, we identified YIL169C in CSS1 as a novel haze gene. Through CSS1 knockout experiments and the correlation between the expansion of CSS1 intragenic repeats and haze positive phenotype amongst brewing strains, we were able to provide further evidence for CSS1 promoting the haze phenotype. Together the results provided herein demonstrate the first evidence that the S. cerevisiae gene, CSS1, has a critical role in promoting haze in dry-hopped beer styles.

Example 6 - Disruption of Css1 and Flo5-Css1 gene fusion results in haze reduction [0091 ] Plasmids, DNA manipulations and Transformation Methods for FLO5-CSS1 disruption: FLO5-CSS1 fusion was disrupted in OYL-011 containing the CSS1 deletion (css/ ) using CRISPR/Cas9 gene editing. The pOY092 (CRISPR/cas9, CSS1 targeting sgRNA, G418 drug resistant cassette) was transformed with a duplex oligonucleotide containing 45 bp homology to the FL05 promoter and CSS1 terminator sequences. The sgRNA break introduced into the FLO5-CSS1 fusion is repaired with the duplex oligonucleotide, resulting in a full deletion of the FLO5-CSS1 fusion. The OYL-011 yeast was transformed with the pOY092 and duplex oligo using standard Li/Ac transformation protocol (Gietz et al.2007). Selection with G418 was used to screen for pOY092 transformants. Subsequently, G418+ transformants were screened by PCR to confirm the successful deletion of the FLO5-CSS1 fusion.

[0092] The following OYL-011 , OYL-011 css1 and OYL-011 flo5-css1/l were assayed for the haze positive phenotype in flask fermentations with dry hop additions. Briefly, brewers wort was inoculated with each yeast isolate at a standard addition rate of 10 million cells/ml. The fermentations proceeded for 7 days at which a dry hop addition (8 g/L) was performed. The resulting fermentations were terminated at day 14 and measurements for haze are displayed in Figure 13.

[0093] As shown in Figure 13, a reduction in haze from 496 NTLIs to 194 NTLIs upon deletion of CSS1 gene (reducing haze by >50%), with a further reduction to 37 NTUs with deletion of both the CSS1 gene and the FLO5-CSS1 gene fusion (reducing haze by >90%).

Example 7 - The long CSS1 allele IHis sufficient for haze formation

[0094] Using CRISPR/cas9 plasmid (pOY092), the native CSS1 locus (short CSS1 allele) was targeted in the haze 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 an allele swap of the short CSS1 allele to the long CSS1 allele (encoding the amino acid sequence set forth 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 parent haze neutral lager strain and the modified strain were assayed for haze phenotype in flask fermentations with dry hopping. The modified strain (OYL-106 + long CSS1 allele) resulted in substantially more haze than the parent strain (OYL-106), confirming the long CSS1 allele is sufficient for haze formation.

[0095] References:

Guthrie, Christine., and Gerald R. Fink. Methods in Enzymology Guide to Yeast Genetics and Molecular Biology : Part B. Burlington: Elsevier Science, 2002.

[0096] Gietz, et al., Nat Protoc 2, 31-34 (2007).

Claims

What is claimed is:

1 . A recombinant organism comprising a heterologous condition specific secretion 1 (CSS1) gene operably linked to a promoter.

2. The recombinant organism of claim 1 , wherein the promoter is a native CSS1 gene promoter.

3. The recombinant organism of claim 1 , wherein the promoter is a heterologous promoter.

4. The recombinant organism of claim 3, wherein the heterologous promoter is a TDH3 promoter, a TDH2 promoter, a CCW12 promoter, a PGK1 promoter, a ADH1 promoter, a ADH2 promoter, a CYC1 promoter, a HHF1 promoter, a HHF2 promoter, aTEF1 promoter, a TEF2 promoter, a HTB2 promoter, a PAB1 promoter, a ALD6 promoter, a RNR1 promoter, a RNR2 promoter, a POP6 promoter, a RAD27 promoter, a PSP2 promoter, a REV1 promoter, a MFA1 promoter, a MFa2 promoter, a GAL1 promoter, a CIIP1 promoter, a MET25 promoter, a ICL1 promoter, a ICL2 promoter, a GAL3 promoter, a HXT1 promoter, a HXT2 promoter, a MAL11 promoter, a MAL31 promoter, a MAL32 promoter, a MAL33 promoter, a MRK1 promoter, or a SUC2 promoter.

5. The recombinant organism of claim 1 , that is yeast.

6. The recombinant organism of claim 2, wherein 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.

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

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

9. The recombinant organism of any one of claims 1-8, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine-rich region at the N-terminus that is composed of at least 45% serine.

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

11 . The recombinant organism of any one of claims 7-10, wherein the serine- rich region at the N-terminus comprises the amino acid sequence set forth in SEQ ID NO: 2 (XXXXSSXSXXSSSX) .

12. The recombinant organism of any one of claims 7-11 , wherein the serine-rich region at the N-terminus comprises one or more amino acid sequences set forth in SEQ ID NOs: 3-19.

13. The recombinant organism of any one of claims 10-12, wherein the serine/threonine-rich region at the C-terminus comprises the amino acid sequence set forth in SEQ ID NO: 20.

14. The recombinant organism of any one of claims 10-13, wherein 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. The recombinant organism of any one of claims 10-14, wherein the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO: 23.

16. The recombinant organism of any one of claims 5-15, comprising detecting the presence of a FLO5-CSS1 gene fusion in the genome of the yeast.

17. The recombinant organism of claim 16, wherein the FLO5-CSS1 gene fusion comprises a nucleotide sequence that encodes an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 25.

18. The recombinant organism of claim 16 or claim 16, wherein the FLO5-CSS1 gene fusion encodes the amino acid sequence set forth in SEQ ID NO: 25.

19. The recombinant organism of any one of claims 10-18, that has a haze positive phenotype as determined by a dry hop-induced haze assay.

20. The recombinant organism of any one of claims 1 -18, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein comprising (a) an amino acid sequence having a serine-rich region at the N-terminus that is expanded by at least 14 amino acids compared to the amino acid sequence set forth in SEQ ID NO: 1 and

(b) an amino acid sequence having a serine/threonine-rich region at the C-terminus that is expanded by at least 36 amino acids compared to the serine/threonine-rich region at the C-terminus of SEQ ID NO: 1 .

21 . A method of identifying a yeast having a haze phenotype, the method comprising

(a) detecting the presence of a CSS1 gene in the genome of the yeast, 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-terminus that is expanded by at least 14 amino acids in length compared to the amino acid sequence set forth in SEQ ID NO: 1 and that is composed of at least 45% serine identifies the yeast as having a haze phenotype.

22. The method of claim 21 , wherein the serine-rich region at the N-terminus 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-terminus of SEQ ID NO: 1 and that is composed of at least 50% serine.

23. The method of claim 21 or claim 22, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein further comprising an amino acid sequence having a serine/threonine-rich region at the C-terminus that is expanded by at least 36 amino acids compared to the serine/threonine-rich region of the C-terminus of SEQ ID NO: 1 .

24. Th method of any one of claims 21-23, wherein the serine-rich region at the N-terminus comprises the amino acid sequence set forth in SEQ ID NO: 2 (XXXXSSXSXXSSSX) .

25. The method of any one of claims 21 -24, wherein the serine-rich region at the N-terminus comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 3- 19.

26. The method of any one of claims 21 -25, wherein the serine/threonine-rich region at the C-terminus comprises the amino acid sequence set forth in SEQ ID NO: 20.

27. The method of any one of claims 21 -26, wherein 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. The method of any one of claims 21 -27, wherein the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO: 23.

29. The method of any one of claims 21 -28, comprising detecting the presence of a FLO5-CSS1 gene fusion in the genome of the yeast.

30. The method of claim 29, wherein the FLO5-CSS1 gene fusion comprises a nucleotide sequence that encodes an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 25.

31 . The method of claim 29 or claim 30, wherein the FLO5-CSS1 gene fusion encodes the amino acid sequence set forth in SEQ ID NO: 25.

32. A method of identifying a yeast having a haze neutral phenotype, the method comprising

(a) detecting the presence of a CSS1 gene in the genome of the yeast, 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 that lacks a serine-rich region at the N-terminus that is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 and that is composed of at least 45% serine identifies the yeast as having a haze neutral phenotype.

33. A method of promoting a haze positive phenotype in a yeast, the method comprising introducing a heterologous CSS1 gene operably linked to a promoter into the genome of the yeast, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine-rich region at the N-terminus that is expanded by at least than 14 amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 , and that is composed of at least 45% serine.

34. The method of claim 33, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein further comprising an amino acid sequence having a serine/threonine-rich region at the C-terminus that is expanded by at least 36 amino acids compared to the serine/threonine-rich region of the C-terminus of SEQ ID NO: 1 .

35. Th method of claim 32 or claim 33, wherein the serine-rich region at the N- terminus comprises the amino acid sequence set forth in SEQ ID NO: 2 (XXXXSSXSXXSSSX) .

36. The method of any one of claims 32-35, wherein the serine-rich region at the N-terminus comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 3- 19.

37. The method of any one of claims 32-35, wherein the serine/threonine-rich region at the C-terminus comprises the amino acid sequence set forth in SEQ ID NO: 20.

38. The method of any one of claims 32-35, wherein 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. The method of any one of claims 32-38, wherein the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO: 23.

40. The method of any one of claims 32-39, further comprising deleting a haze protection factor (HPF1) gene from the genome of the yeast.

41 . A method of promoting a haze positive phenotype in a yeast, the method comprising

(a) identifying the presence of a short form of the CSS1 gene in the genome of the yeast, wherein the short form of the CSS1 gene encodes a Css1 protein comprising an amino acid sequence that lacks a serine-rich region at the N-terminus that is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 , and that is composed of at least 45% serine; and

(b) introducing a heterologous CSS1 gene operably linked to a promoter, wherein the heterologous CSS1 gene encodes a long form of a Css1 protein comprising an amino acid sequence having a serine-rich region at the N-terminus that is expanded by at least 14 amino acids in length compared to the serine-rich region at the N-terminus of SEQ ID NO: 1 , and that is composed of at least 45% serine.

42. The method of claim 41 , wherein the heterologous CSS1 gene encodes a long form of a Css1 protein further comprising an amino acid sequence having a serine/threonine-rich region at the C-terminus that is expanded by at least 36 amino acids compared to the serine/threonine-rich region at the C-terminus of SEQ ID NO: 1 .

43. The method of claim 41 or claim 42, wherein the serine-rich region at the N- terminus comprises the amino acid sequence set forth in SEQ ID NO: 2 (XXXXSSXSXXSSSX) .

44. The method of any one of claims 41 -43, wherein the serine-rich region at the N-terminus comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 3- 19.

45. The method of any one of claims 41 -43, wherein the serine/threonine-rich region at the C-terminus comprises the amino acid sequence set forth in SEQ ID NO: 20.

46. The method of any one of claims 41 -43, wherein the serine/threonine-rich region at the C-terminus comprises one or more amino acid sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22.

47. The method of any one of claims 41 -43, wherein the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO: 23.

48. A method of promoting a haze positive phenotype in a yeast, the method comprising introducing a heterologous FLO5-CSS1 gene fusion operably linked to a promoter into the genome of the yeast.

49. The method of claim 36, wherein the FLO5-CSS1 gene fusion comprises a nucleotide sequence that encodes an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 25.

50. The method of claim 48 or claim 49, wherein the FLO5-CSS1 gene fusion encodes the amino acid sequence set forth in SEQ ID NO: 25.

51 . A method of promoting a haze neutral phenotype in a yeast, the method comprising modifying a CSS1 gene in the genome of the yeast that encodes a long form of a Css1 protein, wherein the modifying step results in inactivation of the CSS1 gene or substitution with a short form of CSS1.

52. The method of claim 51 , wherein the long form of the Css1 protein comprises an amino acid sequence having a serine-rich region at the N-terminus that is expanded by at least 14 amino acids in length compared to the amino acid sequence set forth in SEQ ID NO: 1 and is composed of at least 45% serine.

53. A method of promoting a haze neutral phenotype in a yeast, the method comprising inactivating the FLO5-CSS1 gene fusion from the genome of the yeast.

54. The method of claim 53, wherein the inactivating comprises deleting the FLO5-CSS1 gene fusion from the genome of the yeast.

55. The method of any one of claims 21 -54, wherein the yeast is Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces eubayanus, Saccharomyces paradoxus, Saccharomyces mikitae, Saccharomyces arboricolus Saccharomyces kudriavzevii, Saccharomyces jurei, Saccharomyces pastorianus, Torulaspora delbrueckii, Wickerhamomyces anomoius, Pichia kluyveri, Metschnikowia reukaufii, Hanseniaspora uvarum or Lachancea thermotolerans.