Recombinant yeast with a haze-positive phenotype

Abstract

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

Description

[Technical Field]

[0001] Incorporation by reference of sequence listing This application contains, as a separate part of the disclosure, a Sequence Listing in computer-readable form (Filename: 58543SeqListing.xml, Size: 27,530 bytes; Creation Date: March 11, 2024), which is incorporated herein by reference in its entirety. [Background technology]

[0002] Haze is a well-known phenomenon in the beverage industry. Creating haze in modern pale ale beers is a visual attribute desired by many craft beer enthusiasts. Some yeast strains promote haze better than others. For example, the study disclosed herein classifies strains as either haze-positive or haze-neutral based on their ease of haze production in dry-hopped beers. Yeast strains in the "London Ale3" family of brewer's 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. Therefore, there remains a need in the art for a method to identify yeast with a haze-positive phenotype at the genome level. Summary of the Invention

[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 are methods for identifying yeast having a haze-positive phenotype, the methods comprising: (a) detecting the presence of a CSS1 gene in the genome of the yeast; and (b) determining the length of a protein encoded by the CSS1 gene, wherein the yeast is identified as having a haze-positive phenotype by the presence of a Css1 protein comprising an amino acid sequence having a serine-rich region at its N-terminus that is extended in length by at least 14 amino acids and is composed of at least 45% serine compared to the N-terminal serine-rich region of SEQ ID NO: 1. In some embodiments, the Css1 protein further comprises an amino acid sequence having a serine / threonine-rich region at its C-terminus that is extended by at least 36 amino acids compared to the C-terminal serine / threonine-rich region of SEQ ID NO: 1.

[0006] In another aspect, described herein is a method for 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 a protein encoded by the CSS1 gene, wherein the yeast is identified as having a haze-neutral phenotype by the presence of a Css1 protein comprising an amino acid sequence that is extended by at least 14 amino acids in length compared to the N-terminal serine-rich region of SEQ ID NO: 1 and lacks a serine-rich region at its N-terminus that is at least 45% serine.

[0007] In another aspect, described herein are methods for promoting a haze-positive phenotype in 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 is extended by at least 14 amino acids compared to the amino acid sequence set forth in SEQ ID NO: 1 and lacks a serine-rich region at its N-terminus that is 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 the Css1 protein comprising an amino acid sequence that is extended by at least 14 amino acids compared to the amino acid sequence set forth in SEQ ID NO: 1 and has a serine-rich region at its N-terminus that is 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 extended by at least 36 amino acids compared to the C-terminal serine / threonine-rich region of SEQ ID NO:1.

[0008] In another aspect, described herein is a method of promoting a haze-positive phenotype in 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 yeast, the method comprising modifying, in the genome of the yeast, a CSS1 gene that encodes a long form of the Css1 protein, wherein the modifying step results in inactivation of the CSS1 gene or replacement with a short form of CSS1. [Brief explanation of the drawings]

[0010] [Figure 1]1 shows the results of backcrossing the OYL-011 haze-positive phenotype to the wine line Maxithiol (homozygous diploid) and the resulting haze-positive isolates. [Figure 2] We show that the 0-100 kb region of chromosome 9 exhibited the greatest sequence variation specific to 7A and 7B Haze-positive strains. This region of chromosome 9 contains the putative Haze-positive locus. [Figure 3] Figure 1 shows that the haze-positive phenotype correlates with the long CSS1 allele. [Figure 4] 1 shows that the Haze-positive strain OYL-077 was identified by the long CSS1 allele. [Figure 5] We show that disruption of CSS1 in haze-positive strains results in loss of dry-hop-dependent haze. [Figure 6] Figure 1 shows that the long CSS1 allele is confirmed in additional Haze-positive strains. [Figure 7A] Schematic diagram of the Css1 protein from S288C. [Figure 7B] Schematic representation of the short form of the Css1 protein. [Figure 7C] Schematic representation of the long form of the Css1 protein. [Figures 8A-8C] Characterization of haze phenotype. Figure 8A: Images and haze measurements documenting the haze-positive phenotype of OYL-011. Figure 8B: Images and haze measurements documenting the haze-neutral phenotype of OYL-004. Figure 8C: Haze measurements of a collection of brewing strains dry-hopped on day 7. The average of a minimum of three experimental replicates for each strain is plotted, and error bars represent the standard deviation. The dashed line at 200 NTU indicates the cutoff defining haze-positive and haze-neutral phenotypes. [Figures 9A-9D]Backcrossing of OYL-011 and identification of a candidate haze locus in the left telomeric region of Chr IX. Figure 9A: Schematic of the backcrossing of OYL-011 with a wine strain. Figure 9B: Haze measurements of haze-positive isolates from each backcross. Figure 9C: Haze measurements of the parental strain and two haze-positive and two haze-neutral BC7 isolates. Figure 9D: Variants specific to the two haze-positive BC7 isolates are mapped to a candidate haze locus on the left arm of Chr IX. The ratio of variants found in the BC7 isolate relative to the parental wine strain (y-axis) is plotted in a 50-bp sliding window along the chromosome 9 coordinate of the reference genome S288C (x-axis). [Figures 10A-10D] A large intragenic repeat expansion in the OYL-011 CSS1 allele is associated with haze. Figure 10A: Coverage plot using short reads from Illumina whole-genome sequencing from the parental wine strain (top line) and parental OYL-011 strain (bottom line) mapped to the S288C reference genome. Regions within the N- and C-termini show increased coverage in the OYL-011 strain, indicating a potential repeat expansion in the CSS1 region. Figure 10B: Coverage plot using short reads from Illumina whole-genome sequencing for BC7-A and BC7-B isolates, as well as BC7-C and BC7-D. BC7-A and BC7-B isolates also exhibit increased coverage in the N- and C-terminal regions. Figure 10C: Genotyping of BC7-A spores for the CSS1 N-terminal extension, short allele (approximately 580 bp) and long allele (approximately 2415 bp), and correlation of the long allele with a haze-positive phenotype. Figure 10D: Alignment of S288C, wine strain, and OYL-011 CSS1 alleles. Figure 10E: Schematic of the intragenic repeats in CSS1 of S288C, wine strain, and OYL-011 alleles. The legend indicates the identified repeat motifs along with the putative secretory and GPI anchor sequences. [Figures 11A-11D]N- and C-terminal expansions of CSS1 in a collection of brewing strains. Figure 11A: Schematic of sequence tags used to extract reads from a long read sequencing dataset and determine the N-terminal (tag 1 and tag 2) and C-terminal (tag 2 and tag 3) lengths of CSS1 alleles. Figure 11B: Violin plot showing the size and distribution of the N-terminus (region between tag 1 and tag 2) in long reads from various brewing strains. Haze positives are shown in green and haze neutrals are shown in red. Strains not characterized for the haze phenotype are in white. Figure 11C: Violin plot showing the size and distribution of the C-terminus (region between tag 2 and tag 3) in long reads from various brewing strains. Haze positives are shown in green and haze neutrals are shown in red. Strains not characterized for the haze phenotype are in white. Figure 11D: Haze phenotype of strains identified as carrying a CSS1 N-terminal expansion. [Figures 12A-12C] CSS1 is required for dry-hop-dependent haze formation. Figure 12A: PCR confirmation of complete CRISPR / Cas9 disruption of the CSS1 gene in all css1 strains. Figure 12B: Resulting haze phenotype of css1 strains. Figure 12C: Typical IPA recipe fermented with OYL-011 (left) and OYL-011 css1 (right). [Figure 13] Disruption of the CSS1 gene and the FLO5-CSS1 gene fusion results in reduced haze. [Figure 14] The long CSS1 allele is sufficient for dry-hop-dependent haze formation. Haze produced in the parent haze-neutral lager strain (OYL-106) and in a modified strain in which the native CSS1 allele (short) was replaced with the long CSS1 allele (OYL-106 + long CSS1 allele). DETAILED DESCRIPTION OF THE INVENTION

[0011] This disclosure is based, in part, on the discovery that yeast harboring a long allele of the condition-specific secretion 1 (CSS1) gene (or a FLO5-CSS1 gene fusion) exhibit a haze-positive phenotype. The phrase "haze-positive" phenotype refers to the ability of yeast to produce beer with a cloudy appearance or turbidity after the addition of hop materials during or after fermentation (dry-hopping). Yeast strains that produce turbidity measurements of greater than 200 NTU in dry-hopped beer samples are considered "haze-positive." Turbidity is measured by different photometric methods of turbid media, such as nephelometry, clarification, and nephelometry. It is generally expressed in NTU (Nephelometric Turbidity Units). Units of measurement for turbidity in the brewing industry are the EBC (European Brewing Convention), ASBC (American Society of Brewing Chemists), 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).

[0012] Turbidity measurements are performed using devices such as turbidimeters or nephelometers. These are usually photoelectric receivers that measure the light scattered by the liquid, more specifically the diffusion of light by the suspension, which allows the concentration of suspended substances in the liquid to be evaluated. The devices generally consist of a white light source or an infrared light source. In nephelometry, the scattered light is measured at angles of 90° and 25° to the incident light. In nephelometry, the scattered light is measured by a detector placed on the axis of the incident light.

[0013] 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 9 of the S. cerevisiae genome and encodes an amino acid sequence (Uniprot accession number P40442) that is 995 amino acids long in S. cerevisiae-type strains. Very little is known about the function of the Css1 protein. In contrast to the surprising results demonstrated herein, previous studies have shown that overexpression of CSS1 in wine yeast reduces wine haze (referred to as 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 27-303 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 571-995 of SEQ ID NO:24). It possesses a putative secretion signal at the N-terminus (residues 1-26 of SEQ ID NO:24). Additional S. cerevisiae sequences provided herein (the short CSS1 allele (SEQ ID NO:1) and the long CSS1 allele (SEQ ID NO:23)) reveal an additional hydrophobic region with a putative GPI anchor binding site at the C-terminus. This putative GPI anchor is absent from 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 N-terminal serine-rich region and the C-terminal serine / threonine-rich region correlated with increased dry-hop-dependent haze stability. Deletion of CSS1 in haze-positive brewing yeast resulted in loss of dry-hop-dependent haze stability, demonstrating that the expanded CSS1 allele is required for dry-hop-dependent haze in beer.

[0014] In some embodiments, the heterologous CSS1 gene encodes a long form of the Css1 protein comprising an amino acid sequence having a serine-rich region at its N-terminus that is extended 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 heterologous CSS1 gene encodes a long form of the CSS1 protein comprising an amino acid sequence having a serine-rich region at its N-terminus that is extended 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 heterologous CSS1 gene encodes a long form of the CSS1 protein, comprising an amino acid sequence having an N-terminal serine-rich region that is extended 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 greater 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.

[0015] In some embodiments, the N-terminal serine-rich region comprises the amino acid sequence set forth in SEQ ID NO:2 (XXXXSSXSXXSSSX). In some embodiments, the N-terminal serine-rich region 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 an N-terminal serine-rich region 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 of SEQ ID NOs:2-19.

[0016] In some embodiments, the heterologous CSS1 gene encodes a long form of the CSS1 protein, comprising an amino acid sequence having an N-terminal serine-rich region that is at least 45% serine. In some embodiments, the N-terminal serine-rich region is 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 the CSS1 protein, extended by at least 644 amino acids and comprising an N-terminal serine-rich region that is at least 50% serine.

[0017] In some embodiments, the heterologous CSS1 gene encodes a long form of the Css1 protein, comprising an amino acid sequence having a serine / threonine-rich region at its C-terminus 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-rich region of SEQ ID NO: 1. In some embodiments, the C-terminal serine / threonine-rich region 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 one 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.

[0018] In some embodiments, the heterologous CSS1 gene encodes a long form of the Css1 protein, comprising an amino acid sequence having a serine / threonine-rich region at the C-terminus that is at least 40% serine and threonine-rich. In some embodiments, the C-terminal serine / threonine-rich region is at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65% or more serine and threonine-rich.

[0019] In some embodiments, the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO:23.

[0020] 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 the amino acid sequence set forth in SEQ ID NO: 25.

[0021] In some embodiments, the recombinant organisms described herein have a haze-positive phenotype as determined by the dry hop-induced haze assay (described in Example 1).

[0022] The terms "operably linked" or "operably linked" refer to the association of nucleic acid sequences on a 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" or "associated" with a DNA sequence encoding an RNA or polypeptide when the two sequences are positioned so that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., so that the coding sequence or functional RNA is under the transcriptional control of a promoter). A coding sequence can be operably linked to a regulatory sequence in either a sense or antisense orientation.

[0023] The term "promoter" typically refers to a nucleotide sequence upstream (5') of a coding sequence that controls expression by providing recognition sites for RNA polymerase and other factors necessary for proper transcription. "Promoter" sometimes includes a minimal promoter, a short DNA sequence containing a TATA box and other sequences that help specify the transcription start site, to which regulatory elements are added to enhance expression. "Promoter" also refers to a nucleotide sequence that contains a minimal promoter plus regulatory elements and can control the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter of which are often referred to as enhancers. Thus, an "enhancer" is a DNA sequence that can stimulate promoter activity and can be a native or heterologous element of a promoter inserted to increase the level or tissue specificity of the promoter. It can operate in both orientations (normal or inverted) and can function when moved either upstream or downstream of a 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 may be composed of different elements, derived from different promoters found in nature, or may be composed of synthetic DNA segments.

[0024] A promoter may also contain DNA sequences involved in the binding of protein factors, which control the effectiveness of transcription initiation in response to physiological or developmental states. The "start site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. All other sequences in the gene and its regulatory region are numbered relative to this site. Downstream sequences (i.e., sequences further in the 3' direction that encode proteins) are designated positive, while upstream sequences (the majority of the regulatory region in the 5' direction) are designated negative.

[0025] 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 the 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, an HHF2 promoter, a TEF1 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.

[0026] In some embodiments, the recombinant organism is a yeast. In some embodiments, the yeast is of the genus Saccharomyces. 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.

[0027] Techniques for the recombinant expression of heterologous genes in cells and the genetic modification of recombinant yeast cells are well known to those skilled in the art. Typically, such techniques involve transformation of yeast cells with a nucleic acid construct containing the relevant sequences (e.g., the CSS1 gene). Such methods are known, for example, from standard manuals such as Sambrook and Russell (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 the transformation and genetic modification of fungal host cells are described, for example, in European Patent 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 Patent Application No. EP-A-0481008, European Patent Application No. EP-A-0635574, and U.S. Pat. No. 6,265,186, the disclosures of which are incorporated herein by reference in their entirety.

[0028] Recombinant host cells (e.g., yeast cells) can be cultured using procedures known in the art. For each combination of promoter and host cell, culture conditions conducive to expression of the DNA sequence encoding the polypeptide are available. After a desired cell density or titer of the polypeptide is reached, the culture is stopped and the polypeptide is recovered using known procedures.

[0029] Fermentation media can include known culture media 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 an inorganic nutrient source (e.g., phosphate, magnesium, potassium, zinc, iron, etc.). Optionally, an inducer (depending on the expression construct used) can be included or later added.

[0030] The selection of an 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 that favor the transformed expression host over other potentially contaminating microorganisms.

[0031] Fermentation can be carried out for a period of 0.5 to 30 days. Fermentation can be a batch, continuous, or fed-batch process, at a suitable temperature ranging from 0°C to 45°C, and a pH of, for example, 2 to 10. Preferred fermentation conditions include a temperature ranging from 9°C to 37°C, and / or a pH of 3 to 7. Typically, appropriate conditions are selected based on the choice of fermenting organism and the beverage being fermented.

[0032] Promotion of a haze-positive phenotype In another aspect, described herein is a method of promoting a haze-positive phenotype in 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 the Css1 protein, comprising an amino acid sequence having an N-terminal serine-rich region that is extended by at least 14 amino acids compared to the N-terminal serine-rich region of SEQ ID NO: 1.

[0033] In another aspect, described herein is a method for promoting a haze-positive phenotype in 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 lacking a serine-rich region at its N-terminus that is extended by at least 14 amino acids compared to the N-terminal serine-rich region 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 the Css1 protein comprising an amino acid sequence having a serine-rich region at its N-terminus that is extended by at least 14 amino acids compared to the N-terminal serine-rich region of SEQ ID NO:1.

[0034] In some embodiments, the Css1 protein encodes a short form of the CSS1 gene lacking a serine / threonine-rich region at the C-terminus 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.

[0035] In some embodiments, the heterologous CSS1 gene encodes a long form of the CSS1 protein, comprising an amino acid sequence having a serine-rich region at its N-terminus that is extended 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 heterologous CSS1 gene encodes a long form of the CSS1 protein, comprising an amino acid sequence having a serine-rich region at its N-terminus that is at least 45% serine. In some embodiments, the N-terminal serine-rich region is at least 45%, 46%, 47%, 48%, 49%, or 50% serine-rich. In some embodiments, the N-terminal serine-rich region is extended by at least 14 amino acids in length and is at least 45% serine-rich compared to the N-terminal serine-rich region of SEQ ID NO:1.

[0036] In some embodiments, the N-terminal serine-rich region comprises the amino acid sequence set forth in SEQ ID NO:2 (XXXXSSXSXXSSSX). In some embodiments, the N-terminal serine-rich region 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 an N-terminal serine-rich region 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 of SEQ ID NOs:2-19.

[0037] In some embodiments, the heterologous CSS1 gene encodes a long form of the Css1 protein, comprising an amino acid sequence having an N-terminal serine-rich region that is at least 45% serine. In some embodiments, the N-terminal serine-rich region is at least 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65% or more serine.

[0038] In some embodiments, the heterologous CSS1 gene encodes a long form of the Css1 protein, comprising an amino acid sequence having a serine / threonine-rich region at its C-terminus 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) relative to the C-terminal serine / threonine-rich region of SEQ ID NO: 1. In some embodiments, the C-terminal serine / threonine-rich region 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 one 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.

[0039] In some embodiments, the heterologous CSS1 gene encodes a long form of the Css1 protein, comprising an amino acid sequence having a serine / threonine-rich region at the C-terminus that is at least 40% serine. In some embodiments, the C-terminal serine / threonine-rich region is at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65% or more serine and threonine.

[0040] In another aspect, described herein are methods for promoting a haze-positive phenotype in yeast, the method comprising introducing into the genome of the yeast 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 the amino acid sequence set forth in SEQ ID NO:25.

[0041] In some embodiments, the recombinant organisms described herein (e.g., yeast) are transformed or transfected with a vector containing a 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. A vector may contain a selectable marker for propagation and / or selection in a host. A vector may be incorporated into a host cell by various techniques well known in the art. When introduced into a host cell, the vector may reside in the cytoplasm or be integrated into the genome. In the latter case, it is understood that the vector may further contain nucleic acid sequences that enable homologous recombination or heterologous insertion. A vector can be introduced into a prokaryotic or eukaryotic cell via conventional transformation or transfection techniques. The terms "transformation" and "transfection" can include any one or more of several processes for introducing foreign nucleic acid (e.g., DNA) into host cells, including calcium phosphate, rubidium chloride, or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competency, carbon-based clusters, chemically mediated transfer, protist transformation, electroporation, or particle bombardment (e.g., "gene gun"). Suitable methods for transforming or transfecting 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, the plasmid vector can be introduced by heat shock or electroporation techniques.

[0042] Preferably, the vectors referred to herein are suitable as cloning vectors, i.e., are replicable in microbial systems. Such vectors ensure efficient cloning in bacteria, and preferably in yeast or fungi. These vector systems also preferably contain additional cis-regulatory elements, such as promoters and terminators, and / or selection markers, which can be used to identify suitable transformed host cells or organisms.

[0043] Examples of vectors and processes for constructing vectors suitable for use in the recombinant organisms described herein include those detailed in van den Hondel, C.A.M.J.J., & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi," Applied Molecular Genetics of fungi, J.F. Peberdy et al., Ed., pp. 1-28, Cambridge University Press: Cambridge, or More Gene Manipulations in Fungi (J.W. Bennett & L.L. Lasure, Ed., pp. 396-428: Academic Press: San Diego).

[0044] Expression of the heterologous CSS1 gene can be determined by various techniques, for example, Western blot, Northern blot, or in situ hybridization techniques as described in WO02 / 102970, the entire disclosure of which is incorporated herein by reference.

[0045] In some embodiments, the method further comprises deleting or inactivating the haze protection factor (HPF1) gene (Gene ID, 85410, www.ncbi.nlm.nih.gov / gene / 854010) from the genome of the yeast.

[0046] Promoting a haze-neutral phenotype In another aspect, described herein is a method of promoting a haze-neutral phenotype in yeast, the method comprising modifying, in the genome of the yeast, a CSS1 gene that encodes a long form of the Css1 protein, wherein the modifying step results in inactivation of the CSS1 gene or replacement with a short form of CSS1.

[0047] In some embodiments, a recombinant organism (e.g., yeast) described herein is transformed or transfected with a vector containing a short form of the CSS1 gene. Expression of the short form of the CSS1 gene can be determined by various techniques, such as Western blot, Northern blot, or in situ hybridization techniques described in WO02 / 102970, the entire disclosure of which is incorporated herein by reference.

[0048] In another aspect, described herein are methods of promoting a haze-neutral phenotype in yeast, the method comprising inactivating a 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.

[0049] Screening Method In another aspect, described herein is a method for 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 yeast is identified as having a haze-positive phenotype by the presence of a Css1 protein comprising an amino acid sequence having a serine-rich region at its N-terminus that is extended 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 amino acids in length or more) compared to the N-terminal serine-rich region of SEQ ID NO:1.

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

[0051] In some embodiments, the heterologous CSS1 gene encodes a long form of the Css1 protein, comprising an amino acid sequence having a serine / threonine-rich region at its C-terminus 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. In some embodiments, the C-terminal serine / threonine-rich region 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 comprising at least one 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.

[0052] In some embodiments, the heterologous CSS1 gene encodes a long form of the Css1 protein, comprising an amino acid sequence having a serine / threonine-rich region at the C-terminus that is at least 40% serine. In some embodiments, the C-terminal serine / threonine-rich region is at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65% or more serine and threonine.

[0053] In some embodiments, the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO:23.

[0054] In another aspect, described herein is a method for 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 yeast is identified as having a haze-neutral phenotype by the presence of a CSS1 protein comprising an amino acid sequence lacking a serine-rich region at its N-terminus that is extended by at least 14 amino acids in length compared to the N-terminal serine-rich region of SEQ ID NO: 1. The term "haze-neutral" phenotype refers to the ability of a yeast to produce beer that has a clear appearance or lacks haze after the addition of hop materials during fermentation (dry-hopping). Yeast strains that produce turbidity measurements of less than 200 NTU in dry-hopped beer samples are considered "haze-neutral."

[0055] Exemplary methods for determining whether a yeast has a haze-positive or haze-neutral phenotype are described in the Examples provided herein.

[0056] All patent and non-patent literature referenced herein is hereby incorporated by reference. [Example]

[0057] Example 1 - Identification of the Haze Locus in Yeast Yeast-Dependent Haze Assay: To better understand the mechanisms of haze formation in beer, an assay was developed to screen for yeast strains that contribute to haze formation in dry-hopped beers. The assay involved small-scale fermentations of brewery wort prepared from barley malt inoculated with each yeast strain candidate to 10 million cells / ml. A collection of industrial brewer's yeast strains was assayed in a minimum of three replicate fermentations. Fermentations were dry-hopped with 8 g / L of T90 pelleted hops each day after inoculation until day 7. After a total of 14 days of fermentation, beer samples were centrifuged at 5000 rpm to remove all yeast cells and particulates. The resulting clarified beer samples were measured for haze in an Anton Paar HazeQC turbidity meter. Haze measurements were reported as NTU. Day 7 dry-hopping exhibited the highest haze measurements and were further used to establish criteria for haze-positive and haze-neutral phenotypes. Yeast strains with an average haze measurement of 200 NTU or greater on day 7 of dry hopping were classified as haze-positive. Remaining strains with NTU haze measurements less than 200 on day 7 of dry hopping were classified as haze-neutral. Late-fermentation dry hopping between days 4 and 7 showed the greatest difference in haze measurements in strains OYL-011 and OYL-004 (Figures 6A and 6B, respectively). A collection of brewing strains was phenotypically evaluated using day 7 of dry hopping (Figure 6C). A variety of haze phenotypes were observed, and an arbitrary cutoff of 200 NTU was used to define haze-positive (>200 NTU) and haze-neutral (<200 NTU) strains. Traditional English and American ale strains were among the most haze-positive strains, while Belgian ale strains and German lager strains were the most haze-neutral.

[0058] Yeast genetic backcrossing: Genetic manipulations, including mating, sporulation, and tetrad analysis, were performed using standard procedures (Guthrie et al. 2002). Briefly, a haze-positive tetrad heterozygous industrial brewing strain (OYL-011) was backcrossed to a haze-neutral homozygous diploid wine strain (Maxithiol) for seven generations (Figure 1). At each generation, only the resulting strains with a haze-positive phenotype were used for subsequent backcrosses (Figure 1). After seven backcrosses, two haze-positive and two haze-neutral isolates were obtained that were approximately 99.3% identical to the Maxithiol parent, allowing for the isolation and identification of candidate haze loci. Illumina sequencing was performed to obtain over 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 of the BC7 isolates and wine strains was performed against the reference genome S288C. Variant frequencies were plotted as a ratio between the BC7 isolates and wine strains by their corresponding genomic coordinates. One region (0-30,000 bp) on the left arm of chromosome 9 contained the candidate haze locus, as it was the only region containing variants unique to the BC7-A and BC7-B haze-positive isolates. The wine strain parent or the BC7-C and BC7-D haze-neutral isolates did not contain the candidate haze locus (Figure 9D).

[0059] Variant calling and variant distribution mapping: Complete genome sequences with over 1 Gbp of sequencing data were obtained for the parental strain and four isolates from the backcross experiment. Quality trimming and adapter clipping were performed using Trimmomatic with default parameters. Trimmed raw reads were mapped to the S. cerevisiae reference genome (S288C) using minimap2. Variants were called from the resulting alignment 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 allowed visualization of chromosomal locations on Chr IX, with the highest degree of variance in the S288C reference genome cosegregating with the haze-positive phenotype (Figure 2). This region, corresponding to the haze-positive locus, contains the CSS1 gene.

[0060] The 23,000-26,000 bp region showed several-fold increases in coverage in the OYL-011 parent and the 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- and C-termini (Figure 10A-B). Because the BC7-A and BC7-B isolates were heterozygous for YIL169C, BC7-A was sporulated to obtain meiotic segregants that were homozygous diploids and either haze-positive or haze-neutral. The meiotic segregants were then phenotypically evaluated for haze. Primers were designed to amplify the N-terminus of YIL169C. Two products of different sizes, each carrying the "long" allele corresponding to the haze-positive phenotype, were identified (Figure 10C). Furthermore, PCR products of the full-length OYL-011 "long" allele in YIL169C and the "short" allele in the wine strain were directly sequenced using nanopore sequencing and aligned to the YIL169C allele in S288C, confirming that the Haze-positive OYL-011 allele contains expansions in both the N- and C-terminal regions (Figure 10D). From the amino acid sequence, we noted that the N-terminus was highly enriched for serine (>55% for OYL-011 "long"), and the C-terminus was highly enriched for serine and threonine (>45% for OYL-011 "long"). Further examination of the YIL169C allele identified two repeat motifs: (1) a 14-aa motif at the N-terminus, and (2) a 36-aa motif at the C-terminus (Figure 10E). The N-terminal repeat was expanded 53 times in strain OYL-011, but only 15 times in the S288C lab strain and 7 times in the wine strain. The C-terminal repeat was expanded 17 times in strain OYL-011, but only one time in both the S288C lab strain and the wine strain. Another unexpected finding was the presence of the predicted GPI anchor in the wine parent strain and OYL-011, suggesting the loss of the GPI anchor in the S288C lab strain YIL169C.

[0061] The long allele of CSS1 correlates with the haze phenotype. PCR primers were designed to determine the length of the CSS1 gene in meiotic spores of the resulting seventh-generation backcross haze-positive strain. The backcrossed seventh-generation strain was heterozygous for CSS1, which possesses the haze-positive allele from OYL-011 and the haze-neutral allele from the Maxithiol parent. Primers were designed to amplify the N-terminus of CSS1 from each spore. Two products of different sizes were identified, each carrying the long allele of CSS1, corresponding to the haze-positive phenotype (Figure 3). Furthermore, PCR products obtained using primers flanking the CSS1 long allele sequence were 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 its long CSS1 allele (Figure 4).

[0062] Next, we obtained long-read nanopore sequencing data for the strain collection and extracted reads containing the complete N- and C-terminal sequences using three highly conserved sequences: upstream (tag1 ChrIX: 25,878–26,392), central domain (tag2 ChrIX: 24,727–25,335), and downstream (tag3 ChrIX: 22,324–22,442) of the CSS1 gene (Figure 11A). Because many brewing strains have heterozygous tetrad genomes and often chromosomal aneuploidies, we collected sequence lengths from tag1–tag2 (N-terminus) and tag2–tag3 (C-terminus) to capture all potential CSS1 alleles in each strain. We then plotted the sequence lengths using a violin plot to view both the size and distribution of the N-terminus (Figure 10B) and C-terminus (Figure 10C) in various brewing strains. In our initial screening, we previously identified several strains with long N-terminal regions as haze-positive (OYL-011, OYL-017, OYL-032, and OYL-045). Three haze-neutral strains (OYL-004, OYL-024, and OYL-052) were identified as possessing low frequencies of N-terminally expanded alleles (Figure 11B). Additionally, several haze-positive strains (OYL-061, OYL-043, OYL-015, and OYL-021) did not possess long CSS1 alleles, suggesting that additional genes may be driving haze in these strains (Figure 11B). C-terminal expansions exhibited less variation, with the majority of expansions within a 500-bp distribution (Figure 11C). Interestingly, OYL-001, OYL-009, and OYL-077 were identified as having an N-terminal expansion of CSS1 and were found to be among the most haze-positive strains when assayed for haze, suggesting that the N-terminal length of CSS1 partially predicts the haze-positive phenotype (Figure 11D).

[0063] Violin plot of CSS1 N- and C-terminal lengths: Oxford Nanopore sequencing was used to obtain full-length read genome sequences for the Omega Yeast Collection. Three sequence tags were designed in the most highly conserved regions around and within the CSS1 gene. Minimap2 was used to map all reads to the tag 1 sequence. The resulting paf file was converted to a bed file using a custom Python script, and the reads were trimmed within the Python script and finally extracted using bedtools. The same method was then used to map all tag 1 containing reads to tag 2 sequences, resulting in a final tag-trimmed FASTA file with all reads mapped to tag 1 and tag 2. The sequence-stats bash package was used to obtain the lengths of all trimmed reads for each sample. A violin plot was generated using the ggplot2 package to show the distribution of N-terminal lengths in R. The C-terminal plot was created following the same methodology listed above, except that reads were first mapped to tag 2 and then tag 3 sequences.

[0064] Example 2 - Disruption of CSS1 in a haze-positive strain results in loss of dry-hop-dependent haze Because there is a strong correlation between repeat expansions in CSS1 and the haze phenotype, we used CRISPR / Cas9 to disrupt CSS1 in several of the most haze-positive lines (OYL-011, OYL-009, OYL-077) as well as one of the haze-neutral lines (OYL-004).

[0065] Plasmids, DNA manipulation, and transformation methods for CSS1 / HPF1 disruption: CRISPR / Cas9 gene editing was used to disrupt CSS1 and HPF1 in OYL-004, OYL-011, OYL-009, and OYL-077. For disruption of the endogenous CSS1 and HPF1 genes, plasmids pOY092 (CRISPR / Cas9, CSS1-targeting sgRNA, G418 drug resistance cassette), pOY093 (CRISPR / Cas9, HPF1-targeting sgRNA, G418 drug resistance cassette), and pOY035 (hygromycin B drug resistance cassette) were used. Briefly, primers with 70 bp of homology to the upstream and downstream regions of the CSS1 and HPF1 genes were designed to amplify the hygromycin B drug resistance cassette. The resulting PCR products were transformed into yeast cells with the corresponding CRISPR / Cas9 and sgRNA targeting either the CSS1 or HPF1 gene using a standard Li / Ac transformation protocol (Gietz et al. 2007). pOY092 or pOY093 transformants were screened using G418 selection. G418+ transformants were then screened for hygromycin B resistance. Disruption of CSS1 or HPF1 was confirmed in the corresponding G418+ / HYGB+ transformants using PCR primers specific for the HYGB insertion at the CSS1 or HPF1 locus.

[0066] The following strains were assayed for a haze-positive phenotype during dry-hopped flask fermentation. Briefly, brewery wort was inoculated with each yeast isolate at a standard addition rate of 10 million cells / ml. Fermentation proceeded for 7 days, during which dry-hop addition (8 g / L) was performed. The resulting fermentation was terminated on day 14, and haze measurements were displayed. Control haze-positive strains (OYL-009, OYL-011, OYL-077), containing intact CSS1 and HPF1 strains, exhibited a haze-positive phenotype. CSS1 deletion (css1Δ) in all haze-positive strains resulted in a haze-neutral phenotype, while HPF1 deletion (hpf1Δ) retained the haze-positive phenotype. The OYL-004 haze-neutral strain remained unaffected upon deletion of CSS1 and HPF1. See Figure 5.

[0067] Disruption of each allele was confirmed by PCR by obtaining a product with complete loss of the CSS1 N-terminus and a product indicative of CSS1 disruption (Figure 12A). Each of the resulting css1Δ strains showed a substantial reduction in haze (Figure 12B). Even the OYL-004 haze-neutral strain showed reduced haze upon CSS1 disruption, confirming that CSS1 is required for haze formation in these strains.

[0068] Example 3 - Identification of the long CSS1 allele in additional haze-positive strains De novo complete genome assemblies from long-read and short-read sequencing data were used to determine the CSS1 alleles 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. An expansion of the serine-rich N-terminal repeat domain (an expansion of at least 14 amino acids compared to SEQ ID NO: 1, and at least 45% composed of serine) was noted as a common feature of these long CSS1 alleles. See Figure 6.

[0069] Example 4 - Cloning of CSS1 alleles A haze-positive isolate (OYR-329) from BC7 was excised, and CSS1 tetrads of the OYL-011 and wine strain alleles were PCR-confirmed. Each allele was PCR-amplified and subcloned into a shuttle vector (pOY064) containing AMP and HYG-B drug resistance cassettes. The resulting vector was sequenced by Oxford nanopore long-read sequencing. The OYL-011 CSS1 allele is unstable, exhibiting frequent loss of N- and C-terminal repeat motifs; therefore, PCR products were also sequenced by Oxford nanopore long-read sequencing. In an attempt to modify sequences containing DNA repeats and obtain a stable cloned OYL-011 CSS1 allele, codon-optimized sequences were assembled using HiFi assembly. A clone of the resulting OYL-011 CSS1 allele was obtained containing a slightly modified sequence (pOY112) containing 52 of the 52 N-terminal repeats and 14 of the 17 C-terminal repeats.

[0070] Example 5 - Brewing trials and tetrad testing An IPA wort was prepared from 85% two-row base malt and 15% Munich malt to a 16.9 psi plateau. Hot-side hop addition included 1 g / L Mosaic at the boil for 10 minutes, followed by 2 g / L Citra at the beginning of a 15-minute whirlpool. The wort was cooled to 68°F, aerated with oxygen, and transferred to two fermentation vessels. OYL-011 and OYL-011 css1Δ yeast strains were pitched at 10 million cells / ml, and fermentation was maintained at 70°F. On day 7 of fermentation, the beer was dry-hopped with 16 g / L Citra. Fermentation was completed on day 14, cooled to 32°F for 4 days, and then transferred to serving vessels for carbonation.

[0071] Haze in beers from two fermentation vessels was measured at 428 NTU and 40 NTU for OYL-011 and OYL-011 css1Δ yeast, respectively. This difference was visually striking and presented a very different picture to beer consumers. To prevent panelists from determining which beers had haze and which did not, a tetrad sensory test was conducted in which the beer samples were kept covered in opaque cups. Only one of the 11 panelists was able to identify the correct pair, indicating that aroma, mouthfeel, and taste were not statistically different between beers with and without haze.

[0072] Discussion: Using a classical genetic backcrossing approach, we identified YIL169C of CSS1 as a novel haze gene. Through CSS1 knockout experiments and correlation between the expansion of the CSS1 intragenic repeat and the haze-positive phenotype among brewing strains, we were able to provide further evidence that CSS1 promotes the haze phenotype. Combined with the results provided herein, this demonstrates the first evidence that the S. cerevisiae gene, CSS1, plays an important role in promoting haze in dry-hopped beer styles.

[0073] Example 6 - Disruption of Css1 and Flo5-Css1 gene fusions results in reduced haze Plasmids, DNA manipulations, and transformation methods for FLO5-CSS1 disruption: CRISPR / Cas9 gene editing was used to disrupt the FLO5-CSS1 fusion in OYL-011, which contains a CSS1 deletion (css1Δ). pOY092 (CRISPR / Cas9, CSS1-targeting sgRNA, G418 drug resistance cassette) was transformed with a double-stranded oligonucleotide containing 45 bp of homology to the FLO5 promoter and CSS1 terminator sequences. The sgRNA cleavage introduced into the FLO5-CSS1 fusion was repaired with the double-stranded oligonucleotide, resulting in a complete deletion of the FLO5-CSS1 fusion. OYL-011 yeast was transformed with pOY092 and the double-stranded oligo using a standard Li / Ac transformation protocol (Gietz et al. 2007). pOY092 transformants were screened using G418 selection. G418+ transformants were then screened by PCR to confirm successful deletion of the FLO5-CSS1 fusion.

[0074] The following strains were assayed for a haze-positive phenotype during dry-hopped flask fermentations: OYL-011, OYL-011 css1Δ, and OYL-011 flo5-css1Δ. Briefly, brewery wort was inoculated with each yeast isolate at a standard addition rate of 10 million cells / ml. Fermentation proceeded for 7 days, during which dry-hop addition (8 g / L) was performed. The resulting fermentation was terminated on day 14, and haze measurements are displayed in Figure 13.

[0075] As shown in Figure 13, upon deletion of the CSS1 gene, haze was reduced from 496 NTU to 194 NTU (more than 50% haze reduction), and deletion of both the CSS1 gene and the FLO5-CSS1 gene fusion further reduced haze to 37 NTU (more than 90% haze reduction).

[0076] Example 7 - The long CSS1 allele iH is sufficient for haze formation A CRISPR / cas9 plasmid (pOY092) was used to target the native CSS1 locus (short CSS1 allele) in a haze neutral lager strain. A repair template containing the long CSS1 allele from OYL-011 was used to repair the CRISPR / cas9-targeted CSS1 locus, resulting in an allelic swap of the short CSS1 allele with 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, followed by nanopore sequencing. The parent haze neutral lager strain and the modified strain were assayed for the haze phenotype in flask fermentations using dry hopping. The modified strain (OYL-106 + long CSS1 allele) produced substantially more haze than the parent strain (OYL-106), confirming that the long CSS1 allele is sufficient for haze formation.

[0077] References: Guthrie, Christine., and Gerald R. Fink. Methods in Enzymology Guide to Yeast Genetics and Molecular Biology: Part B. Burlington: Elsevier Science, 2002. Gietz, et al., Nat Protoc 2, 31-34 (2007).

Claims

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 the native CSS1 gene promoter.

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

4. The heterologous promoter is selected from the group consisting of TDH3 promoter, TDH2 promoter, CCW12 promoter, PGK1 promoter, ADH1 promoter, ADH2 promoter, CYC1 promoter, HHF1 promoter, HHF2 promoter, TEF1 promoter, TEF2 promoter, HTB2 promoter, PAB1 promoter, ALD6 promoter, RNR1 promoter, RNR2 promoter, POP6 promoter, RAD27 promoter, PSP2 promoter, 4. The recombinant organism of claim 3, wherein the promoter is a promoter selected from the group consisting of a MET25 promoter, a GAL1 promoter, a REV1 promoter, a MFA1 promoter, a MFa2 promoter, a GAL1 promoter, a CUP1 promoter, a MET25 promoter, an ICL1 promoter, an ICL2 promoter, a GAL3 promoter, a HXT1 promoter, a HXT2 promoter, a MAL11 promoter, a MAL31 promoter, a MAL32 promoter, a MAL33 promoter, an MRK1 promoter, and a SUC2 promoter.

5. The recombinant organism of claim 1, which is a yeast.

6. The yeast is Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces eubayanus, Saccharomyces paradoxus, Saccharomyces micitae, Saccharomyces arboricolus Saccharomyces kudriavzevii, Saccharomyces jurei, Saccharomyces pastorianus, Torulaspora delbrueckii, Wickerhamomyces anomolus, Pichia kluyveri, Metschnikowia 3. The recombinant organism of claim 2, which is Hansonia reukaufii, Hansoniaspora uvarum, or Lachancea thermotolerans.

7. 7. A recombinant organism according to any one of claims 1 to 6, wherein the heterologous CSS1 gene encodes a long form of the 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. 8. The recombinant organism of claim 1, wherein the heterologous CSS1 gene encodes a long form of the CSS1 protein, comprising an amino acid sequence having a serine / threonine-rich region at its C-terminus that is extended by at least 36 amino acids compared to the C-terminal serine / threonine-rich region of SEQ ID NO:

1.

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

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

11. the N-terminal serine-rich region is an amino acid sequence set forth in SEQ ID NO:2 【Chemistry 1】 The recombinant organism according to any one of claims 7 to 10, comprising:

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

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

20.

14. A recombinant organism according to any one of claims 10 to 13, wherein the C-terminal serine / threonine-rich region comprises the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO:

22.

15. A recombinant organism according to any one of claims 10 to 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 to 15, comprising detecting the presence of a FLO5-CSS1 gene fusion in the genome of said yeast.

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

25.

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

25.

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

20. the heterologous CSS1 gene is (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; (b) an amino acid sequence having a serine / threonine-rich region at its C-terminus that is extended by at least 36 amino acids compared to the C-terminal serine / threonine-rich region of SEQ ID NO:

1. A recombinant organism according to any one of claims 1 to 18, encoding a long form of the Css1 protein, comprising:

21. 1. A method for identifying a yeast having a haze phenotype, said method comprising: (a) detecting the presence of the CSS1 gene in the genome of said yeast; (b) determining the length of the protein encoded by the CSS1 gene, wherein the yeast is identified as having a haze phenotype by the presence of a CSS1 protein comprising an amino acid sequence that is extended by at least 14 amino acids compared to the amino acid sequence set forth in SEQ ID NO:1 and has a serine-rich region at its N-terminus that is at least 45% composed of serine.

22. 22. The method of claim 21, wherein the N-terminal serine-rich region is extended by at least 644 amino acids compared to the N-terminal serine-rich region of SEQ ID NO: 1 and comprises an amino acid sequence that is at least 50% serine-based.

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

1.

24. the N-terminal serine-rich region is an amino acid sequence set forth in SEQ ID NO:2 【Chemistry 2】 The method according to any one of claims 21 to 23, comprising:

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

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

20.

27. 27. The method of any one of claims 21 to 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. 28. The method of any one of claims 21 to 27, wherein the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO:

23.

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

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

25.

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

25.

32. 1. A method for identifying a yeast having a haze-neutral phenotype, said method comprising: (a) detecting the presence of the CSS1 gene in the genome of said yeast; (b) determining the length of the protein encoded by the CSS1 gene, wherein the yeast is identified as having a haze-neutral phenotype by the presence of a CSS1 protein comprising an amino acid sequence that is extended by at least 14 amino acids compared to the N-terminal serine-rich region of SEQ ID NO: 1 and lacks a serine-rich region at the N-terminus that is at least 45% serine.

33. A method for promoting a haze-positive phenotype in 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 the CSS1 protein, the long form comprising an amino acid sequence that is extended by at least 14 amino acids compared to the N-terminal serine-rich region of SEQ ID NO: 1 and has a serine-rich region at its N-terminus that is at least 45% serine.

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

1.

35. the N-terminal serine-rich region is an amino acid sequence set forth in SEQ ID NO:2 【Transformation 3】 34. The method of claim 32 or 33, comprising:

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

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

20.

38. 36. The method of any one of claims 32 to 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. 39. The method of any one of claims 32 to 38, wherein the long form of the Css1 protein comprises the amino acid sequence set forth in SEQ ID NO:

23.

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

41. 1. A method for promoting a haze-positive phenotype in yeast, said 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 is extended by at least 14 amino acids compared to the N-terminal serine-rich region of SEQ ID NO: 1 and lacks the N-terminal serine-rich region, which is composed of at least 45% serine; (b) introducing a heterologous CSS1 gene operably linked to a promoter, wherein the heterologous CSS1 gene encodes a long form of the CSS1 protein, the long form comprising an amino acid sequence having a serine-rich region at its N-terminus that is extended 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 the CSS1 protein, further comprising an amino acid sequence having a serine / threonine-rich region at the C-terminus that is extended by at least 36 amino acids compared to the C-terminal serine / threonine-rich region of SEQ ID NO:

1.

43. the N-terminal serine-rich region is an amino acid sequence set forth in SEQ ID NO:2 【Chemistry 4】 43. The method of claim 41 or 42, comprising:

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

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

20.

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

22.

47. 44. The method of any one of claims 41 to 43, wherein 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 haze-positive phenotype in yeast, said method comprising introducing into the genome of said yeast a heterologous FLO5-CSS1 gene fusion operably linked to a promoter.

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

25.

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

25.

51. 1. A method for promoting a haze-neutral phenotype in yeast, the method comprising modifying a CSS1 gene in the genome of the yeast, the CSS1 gene encoding a long form of the CSS1 protein, the modification step resulting in inactivation of the CSS1 gene or replacement 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 that is extended by at least 14 amino acids compared to the amino acid sequence set forth in SEQ ID NO: 1 and has a serine-rich region at the N-terminus that is composed of at least 45% serine.

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

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

55. The yeast is Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces eubayanus, Saccharomyces paradoxus, Saccharomyces Saccharomyces micitae, Saccharomyces arboricolus Saccharomyces kudriavzevii, Saccharomyces jurei, Saccharomyces pastorianus, Torulaspora delbrueckii, Wickerhamomyces anomolus, Pichia kluyveri, Metschnikowia The method of any one of claims 21 to 54, wherein the bacterium is Hansonia reukaufii, Hansoniaspora uvarum, or Lachancea thermotolerans.

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