REDUCING DIACETYL AND DEXTRIN

CROSS-REFERENCE TO RELATED APPLICATIONS AND DOCUMENTS

The present application claims priority from U.S. provisional application 62/733,374 filed September 19, 2018 and herewith incorporated in its entirety. The present application includes a sequence listing entitled 55729550-33PCT_Sequence listing as filed which is also incorporated in its entirety.

TECHNOLOGICAL FIELD

The present disclosure relates to a recombinant yeast host cell expressing heterologous enzymes and acting as a source of enzyme activity in the production of alcoholic beverages.

BACKGROUND

Light beers are one of the largest selling beer products, and include low-calorie light beers and lagers. To produce low-calorie light beers and lagers, low dextrin content is desired since the presence of dextrins significantly contribute to the caloric content of the beverage. However, standard brewing conditions result in a significant amount of non-fermentable short chain dextrins due to the lack of enzymes in the raw materials that break down the carbohydrate.

Ale yeast ( Saccharomyces cerevisiae) typically ferments at warmer temperatures and impart significant flavor profiles, such as esters (fruity flavor). On the other hand, lager yeast (Saccharomyces pasto anus, a hybrid of cerevisiae and eubayanus species) relies on lower temperature profiles to prevent high metabolite creation, and thus impart less flavor compounds contributing to a cleaner, crisper aroma and taste. However at the same time, due to the low temperatures, metabolites that do impart flavors are often slow to be consumed or degraded. One such metabolite is diacetyl, which is a common contributor to off-flavors with a notable buttery or butter-scotch note. To reduce diacetyl content, a“diacetyl rest” is included in lager fermentations as an important step in which the temperatures are temporarily raised to promote the diacetyl uptake and enzymatic conversion by yeast, which metabolize it to acetoin and subsequently butanediol (via butanediol dehydrogenase). However, this extended maturation process can add at least an additional two weeks to the fermentation process. Therefore, lager fermentation is considered more time and energy intensive compared to the ale process.

Because most light beers are lagers, there is, thus, a need to reduce the caloric content and control the production of flavor-imparting metabolites during production of these alcoholic beverages. Furthermore, reduction of lager fermentation and maturation are needed, which would be of great economic importance to the brewing industry. SUMMARY

The present disclosure provides recombinant yeast host cells which have been genetically engineered to reduce dextrin and diacetyl content of a fermented alcoholic beverage.

In a first aspect, the present disclosure provides a recombinant yeast host cell for making an alcoholic beverage obtained by fermentation of a fermentation medium. The recombinant yeast host cell expresses a first heterologous polypeptide having glucoamylase (GA) activity, a variant thereof having GA activity, or a fragment thereof having GA activity. The recombinant yeast host cell also expresses a second heterologous polypeptide having acetolactate decarboxylase (ALDC) activity, a variant thereof having ALDC activity, or a fragment thereof having ALDC activity. In an embodiment, the recombinant yeast host cell has a native a-aceto lactate production pathway. In still another embodiment, the recombinant yeast host cell has one or more heterologous nucleic acid molecules encoding the first and second heterologous polypeptides, variants thereof, or fragments thereof. In a further embodiment, the recombinant yeast host cell has an heterologous nucleic acid molecule encoding the first and second heterologous polypeptides, variants thereof, or fragments thereof. In still another embodiment, the first heterologous polypeptide, variant thereof, or fragment thereof is expressed intracellularly, as a cell-associated first heterologous polypeptide or is secreted. In a further embodiment, the first heterologous polypeptide is a Saccharomycopsis sp. GA, a variant of the Saccharomycopsis sp. GA having GA activity, or a fragment of the Saccharomycopsis sp. GA having GA activity. For example, the first heterologous polypeptide can be a Saccharomycopsis fibuligera GA, a variant of the Saccharomycopsis fibuligera GA having GA activity, or a fragment of the Saccharomycopsis fibuligera GA having GA activity. In certain embodiments, the Saccharomycopsis fibuligera GA has the amino acid sequence of SEQ ID NO: 16, 17, or 18; is a variant of the amino acid sequence of SEQ ID NO: 16, 17, or 18 having GA activity; or is a fragment of the amino acid sequence of SEQ ID NO: 16, 17, or 18 having GA activity. In another embodiment, the second heterologous polypeptide, variant thereof, or fragment thereof is expressed intracellularly, as a cell-associated second heterologous polypeptide or is secreted. In still a further embodiment, the second heterologous polypeptide, the variant thereof and/or the fragment thereof is from Acetobacter, Acetobacterium, Bacillus, Brevibacillus, Clostridium, Desulfovibrio, Enterobacter, Methanosarcina, Paenibacillus, Raoultella, or Sporolactobacillus sp. In an embodiment, the second heterologous polypeptide is from Acetobacter sp. For example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof is an Acetobacter aceti ALDC and have, in certain embodiments, has the amino acid sequence of SEQ ID NO: 6; is a variant of the amino acid sequence of SEQ ID NO: 6 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 6 having ALDC activity. In another embodiment, the second heterologous polypeptide, the variant thereof and/or the fragment thereof is from Acetobacterium sp. For example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof is an Acetobacterium bakii ALDC. In some embodiments, the second heterologous polypeptide has the amino acid sequence of SEQ ID NO: 13; is a variant of the amino acid sequence of SEQ ID NO: 13 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 13 having ALDC activity. In another example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be an Acetobacterium woodii ALDC. In some embodiments, the second heterologous polypeptide has amino acid sequence of SEQ ID NO: 12; is a variant or the amino acid sequence of SEQ ID NO: 12 having ALDC activity or is afragment of the amino acid sequence of SEQ ID NO: 12 having ALDC activity. In yet another embodiment, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be from Bacillus sp. In an example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be a Bacillus amyloliquefaciens ALDC. In some embodiments, the second heterologous polypeptide has amino acid sequence of SEQ ID NO: 3; is a variant of the amino acid sequence of SEQ ID NO: 3 having ALDC activity or is fragment of the amino acid sequence of SEQ ID NO: 3 having ALDC activity. In another example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be a Bacillus cellulasensis ALDC. In some embodiments, the second heterologous polypeptide has amino acid sequence of SEQ ID NO: 7; is a variant of the amino acid sequence of SEQ ID NO: 7 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 7 having ALDC activity. In a further example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be a Bacillus licheniformis ALDC. In some embodiments, the second polypeptide has the amino acid sequence of SEQ ID NO: 4; is a variant of the amino acid sequence of SEQ ID NO: 4 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 4 having ALDC activity. In still a further example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be a Bacillus subtilis ALDC. In some embodiments, the second heterologous polypeptide has the amino acid sequence of SEQ ID NO: 5; is a variant of the amino acid sequence of SEQ ID NO: 5 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 5 having ALDC activity. In another example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be from Brevibacillus sp. For example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be a Brevibacillus brevis ALDC. In some embodiments, the second heterologous polypeptide has the amino acid sequence of SEQ ID NO: 9; is a variant of the amino acid sequence of SEQ ID NO: 9 having ALDC activity or is fragment of the amino acid sequence of SEQ ID NO: 9 having ALDC activity. In yet another embodiment, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be from Clostridium sp. For example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be a Clostridium sp. maddingley ALDC. In some embodiments, the second heterologous polypeptide has the amino acid sequence of SEQ ID NO: 15; is a variant of the amino acid sequence of SEQ ID NO: 15 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 15 having ALDC activity. In another embodiment, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be from Desulfovibho sp. For example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be a Desulfovibho frigidus ALDC. In some embodiments, the second heterologous polypeptide has the amino acid sequence of SEQ ID NO: 1 1 ; is a variant of the amino acid sequence of SEQ ID NO: 11 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 1 1 having ALDC activity. In a further embodiment, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be from Enterobacter sp. For example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be an Enterobacter aerogenes ALDC. In some embodiments, the second heterologous polypeptide has the amino acid sequence of SEQ ID NO: 2; is a variant of the amino acid sequence of SEQ ID NO: 2 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 2 having ALDC activity. In still a further embodiment, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be from Methanosarcina sp. For example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be a Methanosarcina lacustris ALDC. In some embodiments, the second heterologous polypeptide has the amino acid sequence of SEQ ID NO: 14; is a variant of the amino acid sequence of SEQ ID NO: 14 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 14 having ALDC activity. In yet another embodiment, the second heterologous polypeptide, the variant thereof and/or the fragment thereof is from Paenibacillus sp. For example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof is a Paenibacillus pini ALDC. In some embodiments, the second polypeptide has the amino acid sequence of SEQ ID NO: 10; is a variant of the amino acid sequence of SEQ ID NO: 10 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 10 having ALDC activity. In still another embodiment, the second heterologous polypeptide, the variant thereof and/or the fragment thereof is from Raoultella sp. For example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be a Raoultella terrigena ALDC. In some embodiments, the second heterologous polypeptide has the amino acid sequence of SEQ ID NO: 1 ; is a variant of the amino acid sequence of SEQ ID NO: 1 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 1 having ALDC activity. In yet a further embodiment, the second heterologous polypeptide, the variant thereof and/or the fragment thereof is from Sporolactobacillus sp. For example, the second heterologous polypeptide, the variant thereof and/or the fragment thereof can be a Sporolactobacillus inulinus ALDC. In some embodiments, the second heterologous polypeptide has the amino acid sequence of SEQ ID NO: 8; is a variant of the amino acid sequence of SEQ ID NO: 8 having ALDC activity or is a fragment of the amino acid sequence of SEQ ID NO: 8 having ALDC activity. In still another embodiment, the recombinant yeast host cell is from the genus Saccharomyces sp. (Saccharomyces pastohanus and/or Saccharomyces cerevisiae).

In a second aspect, the present disclosure provides a fermenting agent for making a fermented alcoholic beverage comprising or consisting essentially of the recombinant yeast host cell as described herein. In an embodiment, the fermenting agent further comprising a nutrient.

In a third aspect, the present disclosure provides a process for making a fermented alcoholic beverage. The process comprises (i) contacting the recombinant yeast host cell as described herein with a carbohydrate substrate to provide a mixture and (ii) fermenting the mixture. In an embodiment, the process further comprises providing the recombinant yeast host cell as a fermenting agent as defined herein. In another embodiment, the carbohydrate substrate is a wort. In a further embodiment, the recombinant yeast host cell is for reducing/reduces dextrin content of the mixture during fermentation. In another embodiment, the recombinant yeast host cell is for reducing/reduces diacetyl content of the mixture during fermentation. In still another embodiment, the recombinant yeast host cell is for increasing/increases the conversion of a- acetolactate to acetoin during fermentation. In still another embodiment, the alcoholic beverage is a beer. In yet another embodiment, the recombinant yeast host cell is from the species Saccharomyces cerevisiae. In still a further embodiment, the recombinant yeast host cell is from the species Saccharomyces pastorianus and can be used, for example, to make a lager beer. In such embodiment, the recombinant yeast host cell can be used, for example in a process comprising fermenting the mixture at a temperature between 4 to 15°C (1 1 °C for example).

In a fourth aspect, the present disclosure concerns a fermented alcoholic beverage made according to the process defined herein. In some embodiments, the beverage is a beer. In additional embodiments, the beer is a low-calorie beer having, for example, less than 4 g/L of dextrin. In still another embodiment, the beer has less than 0.01 mg/L combined diacetyl and acetolactate.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:

Figure 1A shows a schematic diagram representing Saccharomyces pathway for diacetyl formation and reduction (AHA, acetohydroxy acid; DHA, dihydroxyacid; BCAA, branched chain amino acid) adapted from Korgerus et al. (2013).

Figure 1 B shows pathways for reduction or metabolism of diacetyl (ALDC = acetolactate decarboxylase activity). Figure 2 shows measurements of the degree of polymerization and ethanol production of a lab scale lager fermentation. Results are shown as the concentration of DP4+ (dextrin) sugars (g/L, bars and left axis) and of ethanol (g/L, lozenges and right axis).

Figure 3 shows measurements of diacetyl potential (pg/L, combined free diacetyl and acetolactate) measured by gas chromatography after 96 hour fermentation. Results are shown as the diacetyl potential (pg/L) as function of the heterologous polypeptide(s) expressed in the recombinant yeast.

Figure 4 shows an initial screening assay of heterologous secreted ALDC candidates in the M2390 background. Four candidates (recombinant yeast expressing Enterobacter aerogenes ALDC (M13671), Bacillus subtilis ALDC (M13673), Brevibacillus brevis ALDC (M13676), and Clostridium sp. maddingley ALDC (M13681)) were identified as actively secreting respective heterologous ALDC. Results are shown as the absorbance at 522 nm as function of the heterologous ALDC expressed in the recombinant yeast.

Figure 5 shows a screening assay of heterologous secreted ALDC candidates in the M13175 lager background. Results are shown as the absorbance at 522 nm as function of the heterologous ALDC expressed in the recombinant yeast.

Figure 6 shows measurements of potential diacetyl (pg/L, combined free diacetyl and acetolactate) measured by gas chromatography after a 96 h wort fermentation. Results are shown as the diacetyl potential (pg/L) as function of the heterologous polypeptide(s) expressed in the recombinant yeast.

Figure 7 shows the specific gravity profiles of parent strain M13175 (¨) and ALDC expressing strains (E. aerogenes ALDC strain M15295 (■), B. subtilis ALDC strain M15296 ( ^A ); B. brevis ALDC strain M15297 (X), C. maddingley ALDC strain M15298 (*)) in a lab scale lager fermentation. The results are shown as the specific gravity for each strain in function of fermentation time (hours).

Figure 8 shows a starch assay of glucoamylase expressing lager strains. Results are show as the absorbance at 540 nm as function of the heterologous polypeptide(s) expressed in the recombinant yeast.

Figure 9 shows a cell-associated screening assay of tethered B.subtilis ALDC in the M13175 lager background. Results are shown as the absorbance at 700 nm in function of the tethering moiety used.

Figures 10A and B show a table of amino acid sequences of cloned a-acetolactate decarboxylases and amino acid sequences of glucoamylases.

DETAILED DESCRIPTION The present disclosure relates to recombinant yeast host cells having increased glycolytic activity (for example those expressing heterologous enzymes for breaking down carbohydrates, such as dextrin) to produce low-calorie alcoholic beverages. However, as shown herein, the increase in glycolytic activity by recombinant yeast host cells leads to the accumulation of significant amounts of diacetyl in fermented alcoholic beverages. To avoid increasing diacetyl content, the present disclosure provides recombinant yeast host cells expressing both a first heterologous polypeptide having glucoamylase (GA) activity for reducing the caloric content of alcoholic beverages (for example by reducing the dextrin content of alcoholic beverages) and a second heterologous polypeptide having acetolactate decarboxylase (ALDC) activity for reducing the off-flavors of alcoholic beverages (for example by reducing diacetyl in an alcoholic beverage). In an embodiment, the genetic modifications in the recombinant yeast host cell of the present disclosure comprise, consist essentially of or consist of a first genetic modification for expression of the first heterologous polypeptide and a second genetic modification for expression of the second heterologous polypeptide. In the context of the present disclosure, the expression“the genetic modifications in the recombinant yeast host consist essentially of a first genetic modification for expression of the first heterologous polypeptide and a second genetic modification for expression of the second heterologous polypeptide” refers to the fact that the recombinant yeast host cell can include other genetic modifications which are unrelated to the reduction of dextrin or diacetyl content.

As used in the present disclosure, the expression “diacetyl content” refers to the combined diacetyl and acetolactate content of a mixture, such as a fermented alcoholic beverage.

The term“heterologous” when used in reference to a nucleic acid molecule (such as a promoter or a coding sequence) refers to a nucleic acid molecule that is not natively found in the recombinant host cell.“Heterologous” also includes a native coding region, or portion thereof, that is removed from the source organism and subsequently reintroduced into the source organism in a form that is different from the corresponding native gene, e.g. , not in its natural location in the organism's genome. The heterologous nucleic acid molecule is purposively introduced into the recombinant host cell. The term“heterologous” as used herein also refers to an element (nucleic acid or protein) that is derived from a source other than the endogenous source. Thus, for example, an heterologous element could be derived from a different strain of host cell, or from an organism of a different taxonomic group (e.g. , different kingdom, phylum, class, order, family genus, or species, or any subgroup within one of these classifications). The term "heterologous" is also used synonymously herein with the term“exogenous”.

The first heterologous polypeptide can be an “amylolytic enzyme”, an enzyme involved in amylase digestion, metabolism and/or hydrolysis. The term“amylase” refers to an enzyme that breaks starch down into sugar. All amylases are glycoside hydrolases and act on a-1 ,4- glycosidic bonds. Some amylases, such as g-amylase (glucoamylase), also act on a-1 ,6- glycosidic bonds. Amylase enzymes include a-amylase (EC 3.2.1.1), b-amylase (EC 3.2.1.2), and y-amylase (EC 3.2.1.3). The a-amylases are calcium metalloenzymes, unable to function in the absence of calcium. By acting at random locations along the starch chain, a-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and “limit dextrin” from amylopectin. Because it can act anywhere on the substrate, a-amylase tends to be faster-acting than b-amylase. Another form of amylase, b- amylase is also synthesized by bacteria, fungi, and plants. Working from the non-reducing end, b-amylase catalyzes the hydrolysis of the first a-1 ,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. Another amylolytic enzyme is a-glucosidase that acts on maltose and other short malto-oligosaccharides produced by a-, b-, and g-amylases, converting them to glucose. Another amylolytic enzyme is pullulanase. Pullulanase is a specific kind of glucanase, an amylolytic exoenzyme, that degrades pullulan. Pullulan is regarded as a chain of maltotriose units linked by alpha- 1 ,6-glycosidic bonds. Pullulanase (EC 3.2.1 .41) is also known as pullulan- 6-glucanohydrolase (debranching enzyme). Another amylolytic enzyme, isopullulanase, hydrolyses pullulan to isopanose (6-alpha-maltosylglucose). Isopullulanase (EC 3.2.1.57) is also known as pullulan 4-glucanohydrolase. An“amylase” can be any enzyme involved in amylase digestion, metabolism and/or hydrolysis, including a-amylase, b -amylase, glucoamylase, pullulanase, isopullulanase, and alpha-glucosidase.

The first heterologous polypeptide can be a glucoamylase or have glucoamylase activity. In the context of the present disclosure, the polypeptides having glucoamylase activity include variants of the glucoamylases polypeptides of SEQ ID NO: 16, 17 or 18 (also referred to herein as glucoamylase variants). A variant comprises at least one amino acid difference (substitution or addition) when compared to the amino acid sequence of the glucoamylase polypeptide of SEQ ID NO: 16, 17 or 18. The glucoamylase variants do exhibit glucoamylase activity. In an embodiment, the variant glucoamylase exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the glucoamylase activity of the amino acid of SEQ ID NO: 16, 17 or 18. The glucoamylase variants also have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 16, 17 or 18. The term“percent identity”, as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. The level of identity can be determined conventionally using known computer programs. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y= 10). Default parameters for pairwise alignments using the Clustal method were KTUPLB 1 , GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The variant glucoamylases described herein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative amino acid substitutions are known in the art and are included herein. Non-conservative substitutions, such as replacing a basic amino acid with a hydrophobic one, are also well-known in the art.

A variant glucoamylase can also be a conservative variant or an allelic variant. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the glucoamylase. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the glucoamylase (e.g., the hydrolysis of starch into glucose). For example, the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the glucoamylase.

In an embodiment, a glucoamylase variant has the amino acid sequence of SEQ ID NO: 16, 17 or 18. The glucoamylase of SEQ ID NO: 16 and the glucoamylase variant having SEQ ID NO: 17 are described in WO/2018/002360, the disclosure of which are incorporated herein by reference.

The present disclosure also provide fragments of the glucoamylases polypeptides and glucoamylase variants described herein. A fragment comprises at least one less amino acid residue when compared to the amino acid sequence of the glucoamylase polypeptide or variant and still possess the enzymatic activity of the full-length glucoamylase. In an embodiment, the glucoamylase fragment exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the full-length glucoamylase of the amino acid of SEQ ID NO: 16, 17 or 18. The glucoamylase fragments can also have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 16, 17 or 18. The fragment can be, for example, a truncation of one or more amino acid residues at the amino-terminus, the carboxy terminus or both termini of the glucoamylase polypeptide or variant. Alternatively or in combination, the fragment can be generated from removing one or more internal amino acid residues. In an embodiment, the glucoamylase fragment has at least 100, 150, 200, 250, 300, 350, 400, 450, 500 or more consecutive amino acids of the glucoamylase polypeptide or the variant.

As shown in Figure 1A, diacetyl is formed by the spontaneous decarboxylation of a- acetolactate, a precursor in valine biosynthesis which will leak out of the cell if not efficiently utilized. As shown in Figure 1 B, as a-aceto lactate leaks out of the yeast cells, it spontaneously decarboxylates into diacetyl, which then is taken back up by the yeast and converted to acetoin and butanediol.

Consequently, the second heterologous polypeptide can be an “acetolactate decarboxylase enzyme”, an enzyme involved in converting a-aceto lactate to acetoin, preventing diacetyl formation. Diacetyl is a common contributor to off-flavors with a notable buttery or butter-scotch note with a low flavor threshold of approximately 0.1 ppm. ALDC (EC 4.1 .1 .5) directly converts a-acetolactate into acetoin and carbon dioxide. In some embodiments, the recombinant yeast host cells described herein can have a native a-acetolactate production pathway, and are able to convert pyruvate into a-acetolactate. In another embodiment, the recombinant yeast host cells expresses native acetolactate (AHA) synthases. In a further embodiment, the recombinant yeast host cells of the present disclosure has and expresses native AHA synthases as encoded by ILV2 and/or ILV6 gene.

In the context of the present disclosure, the second heterologous polypeptide having ALDC activity include variants of the ALDC polypeptides of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 (also referred to herein as ALDC variants). A variant comprises at least one amino acid difference (substitution or addition) when compared to the amino acid sequence of the C polypeptides of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15. The ALDC variants do exhibit ALDC activity. In an embodiment, the variant ALDC exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the ALDC activity of the amino acid of SEQ ID NO: 16 or 18. The ALDC variants also have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15. The term“percent identity”, as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. The level of identity can be determined conventionally using known computer programs. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CAB I OS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y= 10). Default parameters for pairwise alignments using the Clustal method were KTUPLB 1 , GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The variant ALDC described herein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative amino acid substitutions are known in the art and are included herein. Non-conservative substitutions, such as replacing a basic amino acid with a hydrophobic one, are also well-known in the art.

A variant ALDC can also be a conservative variant or an allelic variant. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the ALDC. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the ALDC (e.g., catalyzing conversion of a-aceto lactate to acetoin). For example, the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the ALDC.

The present disclosure also provide fragments of the ALDC polypeptides and ALDC variants described herein. A fragment comprises at least one less amino acid residue when compared to the amino acid sequence of the ALDC polypeptide or variant and still possess the enzymatic activity of the full-length ALDC. In an embodiment, the ALDC fragment exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the full-length ALDC having the amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15. The ALDC fragments can also have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15. The fragment can be, for example, a truncation of one or more amino acid residues at the amino- terminus, the carboxy terminus or both termini of the ALDC polypeptide or variant. Alternatively or in combination, the fragment can be generated from removing one or more internal amino acid residues. In an embodiment, the ALDC fragment has at least 100, 150, 200, 250, 300, 350, 400 or more consecutive amino acids of the ALDC polypeptide or the variant.

When expressed in a recombinant host, the polypeptides described herein are encoded on one or more heterologous nucleic acid molecule. In an embodiment, a recombinant yeast host cell has a plurality of heterologous nucleic acid molecules. In an embodiment, a recombinant yeast host cell has the first heterologous polypeptide having ALDC activity encoded on a first heterologous nucleic acid molecule, and the second heterologous polypeptide having GA activity encoded on a second heterologous nucleic acid molecule. In an embodiment, a recombinant yeast host cell has a heterologous nucleic acid molecule. In an embodiment, a recombinant yeast host cell has a heterologous nucleic acid molecule encoding both the first heterologous polypeptide having ALDC activity and the second heterologous polypeptide having GA activity.

Polypeptides having glucoamylase activity

Polypeptides having glucoamylase activity (also referred to as glucoamylases) are exo-acting enzymes capable of terminally hydrolyzing starch to glucose. Glucoamylase activity can be determined by various ways by the person skilled in the art. For example, the glucoamylase activity of a polypeptide can be determined directly by measuring the amount of reducing sugars generated by the polypeptide in an assay in which raw or gelatinized (corn) starch is used as the starting material.

In the context of the present disclosure, the polypeptides having glucoamylase activity can be derived from a yeast, for example, from the genus Saccharomycopsis and, in some instances, from the species Saccharomycopsis fibuligera. The polypeptides having glucoamylase activity can be encoded by the glu0111 gene from S. fibuligera or a glu0111 gene ortholog. An embodiment of glucoamylase polypeptide of the present disclosure is the GLU01 11 polypeptide (GenBank Accession Number: CAC83969.1). The GLU01 11 polypeptide includes the following amino acids (or correspond to the following amino acids) which are associated with glucoamylase include, but are not limited to amino acids located at positions 41 , 237, 470, 473, 479, 485, 487 of SEQ ID NO: 16. It is possible to use a polypeptide which does not comprise its endogenous signal sequence. In an embodiment, the polypeptides having glucoamylase activity include glucoamylase polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 17 or 18. In an embodiment, the polypeptides having glucoamylase activity include glucoamylase polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 17 or 18 and a signal sequence. In an embodiment, the polypeptides having glucoamylase activity include glucoamylase polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 17 or 18 and a tethering moiety.

In the context of the present disclosure, a“glu01 11 gene ortholog” is understood to be a gene in a different species that evolved from a common ancestral gene by speciation. In the context of the present disclosure, a glu0111 ortholog retains the same function, e.g. it can act as a glucoamylase. Glu0111 gene orthologs includes but are not limited to, the nucleic acid sequence of GenBank Accession Number XP_003677629.1 ( Naumovozyma castellii)

XP_003685231.1 ( Tetrapisispora phaffii ), XP_455264.1 ( Kluyveromyces lactis), XP_446481.1 (Candida glabrata), EER33360.1 ( Candida tropicaiis), EEQ36251.1 ( Clavispora iusitaniae), ABN68429.2 ( Scheffersomyces stipitis), AAS51695.2 ( Eremothecium gossypii), EDK43905.1 (Lodderomyces elongisporus), XP_002555474.1 ( Lachancea thermotoierans), EDK37808.2 ( Pichia guilliermondii ), CAA86282 ( Saccharomyces cerevisiae), XP_003680486.1 ( Torulaspora delbrueckii ), XP_503574.1 ( Yarrowia iipoiytica), XP_002496552.1 (Zygosaccharomyces rouxii) , CAX42655.1 ( Candida dubiiniensis), XP_002494017.1 ( Komagataella pastoris) and AET38805.1 ( Eremothecium cymbalariae).

Embodiments of polypeptides having glucoamylase activity have been also been described in PCT/US2012/032443 (published under WO/2012/138942) and PCT/US2011/039192 (published under WO/2011/153516) can also be used in the context of the present disclosure.

The polypeptides having glucoamylase activity, their fragments and their variants can exhibit enzymatic activity towards raw starch. The GLU01 11 polypeptide presented herein as well as glucomylases from Rhizopus oryzae and Corticium rolfsiiare are known to exhibit enzymatic activity towards raw starch.

Still in the context of the present disclosure, it is possible to use a polypeptide which does not comprise its endogenous signal sequence. In an embodiment, the polypeptides having GA activity include GA polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 17 or 18. In an embodiment, the polypeptides having GA activity include GA polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 17 or 18 and a signal sequence. In an embodiment, the polypeptides having GA activity include GA polypeptide comprising the amino acid sequence of SEQ ID NO: 16, 17 or 18 and a tethering moiety.

Polypeptides having acetolactate decarboxylase activity

Polypeptides having ALDC activity (also referred to as acetolactate decarboxylases) include enzymes capable of catalyzing the following reaction.

(S)-2-hydroxy-2-methyl-3-oxobutanoate ^ (R)-2-acetoin + 0O ₂ (Eq. 1)

ALDC belongs to the family of lyases, specifically the carboxy-lyases, which cleave carbon- carbon bonds. The systematic name of this enzyme class is (S)-2-hydroxy-2-methyl-3- oxobutanoate carboxy-lyase [(R)-2-acetoin-forming]. Other names in common use include alpha-acetolactate decarboxylase, and (S)-2-hydroxy-2-methyl-3-oxobutanoate carboxy-lyase. ALDC activity can be determined by various ways by the person skilled in the art. For example, the ALDC activity of a polypeptide can be determined by measuring the diacetyl power of a substance treated with ALDC. As used herein,“diacetyl power” refers to combined free diacetyl and acetolactate content of a substance or mixture.

In the context of the present disclosure, the polypeptides having ALDC activity can be derived from any organisms, but, in some embodiments, from bacteria, for example, from the genus Acetobacter, Acetobacterium, Bacillus, Brevibacillus, Clostridium, Desulfovibrio, Enterobacter, Methanosarcina, Paenibacillus, Raoultella, or Sporolactobacillus. In an embodiment, the polyptides having ALDC activity is derived from Brevibacillus brevis, Bacillus subtilis, Clostridium sp. maddingley, or Enterobacter aerogenes.

In some instances, the polypeptides having ALDC activity can be derived from a bacteria from the genus Acetobacter. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Acetobacter aceti, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 6, a variant of the amino acid sequence of SEQ ID NO: 6, or a fragment of the amino acid sequence of SEQ ID NO: 6.

In some instances, the polypeptides having ALDC activity can be derived from a bacteria from the genus Acetobacterium. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Acetobacterium bakii, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 13, a variant of the amino acid sequence SEQ ID NO: 13, or a fragment of the amino acid sequence of SEQ ID NO: 13. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Acetobacterium woodii, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 12, a variant of the amino acid sequence of SEQ ID NO: 12, or a fragment of the amino acid sequence of SEQ ID NO: 12. In some instances, the polypeptides having ALDC activity can be derived from a bacteria from the genus Bacillus. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Bacillus amyloliquefaciens, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 3, a variant of the amino acid sequence of SEQ ID NO: 3, or a fragment of the amino acid sequence of SEQ ID NO: 3. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Bacillus cellulasensis, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 7, a variant of the amino acid sequence of SEQ ID NO: 7, or a fragment of the amino acid sequence of SEQ ID NO: 7. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Bacillus licheniformis, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 4, a variant of the amino acid sequence of SEQ ID NO: 4, or a fragment of the amino acid sequence of SEQ ID NO: 4. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Bacillus subtilis, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 5, a variant of the amino acid sequence of SEQ ID NO: 5, or a fragment of the amino acid sequence of SEQ ID NO: 5.

In some instances, the polypeptides having ALDC activity can be derived from a bacteria from the genus Brevibacillus. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Brevibacillus brevis, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 9, a variant of the amino acid sequence of SEQ ID NO: 9, or a fragment of the amino acid sequence of SEQ ID NO: 9.

In some instances, the polypeptides having ALDC activity can be derived from a bacteria from the genus Clostridium sp.. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Clostridium sp. maddingley, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 15, a variant of the amino acid sequence of SEQ ID NO: 15, or a fragment of the amino acid sequence of SEQ ID NO: 15.

In some instances, the polypeptides having ALDC activity can be derived from a bacteria from the genus Desulfovibrio. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Desulfovibrio frigidus, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 1 1 , a variant of the amino acid sequence of SEQ ID NO: 1 1 , or a fragment of the amino acid sequence of SEQ ID NO: 1 1 .

In some instances, the polypeptides having ALDC activity can be derived from a bacteria from the genus Enterobacter. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Enterobacter aerogenes, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 2, a variant of the amino acid sequence of SEQ ID NO: 2, or a fragment of the amino acid sequence of SEQ ID NO: 2. In some instances, the polypeptides having ALDC activity can be derived from a bacteria from the genus Methanosarcina. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Methanosarcina lacust s, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 14, a variant of the amino acid sequence of SEQ ID NO: 14, or a fragment of the amino acid sequence of SEQ ID NO: 14.

In some instances, the polypeptides having ALDC activity can be derived from a bacteria from the genus Paenibacillus. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Paenibacillus pini, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 10, a variant of the amino acid sequence of SEQ ID NO: 10, or a fragment of the amino acid sequence of SEQ ID NO: 10.

In some instances, the polypeptides having ALDC activity can be derived from a bacteria from the genus Raoultella. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Raoultella terrigena, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 1 , a variant of the amino acid sequence of SEQ ID NO: 1 , or a fragment of the amino acid sequence of SEQ ID NO: 1 .

In some instances, the polypeptides having ALDC activity can be derived from a bacteria from the genus Sporolactobacillus. In an embodiment, the polypeptides having ALDC activity can be derived from a bacteria from the species Sporolactobacillus inulinus, and include ALDC polypeptides comprising the amino acid sequence of SEQ ID NO: 8, a variant of the amino acid sequence of SEQ ID NO: 8, or a fragment of the amino acid sequence of SEQ ID NO: 8.

Still in the context of the present disclosure, it is possible to use a polypeptide which does not comprise its endogenous signal sequence. In an embodiment, the polypeptides having ALDC activity include ALDC polypeptide comprising the amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15. In an embodiment, the polypeptides having ALDC activity include ALDC polypeptide comprising the amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 and a signal sequence. In an embodiment, the polypeptides having ALDC activity include ALDC polypeptide comprising the amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 and a tethering moiety.

Recombinant host cell

In the context of the present disclosure, the recombinant host cell can be a recombinant yeast host cell. The recombinant yeast host cells of the present disclosure are intended for use in a fermentation process. In an embodiment, the recombinant yeast host cells can be used, for example, in a fermentation process for making alcoholic beverages, such as ale, lager, and light beer. The recombinant yeast host cells of the present disclosure can be provided in an active form (e.g. , liquid, compressed, or fluid-bed dried yeast), in a semi-active form (e.g., liquid, compressed, or fluid-bed dried), in an inactive form ( e.g ., drum- or spray-dried) as well as a mixture therefore.

The present disclosure concerns recombinant yeast host cells that have been genetically engineered. The genetic modification(s) is(are) aimed at increasing the expression of a specific targeted gene (which is considered heterologous to the yeast host cell) and can be made in one or multiple (e.g., 1 , 2, 3, 4, 5, 6, 7, 8 or more) genetic locations. In the context of the present disclosure, when recombinant yeast cell is qualified as being “genetically engineered”, it is understood to mean that it has been manipulated to add at least one or more heterologous or exogenous nucleic acid residue. In some embodiments, the one or more nucleic acid residues that are added can be derived from an heterologous cell or the recombinant host cell itself. In the latter scenario, the nucleic acid residue(s) is (are) added at one or more genomic location which is different than the native genomic location. The genetic manipulations did not occur in nature and are the results of in vitro manipulations of the yeast.

The one or more heterologous nucleic acid molecule present in the recombinant host cell can be integrated in the host cell’s genome. The term“integrated” as used herein refers to genetic elements that are placed, through molecular biology techniques, into the genome of a host cell. For example, genetic elements can be placed into the chromosomes of the host cell as opposed to in a vector such as a plasmid carried by the host cell. Methods for integrating genetic elements into the genome of a host cell are well known in the art and include homologous recombination. The heterologous nucleic acid molecule can be present in one or more copies (e.g., 2, 3, 4, 5, 6, 7, 8 or even more copies) in the yeast host cell’s genome. Alternatively, the heterologous nucleic acid molecule can be independently replicating from the yeast’s genome. In such embodiment, the nucleic acid molecule can be stable and self-replicating.

Suitable recombinant yeast host cells can be, for example, from the genus Saccharomyces, Kluyveromyces, Arxula, Debaryomyces, Candida, Pichia, Phaffia, Schizosaccharomyces, Hansenula, Kioeckera, Schwanniomyces or Yarrowia. Suitable yeast species can include, for example, S. cerevisiae, S. pastorianus, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus or K. fragilis. In some embodiments, the recombinant yeast host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pastorianus, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomyces occidentalis. In some embodiment, the recombinant host cell can be an oleaginous yeast cell. For example, the recombinant oleaginous yeast host cell can be from the genera Biakesiea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. In some alternative embodiments, the recombinant host cell can be an oleaginous microalgae host cell ( e.g ., for example, from the genera Thraustochythum or Schizochytrium). In an embodiment, the recombinant yeast host cell is from the genus Saccharomyces and, in some embodiments, from the species Saccharomyces cerevisiae or Saccharomyces pasto anus. In one particular embodiment, the recombinant yeast host cell is Saccharomyces cerevisiae. In one particular embodiment, the recombinant yeast host cell is Saccharomyces pastorianus.

The recombinant yeast host cells of the present disclosure include one or more heterologous nucleic acid molecule intended to allow the expression (e.g., encoding) of one or more heterologous proteins. In an embodiment, the heterologous protein is an heterologous enzyme. In the context of the present application, the heterologous enzyme can be, without limitation, an heterologous ALDC, and/or heterologous glucoamylase. The recombinant host cell can be further genetically modified to allow for the production of additional heterologous polypeptides. In an embodiment, the recombinant yeast host cell can be used for the production of an enzyme, and especially an enzyme involved in the cleavage or hydrolysis of its substrate (e.g., a lytic enzyme and, in some embodiments, a saccharolytic enzyme). In still another embodiment, the enzyme can be a glycoside hydrolase. In the context of the present disclosure, the term “glycoside hydrolase” refers to an enzyme involved in carbohydrate digestion, metabolism and/or hydrolysis, including amylases (other than those described above), cellulases, hemicellulases, cellulolytic and amylolytic accessory enzymes, inulinases, levanases, trehalases, pectinases, and pentose sugar utilizing enzymes. In another embodiment, the enzyme can be a protease. In the context of the present disclosure, the term “protease” refers to an enzyme involved in protein digestion, metabolism and/or hydrolysis. In yet another embodiment, the enzyme can be an esterase. In the context of the present disclosure, the term“esterase” refers to an enzyme involved in the hydrolysis of an ester from an acid or an alcohol, including phosphatases such as phytases.

The recombinant host cell can be provided as a fermenting agent for making a flavoured alcoholic beverage. In such embodiment, the fermenting agent include, without limitation a nutrient for the fermenting agent (for example, a carbon source).

In order to make the recombinant yeast host cells, heterologous nucleic acid molecules (also referred to as expression cassettes) are made in vitro and introduced into the yeast host cell in order to allow the recombinant expression of the heterologous polypeptides described herein.

The heterologous nucleic acid molecules of the present disclosure comprise a coding region for the heterologous polypeptide, e.g., the heterologous polypeptides described herein. A DNA or RNA “coding region” is a DNA or RNA molecule (preferably a DNA molecule) which is transcribed and/or translated into a heterologous polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences.“Suitable regulatory regions” refer to nucleic acid regions located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing or stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, RNA processing site, effector binding site and stem-loop structure. The boundaries of the coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding region can include, but is not limited to, prokaryotic regions, cDNA from mRNA, genomic DNA molecules, synthetic DNA molecules, or RNA molecules. If the coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding region. In an embodiment, the coding region can be referred to as an open reading frame.“Open reading frame” is abbreviated ORF and means a length of nucleic acid, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.

The nucleic acid molecules described herein can comprise transcriptional and/or translational control regions.“Transcriptional and translational control regions” are DNA regulatory regions, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding region in a host cell. In eukaryotic cells, polyadenylation signals are control regions.

In some embodiments, the heterologous nucleic acid molecules of the present disclosure include a promoter as well as a coding sequence for the heterologous polypeptides described herein. The heterologous nucleic acid sequence can also include a terminator. In the heterologous nucleic acid molecules of the present disclosure, the promoter and the terminator (when present) are operatively linked to the nucleic acid coding sequence of the heterologous polypeptide (including heterologous proteins comprising same), e.g. , they control the expression and the termination of expression of the nucleic acid sequence of the heterologous polypeptide. The heterologous nucleic acid molecules of the present disclosure can also include a nucleic acid coding for a signal peptide, e.g., a short peptide sequence for exporting the heterologous polypeptide outside the host cell. When present, the nucleic acid sequence coding for the signal peptide is directly located upstream and is in frame with the nucleic acid sequence coding for the heterologous polypeptide.

In the heterologous nucleic acid molecule described herein, the promoter and the nucleic acid molecule coding for the heterologous polypeptide are operatively linked to one another. In the context of the present disclosure, the expressions “operatively linked” or “operatively associated” refers to fact that the promoter is physically associated to the nucleotide acid molecule coding for the heterologous polypeptide in a manner that allows, under certain conditions, for expression of the heterologous protein from the nucleic acid molecule. In an embodiment, the promoter can be located upstream (5’) of the nucleic acid sequence coding for the heterologous protein. In still another embodiment, the promoter can be located downstream (3’) of the nucleic acid sequence coding for the heterologous protein. In the context of the present disclosure, one or more than one promoter can be included in the heterologous nucleic acid molecule. When more than one promoter is included in the heterologous nucleic acid molecule, each of the promoters is operatively linked to the nucleic acid sequence coding for the heterologous protein. The promoters can be located, in view of the nucleic acid molecule coding for the heterologous protein, upstream, downstream as well as both upstream and downstream.

“Promoter” refers to a DNA fragment capable of controlling the expression of a coding sequence or functional RNA. The term“expression,” as used herein, refers to the transcription and stable accumulation of sense (mRNA) from the heterologous nucleic acid molecule described herein. Expression may also refer to translation of mRNA into a polypeptide. 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 comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cells at most times at a substantial similar level are commonly referred to as“constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. A promoter is generally bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of the polymerase.

The promoter can be native or heterologous to the nucleic acid molecule encoding the heterologous polypeptide. The promoter can be heterologous or derived from a strain being from the same genus or species as the recombinant host cell. In an embodiment, the promoter is derived from the same genus or species of the yeast host cell and the heterologous polypeptide is derived from a different genus than the host cell. The promoter can be a single promoter or a combination of different promoters.

In the present disclosure, promoters allowing or favoring the expression of the heterologous polypeptides during the propagation phase of the recombinant yeast host cells are preferred. Yeasts that are facultative anaerobes, are capable of respiratory reproduction under aerobic conditions and fermentative reproduction under anaerobic conditions. In many commercial applications, yeast are propagated under aerobic conditions to maximize the conversion of a substrate to biomass. Optionally, the biomass can be used in a subsequent fermentation under anaerobic conditions to produce a desired metabolite. In the context of the present disclosure, it is important that the promoter or combination of promoters present in the heterologous nucleic acid is/are capable of allowing the expression of the heterologous polypeptide during the propagation phase of the recombinant yeast host cell. This will allow the accumulation of the heterologous polypeptides associated with the recombinant yeast host cell prior to fermentation (if any). In some embodiments, the promoter allows the expression of the heterologous polypeptide during propagation, but not during fermentation (if any) of the recombinant yeast host cell.

The promoters can be native or heterologous to the heterologous gene encoding the heterologous protein. The promoters that can be included in the heterologous nucleic acid molecule can be constitutive or inducible. Inducible promoters include, but are not limited to glucose-regulated promoters (e.g., the promoter of the hxt7 gene (referred to as hxt7p); the promoter of the ctt1 gene (referred to as cttl p), a functional variant or a functional fragment thereof; the promoter of the glo1 gene (referred to as glol p), a functional variant or a functional fragment thereof; the promoter of the ygp1 gene (referred to as ygp1 p), a functional variant or a functional fragment thereof; the promoter of the gsy2 gene (referred to as gsy2p, a functional variant or a functional fragment thereof), molasses-regulated promoters (e.g., the promoter of the moll gene (referred to as moU p), a functional variant or a functional fragment thereof), heat shock-regulated promoters (e.g., the promoter of the glo1 gene (referred to as glol p), a functional variant or a functional fragment thereof; the promoter of the sti1 gene (referred to as stil p), a functional variant or a functional fragment thereof; the promoter of the ygp1 gene (referred to as ygpl p), a functional variant or a functional fragment thereof; the promoter of the gsy2 gene (referred to as gsy2p), a functional variant or a functional fragment thereof), oxidative stress response promoters (e.g., the promoter of the cup1 gene (referred to as cupl p), a functional variant or a functional fragment thereof; the promoter of the ctt1 gene (referred to as cttl p), a functional variant or a functional fragment thereof; the promoter of the trx2 gene (referred to as trx2p), a functional variant or a functional fragment thereof; the promoter of the gpd1 gene (referred to as gpdl p), a functional variant or a functional fragment thereof; the promoter of the hsp12 gene (referred to as hsp12p), a functional variant or a functional fragment thereof), osmotic stress response promoters (e.g. , the promoter of the ctt1 gene (referred to as cttl p), a functional variant or a functional fragment thereof; the promoter of the glo1 gene (referred to as glol p), a functional variant or a functional fragment thereof; the promoter of the gpd1 gene (referred to as gpdl p), a functional variant or a functional fragment thereof; the promoter of the ygp1 gene (referred to as ygpl p), a functional variant or a functional fragment thereof) and nitrogen-regulated promoters (e.g., the promoter of the ygp1 gene (referred to as ygp1 p), a functional variant or a functional fragment thereof).

Promoters that can be included in the heterologous nucleic acid molecule of the present disclosure include, without limitation, the promoter of the tdh1 gene, of the hor7 gene, of the hsp150 gene, of the hxt7 gene, of the gpm1 gene, of the pgk1 gene and/or of the stH gene (referred to as stU p, a functional variant or a functional fragment thereof). In an embodiment, the promoter is or comprises the tdhl p and/or the hor7p. In still another embodiment, the promoter comprises or consists essentially of the tdhl p and the hor7p. In a further embodiment, the promoter is the thd 1 p.

In the context of the present disclosure, the promoter controlling the expression of the heterologous polypeptide can be a constitutive promoter (such as, for example, tef2p (e.g. , the promoter of the tef2 gene), cwp2p (e.g., the promoter of the cwp2 gene), ssal p (e.g. , the promoter of the ssa1 gene), end p (e.g. , the promoter of the eno1 gene), hxk1 (e.g., the promoter of the hxk1 gene) and pgkl p (e.g., the promoter of the pgk1 gene). In some embodiment, the promoter is adhl p (e.g. , the promoter of the adh1 gene). However, is some embodiments, it is preferable to limit the expression of the polypeptide. As such, the promoter controlling the expression of the heterologous polypeptide can be an inducible or modulated promoters such as, for example, a glucose-regulated promoter (e.g., the promoter of the hxt7 gene (referred to as hxt7p)) or a sulfite-regulated promoter (e.g., the promoter of the gpd2 gene (referred to as gpd2p or the promoter of the fzf1 gene (referred to as the fzf 1 p)) , the promoter of the ssu1 gene (referred to as ssul p), the promoter of the ssuf-r gene (referred to as ssur1 -rp). In an embodiment, the promoter is an anaerobic-regulated promoters, such as, for example tdhl p (e.g. , the promoter of the tdh1 gene), pau5p (e.g. , the promoter of the pau5 gene), hor7p (e.g., the promoter of the hor7 gene), adhl p (e.g., the promoter of the adh1 gene), tdh2p (e.g., the promoter of the tdh2 gene), tdh3p (e.g., the promoter of the tdh3 gene), gpdl p (e.g., the promoter of the gdp1 gene), cdc19p (e.g. , the promoter of the cdc19 gene), eno2p (e.g. , the promoter of the eno2 gene), pdd p (e.g. , the promoter of the pdd gene), hxt3p (e.g., the promoter of the hxt3 gene), dan1 (e.g. , the promoter of the dan1 gene) and tpM p (e.g. , the promoter of the tpi1 gene).

One or more promoters can be used to allow the expression of each heterologous polypeptides in the recombinant yeast host cell. In the context of the present disclosure, the expression “functional fragment of a promoter” when used in combination to a promoter refers to a shorter nucleic acid sequence than the native promoter which retain the ability to control the expression of the nucleic acid sequence encoding the heterologous polypeptide during the propagation phase of the recombinant yeast host cells. Usually, functional fragments are either 5’ and/or 3’ truncation of one or more nucleic acid residue from the native promoter nucleic acid sequence.

In some embodiments, the nucleic acid molecules include a one or a combination of terminator sequence(s) to end the translation of the heterologous polypeptide. The terminator can be native or heterologous to the nucleic acid sequence encoding the heterologous polypeptide. In some embodiments, one or more terminators can be used. In some embodiments, the terminator comprises the terminator from is from the dit1 gene, from the idp1 gene, from the gpm1 gene, from the pma1 gene, from the tdh3 gene, from the hxt2 gene, from the adh3 gene, from the cyd gene, from the pgk1 gene and/or from the ira2 gene. In an embodiment, the terminator is derived from the dit1 gene. In another embodiment, the terminator comprises or is derived from the adh3 gene. In the context of the present disclosure, the expression“functional variant of a terminator” refers to a nucleic acid sequence that has been substituted in at least one nucleic acid position when compared to the native terminator which retain the ability to end the expression of the nucleic acid sequence coding for the heterologous protein or its corresponding chimera. In the context of the present disclosure, the expression “functional fragment of a terminator” refers to a shorter nucleic acid sequence than the native terminator which retain the ability to end the expression of the nucleic acid sequence coding for the heterologous protein or its corresponding chimera.

The heterologous nucleic acid molecule encoding the heterologous polypeptide, variant or fragment thereof can be integrated in the genome of the yeast host cell. The term“integrated” as used herein refers to genetic elements that are placed, through molecular biology techniques, into the genome of a host cell. For example, genetic elements can be placed into the chromosomes of the host cell as opposed to in a vector such as a plasmid carried by the host cell. Methods for integrating genetic elements into the genome of a host cell are well known in the art and include homologous recombination. The heterologous nucleic acid molecule can be present in one or more copies in the yeast host cell’s genome. Alternatively, the heterologous nucleic acid molecule can be independently replicating from the yeast’s genome. In such embodiment, the nucleic acid molecule can be stable and self-replicating.

The present disclosure also provides nucleic acid molecules for modifying the yeast host cell so as to allow the expression of the heterologous polypeptides, variants or fragments thereof. The nucleic acid molecule may be DNA (such as complementary DNA, synthetic DNA or genomic DNA) or RNA (which includes synthetic RNA) and can be provided in a single stranded (in either the sense or the antisense strand) or a double stranded form. The contemplated nucleic acid molecules can include alterations in the coding regions, non-coding regions, or both. Examples are nucleic acid molecule variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the heterologous polypeptides, variants or fragments thereof.

In some embodiments, the nucleic acid molecules encoding the heterologous polypeptides, fragments or variants that can be introduced into the recombinant host cells are codon- optimized with respect to the intended recipient recombinant host cell. As used herein the term “codon-optimized coding region” means a nucleic acid coding region that has been adapted for expression in the cells of a given organism by replacing at least one, or more than one, codons with one or more codons that are more frequently used in the genes of that organism. In general, highly expressed genes in an organism are biased towards codons that are recognized by the most abundant tRNA species in that organism. One measure of this bias is the“codon adaptation index” or“CAI,” which measures the extent to which the codons used to encode each amino acid in a particular gene are those which occur most frequently in a reference set of highly expressed genes from an organism. The CAI of codon optimized heterologous nucleic acid molecule described herein corresponds to between about 0.8 and 1 .0, between about 0.8 and 0.9, or about 1.0.

The heterologous nucleic acid molecule can be introduced in the host cell using a vector. A “vector,” e.g., a“plasmid”,“cosmid” or“artificial chromosome” (such as, for example, a yeast artificial chromosome) refers to an extra chromosomal element and is usually in the form of a circular double-stranded DNA molecule. Such vectors may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

The present disclosure also provides nucleic acid molecules that are hybridizable to the complement nucleic acid molecules encoding the heterologous polypeptides as well as variants or fragments. A nucleic acid molecule is“hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified, e.g., in Sambrook, J„ Fritsch, E. F. and Maniatis, T. MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 1 1.1 therein. The conditions of temperature and ionic strength determine the“stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50°C for 30 min. For more stringent conditions, washes are performed at higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS are increased to 60°C. Another set of highly stringent conditions uses two final washes in 0.1X SSC, 0.1 % SDS at 65°C. An additional set of highly stringent conditions are defined by hybridization at 0.1 X SSC, 0.1 % SDS, 65°C and washed with 2X SSC, 0.1 % SDS followed by 0.1X SSC, 0.1 % SDS.

Hybridization requires that the two nucleic acid molecules contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived. For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity. In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

Localized expression and tethering moieties

In the context of the present disclosure, the heterologous polypeptide can be expressed intracellularly and only released in the extracellular environment upon the lysis of the recombinant yeast host cell. Also in the context of the present disclosure, the heterologous polypeptide can be“cell-associated” to the recombinant yeast host cell because it is designed to be expressed and remain physically associated with the recombinant yeast host cells. When the heterologous protein is intended to be expressed intracellularly, its signal sequence, if present in the native sequence, can be deleted to allow intracellular expression.

In another embodiment, the heterologous polypeptide of the present disclosure can be expressed in a secreted form. In the context of the present disclosure, when an heterologous polypeptide is expressed in a secreted form, even though it may have a transitory presence inside the cell, it is destined to be transferred outside the cell and exhibit its activity outside the cell. In some embodiments, it can be secreted without being physically associated with the recombinant yeast host cell. In other embodiments, it can remain physically associated with the recombinant yeast host cell. In an embodiment, at least one portion (usually at least one terminus) of the heterologous protein is bound, covalently, non-covalently and/or electrostatically for example, to cell wall (and in some embodiments to the cytoplasmic membrane). For example, the heterologous protein can be modified to bear one or more transmembrane domains, to have one or more lipid modifications (myristoylation, palmitoylation, farnesylation and/or prenylation), to interact with one or more membrane-associated protein and/or to interactions with the cellular lipid rafts. While the heterologous protein may not be directly bound to the cell membrane or cell wall (e.g. , such as when binding occurs via a tethering moiety), the protein is nonetheless considered a“cell-associated” heterologous protein according to the present disclosure.

In some embodiments, the heterologous polypeptide can be expressed to be located at and associated to the cell wall of the recombinant yeast host cell. In some embodiments, the heterologous polypeptide is expressed to be located at and associated to the external surface of the cell wall of the host cell. In some embodiments, the heterologous protein is expressed to be located at and spans across the cell wall of the host cell. In some embodiments, the heterologous polypeptide is expressed to be located at and partially embedded in the cell wall of the host cell. Recombinant yeast host cells all have a cell wall (which includes a cytoplasmic membrane) defining the intracellular (e.g. , internally-facing the nucleus) and extracellular (e.g., externally-facing) environments. The heterologous protein can be located at (and in some embodiments, physically associated to) the external face of the recombinant yeast host’s cell wall and, in further embodiments, to the external face of the recombinant yeast host’s cytoplasmic membrane. In the context of the present disclosure, the expression“associated to the external face of the cell wall/cytoplasmic membrane of the recombinant yeast host cell” refers to the ability of the heterologous protein to physically integrate (in a covalent or non- covalent fashion), at least in part, in the cell wall (and in some embodiments in the cytoplasmic membrane) of the recombinant yeast host cell. The physical integration can be attributed to the presence of, for example, a transmembrane domain on the heterologous protein, a domain capable of interacting with a cytoplasmic membrane protein on the heterologous protein, a post- translational modification made to the heterologous protein (e.g., lipidation), etc.

Some heterologous polypeptide have the intrinsic ability to locate at and associate to the cell wall of a recombinant yeast host cell (e.g., being cell-associated). However, in some circumstances, it may be warranted to increase or provide cell association to some heterologous polypeptides because they exhibit insufficient intrinsic cell association or simply lack intrinsic cell association. In such embodiment, it is possible to provide the heterologous protein as a chimeric construct by combining it with a tethering amino acid moiety which will provide or increase attachment to the cell wall of the recombinant yeast host cell. In such embodiment, the chimeric heterologous polypeptide will be considered“tethered”. It is preferred that the amino acid tethering moiety of the chimeric protein be neutral with respect to the biological activity of the heterologous protein, e.g. , does not interfere with the biological activity (such as, for example, the enzymatic activity) of the heterologous polypeptide. In some embodiments, the association of the amino acid tethering moiety with the heterologous polypeptide can increase the biological activity of the heterologous protein (when compared to the non-tethered,“free” form).

In an embodiment, the recombinant yeast host cell expresses an heterologous polypeptide having ALDC activity which can be a chimeric polypeptide. In another embodiment, the recombinant yeast host cell expresses an heterologous polypeptide having glucoamylase activity which can be a chimeric polypeptide. In some embodiments, the recombinant yeast host cell can express both the ALDC and GA heterologous polypeptides as chimeric polypeptides.

In an embodiment, a tethering moiety can be used to be expressed with the heterologous protein to locate the heterologous protein to the wall of the recombinant yeast host cell. Various tethering amino acid moieties are known in the art and can be used in the chimeric proteins of the present disclosure. The tethering moiety can be a transmembrane domain found on another protein and allow the chimeric protein to have a transmembrane domain. In such embodiment, the tethering moiety can be derived from the FL01 protein (having, for example, the amino acid sequence of SEQ ID NO: 20, a variant thereof or a fragment thereof or being encoded by the nucleic acid sequence of SEQ ID NO: 19).

In still another example, the amino acid tethering moiety can be modified post-translation to include a glycosylphosphatidylinositol (GPI) anchor and allow the chimeric protein to have a GPI anchor. GPI anchors are glycolipids attached to the terminus of a protein (and in some embodiments, to the carboxyl terminus of a protein) which allows the anchoring of the protein to the cytoplasmic membrane of the cell membrane. Tethering amino acid moieties capable of providing a GPI anchor include, but are not limited to those associated with/derived from a SED1 protein (having, for example, the amino acid sequence of SEQ ID NO: 22, a variant thereof or a fragment thereof or being encoded by the nucleic acid sequence of SEQ ID NO: 21), a TIR1 protein (having, for example, the amino acid sequence of SEQ ID NO: 24, a variant thereof or a fragment thereof or being encoded by the nucleic acid sequence of SEQ ID NO: 23), a CWP2 protein (having, for example, the amino acid sequence of SEQ ID NO: 26, a variant thereof or a fragment thereof or being encoded by the nucleic acid sequence of SEQ ID NO: 25), a CCW12 protein (having, for example, the amino acid sequence of SEQ ID NO: 28 or 48, a variant thereof or a fragment thereof or being encoded by the nucleic acid sequence of SEQ ID NO: 27), a SPI1 protein (having, for example, the amino acid sequence of SEQ ID NO: 30 or 38, a variant thereof or a fragment thereof or being encoded by the nucleic acid sequence of SEQ ID NO: 29), a PST1 protein (having, for example, the amino acid sequence of SEQ ID NO: 32, a variant thereof or a fragment thereof or being encoded by the nucleic acid sequence of SEQ ID NO: 31) or a combination of a AGA1 and a AGA2 protein (having, for example, the amino acid sequence of SEQ ID NO: 34, a variant thereof or a fragment thereof or being encoded by the nucleic acid sequence of SEQ ID NO: 33 or having, for example, the amino acid sequence of SEQ ID NO: 36, a variant thereof or a fragment thereof or being encoded by the nucleic acid sequence of SEQ ID NO: 35). In an embodiment, the tethering moiety provides a GPI anchor and, in still a further embodiment, the tethering moiety is derived from the SPI1 protein (having, for example, the amino acid sequence of SEQ ID NO: 30, a variant thereof or a fragment thereof or being encoded by the nucleic acid sequence of SEQ ID NO: 29) or the CCW12 protein (having, for example, the amino acid sequence of SEQ ID NO: 28, a variant thereof or a fragment thereof or being encoded by the nucleic acid sequence of SEQ ID NO: 27).

In an embodiment, the tethering moiety is a fragment of the SPI1 protein that retained its ability to localize to the cell’s membrane. The fragment of the SPI1 protein comprises less than 129 amino acid consecutive residues of the amino acid sequence of SEQ ID NO: 38. For example, the tethering moiety fragment from the SPI1 protein can comprise at least 10, 20, 21 , 30, 40, 50, 51 , 60, 70, 80, 81 , 90, 100, 1 10, 1 1 1 or 120 consecutive amino acid residues from the amino acid sequence of SEQ ID NO: 38. In yet another embodiment, the tethering moiety fragment from the SPI1 protein can comprise or consist essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 40, 42, 44, or 46.

In another embodiment, the tethering moiety is a fragment of a CCW12 protein that retained its ability to localize to the cell’s membrane. The fragment of the CCW12 protein comprises less than 1 12 amino acid consecutive residues of the amino acid sequence of SEQ ID NO: 48. For example, the tethering moiety fragment from the CCW12 protein can comprise at least 10, 20, 24, 30, 40, 49, 50, 60, 70, 74, 80, 90, 99, 100 or 1 10 consecutive amino acid residues from the amino acid sequence of SEQ ID NO: 48. In yet another embodiment, the tethering moiety fragment from the CCW12 protein can comprise or consist essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 50, 52, 54, or 56.

The tethering amino acid moiety can be a variant of a known/native tethering amino acid moiety, for example a variant of the tethering amino acid moiety having the amino acid sequence of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 74, 76, 78, 80, 82, 84, 86, 88, 90 or 92. A variant comprises at least one amino acid difference when compared to the amino acid sequence of the native tethering amino acid moiety. As used herein, a variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the tethering amino acid moiety (e.g., the location on the external face and the anchorage of the heterologous protein in the cytoplasmic membrane). A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the tethering amino acid moiety (e.g. , the location on the external face and the anchorage of the heterologous protein in the cytoplasmic membrane). For example, the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the tethering amino acid moiety. The tethering amino acid moiety variants have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the tethering amino acid moieties described herein. The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. The level of identity can be determined conventionally using known computer programs. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CAB I OS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y= 10). Default parameters for pairwise alignments using the Clustal method were KTUPLB 1 , GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The variant tethering amino acid moieties described herein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide. A“variant” of the tethering amino acid moiety can be a conservative variant or an allelic variant.

The tethering amino acid moiety can be a fragment of a known/native tethering amino acid moiety or fragment of a variant of a known/native tethering amino acid moiety (such as, for example, a fragment of the tethering amino acid moiety having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or a variant thereof). Tethering amino acid moiety“fragments” have at least at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more consecutive amino acids of the tethering amino acid moiety. A fragment comprises at least one less amino acid residue when compared to the amino acid sequence of the known/native tethering amino acid moiety and still possess the biological activity of the full- length tethering amino acid moiety (e.g., the location to the cell wall). In embodiments in which an amino acid tethering moiety is desirable, the heterologous protein can be provided as a chimeric protein expressed by the recombinant yeast host cell and having one of the following formula (I) or (II):

(NH ₂ ) HP - L - TT (COOH) (I)

(NH ₂ ) TT - L - HP (COOH) (II)

In both of these formulae, the residue“HP” refers to the heterologous polypeptide moiety, the residue“L” refers to the presence of an optional linker while the residue“TT” refers to an amino acid tethering moiety for associating the first heterologous polypeptide to a cell wall of the recombinant yeast host cell. In both of these formulae, (NH ₂ ) indicates the amino terminus of the chimeric polypeptide, (COOH) indicates the carboxyl terminus of the chimeric polypeptide, and is an amide linkage. In embodiment, the heterologous polypeptide moiety the polypeptide having ALDC activity, a variant thereof, or a fragment thereof. In embodiment, the heterologous polypeptide moiety the polypeptide having glucoamylase activity, a variant thereof, or a fragment thereof.

When the amino acid linker (L) is absent, the tethering amino acid moiety is directly associated with the heterologous protein. In the chimeras of formula (I), this means that the carboxyl terminus of the heterologous protein moiety is directly associated (with an amide linkage) to the amino terminus of the tethering amino acid moiety. In the chimeras of formula (II), this means that the carboxyl terminus of the tethering amino acid moiety is directly associated (with an amide linkage) to the amino terminus of the heterologous protein.

In some embodiments, the presence of an amino acid linker (L) is desirable either to provide, for example, some flexibility between the heterologous polypeptide moiety and the tethering amino acid moiety or to facilitate the construction of the heterologous nucleic acid molecule. As used in the present disclosure, the“amino acid linker” or“L” refer to a stretch of one or more amino acids separating the heterologous polypeptide moiety HP and the amino acid tethering moiety TT (e.g. , indirectly linking the heterologous polypeptide HP to the amino acid tethering moiety TT). It is preferred that the amino acid linker be neutral, e.g. , does not interfere with the biological activity of the heterologous polypeptide nor with the biological activity of the amino acid tethering moiety. In some embodiments, the amino acid linker L can increase the biological activity of the heterologous polypeptide moiety and/or of the amino acid tethering moiety. In instances in which the linker (L) is present in the chimeras of formula (I), its amino end is associated (with an amide linkage) to the carboxyl end of the heterologous protein moiety and its carboxyl end is associated (with an amide linkage) to the amino end of the amino acid tethering moiety. In instances in which the linker (L) is present in the chimeras of formula (II), its amino end is associated (with an amide linkage) to the carboxyl end of the amino acid tethering moiety and its carboxyl end is associated (with an amide linkage) to the amino end of the heterologous protein moiety. Various amino acid linkers exist and include, without limitations, (G)n, (GS) _n ; (GGS) _n ; (GGGS) _n ; (GGGGS) _n ; (GGSG) _n ; (GSAT) _n , wherein n = is an integer between 1 to 8 (or more). In an embodiment, the amino acid linker L is (GGGGS) _n (also referred to as G4S) and, in still further embodiments, the amino acid linker L comprises more than one G4S (SEQ ID NO: 57) motifs. For example, the amino acid linker L can be (G4S) ₃ and have the amino acid sequence of SEQ ID NO: 58. In another example, the amino acid linker L can be (G) _s and have the amino acid sequence of SEQ ID NO: 59. In still another example, the amino acid linker L can be (G4S) ₈ and have the amino acid sequence of SEQ ID NO: 60.

The amino acid linker can also be, in some embodiments, GSAGSAAGSGEF (SEQ ID NO: 61).

Additional amino acid linkers exist and include, without limitations, (EAAK) _n and (EAAA K) _n , wherein n = is an integer between 1 to 8 (or more). In some embodiments, the one or more (EAAK)n/(EAAAK)n motifs can be separated by one or more additional amino acid residues. In an embodiment, the amino acid linker comprises one or more EA2K (SEQ ID NO: 62) or EA3K (SEQ ID NO: 63) motifs. In an embodiment, the amino acid linker can be (EAAK) ₃ and has the amino acid sequence of SEQ ID NO: 64. In another embodiment, the amino acid linker can be (A(EAAAK)4ALEA(EAAAK)4A) and has the amino acid sequence of SEQ ID NO: 65.

Further amino acid linkers include those having one or more (AP)n motifs wherein n = is an integer between 1 to 10 (or more). In an embodiment, the linker is (AP)10 and has the amino acid of SEQ ID NO: 66.

In some embodiments, the linker also includes one or more HA tag (SEQ ID NO: 67).

Process for making an alcoholic beverage

The recombinant yeast host cell of the present disclosure have been designed to be used in the preparation of a fermented, and in some embodiments a low calorie, alcoholic beverages for human consumption. The present disclosure thus provides a process comprising contacting the recombinant yeast host cell of the present disclosure with a carbohydrate substrate to provide a mixture and fermenting the mixture. The fermentation can be conducted in the presence of and/or by the recombinant yeast host cell described herein.

In some embodiments, it may be advantageous to provide the recombinant yeast host cell of the present disclosure as a fermentation agent. The fermentation agent can include 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the recombinant yeast host cell of the present disclosure. In one embodiment, a fermentation agent for making a fermented alcoholic beverage comprising, consisting essentially or consisting of the recombinant yeast host cell described herein. As used herein, “consisting essentially of or “consist of in reference to a fermentation agent refers to a population of fermenting organisms which do not include a substantial amount of additional fermenting organisms which participate to the fermentation process. In an embodiment, a fermentation agent consisting essentially of the recombinant yeast host cell of the present disclosure is made up of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 99.9% of the recombinant yeast host cell described herein. In still another embodiment, a fermentation agent consisting essentially of the recombinant yeast host cell of the present disclosure is a monoculture of one strain of a recombinant yeast host cell. Alternatively, a fermentation agent comprising of the recombinant yeast host cell of the present disclosure is a combination of more than one strains of the recombinant yeast host cell described herein.

In the process described herein, the recombinant yeast host cells of the present disclosure can be provided in an active form (e.g. , liquid, compressed, or fluid-bed dried yeast), in a semiactive form (e.g. , liquid, compressed, or fluid-bed dried), in an inactive form (e.g. , drum- or spray-dried) as well as a mixture therefore. For example, the recombinant yeast host cells can be a combination of active and semi-active or inactive forms to provide the ratio and dose of the polypeptide required for making fermented alcoholic beverages. In an embodiment, the recombinant yeast host cells are provided in an active and dried form.

The recombinant host cells described herein can be used to hydrolyze (e.g. , saccharify) a complex carbohydrate substrate into glucose. In an embodiment, the recombinant host cells described herein can be used to hydrolyze complex carbohydrate substrate for making alcoholic beverages. The recombinant host cells described herein hydrolyze complex carbohydrate substrates into glucose to allow a concomitant or subsequent fermentation of glucose into ethanol.

A biomass that can be fermented with the recombinant host cell described herein includes any type of biomass known in the art and described herein. For example, the biomass can include, but is not limited to, starch and/or sugar. Starch materials can include, but are not limited to, mashes such as corn, wheat, rye, barley, rice, or milo. Sugar materials can include, but are not limited to, sugar beets, artichoke tubers, sweet sorghum, molasses or cane.

Alternatively, the biomass can include, but not limited, a wort. Wort is a liquid extracted from the mashing process during the brewing of beer or whisky. Wort typically contains sugars, mostly maltose and/or maltotriose, that will be fermented by the brewing yeast to produce alcohol as well as small amounts of non-fermentable larger dextrins. Wort typically also contains amino acids to provide nitrogen to the yeast as well as more complex proteins contributing to beer head retention and flavor. Wort can be made from materials, such as mashes of corn, wheat, rye, barley, rice, or milo.

The process described herein comprises combining a substrate to be hydrolyzed (optionally included in a fermentation medium) with the recombinant host cells expressing the heterologous polypeptides. In some embodiments, the substrate is a biomass comprising starch or sugar. In some embodiments, the substrate is a wort. The recombinant host cells described herein can be used to decrease the caloric content of an alcoholic beverage produced by fermentation, compared with fermentation without addition of an exogenous (purified) amylase to the fermentation mixture. For example, the recombinant yeast host cell can be used to make a low calorie beer. A low-calorie beer can have less than 200, 190, 180, 170, 160, 150, 140, 130, 120, 1 10, 100 or less calories per 354 mL (12 oz) serving. In an embodiment, the recombinant host cells described herein can be used to decrease the dextrin content of an alcoholic beverage produced by fermentation. A low-calorie alcoholic beverage, such as a low calorie beer, can contain at most 5 g/L dextrin, most 1 g/L dextrin, at most 0.5 g/L dextrin, at most 0.1 g/L dextrin, or at most 0.05 g/L dextrin. A dextrin is defined as a hydrolyzed starch polymer greater than three glucose molecules (DP = degree of polymerization, DP3 = maltotriose). In some embodiments, dextrin is provided as a cumulative DP4+ value. In some embodiments, an alcoholic beverage produced using the recombinant host cells described herein has at least 25 g/L ethanol, at least 30 g/L ethanol, at least 35 g/L ethanol, at least 40 g/L ethanol, at least 45 g/L ethanol, at least 50 g/L ethanol, at least 55 g/L ethanol, at least 65 g/L ethanol, at least 70 g/L ethanol, at least 80 g/L ethanol, at least 85 g/L ethanol, at least 90 g/L ethanol, at least 95 g/L ethanol, at least 100 g/L ethanol, at least 105 g/L ethanol, at least 1 10 g/L ethanol, at least 120 g/L ethanol, at least 125 g/L ethanol, at least 130 g/L ethanol, at least 135 g/L ethanol, at least 140 g/L ethanol or at least 150 g/L ethanol. Ethanol production can be measured using any method known in the art. For example, the quantity of ethanol in fermentation samples can be assessed using HPLC analysis. Many ethanol assay kits are commercially available that use, for example, alcohol oxidase enzyme based assays.

The process comprises combining a carbohydrate substrate to be hydrolyzed (optionally included in a fermentation medium) with the recombinant host cells described herein, for the production of a fermented alcoholic beverage. In an embodiment, a carbohydrate substrate is wort. This embodiment is advantageous because it can reduce or eliminate the need to supplement the fermentation medium with external sources of purified enzymes (e.g. , glucoamylase and ALDC) while allowing the fermentation of the biomass during production of an alcoholic beverage. This embodiment is also advantageous because it reduces the time needed for fermentation and maturation. This embodiment is also advantageous because it allows for the production of a low-calorie alcoholic beverage while maintaining low concentration of flavor-imparting metabolites.

In some embodiments, combining a carbohydrate substrate with the recombinant host cells described herein reduces diacetyl content of the resulting mixture during fermentation. The mixture has a diacetyl power of equal to or less than 0.1 mg/L, 90 pg/L, 80 pg/L, 75 pg/L, 70 pg/L, 65 pg/L, 60 pg/L, 55 pg/L, 50, pg/L, 45 pg/L, 40 pg/L, 35 pg/L, 30 pg/L, 25 pg/L, 20 pg/L, 15 pg/L, 10 pg/L, 9 pg/L, 8 pg/L, 7 pg/L, 6 pg/L, 5 pg/L, 3 pg/L, 2 pg/L, 1 pg/L or 0.5 pg/L. In an embodiment, the diacetyl content of the mixture is reduced by increasing the conversion of a- acetolactate to acetoin during fermentation.

In some embodiments, the fermented alcoholic beverage is a beer. In such embodiments, the recombinant host cells described herein is from the species Saccharomyces cerevisiae. A beer can be an ale, a lager, including a light beer. In an embodiment, the fermented alcoholic beverage is a lager. In this embodiment, the recombinant yeast host cell is from the species Saccharomyces pasto anus. This embodiment is also advantageous because it reduces the need for a diacetyl rest to promote diacetyl uptake and enzymatic conversion by yeast, thereby simplifying fermentation and/or reducing the time needed for fermentation and maturation.

The fermentation for the production of an ale beer can be performed at temperatures between about 15 and about 40°C. For example, the fermentation for a production of an ale bear can be performed at a temperature of at least about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21 °C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C or about 40°C. The fermentation for the production of a lager beer can be performed at temperatures at temperatures between about 4°C and about 15°C. For example, the fermentation for the production of a lager beer can be performed at a temperature of less than about 15°C, less than about 14°C, less than about 13°C, less than about 12°C, less than about 1 1 °C, less than about 10°C, less than about 9°C, less than about 8°C, less than about 7°C, less than about 6°C or less than about 5°C.

The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I - HETEROLOGOUS PROTEIN EXPRESSION IN A LAGER STRAIN

BACKGROUND

The M13175 S. pastohanus lager strain and strains derived from M13175 (see Table 1) were added to a lager fermentation at 13°C. The fermentations were performed in duplicate with 12°Plato dry malt extract and 0.01 % hop oil with 0.25 g/L dry cell weight inoculum using 250 mL conical tubes at 175 mL volumes. Table 1. Genotypes of the Saccharomyces strains used in the examples

EXAMPLE II - HETEROLOGOUS GLUCOAMYLASE EXPRESSION IN A LAGER STRAIN

To reduce the caloric content of a lager beer, a glucoamylase from Saccharomycopsis fibuligera was integrated into the M13175 S. pastorianus lager strain to produce the M15301 strain. This M15301 strain was added to lager fermentation as described in Example I, and measurements of the degree of polymerization and ethanol production was measured using high performance liquid chromatography (HPLC) at 144 hours. Referring to Figure 2 and Table 2, the M15301 strain reduced the dextrin content of the lager fermentation product to 0.00 g/L as compared to wild-type M13175 strain, and also slightly higher ethanol production.

Samples were also collected at 96 hours and diacetyl potential was measured as combined free diacetyl and acetolactate using gas chromatography (Figure 3 and Table 2). The samples showed that the M15301 strain also increased the combined free diacetyl and acetolactate content of the lager fermentation product.

Table 2. Dextrin, ethanol and diacetyl potential of lager fermentation using Saccharomyces pastorianus strain M15301

EXAMPLE III - SELECTION OF HETEROLOGOUS ALDC

Referring to Figure 4, and SEQ ID N)s: 1 -15, 15 sequences of a-acetolactate decarboxylases (ALDC) were cloned into the M2390 S. cerevisiae distilling strain. For each sequence, a total of six individual transformants were picked and grown in 600 pi of YPD ₄₀ for 48 h. The supernatant for each clone was screened in the ALDC screening assay as described by Stormer (1975) and subsequently normalized to the M2390 parent control (Figure 4). Of the 15 sequences, only 13 were successfully integrated into the strain and subsequently screened for ALDC activity using a plate based assay measuring acetoin production. As seen in Figure 4, there were four sequences with measureable amounts of acetoin production (above 0.15), indicating a functionally secreted ALDC. The top four candidates were selected and sourced from the organisms Enterobacter aerogenes, Bacillus subtilis, Brevibacillus brevis, and Clostridium sp. maddingley.

The ALDC sequences were then integrated into the S. pastorianus lager strain, M13175 . Transformants were picked and grown in 600 pi of YP-DME ₄₀ for 72 h at room temperature. The cells were separated from the spent media by centrifuging at 3000 rpm for 5 min and the supernatant used in the ALDC assay measuring acetoin production. The transformants were screened for activity in the same plate-based ALDC assay to confirm activity (Figure 5).

The ALDC secreting lager strains were further screened for diacetyl reduction during a lager fermentation. Fermentations were run at 175 ml volumes in 250 ml conical tubes at 13°C. The wort was comprised of 12° Plato dry malt extract with 0.01 % tetra-iso alpha acid hop oil and pitched at 0.25 g/L dry cell weight. Samples were collected at 96 h and analyzed via gas chromatography (GC) for both a-acetolactate and free diacetyl to measure total diacetyl potential. As seen in Figure 6, all four ALDC secreting strains reduced the diacetyl potential by greater than 88% as compared to the parent M13175 lager strain.

The ALDC expressing strains were also evaluated for fermentation kinetics by measuring specific gravity as related to sugar consumption (Figure 7). These strains were added to a lager fermentation as described in Example I. The specific gravity was measured using a brix refractometer at 0 h, 48 h, 96 h, 144 h, and 192 h.

EXAMPLE IV - HETEROLOGOUS GA AND ALDC EXPRESSION IN A LAGER

STRAIN

In order to determine if strains expressing heterologous ALDC could be used to reduce the diacetyl content of strains expressing heterologous GA, additional recombinant strains were engineered. More specifically, the E. aerogenes (SEQ ID NO: 2) and B. subtilis (SEQ ID NO: 5) ALDC sequences were used to design new cassettes co-expressing the respective ALDC and GA from Saccharomycopsis fibuligera, along with a control strain only expressing the same GA. Assays were performed using 1 % gel starch and mixed with supernatant from YPD-propped cultures; reducing sugars were measured using the DNS assay at 540 nm (Figure 8). Once GA activity was confirmed, the GA only, ALDC only, and GA-ALDC co-expressing strains were further evaluated in a lab-scale wort fermentation. Diacetyl potential was again measured via GC and residual dextrins and ethanol measured by HPLC. As seen in Figure 8, the co- expressing strains M15299 and M15300 provided similar diacetyl reduction as the ALDC only expressing strains, M15295 and M15296. As previously discussed, the parent and GA-only expressing strain, M15301 , did not have any reduction in diacetyl. Samples collected at 144 h show that the GA expressing strains completely remove residual dextrins as represented by DP4+ values (Figure 2).

EXAMPLE V - EXPRESSION OF CELL-ASSOCIATED ALDC

Cell-associated ALDC enzymes developed using tethering technology were evaluated. The B. subtilis ALDC sequence was fused to a number of native yeast cell-associated proteins and evaluated for tethered ALDC activity. Six separate transformants were picked and grown in 600 pi of YP-DME 40 g/L for 72 h at room temperature. The cells were separated from the spent media by centrifuging at 3000 rpm for 3 min and the supernatant removed. Cells were washed twice with water and re-suspended in water. The cell slurry was used in the ALDC assay measuring acetoin production.

As seen in Figure 9, the CCW12 and SPI1 tethers proteins provided the most significant cell- associated ALDC activity as compared to the secreted control strain. The cell-association of the ALDC enzyme allows for further expansion of this technology in yeast nutrient products and/or provide an alternative enzyme delivery system.

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