Modulation in production of ester flavor compounds in yeast for flavored beverage production

Abstract

The present disclosure concerns a recombinant yeast host cell for making a flavored solution obtainable or obtained by fermentation of a fermentation medium. The recombinant yeast host cell comprises an upregulated alcohol acetyltransferase 2 (ATF2) and / or an upregulated phenylpyruvate decarboxylase (ARO10); and at least one downregulated native transferase when compared to a wild-type yeast host cell. The at least one downregulated transferase is a native alcohol acetyltransferase 1 (ATF1) and / or a native ethanol acetyl-CoA transferase (EAT1). The present disclosure also concerns process for making a flavored solution which could be used to generate new flavored beverages such as beer, distilled spirits, and wines.

Description

MODULATION IN PRODUCTION OF ESTER FLAVOR COMPOUNDS IN YEAST FOR FLAVORED BEVERAGE PRODUCTIONTECHNOLOGICAL FIELD

[0001] The present disclosure relates to the modulation in production of ester flavor compounds in recombinant yeast host cells for imparting a flavor in the production of flavored beverages and to methods of flavoring a beverage thereof.BACKGROUND

[0002] Volatile esters produced by yeast during fermentation contribute to the fruity and floral organoleptic properties of fermented beverages, including distilled spirits, wines, beer, and sake [1-5], Yeast produces two types of esters, ethyl and acetate esters, which include ethyl acetate (EA), isoamyl acetate (IAA), isobutyl acetate (IBA) and phenethyl acetate (PEA). However, IBA and PEA are generally very low or not detectable in commercial spirits by standard analysis techniques. Increasing the production of IBA and PEA during yeast fermentation represents an important challenge, particularly in the alcoholic beverage industry that relies on yeast fermentation. In contrast, overproduction of EA and IAA may lead to undesirable flavor profiles since above a certain threshold, desirable fruity and floral notes can turn to solvent-like, chemical-like unpleasant aromas [1 , 3], Indeed, while excessively high concentrations of EA and IAA has a negative effect on the organoleptic properties of wine, brandy and beer-type beverages, [1 , 3, 12], controlled overproduction (i.e. modulation) of these compounds was proposed to have the potential of enhancing aroma complexity by providing intense fruity and floral notes by the authors. Thus, modulation of the expression of enzymes involved in the production of ester flavor compounds to arrive at a desirable flavour profile would be highly desirable.

[0003] There is thus a need to develop yeast strains capable of producing acetate esters at desired levels to obtain flavoured solutions having desirable flavour profiles, which could be used in the development of novel flavoured beverages.SUMMARY OF INVENTION

[0004] The present disclosure concerns a recombinant yeast host cell for making a flavored solution and / or to a process for making a flavored solution.

[0005] According to a first aspect, the present invention is directed to a recombinant yeast host cell for making a flavored solution obtainable or obtained by fermentation of a fermentation medium.

[0006] According to a preferred embodiment, the recombinant yeast host cell comprises: a) an upregulated alcohol acetyltransferase 2 (ATF2) and / or an upregulated phenylpyruvate decarboxylase (ARQ10) when compared to a wild-type yeast host cell; and b) at least one downregulated native transferase when compared to a wildtype yeast host cell, wherein the at least one downregulated transferase is a native alcohol acetyltransferase 1 (ATF1) and / or a native ethanol acetyl-CoA transferase (EAT1).

[0007] According to an embodiment, the recombinant yeast host cell comprises a heterologous ATF2 as the upregulated ATF2.

[0008] According to some embodiment, the ATF2 is an alcohol acetyltransferase 2 classified under Enzyme Commission No. 2.3.1 .84; has the ability to convert acetyl- CoA and various alcohol into acetate esters acid; is of prokaryotic or eukaryotic origin; is a ATF2; is encoded by an atf2 gene; is derived from Saccharomyces or Torulaspora species; is derived from Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces cerevisiae x Saccharomyces kudriavzevii, Saccharomyces paradoxus, Saccharomyces mikatae, Torulaspora delbrueckii or Saccharomyces kudriavzevir, has the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20 or be a variant of the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20 having alcohol acetyltransferase 2 activity or be a fragment of the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20 having alcohol acetyltransferase 2 activity; is encoded by a heterologous nucleic acid molecule having a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20; and / or is operably associated to a native or a heterologous promoter.

[0009] According to an embodiment, the recombinant yeast host cell comprises a heterologous ARQ10 as the upregulated ARO10.

[0010] According to some embodiment, the ARQ10 is a phenylpyruvate decarboxylase classified under Enzyme Commission No. 4.1.1.43; has the ability to convert phenylpyruvate into phenylacetaldehyde; is of prokaryotic or eukaryotic origin; is a ARQ10; is encoded by an aro10 gene; is derived from Saccharomyces, Naumovozyma, Maudiozyma, Kazachstania, Arxiozyma, Huiozyma, Nakaseomyces, Naumovozyma, Torulaspora, Zygosaccharomyces or Henningerozyma species; isderived from Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces arboricola, Saccharomyces kudriavzevii, Saccharomyces eubayanus, Naumovozyma castellii, Maudiozyma humilis, Kazachstania Africana, Maudiozyma exigua, Maudiozyma barnettii, Arxiozyma heterogenica, Huiozyma naganishii, Nakaseomyces glabratus, Naumovozyma dairenensis, Torulaspora delbrueckii, Torulaspora globosa, Zygosaccharomyces rouxii Zygosaccharomyces parabailii or Henningerozyma blatae has the amino acid sequence of SEQ ID NO: 5, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 or be a variant of the amino acid sequence of SEQ ID NO: 5, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 having alcohol acetyltransferase 2 activity or be a fragment of the amino acid sequence of SEQ ID NO: 5, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 having alcohol acetyltransferase 2 activity; is encoded by a heterologous nucleic acid molecule having a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42; and / or is operably associated to a native or a heterologous promoter.

[0011] According to an embodiment, the recombinant yeast host cell comprises a heterologous ATF2 as the upregulated ATF2 and a heterologous ARQ10 as the upregulated ARQ10.

[0012] According to an embodiment, the at least one downregulated native transferase is the native ATF1 .

[0013] According to an embodiment, the at least one downregulated native transferase is the native EAT1 .

[0014] According to an embodiment, the at least one downregulated transferase comprise the native alcohol acetyltransferase 1 (ATF1) and the native ethanol acetyl- CoA transferase (EAT1).

[0015] According to an embodiment, the gene encoding the native ATF1 is deleted.

[0016] According to an embodiment, the gene encoding the native EAT1 is deleted.

[0017] According to an embodiment, the recombinant yeast host cell further comprises a downregulated isoamyl acetate-hydrolysing esterase (IAH1) when compared to a wild-type yeast host cell.

[0018] According to an embodiment, the gene encoding the isoamyl acetate- hydrolysing esterase (IAH1) is deleted.

[0019] According to an embodiment, the heterologous promoter is tpil p, tef2p, adhl p, revl p, tdhl p, tirl p, enol p, cyd p, tdh2p, pgkl p, qcr8p, ccw12p, hxt3p, yet3p, sedl p, eno2p, gpml p, hor7p, hsp150p, icll p, ssal p, tdh3p, cprl p, zwfl p, ssd p, pykl p, hxt2p, hsp30p, gpd2p, pfk1 p, pdr12p, danl p, msn4p, hxk2p, set3p, ilv3p, pep4p, cys3p, erglOp, sna3p, erg5p, erg13p, rhol p, erg20p, erg26p, erg9p, cis3p, trx2p, gxp2p, gre2p, tarl p, hpfl p, fas2p, arpl Op, pho23p, reg2p, dtrl p, mip6p, snll p, adh2p, fdh2p, mitl p or gpdl p.

[0020] According to an embodiment, the heterologous promoter is tef2, adhl p, tpil p, revl p, cyd p, qcr8p, yet3p, hsp30p, danl p, msn4p, cys3p, sna3p, gre2p, pho23p, mip6p, or mitl p.

[0021] According to an embodiment, the heterologous promoter is tef2, adhl p, tpil p or revl p.

[0022] According to an embodiment, the recombinant yeast host cell produces, or is capable of producing, one or more ester flavor compound during the fermentation.

[0023] According to an embodiment, the one or more ester flavor compound comprises ethyl acetate (EA), isoamyl acetate (IAA), isobutyl acetate (IBA), phenethyl acetate (PEA), or any combination thereof.

[0024] According to an embodiment, the recombinant yeast host cell produces, or is capable of producing up to about 13 times the amount of EA compared to a wildtype yeast host strain.

[0025] According to an embodiment, the recombinant yeast host cell produces, or is capable of producing up to about 80 times the amount of IAA compared to a wildtype yeast host strain.

[0026] A variety of different yeast strains can be engineered to develop the recombinant yeast host cell of the present disclosure. Suitable recombinant yeast host cells can be, for example, from the genus Saccharomyces, Kluyveromyces, Arxula,Debaryomyces, Candida, Pichia, Phaffia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Torula, Hanseniaspora, Lachancea, Wickerhamomyces or Yarrowia. Suitable yeast species can include, for example, S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, C. utilis, K. lactis, K. marxianus, K. fragilis, Hanseniaspora vineae, Lachancea fermentati, Lachancea thermotolerans, Schizosaccharomyces japonicus and / or Wickerhamomyces anomalus. In some embodiments, the yeast is selected from the group consisting of Saccharomyces cerevisiae, 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 one particular embodiment, the yeast is Saccharomyces cerevisiae. In some embodiment, the host cell can be an oleaginous yeast cell. For example, the oleaginous yeast host cell can be from the genus Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. In some alternative embodiment, the host cell can be an oleaginous microalgae host cell (e.g., for example, from the genus Thraustochytrium or Schizochytriurri). In an embodiment, the recombinant yeast host cell is from the genus Saccharomyces and, in some embodiments, from the species Saccharomyces cerevisiae.

[0027] According to an embodiment, the recombinant yeast host cell is from the genus Saccharomyces.

[0028] According to an embodiment, the recombinant yeast host cell is from the species Saccharomyces cerevisiae.

[0029] According to an embodiment, the recombinant yeast host cell produces, or is capable of producing, up to about 300 g of ethyl acetate per hectoliter of absolute alcohol.

[0030] According to an embodiment, the recombinant yeast host cell produces, or is capable of producing, up to about 140 g of isoamyl acetate per hectoliter of absolute alcohol.

[0031] According to an embodiment, the recombinant yeast host cell produces, or is capable of producing, at least about 0.03 g of isobutyl acetate per hectoliter of absolute alcohol.

[0032] According to an embodiment, the recombinant yeast host cell produces, or is capable of producing between about 5 g to about 300 g of ethyl acetate, and / or between about 5 g to about 140 g of isoamyl acetate per hectoliter of absolute alcohol.

[0033] According to an embodiment, the recombinant yeast host cell produces, or is capable of producing, at least 0.5 g of phenethyl acetate per hectoliter of absolute alcohol.

[0034] According to a second aspect, the present invention is directed to a process for making a flavored solution.

[0035] According to a preferred embodiment, the process comprises:I. contacting the recombinant yeast host cell as described herein with a fermentation medium; andII. fermenting the fermentation medium with the recombinant yeast host cell to produce the flavored solution comprising one or more ester flavor compounds and ethanol.

[0036] According to an embodiment, the flavored solution is a flavored beverage or is made into a flavored beverage.

[0037] According to an embodiment, the flavored beverage is a beer, a distilled spirit, or wine.

[0038] According to an embodiment, the distilled spirit is whisky.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Other and further aspects and advantages of the present invention will be better understood upon the reading of the illustrative embodiments about to be described or will be indicated in the appended claims, and various advantages not referred to herein will occur to one skilled in the art upon employment of the invention in practice. Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, embodiments thereof, and in which:Figure 1 shows the biosynthetic pathways of major acetate esters that provide a variety of aromas in beer and other alcoholic beverages

[0014] ; andFigure 2 shows sensory liking scores depicting the preference of panelists (n = 29) between bourbon-type distillates produced from fermentations with the wild type parental strain, as well as strains SC-1 , and SC-4. The bars represent the dispersion of the liking data for each distillate.DETAILED DESCRIPTION

[0040] Novel recombinant yeast host cells and processes for making a flavored solution will be described hereinafter. Although the invention is described in terms of specific illustrative embodiments, it is to be understood that the embodiments described herein are by way of example only and that the scope of the invention is not intended to be limited thereby.

[0041] The terminology used herein is in accordance with definitions set out below.

[0042] By "about", it is meant that the value can vary within a certain range depending on the margin of error of the method or device used to evaluate or measure. A margin of error of 10% is generally accepted.

[0043] As used herein, a “flavor compound” refers to compounds capable of triggering a flavor sensation in humans. In the context of the present disclosure, the term “flavor compound” is an ester flavor compound that can desirably affect the smell and / or taste of a beverage and the enjoyment associated with drinking the flavored beverage (Burdock et al.).

[0044] As used herein, a “beverage” refers to a potable liquid for human consumption.

[0045] As used herein, a “copy” refers to a copy of a (heterologous) gene by chromosome.

[0046] As used herein, the term “heterologous” when used in reference to a nucleic acid molecule (such as a promoter, a terminator or a coding sequence) or a protein / polypeptide refers to a nucleic acid molecule or a protein / polypeptide that is not natively found in the recombinant yeast cell. “Heterologous” also includes a native coding region / promoter / terminator, or portion thereof, that was 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 yeast's genome.

[0047] As used herein, the term “native” when used in inference to a gene, polypeptide, enzymatic activity, or pathway refers to an unmodified gene, polypeptide, enzymatic activity, or pathway originally found in the recombinant yeast host cell.

[0048] As used herein, “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.

[0049] As used herein, “upregulated” means increased in activity, e.g., increase in enzymatic activity of the enzyme as compared to activity in the wild-type yeast host cell. The upregulation is typically in comparison to the expression in a wild-type cell, unless specified otherwise. In the context of the present disclosure, the wild-type cell is a wild-type yeast strain. The upregulation could be done by the known methods in the art, as for example by the addition of one or more copies of a heterologous gene encoding a heterologous polypeptide in the genome of the recombinant host, increasing activity of the native gene by insertion or deletion of nucleic acids residues, replacement of the native promoter by a heterologous promoter more active or by modulation of a gene regulating the expression of the native gene.

[0050] As used herein, “downregulated” means decreased in activity, e.g., decrease in enzymatic activity of the enzyme as compared to activity in the wild-type yeast host cell. The downregulation is typically in comparison to the expression in a wild-type cell, unless specified otherwise. In the context of the present disclosure, the wild-type cell is a wild-type yeast strain. The downregulation could be done by the known methods in the art, as for example by deletion of the native gene, inactivation of the native gene by insertion or deletion of nucleic acids residues, replacement of the native promoter by a heterologous promoter less active or that does not activate under propagation and / or fermentation conditions, or by modulation of a gene regulating the expression of the native gene.

[0051] As used herein, the unit “g I hL AA” means grams of analyte per hectoliter (100 L) of absolute alcohol (100 %v / v) as described in Commission Regulation (EC) No 2870 / 2000 of 19 December 2000 laying down Community reference methods for the analysis of spirits drinks. This value is converted using the following formula (I):[Analyte in g / hL AA] = ([Analyte in mg / L]*(100 / [EtOH sample in %v / v])) / 10 (I)

[0052] Acetate esters, which contribute to the fruity and floral organoleptic properties of fermented beverages, are produced from a precursor alcohol and acetyl-CoA, by acetyltransferases ATF1 , ATF2, and also EAT1 for ethyl acetate, as shown in scheme 1 .Amino Acidsa- keto acidsAldehydes PyruvateHigher Alcohols Acetyl-CoA\\ / \ Atfl / Atf2 / (Eatl)Acetate EstersScheme 1

[0053] The aroma and flavor conferred by the acetate ester depend on their sensory threshold and their concentration in the alcoholic beverage (Table 1). Moreover, the relative concentrations of the different acetate esters, namely ethyl acetate (EA), isoamyl acetate (IAA), isobutyl acetate (IBA) and 2-phenethyl acetate (PEA), lead to specific flavor profiles.Table 1. List of targeted acetate esters, detection threshold in a variety of food and beverages, and perceived aroma descriptors. [6]Ethyl acetate Ethereal, fruity (pear,5 - 5,000(EA) red apple, pineapple)Isoamyl acetate Fruity (banana, pear,2 - 43(IAA) pear drops)Fruity (exotic,Isobutyl acetate65 - 880 pineapple), floral(I BA)(hyacinth, rose)Phenethyl 3,000Floral (rose), honey acetate (PEA) 5,000 ppb = parts per billion

[0054] Ethyl acetate (EA) is the most abundant of all esters produced during anaerobic ethanolic fermentation and generally the most concentrated ester in commercial distilled spirits (Table 2). Isoamyl acetate (IAA) concentration in final spirits ranges from none (< LOD of 0.01 g / hL AA) to 20 grams per hectoliter of absolute alcohol (g I hL AA) in some high-ester rums (Table 2). Isobutyl acetate (IBA) and phenethyl acetate (PEA) are generally very low or not detectable in commercial spirits by standard analysis techniques (e.g., headspace auto sampler gas chromatography equipped with a flame ionization detector (HS-GC / FID)).

[0055] High ester rums are spirits generally high in ethyl acetate and ethyl esters, their composition being highly dependent on the specific process used. As an example, Jamaican high-ester rums can have ethyl acetate concentrations as high as 1 ,300 g I hL AA. High ester rums are obtained by dunder (stillage) fermentations using a variety of wild yeast and bacteria (aerobic and anaerobic, from the environment). Bacteria produce high concentrations of organic acids [7], which, in combination with alcohols, are precursors to esters. Dunder fermentations are lengthy (15 to 20 days) and difficult to control. High ester rums are usually used for blending rather than as stand-alone product, due to their extremely high ester concentrations. Means of reducing levels of ethyl acetate while increasing levels of phenethyl acetate (PEA) would thus be desirable in the development of beverages having new flavor profiles.

[0056] Baijiu is also known for its high ethyl acetate and organic acid concentrations, and it is also produced by the fermentation with different microorganisms, including yeast, bacteria and molds [8],Table 2. Acetate ester concentration range in different commercial spirits and median sensory thresholds in 20% alcohol by volume (ABV) from laboratory sensory analysis.All values are in gram per hectoliter of absolute alcohol (g / hL AA). Abbreviations: n = number of samples analyzed; <LOD = less than the limit of detection; NA = not measured).

[0057] Ester production during fermentation is dependent on the yeast genetic profile and fermentation parameters, such as carbon and nitrogen sources and availability, temperature, pH and dissolved oxygen [9], The biosynthetic pathways of the major acetate esters are depicted in Figure 1. Acetate esters derive from the condensation between Acetyl-CoA and an alcohol catalyzed by alcohol acetyl transferases (AATases). The alcohol precursor can derive both from amino acids (isoamyl alcohol for IAA, isobutanol for IBA, and 2-phenyl-ethanol for PEA) or from glycolysis intermediates (phosphoenolpyruvate for PEA and pyruvate for EA). Acetate esters produced in the cytosol require the acetyl transferases ATF1 and ATF2 for the condensation of the alcohol precursor and the acetyl CoA (Figure 1A, 1 B and 1 C) [1], While in the cytosol the ethyl acetate is formed by condensation of ethanol and acetyl- CoA catalyzed by ATF1 and ATF2, in the mitochondria, the reaction is catalyzed by EAT1 as depicted in Figure 1 D [1 , 17],

[0058] The reaction depicted in Figure 1 shows the production of isoamyl acetate (IAA). Leucine is the precursor for the synthesis of the banana-scented compound isoamyl acetate. Leucine is deaminated by a branched-chain amino acid transferase (BAT2), decarboxylated by Kid1 / Aro10, and then reduced to produce isoamyl alcohol (a.k.a 3-methyl-1 -butanol) by different alcohol dehydrogenases (ADH). The alcohol acetyltransferases ATF1 and ATF2 catalyze the condensation reaction of isoamyl alcohol and acetyl-CoA to form isoamyl acetate.

[0059] The reaction depicted in Figure 1 B shows the production of phenethyl acetate (PEA). PEA is produced either by phosphoenolpyruvate (from glycolysis) or byphenylalanine, which is deaminated by two amino acid transferases (ARO8 / ARO9) to phenylpyruvate, which is decarboxylated to phenylacetylaldehyde by a pyruvate decarboxylase (ARO10 / PDC) and then reduced to 2-phenylethanol by alcohol dehydrogenases. The acetyl-CoA transferases ATF1 and ATF2 are also responsible for the condensation of 2-phenylethanol and acetyl-CoA.

[0060] The reaction depicted in Figure 1C shows the production of ethyl acetate (EA) in the cytosol. In the case of ethyl acetate, the precursor is pyruvate (glycolysis). Pyruvate is decarboxylated and then reduced to ethanol. ATF1 and ATF2 are again needed for catalysis of the final condensation step with acetyl-CoA in the cytosol. Acetyltransferases are expressed during fermentation and repressed in the presence of oxygen and the presence of fatty acid esters [1 , 10], While ATF1 and ATF2 catalyze the final condensation for the synthesis EA in the cytosol (Figure 1C), the ethanol acetyltransferase EAT1 catalyzes this reaction in the mitochondria (Figure 1 D)

[0011] ,

[0061] The isoamyl acetate-hydrolyzing esterase, IAH1 , hydrolyzes IAA back to isoamyl alcohol [1], thereby affecting the final concentration of IAA and possibly of other esters, such as PEA [3],

[0062] S. cerevisiae encodes two main AATases, ATF1 and ATF2, of which ATF1 controls the majority of acetate ester formation [1 , 12], It was found that purified ATF1 is active on a wide variety of alcohols to generate acetate esters including ethyl acetate, isoamyl acetate, isobutyl acetate, butyl acetate, hexyl acetate, heptyl acetate and octyl acetate [1 , 13],

[0063] Thus, modulation of the expression of ATF1 , ATF2 and / or other enzymes involved in the production of ester flavor compounds to arrive at a desirable flavor profile would be highly desirable. Accordingly, yeast engineering strategies were developed to selectively modulate acetate ester ratios and concentration in fermented and / or distilled beverages. These strains could be used to generate a panoply of unique and intense organoleptic profiles to produce either stand-alone products or products for blending purposes in the beverage industry.

[0064] While overexpression of ATF1 and ATF2 has previously been used to increase acetate ester production in wine, brandy distillate and beer [1 , 3, 12], overexpression of ATF1 during yeast fermentation was shown to yield beverages with unpleasant flavor profiles. Thus, the present disclosure provides means of modulating the relative concentrations of these esters, aiming at enhancing fruity and floralcharacters while avoiding unpleasant off-notes. To achieve these desired flavour profiles, yeast strains showing a reduction in EA and / or IAA while maximizing the production of IBA and / or PEA compared to the previously described yeast strain, SC- 1 (see WO 2019 / 171230 and hereinbelow) overexpressing ATF1 , were developed.

[0065] The present disclosure combines gene upregulation and gene downregulation of genes encoding for key enzymes involved in acetate ester synthesis in order to modulate acetate ester production based on concentration ranges and / or ratios between the different esters to obtain the desired flavor profile while avoiding unpleasant off-notes.

[0066] Having thus introduced the current challenges in making a new flavored beverage as well as the properties that a novel recombinant yeast host cell would be required to have to make such a desired flavored beverage, the description which follows, and the embodiments described therein are provided by way of illustration of an example of particular embodiments of principles and aspects of the present invention. These examples are provided for the purposes of explanation and not of limitation, of those principles of the invention.Recombinant yeast host cells

[0067] According to a first aspect, the present invention is directed to a recombinant yeast host cell for making a flavored solution obtainable or obtained by fermentation of a fermentation medium.

[0068] The recombinant yeast host cells of the present disclosure are intended to be used for making the flavored solution. In an embodiment, the recombinant yeast host cells of the present disclosure are used in a fermentation process (such as, for example, an anaerobic fermentation process) to obtain the flavored solution. In a further embodiment, the flavored solution is a flavored beverage or is made into a flavored beverage. The flavors in the flavored solution may derive at least in part from production of one or more ester flavor compound during the fermentation. According to an embodiment, the recombinant yeast host cell comprises: a) an upregulated alcohol acetyltransferase 2 (ATF2) and / or an upregulated phenylpyruvate decarboxylase (ARO10) when compared to a wild-type yeast host cell; andb) at least one downregulated native transferase when compared to a wild-type yeast host cell, wherein the at least one downregulated transferase is a native alcohol acetyltransferase 1 (ATF1) and / or a native ethanol acetyl-CoA transferase (EAT1).

[0069] According to an embodiment, the recombinant yeast host cell comprises an upregulated alcohol acetyltransferase 2 (ATF2) and / or downregulated native alcohol acetyltransferase 1 (ATF1) when compared to a wild-type yeast host cell. As used herein, the expression "alcohol acetyltransferase 2" (or “ATF2”) or "alcohol acetyltransferase 1 " (or “ATF1 ”) are intended to include enzymes capable of converting acetyl-CoA and various alcohol into acetate esters and includes enzymes that correspond to Enzyme Commission Number EC:2.3.1.84. It is known in the art that while ATF1 and ATF2 are both performing the same reactions, ATF1 is the primary acetyltransferase contributing the most to the conversion of acetyl-CoA and various alcohols into acetate esters in the yeast host cell and that ATF2 is an acetyltransferase having a lesser contribution to the conversion of acetyl-CoA and various alcohol into acetate esters in the yeast host cell when compared to each other. The methods to differentiate alcohol acetyltransferase 1 (ATF1) from alcohol acetyltransferase 2 (ATF2) by genetic deletion studies to identify the predominate contributor to acetate ester formation, for example ethyl acetate and / or isoamyl acetate production, are deemed to be within the scope of those skilled in the art.

[0070] According to an embodiment, the recombinant yeast host cell comprises an upregulated alcohol acetyltransferase 2 (ATF2) when compared to a wild-type yeast host cell. The alcohol acetyltransferase 2 can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native alcohol acetyltransferase 2 and optionally in combination a heterologous alcohol acetyltransferase 2. In some embodiments, the alcohol acetyltransferase 2 is derived from Saccharomyces or Torulaspora species., and in further embodiments from Saccharomyces cerevisiae (for example gene ID: 853088; Saccharomyces Genome Database (SGD):S000003409), Saccharomyces boulardii, Saccharomyces cerevisiae x Saccharomyces kudriavzevii, Saccharomyces paradoxus, Saccharomyces mikatae, Torulaspora delbrueckii or Saccharomyces kudriavzevii. In such embodiments, the alcohol acetyltransferase 2 can have the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20 or be a variant of the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20 having alcohol acetyltransferase 2 activity or be a fragment of the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20 having alcoholacetyltransferase 2 activity. In some embodiment, the alcohol acetyltransferase 2 have at least 37%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20 (a variant thereof or a fragment thereof). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the alcohol acetyltransferase 2. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20 (a variant thereof or a fragment thereof). In one embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 1 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2 (a variant thereof or a fragment thereof).

[0071] According to an embodiment, the recombinant yeast host cell comprises a heterologous ATF2 as the upregulated ATF2.

[0072] According to an embodiment, the recombinant yeast host cell comprises one copy, two copies, three copies or more of a heterologous ATF2 as the upregulated ATF2. In a particular embodiment, the recombinant yeast host cell comprises two copies of a heterologous ATF2 as the upregulated ATF2.

[0073] According to an embodiment, the gene encoding the heterologous ATF2 is operably associated with one or more heterologous promoters.

[0074] “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 bythe 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.

[0075] 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 microbial 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.

[0076] 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), enol p (e.g., the promoter of the enol gene), hxk1 (e.g., the promoter of the hxk1 gene), pgkl p (e.g., the promoter of the pgk1 gene) , revl p (e.g., the promoter of the rev1 gene), cycl p (e.g., the promoter of the cyc1 gene), and ste5p (e.g., the promoter of the ste5 gene). In some embodiment, the promoter is adhl p (e.g., the promoter of the adh1 gene). However, in 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)), a redox-regulated promoter (e.g. the promoter of the gpd2 gene (referred to as gpd2p)), or a sulfite-regulated promoter (e.g., the promoter of the fzf1 gene (referred to as the fzf1 p)), the promoter of the ssu1 gene (referred to as ssu1 p), the promoter of the ssu1-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 pdc1 gene),hxt3p (e.g., the promoter of the hxt3 gene), dan1 (e.g., the promoter of the dan1 gene) and tpil p (e.g., the promoter of the tpi1 gene). In one embodiment, the promoter used to allow the expression of the heterologous polypeptide is tef2p, cwp2p, ssal p, enol p, hxkl p; pgkl p, adhl p, hxt7p, gpd2p, fzfl p, ssul p, ssu1-rp, tdhl p, hor7p, tdh2p, tdh3p, gdpl p, cdc19p, eno2p, pdd p, hxt3p, danl p, tpil p, revl p, cyd p, or ste5p. In an embodiment, the promoter used to allow the expression of the heterologous polypeptide is the adhl p. One or more promoters can be used to allow the expression of each heterologous polypeptides in the recombinant yeast host cell. In some other embodiment, the promoter used to control the expression of the heterologous polypeptide is a weak or moderate promoter to reduce the expression of the heterologous polypeptide by the recombinant yeast host cell such as for example revl p, cyd p, qcr8p, yet3p, hsp30p, danl p, msn4p, cys3p, sna3p, gre2p, pho23p, mip6p, or mitl p.

[0077] In one embodiment, the promoter used to control the expression of the heterologous polypeptide is tpil p (SEQ ID: 3), tef2p (SEQ ID: 6), adhl p (SEQ ID: 7), revl p (SEQ ID: 8), tdhl p (SEQ ID: 89), tirl p (SEQ ID: 90), enol p (SEQ ID: 91), cyd p (SEQ ID: 92), tdh2p (SEQ ID: 93), pgkl p (SEQ ID: 94), qcr8p (SEQ ID: 95), ccw12p (SEQ ID: 96), hxt3p (SEQ ID: 97), yet3p (SEQ ID: 98), sedl p (SEQ ID: 99), eno2p (SEQ ID: 100), gpnd p (SEQ ID: 101), hor7p (SEQ ID: 102), hsp150p (SEQ ID: 103), icll p (SEQ ID: 104), ssal p (SEQ ID: 105), tdh3p (SEQ ID: 106), cpr1 p (SEQ ID: 107), zwf1 p (SEQ ID: 108), ssd p (SEQ ID: 109), pyk1 p (SEQ ID: 110), hxt2p (SEQ ID: 11 1), hsp30p (SEQ ID: 112), gpd2p (SEQ ID: 113), pfkl p (SEQ ID: 114), pdr12p (SEQ ID: 115), danl p (SEQ ID: 116), msn4p (SEQ ID: 1 17), hxk2p (SEQ ID: 118), set3p (SEQ ID: 1 19), ilv3p (SEQ ID: 120), pep4p (SEQ ID: 121), cys3p (SEQ ID: 122), erglOp (SEQ ID:123), sna3p (SEQ ID: 124), erg5p (SEQ ID: 125), erg13p (SEQ ID: 126), rhol p (SEQ ID: 127), erg20p (SEQ ID: 128), erg26p (SEQ ID: 129), erg9p (SEQ ID: 130), cis3p (SEQ ID: 131), trx2p (SEQ ID: 132), gxp2p (SEQ ID: 133), gre2p (SEQ ID: 134), tarl p (SEQ ID: 135), hpfl p (SEQ ID: 136), fas2p (SEQ ID: 137), arp10p(SEQ ID: 138), pho23p (SEQ ID: 139), reg2p (SEQ ID: 140), dtrl p (SEQ ID: 141), mip6p (SEQ ID: 142), snll p (SEQ ID: 143), adh2p (SEQ ID: 144), fdh2p (SEQ ID: 145), mitl p (SEQ ID: 146) or gpdl p (SEQ ID: 147). In some embodiment, the promoter used to control the expression of the heterologous polypeptide is tef2p, adhl p, tpil p, revl p, cyd p, qcr8p, yet3p, hsp30p, danl p, msn4p, cys3p, sna3p, gre2p, pho23p, mip6p, or mitl p. In another embodiment, the promoter used to control the expression of the heterologous polypeptide is tef2, adhl p or tpil p.

[0078] According to an embodiment, the recombinant yeast host cell comprises an heterologous (ATF2) under the control of the promoter from the triose phosphate isomerase (tpiT) gene (SEQ ID: 3), the promoter from the translation elongation factor EF-1 alpha (tef2) gene (SEQ ID: 6), the promoter from the alpha subunit of class I alcohol dehydrogenase (adhT) gene (SEQ ID: 7) or the promoter of the bifunctional DNA-directed DNA polymerase (revf) gene (SEQ ID: 8).

[0079] According to an embodiment, the recombinant yeast host cell comprises an upregulated phenylpyruvate decarboxylase (ARO10) when compared to a wild-type yeast host cell. As used herein, the expression "phenylpyruvate decarboxylase" or “ARO10” is intended to include the enzymes capable of converting phenylpyruvate into phenylacetaldehyde. Phenylpyruvate decarboxylase includes enzymes that correspond to Enzyme Commission Number EC 4.1.1.43. The phenylpyruvate decarboxylase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native phenylpyruvate decarboxylase and optionally in combination a heterologous phenylpyruvate decarboxylase. In some embodiments, the phenylpyruvate decarboxylase is derived from Saccharomyces, Naumovozyma, Maudiozyma, Kazachstania, Arxiozyma, Huiozyma, Nakaseomyces, Naumovozyma, Torulaspora, Zygosaccharomyces or Henningerozyma species, and in further embodiments from Saccharomyces cerevisiae (for example gene ID: 851987; Saccharomyces Genome Database (SGD): S00000278), Saccharomyces pastorianus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces arboricola, Saccharomyces kudriavzevii, Saccharomyces eubayanus, Naumovozyma castellii, Maudiozyma humilis, Kazachstania Africana, Maudiozyma exigua, Maudiozyma barnettii, Arxiozyma heterogenica, Huiozyma naganishii, Nakaseomyces glabratus, Naumovozyma dairenensis, Torulaspora delbrueckii, Torulaspora globosa, Zygosaccharomyces rouxii Zygosaccharomyces parabailii or Henningerozyma blattae. In such embodiments, the phenylpyruvate decarboxylase can have the amino acid sequence of SEQ ID NO: 5, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 be a variant of the amino acid sequence of SEQ ID NO: 5, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 having phenylpyruvate decarboxylase activity or be a fragment of the amino acid sequence of SEQ ID NO: 5 , 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 having phenylpyruvate decarboxylase activity. In some embodiment, the phenylpyruvate decarboxylase have at least 37%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 5, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32,33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 (a variant thereof or a fragment thereof). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the phenylpyruvate decarboxylase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 (a variant thereof or a fragment thereof). In one embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 4, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5 (a variant thereof or a fragment thereof).

[0080] According to an embodiment, the recombinant yeast host cell comprises a heterologous ARQ10 as the upregulated ARQ10.

[0081] According to an embodiment, the recombinant yeast host cell comprises one copy, two copies, three copies or more of a heterologous ARQ10 as the upregulated ARQ10. In a particular embodiment, the recombinant yeast host cell comprises one copy of the heterologous ARQ10.

[0082] According to an embodiment, the gene encoding the heterologous ARQ10 is operably associated with one or more heterologous promoters.

[0083] According to an embodiment, the one or more heterologous promoters operably associated with the heterologous ARQ10 are the promoter from the translation elongation factor EF-1 alpha (tef2) gene (SEQ ID: 6), the alpha subunit of class I alcohol dehydrogenase (adhT) gene (SEQ ID: 7), the triose phosphate isomerase (tp / 7) gene (SEQ ID: 3) , the promoter from the alpha subunit of class I alcohol dehydrogenase (adhT) gene (SEQ ID: 7) , and / orthe bifunctional DNA-directed DNA polymerase (revT) gene (SEQ ID: 8).

[0084] According to another embodiment, the one or more heterologous promoters are the promoter from the translation elongation factor EF-1 alpha (TEF2) gene or the alpha subunit of class I alcohol dehydrogenase (ADH1) gene.

[0085] According to an embodiment, the recombinant yeast host cell comprises a downregulated native alcohol acetyltransferase 1 (ATF1) when compared to a wildtype yeast host cell. Atf1 genes encoding the ATF1 polypeptide include, but are not limited to genes of Saccharomyces species, and in further embodiments from Saccharomyces cerevisiae (for example The atf1 gene Gene ID: 854559; SGD:S000005904; www.yeastaenome.org), Saccharomyces pastorianus, Saccharomyces boulardii, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces arboricola, Saccharomyces eubayanus or Saccharomyces cerevisiae x Saccharomyces kudriavzevii. In such embodiment, the alcohol acetyltransferase 1 can have the amino acid sequence of SEQ ID NO: 10, 43, 44, 45 46, 47, 48, 49 or 50 or be a variant of the amino acid sequence of SEQ ID NO: 10, 43, 44, 45 46, 47, 48, 49 or 50 having alcohol acetyltransferase 1 activity or be a fragment of the amino acid sequence of SEQ ID NO: 10, 43, 44, 45 46, 47, 48, 49 or 50 having alcohol acetyltransferase 1 activity. In some embodiment, the atf1 gene has the nucleic acid sequence of SEQ ID NO: 9, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10. In some embodiment, the alcohol acetyltransferase 1 have at least 37%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 10, 43, 44, 45 46, 47, 48, 49 or 50 (a variant thereof or a fragment thereof). In some embodiments, ATF1 may be downregulated via downregulated expression of the ATF1 mRNA and / or protein, or the atf1 gene may be deleted from the recombinant yeast host cell’s genome. As used in the context of the present disclosure, the downregulation of the native atf1 gene refers to a genetic modification which limits or impedes the expression of the native atf1 gene, when compared to a corresponding yeast strain which does not bear such genetic modification. In some instances, the additional genetic modification reduces but still allows the expression of the native atf1 gene and / or the expression of the native ATF1. In other instances, the genetic modification inhibits the expression of the native atf1 gene and / or the expression of the native ATF1 . The downregulation could be done by the known methods in the art, as for example by deletion of the native atf1 gene, inactivation of the atf1 gene by insertion or deletion of nucleic acids residues, replacement of the native promoter by a heterologous promoter less active or that does not activate under propagation and / or fermentation conditions, or by modulation of a gene regulating the expression of the native atf1 gene. In one embodiment, the genetic modification for downregulated the native atf1 gene is a deletion of the native adh gene.

[0086] According to an embodiment, the recombinant yeast host cell comprises a downregulated native ethanol acetyl-CoA transferase 1 (EAT1). As used herein, the terms "ethanol acetyl-CoA transferase 1" or“EAT1 ” is intended to include the enzymes capable of converting acetyl-coA and ethanol into ethyl acetate. Ethanol acetyl-CoA transferase 1 includes enzymes that correspond to Enzyme Commission Number 2.3.1.268. Eat1 genes encoding the EAT1 polypeptide include, but are not limited togenes of Saccharomyces or Zygosaccharomyces species, and in further embodiments from Saccharomyces cerevisiae (for example Gene ID: 852898; SGD: S000003247;Saccharomyces pastorianus, or Zygosaccharomyces parabailii. In such embodiment, the alcohol acetyltransferase 1 can have the amino acid sequence of SEQ ID NO: 12, 51 or 52 or be a variant of the amino acid sequence of SEQ ID NO: 12, 51 or 52 having ethanol acetyl-CoA transferase 1 activity or be a fragment of the amino acid sequence of SEQ ID NO: 12, 51 or 52 having alcohol ethanol acetyl-CoA transferase 1 . In some embodiment, the eat1 gene has the nucleic acid sequence of SEQ ID NO: 11 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12. In some embodiment, the ethanol acetyl- CoA transferase 1 have at least 37%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 12, 51 or 52 (a variant thereof or a fragment thereof). In some embodiments, EAT1 may be downregulated via downregulated expression of the EAT1 mRNA and / or protein, or the eat1 gene may be deleted from the recombinant yeast host cell’s genome. As used in the context of the present disclosure, the downregulation of the native Eat1 gene refers to a genetic modification which limits or impedes the expression of the native Eat1 gene, when compared to a corresponding yeast strain which does not bear such genetic modification. In some instances, the additional genetic modification reduces but still allows the expression of the native Eat1 gene and / or the expression of the native EAT1. In other instances, the genetic modification inhibits the expression of the native Eat1 gene and / or the expression of the native EAT1. The downregulation could be done by the known methods in the art, as for example by deletion of the native Eat1 gene, inactivation of the Eat1 gene by insertion or deletion of nucleic acids residues, replacement of the native promoter by a heterologous promoter less active or that does not activate under propagation and / or fermentation conditions, or by modulation of a gene regulating the expression of the native Eat1 gene. In one embodiment, the genetic modification for downregulated the native Eat1 gene is a deletion of the native adh gene.

[0087] According to an embodiment, the recombinant yeast host cell comprises an upregulated alcohol acetyltransferase 2 (ATF2) and at least one downregulated native transferase when compared to a wild-type yeast host cell, wherein the at least one downregulated transferase is a native alcohol acetyltransferase 1 (ATF1) and / or a native ethanol acetyl-CoA transferase (EAT1). The at least one downregulated native transferase may be downregulated via downregulated expression or deletion of the gene in the recombinant yeast host cell’s genome.

[0088] According to an embodiment, the recombinant yeast host cell comprises an upregulated phenylpyruvate decarboxylase (ARO10) and at least one downregulated native transferase when compared to a wild-type yeast host cell, wherein the at least one downregulated transferase is a native alcohol acetyltransferase 1 (ATF1) and / or a native ethanol acetyl-CoA transferase (EAT1). The at least one downregulated native transferase may be downregulated via downregulated expression or deletion of the gene in the recombinant yeast host cell’s genome.

[0089] According to an embodiment, the recombinant yeast host cell comprises an upregulated phenylpyruvate decarboxylase (ARO10) and a downregulated native alcohol acetyltransferase 1 (ATF1).

[0090] According to an embodiment, the recombinant yeast host cell comprises an upregulated phenylpyruvate decarboxylase (ARO10) and a downregulated native ethanol acetyl-CoA transferase (EAT1).

[0091] According to an embodiment, the recombinant yeast host cell comprises an upregulated alcohol acetyltransferase 2 (ATF2) and a downregulated native alcohol acetyltransferase 1 (ATF1).

[0092] According to an embodiment, the recombinant yeast host cell comprises an upregulated alcohol acetyltransferase 2 (ATF2) and a downregulated native ethanol acetyl-CoA transferase (EAT1).

[0093] According to an embodiment, the recombinant yeast host cell comprises an upregulated alcohol acetyltransferase 2 (ATF2), an upregulated phenylpyruvate decarboxylase (ARO10), and a downregulated native alcohol acetyltransferase 1 (ATF1).

[0094] According to an embodiment, the recombinant yeast host cell comprises an upregulated alcohol acetyltransferase 2 (ATF2), a downregulated native alcohol acetyltransferase 1 (ATF1), and a downregulated native ethanol acetyl-CoA transferase 1 (EAT1).

[0095] According to an embodiment, the recombinant yeast host cell comprises an upregulated phenylpyruvate decarboxylase 10 (ARO10), a downregulated native alcohol acetyltransferase 1 (ATF1), and a downregulated native ethanol acetyl-CoA transferase 1 (EAT1).

[0096] According to an embodiment, the recombinant yeast host cell comprises an upregulated alcohol acetyltransferase 2 (ATF2), an upregulated phenylpyruvate decarboxylase 10 (ARO10), and a downregulated native ethanol acetyl-CoA transferase 1 (EAT1).

[0097] According to an embodiment, the recombinant yeast host cell comprises two copies of the upregulated ATF2 and a deletion of the atf1 gene such that one copy of the atf2 gene is under the control of tpl1 promoter and a second copy is under the control of the adh1 promoter.

[0098] According to an embodiment, the recombinant yeast host cell comprises an upregulated alcohol acetyltransferase 2 (ATF2) under the control of the TPI1 promoter, an upregulated phenylpyruvate decarboxylase (ARO10), and a deleted native ethanol acetyl-CoA transferase 1 (EAT1).

[0099] According to an embodiment, the recombinant yeast host cell comprises an upregulated alcohol acetyltransferase 2 (ATF2), an upregulated phenylpyruvate decarboxylase 10 (ARO10), a downregulated native alcohol acetyltransferase 1 (ATF1), and a downregulated native ethanol acetyl-CoA transferase 1 (EAT1).

[0100] In yet another embodiment, the recombinant yeast host cell according to any of the above embodiments may further comprise a downregulated isoamyl acetate-hydrolysing esterase 1 (IAH1) when compared to a wild-type yeast host cell. As used herein, the expression "isoamyl acetate-hydrolysing esterase" or “IAH1 ” is intended to include the enzymes capable of converting isoamyl acetate into isoamyl alcohol and acetic acid. Isoamyl acetate-hydrolysing esterase 1 includes enzymes that correspond to Enzyme Commission Number 3.1 .1.112. Iah1 genes encoding the IAH1 polypeptide include, but are not limited to genes of Saccharomyces, Cyberlindnera, Eremothecium, Hanseniaspora, Henningerozyma, Huiozyma, Kazachstania, Kluyveromyces, Lachancea, Maudiozyma, Monosporozyma, Naumovozyma, Torulaspora, Vanderwaltozyma, Wickerhamomyces, Zygosaccharomyces, Zygotorulaspora or Arxiozyma species, and in further embodiments from Saccharomyces cerevisiae (for example Gene ID: 854293; SGD: S000005652;barnetii, Maudiozyma humilis, Monosporozyma servazzii, Naumovozyma castellii, Naumovozyma dairenensis, Saccharomyces arboricola, Saccharomyces cerevisiae x Saccharomyces kudriavzevii, Saccharomyces eubayanus, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomycodes ludwigii, Torulaspora delbrueckii, Torulaspora globosa, Vanderwaltozyma polyspora, Wickerhamomyces anomalus, Wickerhamomyces ciferrii, Zygosaccharomyces parabailii, Zygosaccharomyces rouxii or Zygotorulaspora mrakii. In such embodiment, the isoamyl acetate-hydrolysing esterase 1 can have the amino acid sequence of SEQ ID NO: 14, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or 88 or be a variant of the amino acid sequence of SEQ ID NO: 14, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or 88 having isoamyl acetate- hydrolysing esterase 1 activity or be a fragment of the amino acid sequence of SEQ ID NO: 14, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or 88 having isoamyl acetate- hydrolysing esterase 1. In some embodiment, the isoamyl acetate-hydrolysing esterase 1 have at least 37%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 14, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or 88 (a variant thereof or a fragment thereof). In some embodiment, the iah1 gene has the nucleic acid sequence of SEQ ID NO: 13, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14. In some embodiment, IAH1 may be downregulated via downregulated expression of the IAH1 mRNA and / or protein, or the iah1 gene may be deleted from the recombinant yeast host cell’s genome.

[0101] As the recombinant yeast host cell of the present disclosure has been developed for making a flavored solution obtainable or obtained by fermentation of a fermentation medium, one or more flavor compounds can be found in the flavored solution. Thus, in an embodiment, the recombinant yeast host cell of the present disclosure produces, or is capable of producing, one or more ester flavor compounds during the fermentation.

[0102] In a particular embodiment, the one or more ester flavor compound produced during the fermentation comprises ethyl acetate (EA), isoamyl acetate (IAA), isobutyl acetate (IBA), phenethyl acetate (PEA), or any combination thereof.

[0103] The desired flavour profiles of the flavored beverage required a modulation of acetate ester production such that the concentration of EA and / or IAA is below a certain maximal level to avoid unpleasant solvent-like and chemical-like aromas and / or that concentration of IBA and PEA are present at least above the detection sensory threshold. The methods to determine the concentration of the esters are deemed to be within the scope of those skilled in the art. In some embodiments, the concentration of each acetate ester is determined by gas chromatography of a “low wine” medium obtained from the stripping distillation of a fermentation medium. Low wine stripping distillation indicates the recovery of all the volatile compounds until the concentration of ethanol coming out of the condenser reaches or is below 1 %v / v. The fermentation medium is obtained by a small-scale fermentation carried in about 75 g of grain mash (20-30 % w / w dry solids). The recombinant yeast host cell is pitched at about 0.35 g DCW / L and cultured for about 72 hours at 30-33 °C. The fermentation is supplemented with 200-300 ppm of yeast assimilable nitrogen (YAN) and incubation is carried out with about 150 rpm agitation.

[0104] According to an embodiment, the recombinant yeast host cell produces, or is capable of producing, up to about 300 g, up to about 200 g, up to about 100 g, up to about 90 g, up to about 80 g, up to about 70 g, up to about 60 g, up to about 55 g, up to about 50 g, up to about 40 g, or up to about 30 g of EA per hectoliter of absolute alcohol. In an embodiment, the recombinant yeast host cell produces, or is capable of producing, up to about 60 g of EA per hectoliter of absolute alcohol. Strains capable of producing excessively higher amounts of EA compared to a wild-type yeast host strain resulted in flavored solutions with undesired solvent and chemical notes.

[0105] According to another embodiment, the recombinant yeast host cell produces, or is capable of producing, up to about 13 times, up to about 12 times, up to about 11 times, up to 10 about times, up to 9 about times, up to 8 about times, up to 7 about times, up to 6 about times, up to 5 about times, up to 4 about times, up to 3 about times, or up to 2 about times the amount of EA compared to a wild-type yeast host strain. In an embodiment, the recombinant yeast host cell produces, or is capable of producing, up to 3 times the amount of EA compared to a wild-type yeast host strain.

[0106] In another embodiment, the recombinant yeast host cell produces, or is capable of producing, between about 5 g to about 300 g, about 5 g to about 200 g, about 5 g to about 100 g, about 5 g to about 90 g, about 5 g to about 80 g, about 5 g to about 70 g, about 5 g to about 60 g, about 5 g to about 55 g, about 5 g to about 50 g, about 5 g to about 40 g, about 5 g to about 30 g, about 20 g to about 60 g, about25 g to about 55 g, about 25 g to about 35 g, or about 45 g to about 55 g of EA per hectoliter of absolute alcohol. In an embodiment, the recombinant yeast host cell produces, or is capable of producing, between about 5 g to about 60 g of ethyl acetate per hectoliter of absolute alcohol.

[0107] According to an embodiment, the recombinant yeast host cell produces, or is capable of producing, up to about 140 g, up to about 130 g, up to about 100 g, up to about 90 g, up to about 80 g, up to about 70 g, up to about 60 g, up to about 50g, up to about 40 g, up to about 30 g, up to about 20 g, up to about 15 g, or up to about 10 g of IAA per hectoliter of absolute alcohol. In an embodiment, the recombinant yeast host cell produces, or is capable of producing, up to about 20 g of IAA per hectoliter of absolute alcohol. Strains capable of producing excessively higher amounts of IAA compared to a wild-type yeast host strain resulted in flavored solutions with undesired solvent and chemical notes.

[0108] In another embodiment, the recombinant yeast host cell produces, or is capable of producing, up to about 80 times, up to about 20 times, up to about 10 times, up to about 5 times, up to about 4 times, or up to about 3 times the amount of IAA compared to a wild-type yeast host strain. In an embodiment, the recombinant yeast host cell produces, or is capable of producing, up to about 10 times the amount of IAA compared to a wild-type yeast host strain.

[0109] In another embodiment, the recombinant yeast host cell produces, or is capable of producing, between about 5 g to about 140 g, about 5 g to about 130 g, about 5 g to about 100 g, about 5 g to about 90 g, about 5 g to about 80 g, about 5 g to about 70 g, about 5 g to about 60 g, about 5 g to about 50 g, about 5 g to about 40 g, about 5 g to about 30 g, about 5 g to about 25 g, about 5 g to about 20 g, about 5 g to about 15 g, or about 5 g to about 10 g of IAA per hectoliter of absolute alcohol. In an embodiment, the recombinant yeast host cell produces, or is capable of producing, between about 5 g to about 25 g of isoamyl acetate per hectoliter of absolute alcohol.

[0110] The desired flavour profiles of the flavored beverage required a modulation of acetate ester production to render the IBA and / or PEA concentration at least above the sensory threshold of detection.

[0111] According to another embodiment, the recombinant yeast host cell produces, or is capable of producing, at least about 0.5 g of IBA per hectoliter ofabsolute alcohol. These levels allow production of IBA at least to the sensory threshold of detection.

[0112] According to another embodiment, the recombinant yeast host cell produces, or is capable of producing, at least about 0.03 g, at least about 0.1 g, at least about 0.5 g, at least about 0.6 g, at least about 0.7 g, at least about 1 g, at least about 2 g, at least about 3 g, at least about 5 g, at least about 7 g, or at least about 9 g IBA per hectoliter of absolute alcohol.

[0113] In another embodiment, the recombinant yeast host cell produces, or is capable of producing, at least about 5 times, at least about 6 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 50 times, or at least about 75 times the amount of IBA compared to a wild-type yeast host strain. In an embodiment, the recombinant yeast host cell produces, or is capable of producing, at least about 6 times the amount of IBA compared to a wildtype yeast host strain.

[0114] According to another embodiment, the recombinant yeast host cell produces, or is capable of producing, at least about 1 g of PEA per hectoliter of absolute alcohol. These levels allow production of PEA at least to the sensory threshold of detection.

[0115] According to another embodiment, the recombinant yeast host cell produces, or is capable of producing, at least about 1 g, at least about 1.5 g, at least about 5 g, at least about 10 g, at least about 20 g, at least about 40 g, or at least about 50 g of PEA per hectoliter of absolute alcohol.

[0116] In another embodiment, the recombinant yeast host cell produces, or is capable of producing, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, or at least about 60 times, the amount of PEA compared to a wild-type yeast host strain. In an embodiment, the recombinant yeast host cell produces or is capable of producing at least about 15 times the amount of PEA compared to a wildtype yeast host strain.

[0117] According to another embodiment, the recombinant yeast host cell produces, or is capable of producing, between about 0.03 g to about 10 g, between about 0.5 g to about 10 g or 0.5 g to about 2.5 g of isobutyl acetate. In some embodiment, the recombinant yeast host cell produces, or is capable of producingbetween about 5 and about 30 times the amount of isobutyl acetate compared to an equivalent wild-type yeast host strain during the fermentation.

[0118] According to another embodiment, the recombinant yeast host cell produces, or is capable of producing, between about 0.5 g to about 45 g, about 0.5 g to about 15 g, about 10 g to about 45 g, about 35 g to about 45 g or 4 g to about 45 g of PEA per hectoliter of absolute alcohol. In some embodiment, the recombinant yeast host cell produces, or is capable of producing, between about 10 and about 60 times the amount of PEA compared to an equivalent wild-type yeast host strain during the fermentation.

[0119] The fermentation medium comprising the recombinant yeast host cell can be used in a fermenting step (usually under anaerobic conditions) to allow the production of the desired metabolites (e.g., the ester compound and ethanol). The recombinant yeast host cells can advantageously be easily measured, dosed and formulated for ease of use in fermentation operations.

[0120] The recombinant yeast host cells of the present disclosure can be provided in an active form (e.g., liquid (such as, for example, a cream), compressed, or fluid-bed dried), 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 thereof. In an embodiment, the recombinant yeast host cells are provided in an active and dried form.

[0121] The recombinant yeast host cells of the present disclosure 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. The genetic modification(s) is(are) also aimed at decreasing or removing the expression of a specific targeted gene (which is considered native 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 a recombinant yeast host 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 a heterologous cell or the recombinant yeast host cell itself. In the latter scenario, the nucleic acid residue(s) is (are) added at one or more genomic location which is (are) different than the native genomic location. The genetic manipulations did not occur in nature and are the results of in vitro manipulations ofthe yeast. The genetic modification(s) in the recombinant yeast host cell of the present disclosure comprise, consist essentially of or consist of a genetic modification allowing the expression of a heterologous nucleic acid molecule encoding for one or more heterologous polypeptide forthe production of an ester flavor compound. In the context of the present disclosure, the expression “a genetic modification allowing the expression of a heterologous nucleic acid molecule encoding for one or more heterologous polypeptide for the production of a flavor compound” refers to the fact that the recombinant yeast host cell can include other genetic modifications which are unrelated to the anabolism or the catabolism of the ester flavor compound or ethanol.

[0122] When expressed in a recombinant yeast host cell, the heterologous polypeptides described herein can be encoded on one or more heterologous nucleic acid molecule. The heterologous nucleic acid molecule is purposively introduced into the recombinant yeast host cell. For example, a heterologous element could be derived from a different strain of a yeast 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).

[0123] The heterologous nucleic acid molecule present in the recombinant yeast host cell can be integrated in the recombinant yeast 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 recombinant yeast 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 recombinant yeast host cell’s genome. Alternatively, the heterologous nucleic acid molecule can be independently replicating from the recombinant yeast host cell’s genome. In such embodiment, the nucleic acid molecule can be stable and self— replicating .

[0124] In some embodiments, heterologous polypeptides derived from a different strain of yeast host cells, 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) can be used in the context of the present disclosure.

[0125] The heterologous polypeptide encoded by the heterologous nucleic acid molecule (either for the production of ethanol and / or for the production of the ester flavor compound) can be a variant or a fragment of a known / native polypeptide. A variant comprises at least one amino acid difference when compared to the amino acid sequence of the native polypeptide. As used herein, a variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the polypeptide. 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 polypeptide. The polypeptide variants have at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the polypeptide 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.).

[0126] The variant heterologous polypeptide described herein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or nonconserved 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 maturepolypeptide for purification of the polypeptide. A “variant” of the polypeptide can be a conservative variant or an allelic variant.

[0127] A “variant” can be a fragment of a heterologous wild-type polypeptide or fragment of a variant polypeptide. In some embodiments, polypeptide “fragments” have at least at least 50, 100, 200, 300, 400, 500 or more consecutive amino acids of the corresponding wild-type polypeptide or the variant. A fragment comprises at least one less amino acid residue when compared to the amino acid sequence of the corresponding wild-type heterologous polypeptide or of the variant polypeptide. In some embodiments, the fragment can comprise a N-terminal truncation of the heterologous polypeptide, and / or a C-terminal truncation of the heterologous polypeptides. In some embodiments, the fragments corresponding to the wild-type polypeptide or variant polypeptide to which the signal sequence was removed. In some embodiments, the “fragments” have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the corresponding wild-type polypeptides or variants. In some embodiments, fragments of the polypeptides can be employed for producing the corresponding full-length enzyme by peptide synthesis. Therefore, the fragments can be employed as intermediates for producing the full- length polypeptide.

[0128] In the context of the present disclosure, the fragments exhibit the biological activity of the heterologous wild-type polypeptide or of the variant polypeptide. In an embodiment, the fragment polypeptide exhibits at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological activity of the corresponding heterologous wild-type polypeptide or of the variant. The biological activity of fragments can be determined by methods and assays known in the art.

[0129] In contrast, a substitution, insertion or deletion is said to adversely affect the polypeptide when the altered sequence prevents or disrupts a biological function associated with the polypeptide.

[0130] 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 recombinant yeast host cell in order to allow the recombinant expression of the heterologous polypeptide.

[0131] The heterologous nucleic acid molecules of the present disclosure comprise a coding region for the heterologous polypeptide. A DNA or RNA “codingregion” 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 (such as the recombinant microbial host cell of the present disclosure), 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.

[0132] The heterologous 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 recombinant host cell. In eukaryotic cells, polyadenylation signals are considered control regions.

[0133] In some embodiments, the heterologous nucleic acid molecules of the present disclosure include a promoter as well as a coding sequence for a heterologous polypeptide. 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, 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, thenucleic acid sequence coding for the signal peptide is directly located upstream and in the frame of the nucleic acid sequence coding for the heterologous polypeptide.

[0134] 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 the 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 polypeptide 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.

[0135] 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 retains the ability to control the expression of the nucleic acid sequence encoding the heterologous polypeptide. Usually, functional fragments are either 5’ and / or 3’ truncation of one or more nucleic acid residue from the native promoter nucleic acid sequence.

[0136] 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 derived 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 cyc1 gene, from the pgk1 gene and / or from the ira2 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 retains the ability to end the expressionof the nucleic acid sequence coding for the heterologous protein. 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 retains the ability to end the expression of the nucleic acid sequence coding for the heterologous protein.

[0137] The heterologous nucleic acid molecule encoding the one or more 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 .

[0138] The present disclosure also provides nucleic acid molecules for modifying the yeast host cell so as to allow the expression of the one or more heterologous polypeptide, 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 encoded polypeptide, variants or fragments.

[0139] In some embodiments, the heterologous nucleic acid molecules which can be introduced into the recombinant host cells are codon-optimized with respect to the intended recipient recombinant yeast 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 theextent 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.

[0140] The heterologous nucleic acid molecules can be introduced in the microbial 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.Processes for making the flavoured solution

[0141] The recombinant yeast host cell of the present disclosure has been designed to be used in the preparation of a flavored solution. The present disclosure thus provides a process for making a flavored solution. The process comprises contacting the recombinant yeast host cell as described herein with a fermentation medium and fermenting the fermentation medium with the recombinant yeast host cell to produce the flavored solution comprising one or more ester flavor compounds and ethanol. The recombinant yeast host cells of the present disclosure are intended to be used for making the flavored solution. In certain embodiments, the recombinant yeast host cells of the present disclosure are used in a fermentation process (such as, for example, an anaerobic fermentation process). In an embodiment, the flavored solution is a flavored beverage or is made into a flavored beverage. For example, the fermentation process can be followed by a distillation process of the flavored solution to make a flavored beverage (for example a distilled spirits) and / or by a maturation process, for example in a bottle, a barrel, cask, or other container well-known in the art for beverage maturation, to make the flavored beverage (for example a whisky or a wine).

[0142] In the context of the present disclosure, the production of the ester flavor compound and ethanol usually occurs during the fermentation and, in an embodiment,simultaneously during the fermentation. In an embodiment, the fermentation medium includes a carbohydrates source as fermentable materials which contain C6 sugar as for example fructose, glucose, galactose, sucrose, maltose or starch, as well as their degradation products. As an example, the carbohydrates source can be or comprise a fruit (apple, grape, pears, plums, cherries, peaches), a plant (sugar cane .sugar beet, agave, ginger), a sugar material (honey, molasses), a starchy material (rice, rye, corn, sorghum, millet, barley, wheat, potatoes, cassava) or a derived product (grape must, apple mash, malted grain (or cereal), crushed fruit, fruit puree, fruit juice, fruit must, plant mash, plant juice, gelatinized and saccharified starch from different plant origins as rice, corn, sorghum, wheat, barley). In another embodiment, the fermentation medium or mixture can be or comprise a starchy material. In the context of the present disclosure, a “starchy material” refers to a material that contains starch that could be converted into alcohol by a yeast during alcoholic fermentation. Starchy material could be for example, gelatinized and saccharified starch from cereals, grains (wheat, barley, rice, buckwheat) or grain derived-products (malted grain or a wort) or vegetable (potatoes, cassava). In yet another embodiment, the fermentation medium can be or comprise, but is not limited to, barley, wheat, rye, oats, corn, maize, buckwheat, millet, rice, sorghum, including variants of these cereals that have been subject to the malting, cooking (to rrefi cation) or micronization process, or a combination thereof. In one embodiment, the malted grain (or cereal) is malted barley, malted wheat, malted rye, malted oats, malted corn, malted buckwheat, malted millet, malted rice, and malted sorghum. In another embodiment, the torrefied grain (or cereal) are torrefied barley, torrefied wheat, torrefied rye, torrefied oats, torrefied corn, torrefied buckwheat, torrefied millet, torrefied rice and torrefied sorghum. In yet another embodiment, the micronized grain (or cereal) is micronized barley, micronized wheat, micronized rye, micronized oats, micronized corn, micronized buckwheat, micronized millet, micronized rice and micronized sorghum.

[0143] In some embodiment, the flavored beverage is an alcoholic beverage. In such embodiments, the alcoholic beverage has between 1 to 99 %ABV, between 1 to 80 %ABV, between 5 to 99 %ABV, between 5 to 80 %ABV, between 5 to 60 %ABV, between 5 to 40 %ABV, between 20 to 80 %ABV, between 30 to 80 %ABV, or between 35 to 80 %ABV. Examples of alcoholic beverage products include, but are not limited to beer, brandy, cachaga, Cognac, mezcal, whisky, whiskey (for example bourbon, rye whiskey, wheat whiskey), gin, tequila, rum (for example rhum agricole), wine, mead, sake, baiju, shochu, soju, cider, perry, arrack, jenever, vermouth, Armangnac, korn, raki, pulque, basi, vodka, poitin, akvavit, aquavit, absinthe, spirits, new-make spirit,white dog, or moonshine. The term “flavored beverage” refers to the beverage that has been flavored by the process described in the present application and / or to the beverage that has been flavored by ester flavor compounds produced by the recombinant yeast host cell described in the present application. Examples of flavored alcoholic beverage products include, but are not limited to flavored beer, flavored brandy, flavored cachaga, flavored Cognac, flavored mezcal, flavored whisky, flavored whiskey, flavored bourbon, flavored rye whiskey, flavored wheat whiskey, flavored gin, flavored tequila, flavored rum, flavored rhum agricole, flavored baiju, flavored shochu, flavored soju, flavored arrack, flavored jenever, flavored vermouth, flavored Armangnac, flavored korn, flavored raki, flavored vodka, flavored poitin, flavored akvavit, flavored aquavit, flavored absinthe, flavored spirits, flavored new-make spirit, flavored white dog, or flavored moonshine. In an embodiment, the beverage is a beer or a distilled spirit. In a particular embodiment, the distilled spirit is brandy, whisky, rum, vodka, gin, or tequila. In a more particular embodiment, the distilled spirit is whisky.

[0144] In some embodiments of the present disclosure, the production of the ester flavor compound in the flavored solution occurs during the conversion of a substrate, such as a carbohydrate source during a fermentation.

[0145] As used herein the “fermentation medium” may comprise a carbohydrate source or otherwise may consist essentially of a carbohydrate source. In some embodiment, the “fermentation medium” is a carbohydrate source. In some embodiments, it may be advantageous to provide the recombinant yeast host cell of the present disclosure as a fermentation agent. In one embodiment, a fermenting agent for making the flavored solution comprises or consists essentially of the recombinant yeast host cell described herein. As used herein, “consisting essentially of’ in reference to a fermenting agent refers to a population of fermenting yeasts which do not include a substantial amount of additional fermenting or flavoring organisms which participate to the fermentation process. In an embodiment, a fermenting agent consisting essentially of the recombinant yeast host cell of the present disclosure is made up of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 99.9% of the recombinant yeast host cell described herein. In still another embodiment, a fermenting 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 fermenting agent consisting essentially 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.

[0146] During the fermentation, at least a portion of a carbohydrate source is utilized / converted by the recombinant yeast host cell to make both the ester flavor compound (e.g., to at least a minimal level) and ethanol (to at least a minimal level). The present disclosure provides for a recombinant yeast host cell capable of producing the ester flavor compound in a fermentation medium, so as to accumulate a minimal and / or maximal concentration of the ester flavor compound in the fermentation medium once the carbohydrates source have been converted (e.g., after the conversion of the carbohydrates source). As used herein, the “conversion of the carbohydrates source” or the “carbohydrates source have been converted” is achieved when at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the carbohydrate source is utilized by the microbial biomass. The “conversion of carbohydrates source” or “carbohydrates source have been converted ” can also be achieved when a certain level of ethanol is produced in the fermentation medium, for example when at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, v / v or more of ethanol is produced in the fermentation medium. In some embodiments, the “conversion of carbohydrates source” or “carbohydrates source have been converted” is achieved when a certain level of carbohydrates source remains in the fermentation medium, for example when at most 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 g / L of carbohydrates source remain in the fermentation medium.

[0147] According to an embodiment, the flavored solution is a flavored beverage or is made into a flavored beverage. In an embodiment, the flavored beverage is a beer, a distilled spirit, or wine. In an embodiment, the distilled spirit is whisky.

[0148] In an embodiment, the recombinant yeast host cell of the present disclosure can be used in a distilling process. In such embodiment, the process includes contacting the recombinant yeast host cell (alone or in a combination) of the present disclosure with a fermentation medium, fermenting the fermentation medium to obtain a flavored solution and distilling the flavored solution to obtain a distilled spirit.

[0149] In an embodiment, the distilled spirit is brandy, whisky, rum, vodka, gin, or tequila. In a particular embodiment, the distilled spirit is whisky.

[0150] 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.ExamplesExample 1 : Determination of target acetate ester concentrations for overproduction of acetate esters in distilled spirits

[0151] When the concentration of acetate ester far exceeds a threshold, desirable fruity and floral notes can turn to solvent-like, chemical-like unpleasant aromas [1 , 3], To avoid any unpleasant aromas, the inventors sought to determine acetate ester concentration targets to guide the yeast engineering and the strain selection processes.

[0152] SC-1 has the same genotype as strain M14635 previously described in WO 2019 / 171230 and was first developed from a parental non-engineered strain to overproduce acetate esters via the overexpression of the yeast acetyltransferase gene ATF1 under the control of the strong ADH1 promoter (Table 4). SC-1 was therefore used as a starting point in terms of acetate ester concentration and served as the basis from which the fine tuning of acetate ester in the recombinant yeast host cell would be performed.Table 4. Genotype of yeast strain SC-1.

[0153] The whisky spirit distilled from the flavor solution produced from SC-1 was regarded as aggressive, and the strong solvent and chemical notes were found to be associated with the extremely high concentration of both EA and IAA. Concentration targets were defined based on the sensory profile of spirit of the parental strain (a commercial whisky strain), an acetate ester overproducing strain, SC-1 and blends from those two spirits. Blending distillates of the SC-1 spirit with the control spirit, which had a reduced concentration of acetate esters, resulted in generating more balanced and pleasant spirits. The concentration of ethyl and isoamyl acetate from the preferred blends was used to define a desired concentration approximately 25 g / hL AA to about 60 g / hL AA of EA and approximately 5 g / hL AA to about 30 g / hL AA of IAA indistillates In the case of IBA and PEA it was determined to reach the highest possible concentrations compared to the spirit from the parental strain, since these two congeners are usually not detectable in commercial spirits but have the potential of bringing different aromas and flavor to make more complex and unique spirits. Example 2: Modulation of acetate ester production by engineered strainsStrain engineering

[0154] The strain identifier, loci engineered, genotype and polypeptide overexpressed for each selected strain is described in Table 5. The parental strain used for engineering was DistilaMax® GW (GW), which is a diploid strain. This means that each construct listed in Table 5 was integrated twice, one copy for each chromosome. All promoters used were constitutive promoters, thereby not subject to repression in the presence of oxygen or fatty acid esters compared to the native promoters

[0010] ,Table 5. Strain identifier, loci engineered, genotype and polypeptide overexpressed for each selected strain1“fcy1 ” loci encodes for cytosine deaminaseMaterials and MethodsSmall scale whisky fermentations

[0155] Small scale whisky fermentations were carried out in triplicate in 75 g of corn mash using Ankom units to monitor fermentation kinetics. The corn mash was used at 26.44 % w / w dry solids. Lactoside 247™ (2 ppm) was added to control bacterial contamination. Simultaneous saccharification and fermentation (SSF) was initiated by the addition of 0.280 pl / gDS of DistilaZyme™ GA (microlitre of enzyme per gram of dry solids). Yeast was pitched at 0.35 gDCW / L (grams of dry cell weight per liter) from overnight YPD cultures. Nitrogen was supplemented as 1094 ppm of diammonium sulphate (DAP), corresponding to 232 ppm of yeast assimilable nitrogen (YAN). Incubation was carried out for 70 h at 30 °C and 150 rpm. Small-scale single glass distillation was used to generate low wines (LW) for congener analysis. Fermentation wash (50 mL) from small-scale fermentations was loaded into a 0.5 L round-bottom flask, from which 15 mL of LW distillate at strength between 36.6 and 42.6 %ABV was collected.Medium-scale whisky fermentations

[0156] Medium-scale whisky fermentations for double distillation were carried out in triplicate in 600 g of corn mash using 1 L flasks and the same conditions described above. Medium-scale double glass distillation was performed to obtain new-make spirit (NMS) for sensory analysis. Triplicate wash samples from 600 g fermentations were mixed, split in three identical aliquots and frozen until distillation.DistillationSingle distillation to obtain low wines (LW)

[0157] In a LW distillation (stripping distillation) the volatile molecules (the ethanol and the congeners) were concentrated and separated from the yeast cells, the solids and most of the fermentation media components. When the mash was heated, the chemical components were separated based on their vapor pressure. The condensed vapor from the first distillation is referred to as "low wine" (LW, 160-190 mL). Copper wool (20 g in total) was inserted in the straight column, lyne arm, splash head and the head of the condenser for copper contact. LW samples were used for comparative quantitative analysis of the congeners produced during fermentations.Double distillation to obtain new-make spirits (NMS)

[0158] The procedure was used to obtain new-make spirits for sensory and congener analysis. The process included two batch distillations performed sequentially: a stripping run followed by a spirit run. The stripping distillation to produce LW was described above. The LW from the stripping was then subjected to a second distillation, the spirit run, which further concentrates the distillate. Typically, the first fraction coming out of this distillation was discarded (1 mL, known as heads). The second fraction, the hearts (39 mL), corresponded to the new-make spirit. Tails were not collected.

[0159] Distillate ethanol strength was measured using an Anton-Paar DMA35 density meter. Congener quantification was performed using an Agilent 7820A gas chromatography (GC) system coupled with a 7697A headspace (HS) auto sampler and equipped with a flame ionization detector (FID). The headspace heating zone was maintained at 80 °C, the loop at 110 °C and the transfer line at 120 °C. A CP- Wax 57 CB Agilent column (50 m x 0.25 mm x 0.2 pm) was used for chromatographic separation. The carrier gas was high purity hydrogen with a constant flow rate of 4 mL / min. The injector was set at 220 °C and the split ratio to 30:1. The oven temperature was set at an initial temperature of 40 °C, held for 2 min, raised at 10 °C / min to 120 °C, held for 10 min, raised at 70 °C / min to 200 °C and held for 5 min the total run time was 26 min. The detector temperature was set at 300 °C. A 40 %ABV ethanol calibration curve was used. LW samples were loaded at strength since they were all at %ABV < 40 %. NMS samples were diluted to 40 %ABV. The internal standard 2-pentanol (160 ppm in vial) was added to samples prior to analysis. Samples (100 pL) were added to 20 mL crimp-top headspace vials (23x75 mm) containing 0.5± 0.05 g of sodium chloride. Data was normalized to g / hL of absolute alcohol (g / hL AA) for analysis.

[0160] Quantitative data from congener quantification in LW from fermentation with the candidate strains are shown in Table 6.

[0161] In strains SC-3 and SC-4, modulation of congener production was obtained by combining gene deletions and gene overexpression to selectively limit the overproduction of EA and IAA while maintaining significantly enhanced production of IBA and PEA compared to the parental strain (Table 6). SC-3 and SC-2 were engineered with the same ATF2 overexpression cassette, but strain SC-3 produced 3.7 times more PEA than SC-2 due to the additional overexpression of ARO10. Finally, the highest increase of IBA and PEA was obtained in the spirit from SC-4 from the overexpression of two copies of ATF2 per integration cassette and the deletion of ATF1 (Table 6).Table 6. Congener quantification by gas chromatography with flame ionization detector (GC-FID) in LW distillates from corn mash whisky fermentation with the parental strain (GW), SC-1 and yeast candidates engineered for modulation of acetate ester production.

[0162] Strains SC-3 and SC-4 showed a significant increase in IAA, IBA and PEA compared to the WT strain (Table 7, top) and were within the reduction targets for EA and IAA compared to SC-1 (Table 7, bottom). Strain SC-4 produced the highest concentrations of IBA and PEA compared to the WT strain, followed by SC-3.

[0163] Table 7. Top: Comparison of the fold differences in acetate ester production (LW) between the selected strains (SC-3 and SC-4) and the reference strain DistilaMax ® GW. Bottom: Comparison of the fold differences in acetate ester production (low wines) between SC-1 and the selected strains (SC-3 and SC-4).Sensory Evaluation

[0164] Sensory evaluation was performed with 29 panelists. The panelists were provided with three samples of unaged bourbon spirit derived from fermentation with the control strain (GW), SC-4 and SC-1. The distillates were presented anonymized and in random order. Each glass was filled with distillate adjusted to 20 %ABV with mineral water. Panelists were asked to nose the samples and select their preference in a hedonic scale from 1-7; 1- dislike very much, 2- dislike moderately, 3- dislike slightly, 4- neither like nor dislike, 5- like slightly, 6- like moderately, 7- like very much. The XLSTAT software was used for statistical analysis of the results. The results are shown in Figure 2.

[0165] While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.REFERENCES1. Verstrepen, K.J., etal., Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters. Appl Environ Microbiol, 2003. 69(9): p. 5228-37.2. Takahashi, T., Y. Ohara, and K. Sueno, Breeding of a sake yeast mutant with enhanced ethyl caproate productivity in sake brewing using rice milled at a high polishing ratio. J Biosci Bioeng, 2017. 123(6): p. 707-713.3. Lilly, M., et al., The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates. Yeast, 2006. 23(9): p. 641-59.4. Rice Chaz, S.E., Memmer Nicholas, Cameron Jaren, Carignan Baley Morgan, Freeman Christopher, Brooks Henningsen, Green Hannah Lena, and Argyros Aaron Expression of heterologous enzymes in yeast for flavoures alcoholic beverage production. WO 2019 / 171230 A1 , 2019.5. Dzialo, M.C., et al., Physiology, ecology and industrial applications of aroma formation in yeast. FEMS Microbiol Rev, 2017. 41 (Supp_1): p. S95-S128.6. Burdock, G.A., Fenaroli's Handbook of Flavor Ingredients, (6th ed.). CRC Press. . 2009.7. Nicol, D.A., Rum, in Fermented Beverage Production, A.G.H. Lea and J.R. Piggott, Editors. 2003, Springer US: Boston, MA. p. 263-287.8. Tu, W., et al., Chinese Baijiu: The Perfect Works of Microorganisms. Front Microbiol, 2022. 13: p. 919044.9. GM Walker, C.a., WM Ingledew and C Pilgrim, 6th edition, The alchohol texbook, Chapter 17, in The alchohol texbook. 2017.10. Malcorps, P., et al., A New Model for the Regulation of Ester Synthesis by Alcohol Acetyltransferase in Saccharomyces Cerevisiae during Fermentation. Journal of the American Society of Brewing Chemists, 1991. 49(2): p. 47-53.11. Kruis, A. J., et al., Ethyl acetate production by the elusive alcohol acetyltransferase from yeast. Metab Eng, 2017. 41 : p. 92-101.12. Lilly, M., M.G. Lambrechts, and I.S. Pretorius, Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates. Appl Environ Microbiol, 2000. 66(2): p. 744-53.13. Nancolas, B., et al., Saccharomyces cerevisiae Atflp is an alcohol acetyltransferase and a thioesterase in vitro: Characterization of Atflp. Yeast, 2017. 34.14. Holt, S., et al., The molecular biology of fruity and floral aromas in beer and other alcoholic beverages. FEMS Microbiol Rev, 2019. 43(3): p. 193-222.15. Ichikawa, E., et al., Breeding of a Sake Yeast with Improved Ethyl Caproate Productivity. Agricultural and Biological Chemistry, 1991. 55(8): p. 2153-2154.16. Chadani, T., et al., Genome Editing to Generate Sake Yeast Strains with Eight Mutations That Confer Excellent Brewing Characteristics. Cells, 2021 . 10(6): p. 1299.17. Kruis, A. J. et al., Alcohol Acetyltransferase Eat1 Is Located in Yeast Mitochondria. App and Environ Microbiol, 2018. 84(19): p. 1-11 .

Claims

WHAT IS CLAIMED IS:

1. A recombinant yeast host cell for making a flavored solution obtainable or obtained by fermentation of a fermentation medium, the recombinant yeast host cell comprising: a) an upregulated alcohol acetyltransferase 2 (ATF2) and / or an upregulated phenylpyruvate decarboxylase (ARO10) when compared to a wild-type yeast host cell; and b) at least one downregulated native transferase when compared to a wildtype yeast host cell, wherein the at least one downregulated transferase is a native alcohol acetyltransferase 1 (ATF1) and / or a native ethanol acetyl-CoA transferase (EAT1).

2. The recombinant yeast host cell of claim 1 comprising a heterologous ATF2 as the upregulated ATF2.

3. The recombinant yeast host cell of claim 1 or 2, wherein the ATF2:• is an ATF2 classified under Enzyme Commission No. 2.3.1 .84;• has the ability to convert acetyl-CoA and various alcohol into acetate esters acid;• is of prokaryotic or eukaryotic origin;• is a ATF2;• is encoded by an atf2 gene;• is derived from Saccharomyces or Torulaspora species;• is derived from Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces cerevisiae x Saccharomyces kudriavzevii, Saccharomyces paradoxus, Saccharomyces mikatae, Torulaspora delbrueckii or Saccharomyces kudriavzevir,• has the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20 or be a variant of the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20 having alcohol acetyltransferase 2 activity or be a fragment of the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20 having alcohol acetyltransferase 2 activity;• is encoded by a heterologous nucleic acid molecule having a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2, 15, 16, 17, 18, 19 or 20; and / or• is operably associated to a native or a heterologous promoter.

4. The recombinant yeast host cell of any one of claims 1 to 3, comprising a heterologous ARO10 as the upregulated ARO10.

5. The recombinant yeast host cell of any one of claims 1 to 4, wherein the ARO10:• is an ARO10 classified under Enzyme Commission No. 4.1.1.43;• has the ability to convert phenylpyruvate into phenylacetaldehyde;• is of prokaryotic or eukaryotic origin;• is a AROW;• is encoded by an aro10 gene;• is derived from Saccharomyces, Naumovozyma, Maudiozyma, Kazachstania, Arxiozyma, Huiozyma, Nakaseomyces, Naumovozyma, Torulaspora, Zygosaccharomyces or Henningerozyma species;• is derived from Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces arboricola, Saccharomyces kudriavzevii, Saccharomyces eubayanus, Naumovozyma castellii, Maudiozyma humilis, Kazachstania Africana, Maudiozyma exigua, Maudiozyma barnettii, Arxiozyma heterogenica, Huiozyma naganishii, Nakaseomyces glabratus, Naumovozyma dairenensis, Torulaspora delbrueckii, Torulaspora globosa, Zygosaccharomyces rouxii Zygosaccharomyces parabailii, or Henningerozyma blattae;• has the amino acid sequence of SEQ ID NO: 5, 21 , 22, 23, 24, 25, 26, 27,28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 or be a variant of the amino acid sequence of SEQ ID NO: 5, 21 , 22, 23, 24, 25, 26, 27, 28,29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 having alcohol acetyltransferase 2 activity or be a fragment of the amino acid sequence of SEQ ID NO: 5, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 having alcohol acetyltransferase 2 activity;• is encoded by a heterologous nucleic acid molecule having a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:5, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39,40, 41 or 42; and / or is operably associated to a native or a heterologous promoter.

6. The recombinant yeast host cell of any one of claims 1 to 5 comprising a heterologous ATF2 as the upregulated ATF2 and a heterologous ARO10 as the upregulated ARO10.

7. The recombinant yeast host cell of any one of claims 1 to 6, wherein the at least one downregulated native transferase is the native ATF1 .

8. The recombinant yeast host cell of any one of claims 1 to 6, wherein the at least one downregulated native transferase is the native EAT1 .

9. The recombinant yeast host cell of any one of claims 1 to 6, wherein the at least one downregulated transferase comprises the native alcohol acetyltransferase 1 (ATF1) and the native ethanol acetyl-CoA transferase (EAT1).

10. The recombinant yeast host cell of claim 7 or 9, wherein the gene encoding the native ATF1 is deleted.11 . The recombinant yeast host cell of claim 8 or 9, wherein the gene encoding the native EAT1 is deleted.

12. The recombinant yeast host cell of any one of claims 1 to 11 , further comprising a downregulated isoamyl acetate-hydrolysing esterase (IAH1) when compared to a wild-type yeast host cell.

13. The recombinant yeast host cell of claim 12, wherein the gene encoding the native isoamyl acetate-hydrolysing esterase (IAH1) is deleted.

14. The recombinant yeast host cell of any one of claims 3 to 13, wherein the heterologous promoter is tpip, tef2p, adh1 p, rev1 p, tdh 1 p, tir1 p, enol p, cyc1 p, tdh2p, pgkl p, qcr8p, ccw12p, hxt3p, yet3p, sedl p, eno2p, gpml p, hor7p, hsp150p, icll p, ssal p, tdh3p, cprl p, zwfl p, ssd p, pykl p, hxt2p, hsp30p, gpd2p, pfkl p, pdr12p, danl p, msn4p, hxk2p, set3p, ilv3p, pep4p, cys3p, ergl Op, sna3p, erg5p, erg13p, rhol p, erg20p, erg26p, erg9p, cis3p, trx2p, gxp2p, gre2p, tar1 p, hpf1 p, fas2p, arp1 Op, pho23p, reg2p, dtr1 p, mip6p, sn 11 p, adh2p, fdh2p, mitl p or gpdl p.

15. The recombinant yeast host cell of claim 14, wherein the heterologous promoter is tef2, adhl p, tpil p, revlp, cyd p, qcr8p, yet3p, hsp30p, danl p, msn4p, cys3p, sna3p, gre2p, pho23p, mip6p, or mitl p.

16. The recombinant yeast host cell of claim 15, wherein the heterologous promoter is tef2p, adh1 p, tpi1 p or rev1 p.

17. The recombinant yeast host cell of any one of claims 1 to 16, which produces, or is capable of producing, one or more ester flavor compounds during the fermentation.

18. The recombinant yeast host cell of claim 17, wherein the one or more ester flavor compound comprises ethyl acetate (EA), isoamyl acetate (IAA), isobutyl acetate (IBA), phenethyl acetate (PEA), or any combination thereof.

19. The recombinant yeast host cell of claim 18, which produces, or is capable of producing, up to about 300 g of ethyl acetate per hectoliter of absolute alcohol, and / or up to about 140 g of isoamyl acetate per hectoliter of absolute alcohol.

20. The recombinant yeast host cell of claim 18, which produces, or is capable of producing, up to about 13 times the amount of EA, and / or up to about 80 times the amount of IAA when compared to a wild-type yeast host strain.

21. The recombinant yeast host cell of claim 18, which produces, or is capable of producing, between about 5 g to about 300 g of ethyl acetate per hectoliter of absolute alcohol, and / or between about 5 g to about 140 g of isoamyl acetate per hectoliter of absolute alcohol.

22. The recombinant yeast host cell of any one of claims 18 to 21 , which produces, or is capable of producing, at least about 0.03 g of isobutyl acetate per hectoliter of absolute alcohol, and / or at least about 0.5 g of phenethyl acetate per hectoliter of absolute alcohol.

23. The recombinant yeast host cell of any one of claims 1 to 22 being from the genus Saccharomyces.

24. The recombinant yeast host cell of any one of claims 1 to 23 being from the species Saccharomyces cerevisiae.

25. A process for making a flavored solution, the process comprising:I. contacting the recombinant yeast host cell of any one of claims 1 to 24 with a fermentation medium; andII. fermenting the fermentation medium with the recombinant yeast host cell to produce the flavored solution comprising one or more ester flavor compounds and ethanol.

26. The process of claim 25, wherein the flavored solution is a flavored beverage or is made into a flavored beverage.

27. The process of claim 26, wherein the flavored beverage is a beer, a distilled spirit, or wine.

28. The process of claim 27, wherein the distilled spirit is whisky.

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