Genetically modified yeast for the production of alcohol-free beverages

Abstract

Provided herein are genetically modified yeast cells comprising a genetic modification to reduce sensory detection of one or more wort-related off-flavors in a fermented beverage, and the cells are unable to convert maltose and / or maltotriose to ethanol. Also provided herein are methods of producing a fermented product (e.g., a non-alcoholic fermented beverage) using the genetically modified yeast cells described herein.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 579,269, filed August 28, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application contains a sequence list, which has been electronically submitted in .XML format and is incorporated herein by reference in its entirety. The .XML copy was created on August 27, 2024, named "129085.00043.xml", and is 142,566 bytes in size. The sequence list contained in this .XML file is part of this specification and is incorporated herein by reference in its entirety. Background Technology

[0005] Over the past decade, consumer demand for non-alcoholic (NA) beer has grown dramatically. Total sales of NA beer (generally defined as beer containing less than 0.5% ABV) are projected to approach $25 billion by 2023, up from $13 billion in 2015 (Smith, S. Trends Revealed: The Non-Alcoholic Beer Market Is Bubbling Up. HubSpot: blog.hubspot.com / the-hustle / the-non-alcoholic-beer-market-is-bubbling-up#article (2022)). This near doubling of market size represents a compound annual growth rate (CAGR) of 8.3%, significantly exceeding the 1.8% CAGR of the alcoholic beer industry over the same period (Non-Alcoholic Beer MarketShare, Trends, and Global Sales Analysis to 2030: marketresearchfuture.com / reports / non-alcoholic-beer-market-3912). The rapid popularity of NA beer is mainly attributed to the shift in preferences among traditional beer drinkers.In recent years, numerous studies have found that excessive alcohol consumption poses serious health risks (Rehm, J. et al. The relation between different dimensions of alcohol consumption and burden of disease: an overview. Addiction 105, (2010); White, A. & Hingson, R. The burden of alcohol use: excessive alcohol consumption and related consequences among college students. Alcohol Res. 35, (2013); Behaviors, A. Global status report on alcohol and health 2018: who.int / publications / i / item / 9789241565639(2018)), and as a result, consumers are increasingly motivated to reduce their consumption of alcoholic beverages (Bronin, A. Beyond JAAD January 2023. J. Am. Acad. Dermatol. 88, e1–e4 (2023)). This shift away from alcoholic beverages, coupled with the global trend toward healthier eating habits (International Food Information Council. 2022 Food and Health Survey; Grimmelt, A., Moulton, J., Pandya, C. & Snezhkova, N. Hungry and confused: The winding road to conscious eating.mckinsey.com / industries / consumer-packaged-goods / our-insights / hungry-and-confused-the-winding-road-to-conscious-eating (2022)), is likely the main reason for the recent increase in consumer interest in North American beer.The non-alcoholic beer market is projected to continue its rapid growth in the foreseeable future, with global annual sales expected to exceed $40 billion by 2032 (Pulidindi, K. & Ahuja, K. Non-Alcoholic Beer Market - By Product (Alcohol Free {By Material [Malted Grains, Hops, Yeast and Enzymes], By Technology [Restricted Fermentation and Dealcoholization {Reverse Osmosis}], By Sales Channel (Liquor Stores, Convenience Stores]}, Low Alcohol) & Forecast, 2023-2032, gminsights.com / industry-analysis / non-alcoholic-beer-market (2022)). Summary of the Invention

[0006] On the one hand, this article provides genetically modified yeast cells, such as brewer's yeast cells, that cannot convert maltose and / or maltotriose into ethanol, or have a reduced conversion capacity. In some embodiments, the genetically modified yeast cells contain heterologous nucleic acids encoding enzymes having alcohol acyltransferase (EC 2.3.1.84) activity.

[0007] On the other hand, a liquid fermentation composition is provided comprising: (a) a population of genetically modified yeast cells that are genetically modified to produce one or more acetates and / or ethyl acetates, wherein the yeast cells cannot convert maltose and / or maltotriose into ethanol, or have reduced conversion capacity; (b) a sugar source comprising wort, wherein the total sugar in the wort is attenuated by the population of genetically modified yeast cells by no more than 25%; (c) one or more aldehyde molecules and / or non-aldehyde molecules derived from wort; and (d) no more than 1.0% (v / v) of alcohol.

[0008] On the other hand, a liquid fermentation composition is provided comprising: (a) a population of genetically modified yeast cells that are genetically modified to be unable to convert maltose and / or maltotriose into ethanol, or have reduced conversion capabilities; (b) a sugar source comprising wort, wherein the total sugar in the wort is attenuated by the population of genetically modified yeast cells by no more than 25%; and (c) no more than 1.0% (v / v) of alcohol.

[0009] On the other hand, a method for producing a fermented beverage is provided, comprising: (a) providing genetically modified yeast cells containing functionally disrupted one or more proteins associated with maltose and / or maltotriose transport and / or maltose and / or maltotriose hydrolysis, wherein the functional disruption results in slower growth of the yeast cells when maltose is the sole sugar source, compared to yeast cells without the genetic modification; (b) providing a culture medium containing a sugar source derived from wort; (c) combining the genetically modified yeast cells with the culture medium to form a fermentation composition; and (d) fermenting the fermentation composition to produce a fermented beverage.

[0010] On the other hand, a method for producing fermented beverages is provided, comprising contacting a population of genetically modified yeast cells of the present disclosure with a culture medium containing a sugar source derived from malt extract during fermentation to produce a fermented beverage.

[0011] Various aspects of this disclosure provide genetically modified yeast cells (modified cells) containing one or more genetic modifications to reduce sensory detection of one or more wort-related off-flavors in fermented beverages; wherein the modified cells cannot convert maltose and / or maltotriose into ethanol. In some embodiments, the wort-related off-flavors comprise aldehyde molecules and / or non-aldehyde molecules. In some embodiments, the aldehyde molecules are 2-methylbutyraldehyde, 2-methylpropionaldehyde, hexanal, benzaldehyde, furfural, acetaldehyde, methylthion, phenylacetaldehyde, or 5-hydroxymethylfurfural. In some embodiments, the non-aldehyde molecules are (E)-β-damascone or 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone.

[0012] In some embodiments, the modified cells are capable of converting glucose into ethanol. In some embodiments, the modified cells contain one or more genetic modifications to functionally disrupt one or more proteins associated with the transport of maltose and / or maltotriose into the modified cells and / or the hydrolysis of maltose and / or maltotriose by the modified cells. In some embodiments, the modified cells contain one or more genetic modifications to functionally disrupt maltose and / or maltotriose transporters. In some embodiments, the modified cells are genetically modified to lack MAL31 and MAL11 (AGT1).

[0013] In some embodiments, one or more genetic modifications aimed at reducing sensory detection of one or more wort-related off-flavors result in an increase in the production of one or more acetates and / or ethyl acetates compared to cells without genetic modifications. In some embodiments, the acetates are ethyl acetate, isoamyl acetate, and / or phenethyl acetate. In some embodiments, the ethyl acetates are ethyl hexanoate, ethyl octanoate, and / or ethyl decanoate.

[0014] In some embodiments, one or more genetic modifications that increase the production of one or more acetates and / or ethyl acetates include overexpression of an enzyme having alcohol acyltransferase (AAT) activity. In some embodiments, the enzyme having AAT activity comprises the sequence shown in SEQ ID NO: 5.

[0015] In some embodiments, one or more genetic modifications that increase the production of one or more acetates and / or ethyl acetates include expressing a heterologous enzyme with acyltransferase (AAT) activity. In some embodiments, the heterologous enzyme with AAT activity comprises the sequence shown in SEQ ID NO: 7.

[0016] In some embodiments, one or more genetic modifications aimed at reducing sensory detection of one or more wort-related off-flavors result in increased production of 3-mercaptohexanol (3MH). In some embodiments, the genetic modifications that increase 3MH production include expressing a bacterial enzyme with carbon-sulfur lyase (CSL) activity. In some embodiments, the CSL-active bacterial enzyme comprises the sequence shown in SEQ ID NO: 9.

[0017] In some embodiments, one or more genetic modifications for reducing sensory detection of one or more wort-related off-flavors result in an increase in the production of one or more monoterpenes. In some embodiments, the monoterpenes are linalool, geraniol, and / or citronellol. In some embodiments, the genetic modifications for increasing the production of one or more monoterpenes include expressing one or more of the following: (a) a truncated variant of the yeast HMG1 enzyme; (b) a variant of the ERG20 enzyme; (c) linalool synthase; and / or (d) geraniol synthase. In some embodiments, the truncated variant of the yeast HMG1 enzyme comprises the sequence shown in SEQ ID NO: 11. In some embodiments, the variant of the ERG20 enzyme comprises the sequence shown in SEQ ID NO: 13. In some embodiments, linalool synthase comprises the sequence shown in SEQ ID NO: 15. In some embodiments, geraniol synthase comprises the sequence shown in SEQ ID NO: 17.

[0018] In some embodiments, the yeast cells belong to the genus *Saccharomyces*. In some embodiments, the yeast cells belong to the species *Saccharomyces cerevisiae*. In some embodiments, the yeast cells are *Saccharomyces cerevisiae* Chico Ale yeast, *Saccharomyces cerevisiae* London Ale yeast, *Andechs Lager yeast*, *Augustiner Lageryeast*, or *American Ale yeast*.

[0019] Various aspects of this disclosure provide methods for producing fermented beverages, including contacting any modified cells described herein with a culture medium containing at least one fermentable sugar, wherein the contact is carried out during at least a first fermentation process to produce the fermented beverage. In some embodiments, the fermented beverage is a low-alcohol fermented beverage. In some embodiments, the alcohol content of the fermented beverage is less than or equal to about 1.0% (v / v) of alcohol. In some embodiments, the alcohol content of the fermented beverage is from about 0.01% (v / v) to about 1.0% (v / v). In some embodiments, the alcohol content of the fermented beverage is from about 0.01% (v / v) to about 0.5% (v / v).

[0020] In some embodiments, the method does not include the step of physically removing alcohol from the beverage or prematurely stopping fermentation. In some embodiments, at least one fermentable sugar is provided from at least one sugar source. In some embodiments, the fermentable sugar is glucose, fructose, and / or sucrose. In some embodiments, the first fermentation process results in a reduction of the level of fermentable sugar by at least 15%.

[0021] In some embodiments, the fermented beverage is beer. In some embodiments, the sugar source comprises wort. In some embodiments, the sugar source is wort, and the method further includes producing a culture medium, wherein the culture medium comprises (a) contacting a variety of grains with water; and (b) boiling or soaking the water and grains to produce wort. In some embodiments, the method further includes adding at least one hop variety to the wort to produce hop wort. In some embodiments, the method further includes adding at least one hop variety to the culture medium.

[0022] In some embodiments, the method further includes at least one additional fermentation process. In some embodiments, the method further includes carbonation of the fermentation product.

[0023] Other aspects of this disclosure provide genetically modified brewer's yeast cells (modified cells) containing one or more genetic modifications to functionally disrupt the maltose / maltotriose transporters MAL31 and MAL11 (AGT1).

[0024] Details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will be apparent from the following drawings and detailed description of several embodiments, as well as from the appended claims. Attached Figure Description

[0025] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown in different figures is represented by similar numbers. For clarity, not every component is labeled in every drawing. In the drawings:

[0026] Figure 1 The chemical structures of exemplary fermentable sugars glucose, maltose, and maltotriose are shown.

[0027] Figure 2 This shows the attenuation of wort sugars by the Saccharomyces cerevisiae Chico strain lacking MAL31 and MAL11 (AGT1) (MAL31::ΔAGT1::Δ, also known as BY632 (orange line)) and the wild-type Saccharomyces cerevisiae Chico strain (blue line) in a representative 20 L beer fermentation. Fermentation began at a total wort sugar content of 12 Pareto degrees. The percentage attenuation was calculated as (1 - (Pareto degree at the end of fermentation / Pareto degree at the start of fermentation)) * 100. The BY632 strain resulted in a 21.7% attenuation of wort sugars, while Chico resulted in a 79.1% attenuation.

[0028] Figure 3A The concentrations of ethyl acetate, isoamyl acetate, and phenylethyl acetate in 250 different commercial alcoholic beer samples are shown. The data used in the figure were collected in [reference PMID: 38531860].

[0029] Figure 3BThis chart shows the relative levels of acetate and ethyl acetate in small-scale fermentations of Chico and strains that cannot convert maltose and maltotriose to ethanol (“maltose-negative” (MN) strains). The value reported in each cell represents the change in peak area of ​​each ester measured in fermentation with the specified strain relative to its peak area in fermentation with wild-type Saccharomyces cerevisiae Chico strain, expressed as a log2 factor. All fermentations were repeated twice, and the reported values ​​are the average of these replicates. More positive values ​​indicate higher ester concentrations in fermentation with wild-type Saccharomyces cerevisiae Chico strain, and more negative values ​​indicate lower ester concentrations in fermentation with wild-type Saccharomyces cerevisiae Chico strain. The percentage of attenuation achieved for each strain is shown to the right of the strain name. The genotype of each strain is shown in a table below the heatmap, along with the aroma characteristics detected at the end of fermentation.

[0030] Figure 4 This paper presents a blinded sensory panel evaluation of beers brewed using wild-type Saccharomyces cerevisiae strains Chico, BY632 (MAL31::Δ AGT1::Δ), and Saccharomyces cerevisiae strains Chico BY1505 (MAL31::Δ; AGT1::Δ; YPS3::pNCP1-tmcHMG, pERG11-ERG20(F96W;N127W), pHSP26-tmcMcLIS, pPGK1-tmcObGES). These beers were included in the sensory evaluation along with three other beers brewed using MN strains (not shown). All six beers were scored by five sensory judges on a scale of 1 (best) to 6 (worst). Each dot reports one judge's score for each beer, and the line reports the average score from all judges for each beer. Beer produced by fermentation using strain BY1505 scored significantly better than beer produced using strain BY632 (p=.008, two-sided Wilcoxon rank-sum test), and nominally better than beer produced using wild-type Saccharomyces cerevisiae Chico strain (not significant).

[0031] Figure 5AThis paper presents a blinded sensory panel evaluation of beers brewed using BY632, BY1575, BY1578, BY1576, BY1574, BY1503, BY1573, and BY1504 at different concentrations of isoamyl acetate. All eight beers were rated by twelve sensory judges on a scale of 0 (none) to 10 (most intense), assessing the intensity of tomato, veggie, and grain flavors. Each graph shows the intensity of a wort-related sensory attribute for all eight evaluated NA beers. Each point represents one beer, and the beer's position on the x-axis reflects the concentration of isoamyl acetate present in the beer. The points report the mean intensity score for the specified flavor attribute across all sensory panel members, and the error bar reports the standard deviation of this mean. The blue trend line shows the correlation between isoamyl acetate concentration and the perceived intensity of the specified sensory attribute. All correlation coefficients were statistically significant. Figure 5B A similar diagram of phenethyl acetate is shown. Figure 5C A similar diagram of ethyl acetate is shown.

[0032] Figure 6A This paper presents a blinded sensory panel evaluation of beers brewed using BY632, BY1575, BY1578, BY1576, BY1574, BY1503, BY1573, and BY1504 at different isoamyl acetate concentrations. All eight beers were rated by twelve sensory judges on a scale of 0 (none) to 10 (most intense), assessing the intensity of banana and solvent flavors. Each graph shows the intensity of one sensory attribute for all eight evaluated NA beers. Each point represents one beer, and the beer's position on the x-axis reflects the concentration of isoamyl acetate present in the beer. The points report the mean intensity score for the specified flavor attribute across all sensory panel members, and the error bar reports the standard deviation of that mean. The blue trend line shows the correlation between isoamyl acetate concentration and the perceived intensity of the specified sensory attribute. All correlation coefficients were statistically significant. Figure 6B A similar diagram of phenethyl acetate is shown. Figure 6C A similar diagram of ethyl acetate is shown.

[0033] Figure 7 The sensory panel's blinded ranking preference for five beers brewed using strains BY632, BY1574, BY1503, BY1573, and BY1504 is shown. Box plots show the sensory panel's ranking of each beer, identified by the strain number used to brew them (x-axis, marked with a "B" starting with the strain name).

[0034] Figure 8A-C shows the modeling of acetate ranking preferences to determine the preferred concentration ranges for isoamyl acetate (A), ethyl acetate (B), and phenethyl acetate (C). Black dots report the average preference ranking, and error bars report one standard deviation of the mean. These data are identical to those shown in Figure 3. Horizontal black bars represent the average preference ranking of beer produced by strain y632, which was not engineered for heterologous AAT expression. Parabolas represent the polynomial equations shown in the subtitles of each figure and mathematically describe the relationship between each acetate concentration and its preference ranking. For each molecule, we defined a concentration range that elevates the sensory ranking of the beer above the average ranking of y632. This range is defined as a given acetate concentration that ranks higher than the average ranking of y632 and is higher than the concentration of y632 in beer. The boundaries of this range are represented by vertical dashed lines in each figure, and the concentrations at these boundaries are reported in the text. Detailed Implementation

[0035] Despite increased consumer demand for non-alcoholic (NA) beer (i.e., beer with less than or equal to 0.5% vol / vol) in recent years, the quality of commercially available NA beer is generally considered poor (Blanco, CA, Andrés-Iglesias, C. & Montero, O. Low-alcohol Beers: Flavor Compounds, Defects, and Improvement Strategies. Crit. Rev. Food Sci. Nutr. (2016) doi:10.1080 / 10408398.2012.733979; Gernat, DC, Brouwer, E. & Ottens, M. Aldehydes as Wort Off-Flavours in Alcohol-Free Beers—Origin and Control. Food Bioprocess Technol. 13, 195–216 (2019); Piornos, JA, Koussissi, E., Balagiannis, DP, Brouwer, E. & Parker, JK Alcohol-free and low-alcohol beers: Aromachemistry and sensory characteristics. Compr. Rev. Food Sci. Food Saf. 22,233–259 (2023)). Compared to alcoholic beers, non-alcoholic (NA) beers are typically characterized by a diluted "beer-like" flavor, a thin mouthfeel, and unpleasant aromas. These negative sensory properties of NA beers are well-known due to limitations in the production processes used to make them non-alcoholic (Salanță, LC et al. Non-Alcoholic and CraftBeer Production and Challenges. Processes 8, 1382 (2020)). While several different NA beer production processes exist, they all have drawbacks that negatively impact the flavor and aroma of the resulting beer. Because most commercially available NA beers taste poor, consumers are often forced to choose between a good-tasting alcoholic beer and a healthier but less appealing NA beer.

[0036] This disclosure describes the development of genetically engineered yeast strains capable of producing native (NA) beers with fewer undesirable off-flavors (e.g., wort-related off-flavors) and better aromas. The document also provides modified yeast cells that cannot ferment maltose and / or maltotriose to ethanol or have reduced fermentation capacity, but which also overexpress acyltransferases (AAT) to improve the sensory characteristics of NA beers. Furthermore, the document provides modified yeast cells that cannot ferment maltose and / or maltotriose to ethanol or have reduced fermentation capacity, but which express enzymes with cysteine ​​S-conjugated β-lyase (CSL) activity to improve the sensory characteristics of NA beers. Finally, the document provides modified yeast cells expressing one or more enzymes for the production of monoterpenes, which improve the sensory characteristics of NA beers.

[0037] Traditional processes for producing non-alcoholic beer include: (1) physical dealcoholization after normal fermentation, and (2) stopping fermentation (Muller, C., Neves, LE, Gomes, L., Guimarães, M. & Ghesti, G. Processes for alcohol-free beer production: a review. Food Sci. Technol. 40, 273–281 (2019)). For processes involving physical dealcoholization after normal yeast fermentation, beer is first produced through typical alcoholic fermentation, resulting in a beverage with an ethanol content of 3–10%. After fermentation, beer is treated by one of several methods to remove most of the ethanol, such as thermal treatment processes like evaporation and distillation, or membrane-based processes like dialysis and reverse osmosis (Muller, C., Neves, LE, Gomes, L., Guimarães, M. & Ghesti, G. Processes for alcohol-free beer production: a review. Food Sci. Technol. 40, 273–281 (2019); Kozłowski, R., Dziedziński, M., Stachowiak, B. & Kobus-Cisowska, J. Non- and low-alcoholic beer - popularity and manufacturing techniques. Acta Sci. Pol.Technol. Aliment. 20, (2021)). These processes can produce NA beer with less than 0.01% ABV. However, physical dealcoholization not only removes ethanol, but also removes many of the desired flavor and aroma molecules that give beer its intended flavor and aroma (Areview of methods of low alcohol and alcohol-free beer production. J. FoodEng. 108, 493–506 (2012); Production of Alcohol-Free Beer. in Beer in Health and Disease Prevention 61–75 (Academic Press, 2009)).After dealcoholization, the concentrations of these desired flavor molecules (mainly esters, terpenes, thiols, and fusel oils) can be reduced by more than 80% compared to beer before dealcoholization (Areview of methods of low alcohol and alcohol-free beer production. J. FoodEng. 108, 493–506 (2012)). Combined with the sensory and taste effects of ethanol removal, the loss of these flavor molecules results in a severe lack of "beer-like" flavor and aroma in NA beer.

[0038] Another drawback of physical dealcoholization methods is the need for specialized equipment, which can be expensive and not commonly found in breweries, creating a significant barrier to entry for small and medium-sized breweries (Navrátil, M., Dömény, Z., Sturdík, E., Smogrovicová, D. & Gemeiner, P. Production of non-alcoholicbeer using free and immobilized cells of Saccharomyces cerevisiae deficient in the tricarboxylic acid cycle. Biotechnol. Appl. Biochem. 35, 133–140 (2002)).

[0039] As an alternative to physical dealcoholization, the aborted fermentation (AF) method involves stopping (aborting) the fermentation of the wort before the ethanol concentration exceeds a certain threshold (typically around 0.5% ABV). In practice, this usually means stopping fermentation when the sugar decay (the percentage decrease in wort sugar due to yeast consumption) reaches between 10% and 20%. In contrast, in typical alcoholic beer fermentation, the decay at the end of fermentation will be between 70% and 90%. Since the amount of ethanol produced during fermentation is proportional to the amount of sugar consumed by the yeast, aborted fermentation with only 10-20% sugar consumption can be used to produce beer with an ABV below approximately 0.5%.

[0040] Methods for stopping yeast fermentation at the desired percentage attenuation and ethanol concentration include, for example, cold contact fermentation and fermentation using yeast strains that cannot consume maltose and maltotriose. For cold contact fermentation, wort fermentation is carried out using standard brewing yeast strains, but at a low temperature (approximately 4˚C) for a short period (24–48 hrs) (A review of methods of low alcohol and alcohol-free beer production. J. Food Eng. 108, 493–506 (2012)). Under these conditions, maltose consumption is much lower than in alcoholic fermentation, and the ethanol concentration can be maintained below approximately 0.5% ABV.

[0041] When fermentation is stopped using yeast strains that cannot consume maltose and maltotriose, brewers use special yeast strains that do not express genes necessary for consuming these sugars (Yabaci Karaoglan, S., Jung, R., Gauthier, M., Kinčl, T. & Dostálek, P. Maltose-Negative Yeast in Non-Alcoholic and Low-Alcoholic Beer Production. Fermentation 8, 273 (2022)). Maltose and maltotriose consist of two (maltose) or three (maltotriose) glucose monomers arranged in a linear chain via glycosidic bonds. Figure 1 Since maltose and maltotriose constitute approximately 80-90% of the normal fermentation sugars in brewing wort (He, Y. et al. Wort composition and its impact on the flavour-active higher alcohol andester formation of beer – a review. J. Inst. Brew. 120, 157–163 (2014); Jacques, KA, Lyons, TP & Kelsall, DR The Alcohol Textbook: A Reference for the Beverage, Fuel and Industrial Alcohol Industries. (2003)), fermentation using these special strains allows brewers to limit wort decay to 10-20% and maintain ethanol concentrations at approximately 0.5% v / v. In the brewing industry, yeast strains that cannot consume maltose and maltotriose are generally referred to as “maltose-negative” (MN) strains.

[0042] The production of non-alcoholic (NA) beer using MN yeast does not require expensive or specialized equipment, making this type of aborted fermentation process suitable for breweries of all sizes. However, it has significant drawbacks. For example, non-alcoholic beers produced using currently available MN strains are often considered overly sweet due to the high concentration of unfermented sugars, and lack beer-like flavor due to the reduced level of flavor molecules produced by the yeast during aborted fermentation (Yabaci Karaoglan, S., Jung, R., Gauthier, M., Kinčl, T. & Dostálek, P. Maltose-Negative Yeast in Non-Alcoholic and Low-Alcoholic Beer Production. Fermentation 8, 273 (2022); Piornos, JA et al. Elucidating the Odor-Active Aroma Compounds in Alcohol-Free Beer and Their Contribution to the Worty Flavor. J. Agric. Food Chem. (2020) doi:10.1021 / acs.jafc.0c03902). Furthermore, non-alcoholic beer produced using currently available MN strains has a pronounced "wort" off-flavor and aroma, which impairs the overall sensory characteristics.

[0043] The genetically modified cells and methods of using them described herein enable the production of non-alcoholic fermented products with reduced wort-related off-flavors in sensory detection. In some embodiments, the genetically modified cells and methods of using them described herein result in the production of molecules in the fermented product that mask the detection of wort-related off-flavors (e.g., increase the detection threshold for off-flavors).

[0044] As used herein, "brewing yeast cells" or "brewing yeast strains" refer to yeast strains selected by brewers that possess certain characteristics suitable for brewing alcoholic and non-alcoholic beverages and are genetically distinct from non-brewing yeast strains. Brewing yeast strains include *Saccharomyces cerevisiae* strains, whose genome sequences place them within the "Beer 1," "Beer 2," or "Mixed" phylogenetic branches defined by Gallone et al. (2016) and Priess et al. (2018) (PMIDs 27610566, 30258422). Alternatively, brewing yeast strains can be *Pasteurella* strains, a hybrid species produced during brewing by crossing *Saccharomyces cerevisiae* and *Cypripedium* strains (PMID 26269586). Furthermore, brewing yeast strains have or may have one or more of the following characteristics: 1) they are capable of metabolizing maltotriose, 2) their genomes contain PAD1 and FDC1 alleles that are lost or reduced in function due to loss-of-function mutations, 3) their genomes encode the AGT1 allele of MAL11, and / or they exhibit efficient growth on maltose and maltotriose media. In any embodiment of this disclosure, the genetically modified yeast cells may be derived from brewing yeast strains. Exemplary brewing yeast strains are further disclosed herein.

[0045] As used herein, the term "wheat wort-related off-flavor" refers to undesirable flavors and / or aromas associated with wort and with restricted fermentation of wort using maltose-negative yeast strains. For example, in some embodiments, wort-related off-flavors may be described as grain-like, potato-like, hay-like, stewed apple-like, or tomato-like. Without being bound by any particular theory, approximately ten volatile aldehydes are believed to be associated with undesirable wort-related off-flavors in beer (Gernat, DC, Brouwer, E. & Ottens, M. Aldehydes as Wort Off-Flavors in Alcohol-Free Beers—Origin and Control. Food Bioprocess Technol. 13, 195–216 (2019); Piornos, JA et al. Elucidating the Odor-Active Aroma Compounds in Alcohol-Free Beer and Their Contribution to the Worty Flavor. J. Agric. Food Chem. (2020) doi:10.1021 / acs.jafc.0c03902). In some embodiments, wort-related off-flavors include aldehydes and non-aldehyde molecules. Examples of aldehydes and non-aldehyde molecules that cause wort-related off-flavors include, but are not limited to, 2-methylbutanal, 2-methylpropanal, hexanal, benzaldehyde, furfural, acetaldehyde, methylthionaldehyde, phenylacetaldehyde, or 5-hydroxymethylfurfural, (E)-β-damascone, or 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone.

[0046] Maltose and / or maltotriose-negative (MN) yeast

[0047] For most yeast strains, the ability to consume maltose and maltotriose is conferred by the expression of proteins that: 1) transport maltose and maltotriose molecules into the cell, and 2) hydrolyze the glycosidic bonds linking the individual glucose monomers within these disaccharides and trisaccharides (Needleman, RB Control of Maltase Synthesis in Yeast. (1975)). The result of these transport and hydrolytic activities is the release of glucose, which can then enter the yeast glycolysis pathway and be fermented. Most yeast strains in the genus *Saccharomyces* express transport proteins and glucosidase proteins that perform both functions, and therefore most *Saccharomyces* yeasts are able to metabolize maltose and, to a slightly lower degree, maltotriose (Gallone, B. et al. Domestication and Divergence of *Saccharomyces cerevisiae* Beer Yeasts. Cell 166, 1397–1410.e16 (2016); Warringer, J. et al. Trait variation in yeast is defined by population history. PLoS Genet. 7, e1002111 (2011)). In fact, due to centuries of use in brewing, brewing yeast strains are particularly adept at consuming maltose and maltotriose. During this time, these strains have acquired adaptations—typically the replication of maltose transporter genes and glucosidase genes—that enable them to rapidly ferment maltose and maltotriose in brewing wort (Gallone, B. et al. Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts. Cell166, 1397–1410.e16 (2016)).

[0048] Because most yeast strains, and almost all yeast strains used in brewing, are capable of consuming maltose and maltotriose, brewers have very few brewing yeast strains available that cannot ferment maltose and / or maltotriose into ethanol when using stop fermentation to produce non-alcoholic beer. This scarcity of maltose-negative brewing strains presents a significant challenge for brewers, as non-yeast yeasts, and even non-brewing yeast strains, possess characteristics that make them unsuitable for beer fermentation, such as poor flocculation activity and the production of undesirable flavor molecules. Although there has been some interest in screening non-brewing yeast strains to identify potential strains that are both MN and suitable for beer production, few promising strains have been identified (Yabaci Karaoglan, S., Jung, R., Gauthier, M., Kinčl, T. & Dostálek, P. Maltose-Negative Yeast in Non-Alcoholic and Low-Alcoholic Beer Production. Fermentation 8, 273 (2022)). Therefore, most current methods for stopping fermentation in non-alcoholic beer production using maltose-negative yeasts rely on non-brewing strains that possess flavor or performance characteristics that negatively impact the final beer quality (Yabaci Karaoglan, S., Jung, R., Gauthier, M., Kinčl, T. & Dostálek, P. Maltose-Negative Yeast in Non-Alcoholic and Low-Alcoholic Beer Production. Fermentation 8, 273 (2022); Simões, J. et al. Exploiting Non-Conventional Yeasts for Low-Alcohol Beer Production. Microorganisms 11, (2023)).

[0049] In some embodiments, the modified cells are naturally unable to convert maltose and / or maltotriose to ethanol (e.g., naturally maltose-negative strains). In some embodiments, the modified cells are unable to convert maltose and / or maltotriose to ethanol, or have a reduced conversion ability, because they have been mated with yeast strains that cannot convert maltose and / or maltotriose to ethanol.

[0050] In some embodiments, genetically modified yeast cells that cannot convert maltose and / or maltotriose to ethanol or have a reduced conversion ability contain a heterologous nucleic acid encoding an enzyme with acyltransferase (EC 2.3.1.84) activity. As used herein, “genetically modified yeast cell” and “modified cell” are used interchangeably. As used herein, a reduced ability to convert maltose and / or maltotriose to ethanol means a reduction in the conversion of maltose and / or maltotriose to ethanol. By way of example and not limitation, this reduced ability can be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more of the yeast cell strain from which the genetically modified yeast cell is derived, compared to the genetically modified yeast cell fermented under the same conditions. In some embodiments, the genetically modified yeast cell cannot convert maltose and / or maltotriose to ethanol.

[0051] In some embodiments, the modified cell contains one or more gene modifications to functionally disrupt one or more proteins associated with the transport of maltose and / or maltotriose into the modified cell. Genes encoding these maltose transporters may include any or more of MAL31, MAL11, MPH2, MPH3, MAL21, MAL41, MA61, and AGT1 (PMIDs 9925567, 16332759). Although the nucleotide sequences of any of these genes may differ in different yeast strains, those skilled in the art can identify these genes by sequence homology searches using well-characterized reference sequences as a query. In some embodiments, the modified cell contains one or more gene modifications to functionally disrupt one or more proteins associated with the hydrolysis of maltose and / or maltotriose by the modified cell. Genes encoding proteins involved in maltose hydrolysis include MAL11, MAL12, MAL13, MAL14, MAL16, and STA1 (PMIDs 11598808, 31346683). Although the nucleotide sequences of any of these genes may differ in different yeast strains, those skilled in the art can identify these genes by sequence homology searches using well-characterized reference sequences as a lookup. In some embodiments, the modified cells contain one or more gene modifications to functionally disrupt maltose and / or maltotriose transporters. In some embodiments, the modified cells are genetically modified to lack MAL31 and MAL11 (AGT1). Methods for functionally disrupting genes in yeast cells are known in the art.

[0052] The amino acid sequence of MAL31 (a maltose transporter from Saccharomyces cerevisiae) is provided by the amino acid sequence shown in SEQ ID NO: 1.

[0053] MKGLSSLINRKKDRNDSHLDEIENGVNATEFNSIEMEEQGKKSDFDLSHLEYGPGSLIPNDNNEEVPDLLDEAMQDAKEADESERGMPLMTALKTYPKAAAWSLLVSTTLIQEGYDTAILGAFYALPVFQKKYGSLNSNTGDYEISVSWQIGLCLCYMAGEIVGLQMTGPSVDYMGNRYTLIMALFFLAAFIFILYFCKSLGMIAVGQALCGMPWGCFQCLTVSYASEICPLALRYYLTTYSNLCWAFGQLFAAGIMKNSQNKYANSELGYKLPFALQWIWPLPLAVGIFFAPESPWWLVKKGRIDQARRSLERTLSGKGPEKELLVSMELDKIKTTIEKEQKMSDEGTYWDCVKDGINRRRTRIACLCWIGQCSCGASLIGYSTYFYEKAGVSTDTAFTFSIIQYCLGIAATFISWWASKYCGRFDLYAFGLAFQAIMFFIIGGLGCSDTHGAKMGSGALLMVVAFFYNLGIAPVVFCLVSEIPSSRLRTKTIILARNAYNVIQVVVTVLIMYQLNSEKWNWGAKSGFFWGGFCLATLAWAVVDLPETAGRTFIEINELFRLGVPARKFKSTKVDPFAAAKAAAAEINVKDPKEDLETSVVDEGRNTSSVVNK

[0054] The nucleic acid sequence of MAL31 is provided by the nucleotide sequence shown in SEQ ID NO: 2.

[0055]

[0056] The amino acid sequence of AGT1 (also known as MAL11, a maltose transporter from Saccharomyces cerevisiae) is provided by the amino acid sequence shown in SEQ ID NO: 3.

[0057] MKNIISLVSKKKAASKNEDKNISESSRDIVNQQEVFNTENFEEGKKDSAFELDHLEFTTNSAQLGDSDEDNENVINETNTTDDANEANSEEKSMTLKQALLIYPKAALWSILVSTTLVMEGYDTALLNALYALPVFQRKFGTLNGEGSYEITSQ WQIGLNMCVQCGEIIGLQITPYMVEFMGNRYTMITALGLLTAYVFILYYCKSLAMIAVGQVLSAMPWGCFQGLTVTYASEVCPLALRYYMTSYSNICWLFGQIFASGIMKNSQENLGNSDLGYKLPFALQWIWPAPLMIGIFFAPESPWWLVRK DRVAEARKSLSRILSGKGAEKDIQIDLTLKQIELTIEKERLLASKSGSFFDCFKGVNGRRTRLACLAWVAQNTSGACLLGYSTYFFERAGMATDKAFTFSVIQYCLGLAGTLCSWVISGRVGRWTILTYGLAFQMVCLFVIGGMGFGSGSGASN GAGGLLLALSFFYNAGIGAVVYCIVTEIPSAELRTKTIVLARICYNIMAVINAILTPYMLNVSDWNWGAKTGLYWGGFTAVTLAWVIIDLPETSGRTFSEINELFNQGVPARKFASTVVDPFGKGKTQHDSLADESISQSSSIKQRELNAADKC

[0058] The nucleic acid sequence of AGT1 is provided by the nucleotide sequence shown in SEQ ID NO: 4.

[0059]

[0060] Production of acetates and / or ethyl acetates

[0061] Various aspects of this disclosure relate to modified cells containing genetic modifications that, compared to cells without genetic modifications, reduce the sensory detection of one or more wort-related off-flavors, thereby resulting in an increase in the production of one or more acetates and / or ethyl acetates.

[0062] The wort flavor in stopped-fermentation beer is a result of the combination of aldehydes and non-aldehydes. These aldehydes are present in the unfermented wort, and in normal alcoholic fermentation, yeast either converts them into other non-flavor-contributing molecules or significantly alters the beer's chemistry by producing ethanol and other flavor molecules, rendering these aldehydes imperceptible. In contrast, in stopped-fermentation using maltose-negative strains, it is believed that one or both of these processes occur absent or at reduced levels, thus aldehydes are present and readily perceived as off-flavors in the final beer.

[0063] Esters typically add fresh fruity aromas to beverages. Ethyl acetate, isoamyl acetate, and phenethyl acetate are three acetates commonly produced by yeast during typical beer fermentation and are considered major contributors to the "beer-like" aromas and flavors of alcoholic beers. However, in excess, they can also lead to undesirable off-flavors. During aborted fermentation, the production of these acetates is much lower (i.e., insufficient) due to reduced wort decay and decreased yeast metabolic activity, thus exacerbating the presence and perception of aldehyde- and non-aldehyde-based off-flavors.

[0064] Therefore, this article also provides genetically modified yeast cell compositions and methods for genetically modifying yeast cells to produce balanced volatile ester characteristics, which are essential for achieving the sensory properties required for NA beverages, even in aborted fermentation where the metabolic activity is reduced relative to that of normal alcohol-fermenting yeast.

[0065] Non-limiting examples of acetates that can reduce the sensory detection of one or more wort-related off-flavors include ethyl acetate, isoamyl acetate, phenethyl acetate, and hexyl acetate. Non-limiting examples of ethyl acetates that can reduce the sensory detection of one or more wort-related off-flavors include ethyl hexanoate, ethyl octanoate, and ethyl decanoate.

[0066] In some embodiments, the modified cells overexpress an enzyme with alcohol acyltransferase (AAT) activity. In some embodiments, the modified cells express a heterologous gene encoding an enzyme with alcohol acyltransferase (AAT) activity. As used herein, the term "heterologous gene" refers to a nucleic acid (e.g., DNA) sequence containing genetic instructions that is introduced into and expressed by a host organism (e.g., a gene-modified cell) that does not naturally encode the introduced gene. A heterologous gene may encode an enzyme that the cell does not normally express, a variant of an enzyme that the cell does not normally express (e.g., a mutant enzyme), an extra copy of an enzyme that the cell normally expresses, or a gene that the cell normally expresses but is differently regulated. In some embodiments, the modified cells express an endogenous gene at an increased level compared to expression in unmodified corresponding cells. In some embodiments, the heterologous gene encoding an enzyme with glycosidase activity is a wild-type (naturally occurring) AAT (e.g., a gene isolated from an organism). As used herein, the term “heterologous nucleic acid” means a nucleic acid that meets at least one of the following criteria: (a) the nucleic acid is foreign (“exogenous”) to a given host cell (i.e., not naturally present in a given host cell); (b) the nucleic acid contains a nucleotide sequence naturally present in a given host cell (i.e., “endogenous”) but the amount of the nucleotide sequence in the cell is non-natural (e.g., higher than expected or higher than naturally present); (c) the nucleic acid contains a nucleotide sequence that is different from the endogenous nucleotide sequence, but that nucleotide sequence encodes the same protein (having the same or substantially the same amino acid sequence), and the amount in the cell is non-natural (e.g., higher than expected or higher than naturally present); or (d) the nucleic acid contains two or more nucleotide sequences that do not exist in nature in the same relationship to each other (e.g., the nucleic acid is recombinant).

[0067] In some embodiments, the enzyme with AAT activity is obtained from yeast. In some embodiments, the enzyme with AAT activity is obtained from bacteria. In some embodiments, the enzyme with AAT activity is obtained from plants. In some embodiments, the enzyme with AAT activity is obtained from yeast or fungi.

[0068] In some implementations, the enzyme with AAT activity is derived from Saccharomyces cerevisiae.

[0069] In some embodiments, compared to cells not containing heterologous nucleic acids, genetically modified yeast cells produce an increased amount of one or more acetates and / or ethyl acetates. In some embodiments, compared to cells not containing heterologous nucleic acids, genetically modified yeast cells produce an increased amount of one or more acetates and one or more ethyl acetates. In some embodiments, compared to cells not containing heterologous nucleic acids, genetically modified yeast cells produce an increased amount of one or more acetates and one or more ethyl acetates.

[0070] In some embodiments, one or more acetate esters are selected from the group consisting of ethyl acetate, isoamyl acetate, phenethyl acetate, hexyl acetate, and combinations thereof. In some embodiments, one or more acetate esters comprise ethyl acetate, isoamyl acetate, and phenethyl acetate. In some embodiments, one or more acetate esters comprise isoamyl acetate and phenethyl acetate. In some embodiments, one or more acetate esters comprise isoamyl acetate. In some embodiments, one or more acetate esters comprise phenethyl acetate.

[0071] In some embodiments, one or more ethyl esters are selected from the group consisting of ethyl hexanoate, ethyl octanoate, ethyl decanoate, and combinations thereof. In some embodiments, one or more ethyl esters comprise ethyl hexanoate, ethyl octanoate, and ethyl decanoate. In some embodiments, one or more ethyl esters comprise ethyl hexanoate and ethyl octanoate. In some embodiments, one or more ethyl esters comprise ethyl hexanoate and ethyl decanoate. In some embodiments, one or more ethyl esters comprise ethyl hexanoate. In some embodiments, one or more ethyl esters comprise ethyl octanoate. In some embodiments, one or more ethyl esters comprise ethyl decanoate.

[0072] An exemplary enzyme with AAT activity is ATF1 from Saccharomyces cerevisiae. Saccharomyces cerevisiae ATF1 is provided by the amino acid sequence shown in SEQ ID NO: 5.

[0073] MNEIDEKNQAPVQQECLKEMIQNGHARRMGSVEDLYVALNRQNLYRNFCTYGELSDYCTRDQLTLALREICLKNPTLLHIVLPTRWPNHENYYRSSEYYSRPHPVHDYISVLQELKLSGVVLNEQPEYSAVMKQILEEFKNSKGSYTAKIFKLTTTLTIPYFGPTGPSWRLICLPEEHTEKWKKFIFVSNHCMSDGRSSIHFFHDLRDELNNIKTPPKKLDYIFKYEEDYQLLRKLPEPIEKVIDFRPPYLFIPKSLLSGFIYNHLRFSSKGVCMRMDDVEKTDDVVTEIINISPTEFQAIKANIKSNIQGKCTITPFLHVCWFVSLHKWGKFFKPLNFEWLTDIFIPADCRSQLPDDDEMRQMYRYGANVGFIDFTPWISEFDMNDNKENFWPLIEHYHEVISEALRNKKHLHGLGFNIQGFVQKYVNIDKVMCDRAIGKRRGGTLLSNVGLFNQLEEPDAKYSICDLAFGQFQGSWHQAFSLGVCSTNVKGMNIVVASTKNVVGSQESLEELCSIYKALLLGP

[0074] An exemplary nucleic acid sequence of ATF1 is provided by SEQ ID NO: 6.

[0075]

[0076] An exemplary enzyme with AAT activity is AAT1 from melon (Cucumis melo). Melon AAT1 is provided by the amino acid sequence shown in SEQ ID NO: 7.

[0077] MGETMQTIDFSFHVRKCQPELIAPANPTPYEFKQLSDVDDQQSLRLQLPFVNIYPHNPSLEGRDPVKVIKEAIGKALVFYYPLAGRLREGPGRKLFVECTGEGILFIEADADVSLE EFWDTLPYSLSSMQNNIIHNALNSDEVLNSPLLLIQVTRLKCGGFIFGLCFNHTMADGFGIVQFMKATAEIARGAFAPSILPVWQRALLTARDPPRITFRHYEYDQVVDMKSGLIP VNSKIDQLFFFSQLQISTLRQTLPAHLHDCPSFEVLTAYVWRLRTIALQFKPEEEVRFLCVMNLRSKIDIPLGYYGNAVVVPAVITTAAKLCGNPLGYAVDLIRKAKAKATMEYIK STVDLMVIKGRPYFTVVGSFMMSDLTRIGVENVDFGWGKAIFGGPTTTTGARITRGLVSFCVPFMNRNGEKGTALSLCLPPPAMERFRANVHASLQVKQVVDAVDSHMQTIQSASKGS

[0078] An exemplary nucleic acid sequence of melon AAT1 is provided by SEQ ID NO: 8.

[0079]

[0080] Other examples of enzymes with AAT activity suitable for the compositions and methods provided herein include, but are not limited to, those described in the GenBank accession numbers: NP_001315675.1 (SEQ ID NO: 36), ABO21021.1 (SEQ ID NO: 37), WP_011783747.1 (SEQ ID NO: 38), XP_008462821.2 (SEQ ID NO: 39), EGA72844.1 (SEQ ID NO: 40), NP_011693.1 (SEQ ID NO: 41), NP_011529.1 (SEQ ID NO: 42), ADD16960.1 (SEQ ID NO: 43), AHY74654.1 (SEQ ID NO: 44), NP_001310384.1 (SEQ ID NO: 45), ACT82247.1 (SEQ ID NO: 46). 46), AAG13130.1 (SEQ ID NO: 47), NP_001315389.1 (SEQ ID NO: 48), NP_001295454.1 (SEQ ID NO: 49), AAW31948.1 (SEQ ID NO: 50), XP_007209131.1 (SEQ ID NO: 51), A0A2R6Q326.2 (SEQ ID NO: 52), CAB4309439.1 (SEQ ID NO: 53), P0DO25.1 (SEQ ID NO: 54), WP_004922247.1 (SEQ ID NO: 55) and WP_004920769.1 (SEQ ID NO: 56).

[0081] In some embodiments, cells expressing AAT enzymes are able to increase the production of acetate and / or ethyl acetate compared to cells that do not express AAT or express AAT at low levels (e.g., unmodified cells). In some embodiments, cells expressing AAT enzymes are able to increase the production of acetate and / or ethyl acetate in fermentation products compared to fermentation products produced without using modified cells.

[0082] In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in any one of SEQ ID NO: 5, 7, and 36-56.

[0083] In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 5. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 7. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 36. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 37. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 38.In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 39. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 40. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 41. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 42. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 43.In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 44. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 45. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 46. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 47. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 48.In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 49. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 50. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 51. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 52. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 53.In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 54. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 55. In some embodiments, the enzyme having AAT activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 56.

[0084] The terms “percentage identity,” “sequence identity,” “% identity,” “% sequence identity,” and “% identical” are used interchangeably in this document and refer to a quantitative measure of similarity between two sequences (e.g., nucleic acids or amino acids). Percentage identity can be determined using the algorithms of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified from Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. These algorithms were incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the target protein molecule. When there is a gap between two sequences, the Gapped BLAST described in Altschul et al., NucleicAcids Res. 25(17):3389-3402, 1997, can be used. When using the BLAST and GappedBLAST procedures, the default parameters of the corresponding procedures (e.g., XBLAST and NBLAST) can be used.

[0085] When stating percentage identity or its range (e.g., at least, more than, etc.), unless otherwise specified, the endpoints shall be included, and the range (e.g., at least 70% identity) shall include all ranges within the referenced range (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, to...). The identity of at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% and all its increments (e.g., one-tenth of a percent (i.e., 0.1%), one-hundredth of a percent (i.e., 0.01%), etc.).

[0086] In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in any one of SEQ ID NO: 5, 7, or 36-56. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 37. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 38. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 41. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 43. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 44. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 45. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 46. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 47. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 48. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 51. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 52. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 53. In some embodiments, the enzyme having AAT activity comprises the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, the enzyme having AAT activity comprises the amino acid sequence shown in SEQ ID NO: 55.In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 56. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence shown in SEQ ID NO: 5 or 7. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in any one of SEQ ID NO: 5, 7, or 36-56. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 37. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 38. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 41. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 43. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 44. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 45. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 46. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 47. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 48. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 51. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 52. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 53.In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 55. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 56. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence shown in SEQ ID NO: 5 or 7.

[0087] In some embodiments, the gene encoding an enzyme with AAT activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to the sequence shown in any one of SEQ ID NO: 5, 7, or 36-56. In some embodiments, the gene encoding an enzyme with AAT activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising an amino acid sequence shown in any one of SEQ ID NO: 5, 7, or 36-56.

[0088] In some embodiments, the gene encoding an enzyme with AAT activity comprises a nucleic acid sequence that is at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to the sequence shown in SEQ ID NO: 6 or 8. In some embodiments, the gene encoding an enzyme with AAT activity comprises a nucleic acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 6 or 8.

[0089] Other enzymes possessing or predicted to possess AAT activity can be identified, for example, based on similarity or homology to one or more domains of AAT (such as the AAT provided by any one of SEQ ID NO: 5, 7, and 36-56). In some embodiments, enzymes used in the modified cells and methods described herein can be identified based on similarity or homology to active domains (such as catalytic domains, such as catalytic domains associated with AAT activity). In some embodiments, enzymes used in the modified cells and methods described herein may have a relatively high level of sequence identity with a reference AAT (such as wild-type AAT, such as any one of SEQ ID NO: 5, 7, or 36-56) in the catalytic region, but a relatively low level of sequence identity with the reference AAT based on analysis of a larger portion or the full-length enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein has a catalytic domain region with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity relative to a reference AAT (e.g., SEQ ID NO: 5, 7, or 36-56).

[0090] In some embodiments, the enzymes used in the modified cells and methods described herein have a relatively high level of sequence identity with reference AAT (e.g., SEQ ID NO: 5, 7 or 36-56) in their catalytic domain region, and a relatively low level of sequence identity with reference AAT based on analysis of a larger portion or full-length portion of the enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein, based on a portion or the full-length enzyme, has at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity relative to a reference AAT (e.g., SEQ ID NO: 5, 7, or 36-56).

[0091] Production of 3-mercaptohexanol

[0092] Various aspects of this disclosure relate to modified cells containing genetic modifications that, compared to cells without such modifications, reduce the sensory detection of one or more wort-related off-flavors and result in increased production of 3-mercaptohexanol (3MH). 3MH is a volatile thiol molecule that is typically produced by yeast at very low levels (<50 ng / L) during beer fermentation.

[0093] In some embodiments, the modified cells further comprise a heterologous nucleic acid encoding an enzyme having carbon-sulfur lyase (EC 4.4) activity.

[0094] In some embodiments, modified cells overexpress an enzyme with carbon-sulfur lyase (CSL) activity. In some embodiments, modified cells express a heterologous gene encoding an enzyme with CSL activity. In some embodiments, any of the modified cells described herein are genetically modified to express a heterologous gene encoding an enzyme with CSL activity. In some embodiments, the heterologous gene encoding an enzyme with CSL activity is a wild-type CSL gene (e.g., a gene isolated from an organism). In some embodiments, CSL is obtained from bacteria, fungi, or plants. In some embodiments, CSL is obtained from bacteria.

[0095] In some embodiments, the enzyme having CSL activity is derived from *Citrobacter freundii*. The CSL derived from *Citrobacter freundii* is provided by the amino acid sequence shown in SEQ ID NO: 9.

[0096] MGDNFKHLPEPFRIRVIEPVKRTTREHRNNAIIKSGMNPFLLDSEDVFIDLLTDSGTGAVTQNMQAAMLRGDEAYSGSRSYYALSEAVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRSKMAVFSNYFFDTTQGHSQINGCAVRNVYIKEAFDTGVRYDFKGNFDLDGLERGIQEVGPNNVPYIVATITSNSAGGQPVSLANLKAMYNIAKKYDIPVVMDSARFAENAYFIQKREAEYRDWSIEEITRETYKYADMLAMSAKKDAMVPMGGLLCIKDDTYFDVYTECRTLCVVQEGFPTYGGLEGGAMERLAVGLVDGMNQDWLAYRIAQVQYLVDGLEAIGVTCQQAGGHAAFVDAGKLLPHIPAEQFPAQALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLTIPRATYTQSHMDFIIEAFEHVKENSMNIKGLTFTYEPKVLRFFTAKLKEV

[0097] An exemplary nucleic acid sequence of Citrobacter freundii (C. freundii) CSL is provided by SEQ ID NO: 10.

[0098]

[0099] Other examples of enzymes with CSL activity suitable for the compositions and methods provided herein include, but are not limited to, those referenced by GenBank accession numbers: WP_125339275.1 (SEQ ID NO: 57), WP_105310552.1 (SEQ ID NO: 58), WP_119633472.1 (SEQ ID NO: 59), EDN59205.1 (SEQ ID NO: 60), WP_094789495.1 (SEQ ID NO: 61), and XP_025427068.1 (SEQ ID NO: 62).

[0100] In some embodiments, the heterologous gene encodes an enzyme with CSL activity, enabling cells expressing the enzyme to increase the production of 3-mercaptohexanol (3MH). In some embodiments, the heterologous gene encodes an enzyme with CSL activity, enabling cells expressing the enzyme to increase 3MH production compared to cells not expressing the heterologous gene.

[0101] In some embodiments, the enzyme having CSL activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in any one of SEQ ID NO: 9 and 57-62. In some embodiments, the enzyme having CSL activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 9. In some embodiments, the enzyme having CSL activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 57. In some embodiments, the enzyme having CSL activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 58. In some embodiments, the enzyme having CSL activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 59.In some embodiments, the enzyme having CSL activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 60. In some embodiments, the enzyme having CSL activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 61. In some embodiments, the enzyme with CSL activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 62. In some embodiments, the enzyme with CSL activity comprises the amino acid sequence shown in any one of SEQ ID NO: 9 and 57-62. In some embodiments, the enzyme with CSL activity comprises the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the enzyme with CSL activity comprises the amino acid sequence shown in SEQ ID NO: 57. In some embodiments, the enzyme with CSL activity comprises the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, the enzyme with CSL activity comprises the amino acid sequence shown in SEQ ID NO: 59. In some embodiments, the enzyme with CSL activity comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the enzyme with CSL activity comprises the amino acid sequence shown in SEQ ID NO: 61. In some embodiments, the enzyme with CSL activity comprises the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the enzyme with CSL activity consists of the amino acid sequence shown in any one of SEQ ID NO: 9 and 57-62. In some embodiments, the enzyme with CSL activity consists of the amino acid sequence shown in SEQ ID NO: 9.In some embodiments, the enzyme with CSL activity consists of the amino acid sequence shown in SEQ ID NO: 57. In some embodiments, the enzyme with CSL activity consists of the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, the enzyme with CSL activity consists of the amino acid sequence shown in SEQ ID NO: 59. In some embodiments, the enzyme with CSL activity consists of the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the enzyme with CSL activity consists of the amino acid sequence shown in SEQ ID NO: 61. In some embodiments, the enzyme with CSL activity consists of the amino acid sequence shown in SEQ ID NO: 62.

[0102] In some embodiments, the gene encoding an enzyme with CSL activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to the sequence shown in any one of SEQ ID NO: 9 and 57-62. In some embodiments, the gene encoding an enzyme with CSL activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising an amino acid sequence shown in any one of SEQ ID NO: 9 and 57-62. In some embodiments, the gene encoding an enzyme with CSL activity comprises the nucleic acid sequence shown in SEQ ID NO: 10.

[0103] Other enzymes possessing or predicted to possess CSL activity can be identified, for example, based on similarity or homology to one or more domains of a CSL, such as the CSL provided by SEQ ID NO: 9 or 57-62. In some embodiments, enzymes used in the modified cells and methods described herein can be identified based on similarity or homology to active domains, such as catalytic domains, such as catalytic domains associated with CSL activity. In some embodiments, enzymes used in the modified cells and methods described herein may have a relatively high level of sequence identity with a reference CSL (e.g., wild-type CSL, such as SEQ ID NO: 9 or 57-62) in the catalytic domain region, but the level of sequence identity with the reference CSL may be relatively low based on analysis of a larger portion or the full-length enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity in its catalytic domain region relative to a reference CSL (e.g., SEQ ID NO: 9 or 57-62).

[0104] In some embodiments, the enzyme used in the modified cells and methods described herein has a relatively high level of sequence identity with a reference CSL (e.g., SEQ ID NO: 9 or 57-62) in its catalytic domain region, and a relatively low level of sequence identity with the reference CSL based on analysis of a larger portion or full-length portion of the enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein, based on a portion or the full-length enzyme, has at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity relative to a reference CSL (e.g., SEQ ID NO: 9 or 57-62).

[0105] Monoterpenoid production

[0106] Various aspects of this disclosure relate to genetically modified cells that, compared to cells without genetic modification, reduce the sensory detection of one or more wort-related off-flavors, resulting in increased production of one or more monoterpenes. In some embodiments, one or more monoterpenes include linalool, geraniol, and / or citronellol. Genetic modification of brewing yeast to produce monoterpenes such as linalool, geraniol, and citronellol has been described, for example in PCT Publication No. WO2017 / 100655A1, the entire contents of which are incorporated herein by reference.

[0107] In some embodiments, the modified cells are further genetically modified to produce one or more monoterpenes. In some embodiments, the monoterpenes are selected from the group consisting of linalool, geraniol, and citronellol.

[0108] In some embodiments, the modified cells express one or more of the following (e.g., 1, 2, 3, or 4): (a) a truncated variant of the yeast HMG1 enzyme; (b) a variant of the ERG20 enzyme; (c) linalool synthase; and / or (d) geraniol synthase. In some embodiments, the modified cells express (a) a truncated variant of the yeast HMG1 enzyme; (b) a variant of the ERG20 enzyme; (c) linalool synthase; and (d) geraniol synthase.

[0109] In some embodiments, the modified cell comprises a heterologous nucleic acid encoding an enzyme selected from the group consisting of: (a) a truncated variant of yeast HMG1 enzyme, (b) a variant of ERG20 enzyme or an enzyme having farnesyl diphosphate synthase activity, (c) linalool synthase, (d) geraniol synthase, and (e) combinations thereof. In some embodiments, the modified cell comprises a heterologous nucleic acid encoding: (a) a truncated variant of yeast HMG1 enzyme, (b) a variant of ERG20 enzyme or an enzyme having farnesyl diphosphate synthase activity, (c) linalool synthase, and (d) geraniol synthase.

[0110] In some embodiments, the modified cell comprises a heterologous nucleic acid encoding an enzyme selected from the group consisting of: (a) a truncated variant of yeast HMG1 enzyme, (b) a variant of ERG20 enzyme, (c) linalool synthase, (d) geraniol synthase, and (e) combinations thereof. In some embodiments, the modified cell comprises a heterologous nucleic acid encoding: (a) a truncated variant of yeast HMG1 enzyme, (b) a variant of ERG20 enzyme, (c) linalool synthase, and (d) geraniol synthase.

[0111] Enzymes with HMG-CoA reductase activity

[0112] In some embodiments, any of the modified cells described herein are genetically modified to express a heterologous gene encoding an enzyme with HMG-CoA reductase (3'-hydroxy-3-methylglutaryl-CoA reductase) activity. In some embodiments, the heterologous gene encoding the enzyme with HMG-CoA reductase activity is the wild-type HMG1 gene (e.g., a gene isolated from an organism). In some embodiments, the enzyme with HMG-CoA reductase activity is obtained from bacteria, fungi, or plants. In some embodiments, the enzyme with HMG-CoA reductase activity is obtained from yeast. In some embodiments, the enzyme with HMG-CoA reductase activity is a truncated variant of the enzyme with HMG-CoA reductase activity.

[0113] In some embodiments, the enzyme having HMG-CoA reductase activity is a truncated variant of the yeast HMG1 enzyme. As used herein, “truncated” in relation to HMG1 enzyme or HMG-CoA reductase means an enzyme that is truncated relative to the full-length enzyme sequence. For example, the truncated sequence may be about 5%, 10%, 20%, 30%, 40%, 50%, or more shorter than the full-length reference sequence. By further examples, and not limitation, the truncated enzyme may be about 500 amino acids shorter than the reference enzyme. In some embodiments, the truncation may be N-terminal truncation. As used herein, a “variant” enzyme is an enzyme that has a different sequence from the original sequence, is at least 85%, 90%, 95%, 98%, or 99% sequencely identical to the reference sequence, but retains the functional characteristics of the reference enzyme, although the activity level may be increased or decreased.

[0114] The truncated HMG-CoA reductase (referred to as tHMG) is provided by the amino acid sequence shown in SEQ ID NO: 11.

[0115] DQLVKTEVTKKSFTAPVQKASTPVLTNKTVISGSKVKSLSSAQSSSSGPSSSSEEDDSRDIESLDKKIRPLEELEALLSSGNTKQLKNKEVAALVIHGKLPLYALEKKLGDTTRAVAVRRKALSILAEAPVLASDRLPYKNYDYDRVFGACCENVIGYMPLPVGVIGPLVIDGTSYHIPMATTEGCLVASAMRGCKAINAGGGATTVLTKDGMTRGPVVRFPTLKRSGACKIWLDSEEGQNAIKKAFNSTSRFARLQHIQTCLAGDLLFMRFRTTTGDAMGMNMISKGVEYSLKQMVEEYGWEDMEVVSVSGNYCTDKKPAAINWIEGRGKSVVAEATIPGDVVRKVLKSDVSALVELNIAKNLVGSAMAGSVGGFNAHAANLVTAVFLALGQDPAQNVESSNCITLMKEVDGDLRISVSMPSIEVGTIGGGTVLEPQGAMLDLLGVRGPHATAPGTNARQLARIVACAVLAGELSLCAALAAGHLVQSHMTHNRKPAEPTKPNNLDATDINRLKDGSVTCIKS

[0116] An exemplary nucleic acid sequence of the truncated HMG-CoA reductase is provided by SEQ ID NO: 12.

[0117]

[0118] In some embodiments, the heterologous gene encodes an enzyme with HMG-CoA reductase activity, enabling cells expressing the enzyme to increase the production of one or more monoterpenes. In some embodiments, the heterologous gene encodes an enzyme with HMG-CoA reductase activity, enabling cells expressing the enzyme to increase the production of one or more monoterpenes compared to cells not expressing the heterologous gene.

[0119] In some embodiments, the enzyme having HMG-CoA reductase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 11. In some embodiments, the enzyme having HMG-CoA reductase activity comprises the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the enzyme having HMG-CoA reductase activity consists of the amino acid sequence shown in SEQ ID NO: 11.

[0120] In some embodiments, the gene encoding an enzyme having HMG-CoA reductase activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to the sequence shown in SEQ ID NO: 11. In some embodiments, the gene encoding an enzyme having HMG-CoA reductase activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the gene encoding an enzyme having HMG-CoA reductase activity comprises the nucleic acid sequence shown in SEQ ID NO: 12.

[0121] Other enzymes possessing HMG-CoA reductase activity or predicted to possess HMG-CoA reductase activity can be identified, for example, based on similarity or homology to one or more domains of an HMG-CoA reductase (such as the HMG-CoA reductase provided by SEQ ID NO: 11). In some embodiments, enzymes used in the modified cells and methods described herein can be identified based on similarity or homology to active domains, such as catalytic domains, such as catalytic domains associated with HMG-CoA reductase activity. In some embodiments, enzymes used in the modified cells and methods described herein may have a relatively high level of sequence identity with a reference HMG-CoA reductase, such as a wild-type HMG-CoA reductase, in its catalytic domain region, but a relatively low level of sequence identity with the reference HMG-CoA reductase based on analysis of a larger portion or the full-length enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity in its catalytic domain region relative to a reference HMG-CoA reductase.

[0122] In some embodiments, the enzyme used in the modified cells and methods described herein has a relatively high level of sequence identity in its catalytic domain region relative to a reference HMG-CoA reductase (e.g., SEQ ID NO: 11), and a relatively low level of sequence identity relative to a reference HMG-CoA reductase based on analysis of a larger portion or full-length portion of the enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein, based on a portion or the full length of the enzyme, has at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity relative to a reference HMG-CoA reductase (e.g., SEQ ID NO: 11).

[0123] Enzymes with farnesyl diphosphate synthase (FPPS) activity

[0124] In some embodiments, any of the modified cells described herein are genetically modified to express a heterologous gene encoding an enzyme having farnesyl diphosphate synthase activity. In some embodiments, the heterologous gene encoding an enzyme having farnesyl diphosphate synthase activity is a wild-type farnesyl diphosphate synthase gene (e.g., a gene isolated from an organism). In some embodiments, the enzyme having farnesyl diphosphate synthase reductase activity is obtained from bacteria, fungi, or plants. In some embodiments, the enzyme having farnesyl diphosphate synthase reductase activity is obtained from yeast. In some embodiments, the enzyme having farnesyl diphosphate synthase activity is a variant of an enzyme having farnesyl diphosphate synthase activity, such as a mutant containing one or more amino acid substitutions at any position of F96, N127, and / or K197.

[0125] In some embodiments, the enzyme having farnesyl diphosphate synthase activity is a variant of farnesyl diphosphate synthase with substitution mutations (e.g., F96W and N127W) at the F96 and N127 positions. Farnesyl diphosphate synthase is provided by the amino acid sequence shown in SEQ ID NO:13, and is designated ERG20 (F96W, N127W).

[0126] MASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEACDWYAHSLNYNTPGGKLNRGLSVVDTYAILSNKTVEQLGQEEYEKVAILGWCIELLQAYWLVADDMMDKSITRRGQPCWYKVPEVGEIAIWDAFMLEAAIYKLLKSHFRNEKYYIDITELFHEVTFQTELGQLMDLITAP EDKVDLSKFSLKKHSFIVTFKTAYYSFYLPVALAMYVAGITDEKDLKQARDVLIPLGEYFQIQDDYLDCFGTPEQIGKIGTDIQDNKCSWVINKALELASAEQRKTLDENYGKKDSVAEAKCKKIFNDLKIEQLYHEYEESIAKDLKAKISQVDESRGFKADVLTAFLNKVYKRSK

[0127] An exemplary nucleic acid sequence of farnesyl diphosphate synthase (ERG20 (F96W, N127W)) is provided by SEQ ID NO: 14.

[0128]

[0129] In some embodiments, the heterologous gene encodes an enzyme with farnesyl diphosphate synthase activity, enabling cells expressing the enzyme to increase the production of one or more monoterpenes. In some embodiments, the heterologous gene encodes an enzyme with farnesyl diphosphate synthase activity, enabling cells expressing the enzyme to increase the production of one or more monoterpenes compared to cells not expressing the heterologous gene.

[0130] In some embodiments, the enzyme having farnesyl diphosphate synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 13. In some embodiments, the enzyme having farnesyl diphosphate synthase activity comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the enzyme having farnesyl diphosphate synthase activity consists of the amino acid sequence shown in SEQ ID NO: 13.

[0131] In some embodiments, the gene encoding an enzyme having farnesyl diphosphate synthase activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) identical to the sequence shown in SEQ ID NO: 13. In some embodiments, the gene encoding an enzyme having farnesyl diphosphate synthase activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the gene encoding an enzyme having farnesyl diphosphate synthase activity comprises the nucleic acid sequence shown in SEQ ID NO: 14.

[0132] Other enzymes possessing or predicted to possess farnesyl diphosphate synthase activity can be identified, for example, based on similarity or homology to one or more domains of a farnesyl diphosphate synthase (such as the farnesyl diphosphate synthase provided by SEQ ID NO: 13). In some embodiments, enzymes used in the modified cells and methods described herein can be identified based on similarity or homology to active domains, such as catalytic domains, such as those associated with farnesyl diphosphate synthase activity. In some embodiments, enzymes used in the modified cells and methods described herein may have a relatively high level of sequence identity with a reference farnesyl diphosphate synthase, e.g., in the catalytic domain region, but a relatively low level of sequence identity with the reference farnesyl diphosphate synthase based on analysis of a larger portion or the full-length enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity in its catalytic domain region relative to a reference farnesyl diphosphate synthase (e.g., SEQ ID NO: 13).

[0133] In some embodiments, the enzyme used in the modified cells and methods described herein has a relatively high level of sequence identity in its catalytic domain region relative to a reference farnesyl diphosphate synthase (e.g., SEQ ID NO: 13), and a relatively low level of sequence identity relative to the reference farnesyl diphosphate synthase based on analysis of a larger portion or full-length portion of the enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein, based on a portion or the full-length enzyme, has at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity relative to a reference farnesyl diphosphate synthase (e.g., SEQ ID NO: 13).

[0134] Enzymes with linalool synthase activity

[0135] In some embodiments, any of the modified cells described herein are genetically modified to express a heterologous gene encoding an enzyme with linalool synthase activity. In some embodiments, the heterologous gene encoding the enzyme with linalool synthase activity is a wild-type linalool synthase gene (e.g., a gene isolated from an organism). In some embodiments, the enzyme with linalool synthase activity is obtained from bacteria, fungi, or plants. In some embodiments, the enzyme with linalool synthase activity is obtained from yeast.

[0136] In some embodiments, the enzyme having linalool synthase activity is derived from lemon mint (Mentha citrate) (referred to as McLIS). Linalool synthase is provided by the amino acid sequence shown in SEQ ID NO: 15.

[0137] MTRRSGNYHPSVWDFDFIQSLDTDHYKEEKQLEREEELIMEVKKLLGAKMEATKQLELIDDLQNLGLSYFFRDEIKNILNSIYKIFQNNNSTKVGDLHFTSLGFRLLRQHGFNVSQGVFDCFKNEHGSDFEKTLI GEDTKGVLQLYEASFLLREGEDTLEVARKFSTEFLEEKLKAGIDGDNLSSSIGHSLEIPLHWRIQRLEERWFLDAYSRRKDMNPIIFELAKLDFNIIQATQQEELKDLSRWWNDSSLPQKLPFVRDRLVESYYWA LGLFEAHKFGYERKTAAKIITLITALDDVYDIYGTLDELQLFTHVIRRWDTESATQLPYYLQLFYFVLYNFVSEVAYHILKEEGFISIPFLHRAWVDLVEGYLQEAKWYYTKYTPTMEEYLNYASITIGAPAVIS QIYFMLAKSKEKPVIESFYEYDEIIRLSGMLVRLPDDLGTLPFEMKRGDVAKSIQIYMKEQNATREEAEEHVRFMIREAWKEMNTTMAANSDLRGDVVMAAANLGRDAQFMYLDGDGNHSQLQHRIANLLFKPYV

[0138] An exemplary nucleic acid sequence of linalool synthase is provided by SEQ ID NO: 16.

[0139]

[0140] Other examples of enzymes with linalool synthase activity suitable for the compositions and methods provided herein include, but are not limited to, those referenced in GenBank accessions: Q8H2B4.1 (SEQ ID NO: 63), Q6ZH94 (SEQ ID NO: 64), Q96376 (SEQ ID NO: 65), MN954676 (SEQ ID NO: 66), Q84ZW8.2 (SEQ ID NO: 67), and A0A348B793.1 (SEQ ID NO: 68).

[0141] In some embodiments, the heterologous gene encodes an enzyme with linalool synthase activity, enabling cells expressing the enzyme to increase the production of one or more monoterpenes. In some embodiments, the heterologous gene encodes an enzyme with linalool synthase activity, enabling cells expressing the enzyme to increase the production of one or more monoterpenes compared to cells not expressing the heterologous gene.

[0142] In some embodiments, the enzyme having linalool synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence selected from the group consisting of SEQ ID NO: 15 and 63-68. In some embodiments, the enzyme having linalool synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 15. In some embodiments, the enzyme having linalool synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 63. In some embodiments, the enzyme having linalool synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 64. In some embodiments, the enzyme having linalool synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 65.In some embodiments, the enzyme having linalool synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 66. In some embodiments, the enzyme having linalool synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 67. In some embodiments, the enzyme having linalool synthase activity comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity with the sequence shown in SEQ ID NO: 68. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in any one of SEQ ID NO: 15 and 63-68. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 63. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 64. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 65. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 66. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 67. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 68. In some embodiments, the enzyme having linalool synthase activity consists of the amino acid sequence shown in any one of SEQ ID NO: 15 and 63-68.In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 63. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 64. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 65. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 66. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 67. In some embodiments, the enzyme having linalool synthase activity comprises the amino acid sequence shown in SEQ ID NO: 68.

[0143] In some embodiments, the gene encoding an enzyme having linalool synthase activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising an amino acid sequence having at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) sequence identity with the sequence shown in any one of SEQ ID NO: 15 and 63-68. In some embodiments, the gene encoding an enzyme having linalool synthase activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising an amino acid sequence shown in any one of SEQ ID NO: 15 and 63-68. In some embodiments, the gene encoding an enzyme having linalool synthase activity comprises the nucleic acid sequence shown in SEQ ID NO: 16.

[0144] Other enzymes possessing linalool synthase activity or predicted to possess farnesyl diphosphate synthase activity can be identified, for example, based on similarity or homology to one or more domains of linalool synthase (e.g., linalool synthase provided by any one of SEQ ID NO: 15 and 63-68). In some embodiments, enzymes used in the modified cells and methods described herein can be identified based on similarity or homology to active domains, such as catalytic domains, such as catalytic domains associated with linalool synthase activity. In some embodiments, enzymes used in the modified cells and methods described herein may have a relatively high level of sequence identity with a reference linalool synthase, e.g., in the catalytic domain region, but a relatively low level of sequence identity with the reference linalool synthase based on analysis of a larger portion or the full-length enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity in its catalytic domain region relative to a reference linalool synthase (e.g., SEQ ID NO: 15 or 63-68).

[0145] In some embodiments, the enzyme used in the modified cells and methods described herein has a relatively high level of sequence identity in its catalytic domain region relative to a reference linalool synthase (e.g., SEQ ID NO: 15 or 63-68), and a relatively low level of sequence identity relative to the reference linalool synthase based on analysis of a larger portion or full-length portion of the enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein, based on a portion or the full-length enzyme, has at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity relative to a reference linalool synthase (e.g., SEQ ID NO: 15 or 63-68).

[0146] Enzymes with geraniol synthase activity

[0147] In some embodiments, any of the modified cells described herein are genetically modified to express a heterologous gene encoding an enzyme with geraniol synthase activity. In some embodiments, the heterologous gene encoding an enzyme with geraniol synthase activity is a wild-type geraniol synthase gene (e.g., a gene derived from an organism). In some embodiments, the enzyme with geraniol synthase activity is obtained from bacteria, fungi, or plants. In some embodiments, the enzyme with geraniol synthase activity is obtained from yeast.

[0148] In some embodiments, the enzyme having geraniol synthase activity is derived from basil (Ocimu basilicum) (referred to as ObGES). Geraniol synthase is provided by the amino acid sequence shown in SEQ ID NO: 17.

[0149] MQHMEESSSKRREYLLEETTRKLQRNDTESVEKLKLIDNIQQLGIGYYFEDAINAVLRSPFSTGEEDLFTAALRFRLLRHNGIEISPEIFLKFKDERGKFDESDTLGLLSLYEASNLGVAGEEILE EAMEFAEARLRRSLSEPAAPLHGEVAQALDVPRHLRMARLEARRFIEQYGKQSDHDGDLLELAILDYNQVQAQHQSELTEIIRWWKELGLVDKLSFGRDRPLECFLWTVGLLPEPKYSSVRIELAK AISILLVIDDIFDTYGEMDDLILFTDAIRRWDLEAMEGLPEYMKICYMALYNTTNEVCYKVLRDTGRIVLLNLKSTWIDMIEGFMEEAKWFNGGSAPKLEEYIENGVSTAGAYMAFAHIFFLIGEG VTHQNSQLFTQKPYPKVFSAAGRILRLWDDLGTAKEEQERGDLASCVQLFMKEKSLTEEEARSRILEEIKGLWRDLNGELVYNKNLPLSIIKVALNMARASQVVYKHDQDTYFSSVDNYVDALFFTQ

[0150] An exemplary nucleic acid sequence of geraniol synthase is provided by SEQ ID NO: 18.

[0151]

[0152] Other examples of enzymes with geraniol synthase activity suitable for the compositions and methods provided herein include, but are not limited to, those referenced in GenBank accession numbers: AHE41084.1 (SEQ ID NO: 69), AFD64744.1 (SEQ ID NO: 70), QNQ74216.1 (SEQ ID NO: 71).

[0153] In some embodiments, the heterologous gene encodes an enzyme with geraniol synthase activity, enabling cells expressing the enzyme to increase the production of one or more monoterpenes. In some embodiments, the heterologous gene encodes an enzyme with geraniol synthase activity, enabling cells expressing the enzyme to increase the production of one or more monoterpenes compared to cells not expressing the heterologous gene.

[0154] In some embodiments, the enzyme having geraniol synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in any one of SEQ ID NO: 17 and 69-71. In some embodiments, the enzyme having geraniol synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 17. In some embodiments, the enzyme having geraniol synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 69. In some embodiments, the enzyme having geraniol synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 70. In some embodiments, the enzyme having geraniol synthase activity has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence shown in SEQ ID NO: 71.In some embodiments, the enzyme having geraniol synthase activity comprises the amino acid sequence shown in any one of SEQ ID NO: 17 and 69-71. In some embodiments, the enzyme having geraniol synthase activity comprises the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the enzyme having geraniol synthase activity comprises the amino acid sequence shown in SEQ ID NO: 69. In some embodiments, the enzyme having geraniol synthase activity comprises the amino acid sequence shown in SEQ ID NO: 70. In some embodiments, the enzyme having geraniol synthase activity comprises the amino acid sequence shown in SEQ ID NO: 71. In some embodiments, the enzyme having geraniol synthase activity consists of the amino acid sequence shown in any one of SEQ ID NO: 17 and 69-71. In some embodiments, the enzyme having geraniol synthase activity consists of the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the enzyme having geraniol synthase activity consists of the amino acid sequence shown in SEQ ID NO: 69. In some embodiments, the enzyme having geraniol synthase activity consists of the amino acid sequence shown in SEQ ID NO: 70. In some embodiments, the enzyme having geraniol synthase activity consists of the amino acid sequence shown in SEQ ID NO: 71.

[0155] In some embodiments, the gene encoding an enzyme having geraniol synthase activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising an amino acid sequence having at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) sequence identity with the sequence shown in any one of SEQ ID NO: 17 and 69-71. In some embodiments, the gene encoding an enzyme having geraniol synthase activity comprises a nucleic acid sequence, the enzyme encoded by the nucleic acid sequence comprising an amino acid sequence shown in any one of SEQ ID NO: 17 and 69-71. In some embodiments, the gene encoding an enzyme having geraniol synthase activity comprises the nucleic acid sequence shown in SEQ ID NO: 18.

[0156] Other enzymes possessing geraniol synthase activity or predicted to possess farnesyl diphosphate synthase activity can be identified, for example, based on similarity or homology to one or more domains of geraniol synthases (such as linalool synthases provided by SEQ ID NO: 17 or 69-71). In some embodiments, enzymes used in the modified cells and methods described herein can be identified based on similarity or homology to active domains, such as catalytic domains, such as catalytic domains associated with geraniol synthase activity. In some embodiments, enzymes used in the modified cells and methods described herein may have a relatively high level of sequence identity with a reference geraniol synthase, e.g., in the catalytic domain region, but a relatively low level of sequence identity with the reference geraniol synthase based on analysis of a larger portion or the full-length enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity in its catalytic domain region relative to a reference geraniol synthase (e.g., SEQ ID NO: 17 or 69-71).

[0157] In some embodiments, the enzyme used in the modified cells and methods described herein has a relatively high level of sequence identity in its catalytic domain region relative to a reference geraniol synthase (e.g., SEQ ID NO: 17 or 69-71), and a relatively low level of sequence identity relative to the reference geraniol synthase based on analysis of a larger portion or full-length portion of the enzyme. In some embodiments, the enzyme used in the modified cells and methods described herein, based on a portion or the full-length enzyme, has at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity relative to a reference geraniol synthase (e.g., SEQ ID NO: 17 or 69-71).

[0158] General methods of genetic engineering

[0159] As will be apparent to those skilled in the art, the amino acid position number of selected residues in a protein may differ from that of another protein (e.g., a reference protein). Typically, the corresponding position in another protein can be identified using methods known in the art, for example, by comparing the amino acid sequences of two or more proteins. Software programs and algorithms for aligning amino acid (or nucleic acid) sequences are known and readily available in the art, such as Clustal Omega (Sievers et al., 2011).

[0160] The proteins described herein may further include one or more modifications, such as to specifically alter polypeptide characteristics unrelated to their desired physiological activity. Alternatively, or furthermore, the proteins described herein may include one or more mutations to regulate protein expression and / or activity in cells.

[0161] Mutations in nucleic acids that encode proteins preferably preserve the amino acid reading frames of the coding sequence and preferably do not create regions in the nucleic acid that may hybridize to form secondary structures (such as hairpins or loops), which can be detrimental to protein expression.

[0162] Mutations can be made by selecting amino acid substitutions or by randomly mutagenesis at selected sites in the nucleic acid encoding the polypeptide. As described herein, variant polypeptides can be expressed and one or more activities can be tested to determine which mutation provides the variant polypeptide with the desired properties. Further mutations can be made into variant (or non-variant) polypeptides that silence the amino acid sequence of the polypeptide but provide preferred codons for translation in a particular host (referred to as codon optimization). Preferred codons for translating nucleic acids in, for example, Saccharomyces cerevisiae are well known to those skilled in the art. Other mutations can also be made into the non-coding sequence of a gene or cDNA clone to enhance polypeptide expression. The activity of a protein variant can be tested, as disclosed herein, by cloning a gene encoding the protein variant into an expression vector, introducing the vector into a suitable host cell, expressing the protein variant, and testing the functional ability of the protein.

[0163] The protein described herein may contain amino acid substitutions at one or more positions corresponding to a reference protein (such as a wild-type protein). In some embodiments, the protein contains amino acid substitutions at positions 1, 2, 3, 4, 5, or more corresponding to a reference protein. In some embodiments, the protein is not a naturally occurring protein, e.g., it is genetically modified. In some embodiments, the protein contains amino acid substitutions at positions 1, 2, 3, 4, 5, or more corresponding to a reference protein. In some embodiments, the protein is not a naturally occurring protein, e.g., it is genetically modified.

[0164] In some embodiments, protein variants may also include one or more amino acid substitutions that do not substantially affect the activity and / or structure of the protein. Those skilled in the art will also recognize that conserved amino acid substitutions can be made in proteins to provide functionally equivalent variants of the aforementioned peptides, i.e., variants that retain the functional capabilities of the peptide. As used herein, “conserved amino acid substitution” means an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods known to those skilled in the art for altering peptide sequences, such as those found in references compiling such methods, e.g., *Molecular Cloning: A Laboratory Manual*, J. Sambrook et al., eds., 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or *Current Protocols in Molecular Biology*, FMAusubel et al., eds., John Wiley & Sons, Inc., New York. Exemplary functionally equivalent variants of peptides include conserved amino acid substitutions in the amino acid sequence of the proteins disclosed herein. Conservative substitutions of amino acids include substitutions between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0165] As will be known to those skilled in the art, homologous genes encoding proteins with the desired activity can be obtained from other species and can be identified through homology searches, for example, by protein BLAST searches available on the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov). By comparing the amino acid sequence of a protein with one or more reference proteins, and / or by comparing the secondary or tertiary structures of similar or homologous proteins with one or more reference etane lyases, corresponding amino acid residues in similar or homologous proteins can be determined, and amino acid residues for mutation in similar or homologous proteins can be identified.

[0166] Genes related to this disclosure can be obtained from DNA of any DNA source containing a given gene (e.g., by PCR amplification). In some embodiments, genes related to this invention are synthetic, such as by in vitro chemical synthesis. Any method for obtaining a gene encoding a protein described herein is compatible with the modified cells and methods described herein.

[0167] The disclosure provided herein relates to recombinant expression, functional modification, and variants of genes encoding proteins having desired activities, and their associated uses. Homologs and alleles of nucleic acids related to this invention can be identified using conventional techniques. This invention also covers nucleic acids hybridizing with the nucleic acids described herein under stringent conditions. As used herein, the term “stringent conditions” refers to parameters well known in the art. Nucleic acid hybridization parameters can be found in references that compile such methods, such as *Molecular Cloning: A Laboratory Manual*, J. Sambrook et al., eds., 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or *Current Protocols in Molecular Biology*, FM Ausubel et al., eds., John Wiley & Sons, Inc., New York.

[0168] Other conditions, reagents, etc., exist that offer a similar degree of stringency. Those skilled in the art will be familiar with such conditions, and therefore will not be elaborated upon here. However, it should be understood that a skilled articulator will be able to manipulate the conditions in a manner that allows for clear identification of homologs and alleles of the nucleic acids of this invention (e.g., by using less stringent conditions). A skilled articulator is also familiar with methods for screening cells and libraries to express such molecules, followed by routine isolation of these molecules, and subsequently isolation and sequencing of the associated nucleic acid molecules.

[0169] This invention also includes degenerate nucleic acids containing alternative codons to codons present in natural materials. For example, serine residues are encoded by codons TCA, AGT, TCC, TCG, TCT, and AGC. Each of the six codons is equivalent in terms of encoding serine residues. Therefore, those skilled in the art will understand that any nucleotide triplet encoding serine can be used to guide a protein synthesis apparatus, in vitro or in vivo, to incorporate serine residues into extended polypeptides. Similarly, nucleotide sequence triplets encoding other amino acid residues include, but are not limited to: CCA, CCC, CCG, and CCT (proline codons); CGA, CGC, CGG, CGT, AGA, and AGG (arginine codons); ACA, ACC, ACG, and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC, and ATT (isoleucine codons). Other amino acid residues can similarly be encoded by multiple nucleotide sequences. Therefore, this invention covers degenerate nucleic acids whose codon sequences differ from those of biologically isolated nucleic acids due to the degeneracy of the genetic code. This invention also covers codon optimization to suit the optimal codon usage of the host cell.

[0170] The present invention also provides modified nucleic acid molecules comprising the addition, substitution, and deletion of one or more nucleotides. In a preferred embodiment, these modified nucleic acid molecules and / or the polypeptides they encode retain at least one activity or function, such as enzymatic activity, of the unmodified nucleic acid molecules and / or polypeptides. In some embodiments, the modified nucleic acid molecules encode modified polypeptides, preferably polypeptides having conserved amino acid substitutions as described elsewhere herein. The modified nucleic acid molecules are structurally related to the unmodified nucleic acid molecules, and in a preferred embodiment have sufficient structural relevance to the unmodified nucleic acid molecules such that the modified and unmodified nucleic acid molecules hybridize under stringent conditions known to those skilled in the art.

[0171] For example, modified nucleic acid molecules encoding polypeptides with a single amino acid change can be prepared. Each of these nucleic acid molecules may have one, two, or three nucleotide substitutions, excluding nucleotide changes corresponding to the degeneracy of the genetic code described herein. Similarly, modified nucleic acid molecules encoding polypeptides with two amino acid changes can be prepared, e.g., with 2-6 nucleotide changes. Many such modified nucleic acid molecules will be readily envisioned by those skilled in the art, including, for example, nucleotide substitutions in codons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6, etc. In the foregoing examples, the combination of each two amino acids and all nucleotide substitutions encoding the amino acid substitutions are included in the set of modified nucleic acid molecules. Additional nucleic acid molecules encoding polypeptides with additional substitutions (i.e., three or more), insertions, or deletions (e.g., by introducing a stop codon or splicing site) can also be prepared, and these molecules are also covered by the present invention as will be readily envisioned by those skilled in the art. Any of the foregoing nucleic acids or polypeptides can be tested by conventional experiments for retention of structural relevance or activity to the nucleic acids and / or polypeptides disclosed herein.

[0172] Mutations can be made by selecting amino acid substitutions or by randomly mutagenesis at selected sites in the nucleic acid encoding the polypeptide. As described herein, variant polypeptides can be expressed and one or more activities can be tested to determine which mutation provides the variant polypeptide with the desired properties. Further mutations can be made into variant (or non-variant) polypeptides that silence the amino acid sequence of the polypeptide but provide preferred codons for translation in a particular host (called codon optimization). Preferred codons for translating nucleic acids in, for example, Saccharomyces cerevisiae are well known to those skilled in the art. Other mutations can also be made into the non-coding sequence of a gene or cDNA clone to enhance polypeptide expression.

[0173] In one aspect of this disclosure, one or more genes related to the invention are expressed in a recombinant expression vector. As used herein, a "vector" can be any of a variety of nucleic acids into which a desired sequence can be inserted by restriction enzyme digestion and ligation for transport between different genetic environments or expression in a host cell. Vectors typically include DNA, but RNA vectors are also available. Vectors include, but are not limited to, plasmids, phage plasmids, viral genomes, and artificial chromosomes.

[0174] Cloning vectors are vectors capable of autonomously replicating or integrating into the genome within a host cell. For plasmids, replication of the desired sequence can occur multiple times as the copy number of the plasmid increases in a host cell, such as host bacteria, or it can replicate only once per host before the host reproduces through mitosis. Taking bacteriophages as an example, replication can occur actively during lysis or passively during lysis.

[0175] Expression vectors are vectors into which a desired DNA sequence can be inserted through restriction enzyme digestion and ligation, making it operatively linked to a regulatory sequence and expressable as an RNA transcript. The vector may further contain one or more marker sequences suitable for identifying cells that have been or have not been transformed or transfected by the vector. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, genes encoding proteins whose activity can be detected by standard assays known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase), and genes (e.g., green fluorescent protein) that visibly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques. Preferred vectors are those capable of autonomous replication and expression of structural gene products present in the operatively linked DNA fragment.

[0176] As used herein, when a coding sequence and a regulatory sequence are covalently linked such that the expression or transcription of the coding sequence is influenced or controlled by the regulatory sequence, the coding sequence and the regulatory sequence are said to be "operably" conjoined or operably linked. If the coding sequence is to be translated into a functional protein, if the induction of a promoter in the 5' regulatory sequence leads to transcription of the coding sequence, and if the nature of the connection between the two DNA sequences does not (1) lead to the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein, then the two DNA sequences are said to be operably conjoined or operably linked. Therefore, if a promoter region can influence the transcription of the DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide, then the promoter region is operably linked to the coding sequence.

[0177] When a nucleic acid molecule encoding any of the proteins disclosed herein is expressed in a cell, a variety of transcriptional control sequences (e.g., promoter / enhancer sequences) can be used to guide its expression. In some embodiments, each gene is operatively linked to a promoter (e.g., each gene is linked to a separate promoter). Promoters can be natural promoters, i.e., gene promoters in their endogenous environment that provide normal regulation of gene expression. In some embodiments, promoters can be constitutive promoters, i.e., promoters are unregulated, allowing their associated genes to be continuously transcribed. Various conditional promoters can also be used, such as promoters controlled by the presence or absence of a molecule.

[0178] The exact nature of the regulatory sequences required for gene expression can vary by species or cell type, but will generally include necessary 5' non-transcriptional and 5' non-translational sequences, such as TATA boxes, capping sequences, CAAT sequences, etc., respectively, relating to transcriptional and translation initiation. In particular, such 5' non-transcriptional regulatory sequences will include a promoter region containing a promoter sequence for transcriptionally controlling the operatively conjugated gene. The regulatory sequences may also include enhancer sequences or upstream activator sequences as needed. The vectors of the present invention may optionally include a 5' leader sequence or a signal sequence. The selection and design of suitable vectors are within the competence and discretion of those skilled in the art.

[0179] Expression vectors containing all the necessary expression elements are commercially available and well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, Cold SpringHarbor Laboratory Press, 2012. Cells are genetically engineered by introducing heterologous DNA (RNA) into them. The heterologous DNA (RNA) can be placed under the operative control of transcription elements to allow expression of the heterologous DNA in the host cell. As will be understood by those skilled in the art, any protein described herein can also be expressed in other yeast cells, including yeast strains used to produce wine, mead, sake, cider, etc.

[0180] Nucleic acid molecules encoding proteins having the desired activity disclosed herein can be introduced into one or more cells using methods and techniques standard in the art. For example, nucleic acid molecules can be introduced using standard protocols such as transformation (including chemical transformation and electroporation), transduction, particle bombardment, etc.

[0181] The expression of nucleic acid molecules encoding the proteins claimed in this invention can also be achieved by integrating the nucleic acid molecules into the genome.

[0182] Gene incorporation can be achieved by incorporating protein-coding nucleic acids into the genome of a yeast cell, or by transiently or stably maintaining new protein-coding nucleic acids as episodic elements. In eukaryotic cells, permanent, heritable genetic alterations are typically achieved by introducing DNA into the cellular genome.

[0183] Heterologous genes may also include various transcriptional elements required to express the encoded gene product (e.g., a protein with the desired activity). For example, in some embodiments, any gene described herein may be operatively linked to a promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is active at a specific stage of the fermentation process. For example, in some embodiments, peak expression of the promoter occurs in the early stages of fermentation, such as before >50% of the fermentable sugar has been consumed. In some embodiments, peak expression of the promoter occurs in the later stages of fermentation, such as after 50% of the fermentable sugar has been consumed.

[0184] During fermentation, conditions in the culture medium can change; for example, as sugar sources and oxygen are depleted, the availability of nutrients and oxygen tends to decrease over time. Furthermore, the presence of other factors, such as products of cellular metabolism, can increase these conditions. In some embodiments, the promoter is regulated by one or more conditions during fermentation, such as the presence or absence of one or more factors. In some embodiments, the promoter is regulated by hypoxic conditions. Examples of hypoxia-activated gene promoters are known in the art. See, for example, Zitomer et al. Kidney Int. (1997) 51(2): 507-13; Gonzalez Siso et al. Biotechnol. Letters (2012) 34: 2161-2173.

[0185] In some embodiments, the promoter is a constitutive promoter. Examples of constitutive promoters for yeast cells are known in the art and will be apparent to those skilled in the art. In some embodiments, the promoter is a yeast promoter, such as a natural promoter derived from yeast cells expressing a heterologous or foreign gene.

[0186] Non-limiting examples of promoters used in the gene-modifying cells and methods described herein include: NCP1 promoter (pNCP1), ERG11 promoter (pERG11), HEM13 promoter (pHEM13), SPG1 promoter (pSPG1), PRB1 promoter (pPRB1), QCR10 (pQCR10), PGK1 promoter (pPGK1), OLE1 promoter (pOLE1), ERG25 promoter (pERG25), HHF2 promoter (pHHF2), TDH1 promoter (pTDH1), TDH2 promoter (pTDH2), TDH3 promoter (pTDH3), ENO2 promoter (pENO2), ANT1 promoter (pANT1), PEX11 promoter (pPEX11), or HSP26 promoter (pHSP26).

[0187] NCP1 promoter (pNCP10) – SEQ ID NO: 19

[0188] ATATAACGCTATATACATAAAAGAGAGAAAAAAAAAACTGTTGTTAGCAACAGGTAGTTCAATTGAAGAAATTCCGATGCGCCACGAAGGACGCTCCAGCTTTACATACGCTGATTTTCCCATGATGGATTGCTACTGTGCTTTCGAGCGCCGACTTACAAGATTTGCTCTTCCAGTAAAATTTCACTTTGGGAATCACACCGCGAAACTGGCATACTAACGCATAAGAAAACACAAGAAGAGAAACAACGATAAAATCACGCGGCGACATCTACAGTCCACCTGCCCCTTCCTTCGCTCAATTGCACTTTCCCATGCGCACTACCCATCTATATAGCTATGTATTCTATATCCACGCCTGCAAACGCACACTGTTCGTCCTCAGCCTCTATGCGTACGAGCCAGACCCTTGCTCCCAGTAGATAACGCACGCAAAACTCAAAAGATGGCACACCGGGGGAACAAAGGAGAGTAGAGTCTACCGAACGTCACCGCTGGTACCATGCAGCCATCACTACATACTAGCATCATACTACCACGATCACAGTTCACGACACATCACGTGTGTGCTCGTTTAGCGGTCAACCCGCTATTGTTCTCCAGCCAGCTTTTATCGTTTTGCATTTTTTTTTCGGGCTGCTTTTCGTTCTTCGAGGACAAACGCACCTGTAAAGCTCAGCGCGAGGTTATATATATAGTATAATATGGTGAACAATGTAGCTTAAGTTTGCGTTCCTGTAGTGGTCACCGCTACGCTAGACAACGACCGCGGTTTTCAAGTGATCTACTGTCGCACATAT

[0189] HSP26 promoter (pHSP26) – SEQ ID NO: 20

[0190] CAATATTCTGCGCACATCAATCATTTTCTTACTACATACACTAACATTACTCCTAGTTTAATTTAATTGAATTTTTAACTTTCTTTTCTTTTCATTTGGCAATTTGGCTCCTTGAAAACAAGACTATGGGTCTgTCTCATAAGCCTCAGGGGGGGACCCCAAAAAAATAACGCGGCCATCTTGCATGCACCGTTGAACCTGTAGCTTACAGTAAGCCACAATTCTCTTACCTTCTTGGCAATGTGGCACAAAATAATCTGGTTATGTGTCTTCATTTGGTAATCACTGGGATGTTACTGGGGCAGCAGCAACTCCGTGTGTACCCCTAACTCCGTGTGTACCCCTAAAGAACCTTGCCTGTCAAGGTGCATTGTTGGATCGGAATAGTAACCGTCTTTACATGAACATCCACAACCAACGAAAGTGCTTTTTCAAGCATTGCTTGATTTCTAGAAAGATCGATGGTTATTCCCTCCCCCTTATGCGTCCAAAAATATAGGGTGCTCGTAACAGTAAGGTATTCGCACTTAGCGTGCTCGCAACACAAAATTAAGTAATATGCGAGTTTTAGATGTCCTTGCGGATCTATGCACGTTCTTGAGTGGTATTTCATAACAACGGTTCTTTTTCACCCTTATTCCTAAACATATAAATAGGACCTCCATTAGTTAGAGATCTGTTTTTAATCCATTCACCTTTCATTCTACTCTCTTATACTAATAAAACCACCGATAAAGATATATCAGATCTCTATTAAAACAGGTATCCAAAAAAGCAAACAAACAAACTAAACAAATTAA

[0191] OLE1 promoter (pOLE1) – SEQ ID NO: 21

[0192] AAAAAAGGAAAAAAAAAAAAAAAAAAAATAAATGACACATGGAAATAAGTCAAGGATTAGCGGATATGTAGTTCCAGTCCGGGTTATACCATCACGTGATAATAAATCCAAATGAGAATGAGGGTGTCATATCTAATCATTATGCACGTCAAGATTCTCCGTGACTATGGCTCTTTTCTGAAGCATTTTTCGGGCGCCCGGTGGCCAAAAACTAACTCCGAGCCCGGGCATGTCCCGGGGTTAGCGGGCCCAACAAAGGCGCTTATCTGGTGGGCTTCCGTAGAAGAAAAAAAGCTGTTGAGCGAGCTATTTCGGGTATCCCAGCCTTCTgTGCAGACCGCCCCAGTTGGCTTGGCTCTGGTGCTGTTCGTTAGCATCACATCGCCTGTGACAGGCAGAGGTAATAACGGCTTAAGGTTCTCTTCGCATAGTCGGCAGCTTTCTTTCGGACGTTGAACACTCAACAAACCTTATCTAGTGCCCAACCAGGTGTGCTTCTACGAGTCTTGCTCACTCAGACACACCTATCCCTATTGTTACGGCTATGGGGATGGCACACAAAGGTGGAAATAATAGTAGTTAACAATATATGCAGCAAATCATCGGCTCCTGGCTCATCGAGTCTTGCAAATCAGCATATACATATATATATGGGGGCAGATCTTGATTCATTTATTGTTCTATTTCCATCTTTCCTACTTCTGTTTCCGTTTATATTTTGTATTACGTAGAATAGAACATCATAGTAATAGATAGTTGTGGTGATCATATTATAAACAGCACTAAAACATTACAACAAA

[0193] ERG11 promoter (pERG11) – SEQ ID NO: 22

[0194] GCCGCCTGTCCCGTACAGACGAACAATTGCAACAATGGGCGGTTGTTTAGAGAGGTTTTGTGCCGCGCCCGGGAATTACCGGGGGCACAGCAAAATACGAAATTTCCGGCAAAATGTCTCTGTACGAAAATGAGGCCAGCCTTTATTCCCGACTAAGCCGTACGATTATAGTAATGTCACACGAAGTGGATACTATACGAGCAGCGCACATACAATGTGCGTGCAAGATTTGCCGGGTTGGACAATCTTAAAGGCCGATAATCCACGAAAAACGAGACAAACGAAGCGCTGGCCCTAAACGAAACGAAACGAAACTAAGATTTCAAGATAATCGCTTTTCAGATGACAATAGTCTCTTGCAGAACACAATAAGTGTGGTAGGTATCTTGCAGGAGACATCGATTTTATGCCTGAATTTTTTTTTTTCTTGGTATGAGCACATCCTATTGTGCATCTCCACGAGTTAACGACGTTTACGTACTCGCATGTATTCGAAAAGCCTCTAAAAATTGCCCTCCATGTGTATTCATTATATAAGATAGCGTATTTTCCCTTAGTTTTCTTATTCCCTCTTAATTCTCTCTTTCAATGTCTTTTTTTTTTTCCCTTTTCCTTTTTCCCTTTTCGTTATTGGTAAATTATTTATAAAAAGATTGATAAGCAGTATCGTTCAGCGTGTGTATTTTCGATAGGAAGTAATAATTTTTCATTATATTAATTAATAGTTTCCAGAgAAAATTTTTTTTaCCtTTtACAATTGCAGCAGGCTTGAATAGAAACAGAACAAACGAGTAATACAAG

[0195] PGK1 promoter (pPGK1) – SEQ ID NO: 23

[0196] GTGAGTAAGGAAAGAGTGAGGAACTATCGCATACCTGCATTTAAAGATGCCGATTTGGGCGCGAATCCTTTATTTTGGCTTCACCCTCATACTATTATCAGGGCCAGAAAAAGGAAGTGTTTCCCTCCTTCTTGAATTGATGTTACCCTCATAAAGCACGTGGCCTCTTATCGAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAACTGAAAAAACCCAGACACGCTCGACTTCCTGTCATCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCTAGCGACGGCTCACAGGTTTTGTAACAAGCAATCGAAGGTTCTGGAATGGCGGGAAAGGGTTTAGTACCACATGCTATGATGCCCACTGTGATCTCCAGAGCAAAGTTCGTTCGATCGTACTGTTACTCTCTCTCTTTCAAACAGAATTGTCCGAATCGTGTGACAACAACAGCCTGTTCTCACACACTCTTTTCTTCTAACCAAGGGGGTGGTTTAGTTTAGTAGAACCTCGTGAAACTTACATTTACATATATATAAACTTGCATAAATTGGTCAATGCAAGAAATACATATTTGGTCTTTTCTAATTCGTAGTTTTTCAAGTTCTTAGATGCTTTCTTTTTCTCTTTTTTACAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAATATAAAACA

[0197] HEM13 promoter (pHEM13) – SEQ ID NO: 24

[0198] TAATGTAGAAGGTTGAGAACAACCGGATCTTGCGGTCATTTTTCTTTTCGAGGAAAGTGCAAGTCTGCCACTTTCCAGAAGGCATAGCCTTGCCCTTTTGTTGATATTTCTCCCCACCGTAATTGTTGCATTCGCGATCTTTTCAACAATACATTTTATCATCAAGCCCGCAAATCCTCTGGAGTTTGTCCTCTCGTTCACTGTTGGGAAAAACAATACGCCTAATTCGTGATTAAGATTCTTCAAACCATTTCCTGCGGAGTTTTTACTGTGTGTTGAACGGTTCACAGCGTAAAAAAAAGTTACTATAGGCACGGTATTTTAATTTCAATTGTTTAGAAAGTGCCTTCACACCATTAGCCCCTGGGATTACCGTCATAGGCACTTTCTGCTGAGCTCCTGCGAGATTTCTGCGCTGAAAGAGTAAAAGAAATCTTTCACAGCGGCTCCGCGGGCCCTTCTACTTTTAAACGAGTCGCAGGAACAGAAGCCAAATTTCAAAGAACGCTACGCTTTCGCCTTTTCTGGTTCTCCCACCAATAACGCTCCAGCTTGAACAAAGCATAAGACTGCAACCAAAGCGCTGACGGACGATCCGAAGATAAAGCTTGCTTTGCCCATTGTTCTCGTTTCGAAAGGCTATATAAGGACACGGATTTTCCTTTTTTTTTTCCACCTATTGTCTTTCTTTGTTAAGCTTTTATTCTCCGGGTTTTTTTTTTT

[0199] SPG1 promoter (pSPG1) – SEQ ID NO: 25

[0200] ATGAAGTTCACTTCACATCCAATGAGAAAAACAAAATCCGCAGGGCTATCACCCAGAACATCCTCCACTTCATCTTCTTCAGGACAGAGAAAAGCGCATCACCACCACCATCACCACAACCACGTTTCAAGGACGAAAACTACCGAAAGCACCAAATCAGGCAACAGCAAAAAGGACAGTTCCTCATCCTCAACAAACGACCATCAATTTAAAAGGTCTGAAAAGAAGAAAAAAAGTAAATTTGGCTCGATCTTCAAAAAAGTTTTCGGATGAACCGGATTAATACAAGTAAAATCAGCAAAGATATAGAAGACAAAATAAGCGTGAAAACAATCATAAACCACTCACAACGGGGGTTTTCAGCTGTTACTCCTCCATACATACATTTTGATAAAGATATAATGTTATATTTCTTTTCGTAATTTTGTTTTACTTCGGTTTGCTCTATAGATTTCATCAGCCGCACCGAAAAGGGAGATCAATAAGGTACCCTTTAAAAGGGATAAGAAGCCTAACATCACCCCAATAAATGGAGTAATGGCCAGCATTGGATGAAGAGAAGAATTACGGGATACTGGGATAACACTGTTAAAAATGCTTCGCGACGTGAGGGTCTTATATAAATTGAACTGCCAAATCTCTTTCACATTATCCAGGATAGTTTGGAATGTGTGTTACTGAAAGATCAGAATCAATAAATACAATCAATACAAATATTTAGCGCATAAAATTCAAACAAAGTTTACTGAA

[0201] PRB1 promoter (pPRB1) – SEQ ID NO: 26

[0202] CGAGAAACAGGGGGGGAGAAAAGGGGAAAAGAGAAGGAAAGAAAGACTCATCTATCGCAGATAAGACAATCAACCCTCATGGCGCCTCCAACCACCATCCGCACTAGGGACCAAGCGCTCGCACCGTTAGCAACGCTTGACTCACAAACCAACTGCCGGCTGAAAGAGCTTGTGCAATGGGAGTGCCAATTCAAAGGAGCCGAATACGTCTGTTCGCCTTTTAAGAGGCTTTTTGAACACTGCATTGCACCCGACAAATCAGCCACTAACTACGAGGTCACGGATACATATACCAATAGTTAAAAAATTACATATACTCTATATAGCACAGTAGTGTGATAAATAAAAAATTTTGCCAAGACTTTTTTAAACTGCACCCGACAGATCAGGTCTGTGCCTACTATGCACTTATGCCCGGGGTCCCGGGAGGAGAAAAAACGAGGGCTGGGAAATGTCCGTGGACTTAAAACGCTCCGGGTTAGCAGAGTAGCAGGGCTTTCGGCTTTGGAAATTTAGGTGACTTGTTGAAAAAGCAAAATTTGGGCTCAGTAATGCCACTGCAGTGGCTTATCACGCCAGGACTGCGGGAGTGGCGGGGGCAAACACACCCGCGATAAAGAGCGCGATGAATATAAAAGGGGGCCAATGTTACGTCCCGTTATATTGGAGTTCTTCCCATACAAACTTAAGAGTCCAATTAGCTTCATCGCCAATAAAAAAACAAACTAAACCTAATTCTAACAAGCAAAG

[0203] QCR10 (pQCR10) – SEQ ID NO: 27

[0204] GAGAGCTGGCCAAAAAGAGGGCCGAAGACGGCGTTGAATTTCATTCAAAACTATTTAGAAGGGCAGAGCCAGGTGAGGATTTAGATTATTATATTTACAAGCACATCCCTGAAGGGACCGACAAGCATGAAGAACAGATCAGGAGCATTTTGGAAACTGCCCCGATTTTACCAGGACAGGCATTCACTGAAAAATTTTCTATTCCGGCTTATAAAAAGCATGGAATCCAAAAGAATTAGGCTTCTCATTCTATTTTAATTATACTAGTACGATTTCTCACTCTGTAATTTAATATCAGTGTAATATGCACCTAGTTATGGGTAGTTTTTGCTAACGTTACGAGCCGCGAAACTGTCCTCAATCTTCACCACTACCTCTAATGACTGAAGAATGCTATGCGATATAACGCTGCCGCACTTTGAATATATACTTATATTTACATAGTTTTCAAGTGCGTATTACTATTGCAAAGTAGTATTTTGTCACGTGATTTTGATCCAATTAAAACTAAATATGGTTCAACCCGTTGTTTCCGCATCAAAAAACCATACCATTTATCAAGGGGACGGGATATATCACATAACAGTTTGAATGCATAATTTGTTATAGATATCTTCTGGAATAATCTTCACAGCAAAAGCGCAAGTCGAATAATATATCGATAAATACAATCCATAAGACTTAAAACTAACCTCA

[0205] ERG25 promoter (pERG25) – SEQ ID NO: 28

[0206] ATGTAAAAGTAGATTCGCATCTGAGAATGTTTTCTTCAAATTTTTCTTTTCGGCCTTGGCGCAGAAGACTTTCTCTTCGTCCGCGTCGTATAGGAAACAAAAAAGTGTAAAAAGTAAAAATCGGTATATTACGCAATCCATTCGTTACCCGGTGTAGTCACGTGCGGGTGCGGAGGGCAGCCGTTTAAACCCGTTTGGGCCTTGGTCCGATAGAACCATCTCGCAGGAAAGAGCAGAGCCAGTTATCGGGTGTCGTTTACGCCCAGATGTCGTTTAGCCAGCTCCGCAACGGCCACTGCCACTACCACTGCCTCCCTTCGTATACGGGACACAGAGATCGTATACGGTAAACGACTAGCACTAGGACAATAGCAACTAACAATGACCGATAACAGTCAGCAACCTCAAGGCACCTGGCACAAAACCGTCGAGGAAGAGGCCCTTTGCCACCGTATCGCATCGCTTTCCCACCAATTTTTTTTTTCTTGCTTGCCTCGCCTCTGCTGTGGTCGTATTATTATTATTCATTCTCTGCGCAGTTTAAACTATATACACACGATTAATCCGTTATGCATGTACATTTTCTTGTATGCGTACGCCTGTGTATGCATACTTCTATATAATATATAATACAGGAGGATCTATTCTTTTTTTTCTTCTTCTCTTCTTCTCTCCCTTTTTTCTCTCTCTTCGTTTCTTTCCTTCCAGTAAATCTTTATATTAGTTGTAACTTTTTCTCTTTAGATAGTAGCATAGAGGACTAAGGAAAAGTAGTACAGCCATAAAAAAAAGAGGAAAAG

[0207] HHF2 promoter (pHHF2) – SEQ ID NO: 29

[0208] TGTGGAGTGTTTGCTTGGATTCTTTAGTAAAAGGGGAAGAACAGTTGGAAGGGCCAAAGTGGAAGTCACAAAACAGTGGTCCTATATAAAAGAACAAGAAAAAGATTATTTATATACAACTGCGGTCACAAGAAGCAACGCGAGAGAGCACAACACGCTGTTATCACGCAAACTATGTTTTGACACCGAGCCATAGCCGTGATTGTGCGTCACATTGGGCGATAATGAACGCTAAATGACCAACTCCCATCCGTAGGAGCCCCTTAGGGCGTGCCAATAGTTTCACGCGCTTAATGCGAAGTGCTCGGAACGGACAACTGTGGTCGTTTGGCACCGGGAAAGTGGTACTAGACCGAGAGTTTCGCATTTGTATGGCAGGACGTTCTGGGAGCTTCGCGTCTAAAGCTTTTTCGGGCGCGAAATGCAGACCAGACCAGAACAAAACAACTGACAAGAAGGCGTTTAATTTAATATGTTGTTCACTCGCGCCTGGGCTGTTGTTATTCGGCTAGATACATACGTGTTTGTGCGTATGTAGTTATATCATATATAAGTATATTAGGATGAGGCGGTGAAAGAGATTTTTTTTTTTTCGCTTAATTTATTCTTTTCTCTATCTTTTTTCCTACATCTTGTTCAAAAGAGTAGCAAAAACAACAATCAATACAATAAAATA

[0209] TDH1 promoter (pTDH1) – SEQ ID NO: 30

[0210] GCCCGCTTCTGAAAACTACAGTTGACTTGTATGCTAAAGGGCCAGACTAATGGGAGGAGAAAAAGAAACGAATGTATATGCTCATTTACACTCCATATCACCATATGGAGGATAAGTTGGGTTGAGCTTCTGATCCAATTTATTCTATCCATTAGTTGCTGATATGTCCCACCAGCCAACACTTGATAGTATCTACTCGCCATTCACTTCCAGCAGCGCCAGTAGGGTTGTTGAGCTTAGTAAAAATGTGCGCACCACAAGCCTACATGTCTCCACGTCACATGAAACCACACCGTGGGGCCTTGTTGCGCTAGGAATAGGATATGCGACGAAGACGCTTCTGCTTAGTAACCACACCACATTTTCAGGGGGTCGATCTGCTTGCTTCCTTTACTGTCACGAGCGGCCCATAATCGCGCTTTTTTTTTAAAAGGCGCGAGACAGCAAACAGGAAGCTCGGGTTTCAACCTTCGGAGTGGTCGCAGATCTGGAGACTGGATCTTTACAATACAGTAAGGCAAGCCACCATCTGCTTCTTAGGTGCATGCGACGGTATCCACGTGCAGAACAACATAGTCTGAAGAAGGGGGGAGGAGCATGTTCATTCTCTGTAGCAGTAAGAGCTTGGTGATAATGACCAAAACTGGAGTCTCGAAATCATATAAATAGACAATATATTTTCACACAATGAGATTTGTAGTACAGTTCTATTCTCTCTCTTGCATAAATAAGAAATTCATCAAGAACTTGGTTTGATATTTCACCAACACACACAAAAAACAGTACTTCACTAAATTTACACACAAAACAAA

[0211] TDH2 promoter (pTDH2) – SEQ ID NO: 31

[0212] ATGGAAAGTACCAACATCGGTTGAAACAGTTTTTCATTTACTTATGGTTTATTGGTTTTTCCAGTGAATGATTATTTGTCGTTACCCTTTCGTAAAAGTTCAAACACGTTTTTAAGTATTGTTTAGTTGCTCTTTCGACATATATGATTATCCCTGCGCGGCTAAAGTTAAGGATGCAAAAAACATAAGACAACTGAAGTTAATTTACGTCAATTAAGTTTTCCAGGGTAATGATGTTTTGGGCTTCCACTAATTCAATAAGTATGTCATGAAATACGTTGTGAAGAGCATCCAGAAATAATGAAAAGAAACAACGAAACTGGGTCGGCCTGTTGTTTCTTTTCTTTACCACGTGATCTGCGGCATTTACAGGAAGTCGCGCGTTTTGCGCAGTTGTTGCAACGCAGCTACGGCTAACAAAGCCTAGTGGAACTCGACTGATGTGTTAGGGCCTAAAACTGGTGGTGACAGCTGAAGTGAACTATTCAATCCAATCATGTCATGGCTGTCACAAAGACCTTGCGGACCGCACGTACGAACACATACGTATGCTAATATGTGTTTTGATAGTACCCAGTGATCGCAGACCTGCAATTTTTTTGTAGGTTTGGAAGAATATATAAAGGTTGCACTCATTCAAGATAGTTTTTTTCTTGTGTGTCTATTCATTTTATTATTGTTTGTTTAAATGTTAAAAAAACCAAGAACTTAGTTTCAAATTAAATTCATCACACAAACAAACAAAACAAA

[0213] TDH3 promoter (pTDH3) – SEQ ID NO: 32

[0214] CAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAA

[0215] ENO2 promoter (pENO2) – SEQ ID NO: 33

[0216] ATTGAATACATTAGCAACGCGTCCAGCATTTTTCGGAAGTGTCTCATAAACTTTACTCAAGAGTTAAGTACTGAAAAATTCGACTTTTATGATAGTTCAAGTGTCGACGCTGCGGGTATAGAAAGGGTTCTTTACTCTATAGTGCCTCCTCGCTCAGCATCTGCTTCTTCCCAAAGATGAACGCGGCGTTATGTCACTAACGACGTGCACCAACTTGCGGAAAGTGGAATCCCGTTCCAAAACTGGCATCCACTAATTGATACATCTACACACCGCACGCCTTTTTTCTGAAGCCCACTTTCGTGGACTTTGCCATATGCAAAATTCATGAAGTGTGATACCAAGTCAGCATACACCTCACTAGGGTAGTTTCTTTGGTTGTATTGATCATTTGGTTCATCGTGGTTCATTAATTTTTTTTCTCCATTGCTTTCTGGCTTTGATCTTACTATCATTTGGATTTTTGTCGAAGGTTGTAGAATTGTATGTGACAAGTGGCACCAAGCATATATAAAAAAAAAAGCATTATCTTCCTACCAGAGTTGATTGTTAAAAACGTATTTATAGCAAACGCAATTGTAATTAATTCTTATTTTGTATCTTTTCTTCCCTTGTCTCAATCTTTTATTTTTATTTTATTCTTCTTTTCTTAGTTTCTTTCATAACACCAAGCAACTAATACTATAACATACAATAATA

[0217] ANT1 promoter (pANT1) – SEQ ID NO: 34

[0218] TCCTTGACTTCACTGCCGTTGAACTTTACAGAATTGTATACCGTAGGATTCGGATAAATTTCCTCAGGACCCCAGTTCTTGCGACCTTCTGTACCAAAACAGCGAACTTCTTTCAAAGTCACGGTACCCTTTTCAGAGTCAATATCTTCTAACAGCCCCACATATCTGTTGTCAGTCACAGAGATTAAAGAAATAGTTTTACCGATGTACTGCGACATTGCCTTGCTGCTGTTTTTCGATGAATGCTTCTTCTACGATATCCTCACTATTCGTTTATTGTTCTTTCTGACTGCCTCCACGTTATTCTTTACAATCGCGGGTACAGTTTAATTTTTCCACCGTGTAAGGTGACGGGACCATGGTACTGGACTGAGGAATCATTACATATACGTCAGTAATGGGTATCTTCATAATAAACACCTTTATCCATTTAATATGGAAATCAATTGCACTGTCATTTAAAATAGCGGCATCTATGATACTTAGAGGGCAATTGCATTGATAGAAGTTGTGTCATTTGGTGTTCTCCTCGGATCGAGTTTTAAATCCGATTTTTGCATCTCGGTCCGAGTGACTATTTCAGATAATTGGTAAATATAAGAAGAGCAAGAAGATGTGCAAAAAATAGAGAAAATATGATGCTGCGTAAAAGTACAGACACCCTGGAAGCTAGGCCAAGATTGTTACGAGCATATCATCA

[0219] PEX11 promoter (pPEX11) – SEQ ID NO: 35

[0220] TATATTAGTGTTCGCTGAATTTTTTCCAGTCGAGTCTGCATTGAGATCAATCGATAAACTACTAGCATGATGATTAAGTTCTTGAGCACGTTGCAAAAGTAGCTCACTAACGACATTGGCCAGTTCTTTTTCGCTAAACAACATTATCCATTTTATGGCTAGACTGAAGAAATGCGAATTTATGGAATGCAAGTCCAGAGCCACCGGATCGGTCTTTATAGCGTTCATAACTTTGGTAGAAAACATATCTGGTGGCAGGAGTTCCACAAAAGGTACAGGTTCTTCATCTACCAAGGCTTCGTCACCACCCACAATTGCAAGGATTCTTGCTAACCATAGTGGCAGGTTTAGTTTAGTGTTTTTCGTTATGGGTCGTCCTGGGTTGTTTTCCAAATAACCAAGACCAGGAATATCATATTGAAATTTACAAGGAAACTCTGTCCCATCTGCTAGGACATCATCAATGTCATAGTAACCCATCCTTTCTATGCTTGCCCTTCTATTGTAAAAATATAATACCAAGAACAAAGTTGGCATCAATTCCATTGCGATTTTCACTACGCGTCGGCATTTATCTCGGAGAACCCGAAGCGATGGGTATATCATTTATAGATATAAAAGGGAAGTGACTCCAACAACTGAAAAACCGTTGTCTACATCTACTACTTCAAAGACTTCATCAAGTAATAGTATAATCAAT

[0221] RGI2 promoter (pRGI2) – SEQ ID NO: 72

[0222] GCATCGTCTCATCGGTCTCAAACGATAGGAGCATGCCCGAAGAAATGATTATGGGTGCGTGATCACCAATCTGTGTGAGGCTGTCGAAGATATCTGATAATCAGTAAGCGATAAAAAAAAAATAAAAATAAAAAAATAACGACACCAGCAGGATTTGAACCAGCGCGGGCAGAGCCCAACAGATTTCAAGTCTGTCGCCTTAACCACTCGGCCATAGTGCCTAAAACAATGTAGGTTATTTAAGCAAGTATTGTAGATACTTTTCGTAATAAACTACAATGCACCCACGACTCGCGGTGTAATGATGGCATGAAATCATTGAACGAAGTTTTGCGGCTATACGGCTGAAGGACGAGACTAAAGGGACAGGAATTATTAATGCGGGGTATAATTTGAATAGTATTAACGGGCACTGCCGTTTAGCCATCAAATGCTATTGTTGGGGTATTCTCTCTACTTTTTGTTCTTGGCTTGAACCTTTTCGGCGGTTGGCAATCGTCCGTATATAAGCATCGGCTGTCCCAATCCTCTATTGCCCTTTTCCCTTGCACCTCCTTCTCAATTCTTCGTATCTTTCGCGTAAAGGTAGATCTTGATTCACCTATCTGTCGAAACACGATTAAGTGCAAACGAAACAACGTACAGTATATAACAAAGTATTTTAAATAATAAGATATGTGAGACCTGAGACGGCAT

[0223] ALD6 promoter (pALD6) – SEQ ID NO: 73

[0224] ACACACTATCAGGTCAGGAACTGCCGTCACATACGACACTGCCCCTCACGTAAGGGCATGATAGAATTGGATTATGTAAAAGGTGAAGATACCATTGTAGAAGCAACCAGCACGTCGCCGTGGCTGATGAGGTCTCCTCTTGCCCGGGCCGCAGAAAAGAGGGGCAGTGGCCTGTTTTTCGACATAAATGAGGGGCATGGCCAGCACCGAGACGTCATTGTTGCATATGGCGTATCCAAGCCGAAACGGCGCTCGCCTCATCCCCACGGGAATAAGGCAGCCGACAAAAGAAAAACGACCGAAAAGGAACCAGAAAGAAAAAAGAGGGTGGGCGCGCCGCGGACGTGTAAAAAGATATGCATCCAGCTTCTATATCGCTTTAACTTTACCGTTTTGGGCATCGGGAACGTATGTAACATTGATCTCCTCTTGGGAACGGTGAGTGCAACGAATGCGATATAGCACCGACCATGTGGGCAAATTCGTAATAAATTCGGGGTGAGGGGGATTCAAGACAAGCAACCTTGTTAGTCAGCTCAAACAGCGATTTAACGGTTGAGTAACACATCAAAACACCGTTCGAGGTCAAGCCTGGCGTGTTTAACAAGTTCTTGATATCATATATAAATGTAATAAGAAGTTTGGTAATATTCAATTCGAAGTGTTCAGTCTTTTACTTCTCTTGTTTTATAGAAGAAAAAACATCAAGAAACATCTTTAACATACACAAACACATACTATCAGAATACA

[0225] In some embodiments, a heterologous nucleic acid comprising a gene encoding an enzyme having alcohol acyltransferase activity can be operatively linked to a promoter selected from the group consisting of SEQ ID NO: 19-35 and 72-73. In some embodiments, the promoter is selected from the group consisting of pSPG1 (SEQ ID NO: 26), pHSP26 (SEQ ID NO: 20), pANT1 (SEQ ID NO: 34), pPEX11 (SEQ ID NO: 35), and pALD6 (SEQ ID NO: 73). In some embodiments, the promoter comprises or is composed of SEQ ID NO: 19. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 20. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 21. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 22. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 23. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 24. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 25. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 26. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 27. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 28. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 29. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 30. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 31. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 32. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 33. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 34. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 35. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 72. In some embodiments, the promoter comprises or is composed of SEQ ID NO: 73.

[0226] Genetically modified yeast cells

[0227] This disclosure relates to various aspects of genetically modified yeast cells (modified cells) and the use of such modified cells in methods for producing fermented products (such as fermented beverages). The genetically modified yeast cells described herein are genetically modified to reduce the sensory detection of one or more wort-related off-flavors in fermented beverages. In some embodiments, wort-related off-flavors are aldehyde molecules and / or non-aldehyde molecules. In some embodiments, the genetically modified yeast cells described herein are genetically modified to functionally disrupt one or more proteins associated with the transport of maltose and / or maltotriose into the modified cells and / or the hydrolysis of maltose and / or maltotriose by the modified cells.

[0228] As used herein, “functional disruption,” such as disruption of a target gene, for example, disruption of the function of one or more genes encoding one or more proteins associated with maltose and / or maltotriose transport and / or maltose and / or maltotriose hydrolysis, means that the target gene is altered in a manner that reduces the activity of the protein encoded by the target gene in the host cell. Similarly, “functional disruption” of a target protein, such as disruption of the function of one or more proteins associated with maltose and / or maltotriose transport and / or maltose and / or maltotriose hydrolysis, means that the target protein is altered in a manner that reduces the activity of the protein in the host cell. In some embodiments, the activity of the target protein encoded by the target gene in the host cell is eliminated. In other embodiments, the activity of the target protein encoded by the target gene in the host cell is reduced. Functional disruption of a target gene can be achieved by deleting all or part of the gene, thereby eliminating or reducing gene expression, or eliminating or reducing the activity of the gene product. Disruption of target gene function can also be achieved through regulatory elements of the mutated gene, such as eliminating or reducing expression through the promoter of the mutated gene, or through the coding sequence of the mutated gene, thereby eliminating or reducing the activity of the gene product. In some embodiments, disruption of target gene function results in the removal of the complete open reading frame (ORF) of the target gene. In other embodiments, disruption of function means introducing an early stop codon into the ORF of the target gene. In still other embodiments, disruption of function means inserting a foreign nucleic acid sequence into the ORF of the target gene, or deleting an endogenous nucleic acid sequence from the ORF of the target gene.

[0229] The terms “genetically modified cell,” “genetically modified yeast cell,” and “modified cell” are used interchangeably herein to refer to a eukaryotic cell (e.g., yeast cell) that has been or may be modified by the introduction of a heterologous gene. The term (e.g., modified cell) includes the offspring of the original cell that has been genetically modified by the introduction of a heterologous gene. Those skilled in the art should understand that, due to mutations (i.e., natural, accidental, or intentional alterations to the nucleic acids of the modified cell), the offspring of a single cell may not necessarily be identical to the original parent in morphology or in the complementary composition of its genome or total nucleic acids.

[0230] The yeast cells used in the methods described herein are preferably capable of fermenting sugar sources (e.g., fermentable sugars) and producing ethanol (alcohol) and carbon dioxide. In some embodiments, the yeast cells belong to the genus *Saccharomyces*. The genus *Saccharomyces* comprises nearly 500 different species, many of which are used in food production. One example of a species is *Saccharomyces cerevisiae*, commonly known as "beer yeast" or "bread yeast," and used in the production of wine, bread, beer, and other products. Other members of the genus *Saccharomyces* include, but are not limited to, wild yeast *Saccharomyces paradoxus* (a close species to *Saccharomyces cerevisiae*), *Saccharomyces bayanus*, *Saccharomyces pastorianus*, *Saccharomyces carlsbergensis*, *Saccharomyces uvarum*, *Saccharomyces cerevisiae var boulardii*, and *Saccharomyces eubayanus*. In some embodiments, the yeast is *Saccharomyces cerevisiae*.

[0231] Yeast species can be haploid (i.e., possessing a single set of chromosomes), diploid (i.e., possessing paired sets of chromosomes), or polyploid (i.e., carrying or containing more than two sets of chromosomes). For example, yeast species used in beer brewing are generally divided into two categories: aeroid strains (i.e., *Saccharomyces cerevisiae*), which are top-fermented; and lageroid strains (such as *Saccharomyces pastorianus*, *Saccharomyces carlsbergensis*, and *Saccharomyces uvarum*), which are bottom-fermented. These characteristics reflect their segregation properties in open, rectangular fermenters, as well as other characteristics such as preferred fermentation temperatures and the achieved alcohol concentrations.

[0232] While beer brewing and wine production have traditionally focused on using Saccharomyces cerevisiae strains, other yeast species and genera have also gained attention in the production of fermented beverages. In some implementations, the yeast cells belong to non-yeast genera. See, for example, Crauwels et al. Brewing Science (2015) 68: 110-121; Esteves et al. Microorganisms (2019) 7(11): 478. In some embodiments, the yeast cells belong to the genera Kloeckera, Candida, Starmerella, Hanseniaspora, Kluyveromyces / Lachance, Metschnikowia, Saccharomycodes, Zygosaccharomyces, Dekkera (also known as Brettanomyces), Wickerhamomyces, or Torulaspora.Examples of non-yeast yeasts include, but are not limited to, *Hanseniaspora uvarum*, *Hanseniaspora guillermondii*, *Hanseniaspora vinae*, *Metschnikowia pulcherrima*, *Kluyveromyces / Lachancea thermotolerans*, *Starmerella bacillaris* (formerly known as *Candida stellata* / *Candida zemplinina*), *Saccharomycodes ludwigii*, *Zygosaccharomyces rouxii*, *Dekkera bruxellensis*, *Dekkera anomala*, and *Brettanomyces*. The species include *Custersianus*, *Brettanomyces naardenensis*, *Brettanomyces nanus*, *Wickerhamomyces anomalus*, and *Torulaspora delbrueckii*.

[0233] In some embodiments, the methods described herein involve using more than one genetically modified yeast. For example, in some embodiments, the method may involve using more than one genetically modified yeast belonging to the genus *Saccharomyces*. In some embodiments, the method may involve using more than one genetically modified yeast belonging to a non-*Saccharomyces* genus. In some embodiments, the method may involve using more than one genetically modified yeast belonging to the genus *Saccharomyces* and one genetically modified yeast belonging to a non-*Saccharomyces* genus. Alternatively, or furthermore, any method described herein may involve using one or more genetically modified yeasts and one or more non-genetically modified (wild-type) yeasts.

[0234] In some embodiments, the yeast is a hybrid strain. As will be appreciated by those skilled in the art, the term "hybrid strain" of yeast refers to a yeast strain produced by hybridizing two different yeast strains, for example, to obtain one or more desired characteristics. For example, a hybrid strain may be produced by hybridizing two different yeast strains belonging to the same genus or species. In some embodiments, the hybrid strain is produced by hybridizing a *Saccharomyces cerevisiae* strain and a *Cypripedium spp.* strain. See, for example, Krogerus et al. Microbial Cell Factories (2017) 16: 66.

[0235] In some embodiments, the yeast strain is a wild yeast strain, such as a yeast strain isolated from a natural source and subsequently propagated. Alternatively, in some embodiments, the yeast strain is a domesticated yeast strain. Domesticated yeast strains are artificially selected and cultivated to acquire desired characteristics.

[0236] In some implementations, genetically modified yeast cells can be used in a symbiotic substrate with other yeast or bacterial strains. This symbiotic substrate of yeast cells and bacterial strains can be used, for example, in the production of fermented beverages such as kombucha, kefir, and ginger beer. For example, *Saccharomyces fragilis* is part of a kefir culture and grows on lactose contained in whey. Other bacterial strains that can be used with genetically modified yeast cells in a symbiotic substrate include *Bifidobacterium animalis* subsp. *lactis*, *Bifidobacterium breve*, bacteria of the genus *Lactobacillus*, and bacteria of the genus *Pediococcus*.

[0237] Although many fermented beverages are produced using *Saccharomyces cerevisiae* strains, other yeast genera have also gained attention in the production of fermented beverages and can be used in conjunction with modified cells in a symbiotic matrix. In some embodiments, the other yeast cells belong to non-yeast genera. See, for example, Crauwels et al. *Brewing Science* (2015) 68: 110-121; Esteves et al. *Microorganisms* (2019) 7(11): 478. In some embodiments, other yeast cells belong to the genera Kloeckera, Candida, Starmerella, Hanseniaspora, Kluyveromyces / Lachance, Metschnikowia, Saccharomycodes, Zygosaccharomyces, Dekkera (also known as Brettanomyces), Wickerhamomyces, or Torulaspora.Examples of non-yeast yeasts include, but are not limited to, *Hanseniaspora uvarum*, *Hanseniaspora guillermondii*, *Hanseniaspora vinae*, *Metschnikowia pulcherrima*, *Kluyveromyces / Lachancea thermotolerans*, *Starmerella bacillaris* (formerly known as *Candida stellata* / *Candida zemplinina*), *Saccharomycodes ludwigii*, *Zygosaccharomyces rouxii*, *Dekkera bruxellensis*, *Dekkera anomala*, and *Brettanomyces*. The species include *Custersianus*, *Brettanomyces naardenensis*, *Brettanomyces nanus*, *Wickerhamomyces anomalus*, and *Torulaspora delbrueckii*.

[0238] Methods for genetically modifying yeast cells are known in the art. In some embodiments, the yeast cells are diploid, and a copy of a heterologous gene encoding an enzyme with glycosidase activity as described herein is introduced into the yeast genome.

[0239] In some embodiments, the yeast cell is diploid, and one copy of a heterologous gene encoding an enzyme with the desired activity described herein (e.g., an enzyme with AAT activity, an enzyme with carbon-sulfur-lyase (CSL) activity, HMG1 enzyme, ERG20 enzyme, linalool synthase, and / or geraniol synthase) is introduced into two copies of the yeast genome. In some embodiments, the copies of the heterologous gene are identical. In some embodiments, the copies of the heterologous gene are not identical, but the gene encodes the same enzyme with the desired activity. In some embodiments, the copies of the heterologous gene are not identical, and the gene encodes an enzyme with different activities (e.g., mutants, variants, and fragments thereof). In some embodiments, the cell contains a gene encoding an enzyme with the desired activity, referred to as an endogenous gene, and also contains a second gene encoding an enzyme with the same desired activity, the enzyme of which may be the same as or different from the enzyme encoded by the endogenous gene with the desired activity.

[0240] In some embodiments, the yeast cell is diploid, and one copy of the gene encoding an enzyme having the desired activity (e.g., an enzyme with AAT activity, an enzyme with carbon-sulfur-lyase (CSL) activity, HMG1 enzyme, ERG20 enzyme, linalool synthase, and / or geraniol synthase) is introduced into two copies of the yeast genome. In some embodiments, the copies of the gene encoding the enzyme having the desired activity are identical. In some embodiments, the copies of the gene encoding the enzyme having the desired activity are not identical, but the gene encodes the same enzyme with the same or substantially similar activity. In some embodiments, the copies of the gene encoding the enzyme having the desired activity are not identical, and the gene encodes an enzyme with different activities (e.g., mutants, variants, and fragments thereof). In some embodiments, the cell contains a gene encoding an enzyme having the desired activity, referred to as an endogenous gene, and also contains a second gene encoding an enzyme with the same or substantially similar activity, the enzyme encoded by the second gene may be the same as or different from the enzyme having that activity encoded by the endogenous gene.

[0241] In some embodiments, the yeast cell is tetraploid. A tetraploid yeast cell is a cell that maintains four complete sets of chromosomes (i.e., four copies of complete chromosomes). In some embodiments, the yeast cell is tetraploid, and a copy of the heterologous gene encoding an enzyme having the desired activity (e.g., an enzyme with AAT activity, an enzyme with carbon-sulfur-lyase (CSL) activity, HMG1 enzyme, ERG20 enzyme, linalool synthase, and / or geraniol synthase) is introduced into at least one copy of the genome. In some embodiments, the yeast cell is tetraploid, and a copy of the heterologous gene encoding an enzyme having the desired activity is introduced into more than one copy of the genome. In some embodiments, the yeast cell is tetraploid, and a copy of the heterologous gene encoding an enzyme having the desired activity is introduced into all four copies of the genome. In some embodiments, the copies of the heterologous gene are identical. In some embodiments, the copies of the heterologous gene are not identical, but the gene encodes the same enzyme having the desired activity or substantially similar activity. In some implementations, the copies of the heterologous gene are not identical, and the gene encodes an enzyme with different activities (e.g., mutants, variants, and fragments thereof).

[0242] In some embodiments, the yeast cell is tetraploid, and a copy of the gene encoding an enzyme having the desired activity, as described herein, is incorporated into at least one copy of the genome. In some embodiments, the yeast cell is tetraploid, and a copy of the gene encoding an enzyme having the desired activity, as described herein, is incorporated into more than one copy of the genome. In some embodiments, the yeast cell is tetraploid, and a copy of the gene encoding an enzyme having the desired activity, as described herein, is incorporated into all four copies of the genome. In some embodiments, the copies of the gene encoding an enzyme having the desired activity are identical. In some embodiments, the copies of the gene encoding an enzyme having the desired activity are not identical, but the gene encodes the same enzyme having that activity or substantially similar activity. In some embodiments, the copies of the gene encoding an enzyme having that activity are not identical, and the gene encodes an enzyme with different activities (e.g., mutants, variants, and fragments thereof). In some embodiments, the cell contains a gene encoding an enzyme, referred to as an endogenous gene, and also contains one or more additional copies of a gene encoding an enzyme having the desired activity or substantially similar activity, the enzyme encoded by which the additional copies may encode an enzyme having O-methyltransferase activity that is the same as or different from that encoded by the endogenous gene.

[0243] In some embodiments, the growth rate of the modified cells is not substantially impaired relative to wild-type yeast cells that do not contain genetic modifications. Methods for measuring and comparing the growth rates of the two cell types will be known to those skilled in the art. Non-limiting examples of growth rates that can be measured and compared between two types of cells are replication rate, budding rate, colony-forming units (CFU) produced per unit time, and the amount of decrease in fermentable sugars in the culture medium per unit time. The growth rate of the modified cells is considered "not substantially impaired" relative to the wild-type cells if the measured growth rate is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% of the wild-type cell growth rate.

[0244] Yeast cell strains that can be used with the methods described herein will be known to those skilled in the art and include yeast strains used for brewing the desired fermented beverages as well as commercially available yeast strains. Examples of common beer strains include, but are not limited to, American ale strains, Belgian ale strains, British ale strains, Belgian lambic / sour ale strains, Barleywine / ImperialStout strains, India Pale Ale strains, Brown Ale strains, Kolsch and Altbier strains, Stout and Porter strains, and Wheat beer strains.

[0245] Non-limiting examples of strains that can be used with the genetically modified cells and methods described herein include: Chico American Ale, Wyeast 1056 American Ale 1056, Wyeast 1272 American Ale II 1272, Wyeast Denny's Favorite 50 1450, Wyeast 1332 Northwest Ale 1332, Wyeast 1187 Ringwood Ale 1187, Siebel Inst. BRY 96 American Ale BRY 96, White Labs WLP060 American Ale Yeast Blend, and White Labs V WLP051 California Ale. White Labs WLP051, White Labs WLP001 California Ale, White Labs WLP076 Old Sonoma Ale, White Labs WLP041 Pacific Ale, White Labs WLP008 East Coast Ale, White Labs WLP039 East Midlands Ale, White Labs San Diego Super Yeast, White Labs San Francisco Lager, White Labs Neutral Grain, and Lallemand West Coast Ale BRY-97.American West Coast Ale BRY-97, Lallemand CBC-1 (for secondary fermentation in barrels and bottles), Brewferm Top, Coopers Pure Brewers' Yeast, Fermentis US-05, Real Brewers Yeast Lucky #7, Muntons Premium Gold, Muntons Standard Yeast, East Coast YeastNortheast Ale ECY29, East Coast Yeast Old Newark Ale ECY10, East Coast Yeast Old Newark Beer ECY12, Fermentis Safale US-05, Fermentis Safbrew T-58, Real Brewers Yeast The One, Mangrove Jack US West Coast Yeast, Mangrove Jack Workhorse Beer Yeast, Lallemand Abbaye Belgian Ale, White Labs Abbey IV WLP540, White Labs American Farmhouse Blend WLP670, White Labs Antwerp Ale WLP515, East Coast Yeast Belgian Abbaye ECY09, White Labs Belgian Ale WLP550, Mangrove Jack Belgian Ale Yeast, and Wyeast Belgian Dark Ale 3822-PCWyeast Belgian Dark Ale 3822-PC, Wyeast Belgian Saison 3724, White Labs Belgian Saison I WLP565, White Labs Belgian Saison II WLP566, White Labs Belgian Saison III WLP585, Wyeast Belgian Schelde Ale 3655-PC, Wyeast Belgian Stout 1581-PC, White Labs Belgian Style Ale Yeast Blend WLP575, White... White Labs Belgian Style Saison Ale Blend WLP568, East Coast Yeast Belgian White ECY11, Lallemand Belle Saison, Wyeast Biere de Garde 3725-PC, White Labs Brettanomyces Bruxellensis Trois Vrai WLP648, Brewferm Top, Wyeast Canadian / Belgian Ale 3864-PC, Lallemand CBC-1 (for secondary fermentation in barrels and bottles), Wyeast Farmhouse Ale 3726-PC, East Coast Yeast Farmhouse Brett ECY03. ECY03), Wyeast Golden Ale Yeast 3739-PC (WyeastFlanders Golden Ale 3739-PC), White Labs Flemish Ale Blend WLP665, White Labs French Ale WLP072, Wyeast French Saison 3711, Wyeast Leuven Pale Ale 3538-PC, Fermentis Safbrew T-58, East Coast Yeast Saison Brasserie Blend ECY08, East Coast Yeast Saison Single-Strain ECY14, Real Brewers Yeast TheMonk, Siebel Inst. Trappist Ale BRY 204 Wyeast Trappist Ale (ECY13), Wyeast Trappist Ale (WLP500), Wyeast Trappist Blend 3789-PC, Wyeast British Ale 1098, Wyeast British Ale II 1335, Wyeast British Cask Ale 1026-PC, Wyeast English Special Bitter Ale 1768-PC, and Wyeast Irish Ale 1084. 1084), London Ale, Wyeast London Ale 10281028), Wyeast London Ale III 1318, Wyeast London ESB Ale 1968, Wyeast Ringwood Ale 1187, Wyeast Thames Valley Ale 1275, Wyeast Thames Valley Ale II 1882-PC, Wyeast West Yorkshire Ale 1469, Wyeast Whitbread Ale 1099, Mangrove Jack British Ale Yeast, Mangrove Jack Burton Union Yeast, Mangrove Jack All-Purpose Brewer Yeast, East Coast Yeast British Mild Ale ECY18, East Coast Yeast Northeast Ale ECY29, East Coast Yeast Burton Union Yeast ECY17, East Coast Yeast Old Newark Ale ECY10, White Labs Bedford British Ale WLP006, White Labs British Ale WLP005, White Labs Burton Ale WLP023, White Labs East Midlands Ale WLP039, White Labs British Ale Mixed Yeast WLP085English Ale Blend WLP085), White Labs English Ale WLP002, White Labs Essex Ale Yeast WLP022, White Labs Irish Ale WLP004, White Labs London Ale WLP013, White Labs Manchester Ale WLP038, White Labs Old Sonoma Ale WLP076, White Labs San Diego Super Yeast WLP090, White Labs Whitbread Ale WLP017, White White Labs North Yorkshire Ale WLP037, Coopers Pure Brewers' Yeast, Siebel Inst. English Ale BRY 264, Muntons Premium Gold, Muntons Standard Yeast, Lallemand Nottingham, Fermentis Safale S-04, Fermentis Safbrew T-58, Lallemand Windsor, Real Brewers Yeast Ye Olde English, Brewferm Top, White Labs American Whiskey WLP065, White Labs Dry English Ale WLP007, White Labs Edinburgh Ale WLP028Edinburgh Ale WLP028, Fermentis Safbrew S-33, Wyeast Scottish Ale 1728, East Coast Yeast Scottish Heavy ECY07, White Labs Super High Gravity WLP099, White Labs Whitbread Ale WLP017, Wyeast Belgian Lambic Blend 3278, Wyeast Belgian Schelde Ale 3655-PC, Wyeast Berliner-Weisse Blend 3191-PC, Wyeast Brussels Brett Yeast 5112 Wyeast Brettanomyces Bruxellensis 5112, Wyeast Brettanomyces Lambicus 5526, Wyeast Lactobacillus 5335, Wyeast Pediococcus Cerevisiae 5733, Wyeast Roeselare Ale Blend 3763, Wyeast Trappist Blend 3789-Pc, White Labs Belgian Sour Mix Wlp655, White Labs Berliner Weisse Blend Wlp630, White White Labs “Brussels” Trois yeast WLP644 (White Labs Saccharomyces “Bruxellensis” Trois Wlp644), WhiteWhite Labs Brettanomyces Bruxellensis Wlp650, White Labs Brettanomyces Claussenii Wlp645, White Labs Brettanomyces Lambicus Wlp653, White Labs Flemish Ale Blend Wlp665, East Coast Yeast Berliner Blend Ecy06, East Coast Yeast Brett Anomala Ecy04, East Coast Yeast Brett Bruxelensis Ecy05, and East Coast Yeast Brett Custersianus Ecy19 Ecy19), East Coast Yeast Brett Nanus Ecy16, Strain #2, East Coast Yeast BugCounty ECY20, East Coast Yeast BugFarm ECY01, East Coast Yeast Farmhouse Brett ECY03, East Coast Yeast Flemish Ale ECY02, East Coast Yeast Oud Brune ECY23, Wyeast American Ale 1056, Siebel Inst. American Ale BRY96, White Labs American Ale Yeast Blend WLP060, White Labs Bourbon Yeast WLP070White Labs Bourbon Yeast WLP070, White Labs California Ale VWLP051, White Labs California Ale WLP001, White Labs Dry English Ale WLP007, White Labs East Coast Ale WLP008, White Labs Neutral Grain WLP078, White Labs Super High Gravity WLP099, White Labs Tennessee WLP050, Fermentis US-05, Real Brewers Yeast Lucky #7, Fermentis Safbrew S-33, East Coast Yeast Scottish Heavy Ale ECY07 (East Coast Yeast Scottish Heavy ECY07), Lallemand Windsor (British Ale), Wyeast American Ale 1056, Wyeast American Ale II 1272, Wyeast British Ale 1098, Wyeast British Ale II 1335, Wyeast Denny's Favorite 50 1450, Wyeast London Ale 1028, Wyeast London Ale III 1318, Wyeast London ESB Ale 1968, Wyeast Northwest Ale 1332 Ale1332), WyeastRingwood Ale 1187, Siebel Inst. American Ale BRY 96, White Labs American Ale Yeast Blend WLP060, White Labs Bedford British Ale WLP006, White Labs British Ale WLP005, White Labs Burton Ale WLP023, White Labs California Ale V WLP051, White Labs California Ale WLP001, White Labs East Coast Ale WLP008, White Labs... White Labs English Ale WLP002, White Labs London Ale WLP013, White Labs Essex Ale Yeast WLP022, White Labs Pacific Ale WLP041, White Labs San Diego Super Yeast WLP090, White Labs Whitbread Ale WLP017, BrewfermTop, Mangrove Jack Burton Union Yeast, Mangrove Jack US West Coast Yeast, Mangrove JackMangrove Jack Workhorse Beer Yeast, Coopers Pure Brewers' Yeast, Fermentis US-05, Fermentis Safale S-04, Fermentis Safbrew T-58, RealBrewers Yeast Lucky #7, Real Brewers Yeast The One, Muntons Premium Gold, Muntons Standard Yeast, East Coast Yeast Northeast Ale ECY29, Lallemand Nottingham, Lallemand Windsor, Wyeast American Ale 1056, Wyeast American Ale II 1272, Wyeast British Ale 1098, Wyeast British Ale II 1335 (Wyeast British Ale II 1335), Wyeast Thames Valley Ale 1275, Wyeast Thames Valley Ale II 1882-PC, Wyeast West Yorkshire Ale 1469, Wyeast Whitbread Ale 1099, Wyeast British Cask Ale 1026-PC, Wyeast English Special Bitter Ale 1768-PC, Wyeast London Ale 1028, Wyeast London Ale III 1318 (Wyeast London Ale III 1318), Wyeast London ESB Ale Yeast 1968 (WyeastLondonESB Ale 1968), Wyeast Northwest Ale 1332, Wyeast Ringwood Ale 1187, White Labs American Ale Yeast Blend WLP060, White Labs British Ale WLP005, White Labs Bedford British Ale WLP006, White Labs British Ale WLP005, White Labs Burton Ale WLP023, White Labs California Ale V WLP051, White Labs California Ale WLP001 California Ale WLP001), White Labs East Coast Ale WLP008, White Labs English Ale WLP002, White Labs Essex Ale Yeast WLP022, White Labs French Ale WLP072, White Labs London Ale WLP013, White Labs Pacific Ale WLP041, White Labs Whitbread Ale WLP017, Brewferm Top, East Coast Yeast British Mild Ale ECY18ECY18), Coopers Pure Brewers' Yeast, Muntons Premium Gold, Muntons Standard Yeast, Mangrove Jack Newcastle Dark Ale Yeast, Lallemand CBC-1 (bottle and bottle mature yeast), Lallemand Nottingham, Lallemand Windsor (British Ale), Fermentis Safale S-04, Fermentis US-05, Siebel Inst. American Ale BRY 96, Wyeast American Wheat 1010, Wyeast German Ale 1007, Wyeast Kölsch 2565, Wyeast Kölsch II 2575-PC, WhiteLabs Belgian Lager WLP815, White Labs Düsseldorf Alt WLP036, White Labs European Ale WLP011, White Labs German Ale / Kölsch WLP029, East Coast Yeast Kölschbier ECY21, Mangrove Jack Universal Brewing Yeast, Siebel Inst. Alt Ale BRY 144, Wyeast American Ale 1056, Wyeast American Ale II 1272 1272), Wyeast British Ale 1098, Wyeast British Ale II 1335Wyeast British Ale II 1335, Wyeast Denny's Favorite 50 Yeast 1450, Wyeast English Special Bitter 1768-PC, Wyeast Irish Ale 1084, Wyeast London Ale 1028, Wyeast London Ale III 1318, Wyeast London ESB Ale 1968, Wyeast Northwest Ale 1332, Wyeast Ringwood Ale 1187 1187), Wyeast Thames Valley Ale 1275, Wyeast Thames Valley Ale II 1882-PC, Wyeast West Yorkshire Ale 1469, Wyeast Whitbread Ale 1099, White Labs American Ale Yeast Blend WLP060, White Labs Bedford British Ale WLP006, White Labs British Ale WLP005, White Labs Burton Ale WLP023, White Labs... White Labs California Ale VWLP051 (White Labs California Ale VWLP051), White Labs California Ale WLP001 (White LabsCalifornia Ale WLP001), White Labs East Coast Ale WLP008, White Labs East Midlands Ale WLP039, White Labs English Ale WLP002, White Labs Essex Ale Yeast WLP022, White Labs Irish Ale WLP004, White Labs London Ale WLP013, White Labs Old Sonoma Ale WLP076, White Labs Pacific Ale WLP041, White Labs Whitbread Ale WLP017 WLP017), Coopers Pure Brewers' Yeast, Fermentis US-05, Muntons Premium Gold, Muntons Standard Yeast, Fermentis Safale S-04, Lallemand Nottingham, Lallemand Windsor (British Ale), Siebel Inst. American Ale BRY 96, White Labs American Hefeweizen Ale 320, White Labs Bavarian Weizen Ale 351, White Labs Belgian Wit Ale 400, White Labs Belgian Wit Ale II 410410), White Labs Hefeweizen Ale 300, White Labs Hefeweizen IV Ale 380, Wyeast American Wheat 1010, Wyeast Bavarian Wheat 3638, Wyeast Bavarian Wheat Blend 3056, Wyeast Belgian Ardennes 3522, Wyeast Belgian Wheat 3942, Wyeast Belgian Witbier 3944, Wyeast Canadian / Belgian Ale 3864-PC 3864-PC), Wyeast Forbidden Fruit Yeast 3463, Wyeast German Wheat 3333, Weihenstephan Weizen 3068, Siebel Institute Bavarian Weizen BRY 235, Fermentis Safbrew WB-06, Mangrove Jack Bavarian Wheat, Lallemand Munich (German wheat beer), Brewferm Blanche, Brewferm Lager, East Coast Yeast Belgian White ECY11, Augustiner, Augustiner Lager Yeast Lager), W-34 / 70, Andechs, Andechs Lager, D254, RC212, BO213. In some embodiments, the yeast is the Saccharomyces cerevisiae Chico strain.

[0246] Methods and liquid fermentation compositions (and fermentation products)

[0247] Various aspects of this disclosure relate to methods for producing fermented products using any genetically modified yeast cells described herein. Fermentation utilizes the natural process by which microorganisms convert carbohydrates into alcohols and carbon dioxide. It is a metabolic process that produces chemical changes in an organic substrate through enzymatic action. In the context of food production, fermentation broadly refers to any process by which microbial activity brings about the desired changes in a food or beverage. The conditions for fermentation and the implementation of fermentation are referred to herein as the "fermentation process."

[0248] In some aspects, this disclosure relates to methods for producing fermented products, such as fermented beverages, including contacting any modified cells described herein with a culture medium containing at least one fermentable sugar during a first fermentation process to produce the fermented product. As used herein, "culture medium" refers to a liquid that promotes fermentation, i.e., a liquid that does not inhibit or prevent the fermentation process. In some embodiments, the culture medium is water.

[0249] In some embodiments, a method for producing a fermented beverage includes: (a) providing genetically modified yeast cells comprising functional disruption in one or more enzymes associated with maltose and / or maltotriose transport and / or maltose and / or maltotriose hydrolysis in the yeast cells, wherein the functional disruption results in slower growth of the yeast cells when maltose is the sole sugar source, compared to yeast cells without the genetic modification; (b) providing a culture medium containing a sugar source derived from wort; (c) combining the genetically modified yeast cells with the culture medium to form a fermentation composition; and (d) allowing the fermentation composition to ferment to produce a fermented beverage.

[0250] In some embodiments, a method of producing a fermented beverage includes contacting a population of genetically modified yeast cells according to the present disclosure with a culture medium containing a sugar source derived from malt extract during fermentation to produce the fermented beverage.

[0251] In some embodiments, a fermented beverage produced by the methods disclosed herein is provided. In some embodiments, a fermented beverage comprising an alcohol content not exceeding 1.0% (v / v) and nucleic acids derived from genetically modified yeast cells of this disclosure is provided.

[0252] It should be understood that cells modified in any aspect of this disclosure may be used in the methods of this disclosure.

[0253] In some embodiments, the fermented beverage is a low-alcohol fermented beverage. As used herein, "low-alcohol fermented beverage" means a beverage with a lower alcohol content compared to a beverage fermented using strains that metabolize maltose and maltotriose. In some embodiments, the alcohol content of the fermented beverage is less than or equal to about 1.0% (v / v) alcohol. In some embodiments, the alcohol content of the fermented beverage is less than or equal to about 0.5% (v / v) alcohol.

[0254] In some implementations, the method does not include steps of physically removing alcohol from the beverage or prematurely terminating fermentation.

[0255] In some embodiments, at least one fermentable sugar is provided in the wort-derived sugar source.

[0256] In some implementations, the fermentation process results in a reduction of the wort sugar level by at least 15% but no more than 25%.

[0257] In some implementations, the fermented beverage is beer.

[0258] As used herein, the term "fermentable sugar" refers to a carbohydrate that can be converted into alcohol and carbon dioxide by microorganisms (such as any cells described herein). In some embodiments, fermentable sugars are converted into alcohol and carbon dioxide by enzymes, such as recombinases or cells expressing such enzymes. The genetically modified yeast cells described herein cannot convert maltose and / or maltotriose into ethanol (they cannot ferment maltose and / or maltotriose). Therefore, although maltose and / or maltotriose may be present in a culture medium (e.g., wort), the genetically modified yeast cells do not ferment these sugars into ethanol. Examples of fermentable sugars that can be utilized by the genetically modified yeast cells described herein include, but are not limited to, glucose, fructose, lactose, and sucrose.

[0259] In some embodiments, the fermentable sugar is provided in the sugar source. The sugar source used in the methods of the present invention can depend on, for example, the type of fermentation product and the type of fermentable sugar. Examples of sugar sources include, but are not limited to, wort, cereals / grains, fruit juices (e.g., grape juice and apple juice / cider), honey, sucrose, rice, and koji. Examples of fruits from which fruit juice is available include, but are not limited to, grapes, apples, blueberries, blackberries, raspberries, currants, strawberries, cherries, pears, peaches, nectarines, oranges, pineapples, mangoes, and passion fruit.

[0260] In some embodiments, the modified cells described herein are cultured in an anaerobic or semi-anaerobic environment. Anaerobic cell culture refers to a technique for culturing microorganisms (such as modified yeast cells) in an environment where no oxygen is available. Semi-anaerobic cell culture refers to a technique for culturing microorganisms (such as modified yeast cells) in an environment where oxygen availability is limited, such as in a pre-oxidized culture medium.

[0261] As will be apparent to those skilled in the art, in certain circumstances, it may be necessary to treat the sugar source to make the fermentable sugars available for fermentation. Taking beer production as an example of a fermented beverage, grains (cereals, barley) are soaked in water, causing the grain-water mixture to activate maltase, which converts starch into fermentable sugars—a process known as “saccharification.” The grains are then boiled to concentrate the sugars and the solution is sterilized. As used herein, the term “wort” refers to a liquid produced through the saccharification and boiling processes, containing fermentable sugars. The wort is then exposed to fermenting microorganisms (such as any cells described herein), allowing the enzymes of these microorganisms to convert the sugars in the wort into alcohols and carbon dioxide.

[0262] In some embodiments, the grains are germinated, ungerminated, or a combination of germinated and ungerminated grains. Examples of grains used in the methods described herein include, but are not limited to, barley, oats, corn, rice, rye, sorghum, wheat, karasumugi, and hatomugi.

[0263] In examples of sake production, the sugar source is rice. The rice is cultured with Aspergillus oryzae to convert the rice starch into fermentable sugars, producing koji (fermentation starter). The koji is then exposed to fermenting organisms (e.g., any cells described herein), allowing the enzymes in these organisms to convert the sugars in the koji into alcohols and carbon dioxide.

[0264] In some embodiments, the methods described herein relate to the production of a culture medium, which may involve heating or soaking a sugar source, for example, in water. In some embodiments, the water temperature is at least 50 degrees Celsius (50°C), and the sugar source is incubated for a period of time. In some embodiments, the water temperature is at least 75°C, and the sugar source is incubated for a period of time. In some embodiments, the water temperature is at least 100°C, and the sugar source is incubated for a period of time. Preferably, the culture medium is cooled before adding any of the cells described herein.

[0265] In some embodiments, the methods described herein further include adding at least one (e.g., 1, 2, 3, 4, 5 or more) hop variety during fermentation, for example, by adding it to a culture medium or wort. Hops are the flowers of the hop plant (Humulus lupulus) and are commonly used in fermentation to impart a variety of flavors and aromas to the fermented product. In addition to floral, fruity, and / or citrus flavors and aromas, hops are believed to also impart bitterness and can be characterized according to their intended use. For example, bitter hops impart a certain degree of bitterness to the fermented product due to the presence of alpha acids, while aromatic hops, with their lower alpha acid content, contribute the desired aromas and flavors to the fermented product.

[0266] Whether to add one or more hop varieties to the culture medium and / or wort, and the stage at which the hops are added, can be based on a variety of factors, such as the intended use of the hops. For example, hops intended to impart bitterness to the fermentation product are typically added during wort preparation, such as during wort boiling. In some embodiments, hops intended to impart bitterness to the fermentation product are added to the wort and boiled with the wort for a period of time, such as about 15-60 minutes. In contrast, hops intended to impart desired aromas to the fermentation product are typically added later than hops used for bitterness. In some embodiments, hops intended to impart desired aromas to the fermentation product are added at the end of boiling or after the wort has boiled (i.e., "dry hopping"). In some embodiments, one or more hop varieties may be added multiple times during the process (e.g., at least two, at least three, or more times).

[0267] In some embodiments, hops are added in the form of wet or dry hops and may optionally be boiled with the wort. In some embodiments, hops are in the form of dried hop pellets. In some embodiments, at least one hop variety is added to the culture medium. In some embodiments, the hops are wet (i.e., undried). In some embodiments, the hops are dried and may optionally be further processed before use. In some embodiments, the hops are added to the wort before the fermentation process. In some embodiments, the hops are boiled in the wort. In some embodiments, the hops are boiled with the wort and then cooled with the wort.

[0268] Many hop varieties are known in the art and can be used in the methods described herein. Examples of hop varieties include, but are not limited to: Ahtanum, Amarillo, Apollo, Cascade, Centennial, Chinook, Citra, Cluster, Columbus, Crystal / Chrystal, Eroica, Galena, Glacier, Greenburg, Horizon, Liberty, Millennium, Mosaic, Mount Hood, Mount Rainier, Newport, Nugget, Palisade, Santiam, Simcoe, Sterling, Summit, Tomahawk, Ultra, Vanguard, Warrior, Willamette, Zeus, Admiral, Brewer's Gold, Bullion, Challenger, First Gold, Fuggles, Goldings, Herald, Northdown, Northern Brewer, Phoenix, Pilot, Pioneer, Progress, Target, and WhitbreadGolding. Variety (WGV), Hallertau, Hersbrucker, Saaz, Tettnang, Spalt, Feux-CoeurFrancais, Galaxy, Green Bullet, Motueka, Nelson Sauvin, Pacific Gem, Pacific Jade, Pacifica, Pride of Ringwood, Riwaka, Southern Cross, Lublin, Magnum, Perle, Polnischer Lublin, Saphir, Satus, Select, Strisselspalt, Styrian Goldings, Tardifde Bourgogne, Tradition, Bravo, Calypso, Chelan, Comet, El Dorado, San Juan RubyRed, Sonnet Golding, Super Galena, Tillicum, Bramling Cross, Pilgrim, HallertauerHerkules, Hallertauer Magnum, HallertauerTaurus, Merkur, Opal, Smaragd, HalleratauAroma, Kohatu, Rakau, Stella, Sticklebract, Summer Saaz, Super Alpha, Super Pride, Topaz, Wai-iti, Bor, Junga, Marynka, Premier, Sladek, Styrian Atlas, Styrian Aurora, Styrian Bobek, Styrian Celeia, Sybilla Sorachi Ace, Hallertauer Mittelfrueh, Hallertauer Tradition, Tettnanger, Tahoma, Triple Pearl, Yakima Gold and Michigan Copper.

[0269] In some embodiments, the fermentation process of at least one sugar source comprising at least one fermentable sugar can be carried out for about 1 day to about 31 days. In some embodiments, the fermentation process is carried out for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, or longer. In some embodiments, the fermentation process of one or more fermentable sugars can be carried out at a temperature of about 4°C to about 30°C. In some embodiments, the fermentation process of one or more fermentable sugars can be carried out at a temperature of about 8°C to about 14°C or about 18°C ​​to about 24°C. In some embodiments, the fermentation process of one or more fermentable sugars can be carried out at a temperature of about 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.

[0270] In some embodiments, fermentation leads to a decrease in the amount of fermentable sugars present in the culture medium, referred to as decay. In some embodiments, the decrease in the amount of fermentable sugars occurs at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days or longer after the start of fermentation. In some embodiments, the fermentation method using genetically modified yeast cells as described herein results in a decrease in the amount of fermentable sugars of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35%. In some embodiments, the amount of fermentable sugars fermented by the modified cells is reduced compared to the amount of fermentable sugars fermented by wild-type yeast cells in the same amount of time or during the same fermentation process. In some embodiments, the modified cells ferment up to 90% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the amount of fermentable sugar compared to wild-type yeast cells fermenting in the same amount of time or during the same fermentation process.

[0271] In some embodiments, fermentation results in varying levels of acetate in the fermentation product (e.g., beer). In some embodiments, the fermentation product contains isoamyl acetate at a concentration of about 10 µg / L to about 4000 µg / L. In some embodiments, the fermentation product comprises isoamyl acetate in amounts of about 9.1 µg / L to about 4000 µg / L, about 9.2 µg / L to about 4000 µg / L, about 10 µg / L to about 4000 µg / L, about 20 µg / L to about 4000 µg / L, about 30 µg / L to about 4000 µg / L, about 40 µg / L to about 4000 µg / L, about 50 µg / L to about 4000 µg / L, about 60 µg / L to about 4000 µg / L, about 69.6 µg / L to about 4000 µg / L, about 70 µg / L to about 4000 µg / L, about 80 µg / L to about 4000 µg / L, about 90 µg / L to about 4000 µg / L, about 100 µg / L to about 4000 µg / L, and about 150 µg / L. Approximately 200 µg / L to 4000 µg / L, approximately 250 µg / L to 4000 µg / L, approximately 300 µg / L to 4000 µg / L, approximately 400 µg / L to 4000 µg / L, approximately 500 µg / L to 4000 µg / L, approximately 600 µg / L to 4000 µg / L, approximately 750 µg / L to 4000 µg / L, approximately 800 µg / L to 4000 µg / L, approximately 1000 µg / L to 4000 µg / L, approximately 2000 µg / L to 4000 µg / L, approximately 9.1 µg / L to 3000 µg / L, approximately 9.2 µg / L to 3000 µg / L, approximately 10 µg / L to 3000 µg / L µg / L, about 20 µg / L to about 3000 µg / L, about 30 µg / L to about 3000 µg / L, about 40 µg / L to about 3000 µg / L, about 50 µg / L to about 3000 µg / L, about 60 µg / L to about 3000 µg / L, about 69 µg / L.6 µg / L to about 3000 µg / L, about 70 µg / L to about 3000 µg / L, about 80 µg / L to about 3000 µg / L, about 90 µg / L to about 3000 µg / L, about 100 µg / L to about 3000 µg / L, about 150 µg / L to about 3000 µg / L, about 200 µg / L to about 3000 µg / L, about 250 µg / L to about 3000 µg / L, about 300 µg / L to about 3000 µg / L, about 400 µg / L to about 3000 µg / L, about 500 µg / L to about 3000 µg / L, about 600 µg / L to about 3000 µg / L, about 750 µg / L to about 3000 µg / L, about 800 µg / L to about 3000 µg / L µg / L, about 1000 µg / L to about 3000 µg / L, about 2000 µg / L to about 3000 µg / L, about 9.1 µg / L to about 2000 µg / L, about 9.2 µg / L to about 2000 µg / L, about 10 µg / L to about 2000 µg / L, about 20 µg / L to about 2000 µg / L, about 30 µg / L to about 2000 µg / L, about 40 µg / L to about 2000 µg / L, about 50 µg / L to about 2000 µg / L, about 60 µg / L to about 2000 µg / L, about 69.6 µg / L to about 2000 µg / L, about 70 µg / L to about 2000 µg / L, about 80 µg / L to about 2000 µg / L, about 90 µg / L to about 2000 µg / L µg / L, about 100 µg / L to about 2000 µg / L, about 150 µg / L to about 2000 µg / L, about 200 µg / L to about 2000 µg / L, about 250 µg / L to about 2000 µg / L, about 300 µg / L to about 2000 µg / L, about 400 µg / L to about 2000 µg / L, about 500 µg / L to about 2000 µg / L, about 600 µg / L to about 2000 µg / L, about 750 µg / L to about 2000 µg / L, about 800 µg / L to about 2000 µg / L, about 1000 µg / L to about 2000 µg / L, about 9.1 µg / L to about 1250 µg / L, about 9.2 µg / L to about 1250 µg / L, about 10 About 1250 µg / L, about 20 µg / L to about 1250 µg / L, about 30 µg / L to about 1250 µg / L, about 40 µg / L to about 1250 µg / L, about 50 µg / L to about 1250 µg / L, about 60 µg / L to about 1250 µg / L, about 69 µg / L.6 µg / L to about 1250 µg / L, about 70 µg / L to about 1250 µg / L, about 80 µg / L to about 1250 µg / L, about 90 µg / L to about 1250 µg / L, about 100 µg / L to about 1250 µg / L, about 150 µg / L to about 1250 µg / L, about 200 µg / L to about 1250 µg / L, about 250 µg / L to about 1250 µg / L, about 300 µg / L to about 1250 µg / L, about 400 µg / L to about 1250 µg / L, about 500 µg / L to about 1250 µg / L, about 600 µg / L to about 1250 µg / L, about 750 µg / L to about 1250 µg / L, about 800 µg / L to about 1250 µg / L µg / L, about 1000 µg / L to about 1250 µg / L, about 9.1 µg / L to about 1100 µg / L, about 9.2 µg / L to about 1100 µg / L, about 10 µg / L to about 1100 µg / L, about 20 µg / L to about 1100 µg / L, about 30 µg / L to about 1100 µg / L, about 40 µg / L to about 1100 µg / L, about 50 µg / L to about 1100 µg / L, about 60 µg / L to about 1100 µg / L, about 69.6 µg / L to about 1100 µg / L, about 70 µg / L to about 1100 µg / L, about 80 µg / L to about 1100 µg / L, about 90 µg / L to about 1100 µg / L, about 100 µg / L to about 1100 µg / L µg / L, about 150 µg / L to about 1100 µg / L, about 200 µg / L to about 1100 µg / L, about 250 µg / L to about 1100 µg / L, about 300 µg / L to about 1100 µg / L, about 400 µg / L to about 1100 µg / L, about 500 µg / L to about 1100 µg / L, about 600 µg / L to about 1100 µg / L, about 750 µg / L to about 1100 µg / L, about 800 µg / L to about 1100 µg / L, about 1000 µg / L to about 1100 µg / L, about 9.1 µg / L to about 1023.3 µg / L, about 9.2 µg / L to about 1023.3 µg / L, about 10 µg / L to about 1023.3 µg / L. µg / L, about 20 µg / L to about 1023.3 µg / L, about 30 µg / L to about 1023.3 µg / L, about 40 µg / L to about 1023.3 µg / L, about 50 µg / L to about 1023.3 µg / L, about 60 µg / L to about 1023.3 µg / L, about 69.6 µg / L to about 1023.3 µg / L, and about 70 µg / L to about 1023 µg / L.3 µg / L, about 80 µg / L to about 1023.3 µg / L, about 90 µg / L to about 1023.3 µg / L, about 100 µg / L to about 1023.3 µg / L, about 150 µg / L to about 1023.3 µg / L, about 200 µg / L to about 1023.3 µg / L, about 250 µg / L to about 1023.3 µg / L, about 300 µg / L to about 1023.3 µg / L, about 400 µg / L to about 1023.3 µg / L, about 500 µg / L to about 1023.3 µg / L, about 600 µg / L to about 1023.3 µg / L, about 750 µg / L to about 1023.3 µg / L, about 800 µg / L to about 1023.3 µg / L. µg / L, about 1000 µg / L to about 1023.3 µg / L, about 9.1 µg / L to about 1000 µg / L, about 9.2 µg / L to about 1000 µg / L, about 10 µg / L to about 1000 µg / L, about 20 µg / L to about 1000 µg / L, about 30 µg / L to about 1000 µg / L, about 40 µg / L to about 1000 µg / L, about 50 µg / L to about 1000 µg / L, about 60 µg / L to about 1000 µg / L, about 69.6 µg / L to about 1000 µg / L, about 70 µg / L to about 1000 µg / L, about 80 µg / L to about 1000 µg / L, about 90 µg / L to about 1000 µg / L, about 100 µg / L Approximately 1000 µg / L to 1000 µg / L, approximately 150 µg / L to 1000 µg / L, approximately 200 µg / L to 1000 µg / L, approximately 250 µg / L to 1000 µg / L, approximately 300 µg / L to 1000 µg / L, approximately 400 µg / L to 1000 µg / L, approximately 500 µg / L to 1000 µg / L, approximately 600 µg / L to 1000 µg / L, approximately 750 µg / L to 1000 µg / L, approximately 800 µg / L to 1000 µg / L, approximately 9.1 µg / L to 812.8 µg / L, approximately 9.2 µg / L to 812.8 µg / L, approximately 10 µg / L to 812.8 µg / L, and approximately 20 µg / L to 812.8 µg / L. µg / L, about 30 µg / L to about 812.8 µg / L, about 40 µg / L to about 812.8 µg / L, about 50 µg / L to about 812.8 µg / L, about 60 µg / L to about 812.8 µg / L, about 69.6 µg / L to about 812.8 µg / L, about 70 µg / L to about 812.8 µg / L, about 80 µg / L to about 812.8 µg / L.8 µg / L, about 90 µg / L to about 812.8 µg / L, about 100 µg / L to about 812.8 µg / L, about 150 µg / L to about 812.8 µg / L, about 200 µg / L to about 812.8 µg / L, about 250 µg / L to about 812.8 µg / L, about 300 µg / L to about 812.8 µg / L, about 400 µg / L to about 812.8 µg / L, about 500 µg / L to about 812.8 µg / L, about 600 µg / L to about 812.8 µg / L, about 9.1 µg / L to about 800 µg / L, about 9.2 µg / L to about 800 µg / L, about 10 µg / L to about 800 µg / L, about 20 µg / L to about 800 µg / L, about 30 µg / L Approximately 800 µg / L, approximately 40 µg / L, approximately 50 µg / L, approximately 60 µg / L, approximately 69.6 µg / L, approximately 70 µg / L, approximately 80 µg / L, approximately 90 µg / L, approximately 100 µg / L, approximately 150 µg / L, approximately 200 µg / L, approximately 250 µg / L, approximately 300 µg / L, approximately 400 µg / L, approximately 500 µg / L, approximately 8 ... µg / L, about 600 µg / L to about 800 µg / L, about 750 µg / L to about 800 µg / L, about 9.1 µg / L to about 766 µg / L, about 9.2 µg / L to about 766 µg / L, about 10 µg / L to about 766 µg / L, about 20 µg / L to about 766 µg / L, about 30 µg / L to about 766 µg / L, about 40 µg / L to about 766 µg / L, about 50 µg / L to about 766 µg / L, about 60 µg / L to about 766 µg / L, about 69 µg / L.6 µg / L to about 766 µg / L, about 70 µg / L to about 766 µg / L, about 80 µg / L to about 766 µg / L, about 90 µg / L to about 766 µg / L, about 100 µg / L to about 766 µg / L, about 150 µg / L to about 766 µg / L, about 200 µg / L to about 766 µg / L, about 250 µg / L to about 766 µg / L, about 300 µg / L to about 766 µg / L, about 400 µg / L to about 766 µg / L, about 500 µg / L to about 766 µg / L, about 600 µg / L to about 766 µg / L, about 750 µg / L to about 766 µg / L, about 9.1 µg / L to about 750 µg / L, about 9.2 µg / L to about 750 µg / L µg / L, about 10 µg / L to about 750 µg / L, about 20 µg / L to about 750 µg / L, about 30 µg / L to about 750 µg / L, about 40 µg / L to about 750 µg / L, about 50 µg / L to about 750 µg / L, about 60 µg / L to about 750 µg / L, about 69.6 µg / L to about 750 µg / L, about 70 µg / L to about 750 µg / L, about 80 µg / L to about 750 µg / L, about 90 µg / L to about 750 µg / L, about 100 µg / L to about 750 µg / L, about 150 µg / L to about 750 µg / L, about 200 µg / L to about 750 µg / L, about 250 µg / L to about 750 µg / L, about 300 µg / L About 750 µg / L, about 400 µg / L to about 750 µg / L, about 500 µg / L to about 750 µg / L, about 600 µg / L to about 750 µg / L, about 9.1 µg / L to about 600 µg / L, about 9.2 µg / L to about 600 µg / L, about 10 µg / L to about 600 µg / L, about 20 µg / L to about 600 µg / L, about 30 µg / L to about 600 µg / L, about 40 µg / L to about 600 µg / L, about 50 µg / L to about 600 µg / L, about 60 µg / L to about 600 µg / L, about 69 µg / L.6 µg / L to about 600 µg / L, about 70 µg / L to about 600 µg / L, about 80 µg / L to about 600 µg / L, about 90 µg / L to about 600 µg / L, about 100 µg / L to about 600 µg / L, about 150 µg / L to about 600 µg / L, about 200 µg / L to about 600 µg / L, about 250 µg / L to about 600 µg / L, about 300 µg / L to about 600 µg / L, about 400 µg / L to about 600 µg / L, about 500 µg / L to about 600 µg / L, about 9.1 µg / L to about 500 µg / L, about 9.2 µg / L to about 500 µg / L, about 10 µg / L to about 500 µg / L, about 20 µg / L to about 500 µg / L µg / L, about 30 µg / L to about 500 µg / L, about 40 µg / L to about 500 µg / L, about 50 µg / L to about 500 µg / L, about 60 µg / L to about 500 µg / L, about 69.6 µg / L to about 500 µg / L, about 70 µg / L to about 500 µg / L, about 80 µg / L to about 500 µg / L, about 90 µg / L to about 500 µg / L, about 100 µg / L to about 500 µg / L, about 150 µg / L to about 500 µg / L, about 200 µg / L to about 500 µg / L, about 250 µg / L to about 500 µg / L, about 300 µg / L to about 500 µg / L, about 400 µg / L to about 500 µg / L, about 9.1 Approximately 9.2 µg / L to 400 µg / L, approximately 10 µg / L to 400 µg / L, approximately 20 µg / L to 400 µg / L, approximately 30 µg / L to 400 µg / L, approximately 40 µg / L to 400 µg / L, approximately 50 µg / L to 400 µg / L, approximately 60 µg / L to 400 µg / L, approximately 69.6 µg / L to 400 µg / L, approximately 70 µg / L to 400 µg / L, approximately 80 µg / L to 400 µg / L, approximately 90 µg / L to 400 µg / L, approximately 100 µg / L to 400 µg / L, approximately 150 µg / L to 400 µg / L, approximately 200 µg / L to 400 µg / L µg / L, about 250 µg / L to about 400 µg / L, about 300 µg / L to about 400 µg / L, about 9.1 µg / L to about 300 µg / L, about 9.2 µg / L to about 300 µg / L, about 10 µg / L to about 300 µg / L, about 20 µg / L to about 300 µg / L, about 30 µg / L to about 300 µg / L, about 40 µg / L to about 300 µg / L, about 50 µg / L to about 300 µg / L, about 60 µg / L to about 300 µg / L, about 69.6 µg / L to about 300 µg / L, about 70 µg / L to about 300 µg / L, about 80 µg / L to about 300 µg / L, about 90 µg / L to about 300 µg / L, about 100 µg / L to about 300 µg / L, about 150 µg / L to about 300 µg / L, about 200 µg / L to about 300 µg / L, about 250 µg / L to about 300 µg / L µg / L, about 9.1 µg / L to about 250 µg / L, about 9.2 µg / L to about 250 µg / L, about 10 µg / L to about 250 µg / L, about 20 µg / L to about 250 µg / L, about 30 µg / L to about 250 µg / L, about 40 µg / L to about 250 µg / L, about 50 µg / L to about 250 µg / L, about 60 µg / L to about 250 µg / L, about 69.6 µg / L to about 250 µg / L, about 70 µg / L to about 250 µg / L, about 80 µg / L to about 250 µg / L, about 90 µg / L to about 250 µg / L, about 100 µg / L to about 250 µg / L, about 150 µg / L to about 250 µg / L, about 200 µg / L Approximately 250 µg / L, approximately 9.1 µg / L to approximately 200 µg / L, approximately 9.2 µg / L to approximately 200 µg / L, approximately 10 µg / L to approximately 200 µg / L, approximately 20 µg / L to approximately 200 µg / L, approximately 30 µg / L to approximately 200 µg / L, approximately 40 µg / L to approximately 200 µg / L, approximately 50 µg / L to approximately 200 µg / L, approximately 60 µg / L to approximately 200 µg / L, approximately 69.6 µg / L to approximately 200 µg / L, approximately 80 µg / L to approximately 200 µg / L, approximately 90 µg / L to approximately 200 µg / L, approximately 100 µg / L to approximately 200 µg / L, approximately 150 µg / L to approximately 200 µg / L, approximately 9.1 µg / L to approximately 150 µg / L µg / L, about 9.2 µg / L to about 150 µg / L, about 10 µg / L to about 150 µg / L, about 20 µg / L to about 150 µg / L, about 30 µg / L to about 150 µg / L, about 40 µg / L to about 150 µg / L, about 50 µg / L to about 150 µg / L, about 60 µg / L to about 150 µg / L, about 69 µg / L.6 µg / L to about 150 µg / L, about 70 µg / L to about 200 µg / L, about 80 µg / L to about 150 µg / L, about 90 µg / L to about 150 µg / L, about 100 µg / L to about 150 µg / L, about 9.1 µg / L to about 100 µg / L, about 9.2 µg / L to about 100 µg / L, about 10 µg / L to about 100 µg / L, about 20 µg / L to about 100 µg / L, about 30 µg / L to about 100 µg / L, about 40 µg / L to about 100 µg / L, about 50 µg / L to about 100 µg / L, about 60 µg / L to about 100 µg / L, about 69.6 µg / L to about 100 µg / L, about 70 µg / L to about 100 µg / L µg / L, about 80 µg / L to about 100 µg / L, about 90 µg / L to about 100 µg / L, about 9.1 µg / L to about 90 µg / L, about 9.2 µg / L to about 90 µg / L, about 10 µg / L to about 90 µg / L, about 20 µg / L to about 90 µg / L, about 30 µg / L to about 90 µg / L, about 40 µg / L to about 90 µg / L, about 50 µg / L to about 90 µg / L, about 60 µg / L to about 90 µg / L, about 69.6 µg / L to about 90 µg / L, about 70 µg / L to about 90 µg / L, about 80 µg / L to about 90 µg / L, about 9.1 µg / L to about 80 µg / L, about 9.2 µg / L to about 80 µg / L, about 10 µg / L Approximately 80 µg / L, approximately 20 µg / L, approximately 30 µg / L, approximately 40 µg / L, approximately 50 µg / L, approximately 60 µg / L, approximately 69.6 µg / L, approximately 70 µg / L, approximately 9.1 µg / L, approximately 9.2 µg / L, approximately 10 µg / L, approximately 20 µg / L, approximately 30 µg / L, approximately 40 µg / L, approximately 50 µg / L, approximately 60 µg / L, approximately 69.6 µg / L, approximately 60 ... µg / L to about 70 µg / L, about 9.1 µg / L to about 60 µg / L, about 9.2 µg / L to about 60 µg / L, about 10 µg / L to about 60 µg / L, about 20 µg / L to about 60 µg / L, about 30 µg / L to about 60 µg / L, about 40 µg / L to about 60 µg / L, about 50 µg / L to about 60 µg / L, about 9.1 µg / L to about 50 µg / L, about 9.2 µg / L to about 50 µg / L, about 10 µg / L to about 50 µg / L, about 20 µg / L to about 50 µg / L, about 30 µg / L to about 50 µg / L, about 40 µg / L to about 50 µg / L, about 9.1 µg / L to about 40 µg / L, about 9.2 µg / L to about 40 µg / L, about 10 µg / L to about 40 µg / L, about 20 µg / L to about 40 µg / L, about 30 µg / L to about 6 ... Approximately 40 µg / L, 9.1 µg / L to 30 µg / L, 9.2 µg / L to 30 µg / L, 10 µg / L to 30 µg / L, 20 µg / L to 30 µg / L, 9.1 µg / L to 20 µg / L, 9.2 µg / L to 20 µg / L, and 10 µg / L to 20 µg / L. µg / L or about 9.1, 9.2, 10, 20, 30, 40, 50, 60, 69.6, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 750, 766, 800, 812.8, 1000, 1100, 1023.3, 1250, 2000, 3000, or 4000 µg / L, or any range or value between therewith. In some embodiments, the fermentation product comprises isoamyl acetate in a concentration of about 69.6 µg / L to about 766 µg / L. In some embodiments, the fermentation product comprises isoamyl acetate in a concentration of about 9.2 µg / L to about 812.8 µg / L.

[0272] In some embodiments, the fermentation product contains phenethyl acetate in a concentration of about 2.3 µg / L to about 1500 µg / L. In some embodiments, the fermentation product comprises phenethyl acetate in amounts of about 2.3 µg / L to about 1500 µg / L, 5 µg / L to about 1500 µg / L, about 10 µg / L to about 1500 µg / L, about 20 µg / L to about 1500 µg / L, about 25 µg / L to about 1500 µg / L, about 30 µg / L to about 1500 µg / L, about 40 µg / L to about 1500 µg / L, about 47.2 µg / L to about 1500 µg / L, about 50 µg / L to about 1500 µg / L, about 60 µg / L to about 1500 µg / L, about 70 µg / L to about 1500 µg / L, about 80 µg / L to about 1500 µg / L, about 89.2 µg / L to about 1500 µg / L, and about 90 µg / L. Approximately 100 µg / L to 1500 µg / L, approximately 150 µg / L to 1500 µg / L, approximately 200 µg / L to 1500 µg / L, approximately 250 µg / L to 1500 µg / L, approximately 300 µg / L to 1500 µg / L, approximately 400 µg / L to 1500 µg / L, approximately 500 µg / L to 1500 µg / L, approximately 600 µg / L to 1500 µg / L, approximately 750 µg / L to 1500 µg / L, approximately 800 µg / L to 1500 µg / L, approximately 900 µg / L to 1500 µg / L, approximately 1000 µg / L to 1500 µg / L, and approximately 1100 µg / L to 1500 µg / L. µg / L, about 1200 µg / L to about 1500 µg / L, about 1250 µg / L to about 1500 µg / L, about 1300 µg / L to about 1500 µg / L, about 1400 µg / L to about 1500 µg / L, about 2.3 µg / L to about 1400 µg / L, about 5 µg / L to about 1400 µg / L, about 10 µg / L to about 1400 µg / L, about 20 µg / L to about 1400 µg / L, about 25 µg / L to about 1400 µg / L, about 30 µg / L to about 1400 µg / L, about 40 µg / L to about 1400 µg / L, about 47.2 µg / L to about 1400 µg / L, about 50 µg / L to about 1400 µg / L, about 60 µg / L µg / L to about 1400 µg / L, about 70 µg / L to about 1400 µg / L, about 80 µg / L to about 1400 µg / L, about 89 µg / L.2 µg / L to about 1400 µg / L, about 90 µg / L to about 1400 µg / L, about 100 µg / L to about 1400 µg / L, about 150 µg / L to about 1400 µg / L, about 200 µg / L to about 1400 µg / L, about 250 µg / L to about 1400 µg / L, about 300 µg / L to about 1400 µg / L, about 400 µg / L to about 1400 µg / L, about 500 µg / L to about 1400 µg / L, about 600 µg / L to about 1400 µg / L, about 750 µg / L to about 1400 µg / L, about 800 µg / L to about 1400 µg / L, about 900 µg / L to about 1400 µg / L, about 1000 µg / L to about 1400 µg / L µg / L, about 1100 µg / L to about 1400 µg / L, about 1200 µg / L to about 1400 µg / L, about 1250 µg / L to about 1400 µg / L, about 1300 µg / L to about 1400 µg / L, about 2.3 µg / L to about 1300 µg / L, about 5 µg / L to about 1300 µg / L, about 10 µg / L to about 1300 µg / L, about 20 µg / L to about 1300 µg / L, about 25 µg / L to about 1300 µg / L, about 30 µg / L to about 1300 µg / L, about 40 µg / L to about 1300 µg / L, about 47.2 µg / L to about 1300 µg / L, about 50 µg / L to about 1300 µg / L, about 60 µg / L Approximately 70 µg / L to 1300 µg / L, approximately 80 µg / L to 1300 µg / L, approximately 89.2 µg / L to 1300 µg / L, approximately 90 µg / L to 1300 µg / L, approximately 100 µg / L to 1300 µg / L, approximately 150 µg / L to 1300 µg / L, approximately 200 µg / L to 1300 µg / L, approximately 250 µg / L to 1300 µg / L, approximately 300 µg / L to 1300 µg / L, approximately 400 µg / L to 1300 µg / L, approximately 500 µg / L to 1300 µg / L, approximately 600 µg / L to 1300 µg / L, and approximately 750 µg / L to 1300 µg / L. µg / L, about 800 µg / L to about 1300 µg / L, about 900 µg / L to about 1300 µg / L, about 1000 µg / L to about 1300 µg / L, about 1100 µg / L to about 1300 µg / L, about 1200 µg / L to about 1300 µg / L, about 1250 µg / L to about 1300 µg / L, about 2.3 µg / L to about 1250 µg / L, about 5 µg / L to about 1250 µg / L, about 10 µg / L to about 1250 µg / L, about 20 µg / L to about 1250 µg / L, about 25 µg / L to about 1250 µg / L, about 30 µg / L to about 1250 µg / L, about 40 µg / L to about 1250 µg / L, about 47.2 µg / L to about 1250 µg / L, about 50 µg / L to about 1250 µg / L, about 60 µg / L to about 1250 µg / L, about 70 µg / L to about 1250 µg / L, about 80 µg / L to about 1250 µg / L, about 89.2 µg / L to about 1250 µg / L, about 90 µg / L to about 1250 µg / L, about 100 µg / L Approximately 1250 µg / L to 1250 µg / L, approximately 150 µg / L to 1250 µg / L, approximately 200 µg / L to 1250 µg / L, approximately 250 µg / L to 1250 µg / L, approximately 300 µg / L to 1250 µg / L, approximately 400 µg / L to 1250 µg / L, approximately 500 µg / L to 1250 µg / L, approximately 600 µg / L to 1250 µg / L, approximately 750 µg / L to 1250 µg / L, approximately 800 µg / L to 1250 µg / L, approximately 900 µg / L to 1250 µg / L, approximately 1000 µg / L to 1250 µg / L, approximately 1100 µg / L to 1250 µg / L, and approximately 1200 µg / L to 1250 µg / L. µg / L, about 2.3 µg / L to about 1200 µg / L, about 5 µg / L to about 1200 µg / L, about 10 µg / L to about 1200 µg / L, about 20 µg / L to about 1200 µg / L, about 25 µg / L to about 1200 µg / L, about 30 µg / L to about 1200 µg / L, about 40 µg / L to about 1200 µg / L, about 47.2 µg / L to about 1200 µg / L, about 50 µg / L to about 1200 µg / L, about 60 µg / L to about 1200 µg / L, about 70 µg / L to about 1200 µg / L, about 80 µg / L to about 1200 µg / L, about 89 µg / L.2 µg / L to about 1200 µg / L, about 90 µg / L to about 1200 µg / L, about 100 µg / L to about 1200 µg / L, about 150 µg / L to about 1200 µg / L, about 200 µg / L to about 1200 µg / L, about 250 µg / L to about 1200 µg / L, about 300 µg / L to about 1200 µg / L, about 400 µg / L to about 1200 µg / L, about 500 µg / L to about 1200 µg / L, about 600 µg / L to about 1200 µg / L, about 750 µg / L to about 1200 µg / L, about 800 µg / L to about 1200 µg / L, about 900 µg / L to about 1200 µg / L, about 1000 µg / L to about 1200 µg / L µg / L, about 1100 µg / L to about 1200 µg / L, about 2.3 µg / L to about 1100 µg / L, about 5 µg / L to about 1100 µg / L, about 10 µg / L to about 1100 µg / L, about 20 µg / L to about 1100 µg / L, about 25 µg / L to about 1100 µg / L, about 30 µg / L to about 1100 µg / L, about 40 µg / L to about 1100 µg / L, about 47.2 µg / L to about 1100 µg / L, about 50 µg / L to about 1100 µg / L, about 60 µg / L to about 1100 µg / L, about 70 µg / L to about 1100 µg / L, about 80 µg / L to about 1100 µg / L, about 89.2 µg / L to about 1100 µg / L µg / L, about 90 µg / L to about 1100 µg / L, about 100 µg / L to about 1100 µg / L, about 150 µg / L to about 1100 µg / L, about 200 µg / L to about 1100 µg / L, about 250 µg / L to about 1100 µg / L, about 300 µg / L to about 1100 µg / L, about 400 µg / L to about 1100 µg / L, about 500 µg / L to about 1100 µg / L, about 600 µg / L to about 1100 µg / L, about 750 µg / L to about 1100 µg / L, about 800 µg / L to about 1100 µg / L, about 900 µg / L to about 1100 µg / L, about 1000 µg / L to about 1100 µg / L, about 2.3 About 1000 µg / L, about 5 µg / L to about 1000 µg / L, about 10 µg / L to about 1000 µg / L, about 20 µg / L to about 1000 µg / L, about 25 µg / L to about 1000 µg / L, about 30 µg / L to about 1000 µg / L, about 40 µg / L to about 1000 µg / L, about 47 µg / L.2 µg / L to about 1000 µg / L, about 50 µg / L to about 1000 µg / L, about 60 µg / L to about 1000 µg / L, about 70 µg / L to about 1000 µg / L, about 80 µg / L to about 1000 µg / L, about 89.2 µg / L to about 1000 µg / L, about 90 µg / L to about 1000 µg / L, about 100 µg / L to about 1000 µg / L, about 150 µg / L to about 1000 µg / L, about 200 µg / L to about 1000 µg / L, about 250 µg / L to about 1000 µg / L, about 300 µg / L to about 1000 µg / L, about 400 µg / L to about 1000 µg / L, about 500 µg / L to about 1000 µg / L µg / L, about 600 µg / L to about 1000 µg / L, about 750 µg / L to about 1000 µg / L, about 800 µg / L to about 1000 µg / L, about 900 µg / L to about 1000 µg / L, about 2.3 µg / L to about 900 µg / L, about 5 µg / L to about 900 µg / L, about 10 µg / L to about 900 µg / L, about 20 µg / L to about 900 µg / L, about 25 µg / L to about 900 µg / L, about 30 µg / L to about 900 µg / L, about 40 µg / L to about 900 µg / L, about 47.2 µg / L to about 900 µg / L, about 50 µg / L to about 900 µg / L, about 60 µg / L to about 900 µg / L, about 70 µg / L Approximately 80 µg / L to 900 µg / L, approximately 89.2 µg / L to 900 µg / L, approximately 90 µg / L to 900 µg / L, approximately 100 µg / L to 900 µg / L, approximately 150 µg / L to 900 µg / L, approximately 200 µg / L to 900 µg / L, approximately 250 µg / L to 900 µg / L, approximately 300 µg / L to 900 µg / L, approximately 400 µg / L to 900 µg / L, approximately 500 µg / L to 900 µg / L, approximately 600 µg / L to 900 µg / L, approximately 750 µg / L to 900 µg / L, approximately 800 µg / L to 900 µg / L, approximately 2.3 µg / L to 800 µg / L µg / L, about 5 µg / L to about 800 µg / L, about 10 µg / L to about 800 µg / L, about 20 µg / L to about 800 µg / L, about 25 µg / L to about 800 µg / L, about 30 µg / L to about 800 µg / L, about 40 µg / L to about 800 µg / L, about 47 µg / L.2 µg / L to about 800 µg / L, about 50 µg / L to about 800 µg / L, about 60 µg / L to about 800 µg / L, about 70 µg / L to about 800 µg / L, about 80 µg / L to about 800 µg / L, about 89.2 µg / L to about 800 µg / L, about 90 µg / L to about 800 µg / L, about 100 µg / L to about 800 µg / L, about 150 µg / L to about 800 µg / L, about 200 µg / L to about 800 µg / L, about 250 µg / L to about 800 µg / L, about 300 µg / L to about 800 µg / L, about 400 µg / L to about 800 µg / L, about 500 µg / L to about 800 µg / L, about 600 µg / L Approximately 800 µg / L to 750 µg / L, approximately 2.3 µg / L to 750 µg / L, approximately 5 µg / L to 750 µg / L, approximately 10 µg / L to 750 µg / L, approximately 20 µg / L to 750 µg / L, approximately 25 µg / L to 750 µg / L, approximately 30 µg / L to 750 µg / L, approximately 40 µg / L to 750 µg / L, approximately 47.2 µg / L to 750 µg / L, approximately 50 µg / L to 750 µg / L, approximately 60 µg / L to 750 µg / L, approximately 70 µg / L to 750 µg / L, approximately 80 µg / L to 750 µg / L, approximately 89.2 µg / L to 750 µg / L, approximately 90 µg / L to 750 µg / L, approximately 80 µg / L to 750 µg / L, approximately 90 µg / L to 750 µg / L, approximately 80 µg / L to 750 µg / L, approximately 90 µg / L to 750 µg / L, approximately 80 µg / L to 750 µg / L, approximately 89.2 µg / L to 750 µg / L, approximately 90 µg / L to 750 µg / L, approximately 80 ... Approximately 750 µg / L, approximately 100 µg / L, approximately 150 µg / L, approximately 200 µg / L, approximately 250 µg / L, approximately 300 µg / L, approximately 400 µg / L, approximately 500 µg / L, approximately 600 µg / L, approximately 2.3 µg / L, approximately 5 µg / L, approximately 10 µg / L, approximately 20 µg / L, approximately 25 µg / L, approximately 30 µg / L, approximately 6 ...20 µg / L, approximately 25 µg / L, approximately 20 µg / L, approximately 25 µg / L, approximately 20 µg / L, approximately 25 µg / L, approximately 20 µg / L, approximately 25 µg / L, approximately 20 µg / L, approximately 25 µg / L, approximately 20 µg / L, approximately 25 µg / L, approximately 20 µg / L, approximately 25 µg / L, approximately 20 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, approximately 25 µg / L, µg / L, about 40 µg / L to about 600 µg / L, about 47.2 µg / L to about 600 µg / L, about 50 µg / L to about 600 µg / L, about 60 µg / L to about 600 µg / L, about 70 µg / L to about 600 µg / L, about 80 µg / L to about 600 µg / L, about 89 µg / L.2 µg / L to about 600 µg / L, about 90 µg / L to about 600 µg / L, about 100 µg / L to about 600 µg / L, about 150 µg / L to about 600 µg / L, about 200 µg / L to about 600 µg / L, about 250 µg / L to about 600 µg / L, about 300 µg / L to about 600 µg / L, about 400 µg / L to about 600 µg / L, about 500 µg / L to about 600 µg / L, about 2.3 µg / L to about 594 µg / L, about 5 µg / L to about 594 µg / L, about 10 µg / L to about 594 µg / L, about 20 µg / L to about 594 µg / L, about 25 µg / L to about 594 µg / L, about 30 µg / L to about 594 µg / L µg / L, about 40 µg / L to about 594 µg / L, about 47.2 µg / L to about 594 µg / L, about 50 µg / L to about 594 µg / L, about 60 µg / L to about 594 µg / L, about 70 µg / L to about 594 µg / L, about 80 µg / L to about 594 µg / L, about 89.2 µg / L to about 594 µg / L, about 90 µg / L to about 594 µg / L, about 100 µg / L to about 594 µg / L, about 150 µg / L to about 594 µg / L, about 200 µg / L to about 594 µg / L, about 250 µg / L to about 594 µg / L, about 300 µg / L to about 594 µg / L, about 400 µg / L to about 594 µg / L, about 500 µg / L Approximately 594 µg / L to 500 µg / L, approximately 2.3 µg / L to 500 µg / L, approximately 5 µg / L to 500 µg / L, approximately 10 µg / L to 500 µg / L, approximately 20 µg / L to 500 µg / L, approximately 25 µg / L to 500 µg / L, approximately 30 µg / L to 500 µg / L, approximately 40 µg / L to 500 µg / L, approximately 47.2 µg / L to 500 µg / L, approximately 50 µg / L to 500 µg / L, approximately 60 µg / L to 500 µg / L, approximately 70 µg / L to 500 µg / L, approximately 80 µg / L to 500 µg / L, approximately 89.2 µg / L to 500 µg / L, approximately 90 µg / L to 500 µg / L, approximately 100 µg / L µg / L to about 500 µg / L, about 150 µg / L to about 500 µg / L, about 200 µg / L to about 500 µg / L, about 250 µg / L to about 500 µg / L, about 300 µg / L to about 500 µg / L, about 400 µg / L to about 500 µg / L, about 2.3 µg / L to about 400 µg / L, about 5 µg / L to about 400 µg / L, about 10 µg / L to about 400 µg / L, about 20 µg / L to about 400 µg / L, about 25 µg / L to about 400 µg / L, about 30 µg / L to about 400 µg / L, about 40 µg / L to about 400 µg / L, about 47.2 µg / L to about 400 µg / L, about 50 µg / L to about 400 µg / L, about 60 µg / L to about 400 µg / L, about 70 µg / L to about 400 µg / L, about 80 µg / L to about 400 µg / L, about 89.2 µg / L to about 400 µg / L, about 90 µg / L to about 400 µg / L, about 100 µg / L to about 400 µg / L µg / L, about 150 µg / L to about 400 µg / L, about 200 µg / L to about 400 µg / L, about 250 µg / L to about 400 µg / L, about 300 µg / L to about 400 µg / L, about 2.3 µg / L to about 363.1 µg / L, about 5 µg / L to about 363.1 µg / L, about 10 µg / L to about 363.1 µg / L, about 20 µg / L to about 363.1 µg / L, about 25 µg / L to about 363.1 µg / L, about 30 µg / L to about 363.1 µg / L, about 40 µg / L to about 363.1 µg / L, about 47.2 µg / L to about 363.1 µg / L, about 50 µg / L to about 363.1 µg / L, about 60 µg / L Approximately 363.1 µg / L to 363.1 µg / L, approximately 70 µg / L to 363.1 µg / L, approximately 80 µg / L to 363.1 µg / L, approximately 89.2 µg / L to 363.1 µg / L, approximately 90 µg / L to 363.1 µg / L, approximately 100 µg / L to 363.1 µg / L, approximately 150 µg / L to 363.1 µg / L, approximately 200 µg / L to 363.1 µg / L, approximately 250 µg / L to 363.1 µg / L, approximately 300 µg / L to 363.1 µg / L, approximately 400 µg / L to 363.1 µg / L, approximately 500 µg / L to 363.1 µg / L, approximately 2.3 µg / L to 300 µg / L, approximately 5 µg / L to about 300 µg / L, about 10 µg / L to about 300 µg / L, about 20 µg / L to about 300 µg / L, about 25 µg / L to about 300 µg / L, about 30 µg / L to about 300 µg / L, about 40 µg / L to about 300 µg / L, about 47 µg / L.2 µg / L to about 300 µg / L, about 50 µg / L to about 300 µg / L, about 60 µg / L to about 300 µg / L, about 70 µg / L to about 300 µg / L, about 80 µg / L to about 300 µg / L, about 89.2 µg / L to about 300 µg / L, about 90 µg / L to about 300 µg / L, about 100 µg / L to about 300 µg / L, about 150 µg / L to about 300 µg / L, about 200 µg / L to about 300 µg / L, about 250 µg / L to about 300 µg / L, about 2.3 µg / L to about 281.1 µg / L, about 5 µg / L to about 281.1 µg / L, about 10 µg / L to about 281.1 µg / L, about 20 Approximately 25 µg / L to 281.1 µg / L, approximately 30 µg / L to 281.1 µg / L, approximately 40 µg / L to 281.1 µg / L, approximately 47.2 µg / L to 281.1 µg / L, approximately 50 µg / L to 281.1 µg / L, approximately 60 µg / L to 281.1 µg / L, approximately 70 µg / L to 281.1 µg / L, approximately 80 µg / L to 281.1 µg / L, approximately 89.2 µg / L to 281.1 µg / L, approximately 90 µg / L to 281.1 µg / L, approximately 100 µg / L to 281.1 µg / L, approximately 150 µg / L to 281.1 µg / L, approximately 20 ... Approximately 281.1 µg / L, 250 µg / L, 2.3 µg / L, 5 µg / L, 10 µg / L, 20 µg / L, 25 µg / L, 30 µg / L, 40 µg / L, 47.2 µg / L, 50 µg / L, 60 µg / L, 70 µg / L, 80 µg / L, and 89.2 µg / L to 250 µg / L. µg / L, about 90 µg / L to about 250 µg / L, about 100 µg / L to about 250 µg / L, about 150 µg / L to about 250 µg / L, about 200 µg / L to about 250 µg / L, about 2.3 µg / L to about 200 µg / L, about 5 µg / L to about 200 µg / L, about 10 µg / L to about 200 µg / L, about 20 µg / L to about 200 µg / L, about 25 µg / L to about 200 µg / L, about 30 µg / L to about 200 µg / L, about 40 µg / L to about 200 µg / L, about 47.2 µg / L to about 200 µg / L, about 50 µg / L to about 200 µg / L, about 60 µg / L to about 200 µg / L, about 70 µg / L to about 200 µg / L, about 80 µg / L to about 200 µg / L, about 89.2 µg / L to about 200 µg / L, about 90 µg / L to about 200 µg / L, about 100 µg / L to about 200 µg / L µg / L, about 150 µg / L to about 200 µg / L, about 2.3 µg / L to about 150 µg / L, about 5 µg / L to about 150 µg / L, about 10 µg / L to about 150 µg / L, about 20 µg / L to about 150 µg / L, about 25 µg / L to about 150 µg / L, about 30 µg / L to about 150 µg / L, about 40 µg / L to about 150 µg / L, about 47.2 µg / L to about 150 µg / L, about 50 µg / L to about 150 µg / L, about 60 µg / L to about 150 µg / L, about 70 µg / L to about 150 µg / L, about 80 µg / L to about 150 µg / L, about 89.2 µg / L to about 150 µg / L, about 90 µg / L to about 150 µg / L µg / L, about 100 µg / L to about 150 µg / L, about 2.3 µg / L to about 100 µg / L, about 5 µg / L to about 100 µg / L, about 10 µg / L to about 100 µg / L, about 20 µg / L to about 100 µg / L, about 25 µg / L to about 100 µg / L, about 30 µg / L to about 100 µg / L, about 40 µg / L to about 100 µg / L, about 47.2 µg / L to about 100 µg / L, about 50 µg / L to about 100 µg / L, about 60 µg / L to about 100 µg / L, about 70 µg / L to about 100 µg / L, about 80 µg / L to about 100 µg / L, about 89.2 µg / L to about 100 µg / L, about 90 µg / L to about 100 µg / L µg / L, about 2.3 µg / L to about 90 µg / L, about 5 µg / L to about 90 µg / L, about 10 µg / L to about 90 µg / L, about 20 µg / L to about 90 µg / L, about 25 µg / L to about 90 µg / L, about 30 µg / L to about 90 µg / L, about 40 µg / L to about 90 µg / L, about 47 µg / L.2 µg / L to about 90 µg / L, about 50 µg / L to about 90 µg / L, about 60 µg / L to about 90 µg / L, about 70 µg / L to about 90 µg / L, about 80 µg / L to about 90 µg / L, about 89.2 µg / L to about 90 µg / L, about 2.3 µg / L to about 80 µg / L, about 5 µg / L to about 80 µg / L, about 10 µg / L to about 80 µg / L, about 20 µg / L to about 80 µg / L, about 25 µg / L to about 80 µg / L, about 30 µg / L to about 80 µg / L, about 40 µg / L to about 80 µg / L, about 47.2 µg / L to about 80 µg / L, about 50 µg / L to about 80 µg / L, about 60 µg / L to about 80 µg / L, about 70 µg / L Approximately 80 µg / L to 20 µg / L, approximately 2.3 µg / L to 70 µg / L, approximately 5 µg / L to 70 µg / L, approximately 10 µg / L to 70 µg / L, approximately 20 µg / L to 70 µg / L, approximately 25 µg / L to 70 µg / L, approximately 30 µg / L to 70 µg / L, approximately 40 µg / L to 70 µg / L, approximately 47.2 µg / L to 70 µg / L, approximately 50 µg / L to 70 µg / L, approximately 60 µg / L to 70 µg / L, approximately 2.3 µg / L to 60 µg / L, approximately 5 µg / L to 60 µg / L, approximately 10 µg / L to 60 µg / L, approximately 20 µg / L to 60 µg / L, approximately 25 µg / L to 60 µg / L, approximately 30 µg / L to 7 ... Approximately 40 µg / L to 60 µg / L, approximately 47.2 µg / L to 60 µg / L, approximately 50 µg / L to 60 µg / L, approximately 2.3 µg / L to 50 µg / L, approximately 5 µg / L to 50 µg / L, approximately 10 µg / L to 50 µg / L, approximately 20 µg / L to 50 µg / L, approximately 25 µg / L to 50 µg / L, approximately 30 µg / L to 50 µg / L, approximately 40 µg / L to 50 µg / L, approximately 47.2 µg / L to 50 µg / L, approximately 2.3 µg / L to 40 µg / L, approximately 5 µg / L to 40 µg / L, approximately 10 µg / L to 40 µg / L, approximately 20 µg / L to 40 µg / L, approximately 25 µg / L to 5 ... µg / L to about 40 µg / L, about 30 µg / L to about 40 µg / L, about 2.3 µg / L to about 30 µg / L, about 5 µg / L to about 30 µg / L, about 10 µg / L to about 30 µg / L, about 20 µg / L to about 30 µg / L, about 25 µg / L to about 30 µg / L, about 2.3 µg / L to about 20 µg / L, about 5 µg / L to about 20 µg / L, about 10 µg / L to about 20 µg / L, about 2.3 µg / L to about 10 µg / L, about 5 µg / L to about 10 µg / L, about 2.3 µg / L to about 5 µg / L, or about 2.3, 5, 10, 20, 25, 30, 40, 47.2, 50, 60, 70, 75, 80, 89.2, 90, 100, 150, 200, 250, 281.1, 300, 363.1, 400, 500, 594, 600, 750, 800, 900, 1000, 1100, 1200, 1250, 1300, 1400, or 1500 µg / L, or any range or value thereof. In some embodiments, the fermentation product comprises phenethyl acetate in an amount from about 89.2 µg / L to about 594 µg / L. In some embodiments, the fermentation product comprises phenethyl acetate in an amount from about 2.3 µg / L to about 281.1 µg / L.

[0273] In some embodiments, the fermentation product contains ethyl acetate in a concentration of about 500 µg / L to about 4500 µg / L. In some embodiments, the fermentation product comprises ethyl acetate in amounts of about 500 µg / L to about 4500 µg / L, about 524.8 µg / L to about 4500 µg / L, about 562 µg / L to about 4500 µg / L, about 600 µg / L to about 4500 µg / L, about 700 µg / L to about 4500 µg / L, about 800 µg / L to about 4500 µg / L, about 900 µg / L to about 4500 µg / L, about 1000 µg / L to about 4500 µg / L, about 1250 µg / L to about 4500 µg / L, about 1500 µg / L to about 4500 µg / L, about 1750 µg / L to about 4500 µg / L, about 2000 µg / L to about 4500 µg / L, and about 2500 µg / L. Approximately 4500 µg / L to 3000 µg / L, approximately 3500 µg / L to 4500 µg / L, approximately 4000 µg / L to 4500 µg / L, approximately 500 µg / L to 4073.8 µg / L, approximately 524.8 µg / L to 4073.8 µg / L, approximately 562 µg / L to 4073.8 µg / L, approximately 600 µg / L to 4073.8 µg / L, approximately 700 µg / L to 4073.8 µg / L, approximately 800 µg / L to 4073.8 µg / L, approximately 900 µg / L to 4073.8 µg / L, approximately 1000 µg / L to 4073.8 µg / L, approximately 125 ... µg / L to about 4073.8 µg / L, about 1500 µg / L to about 4073.8 µg / L, about 1750 µg / L to about 4073.8 µg / L, about 2000 µg / L to about 4073.8 µg / L, about 2500 µg / L to about 4073.8 µg / L, about 3000 µg / L to about 4073.8 µg / L, about 3500 µg / L to about 4073.8 µg / L, about 4000 µg / L to about 4073.8 µg / L, about 500 µg / L to about 4000 µg / L, about 524 µg / L.8 µg / L to about 4000 µg / L, about 562 µg / L to about 4000 µg / L, about 600 µg / L to about 4000 µg / L, about 700 µg / L to about 4000 µg / L, about 800 µg / L to about 4000 µg / L, about 900 µg / L to about 4000 µg / L, about 1000 µg / L to about 4000 µg / L, about 1250 µg / L to about 4000 µg / L, about 1500 µg / L to about 4000 µg / L, about 1750 µg / L to about 4000 µg / L, about 2000 µg / L to about 4000 µg / L, about 2500 µg / L to about 4000 µg / L, about 3000 µg / L to about 4000 µg / L, about 3500 µg / L Approximately 4000 µg / L to 4000 µg / L, approximately 500 µg / L to 3800 µg / L, approximately 524.8 µg / L to 3800 µg / L, approximately 562 µg / L to 3800 µg / L, approximately 600 µg / L to 3800 µg / L, approximately 700 µg / L to 3800 µg / L, approximately 800 µg / L to 3800 µg / L, approximately 900 µg / L to 3800 µg / L, approximately 1000 µg / L to 3800 µg / L, approximately 1250 µg / L to 3800 µg / L, approximately 1500 µg / L to 3800 µg / L, approximately 1750 µg / L to 3800 µg / L, approximately 2000 µg / L to 3800 µg / L, approximately 25 ... Approximately 3800 µg / L to 3800 µg / L, approximately 3000 µg / L to 3800 µg / L, approximately 3500 µg / L to 3800 µg / L, approximately 500 µg / L to 3630.8 µg / L, approximately 524.8 µg / L to 3630.8 µg / L, approximately 562 µg / L to 3630.8 µg / L, approximately 600 µg / L to 3630.8 µg / L, approximately 700 µg / L to 3630.8 µg / L, approximately 800 µg / L to 3630.8 µg / L, approximately 900 µg / L to 3630.8 µg / L, approximately 1000 µg / L to 3630.8 µg / L, approximately 1250 µg / L to 3630.8 µg / L, approximately 1500 µg / L to 3800 µg / L, approximately 1500 µg / L to 3800 µg / L, approximately 3000 µg / L to 3800 µg / L, approximately 3000 µg / L to 3800 µg / L, approximately 3500 µg / L to 3800 µg / L, approximately 500 µg / L to 3630.8 µg / L, approximately 1500 µg / L to 3800 µg / L, approximately 30 ... µg / L to about 3630.8 µg / L, about 1750 µg / L to about 3630.8 µg / L, about 2000 µg / L to about 3630.8 µg / L, about 2500 µg / L to about 3630.8 µg / L, about 3000 µg / L to about 3630.8 µg / L, and about 3500 µg / L to about 3630.8 µg / L.8 µg / L, about 500 µg / L to about 3500 µg / L, about 524.8 µg / L to about 3500 µg / L, about 562 µg / L to about 3500 µg / L, about 600 µg / L to about 3500 µg / L, about 700 µg / L to about 3500 µg / L, about 800 µg / L to about 3500 µg / L, about 900 µg / L to about 3500 µg / L, about 1000 µg / L to about 3500 µg / L, about 1250 µg / L to about 3500 µg / L, about 1500 µg / L to about 3500 µg / L, about 1750 µg / L to about 3500 µg / L, about 2000 µg / L to about 3500 µg / L, about 2500 µg / L to about 3500 µg / L µg / L, about 3000 µg / L to about 3500 µg / L, about 500 µg / L to about 3000 µg / L, about 524.8 µg / L to about 3000 µg / L, about 562 µg / L to about 3000 µg / L, about 600 µg / L to about 3000 µg / L, about 700 µg / L to about 3000 µg / L, about 800 µg / L to about 3000 µg / L, about 900 µg / L to about 3000 µg / L, about 1000 µg / L to about 3000 µg / L, about 1250 µg / L to about 3000 µg / L, about 1500 µg / L to about 3000 µg / L, about 1750 µg / L to about 3000 µg / L, about 2000 µg / L to about 3000 µg / L µg / L, about 2500 µg / L to about 3000 µg / L, about 500 µg / L to about 2500 µg / L, about 524.8 µg / L to about 2500 µg / L, about 562 µg / L to about 2500 µg / L, about 600 µg / L to about 2500 µg / L, about 700 µg / L to about 2500 µg / L, about 800 µg / L to about 2500 µg / L, about 900 µg / L to about 2500 µg / L, about 1000 µg / L to about 2500 µg / L, about 1250 µg / L to about 2500 µg / L, about 1500 µg / L to about 2500 µg / L, about 1750 µg / L to about 2500 µg / L, about 2000 µg / L to about 2500 µg / L µg / L, approximately 500 µg / L to approximately 2000 µg / L, approximately 524.8 µg / L to about 2000 µg / L, about 562 µg / L to about 2000 µg / L, about 600 µg / L to about 2000 µg / L, about 700 µg / L to about 2000 µg / L, about 800 µg / L to about 2000 µg / L, about 900 µg / L to about 2000 µg / L, about 1000 µg / L to about 2000 µg / L, about 1250 µg / L to about 2000 µg / L, about 1500 µg / L to about 2000 µg / L, about 1750 µg / L to about 2000 µg / L, about 500 µg / L to about 1750 µg / L, about 524.8 µg / L to about 1750 µg / L, about 562 µg / L to about 1750 µg / L, about 600 µg / L to about 2000 µg / L, about 600 µg / L to about 2000 µg / L, about 600 µg / L to about 2000 µg / L, about 600 µg / L to about 2000 µg / L, about 600 µg / L to about 2000 µg / L, about 5 ... Approximately 1750 µg / L, approximately 700 µg / L, approximately 800 µg / L, approximately 900 µg / L, approximately 1000 µg / L, approximately 1250 µg / L, approximately 1500 µg / L, approximately 500 µg / L, approximately 524.8 µg / L, approximately 562 µg / L, approximately 600 µg / L, approximately 700 µg / L, approximately 800 µg / L, approximately 900 µg / L, approximately 1250 µg / L, approximately 1500 µg / L, approximately 1500 µg / L, approximately 500 µg / L, approximately 524.8 µg / L, approximately 562 µg / L, approximately 600 µg / L, approximately 700 µg / L, approximately 800 µg / L, approximately 900 µg / L, approximately 175 ... Approximately 1500 µg / L to 1500 µg / L, approximately 1000 µg / L to 1500 µg / L, approximately 1250 µg / L to 1500 µg / L, approximately 500 µg / L to 1250 µg / L, approximately 524.8 µg / L to 1250 µg / L, approximately 562 µg / L to 1250 µg / L, approximately 600 µg / L to 1250 µg / L, approximately 700 µg / L to 1250 µg / L, approximately 800 µg / L to 1250 µg / L, approximately 900 µg / L to 1250 µg / L, approximately 1000 µg / L to 1250 µg / L, approximately 500 µg / L to 1000 µg / L, approximately 524.8 µg / L to 1000 µg / L, approximately 562 µg / L to 125 ... About 1000 µg / L, about 600 µg / L to about 1000 µg / L, about 700 µg / L to about 1000 µg / L, about 800 µg / L to about 1000 µg / L, about 900 µg / L to about 1000 µg / L, about 500 µg / L to about 900 µg / L, about 524 µg / L.8 µg / L to about 900 µg / L, about 562 µg / L to about 900 µg / L, about 600 µg / L to about 900 µg / L, about 700 µg / L to about 900 µg / L, about 800 µg / L to about 900 µg / L, about 500 µg / L to about 800 µg / L, about 524.8 µg / L to about 800 µg / L, about 562 µg / L to about 800 µg / L, about 600 µg / L to about 800 µg / L, about 700 µg / L to about 800 µg / L, about 500 µg / L to about 700 µg / L, about 524.8 µg / L to about 700 µg / L, about 562 µg / L to about 700 µg / L, about 600 µg / L to about 700 µg / L, about 5 ... The concentrations are approximately 524.8 µg / L to about 600 µg / L, about 562 µg / L to about 600 µg / L, or any range or value of 500, 524.8, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 3500, 3630.8, 3800, 4000, 4073.8, 4500 µg / L. In some embodiments, the fermentation product comprises ethyl acetate in a concentration of approximately 600 µg / L to about 3800 µg / L. In some embodiments, the fermentation product comprises ethyl acetate in a concentration of approximately 524.8 µg / L to about 3630.8 µg / L.

[0274] In some embodiments, the fermentation product comprises phenethyl acetate and isoamyl acetate in a combined amount of about 10 µg / L to about 5500 µg / L. In some embodiments, the fermentation product comprises phenethyl acetate and isoamyl acetate in combinations of about 10 µg / L to about 5500 µg / L, about 11.4 µg / L to about 5500 µg / L, about 11.5 µg / L to about 5500 µg / L, about 12.5 µg / L to about 5500 µg / L, about 20 µg / L to about 5500 µg / L, about 25 µg / L to about 5500 µg / L, about 30 µg / L to about 5500 µg / L, about 40 µg / L to about 5500 µg / L, about 50 µg / L to about 5500 µg / L, about 60 µg / L to about 5500 µg / L, about 70 µg / L to about 5500 µg / L, about 75 µg / L to about 5500 µg / L, and about 80 µg / L to about 5500 µg / L. µg / L, about 90 µg / L to about 5500 µg / L, about 100 µg / L to about 5500 µg / L, about 125 µg / L to about 5500 µg / L, about 150 µg / L to about 5500 µg / L, about 158.8 µg / L to about 5500 µg / L, about 175 µg / L to about 5500 µg / L, about 200 µg / L to about 5500 µg / L, about 250 µg / L to about 5500 µg / L, about 300 µg / L to about 5500 µg / L, about 400 µg / L to about 5500 µg / L, about 500 µg / L to about 5500 µg / L, about 600 µg / L to about 5500 µg / L, about 700 µg / L to about 5500 µg / L, about 800 µg / L Approximately 5500 µg / L to 5500 µg / L, approximately 900 µg / L to 5500 µg / L, approximately 1000 µg / L to 5500 µg / L, approximately 1100 µg / L to 5500 µg / L, approximately 1200 µg / L to 5500 µg / L, approximately 1250 µg / L to 5500 µg / L, approximately 1300 µg / L to 5500 µg / L, approximately 1400 µg / L to 5500 µg / L, approximately 1500 µg / L to 5500 µg / L, approximately 2000 µg / L to 5500 µg / L, approximately 3000 µg / L to 5500 µg / L, approximately 4000 µg / L to 5500 µg / L, approximately 5000 µg / L to 5500 µg / L, approximately 10 µg / L to about 5000 µg / L, about 11.4 µg / L to about 5000 µg / L, about 11.5 µg / L to about 5000 µg / L, about 12 µg / L.5 µg / L to about 5000 µg / L, about 20 µg / L to about 5000 µg / L, about 25 µg / L to about 5000 µg / L, about 30 µg / L to about 5000 µg / L, about 40 µg / L to about 5000 µg / L, about 50 µg / L to about 5000 µg / L, about 60 µg / L to about 5000 µg / L, about 70 µg / L to about 5000 µg / L, about 75 µg / L to about 5000 µg / L, about 80 µg / L to about 5000 µg / L, about 90 µg / L to about 5000 µg / L, about 100 µg / L to about 5000 µg / L, about 125 µg / L to about 5000 µg / L, about 150 µg / L to about 5000 µg / L, about 158.8 Approximately 175 µg / L to 5000 µg / L, approximately 200 µg / L to 5000 µg / L, approximately 250 µg / L to 5000 µg / L, approximately 300 µg / L to 5000 µg / L, approximately 400 µg / L to 5000 µg / L, approximately 500 µg / L to 5000 µg / L, approximately 600 µg / L to 5000 µg / L, approximately 700 µg / L to 5000 µg / L, approximately 800 µg / L to 5000 µg / L, approximately 900 µg / L to 5000 µg / L, approximately 1000 µg / L to 5000 µg / L, approximately 1100 µg / L to 5000 µg / L, and approximately 1200 µg / L to 5000 µg / L. µg / L, about 1250 µg / L to about 5000 µg / L, about 1300 µg / L to about 5000 µg / L, about 1400 µg / L to about 5000 µg / L, about 1500 µg / L to about 5000 µg / L, about 2000 µg / L to about 5000 µg / L, about 3000 µg / L to about 5000 µg / L, about 4000 µg / L to about 5000 µg / L, about 10 µg / L to about 4000 µg / L, about 11.4 µg / L to about 4000 µg / L, about 11.5 µg / L to about 4000 µg / L, about 12.5 µg / L to about 4000 µg / L, about 20 µg / L to about 4000 µg / L, about 25 µg / L to about 4000 µg / L, about 30 µg / L to about 4000 µg / L, about 40 µg / L to about 4000 µg / L, about 50 µg / L to about 4000 µg / L, about 60 µg / L to about 4000 µg / L, about 70 µg / L to about 4000 µg / L, about 75 µg / L to about 4000 µg / L, about 80 µg / L to about 4000 µg / L, about 90 µg / L to about 4000 µg / L, about 100 µg / L to about 4000 µg / L, about 125 µg / L to about 4000 µg / L, about 150 µg / L to about 4000 µg / L, about 158.8 Approximately 175 µg / L to 4000 µg / L, approximately 200 µg / L to 4000 µg / L, approximately 250 µg / L to 4000 µg / L, approximately 300 µg / L to 4000 µg / L, approximately 400 µg / L to 4000 µg / L, approximately 500 µg / L to 4000 µg / L, approximately 600 µg / L to 4000 µg / L, approximately 700 µg / L to 4000 µg / L, approximately 800 µg / L to 4000 µg / L, approximately 900 µg / L to 4000 µg / L, approximately 1000 µg / L to 4000 µg / L, approximately 1100 µg / L to 4000 µg / L, and approximately 1200 µg / L to 4000 µg / L. µg / L, about 1250 µg / L to about 4000 µg / L, about 1300 µg / L to about 4000 µg / L, about 1400 µg / L to about 4000 µg / L, about 1500 µg / L to about 4000 µg / L, about 2000 µg / L to about 4000 µg / L, about 3000 µg / L to about 4000 µg / L, about 10 µg / L to about 3000 µg / L, about 11.4 µg / L to about 3000 µg / L, about 11.5 µg / L to about 3000 µg / L, about 12 µg / L.5 µg / L to about 3000 µg / L, about 20 µg / L to about 3000 µg / L, about 25 µg / L to about 3000 µg / L, about 30 µg / L to about 3000 µg / L, about 40 µg / L to about 3000 µg / L, about 50 µg / L to about 3000 µg / L, about 60 µg / L to about 3000 µg / L, about 70 µg / L to about 3000 µg / L, about 75 µg / L to about 3000 µg / L, about 80 µg / L to about 3000 µg / L, about 90 µg / L to about 3000 µg / L, about 100 µg / L to about 3000 µg / L, about 125 µg / L to about 3000 µg / L, about 150 µg / L to about 3000 µg / L, about 158.8 Approximately 175 µg / L to 3000 µg / L, approximately 200 µg / L to 3000 µg / L, approximately 250 µg / L to 3000 µg / L, approximately 300 µg / L to 3000 µg / L, approximately 400 µg / L to 3000 µg / L, approximately 500 µg / L to 3000 µg / L, approximately 600 µg / L to 3000 µg / L, approximately 700 µg / L to 3000 µg / L, approximately 800 µg / L to 3000 µg / L, approximately 900 µg / L to 3000 µg / L, approximately 1000 µg / L to 3000 µg / L, approximately 1100 µg / L to 3000 µg / L, and approximately 1200 µg / L to 3000 µg / L. µg / L, about 1250 µg / L to about 3000 µg / L, about 1300 µg / L to about 3000 µg / L, about 1400 µg / L to about 3000 µg / L, about 1500 µg / L to about 3000 µg / L, about 2000 µg / L to about 3000 µg / L, about 10 µg / L to about 2000 µg / L, about 11.4 µg / L to about 2000 µg / L, about 11.5 µg / L to about 2000 µg / L, about 12 µg / L.5 µg / L to about 2000 µg / L, about 20 µg / L to about 2000 µg / L, about 25 µg / L to about 2000 µg / L, about 30 µg / L to about 2000 µg / L, about 40 µg / L to about 2000 µg / L, about 50 µg / L to about 2000 µg / L, about 60 µg / L to about 2000 µg / L, about 70 µg / L to about 2000 µg / L, about 75 µg / L to about 2000 µg / L, about 80 µg / L to about 2000 µg / L, about 90 µg / L to about 2000 µg / L, about 100 µg / L to about 2000 µg / L, about 125 µg / L to about 2000 µg / L, about 150 µg / L to about 2000 µg / L, about 158.8 Approximately 175 µg / L to 2000 µg / L, approximately 200 µg / L to 2000 µg / L, approximately 250 µg / L to 2000 µg / L, approximately 300 µg / L to 2000 µg / L, approximately 400 µg / L to 2000 µg / L, approximately 500 µg / L to 2000 µg / L, approximately 600 µg / L to 2000 µg / L, approximately 700 µg / L to 2000 µg / L, approximately 800 µg / L to 2000 µg / L, approximately 900 µg / L to 2000 µg / L, approximately 1000 µg / L to 2000 µg / L, approximately 1100 µg / L to 2000 µg / L, and approximately 1200 µg / L to 2000 µg / L. µg / L, about 1250 µg / L to about 2000 µg / L, about 1300 µg / L to about 2000 µg / L, about 1400 µg / L to about 2000 µg / L, about 1500 µg / L to about 2000 µg / L, about 10 µg / L to about 1753 µg / L, about 11.4 µg / L to about 1753 µg / L, about 11.5 µg / L to about 1753 µg / L, about 12 µg / L.5 µg / L to about 1753 µg / L, about 20 µg / L to about 1753 µg / L, about 25 µg / L to about 1753 µg / L, about 30 µg / L to about 1753 µg / L, about 40 µg / L to about 1753 µg / L, about 50 µg / L to about 1753 µg / L, about 60 µg / L to about 1753 µg / L, about 70 µg / L to about 1753 µg / L, about 75 µg / L to about 1753 µg / L, about 80 µg / L to about 1753 µg / L, about 90 µg / L to about 1753 µg / L, about 100 µg / L to about 1753 µg / L, about 125 µg / L to about 1753 µg / L, about 150 µg / L to about 1753 µg / L, about 158.8 Approximately 175 µg / L to 1753 µg / L, approximately 200 µg / L to 1753 µg / L, approximately 250 µg / L to 1753 µg / L, approximately 300 µg / L to 1753 µg / L, approximately 400 µg / L to 1753 µg / L, approximately 500 µg / L to 1753 µg / L, approximately 600 µg / L to 1753 µg / L, approximately 700 µg / L to 1753 µg / L, approximately 800 µg / L to 1753 µg / L, approximately 900 µg / L to 1753 µg / L, approximately 1000 µg / L to 1753 µg / L, approximately 1100 µg / L to 1753 µg / L, and approximately 1200 µg / L to 1753 µg / L. µg / L, about 1250 µg / L to about 1753 µg / L, about 1300 µg / L to about 1753 µg / L, about 1400 µg / L to about 1753 µg / L, about 10 µg / L to about 1500 µg / L, about 11.4 µg / L to about 1500 µg / L, about 11.5 µg / L to about 1500 µg / L, about 12 µg / L.5 µg / L to about 1500 µg / L, about 20 µg / L to about 1500 µg / L, about 25 µg / L to about 1500 µg / L, about 30 µg / L to about 1500 µg / L, about 35 µg / L to about 1500 µg / L, about 40 µg / L to about 1500 µg / L, about 50 µg / L to about 1500 µg / L, about 60 µg / L to about 1500 µg / L, about 70 µg / L to about 1500 µg / L, about 75 µg / L to about 1500 µg / L, about 80 µg / L to about 1500 µg / L, about 90 µg / L to about 1500 µg / L, about 100 µg / L to about 1500 µg / L, about 125 µg / L to about 1500 µg / L, about 150 µg / L Approximately 158.8 µg / L to 1500 µg / L, approximately 175 µg / L to 1500 µg / L, approximately 200 µg / L to 1500 µg / L, approximately 250 µg / L to 1500 µg / L, approximately 300 µg / L to 1500 µg / L, approximately 400 µg / L to 1500 µg / L, approximately 500 µg / L to 1500 µg / L, approximately 600 µg / L to 1500 µg / L, approximately 700 µg / L to 1500 µg / L, approximately 800 µg / L to 1500 µg / L, approximately 900 µg / L to 1500 µg / L, approximately 1000 µg / L to 1500 µg / L, approximately 1100 µg / L... About 1500 µg / L, about 1200 µg / L to about 1500 µg / L, about 1250 µg / L to about 1500 µg / L, about 1300 µg / L to about 1500 µg / L, about 1400 µg / L to about 1500 µg / L, about 10 µg / L to about 1400 µg / L, about 11.4 µg / L to about 1400 µg / L, about 11.5 µg / L to about 1400 µg / L, about 12 µg / L.5 µg / L to about 1400 µg / L, about 20 µg / L to about 1400 µg / L, about 25 µg / L to about 1400 µg / L, about 30 µg / L to about 1400 µg / L, about 40 µg / L to about 1400 µg / L, about 50 µg / L to about 1400 µg / L, about 60 µg / L to about 1400 µg / L, about 70 µg / L to about 1400 µg / L, about 75 µg / L to about 1400 µg / L, about 80 µg / L to about 1400 µg / L, about 90 µg / L to about 1400 µg / L, about 100 µg / L to about 1400 µg / L, about 125 µg / L to about 1400 µg / L, about 150 µg / L to about 1400 µg / L, about 158.8 Approximately 175 µg / L to 1400 µg / L, approximately 200 µg / L to 1400 µg / L, approximately 250 µg / L to 1400 µg / L, approximately 300 µg / L to 1400 µg / L, approximately 400 µg / L to 1400 µg / L, approximately 500 µg / L to 1400 µg / L, approximately 600 µg / L to 1400 µg / L, approximately 700 µg / L to 1400 µg / L, approximately 800 µg / L to 1400 µg / L, approximately 900 µg / L to 1400 µg / L, approximately 1000 µg / L to 1400 µg / L, approximately 1100 µg / L to 1400 µg / L, and approximately 1200 µg / L to 1400 µg / L. µg / L, about 1250 µg / L to about 1400 µg / L, about 1300 µg / L to about 1400 µg / L, about 10 µg / L to about 1360 µg / L, about 11.4 µg / L to about 1360 µg / L, about 11.5 µg / L to about 1360 µg / L, about 12.5 µg / L to about 1360 µg / L, about 20 µg / L to about 1360 µg / L, about 25 µg / L to about 1360 µg / L, about 30 µg / L to about 1360 µg / L, about 40 µg / L to about 1360 µg / L, about 50 µg / L to about 1360 µg / L, about 60 µg / L to about 1360 µg / L, about 70 µg / L to about 1360 µg / L, about 75 µg / L About 1360 µg / L, about 80 µg / L to about 1360 µg / L, about 90 µg / L to about 1360 µg / L, about 100 µg / L to about 1360 µg / L, about 125 µg / L to about 1360 µg / L, about 150 µg / L to about 1360 µg / L, about 158 ​​µg / L.8 µg / L to about 1360 µg / L, about 175 µg / L to about 1360 µg / L, about 200 µg / L to about 1360 µg / L, about 250 µg / L to about 1360 µg / L, about 300 µg / L to about 1360 µg / L, about 400 µg / L to about 1360 µg / L, about 500 µg / L to about 1360 µg / L, about 600 µg / L to about 1360 µg / L, about 700 µg / L to about 1360 µg / L, about 800 µg / L to about 1360 µg / L, about 900 µg / L to about 1360 µg / L, about 1000 µg / L to about 1360 µg / L, about 1100 µg / L to about 1360 µg / L, about 1200 µg / L Approximately 10 µg / L to 1300 µg / L, approximately 11.4 µg / L to 1300 µg / L, approximately 11.5 µg / L to 1300 µg / L, approximately 12.5 µg / L to 1300 µg / L, approximately 20 µg / L to 1300 µg / L, approximately 25 µg / L to 1300 µg / L, approximately 30 µg / L to 1300 µg / L, approximately 40 µg / L to 1300 µg / L, approximately 50 µg / L to 1300 µg / L, approximately 60 µg / L to 1300 µg / L, and approximately 70 µg / L to 1300 µg / L. µg / L, about 75 µg / L to about 1300 µg / L, about 80 µg / L to about 1300 µg / L, about 90 µg / L to about 1300 µg / L, about 100 µg / L to about 1300 µg / L, about 125 µg / L to about 1300 µg / L, about 150 µg / L to about 1300 µg / L, about 158 ​​µg / L.8 µg / L to about 1300 µg / L, about 175 µg / L to about 1300 µg / L, about 200 µg / L to about 1300 µg / L, about 250 µg / L to about 1300 µg / L, about 300 µg / L to about 1300 µg / L, about 400 µg / L to about 1300 µg / L, about 500 µg / L to about 1300 µg / L, about 600 µg / L to about 1300 µg / L, about 700 µg / L to about 1300 µg / L, about 800 µg / L to about 1300 µg / L, about 900 µg / L to about 1300 µg / L, about 1000 µg / L to about 1300 µg / L, about 1100 µg / L to about 1300 µg / L, about 1200 µg / L Approximately 10 µg / L to 1250 µg / L, approximately 11.4 µg / L to 1250 µg / L, approximately 11.5 µg / L to 1250 µg / L, approximately 20 µg / L to 1250 µg / L, approximately 25 µg / L to 1250 µg / L, approximately 30 µg / L to 1250 µg / L, approximately 40 µg / L to 1250 µg / L, approximately 50 µg / L to 1250 µg / L, approximately 60 µg / L to 1250 µg / L, approximately 70 µg / L to 1250 µg / L, approximately 75 µg / L to 1250 µg / L, approximately 80 µg / L to 1250 µg / L, approximately 80 µg / L to 1250 µg / L, approximately 1 ... Approximately 1250 µg / L to 1250 µg / L, approximately 90 µg / L to 1250 µg / L, approximately 100 µg / L to 1250 µg / L, approximately 125 µg / L to 1250 µg / L, approximately 150 µg / L to 1250 µg / L, approximately 158.8 µg / L to 1250 µg / L, approximately 175 µg / L to 1250 µg / L, approximately 200 µg / L to 1250 µg / L, approximately 250 µg / L to 1250 µg / L, approximately 300 µg / L to 1250 µg / L, approximately 400 µg / L to 1250 µg / L, approximately 500 µg / L to 1250 µg / L, approximately 600 µg / L to 1250 µg / L, and approximately 700 µg / L to 1250 µg / L. µg / L, about 800 µg / L to about 1250 µg / L, about 900 µg / L to about 1250 µg / L, about 1000 µg / L to about 1250 µg / L, about 1100 µg / L to about 1250 µg / L, about 1200 µg / L to about 1250 µg / L, about 10 µg / L to about 1200 µg / L, about 11.4 µg / L to about 1200 µg / L, about 11.5 µg / L to about 1200 µg / L, about 12.5 µg / L to about 1200 µg / L, about 20 µg / L to about 1200 µg / L, about 25 µg / L to about 1200 µg / L, about 30 µg / L to about 1200 µg / L, about 40 µg / L to about 1200 µg / L, about 50 µg / L to about 1200 µg / L, about 60 µg / L to about 1200 µg / L, about 70 µg / L to about 1200 µg / L, about 75 µg / L to about 1200 µg / L, about 80 µg / L to about 1200 µg / L, about 90 µg / L to about 1200 µg / L, about 100 µg / L to about 1200 µg / L, about 125 µg / L Approximately 150 µg / L to 1200 µg / L, approximately 158.8 µg / L to 1200 µg / L, approximately 175 µg / L to 1200 µg / L, approximately 200 µg / L to 1200 µg / L, approximately 250 µg / L to 1200 µg / L, approximately 300 µg / L to 1200 µg / L, approximately 400 µg / L to 1200 µg / L, approximately 500 µg / L to 1200 µg / L, approximately 600 µg / L to 1200 µg / L, approximately 700 µg / L to 1200 µg / L, approximately 800 µg / L to 1200 µg / L, approximately 900 µg / L to 1200 µg / L, and approximately 1000 µg / L to 1200 µg / L. µg / L, about 1100 µg / L to about 1200 µg / L, about 10 µg / L to about 1100 µg / L, about 11.4 µg / L to about 1100 µg / L, about 11.5 µg / L to about 1100 µg / L, about 12.5 µg / L to about 1100 µg / L, about 20 µg / L to about 1100 µg / L, about 25 µg / L to about 1100 µg / L, about 30 µg / L to about 1100 µg / L, about 40 µg / L to about 1100 µg / L, about 50 µg / L to about 1100 µg / L, about 60 µg / L to about 1100 µg / L, about 70 µg / L to about 1100 µg / L, about 75 µg / L to about 1100 µg / L, about 80 µg / L to about 1100 µg / L µg / L, about 90 µg / L to about 1100 µg / L, about 100 µg / L to about 1100 µg / L, about 125 µg / L to about 1100 µg / L, about 150 µg / L to about 1100 µg / L, about 158 ​​µg / L.8 µg / L to about 1100 µg / L, about 175 µg / L to about 1100 µg / L, about 200 µg / L to about 1100 µg / L, about 250 µg / L to about 1100 µg / L, about 300 µg / L to about 1100 µg / L, about 400 µg / L to about 1100 µg / L, about 500 µg / L to about 1100 µg / L, about 600 µg / L to about 1100 µg / L, about 700 µg / L to about 1100 µg / L, about 800 µg / L to about 1100 µg / L, about 900 µg / L to about 1100 µg / L, about 1000 µg / L to about 1100 µg / L, about 10 µg / L to about 1094.7 µg / L, about 11.4 Approximately 1094.7 µg / L to 1094.7 µg / L, approximately 11.5 µg / L to 1094.7 µg / L, approximately 12.5 µg / L to 1094.7 µg / L, approximately 20 µg / L to 1094.7 µg / L, approximately 25 µg / L to 1094.7 µg / L, approximately 30 µg / L to 1094.7 µg / L, approximately 40 µg / L to 1094.7 µg / L, approximately 50 µg / L to 1094.7 µg / L, approximately 60 µg / L to 1094.7 µg / L, approximately 70 µg / L to 1094.7 µg / L, approximately 75 µg / L to 1094.7 µg / L, approximately 80 µg / L to 1094.7 µg / L, and approximately 90 µg / L to 1094.7 µg / L. µg / L, about 100 µg / L to about 1094.7 µg / L, about 125 µg / L to about 1094.7 µg / L, about 150 µg / L to about 1094.7 µg / L, about 158.8 µg / L to about 1094.7 µg / L, about 175 µg / L to about 1094.7 µg / L, about 200 µg / L to about 1094.7 µg / L, about 250 µg / L to about 1094.7 µg / L, about 300 µg / L to about 1094.7 µg / L, about 400 µg / L to about 1094.7 µg / L, about 500 µg / L to about 1094.7 µg / L, about 600 µg / L to about 1094.7 µg / L, about 700 µg / L to about 1094.7 µg / L. µg / L, about 800 µg / L to about 1094.7 µg / L, about 900 µg / L to about 1094.7 µg / L, about 1000 µg / L to about 1094.7 µg / L, about 10 µg / L to about 1000 µg / L, about 11.4 µg / L to about 1000 µg / L, about 11.5 µg / L to about 1000 µg / L, about 12 µg / L.5 µg / L to about 1000 µg / L, about 20 µg / L to about 1000 µg / L, about 25 µg / L to about 1000 µg / L, about 30 µg / L to about 1000 µg / L, about 40 µg / L to about 1000 µg / L, about 50 µg / L to about 1000 µg / L, about 60 µg / L to about 1000 µg / L, about 70 µg / L to about 1000 µg / L, about 75 µg / L to about 1000 µg / L, about 80 µg / L to about 1000 µg / L, about 90 µg / L to about 1000 µg / L, about 100 µg / L to about 1000 µg / L, about 125 µg / L to about 1000 µg / L, about 150 µg / L to about 1000 µg / L, about 158.8 Approximately 175 µg / L to 1000 µg / L, approximately 200 µg / L to 1000 µg / L, approximately 250 µg / L to 1000 µg / L, approximately 300 µg / L to 1000 µg / L, approximately 400 µg / L to 1000 µg / L, approximately 500 µg / L to 1000 µg / L, approximately 600 µg / L to 1000 µg / L, approximately 700 µg / L to 1000 µg / L, approximately 800 µg / L to 1000 µg / L, approximately 900 µg / L to 1000 µg / L, approximately 10 µg / L to 900 µg / L, approximately 11.4 µg / L to 900 µg / L, and approximately 11.5 µg / L to 900 µg / L. µg / L, about 12.5 µg / L to about 900 µg / L, about 20 µg / L to about 900 µg / L, about 25 µg / L to about 900 µg / L, about 30 µg / L to about 900 µg / L, about 40 µg / L to about 900 µg / L, about 50 µg / L to about 900 µg / L, about 60 µg / L to about 900 µg / L, about 70 µg / L to about 900 µg / L, about 75 µg / L to about 900 µg / L, about 80 µg / L to about 900 µg / L, about 90 µg / L to about 900 µg / L, about 100 µg / L to about 900 µg / L, about 125 µg / L to about 900 µg / L, about 150 µg / L to about 900 µg / L, about 158 ​​µg / L.8 µg / L to about 900 µg / L, about 175 µg / L to about 900 µg / L, about 200 µg / L to about 900 µg / L, about 250 µg / L to about 900 µg / L, about 300 µg / L to about 900 µg / L, about 400 µg / L to about 900 µg / L, about 500 µg / L to about 900 µg / L, about 600 µg / L to about 900 µg / L, about 700 µg / L to about 900 µg / L, about 800 µg / L to about 900 µg / L, about 10 µg / L to about 800 µg / L, about 11.4 µg / L to about 800 µg / L, about 11.5 µg / L to about 800 µg / L, about 12.5 µg / L to about 800 µg / L, about 20 Approximately 800 µg / L, approximately 25 µg / L, approximately 30 µg / L, approximately 40 µg / L, approximately 50 µg / L, approximately 60 µg / L, approximately 70 µg / L, approximately 75 µg / L, approximately 80 µg / L, approximately 90 µg / L, approximately 100 µg / L, approximately 125 µg / L, approximately 150 µg / L, approximately 158.8 µg / L, approximately 175 µg / L, approximately 80 ... µg / L, about 200 µg / L to about 800 µg / L, about 250 µg / L to about 800 µg / L, about 300 µg / L to about 800 µg / L, about 400 µg / L to about 800 µg / L, about 500 µg / L to about 800 µg / L, about 600 µg / L to about 800 µg / L, about 700 µg / L to about 800 µg / L, about 10 µg / L to about 700 µg / L, about 11.4 µg / L to about 700 µg / L, about 11.5 µg / L to about 700 µg / L, about 12 µg / L.5 µg / L to about 700 µg / L, about 20 µg / L to about 700 µg / L, about 25 µg / L to about 700 µg / L, about 30 µg / L to about 700 µg / L, about 40 µg / L to about 700 µg / L, about 50 µg / L to about 700 µg / L, about 60 µg / L to about 700 µg / L, about 70 µg / L to about 700 µg / L, about 75 µg / L to about 700 µg / L, about 80 µg / L to about 700 µg / L, about 90 µg / L to about 700 µg / L, about 100 µg / L to about 700 µg / L, about 125 µg / L to about 700 µg / L, about 150 µg / L to about 700 µg / L, about 158.8 µg / L to about 700 µg / L µg / L, about 175 µg / L to about 700 µg / L, about 200 µg / L to about 700 µg / L, about 250 µg / L to about 700 µg / L, about 300 µg / L to about 700 µg / L, about 400 µg / L to about 700 µg / L, about 500 µg / L to about 700 µg / L, about 600 µg / L to about 700 µg / L, about 10 µg / L to about 600 µg / L, about 11.4 µg / L to about 600 µg / L, about 11.5 µg / L to about 600 µg / L, about 12.5 µg / L to about 600 µg / L, about 20 µg / L to about 600 µg / L, about 25 µg / L to about 600 µg / L, about 30 µg / L to about 600 µg / L, about 40 µg / L Approximately 600 µg / L to 600 µg / L, approximately 50 µg / L to 600 µg / L, approximately 60 µg / L to 600 µg / L, approximately 70 µg / L to 600 µg / L, approximately 75 µg / L to 600 µg / L, approximately 80 µg / L to 600 µg / L, approximately 90 µg / L to 600 µg / L, approximately 100 µg / L to 600 µg / L, approximately 125 µg / L to 600 µg / L, approximately 150 µg / L to 600 µg / L, approximately 158.8 µg / L to 600 µg / L, approximately 175 µg / L to 600 µg / L, approximately 200 µg / L to 600 µg / L, approximately 250 µg / L to 600 µg / L, approximately 300 µg / L to 600 µg / L µg / L, about 400 µg / L to about 600 µg / L, about 500 µg / L to about 600 µg / L, about 10 µg / L to about 500 µg / L, about 11.4 µg / L to about 500 µg / L, about 11.5 µg / L to about 500 µg / L, about 12 µg / L.5 µg / L to about 500 µg / L, about 20 µg / L to about 500 µg / L, about 25 µg / L to about 500 µg / L, about 30 µg / L to about 500 µg / L, about 40 µg / L to about 500 µg / L, about 50 µg / L to about 500 µg / L, about 60 µg / L to about 500 µg / L, about 70 µg / L to about 500 µg / L, about 75 µg / L to about 500 µg / L, about 80 µg / L to about 500 µg / L, about 90 µg / L to about 500 µg / L, about 100 µg / L to about 500 µg / L, about 125 µg / L to about 500 µg / L, about 150 µg / L to about 500 µg / L, about 158.8 µg / L to about 500 µg / L µg / L, about 175 µg / L to about 500 µg / L, about 200 µg / L to about 500 µg / L, about 250 µg / L to about 500 µg / L, about 300 µg / L to about 500 µg / L, about 400 µg / L to about 500 µg / L, about 10 µg / L to about 400 µg / L, about 11.4 µg / L to about 400 µg / L, about 11.5 µg / L to about 400 µg / L, about 12.5 µg / L to about 400 µg / L, about 20 µg / L to about 400 µg / L, about 25 µg / L to about 400 µg / L, about 30 µg / L to about 400 µg / L, about 40 µg / L to about 400 µg / L, about 50 µg / L to about 400 µg / L, about 60 µg / L Approximately 70 µg / L to 400 µg / L, approximately 75 µg / L to 400 µg / L, approximately 80 µg / L to 400 µg / L, approximately 90 µg / L to 400 µg / L, approximately 100 µg / L to 400 µg / L, approximately 125 µg / L to 400 µg / L, approximately 150 µg / L to 400 µg / L, approximately 158.8 µg / L to 400 µg / L, approximately 175 µg / L to 400 µg / L, approximately 200 µg / L to 400 µg / L, approximately 250 µg / L to 400 µg / L, approximately 300 µg / L to 400 µg / L, approximately 10 µg / L to 300 µg / L, and approximately 11.4 µg / L to 300 µg / L. µg / L, about 11.5 µg / L to about 300 µg / L, about 12.5 µg / L to about 300 µg / L, about 20 µg / L to about 300 µg / L, about 25 µg / L to about 300 µg / L, about 30 µg / L to about 300 µg / L, about 40 µg / L to about 300 µg / L, about 50 µg / L to about 300 µg / L, about 60 µg / L to about 300 µg / L, about 70 µg / L to about 300 µg / L, about 75 µg / L to about 300 µg / L, about 80 µg / L to about 300 µg / L, about 90 µg / L to about 300 µg / L, about 100 µg / L to about 300 µg / L, about 125 µg / L to about 300 µg / L, about 150 µg / L to about 300 µg / L, about 158.8 µg / L to about 300 µg / L µg / L, about 175 µg / L to about 300 µg / L, about 200 µg / L to about 300 µg / L, about 250 µg / L to about 300 µg / L, about 10 µg / L to about 250 µg / L, about 11.4 µg / L to about 250 µg / L, about 11.5 µg / L to about 250 µg / L, about 12.5 µg / L to about 250 µg / L, about 20 µg / L to about 250 µg / L, about 25 µg / L to about 250 µg / L, about 30 µg / L to about 250 µg / L, about 40 µg / L to about 250 µg / L, about 50 µg / L to about 250 µg / L, about 60 µg / L to about 250 µg / L, about 70 µg / L to about 250 µg / L, about 75 ... About 250 µg / L, about 80 µg / L to about 250 µg / L, about 90 µg / L to about 250 µg / L, about 100 µg / L to about 250 µg / L, about 125 µg / L to about 250 µg / L, about 150 µg / L to about 250 µg / L, about 158.8 µg / L to about 250 µg / L, about 175 µg / L to about 250 µg / L, about 200 µg / L to about 250 µg / L, about 10 µg / L to about 200 µg / L, about 11.4 µg / L to about 200 µg / L, about 11.5 µg / L to about 200 µg / L, about 12 µg / L.5 µg / L to about 200 µg / L, about 20 µg / L to about 200 µg / L, about 25 µg / L to about 200 µg / L, about 30 µg / L to about 200 µg / L, about 40 µg / L to about 200 µg / L, about 50 µg / L to about 200 µg / L, about 60 µg / L to about 200 µg / L, about 70 µg / L to about 200 µg / L, about 75 µg / L to about 200 µg / L, about 80 µg / L to about 200 µg / L, about 90 µg / L to about 200 µg / L, about 100 µg / L to about 200 µg / L, about 125 µg / L to about 200 µg / L, about 150 µg / L to about 200 µg / L, about 158.8 µg / L to about 200 µg / L µg / L, about 175 µg / L to about 200 µg / L, about 10 µg / L to about 175 µg / L, about 11.4 µg / L to about 175 µg / L, about 11.5 µg / L to about 175 µg / L, about 12.5 µg / L to about 175 µg / L, about 20 µg / L to about 175 µg / L, about 25 µg / L to about 175 µg / L, about 30 µg / L to about 175 µg / L, about 40 µg / L to about 175 µg / L, about 50 µg / L to about 175 µg / L, about 60 µg / L to about 175 µg / L, about 70 µg / L to about 175 µg / L, about 75 µg / L to about 175 µg / L, about 80 µg / L to about 175 µg / L, about 90 µg / L Approximately 100 µg / L to 175 µg / L, approximately 125 µg / L to 175 µg / L, approximately 150 µg / L to 175 µg / L, approximately 158.8 µg / L to 175 µg / L, approximately 10 µg / L to 150 µg / L, approximately 11.4 µg / L to 150 µg / L, approximately 11.5 µg / L to 150 µg / L, approximately 12.5 µg / L to 150 µg / L, approximately 20 µg / L to 150 µg / L, approximately 25 µg / L to 150 µg / L, approximately 30 µg / L to 150 µg / L, approximately 40 µg / L to 150 µg / L, approximately 50 µg / L to 150 µg / L, approximately 60 µg / L to 150 µg / L µg / L, about 70 µg / L to about 150 µg / L, about 75 µg / L to about 150 µg / L, about 80 µg / L to about 150 µg / L, about 90 µg / L to about 150 µg / L, about 100 µg / L to about 150 µg / L, about 125 µg / L to about 150 µg / L, about 10 µg / L to about 125 µg / L, about 11 µg / L.4 µg / L to about 125 µg / L, about 11.5 µg / L to about 125 µg / L, about 12.5 µg / L to about 125 µg / L, about 20 µg / L to about 125 µg / L, about 25 µg / L to about 125 µg / L, about 30 µg / L to about 125 µg / L, about 40 µg / L to about 125 µg / L, about 50 µg / L to about 125 µg / L, about 60 µg / L to about 125 µg / L, about 70 µg / L to about 125 µg / L, about 75 µg / L to about 125 µg / L, about 80 µg / L to about 125 µg / L, about 90 µg / L to about 125 µg / L, about 100 µg / L to about 125 µg / L, about 10 µg / L to about 100 µg / L µg / L, about 11.4 µg / L to about 100 µg / L, about 11.5 µg / L to about 100 µg / L, about 12.5 µg / L to about 100 µg / L, about 20 µg / L to about 100 µg / L, about 25 µg / L to about 100 µg / L, about 30 µg / L to about 100 µg / L, about 40 µg / L to about 100 µg / L, about 50 µg / L to about 100 µg / L, about 60 µg / L to about 100 µg / L, about 70 µg / L to about 100 µg / L, about 75 µg / L to about 100 µg / L, about 80 µg / L to about 100 µg / L, about 90 µg / L to about 100 µg / L, about 10 µg / L to about 90 µg / L, about 11.4 µg / L to about 90 µg / L µg / L, about 11.5 µg / L to about 90 µg / L, about 12.5 µg / L to about 90 µg / L, about 20 µg / L to about 90 µg / L, about 25 µg / L to about 90 µg / L, about 30 µg / L to about 90 µg / L, about 40 µg / L to about 90 µg / L, about 50 µg / L to about 90 µg / L, about 60 µg / L to about 90 µg / L, about 70 µg / L to about 90 µg / L, about 75 µg / L to about 90 µg / L, about 80 µg / L to about 90 µg / L, about 10 µg / L to about 80 µg / L, about 11.4 µg / L to about 80 µg / L, about 11.5 µg / L to about 80 µg / L, about 12.5 µg / L to about 80 µg / L, about 20 µg / L to about 80 µg / L, about 25 µg / L to about 80 µg / L, about 30 µg / L to about 80 µg / L, about 40 µg / L to about 80 µg / L, about 50 µg / L to about 80 µg / L, about 60 µg / L to about 80 µg / L, about 70 µg / L to about 80 µg / L, about 75 µg / L to about 80 µg / L, about 10 µg / L to about 75 µg / L, about 11.4 µg / L to about 75 µg / L, about 11.5 µg / L to about 75 µg / L, about 12.5 µg / L to about 75 µg / L, about 20 µg / L to about 75 µg / L, about 25 µg / L to about 75 µg / L, about 30 µg / L to about 75 µg / L µg / L, about 40 µg / L to about 75 µg / L, about 50 µg / L to about 75 µg / L, about 60 µg / L to about 75 µg / L, about 70 µg / L to about 75 µg / L, about 10 µg / L to about 70 µg / L, about 11.4 µg / L to about 70 µg / L, about 11.5 µg / L to about 70 µg / L, about 12.5 µg / L to about 70 µg / L, about 20 µg / L to about 70 µg / L, about 25 µg / L to about 70 µg / L, about 30 µg / L to about 70 µg / L, about 40 µg / L to about 70 µg / L, about 50 µg / L to about 70 µg / L, about 60 µg / L to about 70 µg / L, about 10 µg / L to about 60 µg / L, about 11.4 µg / L to about 7 ... Approximately 11.5 µg / L to 60 µg / L, approximately 12.5 µg / L to 60 µg / L, approximately 20 µg / L to 60 µg / L, approximately 25 µg / L to 60 µg / L, approximately 30 µg / L to 60 µg / L, approximately 40 µg / L to 60 µg / L, approximately 50 µg / L to 60 µg / L, approximately 10 µg / L to 50 µg / L, approximately 11.4 µg / L to 50 µg / L, approximately 11.5 µg / L to 50 µg / L, approximately 12.5 µg / L to 50 µg / L, approximately 20 µg / L to 50 µg / L, approximately 25 µg / L to 50 µg / L, approximately 30 µg / L to 50 µg / L, approximately 40 µg / L to 50 µg / L µg / L, about 10 µg / L to about 40 µg / L, about 11.4 µg / L to about 40 µg / L, about 11.5 µg / L to about 40 µg / L, about 12 µg / L.5 µg / L to about 40 µg / L, about 20 µg / L to about 40 µg / L, about 25 µg / L to about 40 µg / L, about 30 µg / L to about 40 µg / L, about 10 µg / L to about 30 µg / L, about 11.4 µg / L to about 30 µg / L, about 11.5 µg / L to about 30 µg / L, about 12.5 µg / L to about 30 µg / L, about 20 µg / L to about 30 µg / L, about 25 µg / L to about 30 µg / L, about 10 µg / L to about 25 µg / L, about 11.4 µg / L to about 25 µg / L, about 11.5 µg / L to about 25 µg / L, about 12.5 µg / L to about 25 µg / L, about 20 µg / L to about 25 µg / L, about 10 µg / L to about 20 µg / L µg / L, about 11.4 µg / L to about 20 µg / L, about 11.5 µg / L to about 20 µg / L, about 12.5 µg / L to about 20 µg / L µg / L or approximately 10, 11.4, 11.5, 12.5, 20, 25, 30, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 158.8, 175, 200, 250, 300, 400, 500, 529.2, 600, 700, 800, 900, 1000, 1094.7, 1100, 1200, 1300, 1360, 1386.4, 1400, 1500, 1753, 2000, 3000, 4000, 5000, 5500 µg / L or any range or value thereof. In some embodiments, the fermentation product comprises phenethyl acetate and isoamyl acetate in amounts from about 11.4 µg / L to about 1094.7 µg / L.

[0275] In some embodiments, the fermentation product contains acetates (total phenethyl acetate, isoamyl acetate, and ethyl acetate) in a concentration of about 500 µg / L to about 6500 µg / L. In some embodiments, the fermentation product comprises acetates (total phenethyl acetate, isoamyl acetate, and ethyl acetate) in amounts of about 500 µg / L to about 6500 µg / L, about 536.2 µg / L to about 6500 µg / L, about 536.3 µg / L to about 6500 µg / L, about 574.5 µg / L to about 6500 µg / L, about 600 µg / L to about 6500 µg / L, about 700 µg / L to about 6500 µg / L, about 800 µg / L to about 6500 µg / L, about 900 µg / L to about 6500 µg / L, about 1000 µg / L to about 6500 µg / L, about 1500 µg / L to about 6500 µg / L, about 2000 µg / L to about 6500 µg / L, and about 2500 µg / L. Approximately 6500 µg / L to 3000 µg / L, approximately 3500 µg / L to 6500 µg / L, approximately 4000 µg / L to 6500 µg / L, approximately 4500 µg / L to 6500 µg / L, approximately 5000 µg / L to 6500 µg / L, approximately 5500 µg / L to 6500 µg / L, approximately 6000 µg / L to 6500 µg / L, approximately 500 µg / L to 6219 µg / L, approximately 536.2 µg / L to 6219 µg / L, approximately 536.3 µg / L to 6219 µg / L, approximately 574.5 µg / L to 6219 µg / L, approximately 600 µg / L to 6219 µg / L µg / L, about 700 µg / L to about 6219 µg / L, about 800 µg / L to about 6219 µg / L, about 900 µg / L to about 6219 µg / L, about 1000 µg / L to about 6219 µg / L, about 1500 µg / L to about 6219 µg / L, about 2000 µg / L to about 6219 µg / L, about 2500 µg / L to about 6219 µg / L, about 3000 µg / L to about 6219 µg / L, about 3500 µg / L to about 6219 µg / L, about 4000 µg / L to about 6219 µg / L, about 4500 µg / L to about 6219 µg / L, about 5000 µg / L to about 6219 µg / L, about 5500 µg / L to about 6219 µg / L µg / L, about 6000 µg / L to about 6219 µg / L, about 500 µg / L to about 6000 µg / L, about 536.2 µg / L to about 6000 µg / L, about 536.3 µg / L to about 6000 µg / L, about 574.5 µg / L to about 6000 µg / L, about 600 µg / L to about 6000 µg / L, about 700 µg / L to about 6000 µg / L, about 800 µg / L to about 6000 µg / L, about 900 µg / L to about 6000 µg / L, about 1000 µg / L to about 6000 µg / L, about 1500 µg / L to about 6000 µg / L, about 2000 µg / L to about 6000 µg / L, about 2500 µg / L to about 6000 µg / L, about 3000 µg / L to about 6000 µg / L, about 3500 µg / L to about 6000 µg / L, about 4000 µg / L to about 6000 µg / L, about 4500 µg / L Approximately 5000 µg / L to 6000 µg / L, approximately 5500 µg / L to 6000 µg / L, approximately 500 µg / L to 5500 µg / L, approximately 536.2 µg / L to 5500 µg / L, approximately 536.3 µg / L to 5500 µg / L, approximately 574.5 µg / L to 5500 µg / L, approximately 600 µg / L to 5500 µg / L, approximately 700 µg / L to 5500 µg / L, approximately 800 µg / L to 5500 µg / L, approximately 900 µg / L to 5500 µg / L, approximately 1000 µg / L to 5500 µg / L, approximately 1500 µg / L to 5500 µg / L, approximately 2000 µg / L... µg / L to about 5500 µg / L, about 2500 µg / L to about 5500 µg / L, about 3000 µg / L to about 5500 µg / L, about 3500 µg / L to about 5500 µg / L, about 4000 µg / L to about 5500 µg / L, about 4500 µg / L to about 5500 µg / L, about 5000 µg / L to about 5500 µg / L, about 500 µg / L to about 5000 µg / L, about 536.2 µg / L to about 5000 µg / L, about 536.3 µg / L to about 5000 µg / L, about 574 µg / L.5 µg / L to about 5000 µg / L, about 600 µg / L to about 5000 µg / L, about 700 µg / L to about 5000 µg / L, about 800 µg / L to about 5000 µg / L, about 900 µg / L to about 5000 µg / L, about 1000 µg / L to about 5000 µg / L, about 1500 µg / L to about 5000 µg / L, about 2000 µg / L to about 5000 µg / L, about 2500 µg / L to about 5000 µg / L, about 3000 µg / L to about 5000 µg / L, about 3500 µg / L to about 5000 µg / L, about 4000 µg / L to about 5000 µg / L, about 4500 µg / L to about 5000 µg / L, about 500 µg / L Approximately 4725.4 µg / L, approximately 536.2 µg / L, approximately 536.3 µg / L, approximately 4725.4 µg / L, approximately 574.5 µg / L, approximately 600 µg / L, approximately 700 µg / L, approximately 800 µg / L, approximately 900 µg / L, approximately 1000 µg / L, approximately 1500 µg / L, approximately 2000 µg / L, and approximately 2500 µg / L to 4725.4 µg / L. µg / L, about 3000 µg / L to about 4725.4 µg / L, about 3500 µg / L to about 4725.4 µg / L, about 4000 µg / L to about 4725.4 µg / L, about 4500 µg / L to about 4725.4 µg / L, about 500 µg / L to about 4500 µg / L, about 536.2 µg / L to about 4500 µg / L, about 536.3 µg / L to about 4500 µg / L, about 574 µg / L.5 µg / L to about 4500 µg / L, about 600 µg / L to about 4500 µg / L, about 700 µg / L to about 4500 µg / L, about 800 µg / L to about 4500 µg / L, about 900 µg / L to about 4500 µg / L, about 1000 µg / L to about 4500 µg / L, about 1500 µg / L to about 4500 µg / L, about 2000 µg / L to about 4500 µg / L, about 2500 µg / L to about 4500 µg / L, about 3000 µg / L to about 4500 µg / L, about 3500 µg / L to about 4500 µg / L, about 4000 µg / L to about 4500 µg / L, about 500 µg / L to about 4000 µg / L, about 536.2 Approximately 536.3 µg / L to 4000 µg / L, approximately 574.5 µg / L to 4000 µg / L, approximately 600 µg / L to 4000 µg / L, approximately 700 µg / L to 4000 µg / L, approximately 800 µg / L to 4000 µg / L, approximately 900 µg / L to 4000 µg / L, approximately 1000 µg / L to 4000 µg / L, approximately 1500 µg / L to 4000 µg / L, approximately 2000 µg / L to 4000 µg / L, approximately 2500 µg / L to 4000 µg / L, approximately 3000 µg / L to 4000 µg / L, approximately 3500 µg / L to 4000 µg / L, approximately ... Approximately 536.2 µg / L to 3500 µg / L, approximately 536.3 µg / L to 3500 µg / L, approximately 574.5 µg / L to 3500 µg / L, approximately 600 µg / L to 3500 µg / L, approximately 700 µg / L to 3500 µg / L, approximately 800 µg / L to 3500 µg / L, approximately 900 µg / L to 3500 µg / L, approximately 1000 µg / L to 3500 µg / L, approximately 1500 µg / L to 3500 µg / L, approximately 2000 µg / L to 3500 µg / L, approximately 2500 µg / L to 3500 µg / L, approximately 3000 µg / L to 3500 µg / L, approximately 5 ... µg / L to about 3000 µg / L, about 536.2 µg / L to about 3000 µg / L, about 536.3 µg / L to about 3000 µg / L, about 574 µg / L.5 µg / L to about 3000 µg / L, about 600 µg / L to about 3000 µg / L, about 700 µg / L to about 3000 µg / L, about 800 µg / L to about 3000 µg / L, about 900 µg / L to about 3000 µg / L, about 1000 µg / L to about 3000 µg / L, about 1500 µg / L to about 3000 µg / L, about 2000 µg / L to about 3000 µg / L, about 2500 µg / L to about 3000 µg / L, about 500 µg / L to about 2500 µg / L, about 536.2 µg / L to about 2500 µg / L, about 536.3 µg / L to about 2500 µg / L, about 574.5 µg / L to about 2500 µg / L, about 600 µg / L to about 30 ... Approximately 2500 µg / L to 2500 µg / L, approximately 700 µg / L to 2500 µg / L, approximately 800 µg / L to 2500 µg / L, approximately 900 µg / L to 2500 µg / L, approximately 1000 µg / L to 2500 µg / L, approximately 1500 µg / L to 2500 µg / L, approximately 2000 µg / L to 2500 µg / L, approximately 500 µg / L to 2000 µg / L, approximately 536.2 µg / L to 2000 µg / L, approximately 536.3 µg / L to 2000 µg / L, approximately 574.5 µg / L to 2000 µg / L, approximately 600 µg / L to 2000 µg / L, approximately 700 µg / L to 2000 µg / L, approximately 800 µg / L to 25 ... Approximately 2000 µg / L to 2000 µg / L, approximately 900 µg / L to 2000 µg / L, approximately 1000 µg / L to 2000 µg / L, approximately 1500 µg / L to 2000 µg / L, approximately 500 µg / L to 1500 µg / L, approximately 536.2 µg / L to 1500 µg / L, approximately 536.3 µg / L to 1500 µg / L, approximately 574.5 µg / L to 1500 µg / L, approximately 600 µg / L to 1500 µg / L, approximately 700 µg / L to 1500 µg / L, approximately 800 µg / L to 1500 µg / L, approximately 900 µg / L to 1500 µg / L, approximately 1000 µg / L to 1500 µg / L, approximately 500 µg / L to 20 ... µg / L to about 1000 µg / L, about 536.2 µg / L to about 1000 µg / L, about 536.3 µg / L to about 1000 µg / L, about 574 µg / L.5 µg / L to about 1000 µg / L, about 600 µg / L to about 1000 µg / L, about 700 µg / L to about 1000 µg / L, about 800 µg / L to about 1000 µg / L, about 900 µg / L to about 1000 µg / L, about 500 µg / L to about 900 µg / L, about 536.2 µg / L to about 900 µg / L, about 536.3 µg / L to about 900 µg / L, about 574.5 µg / L to about 900 µg / L, about 600 µg / L to about 900 µg / L, about 700 µg / L to about 900 µg / L, about 800 µg / L to about 900 µg / L, about 500 µg / L to about 800 µg / L, about 536.2 µg / L to about 800 µg / L µg / L, about 536.3 µg / L to about 800 µg / L, about 574.5 µg / L to about 800 µg / L, about 600 µg / L to about 800 µg / L, about 700 µg / L to about 800 µg / L, about 500 µg / L to about 700 µg / L, about 536.2 µg / L to about 700 µg / L, about 536.3 µg / L to about 700 µg / L, about 574.5 µg / L to about 700 µg / L, about 600 µg / L to about 700 µg / L, about 500 µg / L to about 600 µg / L, about 536.2 µg / L to about 600 µg / L, about 536.3 µg / L to about 600 µg / L, about 574.5 µg / L to about 600 µg / L µg / L or about 500, 536.2, 536.3, 574.5, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 4725.4, 5000, 5460.2, 5500, 6000, 6219, or 6500 µg / L or any range or value thereof. In some embodiments, the fermentation product comprises acetate esters (total phenethyl acetate, isoamyl acetate, and ethyl acetate) in an amount from about 536.2 µg / L to about 4725.4 µg / L.

[0276] It should be understood that the fermentation method provided herein can be carried out under conditions sufficient to produce the required amounts of isoamyl acetate, phenethyl acetate, isoamyl acetate and phenethyl acetate or total acetate (isoamyl acetate, phenethyl acetate and isoamyl acetate).

[0277] In some embodiments, a liquid fermentation composition is provided comprising: (a) a population of genetically modified yeast cells to produce one or more acetates and / or ethyl acetates, wherein the genetically modified yeast cells cannot convert maltose and / or maltotriose to ethanol, or have a reduced conversion capacity; (b) a sugar source comprising wort, wherein the total sugar in the wort is attenuated by the population of genetically modified yeast cells by no more than 25%; (c) one or more aldehyde and / or non-aldehyde molecules derived from wort; and (d) no more than about 1.0% (v / v) of alcohol. In some embodiments, the liquid fermentation composition contains no more than about 0.5% (v / v) of alcohol.

[0278] In some embodiments, a liquid fermentation composition is provided comprising: (a) a population of genetically modified yeast cells that are unable to convert maltose and / or maltotriose into ethanol, or have reduced conversion capabilities; (b) a sugar source comprising wort, wherein the total sugars in the wort are attenuated by the population of genetically modified yeast cells by no more than 25%; and (c) no more than 1.0% (v / v) of alcohol. In some embodiments, the liquid fermentation composition contains no more than about 0.5% (v / v) of alcohol.

[0279] It should be understood that throughout this disclosure, the alcohol content provided by volume may include 0% or a minimum or maximum value, such as a minimum of 0.01% alcohol by volume and a maximum of 1.0% alcohol by volume. It should also be understood that in alternative embodiments of this disclosure, the alcohol content by volume of the liquid fermentation composition or fermented beverage and / or product does not exceed 5% or 2.5%.

[0280] It should be understood that the population of genetically modified yeast cells can be modified cells in any way as disclosed in this disclosure.

[0281] In some embodiments, the aldehyde molecules are selected from the group consisting of 2-methylbutyraldehyde, 2-methylpropionaldehyde, hexanal, benzaldehyde, furfural, acetaldehyde, methylthionaldehyde, phenylacetaldehyde, and 5-hydroxymethylfurfural. In some embodiments, the non-aldehyde molecules are selected from the group consisting of (E)-β-damascone and 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone.

[0282] It should also be understood that the levels of acetate and / or ethyl acetate described herein may be characteristic of the liquid fermentation compositions disclosed herein.

[0283] The methods described herein may involve at least one additional fermentation process. Such an additional fermentation method may be referred to as a secondary fermentation process (also known as “aging” or “maturing”). As those skilled in the art will understand, secondary fermentation typically involves transferring the fermented beverage to a second container (e.g., a glass fermentation tank, barrel) and incubating the fermented beverage in that container for a period of time. In some embodiments, secondary fermentation takes from 10 minutes to 12 months. In some implementations, the secondary fermentation is carried out for 10 minutes, 20 minutes, 40 minutes, 40 minutes, 50 minutes, 60 minutes (1 hour), 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. In some embodiments, the additional or secondary fermentation of one or more fermentable sugars can be carried out at a temperature of about 4°C to about 30°C. In some embodiments, the additional or secondary fermentation of one or more fermentable sugars can be carried out at a temperature of about 8°C to about 14°C or about 18°C ​​to about 24°C. In some embodiments, the additional or secondary fermentation of one or more fermentable sugars can be carried out at a temperature of about 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.

[0284] As will be apparent to those skilled in the art, the choice of time and temperature for additional or secondary fermentation processes will depend on factors such as the type of beer, the desired characteristics of the beer, and the yeast strain used in the method.

[0285] In some implementations, one or more additional flavoring agents may be added to the culture medium before or after the fermentation process. Examples include hop oil, hop aroma compounds, hop extracts, hop bittering agents, and isomerized hop extracts.

[0286] Various refining, filtration, and maturation processes can occur after fermentation, after which the liquid is bottled (i.e., filled and sealed in containers for dispensing, storage, or consumption). Any of the methods described herein may further involve distillation, pasteurization, and / or carbonation of the fermentation product. In some embodiments, the method involves carbonation of the fermentation product. Methods for carbonating fermented beverages are known in the art, including, for example, forced carbonation with gases (e.g., carbon dioxide, nitrogen), and natural carbonation by adding further sugar sources to the fermented beverage to promote further fermentation and produce carbon dioxide (e.g., bottle conditioning).

[0287] Fermented products

[0288] This disclosure relates to fermented products produced by any of the methods disclosed herein. In some embodiments, the fermented product is a fermented beverage. In some embodiments, the beverage is beer. In some embodiments, the beverage is sake.

[0289] In some implementations, the fermented product is a fermented food. Examples of fermented foods include, but are not limited to, fermented yogurt, tempeh, miso, kimchi, sauerkraut, fermented sausage, bread, and soy sauce.

[0290] Various aspects of this disclosure relate to sensory detection methods for reducing undesirable molecules such as wort-related off-flavors in fermentation products.

[0291] In some embodiments, the production of acetate and / or ethyl acetate as described herein results in a reduction of approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more in the sensory detection of undesirable wort-related off-odors in fermentation products compared to those produced using yeast cells that do not produce acetate and / or ethyl acetate.

[0292] In some implementations, the production of 3-mercaptohexanol (3MH) as described herein results in a reduction of approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more in the sensory detection of undesirable wort-related off-odors in fermentation products produced using yeast cells that do not produce 3MH.

[0293] In some embodiments, the production of one or more monoterpenes (e.g., linalool, geraniol, and / or citronellol) as described herein results in a reduction of approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more in the sensory detection of undesirable wort-related off-odors in fermentation products produced using yeast cells that do not produce one or more monoterpenes.

[0294] Methods for measuring the levels of wort-related off-flavors or molecules that contribute to wort-related off-flavors will be readily apparent to those skilled in the art and may include sensory assessments (e.g., taste, smell, appearance), such as by a subject sensory panel, including, for example, human taste testers. In some embodiments, gas chromatography-mass spectrometry (GC / MS) is used to measure the levels of wort-related off-flavors or molecules that contribute to wort-related off-flavors. In some embodiments, liquid chromatography-mass spectrometry (LC / MS) is used to measure the levels of wort-related off-flavors or molecules that contribute to wort-related off-flavors.

[0295] In some embodiments, the fermented beverage contains less than or equal to about 0.5% alcohol by volume (also referred to as "ABV", "abv", or "alc / vol"), which is considered "alcohol-free". In some embodiments, the fermented beverage contains less than about 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% alcohol by volume. In some embodiments, the fermented beverage is 0% alcohol by volume (zero alcohol).

[0296] It should be understood that fermented products (such as fermented beverages) of this disclosure may have the properties listed throughout this disclosure for fermented products, such as acetates and / or ethyl acetates, as well as excluded compounds or compounds present at levels below the specified limits.

[0297] Reagent test kit

[0298] Various aspects of this disclosure also provide kits for using genetically modified yeast cells, for example, for producing fermented products such as fermented beverages. In some embodiments, the kit comprises modified cells containing one or more genetic modifications to reduce sensory detection of one or more wort-related off-flavors in fermented beverages, wherein the modified cells cannot convert maltose and / or maltotriose into ethanol.

[0299] In some embodiments, the kit is used for producing fermented beverages. In some embodiments, the kit is used for producing beer. In some embodiments, the kit is used for producing sake.

[0300] The kit may also contain other components for any of the methods described herein or for any of the cell uses described herein. For example, in some embodiments, the kit may contain grains, water, wort, grape juice, yeast, hops, fruit juice, or other sugar sources. In some embodiments, the kit may contain one or more fermentable sugars. In some embodiments, the kit may contain one or more additives, ingredients, or components.

[0301] Instructions for performing the methods described herein may also be included in the kits described herein.

[0302] The kit can be organized to indicate a single-use composition containing any of the modified cells described herein. For example, a single-use composition (e.g., dosage) can be a packaged composition (e.g., modified cells), such as a powder, vial, ampoule, culture tube, tablet, capsule, or pouch containing liquid packaged in a bag (i.e., contained in a bag).

[0303] Compositions (e.g., modified cells) can be provided in dry, lyophilized, frozen, or liquid form. In some embodiments, modified cells are provided as colonies on agar medium. In some embodiments, modified cells are provided as a starting culture that can be directly added to a culture medium. When reagents or components are provided in dry form, they are typically reconstituted by adding a solvent such as a culture medium. The solvent may be provided in another packaging manner and may be selected by those skilled in the art.

[0304] Those skilled in the art are familiar with various packages or kits for dispensing compositions (e.g., modified cells). In some embodiments, the packaging is a labeled blister pack, a dial dispenser pack, a tube, a bag, a drum, or a bottle.

[0305] Any kit described herein may further include one or more containers, such as glass bottles or barrels, for performing the methods described herein.

[0306] General technology

[0307] Unless otherwise stated, the practice of the subject matter of this disclosure will employ conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. Such techniques are well explained in the literature, including but not limited to: *Molecular Cloning: A Laboratory Manual*, J. Sambrook et al., eds., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012; *Oligonucleotide Synthesis* (MJ Gait, ed., 1984); *Methods in Molecular Biology*, Humana Press; *Cell Biology: A Laboratory Notebook* (JE Cellis, ed., 1998), Academic Press; *Animal Cell Culture* (RIFreshney, ed., 1987); *Introduction to Cell and Tissue Culture* (JP Mather and PE Roberts, 1998), Plenum Press; *Cell and Tissue Culture: Laboratory Procedures* (A. Doyle, JB Griffiths, and DG Newell, eds., 1993–8), J. Wiley and Sons; *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (DM Weir and CC Blackwell, Edited.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, edited, 1987); Current Protocols in Molecular Biology (FM Ausubel et al., edited, 1987); PCR: The Polymerase Chain Reaction (Mullis et al., edited, 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991; Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0308] Equivalents and scope

[0309] It should be understood that this disclosure is not limited to any or all of the specific embodiments explicitly described herein, and therefore variations are certainly possible. It should also be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting.

[0310] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this disclosure, preferred methods and materials are described here.

[0311] All publications and patents referenced in this disclosure are for the purpose of disclosing and describing methods and / or materials in connection with the publication to which they are referenced. All such publications and patents are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and / or materials described in the cited publications and patents and does not extend to any dictionary definitions in the cited publications and patents (i.e., any vocabulary definition in the cited publications and patents that is not expressly repeated in this disclosure should not be considered such a definition, nor should it be construed as defining any term appearing in the claims). In the event of any conflict between any incorporated reference and this disclosure, this disclosure shall prevail. Furthermore, any particular embodiment of this disclosure that falls within the scope of the prior art may be expressly excluded from any one or more claims. Since such embodiments are considered known to one of ordinary skill in the art, they may be excluded even if not expressly stated herein. Any particular embodiment of this disclosure may be excluded from any claim for any reason, whether or not related to the existence of the prior art.

[0312] References to any publication are made solely to indicate its prior disclosure prior to the filing date of this application and should not be construed as an admission that this application does not enjoy any rights prior to that publication through previous disclosure. Furthermore, the publication date provided may differ from the actual publication date and may require independent verification.

[0313] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any other several embodiments without departing from the scope or spirit of this disclosure. Any described method may be performed in the order of the described events or in any other logically possible order.

[0314] In the claims, terms such as “a,” “an,” “the,” and “the” may refer to one or more than one unless indicated to the contrary or the context clearly states otherwise. Where pronouns are used herein, regardless of their implied part of speech (e.g., masculine, feminine, neutral, other, etc.), they shall be interpreted as part of speech neutral (i.e., interpreted as equally referring to all parts of speech) unless the context clearly indicates or requires otherwise. Singular words used herein include plurals, and plural words include singulars, unless the context clearly indicates or requires otherwise. If a claim or description includes “or” among the members, the condition is satisfied as long as one, more than one, or all members of the group are present, applied to, or otherwise related to the given product or process, unless indicated to the contrary or the context clearly states otherwise. This disclosure includes embodiments in which exactly one member of the group is present, applied to, or otherwise related to the given product or process. This disclosure includes embodiments in which more than one or all members of the group are present, applied to, or otherwise related to the given product or process.

[0315] Furthermore, this disclosure covers all variations, combinations, and arrangements that incorporate one or more limitations, elements, terms, and descriptive terms from one or more of the listed claims into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations from any other claim dependent on the same basic claim. When elements are presented in list form (e.g., in Markush group form), each subgroup of elements is also disclosed, and any element may be removed from the group. It should be understood that, in general, when this disclosure or an aspect thereof is referred to as including a particular element and / or feature, certain embodiments or aspects of this disclosure consist of or are substantially composed of such elements and / or features. For simplicity, these embodiments are not specifically described herein in their original word-for-word form. It should also be noted that the terms “comprising” and “including” are intended to be open-ended and allow for the inclusion of additional elements or steps. When a scope is given, endpoints are included within that scope unless otherwise stated. Furthermore, unless otherwise indicated or obvious from the context and from the understanding of a person skilled in the art, in different embodiments of this disclosure, values ​​expressed in range form may take any specific value or subrange within that range, up to one-tenth of the lower limit unit of that range, unless the context explicitly specifies otherwise.

[0316] Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments described herein using only conventional experiments. The scope of the embodiments described herein is not intended to be limited to the foregoing description, but rather as set forth in the appended claims. Those skilled in the art will understand that various changes and modifications can be made to this specification without departing from the spirit or scope of this disclosure, as defined in the following claims.

[0317] Implementation

[0318] Example 1: Engineering of maltose-negative yeast strains

[0319] As discussed in this article, the limited number of maltose-negative yeast strains available for brewing presents a significant challenge to the brewing industry. For non-alcoholic beers produced by stopping fermentation using currently available maltose-negative strains, these methods are associated with pronounced wort flavor and aroma. The wort flavor in stopped-fermentation beer results from the presence of a combination of aldehydes and non-aldehydes. These aldehydes are present in the unfermented wort, and in normal alcoholic fermentation, the yeast either converts them into other non-flavor-contributing molecules or significantly alters the beer's chemistry by producing ethanol and other flavor molecules, rendering these molecules imperceptible. In contrast, in stopped fermentation using maltose-negative strains, it is believed that one or both of these processes do not occur or occur at reduced levels, thus aldehydes are present and are very noticeable as off-flavors in the final beer.

[0320] Saccharomyces cerevisiae strains contain several distinct maltose transporter genes responsible for transporting maltose and maltotriose into the cell. Duplication and rearrangement of maltose transporter genes are common in brewing yeasts, and brewing strains typically have more than 10, and sometimes as many as 15, copies of different maltose transporter genes encoded in their genomes. Because maltose transporter loci frequently undergo duplication, deletion, and rearrangement, yeast strains often rely on different combinations of maltose transporter genes to be able to transport maltose and maltotriose into the cell. Therefore, while deletion of maltose transporter genes is expected to prevent yeast from metabolizing certain sugars, it is unclear or unpredictable which gene combinations need to be deleted to make the strain maltose-negative (lacking the conversion of maltose and / or maltotriose to ethanol). Previously, some non-brewing yeast strains (MN) were generated by deleting one or more different combinations of maltose transporters, but these strains have no history of use in brewing beer. If these strains are used in brewing, they may produce off-flavors typically associated with yeast strains that are not adapted to beer fermentation (Yabaci Karaoglan, S., Jung, R., Gauthier, M., Kinčl, T. & Dostálek, P. Maltose-Negative Yeast in Non-Alcoholic and Low-Alcoholic Beer Production. Fermentation 8, 273 (2022); Simões, J. et al. Exploiting Non-Conventional Yeasts for Low-Alcohol Beer Production. Microorganisms 11, (2023)).

[0321] To generate novel MN yeast variants from existing brewing yeast strains, the *Saccharomyces cerevisiae* strain *Chico* was used. Chico is one of the most widely used craft brewing strains, known for producing crisp ales and being a strong flocculant. Unlike other previously engineered yeast strains, *Saccharomyces cerevisiae* strain *Chico* is known to metabolize both maltose and maltotriose. To engineer a *Saccharomyces cerevisiae* strain lacking the ability to ferment maltose and maltotriose, ge...

Claims

1. A genetically modified yeast cell that cannot convert maltose and / or maltotriose into ethanol or has a reduced ability to convert maltose and / or maltotriose into ethanol, said genetically modified yeast cell comprising a heterologous nucleic acid encoding an enzyme having acyltransferase (EC 2.3.1.84) activity.

2. The gene-modified yeast cell according to claim 1, wherein, Compared to cells that do not contain the heterologous nucleic acid, the genetically modified yeast cells produce an increased amount of one or more acetates and / or ethyl acetates.

3. The gene-modified yeast cell according to claim 2, wherein, Acetic esters are selected from the group consisting of ethyl acetate, isoamyl acetate, and phenylethyl acetate.

4. The gene-modified yeast cell according to claim 2, wherein, Ethyl ester is selected from the group consisting of ethyl hexanoate, ethyl octanoate, and ethyl decanoate.

5. The genetically modified yeast cell according to any one of claims 1-4, further comprising a heterologous nucleic acid encoding an enzyme having carbon-sulfur lyase (EC 4.4) activity.

6. The gene-modified yeast cell according to claim 5, wherein, An enzyme having carbon-sulfur lyase activity comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 9 and 57-62.

7. The gene-modified yeast cell according to claim 5, wherein, An enzyme having carbon-sulfur lyase activity comprises a sequence having at least 90% sequence identity with SEQ ID NO:

9.

8. The genetically modified yeast cell according to any one of claims 1-7, further genetically modified to produce one or more monoterpenes.

9. The gene-modified yeast cell according to claim 8, wherein, Monoterpenes were selected from the group consisting of linalool, geraniol, and citronellol.

10. Genetically modified yeast cells according to any one of claims 8-9, wherein, The genetically modified yeast cells contain heterologous nucleic acids encoding enzymes selected from the group consisting of: (a) A truncated variant of the yeast HMG1 enzyme; (b) Variants of the ERG20 enzyme; (c) Linalool synthase; (d) Geraniol synthase; and (e) Their combination.

11. The gene-modified yeast cell according to claim 10, wherein, The truncated variant of the yeast HMG1 enzyme contains a sequence that has at least 90% sequence identity with SEQ ID NO:

11.

12. The gene-modified yeast cell according to claim 10, wherein, Variants of the ERG20 enzyme contain a sequence that has at least 90% sequence identity with SEQ ID NO:

13.

13. The gene-modified yeast cell according to claim 10, wherein, The linalool synthase contains a sequence that has at least 90% sequence identity with any one of SEQ ID NO:15 and 63-68.

14. The gene-modified yeast cell according to claim 10, wherein, The linalool synthase contains a sequence that has at least 90% sequence identity with SEQ ID NO:

15.

15. The gene-modified yeast cell according to claim 10, wherein, Geraniol synthase contains a sequence having at least 90% sequence identity with any one of SEQ ID NO:17 and 69-71.

16. The gene-modified yeast cell according to claim 10, wherein, Geraniol synthase contains a sequence that has at least 90% sequence identity with SEQ ID NO:

17.

17. Genetically modified yeast cells according to any one of claims 10-16, wherein, The genetically modified yeast cells contain heterologous nucleic acids encoding the following: (a) A truncated variant of the yeast HMG1 enzyme; (b) Variants of the ERG20 enzyme; (c) Linalool synthase; and (d) Geraniol synthase.

18. Genetically modified yeast cells according to any one of claims 1-17, wherein, Due to gene modifications of one or more enzymes that functionally disrupt maltose and / or maltotriose transport and / or maltose and / or maltotriose hydrolysis, the genetically modified yeast cells are unable to convert maltose and / or maltotriose into ethanol or have a reduced ability to convert maltose and / or maltotriose into ethanol.

19. The gene-modified yeast cell according to claim 18, wherein, The genetically modified yeast cells contain one or more modifications that functionally disrupt maltose and / or maltotriose transporters.

20. The gene-modified yeast cell according to claim 18, wherein, The modified cells contain one or more modifications that functionally disrupt MAL31 and / or MAL11 (AGT1).

21. The gene-modified yeast cell according to claim 18, wherein, The modified cells contain one or more modifications that functionally disrupt MAL31 and / or MAL11 (AGT1).

22. Genetically modified yeast cells according to any one of claims 1-21, wherein, The modified cells are able to convert glucose into ethanol.

23. Genetically modified yeast cells according to any one of claims 1-22, wherein, The heteronucleotide contains a gene encoding an enzyme with alcohol acyltransferase activity, said gene being operatively linked to a promoter selected from the group consisting of SEQ ID NO: 19-35 and 72-73.

24. The gene-modified yeast cell according to claim 23, wherein, The heteronucleotide contains a gene encoding an enzyme with alcohol acyltransferase activity, said gene being operatively linked to a promoter selected from the group consisting of pSPG1 (SEQ ID NO: 26), pHSP26 (SEQ ID NO: 20), pANT1 (SEQ ID NO: 34), pPEX11 (SEQ ID NO: 35), and pALD6 (SEQ ID NO: 73).

25. The gene-modified yeast cell according to claim 23, wherein, An enzyme having alcohol acyltransferase activity comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 5, 7 and 36-56.

26. The gene-modified yeast cell according to claim 23, wherein, Enzymes with alcohol acyltransferase activity contain a sequence that has at least 90% sequence identity with SEQ ID NO: 5 or SEQ ID NO:

7.

27. The gene-modified yeast cell according to claim 23, wherein, Enzymes with alcohol acyltransferase activity contain a sequence that has at least 90% sequence identity with SEQ ID NO:

5.

28. The gene-modified yeast cell according to claim 23, wherein, Enzymes with alcohol acyltransferase activity contain a sequence that has at least 90% sequence identity with SEQ ID NO:

7.

29. A liquid fermentation composition comprising: (a) A population of genetically modified yeast cells that are genetically modified to produce one or more acetates and / or ethyl acetates, wherein the yeast cells are unable to convert maltose and / or maltotriose into ethanol or have a reduced ability to convert maltose and / or maltotriose into ethanol. (b) A sugar source comprising wort, wherein the total sugar in the wort is attenuated by no more than 25% of the population of the genetically modified yeast cells; (c) One or more aldehyde molecules and / or non-aldehyde molecules derived from wort; and (d) Alcohols not exceeding 1.0% (v / v).

30. The liquid fermentation composition according to claim 29, wherein, The liquid fermentation composition comprises isoamyl acetate, ethyl acetate, and phenylethyl acetate in a total amount of about 500 µg / L to about 5500 µg / L, preferably about 536.2 µg / L to about 4725.4 µg / L.

31. The liquid fermentation composition according to claim 29, comprising isoamyl acetate and phenethyl acetate in an amount of about 10 µg / L to about 1500 µg / L, preferably about 11.4 µg / L to about 1094.7 µg / L.

32. The liquid fermentation composition according to claim 29, comprising an amount of isoamyl acetate of about 9.1 µg / L to about 900 µg / L, preferably about 9.1 µg / L to about 812.8 µg / L, and / or an amount of phenethyl acetate of about 2.3 µg / L to about 500 µg / L, preferably about 2.3 µg / L to about 281.8 µg / L.

33. The liquid fermentation composition according to any one of claims 29-32, wherein, The population of yeast cells comprises the genetically modified yeast cells according to any one of claims 1-28.

34. The liquid fermentation composition according to any one of claims 29-33, wherein, Aldehyde molecules are selected from the group consisting of 2-methylbutanal, 2-methylpropanal, hexanal, benzaldehyde, furfural, acetaldehyde, methylthional, phenylacetaldehyde, and 5-hydroxymethylfurfural.

35. The liquid fermentation composition according to any one of claims 29-34, wherein, Non-aldehyde molecules were selected from the group consisting of (E)-β-damascone and 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone.

36. A liquid fermentation composition comprising: (a) A population of genetically modified yeast cells that are unable to convert maltose and / or maltotriose into ethanol or have a reduced ability to convert maltose and / or maltotriose into ethanol. (b) A sugar source comprising wort, wherein the total sugars in the wort are attenuated by no more than 25% of the population of the genetically modified yeast cells; and (c) Not more than 1.0% (v / v) of alcohol.

37. The liquid fermentation composition according to claim 36, wherein, Due to gene modifications that disrupt one or more enzymes associated with maltose and / or maltotriose transport and / or maltose and / or maltotriose hydrolysis, the genetically modified yeast cells are unable to convert maltose and / or maltotriose into ethanol.

38. The liquid fermentation composition according to claim 37, wherein, The population of yeast cells contains one or more genetic modifications that functionally disrupt maltose and / or maltotriose transporters.

39. The liquid fermentation composition according to claim 37, wherein, The population of yeast cells contains one or more gene modifications that functionally disrupt MAL31 and / or MAL11 (AGT1).

40. A method for producing a fermented beverage, comprising: (a) A genetically modified yeast cell comprising functional disruption of one or more enzymes associated with maltose and / or maltotriose transport and / or maltose and / or maltotriose hydrolysis, wherein, compared with yeast cells not containing the genetic modification, the functional disruption results in slower growth of the yeast cell when maltose is the sole sugar source. (b) Provide a culture medium containing sugars derived from wort; (c) Combining the genetically modified yeast cells with the culture medium to form a fermentation composition; and (d) Ferment the fermentation composition to produce a fermented beverage.

41. The method according to claim 40, wherein, Genetically modified yeast cells contain one or more gene modifications that functionally disrupt maltose and / or maltotriose transporters.

42. The method according to claim 40, wherein, Genetically modified yeast cells contain one or more gene modifications that functionally disrupt MAL31 and / or MAL11 (AGT1).

43. A method for producing a fermented beverage, comprising contacting a population of genetically modified yeast cells according to any one of claims 1-28 with a culture medium containing a sugar source derived from malt extract during fermentation to produce the fermented beverage.

44. The method according to any one of claims 40-43, wherein, Fermented beverages are low-alcohol fermented beverages.

45. The method according to any one of claims 40-44, wherein, Fermented beverages have an alcohol content of less than or equal to about 0.5% (v / v) alcohol.

46. ​​The method according to any one of claims 40-45, wherein, The method does not include steps for physically removing alcohol from the beverage or prematurely terminating fermentation.

47. The method according to any one of claims 40-46, wherein, At least one fermentable sugar is provided in the wort-derived sugar source.

48. The method according to any one of claims 40-47, wherein, The fermentation process reduces the sugar level in the wort by at least 15% but no more than 25%.

49. The method according to any one of claims 40-48, wherein, Fermented beverages include beer.

50. The method according to any one of claims 40-49, wherein, The fermented beverage contains isoamyl acetate, ethyl acetate, and phenylethyl acetate in a total amount of about 500 µg / L to about 6500 µg / L, preferably about 536.2 µg / L to about 4725.4 µg / L.

51. The method according to any one of claims 40-49, wherein, The fermented beverage contains isoamyl acetate and phenethyl acetate in amounts from about 10 µg / L to about 1500 µg / L.

52. The method according to any one of claims 40-49, wherein, The fermented beverage contains isoamyl acetate in an amount of about 9 µg / L to about 900 µg / L, preferably about 9.1 µg / L to about 812.8 µg / L, and / or phenethyl acetate in an amount of about 2.3 µg / L to about 500 µg / L, preferably about 2.3 µg / L to about 281.8 µg / L.

53. The method according to any one of claims 40-52, further comprising producing a culture medium, wherein producing the culture medium comprises: (a) Contacting various grains with water; as well as (b) Boil or soak the water and grains to produce wort.

54. The method of claim 53, further comprising adding at least one hop variety to the wort to produce hop wort.

55. The method according to any one of claims 40-54, further comprising adding at least one hop variety to the culture medium.

56. The genetically modified yeast cells according to any one of claims 1-28, the liquid fermentation composition according to any one of claims 29-39, or the method according to any one of claims 40-55, wherein, Yeast cells belong to the genus *Yeast*.

57. The genetically modified yeast cells according to any one of claims 1-28, the liquid fermentation composition according to any one of claims 29-39, or the method according to any one of claims 40-55, wherein, The yeast cells belong to the Saccharomyces cerevisiae species.

58. The genetically modified yeast cells according to any one of claims 1-28, the liquid fermentation composition according to any one of claims 29-39, or the method according to any one of claims 40-55, wherein, The yeast cells are Saccharomyces cerevisiae, Saccharomyces lonelii, Saccharomyces d'Andex, Saccharomyces augustinii, or Saccharomyces d'Americane.

59. A genetically modified yeast cell (modified cell), comprising: One or more genetic modifications for sensory detection of reducing one or more wort-related off-flavors in fermented beverages; The modified cells described herein cannot convert maltose and / or maltotriose into ethanol.

60. The gene-modified cell according to claim 59, wherein, Malt wort-related off-odors contain aldehyde molecules and / or non-aldehyde molecules.

61. The gene-modified cell according to claim 60, (a) wherein the aldehyde molecule is 2-methylbutyraldehyde, 2-methylpropionaldehyde, hexanal, benzaldehyde, furfural, acetaldehyde, methylthion, phenylacetaldehyde, or 5-hydroxymethylfurfural, and / or (b) wherein the non- Aldehyde molecules are (E)-β-damascone or 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone.

62. The gene-modified cell according to any one of claims 59-61, wherein, The modified cells are able to convert glucose into ethanol.

63. The gene-modified cell according to any one of claims 59-61, wherein, The modified cell contains one or more gene modifications whose functional disruption is associated with one or more proteins related to the transport of maltose and / or maltotriose into the modified cell and / or the hydrolysis of maltose and / or maltotriose by the modified cell.

64. The gene-modified cell according to claim 63, wherein, The modified cells contain one or more gene modifications that functionally disrupt maltose and / or maltotriose transporters.

65. The gene-modified cell according to claim 64, wherein, The modified cells contain one or more gene modifications that functionally disrupt MAL31 and MAL11 (AGT1).

66. The gene-modified cell according to any one of claims 59-65, wherein, Compared to cells that do not contain the aforementioned genetic modifications, one or more genetic modifications that reduce the sensory detection of one or more wort-related off-flavors result in an increase in the production of one or more acetates and / or ethyl acetates.

67. The gene-modified cell according to claim 66, wherein, Acetic esters are ethyl acetate, isoamyl acetate, and / or phenylethyl acetate.

68. The gene-modified cell according to claim 66, wherein, Ethyl esters are ethyl hexanoate, ethyl octanoate, and / or ethyl decanoate.

69. The gene-modified cell according to any one of claims 66-68, wherein, One or more gene modifications that increase the production of one or more acetates and / or ethyl acetates include overexpression of enzymes with alcohol acyltransferase (AAT) activity.

70. The gene-modified cell according to claim 69, wherein, Enzymes with AAT activity contain the sequence shown in SEQ ID NO:

5.

71. The gene-modified cell according to any one of claims 66-68, wherein, One or more gene modifications that increase the production of one or more acetates and / or ethyl acetates include the expression of a heterologous enzyme with alcohol acyltransferase (AAT) activity.

72. The gene-modified cell according to claim 71, wherein, Heterozymes with AAT activity contain the sequence shown in SEQ ID NO:

7.

73. The gene-modified cell according to any one of claims 59-65, wherein, One or more genetic modifications that reduce the sensory detection of one or more wort-related off-flavors lead to increased production of 3-mercaptohexanol (3MH).

74. The gene-modified cell according to claim 73, wherein, One or more genetic modifications that increase 3MH production include expressing bacterial enzymes with carbon-sulfur lyase (CSL) activity.

75. The gene-modified cell according to claim 74, wherein, Bacterial enzymes with CSL activity contain the sequence shown in SEQ ID NO:

9.

76. The gene-modified cell according to any one of claims 59-65, wherein, One or more genetic modifications that reduce sensory detection of one or more wort-related off-flavors lead to an increase in the production of one or more monoterpenes.

77. The gene-modified cell according to claim 76, wherein, Monoterpenes are linalool, geraniol and / or citronellol.

78. The gene-modified cell according to claim 76 or 77, wherein, One or more gene modifications that increase the production of one or more monoterpenes include expressing one or more of the following: (a) A truncated variant of the yeast HMG1 enzyme; (b) Variants of the ERG20 enzyme; (c) linalool synthase; and / or (d) Geraniol synthase.

79. The gene-modified cell according to claim 78, wherein, A truncated variant of the yeast HMG1 enzyme contains the sequence shown in SEQ ID NO:

11.

80. The gene-modified cell according to claim 78 or 79, wherein, Variants of the ERG20 enzyme contain the sequence shown in SEQ ID NO:

13.

81. The gene-modified cell according to any one of claims 78-80, wherein, Linalool synthase contains the sequence shown in SEQ ID NO:

14.

82. The gene-modified cell according to any one of claims 78-81, wherein, Geraniol synthase contains the sequence shown in SEQ ID NO:

16.

83. The gene-modified cell according to any one of claims 59-82, wherein, Yeast cells belong to the genus *Yeast*.

84. The gene-modified cell according to claim 83, wherein, The yeast cells belong to the Saccharomyces cerevisiae species.

85. The gene-modified cell according to claim 84, wherein, The yeast cells are Saccharomyces cerevisiae, Saccharomyces lonelii, Saccharomyces d'Andex, Saccharomyces augustinii, or Saccharomyces d'Americane.

86. A method for producing a fermented beverage, comprising: The modified cells according to any one of claims 59-85 are contacted with a culture medium containing at least one fermentable sugar. The contact is carried out during at least the first fermentation process to produce a fermented beverage.

87. The method according to claim 86, wherein, Fermented beverages are low-alcohol fermented beverages.

88. The method according to claim 86 or 87, wherein, Fermented beverages have an alcohol content of less than 0.5% (v / v) alcohol.

89. The method according to any one of claims 86-88, wherein, The method does not include steps for physically removing alcohol from the beverage or prematurely terminating fermentation.

90. The method according to any one of claims 86-89, wherein, At least one fermentable sugar is provided in at least one sugar source.

91. The method according to claim 90, wherein, Fermentable sugars are glucose, fructose, and / or sucrose.

92. The method according to any one of claims 86-91, wherein, The first fermentation process reduces the level of fermentable sugars by at least 15%.

93. The method according to any one of claims 86-92, wherein, Fermented beverages include beer.

94. The method according to any one of claims 86-93, wherein, The sugar source includes malt extract.

95. The method according to claim 94, wherein, The sugar source is wort, and the method further includes producing a culture medium, wherein producing the culture medium comprises: (a) Contacting various grains with water; and (b) Boil or soak the water and grains to produce wort.

96. The method of claim 95, further comprising adding at least one hop variety to the wort to produce hop wort.

97. The method according to any one of claims 86-96, further comprising adding at least one hop variety to the culture medium.

98. The method according to any one of claims 86-97, further comprising at least one additional fermentation process.

99. The method according to any one of claims 86-98, further comprising carbonating the fermentation product.

100. A genetically modified brewing yeast cell (modified cell), comprising: One or more gene modifications for functionally disrupting the maltose / maltotriose transporters MAL31 and MAL11 (AGT1).

101. A fermented beverage produced by the method according to any one of claims 40-58.

102. A fermented beverage comprising not more than 1.0% (v / v) of alcohol and nucleic acids from genetically modified yeast cells according to any one of claims 1-28.

103. The fermented beverage according to claim 102, wherein, The fermented beverage contains no more than 0.5% (v / v) of alcohol.

104. The genetically modified yeast cells according to any one of claims 1-28 or 56-85, the liquid fermentation composition according to any one of claims 29-39, or the method according to any one of claims 40-55 and 86-99, wherein, The yeast cells mentioned are brewing yeast cells.

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