WO2014022692A1

WO2014022692A1 - Prion-based manipulation of yeast fermentation and growth

Info

Publication number: WO2014022692A1
Application number: PCT/US2013/053272
Authority: WO
Inventors: Daniel JAROSZ; Jessica C.S. BROWN; Susan Lindquist
Original assignee: Whitehead Institute for Biomedical Research
Current assignee: Whitehead Institute for Biomedical Research
Priority date: 2012-08-01
Filing date: 2013-08-01
Publication date: 2014-02-06
Anticipated expiration: 2015-02-01

Description

PRION-BASED MANIPULATION OF YEAST FERMENTATION AND GROWTH

Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No.

61/678,610, filed on August 1, 2012. The entire teachings of the above application(s) are incorporated herein by reference.

Background

[0002] Prion proteins have the unusual ability to stably adopt multiple conformations, at least one of which is self-perpetuating. Prions have been identified in a wide variety of eukaryotic organisms, ranging from yeast to humans. In mammals, prions are responsible for a number of diseases such as mammalian spongiform encephalopathies (TSEs). In yeast, the ability of prions to convert between structurally and functionally distinct states, one or more of which is transmissible, allows them to act as non-Mendelian elements of phenotypic inheritance.

Summary

[0003] The ability of prions to convert between structurally and functionally distinct states, one or more of which is transmissible, provides the basis for a mode of inheritance in which biological traits are inherited based on self-templating changes in protein structure rather than on changes in nucleic acid sequence. In some aspects the invention relates to modulating acquisition, maintenance, or loss of a prion to in order to manipulate a phenotype of interest in a fungal cell, e.g., a yeast cell.

[0004] In some aspects, the invention provides methods of manipulation of yeast fermentation and/or growth comprising modulating a yeast prion named [GAR+]. In some aspects, the invention provides methods of altering alcohol production in yeast fermentations by modulating a yeast prion named [GAR+]. In some aspects, methods for modulating

[GAR+], e.g., inhibiting [GAR+] or inducing [GAR+] are provided.

[0005] In some aspects, the [GAR ] prion alters carbon source utilization by the yeast Saccharomyces cerevisiae. In some aspects, [GAR+] allows yeast to use non-preferred carbon sources in the presence of a preferred carbon source, glucose, which results in faster growth and/or higher bio mass on complex mixtures of carbon sources, in some embodiments a complex mixture comprises or is derived from a fruit or grain. Complex mixtures such as molasses or grape must are frequently used in industrial processes, e.g., because they are less expensive and/or easier to obtain or more suitable than pure glucose. In some aspects,

[GAR+] thus increases efficiency of using yeast to produce virtually any small molecule, e.g., a fine chemical or a therapeutic agent. In some aspects, [GAR+] is of use in biofuel production.

[0006] In some aspects, [GAR+] decreases the final ethanol content of fermentations, which, in some embodiments, is useful in producing lower alcohol content products (e.g., reduced alcohol content beer, wine, or other fermented beverage) or allowing greater control over the fermentation process.

[0007] In some aspects, [GAR+] allows yeast cells to tolerate higher ethanol

concentrations than isogenic [gar-] cells which, in some embodiments, is useful in producing ethanol, e.g., as a biofuel. For example, [GAR+] cells survived high ethanol concentrations better than isogenic [gar-] cells, a property that would, for example, give [GAR+] cells an advantage for growth in late fermentations. In some embodiments inhibiting [GAR+] helps increase alcohol production as a biofuel. For example, biofuel fermentations are often not sterile and bacteria can switch on [GAR+], causing the cells to make less alcohol. In some embodiments inhibiting [GAR+] induction is useful in production of ethanol, e.g., as a biofuel. In some embodiments such production uses a cellulosic or non-cellulosic biomass as a feedstock.

[0008] In some aspects, [GAR+] decreases alcohol production and/or reduces the final ethanol content of fermentations, which, in some embodiments, is useful in producing lower alcohol content products (e.g., reduced alcohol content beer, wine, or other fermented beverage) or allowing greater control over the fermentation process.

[0009] [GAR+] can be induced by a wide variety of bacteria, including a variety of bacteria that are found in wine fermentations. Among bacterial species tested, roughly 15% were able to induce [GAR+] however, a marked enrichment for bacteria capable of [GAR+] induction in species found by in arrested wine fermentions (e.g. Pediococcus damnosis and Lactobacillus kunkeii) compared to the mix of species commonly found in wine

fermentations.

[0010] In some aspects, inhibiting [GAR+] or avoiding acquisition of [GAR+] is of use to inhibit or prevent stuck fermentation (i.e., non-intentionally and/or unwanted arrested fermentation). Stuck fermentation is a condition that occurs, e.g., in winemaking, in which fermentation substantially stops without intentional intervention by man. For example, fermentation may undesirably stop before all or at least a desired amount of the sugar in a medium is consumed

[0011] In some aspects, a variety of yeast mutants that have enhanced ability to undergo the switch to the [GAR+] state in the presence of bacteria that are capable of inducing

[GAR+] in wild type cells are disclosed herein. Such mutants or other strains in which the same genes are functionally inactivated, e.g., by at least partial deletion or insertion or using RNA interference (RNAi) are useful in a wide variety of industrial processes and/or for producing a wide variety of products.

[0012| In some aspects, a variety of yeast mutants that arc impaired in ability to undergo the switch to the [GAR+] state in the presence of bacteria that are capable of inducing

[GAR+] in wild type cells are disclosed herein. Such mutants or other strains in which the same genes are functionally inactivated, e.g., by at least partial deletion or insertion of a nucleic acid into the gene or, in some embodiments, using RNA interference (RNAi), are useful in a wide variety of industrial processes and/or for producing a wide variety of products.

[0013] In some embodiments, methods of generating or selecting yeast strains that have enhanced or impaired acquisition (e.g., induction) of [GAR+] are disclosed herein. For example, in some embodiments standard methods of yeast genetics can be used to construct deletion mutants. In some aspects, resulting yeast are used in producing a product or performing at least one step of an industrial process.

[0014] In some aspects, a method comprises monitoring the appearance of [GAR+] or the proportion of yeast cells that are [GAR+] during the growth of a yeast culture by detecting or measuring RNA whose transcription is altered in [GAR+] cells as compared with [gar-] cells. In some embodiments the RNA is HXT3 RNA. In some embodiments quantitative PCR for the RNA is performed. In some embodiments a sample comprising cells is removed from a culture at one or more time points and tested for [GAR+] cells.

[0015] In some aspects, a method comprises eliminating [GAR+] by growing cells on or in medium comprising a [GAR+] inhibitor. In some embodiments a [GAR+] inhibitor comprises a glutamine analog. The glutamine analog may be non-metabolizable by the cell, at least non-metabolizable by the pathways that metabolize glutamine and/or may not be usable as an energy source. In some embodiments the glutamine analog is azaserine (0-(2- Diazoacetyl)-L-serine).

[0016] In some embodiments a [GAR+] inhibitor comprises a flavonol. In some embodiments the flavonol is myrecitin (3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)- 4- chromenone). In some embodiments the flavonol is quercetin (2-(3,4-dihydroxyphenyl)- 3,5,7-trihydroxy-4H-chromen-4-one).

[0017] In some embodiments a [GAR+] inhibitor comprises an Hsp70 inhibitor. In some embodiments the Hsp70 inhibitor is a flavonol. In some embodiments an Hsp70 inhibitor, e.g., a flavonol, is a compound that occurs naturally in a composition of interest herein, e.g., a wine. In some embodiments an Hsp70 inhibitor that occurs naturally in a composition is used at a concentration greater than that at which it occurs naturally in the type of composition in which it is used, e.g., a wine, or in a form distinct from that in which it naturally occurs in the type of composition in which it is used. In some embodiments the concentration of a flavonol or the amount added to a culture medium or composition is at least 20, 30, 40, 50, 75, or lOO mg/L.

[0018] In some embodiments the Hsp70 inhibitor is a compound of the following formula:

[0019] wherein Ri, R₂, R₃, R4 and R₅ are the same or different and represent a radical selected from the group of hydrogen, optionally substituted alkyl, hydro xyl, alkoxy, thio, alkylthio, halogen, amino, monoalkylamino, dialkylamino, amido, nitro, carboxyl, alkoxycarbonyl, alkylcarbonyl, alkylcarbonyloxy, guanidino, phosphate, sulfamido and sulfonamido; Q represents a divalent linking moiety selected from the group consisting of— C(R₆R₇)-C(R₈R9)~, -CRio=CRii--, and -C≡C-, wherein R₆, R₇, R₈, R₉, Rio, and R_n represent a radical selected from the group consisting of hydrogen and optionally substituted alkyl; R_a and Rb are the same or different and represent hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, hydroxyl, alkoxy, amino, monoalkylamino, dialkylamino, carboxy, alkylcarbonyl, and alkyloxycarbonyl; or, optionally, Rj, R₂, R₃, R4, R5, R_a and R_b are independently substituted with one member of a specific binding pair or a targeting ligand to facilitate targeting of the compound to a surface or matrix. In some embodiments Q represents ~C≡C-, and Ri, R₂, R₃, R4, R5, R_a and R_b each represents hydrogen or C_1-6 alkyl, e.g., C_1-4 alkyl, e.g., methyl, ethyl, or propyl. In some embodiments Q represents— C≡C— , and Ri, R₂, R₃, R4, R5, R_a and R_b each represents hydrogen. In some embodiments Q represents— C≡C— , exactly one or at least one of Ru R₂, R₃, R4, and R₅ represents halogen, and either or both of R_a and R optionally is hydrogen. In some embodiments Q represents -C≡C— , exactly one or at least one of R_l5 R₂, R₃, R4, and R₅ represents halogen, and R_a, R_b or both is hydrogen. In some embodiments the compound is of the following formula:

[0020] wherein R represents a substituent selected from the group consisting of chloro, fluoro, Ci-4 alkyl, e.g., methyl, ethyl, or propyl alkyl, trifluoromethyl, amino, carboxy, hydroxyl and methoxy; and R and R" are the same or different and represent a radical selected from the group of hydrogen, optionally substituted Ci_-6 alkyl, hydroxyl, alkoxy, thio, alkylthio, halogen, amino, monoalkylamino, dialkylamino, amido, nitro, carboxy,

alkoxycarbonyl, alkylcarbonyl and alkylcarbonyloxy; R_a and R_b are the same or different and represent a radical selected from the group of hydrogen, hydroxyl, alkoxy, amino,

monoalkylamino, dialkylamino, carboxy, alkoxycarbonyl, alkylcarbonyl and optionally substituted Ci_-6 alkyl, said alkyl substituent being at least one selected from the group consisting of hydroxyl, thio, alkoxy, alkylthio, halogen, amino, monalkylamino,

dialkylamino, guanidino, phosphate, amido, nitro, carboxyl, sulfamido, sulfonamido, alkoxycarbonyl, alkylcarbonyl, and alkylcarbonyloxy. In some embodiments R, R", R_a and Rb are hydrogen, and R is a radical selected from the group consisting of chloro, fluoro, amino, carboxy, hydroxy and methoxy. Exemplary compounds of Formula I, II, and III are described in US Pat. Pub. No. 201 10189125. In some embodiments the Hsp70 inhibitor of Formula I is 2-phenylethynesulfonamide (PES, also referred to as pifithrin-μ) (Liu, JI, et al., (2009) Molecular Cell, 36: 15-27; US Pat. Pub. No. 201 10189125). In some embodiments the Hsp70 inhibitor of Formula I or II is 2-(3-chlorophenyl) ethynesulfonamide.

[0021] In some embodiments the Hsp70 inhibitoris 2-aminopurine.

[0022] In some embodiments the Hsp70 inhibitor is a benzylidene lactam compound such as N-form l-3,4-methylenedioxy-benzylidene-ybutyrolactam (also known as KNK437) or a derivative thereof, such as those described in Mosser, D. D., et al, (1997) Mol. Cell. Biol, 17: 5317-5327. [0023] In some embodiments a population of [GAR+] yeast are contacted with a [GAR+] inhibitor in an amount and for a time sufficient to convert the population to [gar-]. In some embodiments yeast are contacted with a [GAR+] inhibitor in an amount and for a time sufficient to reduce the number of [GAR+] cells by a factor of at least 10, at least 10 , at least 10³, at least 10⁴, at least 10⁵ , at least 10⁶ , or more, or any intervening range or value. In some embodiments yeast are contacted with a [GAR+] inhibitor in an amount and for a time sufficient to reduce the number of [GAR+] cells by a factor of between 10 and 10², between 10² and 10³, between 10³ and 10⁴, between 10⁴ and 10⁵, or between 10⁵ and 10⁶.

[0024] In some embodiments a [GAR -] inhibitor or inducer is substantially non-toxic to yeast at the concentrations in which it usefully inhibits or induces [GAR+]. In some embodiments a [GAR+] inhibitor or inducer is substantially non-toxic to mammalian cells at such concentrations. In some embodiments a [GAR+] inhibitor or inducer is substantially non-toxic to mammals when humans are exposed to the [GAR+] inhibitor or inducer in the quantities in which it may be found in a product produced using a yeast culture to which the [GAR+] inhibitor or inducer has been added. In some embodiments a product is purified from a culture such that a [GAR+] inhibitor or inducer is not detectably present in the product. In some embodiments a [GAR+] inhibitor or inducer is at least in part inactivated or removed from a culture. In some embodiments inactivation or removal is sufficient to render the [GAR+] inhibitor or inducer undetectable or to reduce its level such that it does not have a significant effect on [GAR+].

[0025] In some embodiments the difference in the number of cells that grow on rich medium (e.g., YPD or a similarly rich medium) versus rich medium containing a [GAR+] inhibitor such as azaserine is used to measure the fraction of [GAR+] cells in a culture or other composition.

[0026] In some embodiments a culture or composition comprising yeast cells is tested for [GAR+] cells at one or more time points. In some embodiments one or more samples is removed from a culture or composition at one or more time points. The culture, composition, or sample is tested for [GAR+] cells or for a modulator of [GAR+]. In some embodiments the modulator of [GAR+] is a bacterium or bacterial product that induces [GAR+]. In some embodiments the culture or composition is tested at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more time points during its use. In some embodiments the culture or composition is tested at reasonably regular intervals, e.g., about every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 16, 20, or 24 hours during at least part of its use. In some embodiments a continuous or semi-continuous monitoring method is used. For example, sample can be removed continuously and tested, e.g., in a flow cell, or the culture or composition can be monitored using an external monitoring system such as an optical or spectroscopic external monitoring system.

[0027] In some embodiments a culture medium is inoculated with a "starter culture" (or sample thereof) or comprising yeast cells. In some embodiments a starter culture may be tested for [GAR+] cells. In some embodiments a starter culture containing at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%, 99.99%, 99.995%, 99.999%), or more [GAR+] cells may be used, e.g., to inoculate a larger culture. In some embodiments a starter culture contains a single yeast species or a single yeast strain. In some embodiments 2, 3, 4, 5, or more starter cultures may be used. In some embodiments a starter culture containing at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%, 99.99%, 99.995%, 99.999%, or more [gar+] cells may be used, e.g., to inoculate a larger culture. In some embodiments a starter culture may be exposed to a [GAR+] modulator, e.g., a [GAR+] inducer or [GAR+] inhibitor. In some embodiments a [GAR+] species or strain, e.g., in a starter culture, is converted to [gar-] (e.g., induced to become prior [gar-]; cured of [GAR+]) prior to being used to inoculate a larger culture. In some embodiments a [gar-] species or strain, e.g., in a starter culture, is converted to [GAR+] (e.g., induced to become [GAR+]) prior to being used to inoculate a larger culture. In some embodiments the volume of a starter culture is no more than about 0.1%, 0.5%, 1%, 2%, 5%, or 10% of the larger culture. In some embodiments the volume of a starter culture or the amount of starter culture added to a larger culture is no more than 1 mL, 2.5 mL, 5 mL, 10 mL, 25 mL, 50 mL, 100 mL, 250 mL, 500 mL, or 1000 mL. In some embodiments the volume of a starter culture or the amount of starter culture added to a larger culture is no more than 1 L, 2.5 L, 5L, or 10L. In some embodiments a larger culture is inoculated to an ODgoo of under about 0.01, 0.025., 05, 0.1 , 0.125, 0.15, 0.175, or 0.20. It will be understood that a starter culture may use a different culture medium to the culture medium in the culture it is used to inoculate. In some embodiments a starter culture may use a peptone-based yeast culture medium such as YPD, or YP plus a different carbon source. In some embodiments a culture may be inoculated at a concentration between

4 8 5 7 6 7

10" and 10° cells per ml, e.g., between 10 and 10 cells per ml, e.g., between 10 and 10 cells cells per ml or any intervening range or value. For example, in some embodiments a culture may be inoculated at a concentration 3 x 10⁶ cells per ml.

[0028] In some embodiments yeast, e.g., yeast may be provided in liquid medium. In some embodiments yeast may be provided in dry form. In some embodiments yeast, e.g., yeast used or to be used to inoculate a culture, may be washed, concentrated, compressed, powdered, dried (e.g., freeze dried), encapsulated, frozen, or otherwise processed. In some embodiments yeast may be shaped into particles. In some embodiment yeast may be shaped, e.g., compressed, to form a macroscopic object such as a cake or bar. In some embodiments yeast may be in active, dried form. In some embodiments yeast may be in inactive dried form.

100291 In some embodiments a culture or composition is modified based at least in part on results of testing for [GAR+] cells or for a [GAR+] modulator, e.g, a [GAR+] inducer such as bacteria or a small molecule that induces or inhibits [GAR+]. Thus in some embodiments a method comprises modifying a culture or composition based at least in part on results of testing for [GAR+] cells or for a [GAR+] modulator, e.g, a [GAR+] inducer such as bacteria or a small molecule that induces or inhibits [GAR+]. In some embodiments modifying the culture or composition comprises altering the overall content of the culture or composition so as to enhance [GAR+] acquisition or maintenance in situations where [GAR+] is useful or desired. In some embodiments modifying the culture or composition comprises altering the overall content of the culture or composition so as to inhibit [GAR+] acquisition or maintenance in situations where [GAR+] is deleterious or not desired. In some embodiments modifying the culture or composition comprises adding a nutrient to the composition. In some embodiments the nutrient is one or more carbon sources, e.g., one or more sugars, e.g., glucose. In some embodiments the nutrient is one or more amino acids or nitrogen sources. In some embodiments modifying the culture or composition comprises adding yeast cells that are [gar-] or adding a [GAR+] inhibitor if the results reveal presence of [GAR+] cells or presence of a [GAR+] inducer in circumstances where [GAR+] is not desired or useful. In some embodiments modifying the culture or composition comprises adding yeast cells that are [GAR+] or adding a [GAR+] inducer if the results reveal presence of [gar-] cells or a [GAR+] inhibitor in circumstances where [GAR+] is desired or useful. In some embodiments modifying the culture or composition comprises changing the temperature or pH.

[0030] Examples of bacteria that induce [GAR+] are listed in Table A and/or Figure 14. In some embodiments a bacterium is a strong inducer. In some embodiments a bacterium is an intermediate inducer. In some embodiments a bacterium is a weak inducer. See Figure 14 for representative examples of strong, intermediate, and weak inducers, and a method of testing induction. Whether a given bacterium is a weak, intermediate, or strong inducer, or a non-inducer, e.g., whether it falls within the range of strong, intermediate, or weak inducers pictured in Figure 14, may be readily determined. In some embodiments bacteria can be tested using typical microbiological techniques such as culturing them on appropriate medium, visualizing the bacteria or bacterial colonies thereof, staining them with appropriate stains, immunological methods, DNA or RNA analysis, etc., or any other means of identifying the bacteria known in the art, to determine whether they are of a species capable of inducing [GAR+]. In some embodiments a bioassay is used. In some embodiments the bioassay comprises assessing the ability of the bacteria to induce [GAR+] in yeast, e.g., S. cerevesiae. In some embodiments a substance to be used in an industrial process is tested for presence or amount of bacteria capable of inducing [GAR+] or for presence or amount of a small molecule capable of inducing [GAR+].

[0031] In some aspects the invention relates to interkingdom communication as a mode of altering prion acquisition (e.g., prion induction), maintenance, or loss. In certain embodiments the interkingdom communication is between a prokaryote and a eukaryote. In some embodiments the prokaryote is a bacterium. In some embodiments the eukaryote is a fungus. In some embodiments the fungus is a yeast. In some embodiments the yeast is a budding yeast. In some embodiments interkingdom communication comprises secretion of a small molecule by a bacterium, wherein the small molecule modulates (e.g., induces or inhibits) acquisition of a prion by a non-bacterial cell, e.g., a fungal cell. In some

embodiments the fungal cell is a yeast cell. In some embodiments the interkingdom communication is between first and second microorganisms (e.g., a fungus and a bacterium) that are commonly found in a community, e.g., a community that exists in nature or a community that exists in a composition used in an industrial process that utilizes one or more components comprising living organisms (e.g., a plant or portion thereof such as a fruit, grain, root, seed, leaf, bark, trunk, etc.), wherein at least one of the components is not substantially sterilized before use in the industrial process. In some embodiments two or more species in a community are found in close association with each other such that a sample of about 1 cubic centimeter (cc) volume would recover at least 10, at least 100, at least 1000, at least 10⁴, or at least 10⁵ individuals or colony forming units of each

microorganism species. In some embodiments the interkingdom communication between a bacterium and a fungal cell modulates, e.g., induces, acquisition of a prion by the fungal cell, wherein acquisition of the prion is beneficial to the fungal cell, the bacterial cell, or both, under at least some environmental conditions.

[00321 In some aspects the invention relates to the recognition that small organic molecules, e.g., small organic molecules produced by bacteria and, in some embodiments, secreted by bacteria, can modulate prion acquisition (e.g., prion induction), maintenance, or loss, e.g., in fungal cells, e.g., in yeast cells. In some embodiments, a small organic molecule acts as a prion inducer, i.e., induces prion acquisition. In some aspects, "induces prion acquisition" refers to causing prion acquisition to occur at a rate exceeding that which would be predicted based on spontaneous mutation frequency and/or that which exists in the absence of the inducer. In some embodiments the rate of prion acquisition is increased by a factor of at least 10; 100; 10³; or 10⁴, e.g., between 10 and 100-fold, between 100 and 10³- fold, between 10³-fold and 10⁴-fold, between 10⁴-fold and 10⁵-fold, or between 10⁵-fold and 10⁶-fold, between 10⁶-fold and 10⁷-fold.

[0033] In some aspects, small molecules that modulate prion acquisition (e.g., prion induction), maintenance, or loss, e.g., that induce or inhibit prion acquisition may be identified by screening culture medium conditioned by a microorganism, e.g., a bacteria, or a cell lysate prepared from the microorganism. In some embodiments the microorganism is one that commonly exists in a community with a fungal cell. The culture medium may be fractionated, and fractions may be tested for prion modulating ability, e.g., prion inducing or inhibiting ability. Fractions that are enriched for such activity can be further fractionated until, e.g., a relatively pure preparation of small molecule is obtained. Fractionation can be performed using any method known in the art. It may be based on one or more physical or chemical properties. In some embodiments fractionated is based at least in part on size, affinity, charge, solubility in any of a variety of solvents, etc. The chemical identity (e.g., structure) of the small molecule may be identified using methods such as mass spectrometry, nuclear magnetic resonance spectrometry, liquid and/or gas chromatography, FTIR spectrometry, or other methods known in the art. Once a small molecule is identified the small molecule may subsequently be prepared using any suitable method (e.g., by purifying from cultured medium, or synthetically or semi-synthetically) and used to modulate the prion.

[0034] In some aspects, the invention provides an isolated small molecule characterized in that: (a) S. hominis bacteria are capable of producing and secreting the molecule; and (b) the molecule is capable of inducing [GAR+] in S. cerevesiae. In some aspects, the molecule is further characterized in that (c) it is not an acyl-homoserine lactone, farnesol, or 2- phenylethanol and is stable to boiling, pH extremes, and freeze/thaw cycles. In some embodiments a pH extreme refers to a pH below about 4.0, below about 3.0, below about 2.0, above about 10.0, above about 11.0, or above about 12.0. In some aspects, the invention provides methods of using the small molecule to incude [GAR+] in yeast, e.g., S. cerevesiae. In some embodiments the small molecule is used as a component of a yeast culture. In some embodiments the yeast culture is used to produce a product. In some embodiments the yeast culture is used in an industrial process, e.g., to perform at least one step of an industrial process.

Brief Description of the Drawings

[0035] Fig. 1. [GAR+] shares the genetic characteristics of yeast prions. (A) Mating of [gar-] MATa to [GAR+] MATa in the W303 background. Resultant diploids show semidominant [GAR+] with a mixed population of large colonies ("strong") and small colonies ("weak"). All spot tests shown are fivefold dilutions. Diploids are selected prior to plating to ensure that they are a pure population. (B) Tetrad spores from the "strong"

[GAR+]. Diploids in A show non-Mendelian segregation of [GAR+]. (C). Cytoduction shows cytoplasmic inheritance of [GAR+]. The [GAR+] donor is 10B URA3+his3- p+karl-1 and the acceptor is W303 ura3-HIS3+ pOKARl . The [GAR+] donor is therefore capable of growing on glycerol but the [gar-] acceptor is not; "mixed" cells were selected for growth on glycerol ([GAR+] cytoplasm) and SD-his 5-FOA ([gar-] nucleus and counterselection against the [GAR+] nucleus). (D). [GAR+] frequency in various laboratory strains. Data are shown as mean ± standard deviation (n = 6). (E). Tetrad spores from a [GAR+] diploid with the genotype h sp 104 : : LEU2/H S P 104. Ahspl04 spores are still [GAR+]. (F). Tetrad spores from a [GAR+] diploid with the genotype ssal : :HIS3/SSAl ssa2::LEU2/SSA2. AssalAssa2 spores are no longer [GAR+].

[0036] Fig. 2. The Snf3/Rgt2 glucose signaling pathway affects [GAR+]. (A) Hxt3-GFP signal in [gar-] and [GAR+] cells (S288c background) by fluorescence microscopy. (B) Frequency of [GAR+] in knockouts of members of the Snf3/Rgt2 glucose signaling pathway. Asnfi is completely resistant to glucosamine, and therefore [GAR+] frequency could not be measured. Furthermore, the frequency of spontaneous glucosamine-resistant colonies in the Argtl , Astdl , and Amthsl strains was close to the rate of genetic mutation, and therefore these colonies might not carry the actual [GAR+] element. Overall, this pathway is enriched for genes that alter [GAR+] frequency when knocked out relative to the library of

nonessential genes (P = 8 ^χ 10-6, Fisher's exact test). (C) The SnB/Rgt2 glucose signaling pathway. (Adapted with permission from Moriya and Johnston 2004; ©2004 National Academy of Sciences, USA.) (D) Measurement of [GAR+] frequency following

overexpression of SnD/Rgt2 pathway members. Data are shown as mean ± standard deviation (n = 6). STD1 strongly induces conversion to [GAR+] and MTH1 blocks it. (E, top) Tetrad spores from a [GAR+] diploid with the genotype stdl -kanMX/STDl . (Bottom) Spores from top crossed to a [gar-] strain with a wild-type STD1 allele.

[0037] Fig. 3. Pmal is involved in [GAR+]. (A) Native gel of Pmal, Stdl , and Mthl in [gar-] and [GAR+]. Either Stdl (left) or Mthl (right) was tagged with six tandem HA tags and samples were processed as described below from [gar-] and [GAR+] strains of each background. (Bottom right) Total, supernatant (sup.), digitonin soluble (det. sol.), and digitonin-insoluble (insol.) fractions were run on SDS gels and probed for Pmal and Stdl or Mthl as a fractionation control. No differences in Pmal, Stdl, or Mthl levels or localization were detected between [gar-] and [GAR+]. (Top right) Blots of the total fraction were stained with Ponceau Red to confirm equal amounts of starting material. (B) Measurement of

[GAR+] frequency in knockout mutants of genes previously shown to affect (Asur4, Alstl) (Roberg et al. 1999; Eisenkolb et al. 2002) or not affect (Alcb3, Alcb4, Adpll, Aatgl9) (Gaigg et al. 2005; Mazon et al. 2007) attributes of wild-type Pmal . Graph represents the mean ± standard deviation (n = 6). (C) Mutants in phosphorylation sites at the C terminus of Pmal affect [GAR+] frequency. Starting strain is haploid, [gar-], genotype pmal "kanMX with p316-PMAl . p314-PMAl carrying wild-type PMAl or mutants of interest were transformed into the starting strain and then p316-PMAl plasmid selected against by growth on 5-FOA. Graph represents the mean ± standard deviation (n - 6). P-values are the binomial distribution of the mean. (D) Pmal mutants that increase [GAR+] frequency show decreased levels of Hxt3-GFP. Graph represents the mean ± standard deviation (n > 6) and P-values were determined using the χ2 test. Strain background is a hybrid of W303 and S288C.

[0038] Fig. 4. Alterations to Pmal affect [GAR+]. (A) [GAR+] induction by transient overexpression of PMAl in a wild-type background. Data are shown as the mean of [GAR+] frequency ± standard deviation (n = 6). Western is total protein probed with aPmal antibody and quantified using Scion Image. (Right) The blot was stained with Ponceau Red to confirm equal loading. (B) Propagation of [GAR+] is impaired in ΡΜΑ1 Δ40Ν Astdl double mutants. Tetrad spores from a [GAR+] diploid with the genotype GAL-PMA I Δ40Ν/Ρ A 1 stdl -kanMX/STDl were crossed to a [gar-] strain with wild-type PMAl and STD1 alleles. ΡΜΑ1Δ40Ν Astdl spores cannot propagate [GAR+] to wild-type [gar-] yeast. The few glucosamine-resistant colonies found in the ΡΜΑ1Δ40Ν Astdl background exhibit standard, Mendelian inheritance of the glucosamine resistance phenotype and thus do not carry the [GAR+] element. [0039] Fig. 5. [GAR+] exhibits a Pmal -dependent species barrier. (A) [GAR+] frequency of S. bayanus and S. paradoxus cells grown at 30°C (left), the optimal growth temperature of S. paradoxus, or 23°C (right), the optimal growth temperature of S. bayanus. Data are shown as the mean of [GAR+] frequency ± standard deviation (n = 6). (B) Substitution of PMA1 from S. cerevisiae with PMA1 from S. bayanus or S. paradoxus prevents [GAR+]

propagation. Starting strain is haploid, [GAR+], genotype pmal -kanMX with p3 16-PMA 1 S. cerevisiae as a covering plasmid. p314-PMAl carrying PMA1 from S. cerevisiae (S.c, top), S. paradoxus (S.par., middle), or S. bayanus (S.bay., bottom) was transformed into the starting strain and p3 16-PMAl S.c. selected against by replica plating to 5-FOA (S.c. IN, S.p. IN, or S.b. IN). These haploids were mated to a wild-type S. cerevisiae [gar-] background, restreaked twice, and tested for [GAR+]. Representative data from three independent experiments are shown.

100401 Fig. 6. Pmal and the Rgt2/Snf3 glucose signaling pathway. We propose that Pmal acts as a part of the Rgt2/Snf3 signaling pathway. (A) In [g r^~] glucose-grown cells, Pmal associates with Mthl . The glucose signal is propagated through Snf3 and Rgt2 to Yckl and Yck2, which phosphorylate Mthl and Stdl . This phosphorylation marks Mthl and Stdl for degredation, leaving their interacting partner, Rgtl, free in the cytosol, where it does not repress transcription at the HXT3 locus. (B) Under [GAR+] conditions, HXT3 transcription is repressed, which resembles that of cells grown in a carbon source other than glucose. Pmal associates with Stdl, which somehow facilitates the repression of HXT3, possibly by altering the affinity of Stdl for Rgtl . Association with Stdl has been shown previously to facilitate the binding of Rgtl to DNA (Lakshmanan et al. 2003). The association between Pmal can either be transient or stable, but either way it aids in the establishment of an altered signaling pathway. This altered pathway is then maintained either by the contained association between Stdl and Pmal or by a feedback loop within the signaling cascade itself.

[0041] Fig. 7. A prion-based reversal of glucose repression. Glucose represses transcription of genes involved in utilization of alternative carbon sources.

[0042] Fig. 8. (A) [GAR⁺] likely arises from rewiring of the Snf3/Rgt2 glucose signaling pathway. -40-fold reduced HXT3 transcripts in [GAR+], Change in protease susceptibility and protein-protein interactions of Pmal - physiology consistent with gain-of-function. No known participation of amyloid (or l isp 104 dependence). (B) [GAR+]: Escape from glucose repression. Upper panel: Non-Mendelian inheritance. Lower panel: Depends on protein folding machinery.

1 043] Fig. 9. [GAR⁺] appears frequently in wild yeast strains. [0044] Fig. 10. [GAR⁺] is beneficial in mixed carbon sources.

[0045] Fig. 11. Glucose repression re-evolved. Colonies arise on glycerol glucosamine at—1 : 10,000 (1.2 ± 0.4 x 10^"4). Monosporic derivatives are also glucosamine resistant.

[0046] Fig. 12. Life occurs in communities.

[0047] Fig. 13. Bacteria elicit [GAR⁺].

[0048] Fig. 14. Bacteria elicit [GAR ] .

[0049] Fig. 15. Bacteria elicit [GAR⁺] by secreting a prion-inducing factor.

[0050] Fig. 16. [GAR⁺] confers advantages to yeast and bacteria alike: long-term survival for yeast.

[0051] Fig. 17. [GAR⁺] confers advantages to yeast and bacteria alike: reduced ethanol production for bacteria.

[0052] Fig. 18. [GAR⁺] confers advantages to yeast and bacteria alike: reduced ethanol production for bacteria.

[00531 Fig. 19. As a result, [GAR⁺] shifts the outcome of microbial competition in fermentations. Left panel: [gar-]. Right panel: [GAR⁺]. 100X more bacteria present in [GAR⁺] fermentation. No loss of yeast density (and greater recovery of colony forming units from the lees.

[0054] Fig. 20. Why has [GAR⁺] been conserved?

[0055] Fig. 21. A model for [GAR+] conservation.

[0056] Fig. 22. A model for [GAR+] conservation.

[0057] Fig. 23. Spontaneous glucosamine-resistant colonies. Exponential phase yeast grown in YPD (2% glucose) were plate to 2% glucose (left) or 2% glycerol + 0.05% glucosamine (GGM; right). Spontaneous gluocosamine- resistant colonies are visible on the GGM plate. These are restreaked then used in [GAR+] studies.

[0058] Fig. 24. "Strong" and "weak" [GAR+] strains (A) Spot tests of "strong" and "weak" [GAR+] strains demonstrate that the different strains result in different colonies sizes on GGM. All plates were incubated at 30°C for the same amount of time. (B) "Weak"

[GAR+] diploids result in predominantly "strong" [GAR+] spores following meiosis (top). A "weak" diploid occasionally gives rise to a four "weak" spores following meiosis (bottom). All spot tests are incubated at 30°C for the same amount of time.

[0059] Fig. 25. Hsp70-dependent curing of [GAR⁺] is reversible. (A) The crosses involved in a [GAR⁺] propagation assay are shown. Cells carrying [GAR⁺] were mated to [gar^'] cells carrying a mutation of interest ("Δ"), here AssalAssa2. Diploids were selected for glucosamine-resistance, then sporulated. These spores ("haploids") were then crossed to wild- type [gar ] cells and we then selected for the resultant diploids ("diploids"). Both haploids and diploids were tested for glucosamine resistance; if diploids were sensitive to glucosamine, then the [GAR⁺] heritable element cannot be propagated through the mutant of interest and [GAR⁺] is therefore "cured" to [gar^']. (B) [GAR⁺] frequency within a population of wild-type [gar^'] cells or cells "cured" of [GAR⁺] by deletion of ssal and ssa2, then crossed to [gar^']. The final cross to [gar ] demonstrates whether [GAR*] can propagate through AssalAssa.2 mutants, as outlined in part a. [GAR⁺] frequency is measured in the cells that result from this cross. Because [GAR⁺] appears spontaneously at the same frequency as wild- type, AssalAssa2 mutants reversibly cure [GAR*]. Also, this demonstrates that [GAR^'] is not "cryptic" in AssalAssa2 mutants, otherwise all cells would be [GAR⁺] and the measured frequency approaching 1.0.

[0060] Fig. 26. Transcriptional profiling of [gar^'] and [GAR⁺] cells. A significance analysis of microarrays (SAM) plot of Affymetrix microarrays comparing [gar ] and [GAR*] cells grown in glucose. The X-axis represents the expected difference for each gene between [gar ] and [GAR⁺] and the Y-axis the observed difference. 1000 permutations were run. A single point (green) in the bottom left corner represents the only transcript that exhibits a significant change in abundance: YDR345C (HXT3).

[0061] Fig. 27. Knockout mutants of Rgt2/Snf3 pathway members propagate [GAR*], [gar^'] strains in which various members of the Rgt2/Snf3 pathway were knocked out were crossed to [GAR⁺] cells, then sporulated and dissected. These spores ("IN") were tested for glucosamine resistance and then crossed to [gar ] haploids to determine whether

[GAR⁺] can be propagated through these mutants ("2N") (see Figure S3 for outline of crosses). Argtl IN cells are not glucosamine-resistant but 2N cells are, demonstrating that [GAR*] is cryptic in Argtl haploid cells. However, RGT1 is not the causal agent of [GAR*] because [GAR*] can be propagated from Argtl to wild-type cells.

[0062] Fig. 28. Induction of [GAR*] by STD1 and DOG2. STD1 and DOG2 were identified from a screen for genes that induce conversion to [GAR*] from [gar ] following transient overexpression. The original screen was performed using a library of plasmids under control of the GAL1 promoter. Genes identified during the first round of screen were retested under control of a GPD promoter. STD1 and DOG2 were identified by this method. DOG2 overexpression induces [GAR ] conversion at a rate 10- fold higher than vector and STD1 induces [GAR⁺] conversion at a rate over 1000 fold higher than vector. Error bars represent the standard deviation, n = 6.

[0063] Fig. 29. Immunoprecipitation of Stdl-6HA from [gar ] and [GAR⁺] cells.

Immunoprecipitation of Stdl -6HA from Astdl, [gar ], and [GAR⁺] strains. The total protein lysate is shown on the left and the immunoprecipitation samples on the right. One band (arrow) was found in the immunoprecipitation of Stdl-6HA from [GAR⁺] but not [gar ] or Astdl samples. This was analyzed by mass spectrometry and found to be Pmal . Coverage was >20% of the 918 amino acid protein.

[0064] Fig. 30. Pmal from [GAR⁺] is more sensitive to trypsin than [gar ] Pmal . Trypsin digestion of Pmal (left) or Sec61 (right) from [gar ] (top) or [GAR⁺] (middle). A total of six blots were averaged (bottom) and the amount of uncut Pmal or Sec61 measured and graphed relative to t = 0. The graph depicts the mean (n = 6) of (t=n)/(t=0) and p- value was calculated using a paired Wilcoxon test. White bars represent [gar ] protein samples and black bars represent [GAR⁺] protein samples. A red asterisks marks statistically significant points (p = 0.03).

[0065] Fig. 31. Asurl and Alstl alter Pmal oligomers but still propagate the [GAR' ] element (A) Native gel blotted for Pmal from knockout mutants of genes previously shown to affect (Asur4, Alstl) (Roberg et al. 1999; Eisenkolb et al. 2002) or not affect (Alcb3, Alcb4, Adpll) (Gaigg et al. 2005) attributes of wild-type Pmal (left). SDS gels of total, supernatant (sup.), digitonin soluble (det. sol.), and digitonin insoluble (insol.) fractions were probed with ccPmal antibody following blotting (right). The "total" blot was also stained with Ponceau Red to confirm equal amounts of starting material (bottom right). (B) [gar ] strains in which either Istl (top) or sur4 (bottom) were knocked out were crossed to [GAR⁺] cells, then sporulated and dissected. These spores were tested for glucosamine resistance. All spores grown on glycerol-glucosamine plates, demonstrating that Alstl and Asur4 can hold [GAR⁺]. Asur4 was also identified in our deletion library screen for mutants that exhibit a low frequency of [GAR⁺] appearance.

[0066] Fig. 32. PMA1 nonsense mutations do not induce [GAR⁺]. The PMA1 ORF containing nonsense mutations at Q23 or E59 was transiently overexpressed. This did not induce [GAl ] relative to vector, demonstrating that the increase in [GAR' \ due to PMA1 overexpression (figure 4a) is specific to the Pmal protein. [0067] Fig. 33. ΡΜΑ1Δ40Ν propagates [GAR⁺]. Top: tetrad spores from a [GAR⁺] diploid with the genotype GAL- PMA1A40N/PMA1. The pmal mutation is marked with His⁺. Wild-type spores grown on glucosamine-containing medium but pmal mutants cannot grown on any medium lacking galactose, so grown on glycerol-glucosamine cannot be measured. Bottom: spores from top crossed to a [gar^' strain containing a wild-type PMA1 allele. PMA1A40N spores grow on glycerol-glucosamine medium and therefore can propagate

[GAR⁺] to wild-type [gar^'] yeast.

[0068] Fig. 34. Pmal and Stdl do not change localization between [gar ] and [GAR⁺] cells, (A) Detection of Pmal-GFP in [gar ] and [GAR⁺] cells. Pmal-GFP is found at the plasma membrane and in the vacuole in both [gar ] and [GAR⁺] cells. These data are supported by the Native gel fractions, which do not show any difference in Pmal or Stdl between supernatant, soluble, and insoluble fractions (Figure 3A). (B) Detection of Stdl- 6HA by indirect immunofluorescence. Stdl was too scarce to be detected by fluorescent protein fusions at the chromosomal locus. Stdl-6HA is predominantly in the nucleus in both [gar ] and [GAR⁺] cells, which is consistent with previous reports (Schmidt et al l 999).

[0069] Fig. 35. Pmal and Stdl do not form SDS-resistant species in [gar ] or [GAR⁺] cells. (A) SDS-treated protein samples from [psi^'] and [PSf] (left) and [gar ] and [GAR⁺] (right) were run on Blue Native gels. Samples were incubated 10 min in 4% SDS at 37°C before running, transferred by standard Western techniques, then probed with aSup35 (left) or Pmal antibodies. Sup35 shows protein in the well in [PSf^~] but not in [psi], indicated a difference in SDS-solubility. This is expected because Sup35 forms amyloid in [PSf]. Pmal , however, does not show any difference in SDS-solubility between [gar ] and [GAR⁺], indicating that Pmal does not enter into an amyloid state, b) Samples run on SDS gels and blotted for the protein of interest (top) or stained with Ponceau as a loading control (bottom). [gar ] and [GAR⁺] samples were probed with aPmal (far left) or cxHA (second left; to detect Stdl-6HA). There were no differences in mobility in Pmal or Stdl between [gar ] and

[GAR⁺] samples following incubation in 4% SDS for 10 min at 37°C. When [psi^'] and [PSf] protein samples were treated this way, however, (far right: 37°C for lOmin), Sup35 protein from [PSf] runs higher than that from [psi^'] and does not resolve well. When protein samples are boiled, however (second right), Sup35 shows no difference in mobility between [psi] and [PSf ] . Sup35 therefore behaves like an amyloid in [PSf] whereas neither Pmal nor Stdl exhibit the SDS resistance characteristic of amyloids in [gar ] or [GAR⁺]. [0070] Fig. 36. [PSf ], [URE3], and [RNQ⁺] do not alter [GAR⁺] frequencies. We measured [GAR⁺] frequencies in a number of strain backgrounds carrying different states of the PSI, RNQ, and URE3 prions. [GAR^{^}] frequency varied more with strain background than with prion state of the strain. In the case of the PSI prion, strains carrying [PSf] sometimes showed a lower [GAR⁺] frequency (BY) and sometimes a higher one (W303 and 74D).

However, the variation in [GAR⁺] frequency in these strains is two fold or less.

[0071] Fig. 37. 2D gel analysis of [gar ] and [GAR '] protein samples does not reveal any proteins that change solubility, [gar ] (top) and [GAR⁺] (bottom) protein samples were separated into soluble (supernatant; left) and insoluble (pellet; right) fractions, then analyzed by 2D gel electrophoresis. No difference in localization of any protein spot was detected.

[0072] Fig. 38. Pmal alignment. Alignment of Pmal from S. cerevisiae, S. paradoxus, and S. bayanus. Identical amino acids are marked in blue and different amino acids in red. Red asterisks mark the location of varying amino acids. Red dots mark gaps.

[0073] Fig. 39. Stdl alignment. Alignment of Stdl from S. cerevisiae, S. paradoxus, and S. bayanus. Identical amino acids are marked in blue and different amino acids in red. Red asterisks mark the location of varying amino acids. Red dots mark gaps. Note that the N- terminus of S. paradoxus Stdl is missing.

[0074]

Detailed Description of Certain Embodiments

[0075] I. Glossary and References

[0076] The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of molecular biology, cell culture, recombinant nucleic acid (e.g., DNA) technology, immunology, nucleic acid and polypeptide detection, manipulation, and quantification that are within the skill of the art. Non-limiting descriptions of certain of these techniques are found in the following publications: Xiao, W. (ed.) Yeast protocols (Methods in molecular biology) (Clifton, N.J.); v. 313. Totowa, N.J.: Humana Press, Clifton, NJ, 2006; Guthrie, C, and Fink, G. (eds.) Guide to Yeast Genetics and

Molecular Cell Biology, Part B, Volume 350 (Methods in Enzymology), Academic Press, 2002; Amberg, D., et al., (eds.) Methods in Yeast Genetics: A Cold Spring Harbor

Laboratory Course Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2005; Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3^r ed., Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, 2001 ; Harlow, E. and Lane, D., Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988. All patents, patent applications, and other publications and references mentioned herein are incorporated by reference in their entirety. In the event of a conflict between the

specification and any of the incorporated references the specification shall control but may be expressly amended based on any of the incorporated references and/or to correct obvious errors. Information relating to fermentation technology of use in various embodiments may be found in, El-Mansi, M., et al. (eds.) Fermentation Microbiology and Biotechnology, CRC Press; 3 edition (201 1); Fermentation and Biochemical Engineering Handbook, 2nd Ed., Second Edition: Principles, Process Design and Equipment by Henry C. Vogel and Celeste C. Todaro, William Andrew (publisher); 2nd edition (2007).

[0077] Information relating to various foods and beverages prepared at least in part using fermentation and/or relating to use of fermentation in preparing various foods and beverages may be found in, e.g., Robinson, Jancis (2006) The Oxford Companion to Wine (3rd ed.) Oxford University Press, USA; Oliver, Garrett (201 1) The Oxford Companion to Beer Oxford University Press, USA; 1st edition; Beer and wine production: Analysis,

characterization, and technological advances, edited by Barry H. Gump and David J. Pruett, ACS Symposium Series No. 536 American Chemical Society, Washington, DC, 1993;

Hough, D., The Biotechnology of Malting and Brewing (Cambridge Studies in

Biotechnology). Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

[0078] The following definitions and related information are provided here for convenience. Art-accepted abbreviations are used herein unless otherwise indicated.

[0079] "About" in reference to a numerical value generally refers to a range of values that fall within ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5% of the value unless otherwise stated or otherwise evident from the context.

[0080] "Antibody" as used herein refers to immunoglobulin molecules or portions thereof capable of specifically binding to an antigen. An antibody can be polyclonal or monoclonal. Antibodies or purified fragments having an antigen binding region, e.g., fragments such as Fv, Fab', F(ab')2, Fab fragments, single chain antibodies (which typically include the variable regions of the heavy and light chains of an immunoglobulin, linked together with a short (usually serine, glycine) linker, chimeric, humanized, or fully human antibodies are encompassed. An antibody may be identified and prepared by conventional procedures. An antibody may be of mammalian origin, e.g., rodent (e.g., murine) or human, or avian (e.g., chicken) origin and could be of any of the various immunoglobulin classes or subclasses known in the art.

[0081] An "expression control element" as used herein can be any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, that facilitates the expression of a nucleic acid. The expression control element may, for example, be a yeast, bacterial, mammalian or viral (e.g., phage) promoter. An expression control element, e.g., promoter, can be constitutive or conditional, e.g., regulatable (e.g., inducible or repressible). Inducible promoters direct expression in the presence of an inducing agent (e.g., an appropriate small molecule) or inducing condition (e.g., increased temperature), while in the absence of such agent or condition expression is usually much lower or undetectable above background. In some embodiments the promoter is titratable, e.g., the level of expression can be regulated by varying the concentration of an inducing or repressing agent. For example, a higher concentration of inducing agent typically results in higher expression level. It will be understood that induction in some instances may be achieved by relieving repression. Tetracycline controlled transcriptional activation is a method of inducible expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or a derivative (e.g., doxycycline). Two "Tet" systems (Tet-off and Tet-on) are widely used. Expression control elements capable of directing transcription in cells are known in the art. Exemplary expression control elements are mentioned herein. In some embodiments of the invention, transcription of a sequence of interest can be irreversibly turned on or off using the Cre/Lox or Flp/FRT recombinase system. For example, a nucleic acid "stuffer sequence" can be positioned between sites for a recombinase. Delivering the recombinase to a cell (e.g., by expressing it therein or by introducing it from outside the cell), results in excision of the stuffer sequence. Such excision can bring an expression control element, e.g., a promoter, into operable association with a nucleic acid segment of interest, resulting in its transcription.

[0082] "Identity" refers to the extent to which the sequence of two or more nucleic acids or polypeptides is the same. The percent identity between a sequence of interest A and a second sequence B may be computed by aligning the sequences, allowing the introduction of gaps to maximize identity, determining the number of residues (nucleotides or amino acids) that are opposite an identical residue, dividing by the minimum of TGA and TGB (here TGA and TGB are the sum of the number of residues and internal gap positions in sequences A and B in the alignment), and multiplying by 100. When computing the number of identical residues needed to achieve a particular percent identity, fractions are to be rounded to the nearest whole number. Sequences can be aligned with the use of a variety of computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generate alignments. The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990) modified as in Karlin and Altschul, Proc. Natl. Acad Sci. USA 90:5873-5877,1993 is incorporated into the NBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J. Mol. Biol. 215:403- 410, 1990). In some embodiments, to obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs may be used. A PAM250 or BLOSUM62 matrix may be used. See the Web site having URL www.ncbi.nlm.nih.gov. Other suitable programs include CLUSTALW (Thompson JD, Higgins DG, Gibson TJ, Nuc Ac Res, 22:4673-4680, 1994) and GAP (GCG Version 9.1 ; which implements the Needleman & Wunsch, 1970 algorithm (Needleman SB, Wunsch CD, J Mol Biol, 48:443-453, 1970.)

[0083] As used herein, "non-endogenous" refers to genes, molecules, pathways, processes, that are not naturally found in a particular context, e.g., in or associated with a cell or organism. For example, a "non-endogenous" nucleic acid could be derived at least in part from a different organism or could be at least in part invented by man and not found in nature. "Non-endogenous" can include modifying an endogenous molecule. For example, homologous recombination could be used to modify an endogenous gene (e.g., alter its sequence), with resulting gene being considered "non-endogenous". "Non-endogenous" also encompasses introducing a nucleic acid that has the same sequence as an endogenous nucleic acid into a cell, wherein said introduction genetically modifies the recipient cell. For example, the introduced nucleic acid may be joined to a nucleic acid to which it is not joined in nature, e.g., an expression control element, or integrated into the genome in a position in which it is not found in nature.

[0084] As used herein, the term "nucleic acid" is used to mean one or more nucleotides, i.e. a molecule comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and organic base, which may be a substituted pyrimidine (e.g. cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)). The term "nucleic acid" is used interchangeably with "polynucleotide" or "oligonucleotide" as those terms are ordinarily used in the art, i.e., polymers of nucleotides, where oligonucleotides are generally shorter in length than polynucleotides (e.g., 60 nucleotides or less). A series of nucleotides bonded together, i.e., within a polynucleotide or an oligonucleotide can be referred to as a "nucleic acid sequence" or "nucleotide sequence", and the nucleotide subunits are typically indicated using the abbreviation of the base, e.g., A, G, C, T, U. Where the present invention provides a nucleotide sequence, it is understood that the complementary sequence is also provided, and both single- and double-stranded forms are provided. Purines and pyrimidines include, but are not limited to, natural nucleosides (for example, adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine), nucleoside analogs, chemically or biologically modified bases (for example, methylated bases), modified sugars (2'-fluororibose, arabinose, or hexose), modified phosphate groups (for example, phosphorothioates or 5'-N-phosphoramidite linkages), and other naturally and non-naturally occurring nucleobases, including substituted and

unsubstituted aromatic moieties. Other modifications are well-known to those of skill in the art. In some embodiments a nucleic acid comprises non-nucleotide material, such as at the end(s) or internally (at one or more nucleotides). A nucleic acid can be single-stranded, double-stranded, or partially double-stranded. In some embodiments a nucleic acid is composed of RNA. In some embodiments a nucleic acid is composed of DNA. In various embodiments a double-stranded nucleic acid may have one or more overhangs (5 ' and/or 3 ' overhangs). In some embodiments a nucleic acid comprises standard nucleotides (A, G, C, T, U). In other embodiments a nucleic acid comprises one or more non-standard nucleotides. In some embodiments, one or more nucleotides are non-naturally occurring. A nucleic acid may comprise a detectable label, e.g., a fluorescent dye.

[00851 A "polypeptide" refers to a polymer of amino acids. A protein is a molecule comprising one or more polypeptides. A peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length. The terms "protein", "polypeptide", and "peptide" may be used interchangeably. Polypeptides of interest herein typically contain standard amino acids (the 20 L-amino acids that are most commonly found in nature in proteins). However, other amino acids and/or amino acid analogs known in the art can be used in certain embodiments of the invention. One or more of the amino acids in a polypeptide may be modified, for example, by addition, e.g., covalent linkage, of a non- peptide moiety, such as a carbohydrate group, a phosphate group, a linker for conjugation, etc. A polypeptide sequence presented herein is presented in an N-terminal to C-terminal direction unless otherwise indicated. "Polypeptide domain" refers to a segment of amino acids within a longer polypeptide. A polypeptide domain may exhibit one or more discrete binding or functional properties, e.g., a catalytic activity. Often a domain is recognizable by its conservation among polypeptides found in multiple different species.

[0086] "Purified" or "substantially purified" may be used herein to refer to an isolated nucleic acid or polypeptide that is present in the substantial absence of other biological macromolecules, e.g., other nucleic acids and or polypeptides. In some embodiments a purified nucleic acid (or nucleic acids) is substantially separated from cellular polypeptides. In some embodiments, the ratio of nucleic acid to polypeptide is at least 5:1 or at least 10: 1 by dry weight. In some embodiments a purified polypeptide is separated from cellular nucleic acids. In some embodiments, the ratio of nucleic acid to polypeptide is at least 5: 1 or at least 10: 1 by dry weight. In some embodiments, a nucleic acid or polypeptide is purified such that it constitutes at least 75%, 80%, 85%, or 90% by weight, e.g., at least 95% by weight, e.g., at least 99% by weight, or more, of the total nucleic acid or polypeptide material present. In some embodiments, water, buffers, ions, and/or small molecules (e.g., precursors such as nucleotides or amino acids), can optionally be present in a purified preparation. A purified molecule may be prepared by separating it from other substances (e.g., other cellular materials), or by producing it in such a manner to achieve purity. In some embodiments, a purified molecule or composition refers to a molecule or composition comprising one or more molecules, that is prepared using any art-accepted method of purification. In some embodiments "partially purified" means that a molecule produced by a cell is no longer present within the cell, e.g., the cell has been lysed and, optionally, at least some of the cellular material (e.g., cell wall, cell membrane(s), cell organelle(s)) has been removed.

[0087] A "variant" of a particular polypeptide or polynucleotide has one or more alterations (e.g., amino acid or nucleotide additions, substitutions, and/or deletions, which may be referred to collectively as "mutations") with respect to the polypeptide or

polynucleotide, which may be referred to as the "original polypeptide or polynucleotide". Thus a variant can be shorter or longer than the polypeptide or polynucleotide of which it is a variant. In some embodiments a "variant" comprises a "fragment". The term "fragment" refers to a portion of a polynucleotide or polypeptide that is shorter than the original polynucleotide or polypeptide. In certain embodiments of the invention a variant comprises a portion that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more sequence identity to the original polypeptide or polynucleotide over a portion of the original polypeptide or polynucleotide having a length at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, of the length of the original polypeptide or polynucleotide. In a non-limiting embodiment a variant polypeptide has at least 80%o, 90%, 95%, 96%, 97%, 98%, or 99% identity to the original polypeptide over a portion of the original polypeptide having a length at least 100 amino acids. In a non-limiting embodiment a variant polypeptide has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the original polypeptide over a functional domain of the original polypeptide. In some embodiments, a variant

polynucleotide or polypeptide is generated using recombinant DNA techniques. In some embodiments amino acid "substitutions" replace one amino acid with another amino acid having similar structural and/or chemical properties, e.g., conservative amino acid replacements. "Conservative", amino acid substitutions may be made on the basis of similarity in any of a variety or properties such as side chain size, polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathicity of the residues involved. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan and methionine. The polar (hydrophilic), neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Insertions or deletions may range in size from about 1 to 20 amino acids, e.g., 1 to 10 amino acids. In some instances larger domains may be removed without substantially affecting function. In certain embodiments, the sequence of a variant can be obtained by making no more than a total of 5, 10, 15, or 20 amino acid additions, deletions, or substitutions to the sequence of a naturally occurring enzyme. In some embodiments, not more than 1%, 5%, 10%, or 20% of the amino acids in a polypeptide are insertions, deletions, or substitutions relative to the original polypeptide. Guidance in determining which amino acid residues may be replaced, added, or deleted without eliminating or substantially reducing an activity of interest, may be obtained, e.g., by aligning and comparing the sequence of the particular polypeptide with that of homologous functional polypeptides (e.g., orthologs from other organisms). One of skill in the art will be aware that amino acid residues that are conserved among various species are, in general, more likely to be important for activity than amino acids that are not conserved. 10088 J "Isolated" as used herein refers to a molecule, e.g., a nucleic acid or polypeptide, separated from at least some other components (e.g., nucleic acid or polypeptide) that are present with the nucleic acid or polypeptide as found in its natural source (or a molecule produced from such an isolated molecule) and/or a molecule prepared at least in part by the hand of man. In some embodiments an isolated nucleic acid or polypeptide is at least in part synthesized using recombinant DNA technology, e.g., using in vitro transcription or translation, respectively, or an isolated nucleic acid sequence is synthesized using amplification (e.g., PCR). In some embodiments an isolated nucleic acid or polypeptide is chemically synthesized. In some embodiments, an isolated nucleic acid is removed from its genomic context. In some embodiments, an isolated nucleic acid is joined to a nucleic acid to which it is not joined in nature. For example, an isolated nucleic acid may be joined to a sequence comprising an expression control element to which the nucleic acid is not operably linked in nature. In some embodiments, an isolated nucleic acid is present in a vector which, in some embodiments, is not a sequencing vector. "Isolated" can also refer to a cell that is removed from its natural habitat, e.g., a cell maintained in a laboratory, e.g., in culture, or a descendant of the cell.

[0089] As used herein, the term "selectable marker" (sometimes termed "marker" herein) typically refers to a gene that encodes an enzymatic or other activity that confers on a cell the ability to grow in medium lacking what would otherwise be an essential nutrient or confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed or otherwise renders a cell specifically detectable or selectable. The term "selectable marker" can also refer to the gene product itself. In some embodiments expression of a selectable marker by a cell confers a significant growth or survival advantage on the cell (relative to cells not expressing the marker) under certain defined culture conditions (selective conditions) such that maintaining the cell under such conditions allows the identification (and optionally the isolation) or elimination of cells that express the marker. Antibiotic resistance markers include genes encoding enzymes that provide resistance to neomycin, zeocin, hygromycin, kanamycin, puromycin, chloramphenicol, etc. A second non-limiting class of selectable markers is nutritional markers. Such markers are generally enzymes that function in a biosynthetic pathway to produce a compound that is needed for cell growth or survival. Examples in yeast include enzymes that participate in biosynthetic pathways for synthesis of amino acids such as uracil, leucine, histidine, tryptophan, etc. It will be appreciated that selectable markers encompass those in which negative selection is employed. Optically detectable molecules, e.g., fluorescent or luminescent proteins, are another class of marker, sometimes termed "detectable marker". Enzymes with a readily assayed activity such as alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS),

chloramphenicol acetyltransferase (CAT), horseradish peroxidase (HRP), lucifera.se (Luc) can also be used. Such genes can also be used as reporters or controls, e.g., to assess the presence of a prion, e.g., [GAR+].

[0090] As used herein, a first sequence is "substantially complementary" to a second sequence if at least 75% of the nucleotides in the two sequences are capable of forming hydrogen bonded base pairs (bp) with oppositely located nucleotides (i.e., a nucleotide is capable of base pairing with a nucleotide located at the opposite position in the other strand) when the sequences are aligned in opposite orientation. In some embodiments, the two sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%

complementary. As known in the art, in the canonical Watson-Crick base pairing, adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). Non- Watson-Crick base pairing with alternate hydrogen bonding patterns also occur, especially in RNA; common among such patterns are Hoogsteen base pairs and wobble base pairs. In some embodiments of the invention, a dsRNA or siRNA comprises only Watson-Crick base pairs, while in other embodiments at least some of the base pairs are non- Watson-Crick base pairs.

[0091] A "small interfering RNA" or "siRNA" as used herein, refers in some

embodiments to an RNA molecule derived from the successive cleavage of longer double- stranded RNA (dsRNA), e.g., within a cell by an enzyme comprising an RNase III domain, to produce an RNA molecule composed of two at least substantially complementary strands generally having a length of between 15 and 30 nucleotides, and more often between 20 and 25 nucleotides, e.g., 20, 21 , 22, 23, 24, or 25 nucleotides, wherein each strand typically comprises a 5' phosphate group and a 3 ' hydroxyl (-OH) group. Naturally occurring siRNAs typically comprise a duplex structure between about 18 and 23 base pairs (bp) long, e.g., 18, 19, 20, 21 , 22, 23 bp long. Often the portions of the strands that form the duplex are perfectly (100%o complementary), but in some embodiments the strands of the duplex are, e.g., at least 80%o, 90%), or 95%o complementary, e.g., the duplex comprises between 1 -5 mismatches, e.g., 1 , 2, 3, 4, 5 mismatches (referring to a pair of nucleotides located opposite one another that do not form a base pair) or bulges, which mismatches or bulges may be located, e.g., near one or both ends of the duplex. The term "siRNA" also encompasses molecules of similar structure that are generated extracellularly, e.g., in a cell extract, in a composition comprising an isolated Dicer polypeptide, or using chemical synthesis. Such siRNAs, e.g., those generated using chemical synthesis, can comprise a variety of different nucleotides and internucleoside linkages, as known in the art. siRNAs can be blunt-ended or have overhangs, e.g., 3 ' overhangs. In some embodiments an overhang is from 1 - 10 nucleotides in length, e.g., 1 , 2, 3, 4, or 5 nucleotides long, e.g., 2 nucleotides long. One of skill in the art will be aware of various approaches to generating synthetic siRNAs that have, for example, increased resistance to degradation. In some embodiments, one or more nucleotides at the 3 ' end of an siRNA, e.g., 2 nucleotides, is/are deoxyribonucleotide(s), e.g., dT. [0092] "Transfection" refers to the introduction of a nucleic acid into a cell. The term is intended to encompass nucleic acid transfer into prokaryotic (e.g., bacterial), fungal, and plant cells (sometimes termed "transformation"). Cells may be transiently or stably transfected. Stable cell lines can be generated using standard selection methods. A cell has been "stably transfected" with a nucleic acid construct when the nucleic acid construct is capable of being inherited by daughter cells over many generations, e.g., is integrated into the genome of the cell. "Transient transfection" refers to cases where exogenous nucleic acid does not integrate into the genome of a transfected cell and is progressively lost as cells divide.

[0093] A "vector" as used herein, refers to a nucleic acid or a virus or portion thereof (e.g., a viral capsid) capable of mediating entry of, e.g., transferring, transporting, etc., a nucleic acid molecule into a cell. Where the vector is a nucleic acid, the nucleic acid molecule to be transferred is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A nucleic acid vector may include sequences that direct autonomous replication (e.g., an origin of replication) in a cell and/or may include sequences sufficient to allow integration of part or all of the nucleic acid into host cell DNA. Useful nucleic acid vectors include, for example, DNA or RNA. plasmids, cosmids, artificial chromosomes, and naturally occurring or modified viral genomes or portions thereof or nucleic acids (DNA or RNA) that can be packaged into viral capsids. Vectors often include one or more selectable markers. "Expression vectors" typically include regulatory sequence(s), e.g., expression control sequences such as a promoter, sufficient to direct transcription of an operably linked nucleic acid. An expression vector often comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in vitro expression system. Vectors often include one or more appropriately positioned sites for restriction enzymes, e.g., to facilitate introduction of the nucleic acid to be transported or expressed into the vector.

[0094] II. Compositions and Methods Relating to [GAR+]

[0095] Yeast prions provide a mechanism for generating heritable phenotypic diversity that promotes survival in fluctuating environments and the evolution of new traits.

[0096] Prions are found in laboratory and wild strains of yeast, e.g., Saccharomyces. They confer diverse phenotypes that are frequently beneficial, e.g., under selective conditions. The present invention encompasses the recognition that prion modulation, e.g., modulating the acquisition, maintenance, or loss, of a prion, e.g., [GAR+] is useful in a variety of industrial processes, e.g., in production of a variety of products. [0097] Applicants previously reported the discovery of a prion that makes yeast cells resistant to the glucose-associated repression of alternative carbon sources and named it

[GAR+] (for "resistant to glucose-associated repression," with capital letters indicating dominance and brackets indicating its non-Mendelian character) (2). [GAR+] appears spontaneously at a high rate and is transmissible by non-Mendelian, cytoplasmic inheritance. As described further in Example 1 , [GAR+] shows non-Mendelian, cytoplasmic inheritance and is transmissible by transfer of cytoplasmic material (cytoduction), i.e., is "infectious". [GAR+] appears spontaneously at high frequency in a variety of genetic backgrounds and is curable by transient changes in chaperone protein levels. Several lines of evidence indicate that the [GAR+] prion state involves a complex between a small fraction of the cellular complement of Pmal , the major plasma membrane proton pump in S. cerevesiae, and Stdl, a much lower-abundance protein that participates in glucose signaling. The Pmal proteins from closely related Saccharomyces species are also associated with the appearance of [GAR+]. Pmal and Stdl can create a transmission barrier for [GAR+] propagation and induction in S. cerevisiae. The discovery that yeast cells employ a prion-based mechanism for heritably switching between distinct carbon source utilization strategies, and employ the plasma membrane proton pump to do so, expanded the biological framework in which self- propagating protein-based elements of inheritance operate.

[0098] The present disclosure encompasses the recognition that [GAR+] can be modulated for a variety of useful purposes. The invention provides, among other things, methods of modulating [GAR+], methods of producing cells with altered acquisition of

[GAR+], methods of producing products using cells that are [GAR+] or [gar-], methods of using cells that are [GAR+] or [gar-] in industrial processes, and related compositions.

[0099] [GAR+] allows yeast to use non-preferred carbon sources in the presence of the preferred carbon source, glucose. In some aspects, the invention provides the recognition that acquisition of [GAR+] results in faster growth and higher biomass on complex mixtures of carbon sources. Complex mixtures such as molasses or grape must are frequently used in industrial processes because they are cheaper than pure glucose. [GAR+] thus increases efficiency of using yeast to produce virtually any small molecule, which would be of considerable interest to pharma/biotech because small molecule pharmaceuticals are increasingly produced in yeast. [GAR+] could also be useful in biofuel production. [GAR+] also decreases the final ethanol content of fermentations, which could be useful for winemakers in producing lower alcohol content products or allowing greater control over the fermentation process. 1001001 In some aspects [GAR+] confers on yeast an improved ability to grow under conditions in which one or more nutrients is limited. In some aspects [GAR+] confers on yeast an improved ability to grow under conditions in which one or more amino acids is limited. In some aspects [GAR+] confers on yeast an improved ability to grow under conditions in which nitrogen supply is limited. In some embodiments a method comprises inducing [GAR+] or providing yeast cells in which [GAR+] is induced or that have enhanced [GAR+] induction; and (b) culturing the yeast cells in culture medium in which one or more nutrients, e.g., one or more amino acids, is limited. In some embodiments a nutrient is considered "limited" in a medium if addition of the nutrient to the medium results in an increased growth rate of a [gar-] yeast strain of interest. In some embodiments a nutrient is considered "limited" in a medium if it is present in amounts less than that found in typical "rich" growth medium, such as YPD (also termed YEPD; yeast extract peptone dextrose). The agar version of YEPD typically consists of 1% (mass/volume) yeast extract, 2% peptone, 2% glucose/dextrose, 2% agar, with the rest being water. The liquid version of YEPD typically contains 1% yeast extract, 2% peptone, 1% glucose/dextrose, and the rest is distilled water.

[00101] The term "culture medium" is used in a broad sense to refer to any nutrient- containing composition useful to culture cells, e.g., yeast cells, bacterial cells, mammalian cells. Culture media thus include (i) compositions used as culture media in laboratory purposes, (ii)xompositions used as culture media in industrial processes where a product produced at least in part by cultured cells is to be isolated from the culture medium, and (iii) media that are to be used in or as a product or a component of a product (such as a grape juice that will be used to make wine). A culture medium may be liquid or solid (which includes media having a semi-solid or gel-like consistency). In some embodiments a solid medium comprises agar or another solidifying or gelling agent.

[00102] In some embodiments a culture medium comprises material derived from grapes. In some embodiments grapes are grown in Argentina, Australia, the United States (e.g., California), Chile, China, France, Germany, Greece, Italy, Maldova Portugal, Romania, Russia, Spain, South Africa, or New Zealand. In some embodiments at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, e.g., 100% of the grapes from which grape-derived material is obtained are grown in one of the afore-mentioned countries. In some embodiments at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, e.g., 100% of the material is derived from grapes grown in a particular country, e.g., any of the foregoing countries. In some embodiments at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%), 99%>, or more, e.g., 100% of the grapes from which grape-derived material is obtained are grown in Napa Valley or Sonoma Valley in California; Willamette Valley in Oregon; Columbia Valley in Washington; Barossa Valley in South Australia;

Hunter Valley in New South Wales; Lujan de Cuyo in Argentina; Central Valley in Chile; Vale dos Vinhedos in Brazil; Hawke's Bay or Marlborough in New Zealand; Okanagan Valley or Niagara Peninsula in Canada, or any American Viticultural Area. In some embodiments at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, e.g., 100%) of the grapes from which grape-derived material is obtained are grown in a particular year. In some embodiments grapes or grape-derived material may be measured by volume. In some embodiments grapes or grape-derived material may be measured by weight. In some embodiments grape-derived material includes grape juice and at least some grape- derived solid material (grape skin, seeds, and/or stems). In some embodiments grape-derived material lacks at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more of the solid material that would be present in the whole grapes. Material derived from grapes may be filtered or otherwise processed to remove solid material. In some embodiments grapes are red, purple, or green grapes. In some embodiments at least 50%>, 60%, 70%, 75%, 80%o, 85%o, 90%, 95%, 98%, 99%>, or more of the grapes, e.g., 100%, are red. In some embodiments at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more of the grapes, e.g., 100%, are purple. In some embodiments at least 50%>, 60%o, 70%>, 75%>, 80%, 85%, 90%, 95%, 98%, 99%, or more of the grapes, e.g., 100%, are green.

[00103] In some embodiments a culture medium comprises material derived from sugar cane, corn, sorghum, grass, or wood. In some embodiments a culture medium comprises a lignocellulosic material such as wood, bagasse, or straw. In some embodiments a culture medium comprises sugar derived from a lignocellulosic material such as wood, bagasse, or straw, which has been subjected to cellulolysis.

[00104] In some embodiments a method of testing for a prion comprises measuring the level of expression of a gene, wherein the level of expression of the gene is regulated directly or indirectly by the prion. The level of gene expression may be quantified at the level of RNA (e.g., mRNA) or protein. Standard methods for measuring the level of a gene product can be used, e.g., hybridization or amplification-based methods can be used for RNA, e.g., RNA solution hybridization, nuclease protection, Northern blots, reverse transcription, microarrays, or PCR (e.g., quantitative PGR such as Taqman PCR). For proteins, antibody or other affinity-based methods can be used, e.g., Western blots, enzyme linked immunosorbent assay (ELISA), Western blotting. For proteins that are readily detectable, e.g., fluroscent or having an enzymatic activity, appropriate methods such as fluorescence activated cell sorting (FACS) or enzymatic detection may be used. In some embodiments, an alteration in gene expression results in a change in morphology (e.g., cell shape) or cell properties that may be detected using visual observation (e.g., using a microscope). In some embodiments a method of testing for a prion comprises detecting a prion confirmation using a prion-specific antibody.

[00105] A variety of different yeast are of use in various embodiments of any aspect of the invention. In some embodiments a yeast is a budding yeast. In some embodiments a budding yeast is a member of the subphylum Saccharomycotina. In some embodiments, a budding yeast is a member of the genus Saccharomyces, e.g., S. cerevesiae, the genus Kluveromyces, e.g., Kluveromyces polysporus, the genus Candida, e.g., Candida albicans, or the genus Pichia, e.g., Pichia pastoris. In some embodiments a budding yeast is a member of the Saccharomyces sensu stricto. As used herein, the Saccharomyces sensu stricto genus includes S. cerevisiae, and at least seven other natural species (S. paradoxus, S. cariocanus, S.

mikatae, S. arboricolus, S. kudriavzevii, S. uvarum, and S. bayanus) and at least one hybrid species (S. pastorianus). In some embodiments a budding yeast is Naumovozyma castellii (also referred to as Saccharomyces castellii). In some embodiments, a yeast is of a species or strain used in wine-making, brewing, food production, or biofuel production. In some embodiments, a yeast is dimorphic. Such yeast exhibits budding under some environmental conditions. For example, Arxula adeninivorans (Blastobotrys adeninivorans) is a dimorphic yeast useful in various biotechnological applications. In some embodiments a strain is a wild strain, as recognized in the art. In some embodiments a yeast is a fission yeast such as fission yeast Schizosaccharomyces pombe. In some embodiments a strain is a clinical isolate, e.g., isolated from a mammalian, e.g., human, subject suffering from a disease, e.g., clinical or subclinical infection by the yeast. In some embodiments a yeast culture is relatively pure, e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the yeast cells are of a particular species or strain. In some embodiments a culture comprises two or more different yeast strains or species, each contributing at least 1%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, or 50% of the yeast cells in the culture.

[00106] In some embodiments, the yeast is a laboratory strain. Exemplary laboratory strains of S. cerevesiae include strains S288c, W303, and derivatives thereof. See, e.g., Sherman, F., Getting started with yeast, Methods Enzymol. 350, 3-41 (2002); Mortimer and Johnston, Genetics 1 13 :35-43 (1986); van Dijken et al, Enzyme Microb Technol 26:706-714 (2000); Winzeler et al., Genetics 163 :79-89 (2003). In some embodiments the yeast is a strain that is present in the American Type Culture Collection (ATCC) yeast collection, e.g., a strain listed in the Yeast Genetics Stock Center catalog, 10^th ed. (1999). In some embodiments the yeast is a member of a species or strain whose genome has at least in part been sequenced. See, e.g., http://www.ncbi.nlm.nih.gov/sites/entrez under "Genome Project". See also, Yeast Gene Order Browser, available at http://wolfe.gen.tcd.ie/ygob/ (e.g., Version 3.0). See Byrne P and Wolfe KH, The Yeast Gene Order Browser:

combining curated homology and syntenic context reveals gene fate in polyploid species, Genome Research, 15(10): 1456-61 , 2005. The Candida Genome Browser

(http://www.candidagenome.org/) is also of use. In some embodiments a yeast is a wild strain. In some embodiments a yeast is a strain derived by crossing a laboratory strain and a wild strain. In some embodiments a yeast is of an industrially important species or strain. In some embodiments a yeast is polyploid. In some embodiments a yeast is aneuploid. In some embodiments a yeast is diploid. In some embodiments a yeast strain is a strain that is available from the Centraalbureau voor Schimmelcultures (CBS), the ATCC, the Phaff Yeast Culture Collection (PYCC), the National Collection of Yeast Cultures (NCYC), or any culture collection described in Boundy-Mill, K., J Ind Microbiol Biotechnol (2012). In some embodiments the yeast is a wine yeast. In some embodiments a yeast, e.g., a wine yeast, is available from the Enology Culture Collection, housed in the Department of Viticulture and Enology, University of California, Davis

(http://wineserver.ucdavis.edu/collection/index.php). For example, in some embodiments a wine yeast is listed in Table 1 or Table 2. Yeast strains may be obtained from any of a number of commercial suppliers such as Lallemand (corporate office Montreal, Quebec, Canada; http://www.lallemand.com), Anchor (Johannesburg, South Africa;

http://www.anchor.co.za/), DSM (Heerlen, Netherlands;

http://www.dsm.com/corporate/home.html), AB Mauri (Peterborough, UK;

http://www.abmauri.com/), Lesaffre (Marcq-en-Baroeulm, France; http://www.lesaffre.com), Laffort (Bordeaux, France; http://www.laffort.com/en) or Scott Laboratories (Petaluma, California and Pickering, Ontario; www.scottlab.com). In some embodiments a strain available from one or more of these suppliers is used. Catalogs of the afore-mentioned noncommercial and commercial sources and suppliers are incorporated herein by reference. [00107] Table 1 : Selected yeast strains

Yeast 554 Saccharomyces cerevisiae B-3c Sherry wine

Yeast 555 Saccharomyces cerevisiae B-3D Sherry wine

Yeast 556 ^{" "} Saccharomyces cerevisiae B3-D ^" Sherry wine

Yeast 557 Saccharomyces cerevisiae J1934-3A (Sherry wine

Yeast 558 Saccharomyces cerevisiae J1934-3B Sherry wine

Yeast 561 Saccharomyces cerevisiae J1934-3a Sherry wine

Yeast 562 ^" Saccharomyces cerevisiae J1935-I-3a (Sherry wine

Yeast 563 Saccharomyces cerevisiae J1935-I— 3b Sherry wine

Yeast 565 Saccharomyces cerevisiae J-F P-3a Sherry wine

Yeast 567 Saccharomyces cerevisiae J-F P-3c Sherry wine

Yeast 568 Saccharomyces cerevisiae J-F P-3d Sherry wine j

Yeast 569 Saccharomyces cerevisiae J-F P-3b Sherry wine

Yeast 571 Saccharomyces cerevisiae J-F P-3b [Sherry wine

Yeast 574 Saccharomyces cerevisiae wine

Sylvaner

Yeast 575 Saccharomyces cerevisiae Mittelbergehim

wine membranifacien Riesling

Yeast 576 Pichia Wolxheim

s wine

Yeast 577 Saccharomyces cerevisiae Wolxheim Muscat wine iGewurtrami

Yeast 578 Saccharomyces cerevisiae Dambach

ner wine commercial

Yeast 579 Saccharomyces cerevisiae Montrachet dry wine yeast

[Flor Yeast,

Yeast 580 Saccharomyces cerevisiae Flor Yeast

! Sherry wine

Schizosaccharo

Yeast 584 pombe Rankine #442 wine?

myces

Yeast 586 Saccharomyces cerevisiae 350 36029 wine

Yeast Saccharomyces cerevisiae Wine

wine,

Yeast 591 Saccharomyces cerevisiae

German

Schizosaccharo

Yeast 592 octosporus H105 Wine

myces

cerevisiae race "prise de Champagne

Yeast 594 Saccharomyces

bayanus mousse" wine, France commercial

Yeast 595 Saccharomyces cerevisiae Champagne dry wine yeast

Yeast 596 Pichia vini 624 wine

wine yeast

Yeast 773 Saccharomyces cerevisiae 142 wine yeast

wine yeast

experiment I

dry wine lyeast

experiment wine,

Yeast 2029 Brettanomyces bruxellensis

cabernet

Wine, 1972

Yeast 2030 Brettanomyces bruxellensis Cabernet

Sauvignon j

Commercial

Yeast 2035 Saccharomyces cerevisiae CHOOl dry wine yeast

1 Commercial

Yeast 2036 Saccharomyces cerevisiae CH003 dry wine I

1

ί yeast

Yeast 2037 Saccharomyces cerevisiae wine

i Commercial

Yeast 2038 Saccharomyces cerevisiae BM 45 ;dry wine culture

Commercial

Yeast 2039 Saccharomyces cerevisiae W15 dry wine

'yeast

Pinot Noir

Yeast 2040 Candida parapsilosis wine,

California '

Brettanomyces/ j Wine. Fruit,

Yeast 2041 bruxellensis

Dekkera I ; Thailand

Brettanomyces/ wine,

Yeast 2042 bruxellensis B-l

Dekkera California

Brettanomyces/ wine.

Yeast 2043 bruxellensis B-2

Dekkera : California wine,

Yeast 2044 Pichia guilliermondii FPW

jCalifornia wine,

Yeast 2045 Pichia guilliermondii FW2

California

Wine,

Brettanomyces/

Yeast 2046 bruxellensis Merlot,

Dekkera

jCalifornia

Wine,

Brettanomyces/ Cabernet

Yeast 2047 bruxellensis

Dekkera Sauvignon,

California iWine, Pinot

Brettanomyces/

Yeast 2048 bruxellensis Noir.

Dekkera

California

Brettanomyces/ Wine, Pinot

Yeast 2049 bruxellensis

Dekkera Noir, New

Yeast 12089 (Saccharomyces cerevisiae SF4 Stuck _; fermentation j

!, wine j

Stuck

Yeast 2090 Saccharomyces cerevisiae SF5 fermentation ^'

;, wine

Wine,

Brettanomyces/ (Bordeaux,

Yeast 2091 bruxellensis CBS 2797 j

Dekkera (France,

1957 1 jWine, sparkling

Brettanomyces/

Y east i y bruxeliensis CBS 2796 mosselle,

Dekkera

Germany, i 1957 !

Brettanomyces/ Wine. New _\

Yeast 2093 bruxellensis

Dekkera i York

Brettanomyces/

Yeast 2094 bruxellensis

Dekkera j Wine :

Yeast 2095 Trigonopsis variabilis iwine

Schizosaccharo w ine

Yeast 2096 pombe \

myces j fermentation

^■Botrytis

Yeast 2097 Candida zemplinina EJ1

I wine cerevisiae race William- Commercial

Yeast 2099 Saccharomyces

bayanus Selyem wine yeast

Wine, Barrel

Yeast 2120 Saccharomyces cerevisiae

fermentation

Yeast 2176 Saccharomyces cerevisiae BBL1 [wine, barrel

Yeast 2Ϊ77 Rhodotorula mucilaginosa BBL4 [wine, barrel

Yeast 2199 Saccharomyces cerevisiae #3 LWL J wine

iwine or

Yeast 2201 Saccharomyces cerevisiae #23 LWL

imust

Iwine or

Yeast 2205 Saccharomyces cerevisiae 1576

must

Yeast 2206 Saccharomyces cerevisiae LWL 2973 wine

Premier Cuvee commercial

Yeast 2212 Saccharomyces cerevisiae

LWL wine yeast

Yeast 22Ϊ Saccharomyces cerevisiae RH Phillips |wine

French White commercial

Yeast 2214 Saccharomyces cerevisiae

LWL wine yeast

Montrachet commercial

Yeast 2215 Saccharomyces cerevisiae

LWL wine yeast

Yeast 2216 Saccharomyces cerevisiae Simi white commercial

yeast

bayanus ;5005 dry wine yeast

commercial

Saccharomyces cerevisiae Fermicru VR5 dry wine yeast commercial

Fermirouge

Saccharomyces cerevisiae dry wine

5012

yeast commercial

Saccharomyces cerevisiae Fermivin 7013 dry wine yeast

Commercial

Yeast 2496 Saccharomyces cerevisiae Uvaferm CEG dry wine :

j Yeast i

Commercial Yeast 2497 Saccharomyces cerevisiae CY3079 dry wine yeast

Commercial j cerevisiae var.

Yeast 2498 ; Saccharomyces DV10 dry wine bayanus

Yeast

Commercial

Yeast 2499 Saccharomyces cerevisiae ICV-D254 dry wine yeast

Commercial

Yeast 2500 Saccharomyces cerevisiae Enoferm Ml dry wine

Yeast

Commercial

Enoferm Simi

Yeast (2501 Saccharomyces cerevisiae dry wine

White

Yeast

Commercial

Yeast 2502 Saccharomyces cerevisiae IT306 dry wine yeast

2 2 Brettanomyces/ Wine, South

I Yeast bruxellensis SA-2

Dekkera Africa

~_{e n A} iBrettanomyces/ !, Wine, South

Yeast 2504 ^! , , ^J jbruxellensis SA-3 i

iDekkera ι Africa

(Brettanomyces/ Wine, South Yeast bruxellensis SA-4 i

jDekkera Africa

Brettanomyces/ ;Wine, South

Yeast 2506 bruxellensis SA-5

Dekkera (Africa

2 Qj Brettanomyces/

Yeast bruxellensis 24/04A ^

IDekkera [Uruguay

Brettanomyces/ Wine,

Yeast 2508 „ , , ibruxellensis 24/04B 1

Dekkera | Uruguay

Brettanomyces/ L Wine.

Yeast 2509 , , Ibruxellensis 35/02C ^■

Dekkera (Uruguay wine,

Saccharomycod Cabernet Yeast 2510 ludwigii

es Sauvignon,

California

Brettanomyces / Wine, red,

Yeast 251 1 bruxellensis

Dekkera Virginia

Brettanomyces/ Wine,

Yeast 2513 „ , . ibruxellensis

Dekkera | Blueberry Yeast 2514 Bullera coprosmaensis P2E wine, Shiraz Malvasia j

Bianca,

Yeast 2518 Issatchenkia orientalis MB1

wine,

California

Malvasia bianca wine,

Yeast 2519 Pichia manshurica MB-3

N.

California

Malvasia bianca wine,

Yeast 2520 Acremonium strictum MB-4

N.

California commercial

Yeast 2521 Saccharomyces cerevisiae VRB dry wine yeast commercial ;

Yeast 2522 Saccharomyces cerevisiae Enoferm Ml dry wine yeast commercial

Yeast 2523 Saccharomyces cerevisiae Chardonnay dry wine yeast commercial

Yeast 2524 Saccharomyces cerevisiae CSM dry wine yeast commercial

Yeast 2525 Saccharomyces cerevisiae GRE dry wine yeast commercial

Yeast 2526 Saccharomyces cerevisiae 2323 dry wine yeast commercial

Yeast 2527 Saccharomyces cerevisiae W46 dry wine yeast commercial

Yeast 2528 Saccharomyces cerevisiae R-HST dry wine yeast commercial dry wine yeast commercial dry wine

^'yeast

commercial

Yeast 2531 Saccharomyces cerevisiae M05 dry wine yeast ⁽commercial

Yeast [Saccharomyces ^:cerevisiae Enoferm, QA23 ry wine

[yeast commercial

Yeast (2533 (Saccharomyces cerevisiae dry wine yeast commercial

Yeast 2534 Saccharomyces cerevisiae dry wine yeast commercial

Yeast 2535 Saccharomyces cerevisiae Syrah dry wine yeast commercial

Yeast 12536 Saccharomyces (cerevisiae WAM dry wine yeast commercial

Yeast (2537 Saccharomyces cerevisiae V1 1 16 dry wine yeast commercial

Yeast 2538 (Saccharomyces (cerevisiae BDX dry wine yeast commercial

Yeast 2539 Saccharomyces cerevisiae MT dry wine

(yeast commercial

Yeast 2540 Saccharomyces (cerevisiae BA1 1 dry wine

(yeast commercial

Yeast 2541 Saccharomyces cerevisiae 58W3 dry wine yeast commercial

Yeast 2542 Saccharomyces (cerevisiae BGY dry wine yeast commercial

Yeast 2543 Saccharomyces (cerevisiae SLO dry wine yeast commercial

Yeast [2544 Saccharomyces (cerevisiae dry wine yeast commercial

I Yeast 2545 Saccharomyces .cerevisiae L2226 dry wine yeast commercial

Yeast 12546 Saccharomyces cerevisiae NA33 ;dry wine

(yeast

Pennsylvani

California

California

[00108] Table 2: Selected yeast strains

Ba30 J grapes, vineyard,

Yeast 935 Saccharomyces cerevisiae

Lambrusco 1 Italy

grapes, vineyard,

Yeast 936 Saccharomyces cerevisiae Ba56

Italy

Ba69 I grapes, vineyard,

Yeast 937 Saccharomyces cerevisiae

Lambrusco Italy

Ba86 grapes, vineyard,

Yeast 938 Saccharomyces cerevisiae

Lambrusco Italy

_Ba99 (grapes, vineyard,

Yeast 939 Saccharomyces cerevisiae

Lambrusco Italy

grapes, vineyard,

Yeast 940 Saccharomyces cerevisiae Bal 11 Albana

Italy

Bail3 grapes, vineyard,

Yeast 941 Saccharomyces cerevisiae

Sangiovese Italy

Bal26 grapes, vineyard,

Yeast 942 Saccharomyces cerevisiae

Trebbiano j Italy

Bal27 grapes, vineyard, j

Yeast 943 Saccharomyces cerevisiae

Trebbiano Italy

Bal37 grapes, vineyard,

Yeast 944 Saccharomyces cerevisiae

Trebbiano Italy

Bal48 grapes, vineyard,

Yeast 945 Saccharomyces cerevisiae

Malvasia Italy

Bal50 grapes, vineyard,

Yeast 946 Saccharomyces cerevisiae

Monterosso Italy

Bal54 grapes, vineyard,

Yeast 947 Saccharomyces cerevisiae

Monterosso Italy

Bal 79 grapes, vineyard,

Yeast 948 Saccharomyces cerevisiae

Gutturnio j Italy

Bal 94 grapes, vineyard,

Yeast 949 Saccharomyces cerevisiae

Sangiovese Italy

Bal96 Uva ¹ grapes, vineyard,

Yeast 950 Saccharomyces cerevisiae

d'Oro Italy

Ba205 | grapes, vineyard,

Yeast 951 Saccharomyces cerevisiae

Sangiovese Italy

Ba 215 Uva grapes, vineyard,

Yeast 952 Saccharomyces cerevisiae

d'Oro Italy

Ba218 Uva grapes, vineyard,

Yeast 953 Saccharomyces cerevisiae

d'Oro Italy

Ba220 Uva grapes, vineyard,

Yeast 954 Saccharomyces cerevisiae

d'Oro Italy

Ba223 j grapes, vineyard,

Yeast 955 Saccharomyces cerevisiae

Scandiano Italy

Yeast 956 Saccharomyces cerevisiae Ba224 Pinot ^! grapes, vineyard,

[00109] In some embodiments a yeast strain, e.g., a yeast strain used in wine production, is ATCC 26249, ATCC 114, or NCYC numbers 3266, 3290, 33 14, 33 18, 33 19, 3445. 3469, 3470; T73, WE372, Y- 1 2649; Y-162; Y-2034; Y-241 1 ; Y-266; Y-269; Y-584; Y-71 1 5; Y- 865; UCD 2778; UCD 2780; UCD 932, ECl 1 18, or Y-162. In some embodiments a yeast strain, e.g., a yeast strain used in wine production, is AWRI 350, AWRI 796, AWRI 1503, AWRI FUSION (formerly 1502), AWRI R2, BP 725, Cru-Blanc, Elegance, EP2, Maurivin B, PDM, Primeur, Sauvignon, UCD522, or UOA MaxiThiol (all available from AB Mauri). In some embodiments a yeast strain, e.g., a yeast strain used in brewing, is Ale 514, Lager 497, or Weiss (all available from AB Mauri). In some embodiments a yeast strain, e.g., a yeast strain used in wine production, is NT 202, NT 50, NT 1 16 White, NT 1 16 Red, NT 1 12, NT 45, VIN 2000, VIN 13, VIN 7, WE 372, WE 14, N 96, 228 (all available from Anchor). In some embodiments a yeast strain, e.g., a yeast strain used in wine production, is an Enoferm®, Lalvin®, or Uvaferm® strain (all available from Lallemand). In some embodiments a yeast strain, e.g., a yeast strain used in wine production, is a Prise de Mousse strain such as S92. In some embodiments a Zymoflore® yeast strain or Actiflore® yeast strain (available from Laffort) may be used, e.g., in wine production. In some embodiments a yeast blend may be used. For example, an Anchor Alchemy I or II yeast blend may be used in wine production. In some embodiments a yeast strain, e.g., a yeast strain used in biofuel production, is Ethanol Red® (available from Lesaffre). In some embodiments a yeast strain, e.g., a yeast strain used in biofuel production, is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil or a derivative thereof such as JAY270 or JAY291 (Argueso JL, et al., Genome Res. (2009) 19(12):2258-70). In some embodiments a yeast strain, e.g., a yeast strain used in biofuel production, BG-1 , CAT-1, PE- 2, SA-l ,and VR-1 distributed initially by Lallemand Inc. and more recently by LNF Latino Americana Ltda. (http://www.lnf.com.br/). In some embodiments a yeast strain, e.g., a yeast strain used in biofuel production, is YE1358 or YE1615 (USSN 12/853,796). In some embodiments a yeast strain used in biofuel production has an amplification of the telomeric SNO and/or SNZ genes, which are involved in the biosynthesis of vitamins B6 (pyridoxine) and Bl (thiamin). It will be understood that strains derived from any of the strains disclosed herein may be used in various embodiments.

[00110] Bacterial cells of interest in various embodiments can be gram positive, gram negative, or acid-fast and can have various morphologies, e.g., spherical (cocci) or rod- shaped. They can be laboratory strains or isolated from nature. In some embodiments a bacterium is listed in Table A and/or Figure 14.

[00111] In some aspects, modulating [GAR+] acquisition (e.g., by using a [GAR+] modulator such as a [GAR+] inhibitor or inducer or by using a yeast strain that has impaired or enhanced [GAR+] acquisition) is used in an industrial biotransformation, industrial process, and/or or production of a product. In some aspects, an "industrial

biotransformation" refers to the intentional use of one or more microorganisms such as bacteria, fungi (e.g., yeast), or both to carry out a biochemical reaction or series of reactions to make one or more products useful to humans. In some embodiments a product is an end product to be used directly by humans, e.g., consumed, used as a medication, or used as fuel. In some embodiments a product is an intermediate that will be subjected to one or more further processing steps (e.g., one or more chemical reactions) and/or combined with one or more other substances to produce an end product to be used directly by humans. In some embodiments a product is packaged in a suitable container after production. For example, a beverage may be packaged in a bottle (e.g., wine) or can (e.g., beer). A food may be packaged in a bag, jar, box, etc. A pharmaceutical compound (therapeutic agent) may be packaged in a bottle, blister pack, vial, ampoule, etc. A fine or bulk chemical may be packaged in a bottle or jar, etc. The container may be labeled with or contain one or more labels with information such as the name of the product, amount, ingredients, etc.

[00112] In some embodiments an industrial process or industrial biotransformation refers to a process in which at least 0.1 liter, at least 1.0 liters, at least 10 liters, at least 100 liters, at least 1000 liters or more of the relevant product is produced. In some embodiments an industrial process or industrial biotransformation refers to a process in which at least 100 grams, at least 1 kilogram at least 10 kilograms, at least 100 kilograms, or at least 1,000 kilogram of the relevant product is produced. In some embodiments a product is one that is regulated by a government agency, e.g., as to one or more of the following: alcohol content, labeling, safety, efficacy, purity, transportation, sale, prescription, etc. In some embodiments a product is one that is traded in interstate or international commerce and/or of which had at least $1000, at least $10,000, at least $100,000, at least $1,000,000, at least $10,000,000, or more average annual sales in the United States averaged over the years 2000-2009, inclusive.

[00113] In some embodiments an industrial process or industrial biotransformation takes place outside a laboratory setting and/or is primarily performed for purposes of producing a product to be provided or sold to a consumer or to be used in further production, e.g., manufacturing, of a product to be sold to a consumer. In some embodiments a product is a beverage, e.g., a fermented beverage such as wine, beer, cider, sake, mead, or the like. In some embodiments a wine is a red wine. In some embodiments a wine is a white wine. In some embodiments a wine is a rose (a type of wine that incorporates some of the color from the grape skins, but not enough to qualify it as a red wine). In some embodiments a wine is a sparkling wine, such as champagne. A sparkling wine contains significant amounts of carbon dioxide (e.g., enough to give it a fizzy quality), which may be produced naturally from fermentation or added, e.g., by force-injecting, in some embodiments a product is produced at least in part in a winery or brewery.

[00114] In some embodiments a beverage (e.g., a wine or beer) or other product is entitled to a particular appellation. An appellation is a legally defined and protected geographical indication used to identify where the grapes for a wine were grown. Restrictions other than geographical boundaries, such as what grapes may be grown, maximum grape yields, alcohol level, and other quality factors, may also apply before an appellation name may legally appear on a wine bottle label. The rules that govern appellations are dependent on the country in which the wine was produced. In some embodiments a product, e.g., a beverage, e.g., a wine, bears a label indicating a particular appellation that applies to the wine. In some embodiments an appellation is defined by the French Institut National des Appellations d'Origine (INAO), now called the Institut national de l'origine et de la qualite (INAO). In some embodiments a wine is entitled to use the designation Appellation d'Origine Controlee (AOC) on its label. There are currently over 300 appellations acknowledged by the INAO.

[00115] In some embodiments a wine is a Bordeaux (produced in the Bordeaux region), Burgundy, Pinot (e.g., Pinot Noir, Pinot Grigio), Merlot, Syrah, Chardonnay, Chianti, Cabernet Sauvignon, Sauvignon blanc, Riesling, Muller Thurgau, Kerner, Sylvanor, Chenin blanc, or Semillon.

[00116] In some embodiments grapes, e.g., grapes used in winemaking, may be of the species Vitis vinifera. In some embodiments grapes, e.g., grapes used in winemaking, may be of other species or may be hybrids, created by the genetic crossing of two species, e.g., Vitis vinifera crossed with a different species, e.g., V. labrusca, V. aestivalis, V. ruprestris, V. rotimdifolia or V. riparia. In some embodiments grapes are red grapes. In some

embodiments red grapes are of any of the following varieties: Barbera, Bonarda, Cabernet franc, Cabernet sauvignon, Carnemere, Durif (also called Petit Syrah), Gamay, Grenache, Merlot, Mourvedre, Muscat, Peloursin, Pinotage, Pinot noir, Sangiovese, Shiraz (also called Syrah), Tannat, Tempranillo, Zinfandel (also called Primitivo). In some embodiments grapes are white grapes. In some embodiments white grapes are of any of the following varieties: Chardonnay, Chenin blanc, Colombard, Gewurztraminer, Pinot gris (also called Pinot grigio), Riesling, Sauvignon blanc, Semillon, Torrentes, Trebbiano,

Verdelho,Vermentino (also called Rolle), Viognier. In some embodiments grapes are purple or black grapes, e.g., Malbec, Mustadine.

[00117] In some embodiments, fermentation by [GAR+] yeast results in a product with a lower alcohol content than fermentation by isogenic [gar-] yeast (or [gar-] yeast that are isogenic except with respect to a gene that modulates [GAR+] acquisition) under the same conditions. In some embodiments the content is lower by at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 7.5%, 10%, or more (alcohol by volume; ABV). In some embodiments, inducing or enhancing [GAR+] results in a product with a lower alcohol content than would be the case in the absence of such inducing or enhancing. In some embodiments the content is lower by at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 7.5%, 10%, or more (alcohol by volume; ABV). In some embodiments the product is a beverage, e.g., wine or beer. In some embodiments a low alcohol wine has an alcohol content of below about 12% ABV, e.g., between 5% and 1 1% ABV. In some embodiments a low alcohol beer has an alcohol content of 0.05%-1.2% ABV.

[00118] In some embodiments, fermentation by [gar-] yeast results in a product with a higher alcohol content than fermentation by isogenic [GAR+] yeast (or [GAR+] yeast that are isogenic except with respect to a gene that modulates [GAR+] acquisition) under the same conditions. In some embodiments the content is higher by at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%), 4%, 4.5%), 5%, 7.5%», 10%, or more. In some embodiments, inhibiting or repressing [GAR+] results in a product with a higher alcohol content than would be the case in the absence of such inducing or enhancing. In some embodiments the content is higher by at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 7.5%, 10%, or more. In some embodiments the product is a biofuel, e.g., ethanol.

[00119] In some embodiments an industrial biotransformation, e.g., an industrial fermentation, has at least two stages. The stages may in some embodiments be distinguished by presence or addition of different yeast, bacteria, or combinations of yeast and bacteria in the different stages. In some embodiments [GAR+] is modulated during at least one of the stages.

[00120] In some embodiments an industrial process or industrial biotransformation takes place in a large container, e.g., a vat, fermenter, etc., having a capacity of at least 10 liters, at least 100 liters, at least 1000 liters, or more. The container may be equipped with instruments to, e.g., automatically monitor the process, remove product, add medium or medium components, etc. In some embodiments a container, or at least the inner walls of the container, is made of steel or wood. In some embodiments a fermentation is conducted in a closed container. In some embodiments a fermentation is conducted in an open container. In some embodiments a fermentation is conducted inside a wine bottle.

[00121] In some aspects, "industrial fermentation" refers to the intentional use of fermentation by one or more microorganisms such as bacteria, fungi (e.g., yeast), or both, to make one or more products useful to humans. Thus, an industrial fermentation is an example of an industrial biotransformation.

1001221 In some aspects, a fermentation is an ATP-generating process involving the oxidation of organic compounds, such as carbohydrates, using an organic compound, e.g., an endogenous organic compound, as an electron acceptor. Fermentation is important in anaerobic conditions, in which oxidative phosphorylation camiot take place to maintain the production of ATP (adenosine triphosphate). However, fermentation can be and often is carried out in an anaerobic environment. For example, yeast cells typically prefer fermentation to oxidative phosphorylation even in the presence of abundant oxygen, as long as sugars are readily available for consumption. Alcoholic fermentation is a fermentation in which carbon sources, e.g., sugars, are converted into ethanol and carbon dioxide. Sugars are the most common substrate of fermentation, and typical examples of fermentation products are ethanol, lactic acid, lactose, and hydrogen. Other compounds of interest that can be produced by fermentation include organic acids, such as butyric acid, and ketones such as acetone. In some embodiments a fermentation takes place over a period of between 6 and 12 hours, 12 and 24 hours, 24 hours to 3 days. In some embodiments a fermentation takes place over a period of between 1 and 20 days, e.g., between 3 and 5 days, between 5 and 10 days, between 10 and 15 days. In some embodiments a first period of fermentation may be followed by a second period of fermentation . In some embodiments a first period of fermentation may take place in aerobic conditions, and a second period of fermentation may take place under anaerobic conditions. In some embodiments a first period fermentation may be followed by a second period of fermentation, which may take place in the same container or a different container.

[00123] In some embodiments a [GAR+] modulator is used (e.g., is present in a composition or culture) at a concentration between about 1 pg/ml and about 10 mg/ml. In some embodiments a [GAR+] modulator is used at a concentration between about 1 ng/ml and about 1 mg/ml. In some embodiments a [GAR+] modulator is used at a concentration of at least about 10 ng/ml, 100 ng/ml, 1 microgram/ml, 10 micrograms/ml, or 100 micrograms per ml, up to about about 1 mg/ml or about 10 mg/ml. An optimum or suitable concentration for a particular use may be readily determined by, e.g., testing various concentrations or ranges for, e.g., their effect on [GAR+] acquisition, maintenance, or loss, or on a process in which yeast cells are used. [GAR+] may be modulated at any time before or during a fermentation. In some embodiments a [GAR+] modulator may be covalently or

noncovalently attached to a surface or matrix that is in contact with yeast. In some embodiments yeast may be immobilized to a surface or matrix. In some embodiments the matrix comprises particles such as beads. In some embodiments a surface is an inner wall or floor of a container in which yeast are cultured. In some embodiments a matrix comprises particles such as beads.

[00124] In some aspects, the invention provides isolated nucleic acids and vectors useful to delete or otherwise functionally inactivate a gene that affect [GAR+] acquisition, maintenance, or loss, e.g., a DRGA or DEGA gene (see Tables B and C for examples). Sequences of the DRGA and/or DEGA genes or other genes mentioned herein may be found in publicly available databases such as those available at the NCBI, e.g., Gene, Protein, Nucleotide, RefSeq. In some embodiments a RefSeq sequence is used. Polymorphic variants, e.g., variants that exist among a population, are encompassed in certain

embodiments.

[00125] In some embodiments an isolated nucleic acid is in a vector used in the art in genetic engineering of a fungus, e.g., a yeast, e.g., a budding yeast. In some embodiments the vector is a plasmid. Other vectors include artificial chromosomes and linear nucleic acid molecules that are distinct from linearized plasmids. In some embodiments the vector is an integrating vector. In some embodiments the vector comprises an expression control element operably linked to a nucleic acid to be transcribed (e.g., a nucleic acid that encodes a polypeptide of the invention or that provides a template for transcription of a dsRNA). Three well known plasmid systems used for recombinant expression and replication in yeast cells include integrative plasmids, low-copy-number ARS-CEN plasmids, and high-copy- number 2μ plasmids. See, e.g., Christianson TW, et al., "Multifunctional yeast high-copy-number shuttle vectors". Gene. 1 10: 1 19-22 (1992); Sikorski, "Extrachromosomal cloning vectors of Saccharomyces cerevisiae", in Plasmid, A Practical Approach, Ed. K. G. Hardy, IRL Press, 1993; Parent, S.A., and Bostian, K.A., Recombinant DNA technology: yeast vectors, p. 121 - 178. In Wheals, A.E., et al. (eds.) The yeasts, vol. 6. Yeast genetics. Academic Press, Longon, UK (1995). An example of integrating plasmids of use in budding yeast are YIp plasmids, which are maintained at one copy per haploid genome and inherited in Mendelian fashion. Such a plasmid, containing a nucleic acid of interest, a bacterial origin of replication and a selectable gene (typically an antibiotic-resistance marker), is typically produced in bacteria. The purified vector may be linearized and used to transform competent yeast cells. YCp plasmids, which contain the autonomous replicating sequence (ARSl) and a centromeric sequence (CEN4), are examples of low-copy-number ARS-CEN plasmids. These plasmids are usually present at 1 -2 copies per cell. An example of the high-copy-number 2μ plasmids are YEp plasmids, which contain a sequence approximately 1 kb in length (named the 2μ sequence). The 2μ sequence acts as a yeast replicon giving rise to higher plasmid copy number. These plasmids may require selection for maintenance.

[00126] In some embodiments, an integrating plasmid is a pRS plasmid (e.g., pRS303, pRS304, pRS305 or pRS306 or other integrative plasmids). In some embodiments, the plasmid is an extrachromosomal plasmid (e.g., pRS313, pRS314, pRS315, pRS316, pRS413, pRS414, pRS415, pRS416, pRS423, pRS424, pRS425, pRS426). In some embodiments the plasmid is a member of the YES™ Vector Collection, e.g., pYES (Invitrogen, Carlsbad, CA). In some embodiments, the plasmid is a Gateway plasmid. See, e.g., Geiser JR. Recombinational cloning vectors for regulated expression in Saccharomyces cerevisiae. Biotechniques, 38:378-382 (2005); Van Mullem V, et al., Construction of a set of

Saccharomyces cerevisiae vectors designed for recombinational cloning. Yeast. 20:739-46 (2003); Alberti, S., et al., A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast, 24(10):913-9 (2007).

[00127] A nucleic acid may be introduced into a cell, e.g., a yeast cell, using any suitable method. Yeast cells are often transformed by chemical methods (e.g., as described by Rose et al, 1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The cells are typically treated with lithium acetate to achieve transformation efficiencies of approximately 10⁴ colony- forming units (transformed cells)^g of DNA. In some embodiments, yeast perform homologous recombination such that the cut, selectable marker recombines with the mutated (usually a point mutation or a small deletion) host gene to restore function. Transformed cells are then isolated on selective media. Of course, any suitable means of introducing nucleic acids into yeast cells can be used, such as

electroporation. See, e.g., Gietz, R.D. and Woods, R.A., Genetic transformation of yeast. BioTechniques, 30:816-820; 822-826, 828 (2001). Many yeast vectors (e.g., plasmids) typically contain a yeast origin of replication, an antibiotic resistance gene, a bacterial origin of replication (for propagation in bacterial cells), multiple cloning sites, and a yeast nutritional marker gene to promote maintenance and/or genomic integration in yeast cells. The yeast nutritional gene (or "auxotrophic marker") is often one of the following: 1) TRP1 (Phosphoribosylanthranilate isomerase); 2) URA3 (Orotidine-5 '-phosphate decarboxylase); 3) LEU2 (3-Isopropylmalate dehydrogenase); 4) HIS3 (Imidazoleglycerolphosphate dehydratase or IGP dehydratase); or 5) LYS2 (a-aminoadipate-semialdehyde

dehydrogenase). An antibiotic resistance gene can facilitate maintenance and propagation of the plasmid in bacteria and/or to identify yeast transformants and/or promote maintenance of the plasmid in yeast. Exemplary antibiotic resistance markers include the kanamycin (G418) resistance gene, chloramphenicol resistance gene, and hygromycin resistance gene. See, e.g., U.S. Pat. No. 6,214,577. A number of other selectable markers of use in yeast are known. See, e.g., U.S. Pat. No. 4,626,505. The AR04-OFP and FZF1 -4 genes, which confer p- fluoro-DL-phenylalanine resistance and sulfite resistance, respectively, may also be used as dominant selectable markers, e.g., in laboratory and wine yeast S. cerevisiae strains

(Cebollero, E. and Gonzalez, R. Applied and Environmental Microbiology, 70 (12): 7018- 7023, 2004). One of ordinary skill in the art will select an appropriate marker based on considerations such as whether the yeast is auxotrophic or prototrophic, convenience, and the particular application.

[00128] In some embodiments a yeast vector contains one or more expression control sequences, e.g., promoter sequences. As known in the art, a "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and transcription factors, to initiate the transcription of a nucleic acid sequence. The phrase "operably linked" indicates that an expression control element, e.g., a promoter, is in an appropriate location and/or orientation in relation to a nucleic acid to control transcriptional initiation and/or expression of the nucleic acid. A promoter may be one that is naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment. Alternatively, a promoter may be a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid segment in its natural environment. Such promoters may include promoters of other genes and promoters that are not naturally occurring. An expression control element may be derived from a yeast of the species or strain in which an operably linked nucleic acid is to be expressed. For example, if a nucleic acid is to be expressed in S. cerevesiae, an S. cerevesiae promoter may be used to direct expression of a dsRNA. However, any expression control element capable of directing transcription in the cell of interest may be used.

[00129] In some embodiments a constitutive promoter is used. In some embodiments a regulatable, e.g., inducible, promoter is used. Examples of inducible yeast promoters include GAL1- 10, GAL1, GALL, GALS, TET, CUP1 , VP 16 and VP16-ER. Examples of repressible yeast promoters include Met25. Examples of constitutive yeast promoters include glyceraldehyde 3 -phosphate dehydrogenase promoter (GPD), phosphoglycerate kinase (PGK), alcohol dehydrogenase promoter (ADH), translation-elongation factor- 1 -alpha promoter (TEF), cytochrome c-oxidase promoter (CYC1), and MRP7. Promoters containing steroid response elements (e.g., glucocorticoid response element) inducible by glucocorticoid or other steroid hormones can also direct expression in yeast. Yet other yeast constitutive or inducible promoters, such as those of the genes for alpha factor, phosphate pathway genes (e.g., PH05), or alcohol oxidase may be used. In some embodiments a vector comprises an expression control element known as an upstream activating sequence (UAS). Such elements, which are considered functional equivalents of metazoan enhancers, can activate gene transcription from remote positions, e.g., up to about 1,000 - 1,200 bp from the promoter. See, e.g., Petrascheck, M., et al., Nucleic Acids Res., 33(12): 3743-3750, 2005, for discussion. The level of expression achieved using an inducible promoter can be regulated, e.g., by controlling the amount of inducing agent or the length of exposure. Further, mutant promoters that result in lower expression levels than a wild type promoter can be used. In some embodiments, an expression control element originates from a species in which the expression control element is to be used to direct expression while in other embodiments the expression control element originates from a different species.

[00130] In some aspects, the invention provides vectors suitable for mutating, e.g., at least in part deleting or creating an insertion in a DEGA or DRGA gene of a yeast. In some embodiments such mutation renders the gene or encoded polypeptide non- functional.

[00131] In some embodiments a vector includes a cloning site for insertion of a nucleic acid of interest (e.g., a nucleic acid to be used to functionally inactivate a DEGA or DRGA gene. In general, any restriction enzyme site may serve this purpose. Certain embodiments include a multiple cloning site, or polylinker. In some embodiments, the cloning site is positioned so that an inserted nucleic acid is operably linked to expression control element(s), e.g., a promoter, already present in the vector. In other embodiments, a nucleic acid cassette comprising one or more expression control elements and a nucleic acid to be transcribed is inserted into a vector. The vector or nucleic acid cassette may further comprise a

transcriptional terminator (e.g., the yeast CYC1 terminator).

[00132] In some embodiments a nucleic acid, nucleic acid cassette, or vector comprises a portion that encodes a reporter protein or tag. The reporter protein or tag may be useful for, e.g., enhancing expression, detection, and/or purification of a polypeptide. For example,a tag can be an affinity tag (e.g., HA, TAP, Myc, His, Flag, GST), solubility-enhancing and/or expression-enhancing tag (e.g., a SUMO tag, NUS A tag, SNUT tag, or a monomeric mutant of the Ocr protein of bacteriophage T7). See, e.g., Esposito D and Chatterjee DK. Curr Opin Biotechnol.; 17(4):353-8 (2006). A tag is often relatively small, e.g., ranging from a few amino acids up to about 100 amino acids long. In some embodiments a reporter comprises a fluorescent protein (e.g., GFP, CFP, or related proteins (including enhanced versions such as EGFP, ECFP, EYFP), Cerulean, DsRed, mCherry, mTomato), a luciferase (e.g., Renilla or Gaussia or Metridia luciferase or similar proteins). In some embodiments a reporter or tag is more than 100 amino acids long, e.g., up to about 500 amino acids long. In some embodiments a reporter tag is located at the N-terminus or C-terminus of a polypeptide. A polypeptide may comprise multiple tags. [00133] In some aspects, the invention provides a kit comprising any one or more of the following: (1) one or more naturally occurring or genetically engineered yeast strains that have altered acquisition, induction, maintenance, or loss of [GAR+]; (2) one or more isolated nucleic acids, vectors, or RNAi agents useful for generating a yeast strain that has altered induction of [GAR+], e.g., that has a functionally inactivated "Deletions that Enhance GAR Acquisition" (DEGA) genes or a functionally inactivated "Deletions that Reduce GAR Acquisition" (DRGA) genes; (3) a bacterium that induces [GAR+]; (4) a small molecule that induces or inhibits [GAR+]; (5) a primer, probe, or reporter molecule useful for testing for [GAR+] cells or useful for testing for [GAR+] induction or for a [GAR+] inducer. In some aspects, a kit comprises yeast that have been tested to determine whether they are [GAR+] or [gar-}. In some embodiments the kit bears a label indicating that the yeast are [GAR+] or indicating that the yeast are [gar-] or is associated with information indicating that the yeast are [GAR+] or indicating that the yeast are [gar-]. In some embodiments a kit comprises or is associated with instructions for use of the kit or component(s) thereof for one or more purposes or in one or more methods described herein. For example, the kit may comprise instructions for (i) using the yeast in an industrial process, e.g., to produce a product, e.g., a beverage (e.g., wine), biofuel, small molecule, or fine chemical; (ii) inducing or inhibiting [GAR+]; (iii) generating a yeast strain that has altered induction of [GAR+]; (iv) testing for [GAR+]; or (v) any combination of the foregoing. In some embodiments a kit is associated with instructions or information if the instructions or information are posted on a website together with or reachable via a link from a name or description of the kit or its catalog number.

[00134] In some embodiments a kit comprises one or more items useful for control purposes, e.g., a control plasmid, control primer(s).

[00135] Components of a kit can be packaged together in a single container or may be provided in multiple containers. A composition may be provided in concentrated form (e.g., as a 5X, 10X, 50X concentrate), which can be diluted to IX to provide a suitable

concentration for the intended use. In some embodiments, two or more individual kits (which may be packaged together in a single larger container) are provided.

[00136] Any gene of interest can be overexpressed or functionally inactivated in various embodiments of the invention, provided that in at least some embodiments doing so is not lethal to a cell. Overexpression or functionally inactivating a gene may be useful to improve production of a product by a yeast or may enable the use of nutrients or other starting materials that could not otherwise by productively utilized by the yeast. The gene can be an endogenous gene or a non-endogenous gene. In some embodiments a gene encodes a protein. In some embodiments a gene encodes a RNA or protein of unknown function. In some embodiments a gene encodes a protein that has at least one known function. In some embodiments the protein is an enzyme. In some embodiments the enzyme is of any of the following classes as classified in accordance with the International Union of Biochemistry and Molecular Biology nomenclature for enzymes: EC 1 Oxidoreductases: catalyze oxidation/reduction reactions; EC 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group); EC 3 Hydrolases: catalyze the hydrolysis of various bonds; EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation; EC 5 Isomerases:

catalyze isomerization changes within a single molecule; EC 6 Ligases: join two molecules with covalent bonds. According to this nomenclature, each enzyme is described by a sequence of four numbers preceded by "EC", in which the first number broadly classifies the enzyme based on its mechanism. In some embodiments the enzyme participates in a biosynthetic pathway that produces a product of use to humans. RNAi enables a constitutive or inducible knock-down system that provides an alternative to existing technologies for generating yeast with reduced or absent expression, such as technologies that involve genetically altering a gene, e.g., by disrupting or at least in part deleting the gene. RNAi may also be used together with such technologies for any purpose herein.

[00137] In some aspects, methods of identifying agents that modulate prions, e.g.,

[GAR+], are provided. Various types of agents may be screened, identified, or evaluated using the methods described herein, such as small organic molecules, inorganic molecules, nucleic acids, polypeptides, and peptidomimetics (e.g., peptoids). Small organic molecules typically have a molecular weight in the range of 50 daltons to 3,000 daltons. These compounds often contain multiple carbon-carbon bonds and can comprise functional groups important for structural interaction with proteins (e.g., hydrogen bonding), and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two of the functional chemical groups. These compounds often comprise one or more cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups. Compounds may comprise nucleotides, amino acids, sugars, fatty acids, and derivatives or structural analogs thereof. Nucleotides and amino acids may be standard or non-standard. If non-standard, they may be naturally occurring or non-naturally occurring (i.e., not found in nature). Similarly, nucleic acids and polypeptides may comprise standard or non-standard nucleotides and amino acids, respectively, and may have non-standard inter-subunit linkages. [00138] Compounds can be members of, e.g., chemical libraries, natural product libraries, combinatorial libraries, etc. Chemical libraries can comprise diverse chemical structures, some of which may be known compounds, analogs of known compounds, or analogs or compounds that have been identified as "hits" or "leads" in other drug discovery screens, while others are derived from natural products, and still others arise from non-directed synthetic organic chemistry. Compounds from chemical libraries are often arrayed in mult- well plates (e.g., 96- or 384-well plates). Natural product libraries can be prepared from collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by, e.g.: (1) fermentation and extraction of broths from soil, plant or marine microorganisms, or (2) extraction of plants or marine organisms. Compound libraries are commercially available from a number of companies. In addition, various government and non-profit research institution have compound libraries that are available to the scientific community. For example, the Molecular Libraries Small Molecule Repository (MLSMR), a component of the National Institutes of Health (NIH) Molecular Libraries Program is designed to identify, acquire, maintain, and distribute a collection of >300,000 chemically diverse compounds with known and unknown biological activities for use, e.g., in high-throughput screening (HTS) assays (see https://mli.nih.gov/mli/).

[00139] In some embodiments, methods that involve contacting a cell, e.g., a fungal cell, with an agent are optionally carried out in cells bearing mutations in or deletions of the one or more genes that affects membrane efflux pumps and/or that alters permeability for drugs, so as to reduce efflux and/or increase permeability. For example, in some embodiments, methods that involve contacting a yeast cell with an agent are optionally carried out in yeast strains bearing mutations in or deletions of the ERG6 gene, the PDR1 gene, the PDR3 gene, the PDR5 gene, the SNQ2 gene, and/or any other gene which affects membrane efflux pumps and/or alters permeability for drugs, so as to reduce efflux and/or increase permeability.

[00140] Budding yeast are used to produce a wide variety of compounds of interest. For example, various strains of S. cerevesiae or strains whose genome is at least in part derived from S. cerevesiae are used extensively in fermentative production processes. In addition to S. cerevesiae, industrially important yeast include S. pastorianus, and Kluyveromyces lactis. See, e.g., Satyanarayana, T. and Kunze, G. (eds.) Yeast biotechnology: diversity and applications; Springer, 2009, and references therein. In some embodiments of the invention, RNAi is used in metabolic engineering of yeast, e.g., budding yeast, e.g., industrially important budding yeast, to improve cellular activities by manipulating, e.g., enzymatic, transport, and/or regulatory functions with the use of recombinant nucleic acid (e.g., recombinant DNA) technology. Metabolic engineering can result in a product with improved quality, or result in time and/or cost savings, etc. See, e.g., Nevoigt, E., Microbiology and Molecular Biology Reviews, 72(3): 379-412 (2008) and references therein, all of which are incorporated herein by reference.) "Cellular activities" can comprise product formation or cell properties such as stress tolerance (e.g., tolerance to extremes of temperature (e.g., heat stress), osmotic stress, oxidative stress, pH, intracellular or extracellular accumulation of a product), or ability to utilize particular nutrients or substrates.

[00141] In some embodiments, prion modulation is used together with existing techniques useful for metabolic engineering, such as global transcription machinery engineering (see, e.g., PCT/US2006/037597, published as WO/2007/038564).

[00142] In some embodiments, prion modulation is used together with RNAi in production of a product of interest or to metabolize (e.g., break down, degrade) a product of interest. In some embodiments prion modulation is used in an industrially important yeast, e.g., a yeast species or strain that is used to produce a product of interest sold or traded in interstate commerce in the U.S. or internationally. In some embodiments prion modulation is used in a yeast species or strain that has been given GRAS (generally recognized as safe) status by the FDA.

[00143] S. cerevesiae and various other yeasts are used extensively in the baking, wine, and brewing industries, in the production of products of interest such as biofuels (e.g., ethanol), fine and bulk chemicals such as glycerol, propanediol, organic acids, sugar alcohols, L-G3P, ergosterol and other steroids, and isoprenoids, to name a few. In some embodiments, prion modulation is used to improve the production of a food, nutritional supplement, beverage, or component thereof. In some embodiments prion modulation is used in a baker's, wine, brewer's, sake, or distiller's yeast, e.g., S. cerevesiae or S. pastorianus. In some embodiments prion modulation is used in a yeast species or strain that has been given GRAS (generally recognized as safe) status by the FDA. In some embodiments prion modulation is used in a yeast that has been genetically engineered to improve one or more cellular activities by deleting, mutating, or expressing (e.g., overexpressing) a gene. For example, the yeast may express one or more heterologous gene(s) from a different yeast or other fungus, from bacteria, or from a non-fungal eukaryote. For example, Saccharomyces yeasts have been genetically engineered to ferment pentose(s), e.g., xylose, one of the major fermentable sugars present in cellulosic biomasses, so that ethanol can be efficiently produced from such feedstocks. In some embodiments a yeast is Dekkera bruxelle is. [00144] In some embodiments, the yeast is of the genus Kluveromyces. For example, Kluveromyces lactis and Kluyveromyces marxianus are of use in a variety of biotechnological processes. In some embodiments, the yeast has increased tolerance to an environmental condition, e.g., heat, cold, osmolarity (e.g., salt concentration) relative to S. cerevesiae. In some embodiments, the yeast is of the genus Debaryomyces, e.g., Debaryomyces hansenii, which is a cryotolerant, marine yeast that can tolerate salinity levels up to 24%. Cryo- and osmotolerance account for its important role in several agro-food processes. D. hansenii is common in cheeses (wherein it provides proteolytic and lipolytic activities during cheese ripening) and is also found in dairies and in brine because it is able to grow in the presence of salt at low temperature and to metabolize lactic and citric acids.

[00145] In some embodiments a strain of yeast that can reduce the acidity of a culture medium such as grape must may be used, e.g., a yeast that can convert L-malate to L-lactate during alcohol fermentation. For example, the yeast may be Saccharomyces cerevisiae strain ML01 , which is derived from parental strain S92 and carries a gene encoding malolactic enzyme (mleA) from Oenococcus oeni and a gene encoding malate permease (mael) from Schizosaccharomyce pombe (Husnik, JI, et al., Am. J. Enol. Vitic. (2007) 58: 1, pp. 42 - 52). In some embodiments yeast strains derived from other parental strains, e.g., other strains disclosed herein or known in the art may be used. Such strains may harbor the same genes or homologs thereof or genes encoding proteins having the same or similar function.

[00146] In some embodiments, the product of interest is a recombinant protein.

Exemplary proteins that can be produced in yeast are antibodies, vaccine components, interferons, and insulin. In some embodiments, the product of interest is a pharmaceutical agent, which may be a recombinant protein or a non-protein biomolecule. In some embodiments the product of interest is a small organic molecule. In some embodiments the product of interest is a precursor that may be subsequently used in a process that may, but need not, involve yeast.

[00147] In some embodiments, the product of interest is a biofuel. Biofuel is defined as solid, liquid or gaseous fuel obtained from relatively recently lifeless or living biological material and is different from fossil fuels, which are derived from long dead biological material. In some embodiments the biofuel is an alcohol. In some embodiments, the biofuel is a bio-oil. Ethanol is an exemplary biofuel. S. cerevesiae has traditionally been used for ethanol production (Nevoit, supra). Approaches for improving yeast bioethanol production can include, e.g., (i) efforts to improve processes that use starch or sugar as a starting material; (ii) efforts to improve processes that use lignocellulosic biomass substrate, and/or (iii) efforts to improve sugar-to-ethanol conversion efficiency and/or yeast ethanol tolerance. In some embodiments, RNAi is used in yeast to silence genes whose silencing improves ethanol tolerance, increases ethanol yield, and/or allows the use of a broader range of substrates for ethanol production. For example, deregulating glucose repression of galactose utilization can improve galactose utilization in the production of ethanol. Simultaneous deletion of GAL6, GAL80, and MIG1 was shown to result in an increase in specific galactose uptake rate (Ostergaard, S., et al., Nat. Biotechnol, 18: 1283-1286, 2000). The present invention envisions silencing these genes by RNAi. In some embodiments, RNAi is used to improve ethanol production in a yeast that naturally utilizes pentoses, e.g., xylose, such as P. stipitis.

[00148] In some embodiments, a product of interest is a lipid. In some embodiments the yeast is an oleaginous yeast. In some embodiments the yeast is a Yarrowia. Yarrowia lipolytica is an exemplary yeast that has developed efficient mechanisms for breaking down and using hydrophobic substrates. It has an ability to accumulate large amounts of lipids and has a variety of biotechnologieal applications.

[00149] In some embodiments, a yeast is used to remediate waste or in environmental cleanup. For example a yeast may be used to degrade oil after an oil spill or otherwise decontaminate areas that have accumulation of undesired substances, e.g., pollutants, that can be metabolized by the yeast.

[00150] In some embodiments, prion modulation is used in combination with modulation of a gene that affects tolerance to a metabolite or toxin. In some embodiments the metabolite is ethanol. In some embodiments the metabolite is a byproduct of a metabolic reaction useful to produce a product of interest. In some embodiments, the toxin is a molecule produced by a species that exists in a culture with a fungal species or strain of interest, wherein the toxin exerts deleterious effects on the fungal species or strain.

[00151] A gene whose modulation affects prion acquisition, maintenance, or loss can be mutated or deleted using standard genetic engineering approaches (in a strain for which such approaches are available), or a screen can be performed to identify a strain having a mutant allele of the gene. The resulting mutant can be used, e.g., to produce a product of interest. This approach may be of use in situations where it is desired to utilize a non-genetically engineered yeast.

[00152] In some embodiments RNAi is used to modulate a prion or to modulate a gene whose modulation is useful in combination with a prion-based method of the present invention. In some embodiments, the cell has a functional endogenous RNAi pathway. In some embodiments the cell lacks a functional endogenous RNAi pathway and is engineered to have a functional RNAi pathway. For example, in some embodiments the cell lacks a functional Dicer protein, a functional Argonaute protein, or both. The cell is engineered to express at least a portion of the RNAi pathway protein(s) that the cell lacks, such that the resulting cell has a functional RNAi pathway. Standard vectors and methods used in the art for introducing genetic constructs into cells can be used to introduce a nucleic acid encoding at least a portion of an RNAi pathway protein into a cell. RNAi in budding yeast is described, e.g., in PCT/US2010/002469 (WO/2011/031319).

[00153] In some aspects, the invention provides a fungal strain that is selected or genetically engineered to maintain a stable [GAR+] phenotype. In some embodiments, such a strain exhibits less variability over time, e g., it may have improved maintenance of its ability to produce a product of interest over time, relative to a comparable fungal strain that has not been so selected or engineered, e.g., an otherwise isogenic fungal strain. In some embodiments, this aspect may allow the use of certain species or strains in one or more processes, e.g., one or more industrial processes, for which use they would otherwise be less well suited or unsuitable as a result of reversion to a [gar-] phenotype. The invention encompasses use of prion stabilization to stabilize a fungal strain or fungal culture, e.g., to inhibit the strain or culture from changing one or more properties of interest over time.

[00154] Once a gene that modulates a prion of interest is identified in a first species, e.g., a yeast species, e.g., S. cerevesiae, homologs of the gene can be identified in one or more second species, e.g., another eukaryote (e.g., other fungi, e.g., other yeast species). Publicly available databases can be searched using at least a portion of a DN A, RNA, or protein sequence and homologous sequences identified. In some embodiments, manipulating such genes or their encoded gene products can be used to modulate the corresponding prion in the second species.

|001 5] In certain embodiments, the invention provides a method of screening for agents that modulate prion acquisition, induction, maintenance, or loss. Certain of the methods comprise: (a) contacting a yeast cell or yeast culture with an agent; (b) assessing the yeast for prion acquisition, induction, maintenance, or loss; and (c) identifying the test agent as an agent that modulates prion acquisition, induction, maintenance, or loss if prion acquisition, induction, maintenance, or loss is altered as compared with a yeast cell or yeast culture that has not been contacted with the test agent. An agent identified in a screen may be used to modulate acquisition, induction, maintenance, or loss or the relevant prion by contacting yeast cells with the agent, e.g., by adding the agent to culture medium prior to or after inoculating the culture medium with yeast. In some embodiments the prion is [GAR+] .

[00156] Compounds disclosed herein can exist as their corresponding salt, ester, or prodrug. As used herein, the term "salts" refers to salts or zwitterionic forms of the compounds disclosed herein. Salts of such compounds can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation. Suitable cations include alkali metal (e.g., sodium or potassium) and alkaline earth metal (e.g., calcium or magnesium) cations. In addition, salts of the compounds that contain a basic center are acid addition salts formed with acceptable acids. Examples of acids which can be employed include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, malonic, lactic and citric. Nonlimiting examples of salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, glycerolphosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, malonate, fumarate, maleate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate. oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, glutamate, bicarbonate, undecanoate, lactate, citrate, tartrate, gluconate, benzene sulphonate, and p- toluenesulphonate salts. In addition, available amino groups present in the compounds can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. A reference to compounds appearing herein is intended to include compounds disclosed herein as well as acceptable salts, solvates (e.g., hydrates), esters, or prodrugs thereof. Acceptable salts may be prepared using procedures that are familiar to those of skill in the art. "Prodrug" in the context of the present disclosure refers to a compound that can be converted in yeast or in yeast culture medium to a compound disclosed herein. In some aspects, "acceptable salts, solvates (e.g., hydrates), esters, or prodrugs" refers to salts, solvates, esters, or prodrugs that are sufficiently non-toxic to yeast that they can reasonably be used for purposes described herein. In some

embodiments such compounds are sufficiently non-toxic to mammals, e.g., humans, that they can reasonably be used in a method described herein that comprises producing a product to be consumed or used by mammals, e.g., humans. Where compounds described herein may exist in particular geometric or stereoisomeric forms, all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, are considered to fall within the scope of the disclosure. Certain embodiments may be directed to any particular isomer or mixture

[00157] It should be noted that [GAR+] is exemplified herein, but embodiments of the invention may be applied in the context of any prion of interest, such embodiments being within the scope of the present disclosure. Exemplary prions are described in, e.g.,

Halfmann, R, et al.,(2012) (cited below), Alberti S, et al. (2009) (cited below).

Examples

Example 1: Identification and Characterization of [GAR+]

[00158 J Identification of [GAR+]

[00159] We searched the literature for Saccharomyces cerevisiae phenotypes with prion- like inheritance patterns. One was described many years ago in a screen for cells with an alteration in carbon source utilization (Ball et al. 1976). The basis of the screen was the extreme preference of S. cerevisiae for glucose as a carbon source. In glucose media, cells repress genes necessary to process other carbon sources such as glycerol (Santangelo 2006). Glucosamine, a nonmetabolizable glucose mimetic, induces a similar repression. Therefore, yeast cells cannot use glycerol as a carbon source if even small amounts of glucosamine are present (Hockney and Freeman 1980; Nevado and Heredia 1996). Some cells spontaneously acquire the ability to use glycerol in the presence of glucosamine, presumably due to defects in glucose repression. Some of these exhibit dominant, non-Mendelian inheritance (Ball et al. 1976). Further, the phenotype is neither carried by the mitochondrial genome nor by a plasmid (Kunz and Ball 1977). Employing a variety of methods, this Example shows that this factor, which we named [GAR+] (for "resistant to glucose-associated repression," with capital letters indicating dominance and brackets indicating its non-Mendelian character), exhibits all of the genetic characteristics of a yeast prion, and describes use of biochemical and genetic methods to identify certain proteins that play important roles in [GAR+] inheritance.

[00160] [GAR+] shows non-Mendelian, infectious inheritance

[00161] We obtained cells able to use glycerol as a carbon source despite the presence of glucosamine, as did Ball and colleagues (Ball et al. 1976; Kunz and Ball 1977), by selecting for cells that could grow in 2% glycerol in the presence of 0.05% glucosamine. Colonies appeared at a frequency of approximately five in 10⁴ cells in the W303 genetic background, well above the predicted mutational frequency (Fig. 23). Some recessive mutations allow growth on glycerol in the presence of glucosamine (see Table SI ; Ball et al. 1976), but the novel phenotypes described by Ball and colleagues (Ball et al. 1976; Kunz and Ball 1977) were dominant. Therefore, we first crossed our cells to wild-type cells. All diploids exhibited an unstable semidominant phenotype (Fig. 1 A). Specifically, a mixed population was produced in which some diploids showed "strong" phenotypes (large colonies) and others showed "weak" phenotypes (small colonies) (Fig. 24A). Cells with weak phenotypes invariably converted to strong over ~25 generations (data not shown). Notably, both mammalian and fungal prions exhibit "strong" and "weak" strains (Aguzzi et al. 2007).

[00162] In yeast, chromosomally inherited traits show 2:2 segregation following meiosis. Both strong and weak [GAR+] phenotypes, however, exhibited non-Mendelian 4:0

[GAR+]:[gar^~] non-Mendelian segregation (Fig. IB). That is, all meiotic progeny exhibited a capacity to grow on glucose in the presence of glucosamine. Spores produced from cells with weak phenotypes generally converted to strong phenotypes (Fig. 24B, bottom). We named the responsible genetic element responsible for this trait [GAR+].

[00163] To determine whether [GAR i J is transmissible by cytoduction (that is,

"infectious"), we used a mutant defective in nuclear fusion (karl-1). During mating, karl cells fuse but nuclei do not (Conde and Fink 1976). Selecting for a particular nucleus and cytoplasm of interest after mating accomplishes cytoplasmic exchange without the transfer of nuclear material. We mated a [GAR+] strain carrying the nuclear markers URA3⁺ hi$3^~ and the cytoplasmic marker p⁺ to a karl-1 [gar^~] strain that was ura3^~ HIS3⁺ and p°. We then selected for cells containing the nucleus originally associated with [gar^~] cells and the cytoplasm originally associated with [GAR+] cells. All 10 strains tested were [GAR+] (Fig. 1C). Thus, [GAR+] exhibits an "infectious," nonnuclear pattern of inheritance.

[00164] [GAR+] appears at high frequency in a variety of genetic backgrounds.

[00165] We next asked whether [GAR+] was an oddity of specific strains or could appear in diverse genotypes. Cells able to use glycerol in the presence of glucosamine appeared at a frequency of approximately nine in 10⁵ cells in the BY background, approximately one in 10⁴ cells in 74D, approximately five in 10⁴ cells in W303, and approximately seven in 10⁴ cells in Sigma. In the SKI background, [GAR+] appeared at the astonishingly high rate of approximately one in 10 cells (Fig. ID). In comparison, the frequency of heritable phenotypic change due to genetic mutation is generally approximately one in 10⁶ haploid cells (Ohnishi et al. 2004).

[00166] We tested dozens of variants from each background for dominance. All exhibited the semidominant pattern observed in W303 (Fig. IB; data not shown). [GAR+] cells of the 74D background did not sporulate, preventing us from testing segregation pattern. In W303 and W303/BY hybrids, [GAR+] only delayed sporulation (data not shown). In every tetrad tested from these backgrounds (>25 of each genotype), [GAR+] showed 4:0 [GAR+]:[gar^~] segregation (Fig. IB; data not shown). Together, these data establish that yeast strains of diverse genetic backgrounds commonly switch carbon utilization strategies in a heritable way by acquiring a non-Mendelian element of inheritance.

[00167] [GL R+] is curable by transient changes in chaperone protein levels

|00168] The inheritance o prions is based on self-perpetuating changes in protein conformations. In contrast to other non-Mendelian elements, a hallmark of prion phenotypes is the ability of transient changes in the expression of chaperones to cause a heritable loss of the phenotype. Other yeast prions, as well as 18 of 19 newly identified protein domains with prion-forming capability, require Hspl04 for propagation (Chernoff et al. 1995; Derkatch et al. 1997; Moriyama et al. 2000; Shorter and Lindquist 2004; Jones and Tuite 2005; Du et al. 2008; Alberti et al. 2009; Patel et al. 2009). To test the influence of Hspl 04 on [GAR+], we crossed [GAR+] cells to cells carrying a knockout of hspl04 and sporulated them, lisp 104 was not required for [GAR+] inheritance: Ahsp 104 segregants remained [GAR ⁾ ] (Fig. IE). [GAR+] was also not curable by growth on guanidinium hydrochloride, which inhibits 1 lspl ()4's ATPase activity (Ferreira et al. 2001; Jung and Masison 2001), nor by

overexpression of HSP104 (data not shown).

[00169] We next tested the Hsp70 proteins Ssal and Ssa2 (Werner- Washburne et al.

1987), mutations in which affect the inheritance of other prions (Sweeny and Shorter 2008). These mutations are also a good measure of general chaperone sensitivity, as they induce production of most chaperone proteins (Oka et al. 1997). Strikingly, all Assa 1 Assa2 meiotic products lost the ability to grow on glycerol in the presence of glucosamine (Fig. IF). To test whether this was due to curing of the [GAR ¹ \ genetic element, or whether the AssalAssa2 mutations simply masked the phenotype, we restored SSA1 and SSA2 to the glucosamine- sensitive AssalAssa2 progeny by mating them back to wild-type [gar-] cells (see Fig. 25 A for diagram of cross). Restoring Hsp70 function did not result in the reappearance of the [GAR+] phenotype (data not shown). However, when the cells were plated on medium with glucosamine, colonies able to grow on glycerol could be recovered at normal frequencies (Fig. 2513 ). Thus, a transient change in chaperone proteins was sufficient to cure cells of [GAR+] and this curing was reversible, both hallmarks of prion biology (Wickner 1994). [GAR+] therefore exhibits all of the distinguishing genetic characteristics of yeast prions.

[00170] [GAR+] is regulated by the Rgt2/Sn glucose signaling pathway [00171] We performed gene expression profiling to identify transcriptional consequences of [GAR+]. In glucose-grown cultures tested just prior to the diauxic shift, only one gene showed a detectable difference between [gar-] cells and [GAR+] cells on our arrays, but that gene was very strongly affected. Hexose Transporter 3 (HXT3) was ~36-fold down-regulated in [GAR+] cells compared with [gar-] cells (Fig. 26). No other transcript exhibited more than a twofold change. We used an Hxt3-GFP fusion protein under the control of the endogenous HXT3 promoter to examine protein levels. Hxt3-GFP was easily visible at the plasma membrane in late log phase [gar-] cells, but extremely difficult to detect in [GAR+] cells (Fig. 2A). The loss of HXT3 expression (Ahxt3) alone did not allow cells to use glycerol in the presence of glucosamine (Fig. 2B), and thus does not explain the [GAR+] phenotype. However, it led us to hypothesize that the causal agent of [GAR+] is a regulator of HXT3 expression.

[00172] To define the protein(s) required for [GAR+] inheritance, we took advantage of two things. First, transient overexpression of each of the known prion proteins dramatically increases the frequency at which the corresponding prion appears (Uptain and Lindquist 2002). Second, the [GAR+] determinant exerts a strong effect on HXT3 expression, and HXT3 predominantly controlled by the Snf3/Rgt2 pathway (Kim et al. 2003; Santangelo 2006). When glucose is present, transmembrane glucose sensors Snf3 and Rgt2 transmit a signal to the Yckl and Yck2 complex, which then phosphorylates Mthl and Stdl , marking them for degradation (Fig. 2C; Moriya and Johnston 2004). When glucose is not present, Mthl and Stdl accumulate and interact with Rgtl . This complex then binds to the HXT3 promoter and represses transcription of HXT3 (Lakshmanan et al. 2003).

[00173] We tested each gene in the Snf3/Rgt2 regulatory pathway for induction of [GAR+] when overexpressed from a plasmid with a strong constitutive promoter, GPD (Fig. 2D). In every strain test, 5^*7737 caused an extraordinary increase in the appearance of colonies able to grow on glycerol in the presence of glucosamine. In W303, for example, the increase was ~900-fold over empty vector; more than one in 10 cells in these cultures converted to

[GAR+], This is at the high end of prion inductions obtained by analogous experiments with other proteins (Masison and Wickner 1995; Derkatch et al. 1996). While no other gene in this pathway induced [GAR+], overexpression of the STDl paralog MTH1 blocked its

appearance, further confirming the importance of members of this pathway in [GAR+] biology. [00174] Transient STD1 overexpression induces [GAR+] but is not required for maintenance

[001751 Next, we asked if transient expression of STD1 was sufficient to create a heritable change in phenotype, a defining feature of prion biology. When—100 cells that had lost the overexpression plasmid were tested, all retained the [GAR+] phenotype (confirmed by marker loss) (data not shown). Thus, STD1 is not simply a dynamic regulator of glucose repression. Rather, its transient overexpression induces a new, heritable state of carbon utilization.

[00176] These data suggested that Stdl is the determinant of the [GAR+] prion, but further data indicated it could not be the sole determinant. First, most prion phenotypes mimic loss- of -function phenotypes of their prion determinants. However, Astdl strains derived from a [gar^~] background were not able to grow on glycerol in the presence of glucosamine (Fig. 2B; data not shown). Furthermore, Astdl cells derived from a [GAR+] background were able to do so, indicating that they kept the prion (data not shown). Finally, such cells were able to pass the [GAR] element onto progeny in tester crosses for inheritance of the prion element (Fig. 2E). Therefore, [GAR+] maintenance does not require STD1. This makes [GAR+] unusual among yeast prions in that its transient inducing agent is not required for

propagation.

[00177] We next examined all other members of the Rgt2/Snf3 pathway. None behaved as would be expected for the causal agent of [GAR+]. All knockouts were capable of propagating [GAR+] (Fig. 27). Cells with rgtl knockouts did not exhibit the prion phenotype, but they maintained it in a "cryptic" form. It reappeared when cells were crossed to [gar^~] RGT1 cells. Therefore, RGT1 is required for the manifestation of the [GAR+] phenotype but is not necessary for its propagation.

100178] Identification of genes that modify the frequency of [GAR+] appearance

[00179] We conducted genome-wide screens for affecters of [GAR+] induction. We screened the S. cerevisiae haploid deletion library (Giaever et al. 2002) for mutants that were incapable of inducing [GAR+] (Table S2), caused a high frequency of appearance of [GAR+] (Table S3), or that themselves exhibited an ability to grow on glycerol in the presence of glucosamine (Table SI). Four of the eight members of the Snf3/Rgt2 pathway showed a phenotype in this screen (P = 8 x 10^~6; Fisher's exact test). Asnf3 grows on glycerol with glucosamine (Table S I), and Astdl, Amthl, and Argtl exhibited lower than normal [GAR+] induction (Fig. 2B; Table S2). However, none of these genes were required for the maintenance of [GAR^■ ] in strains already carrying the element (Fig. 27). [00180] Finally, we screened a library of -5000 ORFs (-85% of yeast ORFs) on a galactose-inducible single-copy plasmid (Leonardo et al. 2002) to find genes that induce [GAR+] following overexpression. STDl was the only clone that caused strong [GAR+] induction, ~ 1000-fold when retested under the regulation of the GPD promoter. A second gene, DOG2, caused a 10-fold induction (Fig. 28).

[00181] Pmal associates with Stdl and is a component of [GAR+]

[00182] Since neither the deletion nor the overexpression screen identified a protein that by itself could embody the [GAR+] prion, we turned to biochemical methods. STDl had been implicated in [GAR+] in three ways: (1) The highly specific down-regulation of HXT3 pointed to members of the Rgt2/Snf3 glucose signaling pathway, (2) transient STDl overexpression caused huge increases in [GAR+] appearance, and (3) deletion of stdl reduced the spontaneous appearance of [GAR+] to the frequency of genetic mutations. We hypothesized, therefore, that Stdl might physically interact with an unknown propagating agent.

[00183] We sought proteins that interacted with Stdl by coimmunoprecipitation with an HA-tagged derivative. A high-molecular-weight (HMW) band was recovered from [GAR+] protein lysates but not from \g r^~] lysates (Fig. 29). Mass spectrometry analysis identified the protein as Pmal, a large, highly abundant P-type ATPase with 10 transmembrane domains that is the major controller of membrane potential and cytoplasmic pH (Morsomme et al. 2000). When the same assay was performed with isogenic Astdl cells, Pmal was not detected. Notably, if Pmal is indeed a constituent of the prion, we would not have identified it in our genetic screens. It is essential (Serrano et al. 1986), and therefore it is absent from the deletion library. Moreover, it is already the most abundant membrane protein in yeast and is notoriously difficult to overexpress (Eraso et al. 1987).

[00184] Transient overexpression of STDl induced [GAR+] and transient overexpression of its paralog, MTH1, inhibited [GAR+] conversion. We therefore asked whether Pmal exhibited heritable differences in association with Stdl and Mthl in [gar^"] and [GAR+] cells. As a multipass transmembrane protein, Pmal is intractable to most methods of analyzing protein complexes, but it migrates as an oligomeric species when digitonin lysates are separated on Blue Native gels (Gaigg et al. 2005). Most Pmal in [GAR+] and [gar^~] cells migrated as heterogenous HMW complexes, but a smaller fraction migrated as two distinct complexes of (very roughly) 600 and 700 kDa (Fig. 3A, top). The lower bands (especially the 600-kDa species) were associated with Stdl in [GAR+] cells but with Mthl in [gar^"] cells (Fig. 3 A, bottom). Stdl is much less abundant than Pmal . Consistent with the fact that only a small fraction of Pmal is associated with Stdl in [GAR+] cells, Pmal showed a minor but statistically significant change in protease sensitivity between [gar^"] and [GAR+] cells (Fig. 30).

[00185] Next, we asked whether mutations that affect Pmal oligomerization and trafficking to the plasma membrane alter [GAR+] frequency. Mutants that affect phospholipid synthesis and protein trafficking but not Pmal oligomerization— LCB3, LCB4, DPL1, and

ATG19 (Lee et al. 2002; Mazon et al. 2007)— did not change the appearance of [GAR+] (Fig.

3B; Fig. 31 A). Mutants that do affect Pmal oligomerization and trafficking— SUR4 and

LST1 (Roberg et al. 1999; Lee et al. 2002)— decreased the appearance of [GAR+] (Fig. 3B;

Fig. 31 A). These genes were not, however, required for [GAR+] maintenance (Fig. 3 IB).

[00186] We explored the relationship between Pmal , [GAR+], and the Rgt2/Snf3 glucose signaling pathway. Carbon sources regulate Pmal's phosphorylation state (Lecchi et al.

2005), its ATPase activity (Serrano 1983), and its conformation (Miranda et al. 2002) through residues S899, S911, and T912 in the C-terminal tail, which faces the cytosol (Eraso et al.

2006; Lecchi et al. 2007). We mutated S899, S911, and T912 to alanine, which cannot be phosphorylated, or to aspartic acid, which mimics constitutive phosphorylation.

(Phosphorylated S91 1 and T912 are commonly observed in glucose media and the nonphosphorylated forms when cells are starved of glucose [Lecchi et al. 2007].) S899 mutations and S91 ID and/or T912D mutations had no effect on [GAR+] frequency.

However, S91 1A and S91 1A/T912A increased the frequency of [GAR+] appearance by several-fold (Fig. 3C). Notably, these same mutants also reduced levels of an Hxt3-GFP reporter, both a readout for the Rgt2/Snf3 pathway and the only change in gene expression detected in [GAR^■ ] cells (Fig. 3D). These results indicate that Pmal affects glucose signaling to regulate HXT3. In any case, the fact that such subtle mutations in the Pmal protein affect

[GAR+] induction confirms that Pmal plays a key role in [GAR+] biology.

[00187] The unstructured N terminus of Pmal is involved in [(7/4/?+] propagation

[00188] A characteristic of prions is that transient overexpression is sufficient for induction. However, Pmal is the most abundant plasma membrane protein in yeast

(Morsomme et al. 2000), and overexpression is not well tolerated (Eraso et al. 1987). We found that we could obtain a threefold increase in Pmal protein levels with a EjVplasmid and a GPD promoter. This caused a corresponding increase in [GAR+] frequency (Fig. 4A).

Introducing stop codons at amino acid positions 23 or 59 eliminated this effect (Fig. 32).

Thus, it is not the nucleic acid sequence but the Pmal protein that contributes to [GAR+] induction. Finally, when the inducing GPD PMA1 plasmid was lost, the cells remained GAR+]. Thus, a transient increase in PMA1 was sufficient to induce a heritable change in phenotype.

[00189] Pmal's N and C termini face the cytosol. The C terminus is predicted to be a- helical and the N terminus unstructured (Morsomme et al. 2000), the latter a characteristic of prions. An N-terminally truncated (Δ40) mutant of PMA1 did not increase [GAR+] appearance although the protein was expressed at wild-type levels (Fig. 4A). A C-terminally truncated PMA1 did increase [GAR+] induction, even though its levels were reduced.

[00190] [GAR+] could be propagated through cells whose only source of Pmal was a G^ZJ-regulated N-terminal deletion, Ρ 47Δ40Ν (Fig. 33 ). Strikingly, however, it did not propagate through a double mutant of ΡΜΑ1Δ40Ν and stdl, and it did not reappear when wild-type PMA1 and STD1 function were restored with crosses (Fig. 4B). (The few glucosamine-resistant colonies that remained were not [GAR+] but contained conventional recessive; data not shown.) Thus, once [GAR+] has been established, it is maintained in the absence of either Stdl or the N terminus of Pmal , but not in the absence of both.

[00191] [GAR+] is sensitive to a Pmal-dependent 'species barrier'

[00192] Previously described yeast prion proteins exhibit changes in localization and solubility in the prion state (Uptain and Lindquist 2002) and affect the induction of other prions by cross-templating (Derkatch et al. 2000, 2001). There was no difference in localization of Pmal or Stdl between \gar ^~] and [GAR+] (Fig. 34). Neither formed a detectable SDS-resistant species in [GAR+] (Fig. 35). Furthermore, the frequency of [GAR+] appearance did not change in backgrounds carrying [PSf], [RNQ⁺], or [URE3], prions that broadly affect the appearance of amyloid-based prions (Fig. 36). Analysis of protein extracts by two-dimensional (2D) gel electrophoresis did not reveal any proteins that changed solubility between [gar^~] and [GAR+] (Fig. 37). [GAR+] was not affected by Hspl04 expression (Fig. IE). Whatever the manner by which Pmal and Stdl contribute to the prion state, it is not likely by forming amyloid.

[00193] The extremely stable nature of amyloids allows them to be confirmed as prion determinants by "protein only" transformation (Maddelein et al. 2002; Tanaka et al. 2004). The lack of an identifiable amyloid in [GAR+] cells precluded the use of this procedure for [GAR+]. Instead, to rigorously test the relation between Pmal , Stdl , and [GAR+], we performed a classic "transmission barrier" experiment. Small differences in amino acid sequence cause prions that originate in one species to fail in transmission to another (Santoso et al. 2000; Bagriantsev and Liebman 2004; Chen et al. 2007). If Pmal and Stdl contribute to a transmission barrier for [GAR+], it would establish that they are integral to the propagating element.

[00194] We chose to study a possible [GAR+] transmission barrier using Saccharomyces bayanus and Saccharomyces paradoxus, two closely related sensu stricto species that also exhibit glucose-mediated repression of the utilization of other carbon sources. First, we asked whether diploids of these species could also acquire the ability to use glycerol in the presence of glucosamine (Fig. 5A). They could, and they did so at a higher frequency than expected for mutation. Indeed, [GAR+] appeared in S. bayanus at an astonishingly high rate (greater than one in 1000 cells). Moreover, the [GAR+] phenotype was very stable in these cells. Thus, the ability to heritably switch carbon utilization strategies through this prion is broadly used.

[00195] We asked whether the Pmal proteins from S. bayanus and S. paradoxus can propagate [GAR+] in S. cerevisiae. Sequence differences between the species are slight (Fig. 38): S. bayanus Pmal and S. paradoxus Pmal are 96% and 99% identical to S. cerevisiae Pmal, respectively. Most of these changes are in the N-terminal region, which is required for prion induction

[00196] First, we transformed S. bayanus or S. paradoxus PMAl plasmids into an S.

cerevisiae strain in which a deletion of the essential PMAl gene was covered by a plasmid encoding S. cerevisiae Pmal . The S. cerevisiae PMAl plasmid was then selected against. All cells grew at the same rate on glucose, indicating that the Pmal protein from these species was fully functional in S. cerevisiae. However, when [GAR+] cells were selected by plating these cells to glycerol-glucosamine medium, the resultant phenotypes were weak, unstable, and appeared at a low frequency. When putative [GAR+] cells were passaged on nonselective medium and then plated back onto glucosamine-containing medium, many fewer cells with S. bayanus or S. paradoxus PMAl maintained the resistant phenotype than cells with S.

cerevisiae PMAl (data not shown). Thus, in a background where the entire genome otherwise remains the same, changing the species of origin for Pmal had a critical effect on [GAR+] induction and propagation.

[00197] Next, we asked whether the S. bayanus or S. paradoxus Pmal proteins could propagate a [GAR+] state received from the S. cerevisiae protein. We performed another plasmid shuffle, this time starting with cells already carrying a strong S. cerevisiae [GAR+] element. We selected against the plasmid carrying the S. cerevisiae PMAl after ~25 generations. After another 25 generations, cells were tested for the ability to grow on glycerol in the presence of glucosamine. Most retained a strong [GViR i | phenotype. Thus, strains with S. bayanus and S. paradoxus PMAl were capable of accepting and propagating [GAR+] from strains with S. cerevisiae PMAl (Fig. 5B), at least after coexpression of both proteins for 25 generations.

[00198] Finally, we tested how efficiently [GAR+] elements from cells expressing S.

bayanus or S. paradoxus PMAl could be transmitted back to cells expressing only S.

cerevisiae PMAl. Multiple [GAR+] strains carrying the three PMAl genes were mated to wild-type [gar^~] cells. Cells expressing PMAl from S. paradoxus could not transmit [GAR+] at all, and cells expressing PMAl from S. bayanus transmitted it very inefficiently. Controls expressing S. cerevisiae PMAl transmitted [GAR+] efficiently (Fig. 5B). Thus, the PMAl species of origin creates a strong transmission barrier for [GAR+] propagation.

[00199] Might Stdl , the [GAR+] induction factor that is complexed with Pmal in [GAR+] cells, create an induction barrier? Stdl is 81% identical between ,S'. cerevisiae and S. bayanus but much more divergent in S. paradoxus (Supplementaql Fig. 39). We transiently overexpressed STD1 from each organism in [gar^~] S. cerevisiae cells carrying each of the three Pmal genes. STD1 alleles of S. cerevisiae and S. bayanus acted as general inducers. They increased the appearance of [GAR+] ~ 1000-fold in strains producing the Pmal protein of any of the three species (Fig. 5C). In contrast, S. paradoxus STD1 did not induce [GAR+] in any. Presumably, some other factor contributes to [GAR+] induction in S. paradoxus. Most importantly, however, this experiment demonstrates that Stdl creates a strong species barrier for [GAR+] induction, confirming its intimate involvement in the prion.

[00200] Discussion

[002011 The ability of cells to sense and adapt to nutrients is crucial to survival in highly competitive and rapidly fluctuating environments. Here, we describe a cytoplasmically inherited element, [GAR+], that is involved in the fundamental processes of glucose sensing and signaling and carbon source utilization. [GAR+] arises spontaneously in every S.

cerevisiae strain tested, as well as sibling species separated by ~5 million years of evolution (Kellis et al. 2004)— S. paradoxus and S. bayanus— at frequencies much higher than genetic mutations.

[00202] [GAR+] fulfills all of the genetic criteria established for prions: It is dominant (or at least semidominant). It exhibits non-Mendelian inheritance. It can be transferred via cytoplasmic exchange. Transient changes in the levels of chaperone proteins are sufficient to heritably cure cells of the [GAR+] state. Transient changes in the expression of proteinaceous determinants heritably induce [GAR+]. The non-Mendelian mechanism of inheritance that best describes [GAR+] is that of a prion. [00203] In other ways, however, [GAR+] seems very different from previously described yeast prions. It has at least two components: the plasma membrane proton pump Pmal, and the glucose signaling factor Stdl . Transient overexpression of either PMA1 or STD1 is sufficient to establish a heritable conversion to [GAR+], yet once [GAR+] is established, either is sufficient for propagation. Cells lacking stdl and also carrying a small deletion in the N terminus of Pmal cannot propagate [GAR+] at all. Pmal and Stdl associate in an oligomeric complex in [GAR+] cells, but this complex is barely detectable in gar^~] cells. The integral relationship between these proteins and the [GAR+] state was tested and confirmed by transmission barrier experiments. Substituting the PMA1 gene from S. bayanus or & paradoxus for that of S. cerevisiae blocked propagation of [GAR+] to S. cerevisiae Pmal . Substituting Stdl from S. paradoxus eliminated its potency in [GAR+] induction.

[00204] [GAR+] does not involve a detectable amyloid form, at least of the Pmal or Stdl proteins. It also is not sensitive to overexpression or deletion of the general amyloid remodeling protein Hspl04. l isp 104 severs amyloid filaments to ensure orderly inheritance of prion templates to daughter cells. It is required for the propagation of all known prions as well as for 18 of 19 recently discovered prion candidates (Chernoff et al. 1995; Patino et al. 1996; Derkatch et al. 1997; Ness et al. 2002; Cox et al. 2003; Kryndushkin et al. 2003;

Shorter and Lindquist 2004, 2006; Jones and Tuite 2005; Tipton et al. 2008; Alberti et al. 2009). Thus, the absence of dependence on Hspl04 makes it rather unlikely that [GAR+] involves any amyloid-based element.

[00205] One possibility is that [GAR+] inheritance and propagation result from heritable alterations in Rgt2/Snf3 signaling involving a self-sustaining feedback loop. Indeed, Stdl and its paralog, Mthl , are subject to many feedback mechanisms involving their own

transcription and degradation (Lakshmanan et al. 2003; Moriya and Johnston 2004; Polish et al. 2005; Kim et al. 2006), and Stdl is found both in the nucleus and on the plasma membrane (Schmidt et al. 1999). Furthermore, Pmal is very abundant and Stdl is extremely scarce (Morsomme et al. 2000). Our data suggest that only a small fraction of Pmal contributes to [GAR+] and that Stdl is the limiting factor. This would be consistent with altered signaling, as only small amounts of the Stdl protein would be necessary to shift the activity of a fraction of Pmal . However, if [GAR+] is simply due to altered signaling, the mechanism that maintains it must be remarkably robust, as it has been maintained in a highly stable state in some of our strains for 6 years now, with repeated dilutions into log phase, storage in the freezer and refrigeration, transitions back to room temperature, and growth in liquid and on plates, in a wide variety of different media, through repeated rounds of growth into stationary phase (wherein most aspects of carbon metabolism undergo profound changes), and through starvation-induced meiosis.

[00206] Another possibility is that [GAR+] starts with a change in the association of Stdl and Pmal that induces a conformational change in oligomeric species of each. These can then be maintained in the absence of either Stdl or the Pmal N terminus, but not in the absence of both (Fig. 6). We do not exclude the possibility that another protein contributes to the

[GAR+] state. Indeed, our observations that S. paradoxus acquires [GAR+] at a high frequency, but that the Pmal and Stdl proteins of S. paradoxus do not reconstitute [GAR+] in S. cerevisiae, suggesting the involvement of another protein. (This protein might even form amyloid, but if so it does not require Hspl04 and has escaped detection in our genetic screens.)

[00207] Of course, models involving self-perpetuating signaling loops and conformational changes are not mutually exclusive. Associations between Pmal and Stdl might result in a conformational change that alters signaling and sets up a robust feedback loop that helps maintain the association, either between those same molecules of Pmal and Stdl or between other molecules and these proteins (Fig. 6). Another remaining question is the precise reason why cells carrying [GAR+] are able to grow on glycerol in the presence of glucosamine. We hypothesize that the [GAR+] phenotype involves altered signaling through a glucose-sensing pathway, likely through Stdl's previously reported ability to interact with the DNA-binding protein Rgtl (Fig. 6; Lakshmanan et al. 2003).

[00208] Materials and Methods Used in Example 1

[00209] Yeast strains and genetic manipulations

[00210] Strain construction and manipulation followed standard yeast techniques. A list of strains and plasmids used in this study is available in Tables SI and S2. Unless otherwise stated, data shown are from genetic background W303. Fivefold dilutions were used for all spotting assays. Media used were yeast peptone-based medium containing the designated carbon source (YPD, YPglycerol, and YPgalactose), synthetic medium lacking a particular amino acid (SD), or glycerol glucosamine medium (GGM; 1% yeast extract, 2% peptone, 2% glycerol, 0.05% D-[+] -glucosamine [Sigma G4875]).

[00211] [GAR+] frequenc assays and isolation of [GAR+\

[00212] Cultures for [GAR+ frequency assays were grown overnight in 2% glucose, either YPD or SD, subcultured in the same, then grown to early exponential phase (OD₆₀o ⁼ 0.2-0.4). Cultures were plated straight to GGM and diluted 10^~~4 for plating to YPD. To isolate [GAR+] for further study, colonies from GGM were restreaked once to GGM then used in downstream applications. Unless otherwise stated, error bars in [GAR+] frequency assays represent the standard deviation and P-values are the binomial distribution of the mean. In all assays for [GAR+] propagation, cells were passaged for >100 generations before testing for growth on glycerol in the presence of glucosamine. Sporulation was performed by growing to diauxic shift in YPD or SD, plating to sporulation plates (1% potassium acetate, 0.05% dextrose, 0.1% yeast extract, 0.01% complete amino acid mix [Biol 01]), and incubating at 23 °C until sporulated.

[00213] Genetic, biochemical, and cell biological analysis

[00214] Gene expression profiling, Western blotting, immunoprecipitation, fluorescent microscopy, Blue Native gel analysis, protease sensitivity analysis, and genetic screens were all performed using standard procedures. Detailed descriptions are available in the

Supplemental Material.

[00215| Supplementary Materials and Methods

[00216] Western blotting

[00217] Protein samples were run on 4-12% SDS gels from Invitrogen and blotted to PVDF using standard techniques. All samples to be tested for Pmal were incubated in loading buffer (4% SDS, 50mM Tris pH 6.8, 2% β-mercaptoethanol, 10% glycerol) for lOmin at 37°C prior to loading. Monoclonal a Pmal mouse antibody was obtained from EnCor Biotechnology. Polyclonal a Pmal rabbit antibody was a gift from Amy Chang. Polyclonal Sec61 antibody was a gift from Tom Rapaport. Immune complexes were visualized by ECL.

[00218] Gene expression profiling

[00219] PolyA RNA was produced using standard methods (Schmitt et al. 1990) from cells grown in 2% glucose that were about to undergo diauxic shift. Samples were labeled and hybridized to Affymetrix S98 arrays by the Whitehead Center for Microarray Technology using standard methods. Data was analyzed using Genespring and TIGR Multiexperiment Viewer. Data was deposited at NCBI GEO (http://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE12479).

[00220] Hx(3-GFP analysis

[00221] Hxt3-GFP signal was observed starting at OD₆oo ⁼ 0.7 in an S288C background. Microscopy was performed on a Zeiss axioplan using Metamorph software.

[00222 J Immunoprecipitation

[00223] IPs were performed using standard procedures in IP buffer (50mM HEPES pH 7.5, 150mM NaCl, 2.5mM EDTA, 1% V/VTriton X-100, 40mM NEM, 3mM PMSF, 1 Protease Inhibitor Cocktail Tablet per 5ml buffer [Roche]). Cells were lysed either by bead beating (9 x 30sec with 15sec on ice between) or spheroplasting (30min at 30°C in 1M D-sorbitol, 0.1M EDTA, 0.5mg/ml zymolase) with comparable results. Lysates were adjusted for protein concentration, incubated with protein G agarose beads (Roche) for 30min at 4°C, centrifuged at 3300 x g for 2min, and the supernatant collected. The supernatant was then incubated with ^g mouse a HA antibody (Sigma) for 1 hour at 4°C followed by incubation with 50μ1 protein G beads (Roche) for 1 hour at 4°C. Samples then washed six times in chilled IP buffer, boiled to elute, and run on a 4-12% SDS gel. Gels were either stained with colloidal Coomassie (Invitrogen) or blotted for Pmal . Stdl- and Mthl -tagged strains were shown to acquire and stably maintain the [GAR⁺] element (data not shown).

[00224] Blue Native gels

[00225] Midlog cultures (150ml, OD₆oo~0.5) were lysed by bead beating (9 x 30sec with 15sec on ice between) into sorbitol buffer (250mM sorbitol, 50mM Tris pH 7.5, 3mM PMSF, 1 Protease Inhibitor Cocktail Tablet per 5ml buffer [Roche]). Samples were equalized at a concentration of 15μ^ 1 in 650μ1, a "total" cellular protein sample collected, and centrifuged at 16000 x g for 30min at 4°C. The supernatant was removed, a sample saved for downstream analysis, and the pellet washed once in sorbitol buffer. The pellet was resuspended in sorbitol buffer (200μ1), and an aliquot (95μ1) incubated 20min on ice with digitonin to 1% (Calbiochem). These samples were then centrifuged at 16000 x g at 4°C for 30min and separated into supernatant ("digitonin soluble") and pellet ("digitonin insoluble") fractions. 15μ1 of the soluble fraction was incubated with Coomassie G-250 at a detergent to dye ratio of 8: 1 for lOmin on ice then loaded onto 3-12% Blue Native gel (Invitrogen) and run at 4°C as per the manufacturer's instructions.

[00226] Trypsin digestion

[00227] Cells were grown to mid exponential phase (OD₆oo~0.5), washed three times in water, then lysed by bead beating (9 x 30sec with 15sec on ice between) into sorbitol buffer (250mM sorbitol, 50mM Tris pH 7.5, 3mM PMSF, 1 Protease Inhibitor Cocktail Tablet per 5ml buffer [Roche]). Samples were centrifuged at 16000 x g for 30min at 4°C, the supernatant removed, then washed three times in sorbitol buffer with protease inhibitors and three times in sorbitol buffer without protease inhibitors. For trypsin reactions, 10μg protein and 4μg trypsin (Worthington) were used in a total volume of 20μ1. Reactions were incubated at 30°C and stopped after the designated point in time by addition of 2μ1 soybean trypsin inhibitor (lOmg/ml stock, from Sigma) then immediately frozen in an ethanol/dry ice bath. Samples were run on gels as described above, probed with monoclonal ocPmal, stripped, and re-probed with polyclonal aSec61.

[00228] Screen of S. cerevisiae deletion library for [GAR⁺] induction-deficient knockout mutants

[00229] Library plates were inoculated into 96- well plates containing 200μ1 YPD and grown 48 hours at 30°C. Cells were then resuspended and plated to media containing 2% glucose, 2% glycerol, or 2% glycerol + 0.03% glucosamine (optimal concentration for the BY strain background). Plates were photographed every 24 hours for seven days. Wild-type controls showed the appearance of glucosamine-resistant colonies after five days. Mutants that exhibited earlier appearance of glucosamine-resistant colonies were either completely resistant to glucosamine (when every cell in the population grew on glucosamine medium) or exhibited high rates of appearance of [GAR⁺] (when a subset of the population grew on glucosamine medium). Mutants that showed few or no glucosamine-resistant colonies after seven days were considered deficient in induction or maintenance of [GAR⁺]. Knockout mutants that exhibited a growth defect on glucose- or glycerol-based media were excluded from the analysis. Data were obtained from two replicates of two independent experiments.

[00230] Screen for ORFs that induce [GAR ^'] following transient overexpression

[00231] A library of plasmids, each containing a single S. cerevisiae ORF under control of the inducible GAL1 promoter, was mated to a strain containing a GAL- estradiol fusion plamid (Quintero et al. 2007). The latter allows induction of GAL I promoters by growth on estradiol without galactose. We selected for diploids carrying both plasmids on glucose medium lacking histidine (estradiol plasmid marker) and uracil (GAL1 plasmid marker). Following this selection, cells were grown in selective medium containing ImM estradiol, which induces gene expression (Quintero et al. 2007), for 24 hours. These cells were harvested, washed once in I TO, resuspended in ¾0, and spotted to media containing either 2% glucose or 2% glycerol + 0.05% glucosamine. Plates were imaged every 24 hours for seven days. Control spots containing cells carrying the empty induction plasmid exhibited glucosamine-resistant colonies after five days. Data were collected from two independent screens. Spots that showed growth on glucosamine- medium prior to five days were retested individually.

[00232] 2D gel electrophoresis [00233] 2D gels were performed as previously described (Gorg et al. 2004) with the following modifications. Mid-expontential phase yeast cell were lysed by spheroplasting (0.5mg/ml zymolase), resuspending in buffer (50mM HEPES, 150mM NaCl, 2.5mM EDTA, 1%(V/V) TritonX-100, and protease inhibitors) then running through a 21G needle. Protein samples were separated into supernatant and pellet fractions by centrifuging at 14,000g. Samples were diluted in rehydration solution and IPG buffer (GE Healthcare) and lmg total protein was loaded onto 1 1cm IPG DryStrip pH 3-1 1 nonlinear (GE Healthcare). IPG strips were rehydrated overnight then run on a Multiphor II electrophoresis apparatus. The second dimension was run on 4-12% gradient SDS gels from GE Healthcare. Gels were visualized using a Colloidal Blue Straining Kit (Invitrogen).

[00234] Indirect Immuno flour escence

[00235] Immunofluorescence experiments were performed as previously described (Amberg et al. 2005). Anti-HA antibody (Sigma) was used at 1 : 100 dilution. Anti-mouse Texas Red (Molecular Probes) was used at 1 : 100 dilution.

[00236] SDS solubility

[00237] Protein samples for measuring the SDS solubility of Pmal, Stdl, and Sup35 extracted as described in the Native gel protocol in Materials and Methods. Total protein was diluted in loading buffer to a final SDS concentration on 4% then incubated lOmin at 37°C or boiled for 5min, as indicated. Transfer to PVDF membrane and Western blotting was as described.

[00238] Table S 1 : Knockout mutants able to grow on glycerol in the absence of glucosamine

[00239] Table S2: Knockout mutants with a reduced frequency of [GAR ] appearance

[00240] Table S3: Knockout mutants that switch to [GAR+] at high frequency

[00241] Table S4: Yeast strains used in this study

[00242] Table S5: Plasmids used in this study

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Supplemental References

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Eisenkolb, M., Zenzmaier, C, Leitner, E., and Schneiter, R. 2002. A Specific Structural Requirement for Ergosterol in Long-chain Fatty Acid Synthesis Mutants Important for Maintaining Raft Domains in Yeast. Mol Biol Cell 13(12): 4414-4428.

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sphingolipidswith very long chain fatty acids but not ergosterol is required for routing of newly synthesized plasma membrane ATPase to the cell surface of yeast. J Biol Chem 280(23): 22515-22522.

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Schmidt, M.C., McCartney, R.R., Zhang, X., Tillman. T.S., Solimco. I I.. Wo 111, S., Almonte, C, and Watkins, S.C. 1999. Stdl and Mthl Proteins Interact with the Glucose Sensors To Control Glucose- Regulated Gene Expression in Saccharomyces cerevisiae. Mol Cell Biol 19(7): 4561-4571.

Example 2: [GAR+\ is elicited by interaction with bacteria

[00244] Through a serendipitous bacterial contamination of a selective yeast plate, we discovered that Staphylococcus hominis induces the [GAR+] prion in yeast cells. Yeast cells normally cannot grow on glycerol medium (GLY) containing trace quantities of glucosamine (GlcN), a nonmetabolizable glucose mimetic. This is because, even when present in very small quantities, GlcN triggers 'glucose repression' and prevents yeasts from using glycerol. [GAR+] relieves glucose repression and allows them to grow. Prion minus, [gar-], yeast cells surrounding an S. hominis bacterial contaminant grew well on GLY/GlcN. After this accidental finding, we directly tested the ability of S. hominis to induce yeast growth on glycerol media with trace glucosamine (GLY/GlcN). The induction was robust and highly reproducible (Fig. 7).

[00245] To determine whether these yeast cells truly harbored the [GAR+] prion, we isolated multiple yeast colonies from GLY/GlcN plates containing S. hominis, propagated them for hundreds generations on glycerol media without glucosamine, and then transferred them back to GLY/GlcN. Through all these doublings in the absence of bacteria, the cells retained the glucosamine-resistant trait. Next we tested them for two defining characteristics of prion-based genetic traits: cytoplasmic inheritance and sensitivity to changes in protein chaperone function. In genetic crosses, the glucosamine-resistant trait that had been induced by the bacteria had the same dominant, cytoplasmic pattern of inheritance as [GAR+]. The glucosamine resistance of these colonies was also eliminated by transient reductions in the function of the Hsp70 chaperone. This same treatment eliminates spontaneous [GAR+], but not glucosamine resistance caused by genetic mutations. These and other characteristics of the cells established that their glucosamine-resistant trait was due to the acquisition of the prion we have previously characterized as [GAR+]².

Example 3: Prion induction is driven by interkingdom chemical communication

[00246] Bacterial induction of [GAR+] exhibited a steep spatial gradient on agar plates. This suggested it might be mediated by a diffusible factor. We tested this directly by pre- growing inducing bacteria in liquid GLY/GlcN medium to late exponential phase and removing the bacteria by filtration. We exposed exponential-phase [gar-] yeast cells to this sterile conditioned medium, washed the cells, and plated them to select for [GAR+].

Exposure to conditioned medium greatly increased the number of [GAR+] colonies. This was apparent even with very brief incubations (1-4 h). Thus, conditioned medium does not simply enrich for the growth of pre-existing [GAR+] cells, but induces appearance of the prion.

[00247] The bacterial induction of [GAR+] was not due to a change in the pH of the growth medium, nor to molecules previously known to mediate microbial signaling (e.g. acyl-homoserine lactones, farnesols, 2-phenylethanol). The inducing activity was stable to boiling, pH extremes, freeze/thaw cycles, and protease digestion. It could also be recovered in the flow through from 3 kDa filters and extracted into polar organic solvents. Thus, bacteria induce the yeast prion [GAR+] via small-molecule chemical communication.

Example 4: Overlapping pathways drive both spontaneous and

bacterially-indueed [GAR+]

[00248] To identify yeast genes that are involved in perceiving the inducing signal and transmuting it into a heritable trait, we performed a genome-wide screen. We employed a library of -4800 isogenic yeast strains containing precise deletions of virtually all nonessential open reading frames (ORFs) in the genome⁹. Using robotic pinning, we

interspersed rows of such yeast mutants with rows of S. hominis and grew them on

GLY/GlcN plates. We identified 59 yeast mutants that reduced [GAR+] induction and 30 that enhanced it (Tables B and C). The proteins encoded by these gene sets were strongly enriched for interactions with Pmal (P < 0.01 by Chi-squared test), the key determinant of spontaneous [GAR+]. Furthermore, there was a strong congruence (Fisher exact test) between those genes that affected the bacterial induction of [GAR+] and its spontaneous appearance². Hence, overlapping genetic networks are involved in both processes.

[00249] There were, however, a number of genes that strongly influenced the bacterial induction of [GAR+] but had no effect on the spontaneous appearance of the prion (Tables B and C; see also Supplementary Tables in Example 1 for genes that influence spontaneous appearance of [GAR+ ). The recent advent of genome- wide analyses in S. cerevisiae has ensured that every gene we analyzed in this collection has previously been examined in more than 400 diverse conditions. Yet despite the exhaustive nature of these analyses, several of the genes identified in our screen have had no previously reported effect on growth. They might well exist at least in part for the purpose of such social communication.

[00250] Table B : Deletions that Enhance GAR Acquisition (DEGA Genes)

Table C: Deletions that Reduce GAR Acquisition (DRGA Genes)

- I l l -

Example 5: Evolutionarily conservation of [GAR+] induction

[00252J Next we asked if the capacity of bacteria to secrete a prion-inducing factor was an idiosycratic property of Staphylococcus. We assembled more than one hundred wild isolates of diverse bacterial species and tested them for [GAR+] inducing capacity. Roughly fifteen percent of the bacteria we tested induced [GAR+] when yeast cells were spotted adjacent to them on selective agar plates (Fig. 14; Table A). Different species induced the prion with different efficiencies, ranging up to a remarkable 50,000-fold increase over the spontaneous frequency of [GAR+] . Inducing bacteria included both Gram positive and Gram negative organisms and, within these groups, did not cluster by clade. There was, however, a marked enrichment for bacteria capable of [GAR⁺] induction in species found by in arrested wine fermentions (e.g. Pediococcus damnosis and Lactobacillus kunkeii) compared to the mix of species commonly found in wine fermentations.

[00253] Table A: Bacteria that induce \GAR+\ :

Wine bacteria that induce \GAR+] :

Pediococcus damnosus

Pediococccus ethanolidurans

Pediococcus parvus

Lactobacillus casei

Lactobacillus hilagardi

Paenibacillus barcinonensis

Staphylococcus pasteuri

Staphylococcus warneri

Bacillus megaterium

Gluconobacter cerinus

Other bacteria that induce IGAR+] :

Bacillus megaterium

Bacillus thuringensis

Bacillus armyloliquefaciens

Bacillus atrophaeus

Bacillus circulans

Bacillus lichenformis

Bacillus subtilis

Bacillus thuringensis

Geobacillus steathermophilus

Listeria innocua

Micrococcus luteus

Serratia marcesens

Shewanella baltica

Sinorhizobium meliloti Staphylococcus gallinarium

Staphylococcus hominis

Stropmyces griseus

Escherichia coli (wild strains)

[00254] We assembled a panel of 15 genetically diverse yeast strains from distinct wild origins: wine, beer, sake, soil, oak, and infected human patients. Collectively, these strains harbor at least 100,000 polymorphisms. We grew each of these strains to mid-exponential phase and plated them in five- fold serial dilutions adjacent to rows of bacteria (S. hominis) on GLY + GlcN plates. In each strain this exposure induced in some cells a heritable capacity to circumvent glucose repression on glycerol, confirming that the ability of yeast to respond to this bacterial signal and acquire the ability to grow on glycerol in the presence of

glucosamine is broadly distributed. Thus, this cross-kingdom communication between bacteria and yeast has been broadly conserved over the evolutionary history of the species.

Example 6: [GAR+] induction confers selective advantages to bacteria and yeast alike [00255] We wondered what advantages this chemical dialog might confer to a bacterial cell. The major difference in gene expression between [GAR+] and [gar-] cells is a ~ 40-fold reduction in the transcription of HXT3, a hexose transporter in [GAR+] cells . We tested whether glucose uptake was reduced in [GAR-] cells. It was not, likely because yeast have many hexose transporters^{1 1}. However, Hxt3 mutants can have a unique influence on fermentation kinetics and ethanol production¹². Thus, [GAR+] might reduce the production of ethanol by yeast cells, providing a less hostile environment for bacterial growth.

[00256] Indeed, when cells were grown on glucose medium, [GAR+] cells produced substantially lower amounts of ethanol than isogenic [gar-] cells, 6% vs 10%. This result was highly reproducible, and also held true in a grape must medium that recapitulates the environment of early wine fermentation. Next, we tested the ability of evolutionarily diverse bacterial cells to survive exposure to a wide range of ethanol concentrations. In exposures ranging from 1 to 72 hrs, differences in ethanol exposure of this magnitude had a marked effect on the growth and survival of the 30 diverse bacterial species we tested.

[00257] Why might yeast conserve an ability to switch to the [GAR+] state, given that that the bacteria in their environment might exploit this switch to their advantage? Since glucose repression limits the utilization of other carbon sources, we measured the growth of [gar-] and [GAR+] cells across a wide range o carbon sources. In pure carbo sources (e.g.

glucose, sucrose, maltose, fructose, galactose, acetate, glycerol, and ethanol) [GAR+] provided no growth advantage. Indeed, a very modest detrimental effect could be discerned when [GAR+] and [gar-] cells were grown in competition in sucrose alone or glycerol alone (Fig. 10). However, in nature the carbon sources yeast exploit are generally mixed. In such mixtures [GAR+] cells frequently grew substantially better than [gar-] cells. This advantage also held true with commercially important substrates for fermentation (e.g. grape must and molasses) and was particularly evident in direct competitions between [GAR+] and [gar-] cells (Fig. 10). As a frame of reference, the disadvantage [gar-] cells exhibited in mixed carbon sources was frequently greater than has been reported for 30% of yeast strains in the genome wide deletion collection¹³. Thus, [GAR+] provides mutual selective advantages to the bacteria that induce the prion and the yeast that acquire it.

Example 7: Cellular dynamics of the interkingdom interaction

[00258] To investigate the dynamics of this interkingdom interaction on a single-cell level, we took advantage of droplet microfiuidics. This technique enables rapid analysis of many cell types in gas-permeable microdroplets¹⁴. We encapsulated between one and three yeast cells per droplet in nonselective medium (rich liquid GLY with a droplet volume of 65 pL) and analyzed millions of such growth chambers. Both [gar-] and [GAR+] cells grew in droplets at the same rates, and to the same final culture densities, as they do in bulk culture. To examine [GAR+] induction rates, we encapsulated cells in droplets with [GAR+] selection medium (liquid GLY/GlcN). The cells harbored a constitutive fluorescent marker (mOrange) to report on cell number and an Hxt3-GFP fusion to report on prion status. In this medium, [GAR+] cells grew well, but most [gar-] cells underwent at most one doubling. However, approximately one in 100,000 droplets of [gar-] cells grew well. Cells in these droplets proved to have switched on the prion reporter. The frequency with which such cells appeared in microdroplets was equivalent to the frequency of [GAR+] colony formation on GLY/GlcN plates.

[00259] Next we co-encapsulated -ten cells of an inducing bacteria (a wild strain of E. coif) with each yeast cell. Due to the much smaller bacterial cell size, this translates to a 100- fold excess of yeast cell mass. The bacterial cells were labeled with a different fluorescent marker (m ate2), enabling independent detection of their growth. Even at early time points (12h post encapsulation), a significant portion of the droplets had a fluorescent signal characteristic of [GAR+] yeast. After 48h, cells in -80% of droplets had switched to the

[GAR+] state, an 800,000 fold increase over the spontaneous switching rate. The induction of [GAR+] requires neither the concerted action of millions of bacteria to produce the signal nor of millions of yeast to perceive it. Rather, the interaction between small numbers of cells is sufficient to elicit the prion with high efficiency and this switch takes place rapidly, in the course of just a few cell doublings.

Example 8: [GAR+] transforms the community dynamics of natural fermentations

[00260] In natural wine fermentations the microbiological ecology and physical environment is dynamic and complex. In "successful" wine fermentations S. cerevisiae eventually comes to dominate other microbes. Several mechanisms are involved, including nutrient depletion, and the production of H₂S, heat, and ethanol¹⁵. These conditions are toxic to most bacteria, but yeast are remarkably tolerant of them. We investigated what effects [GAR+] has in such circumstance. We performed wine fermentations with unsterilized Chardonnay must produced by the vineyard at the University of California Davis. We inoculated six gallon fermentation vessels with either [GAR+] or [gar-] versions of a commonly used wine strain (UCD#932) at a concentration 3 x 10⁶ cells per ml. The [gar-] cells vigorously fermented the Chardonnay must, dominating the microbial competition with typical fermentation kinetics. In contrast the [GAR+] cells displayed kinetics typical of those found in "unsuccessful" naturally-arrested fermentations, producing much less ethanol (see, e.g., Figs. 17 and 18 for comparison of ethanol production by [GAR+] and [gar-] yeast). As a consequence, other wine microorganisms flourished, particularly lactic acid bacteria (see, e.g., Fig. 19).

[00261] Surprisingly, proliferation of the bacteria did not come at the expense of yeast biomass. In all three fermentations, we observed similar concentrations of yeast cells in [gar- ] and [GAR+] fermentations. Furthermore, [GAR+] yeast remained more viable than [gar-] yeast during the latter part of the three week fermentation. In separate experiments, investigating possible explanations, we found that [GAR+] survived high ethanol

concentrations better than isogenic [gar-] cells (Fig. 4c), a property that would give [GAR r] cells an advantage for growth in late fermentations. Another stress yeast encounter in fermentation is starvation for amino acids. To test whether [GAR+] alters amino acid uptake we tested a laboratory strain that was auxotrophic for tryptophan. Uptake of this amino acid is particularly dependent upon the function of Pmal , one of the [GAR+] determinants. We found that [GAR+] cells grew far better in media with limiting tryptophan than isogenic [gar- ] cells. Thus, the [GAR - ] prion couples microbial dynamics to S. cerevisiae metabolism in a manner that might act to the frustration of man, but is beneficial to yeast and bacteria alike. [00262] References cited in Examples 2-8

[00263] 1. Mortimer, R. K. Evolution and variation of the yeast (Saccharomyces) genome. Genome Res 10, 403-409 (2000).

[00264] 2. Brown, J. C. & Lindquist, S. A heritable switch in carbon source utilization driven by an unusual yeast prion. Genes & development 23, 2320-2332 (2009).

[00265] 3. Shorter, J. & Lindquist, S. Prions as adaptive conduits of memory and inheritance. Nat Rev Genet 6, 435-450 (2005).

[00266] 4. 1 lalfmann. R. & Lindquist, S. Epigenetics in the extreme: prions and the inheritance of environmentally acquired traits. Science 330, 629-632 (2010).

[00267] 5. Prusiner, S. B. Novel proteinaceous infectious particles cause scrapie. Science 216, 136-144 (1982).

[00268] 6. Shorter, J. & Lindquist, S. Hspl04, Hsp70 and Hsp40 interplay regulates formation, growth and elimination of Sup35 prions. Embo J 21, 2712-2724 (2008).

[00269] 7. Chernoff, Y. O. Stress and prions: lessons from the yeast model. FEBS Lett 581, 3695-3701 (2007).

[00270] 8. Halfmann, R.. Jarosz, D. P., Jones, S. K., Chang, A., Lancaster, A. K. & Lindquist, S. Prions are a common mechanism for phenotypic inheritance in wild yeasts. Nature 482, 363-368 (2012).

[00271] 9. Winzeler, E. A. et al Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901-906 (1999).

[00272J 10. Ng, W. L. & Bassler, B. L. Bacterial quorum-sensing network architectures. Annual review of genetics 43, 197-222 (2009).

[00273] 1 1. Leandro, M. J., Fonseca, C. & Goncalves, P. Hexose and pentose transport in ascomycetous yeasts: an overview. FEMS Yeast Res 9, 511-525 (2009).

[00274] 12. Karpel, J. E., Place. W. R. & Bisson, L. F. Analysis of the Major Hexose Transporter Genes in Wine Strains of Saccharomyces cerevisiae. American Journal of Enology and Viticulture 59, 265-275 (2008).

[00275] 13. Breslow, D. K. et al. A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nature methods 5, 71 1-718 (2008).

[00276] 14. Koster, S. et al. Drop-based microfluidic devices for encapsulation of single cells. Lab on a chip 8, 1 1 10-1 1 15 (2008).

[00277] 15. Bisson, L. P., Karpel, J. E., Ramakrishnan, V. & Joseph, L. Functional genomics of wine yeast Saccharomyces cerevisiae. Advances in food and nutrition research 53, 65- 121 (2007).

[00278] 16. Hogan, D. A. & Kolter, R. Pseudomonas-Candida interactions: an ecological role for virulence factors. Science 296, 2229-2232 (2002).

[00279] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the embodiments described above, but rather is as set forth in the claims. The invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

[00280] Articles such as "a" and "an", and the like, may mean one or more than one unless indicated to the contrary or otherwise evident from the context.

[00281] The phrase "and/or" as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause. As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when used in a list of elements, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but optionally more than one, of list of elements, and, optionally, additional unlisted elements. Only terms clearly indicative to the contrary, such as "only one of "or "exactly one of will refer to the inclusion of exactly one element of a number or list of elements. Thus claims that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present, employed in, or otherwise relevant to a given product or process unless indicated to the contrary. The invention provides embodiments in which exactly one member of the group is present, employed in, or otherwise relevant to a given product or process. The invention also provides embodiments in which more than one, or all of the group members are present, employed in, or otherwise relevant to a given product or process. It is to be understood that the invention encompasses embodiments in which one or more limitations, elements, clauses, descriptive terms, etc., of a claim is introduced into another claim. For example, and without limitation, a claim that is dependent on another claim can be modified to include one or more elements or limitations found in any other claim that is dependent on the same base claim.

[00282] Where the claims recite a composition, it is understood that methods of using the composition as disclosed herein are provided, and methods of making the composition according to any of the methods of making disclosed herein are provided. Where the claims recite a method, it is understood that a composition for performing the method is provided and/or a product produced using the method are provided. Where elements are presented as lists or groups, each subgroup is also disclosed. It should also be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist of, or consist essentially of, such elements, features, etc.

[00283] It is contemplated that aspects and embodiments herein may be freely combined. Resulting aspects and embodiments are within the scope of the invention. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited although such embodiments are, of course, encompassed.

[00284] Where ranges are given herein, the invention provides embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and

embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. In any embodiment of the invention in which a numerical value is prefaced by "about", the invention provides an embodiment in which the exact value is recited. In any embodiment of the invention in which a numerical value is not prefaced by "about", the invention provides an embodiment in which the value is prefaced by "about". Where the phrase "at least" precedes a series of numbers, it is to be understood that the phrase applies to each number in the list (it being understood that, depending on the context, such as in describing percent identity, 100% of a value may be an upper limit.) It is also understood that any one or more embodiments, features, or aspects of the present invention, or any combination thereof, may be explicitly excluded from any one or more of the claims.

Claims

We claim:

1. A method of using a yeast cell to produce a product, the method comprising (a) providing a yeast cell to be used in production of a product; (b) modulating acquisition, maintenance, or loss of a prion in the yeast cell, wherein the prion affects yeast fermentation or growth; and (c) using the yeast cell to produce the product.

2. The method of claim 1, wherein producing the product comprises using the yeast cell to perform fermentation.

3. The method of claim 1 , wherein modulating acquisition, maintenance, or loss of a prion comprises enhancing the acquisition, maintenance, or loss of the prion.

4. The method of claim 1, wherein modulating acquisition, maintenance, or loss of a prion comprises inhibiting the acquisition, maintenance, or loss of the prion.

5. The method of claim 1, wherein modulating acquisition of the prion comprises modulating induction of the prion.

6. The method of claim 1 , wherein modulating acquisition o the prion comprises inducing the prion.

7. The method of claim 1, wherein modulating acquisition of the prion comprises inhibiting induction of the prion.

8. The method of claim 1, wherein the prion alters alcohol production by the yeast cell.

9. The method of claim 1, wherein the prion decreases alcohol production by the yeast cell.

10. The method of claim 1 , wherein the prion alters alcohol tolerance of the yeast cell.

1 1. The method of claim 1 , wherein the prion increases alcohol tolerance of the yeast cell.

12. The method of claim 1, wherein the prion alters utilization of at least one carbon source by the yeast cell.

13. The method of claim 1 , wherein the prion allows the yeast cell to use at least one non- preferred carbon source in the presence of a preferred carbon source.

14. The method of claim 1 , wherein the prion allows the yeast cell to use at least one non- preferred carbon source in the presence of glucose.

15. The method of claim 1 , wherein the prion allows the yeast cell to use at least one non- preferred sugar as a carbon source in the presence of glucose.

16. The method of claim 1, wherein the prion increases the growth rate of the yeast cell.

17. The method of claim 1 , wherein the prion increases the life span of the yeast cell.

18. The method of claim 1, wherein the prion increases the total biomass achievable in a yeast culture.

19. The method of claim 1 , wherein the product is a beverage or food.

20. The method of claim 1, wherein the product is a wine, a beer, a mead, a sake (nihonshu), a dough, or a batter.

21. The method of claim 1 , wherein the product is a biofuel.

22. The method of claim 1, wherein the product is ethanol.

23. The method of claim 1, wherein the product is a small molecule, a fine chemical, a therapeutic agent, or a protein.

24. The method of claim 1, wherein using the yeast cell in producing the product comprises culturing the yeast cell in a medium comprising at least two or at least three different sugars, each present at at least 0.1% (w/v).

25. The method of claim 1, wherein using the yeast cell in producing the product comprises culturing the yeast cell in a medium comprising no more than 1 % glucose (w/v).

26. The method of claim 1, wherein using the yeast cell in producing the product comprises culturing the yeast cell in a medium comprising or derived at least in part from grape must, wort, molasses, rice, honey, a grain (e.g., wheat, corn, rice, barley), or potato.

27. The method of any of the preceding claims, wherein modulating acquisition of the prion comprises contacting the yeast cell with a bacterium or secreted bacterial product that modulates acquisition of the prion.

28. The method of any of the preceding claims, wherein modulating acquisition of the prion comprises inducing the prion, and wherein inducing the prion comprises contacting the yeast cell with a bacterium or secreted bacterial product that induces the prion.

29. The method of any of the preceding claims, wherein the yeast cell is not a laboratory strain.

30. The method of any of the preceding claims, wherein the yeast cell is not genetically engineered.

31. The method of any of the preceding claims, wherein the yeast cell is genetically engineered.

32. The method of any of the preceding claims, wherein the yeast cell is a member of a species that exhibits glucose-mediated repression of the utilization of other carbon sources.

33. The method any of the preceding claims, wherein the yeast cell is a Saccharomyces cell.

34. The method of any of the preceding claims, wherein the yeast cell is a Saccharomyces cerevesiae cell.

35. The method of any of the preceding claims, wherein the yeast cell is a member of a wild strain.

36. The method of any of the preceding claims, wherein the yeast cell is a wine yeast, a brewer's yeast, or a baker's yeast.

37. The method of any of the preceding claims, wherein the yeast cell is a member of a yeast culture, and wherein using the yeast cell to produce the product comprises using the yeast cell culture to produce the product.

38. The method of any of the preceding claims, wherein the yeast cell is a member of a yeast culture, and wherein the method comprises testing the yeast cell or yeast cell culture for the prion status at least once during production of the product.

39. The method of any of the preceding claims, wherein the yeast cell is a member of a yeast culture, and wherein the method comprises testing the yeast cell or yeast cell culture for prion status at least once during production of the product, and wherein testing the yeast cell or yeast cell culture for prion status comprises measuring the level of expression of a gene that is regulated by the prion.

40. The method of any of the preceding claims except claim 30, wherein the yeast cell is a member of a yeast culture, and wherein the method comprises testing the yeast cell or yeast culture for prion status at least once during production of the product, and wherein testing the yeast cell or yeast culture for prion status comprises measuring the level of expression of a gene that is regulated by the prion, wherein the gene is a reporter gene.

41. The method of any of claims 38 to 40, wherein the method further comprises modifying the culture or culture conditions based at least in part on the prion status.

42. The method of claim 41, wherein altering the culture or culture conditions comprises adding additional yeast cells to the culture, changing the composition of the culture medium, adding an additional carbon source to the culture, adding an inducer or inhibitor to the culture, or altering the temperature or pH of the culture.

43. The method of any of the preceding claims, wherein the prion is [GAR f ].

44. A method of performing at least one step of an industrial process, the method

comprising: (a) providing a yeast culture capable of performing at least one step of an industrial process; (b) modulating the [GAR+] prion in at least some yeast cells in the culture; and (c) using the yeast culture to perform at least one step of the industrial process.

45. The method of claim 44 wherein the industrial process is production of a beverage, food, small molecule, fine chemical, biofuel, therapeutic agent, protein, or component of any of the foregoing, and wherein modulating the [GAR+] prion comprises inducing or stabilizing [GAR+].

46. The method of claim 44 wherein the industrial process is production of a reduced alcohol beverage, and modulating the [GAR+] prion comprises inhibiting acquisition of [GAR+] or eliminating [GAR+].

47. The method of claim 44 wherein the yeast culture comprises at least some yeast cells that are not a laboratory strain.

48. The method of claim 44 wherein the the yeast culture comprises at least some yeast cells that are not genetically engineered.

49. The method of claim 44 wherein the yeast culture comprises at least some yeast cells that are genetically engineered.

50. The method of claim 44 the yeast culture comprises at least some yeast cells that are members of a species that exhibits glucose-mediated repression of the utilization of other carbon sources.

51. The method of claim 44 wherein the yeast culture comprises at least some yeast cells that are Saccharomyces cells.

52. The method of claim 44 wherein the yeast culture comprises at least some yeast cells that are Saccharomyces cerevesiae cells.

53. The method of claim 44 wherein the yeast culture comprises at least some yeast cells that are of a wild strain.

54. The method of claim 44 wherein the yeast culture comprises at least some yeast cells that are wine yeast, brewer's yeast, or baker's yeast.

55. The method of claim 44, wherein the method comprises testing the yeast culture for [GAR+] status or for a [ GAR+] modulating agent at least once prior to or during performance of the industrial process.

56. The method of claim 44, wherein the method comprises testing the yeast culture for [GAR+] status at least once prior to or during performance of the industrial process, and wherein testing the yeast culture for [GAR+] status comprises measuring the level of expression of a gene in at least one yeast cell of the culture, wherein the gene is regulated by [GAR+].

57. The method of claim 44, wherein the method comprises testing the yeast culture for [GAR+] status at least once prior to or during performance of the industrial process, and wherein testing the yeast cell culture for [GAR+] status comprises measuring the level of expression in at least one of a gene in at least one yeast cells of the yeast culture, wherein the gene is regulated by [GAR+], and wherein the gene is a reporter gene.

58. The method of claim 44, wherein the method comprises testing the yeast culture for [GAR+] status or for a [GAR+] modulating agent at least once prior to or during performance of the at least one step of the industrial process, and wherein testing the yeast culture for a [GAR+] modulating agent comprises testing the culture for a [GAR+] inducing bacterium or for a

[GAR+] inducing molecule secreted by a [GAR+] inducing bacterium.

59. The method of claim 58, wherein the [GAR+] inducing bacterium is a member of a genus having at least one species listed in Table A.

60. The method of claim 58, wherein the [GAR+] inducing bacterium is listed in Table A.

61. The method of claim 58, wherein the [GAR+] inducing bacterium is a wine bacterium listed in Table A.

62. The method of claim 58, wherein the [GAR+] inducing bacterium is a wine bacterium listed in Table A, and wherein the industrial process is production of a wine.

63. The method of any of claims 47 to 62 wherein the method further comprises modifying the culture or culture conditions based at least in part on the [GAR+] status or based at least in part on presence, absence, or amount of a [GAR+] modulating agent.

64. The method of claim 63, wherein altering the culture or culture conditions comprises adding additional yeast cells to the culture, changing the composition of the culture medium, adding an additional carbon source to the culture, adding an inducer or inhibitor to the culture, or altering the temperature or pH of the culture.

65. A method of performing at least one step of an industrial process, the method comprising: (a) providing a yeast cell culture capable of performing at least one step of an industrial process, wherein the yeast culture comprises yeast cells that are selected or genetically engineered to have enhanced acquisition or stabilized maintenance of [GAR+]; and (b) using the yeast culture to perform at least one step of the industrial process.

66. The method of claim 65, wherein the yeast cells that are selected or genetically engineered to have enhanced acquisition or stabilized maintenance of [GAR+] have at least one functionally inactivated gene, wherein the functionally inactivated gene is a DEGA gene, e.g., a DEGA gene listed in Table B.

67. A method of performing at least one step of an industrial process, the method comprising: (a) providing a yeast culture capable of performing at least one step of an industrial process, wherein the yeast culture comprises yeast cells that are selected or genetically engineered to have impaired acquisition of [GAR+ and (b) using the yeast culture to perform at least one step of the industrial process.

68. The method of claim 67, wherein the yeast cells that are selected or genetically engineered to have impaired acquisition of [GAR+] have at least one functionally inactivated gene, wherein the functionally inactivated gene is a DRGA gene, e.g., a DRGA gene listed in Table C.

69. A method of generating a yeast cell that has enhanced acquisition of [GAR+l the method comprising functionally inactivating at least one DEGA gene in the yeast cell, wherein the DEGA gene is listed in Table B; and (b) verifying that the yeast cell has enhanced acquisition of [GAR+l

70. A method of generating a yeast cell that has impaired acquisition of [GAR+], the method comprising functionally inactivating at least one DRGA gene in the yeast cell, wherein the DRGA gene is listed in Table C; and (b) verifying that the yeast cell has impaired acquisition of [GAR+l

71. The method of claim 69 or 70, further comprising preparing a yeast culture from the yeast cell.

72. The method of claim 69 or 70, further comprising preparing a yeast culture from the yeast cell and using the yeast culture to produce a product.

73. The method of claim 69 or 70, further comprising preparing a yeast culture from the yeast cell and using the yeast culture to produce a beverage, food, small molecule, fine chemical, biofuel, therapeutic agent, or protein.

74. The method of claim 69 or 70, further comprising preparing a yeast culture from the yeast cell and using the yeast culture to produce a wine, beer, mead, sake, dough, or batter.

75. The method of claim 69 or 70, further comprising preparing a yeast culture from the yeast cell and using the yeast culture to perform at least one step of an industrial process.

76. A method that comprises inducing or enhancing acquistition of [GAR+] in a cell, e.g., a yeast cell, wherein the method comprises overexpressing, e.g., transiently overexpressing, at least a portion of the STD1. DOG2, or PMA1 gene in the cell or functionally inactivating at least one DEGA gene.

77. A method that comprises inhibiting induction of [GAR+] in a cell, e.g., a yeast cell, wherein the method comprises overexpressing at least a portion of the MTH1 gene in the cell or functionally inactivating at least one DRGA gene.

78. A yeast cell with a stable [gar^~] phenotype, wherein the yeast cell optionally lacks a functional STD1, RGT1, MTH1, SUR4 or LST1 gene or optionally lacks a functional DRGA gene.

79. A method of generating a yeast cell that has enhanced acquisition of [GAR+], the method comprising functionally inactivating at least one DEG ("Deletions Enhance [GAR+]") gene in the yeast cell, wherein the DEG gene is listed in Table B; and (b) verifying that the yeast cell has enhanced acquisition of [GAR+].

80. A method of determining the [GAR+] status of a yeast cell comprising assessing the ability of the yeast cell to grow on glycerol in the presence of glucosamine, wherein loss of the ability to grow on glycerol in the presence of glucosamine indicates that the yeast cell lacks the [GAR+] prion.

81. A method of determining the [GAR+] status of a yeast cell comprising measuring the expression of at least one gene whose expression is regulated directly or indirectly by [GAR+].

82. A method of determining the [GAR+] status of a yeast cell comprising measuring the expression of the gene that encodes Hexose Transporter 3 (HXT3), wherein decreased expression of the gene indicates that the yeast cell is [GAR^■ \.

83. A method of inhibiting or reducing the likelihood of stuck fermentation or spoilage in a yeast culture, the method comprising inhibiting acquisition of [GAR+] by yeast cells in the yeast culture.

84. A method of testing a yeast culture, culture medium, or culture medium componen t to assess its suitability for use in a process of interest, the method comprising testing the culture for presence of at least one bacterial species or substance capable of inducing [GAR+] in yeast cells.

85. The method of claim 84, wherein the bacterial species is listed in Table A or is a member of a genus of which at least one species is listed in Table A. ου. i lie mciiiuu υι c iiin ot, wnerem me cuiiure mcuium or culture medium component comprises or is derived at least in part from grape must, wort, molasses, rice, honey, a grain (e.g., wheat, corn, rice, barley), or potato.

87. A method of preparing a yeast culture or culture medium, the method comprising (a) providing at least one standard yeast culture medium component; and (b) combining a [GAR+] modulator with the at least one standard yeast culture medium component.

88. The method of claim 87, wherein the at least one standard yeast culture medium component comprises at least one sugar.

89. The method of claim 87, wherein the [GAR+] modulator is a [GAR+] inducer.

90. The method of claim 87, wherein the [GAR+] modulator is a [GAR+] inducer comprising bacteria, wherein the bacteria are of a bacterial species listed in Table A or are of a genus of which at least one species is listed in Table A.

91. The method of claim 87, wherein the [GAR+] modulator is a [GAR+] inducer comprising a small molecule secreted by bacteria, wherein the bacteria are of a bacterial species listed in Table A or are of a genus of which at least one species is listed in Table A.

92. A method of identifying an agent that modulates acquisition or loss of a prion of interest by a cell, the method comprising: (a) providing a cell, wherein the cell is of a species known to be capable of harboring the prion of interest; (b) contacting the cell with a test agent other than the prion of interest or a variant thereof; and (c) assessing the cell for acquisition or loss of the prion of interest, wherein acquisition or loss by the cell of the prion of interest indicates that the test agent modulates acquisition or loss of the prion of interest by the cell.

93. The method of claim 92, wherein the cell does not harbor the prion of interest and step (c) comprises assessing the cell for acquisition of the prion of interest.

94. The method of claim 92, wherein the cell harbors the prion of interest and step (c) comprises assessing the cell for loss of the prion of interest.

95. The method of claim 92, wherein the cell is a fungal cell.

96. The method of claim 92, wherein the cell is a yeast cell.

The method of claim 92, wherein the test agent is a small molecule. V8. ine method ot claim 92, wherein the test agent is a microorganism, a culture medium conditioned by a microorganism, or one or more molecules or fractions obtained from a culture medium conditioned by a microorganism.

99. The method of claim 92, wherein the test agent is a microorganism, a culture medium conditioned by a microorganism, or one or more molecules or fractions obtained from a culture medium conditioned by a microorganism, wherein the microorganism occurs naturally in association with the cell.

100. The method of claim 92, wherein the prion of interest confers a phenotype on the cell, wherein the phenotype comprises a survival advantage under at least some conditions or an alteration in metabolism

101. The method of claim 92, wherein assessing the cell for acquisition or loss of the prion of interest comprises testing the cell to determine whether the cell exhibits at least one phenotype conferred by the prion of interest.

102. The method of claim 92, wherein the prion is [GAR+].

103. A yeast cell culture comprising yeast cells and an inducer or inhibitor of [GAR+].

104. The yeast cell culture of claim 103, wherein the yeast cells comprise non-laboratory strain yeast cells, S. cerevesiae, wine yeast, brewer's yeast, wild yeast, or a combination thereof.

105. The yeast cell culture of claim 103 or 104 and a culture medium useful for producing a wine, beer, mead, batter, dough, biofuel, small molecule, fine chemical, or therapeutic agent.