WO2014022692A1 - Manipulation à base de prions de la fermentation et la croissance de levures - Google Patents

Manipulation à base de prions de la fermentation et la croissance de levures Download PDF

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WO2014022692A1
WO2014022692A1 PCT/US2013/053272 US2013053272W WO2014022692A1 WO 2014022692 A1 WO2014022692 A1 WO 2014022692A1 US 2013053272 W US2013053272 W US 2013053272W WO 2014022692 A1 WO2014022692 A1 WO 2014022692A1
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yeast
gar
culture
prion
cell
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PCT/US2013/053272
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English (en)
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Daniel JAROSZ
Jessica C.S. BROWN
Susan Lindquist
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Whitehead Institute For Biomedical Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells

Definitions

  • Prions 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.
  • TSEs mammalian spongiform encephalopathies
  • the ability of prions to convert between structurally and functionally distinct states 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.
  • 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.
  • 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.
  • the [GAR ] prion alters carbon source utilization by the yeast Saccharomyces cerevisiae.
  • [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.
  • [GAR+] thus increases efficiency of using yeast to produce virtually any small molecule, e.g., a fine chemical or a therapeutic agent.
  • [GAR+] is of use in biofuel production.
  • [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.
  • lower alcohol content products e.g., reduced alcohol content beer, wine, or other fermented beverage
  • [GAR+] allows yeast cells to tolerate higher ethanol
  • inhibiting [GAR+] helps increase alcohol production as a biofuel.
  • biofuel fermentations are often not sterile and bacteria can switch on [GAR+], causing the cells to make less alcohol.
  • inhibiting [GAR+] induction is useful in production of ethanol, e.g., as a biofuel.
  • such production uses a cellulosic or non-cellulosic biomass as a feedstock.
  • [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.
  • lower alcohol content products e.g., reduced alcohol content beer, wine, or other fermented beverage
  • [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
  • 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
  • yeast mutants that have enhanced ability to undergo the switch to the [GAR+] state in the presence of bacteria that are capable of inducing
  • RNA interference RNA interference
  • 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.
  • yeast strains that have enhanced or impaired acquisition (e.g., induction) of [GAR+] are disclosed herein.
  • standard methods of yeast genetics can be used to construct deletion mutants.
  • resulting yeast are used in producing a product or performing at least one step of an industrial process.
  • 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.
  • the RNA is HXT3 RNA.
  • quantitative PCR for the RNA is performed.
  • a sample comprising cells is removed from a culture at one or more time points and tested for [GAR+] cells.
  • a method comprises eliminating [GAR+] by growing cells on or in medium comprising a [GAR+] inhibitor.
  • 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.
  • the glutamine analog is azaserine (0-(2- Diazoacetyl)-L-serine).
  • a [GAR+] inhibitor comprises a flavonol.
  • the flavonol is myrecitin (3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)- 4- chromenone).
  • the flavonol is quercetin (2-(3,4-dihydroxyphenyl)- 3,5,7-trihydroxy-4H-chromen-4-one).
  • a [GAR+] inhibitor comprises an Hsp70 inhibitor.
  • the Hsp70 inhibitor is a flavonol.
  • an Hsp70 inhibitor, e.g., a flavonol is a compound that occurs naturally in a composition of interest herein, e.g., a wine.
  • 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.
  • 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.
  • the Hsp70 inhibitor is a compound of the following formula:
  • Ri, R 2 , R 3 , R4 and R 5 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;
  • R a and Rb are the same or different and
  • Q represents ⁇ C ⁇ C-, and Ri, R 2 , R 3 , 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.
  • Q represents— C ⁇ C—
  • Q represents— C ⁇ C— , exactly one or at least one of Ru R 2 , R 3 , R4, and R 5 represents halogen, and either or both of R a and R optionally is hydrogen.
  • Q represents -C ⁇ C— , exactly one or at least one of R l5 R 2 , R 3 , R4, and R 5 represents halogen, and R a , R b or both is hydrogen.
  • the compound is of the following formula:
  • 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,
  • R a and R b are the same or different and represent a radical selected from the group of hydrogen, hydroxyl, alkoxy, amino,
  • alkyl substituent being at least one selected from the group consisting of hydroxyl, thio, alkoxy, alkylthio, halogen, amino, monalkylamino,
  • 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.
  • the Hsp70 inhibitor of Formula I is 2-phenylethynesulfonamide (PES, also referred to as pifithrin- ⁇ ) (Liu, JI, et al., (2009) Molecular Cell, 36: 15-27; US Pat. Pub. No. 201 10189125).
  • the Hsp70 inhibitor of Formula I or II is 2-(3-chlorophenyl) ethynesulfonamide.
  • the Hsp70 inhibitor is 2-aminopurine.
  • 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.
  • 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-].
  • 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 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , or more, or any intervening range or value.
  • 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 10 2 , between 10 2 and 10 3 , between 10 3 and 10 4 , between 10 4 and 10 5 , or between 10 5 and 10 6 .
  • a [GAR -] inhibitor or inducer is substantially non-toxic to yeast at the concentrations in which it usefully inhibits or induces [GAR+].
  • a [GAR+] inhibitor or inducer is substantially non-toxic to mammalian cells at such concentrations.
  • 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.
  • a product is purified from a culture such that a [GAR+] inhibitor or inducer is not detectably present in the product.
  • 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+].
  • the difference in the number of cells that grow on rich medium e.g., YPD or a similarly rich medium
  • rich medium containing a [GAR+] inhibitor such as azaserine is used to measure the fraction of [GAR+] cells in a culture or other composition.
  • a culture or composition comprising yeast cells is tested for [GAR+] cells at one or more time points.
  • 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+].
  • the modulator of [GAR+] is a bacterium or bacterial product that induces [GAR+].
  • 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.
  • 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.
  • a continuous or semi-continuous monitoring method is used.
  • 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.
  • a culture medium is inoculated with a "starter culture” (or sample thereof) or comprising yeast cells.
  • a starter culture may be tested for [GAR+] cells.
  • 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.
  • 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.
  • 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.
  • a starter culture may be exposed to a [GAR+] modulator, e.g., a [GAR+] inducer or [GAR+] inhibitor.
  • 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.
  • 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.
  • the volume of a starter culture is no more than about 0.1%, 0.5%, 1%, 2%, 5%, or 10% of the larger culture.
  • 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.
  • a starter culture may use a different culture medium to the culture medium in the culture it is used to inoculate.
  • a starter culture may use a peptone-based yeast culture medium such as YPD, or YP plus a different carbon source.
  • a culture may be inoculated at a concentration between
  • a culture may be inoculated at a concentration 3 x 10 6 cells per ml.
  • 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.
  • 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+].
  • 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+].
  • 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.
  • 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.
  • modifying the culture or composition comprises adding a nutrient to the composition.
  • the nutrient is one or more carbon sources, e.g., one or more sugars, e.g., glucose.
  • the nutrient is one or more amino acids or nitrogen sources.
  • 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.
  • 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.
  • modifying the culture or composition comprises changing the temperature or pH.
  • a bacterium is a strong inducer.
  • a bacterium is an intermediate inducer.
  • 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.
  • 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+].
  • a bioassay is used.
  • the bioassay comprises assessing the ability of the bacteria to induce [GAR+] in yeast, e.g., S. cerevesiae.
  • 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+].
  • the invention relates to interkingdom communication as a mode of altering prion acquisition (e.g., prion induction), maintenance, or loss.
  • the interkingdom communication is between a prokaryote and a eukaryote.
  • the prokaryote is a bacterium.
  • the eukaryote is a fungus.
  • the fungus is a yeast.
  • the yeast is a budding yeast.
  • 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.
  • a non-bacterial cell e.g., a fungal cell.
  • the fungal cell is a yeast cell.
  • 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.
  • first and second microorganisms e.g., a fungus and a bacterium
  • 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.), where
  • 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 10 4 , or at least 10 5 individuals or colony forming units of each
  • 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.
  • 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.
  • a small organic molecule acts as a prion inducer, i.e., induces prion acquisition.
  • inducer 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.
  • the rate of prion acquisition is increased by a factor of at least 10; 100; 10 3 ; or 10 4 , e.g., between 10 and 100-fold, between 100 and 10 3 - fold, between 10 3 -fold and 10 4 -fold, between 10 4 -fold and 10 5 -fold, or between 10 5 -fold and 10 6 -fold, between 10 6 -fold and 10 7 -fold.
  • small molecules that modulate 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.
  • a microorganism e.g., a bacteria, or a cell lysate prepared from the microorganism.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention provides methods of using the small molecule to incude [GAR+] in yeast, e.g., S. cerevesiae.
  • yeast e.g., S. cerevesiae.
  • the small molecule is used as a component of a yeast culture.
  • the yeast culture is used to produce a product.
  • the yeast culture is used in an industrial process, e.g., to perform at least one step of an industrial process.
  • 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"
  • 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.
  • 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
  • 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+].
  • 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 .
  • 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 ⁇ 2 test.
  • Strain background is a hybrid of W303 and S288C.
  • FIG. 4 Alterations to Pmal affect [GAR+].
  • B Propagation of [GAR+] is impaired in ⁇ 1 ⁇ 40 ⁇ Astdl double mutants.
  • 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).
  • Fig. 7. A prion-based reversal of glucose repression. Glucose represses transcription of genes involved in utilization of alternative carbon sources.
  • FIG. 8. [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).
  • Fig. 15 Bacteria elicit [GAR + ] by secreting a prion-inducing factor.
  • Fig. 17. confers advantages to yeast and bacteria alike: reduced ethanol production for bacteria.
  • [GAR + ] shifts the outcome of microbial competition in fermentations.
  • Fig. 21 A model for [GAR+] conservation.
  • Fig. 22 A model for [GAR+] conservation.
  • 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.
  • [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.
  • 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 (" ⁇ "), 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").
  • Fig. 26 Transcriptional profiling of [gar ' ] and [GAR + ] cells.
  • SAM microarrays
  • 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).
  • 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.
  • RGT1 is not the causal agent of [GAR*] because [GAR*] can be propagated from Argtl to wild-type cells.
  • Fig. 28 Induction of [GAR*] by STD1 and DOG2.
  • Fig. 29 Immunoprecipitation of Stdl-6HA from [gar ] and [GAR + ] cells.
  • 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).
  • 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.
  • Fig. 33 ⁇ 1 ⁇ 40 ⁇ propagates [GAR + ]. Top: tetrad spores from a [GAR + ] diploid with the genotype GAL- PMA1A40N/PMA1. The pmal mutation is marked with His + .
  • Fig. 34 Pmal and Stdl do not change localization between [gar ] and [GAR + ] cells
  • Fig. 35 Pmal and Stdl do not form SDS-resistant species in [gar ] or [GAR + ] cells.
  • 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].
  • [PSf ], [URE3], and [RNQ + ] do not alter [GAR + ] frequencies.
  • [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.
  • strains carrying [PSf] sometimes showed a lower [GAR + ] frequency (BY) and sometimes a higher one (W303 and 74D).
  • 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.
  • 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.
  • 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.
  • Antibody 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.
  • rodent e.g., murine
  • avian e.g., chicken
  • 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.
  • 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.
  • transcription of a sequence of interest can be irreversibly turned on or off using the Cre/Lox or Flp/FRT recombinase system.
  • a nucleic acid "stuffer sequence" can be positioned between sites for a recombinase.
  • Delivering the recombinase to a cell results in excision of the stuffer sequence.
  • 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.
  • 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.
  • TGA and TGB are the sum of the number of residues and internal gap positions in sequences A and B in the alignment
  • 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).
  • Gapped BLAST is utilized as described in Altschul et al. (Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997).
  • Altschul et al. Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • a substituted pyrimidine e.g. cytosine (C), thymidine (T) or uracil (U)
  • purine e.g. adenine (A) or guanine (G)
  • 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).
  • nucleic acid sequence or nucleotide sequence
  • nucleotide subunits are typically indicated using the abbreviation of the base, e.g., A, G, C, T, U.
  • 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
  • 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.
  • a nucleic acid is composed of RNA.
  • a nucleic acid is composed of DNA.
  • a double-stranded nucleic acid may have one or more overhangs (5 ' and/or 3 ' overhangs).
  • a nucleic acid comprises standard nucleotides (A, G, C, T, U).
  • 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.
  • 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.
  • 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.
  • 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.
  • a purified nucleic acid or nucleic acids
  • a purified polypeptide is substantially separated from cellular polypeptides.
  • the ratio of nucleic acid to polypeptide is at least 5:1 or at least 10: 1 by dry weight.
  • a purified polypeptide is separated from cellular nucleic acids.
  • the ratio of nucleic acid to polypeptide is at least 5: 1 or at least 10: 1 by dry weight.
  • 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.
  • water, buffers, ions, and/or small molecules e.g., precursors such as nucleotides or amino acids
  • 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.
  • 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.
  • "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.
  • 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
  • a variant can be shorter or longer than the polypeptide or polynucleotide of which it is a variant.
  • a "variant" comprises a "fragment".
  • fragment refers to a portion of a polynucleotide or polypeptide that is shorter than the original polynucleotide or polypeptide.
  • 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.
  • 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.
  • 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.
  • a variant polypeptide has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%
  • polynucleotide or polypeptide is generated using recombinant DNA techniques.
  • amino acid substitutions replace one amino acid with another amino acid having similar structural and/or chemical properties, e.g., conservative amino acid replacements.
  • Constant 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.
  • 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.
  • 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.
  • 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).
  • homologous functional polypeptides e.g., orthologs from other organisms.
  • isolated 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.
  • 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).
  • an isolated nucleic acid or polypeptide is chemically synthesized.
  • an isolated nucleic acid is removed from its genomic context.
  • an isolated nucleic acid is joined to a nucleic acid to which it is not joined in nature.
  • 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.
  • 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.
  • selectable marker 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.
  • selectable marker can also refer to the gene product itself.
  • 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.
  • yeast examples 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),
  • AP alkaline phosphatase
  • LacZ beta galactosidase
  • GUS beta glucoronidase
  • chloramphenicol acetyltransferase CAT
  • HRP horseradish peroxidase
  • Luc lucifera.se
  • genes can also be used as reporters or controls, e.g., to assess the presence of a prion, e.g., [GAR+].
  • 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.
  • the two sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
  • adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA.
  • 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.
  • 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.
  • RNA small interfering RNA
  • siRNA refers in some
  • RNA molecule derived from the successive cleavage of longer double- stranded RNA e.g., within a cell by an enzyme comprising an RNase III domain
  • dsRNA double- stranded RNA
  • an enzyme comprising an RNase III domain
  • 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.
  • 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 or more nucleotides at the 3 ' end of an siRNA is/are deoxyribonucleotide(s), e.g., dT.
  • 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.
  • 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.
  • 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.
  • Yeast prions provide a mechanism for generating heritable phenotypic diversity that promotes survival in fluctuating environments and the evolution of new traits.
  • 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.
  • 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.
  • [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.
  • 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+] allows yeast to use non-preferred carbon sources in the presence of the preferred carbon source, glucose.
  • 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.
  • [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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • a solid medium comprises agar or another solidifying or gelling agent.
  • a culture medium comprises material derived from grapes.
  • 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.
  • 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.
  • grapes or grape-derived material may be measured by volume. In some embodiments grapes or grape-derived material may be measured by weight.
  • 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.
  • 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.
  • a culture medium comprises material derived from sugar cane, corn, sorghum, grass, or wood.
  • a culture medium comprises a lignocellulosic material such as wood, bagasse, or straw.
  • a culture medium comprises sugar derived from a lignocellulosic material such as wood, bagasse, or straw, which has been subjected to cellulolysis.
  • 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).
  • antibody or other affinity-based methods can be used, e.g., Western blots, enzyme linked immunosorbent assay (ELISA), Western blotting.
  • ELISA enzyme linked immunosorbent assay
  • 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.
  • FACS fluorescence activated cell sorting
  • 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).
  • a method of testing for a prion comprises detecting a prion confirmation using a prion-specific antibody.
  • a yeast is a budding yeast.
  • a budding yeast is a member of the subphylum Saccharomycotina.
  • 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.
  • a budding yeast is a member of the Saccharomyces sensu stricto.
  • the Saccharomyces sensu stricto genus includes S. cerevisiae, and at least seven other natural species (S. paradoxus, S. cariocanus, S.
  • a budding yeast is Naumovozyma castellii (also referred to as Saccharomyces castellii).
  • a yeast is of a species or strain used in wine-making, brewing, food production, or biofuel production.
  • a yeast is dimorphic. Such yeast exhibits budding under some environmental conditions.
  • Arxula adeninivorans (Blastobotrys adeninivorans) is a dimorphic yeast useful in various biotechnological applications.
  • a strain is a wild strain, as recognized in the art.
  • a yeast is a fission yeast such as fission yeast Schizosaccharomyces pombe.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • ATCC American Type Culture Collection
  • 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:
  • 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.
  • 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).
  • the yeast is a wine yeast.
  • 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
  • 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;
  • Yeast 12089 Sacharomyces cerevisiae SF4 Stuck ; fermentation j
  • Rhodotorula mucilaginosa BBL4 [wine, barrel
  • Yeast (2533 (Saccharomyces cerevisiae dry wine yeast commercial
  • Yeast 2538 Sacharomyces (cerevisiae BDX dry wine yeast commercial
  • 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.
  • 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).
  • a yeast strain e.g., a yeast strain used in brewing, is Ale 514, Lager 497, or Weiss (all available from AB Mauri).
  • a yeast strain e.g., a yeast strain used in wine production
  • 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).
  • a yeast strain e.g., a yeast strain used in wine production
  • a yeast strain e.g., a yeast strain used in wine production
  • Prise de Mousse strain such as S92.
  • a Zymoflore® yeast strain or Actiflore® yeast strain may be used, e.g., in wine production.
  • a yeast blend may be used.
  • an Anchor Alchemy I or II yeast blend may be used in wine production.
  • a yeast strain e.g., a yeast strain used in biofuel production, is Ethanol Red® (available from Lesaffre).
  • a yeast strain e.g., a yeast strain used in biofuel production
  • 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/).
  • a yeast strain e.g., a yeast strain used in biofuel production
  • 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.
  • 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.
  • 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
  • a [GAR+] modulator such as a [GAR+] inhibitor or inducer or by using a yeast strain that has impaired or enhanced [GAR+] acquisition
  • an "industrial" modulator such as a [GAR+] inhibitor or inducer or by using a yeast strain that has impaired or enhanced [GAR+] acquisition
  • 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.
  • a product is an end product to be used directly by humans, e.g., consumed, used as a medication, or used as fuel.
  • 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.
  • a product is packaged in a suitable container after production.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a product is a beverage, e.g., a fermented beverage such as wine, beer, cider, sake, mead, or the like.
  • a wine is a red wine.
  • a wine is a white wine.
  • 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).
  • 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.
  • a beverage e.g., a wine or beer
  • 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.
  • a product e.g., a beverage, e.g., a wine, bears a label indicating a particular appellation that applies to the wine.
  • an appellation is defined by the French Institut National des Appellations d'Origine (INAO), now called the Institut national de l'rare et de la qualite (INAO).
  • INAO French Institut National des Appellations d'Origine
  • AOC Appellation d'Origine Controlee
  • 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.
  • Pinot e.g., Pinot Noir, Pinot Grigio
  • Merlot Syrah
  • Chardonnay Chianti
  • Cabernet Sauvignon Cabernet Sauvignon blanc
  • Riesling Muller Thurgau
  • Kerner Kerner
  • Sylvanor Chenin blanc
  • Semillon Semillon
  • grapes e.g., grapes used in winemaking
  • grapes may be of the species Vitis vinifera.
  • 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.
  • grapes are red grapes.
  • 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).
  • grapes are white grapes.
  • 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
  • grapes are purple or black grapes, e.g., Malbec, Mustadine.
  • 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.
  • 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).
  • 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.
  • 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).
  • the product is a beverage, e.g., wine or beer.
  • a low alcohol wine has an alcohol content of below about 12% ABV, e.g., between 5% and 1 1% ABV.
  • a low alcohol beer has an alcohol content of 0.05%-1.2% ABV.
  • 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.
  • 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.
  • 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.
  • 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.
  • the product is a biofuel, e.g., ethanol.
  • an industrial biotransformation e.g., an industrial fermentation
  • 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.
  • [GAR+] is modulated during at least one of the stages.
  • 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.
  • a container, or at least the inner walls of the container is made of steel or wood.
  • a fermentation is conducted in a closed container.
  • a fermentation is conducted in an open container.
  • a fermentation is conducted inside a wine bottle.
  • 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.
  • microorganisms such as bacteria, fungi (e.g., yeast), or both.
  • an industrial fermentation is an example of an industrial biotransformation.
  • 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).
  • ATP adenosine triphosphate
  • fermentation can be and often is carried out in an anaerobic environment.
  • 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.
  • a fermentation takes place over a period of between 6 and 12 hours, 12 and 24 hours, 24 hours to 3 days.
  • 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.
  • a first period of fermentation may be followed by a second period of fermentation .
  • 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.
  • 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.
  • 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.
  • a [GAR+] modulator may be covalently or
  • yeast may be immobilized to a surface or matrix.
  • the matrix comprises particles such as beads.
  • a surface is an inner wall or floor of a container in which yeast are cultured.
  • a matrix comprises particles such as beads.
  • 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
  • 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.
  • the vector is a plasmid.
  • Other vectors include artificial chromosomes and linear nucleic acid molecules that are distinct from linearized plasmids.
  • the vector is an integrating vector.
  • 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 2 ⁇ 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.
  • plasmids of use in budding yeast are YIp plasmids, which are maintained at one copy per haploid genome and inherited in Mendelian fashion.
  • 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 2 ⁇ plasmids are YEp plasmids, which contain a sequence approximately 1 kb in length (named the 2 ⁇ sequence). The 2 ⁇ sequence acts as a yeast replicon giving rise to higher plasmid copy number. These plasmids may require selection for maintenance.
  • an integrating plasmid is a pRS plasmid (e.g., pRS303, pRS304, pRS305 or pRS306 or other integrative plasmids).
  • the plasmid is an extrachromosomal plasmid (e.g., pRS313, pRS314, pRS315, pRS316, pRS413, pRS414, pRS415, pRS416, pRS423, pRS424, pRS425, pRS426).
  • the plasmid is a member of the YESTM Vector Collection, e.g., pYES (Invitrogen, Carlsbad, CA).
  • 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).
  • 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 10 4 colony- forming units (transformed cells) ⁇ g of DNA.
  • 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.
  • any suitable means of introducing nucleic acids into yeast cells can be used, such as
  • yeast vectors 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
  • 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
  • a yeast vector contains one or more expression control sequences, e.g., promoter sequences.
  • 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.
  • 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.
  • a constitutive promoter is used.
  • a regulatable, e.g., inducible, promoter is used.
  • inducible yeast promoters include GAL1- 10, GAL1, GALL, GALS, TET, CUP1 , VP 16 and VP16-ER.
  • repressible yeast promoters include Met25.
  • constitutive yeast promoters examples 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.
  • GPD glyceraldehyde 3 -phosphate dehydrogenase promoter
  • PGK phosphoglycerate kinase
  • ADH alcohol dehydrogenase promoter
  • TEZ translation-elongation factor- 1 -alpha promoter
  • CYC1 cytochrome c-oxidase promoter
  • MRP7 MRP7.
  • Promoters containing steroid response elements e.g., glucocorticoid response element inducible by glucocorticoid or other steroid hormones can also direct expression
  • 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.
  • a vector comprises an expression control element known as an upstream activating sequence (UAS).
  • UAS upstream activating sequence
  • 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.
  • 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.
  • 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.
  • 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.
  • a nucleic acid of interest e.g., a nucleic acid to be used to functionally inactivate a DEGA or DRGA gene.
  • any restriction enzyme site may serve this purpose.
  • Certain embodiments include a multiple cloning site, or polylinker.
  • 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.
  • 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
  • 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.
  • 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.
  • a tag is often relatively small, e.g., ranging from a few amino acids up to about 100 amino acids long.
  • 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).
  • a reporter or tag is more than 100 amino acids long, e.g., up to about 500 amino acids long.
  • a reporter tag is located at the N-terminus or C-terminus of a polypeptide.
  • a polypeptide may comprise multiple tags.
  • 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+
  • a kit comprises yeast that have been tested to determine whether they are [GAR+] or [gar- ⁇ .
  • 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-].
  • 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.
  • 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.
  • 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.
  • kits comprises one or more items useful for control purposes, e.g., a control plasmid, control primer(s).
  • compositions 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
  • kits which may be packaged together in a single larger container.
  • 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.
  • a gene encodes a protein.
  • a gene encodes a RNA or protein of unknown function.
  • a gene encodes a protein that has at least one known function.
  • the protein is an enzyme.
  • 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:
  • 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.
  • [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.
  • 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.
  • MLSMR Molecular Libraries Small Molecule Repository
  • NASH National Institutes of Health
  • HTS high-throughput screening
  • 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.
  • 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.
  • Budding yeast are used to produce a wide variety of compounds of interest.
  • 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.
  • 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.
  • 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.
  • yeast e.g., budding yeast, e.g., industrially important budding yeast
  • Metabolic engineering can result in a product with improved quality, or result in time and/or cost savings, etc.
  • 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.
  • 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
  • ability to utilize particular nutrients or substrates e.g., tolerance to extremes of temperature (e.g., heat stress), osmotic stress, oxidative stress, pH, intracellular or extracellular accumulation of a product.
  • 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).
  • 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.
  • 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.
  • prion modulation is used in a yeast species or strain that has been given GRAS (generally recognized as safe) status by the FDA.
  • 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.
  • 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.
  • prion modulation is used to improve the production of a food, nutritional supplement, beverage, or component thereof.
  • prion modulation is used in a baker's, wine, brewer's, sake, or distiller's yeast, e.g., S. cerevesiae or S. pastorianus.
  • prion modulation is used in a yeast species or strain that has been given GRAS (generally recognized as safe) status by the FDA.
  • 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.
  • 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.
  • 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.
  • a yeast is Dekkera bruxelle is.
  • the yeast is of the genus Kluveromyces.
  • Kluveromyces lactis and Kluyveromyces marxianus are of use in a variety of biotechnological processes.
  • the yeast has increased tolerance to an environmental condition, e.g., heat, cold, osmolarity (e.g., salt concentration) relative to S.
  • 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.
  • a strain of yeast that can reduce the acidity of a culture medium such as grape must be used, e.g., a yeast that can convert L-malate to L-lactate during alcohol fermentation.
  • 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).
  • 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.
  • the product of interest is a recombinant protein.
  • Exemplary proteins that can be produced in yeast are antibodies, vaccine components, interferons, and insulin.
  • the product of interest is a pharmaceutical agent, which may be a recombinant protein or a non-protein biomolecule.
  • the product of interest is a small organic molecule.
  • the product of interest is a precursor that may be subsequently used in a process that may, but need not, involve yeast.
  • 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.
  • the biofuel is an alcohol.
  • the biofuel is a bio-oil.
  • Ethanol is an exemplary biofuel. S. cerevesiae has traditionally been used for ethanol production (Nevoit, supra).
  • 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.
  • RNAi is used to improve ethanol production in a yeast that naturally utilizes pentoses, e.g., xylose, such as P. stipitis.
  • a product of interest is a lipid.
  • the yeast is an oleaginous yeast.
  • 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.
  • a yeast is used to remediate waste or in environmental cleanup.
  • 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.
  • prion modulation is used in combination with modulation of a gene that affects tolerance to a metabolite or toxin.
  • the metabolite is ethanol.
  • the metabolite is a byproduct of a metabolic reaction useful to produce a product of interest.
  • 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.
  • 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.
  • 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.
  • the cell has a functional endogenous RNAi pathway.
  • the cell lacks a functional endogenous RNAi pathway and is engineered to have a functional RNAi pathway.
  • 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).
  • the invention provides a fungal strain that is selected or genetically engineered to maintain a stable [GAR+] phenotype.
  • a fungal 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.
  • 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.
  • 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).
  • 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.
  • manipulating such genes or their encoded gene products can be used to modulate the corresponding prion in the second species.
  • 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.
  • the prion is [GAR+] .
  • salts can exist as their corresponding salt, ester, or prodrug.
  • 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.
  • salts of the compounds that contain a basic center are acid addition salts formed with acceptable acids.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a method described herein that comprises producing a product to be consumed or used by mammals, e.g., humans.
  • 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
  • [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 10 5 cells in the BY background, approximately one in 10 4 cells in 74D, approximately five in 10 4 cells in W303, and approximately seven in 10 4 cells in Sigma.
  • [GAR+] appeared at the astonishingly high rate of approximately one in 10 cells (Fig. ID).
  • the frequency of heritable phenotypic change due to genetic mutation is generally approximately one in 10 6 haploid cells (Ohnishi et al. 2004).
  • HXT3 Hexose Transporter 3
  • 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 causal agent of [GAR+] is a regulator of HXT3 expression.
  • Stdl is the determinant of the [GAR+] prion, but further data indicated it could not be the sole determinant.
  • most prion phenotypes mimic loss- of -function phenotypes of their prion determinants.
  • Astdl strains derived from a [gar ⁇ ] background were not able to grow on glycerol in the presence of glucosamine (Fig. 2B; data not shown).
  • Astdl cells derived from a [GAR+] background were able to do so, indicating that they kept the prion (data not shown).
  • prions A characteristic of prions is that transient overexpression is sufficient for induction.
  • Pmal is the most abundant plasma membrane protein in yeast
  • 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 ( ⁇ 40) 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.
  • [GAR+] could be propagated through cells whose only source of Pmal was a G ⁇ ZJ-regulated N-terminal deletion, ⁇ 47 ⁇ 40 ⁇ (Fig. 33 ). Strikingly, however, it did not propagate through a double mutant of ⁇ 1 ⁇ 40 ⁇ 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.
  • 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).
  • 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).
  • 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.
  • [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.
  • [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 .
  • [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;
  • [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
  • [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
  • 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]).
  • 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.
  • 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% ⁇ -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.
  • 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 50 ⁇ 1 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).
  • the pellet was resuspended in sorbitol buffer (200 ⁇ 1), and an aliquot (95 ⁇ 1) 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. 15 ⁇ 1 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.
  • 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.
  • 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).
  • GAL- estradiol fusion plamid The latter allows induction of GAL I promoters by growth on estradiol without galactose.
  • 2D gel electrophoresis 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.
  • 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.
  • Table S3 Knockout mutants that switch to [GAR+] at high frequency
  • Table S4 Yeast strains used in this study
  • Multicopy SUP35 gene induces de- no vo appearance of psi-like factors in the yeast Saccharomyces cerevisiae. Curr Genet 24: 268- 270.
  • Eisenkolb M Zenzmaier C, Leitner E, 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: 4414 ⁇ 1428. Eraso P, Cid A, Serrano R. 1987. Tight control of the amount of yeast plasma membrane ATPase during changes in growth conditions and gene dosage. FEBS Lett 224: 193-197.
  • Saccharomyces cerevisiae KAR2 (BiP) gene expression is induced by loss of cytosolic HSP70/Ssalp through a heat shock element- mediated pathway. J Biochem 121 : 578-584.
  • Patino MM Liu J- J, Glover JR, Lindquist S. 1996. Support for the prion hypothesis for inheritance of a phenotypic trait in yeast. Science 273: 622-626.
  • Paushkin SV Kushnirov VV, Smirnov VN, Ter-Avanesyan MD. 1996. Propagation of the yeast prion-like [psi+] determinant is mediated by oligomerization of the SUP35-encoded polypeptide chain release factor.
  • Polish JA Kim J-H, Johnston M. 2005, How the Rgtl transcription factor of Saccharomyces cerevisiae is regulated by glucose. Genetics 169: 583-594.
  • LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum. J Cell Biol 145: 659-672.
  • LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum. The Journal of cell biology 145(4): 659-672.
  • yeast cells 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+].
  • Example 3 Prion induction is driven by interkingdom chemical communication
  • 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.
  • yeast genes that are involved in perceiving the inducing signal and transmuting it into a heritable trait.
  • ORFs nonessential open reading frames
  • Table B Deletions that Enhance GAR Acquisition (DEGA Genes)
  • Table A Bacteria that induce ⁇ GAR+ ⁇ :
  • glucosamine is broadly distributed. Thus, this cross-kingdom communication between bacteria and yeast has been broadly conserved over the evolutionary history of the species.
  • [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).
  • a constitutive fluorescent marker (mOrange) to report on cell number
  • Hxt3-GFP fusion to report on prion status.
  • 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.
  • [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.
  • 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.
  • 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.

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Abstract

L'invention concerne, selon certains aspects, des procédés de modulation de la fermentation ou de la croissance de levures par modulation du prion [GAR+]. Selon certains modes de réalisation, [GAR+] est induit par mise en contact de cellules de levure avec une bactérie qui produit un inducteur à petite molécule de [GAR+] ou par mise en contact de cellules de levure avec la petite molécule ou le milieu conditionné comprenant la petite molécule ou une fraction d'un tel milieu qui comprend la petite molécule. Selon certains aspects, l'invention concerne des procédés d'identification d'un modulateur d'un prion d'intérêt. Selon certains modes de réalisation, un procédé d'identification d'un modulateur d'un prion comprend (a) la préparation d'un milieu conditionné par des bactéries; (b) la mise en contact d'au moins une partie du milieu conditionné avec des bactéries avec une cellule non bactérienne; et (c) la surveillance de la cellule au regard d'une modification d'un phénotype associé avec un prion d'intérêt. Selon certains modes de réalisation, la cellule non bactérienne est une cellule de levure. Selon certains modes de réalisation, le prion est [GAR+].
PCT/US2013/053272 2012-08-01 2013-08-01 Manipulation à base de prions de la fermentation et la croissance de levures WO2014022692A1 (fr)

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CN105087631A (zh) * 2015-08-07 2015-11-25 山东大学 一种提高酿酒酵母木糖吸收和利用能力的方法
WO2019006341A1 (fr) * 2017-06-30 2019-01-03 Zimitech, Inc. Microorganismes modifiés en vue d'une utilisation améliorée des oligosaccharides
WO2021209942A1 (fr) * 2020-04-15 2021-10-21 New Life Biosciences Llc Traitement microbien pour des systèmes d'eau et l'assainissement du sol

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087631A (zh) * 2015-08-07 2015-11-25 山东大学 一种提高酿酒酵母木糖吸收和利用能力的方法
WO2019006341A1 (fr) * 2017-06-30 2019-01-03 Zimitech, Inc. Microorganismes modifiés en vue d'une utilisation améliorée des oligosaccharides
CN111051498A (zh) * 2017-06-30 2020-04-21 齐米科技股份有限公司 用于增强低聚糖利用的工程化的微生物
US11597938B2 (en) 2017-06-30 2023-03-07 Zimitech, Inc. Engineered microorganisms for enhanced use of oligosaccharides
WO2021209942A1 (fr) * 2020-04-15 2021-10-21 New Life Biosciences Llc Traitement microbien pour des systèmes d'eau et l'assainissement du sol
GB2611433A (en) * 2020-04-15 2023-04-05 New Life Biosciences Llc Microbial treatment for water systems and soil remediation

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