WO2003091399A2 - Production et utilisation d'organismes tolerants au sel et a la densite de culture - Google Patents

Production et utilisation d'organismes tolerants au sel et a la densite de culture Download PDF

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Publication number
WO2003091399A2
WO2003091399A2 PCT/US2003/012686 US0312686W WO03091399A2 WO 2003091399 A2 WO2003091399 A2 WO 2003091399A2 US 0312686 W US0312686 W US 0312686W WO 03091399 A2 WO03091399 A2 WO 03091399A2
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Prior art keywords
cells
cell
genes
ygl039w
ygl157w
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PCT/US2003/012686
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English (en)
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WO2003091399A3 (fr
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Jonathan Rosenblum
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Activx Biosciences, Inc.
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Priority to JP2003587935A priority Critical patent/JP2005523697A/ja
Priority to AU2003225134A priority patent/AU2003225134A1/en
Publication of WO2003091399A2 publication Critical patent/WO2003091399A2/fr
Publication of WO2003091399A3 publication Critical patent/WO2003091399A3/fr

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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • 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

Definitions

  • the present invention relates generally to the field of organisms that tolerate environmental stress conditions, in particular high salt and/or high culture density conditions.
  • antiporter genes can also provide salt tolerance.
  • creation of salt tolerance in plants by expression of sodium antiporters has been demonstrated.
  • Zhang et al., Proc. Natl. Acad. Sci. USA 98:12832- 12836 (2001) described sodium antiporter expression and salt tolerance in Brassica napus and Zhang et al., Nat. Biotechnol. 19:765-768 (2001) described similar effects in tomato.
  • Apse et al, Science 285:1256-1258 (1999) described sodium antiporter expression and salt tolerance in Arabidopsis.
  • Another approach involves the application of a substance that induces salt tolerance.
  • a substance that induces salt tolerance is gibberellin. Zhao Ke-fu et al., Aust. J.
  • Yeast can also be obtained that are salt tolerant.
  • salt tolerant for example, Park et al., J.
  • the present invention provides methods for producing and using cells that tolerate environmental stress conditions, particularly high salt and/or high culture density conditions, as well as the cells themselves, and related compositions .
  • Such cells are generally provided by inactivating particular genes.
  • This allows modified strains to be created from many different cells and organisms, for example, yeasts and plants.
  • the resulting modified cells and organisms can be used in a large variety of processes in which tolerance to high salt, high culture density, and/or other environmental stress conditions is advantageous.
  • the invention provides a cell(s), preferably an isolated or purified cell, that has at least one gene functionally homologous to YGL039w, or functionally homologous to YGL157w or both inactivated.
  • the cell has greater tolerance to high salt, e.g., sodium chloride (and preferably additional dissolved materials and/or high culture density), concentration than a corresponding cell that has an active form of the gene.
  • high salt e.g., sodium chloride (and preferably additional dissolved materials and/or high culture density)
  • concentration e.g., sodium chloride (and preferably additional dissolved materials and/or high culture density)
  • a plurality of genes are inactivated, e.g., 2, 3, 4, or even more genes.
  • the cell is a yeast cell, preferably a Saccharomyces cell, most preferably a Saccharomyces cerevisiae cell.
  • the cell may be from an AspergiUus species. More generally the cell can be a fungal cell or a plant cell.
  • the cell is in a cell culture, preferably in a population of such cells.
  • the cell culture is a liquid culture.
  • the cell culture is a high density cell culture.
  • the cell is in high salt culture conditions; the cell(s) further comprises at least one additional genetic characteristic providing increased salt tolerance, e.g., the additional genetic characteristic can be a mutation(s) or a heterologous gene(s) that may, for example, provide increased sodium pump (antiporter) activity; in addition to increased salt tolerance, the cell is also tolerant to increased culture density and/or starvation conditions as compared to corresponding cells that have active forms of the genes functionally homologous to YGL039w and/or
  • YGL157w preferably tolerating increased culture densities where the density is increased at least 25%, 50%, 100%, 150%, 200%, 300%, 400%, 500%, 700%, 1000%, or even more; and/or the cells maintain active growth to a higher density than cells with active forms of the genes, e.g., the cells can maintain exponential growth phase to a higher density.
  • the cell (or culture or population) is in a culture that is both high salt and high density.
  • the invention provides a population of salt tolerant cells in a high salt environment, where the cells have at least one inactivated gene functionally homologous to YGL039w or YGL157w or both, thereby providing salt tolerance to the cells.
  • the salt tolerant cells will therefore grow in the high salt environment.
  • the invention provides a salt tolerant organism that has cells in which at least one gene functionally homologous to YGL039w or YGL157w or both are inactivated.
  • the inactivation of the genes confers salt tolerance and/or an ability to grow to high density and/or tolerance to solutions with elevated dissolved components (e.g., various ions (such as various minerals) and/or products of cell or organism culture).
  • the invention provides yeast that have at least one artificial genetic alteration, where the yeast is more salt tolerant and actively grows to higher culture density than an isogeneic yeast without the at least one artificial genetic alteration.
  • Such alterations include those described above, e.g., an alteration inactivating at least one gene.
  • the invention provides a yeast strain that is more salt tolerant and grows to a higher density in rich media than the corresponding wild type strain.
  • the corresponding wild type strain is a designated standard wild type strain deposited in an approved depository.
  • the invention includes a Saccharomyces cerevisiae yeast strain that grows to an OD600 of at least 15 in YPD medium. Preferably the strain actively grows to higher density in YPD medium with 0.9M NaCl than strain BY4743 [0019] Yet another related aspect concerns a Saccharomyces cerevisiae yeast strain that grows to a higher density and/or faster in YPD medium with 0.9M NaCl than strain BY4743.
  • Another related aspect concerns a mutated cell that is more tolerant to the presence of salt and grows to higher density under growth conditions than the parent cell.
  • the mutation can include inactivation of at least one gene, e.g., a gene functionally homologous to YGL039w and/or YGL157w.
  • the invention provides a culture kit that includes a plurality of cells as described for an aspect above, e.g., cells comprising at least one inactivated gene functionally homologous to YGL039w and/or YGL157w, packaged in a storage device.
  • the kit also includes instructions for growing the cells and/or one or more culture media suitable for growing the cells.
  • a medium may, for example, be provided in liquid form (concentrated or at normal use concentration), in dry form, or in the form of a plurality of components that are combined to at least partially provide a medium.
  • a medium may also be a high salt medium.
  • the cells are of a type as described for an aspect above, or otherwise described herein as suitable for use in the present invention.
  • the fungal cells may be spores.
  • the plant cells may be seeds, seedlings, cuttings, or other plant form capable of growth.
  • the storage device may, for example, include a box(es), vial(s), tube(s), envelope(s), can(s), and/or bag(s).
  • the invention provides a method for growing cells to high density, by culturing cells that have at least one inactivated gene functionally homologous to genes YGL039w and/or YGL157w under growth conditions, until high cell density is obtained during exponential growth phase. That high cell density is greater than that obtained in exponential growth phase under the same growth conditions for cells that are isogeneic except for having active genes functionally homologous to genes YGL039w and YGL157w, preferably at least 25%, 50%, 80%, 100%, 200% greater, or even higher.
  • the cells are of a type described for an aspect above, or otherwise described herein as suitable for use in the present invention.
  • the cells are grown to stationary phase.
  • the cell density obtained at stationary phase is greater than that obtained at stationary phase under the same growth conditions for cells that are isogeneic except for having active genes functionally homologous to genes YGL039w and YGL157w, e.g., at least 25%, 50%, 80%, 100%, or 200% higher, or even greater.
  • the ability of cells of the present invention to grow to higher densities provides a method for providing increased yield of a cell product.
  • the method involves culturing cells that have at least one inactivated gene functionally homologous to genes YGL039w and/or YGL157w under growth conditions, and purifying the desired product or products.
  • the cells are grown in exponential growth phase to a cell density at least 25%, 50%, 80%, 100%, or 200% greater (or even more) than that obtained in exponential growth phase under the same growth conditions for cells that are isogeneic except for having active genes functionally homologous to genes YGL039w and/or YGL157w; the cells are grown to stationary phase, preferably the density of said cells at stationary phase is greater than that obtained at stationary phase under the same growth conditions for cells that are isogeneic except for having active genes homologous to genes YGL039w and YGL157w, e.g., at least 25%, 50%, 80%, 100%, or 200% greater.
  • the cells are of a types indicated for an aspect above, or otherwise indicated herein as suitable for use in the present invention.
  • the invention provides a method for growing cells in high salt conditions, by culturing cells that have at least one inactivated gene functionally homologous to genes YGL039w and/or YGL157w under high salt growth conditions, where the high salt growth conditions inhibit growth of cells that are isogeneic except for having active genes functionally homologous to genes YGL039w and/or YGL157w.
  • the level or concentration of ions, e.g., sodium ions, in the high salt conditions can vary depending on the particular type of cells.
  • the sodium concentration e.g., from sodium chloride
  • cells are of a type indicated herein as suitable for the present invention.
  • the invention provides a method for producing a comestible product in a process utilizing cell culture, involving culturing cells that have at least one inactivated gene functionally homologous to YGL039w and or YGL157w in that process.
  • the cells are as described for an aspect above.
  • comestible products that utilize a cell culture process include soy sauce (fermented), beer, wine, cheese, and certain nutritional supplements, e.g., yeast-based nutritional supplements.
  • the cells used in the process can be of various types, selected as suitable for the particular process, including cells of types described herein as suitable for use in the present invention.
  • the process is a fermentation.
  • the culturing is carried out in liquid culture.
  • the culturing can be performed as continuous active culture, or carried out in batch culture, e.g., to stationary phase.
  • the cell density in a batch culture, e.g., at stationary phase, or in the continuous active culture is greater than that obtained under the same growth conditions by cells that are isogeneic except for having active genes functionally homologous to genes YGL039w and/or YGL157w.
  • Two related aspects provide a method for producing a cell tolerant to high levels of dissolved materials, and a method for producing a cell tolerant to high culture density.
  • the methods involve inactivating forms of genes having sequence match to YGL039w and/or YGL157w in the cell; and testing those cells for tolerance to high levels of dissolved material, e.g., salts such as sodium chloride, and/or high culture density.
  • dissolved material e.g., salts such as sodium chloride
  • the type of cells can be of many different types, e.g., as indicated herein for use in the present invention.
  • the gene inactivations can be accomplished in various ways. Examples include, without limitation, deletion of at least a part of the genes; creating insertions in the genes, typically heterologous sequence insertions; inhibiting transcription; inhibiting transcript processing; and inhibiting translation, as well as inhibition of the polypeptide or protein. For use in production of comestible products and other applications in which long term stability of the inactivation is advantageous, preferably deletion or insertion inactivations are used.
  • Different types of cells can have a plurality of genes, e.g., 2 or more than 2 genes with sequence match to YGW39w and/or YGL157w, for example, 3, 4, 5, or more genes (and/or gene products) with matching sequences.
  • S. cerevisiae contains 4 different genes having matching sequences, but YGL039w and YGL157w are the genes found to provide tolerance to salt and high culture density when inactivated.
  • the cell contains at least 2, 3, 4, or 5 different genes with a sequence match to YGL039w and/or YGL157w.
  • Cells with different combinations of those genes inactivated, e.g., pairwise combinations) are tested to identify the functional homologs.
  • Preferably cells with at least all pairwise combinations of inactivated genes are tested, and more preferably with all combinations of inactivated genes are tested.
  • the high levels of dissolved materials can include a variety of different components, e.g., high levels of sodium chloride and/or high levels of cell culture products.
  • the method can also include identifying genes and/or gene products with sequence match to YGL039w or YGL157w or both (or the corresponding gene products). Such identifications can be done in a number of different ways. Appropriate methods are well-known to those in the art. Examples include performing sequence alignments, using low stringency PCR to amplify sequences with sequence match to sequences of genes YGL039w and YGL157w, and using activity-based probes that react with YGL039w and/or Ygll57w. In preferred embodiments using activity-based probes, the probe reacts on a tyrosine in YGL039w and/or Ygll57w peptides. The various methods can also be used in any combination.
  • the invention also provides a method for growing an organism under elevated salt conditions in another aspect.
  • the method involves culturing an organism that has at least one gene functionally homologous to genes YGL039w and/or YGL157w inactivated, under high salt growth conditions, where the high salt growth conditions inhibit growth of an organism that is isogeneic except for having active forms of the inactivated genes.
  • the high salt conditions can be at various levels or concentrations, e.g., depending on the type of organism.
  • concentration of sodium ion e.g., from sodium chloride
  • the concentration of sodium ion is at least 0.3M, 0.4M, 0.5M, 0.7M, 0.8M at least 0.9 M ⁇ 0.1 M sodium, or is at least 0.9 M sodium.
  • the sodium ion concentration dissolved in the water or medium is preferably at least 0.02M, 0.04M, 0.05M, 0.08M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, or even higher.
  • the organism can be of any type that has the indicated genes, e.g., a yeast, fungus, or plant, or other organism indicated as suitable for this invention.
  • Plants can, for example, include forage plants, such as alfalfa and grasses, grain plants, such as wheat, barley, oat, rice, and corn, and vegetables, as well as many other types of plants
  • a method for soil improvement involves growing at least one plant having at least one inactivated gene functionally homologous to YGL039w and/or YGL157w in soil containing a high level of salt, and removing the plants, or at least portions of the plants, following a growth period of at least 10 days. Removed plant portions contain elevated levels of salt. Thus, removal of the plants or plant parts removes excess salt.
  • the concentration of dissolved sodium resulting from the combination of sodium in the soil and in any water provided is as shown in the preceding aspect.
  • the plants will be grown for longer times, e.g., at least 30 days, 60 days, 90 days, 180 days, 1 year, 2 years, 5 years, 10 years, or even longer.
  • the plants are annuals or perennials.
  • the growing and removing can be done repetitively, either with replanting and/or with plants that re-grow after cutting. In particular embodiments, the repetition is done at least 2, 5, 10, 20, 40, or more times.
  • isolated means that a cell has been removed from an earlier environment, e.g., a natural environment, or from a mixed population.
  • the term "significant" is used to indicate that the level of increase is useful to the person making such an increase, and is preferably at least 2-fold, more preferably at least 5- to 10-fold or even more.
  • a cell be in purified form.
  • purified in reference to cells does not require absolute purity (such as a homogeneous culture). Instead, it represents an indication that the cell is relatively more pure than a prior environment, e.g., in the natural environment. Compared to the prior level, this level should be at least 2-5 fold greater, e.g., in terms of cells/mL or cell/cm 3 .
  • purified cells do not have other cells present at a level detectable by isolation methods appropriate for the type of cells that may be present.
  • the term "inactivated", in reference to a gene, means that gene product from a gene that normally encodes a functional product has been modified or treated such that the gene produces no product, completely inactive product, or sufficiently low level of functional product so that the effects described for this invention are obtained, or that the expression or activity of a gene product has been sufficiently inhibited such that the effect described for this invention are obtained (for genes useful in this invention).
  • the reduction in gene product activity is at least 80%, more preferably at least 90%, still more preferably at least 95%, and most preferably at least 97, 98, 99, or even 100% (no detectable activity).
  • co-inactivation of more than one gene may produce the desired effect.
  • Such inactivation can be accomplished in various ways, e.g., by deletion of all or part of the gene; inserting another sequence or sequences in the gene; by contacting the gene or corresponding niRNA with sense, antisense, ribozyme, or triple-helix-forming sequences, or combinations thereof; by downregulating expression of the gene; by inhibiting processing of transcript; by inhibiting translation of an mRNA; and/or by inhibiting action of a polypeptide or protein.
  • Reference to inactivation of a gene thus can involve inactivation at the DNA, RNA, and/or protein level unless clearly indicated to be more limited.
  • YGL039w refers to the S. cerevisiae coding sequence, gene, and encoded polypeptide represented by SEQ ID NOs. 1 and 2 in Table 1.
  • Ygl039w refers to the encoded polypeptide.
  • Ygll57w refers to the S. cerevisiae coding sequence, gene, and encoded polypeptide represented by SEQ ID NOs. 3 and 4 in Table 2.
  • Ygll57w refers to the encoded polypeptide.
  • sequence match and “matching sequence” and terms of the like import when used without modification indicate that a sequence has at least a minimum level of nucleotide or amino sequence identity (or similarity) to a reference sequence.
  • Matching nucleotide sequences e.g., gene sequences or coding sequences
  • sequence identity as defined by the maximal base match in a computer-generated alignment of two or more nucleic acid sequences
  • 105, 120, 150, or 200 nucleotides, or over an entire ORF or entire gene, more preferably at least 80% or 85%, still more preferably at least 90%, and most preferably at least 95%.
  • a polypeptide with sequence match (typically a product of a gene with sequence matching) has at least 20% identity over a window of at least 20 contiguous amino acid residues (or larger window as indicated below), preferably at least 25, 30, 35, 40, 45, or 50 amino acids, more preferably over at least 100 amino acids with the sequence of a reference polypeptide.
  • the matching sequence has at least 20% identity over the full-length polypeptide encoded by an ORF or entire gene.
  • the comparison is against YGL039w and/or Ygll 57w polypeptide (or other functionally homologous polypeptide(s)). Highly preferred are amino acid identity levels of 25%, 27%, 30%, 32%, 35%, 37%, or even higher.
  • similarity may also be used to characterize polypeptide sequence matching.
  • the term refers to a measure of sequence matching which matches both identical amino acids and conservatively changed amino acids.
  • similar refers to amino acids in which the substituting amino acid has chemico-physical properties which are similar to that of the substituted amino acid.
  • the similar chemico-physical properties include similarities in charge, bulkiness, hydrophobicity, hydrophilicity and the like.
  • amino acid similarity is used in the homology comparison, the similarity level is at least 45%, more preferably at least 50%, 55%, 60%, 65%, 70%, or even higher.
  • % sequence identity For nucleotide or amino acid sequence comparisons where a sequence match is defined by a % sequence identity, the percentage may be determined using BLAST programs (with default parameters (Altschul et al., 1997, "Gapped BLAST and PSI-
  • BLAST a new generation of protein database search programs, Nucleic Acid Res. 25:3389-3402
  • Any of a variety of algorithms known in the art which provide comparable results can also be used, with parameters adjusted to provide equivalent results. Performance characteristics for three different algorithms in sequence searching is described in Salamov et al., 1999, "Combining sensitive database searches with multiple intermediates to detect distant homologues.” Protein Eng. 12:95-100.
  • Another exemplary program package is the GCGTM package from the University of
  • sequence matches can be defined as the maximum number of identical (or similar) nucleotide or amino acid residues in a comparison sequence compared to a reference sequence for two equal length sequences when the two sequences are aligned to provide maximum match, without the incorporation of gap penalties or other calculations.
  • % identity is used as the measure of sequence match, the % identity is calculated as (number of identical residues) ⁇ (total number of residues in each sequence) x (100).
  • similarity is used, the % sequence match (or similarity) is calculated as (number of identical residues + number of similar residues) ⁇ (total number of residues in each sequence) x
  • sequence matching may also, or in addition, be characterized by the ability of two complementary nucleic acid strands to hybridize to each other under appropriately stringent conditions that allows hybridization at sequence match levels as described above.
  • matching nucleotide sequences will distinguishably hybridize with a reference sequence with up to three mismatches in ten (i.e., at least 70% base match in two sequences of equal length).
  • the allowable mismatch level is up to two mismatches in 10, or up to one mismatch in ten, more preferably up to one mismatch in twenty.
  • Hybridizations are typically and preferably conducted with probe- length nucleic acid molecules, preferably 20-100 nucleotides in length. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not.
  • hybridization conditions and parameters see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor University Press, Cold Spring, N.Y.; Ausubel, F.M. et al. (1994) Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, N. J. Matching gene sequences may thus be identified using any nucleic acid sequence of interest.
  • a typical hybridization utilizes, besides the labeled probe of interest, a salt solution such as 6X SSC (NaCl and Sodium Citrate base) to stabilize nucleic acid strand interaction, a mild detergent such as 0.5% SDS, together with other typical additives such as Denhardt's solution and salmon sperm DNA.
  • a salt solution such as 6X SSC (NaCl and Sodium Citrate base) to stabilize nucleic acid strand interaction
  • a mild detergent such as 0.5% SDS
  • Other typical additives such as Denhardt's solution and salmon sperm DNA.
  • the solution is added to the immobilized sequence to be probed and incubated at suitable temperatures to preferably permit specific binding while minimizing nonspecific binding.
  • the temperature of the incubations and ensuing washes is critical to the success and clarity of the hybridization.
  • Stringent conditions employ relatively higher temperatures, lower salt concentrations, and or more detergent than do non-stringent conditions.
  • Hybridization temperatures also depend on the length, complementarity level, and nature (i.e., "GC content") of the sequences to be tested. Typical stringent hybridizations and washes are conducted at temperatures of at least 40°C, while lower stringency hybridizations and washes are typically conducted at 37°C down to room temperature ( ⁇ 25°C).
  • GC content i.e., "GC content"
  • stringent hybridization conditions hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5X SSC, 50 mM NaH 2 PO 4 , pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5X Denhart's solution at 42°C overnight; washing with 2X SSC, 0.1% SDS at 45°C; and washing with 0.2X SSC, 0.1% SDS at 45°C.
  • stringent hybridization conditions should not allow for hybridization of two nucleic acids which differ over a stretch of 20 contiguous nucleotides by more than two bases.
  • tolerant refers to the ability of a cell or organism to survive and grow better in a given environmental condition better than a reference cell or organism.
  • the reference cell or organism is a wild type cell or organism, or a cell or organism that is isogeneic except for a specified genetic difference.
  • salt tolerance includes tolerance to elevated levels of dissolved salts, e.g., NaCl, but can also include tolerance to elevated levels of other dissolved salts, e.g., potassium, calcium, and magnesium salts.
  • corresponding cell and “isogeneic” refer to a cell or organism that is the same as a cell or organism of interest except for some particular, specified genetic difference.
  • a wild type cell is isogeneic to a mutant of that cell except for that mutation(s).
  • isogeneic means that that a cell or organism has the same genetic complement (except for individual variation) as a reference cell or organism.
  • the term is used to indicate that a mutated or derivative cell or organism has the same genetic background as a reference cell, differing only with respect to a specific genetic change.
  • active forms indicates that the particular gene is present and expressed in an organism, and the gene product has its normal biological activity.
  • the term "cell” or “cells” includes the conventional biological understanding of cells, but also includes biological entities capable of developing into cells and/or complex organisms, e.g., seeds and spores, and reproductive entities, e.g., pollen. Unless indicated to the contrary, the term includes cells in all contexts, for example, single cells, cultures of single cells, and cells in multi-celled organisms.
  • organism has its usual biological meaning, referring to a self- replicating, living entity that is in a natural organizational form.
  • yeast is used in its usual biological meamng to refer to fungi that are single-celled during at least part of the life cycle. In some cases yeasts form pseudohyphae consisting of linear chains of incompletely separated budded cells.
  • fungus is used in it usual biological meaning, and includes both yeasts and molds. Some fungi have multi-nucleate structures.
  • spores refers to a natural storage form of an organism, capable to developing into an active organism under appropriate conditions. Examples include fungal spores.
  • plant is used in its usual biological meaning.
  • seeds is used in its usual biological meaning to refer to a structure formed from the ovule of seed plants following fertilization.
  • culturing refers to a process of growing cells or organisms under conditions that allow increase in size and/or number of cells or organisms, or that are intended to test for such increase in size and/or number.
  • culture includes growth of yeast cells in liquid or solid media culture, as well as growth of plants in soil.
  • culturing is distinguished from mere storage of cells or organisms.
  • cell culture is used to refer to culture of cells as distinguished from culturing multicellular organisms, such as plants. That is, the cells are present as generally separated cells without organization into natural complex structures such as tissues. Commonly, cell culture is carried out with liquid media, with the cells either on a surface or surfaces and bathed by the media, or suspended in the media.
  • culture density refers to the density of cells (for a cell culture) or the density of organisms in a culture. Typically, the term is applied to cell cultures, e.g., cells/mL.
  • high density or “high cell density” refers to a density of cells (or organisms) that is greater than that of a reference condition, generally the density provided by a cell that is isogenic except for specified genetic changes, e.g., inactivation of genes functionally homologous to YGL039w and/or YGL157w.
  • high salt conditions refers to the presence of salt, e.g., sodium chloride (or sodium ion) in solution or in position to become solubihzed at a concentration higher than normal for a particular cell type or organisms of interest.
  • salt e.g., sodium chloride (or sodium ion)
  • the term refers to the salt concentration in the liquid medium
  • the term refers to the salt concentration in the soil and/or in water available to the plant.
  • kits refers to a combination of two or more items selected to be suitable for a particular use or combination of uses and packaged together. Preferably a kit is prepared to be suitable for commercial sale.
  • storage device means a container able to hold and protect the contents from loss under expected condition. Examples include without limitation, bottles, bags, sleeves, envelopes, vials, tubes, and cans.
  • growth conditions refers to conditions that allow growth, preferably including increase in numbers, of a reference cell or organism.
  • exponential growth phase is used in its usual biological sense to refer to the period of growth of cells (e.g., yeasts) in non-replenished medium during which active growth occurs.
  • cells e.g., yeasts
  • the exponential growth phase is shown as a generally linear section of the curve, typically between an upward curving initial growth period (generally representing a lag phase and induction of growth) and a later portion of the curve where the slope decreases as growth in the number of cells substantially slows and usually essentially stops (stationary phase).
  • “stationary phase” refers to the period in growth of cells in non-replenished medium during which the increase in the number of cells substantially slows and typically stops. Cells can also be maintained in exponential growth phase in continuous culture, e.g., by replenishment of media and removal of cells.
  • the term "increased yield” means that a culture produces a greater amount of the product than a reference culture, or a greater amount in a specified time period.
  • the increase may, for example, be due to the presence of a greater density (number) of cells in a particular volume of culture.
  • the greater amount is at least 20%, more preferably at least 50%, still more preferably at least 75%, and most preferably at least 100% more.
  • the increase can be even greater, e.g., 200%, 300%, 400%, 500%, or more.
  • cell product refers to a product produced by the biological actions of a cell or organism. Generally a cell product of interest will be a molecule produced by the cell. The product may be a natural product of the cell, or may be produced due to the presence of exogenous genes or gene mutations.
  • purifying refers to a process of separating a particular composition (e.g., a molecule or a cell) from at least some of the other compositions with which it is found. While the particular compositions may be purified to homogeneity, the term also includes lesser levels of separation.
  • the terms “inhibit”, “inhibit growth”, and like terms refer to a reduction in activity or growth rate resulting from the presence of an agent(s) or environmental condition.
  • compositions refers to a product that is generally regarded as edible or drinkable by humans. Thus, the term includes both food and drink products.
  • process utilizing cell culture refers to a process in which the presence of the cells is intended and provides a significant and desired effect in the process. Examples include the presence of yeasts or other fungi in fermentation processes in producing beer, wine, soy sauce, raised bread, and cheese. Thus, the term does not include the incidental presence of cells in a process, or the unintended presence of cells that produce an undesirable effect.
  • fermentation has its usual meaning in referring to a metabolic process (and the associated culture process) that is not principally a respiration process. Thus, fermentation is a generally anaerobic process.
  • liquid culture refers to a culture of cells or organisms that is carried out with the cells or organisms primarily suspended in a liquid growth medium.
  • deletion refers to the removal of one or more nucleotides of a prior nucleic acid sequence.
  • the term can include the removal of most or all of a coding sequence, or an entire gene.
  • Such deletion can also result in the removal of one or more amino acid residues from an encoded polypeptide.
  • a deletion preferably inactivates the gene in which the deletion occurs.
  • insertion refers to the addition of one or more nucleotides in a nucleic acid sequence. In a coding sequence, such insertions can result in a chimeric product if inserted in-frame, or can result in frame shifts. As used in the present invention, insertions are usually heterologous sequence insertions, but may also be autologous sequence insertions. Insertions may be performed either with or without accompanying deletions. As used in this invention, insertions preferably inactivate the gene in which the insertion occurs and/or provides a tag for an encoded product.
  • sequence alignment has its usual meaning in referring to a process of matching identical or similar residues in two (or more) sequences to identify the maximum number of matching residues. While the process can be done manually, typically such alignment is performed on a computer using widely available software.
  • low stringency PCR refers to a polymerase chain reaction (PCR) process in which amplification occurs even when one or more of the primers used have substantial mismatch from the target sequence.
  • PCR polymerase chain reaction
  • the term "activity-based probe” refers to a molecule that is used to identify proteins (generally enzymes) of a particular class by reacting at the active site of the target molecule, thereby identifying active members of the class.
  • the probe has a direct or indirect tag (e.g., biotin or a fluorescent moiety, or a light scattering moiety).
  • Activity-based probes and their use are described, for example, in PCT Application
  • PCT/US02/03808 entitled “Activity Based Probe Analysis
  • U.S. Provisional Application No. 60/363,762 entitled “Tethered Activity-Based Probes and Uses Therefore”
  • PCT Application No. PCT/US00/34187 WO 01/77684
  • FIG. 1 shows images of a set of two culture plates showing the comparative growth of S. cerevisiae strains that are respectively (from left to right) wild type, 7GEO3 w/YGL157w double knockout, YGW39w single knockout, and YGL157w single knockout, in rich medium (YPD) and under high NaCl conditions.
  • the double knockout strain grows to a higher density in the high salt conditions than the wild type or single knockout strains.
  • FIG. 2 is a graph showing the growth curves (optical density) in rich medium for S. cerevisiae strains that are respectively wild type, 7GE03°w/YGL157w double knockout, YGL039w single knockout, and YGL157w single knockout. As shown in the graph, the double knockout strain grows to significantly higher density than the wild type and single knockout strains.
  • the present invention is based on the surprising discovery that cells can be created that are both salt tolerant and will grow to higher densities than parent cells, e.g., wild type cells. For example, inactivation of specific genes results in the organism being salt tolerant, and/or tolerant to the environmental stress produced by high culture density. The ability to tolerate higher culture density demonstrates that the cells are able to continue actively growing beyond the point where feedback from the enviromnent would cause normal cells to stop growing, e.g., starvation or accumulation of metabolic products. Thus, the cells can be tolerant to starvation conditions and/or higher culture density.
  • Exemplary genes whose inactivation provides the salt tolerance and/or tolerance to high culture density were identified in Saccharomyces cerevisiae.
  • the two genes were identified as YGL039w and YGL157w under the standard nomenclature used for identified open reading frames in S. cerevisiae.
  • the sequences of the two genes, along with the encoded polypeptides are provided in Tables 1 and 2 as SEQ ID NOs. 1-4. Also provided are the sequence accession numbers for the sequences. These two genes had a high level of homology, with greater than 70% identity between the encoded polypeptides.
  • Additional functional homologs can be identified and characterized in other organisms using known methods.
  • the surprising determination that the double gene knockout had salt tolerance and actively grew to higher density than wild type provides a beneficial method for providing modified cells with commercially valuable properties without creating transgenic organisms.
  • the salt tolerance allows a double knockout organism to actively grow in a broader range of conditions than the wild type organism, while the tolerance of high culture density provides more efficient use in fermentations and other culture processes.
  • the corresponding nucleic acid sequences were identified as YGL039w and YGL157w. As the genome of S. cerevisiae has been sequenced, this identification was done by computer searching. To characterize these gene products, the genes were each cloned and overexpressed in bacteria. It was confirmed that both of the gene products were labeled by the fluorophosphonate probes. The specific labeled amino acids from each protein were identified using conventional mass spectrometry methods. Each protein was labeled on a tyrosine residue (in the motif fYny(c/s): the capitalized Y is the tyrosine that gets modified).
  • the double knock-out yeast that resulted were viable and had normal-appearing colony morphology and microscopic morphology. J-n looking for phenotypes of the double knockout, the yeast were grown under various conditions, looking for conditions where the double knockout was less robust than wild-type yeast. However, the opposite was found; the double knockout was able to grow to higher density than the wild type yeast. Additionally, the salt resistance of the double knockout strain was greater than that of the wild type strain, and greater than that of either of the single knockout strains.
  • the double knockout provided a synergistic contribution to salt tolerance.
  • the YGL039w single knockout strain was only slightly (if at all) more tolerant to 0.9 M NaCl than the wild type strain, while the YGL157w single knockout grew to approximately lOx the number of cells in rich medium with 0.9 M NaCl as the wild type. In contrast, the double knockout grew to approximately lOOx the number of cells as wild type.
  • the double knockout strain also grew to high densities during active growth. In effect, the double knockout strain continued in exponential growth phase to substantially higher cell density than either the wild type or the single gene knockouts.
  • genes having sequence match to YGL039w and YGL157w can be located in a variety of different organisms and inactivated and tested for use in the present invention.
  • Sequence matching genes can be identified using various known techniques. In locating sequence matching genes, the simplest method is to use sequence comparisons of known sequences. Preferably, the sequence comparison is carried out at the amino acid level, but can also be done using the corresponding nucleotide sequences. While such comparisons can be done manually, highly preferably computer-based analysis is used. In this analysis, two sequences are aligned to maximize the matching amino acids (or nucleotides). Usually the matches are for identity, but for amino acids, similarity can be used.
  • an amino acid sequence identified as a sequence match has at least 20% identity over a window of at least 50 amino acids, preferably over at least 100 amino acids, and most preferably over the full-length polypeptide encoded by an ORF compared against Ygl039w and/or
  • Ygll57w (or other functionally homologous polypeptide). Highly preferred are amino acid identity levels of 25%, 27%, 30%, 32%, 35%, 37%, or even higher. If amino acid similarity is used, the similarity level is at least 45%, more preferably at least 50%, 55%, 60%, 65%, 70%, or even higher.
  • polypeptides with sequence matching are identified in an organism, those polypeptides can be tested, as described below, to determine which constitute functional homologs, i.e., which sequence matches produce the effects demonstrated for Ygl039w and Ygll57w inactivation.
  • sequence alignment and sequence match determinations will generally be performed on sequence matches (or other potential functional homologs) revealed by other techniques.
  • sequence matches can be identified using low stringency PCR and sequencing of the amplified sequences.
  • potential functional homologs can be identified using activity-based probes, followed by characterization of the protein (e.g., by sequencing).
  • the corresponding coding sequences can then be identified, e.g., by searching for a nucleic acid sequence encoding the particular polypeptide or portion thereof, or by using degenerate probes and/or low stringency PCT to obtain corresponding coding sequences.
  • hybridization probe screening is performed using cDNA libraries, while PCR may be performed using genomic and or cDNA libraries.
  • the various techniques can also be used in combination.
  • low stringency PCR can be used to identify matching sequences, a well-known approach.
  • this method involves the use of PCR primers that are designed based on a known sequence, in this case the nucleotide sequences of YGL039w and YGL157w genes (or identified functional homologs).
  • Hybridization conditions are used that allow a selected level of mismatches of the primers.
  • primers can be tested to provide primer set(s) that provide appropriate amplification.
  • Primers can be selected corresponding to unique or low redundancy regions. Additionally, once sequence matches are identified in particular cells or organisms, the sequences of those sequence matches can be used in further primer (or probe) design.
  • sequence matches in an organism can be demonstrated using hybridization of sequence matches to a reference sequence (e.g.,
  • YGL039w and/or YGL157w gene sequences i.e., hybridization probes.
  • coding sequences are used.
  • Detection of hybridized sequences can be performed in various ways. When the hybridization is carried out using non-amplified nucleic acid from an organism, the detection method should be highly sensitive, e.g., capable of detecting one or a few copies. An example of such a detection method is the use of light scattering particles (e.g., gold and/or silver). See, e.g., Yguerabide et al. U.S. Patent 6,214,560 Alternatively, hybridization can be used in conjunction with low stringency PCR. In this case the detection method does not need to be as sensitive.
  • probes can be selected corresponding to unique or low redundancy target regions.
  • probes can be selected corresponding to unique or low redundancy target regions.
  • Sufficient material for sequencing can be isolated using hybridization (effectively used as affinity chromatography), but preferably is provided by amplification. Such amplification can be performed as low stringency PCR. Primers can be based on the hybridization probes.
  • the initial sequence match determination provides full-length coding sequence, or even full-length gene. If full-length sequence is not provided initially, full-length sequence can be obtained using the partial sequence already provided to provide exact match probes to isolate larger fragments or cDNAs, providing the missing sequence portions .
  • the partial sequence can be used to provide probes suitable for high stringency hybridization (preferably providing perfect match).
  • probes can be used to detect genomic or cDNA clone inserts to identify a clone that includes the full coding sequence or full gene.
  • the clone preferably is constructed with adjacent sequences that allow convenient isolation and sequencing of the insert.
  • the gene product can be inactivated in the process of identifying functional homologs, as described below.
  • Possible functional homologs can also be identified, at least initially, using activity-based probes. As described Ygl039w and Ygll 57w were initially identified as being of interest using fluorophosphonate activity-based probes that usually react with the active site serine hydrolases. In this case, the probe reacted with anomalously nucleophilic tyrosines in these two gene products. Thus, such probes, or other activity- based probes that react with the Ygl039w or Ygll57w gene products (or identified functional homolog) can be used to react with possible functional homologs. Such possible functional homologs can be at least partially sequenced as was done for Ygl039w and Ygll57w, and the residue with which the probe reacted identified using conventional mass spectrometry techniques.
  • the coding sequence can be identified.
  • the coding sequence identification can be done immediately by computer sequence search if the coding sequence is available on computer. If not available on computer, the coding sequence can be obtained using conventional methods involving hybridization and/or PCR using degenerate probes and/or primers to locate and/or amplify the homologous nucleotide sequence.
  • the nucleotide sequence can then be sequenced by conventional methods.
  • the polypeptide sequence encoded by the nucleotide sequence can then be checked to confirm that the correct nucleotide sequence has been obtained.
  • sequence matches can be inactivated to test for the creation of salt tolerance and/or tolerance to high culture density.
  • S. cerevisiae was found to contain four sequence matches, but only Ygl039w and Ygll57w provided the salt tolerance and culture density tolerance when inactivated.
  • Ygl039w and Ygll57w provided the salt tolerance and culture density tolerance when inactivated.
  • This discrimination can be done in a systematic manner, e.g., by inactivating the sequence matches in all combinations.
  • other schemes can be followed.
  • all identified matching sequence genes can be inactivated in a single strain, thereby establishing whether the necessary functional homologs have been included in the set of sequence matches (so long as inactivation of the full set of sequence match genes does not result in the cell or organism being non- viable or having a phenotype that interferes with the salt tolerance and culture density determinations.
  • overlapping subsets of the sequence match genes can be inactivated, so that the identity of functional homologs can be further narrowed.
  • Gene inactivation can be carried out in a number of different ways.
  • An exemplary method is the use of homologous recombination to create inactivating deletions in a target gene, often with accompanying insertion of heterologous sequence.
  • a system that is often used for plants utilizes the Cre-lox system.
  • a description of the use of this system is provided in Bayley et al., "Exchange of Gene Activity in Transgenic Plants Catalyzed by the Cre-Lox Site-Specific Recombination System", Plant Molecular Biology 18:353-361 (1992). Further examples are provided in Qin, et al., Proc. Natl. Acad. Sci. USA 91:1706-1710 (1994); Sauer, Methods in Enzymology 225:890-900 (1993).
  • Another exemplary method that can be used is based on inactivating insertions, e.g., transposon insertions or insertions by Ag obacterium tumefaciens T-DNA (or similar element).
  • insertions e.g., transposon insertions or insertions by Ag obacterium tumefaciens T-DNA (or similar element).
  • Ag obacterium tumefaciens T-DNA or similar element.
  • An example of a systematic insertional knockout approach is provided by the Arabidopsis Knockout Facility creation of knockouts of Arabidopsis thaliana.
  • R ⁇ A interference R ⁇ A interference
  • this method involves the introduction of dsR ⁇ A-expressing constructs targeted to a particular gene into a cell.
  • the introduced constructs can be designed to be present in extrachromasomal vectors, or to integrate into the host cell genome.
  • ribozymes or other catalytic nucleotide-containing molecule, such as catalytic DNA (all referred to herein as ribozymes)
  • ribozymes are either expressed in the cell, e.g., from a vector, or stablilized ribozymes are introduced exogenously, e.g., using liposome delivery.
  • stablilized ribozymes are introduced exogenously, e.g., using liposome delivery.
  • antisense molecules are nucleotide- containing molecules that are either expressed in a cell, or are introduced into a cell exogenously.
  • the antisense molecules or ribozymes
  • Such antisense molecules generally act by blocking translation and/or by inducing cleavage of the message by endogenous cell components.
  • An example of this approach is described in Baucher et al., Plant Mol. Biol. 39:437-447 (1999). Additional inhibitions are described in Lapierre et al., Plant Physiol. 119:153-164 (1999) and MacKay et al., Science 277:235-239 (1997).
  • the ribozyme or antisense methods will not completely eliminate expression, but will reduce the level of expression sufficiently to provide an indicator of whether a sequence match is a functional homolog.
  • testing can proceed in various ways. For example, in order to confirm the presence of functional homologs, ribozymes or antisense molecules can be targeted to sequences that are identical between some or all of the sequence matches. Once the presence of functional homologs is confirmed, different combination can be tested to select the functional homologs, with the ribozymes or antisense molecules targeted to sequences that differ sufficiently between sequence matches to provide either specific, or specific combination, inhibition. Similarly, initial targeting can be to sequences that differ sufficiently to provide first stage testing of combinations of sequence matches. Also, for both ribozymes and antisense molecules, multiple ribozymes and/or antisense molecules can be targeted to a particular transcript, thereby providing a higher level of inhibition.
  • Expression inhibition can also be performed by inhibiting transcription by forming triple helix structures.
  • Methods and constructs for performing such inhibition are known in the art. (See, e.g., Blume et al, 1992, Nucl. Acids Res. 20; 1777;
  • Methods such as those mentioned above or other techniques for inhibiting or blocking expression of a gene can be used in the present invention. Combinations of two or more of the different techniques can also be used for a particular gene or genes in a particular organism. Such combinations can be particularly useful if difficulty is encountered in inactivating or sufficiently inhibiting particular genes.
  • the inactivation and testing of sequence match genes provides selection of the sequence matches that are also functional homologs. With that selection, cell or organism strains are provided (or can be provided by mutational inactivation) and can be used in the various processes for which the original strain was appropriate, as well as in new processes made practical by the increased salt tolerance and/or increased cell density tolerance.
  • yeasts and other fungi generally can typically be cultured in liquid media or on solid media, for inactivation, testing, and/or culture in connection with complex organisms, it is often desirable to utilize cell or tissue culture.
  • Appropriate techniques for providing cell and tissue cultures from plants are well-known. Exemplary descriptions are provided in Maki, et al., "Procedures for Introducing Foreign DNA into
  • Yeast cells can, for example, be transformed by converting yeast cells into protoplasts, e.g., using zymolyase, lyticase, or glusulase, followed by addition of the nucleic acid and polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • An exemplary method for the transformation of S. cerevisiae is as follows. Yeast strains are cultured overnight in YPD (yeast extract, peptone, dextrose) medium at about 30°C. The resulting culture is diluted to an A600 of about 0.2 in about 200 ml YPD medium and incubated at about 30°C. until the A.sub.600 reaches approximately 0.8. The cells are pelleted by centrifugation and are washed in about 20 ml sterile water. The pelleted yeast cells are then resuspended in about 10 ml TEL (10 mM Tris ⁇ H7.5, 1 mM EDTA, 0.1 M LiAcetate pH 7.5) buffer.
  • YPD yeast extract, peptone, dextrose
  • the cells are pelleted by centrifugation and again resuspended in about 2 ml TEL.
  • About 100 microgram of well-sheared single stranded DNA and plasmid DNA are added to an eppendorf tube.
  • To this tube is added about 100 microliter of competent yeast cells, followed by mixing.
  • To the cell/DNA mixture is added about 0.8 ml of 40% PEG-3350 in TEL, followed by thorough mixing. This mixture is incubated for about 30 minutes at 30°C, followed by a heat shock for 20 minutes at 42°C.
  • the mixture is centrifuged to remove the supernatant and pellet the cells.
  • the yeast cell pellet is washed with about 1 ml TE, pelleted again by centrifugation, and then plated on selective media.
  • Agrobacterium mediated transformation for example, Agrobacterium tumefaciens-mediated transformation
  • Agrobacterium tumefaciens-mediated transformation preferably with T-DNA flanking regions
  • Agrobacterium mediated transformation for example, Agrobacterium tumefaciens-mediated transformation
  • T-DNA flanking regions e.g., Horsch et al., Science 233:496-498 (1984); Fraley et al., Proc. N ⁇ tl. Ac ⁇ d. Sci. USA 80:4803 (1983); Hiei et al, Plant J. 6:271-282 (1994); Rogers et al., Methods in Enzymology 153:253-305 (1987)
  • General descriptions of plant expression vectors and reporter genes and transformation protocols can be found in Gruber, et al., "Nectors for
  • regenerate whole plants In the case of plant cells and tissues, following transformation, it can be useful to regenerate whole plants. Such regeneration can be carried out by known methods suitable for the particular plant type. In many cases regeneration is performed using embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods or stems. An example of a regeneration method is described in
  • a single knockout strain can provide a useful level of salt tolerance compared to wild type.
  • the YGL157w single knockout strain provided approximately lOx the number of cells as either the wild type strain or the YGL039w single knockout in 0.9M ⁇ aCl.
  • the single knockout result indicates that single knockouts of functional homologs in other organisms is also useful for creating salt tolerant cells and organisms.
  • gene inactivation methods can be used either directly with single gene knockouts of the individual sequence match genes, preferably of all sequence match genes in a particular cell or organism, or by focusing from strains bearing multiple knockouts shown to include one or more functional homologs.
  • testing of cells with inactivated sequence match genes can be done using conventional growth assays. Thus, cells are grown in an appropriate growth media for the cell type, but with the salt concentration (or other dissolved component or components) at higher than normal concentration. The ability of the strains with inactivated sequence matches to grow in the stress condition is determined and compared to the ability of a reference strain to grow under those conditions.
  • Testing for increased culture density can be performed generally as described herein for the exemplary S cerevisiae strains.
  • the number of the respective strains are normalized and the respective cultures are grown under the same conditions for a period sufficient for at least the wild type, parent, or other reference strain to go through active growth and into stationary phase (or significantly reduced growth rate).
  • the growth is continued for sufficient time that all strains pass to stationary phase.
  • At least the maximum cell densities are determined (e.g., as optical density or viable cell numbers or densities or concentrations).
  • the cell densities are determined at multiple time points, thereby allowing construction of growth curves.
  • the terminal densities (or total numbers) and/or growth curves provide comparative growth indicators.
  • culture conditions should be selected as appropriate for the particular cells or organisms.
  • exemplary media includes YPD (1% yeast extract, 2% peptone, 2% dextrose, water (weight %s)).
  • Either single, double, or other knockout combinations can be used in various ways, depending on the type of organism or cell.
  • the knockout strains are useful in any process in which it would be useful to have cells or organisms grow in a higher than normal concentration of salt (or other dissolved components), or actively grow to higher concentration than the wild type or other reference strain), or both.
  • tolerance of such stress conditions can open additional new applications for particular types of organisms.
  • the strains or cell types of the present invention can reduce costs and/or increase output of a desired product.
  • tolerance to high culture density allows the culture to be effectively run with a denser culture. This applies to both batch culture and continuous culture applications.
  • the tolerant organisms allow the culture to be run to higher cell density, generally providing a greater amount of product for that batch and/or faster output.
  • the tolerant strain culture can be run at higher culture density while still maintaining active cell growth. Those higher call densities provide greater cell product output from the culture.
  • the cells and organisms can be used in many different production processes, including production of comestible products.
  • the strains routinely used can be modified by inactivating the respective functionally homologous genes in that strain, thereby providing a strain more tolerant to culture stresses.
  • the modified strains can be cultured in the process at higher density while maintaining active growth.
  • Examples of such established processes include those used in production of beer, wine, fermented soy sauce, and cheese. Those processes are well-known in the respective fields.
  • the use of the modified organisms is also very useful in cell culture production of products that are isolated from the culture. Some products are isolated from the medium, while others are isolated from the organisms after the cells are lysed or broken. The present invention is applicable in both cases.
  • the present strains allow growth in conditions with high levels of salt in the soil and/or water. This properly provides at least two types of applications. In the first, the plants will still grow when water containing elevated levels of salt (e.g., NaCl) and/or other dissolved minerals and other ions is supplied, thus allowing use of slightly brackish water.
  • salt e.g., NaCl
  • plants can be grown in soil that contains a high level of salt or other components that can mobilize in the water, allowing growth in marginal soil. Further, if a plant is selected that both grows in the high salt conditions and has a high salt concentration in at least some tissues, the plants can be grown and harvested. Harvesting removes the salt concentration in the plant, thus removing salt from the soil. Repetition of the process can remove substantial amounts of salt from the soil. This process can be carried out over a number of years. In such use the plants both stabilize the soil and improve the soil by the salt removal.
  • the plant is a peremiial that provides a plurality of harvestings each year.
  • the crop can itself be a commercial crop, etc., alfalfa, wheat, corn, barley, and rice, among others. This process is also applicable to removal of other soil components that can mobilize in water and be taken up by the plants.
  • Example 1 Labeling and Identification of Ygl039 and YgU57w
  • a culture of yeast strain BY4743 was grown in rich media to an OD600 of approximately 5.
  • Cells were sedimented and resuspended in phosphate buffered saline, pH 7.4. Following reduction of disulfide bonds in the cell wall with DTT, cells were lysed with a high-pressure homogenizer. Cell lysates were spun at 15,000xg, and the supernatent from the 15,000xg spin was spun at 100,000xg. The fraction from which Ygl039w and Ygll57w were purified was the supernatant from the 100,000 x g centrifugation. lOmg of lysate was reacted with 4microM of FP- tetramethylrhodamine (an activity-based probe for serine hydrolases) for lh at room temperature.
  • proteins were denatured with urea, disulfide bonds were reduced with DTT and cysteines alkylated with iodoacetamide.
  • labeled proteins were isolated by chromatography. Protein samples that have been labeled with an activity-based probe were passed through an anti-rhodamine antibodies affinity purification column (Sepaharose), where the bound antibodies recognize the rhodamine probes attached to the labeled proteins. Labeled proteins that are purified in this manner were separated by standard 1- dimensional SDS-PAGE (12.5%) and visualized using laser-induced fluorescence from a flat-bed laser scanner. Fluorescent proteins are physically removed from the gel using a spot picker (Amersham).
  • the mass spectrometer provides the means to observe the masses of the intact, -1, -2 and —3 fragments. Since the masses of all the amino acids is known, this method enables peptide sequencing in the mass spectrometer. Data generated from mass spectrometer was compared to databases of protein sequences generated from S. cerevisiae, from which the sample was derived, to identify proteins that contained the predicted peptide sequence. Two proteins identified from a 37kD region in this manner were Ygl039w and Ygll57w.
  • identification and/or sequencing can be performed for other proteins, e.g., from other organisms.
  • the amino acid sequence preferably the entire polypeptide
  • the amino acid sequence can be used to obtain the corresponding nucleic acid sequence by conventional methods.
  • corresponding coding sequences or genes can be obtained using degenerate hybridization probe sets and/or using low stringency PCR
  • probe and/or primers preferably based on the amino acid sequence.
  • the probes and/or primers are designed to be complementary to unique or low degeneracy regions.
  • Example 2 Inactivating Ygl039w and YgU57w
  • a PCR product that contained the URA2 sequence (amplified from pYES2 from Invitrogen) flanked by sequence from the beginning and the end of YGL 157w was made. Transformation of the PCR product into the YGL039w knockout strain led to recombination of the flanking sequences, resulting in the replacement of almost the entire YGL157w gene with that of URA2 ⁇ allowing for the selection of recombinants and the deletion of YGL157w. Proper integration was verified by PCR.
  • the oligonucleotides used for the deletion PCR were:
  • the 5' primer is listed first. For each, the capital letters show sequence match to parts of YGL157w, while the lower case sequence anneals to sequences around the URA2 gene.
  • Example 3 Characterization of Cells with Inactivated Ygl039w and Ygll57w
  • Two assays were performed to look for growth phenotypes of the double deletion strain relative to the two single deletion strains and wild-type yeast.
  • the four strains were grown in YPD overnight and diluted to an OD600 of 5.0.
  • One to ten serial dilutions of the OD600 5.0 stock were made down to 1/10,000.
  • Three microliters of each dilution were spotted onto a YPD plate, and a YPD plate supplemented with 0.9M NaCl. The plates were incubated at 30°C for 2-4d. The results are shown in Figure 1, demonstrating that the double deletion strain grew significantly better in the 0.9 M NaCl supplemented medium compared to the wild type and to the single deletion strains.
  • the YGL157w single deletion strain grew significantly better than either the wild type or the YGL039w single deletion strain.
  • the four strains were grown overnight in YPD, diluted to normalize their OD600 and grown in YPD at 30degC. Over a period of approximately one day, samples of each culture were removed at several time points and the OD600 of the culture was taken. The results were plotted and are shown in Figure 2. As shown in the figure, the double deletion strain grew to significantly greater density than any of the other 3 strains. All the other strains grew to quite similar densities.
  • Example 4 Identification of Sequence Matches of Ygl039w and Ygll57w in other species [0145]
  • a basic protein-protein BLAST search was conducted using Ygl039w as a query sequence.
  • Sequence similar proteins were found in many species of plants, including rice, corn and Arabidopsis. Exemplary sequence matches are identified above. These proteins are typically on the order of 25% identical over 250-300 amino acids. As such, the proteins are considered to be highly sequence similar and most likely are functionally similar.
  • ATGACTACTG AAAAAACCGT TGTTTTTGTT TCTGGTGCTA CTGGTTTCAT TGCTCTACAC GTAGTGGACG ATTTATTAAA AACTGGTTAC AAGGTCATCG

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Abstract

L'invention concerne des cellules et des organismes tolérants à la forte densité de culture et/ou au sel. Ces cellules présentent deux gènes particuliers inactivés, ce qui se traduit par une tolérance aux contraintes de l'environnement. L'invention traite aussi de procédés de création et d'utilisation de ces cellules et ces organismes. Les cellules et les organismes peuvent être utilisés dans bon nombre d'applications différentes où la tolérance à une teneur élevée en sel et/ou une forte densité est avantageuse. Ces applications concernent des processus de production alimentaire et industrielle, comprenant des cultures de cellules, y compris la culture de levure.
PCT/US2003/012686 2002-04-23 2003-04-23 Production et utilisation d'organismes tolerants au sel et a la densite de culture WO2003091399A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2003587935A JP2005523697A (ja) 2002-04-23 2003-04-23 塩耐性および培養密度耐性生物の生成および使用
AU2003225134A AU2003225134A1 (en) 2002-04-23 2003-04-23 Production and use of salt tolerant and culture density tolerant organisms

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US37508302P 2002-04-23 2002-04-23
US60/375,083 2002-04-23

Publications (2)

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WO2003091399A2 true WO2003091399A2 (fr) 2003-11-06
WO2003091399A3 WO2003091399A3 (fr) 2004-08-26

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JP (1) JP2005523697A (fr)
AU (1) AU2003225134A1 (fr)
WO (1) WO2003091399A2 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001071045A2 (fr) * 2000-03-23 2001-09-27 Millennium Pharmaceuticals, Inc. Criblage a debit eleve relatif a des inhibiteurs de la synthese d'acides gras, d'ergosterols, de sphingolipides ou de phospholipides dans des champignons

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001071045A2 (fr) * 2000-03-23 2001-09-27 Millennium Pharmaceuticals, Inc. Criblage a debit eleve relatif a des inhibiteurs de la synthese d'acides gras, d'ergosterols, de sphingolipides ou de phospholipides dans des champignons

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [Online] May 1996 HEBLING ET AL, XP002978254 Retrieved from NCBI Database accession no. (Z72561) *
DATABASE GENBANK [Online] May 1996 JAMES ET AL, XP002978253 Retrieved from NCBI Database accession no. (Z72679) *

Also Published As

Publication number Publication date
WO2003091399A3 (fr) 2004-08-26
AU2003225134A1 (en) 2003-11-10
AU2003225134A8 (en) 2003-11-10
JP2005523697A (ja) 2005-08-11

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