WO2022072833A2 - Constructions d'expression et méthodes de modification génétique de cellules - Google Patents

Constructions d'expression et méthodes de modification génétique de cellules Download PDF

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WO2022072833A2
WO2022072833A2 PCT/US2021/053178 US2021053178W WO2022072833A2 WO 2022072833 A2 WO2022072833 A2 WO 2022072833A2 US 2021053178 W US2021053178 W US 2021053178W WO 2022072833 A2 WO2022072833 A2 WO 2022072833A2
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promoter element
cell
protein
group
plant cell
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PCT/US2021/053178
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WO2022072833A3 (fr
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Ranjani VARADAN
Martin Andrew HOYT
Allen HENDERSON
Rachel FRASER
Sergey SOLOMATIN
Xin Li
Celeste HOLZ-SCHIETINGER
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Impossible Foods Inc.
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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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon

Definitions

  • This disclosure generally relates to DNA constructs and methods of using such DNA constructs to genetically engineer cells.
  • a plant cell including a first nucleic acid molecule encoding a transcriptional activator operably linked to a first promoter element, and a second nucleic acid molecule encoding a target protein.
  • the second nucleic acid molecule can be operably linked to the first promoter element or a second promoter element, wherein the second promoter element can be positively regulated by the transcriptional activator.
  • the first promoter element, the second promoter element, or both the first promoter element and the second promoter element can be constitutive promoter elements.
  • the first promoter element, the second promoter element, or both the first promoter element and the second promoter element can be inducible promoter elements.
  • the first promoter element, the second promoter element, or both the first promoter element and the second promoter element can be tissue-specific promoter elements.
  • the first promoter element, the second promoter element, or both the first promoter element and the second promoter element can be seed-specific promoter elements.
  • the promoter element can be selected from: (1) a promoter element from a gene selected from the group consisting of soy beta-conglycinin, G1 -Glycinin, KTI, P24, LEC1, suspensor G564, maize MAC1, maize Cat3, Arabidopsis viviparous- 1, Arabidopsis atmycl, a Brassica napus napin (e.g., napA), mannopine synthase, actin, ubiquitin-1, isocitrate lyase, malate synthase, class 10-1,3-glucanase B, canola cDNAs CA25, CA8, AX92, lipid transfer protein, serine carboxypeptidase, a repetitive proline-rich cell wall protein gene, alphaamylase, (2) a cauliflower mosaic virus 35S promoter element, a figwort mosaic virus 34S promoter element, an Arabidopsis PR1 promoter element, and a combination thereof
  • the promoter element can be a Cauliflower mosaic virus 35S promoter element, and the transcriptional activator can be selected from the group consisting of TFIID, ASF-1, OCTSF, and a combination thereof.
  • the promoter element can be an Arabidopsis PR1 promoter element, and the transcriptional activator can be selected from the group consisting of AtWRKY50, a TGA transcription factor, and a combination thereof.
  • the promoter element can be a promoter element from an isocitrate lyase gene, and the transcriptional activator can be KlCat8p.
  • the promoter element can be a promoter element from a malate synthase gene, and the transcriptional activator can be PaHB5.
  • the promoter element can be a promoter element from an alpha-amylase gene, and the transcriptional activator can be selected from the group consisting of OsMYBSl, OsMYBS2, OsMYBS3, and a combination thereof.
  • the target protein can be a heme-containing protein. In some embodiments, the target protein can be a protein that modifies other proteins. In some embodiments, the target protein includes one or more enzymes in the biosynthetic pathway of a molecule of interest. In some embodiments, the molecule of interest can be a pharmaceutical, a porphyrin or derivative thereof, or a carbohydrate. In some embodiments, the molecule of interest can be a heme. In some embodiments, the molecule of interest can be methylcellulose. In some embodiments, the target protein can be a self-assembling protein. In some embodiments, the selfassembling protein can be selected from the group consisting of tubulin, actin, casein, keratin, or a combination thereof.
  • the target protein can be an animal protein. In some embodiments, the target protein has an artificial amino acid sequence. In some embodiments, the target protein has an amino acid sequence with portions from multiple organisms. In some embodiments, the target protein has a flavor. In some embodiments, the target protein has a color. In some embodiments, the surface activity of the target protein has been engineered. In some embodiments, the target protein sequence has an amino acid sequence, such that when heated with heme, a meat flavor can be produced. In some embodiments, the cell can be engineered to down-regulate a compound that results in an off-flavor and/or off-aroma. In some embodiments, the target protein can be engineered to bind or to release a particular ligand.
  • the multicellular group can be a plant or plant component.
  • the multicellular group can be a bean or a seed.
  • the bean or the seed has a low polyunsaturated fatty acid content.
  • the bean or the seed has a low polyunsaturated fatty acid content.
  • the bean or the seed has a low linolenic acid content.
  • the bean or the seed has a high monounsaturated fatty acid content.
  • the bean or the seed has a high oleic acid content.
  • the bean or the seed has a low unsaturated fatty acid content.
  • the bean or the seed has a high saturated fatty acid content.
  • the product can be a protein isolate, a protein concentrate, a textured protein, or a combination thereof. In some embodiments, at least 20% of the proteins in the product can be functional. In some embodiments, the product can be a low-flavor product.
  • the processing can include extracting the product with supercritical CO2, extracting the product with supercritical CO2 and a subsequent extraction with an organic solvent, fractionating the product with ammonium sulfate, and treating the product with a cyclodextrin.
  • a food product including any of the plant cells provided herein. Also provided herein is a food product including any of the multicellular groups provided herein. Also provided herein is a food product including the any of the products provided herein. In some embodiments, the food product can be a meat substitute. Also provided herein is a method for expressing a target protein in a cell, the method including providing any of the plant cells provided herein, and culturing the plant cell under conditions suitable for expression of the first nucleic acid molecule and the second nucleic acid molecule, thereby expressing the target protein. In some embodiments, after the culturing step, at least 10% of the cell, by dry weight, can be the target protein. In some embodiments, after the culturing step, at least 10% of the cytosolic protein cell, by dry weight, can be the target protein.
  • a cell including a first nucleic acid molecule encoding a transcriptional activator operably linked to a first promoter element, and a second nucleic acid molecule encoding a target protein, wherein the target protein can be a protein that modifies other proteins, is a pharmaceutical, is an enzyme in the biosynthetic pathway of a molecule of interest, is a self-assembling protein, is an animal protein, has an artificial amino acid sequence, has an amino acid sequence with portions from multiple organisms, has a flavor, has a color, has an engineered surface activity, has an amino acid sequence, such that when heated with heme, a meat flavor can be produced, and/or has been engineered to bind or to release a particular ligand.
  • the target protein can be a protein that modifies other proteins, is a pharmaceutical, is an enzyme in the biosynthetic pathway of a molecule of interest, is a self-assembling protein, is an animal protein, has an artificial amino acid sequence, has an amino acid sequence with portions from multiple organisms
  • the second nucleic acid molecule can be operably linked to the first promoter element or a second promoter element, wherein the second promoter element can be positively regulated by the transcriptional activator.
  • the first promoter element, the second promoter element, or both the first promoter element and the second promoter element can be constitutive promoter elements.
  • the first promoter element, the second promoter element, or both the first promoter element and the second promoter element can be inducible promoter elements.
  • the first promoter element, the second promoter element, or both the first promoter element and the second promoter element can be tissue-specific promoter elements.
  • the second promoter element, or both the first promoter element and the second promoter element can be seed-specific promoter elements.
  • the promoter element can be selected from: (1) a promoter element from a gene selected from the group consisting of soy beta-conglycinin, G1 -Glycinin, KTI, P24, LEC1, suspensor G564, maize MAC1, maize Cat3, Arabidopsis viviparous- 1, Arabidopsis atmycl, a Brassica napus napin (e.g., napA), mannopine synthase, actin, ubiquitin-1, isocitrate lyase, malate synthase, class I P-l,3-glucanase B, canola cDNAs CA25, CA8, AX92, lipid transfer protein, serine carboxypeptidase, a repetitive proline-rich cell wall protein gene, alpha- amylase, (2) a cauliflower mosaic virus 35S promoter element, a figwort mosaic virus 34S promoter element, an arabidopsis PR1 promoter element, and a combination
  • the promoter element can be a promoter element from a Brassica napus napin, and the transcriptional activator can be ABB.
  • the promoter element can be a Cauliflower mosaic virus 35S promoter element, and the transcriptional activator can be selected from the group consisting of TFIID, ASF-1, OCTSF, and a combination thereof
  • the promoter element can be an Arabidopsis PR1 promoter element, and the transcriptional activator can be selected from the group consisting of AtWRKY50, a TGA transcription factor, and a combination thereof.
  • the promoter element can be a promoter element from an isocitrate lyase gene, and the transcriptional activator can be KlCat8p. In some embodiments, the promoter element can be a promoter element from a malate synthase gene, and the transcriptional activator can be PaHB5. In some embodiments, the promoter element can be a promoter element from an alpha-amylase gene, and the transcriptional activator can be selected from the group consisting of OsMYBSl, OsMYBS2, OsMYBS3, and a combination thereof.
  • the molecule of interest can be a pharmaceutical, a porphyrin or derivative thereof, or a carbohydrate. In some embodiments, the molecule of interest can be a heme. In some embodiments, the molecule of interest can be methylcellulose. In some embodiments, the self-assembling protein can be selected from the group consisting of tubulin, actin, casein, keratin, or a combination thereof. In some embodiments, the cell can be engineered to down-regulate a compound that results in an off-flavor and/or off-aroma.
  • the multicellular group can be a plant or plant component.
  • the multicellular group can be a bean or a seed.
  • the bean or the seed has a low polyunsaturated fatty acid content.
  • the bean or the seed has a low linolenic acid content.
  • the bean or the seed has a high monounsaturated fatty acid content.
  • the bean or the seed has a high oleic acid content.
  • the bean or the seed has a low unsaturated fatty acid content.
  • the bean or the seed has a high saturated fatty acid content.
  • the product can be a protein isolate, a protein concentrate, a textured protein, or a combination thereof. In some embodiments, at least 20% of the proteins in the product can be functional. In some embodiments, the product can be a low-flavor product.
  • a food product including any of the cells provided herein. Also provided herein is a food product including any of the multicellular groups provided herein. Also provided herein is a food product including the any of the products provided herein. In some embodiments, the food product can be a meat substitute.
  • Also provided herein is a method for expressing a target protein in a cell, the method including providing any of the cells provided herein, and culturing the cell under conditions suitable for expression of the first nucleic acid molecule and the second nucleic acid molecule, thereby expressing the target protein.
  • at least 10% of the cell, by dry weight can be the target protein.
  • at least 10% of the cytosolic protein cell, by dry weight can be the target protein.
  • Nucleic acid constructs are provided herein that allow for genetically engineering a cell to increase the recombinant expression of one or more polypeptides. Without being bound by any particular mechanism, the methods described herein can create a positive feedback loop where expression of a transcriptional activator induces a promoter that is operably linked to a nucleic acid encoding the transcriptional activator. This can lead to increased expression of the transcriptional activator as well as one or more target proteins that are operably linked to promoter elements positively regulated by the transcriptional activator. Nucleic acid constructs are provided herein that allow for genetically engineering a cell.
  • a cell can be any appropriate cell, for example, a bacterial cell, a fungal (e.g., yeast) cell, an algal cell, an archaeal cell, or a plant cell.
  • a cell is a bacterial cell.
  • a cell is a fungal cell (e.g., yeast cell).
  • a cell is a plant cell.
  • a cell is a soy cell.
  • Genetically engineering a cell typically includes introducing a recombinant nucleic acid molecule into the cell.
  • General genetic engineering methods are known in the art.
  • Nucleic acid molecules used in the methods described herein are typically DNA, but RNA molecules can be used under the appropriate circumstances.
  • exogenous refers to any nucleic acid sequence that is introduced into a cell from, for example, the same or a different organism or a nucleic acid generated synthetically (e.g., a codon-optimized nucleic acid sequence).
  • an exogenous nucleic acid can be a nucleic acid from one microorganism (e.g., one genus or species of methylotrophic yeast) that is introduced into a different genus or species of methylotrophic yeast; however, an exogenous nucleic acid also can be a nucleic acid from a methylotrophic yeast that is introduced recombinantly into a methylotrophic yeast as an additional copy despite the presence of a corresponding native nucleic acid sequence, or a nucleic acid from a methylotrophic yeast that is introduced recombinantly into a methylotrophic yeast containing one or more mutations, insertions, or deletions compared to the sequence native to the methylotrophic yeast.
  • one microorganism e.g., one genus or species of methylotrophic yeast
  • an exogenous nucleic acid also can be a nucleic acid from a methylotrophic yeast that is introduced recombinant
  • P. pastoris contains an endogenous nucleic acid encoding an AL AS; an additional copy of the P. pastoris ALAS nucleic acid (e.g., introduced recombinantly into / pastoris') is considered to be exogenous.
  • an “exogenous” protein is a protein encoded by an exogenous nucleic acid.
  • an exogenous nucleic acid can be a heterologous nucleic acid.
  • a heterologous nucleic acid refers to any nucleic acid sequence that is not native to an organism (e.g., a heterologous nucleic acid can be a nucleic acid from one microorganism (e.g., one genus or species of methylotrophic yeast, whether or not it has been codon-optimized) that is introduced into a different genus or species of methylotrophic yeast).
  • a heterologous” protein is a protein encoded by a heterologous nucleic acid.
  • a nucleic acid molecule is considered to be exogenous to a host organism when any portion thereof (e.g., a promoter sequence or a sequence of an encoded protein) is exogenous to the host organism.
  • a nucleic acid molecule is considered to be heterologous to a host organism when any portion thereof (e.g., a promoter sequence or a sequence of an encoded protein) is heterologous to the host organism.
  • Transcriptional activators and nucleic acids encoding transcriptional activators (e.g., exogenous nucleic acids encoding transcriptional activators), are known in the art. Transcriptional activators may be normally expressed at low levels. Therefore, it may be desirable to place the nucleic acid encoding the transcriptional activator under control of a promoter that is inducible.
  • operably linked means that a promoter or other expression element(s) are positioned relative to a nucleic acid coding sequence in such a way as to direct or regulate expression of the nucleic acid (e.g., in-frame).
  • the recombinant nucleic acid molecule described herein can be stably integrated into the genome of the cell, or can be extrachromosomally expressed from a replication-competent plasmid. Methods of achieving both are well known and routinely used in the art, and may be influenced by the choice of cell.
  • transcriptional activator Typically, the choice of transcriptional activator and promoter element are linked, as a desired feedback loop is achieved if the transcriptional activator drives its own expression.
  • a promoter element can be provided in the form of a portion of a promoter sequence or an entire promoter sequence.
  • a promoter element to which an encoded protein is operably linked typically includes both a region to which the transcriptional activator binds as well as a minimal sequence necessary for assembly of a transcription complex required for transcription initiation (sometimes also called a core promoter element or a basal promoter element).
  • Basal promoter elements frequently include a "TATA box" element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation.
  • Basal promoter elements also may include a "CCAAT box” element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site.
  • CCAAT box typically the sequence CCAAT
  • GGGCG sequence typically the sequence CGGCG sequence
  • the promoter element can be a seed-specific promoter element.
  • a seed specific promoter element can be from the soy beta-conglycinin gene (see, for example, Chen, et al., Dev Genet., 10(2): 112-22 (1989)) or beta-conglycinin alpha prime subunit 2, a Gl-Glycinin seed specific promoter element (Ding, et al., Biotechnol Lett., 28(12):869-75 (2006)), a KTI promoter element (see, for example, Perez-Grau and Goldberg, Plant Cell.
  • promoter elements from the following seed-genes: zygote and embryo LEC1; suspensor G564; maize MAC1 (see, Sheridan, Genetics 142: 1009-1020 (1996)); maize Cat3, (see, GenBank No. L05934, Abler, Plant Mol. Biol. 22:10131-1038, (1993)); Arabidopsis viviparous- 1, (see, Genbank No.
  • the promoter element can be a constitutive promoter element such as a cauliflower mosaic virus (CaMV) 35S promoter element, a mannopine synthase (MAS) promoter element, a 1' or 2' promoter element derived from T-DNA of Agrobacterium tumefaciens, a figwort mosaic virus 34S promoter element, actin promoter elements such as a rice actin promoter element, or a ubiquitin promoter element such as a maize ubiquitin-1 promoter element. See also U.S. Patent No. 8,115,058 for additional constitutive promoters.
  • a constitutive promoter element such as a cauliflower mosaic virus (CaMV) 35S promoter element, a mannopine synthase (MAS) promoter element, a 1' or 2' promoter element derived from T-DNA of Agrobacterium tumefaciens, a figwort mosaic virus 34S promoter element, actin promoter elements such as a rice act
  • an inducible promoter element can be a modified cauliflower mosaic virus (CaMV) 35S promoter element that is responsive to tetracycline. See, Gatz, et al., Plant J. 2, 397- 404 (1992), and Weinmann, et al., Plant J., 5, 559-569 (1994).
  • an inducible promoter element can be dexamethasone-inducible, or dexamethasone-inducible and tetracycline- inactivatable.
  • an inducible promoter element can be responsive to copper. See, Mett, etal., Proc. Natl Acad. Sci. USA, 90, 4567-4571 (1993).
  • an inducible promoter element can be responsive to an insecticide (e.g., tebufenozide or methoxyfenozide). See, Koo, et al., Plant J., 37, 439-448 (2004); Martinez, et al., Plant J., 5, 559-569 (1999); and Padidam, et al, Transgenic Res., 12, 101-109 (2003).
  • an inducible promoter element can be estrogen responsive (e.g., 17 beta estradiol). See, Bruce, et al., (2000) Plant Cell, 12, 65-80 (2000); and Zuo, etal., Plant J., 24, 265-273 (2000).
  • an inducible promoter element can be responsive to salicylic acid, ethylene, or jasmonic acid.
  • Salicylic acid SA
  • SARE SA-responsive elements
  • Ethylene Ethylene
  • Ethylene Ethylene
  • Ethyl jasmonate interacts with jasmonic acid responsive element (JAR) which drives the expression of the nucleic acid of interest.
  • the inducer e.g., ethylene gas
  • the inducer can be added to the malting chamber to induce expression of the nucleic acid.
  • an inducible promoter element can be ethanol responsive (e.g., ethanol or acetaldehyde responsive).
  • ethanol responsive e.g., ethanol or acetaldehyde responsive
  • the Aspergillus nidulans ALCR transcription factor alcR
  • the palcA promoter is positioned upstream of a target DNA for expression.
  • ethanol-inducible expression can be based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. See, Werner, et al., Proc Natl AcadSci USA, 108(34): 14061-14066 (2011).
  • the promoter element can be a germination specific promoter element.
  • a promoter element typically results in expression of the target product during germination and/or early seedling growth in one or more of the radical, hypocotyl, cotyledons, epicotyl, root tip, shoot tip, meristematic cells, seed coat, endosperm, true leaves, internodal tissue, and nodal tissue.
  • promoter elements from genes encoding the glyoxysomal enzymes isocitrate lyase (ICL) and malate synthase (MS) from several plant species (Zhang et al., Plant Physiol. 104: 857-864, 1994); Reynolds and Smith, Plant Mol. Biol.
  • Promoter elements also can be from other genes whose mRNAs appear to accumulate specifically during the germination process, for example class I P-l,3-glucanase B from tobacco (Vogeli-Lange et al, Plant J., 5: 273-278, 1994); canola cDNAs CA25, CA8, AX92 (Harada et al., Mol. Gen. Genet., 212: 466-473, 1988); Dietrich et al., J.
  • lipid transfer protein Sossountzove et al, Plant Cell, 3: 923-933, 1991
  • rice serine carboxypeptidases Wango et al., Plant Phys., 105: 1275-1280, 1994
  • repetitive proline-rich cell wall protein genes Datta el a!.. Plant Mol. Biol. 14: 285-286, 1990.
  • An a-amylase promoter element also can be used as a germination specific promoter element. See, Eskelin, et al., Plant Biotechnology Journal, 7: 657-672 (2009).
  • promoters can include aleurone-specific promoters, endosperm-specific promoters, embryo-specific promoters, leaf-and-stem-specific promoters, panicle-specific promoters, rootspecific promoters, and pollen-specific promoters (see, for example U.S. Patent No. 8,115,058).
  • Table 1 provides some exemplary promoter element-transcriptional activator pairings.
  • a feedback loop created using a transcriptional activator such as those described herein can be used to drive expression of one or more target proteins.
  • a cell can include a second nucleic acid molecule encoding a target protein operably linked to a promoter element positively regulated by the transcription factor.
  • a target protein can be any appropriate target protein.
  • a target protein can be a heme-containing protein.
  • the term “heme-containing protein” can be used interchangeably with “heme-containing polypeptide” or “heme protein” or “heme polypeptide” or “heme-loaded heme-containing polypeptide” and includes any polypeptide that can covalently or noncovalently bind to a heme moiety.
  • the terms “heme cofactor” and “heme” are used interchangeably and refer to the iron-containing (Fe 2+ or Fe 3+ ) compound of the porphyrin class which forms a nonprotein part of the heme-containing protein.
  • a heme-containing protein can be an androglobin, a cytoglobin, a globin E, a globin X, a globin Y, a hemoglobin, a leghemoglobin, a flavohemoglobin, Hell's gate globin I, a myoglobin, an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, a cytoglobin, a histoglobin, a neuroglobins, a chlorocruorin, a truncated hemoglobin (e.g., HbN or HbO), a truncated 2/2 globin, a hemoglobin 3 (e.g., Glb3), a cytochrome, or a peroxidase.
  • a truncated hemoglobin e.g., HbN or HbO
  • a target protein can be a protein that modifies other proteins (e.g., a glutaminase).
  • a target protein can be a pharmaceutical (e.g., an antibody or an antigen-binding fragment thereof, vaccine or antigen, blood factor, thrombolytic agent, hormone, hematopoietic growth factor, interferon, interleukin, fusion protein, biologic, biosimilar, or a therapeutic enzyme such as an L-asparaginase).
  • a target protein can include one or more enzymes in the biosynthetic pathway of a molecule of interest (e.g., a pharmaceutical (e.g., a polyketide), a porphyrin or derivative thereof (e.g., a heme), or a carbohydrate (e.g., methylcellulose)).
  • a pharmaceutical e.g., a polyketide
  • a porphyrin or derivative thereof e.g., a heme
  • carbohydrate e.g., methylcellulose
  • a pharmaceutical can include a protein (e.g., an antibody or antigen-binding fragment thereof, vaccine or antigen, blood factor, thrombolytic agent, hormone, hematopoietic growth factor, interferon, interleukin, fusion protein, biologic, biosimilar, or a therapeutic enzyme, such as an 1- asparaginase).
  • a pharmaceutical can include an organic and/or inorganic compound (e.g., a polyketide such as erythromycin).
  • a target protein can be an animal protein (e.g., casein).
  • a target protein can be a self-assembling protein (e.g., tubulin, actin, casein, or keratin).
  • a target protein can be a silk protein.
  • a target protein can have an artificial amino acid sequence and/or an amino acid sequence with portions from multiple organisms.
  • a target protein can be engineered to have an altered isoelectric point (e.g., an isoelectric point below about pH 4 or above about pH 7).
  • a target protein can further include a targeting sequence (e.g., such that the target protein is trafficked to a subcellular location, such as the mitochondria, nucleus, or so forth).
  • a target protein can be a protein that has a flavor (e.g., has a flavor of an animal meat).
  • a target protein can be a protein that has a color (e.g., red, orange, yellow, green, cyan, blue, violet, black), for example, fluorescent proteins, luminescent proteins, chromoproteins, metalloproteins, phycobiliproteins, hemocyanins, hemerythrin, or globins.
  • the color of a target protein can be provided from a chromophore, a cofactor, or a prosthetic group.
  • a target protein can be engineered (e.g., via a level of surface activity) have different taste profiles.
  • a target protein can be engineered to bind or release a particular ligand (e.g., a cofactor, a metal ion, oxygen, small molecule, oil, and/or water).
  • a particular ligand e.g., a cofactor, a metal ion, oxygen, small molecule, oil, and/or water.
  • a target protein for a meat application, it may be beneficial for a target protein to hold water.
  • a target protein to release water.
  • a target protein can be, for example and without limitation, a dehydrin, a phytase, a protease, a catalase, a lipase, a peroxidase, an amylase, a transglutaminase, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, or an antibody against any such polypeptides.
  • a heterologous nucleic acid can encode one or more enzymes involved in the pathway for production of small molecules, such as ethanol, lactic acid, butanol, adipic acid, or succinic acid.
  • a nucleic acid encoding a target protein can be separate from the nucleic acid molecule encoding a transcriptional activator operably linked to a promoter element, or can be contiguous with the nucleic acid encoding a transcriptional activator operably linked to a promoter element. It also would be appreciated by a skilled artisan that a single promoter, or promoter element therefrom, can be used to drive transcription of both or all of the genes (e.g., the nucleic acid encoding the transcriptional activator as well as the one or more nucleic acids encoding the target protein).
  • a cell can include 10% or more (e g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more) by dry weight of the one or more target proteins.
  • cytosol of cell can include 10% or more (e g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more) by dry weight of the one or more target proteins.
  • multicellular groups of the cells provided herein are cellular culture of the cells provided herein.
  • plants or plant components e.g., stalks, stems, leaves, fruits, or seeds (e.g., beans) of the cells provided herein.
  • the cells provided herein can be used in a food product (e.g., a meat substitute).
  • multicellular groups (e.g., plants or plant components) of the cells provided herein can be used in a food product.
  • a bean including cells (or primarily including cells) as described herein can be used in a food product.
  • a bean including cells (or primarily including cells) as described herein can be processed to be used in a food product (e.g., by making a protein concentrate, a protein isolate and/or a textured protein).
  • cells provided herein can provide benefits to a food product, such as, without limitation, decreased off-flavor, decreased off-color, increased on-target (e.g., animal) flavor, or having low- flavor.
  • “low flavor” with respect to a plant and/or protein product means that the plant and/or protein product has less flavor than the source of the plant and/or protein product (e.g., soy, if a soy plant and/or protein product is described). For example, less of one or more compounds that give rise to a distinguishing flavor associated with the source of the protein.
  • a low flavor plant and/or protein product can have little flavor of its own.
  • a low flavor plant and/or protein product has less flavor than a known plant and/or protein product (e.g., a commercial soy protein isolate, such as those described herein). Having less flavor can be determined, for example, by a trained human panelist, or, for example, by measurement of one or more volatile compounds commonly understood to impart flavor and/or aroma.
  • a known plant and/or protein product e.g., a commercial soy protein isolate, such as those described herein.
  • Having less flavor can be determined, for example, by a trained human panelist, or, for example, by measurement of one or more volatile compounds commonly understood to impart flavor and/or aroma.
  • proteins from the cells provided herein can be used in a food product.
  • Exemplary food products are described in, for example, U.S. Publication Nos. 2014/0193547 and 2015/0305361, and U.S. Patent Nos. 10,039,306, 9,700,067, 10,172,380, and 9,011,949.
  • Additional methods for the production of soy protein products having reduced off-flavors can include an extraction using supercritical carbon dioxide (optionally also with an organic solvent) (see, for example, U.S. Patent No. 7,638,155 and U.S. Publication No. 2008/0145511), salting-out methods (for example ammonium sulfate fractionation of phospholipid-bound proteins), and/or treatment with a cyclodextrin (e.g., a p-cyclodextrin) than can optionally include changes to physical or chemical properties of the treatment - e.g., pH, temperature, or redox potential (see, for example, Damoong and Arora, Annu. Rev. Food Sci. Technol. 2013. 4:327- 346).
  • a cyclodextrin e.g., a p-cyclodextrin
  • the cells e.g., multicellular group (e.g., plant or plant component (e.g., bean or seed))
  • a product produced therefrom e.g., a protein isolate, a protein concentrate, and/or a textured protein
  • the amino acid profile of the cells can be engineered to produce a specific flavor profile.
  • one or more compounds e.g., small molecules and/or proteins that result in off-flavors and/or off-aromas can be downregulated.
  • a specific desired flavor profile can be that of a meat (e.g., beef, chicken, pork, or fish).
  • a compound that results in an off-flavor and/or off-aroma can be a compound that has a flavor and/or aroma of the cells (e.g., multicellular group (e.g., plant or plant component (e.g., bean or seed))), such as soy.
  • At least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of the proteins in the cells provided herein are functional.
  • functional proteins can have one or more of the following properties: non-denatured; capable of forming a gel upon heating (e.g., a suspension of about 25 to about 250 mg/mL (e.g., about 25 to about 50 mg/mL, about 25 to about 100 mg/mL, about 25 to about 150 mg/mL, about 25 to about 200 mg/mL, about 50 to about 250 mg/mL, about 100 to about 250 mg/mL, about 150 to about 250 mg/mL, or about 200 to about 250 mg/mL) at a pH of about 7.0) thermally transitions to a gel upon heating to about 65°C); thermally denatures during incubation between about 50°C and about 85°C, with greater than about 80% of the protein denaturing after about 20 minutes
  • multicellular groups of cells can be processed into a product (e.g., a protein concentrate, a protein isolate, and/or textured protein).
  • a product can be produced by any appropriate method.
  • a product can be produced such that it includes functional protein.
  • at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of the proteins in a product (e.g., a protein isolate, a protein concentrate and/or textured protein) produced from cells provided herein are functional.
  • a product can be produced such that it includes denatured protein.
  • a product can be produced such that it is enriched in proteins from certain subcellular locations (e.g., cytosol, nucleus, mitochondria, a plastid, and so forth).
  • Suitable plants can produce seeds or beans of any color, including black, brown, red, yellow, or green, and can be variegated or bicolored.
  • the color of a seed or a bean can be influenced by a target protein (e.g., a heme-containing protein) and/or environmental conditions (e.g., temperature and/or pH).
  • a plant can be used that produces an oil with a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content.
  • U.S. Patent No. 5,981,781 which describes soy plants that produce an oil with an oleic acid content of greater than 75% and a polyunsaturated fatty acid content of less than 10%.
  • U.S. Patent No. 9,918,485 which describes soy plants that produce an oil with an oleic acid content of at least 60%.
  • the Plenish® high oleic soybeans from Pioneer for example, see U.S. Publication No. 2008/0312082.
  • Typical soybean plants produce oils with high levels of polyunsaturated fatty acids, and the oil is more prone to oxidation than oils with higher levels of monounsaturated and saturated fatty acids.
  • a plant can be used that produces an oil with a high saturated fatty acid content.
  • a plant can be used that produces an oil with a high saturated fatty acid content.
  • soy plants that produce an oil with 40% or more stearic acid (e.g., 50% stearic acid).
  • U.S. Publication No. 2004/0049813 describes soy plants that produce an oil with a combined palmitic acid and stearic acid content of greater than 21%, an oleic acid content of greater than 60% and a polyunsaturated fatty acid content of less than 7%.
  • Higher saturated fatty acid content typically is associated with animal fat (typically 22-37% C16:0 and 8-30% C18:0) and/or palm oil (typically 39-48% C16:0 and 3.5-6% C18:0), and may impart a different flavor profile to the heme or other proteins isolated from the plant.
  • a soy plant can be used that has a higher protein content in seeds than in the seeds of a reference soybean.
  • International Publication No. WO 2020/106488 describes a soy plant with decreased expression or activity of one or more HECT E3 ligase (HEL) that has an increased protein content.
  • HELI HECT E3 ligase
  • knocking out HELI, or both HELI and HEL2 increased seed protein content significantly compared to wild type plants, and knocking out both HELI and HEL2 showed higher seed protein content than the HELI knockout.
  • the knockout of HELI showed increased oleic and stearic acid contents and reduced linolenic, palmitic and stachyose contents.
  • HELI and HEL2 increased oleic content and reduced linoleic, linolenic, palmitic, stearic, stachyose, and total soluble carbohydrate contents.
  • HECT E3 ligases also called E3 ubiquitin ligases, changes protein degradation in cells, altering plant traits such as those described above.
  • the fatty acid profile of a plant can be modified by breeding and/or genetic engineering. See, for example, Nguyen, etal., Curr Genomics, 17(3):241-260 (2016), and Clemente and Cahoon, Plant Physiol., 151(3): 1030-1040 (2009), and U.S. Patent No. 6,323,392.
  • a plant can have decreased activity of delta-12 desaturase, which converts oleic acid to linoleic acid.
  • the plants can be modified such that they contain increased oleoyl- or stearoyl-ACP thioesterase activity and decreased fatty acid desaturase activities, including delta-9, delta-12, and delta-15 desaturase activities.
  • Plants also can be modified such that they contain increased 3 -ketoacyl -ACP synthase II (KAS II). Increased thioesterase activity may not be necessary if delta-9 desaturase activity is completely inhibited. Plants also can exhibit increased palmitoyl-ACP thioesterase activity.
  • the expression and/or activity of one or more enzymes that act on lipids or fatty acids in a plant can be modified by breeding or genetic engineering.
  • lipases hydrolyze lipids into free fatty acids, which can generate off-flavors upon oxidation.
  • Lipoxygenases such as 9-lipoxygenases or 13 -lipoxygenases, catalyze the di oxygenation of polyunsaturated fatty acids, such as linoleic acid, alpha-linolenic acid, or arachidonic acid, into hydroperoxides.
  • Hydroperoxide lyases can catalyze the cleavage of C-C bonds in hydroperoxides of fatty acids, which can result in the formation of aldehydes.
  • aldehydes for example, hexenal, hexanal, nonenal, nonanal, or nonadienal, can contribute off-flavors or aromas or undergo isomerization, dehydrogenation, reduction to alcohols, or oxidation to esters.
  • a plant can be modified to have decreased expression and/or activity of one or more lipases, lipoxygenases, and/or hydroperoxide lyases.
  • plants can have a desired fatty acid profile (e.g., a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content, a high medium chain fatty acid content, or a high saturated fatty acid content) and decreased expression and/or activity of one or more lipases, lipoxygenases, and/or hydroperoxide lyases.
  • a low lipase, lipoxygenase, and/or hydroperoxide lyase activity can yield a low flavor plant (and/or protein product produced therefrom). See, for example International Publication No. WO 2017/218883.
  • hybrids of plants with modified FAD-2 or FAD-3 enzymes can result in altered fatty acid profiles.
  • a hybrid between a female Brassica napus parent with a homozygous modified FAD-2 (delta-12 fatty acid desaturase 2) in either the A- or C- genome and a male Brassica napus plant with a homozygous modified FAD-2 in both the A- and C-genomes can result in progeny that produce elevated oleic acid (Cl 8: 1) of at least 80% by weight and reduced linolenic acid (C 18:3) content of no more than 3% by weight of total fatty acid content in their seeds. See, for example, U.S. Patent No. 6,323,392.
  • oleic acid content of seeds can be increased to between 70% and 90% and linolenic acid content is decreased to less than about 3% by weight of the total fatty acids in the seed of a plant (e.g., a soybean, canola, or sunflower plant) with four or more modifications to the genes encoding a FAD2 and a FAD3 polypeptide, such as FAD2-1A, FAD2-1B, FAD3a, and FAD3b compared to the seed of a control plant without the introduced genetic modifications.
  • a plant e.g., a soybean, canola, or sunflower plant
  • At least one characteristic selected from: (A) increased oleic acid content, (B) decreased linoleic acid content, (C) decreased linolenic acid content, (D) decreased stearic acid content, and (E) decreased palmitic acid content can be obtained in seeds of plants by altering the expression or activity, or both, of one or more HECT E3 ligase (HEL) polypeptides (e.g., decreasing by expression or activity). Modifications can include, for example, a deletion, an insertion, or a substitution in a HECT E3 ligase gene. See, for example, International Publication No. WO 2020/106488.
  • a thioesterase e.g., the Umbellularia californica Nutt. FATB1
  • medium-chains fatty acids such as C12:0 and C14:0 from the plastid fatty acid synthetic pathway
  • a plant e.g., a soy plant. See, for example, Hu, et al., PLosS One, 12(2) :e0172296 (2017), and Voelker, et al, Science, 257(5066):72-4 (1992).
  • plants can have a desired fatty acid profile.
  • Non-limiting examples include high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content such as the Plenish® soybeans (see, e.g., U.S. Publication No. 2008/0312082), a high medium chain fatty acid content, modified fatty acid metabolism (e.g., pea cultivar Snak Hero (SL3192), see, for example, U.S. Patent No. 10,660,297), or a high saturated fatty acid content.
  • Plenish® soybeans see, e.g., U.S. Publication No. 2008/0312082
  • modified fatty acid metabolism e.g., pea cultivar Snak Hero (SL3192), see, for example, U.S. Patent No. 10,660,297
  • SL3192 pea cultivar Snak Hero
  • Such plants can be used as a source for protein (e.g., protein concentrate, protein isolate, or textured vegetable protein such as textured soy protein) to improve the flavor profde and/or functional characteristics of the protein (e.g., having one or more of reduced off-flavors, altered gel-strength, altered viscosity, improved oxidative stability, and improved whiteness).
  • protein e.g., protein concentrate, protein isolate, or textured vegetable protein such as textured soy protein
  • functional characteristics of the protein e.g., having one or more of reduced off-flavors, altered gel-strength, altered viscosity, improved oxidative stability, and improved whiteness.
  • Methods for making textured vegetable protein (e.g., textured soy protein) having reduced off-flavors can include isolating protein from soybeans that yield an oil having a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content and processing the soy protein to obtain textured soy protein (e.g., forming the soy protein into desired shape by extrusion, or cooking the soy protein and spray drying).
  • a plant has a low polyunsaturated fatty acid (e.g., linolenic acid and/or linoleic acid) content. In some embodiments, a plant has a low linolenic acid content. In some embodiments, a plant has a high monounsaturated fatty acid content. In some embodiments, a plant has a high oleic acid content. In some embodiments, a plant has a low polyunsaturated fatty acid content and a high monounsaturated fatty acid content. In some embodiments, a plant has a low linolenic acid content and a high oleic acid content.
  • a plant has a low polyunsaturated fatty acid content and a high oleic acid content. In some embodiments, a plant has a low linolenic acid content and a high monounsaturated fatty acid content. In some embodiments, a plant has a low unsaturated fatty acid content. In some embodiments, a plant has a high saturated fatty acid content.
  • Methods of introducing nucleic acids into plants or cells include, without limitation, transduction, electroporation, biolistic particle delivery, and transformation.
  • Agrobacterium-mediated transformation, viral vector-mediated transformation, chemical transformation, electroporation, or particle gun transformation can be used for introducing nucleic acids into monocotyledonous or dicotyledonous plants. See, for example, U.S. Patent Nos. 5,538,880; 5,204,253; 6,329,571 and 6,013,863.
  • gene editing techniques using site-specific nucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENS), or the clustered regularly interspaced short palindromic repeat (CRISPR)- Cas9 system can be used to produce plants or cells having altered characteristics including one or more of fatty acid profile, flavor profile, or sugar content. See, for example, Aroroa and Narula, Front. Plant Sc. 8: 1932 (2017). If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
  • a “purified” polypeptide is a polypeptide that has been separated or purified from cellular components that naturally accompany it. Typically, the polypeptide is considered “purified” when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight, free from the polypeptides and naturally occurring molecules with which it is naturally associated. Since a polypeptide that is chemically synthesized is, by nature, separated from the components that naturally accompany it, a synthetic polypeptide is “purified.”
  • an “enriched” protein is a protein that accounts for at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more) by dry weight, of the mass of the production cell, or at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99%) by dry weight, the mass of the production cell lysate (e.g., excluding cell wall or membrane material).
  • a “purified” protein is a protein that has been separated from cellular components that naturally accompany it. Typically, the protein is considered “purified” when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight, free from other proteins and naturally occurring molecules with which it is naturally associated.
  • heme-containing proteins can be produced before purification in methylotrophic yeast such as Pichia (e.g., P. pastoris) or in transgenic plants, transgenic cells, or transgenic seeds using inducible promoters and/or a positive feedback loop such as those disclosed in U.S. Publication No. 2018/0127764.
  • methylotrophic yeast such as Pichia (e.g., P. pastoris)
  • transgenic plants, transgenic cells, or transgenic seeds using inducible promoters and/or a positive feedback loop such as those disclosed in U.S. Publication No. 2018/0127764.
  • nucleic acids can include DNA and RNA, and includes nucleic acids that contain one or more nucleotide analogs or backbone modifications.
  • a nucleic acid can be single stranded or double stranded, which usually depends upon its intended use. Also provided are nucleic acids and polypeptides that differ from a given sequence.
  • Nucleic acids and polypeptides can have at least 50% sequence identity (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to a given nucleic acid or polypeptide sequence.
  • a nucleic acid or polypeptide can have 100% sequence identity to a given nucleic acid or polypeptide sequence.
  • two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined.
  • the number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value.
  • the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence.
  • a single sequence can align with more than one other sequence and hence, can have different percent sequence identity values over each aligned region.
  • the alignment of two or more sequences to determine percent sequence identity can be performed using the computer program ClustalW and default parameters, which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res., 3 l(13):3497-500. ClustalW calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments.
  • the default parameters can be used (i.e., word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5); for an alignment of multiple nucleic acid sequences, the following parameters can be used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes.
  • word size 1; window size: 5; scoring method: percentage; number of top diagonals: 5; and gap penalty: 3.
  • ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher website or at the European Bioinformatics Institute website on the World Wide Web.
  • Changes can be introduced into a nucleic acid molecule, thereby leading to changes in the amino acid sequence of the encoded polypeptide.
  • changes can be introduced into nucleic acid coding sequences using mutagenesis (e.g., site-directed mutagenesis, PCR-mediated mutagenesis, transposon mutagenesis, chemical mutagenesis, UV mutagenesis or radiation induced mutagenesis) or by chemically synthesizing a nucleic acid molecule having such changes.
  • mutagenesis e.g., site-directed mutagenesis, PCR-mediated mutagenesis, transposon mutagenesis, chemical mutagenesis, UV mutagenesis or radiation induced mutagenesis
  • Such nucleic acid changes can lead to conservative and/or non-conservative amino acid substitutions at one or more amino acid residues.
  • a “conservative amino acid substitution” is one in which one amino acid residue is replaced with a different amino acid residue having a similar side chain (see, for example, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5(Suppl. 3): 345-352, which provides frequency tables for amino acid substitutions), and a nonconservative substitution is one in which an amino acid residue is replaced with an amino acid residue that does not have a similar side chain.
  • Nucleic acid and/or polypeptide sequences may be modified as described herein to improve one or more properties such as, without limitation, increased expression (e.g., transcription and/or translation), tighter regulation, deregulation, loss of catabolite repression, modified specificity, secretion, thermostability, solvent stability, oxidative stability, protease resistance, catalytic activity, and/or color.
  • an “isolated” nucleic acid molecule is a nucleic acid molecule that is free of sequences that naturally flank one or both ends of the nucleic acid in the genome of the organism from which the isolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease digestion). Such an isolated nucleic acid molecule is generally introduced into a vector (e.g., a cloning vector, or an expression vector) for convenience of manipulation or to generate a fusion nucleic acid molecule, discussed in more detail below.
  • an isolated nucleic acid molecule can include an engineered nucleic acid molecule such as a recombinant or a synthetic nucleic acid molecule.
  • Vectors as described herein can be introduced into a cell (e.g., a host cell).
  • host cell refers to the particular cell into which the nucleic acid is introduced and also includes the progeny of such a cell that carry the vector.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • nucleic acids can be expressed in bacterial cells such as E. coli, or in insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • nucleic acids are well known to those skilled in the art and include, without limitation, electroporation, calcium phosphate precipitation, polyethylene glycol (PEG) transformation, heat shock, lipofection, microinjection, and viral - mediated nucleic acid transfer.
  • electroporation calcium phosphate precipitation
  • PEG polyethylene glycol
  • Nucleic acids can be isolated using techniques routine in the art. For example, nucleic acids can be isolated using any method including, without limitation, recombinant nucleic acid technology, and/or the polymerase chain reaction (PCR). General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides.
  • PCR polymerase chain reaction
  • Polypeptides can be purified from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography.
  • a polypeptide also can be purified, for example, by expressing a nucleic acid in an expression vector.
  • a purified polypeptide can be obtained by chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • a construct or vector containing a nucleic acid construct as described herein (e.g., a nucleotide sequence that encodes a polypeptide operably linked to a promoter element as described herein) also is provided.
  • Constructs or vectors, including expression constructs or vectors, are commercially available or can be produced by recombinant DNA techniques routine in the art.
  • a construct or vector containing a nucleic acid can have expression elements operably linked to such a nucleic acid, and further can include sequences such as those encoding a selectable marker (e.g., an antibiotic resistance gene).
  • a construct or vector containing a nucleic acid can encode a chimeric or fusion polypeptide (i.e., a polypeptide operatively linked to a heterologous polypeptide, which can be at either the N-terminus or C-terminus of the polypeptide).
  • a heterologous polypeptide i.e., a polypeptide operatively linked to a heterologous polypeptide, which can be at either the N-terminus or C-terminus of the polypeptide.
  • heterologous polypeptides are those that can be used in purification of the encoded polypeptide (e.g., 6xHis tag, glutathione S-transferase (GST)).
  • Nucleic acids also can be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sections 7.37-7.57, 9.47-9.57, 11.7-11.8, and 11.45-11.57). Sambrook et al. discloses suitable Southern blot conditions for oligonucleotide probes less than about 100 nucleotides (Sections 11.45-11.46).
  • the Tm between a sequence that is less than 100 nucleotides in length and a second sequence can be calculated using the formula provided in Section 11.46.
  • Sambrook et al. additionally discloses Southern blot conditions for oligonucleotide probes greater than about 100 nucleotides (see Sections 9.47-9.54).
  • the Tm between a sequence greater than 100 nucleotides in length and a second sequence can be calculated using the formula provided in Sections 9.50-9.51 of Sambrook et al.
  • the conditions under which membranes containing nucleic acids are prehybridized and hybridized, as well as the conditions under which membranes containing nucleic acids are washed to remove excess and non-specifically bound probe, can play a significant role in the stringency of the hybridization.
  • Such hybridizations and washes can be performed, where appropriate, under moderate or high stringency conditions.
  • washing conditions can be made more stringent by decreasing the salt concentration in the wash solutions and/or by increasing the temperature at which the washes are performed.
  • high stringency conditions typically include a wash of the membranes in 0.2X SSC at 65°C.
  • interpreting the amount of hybridization can be affected, for example, by the specific activity of the labeled oligonucleotide probe, by the number of probe-binding sites on the template nucleic acid to which the probe has hybridized, and by the amount of exposure of an autoradiograph or other detection medium.
  • any number of hybridization and washing conditions can be used to examine hybridization of a probe nucleic acid molecule to immobilized target nucleic acids, it is more important to examine hybridization of a probe to target nucleic acids under identical hybridization, washing, and exposure conditions.
  • the target nucleic acids are on the same membrane.
  • a nucleic acid molecule is deemed to hybridize to a nucleic acid but not to another nucleic acid if hybridization to a nucleic acid is at least 5-fold (e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold) greater than hybridization to another nucleic acid.
  • the amount of hybridization can be quantitated directly on a membrane or from an autoradiograph using, for example, a Phosphorlmager or a Densitometer (Molecular Dynamics, Sunnyvale, CA). Polypeptides can be detected using antibodies.
  • Techniques for detecting polypeptides using antibodies include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • An antibody can be polyclonal or monoclonal.
  • An antibody having specific binding affinity for a polypeptide can be generated using methods well known in the art.
  • the antibody can be attached to a solid support such as a microtiter plate using methods known in the art. In the presence of a polypeptide, an antibody -polypeptide complex is formed.
  • Detection e.g., of an amplification product, a hybridization complex, or a polypeptide is usually accomplished using detectable labels.
  • label is intended to encompass the use of direct labels as well as indirect labels.
  • Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • Methods are described herein that can be used to generate a strain that lacks sequences for selection (i.e., that lacks a selectable marker). These methods include using a circular plasmid DNA vector and a linear DNA sequence; the circular plasmid DNA vector contains a selection marker and an origin of DNA replication (also known as an autonomously replicating sequence (ARS)), and the linear DNA sequence contains sequences for integration into the Pichia genome by homologous recombination.
  • ARS autonomously replicating sequence
  • a linear DNA molecule additionally can include nucleic acid sequences encoding one or more proteins of interest such as, without limitation, heme-bound LegH, a dehydrin, a phytase, a protease a catalase, a lipase, a peroxidase, an amylase, a transglutaminase, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, one or more enzymes involved in the pathway for production of small molecules, such as ethanol, lactic acid, butanol, adipic acid or succinic acid, or an antibody against any such proteins.
  • Cells can be transformed with both DNA molecules and the transformants selected by the presence of the selectable marker on the circular plasmid. Transformants then can be screened for integration of the linear DNA molecule into the genome using, for example, PCR. Once transformants with the correct integration of the marker-free linear DNA molecule are identified, the cells can be grown in the absence of selection for the circular plasmid. Because the markerbearing plasmid is not stably maintained in the absence of selection, the plasmid is lost, often very quickly, after selection is relaxed. The resulting strain carries the integrated linear DNA in the absence of heterologous sequences for selection. Therefore, this approach can be used to construct strains that lack a selectable marker with little to no impact on recombinant product (e.g., protein) yield.
  • recombinant product e.g., protein

Abstract

L'invention concerne des méthodes et du matériel destinés à la modification génétique de cellules.
PCT/US2021/053178 2020-10-02 2021-10-01 Constructions d'expression et méthodes de modification génétique de cellules WO2022072833A2 (fr)

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US11359206B2 (en) 2020-10-28 2022-06-14 Pioneer Hi-Bred International, Inc. Leghemoglobin in soybean
US11401526B2 (en) 2020-09-30 2022-08-02 Nobell Foods, Inc. Recombinant fusion proteins for producing milk proteins in plants
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