US Patent No. 9,057,063 titled Genetically modified biological cells

US 9,057,063, inventor Theresa O'Keefe, titled Genetically modified biological cells


first claim:

A recombinant genetically modified bacterial cell comprising: a recombinant chromosomally integrated expression cassette, the recombinant chromosomally integrated expression cassette comprising a first nucleic acid molecule encoding a protein of interest and a second nucleic acid molecule encoding a positive selectable marker, said positive selectable marker being an enzyme necessary for the production of an essential vitamin B12, which is the enzyme with EC 1.3.1.54 or EC 2.1.1.133, wherein the expression of the positive selectable marker is necessary for survival of the genetically modified bacterial cell in media lacking said vitamin, and wherein promoter of the expression cassette drives expression of positive selectable marker and expression of the protein of interest.

From the specification:


The present invention is based, in part, on our discovery of a way to configure expression cassettes so that the expression of a selectable marker protein, which is critical for the growth or survival of a cell, also results in the expression of a protein of interest in a biological cell. Accordingly, in one aspect, the invention features a genetically modified cell (e.g., a bacterial cell) that includes a chromosomally integrated or cytoplasmic expression cassette that includes a first nucleic acid sequence encoding a protein of interest and a second nucleic acid sequence encoding a selectable marker protein. The regulatory sequence (e.g., the sequence encoding a functional promoter) that drives expression of the required selectable marker protein also drives expression of the protein of interest. For that reason, we may refer to their expression as being "linked" or "functionally coupled."

In some embodiments, a selectable marker is defined as an amino acid sequence that, in a population of cells derived from a parent cell to which a nucleic acid encoding the selectable marker was first introduced, was capable of acting as a selectable marker in the parent cell given the genomic constitution of the parent cell at the time the nucleic acid encoding the selectable marker was introduced into the parent cell.

In some embodiments, the protein of interest is not a purification or epitope tag.

The genetically modified cell can be a bacterial cell (e.g., a cyanobacterium). For example, the genetically modified bacterial cell can belong to one of the following species: Anabaena species (sp.), Anabaenopsis sp., Aphanizomenon sp., Arthrospira sp., Calothrix sp., Chamaesiphon sp., Chlorogloeopsis sp., Chroococcidiopsis sp., Chroococcus sp., Cyanothece sp., Cylindrospermum sp., Dactylococcopsis sp., Dermocarpella sp., Fischerella sp., Geitlerinema sp., Gloeobacter sp., Gloeocapsa sp., Gloeothece sp., Leptolyngbya sp., Lyngbya sp., Microchaete sp., Microcoleus sp., Microcystis sp., Myxosarcina sp., Nodularia sp., Nostoc sp., Oscillatoria sp., Pleurocapsa sp., Pseudanabaena sp., Scytonema sp., Spirulina sp., Stanieria sp., Symploca sp., Synechococcus sp., Synechocystis sp., Tolypothrix sp., or Xenococcus sp. In some embodiments, the genetically modified bacterial cell can belong to one of the following species: Calothrix sp., Geitlerinema sp., Myxosarcina sp., Pleurocapsa sp., or Stanieria sp.

The protein of interest that is expressed by the genetically modified bacterial cell can be a bacterial, plant, or mammalian protein and the nucleic acid sequence from which a protein of interest is expressed can be codon optimized so that it is optimally expressed in the genetically modified bacterial cell. The protein of interest can be a metal-binding protein. For example, the metal-binding protein can be a metallothionein, a transcription factor, or an enzyme. The protein of interest can be an enzyme. For example, the enzyme can be a protease, an oxidase, a phytase, a chitinase, an invertase, a lipase, a cellulase, a xylenase, a kinase, a phosphatase, an enzyme within a biosynthetic pathway that results in the production of an oil, an enzyme active in the synthesis of fatty acids 10-16 carbons in length, an enzyme that accelerates fructose metabolism, an enzyme that accelerates glycerol metabolism (e.g., glycerol kinase or glycerol 3-phosphate dehydrogenase), or an enzyme that converts lactic acid to pyruvate (e.g., lactate dehydrogenase M4). The protein of interest can be an enzyme active in the synthesis of fatty acids 10-16 carbons in length, for example, pyruvate dehydrogenase, dihydrolipoyl dehydrogenase, dihydrolipoyllysine-residue acetyltransferase, acteyl-CoA carboxylase, malonyl-CoA:ACP transacylase, and fatty acid synthase.

The protein of interest can be a protein that accelerates fructose metabolism or a protein that increases fructose transport into a cell, for example, hepatic fructokinase, aldolase B, glyceraldehyde kinase, or a protein belonging to the ATP-binding cassette transporter (ABC-transporter) family and that transports fructose (e.g., phosphoenolpyruvate-protein phosphotransferase, HPr protein or protein-N.pi.-phosphohistidine-sugar phosphotransferase (fructose-specific)).

The protein of interest can be an immunogen, a soluble protein, an anti-toxin, a plant protein (e.g., zein), a mammalian protein, a hormone, a transcription factor, a growth factor, an anticoagulant, a lysosomal protein, an enzyme substrate, a receptor or a subunit thereof, a heavy chain of an immunoglobulin, a light chain of an immunoglobulin, or a single chain antibody. In certain embodiments, the protein of interest is an immunogen which is part of a fusion protein. In some embodiments, the protein of interest is a mammalian protein (e.g., a mammalian hormone, a mammalian transcription factor, a mammalian growth factor, a mammalian anticoagulant, a mammalian lysosomal protein, a mammalian enzyme substrate, a mammalian receptor or a subunit thereof, a mammalian heavy chain of an immunoglobulin, a mammalian light chain of an immunoglobulin, or a mammalian single chain antibody).

In some embodiments, the genetically modified bacterial cell express a positive selectable marker protein, for example, an enzyme within a biosynthetic pathway that produces an essential nutrient or an enzyme within a biosynthetic pathway that produces a vitamin. In some embodiments, the positive selectable marker is an enzyme within a biosynthetic pathway that produces any one of the following essential nutrients: ascorbic acid (vitamin C), biotin (vitamin B7), choline, folic acid (vitamin B9), inositol, nicotinic acid/niacin (vitamin B3), para-aminobenzoic acid (PABA), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), riboflavin (vitamin B2), thiamine (vitamin B1), thymidine retinoid (vitamin A), tocopherol (vitamin E), naphthoquinon (vitamin K), ergocalciferol (vitamin D), or the pantothenic acid derivative coenzyme A. In certain embodiments, the bacterial cell expresses an enzyme within a biosynthetic pathway that produces vitamin B12. The enzymes found at the following genomic loci, for example, can be used as positive selectable markers: cobA, cobI, cobG, cobJ, cobM, cobF, cobK, cobL, cobH, cobB, cobN, cysG, cbiK, cbiX, cbiL, cbiH, cbiF, cbiD, cbiJ, cbiE, cbiT, cbiC, cbiA, cob(II) a,c-diamide reductase, cobO, cobP, BtuR, cobQ, cbiP, cobC, codD, cbiB, cobU, cobV and cobS.

The protein of interest may include a purification tag such as a histidine tag, a calmodulin binding peptide (CBP) tag, a immunoglobulin (Ig) tag, a maltose-binding protein (MBP) tag, a Chitin binding domain (CBD) tag or a glutathione-S-transferase (GST) tag.

In some embodiments, the genetically modified bacterial cell carries an expression cassette that includes a nucleic acid sequence encoding a purification tag that is functionally coupled to the first nucleic acid sequence. The purification tag can be a histidine tag, a calmodulin binding peptide (CBP) tag, a immunoglobulin (Ig) tag, a maltose-binding protein (MBP) tag, a Chitin binding domain (CBD) tag or a glutathione-S-transferase (GST) tag. The expression cassette may include a single promoter inserted upstream from the first nucleic acid sequence. The promoter may be naturally associated with the second nucleic acid sequence. The genetically modified bacterial cell may carry an expression cassette that includes a sequence encoding a linker between the protein of interest and the selectable marker protein and the linker may, or may not be, cleavable. In addition, the expression cassette may include a third nucleic acid sequence encoding a transcription fact and upstream from the first nucleic acid sequence, a promoter that is driven by that transcription factor.

In one aspect, this invention includes an expression cassette including a first nucleic acid sequence encoding a protein of interest and a second nucleic acid sequence encoding a selectable marker protein. In some embodiments, the expression cassette includes a single promoter inserted upstream from the first nucleic acid sequence. In some embodiments, the promoter is one that is naturally associated with the second nucleic acid sequence. The expression cassette may further include a nucleic acid sequence encoding a linker between the protein of interest and the selectable marker protein and this linker may be cleavable. The expression cassette may further include a nucleic acid sequence encoding a purification tag that is functionally coupled to the protein of interest. In some embodiments, the expression cassette includes a third nucleic acid sequence encoding a transcription factor and, upstream from the first nucleic acid sequence, a promoter that is driven by the transcription factor. In some embodiments, the expression cassette includes a first nucleic acid sequence encoding a protein of interest and a second nucleic acid sequence encoding an enzyme within a biosynthetic pathway that produces an essential nutrient. In these embodiments, the essential nutrient can be a vitamin, for example, ascorbic acid (vitamin C), biotin (vitamin B7), choline, folic acid (vitamin B9), inositol, nicotinic acid/niacin (vitamin B3), para-aminobenzoic acid (PABA), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), riboflavin (vitamin B2), thiamine (vitamin B1), thymidine retinoid (vitamin A), tocopherol (vitamin E), naphthoquinon (vitamin K), ergocalciferol (vitamin D), the pantothenic acid derivative coenzyme A, or vitamin B12. In some embodiments, the expression cassette includes a nucleic acid sequence encoding a purification tag that is functionally coupled to the protein of interest. In some embodiments, the expression cassette includes a single promoter inserted upstream from a nucleic acid encoding the protein of interest. In some embodiments, the expression cassette is carried in an expression vector.

In one aspect, this features a fusion protein that includes a protein of interest and an enzyme within a biosynthetic pathway that produces an essential nutrient. The protein of interest and the enzyme are optionally linked by a cleavable or non-cleavable linker.

In one aspect, this invention features methods of genetically modifying a cyanobacterium. The method includes steps of providing a cyanobacterial cell and performing targeted replacement of a nucleic acid sequence within the cyanobacterial cell that encodes a positive selectable marker (i.e., a protein critical for cell growth or survival) with a nucleic acid sequence that encodes a negative selectable marker. The method can optionally include a further step of performing targeted replacement of the nucleic acid sequence that encodes the negative selectable marker with a nucleic acid sequence encoding (i) a protein of interest and (ii) a nucleic acid sequence encoding the positive selectable marker. The positive selectable marker can be an enzyme within a biosynthetic pathway that produces an essential nutrient or a gene critical for cell growth and survival under specified conditions. The negative selectable marker can be a protein that confers antibiotic resistance.

In one aspect, this invention features genetically modified cells (e.g., cyanobacteria) that are made by the methods described herein.

In one aspect, this invention features methods of producing a protein of interest including the steps of (a) culturing a genetically modified cyanobacterium made by the methods described herein under conditions that permit expression of a protein of interest; and (b) isolating the protein of interest from the cyanobacterium.

In one aspect, this invention includes methods of removing a metal from a metal-containing material. One or more of the methods of this invention may include steps of contacting the metal-containing material with a genetically modified cell expressing a metal-binding protein. Alternatively, the method may include steps of contacting the metal-containing material with a metal-binding protein produced by the methods described herein. These methods may be used to remove metal from soil or water. In additions, some or all of these methods may be used to remove metal-containing material from within a patient.

In one aspect, this invention includes methods of accelerating fructose metabolism. One or more of the methods of this invention may include steps of contacting a fructose-containing material with a cell expressing an enzyme that accelerates fructose metabolism or increases cellular uptake of fructose as described herein. The cell may express hepatic fructokinase, aldolase B, or glyceraldehyde kinase. The fructose-containing material can include water.

In some embodiments of this invention, the nucleic acid sequences that are contained in the expression cassettes are not immediately upstream or downstream from one another in a naturally occurring bacterial cell.