Metabolic engineering of lipid metabolism by improving fatty acid binding and transport

Abstract

CHI like fatty acid binding proteins and genes, recombinant cells and organisms, methods of metabolic pathway engineering to improve lipid production in cells, Crystal structures of CHI like fatty acid binding proteins, methods of engineering CHI like fatty acid binding proteins and systems thereof are provided.

1 . A recombinant cell that expresses a heterologous chalcone isomerase like fatty acid binding protein gene, which gene encodes a chalcone isomerase like fatty acid binding protein that binds to a fatty acid in the cell. 
     
     
         2 . The recombinant cell of  claim 1 , wherein the gene encodes At287 (At1g53520), At279 (At3g63170), At396 (At2g26310) or a homolog thereof. 
     
     
         3 . The recombinant cell of  claim 2 , wherein the gene encodes one or more protein identified in Tables 3-5. 
     
     
         4 . The recombinant cell of  claim 1 , wherein the gene encodes a chalcone isomerase like fatty acid binding protein that is at least 25% identical to At287, At279, or At396 and that encodes a conserved Arg amino acid residue in a position corresponding to Arg 103 of At279 or Arg 114 of At287, and that encodes a conserved Tyr residue in a position corresponding to Tyr 116 of At279 or Tyr 126 of At287, which conserved Arg and conserved Tyr residues participate in sequestering a carboxylic acid moiety on the fatty acid when the fatty acid is bound to the protein. 
     
     
         5 . The recombinant cell of  claim 4 , wherein the gene is at least 60% identical to At287, At279, or At396. 
     
     
         6 . The recombinant cell of  claim 1 , wherein the gene is more highly expressed than a corresponding native chalcone isomerase like fatty acid binding protein gene of the cell, thereby increasing lipid content of the recombinant cell as compared to a corresponding cell that does not express the gene. 
     
     
         7 . The recombinant cell of  claim 1 , wherein the protein regulates transport of the fatty acid from an inner thylakoid membrane of the cell to an outer membrane of a plastid of the cell. 
     
     
         8 . The recombinant cell of  claim 1 , wherein the cell is a plant cell. 
     
     
         9 . The recombinant cell of  claim 1 , wherein the cell is a cell of a recombinant plant. 
     
     
         10 . The recombinant cell of  claim 9 , wherein the plant is a member of a family selected from: Graminae, Leguminosae, Compositae and Rosaciae, or wherein the plant is a member of a genus selected from  Agrostis, Allium, Antirrhinum, Apium, Arachis, Asparagus, Atropa, Avena, Bambusa, Brassica, Bromus, Browaalia, Camellia, Cannabis, Capsicum, Cicer, Chenopodium, Chichorium, Citrus, Coffea, Coix, Cucumis, Curcubita, Cynodon, Dactylis, Datura, Daucus, Digitalis, Dioscorea, Elaeis, Eleusine, Festuca, Fragaria, Geranium, Glycine, Helianthus, Heterocallis, Hevea, Hordeum, Hyoscyamus, Ipomoea, Lactuca, Lens, Lilium, Linum, Lolium, Lotus, Lycopersicon, Majorana, Malus, Mangifera, Manihot, Medicago, Nemesia,    Nicotiana, Onobrychis, Oryza, Panicum, Pelargonium, Pennisetum, Petunia, Pisum, Phaseolus, Phleum, Poa, Prunus, Ranunculus, Raphanus, Ribes, Ricinus, Rubus, Saccharum, Salpiglossis, Secale, Senecio, Setaria, Sinapis, Solanum, Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonella, Triticum, Vicia, Vigna, Vitis, Zea , the Olyreae, and the Pharoideae. 
     
     
         11 . The recombinant cell of  claim 9 , wherein the plant is a  Zea mays , soybean, cotton,  Brassica naupus, Brassica juncea , tobacco, sunflower, safflower, rapeseed, canola, olive or  Arabidopsis thalina  plant. 
     
     
         12 . The recombinant cell of  claim 1 , wherein the fatty acid is oleic acid, lauric acid, myristic acid, palmitic acid or steric acid. 
     
     
         13 . The recombinant cell of  claim 1 , wherein the fatty acid is saturated. 
     
     
         14 . The recombinant cell of  claim 1 , wherein the fatty acid is unsaturated. 
     
     
         15 . A recombinant cell that expresses a heterologous regulator of a chalcone isomerase like fatty acid binding protein gene. 
     
     
         16 . The recombinant cell of  claim 15 , wherein the regulator is selected from the group consisting of: a transcription factor that regulates expression of the gene, an anti-sense nucleic acid that inhibits transcription or translation of an mRNA encoded by the gene, an siRNA that inhibits translation of an mRNA encoded by the gene, and an miRNA that inhibits translation of an mRNA encoded by the gene. 
     
     
         17 . The recombinant cell of  claim 15 , wherein the regulator increases production of a chalcone isomerase like fatty acid binding protein in the cell, thereby increasing lipid content of the cell. 
     
     
         18 . The recombinant cell of  claim 15 , wherein the regulator decreases production of a chalcone isomerase like fatty acid binding protein in the cell, thereby decreasing lipid content of the cell. 
     
     
         19 . An expression vector that encodes a chalcone isomerase like fatty acid binding protein. 
     
     
         20 . The expression vector of  claim 19 , wherein the gene encodes At287, At279, At396 or a homolog thereof. 
     
     
         21 . The expression vector of  claim 19 , wherein the expression vector is expressible in a plant, bacteria, fungi or mammalian cell. 
     
     
         22 . An isolated chalcone isomerase like fatty acid binding protein. 
     
     
         23 . The isolated protein of  claim 22 , wherein the protein is At287, At279, or a homolog thereof. 
     
     
         24 . A crystal comprising a chalcone isomerase like fatty acid binding protein. 
     
     
         25 . The crystal of  claim 24 , wherein the crystal comprises At287, At279, At396 or a homolog thereof. 
     
     
         26 . The crystal of  claim 24 , wherein the crystal comprises a fatty acid bound to the protein. 
     
     
         27 . A method of making a recombinant cell, the method comprising:
 introducing a recombinant gene into a cell, which recombinant gene encodes a recombinant chalcone isomerase like fatty acid binding protein; and,   expressing the recombinant gene in the resulting recombinant cell.   
     
     
         28 . The method of  claim 27 , wherein the gene encodes At287, At279, At396 or a homolog thereof. 
     
     
         29 . The method of  claim 27 , wherein the cell is a plant cell. 
     
     
         30 . The method of  claim 27 , wherein the recombinant chalcone isomerase like fatty acid binding protein is more highly expressed than a native chalcone isomerase like fatty acid binding protein homolog. 
     
     
         31 . The method of  claim 27 , wherein expression of the recombinant chalcone isomerase like fatty acid binding protein increases lipid content of the cell. 
     
     
         32 . A recombinant plant cell made by the method of  claim 29 . 
     
     
         33 . A method of modulating lipid content of a cell, the method comprising:
 expressing a recombinant chalcone isomerase like fatty acid protein gene in the cell, or expressing a heterologous modulator of a chalcone isomerase like fatty acid protein gene in the cell; wherein expression of the recombinant gene or heterologous modulator modulates lipid content of the cell.   
     
     
         34 . The method of  claim 33 , wherein expression of the recombinant chalcone isomerase like fatty acid protein gene increases lipid content of the cell. 
     
     
         35 . The method of  claim 33 , wherein expression of the modulator increases lipid content of the cell. 
     
     
         36 . The method of  claim 33 , wherein expression of the modulator decreases lipid content of the cell. 
     
     
         37 . A cell made by the method of  claim 33 . 
     
     
         38 . A method of selecting a plant for lipid content, the method comprising:
 identifying a polymorphism in a plant population that correlates with a phenotype encoded by a chalcone isomerase like fatty acid binding protein gene; and,   performing marker assisted selection of the population to select a plant in the population for the polymorphism.   
     
     
         39 . The recombinant cell of  claim 38 , wherein the gene is the same as or homologous to a gene that encodes At287, At279, or At396. 
     
     
         40 . A plant produced by the method of  claim 38 . 
     
     
         41 . A method of modifying a chalcone isomerase like fatty acid binding protein, the method comprising:
 accessing an information set derived from a crystal structure of the protein or homolog thereof, and, based on information in the information set, predicting whether making a change to the structure of the protein will increase or decrease binding to a fatty acid binding protein ligand; and,   modifying the protein based upon on said predicting.   
     
     
         42 . The method of  claim 41 , wherein the information set comprises crystal structure coordinate information in  FIGS. 5-6 . 
     
     
         43 . A system comprising an information storage module comprising an information set derived from a crystal structure of a chalcone isomerase like fatty acid binding protein.