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

This application claims priority to and full benefit of U.S. Ser. No. 60/831,046 filed Jul. 13, 2006, METABOLIC ENGINEERING OF LIPID METABOLISM BY IMPROVING FATTY ACID BINDING AND TRANSPORT by Pojer et al., the full disclosure of which is incorporated herein by reference in its entirety.

The application was supported in part by National Science Foundation MCB-0236027. The government may have certain rights in the invention.

This invention is in the field of metabolic pathway engineering of lipids in plants and in the field of protein crystallography and design.

Currently, the majority of vegetable oil production (estimated at 87 million metric tons with approximate market value of 40 billion U.S. dollars) goes into human consumption, with as much as 25% of human caloric intake in developed countries being derived from plant fatty acids. In addition to their importance in human nutrition, plant fatty acids are also major ingredients of nonfood products such as soaps, detergents, lubricants, biofuels, cosmetics, and paints. With the accelerating costs of petroleum, vegetable oils provide an increasingly cost-effective alternate source for raw materials.

Selecting plants for increased (and decreased) oil production by classical genetic selection methods has been ongoing for at least a century. Indeed, the complexity in determining trait-genotype associations for the seemingly simple trait of oil production has been demonstrated. For example, Laurie et al. (2004) “The Genetic Architecture of Response to Long-Term Artificial Selection for Oil Concentration in the Maize Kernel” Genetics 168:2141-2155 describe an association study that involved selection of the maize kernel for the simple phenotype of altered oil concentration, over a period of more than a century (one of the longest running selection experiments in biology). The association study detected about 50 “quantitative trait loci” (QTL) that contributed to changes in oil concentration over the 100+ year period, together accounting for only about 50% of the observed variance (suggesting that even more than the 50 identified QTL influence the oil concentration phenotype). The individual QTL effect estimates for the identified QTL were small and largely additive. In the oil phenotype experiment described by Laurie et al., the populations changed from a 4.7% oil content at the beginning of the experiment to a 19.3% oil content at the end, among the lines selected for high oil content, and a 1.1% oil content in the lines selected for low oil content.

The biochemical study of de novo fatty acid biosynthesis in plants is, thus, fundamentally important and practically essential for the metabolic engineering of fatty acid biosynthesis in agronomically important crops (see also, Thelen and Ohlrogge (2002) “Metabolic engineering of fatty acid biosynthesis in Plants,” Metabolic Engineering 4: 12-21; Broun et al. (1999) “Genetic engineering of plant lipids,” Annu. Rev. Nutr. 19: 197-216). In plants, the majority of fatty acids are biosynthesized in the plastid. Over the last two decades nearly all aspects of fatty acid metabolism in plants have been uncovered. However, one of the remaining questions that has thus far resisted elucidation is how free fatty acids are transferred from an inner thylakoid membrane to an outer envelope of a plastid, where they are reactivated to acyl-CoAs for utilization in cytosolic glycerolipid synthesis. Without knowledge of how this mechanism works, efforts to increase flux through fatty acid synthesis pathways by metabolic engineering have been hampered.

The present invention overcomes these previous difficulties, by providing a new family of chalcone-isomerase like genes that encode fatty acid binding proteins that, e.g., assist in transport of fatty acids from the thylakoid membrane to the outer plastid envelope. These and other features of the invention will be apparent upon review of the following.

The present invention provides the discovery that chalcone isomerase like fatty acid binding proteins are likely fatty acid transporters that facilitate transport of fatty acids from the thylakoid membrane to the outer plastid envelope. This discovery provides a target for engineering lipid metabolism, e.g., in plants. For example, lipid production is likely to be increased by overexpressing chalcone isomerase like fatty acid binding proteins in cells of the plants. In addition, the complete crystal structures of two of these proteins are provided. This crystal structure information makes it possible to engineer the proteins to modulate fatty acid binding and plastid transport, e.g., to increase transport activity.

Accordingly, in a first aspect, the invention provides a recombinant cell that expresses a heterologous chalcone isomerase like fatty acid binding protein gene, which encodes a chalcone isomerase like fatty acid binding protein that binds to a fatty acid in the cell.

A variety of examples of such genes and proteins are provided, including At287 (At1g53520), At279 (At3g63170), At396 (At2g26310) or homologs thereof, e.g., those identified in Example 2. Homologs include chalcone isomerase like fatty acid binding proteins that are 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. Homologs with higher levels of identity are also a feature of the invention, including genes that are at least 60% identical to At287, At279, or At396.

Optionally, the gene is highly expressed, e.g., more highly expressed than a corresponding native chalcone isomerase like fatty acid binding protein gene of the cell. This high level of expression increases lipid content of the recombinant cell as compared to a corresponding cell that does not express the gene. That is, the protein may regulate transport of the fatty acid from an inner thylakoid membrane of the cell to an outer membrane of a plastid of the cell, and over expression of the protein may increase plastid transport.

The cell is optionally a plant cell and can be part of a recombinant plant. Examples of suitable plants that can be made recombinant include plants that are members 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. For example, the plant can be a Zea mays, soybean, cotton, Brassica naupus, Brassica juncea, tobacco, sunflower, safflower, rapeseed, canola, olive or Arabidopsis thalina plant.

The CHI like fatty acid binding protein can bind any of a variety of fatty acids, including oleic acid, lauric acid, myristic acid, palmitic acid and/or steric acid. The fatty acid can be saturated or unsaturated.

In a related aspect, the invention includes a recombinant cell that expresses a heterologous regulator of a chalcone isomerase like fatty acid binding protein gene. Such regulators include transcription factors that regulate expression of the gene, anti-sense nucleic acids that inhibit transcription or translation of an mRNA encoded by the gene, siRNAs that inhibits translation of an mRNA encoded by the gene, and miRNAs that inhibit translation of an mRNA encoded by the gene. The regulator can increase or decrease production of a chalcone isomerase like fatty acid binding protein in the cell, thereby increasing or decreasing lipid content of the cell. All of the above features apply to this embodiment as well, e.g., with respect to cells, plants, fatty acids, etc.