The shikimate pathway, present in many organisms including plants, links metabolism of carbohydrates to biosynthesis of aromatic compounds. In a sequence of several metabolic steps, primary carbon is converted via shikimate into chorismate, (upper part of FIG. 1). Chorismate then serves as a precursor for the synthesis of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan (bottom part of FIG. 1). In plants, the products of the shikimate pathway itself as well as the aromatic amino acids produced downstream are involved in the production of multiple secondary metabolites, such as alkaloids, flavonoids, lignin, coumarins, indole derivatives and other phenolic compounds. Thus, the Shikimate pathway serves as a bridge between primary and secondary metabolism.
The first committed enzyme of the Shikimate pathway is 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS), which converts hosphoenolpyruvate (PEP) and Erythrose 4-Phosphate (E-4P) into 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate (DAHP) (FIG. 1). Escherichia (E.) coli has three different DAHPS isoenzymes encoded by the AroF, AroG and AroH genes, which encode proteins that are feedback inhibited by the individual aromatic amino acids tyrosine (Tyr) phenylalanine (Phe) and tryptophan (Trp), respectively (Brown, K. Genetics, 1968. 60(1):31-48). The major Phe-sensitive DAHPS isoform, which makes up to 80% of the total E. coli DAHPS activity, is a homo-tetramer protein encoded by the AroG gene (Hu, C. et al., J Basic Microbiol, 2003. 43(5): p. 399-406; Wallace, B. and J. Pittard, J Bacteriol, 1967. 93:237-244). It has also been suggested that the N-terminus as well as the interior regions of this AroG-encoded DAHPS are involved in its feedback inhibition by phenylalanine (Hu et al. 2003, supra; Xu, J. et al., J Basic Microbiol, 2004. 44(5):400-406: Ger, Y. et al., J Biochem, 1994. 116(5):986-990). Mutations replacing a number of amino acids in the AroG-encoded DAHPS, including proline at position 150 to leucine, leucie at position 175 to glutamine, leucine at position 179 to aspartic acid and phenylalanine at position 209 to alanine, significantly reduced the extent of feedback inhibition of 1 mM of phenylalnine while showing kinetic properties similar to the wild type (Xu et al. 2004, supra). These mutations were also associated with significant overproduction of phenylalanine in the bacterial cells (Hu et al. 2003, supra).
European Patent No. 1270721 discloses recombinant DNA sequences encoding feedback inhibition released enzymes, particularly a mutated AroG gene encoding 3-deoxy-D-arabinoheptulonic acid-7-phosphate synthase, wherein the proline residue 150 is substituted by a leucine residue, plasmids containing these recombinant DNA sequences, microorganisms transformed with these plasmids, and a process for preparing L-tryptophan, L-phenylalanine and L-tyrosine by fermentation.
In contrast to E. coli and many other bacterial species, the allosteric regulation of plant DAHPS is still questionable. (Gilchrist, D. and T. Kosuge, In: B. N. Miflin, ed, the Biochemistry of Plants, Academic Press, New York, 1980. 5:507-531; Herrmann, K. M. and L. M. Weaver, Annu Rev Plant Physiol Plant Mol Biol, 1999. 50:473-503). It has been described that the in vitro activities of DAHPS from different plant species may be weakly inhibited by Trp and Tyr or weakly activated by either Trp or Tyr. In addition, the activity of the bean (Vigna radiate) DAHPS is weakly inhibited by prephenate and arogenate, the precursors of Phe and Tyr biosynthesis. It is still unknown, however, whether this feedback is due to inhibition of the enzyme expression or inhibition of its activity.
Arabidopsis plants possess two DAHPS genes, DHS1 (At4G39980) and DHS2 (At4G33510) in addition to one putative gene (At1g22410) with high similarity to DHS1. The expression of DHS1 in Arabidopsis is induced by physical wounding or by infiltration with pathogenic Pseudomonas syringae strains (Keith, B. et al., Proc Natl Acad Sci USA, 1991. 88(19):8821-8825). The presence of amino-terminal extensions characteristic of chloroplast transit peptides in the Arabidopsis proteins encoded by DHS1 and DHS2 supports the notion that both proteins are localized in the chloroplast. Tomato (Solanum esculentum) also contains two distinct DHAPS encoding genes that contain plastid transit peptides and are differentially expressed (Gorlach, J. et al., Plant Mol Biol, 1993. 23(4):707-16). Nevertheless, despite the available information on DAHPS expression and activity in plants, it is still unknown whether this enzyme serves as a major regulator of the flux through the Shikimate pathway and hence whether DAHPS is a key regulatory enzyme bridging between primary and secondary metabolism.
U.S. Pat. No. 5,906,925 discloses methods for increasing the yield of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) in microorganisms through genetic alterations, particularly by overexpression of phosphoenol pyruvate synthase.
U.S. Pat. No. 6,911,331 discloses isolated nucleic acid fragment encoding a DAHP synthetase. The patent also discloses the construction of a chimeric gene encoding all or a portion of the DAHP synthetase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the DAHP synthetase in a transformed host cell, including plant cell.
U.S. Pat. No. 5,776,736 discloses the enzymes 3-dehydroquinate synthase, shikimate kinase, 5-enolpyruvoyl-Shikimate-3-phosphate synthase and chorismate synthase as rate-limiting enzymes in the common pathway of aromatic amino acid biosynthesis in prokaryotes. Transforming prokaryotic cells with exogenous DNA sequences encoding these enzymes resulted in a significant increase in the end product production.
U.S. Pat. No. 7,790,431 discloses enzymes and enzymatic pathways for the pyruvate-based synthesis of shikimate or at least one intermediate thereto or derivative thereof. The patent also discloses nucleic acids encoding the enzymes, cells transformed therewith, and kits containing said enzymes, cells, or nucleic acid. A KDPGa1 aldolase is used to perform condensation of pyruvate with D-erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP); a 3-dehydroquinate synthase is used to convert the DAHP to 3-dehydroquinate (DHQ); DHQ dehydratase can then convert DHQ to the key shikimate intermediate, 3-dehydroshikimate.
International (PCT) Application Publication No. WO 2009/072118 to the inventors of the present invention discloses that transforming plant cells with polynucleotide(s) encoding feedback-insensitive chorismate mutase and prephenate dehydrates results in altered content of at least one of the aromatic amino acids phenylalanine, tyrosine and tryptophan. However, these enzymes are located downstream the Shikimate pathway, and thus the amount of available chorismate is a “bottle neck” dictating the maximum levels of aromatic acids that may be produced.
Thus, means for modulation of the Shikimate pathway in plants towards the production of increased rates of the aromatic amino acids phenylalanine, tyrosine and tryptophan is highly desired, as these amino acids are both essential components of protein synthesis and also serve as precursors for a wide range of secondary metabolites that are important for plant growth as well as for human nutrition and health.