MXPA97009351A

MXPA97009351A - Alpha-hordotionine derivatives with high content treon

Info

Publication number: MXPA97009351A
Application number: MXPA/A/1997/009351A
Authority: MX
Inventors: A Rao Gururaj
Original assignee: Pioneer Hibred International Inc
Priority date: 1995-06-02
Filing date: 1997-12-01
Publication date: 1998-02-01

Abstract

Alpha-hordothionine derivatives made by site-specific substitution with threonine residues provide threonine in plant

Description

O-HORDOTIONINE DERIVATIVES WITH HIGH TREONINE CONTENT DESCRIPTION OF THE INVENTION This invention relates to the improvement of food formulations. Specifically, this invention relates to a-hordothionine derivatives that provide high percentages of threonine in plants. Food formulations are required to provide the animals with essential nutrients critical for growth. However, crop plants generally become food sources with low nutritional quality because they contain low portions of several amino acids that are essential for animals but can not be synthesized by them. For many years researchers have tried to improve the balance of essential amino acids in proteins of important crops through breeding programs. As more is known about the storage of proteins and the expression of the genes that encode those proteins, and transformation systems are developed for a greater variety of plants, molecular tests to improve the quality of seed proteins can provide alternatives to more conventional attempts. Thus, the specific levels of amino acids in a given culture can be improved by means of biotechnology.

An alternative method is to express a heterologous protein of a favorable amino acid composition at levels sufficient to improve enrichment of the feed or forage. For example, several amino acids rich in sulfur have been identified. A key to the good expression of these proteins includes efficient expression cartridges with specific promoters. Not only the regions that control the genes direct the synthesis of high levels of mRNA, the mRNA must be transferred to a stable protein. Among the essential amino acids necessary for animal nutrition, often scarce in crop plants, it is found in methionine, trionine and lysine. Attempts to increase the levels of these free amino acids by means of reproduction, mutant selection and / or changing the composition of accumulated storage proteins in the crop plants have had reduced success, generally the expression of the transgenic storage protein was too much. low. The expression cartridge of the Brazil nut promoted with 2S phaseolin is an effect of a chimeric gene specific to the effective seed. However, even through the protein of the Brazil nut increases the total amount of methionine and bound methionine, improving the nutritional value, there seems to be a threshold limit in regard to the total amount of methionine accumulated in the seeds . The seeds are still insufficient sources of methionine.

An alternative to improve the specific amino acid levels by altering the levels of proteins containing the desired amino acids is the modification of the amino acid biosynthesis. The technology of recombinant DNA and generic transfer technologies have been applied to alter the enzymatic activity by catalyzing key stages in the biosynthetic path of the amino acids. Glass an, in U.S. Pat. No. 5,258,300; Galili et al. In the European patent application no. 485870; [1992]. However, the modification of the amino acid levels in the seeds is not always correlated with changes in the level of proteins that incorporate these amino acids. Burrow et al., Mol. Gen.Genet. : vol. 241; p. 431-439 (1993). Although significant increases in the levels of free lysine in the leaves have been obtained by means of the selection of the DHSPS mutants or by expressing E.coli DHDPS in plants, it remains shown that these alterations can increase the amino acids, linked targets, that represent approximately 90% or more of the total amino acids. Thus there is a minimal impact on the nutritional value of the seeds. Based on the above there is a need for methods to increase the levels of essential amino acids, threonine, methionine and lysine in the seeds of plants. It is therefore an object of the present invention to provide methods for genetically modifying plants to increase the levels of the essential amino acid threonine in plants. It is another object of the present invention to provide the seeds for food and / or forages with higher levels of the essential amino acid threonine than the wild species of the same seeds. It has been determined that a class of compounds, the a-hordothionin is a protein of 45 amino acids that has been well characterized. It can be isolated from barley seeds [Hordeum vulgare]. The molecule is stabilized by four disulfide bonds resulting from eight cysteine residues. The amino acid sequence as provided in sequence identification no. 1 In its natural form it is especially rich in arginine and lysine, containing 5 residues [10%] of each. However, it contains only 3 residues (7%) of the essential amino acid threonine. The protein has been synthesized and the three-dimensional structure has been determined by means of computer modeling. The molding of the protein predicts that the ten charged residues [arginine at positions 5,10,17,19 and 30, and lysine at positions 1,23,32,38 and 45] all are made on the surface of the molecule. The polar amino acid side chains [asparagine in position 11, glutamine in position 22 and threonine in position 41 [also present on the surface of the molecule. In addition, the hydrophobic amino acids [such as the leucine side chains at positions 8, 15, 24 and 44 and valine at position 18] are also accessible for solvents. The three-dimensional molding of the protein indicates that the arginine residue at position 10 is critical for the retention of the appropriate three-dimensional structure and possible doubling through the hydrogen bonding interactions with the terminal residue. A substitution of threonine at that point will disrupt hydrogen bonds that include arginine at position 10, serine at position 2 and lysine at position 45, leading to the destabilization of the structure. The synthetic peptide that has this substitution could not bend correctly, which supports this analysis. The conservation of the arginine residues at position 10 provided a protein that was correctly bent. Since threonine is a polar amino acid, the surface polar amino acid residues, asparagine in position 11 and glutamine in portion 22, were replaced; and the charged amino acids, lysine at positions 1,23,32 and 38 and arginine at positions 5,17,19 and 30, were replaced with threonine. the resulting compound has the sequence indicated in the SEQUENTIAL IDENTIFIED NO. 2. The molecule can be synthesized by synthesis of peptides in solid phase and bent into a stable structure. It has 13 threonine residues [29%]. While the IDENTIFIED SEQUENCE does not. 2 is illustrative of the present invention, is not intended to be a limitation. Substitutions of threonine can also be made in positions containing charged amino acids. Only the arginine in position 10 and the lysine in position 45 are critical to maintain the structure of the protein. It can also be substituted at points that have hydrophobic amino acids. It can also be substituted in places that have hydrophobic amino acids. These include positions 8.15.18 and 24. The resulting compound has the sequence indicated in SEQUENCE IDENTIFIED NO. 3. The synthesis of the compounds is carried out according to the peptide synthesis methods that are well known in the art and therefore do not form part of the invention. In vitro the compounds have been synthesized and applied to an Applied Biosystems 413a peptide synthesizer using fastmoc chemicals "including hbtu [2- (l-benzothreiazol-1-yl-1, 2,3,3-tetramethylurin hexafluorophosphate published by Rao et al Int.J.Pep.Prot.Res.: vol 40, pp. 508-514; [1992] The peptides are divided according to standard protocols and purified by means of reverse phase chromatography using methods The sequence to inoactiva of each peptide was confirmed by means of automatic Edman degradation in a protein sequencer / analyzer 120a pth 477a from Applied Biosystems .. More preferably, however, the compounds of the invention are synthesized in vitro by means of bacterial or plant cells that have been transformed by inserting an expression cartridge containing a synthetic gene when transcribed and translated give the desired compound.These empty expression cartridges, which provide the s regulatory sequences appropriate for the plant or bacterial expression of the desired sequence, are also well known, and the nucleotide sequence for the synthetic gene, either RNA or DNA, can be easily derived from the amino acid sequence for the protein using standard reference texts , preferably those synthetic genes employ codons preferred by plants to improve the expression of the desired protein. The following description exemplifies the compositions of this invention and the methods of preparing and using them. However, it will be understood that other known methods that those of ordinary skill in the art know to be equivalent could also be employed. The genes encoding these compounds can be inserted into appropriate expression cartridges and introduced into cells of plant species. Thus, an especially preferred embodiment of this method includes inserting into the plant genome a DNA sequence encoding a compound of this invention in an appropriate reading frame, together with the transcription initiator and promoter sequences active in the plant. The transcription and translation of the DNA sequence under control of the regulatory sequences causes the expression of the protein sequence at levels that provide a high amount of a protein in the tissues of the plant. Preferred plants to be transformed according to the methods of the invention are cereals including corn, rye, barley, wheat, sorghum, oats, millet, rice, sunflower, alfalfa, rapeseed and soybean. DNA sequences encoding the appropriate amino acid sequence can then be prepared and this synthetic DNA sequence can be inserted into a suitable plant expression cartridge. Likewise, various cartridges and plant expression vectors are well known in the art. With the term "expression cartridge" a complete set of control sequences including starter sequences is implied, promoters and terminals that work in a plant cell when crossing a structural gene in the proper reading frame. The cartridges of frequent expression and preferably contain any assortment of restriction sites suitable for the division and insertion of any desired structural gene. It is important that the cloned gene has a start codon in the reading frame suitable for the structural sequence. In addition, the plant expression cartridge preferably includes a strong promoter sequence at one end to cause the ene to be transcribed at a high frequency, and a poly-a recognition sequence at the other end for proper processing and transport of the RNA delivery courier. An example of that preferred expression cartridge [void] in which the cDNA of the present invention can be inserted into the plasmid pPHI414 developed by Beach, et.al., of Pioneer Hi-Bred International, Inc., Johnston, IA, described in the patent application of ee. uu DO NOT. 07 / 785,648 [1991]. HE They will design highly preferred plant expression cartridges that include one or more selectable marker genes, such as the kanamycin resistance or herbicide tolerance genes. The term "vector" implies a DNA sequence that is capable of replicating and expressing a foreign gene in a host cell. Typically, the vector has one or more endocrucleasease recognition sites that can be cut in a predictable manner by the use of the appropriate enzyme, such vectors are preferably constructed to include structural genetic sequences that impart resistance to antibiotics or herbicides which then serve as markers to identify and separate transformed cells. Preferred labeling / labeling agents include kanamycin, chlorosulfuron, fosonomycin, hygromycin and methotrezate. A cell in which the foreign genetic material in a vector is functionally expressed has been "transformed" by the vector and is called "transformant". A particularly preferred vector is a plasmid, which involves a circular two-stranded DNA molecule that is not part of the chromosomes of the cell. As mentioned above, both genomic and cDNA encoding the gene of interest in this invention can be used. The vector of interest can be partially constructed from a cDNA clone and partially from a genomic clone. when the gene of interest has been isolated, genetic constructs are made that contain the necessary regulatory sequences that provide efficient expression of the gene in the host cell. According to the invention, the genetic construct will contain [a] a first genetic sequence encoding the protein or characteristic of interest and [b] one or more regulatory sequences that are linked to either side of the structural gene of interest. Typically, the regulatory sequences will be selected from the group comprising the promoters and terminators. The regulatory sequences may be from autologous or heterologous sources. Promoters that can be used in the genetic sequence include the NOS, OCS and CaMV promoters. An efficient plant promoter that can be used is an overproducing plant promoter. The overproducting plant promoters that can be used in the invention include the promoter of the chlorophyll binding protein a = β and the promoter of the small subunit [ss] of soybean ribulose-1,5-bisphosphate carboxylase. See for example Berry-Lowe, et. al., J.Molecular and App.Gen .: vol. 1; p. 483-498; [1982]. These two promoters are known to be induced by light in eukaryotic plant cells. See for example An Asricultural Perspective. A. Cashmnore, Pelham, New York; p. 29-38; (1983); G. Coruzzi, et. to the. J.Biol.Chem.; vol. 258; p. 1399; (1983); and P. Dunsmuir et. al., J. Molecular and App.Gen .; vol. 2; p. 285, [1983]. The expression cartridge contains the structural gene of the protein of this invention operably linked to the desired control sequences can be ligated into a suitable cloning vector. In general, plasmid or viral vectors [bacteriophages] are used that contain replication and control sequences derived from the species compatible with the host cell. The cloning vector typically carries a replication origin if as specific genes that are capable of providing phenotypic selection markers in the transformed host cells. Typically, genes conferring resistance to selected antibiotics or herbicides are used. After the genetic material is introduced into the target cells, the transformed cells and / or colonies of the cells can be isolated by selection based on those markers. Typically, an intermediate host cell will be used in the practice of the invention to increase the number of copies of the cloning vector. With a large number of copies, the sector containing the gene of interest can be isolated in important amounts to be introduced into the plant cells, the plant cells that can be used in the practice of the invention include prokaryotes, including bacterial hosts such as E.Coli. S.T? Phimurium. and Serratia arcessens. Eukaryotic hosts such as yeast or fungal filaments can also be used in this invention. And that these hosts are also microorganisms will be essential to ensure that plant promoters that do not cause the expression of the protein in the bacteria are used in the vector. The isolated cloning vector will then be introduced into the plant cell using any convenient technique including electroporation [in protoplasts], retroviruses, bombardment and microinjection in cells of monocotyledone or dicotilendoneous plants in cell or tissue cultures to provide transformed plant cells containing as foreign DNA at least one copy of the DNA sequence of the plant expression cartridge. Preferably the monocotyledonous species will be selected from corn, sorghum, wheat or rice, and the dicotyledonous species will be selected from soybeans, alfalfa, rapeseed, sunflower or tomato. Using known techniques the protoplasts can be regenerated and the cell or tissue culture can be regenerated to form whole fertile plants which carry and express the gene for a protein according to the invention. Accordingly, a highly preferred embodiment of the present invention is a transformed corn plant, which cells contain as foreign DNA at least one copy of the DNA sequence of an expression cartridge of this invention. Also those of ordinary skill in the art will appreciate that the plant vectors provided herein can be incorporated into agrobacterium tumefaciens, which can be used to transfer the vector into susceptible plant cells, mainly of dicotyledonous species in which the expression cartridges are introduced into the cells at infecting the cells with agrobacterium tumefaciens, of which a plasmid has been modified to include a plant expression cartridge of the invention. LIST OF THE SEQUENCE [1] GENERAL INFORMATION: [i] APPLICANT: PIONNER HI-BRED INTERNATIONAL, INC. [ii] TITLE OF THE INVENTION: DERIVATIVES OF O-HORDOTIONIN WITH HIGH CONTENT OF TREONINE [Üi] NUMBER OF SEQUENCES: 3 [iv] ADDRESS FOR CORRESPONDENCE [TO] RECIPIENT: PIONNER HI-BRED INTERNATIONAL, INC. [B] ADDRESS: 700 Capital Square, 400 Locust Street [C] CITY: Des Moines [D] STATE: Io a [E] COUNTRY: USA [F] POSTAL CODE: 50309 [V] LEGIBLE FORM FOR THE COMPUTER: [A] TYPE OF MEDIUM: Flexible disk [B] COMPUTER: IBM compatible PC [C] OPERATING SYSTEM: PC-DOS / MS-DOS [D] SOFTWARE: Patentln Realease # 1.0, version # 1.30 [i] CURRENT APPLICATION DATA: [A] APPLICATION NUMBER: PCT: [B] SUBMISSION DATE: [C] CLASSIFICATION: [VÜ] INFORMATION ABOUT THE LAWYER / AGENT: [A] NAME: Simon, Soma G. [B] REGISTRATION NUMBER: 37,444 [C] REFERENCE NUMBER / DOCUMENT: 354-PCT [2] INFORMATION FOR SEQ ID NO.:l: [i] CHARACTERISTICS OF THE SEQUENCE : [A] LENGTH: 45 amino acids [B] TYPE: amino acid [C] TOPOLOGY: linear [XI] DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 1: Lys Ser Cys Cys Arg Ser Thr Leu Gly Arg Asn Cys Tyr Asn Leu Cys 1 5 10 15 Arg Val Arg Gly Wing Gln Lys Law Cys Wing Gly Val Cys Arg Cys Lys 20 25 30 Leu Thr Ser Ser Gly Lys Cys Pro Thr Gly Phe Pro Lys 35 40 45 [2] INFORMATION FOR SEQ ID NO.:2: [i] ] CHARACTERISTICS OF THE SEQUENCE: [A] LENGTH: 45 amino acids [B] TYPE: amino acid [D] TOPOLOGY: linear [XI] DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 2: Thr Ser Cys Cys Thr Ser Thr Leu Gly Arg Thr Cys Tyr Asn Leu Cys 1 5 10 15 Thr Val Thr Gly Wing Thr Thr Law Cys Wing Gly Val Cys Thr Cys Thr 20 25 30 Leu Thr Ser Ser Gly Thr Cys Pro Thr Gly Phe Pro Lys 35 40 45 [2] INFORMATION FOR SEQ ID NO. : 3: [A] LENGTH: 45 amino acids [B] TYPE: amino acid [D] TOPOLOGY: linear [XI] DESCRIPTION OF SEQUENCE: SEQ ID NO. 3: Thr Ser Cys Cys Thr Ser Thr Thr Gly Lys Thr Cys Tyr Asn Thr Cys 1 5 10 15 Thr Thr Arg Wing Thr Thr Thr Cys Wing Gly Val Cys Thr Cys Thr 20 25 30 Leu Thr Ser Ser Gly Thr Cys Pro Thr Gly Phe Pro Lys 35 40 45

Claims

CLAIMS 1.- A protein that has the sequence with the SEQUENCE IDENTIFICATION NO. 3 in which the amino acid residues in one or more of positions 1,5,7,8,11,1,5,17, 18,19,22,23,24,30,32,34,38 and 41 are Threonine and the remainder of the residues in those positions are the residues of the corresponding positions in the SEQUENCE IDENTIFICATION NO. 1.
2. A protein according to claim 1 in which one or more of the amino acid residues in 1,5,7,11,17,19, 22,2,3,30,32,34,38 and 41 are threonine.
3. A protein according to claim 2 wherein at least 5 of the amino acid residues at positions 1,5,7,11,17,19,22,2,3,30,32,34,38 and 41 are threonine.
4. A protein according to claim 3 in which at least 7 of the amino acid residues at positions 1,5,7,11,17,19,22,2,3,30,32,34,38 and 41 are threonine.
5. A nucleotide sequence that encodes a protein that has the sequence corresponding to the SEQUENCE IDENTIFICATION NO. 3, in which the amino acid residues in one or more of the positions 1,5,7,8,11,1,5,17,18,19,22,23,24,30, 32,34,38 and 41 are threonine and the rest of the residues in those positions are the residues of the corresponding positions in the IDENTIFICATION OF SEQUENCE NO. 1.
6.- An RNA sequence that encodes a protein that has the sequence corresponding to the SEQUENCE IDENTIFICATION NO. 3, in which the amino acid residues in one or more of the positions 1,5,7,8,11,1,5,17,18,19,22,23,24,30, 32,34,38 and 41 are threonine and the rest of the residues in those positions are the residues of the corresponding positions in the IDENTIFICATION OF SEQUENCE NO. 1.
7.- A DNA sequence that encodes a protein that has the sequence corresponding to the SEQUENCE IDENTIFICATION NO. 3, in which the amino acid residues in one or more of the positions 1,5,7,8,11,1,5,17,18,19,22,23,24,30, 32,34,38 and 41 are threonine and the rest of the residues in those positions are the residues of the corresponding positions in the IDENTIFICATION OF SEQUENCE NO. 1.
8. An expression cartridge containing the DNA sequence according to claim 7 operably linked to the regulatory sequences of the plant that causes the expression of the DNA sequence in the cells of the plant.
9. A bacterial transformation vector consisting of an expression cartridge according to claim 8, operatively linked to the regulatory sequences of bacterial expression that cause the replication of the expression cartridge in the bacterial cells.
10. Bacterial cells containing as foreign plasmid at least one copy of a bacterial transformation vector according to claim 9.
11. Transformed plant cells containing at least one copy of the expression cartridge according to claim 8.
12. The transformed plant cells according to claim 11, wherein the cells are of a monocotyledone species.
13. The transformed plant cells according to claim 11, wherein the cells are selected from the group consisting of corn, sorghum, wheat and rice cells.
14. - The transformed plant cells according to claim 11, wherein the cells are of a dicotyledonous species.
15. The transformed plant cells according to claim 14, wherein the cells are selected from the group consisting of soy, alfalfa, rapeseed, sunflower, tobacco and tomato cells.
16. A cell or tissue culture of corn consisting of cells according to claim 13.
17. A method for improving the threonine content of a plant cell or seed comprising the step of expressing a protein according to claim 1 in the cell or seed.
18. A method according to claim 17 in which the plant is a dicotyledonous plant.
19. A method according to claim 17 in which the plant is a monocotyledone plant.
20. - The method according to claim 19 by means of which the threonine content of the seed of the plant is improved. BEs The a-hordothionine derivatives made by substitution specific to a position with threonine residues provide threonine in plants.