Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-08-19
2001-10-02
Benzion, Gary (Department: 1638)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S419000, C435S471000, C536S023600
Reexamination Certificate
active
06297055
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding amino acid decarboxylases in plants and seeds.
BACKGROUND OF THE INVENTION
In addition to their role as protein monomeric units, amino acids are energy metabolites and precursors of many biologically important nitrogen-containing compounds, such as heme, physiologically active amines, glutathione, other amino acids, nucleotides, and nucleotide coenzymes. Excess dietary amino acids are neither stored for future use nor excreted. Rather they are converted to common metabolic intermediates such as pyruvate, oxaloacetate and alpha-ketoglutarate. Consequently, amino acids are also precursors of glucose, fatty acids and ketone bodies and are therefore metabolic fuels.
Amino acid decarboxylases are induced in cells as a response to various forms of stress. In
Salmonella typhimurium
lysine decarboxylase (EC 4.1.1.18) is induced by low pH, is required for acid tolerance, and contributes significantly to pH homeostasis in environments as low as pH 3.0 (Park et al. (1996)
Mol. Microbiol.
20:605-611). At least two different lysine decarboxylases exist in
Escherichia coli
: an extensively characterized inducible decarboxylase, and a decarboxylase which is present in low amounts upon derepression by an as yet undetermined factor (Lemonnier and Lane (1998)
Microbiology
144:751-760). A monomeric lysine decarboxylase from soybean has been purified and characterized, but its sequence has not yet been determined (Kim et al. (1998)
Arch. Biochem. Biophys.
354:40-46).
Two similar but distinct enzymes are referred to as dopa decarboxylase:tyrosine decarboxylase and tryptophan decarboxylase. Dopa decarboxylase:tyrosine decarboxylase has been shown to be involved in several different pathways such as histidine metabolism, tyrosine metabolism, tryptophan metabolism, phenylalanine metabolism, and alkaloid biosynthesis. In the eastern tiger swallowtail butterfly
Papilio glaucus
dopa decarboxylase:tyrosine decarboxylase provides dopamine to the two major color pigments, papiliochrome (yellow) and melanin (black). Dopa decarboxylase:tyrosine decarboxylase activity is spatially and temporally regulated, being utilized early in presumptive yellow tissues and later in black, forming part of a developmental switch between yellow or black (Koch et al. (1998)
Development
125:2303-2313).
L-tyrosine decarboxylase (EC 4.1.1.25) is involved in an early, and potential rate-limiting step, in the biosynthesis of isoquinoline alkaloids, such as morphine and codeine, in opium poppy (P
apaver somniferum
). This enzyme catalyzes the conversion of L-tyrosine to tyramine and carbon dioxide. Several members of the tyrosine decarboxylase family, differentially expressed in various tissues, have been identified in poppy (Facchini and De Luca (1994)
J. Biol. Chem.
269:26684-26690). Four parsley (
Petroselinum crispum
) tyrosine decarboxylases have been identified from cDNAs representing genes that are transcriptionally activated upon fungal infection or elicitor treatment. The deduced protein sequences share extensive similarity with two functionally related enzymes, tryptophan decarboxylase from periwinkle and dopa decarboxylase:tyrosine decarboxylase from
Drosophila melanogaster
(Kawalleck et al. (1993)
J. Biol. Chem.
268:2189-2194).
Tryptophan decarboxylase (EC 4.1.1.28) catalyzes a key step in the biosynthesis of terpenoid indole alkaloids catalyzing the conversion of tryptophan to tryptamine and carbon dioxide. Chimeric gene constructs in which a tryptophan decarboxylase cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator have been expressed in callus and cell suspension cultures. Calluses harboring the tryptophan decarboxylase sense construct showed increased levels of tryptophan decarboxylase protein and activity, as well as the tryptamine content, but no significant increase in terpenoid indole alkaloid (Goddijn et al. (1995)
Transgenic Res.
4:315-323). Tryptophan decarboxylase supplies tryptamine for the indole moiety of Camptothecin, a valuable anti-cancer monoterpene alkaloid, and its derivatives. Tryptophan decarboxylase is considered a key step in monoterpene indole alkaloid biosynthesis as it links primary and secondary metabolism. Two autonomously regulated tryptophan decarboxylase genes from Camptotheca have been identified and isolated. One of these genes is part of a developmentally regulated chemical defense system while the other gene serves as part of a defense system induced during pathogen challenge. When expressed in
Escherichia coli
, the product of each gene will decarboxylate tryptophan, but is inactive against tyrosine, phenylalanine and 3,4-dihydroxyphenylalanine (dopa) (Lopez-Meyer and Nessler (1997)
Plant J.
11:1167-1175).
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding amino acid decarboxylases. Specifically, this invention concerns an isolated nucleic acid fragment encoding a lysine decarboxylase, a tyrosine decarboxylase or a tryptophan decarboxylase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a lysine decarboxylase, a tyrosine decarboxylase or a tryptophan decarboxylase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding lysine decarboxylase, tyrosine decarboxylase or tryptophan decarboxylase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of an amino acid decarboxylase selected from the group consisting of lysine decarboxylase, tyrosine decarboxylase and tryptophan decarboxylase.
In another embodiment, the instant invention relates to a chimeric gene encoding a lysine decarboxylase, a tyrosine decarboxylase or a tryptophan decarboxylase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a lysine decarboxylase, a tyrosine decarboxylase or a tryptophan decarboxylase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a lysine decarboxylase, a tyrosine decarboxylase or a tryptophan decarboxylase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of a lysine decarboxylase, a tyrosine decarboxylase or a tryptophan decarboxylase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a lysine decarboxylase, a tyrosine decarboxylase or a tryptophan decarboxylase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of lysine decarboxylase, tyrosine decarboxylase or tryptophan decarboxylase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a lysine decarboxylase, a tyrosine decarboxylase or a tryptophan decarboxylase.
A
Falco Saverio Carl
Famodu Layo O.
Orozco, Jr. Emil M.
Benzion Gary
E. I. Du Pont de Nemours and Company
Mehta Ashwin S.
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