Transgenic plants containing altered levels of steroid compounds

A recombinant vector comprising operably linked in the 5′ to 3′ direction,

• a promoter, a DNA sequence encoding a polypeptide having 3-hydroxy-3-methylglutaryl-Coenzyme A reductase enzyme activity, and a transcription termination signal sequence;
• a promoter, a DNA sequence encoding at least one polypeptide having steroid pathway enzyme activity selected from the group consisting of
• squalene epoxidase enzyme acitivity,
• sterol methyl transferase I enzyme activity,
• sterol C4-demethylase enzyme activity,
• obtusifoliol C14α-demethylase enzyme activity,
• sterol C5-desaturase enzyme activity, and
• sterol methyl transferase II enzyme activity, and a transcription termination signal sequence.

A transformed host cell comprising a plant expression vector comprising:

• (a) as operably linked components in the 5′ to 3′ direction, a promoter, a DNA sequence encoding a polypeptide having 3-hydroxy-3-methylglutaryl-Coenzyme A reductase enzyme activity, and a transcription termination signal sequence;
• and
• (b) as operably linked components in the 5′ to 3′ direction, a promoter, a DNA sequence encoding at least one polypeptide having steroid pathway enzyme activity selected from the group consisting of
• squalene epoxidase enzyme activity,
• sterol methyl transferase I enzyme activity,
• sterol C4-demethylase enzyme activity,
• obtusifoliol C14α-demethylase enzyme activity,
• sterol C5-desaturase enzyme activity, and
• sterol methyl transferase II enzyme activity, and a transcription termination signal sequence.

A storage organ including at least one transformed host cell containing a recombinant vector comprising:

• (a) as operably linked components in the 5′ to 3′ direction, a promoter, a DNA sequence encoding at least one polypeptide having 3-hydroxy-3-methylglutaryl-Coenzyme A reductase enzyme activity, and a transcription termination signal sequence;
• and
• (b) as operably linked components in the 5′ to 3′ direction, a promoter, a DNA sequence encoding a polypeptide having steroid pathway enzyme activity selected from the group consisting of
• squalene epoxidase enzyme activity,
• sterol methyl transferase I enzyme activity,
• sterol C4-demethylase enzyme activity,
• obtusifoliol C14α-demethylase enzyme activity,
• sterol C5-desaturase enzyme activity, and
• sterol methyl transferase II enzyme activity, and a transcription termination signal sequence.

A process of increasing the formation of steroid pathway products in a transformed host cell as compared to an otherwise identical non-transformed host cell comprising:

• (1) transforming a host cell with a recombinant vector comprising
• (a) as operably linked components in the 5′ to 3′ direction, a promoter, a DNA sequence encoding a first polypeptide having 3-hydroxy-3-methylglutaryl-Coenzyme A reductase enzyme activity, and a transcription termination signal sequence;
• and
• (b) as operably linked components in the 5′ to 3′ direction, a promoter, a DNA sequence encoding at least one polypeptide having steroid pathway enzyme activity selected from the group consisting of
• squalene epoxidase enzyme activity,
• sterol methyl transferase I enzyme activity,
• sterol C4-demethylase enzyme activity,
• obtusifoliol C14α-demethylase enzyme activity,
• sterol C5-desaturase enzyme activity, and
• sterol methyl transferase II enzyme activity,
• and a transcription termination signal sequence, and
• (2) regenerating said transformed plant cell into said transgenic plant.

A transgenic plant seed transformed with a vector comprising a DNA segment that encodes a polypeptide having 3-hydroxy-3-methylglutaryl-Coenzyme A reductase activity, and a DNA segment that encodes a polypeptide having a steroid pathway enzyme activity selected from the group consisting of

• squalene epoxidase enzyme activity,
• sterol methyl transferase I enzyme activity,
• sterol C4-demethylase enzyme activity,
• obtusifoliol C14α-demethylase enzyme activity,
• sterol C5-desaturase enzyme activity, and
• sterol methyl transferase II enzyme activity, and a promoter suitable for driving expression of said polypeptides in said plant cell, wherein said transgenic plant seed is capable of germinating into a transgenic plant that over-accumulates steroid pathway products relative to a non-transformed plant of the same strain;
• and mutants, recombinants, genetically engineered derivatives thereof and hybrids derived therefrom, wherein said mutants, recombinants, genetically engineer derivatives thereof and hybrids derived therefrom maintain the ability to overaccumulate steroid pathway products.

A plant, the genome of which includes introduced DNA comprising:

• DNA encoding a polypeptide having 3-hydroxy-3-methyulglutaryl-Coenzyme A reductase enzyme activity, wherein said plant contains an elevated level of total accumulated sterol, compared to an otherwise identical plant, the genome of which does not comprise said introduced DNA encoding a polypeptide encoding 3-hydroxy-3-methyulglutaryl-Coenzyme A reductase enzyme activity;
• and
• further comprising introduced DNA encoding at least one polypeptide having steroid pathway enzyme activity selected from the group consisting of
• squalene epoxidase enzyme activity,
• sterol methyl transferase I enzyme activity,
• sterol C4-demethylase enzyme activity,
• obtusifoliol C14α-demethylase enzyme activity,
• sterol C5-desaturase enzyme activity, and
• sterol methyl transferase II enzyme activity,
• wherein said introduced DNAs are operatively linked to regulatory signals that cause seed-specific expression of said introduced DNAs, and wherein seeds of said plant contain a reduced level of squalene, cycloartenol, 24-methylene cycloartenol, obtusifoliol, stigmasta-7-enol, or campesterol compared to the seeds of an otherwise identical plant whose genome does not contain introduced DNA encoding said at least one polypeptide having steroid pathway enzyme activity.

An isolated DNA molecule, having a nucleotide sequence selected from the group consisting of:

• (a) disclosure squalene epoxidase SEQ ID NO:4, 6, 8, 10, or the complement thereof;
• (b) a nucleotide sequence that hybridizes to said nucleotide sequence of (a) under a wash stringency equivalent to 0.5X SSC to 2X SSC, 0.1% SDS, at 55-65° C., and which encodes a polypeptide having squalene epoxidase enzymatic activity substantially similar to that of the disclosed squalene epoxidase;
• (c) a nucleotide sequence encoding the same genetic information as said nucleotide sequence of (a), but which is degenerate in accordance with the degeneracy of the genetic code;
• and
• (d) a nucleotide sequence encoding the same genetic information as said nucleotide sequence of (b), but which is degenerate in accordance with the degeneracy of the genetic code.

An isolated DNA molecule, having a nucleotide sequence selected from the group consisting of:

• (a) disclosure obtusifoliol C14α-demethylase SEQ ID NO:12, 14, or 17, or the complement thereof;
• (b) a nucleotide sequence that hybridizes to said nucleotide sequence of (a) under a wash stringency equivalent to 0.5X SSC to 2X SSC, 0.1% SDS, at 55-650C., and which encodes a polypeptide having obtusifoliol C14α-demethylase enzymatic activity substantially similar to that of the disclosed obtusifoliol C14α-demethylase;
• (c) a nucleotide sequence encoding the same genetic information as said nucleotide sequence of (a), but which is degenerate in accordance with the degeneracy of the genetic code;
• and
• (d) a nucleotide sequence encoding the same genetic information as said nucleotide sequence of (b), but which is degenerate in accordance with the degeneracy of the genetic code.

A process of increasing the formation of steroid pathway products and tocopherols in a transformed host cell as compared to an otherwise identical non-transformed host cell comprising,

• (A) transforming a host cell with:
• (1) a recombinant vector comprising
• (a) as operably linked components in the 5′ to 3′ direction, a promoter, a DNA sequence encoding a first polypeptide having 3-hydroxy-3-methylglutaryl-Coenzyme A reductase enzyme activity, and a transcription termination signal sequence;
• and
• (b) as operably linked components in the 5′ to 3′ direction, a promoter, a DNA sequence encoding at least one polypeptide having steroid pathway enzyme activity selected from the group consisting of
• squalene epoxidase enzyme activity,
• sterol methyl transferase I enzyme activity,
• sterol C4-demethylase enzyme activity,
• obtusifoliol C14α-demethylase enzyme activity,
• sterol C5-desaturase enzyme activity, and
• sterol methyl transferase II enzyme activity,
• and a transcription termination signal sequence;
• (2) a recombinant vector comprising as operably linked components, a promoter, a DNA sequence encoding at least one tocopherol synthesis pathway enzyme selected from the group consisting of
• 3-deoxy-D-arabino-heptulosonate-7-P synthase (DAHP synthase),
• shikimate kinase,
• a prephenate dehydrogenase,
• 1-deoxy-d-xylulose 5-phosphate synthetase (DXS),
• 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR),
• 4-diphosphocytidyl-2C-methyl-d-erythritol synthase (YgbP),
• 4-diphosphocytidyl-2C-methyl-d-erythritol kinase (YchB),
• 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (YgbB),
• the gene product of GcpE, LytB,
• geranylgeranylpyrophosphate synthase (GGPPS),
• geranylgeranylpyrophosphate hydrogenase (GGH),
• phytyl/prenyltransferase (PPT),
• 4-hydroxy-phenylpyruvate dioxygenase (HPPD),
• 2-methyl-6-phytylplastooquinol,
• tocopherol methyltransferase I (MTI),
• tocopherol cyclase and
• γ-tocopherol methyltransferase (GMT);
• 4-amino-4-dexoyprephenate dehydrogenase (TryA);
• slr 1736;
• slr 1737
• ATPT2;
• AANT1;
• and
• and a transcription termination sequence;
• and
• (B) regenerating said transformed plant cell into said transgenic plant.