Transgenic plants containing altered levels of steroid compounds Number:6,822,142 from the United States Patent and Trademark Office (PTO) owispatent Disclosed are constructs comprising sequences encoding 3-hydroxy-3methylglutaryl-Coenzyme A reductase and at least one other sterol synthesis pathway enzyme. Also disclosed are methods for using such constructs to alter sterol production and content in cells, plants, seeds and storage organs of plants. Also provided are oils and compositions containing altered sterol levels produced by use of the disclosed constructs. Novel nucleotide sequences useful in the alteration of sterol production are also provided. Also provided are cells, plants, seeds and storage organs of plants comprising sequences encoding 3-hydroxy-3methylglutaryl-Coenzyme A reductase, at least one other sterol synthesis pathway enzyme and at least one tocopherol synthesis enzyme.<H containing, Transgenic, Transgenic, plan, 6822142.html, Patent, pto, 6822142

Title: Transgenic plants containing altered levels of steroid compounds

Abstract: Disclosed are constructs comprising sequences encoding 3-hydroxy-3methylglutaryl-Coenzyme A reductase and at least one other sterol synthesis pathway enzyme. Also disclosed are methods for using such constructs to alter sterol production and content in cells, plants, seeds and storage organs of plants. Also provided are oils and compositions containing altered sterol levels produced by use of the disclosed constructs. Novel nucleotide sequences useful in the alteration of sterol production are also provided. Also provided are cells, plants, seeds and storage organs of plants comprising sequences encoding 3-hydroxy-3methylglutaryl-Coenzyme A reductase, at least one other sterol synthesis pathway enzyme and at least one tocopherol synthesis enzyme.

Patent Number: 6,822,142 Issued on 11/23/2004 to Karunanandaa, et al.

Inventors: Karunanandaa; Balasulojini (St. Louis, MO); Post-Beittenmiller; Martha (St. Louis, MO); Venkatramesh; Mylavarapu (St. Louis, MO); Kishore; Ganesh M. (St. Louis, MO); Thorne; Gregory M. (St. Louis, MO); LeDeaux; John R. (St. Louis, MO)
Assignee: Monsanto Company (St. Louis, MO)

Appl. No.: 885723

Filed: June 20, 2001

Current U.S. Class: 800/298 ; 435/320.1; 435/419; 536/23.6; 800/278

Field of Search: 800/278,288,295,298 435/320.1,410,419,183,468 536/23.1,23.2,23.5,23.6,23.7,23.74

References Cited [Referenced By]

U.S. Patent Documents


5306862	April 1994	Chappell et al.
5349126	September 1994	Chappell et al.
5365017	November 1994	Chappell et al.
5460949	October 1995	Saunders et al.
5480805	January 1996	Wolf et al.
5589619	December 1996	Chappell
6153815	November 2000	Covello

Foreign Patent Documents


0480730	Apr., 1992	EP
0486290	May., 1992	EP
09121863	May., 1997	JP
WO 93/02187	Feb., 1993	WO
WO 97/03202	Jan., 1997	WO
WO 97/34003	Sep., 1997	WO
WO 98/45457	Oct., 1998	WO
WO 99/04622	Feb., 1999	WO
WO 00/61771	Oct., 2000	WO
WO 01/31027	Mar., 2001	WO

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Primary Examiner: Fox; David T.
Assistant Examiner: Kallis; Russell
Attorney, Agent or Firm: Fulbright & Jaworski L.L.P.

Claims

What is claimed is:

1. A recombinant construct comprising: (a) a DNA sequence encoding a polypeptide having 3-hydroxy-3-methylglutaryl-Coenzyme A reductase enzyme activity, and (b) a DNA sequence encoding a polypeptide having squalene epoxidase enzyme activity.

2. The recombinant construct of claim 1, further comprising at least one promoter operably linked to said coding regions.

3. The recombinant construct of claim 1, further comprising a first promoter operably linked to said DNA sequence encoding a polypeptide having 3-hydroxy-3-methylglutaryl-Coenzyme A reductase enzyme activity and a second promoter operably linked to said DNA sequence encoding squalene epoxidase enzyme activity, wherein said first and second promoters may or may not be the same.

4. The recombinant construct of claim 2 or 3 further comprising an operably linked transcription termination sequence located 3' to each coding region.

5. A recombinant construct according to claim 3 wherein the promoters are selected from the group consisting of seed-specific promoters, organ specific promoters and constitutive promoters.

6. A recombinant vector comprising operably linked in the 5' to 3' direction, a promoter, a DNA sequence encoding a polypeptide having a 3-hydroxy-3-methylglutaryl-Coenzyme A reductase enzyme activity, and a transcription termination signal sequence; a promoter, a DNA sequence encoding squalene epoxidase enzyme activity, and a transcription termination signal sequence.

7. The recombinant vector of claim 6 wherein said vector is a plant expression vector.

8. A transformed host cell comprising a recombinant construct of claim 1.

9. The transformed host cell of claim 8 wherein said cell is a plant cell.

10. A transformed host cell comprising a recombinant vector of claim 6.

11. The transfonued host cell according to claim 10 wherein said host cell is a plant cell.

12. 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 a 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 squalene epoxidase enzyme activity, and a transcription termination signal sequence.

13. The transformed host cell according to claim 12 wherein said host cell is a plant cell.

14. A cell culture comprising transformed host cells according to any one of claims 8-13.

15. A transformed plant comprising at least one transformed host cell of any one of claim 9 or 11.

16. A transformed plant storage organ, comprising at least one transformed host cell according to any one of claim 9 or 11.

17. A transformed plant storage organ including at least one transformed plant 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 squalene epoxidase activity, and a transcription termination signal sequence.

18. A process of increasing the formation of steroid pathway products in a transformed plant as compared to an otherwise identical non-transformed plant comprising: (1) transforming a host plant 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 squalene epoxidase enzyme activity, and a transcription termination signal sequence, and (2) regenerating the transformed host plant cell into said transgenic plant.

Description

TECHNICAL FIELD

The present invention relates to biotechnology with an emphasis on plant biotechnology, and particularly biotechnology affecting the biosynthesis of steroid compounds.

BACKGROUND

Enhancement of the nutritional or health benefits of oils through genetic engineering is being addressed throughout the agricultural community. Several approaches involve manipulation of already present cellular biosynthetic pathways. Steroid biosynthetic pathways are of current interest, particularly for the enhancement of health benefits from food oils.

Several related U.S. patents address increasing sterol accumulation in higher plants. Those patents include U.S. Pat. No. 5,589,619 "Process and Composition for increasing squalene and sterol accumulation in higher plants" (accumulation of squalene in transgenic plants by increasing HMGR activity) and U.S. Pat. No. 5,306,862 "Method and composition for increasing sterol accumulation in higher plants" (increasing HMGR activity to increase plant sterol accumulation--including sterol and cycloartenol, which affects insect resistance--in tobacco, tomato, corn, carrot, soybean, cotton, barley, arabidopsis, guayule and petunia; seeds with elevated sterol/cycloartenol, 7S promoter and CaMV promoters), U.S. Pat. No. 5,365,017 "Method and composition for increasing sterol accumulation in higher plants" (DNA construct with HMGR-CaMV 35S, transgenic plants, hybrid plants, corn, soy, barley, tomato, Arabidopsis), U.S. Pat. No. 5,349,126 "Process and composition for increasing squalene and sterol accumulation in higher plants" (increase in squalene and sterol accumulation by increasing HMGR activity in transgenic tobacco, cotton, soybean, tomato, alfalfa, Arabidopsis, corn, barley, carrot and guayule plants), and EP 486290 (enhancement of squalene and specific sterol.[squalene zymosterol, cholest-7,24-dienol, cholest-5,7,24-trienol] accumulation in yeast by increasing HMGR activity in yeast deficient in enzymes that convert squalene to ergosterol).

In those patents, the amount of a protein exhibiting 3-hydroxy-3-methylglutaryl Coenzyme-A reductase (HMGR) activity is typically increased. HMGR widens a "bottleneck" near the beginning of a biosynthetic path to steroid production, permitting a higher carbon flux through steroid biosynthetic pathways and resulting in increased sterol accumulation.

U.S. Pat. No. 5,480,805 "Composition for modulating sterols in yeast" (enhancement of delta 8-7 isomerase activity-ERG2 enhances accumulation of specific sterols in yeast).

U.S. Pat. No. 5,460,949 "Method and composition for increasing the accumulation of squalene and specific sterols in yeast" (increasing squalene, zymosterol and specific sterols in yeast by increasing HMGR in yeast having decreased erg5 and erg6 activity--Sc and hamster HMGR).

WO 9845457 (SMTI, Erg6 from A.t., corn, yeast; transgenic plants with altered sterol levels_using DNA encoding an enzyme binding a first sterol and producing a second sterol--altered carotenoid, tocopherol, modified FA levels--HMGR, 5.alpha.-reductase, geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, isopentenyl diphosphate isomerase).

Acetate is the metabolic precursor of a vast array of compounds vital for cell and organism viability. Acetyl coenzyme A (CoA) reacts with acetoacetyl CoA to form 3-hydroxy-3 methylglutaryl CoA (HMG-CoA). HMG-CoA is reduced to mevalonate in an irreversible reaction catalyzed by the enzyme HMG-CoA reductase. Mevalonate is phosphorylated and decarboxylated to isopentenyl-pyrophosphate (IPP). Through the sequential steps of isomerization, condensation and dehydrogenation, IPP is converted to geranyl pyrophosphate (GPP). GPP combines with IPP to form farnesyl pyrophosphate (FPP), two molecules of which are reductively condensed to form squalene, a 30-carbon precursor of sterols.

A key enzyme in sterol biosynthesis is 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoA reductase or HMGR). Schaller et al. (Plant Physiol. 109: 761-770, 1995) found that over-expression of rubber HMGR (hmg1) genomic DNA in tobacco leads to the overproduction of sterol end-products (sitosterol, campesterol and stigmasterol) up to 6-fold in leaves. Further, the excess sterol was stored as steryl-esters in lipid bodies. HMGR activity was increased by 4- to 8-fold.

Sterols are derivatives of a fused, reduced ring system, cyclopenta-[a]-phenanthrene, comprising three fused cyclohexane rings (A, B, and C) in a phenanthrene arrangement, and a terminal cyclopentane ring (D) having the formula (I) and carbon atom position numbering shown below: ##STR1##

where R is an 8 to 10 carbon-atom side chain.

In plants, squalene is converted to squalene epoxide, which is then cyclized to form cycloartenol (4,4,14.alpha.-trimethyl-9.beta.,19-cyclo-5.alpha.-cholest-24-en-3.beta.-o l). Cycloartenol has two methyl groups at position 4, a methyl group at position 14, a methylene bridge between the carbon atoms at positions 9 and 19 that forms a disubstituted cyclopropyl group at those positions, and includes an 8-carbon sidechain of the formula: CH.sub.3 CH(CH.sub.2).sub.2 CH.dbd.C(CH.sub.3).sub.2. Squalene epoxide can alternatively be converted into pentacyclic sterols, containing five instead of four rings. Exemplary pentacyclic sterols include the phytoalexins and saponins.

Being one of the first sterols in the higher plant biosynthetic pathway, cycloartenol serves as a precursor for the production of numerous other sterols. In normal plants, cycloartenol is converted to predominantly 24-methylene cycloartenol (4,4,14.alpha.-dimethyl-9.beta., 19-cyclo-22,23-dihydro-ergosta-24(28)-en-3-.beta.-ol), cycloeucalenol, (4,14.alpha.-trimethyl-9.beta.,19 cyclo-5.alpha.-ergosta-24(28)-en-3.beta.-ol), isofucosterol (5.alpha.-stigmasta-5-24(28)-dien-3.beta.-ol), sitosterol (5.alpha.-stigmasta-5-en-3.beta.-ol), stigmasterol-(stigmasta-5,-22-dien-3.beta.-ol), campesterol (5.alpha.-ergosta-5-en-3.beta.-ol), and cholesterol (5.alpha.-cholesta-5-en-3.beta.-ol). These transformations are illustrated in FIG. 1.

Although sterols produced by plants, and particularly higher (vascular) plants, can be grouped by the presence or absence of one or more of several functionalities, plant sterols are classified into two general groups herein; i.e., those containing a double bond between the carbon atoms at positions 5 and 6 (delta-5 or .DELTA.5 sterols) and those not containing a double bond between the carbon atoms at positions 5 and 6 (non-delta-5 sterols).

Exemplary naturally-occurring delta-5 plant sterols are isofucosterol, sitosterol, stigmasterol, campesterol, cholesterol, and dihydrobrassicasterol. Exemplary naturally occurring non-delta-5 plant sterols are cycloartenol, 24-methylene cycloartenol, cycloeucalenol, and obtusifoliol. The most abundant sterols of vascular plants are campesterol, sitosterol, and stigmasterol, all of which contain a double bond between the carbon atoms at positions 5 and 6 are classified as delta-5 sterols.

The HMG-CoA reductase enzymes of animals and yeasts are integral membrane glycoproteins of the endoplasmic reticulum. The intact enzyme comprises three regions: a catalytic region containing the active site of the enzyme; a membrane binding region anchoring the enzyme to the endoplasmic reticulum; and a linker region joining the catalytic and membrane binding regions of the enzymes. The membrane binding region occupies the amino-terminal (N-terminal) portion of the intact protein, whereas the catalytic region occupies the carboxy-terminal (C-terminal) portion of the protein, with the linker region constituting the remaining portion. M.E. Basson et al., Mol. Cell Biol., 8(9):3797-3808 (1988).

The activity of HMG-CoA reductase in animals and yeasts is known to be subject to feedback inhibition by sterols. Such feedback inhibition requires the presence of the membrane binding region of the enzyme. See, e.g., G. Gil et al, Cell, 41:249-258 (1985); M. Bard and J. F. Downing, J. Gen. Microbiol., 124:415-420 (1981).

Given that mevalonate is the precursor for sterols and other isoprenoids, it might be expected that increases in the amount or activity of HMG-CoA reductase would lead to increases in the accumulation of both sterols and other isoprenoids.

In mutant strains of the yeast Saccharomyces cerevisiae (S. cerevisiae) having abnormally high levels of HMG-CoA reductase activity, the production of two sterols, 4,14-dimethylzymosterol and 14-methylfucosterol is markedly increased above normal. Downing, et al., Biochem. Biophys. Res. Comm., 94(3): 874-979 (1980).

When HMG-CoA reductase activity was increased by illumination in non-photosynthetic microorganisms, isoprenoid (carotenoid), but not sterol (ergosterol), synthesis was enhanced. Tada, et al., Plant and Cell Physiology, 23(4):615-621 (1982).

WO 9703202 discloses a method for identifying agents modulating sterol biosynthesis using a yeast acetoacetyl CoA thiolase (ERG10) gene linked to a reporter system to evaluate compounds, such as lovastatin and other HMG-CoA synthase inhibitors, that affect cholesterol biosynthesis.

U.S. Pat. No. 5,668,001 teaches a recombinant avian HMG-CoA synthase preparation useful for evaluating drugs that inhibit cholesterol biosythesis.

JP 09121863 discloses a plant with increased 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity as a result of increasing the expression of a mutant protein kinase gene that regulates expression of the HMGR gene. The increased HMGR activity increases squalene, zymosterol, cholesta-7,24-dienol and cholest-5,7,24-trenol accumulation in yeast with ERG5 and ERG6 mutants.

EP 480730 "Plant-sterol accumulation and pest resistance-by increasing copy number of 3-hydroxy-3-methyl glutaryl coenzyme-A reductase gene in tobacco, tomato and corn.

WO 9913086 "Human Delta 7-sterol reductase polypeptide-useful for diagnosis or treatment of genetic defects e.g. hereditary Smith-Lemli-Opitz syndrome" teaches making and using the recombinant polypeptide with humans.

Chappell et al. U.S. Pat. No. 5,589,619 teaches that transformation of higher plants with truncated HMG-CoA reductase enhanced the production of squalene, cycloartenol and certain sterols, particularly compounds having unsaturations at the 5-position. Several intermediate sterols as are shown in FIG. 1 were also produced. It would be beneficial if the production of sitosterol and stigmasterol could be enhanced while lessening the accumulation of the intermediate sterols. The present invention provides avenues for enhancing production of sitosterol and stigmasterol and lessening the accumulation of the intermediate sterols.

Gonzalez et al. (Abstract of poster at Third Terpnet Meeting of the European Network on Plant Isoprenoids, May 29-30, 1997, Poitiers, France) over-expressed the Arabidopsis HMGR cDNA (hmg1 and hmg2) and found sterol overproduction with hmg1 only. They used two forms of the hmg1 gene, a full-length form and a truncated form containing only the catalytic domain. HMGRs have three domains, an N-terminal membrane spanning domain, a short linker domain, and a C-terminal catalytic domain. In this case the transgenic plants were also Arabidopsis. The difference between the full-length and truncated forms was a greater accumulation of pathway intermediates in the case of the truncated form. More importantly, the intermediates demonstrated as accumulating were cycloartenol, 24-methylenecycloartanol and obtusifoliol.

Finally, U.S. Pat. Nos. 5,365,017 and 5,306,862, both assigned to Amoco Corp., disclose a method for increasing sterol accumulation in plants by increasing the copy number of a gene having HMG-CoA reductase activity. These inventions disclose a method using hamster truncated HMGR that consisted of the catalytic domain and the linker domain. According to the claims the linker domain was essential for activity. They also demonstrated a greater accumulation of pathway intermediates such as cycloartenol.

BRIEF SUMMARY

The present invention relates to transgenic plants and their progeny having improved nutritional characteristics. More particularly, the present invention relates to transgenic plants and their progeny, the storage organs (e.g. seed, fruit and vegetable parts) of which contain modified levels of steroid compounds, such as (i) elevated levels of beneficial phytosterols (e.g., sitosterol), phytostanols (e.g., sitostanol), and esters thereof, relative to an otherwise identical plant transformed only with a truncated HMG-CoA reductase gene or a wild-type plant, and (ii) reduced levels of steroid pathway intermediate compounds (e.g. one or more of squalene, cycloartenol, 24-methylene cycloartenol, obtusifoliol, stigmasta-7-enol and campesterol) in their storage organs relative to an otherwise identical transgenic plant transformed only with a truncated HMG-CoA reductase gene. Nucleic acid sequences encoding enzymes that affect the biosynthesis and accumulation of steroid compounds in plants (HMG-CoA reductase and a steroid pathway enzyme), and methods for using these sequences to produce such transgenic plants, are also provided. These methods comprise, for example, introducing into cells nucleic acid sequences encoding enzymes that affect the levels of accumulated steroid pathway end products.

The present invention contemplates a recombinant construct or a recombinant vector that contains 2 DNA sequences. The first encodes a polypeptide exhibiting 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase activity. The second DNA sequence encodes a polypeptide exhibiting the activity of another steroid pathway enzyme. Each polypeptide-encoding DNA sequence is operably linked in the 5' to 3' direction to a promoter and a transcription termination signal sequence independent of the other sequence. The promoter is located upstream and the termination sequence downstream of each polypeptide-encoding DNA sequence. The second DNA sequence encoding a steroid pathway enzyme can code for a squalene epoxidase enzyme, a sterol methyl transferase I enzyme, a sterol C4-demethylase enzyme, a obtusifoliol C14.alpha.-demethylase enzyme, a sterol C5-desaturase enzyme, or a sterol methyl transferase II enzyme. It is contemplated that HMG-CoA reductase and the steroid pathway enzyme activity comes from a mutant or truncated form of those enzymes, such as a truncated HMG-CoA reductase lacking the transmembrane region while retaining a functional catalytic domain. Examples of such preferred HMG CoA reductases include the truncated rubber and Arabidopsis HMG CoA reductases disclosed herein.

Preferably, the regulatory function of a promoter is substantially unaffected by cellular levels of squalene such as the CaMV 35S promoter. In one aspect, a promoter is seed-specific. In another aspect, a promoter is derived from a species in a different order from a host cell. In another aspect, the HMG-CoA reductase or steroid pathway enzymes is from a species in a different order from the order that of the host cell. The invention contemplates a construct or recombinant vector having more than one DNA sequence encoding a steroid pathway enzyme that do not have to be under the control of the same promoter. Preferably, a recombinant vector is a plant expression vector.

In another aspect of the invention, a transformed host cell comprises a recombinant construct or vector as described above. Preferably, a host cell is plant cell, preferably that plant cell is from canola, soybean, corn, maize, tobacco, cotton, rape, tomato or alfalfa. The invention contemplates a host cell in a cell culture, plants derived from transformed host cells, and storage organs (seeds, fruits and vegetable parts) from transgenic plants.

In addition to contemplating transgenic plants and seeds, the invention contemplates transgenic plant seeds capable of germinating into a transgenic plant and mutants, recombinants, genetically engineered derivatives thereof and hybrids derived therefrom. The plant over-accumulates steroid pathway products relative to a native, non-transgenic plant of the same strain, wherein said mutants, recombinants, genetically engineered derivatives thereof and hybrids derived therefrom maintain the ability to overaccumulate steroid pathway products.

The invention contemplates 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. Contemplated processes use the described recombinant constructs and vectors to transform host cells, then growing the host cells or regenerating transgenic plants therefrom.

In one aspect of the invention, the genome of a contemplated plant, its progeny, seeds or cell culture, comprises introduced DNA encoding an HMG-CoA reductase activity and introduced DNA encoding a steroid pathway enzyme that is a squalene epoxidase enzyme, a sterol methyl transferase I enzyme, a sterol C4-demethylase enzyme, a obtusifoliol C14.alpha.-demethylase enzyme, a sterol C5-desaturase enzyme, or a sterol methyl transferase II enzyme. The storage organs of such a plant contain an elevated level of total accumulated sterol, compared to storage organs of an otherwise identical plant, the genome of which does not comprise said introduced DNA. Further, the storage organs of the 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 or a plant comprising an introduced DNA encoding an HMG-CoA reductase enzyme.

The invention contemplates a method of producing a plant that accumulates an elevated level of sterol pathway products compared to a corresponding plant comprising no introduced DNA encoding a peptide, polypeptide, or protein that affects the biosynthesis and accumulation of a sterol pathway product, comprising sexually crossing plants to arrive at a plant comprising nucleic acid encoding an HMG CoA reductase and a steroid pathway enzyme, including crosses with a nurse cultivar. The plants, including apomicitic plants, uniform populations of the plants and their seeds and parts other than seeds are contemplated.

Another aspect of the invention is oils containing at least one sterol pathway product, extracted from the seeds of a contemplated plant. Preferably sitosterol, at least one sitosterol ester, or mixtures thereof, comprise at least about 57% by weight of the total sterol compounds of a contemplated oil. Preferably sitosterol, that at least one sitosterol ester, or mixtures thereof, comprise at least about 0.08% of the dry weight of a contemplated seed. Preferably, the oil has a reduced amount of squalene, cycloartenol, 24-methylene cycloartenol, obtusifoliol, stigmasta-7-enol, campesterol, or combinations thereof, compared to oil from a corresponding transgenic plant that does not contain introduced DNA encoding a squalene epoxidase enzyme, a sterol methyl transferase I enzyme, a sterol C4-demethylase enzyme, a obtusifoliol C14.alpha.-demethylase enzyme, a sterol C5-desaturase enzyme, a sterol methyl transferase II enzyme, or mixture thereof; wherein the reduction is in the range of from about 10% to about 100%.

Sitosterol ester compositions derived from transgenic plants of the present invention, their progeny or their seeds are also contemplated, preferably wherein an esterifying fatty acid has 2 to 22 carbon atoms in the main chain.

A further aspect of the invention is cholesterol-lowering compositions comprising contemplated oils and sitosterol ester compositions. Another further aspect of the invention is foods, food ingredients, or food compositions comprising contemplated oils.

Still further, the invention contemplates pharmaceutical compositions comprising a cholesterol-lowering effective amount of a contemplated oil, and a pharmaceutically acceptable carrier, excipient, or diluent.

A method of lowering the plasma concentration of low density lipoprotein cholesterol is contemplated, comprising orally administering to a human or animal subject an effective amount of an above composition. Also contemplated is a method of treating or preventing an elevated plasma concentration of low-density lipoprotein cholesterol, comprising orally administering to a human or animal subject an effective amount of a contemplated composition.

A related aspect of the invention is a method of making a food additive composition, comprising obtaining oil containing a sterol pathway product compound from seed of a contemplated transgenic plant and mixing the oil with an edible solubilizing agent, an effective amount of a dispersant, and optionally, an effective amount of an antioxidant.

Novel forms of two sterol pathway enzymes and the nucleic acids that encode them are disclosed: an Arabidopsis enzyme having nucleic acid similarity to a squalene epoxidase, and an Arabidopsis enzyme having nucleic acid similarity to an obtusifoliol C14.alpha.-demethylase enzyme. Thus, the invention contemplates an isolated DNA molecule having a nucleotide sequence of disclosure SEQ ID NO: 4, 6, 8, 10, 14, 15, 17 or the complements thereof. Also contemplated is a nucleotide sequence that hybridizes to the nucleotide sequence of SEQ ID NO:4, 6, 8, 10, 14, 15, 17 or their complements under a wash stringency equivalent to 0.5X SSC to 2X SSC, 0.1% SDS, at 55-65.degree. C., and that encode a polypeptide having squalene epoxidase or obtusifoliol C14.alpha.-demthylase enzymatic activity. Preferably, that enzymatic activity is substantially similar to that of a disclosed squalene epoxidase or obtusifoliol C14.alpha.-demethylase, respectively. By substantially smiliar is meant having enzymatic activity differing from that of the disclosed enzymes by about 30% or less, preferably by about 20% or less, and more preferably by about 10% or less when assayed by standard enzymatic assays. Also contemplated is a nucleotide sequence encoding the same genetic information as said nucleotide sequence of SEQ ID NO: 4, 6, 8, 10, 14, 15, 17 or their complements or that hybridize as described above, but which is degenerate in accordance with the degeneracy of the genetic code. Recombinant constructs, vectors and transformed host cells comprising the novel isolated and purified nucleic acid sequences are also contemplated. In one embodiment, the vector is a plant vector and the host cell is a plant cell. Methods of producing the disclosed squalene epoxidase or obtusifoliol C14.alpha.-demethylase enzymes are also contemplated comprising culturing a transformed host cell for a time and under conditions conductive to the production of the squalene epoxidase or obtusifoliol C14.alpha.-demethylase enzyme, and recovering the produced squalene epoxidase or obtusifoliol C14.alpha.-demethylase enzyme.

Yet another aspect provides any of the above described transformed host cells, further comprising a recombinant construct or expression vector encoding a tocopheral synthesis pathway enzyme, and in particular, S-adenosylmethionine-dependent .alpha.-tocopherol methyltransferase. Also included are plants, seeds and storage organs comprising the transformed host cells.

Another aspect provides, 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 (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 C14.alpha.-demethylase enzyme activity, sterol C5-desaturase enzyme activity, and sterol methyl transferase II enzyme activity, and a transcription termination signal sequence; (2) transforming the host cell of (1) with a recombinant vector comprising as operably linked components, a promoter, a DNA sequence encoding a tocopherol synthesis pathway enzyme, and a transcription termination sequence; and (3) regenerating said transformed plant cell into said transgenic plant.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims and accompanying figures where:

FIG. 1 is an abbreviated version of a plant steroid compound biosynthetic pathway that shows the enzymes affecting steroid compound biosynthesis and accumulation. These include: HMG-CoA reductase, squalene epoxidase, sterol methyl transferase I, sterol C4-demethylase, obtusifoliol C14.alpha.-demethylase, sterol C5 desaturase and sterol methyl transferase II.

FIG. 2 depicts the forms of Arabidopsis and rubber HMGR1 tested in Arabidopsis and yeast to compare expression, activity and sterol production.

FIG. 3 is a map showing the structure of construct pMON29920. pMON29920 is a binary transformation vector with P-7S/E9 3' cassette and the KAN gene flanked by the two borders where P-7S is the promoter of alpha' beta conglycinin protein from soybean, E9 3' is the 3' end of pea rbc E9 gene and KAN is the coding sequence for NPTII that confers resistance to kanamycin. The NPTII gene is driven by the 35S promoter from cauliflower mosaic virus. Spc.Str is the coding region for Tn7 adenylyltransferase conferring resistance to spectinomycin and streptomycin; ori-V: the vegetative origin of replication; rop: coding region for repressor of primer; ori-322: minimum known sequence required for a functional origin of replication; NOS 3': the 3' termination end of nopaline synthase coding region.

FIG. 4 is a map showing the structure of construct pMON43800. pMON43800 is a recombinant binary vector for Agrobacterium-mediated transformation, carrying the rubber HMGR1 gene cassette. The HMGR1 gene is driven by the 7S alpha' beta conglycinin promoter from soybean. P-7S: 7S promoter, rubber HMGR1 gene: coding sequence for 3-hydroxy-3-methylglutaryl reductase from Hevea brasiliensis; E9 3': 3' end of pea rbcS E9 gene; P-35S: 35S promoter from cauliflower mosaic virus; KAN: coding region for NPTII gene conferring resistance for kanamycin; NOS 3': 3' termination end of nopaline synthase coding region: Left Border: Octapine left border from Octapine Ti plasmid pTiA6; ori-V: the vegetative origin of replication; rop: coding region for repressor of primer; Spc/Str: coding region for Tn7 adenylyltransferase conferring resistance to spectinomycinand streptomycin.

FIG. 5 is a map showing the structure of construct pMON23616. pMON23616 is a plant expression vector containing P-NOS/ORF-7/KAN/NOS-3' cassette. P-NOS: NOS promoter from Agrobacterium tumefaciens pTiT37; ORF-7: a short open reading frame that attenuates expression of KAN in plants; KAN: coding sequence of NPTII gene that confers resistance to kanamycin and neomycin; ble: confers resistance to bleomycin; NOS 3': 3' termination end of nopaline synthase coding region; Left Border: Octapine left border from Octapine Ti plasmid pTiA6; ori-V: the vegetative origin of replication; rop: coding region for repressor of primer; Spc/Str: coding region for Tn7 adenylyltransferase conferring resistance to spectinomycin and streptomycin.

FIG. 6 is a map showing the structure of construct pMON43818. pMON43818 is a recombinant binary vector carrying the gene encoding rubber hydroxymethyl glutaryl CoA reductase1 (HMGR1) in sense orientation driven by the soybean alpha' beta conglycinin promoter. P-NOS: nopaline synthase gene promoter; kan: coding region for neomycin phospho transferase protein to confer resistance to kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Soy Alpha' Beta Conglycinin: 7S alpha' beta conglycinin gene promoter from soybean; Rubber HMGR1 gene: coding sequence for HMGR1 gene from Hevea brasiliensis; E9 3': 3' end of pea rbcS E9 gene; Left border: octopine left border, sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; Ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence of T-DNA essential for integration into Agrobacterium.

FIG. 7 is a map showing the structure of construct pMON43052. pMON43052 is a recombinant shuttle vector, carrying the cDNA fragment encoding the catalytic domain of Arabidopsis HMGR1 in sense orientation driven by the soybean alpha' beta conglycinin promoter. P-NOS: nopaline synthase gene promoter; kan: coding region for neomycin phosphotransferase protein to confer resistance to kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Soy Alpha' Beta Conglycinin: 7S alpha' beta conglycinin gene promoter from soybean; Arabidopsis HMGR catalytic domain: coding sequence for the catalytic domain of Arabidopsis HMGR1 protein; E9 3': 3' end of pea rbcS E9 gene; Left border: octopine left border, sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; Ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence of T-DNA essential for integration into Agrobacterium.

FIG. 8 is a map showing the structure of construct pMON51850. pMON51850 is a binary vector for Agrobacterium mediated transformation of soybean. P-NOS: nopaline synthase gene promoter; kan: coding region for neomycin phosphotransferase protein to confer resistance to kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Left border: octopine left border sequence essential for transfer of T-DNA into Agrobacterium; ori-v: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence of T-DNA essential for integration into Agrobacterium.

FIG. 9 is a map showing the structure of construct pMON43057. pMON43057 is a recombinant binary vector for Agrobacterium mediated transformation of soybean, carrying the gene cassette for expressing catalytic domain of HMGR1 from Arabidopsis thaliana. The catalytic domain of the HMGR1 cDNA is driven by soybean 7S alpha' beta conglycinin promoter. P-NOS: nopaline synthase gene promoter; kan: coding region for neomycin phosphotransferase protein to confer resistance to kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Left border: octopine left border sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence essential for transfer of T-DNA into Agrobacterium; Soy Alpha' Beta Conglycinin: soybean 7S alpha' beta conglycinin gene promoter; Arabidopsis HMGR catalytic domain: coding sequence for Arabidopsis HMGR1 catalytic domain; E9 3': 3' end of pea rbcS E9 gene.

FIG. 10 is a map showing the structure of construct pMON43058. pMON43058 is a recombinant binary vector for Agrobacterium-mediated soybean transformation, carrying gene expression cassettes for catalytic domain of HMGR1 from Arabidopsis thaliana and SMTII from Arabidopsis thaliana. P-NOS: nopaline synthase gene promoter; kan: coding region for neomycin phosphotransferase protein to confer resistance to kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Left border: octopine left border sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence essential for transfer of T-DNA into Agrobacterium; Soy Alpha' Beta Conglycinin: 7S alpha' beta conglycinin gene promoter from soybean; Arabidopsis HMGR catalytic domain: sequence encoding the catalytic domain of Arabidopsis HMGR1; E9 3': 3' end of pea rbcS E9 gene; Soy Alpha' Beta Conglycinin: soybean 7S alpha' beta conglycinin gene promoter; Arabidopsis SMT2: cDNA encoding sterol methyl transferase II enzyme from Arabidopsis thaliana (accession no: X89867); NOS 3': 3' termination end of nopaline synthase coding region.

FIG. 11 is profile (histogram) of the sterol composition of R1 transgenic soybean seeds when Arabidopsis truncated HMGR (catalytic domain without linker) was overexpressed using seed-specific 7S promoter (data from pMON43057:p7S::At HMGR truncated).

FIG. 12 is a profile (histogram) of the sterol composition of R1 transgenic soybean seeds when Arabidopsis truncated HMGR (catalytic domain without linker) and Arabidopsis SMTII were overexpresed (data from pMON43058:p7S::At HMGR truncated and p7S::At SMTII). The expression of the genes is controlled by the seed-specific 7S promoter.

FIG. 13 is a map showing the structure of construct pMON53733. pMON53733 is a recombinant binary vector carrying the cDNA encoding full-length form of Arabidopsis hydroxymethyl glutaryl CoA reductase1(HMGR1) in sense orientation driven by the enhanced cauliflower mosaic virus 35S promoter. P-35S: 35S promoter from cauliflower mosaic virus; kan: confers resistance to neomycin and kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Left border: octopine left border, sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence of T-DNA essential for integration into Agrobacterium; P-e35S: enhanced cauliflower mosaic virus promoter; Arabidopsis HMGR1: cDNA sequence encoding full-length form of Arabidopsis HMGR1; E9 3': 3' end of pea rbcS E9 gene.

FIG. 14 is a map showing the structure of construct pMON53734. pMON53734 is a recombinant binary vector carrying the cDNA encoding catalytic domain with linker region of Arabidopsis hydroxymethyl glutaryl CoA reductase1 (HMGR1) in sense orientation driven by the enhanced cauliflower mosaic virus 35S promoter. P-35S: 35S promoter from cauliflower mosaic virus; kan: confers resistance to neomycin and kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Left border: octopine left border, sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence of T-DNA essential for integration into Agrobacterium; P-e35S: enhanced cauliflower mosaic virus promoter; Arabidopsis tHMGR1: cDNA sequence encoding catalytic domain with linker region of Arabidopsis HMGR1; E9 3': 3' end of pea rbcS E9 gene.

FIG. 15 is a map showing the structure of construct pMON53735. pMON53735 is a recombinant binary vector carrying the cDNA encoding catalytic domain without the linker region of Arabidopsis hydroxymethyl glutaryl CoA reductase1 (HMGR1) in sense orientation driven by the enhanced cauliflower mosaic virus 35S promoter. P-35S: 35S promoter from cauliflower mosaic virus; kan: confers resistance to neomycin and kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Left border: octopine left border, sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence of T-DNA essential for integration into Agrobacterium; P-e35S: enhanced cauliflower mosaic virus promoter; Arabidopsis cHMGR1: cDNA sequence encoding catalytic domain without the linker region of Arabidopsis HMGR1; E9 3': 3' end of pea rbcS E9 gene.

FIG. 16 is a map showing the structure of construct pMON53736. pMON53736 is a recombinant binary vector carrying the cDNA encoding full-length form of rubber (Hevea brasiliensis) hydroxymethyl glutaryl CoA reductase1 (HMGR1) in sense orientation driven by the enhanced cauliflower mosaic virus 35S promoter. P-35S: 35S promoter from cauliflower mosaic virus; kan: confers resistance to neomycin and kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Left border: octopine left border, sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence of T-DNA essential for integration into Agrobacterium; P-e35S: enhanced cauliflower mosaic virus promoter; Hevea HMGR1: cDNA sequence encoding full-length form of rubber HMGR1; E9 3': 3" end of pea rbcS E9 gene.

FIG. 17 is a map showing the structures of construct pMON53737. pMON53737 is a recombinant binary vector carrying the cDNA encoding catalytic domain with linker region of rubber (Hevea brasiliensis) hydroxymethyl glutaryl CoA reductase1 (HMGR1) in sense orientation_driven by the enhanced cauliflower mosaic virus 35S promoter. P-35S: 35S promoter from cauliflower mosaic virus; kan: confers resistance to neomycin and kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Left border: octopine left border, sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence of T-DNA essential for integration into Agrobacterium; P-e35S: enhanced cauliflower mosaic virus promoter; rubber tHMGR1: cDNA sequence encoding catalytic domain with linker region of rubber HMGR1; E9 3': 3" end of pea rbcS E9 gene.

FIG. 18 is a map showing the structure of construct pMON53738. pMON53738 is a recombinant binary vector carrying the cDNA encoding mutant form of rubber (Hevea brasiliensis) hydroxymethyl glutaryl CoA reductase1 (HMGR1) in sense orientation driven by the enhanced cauliflower mosaic virus 35S promoter. In the mutant rubber HMGR1 the putative phosphorylation site, the serine amino acid residue at position 566 is changed to alanine amino acid residue (SEQ ID 23). P-35S: 35S promoter from cauliflower mosaic virus; kan: confers resistance to neomycin and kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Left border: octopine left border, sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence of T-DNA essential for integration into Agrobacterium; P-e35S: enhanced cauliflower mosaic virus promoter; rubber tHMGR1 Ala 566: cDNA sequence encoding catalytic domain with linker region of rubber HMGR1 in which serine amino acid residue at position 566 is changed to alanine amino acid residue using site directed mutagenesis; E9 3': 3' end of pea rbcS E9 gene.

FIG. 19 is a map showing the structure of construct pMON53739. pMON53739 is a recombinant binary vector carrying the cDNA encoding mutant form of rubber (Hevea brasiliensis) hydroxymethyl glutaryl CoA reductase1 (HMGR1) in sense orientation driven by the enhanced cauliflower mosaic virus 35S promoter. In the mutant rubber HMGR1 the putative phosphorylation site, the serine amino acid residue at position 567 is changed to alanine amino acid residue (SEQ ID 24). P-35S: 35S promoter from cauliflower mosaic virus; kan: confers resistance to neomycin and kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Left border: octopine left border, sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence of T-DNA essential for integration into Agrobacterium; P-e35S: enhanced cauliflower mosaic virus promoter; rubber tHMGR1 Ala 567: cDNA sequence encoding catalytic domain with linker region of rubber HMGR1 in which serine amino acid residue at position 567 is changed to alanine amino acid residue using site directed mutagenesis; E9 3': 3' end of pea rbcS E9 gene.

FIG. 20 is a map showing the structure of construct pMON53740. pMON53740 is a recombinant binary vector carrying the cDNA encoding catalytic domain without linker region of rubber (Hevea brasiliensis) hydroxymethyl glutaryl CoA reductase1 (HMGR1) in sense orientation driven by the enhanced cauliflower mosaic virus 35S promoter. P-35S: 35S promoter from cauliflower mosaic virus; kan: confers resistance to neomycin and kanamycin; NOS 3': 3' termination end of nopaline synthase coding region; Left border: octopine left border, sequence essential for transfer of T-DNA into Agrobacterium; ori-V: plasmid origin of replication in Agrobacterium; rop: coding sequence for repressor of primer; ori-322: origin of replication in E. coli; Spc/Str: coding region for Tn7 adenylyltransferase (AAD(3")) conferring resistance to spectinomycin and streptomycin; Right Border: right border sequence of T-DNA essential for integration into Agrobacterium; P-e35S: enhanced cauliflower mosaic virus promoter; rubber cHMGR1: cDNA sequence encoding catalytic domain without linker region of rubber HMGR1; E9 3': 3' end of pea rbcS E9 gene.

FIG. 21 is a graph comparing the cycloartenol content in micrograms of steroid compound per gram of seeds analyzed in transgenic Arabidopsis plants transformed with pMON53733 through pMON53740 compared to control plants.

FIG. 22 is a graph comparing the 24-methylene cycloartenol content in micrograms of steroid compound per gram of seeds analyzed in transgenic Arabidopsis plants transformed with pMON53733 through pMON53740 compared to control plants.

FIG. 23 is a graph comparing the obtusifoliol content in micrograms of steroid compound per gram of seeds analyzed in transgenic Arabidopsis plants transformed with pMON53733 through pMON53740 compared to control plants.

FIG. 24 is a graph comparing the campesterol content in micrograms of steroid compound per gram of seeds analyzed in transgenic Arabidopsis plants transformed with pMON53733 through pMON53740 compared to control plants.

FIG. 25 is a graph comparing the sitosterol content in micrograms of steroid compound per gram of seeds analyzed in transgenic Arabidopsis plants transformed with pMON53733 through pMON53740 compared to control plants.

FIG. 26 is a graph comparing the sitostanol content in micrograms of steroid compound per gram of seeds analyzed in transgenic Arabidopsis plants transformed with pMON53733 through pMON53740 compared to control plants.

FIG. 27 is a sterol profile (histogram) of transgenic Arabidopsis harboring different forms of rubber HMGR.

FIG. 28 is a graph of the squalene, zymosterol and erogosterol content in micrograms of sterol per milligram of cell dry weight from HMGR constructs in yeast HMGR1 knockout mutants for constructs having full length and truncated HMG CoA reductase (HMGR) sequences. The truncated sequences contain substantial portions of the catalytic region but lack the linker region and the transmembrane region of HMGR. These sequences are derived from Arabidopsis and rubber plants.

FIG. 29 is a map showing the structure of construct pMON43842. pMON43842 is a yeast expression vector carrying cDNA encoding Arabidopsis putative obtusifoliol C14.alpha.-demethylase (AC002329) in sense orientation driven by the p423Gall promoter. Sc.His3: HIS3 region from Saccharomyces cerevisiae encoding imidazoleglycerol-phosphate dehydratase for histidine synthesis; Ori-f1: bacteriophage f1 origin of replication; LAC: contains partial lacI coding sequence, promoter Plac, promoter Pt7, promoter Pt3, KS polylinker, and partial lacZ coding sequence; lacZ: partial coding sequence for beta-d-galactosidase or lacZ protein; T-Sc.Cyc1: a terminator from Cyc1- iso-1-cytochrome c from Saccharomyces cerevisiae to terminate transcription; obtus. C14.alpha..demethylase (AC002329): cDNA encoding Arabidopsis putative obtusifoliol C14.alpha.-demethylase; P-Sc.Gall: a promoter from Gal1- galactokinase of Saccharomyces cerevisiae to direct expression with galactose induction; LacZ-alpha: partial coding sequence for beta-d-galactosidase or lacZ protein; Ori-pUC: minimum sequence required for a functional origin of replication, sequence downstream of this region is known to affect copy number when expressed in bacteria; AMP: contains the P3 promoter and the beta-lactamase coding sequence, conferring resistance to ampicillin, penicillin, and carbenicillin; Sc.2 micron: 2 micron origin of replication.

FIG. 30 is a map showing the structure of construct pMON43843. pMON43843 is a yeast expression vector carrying cDNA encoding Arabidopsis putative squalene epoxidase 1 (ATA506263) in sense orientation driven by the p423Gal1 promoter. Sc.His3: HIS3 region from Saccharomyces cerevisiae encoding imidazoleglycerol-phosphate dehydratase for histidine synthesis; Ori-f1: bacteriophage f1 origin of replication; LAC: contains partial lacI coding sequence, promoter Plac, promoter Pt7, promoter Pt3, KS polylinker, and partial lacZ coding sequence; lacZ: partial coding sequence for beta-d-galactosidase or lacZ protein; T-Sc.Cyc1: a terminator from Cyc1- iso-1-cytochrome c from Saccharomyces cerevisiae to terminates transcription; Squalene epoxidase 1 (ATA506263): cDNA encoding Arabidopsis putative squalene epoxidase 1 (ATA506263); P-Sc.Gal1: a promoter from Gal1- galactokinase of Saccharomyces cerevisiae to direct expression with galactose induction; LacZ-alpha: partial coding sequence for beta-d-galactosidase or lacZ protein; Ori-pUC: minimum sequence required for a function