DETAILED DESCRIPTION OF THE INVENTION

[0045] It will be readily apparent to one skilled in the art that various embodiments and modifications may be made in the invention disclosed herein without departing from the scope and spirit of the invention.

[0046] As used in the specification, “a” or “an” may mean one or more. As used in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more.

[0047] The technology of the present invention is related to the invention described in the U.S. patent application entitled, “ Ginkgo biloba Levopimaradiene Synthase” filed on the same day and incorporated by reference herein.

[0048] I. Definitions

[0049] The term “diterpene” as used herein is defined as a terpene molecule having four isoprene units (C ₂₀ compounds).

[0050] The term “diterpene precursor” as used herein is defined as a metabolite in a pathway that serves as a synthetic or biosynthetic precursor to the production of a diterpene. A preferred diterpene precursor is geranylgeranyl pyrophosphate but also includes farnesyl pyrophosphate and isopentenyl pyrophosphate.

[0051] The term “diterpene synthase” as used herein is defined as an enzyme that catalyzes biosynthesis of a diterpene. In a specific embodiment, the term “diterpene cyclase” is used herein to refer to a diterpene synthase that effects a cyclization reaction to produce a diterpene having at least one cyclic structure. A non-limiting example of a diterpene comprising three cyclic structures is abietadiene.

[0052] The term “downregulated” as used herein refers to the state of a metabolic pathway being altered in which a step or process in the pathway is decreased or downregulated, such as in activity of an enzyme or expression of a nucleic acid sequence, respectively. In a specific embodiment, the modification is the result of an alteration in a nucleic acid sequence which encodes an enzyme in the pathway, an alteration in expression of a nucleic acid sequence which encodes an enzyme in the pathway, or an alteration in translation or proteolysis of an enzyme in the pathway, or a combination thereof. A skilled artisan recognizes that there are commonly used standard methods in the art to obtain the alterations, such as by mutation.

[0053] The terms “exogenous nucleic acid sequence” and “exogenous polynucleotide” refer to a nucleic acid sequence or polynucleotide that has been prepared and provided to a cell. In one aspect, the nucleic acid sequence has been isolated and purified by methods well-known in the art and, optionally, modified. This nucleic acid sequence is then provided to a cell employing methods known in the art, thereby producing a recombinant cell. In another aspect, the terms refer to a non-native nucleic acid sequence that has been provided to a cell. For example, an allele that confers an increase in a flux of a metabolic pathway has been added to the cell of the present invention by conventional methods such as genetic cross, and, thus, represents an exogenous sequence as compared to the native cell.

[0054] The term “GGPP” as used herein is defined as geranylgeranyl pyrophosphate and employed interchangably in the art with geranlygeranyl diphosphate (e.g., “GGDP”). The acyclic carbon structure possesses four double bonds, which preferably at least one is in the E configuration.

[0055] The term “HMGR” as used herein is defined as HMG-CoA reductase. A skilled artisan is aware that HMGR catalyzes the reduction of 3-hydroxy-3-methylglutaryl Coenzyme A to mevalonate, which is interchangeably referred to as mevalonic acid (see FIG. 6 ).

[0056] The term “isoprene” as used herein is defined as a C ₅ chemical unit as shown in FIG. 1 .

[0057] The term “inducer” as used herein is defined as a compound, molecule or structure such as a promoter, that controls and effects a process. Specifically, one inducer of the present invention is galactose, which controls expression of a nucleic acid sequence of the present invention.

[0058] The terms “modified” or “modification” as used herein refer to the state of a metabolic pathway being altered in which a step or process in the pathway is decreased or downregulated or increased or upregulated, such as in activity of an enzyme or expression of a nucleic acid sequence. In a specific embodiment, the modification is the result of an alteration in a nucleic acid sequence which encodes an enzyme in the pathway, an alteration in expression of a nucleic acid sequence which encodes an enzyme in the pathway, or an alteration in translation or proteolysis of an enzyme in the pathway, or a combination thereof. Further, the modification is the result of introducing or exogenously providing to a cell having a metabolic pathway a nucleic acid sequence the effects a desired modification of the pathway. A skilled artisan recognizes that there are commonly used standard methods in the art to obtain the alterations, such as by mutation.

[0059] The term “monoterpene” as used herein is defined as a terpene having two isoprene units (C ₁₀ compounds), wherein the monoterpene is a metabolite of geranyl diphosphate or geranyl pyrophosphate.

[0060] The term “sesquiterpene” as used herein is defined as a terpene having three isoprene units (C ₁₅ compounds), wherein the sesquiterpene is a metabolite of farnesyl diphosphate or farnesyl pyrophosphate.

[0061] The term “soluble form” as used herein is defined as a form, such as an amino acid sequence, that demonstrates HMG-CoA reductase activity. In a specific embodiment, the soluble form contains no more than about three transmembrane domains.

[0062] The term “terpene” as used herein is defined a material comprising isopentene (also called isoprene) units. The structure of the isoprene unit comprising terpenes is shown in FIG. 1 .

[0063] The term “triterpene” as used herein is defined as a terpene having six isoprene units (C ₃₀ compounds), wherein the triterpene is a metabolite of squalene or oxidosqualene.

[0064] The term “under conditions wherein said diterpene is produced” as used herein is defined as an environment wherein a diterpene is produced, wherein such parameters as temperature, such as between about 28° C. and about 32° C., but preferably about 30° C., growth media content, which is well known in the art, availability of an inducer for an inducible promoter, and the like are provided to produce the diterpene.

[0065] The term “under conditions wherein said geranylgeranyl pyrophosphate is produced” and “under conditions wherein said GGPP is produced” as used herein is defined as an environment wherein geranylgeranyl pyrophosphate is produced. In specific embodiments, the conditions includes cultivation temperature, such as between about 28° C. and about 32° C., but preferably about 30° C. unless an alteration comprising a temperature-sensitive mutation has been employed, growth media content, which is well known in the art, availability of an inducer for an inducible promoter, and the like are provided to produce the geranylgeranyl pyrophosphate (GGPP).

[0066] The term “under conditions wherein said geranylgeraniol is produced” and “under conditions wherein said GGOH is produced” as used herein is defined as an environment wherein geranylgeraniol is produced. In specific embodiments, the conditions includes cultivation temperature, such as between about 28° C. and about 32° C., but preferably about 30° C. unless an alteration comprising a temperature-sensitive mutation has been employed, growth media content, which is well known in the art, availability of an inducer for an inducible promoter, and the like are provided to produce the geranylgeraniol (GGOH).

[0067] The term “unicellular organism” as used herein is defined as a non-human organism which is a single cell and is incapable of development into a multicellular organism. In a specific embodiment, this includes bacteria, such as Escherichia coli , and yeast, such as Saccharomyces. Preferably the unicellular organism comprises an isoprenoid biosynthetic pathway and/or a sterol biosynthetic pathway.

[0068] The term “upregulated” as used herein is defined as increased in expression of a particular nucleic acid sequence over native or wild type expression levels. The upregulation results from, for example, an increase in transcription of the sequence, an increase in stability of a messenger RNA of the sequence, a combination thereof, or through other means known in the art which increase levels of expression levels of a nucleic acid sequence. In a specific embodiment, the increase in expression is the result of a promoter operatively linked to the nucleic acid sequence which is not native to the nucleic acid sequence. In another specific embodiment, the increase in expression is the result of an inducible or constitutive promoter which regulates the nucleic acid sequence. In another specific embodiment, the increase in a cognate protein level of the nucleic acid sequence is the result of a promoter operatively linked to the amino acid sequence which is not native to the amino acid sequence.

[0069] II. The Present Invention

[0070] The present invention is directed to recombinant yeast, such as Saccharomyces, which is modified at at least one step in the sterol biosynthetic pathway to produce high levels of diterpene hydrocarbon(s) or a diterpene precursor such as GGPP. Yeast are readily genetically manipulated, and the metabolism of its major sterol, ergosterol, is well understood. Therefore, the yeast serve as a general production system, particularly because the yeast system is uniquely adaptable to further modify and biosynthesize other terpenes (including monoterpenes, sesquiterpenes, diterpenes and triterpenes) in vivo.

[0071] Another embodiment of the present invention is a recombinant bacteria, such as E. coli , which is modified at at least one step in the isoprenoid biosynthetic pathway to produce high levels of diterpene hydrocarbon(s) or a diterpene precursor such as GGPP. Bacteria are readily genetically manipulated and cultivated, and the isoprenoid metabolism of hopanoids, the prokaryotic cellular analog to sterols, is understood to comprise production in vivo of GGPP that is not a commecially feasible level.

[0072] Standard methods and reagents in the field of yeast molecular genetics, particularly regarding Saccharomyces cerevisiae , are well known in the art. References for such methods include Methods in Yeast Genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual (Burke et al., 2000) and Current Protocols in Molecular Biology , Chapter 13 (Ausubel et al., 1994), both incorporated by reference herein. A skilled artisan is aware that Saccharomyces is the yeast of choice, which includes many known species such as S. cerevisiae, S. italicus, S. oviformis, S. capensis, S. chevalieri, S. douglasii, S. paradoxus, S. cariocanus, S. kudriavzevii, S. mikatae, S. bayanus and S. pastorianus . In another embodiment, it is contemplated that filamentous fungi, such as Aspergillus, is used instead of a unicellular organism as defined herein. However, a skilled artisan recognizes that filamentous fingi, which normally develop into a multicellular septate organism, are unicellular in a pre-septate developmental stage.

[0073] In a preferred embodiment, the diterpene producing strain overexpresses a geranylgeranyl pyrophosphate synthase, overexpresses a diterpene synthase, overexpresses a soluble form of 3-hydroxy-3-methylglutaryl Co-A reductase, and contains a nucleic acid sequence that encodes a gene that confers an increase in sterol metabolic flux to the cell as compared to native sterol metabolic flux levels. That is, coexpression of a 3-hydroxy-3-methylglutaryl Co-A reductase or other enzymes that yield geranylgeranyl pyrophosphate allows production of geranylgeranyl pyrophosphate metabolites which include geranylgeraniol, diterpene hydrocarbon(s), further metabolites and related compounds. One non-limiting example of a nucleic acid sequence the encodes a gene that confers an increase in sterol metabolic flux is upc2-1. Genes that effect similar increases in sterol metabolic flux to the cell are contemplated as their effect is expected to increase the amount of diterpene produced in the recombinant cell. Incubation in the presence of a polyaromatic resin allows the product(s) to be adsorbed extracellularly, which greatly simplifies recovery and increases isolated yields. In specific embodiments, one or more additional enzymes are employed to further metabolize the diterpene synthase product (i.e., an oxidoreductase). In a preferred embodiment, the engineered unicellular organism is grown in the presence of at least about 5% (w/v) sterile polyaromatic resin-supplemented media.

[0074] A yeast cell of the present invention to produce a diterpene precursor preferably has at least one of the following: an exogenous polynucleotide encoding a polypeptide of amino acid sequence of a geranylgeranyl pyrophosphate synthase under the control of a promoter operable in the yeast, an exogenous nucleic acid sequence encoding a polypeptide of amino acid sequence of a HMG-CoA reductase under control of a promoter operable in the yeast, and/or an exogenous polynucleotide encoding a polypeptide that confers an increase in sterol metabolic flux to the cell as compared to native sterol metabolic flux levels. It is understood that a yeast cell that is desired to produce diterpenes in vivo must further comprise an exogenous polynucleotide encoding a polypeptide of amino acid sequence of a diterpene synthase under the control of a promoter operable in the yeast.

[0075] In specific embodiments, the yeast cell comprises a yeast geranylgeranyl pyrophosphate synthase, a diterpene synthase, a truncated form of HMG-CoA reductase, and a nucleic acid sequence encoding a gene that confers an increase in sterol metabolic flux. The geranylgeranyl pyrophosphate synthase originated in other organisms are also contemplated in specific embodiments. In one embodiment, a yeast cell is manipulated by standard molecular genetics methods to additionally contain BTS1, and/or the truncated HMG-CoA reductase, and/or the nucleic acid sequence that confers an increase in sterol metabolic flux. The nucleic acids are chromosome bound to minimize antibiotic selection requirements or are episomally borne and maintained in the cell by a selection or a functional means. Furthermore, the nucleic acid sequences of the present invention are preferably regulated by an inducible promoter, such as GAL1, CUP1 or MET3, to provide a means of external control of GGPP biosynthesis. In an alternative embodiment, a constitutive promoter such as the PGK promoter or the ADH promoter is utilized. In a specific embodiment, the constitutive promoter is employed to control expression of the diterpene synthase. In a preferred embodiment, the constitutive promoters are strong promoters.

[0076] In the specific embodiment wherein one or more nucleic acid sequences are included in the yeast cell to modify GGPP and/or utilize GGPP as a substrate for subsequent syntheses, this sequence(s) is contained on a multicopy plasmid bearing a selection means. In the specific embodiment wherein abietadiene is generated from the GGPP biosynthesis pathway, the abietadiene cyclase (e.g., diterpene synthase) is plasmid-borne within the cell and selected for by standard means.

[0077] In a specific embodiment, a normative nucleic acid sequence is incorporated into a yeast cell. Advantages of employing native nucleic acid and amino acid sequences includes, for example, cellular recognition of the recombinant structure. However, as a skilled artisan is aware, the cellular recognition has a disadvantage in that, for example, the recombinant structure is a highly regulated structure in the cell. Thus, accumulating the structure in vivo effects metabolic and regulatory mechanisms that are adverse to diterpene and diterpene precursor production.

[0078] A skilled artisan is aware that in the specific embodiments wherein there is variability in isolated and/or production yields, yet still highly improved over yields generated in the absence of a GGPP synthase, a soluble form of HMG-CoA reductase, and a nucleic acid encoding a gene that confers an increase in the sterol metabolic flux. This variability is due, for example, to the initiation of native regulatory mechanisms, the accumulation of hydrolyzed diterpene precursor, the transportation mechanisms responsible for molecular exportation, and/or other unknown mechanistic events controlling sterol biosynthesis.

[0079] In the specific embodiment wherein a plant nucleic acid sequence is utilized, a skilled artisan is aware that, for instance, such as is required by E. coli , a plastidyl targeting sequence is be identified and removed. If the targeting sequence does not occur, the structural products are vulnerable to incorporation into inclusion bodies. However, the significant surplus of precursor generated by the compositions and methods of the present invention allow use of the full length plant nucleic acid sequence, which is a significant advantage of the present invention. In particular, this is advantageous in the methods of the present invention wherein a diterpene synthase is identified by enzymatic activity in the compositions of the present invention.

[0080] Alternatively, a unicellular organism comprising an isolated polypeptide encoding an amino acid sequence of a GGPP synthase, the soluble form of HMG-CoA reductase, the diterpene synthase, a gene that confers an increase in sterol metabolic flux, the squalene synthase, the hexaprenylpyrophosphate synthetase and/or the prenyltransferase are within the scope of the present invention. Non-limiting examples of amino acid sequences are provided herein.

[0081] In other embodiments, a modification is made that decreases, downregulates, diminishes or removes biosynthetic pathways that compete for GGPP bioavailability. In another specific embodiment, a hexaprenylpyrophosphate synthetase is modified to increase FPP flux into the engineered GGPP biosynthesis pathway. An example of a hexaprenylpyrophosphate synthetase is COQ1 (GenBank Accession No. J05547; SEQ ID NO:401). Hexaprenylpyrophosphate is the committed step of pathways which produce dolichols and ubiquinones. In an additional specific embodiment, a prenyltransferase is modified. Prenyltransferases are well known in the art. In a specific embodiment, the site of inhibition is protein farnesyltransferase (such as STE14; GenBank Accession No. L15442 (SEQ ID NO:402) or GenBank Accession No. L07952 (SEQ ID NO:403)), protein geranylgeranyltransferase I alpha subunit (such as CDC43; GenBank Accession No. M31114; SEQ ID NO:404), protein geranylgeranyltransferase I beta subunit (such as RAM2; GenBank Accession No. M88584; SEQ ID NO:405), protein geranylgeranyltransferase II alpha subunit (such as BET2; GenBank Accession No. M26597; SEQ ID NO:406), protein geranylgeranyltransferase II beta subunit (such as BET4; GenBank Accession No. U14132; SEQ ID NO:407), or a combination thereof.

[0082] A skilled artisan is aware of sequence repositories, such as GenBank, to obtain nucleic acid and amino acid sequences utilized in the present invention. Examples of geranylgeranyl pyrophosphate synthase nucleic acid sequences for the present invention include the following: U31632 (SEQ ID NO:1); AF049658 (SEQ ID NO:2); AK025139 (SEQ ID NO:3); AB000835 (SEQ ID NO:4); AJ276129 (SEQ ID NO:5); AB034250 (SEQ ID NO:6); AB034249 (SEQ ID NO:7); AW132388 (SEQ ID NO:8); AW034766 (SEQ ID NO:9); AI496168 (SEQ ID NO:10); AF081514 (SEQ ID NO:11); AF020041 (SEQ ID NO:12); X98795 (SEQ ID NO:13); X92893 (SEQ ID NO:14); X80267 (SEQ ID NO:15); L37405 (SEQ ID NO:16); U15778 (SEQ ID NO:17); L40577 (SEQ ID NO:18); M87280 (SEQ ID NO:19); L25813 (SEQ ID NO:20); and AF049659 (SEQ ID NO:21). A skilled artisan is aware that sequences unrelated to geranylgeranyl pyrophosphate synthase in those sequences which comprise large regions of the genome of a particular organism are not within the scope of the invention. In a preferred embodiment, SEQ ID NO:1 is utilized as a geranylgeranyl pyrophosphate synthase nucleic acid sequence in the cell of the invention.

[0083] Examples of geranylgeranyl pyrophosphate synthase amino acid sequences for the present invention include the following: AAA83262.1 (SEQ ID NO:22); AAC05595.1 (SEQ ID NO:23); AAC05273.1 (SEQ ID NO:24); NP _— 043281.1 (SEQ ID NO:25); BAB18334.1 (SEQ ID NO:26); AAC68232.1 (SEQ ID NO:27); CAC12434.1 (SEQ ID NO:28); BAB02385.1 (SEQ ID NO:29); CAB94793.1 (SEQ ID NO:30); AAF38891.1 (SEQ ID NO:31); BAA23157.1 (SEQ ID NO:32); BAA19583.1 (SEQ ID NO:33); CAB89115.1 (SEQ ID NO:34); AAD12206.1 (SEQ ID NO:35); AAD08933.1 (SEQ ID NO:36); CAB80510 (SEQ ID NO:37); CAB80347.1 (SEQ ID NO:38); CAB38744.1 (SEQ ID NO:39); BAA16690.1 (SEQ ID NO:40); AAD38295.1 (SEQ ID NO:41); BAA86285.1 (SEQ ID NO:42); BAA86284.1 (SEQ ID NO:43); CAB53152.1 (SEQ ID NO:44); CAB56064.1 (SEQ ID NO:45); BAA77251 (SEQ ID NO:46); CAB16803.1 (SEQ ID NO:47); CAB37502.1 (SEQ ID NO:48); AAD16018.1 (SEQ ID NO:49); AAC77874.1 (SEQ ID NO:50); CAA17477.1 (SEQ ID NO:51); AAC06913.1 (SEQ ID NO:52); CAA67330.1 (SEQ ID NO:53); AAB67731.1 (SEQ ID NO:54); CAA63486.1 (SEQ ID NO:55); CAA56554.1 (SEQ ID NO:56); AAA96328.1 (SEQ ID NO:57); AAA91949.1 (SEQ ID NO:58); AAA86688.1 (SEQ ID NO:59); AAA81879.1 (SEQ ID NO:60); AAA81312.1 (SEQ ID NO:61); AAA32797.1 (SEQ ID NO:62); BAB01876 (SEQ ID NO:63); BAA23157 (SEQ ID NO:64); AAD43148 (SEQ ID NO:65); NP _— 043281 (SEQ ID NO:66); BAB18334 (SEQ ID NO:67); E81650 (SEQ ID NO:68); T36967 (SEQ ID NO:69); S76966 (SEQ ID NO:70); A72041 (SEQ ID NO:71); T02429 (SEQ ID NO:72); S74538 (SEQ ID NO:73); S71230 (SEQ ID NO:74); S71231 (SEQ ID NO:75); AAC05595 (SEQ ID NO:76); AAC05273 (SEQ ID NO:77); BAB02387 (SEQ ID NO:78); BAB01936 (SEQ ID NO:79); AAF39709 (SEQ ID NO:80); BAA23158 (SEQ ID NO:81); E70365 (SEQ ID NO:82); S49625 (SEQ ID NO:83); P34802 (SEQ ID NO:84); and P80042 (SEQ ID NO:85). In a preferred embodiment, SEQ ID NO:22 is utilized as a geranylgeranyl pyrophosphate synthase amino acid sequence in the cell.

[0084] One non-limiting example of a gene that confers an increase to sterol metabolic flux as compared to native sterol metabolic flux levels is the upc2-1 allele. The upc2-1 allele comprises a guanine to adenine transition in the open reading frame designated YDR213W on chromosome IV (Leak et al., 1999; incorporated by reference herein in its entirety). The nucleic acid sequence is known and/or obtained through GenBank Accession No. Z68194 (SEQ ID NO:399), and Leak et al. (1999) describe the mutations associated with the upc2-1 allele. Incorporation of the upc2-1 allele conferred an increase in sterol metabolic flux as compared to native sterol metabolic flux levels, and thus, demonstrates that other such genes that confer the same biological activity, e.g., increase sterol metabolic flux levels, are expected to increase production in vivo of a diterpene and a diterpene precursor.

[0085] In a preferred embodiment of the present invention, a soluble form of HMG-CoA reductase is utilized. A skilled artisan is aware that this requires removal of hydrophobic sequences responsible for conferring insolubility to the gene product, such as transmembrane domains, and is furthermore aware of standard methods to achieve such removal from the sequence. Examples of HMG-CoA reductase nucleic acid sequences, which in specific embodiments may be altered to achieve solubility of the reductase for the present invention, include the Saccharomyces cerevisiae open reading frame found on chromosome XIII at locus YML075C (SEQ ID NO:86); NM _— 000859 (SEQ ID NO:87); X00494 (SEQ ID NO:88); AF273765 (SEQ ID NO:89); AF273764 (SEQ ID NO:90); AF273763 (SEQ ID NO:91); AF273762 (SEQ ID NO:92); AF273761 (SEQ ID NO:93); AF273760 (SEQ ID NO:94); AF273759 (SEQ ID NO:95); AF273758 (SEQ ID NO:96); AF273757 (SEQ ID NO:97); AF273756 (SEQ ID NO:98); AF273755 (SEQ ID NO:99); AF273754 (SEQ ID NO:100); AF290098 (SEQ ID NO:101); AF290096 (SEQ ID NO:102); AF290090 (SEQ ID NO:103); AF290088 (SEQ ID NO:104); AF290086 (SEQ ID NO:105); AF071750 (SEQ ID NO:106); AB037907 (SEQ ID NO:107); AF155593 (SEQ ID NO:108); X58370 (SEQ ID NO:109); AF162705 (SEQ ID NO:110); AF159136 (SEQ ID NO:111); AF159138 (SEQ ID NO:112); AB015627 (SEQ ID NO:113); AB015626 (SEQ ID NO:114); AV374599 (SEQ ID NO:115); AV317420 (SEQ ID NO:116); AV317328 (SEQ ID NO:117); AV317132 (SEQ ID NO:118); AV277976 (SEQ ID NO:119); AV259312 (SEQ ID NO:120); AV237573 (SEQ ID NO:121); AF142473 (SEQ ID NO:122); E17178 (SEQ ID NO:123); E17177 (SEQ ID NO:124); AF110382 (SEQ ID NO:125); AB021862 (SEQ ID NO:126); U97683 (SEQ ID NO:127); A1326595 (SEQ ID NO:128); U33178 (SEQ ID NO:129); U30179 (SEQ ID NO:130); L34829 (SEQ ID NO:131); L34824 (SEQ ID NO:132); AB012603 (SEQ ID NO:133); AA982887 (SEQ ID NO:134); AF038045 (SEQ ID NO:135); AA710790 (SEQ ID NO:136); AA597171 (SEQ ID NO:137); AA517939 (SEQ ID NO:138); U51986 (SEQ ID NO:139); U51985 (SEQ ID NO:140); AA260731 (SEQ ID NO:141); AA109510 (SEQ ID NO:142); L76979 (SEQ ID NO:143); X70034 (SEQ ID NO:144); X94308 (SEQ ID NO:145); X68651 (SEQ ID NO:146); X94307 (SEQ ID NO:147); A10474 (SEQ ID NO:148); A10471 (SEQ ID NO:149); A10468 (SEQ ID NO:150); A10465 (SEQ ID NO:151); A10462 (SEQ ID NO:152); X55286 (SEQ ID NO:153); J04537 (SEQ ID NO:154); A10473 (SEQ ID NO:155); A10470 (SEQ ID NO:156); A10467 (SEQ ID NO:157); M15959 (SEQ ID NO:158); M62633 (SEQ ID NO:159); M62766 (SEQ ID NO:160); M12705 (SEQ ID NO:161); M22002 (SEQ ID NO:162); L19261 (SEQ ID NO:163); J04200 (SEQ ID NO:164); J03523 (SEQ ID NO:165); M27294 (SEQ ID NO:166); M24015 (SEQ ID NO:167); or a combination thereof.

[0086] Examples of HMG-CoA reductase amino acid sequences that are subsequently altered to achieve solubility of the reductase for the present invention include the following: NP _— 013636.1 (SEQ ID NO:168); NP _— 000850.1 (SEQ ID NO:169); CAA25189.1 (SEQ ID NO:170); AAG02454.1 (SEQ ID NO:171); AAG02449.1 (SEQ ID NO:172); AAG02434.1 (SEQ ID NO:173); AAG02429 (SEQ ID NO:174); AAG02423.1 (SEQ ID NO:175); AAD20975.2 (SEQ ID NO:176); BAB07821.1 (SEQ ID NO:177); AAD38406.1 (SEQ ID NO:178); CAA41261.1 (SEQ ID NO:179); AAF80475.1 (SEQ ID NO:180); AAF80374.1 (SEQ ID NO:181); BAA74566.1 (SEQ ID NO:182); BAA74565 (SEQ ID NO:183); AAD47596.1 (SEQ ID NO:184); AAD38873.1 (SEQ ID NO:185); BAA36291.1 (SEQ ID NO:186); AAD09278 (SEQ ID NO:187); AAC46885.1 (SEQ ID NO:188); AAC37437.1 (SEQ ID NO:189); AAC37436.1 (SEQ ID NO:190); AAC37435.1 (SEQ ID NO:191); AAC37434.1 (SEQ ID NO:192); AAC37433.1 (SEQ ID NO:193); AAC37432.1 (SEQ ID NO:194); AAC37431.1 (SEQ ID NO:195); BAA31937.1 (SEQ ID NO:196); AAC05089.1 (SEQ ID NO:197); AAC05088.1 (SEQ ID NO:198); AAB67527.1 (SEQ ID NO:199); BAA06492.1 (SEQ ID NO:200); AAB52552.1 (SEQ ID NO:201); AAB52551.1 (SEQ ID NO:202); AAB39277.1 (SEQ ID NO:203); CAA49628.1 (SEQ ID NO:204); CAA63971.1 (SEQ ID NO:205); CAA48610.1 (SEQ ID NO:206); CAA63970.1 (SEQ ID NO:207); CAA39001.1 (SEQ ID NO:208); AAA76821.1 (SEQ ID NO:209); CAA00908.1 (SEQ ID NO:210); CAA00907.1 (SEQ ID NO:211); CAA00906.1 (SEQ ID NO:212); CAA00905.1 (SEQ ID NO:213); CAA00904.1 (SEQ ID NO:214); AAA67317.1 (SEQ ID NO:215); AAA37819.1 (SEQ ID NO:216); AAA37077.1 (SEQ ID NO:217); AAA34677.1 (SEQ ID NO:218); AAA32814.1 (SEQ ID NO:219); AAA30060.1 (SEQ ID NO:220); AAA29896.1 (SEQ ID NO:221); AAA25894.1 (SEQ ID NO:222); AAA25837.1 (SEQ ID NO:223); P43256 (SEQ ID NO:224); A23586 (SEQ ID NO:225); S12554 (SEQ ID NO:226); S72194 (SEQ ID NO:227); T07112 (SEQ ID NO:228); S56715 (SEQ ID NO:229); S56714 (SEQ ID NO:230); S56712 (SEQ ID NO:231); S56711 (SEQ ID NO:232); S56710 (SEQ ID NO:233); S33175 (SEQ ID NO:234); 028538 (SEQ ID NO:235); AAA25837 (SEQ ID NO:236); O26662 (SEQ ID NO:237); Q58116 (SEQ ID NO:238); Q59468 (SEQ ID NO:239); P54960 (SEQ ID NO:240); P48019 (SEQ ID NO:241); P48020 (SEQ ID NO:242); Q01559 (SEQ ID NO:243); Q03163 (SEQ ID NO:244); Q00583 (SEQ ID NO:245); P13702 (SEQ ID NO:246); P14891 (SEQ ID NO:247); Q9YAS4 (SEQ ID NO:248); Q9Y7D2 (SEQ ID NO:249); Q9XHL5 (SEQ ID NO:250); Q9XEL8 (SEQ ID NO:251); Q9V1R3 (SEQ ID NO:252); Q9V1R3 (SEQ ID NO:253); Q41437 (SEQ ID NO:254); 076819 (SEQ ID NO:255); O74164 (SEQ ID NO:256); O64967 (SEQ ID NO:257); O64966 (SEQ ID NO:258); O59469 (SEQ ID NO:259); O51628 (SEQ ID NO:260); O24594 (SEQ ID NO:261); NP _— 000850 (SEQ ID NO:262); CAA25189 (SEQ ID NO:263); NP _— 013555 (SEQ ID NO:264); NP _— 013308 (SEQ ID NO:265); AAA36989 (SEQ ID NO:266); Q12649 (SEQ ID NO:267); PO ₄₀₃₅ (SEQ ID NO:268); AAG21343 (SEQ ID NO:269); AAG02454 (SEQ ID NO:270); AAG02449 (SEQ ID NO:271); AAG02434 (SEQ ID NO:272); AAG02429 (SEQ ID NO:273); AAG02423 (SEQ ID NO:274); AAD20975 (SEQ ID NO:275); BAB07821 (SEQ ID NO:276); AAD38406 (SEQ ID NO:277); AAF80475 (SEQ ID NO:278); AAF80374 (SEQ ID NO:279); AAF80373 (SEQ ID NO:280); Q12577 (SEQ ID NO:281); BAA74566 (SEQ ID NO:282); BAA74565 (SEQ ID NO:283); P54869 (SEQ ID NO:284); O02734 (SEQ ID NO:285); O08424 (SEQ ID NO:286); Q10283 (SEQ ID NO:287); Q29512 (SEQ ID NO:288); P51639 (SEQ ID NO:289); P54839 (SEQ ID NO:290); P54874 (SEQ ID NO:291); Q01581 (SEQ ID NO:292); P54872 (SEQ ID NO:293); P54871 (SEQ ID NO:294); P54873 (SEQ ID NO:295); P54868 (SEQ ID NO:296); P54870 (SEQ ID NO:297); P54961 (SEQ ID NO:298); P48021 (SEQ ID NO:299); P48022 (SEQ ID NO:300); P34136 (SEQ ID NO:301); P34135 (SEQ ID NO:302); Q01237 (SEQ ID NO:303); P20715 (SEQ ID NO:304); P16237 (SEQ ID NO:305); P09610 (SEQ ID NO:306); P14773 (SEQ ID NO:307); P00347 (SEQ ID NO:308); P12684 (SEQ ID NO:309); P29058 (SEQ ID NO:310); P12683 (SEQ ID NO:311); P29057 (SEQ ID NO:312); P17425 (SEQ ID NO:313); P13704 (SEQ ID NO:314); P23228 (SEQ ID NO:315); P22791 (SEQ ID NO:316); AAD47596 (SEQ ID NO.317); 5542336 (SEQ ID NO:318); 5542335 (SEQ ID NO:319); 5542334 (SEQ ID NO:320); 5542333 (SEQ ID NO:321); AAD38873 (SEQ ID NO:322); BAA36291 (SEQ ID NO:323); AAD09278 (SEQ ID NO:324); AAC46885 (SEQ ID NO:325); AAC37437 (SEQ ID NO:326); AAC37435 (SEQ ID NO:327); AAC37434 (SEQ ID NO:328); AAC37433 (SEQ ID NO:329); AAC37432 (SEQ ID NO:330); AAC37431 (SEQ ID NO:331); AAC37436 (SEQ ID NO:332); BAA31937 (SEQ ID NO:333); AAC05089 (SEQ ID NO:334); AAC05088 (SEQ ID NO:335); AAB67527 (SEQ ID NO:336); AAB52552 (SEQ ID NO:337); AAB52551 (SEQ ID NO:338); AAB39277 (SEQ ID NO:339); CAA49628 (SEQ ID NO:340); 2116416F (SEQ ID NO:341); 2116416E (SEQ ID NO:342); 2116416D (SEQ ID NO:343); 2116416C (SEQ ID NO:344); 2116416B (SEQ ID NO:345); 2116416A (SEQ ID NO:346); CAA63971 (SEQ ID NO:347); CAA63970 (SEQ ID NO:348); CAA39001 (SEQ ID NO:349); CAA00906 (SEQ ID NO:350); CAA00907 (SEQ ID NO:351); CAA00908 (SEQ ID NO:352); CAA00904 (SEQ ID NO:353); AAA67317 (SEQ ID NO:354); AAA37819 (SEQ ID NO:355); AAA37077 (SEQ ID NO:356); AAA32814 (SEQ ID NO:357); AAA29896 (SEQ ID NO:358); RDHYE (SEQ ID NO.359); and AAA25894 (SEQ ID NO:360).

[0087] Diterpene synthase nucleic acid sequences that are useful in the present invention include Stevia rebaudiana kaurene synthase (KS22-1) (GenBank Accession number AF097311; SEQ ID NO:361); Stevia rebaudiana kaurene synthase (KS1-1) (GenBank Accession number AF097310; SEQ ID NO:362); Stevia rebaudiana copalyl pyrophosphate synthase (Cpps1) (GenBank Accession No. AF034545; SEQ ID NO:412); Taxus brevifolia taxadiene synthase (TDC1) (GenBank Accession No. U48796; SEQ ID NO:363); Phaeosphaeria sp. L487 mRNA for ent-kaurene synthase (GenBank Accession No. AB003395; SEQ ID NO:364); Abies grandis abietadiene synthase (ac22) (GenBank Accession No. U50768; SEQ ID NO:365) (Stoffer-Vogel et al., 1996); Ricinus communis casbene synthase (GenBank Accession No. L32134; SEQ ID NO:366) (Hill et al., 1996); Cucumis sativus KS mRNA for ent-kaurene synthase (GenBank Accession No. AB045310; SEQ ID NO:367); Lactuca sativa LsKS1 mRNA for ent-kaurene synthase No1 (GenBank Accession No. AB031205; SEQ ID NO:368); Glycine max sequence GenBank Accession No. BE473763 (SEQ ID NO:369); Glycine max sequence GenBank Accession No. AW759166 (SEQ ID NO:370); Gibberella fujikuroi mRNA for GfCPS/KS (GenBank Accession No. AB013295; SEQ ID NO:371); Lotus japonicus cDNA (GenBank Accession No. A1967851; SEQ ID NO:372); Glycine max sequence (GenBank Accession No. AI940878; SEQ ID NO:373); Homo sapiens sequence (GenBank Accession No. A1809939; SEQ ID NO:374); Zea mays kaurene synthase (KS) mRNA (GenBank Accession No. AF105149; SEQ ID NO:375); Arabidopsis thaliana chromosome 1 BAC T8K14 sequence (GenBank Accession No. AC007202; SEQ ID NO:376); Arabidopsis thaliana ent-kaurene synthase (GA2) mRNA (GenBank Accession No. AF034774; SEQ ID NO:377) (Sun and Kamiya, 1994); unknown source cDNA encoding ent-kaurene synthase A (GenBank Accession No. E12936; SEQ ID NO:378); Mycobacterium tuberculosis sequence (GenBank Accession No. AL009198; SEQ ID NO:379); Pisum sativum ent-kaurene synthase A (LS) mRNA (GenBank Accession No. U63652; SEQ ID NO:380); Cucurbita maxima ent-kaurene synthase B mRNA (GenBank Accession No. U43904; SEQ ID NO:381) (Yamaguchi et al., 1996); and Zea mays kaurene synthase A (An1) mRNA (GenBank Accession No. L37750; SEQ ID NO:382) (Bensen et al., 1995).

[0088] Corresponding diterpene synthase amino acid sequences include Stevia rebaudiana kaurene synthase (KS22-1) (GenBank Accession number AAD34295.1 (SEQ ID NO:383); Stevia rebaudiana kaurene synthase (KS1-1) (GenBank Accession number AAD34294.1; SEQ ID NO:384); Stevia rebaudiana copalyl pyrophosphate synthase (Cpps1) (GenBank Accession No. AAB87091.1; SEQ ID NO:385); Taxus brevifolia taxadiene synthase (TDC1) (GenBank Accession No. AAC49310.1; SEQ ID NO:386); Phaeosphaeria sp. L487 mRNA for ent-kaurene synthase (GenBank Accession No. BAA22426.1; SEQ ID NO:387); Abies grandis abietadiene synthase (ac22) (GenBank Accession No. AAB05407.1; SEQ ID NO:388); Cucumis sativus KS mRNA for ent-kaurene synthase (GenBank Accession No. BAB19275.1; SEQ ID NO:389); Lactuca sativa LsKS1 mRNA for ent-kaurene synthase No1 (GenBank Accession No. BAB12441.1; SEQ ID NO:390); Gibberella fujikuroi mRNA for GfCPS/KS (GenBank Accession No. BAA84917.1; SEQ ID NO:391); Zea mays kaurene synthase (KS) mRNA (GenBank Accession No. AAD34319.1; SEQ ID NO:392); Mycobacterium tuberculosis sequence (GenBank Accession No. CAA15731.1; SEQ ID NO:393); Pisum sativum ent-kaurene synthase A (LS) mRNA (GenBank Accession No. AAB58822.1; SEQ ID NO:394); Cucurbita maxima ent-kaurene synthase B mRNA (GenBank Accession No. AAB39482.1; SEQ ID NO:395); Zea mays kaurene synthase A (An1) mRNA (GenBank Accession No. AAA73960.1; SEQ ID NO:396).

[0089] In a specific embodiment, a Ginkgo biloba levopimaradiene synthase nucleic acid sequence (SEQ ID NO:397), which encodes the amino acid sequence of SEQ ID NO:398, is utilized for a diterpene synthase in the present invention, wherein the sequences are the subject of a U.S. patent application filed on the same day as this present application and is entitled, “ Ginkgo biloba Levopimaradiene Synthase,” incorporated by reference herein.

[0090] III. Terpenes

[0091] Terpenes are well known in the art, including geraniol or limonene (monoterpenes), farnesol or γ-bisabolene (sesquiterpenes), and squalene or β-amyrin (a triterpene). They are naturally-occurring compounds and are the most abundant components of essential oils of many plants and flowers. Terpenes are extracted from plants and flowers for a variety of purposes by distilling the plants with water. In a specific embodiment, terpenes are biosynthesized from acetyl-CoA (e.g., a derivitized acetate) and isopentenyl pyrophosphate. In one specific embodiment, terpenes are open chain systems or acyclic, such as geraniol, farnesol, geranylgeraniol and citronellal. Other terpenes are monocyclic, such as menthol and zingiberene, although the majority of terpenes are cyclic, such as β-santalol, β-cadinene, matricarin, and copaene. Carotenoids such as β-carotene, a precursor for vitamin A, and lycopene are also terpenes.

[0092] A major class of terpenes includes the sterols. A skilled artisan is aware of many reviews in the field of yeast sterol biosynthesis, such as Parks et al. (1995), Parks and Casey (1995), Paultauf and Kohlwein (1992), and Goldstein and Brown (1990), all of which are incorporated by reference herein in their entirety. The catalytic processes leading to the formation of FPP are commonly referred to as the isoprenoid pathway. The name originates from the isoprene unit (C ₅ ), which subsequent to activation with a pyrophosphate, functions as the building blocks of terpenes. Easily detected by the integral number of C ₅ units in their hydrocarbon skeleton, terpenes (i.e., isoprenoids) contribute to critical physiological roles in the cell, including tRNA modification, ubiquinone and dolichol biosynthesis, protein prenylation, and heme A biosynthesis.

[0093] IV. The upc2-1 Allele

[0094] In one aspect of the present invention, a mechanism that effects sterol metabolic flux is controlled. A nucleic acid sequence that encodes a gene that confers an increase on sterol metabolic flux was provided to a cell of a unicellular organism, and the amount of diterpene and diterpene alcohol was measured to determine the increase in sterol metabolic flux as compared to native sterol metabolic flux levels. The increase observed demonstrated that incorporating such nucleic acid sequences for expression in a resulting recombinant cell improves and enhances diterpene levels produced in vivo.

[0095] The representative example employed herein was a sterol uptake control mutant (upc ⁻ ) that was isolated via ethylmethanesulfonate mutagenesis from wild-type Saccharomyces cerevisiae (Lewis et al., 1998). The sterol uptake control UPC2 allele upc2-1 (SEQ ID NO:399) increases the metabolic flux of sterol biosynthesis. It was originally cloned by calcium sensitivity, and the protein contains a DNA binding motif. The upc2-1 allele confers Erg ⁻ Hem ⁺ prototrophy and is a semi-dominant mutation. The mutation is a point mutation that results in an Asp residue instead of a Gly residue at amino acid 888. The upc2-1 allele (Crowley et al., 1998; Leak et al., 1999; both incorporated by reference in their entirety herein) is utilized in the compositions and methods of the present invention for both overcoming control of sterol importation uptake and increasing sterol biosynthesis (increasing metabolic flux). Another example of a gene that confers such activity is SUT 1 (SEQ ID NO:414; Karst et al., 2001). In another specific embodiment, two separate alleles which confer both phenotypes, or a different single allele which confers both phenotypes, are utilized in lieu of the upc2-1 allele.

[0096] V. HMG-CoA Reductase

[0097] Yeast have two isozymes of HMG-CoA reductase, Hmg1p and Hmg2p, produced from genes on separate chromosomes (Basson et al., 1986), although the vast majority of reductase activity under normal conditions is the result of Hmg1p activity. Null mutations in both genes cause lethality, yet null mutations in either gene alone are viable although survival is reduced (Basson et al., 1987). In a specific embodiment of the present invention, endogenous copies of both HMG1 and HMG2 remain intact in the cell which harbors the recombinant nucleic acid sequence encoding the soluble form of HMG-CoA reductase.

[0098] The cells of the present invention preferably comprise HMG-CoA reductase to improve production of diterpenes and diterpene precursors. HMG-CoA reductase is a rate-limiting enzyme in early sterol biosynthesis in eukaryotic cells. A skilled artisan is aware that increasing significant levels of HMG-CoA reductase in a yeast cell, which is membrane-bound in most organisms, results in generation of extensive membrane structures (Profant et al., 1999) that is detrimental to diterpene and diterpene precursor biosyntheses. Therefore, it is preferred that the form of HMG-CoA reductase utilized in the compositions of the present invention lack sequences responsible or associated with transmembrane domains. These structures are easily identified by standard means in the art, such as commercially available computer programs including Genetics Computer Group® (Madison, Wis.). To eliminate the rate limitation associated with this enzyme in the yeast Saccharomyces cerevisiae , a truncated HMG1 gene producing a form of the enzyme that lacks the membrane-binding region (i.e. amino acids 1-552; SEQ ID NO:400) (Polakowski et al., 1998) was utilized in the preferred embodiments.

[0099] A skilled artisan is aware that there are structurally distinct HMG-CoA reductases depending on the organism. For example, Arabidopsis HMG-CoA reductase lacks the membrane-spanning architecture present in other organisms, yet overexpression of the Arabidopsis nucleic acid sequence encoding HMG-CoA reductase in a yeast mutant suppresses its growth defect, suggesting the sequence is functionally interchangeable between the two organisms (Learned and Fink, 1989; incorporated by reference herein in its entirety). A similar experiment demonstrated restoration of normal growth to a CHO cell line which was HMG-CoA reductase-deficient (Goldstein and Brown, 1990). Thus, a skilled artisan is aware by the methods and design of Learned and Fink (1989) and by methods well known in the art how to test other HMG-CoA reductase sequences for functional complementation of a yeast HMG-CoA reductase defect. A skilled artisan is also aware that although structural differences exist between different organisms, the preferred aspects of the sequence are intracellular solubility and reductase activity. Therefore, in specific embodiments of the present invention, the nucleic acid sequence encoding a HMG-CoA reductase contains a deletion corresponding to an N-terminal sequence.

[0100] It is well known that there are two native S. cerevisiae HMG-CoA reductases, both of which have a N-terminus transmembrane spanning domain (1.6 kb). Thus, in a preferred embodiment, a yeast HMG-CoA reductase lacking at least part of this domain is utilized in the compositions and methods of the present invention.

[0101] VI. Geranylgeranyl Pyrophosphate

[0102] The BTS1 gene in Saccharomyces cerevisiae was cloned as a suppressor of a bet2-1 mutant, which is defective for the β-subunit of the type II geranylgeranyltransferase (Jiang et al., 1990). BTS1 suppresses a growth defect of bet2-1 whether expressed on a low (CEN) or multiple (2 um) copy vector. Furthermore, the BTS1 gene product demonstrates functional activity of a geranylgeranyl pyrophosphate (GGPP) synthase, such as functionally substituting for a bacterial GGPP synthase. The BET2 gene product is important for geranylgeranylation of a multitude of proteins in a variety of cellular processes, such as small GTP-binding proteins of the Ras superfamily and nuclear lamins. Up to 0.5% of cellular proteins are estimated to be prenylated which increases hydrophobicity and permits protein association with cellular membranes. Geranylgeranylation occurs from covalent attachment of all-trans geranylgeranyl diphosphate to proteins comprising terminal cysteines within CAAL, CC, or CXC sequence motifs. GGPP biosynthesis is critical for cell viability, and modifications in the prenylation pathway are contemplated to preferably include a reduced rate of GGPP consumption wherein the reduced rate is sufficient to maintain integrity of cellular homeostasis.

[0103] VII. Nucleic Acid-Based Expression Systems

[0104] A. Vectors

[0105] The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference.

[0106] The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.

[0107] 1. Promoters and Enhancers

[0108] A “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0109] A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0110] Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (1989), incorporated herein by reference. The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

[0111] Table 1 lists several elements/promoters that may be employed, in the context of the present invention, to regulate the expression of a gene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of expression but, merely, to be exemplary thereof. Table 2 provides examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus. 1