WO2006079020A2 - Identification fonctionnelle du gene de muticus hyoscyamus codant pour l'activite de la premnaspirodiene hydroxylase - Google Patents

Identification fonctionnelle du gene de muticus hyoscyamus codant pour l'activite de la premnaspirodiene hydroxylase Download PDF

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WO2006079020A2
WO2006079020A2 PCT/US2006/002265 US2006002265W WO2006079020A2 WO 2006079020 A2 WO2006079020 A2 WO 2006079020A2 US 2006002265 W US2006002265 W US 2006002265W WO 2006079020 A2 WO2006079020 A2 WO 2006079020A2
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protein
isolated
host cell
terpene
amino acid
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PCT/US2006/002265
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WO2006079020A3 (fr
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Joe Chappell
Yunsoo Yeo
Shunji Takahashji
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University Of Kentucky Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)

Definitions

  • the present invention relates to the functional identification of a gene which encodes for an enzyme that catalyzes the regio-and stereo-specific hydroxylation of sesquiterpene scaffolds. More particularly, the present invention discloses the DNA sequence for the Hyoscyamus muticus premnaspirodiene synthase gene, HPO, which when expressed in a heterologous host such as yeast, provides an enzyme activity that catalyzes mono-and successive hydroxylation of premnaspirodiene and other sesquiterpene scaffolds.
  • HPO Hyoscyamus muticus premnaspirodiene synthase gene
  • Terpenes are a diverse family of compounds with carbon skeletons composed of five-carbon isoprene units. Approximately 20,000 different terpenes and terpenoids (compounds of terpene origin whose carbon skeleton has been altered or rearranged) have been identified to date, representing only a small fraction of the estimated natural variation. Terpenes are commonly isolated from the essential oils of plants. Essential oils often have pleasant tastes or aromas, and they are widely used as flavorings, deodorants, and medicines. [0004] Sesquiterpenes are terpenes with 15 carbon atoms (three isoprene units). The plant kingdom contains the highest diversity of sesquiterpenes. Often they play a role in defense of the plants against pathogens, insects and herbivores and for attraction of pollinating insects.
  • Valencene (1 ,2,3,5,6,7,8,8a-octahydro-7-isopropenyl-1 ,8a- dimethyl-naphthalene) and nootkatone (4,4a,5,6,7,8-hexahydro-6-isopropenyl- 4,4a-dimethyl-2(3H)-naphtalenone) are just two examples of sesquiterpenes that are derived from cyclization of the ubiquitous pyrophosphate intermediate farnesyl diphosphate. Nootkatone is formed by the oxidation of valencene.
  • Valencene and nootkatone are compounds of natural origin, and are natural constituents of citrus oils, such as orange and grapefruit. Because of its excellent organoleptic qualities and in particular its typical grapefruit taste, nootkatone is a widely used ingredient in perfumery and the flavor industry. Alternatively, nootkatone may be used as an insecticide. Valencene, the starting material for the generation of nootkatone (either biologically or chemically), is also used as a flavorant and fragrance.
  • Nootkatone is a high demand, high value flavorant added to many of the commercial soft drinks sold worldwide.
  • the practice of extracting nootkatone from citrus pulp and rind is considered an expensive and somewhat unreliable process.
  • Nootkatone can be synthesized by the oxidation of valencene.
  • the valencene starting material is expensive and is easily degraded during evaporative heat concentration processes typically used to remove the bulk of water from the feed juice.
  • current methods to purify valencene from citrus fruits are costly, difficult, and are limited by what the fruit can deliver.
  • such methods are vulnerable to interruptions in the supply of citrus fruits, which is dependent on the weather.
  • HPO gene of the present invention now provides an alternative means of generating important stereochemical ⁇ pure starting materials for the reliable and cost effective production of nootkatone and other high value sesquiterpenes.
  • the co-expression of a valencene synthase gene along with the HPO gene in transgenic plants or microbial cells provides for large scale production of nootkatone.
  • other terpene synthase genes can be used in combination with the HPO gene to generate other novel terpene moieties, which could be of value for pharmaceutical, agricultural and other industrial applications.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a functional Hyoscyamus mutic ⁇ s premnaspirodiene oxidase protein such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone.
  • the present invention relates to an isolated and purified DNA sequence wherein the sequence is:
  • the present invention relates to an isolated and purified DNA sequence wherein the sequence encodes a protein of the sequence:
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1 , provided that the nucleic acid sequence is translated into a protein encoding a functional Hyoscyamus muticus premnaspirodiene synthase protein such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is a nucleic acid sequence that is at least 97.5% identical to SEQ ID NO: 1.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 1.
  • the present invention relates to an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the DNA sequence is selected from the group consisting of:
  • the present invention relates to an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the DNA sequence is selected from the group consisting of:
  • the present invention relates to an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the DNA sequence is selected from the group consisting of:
  • the present invention relates to an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the DNA sequence is selected from the group consisting of:
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 480 is changed from valine to serine.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 482 is changed from valine to isoleucine.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 484 is changed from alanine to isoleucine.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 366 is changed from valine to serine.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein such that the protein has a catalytic activity of successfully hydroxylating valencene at C2 first to nootkatol and then to nootkatone.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is SEQ ID NO: 1.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence encodes a protein of the sequence SEQ ID NO: 2.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is at least 97.5% identical to SEQ ID NO: 1.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is at least 99% identical to SEQ ID NO: 1.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of: (a) SEQ ID NO: 1 ; and (b) a DNA sequence encoding a protein differing by from one to 20 conservative amino acid substitutions from SEQ ID NO: 2, wherein the protein encoded is a functional Hyoscyamus muticus premnaspirodiene synthase protein such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone and wherein a conservative amino acid substitution is one of the following substitutions: Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; G
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 480 is changed from valine to serine.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 482 is changed from valine to isoleucine.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 484 is changed from alanine to isoleucine.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 366 is changed from valine to serine.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein such that the protein has a catalytic activity of successfully hydroxylating valencene at C2 first to nootkatol and then to nootkatone.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is SEQ ID NO: 1.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence encodes a protein of the sequence SEQ ID NO: 2.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is at least 97.5% identical to SEQ ID NO: 1.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is at least 99% identical to SEQ ID NO: 1.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 480 is changed from valine to serine.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 482 is changed from valine to isoleucine.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 484 is changed from alanine to isoleucine.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 366 is changed from valine to serine.
  • the present invention relates to a method of producing an isolated protein having Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone comprising the steps of:
  • the present invention relates to a method of producing an isolated protein having Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone comprising the steps of:
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone.
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone, wherein the protein has the amino acid sequence of SEQ ID NO: 2.
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone, wherein the protein ahs an amino acid sequence selected from the group consisting of:
  • a conservative amino acid substitution is one of the following substitutions: Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone, wherein the protein ahs an amino acid sequence selected from the group consisting of:
  • a conservative amino acid substitution is one of the following substitutions: Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; G
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone, wherein the protein ahs an amino acid sequence selected from the group consisting of:
  • a conservative amino acid substitution is one of the following substitutions: Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; G
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone, wherein the protein ahs an amino acid sequence selected from the group consisting of:
  • a conservative amino acid substitution is one of the following substitutions: Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; G
  • the present invention relates to an isolated and purified protein molecule having a functional terpene hydroxylase activity wherein the protein molecule has the sequence of SEQ ID NO: 2 wherein residue 480 is changed from valine to serine.
  • the present invention relates to a method of producing nootkatone comprising the step of reacting the isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity with valencene under conditions in which the protein molecule catalyzes the successive oxidation of the valencene first to nootkatol and then to nootkatone.
  • the present invention relates to a method of producing nootkatone comprising the step of reacting the isolated and purified protein molecule of SEQ ID NO: 2 with valencene under conditions in which the protein molecule catalyzes the successive oxidation of the valencene first to nootkatol and then to nootkatone.
  • the present invention relates to a method of producing nootkatone comprising the step of reacting the isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity with valencene under conditions in which the protein molecule catalyzes the successive oxidation of the valencene first to nootkatol and then to nootkatone, wherein the isolated and purified protein molecule has an amino acid sequence selected from the group consisting of:
  • a conservative amino acid substitution is one of the following substitutions: Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; G
  • the present invention relates to a yeast cell transformed or transfected with:
  • a first vector including therein a DNA molecule encoding functional Hyoscyamus muticus premnaspirodiene oxidase protein such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone;
  • a third vector including therein a DNA molecule encoding a functional Hyoscyamus muticus premnaspirodiene protein synthase; such that the yeast cell expresses: (1 ) the functional Hyoscyamus muticus premnaspirodiene oxidase protein in a quantity sufficient to hydroxylate valencene; (2) the functional P450 reductase protein in a quantity sufficient to supply reducing equivalents for the Hyoscyamus muticus premnaspirodiene oxidase protein; and (3) the functional Hyoscyamus muticus premnaspirodiene protein synthase in a quantity sufficient to produce premnaspirodiene; and such that the premnaspirodiene is converted by the cell to solavetivone.
  • the present invention relates to a method of producing an oxidized terpene from unoxidized terpene substrate comprising the steps of:
  • the present invention relates to a method of producing an oxidized terpene from an unoxidized terpene substrate comprising the steps of: (a) providing the unoxidized terpene substrate to the host cell transformed or transfected with a vector comprising isolated and purified DNA sequence of SEQ ID NO: 1 ;
  • the present invention relates to a method of producing an oxidized terpene from an unoxidized terpene substrate comprising the steps of:
  • the present invention relates to a method of producing an oxidized terpene from an unoxidized terpene substrate comprising the steps of:
  • the present invention relates to a method of producing an oxidized terpene from an unoxidized terpene substrate comprising the steps of:
  • the present invention relates to a method of producing an oxidized terpene from an unoxidized terpene substrate comprising the steps of:
  • the present invention relates to a method for producing a mutein of Hyoscyamus mutic ⁇ s premnaspirodiene oxidase with at least one altered property selected from the group consisting of regiospecificity and stereospecificity comprising the steps of:
  • Figure 1 illustrates biosynthetic transformations catalyzed by the Hyoscyamus muticus premnaspirodiene synthase, showing that the HPO enzyme catalyzes the successive hydroxylation of sesquiterpene scaffolds, thus generating a first mono-hydroxylated form, followed by the subsequent ketone form.
  • Figure 2 illustrates that the PCR primers which were used to isolate the HPO gene were initially designed relative to the EAH gene (71 D20, GenBank accession number AF368376), and subsequently to other 71 D family members whose expression are inducible by biological and abiotic stress (71 D4, accession # AJ296346; 71 D6, U48434; 71 D7, U48435; 71 D16, AF166332).
  • Figure 3 illustrates the results of GC-MS analysis of reaction products generated by in vitro assays with microsomes from yeast over-expressing the HPO gene and incubated with premnaspirodiene as substrate (total ion chromatogram).
  • Figure 4 illustrates the results of GC-MS analysis of reaction products generated by in vitro assays with microsomes from yeast over- expressing the HPO gene and incubated with valencene as substrate. Total ion chromatogram.
  • Figure 6 shows that site-directed mutagenesis was used to alter the DNA sequence of the HPO gene corresponding to those codons for the indicated amino acids, the mutant genes over-expressed in yeast and isolated microsomes used to biochemically characterize the mutant enzyme activities.
  • Figure 7 provides a cartoon depiction of the engineering of a yeast line to effect sesquiterpene hydrocarbon and oxygenated terpene biosynthesis (A) and an ethyl acetate extract of P450 reductase, HPS and HPO engineered yeast cells assessed by GC-MS (B).
  • Figure 8 illustrates a sequence alignment of amino acids lining the active site (1st tier) and those within 3 A of the active site residues (2nd tier) of HPH (SEQ ID NO: 2) with the corresponding positions of TEAH (SEQ ID NO: 19).
  • Figure 9 illustrates the mechanisms of reactions catalyzed by HPO relative to EAH, wherein the differing font size visually depicts relative catalytic rates.
  • Figure 10 illustrates the CO difference spectrum for HPO expression in yeast, wherein the absolute absorbance value can be used to calculate the absolute level of the HPO protein in the microsome preparation.
  • Figure 11 illustrates the reactions carried out with the substrate premnaspirodiene, indicating successive hydroxylation to 4 ⁇ -solavetivol and then to solavetivone.
  • Figure 12 illustrates the reactions carried out with the substrate valencene, indicating successive hydroxylation to ⁇ -nootkatol and then to nootkatone.
  • Figure 13 illustrates the reaction carried out with valencene with a reaction time of 1 minute.
  • Figure 14 illustrates the reaction carried out with 5-epi- aristolochene with a reaction time of 0, 1 , 2, 5 or 10 minutes.
  • Figure 15 illustrates the reaction carried out with 4- epieremophilene and its double-bond isomer with a reaction time of 5 minutes (upper panel), as well as the reaction with ⁇ -cedrene with a reaction time of 5 minutes (lower panel).
  • Figure 16 is a table showing a comparison of enzyme kinetics of HPO for various substrates relative to the previously characterized EAH (5-epi- aristolochene dihydroxylase) hydroxylases. K m , k oa t, and k ca t/Km are shown.
  • Figure 17 is a schematic depiction of a method for converting HPO into an enzyme with the same activity as EAH.
  • Figure 18 shows the EAH activity of a number of muteins, including those produced by domain swapping between EAH and HPO, and those produced by domain swapping between EAH and HPO with additional mutations in the HPO domain, including: V482I/A484I (a double mutation); V366S/V482I/A484I (a triple mutation); G280T/G281S/V366S ⁇ /482I/A484I (a quintuple mutation); I294V/F296V/V366S/V482I/A484I (a quintuple mutation); I294V/F296V/V366S/V482I/A484I (a septuple mutation); G280T/G281S/V366S ⁇ /480S/V482I (a quintuple mutation); I294V/F296V/366S/V480S/V482I (a quintuple mutation; and I294V/F296
  • Figure 19 shows the general strategy of using domain-swapping mutations based on substrate recognition sequences (SRS) 1 reciprocal site- directed mutagenesis based on homology modeling with mammalian P450s, and a combination of domain-swapping and site-directed mutagenesis.
  • SRS substrate recognition sequences
  • Figure 20 shows the results of homology modeling and site- directed mutagenesis in generating muteins of HPO indicated by the amino acid in the native (wildtype) enzyme, the amino acid position, then the mutant amino acid.
  • V366S indicates that the valine in position 366 of the wildtype enzyme has been mutated to serine.
  • Mutants affecting SRS 4, 5, and 6 include: S308T/V366S/V480S/V482l/A484l; S308T/V366S/V480S/A484l; S308T/V366S/V482l/A484l; S308T/V366S/V480S ⁇ /482l/; S308T/V366S/A484l;S308T/V366S/V482l; and S308T ⁇ /366S/V480S.
  • Mutants affecting SRS 4 and 5 include S308T7V366S.
  • Mutants affecting SRS 5 and 6 include: V366S/V480S ⁇ /482I/A484I; V366S/V480S/A484I; V366S/V482I/A484I (designated the M3 mutant); V366S/V480S/V482I; V366S/A484I; and V366S/V480S.
  • Mutants affecting SRS 5 include: V366S.
  • Mutants affecting SRS6 include: V480S/V482I/A484I; V482I/A484I; V480S/A484I; V480S/V482I; A484I; V482I; and V480S. The 5-epiaristolochene hydroxylase activity of these enzymes is shown.
  • Figure 21 is similar to Figure 20, but depicts changes in SRS 1 and/or SRS 2 based on M3 ( Figure 20). Mutants include: L52E/M3, L52E/G209E/M3, G209E/M3, C119S/M3, R113Q/M3, V109E/M3, E107D/C119S/M3, E107D/M3, P106M/C119S/M3, P106M/M3, L104V/C119S/M3, L104V/P106M/E107D/M3, L104V/M3,
  • the 5-epiaristolochene hydroxylase activity of these enzymes is shown.
  • Figure 22 shows the results from a combination of domain- swapping mutations and site-directed mutagenesis; the mutation of V366S greatly diminishes the 5-epiaristolochene hydroxylase activity.
  • FIG 23 shows additional results from site-directed mutagenesis in SRS 4 as well as domain swapping. Mutants include: G280T/G281S/I294V/F296V/M3, I294V/F296V/M3, G280T/G281S/M3, V482I/A484I; V366S/V482I/A484I, and I294V/F296V/V366S/A482I/A484I. EAH activity is shown in terms of both 5-epiaristolochene hydroxylase activity and 1 ⁇ (OH)EA hydroxylase activity.
  • substantially identical means that a relevant sequence is at least 70%, 75%, 80%, 85%, 90%, 92%, 95% 96%, 97%, 98%, or 99% identical to a given sequence.
  • sequences may be allelic variants, sequences derived from various species, or they may be derived from the given sequence by truncation, deletion, amino acid substitution or addition. Percent identity between two sequences is determined by standard alignment algorithms such as ClustalX when the two sequences are in best alignment according to the alignment algorithm.
  • hybridization or “hybridizes” under certain conditions is intended to describe conditions for hybridization and washes under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other.
  • Appropriate hybridization conditions can be selected by those skilled in the art with minimal experimentation as exemplified in Ausubel, F. A., et al., eds., Current Protocols in Molecular Biology Vol. 2, John Wiley and Sons, Inc., New York (1995). Additionally, stringency conditions are described in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989). Variations on the conditions for low, moderate, and high stringency are well known in the art and may be used with the current invention.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide oligonucleotide or polynucleotide, including single- or double- stranded forms, and coding or non-coding (e.g., "antisense") forms.
  • the term encompasses nucleic acids containing known analogues of natural nucleotides.
  • the term also encompasses nucleic acids including modified or substituted bases as long as the modified or substituted bases interfere neither with the Watson-Crick binding of complementary nucleotides or with the binding of the nucleotide sequence by proteins that bind specifically.
  • the term also encompasses nucleic-acid-like structures with synthetic backbones.
  • DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N- carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991 ); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem.
  • PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described, e.g., by U.S. Pat. Nos. 6,031 ,092; 6,001 ,982; 5,684,148; see also, WO 97/03211 ; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197.
  • Other synthetic backbones encompassed by the term include methylphosphonate linkages or alternating methylphosphonate and phosphodiester linkages (see, e.g., U.S. Pat. No.
  • nucleotide sequence or molecule may also be referred to as a "nucleotide probe.”
  • nucleic acid molecules of the invention are derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequence by standard biochemical methods. Examples of such methods, including methods for PCR protocols that may be used herein, are disclosed in Sambrook et al.
  • nucleic acid molecule also includes its complement as determined by the standard Watson- Crick base-pairing rules, with uracil (U) in RNA replacing thymine (T) in DNA, unless the complement is specifically excluded.
  • amino acids which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three-letter or one-letter abbreviations.
  • nucleotides which occur in the various DNA fragments, are designated with the standard single-letter designations used routinely in the art.
  • the nucleic acid molecules of the invention include DNA in both single-stranded and double-stranded form, as well as the DNA or RNA complement thereof.
  • DNA includes, for example, DNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof.
  • Genomic DNA, including translated, non-translated and control regions, may be isolated by conventional techniques, e.g., using any one of the cDNAs of the invention, or suitable fragments thereof, as a probe, to identify a piece of genomic DNA which can then be cloned using methods commonly known in the art.
  • nucleic acids of the invention are encompassed by the invention.
  • reference to a nucleic acid "encoding" a protein or polypeptide encompasses not only cDNAs and other intronless nucleic acids, but also DNAs, such as genomic DNA, with introns, on the assumption that the introns included have appropriate splice donor and acceptor sites that will ensure that the introns are spliced out of the corresponding transcript when the transcript is processed in a eukaryotic cell. Due to the degeneracy of the genetic code wherein more than one codon can encode the same amino acid, multiple DNA sequences can code for the same polypeptide.
  • Such variant DNA sequences can result from genetic drift or artificial manipulation (e.g., occurring during PCR amplification or as the product of deliberate mutagenesis of a native sequence).
  • Deliberate mutagenesis of a native sequence can be carried out using numerous techniques well known in the art. For example, oligonucleotide-directed site-specific mutagenesis procedures can be employed, particularly where it is desired to mutate a gene such that predetermined restriction nucleotides or codons are altered by substitution, deletion or insertion. Exemplary methods of making such alterations are disclosed by Walder et al. (Gene 42:133,1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 12-19, 1985); Smith et al.
  • the present invention thus encompasses any nucleic acid capable of encoding a protein of the current invention.
  • DNA sequences encoding the polypeptides or proteins of the invention can be obtained by several methods.
  • the DNA can be isolated using hybridization procedures that are well known in the art. These include, but are not limited to: (1) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences; (2) antibody screening of expression libraries to detect shared structural features; and (3) synthesis by the polymerase chain reaction (PCR).
  • RNA sequences of the invention can be obtained by methods known in the art (See, for example, Current Protocols in Molecular Biology, Ausubel, et al., Eds., 1989).
  • DNA sequences encoding proteins or polypeptides of the invention can be obtained by: (1 ) isolation of a double- stranded DNA sequence from the genomic DNA; (2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and (3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
  • the isolation of genomic DNA is the least common.
  • nucleotide sequences that are within the scope of the invention, all nucleotide sequences encoding the proteins or polypeptides that are embodiments of the invention as described are included in nucleotide sequences that are within the scope of the invention. This further includes all nucleotide sequences that encode polypeptides according to the invention that incorporate conservative amino acid substitutions as defined above. This further includes nucleotide sequences that encode larger proteins incorporating the proteins or polypeptides, including fusion proteins, and proteins that incorporate amino-terminal or carboxyl-terminal flanking sequences.
  • Nucleic acid sequences of the present invention further include nucleic acid sequences that are at least 95% identical to the sequences above, with the proviso that the nucleic acid sequences retain the activity of the sequences before substitutions of bases are made, including any activity of proteins that are encoded by the nucleotide sequences and any activity of the nucleotide sequences that is expressed at the nucleic acid level, such as the binding sites for proteins affecting transcription.
  • the nucleic acid sequences are at least 97.5% identical. More preferably, they are at least 99% identical.
  • “identity” is defined according to the Needleman- Wunsch algorithm (S.B. Needleman & CD. Wunsch, "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins," J. MoI. Biol. 48: 443-453 (1970)).
  • polypeptides refers to a genus of polypeptide or peptide fragments that encompass the amino acid sequences identified herein, as well as smaller fragments.
  • a polypeptide may be defined in terms of its antigenic relatedness to any peptide encoded by the nucleic acid sequences of the invention.
  • a polypeptide within the scope of the invention is defined as an amino acid sequence comprising a linear or 3- dimensional epitope shared with any peptide encoded by the nucleic acid sequences of the invention.
  • a polypeptide within the scope of the invention is recognized by an antibody that specifically recognizes any peptide encoded by the nucleic acid sequences of the invention.
  • Antibodies are defined to be specifically binding if they bind polypeptides of the invention with a K a of greater than or equal to about 10 7 M "1 , such as greater than or equal to 10 8 M "1 .
  • the term "isolated,” in reference to polypeptides or proteins, means that the polypeptide or protein is substantially removed from polypeptides, proteins, nucleic acids, or other macromolecules with which it, or its analogues, occurs in nature.
  • a polypeptide "variant” as referred to herein means a polypeptide substantially homologous to a native polypeptide, but which has an amino acid sequence different from that encoded by any of the nucleic acid sequences of the invention because of one or more deletions, insertions or substitutions. Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. See Zubay, Biochemistry, Addison-Wesley Pub. Co., (1983).
  • suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule.
  • Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g. Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, Benjamin/Cummings, p. 224).
  • such a conservative variant has a modified amino acid sequence, such that the change(s) do not substantially alter the protein's (the conservative variant's) structure and/or activity, e.g., antibody activity, enzymatic activity, or receptor activity.
  • amino acid sequence i.e., amino acid substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity.
  • amino acids having similar properties e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.
  • one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; Gly/Ala or Pro; His/Asn or GIn; lie/Leu or VaI; Leu/lle or VaI; Lys/Arg or GIn or GIu; Met/Leu or Tyr or lie; Phe/Met or Leu or Tyr; Ser/Thr; Thr/Ser; Trp/Tyr; Tyr/Trp or Phe; Val/lle or Leu.
  • An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: (1 ) alanine (A or Ala), serine (S or Ser), threonine (T orThr); (2) aspartic acid (D or Asp), glutamic acid (E or GIu); (3) asparagine (N or Asn), glutamine (Q or GIn); (4) arginine (R or Arg), lysine (K or Lys); (5) isoleucine (I or lie), leucine (L or Leu), methionine (M or Met), valine (V or VaI); and (6) phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp); (see also, e.g., Creighton (1984) Proteins, W.
  • substitutions are not the only possible conservative substitutions. For example, for some purposes, one may regard all charged amino acids as conservative substitutions for each other whether they are positive or negative.
  • individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered "conservatively modified variations" when the three-dimensional structure and the function of the protein to be delivered are conserved by such a variation.
  • substitution score matrices such PAM 120, PAM-200, and PAM-250 as discussed in Altschul, (J. MoI. Biol. 219:55565 (1991 )).
  • Other such conservative substitutions for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • Naturally-occurring peptide variants are also encompassed by the invention.
  • examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the polypeptides described herein.
  • Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptides encoded by the sequences of the invention.
  • Variants of the valencene synthase of the invention may be used to attain desired enhanced or reduced enzymatic activity, modified regiochemistry or stereochemistry, or altered substrate utilization or product distribution.
  • a variant or site direct mutant may be made by any methods known in the art.
  • Variants and derivatives of native polypeptides can be obtained by isolating naturally-occurring variants, or the nucleotide sequence of variants, of other or same plant lines or species, or by artificially programming mutations of nucleotide sequences coding for native citrus polypeptides. Methods for site- directed mutagenesis are well-known in the art and include, but are not limited to, the methods described in J. Sambrook & D.W. Russell, "Molecular Cloning: A Laboratory Manual (3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001), ch. 13, incorporated herein by this reference.
  • the invention contemplates: vectors comprising the nucleic acids of the invention.
  • vector refers to a plasmid, virus, phagemid, or other vehicle known in the art that has been manipulated by insertion or incorporation of heterologous DNA, such as nucleic acid encoding proteins or polypeptides according to the present invention.
  • Vectors include, but are not limited to, expression vectors; vectors suitable for other purposes that are not expression vectors are known in the art.
  • Such expression vectors typically contain a promoter sequence for efficient transcription of the inserted nucleic acid in a cell.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes that permit phenotypic selection of transformed cells.
  • Expression vectors containing a nucleic acid sequence of the invention can be prepared using well known methods and include a cDNA sequence encoding the polypeptide operably linked to suitable transcriptional or translational regulatory nucleotide sequences.
  • suitable transcriptional or translational regulatory nucleotide sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation initiation and termination.
  • Nucleotide sequences are "operably linked" when the regulatory sequence functionally relates to the cDNA sequence of the invention. Expression vectors, regulatory elements and the construction thereof are well known in the art, and therefore are not limited to those recited above.
  • sequences encoding appropriate signal peptides that are not naturally associated with the polypeptides of the invention can be incorporated into expression vectors.
  • a DNA sequence for a signal peptide (secretory) leader can be fused in-frame to a nucleotide sequence of the invention so that the polypeptide of the invention is initially translated as a fusion protein comprising the signal peptide.
  • a signal peptide that is functional in the intended host cells enhances extracellular secretion of the expressed polypeptide.
  • the signal peptide can be cleaved from the polypeptide upon secretion from the cell. In some cases, signal peptides are cleaved in two or more stages; this is also within the scope of the invention where appropriate.
  • Fusions of additional peptide sequences at the amino and carboxyl terminal ends of the polypeptides of the invention can be used with the current invention.
  • the invention includes a host cell comprising a nucleic acid of the invention.
  • the term "host cell” refers to a cell in which a vector can be propagated and its DNA expressed.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Such progeny are included when the term "host cell” is used. Methods of stable transfer where the foreign DNA is continuously maintained in the host are known in the art.
  • Another embodiment of the invention is a method of making a recombinant host cell comprising introducing the vectors of the invention, into a host cell.
  • a method of producing a polypeptide comprising culturing the host cells of the invention under conditions to produce the polypeptide is contemplated. In one embodiment the polypeptide is recovered.
  • Suitable host cells for expression of polypeptides of the invention are well known in the art, and include, but are not limited to, prokaryotes, yeast, higher eukaryotic cells, or combinations thereof. (See for example, Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York (1985)).
  • a particularly preferred host cell is yeast.
  • Cell-free translation systems also well known in the art, could also be employed to produce the disclosed polypeptides using RNAs derived from DNA constructs disclosed herein.
  • Host cells may be modified by any methods known in the art for gene transfer including, for example, the use of delivery devices such as lipids and viral vectors, naked DNA, electroporation and particle-mediated gene transfer.
  • delivery devices such as lipids and viral vectors, naked DNA, electroporation and particle-mediated gene transfer.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as Escherichia coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCI 2 method by procedures well known in the art.
  • CaCI 2 or RbCI can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
  • the host is a eukaryote
  • methods of transfection of DNA as calcium phosphate co-precipitates conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used.
  • a variety of host-expression vector systems may be utilized to express proteins or polypeptides according to the present invention. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an appropriate coding sequence; yeast transformed with recombinant yeast expression vectors containing the appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus) containing an appropriate coding sequence, or
  • any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter, et al., Methods in Enzymology, 153:516-544, 1987).
  • inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
  • promoters derived from the genome of mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted polypeptide coding sequence.
  • yeast such as Saccharomyces cerevisiae
  • vectors containing constitutive or inducible promoters may be used.
  • vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome. Cloning and expression in yeast is further described in T.A. Brown, "Gene Cloning and DNA Analysis” (4 th ed., Blackwell, 2001 ), pp. 286-288. Other species of yeast such as Pichia pastoris can be used.
  • the expression of a polypeptide coding sequence may be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson, et al., Nature, 310:511-514, 1984), or the coat protein promoter to TMV (Takamatsu, et al., EMBO J., 6:307-311 , 1987) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi, et al., EMBO J.
  • An alternative expression system that can be used to express a protein of the invention is an insect system.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the polypeptide coding sequence may be cloned into non-essential regions (in Spodoptera frugiperda, for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • telomeres For long-term, high-yield production of recombinant proteins, stable expression is preferred. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the a cDNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • engineered cells may be allowed to grow for 1-2 days in enriched media, and then are switched to a selective media.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., Cell 11 :223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci.
  • adenine phosphoribosyltransferase genes which can be employed in tk “ , hgprt " or aprt " cells respectively.
  • antimetabolite resistance-conferring genes can be used as the basis of selection; for example, the genes for dhfr, which confer resistance to methotrexate (Wigler, et al., Natl. Acad. Sci. USA,77:3567, 1980; O'Hare, et al., Proc. Natl. Acad. Sci.
  • gpt which confers resistance to mycophenolic acid
  • neo which confers resistance to the aminoglycoside G418
  • hygro which confers resistance to hygromycin
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC ornithine decarboxylase
  • DFMO 2-(difluoromethyl)-DL-omithine
  • Isolation and purification of protein expressed in mammalian, plant, yeast, or bacterial cells may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
  • Antibodies provided in the present invention are immunoreactive with a polypeptide according to the present invention.
  • Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.
  • Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).
  • the cDNAs of the invention may be expressed in such a way as to produce either sense or antisense RNA.
  • the expression of antisense RNA can be used to down-modulate the expression of the protein encoded by the mRNA to which the antisense RNA is complementary.
  • a further embodiment of the invention is methods of making terpenoids and sesquiterpene compounds, for example, using the nucleotides and polypeptides of the invention.
  • an acyclic pyrophosphate terpene precursor is any acyclic pyrophosphate compound that is a precursor to the production of at least one terpene including but not limited to geranyl-pyrophosphate (GPP), farnesyl- diphosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP).
  • GPP geranyl-pyrophosphate
  • FPP farnesyl- diphosphate
  • GGPP geranylgeranyl-pyrophosphate
  • an organism e.g., microorganism or plant
  • a platform for high level production of a substrate of sesquiterpene synthases e.g., FPP
  • FPP sesquiterpene synthases
  • nucleic acids of the invention that are DNA encompass both cDNA (DNA reverse transcribed from mRNA and lacking introns) and isolated genomic DNA (DNA that can contain introns.)
  • the nucleic acids of the invention are used to create other nucleic acids coding for sesquiterpene synthases.
  • the invention provides for a method of identifying a sesquiterpene synthases comprising constructing a DNA library using the nucleic acids of the invention, screening the library for nucleic acids which encode for at least one sesquiterpene synthase.
  • the DNA library using the nucleic acids of the invention may be constructed by any process known in the art where DNA sequences are created using the nucleic acids of the invention as a starting point, including but not limited to DNA shuffling.
  • the library may be screened for sesquiterpene synthases using a functional assay to find a target nucleic acid that encodes a sesquiterpene synthase.
  • the activity of a sesquiterpene synthase may be analyzed using, for example, the methods described herein. In one embodiment, high through put screening is utilized to analyze the activity of the encoded polypeptides.
  • nucleotide probe is defined as an oligonucleotide or polynucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, through complementary base pairing, or through hydrogen bond formation.
  • a "target nucleic acid” herein refers to a nucleic acid to which the nucleotide probe or molecule can specifically hybridize.
  • the probe is designed to determine the presence or absence of the target nucleic acid, and the amount of target nucleic acid.
  • the target nucleic acid has a sequence that is significantly complementary to the nucleic acid sequence of the corresponding probe directed to the target so that the probe and the target nucleic acid can hybridize.
  • the hybridization conditions are such that hybridization of the probe is specific for the target nucleic acid.
  • the probe may also contain additional nucleic acids or other moieties, such as labels, which may not specifically hybridize to the target.
  • the term target nucleic acid may refer to the specific nucleotide sequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA).
  • the term target nucleic acid may refer to the specific nucleotide sequence of a larger nucleic acid to which the probe is directed
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone.
  • the nucleic acid sequence is typically DNA.
  • the DNA encodes a protein of the sequence
  • the DNA sequence can alternatively encode a protein including SEQ ID NO: 2 therein, such as a protein that has additional amino-terminal or carboxyl-terminal flanking sequences, or a fusion protein coupling the amino acid sequence of SEQ ID NO: 2 with one or more additional domains.
  • SEQ ID NO: 1 A particularly preferred DNA sequence is SEQ ID NO: 1 , below.
  • the present invention relates to an isolated and purified DNA sequence wherein the sequence is: ATGCAATTCTTCAGCTTGGTTTCCATCTTCCTTTTTCTATCTTTTTTGTTTTTG TTAAGGAAATGGAAGAACTCCAATAGCCAGTCCAAGAAATTGCCTCCAGGT CCATGGAAACTTCCATTACTAGGTAGCATGCTTCATATGGTTGGTGGACTTC CACATCATGTACTTAGAGATTTAGCAAAAAAATATGGACCACTTATGCATCTT CAACTTGGTGAAGTTTCTGCTGTTGTTGTTACTTCTCCTGATATGGCAAAAG AAGTACTAAAAACTCATGACATTGCGTTCGCGTCTAGGCCTAAACTTTTAGC CCCAGATTGTATGTTACAACAGGTCTGACATTGCGTTTTGCCCTTATGGT GATTACTGGAGACAAATGCGTAAAATTTGTCTTGGAAGTGTTGAGTGCCA AGAATGTTAGGTCATTCAGCTCTCTTTCGTCTAGTGGAGACAACAGGTCTGACATTGCGTTTTGCCCTTATGGT
  • the present invention relates to an isolated and purified DNA sequence wherein the sequence encodes a protein of the sequence:
  • LMLVATPYQPSRE SEQ ID NO: 2.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1 , provided that the nucleic acid sequence is translated into a protein encoding a functional Hyoscyamus muticus premnaspirodiene synthase protein such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is a nucleic acid sequence that is at least 97.5% identical to SEQ ID NO: 1.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 1.
  • the present invention relates to an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the DNA sequence is selected from the group consisting of:
  • the present invention relates to an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the DNA sequence is selected from the group consisting of:
  • the present invention relates to an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the DNA sequence is selected from the group consisting of:
  • the present invention relates to an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the DNA sequence is selected from the group consisting of:
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 480 is changed from valine to serine, designated herein as V480S.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 482 is changed from valine to isoleucine, designated herein as V482I.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 484 is changed from alanine to isoleucine, designated herein as A484I.
  • the present invention relates to an isolated and purified nucleic acid sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 366 is changed from valine to serine, designated herein as V366S.
  • mutated proteins that are within the scope of the present invention are designated and described by reciting first the wild-type amino acid, the position at which the mutation occurs, then the mutated amino acid introduced, such as V366S, described above.
  • Mutated nucleic acid sequences and proteins encoded by the mutated nucleic acid sequences that are within the scope of the invention include multiple mutations that include more than one of the mutations described above or additional mutations that are described more particularly in Figure 5. Up to at least seven mutations are possible in a single mutein.
  • mutated nucleic acid sequences and proteins encoded by the mutated nucleic acid sequences are within the scope of the invention and are described more particularly in Example 5. These include the following: V482I/A484I (a double mutation); V366S/V482I/A484I (a triple mutation); G280T/G281S/V366S/V482I/A484I (a quintuple mutation); I294V/F296V/V366S/V482I/A484I (a quintuple mutation); I294V/F296V/V366S/V482I/A484I (a septuple mutation); G280T/G281S/V366S/V480S/V482I (a quintuple mutation); I294V/F296V/366S ⁇ /480S/V482I (a quintuple mutation; I294V/F296V/V366S/V
  • L52E/M3 (where M3 is the triple mutant described above and the other mutations are included in an M3 background); L52E/G209E/M3; G209E/M3; C119S/M3; R113Q/M3; V109E/M3; E107D/C119S/M3; E107D/M3; P106M/C119S/M3; P106M/M3; L104V/C119S/M3; L104V/P106M/E107D/M3; L104V/M3; L103I/L104V/C119S/M3; L103I/L104V/M3; L103I/C119S/M3; L103I/M3; G280T/G281S/I294V/F296V/M3, I294V/F296V/M3, and G280T/G281 S/M3. Other combinations of these mutations are within the scope of the invention.
  • Another aspect of the present invention relates to the generation of functional hydroxylases by domain swapping between the epiaristolochene hydroxylase (EAH) and HPO enzymes.
  • EAH epiaristolochene hydroxylase
  • HPO enzymes have a high degree of sequence identity and sequence similarity. Specifically, a total of six domains that are responsible for substrate specificity and/or recognition have been identified in the HPO enzyme (Example 5). These domains are designated SRS (for substrate recognition sequence), and are referred to herein as SRS 1 , SRS 2, SRS 3, SRS 4, SRS 5, and SRS 6. Comparable domains are located in EAH.
  • hydroxylases generated by domain swapping that have either EAH activity (the production of capsidiol or 1 ⁇ (OH)EA)) or HPO activity, or a combination of both activities.
  • EAH activity the production of capsidiol or 1 ⁇ (OH)EA
  • HPO activity the production of capsidiol or 1 ⁇ (OH)EA
  • a functional hydroxylase generated by domain swapping that includes SRS 1, SRS 2, and SRS 3 from HPO and SRS 4, SRS 5, and SRS 6 from EAH
  • a functional hydroxylase generated by domain swapping that includes SRS 1 , SRS 2, SRS 3, SRS 4, and SRS 5 from EAH and SRS 6 from HPO
  • a functional hydroxylase generated by domain swapping that includes SRS 1 , SRS 2, SRS 3, and SRS 6 from EAH and SRS 4 and S
  • Hydroxylases (oxidases) produced by domain swapping and within the scope of the invention can be further modified by mutagenesis, particularly mutagenesis that results in the introduction of one or more point mutations such as are described above and set forth in greater detail in Figure 5. These muteins of hydrolases produced by domain swapping and subsequent mutagenesis are within the scope of the invention. Specific mutations are those of Example 5.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein such that the protein has a catalytic activity of successfully hydroxylating valencene at C2 first to nootkatol and then to nootkatone.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is SEQ ID NO: 1.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence encodes a protein of the sequence SEQ ID NO: 2.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is at least 97.5% identical to SEQ ID NO: 1.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is at least 99% identical to SEQ ID NO: 1.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 480 is changed from valine to serine.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 482 is changed from valine to isoleucine.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 484 is changed from alanine to isoleucine.
  • the present invention relates to a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 366 is changed from valine to serine.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein such that the protein has a catalytic activity of successfully hydroxylating valencene at C2 first to nootkatol and then to nootkatone.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is SEQ ID NO: 1.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence encodes a protein of the sequence SEQ ID NO: 2.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is at least 97.5% identical to SEQ ID NO: 1.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is at least 99% identical to SEQ ID NO: 1.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a functional Hyoscyamus muticus premnaspirodiene oxidase protein, wherein the sequence is selected from the group consisting of:
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 480 is changed from valine to serine.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 482 is changed from valine to isoleucine.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 484 is changed from alanine to isoleucine.
  • the present invention relates to a host cell transformed or transfected with a vector comprising an isolated and purified DNA sequence encoding a protein having the sequence of SEQ ID NO: 2 wherein residue 366 is changed from valine to serine.
  • the present invention relates to a method of producing an isolated protein having Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone comprising the steps of:
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone.
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone, wherein the protein has the amino acid sequence of SEQ ID NO: 2.
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone, wherein the protein ahs an amino acid sequence selected from the group consisting of:
  • a conservative amino acid substitution is one of the following substitutions: Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; G
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone, wherein the protein ahs an amino acid sequence selected from the group consisting of:
  • a conservative amino acid substitution is one of the following substitutions: Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; G
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone, wherein the protein ahs an amino acid sequence selected from the group consisting of:
  • a conservative amino acid substitution is one of the following substitutions: Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; G
  • the present invention relates to an isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone, wherein the protein ahs an amino acid sequence selected from the group consisting of:
  • a conservative amino acid substitution is one of the following substitutions: Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; G
  • the present invention relates to an isolated and purified protein molecule having a functional terpene hydroxylase activity wherein the protein molecule has the sequence of SEQ ID NO: 2 wherein residue 480 is changed from valine to serine.
  • the present invention relates to a method of producing nootkatone comprising the step of reacting the isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity with valencene under conditions in which the protein molecule catalyzes the successive oxidation of the valencene first to nootkatol and then to nootkatone.
  • the present invention relates to a method of producing nootkatone comprising the step of reacting the isolated and purified protein molecule of SEQ ID NO: 2 with valencene under conditions in which the protein molecule catalyzes the successive oxidation of the valencene first to nootkatol and then to nootkatone.
  • the present invention relates to a method of producing nootkatone comprising the step of reacting the isolated and purified protein molecule having functional Hyoscyamus muticus premnaspirodiene synthase activity with valencene under conditions in which the protein molecule catalyzes the successive oxidation of the valencene first to nootkatol and then to nootkatone, wherein the isolated and purified protein molecule has an amino acid sequence selected from the group consisting of:
  • the present invention relates to a yeast cell transformed or transfected with:
  • a first vector including therein a DNA molecule encoding functional Hyoscyamus muticus premnaspirodiene oxidase protein such that the protein has a catalytic activity of successively hydroxylating valencene at C2 first to nootkatol and then to nootkatone;
  • a third vector including therein a DNA molecule encoding a functional Hyoscyamus muticus premnaspirodiene protein synthase; such that the yeast cell expresses: (1) the functional Hyoscyamus muticus premnaspirodiene oxidase protein in a quantity sufficient to hydroxylate valencene; (2) the functional P450 reductase protein in a quantity sufficient to supply reducing equivalents for the Hyoscyamus muticus premnaspirodiene oxidase protein; and (3) the functional Hyoscyamus muticus premnaspirodiene protein synthase in a quantity sufficient to produce premnaspirodiene; and such that the premnaspirodiene is converted by the cell to solavetivone.
  • the present invention relates to a method of producing an oxidized terpene from unoxidized terpene substrate comprising the steps of:
  • the present invention relates to a method of producing an oxidized terpene from an unoxidized terpene substrate comprising the steps of:
  • the present invention relates to a method of producing an oxidized terpene from an unoxidized terpene substrate comprising the steps of:
  • the present invention relates to a method of producing an oxidized terpene from an unoxidized terpene substrate comprising the steps of:
  • the present invention relates to a method of producing an oxidized terpene from an unoxidized terpene substrate comprising the steps of:
  • the present invention relates to a method of producing an oxidized terpene from an unoxidized terpene substrate comprising the steps of:
  • the present invention relates to a method for producing a mutein of Hyoscyamus mutic ⁇ s premnaspirodiene oxidase with at least one altered property selected from the group consisting of regiospecificity and stereospecificity comprising the steps of:
  • the HPO gene now provides an alternative means of generating important stereochemically pure starting materials for the reliable and cost effective production of nootkatone and other high value sesquiterpenes.
  • the co-expression of a valencene synthase gene along with the HPO gene in transgenic plants or microbial cells could providing for large scale production of nootkatone.
  • other terpene synthase genes could be used in combination with the HPO gene to generate other novel terpene moieties, which could be of value for pharmaceutical, agricultural and other industrial applications.
  • Figure 1 shows biosynthetic transformations catalyzed by the Hyoscyamus muticus premnaspirodiene synthase.
  • Evidence provided below demonstrates that the HPO enzyme catalyzes the successive hydroxylation of sesquiterpene scaffolds, thus generating a first mono-hydroxylated form, followed by the subsequent ketone form.
  • the HPO enzyme can utilize a broad range of structurally diverse substrates, such as the vetispirane (A) or eremophilane (B) class of sesquiterpenes.
  • Example 1 Isolation of the HPO gene
  • the HPO gene was isolated using an RT-PCR strategy relying on the design of PCR primers derived from comparison to already cloned terpene hydroxylase genes, including the epi-aristolochene dihydroxylase gene (Ralston et al., 2001 ) and limonene-6-hydroxylase (Lupien et al., 1999).
  • First stand cDNA was generated by reverse transcription of mRNA isolated from elicitor-induced Hyoscyamus muticus roots using oligo-dT as the initial primer, followed by PCR amplification using Forward primer 1 and Reverse primer 1 shown in Figure 2. Both RT and PCR reactions were performed under standard assay conditions.
  • PCR primers used to isolate the HPO gene were initially designed relative to the EAH gene (71 D20, GenBank accession number AF368376), and subsequently to other 71 D family members whose expression are inducible by biological and abiotic stress (71 D4, accession # AJ296346; 71 D6, U48434; 71 D7, U48435; 71 D16, AF166332) (SEQ ID NOs: 3-7 and 11- 15). These GenBank accession numbers and the documentation associated with these numbers are incorporated herein by this reference. The starting position is indicated in Figure 2.
  • F1 is ATGCAATTCTTCAGCTTGGTTTCC (SEQ ID NO: 8).
  • F2 is TTGGYTTCCATYTTCCTWTT (SEQ ID NO: 9).
  • F3 is TTTYTGTTRAGGAAATGGAA (SEQ ID NO: 10).
  • R1 is GGAATAGTTGGAAGAGCTCATT (SEQ ID NO: 16)
  • R2 is TGAGGAATASTTGGAAGA (SEQ ID NO: 17).
  • R3 is ACGGTGAGGAATASTTGGAA (SEQ ID NO: 18).
  • Y is cytosine (C) or thymine (T).
  • W is adenine (A) or ' thymine (T).
  • S is guanine (G) or cytosine (C).
  • R is adenine (A) or guanine (G).
  • RT-PCR amplified DNA was cloned into the pGEM plasmid vector and sequenced according to standard procedures.
  • the DNA sequence and deduced amino acid sequence for the encoded protein are shown below.
  • amino acid sequence for the HPO protein deduced from the open reading frame of the HPO DNA sequence is:
  • HPO protein is highly homologous to previously characterized terpene hydroxylases.
  • HPO is 81 % identical to EAH (Ralston et al., 2001) with approximately 91 amino acid substitutions, and 50% identical to limonene 6-hydroxylase (GenBank accession # AF124815, Lupien et al., 1999).
  • the HPO gene was functionally characterized by cloning the cDNA into a yeast expression vector (YePD-60), introducing the recombinant vector into yeast containing a suitable P450 reductase gene, such as line WAT11 (Urban et al., 1997), and inducing expression of the HPO gene by addition of galactose to the culture media (as per Ralston et al., 2001 ).
  • the microsome fraction was subsequently isolated from the collected yeast cells and assayed for hydroxylase activity by incubation with sesquiterpene substrates and reducing equivalents (NADPH) for various lengths of time.
  • Figure 3 demonstrates the time dependent conversion of premnaspirodiene to solavetivol and solavetivone by microsomes from yeast over-expressing the HPO gene.
  • the solavetivol and solavetivone reaction products were identified by identical retention time with authentic standards and by MS comparisons of the reaction products to authentic standards.
  • the HPO enzyme also exhibits broad substrate specificity, but maintains regio-and stereo-specificity.
  • incubation of microsomes from yeast over-expressing the HPO gene with valencene yields time dependent biosynthesis of ⁇ -nootkatol and nootkatone (Fig. 4). Reaction products were identified by comparison of retention time and MS to authentic standards (Fig. 5).
  • the wildtype HPO enzyme exhibits broad substrate specificity
  • the regio-and stereo-specificity of the enzyme can be manipulated by selective site-directed mutagenesis.
  • the HPO amino acid sequence was threaded onto the 3-Dimensional structure of the mammalian 2B4 hydroxylase (Scott et al., 2004) and several sesquiterpene substrate structures docked into the predicted active site pocket. Amino acid residues within 13 A of the modeled substrate molecules were mapped and rationalized with predicted chemical transformations catalyzed by the HPO enzyme.
  • Residues capable of steric, ionic, electronic and hydrophobic interactions with the substrate molecule were thus identified for their potential for substrate binding and positioning of the substrate molecule relative to the heme-catalytic center. These residues included V482, A484, V480 and others.
  • Site-directed mutagenesis was used to introduce changes into the HPO gene that resulted in amino acid substitutions within the HPO enzyme. For example, the codon corresponding to position 480 coding for valine was changed to that coding for serine, V480S.
  • the mutant gene was over-expressed in yeast and isolated microsomes used in in vitro assays to assess the catalytic activity of the mutant enzyme relative to the wildtype enzyme.
  • Figure 6 Several examples of how site-directed mutagenesis was used to alter the catalytic activity and regiospecificity of the HPO enzyme are illustrated in Figure 6.
  • the HPO enzyme activity requires reducing equivalents in the form of electrons for catalytic activity, which are supplied by P450 reductase proteins.
  • Yeast cells were first engineered with a P450 reductase gene, inserted into the genome of the yeast cell under the control of a strong expression promoter and using an auxotrophic selection marker such as the TRP1 gene (Urban et al., 1997).
  • the HPO and Hyoscyamus muticus premnaspirodiene synthase, HPS, genes were then inserted into separate yeast expression vectors and transformed into yeast harboring the P450 reductase gene, creating yeast lines containing 3 engineered genes as illustrated in Figure 7A.
  • yeast lines were grown under standard culture conditions before addition of galactose to the culture media to induce expression of the engineered genes.
  • Yeast cells were subsequently extracted with ethyl acetate and the organic extract examined by GC-MS.
  • Figure 7B illustrates a typical GC chromatogram for the organic extracts and demonstrates the accumulation of premnaspirodiene, solavetivol and solavetivone by the engineered yeast cultures. Neither wildtype yeast nor engineered yeast lines grown without induction treatment accumulated any of these compounds. Compounds were identified by MS comparisons to authentic standards.
  • Figure 7 is a cartoon depiction of the engineering of a yeast line to effect sesquiterpene hydrocarbon and oxygenated terpene biosynthesis (A).
  • Example 4 The assays of Example 4 were performed in vitro by overexpressing the HPO gene in a yeast host (WAT 11 ), isolating microsomes from the yeast, and then incubating microsomes with the indicated substrates.
  • Figure 8 shows the amino acid alignment between HPO (SEQ ID NO: 2) and EAH (SEQ ID NO: 19). The difference is 91 amino acids and two spaces.
  • Figure 9 shows a summary of the reactions catalyzed by HPO relative to EAH.
  • the font size depicts the relative catalytic rates.
  • Figure 10 shows the CO difference spectrum for HPO expressed in yeast. To calculate exactly how much HPO protein is expressed and properly inserted into the membranes of yeast (the microsomal fraction), a CO (carbon monoxide) difference spectrum is obtained. The peak at 450 nm is specific for the heterologous expressed P450 gene and the absolute absorbance value can be used to calculate the absolute level of the HPO protein in the microsome preparation. This is necessary to calculate the kinetic constants of Figure 16, below.
  • Figure 11 shows the reactions carried out with the substrate premnaspirodiene, indicating successive hydroxylation to 4 ⁇ -solavetivol and then to solavetivone.
  • Figure 12 shows the reactions carried out with the substrate valencene, indicating successive hydroxylation to ⁇ -nootkatol and then to nootkatone. There is a side reaction resulting in the formation of ⁇ -nootkatol, but the relative specificity constant based on k ca t/Km values favors the formation of ⁇ - nootkatol over ⁇ -nootkatol by a ratio of 70:1.
  • Figure 13 shows the reaction carried out with valencene with a reaction time of 1 minute.
  • Figure 14 shows the reaction carried out with 5-epi-aristolochene with a reaction time of 0, 1 , 2, 5 or 10 minutes.
  • the principal product is 2 ⁇ (OH)EA, but there is a minor side product of 2 ⁇ (OH)EA.
  • Figure 15 shows the reaction carried out with 4-epieremophilene and its double-bond isomer with a reaction time of 5 minutes (upper panel), as well as the reaction with ⁇ -cedrene with a reaction time of 5 minutes (lower panel).
  • Figure 16 is a table showing a comparison of enzyme kinetics of HPO for various substrates relative to the previously characterized EAH (5-epi- aristolochene dihydroxylase) hydroxylases. K m> k ca t, and k ca t/K m are shown.
  • Example 5 Generation of additional muteins having altered enzymatic activity
  • the strategy is to obtain stepwise mutants that gain new functionality.
  • Figure 18 shows the EAH activity of a number of muteins, including those produced by domain swapping between EAH and HPO, and those produced by domain swapping between EAH and HPO with additional mutations in the HPO domain, including: V482I/A484I (a double mutation); V366S/V482I/A484I (a triple mutation); G280T/G281S/V366S/V482I/A484I (a quintuple mutation); I294V/F296V/V366SA/482I/A484I (a quintuple mutation); I294V/F296V/V366S/V482I/A484I (a septuple mutation); G280T/G281S ⁇ /366S/V480S/V482I (a quintuple mutation); I294V/F296V/366S/V480S/V482I (a quintuple mutation; and I294V/F296V/3
  • Figure 19 shows the general strategy of using domain-swapping mutations based on substrate recognition sequences (SRS), reciprocal site- directed mutagenesis based on homology modeling with mammalian P450s, and a combination of domain-swapping and site-directed mutagenesis. The six substrate recognition sites are shown along with EAH activity.
  • SRS substrate recognition sequences
  • reciprocal site- directed mutagenesis based on homology modeling with mammalian P450s
  • site-directed mutagenesis The six substrate recognition sites are shown along with EAH activity.
  • Figure 20 shows the results of homology modeling and site- directed mutagenesis in generating muteins of HPO indicated by the amino acid in the native (wildtype) enzyme, the amino acid position, then the mutant amino acid.
  • V366S indicates that the valine in position 366 of the wildtype enzyme has been mutated to serine.
  • Mutants affecting SRS 4, 5, and 6 include: S308T/V366S/V480S/V482I/A484I ; S308T7V366S/V480S/A484I ; S308T/V366S/V482l/A484l; S308T/V366S/V480S/V482l/; S308T ⁇ /366S/A484l;S308T/V366S ⁇ /482l; and S308T ⁇ /366S/V480S.
  • Mutants affecting SRS 4 and 5 include S308T7V366S.
  • Mutants affecting SRS 5 and 6 include: V366S/V480S/V482I/A484I; V366S/V480S/A484I; V366S/V482I/A484I (designated the M3 mutant); V366S/V480S/V482I; V366S/A484I; and V366S/V480S.
  • Mutants affecting SRS 5 include: V366S.
  • Mutants affecting SRS6 include: V480S/V482I/A484I; V482I/A484I; V480S/A484I; V480S ⁇ /482I; A484I; V482I; and V480S. The 5-epiaristolochene hydroxylase activity of these enzymes is shown.
  • Figure 21 is similar to Figure 20, but depicts changes in SRS 1 and/or SRS 2 based on M3 ( Figure 20). Mutants include: L52E/M3, L52E/G209E/M3, G209E/M3, C119S/M3, R113Q/M3, V109E/M3, E107D/C119S/M3, E107D/M3, P106M/C119S/M3, P106M/M3, L104V/C119S/M3, L104V/P106M/E107D/M3, L104V/M3,
  • the 5-epiaristolochene hydroxylase activity of these enzymes is shown.
  • Figure 22 shows the results from a combination of domain- swapping mutations and site-directed mutagenesis; the mutation of V366S greatly diminishes the 5-epiaristolochene hydroxylase activity.
  • FIG 23 shows additional results from site-directed mutagenesis in SRS 4 as well as domain swapping. Mutants include: G280T/G281S/I294V/F296V/M3, I294V/F296V/M3, G280T/G281S/M3, V482I/A484I; V366S/V482I/A484I, and I294V/F296VA/366S/A482I/A484I. EAH activity is shown in terms of both 5-epiaristolochene hydroxylase activity and 1 P(OH)EA hydroxylase activity.
  • Nucleic acids and polypeptides according to the invention provide an efficient, rapid, and cost-effective route for the production of large quantities of oxidized terpenes, many of which are of high commercial value, including the beverage flavoring nootkatone. They obviate the necessity of isolating enzymes from natural products while increasing the uniformity of the process and reducing the reliance of the process on biological starting material.
  • nucleic acids, polypeptides, vectors, host cells, and processes according to the invention have industrial applicability.
  • the invention encompasses each intervening value between the upper and lower limits of the range to at least a tenth of the lower limit's unit, unless the context clearly indicates otherwise. Moreover, the invention encompasses any other stated intervening values and ranges including either or both of the upper and lower limits of the range, unless specifically excluded from the stated range.

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Abstract

L'invention concerne des molécules d'acide nucléique isolées et purifiées codant pour la premnaspirodiène oxydase de Hyoscyamus muticus (HPO), préparées par clonage moléculaire. Ces molécules d'acide nucléique codent pour l'enzyme oxydase qui peut catalyser l'hydroxylation de la valencène en bêta-nootkatol et en nootkatone, présentant un intérêt sur le plan industriel et utilisé dans les produits alimentaires. L'invention concerne également des vecteurs renfermant ces molécules d'acide nucléique, des cellules hôtes transformées ou transfectées par les vecteurs, ainsi que des méthodes destinées à la production de protéines par expression des molécules d'acide nucléique dans les cellules hôtes. L'invention concerne également des méthodes destinées à la production d'un substrat de terpène oxydé à partir d'un substrat de terpène non oxydé, dans lesquelles sont utilisées des protéines ou des molécules d'acide nucléique exprimées selon l'invention.
PCT/US2006/002265 2005-01-19 2006-01-19 Identification fonctionnelle du gene de muticus hyoscyamus codant pour l'activite de la premnaspirodiene hydroxylase WO2006079020A2 (fr)

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Cited By (11)

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WO2008116056A2 (fr) 2007-03-20 2008-09-25 Allylix, Inc. Nouveaux procédés de production de 5-épi-β-vétivone, de 2-isopropyl-6,10-diméthyl-spiro[4.5]déca-2,6-dién-8-one et de 2-isopropyl-6,10-diméthyl-spiro[4.5]déca-1,6-dién-8-one
WO2011074954A2 (fr) 2009-12-16 2011-06-23 Isobionics B.V. Valencène synthase
US8106260B2 (en) 1996-04-12 2012-01-31 The Board Of Trustees Of The University Of Kentucky Chimeric isoprenoid synthases and uses thereof
US8124811B2 (en) 2007-03-20 2012-02-28 Allylix, Inc. Fragrance and methods for production of 5-epi-β-vetivone, 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one, and 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one
WO2012058636A1 (fr) * 2010-10-29 2012-05-03 Allylix, Inc. Polypeptides modifiés de valencène synthase, molécules d'acide nucléique codant pour ceux-ci, et leurs utilisations
US8192950B2 (en) 2003-07-24 2012-06-05 The University Of Kentucky Research Foundation Sesquiterpene synthase gene and protein
EP2537926A1 (fr) 2011-06-21 2012-12-26 Isobionics B.V. Valencène synthase
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WO2016029187A3 (fr) * 2014-08-21 2016-05-06 Givaudan Sa Procédés de production de terpènes oxygénés
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US8192950B2 (en) 2003-07-24 2012-06-05 The University Of Kentucky Research Foundation Sesquiterpene synthase gene and protein
US9534237B2 (en) 2003-07-24 2017-01-03 University Of Kentucky Research Foundation Sesquiterpene synthase gene and protein
US8835131B2 (en) 2003-07-24 2014-09-16 University Of Kentucky Research Foundation Sesquiterpene synthase gene and protein
AU2012204126B2 (en) * 2007-03-20 2014-05-15 Evolva, Inc. Novel Methods for Production of 5-epi-beta-vetivone, 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one, and 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one
EP2137127A2 (fr) * 2007-03-20 2009-12-30 Allylix, Inc. Nouveaux procédés de production de 5-épi-beta -vétivone, de 2-isopropyl-6,10-diméthyl-spiro[4.5]déca-2,6-dién-8-one et de 2-isopropyl-6,10-diméthyl-spiro[4.5]déca-1,6-dién-8-one
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WO2008116056A3 (fr) * 2007-03-20 2008-12-18 Allylix Inc Nouveaux procédés de production de 5-épi-β-vétivone, de 2-isopropyl-6,10-diméthyl-spiro[4.5]déca-2,6-dién-8-one et de 2-isopropyl-6,10-diméthyl-spiro[4.5]déca-1,6-dién-8-one
EP2137127A4 (fr) * 2007-03-20 2012-02-01 Allylix Inc Nouveaux procédés de production de 5-épi-beta -vétivone, de 2-isopropyl-6,10-diméthyl-spiro[4.5]déca-2,6-dién-8-one et de 2-isopropyl-6,10-diméthyl-spiro[4.5]déca-1,6-dién-8-one
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US8362309B2 (en) 2007-03-20 2013-01-29 Allylix, Inc. Fragrance and methods for production of 5-epi-β-vetivone, 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-2,6-dien-8-one, and 2-isopropyl-6,10-dimethyl-spiro[4.5]deca-1,6-dien-8-one
US8642815B2 (en) 2007-03-20 2014-02-04 Allylix, Inc. Fragrance and methods for production of 5-epi-β-vetivone, 2-isopropyl-6, 10-dimethyl-spiro[4.5]deca-2,6-dien-8-one, and 2-isopropyl-6, 10-dimethyl-spiro[4.5]deca-1, 6-dien-8-one
WO2008116056A2 (fr) 2007-03-20 2008-09-25 Allylix, Inc. Nouveaux procédés de production de 5-épi-β-vétivone, de 2-isopropyl-6,10-diméthyl-spiro[4.5]déca-2,6-dién-8-one et de 2-isopropyl-6,10-diméthyl-spiro[4.5]déca-1,6-dién-8-one
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AU2011320127B2 (en) * 2010-10-29 2015-10-01 Evolva, Inc. Modified valencene synthase polypeptides, encoding nucleic acid molecules and uses thereof
US9303252B2 (en) 2010-10-29 2016-04-05 Evolva, Inc. Modified valencene synthase polypeptides, encoding nucleic acid molecules and uses thereof
WO2012058636A1 (fr) * 2010-10-29 2012-05-03 Allylix, Inc. Polypeptides modifiés de valencène synthase, molécules d'acide nucléique codant pour ceux-ci, et leurs utilisations
EP2537926A1 (fr) 2011-06-21 2012-12-26 Isobionics B.V. Valencène synthase
WO2012177129A2 (fr) 2011-06-21 2012-12-27 Isobionics B.V. Valencène synthase
US9839214B2 (en) 2012-12-18 2017-12-12 Evolva, Inc. Solavetivone and 5-epi-beta-vertivone as pest repellants and pesticides
US10206393B2 (en) 2012-12-18 2019-02-19 Evolva, Inc. Solavetivone and 5-epi-β-vetivone as pest repellants and pesticides
WO2016029187A3 (fr) * 2014-08-21 2016-05-06 Givaudan Sa Procédés de production de terpènes oxygénés
CN107002109A (zh) * 2014-08-21 2017-08-01 马努斯生物合成股份有限公司 含氧萜烯的生产方法
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US10934564B2 (en) 2014-08-21 2021-03-02 Manus Bio Inc. Methods for production of oxygenated terpenes
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US11952608B2 (en) 2014-08-21 2024-04-09 Manus Bio Inc. Methods for production of oxygenated terpenes
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