WO2015040197A1 - Method for producing fragrant alcohols - Google Patents
Method for producing fragrant alcohols Download PDFInfo
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- WO2015040197A1 WO2015040197A1 PCT/EP2014/070060 EP2014070060W WO2015040197A1 WO 2015040197 A1 WO2015040197 A1 WO 2015040197A1 EP 2014070060 W EP2014070060 W EP 2014070060W WO 2015040197 A1 WO2015040197 A1 WO 2015040197A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
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- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
- C12N9/0079—Steroid 11 beta monooxygenase (P-450 protein)(1.14.15.4)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
- C12Y114/14001—Unspecific monooxygenase (1.14.14.1)
Definitions
- the field relates to cytochrome P450s and uses to produce sesquiterpene alcohols.
- Terpenes hydrocarbons such as alpha and beta santalenes have been produced via biochemical processes for example such as through genetically altered cells. These terpenes and the alcohol derived from them are major constituents of sandalwood oil and the alchohols are important perfumery ingredients typically obtained commercially through the distillation of the heartwood of Santalum species (e.g., Sandalwood).
- alcohols examples include -sinensol, ?-sinensol, -santalol, ?-santalol, a-trans- bergamotol and e/?z ' - ?-santalol.
- biochemical pathway it is further desirable to use a biochemical pathway to not only generate such alcohols but it is further desirable to selectively produce, via a biochemical pathway, cz ' s-isomers of the alcohols such as iso- -sinensol, iso-fi-sinensol, (Z)- - santalol, (Z)- ?-santalol, (Z)- -ira «5-bergamotol and (Z)-e/?z ' - ?-santalol.
- Cytochrome P450s represent a family of enzymes of oxidases. P450s commonly catalyze a monooxygenase reaction. Cytochrome P450 enzymes are classified into families and subfamilies based on the amino acid sequences homology. Members of a same subfamily share over 55% amino acid sequence identity and have usually similar enzymatic activities (substrate and/or product selectivity).
- CYP71AV1 NCBI accession No ABB82944.1, SEQ ID No. 51 and 52
- CYP71AV8 NCBI accession No
- ADM86719.1, SEQ ID No. 1 and 2 are two members of the CYP71AV sub-family and shares 78 % sequence identity.
- CYP71AV1 has previously been shown to oxidize amorphadiene (Teoh et al, FEBS letters 580 (2006) 1411-1416).
- CYP71AV8 has previously been shown to oxidize ( + )-valencene, germacrene A and amorphadiene (Cankar et al, FEBS Lett. 585(1), 178-182 (2011)).
- polypeptide having an amino acid sequence having at least, or at least about, 45% of sequence identify to a polypeptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 79, and SEQ ID NO: 81; and ii) optionaly isolating the alcohol wherein R is a saturated, mono-unsaturated or poly-uns
- a sesquiterpene comprising a- sinensol, ?-sinensol, -santalol, ?-santalol, a-£ra «s-bergamotol,e/?z ' - ?-santalol, lancelol and/or or mixtures thereof comprising:
- Also provided herein is a method of producing a-sinensol, ?-sinensol, a-santalol,?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting a-farnesene, ⁇ -farnesene , a-santalene, ?-santalene, a-trans- bergamotene and/or e/?z ' - ?-santalene, with a polypeptide having a P450 monooxygenase activity wherein the sesquiterpene alcohol produced comprises at least, or at least about, 36% of a cis isomer.
- an isolated polypeptide having monooxygenase activity comprising an amino acid sequence that is at least, or at least about 45%, 50 %, 55%, 50%, 65%, 70%, 80%, 90%, 95%, 98% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 71, and SEQ ID NO:73.
- an isolated polypeptide having monooxygenase activity comprising an amino acid sequence that is at least, or at least about 45%, 50 %, 55%, 50%, 65%, 70%, 80%, 90%, 95%, 98% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 79, and SEQ ID NO: 81.
- an isolated polypeptide having monooxygenase activity comprising an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO:71, SEQ ID NO:73 SEQ ID NO: 79, and SEQ ID NO: 81.
- a sesquiterpene alcohol selected from the group consisting of ⁇ -sinensol, ?-sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, and lancelol or mixtures thereof :
- a) produces a acylic pyrophosphate terpene precursor; b) expresses a P450 reductase, c) expresses a polypeptide that has a-farnesene, ⁇ -farnesene , a-santalene, ?-santalene, a-fraws-bergamotene and/or epi- ?-santalene, synthase activity and produces ⁇ -farnesene, ⁇ -farnesene , a-santalene, ?-santalene, a-fraws-bergamotene and/or e/?z ' - ?-santalene and d) expresses a polypeptide with an amino acid sequence having at least, or at least about, 45% of sequence identify to a polypeptide selected from the group
- Figure 3 GCMS analysis of the conversion of sesquiterpenes by E. Coli cells expressing the CYP71AV8 and the CPRm proteins.
- A Bioconversion of (+)-alpha-santalene.
- B Bioconversion of a (+)-alpha-santalene/(-)-beta-santalene mixture.
- Figure 4. Organisation of the synthetic bi-cistronic operon containing a P450 and a CPR cDNA.
- Figure 6 GCMS analysis of the sesquiterpene molecules produced by E. Coli cells expressing CYP71AV8, CPRm, an alpha-santalene synthase (A) or a alpha- santalene/beta-santalene synthase (B), and mevalonate pathway enzymes.
- A alpha-santalene synthase
- B alpha-santalene/beta-santalene synthase
- mevalonate pathway enzymes 1, (+ )-a- santalene; 2 f-j-a-fraws-bergamotene; 3, (+ )-e/?z ' - ?-santalene; 4, f-)- ?-santalene.
- Figure 7 Oxidation of (+ j- -santalene by CYP71AV8 wild type (A) and mutant L-358 (B).
- the cultivations were performed in TB medium containing 3% glycerol as carbon source.
- the different products were identified as a-santalene (1), (E)- a-santalal(2), fZj-a-santalol (4), and (3 ⁇ 4)-a-santalol (3).
- FIG. 8 GC-MS profiles of the sesquiterpene products generated by E. Coli KRX cells expressing CPRm, SaSAS, the mevalonate pathway enzymes and CYP71AV8-L358F. The cultivations were performed in TB medium containing 3% glycerol as carbon source. The different products identified by their mass spectra are indicated.
- Figure 10 GC analysis of the in vivo conversion of (+ j-a-santalene to fZj-a-santalol by a P450-BM3 double-mutant (variant #17).
- Solvent extracts of cultures of recombinant E. coli cells co-expressing a Clausena lansium a-santalene synthase and either the wild-type P450-BM3 (A) or the P450-BM3 variant #17 (B) were analyzed as described in example 11.
- Figure 11 GC analysis of the in vivo conversion of (+ j-a-santalene, f-)- ?-santalene, (-)- ⁇ -fraws-bergamotene and ( + j-e/?z ' - ?-santalene by a P450-BM3 double-mutant.
- Solvent extracts of cultures of recombinant E. coli cells co-expressing an alpha-santalene/beta- santalene synthase from Santalum album and either the wild-type P450-BM3 (A) or the P450-BM3 variant #17 (B) were analyzed as described in example 11.
- Figure 13 GCMS analysis of the conversion of (+ j- -santalene, f-j- ?-santalene, (-)-a- fraws-bergamotene and ( + j-e/?z ' - ?-santalene by the recombinant SaCP816 enzyme.
- C Sandalwood oil for comparison of the retention times. All assays were performed in-vitro as described in example 4.
- Figure 14 GCMS analysis of the molecules produced by E. Coli engineered to produced sesquiterpenes and expressing SaCP816, CPRm, an alpha-santalene synthase (CIASS) (A) or a alpha-santalene/beta-santalene synthase (SaSAS) (B).
- CIASS alpha-santalene synthase
- SaSAS alpha-santalene/beta-santalene synthase
- Figure 15 GCMS analysis of the conversion of (+)-a-santalene (21) by the recombinant SaCP10374 P450 enzyme.
- Figure 16 GCMS analysis of the conversion of a mixture composed of (+ j-a-santalene (21), (-)-a-trans-bergamotene (17); (+)-e/?z ' - ?-santalene and f-)- ?-santalene (25) (prepared using the SaTp8201 recombinant protein, example 4) by the recombinant SaCP10374 P450s enzymes.
- B Assay with E. coli crude protein extract containing the recombinant SaCP10374 protein. The numbers indicated on the chromatograms refer to the structures presented in figure 27.
- Figure 17 GCMS analysis of the conversion of ?-farnesene (1) by the recombinant S. album P450s enzymes.
- A Control without the recombinant P450 enzyme.
- B Assay with E. coli crude protein extract containing the recombinant SaCP10374 protein.
- C Assay with E. coli crude protein extract containing the recombinant SaCP816 protein.
- the numbers indicated on the chromatograms refer to the structures presented in figure 27.
- Figure 18 GCMS analysis of the conversion of -farnesene (5) by the recombinant S. album P450s enzymes.
- A Control without the recombinant P450 enzyme.
- B Assay with E. coli crude protein extract containing the recombinant SaCP10374 protein.
- C Assay with E. coli crude protein extract containing the recombinant SaCP816 protein.
- the numbers indicated on the chromatograms refer to the structures presented in figure 27.
- Figure 19 GCMS analysis of the conversion of (-)-sesquisabinene B (9) by the recombinant S. album P450s enzymes. A. Control without the recombinant P450 enzyme.
- Figure 20 GCMS analysis of the conversion of (- )- ?-bisabolene (13) by the recombinant S. album P450s enzymes. A. Control without the recombinant P450 enzyme.
- the numbers indicated on the chromatograms refer to the structures presented in figure 27
- Figure 21 GCMS analysis of the conversion of (-)-a-bergamotene (17) by the recombinant S. album P450s enzymes.
- A Control without the recombinant P450 enzyme.
- B Assay with E. coli crude protein extract containing the recombinant SaCP10374 protein.
- C Assay with E. coli crude protein extract containing the recombinant SaCP816 protein.
- the numbers indicated on the chromatograms refer to the structures presented in figure 27.
- Figure 22 GCMS analysis of the products generated in-vivo as described in example 23 by E. Coli KRX cells transformed with the plasmids pACYC-29258-4506 and the plasmid pD444-SR-AaBFS (A), SaCP10374-CPRm-AaBFS-pCWori (B), or SaCP816-CPRm-AaBFS-pCWori (C).
- the chromatograms show the formation of ( ⁇ )- ⁇ - farnesene (1) as well as oxidized derivatives (2-3) (see figure 27 for corresponding structures).
- Figure 23 GCMS analysis of the products generated in-vivo as described in example 23 by E. Coli KRX cells transformed with the plasmids pACYC-29258-4506 and the plasmid pD444-SR-PaBAFS (A), SaCP10374-CPRm-PaAFS-pCWori (B), or SaCP816-CPRm-PaAFS-pCWori (C).
- the chromatograms show the formation of (E,E)- or-farnesene (5) as well as oxidized derivatives (6-8) (see figure 27 for corresponding structures). The peak of farnesol resulting from the hydrolysis of excess FPP is inducated on each chromatogram.
- Figure 24 GCMS analysis of the products generated in-vivo as described in example 23 by E. Coli KRX cells transformed with the plasmids pACYC-29258-4506 and the plasmid pETDuet-SaTps647 (A), SaCP10374-CPRm-SaTps647-pCWori (B), or SaCP816-CPRm-SaTPS647-pCWori(C).
- the chromatograms show the formation of (-)- sesquisabinene B (9) as well as oxidized derivatives (10-12) (see figure 27 for corresponding structures).
- Figure 25 GCMS analysis of the products generated in-vivo as described in example 23 by E. Coli KRX cells transformed with the plasmids pACYC-29258-4506 and the plasmid pETDuet-ClTps2 (A) or SaCP10374-CPRm-ClTps2-pCWori (B).
- the chromatograms show the formation of (+)- or-santalene (21) as well as oxidized derivatives (23-24) (see figure 27 for corresponding structures).
- Figure 26 GCMS analysis of the products generated in-vivo as described in example 23 by E.
- the chromatograms show the formation of f+j-or-santalene (21), f-)- ?-santalene (25) and f-j-fraws-a-Bergamotene (17) as well as oxidized derivatives (19, 20, 23, 24, 27 and 28) (see figure 27 for corresponding structures).
- a method of producing a sesquiterpene comprising -sinensol, ?-sinensol, -santalol, ?-santalol, a-fraws-bergamotol, epi- ⁇ - santalol, and lancelol and/or mixtures thereof comprising contacting -farnesene, ⁇ - farnesene , a-santalene, ?-santalene, a-fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 2.
- the method comprises a cell that expresses the polypeptide.
- a method of producing a a-sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting a-farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 4.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 6.
- the method comprises a cell that expresses the polypeptide.
- a method of producing -sinensol, ⁇ - sinensol, -santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting -farnesene, ⁇ -farnesene , -santalene, ⁇ - santalene, a-fraHs-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 8.
- the method comprises a cell that expresses the
- a method of producing a-sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting a-farnesene, ⁇ -farnesene , a-santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 28.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 30.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 32.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 34.
- the method comprises a cell that expresses the polypeptide.
- a method of producing -sinensol, ⁇ - sinensol, -santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting -farnesene, ⁇ -farnesene , -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 36.
- the method comprises a cell that expresses the polypeptide.
- a method of producing a-sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting a-farnesene, ⁇ -farnesene , a-santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 38.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 40.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 42.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting -farnesene, ⁇ -farnesene , -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 44.
- the method comprises a cell that expresses the polypeptide.
- a method of producing -sinensol, ⁇ - sinensol, -santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting a-farnesene, ⁇ -farnesene , a-santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 50.
- the method comprises a cell that expresses the polypeptide.
- a method of producing a-sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 52.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 54.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 58.
- the method comprises a cell that expresses the polypeptide.
- a method of producing -sinensol, ⁇ - sinensol, -santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting -farnesene, ⁇ -farnesene , -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO:60.
- the method comprises a cell that expresses the polypeptide.
- a method of producing a-sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting a-farnesene, ⁇ -farnesene , a-santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 62.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 64.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 66.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 68.
- the method comprises a cell that expresses the polypeptide.
- a method of producing -sinensol, ⁇ - sinensol, -santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting -farnesene, ⁇ -farnesene , -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 71.
- the method comprises a cell that expresses the polypeptide..
- a method of producing a-sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting a-farnesene, ⁇ -farnesene , a-santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 73.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 79.
- the method comprises a cell that expresses the polypeptide.
- a method of producing ⁇ -sinensol, ⁇ - sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol and/or mixtures thereof comprising contacting ⁇ -farnesene, ⁇ -farnesene , ⁇ -santalene, ⁇ - santalene, ⁇ -fraws-bergamotene and/or e/?z ' - ?-santalene, with a polypeptide comprising an amino acid sequence having at least, or at least about, 45%, 50 %, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 98% % sequence identify to SEQ ID NO: 81.
- the method comprises a cell that expresses the polypeptide.
- nucleotide sequences provided herein for producing a polypeptide for use in producing an alcohol have a nucleic acid sequence at least, or at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 70 SEQ ID NO: 72, SEQ ID NO: 78 and S
- nucleotide sequences provided herein are heterologous in that they are not typically or normally produced by a cell in which it is expressed herein and is generally not endogenous to the cell into which it is introduced - it being typically obtained from another cell or could be made synthetically.
- a method of producing a sesquiterpene alcohol comprising -sinensol, ?-sinensol, -santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol, and/or mixtures thereof comprising contacting fraws-a-farnesene trans- ⁇ -farnesene , -santalene, ?-santalene, a-fraws-bergamotene, e/?z ' - ?-santalene, and/ or ?-bisabolene with a polypeptide having a P450 monoxygenase activity wherein the alcohol produced comprises at least, or at least about, 36%, of a cis isomer and wherein the polpeptide e comprises an amino acid sequence having at least or at least about 45%, 50 %, 55%, 60%,
- a sesquiterpene alcohol comprising a-sinensol, ?-sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol, and/or mixtures thereof comprising contacting fraws-a-farnesene trans- ⁇ -farnesene , a-santalene, ?-santalene, a-fraws-bergamotene, e/?z ' - ?-santalene, and/ or ?-bisabolene with a polypeptide having a P450 monoxygenase activity wherein the alcohol produced comprises at least, or at least about, 46%, of a cis isomer and wherein the polpeptide e comprises an amino acid sequence having at least or at least about 45%, 50 %, 55%,
- a method of producing a sesquiterpene alcohol comprising -sinensol, ?-sinensol, -santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol, and/or mixtures thereof comprising contacting fraws-a-farnesene trans- ⁇ -farnesene , -santalene, ?-santalene, a-fraws-bergamotene, e/?z ' - ?-santalene, and/ or ?-bisabolene with a polypeptide having a P450 monoxygenase activity wherein the alcohol produced comprises at least, or at least about, 50%, of a cis isomer and wherein the polpeptide e comprises an amino acid sequence having at least or at least about 45%, 50 %, 55%, 60%, 65%
- a method of producing a sesquiterpene alcohol comprising a-sinensol, ?-sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol, and/or mixtures thereof comprising contacting fraws-a-farnesene trans- ⁇ -farnesene , a-santalene, ?-santalene, a-fraws-bergamotene, e/?z ' - ?-santalene, and/ or ?-bisabolene with a polypeptide having a P450 monoxygenase activity wherein the alcohol produced comprises at least, or at least about, 72%, of a cis isomer and wherein the polpeptide e comprises an amino acid sequence having at least or at least about 45%, 50 %, 55%,
- a sesquiterpene alcohol comprising ⁇ -sinensol, ?-sinensol, a-santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol, and/or mixtures thereof comprising contacting fraws-a-farnesene trans- ⁇ -farnesene , ⁇ -santalene, ?-santalene, a-fraws-bergamotene, e/?z ' - ?-santalene, and/ or ?-bisabolene with a polypeptide having a P450 monoxygenase activity wherein the alcohol produced comprises at least, or at least about, 96%, of a cis isomer and wherein the polpeptide e comprises an amino acid sequence having at least or at least about 45%, 50 %, 55%,
- a method of producing a sesquiterpene alcohol comprising -sinensol, ?-sinensol, -santalol, ?-santalol, a-fraws-bergamotol, e/?z ' - ?-santalol, lancelol, and/or mixtures thereof comprising contacting fraws-a-farnesene trans- ⁇ -farnesene , a-santalene, ?-santalene, a-fraws-bergamotene, e/?z ' - ?-santalene, and/ or ?-bisabolene with a polypeptide having a P450 monoxygenase activity wherein the alcohol produced comprises at least, or at least about, 100%, of a cis isomer and wherein the polpeptide e comprises an amino acid sequence having at least or at least about 45%, 50 %, 55%, 60%,
- nucleic acid molecule selected from the group consisting of: i) a nucleic acid having an nucleic acid sequence selected from the group consisting SEQ ID. NO: 70 and 72; and ii) a nucleic acid molecule that encodes a polypeptide having p450 monooxygenase activity wherein the polypeptide comprises an amino acid sequence that is at least, or at least about 45%, 50 %, 55%, 50%, 65%, 70%, 80%, 90%, 95%, or 98% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71, and SEQ ID NO: 73. More particularly the polypeptide encoded has the sequence selected from the group consisting of SEQ ID NOs: 71, and SEQ ID NO: 73.
- nucleic acid molecule selected from the group consisting of: i) a nucleic acid having an nucleic acid sequence selected from the group consisting SEQ ID. NO: 78 and 80; and ii) a nucleic acid molecule that encodes a polypeptide having p450 monooxygenase activity wherein the polypeptide comprises an amino acid sequence that is at least, or at least about 45%, 50 %, 55%, 50%, 65%, 70%, 80%, 90%, 95%, or 98% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, and SEQ ID NO: 82. More particularly the polypeptide encoded has the sequence selected from the group consisting of SEQ ID NOs: 79, and SEQ ID NO: 82.
- nucleic acid molecule selected from the group consisting of: i) a nucleic acid having an nucleic acid sequence selected from the group consisting SEQ ID. NO: 27, 29, 31, 33, and 35; and ii) a nucleic acid molecule that encodes a polypeptide having p450 monooxygenase activity wherein the polypeptide has the sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36.
- a method for producing a polypeptide having P450 monoxygenase activity comprising the steps of transforming a host cell or non-human organism with a nucleic acid encoding a polypeptide having at least, or at least about, 45%, 50 %, 55%, 50%, 65%, 70%, 80%, 90%, 95%, or 98% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 71, and SEQ ID NO: 73 and culturing the host cell or organism under conditions that allow for the production of the polypeptide.
- a method for producing a polypeptide having P450 monoxygenase activity comprising the steps of transforming a host cell or non-human organism with a nucleic acid encoding a polypeptide having the sequence selected from the group consisting of SEQ ID NO: 71, and SEQ ID NO: 73 and culturing the host cell or organism under conditions that allow for the production of the polypeptide.
- a method for producing a polypeptide having P450 monoxygenase activity comprising the steps of transforming a host cell or non-human organism with a nucleic acid encoding a polypeptide having at least, or at least about, 45%, 50 %, 55%, 50%, 65%, 70%, 80%, 90%, 95%, or 98% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 79, and SEQ ID NO: 81 and culturing the host cell or organism under conditions that allow for the production of the polypeptide.
- a method for producing a polypeptide having P450 monoxygenase activity comprising the steps of transforming a host cell or non-human organism with a nucleic acid encoding a polypeptide having the sequence selected from the group consisting of SEQ ID NO: 79, and SEQ ID NO: 81 and culturing the host cell or organism under conditions that allow for the production of the polypeptide.
- the alcohols can be converted to aldehydes or acids such as but not limited to sinensals, santalals, bergamotenals, and lanceals.
- the alcohols, aldehydes or acids can be further converted to derivatives such as, but not limited to esters, amides, glycosides, ethers or acetals.
- Nucleic acid and polypeptides described herein may be isolated for example from Cichorium intybus L., Bacillus megaterium, Santalum Album and Artemisia annua.
- CYP71AV8, P450-BM3 (CYP102A1), and CYP71AV1 including variants are described herein.
- CYP71AV8 from the plant Cichorium intybus L. was previously characterized as a P450 mono-oxygenase able to oxidize region-selectively ( + j-valencene producing fraws-nootkatol, cz ' s-nootkatol and (+ j-nootkatone.
- CYP71AV8 was also found to catalyse the oxidation of germacrene A and amorpha-4,11-diene in the C-12 position (Cankar et al, FEBS Lett. 585(1), 178-182 (2011)).
- the amino acid sequence of the wild type enzyme (NCBI accession No ADM86719.1, SEQ ID No 1 and 2) was used to design a cDNA sequence optimized for expression in E. coli.
- the P450 monooxygenases are membrane -bound proteins and the N- terminal sequence of these proteins constitute a membrane anchor essential for the membrane localization of these enzymes.
- This part of the protein usually delimited by a proline-rich domaine, is not essential for the control of the specificity of the enzymatic activity.
- This region can thus be modified by deletion, insertion or mutation without effect on the catalytic activity.
- specific modification of the N-terminal region of eukaryotic P450s, including plant P450s have been shown to have a positive effect on the levels of functional recombinant proteins when expressed in microorganisms (Halkier et al (1995) Arch. Biochem. Biophys. 322, 369-377; Haudenschield et al (2000) Arch.
- P450 monooxygenases the recognition and binding of the substrate is controlled by several amino acid residues distributed in different regions along the protein amino acid sequences. These regions, defined as substrate recognition sites (SRS), can be localized in the amino acid sequence of any P450 by simple sequence alignment based for example on the work made by Gotoh (Gotoh O (1992) J. Biol. Chem. 267(1), 83-90).
- SRS substrate recognition sites
- residues in the CYP71AV8 protein that interact with the substrate and can influence the regioselectivity of the hydroxylation reaction are the amino acids Asn98 to Glyl21, Thrl98 to Leu205, Lys232 to He 240, Asn282 to Ala300, His355 to Arg367 and Thr469 to Val 476.
- the modification of one or more residues in these regions can potentially alter the substrate specificity, the stereochemistry of the reaction or its regioselectivity.
- alteration of the regioselectivity of the reaction catalyzed by a P450 can be found in Schalk et al (2002) Proc. Natl. Acad. Sci. USA 97(22), 11948-11953. In this publication a single residue change in plant P450 enzymes led to a complete conversion to the regiospecificity of the enzymatique reaction.
- a “sesquiterpene synthase” or a “polypeptide having a sesquiterpene synthase activity” is intended for the purpose of the present application as a polypeptide capable of catalyzing the synthesis of a sesquiterpene molecule or of a mixture of sesquiterpene molecules from a acyclic pyrophosphate terpene precursor selected from the group consisting of geranyl-pyrophosphate (GPP), farnesy-diphosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP) .
- GPP geranyl-pyrophosphate
- FPP farnesy-diphosphate
- GGPP geranylgeranyl-pyrophosphate
- Alpha santalene, beta-santalene, alpha-trans-bergamotene, and/or epi-beta santalene may be prepared using the synthases described for example in U.S. Patent Publication No.: 2011-0008836, published January 13, 20111 and in U.S. Patent Publication No.: 2011-0281257, published November 27, 2011, both of which are incorporated herein in their entirety.
- polypeptides are also meant to include truncated polypeptides provided that they keep their P450 monooxygenase activity as defined in any of the above embodiments..
- the percentage of identity between two peptidic or nucleotidic sequences is a function of the number of amino acids or nucleotide residues that are identical in the two sequences when an alignment of these two sequences has been generated. Identical residues are defined as residues that are the same in the two sequences in a given position of the alignment.
- the percentage of sequence identity is calculated from the optimal alignment by taking the number of residues identical between two sequences dividing it by the total number of residues in the shortest sequence and multiplying by 100.
- the optimal alignment is the alignment in which the percentage of identity is the highest possible. Gaps may be introduced into one or both sequences in one or more positions of the alignment to obtain the optimal alignment. These gaps are then taken into account as non-identical residues for the calculation of the percentage of sequence identity.
- Alignment for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways using computer programs and for instance publicly available computer programs available on the world wide web.
- the BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247- 250, 1999) set to the default parameters, available from the National Center for Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi, can be used to obtain an optimal alignment of peptidic or nucleotidic sequences and to calculate the percentage of sequence identity.
- NCBI National Center for Biotechnology Information
- a particular organism or cell is meant to be “capable of producing FPP” when it produces FPP naturally or when it does not produce FPP naturally but is transformed to produce FPP, either prior to the transformation with a nucleic acid as described herein or together with said nucleic acid.
- Organisms or cells transformed to produce a higher amount of FPP than the naturally occurring organism or cell are also encompassed by the "organisms or cells capable of producing FPP". Methods to transform organisms, for example microorganisms, so that they produce FPP are already known in the art.
- Non-human host organisms suitable to carry out the method described herein in vivo may be any non-human multicellular or unicellular organisms.
- the non-human host organism used to carry out the invention in vivo is a plant, a prokaryote or a fungus. Any plant, prokaryote or fungus can be used. Particularly useful plants are those that naturally produce high amounts of terpenes.
- the plant is selected from the family of Solanaceae, Poaceae, Brassicaceae, Fabaceae, Malvaceae, Asteraceae or Lamiaceae.
- the plant is selected from the genera Nicotiana, Solanum, Sorghum, Arabidopsis, Brassica (rape), Medicago (alfalfa), Gossypium (cotton), Artemisia, Salvia and Mentha.
- the plant belongs to the species of Nicotiana tabacum.
- the non-human host organism used to carry out the method of the invention in vivo is a microorganism.
- Any microorganism can be used but according to an even more particular embodiment said microorganism is a bacteria or yeast.
- said bacteria is E. coli and said yeast is Saccharomyces cerevisiae. Some of these organisms do not produce FPP naturally.
- these organisms have to be transformed to produce said precursor. They can be so transformed either before the modification with the nucleic acid described according to any of the above embodiments or simultaneously, as explained above.
- Isolated higher eukaryotic cells can also be used, instead of complete organisms, as hosts to carry out the method of the invention in vivo.
- Suitable eukaryotic cells may be any non-human cell, but are particularly plant or fungal cells.
- polypeptide is intended as a polypeptide or peptide fragment that encompasses the amino acid sequences identified herein, as well as truncated or variant polypeptides, provided that they keep their P450 monooxygenaseactivity as defined above and that they share at least the defined percentage of identity with the corresponding polypeptide.
- variant polypeptides are naturally occurring 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 of the invention. Polypeptides encoded by a nucleic acid obtained by natural or artificial mutation of a nucleic acid of the invention, as described thereafter, are also encompassed by the invention.
- Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends can also be used in the methods of the invention.
- a fusion can enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system.
- additional peptide sequences may be signal peptides, for example.
- the present invention encompasses methods using variant polypeptides, such as those obtained by fusion with other oligo- or polypeptides and/or those which are linked to signal peptides.
- Polypeptides resulting from a fusion with another functional protein, such as another protein from the terpene biosynthesis pathway can also be advantageously be used in the methods of the invention.
- polypeptide is intended as a polypeptide or peptide fragment that encompasses the amino acid sequence identified herein, as well as truncated or variant polypeptides, provided that they keep their activity as defined above.
- variant polypeptides are naturally occurring 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 of the invention. Polypeptides encoded by a nucleic acid obtained by natural or artificial mutation of a nucleic acid of the invention, as described thereafter, are also encompassed by the invention.
- Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends are also encompassed by the polypeptides of the invention.
- a fusion can enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system.
- additional peptide sequences may be signal peptides, for example.
- the present invention encompasses variants of the polypeptides of the invention, such as those obtained by fusion with other oligo- or polypeptides and/or those which are linked to signal peptides.
- Polypeptides resulting from a fusion with another functional protein, such as another protein from the terpene biosynthesis pathway are also encompassed by the polypeptides of the invention.
- nucleic acid of the invention can be defined as including deoxyribonucleotide or ribonucleotide polymers in either single- or double- stranded form (DNA and/or RNA).
- nucleotide sequence should also be understood as comprising a polynucleotide molecule or an oligonucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid.
- Nucleic acids of the invention also encompass certain isolated nucleotide sequences including those that are substantially free from contaminating endogenous material.
- the nucleic acid of the invention may be truncated, provided that it encodes a polypeptide encompassed by the present invention, as described above.
- Another important tool for transforming host organisms or cells suitable to carry out the method of the invention in vivo is an expression vector comprising a nucleic acid according to any embodiment of the invention. Such a vector is therefore also an object of the present invention.
- an "expression vector” as used herein includes any linear or circular recombinant vector including but not limited to viral vectors, bacteriophages and plasmids. The skilled person is capable of selecting a suitable vector according to the expression system.
- the expression vector includes the nucleic acid of the invention operably linked to at least one regulatory sequence, which controls transcription, translation, initiation and termination, such as a transcriptional promoter, operator or enhancer, or an mRNA ribosomal binding site and, optionally, including at least one selection marker. Nucleotide sequences are "operably linked" when the regulatory sequence functionally relates to the nucleic acid of the invention.
- the expression vectors of the present invention may be used in the methods for preparing a genetically transformed host organism and/or cell, in host organisms and/or cells harboring the nucleic acids of the invention and in the methods for making polypeptides having a P450 monooxygenase activity, as disclosed further below.
- Recombinant non-human host organisms and cells transformed to harbor at least one nucleic acid of the invention so that it heterologously expresses or over-expresses at least one polypeptide of the invention are also very useful tools to carry out the method of the invention.
- Such non-human host organisms and cells are therefore another object of the present invention.
- a nucleic acid according to any of the above-described embodiments can be used to transform the non-human host organisms and cells and the expressed polypeptide can be any of the above-described polypeptides.
- Non-human host organisms of the invention may be any non-human multicellular or unicellular organisms.
- the non-human host organism is a plant, a prokaryote or a fungus. Any plant, prokaryote or fungus is suitable to be transformed according to the present invention. Particularly useful plants are those that naturally produce high amounts of terpenes.
- the plant is selected from the family of Solanaceae, Poaceae, Brassicaceae, Fabaceae, Malvaceae, Asteraceae or Lamiaceae.
- the plant is selected from the genera Nicotiana, Solanum, Sorghum, Arabidopsis, Brassica (rape), Medicago (alfalfa), Gossypium (cotton), Artemisia, Salvia and Mentha.
- the plant belongs to the species of Nicotiana tabacum.
- the non-human host organism is a microorganism.
- Any microorganism is suitable for the present invention, but according to an even more particular embodiment said microorganism is a bacteria or yeast.
- said bacteria is E. coli and said yeast is Saccharomyces cerevisiae.
- Isolated higher eukaryotic cells can also be transformed, instead of complete organisms.
- higher eukaryotic cells we mean here any non-human eukaryotic cell except yeast cells.
- Particular higher eukaryotic cells are plant cells or fungal cells.
- transformed refers to the fact that the host was subjected to genetic engineering to comprise one, two or more copies of each of the nucleic acids required in any of the above-described embodiment.
- the term “transformed” relates to hosts heterologously expressing the polypeptides encoded by the nucleic acid with which they are transformed, as well as over-expressing said polypeptides. Accordingly, in an embodiment, the present invention provides a transformed organism, in which the polypeptides are expressed in higher quantity than in the same organism not so transformed.
- transgenic host organisms or cells such as plants, fungi, prokaryotes, or cultures of higher eukaryotic cells.
- Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, plant and mammalian cellular hosts are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, Elsevier, New York and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd edition, 1989, Cold Spring Harbor Laboratory Press.
- Cloning and expression vectors for higher plants and/or plant cells in particular are available to the skilled person. See for example Schardl et al. Gene 61: 1- 11, 1987.
- transgenic plants Methods for transforming host organisms or cells to harbor transgenic nucleic acids are familiar to the skilled person.
- current methods include: electroporation of plant protoplasts, liposome-mediated transformation, agrobacterium-mediated transformation, polyethylene-glycol-mediated transformation, particle bombardement, microinjection of plant cells, and transformation using viruses.
- transformed DNA is integrated into a chromosome of a non- human host organism and/or cell such that a stable recombinant system results.
- Any chromosomal integration method known in the art may be used in the practice of the invention, including but not limited to recombinase-mediated cassette exchange (RMCE), viral site-specific chromosomal insertion, adenovirus and pronuclear injection.
- RMCE recombinase-mediated cassette exchange
- viral site-specific chromosomal insertion adenovirus and pronuclear injection.
- polypeptide variant as referred to herein means a polypeptide having the above described activity and being substantially homologous to the polypeptide according to any of the above embodiments, but having 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.
- conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. See Zubay, Biochemistry, 1983, Addison-Wesley Pub. Co.
- substitutions can be calculated using substitution score matrices such a PAM-120, PAM-200, and PAM-250 as discussed in Altschul, J. Mol. Biol., 1991, 219, 555-565.
- 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 acid from the polypeptides encoded by the sequences of the invention.
- Variants of the polypeptides of the invention may be used to attain for example desired enhanced or reduced enzymatic activity, modified regiochemistry or stereochemistry, or altered substrate utilization or product distribution, increased affinity for the substrate, improved specificity for the production of one or more desired compounds, increased velocity of the enzyme reaction, higher activity or stability in a specific environment (pH, temperature, solvent, etc), or improved expression level in a desired expression system.
- a variant or site directed mutant may be made by any method 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, for examples plants from the Santalum species, or by artificially programming mutations of nucleotide sequences coding for the polypeptides of the invention. Alterations of the native amino acid sequence can be accomplished by any of a number of conventional methods.
- Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends of the polypeptides of the invention can be used to enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system.
- additional peptide sequences may be signal peptides, for example.
- the present invention encompasses variants of the polypeptides of the invention, such as those obtained by fusion with other oligo- or polypeptides and/or those which are linked to signal peptides.
- Fusion polypeptide encompassed by the invention also comprise fusion polypeptides resulting from a fusion of other functional proteins, such as other proteins from the terpene biosynthesis pathway.
- the alcohols produced herein may be isolated by extraction for example using known methods to extract the alcohols generated in nature (e.g., extraction from Sandalwood).
- the alcohols produced herein have use as fragrant compounds that may be used in perfumery.
- the membrane anchor region of CYP71AV8 was redesigned to introduce the modifications detailed bellow.
- the 5'-end was modified to replace the first amino acids of the membrane anchor region with a peptide sequence shown to improve the heterologous expression of membrane-bound P450s in bacterial cells (Alkier, B.A. et al. Arch. Biochem. Biophys. 322, 369-377 (1995), Haudenschield, et al Arch. Biochem. Biophys. 379, 127-136 (2000)).
- the codon usage was adapted to match the E. Coli codon usage.
- several cDNA were designed for CYP71AV8 with different 3'-end modifications and optimizations:
- CYP71AV8-65188 in this construct the 22 first codons were replaced by a sequence coding for the M ALLLA VFWS ALIIL V peptide (SEQ ID NO 3 and 4).
- CYP71AV8-P2 the entire anchor-encoding sequence was replaced by the anchor sequence of an optimized limonene-hydroxylase from mint (PM2 in
- CYP71AV8-P20 this construct encodes for the same protein as the previous one but the membrane anchor region was further codon optimize (SEQ ID NO 7 and 8).
- the Figure 1 compares the amino acid sequences of the N-terminal regions of the different CYP71AV8 variants and Figure 2 compares the DNA sequences of the 3 constructs.
- the three optimized CYP71AV8 cDNAs were synthesized in-vitro (DNA2.0, Menlo Park, CA, USA) and cloned as Ndel-HindlW fragment into the pCWori+ expression plasmid (Barnes, H.J. Method Enzymol. 272, 3-14; (1996)).
- CYP71AV8 Functional expression of CYP71AV8 in bacterial cells
- the JM109 E. coli cells were transformed with the CYP71AV8 expression plasmids (example 1). Single colonies of transformants were used to inoculated cultures of 5 mL LB medium containing 50 ⁇ g/mL ampicillin. The cells are grown for 10 to 12 hours at 37°C. The cultures were then used to inoculate 250 mL TB Medium (Terrific Broth) supplemented with 50 ⁇ g/mL ampicillin and 1 mM Thiamine HCL.
- the cultures were incubated at 28°C for 3-4 h with moderate shaking (200 rpm) before 75 mg/L ⁇ -aminolevulinic acid (sigma) and 1 mM IPTG (Isopropyl ⁇ -D-l- thiogalactopyranoside) was added, and the cultures were maintained at 28°C for 24-48h with 200 rpm shaking.
- moderate shaking 200 rpm
- IPTG Isopropyl ⁇ -D-l- thiogalactopyranoside
- the expression of the P450 enzymes can be evaluated qualitatively and quantitatively by measuring the CO-binding spectrum (Omura, T. & Sato, R. (1964) /. Biol. Chem. 239, 2379-2387) in the E. coli protein fractions.
- the cells are centrifuged (10 min, 5000 g, 4°C) and resuspended in 35 mL ice-cold buffer 1 (100 mM Tris-HCl pH 7.5, 20% glycerol, 0.5 mM EDTA).
- ice-cold buffer 1 100 mM Tris-HCl pH 7.5, 20% glycerol, 0.5 mM EDTA.
- One volume of 0.3 mg/ml lysozyme (Sigma- Aldrich) in water was added and the suspension left 10-15 min at 4°C with agitation.
- the suspension is centrifuged 10 min at 7000 g and 4°C and the pellet is resuspended in 20 mL buffer 2 (25 mM KP0 4 pH 7.4, 0.1 mM EDTA, 0.1 mM DTT, 20% glycerol).
- the suspension is subject to one cycle of freeze-thaw at -80°C, 0.5 mM PMSF (phenylmethylsulfonyl fluoride, Sigma-Aldrich) is added and the suspension is sonicated 3 times for 20 sec.
- the suspension is centrifuged 10 min at 10000 g (to remove cell debris) and the supernatant is recovered and centrifuged 2 hours at 100,000 g.
- PMSF phenylmethylsulfonyl fluoride
- the pellet (membrane protein fraction) is resuspended in 2-3 ml of buffer 3 (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 20% glycerol).
- buffer 3 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 20% glycerol.
- the protein fraction is diluted (1/10) in buffer 3 to a final volume of 2 mL.
- Some crystals of sodium dithionite (Na 2 S 2 0 4 ) are added, the sample is divided into two cuvettes and the baseline recorded between 370 and 500 nm. The sample cuvette is then saturated with carbon monoxide and the difference spectrum is recorded.
- the concentration of P450 enzyme can be estimated from the amplitude of the peak at 450 nm using the extension coefficient for the reduced CO complex of 91 mM "1 TM "1 (Omura, T. & Sato, R. (1964) /. Biol. Chem. 239, 2379- 2387). Following this procedure, typical CO-spectra with a maximum absorbance at 450 nm were measured for the recombinant CYP71AV8, attesting for a proper folding into functional P450 enzymes.
- CPR P450-reductase
- CPRm previously isolated from Mentha piperita (CPRm, unpublished data, SEQ ID NO 10), optimized the codon usage of the full-lengh cDNA (SEQ ID No 9) and cloned it into the Ncol and Hindlll restriction sites of the pACYCDuet-1 expression plasmid (Novagen) providing the plasmid pACYC- CPRm.
- CYP71AV8 and CPRm were co-expressed in E. Coli cells using the two plasmids pCWori-CYP71AV8-65188 and pACYCDuet-CPRm.
- BL21 StarTM(DE3) E. coli cells (Invitrogen, Carlsbad, CA) were co-transformed with these two plasmids. Transformed cells were selected on carbenicillin (50 ⁇ g/ml) and chloramphenicol (34 ⁇ g/ml) LB- agarose plates. Single colonies were used to inoculate 5 mL liquid LB medium supplemented with the same antibiotics. The culture was incubated overnight at 37°C.
- the different sequiterpene hydrocarbons used as substrates in the bioconversion assays were prepared as described previously using E. coli cells engineered to produced farnesyl diphosphate (FPP) from an heterologous mevalonate pathway and expressing a plant derived sesquiterpene synthase.
- FPP farnesyl diphosphate
- the engineering and use of the E. coli host cells was described in patent WO2013064411 or in Schalk et al (2013) /. Am. Chem. Soc. 134, 18900-18903. Briefly, an expression plasmid was prepared containing two operons composed of the genes encoding the enzymes for a complete mevalonate pathway.
- a first synthetic operon consisting of an E. coli acetoacetyl-CoA thiolase (atoB), a
- Staphylococcus aureus HMG-CoA synthase (mvaS), a Staphylococcus aureus HMG-CoA reductase (mvaA) and a Saccharomyces cerevisiae FPP synthase (ERG20) genes was synthetized in-vitro (DNA2.0, Menlo Park, CA, USA) and ligated into the Ncol-Bam l digested pACYCDuet-1 vector (Invitrogen) yielding pACYC-29258.
- a second operon containing a mevalonate kinase (MvaKl), a phosphomevalonate kinase (MvaK2), a mevalonate diphosphate decarboxylase (MvaD), and an isopentenyl diphosphate isomerase (idi) was amplified from genomic DNA of Streptococcus pneumoniae (ATCC BAA-334) and ligated into the second multicloning site of pACYC-29258 providing the plasmid pACYC-29258-4506.
- This plasmid thus contains the genes encoding all enzymes of the biosynthetic pathway leading from acetyl-coenzyme A to FPP.
- coli cells (BL21 StarTM(DE3), Invitrogen) were co-transformed with the plasmid pACYC-29258-4506 and either the plasmid pET101-Cont2_l (containing a cDNA encoding for the Clausena lansium (+)- -santalene synthase (C1ASS), WO2009109597) or the plasmid pETDuet- SCH10-Tps8201-opt (containing a cDNA encoding for a Santalum album ( + )-a- santalene/(-)-/?-santalene synthase (SaSAS), WO2010067309) and this cells were used to produce and purify (+)- -santalene or a mixture of (+)- -santalene, f-)- ?-santalene, (-)-a- fraws-bergamotene and ( + )-e/?z
- CYP71AV8 The enzymatic activity of CYP71AV8 was evaluated by bioconversion in E. coli cells using the sesquiterpene molecules listed above as substrates.
- BL21 StarTM(DE3) E. coli cells (Invitrogen) transformed with the plasmids pACYCDuet- CPRm and pCWori- CYP71AV8-65188 were cultivated and harvested as described in example 3.
- the substrates sesquiterpene hydrocarbons
- the conversion was allowed to proceed for 24 hours at 20°C with moderate shaking.
- the media were extracted with 2 volumes of MTBE (Methyl ferf-buthyl ether, Sigma) and the extracts were analyzed by GCMS on an Agilent 6890 Series GC system connected to an Agilent 5975 mass detector.
- the GC was equipped with 0.25 mm inner diameter by 30 m SPB-1 capillary column (Supelco, Bellefonte, PA).
- the carrier gas was He at a constant flow of 1 mL/min.
- the initial oven temperature was 80°C (1 min hold) followed by a gradient of 10°C/min to 300°C.
- the identification of the products was based on the comparison of the mass spectra and retention indices with authentic standards and internal databases.
- the 6 plasmids were transferred into E. Coli BL21 StarTM(DE3) cells and the recombinant cells were used in bio-conversion assays as described in example 4.
- the (+ j-a-santalene and the (+ j-a-santalene, f-j- ?-santalene, (-)- ⁇ -fraws-bergamotene and ( + j-e/?z ' - ?-santalene mixture were used as substrates and quantities of total oxygenated sesquiterpene products were evaluated.
- CYP71AV8-P2 or CYP71AV8-P20 cDNA was used and for the terpene synthase, the Clausena lansium (+ j-a-santalene synthase cDNA (C1ASS) (WO2009109597) or a cDNA encoding for a Santalum album ( + )-a-santalene/( - - ?-santalene synthase (SaSAS) (WO2010067309) was used.
- Four plasmids were thus constructed using the following procedure.
- a codon optimized version of the C1ASS cDNA (SEQ ID NO 19-20) was designed and synthesized (DNA 2.0) and cloned in the Ndel-Kpnl sites of the pETDUET- 1 plasmid (Novagen) providing the plasmid pETDuet-Tps2opt.
- For SaSAS an optimized full-length cDNA was designed (SEQ ID NO 21-22), synthesized and cloned in the pjexpress414 plasmid (DNA2.0) providing the plasmid pJ414-SaTps8201-l-FLopt.
- primer were designed for cloning using the In- Fusion ® technique (Clontech, Takara Bio Europe).
- the optimized C1ASS cDNA and the optimized SaSAS cDNA were amplified using these primers and the pETDuet-Tps2opt and pJ414- SaTps8201-l-FLopt plasmids as template, respectively.
- the two PCR products were ligated in the plasmids pCWori-CYP71AV8-P2-CPRm or pCWori-CYP71AV8-P20- CPRm digested with the Hin Ull restriction enzyme and using the In-Fusion ® Dry-Down PCR Cloning Kit (Clontech, Takara Bio Europe), providing four new plasmids : pCWori- CYP71AV8-P2-CPRm-ClASS, pCWori-CYP71AV8-P2-CPRm-SaSAS, pCWori- CYP71AV8-P20-CPRm-ClASS, and pCWori-CYP71AV8-P20-CPRm-SaSAS (SEQ ID NO 23-26).
- Transformed cells were selected on carbenicillin (50 ⁇ g/ml) and chloramphenicol (34 ⁇ g/ml) LB-agarose plates. Single colonies were used to inoculate 5 mL of LB medium supplemented with appropriate antibiotics. Cultures were incubated overnight at 37°C and 250 rpm.
- CYP71AV8 is highly selective for the 'terminal trans carbon' of (+)- -santalene and f-)- ?-santalene and produced exclusively (3 ⁇ 4)-a-santalol, (3 ⁇ 4)- ?-santalol.
- L358 was first selected as an active site residue controlling the enzyme activity.
- a series of variant of CYP71AV8 were generated by replacing the codon encoding for L358 by codons encoding for other amino acids. The mutation was introduced in a two-step PCR procedure using a combination of degenerated
- a first PCR was performed to amplify the 5' portion of the cDNA using the mutagenesis reverse primer AV8-L358- rev (5'-
- the pCWori-CYP71AV8-P2-CPRm-ClASS was use for the template.
- a second round of amplification was performed using the two above PCR products as template and the primers AV8-L358-fw + AV8-CPR-rev and allowed to amplify the full- length CYP71AV8 variant cDNAs. All the PCR reactions were perfomed using the PfuUltra II fusion HS DNA polymerase (Stratagene) following the manufacturer instruction.
- the modified cDNA were ligated into the Ndel-Sall digested pCWori- CYP71 AV8-P2-CPRm-ClASS using the Gibson Assembly Master Mix (New England Biolabs).
- each CYP71AV8 variant was performed using the in-vivo sesquiterpene production method described in example 6. Briefly, the pC Wori+ plasmid containing one of the CYP71AV8 variant cDNA, the CPRm cDNA and the CIASS cDNA was co-transformed with the pACYC-29258 plasmid into KRX E. Coli cells (Promega). The transformed cells were selected, cultivated and the production of sesquiterpenes was evaluated as described in example 6. As shown in figure 7, compared to the wild type P450 enzyme, with some of the variants (Zj-a-santalol was produced in addition to the trans oxidation products.
- CYP71AV8 can be engineered and used to produce the (Zj-a-santalol.
- the L358T, L358S, L358A and L358F variants can be used for the terminal oxidation of (+ j- -santalene with a selectivity up to 46 % for the cis terminal carbon.
- the variants of CYP71AV8 were evaluated for the production of (Z)- ?-santalol.
- New plasmids were prepared by replacing the CIASS cDNA in the above plasmid by the SaSAS cDNA.
- the plasmid pCW-CYP71AV8-L358F- CPRm-ClASS was digested with the restriction enzymes Hindlll and EcoRI to remove the CIASS cDNA.
- the pCWori-CYP71AV8-P2-CPRm-SaSAS was digested with the same enzymes to recover the SaSAS cDNA with the compatible cohesive ends.
- the linearized vector and the digested insert were ligated using the T4 DNA ligase (New England Biolabs).
- the plasmid thus obtained was used for in-vivo production of oxygenated sesquiterpenes in E. coli cells in the same condition as described above.
- the figure 8 present the GCMS profile of the analysis of the products formed by CYP71AV8- L358F and shows that modified CYP71AV8 enzymes can also be used to produce ( ⁇ )- ⁇ - santalol.
- CYP71AV1 NCBI accession No ABB82944.1 was evaluated for the oxidation of sesquiterpenes with the santalene skeleton.
- a plasmid was prepared with a configuration similar to the plasmids described in example 5 : a bi-cistronic operon containing an optimized cDNA encoding for an N-terminal modified CYP71AV1 protein (SEQ ID NO 53 and 54) and the aaCPR cDNA (example 5) was designed, synthesized in- vitro
- CYP71AV1 a synthetic operon containing the CYP71AV1 cDNA, the aaCPR and the ( + j-a-santalene synthase cDNA (CIASS) was prepared.
- the CYP71AV1 cDNA was recovered from the bi-cistronic operon described in the previous paragraph by digestion with the same enzymes and ligated, using the T4 DNA ligase (New England Biolabs), into the digested pCWori plasmid described above yielding the plasmid pCWori-CYP71AVl-CPRm-ClASS.
- This plasmid together with the plasmid pACYC- 29258-4506 were used to co-transform E coli BL21 StarTM(DE3) (Invitrogen) cells.
- the recombinant cells were cultivated in conditions allowing the production of sesquiterpene molecules as described in example 6.
- a P450-BM3 mutant library of 24 variants was constructed by systematically combining five hydrophobic amino-acids (alanine, valine, phenylalanine, leucine and isoleucine) in two positions located close to the centre of the heme group of P450-BM3. Altering the side chain size of these two amino acids has been shown to drastically change the shape of the substrate binding cavity in close proximity of the heme group (Appl Microbiol Biotechnol 2006, 70:53; Adv Synth Catal 2006, 348:763).
- the first hot spot (Phe 87) is known to alter substrate specificity and regioselectivity while the second position (Ala 328) has been predicted to interact with all substrates during oxidation (ChemBiochem 2009, 10:853).
- the P450-BM3 variants were either generated using the QuickChangeTM site-directed mutagenesis kit (Invitrogen, Carlsbad, CA) or were chemically synthetized by DNA2.0 (Menlo Park, CA).
- the P450-BM3 variants and wild- type were subcloned into the bacteria expression plasmids pET22b, pET28+, pETDuet-1 and pCDFDuet-1 (Novagen, Madison, WI) and were transformed in Escherichia coli BL21(DE3) or BL21StarTM(DE3) (Invitrogen, Carlsbad, CA).
- A/pfta-santalene in vitro screening of the P450-BM3 Library
- the 24 P450-BM3 mutants and the wild-type version of the enzyme were heterologously expressed in E. coli BL21(DE3) cells as reported previously (Adv. Synth. Catal. 2003, 345:802).
- a single colony of transformed cells was used to inoculate 2 ml of Luria-Bertani (LB) medium supplemented with 30 ⁇ g/ml kanamycin and grown at 37°C with orbital shaking (150 rpm) until OD5 78 reaches a value of 0.6 to 1.0.
- This pre-culture was used to inoculate 200 ml of LB medium containing 30 ⁇ g/ml kanamycin.
- the cells were grown at 37°C with orbital shaking at 160 rpm to an OD57 8 of 0.8. Expression of the protein was then induced by the addition of 0.35mM isopropyl ⁇ -D-l- thiogalactopyranoside (IPTG). After 6 hours of growth at 30°C under agitation, the cells were harvested by centrifugation and lysed by sonication.
- IPTG isopropyl ⁇ -D-l- thiogalactopyranoside
- the alpha-santalene used as substrate in the bioconversion assays was prepared as described in Example 4. The conversions were carried out in 1 ml of 50 mM potassium phosphate buffer containing -0.5 ⁇ CYP enzyme, 2% (v/v) DMSO, and 0.2 mM ⁇ - santalene substrate. Reaction was started by adding 0.1 mM NADPH and was carried out for 22h at room temperature with moderate shaking.
- the P450-BM3 mutant library was also screened in vivo using a bacteria strain engineered to produce (+)- -santalene from a simple carbon source.
- the FPP- overproducing strain described in Example 4 was transformed with a pETDuet-1 plasmid containing a codon-optimized version of a (+ )- -santalene synthase from Clausena lansium (C1ASS) (WO2009109597) (SEQ ID No 19 and 20) and each of the P450-BM3 variants cloned into the first and second multiple cloning sites (MCS) of the vector, respectively.
- C1ASS Clausena lansium
- the ( + )- -santalene synthase cDNA was cloned into the pETlOlexpression plasmid (Novagen) and each of the P450-BM3 mutants from the library into the pCDFDuet-1 vector (Novagen).
- the resulting recombinant vectors were co-transformed in the FPP-overproducing strain. Single colonies of transformed cells were used to inoculate 5 mL of LB medium supplemented with the appropriate antibiotics. Cultures were then incubated overnight at 37°C and 250 rpm. The following day, 2 mL of Terrific Broth (TB) medium
- GC/MS methyl ieri-butyl ether
- sesquiterpene hydrocarbons consisting of (+ )-a-santalene, ( - )- ?-santalene, ( - )-a-trans- bergamotene and ( + )-e/?z ' - ?-santalene.
- the FPP-overproducing bacteria strain described in Example 4 was transformed with a recombinant pETDuet- 1 expression
- (+)-a-santalene, f-)- ?-santalene, (-)-a-trans -bergamotene and ( + )-e/?z ' - ?-santalene were efficiently oxidized by the P450-BM3 double-mutant to yield fZj-a-santalol, (Z)- ?-santalol, (Zj-a-fraws-bergamotol and (Z)-e/?z ' - ?-santalol.
- the seeds of S. album were obtained from B&T World Seeds (Aigues-Vives, France) and from Sandeman Seeds (Lalongue, France). The seeds were first surface sterilised in 2.5% Hypochlorous acid (HCIO) for 120 min, and rinsed 3 times in sterile ultrapure water. The seeds were then shelled and placed on MS basal medium (Murashige & Skoog, 1962, Physiologia Plantarum 15, 473-497) supplemented with 15 g/L sucrose and 7.8 g/L agar, pH 5.7. Germination was typically observed after 9 to 18 days with a yield of approximately 40%.
- MS basal medium Merashige & Skoog, 1962, Physiologia Plantarum 15, 473-497
- the whole transcriptome was sequenced using the Illumina Total RNA-Seq technique and the Illumina HiSeq 2000 sequencer. A total of 108.7 millions of paired- reads of 2x100 bp were generated. The reads were assembled using the De Novo Assembly application of CLC-Bio Genomic Workbench (CLCBo, Denmark). A total 82' 479 of contigs with an average size of 683 bp were assembled. The contigs were search using the tBlastn algorithm (Altschul et al, J. Mol. Biol. 215, 403-410, 1990) and using as query sequence known P450 amino acid sequences such as the sequence of CYP71AV1 (NCBI accession No ABB82944.1).
- SCH37-Q816 contained a 1503 bp length open reading frame (ORF) (SEQ ID NO 70) encoding for a 500 amino acid protein, SaCP816 (SEQ ID NO 71). This amino acid showed homology with know cytochrome P450 sequences the closest sequence being a P450 from Vitis vinifera, CYP71D10 (NCBI accession No AAB94588.1) sharing 62% amino acid sequence identity.
- the protein was heterologously expressed in E. coli cells.
- the ORF sequence was modified for improved expression in E. Coli: the first 17 codons were replaced by the codons encoding for the MALLLAVFWSALIILV peptide and the codon usage of the whole ORF sequence was modified to match the E. coli codon usage.
- This cDNA (SaCP120293 (SEQ ID NO 72) encoding for the modified SaCP816 (SEQ ID NO 73) was synthesized in-vitro (DNA2.0) and cloned in the pJExpress404 plasmid (DNA2.0). The heterologous expression was performed as described in example 2.
- a bicistronic operons was designed to express the P450 enzyme and a CPR from a single plasmid and under the control of a unique promoter.
- the optimized SaCP120293 cDNA was combined with the CPRm cDNA (SEQ ID No 9, Example 3) to prepare a bicistronic construct (SEQ ID NO 74) containing successively the P450 cDNA a linker sequence including a ribosome binding site (RBS) and the CPRm cDNA.
- This construct was prepared by PCR by amplifying the P450 and CPR cDNAs separately and with 5' and 3 ' overhangs suitable for the cloning using the In-Fusion ® procedure (Clotech) in the Ndel-Hindlll sites of the pCWori+ plasmid (Barnes H.J (1996) Method Enzymol. 272, 3- 14) providing the plasmid SaCP816-CPRm-pCWori (SEQ ID NO 74).
- the JM109 E. coli cells were transformed with the SaCP816-CPRm-pCWori expression plasmid.
- the transformed cells were grown and the cell-free extract containing the recombinant proteins were prepared as described in example 2. This protein fraction was used for the evaluation the enzymatique conversion of sesquiterpene molecules (example 16).
- Example 16 The JM109 E. coli cells were transformed with the SaCP816-CPRm-pCWori expression plasmid.
- the transformed cells were grown and the cell-free extract containing the recombinant proteins were prepared as described in example 2. This protein fraction was used for the evaluation the enzymatique conversion of sesquiterpene molecules (example 16).
- Example 16 Example 16
- the assays were performed in 1 mL of 100 mM Tris-HCL pH 7.4 buffer containing 20 to 50 microL protein extract, 500 microM NADPH (reduced Nicotinamide adenine dinucleotide phosphate), 5 microM FAD (Flavine adenine dinucleotide), 5 microM FMN (flavine mononucleotide), and 300 microM of
- sesquiterpenes either (a)-santalene or a mixture of (+)- -santalene, f-)- ?-santalene, (-)-a- fraws-bergamotene and (+)-e/?z ' - ?-santalene).
- Figure 13 shows that (+)-a- santalene, ( - )- ?-santalene, ( - j-a-fraws-bergamotene and ( + )-e/?z ' - ?-santalene were oxidized by SaCP816 to forme fZj-a-santalol, (Z)- ?-santalol, (Z)-a- fraws-bergamotol and (Z)-epi-?-santalol.
- (+)- -santalene and the (+)- -santalene, f-)- ?-santalene, ( - )-a-trans -bergamotene, ( + )-e/?z ' - ?-santalene or other structurally similar molecules can also be produced directly in E. Coli cells engineered to produce sesquiterpenes from a carbon source such as glucose or glycerol.
- Plasmids were prepared consisting of the pCWori+ plasmid containing a synthetic operon composed of the SaCP120293 cDNA (SEQ ID No 72), the CPRm cDNA (SEQ ID No 9) and a terpene synthase encoding cDNA.
- the terpene synthase the Clausena lansium ( + j- -santalene synthase cDNA (C1ASS) (WO2009109597) or a cDNA encoding for a Santalum album f+)- -santalene /f-j- ⁇ -santalene synthase (SaSAS) (WO2010067309) was used.
- Two plasmids were thus constructed using a procedure similar to the procedure described in example 6.
- the codon optimized ( + )- -santalene synthase cDNA (SEQ ID NO 19) and the (+ )-a-santalene/(-)- ?-santalene synthase cDNA (SEQ ID NO 21) were amplified as described in example 6 and ligated using the In-Fusion ® Dry-Down PCR Cloning Kit (Clontech, Takara Bio Europe) in the plasmids SaCP816-CPRm-pCWori digested with the Hin HII restriction enzyme providing the two new plasmids SaCP816- CPRm-ClASS-pCWori (SEQ ID NO 75) and SaCP816-CPRm-SaSAS-pCWori (SEQ ID NO 76).
- E. coli XRX cells Promega co-transformed with either of these 2 plasmids and with the plasmid pACYC-29258-4506 carrying a complete mevalonate pathway (example 4).
- Transformed cells were selected on carbenicillin (50 ⁇ g/ml) and chloramphenicol (34 ⁇ g/ml) LB- agarose plates. Single colonies were used to inoculate 5 mL of LB medium supplemented with appropriate antibiotics. Cultures were incubated overnight at 37°C and 250 rpm.
- the culture were cooled down to 20°C and the expression of the proteins was induced with 0.1 mM IPTG (Isopropyl ⁇ -D- l- thiogalactopyranoside) and 0.1 % Rhamnose, and 75 ⁇ g/L ⁇ -aminolevulinic acid (sigma) and 2% (v/v) of decane were added.
- IPTG Isopropyl ⁇ -D- l- thiogalactopyranoside
- Rhamnose 75 ⁇ g/L ⁇ -aminolevulinic acid (sigma) and 2% (v/v) of decane were added.
- the whole culture broth was extracted with 1 volume of MTBE and analyzed by GCMS as described in example 4.
- the protein was heterologously expressed in E. coli cells.
- the ORFs sequence were modified to improve the expression in E. Coli: the 18 first codons were replaced by the codons encoding for the MALLLAVFWSALII peptide and the codon usage of the whole ORF sequence was optimized.
- the new cDNA, SaCP120292 (SEQ ID NO 80), encoding for the modified SaCP10374 (SEQ ID NO 81) was synthesized in-vitro (DNA2.0) and cloned in the pJExpress404 plasmid (DNA2.0).
- heterologous expression was performed as described in example 2. Following this procedure, typical CO-spectra with a maximum absorbance at 450 nm was measured for this new recombinant S. abum P450, attesting for a proper folding into functional P450 enzymes.
- a P450 reductase (CPR) was coexpressed.
- CPR P450 reductase
- a bicistronic operons was designed similarly as described in example 15 to express SaCP10374 and CPRm (a mint P450 reductase) from a single plasmid and under the control of a unique promoter.
- the optimized SaCP 12092 cDNA was combined with the CPRm cDNA to prepare the bicistronic constructs (SEQ ID NO 82) containing successively the P450 cDNA a linker sequence including a ribosome binding site (RBS) and the CPRm cDNA.
- This construct was prepared by PCR as described in example 15. and cloned in the pCWori+ plasmid (Barnes H.J (1996) Method Enzymol. 272, 3-14) providing the plasmid SaCP10374-CPRm-pCWori.
- the JM109 E. coli cells were transformed with these bicistronic expression plasmid.
- the transformed cells were grown and the cell-free extract containing the recombinant proteins were prepared as described in example 2.
- the membrane protein fractions were used for the evaluation the enzymatique conversion of sesquiterpene molecules (example 21)
- Figure 15 and 16 show that (+)- -santalene, f-)- ?-santalene, f-j-a-fraws-bergamotene and (+)- ⁇ - ⁇ - santalene were oxidized by SaCP10374 to form (.Ej-a-santalol, (3 ⁇ 4)- ?-santalol, (E)-a- fraws-bergamotol and (E)-epi- ?-santalol.
- the (- j-sesquisabinene B and (-)- ⁇ - bisabolene were produced using the pETDuet expression plasmid containing either a cDNA encoding for SaTps647, a Santalum album (- j-sesquisabinene B synthase (NCBI accession No. ADP37190.1) or a cDNA encoding for SaTps30, a Santalum album (-)- ⁇ - bisabolene synthase (NCBI accession No.
- ADP37189.1 in combination with the pACYC-29258-4506 plasmid described in example 4.
- the ⁇ -farnesene was obtained from Bedoukian (Dambury, Ct, USA), oc-farnesene was from Treatt (Suffolk, UK) and (-)-a- fraws-bergamotene was purified from citrus oil.
- album P450s are regioselective for one of the two carbons of the terminal gem-dimethyl group (Rl or R2 in figure 27) : SaCP816 catalyzes the selective oxidation of the carbon atom of the methyl in cis position relative to the terminal doubel bond (Rl in figure 27), whereas SaCP10374 catalyzes the oxidation of the same substrates exclusively on the carbon atom of the methyl group in trans relative to the terminal doubel bond (R2 in figure 27).
- the trans and cis isomers of each sesquiterpene alcohol are easily separated in the chromatographic conditions used in these assays.
- the formation of the corresponding aldehyde when the trans-methyl group is oxidyzes is attributed to E. coli endogenous alcohol dehydrogenase activity.
- SaCP10374 isolated from Santalum album can be used for the selective hydroxylation of the cis-terminal and trans-terminal carbon, respectivly, of various sesquiterpene molecules have structure similarities with ⁇ -farnesene, -farnesene, (+ j- -santalene, (-)- ?-santalene, (-)-a- fraws-bergamotene, (-)-sesquisabinene B or (- )- ?-bisabolene.
- the oxidized sesquiterpene molecules described in example 21 and 22 can also be produced directly using whole cells, such as for example E. coli cells engineered to produce sesquiterpenes from a carbon source such as glucose or glycerol.
- Plasmids were prepared consisting of the pCWori+ plasmid containing a synthetic operon composed of the SaCP120293 cDNA (SEQ ID No 72), or the SaCP120292 (SEQ ID No 80), the CPRm cDNA (SEQ ID No 9) and a terpene synthase encoding cDNA (encoding either for an Artemisia annua ?-farnesene synthase cDNA (NCBI accession No AAX39387.1.1), a Picea abies -farnesene synthase (NCBI accession No AAS47697.1), a S.
- album (-)- Sesquisabinene B (NCBI accession No ADP37190.1), a S. album (-)-fi-Bisabolene synthase (NCBI accession No ADP37189.1), a Clausena lansium oc-santalene synthase (NCBI accession No ADR71055.1) or a S. album or-/?-santalene synthase(NCBI accession No ADP30867.1)).
- the plasmids carrying the different combinations of synthetic operons were prepared using the following procedure.
- the plasmid pD444-SR-AaBFS (containing an optimized cDNA encoding for AaBFS, an Artemisia annua (3 ⁇ 4)- ?-farnesene synthase (NCBI accession No AAX39387.1), the plasmid pD444-SR-PaAFS (containing an optimized cDNA encoding for PaAFS, a Picea abies (.Ej-a-farnesene synthase (NCBI accession No.
- AAS47697.1 were used to amplify by PCR the (3 ⁇ 4)- ?-farnesene synthase and (3 ⁇ 4)-a-farnesene synthase cDNAs, respectively.
- the plasmids pETDuet-SaTps647and pETDuet-SaTps30 were used as template to amplify by PCR the sesquisabinene B synthase and the bisabolene synthase cDNAs, respectively.
- primer were designed for the cloning using the In-Fusion ® technique
- the AaBFS cDNA was amplified using the foward primer CPRm_aaBFS_Inf 1
- the PaAFS cDNA was amplified using the foward primer
- the SaTps647 cDNA was amplified using the primer foward CPRm_Tps647_inf 1 (5 ' GCGTGATGTGTGGTAATAAAAGCTTAGGAGGTAAAAAT GGCGACCGTTGTGGATGATTCT-3 ' ) and the primer reverse Tps647_Inf2
- the SaTps30 cDNA was amplified using the primer foward CPRm_Tps30_Infl-
- Tps30_Inf2 (GTGATGTGTGGTAATAAAAAGCTGAATTCTTAGTCCTCTTCATTCA GCGGGATCGGGTG).
- SaCP10374-CPRm-pCWOri (SEQ ID NO 82) digested with the HindSR restriction enzyme and using the In-Fusion ® Dry-Down PCR Cloning Kit (Clontech, Takara Bio Europe), providing the new plasmids SaCP816-CPRm-SaTPS647-pCWori (SEQ ID NO 83), SaCP10374-CPRm-SaTPS647-pCWori (SEQ ID NO 84), SaCP816- CPRm-SaTPS30-pCWori (SEQ ID NO 85), SaCP10374-CPRm-SaTPS30-pCWori (SEQ ID NO 86), SaCP816-CPRm-AaBFS-pCWori (SEQ ID NO 87), SaCP10374-CPRm- AaBFS-pCWori (SEQ ID NO 88), SaCP816-CPRm-PaAFS-pCWori (SEQ ID NO 89), Sa
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WO2018160066A1 (en) | 2017-03-02 | 2018-09-07 | Isobionics B.V. | Santalene synthase |
WO2021130241A1 (en) | 2019-12-23 | 2021-07-01 | Firmenich Sa | Biochemically produced sandalwood oil |
EP3746551A4 (en) * | 2018-01-31 | 2021-11-03 | The Regents Of The University Of Michigan | Biocatalyst and methods for synthesizing mixed disulfide conjugates of thienopyridine compounds |
EP3818153A4 (en) * | 2018-07-06 | 2022-04-06 | The Regents Of The University Of Colorado | Genetically encoded system for constructing and detecting biologically active agents |
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CA2924541A1 (en) * | 2013-09-19 | 2015-03-26 | Firmenich Sa | Method for producing fragrant alcohols |
WO2018015453A2 (en) * | 2016-07-20 | 2018-01-25 | Firmenich Sa | Vetiver |
MX2020005258A (en) * | 2018-01-18 | 2020-08-24 | Firmenich & Cie | Cytochrome p450 monooxygenase catalyzed oxidation of sesquiterpenes. |
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NL2023931B1 (en) * | 2019-10-02 | 2021-06-01 | Isobionics B V | Oxidation of santalene to santalol |
CN115176023A (en) * | 2020-01-23 | 2022-10-11 | 阿米瑞斯公司 | Amorpha-4, 11-diene 12-monooxygenase variants and uses thereof |
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WO2018160066A1 (en) | 2017-03-02 | 2018-09-07 | Isobionics B.V. | Santalene synthase |
EP3746551A4 (en) * | 2018-01-31 | 2021-11-03 | The Regents Of The University Of Michigan | Biocatalyst and methods for synthesizing mixed disulfide conjugates of thienopyridine compounds |
EP3818153A4 (en) * | 2018-07-06 | 2022-04-06 | The Regents Of The University Of Colorado | Genetically encoded system for constructing and detecting biologically active agents |
US11472847B2 (en) | 2018-07-06 | 2022-10-18 | The Regents Of The University Of Colorado | Genetically encoded system for constructing and detecting biologically active agents |
US11993635B2 (en) | 2018-07-06 | 2024-05-28 | The Regents Of The University Of Colorado, A Body Corporate | Genetically encoded system for constructing and detecting biologically active agents |
WO2021130241A1 (en) | 2019-12-23 | 2021-07-01 | Firmenich Sa | Biochemically produced sandalwood oil |
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