METHOD FOR PRODUCING PATCHOULOL AND 7-EPI-ALPHA-SELINENE
20170283841 ยท 2017-10-05
Assignee
Inventors
Cpc classification
C12N15/8243
CHEMISTRY; METALLURGY
International classification
C12P15/00
CHEMISTRY; METALLURGY
C12P5/00
CHEMISTRY; METALLURGY
C12N15/82
CHEMISTRY; METALLURGY
Abstract
The present invention provides a method of producing patchoulol and 7-epi--selinene, said method comprising contacting at least one polypeptide with farnesyl phyrophosphate (FPP). In particular, said method may be carried out in vitro or in vivo to produce patchoulol and 7-epi--selinene, compounds which can be useful in the field of perfumery. The present invention also provides the amino acid sequence of a polypeptide useful in the method of the invention. A nucleic acid encoding the polypeptide of the invention and an expression vector containing said nucleic acid are also part of the present invention. A non-human host organism or a cell transformed to be used in the method of producing patchoulol and 7-epi--selinene is also an object of the present invention.
Claims
1. A recombinant polypeptide having sesquiterpene synthase activity and comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the polypeptide when expressed produces a mixture of terpenes comprising one or more of patchoulol and 7-epi--selinene.
2. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 95% or 98% sequence identity to SEQ ID NO: 1.
3. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
4. An isolated recombinant nucleic acid molecule comprising a) a nucleotide sequence encoding the polypeptide of claim 1; b) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2 or the complement thereof.
5. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2 or the complement thereof.
6. An expression vector comprising the nucleic acid molecule of claim 4 or a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2 or the complement thereof.
7. The expression vector of claim 6, wherein the vector is in the form of a viral vector, a bacteriophage or a plasmid.
8. The expression vector of claim 6, wherein the vector further comprises at least one regulatory sequence operably linked to the nucleic acid, and optionally at least one selection marker.
9. The expression vector of claim 8, wherein the at least one regulatory sequence controls transcription, translation initiation or termination, and wherein the at least one regulatory sequence comprises a transcriptional promoter, operator or enhancer or an mRNA ribosomal binding site.
10. A non-human host organism or a host cell transformed to harbor at least one nucleic acid molecule of claim 4 or a vector comprising said nucleic acid, so that it heterologously expresses or over-expresses at least one polypeptide encoded by the nucleic acid molecule.
11. The non-human host organism of claim 10, wherein the non-human host organism or host cell is a plant, a microorganism, a prokaryote, or a fungus.
12. The non-human host organism or host cell of claim 11, wherein the non-human host organism or host cell is a bacteria or a yeast.
13. The non-human host organism or host cell of claim 12, wherein the bacteria is E. coli and the yeast is Saccharomyces cerevisiae.
14. The host cell of claim 10, wherein the host cell is a plant cell or a fungal cell.
15. A method for producing at least one polypeptide having sesquiterpene synthase activity comprising a) culturing a non-human host organism or a host cell transformed to express or over-express the polypeptide of claim 1 under conditions that allow for the production of the polypeptide; and b) optionally isolating the polypeptide from the non-human host organism or cell cultured in step a) or from culture media used in culturing the non-human host organism or a host cell.
16. The method of claim 15, further comprising, prior to step a), transforming a non-human host organism or host cell with an expression vector comprising the nucleic acid molecule of claim 4, so that it harbors said nucleic acid and expresses or over-expresses a polypeptide encoded by said nucleic acid.
17. A method for preparing a variant polypeptide having sesquiterpene synthase activity comprising a patchoulol and 7-epi--selinene synthase activity, the method comprising: (a) selecting a nucleic acid according to claim 4; (b) modifying the selected nucleic acid to obtain at least one mutant nucleic acid; (c) transforming host cells or unicellular organisms with the mutant nucleic acid sequence to express a polypeptide encoded by the mutant nucleic acid sequence; (d) screening the polypeptide for at least one modified property; and, (e) optionally, if the polypeptide has no desired variant patchoulol and 7-epi--selinene synthase activity, repeating the process steps (a) to (d) until a polypeptide with a desired variant patchoulol and 7-epi--selinene synthase activity is obtained; (f) optionally, if a polypeptide having a desired variant patchoulol and 7-epi--selinene synthase activity was identified in step d), isolating the corresponding mutant nucleic acid obtained in step (c).
Description
DESCRIPTION OF THE DRAWINGS
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SPECIFIC EMBODIMENTS OF THE INVENTION OR EXAMPLES
[0128] The invention will now be described in further detail by way of the following Examples.
Example 1
[0129] Isolation of a Sesquitepene Synthase cDNA from Valeriana Jatamansi Roots
[0130] Valeriana jatamansi (synonym: Valeriana walichii) plants were obtained from B & T World Seeds (Paguignan, Aigues-Vives, France). The plants were cultivated in a green house at minimal temperature of 14 C. Rhizomes, young and mature roots were collected separately and immediately frozen in liquid nitrogen. The material was crushed and granddad to a fine powder in liquid nitrogen using a mortar and pestle. Total RNA was extracted using the CONCERT Plant RNA Reagent from Invitrogen following the manufacturer instruction except for the isopropanol precipitation which was replaced by a 2M LiCl precipitation. The quality of the RNA was evaluated on an agarose gel by verifying the integrity of the ribosomal RNA bands. The mRNA was purified from the total RNA by oligodT-cellulose affinity chromatography using the FASTTRACK 2.0 mRNA isolation Kit (Invitrogen) following the manufacturer's instructions.
[0131] Degenerated oligonucleotides were designed to conserved motifs observed in alignments of the amino acid sequences of plant sesquiterpene synthases (Deguerry et al, 2006, Arch Biochem Biophys. 454(2), 123-36). RT-PCR with these sesquiterpene synthases-specific oligonucleotides were performed using the Qiagen OneStep RT-PCR Kit and an Eppendorf Mastercycler gradient thermal cycler. Typical reaction mixtures contain 10 l SX Qiagen OneStep RT-PCR buffer, 200 M each dNTP, 0.4 M each primer, 2 l Qiagen OneStep RT-PCR Enzyme Mix, 1 l RNASIN Ribonuclease Inhibitor (Promega Co.) and 1 g total RNA in a final volume of 50 l. The thermal cycler conditions were: 30 min at 50 C. (reverse transcription); 15 min at 95 C. (DNA polymerase activation); 35 cycles of 30 sec at 94 C., 30 sec at 41 to 51 C. and 1 min at 72 C. The sizes of the PCR products were evaluated on a 1.2% agarose gel. The bands corresponding to the expected size were excised from the gel, purified using the QIAQUICK Gel Extraction Kit (Qiagen) and cloned in the pCR 2.1-TOPO vector using the TOPO TA cloning Kit (Invitrogen). Inserted cDNAs were then subject to DNA sequencing and the sequence compared against the GenBank non-redundant protein database (NCBI) using the BLASTX algorithm (Altschul et al 1990, J. Mol. Biol. 215, 403-410). The combination of the forward primer TpsCF2 (SEQ ID NO:3) (5-GGGA(A/T)(A/T)G(A/T)(A/T/G/C)(A/T)(C/T/G)GTTGAA(T/G)(T/G)TTATTTTTGG-3) and the reverse primer TpsCR3 (SEQ ID NO:4) (5-GT(A/T)(C/G)CGTG(A/T/G/C/)G(A/C/T)GTCGTA(A/C/T)G(T/G)GTCATC-3) provided a fragment of 81 bp presenting sequence homology with known plant sesquiterpene synthases.
[0132] A combination of 3 and 5 Rapid Amplification of cDNA Ends (RACE) was then used to obtain the full-length sequence of the cDNA corresponding to this fragment. For the 3RACE, two sens oligonucleotides were deduced from the 81 bp sequence obtained by RT-PCR: 20-3R1 (SEQ ID NO:5) and 20-3R2 (SEQ ID NO:6). An adaptor-ligated double stranded cDNA was prepared using the MARATHON cDNA Amplification Kit (Clontech) following the manufacturer's protocol. This cDNA was prepared from mRNA purified from V. jatamansi rhizome total RNA. Typical RACE reaction mixtures contain, in a final volume of 50 l, 5 l 10PCR Reaction Buffer (Clontech), 200 M each dNTP, 1 l ADVANTAGE 2 Polymerase Mix, 200 nM each primer and 5 l of 250 fold diluted cDNA. Amplification was performed on an Eppendorf Mastercycler gradient thermal cycler. The thermal Cycling conditions were as follows: 1 min at 94 C., 5 cycles of 5 sec at 94 C. and 3 min at 72 C., 5 cycles of 5 sec at 94 C. and 3 min at 70 C., 20 cycles of 5 sec at 94 C. and 3 min at 68 C. The amplification products were evaluated, sub-cloned, and the sequence analyzed as described above. The 3-end of the cDNA was obtained after a first round amplification with the primer 20-3R1 and the AP1 primer (clontech) and a second round amplification with the primer 20-3R2 and the AP2 primer (Clontech).
[0133] Two reverse primers were deduced from the sequence obtained by 3RACE: 20-5R1 (SEQ ID NO:7) and 20-5R2 (SEQ ID NO:8). The SMART RACE cDNA amplification Kit (Clontech) was used to prepare a 5RACE-ready cDNA from V. jatamansi root total RNA. The 5RACE was performed in 50 l of 1 advantage 2 PCR buffer containing 200 M dNTPs, 5 l Universal Primer Mix (Clontech), 2 M of the first gene specific primer (20-5R1), 2.5 l 5RACE-ready cDNA and 1 l ADVANTAGE 2 Polymerase Mix. A second round of amplification was performed in the same condition with 1 l of the first amplification, the nested gene specific primer (20-5R2) and the Nested Universal primer (Clontech). The temperature cycling condition and DNA fragment analysis were as described above for the in 3RACE.
[0134] The combination of 3 and 5-RACE allowed the reconstitution of the full-length sequence of a cDNA which was named ValR20 (SEQ ID NO:9). The deduced amino acid sequence (SEQ ID NO: 10) showed homology with plant sesquiterpene synthases and contained typical terpene synthases amino acid motifs such as the DDxxD motif. The closest sequences were a vetispiradiene synthase amino acid sequence from S. tuberosum (49% identity) and a putative sesquiterpene synthase from V. vinifera (up to 46% identity).
Example 2
[0135] Heterologous Expression and Characterization of ValR20 as a 7-epi--Selinene and Patchoulol Synthase
[0136] Two oligonucleotides, Val-R20-topo-start (SEQ ID NO: 11) and Val-R20-stop (SEQ ID NO: 12), where designed from the start and stop regions of ValR20 and used to amplify the full-length sequence of this cDNA. The Val-R20-topo-start was designed according to the CHAMPION pET Directional TOPO Expression Kits (invitrogen). The amplification was performed with the Pfu DNA polymerase (Promega) from the 5-RACE-Ready cDNA pool prepared with the Smart RACE cDNA amplification kit (clontech). The thermal cycling conditions were as follows: 2 min at 95 C.; 32 cycles of 5 sec at 94 C., 20 sec at 53 C. and 3 min at 72 C. The PCR products were purified on an agarose gel and eluted using the QIAQUICK Gel Extraction Kit (Qiagen, Valencia, Calif.). The PCR product was ligated into the pET101 plasmid following the manufacturer protocol (Invitrogen). The constructs were verified by DNA sequencing. One clone, 504-ValR20 (SEQ ID NO:2), was selected for the subsequent heterologous expression and enzyme assay experiments. Compared to the sequences reconstituted from the RACE, the sequence of 504-ValR20 (SEQ ID NO:2) showed four nucleotide differences leading to three amino acid changes. The amino acid sequence encoded by 504-ValR20 is provided in SEQ ID NO: 1.
[0137] The plasmid was transferred into BL21 STAR (DE3) E. coli cells (Invitrogen). Single colonies of transformed cells were used to inoculate 5 mL LB medium. After reaching an OD of 0.3, the 5 mL cultures were used to inoculate 25 mL of LB medium. The cultures were incubated at 37 C. until reaching an OD of 0.5 and they were then transferred to a 20 C. incubator. After 1 hour equilibration, the expression of the proteins was induced by addition of 1 mM IPTG and the culture were incubated over-night at 20 C. The next day, the cells were collected by centrifugation, resuspended in 1 mL Extraction Buffer (50 mM MOPSO, pH 7.4, 1 mM DTT, 10% glycerol) and disrupted by sonication. The cell debris were sedimented by centrifugation 30 min at 18,000 g and the supernatant containing the soluble proteins was recovered.
[0138] The enzymatic assays were performed in Teflon sealed glass tubes using 250 L protein extract in a final volume of 5 mL Extraction Buffer supplemented with 10 mM MgCl.sub.2 and 100 to 250 M farnesyl pyrophosphate (prepared as described by Keller and Thompson, J. Chromatogr 645(1), 161-167, 1993). The assays were overlaid with 3 mL pentane and the tubes incubated overnight at 30 C. The pentane phase was recovered and the medium extracted with a second volume of pentane. The combined pentane fractions were concentrated under nitrogen and analyzed by GC-MS on a Hewlett-Packard 6890N GC-quadrupole mass selective detector system, equipped with a 0.25 mm inner diameter by 30 m length DB-IMS (J&W Scientific) capillary column. The oven temperature was programmed from 50 C. (1 min hold) to 280 C. at 10 C./min. The carrier gas was He at a constant flow of 1 mL/min. The identity of the products was confirmed based on the concordance of the retention indexes and mass spectra of authentic standards when available or based on published data (Joulain and Koenig, 1998). Negative controls were performed with proteins obtained from E coli transformed with the pET101 plasmid without insert.
[0139] The recombinant enzyme (SEQ ID NO:1) was found to be a multiple product sesquiterpene synthase converting farnesyl-pyrophosphate to at least 11 products. The major product was 7-epi--selinene which accounted for 34.2% of the total sesquiterpene mixture. Patchoulol was the second most abound product of the enzyme (16.4%). Other sesquiterpene produced were germacrene A (12.2%), -guaiene (2.4%), seychellene (4.6%), -humulene (5.3%), -patchoulene (2.4%), -selinene (9.6%), -bulnesene (6.3%), (E)-nerolidol (2.5%) and an unidentified sesquiterpene alcohol (4.1%).