METHOD FOR PRODUCING BETA-SANTALENE
20180105837 ยท 2018-04-19
Assignee
Inventors
Cpc classification
C12P5/007
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides a method of producing -santalene, said method comprising contacting at least one polypeptide with farnesyl pyrophosphate (FPP). In particular, said method may be carried out in vitro or in vivo to produce -santalene, a very useful compound in the fields of perfumery and flavoring. 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 -santalene is also an object of the present invention.
Claims
1. A recombinant polypeptide having a -santalene synthase activity and comprising an amino acid sequence at least 90% identical to SEQ ID NO: 15.
2. An isolated polypeptide having a -santalene synthase activity and comprising the amino acid sequence of SEQ ID NO: 27.
3. An isolated nucleic acid encoding a polypeptide comprising the amino acid sequence according to claim 2.
4. An isolated nucleic acid molecule comprising a) the cDNA sequence of SEQ ID NO: 14 or the complement thereof; b) a modified sequence of SEQ ID NO: 14, wherein the first 23 codons of SEQ ID NO: 14 were removed; or c) the modified sequence of b) optimized for E. coli expression.
5. An expression vector comprising a) the nucleic acid molecule of claim 4; b) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 15, or SEQ ID NO: 27 and having a -santalene synthase activity; or c) the nucleic acid of b) comprising a nucleotide sequence at least 75% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 26 or the complement thereof.
6. A non-human host organism or cell a) transformed to harbor i) the nucleic acid of claim 4; ii) a nucleic acid encoding a polypeptide having a -santalene synthase activity comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 15, or SEQ ID NO: 27; or iii) the nucleic acid of ii) comprising a nucleotide sequence having at least 75% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 26 or the complement thereof; or b) comprising an expression vector comprising the nucleic acid of i), ii), or iii) above; so that the non-human host organism or cell heterologously expresses or over-expresses at least one polypeptide having a -santalene synthase activity.
7. The non-human host organism or cell of claim 6, wherein said non-human host organism or cell is a plant, a prokaryote, a fungus, a plant cell, or a fungal cell.
8. The non-human host organism or cell of claim 6, wherein said non-human host organism is a microorganism.
9. The non-human host organism of claim 8, wherein said microorganism is a bacteria or yeast.
10. The non-human host organism of claim 9, wherein the bacteria is E. coli and the yeast is Saccharomyces cerevisiae.
11. The non-human organism or cell of claim 6, wherein the non-human organism or cell comprises a) a nucleic acid encoding a polypeptide having a -santalene synthase activity comprising an amino acid sequence having at least 95%, 98% or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 15, or SEQ ID NO: 27; or b) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 26 or the complement thereof.
12. The expression vector of claim 5, wherein the expression vector comprises a) a nucleic acid encoding a polypeptide having a -santalene synthase activity comprising an amino acid sequence having at least 95%, 98% or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 15, or SEQ ID NO: 27; or b) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 26 or the complement thereof.
13. The non-human organism or cell of claim 6, wherein the non-human organism or cell is capable of producing farnesyl pyrophosphate (FPP).
14. A method for producing -santalene comprising a) cultivating the non-human organism or cell of claim 13 under conditions conducive to the production of -santalene; and b) optionally, recovering the -santalene produced in a).
15. The method of claim 14, further comprising processing the -santalene to a -santalene derivative using a chemical or biochemical synthesis or a combination of both.
16. The method of claim 15, wherein the derivative comprises -santalol.
Description
DESCRIPTION OF THE DRAWINGS
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SPECIFIC EMBODIMENTS OF THE INVENTION OR EXAMPLES
[0150] The invention will now be described in further detail by way of the following Examples.
Example 1
DNA Library Construction, Sequencing and Extraction of Terpene Synthase Related Sequences
[0151] Young hypocotyls segments obtained from aseptically germinated seeds of Santalum album L. (5 weeks old) were used to induce callus formation. 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% HClO for 120 minutes, and rinsed three 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%. The plantlets were allowed to grow in-vitro for 2 to 3 months in a cultivation room at a temperature of 27 C., with cool, white fluorescent light and with a 16 hours photoperiod. To induce the formation of green callus, the hypocotyls segments were cut into 3-4 mm transverse segments which were placed on Gbg basal medium (Gamborg & al, 1968, Exp Cell Res. 50(1), 151-158) supplemented with 0.5 M 2,4D (2,4-Dichlorophenoxyacetic acid, Sigma-Aldrich Co.) and 10 M Kin (Kinetin, Sigma-Aldrich Co.) in Petri dishes. The growth of the callus was perpetuated by transferring the tissue every four weeks to fresh medium in Petri dishes. All callus cultures were performed in a growth chamber in the same conditions as above.
[0152] Callus obtained after one month of culture in Gbg medium containing 5 M Kin and 2 mM ACC were used for the RNA extraction and cDNA library construction. Total RNA were extracted following the protocol described by Lefort and Douglas (Ann. For. Sci. 56 (1999), 259-263) except that the RNase treatment was omitted. The pellet was resuspended in 200 l RNase-free water and centrifuged twice for 10 minutes at 20000 g to remove the polysaccharides. Approximately 125 g total RNA were obtained from 2.2 g of cells. The mRNAs were purified using the FastTrack 2.0 mRNA Isolation Kit (Invitrogen) and a cDNA library was made using the SMART PCR cDNA Synthesis Kit (Clontech Laboratories, Inc.) following the manufacturer's instructions.
[0153] The technology of massive parallel sequencing of small DNA fragments developed by Illumina (San Diego, Calif.) was used to sequence the whole cDNA library. The preparation of the DNA for sequencing, the sequencing and the assembling of the reads were performed by Fasteris SA (Plan-les-Ouates, Switzerland). The cDNA library was treated following the Genomic Sample Prep Kit (Illumina) and sequenced on the Genome Analyzer system (Illumina). A total of 4.03 millions of 35 bp sequences (reads) were obtained. These reads were assembled using EDENA 2.1.1, a software finding overlaps between the reads and assembling de novo contigs (Hernandez et al, De novo bacterial genome sequencing: Millions of very short reads assembled on a desktop computer. Genome Res. 2008;18:802-809). The assembling was run with minimum matches of 26 to 20 bases. After eliminating contigs shorter than 100 bases, 1983 to 3473 unique contigs were obtained with a maximum length of 1331 to 1914 depending of the parameters selected for the assembling. Another assembling was performed using the Velvet 1.0 program (Zerbino and Birney (2008), Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18(5), 821-829), providing 5905 unique contigs of length between 100 and 1616 bases.
[0154] All the contigs generated were compared against a protein sequences data base (non-redundant protein sequences, NCBI, http://www.ncbi.nlm.nih.gov) using the Blastx algorithm (Altschul et al, J. Mol. Biol. 215, 403-410, 1990; http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The contigs showing significant sequence homology with plant sesquiterpene synthases were retained. A total of 46 contigs with a length of 100 to 621 bases were thus selected. These contigs were then processed using the CAP program (Huang, Genomics 14(1), 18-25, 1992) to assemble them and generate longer sequences. Five unique contigs of length of 445 to 1064 were thus assembled. The deduced amino acid sequences showed significant homology with plant terpene synthases and especially with sequences described or annotated as monoterpene synthases. Alignment of these amino acid sequences showed that at least two distinct cDNAs were present (two sequences were found in most of the positions across the alignment). This alignment showed also that at least one N-terminal and one C-terminal sequence was present. To obtain the full length sequences and to assign the exact 5-end and 3-end sequences to each cDNA, a rapid amplification of cDNA ends experiment (RACE) was employed.
Example 2
Amplification of the Full-Length Sequences of a Terpene Synthase cDNA
[0155] For the RACE experiments, a set of primers was designed from one out of the five contigs obtained as described above. Thus the forward primers SCH5-Ct58-R1 (SEQ ID NO:6) and SCH5-Ct58-R2 (SEQ ID NO:7) and the reverse primers SCH5-Ct58-F3 (SEQ ID NO:8) and SCH5-Ct58-F4 (SEQ ID NO:9) were deduced from SCH5-contig-5 (SEQ ID NO:5).
[0156] The PCR were performed with the Universal Primer A Mix (UPM) (SMART RACE cDNA Amplification Kit, Clontech Laboratories, Inc.) in 50 l final volume containing 200 M dNTPs mix, 5 l cDNA library (Example 1), 0.2 M gene-specific primer, 0.2 M UPM Primer Mix (Clontech Laboratories, Inc.), 1 l Advantage 2 Polymerase Mix (Clontech Laboratories, Inc.) and 5 l 10 cDNA PCR Reaction Buffer (Clontech Laboratories, Inc.). The thermal cycling conditions were as follows: 3 minutes at 94 C.; 5 cycles of 30 sec at 94 C. and 3 minutes at 72 C.; 5 cycles of 30 sec at 94 C. and 3 minutes at 70 C.; 5 cycles of 30 sec at 94 C. and 3 minutes at 68 C.; 3 minutes at 72 C. With the 5RACE, a 610 bp DNA fragment (SCH5-Ct58 RR1, SEQ ID NO:10) including the 5end of the cDNA was obtained. With the 3RACE a 1049 bp fragment (SCH5-Ct58-RF4, SEQ ID NO:11) was obtained and the combination of the two RACE products with the SCH5-contig-5 sequence (SEQ ID NO:5) allowed the reconstitution of a new full-length cDNA (SCH5-Ct58, SEQ ID NO:12). The 2157 bp SCH5-Ct58 cDNA encoded for a 569 amino acid protein (SEQ ID NO:13) showing homology with plant terpene synthases sequences and containing motifs characteristic of terpene synthases such as the DDxxD motif present in all monoterpene and sesquiterpene synthases. Interestingly the amino acid sequence showed higher similarity to monoterpene synthases than to sesquiterpene synthases. However the presence of chloroplast peptide signal, a common feature in plant monoterpene synthases, was not predicted from the analysis of the N-terminal sequence (Emanuelsson, O., Nielsen, N., and von Heijne, G. 1999. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Science 8, 978-984).
Example 3
Heterologous Expression and In-Vitro enzymatic Activity of SCH5-Ct58
[0157] We decided to modify the DNA sequence of SCH5-Ct58 (SEQ ID NO:12) and to redesign the sequence for optimal heterologous expression in E coli cells. To start with the true amino acid sequence, the exact nucleotidic sequence of SCH5-Ct58 in the cDNA library had first to be established. The Eland Software (Illumina) was used to retrieve all reads matching with the SCH5-Ct58 sequence (SEQ ID NO:12) with a maximum of 2 mismatches. A total of 5224 reads were recovered and were aligned using the CAP program (Huang, Genomics 14(1), 18-25, 1992) with the SCH5-Ct58 DNA sequence (SEQ ID NO:12) as a reference. The average coverage over the whole sequence was above 100 allowing for the unambiguous deduction of the new cDNA sequence SCH5-Ct94 (SEQ ID NO:14). In this new sequence 5 bases were corrected compared to the SCH5-Ct58 sequence (SEQ ID NO:12) deduced from the RACE results and those corrections resulted in a new amino acid sequence (SCH5-Ct94, SEQ ID NO:15) with a two-residues difference. For heterologous expression, the DNA sequence of SCH5-Ct94 (SEQ ID NO:14) was modified to remove the first 23 codons and replace by the ATGGCT sequence and the codon usage was changed to optimize the sequence for E coli expression (DNA 2.0, Menlo Park, Calif., USA). The cDNA thus designed (SCH5-Ct94-opt, SEQ ID NO:2) was synthesized (DNA 2.0, Menlo Park, Calif., USA) and sub-cloned into the NdeI-KpnI sites of the pETDuet-1 plasmid providing the plasmid Ct94-pETDuet. This optimized cDNA sequence encoded for the polypeptide SCH5-Ct94-opt (SEQ ID NO:1).
[0158] Heterologous expression of Ct94 was performed in E coli BL21(DE3) cells using the plasmid Ct94-pETDuet. In-vitro enzyme assays were performed with FPP as substrate in the conditions described above and sesquiterpene synthase activity was observed with formation of a mixture of five sesquiterpenes. The identity of these sesquiterpenes was confirmed by GC-MS as being the sesquiterpene characteristic of santalum album: -santalene, trans--bergamotene, epi--santalene, -santalene and -famesene (
TABLE-US-00001 TABLE 1 Effect of the concentration of Mg.sup.2+ and Mn.sup.2+ ions on the composition of the mixture of sesquiterpenes obtained by contacting SEQ ID NO: 1 with FPP Percentage, relative to the whole product mixture 15 mM 2 mM 0.75 mM 0.75 mM MgCl.sub.2 + MgCl.sub.2 MgCl.sub.2 MgCl.sub.2 1 mM MnCl.sub.2 -santalene 38.0 33.0 36.5 24.5 trans-- 18.2 11.8 12.6 35.4 bergamotene epi--santalene 5.7 6.4 5.6 4.1 -santalene 36.7 47.5 44.1 33.3 -farnesene 1.3 1.3 1.1 2.75
Example 4
In-Vivo Production of Sesquiterpenes in E coli using the Ct94 cDNA
[0159] The use of the S. album santalene synthase for the in-vivo production of sesquiterpenes in E coli cells was evaluated by coexpressing the enzymes of a five step biosynthetic pathway converting mevalonic acid to FPP.
[0160] The yeast FPP synthase gene was amplified from S. cerevisiae genomic DNA using the primers FPPy_Ncol (SEQ ID NO:16) AND fppY-Eco (SEQ ID NO:17). The amplified DNA was ligated as NdeI-EcorI fragment in the first multi cloning site (MCS1) of the pACYCDuet-1 plasmid providing the plasmid FPPs-pACYCDuet harbouring the FPPs gene under the control of the T7 promoter. An operon including the genes encoding for a mevalonate kinase (mvaK1), a phosphomevalonate kinase (mvaK2), a mevalonate diphosphate decarboxylase (MvaD) and a isopentenyl diphospahte isomerase (idi) was amplified from genomic DNA of Streptococcus pneumoniae (ATCC BAA-334) with the primers MVA-up1-start (SEQ ID NO:18) and MVA-up2-stop (SEQ ID NO:19). The PCR was performed using the PfuUltra II Fusion HS DNA polymerase (Stratagen). The composition of the PCR mix was according to the manufacturer instructions. The thermal cycling condition were 2 minutes at 95 C.; 30 cycles of 20 sec at 95 C., 20 sec at 58 C. and 90 sec at 72 C.; and 3 minutes at 72 C. The 3.8 Kb fragment was purified on an agarose gel and ligated using the In-Fusion Dry-Down PCR Cloning Kit (clontech) into the second MCS of the FPPs-pACYCDuet plasmid digested with NdeI and XhoI providing the plasmid pACYCDuet-4506. The sequences of the two inserts were fully sequenced to exclude any mutation.
[0161] BL21 Star(DE3) E. coli cells (Invitrogen) were co-transformed with the plasmids pACYCDuet-4506 and Ct94-pETDuet and transformed cells were selected on carbenicillin (50 g/ml) chloramphenicol (34 g/ml) LB-agarose plates. Single colonies were used to inoculate 5 mL LB medium with 50 g/ml carbenicilin and 34 g/ml chloramphenicol. The culture was incubated overnight at 37 C. The next day 2 mL of TB medium supplemented with the same antibiotics were inoculated with 0.2 mL of the overnight culture. After 6 hours incubation at 37 C., the culture was cooled down to 28 C. and 1 mM IPTG, 2 mg/mL mevalonate (prepared by dissolving mevalonolactone (Sigma) in 0.5 N NaOH at a concentration of 1 g/mL and incubating the solution for 30 minutes at 37 C.) and 0.2 mL decane were added to each tube. The cultures were incubated for 48 hours at 28 C. The cultures were then extracted twice with 2 volumes of ethyl acetate, the organic phase was concentrated to 500 l , and analyzed by GC-MS as described above in Example 3. In these conditions sesquiterpene production above 200 mg/L was routinely achieved. Beta-santalene was produced.
Example 5
In-Vivo Production of Sesquiterpenes in S. cerevisiae using the Ct94 cDNA
[0162] For in-vivo production of sesquiterpenes in yeast cells, a saccharomyces cerevisiae strain YNP5 in which the ERG9 gene (coding for the squalene synthase, the enzyme converting FPP to squalene) has been down-regulated by replacing the native ERG9 promoter with the regulable MET3 promoter. In previous work with plant sesquiterpene synthases, this strategy led to a reduced ergosterol biosynthesis in the cells and an accumulation of FPP available for sesquiterpene synthases (Asadollahi, Biotechnology and Bioengineering, 99(3), 666-677, 2008).
[0163] The SCH5-Ct94-opt cDNA (SEQ ID NO:2) was amplified from the Ct94-pETDuet with the primers Ct94_BamHI (SEQ ID NO:20) and T7term (SEQ ID NO:21). The PCR was performed with the Pfu DNA Polymerase (Promega) using the following thermal cycling condition: 90 sec at 94 C.; 35 cycles of 45 sec at 94 C., 45 sec at 55 C., 4 minutes at 72 C.; and 10 minutes at 72 C. The amplified cDNA was digested with the BamHI and XhoI restriction sites and ligated in the corresponding sites of the pESC-URA plasmid (Stratagen) providing the plasmid Ct94-pESC-ura. S.c. The YNPS cells were transformed using the S.c. EasyComp Transformation Kit (Invitrogen).
[0164] One single colony of transformed yeast strains were used to inciluate 20 ml of YNB medium (5 g/L (NH.sub.4).sub.2SO.sub.4; 3 g/L KH.sub.2PO.sub.4; 0.5 g/L MgSO.sub.4. 7 H.sub.2O; 1 mL/L trace metal solution) supplemented with 2% glucose. The culture was incubated for 24 hours at 28 C. The cells were recovered by centrifugation and resuspended in 20 mL of YNB medium supplemented with 2% galacoste. After on 1 hour culture, methionine at 0.5 mM final concentration and 2 mL decane were added to the culture. After 24 hours incubation at 28 C., the cultures were extracted with ethyl acetate and analysed by GC-MS as described in Example 4. The total quantity of sesquiterpenes produced by the yeast cells in these conditions was estimated at 50 mg/L.
Example 6
Isolation of a Santalene Synthase from Santalum Album Roots
[0165] Seedlings of Santalum album obtained from aseptically germinated seeds were transferred to soil 5 to 10 weeks after germination. Since santalum species are root hemiparasites, the soil adaptation was made in close contact with 6-months to 1-year old citrus (Citrus sinensis) plants. The roots of the santalum plants were harvested, 2-3 years after the transfer to the soils and separated from the host plant roots. GC-MS analysis of an extract of these roots showed the presence of the sandalwood oil characteristic sesquiterpenes. Total RNA was extracted from the roots using the Concert Plant RNA Reagent (Invitrogen). From 12 g of tissue, 640 g of total RNA were isolated. The mRNA were purified using the FastTrack 2.0 mRNA Isolation Kit (Invitrogen) and a cDNA library was made using the Marathon cDNA Amplification Kit (Clontech Laboratories, Inc.) following the manufacturer instructions.
[0166] An amount of 1 g of cDNA was used for sequencing using the Genome Analyzer System (Illumina). A total of 10.3 millions of 35bp-length reads were obtained. These reads were assembled using in parallel the Edena (Hernandez et al, 2008, Genome Res. 18, 802-809) and the Velvet (Zerbinoa and Birney, 2008, Genome Res. 18: 821-829) assembler softwares resulting in 18937 and 22414 unique contigs with an average range of 242 and 211 bp. The reads were searched using the tBlastn program (Altschul et al, 1990, J. Mol. Biol. 215, 403-410) with the SCH5-CT94 amino acid sequence (SEQ ID NO:15) as query sequence. Fifteen contigs were selected showing significant homology of their deduced amino acid sequences with plant sesquiterpene synthases. These selected contigs were reassembled into two distinct sequences, of which SCH10-Ct8201 (SEQ ID NO:22) was 383 bp in length and showed the highest homology with SCH5-CT94 DNA sequence (SEQ ID NO:14). The forward primer SCH10-Ctg8201-F2 (SEQ ID NO:23) was designed from the SCH10-Ct8201 sequence and successfully used for 3RACE using the Marathon cDNA Amplification Kit (Clontech Laboratories, Inc.). From the sequence of the 3RACE product thus obtained, two reverse primers (SCH10-Ct19779-R3 (SEQ ID NO:24) and SCH10-Ct19779-R4 (SEQ ID NO:25)) were designed and successfully used for the amplification by 5RACE of the 5end of the corresponding cDNA. From the sequences of the 3RACE and 5RACE a full-length sequence of a new terpene synthase was thus reconstituted. In order to verify the sequence, the MAQ program (Li et al, 2008, Genome Res. 18(11), 1851-1858) was used to search and map all the reads with a maximum of 2 mismatches. This approach provided a 1725 bp-length DNA sequence (SEQ ID NO:26) encoding for a 570 amino acid-length protein (SEQ ID NO:27) having 91.9% identity with the amino acid sequence of SCH5-Ct94 (SEQ ID NO:15).
[0167] For heterologous expression in E coli, an optimized cDNA was designed by deleting the 21 first codons, adding the sequence ATGGCTACC as the first 3 codons and optimizing the codon usage for E coli. This optimized sequence (SCH10-Ct8201-opt, SEQ ID NO:4) encoding for the N-terminal modified protein SCH10-Tps8201-opt (SEQ ID NO:3) was synthesized (DNA 2.0; Menlo Park, Calif., USA) and sub-cloned in the NdeI-KpnI sites of the pETDuet-1 expression plasmid (Novagen). Heterologous expression and enzymatic characterization of SCH10-Tps8201-opt (SEQ ID NO:3) was performed as described in Example 3. The recombinant protein showed sesquiterpene synthase activity and produced from FPP the same mixture of sesquiterpenes as the SCH5-CT94-opt recombinant protein (SEQ ID NO:1, Example 3) with the same relative proportions.