SANTALENE SYNTHASE

Abstract

The invention is directed to a santalene synthase, to a nucleic acid encoding said santalene synthase, to an expression vector comprising said nucleic acid, to a host cell comprising said expression vector, to a method of preparing santalene, to a method of preparing santalol and to a method of preparing a santalene synthase. The invention is further directed to an antibody specific for the santalane synthase.

Claims

1. Santalene synthase comprising an amino acid sequence as shown in SEQ ID NO: 3 or a functional homologue thereof, said homologue being a santalene synthase comprising an amino acid sequence which has a sequence identity of at least 60% with SEQ ID NO: 3.

2. Santalene synthase according to claim 1, having at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 3.

3. Nucleic acid, comprising a nucleic acid sequence encoding a santalene synthase according to claim 1, or a complementary sequence thereof.

4. Nucleic acid according to claim 3, wherein the nucleic acid comprises a nucleic acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleic acid sequence having a sequence identity of at least 60%, at least 65%, at least 75%, at least 85%, at least 90% or at least 95% with a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary sequence of any of these sequences.

5. Expression vector comprising a nucleic acid according to claim 3.

6. A host cell, which may be an organism per se or part of a multi-cellular organism, said host cell comprising an expression vector comprising a heterologous nucleic acid sequence according to claim 3.

7. A host cell according to claim 6, wherein the host cell is a bacterial cell selected from the group of Gram negative bacteria, in particular from the group of Rhodobacter, Paracoccus and Escherichia.

8. A host cell according to claim 6, wherein the host cell is a fungal cell selected from the group of Aspergillus, Blakeslea, Penicillium, Phaffia (Xanthophyllomyces), Pichia, Saccharomyces, and Yarrowia.

9. Transgenic plant or culture comprising transgenic plant cells, said plant or culture comprising host cells according to claim 6, wherein the host cell is of a transgenic plant selected from Nicotiana spp, Solanum spp, Cichorum intybus, Lactuca sativa, Mentha spp, Artemisia annua, tuber forming plants, oil crops, liquid culture plants, tobacco BY2 cells, Physcomitrella patens, and trees.

10. Transgenic mushroom or culture comprising transgenic mushroom cells, said mushroom or culture comprising host cells according to claim 6, wherein the host cell is selected from Schizophyllum, Agaricus and Pleurotis.

11. Method for preparing santalene, comprising converting a farnesyl diphosphate to santalene in the presence of a santalene synthase according to claim 1.

12. Method for preparing santalene according to claim 11, wherein the santalene is prepared in a host cell, a plant or plant culture, or a mushroom or mushroom cultureexpressing said santalene synthase.

13. Method according to claim 11, further comprising isolating the santalene.

14. Method according to any one of claims 11, wherein the ratio -santalene to -bergamotene ratio is higher than 1.

15. Method according to any one of claims 11, wherein the -santalene to -bergamotene ratio is higher than 0.5:1.

16. Method according to claim 11, wherein the ratio of santalenes (- and -santalene) to -bergamotene is higher than 2:1.

17. Method for preparing santalol, comprising converting FPP to santalene in the presence of a santalene synthase according to claim 1, further comprising converting the santalene into santalol.

18. Method for preparing santalol, preferably -santalol, according to claim 17, wherein the santalene is prepared in a host cell, a plant or plant culture, or a mushroom or mushroom culture, expressing said santalene synthase.

19. Method according to claim 17, further comprising isolating the santalol, preferably the -santalol.

20. Antibody that specifically binds to a santalene synthase according to claim 1.

Description

FIGURE LEGENDS

[0134] FIG. 1 Map of plasmid p-m-LPppa-CiCaSSy-mpmii alt.

[0135] FIG. 2 Map of plasmid p-m-SPppa-MBP-CiCaSSy-mpmii alt.

[0136] FIG. 3 Map of plasmid p-m-PcrtE-TRX-CiCaSSy-mpmii alt.

[0137] FIG. 4 Map of plasmid p-m-PcrtE-TRX-SaSSy-mpmii alt.

[0138] FIG. 5 GC chromatogram of terpene species produced by CiCaSSy in R. sphaeroides. The compounds identified by GC-MS are: -santalene (Retention time: 5.3 min), trans--bergamotene (Rt: 5.45 min), epi--santalene (Rt: 5.75 min) and -santalene (Rt: 5.95 min).

[0139] FIG. 6 Production of total terpenes (A), -santalene (B), -santalene (C), and trans--bergamotene (D) during fed-batch fermentation of Rhodobacter sphaeroides Rs265-9c strains harbouring plasmids with either the gene encoding SaSSy or CiCaSSy santalene synthase. The product ratios for the individual terpenes (B, C and D) are represented as the amount (area) relative to the area total of the components as indicated in the GC chromatogram of FIG. 5.

[0140] FIG. 7 Alignment of CiCaSSY with relevant proteins The CiCaSSY (TS23-3) protein sequence (SEQ ID NO: 3) was aligned to the protein sequences of its nearest variants TS23-1 (SEQ ID NO: 10) and TS23-2 (SEQ IL) NO: 11) found in C. camphora. The 21 out of 553 residues which were different between the protein of TS23-1 and CiCaSSY are highlighted.

[0141] FIG. 8 GC-MS analysis of terpene production in E. coli (example 5), vector control sample: E. coli transformed with pACYCDUET.

[0142] FIG. 9 GC-MS analysis of terpene production in E. coli (example 5), clone TS23-1 sample: E. coli transformed with pACYCDuet_TS23-1

[0143] FIG. 10 GC-MS analysis of terpene production in E. coli (example 5), clone TS23-3 sample: E. coli transformed with pACYCDuet_TS23-3 (CicaSSy)

[0144] FIG. 11 GC-MS analysis of terpene production in E. coli (example 5), clone SaSSy sample: E. coli transformed with pACYCDuet_SaSSy)

EXAMPLES:

Example 1

[0145] GC-MS Analysis of Cinnamomum camphora

[0146] A Cinnamomum camphora plant of about 30 cm tall was purchased from Planfor (Ppinires PLANFOR, RD 651 40090 UCHACQFRANCE). Cinnamomum camphora is known to occur in several chemotypes. In particular the cineole type appears to contain santalene (Stubbs et al., 2004, Pelissier et al., 1995), while other chemotypes (camphor, linalool) have not been reported to contain santalene (e.g. Frizzo 2000; Pino 1998). The plant was dissected in leaf, stem and root material. 0.5 g of plant material was weighed in a precooled glass tube, and 2 mL of dichloromethane was added. The suspension was vortexed for 1 min, sonicated for 5 min in an ultrasonic bath and centrifuged for 5 min at 1500 g at room temperature. The supernatant was collected and filtered over a column of 1 g sodium sulphate. About 2 L was analysed by GC/MS using a gas chromatograph as described in detail by Cankar et al. (2015). Santalenes were identified by the comparison of retention times and mass spectra to those of sandalwood oil (Sigma-Aldrich).

[0147] Results:

[0148] The roots, leaves and stem of C. camphora appeared to contain compounds that correspond to -santalene (Rt 13.17 min), -bergamotene (Rt 13.34 min), epi--santalene (Rt 13.54 min) and B-santalene (Rt 13.69 min). The concentration of santalenes was highest in the roots. Other compounds found in the roots of the Cinnamomum plant were identified as guaiol, guaiadiene, intermedeol, eremoligenol, germacrene D, isolepidozene, saffrol, limonene, pinene, camphene, myrcene, sabinene, 1,8-cineol and camphor. Therefore, this tissue was further taken for extraction of RNA.

Example 2

[0149] RNA Extraction and Analysis

[0150] The RNA of C. camphora root material was isolated as follows: About 15 mL extraction buffer (2% hexadecyl-trimethylammonium bromide, 2% polyvinylpyrrolidinone K 30, 100 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2.0 M NaCl, 0.5 g/L spermidine and 2% -mercaptoethanol) was warmed to 65 C., after which 3 g ground tissue was added and mixed. The mixture was extracted two times with an equal volume of chloroform:isoamylalcohol (1:24), and one-fourth volume of 10 M LiCl was added to the supernatant and mixed. The RNA was precipitated overnight at 4 C. and harvested by centrifugation at 10 000 g for 20 min. The pellet was dissolved in 500 L of SSTE [1.0 M NaCl, 0.5% SDS, 10 mM Tris-HCl (pH 8,0), 1 mM EDTA (pH 8.0)] and extracted once with an equal volume of chloroform: isoamylalcohol. Two volumes of ethanol were added to the supernatant, incubated for at least 2 h at 20 C., centrifuged at 13 000 g and the supernatant removed. The pellet was air-dried and resuspended in water. Total RNA (60 g) was shipped to Vertis Biotechnology AG (Freising, Germany). Poly-A+ RNA was isolated, random primed cDNA synthesized using a randomized N6 adapter primer and M-MLV H-reverse transcriptase. cDNA was sheared and fractionated, and fragments of a size of 500 bp were used for further analysis. The cDNAs carry attached to their 5 and 3 ends the adaptor sequences A and B as specified by Illumina. The material was subsequently analysed on an Illumina MiSeq Sequencing device. In total, 27,919,287 sequences were read by the MiSeq, with a total sequence length of 10,592,407,803 basepairs. Trimmomatic-0.32 was used to trim sequences from Illumina sequencing adapters, Seqprep was used to overlap paired end sequences, and bowtie2 (version 2.2.1) was used to remove phiX contamination (phiX DNA is used as a spike-in control, usually present in <1%). Paired end reads and single reads were used in a Trinity assembly (trinityrnaseq-2.0.2). A total number of 160871 contigs were assembled by Trinity.

[0151] In order to identify sesquiterpene synthases, the C. camphora contigs were used to create a database of cDNA sequences. In this database, the TBLASTN program was deployed to identify cDNA sequences that encode proteins that show identity with protein sequences of sesquiterpene synthases, including santalene synthases from Santalum album (GenBank accession E3W202). Clausena lansium (ADR71055) and Solanum habrochaites (ACJ38409), valencene synthase from Callitropsis nootkatensis (CDM55287) and trans--bergamotene synthase from Phyla dulcis (AFR23371). In total 95 contigs in the C. camphora cDNA database were identified which have significant homology to sesquiterpene synthases. The contigs were grouped into 28 groups according to their overlap in sequence. These 28 contigs were further characterized by analyzing them using the BLASTX program to align them to protein sequences present in the UniProt database (downloaded Aug. 28, 2015), and 14 of them were identified as putative sesquiterpene synthase sequences and other 14 as putative monoterpene synthases, according to their homology to terpene synthases sequences present in UniProt.

[0152] Contigs were screened for open reading frames encoding the full-length terpene synthase proteins, based on the alignments provided by the BLASTX analysis. The following criterion for identifying a protein full length was used: both sesquiterpene synthases and monoterpene synthases carry a RRxxxxxxxxW motif (RRX8W) close to their N-terminal start. An in-frame ATG codon should map 20-70 codons upstream from the region encoding the RRX8W motiv. or its orthologous position, to be identified as a startcodon.

Example 3

Cloning of Cinnamomum camphora Santalene Synthase (CiCaSSy)

[0153] Full length open reading frames were amplified from the cDNA of C. camphora. Forward and reverse primers as shown in Table 1 were designed and used to amplify total open reading frames in such a way that the reading frame was fused to the C-terminus of a His-6 tag in the plasmid pCDF-DUET-1 (Novagen corporation). A total of 37 different terpene synthase ORFs were cloned. Using the primers TS23fw and TS23re (Table 1), three different closely related cDNAs were obtained, which encoded proteins with SEQ ID NO:10 (TS23-1), SEQ ID NO:11 (TS23-2), and SEQ ID NO:3 (T523-3).

TABLE-US-00001 TABLE1 name Sequence clones TS23fw atatggatcctATGGACTCCATGGAGGTACGCCGT TS23-1, CTG(SEQIDNO:8) TS23-2, TS23re atatgcggccgcTCATCCCAAGTTGATGGATTCCT TS23-3 TCAATGGCACTG (SEQIDNO:9)

[0154] The cloned variants were analysed by sequencing the TS insert. Different variants were introduced into chemical competent E. coli BL21-RIL (Stratagene), by heat shock transformation, and selected on LB-agar with 1% glucose, 50 ug/ml spectinomycin and 50 ul/ml chloramphenicol. Transformants were transferred to 5 ml LB liquid medium with 1% glucose 50 ug/ml spectinomycin and 50 ug/ml chloramphenicol and grown overnight at 37 oC and 250 rpm.

[0155] 200 L of those cultures was transferred to 20 mL of LB medium with the appropriate antibiotic in a 100 mL Erlenmeyer flask, and incubated at 37 C., 250 rpm until the A600 was 0.4 to 0.6. Subsequently, 1 mM IPTG was added and cultures were incubated overnight at 18 C. and 250 rpm. The next day, cells were harvested by centrifugation (10 min 8000g). medium was removed, and cells were resuspended in 1 mL Resuspension buffer (50 mM Tris-HCl pH=7.5, 1.4 mM -mercaptoethanol; 4 C.). Cells were disrupted by shaking 2 times for 10 seconds with 0.2 g zirconium sand in a Fastprep machine at speed 6.5. Insoluble particles were subsequently removed by centrifugation (10 min 13,000g, 4 C.). Soluble protein was immediately used for enzyme assays.

Example 4

In Vitro Enzyme Assay

[0156] For enzyme assays, in a glass tube a mix was made of 800 L of MOPSO buffer (15 mM MOPSO (3-[N-morpholino]-2-hydroxypropane sulphonic acid) pH=7.0, 12.5% glycerol, 1 mM MgCl2. 0.1% tween 20, 1 mM ascorbic acid, 1 mM dithiothreitol). 100 L of purified enzyme solution and 5 L of farnesyl diphosphate or geranyl diphosphate (10 mM, Sigma FPP dry-evaporated and dissolved in 50% ethanol) and 20 L Na-orthovanadate 250 mM. This mix was incubated at 30 C. with mild agitation for 2 hours. Subsequently, the water-phase was extracted with 2 mL ethylacetate. Ethylacetate phase was collected, centrifuged at 1200g, dried over a sodium sulphate column and analyzed by GC-MS.

[0157] The GC-MS analysis was performed on an Agilent Technologies system, comprising a 7980A GC system, a 597 C inert MSD detector (70 eV), a 7683 auto-sampler and injector and a Phenomenex Zebron ZB-5ms column of 30 m length0.25 mm internal diameter and 0.25 m stationary phase, with a Guardian precolumn (5 m). In this system, 1 L of the sample was injected. The injection chamber was at 250 C., the injection was splitless, and the ZB5 column was maintained at 45 C. for 2 minutes after which a gradient of 10 C. per minute was started, until 300 C. Peaks were detected in chromatograms of the total ion count. Compounds were identified by their retention index and by their mass spectrum in combination with comparison of the mass spectrum to libraries (NIST8 and in-house).

[0158] Clone TS23-3 (SEQ ID NO: 3) was found to produce Santalenes in this in vitro assay, and thus to encode a santalene synthase, and was termed CiCaSSY.

[0159] The closely related clones TS23-1 (SEQ ID NO: 10) and TS23-2 (SEQ ID NO: 11) did not produce any santalenes or other sesquiterpenes in the in vitro assay.

Example 5

[0160] Expression of santalene synthase in E. coli

[0161] For the production of sesquiterpenes in E. coli, the terpene synthase has to be provided with the substrate FPP. Cicassy was therefore co-expressed with a plasmid containing all genes necessary for the synthesis of FPP (pBbA5c-MevT-MBIS-NPtII). This plasmid is a variant of plasmid pBbA5c-MevT(CO)-MBIS(CO, IspA) (Peralta-Yalta et al., 2011), in which the chloramphenicol resistance marker has been exchanged for a kanamycin resistance marker (NptII). From this plasmid, a 728 basepair fragment ranging from the Apal site to the start of the chloramphenicol acyltransferase (CAT) was amplified using Phusion polymerase and primers P7 GCTGTTAGCGGGCCCATTAAG (SEQ ID NO: 12) and P2 GATATTCTCATTTTAGCCATTTTAGCTTCCTTAGCTCCTG (SEQ ID NO: 13). and the neomycin phosphotransferase II (NptII) gene from pBINPlus (van Engelen 1995) by using primers P5 CAGGAGCTAAGGAAGCTAAAATGGCTAAAATGAGAATATC (SEQ ID NO: 14) and P6 CCAAGCGAGCTCGATATCAAACTA_AAACAATTCATCCAG (SEQ ID NO: 15). Fragments were isolated from gel and used as template for a fusion PCR, using primers PG and P7 and amplified a 1524 bp fusion fragment. This fragment, and pBbA5c-MevT(CO)-MBIS(CO, IspA) were both digested with Apal and Sad restriction enzymes, and the vector fragment of pBbA5c-MevT(CO)-MBIS(CO) and the digested fusion PCR fragment were ligated and transformed into E. coli DH5alpha by electroporation, and recombinant colonies were selected on LB+kanamycin. Presence of the genetic elements including the MEV pathway operon and the NptII gene was confirmed by isolating miniprep plasmid DNA and analysis of this DNA by digestion with ApaI and Sad, yielding bands of approximately 12000 bp and 1500 bp. The resulting plasmid was called pBbA5Sc-MevT-MBIS-NptII.

[0162] In addition, the 1670 by BamHI Notl fragment of pCDF-DUET-1 vector in which CiCaSSY had been cloned was transferred to pACYC-DUET-1 (Novagen corporation), for a fair comparison to SaSSY, which had also been introduced in pACYC-DUET-1 in this way.

[0163] First plasmid pBbA5c-MevT-MBIS-NPtII was transformed by heat shock to commercially available competent BL21DE3 cells (New England Biolabs cat C2527). Transformants were selected on LB plates containing kanamycin 50 ug/ml and glucose 1%.

[0164] A pBbA5c-MevT-MBIS-NPtII transformant was grown and competent cells were made with the CaCl.sub.2 method. Briefly, 10 ml culture of this transformant in LB+1% glucose+50 ug/ml Kanamycin was grown until A600=0.5. Subsequently, cells were centrifuged at 8000g for 5 min, resuspended in 1 ml ice-cold 100 mM CaCl.sub.2, centrifuged again for 5 minutes at 8000g, supernatant was discarded and cells were resuspended in 1 ml ice-cold 100 mM CaCl.sub.2 and 50 ul of these cells were used for transformation. The cells were transformed by heat-shock with 50 ng of plasmids pACYCDuet, pACYCDuet_TS23-1, pACYCDuet_CicaSSy and pACYCDuet_Sassy. Transformants were selected on LB plates containing kanamycin 50 ug/ml, chloroamphenicol 50 ug/ml and glucose 1%.

[0165] A tube with 5 ml LB medium with kanamycin 50 ug/ml,l chloroamphenicol 50 ug/ml and glucose 1% was inoculated with a colony containing both plasmids and grown overnight at 37 C. The overnight culture was used to inoculate 20 ml LB plus antibiotics (but no glucose) to an OD of 0.1. The culture was grown to OD 0.45-0.55 and then induced with 20 ul 1M IPTG. The culture was overlaid with 2 ml of dodecane to prevent evaporation of sesquiterpenes from the flask and grown overnight at 28 C. and 250 rpm.

[0166] For GC-MS analysis the dodecane was separated from the culture by centrifugation and diluted 200 times with ethyl acetate. 2 L were analysed by GC/MS using a gas chromatograph as described in detail by Cankar et al. (2015).

[0167] Ts23-1 did not produce a detectable amount of any terpene in this system (FIG. 9). The major product of SaSSY in this system was found to be trans -bergamotene (FIG. 11); the major product of CiCaSSY was found to be -santalene (FIG. 10).

Example 6

[0168] RS102 Medium

[0169] 20 g/L Yeast extract (Gistex, DSM) and 0.5 g/L NaCl are dissolved in distilled water, pH is brought to 7.4 with NaOH, distilled water is added to a volume of 930 ml, and the medium is steam sterilised.

[0170] One ml of sterile 0.5 g/ml MgSO.sub.4-7H.sub.2O, 2 ml of sterie filtered microelements (80 g/L (NH.sub.4).sub.2Fe(SO.sub.4).sub.2-p6H.sub.2O; 6 g/L ZnSO.sub.4-7H.sub.2O; 2 g/L MnSO.sub.4-H.sub.2O; (0.2 g/L NiSO.sub.4-6H.sub.2O, optionally); 2 g/L Vitamin C), and 2 ml of autoclaved CaFe solution (75 g/L CaCl.sub.2-2H.sub.2O; 5 g/L FeCl.sub.3-6H.sub.2O; 3.75 ml HCl (37%)) and 66 ml of glucose solution are added to the sterilised medium,

Example 7

[0171] Bacteria and Culture Conditions

[0172] Rhodobacter sphaeroides strain Rs265-9c was obtained from Rhodobacter sphaeroides strain ATCC 35053 [purchased from the American Type Culture Collection (ATCCManassas, Va., USAwww.atcc.org); number 35053; Rhodobacter sphaeroides (van Niel) Imhoff et al., isolated from a sewage settling pond in Indiana and deposited as Rhodopseudomonas sphaeroides van Niel] after two rounds of mutagenesis and was used as the base host for construction of recombinant strains having improved production of santalene. All R. sphaeroides strains were grown at 30 C. in medium RS102 unless otherwise stated.

[0173] E. coli strains were grown at 37 C. in LB medium (Becton Dickinson, Sparks, Md., USA). For maintenance of plasmids in recombinant E. coli and R. sphaeroides strains, ampicillin (100 mg/L), chloramphenicol (30 mg/L) and/or neomycin (25-50 mg/L, depending on the plasmid) were added to the culture medium. Liquid cultures were routinely grown aerobically in a rotary shaker. When solid media were required, agar (1.5% final concentration) was added.

Example 8

Cloning of Santalene Synthase (CiCaSSy) and Construction of Plasmid p-m-LPppa-CiCaSSy-mpmii alt

[0174] For the expression of Cinnamomum camphora santalene synthase in R. sphaeroides, the full length ORF was custom synthesised and optimised in terms of codon usage for R. sphaeroides by Genscript USA Inc. (Piscataway, N.J., USA). Additionally, the sequence for the promoter LPppa was added at the 5of the gene (SEQ ID NO:2). The construct was delivered cloned into plasmid pUC57. The complete construct was excised from the plasmid using the restriction enzymes EcoRI and BamHI (at the 5 and 3, respectively). The fragment containing the promoter and the gene (1758 bp) was ligated to the p-m-mpmii alt vector previously digested with the same two restriction enzymes. The ligation mixture was transformed into E. coli 517-1 cells. Transfer of p-m-LPppa-CiCaSSy-mpmii alt (FIG. 1) from S17-1 to R. sphaeroides Rs265-9c by conjugation was performed using standard procedures (Patent US 9260709B2). The nucleotide sequence of the construct LPppa-CiCaSSy is given in SEQ ID NO:2; the protein sequence is represented in SEQ ID NO:3.

Example 9

[0175] Construction of plasmid p-m-SPppa-MBP-CiCaSSy-mpmii alt

[0176] For the expression of the CiCaSSy gene in combination with the MBP tag under the regulation of promoter SPppa, the construct SPppa-MBP was synthesized by Genscript USA Inc. (Piscataway, N.J., USA). The gene coding MBP was codon optimized for the expression in R. sphaeroides. The construct SPppa-MBP was then fused to the CiCaSSy sequence and cloned in the p-m-mpmii alt vector previously digested with EcoRI and Band-H. Briefly, the construct SPppa-MBP was amplified using the primers 5-CTGTCCATGATCTTGTCGTCGTC -3 (SEQ ID NC): 16) and 5-CTGGCCTCAGAATTCAAATTTATTTGCTTTGTGAGCCCATAAC-3 (SEQ ID NO: 17), and CiCaSSy was amplified with primers 5-CAAGATCATGGACAGCATGCTC-3 (SEQ ID NO: 18) and 5-TTTATGATTTGGATCCTCAGCCCAGGTT-3 (SEQ ID NO: 19). The amplicons and the digested vectors were then assembled using the InFusion enzyme mix from Clontech. The reaction mixture was transformed into E. coli S17-1 cells. Transfer of p-m-SPppa-MBP-CiCaSSy-mpmii alt (FIG. 2) from S17-1 to R. sphaeroides Rs265-9c by conjugation was performed using standard procedures (U.S. Pat. No. 9,260,709B2). The nucleotide sequence of the construct SPppa-MBP-iCaSSy is given in SEQ ID NO:4; the protein sequence is represented in SEQ ID NO:5.

Example 10

[0177] Construction of plasmid p-m-PcrtE-TRX-CiCaSSy-mpmii alt For the expression of the CiCaSSy gene in combination with the TRX tag under the regulation of promoter PcrtE, the construct PcrtE-TRX was synthesized by Cienscript USA Inc. (Piscataway, N.J., USA). The gene coding TRX from E. coli was codon optimized for the expression in R. sphaeroides. The construct PcrtE-TRX was then fused to the CiCaSSy sequence and cloned in the p-m-mpmii alt vector previously digested with EcoRI and BamHI. Briefly, the construct PcrtE-TRX was amplified using the primers 5-CTGTCCATAATCTTGTCGTCGTCAT-3 (SEQ ID NO: 20) and 5- ACTGGCCTCAGATTCCCTCTGCTGAACG-3 (SEQ ID NO: 21), and CiCaSSy was amplified with primers 5-CAAGATTATGGACAGCATGGAAGTCC -3 (SEQ ID NO: 22) and 5-TTTATGATTTGGATCCTCAGCCAGGTT-3 (SEQ ID NO: 23). The amplicons and the digested vectors were then assembled using the InFusion enzyme mix from Clontech. The reaction mixture was transformed into E. coli S17-1 cells.

[0178] Transfer of p-m-PcrtE-TRX-CiCaSSy-mpmii alt (FIG. 3) from S17-1 to R. sphaeroides Rs265-9c by conjugation was performed using standard procedures (U.S. Pat. No. 9,260,709B2).

[0179] The nucleotide sequence of the construct PcrtE-TRX-CiCaSSy is given in SEQ ID NO:6; the protein sequence is represented in SEQ ID NO:7

Example 11

Construction of Plasmid p-m-PcrtE-TRX-SaSSy-mpmii alt

[0180] For the expression of Santalum album santalene synthase (SaSSy) in R. sphaeroides in combination with the TRX tag under the regulation of promoter PcrtE, the full length ORF together with the TRX from E. coli was custom synthesised and optimised in terms of codon usage for R. sphaeroides by Genscript USA Inc. (Piscataway, N.J., USA). Additionally, the sequence for the promoter PcrtE was added at the 5 of the gene (SEQ ID NO: 24). The construct was delivered cloned into plasmid pUC57. The complete construct was excised from the plasmid using the restriction enzymes EcoRI and BamHI (at the 5 and 3, respectively). The fragment containing the promoter and the genes (2335 bp) was ligated to the p-m-mpmii alt vector previously digested with the same two restriction enzymes. The ligation mixture was transformed into E. coli S17-1 cells.

[0181] Transfer of p-m-PcrtE-TRX-SaSSy-mpmii alt (FIG. 4) from S17-1 to R. sphaeroides Rs265-9c by conjugation was performed using standard procedures (U.S. Pat. No. 9,260,709B2).

[0182] The nucleotide sequence of the construct PcrtE-TRX-SaSSy is given in SEQ ID NO: 24; the protein sequence is represented in SEQ ID NO: 25.

Example 12

[0183] Growth conditions shake flasks Seed cultures were performed in 100 ml shake flasks without baffles with 20 ml RS102 medium with 100 mg/L neomycin and a loop of glycerol stock. Seed culture flasks were grown for 72 hours at 30 C. in a shaking incubator with an orbit of 50 mm at 110 rpm.

[0184] At the end of the 72 hours, the OD600 of the culture was assessed in order to calculate the exact volume of culture to be transferred to the larger flasks.

[0185] Shake flask experiments were performed in 300 ml shake flasks with 2 bottom baffles. Twenty nil of RS102 medium and neomycin to a final concentration of 100 mg/L were added to the flask together with 2 ml of sterile n-dodecane. The volume of the inoculum was adjusted to obtain a final OD600 value of 0.05 in 20 ml medium. The flasks were kept for 72 hours at 30 C. in a shaking incubator with an orbit of 50 mm at 110 rpm. Shake flask experiments were performed in duplicates.

Example 13

[0186] Sample Preparation for Analysis of Isoprenoid Content in Organic Phase

[0187] Cultures were collected 72 hours after inoculation in pre-weighted 50 ml PP tubes which were then centrifuged at 4500g for 20 minutes. The n-dodecane layer was transferred to a microcentrifuge tube for later GC analysis.

[0188] Ten microliters of ethyl laureate were weighed in a 10-ml glass vial to which 800 l of the isolated dodecane solution were added and weighed. Subsequently, 8 ml of acetone were added to the vial to dilute the dodecane concentration for a more accurate GC analysis. Approximately, 1.5 ml of the terpene-containing dodecane in acetone solution were transferred to a chromatography vial.

Example 14

[0189] Gas Chromatography

[0190] Gas chromatography was performed on a Shimadzu GC2010 Plus equipped with a Restek RTX-5Sil MS capillary column (30 m0.25 mm, 0.5 pm). The injector and FID detector temperatures were set to 280 C. and 300 C., respectively. Gas flow through the column was set at 40 ml/min. The oven initial temperature was 160 C., increased to 180 C. at a rate of 2 C./min, further increased to 300 C. at a rate of 50 C./min, and held at that temperature for 3 min. Injected sample volume was 1 L with a 1:50 split-ratio, and the nitrogen makeup flow was 30 ml/min 30

Example 15

[0191] Analysis of Terpenes Produced by CiCaSSy in R. sphaeroides

[0192] FIG. 5 shows the chromatogram obtained by analysing the organic phase isolated from all R. sphaeroides cultures expressing the CiCaSSy gene (strains from example 9. 10 and 11). Four principal compounds were identified: -santalene (A), trans--bergamotene (B), epi--santalene (C) and -santalene (B). The most abundant terpene was -santalene, followed by trans--bergamotene and -santalene. Since no purified santalenes are available to be used as standards, the terpene titre was calculated based on the GC response factor obtained with the terpene valencene. The total terpene production (cumulative area under the curve for all 4 terpenes) obtained in the strains from example 9, 10 and 11 are 5.30.12 g/kg dodecane, 5.80.29 g/kg dodecane and 3.40.09 g/kg dodecane, respectively. The ratio for the four terpene species was conserved in all the strains.

Example 16

[0193] Seed Medium for Fed-Batch Cultivation

[0194] The following components were dissolved in 1 L of water: 20.8 g yeast extract, 10.3 g MgSO.sub.4.7SO.sub.4.7H.sub.2O, 86 mg ZnSO.sub.4.7H.sub.2O, 30 mg MnSO.sub.4.H.sub.2O, 1.1 g CaCl.sub.2.2H.sub.2O, 0.96 g FeSO.sub.4.7H.sub.2O, 1.44 g KH.sub.2PO.sub.4, and 1.44 g K.sub.2HPO.sub.4. The pH was adjusted to 7.4 with 10 M NaOH. After sterilisation (121 C., 20 min), 50 ml of 50% (w/w) glucose, and 1 ml of 0.1 mg/ml Neomycin sulfate is added per liter of medium.

Example 17

[0195] Medium for Fed-Batch Cultivation

[0196] The following components were dissolved in 1 L of water: 25 g Yeast extract. 1.7 g MgSO.sub.4.7H.sub.2O, 0.10 g ZnSO.sub.4.7H.sub.2O, 35 mg MnSO.sub.4.H.sub.2O, 1.3 g CaCl.sub.2.2H.sub.2O, 0.17 g FeCl.sub.3.6H.sub.2O, 1.7 g K.sub.2HPO.sub.4, 1.7 KH.sub.2PO.sub.4, 1.1 g (NH.sub.4).sub.2Fe(SO.sub.4).sub.2.6H.sub.2O, 2.8 g (NH.sub.4).sub.2SO.sub.4, 1.1 g (NH.sub.4)H.sub.2PO.sub.4, 1.9 g MgCl.sub.2.6H.sub.2O, and 1 mL antifoam.

[0197] After sterilisation (121 C., 20 min), the pH is adjusted to 7.0 with 25% Ammonium hydroxide solution. Per liter of sterile medium, 60 mL of 50% (w/w) glucose. 1 ml of 0.1 mg/ml Neomycine sulfate, 4 mg Niacin, 8 mg Thiamin.HCl. 4 mg Nicotinamide, 0.2 mg Biotin, and 150 mL of n-dodecane were added.

Example 18

[0198] Cultivation Conditions Fed-Batch Fermentation

[0199] Seed cultures for the fed-batch cultivations were prepared by inoculation of 500 ml of sterile seed medium (example 16) by adding 1 ml of glycerol stock of the appropriate Rhodobacter sphaeroides strain, and incubation for 48 hours at 30 C.

[0200] In a 1 L fermenter vessel. 350 mL of fed-batch medium was sterilized and supplemented with (filter) sterilized glucose, Neomycin, vitamins, and n-dodecane as indicated (example 17). By addition of 50 ml of seed culture, the medium was inoculated and incubated for approximately 24 hours at 30 C., agitation of 600 rpm, air flow of 0.3 vvm, and a pH of 7 (adjusted by automated addition of 12.5% Ammonium hydroxide solution). After 24 hours, the agition was increased to 1200 rpm, and 450-500 g of 50 (w/w)% glucose was fed to the fermenter within 100 hours.

Example 19

[0201] GC Sample Preparation Fed-Batch Fermentation

[0202] Broth samples of approximately 20 mL were collected during a fed-batch cultivation. Ten microliters of ethyl laureate were weighed in a 10 ml glass vial to which 0.8 ml of broth sample added and weighed. Subsequently, 8 ml of acetone were added to the vial and the aceton-broth mixture was incubated at room temperature for 25 minutes while shaken at 400 rpm. Approximately 1.5 ml of the terpene-containing aceton-broth mixture was transferred to a chromatography vial and used for analysis according to example 14.

[0203] Example 20

[0204] Comparison of Terpene Production by Rhodobacter spaeroides Strains Harbouring CiCassy and SaSSy

[0205] Rhodobacter spaeroides strains harbouring either CiCaSSy (p-m-SPppa-MBP-CiCaSSy-mpmii alt, example 10) or SaSSy (p-m-PcrtE-TRX-CiCaSSy-mpmii alt, example 11) were cultivated in fed-batch mode according to example 18. Samples were withdrawn from the fermentation broth at various time points and were analyzed for the production of terpenes according to example 19 and 14. The concentration of total terpenes produced (cumulative areas of the peaks A-D in FIG. 5) were nearly equal for both strains throughout the cultivation (FIG. 6A), and a final terpene concentration of approximately 5.5 g/kg of broth was obtained. Throughout 120 hours of fed-batch cultivation, the terpenes produced by the CiCaSSy strain consisted of approximately 49% of -santalene (FIG. 6B), 21% of -santalene (FIG. 3C), and 25% of trans--bergamotene (FIG. 6D), whereas the SaSSy strain produced approximately 28% of -santalene (FIG. 6B), 14% of -santalene (FIG. 6C), and 52% of trans--bergamotene (FIG. 6D). The percentage of -santalene and -santalene produced by the CiCaSSy strain were respectively 1.8 fold and 1.5 fold higher than that of the SaSSy strain. In contrast, the trans--bergamotene fraction produced by the CiCaSSy strain was 2 fold lower than that of the SaSSy strain.

REFERENCES

[0206] Amick, J. D., Julien, B. N., 2015. Modified Santalene synthase polypeptides, encoding nucleic acid molecules and uses thereof. WO/2015/153501

[0207] Baldovini, N., Delasalle, C., Joulain, D., 2011. Phytochemistry of the heartwood from fragrant Santalum species: a review. Flavour Fragr. J. 26, 7-26.

[0208] Cankar, Jongedijk, Klompmaker, Majdic, Mumm, Bouwmeester, Bosch & Beekwilder (2015) (+)-Valencene production in Nicotiana benthamiana is increased by down-regulation of competing pathways. Biotechnol. J. 10. 180-189

[0209] Chapuis, C., 2012. Intermediates for the preparation of Beta-Santalol. WO/2012/110375

[0210] Daviet, L., ROCCI, L., and Schalk, M. (2015). Method for producing fragrant alcohols. (WO2015040197)

[0211] Diaz-Chavez, M. L., Moniodis, J., Madilao, L. L., Jancsik, S., Keeling, C. I., Barbour, E. L., Ghisalberti, E. L., Plummer, J. A., Jones, C. G., Bohlmann, J., 2013. Biosynthesis of Sandalwood Oil: Santalum album CYP76F Cytochromes P450 Produce Santalols and Bergamotol. FLoS ONE 8.

[0212] Frizzo, Caren D. et al. Essential oils of camphor tree (cinnamomum camphora nees & eberm) cultivated in Southern Brazil. Braz. arch. biol. technol. [online]. 2000, vol.43, n.3 [cited 2016-02-26], pp. 313-316.

[0213] Jones C G, Moniodis J, Zulak K G, Scaffidi A, Plummer J A, Ghisalberti E L, Barbour E L, Bohlmann J. (2011) Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases. J Biol Chem. 286(20):17445-54.

[0214] Ngo, K.-S., and Brown, G. D. (2000). Autoxidation of -santalene. Journal of Chemical Research 2000, 68-70.

[0215] Pelissier, Y., Chantal, M., Prunac, S., Bessiere, J. 1995. Volatile components of leafs, stem and bark of Cinnamomum camphora Nees et Ebermaier. J. Essent. Oil Res. 7, 313-315.

[0216] Peralta-Yahya P P, Ouellet M, Chan R, Mukhopadhyay A, Keasling J D, Lee T S. (2011) Identification and microbial production of a terpene-based advanced biofuel. Nat Commun. 2:483.

[0217] Pino J A & Fuentes V. 1998, Leaf Oil of Cinnamomum camphora (L.) J. Presl. from Cuba. Journal of Essential Oil Research 10, 531-532

[0218] Schalk, M., 2016. Method for producing -santalene. U.S. Pat. No. 9,297,004.

[0219] Schalk, M., 2014. Method for producing beta-santalene. EP2376643.

[0220] Schalk, M., 2006. Novel sesquiterpene synthases and methods of their use. WO/2006/13452.

[0221] Stubbs, B. J., Specht, A., and Brushett, D. 2004. The Essential Oil of Cinnamomum camphora (L.) Nees and Eberm.Variation in Oil Composition Throughout the Tree in Two Chemotypes from Eastern Australia. Journal of Essential Oil Research 16, 9-14.

[0222] Teixeira da Silva, J. A., Kher, M. M., Soner, D., Page, T., Zhang, X., Nataraj, M., Ma, G., 2016. Sandalwood: basic biology, tissue culture, and genetic transformation. Planta 243, 847-887.

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[0225] Zulak, K., Jones, C., Moniodis, J., Bohlmann, J., 2016. Terpene synthases from Santalum. U.S. Pat. No. 9,260,728.

TABLE-US-00002 SEQUENCES SEQIDNO:1 CiCaSSy-Cinnamomumcamphora Nucleotidesequence ATGGACTCCATGGAGGTACGCCGCTCTGCAATCTATCACTCGACCTTTTGGGATATTGATAGCATTCGCGCCCTGCTCGCAA GAAGAGACTGCACTGCTGCAGCTGCATTGAGTCCTGACCATCACAAAAGACTCAAGGAAAGAATTCAGCGCCGGCTACAAGA CATCACACAGCCACACCATCTGCTTGGATTGATCGACGCTGTCCAACGCCTCGGTGTGGCCTACCAGTTTGAGGAAGAAATC AGTGACGCACTGCATGGGCTTCACTCAGAGAACACGGAGCATGCAATTAAGGACAGTCTGCACCACACATCTCTCTATTTTA GATTGCTTAGGCAACATGGGTGTAACCTTTCATCAGACATATTCAACAAATTTAAGAAGGAAGGAGGAGGTTTCAAGGCAAG CCTATGTGAGGATGCAATGGGACTTTTGAGCTTGTATGAGGCTGTACGTCTTAGCGTCAAAGGTGAAGCCATCTTGGAGGAA GCTCAGGTCTTCTCGATCGCGAATTTGAAGATTCTGATGGAAAGGGTGGAGAGGAAGCTGGCAGATAGAATAGAACATGCCT TGGAGATCCCCTTGTATTGGAGGGCGCCGAGACTGGAAGCTAGATGGTACATAGATGTATATGAAAAGGAAGATGGGAGGAT TGATGACTTGCTTGATTTTGCAAAGCTAGATTTCAACAGGGTGCAAATGTTGTACCAAACCGAACTGAAGGAATTATCAATG TGGTGGGAATTGCTGGGGTTACCAGCGAAGATGGGGTTCTTCCGAGACAGACTATTGGAGAACCATCTCTTTTCAATTGCAG TGGTTGTCGAGCCTCAATACTCCCAGTGTAGAGTAGCAATTACAAAAGCCATAGTCCTTATGACAGCAATGGATGACTTTTA TGATGTGCATGGTTTGCCAGATGAGCTAAAAGTCTTCACGGACACCGTTAATCGGTGGGATTTAGAGGGAATTGATCAACTA CCAGAGTATATGAAGCTGTACTACTTGGCGTTATATAATACAACCATGAGACCGCATACATCATCCTCAAGGAGAAGGGATT CAATGCTACACATTATCTGAAGAAACTGTGGGCAATGCAAAGTAACGCGTACTTTCGGGAAGCTCAATGGTTCAACAGTGGT TACATACCTAAATTTGATGAGTATTTAGACAATGCTTTAGTCTCAGTTGGGGCGCCCTTTGTATTGGGTCTCTCATACCCCA TGATACAACAACAAATATCAAAGGAGGAAATTGATTTAATCCCCGAAGATCTAAATCTCCTCCGTTGGGCATCGATCATATT TCGACTATATGATGATTTGGCCACTTCAAAGGCTGAGCAACAACGTGGGGACGTGCCAAAATCCATCCAATGTTATATGCAT GAAACTGGTAGTTCGGAGGAAGTTGCAGCAAACCATATCAGGGACCTCATCAGTGATGCTTGGAAGGAAGTGAATGCAGAGT GTTTGAAACCTACTTCTCTGTCAAAGCATTACGTGGGAGTAGCTCCAAATTCGGCTAGGTCTGGAGTGCTGATGTACCATCA TGACTTTGATGGCTTTGCAAGTCCCCATGGCAGGACTAATGCTCATATCACGTCAATATTTTTGAACCAGTGCCATTGAAGG AATCCATCAACTTGGGATGA SEQIDNO:2 LPppa-CiCaSSy-Cinnamomumcamphora Nucleotidesequence(codonoptimized) AAATCATAAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATTAGATTCACCGGCGAGCCAGCAGGAATTTCACTTAGAT GACAGGAGGGACATATGGACAGCATGGAAGTCCGGCGGTCGGCGATCTACCACAGCACGTTCTGGGACATCGACAGCATCCG GGCGCTCCTGGCGCGGCGGGACTGCACGGCGGCCGCGGCCCTCTCGCCCGACCACCATAAGCGCCTGAAGGAGCGCATCCAG CGCCGCCTCCAGGACATCACCCAGCCCCACCATCTGCTCGGCCTCATCGACGCCGTGCAGCGCCTGGGCGTGGCCTACCAGT TCGAGGAAGAGATCTCGGACGCGCTGCACGGCCTCCATTCGGAGAACACCGAGCACGCCATCAAGGACTCGCTGCACCATAC GTCGCTCTATTTCCGCCTGCTCCGCCAGCATGGCTGCAACCTGTCGTCGGACATCTTCAACAAGTTCAAGAAGGAAGGCGGC GGCTTCAAGGCCTCGCTCTGCGAGGACGCCATGGGCCTGCTCTCGCTGTATGAGGCCGTGCGCCTCTCGGTGAAGGGCGAGG CCATCCTGGAGGAAGCCCAGGTGTTCTCGATCGCCAACCTGAAGATCCTCATGGAGCGCGTGGAGCGCAAGCTCGCCGACCG CATCGAGCATGCCTGGAGATCCCGCTCTATTGGCGCGCCCCGCGTCTGGAGGCCCGCTGGTACATCGACGTGTATGAGAAGG AAGACGGCCGCATCGACGACCTGCTCGACTTCGCGAAGCTGGACTTCAACCGCGTGCAGATGCTCTATCAGACCGAGCTGAA GGAGCTCTCGATGTGGTGGGAGCTGCTGGGCCTGCCCGCCAAGATGGGCTTCTTCCGCGACCGCCTGCTCGAGAACCACCTC TTCTCGATCGCCGTGGTGGTGGAGCCCAGTACTCGCAGTGCCGCGTGGCCATCACCAAGGCGATCGTGCTGATGACGGCGAT GGACGACTTCTATGACGTGCATGGCCTGCCGGACGAGCTCAAGGTGTTCACCGACACGGTGAACCGCTGGGACCTGGAGGGC ATCGACCAGCTCCCCGAGTACATGAAGCTGTACTATCTGGCGCTCTACAACACCACGAACGAGACGGCCTATATCATCCTGA AGGAGAAGGGCTTCAACGCCACGCATTACCTGAAGAAGCTCTGGGCCATGCAGTCGAACGCGTATTTCCGCGAGGCCCAGTG GTTCAACTCGGGCTACATCCCGAAGTTCGACGAGTATCTGGACAACGCCCTCGTGTCGGTGGGCGCCCCGTTCGTGCTGGGC CTCTCGTATCCCATGATCCAGCAGCAGATCTCGAAGGAAGAGATCGACCTGATCCCCGAGGACCTCAACCTGCTCCGCTGGG CCTCGATCATCTTCCGCCTGTACGACGACCTGGCCACCTCGAAGGCCGAGCAGCAGCGCGGCGACGTGCCCAAGTCGATCCA GTGCTATATGCATGAGACGGGCTCGTCGGAGGAAGTGGCGGCCAACCATATCCGCGACCTGATCTCGGACGCGTGGAAGGAA GTGAACGCCGAGTGCCTGAAGCCGACCTCGCTCTCGAAGCACTACGTGGGCGTGGCCCCCAACTCGGCCCGCTCGGGCGTGC TCATGTATCACCATGACTTCGACGGCTTCGCGTCGCCCCATGGCCGCACGAACGCCCACATCACGAGCATCTTCTTCGAGCC GGTCCCCTCAAGGAGAGCATCAACCTGGGCTGA SequenceinItalicsistheLPppapromoter. SEQIDNO:3 CiCaSSy-Cinnamomumcamphora Aminoacidsequence MDSMEVRRSAIYHSTFWDIDISRALLARRDCTAAAALSPDHHKRLKERIQRRLQDITQPHHLLGLIDAVQRLGVAYQFEEEI SDALHGLHSENTEHAIKDSLHHTSLYFRLLRQHGCNLSSDIFNKFKKEGGGFKASLCEDAMGLLSLYEAVRLSVKGEAILEE AQVFSIANLKILMERVERKLADRIEHALEIPLYWRAPRLEARWYIDVYEKEDGRIDDLLDFAKLDFNRVQMLYQTELKELSM WWELLGLPAKMGFFRDRLLENHLFSIAVVVEPQYSQCRVAITKAIVLMTAMDDFYDVHGLPDELKVFTDTVNRWDLEGIDQL PEYMKLYYLALYNTTNETAYIILKEKGFNATHYLKKLWAMQSNAYFREAQWFNSGYIPKFDYLDNALVSVGAPFVLGLSYPM IQQQISKEEIDLIPEDLNLLRWASHFRLYDDLATSKAEQQRGDVPKSIQCYMHETGSSEVVAANHIRDLISDAWKEVNAECL LPTSLSKHYVGVAPNSARGVLMYHHDFDGFASPHGRTNAHITSIFFEPVPLKESINLG SEQIDNO:4 SPppa-MBP-CiCaSSy-Nucleotidesequence AAATTTATTTGCTTTGTGAGCGGATAACAATTATTAGATTCACCGGCGAGCCAGCAGGAATTTCACTCTAGATGACAGGAGG GACATCATACGACGACGACAAGATCTTCCAGGACAAGCTCTATCCCTTCAGCGTGGGACGCCGTGCGCTACAACGGCAAGCT GATCGCGTATCCCATCGCGGTGGAGGCCCTGTCGCTCATCTATAACAAGGACCTGCTCCCGAACCCGCCCAAGACCTGGGAG GAGATCCCCGCCCTCGACAAGGAGCTGAAGGCCAAGGGCAAGTCGGCGCTCATGTTCAACCTGCAGGAGCCGTACTTCACCT GGCCCCTGATCGCGGCCGACGGCGGCTACGCGTTCAAGTATGAGAACGGCAAGTATGACATCAAGGACGTGGGCGTGGACAA CGCGGGCGCCAAGGCCGGCCTGACCTTCCTCGTGGACCTGATCAAGAACAAGCACATGAACGCCGACACGGACTACTCGATC GCGGAGGCCGCGTTCAACAAGGGCGAGACCGCCATGACGATCAACGGCCCGTGGGCGTGGTCGAACATCGACACCTCGAAGG TGAACTATGGCGTGACCGTGCTCCCCACGTTCAAGGGCCAGCCCTCGAAGCCCTTCGTGGGCGTGCTGTCGGCGGGCATCAA CGCCGCGTCGCCGAACAAGGAGCTCGCGAAGGAGTTCCTGGAGAACTACCTGCTCACCGACGAGGGCCTGGAGGCCGTGAAC AAGGACAAGCCCCTGGGCGCCGTGGCCCTGAAGTCGTATGAGGAAGAGCTGGTGAAGGACCCGCGCATCGCGGCCACCATGG AGAACGCGCAGAAGGGCGAGATCATGCCGAACATCCCCCAGATGTCGGCCTTCTGGTATGCGGTGCGCACCGCCGTGATCAA CGCGGCCTCGGGCCGCCAGACCGTGGACGAGGCCCTCAAGGACGCCCAGACCGGCGACGACGACGACAAGATCATGGACAGC ATGGAAGTCCGGCGGTCGGCGATCTACCACAGCACGTTCTGGGACATCGACAGCATCCGGGCGCTCCTGGCGCGGCGGACTG CACGGCGGCCGCGGCCCTCTCGCCCGACCACCATAAGCGCCTGAAGGAGCGCATCCAGCGCCGCCTCCAGGACATCACCCAG CCCCACCATCTGCTCGGCCTCATCGACGCCGTGCAGCGCCTGGGCGTGGCCTACCAGTTCGAGGAAGAGATCTCGGACGCGC TGCACGGCCTCCATTCGGAGAACACCGAGCACGCCATCAAGGACTCGCTGCACCATACGTCGCTCTATTTCCGCCTGCTCCG CCAGCATGGCTGCAACCTGTCGTCGGACATCTTCAACAAGTTCAAGAAGGAAGGCGGCGGCTTCAAGGCCTCGCTCTGCGAG GACGCCATGGGCCTGCTCTCGCTGTATGAGGCCGTGCGCCTCTCGGTGAAGGGCGAGGCCATCCTGGAGGAAGCCCAGGTGT TCTCGATCGCCAACCTGAAGATCCTCATGGAGCGCGTGGAGCGCAAGCTCGCCGACCGCATCGAGCATGCCCTGGAGATCCC GCTCTATTGGCGCGCCCCGCGTCTGGAGGCCCGCTGGTACATCGACGTGTATGAGAAGGAAGACGGCCGCATCGACGACCTG CTCGACTTCGCGAAGCTGGACTTCAACCGCGTGCAGATGCTCTATCAGACCGAGCTGAAGGACTCTCGATGTGGTGGGAGCT GCTGGGCCTGCCCGCCAAGATGGGCTTCTTCCGCGACCGCCTGCTCGAGAACCACCTCTTCTCGATCGCCGTGGTGGTGGAG CCCCAGTACTCGCAGTGCCGCGTGGCCATCACCAAGGCGATCGTGCTGGATGACGGCGATGGACGACTTCTATGACGTGCAT GGCCTGCCGGACGAGCTCAAGGTGTTCACCGACACGGTGAACCGCTGGGACCTGGAGGGCATCGACCAGCTCCCCGAGTACA TGAAGCTGTACTATCTGGCGCTCTACAACACCACGAACGAGACGGCCTATATCATCCTGAAGGAGAAGGGCTTCAACGCCAC GCATTACCTGAAGAAGCTCTGGGCCATGCAGTCGAACGCGTATTTCCGCGAGGCCCAGTGGTTCAACTCGGGCTACATCCCG AAGTTCGACGAGTATCTGGACAACGCCCTCGTGTCGGTGGGCGCCCCGTTCGTGCTGGGCCTCTCGTATCCCATGATCCAGC AGCAGATCTCGAAGGAAGAGATCGACCTGATCCCCGAGGACCTCAACCTGCTCCGCTGGGCCTCGATCATCTTCCGCCTGTA CGACGACCTGGCCACCTCGAAGGCCGAGCAGCAGCGCGGCGACGTGCCCAAGTCGATCCAGTGCTATATGCATGAGACGGGC TCGTCGGAGGAAGTGGCGGCCAACCATATCCGCGACCTGATCTCGGACGCGTGGAAGGAAGTGAACGCCGAGTGCCTGAAGC CGACCTCGCTCTCGAAGCACTACGTGGGCGTGGCCCCCAACTCGGCCCGCTCGGGCGTGCTCATGTATCACCATGACTTCGA CGGCTTCGCGTCGCCCCATGGCCGCACGAACGCCCACATCACGAGCATCTTCTTCGAGCCGGTCCCCCTCAAGGAGAGCATC AACCTGGGCTGA SequenceinItalicsistheSPppapromoter;theunderlinedsequenceisthecodon optimizedMBP. SEQIDNO:5 MBP-CiCaSSy-Proteinsequence MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITP DKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSEAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVT VLPTFKGQPSKPFVGVLSAGINAAPSNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKG EIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGDDDDKIMDSMEVRRSAIYHSTFWDIDSIRALLARRDCTAAAA LSPDHHKRLKERIQRRLQDITQPHHLLGLIDAVQRLGVAYQFEEEISDALHGLHSENTEHAIKDSLHHTSLYFRLLRQHQHG CNLSSDIFNKFKKEGGGFKASLCEDAMGLLSLYEAVRLSVKGEAILEEAQVFSIANLKILMERVERKLADRIEHALEIPLYW RAPRLEARWYIDVYEKEDGRIDDLLDFAKLDFNRVQMLYQTELKELSMWWELLGLPAKMGFFRDRLLENHLFSIAVVVEPQY SQCRVAITKAIVLMTAMDDFDVHGLPDELKVFTDTVNRWDLEGIDQLPEYMKLYYLALYNTTNETAYIILKEKGFNATHYLK KLWAMQSNAYFREAQWFNSGYIPKFDEYLDNALVSVGAPFVLGLSYPMIQQQISKEEIDLIPEDLNLLRWASIIFRLYDDLA TSKAEQQRGDVPKSIQCYMHETGSSEEVAANHIRDLISDAWKEVNAECLKPTSLSKHYVGVAPNSARSGVLMYHHDFDGFAS PHGRTNAHITSIFFEPVPLKESINLG TheunderlinedsequenceintheMBP. SEQIDNO:6 PcrtE-TRX-CiCaSSy-Nucleotidesequence CGCTGCTGAACGCGATGGCGGCGCGGGGCGCGACGCGCGGGGCCGCATCCGTCTGCATCGGCGGGGGCGAGGCGACGGCCAT CGCGCTGGAACGGCTGAGCTAATTCATTTGCGCGAATCCGCGTTTTTCGTGCACGATGGGGGAACCGGAAACGGCCACGCCT GTTGTGGTTGCGTCGACCTGTCTTCGGGCCATGCCCGTGACGCGATGTGGCAGGCGCATGGGGCGTTGCCGATCCGGTCGCA TGACTGACGCAACGAAGGCACATATGTCGGACAAGATCATCCACCTGACCGACGACAGCTTCGACACCGACGTGCTGAAGGC CGACGGCGCCATCCTCGTCGATTTCTGGGCCGAATGGTGCGGCCCCTGCAAGATGATCGCGCCGATCCTCGACGAGATCGCC GACGAATATCAGGGCAAGCTGACCGTCGCCAAGCTGAACATCGACCAGAACCCGGGCACGGCGCCGAAATACGGCATCCGCG GCATCCCGACGCTGCTGCTCTTCAAGAACGGCGAGGTGGCGGCCACCAAGGTCGGCGCGCTGTCGAAGGGCCAGCTGAAGGA GTTCCTCGATGCGAACCTCGCCGGTGGTGATGACGACGACAAGATTATGGACAGCATGGAAGTCCGGCGGTCGGCGATCTAC CACAGCACGTTCTGGGACATCGACAGCATCCGGGCGCTCCTGGCGCGGCGGGACTGCACGGCGGCCGCGGCCCTCTCGCCCG ACCACCATAAGCGCCTGAAGGAGCGCATCCAGCGCCGCCTCCAGGACATCACCCAGCCCCACCATCTGCTCGGCCTCATCGA CGCCGTGCAGCGCCTGGGCGTGGCCTACCAGTTCGAGGAAGAGATCTCGGACGCGCTGCACGGCCTCCATTCGGAGAACACC GAGCACGCCATCAAGGACTCGCTGCACCATACGTCGCTCTATTTCCGCCTGCTCCGCCAGCATGGCTGCAACCTGTCGTCGG ACATCTTCAACAAGTTCAAGAAGGAAGGCGGCGGCTTCAAGGCCTCGCTCTGCGAGGACGCCATGGGCCTGCTCTCGCTGTA TGAGGCCGTGCGCCTCTCGGTGAAGGGCGAGGCCATCCTGGAGGAAGCCCAGGTGTTCTCGATCGCCAACCTGAAGATCCTC ATGGAGCGCGTGGAGCGCAAGCTCGCCGACCGCATCGAGCATGCCCTGGAGATCCCGCTCTATTGGCGCGCCCCGCGTCTGG AGGCCCGCTGGTACATCGACGTGTATGAGAAGGAAGACGGCCGCATCGACGACCTGCTCGACTTCGCGAAGCTGGACTTCAA CCGCGTGCAGATGCTCTATCAGACCGAGCTGAAGGAGCTCTCGATGTGGTGGGAGCTGCTGGGCCTGCCCGCCAAGATGGGC TTCTTCCGCGACCGCCTGCTCGAGAACCACCTCTTCTCGATCGCCGTGGTGGTGGAGCCCCAGTACTCGCAGTGCCGCGTGG CCATCACCAAGGCGATCGTGCTGATGACGGCGATGGACGACTTCTATGACGTGCATGGCCTGCCGGACGAGCTCAAGGTGTT CACCGACACGGTGAACCGCTGGGACCTGGAGGGCATCGACCAGCTCCCCGAGTACATGAAGCTGTACTATCTGGCGCTCTAC AACACCACGAACGAGACGGCCTATATCATCCTGAAGGAGAAGGGCTTCAACGCCACGCATTACCTGAAGAAGCTCTGGGCCA TGCAGTCGAACGCGTATTTCCGCGAGGCCCAGTGGTTCAACTCGGGCTACATCCCGAAGTTCGACGAGTATCTGGACAACGC CCTCGTGTCGGTGGGCGCCCCGTTCGTGCTGGGCCTCTCGTATCCCATGATCCAGCAGCAGATCTCGAAGGAAGAGATCGAC CTGATCCCCGAGGACCTCAACCTGCTCCGCTGGGCCTCGATCATCTTCCGCCTGTACGACGACCTGGCCACCTCGAAGGCCG AGCAGCAGCGCGGCGACGTGCCCAAGTCGATCCAGTGCTATATGCATGAGACGGGCTCGTCGGAGGAAGTGGCGGCCAACCA TATCCGCGACCTGATCTCGGACGCGTGGAAGGAAGTGAACGCCGAGTGCCTGAAGCCGACCTCGCTCTCGAAGCACTACGTG GGCGTGGCCCCCAACTCGGCCCGCTCGGGCGTGCTCATGTATCACCATGACTTCGACGGCTTCGCGTCGCCCCATGGCCGCA CGAACGCCCACATCACGAGCATCTTCTTCGAGCCGGTCCCCCTCAAGGAGAGCATCAACCTGGGCTGA SequenceinItalicsisthePcrtEpromoter;theunderlinedsequenceisthecodon optimizedTRX. SEQIDNO:7 TRX-CiCaSSy-Proteinsequence MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLF KNGEVAATKVGALSKGQLKEFLDANLAGGDDDDKIMDSMEVRRSAIYHSTFWDIDSIRALLARRDCTAAAALSPDHHKRLKE RIQRRLQDITQPHHLLGLIDAVQRLGVAYQFEEEISDALHGLHSENTEHAIKDSLHHTSLYFRLLRQHGCNLSSDIFNKFKK EGGGFKASLCEDAMGLLSLYEAVRLSVKGEAILEEAQVFSIANLKILMERVERKLADRIEHALEIPLYWRAPRLEARWYIDV YEKEDGRIDDLLDFAKLDFNRVQMLYQTELKELSMWWELLGLPAKMGFFRDRLLENHLFSIAVVVEPQYSQCRVAITKAIVL MTAMDDFYDVHGLPDELKVFTDTVNRWDLEGIDQLPEYMKLYYLALYNTTNETAYIILKEKGFNATHYLKKLWAMQSNAYFR EAQWFNSGYIPKFDEYLDNALVSVGAPFVLGLSYPMIQQQISKEEIDLIPEDLNLLRWASIIFRLYDDLATSKAEQQRGDVP KSIQCYMHETGSSEEVAANHIRDLISDAWKEVNAECLKPTSLSKHYVGVAPNSARSGVLMYHHDFDGFASPHGRTNAHITSI FFEPVPLKESINLG TheunderlinedsequenceistheTRX. SEQIDNO:8 TS23FW-Nucleotidesequence atatggatcctATGGACTCCATGGAGGTACGCCGCTCTG SEQIDNO:9 TS23RE-Nucleotidesequence atatgcggccgcTCATCCCAAGTTGATGGATTCCTTCAATGGCACTG SEQIDNO:10 TS23-1-Proteinsequence MDSMEVRRSANYHSTFWDIDSIRALLARRDCTVAAALSHDHHHKRLKERIQRRLQDITQPHHLLGLIDAVQRLGVAYQFEEE ISDALHGLHSENTEHAIKDSLHHTSLYFRLLRQHGCNLSSDIFNKFKKEGGGFKASLCEDAMGLLSLYEAAHLGVKSEAILE EAQVFSTSNLKILMERVERKLADRIDHALEIPLYWRAPRVEARWIYDVYEKEDGRIDDLLDFAKLDFNRVQMLYQTELKELS MWWELLGLPEKMGFFRDRLLESHLFSIGVVVEPQYSQCRVAITKALVLFTAMDDFYDVHGLPEELKVFTDTVNRWDLEGIDQ LPEYMKLYYLALYNTTNETAYIILKEKGFNATHYLKKLWAMQSNSYFREAQWFNSGYIPKFDEYLDNALVSVGVPLLLGLSY PMIQQHISKAEIDLIPEDLNLLRWASIIFRLYNDLATSKAEQQRGDVPKSIQCYMHETGSSEVVAANHIRDLISDAWKELNA ECLKPTSLSKIIYVGVAPNSARSGVLMYIIIIDFDGFASPIISRTNAIITSIFFEPVPLKSEINLG SEQIDNO:11 TS23-2-Proteinsequence MDMSEVRRSANYHSTFWDIDSIRALLARRDCTVAAALSHDHHKRLKERIQRRLQDITQPHHLLGLIDAVQRLGVAYQFEEEI SDALHGLHSENTEHAVKDSLHHTSLYFRLLRQHGCNLSTDIFNKFKKEGGGFKASLCEDAMGLLSLYEAAHLGVKSEAILEE AQVFSTSNLKILMERVERKLADRIDHALEIPLYWRAPRVEARWYIDVYEKEDGRIDDLLDFAKLDFNRVQMLYQTELKELSM WWELLGLPEKMGFFRDRLLESHLFSIGVVVEPQYSQCRVAITKALVLFTAMDDFYDVHGLPEELKVFTDTVNRWDLEGIDQL PEYMKLYYLALYNTTNETAYIILKEKGFNATHYLKKLWAMQSNSYFREAQWFNSGYIPKFDEYLDNALVSVGVPLLLGLSYP MIQQHISKAEIDLIPEDLNLLRWASIIFRLYNDLATSKAEQQRGDVPKSIQCYMHETGSSEEVAANHIRDLISDAWKEVNAE CLKPTSLSKHYVGVAPNSARSGVLMYHHDFDGFASPHSRTNAHITSIFFEPVPLKESINLG SEQIDNO:12 P7-Nucleotidesequence GCTGTTAGCGGGCCCATTAAG SEQIDNO:13 P2-Nucleotidesequence GATATTCTCATTTTAGCCATTTTAGCTTCCTTAGCTCCTG SEQIDNO:14 P5-Nucleotidesequence CAGGAGCTAAGGAAGCTAAAATGGCTAAAATGAGAATATC SEQIDNO:15 P6-Nucleotidesequence CCAAGCGAGCTCGATATCAAACTAAAACAATTCATCCAG SEQIDNO:16 Nucleotidesequence CTGTCCATGATCTTGTCGTCGTC SEQIDNO:17 Nucleotidesequence ACTGGCCTCAGAATTCAAATTTATTTGCTTTGTGAGCGGATAAC SEQIDNO:18 Nucleotidesequence CAAGATCATGGACAGCATGGAAGTC SEQIDNO:19 Nucleotidesequence TTTATGATTTGGATCCTCAGCCCAGGTT SEQIDNO:20 Nucleotidesequence CTGTCCATAATCTTGTCGTCGTCAT SEQIDNO:21 Nucleotidesequence ACTGGCCTCAGAATTCCGCTGCTGAACG SEQIDNO:22 Nucleotidesequence CAAGATTATGGACAGCATGGAAGTCC SEQIDNO:23 Nucleotidesequence TTTATGATTTGGATCCTCAGCCCAGGTT SEQIDNO:24 PcrtE-TRX-SaSSy-Nucleotidesequence CGCTGCTGAACGCGATGGCGGCGCGGGGCGCGACGCGCGGGGCCGCATCCGTCTGCATCGGCGGGGGCGAGGCGACGGCCAT CGCGCTGGAACGGCTGAGCTAATTCATTTGCGCGAATCCGCGTTTTTCGTGCACGATGGGGGAACCGGAAACGGCCACGCCT GTTGTGGTTGCGTCGACCTGTCTTCGGGCCATGCCCGTGACGCGATGTGGCAGGCGCATGGGGCGTTGCCGATCCGGTCGCA TGACTGACGCAACGAAGGCACATATGTCGGACAAGATCATCCACCTGACCGACGACAGCTTCGACACCGACGTGCTGAAGGC CGACGGCGCCATCCTCGTCGATTTCTGGGCCGAATGGTGCGGCCCCTGCAAGATGATCGCGCCGATCCTCGACGAGATCGCC GACGAATATCAGGGCAAGCTGACCGTCGCCAAGCTGAACATCGACCAGAACCCGGGCACGGCGCCGAAATACGGCATCCGCG GCATCCCGACGCTGCTGCTCTTCAAGAACGGCGAGGTGGCGGCCACCAAGGTCGGCGCGCTGTCGAAGGGCCAGCTGAAGGA GTTCCTCGATGCGAACCTCGCCGGTGGTGATGACGACGACAAGATTATGGACAGCAGCACCGCGACCGCCATGACCGCCCCC TTCATCGACCCCACCGACCACGTGAACCTCAAGACCGACACCGACGCCAGCGAGAACCGTCGCATGGGCAACTACAAGCCGT CGATCTGGAACTATGACTTCCTGCAGAGCCTCGCCACCCACCATAACATCGTGGAGGAGCGCCACCTCAAGCTGGCGGAGAA GCTGAAGGGCCAGGTCAAGTTCATGTTCGGCGCCCCTATGGAGCCCCTCGCGAAGCTCGAGCTGGTCGACGTGGTCCAGCGG CTCGGCCTGAACCACCTGTTCGAGACCGAGATCAAGGAAGCCCTCTTCTCGATCTACAAGGACGGCAGCAACGGGTGGTGGT TCGGCCACCTGCATGCGACGTCGCTCCGCTTCCGGCTGCTCCGCCAGTGCGGCCTGTTCATCCCCCAGGACGTGTTCAAGAC CTTCCAGAACAAGACGGGCGAGTTCGACATGAAGCTCTGCGACAACGTCAAGGGCCTGCTCTCGCTGTACGAGGCCAGCTAT CTCGGCTGGAAGGGCGAGAACATCCTGGACGAGGCCAAGGCGTTCACCACGAAGTGCCTCAAGTCGGCCTGGGAGAACATCA GCGAGAAGTGGCTGGCGAAGCGCGTGAAGCACGCCCTCGCGCTGCCGCTCCATTGGCGCGTCCCCCGGATCGAGGCGCGGTG GTTCATCGAGGCCTATGAGCAGGAAGCCAACATGAACCCCACCCTGCTCAAGCTGGCCAAGCTCGACTTCAACATGGTGCAG TCGATCCACCAGAAGGAGATCGGCGAGCTGGCGCGGTGGTGGGTCACCACGGGCCTGGACAAGCTCGCCTTCGCGCGGAACA ACCTGCTCCAGTCGTACATGTGGAGCTGCGCCATCGCGTCGGACCCCAAGTTCAAGCTGGCCCGCGAGACCATCGTGGAGAT CGGCTCGGTCCTCACGGTGGTCGACGACGGCTACGACGTGTATGGCAGCATCGACGAGCTGGACCTCTATACCTCGAGCGTG GAGCGGTGGTCGTGCGTCGAGATCGACAAGCTCCCGAACACCCTGAAGCTCATCTTCATGTCGATGTTCAACAAGACGAACG AGGTCGGCCTCCGCGTCCAGCATGAGCGGGGCTACAACTCGATCCCCACGTTCATCAAGGCCTGGGTGGAGCAGTGCAAGTC GTATCAGAAGGAAGCCCGCTGGTTCCACGGTGGCCATACCCCGCCCCTGGAGGAGTACTCGCTGAACGGCCTCGTGAGCATC GGCTTCCCGCTGCTCCTGATCACGGGCTATGTGGCCATCGCGGAGAACGAGGCCGCGCTGGACAAGGTCCACCCGCTCCCCG ACCTGCTGCATTACTCGAGCCTCCTGTCGCGCCTGATCAACGACATCGGCACCAGCCCCGACGAGATGGCGCGGGGCGACAA CCTCAAGTCGATCCACTGCTATATGAACGAGACGGGCGCCAGCGAGGAAGTGGCGCGCGAGCATATCAAGGGCGTCATCGAG GAGAACTGGAAGATCCTGAACCAGTGCTGCTTCGACCAGTCGCAGTTCCAGGAGCCGTTCATCACCTTCAACCTCAACTCGG TGCGCGGCAGCCACTTCTTCTACGAGTTCGGCGACGGCTTCGGCGTCACGGACTCGTGGACGAAGGTGGACATGAAGAGCGT GCTGATCGACCCCATCCCCCTGGGCGAGGAGTGA SequenceinItalicsisthePcrtEpromoter;theunderlinedsequenceisthecodon optimizedTRX. SEQIDNO:25 TRX-SaSSy-Proteinsequence MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLF KNGEVAATKVGALSKGQLKEFLDANLAGGDDDDKIMDSSTATAMTAPFIDPTDHVNLKTDTDASENRRMGVYKPSIWNYDFL QSLATHHNIVEERHLKLAEKLKGQVKFMFGAPMEPLAKLELVDVVQRLGLNHLFETEIKEALFSIYKDGSNGWWFGHLHATS LRFRLLRQCGLFIPQDVFKTFQNKTGEFDMKLCDNVKGLLSLYEASYLGWKGENILDEAKAFTTKCLKSAWENISEKWLAKR VKHALALPLHWRVPRIEARWFIEAYEQEANMNPTLLKLAKLDFNMVQSIHQKEIGELARWWVTTGLDKLAFARNNLLQSYMW SCAIASDPKFKLARETIVEIGSVLTVVDDGYDVYGSIDELDLYTSSVERWSCVEIDKLPNTLKLIFMSFNKTNEVGLRVQHE RGYNSIPTFIKAWVEQCKSYQKEARWFHGGHTPPLEEYSLNGLVSIGFPLLLITGYVAIAENEAALDKVHPLPDLLHYSSLL SRLINDIGTSPDEMARGDNLKSIHCYMNETGASEEVAREHIKGVIEENWKILNQCCFDQSQFQEPFITFNLNSVRGSHFFYE FGDGFGVTDSWTKVDMKSVLIDPIPLGEE TheunderlinedsequenceistheTRX. SEQIDNO:26 NusA-Proteinsequence MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDVRVQIDSRKSGDFDTFRRWLVVDEVTQPTKEITLEAAR YEDESLNLGDYVEDQIESVTFDRITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVIL REDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEIKAAARDPGSRAKIAVKTNDKRI DPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRL ASQLSGWELNVMTVDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLDEPTVEALRER AKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDLAEQGIDDLADIEGLTDEKAGALIMAARNICWF GDEA SEQIDNO:27 TRX-Proteinsequence MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLF KNGEVAATKVGALSKGQLKEFLDANLAGGDDDDKI SEQIDNO:28 MBP-Proteinsequence MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWHADRFGGYAQSGLLAEITP DKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVT VLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKG EINPNIPQMSAFWYAVRTAVINAAWGRQTVDEALKDAQTGDDDDKI SEQIDNO:29 SET-Proteinsequence EEASVTSTEETLTPAQEAARTRAANKARKEAELAAATAEQ SEQIDNO:30 NusA-CiCaSSy-Nucleotidesequence ATGAACAAGGAGATCCTCGCGGTGGTGGAGGCGGTGTCGAACGAGAAGGCGCTGCCCCGCGAGAAGATCTTCGAGGCCCTGG AGTCGGCCCTGGCCACCGCGACCAAGAAGAAGTACGAGCAGGAGATCGACGTGCGCGTGCAGATCGACCGCAAGTCGGGCGA CTTCGACACGTTCCGCCGCTGGCTCGTGGTGGACGAGGTGACCCAGCCCACGAAGGAGATCACCCTGGAGGCGGCCCGCTAT GAGGACGAGTCGCTGAACCTCGGCGACTATGTGGAGGACCAGATCGAGTCGGTGACCTTCGACCGCATCACCACGCAGACGG CGAAGCAGGTGATCGTGCAGAAGGTGCGCGAGGCCGAGCGCGCCATGGTGGTGGACCAGTTCCGCGAGCACGAGGGCGAGAT CATCACCGGCGTGGTGAAGAAGGTGAACCGCGACAACATCTCGCTGGACCTGGGCAACAACGCGGAGGCCGTGATCCTGCGC GAGGACATGCTCCCGCGCGAGAACTTCCGCCCGGGCGACCGCGTGCGCGGCGTGCTCTATTCGGTGCGCCCCGAGGCCCGTG GCGCCCAGCTGTTCGTGACCCGCTCGAAGCCGGAGATGCTGATCGAGCTCTTCCGCATCGAGGTGCCCGAGATCGGCGAGGA AGTGATCGAGATCAAGGCGGCCGCCCGCGACCCGGGCTCGCGCGCGAAGATCGCCGTGAAGACCAACGACAAGCGCATCGAC CCCGTGGGCGCCTGCGTGGGCATGCGTGGCGCCCGCGTGCAGGCCGTGTCGACCGAGCTCGGCGGCGAGCGCATCGACATCG TGCTGTGGGACGACAACCCGGCGCAGTTCGTGATCAACGCCATGGCCCCGGCGGACGTGGCCTCGATCGTGGTGGACGAGGA CAAGCATACCATGGACATCGCCGTGGAGGCGGGCAACCTGGCCCAGGCCATCGGCCGCAACGGCCAGAACGTGCGCCTGGCC TCGCAGCTCTCGGGCTGGGAGCTGAACGTGATGACGGTGGACGACCTGCAGGCCAAGCATCAGGCCGAGGCCCATGCCGCCA TCGACACCTTCACGAAGTACCTCGACATCGACGAGGACTTCGCGACCGTGCTCGTGGAGGAAGGCTTCTCGACGCTGGAGGA GCTCGCCTATGTGCCGATGAAGGAGCTGCTCGAGATCGAGGGCCTGGACGAGCCGACGGTGGAGGCGCTCCGCGAGCGCGCC AAGAACGCCCTGGCCACCATCGCCCAGGCCCAGGAAGAGTCGCTGGGCGACAACAAGCCGGCCGACGACCTGCTCAACCTGG AGGGCGTGGACCGCGACCTGGCCTTCAAGCTCGCCGCCCGCGGCGTGTGCACGCTCGAGGACCTGGCCGAGCAGGGCATCGA CGACCTGGCCGACATCGAGGGCCTCACCGACGAGAAGGCCGGCGCCCTGATCATGGCCGCCCGCAACATCTGCTGGTTCGGC GACGAGGCGATGGACAGCATGGAAGTCCGGCGGTCGGCGATCTACCACAGCACGTTCTGGGACATCGACAGCATCCGGGCGC TCCTGGCGCGGCGGGACTGCACGGCGGCCGCGGCCCTCTCGCCCGACCACCATAAGCGCCTGAAGGAGCGCATCCAGCGCCG CCTCCAGGACATCACCCAGCCCCACCATCTGCTCGGCCTCATCGACGCCGTGCAGCGCCTGGGCGTGGCCTACCAGTTCGAG GAAGAGATCTCGGACGCGCTGCACGGCCTCCATTCGGAGAACACCGAGCACGCCATCAAGGACTCGCTGCACCATACGTCGC TCTATTTCCGCCTGCTCCGCCAGCATGGCTGCAACCTGTCGTCGGACATCTTCAACAAGTTCAAGAAGGAAGGCGGCGGCTT CAAGGCCTCGCTCTGCAGAGGACGCCATGGGCCTGCTCTCGCTGTATGAGGCCGTGCGCCTCTCGGTGAAGGGCGAGGCCAT CCTGGAGGAAGCCCAGGTGTTCTCGATCGCCAACCTGAAGATCCTCATGGAGCGCGTGGAGCGCAAGCTCGCCGACCGCATC GAGCATGCCCTGGAGATCCCGCTCTATTGGCGCGCCCCGCGTCTGGAGGCCCGCTGGTACATCGACGTGTATGAGAAGGAAG ACGGCCGCATCGACGACCTGCTCGACTTCGCGAAGCTGGACTTCAACCGCGTGCAGATGCTCTATCAGACCGAGCTGAAGGA GCTCTCGATGTGGTGGGAGCTGCTGGGCCTGCCCGCCAAGATGGGCTTCTTCCGCGACCGCCTGCTCGAGAACCACCTCTTC TCGATCGCCGTGGTGGTGGAGCCCCAGTACTCGCAGTGCCGCGTGGCCATCACCAAGGCGATCGTGCTGATGACGGCGATGG ACGACTTCTATGACGTGCATGGCCTGCCGGACGAGCTCAAGGTGTTCACCGACACGGTGAACCGCTGGGACCTGGAGGGCAT CGACCAGCTCCCCGAGTACATGAAGCTGTACTATCTGGCGCTCTACAACACCACGAACGAGACGGCCTATATCATCCTGAAG GAGAAGGGCTTCAACGCCACGCATTACCTGAAGAAGCTCTGGGCCATGCAGTCGAACGCGTATTTCCGCGAGGCCCAGTGGT TCAACTCGGGCTACATCCCGAAGTTCGACGAGTATCTGGACAACGCCCTCGTGTCGGTGGGCGCCCCGTTCGTGCTGGGCCT CTCGTATCCCATGATCCAGCAGCAGATCTCGAAGGAAGAGATCGACCTGATCCCCGAGGACCTCAACCTGCTCCGCTGGGCC TCGATCATCTTCCGCCTGTACGACGACCTGGCCACCTCGAAGGCCGAGCAGCAGCGCGGCGACGTGCCCAAGTCGATCCAGT GCTATATGCATGAGACGGGCTCGTCGGAGGAAGTGGCGGCCAACCATATCCGCGACCTGATCTCGGACGCGTGGAAGGAAGT GAACGCCGAGTGCCTGAAGCCGACCTCGCTCTCGAAGCACTACGTGGGCGTGGCCCCCAACTCGGCCCGCTCGGGCGTGCTC ATGTATCACCATGACTTCGACGGCTTCGCGTCGCCCCATGGCCGCACGAACGCCCACATCACGAGCATCTTCTTCGAGCCGG TCCCCTCAAGGAGAGCATCAACCTGGGCTGA TheunderlinedsequenceisthecodonoptimizedNusA SEQIDNO:31 SET-CiCaSSy-Nucleotidesequence ATGGAGGAGGCCAGCGTGACCAGCACCGAGGAGACCCTGACCCCGGCCCAGGAGGCCGCCCGCACCCGCGCCGCCAACAAGG CCCGCAAGGAGGCCGAGCTGGCCGCCGCCACCGCCGAGCAGGCCGCCATGGACAGCATGGAAGTCCGGCGGTCGGCGATCTA CCACAGCACGTTCTGGGACATCGACGCATCCGGGCGCTCCTGGCGCGGCGGGACTGCACGGCGGCCGCGGCCCTCTCGCCCG ACCACCATAAGCGCCTGAAGGAGCGCATCCAGCGCCGCCTCCAGGACATCACCCAGCCCCACCATCTGCTCGGCCTCATCGA CGCCGTGCAGCGCCTGGGCGTGGCCTACCAGTTCGAGGAAGAGATCTCGGAGCGCGCTGCACGGCCTCCATTCGGAGAACAC CGAGCACGCCATCAAGGACTCGCTGCACCATACGTCGCTCTATTTCCGCCTGCTCCGCCAGCATGGCTGCAACCTGTCGTCG GACATCTTCAACAAGTTCAAGAAGGAAGGCGGCGGCTTCAAGGCCTCGCTCTGCGAGGACGCCATGGGCCTGCTCTCGCTGT ATGAGGCCGTGCGCCTCTCGGTGAAGGGCGAGGCCATCCTGGAGGAAGCCCAGGTGTTCTCGATCGCCAACCTGAAGATCCT CATGGAGCGCGTGGAGCGCAAGCTCGCCGACCGCATCGAGCATGCCCTGGAGATCCCGCTCTATTGGCGCGCCCCGCGTCTG GAGGCCCGCTGGTACATCGACGTGTATGAGAAGGAAGACGGCCGCATCGACGACCTGCTCGACTTCGCGAAGCTGGACTTCA ACCGCGTGCAGATGCTCTATCAGACCGAGCTGAAGGAGCTCTCGATGTGGTGGGAGCTGCTGGGCCTGCCCGCCAAGATGGG CTTCTTCCGCGACCGCCTGCTCGAGAACCACCTCTTCTCGATCGCCGTGGTGGTGGAGCCCCAGTACTCGCAGTGCCGCGTG GCCATCACCAAGGCGATCGTGCTGATGACGGCGATGGACGACTTCTATGACGTGCATGGCCTGCCGGACGAGCTCAAGGTGT TCACCGACACGGTGAACCGCTGGGACCTGGAGGGCATCGACCAGCTCCCCGAGTACATGAAGCTGTACTATCTGGCGCTCTA CAACACCACGAACGAGACGGCCTATATCATCCTGAAGGAGAAGGGCTTCAACGCCACGCATTACCTGAAGAAGCTCTGGGCC ATGCAGTCGAACGCGTATTTCCGCGAGGCCCAGTGGTTCAACTCGGGCTACATCCCGAAGTTCGACGAGTATCTGGACAACG CCCTCGTGTCGGTGGGCGCCCCGTTCGTGCTGGGCCTCTCGTATCCCATGATCCAGCAGCAGATCTCGAAGGAAGAGATCGA CCTGATCCCCGAGGACCTCAACCTGCTCCGCTGGGCCTCGATCATCTTCCGCCTGTACGACGACCTGGCCACCTCGAAGGCC GAGCAGCAGCGCGGCGACGTGCCCAAGTCGATCCAGTGCTATATGCATGAGACGGGCTCGTCGGAGGAAGTGGCGGCCAACC ATATCCGCGACCTGATCTCGGACGCGTGGAAGGAAGTGAACGCCGAGTGCCTGAAGCCGACCTCGCTCTCGAAGCACTACGT GGGCGTGGCCCCCAACTCGGCCCGCTCGGGCGTGCTATGTATCACCATGACTTCGACGGCTTCGCGTCGCCCCATGGCCGCA CGAACGCCCACATCACGAGCATCTTCTTCGAGCCGGTCCCCCTCAAGGAGAGCATCAACCTGGGCTGA TheunderlinedsequenceisthecodonoptimizedSETTag SEQIDNO:32 NusA-CiCaSSy-Proteinsequence MNKEILAVVEAVSNEKALPREKIFEALESLALATATKKKYEQEIDVRVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAAR YEDESLNLGDYVEDQIESVTFDRITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVIL REDMLPRENFRPGDRVRGVLYSVRPEARGQALFVTRSKPEMLIELFRIEVPEIGEEVIEIKAAARDPGSRAKIAVKTNDKRI DPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRL ASQLSGWELNVMTVDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAVYVPMKELLEIEGLDEPTVEALRE RAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDLAEQGIDDLADIEGLTDEKAGALIMAARNICW FGDEAMDSMEVRRSAIYHSTFWDIDSIRALLARRDCTAAAALSPDHHKRLKERIQRRLQDITQPHHLLGLIDAVQRLGVAYQ FEEEISDALHGLHSENTEHAIKDSLHHTSLYFRLLRQHGCNLSSDIFNKFKKEGGGFKASLCEDAMGLLSLYEAVRLSVKGE AILEEAQVFSIANLKILMERVERKLADRIEHALEIPLYWRAPRLEARWYIDVYEKEDGRIDDLLDFAKLDFNRVQMLYQTEL KELSMWWELLGLPAKMGFFRDRLLENHLFSIAVVVEPQYSQVRVAITKIAVLMTAMDDFYDVHGLPDELKVFTDTVNRWDLE GIDQLPEYMKLYYLALYNTTNETAYIILKEKGFNATIIYLKKLWAMQSNAYFREAQWFNSGYIPKFDEYLDNALVSVGAPFV LGLSYPMIQQQISKEEIDLIPEDLNLLRWASIIFRLYDDLATSKAEQQRGDVPKSIQCYMHETGSSEEVAANHIRDLISDAW KEVNAECLKPTSLSKHYVGVAPNSARSGVLMYHHDFDGFASPHGRTNAHITSIFFEPVPLKESINLG TheunderlinedsequenceisNusA SEQIDNO:33 SET-CiCaSSy-Proteinsequence MEEASVTSTEETLTPAQEAARTRAANKARKEAELAAATAEQMDSMEVRRSAIYHSTFWDIDSIRALLARRDCTAAAALSPDH HKRLKERIQRRLQDITQPHHLLGLIDAVQRLGVAYQFEEEISDALHGLHSENTEHAIKDSLHHTSLYFRLLRQHGCNLSSDI FNKFKKEGGGFKASLCEDAMGLLSLYEAVRLSVKGEAILEEAQVFSIANLKILMERVERKLADRIEIIALEIPLYWRAPRLE ARWYIDVYEKEDGRIDDLLDFAKLDFNRVQMLYQTELKELSMWWELLGLPAKMGFFRDRLLENHLFSIAVVVEPQYSQCRVA ITKAIVLMTAMDDFYDVHGLPDELKVFTFTVNRWDLEGIDQLPEYMKLYYLALYNTTNETAYIILKEKGFNATHYLKKLWAM QSNAYFREAQWFNSGYIPKFDEYLDNALVSVGAPFVLGLSYPMIQQQISKEEIDLIPEDLNLLRWASIIFRLYDDLATSKAE QQRGDVPKSIQCYMHETGSSEEVAANHIRDLISDAWKEVNAECLKPTSLSKHYVGVAPNSARSGVLMYHHDFDGFASPHGRT NAHITSIFFEPVPLKESINLG TheunderlinedsequenceisSET