VALENCENE SYNTHASE

Abstract

The present invention relates to a novel valencene synthase, to a nucleic acid encoding such valencene synthase, to a host cell comprising said encoding nucleic acid sequence and to a method or preparing valencene, comprising converting farnesyl diphosphate to valencene in the presence of a valencene synthase according to the invention.

Claims

1. Valencene synthase, which valence synthase has an increased productivity towards the conversion of farnesyl diphosphate into valencene (expressed as molar amount of valencene formed per hour) compared to a valencene synthase represented by SEQ ID NO: 2, or which valencene synthase comprises an amino acid sequence represented by SEQ ID NO: 3 provided that at least one position marked X in SEQ ID NO: 3 is different from the corresponding position in SEQ ID NO: 2.

2. Valencene synthase according to claim 1, wherein the valencene synthase has an increased specific productivity, increased stability, increased product specificity (relative to the conversion of farnesyl diphosphate into Germacrene A) or an increased expression in a host cell, compared to a valencene synthase represented by SEQ ID NO: 2.

3. Valencene synthase according to claim 2, wherein the product specificity, expressed as the molar ratio valencene formed from farnesyl diphospate to Germacrene A formed from farnesyl diphosphate (under test conditions), is 10 or more, preferably 13-30, in particular 15-25.

4. Valencene synthase according to claim 1, wherein the specific productivity of the valencene synthase, expressed as the molar amount of valencene formed per hour per amount of valencene synthase is at least 1.5 times, preferably 2.0 to 10 times, in particular 2.5 to 5 times the specific productivity of the valencene synthase represented by SEQ ID NO: 2.

5. Valencence synthase according to claim 1, wherein the valencene synthase has at least one modification, in particular at least one substitution, in the second shell of the valencene synthase or at least one modification, in particular at least one substitution, in the first shell of the valencene synthase, compared to the valencene synthase represented by SEQ ID NO: 2.

6. Valencene synthase according to claim 1, wherein at a position corresponding to a position having a cysteine in SEQ ID NO: 2 a different amino acid is present.

7. Valencene synthase according to claim 1, wherein the valencene synthase has one or more modifications, in particular one or more substitutions, compared to the valence synthase represented by SEQ ID NO: 2, at an amino acid position corresponding to a position selected from the group of 16, 128, 171, 187, 225, 244, 300, 302, 307, 319, 323, 327, 331, 334, 398, 405, 409, 410, 412, 436, 438, 439, 444, 448, 449, 450, 463, 488, 490, 492, 502, 503, 507, 527, 556, 559, 560, 566, 568, 569, and 570 of SEQ ID NO: 2.

8. Valencene synthase according to claim 6, wherein one or more modifications are selected from the group of 16A, 16T, 16S, 128L, 171R, 187K, 225S, 244S. 244T, 300Y, 302D, 307T, 307A, 319Q, 323A, 327L, 331G, 334L, 3981, 398M, 398T, 405T, 405V, 409F, 410F, 410V, 410L, 412G, 436L, 436K, 436T, 436W, 438T, 439G, 439A, 4441, 444V, 448S, 449F, 4491, 449Y, 450L, 450M, 450V, 463E, 463S, 463G, 463W, 488Y, 488H, 488S, 490N, 490A, 490T, 490F, 492A, 492K, 502Q, 503S, 507E, 507Q, 527T, 527S, 527A, 556T, 559H, 559L, 559V, 560L, 566S, 566A, 566G, 568S, 5691, 569V, 570T, 570G, 570A and 570P.

9. Valencene synthase according to claim 1, wherein the valencene synthase comprises an amino acid sequence having at least 55%, at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 2.

10. Valencene synthase according to claim 1, wherein the valencene synthase comprises a first polypeptide segment and a second polypeptide segment, the first segment comprising a tag-peptide and the second segment comprising a polypeptide having valencene synthase activity.

11. Nucleic acid, comprising a nucleic acid sequence encoding a valencene synthase according to claim 1, a complementary sequence thereof, or comprising a nucleic acid sequence hybridising with a nucleic acid sequence encoding a valencene synthase according to any of the preceding claims under stringent conditions.

12. Expression vector comprising a nucleic acid according to claim 11.

13. Antibody having specific binding activity to a valencene synthase according to claim 1, or a protein having specific binding affinity to an antigen binding part of said antibody.

14. A host cell, which may be an organism per se or part of a multi-cellular organism, said host cell comprising an expression vector according to claim 12, which host cell preferably is selected from the group of bacterial cells, fungal cells and plant cells, and more preferably is: a bacterial cell selected from the group of gram negative bacteria, such as Rhodobacter, Paracoccus or Escherichia; a fungal cell selected from the group of Aspergillus, Blakeslea, Penicillium, Phaffia (Xanthophyllomyces), Pichia, Saccharomyces, and Yarrowia; a transgenic plant or culture comprising transgenic plant cells, wherein the host cell is of a transgenic plant selected from Nicotiana spp, Solarium spp, Cichorum intybus, Lactuca sativa, Mentha spp, Artemisia annua, tuber forming plants, oil crops and trees; or a transgenic mushroom or culture comprising transgenic mushroom cells, wherein the host cell is selected from Schizophyllum, Agaricus and Pleurotisi

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

16. Method for preparing nootkatone, wherein valencene prepared in a method according to claim 15 is converted into nootkatone, which conversion may comprise a regiospecific hydroxylation of valencene followed by oxidation thereby forming nootkatone.

Description

EXAMPLES

Example 1

Preparation of Valencene Synthase Mutants for Activity Testing

[0179] Synthetic DNA fragments with the genes encoding the Valencene Synthase mutants according to the current invention (ValC_mutant) and with the gene encoding the Valencene Synthase wild type (ValC_wt, SEQ ID NO: 2) were obtained from DNA2.0 (Menlo Park, Calif., USA) in expression vector pACYCDuet-1 (Novagen, Merck, KGaA, Darmstadt, Germany). The Valencene Synthase genes had been codon optimized for improved expression in Rhodobacter by DNA2.0. The plasmids were designated pACYCDuet:ValC_mutant or pACYCDuet:Valc_wt. The nucleotide sequence of pACYCDuet:ValC_wt is shown in SEQ ID NO: 5. The nucleotide sequence of the pACYCDuet:ValC_mutant plasmids is identical to that of pACYCDuet:ValC_wt, except for the nucleotide substitutions needed for the formation of the Valencene Synthase mutants.

[0180] The expression of these pA_CYCDuet-1 based recombinant vectors in E. coli resulted in the formation of the valencene synthase (including its N-terminal methionine residue) with the following, pACYC-Duet-1 derived, N-terminal extension: NH.sub.2-MGSSHHHHHHSQDPH-COOH.

[0181] The freeze-dried pACYC-Duet-1 based vectors obtained from DNA2.0 were redissolved in 50 L of sterile water each and incubated at 37 C. for 1.5 h. Subsequently, commercially available chemically competent E. coli BL21(DE3) cells in 96-well format were transformed with 1 L redissolved plasmid solution per well applying the supplier's protocol (Novagen; protocol TB 313 Rev.B0304). Each transformation mixture (50 L plus 50 L SOC medium to facilitate uniform distribution of the liquid over the plate) were plated onto selective plates (LB-medium with 1% (w/v) glucose and 30 chloramphenicol) and incubated at 37 C. for 16 h. All selective plates contained colonies, but the number of transformants obtained greatly varied from 1 colony per plate to over 50. Plates were stored at 4 C. until further use.

[0182] Single colonies of each individual transformant were inoculated in 5 mL of LB medium with 1% (w/V) glucose and 50 g/mL chloramphenicol, and grown overnight at 37 C. These overnight cultures were used to prepare glycerol stocks, which were stored at 70 C. In addition, 200 L of each overnight culture were transferred to a 100 mL Erlenmeyer flask with 20 mL 2YT medium with 50 g/mL chloramphenicol. Flasks were closed with Rotilabo culture plugs (Carl Roth GmbH +Co. KG, Karlsruhe, Germany), and incubated at 250 rpm and 37 C. until the OD600 nm was 0.6-0.8. Then, 20 L of a 1 M IPTG solution was added and incubation was continued overnight at 250 rpm and 18 C. The next day, cultures were harvested by centrifugation in a 50 mL tube at 3,400 rpm for 15 min, after which the culture medium was removed. Subsequently, cell pellets were stored frozen at 20 C. As a reference, E. coli BL21(DE3) transformed with pACYC-Duet-1 containing the gene encoding the wild type valencene synthase (SEQ ID NO: 2) was treated accordingly.

[0183] For preparation of cleared lysates, the cell pellets were thawed on ice and resuspended in 1 mL of 50 mM Tris-HCl (pH 7.5) buffer. Then about 0.2 g of Zirconia/Silica beads 0.1 mm (BioSpec Products Inc.; http://www.biospec.com/) were added and lysis was effected by shaking for 10 seconds in a Bio101/Savant FastPrep FP 120 machine (MP Biomedicals LLC., Illkirch, France) at speed 6.5, followed by transfer of the tubes to ice for 2 min., and another round of shaking for 10 seconds at speed 6.5. Subsequently, the lysates wore centrifuged for 10 min. at 13,000g and 4 C., and supernatants (=cleared lysate) were immediately used for enzyme assays.

[0184] Because the number of valencene synthase variants to be tested was larger than the number of cleared lysates that could be produced per day, each day also a cleared lysate was prepared from the cells expressing the wild type valencene synthase as reference.

Example 2

Determination of (Specific) Productivity and Product Specificity of Valencene Synthase Mutants

[0185] For determination of the valencene productivity of the mutant valencene synthases, the following assay was used.

[0186] In a glass tube were mixed: [0187] 65 L of 50 mM Tris-HCl, pH 7.5; [0188] 800 L of Assay buffer (15 mM MOPSO (3-[N-morpholino]-2-hydroxypropane sulphonic acid) pH=7.0; 12.5% (v/v) glycerol; 1 mM ascorbic acid; 0.1% Tween 20; 1 mM MgCl.sub.2; 2 mM dithiothreitol [added just before use]; pH to 7.0 with NaOH); [0189] 20 L of 250 mM Na-orthovanadate; [0190] and 5 L of 10 mM farnesyl diphosphate (FPP, dry-evaporated and dissolved in 0.2 M ammonium carbamate and 50% ethanol, Sigma);

[0191] The reaction was started by the addition of 35 of cleared lysate. After mixing, the glass tube was incubated at 30 C. with mild agitation for 2 hours. The reaction mixture was then extracted with 2 mL ethylacetate and vortexed for 10 sec. After centrifugation for 10 min. at 1,200g, the ethylacetate layer was collected, dried over a sodium sulphate column and used for GC-MS analysis. To this end, analytes from 1 L of the ethylacetate layer were separated using gas chromatography on a 7890A GC system (Agilent Technologies) equipped with a 30 m0.25 mm, 0.25 mm film thickness column (ZB-5, Phenomenex) using helium as carrier gas at a flow rate of 1 mL/min. The injector (7683B Series, Agilent Technologies) was used in splitless mode with the inlet temperature set to 250 C. The initial oven temperature of 55 C. was increased after 1 min to 300 C. at a rate of 15 C./min and held for 5 min. at 300 C. The GC was coupled to a mass-selective detector (model 5975C, Agilent Technologies). Compounds were identified and quantified by their mass spectra, retention times and surface area of MS chromatograms in comparison with those of an authentic standard of valencene. Germacrene A was identified by the incidence of its Cope-reaction product -elemene, and quantified by comparison of its total-ion-count surface area to that of valencene.

[0192] The amount of cleared lysate (35 L) and the reaction time (2 hours) in this assay were chosen in the range where the amount of valencene termed is linearly dependent on the amount of lysate. This approach secured that valencene synthase mutants with improved productivity are identifiable by this assay.

[0193] To compensate the valencene productivities of the different valencene synthase mutants for differences in their expression level, the relative amount of the valencene synthase protein in each cleared lysate was quantified. From each cleared lysate, 60 L was added to 20 L of 4 sample buffer (composition below), incubated for 2 min. at 100 C., and stored frozen. For the analysis, this stock solution was subsequently diluted 1:50 in 1 sample buffer, incubated for 2 min. at 100 C., and briefly centrifuged. 10 L was then loaded on a 12-wells RunBlue SDS-PAGE gel (4-20%) (Expedeon, Harston, Cambridgeshire, UK). On each gel, also a negative control (cleared lysate of E. coli BL21(DE3) containing an empty pACYCDuet-1 vector) and a positive control (cleared lysate of E. coli BL21(DE3) containing pACYCDuets1 with the gene encoding the wild type valencene synthase) were loaded, Gels were run in running buffer (composition below) under cooled conditions for 30 min. at 25 mA/gel, followed by 60 min. at 50 mA/gel. Gels were stained using SYPRO Ruby protein gel stain (Invitrogen, Breda, The Netherlands). The staining procedure included fixation for 30 min. in 40% (v/v) ethanol+10% (v/v) acetic acid solution, a single wash step in demi water, followed by 4 hours incubation in non-diluted SYPRO Ruby protein gel stain in the dark. Subsequently gels were washed for 1 hour in 10% (ply) ethanol+7% (v/v) acetic acid solution, and then briefly washed in demi water. This staining procedure did yield a linear response curve.

[0194] Protein bands were quantified by scanning the gels on an Ettan DIGE Imager (GE Healthcare, Diegem, Belgium) using Poststain mode, 100 m pixels, Matrix type: Gel, and the Sypro Ruby 1 channel (Excitation Filter 480/30 nm, Emission Filter 595/25 nm, exp 0.2). Bands were detected by ImageQuant TL software package (GE Healthcare, Diegem, Belgium) using manual lane creation, rolling-hall background subtraction and manual peak detection.

[0195] Lanes with the positive control always showed an extra band that ran slightly faster than the bovine serum albumin (BSA, 66 kW) band in the lane with the molecular weight marker. Because this extra band was consistently absent from the negative control, and present in the lanes loaded with the cleared lysate from the E. coli clones expressing the mutant valencene synthases according to the invention, it was concluded that this band represents the valencene synthase, although the calculated molecular weight of valencene synthase (including the N-terminal His.sub.6-tag) is 70,967 Da. Quantification of the valencene synthase band was then done relative towards the intensity of the corresponding band in the lane with the positive control, with subtraction of the background intensity in the lane with the negative control.

[0196] The composition of the sample buffer (1) was: [0197] 10% (v/v) Glycerol [0198] 1% (w v) Sodium Dodecyl Sulfate (SIDS) [0199] 0.2 M Triethanolamine-IICl, pII 7.6 [0200] 1% (w/v) Ficoll-400 [0201] 0.006% (w/v) Phenol Red [0202] 0.006% (w/v) Coomassie Brilliant Blue G250 [0203] 0.5 mM EDTA.

[0204] The composition of the running buffer was: [0205] 0.04 M Tricine [0206] 0.06 M Tris [0207] 0.1% (w/v) Sodium Dodecyl Sulfate (SDS) [0208] 2.5 mM Sodium Bisulfite [0209] pH=8.2

[0210] The results of these in vitro tests for the valencene synthase mutants with a single amino acid modification are presented in Table 1.

TABLE-US-00001 TABLE 1 Results obtained by in-vitro testing valencene synthase mutants with a singly point mutation compared to SEQ ID NO: 2, and overview of location of the mutated amino acid positions in the 3D structural model of the valencene synthase. Prod. Spec. Prod. Rel. Prot. Distance Location WT Mutant (% rel. (% rel. Band Ratio to nearest in model Position.sup.a AA AA to wt).sup.b to wt).sup.c Intensity.sup.d Val/Ger-A.sup.e FPP atom structure.sup.f WT.sup.g 100.0 100.0 8.6 16 C A 166.5 82.5 1.31 9.7 40 Cys T 133.1 86.8 1.31 7.8 S 148.6 95.2 1.43 7.9 128 I L only tested in double mutant 28.6 far 171 K R 107.0 98.6 1.75 7.7 25.3 far 187 R K only tested in double mutant 22.5 far 225 C S 117.2 92.9 1.66 5.3 Cys Cys 244 C S 188.9 77.7 1.77 10.8 Cys Cys T 127.1 105.2 1.59 8.0 300 F Y 237.5 150.8 1.34 4.7 8.4 2nd 302 H D 103.9 42.4 2.25 3.4 11.4 2nd 307 S T 198.1 59.0 2.44 11.6 6.4 1st A 243.6 143.4 1.56 2.7 319 E Q 103.6 62.2 1.53 9.3 22.6 far 323 C A 180.2 119.5 1.98 8.2 Cys Cys 327 C L 284.4 243.4 1.88 6.9 Cys Cys 331 S G 155.2 89.2 1.13 4.9 3.5 1st 334 M L 174.2 59.4 2.50 2.6 3.9 1st 398 V M only tested in double mutant 20.6 far I 114.2 83.3 2.21 6.6 T only tested in double mutant 405 C T 154.7 103.7 1.37 9.2 Cys Cys V 123.0 75.6 1.49 9.5 409 Y F 187.6 224.1 1.40 6.0 7.3 1st 410 I F 397.8 261.2 0.99 6.6 11.2 2nd V 314.6 162.7 2.54 7.6 L 155.4 291.0 0.89 7.6 412 A G 184.3 175.5 1.69 8.9 11.4 2nd 436 V L 223.9 175.7 0.83 8.6 8.1 2nd K 192.4 80.8 1.55 7.8 T 168.3 151.2 1.86 9.1 W only tested in double mutant 438 S T 210.0 90.2 1.98 3.9 4.4 1st 439 S G 249.2 85.0 2.13 11.7 3.7 1st A 159.9 120.3 1.22 8.7 444 L I 126.6 52.2 1.58 23.7 5.5 1st V 107.9 120.0 1.50 16.7 448 A S 119.2 104.1 1.05 7.8 14.1 2nd 449 L F 158.5 175.4 1.46 9.9 17.7 far Y only tested in double mutant I only tested in double mutant 450 I L 168.1 93.9 1.64 8.1 17.8 far M 120.2 103.9 0.98 8.5 V only tested in double mutant F only tested in double mutant 463 Q S only tested in double mutant 22 far E 123.0 60.4 1.48 8.9 G only tested in double mutant W only tested in double mutant 488 F Y 152.2 84.3 1.30 6.4 10 2nd S 242.4 216.9 1.87 3.3 H 277.5 132.2 3.50 6.2 490 D N 302.7 258.3 1.96 4.1 9.6 2nd A 231.1 188.3 2.05 2.4 T 207.2 134.4 2.03 2.0 F 133.5 66.1 1.47 4.8 492 Q A 161.2 175.8 1.48 7.3 12 2nd K 264.4 184.7 0.93 8.5 502 E Q 319.1 148.1 1.40 9.7 14.4 2nd 503 C S 198.8 117.8 1.22 7.0 Cys Cys 507 D E 264.8 195.8 1.24 8.1 20.5 far Q 165.3 116.9 1.86 8.5 527 C T 173.5 66.5 1.69 9.7 Cys Cys S 134.8 99.2 1.25 7.5 A 127.3 40.1 2.29 7.4 556 V T 272.2 507.2 0.86 4.3 6.8 1st 559 F H 152.5 94.1 1.38 3.1 10.2 2nd L 183.7 103.7 1.62 3.3 V 126.2 58.3 1.84 5.3 560 M L 199.9 73.9 1.97 5.0 3.8 1st 566 L S 317.3 150.2 1.52 5.7 8.7 2nd A 195.1 119.4 1.06 5.7 G 141.3 141.8 1.66 4.8 568 T S 321.0 130.3 1.60 3.6 10.1 2nd 569 H I 362.7 147.1 1.89 8.1 5.7 1st V 203.4 142.8 1.21 5.1 570 S T 258.5 200.5 2.15 2.3 10.1 2nd G 288.3 271.1 1.78 3.4 A 116.2 44.1 1.92 7.6 P 140.3 43.6 2.32 1.8 .sup.aPosition of the Valencene Synthase amino acid residue mutated; residue numbering starting from the N-terminal methionine residue in SEQ ID NO: 2 (=Met-1) .sup.bValencene productivity relative to that obtained with wild type valence synthase (=control) {(Val[Sample]/Val[Control]) 100%} .sup.cSpecific valencene productivity relative to that of the wild type valence synthase (=control) {(Val[Sample]/Prot[Sample)/(Val[Control]/Prot[Control])} 100% .sup.dIntensity of the valencene synthase protein band on an SDS-PAGE gel relative to the intensity of that band in the positive control .sup.eRatio valencene to Germacrene A formed from FPP in the standard productivity assay. Nb. Under the GC conditions applied, Germacrene A is actually detected as its thermal rearrangement product -elemene .sup.f1st: first shell amino acid residue; 2nd: second shell amino acid residue; far: amino acid residues that are not residing in the first and second shell; Cys: cysteine residue .sup.gValencene Synthase wild-type (SEQ ID NO: 2)

[0211] The results in Table 1 show that the valencene synthase mutants with a single point mutation according to the invention demonstrate clearly improved in-vitro valencene productivities. The valencene productivity of these mutants relative to that obtained in this test system with the wild type valencene synthase ranges from 103.6% (ValC:E319Q) to 397.8% (ValC:I410F). For many of the valencene synthase mutants tested this improved valencene productivity goes together with an improved specific valencene productivity pointing to the fact that the improved valencene productivity is not primarily the result of an increased expression of these mutants in E. coli. Other valencene synthase mutants according to the invention are clear expression mutants as the intensity of the valencene synthase band on SDS-PAGE gel compared to a positive control is much higher than that of the band belonging to the wild type valencene synthase. Finally, valencene synthase mutants have been obtained that produce much less germacrene A compared to valencene as the wild type valencene synthase.

[0212] The data in Table 2 prove that also the valencene synthase mutants containing at least two point mutations according to the invention show clearly improved in-vitro valencene productivities, ranging from 100.6% (ValC:V436W,L449Y) to 266.7% (ValC:Q463S,F488S) compared to the wild type valencene synthase.

TABLE-US-00002 TABLE 2 Results obtained by in-vitro testing valencene synthase mutants with two point mutations compared to SEQ ID NO: 2.sup.a. WT WT Productivity Specific Rel. Prot. Position amino Position amino Mutation Mutation (% rel. to Producitvity Band Ratio 1 acid 2 acid position 1 position 2 weight).sup.b (% rel to wt) Intensity Val/Ger-A 128 I 302 H L D 114.1 69.0 1.07 4.5 398 V 449 L I Y 116.8 118.0 0.71 6.7 T I 116.4 51.7 1.46 9.0 463 Q 488 F S S 266.7 178.0 1.97 3.4 E Y 226.9 122.8 2.43 6.5 G Y 144.0 99.4 1.91 6.4 W H 137.8 99.9 0.99 5.1 436 V 449 L L F 133.6 123.0 1.43 6.9 K I 114.5 89.2 1.69 5.6 W Y 100.6 140.0 1.16 3.3 436 V 450 I L V 190.4 109.6 1.59 8.4 K L 177.2 212.2 1.35 8.3 W F 133.0 92.1 1.04 7.3 187 R 398 V K M 47.8 170.5 0.45 3024.9 .sup.aFor the meaning of the different column headers, see table 1.

Example 3

Construction of Rhodobacter sphaeroides Strains Expressing Improved Valencene Synthase Mutants

[0213] Description of Novel Synthetic Mevalonate Operon in Plasmid pJ241-59440-mev A2415 T4088 mod1

[0214] A synthetic DNA fragment containing a modified version of the mevalonate operon from P. zeaxanthinifaciens (described in WO 06/018211 and Hmbelin, M., et al., Gene (2002) 297: 129-139 [Accession AJ431690]) was purchased from DNA2.0 inserted in pJ241 (proprietary plasmid from DNA2.0). The plasmid was designated pJ241-59440-mev_A2415.sub.T4088_mod1 and its nucleotide sequence is shown in SEQ ID NO: 6. The modifications compared to the wild type nucleotide sequence, which were meant to facilitate further cloning steps and were effected by silent mutations, involved the removal of the recognition sites for restriction enzymes BamHI, BglII, EcoRI and NdeI and introduction of unique recognition sites for restriction enzymes KpnI and XhoI within the mevalonate operon insert (position 1240 to position 7875 in SEQ ID NO: 6).

Subcloning of the Novel Synthetic Mevalonate Operon into pBBR-Based Plasmid Resulting in Plasmid p-mevAT-PcrtE-trx

[0215] Plasmid pBBR-K-mev-op-4-89-PcrtE-trx-valFpoR-rev (described in the patent applications International patent application number PCT/NL2010/050848 and European patent application number EP00174999.0) was digested with AseI and the larger fragment was isolated and self-ligated resulting in plasmid p-m-489-PcrtE-trx. The new plasmid was cut with ZraI and the 6,636 bp fragment was isolated. Plasmid pJ241-59440-mev_A2415_T4088_mod1 was also cut with ZraI and the 5,569 bp fragment was isolated and ligated with the 6,636 bp fragment originating from p-m-489-PcrtE-trx. The new construct was checked for insertion in the correct orientation and designated p-mevAT-PcrtE-trx.

Construction of Plasmids p-mevAAT-PcrtE-trx-ValC Mutant and p-mevAT-PcrtE-trx-ValC wt by Insertion of the Genes Encoding the Valencene Synthase Mutants According to the Invention and the Wild Type Valencene Synthase into Plasmid p-mevAT-PcrtE-trx

[0216] Synthetic DNA fragments with the genes encoding the Valencene Synthase mutants according to the current invention (ValC_mutant) and with the gene encoding the Valencene Synthase wild type (ValC_wt, SEQ ID NO: 2) were obtained from DNA2.0 (Menlo Park, Calif., USA) in general cloning vector pJ201 (proprietary vector DNA2.0). The Valencene Synthase genes had been codon optimized by DNA2.0 for improved expression in Rhodobacter. The plasmids were designated pJ201:ValC_mutant or pJ201:ValC_wt. The nucleotide sequence of pJ201:ValC_wt is shown in SEQ ID NO: 7. The nucleotide sequence of the pJ201:ValC.sub.mutant plasmids is identical to that of pJ201:ValC_wt except for the nucleotide substitutions needed for the formation of the Valencene Synthase mutants according to the current invention.

[0217] Plasmid p-mevAT-PcrtE-trx was cut with AseI and BamHI and the 12,136 bp fragment was isolated. Plasmids pJ201:ValC_mutant (see Table 3, column 2) and plasmid pJ201:ValC_wt were cut with NdeI and BamHI and the 1,777 bp fragments were isolated and ligated with the 12,136 by fragment from p-mevAT-PcrtE-trx, resulting in the 18 p-mevAT-PcrtE-trx-ValC_mutant plasmids listed in Table 3, column 3, and in p-mevAT-PcrtE-trx-ValC_wt.

Construction of Plasmids p-m-4-89-PcrtE-trx-ValC Mutant by Replacing the Synthetic Mevalonate Operon mev_A2415_T4088_mod1 in Plasmids p-mevAT-PcrtE-trx-ValC Mutant with Mevalonate Operon mv-op-4-89

[0218] Plasmid p-m-4-89-PcrtE-trx-ValC-opt is essentially the same plasmid as pBBR-K-mev-op-4-89-PcrtE-trx-valFpoR-rev except for the fact that the valF coding region from pBBR-K-mev-op-4-89-PcrtE-trx-valFpoR-rev was replaced by the valC-opt coding region (SEQ ID NO: 18 in PCT/NL2010/050848 and EP09174999, which sequence is incorporated herein by reference).

[0219] Plasmid p-m-4-89-PcrtE-trx-valC-opt was cut with ZraI, BlpI and SacI and the 6.739 bp ZraI-BlpI fragment was isolated. The p-mevAT-PcrtE-trx-ValC_mutant plasmids (Table 3 column 3) were cut with ZraI, BlpI and AsiSI and the 7,234 bp ZraI-BlpI fragments were isolated. These fragments were subsequently ligated with the 6,739 by fragment from p-m-4-89-PcrtE-trx-valC-opt leading to the plasmids p-m-4-89-PcrtE-trx-ValC_mutant (Table 4, column 3).

Construction of Rhodobacter sphaeroides Rs265-9c Strains Expressing the Mevalonate Pathway and Mutant Valencene Synthases According to the Invention

[0220] E. coli S17-1 was transformed with the plasmids shown in Table 4, column 3, and the resulting strains were used to transfer the plasmids into R. sphaeroides Rs265-9c by conjugation as described in PCT/NL2010/050848 and EP09174999.

TABLE-US-00003 TABLE 3 Nomenclature of plasmids constructed from the 12,136 bp p-mevAT-PcrtE-trx fragment and the 1,777 bp fragments originating from the pJ201: mutant plasmids Recipient plasmid Origin of insert (1,777 (12,136 bp AseI - bp NdeI - BamHI fragment BamHI fragment) comprising valC-mut) New plasmid constructed p-mevAT-PcrtE-trx pJ201: V556T p-mevAT-PcrtE-trx-ValC_V556T pJ201: I410L p-mevAT-PcrtE-trx-ValC_I410L pJ201: S570G p-mevAT-PcrtE-trx-ValC_S570G pJ201: I410F p-mevAT-PcrtE-trx-ValC_I410F pJ201: D490N p-mevAT-PcrtE-trx-ValC_D490N pJ201: C327L p-mevAT-PcrtE-trx-ValC_C327L pJ201: I410V p-mevAT-PcrtE-trx-ValC_I410V pJ201: L566S p-mevAT-PcrtE-trx-ValC_L566S pJ201: E502Q p-mevAT-PcrtE-trx-ValC_E502Q pJ201: H569I p-mevAT-PcrtE-trx-ValC_H569I pJ201: F488H p-mevAT-PcrtE-trx-ValC_F488H pJ201: T568S p-mevAT-PcrtE-trx-ValC_T568S pJ201: Q492K p-mevAT-PcrtE-trx-ValC_Q492K pJ201: Q463S-F488S p-mevAT-PcrtE-trx-ValC_Q463S-F488S pJ201: F300Y p-mevAT-PcrtE-trx-ValC_F300Y pJ201: Q463E-F488Y p-mevAT-PcrtE-trx-ValC_Q463E-F488Y pJ201: S439G p-mevAT-PcrtE-trx-ValC_S439G pJ201: C503S p-mevAT-PcrtE-trx-ValC_C503S

TABLE-US-00004 TABLE 4 Nomenclature of plasmids constructed from the 6,739 bp p-m-4-89-PcrtE-trx-valC-opt fragment and the 7,234 bp fragments originating from the p-mevAT-PcrtE-trx-ValC_Mutant plasmids Recipient plasmid (6,739 Origin of insert (7,234 bp ZraI - BlpI fragment) bp ZraI - BlpI fragment) New plasmid constructed p-m-4-89-PcrtE-trx-valC-opt p-mevAT-PcrtE-trx-ValC_V556T p-m-4-89-PcrtE-trx-ValC_V556T p-mevAT-PcrtE-trx-ValC_I410L p-m-4-89-PcrtE-trx-ValC_I410L p-mevAT-PcrtE-trx-ValC_S570G p-m-4-89-PcrtE-trx-ValC_S570G p-mevAT-PcrtE-trx-ValC_I410F p-m-4-89-PcrtE-trx-ValC_I410F p-mevAT-PcrtE-trx-ValC_D490N p-m-4-89-PcrtE-trx-ValC_D490N p-mevAT-PcrtE-trx-ValC_C327L p-m-4-89-PcrtE-trx-ValC_C327L p-mevAT-PcrtE-trx-ValC_I410V p-m-4-89-PcrtE-trx-ValC_I410V p-mevAT-PcrtE-trx-ValC_L566S p-m-4-89-PcrtE-trx-ValC_L566S p-mevAT-PcrtE-trx-ValC_E502Q p-m-4-89-PcrtE-trx-ValC_E502Q p-mevAT-PcrtE-trx-ValC_H569I p-m-4-89-PcrtE-trx-ValC_H569I p-mevAT-PcrtE-trx-ValC_F488H p-m-4-89-PcrtE-trx-ValC_F488H p-mevAT-PcrtE-trx-ValC_T568S p-m-4-89-PcrtE-trx-ValC_T568S p-mevAT-PcrtE-trx-ValC_Q492K p-m-4-89-PcrtE-trx-ValC_Q492K p-mevAT-PcrtE-trx-ValC_Q463S-F488S p-m-4-89-PcrtE-trx-ValC_Q463S-F488S p-mevAT-PcrtE-trx-ValC_F300Y p-m-4-89-PcrtE-trx-ValC_F300Y p-mevAT-PcrtE-trx-ValC_Q463E-F488Y p-m-4-89-PcrtE-trx-ValC_Q463E-F488Y p-mevAT-PcrtE-trx-ValC_S439G p-m-4-89-PcrtE-trx-ValC_S439G p-mevAT-PcrtE-trx-ValC_C503S p-m-4-89-PcrtE-trx-ValC_C503S

Example 4

Cultivation of Rhodobacter sphaeroides Strains Under Standard Shake-Flask Conditions and Evaluation of Valencene Production

Preparation of Frozen Cell Stocks

[0221] Frozen cell stocks of R. sphaeroides strains were prepared by introducing a loop-full of frozen coils into 2 mL RS102 medium containing 50 mg/L kanamycin (if applicable for plasmid maintenance). The preculture was grown at 80 C. with agitation at 220 rpm for 24 h. A 250 L aliquot of preculture was transferred to 25 mL of RS102 medium containing 50 mg/L kanamycin to initiate (t=0) growth. The 25 mL main culture was grown in a 250-mL baffled Erlenmeyer flasks at 30 C. with agitation at 220 rpm for about 24 h. Bacterial cell cultures were mixed with sterile anhydrous glycerol and sterile water so as to reach a final glycerol content of 25% and a final optical density at 660 nanometers (OD.sub.660) of 12. The resulting cell suspension was aseptically distributed in 1.2 mL-aliquots into 2 mL-cryovials then frozen at 80 C. until used.

Shake-Flask Procedure

[0222] Inoculants of R. sphaeroides strains were started by introducing 250 L of a thawed and homogenized frozen cell stock into 25 mL of RS102 medium containing 50 mg/L of kanamycin (if applicable for plasmid maintenance). Precultures were grown in 250-mL baffled Erlenmeyer flasks for 24-28 h at 30 C. with agitation at 220 rpm. A suitable aliquot of preculture was transferred to 22.5 mL of RS102 medium containing 50 mg/L of kanamycin (if applicable for plasmid maintenance) to initiate (t=0) shake-flask experiments with an initial optical density at 660 nm (OD.sub.660) of 0.16. Main cultures were grown in 250-mL baffled Erlenmeyer flasks at 30 C. with agitation at 220 rpm. After 8 h cultivation, 2.5 mL of n-dodecane were added to the bacterial culture. Shake-flask cultivation continued at 30 C. with agitation at 220 rpm for 72 h from inoculation. Each seed culture served to inoculate two duplicate shake-flasks with a final volume of 25 mL whole broth, composed of culture medium and n-dodecane for in situ product recovery. Samples (0.5 mL) of biphasic culture broth were removed at 24 h intervals and analyzed for growth (OD.sub.660), pH, and glucose in supernatant. At the end of the experiments (t=72 h), the biphasic culture broth was analyzed for presence of valencene (see analytical methods below). At the end of the experiments, 10 l, of culture broth were aseptically plated on general cultivation count agar plates (Becton Dickinson GmbH, Heidelberg, Germany) and incubated at 37 C. for 24 h to test for contamination.

Analytical Methods

Sample Preparation for Analysis of Isoprenoid Content in Whole Broth

[0223] In a typical procedure, 400 L whole broth samples are transferred to a disposable sterile 15 mL polypropylene conical tube, treated with 4 acetone, vigorously shaken on an IKA Vibrax orbital shaker at 1,500 rpm for 20 min, then incubated in a bench top ultrasonic bath for 30 min at ambient temperature. Finally samples are centrifuged at maximum speed and the supernatant transferred to amber chromatography vials for analysis by gas chromatography (see below). Product yields are determined based on calibration curves established using a standard solution of authentic valencene prepared as follows: 0.5 mL of authentic valencene are added into a 10 mL volumetric flask and dissolved with analytical grade n-dodecane. Aliquots of valencene standard solution (20, 40 and 80 l) are transferred to disposable sterile 15 mL polypropylene conical tubes, treated with deionized sterile water (380, 360, and 320 L, respectively) and 4 mL acetone. Each mixture is homogenized vigorously on a vortex shaker then transferred to amber chromatography vials for analysis by gas chromatography, wherefrom a calibration curve is derived.

Gas Chromatography

[0224] Gas chromatography is performed on a Hewlett-Packard GC 6890 instrument equipped with a Restek Rtx-5Sil MS capillary column (30.0 m0.28 mm0.5 m). The injector and FID detector temperatures are set to 800 C. and 250 C., respectively. Gas flow through the column is set at 1.5 mL/min. The oven initial temperature is held at 70 C. for 0.5 min, increased to 150 C. at a rate of 20 C./min, then increased to 205 C. at a rate of 5 C./min, further increased to 300 C. at a rate of 40 C./min, then cooled down to 60 C. and held at that temperature for 3 min until the next injection. Injected sample volume is 1 L with a 4:1 split-ratio. Product yields are determined based on calibration curves established for authentic samples. Germacrene A is detected as -Elemene and quantified with the response factor determined for valencene. The content of Valencene and -Elemene in the organic phase wis extrapolated from whole broth analyses. The measured concentration of Valencene and -Elemene in whole broth is set in relation to the one found for n-dodecane.

Example 5

In Vivo Expression of C. nootkatensis Valencene Synthase in Yeast

[0225] The full length open reacting frame encoding the C. nootkatensis valencene synthase (ValC, SEQ ID: NO:2) was amplified from plasmid pAC-65-3 with the primers 65-3ATGDuetFw 5-tatatggatccATGCTGAAATGTTTAATGGAAATTCCAGC-3 (BamHI recognition site underlined), and DuetAS1 5-GATTATGCGGCCGTGTACAA-3 in a manner as described in Example 9 of International patent application number PCT/ML2010/050848, which Example is incorporated herein by reference. Variants of this valenene synthase, such as valencence synthases comprising a sequence as shown SEQ ID NO: 3 or 4 can be expressed in yeast in a similar manner, using common general knowledge and the information disclosed herein.

Example 6

Expression of ValC in Plants

[0226] The valencene synthase comprising a sequence according to SEQ ID NO: 2 was expressed in a plant in a manner as described in Example 10 of International patent application number PCT/NL2010/050848, which Example is incorporated herein by reference. Variants of this valenene synthase, such as valencence synthases comprising a sequence as shown in SEQ ID NO: 3 or 4 can be expressed in a plant in a similar manner, using common general knowledge, and the information disclosed herein.

TABLE-US-00005 SEQUENCES SEQIDNO:1 valC Chamaecyparisnootkatensis Nucleotidesequence ATGGCTGAAATGTTTAATGGAAATTCCAGCAATGATGGAAGTTCTTGCATGCCCGTGAAGGACGCCCTTCGTC GGACTGGAAATCATCATCCTAACTTGTGGACTGATGATTTCATACAGTCCCTCAATTCTCCATATTCGGATTC TTCATACCATAAACATAGGGAAATACTAATTGATGAGATTCGTGATATGTTTTCTAATGGAGAAGGCGATGAG TTCGGTGTACTTGAAAATATTTGGTTTGTTGATGTTGTACAACGTTTGGGAATAGATCGACATTTTCAAGAGG AAATCAAAACTGCACTTGATTATATCTACAAGTTCTGGAATCATGATAGTATTTTTGGCGATCTCAACATGGT GGCTCTAGGATTTCGGATACTACGACTGAATAGATATGTCGCTTCTTCAGATGTTTTTAAAAAGTTCAAAGGT GAAGAAGGACAATTCTCTGGTTTTGAATCTAGCGATCAAGATGCAAAATTAGAAATGATGTTAAATTTATATA AAGCTTCAGAATTAGATTTTCCTGATGAAGATATCTTAAAAGAAGCAAGAGCGTTTGCTTCTATGTACCTGAA ACATGTTATCAAAGAATATGGTGACATACAAGAATCAAAAAATCCACTTCTAATGGAGATAGAGTACACTTTT AAATATCCTTGGAGATGTAGGCTTCCAAGGTTGGAGGCTTGGAACTTTATTCATATAATGAGACAACAAGATT GCAATATATCACTTGCCAATAACCTTTATAAAATTCCAAAAATATATATGAAAAAGATATTGGAACTAGCAAT ACTGGACTTCAATATTTTGCAGTCACAACATCAACATGAAATGAAATTAATATCCACATGGTGGAAAAATTCA AGTGCAATTCAATTGGATTTCTTTCGGCATCGTCACATAGAAAGTTATTTTTGGTGGGCTAGTCCATTATTTG AACCTGAGTTCAGTACATGTAGAATTAATTGTACCAAATTATCTACAAAAATGTTCCTCCTTGACGATATTTA TGACACATATGGGACTGTTGAGGAATTGAAACCATTCACAACAACATTAACAAGATGGGATGTTTCCACAGTT GATAATCATCCAGACTACATGAAAATTGCTTTCAATTTTTCATATGAGATATATAAGGAAATTGCAAGTGAAG CCGAAAGAAAGCATGGTCCCTTTGTTTACAAATACCTTCAATCTTGCTGGAAGAGTTATATCGAGGCTTATAT GCAAGAAGCAGAATGGATAGCTTCTAATCATATACCAGGTTTTGATGAATACTTGATGAATGGAGTAAAAAGT AGCGGCATGCGAATTCTAATGATACATGCAGTAATACTAATGGATACTCCTTTATCTGATGAAATTTTGGAGC AACTTGATATCCCATCATCCAAGTCGCAAGCTCTTCTATCATTAATTACTCGACTAGTGGATGATGTCAAAGA CTTTGAGGATGAACAAGCTCATGGGGAGATGGCATCAAGTATAGAGTGCTACATGAAAGACAACCATGGTTCT ACAAGGGAAGATGCTTTGAATTATCTCAAAATTCGTATAGAGAGTTGTGTGCAAGAGTTAAATAAGGAGCTTC TCGAGCCTTCAAATATGCATGGATCTTTTAGAAACCTATATCTCAATGTTGGCATGCGAGTAATATTTTTTAT GCTCAATGATGGTGATCTCTTTACACACTCCAATAGAAAAGAGATACAAGATGCAATAACAAAATTTTTTGTG GAACTAATCATTCCATAG SEQIDNO:2 ValC Chamaecyparisnootkatenisis Aminoacidsequence MAEMFNGNSSNDGSSCMPVKDALRRTGNHHPNLWTDDFIQSLNSPYSDSSYHKHREILIDEIRDMFSNGEGDE FGVLENIWFVDVVQRLGIDRHFQEEIKTALDYIYKFWNHDSIFGDLNMVALGFRILRLNRYVASSDVFKKFKG EEGQFSGFESSDQDAKLEMMLNLYKASELDFPDEDILKEARAFASMYLKHVIKEYGDIQESKNPLLMEIEYTF KYPWRCRLPRLEAWKFIHIMRQQDCNISLANNLYKIPKIYMKKILELAILDFNILQSQHCHEMKLISTWWKNS SAIQLDFFRHRHIESYFWWASPLFEPSFSTCRINCTKLSTKMFLLDDIYDTYGTVEELKPFTTTLTRWDVSTV DNHPDYMKIAFNFSYEIYKEIASEAERKHGPFVYKYLQSCWKSYIEAYMQEAEWIASNHIPGFDEYLMNGVKS SGMRILMIHALILMDTPLSDEILEQLDIPSSKSQALLSLITRLVDDVKDFEDEQAHGEMASSIECYMKDNHGS TREDALNYLKIRIESCVQELNKELLEPSNMHGSFRNLYLNVGMRVIFFMLNDGDLFTHSNRKEIQDAITKFFV EPIIP SEQIDNO:3 MAEMFNGNSSNDGSSXMPVKDALRRTGNHHPNLWTDDFIQSLNSPYSDSSYHKHREILIDEIRDMFSNGEGDE FGVLENIWFVDVVQRLGIDRHFQEEIKTALDYIYKFWNHDSIFGDLKMVALGFRXLRLNRYVASSDVFKKFKG EEGQFSGFESSDQDAKLEMMLNLYXASELDFPDEDILKEAXAFASMYLKHVIKEYGDIQESKNPLLMEIEYTF KYPWRXRLPRLEAWNFIHIMRQQDXNISLANNLYKIPKIYMKKILELAILDFNILQSQHQHEMKLISTWWKNS SAIQLDFXRXRHIEXYFWWASPLFERXFSTXRINXTKLXTKXFLLDDIYDTYGTVEELKPFTTTLTRWDVSTV DNHPDYMKIAFNFSYEIYKEIASEAERKHGPFXYKYLQSXWKSXXEXYMQEAEWIASNHIPGFDEYLMNGXKX XGMRIXMIHXXXLMDTPLSDEILEXLDIPSSKSQALLSLITRLVDDVKDXSXEXAHGEMASSIXXYMXXNHGS TREDALNYLKIRIESXVQELNKELLEPSNMHGSFRNLYLNVGMRXIFXXLNDGDXFXXXNRKEIQDAITKFFV EPIIP SEQIDNO:4 MAEMFNGNSSNDGSS(CATS)MPVKDALRRTGNHHPNLWTDDFIQSLNSPYSDSSYHKHREILIDEIRDMFSN GEGDEFGVLENIWFVDVVQRLGIDRHFQEEIKTALDYIYKFWNHDSIFGDLNMVALGFR(IL)LRLNRYVASS DVFKKFKGEEGQFSGFESSDQDAKLEMMLNLY(KR)ASELDFPDEDILKEA(RK)AFASMYLKHVIKSYGDIQ ESKNPLLMEIEYTFKYPWR(CS)RLPRLEAWNFIHIMRQQD(CST)NISLANNLYKIPKIYMKKILELAILDF NILQSQHQHEMKLISTWWKNSSAIQLDF(FY)R(HD)RHIE(STA)YFWWASPLFEP(EQ)FST(CA)RIN(C L)TKL(SG)TK(ML)FLLDDIYDTYGTVEELKPFTTTLTRWDVSTVDNHPDYMKIAFNFSYEIYKEIASEAER KHGPF(VIMT)YKYLQS(CTV)WKS(YF)(IFVL)E(AG)YMQEAEWIASNHIPGFDEYLMNG(VLKTW)K(S T)(SGA)GMRI(LIV)MIH(AS)(LFIY)(ILMV)LMDTPLSDEILE(QESGW)LDIPSSKSCALLSLITRLV DDVKD(FYHS)E(DNATF)E(QAK)AHGEMASSI(EQ)(CS)YMK(DEQ)NHGSTREDALNYLKIRIES(CTS A)VQELNKELLEPSNMHGSFRNLYLNVGMR(VT)IF(FHLV)(ML)LNDGD(LSAG)F(TS)(HIV)(SGAP T)NRKEIQDAITKFFVEPIIP SEQIDNO:5 pACYCDuet:ValC_wt E.coliexpressionvectorpACYCDuet-1withthecodonoptimizedgene (valC-cpt)encodingthewildtypeValenceneSynthase Nucleotidesequence GGGGAATTGTGAGCGGATAACAATTCCCCTGTAGAAATAATTTTGTTTAACTTTAATAAGGAGATATACCATG GGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGCATATGGCCGAAATGTTCAATGGCAATTCCAGCA ATGATGGCAGCTCCTGCATGCCGGTCAAGGACGCGCTGCGCCGCACCGGGAACCACCATCCGAACCTCTGGAC CGACGATTTCATCCAGTCGCTGAACTCCCCCTATTCGGATTCCTCGTATCATAAACATCGCGAGATCCTGATC GATGAGATCCGGGACATGTTCTCCAACGGCGAGGGGGATGAGTTCGGGGTCCTCGAGAACATCTGGTTCGTCG ACGTGGTCCAGCGGCTGGGCATCGATCGGCACTTCCAGGAAGAGATCAAGACGGCCCTGGATTATATCTATAA GTTCTGGAACCATGATAGCATCTTCGGCGACCTCAACATGGTGGCGCTGGGGTTCCGCATCCTGCGGCTCAAT CGCTACGTGGCGTCGTCGGACGTGTTCAAGAAGTTCAAGGGCGAGGAGGGCCAGTTCTCGGGGTTCGAGAGCA GCGATCAGGACGCCAAGCTGGAGATGATGCTGAACCTCTACAAGGCCTCGGAACTCGACTTCCCGGATGAGGA CATCCTCAAGGAAGCGCGGGCCTTCGCGTCGATGTATCTCAAGCATGTCATCAAGGAGTATGGGGACATCCAG GAATCGAAGAACCCCCTGCTCATGGAGATCGAGTACACCTTCAAGTACCCCTGGCGCTGCCGCCTCCCGCGGC TGGAGGCGTGGAACTTCATCCACATCATGCGGCAGCAGGACTGCAATATCTCGCTCGCCAACAACCTCTATAA GATCCCGAAGATCTATATGAAGAAGATCCTGGAGCTGGCGATCCTCGACTTCAACATCCTCCAGAGCCAGCAT CAGCATGAGATGAAACTGATCAGCACGTGGTGGAAGAACTCGTCCGCGATCCAGCTCGACTTCTTCCGCCACC GCCATATCGAGAGGTACTTCTGGTGGGCCAGCCCGCTGTTCGAGCCCGAGTTCTCCACCTGCCGCATCAACTG CACCAAGCTGTCCACCAAGATGTTCCTCCTGGACGACATCTATGACACGTACGGGACCGTCGAGGAACTCAAG CCGTTCACGACCACCCTCACGCGCTGGGATGTCAGCACGGTGGACAATCACCCGGACTACATGAAGATCGCGT TCAATTTCTCCTACGAGATCTACAAGGAGATCGCGTCCGAGGCCGAGCGCAAGCACGGCCCGTTCGTGTATAA GTATCTCCAGTCGTGCTGGAAGTCGTATATCGAGGCGTATATGCAGGAGGCCGAGTGGATCGCCTCCAACCAC ATCCCCGGCTTCGACGAGTACCTGATGAATGGCGTGAAGAGCTCGGGGATGCGCATCCTCATGATCCATGCGC TGATCCTGATGGATACGCCCCTGTCCGACGAGATCCTCGAGCAGCTCGACATCCCGAGCAGCAAGAGCCAGGC CCTGCTGTCGCTCATCACGCGGCTCGTCGATGATGTGAAGGATTTCGAGGACGAGCAGGCGCATGGGGAGATG GCCTCGTCGATCGAATGCTATATGAAGGATAATCACGGCTCCACGCGCGAGGACGCCCTGAACTACCTGAAAA TCCGCATCGAGAGCTGCGTGCAGGAGCTCAACAAGGAACTCCTCGAACCGAGCAACATGCATGGCAGCTTCCG CAACCTGTACCTCAACGTGGGCATGCGGGTGATCTTCTTCATGCTGAACGACGGGGACCTCTTCACCCATTCG AATCGGAAGGAGATCCAGGATGCGATCACGAAGTTCTTCGTGGAACCGATCATCCCGTGATAAGGATCCCTGC AGGTCGACAAGCTTGCGGCCGCATAATGCTTAAGTCGAACAGAAAGTAATCGTATTGTACACGGCCGCATAAT CGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCATCTTAGTATATTAGTTAAGT ATAAGAAGGAGATATACATATGGCAGATCTCAATTGGATATCGGCCGGCCACGCGATCGCTGACGTCGGTACC CTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAGCACATGGACTCGTCTACTAGCGCAGCTT AATTAACCTAGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAG GGGTTTTTTGCTGAAACCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTA AACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGA ATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCACCAGGCGTTTAAGGGCACCAATAACTGC CTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATG GAAGCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATAT TTGCCCATAGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCA CCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTA ACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAA AACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTT TCATTGCCATACGGAACTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTT GTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGA GCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGA TTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCT TATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAG GGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATT CGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAG TGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGC CGGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTC ATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCG CTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCC TGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGC CCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACC AGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTG TTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGT ATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAA GACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCG GTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTT GGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGC AGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCTAGATTTCAGTGCAATTTATCT CTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTCATGTTAGTCATGCCCCGCGCC CACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCT AACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATG AATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGAC GGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCC AGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATC CCACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCGAGCGCCATCTG ATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGAC ATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAG CCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGAC CAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAG ACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGAT AGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCT TCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGC GACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTG CCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTG GCTGGCCTGGTTCACCAGGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTT ACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGC GCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAATTAATACGACTCA CTATA SEQIDNO:6 pJ241-59440-mev_A2415_T4088_mod1 SyntheticDNAfragmentwithamodifiedversionoftheP. zeaxanthinifaciensmevalonateoperonclonedintovectorpJ241 Nucleotidesequence TAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAA AAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTC TGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAG AAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAA CAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTG AGCGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAGTGCAACCGGCGCAGGAAC ACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAACGCTGTTTTTCCGG GGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGTGGCATAAA TTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGA AACAACTCTGGCGCATCGGGCTTCCCATACAAGCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAG CCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTTGAATATG GCTCATATTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTT GAATGTATTTAGAAAAATAAACAAATAGGGGTCAGTGTTACAACGAATTAACCAATTCTGAACATTATCGCGA GCCCATTTATACCTGAATATGGCTCATAACACCCCTTGTTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTG ACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGACTCCCCATGCGAGAGTAGG GAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGCCCGGGCTAATTAGGGGG TGTCGCCCTTATTCGACTCTATAGTGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCTGAAGTGGGGACGT CTCGATTCTGCCCGCGAAGAATGCGATGCATCCAGATGATGCAGAACGAAGAAGCGGAAGCGCCCGTGAAAGA CCAGATGATTTCCCATACCCCGGTGCCCACGCAATGGGTCGGCCCGATCCTGTTCCGCGGCCCCGTCGTCGAG GGCCCGATCAGCGCGCCGCTGGCCACCTACGAGACGCCGCTCTGGCCCTCGACCGCGCGGGGGGCAGGGGTTT CCCGGCATTCGGGCGGCATCCAGGTCTCGCTGGTCGACGAACGCATGAGCCGCTCGATCGCGCTGCGGGCGCA TGACGGGGCGGCGGCGACCGCCGCCTGGCAGTCGATCAAGGCCCGCCAGGAAGAGGTCGCGGCCGTGGTCGCC ACCACCAGCCGCTTCGCCCGCCTTGTCGAGCTGAATCGCCAGATCGTGGGCAACCTGCTTTACATCCGCATCG AATGCGTGACGGGCGACGCCTCGGGTCACAACATGGTCACCAAGGCCGCCGAGGCCGTGCAGGGCTGGATTCT GTCGGAATACCCGATGCTGGCCTATTCCACGATCTCGGGGAACCTGTGCACCGACAAGAAGGCGTCGGCGGTC AACGGCATCCTGGGCCGCGGCAAATACGCCGTCGCCGAGGTCGAGATCCCGCGCAAGATCCTGACCCGCGTGC TGCGCACCAGCGCCGAGAAGATGGTCCGCCTGAACTACGAGAAGAACTATGTCGGGGGTACGCTGGCGGGGTC GCTGCGCAGTGCGAACGCGCATTTCGCCAACATGCTGCTGGGCTTCTACCTGGCGACGGGGCAGGACGCGGCC AACATCATCGAGGCCAGCCAGGGCTTCGTCCATTGCGAGGCCCGCGGCGAGGATCTGTATTTCTCGTGCACGC TGCCCAACCTCATCATGGGCTCGGTCGGTGCCGGCAAGGGCATCCCCTCGATCGAGGAGAACCTGTCGCGGAT GGGCTGCCGCCAGCCGGGCGAACCCGGCGACAACGCGCGCCGTCTTGCGGCGATCTGCGCGGGCGTCGTGCTG TGTGGTGAATTGTCGCTGCTTGCGGCCCAGACCAACCCCGGAGAGTTGGTCCGCACCCACATGGAGATGGAGC GATGACCGACAGCAAGGATCACCATGTCGCGGGGCGCAAGCTGGACCATCTGCGTGCATTGGACGACGATGCG GATATCGACCGGGGCGACAGCGGCTTCGACCGCATCGCGCTGACCCATCGCGCCCTGCCCGAGGTGGATTTCG ACGCCATCGACACGGCGACCAGCTTCCTGGGCCGTGAACTGTCCTTCCCGCTGCTGATCTCGTCCATGACCGG CGGCACCGGCGAGGAGATCGAGCGCATCAACCGCAACCTGGCCGCTGGTGCCGAGGAGGCCCGCGTCGCCATG GCGGTGGGCTCGCAGCGCGTGATGTTCACCGACCCCTCGGCGCGGGCCAGCTTCGACCTGCGCGCCCATGCGC CCACCGTGCCGCTGCTGGCCAATATCGGCGCGGTGCAGCTGAACATGGGGCTGGGGCTGAAGGAATGCCTGGC CGCGATCGAGGTGCTGCAGGCGGACGGCCTGTATCTGCACCTGAACCCCCTGCAAGAGGCCGTCCAGCCCGAG GGGGATCGCGACTTTGCCGATCTGGGCAGCAAGATCGCGGCCATCGCCCGCGACGTTCCCGTGCCCGTCCTGC TGAAGGAGGTGGGCTGCGGCCTGTCGGCGGCCGATATCGCCATCGGGCTGCGCGCCGGCATCCGGCATTTCGA CGTGGCCGGTCGCGGCGGCACATCCTGGAGCCGGATCGAGTATCGCCGCCGCCAGCGGGCCGATGACGACCTG GGCCTGGTCTTCCAGGACTGGGGCCTGCAGACCGTGGACGCCCTGCGCGAGGCGCGGCCCGCGCTTGCGGCCC ATGATGGAACCAGCGTGCTGATCGCCAGCGGCGGCATCCGCAACGGTGTCGACATGGCGAAATGCGTCATCCT GGGGGCCGACATGTGCGGGGTCGCCGCGCCCCTGCTGAAAGCGGCCCAAAACTCGCGCGAGGCGGTTGTATCC GCCATCCGGAAACTGCATCTGGAGTTCCGGACAGCCATGTTCCTCCTGGGTTGCGGCACGCTTGCCGACCTGA AGGACAATTCCTCGCTTATCCGTCAATGAAAGTGCCTAAGATGACCGTGACAGGAATCGAAGCGATCAGCTTC TACACCCCCCAGAACTACGTGGGACTGGATATCCTTGCCGCGCATCACGGGATCGACCCCGAGAAGTTCTCGA AGGGGATCGGGCAGGAGAAAATCGCACTGCCCGGCCATGACGAGGATATCGTGACCATGGCCGCCGAGGCCGC GCTGCCGATCATCGAACGCGCGGGTACCCAGGGCATCGACACGGTTCTGTTCGCCACCGAGAGCGGGATCGAC AAGTCGAAGGCCGCCGCCATCTATCTGCGCCGCCTGCTGGACCTGTCGCCCAACTGCCGTTGCGTCGAGCTGA AGCAGGCCTGCTATTCCGCGACGGCGGCGCTGCAGATGGCCTGCGCGCATGTCGCCCGCAAGCCCGACCGCAA GGTGCTGGTGATCGCGTCCGATGTCGCGCGCTATGACCGCGAAAGCTCGGGCGAGGCGACGCAGGGTGCGGGC GCCGTCGCCATCCTTGTCAGCGCCGATCCCAAGGTGGCCGAGATCGGCACCGTCTCGGGGCTGTTCACCGAGG ATATCATGGATTTCTGGCGGCCGAACCACCGCCGCACGCCCCTGTTCGACGGCAAGGCATCGACGCTGCGCTA TCTGAACGCGCTGGTCGAGGCGTGGAACGACTATCGCGCGAATGGCGGCCACGAGTTCGCCGATTTCGCGCAT TTCTGCTATCACGTGCCGTTCTCGCGGATGGGCGAGAAGGCGAACAGCCACCTGGCCAAGGCGAACAAGACGC CGGTGGACATGGGGCAGGTGCAGACGGGCCTGATCTACAACCGGCAGGTCGGGAACTGCTATACCGGGTCGAT CTACCTGGCATTCGCCTCGCTGCTGGAGAACGCTCAGGAGGACCTGACCGGCGCGCTGGTCGGTCTGTTCAGC TATGGCTCGGGTGCGACGGGCGAGTTCTTCGATGCGCGGATCGCGCCCGGTTACCGCGACCACCTGTTCGCGG AACGCGATCGCGAATTGCTGCAGGATCGCACGCCCGTCACGTATGACGAATACGTTGCCCTGTGGGACGAGAT CGACCTGACGCAGGGCGCGCCCGACAAGGCGCGCGGTCGTTTCAGGCTGGCAGGTATCGAGGACGAGAAGCGC ATCTATGTCGACCGGCAGGCCTGAAGCAGGCGCCCATGCCCCGGGCAAGCTGATCCTGTCCGGGGAACATTCC GTGCTCTATGGTGCGCCCGCGCTTGCCATGGCCATCGCCCGCTATACCGAGGTGTGGTTCACGCCGCTTGGCA TTGGCGAGGGGATACGCACGACATTCGCCAATCTCTCGGGCGGGGCGACCTATTCGCTGAAGCTGCTGTCGGG GTTCAAGTCGCGGCTGGACCGCCGGTTCGAGCAGTTCCTGAACGGCGACCTAAAGGTGCACAAGGTCCTGACC CATCCCGACGATCTGGCGGTCTATGCGCTGGCGTCGCTTCTGCACGACAAGCCGCCGGGGACCGCCGCGATGC CGGGCATCGGCGCGATGCACCACCTGCCGCGACCGGGTGAGCTGGGCAGCCGGACGGAGCTGCCCATCGGCGC GGGCATGGGGTCGTCTGCGGCCATCGTCGCGGCCACCACGGTCCTGTTCGAGACGCTGCTGGACCGGCCCAAG ACGCCCGAACAGCGCTTCGACCGCGTCCGCTTCTGCGAGCGGTTGAAGCACGGCAAGGCCGGTCCCATCGACG CGGCCAGCGTCGTGCGCGGCGGGCTTGTCCGCGTGGGCGGGAACGGGCCGGGTTCGATCAGCAGCTTCGATTT GCCCGAGGATCACGACCTTGTCGCGGGACGCGGCTGGTACTGGGTACTGCACGGGCGCCCCGTCAGCGGGACC GGCGAATGCGTCAGCGCGGTCGCGGCGGCGCATGGTCGCGATGCGGCGCTGTGGGACGCCTTCGCAGTCTGCA CCCGCGCGTTGGAGGCCGCGCTGCTGTCTGGGGGCAGCCCCGACGCCGCCATCACCGAGAACCAGCGCCTGCT CGAGCGCATCGGCGTCGTGCCGGCAGCGACGCAGGCCCTCGTGGCCCAGATCGAGGAGGCGGGTGGCGCGGCC AAAATCTGCGGCGCAGGTTCCGTGCGGGGCGATCACGGCGGGGCGGTCCTCGTGCGGATTGACGACGCGCAGG CGATGGCTTCGGTCATGGCGCGCCATCCCGACCTCGACTGGGCGCCCCTGCGCATGTCGCGCACGGGGGCGGC ACCCGGCCCCGCGCCGCGTGCGCAACCGCTGCCGGGGCAGGGCTGATGGATCAGGTCATCCGCGCCAGCGCGC CGGGTTCGGTCATGATCACGGGCGAACATGCCGTGGTCTATGGACACCGCGCCATCGTCGCCGGGATCGAGCA GCGCGCCCATGTGACGATCGTCCCGCGTGCCGACCGCATGTTTCGCATCACCTCGCAGATCGGGGCGCCGCAG CAGGGGTCGCTGGACGATCTGCCTGCGGGCGGGACCTATCGCTTCGTGCTGGCCGCCATCGCGCGACACGCGC CGGACCTGCCTTGCGGGTTCGACATGGACATCACCTCGGGGATCGATCCGAGGCTCGGGCTTGGGTCCTCGGC GGCGGTGACGGTCGCCTGCCTCGGCGCGCTGTCGCGGCTGGCGGGGCGGGGGACCGAGGGGCTGCATGACGAC GCGCTGCGCATCGTCCGCGCCATCCAGGGCAGGGGCAGCGGGGCCGATCTGGCGGCCAGCCTGCATGGCGGCT TCGTCGCCTATCGCGCGCCCGATGGCGGTGCCGCGCAGATCGAGGCGCTTCCGGTGCCGCCGGGGCCGTTCGG CCTGCGCTATGCGGGCTACAAGACCCCGACAGCCGAGGTGCTGCGCCTTGTGGCCGATCGGATGGCGGGCAAC GAGGCCGCTTTCGACGCGCTCTACTCCCGGATGGGOGCAAGCGCAGATGCCGCGATCCGCGCGGCGCAAGGGC TGGACTGGGCTGCATTCCACGACGCGCTGAACGAATACCAGCGCCTGATGGAGCAGCTGGGCGTGTCCGACGA CACGCTGGACGCGATCATCCGCGAGGCGCGCGACGCGGGCGCCGCAGTCGCCAAAATCTCCGGCTCGGGGCTG GGGGATTGCGTGCTGGCACTGGGCGACCAGCCCAAGGGTTTCGTGCCCGCAAGCATTGCCGAGAAGGGACTTG TTTTCGATGACTGATGCCGTCCGCGACATGATCGCCCGTGCCATGGCGGGCGCGACCGACATCCGAGCAGCCG AGGCTTATGCGCCCAGCAACATCGCGCTGTCGAAATACTGGGGCAAGCGCGACGCCGCGCGGAACCTTCCGCT GAACAGCTCCGTCTCGATCTCGTTGGCGAACTGGGGCTCTCATACGCGGGTCGAGGGGTCCGGCACGGGCCAC GACGAGGTGCATCACAACGGCACGCTGCTCGATCCGGGCGACGCCTTCGCGCGCCGCGCGTTGGCATTCGCTG ACCTGTTCGGGGGGGGGAGGCACCTGCCGCTGCGGATCACGACGCAGAACTCGATCCCGACGGCGGCGGGGCT TGCCTCGTCGGCCTCGGGGTTCGCGGCGCTGACCCGTGCGCTGGCGGGGGCGTTCGGGCTGGATCTGGACGAC ACGGATCTGAGCCGCATCGCCCGGATCGGCAGTGGCAGCGCCGCCCGCTCGATCTGGCACGGCTTCGTCCGCT GGAACCGGGGCGAGGCCGAGGATGGGCATGACAGCCACGGCGTCCCGCTGGACCTGCGCTGGCCCGGCTTCCG CAICGCGATCGTGGCCGTGGACAAGGGGCCCAAGCCTTTCAGTTCGCGCGACGGCATGAACCACACGGTCGAG ACCAGCCCGCTGTTCCCGCCCTGGCCTGCGCAGGCGGAAGCGGATTGCCGCGTCATCGAGGATGCGATCGCCG CCCGCGACATGGCCGCCCTGGGICCGCGGGTCGAGGCGAACGCCCTTGCGATGCACGCCACGATGATGGCCGC GCGCCCGCCGCTCTGCTACCTGACGGGCGGCAGCTGGCAGGTGCTGGAACGCCTGTGGCAGGCCCGCGCGGAC GGGCTTGCGGCCTTTGCGACGATGGATGCCGGCCCGAACGTCAAGCTGATCTTCGAGGAAAGCAGCGCCGCCG ACGTGCTGTACCTGTTCCCCGACGCCAGCCTGATCGCGCCGTTCGAGGGGCGTTGAACGCGTAAGACGACCAC TGGGTAAGGTTCTGCCGCGCGTGGTCTCGACTGCCTGCAAAGAGGTGCTTGAGTTGCCGCGTGACTGCGGCGG CCGACTTCGTGGGACTTGCCCGCCACGCTGACGAAGGGCGTAATCCAGCACACTGGCGGCCGTTACTAGTTTC AGAGCGGCCGCCACCGCGGTGGAGGGCGGCACCTCGCTAACGGATTCACCGTTTTTATCAGGCTCTGGGAGGC AGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGAATTCAAAA TGAAGTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCTATAGTGAGTCGAATAAGGGCGACACAAAATTT ATTCTAAATGCATAATAAATACTGATAACATCTTATAGTTTGTATTATATTTTGTATTATCGTTGACATGTAT AATTTTGATATCAAAAACTGATTTTCCCTTTATTATTTTCGAGATTTATTTTCTTAATTCTCTTTAACAAACT AGAAATATTGTATATACAAAAAATCATAAATAATAGATGAATAGTTTAATTATAGGTGTTCATCAATCGAAAA AGCAACGTATCTTATTTAAAGTGCGTTGCTTTTTTCTCATITATAAGGTTAAATAATTCTCATAIATCAAGCA AAGTGACAGGCGCCCTTAAATATTCTGACAAAIGCTCTTTCCCTAAACTCCCCCCATAAAAAAACCCGCCGAA GCGGGTTTTTACGTTATTTGCGGATTAACGATTACTCGTTATCAGAACCGCCCAGGGGGCCCGAGCTIAAGAC TGGCCGTCGTTTTACAACACAGAAAGAGTTTGTAGAAACGCAAAAAGGCCATCCGTCAGGGGCCTTCTGCTTA GTTTGATGCCTGGCAGTTCCCTACTCTCGCCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCG GCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGA AAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATA GGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATA AAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA CTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGC AGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGGCTAACTACGGCTACACTAGAAGAACAG TATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACA AACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA GATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGACGCGCGCGTAACTCACGTTAAGGGATTTT GGTCATGAGCTTGCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCTTT SEQIDNO:7 pJ201:ValC_Mt E.colicloningvectorpJ201withthecodonoptimizedqene(valC-opt) encodingthewildtypeValenceneSynthase Nucleotidesequence TAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAA AAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTC TGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAG AAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAA CAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTG AGCGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAGTGCAACCGGCGCAGGAAC ACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAACGCTGTTTTTCCGG GGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGTGGCATAAA TTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGA AACAACTCTGGCGCATCGGGCTTCCCATACAAGCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAG CCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTTGAATATG GCTCATATTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTT GAATGTATTTAGAAAAATAAACAAATAGGGGTCAGTGTTACAACCAATTAACCAATTCTGAACATTATCGCGA GCCCATTTATACCTGAATATGGCTCATAACACCCCTTGTTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTG ACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGACTCCCCATGCGAGAGTAGG GAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGCCCGGGCTAATTAGGGGG TGTCGCCCTTAGGTACGAACTCGATTGACGGGTCTCAAGCTTGTCGACCTGCAGGGATCCTTATCACGGGATG ATCGGTTCCACGAAGAACTTCGTGATCGCATCCTGGATCTCCTTCCGATTCGAATGGGTGAAGAGGTCCCCGT CGTTCAGCATGAAGAAGATCACCCGCATGCCCACGTTGAGGTACAGGTTGCGGAAGCTGCCATGCATGTTGCT CGGTTCGAGGAGTTCCTTGTTGAGCTCCTGCACGCAGCTCTCGATGCGGATTTTCAGGTAGTTCAGGGCGTCC TCGCGCGTGGAGCCGTGATTATCCTTCATATAGCATTCGATCGACGAGGCCATCTCCCCATGCGCCTGCTCGT CCTCGAAATCCTTCACATCATCGACGAGCCGCGTGATGAGCGACAGCAGGGCCTGGCTCTTGCTGCTCGGGAT GTCGAGCTGCTCGAGGATCTCGTCGGACAGGGGCGTATCCATCAGGATCAGCGCATGGATCATGAGGATGCGC ATCCCCGAGCTCTTCACGCCATTCATCAGGTACTCGTCGAAGCCGGGGATGTGGTTGGAGGCGATCCACTCGG CCTCCTGCATATACGCCTCGATATACGACTTCCAGCACGACTGGAGATACTTATACACGAACGGGCCGTGCTT GCGCTCGGCCTCGGACGCGATCTCCTTGTAGATCTCGTAGGAGAAATTGAACGCGATCTTCATGTAGTCCGGG TGATTGTCCACCGTGCTGACATCCCAGCGCGTGAGGGTGGTCGTGAACGGCTTGAGTTCCTCGACGGTCCCGT ACGTGTCATAGATGTCGTCCAGGAGGAACATCTTGGTGGACAGCTTGGTGCAGTTGATGCGGCAGGTGGAGAA CTCGGGCTCGAACAGCGGGCTGGCCCACCAGAAGTAGCTCTCGATATGGCGGTGGCGGAAGAAGTCGAGCTGG ATCGCGGACGAGTTCTTCCACCACGTGCTGATCAGTTTCATCTCATGCTGATGCTGGCTCTGGAGGAIGTTGA AGTCGAGGATCGCCAGCTCCAGGATCTTCTTCATATAGATCTTCGGGATCTTATAGAGGTTGTTGGCGAGCGA GATATTGCAGTCCTGCTGCCGCATGATGTGGATGAAGTTCCACGCCTCCAGCCGCGGGAGGCGGCAGCGCCAG GGGTACTTGAAGGTGTACTCGATCTCCATGAGCAGGGGGTTCTTCGATTCCTGGATGTCCCCATACTCCTTGA TGACATGCTTGAGATACATCGACGCGAAGGCCCGCGCTTCCTTGAGGATGTCCTCATCCGGGAAGTCGAGTTC CGAGGCCTTGTAGAGGTTCAGCATCATCTCCAGCTTGGCGTCCTGATCGCTGCTCTCGAACCCCGAGAACTGG CCCTCCTCGCCCTTGAACTTCTTGAACACGTCCGACGACGCCACGTAGCGATTGAGCCGCAGGATGCGGAACC CCAGCGCCACCATGTTGAGGTCGCCGAAGATGCTATCATGGTTCCAGAACTTATAGATATAATCCAGGGCCGT CTTGATCTCTTCCTGGAAGTGCCGATCGATGCCCAGCCGCTGGACCACGTCGACGAACCAGATGTTCTCGAGG ACCCCGAACTCATCCCCCTCGCCGTTGGAGAACATGTCCCGGATCTCATCGATCAGGATCTCGCGATGTTTAT GATACGAGGAATCCGAATAGGGGGAGTTCAGCGACTGGATGAAATCGTCGGTCCAGAGGTTCGGATGGTGGTT CCCGGTGCGGCGCAGCGCGTCCTTGACCGGCATGCAGGAGCTGCCATCATTGCTGGAATTGCCATTGAACATT TCGGCCATATGCGGATCTGAGACCTTCAGCGACTAAGTACGTGTAAAGGGCGACACAAAATTTATTCTAAATG CATAATAAATACTGATAACATCTTATAGTTTGTATTATATTTTGTATTATCGTTGACATGTATAATTTTGATA TCAAAAACTGATTTTCCCTTTATTATTTTCGAGATTTATTTTCTTAATTCTCTTTAACAAACTAGAAATATTG TATATACAAAAAATCATAAATAATAGATGAATAGTTTAATTATAGGTGTTCATCAATCGAAAAAGCAACGTAT CTTATTTAAAGTGCGTTGCTTTTTTCTCATTTATAAGGTTAAATAATTCTCATATATCAAGCAAAGTGACAGG CGCCCTTAAATATTCTGACAAATGCTCTTTCCCTAAACTCCCCCCATAAAAAAACCCGCCGAAGCGGGTTTTT ACGTTATTTGCGGATTAACGATTACTCGTTATCAGAACCGCCCAGGGGGCCCGAGCTTAAGACTGGCCGTCGT TTTACAACACAGAAAGAGTTTGTAGAAACGCAAAAAGGCCATCCGTCAGGGGCCTTCTGCTTAGTTTGATGCC TGGCAGTTCCCTACTCTCGCCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA GCGGTATCAGCTGACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCC CCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG GCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT TTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCG CTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT ATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGGCTAACTACGGCTACACTAGAAGAACAGTATTTGGTAT CTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCT GGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGA TCTTTTCTACGGGGTCTGACGCTCAGTGGAACGACGCGCGCGTAACTCACGTTAAGGGATTTTGGTCATGAGC TTGCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCTTT