C1[C:6]([C@@H:7](O)[C@H:8](NC(C(Cl)Cl)=O)[CH2:9]O)=CC=C([N+]([O-])=O)C=1.[CH3:21]C(S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O)C.[C:36]([O-])(=O)[CH2:37][C@:38]([CH2:41][CH2:42]O)([CH3:40])O.[CH3:46][C:47]1(O)[CH2:53]C(=O)OC[CH2:48]1.CCCCCCCCCC>[OH-].[Na+]>[CH3:48][C:47]([CH3:53])=[CH:46][CH2:42][CH2:41][C@@:38]1([CH3:21])[C:37](=[CH2:36])[C@H:7]2[CH2:6][C@@H:40]1[CH2:9][CH2:8]2
BL21 Star™ (DE3) E. coli cells (Invitrogen) were co-transformed with the plasmids pACYCDuet-4506 and Ct94-pETDuet and transformed cells were selected on carbenicillin (50 μg/ml) chloramphenicol (34 μg/ml) LB-agarose plates. Single colonies were used to inoculate 5 mL LB medium with 50 μg/ml carbenicilin and 34 μg/ml chloramphenicol. The culture was incubated overnight at 37° C. The next day 2 mL of TB medium supplemented with the same antibiotics were inoculated with 0.2 mL of the overnight culture. After 6 hours incubation at 37° C., the culture was cooled down to 28° C. and 1 mM IPTG, 2 mg/mL mevalonate (prepared by dissolving mevalonolactone (Sigma) in 0.5N NaOH at a concentration of 1 g/mL and incubating the solution for 30 minutes at 37° C.) and 0.2 mL decane were added to each tube. The cultures were incubated for 48 hours at 28° C. The cultures were then extracted twice with 2 volumes of ethyl acetate, the organic phase was concentrated to 500 μL and analyzed by GC-MS as described above in Example 3. In these conditions sesquiterpene production above 200 mg/L was routinely achieved. Beta-santalene was produced.
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