反应详情

温度

28 °C

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.

1. customwere inoculated with 0.2 mL of the overnight culture
2. waitAfter 6 hours incubation at 37° C.
3. customfor 30 minutes
4. customat 37° C.
5. additionwere added to each tube
6. waitThe cultures were incubated for 48 hours at 28° C
7. extractionThe cultures were then extracted twice with 2 volumes of ethyl acetate
8. concentrationthe organic phase was concentrated to 500 μL