反应详情

温度

28 °C

BL21 Star™(DE3) E. coli cells (Invitrogen, Carlsbad, Calif.) were transformed with the plasmids pET101-cont2_1 (SEQ ID NO:2) prepared as described in Example 3 and with the plasmid pACYCDuet-4506. Transformed cells were selected on carbenicillin (50 μg/ml) and chloramphenicol (34 μg/ml) LB-agarose plates. Single colonies were used to inoculate 5 mL liquid LB medium supplemented with the same antibiotics. 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 min 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 4. In these conditions the cells produced (+)-α-santalene at 250 mg/L culture in 48 hours.

1. custom(SEQ ID NO:2) prepared
2. customwere inoculated with 0.2 mL of the overnight culture
3. waitAfter 6 hours incubation at 37° C.
4. customfor 30 min
5. customat 37° C.
6. additionwere added to each tube
7. waitThe cultures were incubated for 48 hours at 28° C
8. extractionThe cultures were then extracted twice with 2 volumes of ethyl-acetate
9. concentrationthe organic phase was concentrated to 500 μL