Protocols often seem to use regulatable promoters when expressing a heterologous gene in a host like yeast where they add something like IPTG to induce expression from (say) the lac promoter.
Is there a reason to do this as opposed to using a constitutive promoter? i.e. Is there reason to think that regulatable (inducible) promoters have higher expression levels than constitutive promoters?
In other words, what utility is there in having an external "switch", i.e. the IPTG to turn on expression on-demand rather than just boost the default expression level using a promoter not requiring induction?
The goal here is small biomolecule synthesis by industrial fermentation of a transgenic yeast.
Example Protocol:
L21 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.
