Increase of copy number of HMG-CoA reductase and FPP synthase genes improves the amorpha-4,11-diene production in engineered yeast

The gene encoding amorpha-4,11-diene synthase was cloned from Artemisia annua L. Other two genes encoding the FPP synthase (FPPS) and HMG-CoA reductase (HMGR) were cloned from Saccharomyces cerevisiae. The cloned cDNAs were confirmed by DNA sequencing. Two expression vectors were constructed, one is named pGBT9/A/HMG/FPP harboring genes for HMG-CoA reductase and FPP synthase and the other is pYeDP60/G/AS, containing the gene encoding amorpha-4,11-diene synthase. Two kinds of engineered yeast were constructed: the first was named WHT［AS］,which contained the plasmid pYeDP60/G/AS; the second was WHT［HMG+FPP+AS］, in which the vectors pGBT9/A/HMG/FPP and pYeDP60/G/AS were introduced by cotransformation mediated with LiOAc and PEG4000. The positive clones were identified for further fermentations. The samples from fermentations were analyzed by GC-MS for amorpha-4,11-diene. The results show that engineered yeasts could produce amorpha-4,11-diene. Moreover, the amorpha-4,11-diene production of WHT［HMG+FPP+AS］ and WHT［AS］ were 23.6 mg·L^-1 and 10 μg·L^-1, respectively. Its concentrations were reported as equivalents of valencene. The results showed the copy number increase of HMGR and FPPS genes can improve the production of amorpha-4,11-diene in the fermentation of engineered yeasts.