Improved production of germacrene A, a direct precursor of ß-elemene, in engineered Saccharomyces cerevisiae by expressing a cyanobacterial germacrene A synthase

BACKGROUND: The sesquiterpene germacrene A is a direct precursor of ß-elemene that is a major component of the Chinese medicinal herb Curcuma wenyujin with prominent antitumor activity. The microbial platform for germacrene A production was previously established in Saccharomyces cerevisiae using the germacrene A synthase (LTC2) of Lactuca sativa. RESULTS: We evaluated the performance of LTC2 (LsGAS) as well as nine other identified or putative germacrene A synthases from different sources for the production of germacrene A. AvGAS, a synthase of Anabaena variabilis, was found to be the most efficient in germacrene A production in yeast. AvGAS expression alone in S. cerevisiae CEN.PK2-1D already resulted in a substantial production of germacrene A while LTC2 expression did not. Further metabolic engineering the yeast using known strategies including overexpression of tHMGR1 and repression of squalene synthesis pathway led to an 11-fold increase in germacrene A production. Site-directed mutagenesis of AvGAS revealed that while changes of several residues located within the active site cavity severely compromised germacrene A production, substitution of Phe23 located on the lateral surface with tryptophan or valine led to a 35.2% and 21.8% increase in germacrene A production, respectively. Finally, the highest production titer of germacrene A reached 309.8 mg/L in shake-flask batch culture. CONCLUSIONS: Our study highlights the potential of applying bacterial sesquiterpene synthases with improved performance by mutagenesis engineering in producing germacrene A.