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.

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The industrial fungus Trichoderma reesei has an outstanding capability of secreting an enzyme cocktail comprising multiple plant biomass-degrading enzymes. Herein, the overexpression of XYR1, the master transactivator controlling (hemi)cellulase gene expression, was performed in T. reesei lacking four main cellulase-encoding genes. The resultant strain Δ4celOExyr1 was able to produce a dramatically different profile of secretory proteins on soluble glucose or lactose compared with that of the wild-type T. reesei. The Δ4celOExyr1 secretome included cellulases EGIII and BGLI as well as several hemicellulases and nonhydrolytic cellulose degradation-associated proteins that are not preferentially induced in the wild-type T. reesei strain. Δ4celOExyr1 produced a significant amount of α-arabinofuranosidase I on lactose, and the crude enzyme cocktail of Δ4celOExyr1 not only released a considerable quantity of glucose but also exhibited remarkable performance in the hydrolytic release of xylose, arabinose, and mannose from un-pretreated corn fiber. These results showed that the engineered T. reesei strain holds great potential for improving the saccharification efficiency of the hemicellulosic constituents within corn fiber.

BackgroundThe 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. ResultsWe evaluated the performance of LTC2 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, repression of squalene synthesis pathway and decreasing farnesol synthesis, 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. ConclusionsOur study highlights the potential of applying bacterial sesquiterpene synthases with improved performance by mutagenesis engineering in producing germacrene A.