tailieunhanh - Báo cáo khoa học: Engineering triterpene production in Saccharomyces cerevisiae – b-amyrin synthase from Artemisia annua

Using a degenerate primer designed from triterpene synthase sequences, we have isolated a new gene from the medicinal plantArtemisia annua. The predicted protein is highly similar to b-amyrin synthases (EC .–), sharing amino acid sequence identities of up to 86%. | ễFEBS Journal Engineering triterpene production in Saccharomyces cerevisiae - b-amyrin synthase from Artemisia annua James Kirby1 Dante W. Romanini2 Eric M. Paradise1 3 and Jay D. Keasling1 3 4 5 1 California Institute for Quantitative BiomedicalResearch University of California Berkeley CA USA 2 Department of Chemistry University of California Berkeley CA USA 3 Department of ChemicalEngineering University of California Berkeley CA USA 4 Department of Bioengineering University of California Berkeley CA USA 5 PhysicalBiosciences Division Lawrence Berkeley NationalLaboratory Berkeley CA USA Keywords Artemisia annua isoprenoids metabolic engineering Saccharomyces cerevisiae b-amyrin synthase Correspondence J. D. Keasling Berkeley Center for Synthetic Biology 717 Potter Street Building 977 Mail code 3224 University of California Berkeley CA 94720-3224 USA Fax 1 510 495 2630 Tel 1 510 495 2620 E-mail keasling@ Received 12 December 2007 revised 11 February 2008 accepted 18 February 2008 Using a degenerate primer designed from triterpene synthase sequences we have isolated a new gene from the medicinal plant Artemisia annua. The predicted protein is highly similar to b-amyrin synthases EC sharing amino acid sequence identities of up to 86 . Expression of the gene designated AaBAS in Saccharomyces cerevisiae followed by GC MS analysis confirmed the encoded enzyme as a b-amyrin synthase. Through engineering the sterol pathway in S. cerevisiae we explore strategies for increasing triterpene production using AaBAS as a test case. By manipulation of two key enzymes in the pathway 3-hydroxy-3-methylglutaryl-CoA reductase and lanosterol synthase we have improved b-amyrin production by 50 achieving levels of 6 mg L-1 culture. As we have observed a 12-fold increase in squalene levels it appears that this strain has the capacity for even higher b-amyrin production. Options for further engineering efforts are explored. doi .