title: Engineering triterpene production in Saccharomyces cerevisiae-beta-amyrin synthase from Artemisia annua.
abstract: 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 beta-amyrin synthases (EC 5.4.99.-), 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 beta-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 beta-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 beta-amyrin production. Options for further engineering efforts are explored.
authors.affiliation: California Institute for Quantitative Biomedical Research, University of California, Berkeley, CA 94720, USA.
authors.forename: James
authors.initials: J
authors.lastname: Kirby
authors.forename: Dante W
authors.initials: DW
authors.lastname: Romanini
authors.forename: Eric M
authors.initials: EM
authors.lastname: Paradise
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D000595:Amino Acid Sequence; D031172:Artemisia annua / Q000201:enzymology* / Q000235:genetics; D017124:Conserved Sequence; D008401:Gas Chromatography-Mass Spectrometry; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014315:Triterpenes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014315:Triterpenes; D019751:Intramolecular Transferases; C039924:2,3-oxidosqualene-beta-amyrin-cyclase