Engineering triterpene production in Saccharomyces cerevisiae-beta-amyrin synthase from Artemisia annua

FEBS J. 2008 Apr;275(8):1852-9. doi: 10.1111/j.1742-4658.2008.06343.x. Epub 2008 Mar 8.

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.

MeSH terms

  • Amino Acid Sequence
  • Artemisia annua / enzymology*
  • Artemisia annua / genetics
  • Conserved Sequence
  • Gas Chromatography-Mass Spectrometry
  • Intramolecular Transferases / chemistry
  • Intramolecular Transferases / genetics
  • Intramolecular Transferases / isolation & purification
  • Intramolecular Transferases / metabolism*
  • Molecular Sequence Data
  • Protein Engineering
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Sequence Alignment
  • Triterpenes / chemistry
  • Triterpenes / metabolism*

Substances

  • Triterpenes
  • Intramolecular Transferases
  • 2,3-oxidosqualene-beta-amyrin-cyclase