title: Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids.
abstract: BACKGROUND
  Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway.
  
  
  RESULTS
  S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production.
  
  
  CONCLUSIONS
  Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields.
authors.affiliation: Department of Natural Products and Biotechnology, Centre International de Hautes Etudes Agronomiques Méditerranéennes, Mediterranean Agronomic Institute of Chania, PO Box 85, Chania 73100, Greece.
authors.forename: Codruta
authors.initials: C
authors.lastname: Ignea
authors.forename: Ivana
authors.initials: I
authors.lastname: Cvetkovic
authors.forename: Sofia
authors.initials: S
authors.lastname: Loupassaki
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Kefalas
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Johnson
authors.forename: Sotirios C
authors.initials: SC
authors.lastname: Kampranis
authors.forename: Antonios M
authors.initials: AM
authors.lastname: Makris
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D016254:Mutagenesis, Insertional / Q000379:methods*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D027543:Salvia officinalis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D013729:Terpenes / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D013729:Terpenes; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase