Figure 4

Improved yeast strains generated by strong promoter integration and single allele deletion for ERG9. (A) Map of the COD2 cassette, designed for integration of the galactose inducible promoter into promoter regions of endogenous genes. Subsequent to integration, the loxP-URA3-loxP part of the cassette is removed by excision from the enzyme Cre recombinase; (B) Map of the COD3 cassette, designed for the integration of the constitutive ADH1 promoter; C) Cineole production increase in AM68-01 cells, which overexpress ERG20, under the galactose promoter. AM70-01 cells, which are additionally haploinsufficient for ERG9, present substantially reduced levels of cineole production; (D) Expression of a sesquiterpene synthase P330 from S. pomifera in AM68-02 cells; peak 1, α-cubebene; peak 2, α-copaene; peak 3, trans-β-caryophyllene; peak 4, δ-cadinene; (E) Trans-β-caryophyllene standard used for quantification (peak 1);. (F) Copaene standard used for quantification (peak 1); (G) Sesquiterpene yields in the newly developed yeast strains. AM70-02 cells exhibit a substantial yield increase compared to wild type EG60-03 cells;