Figure 5

Development of improved yeast strains for sesquiterpene production. (A) Structure of the recyclable integration cassette COD7 used for integration of extra gene copies of HMG2 (K6R) in to desirable loci in the genome (Promoter P_TDH3- cloning site-termination sequence-LoxP-HIS5-LoxP). Subsequent to integration of the cassette the HIS5 selection marker can be excised by the action of Cre recombinase; (B) Yields of caryophyllene measured after terpene extraction in EG60 wild type cells, AM94 cells which provided the basis for improvement and the final AM109 strain carrying ubc7, ssm4(doa10), pho86, erg9 heterozygous deletions; (C) Freshly grown AM109 (Mat a/α, P_Gal1-HMG2(K6R):: HOX2, ura3, trp1, his3, P_TDH3-HMG2(K6R)X 2-::leu2 ERG9/erg9, UBC7/ubc7, SSM4/ssm4, PHO86/pho86), AMD7 (Mat α, ura3, his3, leu2, ubi4:: G418, ubc7:: HIS5), AMD14 (Mat α, ura3, his3, leu2, ssm4:: G418, ubc7:: HIS5) and AMD21 (Mat α, ura3, his3, leu2, pho86:: G418, ubc7:: HIS5) cells were resuspended in fresh medium at the same OD₆₀₀. The cultures were incubated shaking at 30°C. Samples were taken every 2 hours for a period of 24 hours and the OD₆₀₀ was measured.