Figure 4

Limonene biosynthesis in 50 mL cultures. S. cerevisiae EPY210C expressing the C. limon (CLLS) limonene synthase, M. spicata limonene synthase (MSLS), or carrying an empty expression vector (−ve) were cultured for 120 h in 50 mL of culture medium. Initially strains were cultured in supplemented YP medium (YP+) and the optical density (A) and reaction rate of the dodecane phase with DPPH (B) were recorded. This experiment was repeated with –ve and CLLS cultures in several defined medium compositions ( panels C and D): SD medium containing extra metals (SD + M), pH adjusted SD medium (SD(pH)), pH adjusted SD medium with extra metals (SD(pH) + M), extra nitrogen (SD(pH) + N), or extra metals and nitrogen (SD(pH) + MN), or supplemented YP medium (YP+). Limonene titres (E) were calculated following GC-MS analysis of dodecane extracts. All data shown are calculated from n = 3 biological replicates, showing mean ± 1 SD. ND = not detected, NQ = not quantifiable. Results were compared to negative controls using an unpaired Student’s t-test (** = p < 0.01, *** = p < 0.001).