Assessing the state of the mevalonate pathway in engineered strains. (A) The transmembrane domain (1–667) of HMG1 which is regulated by proteolytic degradation was fused to EYFP and expressed by a stable promoter. The mean fluorescence from 50.000 cells per culture in triplicate was measured by flow cytometry. Increased Hmgp stability does not play a role in observed strain improvements; (B) Excess FPP and GGPP which is not converted by CLS to 8OH-CPP can be released in the medium and hydrolyzed to farnesol (FOH) and nerolidol (NOH) from FPP, and geranyllinalool (GLOH) and geranylgeraniol (GGOH) from GGPP. These metabolites were quantified in the engineered strains. Cells carrying empty vector show a small increase in NOH, FOH levels at each successive modification. Expression of CLS-ERG20 (F96C) leads a substantial increase subsequent to the second genetic perturbation (AM229); (C) GLOH + GGOH are increasing after the second perturbation; (D) co-expression of CLS-ERG20 (F96C) with SCLS reduced the levels of released GLOH + GGOH, as substrate is efficiently converted to sclareol.