Figure 2

GC-FID and GC-MS detection and quantification of sesquiterpenoids in Synechocystis. (a) Strains expressing mVenus, AgBIS, MrBBS and PcPs were cultivated for 4 days in 6-well plates with 20% [v/v] dodecane overlay, which was directly subjected to quantitative GC-FID analysis, deploying β-caryophyllene as internal standard (IS). (b) mVenus accumulation profile at time points 48 h and 96 h from cultures treated with 2 µM CuSO₄ (+Cu) and non-induced cultures (−Cu). Data from strain AgBIS is shown as negative fluorescence control. Relative fluorescence units (RFU) were normalized to the corresponding OD₇₅₀ data and are depicted as mean values of sextuplicates with SD. (c–e) GC elution profiles of bisabolene, patchoulol and bisabolol, respectively. Sesquiterpene identity was verified by alignment of retention times to commercial references; overlays from cultures expressing mVenus as well as ‘blank’ dodecane samples (IS only) served as negative controls. Signals at 7.80 min correspond to the IS. Signals at 10.85 min, 16.98 min and 17.43 min correspond to bisabolene, patchoulol and bisabolol, respectively. (f–h) Mass spectra of bisabolene, patchoulol and bisabolol detected by GC-MS analysis of dodecane samples. Signal spectra obtained with the corresponding reference compound are depicted in the lower panel. (i–k) Volumetric titers from induced (+Cu) and non-induced (−Cu) cultures. Quantitative diagrams show individual values from biological sextuplicates and their mean with SD. The figures were prepared using Graphpad Prism 8.2.1. (https://www.graphpad.com/scientific-software/prism/). The photograph in (a) was taken with a “Fairphone 2” camera.