Fig. 4
From: Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric substrate
Engineering SfCinS1 to become specific for NPP. a A library of 19 different SfCinS variants was evaluated in yeast cells producing either only GPP, or both GPP and NPP. To identify variants with improved specificity for NPP, the ratio of monoterpene product titers obtained with and without the NPP pathway was determined and shown here in logarithmic scale. Samples were analyzed in triplicate originating from independent yeast transformations (n = 3 biologically independent samples). Errors correspond to the mean absolute deviation (MAD) around the mean. b Proposed GPP and NPP cyclization mechanism leading to the synthesis of 1,8-cineole by SfCinS1. Initially, GPP binds at the extended conformation. Following syn migration of the diphosphate, the transoid extended conformation of LPP transitions first to the cisoid form and then to the catalytically competent cisoid closed conformation44. In NPP, the C-2,3 bond is already in the cis-conformation, bypassing the requirement for the transoid to cisoid transition. The insets show models of the active site of SfCinS1 with two proposed conformers of 2-fluorolinalyl diphosphate (2F-LPP) superimposed. The two conformations have been obtained by soaking crystals of mint limonene synthase with 2F-LPP or 2-fluoro-geranyl diphosphate (2F-GPP). In the crystal, 2F-GPP is converted to the transoid 2F-LPP form, while 2F-LPP assumes a conformation that resembles the extended cisoid form of LPP. For the GPP reaction to proceed, extensive conformational changes of the substrate are needed for the conversion from transoid to cisoid. F571 is positioned in the center of this transition. Red arrows indicate chemical bond rotation. Graphics produced with UCSF Chimera68. Source data of a are provided as a Source Data file
