Fig. 5
From: Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric substrate
Monoterpene profile of the terpene synthase variants. Variants of ClLimS (a), SpSabS (b), SeCamS (c), and PtPinS (d), carrying different substitutions in the residue homologous to SfCinS1(F571), were analyzed in yeast cells producing NPP versus non-NPP-producing cells. On the left-hand side of panels a–d, the bar charts show the ratio of the total monoterpene production titer when both the NPP and GPP pathways are present divided by the titer of monoterpene production by the same variant when only the GPP pathway is present. The bar charts on the right-hand side of panels a–d show the total monoterpene titer obtained by each mutant synthase when the NPP pathway was present in comparison to the corresponding wild-type enzyme titer (normalized to 1), indicating the overall efficiency of each enzyme variant. e SeCamS variants H583F and H583V switch specificity from a camphene synthase to a highly efficient limonene synthase. f The overall improvements in pathway performance enable the introduction of an additional downstream modification step. Yeast cells co-expressing SpSabS and CYP750B1 produce detectable amounts of trans-sabin-3-ol only when the NPP pathway is present. Replacement of wild-type SpSabS by the NPP-specific variant SpSabS(H561F) improved trans-sabin-3-ol synthesis even further. g Introduction of PERG1 upstream of ERG20 in strain MIC3 installs dynamic control of fluxes through Erg20p. In combination with the NPP-specific variants ClLimS(H570Y) and SpSabS(H561F), this leads to further improvement in limonene and sabinene titers, respectively. Strains denoted by GPP express Erg20p(N127W) and produce only GPP, while strains denoted by GPP + NPP express Erg20p(N127W)-SlNPPS1 and produce both GPP and NPP. Samples were prepared independently from three different yeast transformations, processed and analyzed separately (n = 3 biologically independent samples). The average yield of each compound between the samples was calculated, and the mentioned ratios and fold improvements in the product profile were deduced. Error bars in panels a–d correspond to the geometric mean of the original two relative MAD values. Source data of a–d and g are provided as a Source Data file
