Catalytic promiscuity, as a modern and imperfect understanding concept in enzyme catalysis community, is prevailing in plant-derived sesquiterpene cyclases (FPPC) and highly related to the chemical diversity of sesquiterpenoid natural products. Both the Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) and Aspergillus terreus aristolochene synthase (ATAS) belong to FPPC, involve the same reaction pathway, and yield their ultimate main product (aristolochene) with different stereochemistries. The catalytic promiscuity of TEAS and fidelity of ATAS have been observed in previous experimental studies, but the detailed catalytic mechanism is still not clear. Herein, by employing the quantum classical multiscale molecular dynamics simulations and site-directed mutagenesis experiments, the complete enzyme catalytic pathways from the substrate to aristolochene and various side products (in TEAS) are investigated and the important mechanism insights are included: (1) the PPi moiety (diphosphate group, released from the substrate) would further act as the general acid/base in both TEAS and ATAS enzyme catalysis, which likely plays a general role in FPPC. (2) The Asp444−Tyr520 dyad acts as an additional general acid/base residue pair to increase promiscuity in TEAS. (3) The enriched aromatic residues are essential for the catalytic fidelity of ATAS. Finally, we further discuss the three critical chemical control factors which are proposed to be responsible for the catalytic promiscuity and fidelity in most FPPC, that is, substrate folding mode, intermediate flexibility, and key residue, owing to the more or less plasticity of the active pocket in various FPPC.

Sandalwood oil has been widely used in perfumery industries and aromatherapy. Santalols are its major components. Herein, we attempted to construct santalol-producing yeasts. To alter flux from predominant triterpenoid/ steroid biosynthesis to sesquiterpenoid production, expression of ERG9 (encoding yeast squalene synthase) was depressed by replacing its innate promotor with P HXT1 and fermenting the resulting strains in galactose-rich media. And the genes related to santalol biosynthesis were overexpressed under control of GAL promotors, which linked santalol biosynthesis to GAL regulatory system. GAL4 (a transcriptional activator of GAL promotors) and PGM2 (a yeast phosphoglucomutase) were overexpressed to overall promote this artificial santalol biosynthetic pathway and enhance galactose uptake. 1.3 g/L santalols and 1.2 g/L Z-α-santalol were achieved in the strain WL17 expressing SaSS (α-santalene synthase from Santalum album) and WL19 expressing SanSyn (α-santalene synthase from Clausena lansium) by fed-batch fermentation, respectively. This study constructed the microbial santalol-producing platform for the first time.

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Four terpene synthases for the biosynthesis of volatile terpenoids were identified from the transcriptome of Stellera chamaejasme L. flowers, including SchTPS1, SchTPS2, SchTPS3, and SchTPS4. Their functions were characterized by synthetic biology approaches in Escherichia coli and in vitro enzymatic assays. SchTPS1, SchTPS2, and SchTPS3 are guaiene synthases, while SchTPS4 is an (E,E)-geranyl linalool synthase. Next, SchTPS1 and α-guaiene 2-oxidase VvSTO2 were co-expressed in Saccharomyces cerevisiae to reconstruct the biosynthetic pathway of (−)-rotundone, which is a unique aroma compound in fruits, vegetables, and wines. This is the first report for the construction of a (−)-rotundone-producing microbial platform.

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