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.

Plant essential oils (PEOs) are widely used in cosmetic and nutraceutical industries. The component ratios of PEOs determine their qualities. Controlling the component ratios is challenging in construction of PEO biotechnological platforms. Here, we explore the catalytic reaction pathways of both product-promiscuous and product-specific santalene synthases (i.e., SaSSy and SanSyn) by multiscale simulations. F441 of SanSyn is found as a key residue restricting the conformational dynamics of the intermediates, and thereby the direct deprotonation by the general base T298 dominantly produce α-santalene. The subsequent mutagenesis of this plastic residue leads to generation of a mutant enzyme SanSynF441V which can produce both α- and β-santalenes. Through metabolic engineering efforts, the santalene/santalol titer reaches 704.2 mg/L and the component ratio well matches the ISO 3518:2002 standard. This study represents a paradigm of constructing biotechnological platforms of PEOs with desirable component ratios by the combination of metabolic and enzymatic engineering.

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Betulinic acid (BA) and its derivatives possess potent pharmacological activity against cancer and HIV. As with many phytochemicals, access to BA is limited by the requirement for laborious extraction from plant biomass where it is found in low amounts. This might be alleviated by metabolically engineering production of BA into an industrially relevant microbe such as Saccharomyces cerevisiae (yeast), which requires complete elucidation of the corresponding biosynthetic pathway. However, while cytochrome P450 enzymes (CYPs) that can oxidize lupeol into BA have been previously identified from the CYP716A subfamily, these generally do not seem to be specific to such biosynthesis and, in any case, have not been shown to enable high-yielding metabolic engineering. Here RoCYP01 (CYP716A155) was identified from the BA-producing plant Rosmarinus officinalis (rosemary) and demonstrated to effectively convert lupeol into BA, with strong correlation of its expression and BA accumulation. This was further utilized to construct a yeast strain that yields > 1 g/L of BA, providing a viable route for biotechnological production of this valuable triterpenoid.