Construction of Saccharomyces Cerevisiae Cell Factory for β-Amyrin Production

The production of terpenoids in microorganisms especially yeast can be a consistent and sustainable way compared with other methods such as extraction from plants and chemical synthesis. Here, an engineered S. cerevisiae capable of producing β-amyrin, a commercially valuable compound and precursor of triterpenoids, was constructed via various refactoring approaches beginning with the heterogenous expression of β-amyrin synthase from Glycyrrhiza glabra. The titer of β-amyrin was increased 33 folds by introducing a heterogenous squalene monooxygenase from Candida albicans with high squalene affinity, which efficiently dragged intermediate metabolite squalene toward β-amyrin. Squalene biosynthesis module was further strengthened via introducing isopentenyl pyrophosphate isomerase from Escherichia coli along with overexpressing farnesyl diphosphate synthase and squalene synthase from S. cerevisiae, which consequently increased 49 folds of β-amyrin titer. Through reconstructing the promoters with the binding site of transcription factor UPC2, directed transcriptional regulation on the engineered pathway was availably achieved, resulting in β-amyrin titer increased by 65-folds. Ethanol fed-batch fermentation was also employed to enhance β-amyrin synthesis. After optimization, β-amyrin production was finally up to 138.80 mg/L with a yield of 16.30 mg/g DCW in the fermenter experiments, which was increased by 185 and 232 times compared with the initially engineered strain, respectively. Our refactored yeast strain can serve as a cell factory for β-amyrin production as well as the biosynthesis of various β-amyrin derived triterpenoids such as glycyrrhizin and soysaponin.

Keywords: β-Amyrin, Triterpenoids, Transcriptional regulation, Saccharomyces cerevisiae, cell factory