(670f) Efficiently Refactoring β-Amyrin Synthesis in Saccharomyces Cerevisiae

Triterpenoids are a highly diverse group of natural products widely distributed in plants. In general, they are biosynthesized in plants via the cytosolic mevalonic acid (MVA) pathway and have been verified to require a key precursor triterpene β-amyrin that is derived from the first cyclized product 2,3-oxidosqualene after a series of processes mediated by 2,3-oxidosqualene cyclase and β-amyrin synthase. Based on genetic and bioactivity analyses performed to date, β-amyrin not only serves as the triterpenoid precursor to a wide range of downstream products such as glycyrrhizin, soyasaponin, but also reflects the potential anti-inflammatory, antihyperglycemic and hypolipidemic effects. However, β-amyrin is currently limited in widespread use by scarce availability extracted from natural plant sources and inefficiencies in chemical synthesis caused by its structural complexity. As a result, screening of untapped biological process for β-amyrin production is expected to be continued in the future.

An efficient S. cerevisiae strain capable of reaching relevant titers of β-amyrin was thus constructed. The necessary precursors can be supplied from the wild yeast, thus the β-amyrin production in S. cerevisiae was initially evaluated by introducing the multi-copy expression plasmid pRS42K containing a β-amyrin synthase gene (bAS) derived from Glycyrrhiza glabra under control of FBA1 promoter into S. cerevisiae resulting in strain SpKbS. The transformed strain was able to synthesize a low amount of β-amyrin confirmed by GC-MS. In order to increase the supply of direct precursor 2,3-oxidosqualene for β-amyrin synthesis, one of main regulatory steps in MVA pathway was strengthened by introducing a heterogenous squalene monooxygenase (SE) from Candida albicans. The research displayed that upregulating SE availably results in enhanced β-amyrin production. To increase FPP and squalene flux for β-amyrin production over-expressions of enzymes from IPP to squalene synthesis yield the titer of 31.5 mg/l. Another approach used for enhancing the β-amyrin production in yeast is to regulate transcriptional activation of enzymes. By refactoring the hired promoters via introduced a 7-bp sterol regulatory element (SRE) responsible for binding upc2-1, a semi-dominant mutant allele that enhances the activity of UPC2 (a global transcription factor regulating the biosynthesis of sterols in S. cerevisiae, the resulting yeast was able to produce 45.0 mg/l β-amyrin which reach to 120.0 mg/l in fed-batch fermentation. This titer was about 160 folds compared with that of the strain SpKbS.The present study evaluated this refactoring yeast cell for the production of β-amyrin. And we believe this refactored yeast cell would be promising for further production of a wide range of triterpenoids-derived from β-amyrin.

Acknowledgements

This work was financially supported by National High Technology Research and Development Program of China (863 Program No.2012AA02A704), and by National Science Foundation of China (No. 21376028).