Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum

Abstract

The saikosaponins comprise oleanane-and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1, encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16α hydroxylation of oleanane-and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated β-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.cyclodextrin | amyrin | metabolic engineering | triterpenoid

OSC2 and CYP716A14v2 Catalyze the Biosynthesis of Triterpenoids for the Cuticle of Aerial Organs of Artemisia annua

“…To determine the cyclization specificity of OSC2, we expressed its gene in a sterol engineered S. cerevisiae strain TM1 (Moses et al, 2014). Strain TM50, expressing OSC2 from a galactoseinducible promoter, was generated from strain TM1 and compared with a control strain (TM5).…”

“…The genotypes of all yeast strains generated and used in this study are provided in Supplemental Table 2. Strains TM50 and TM5 were cultured in medium containing galactose and methyl-b-cyclodextrin (MbCD), which sequesters triterpenes from yeast cells and transports them to the growth medium (Moses et al, 2014). Organic extracts of their spent media were compared by gas chromatography-mass spectrometry (GC-MS) analysis.…”

“…CYP716A12 from Medicago truncatula (Carelli et al, 2011), CYP716A15 and CYP716A17 from Vitis vinifera (Fukushima et al, 2011), CYP716AL1 from C. roseus (Huang et al, 2012), CYP716A52v2 from P. ginseng , and CYP716A75 from Maesa lanceolata (Moses et al, 2015) are C-28 oxidases of a-amyrin, b-amyrin, and lupeol. CYP716Y1 from Bupleurum falcatum is a C-16a oxidase of a-amyrin and b-amyrin (Moses et al, 2014), and CYP716A47 and CYP716A53v2 from P. ginseng are C-12 and C-6 oxidases of dammaranes, respectively (Han et al, 2011(Han et al, , 2012. The CYP716A12 protein was used to screen for candidate CYP716 family P450s in the A. annua transcriptome data set.…”

“…To explore the multifunctionality of CYP716A14v2, we expressed its gene in yeast strain TM6 (Moses et al, 2014), which produces the pentacyclic triterpenes lupeol and lupanediol. Yeast strain TM56 (Supplemental Table 2) was derived from strain TM6 by transforming the latter with CYP716A14v2 and At-ATR1.…”

“…In addition, strain TM111 containing an empty vector control was also generated (Supplemental Table 2). These yeast strains were cultured for 72 h in synthetic medium containing galactose and MbCD as described (Moses et al, 2014), and the resulting spent medium was used for metabolite extraction and GC-MS analysis. (A) and (B) qPCR analysis of transgenic A. annua plants revealed silencing of OSC2 (OSC) (A) and CYP716A14v2 (CYP) (B).…”