Combinatorial Biosynthesis of Sapogenins and Saponins in Saccharomyces Cerevisiae Using a C-16 Α Hydroxylase from Bupleurum
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Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum Tessa Mosesa,b,c,d, Jacob Polliera,b, Lorena Almagroe, Dieter Buystf, Marc Van Montagua,b,1, María A. Pedreñoe, José C. Martinsf, Johan M. Theveleinc,d, and Alain Goossensa,b,1 aDepartment of Plant Systems Biology, VIB, 9052 Gent, Belgium; bDepartment of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; cDepartment of Molecular Microbiology, VIB, 3001 Leuven-Heverlee, Belgium; dLaboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, 3001 Leuven-Heverlee, Belgium; eDepartment of Plant Biology, Faculty of Biology, Universidad de Murcia, 30100 Murcia, Spain; and fDepartment of Organic Chemistry, Ghent University, 9000 Gent, Belgium Contributed by Marc Van Montagu, December 17, 2013 (sent for review April 29, 2013) The saikosaponins comprise oleanane- and ursane-type triterpene P450s that modify the β-amyrin backbone on C-11; C-12,13; saponins that are abundantly present in the roots of the genus C-16; C-22; C-23; C-28 or C-30 have been characterized from Bupleurum widely used in Asian traditional medicine. Here we Glycyrrhiza uralensis, Avena strigosa, Medicago truncatula, Glycine identified a gene, designated CYP716Y1, encoding a cytochrome max, Vitis vinifera,andCatharanthus roseus (11–18). Hydroxylases P450 monooxygenase from Bupleurum falcatum that catalyzes from Panax ginseng that oxidize the dammarenediol-II backbone the C-16α hydroxylation of oleanane- and ursane-type triterpenes. on C-6, C-12, or C-28 (19–21), and a C-20 hydroxylase from Lotus Exploiting this hitherto unavailable enzymatic activity, we launched japonicus (22) that modifies lupeol, have also been identified. To a combinatorial synthetic biology program in which we combined characterize these P450s, they have been ectopically expressed in CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransfer- yeast strains either producing β-amyrin or externally supplied with ase genes available from other plant species and reconstituted the candidate substrates. Similarly, several OSCs have been produced synthesis of monoglycosylated saponins in yeast. Additionally, and functionally analyzed in yeast. From these studies, it is clear we established a culturing strategy in which applying methylated that yeast cells cannot only be used for the characterization of PLANT BIOLOGY β-cyclodextrin to the culture medium allows the sequestration of novel enzymes, but possibly also as a heterologous host for the heterologous nonvolatile hydrophobic terpenes, such as triterpene production of triterpene sapogenins (23). To date only two pilot sapogenins, from engineered yeast cells into the growth medium, studies have aimed at engineering of β-amyrin production in yeast thereby greatly enhancing productivity. Together, our findings (24, 25), but no efforts toward engineering of sustainable pro- provide a sound base for the development of a synthetic biology duction of sapogenins or saponins in yeast have been reported. platform for the production of bioactive triterpene sapo(ge)nins. Here, we identified and characterized CYP716Y1, a P450 from Bupleurum falcatum that corresponds to a C-16α oxidase, desig- cyclodextrin | amyrin | metabolic engineering | triterpenoid nated according to Nelson’s nomenclature (http://drnelson.uthsc. edu/cytochromeP450.html). By designing triterpene-hyperproducing riterpene saponins are secondary metabolites that exhibit Ta large structural diversity and wide range of biological ac- Significance tivities in many plant species (1, 2). Saponins are glycosides of sapogenins, which are composed of 30 carbon atoms arranged in “ ” Saponins are plant molecules that are produced as a chemical 4- or 5-ring structures that are decorated by functional groups. defense against herbivores and eukaryotic pathogens. They Saponins are synthesized by multiple glycosylations of the sapogenin constitute structurally diverse, bioactive compounds composed building blocks that are produced by multiple cytochrome P450- of a 30-carbon triterpene backbone adorned with multiple dependent monooxygenase (P450) or oxidoreductase-mediated functional groups and sugars. Saikosaponins are abundant β modifications of basic backbones, such as -amyrin (oleanane type), saponins accumulating in the Asian medicinal plant Bupleurum α -amyrin (ursane type), lupeol, and dammarenediol. These back- falcatum, but none of the enzymes involved in their bio- bones are generated by oxidosqualene cyclase (OSC)-mediated cy- synthesis had been characterized. We identified a cytochrome clization of 2,3-oxidosqualene, which is also an intermediate in the P450 involved in the oxidation of saikosaponins, thereby synthesis of sterols in eukaryotes (3, 4). Both saponins and sap- expanding the enzyme compendium that can generate plant ogenins include biologically active compounds or serve as starter saponins with an extra activity. Using this enzyme compen- molecules for the generation of novel, potentially bioactive struc- dium, we established a synthetic biology program to reconstitute tures by synthetic modification (5–7). saponin biosynthesis in the yeast Saccharomyces cerevisiae The genus Bupleurum consists of perennial herbs that are used and developed a cyclodextrin-based culturing strategy to se- in Asian traditional medicine, either alone or in combination quester triterpenes from engineered yeast cells and enhance with other ingredients, for the treatment of common colds, fever, their productivity. and inflammatory disorders (8). Saikosaponins constitute the largest class of secondary metabolites in Bupleurum and can Author contributions: T.M., J.P., M.V.M., J.M.T., and A.G. designed research; T.M., J.P., account for up to ∼7% of root dry weight. Their accumulation L.A., and D.B. performed research; M.A.P. and J.C.M. contributed new reagents/analytic tools; T.M., J.P., D.B., J.C.M., and A.G. analyzed data; and T.M., J.P., D.B., J.C.M., and A.G. can be further stimulated by jasmonate treatment (9). More than wrote the paper. 120 closely related oleanane- and ursane-type saikosaponins The authors declare no conflict of interest. have been identified from this genus and the oxidations at var- Data deposition: The sequence reported in this paper has been deposited in the GenBank ious positions suggest the presence of multiple enzymes, mainly database [accession no. KC963423 (CYP716Y1)]. P450s, capable of catalyzing specific modifications on the amyrin 1To whom correspondence may be addressed. E-mail: [email protected] or backbones (8, 10). To date, no P450 or oxidoreductase involved [email protected]. in triterpene saponin biosynthesis has been identified from This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Bupleurum species. 1073/pnas.1323369111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1323369111 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 starter strains, optimizing culturing conditions for triterpene syn- thesis, and using the CYP716Y1 gene in a combinatorial synthetic biology program, we established a platform that allows us to pro- duce and sequester triterpene sapogenins in culture medium and to reconstitute a full saponin synthetic pathway in yeast cells. Results Transcript Profiling of Jasmonate-Treated B. falcatum Roots Reveals CYP716Y1. To identify saponin biosynthetic genes, expression analysis on roots of methyl jasmonate (MeJA)-treated B. falca- tum plants was performed with cDNA-amplified fragment length polymorphism–based transcript profiling (26). The expression of 18,695 transcript tags was monitored over time and 1,771 MeJA- responsive tags (hereafter referred to as “BF tags”) were isolated. Direct sequencing of the reamplified BF tags gave good-quality sequences for 1,217 fragments. BF tags corresponding to genes encoding enzymes known to catalyze early steps in triterpene saponin biosynthesis, such as β-amyrin synthase (bAS), displayed similar expression patterns, suggesting tight coregulation, and reached maximum expression levels 4–24 h post-MeJA elici- tation (Fig. 1A). The gene corresponding to tag BF567 was coregulated with these genes and matched a P450 with 48% and 47% sequence similarity with the P. ginseng CYP716A53v2 and M. truncatula CYP716A12, respectively. The full-length open reading frame corresponding to the BF567 tag (hereafter called “CYP716Y1”) was cloned from a B. falcatum cDNA library (27). Phylogenetic analysis confirmed that CYP716Y1 belongs to the CYP716 family of P450s involved in triterpene saponin bio- synthesis (Fig. 1B and Fig. S1). Generation of Triterpene-Producing Yeast Strains. To determine the Fig. 1. Identification of CYP716Y1 by transcript profiling of MeJA-treated in vivo enzymatic activity of CYP716Y1, we engineered Sac- B. falcatum roots. (A) Coregulation of CYP716Y1 (in green) with other tri- charomyces cerevisiae strains for hyperproduction of triterpenes, terpene saponin biosynthetic enzymes. Cluster of the B. falcatum tran- analogous to a previously described strain (24). All yeast strains scriptome comprising tags corresponding to genes reported to be involved in triterpene biosynthesis. Treatments and time points (in hours) are in- generated in this study are listed in Table S1. First, we replaced dicated at the top. Transcriptional activation and repression relative to the the native promoter of lanosterol synthase with a methionine-