Combinatorial Biosynthesis of Legume Natural and Rare Triterpenoids in Engineered Yeast

Abstract

Triterpenoid saponins are a diverse group of specialized (secondary) metabolites with many biological properties. The model legume Medicago truncatula has an interesting profile of triterpenoid saponins from which sapogenins are differentiated into hemolytic and non-hemolytic types according to the position of their functional groups and hemolytic properties. Gene co-expression analysis confirmed the presence of candidate P450s whose gene expression correlated highly with that of β-amyrin synthase (bAS). Among these, we identified CYP716A12 and CYP93E2 as key enzymes in hemolytic and non-hemolytic sapogenin biosynthetic pathways. The other candidate P450s showed no β-amyrin oxidation activity. However, among the remaining candidate P450s, CYP72A61v2 expression highly correlated with that of CYP93E2, and CYP72A68v2 expression highly correlated with that of CYP716A12. These correlation values were higher than occurred with bAS expression. We generated yeast strains expressing bAS, CPR, CYP93E2 and CYP72A61v2, and bAS, CPR, CYP716A12 and CYP72A68v2. These transgenic yeast strains produced soyasapogenol B and gypsogenic acid, respectively. We were therefore able to identify two CYP72A subfamily enzymes: CYP72A61v2, which modifies 24-OH-β-amyrin, and CYP72A68v2, which modifies oleanolic acid. Additionally, P450s that seemed not to work together in planta were combinatorially expressed in transgenic yeast. The yeast strains (expressing bAS, CPR, CYP72A63 and CYP93E2 or CYP716A12) produced rare triterpenoids that do not occur in M. truncatula. These results show the potential for combinatorial synthesis of diverse triterpenoid structures and enable identification of the enzymes involved in their biosynthesis.

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

“…Through transcript profiling of MeJA-treated B. falcatum roots, we identified CYP716Y1, encoding a P450 that is transcriptionally coregulated with known triterpene saponin biosynthetic genes and that catalyzes the C-16α hydroxylation of amyrin, a catalytic activity previously not linked to any gene product in the plant kingdom. Hence, the P450 compendium that can oxidize the amyrin backbone is expanded, now covering seven positions on the triterpene backbone (11)(12)(13)(14)(15)(16)(17)(18) and including two enzymes that catalyze hydroxylation at C-16, in β-(CYP51H10) (12) and α-configuration (CYP716Y1). Like many reported P450s (15,17), CYP716Y1 can hydroxylate α-and β-amyrin, both pentacyclic triterpenes.…”

“…P450s that modify the β-amyrin backbone on C-11; C-12,13; C-16; C-22; C-23; C-28 or C-30 have been characterized from Glycyrrhiza uralensis, Avena strigosa, Medicago truncatula, Glycine max, Vitis vinifera, and Catharanthus roseus (11)(12)(13)(14)(15)(16)(17)(18). Hydroxylases from Panax ginseng that oxidize the dammarenediol-II backbone on C-6, C-12, or C-28 (19)(20)(21), and a C-20 hydroxylase from Lotus japonicus (22) that modifies lupeol, have also been identified.…”

Enhanced Secondary- and Hormone Metabolism in Leaves of Arbuscular Mycorrhizal Medicago truncatula

“…b-Amyrin synthase is committed to the first step in the biosynthesis of saponins, which are glycosylated triterpenoids protecting plants against pathogens and pests (Morita et al, 2000;Suzuki et al, 2002;Thimmappa et al, 2014). In addition, we found three genes for cytochromes P450: CYP76A61 and CYP93E2 (b-amyrin hydrolase), catalyzing the formation of the nonhemolytic sapogenin soyasapogenol B (Soya_B), and CYP72a67v2, which is a putative b-amyrin oxidase (Fukushima et al, 2013). Finally, UGT73K1 encodes a triterpene UDP-glucosyl transferase dedicated to linking a sugar moiety to the triterpene aglycones hederagenin and soyasapogenols B and E to produce saponins (Achnine et al, 2005).…”

Recent advances in steroidal saponins biosynthesis and in vitro production

“…Transgenic yeast strains expressing β-amyrin synthase (bAS), P450 reductase, CyP93E2 and CyP72A61v2 were able to produce soyasapogenol B, and yeast strain over expressing bAS, P450 reductase, CyP716A12 and CyP72A68v2 produce gypsogenic acid. Additionally, P450 s that seemed not to work together in planta were combinatorially expressed in transgenic yeast for the production of saponins (Fukushima et al 2013). Combinatorial synthetic biology program was also successful for the synthesis of monoglycosylated saponins in yeast by combining CyP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes from different plant species (Moses et al 2014a, b).…”