1tables and figures Supplementary Table 1 Genes potentially implicated in Medicago truncatula triterpenoid biosynthesis correlated with bAS NameProbeset IDCorrelation bAS Mtr S1_s_a t CYP72A68Mtr S1_at CYP72A67Mtr S1_at Anthocyanidin 3-O- glucosyltransferase Mtr S1_at0.847 CYP72A67Mtr S1_at0.827 bASMtr S1_at CYP72A65Mtr S1_at Cytokinin-O-glucosyltransferaseMtr S1_at CYP716A12 Mtr S1_s_a t Anthocyanidin 3-O- glucosyltransferase Mtr S1_at Cytokinin-O-glucosyltransferaseMtr S1_at CYP716A12Mtr S1_at CYP89A2 Mtr S1_x_a t CYP89A2Mtr S1_at UGT73K1Mtr S1_at CYP72A61Mtr S1_at CYP93E2Mtr S1_at P450s correlated with bAS (Mtr S1_at) based on a Pearson correlation coefficient cut-off 0.4.

2tables and figures Supplementary Table 2 Estimated product yields of bAS/CPR/CYP716A12/CYP72A68v2- and bAS/CPR/CYP93E2/CYP72A61v2-expressing yeast Compound (peak no.)Estimated yield (mg/L) bAS/CPR/CYP93E2/CYP72A61-expressing yeast β-Amyrin (1) OH-β-Amyrin (2)0.27 Soyasapogenol B (3)1.35 bAS/CPR/CYP716A12/CYP72A68-expressing yeast β-Amyrin (1)0.55 Erythrodiol (4)0.09 Gypsogenic acid (8)0.96 Estimation was performed based on peak areas from the mass chromatograms shown in Figs. 2 and 3, by comparing the areas of internal standard and authentic compounds with known concentrations.

3 Supplementary Fig. S1 Expression profiles of P450 genes potentially involved in soyasapogenol B biosynthesis. Co-expression analysis was performed using the gene co-expression tool for Medicago truncatula ( Expression profiles for bAS, CYP93E2, CYP72A61, and UGT73K1 are shown. Corresponding probe IDs and correlation values are indicated in Table 1. CYP93E2bASCYP72A61UGT73K 1

4 Supplementary Fig. S2 Expression profiles of P450 genes potentially involved in gypsogenic acid biosynthesis. Co-expression analysis was performed using the gene co-expression tool for Medicago truncatula ( Expressions of bAS, CYP716A12, CYP72A68, and UGT73F3 are shown. Corresponding probe IDs are indicated in Table 2. CYP716A12bASCYP72A68UGT73F3

5tables and figures Supplementary Fig. S4 Mass spectrum of product resulting from transgenic yeast co- expressing bAS, CPR, CYP716A12, and CYP72A68. The mass spectrum of peak 4 shown in Fig. 3A matches up with that of authentic erythrodiol (4). Peak 4 in Fig. 3A Authentic erythrodiol (4) Supplementary Fig. S3 Mass spectrum of product resulting from transgenic yeast co- expressing bAS, CPR, CYP93E2, and CYP72A61. The mass spectrum of peak 2 shown in Fig. 2A matches up with that of authentic 24-OH-β- amyrin (2). Peak 2 in Fig. 2A Authentic 24-OH-β-amyrin (2)

6tables and figures Peak 4 in Fig. 4A Peak 5 in Fig. 4A Authentic erythrodiol (4) Authentic oleanolic acid (5) Supplementary Fig. S5 Mass spectra of products resulting from transgenic yeast co- expressing bAS, CPR, CYP93E2, and CYP716A12. The mass spectra of peaks 2, 4, and 5 shown in Fig. 4A match up with those of authentic 24-OH-β-amyrin (2), erythrodiol (4), and oleanolic acid (5), respectively. Authentic 24-OH-β-amyrin (2) Peak 2 in Fig. 4A

7tables and figures Supplementary Fig. S6 In vivo production of queretaroic acid and other β-amyrin derivatives in transgenic yeast co-expressing bAS, CPR, CYP716A12, and CYP72A63. (A) and (B) show GC-MS analysis (TIC) of ethyl acetate extracts (using DB-1 MS column) from yeast cultures. (A) The yeast strains were engineered to express bAS, CPR, CYP716A12, and CYP72A63; (B) bAS, CPR, and CYP716A12 as controls. (C) The structures were deduced from mass spectra because authentic standards were not available. Queretaroic acid was identified by comparison with bibliographic data (Burnouf- Radosevich et al. 1985). bAS/CPR/CYP716A12 Relative ion intensity (%) Retention time (min) bAS/CPR/CYP716A12/CYP72A63 A B

8tables and figures Peak 14 in (A) Peak 15 in (A) Authentic 30-OH-β-amyrin Peak 18 in (A) Peak 16 in (A) Peak 17 in (A) Queretaroic acid C Peak 4 in (A) Peak 5 in (A) Authentic erythrodiol (4) Authentic oleanolic acid (5) Supplementary Fig. S6 (Cont.)

9tables and figures Supplementary Fig. S7 In vivo production of rare triterpenoids in transgenic yeast co- expressing bAS, CPR, CYP93E2, and CYP72A63. (A) and (B) show GC-MS analysis (TIC) of ethyl acetate extracts (using DB-1 MS column) from yeast cultures. (A) The yeast strains were engineered to express bAS, CPR, CYP93E2, and CYP72A63; (B) bAS, CPR, and CYP93E2 as controls. (C) The structures were deduced from mass spectra. bAS/CPR/CYP93E2 Relative ion intensity (%) Retention time (min) 20 β-Amyrin (1) bAS/CPR/CYP93E2/CYP72A63 A B

10tables and figures Peak 2 in (A) Authentic 24-OH-β-amyrin (6) Peak 19 in (A) C Peak 20 in (A) Peak 14 in (A) Peak 15 in (A) Authentic 30-OH-β-amyrin (14) Authentic 11-deoxo-glycyrrhetinic acid(15) Peak 21 in (A) Peak 22 in (A) Supplementary Fig. S7 (Cont.)