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Supplementary Table 1 Genes potentially implicated in Medicago truncatula triterpenoid biosynthesis correlated with bAS

Name Probeset ID Correlation

bAS Mtr.32384.1.S1_s_at 0.9929

CYP72A68 Mtr.37298.1.S1_at 0.9036

CYP72A67 Mtr.37299.1.S1_at 0.9027

Anthocyanidin 3-O-glucosyltransferase Mtr.40639.1.S1_at 0.847

CYP72A67 Mtr.28860.1.S1_at 0.827

bAS Mtr.32384.1.S1_at 0.8215

CYP72A65 Mtr.24379.1.S1_at 0.7903

Cytokinin-O-glucosyltransferase Mtr.13275.1.S1_at 0.7838

CYP716A12 Mtr.31199.1.S1_s_at 0.7525

Anthocyanidin 3-O-glucosyltransferase Mtr.41162.1.S1_at 0.7504

Cytokinin-O-glucosyltransferase Mtr.43628.1.S1_at 0.7391

CYP716A12 Mtr.43018.1.S1_at 0.6951

CYP89A2 Mtr.51061.1.S1_x_at 0.6693

CYP89A2 Mtr.51061.1.S1_at 0.6693

UGT73K1 Mtr.37228.1.S1_at 0.5747

CYP72A61 Mtr.43117.1.S1_at 0.5452

CYP93E2 Mtr.8618.1.S1_at 0.4834

P450s correlated with bAS (Mtr.18630.1.S1_at) based on a Pearson correlation coefficient cut-off 0.4.

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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) 1.07

24-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.

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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 (http://mtgea.noble.org/v2/correlation_search_form.php). Expression profiles for bAS, CYP93E2, CYP72A61, and UGT73K1 are shown. Corresponding probe IDs and correlation values are indicated in Table 1.

CYP93E2bAS CYP72A61 UGT73K1

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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 (http://mtgea.noble.org/v2/correlation_search_form.php). Expressions of bAS, CYP716A12, CYP72A68, and UGT73F3 are shown. Corresponding probe IDs are indicated in Table 2.

CYP716A12bAS CYP72A68 UGT73F3

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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)

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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

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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

Rel

ativ

e io

n in

tens

ity (%

)

Retention time (min)

bAS/CPR/CYP716A12/CYP72A63A

B

14 15 16 1817

19 20 21 23 2422 2518 26

54

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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.)

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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

Rel

ativ

e io

n in

tens

ity (%

)

Retention time (min)

20

β-Amyrin (1)bAS/CPR/CYP93E2/CYP72A63

A

B

21

22

19 20 21 23 2422 2518

2 14

17 26

15

27 28

19

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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.)