Supporting Information

Allylic hydroxylation of triterpenoids by a plant cytochrome P450 triggers key chemical transformations that produce a variety of bitter compounds

Shohei Takase1, Kota Kera2, Yoshiki Nagashima2, Kazuto Mannen2, Tsutomu Hosouchi2, Sayaka Shinpo2, Moeka Kawashima1, Yuki Kotake1, Hiroki Yamada1, Yusuke Saga1, Junnosuke Otaka1, Hiroshi Araya1, Masaaki Kotera3, Hideyuki Suzuki2*, and Tetsuo Kushiro1*

1School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan; 2Department of Research and Development, Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan; 3Development Department of Chemical System Engineering, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan

S1

Experimental

Classification of 27,127 total contigs for RNA seq analysis 27,127 total contigs derived from the M. charantia, transcriptome assembly were subjected to additional validation and annotation. BLASTx program based homology search was conducted against an NCBI non-redundant (nr) protein database for all unigenes, and best aligning results were selected to annotate the unigenes. In order to compare the gene expression patterns among the ten tissues, we performed a series of analysis as explained below. First, RNA-seq total genes from ten tissues obtained by three sequencers (GAIIx, HiSeq and Rapid) were independently normalized by centering and scaling using average and standard deviation, respectively. The average of the three normalized values were regarded as the relative expression level of the genes, and were used in the following analyses. Consequently, principal component analysis (PCA) and hierarchical clustering was carried out to overview the relationship among plant tissues in the viewpoint of expression levels. We used Scikit-learn, an open-source machine-learning library https://scikit-learn.org/. In the hierarchical clustering, we adopted the weighted method and correlation metric. The first principal component clearly separated leaves and others, whilst the second principal component clearly separated flowers and others. Plot of plant tissues on the first and the third principal component scores, clearly separated fruits and others. To sum up, the ten plant tissues were classified into four, i.e., leaves, flowers, fruits and others. Cumulative contribution rates of the principal components showed that the first three components gave as sufficient variance as about 80%. The hierarchical clustering provided the same conclusion as that of PCA, that is, the ten plant tissues were classified into four, i.e., leaves, flowers, fruits and others. In order to visualize the distribution of the annotation, we used KAAS (KEGG Automatic Annotation Server, https://www.genome.jp/kegg/kaas/) applying the SBH (single-directional best hit) approach against the default set of genome-sequenced organisms. The genes were grouped according to the annotations using KAAS, and their expression levels were calculated by summing up the normalized expression levels of the corresponding genes. We focused on the enzyme classification, CYP classification, glycosyltransferase classification and metabolic pathways. Expression levels of the annotated genes were classified according to the enzyme classification provided as the IUBMB's Enzyme List (also known as the EC numbers). Some oxidoreductases (such as EC 1.1, EC 1.11, and EC 1.14), transferases (such as EC 2.1, EC 2.3, EC 2.4, and EC 2.7), hydrolases (such as EC 3.1, EC 3.4, EC 3.6) and lyases (such as EC4.1 and EC 4.2) were highly expressed in most plant tissues. Among them, expression levels of some enzymes (such as EC 1.11, EC 2.3, EC 3.4, and EC 4.1) were shown to be tissue-specific. Among the CYP genes, CYP88 family genes were expressed significantly, but they depended on the plant tissues. CYP73, CYP74 and CYP75 family genes also showed relatively high expressions. Most glycosyltransferases belonged to those for storage polysaccharides and structural polysaccharides, but these also depended on plant tissues. Glycosyltransferases that act on hydrophobic molecules and trehalose were also shown to be expressed relatively high. Regarding metabolic pathways, leaves generally express genes for energy metabolism. Other significant expressions include those for carbohydrate metabolism and amino acid metabolism. It was also found that the expression levels differed significantly dependent on the plant tissues for energy metabolism and biosynthesis of secondary metabolism. Leaves were also found to highly express genes related to photosynthesis.

S2

Table S1 The list of prim

ers used in this study

S3

Nam

e (for sub-cloning)Sequence

VectorM

ulti cloning siteM

cCBS_Bam

HI-N

5'-GAG

AGG

ATCC

ATGTG

GAG

GTTAAAG

GTG

GG

AGC

-3'M

cCBS_SalI-C

5'-TTCC

GTC

GAC

TTATTCG

GTC

AAAACC

CTATG

GC

-3'C

YP81AQ19_N

otI_N5'-AAG

GG

CG

GC

CG

CATG

GAG

AATATTTTGC

TGTATTTC

TC-3'

CYP81AQ

19_NotI_C

5'-TAGTG

CG

GC

CG

CTC

ATTTTCTAAC

AAGAC

CAAC

TTC-3'

CYP88L7_N

otI_N5'-AAG

CTTG

CG

GC

CG

CATG

GAAC

TTTTGAG

CAATTTTG

GG

GC

C-3'

CYP88L7_SpeI_C

5'-AAGC

TTACTAG

TCTAATAAC

TTGG

GAG

CTTAG

TTATGTTG

G-3'

CYP88L8_N

otI_N5'-AG

CTG

CG

GC

CG

CATG

GAAATAC

TGAAC

AATTTTTGG

GC

TC-3'

CYP88L8_SpeI_C

5'-AGC

TACTAG

TCTAATAAC

TTTGG

AGTTTAG

TTATTTTGG

TGAG

-3'C

YP81AQ19_Bam

HI_N

5'-AAACC

CG

GATC

CATG

GAG

AATATTTTGC

TGTATTTC

TCAC

TCTC

-3'C

YP81AQ19_SalI_C

5'-AAACC

CG

TCG

ACTC

ATTTTCTAAC

AAGAC

CAAC

TTCTTC

CAC

C-3'

LjCR

P_NotI_N

5'-AAGG

GC

GG

CC

GC

ATGG

AAGAATC

AAGC

TCC

ATGAAG

-3'LjC

RP_PacI_C

5'-TTAATTAATCAC

CATAC

ATCAC

GC

AAATAC-3'

Nam

e (for RT-PC

R)

SequenceM

cCBS_Fw

5'-ACG

GC

AAGTG

TGG

GAG

TTCTG

-3'M

cCBS_R

v5'-TC

GG

AAGTTTG

CTTTC

GATG

G-3'

CYP81AQ

19_Fw5'-G

ATGTC

TCATTTG

CTC

AACAATC

CA-3'

CYP81AQ

19_Rv

5'-TCG

CAATG

TCTC

GG

CG

ATTA-3'C

YP88L7_Fw5'-G

GTG

GAG

GG

TTTGG

AGG

AA-3'C

YP88L7_Rv

5'-GAAAAC

AGG

CC

CC

AAGAAAAC

-3'M

cActin_Fw5'-C

ACTC

AACC

CAAAG

GC

TAACAG

AGA-3'

McActin_R

v5'-C

CATC

ACC

AGAATC

CAG

CAC

A-3'

pESC-U

RA

pESC-LEU

211121

Table S2 C

ontigs from R

NA

-seq analysis of M. charantia highly correlated w

ith McC

BS gene in

ConfeitoG

UIplus analysis. These contigs w

ere annotated by BLA

STX search.

S4

Feature IDR

ename

Description

M01391

McC

BSgb|AEM

42982.1| cucurbitadienol synthase [Siraitia grosvenorii]

M00873

CYP88L8

ref|XP_004164374.1| PRED

ICTED

: beta-amyrin 11-oxidase-like [C

ucumis sativus]

M01465

CYP81AQ

19ref|XP_004151921.1| PR

EDIC

TED: LO

W Q

UALITY PR

OTEIN

: cytochrome P450 81D

1-like [Cucum

is sativus]M

03390ref|XP_004133794.1| PR

EDIC

TED: cytochrom

e b5-like [Cucum

is sativus]M

03394ref|XP_004136172.1| PR

EDIC

TED: ABC

transporter C fam

ily mem

ber 4-like [Cucum

is sativus]M

04110C

YP88L7ref|XP_004164374.1| PR

EDIC

TED: beta-am

yrin 11-oxidase-like [Cucum

is sativus]M

04600em

b|CAN

72427.1| hypothetical protein VITISV_008825 [Vitis vinifera]M

05208ref|XP_004146353.1| PR

EDIC

TED: probable 1-deoxy-D

-xylulose-5-phosphate synthase 2, chloroplastic-like [Cucum

is sativus]M

05234ref|XP_004142907.1| PR

EDIC

TED: squalene m

onooxygenase-like [Cucum

is sativus]M

08299em

b|CAN

72427.1| hypothetical protein VITISV_008825 [Vitis vinifera]M

11752N

o hits foundM

12380N

o hits foundM

14136N

o hits foundM

14201ref|XP_004161745.1| PR

EDIC

TED: LO

W Q

UALITY PR

OTEIN

: uncharacterized protein C5H

10.03-like [Cucum

is sativus]M

16398ref|XP_004135271.1| PR

EDIC

TED: inorganic phosphate transporter 2-1, chloroplastic-like [C

ucumis sativus]

M19783

ref|XP_004134262.1| PRED

ICTED

: protein FEZ-like [Cucum

is sativus]M

23291N

o hits foundM

25354N

o hits foundM

25927N

o hits found

Figure S1 LC/MS-MS chromatogram of yeast extracts from expression of CYP81AQ19 and other CYP81As (CYP81A_1 ~ CYP 81A_6), identified from RNA-seq data of M. charantia, together with McCBS and LjCPR. Only CYP81AQ19 was shown to produce a hydroxylated product 2. DOS: dioxidosqualene, ES: ergosterol, OS: oxidosqualene, CB: cucurbitadienol.

0 10 20 30 40 50 60 70

Time (min)

0

100

0

100

0

100

0

100

0

100

0

100

0

10053.72

33.21 50.2263.7847.0423.18

53.85

47.1850.09

47.28 53.82

33.29 58.40

54.0147.28

50.38

53.8847.29

50.3333.289.27

53.8747.34

50.42 63.8058.4533.40

53.6547.07

50.26 58.2833.2127.39

CYP81AQ19

CYP81A_1!

CYP81A_2!

CYP81A_3!

CYP81A_4!

CYP81A_5!

CYP81A_6!

CBOSESDOS2

S5

DOS：dioxidosqualene!ES：ergosterol!OS：oxidosqualeneCB：cucurbitadienol

Figure S2 MS spectra of peaks obtained from LC/MS-MS analysis of yeast extracts from CYP81AQ19 expression with McCBS.

ES

DOS

CB

OS

CYP81AQ19product (2)

[M + H - H2O]+

[M + H]+=443.39O

O

[M + H]+=427.39

[M + H]+=397.35

[M + H]+=427.39

O

HO

H H

HOHH

S6

S7

Figure S3 Enlarged LC/MS-MS chromatogram (Fig. 3) of yeast extracts from expression of CYP81AQ19, CYP88L7, and co-expression of CYP88L7 and CYP81AQ19 along with McCBS. MS spectra of each circled peaks are shown in Fig. S3.

McCBS + CYP81AQ192

McCBS + CYP88L7

McCBS + CYP88L7 + CYP81AQ19

S8

Figure S4 MS spectra of peaks obtained from LC/MS-MS analysis of yeast extracts from expression of CYP88L7 and co-expression of CYP88L7 and CYP81AQ19. Each circled number corresponds to peaks shown in Fig. S2.

6

5

10 or 11

7

10 or 11

Cucurbita-5,24-diene-3β,23α-diol (2)Figure S5 Structure and 1H- and 13C-NMR assignments of cucurbita-5,24-diene-3β,23α-diol (2) in CDCl3. Arrows indicate a correlation observed by HMBC.

S9

HO

OHH H

H

1

2

3

4

56

7

89

10

19

11

12

14 15

161317

1820

30

21 22 23

24

2526

27

2829

Position 1H-NMR 13C-NMR

1 21.12 28.83 3.473, 1H, brs 76.64 41.45 141.26 5.582, 1H, d (J = 6.0 Hz) 121.57 24.38 43.69 34.410 37.811 32.312 34.713 46.314 49.215 30.416 28.117 50.918 0.874, 3H, s 15.419 0.909, 3H, s 28.020 32.621 0.962, 3H, d (J = 6.4 Hz) 18.722 44.423 4.460, 1H, td (J = 7.6, 2.8 Hz) 66.024 5.187, 1H, d (J = 8.7 Hz) 128.925 133.926 1.676, 3H, s 18.127 1.698, 3H, s 25.728 1.016, 3H, s 27.229 1.130, 3H, s 25.430 0.790, 3H, s 17.7

Figure S6 1H-NMR spectrum of cucurbita-5,24-diene-3β,23α-diol (2) measured in CDCl3.

Figure S7 13C-NMR spectrum of cucurbita-5,24-diene-3β,23α-diol (2) measured in CDCl3.

S10

HO

OH

(2)

HO

OH

(2)

PPM

5.0 4.0 3.0 2.0 1.0

5.5880

5.5761

5.1959

5.1785

4.4740

4.4602

4.4438

3.4726

1.6980

1.6779

1.6761

1.2473

1.1300

1.0155

1.0027

0.9679

0.9551

0.9092

0.8735

0.7901

PPM

140 130 120 110 100 90 80 70 60 50 40 30 20 10

141.

4180

134.

1460

129.

2014

121.

7538

77.

5113

77.

2518

77.

0000

76.

8779

66.

2026

51.

1931

49.

4381

46.

5842

44.

6994

43.

8066

41.

6624

38.

0302

34.

9322

34.

6651

32.

8871

32.

5209

30.

6895

29.

9493

29.

5678

29.

0947

28.

3622

28.

2859

27.

4617

25.

9890

25.

6991

24.

5774

21.

3725

18.

9307

18.

3736

17.

9845

15.

6418

14.

3828

Figure S8 HMBC spectrum of cucurbita-5,24-diene-3β,23α-diol (2) measured in CDCl3.

Figure S9 HMQC spectrum of cucurbita-5,24-diene-3β,23α-diol (2) measured in CDCl3.

S11

4 2

PPM

10050

PPM

4 2

PPM

140130

120110

10090

8070

6050

4030

20

PPM

Figure S10 Determination of absolute configuration of C23-hydroxyl group in cucurbita-5,24-diene-3β,23α-diol (2) using modified Mosher’s method. 1H-NMR of (S)- or (R)-MTPA esterified 2 were recorded in CDCl3.

OMTPA

+0.08+0.04

-0.13 -0.04

-0.01+0.003+0.070 -0.144

-0.028-0.005

18

2124

26

27

S12

Position !S-ester (ppm) ! -ester (ppm) "!SR (=!S-!R)18 0.8524 0.8497 0.002721 0.9656 0.8960 0.069624 5.0347 5.1785 -0.143826 1.6971 1.7246 -0.027527 1.7814 1.7860 -0.0046

Figure S11 1H-NMR spectrum of (R)-MTPA-conjugated cucurbita-5,24-diene-3β,23α-diol (2) measured in CDCl3.

Figure S12 1H-NMR spectrum of (S)-MTPA-conjugated cucurbita-5,24-diene-3β,23α-diol (2) measured in CDCl3.

S13

HO

O-(S)-MTPA

HO

O-(R)-MTPA

PPM

5.0 4.0 3.0 2.0 1.0

5.5871

5.5834

5.5807

5.5715

5.1785

4.8478

3.5450

3.4900

3.4726

3.3700

3.3673

1.7878

1.7860

1.7246

1.5744

1.3050

1.1337

1.1236

1.0201

1.0045

0.9908

0.9120

0.9065

0.8973

0.8799

0.8680

0.8561

0.8497

0.7901

0.7471

0.6683

PPM

5.0 4.0 3.0 2.0 1.0

5.5899

5.5862

5.5834

5.5743

5.0347

4.8505

3.5194

3.4928

3.4754

1.7814

1.7787

1.6999

1.6971

1.5771

1.3096

1.3078

1.2134

1.1914

1.1364

1.0228

1.0155

0.9715

0.9596

0.9147

0.8708

0.8589

0.8524

0.7929

0.7874

0.7801

Cucurbita-5,23,25-trien-3β-ol (3)

Figure S13 Structure and 1H- and 13C-NMR assignments of cucurbita-5,23,25-trien-3β-ol (3) in CDCl3. Arrows indicate a correlation observed by HMBC.

S14

HO

H H

HPosition 1H-NMR 13C-NMR

1 21.12 28.93 3.474, 1H, brs 76.64 41.45 141.26 5.587, 1H, d (J = 5.1 Hz) 121.47 24.48 43.69 34.510 37.811 32.312 34.813 46.314 49.215 30.316 27.917 50.418 0.876, 3H, s 15.419 0.912, 3H, s 28.020 36.721 0.887, 3H, d (J = 6.0 Hz) 18.822 39.823 5.622, 1H, m 129.524 6.109, 1H, d (J = 16.1 Hz) 134.025 142.226 4.848, 2H, brs 114.027 1.835, 3H, s 18.828 1.022, 3H, s 27.329 1.135, 3H, s 25.430 0.798, 3H, s 17.8

Figure S14 1H-NMR spectrum of cucurbita-5,23,25-trien-3β-ol (3) measured in CDCl3.

HO(3)

PPM

6.0 5.0 4.0 3.0 2.0 1.0

6.1249

6.0928

5.6522

5.6393

5.6219

5.6045

5.5917

5.5816

4.8478

4.3054

4.2917

4.2788

3.4735

1.8346

1.5725

1.2482

1.1346

1.0219

0.9120

0.8928

0.8808

0.8763

0.7975

Figure S15 13C-NMR spectrum of cucurbita-5,23,25-trien-3β-ol (3) measured in CDCl3.

S15

Cucurbita-5,23-dien-3β,25-diol (4)

Figure S16 Structure and 1H- and 13C-NMR assignments of cucurbita-5,23-dien-3β,25-diol (4) in CDCl3.

S16

HO

OH

H H

HPosition 1H-NMR 13C-NMR

1 21.12 28.83 3.474, 1H, brs 76.54 41.45 141.26 5.581, 1H, brs 121.47 24.38 43.69 34.410 37.811 30.312 34.713 46.214 49.115 29.916 28.017 50.118 0.850, 3H, s 15.419 0.913, 3H, s 27.820 32.321 0.874, 3H, d (J = 6.0 Hz) 18.722 39.123 5.581, 1H, brs 125.524 5.581, 1H, brs 139.325 70.826 1.306, 6H, s 29.727 1.306, 6H, s 29.828 1.020, 3H, s 27.229 1.134, 3H, s 25.430 0.790, 3H, s 17.8

Figure S17 1H-NMR spectrum of cucurbita-5,23-dien-3β,25-diol (4) measured in CDCl3.

Figure S18 13C-NMR spectrum of cucurbita-5,23-dien-3β,25-diol (4) measured in CDCl3.

S17

(Tho

usan

ds)

00.

10.

20.

30.

40.

50.

60.

70.

80.

91.

01.

11.

21.

31.

41.

51.

61.

71.

81.

92.

02.

12.

22.

32.

42.

5

X : parts per Million : 1H

7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

7.

260

7.

233

5.

581

3.

474

1.

306

1.

250

1.

134

1.

020

0.

913

0.

890

0.

880

0.

877

0.

868

0.

850

0.

790

(Tho

usan

ds)

010

.020

.0

X : parts per Million : 13C

160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

141

.178

139

.338

125

.464

121

.448

77.

495

77.

251

77.

000

76.

743

76.

468

70.

753

50.

118

49.

141

46.

225

43.

603

41.

415

39.

123

37.

784

36.

219

34.

740

34.

435

32.

259

30.

315

29.

936

29.

844

29.

691

28.

848

28.

035

27.

809

27.

234

25.

443

24.

343

21.

116

18.

677

17.

754

15.

413

HO

OH

(4)

HO

OH

(4)

Cucurbita-5,24-diene-3β,19-diol (5) Figure S19 Structure and 1H- and 13C-NMR assignments of cucurbita-5,24-diene-3β,19-diol (5) in CDCl3. Arrows indicate a correlation observed by HMBC.

S18

HO

HOH2C H

H

Position 1H-NMR 13C-NMR

1 17.92 29.33 3.475, 1H, brs 76.54 37.25 141.36 5.641, 1H, d (J = 6.0 Hz) 122.57 24.78 38.89 41.310 36.011 30.912 31.913 45.814 48.915 34.816 29.717 50.418 0.861, 3H, s 14.8

193.363, 1H, d (J = 10.5 Hz)3.534, 1H, d (J = 10.5 Hz) 68.9

20 35.721 0.886, 3H, d (J = 6.5 Hz) 18.622 36.323 24.824 5.073, 1H, t (J = 7.1 Hz) 125.125 130.926 1.580, 3H, s 17.627 1.662, 3H, s 25.728 1.002, 3H, s 27.029 1.122, 3H, s 26.430 0.810, 3H, s 20.2

Figure S20 1H-NMR spectrum of cucurbita-5,24-diene-3β,19-diol (5) measured in CDCl3.

Figure S21 13C-NMR spectrum of cucurbita-5,24-diene-3β,19-diol (5) measured in CDCl3.

S19

abun

danc

e0

100.

020

0.0

300.

040

0.0

500.

060

0.0

700.

080

0.0

900.

0

X : parts per Million : 1H

6.0 5.0 4.0 3.0 2.0 1.0

5.

647

5.

635

5.

073

4.

276

4.

259

3.

544

3.

523

3.

475

3.

373

3.

352

2.

162

1.

662

1.

580

1.

232

1.

122

1.

002

0.

892

0.

879

0.

861

0.

810

(Tho

usan

ds)

010

.020

.030

.040

.050

.060

.070

.0

X : parts per Million : 13C

140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0

141

.301

130

.977

130

.922

125

.128

122

.536

77.

251

77.

000

76.

743

76.

456

68.

883

50.

400

48.

920

45.

785

41.

305

37.

179

36.

348

35.

724

31.

880

30.

938

29.

667

29.

331

26.

978

25.

706

24.

771

24.

710

22.

656

18.

598

17.

883

17.

589

14.

759

HO

HOH2C

(5)

HO

HOH2C

(5)

Figure S22 HMBC spectrum of cucurbita-5,24-diene-3β,19-diol (5) measured in CDCl3.

Figure S23 HMQC spectrum of cucurbita-5,24-diene-3β,19-diol (5) measured in CDCl3.

S20

X : parts per Million : 1H5.0 4.0 3.0 2.0 1.0

Y :

parts

per

Mill

ion

: 13C

150.

014

0.0

130.

012

0.0

110.

010

0.0

90.0

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

(Thousands)0 20.0 40.0 60.0

(Tho

usan

ds)

01.

0

X : parts per Million : 1H5.0 4.0 3.0 2.0 1.0

Y :

parts

per

Mill

ion

: 13C

160.

015

0.0

140.

013

0.0

120.

011

0.0

100.

090

.080

.070

.060

.050

.040

.030

.020

.010

.0

(Thousands)0 20.0 40.0 60.0

(Tho

usan

ds)

01.

0

5β,19-Epoxy-cucurbita-6,24-dien-3β-ol (6)

Figure S24 Structure and 1H- and 13C-NMR assignments of 5β,19-epoxy-cucurbita-6,24-dien-3β-ol (6) in CDCl3. Arrows indicate a correlation observed by HMBC.

S21

HOO

H

H

Position 1H-NMR 13C-NMR

1 17.62 23.53 3.404, 1H, brs 76.24 37.15 87.56 6.039, 1H, d (J = 10.0 Hz) 132.17 5.637, 1H, dd (J = 9.5, 3.5 Hz) 131.68 52.09 45.410 38.711 27.312 31.013 45.214 48.515 33.116 28.017 50.318 0.863, 3H, s 14.8

193.513, 1H, d (J = 8.5 Hz)3.670, 1H, d (J = 8.5 Hz) 79.8

20 35.721 0.884, 3H, d (J = 7.0 Hz) 18.522 36.323 24.824 5.092, 1H, t (J = 7.1 Hz) 125.025 131.126 1.602, 3H, s 17.527 1.682, 3H, s 25.728 0.896, 3H, s 24.529 1.200, 3H, s 20.530 0.855, 3H, s 20.0

Figure S25 1H-NMR spectrum of 5β,19-epoxy-cucurbita-6,24-dien-3β-ol (6) measured in CDCl3.

Figure S26 13C-NMR spectrum of 5β,19-epoxy-cucurbita-6,24-dien-3β-ol (6) measured in CDCl3.

S22

(Tho

usan

ds)

00.

10.

20.

30.

40.

50.

60.

70.

80.

91.

0

X : parts per Million : 1H

6.0 5.0 4.0 3.0 2.0 1.0

6.

049

6.

029

5.

650

5.

643

5.

631

5.

624

5.

092

3.

678

3.

661

3.

521

3.

504

3.

404

1.

682

1.

602

1.

249

1.

200

0.

896

0.

891

0.

877

0.

863

0.

855

(Tho

usan

ds)

-5.0

-3.0

-1.0

1.0

3.0

5.0

7.0

9.0

11.0

13.0

15.0

17.0

19.0

X : parts per Million : 13C

140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0

132

.102

131

.594

131

.063

125

.024

87.

507

79.

824

77.

257

77.

000

76.

743

76.

163

51.

970

50.

320

48.

535

45.

418

45.

186

35.

669

33.

108

31.

910

30.

944

29.

698

29.

478

29.

423

29.

355

27.

283

24.

826

24.

502

23.

549

22.

681

20.

462

17.

540

14.

124

HOO (6)

HOO (6)

Figure S27 HMBC spectrum of 5β,19-epoxy-cucurbita-6,24-dien-3β-ol (6) measured in CDCl3.

Figure S28 HMQC spectrum of 5β,19-epoxy-cucurbita-6,24-dien-3β-ol (6) measured in CDCl3.

S23

X : parts per Million : 1H5.0 4.0 3.0 2.0 1.0

Y :

parts

per

Mill

ion

: 13C

150.

014

0.0

130.

012

0.0

110.

010

0.0

90.0

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

(Thousands)0 20.0 40.0 60.0

(Tho

usan

ds)

01.

0

X : parts per Million : 1H5.0 4.0 3.0 2.0 1.0

Y :

parts

per

Mill

ion

: 13C

160.

015

0.0

140.

013

0.0

120.

011

0.0

100.

090

.080

.070

.060

.050

.040

.030

.020

.010

.0

(Thousands)0 20.0 40.0 60.0

(Tho

usan

ds)

01.

0

Figure S29 1H-NMR spectrum of semi-purified sample containing mixture of (7), (8), and (9) in CDCl3.

Figure S30 13C-NMR spectrum of semi-purified sample containing mixture of (7), (8), and (9) in CDCl3.

S24

PPM

9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

9.7318

5.8949

5.8830

5.6567

5.6457

5.5779

5.3379

5.1932

5.1758

4.4712

4.4538

4.4346

4.2743

4.1808

4.0901

3.9738

3.9628

3.7090

3.5688

3.5542

3.5322

3.4836

3.3829

3.3609

1.7035

1.6990

1.6962

1.6742

1.6083

1.5899

1.2995

1.2427

1.1878

1.1438

1.1310

1.0457

1.0109

0.9697

0.9569

0.9019

0.8845

0.8708

0.8570

0.8149

0.7370

PPM

140 130 120 110 100 90 80 70 60 50 40 30 20 10

141.

3112

139.

5409

134.

9167

134.

8099

133.

8026

129.

9720

128.

9114

128.

8351

125.

0732

125.

0197

124.

3940

123.

7836

122.

5779

78.

2285

77.

2518

77.

0000

76.

7482

76.

4964

76.

1148

70.

7429

69.

0336

66.

1263

65.

9203

65.

8287

50.

9642

50.

6895

49.

8501

49.

0184

47.

6830

47.

4770

45.

9356

45.

2412

44.

4018

44.

3026

41.

3496

41.

3267

39.

6098

38.

9993

38.

8543

37.

1985

36.

7788

36.

5041

36.

1989

36.

0920

34.

7872

34.

6193

32.

5896

32.

5438

31.

9028

29.

9112

29.

6899

29.

3465

29.

3007

29.

0871

28.

8582

28.

3469

28.

1714

28.

0188

27.

6754

27.

3855

27.

1871

27.

0039

26.

4240

26.

3553

25.

7220

25.

4549

25.

3633

25.

2031

24.

7529

23.

3794

23.

1581

22.

6773

21.

1512

20.

9681

20.

2508

19.

6174

18.

6789

18.

1066

17.

8471

15.

9547

15.

8860

14.

8483

14.

1234

HO

HOH2COH

(7)

HO

OHC

OH

OH

(8)

HO

OHC

OH

OH

(9)

HO

HOH2COH

(7)

HO

OHC

OH

OH

(8)

HO

OHC

OH

OH

(9)

Figure S31 HMBC spectrum of semi-purified sample containing mixture of (7), (8), and (9) in CDCl3.

Figure S32 HMQC spectrum of semi-purified sample containing mixture of (7), (8), and (9) in CDCl3.

S25

9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

PPM

140130

120110

10090

8070

6050

4030

2010

0

PPM

7.5 5.0 2.5

PPM

140130

120110

10090

8070

6050

4030

2010

PPM

Cucurbita-5,24-diene-3β,19,23α-triol (7)

Figure S33 Structure and 1H- and 13C-NMR assignments of cucurbita-5,24-diene-3β,19,23α-triol (7) in CDCl3. Arrows indicate a correlation observed by HMBC.

Figure S34 1H-NMR spectrum of cucurbita-5,24-diene-3β,19,23α-triol (7) measured in CDCl3.

S26

HO

HOH2COH

H

H

PPM

6.0 5.0 4.0 3.0 2.0 1.0

5.6641

5.6531

5.1977

5.1813

4.4822

4.4767

4.4593

4.4456

4.4401

4.2898

3.5597

3.5386

3.4873

3.3957

3.3746

2.4007

2.3787

2.2514

2.2358

2.0105

1.7026

1.6816

1.6128

1.5945

1.1364

1.0173

0.9752

0.9624

0.9092

0.8213

HO

HOH2COH

(7)

Position 1H-NMR 13C-NMR

1 18.12 28.93 3.487, 1H, brs 76.54 41.45 141.46 5.659, 1H, d (J = 5.5 Hz) 122.67 25.58 38.99 49.010 37.211 27.012 29.913 46.014 51.015 34.816 28.017 51.018 0.909, 3H, s 14.8

193.385, 1H, d (J = 10.6 Hz)3.549, 1H, d (J = 10.6 Hz) 69.0

20 32.621 0.969, 3H, d (J = 6.4 Hz) 20.222 44.423 4.461, 1H, td (J = 9.2, 2.8 Hz) 65.924 5.190, 1H, d (J = 8.2 Hz) 128.925 134.126 1.703, 3H, s 26.427 1.681, 3H, s 17.928 1.017, 3H, s 28.029 1.136, 3H, s 25.730 0.821, 3H, s 18.7

Figure S35 13C-NMR spectrum of cucurbita-5,24-diene-3β,19,23α-triol (7) measured in CDCl3.

Figure S36 HMBC spectrum of cucurbita-5,24-diene-3β,19,23α-triol (7) measured in CDCl3.

S27

PPM

140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0

141.

1052

128.

6748

122.

3414

99.

6249

77.

0000

76.

7482

76.

4887

76.

2217

68.

7284

65.

6761

50.

7276

48.

7666

45.

6990

44.

1653

41.

1054

38.

6254

36.

9619

35.

8479

34.

5507

32.

3530

29.

6747

28.

5987

27.

7899

26.

7369

26.

1417

25.

4778

25.

2031

19.

9914

18.

4423

17.

8624

17.

6106

14.

5965

HO

HOH2COH

(7)

4 2

PPM

10050

PPM

3β,7,23α-Trihydroxy-cucurbita-5,24-dien-19-al (8)

Figure S37 Structure and 1H- and 13C-NMR assignments of 3β,7,23α-trihydroxy-cucurbita-5,24-dien-19-al (8) in CDCl3. Arrows indicate a correlation observed by HMBC.

Figure S38 1H-NMR spectrum of 3β,7,23α-trihydroxy-cucurbita-5,24-dien-19-al (8) measured in CDCl3.

S28

HO

OHCOH

OH

H

H

PPM

9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

9.7015

7.2600

5.8885

5.1977

5.1813

4.4612

3.9729

3.9628

3.5679

3.4671

2.0123

1.7054

1.6834

1.5689

1.2409

1.0549

0.9926

0.9798

0.8387

0.7498

HO

OHC

OH

OH

(8)

Position 1H-NMR 13C-NMR

1 21.22 28.33 3.568, 1H, brs 76.54 41.35 145.76 5.889, 1H, d (J = 3.2 Hz) 124.47 3.968, 1H, d (J = 5.1 Hz) 66.18 47.79 49.910 36.511 22.712 31.913 45.914 49.015 34.816 28.217 51.018 0.912, 3H, s 14.819 9.702, 1H, s 187.820 32.621 0.986, 3H, d (J = 6.2 Hz) 18.722 44.423 4.461, 1H, td (J = 9.2, 2.8 Hz) 65.924 5.190, 1H, d (J = 8.2 Hz) 128.925 133.826 1.705, 3H, s 25.727 1.683, 3H, s 18.728 1.055, 3H, s 27.429 1.241, 3H, s 25.530 0.750, 3H, s 18.1

3β,7,25-Trihydroxy-cucurbita-5,23-dien-19-al (9)

Figure S39 Structure and 1H- and 13C-NMR assignments of 3β,7,25-trihydroxy-cucurbita-5,23-dien-19-al (9) in CDCl3. Arrows indicate a correlation observed by HMBC.

Figure S40 1H-NMR spectrum of 3β,7,25-trihydroxy-cucurbita-5,23-dien-19-al (9) measured in CDCl3.

S29

HO

OHC

OH

OH

H

H

PPM

9 8 7 6 5 4 3 2 1

9.7070

7.2600

5.8968

5.8904

5.5917

5.5880

5.5816

5.5725

3.9747

3.9646

3.5697

2.0957

2.0095

1.3069

1.2418

1.0558

0.9074

0.8955

0.8854

0.7471

HO

OHC

OH

OH

(9)

Position 1H-NMR 13C-NMR

1 21.22 28.33 3.570, 1H, brs 76.54 41.35 145.76 5.894, 1H, d (J = 3.2 Hz) 124.47 3.970, 1H, d (J = 5.1 Hz) 66.18 47.79 49.910 36.511 22.712 31.913 45.914 49.015 34.816 28.217 51.018 0.885, 3H, s 14.119 9.707, 1H, s 187.820 36.221 0.901, 3H, d (J = 6.0 Hz) 18.722 39.023 5.582, 1H, brs 125.124 5.588, 1H, brs 139.525 70.726 1.307, 6H, s 29.927 1.307, 6H, s 29.928 1.056, 3H, s 27.429 1.242, 3H, s 25.530 0.747, 3H, s 18.1

5β,19-Epoxy-cucurbita-6,24-diene-3β,23α-diol (10) Figure S41 Structure and 1H- and 13C-NMR assignments of 5β,19-epoxy-cucurbita-6,24-diene-3β,23α-diol (10) in CDCl3. Arrows indicate a correlation observed by HMBC.

5β,19-Epoxy-cucurbita-6,23-diene-3β,25-diol (11) Figure S42 Structure and 1H- and 13C-NMR assignments of 5β,19-epoxy-cucurbita-6,23-diene-3β,25-diol (11) in CDCl3. Arrows indicate a correlation observed by HMBC.

S30

HOO

OHH

H

HOO

OH

H

H

Position 1H-NMR 13C-NMR

1 17.62 23.53 3.405, 1H, brd 76.14 37.25 87.56 6.038, 1H, d (J = 10.1 Hz) 131.77 5.634, 1H, dd (J = 9.6, 3.7 Hz) 131.58 52.09 45.410 38.811 24.512 30.713 45.214 48.615 33.116 27.917 50.818 0.862, 3H, s 14.9

193.511, 1H, d (J = 8.3 Hz)3.669, 1H, d (J = 8.3 Hz) 79.8

20 32.521 0.972, 3H, d (J = 6.5 Hz) 18.622 44.423 4.469, 1H, td (J = 7.6, 2.8 Hz) 65.924 5.195, 1H, d (J = 8.2 Hz) 128.925 133.926 1.708, 3H, s 25.727 1.687, 3H, s 18.128 0.893, 3H, s 24.529 1.199, 3H, s 20.530 0.858, 3H, s 20.0

Position 1H-NMR 13C-NMR

1 17.62 23.53 3.405, 1H, brd 76.14 37.25 87.56 6.038, 1H, d (J = 10.1 Hz) 131.77 5.634, 1H, dd (J = 9.6, 3.7 Hz) 131.58 52.09 45.410 38.811 24.512 30.713 45.214 48.615 33.116 27.917 50.018 0.862, 3H, s 14.9

193.511, 1H, d (J = 8.3 Hz)3.669, 1H, d (J = 8.3 Hz) 79.8

20 36.121 0.883, 3H, d (J = 6.0 Hz) 18.622 39.023 5.585, 1H, brs 125.224 5.592, 1H, brs 139.525 70.726 1.311, 6H, s 29.727 1.311, 6H, s 29.728 0.893, 3H, s 24.529 1.199, 3H, s 20.530 0.858, 3H, s 20.0

Figure S43 1H-NMR spectrum of the mixture of (10) and (11) measured in CDCl3.

Figure S44 13C-NMR spectrum of the mixture of (10) and (11) measured in CDCl3.

S31

PPM

6.0 5.0 4.0 3.0 2.0 1.0

6.0479

6.0278

5.6476

5.6402

5.6283

5.6210

5.5917

5.5853

5.5761

5.2032

5.1868

4.4831

4.4685

4.4520

4.0260

4.0205

4.0058

4.0013

3.6769

3.6604

3.6440

3.5194

3.5029

3.4149

3.3948

1.7081

1.6871

1.3114

1.3096

1.2501

1.1987

0.9789

0.9660

0.8928

0.8891

0.8616

0.8579

PPM

140 130 120 110 100 90 80 70 60 50 40 30 20 10

139.

5256

131.

7195

131.

6889

131.

5363

128.

8961

125.

2181

87.

4922

79.

8233

77.

2518

77.

0000

76.

7482

76.

1301

65.

8745

51.

9714

51.

9485

50.

8039

49.

9722

48.

5682

45.

4243

45.

3175

45.

2030

44.

3789

39.

0451

38.

7780

37.

1527

36.

1378

33.

1390

33.

0932

32.

5438

31.

9181

30.

8269

30.

7048

29.

9722

29.

8502

29.

6975

28.

2325

27.

9425

27.

3244

25.

7372

24.

5240

23.

5472

20.

4721

20.

0142

19.

9913

18.

5720

18.

1218

17.

5572

14.

9093

14.

8864

HO

OH

O (11)

HOO

OH

(10)

HO

OH

O (11)

HOO

OH

(10)

Figure S45 HMBC spectrum of the mixture of (10) and (11) measured in CDCl3.

Figure S46 HMQC spectrum of the mixture of (10) and (11) measured in CDCl3.

S32

6 4 2

PPM

150140

130120

110100

9080

7060

5040

3020

10

PPM

6 4 2

PPM

10050

PPM

Cucurbita-5,24-diene-3β,7β-diol (12)

Figure S47 Structure and 1H- and 13C-NMR assignments of cucurbita-5,24-diene-3β,7β-diol (12) in CDCl3. Arrows indicate a correlation observed by HMBC.

Figure S48 Stereochemistry of C7 hydroxy group of cucurbita-5,24-diene-3β,7β-diol (12) determined by NOE measurements. Arrows indicate the observed NOE effects.

S33

H

HO

H3C

CH3

H

H

H

HOH3C

H

H3C

CH3

7

HO OH

H H

H

Position 1H-NMR 13C-NMR

1 21.02 28.73 3.537, 1H, brs 76.74 41.55 146.66 5.811, 1H, d (J = 5.5 Hz) 122.57 3.936, 1H, d (J = 5.5 Hz) 68.18 53.09 33.810 38.511 32.612 30.013 45.814 48.115 34.616 27.817 50.118 0.881, 3H, s 15.319 1.054, 3H, s 29.720 35.821 0.889, 3H, d (J = 7.0 Hz) 18.622 36.423 24.824 5.078, 1H, t (J = 7.1 Hz) 125.125 131.026 1.586, 3H, s 17.627 1.668, 3H, s 25.428 1.019, 3H, s 27.629 1.192, 3H, s 25.730 0.683, 3H, s 17.8

Figure S49 1H-NMR spectrum of cucurbita-5,24-diene-3β,7β-diol (12) measured in CDCl3.

Figure S50 13C-NMR spectrum of cucurbita-5,24-diene-3β,7β-diol (12) measured in CDCl3.

S34

PPM

10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

10.0

341

7.2

600

5.8

161

5.8

052

5.0

924

5.0

777

5.0

640

3.9

417

3.9

307

3.5

368

1.9

876

1.6

678

1.6

458

1.6

046

1.5

854

1.2

620

1.2

418

1.1

914

1.0

531

1.0

183

0.8

964

0.8

808

0.8

689

0.6

820

PPM

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20

185.

2638

146.

6221

130.

9564

125.

0808

122.

4940

77.

2518

77.

0000

76.

7482

68.

1255

53.

0321

50.

1324

48.

0951

45.

8135

41.

4564

38.

5415

36.

3515

35.

7563

34.

5506

33.

8334

32.

6125

30.

0409

29.

6670

29.

5755

28.

6521

27.

7594

27.

6296

25.

6991

25.

3710

24.

7834

20.

9757

18.

6026

17.

7937

17.

5953

15.

2756

HO OH(12)

HO OH(12)

Figure S51 HMBC spectrum of cucurbita-5,24-diene-3β,7β-diol (12) measured in CDCl3.

Figure S52 HMQC spectrum of cucurbita-5,24-diene-3β,7β-diol (12) measured in CDCl3.

S35

10.0 7.5 5.0 2.5

PPM

190180

170160

150140

130120

110100

9080

7060

5040

3020

PPM

10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

PPM

180170

160150

140130

120110

10090

8070

6050

4030

20

PPM

Figure S53 NOE difference spectrum (radiation at 3.94 ppm) of cucurbita-5,24-diene-3β,7β-diol (12) measured in CDCl3.

Figure S54 NOE difference spectrum (radiation at 0.68 ppm) of cucurbita-5,24-diene-3β,7β-diol (12) measured in CDCl3.

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HO OH(12)

HO OH(12)

Figure S55 Phylogenetic tree of three P450s identified in this study (denoted in red) with other related P450s involved in triterpene oxidations. Accession numbers are; CYP716A47 (JN604537), CYP716A53v2 (JX036031), CYP716E26 (XM_004241773), CYP716A12 (DQ335781), CYP716A94 (ALO23117), CYP716Y1 (KC963423), CYP51H10 (DQ680852), CYP714E19 (KF004520), CYP72A61 (DQ335793), CYP72A154 (AB558153), CYP72A397 (ALO23113), CYP93E1 (NC016095), CYP88D6 (AB433179), CYP87D16 (KF318735), CYP87D18 (HQ128571). The followings are from CuGenDB (http://cucurbitgenomics.org/). CYP81Q58 (Csa6G088160), Cl180 (Cla007079), Cm180 (Melo3C022375), CYP88L2 (Csa3G903540), Cl890 (Cla008355), Cm890 (Melo3C002192), CYP87D20 (Csa1G044890).

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