Supporting Information for

Qualitative metabolomics-based discovery of a phenolic UDP-

xylosyltransferase with a broad substrate spectrum from Lentinus brumalis

Eunah Jeonga,b, Wonyong Kimc, Seungju Sona, Sungyeon Yanga, Dasom Gwona,b, Jihee Honga,b,
Yoonhee Chod, Chang-Young Janga,b, Martin Steineggerd,e, Young Woon Limd,f, Kyo Bin Kanga,b,1
a
College of Pharmacy, Sookmyung Women's University, Seoul 04310, Korea
b
Research Institute of Pharmaceutical Sciences and Muscle Physiome Research Center, Sookmyung
Women's University, Seoul 04310, Korea
c
Korean Lichen Research Institute, Sunchon National University, Suncheon 57922, Korea
d
School of Biological Sciences, Seoul National University, Seoul 08826, Korea
e
Artificial Intelligence Institute and Institute of Molecular Biology and Genetics, Seoul National University,
Seoul 08826, Korea
f
Institute of Microbiology, Seoul National University, Seoul 08826, Korea
1
Corresponding author: Kyo Bin Kang
Email: kbkang@sookmyung.ac.kr

This PDF file includes:

SI Results
SI Materials and Methods
Figures S1 to S11
Tables S1 to S5
Legends for Datasets S1 to S3
SI References

Other supporting materials for this manuscript include the following:

Datasets S1 to S3

1
SI Results

Isolation of compounds 2a–2g, 11a, 12a, and 12b. A total of 8 L culture of L. brumalis

supplemented with 1 (357.1 mg). After 5 days of culture, the fermentation broth was collected and

extracted twice with equal volumes of EtOAc. The extract was fractionated on a preparative

Waters 600 HPLC system with a Waters 996 Photodiode Array Detector using a Hector-M C18

column (250 × 21.1 mm, 5 μm) and a Spursil C18-EP (250 × 10.0 mm, 5 μm). The mobile phase

was 0.1 % formic acid and acetonitrile (MeCN). The crude extract (859.7 mg) was purified with a

flow rate set to 12 mL/min, and a linear gradient of B 35% to 100% was applied to yield nine

fractions A−I and a purified compound 2c (24.4 mg, tR = 21.5 min). Compounds 2a (0.8 mg), 2d

(1.5 mg) and 2f (1.3 mg) were precipitated from fractions D, G and H, respectively, due to their

low solubility in MeOH. Fraction I was purified by preparative HPLC (Spursil C18-EP, 4 mL/min,

MeCN-0.1 % formic acid in H2O, 37:63 in isocratic mode) to yield 2b (1.2 mg, tR 10.5 min).

Compound 2e (3.9 mg, tR 21.5 min) was isolated from fraction A via preparative HPLC (Spursil

C18-EP, 4 mL/min, MeCN-0.1 % formic acid in H2O, 25:75 → 35:65). Fraction E was purified by

preparative HPLC (Spursil C18-EP, 4 mL/min, MeCN-0.1 % formic acid in H2O, 25:75 → 34:66)

to afford 2g (0.6 mg, tR 23.0 min).

Cultures (2 L) of L. brumalis were supplemented with magnolol (55.9 mg) and fermented for 5

days, then extracted with EtOAc. The crude extract (153.4 mg) was purified on a preparative HPLC

(Hector-M C18 column, 4 mL/min, MeCN-0.1 % formic acid in H2O, 20:80 → 100:0) to yield 11a

(25.2 mg, tR 22.5 min).

Cultures (2 L) of L. brumalis supplemented with honokiol (55.9 mg) were fermented for 5 days,

then extracted with EtOAc. The total 137.9 mg of crude extract were purified on a preparative

2
HPLC (Hector-M C18 column, 4 mL/min, MeCN-0.1 % formic acid in H2O, 50:80 → 100:0) to

yield compounds 12a (1.2 mg, tR 10.5 min) and 12b (0.7 mg, tR 23.5 min).

Structural elucidation of the isolated metabolites. The MS/MS spectra of 2a–2d exhibited

neutral losses of 132.04 Da, which was suggested to be a loss of pentose moiety. From the m/z

values of the fragment ion yielded by the loss of a pentose from the precursor ions (2a, m/z

269.0439; 2b, m/z 253.0492; 2c, m/z 283.0603; 2d, m/z 299.0544), the aglycone structures of 2a–

2d were deduced to be baicalein (2a), chrysin (2b), oroxylin A (2c), and 5,8-dihydroxy-6-

methoxyflavone (2d). These were confirmed by comparison of 1H and 13

C NMR data with the

references (1–3).

The 1H-1H COSY and HMBC NMR spectra of 2a suggested that the anomeric proton signal at

δH 5.02 (d, J = 7.5 Hz) was associated with the 13C signals of the sugar moiety at δC 101.3 (CH),

72.9 (CH), 75.7 (CH), 69.3 (CH), and 65.9 (CH2), which were consistent with the presence of a β-

xylopyranosyl unit in 2a (4–6). The acid hydrolysis of 2a followed by the TLC analysis (Fig S2)

confirmed the identification of the xylosyl moiety. The HPLC analysis after arylthiocarbamoyl-

thiazolidine derivatization (7) also confirmed the identification of the xylosyl moiety, and it further

suggested that the absolute configuration of the xylose to be D-form (Fig S3). The HMBC

correlation from the anomeric proton to C-7 (δC 151.5) revealed the substitution of the xylosyl

moiety at C-7. Taken together, compound 2a was determined to be baicalein 7-O-β-D-

xylopyranoside, which is a previously unknown compound. Using the same method, the pentosyl

moieties of 2b, 2c, and 2d were also identified as β-D-xylopyranosyl group, and the anomeric

proton in each compound showed HMBC correlation to C-7. Thus, compound 2b was determined

3
as chrysin 7-O-β-D-xylopyranoside, while 2c was oroxylin A 7-O-β-D-xylopyranoside and 2d was

5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranoside.

The m/z values of the deprotonated molecular ions and two successive steps of 132.04 Da neutral

losses in MS/MS spectra suggested compounds 2e–2g as dipentosylated derivatives. Similar to the

cases of 2a–2d, the aglycones of 2e–2g were identified as baicalein (2e), oroxylin A (2f), and 5,8-

dihydroxy-6-methoxyflavone (2g) based on the MS/MS fragment ions, and 1H and 13

C NMR

spectral data.

In the 1H-1H COSY and HMBC NMR spectra of 2e, an anomeric proton signal at δH 4.93 (d, J

= 6.6 Hz) formed a spin system of a pentose moiety with the 13C signals at δC 103.7 (CH), 73.3

(CH), 75.4 (CH), 69.4 (CH), and 65.6 (CH2), while another anomeric proton at δH 5.08 (d, J = 7.2

Hz) was associated with the 13C resonances at δC 101.3 (CH), 73.0 (CH), 75.6 (CH), 69.2 (CH),

and 65.9 (CH2). These NMR resonances suggested the presence of two β-D-xylopyranosyl units in

2e, and it was confirmed by the TLC and HPLC analysis on the hydrolysate of 2e Figs S2 and S3).

The HMBC spectrum showed HMBC correlations from the anomeric proton at δH 4.93 to C-6 (δC

132.6), and from the other anomeric proton at δH 5.08 to C-7 (δC 156.0), suggesting compound 2e

to be baicalein 6,7-di-O-β-D-xylopyranoside. Differently from 2e, compounds 2f and 2g exhibited

HMBC correlations from an anomeric proton (δH 4.48, 2f; 4.54, 2g) to C-3 of the other xylosyl

moiety (δC 85.4, 2f and 2g), which suggested a presence of β-D-xylopyranosyl-(1→3)-β-D-

xylopyranosyl group in each compound. The positions of the dixylosyl group were confirmed as

C-7 in both 2f and 2g by the HMBC correlations from the anomeric protons to C-7. Accordingly,

compound 2f was determined as oroxylin A 7-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranoside

while 2g was 5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranosyl-(1→3)-β-D-

xylopyranoside.
4
 The MS/MS spectra of 11a, 12a, and 12b also exhibited neutral losses of 132.04 Da, suggesting

the presence of the xylose moiety. The fragment ion of m/z 265.1227 (calcd. for [C18H17O2]−,

265.1230) were observed in MS/MS spectra of 11a, 12a, and 12b, which suggested that the

aglycone structures of 11a, 12a and 12b were same as the originally supplemented compounds,

magnolol (11a) and honokiol (12a, 12b). These were confirmed by comparison of 1H and 13
C

NMR data with the ones of the reference standards.

The 1H-1H COSY and HMBC NMR analysis showed the association of five 13
C NMR

resonances δC 102.9 (CH), 74.4 (CH), 77.1 (CH), 70.8 (CH), and 66.7 (CH2) with an anomeric

proton signal at δH 4.96 (d, J = 7.2 Hz), which suggested the presence of a β-D-xylopyranosyl

moiety in 11a. It was confirmed by the TLC analysis (Fig S4) and the arylthiocarbamoyl-

thiazolidine derivatization coupled with HPLC (Fig S5) on the hydrolysate of 11a. The HMBC

correlation from the anomeric proton to C-2 (δC 153.1) suggested the presence of the xylosyl

moiety at C-2. Taken together, compound 11a was determined as magnolol 2-O-β-D-

xylopyranoside. With the same method, compounds 12a and 12b were identified as honokiol 4′-

O-β-D-xylopyranoside (12a) and honokiol 2-O-β-D-xylopyranoside (12b), respectively.

Phylogenetic analysis on the UGTs annotated from the reference genome of the tested

species. deduced amino acid sequences of the 19 species were scanned against the Pfam database

(version 34). A total of 138 sequences containing a Pfam domain for UDP-glucoronosyl and UDP-

glucosyl transferase (PF00201) were found in the 19 species. The number of UGTs in wood-

decaying fungi was highly variable, with gene count ranging from 0 to 25. Trametes coccinea

strain CIRM-BRFM-310 (syn. Pycnoporus coccineus) possessed 25 UGTs, showing the greatest

number of UGTs, whereas the genomes of Neolentinus lepideus did not contain any sequences

5
containing PF00201 (SI Appendix, Table S5). In general, species belonging to the genus Trametes

had a large number of UGTs. Eight putative UGTs were found in L. brumalis. A phylogenetic tree

for the 138 UGTs indicated species-level gene duplication and divergence of UGTs in many wood-

decaying fungi, which may be one of the evolutionary outcomes from counteracting structural

diversification of plant phenolics (SI Appendix, Figure S7). Eight UGTs found in the genome of

L. brumalis can be classified into three distinct groups, suggesting their divergent roles in

glycosylation. We expected that we could find a certain UGT forming an enzyme family with other

enzymes of the species which showed similar spectra of pentosylation acceptors; however, six

among eight UGTs formed clades with UGTs of Fomes fomentarius, Perenniporia fraxinea, and

Poriella subacida, which were the genetically closest species and showed similar glycosylation

patterns in the phenotypic assay. Thus, the candidate UGTs could not be further prioritized by this

analysis.

SI Materials and Methods

Structural identification of the isolated metabolites. Optical rotations were measured using

a Jasco P-200 digital polarimeter (Jasco, Tokyo, Japan). Nuclear magnetic resonance (NMR)

spectra were obtained using a Bruker Avance Ⅲ HD 500 spectrometer (Bruker BioSpin Corp.,

Billerica, MA, USA).

Baicalein 7-O-β-D-xylopyranoside (2a): amorphous solid, C20H18O9; [α]20

D −9.5 (c 0.01,

MeOH); 1H NMR and 13C NMR data, see Table S1; HRESIMS m/z 401.0861 [M − H]− (calcd for

C20H17O9, 401.0878); the raw MS/MS spectrum is deposited in the GNPS spectral library,

https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB0001012868

6#%7B%7D.
6
 Chrysin 7-O-β-D-xylopyranoside (2b): amorphous solid, C20H18O8; [α]20
D −4.1 (c 0.01, MeOH);

1
H NMR and 13C NMR data, see Table S1; HRESIMS m/z 385.0917 [M − H]− (calcd for C20H17O8,

385.0930); the raw MS/MS spectrum is deposited in the GNPS spectral library,

https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB0001012868

7#%7B%7D.

Oroxylin A 7-O-β-D-xylopyranoside (2c): amorphous solid, C21H20O9; [α]20

D −26.1 (c 0.01,

MeOH); 1H NMR and 13C NMR data, see Table S1; HRESIMS m/z 415.1022 [M − H]− (calcd for

C21H19O9, 415.1034); the raw MS/MS spectrum is deposited in the GNPS spectral library,

https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB0001012868

8#%7B%7D.

5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranoside (2d): amorphous solid, C21H20O10;

1 13
[α]20
D +70.2 (c 0.01, MeOH); H NMR and C NMR data, see Table S1; HRESIMS m/z 431.0973

[M − H]− (calcd for C21H19O10, 431.0984); the raw MS/MS spectrum is deposited in the GNPS

spectral library,

https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB0001012868

9#%7B%7D.

Baicalein 6,7-di-O-β-D-xylopyranoside (2e): amorphous solid, C25H26O13; [α]20

D +3.6 (c 0.01,

MeOH); 1H NMR and 13C NMR data, see Table S2; HRESIMS m/z 533.1285 [M − H]− (calcd for

C25H25O13, 533.1301); the raw MS/MS spectrum is deposited in the GNPS spectral library,

https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB0001012869

0#%7B%7D.

Oroxylin A 7-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranoside (2f): amorphous solid,

1 13
C26H28O13; [α]20
D +25.4 (c 0.005, MeOH); H NMR and C NMR data, see Table S2; HRESIMS
7
m/z 547.1448 [M − H]− (calcd for C26H27O13, 547.1457); the raw MS/MS spectrum is deposited in

the GNPS spectral library,

https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB0001012869

1#%7B%7D.

5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranoside (2g):

1 13
amorphous solid, C26H27O14; [α]20
D +92.8 (c 0.005, MeOH); H NMR and C NMR data, see Table

S2; HRESIMS m/z 563.1388 [M − H]− (calcd for C26H27O14, 563.1406); the raw MS/MS spectrum

is deposited in the GNPS spectral library,

https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB0001012869

2#%7B%7D.

Magnolol 2-O-β-D-xylopyranoside (11a): amorphous solid, C23H26O6; [α]20

D −10.6 (c 0.01,

MeOH); 1H NMR and 13C NMR data, see Table S3; HRESIMS m/z 397.1641 [M − H]− (calcd for

C23H25O6, 397.1657); the raw MS/MS spectrum is deposited in the GNPS spectral library,

https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB0001012869

3#%7B%7D.

Honokiol 4′-O-β-D-xylopyranoside (12a): amorphous solid, C23H26O6; [α]20

D −7.8 (c 0.01,

MeOH); 1H NMR and 13C NMR data, see Table S3; HRESIMS m/z 397.1649 [M − H]− (calcd for

C23H25O6, 397.1657); the raw MS/MS spectrum is deposited in the GNPS spectral library,

https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB0001012869

4#%7B%7D.

Honokiol 2-O-β-D-xylopyranoside (12b): amorphous solid, C23H26O6; [α]20

D −6.8 (c 0.005,

MeOH); 1H NMR and 13C NMR data, see Table S3; HRESIMS m/z 397.1650 [M − H]− (calcd for

C23H25O6, 397.1657); the raw MS/MS spectrum is deposited in the GNPS spectral library,
8
https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB0001012869

5#%7B%7D.

Raw NMR FID data are available at https://doi.org/10.5281/zenodo.7376079

Determination of sugar moieties of the isolated compounds. Approximately 0.5 mg of each

compound was separately hydrolyzed with 1N HCl (1.0 mL) at 80 °C for 2 h. The hydrolysates

were extracted with EA (2 × 0.5 mL) to remove aglycone. The aqueous layer was concentrated

and compared with D-ribose, D-arabinose, and D-xylose on silica gel TLC plates with n-BUOH-

acetone-pyridine-H2O (2:2:1:1), visualizing with p-anisaldehyde and heating. The absolute

configurations of xyloses were analyzed after arylthiocarbamoyl-thiazolidine derivatization. The

aqueous layer was concentrated and dissolved in pyridine (100 μL) containing L-cysteine methyl

ester hydrochloride (0.5 mg) and heated 60 °C for 1 h; then o-toylisothiocyanate (100 μL) was

added and heated 60 ℃ for additional 1 h. Each reaction mixture was directly analyzed by an

HPLC (Agilent 1100 system) eluted by a mobile phase consisting of solvents A (0.1 % formic acid

in H2O) and B (acetonitrile). The flow rate was set to 0.3 mL/min. A linear gradient was set to be

10–100 % B (0–12 min) followed by 3 min washout phase at 100 % B and 3 min re-equilibration

phase at 10 % B, successively.

Phylogenetic analysis on 138 putative UGTs. A maximum likelihood tree was constructed

using the RAxML program (v8.2) (8) and annotated using iTOL (https://itol.embl.de) (9). Nodal

supports were evaluated by 1,000 bootstrap replications.

RNA isolation. TRIzol (1 mL; Sigma Aldrich) was added to 100 mL of the lyophilized samples,

mixed by vortexing, and then incubated at room temperature for 5 min. Chloroform (200 μL) was

added to each TRIzol slurry and the tubes mixed by inversion, then incubated at room temperature
9
for 5 min. The samples were centrifuged at 12,000 g for 15 min at 4°C. The supernatants were

collected, phenol (300 μL, pH 4.6) and chloroform:isoamyl alcohol (24:1 v/v) (300 μL) were added

and the tubes were incubated at room temperature for 5 min. The samples were centrifuged at

12,000 g for 10 min at 4°C. The supernatants were collected, and chloroform (450 μL) was added,

and the samples were incubated at room temperature for 1 min. The samples were centrifuged at

12,000 g for 5 min at 4 °C. The upper aqueous layer was collected, 3M sodium acetate (80 μL)

and ice-cold 100% isopropanol (400 μL) were added, and the samples were incubated at room

temperature for 10 min and then incubated for 2 h at –20°C. Each sample was centrifuged for 15

min at 12,000g, 4°C. After discarding the supernatant, 1 mL of ice-cold 75% EtOH was added and

the samples were centrifuged for 10 min at 7,500g, 4 °C, then the supernatant was removed. The

sample was resuspended in RNase-free water and DNase I digestion was performed with an

RNase-Free DNase Set (Qiagen, Hilden, Germany) and with an RNA clean-up step using an RNA

concentrator-25 following manufacturer’s instructions. The quality and quantity of total RNA were

assessed using a NanoDrop 1000 spectrophotometer and an Agilent 2100 bioanalyzer (Agilent

Technologies, Santa Clara, CA, USA).

10
Fig. S1. Time-dependent analysis of the 1-supplemented culture broth of L. brumalis. a. Base peak

ion (BPI) chromatograms of the culture broths of L. brumalis, collected in day 0, 1, 2, 4, 6, and 8

after treatment of 1. It should be noted that the other xylosylated compounds found in this study,

such as 2b, 2c, 2f, or 2g were also detected, but they are not clearly observed in the BPI

chromatograms due to the relatively low ion intensities.

11
Fig. S2. Thin layer chromatography (TLC) analysis confirmed that xylose is the sugar moiety
attached to the compounds 2a–2g.

12
Fig. S3. Arylthiocarbamoyl-thiazolidine derivatization followed by HPLC analysis confirmed the
absolute configurations of xylosyl moieties in 2a–2g to be D-xylose.

13
Fig. S4. Thin layer chromatography (TLC) analysis confirmed that xylose is the sugar moiety
attached to the compounds 11a, 12a, and 12b.

14
Fig. S5. Arylthiocarbamoyl-thiazolidine derivatization followed by HPLC analysis confirmed the
absolute configurations of xylosyl moieties in 11a, 12a, and 12b to be D-xylose.

15
Fig. S6. The UGT catalyzing O-xylosylation in L. brumalis is not an extracellularly secreted
enzyme, while the β-glucuronidase is.

16
Fig. S7. Diversification of UDP-glycosyltransferase in wood-decaying fungi. A maximum-

likelihood tree was reconstructed for 138 UDP-glycosyltransferases (UGTs) identified in 19 wood-

decaying fungi. Two previously characterized UGTs, UGT58A1 from Absidia caerulea (NCBI

accession: KX610760) and UGT59A1 from Rhizopus japonicus (KX610761), were set as an

outgroup. The numbers at the internal nodes indicate bootstrap values greater than 85% from 1,000

bootstrap replications. Branch lengths are proportional to the inferred amount of evolutionary

change, and the scale represents 0.2 amino acid sequence substitutions per site. Highly bootstrap-

supported clades that consist of UGTs from a single species or a group of closely related species
17
(Trametes gibbosa, T. pubescens, and T. versicolor) were shaded with different colors. Eight UGTs

(UGT1–UGT8) in L. brumalis were depicted.

18
Fig. S8. Confirmation of recombinant LbUGT3 catalyzing O-xylosylation against 11 and 12.
Detection of xylosylated metabolites (11a, 12a, and 12b) and hexosylated metabolites of 11 and
12 in the reaction mixtures of the purified LbUGT3, with 11 or 12 and UDP-xylose, UDP-glucose
or together.

19
Fig. S9. MS/MS spectra of metabolites tentatively annotated as xylosylated 3–8, 10, 15, 16, and
18 (marked with asterisks* in Figs. 2D and 3B).

20
Fig. S10. The predicted structure of UGT66A1. (A) The protein structure of UGT66A1 predicted

by AlphaFold2 through ColabFold, colored by pLDDT. (B) pLDDT plot illustrating the prediction

quality for all five AlphaFold PTM models. (C) Predicted alignment plot displaying the agreement

among all five models. (D) The predicted structure of UGT66A1 (light blue) overlapped with an

UGT annotated from the draft genome of Trametes pubescens (left, yellow; Uniprot:

A0A1M2VXJ8; TM-score: 0.985) and UGT74AC1 from Siraitia grosvenorii (right, yellow; PDB:

6l8w chain A; TM-score: 0.799).

21
Fig. S11. Antifungal activity test of compounds 2, 11, and 12 against L. brumalis showed that L.

brumalis is resistant to these compounds. The data presented as the mean ± SD of the diameters of

colonies (n=3) from 36 h to 60 h after inoculation; *p < 0.05, **p < 0.01, and ***p < 0.005

compared with the vehicle. Itraconazole was used as a positive control

22
Table S1. 1H (500 MHz) and 13C (125 MHz) NMR Data of Compounds 2a–2d
(in DMSO-d6)
2a 2b 2c 2d
Positio
H H H H
n C C C C
(J in Hz) (J in Hz) (J in Hz) (J in Hz)
2 163.5 163.8 163.7 162.9

3 104.7 7.01, s 105.4 7.07, s 104.9 7.04, s 104.4 7.02, s

4 182.6 182.2 182.5 182.3

4a 106.1 105.4 106.1 102.8

5 149.2 161.1 152.5 149.2

6 130.7 99.7 6.45, s 132.7 152.3

7 151.5 163.0 156.5 125.3

8 94.0 7.01, s 94.8 6.89, s 94.3 7.06, s 131.6

8a 146.7 n.d. 152.3 145.5

OCH3 60.3 3.77, s 60.1 3.77, s

130.8 130.6 130.6 130.7

126.5 8.11, m 126.6 8.12, m 126.5 8.11, d (7.3) 126.6 8.25, m

129.2 7.59, m 129.2 7.60, m 129.2 7.59, m 129.1 7.58, m

132.1 7.62, m 132.2 7.63, m 132.2 7.63, m 132.1 7.61, m

129.2 7.59, m 129.2 7.60, m 129.2 7.59, m 129.1 7.58, m

126.5 8.11, m 126.6 8.12, m 126.5 8.11, d (7.3) 126.6 8.25, m

Xyl-1 101.3 5.02, d (7.5) 100.2 5.09, d (5.3) 100.5 5.15, d (7.3) 106.4 4.73, d (7.5)

Xyl-2 72.9 3.37, m 72.9 3.26, m 72.9 3.36, m 73.9 3.44, m

Xyl-3 75.7 3.29, m 76.3 3.26, m 76.4 3.30, m 75.9 3.25, m

Xyl-4 69.3 3.42, m 69.2 3.39, m 69.2 3.43, m 69.3 3.46, m

3.82, dd
3.82, m 3.77, m 3.81, m (11.3, 5.2)
Xyl-5 65.9 65.9 65.9 66.2
3.42, m 3.39, m 3.43, m 3.14, dd
(11.3, 10.3)
5-OH 12.57, s 12.80, s 12.79, s 12.79, s

2
Table S2. 1H (500 MHz) and 13C (125 MHz) NMR Data of Compounds 2e–2g (in DMSO-
d6)

2e 2f 2g
position H H H
C C C
(J in Hz) (J in Hz) (J in Hz)
2 163.7 163.8 159.9
3 105.0 7.05, s 105.1 7.07, s 103.4 6.65, s
4 182.5 182.6 179.6
4a 106.2 106.2 96.1
5 n.d. 152.6 n.d.
6 129.8 132.6 134.4
7 156.0 156.2 128.5
8 94.5 7.05, s 94.4 7.07, s n.d.
8a 152.7 152.6 148.8
OCH3 60.3 3.77, s 58.6 3.65, s
130.6 130.6 131.7
126.6 8.13, m 126.5 8.13, m 125.9 8.15, m
129.2 7.60, m 129.2 7.61, m 128.8 7.53, m
132.2 7.63, m 132.2 7.64, m 131.0 7.53, m
129.2 7.60, m 129.2 7.61, m 128.8 7.53, m
126.6 8.13, m 126.5 8.13, m 126.0 8.15, m
Xyl-1 103.7 4.93, d (6.6) 99.9 5.25, d (7.3) 109.3 4.32, d (7.5)
Xyl-2 73.3 3.35, m 72.1 3.60, m 73.6 3.41, m
Xyl-3 75.4 3.23, m 85.4 3.53, m 85.4 3.41, m
Xyl-4 69.4 3.41, m 67.5 3.53, m 67.6 3.48, m
3.79, dd
3.88, m 3.86, m
(11.6, 5.0)
Xyl-5 65.6 65.4 66.2
3.03, dd
3.49, m 3.20, m
(11.6, 9.0)
Xyl-1′ 101.3 5.08, d (7.2) 104.4 4.48, d (7.4) 104.8 4.54, d (7.4)
Xyl-2′ 73.0 3.38, m 73.8 3.11, m 74.4 3.06, m
Xyl-3′ 75.6 3.29, m 76.1 3.17, m 76.4 3.15, m
Xyl-4′ 69.2 3.41, m 69.4 3.32, m 69.5 3.29, m
3.83, m 3.78, m 3.72, m
Xyl-5′ 65.9 65.8 65.7
3.42, m 3.11, m 3.03, m
5-OH 12.82, s 12.62, s

3
Table S3. 1H (500 MHz), 13C (125 MHz) NMR Data of Compounds 11a, 12a, and 12b
(in DMSO-d6)
11a 12a 12b
position H H H
C C C
(J in Hz) (J in Hz) (J in Hz)
1 129.7 127.2 127.3
2 153.1 152.5 152.3
3 117.6 6.85, d (8.2) 116.0 6.83, d (8.2) 115.7 7.08, d (8.5)
4 129.7 7.03, dd (8.2, 2.3) 128.1 6.92, dd (8.2, 2.3) 127.9 7.02, dd (8.5, 2.2)
5 133.1 132.5 129.2
6 132.7 6.98, d (2.3) 130.1 6.98, d (2.3) 130.6 7.05, d (2.2)
7 40.4 38.7 3.27, m 39.2 3.32, m
8 139.4 5.99, m 138.3 5.93, m 138.5 5.94, m
5.00, m 5.00, m
9 115.9 5.05, m 115.3 116.0
5.05, m 5.05, m
128.0 128.7 125.3
154.2 128.0 7.31, dd (8.5, 2.3) 128.6 7.28, dd (8.3, 2.3)
115.6 7.11, d (8.5) 114.9 7.05, d (8.5) 114.9 6.80, d (8.3)
130.0 7.15, dd (8.5, 2.2) 153.7 153.7
135.3 127.3 125.1
132.9 7.05, d (2.2) 130.2 7.26, d (2.3) 131.4 7.32, d (2.3)
40.4 33.9 3.41, m 34.3 3.31, m
139.1 5.99, m 137.3 5.99, m 137.8 5.98, m
5.00, m 5.04, m
115.8 5.05, m 115.7 116.0
5.10, m 5.08, m
Xyl-1 102.9 4.96, d (7.2) 101.9 4.81, d (7.3) 101.3 4.97, d (7.5)
Xyl-2 74.4 3.33, m 73.3 3.28, m 73.7 3.17, m
Xyl-3 77.1 3.43, m 76.5 3.24, m 77.2 3.22, m
Xyl-4 70.8 3.53, m 69.4 3.39, m 69.9 3.33, m
3.92, m 3.76, m 3.70, m
Xyl-5 66.7 65.7 66.2
3.36, m 3.24, m 3.22, m

4
Table S4. Fungal strains used in this study.

Species name Strain accession ID Reference genome ID

Amaropostia stiptica KMRB19032901 11775661

Cerrena unicolor KMRB16092759 10061441

Fomes fomentarius KMRB18072701 11275901

Fomitiporia punctata KMRB15102101 (not available)

Fomitopsis pinicola KMRB19090114 11848881

Grifola frondosa KMRB18092931 GCA_001683735.12

Irpex lacteus KMRB19073146 11124241

Laetiporus sulphureus KMRB02062001 10069171

Lentinus brumalis KMRB15102011 10515661

Neolentinus lepideus KMRB18111330 10069211

Perenniporia fraxinea KMRB18091336 12973531

Phlebiopsis gigantea KMRB19040322 11848161

Poriella subacida KMRB17091608 12405741

Steccherinum sp. KMRB19032111 (not available)

Trametes coccinea KMRB19080521 10176851

Trametes gibbosa KMRB17111316 11112291

Trametes hirsuta KMRB19071524 GCA_001302255.22,3

Trametes pubescens KMRB17121406 GCA_001895945.12

Trametes versicolor KMRB17121408 11848271

Trametopsis cervina KMRB19040307 10515721

Trichaptum abietinum KMRB19032112 10069251

Xylodon flaviporus KMRB16061440 10185971

1
JGI project ID 2Genbank accession number 3This sequence was excluded due to a lack of gene annotation.

5
Table S5. The number of genes annotated as regions encoding UDP-glucoronosyl and

UDP-glucosyl transferase (PF00201) in the genomes of 19 species analyzed in this study.

Species name The number of UGTs

Amaropostia stiptica 9
Cerrena unicolor 6
Fomes fomentarius 7
Fomitiporia punctata N/A
Fomitopsis pinicola 3
Grifola frondosa 1
Irpex lacteus 2
Laetiporus sulphureus 3
Lentinus brumalis 8
Neolentinus lepideus 0
Perenniporia fraxinea 6
Phlebiopsis gigantea 2
Poriella subacida 16
Steccherinum sp. N/A
Trametes coccinea 25
Trametes gibbosa 12
Trametes hirsuta N/A
Trametes pubescens 14
Trametes versicolor 19
Trametopsis cervina 2
Trichaptum abietinum 2
Xylodon flaviporus 1

6
Dataset S1 (separate file). A full list of metabolites ions observed in theLC-MS/MS
analysis.

Dataset S2 (separate file). A full list of 138 UGTs annotated from the publicly available
genomes of 19 wood decaying fungi.

Dataset S3 (separate file). The RPKM values of entire genes in the RNAseq data.

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