Supplementary Information The biosynthetic pathway for a thousand-year-old natural food colorant and citrinin in Penicillium marneffei Patrick C. Y. Woo 1,2,3,4* †, Ching-Wan Lam 5 † Emily W. T. Tam 1 †, Kim-Chung Lee 5 , Karrie K. Y. Yung 5 , Chris K. F. Leung 5 , Kong-Hung Sze 1,2,3,4 , Susanna K. P. Lau 1,2,3,4* and Kwok-Yung Yuen 1,2,3,4 1 Department of Microbiology, The University of Hong Kong, Hong Kong, 2 State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, 3 Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong, 4 Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, 5 Department of Pathology, The University of Hong Kong, Hong Kong
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Supplementary Information
The biosynthetic pathway for a thousand-year-old natural
food colorant and citrinin in Penicillium marneffei
Patrick C. Y. Woo1,2,3,4*†, Ching-Wan Lam5† Emily W. T. Tam1†, Kim-Chung Lee5, Karrie K. Y. Yung5, Chris K. F. Leung5, Kong-Hung Sze1,2,3,4, Susanna K. P. Lau1,2,3,4* and Kwok-Yung Yuen1,2,3,4
1Department of Microbiology, The University of Hong Kong, Hong Kong, 2State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, 3Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong, 4Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, 5Department of Pathology, The University of Hong Kong, Hong Kong
wild type
orf1 expression
actin expression
orf1KD
P. marneffei strains
wild type
orf2 expression
actin expression
orf2KD
wild type
orf3 expression
actin expression
orf3KD
wild type
orf4 expression
actin expression
orf4KD
wild type
orf5 expression
actin expression
orf5KD
wild type
rp3 expression
actin expression
rp3KD
P. marneffei strains
wild type
rp4 expression
actin expression
rp4KD
wild type
orf6 expression
actin expression
orf6KD
wild type
orf7 expression
actin expression
orf7KD
wild type
orf8 expression
actin expression
orf8KD
wild type
pks3 expression
actin expression
pks3KD
wild type
rp1 expression
actin expression
rp1KD
wild type
rp2 expression
actin expression
rp2KD
wild type
orf9 expression
actin expression
orf9KD
wild type
orf10 expression
actin expression
orf10KD
Supplementary Figure S1: Expression of pks3 and the 14 genes upstream and
downstream of pks3 in the corresponding knockdown mutants of Penicillium
marneffei.
Acquisition time (min)
102
15 15.2 15.4 15.6 15.8 16 16.2
0
16.4
EIC (561.2684), wild type
EIC (561.2684), rp4 knockdown
EIC (561.2684), rp3 knockdown
EIC (561.2684), rp2 knockdown
EIC (561.2684), rp1 knockdown
EIC (561.2684), pks3 knockdown
16.6 16.8 17
102 mAU
250 300 350 400 450 500 550
-0.2
0.2
0.6
0.4
1.0
0.8
Wavelength (nm)
0
-0.4
-0.6
600 650
a
b
c
Supplementary Figure S2: Detection of arginine conjugated with monascorubrin in P.
marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of arginine-monascorubrin conjugate detected in
wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
102 mAU
250 300 350 400 450 500 550
-0.2
0.2
0.6
0.4
0.8
0
600 650
a
b
c
Acquisition time (min)
102
16 16.2 16.4 16.6 16.8 17 17.2
0
17.4
EIC (511.2803), wild type
EIC (511.2803), rp4 knockdown
EIC (511.2803), rp3 knockdown
EIC (511.2803), rp2 knockdown
EIC (511.2803), rp1 knockdown
EIC (511.2803), pks3 knockdown
17.6 17.8
Wavelength (nm)
Supplementary Figure S3: Detection of lysine conjugated with monascorubrin in P.
marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of lysine-monascorubrin conjugate detected in
wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
Acquisition time (min)
102
16 16.5
0
17
EIC (497.2282), wild type
EIC (497.2282), rp4 knockdown
EIC (497.2282), rp3 knockdown
EIC (497.2282), rp2 knockdown
EIC (497.2282), rp1 knockdown
EIC (497.2282), pks3 knockdown
17.5 18
102 mAU
250 300 350 400 450 500 550
0.5
1.0
Wavelength (nm)
0
600 650
a
b
c
Supplementary Figure S4: Detection of asparagine conjugated with monascorubrin in P.
marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of asparagine-monascorubrin conjugate detected
in wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
a
b
c
Acquisition time (min)
102
0
EIC (470.2173), wild type
EIC (470.2173), rp4 knockdown
EIC (470.2173), rp3 knockdown
EIC (470.2173), rp2 knockdown
EIC (470.2173), rp1 knockdown
EIC (470.2173), pks3 knockdown
17.5 18 18.5 19 19.5
101 mAU
250 300 350 400 450 500 550
1
2
Wavelength (nm)
0
600
Supplementary Figure S5: Detection of serine conjugated with monascorubrin in P.
marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of serine-monascorubrin conjugate detected in
wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
c
a
b Acquisition time (min)
102
18.4 18.6
0
18.8
EIC (534.2098), wild type
EIC (534.2098), rp4 knockdown
EIC (534.2098), rp3 knockdown
EIC (534.2098), rp2 knockdown
EIC (534.2098), rp1 knockdown
EIC (534.2098), pks3 knockdown
19 19.2 19.4
101 mAU
250 300 350 400 450 500 550
1
2
Wavelength (nm)
0
600 650
3
Supplementary Figure S6: Detection of glutamic acid conjugated with monascorubrin in
P. marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of glutamic-monascorubrin conjugate detected in
wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
c
a
b Acquisition time (min)
102
17.5 18
0
18.5
EIC (546.2486), wild type
EIC (546.2486), rp4 knockdown
EIC (546.2486), rp3 knockdown
EIC (546.2486), rp2 knockdown
EIC (546.2486), rp1 knockdown
EIC (546.2486), pks3 knockdown
19 19.5 21 20 20.5
101 mAU
250 300 350 400 450 500 550
1
2
Wavelength (nm)
0
600 650
3
-1
Supplementary Figure S7: Detection of tyrosine conjugated with monascorubrin in P.
marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of tyrosine-monascorubrin conjugate detected in
wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
c
a
b Acquisition time (min)
102
18
0
18.5
EIC (440.2068), wild type
EIC (440.2068), rp4 knockdown
EIC (440.2068), rp3 knockdown
EIC (440.2068), rp2 knockdown
EIC (440.2068), rp1 knockdown
EIC (440.2068), pks3 knockdown
19 19.5 21 20 20.5
101 mAU
300 400 500 600
1
2
Wavelength (nm)
0
3
Supplementary Figure S8: Detection of glycine conjugated with monascorubrin in P.
marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of glycine-monascorubrin conjugate detected in
wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
c
a
b Acquisition time (min)
101
18.4 18.6
0
18.8
EIC (498.2122), wild type
EIC (498.2122), rp4 knockdown
EIC (498.2122), rp3 knockdown
EIC (498.2122), rp2 knockdown
EIC (498.2122), rp1 knockdown
EIC (498.2122), pks3 knockdown
19 19.2 19.4
101 mAU
250 300 350 400 450 500 550
Wavelength (nm)
0
600 650
1
2
3
Supplementary Figure S9: Detection of aspartic acid conjugated with monascorubrin in
P. marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of aspartic acid-monascorubrin conjugate
detected in wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
a
b
c
Acquisition time (min)
102
20 20.5
0
21
EIC (591.2466), wild type
EIC (591.2466), rp4 knockdown
EIC (591.2466), rp3 knockdown
EIC (591.2466), rp2 knockdown
EIC (591.2466), rp1 knockdown
EIC (591.2466), pks3 knockdown
21.5 22 22.5 23
101 mAU
300 400 500
Wavelength (nm)
0
600
0.5
1
Supplementary Figure S10: Detection of tryptophan conjugated with monascorubrin in
P. marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of tryptophan-monascorubrin conjugate detected
in wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
a
b
c
Acquisition time (min)
102
19 19.2
0
19.4
EIC (482.2537), wild type
EIC (482.2537), rp4 knockdown
EIC (482.2537), rp3 knockdown
EIC (482.2537), rp2 knockdown
EIC (482.2537), rp1 knockdown
EIC (482.2537), pks3 knockdown
19.6 19.8 20
101 mAU
300 400 500
Wavelength (nm)
0
600
0.5
1
Supplementary Figure S11: Detection of valine conjugated with monascorubrin in P.
marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of valine-monascorubrin conjugate detected in
wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
c
a
b Acquisition time (min)
102
21.2
0
21.6
EIC (536.2077),
wild type
EIC (536.2077), rp4 knockdown
EIC (536.2077), rp3 knockdown
EIC (536.2077), rp2 knockdown
EIC (536.2077), rp1 knockdown
EIC (536.2077), pks3 knockdown
21.8 22 22.6 21.6 22.2 22.4 22.8 23
101 mAU
300 400 500
Wavelength (nm)
0
600
0.2
1
-0.2
-0.4
0.6
0.8
0.4
Supplementary Figure S12: Detection of methionine conjugated with monascorubrin in
P. marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of methionine-monascorubrin conjugate detected
in wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
c
a
b Acquisition time (min)
21.5
0
EIC (496.2694),
wild type
EIC (496.2694), rp4 knockdown
EIC (496.2694), rp3 knockdown
EIC (496.2694), rp2 knockdown
EIC (496.2694), rp1 knockdown
EIC (496.2694), pks3 knockdown
23.5 22 22.5 24 24.5 23
102
101 mAU
300 400 500
Wavelength (nm)
0
600
1
Supplementary Figure S13: Detection of leucine conjugated with monascorubrin in P.
marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of leucine-monascorubrin conjugate detected in
wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
c
a
b Acquisition time (min)
21.5
0
EIC (496.2694),
wild type
EIC (496.2694), rp4 knockdown
EIC (496.2694), rp3 knockdown
EIC (496.2694), rp2 knockdown
EIC (496.2694), rp1 knockdown
EIC (496.2694), pks3 knockdown
23.5 22 22.5 24
23
102
101 mAU
300 400 500
Wavelength (nm)
0
600
1
Supplementary Figure S14: Detection of isoleucine conjugated with monascorubrin in P.
marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of isoleucine-monascorubrin conjugate detected
in wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
c
a
b Acquisition time (min)
102
24 21.5 22 22.5
0
23
EIC (552.2357), wild type
EIC (552.2357), rp4 knockdown
EIC (552.2357), rp3 knockdown
EIC (552.2357), rp2 knockdown
EIC (552.2357), rp1 knockdown
EIC (552.2357), pks3 knockdown
23.5
101 mAU
300 400 500
Wavelength (nm)
0
600
1
Supplementary Figure S15: Detection of phenylalanine conjugated with monascorubrin
in P. marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of phenylalanine-monascorubrin conjugate
detected in wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
c
Acquisition time (min)
101
14.2 14.4 12.6 12.8 13 13.2
0
13.4
EIC (533.2171), wild type
EIC (533.2171), rp4 knockdown
EIC (533.2171), rp3 knockdown
EIC (533.2171), rp2 knockdown
EIC (533.2171), rp1 knockdown
EIC (533.2171), pks3 knockdown
13.6 13.8 14
Wavelength (nm)
101 mAU
300 350 400 450 500 550
0.5
0
a
b
Supplementary Figure S16: Detection of arginine conjugated with rubropunctatin in P.
marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of arginine-rubropunctatin conjugate detected in
wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
a
b
c
Acquisition time (min)
102
20.4 20.6 19 19.2 19.4 19.6
0
19.8
EIC (524.2044), wild type
EIC (524.2044), rp4 knockdown
EIC (524.2044), rp3 knockdown
EIC (524.2044), rp2 knockdown
EIC (524.2044), rp1 knockdown
EIC (524.2044), pks3 knockdown
20 20.2
Wavelength (nm)
101 mAU
300 400 500 600
1
0
Supplementary Figure S17: Detection of phenylalanine conjugated with rubropunctatin
in P. marneffei. (a) Extracted ion chromatogram, (b) UV absorption spectrum and (c) MS/MS
fragmentation pattern showing the presence of phenylalanine-rubropunctatin conjugate
detected in wild type P. marneffei but not in pks3 or rp1 to rp4 knockdown mutants.
Supplementary Table S1: Relative expression of all genes involved in biosynthesis
of red pigment in different knockdown mutants of Penicillium marneffei.
P. marneffei strains Relative expression
pks3 rp1 rp2 rp3 rp4
Wild type 1 1 1 1 1
pks3 KD mutant <0.01 0.13 0.08 0.05 0.05
rp1 KD mutant 0.05 0.15 0.12 0.01 0.10
rp2 KD mutant 0.54 0.91 0.23 0.90 0.52
rp3 KD mutant 0.98 1.06 0.30 0.33 0.73
rp4 KD mutant 0.44 0.62 0.22 2.10 0.29
Sample Absorption maxima
wild type 373 405 499 553
orf1 knockdown 373 411 501 553
orf2 knockdown 373 416 499 556
orf3 knockdown 373 416 500 558
orf4 knockdown 373 404 498 556
orf5 knockdown 372 414 503 558
pks3 knockdown --- --- --- 560
rp1 knockdown --- --- --- 559
rp2 knockdown 372 404 --- 561
rp3 knockdown 372 404 --- 561
rp4 knockdown 372 411 --- 559
orf6 knockdown 373 417 500 557
orf7 knockdown 372 417 496 553
orf8 knockdown 374 416 499 554
orf9 knockdown 376 417 499 556
orf10 knockdown 373 409 494 557
medium --- --- --- 560
Supplementary Table S2: UV-Vis spectroscopic absorption maxima of culture
filtrates in Penicillium marneffei.
Supplementary Table S3: Molecular formula, mass-to-charge ratio and
retention time of different amino acids conjugated with monascorubrin or
rubropunctatin in culture filtrate of Penicillium marneffei detected by