Supplementry Informtion The iosyntheti pthwy for thousnd-yer-old nturl food olornt nd itrinin in Peniillium mrneffei Ptrik C. Y. Woo,2,3,4*, Ching-Wn Lm 5 Emily W. T. Tm, Kim-Chung Lee 5, Krrie K. Y. Yung 5, Chris K. F. Leung 5, Kong-Hung Sze,2,3,4, Susnn K. P. Lu,2,3,4* nd Kwok-Yung Yuen,2,3,4 Deprtment of Miroiology, The University of Hong Kong, Hong Kong, 2 Stte Key Lortory of Emerging Infetious Diseses, The University of Hong Kong, Hong Kong, 3 Reserh Centre of Infetion nd Immunology, The University of Hong Kong, Hong Kong, 4 Crol Yu Centre for Infetion, The University of Hong Kong, Hong Kong, 5 Deprtment of Pthology, The University of Hong Kong, Hong Kong
P. mrneffei strins orfkd P. mrneffei strins rp3kd orf expression rp3 expression orf2kd rp4kd orf2 expression rp4 expression orf3kd orf6kd orf3 expression orf6 expression orf4kd orf7kd orf4 expression orf7 expression orf5kd orf8kd orf5 expression orf8 expression pks3kd orf9kd pks3 expression orf9 expression rpkd orfkd rp expression orf expression rp2kd rp2 expression Supplementry Figure S: Expression of pks3 nd the 4 genes upstrem nd downstrem of pks3 in the orresponding knokdown mutnts of Peniillium mrneffei.
2 EIC (56.2684), pks3 knokdown EIC (56.2684), rp knokdown EIC (56.2684), rp2 knokdown EIC (56.2684), rp3 knokdown EIC (56.2684), rp4 knokdown EIC (56.2684), 5 5.2 5.4 5.6 5.8 6 6.2 2 mau..8.6.4.2 -.2 -.4 -.6 6.4 6.6 6.8 7 25 3 35 4 45 5 55 Wvelength (nm) 6 65 Supplementry Figure S2: Detetion of rginine onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of rginine-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (5.283), pks3 knokdown EIC (5.283), rp knokdown EIC (5.283), rp2 knokdown EIC (5.283), rp3 knokdown EIC (5.283), rp4 knokdown EIC (5.283), 6 6.2 6.4 6.6 6.8 7 7.2 2 mau 7.4 7.6 7.8.8.6.4.2 -.2 25 3 35 4 45 5 55 Wvelength (nm) 6 65 Supplementry Figure S3: Detetion of lysine onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of lysine-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (497.2282), pks3 knokdown EIC (497.2282), rp knokdown EIC (497.2282), rp2 knokdown EIC (497.2282), rp3 knokdown EIC (497.2282), rp4 knokdown EIC (497.2282), 2 mau 6 6.5 7 7.5 8..5 25 3 35 4 45 5 55 Wvelength (nm) 6 65 Supplementry Figure S4: Detetion of sprgine onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of sprgine-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (47.273), pks3 knokdown EIC (47.273), rp knokdown EIC (47.273), rp2 knokdown EIC (47.273), rp3 knokdown EIC (47.273), rp4 knokdown EIC (47.273), mau 7.5 8 8.5 9 9.5 2 25 3 35 4 45 5 55 Wvelength (nm) 6 Supplementry Figure S5: Detetion of serine onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of serine-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (534.298), pks3 knokdown EIC (534.298), rp knokdown EIC (534.298), rp2 knokdown EIC (534.298), rp3 knokdown EIC (534.298), rp4 knokdown EIC (534.298), mau 8.4 8.6 8.8 9 9.2 9.4 3 2 25 3 35 4 45 5 55 Wvelength (nm) 6 65 Supplementry Figure S6: Detetion of glutmi id onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of glutmi-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (546.2486), pks3 knokdown EIC (546.2486), rp knokdown EIC (546.2486), rp2 knokdown EIC (546.2486), rp3 knokdown EIC (546.2486), rp4 knokdown EIC (546.2486), mau 7.5 8 8.5 9 9.5 2 2.5 2 3 2-25 3 35 4 45 5 55 Wvelength (nm) 6 65 Supplementry Figure S7: Detetion of tyrosine onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of tyrosine-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (44.268), pks3 knokdown EIC (44.268), rp knokdown EIC (44.268), rp2 knokdown EIC (44.268), rp3 knokdown EIC (44.268), rp4 knokdown EIC (44.268), mau 8 8.5 9 9.5 2 2.5 2 3 2 3 4 5 6 Wvelength (nm) Supplementry Figure S8: Detetion of glyine onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of glyine-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
EIC (498.222), pks3 knokdown EIC (498.222), rp knokdown EIC (498.222), rp2 knokdown EIC (498.222), rp3 knokdown EIC (498.222), rp4 knokdown EIC (498.222), mau 8.4 8.6 8.8 9 9.2 9.4 3 2 25 3 35 4 45 5 55 Wvelength (nm) 6 65 Supplementry Figure S9: Detetion of sprti id onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of sprti id-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (59.2466), pks3 knokdown EIC (59.2466), rp knokdown EIC (59.2466), rp2 knokdown EIC (59.2466), rp3 knokdown EIC (59.2466), rp4 knokdown EIC (59.2466), mau 2 2.5 2 2.5 22 22.5 23.5 3 4 5 Wvelength (nm) 6 Supplementry Figure S: Detetion of tryptophn onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of tryptophn-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (482.2537), pks3 knokdown EIC (482.2537), rp knokdown EIC (482.2537), rp2 knokdown EIC (482.2537), rp3 knokdown EIC (482.2537), rp4 knokdown EIC (482.2537), mau 9 9.2 9.4 9.6 9.8 2.5 3 4 5 Wvelength (nm) 6 Supplementry Figure S: Detetion of vline onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of vline-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (536.277), pks3 knokdown EIC (536.277), rp knokdown EIC (536.277), rp2 knokdown EIC (536.277), rp3 knokdown EIC (536.277), rp4 knokdown mau EIC (536.277), 2.2 2.6 2.6 2.8 22 22.2 22.4 22.6 22.8 23.8.6.4.2 -.2 -.4 3 4 5 Wvelength (nm) 6 Supplementry Figure S2: Detetion of methionine onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of methionine-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (496.2694), pks3 knokdown EIC (496.2694), rp knokdown EIC (496.2694), rp2 knokdown EIC (496.2694), rp3 knokdown EIC (496.2694), rp4 knokdown EIC (496.2694), mau 2.5 22 22.5 23 23.5 24 24.5 3 4 5 Wvelength (nm) 6 Supplementry Figure S3: Detetion of leuine onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of leuine-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (496.2694), pks3 knokdown EIC (496.2694), rp knokdown EIC (496.2694), rp2 knokdown EIC (496.2694), rp3 knokdown EIC (496.2694), rp4 knokdown EIC (496.2694), mau 2.5 22 22.5 23 23.5 24 3 4 5 Wvelength (nm) 6 Supplementry Figure S4: Detetion of isoleuine onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of isoleuine-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (552.2357), pks3 knokdown EIC (552.2357), rp knokdown EIC (552.2357), rp2 knokdown EIC (552.2357), rp3 knokdown EIC (552.2357), rp4 knokdown EIC (552.2357), mau 2.5 22 22.5 23 23.5 24 3 4 5 Wvelength (nm) 6 Supplementry Figure S5: Detetion of phenyllnine onjugted with monsorurin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of phenyllnine-monsorurin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
EIC (533.27), pks3 knokdown EIC (533.27), rp knokdown EIC (533.27), rp2 knokdown EIC (533.27), rp3 knokdown EIC (533.27), rp4 knokdown EIC (533.27), 2.6 2.8 3 3.2 3.4 3.6 3.8 4 mau 4.2 4.4.5 3 35 4 45 5 55 Wvelength (nm) Supplementry Figure S6: Detetion of rginine onjugted with ruropunttin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of rginine-ruropunttin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
2 EIC (524.244), pks3 knokdown EIC (524.244), rp knokdown EIC (524.244), rp2 knokdown EIC (524.244), rp3 knokdown EIC (524.244), rp4 knokdown EIC (524.244), 9 9.2 9.4 9.6 9.8 2 2.2 mau 2.4 2.6 3 4 5 6 Wvelength (nm) Supplementry Figure S7: Detetion of phenyllnine onjugted with ruropunttin in P. mrneffei. () Extrted ion hromtogrm, () UV sorption spetrum nd () MS/MS frgmenttion pttern showing the presene of phenyllnine-ruropunttin onjugte deteted in P. mrneffei ut not in pks3 or rp to rp4 knokdown mutnts.
Supplementry Tle S: Reltive expression of ll genes involved in iosynthesis of red pigment in different knokdown mutnts of Peniillium mrneffei. P. mrneffei strins Reltive expression pks3 rp rp2 rp3 rp4 Wild type pks3 KD mutnt <..3.8.5.5 rp KD mutnt.5.5.2.. rp2 KD mutnt.54.9.23.9.52 rp3 KD mutnt.98.6.3.33.73 rp4 KD mutnt.44.62.22 2..29
Supplementry Tle S2: UV-Vis spetrosopi sorption mxim of ulture filtrtes in Peniillium mrneffei. Smple Asorption mxim 373 45 499 553 orf knokdown 373 4 5 553 orf2 knokdown 373 46 499 556 orf3 knokdown 373 46 5 558 orf4 knokdown 373 44 498 556 orf5 knokdown 372 44 53 558 pks3 knokdown --- --- --- 56 rp knokdown --- --- --- 559 rp2 knokdown 372 44 --- 56 rp3 knokdown 372 44 --- 56 rp4 knokdown 372 4 --- 559 orf6 knokdown 373 47 5 557 orf7 knokdown 372 47 496 553 orf8 knokdown 374 46 499 554 orf9 knokdown 376 47 499 556 orf knokdown 373 49 494 557 medium --- --- --- 56
Supplementry Tle S3: Moleulr formul, mss-to-hrge rtio nd retention time of different mino ids onjugted with monsorurin or ruropunttin in ulture filtrte of Peniillium mrneffei deteted y UHPLC-MS nlysis. Nme Moleulr Formul Mss-to-hrge rtio Retention time (min) Arginine-monsorurin C 29 H 38 N 4 O 6 538.279 6.37 Arginine-ruropunttin C 27 H 34 N 4 O 6 5.2478 3.47 Asprgine-monsorurin C 27 H 32 N 2 O 7 496.2282 8.9 Asprti id-monsorurin C 27 H 3 NO 8 497.25 9.584 Glutmi id-monsorurin C 28 H 33 NO 8 5.226 9.25 Glyine-monsorurin C 25 H 29 NO 6 439.995 9.533 Isoleuine-monsorurin C 29 H 37 NO 6 495.262 23.97 Leuine-monsorurin C 29 H 37 NO 6 495.262 22.94 Lysine-monsorurin C 29 H 38 N 2 O 6 5.273 6.93 Methionine-monsorurin C 28 H 35 NSO 6 53.285 22.279 Phenylline-monsorurin C 32 H 35 NO 6 529.2464 23.29 Phenylline ruropunttin C 3 H 3 NO 6 52.2224 9.73 Serine-monsorurin C 26 H 3 NO 7 469.2 8.9 Tryptophn-monsorurin C 34 H 36 N 2 O 6 568.2573 2.696 Tyrosine-monsorurin C 32 H 35 NO 7 545.244 9.348 Vline-monsorurin C 28 H 35 NO 6 48.2464 22.57
Supplementry Tle S4: Primers nd plsmids used for gene knokdown experiments in the present study. Gene Primer RE site F/R Sequene (5-3 ) Plsmid orf LPW7686 XhoI F CCGCTCGAGTATGTCCGGGCACAGACAAG ppw2284 LPW7687 HindIII R CCCAAGCTTGGGAGTGGGACGCAGTTGACACC LPW7688 KpnI F GGGGTACCTATGTCCGGGCACAGACAAG LPW7689 BglII R GAAGATCTAGTGGGACGCAGTTGACACC orf2 LPW769 XhoI F CCGCTCGAGGGGAGAACGTGCTTTTGCTC ppw2285 LPW769 HindIII R CCCAAGCTTGGGGAAGGAGCCGGTGACTTCAA LPW7692 KpnI F GGGGTACCGGGAGAACGTGCTTTTGCTC LPW7693 BglII R GAAGATCTGAAGGAGCCGGTGACTTCAA orf3 LPW7694 XhoI F CCGCTCGAGCGACACATACGATCATCCAGCA ppw2286 LPW7695 HindIII R CCCAAGCTTGGGTGTCGCTACCAGTTGGCTCA LPW7696 KpnI F GGGGTACCCGACACATACGATCATCCAGCA LPW7697 BglII R GAAGATCTTGTCGCTACCAGTTGGCTCA orf4 LPW7698 XhoI F CCGCTCGAGCGATACTCCGTGTGAAAGACTGC ppw2287 LPW7699 HindIII R CCCAAGCTTGGGGTGGCTGTGGTCAAGCAGTG LPW77 KpnI F GGGGTACCCGATACTCCGTGTGAAAGACTGC LPW77 BglII R GAAGATCTGTGGCTGTGGTCAAGCAGTG orf5 LPW772 XhoI F CCGCTCGAGCGTTTTCGTTACCGGAGGTT ppw2288 LPW773 HindIII R CCCAAGCTTGGGAGCATGGGTAGGAATGACCG LPW774 KpnI F GGGGTACCCGTTTTCGTTACCGGAGGTT LPW775 BglII R GAAGATCTAGCATGGGTAGGAATGACCG pks3 LPW9873 XhoI F CCGCTCGAGCCTTCTCTTTCGGATCTCTTC ppw294 LPW9874 KpnI F GGGGTACCCCTTCTCTTTCGGATCTCTTC LPW9875 BglII R GAAGATCTGCCTAATGTCAAGCTTTTCG rp LPW 826 XhoI F CCGCTCGAGCTGCTGGCGATACCGAGTTC ppw2422 LPW 827 HindIII R CCCAAGCTTGGGGCAAGGCATCAGCTCAATGA LPW 828 KpnI F GGGGTACCCTGCTGGCGATACCGAGTTC LPW 829 BglII R GAAGATCTGCAAGGCATCAGCTCAATGA rp2 LPW 776 XhoI F CCGCTCGAGCATGGGCTACTCGGTTTGGA ppw2289 LPW 777 HindIII R CCCAAGCTTGGGGTTCGCCTTTGGAGTTCTGC LPW 778 KpnI F GGGGTACCCATGGGCTACTCGGTTTGGA LPW 779 BglII R GAAGATCTGTTCGCCTTTGGAGTTCTGC rp3 LPW 82 XhoI F CCGCTCGAGGCAGTAATCGGTTGGGTTCG ppw2423 LPW 82 HindIII R CCCAAGCTTGGGCGCATGGAACTGAAGGATGA LPW 822 KpnI F GGGGTACCGCAGTAATCGGTTGGGTTCG LPW 823 BglII R GAAGATCTCGCATGGAACTGAAGGATGA rp4 LPW 824 XhoI F CCGCTCGAGAAAGTCAATGACCCTGCCGA ppw2424 LPW 825 HindIII R CCCAAGCTTGGGGTCAAAGACCTGGCTGGCAC LPW 826 KpnI F GGGGTACCAAAGTCAATGACCCTGCCGA LPW 827 BglII R GAAGATCTGTCAAAGACCTGGCTGGCAC
Gene Primer RE site F/R Sequene (5-3 ) Plsmid orf6 LPW8683 XhoI F CCGCTCGAGATAGACCTGAAATGGTTCCGATGG ppw2425 LPW8684 HindIII R CCCAAGCTTGGGGTGATGTAAATCCTCATCGAGCAG LPW8685 KpnI F GGGGTACCATAGACCTGAAATGGTTCCGATGG LPW8686 BglII F GAAGATCTGTGATGTAAATCCTCATCGAGCAG orf7 LPW8426 XhoI R CCGCTCGAGAGGCTCCTTTCTCCTCCGAT ppw2426 LPW8427 HindIII F CCCAAGCTTGGGTGAACGTGGATTGTGGGAAG LPW8428 KpnI R GGGGTACCAGGCTCCTTTCTCCTCCGAT LPW8429 BglII F GAAGATCTTGAACGTGGATTGTGGGAAG orf8 LPW843 XhoI R CCGCTCGAGCGCTGTGACCGAAATGGAT ppw2427 LPW843 HindIII F CCCAAGCTTGGGGGAGTCACACCGCAGATAATGA LPW8432 KpnI R GGGGTACCCGCTGTGACCGAAATGGAT LPW8433 BglII F GAAGATCTGGAGTCACACCGCAGATAATGA orf9 LPW8434 XhoI F CCGCTCGAGCTGGTTGGAGGTGCCTTGA ppw2428 LPW8435 HindIII R CCCAAGCTTGGGGCCCATACTGTGCGAATCGTA LPW8436 KpnI F GGGGTACCCTGGTTGGAGGTGCCTTGA LPW8437 BglII R GAAGATCTGCCCATACTGTGCGAATCGTA orf LPW8438 XhoI F CCGCTCGAGGAGGAGGAAATTGACCAGACTGG ppw2429 LPW8439 HindIII R CCCAAGCTTGGGGGTCAGACCTTCCTTGCCGT LPW844 KpnI F GGGGTACCGAGGAGGAAATTGACCAGACTGG LPW844 BglII R GAAGATCTGGTCAGACCTTCCTTGCCGT