Electronic Supplementary Material
Chemical induction of silent biosynthetic pathway transcription in Aspergillus niger
K. M. Fisch · A. F. Gillaspy · M. Gipson · J. C. Henrikson · A. R. Hoover · L. Jackson ·
F. Z. Najar · H. Wägele · R. H. Cichewicz
e-mail:
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Contents
Fig. S1Analysis of 2-CT values (mean and range of six replicates) for three housekeeping genes in control and treated (SAHA and 5-AZA) A. niger ATCC 1015 cultures
Table S1Scaffold alignments for A. niger ATCC 1015 versus A. niger CBS 513.88
Table S2 List of epigenetic modifiers and their effects on mycelial growth and secondary metabolite production in A. niger ATCC 1015
Table S3 Primers used to probe the expression of PKS, NRPS, HPN and housekeeping genes in A. niger ATCC 1015
Table S4 Accession numbers of the proteins included in the phylogenetic analysis of KS domains
Table S5 Identification of expressed A. niger secondary metabolite pathways in a comprehensive EST library
Fig. S1Analysis of 2-CT values (mean and range of six replicates) for three housekeeping genes in control and treated (SAHA and 5-AZA) A. niger ATCC 1015 cultures
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Table S2 List of epigenetic modifiers and their effects on mycelial growth and secondary metabolite production in A. niger ATCC 1015
Compound(mode of action) / Effect on cell growth
(log [inhibitor], M)a / Secondary metabolite diversityb / Notes
-3 / -4 / -5 / -6 / -7
Acetohydroxamic acid
(presumed HDAC inhibitor) / ++ / +++ / +++ / +++ / +++ / ND / No significant changes in growth
5-Azacytidine (5-AZA)
(DNMT inhibitor) / + / ++ / +++ / +++ / +++ / 10 / Culture turned pale yellow; new metabolites confirmed by HPLC
5-Aza-2'-deoxycytidine
(DNMT inhibitor) / ++ / ++ / +++ / +++ / +++ / 8 / Results similar to 5-azacytidine
Benzohydroxamic acid
(presumed HDAC inhibitor) / - / + / ++ / +++ / +++ / 8 / Strong color change (yellow) at 10 µM
Coumarin
(SIR2 deacetylase inhibitor) / - / ++ / ++ / +++ / +++ / ND / Culture phenotype altered (small pellets)
Dihydrocoumarin
(SIR2 deacetylase inhibitor) / - / + / ++ / ++ / +++ / 11 / Culture phenotype altered (small pellets)
7,8-Dihydroxycoumarin
(SIR2 deacetylase inhibitor) / ++ / ++ / +++ / +++ / +++ / ND / Little toxicity, culture phenotype altered (small pellets)
Eosin Y
(histone methyltransferase inhibitor) / - / + / ++ / ++ / +++ / 9 / Cells exhibited strong discoloration from dye
Eosin Y disodium salt
(histone methyltransferase inhibitor) / - / + / + / ++ / +++ / 10 / Cells exhibited strong discoloration from dye
Hydralazine hydrochloride
(DNMT inhibitor) / + / ++ / ++ / +++ / +++ / 11 / Culture phenotype altered (small pellets); strong color change (yellow) at 10 µM
Nicotinamide
(SIR2 deacetylase inhibitor) / + / ++ / ++ / +++ / +++ / 10 / Strong color change (yellow) at 100 µM
Nicotinic acid hydroxamate
(presumed SIR2 deacetylase/HDAC inhibitor) / + / ++ / ++ / +++ / +++ / 8 / Strong color change (yellow) at 100 µM
Procainamide hydrochloride
(DNMT inhibitor) / ++ / ++ / +++ / +++ / +++ / ND / No significant changes in growth
Procaine hydrochloride
(DNMT inhibitor) / ++ / ++ / +++ / +++ / +++ / ND / No significant changes in growth
Sodium butyrate
(HDAC inhibitor) / + / ++ / ++ / +++ / +++ / 8 / No significant changes in growth
Splitomicin
(HDAC/sirtuin inhibitor) / - / + / ++ / +++ / +++ / 7 / Poor solubility of splitomicin in aqueous medium
Suberohydroxamic acid
(HDAC inhibitor) / + / ++ / +++ / +++ / +++ / 4 / Culture turned pale yellow
Suberoylanilide hydroxamic acid (SAHA)
(HDAC inhibitor) / + / ++ / ++ / +++ / +++ / 9 / Culture phenotype altered; strong color change at 100 µM; new metabolites confirmed by HPLC
a Growth of A. niger was qualitatively assessed by visual inspection of replicate cultures. Fungal growth was scored as “+++” (growth equivalent to control cultures), “++” (<50% reduction in growth), “+” (≥50% reduction in growth), or “-” (no growth observed). Growth scores indicated in bold and underlined represent the concentration of epigenetic modifier used for assessing secondary metabolite chemical diversity
b Chemical diversity was determined as the number of new spots observed from crude culture extracts by silica gel TLC. Plates were developed with toluene-ethyl acetate-acetic acid (50:50:1 by volume). A designation of “ND” indicates that the chemical diversity of that particular treatment was not determined
Table S3 Primers used to probe the expression of PKS, NRPS, HPN and housekeeping genes in A. niger ATCC 1015a
Gene name / Forward primer (5'-3') / Reverse primer (5'-3') / Primer length (nt) / Product size (bp)PKS 1 / CAGCGCCAACAATCAGAACCTC / TTGCTCGGGCTGAACTGTGTATC / 21 / 179
PKS 2 / GGACCTCCATGCGAAAGATAC
GCTGTTCACCCTACGACCATTGA / GGGCCTCTCAAGTCGTATTCGT
ACTGCATCATTGTCACTCCTG / 21
23 / 108
149
PKS 3 / TTACCCCGCGACCAGAAACAC
AAACCCCATTGCTACTAAAGG / TAACACGTCGGGGCGGAATAC
CCCCGAAAAGAGCTACCATA / 21
21 / 179
286
PKS 4 / AACCCGTCCCGCTCAAGTCATC / CACCCAATTACGGCACCACAAA / 22 / 165
PKS 6 / TGCAGGCGATTAAAGGTTCAAG
ACAACCGGTGGGACAATCTG / GTTCATCGGTCATTTTCTCAAG
CTCGACCGCCGCGTTTTCAA / 22
20 / 106
75
PKS 8 / TGCATGGCGAAATATCCTGAAG
GAAGAGGTCGGTCCACAC / CAACGGCTGGAAATACGACAAG
CATTGTCTTGGTTCGTTCC / 22
18 / 86
105
PKS 9 / TTGTGGCCGATGAAAAGGAAG
CCCGGGTGGCAAGGCTGTTA / GATGTCGAAGGGGATGGTCTT
TGCGGCGTAAAAGGGTCATCT / 21
20 / 143
224
PKS 12 / GGCAGGAAACAAGGGTTATGAG
GGAAGTGCGAATGAACTGATAGTG
GATACGGAGCCCCCATTCAG / TATCCTCCAAGTCGGGTAAAGTTC
GTGCGGCGATAATCCATCAAG
CGGCGTTGTACTCCCCCATAG / 22
24
20 / 110
90
186
PKS 13 / CCGACCTTAGCTGGAGACTTA / TAGCGGGCGTTTGGATACTTG / 21 / 263
PKS 14 / TGAATTGGACGCCCGATGAGAT
TCGCTGGCGTGTTCGTGGTA / GTCCGCAAAAGTCCAGTCGTAG
GTTGCCGGCTGCGTAGTTG / 22
20 / 136
279
PKS 15 / ACAGCGACGGGAAGACATC
CAGATGATGGAGGTGGTGAGAG / CTTGTACCGGCTCTGTCGGA
CTCCAGCCAGTCCTCCCCGTAAC / 19
22 / 111
108
PKS 20 / TTGTTGCCCCATCAGCCAGACG
CGGGCAAATCCTACGACAACTAAG / AGCCGCCAGCACCAGTTGAG
ATGCGGGCCAACCTCAATCACTA / 22
24 / 256
186
PKS 25 / GCGTGACTGGCAAAGTCGTTAG
GCAGCTGCAACTCGCCATCTG / AGAGAACTGCACTGGCGATACC
ACCCAGGTGAGTTGGATGATTC / 22
21 / 86
82
PKS 26 / CGATCGGCGAAAATGGTAGTAGA / CTCAAGCGGATTCCAGTCATTC / 21 / 156
PKS 27 / CCTGAGAATCGGCGCAACGGTG / GGTAGGGCGGCAGGTCAATAG / 22 / 275
PKS 30 / CGCGGCGACATACGAGATTGAT
TCCACCGCGCCGACAAATAC / CAGGTTTGCCCGCGAGTGAT
CCCAGGAGCGAAGCAAGTCTAC / 22
20 / 130
165
PKS 32 / GAAGCCGTCATTCGGGAAGTAG
GGCCGTTGTTGGACATTC
CTCTTGCGGCCTGGTGGTTAC / AGCACGGGCATAAGTATCTCTA
TAGAATCCTCGGTAGTATGC
GCATGGGGGAATTGGTCTCA / 22
18
21 / 109
103
205
PKS 33 / CTACGCCGCGAAGAAGATAC
ATGTCGCCTCGGGAAGCACT / GAGCCGCGGTTGTCAGGTA
GTTACCGCCAGGGATGAAATAG / 20
20 / 256
213
PKS 34 / CTTGGGGCCTCACGAACAGT / GCTCCCGAAGAAGATGGTC / 20 / 235
PKS 36 / CGCCACATTATTCGCTTATCTGA
GCCGAGCCACTATTCTATACATTC / GCCACTTGCCGCATGCCCTCTA
TCCCCGCAACTGCTGAACATC / 23
24 / 119
74
PKS 37 / GGGACCGGTTAAAAGCATTGAC / TAAGCGTAGCATCCGAGTGGTG / 22 / 224
PKS 38 / GGTGCGAGGGTAAACCAAGTC
TTCGTGCCGCTGGATTTAC
CGCGATGCCCGGTTTGCTAT
TCGCAATGCCGATGGGTATG / ATCGCCAAGGAGCCAAGTAG
GGGCCTAGAGATGATGGTGAAT
ACCGGCCGCCTCTTCATCAGT
CGCCTGTTTGTATGCCGTTCTTA / 21
19
20
20 / 76
89
225
202
Table S3 (Continued)
Gene name / Forward primer (5'-3') / Reverse primer (5'-3') / Primer length (nt) / Product size (bp)PKS 39 / CGTGCTCCGGATCAATAGTCG / CTTCGCCGCGGCAATACC / 21 / 201
PKS 40 / TCATACAGGGCAGGCAAACTAC
ATTGCGTCGGCCAGGTTCG / GCGGCGAGATGGAAAACAATG
CCGGTTAGCGCAGTAGGTTC / 22
19 / 292
220
PKS 42 / AAGCGGGAGGGCCAAAACTGATAG / AGGCCAACTGACCCACAACATC / 24 / 153
PKS 43 / CGCGAAGAGGAAAGAGACAATA / ATCGGGCAAATTTCGGCATAC / 22 / 222
PKS 44 / ACGGCCCAGAAATGTCAACTC
CCGACTTCTTGACCGACTGCT / GTGTCAGGCCGGGAAACCAA
CGACGAACCCACCGACTCCTTG / 21
21 / 238
178
PKS 45 / CATGAGTACAGCGTTGAAGTC
CGAAGCGCACAATCAAGAAG / CCGAAAGTGCTGAGTGTGG
GCTCTCGCATTTAGGGTCAT / 21
20 / 83
94
PKS 46 / AGGGCATCCAGAATACGCACACTC / TGGCCGGCCATGAACAACC / 24 / 106
PKS 48 / CCACAACACCCGCAACATTAC / GTGCCAGCATCTCGTCGTC / 21 / 275
PKS 53 / TGTCCGGCGGTGTTAATGTGAT / TTTACGGCGGAGGCAGCGATGA / 22 / 217
PKS 54 / TCAGCGCGGCGGGAGGAAATA / GCGATGCGGTGTTTGTGATG / 21 / 235
PKS 55 / GGGGCATTTGTACGTCACTTTC / ATCCTCATTTGCGGGGTTTTGT / 21 / 210
NRPS 4 / CGCGGATTACAAGTCGTCGTTC / TGCGGCCAATTCATCCAAGTTTAG / 22 / 288
NRPS 6 / GGGGATGTCACCTTCGTTATG / ACAGCTTCTTCGTCGGCAGTCA / 21 / 272
NRPS 8 / AGGTTGCCGTTGAGGTTGTTAC / AGGCGCCGCATGGGGAAGTA / 21 / 223
NRPS 9 / GTCCGCATGGCATTCTTTACAC
ACCGCTATGCCGCTGTTCTGTA / GAGGTCGCGATCCACTACACAG
GCTGTAGGGGATATTGCTGATTG / 22
22 / 296
185
NRPS 12 / CGACGCGGAATGAATCTGAG
ATTCTACTCTTGCCGCCTTCACTG / GCGTCGGGGCAATGTAACCA
CGCATGCCACGCCCTTACCAAC / 24 / 236
NRPS 14 / TTGCGGCCACAATAACACCTA / CGCCCCTGCCGTACCAAACTT / 21 / 288
NRPS 15 / TCTGCAGCCCTTCCGATTTACAT / AGGACCGCTCCATACTGATTTG / 23 / 262
NRPS 17 / CTCCGCGTCAATCTCACTACCTC / CGGAATCATCGCGACAAAATCTC / 23 / 227
NRPS 18 / GGCCCTCCATCACGCTCTTTAT
TTCGCCAGTTACACCTTTGATAC / CGCTGTTCTTGGCCTCGTATGT
TCCGGGCGCATGAGACTAATAA / 22
23 / 233
179
NRPS 22 / AATCGCCGCTGGTTTGTGCTGA
CAAGCCGCTGGGAAACACTCACT / CCGAGGGAAGAGATGGGAATG
GACAGCTCCGCCACAACATCTC / 22
23 / 235
229
NRPS 23 / TGCCGTCGGAAGCCACAGTCAT
TCAATGCGCCGTCCCTCTCAGT / TCCGCATCAGCCTTGGTCAGTG
CAATGCGCCGTCCCTCTCAGT / 22
22 / 175
177
NRPS 26 / TCGGGCAGCACAGGCAAACC / GGGCGGAGAAGAGCGAGAAGAA / 20 / 163
NRPS 29 / GCACCCACTTTCCCCTCCTTAC / CGGTCAATGCGAACACGAACTG / 22 / 275
NRPS 30 / TCCGCCTTGACTTTGCTTTGAC / GCCATACCCGCCTTGCTAATAC / 22 / 208
NRPS 31 / CCGATATTGCGCAGCGTCTTTG / GCGAGCAGCATCAGGTTATTTTG / 22 / 167
HPN 7 / GGCAAGTTCATGATGGGGATAA / CTTTTGTGATGCGCTGGGAGAA / 22 / 189
HPN 10 / GGAGACTCTGCTGCCAAACCTA / TCTCTCCGCTGCCTCCGACAAGT / 22 / 214
Table S3 (Continued)
Gene name / Forward primer (5'-3') / Reverse primer (5'-3') / Primer length (nt) / Product size (bp)HPN 11 / TAGTGATGCCGCGGGTCTTGAT
ATTGTTCCGGTTTGGTTTCAG / TTCGGCTTCTGCGCATTGTC
GCCGTCAGGTGGTCATACAGAG / 22
21 / 159
139
HPN 16 / CCGACGATTTGCCCTTATTTCC / TAGCGTGGGCGATTCCGAGACAG / 22 / 234
HPN 23 / TCAGTGGCCGCGTCCTTAG
CGCGCCGTCTGAGTATTGTGTC
TCAATGCGCCGTCCCTCTCAGT / GCGTAGGCTGGATGACAATGATA
TTTGCAAAGGCCTCACCACCAC
CAATGCGCCGTCCCTCTCAGT / 19
22
22 / 279
102
177
HPN 28 / AGCGGAAAAGATCGAACTCAAC
AGATGGCCGCAGACCGTTGTTC
GGCCTCTTCCGATTGGTGTTC
CTGCTGGTGGGCTCTATCAAAAC / ATAGACCGGCGGGCACTGTTCA
GCGGCCCATATCGTTGTAGGT
TTCCGCAAGTAGCCGTAGTCTC
AAGGCGCCGTAATATGGAAGAA / 22
22
21
23 / 249
114
190
161
HPN 29 / GGCCGCTGAGGATTGGTTCGT
CCGGTGCCAAAACTGTATGAG
CCGTCGCAGAAACCATTGTG / GGAGCAGTCGGATCGTTTGTTC
CGGGTCCGGGTCGTAAATG
TCAGCGAACAGGACAGGATTG / 21
21
20 / 223
276
139
HPN 31 / CCAGCCTGAAGCATCGTCC / GGGTTCTCAGGCACTTGGTC / 19 / 229
HPN 47 / ATGGCAACCCCGTCCGAGTATTC
CTGCGGCCAAGGCTGCTATT / AGGACCGGCAGGGATGAGAGTA
TGCTCCTCAACCGCCTTCTGTAG / 23
20 / 249
235
actin / GGTCTGGAGAGCGGTGGTAT / GAAGAAGGAGCAAGAGCAGTG / 20 / 231
sarA / ACGGGTAAGGGCAAGGTTC / TCGCAACAATAAAGTCAACAGC / 19 / 227
cox5 / GACCAAGGAGTGGCAGGAG / GGTGGGAGGCAGCAGTTC / 19 / 313
aValues that are “struck-through” with a line indicate primers that exhibited dimerization during initial PCR tests and were subsequently replaced by non-dimerizing primers.
Table S4 Accession numbers of the proteins included in the phylogenetic analysis of KS domainsa
KS protein sequence name used in the tree / Protein accession numberAphanizomenon ovalisporum / AAM33468.1
Aspergillus fumigatus alb1 / AAC39471.1 and XP_756095.1
Aspergillus fumigatus Af293 Q4WFD7 / Q4WFD7
Aspergillus fumigatus A1163 EDP48973.1 / EDP48973.1
Aspergillus nidulans AN0150.2 / XP_657754.1
Aspergillus nidulans AN1034.2 / XP_658638.1
Aspergillus nidulans AN1036.2 / XP_658640.1
Aspergillus nidulans AN1784.2 / XP_659388.1
Aspergillus nidulans AN3386.2 / XP_660990.1
Aspergillus nidulans AN3612.2 / XP_661216.1
Aspergillus nidulans AN6791.2 / XP_664395.1
Aspergillus nidulans pksST / AAA81586.1
Aspergillus nidulans wA / CAA46695.2
Aspergillus parasiticus pksL1 / AAC41675.1
Aspergillus parasiticus pksL2 / AAC23536.1
Aspergillus terreus at1 / BAB88688.1
Aspergillus terreus at4 / BAB88689.1
Aspergillus terreus at5 / BAB88752.1
Aspergillus terreus LDKS / AAD34559.1
Aspergillus terreus LNKS / AAD39830.1
Aspergillus terreus pksM / AAC49814.1 and BAA20102.2
Botryotinia fuckeliana PKS1 / AAR90237.1
Botryotinia fuckeliana PKS3 / AAR90239.1
Botryotinia fuckeliana PKS8 / AAR90244.1
Botryotinia fuckeliana PKS12 / AAR90248.1
Botryotinia fuckeliana PKS14 / AAR90250.1
Botryotinia fuckeliana PKS16 / AAR90252.1
Botryotinia fuckeliana PKS17 / AAR90253.1
Caenorhabditis elegans / NP_492417.1
Chaetomium globosum CBS 148.51 CHGG_05113 / XP_001224327.1
Cochliobolus heterostrophus PKS1 / AAB08104.2
Cochliobolus heterostrophus PKS2 / AAR90257.1 and ABB76806.1
Cochliobolus heterostrophus PKS22 / AAR90276.1
Cochliobolus heterostrophus PKS24 / AAR90278.1
Colletotrichum lagenarium PKS1 / BAA18956.1
Cordyceps bassiana TenS / CAL69597.1
Cylindrospermopsis raciborskii AWT205 / ABX60161.1
Dothistroma septosporum pksA / AAZ95017.1
Fusarium heterosporum Eqis / AAV66106.1
Gallus gallus / NP_990486.1
Gibberella moniliformis PKS1 / AAR92208.1
Gibberella moniliformis PKS2 / AAR92209.1
Gibberella moniliformis PKS3 / CAC88775.1
Gibberella moniliformis PKS4 / CAB92399.1
Table S4 (Continued)
KS protein sequence name used in the tree / Protein accession numberGibberella moniliformis PKS5 / AAR92212.1
Gibberella moniliformis PKS6 / AAR92213.1
Gibberella moniliformis PKS7 / AAR92214.1
Gibberella moniliformis PKS8 / AAR92215.1
Gibberella moniliformis PKS9 / AAR92216.1
Gibberella moniliformis PKS10 / AAR92217.1
Gibberella moniliformis PKS11 / AAR92218.1
Gibberella moniliformis PKS12 / AAR92219.1
Gibberella moniliformis PKS13 / AAR92220.1
Gibberella moniliformis PKS14 / AAR92221.1
Gibberella moniliformis PKS15 / AAR92222.1
Gibberella zeae PKS13 / ABB90282.1
Glarea lozoyensis PKS1 / AAN59953.1
Glarea lozoyensis PKS2 / AAX35547.1
Homo sapiens / NP_004095.4
Monascus purpureus citrinin / BAD44749.1 and AAY33862.1
Monascus purpureus PKS / CAC94008.1 and AAY33863.1
Penicillium citrinum mlcA / BAC20564.1
Penicillium citrinum mlcB / BAC20566.1
Penicillium patulum 6MSAS / P22367.1
Wangiella dermatitidis WdPKS1 / AAD31436.3
Bae 3 / YP_001421292.1 (BaeJ KS3)
Bae 4 / YP_001421292.1 (BaeJ KS4)
Bae 5 / YP_001421292.1 (BaeJ KS5)
Bae 6 / YP_001421293.1 (BaeL KS6)
Bae 7 / YP_001421293.1 (BaeL KS7)
Bae 8 / YP_001421293.1 (BaeL KS8)
Bae 9 / YP_001421293.1 (BaeL KS9)
Bae 10 / YP_001421294.1 (BaeM KS10)
Bae 11 / YP_001421294.1 (BaeM KS11)
Bae 13 / YP_001421295.1 (BaeN KS13)
Bae 14 / YP_001421295.1 (BaeN KS14)
Bae 15 / YP_001421295.1 (BaeN KS15)
EpoA / AAF26919.1
EpoB / AAF26920.1
EpoC2 / AAF26921.1 first KS
EpoC3 / AAF26921.1 second KS
EpoC4 / AAF26921.1 third KS
EpoC5 / AAF26921.1 fourth KS
EpoD6 / AAF26922.1 first KS
EpoD7 / AAF26922.1 second KS
EpoE / AAF26923.1
a For Aspergillus oryzae, A. fumigatus Af293, Phoma sp. C2932, and partial KS protein sequences, the corresponding protein accession numbers are given in the trees. For all other species, protein accession numbers were used as presented in this table.
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