YFGBI 2219 No.

of Pages 8, Model 5G
3 May 2010
ARTICLE IN PRESS

Fungal Genetics and Biology xxx (2010) xxx–xxx

1

Contents lists available at ScienceDirect

Fungal Genetics and Biology

journal homepage: www.elsevier.com/locate/yfgbi

2 Review

Fungal secondary metabolites – Strategies to activate silent gene clusters

F
3

4 Axel A. Brakhage a,b,*, Volker Schroeckh a

OO
5 a
Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI),
6 Beutenbergstr. 11a, 07745 Jena, Germany
7 b
Department of Microbiology and Molecular Biology, Institute for Microbiology, Friedrich Schiller University Jena, Germany

PR
a r t i c l e i n f o a b s t r a c t
1 5
2 0
11 Article history: Filamentous fungi produce a multitude of low molecular weight bioactive compounds. The increasing 26
12 Received 17 March 2010 number of fungal genome sequences impressively demonstrated that their biosynthetic potential is far 27
13 Accepted 21 April 2010 from being exploited. In fungi, the genes required for the biosynthesis of a secondary metabolite are clus- 28
14 Available online xxxx
tered. Many of these bioinformatically newly discovered secondary metabolism gene clusters are silent 29
under standard laboratory conditions. Consequently, no product can be found. This review summarizes 30
15 Keywords: D
the current strategies that have been successfully applied during the last years to activate these silent 31
16 Natural product
gene clusters in filamentous fungi, especially in the genus Aspergillus. 32
17 Secondary metabolite
18 Polyketide synthase
The techniques take advantage of genome mining, vary from the simple search for compounds with 33
TE
19 Nonribosomal peptide synthetase bioinformatically predicted physicochemical properties up to methods that exploit a probable interaction 34
20 Aspergillus of microorganisms. Until now, the majority of successful approaches have been based on molecular biol- 35
21 Epigenetics ogy like the generation of gene ‘‘knock outs”, promoter exchange, overexpression of transcription factors 36
22 Gene cluster or other pleiotropic regulators. Moreover, strategies based on epigenetics opened a new avenue for the 37
23 Microbial communication elucidation of the regulation of secondary metabolite formation and will certainly continue to play a sig- 38
24
nificant role for the elucidation of cryptic natural products. The conditions under which a given gene clus- 39
EC

ter is naturally expressed are largely unknown. One technique is to attempt to simulate the natural 40
habitat by co-cultivation of microorganisms from the same ecosystem. This has already led to the activa- 41
tion of silent gene clusters and the identification of novel compounds in Aspergillus nidulans. These sim- 42
ulation strategies will help discover new natural products in the future, and may also provide 43
fundamental new insights into microbial communication. 44
RR

Ó 2010 Published by Elsevier Inc. 45

46
47
48 1. Introduction with other organisms ranging from bacteria, fungi and algae to 63
protozoans and even metazoans (Goh et al., 2002; Losada et al., 64
CO

49 Although natural products have been empirically used in an- 2009). The evolution of these so-called secondary metabolites over 65
50 cient human populations (Bassett et al., 1980), it was only in the millions of years was most likely accomplished because microor- 66
51 20th century that we have started to identify and characterise sys- ganisms used them as chemical signals for communication, to de- 67
52 tematically these important compounds. Until today, besides their fend the habitat or to inhibit the growth of competitors. Although 68
53 protective functions e.g. against UV radiation as pigments of spores numerous natural products have been identified during the last 69
54 (Langfelder et al., 2003), natural products have been of major decades it is obvious that a plethora of compounds still await dis- 70
UN

55 interest because their suitability for the treatment of infectious covery. This assumption results not only from the fact that only a 71
56 diseases, cancer, as immunosuppressants, and, finally, as a persis- small number of microorganisms have been cultivated and discov- 72
57 tent source of new and innovative therapeutic agents and drug ered yet (reviewed in Yin et al. (2007)) but also from recent gen- 73
58 leads (Newman and Cragg, 2007). A multitude of these structurally ome sequencing projects (see below). A successful cultivation of 74
59 heterogenic low molecular weight molecules is produced by a new fungal strain is often sufficient to identify new natural prod- 75
60 microorganisms, especially by soil-dwelling bacteria and fungi. ucts. Examples are the isolation of the arugosins from the marine- 76
61 This is not a surprise taking into account that these organisms live derived fungus Emericella nidulans var. acristata (Kralj et al., 2006) 77
62 in complex ecosystems where they compete and communicate or the finding of deoxypodophyllotoxin, a pro-drug of the antican- 78
cer drug podophyllotoxin, in the endophytic fungus Aspergillus 79
fumigatus Fresenius (Kusari et al., 2009). 80
* Corresponding author at: Department of Molecular and Applied Microbiology,
Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll
The analysis of the growing number of sequenced fungal gen- 81

Institute (HKI), Beutenbergstr. 11a, 07745 Jena, Germany. Fax: +49 3641 5320802. omes revealed two interesting aspects: (i) the discovery of a large 82
E-mail address: axel.brakhage@hki-jena.de (A.A. Brakhage). number of genes by bioinformatic analyses based on the highly 83

1087-1845/$ - see front matter Ó 2010 Published by Elsevier Inc.

doi:10.1016/j.fgb.2010.04.004

Please cite this article in press as: Brakhage, A.A., Schroeckh, V. Fungal secondary metabolites – Strategies to activate silent gene clusters. Fungal Genet.
Biol. (2010), doi:10.1016/j.fgb.2010.04.004
 YFGBI 2219 No. of Pages 8, Model 5G
3 May 2010
ARTICLE IN PRESS

2 A.A. Brakhage, V. Schroeckh / Fungal Genetics and Biology xxx (2010) xxx–xxx

84 conserved PKS and NRPS genes, putatively involved in secondary as peptidyl carrier protein (PCP) that serves as an anchor for the 147
85 metabolite biosynthesis which is much greater than anticipated growing peptide chain. Additionally, a variety of optional [e.g. 148
86 even in strains that have been extensively studied for the forma- methyltransferase (MT) or epimerisation (E)] domains may be 149
87 tion of natural products and (ii) the confirmation of an earlier dis- present (Walsh et al., 2001). Similarly, three domains are the basic 150
88 covery, namely that most of these genes are located in clusters. equipment of most PKS elongation modules, i.e., an acyltransferase 151
89 Thus, the true biosynthetic potential of most secondary metabolite (AT) domain for extender unit selection and transfer, an acyl carrier 152
90 producing fungi remains to be determined. Two main challenges protein (ACP) for extender unit loading and a ketoacyl synthase 153
91 limit the access to these potentially new compounds: the inability (KS) domain for decarboxylative condensation of the extender unit 154
92 to cultivate diverse potentially interesting producers of natural (usually malonyl-CoA) with an acyl thioester. The resulting b-keto 155
93 products in the laboratory and the perception that the majority thioester may subsequently be processed by b-ketoacyl reductase 156
94 of secondary metabolite biosynthesis gene clusters is silent under (KR) domains, dehydratase (DH) domains, enoyl reductase (ER) 157
95 standard laboratory conditions (Hertweck, 2009). As long as the domains and MT domains (Cane, 1997). Modular PKS and NRPS 158

F
96 triggers for their induction have not been identified, these clusters systems can also closely cooperate to form so-called hybrid prod- 159
97 remain silent (Brakhage et al., 2008). Consistently, nearly none of ucts like rapamycin or yersiniabactin (Cane and Walsh, 1999), 160

OO
98 the transcripts of the predicted secondary metabolite gene clusters the antibiotic TA (Paitan et al., 1999), myxothiazol (Silakowski 161
99 in the Aspergillus niger strain CBS 513.88 were detected under stan- et al., 1999) and myxochromides S13 (Wenzel et al., 2005). 162
100 dard fermentation conditions (Pel et al., 2007). Until now, the genomes of eight Aspergillus species have been 163
101 This review summarizes the various strategies that have been published, i.e., Aspergillus clavatus (Fedorova et al., 2008), Aspergil- 164
102 successfully applied during the last years to activate silent gene lus flavus (Payne et al., 2006), A. fumigatus (Nierman et al., 2005), 165
103 clusters in filamentous fungi, especially in the genus Aspergillus. Aspergillus oryzae (Machida et al., 2005), Aspergillus nidulans (Gala- 166

PR
104 Furthermore, we also discuss the recent identification of poten- gan et al., 2005), A. niger (Pel et al., 2007), Aspergillus terreus (Broad/ 167
105 tially naturally occurring inducing conditions. There are several MIT) and Neosartorya fischeri (Fedorova et al., 2008). Sequencing of 168
106 other recently published excellent reviews dealing with various as- several other species and further strains of the above named Asper- 169
107 pects of secondary metabolite gene clusters in microorganisms gilli are under progress, e.g. of Aspergillus parasiticus (Univ. Okla- 170
108 Q1 (Zerikly and Challis, 2009; Scherlach and Hertweck, 2009; homa), Aspergillus aculeatus or Aspergillus carbonarius (DOE Joint 171
109 Hertweck, 2009; Chiang et al., 2009a,b; Brakhage et al., 2009a,b). D Genome Institute). The availability of the genome sequences has 172
led to rapid progress in identifying genes putatively responsible 173
for secondary metabolite production. An exciting result of the sub- 174
110 2. Genomics and secondary metabolism in Aspergillus sequent genome annotations was the high number of secondary 175
TE
metabolite gene clusters in Aspergilli, typically between 30 and 176
111 Filamentous fungi are well known producers of secondary 40 per species. This confirms the one-strain-many-compounds 177
112 metabolites (Hoffmeister and Keller, 2007). These compounds in- (OSMAC) approach put forward by Zeeck (Bode et al., 2002). The 178
113 clude e.g. the antibiotic penicillin (Brakhage, 1998), the immuno- vast majority of the genetically encoded metabolites, however, is 179
114 suppressant cyclosporine or the anti-hypercholesterolemic agent unknown and the corresponding gene clusters were therefore re- 180
EC

115 lovastatin (Keller et al., 2005). A literature survey covering more ferred to as ‘‘cryptic” or ‘‘orphan”. Moreover, only a small propor- 181
116 than 23,000 bioactive microbial products, i.e., antifungal, antibac- tion of the identified gene clusters has been found to be 182
117 terial, antiviral, cytotoxic and immunosuppressive agents, shows conserved between different species. To gain access to this unex- 183
118 that the producing strains are mainly from the fungal kingdom plored reservoir of metabolites, their biosyntheses need to be in- 184
119 (ca. 42%), followed by strains belonging to the genus Streptomyces duced. For that purpose, various strategies have been developed 185
RR

120 (32.1%) (Lazzarini et al., 2000). Consequently, fungi have been of and successfully applied (see below). These include the bioinfor- 186
121 interest to researchers interested in secondary metabolism for a matic analysis of fungal genomes with regard to cluster organiza- 187
122 long time. The publications of the pre-genomics era mainly dealt tion and the structure of multi-modular enzymes like PKSs and 188
123 with the optimization of the production conditions and on struc- NRPSs. In addition, the deduced putative secondary metabolome 189
124 ture determination. Studies on the genetic regulation were per- needs to be analysed which will lead to the discovery of novel com- 190
125 formed on a rather limited number of metabolites, such as pounds that may have been hitherto overlooked, e.g. due to very 191
CO

126 penicillins (Brakhage, 1998), cephalosporins (Schmitt et al., 2004) low levels or physicochemical properties that make their isolation 192
127 and aflatoxins (Keller and Hohn, 1997). These investigations also difficult. 193
128 showed that the biosynthesis genes for a particular pathway were Based on genome mining, several strategies have been proposed 194
129 located in clusters that can span a few tens of kbs. Another striking to induce expression of cryptic secondary metabolite gene clusters 195
130 finding was the involvement of large, multidomain and in fungi that can be subdivided into three categories: (i) methods 196
UN

131 multi-modular enzymes in the biosynthesis of many secondary that are mainly based on the bioinformatic prediction of the PKS 197
132 metabolites. These enzymes include polyketide synthases (PKSs), or NRPS, their substrates or their resulting metabolite product, 198
133 nonribosomal peptide synthetases (NRPSs), prenyltransferases (ii) molecular and epigenetics based methods and, (iii) methods 199
134 and terpene cyclases. Later on, genome analyses revealed that that attempt to predict natural conditions that lead to the 200
135 the adjacent genes belonging to the previously identified clusters activation. 201
136 encode mostly regulatory proteins, oxidases, hydroxylases and
137 transporters. PKSs and NRPSs utilize simple malonyl (PKS) or ami-
138 no acid (NRPS) building blocks or derivatives thereof. Although the 3. Genome mining for identification of cryptic secondary 202
139 substrates can differ considerably, these multi-modular enzymes metabolite gene clusters 203
140 show striking similarities in the architecture as well as in the
141 mechanisms used for product assembly. Each module is responsi- 3.1. Identification of metabolites by bioinformatic prediction of their 204
142 ble for one or more chain-elongation steps and can be subdivided physicochemical properties 205
143 into domains controlling the choice of the extender unit and con-
144 nection with the growing peptide chain. A typical NRPS module The principles underlying the synthesis of secondary metabolites 206
145 minimally consists of an adenylation (A) domain responsible for by multi-modular enzymes allow the design of bioinformatic tools 207
146 amino acid activation, and a thiolation (T) domain – also known that help to predict not only the assembly line of a given PKS or 208

Please cite this article in press as: Brakhage, A.A., Schroeckh, V. Fungal secondary metabolites – Strategies to activate silent gene clusters. Fungal Genet.
Biol. (2010), doi:10.1016/j.fgb.2010.04.004
 YFGBI 2219 No. of Pages 8, Model 5G
3 May 2010
ARTICLE IN PRESS

A.A. Brakhage, V. Schroeckh / Fungal Genetics and Biology xxx (2010) xxx–xxx 3

209 NRPS, but also its substrates and, hence, the physicochemical prop- bolic profile analysis of the mutant and the wild type, e.g. by 237
210 erties of the potential products (Fig. 1). This facilitates the subse- HPLC or LC-MS. This strategy was successfully followed e.g. by 238
211 quent isolation of metabolites from a respective microbial strain. Chiang et al. (2008) who discovered the emericellamide biosynthe- 239
212 An example of combining bioinformatic data with a systematic var- sis gene cluster in A. nidulans and subsequently elucidated its bio- 240
213 iation of the cultivation parameters is the discovery of the aspoqui- synthesis pathway that involves both a PKS and an NRPS (Fig. 2A). 241
214 nolones A–D (Fig. 1) (Scherlach and Hertweck, 2006). A more However, a major draw back of this strategy is that it is restricted 242
215 sophisticated extension of this method represents the so-called to the small number of metabolites that are produced under stan- 243
216 ‘‘genomisotopic approach”. It requires the feeding of labeled sub- dard cultivation conditions and clearly not applicable when the 244
217 strates, the search for labeled metabolites with the predicted prop- gene cluster of interest is silent. Therefore, alternative strategies 245
218 erties and the ‘‘in vitro reconstitution approach”. The later approach were based on the heterologous expression of genes controlled 246
219 is based on the heterologous production of the predicted biosynthe- by defined promoters (see above, Corre et al., 2008) or on the ex- 247
220 sis enzymes which then can be applied in vitro with the predicted change of promoters of secondary metabolite biosynthesis genes. 248

F
221 substrate to get the desired products (reviewed in Zerikly and Chal- Both methods would be feasible in particular for bacterial gene 249
222 lis, 2009). In summary, the described method is characterized by the clusters with polycistronic gene organization. In the case of fungal 250

OO
223 fact that on one hand there is no or only little genetic manipulation secondary metabolism gene clusters, however, promoter ex- 251
224 necessary, on the other hand, however, the required prediction steps changes can be cumbersome because they need to be employed 252
225 need a high degree of reliability to discriminate the putative second- for individual genes (Kennedy and Turner, 1996). Other drawbacks 253
226 ary metabolites from other molecules with similar physicochemical are the requirement of genetic techniques for the fungus investi- 254
227 properties. gated, e.g., when the low frequency of homologous recombination 255
in many fungi is considered, the cumbersome handling of large 256

PR
228 3.2. Methods based on gene knock outs and epigenetics gene constructs or, that overexpression of a single gene of a cluster 257
often does not overcome the limitation of another gene product of 258
229 As described above, systematic mining of the sequenced Asper- the same cluster. E.g., in the penicillin biosynthesis gene cluster of 259
230 gillus genomes had revealed the existence of nearly 40 cryptic bio- A. nidulans, only overexpression of the acvA gene encoding the 260
231 synthesis gene clusters for secondary natural products per genome. NRPS leads to significantly increased penicillin titres but not the 261
232 In A. nidulans, the deduced gene products imply that the fungus is overexpression of the two other genes of the cluster (Kennedy 262
233
234
able to generate up to 32 polyketides, 14 nonribosomal peptides
and two indole alkaloids (Brakhage et al., 2008; Rank et al., 2010).
D and Turner, 1996; Brakhage, 1998). These problems can be over-
come at least in part by the manipulation of pleiotropic regulators
263
264
235 The ‘‘classical” way to find a new metabolite relies on the inac- of secondary metabolism (see below) or in the overexpression of 265
TE
236 tivation of a biosynthesis gene followed by a comparative meta- pathway-specific regulatory genes, that are present in many 266
EC
RR
CO
UN

Fig. 1. Discovery of new secondary metabolites by predicting the physicochemical properties of a putative secondary metabolite. Prediction of the modular composition of
the PKS/NRPS was used to predict the most likely substrates and, hence, the physicochemical properties of the sought-after metabolite. The discovery of aspoquinolones by
combining bioinformatic data with a systematic variation of the cultivation parameters exemplifies this approach (Scherlach and Hertweck, 2006).

Please cite this article in press as: Brakhage, A.A., Schroeckh, V. Fungal secondary metabolites – Strategies to activate silent gene clusters. Fungal Genet.
Biol. (2010), doi:10.1016/j.fgb.2010.04.004
 YFGBI 2219 No. of Pages 8, Model 5G
3 May 2010
ARTICLE IN PRESS

4 A.A. Brakhage, V. Schroeckh / Fungal Genetics and Biology xxx (2010) xxx–xxx

F
OO
PR
D
Fig. 2. Discovery of new secondary metabolites by molecular biology based strategies. A. Knock out of a putative PKS or NRPS gene. A double knock out of the genes easA and
TE
easB encoding an NRPS and a PKS, respectively, led to the discovery of the emericellamides (Chiang et al., 2008). B. Overexpression of a gene cluster-associated transcription
factor gene. The overexpression of the regulator gene apdR under control of the alcohol dehydrogenase gene promoter alcA induced the formation of the aspyridones A and B
(Bergmann et al., 2007). C. Exchange of the promoter of a putative PKS or NRPS gene. The substitution of the PKS gene afoE promoter by the strong, regulatable alcA promoter
led to the production of asperfuranone (Chiang et al., 2009a,b).
EC

267 secondary metabolite gene clusters. The latter approach is feasible gests a link between production of secondary metabolites and 299
268 because all of the genes required for the biosynthesis of a typical morphological differentiation. Deletion of laeA decreased sterig- 300
269 secondary metabolite are clustered and in some cases, a single reg- matocystin and penicillin formation in A. nidulans and gliotoxin 301
270 ulator controls the expression of all members of the cluster to a production in A. fumigatus. By contrast, overexpression of laeA pos- 302
271 certain extent (McAlpine et al., 2005). However, potentially re- itively triggered the production of these natural products (Bok and 303
RR

272 quired post-translational modifications of cluster-specific regula- Keller, 2004). Comparative metabolite profiling of both the dele- 304
273 tors could lower the productivity of these approaches. For tion as well as the overexpression mutants led to the discovery 305
274 example, it has been shown that the sterigmatocystin gene cluster of the terrequinone A gene cluster in A. nidulans (Fig. 3A) (Bok 306
275 specific transcription factor AflR in A. nidulans needs to be phos- et al., 2006). Although 13 of 22 secondary metabolite gene clusters 307
276 phorylated to be efficiently transported into the nucleus (Shimizu of A. fumigatus analysed are positively regulated by LaeA (Perrin 308
277 et al., 2003). Despite these restrictions, the successful induction et al., 2007), no previously unknown secondary metabolite has 309
CO

278 of a silent metabolic pathway in A. nidulans by the overexpression been demonstrated in this fungus yet to be formed through awak- 310
279 of a pathway specific transcription factor was reported. Bergmann ening of a silent gene cluster by LaeA. 311
280 et al. (2007) overexpressed in A. nidulans a pathway-specific regu- Another approach comprising a pleiotropic effector is based on 312
281 latory gene of A. nidulans designated apdR which encodes a Zn2Cys6 the requirement of a 40 -phosphopantetheinyl transferase (PPTase) 313
282 transcription factor. Overexpression of the apdR gene led to activa- for activity of both PKSs and NRPSs. Comparative metabolic profiling 314
UN

283 tion of the apdR containing silent gene cluster including the endog- of a conditional mutant of the PPTase CfwA and a cfwA null mutant 315
284 enous apdR gene. As a result of this activation, two novel PKS–NRPS led to the identification of the polyketides shamixanthone, emeri- 316
285 hybrid metabolites, named aspyridones A and B, were discovered cellin, and dehydroaustinol in A. nidulans (Márquez-Fernández 317
286 (Fig. 2B). The advantage of this technique is that only a small gene et al., 2007). 318
287 needs to be handled, and that ectopic integration into the genome Because of its sequence similarity to histone and arginine meth- 319
288 of a construct existing of the regulatory gene fused with an induc- yltransferases, LaeA was suggested to act via chromatin remodel- 320
289 ible promoter would be sufficient, bypassing all limitations poten- ing (Keller et al., 2005). Accordingly, Keller and coworkers 321
290 tially based on a low rate of homologous recombination. Similarly, demonstrated the importance of histone deacetylase (HDA) activ- 322
291 the exchange of the promoter of a CtnR-like fungal transcriptional ity on the chemical diversity in Aspergillus (Shwab et al., 2007). Dis- 323
292 activator gene by the inducible alcA promoter derived from alcohol ruption of the hda gene in A. nidulans led to the transcriptional 324
293 dehydrogenase enabled the production of the polyketide asperf- activation of gene clusters for the production of sterigmatocystin 325
294 uranone in A. nidulans (Chiang et al., 2009a,b) (Fig. 2C). and penicillin. Based on this data, it was conceivable that chemical 326
295 A pleiotropic regulator of secondary metabolism in the genus epigenetics caused by the addition of chromatin-modulating 327
296 Aspergillus is the nuclear protein LaeA (Bok and Keller, 2004). The agents to fungi would allow to induce cryptic fungal gene clusters. 328
297 finding that LaeA and the light-regulated developmental factor This technique does not require strain-dependent genetic manipu- 329
298 VeA are both part of a nuclear complex (Bayram et al., 2008) sug- lation and can thus be applied to any fungal strain. Williams et al. 330

Please cite this article in press as: Brakhage, A.A., Schroeckh, V. Fungal secondary metabolites – Strategies to activate silent gene clusters. Fungal Genet.
Biol. (2010), doi:10.1016/j.fgb.2010.04.004
 YFGBI 2219 No. of Pages 8, Model 5G
3 May 2010
ARTICLE IN PRESS

A.A. Brakhage, V. Schroeckh / Fungal Genetics and Biology xxx (2010) xxx–xxx 5

F
OO
PR
D
TE
Fig. 3. Discovery of new secondary metabolites by epigenetics based methods. A. Overproduction of a pleiotropic regulator. The overexpression of the laeA gene encoding a
putative methyltransferase led to terrquinone A formation (Bok et al., 2006). B. Deletion of genes involved in histone modification positively influence the production of the
secondary metabolites. Deletion of the single A. nidulans sumoylation gene sumO induced asperthecin production (Bok et al., 2009). Deletion of the cclA gene, encoding one of
the eight COMPASS proteins in A. nidulans, led to the identification of monodiyctiphenone and emodins (Bok et al., 2009). The molecular basis on how these two deletions led
to gene cluster induction, remains to be elucidated. C. Feeding of compounds that modify the histone acetylation status, e.g. of histone deacetylase (HDAC) or DNA
methyltransferase (DMAT) inhibitors. The HDAC inhibitor suberoylanilide hydoxamic acid (SAHA) stimulated the production of nygerone A (Henrikson et al., 2009). By
EC

contrast, feeding the DMAT inhibitor 5-azacytidine elicited oxylipin biosynthesis (Williams et al., 2008).

331 (2008) confirmed the hypothesis that small-molecule epigenetic tion) revealed the genes responsible for asperthecin biosynthesis 361
332 modifiers could be rationally employed for accessing silent natural (Fig. 3B) (Szewczyk et al., 2008). Recently, the groups of Keller and 362
333 product pathways. Histone deacetylase (HDAC) or DNA methyl- Strauss reported experimental support for the regulation of second- 363
RR

334 transferase (DMAT) inhibitors were added to cultures. Among oth- ary metabolite gene clusters by metabolism-dependent, reversible 364
335 ers the HDAC inhibitor suberoylanilide hydoxamic acid (SAHA) formation of heterochromatin, in which LaeA plays a role in revert- 365
336 stimulated the production of new cladochromes and of calphostin ing the heterochromatic state (Reyes-Dominguez et al., 2010). 366
337 B in Cladosporium cladosporioides (Williams et al., 2008). By con- Yet another approach to discover natural products and their 367
338 trast, feeding the DMAT inhibitor 5-azacytidine elicited the de novo biosynthesis pathways stems from proteome analyses (Bumpus Q2 368
339 production of several oxylipins (Fig. 3C) by the same microorgan- et al., 2009). A mass spectrometry-based method, called PrISM 369
CO

340 ism and of lunalides in a Diatrype sp. culture (Williams et al., (Proteomic Investigation of Secondary Metabolism), permits the 370
341 2008). Another example that demonstrates the potential of this simultaneous identification of peptides and polyketides produced 371
342 strategy is the isolation of nygerone A from A. niger when cultured by NRPSs and PKSs enzymes, respectively, and of the responsible 372
343 with SAHA (Fig. 3C) (Henrikson et al., 2009). gene cluster. Although the method has not been applied to fungal 373
344 Histones are substrates for numerous modifications. Besides species, it appears to represent an impressive tool for the identifi- 374
UN

345 acetylation, methylation, phosphorylation, ubiquitination, sumoy- cation of new metabolites in Bacillus and Streptomyces species. 375
346 lation and neddylation belong to the mechanisms by which a certain
347 gene expression status is controlled. Methylation of lysine 4 on his- 3.3. Novel metabolites produced through simulation of natural gene 376
348 tone 3 is controlled by the COMPASS (complex associated with Set1) cluster activating conditions 377
349 complex. Deletion of the cclA gene, encoding one of the eight COM-
350 PASS proteins in A. nidulans, and subsequent chemical profiling of The functional role of natural products in the environment is 378
351 the fungal metabolites led to the identification of the gene clusters largely unknown and a matter of debate (Goh et al., 2002; Yin 379
352 responsible for monodyctiphenone, emodin and derivatives thereof et al., 2007; Aminov, 2009). The current opinions range from no 380
353 (Fig. 3B) as well as for the biosynthesis enzymes for the compounds role for secondary metabolites in increasing the fitness of an organ- 381
354 F-9775A and F-9775B (Bok et al., 2009). Deletion of the single A. ism to the opposite view, that every secondary metabolite is made 382
355 nidulans sumoylation gene sumO influenced the formation of several because it confers a biological advantage for the producer in a par- 383
356 secondary metabolites. Whereas a lower amount of austinol and ticular habitat (Firn and Jones, 2000; Brakhage et al., 2009a,b). 384
357 dehydroaustinol was formed, a strong increase in asperthecin pro- Bacteria and fungi co-inhabit a wide variety of environments, 385
358 duction occurred. The molecular mechanism behind these effects including soil, the rhizosphere, plants, mucosal membranes and 386
359 has not been elucidated yet, but genetic analysis of double knock the gut (Kobayashi and Crouch, 2009). Special forms of interactions 387
360 out mutants (each contain the sumO and a further PKS gene dele- encompass bacterial endosymbionts in fungi (Partida-Martinez and 388

Please cite this article in press as: Brakhage, A.A., Schroeckh, V. Fungal secondary metabolites – Strategies to activate silent gene clusters. Fungal Genet.
Biol. (2010), doi:10.1016/j.fgb.2010.04.004
 YFGBI 2219 No. of Pages 8, Model 5G
3 May 2010
ARTICLE IN PRESS

6 A.A. Brakhage, V. Schroeckh / Fungal Genetics and Biology xxx (2010) xxx–xxx

389 Hertweck, 2005), pathogenic interactions, the association of bacteria olite elucidation, the molecular and chemical nature behind this 428
390 with mykorrhiza (Bonfanta and Anca, 2009) or mutualistic interac- interspecies cross-talk remains largely unknown. Together with 429
391 tions in lichen holobionts (Grube and Berg, 2009). Furthermore, the group of Hertweck, we employed for the first time microarray 430
392 metagenomes isolated from environments such as soil, have been technology to monitor the selective induction of silent fungal bio- 431
393 shown to be rich sources for novel natural products further support- synthesis genes through bacterial–fungal interactions (Schroeckh 432
394 ing the view that close interaction of microorganisms is favorable for et al., 2009). Through individual co-cultivation of A. nidulans with 433
395 secondary metabolite production (Daniel, 2004). From this observa- a collection of 58 actinomycetes we identified a bacterium, Strepto- 434
396 tion and from the assumption that microorganisms interact with myces rapamycinicus, that selectively triggered the expression of 435
397 each other in their natural environment and that these interactions gene clusters that are silent under standard laboratory conditions. 436
398 constitute a driving force for the production of secondary metabo- Chemical analysis, qRT-PCR and gene inactivation revealed that the 437
399 lites, simulation of these habitats, e.g. by culturing two or more induced cryptic PKS gene codes for the long sought-after orsellinic 438
400 strains together, should be a rational way to detect new molecules. acid synthase. Orthologs of this synthase are widespread among 439

F
401 It is generally accepted that this molecular interplay can have the fungal domain including lichen mycobionts. Apart from this 440
402 drastic effects on the partners. For example, extracts of different archetypal polyketide, A. nidulans also produces a typical lichen 441

OO
403 Aspergillus strains have greater antifungal activity when grown in metabolite, the orsellinic acid derivative lecanoric acid (Fig. 4). It 442
404 the presence of another competing Aspergillus (Losada et al., is intriguing that this compound is usually found in a fungal/bacte- 443
405 2009). The presence of rhizoxin producing endobacterium Burk- rial mutualism, and thus likely plays a role in microbial communi- 444
406 holderia sp. is obligatory for sporulation of its host Rhizopus cation. On the other hand, because lecanoric acid inhibits ATP 445
407 microsporus (Partida-Martinez et al., 2007) and is required for pro- synthesis and electron transfer, it is also conceivable that the bac- 446
408 Q3 duction of the toxin rhizoxin by the fungus (Partida-Matinez and terium has elicited a fungal defense strategy affecting organisms 447

PR
409 Hertweck, 2005). Co-cultivation of two marine microorganisms, that are susceptible to lecanoric acid-mediated energy breakdown. 448
410 the fungus Emericella sp. and the actinomycete Salinispora arenicola Because the inducing bacterium is not affected by lecanoric acid, 449
411 enhanced the expression of the emericellamids biosynthesis gene one may even speculate about a symbiotic relation between the 450
412 cluster 100-fold (Oh et al., 2007). Their biosynthesis gene cluster fungus and the bacterium to defend against other microorganisms. 451
413 was identified in A. nidulans (Chiang et al., 2008). Pestalone, a po- The orsellinic acid synthase belongs to the large number of 6- 452
414 tent antibiotic against methicillin-resistant Staphylococcus aureus, D methylsalicylic acid-type PKSs found in lichen-forming fungi as 453
415 was isolated from a co-culture of another marine fungus, Pestalotia well as in actinomycetes. Investigations raising questions about 454
416 sp., with a yet unknown a-proteobacterium (Cueto et al., 2001). how the biosynthesis enzymes for typical lichen compounds, such 455
417 Fungal endophytes of the genus Epichloe synthesize the nonribos- as orsellinic acid derivatives have evolved in such unrelated organ- 456
TE
418 omal peptide and potent insect feeding deterrent peramine after isms support a horizontal gene transfer event from an ancient 457
419 colonizing perennial ryegrass thereby protecting these plants from actinobacterium to an ascomycete (Schmitt and Lumbsch, 2009). 458
420 Listronotus bonariensis feeding damage (Tanaka et al., 2005). It was Surprisingly, despite the many extracellular compounds and 459
421 demonstrated that loss of the laeA gene in A. nidulans caused a dis- proteins produced by Streptomyces (Chater et al., 2010), the expres- 460
422 advantage to the fungus when under the attack of the fungivorous sion of orsellinic and lecanoric acid biosynthesis genes in A. nidu- 461
EC

423 springtail Folsomia candida (Insecta: Collembola). The springtails lans does not rely on diffusible chemical signals secreted by the 462
424 preferentially fed on the laeA mutant compared with the wild-type streptomycete. Dialysis experiments and electron microscopy un- 463
425 strain (Rohlfs et al., 2007). veiled that an intimate, physical contact of the bacterium and the 464
426 Although these and other studies illustrated the potential of fungus is required to elicit the specific response (Fig. 4). This 465
427 mixed cultivations of microorganisms for novel secondary metab- unprecedented intimate trans domain interaction of Aspergillus 466
RR
CO
UN

Fig. 4. Formation of new secondary metabolites via interspecies cross-talk. A distinct actinomycete, the soil bacterium Streptomyces rapamycinicus induced the production of
orsellinic acid, the typical lichen compound lecanoric acid, and the cathepsin K inhibitors F-9775A and F-9775B in the filamentous fungus Aspergillus nidulans by a yet
unidentified mechanism that involves an intimate contact between the partner organisms (Schroeckh et al., 2009).

Please cite this article in press as: Brakhage, A.A., Schroeckh, V. Fungal secondary metabolites – Strategies to activate silent gene clusters. Fungal Genet.
Biol. (2010), doi:10.1016/j.fgb.2010.04.004
 YFGBI 2219 No. of Pages 8, Model 5G
3 May 2010
ARTICLE IN PRESS

A.A. Brakhage, V. Schroeckh / Fungal Genetics and Biology xxx (2010) xxx–xxx 7

467 and Streptomyces very likely simulates a scenario occurring in the Bayram, O., Krappmann, S., Ni, M., Bok, J.W., Helmstaedt, K., Valerius, O., Braus- 526
Stromeyer, S., Kwon, N.J., Keller, N.P., Yu, J.H., Braus, G.H., 2008. VelB/VeA/LaeA 527
468 field and raises important questions with regard to the molecular 528
complex coordinates light signal with fungal development and secondary
469 basis of such microbial interactions as well as to fungal signal metabolism. Science 320, 1504–1506. 529
470 transduction. Indeed, such close physical contacts seem to play a Bergmann, S., Schümann, J., Scherlach, K., Lange, C., Brakhage, A.A., Hertweck, C., 530
2007. Genomics-driven discovery of PKS–NRPS hybrid metabolites from 531
471 considerable role in microbial interactions in nature. Co-cultures 532
Aspergillus nidulans. Nat. Chem. Biol. 3, 213–217.
472 of Aspergillus proliferans and Streptomyces olivaceoviridis secreting Bode, H.B., Höfs, R., Bethe, B., Zeeck, A., 2002. Big effects from small changes: possible 533
473 the chitin-binding protein (CHB) form a closely tangled network ways to explore naturés chemical diversity. ChemBioChem. 3, 619–627. 534
Bok, J.W., Keller, N.P., 2004. LaeA, a regulator of secondary metabolism in 535
474 of fungal and streptomycete hyphae maintained by CHB molecules
Aspergillus. Euk. Cell 3, 527–535. 536
475 and aggregated to an extracellular matrix (Siemieniewicz and Bok, J.W., Hoffmeister, D., Maggio-Hall, L.A., Murillo, R., Glasner, J.D., Keller, N.P., 537
476 Schrempf, 2007). Furthermore, rhizosphere bacteria adhere to the 2006. Genomic mining for Aspergillus natural products. Chem. Biol. 13, 31–37. 538
477 cell surface of mycorrhizal fungi and cause changes in fungal gene Bok, J.W., Chiang, Y., Szewczyk, E., Davidson, A.D., Sanchez, J.F., Lo, H.C., Watanabe, 539
K., Oakley, B.R., Wang, C.C.C., Keller, N.P., 2009. Chromatin-level regulation of 540
478 expression (Bonfanta and Anca, 2009). The effector molecules are

F
cryptic biosynthetic gene clusters in Aspergillus nidulans. Nat. Chem. Biol. 5, 541
479 released by the bacteria that degrade the fungal cell walls or that 462–464. 542
480 are perceived by the fungus. Also, the injection of molecules via Bonfanta, P., Anca, I.-A., 2009. Plants, mycorrhizal fungi, and bacteria: a network of 543

OO
interactions. Annu. Rev. Microbiol. 63, 863–883. 544
481 the type III secretion system and the direct binding to the fungal Brakhage, A.A., 1998. Molecular regulation of beta-lactam biosynthesis in 545
482 cell wall via lectins appears to occur (Bonfanta and Anca, 2009). filamentous fungi. Microbiol. Mol. Rev. 62, 547–585. 546
483 Consequently, the concept of interspecies cross-talk leading to Brakhage, A.A., Schuemann, J., Bergmann, S., Scherlach, K., Schroeckh, V., Hertweck, 547
C., 2008. Activation of fungal silent gene clusters: a new avenue to drug 548
484 chemical diversity provides a conceptual framework for the eluci- 549
discovery. Prog. Drug Res. 66, 3–12.
485 dation of secondary metabolites and its function as infochemicals. Brakhage, A.A., Bergmann, S., Schuemann, J., Scherlach, K., Schroeckh, V., Hertweck, 550
C., 2009a. Fungal genome mining and activation of silent gene clusters. In: 551

PR
Esser, K. (Ed.), The Mycota XV. Springer-Verlag, Heidelberg, pp. 297–303. 552
486 4. Conclusions 553
Brakhage, A.A., Gierl, A., Hartmann, T., Strack, D., 2009b. Evolution of metabolic
diversity. Phytochemistry 70, 1619–1620. 554
487 The availability of a growing number of fungal genome se- Cane, D.E., 1997. Introduction: polyketide and nonribosomal polypeptide 555
biosynthesis. From collie to coli. Chem. Rev. 97, 2463–2464. 556
488 quences offers excellent prospects to discover numerous novel sec-
Cane, D.E., Walsh, C.T., 1999. The parallel and convergent universes of polyketide 557
489 ondary metabolites by genome mining. As we reported here, synthases and nonribosomal peptide synthetases. Chem. Biol. 6, R319–R325. 558
490 several successful strategies exist to uncover the up to 50 putative Chater, K.F., Biró, S., Lee, K.J., Palmer, T., Schrempf, H., 2010. The complex 559
D extracellular biology of Streptomyces. FEMS Microbiol. Rev. 34, 171–198. 560
491 secondary metabolites of a particular fungal species. The tech-
Chiang, Y.-M., Szewczyk, E., Nayak, T., Sanchez, J.F., Lo, H., Simityan, H., Kuo, E., 561
492 niques vary from the simple search for compounds with bioinfor- Praseuth, A., Watanabe, K., Oakley, B.R., Wang, C.C.C., 2008. Discovery of the 562
493 matically predicted physicochemical properties up to methods emericellamide gene cluster: an efficient gene targeting system for the genomic 563
TE
494 that exploit a probable interaction of microorganisms. Until now, mining of natural products in Aspergillus nidulans. Chem. Biol. 15, 527–532. 564
Chiang, Y.-M., Szewczyk, E., Davidson, A.D., Keller, N.-P., Oakley, B.R., Wang, C.C.C., 565
495 the majority of successful approaches have been based on molecu- 2009a. A gene cluster containing two fungal polyketide synthases encodes the 566
496 lar biology like the generation of gene ‘‘knock outs”, promoter ex- biosynthetic pathway for a polyketide, asperfuranone, in Aspergillus nidulans. J. 567
497 change, overexpression of transcription factors or other pleiotropic Am. Chem. Soc. 131, 2965–2970. 568
Chiang, Y.-M., Lee, K.-H., Sanchez, J.F., Keller, N.P., Wang, C.C.C., 2009b. Unkocking 569
498 regulators. Moreover, strategies based on epigenetics opened a 570
EC

fungal cryptic natural products. Nat. Prod. Commun. 4, 1505–1510.

499 new avenue for the elucidation of the regulation of secondary Corre, C., Song, L., O’Rourke, S., Chater, K.F., Challis, G.L., 2008. 2-Alkyl-4- 571
500 metabolite formation and will certainly continue to play a signifi- hydroxymethylfuran-3-carboxylic acids, antibiotic production inducers 572
discovered by Streptomyces coelicolor genome mining. Proc. Natl. Acad. Sci. 573
501 cant role for the elucidation of cryptic natural products. 574
USA 105, 17510–17515.
502 The various tools for genome mining have both strengths and Cueto, M., Jensen, P.R., Kauffman, C., Fenical, W., Lobkovsky, E., Clardy, J., 2001. 575
503 weaknesses. The main obstacle is that the conditions under which Pestalone, a new antibiotic produced by a marine fungus in response to 576
RR

bacterial challenge. J. Nat. Prod. 64, 1444–1446. 577

504 a given gene cluster is expressed are largely unknown. Hence, the
Daniel, R., 2004. The soil metagenome – a rich source for the discovery of novel 578
505 development and application of strategies that simulate the natu- natural products. Curr. Opin. Biotechnol. 15, 199–204. 579
506 ral habitat of cluster-bearing microorganisms, e.g. by co-cultiva- Fedorova, N.D., Khaldi, N., Joardar, V.S., Maiti, R., Amedeo, P., Anderson, M.J., 580
Crabtree, J., Silva, J.C., Badger, J.H., Albarraq, A., Angiuoli, S., Bussey, H., Bowyer, 581
507 tion of one or more microorganisms from the same ecosystem,
P., Cotty, P.J., Dyer, P.S., Egan, A., Galens, K., Fraser-Liggett, C.M., Haas, B.J., 582
508 are required and have already demonstrated their potential (Oh Inman, J.M., Kent, R., Lemieux, S., Malavazi, I., Orvis, J., Roemer, T., Ronning, C.M., 583
509 et al., 2007; Schroeckh et al., 2009). Studies on such systems will 584
CO

Sundaram, J.P., Sutton, G., Turner, G., Venter, J.C., White, O.R., Whitty, B.R.,
510 not only provide fundamental insights into microbial communica- Youngman, P., Wolfe, K.H., Goldman, G.H., Wortman, J.R., Jiang, B., Denning, 585
D.W., Nierman, W.C., 2008. Genomic islands in the pathogenic filamentous 586
511 tion, but will also become an integral part of research for natural fungus Aspergillus fumigatus. PLoS Genet. 4, e1000046. 587
512 product discovery in the future. Firn, R.D., Jones, C.G., 2000. The evolution of secondary metabolism – a unifying 588
model. Mol. Microbiol. 37, 989–994. 589
Galagan, J.E., Calvo, S.E., Cuomo, C., Ma, L.J., Wortman, J.R., Batzoglou, S., Lee, S.I., 590
513 5. Uncited reference 591
Basturkmen, M., Spevak, C.C., Clutterbuck, J., Kapitonov, V., Jurka, J., Scazzocchio,
UN

C., Farman, M., Butler, J., Purcell, S., Harris, S., Braus, G.H., Draht, O., Busch, S., 592
514 Q4 Yim and Wang (2008). D’Enfert, C., Bouchier, C., Goldman, G.H., Bell-Pedersen, D., Griffiths-Jones, S., 593
Doonan, J.H., Yu, J., Vienken, K., Pain, A., Freitag, M., Selker, E.U., Archer, D.B., 594
Penalva, M.A., Oakley, B.R., Momany, M., Tanaka, T., Kumagai, T., Asai, K., 595
515 Acknowledgments Machida, M., Nierman, W.C., Denning, D.W., Caddick, M., Hynes, M., Paoletti, M., 596
Fischer, R., Miller, B., Dyer, P., Sachs, M.S., Osmani, S.A., Birren, B.W., 2005. 597
516 Work in the authors’ laboratory was supported by the Excel- Sequencing of Aspergillus nidulans and comparative analysis with A Fumigatus 598
and A. oryzae. Nature 438, 1105–1115. 599
517 lence Graduate School Jena School for Microbial Communication, 600
Goh, E.B., Yim, G., Tsui, W., McClure, J., Surette, M.G., Davies, J., 2002. Transcriptional
518 the International Leibniz Research School for Microbial and Molecular modulation of bacterial gene expression by subinhibitory concentrations of 601
519 Interactions, and the Leibniz Institute for Natural Product Research antibiotics. Proc. Natl. Acad. Sci. USA 99, 17025–17030. 602
Grube, M., Berg, G. 2009. Microbial consortia of bacteria and fungi with focus on the 603
520 and Infection Biology – Hans Knoell Institute (HKI). 604
lichen symbiosis. Fungal Biol. Rev. doi:10.1016/j.fbr.2009.10.001.
Henrikson, J.C., Hoover, A.R., Joyner, P.M., Cichewicz, R.H., 2009. A chemical 605
521 References epigenetics approach for engineering the in situ biosynthesis of a cryptic 606
natural product from Aspergillus niger. Org. Biomol. Chem. 7, 435–438. 607
Hertweck, C., 2009. Hidden biosynthetic treasures brought to light. Nat. Chem. Biol. 608
522 Aminov, R.I., 2009. The role of antibiotics and antibiotic resistance in nature.
5, 450–452. 609
523 Environ. Microbiol. 11, 2970–2988.
Hoffmeister, D., Keller, N.P., 2007. Natural products of filamentous fungi: enzymes, 610
524 Bassett, E.J., Keith, M.S., Armelagos, G.J., Martin, D.L., 1980. Tetracycline-labeled
genes, and their regulation. Nat. Prod. Rep. 24, 393–416. 611
525 human bone from ancient Sudanese Nubia (AD 350). Science 209, 1532–1534.

Please cite this article in press as: Brakhage, A.A., Schroeckh, V. Fungal secondary metabolites – Strategies to activate silent gene clusters. Fungal Genet.
Biol. (2010), doi:10.1016/j.fgb.2010.04.004
 YFGBI 2219 No. of Pages 8, Model 5G
3 May 2010
ARTICLE IN PRESS

8 A.A. Brakhage, V. Schroeckh / Fungal Genetics and Biology xxx (2010) xxx–xxx

612 Keller, N.P., Hohn, T.M., 1997. Metabolic pathway gene clusters in filamentous Pel, H.J., de Winde, J.H., Archer, D.B., Dyer, P.S., Hofmann, G., Schaap, P.J., Turner, G., 691
613 fungi. Fungal Genet. Biol. 21, 17–29. de Vries, R.P., Albang, R., Albermann, K., Andersen, M.R., Bendtsen, J.D., Benen, 692
614 Keller, N.P., Turner, G., Bennett, J.W., 2005. Fungal secondary metabolism – from J.A., van den Berg, M., Breestraat, S., Caddick, M.X., Contreras, R., Cornell, M., 693
615 biochemistry to genomics. Nat. Rev. Microbiol. 3, 937–947. Coutinho, P.M., Danchin, E.G., Debets, A.J., Dekker, P., van Dijck, P.W., van Dijk, 694
616 Kennedy, J., Turner, G., 1996. [delta]-(L-[alpha]-aminoadipyl)-L-cysteinyl-D-valine A., Dijkhuizen, L., Driessen, A.J., d”Enfert, C., Geysens, S., Goosen, C., Groot, G.S., 695
617 synthetase is a rate limiting enzyme for penicillin production in Aspergillus de Groot, P.W., Guillemette, T., Henrissat, B., Herweijer, M., van den Hombergh, 696
618 nidulans. Mol. Gen. Genet. 253, 189–197. J.P., van den Hondel, C.A., van der Heijden, R.T., van der Kaaij, R.M., Klis, F.M., 697
619 Kobayashi, D.Y., Crouch, J.A., 2009. Bacterial/fungal interactions: from pathogens to Kools, H.J., Kubicek, C.P., van Kuyk, P.A., Lauber, J., Lu, X., van der Maarel, M.J., 698
620 mutualistic endosymbionts. Annu. Rev. Phytopathol. 47, 63–82. Meulenberg, R., Menke, H., Mortimer, M.A., Nielsen, J., Oliver, S.G., Olsthoorn, M., 699
621 Kralj, A., Kehraus, S., Krick, A., Eguereva, E., Kelter, G., Maurer, M., Wortmann, A., Pal, K., van Peij, N.N., Ram, A.F., Rinas, U., Roubos, J.A., Sagt, C.M., Schmoll, M., 700
622 Fiebig, H.H., Konig, G.M., 2006. Arugosins G and H: prenylated polyketides from Sun, J., Ussery, D., Varga, J., Vervecken, W., van de Vondervoort, P.J., Wedler, H., 701
623 the marine-derived fungus Emericella nidulans var. acristata. J. Nat. Prod. 69, Wosten, H.A., Zeng, A.P., van Ooyen, A.J., Visser, J., Stam, H., 2007. Genome 702
624 995–1000. sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. 703
625 Kusari, S., Lamshöft, M., Spiteller, M., 2009. Aspergillus fumigatus Fresenius, an Nat. Biotechnol. 25, 221–231. 704
626 endophytic fungus from Juniperus communis L. Horstmann as a novel source of Perrin, R.M., Fedorova, N.D., Bok, J.W., Cramer, R.A., Wortman, J.R., Kim, H.S., 705

F
627 the anticancer pro-drug deoxypodophyllotoxin. J. Appl. Microbiol. 107, 1019– Nierman, W.C., Keller, N.P., 2007. Transcriptional regulation of chemical 706
628 1030. diversity in Aspergillus fumigatus by LaeA. PLoS Pathog. 3, e50. 707
629 Langfelder, K., Streibel, M., Jahn, B.J., Haase, G., Brakhage, A.A., 2003. Melanin Rank, C., Larsen, T.O., Frisvad, J.C., 2010. Functional systems biology of Aspergillus. 708

OO
630 biosynthesis and virulence of human pathogenic fungi. Fungal Genet. Biol. 38, In: Machida, M., Gomi, K. (Eds.), Aspergillus. Molecular Biology and Genomics. 709
631 143–158. Caister Academic Press, Norfolk, pp. 173–198. 710
632 Lazzarini, A., Cavaletti, L., Toppo, G., Marinelli, F., 2000. Rare genera of Reyes-Dominguez, Y., Bok, J.W., Berger, H., Shwab, E.K., Basheer, A., Gallmetzer, A., 711
633 actinomycetes as potential producers of new antibiotics. Anton Van Lee 78, Scazzocchio, C., Keller, N.P., Strauss, J. 2010. Heterochromatic marks are 712
634 399–405. associated with the repression of secondary metabolism clusters in Aspergillus 713
635 Losada, L., Ajayi, O., Frisvad, J.C., Yu, J.J., Nierman, W.C., 2009. Effect of competition nidulans. Mol. Microbiol. doi:10.1111/j.1365-2958.2010.07051.x. 714
636 on the production and activity of secondary metabolites in Aspergillus species. Rohlfs, M., Albert, M., Keller, N.P., Kempken, F., 2007. Secondary chemicals protect 715
637 Med. Mycol. 47, S88–S96. mould from fungivory. Biol. Lett. 3, 523–525. 716

PR
638 Machida, M., Asai, K., Sano, M., Tanaka, T., Kumagai, T., Terai, G., Kusumoto, K., Arima, Scherlach, K., Hertweck, C., 2006. Discovery of aspoquinolones A–D, prenylated 717
639 T., Akita, O., Kashiwagi, Y., Abe, K., Gomi, K., Horiuchi, H., Kitamoto, K., Kobayashi, quinoline-2-one alkaloids from Aspergillus nidulans, motivated by genome 718
640 T., Takeuchi, M., Denning, D.W., Galagan, J.E., Nierman, W.C., Yu, J., Archer, D.B., mining. Org. Biomol. Chem. 4, 3517–3520. 719
641 Bennett, J.W., Bhatnagar, D., Cleveland, T.E., Fedorova, N.D., Gotoh, O., Horikawa, Scherlach, K., Hertweck, C., 2009. Triggering cryptic natural product biosynthesis in 720
642 H., Hosoyama, A., Ichinomiya, M., Igarashi, R., Iwashita, K., Juvvadi, P.R., Kato, M., microorganisms. Org. Biomol. Chem. 7, 1753–1760. 721
643 Kato, Y., Kin, T., Kokubun, A., Maeda, H., Maeyama, N., Maruyama, J., Nagasaki, H., Schmitt, I., Lumbsch, H.T., 2009. Ancient horizontal gene transfer from bacteria 722
644 Nakajima, T., Oda, K., Okada, K., Paulsen, I., Sakamoto, K., Sawano, T., Takahashi, enhances biosynthetic capabilities of fungi. PLoS ONE 4, e4437. 723
645 M., Takase, K., Terabayashi, Y., Wortman, J.R., Yamada, O., Yamagata, Y., Anazawa, Schmitt, E.K., Hoff, B., Kück, U., 2004. Regulation of cephalosporin biosynthesis. Adv. 724
646 H., Hata, Y., Koide, Y., Komori, T., Koyama, Y., Minetoki, T., Suharnan, S., Tanaka, A., Biochem. Eng./Biotechnol. 88, 1–43. 725
647 Isono, K., Kuhara, S., Ogasawara, N., Kikuchi, H., 2005. Genome sequencing and
D
Schroeckh, V., Scherlach, K., Nützmann, H.-W., Shelest, E., Schmidt-Heck, W., 726
648 analysis of Aspergillus oryzae. Nature 438, 1157–1161. Schuemann, J., Martin, K., Hertweck, C., Brakhage, A.A., 2009. Intimate bacterial- 727
649 Márquez-Fernández, O., Trigos, A., Ramos-Balderas, J.L., Viniegra-González, G., fungal interaction triggers biosynthesis of archetypal polyketides in Aspergillus 728
TE
650 Deising, H.B., Aguirre, J., 2007. Phosphopantetheinyl transferase CfwA/NpgA is nidulans. Proc. Natl. Acad. Sci. USA 106, 14558–14563. 729
651 required for Aspergillus nidulans secondary metabolism and asexual Shimizu, K., Hicks, J.K., Huang, T.-P., Keller, N.P., 2003. Pka, Ras and RGS protein 730
652 development. Euk. Cell 6, 710–720. interactions regulate activity of AflR, a Zn(II)2Cys6 transcription factor in 731
653 McAlpine, J.B., Bachmann, B.O., Piraee, M., Tremblay, S., Alarco, A.M., Zazopoulos, E., Aspergillus nidulans. Genetics 165, 1095–1104. 732
654 Farnet, C.M., 2005. Microbial genomics as a guide to drug discovery and Shwab, E.K., Bok, J.W., Tribus, M., Galehr, J., Graessle, S., Keller, N.P., 2007. Histone 733
655 structural elucidation: ECO-02301, a novel antifungal agent, as an example. J. deacetylase activity regulates chemical diversity in Aspergillus. Euk. Cell 6, 734
656 735
EC

Nat. Prod. 68, 493–496. 1656–1664.

657 Newman, D.J., Cragg, G.M., 2007. Natural products as sources of new drugs over the Siemieniewicz, K.W., Schrempf, H., 2007. Concerted responses between the chitin- 736
658 last 25 years. J. Nat. Prod. 70, 461–477. binding protein secreting Streptomyces olivaceoviridis and Aspergillus proliferans. 737
659 Nierman, W.C., Pain, A., Anderson, M.J., Wortman, J.R., Kim, H.S., Arroyo, J., Berriman, Microbiology 153, 593–600. 738
660 M., Abe, K., Archer, D.B., Bermejo, C., Bennett, J., Bowyer, P., Chen, D., Collins, M., Silakowski, B., Schairer, H.U., Ehret, H., Kunze, B., Weinig, S., Nordsiek, G., Brandt, P., 739
661 Coulsen, R., Davies, R., Dyer, P.S., Farman, M., Fedorova, N., Fedorova, N., Blocker, H., Hofle, G., Beyer, S., Muller, R., 1999. New lessons for combinatorial 740
662 Feldblyum, T.V., Fischer, R., Fosker, N., Fraser, A., Garcia, J.L., Garcia, M.J., Goble, biosynthesis from myxobacteria. The myxothiazol biosynthetic gene cluster of 741
RR

663 A., Goldman, G.H., Gomi, K., Griffith-Jones, S., Gwilliam, R., Haas, B., Haas, H., Stigmatella aurantiaca DW4/3–1. J. Biol. Chem. 274, 37391–37399. 742
664 Harris, D., Horiuchi, H., Huang, J., Humphray, S., Jimenez, J., Keller, N., Khouri, H., Szewczyk, E., Chiang, Y., Oakley, E., Davidson, A.D., Wang, C.C.C., Oakley, B.R., 2008. 743
665 Kitamoto, K., Kobayashi, T., Konzack, S., Kulkarni, R., Kumagai, T., Lafon, A., SUMO regulation of secondary metabolite production in Aspergillus nidulans: 744
666 Latge, J.P., Li, W., Lord, A., Lu, C., Majoros, W.H., May, G.S., Miller, B.L., identification of the asperthecin gene cluster. Appl. Environ. Microbiol. 74, 745
667 Mohamoud, Y., Molina, M., Monod, M., Mouyna, I., Mulligan, S., Murphy, L., 7607–7612. 746
668 O’Neil, S., Paulsen, I., Penalva, M.A., Pertea, M., Price, C., Pritchard, B.L., Quail, Tanaka, A., Tapper, B.A., Popay, A., Parker, E.J., Scott, B., 2005. A symbiosis expressed 747
669 M.A., Rabbinowitsch, E., Rawlins, N., Rajandream, M.A., Reichard, U., Renauld, H., non-ribosomal peptide synthetase from a mutualistic fungal endophyte of 748
670 Robson, G.D., Rodriguez de, C.S., Rodriguez-Pena, J.M., Ronning, C.M., Rutter, S., 749
CO

perennial ryegrass confers protection to the symbiotum from insect herbivory.

671 Salzberg, S.L., Sanchez, M., Sanchez-Ferrero, J.C., Saunders, D., Seeger, K., Mol. Microbiol. 57, 1036–1050. 750
672 Squares, R., Squares, S., Takeuchi, M., Tekaia, F., Turner, G., Vazquez de Walsh, C.T., Chen, H., Keating, T.A., Hubbard, B.K., Losey, H.C., Luo, L., Marshall, C.G., 751
673 Aldana, C.R., Weidman, J., White, O., Woodward, J., Yu, J.H., Fraser, C., Galagan, Miller, D.A., Patel, H.M., 2001. Tailoring enzymes that modify nonribosomal 752
674 J.E., Asai, K., Machida, M., Hall, N., Barrell, B., Denning, D.W., 2005. Genomic peptides during and after chain elongation on NRPS assembly lines. Curr. Opin. 753
675 sequence of the pathogenic and allergenic filamentous fungus Aspergillus Chem. Biol. 5, 525–534. 754
676 fumigatus. Nature 438, 1151–1156. Wenzel, S.C., Kunze, B., Höfle, G., Silakowski, B., Scharfe, M., Blöcker, H., Müller, R., 755
677 Oh, D.C., Kauffman, C.A., Jensen, P.R., Fenical, W., 2007. Induced production of 2005. Structure and biosynthesis of myxochromides S1–3 in Stigmatella 756
UN

678 emericellamides A and B from the marine-derived fungus Emericella sp. In aurantiaca: evidence for an iterative bacterial type I polyketide synthase and 757
679 competing co-culture. J. Nat. Prod. 70, 515–520. for module skipping in nonribosomal peptide biosynthesis. ChemBioChem. 6, 758
680 Paitan, Y., Alon, G., Orr, E., Ron, E.Z., Rosenberg, E., 1999. The first gene in the 375–385. 759
681 biosynthesis of the polyketide antibiotic TA of Myxococcus xanthus codes for a Williams, R.B., Henrikson, J.C., Hoover, A.R., Lee, A.E., Cichewicz, R.H., 2008. 760
682 unique PKS module coupled to a peptide synthetase. J. Mol. Biol. 286, 465–474. Epigenetic remodeling of the fungal secondary metabolome. Org. Biomol. 761
683 Partida-Martinez, L.P., Hertweck, C., 2005. Pathogenic fungus harbours Chem. 6, 1895–1897. 762
684 endosymbiotic bacteria for toxin production. Nature 437, 884–888. Yim, G., Wang, H.H., 2008. Antibiotics as signalling molecules. Philos. Trans. R. Soc. 763
685 Partida-Martinez, L.P., Monajembashi, S., Greulich, K.O., Hertweck, C., 2007. London, B Biol. Sci. 362, 1195–1200. 764
686 Endosymbiont-dependent host reproduction maintains bacterial-fungal Yin, J., Straight, P.D., Hrvatin, S., Dorrestein, P.C., Bumpus, S.B., Jao, C., Kelleher, N.L., 765
687 mutualism. Curr. Biol. 17, 773–777. Kolter, R., Walsh, C.T., 2007. Genome-wide high-throughput mining of natural- 766
688 Payne, G.A., Nierman, W.C., Wortman, J.R., Pritchard, B.L., Brown, D., Dean, R.A., product biosynthetic gene clusters by phage display. Chem. Biol. 14, 303–312. 767
689 Bhatnagar, D., Cleveland, T.E., Machida, M., Yu, J., 2006. Whole genome Zerikly, M., Challis, G.L., 2009. Strategies for the discovery of new natural products 768
690 comparison of Aspergillus flavus and A. Oryzae. Med. Mycol. 44 (Suppl), 9–11. by genome mining. Chembiochem. 10, 625–633. 769
770

Please cite this article in press as: Brakhage, A.A., Schroeckh, V. Fungal secondary metabolites – Strategies to activate silent gene clusters. Fungal Genet.
Biol. (2010), doi:10.1016/j.fgb.2010.04.004