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Application of genome-wide strategies for the mining of secondary metabolite biosynthesis pathways in filamentous fungi

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Application of genome-wide strategies for the mining of secondary metabolite biosynthesis pathways in filamentous fungi

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APPLICATION OF GENOME-WIDE STRATEGIES FOR THE
MINING OF SECONDARY METABOLITE BIOSYNTHESIS
PATHWAYS IN FILAMENTOUS FUNGI

by
Junko Yaegashi

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)

May 2015

Copyright 2015 Junko Yaegashi
i

DEDICATION

I dedicate this work to my parents, Junichi and Kazuko Yaegashi,
for their immense love, support, and understanding.

ii

ACKNOWLEDGEMENTS
I would like to thank my mentor and advisor, Professor Clay C.C. Wang, for his guidance and
support. When I first joined the PIBBS program, I had no idea I would be working on fungi, but
when I talked to Dr. Wang, his enthusiasm and wealth of knowledge drew me into the world of
fungal natural product research. His style of mentoring has trained me in critical thinking with
great opportunities to conduct independent research, which will be a valuable asset for me as a
scientist.
I would also like to thank the professors who served on my committee, Professor Curtis
Okamoto, Professor Bogdan Olenyuk, Professor Bangyan Stiles, and Professor Chao Zhang for
their insightful comments and encouragement.
Furthermore, I would like to show my appreciation to all the former and current members of the
Wang lab for all their help and support. In particular, I would like to show my gratitude to Dr.
Yi-Ming Chiang, Dr. Shu-Lin Chang, and Dr. James Sanchez, who assisted me throughout my
research endeavors. I’d also like to thank my original lunch buddy, Mike Praseuth, who showed
me that you can “chillax” and have a little fun even in grad school, and also my 2
nd
generation
lunch buddies/ 7
th
floor inhabitants John Gallagher and Dr. James Sanchez for their great
friendship and support.
Chapter 1 is incorporated from Yaegashi, J., Oakley, B.R., and Wang, C.C.C. (2014). Recent
advances in genome mining of secondary metabolite biosynthetic gene clusters and the
development of heterologous expression systems in Aspergillus nidulans. Journal of Industrial
Microbiology & Biotechnology 41, 433-442.
iii

Chapter 2 is incorporated from Sanchez, J.F., Entwistle, R., Hung, J.H., Yaegashi, J., Jain, S.,
Chiang, Y.M., Wang, C.C.C., and Oakley, B.R. (2011). Genome-Based Deletion Analysis
Reveals the Prenyl Xanthone Biosynthesis Pathway in Aspergillus nidulans. Journal of the
American Chemical Society 133, 4010-4017.
Chapter 3 is incorporated from Yaegashi, J., Praseuth, M.B., Tyan, S.-W., Sanchez, J.F.,
Entwistle, R., Chiang, Y.-M., Oakley, B.R., and Wang, C.C.C. (2013). Molecular Genetic
Characterization of the Biosynthesis Cluster of a Prenylated Isoindolinone Alkaloid Aspernidine
A in Aspergillus nidulans. Organic Letters 15, 2862-2865.
Chapter 4 and 5 are manuscripts currently in preparation for submission to Journal of the
American Chemical Society and Organic Letters, respectively.
The author has made the most contributions to the review in Chapter 1 and the study
described in Chapter 3, 4, and 5 and some contributions to the study in Chapter 2.

iv

TABLE OF CONTENTS

DEDICATION i
ACKNOWLEGEMENTS ii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT xi
CHAPTER I: Introduction 1
1.1 Recent advances in genome mining of secondary metabolite biosynthetic
gene clusters and the development of heterologous expression systems in
Aspergillus nidulans 1
1.1.1 Abstract 1
1.1.2 Introduction 1
1.1.3 The status of annotating secondary metabolite genes in A.
nidulans 3
1.1.4 Bioinformatic advances 4
1.1.5 Genome-wide kinase knock-outs 6
1.1.6 Genome-wide analysis of all non-reduced polyketide synthases
and NRPS-like enzymes in A. nidulans 8
1.1.7 Use of A. nidulans as a host for heterologous expression of SM
genes from other Aspergillus species 9
1.1.8 Conclusions 11
1.2 Application of the genome mining strategy to Penicillium species 18
1.2.1 Significance of Penicillium 18
1.2.2 The status of secondary metabolite gene cluster elucidation in
Penicillium 20
1.2.3 Conclusion 22
CHAPTER II: Genome-based deletion analysis reveals the prenyl xanthone
biosynthesis pathway in Aspergillus nidulans 23
2.1 Abstract 23
2.2 Introduction 25
2.3 Results 28
2.4 Discussion 35
2.5 Conclusion 40
2.6 Materials and Methods 41
2.7 Supplementary Information 44

v

CHAPTER III: Molecular genetic characterization of the biosynthesis cluster of a
prenylated isoindolinone alkaloid aspernidine A in Aspergillus nidulans 82
3.1 Abstract 82
3.2 Introduction 83
3.3 Results and Discussion 85
3.4 Materials and Methods 89
3.5 Supplementary Information 93

CHAPTER IV: Application of an efficient gene targeting system linking secondary
metabolites to their biosynthetic genes in Penicillium canescens 115
4.1 Abstract 115
4.2 Introduction 116
4.3 Results and Discussion 118
4.4 Materials and Methods 126
CHAPTER V: Molecular genetic analysis of a diterpenic meroterpenoid 15-
deoxyoxalicine B biosynthesis gene cluster in Penicillium canescens 151
5.1 Abstract 151
5.2 Introduction 152
5.3 Results 155
5.4 Discussion 159
5.5 Materials and Methods 163
5.6 Supplementary Information 166

CHAPTER VI: Conclusion and perspective 191

BIBLIOGRAPHY 196

vi

LIST OF TABLES
Table 1-1. Secondary metabolism gene clusters in A. nidulans 13
Table 2-1. Gene designations of the text and corresponding annotations from two
websites 52
Table 2-2. Primers used in this study 53
Table 2-3. A. nidulans strains used in this study 58
Table 3-1. Putative function of genes within the aspernidine A cluster 98
Table 3-2. Primers used in this study 99
Table 3-3. A.nidulans strains used in this study 101
Table 4-1. List of all putative PKS and NRPS genes in P. canescens 128
Table 4-2. Primers used in this study 129
Table 5-1. Putative function of genes within the 15-deoxyoxalicine B cluster 170
Table 5-2. Primers used in this study 171
Table 5-3.
1
H-NMR Data for Compounds 1-3 174
Table 5-4. .
1
H-NMR Data for Compounds 4 and 6 175
Table 5-5.
1
H-NMR Data for Compound 7 176
vii

Table 5-6.
1
H-NMR Data for Compound 8 177
viii

LIST OF FIGURES
Figure 1-1. Structures of compounds isolated from A. nidulans 17
Figure 2-1. The prenyl xanthones and the compounds that emerged from the study
of targeted gene deletions 61
Figure 2-2. HPLC profiles of extracts from stcJ, cclA, mdpG detected by UV
absorption at 254 nm 62
Figure 2-3. Organization of the genes surrounding the PKS mdpG 63
Figure 2-4. HPLC extracts of strains in the cluster as detected by UV absorbance 64
Figure 2-5. HPLC extracts of xptA, sptB, and xptC as detected by UV absorption 65
Figure 2-6. Proposed biosynthesis of prenyl xanthones 66
Figure 2-7. UV/Vis and ESI-MS of 1-6 67
Figure 2-8. UV/Vis and ESI-MS of 7-17 68
Figure 2-9. to Figure 2-21
1
H NMR and
13
C NMR spectra 69
Figure 3-1. HPLC profiles of extracts of strains as detected by UV absorbance at
254 nm 102
Figure 3-2. Structures of compounds elucidated throughout the study 103
Figure 3-3. Organization of the genes surrounding the PKS pkfA 104
ix

Figure 3-4. HPLC profile of extracts of strains in the cluster as detected by UV
absorption at 254 nm 105
Figure 3-5. Proposed biosynthesis pathway of aspernidine A 106
Figure 3-6. UV/Vis and ESI-MS of isolated compounds 8-11 107
Figure 3-7. to Figure 3-12.
1
H NMR and
13
C NMR spectra 108
Figure 3-13. Putative aspernidine A cluster embedded within a conserved syntenic
region of Aspergillus genome 114
Figure 4-1. Structures of compounds identified in this study 138
Figure 4-2. Strategy for ku70 deletion in P. canescens using hygromycin
resistance marker (hph) 139
Figure 4-3. Strategy for pyrG deletion 140
Figure 4-4. Morphological difference between control and mutants carrying
Protein ID 366620 deletion 141
Figure 4-5. HPLC profile of extracts from control strain and Protein ID 243077
deletant 142
Figure 4-6. HPLC profile of extracts from control strain and Protein ID 308305
deletant 143
x

Figure 4-7. HPLC profile of extracts from control strain and Protein ID 371741
deletant 144
Figure 4-8. HPLC profile of extracts from control strain and Protein ID 400488
deletant 145
Figure 4-9. UV-Vis and ESIMS spectra of compounds 1 to 4. 146
Figure 4-10.
1
H NMR spectrum of griseofulvin (1) 147
Figure 4-11.
1
H NMR spectrum of xanthoepocin (2) 148
Figure 4-12.
1
H NMR spectrum of 15-deoxyoxalicine B (3) 149
Figure 4-13.
1
H NMR spectrum of amauromine (4) 150
Figure 5-1. Structurally related meroterpenoids 178
Figure 5-2. Structures of novel compounds isolated and characterized from this
study 179
Figure 5-3. HPLC profiles of extracts from control and olcA ∆ strains 180
Figure 5-4. Orientation of the genes surrounding the PKS olcA involved in 15-
deoxyxoalicine B biosynthesis 181
Figure 5-5. HPLC extracts of strains in the cluster as detected by UV absorbance
at 254 nm 182
Figure 5-6. Proposed biosynthesis pathway for 15-deoxyoxalicine B 183
Figure 5-7. UV-Vis and ESI-MS spectra of compounds isolated from this study 184
xi

Figure 5-8.
1
H NMR spectrum of decaturin A (3) 186
Figure 5-9.
1
H NMR spectrum of decaturin C (4) 187
Figure 5-10.
1
H NMR spectrum of decaturin F (6) 188
Figure 5-11.
1
H NMR spectrum of predecaturin E (7) 189
Figure 5-12.
1
H NMR spectrum of decaturin G (8) 190

xii

ABSTRACT
Genome projects of filamentous fungi have generated an unprecedented amount of information,
and in a moment in time often referred to as the “post-genomic era”, it is up to us to find ways to
provide meaning to this immense quantity of genome sequence data. Fungi have long been
known to be prolific producers of bioactive secondary metabolites, but bioinformatic analysis has
showed that these organisms harbor the potential to produce far more secondary metabolites than
are currently known. The work herein describes genome-wide strategies to approach two main
challenges we now face: 1) finding ways to access the hidden natural products and 2) developing
and/ or using genetic systems in fungal species to link secondary metabolites to their
biosynthesis genes and elucidate their biosynthesis pathways. We have demonstrated the power
of combining bioinformatics, molecular gene targeting, and natural product chemistry in
secondary metabolite biosynthesis research.
We initally used A. nidulans as the target species and took advantage of its well-established gene
targeting system. First, genome-based deletion analysis in A. nidulans led us to find genes
involved in the biosynthesis of xanthones located in at least three distinct loci in the genome.
This was particularly interesting because it contradicted the general notion that fungal secondary
metabolites are clustered. This highlighted the utility of genomics combined with efficient gene
targeting to identify these dispersed genes. Next, we hypothesized that altering the expression of
kinases would have an effect on secondary metabolite production and may activate silent gene
clusters. We used a genome-wide kinase knockout library and screened its secondary metabolite
profile and found that the mpkA deletion strain produced aspernidine A, a compound not
previously isolated from A. nidulans. We performed additional gene deletions and determined
xiii

the border of the biosynthesis gene cluster and proposed the biosynthesis pathway for
aspernidine A.
We then took advantage of the recent advances in genome sequencing of Penicillium species and
chose Penicillium canescens as the target species. This species is a prolific producer of
secondary metabolites but no previous work has been reported to link their metabolites to
biosynthesis genes. This is due in large part to the lack of an efficient gene targeting system in
this organism. We therefore developed a genetic system in P. canescens that would allow us to
perform targeted gene manipulations rapidly and efficiently. We then applied this system and
generated a genome-wide deletion library of polyketide synthase (PKS) genes and nonribosomal
peptide synthetase (NRPS) genes. Using this library, we successfully linked four secondary
metabolites, griseofulvin, xanthoepocin, 15-deoxyoxalicine B, and amauromine, to their core
secondary metabolite biosynthesis genes. Interestingly, 15-deoxyoxalicine B is a structurally
unique diterpenic meroterpenoid, a class of fungal metabolites whose biosynthesis mechanism
remains largely unknown. We performed additional gene deletions and determined the gene
cluster involved in its biosynthesis. We were able to isolate and characterize intermediates from
the gene deletants, which allowed us to propose a biosynthesis pathway. We believe the gene
targeting system and genome-wide PKS and NRPS deletion libraries will be important resources
towards a systematic understanding of secondary metabolite biosynthesis in P. canescens.

1

CHAPTER I: Introduction

1.1 Recent advances in genome mining of secondary metabolite biosynthetic gene
clusters and the development of heterologous expression systems in Aspergillus nidulans

1.1.1 Abstract
Fungi are prolific producers of secondary metabolites (SMs) that show a variety of
biological activities. Recent advances in genome sequencing have shown that fungal
genomes harbor far more SM gene clusters than are expressed under conventional
laboratory conditions. Activation of these “silent” gene clusters is a major challenge, and
many approaches have been taken to attempt to activate them and, thus, unlock the vast
treasure chest of fungal SMs. This review will cover recent advances in genome mining of
SMs in Aspergillus nidulans. We will also discuss current updates in gene annotation of A.
nidulans and recent developments in A. nidulans as a molecular genetic system, both of
which are essential for rapid and efficient experimental verification of SM gene clusters on
a genome-wide scale. Finally, we will describe advances in the use of A. nidulans as a
heterologous expression system to aid in the analysis of SM gene clusters from other fungal
species that do not have an established molecular genetic system.
1.1.2 Introduction
Filamentous fungi have played an important role in the history of drug discovery and
development. The secondary metabolites (SMs) that these organisms produce have served
2

as a source of low molecular weight molecules with a variety of biological activities.
Examples of these are antibiotics such as penicillin, immunosuppressants such as
cyclosporine, antifungals such as griseofulvin and the echinocandins, and
antihypercholesterolemic drugs such as lovastatin (Brakhage et al., 2008; Hoffmeister et
al., 2007; Newman et al., 2012). Many of the bioactive SMs that are easily accessible under
conventional laboratory conditions have already been isolated and patented for drug
development. However, advances in genome sequencing (Galagan et al., 2005; Machida et
al., 2005; Nierman et al., 2005; Pel et al., 2007) revealed that fungal species harbor an
abundance of SM gene clusters and these far exceed the number of known metabolites
produced by the species (Sanchez et al., 2012b). This potential abundance of SMs may
reflect their importance in nature as a chemical arsenal for niche security (Rohlfs et al.,
2007). The carefully controlled growth conditions in laboratory culture settings prevent any
competition or life-threatening circumstances that would trigger the production of SMs,
thereby leaving many of the gene clusters dormant. Activating these silent gene clusters,
revealing their biosynthetic pathways, and isolating the SMs produced by these pathways is
a major challenge in the search for new SMs.
Various approaches have been taken in attempts to activate silent SM gene clusters (Chiang
et al., 2011), including fusing of regulatable promoters to a pathway-specific transcription
factor (Bergmann et al., 2007; Chiang et al., 2009), removal of genes required for
heterochromatin formation (Bok et al., 2009), genome-wide analysis of mutants of LaeA, a
global regulator of SM (Bok et al., 2006) , co-incubation with microorganisms to mimic
conditions in nature (Schroeckh et al., 2009), and the “one strain many compounds”
(OSMAC) strategy (Bode et al., 2002). Most of these approaches were developed in A.
3

nidulans due to the availability of highly efficient gene-targeting systems in this model
organism. The developed approaches are often subsequently applied to other filamentous
fungi.
In this review we focus on recent advances in genome mining of secondary metabolism
genes in A. nidulans. We also describe the current status of the annotation of the products
of secondary metabolism genes in A. nidulans. We would also like to direct readers to the
accompanying review in this issue by our collaborators Nancy Keller and Philipp Wiemann
on general strategies for mining fungal natural products and to other recent reviews on this
subject (Brakhage, 2013; Tsunematsu et al., 2013; Yin and Keller, 2011).
1.1.3 The status of annotating secondary metabolite genes in A. nidulans
Among the Aspergillus species, A. nidulans has been used as a model organism, making it
the most comprehensively studied and best characterized species in the genus with the
largest body of literature. Most studies of secondary metabolite biosynthesis in A. nidulans
have used strains derived from a common reference strain, A. nidulans FGSC A4. A.
nidulans FGSC A4 was initially sequenced by Cereon Genomics (Monsanto) in 1998 to
three-fold genome equivalent coverage and the sequence was publicly released in 2003.
Shortly thereafter, additional sequencing was completed at the Whitehead Institute/MIT
Center for Genomic Research to give a total of 13 genome-equivalent coverage. The
seminal paper describing the A. nidulans genome was published in 2005 (Galagan et al.,
2005). Access to this sequenced genome has allowed investigators to use sequence
similarity to known genes from other species to mine for core genes that are involved in
secondary metabolism in A. nidulans. Algorithms such as SMURF (Secondary Metabolite
4

Unknown Regions Finder) (Khaldi et al., 2010) and antiSMASH (antibiotics and
Secondary Metabolite Analysis Shell) (Medema et al., 2011) are extremely useful in
predicting the core SM biosynthetic genes. Taking into consideration the most recent
annotation and additional analysis of available genomic data, our group’s most recent
estimate is that the A. nidulans genome contains 56 putative secondary metabolism core
genes including 27 polyketide synthase genes (PKS), 2 polyketide synthase-like genes
(PKS-like), 11 nonribosomal peptide synthetase genes (NRPS), 15 NRPS-like genes, and 1
hybrid NRPS-PKS gene. Table 1 and Figure 1 show our current understanding of the
products of these genes and the products from the pathways.
1.1.4 Bioinformatic advances
Since the original publication of the genome sequence data (Galagan et al., 2005), A.
nidulans gene annotations have been refined repeatedly to correct incomplete or inaccurate
content (Arnaud et al., 2012; Arnaud et al., 2010; Inglis et al., 2013; Nitsche et al., 2011;
Wortman et al., 2009). The Aspergillus Genome Database (AspGD; http://www.aspgd.org/)
provides gene and protein sequence data that are curated based on submitted information
and published literature. Although the wealth of data and the availability of the algorithms
mentioned previously have provided accurate predictions of core SM biosynthetic genes, it
is still not possible to predict with accuracy the boundaries of secondary metabolite gene
clusters or the functions of each member of the clusters based solely on genome sequence
data. This is due to the fact that many of the genes surrounding the core SM biosynthetic
genes often have unknown functions, making predictions of their involvement in the
biosynthetic process of the SM almost impossible. Elucidation of biosynthetic gene clusters
5

have thus been heavily dependent on experimental verification, a laborious process that
involves single gene deletion of each gene with a suspected role in SM biosynthesis,
followed by identification and characterization of SMs produced by the deletion strains.
Improvements in “omics”-based methods for accurate prediction of SM gene cluster
members and the availability of more precise annotations are desirable for a more rapid and
efficient experimental verification of novel SM gene clusters.
Andersen et al. recently published a novel strategy for the accurate prediction of SM gene
cluster boundaries (Andersen et al., 2013) based on the fact that expression of genes of a
given SM cluster is coordinately regulated. A DNA expression microarray was used to
identify genes that were co-regulated with SM gene cluster backbone enzymes. A variety
of culture media were selected that, based on SM profiling experiments, would elicit
expression of as many gene clusters as possible. Samples were then taken from A. nidulans
growing on the selected culture media for transcriptional profiling, and the generated data
were combined with previously published data to form a superset of a total of 44
expression conditions for analysis. Andersen et al. developed clustering scores (CSs) that
reflected the degree to which each gene was co-regulated with its neighbors. They
developed statistical guidelines for identifying the extent of gene clusters, which were
applied to the microarray data to generate cluster predictions. Comparisons with published
data demonstrated that their algorithm predicted gene clusters with high accuracy and can
even predict gene clusters that are scattered across different chromosomes. Using this
algorithm, a list of 58 predicted SM gene clusters was generated.
6

These data have been curated at AspGD and applied as a criterion for the manual
annotation of computationally predicted gene clusters as a part of a continued effort to
improve and refine the prediction of SM gene cluster boundaries(Inglis et al., 2013). This
updated gene cluster boundary annotation also incorporates published experimental data,
synteny between clustered genes among different species, functional annotation of putative
gene cluster members, and increase in the distance between predicted boundary genes and
genes that are directly adjacent to it but not included in the cluster. This new and improved
set of comprehensive SM gene cluster predictions will aid in facilitating the future
investigation of novel Aspergillus SMs.
1.1.5 Genome-wide kinase knock-outs
The molecular genetic system of A. nidulans is powerful and technical advances in recent
years have made genome-wide, systematic approaches more feasible. The Fungal Genetics
Stock Center (FGSC) provides a systematic gene deletion construct collection, a valuable
experimental resource for the A. nidulans research community. De Souza et al. have
generated a set of gene deletion constructs for 9,851 genes, which represents 93.3% of the
encoding genome (Colin P. De Souza, 2013). Mutant strains generated with the cassettes
are deposited with the FGSC after construction.
Using this deletion construct resource, a genome-wide kinase knock-out library consisting
of deletion strains of most A. nidulans non-essential kinase genes was generated and
deposited at the FGSC (Colin P. De Souza, 2013). The kinase deletion strains were used for
genome-wide functional analysis of kinases, resulting in identification of many previously
unknown functions for kinases(Colin P. De Souza, 2013). This kinase knock-out library
7

was screened to test the hypothesis that manipulation of kinase expression has the potential
to activate silent SM gene clusters (Yaegashi et al., 2013). This led to the discovery of an
mpkA deletant that produced aspernidine A, a compound that had been discovered
previously in A. nidulans (Scherlach et al., 2010b) but the biosynthetic pathway remained
unknown. The mpkA deletant produced a sufficient amount of aspernidine A to allow the
identification and analysis of the gene cluster involved in its biosynthesis. From the
chemical structure of aspernidine A combined with previous data (Ahuja et al., 2012), it
was predicted that a nonreducing polyketide synthase (NR-PKS) gene, pkfA (AN3230) is
involved in the biosynthesis of aspernidine A. Deletion of pkfA confirmed this, and the
boundary of the gene cluster was identified through a series of gene deletions of the
surrounding genes of pkfA. Analysis of the SMs produced by mpkA deletion strains resulted
in isolation and characterization of novel intermediates that aided in generating a proposed
pathway for aspernidine A.
A similar deletion set of 28 protein phosphatase genes was generated and used to identify
four essential phosphatases and four required for normal growth (Son and Osmani, 2009).
The deposited deletion constructs were also used in a study that identified multiple kinases
and phosphatases involved in the sensing of carbon and energetic status, and also
contributed to the understanding of the signaling cascades that result in regulation of CreA
derepression and hydrolytic enzyme production (Brown et al., 2013).

8

1.1.6 Genome-wide analysis of all non-reduced polyketide synthases and NRPS-like
enzymes in A. nidulans
Despite the success of various strategies to activate silent gene clusters, a large number of
potential SM gene clusters remain untapped. To analyze clusters resistant to activation
through existing approaches, a strategy was developed that completely bypasses normal
regulation (Ahuja et al., 2012). It takes advantage of recent advances in the construction of
transforming fragments by fusion PCR and effective gene targeting to replace promoters of
SM genes with the regulatable alcA promoter. It was applied to obtain a comprehensive
understanding of the products of nonreducing polyketide synthase (NR-PKS) genes, a class
of key genes of SM biosynthetic pathways (Ahuja et al., 2012). The A. nidulans genome
harbors 14 NR-PKS genes, and combined efforts by several groups over the years led to the
identification of the chemical products of six of them (Bok et al., 2009; Brown et al.,
1996b; Chiang et al., 2010a; Chiang et al., 2009; Li et al., 2011; Sanchez et al., 2010;
Schroeckh et al., 2009; Szewczyk et al., 2008; Watanabe et al., 1999; Yu and Leonard,
1995). To determine the products of the remaining eight NR-PKS genes, the native
promoters for each NR-PKS and other genes necessary for product formation or release
were replaced with the alcA promoter. Induction of expression resulted in the production
and release of compounds from each of the NR-PKS and allowed the completion of the
determination of the products of NR-PKS genes of A. nidulans.
This approach can be applied to the discovery of other classes of SM biosynthetic gene
clusters. This was demonstrated by systematically targeting nonribosomal peptide
synthetase (NRPS)-like genes for promoter replacement, resulting in the discovery that one
9

of the NRPS-like genes, micA, is the sole gene responsible for the biosynthesis of the
metabolite microperfuranone (Yeh et al., 2012).
In another strategy carried out by Nielsen et al., a genome-wide PKS deletion library was
constructed by systematically deleting all 32 putative PKS genes (Nielsen et al., 2011). A
reference strain was cultured on an array of culture media to find conditions that would
induce production of SMs that were not previously linked to a gene cluster, and this was
followed by screening of the genome-wide PKS deletion library to establish the genetic
link to the SMs. This approach provided novel links between PKS genes and SMs,
demonstrating its strength and the potential usefulness of the deletion library as a resource
for further PKS studies.
1.1.7 Use of A. nidulans as a host for heterologous expression of SM genes from other
Aspergillus species
The highly advanced and established molecular genetic system of A. nidulans can be
applied to the study of SM production of other fungal species that have poor or nonexistent
molecular genetic systems (Yin et al., 2013). Heterologous expression of fungal genes in
other fungi has been used and with some success, but this approach is not without
limitations including finding a suitable host and the difficulty of handling large genes and
gene clusters. An advantage of fungal systems over bacterial for expressing fungal
secondary metabolism genes is that fungi can correctly splice introns of secondary
metabolism genes from other fungi resulting in successful expression (Chaing et al., 2013;
Fujii et al., 2001; Kasahara et al., 2006). Since many fungal SM genes are quite large and
contain introns (often several introns) this is of considerable benefit.
10

Major advances have recently been made in establishing A. nidulans as a host for
heterologous expression of fungal SMs. First, entire SM gene clusters have been deleted to
eliminate production of unwanted A. nidulans SMs, resulting in reduced SM background
and facilitating detection and isolation of compounds produced by the heterologously
expressed genes (Chiang et al., 2013).
Second, a system for transferring SM genes from other fungi while placing them under
control of the alcA promoter has been developed (Chaing et al., 2013; Maiya et al., 2006).
This system uses a strategy that involves 1) PCR amplification of each gene, 2) the use of
fusion PCR to place each gene under control of the alcA promoter and to construct a
transforming fragment, and 3) integration of the fragment into a target A. nidulans locus.
For larger clusters several genes must be transferred into A. nidulans and, to avoid running
out of selectable markers for transformation, a marker recycling strategy was developed
(Chiang et al., 2013). Each time a new gene is introduced into A. nidulans a selectable
marker is evicted and this marker can be used in the subsequent transformation. This
strategy allows an unlimited number of genes to be transferred into and expressed in A.
nidulans. The use of this approach resulted in the successful expression of all six genes of
the gene cluster that encodes the production of asperfuranone, a cryptic gene cluster from
A. terreus. Furthermore, various combinations of expression genes were tested, leading to
clarification of the asperfuranone biosynthetic pathway.
Another recent approach to transfer members of entire SM gene clusters is to assemble the
PCR amplified individual cluster fragments into a single large transforming fragment using
USER fusion, followed by insertion into the integration vector by USER cloning (Nielsen
et al., 2013). Using this technique, a total of 13 genes of a putative gene cluster responsible
11

for geodin biosynthesis from A. terreus were transferred into A. nidulans in a two-step
process, successfully enabling geodin biosynthesis in A. nidulans.
1.1.8 Conclusions
Advances in genome sequencing in fungi have provided us with a wealth of information
that suggests that the number of SM gene clusters far exceeds the number of discovered
compounds. A combination of bioinformatics and experimental verification is fundamental
to elucidating the SM biosynthetic pathways that these SM gene clusters encode. Among
the many species of Aspergillus, A. nidulans is used as a model organism and it is the
species with the most abundant literature by far and the most advanced, highly efficient
molecular genetic system. Recent advances in development of prediction algorithms in A.
nidulans and updated curation by AspGD have given us access to improved SM gene
cluster predictions, which we can use as a basis for subsequent experimental verification.
Advances in transforming fragment construction techniques and effective gene targeting
expedite the experimental verification process. These advances, in combination, have
enabled quick and systematic approaches to uncover the potential of SM production by A.
nidulans. The application of these advances is not limited to the SMs of A. nidulans.
Combined efforts such as the “1000 Fungal Genomes Project
(http://1000.fungalgenomes.org/home/ )” by the DOE Joint Genome Institute (JGI) are
dedicated to sequencing numerous different species of fungi and providing a database for
the research community. Many of these fungi do not have good molecular genetic systems,
which makes experimental verification a big challenge. Heterologous expression of fungal
genes in other host fungi is one approach that is being used, and major advances have been
made to establish A. nidulans as a host. Newly developed methods in constructing
12

transforming fragments and improved transformation strategies have made it possible for
large or multiple genes to be transformed into A. nidulans. These approaches will
contribute greatly to uncovering the untapped resources of SMs that the fungal genomes
encode.

13

Table 1-1. Secondary metabolism gene clusters in A. nidulans
No
AspGD
Designation
Core
Gene
Name
Gene
type
a

Metabolites isolated from A. nidulans References
1 AN0016 pes1 NRPS
2 AN0150 mdpG NR-PKS emodin (1), emodin analogs (2-10),
shamixanthone (11), epishamixanthone (12),
variecoxanthone A (13), emericellin (14),
atrochrysone carboxylic acid (15), 1-hydroxy-
6-methyl-8-hydroxymethylxanthone (16),
paeciloxanthone (17), monodictyphenone
(18), 3-(2,6-dihydroxyphenyl)-4-hydroxy-6-
methyl-1(3H)-isobenzofuranone (19),
arugosin A, H (20,21)
(Bok et al.,
2009; Chiang
et al., 2010a;
Sanchez et
al., 2011b;
Scherlach et
al., 2011;
Soukup et al.,
2012)
3 AN0523 pkdA NR-PKS 2-ethyl-4,6-dihydroxy-3,5-
dimethylbenzaldehyde (22)
(Ahuja et al.,
2012)
4 AN0607 sidC NRPS ferricrocin (Eisendle et
al., 2003)
5 AN1034 afoE NR-PKS (2Z,4Z)-4,6-dimethylocta-2,4-dienoic aicd
(23), asperfuranone (24), 6-[(3E,5E)-5,7-
dimethyl-2-oxonona-3,5-dienyl-2,4-
dihydroxy-3-methylbenzaldehyde (25),
preasperpyranone (26), asperpyranone (27),
Proasperfuranone A, B (28, 29)
(Chiang et
al., 2009;
Somoza et
al., 2012)
6 AN1036 afoG HR-PKS (2Z,4Z)-4,6-dimethylocta-2,4-dienoic aicd
(23), asperfuranone (24), 6-[(3E,5E)-5,7-
dimethyl-2-oxonona-3,5-dienyl-2,4-
dihydroxy-3-methylbenzaldehyde (25),
preasperpyranone (26), asperpyranone (27),
Proasperfuranone A, B (28, 29)
(Chiang et
al., 2009;
Somoza et
al., 2012)
7 AN1242 NRPS nidulanin A (31) (Andersen et
al., 2013)
8 AN1680 NRPS-
like

9 AN1784 pkjA HR-PKS
10 AN2032 pkhA NR-PKS 2,4-dihydroxy-6-[(3E,5E,7E)-2-oxonona-
3,5,7-trienyl]benzaldehyde (30)
(Ahuja et al.,
2012)
11 AN2035 pkhB HR-PKS 2,4-dihydroxy-6-[(3E,5E,7E)-2-oxonona-
3,5,7-trienyl]benzaldehyde (30)
(Ahuja et al.,
2012)
12 AN2064 NRPS-
like

13 AN2545 easA NRPS emericellamides (32-36) (Chiang et
al., 2008)
14 AN2547 easB HR-PKS emericellamides (32-36) (Chiang et
al., 2008)
15 AN2621 acvA NRPS penicillin (Maccabe et
al., 1991;
Soukup et al.,
2012)

14

Table 1-1 (continued). Secondary metabolism gene clusters in A. nidulans
No
AspGD
Designation
Core
Gene
Name
Gene
type
a

Metabolites isolated from A. nidulans References
16 AN2924 NRPS-
like

17 AN3230 pkfA NR-PKS orsellinaldehyde (37), 3-(2,4-dihydroxy-6-
methylbenzyl)-orsellinaldehyde (38),
aspernidine A-E (41-45)
(Ahuja et al.,
2012;
Scherlach et
al., 2010a;
Yaegashi et
al., 2013),
18 AN3386 pkiA NR-PKS 7-methyl-3-nonylisoquinoline-6,8-diol (39),
6-hydroxy-7-methyl-3-nonylisoquinoline-5,8-
dione (40), 2,4-dihydroxy-3-methyl-6-(2-
oxoundecyl)benzaldehyde (46), 4-hydroxy-3-
methyl-6-(2-oxoundecyl)-2-pyrone (47)
(Ahuja et al.,
2012)
19 AN3396 micA NRPS-
like
microperfuranone (48) (Yeh et al.,
2012)
20 AN3495 inpA NRPS-
like

21 AN3496 inpB NRPS
22 AN3612 HR-PKS
23 AN4827 NRPS-
like

24 AN5318 NRPS-
like

25 AN6000 aptA NR-PKS asperthecin (49), 2,3,6,8,9-pentahydroxy-1-
oxo-3-(2-oxopropyl)-1,2,3,4-
tetrahydroanthracene-2-carboxylic acid (50)
(Ahuja et al.,
2012; Li et
al., 2011;
Szewczyk et
al., 2008)
26 AN6236 sidD NRPS
27 AN6431 HR-PKS
28 AN6444 NRPS-
like

29 AN6448 pkbA NR-PKS 2,5-dimethylresorcinol (51), 3-
methylorsellinic acid (52), cichorine (53),
nidulol (54),
(Ahuja et al.,
2012;
Sanchez et
al., 2012a)
30 AN6791 HR-PKS
31 AN7071 pkgA NR-PKS alternariol (55), citreoisocoumarin (56),
analogs of citreoisocoumarin (57-60)
(Ahuja et al.,
2012)
32 AN7084 PKS-like
33 AN7489 PKS-like

15

Table 1-1 (continued). Secondary metabolism gene clusters in A. nidulans
No
AspGD
Designation
Core
Gene
Name
Gene
type
a

Metabolites isolated from A. nidulans References
34 AN7825 stcA
(pksST)
NR-PKS norsolorinic acid (61), norsolorinic acid
anthrone (62), sterigmatocystin (63)
(Ahuja et al.,
2012; Brown
et al., 1996b;
Wang et al.,
2008; Yu and
Leonard,
1995)
35 AN7837+
AN7838
HR-PKS
36 AN7884 NRPS
37 AN7903 pkeA NR-PKS 2,4-dihydroxy-3-methyl-6-(2-
oxopropyl)benzaldehyde (64)
(Ahuja et al.,
2012)
38 AN7909 orsA NR-PKS F9775A, B (65, 66), orsellinic acid (67),
diorcinol (68), gerfelin (69), 10-deoxygerfelin
(70)
(Bode et al.,
2002; Bok et
al., 2009;
Sanchez et
al., 2010;
Scherlach et
al., 2011;
Schroeckh et
al., 2009)
39 AN8105 NRPS-
like

40 AN8209 wA NR-PKS 2-acetoacetyl T4HN (71), naphthopyrone
YWA1 (72)
(Ahuja et al.,
2012; Fujii et
al., 2001;
Watanabe et
al., 1999)
41 AN8383 ausA NR-PKS isoaustinone (73), analogs of isoaustinone
(74, 75), austinol (76), dehydroausitnol (77),
protoaustinoid (78), preaustinoid A3-A5 (79-
81), austinoneol A (82), neoaustinone (83),
austinolide (84)
(Ahuja et al.,
2012; Lo et
al., 2012;
Nielsen et al.,
2011)
42 AN8412 apdA Hybrid aspyridone A, B (85, 86) (Bergmann et
al., 2007)
43 AN8513 tdiA NRPS-
like
terrequinone A (87) (Bouhired et
al., 2007;
Soukup et al.,
2012)
44 AN8910 HR-PKS
45 AN9005 HR-PKS
46 AN9129 NRPS-
like

47 AN9226 nrpA NRPS
48 AN9243 NRPS-
like

16

Table 1-1 (continued). Secondary metabolism gene clusters in A. nidulans
No
AspGD
Designation
Core
Gene
Name
Gene
type
a

Metabolites isolated from A. nidulans References
49 AN9244 NRPS
50 AN9291 NRPS-
like

51 AN10297 NRPS-
like

52 AN10430 HR-PKS
53 AN10486 NRPS-
like

54 AN10576 ivoA NRPS
55 AN11191 pkkA HR-PKS
56 AN12440 NR-PKS
a
Abbreviations: polyketide synthase (PKS), non ribosomal peptide synthetase (NRPS), hybrid PKS-NRPS (Hybrid),
nonreduced polyketide synthase (NR-PKS), highly reduced polyketide synthase (HR-PKS)
17

Figure 1-1. Structures of compounds isolated from A. nidulans
18

1.2 Application of the genome mining strategy to Penicillium species
1.2.1 Significance of Penicillium
Penicillium is a genus of filamentous fungi known for its commercial, agricultural, and
medical importance. Species in Penicillium, such as P, camemberti and P. roqueforti, play
fundamental roles in the ripening process of cheeses. Other species are plant pathogens,
such as P. expansum, a major agricultural problem in apples and other harvest. P.
expansum is known to produce a carcinogenic metabolite patulin, a neurotoxin that
contaminates apple juice and other apple products that poses health concerns.
Fungi of the genus Penicillium are known to produce a vast variety of chemical compounds
as secondary metabolites. These chemical compounds include polyketides,
diketopiperazines, ergot alkaloids, benzodiazepines, quinolones, and quinazolines
(Kozlovskii et al., 2012). Many of these compounds have been found to have various
bioactivies. Penicillin, an antibiotic and perhaps the most famous of these bioactive
compounds, is produced industrially by Penicillium chrysogenum (Raper et al., 1949).
Mycophenolic acid is an immunosuppressant used in transplant patients that is produced by
P. breviocompactum, and P. griseofulvum and other Penicillium species produce
griseofulvin, an orally administered antifungal drug.
Historically, the development of industrial strains for the production of SMs that are
beneficial to humans have relied on random mutations and extensive rounds of selection.
Penicillin, for example, was produced in very low amounts (about 1.2µg/mL) in Alexander
Fleming’s original P. nonatum strain (NRRL1249B21). The discovery of P. chrysogenum
NRRL1951, a better producer of penicillin (60-150µg/mL), marked the start of industrial
19

strain improvement programs to generate strains with improved penicillin production. After
decades of classical mutagenesis such as x-ray irradiation, UV irradiation, and nitrogen
mustard treatment, each followed by extensive selection to select for strains with improved
penicillin production capabilities, the overproducer mutants currently used in industrial
production of penicillin are able to produce more than 50,000µg/mL in fed batch cultures.
However, the molecular basis for this improved productivity remained obscure. The
publication of the P. chrysogenum genome was able shed some light on this issue, along
with the advance of other –omics such as transcriptomics and proteomics.
In 2011, the Joint Genome Institute (JGI) of the United States Department of Energy
launched a Fungal Genomics Program, aiming to scale up sequencing and analysis of
fungal genomes. Early in 2014, JGI released the complete genome sequence of over ten
Penicillium species. MycoCosm is JGI’s web-based fungal genomics portal that allows
analysis of fungal genome sequences and other –omics data that is publicly available to the
research community. With the wealth of information now readily available, we are now
able to tap into the treasure chest of the fungal genome and perform in silico analysis. Our
group analyzed the newly released genome sequences of Penicillium species and found
that, much like Aspergillus species, these species also harbor a larger number of potential
SM biosynthesis genes that far exceed the number of SMs they are known to produce.
Using the strategies applied to study silent SM gene clusters in A. nidulans, there is great
potential to induce the generation of previously unknown natural products in Penicillium.
Even for compounds that have already been reported to be produced in these species, the
availability of genomic data will be instrumental in identifying genes involved in
20

biosynthesis at a rapid pace. Development of an efficient gene targeting system for
Penicillium species will greatly contribute to providing molecular and genetic basis for the
biosynthesis of SMs.
1.2.2 The status of secondary metabolite gene cluster elucidation in Penicillium
Considering its importance as an antibiotic, it is understandable that one of the first SMs
studied to elucidate their biosynthesis mechanism in Penicillium was penicillin. Molecular
and genetic characterization of the core enzyme isopenicillin N synthetase of P.
chrysogenum was first reported in 1986 (Carr et al., 1986). Since then the molecular
mechanisms of penicillin biosynthesis was revealed through molecular cloning, analysis,
and expression of the genes (Barredo et al., 1989; Diez et al., 1989; Maccabe et al., 1991;
Martin, 1987; Muller et al., 1991). Genome sequencing has advanced tremendously,
making it possible to comapre between strains that produce low amounts of penicillin and
industrial high-producing strains. Combined with the knowledge of the mechanism of
biosynthesis, further improvements to the fungal strains have been applied to increase the
production of penicillin in P. chrysogenum (Ozcengiz and Demain, 2013; van den Berg,
2011).
Two polyketide metabolites, griseofulvin and viridicatumtoxin, and one NRP,
tryptopquialanine, were associated with their corresponding gene clusters in P. aethiopicum
(Chooi et al., 2010; Gao et al., 2011). This was accomplished through the combined use of
bioinformatics and the gene knockout approach. These studies also found that the gene
clusters were localized within conserved syntenic regions of the P. aethiopicum genome,
raising an interesting perspective into the evolution of clustering of SM genes in fungi.
21

Gene silencing by RNAi was used in P. chrysogenum to study the genes encoding the
biosynthesis of roquefortine C and meleagrin (Garcia-Estrada et al., 2011). Transcriptomic
data was used to define the border of the biosynthesis gene cluster. Results of this study
showed that the the genes involved in the biosynthesis of the two compounds are
coregulated and that they derive from a single pathway.
The core PKS gene of mycophenolic acid (MPA) biosynthesis in P. brevicompactum was
localized by bioinformatics analysis targeting putative resistance genes. The rationale was
that gene clusters of biologically active metabolites typically harbor resistance genes that
makes the organism resistant to the SM it produces. MPA is known to inhibit IMP
dehydrogenase (IMPDH), which is the rate-controlling enzyme in GMP biosynthesis. The
hypothesis was that the biosynthesis gene cluster for MPA may contain a gene encoding an
IMPDH homologue to confer resistance. Using this rationale, the MPA biosynthesis gene
cluster was located and the involvement of the core PKS gene mpaC was confirmed by
targeted gene deletion. The mpaC gene was heterologously expressed in A. nidulans to
further confirm that it indeed encodes a PKS that produces 5-methylorsellinic acid (5-
MOA), which is the first intermediate in mycophenolic acid biosynthesis (Hansen et al.,
2011).
Finally, the most recent report is the finding of the biosynthesis gene cluster that encodes
communesins in P. expansum. Communesins are indole alkaloids that have structurally
unique characteristics that have inspired numerous synthetic studies. The structure of
communesin suggested the involvement of at least three enzymes: a highly-reducing (HR)
PKS, an enzyme homologous to 4-dimethylallyl tryptophan synthase (DMATS), and a
22

tryptophan decarboxylase (TDC). The whole genome sequence of P. expansum was used
for bioinformatics analysis to search for a gene cluster that contained an HR-PKS,
DMATS, and TDC in close proximity to each other. The involvement of each gene on the
candidate gene cluster in the biosynthesis of communesin was then verified by the gene
knockout approach. Intermediates were isolated and characterized, enabling the
biosynthesis pathway to be mapped out.
1.2.3 Conclusion
Penicillium species produce a large number of bioactive SMs. In 2004, Frisvad et al.
reported that the 58 species in Penicillium subgenus Penicillium produces a total of 132
SM families with an average of 5 SM families per species (Frisvad et al., 2004). This
number, however, is an underestimate because it does not include several pigments,
volatiles, and uncharacterized SMs. Furthermore, genome sequence data has become
available for several species, and bioinformatics analysis has revealed that every species
harbors a large number of core SM genes that far exceeds the number of known SMs.
However, there are only a handful of SMs that have been linked to their biosynthesis gene
clusters in Penicillium. This species has clearly been understudied compared to species
such as Aspergillus. This is due in part to the absence of an established tool that would
allow for rapid and efficient gene manipulations. We therefore believe that the
development of an efficient gene targeting system in Penicillium species will provide a
useful that will aid in unveiling the link between genes and metabolites and also in
uncovering the products of silent gene clusters.

23

CHAPTER II: Genome-based deletion analysis reveals the prenyl xanthone
biosynthesis pathway in Aspergillus nidulans
2.1 Abstract
Xanthones are a class of molecules that bind to a number of drug targets and possess a
myriad of biological properties. An understanding of xanthone biosynthesis at the genetic
level should facilitate engineering of second-generation molecules and increasing
production of first-generation compounds. The filamentous fungus Aspergillus
nidulans has been found to produce two prenylated xanthones, shamixanthone and
emericellin, and we report the discovery of two more, variecoxanthone A and
epishamixanthone. Using targeted deletions that we created, we determined that a cluster of
10 genes including a polyketide synthase gene, mdpG, is required for prenyl xanthone
biosynthesis. mdpG was shown to be required for the synthesis of the anthraquinone
emodin, monodictyphenone, and related compounds, and our data indicate that emodin and
monodictyphenone are precursors of prenyl xanthones. Isolation of intermediate
compounds from the deletion strains provided valuable clues as to the biosynthetic
pathway, but no genes accounting for the prenylations were located within the cluster. To
find the genes responsible for prenylation, we identified and deleted seven putative
prenyltransferases in the A. nidulans genome. We found that two prenyltransferase genes,
distant from the cluster, were necessary for prenyl xanthone synthesis. These genes belong
to the fungal indole prenyltransferase family that had previously been shown to be
responsible for the prenylation of amino acid derivatives. In addition, another prenyl
xanthone biosynthesis gene is proximal to one of the prenyltransferase genes. Our data, in
24

aggregate, allow us to propose a complete biosynthetic pathway for the A.
nidulans xanthones.

25

2.2 Introduction
Xanthones are polyphenolic compounds produced by higher plants, lichens, and fungi. In
common they share a 9H-xanthen-9-one scaffold, but the class is highly diverse owing to
functionalization with a range of substituents at various positions (El-Seedi et al., 2010). As
a consequence, the xanthone core has been described as a “privileged structure,” (Lesch
and Brase, 2004) with members of this group exhibiting the potential to bind to a variety of
targets. Indeed, xanthones have proven to be an important class of secondary metabolites.
Over 250 of them have been shown to possess biological activities, including antimicrobial,
antioxidant, cytotoxic, and neuropharmacological activities (El-Seedi et al., 2010).
In many instances xanthones are functionalized with prenyl groups, commonly the five
carbon dimethylallyl moieties. Examples of prenylated xanthones include mangostanin and
α-mangostin, which are strongly inhibitory to both sensitive and methicillin-resistant
strains of Staphylococcus aureus (Panthong et al., 2006). γ-Mangostin also displays potent
inhibitory activity against mediators of prostaglandin release, COX-1 and COX-2
(Nakatani et al., 2002). Other prenylated xanthones have been found to be strongly active
against Bacillus subtilis, Staphylococcus faecalis, Staphylococcus typhi, and Candida
glabrata (Saleem et al., 2010). By themselves, prenyl groups are important contributors to
the outstanding structural diversity of natural products (Heide, 2009).Prenylated secondary
metabolites isolated from plants, bacteria, and fungi display a large variety of medicinal
properties, including antitumor, antiretroviral, and psychotrophic activities, that are often
distinct from their nonprenylated precursors (Botta et al., 2005).
26

Molecular genetic analysis of the biosynthesis of prenylated and nonprenylated xanthones,
as well as prenylations in general, would greatly advance our understanding of fungal
secondary metabolite biosynthesis and, by relating gene sequence to function, facilitate
prediction of the function of homologous genes. Moreover, identification of the genes
involved in the production of particular secondary metabolites offers the possibility of
enhanced production through replacement of their native promoters with strong or
inducible promoters. In addition, semisynthesis, mutasynthesis, and chemoenzymatic
synthesis that lead to second-generation compounds with improved pharmacodynamic and
pharmacokinetic properties should greatly benefit from identification and manipulation of
the genes involved in secondary metabolite production.
The filamentous fungus Aspergillus nidulans is, in principle, an excellent organism in
which to study the biosynthesis of prenylated xanthones. It is known to produce two
prenylated xanthones, shamixanthone (2) and emericellin (3) (Figure 2-1) (Ishida et al.,
1975; Marquez-Fernandez et al., 2007). Its genome has been sequenced and is reasonably
well annotated (Galagan et al., 2005; Wortman et al., 2009), and development of an
efficient gene targeting system (Nayak et al., 2006; Szewczyk et al., 2006) has greatly
facilitated the targeted deletion of secondary metabolite genes (Chiang et al., 2008;
Sanchez et al., 2010). We have taken advantage of these attributes to identify and
characterize the prenyl xanthone biosynthesis pathway in A. nidulans.
We previously identified, using a set of deletions of the aromatic, nonreducing PKS genes
in a chromatin remodeling mutant background, a PKS gene,mdpG, that is responsible for
synthesis of the related polyketides monodictyphenone and emodin (Bok et al., 2009;
27

Chiang et al., 2010a). Here, we employed the same set of deletions in the investigation of
prenyl xanthone synthesis and found that, again, mdpG is responsible. Deletion of genes
flanking mdpG revealed that the prenylated xanthone biosynthetic pathway is complex,
involving the products of nine genes that cluster with mdpG. Identification of intermediate
compounds that accumulate in the deletion strains provides important clues as to the steps
in the xanthone biosynthesis pathway. Many of these intermediates have not previously
been identified from A. nidulans and are of potential medical interest. Surprisingly and
interestingly, however, the prenyltransferase genes required for prenyl xanthone
biosynthesis are not clustered with the PKS gene. Bioinformatic analysis of the A.
nidulans genome allowed us to identify putative prenyltransferase genes elsewhere in the
genome whose products might be required for prenylation of the xanthones. By deleting
each of these candidate genes, we found two genes that are required for prenylation of
xanthones. They are distant from the mdpG cluster, and it is unlikely that they could have
been identified by traditional approaches. Interestingly, the products are members of a
family of prenyltransferases that are recognized to prenylate amino acids and their
derivatives, as opposed to polyketides. Lastly, we discovered a gene proximal to one of the
prenyltransferase genes that, upon deletion, leads to the loss of two of the four prenyl
xanthones that we identified. Our data, in aggregate, allowed us to unravel the complex
xanthone biosynthesis pathway in A. nidulans, and they highlight the utility of genomics
coupled with powerful molecular genetic methods for determining biosynthetic pathways,
especially when the cluster is complex and critical genes are distant from the primary
cluster.

28

2.3 Results
Characterization of Prenylated Xanthones from A. nidulans.
LO2026, an A. nidulans strain carrying a deletion of the stcJ gene (stcJ Δ), which is
required for the biosynthesis of the carcinogenic secondary metabolite sterigmatocystin
(Brown et al., 1996a), was cultivated on Yeast Extract Sucrose (YES) agar for 5 days at
37 °C. The rationale for the use of an stcJ Δ strain is that elimination of sterigmatocystin
frees up the common polyketide precursor malonyl-CoA and facilitates detection and
isolation of other metabolites. The strain also carries nkuA Δ to facilitate gene targeting
(Nayak et al., 2006). Reverse-phase LC/MS analysis of the crude organic extract revealed
the presence of several late-eluting, nonpolar metabolites (Figure 2-2). For full
characterization, we cultivated the strain on YES plates at a larger scale and purified the
compounds using flash chromatography, HPLC, and preparatory TLC when necessary.
NMR characterization revealed the molecules to be shamixanthone (2) and emericellin (3),
as well as, in lesser amounts, two prenylated xanthones not previously observed from this
species, variecoxanthone A (1) (Chexal et al., 1975) and epishamixanthone (4) (Ishida et
al., 1976). (Figure 2-1; NMR data available in the Supporting Information, Structural
Characterization and Figures 2-9 to 2-21). We also found that other solid media including
Yeast Agar Glucose (YAG) and shredded wheat are conductive to xanthone production.
Analysis of Prenyl Xanthone Biogenesis through Targeted Deletions
The structural similarity of the prenyl xanthones (Figure 2-1) suggests a common
biosynthetic origin, and the presence of aromatic rings is indicative of formation catalyzed
29

by a nonreduced polyketide synthase (NR-PKS) (Cox, 2007). Analysis of the sequenced
genome of A. nidulans suggests there are 12 NR-PKS genes in this species. The products of
two of the genes, stcA encoding the sterigmatocystin PKS and wA, a spore pigment PKS,
were already determined (Minto and Townsend, 1997; Watanabe et al., 1999), leading us to
focus on the 10 remaining NR-PKSs. In a previous project, we discovered that deletion
of cclA, a bre2 homologue orchestrating histone H3 lysine 4 methylation, resulted in the
synthesis of related aromatic compounds including monodictyphenone and emodin under
conditions in which these molecules were normally not observed (Bok et al., 2009). For
that project we deleted the 10 annotated NR-PKS genes in a cclA Δ background and
discovered that deletion of one NR-PKS gene, AN0150.4 (using the nomenclature of the
Central Aspergillus Data Repository, CADRE,http://www.cadre-genomes.org.uk/, and
the Aspergillus genome database,http://www.aspgd.org/) in a cclA Δ background, resulted
in the elimination of these compounds (Chiang et al., 2010a). We designated
AN0150.4 mdpG and the PKS it encodes MdpG. (See Table 2-1 for our gene designations
and the corresponding annotations from CADRE/AspGD and the Broad Institute
Aspergillus Comparative Database.) For the current project we utilized the same 10 NR-
PKS deletant strains to learn whether any of them were required for the synthesis of the
prenyl xanthones in A. nidulans. We were surprised to discover that, in addition to
monodictyphenone and its related molecules, the xanthones disappeared in the mdpG Δ
strain (Figure 2-2) and not in the other deletant strains (data not shown). This reveals that
MdpG is the PKS responsible for the synthesis of not only emodin, monodictyphenone, and
related compounds but also the xanthones.
30

Next, we tried to identify additional genes involved in A. nidulans prenyl xanthone
biosynthesis. Facilitating the search, secondary metabolism genes inA. nidulans are usually
clustered, prompting us to focus on the genes surrounding mdpG (AN10039 to AN0153)
(Figure 2-3). To study the synthesis of the xanthones as it would occur naturally, without
the influence of a potentially powerful chromatin remodeling mutation, we deleted these
genes in the LO2026 background carrying the wild-type cclA gene (cclA+). All deletions
were verified by diagnostic PCR (using three different primer sets for each gene) (see
Materials and Methods).
Attempted deletion of AN0153, homologous to a DNA-binding protein, failed to yield any
viable transformants, in agreement with previous observations (Chiang et al., 2010a). This
gene appears to be essential for viability and unrelated to secondary metabolism and was
thus excluded from further study here. Deletion of the other genes significantly reduced or
eliminated prenyl xanthone formation except for AN10039, mdpE Δ, and mdpI Δ (Figures 2-
3 and 2-4). Two differences were observed in relation to our investigation using cclA Δ: In
the cclA Δ background, deletion of the transcriptional regulator gene mdpE eliminates the
synthesis of monodictyphenone and related compounds; however, for the prenyl xanthones
this gene does not appear consequential (Figure 2-3). Additionally, the monooxygenase
gene mdpD was not found to be important for the production of monodictyphenone and
analogous products in the cclA Δ background, whereas it plays a crucial role in prenyl
xanthone formation.
The deletant strains that reduced prenyl xanthone production were examined for the
presence of any new metabolites that might represent intermediates in the biosynthetic
31

pathway of the xanthones. Extracts from strains carrying deletions of mdpG, mdpA,
and mdpF contained no obvious intermediates from the prenyl xanthone biosynthesis
pathways. A strain carrying a deletion ofmdpH did not display new metabolites in
significant amounts, but a compound with the mass and retention time of endocrocin (10),
recently identified from anmdpH/cclA double-deletant strain (Chiang et al., 2010a), was
observed.
The mdpL Δ strain, the extract of which displayed the greatest number of significant peaks
in the chromatogram, was grown in large scale on YES plates, and the major metabolites
were isolated by flash chromatography and HPLC and characterized primarily using NMR.
The compounds were determined to be 2, ω-dihydroxyemodin (5), ω-hydroxyemodin (6),
emodin (7), aloe-emodin (11), and chrysophanol (12) (Figure 2-4; NMR data shown in the
Supporting Information). Also, mdpC Δ, mdpJ Δ, and mdpK Δ extracts each exhibited 5, 6,
and 7, as determined by their masses and retention times in comparison to the mdpL Δ
extract, but little or no 11 or 12. Chromatograms ofmdpB Δ extracts similarly displayed the
three metabolites, along with a number of small, indistinct peaks.
The mdpD Δ extract also yielded 5, 6, and 7 but with addition of monodictyphenone (16), a
core xanthone structure (8), and the C-prenylated analog paeciloxanthone (9). Finally, the
polyketide cichorine (13) and the meroterpenoids austinol (14) and dehydroaustinol (15)
could be detected in all strains tested, indicating they are unrelated to prenyl xanthone
biosynthesis.

32

Identification of Two Genes Required for Prenylation of the Xanthone Scaffold
Although our results revealed a gene cluster responsible for synthesis of A. nidulans prenyl
xanthones, the lack of a prenyltransferase gene in this cluster made it clear that the required
prenyltransferase gene(s) must be located elsewhere in the genome. On the basis of
homology to known genes of this class (Wortman et al., 2009), we were able to identify
several putative prenyltransferase genes in the A. nidulans genome: AN6784.4, AN8514.4
[tdiB (Bok et al., 2006)], AN10289.4, AN11080.4, AN11194.4, and AN11202.4. We
deleted each of these genes, and the deletant strains were cultured on YES plates. LC/MS
analysis revealed that deletion of AN6784.4, but not the other prenyltransferase genes,
resulted in the elimination of 2−4 (Figure 2-5a) and accumulation of a compound which
upon large-scale isolation and characterization was confirmed to be variecoxanthone A (1).
Because AN6784.4 is a prenyltransferase gene required for synthesis of the xanthones, we
hereby designate it xptA (xanthone prenyltransferase A).
Since the A. nidulans xanthones contain two prenyl groups (except for variecoxanthone A,
which only has one), it was apparent that an additional prenyltransferase gene was required.
We hypothesized that the genome must contain an additional, unannotated
prenyltransferase gene. We performed a local BLAST search of the Broad Institute
Aspergillus Comparative Database
(http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html)
using AN6784.4 as a query sequence. We identified two annotated partial gene fragments
designated ANID_12402.1 and ANID_12430.1. Inspection of the two fragments revealed
that the 3 ′-end of ANID_12402.1 overlaps with the 5 ′-end of ANID_12430.1 by ∼600 bp.
33

Further, the Web site predicted that the sequence of ANID_12402 is prematurely truncated
at its 3′-end. For these reasons we concluded that the nonoverlapping portion of
ANID_12430 was in fact the continuation of ANID_12402 and that the 5 ′-end of
ANID_12402 and the 3 ′-end of ANID_12430 are the boundaries of a single gene. Indeed,
the sequence of this putative gene is found intact in CADRE/AspGD but without
annotation. Deletion of this gene led to the elimination of the xanthones and accumulation
of several metabolites, including 7, 12, 16, and a putative monodictyphenone derivative
(17) (Figure 2-5a). We designate this new gene xptB (xanthone prenyltransferase B).
Identification of an Additional Prenyl Xanthone Biosynthesis Gene
The data were suggestive of a biosynthesis in which emericellin (3) is a precursor to
shamixanthone (2) and epishamixanthone (4) (see Discussion). Even after identification
of xptA and xptB it was clear that a gene encoding the enzyme responsible for the
transformation from emericellin to shamixanthone and epishamixanthone was required but
not yet identified. Given the tendency of fungal secondary metabolite genes to exist in
clusters, we reasoned that the missing gene might reside proximate to xptA or xptB. An
analysis of the putative function of the genes neighboring xptA revealed none that were
likely involved in secondary metabolism; however, AN7998.4 and AN7999.4 upstream
of xptB were possible candidates. Deletion of AN7998.4 yielded a metabolite profile that
continued to display variecoxanthone A (1) and 3 but lacked 2 and 4 (Figure 2-5b and 2-
5c), revealing that AN7998.4 is the missing gene in the biosynthesis of the two prenyl
xanthones. We designate AN7998.4 as xptC. The amino acid sequence indicates that it is a
member of the glucose −methanol −choline (GMC) oxidoreductase superfamily, a broad
34

class that includes cellobiose dehydrogenase, choline dehydrogenase, and methanol oxidase
(Cavener, 1992). Deletion of AN7999.4 had no obvious effect on prenyl xanthone
biosynthesis.

35

2.4 Discussion
We used a combination of genomics, efficient gene targeting, and natural products
chemistry to elucidate the complex prenyl xanthone biosynthesis pathway in A. nidulans,
the first such pathway to be deciphered in a fungus. This pathway is complex, involving the
products of 10 clustered genes and three genes located apart from the main cluster. The
genes mdpA throughmdpL are located on the right arm of Chromosome VIII,
whereas xptA is located on the right arm of Chromosome I and xptB and xptC are found on
the left arm of Chromosome II. In all cases the genes are ∼0.5 Mb from a telomere. Other
uncommon examples of separated fungal secondary metabolism genes include the genes
for T-toxin biosynthesis in Cochliobolus heterostrophus, with 9 genes in two unlinked loci,
and the nonclustered acetyltransferase gene Tri101 involved in trichothecene biosynthesis
inFusarium species, having a different evolutionary history from other trichothecene genes
(Inderbitzin et al., 2010; O'Donnell et al., 2000). Without genomics and efficient gene
targeting it would have been difficult, if not impossible, to identify all of the genes in the
pathway. In particular, it would have probably been extremely difficult to identify the three
genes that are separated in the genome from the bulk of the xanthone biosynthesis genes in
the absence of the ability to identify candidate genes and to delete them en masse.
The two prenyltransferases, encoded by the xptA and xptB genes, are particularly
interesting. They are both homologous to members of a class of enzymes known as fungal
indole prenyltransferases (Steffan et al., 2009). For example, XptA is 27% identical at the
amino acid level to SirD from Leptosphaeria maculans (Kremer and Li, 2010) and 24%
identical to FgaPT2 from Aspergillus fumigatus (Unsold and Li, 2005). XptB is 29% and
36

27% identical to these enzymes, respectively, and XptA and XptB are 37% identical to
each other. Unlike many other prenyltransferase enzymes, the fungal indole
prenyltransferases are soluble, lack (N/D)DxxD motifs, and continue to be active in the
absence of Mg
2+
(Steffan et al., 2009). The fungal indole prenyltransferases are capable of
regular and reverse C-prenylations as well as N- and O-prenylations. Their solubility may
be useful for chemoenzymatic synthesis in terms of ease of manipulation and efficiency of
catalysis. All members of this class studied in detail to date prenylate compounds of amino
acid origin, whereas our data reveal that XptA and XptB clearly prenylate polyketides.
There were hints from the literature that this class might have a range of substrates
extending to polyketides. For example, the X-ray structure of dimethylallyl tryptophan
synthase (DMATS) from A. fumigatus revealed a common architecture (but no significant
primary amino acid similarity) with the bacterial enzyme NphB that catalyzes addition of a
10-carbon geranyl moiety to a polyketide-based aromatic scaffold (Metzger et al., 2009). In
addition, a gene in Penicillium aethiopicum that is homologous to indole prenyltransferases
is proximal to a polyketide gene necessary for the formation of viridicatumtoxin and was
proposed to contribute to addition of a geranyl group to that metabolite (Chooi et al., 2010).
Our findings provide the first direct evidence, however, that this class of prenyltransferases
can prenylate polyketides, and the possibility must be considered that XptA and XptB may
prenylate polyketide substrates beyond those reported in this study.
Our deletions reveal the genes required for prenyl xanthone synthesis, but correlation of the
deleted genes with the intermediates that accumulate in the deletion strains allows us to
propose a biosynthetic pathway for the A. nidulansprenyl xanthones (Figure 2-1). We
previously detailed the synthesis of monodictyphenone (16) in a cclA Δ background, which
37

was dependent on many of the same secondary metabolite genes also responsible for the
prenyl xanthones (Chiang et al., 2010a). We propose that 16 is an intermediate en route to
prenyl xanthones (Figure 2-6). One new observation, however, is that from the mdpL Δ
strain we have now isolated chrysophanol (12).
Earlier work by others used isotopic precursors to propose the biosynthesis of2 and a
similar metabolite, tajixanthone, in Aspergillus variecolor (Ahmed et al., 1992). The
labeling patterns from [1-
13
C]- and [2-
13
C]-acetate incorporation suggested an octaketide
precursor, which could condense into an anthrone and possibly autooxidize to an
anthraquinone. Interestingly, the earlier work also found that isotopically labeled 12 was
incorporated into tajixanthone; it was proposed that xanthone formation proceeds from an
octaketide to 7 and further to 12. Because our earlier analysis (Chiang et al.,
2010a) indicates that 12 is not in the direct biosynthetic pathway from 7 to 16, we propose
that it is a shunt metabolite; more specifically, the mdpL knockout may yield an unstable
intermediate that transforms to chrysophanol. 7 might not be converted to 12 when the
biosynthesis is uninterrupted. An alternative explanation for incorporation of 12into
tajixanthone is that 12 is metabolized to 7 at a low rate through an endogenous mechanism.
Aloe-emodin (11), the ω-hydroxylated analog of 12, may be a side product arising from
oxidation by an endogenous cytochrome P450 enzyme (Kelly et al., 2009).
Elucidating the enzymatic conversion of 7 to 16 is complicated by the fact that the process
appears to be dependent on five of the clustered genes, mdpB,mdpC, mdpJ, mdpK,
and mdpL, and that the metabolite profiles from each of the corresponding deletion strains
are similar. As a further complication, the presence of several metabolites in a deletant
38

strain (for example, five major compounds from mdpL Δ) makes it difficult to ascribe their
roles in the biosynthesis and their relation to each other. We refer the reader to (Chiang et
al., 2010a) for a discussion of the possible functions of mdpB, mdpC, mdpJ, mdpK,
and mdpLin monodictyphenone biosynthesis. Our data allow us, however, to deduce the
remainder of the prenyl xanthone biosynthesis pathway with considerable confidence.
In this study we show conclusively that four genes, mdpD, xptA, xptB, andxptC are, at a
minimum, responsible for conversion of monodictyphenone to the prenyl xanthones. It is
easiest to deduce the steps of the biosynthetic pathway by working back from the final
products, shamixanthone (2) and epishamixanthone (4). The xptC deletion does not
accumulate 2 or 4 but does accumulate emericellin (3) and variecoxanthone A (1). This
allows us to deduce that XptC is required for conversion of 3 or 1 to 2 and 4.
The xptAdeletion strains accumulate 1 but not 3, 2, or 4. This result allows us to deduce
that XptA is required for the C-prenylation of 1 to form 3 and also allows us to deduce
that 3 is the compound that is converted by XptC to 2 and 4. Given the similarity of XptC
to oxidative enzymes, we suggest that oxidation of the primary alcohol of 3 to an aldehyde,
followed by ene cyclization (Chexal et al., 1975), may yield 2 and its epimer 4. From the
facts that XptA catalyzes the C-prenylation of 1 and no prenylated xanthones are found
in xptB deletion strains, we deduce that XptB catalyzes the O-prenylation required for
formation of 1 and that the O-prenylation occurs before the C-prenylation and is a
prerequisite for the C-prenylation. From the mdpD deletant we isolated compound 8, which
contains the xanthone core but does not bear functionality at the 2 position, at which the
final products feature the O-prenyl group. Oxidation of the xanthone core at the C-2
position is necessary for O-prenylation, so we postulate that MdpD, homologous to known
39

monooxygenases, catalyzes oxidation prior to O-prenylation by XptB. In the absence of the
C-2 oxygen, XptA is able to yield the C-prenylated product 9 in small amounts. In
the xptB deletant strain, we were able to detect compound 17, which we suspect to be a
shunt metabolite arising from reduction of the carbonyl group of monodictyphenone (16)
and condensation with the carboxylic acid.
In addition to assisting in elucidating the biosynthetic pathway, many of the shunt products
and intermediate molecules are bioactive. For instance, 11 is cytotoxic to neuroblastoma,
Ewing sarcoma, and pPNET cell lines and inhibits neuroblastoma tumors in vivo but is not
toxic to normal cells nor animal models (Pecere et al., 2000). 9 displays antimicrobial and
antiacetylcholineesterase activity and is also cytotoxic against hepG2 cells (Wen et al.,
2008). Compound 8 has antifungal and antibacterial activities (Hein et al., 1998). These
findings indicate that a benefit of targeted deletions of genes is the accumulation of useful
quantities of biosynthetic intermediates that may themselves possess noteworthy biological
properties.

40

2.5 Conclusion
Previous work demonstrated that a cluster of 10 genes was responsible for several related
compounds that emerged in a chromatin deletant strain of A. nidulans, including
monodictyphenone (Chiang et al., 2010a). The current work reveals that four prenyl
xanthones rely on most of these genes for their synthesis but in addition require a
monooxygenase gene within the cluster and three genes outside of the cluster, including
two belonging to a family of indole prenyltransferase genes and one homologous to GMC
oxidoreductase genes. The combined data allow us to propose that these latter genes are
involved in the later stages of prenyl xanthone formation to complete a biosynthesis in
which monodictyphenone is a precursor.

41

2.6 Materials and Methods
Generation of Fusion PCR Fragments, A. nidulans Protoplasting, and Transformation
All gene deletions were carried out according to the gene targeting procedures of Szewczyk
et al. Two ∼1000 base pair fragments upstream and downstream of every targeted gene
were amplified from A. nidulans genomic DNA using PCR. Primers used in this study are
listed in Table 2-2, Supporting Information. The two amplified flanking sequences and
an A. fumigatus pyrG selectable marker cassette were fused together by PCR using nested
primers.A. nidulans strains in this study are displayed in Table 2-3, Supporting
Information. Protoplast generation and transformation were performed as described
(Szewczyk et al., 2006). The strain LO2026 carrying a deletion of the stcJ gene that
eliminates sterigmatocystin production was used as the recipient strain. Diagnostic PCR of
the deletant strains was carried out using the external primers from the first round of PCR.
The difference in the size between the gene replaced by the selective marker and the native
gene allowed us to establish if the transformants carried correct gene replacements. For
further verification, diagnostic PCR was performed two more times, using one of the
external primers and a primer located inside the marker gene, then the other external primer
and an internal primer. In these instances, the deletants yielded the PCR product of the
expected size whereas no product was present in nondeletants.
Media and Cultivation of Strains
YES media was prepared by combining 20 g of yeast extract, 120 g of sucrose, 20 g of
agar, and 2 mL of trace element solution (Sanchez et al., 2010) in 1 L H2O. For LC/MS
42

screening, spores of LO2026 (the control strain) and three strains of each gene deletant
were individually cultivated (1 × 10
7
spores) on 10 × 150 mm Petri dishes containing YES
agar and cultivated at 37 °C for 5 days. The agar was chopped into ∼2 cm
2
pieces, and the
material was extracted using sonication with methanol, followed by 1:1
methanol:dichloromethane. The organic solvents were removed in vacuo, and the
remaining material was partitioned between H2O (25 mL) and ethyl acetate (25 mL × 2).
The combined ethyl acetate layers were evaporated, and the crude material was redissolved
at a concentration of 20 mg/mL in DMSO and then diluted 5-fold in methanol.
LC/MS Analysis
LC/MS was carried out using a ThermoFinnigan LCQ Advantage ion trap mass
spectrometer with an RP C18 column (Alltech Prevail; 2.1 × 100 mm with a 3 μm particle
size) at a flow rate of 125 μL/min and monitored by a UV detector at 254 nm. The solvent
gradient was 95% MeCN −H2O (solvent B) in 5% MeCN −H2O (solvent A) both containing
0.05% formic acid:0% B from 0 to 5 min, 0 to 100% B from 5 to 35 min, 100% B from 35
to 40 min, 100% B to 0% B from 40 to 45 min, and reequilibration with 0% B from 45 to
50 min.
Isolation of Metabolites
The LO2026 (stcJ Δ), LO3387 (mdpL Δ), LO3337 (mdpD Δ), LO3896 (xptA Δ), and LO4178
(xptB Δ) strains were each cultivated in 25 × 150 mm Petri dishes containing 2 L YES
media and extracted in the same manner as above. The crude material was subjected to
silica gel column chromatography, using ethyl acetate and hexanes as the eluent. The
43

materials were further separated by preparative HPLC [Phenomenex Luna 5 μm C18 (2),
250 × 21.2 mm] with a flow rate of 5.0 mL/min and measured by a UV detector at 250 nm.
Shamixanthone (2), emericellin (3), epishamixanthone (4), and paeciloxanthone (9)
required further purification using preparative TLC. See Supporting Information for more
details about isolation.

44

2.7 Supplementary Information
Supplementary Methods
NMR spectra were run on a Varian Mercury Plus 400 spectrometer. IR spectra were
obtained on a Bruker Vertex 80 FTIR spectrometer. For all structural isolations and
elucidations twenty-five 150x15 mm petri dishes containing solid YES media were
inoculated with an A. nidulans strain. All compounds, from ~1.5g crude extract, were
initially applied to a SiO2 column (Merck 230-400 mesh, ASTM) and eluted with 1 L
fractions. All collected fractions were further purified by reverse phase HPLC
[Phenomenex Luna 5 μm C18 (2), 250 x 10 mm] with a flow rate of 10.0 mL/min and
measured by a UV detector at 250 nm. Solvent A was 5% acetonitrile in water with 0.05%
trifluoroacetic acid. Solvent B was 100% acetonitrile with 0.05% trifluoroacetic acid.
For the isolation of compounds 1-4 LO2026 (stcJ ∆) was cultivated, and 10:90 ethyl
acetate:hexanes was the eluent for silica gel chromatography. The HPLC gradient system
was 70 to 100% B from 0 to 40 min, 100% B from 40 to 46 min, 100% B to 70% B from
46 to 47 min, and re-equilibration with 70% B from 47 to 52 min. Shamixanthone (2) and
emericellin (3) co-eluted and were separated using preparative TLC with 7.5:92.5 ethyl
acetate:hexanes as eluent. Epishamixanthone (4) required further purification and was
isolated using preparative TLC with 7.5:92.5 ethyl acetate:hexanes as eluent.
For the isolation of 2, ω-dihydroxyemodin (5), ω-hydroxyemodin (6), emodin (7),
aloeemodin (11) and chrysophanol (12) LO3387 (mdpL ∆) was utilized. A 10:90 ethyl
acetate:hexanes fraction afforded semi-pure material containing chrysophanol (12). The
45

HPLC gradient system was 50 to 75% B from 0 to 40 min, 75% B to 100%B from 40 to 41
min, 100% B from 41 to 46 min, 100% B to 50% B from 46 to 47 min, and reequilibration
with 50% B from 47 to 52 min. A 20:80 ethyl acetate:hexanes fraction afforded semi-pure
material containing aloe-emodin (11) and emodin (7). The HPLC gradient system was 40
to 65% B from 0 to 40 min, 65% B to 100%B from 40 to 41 min, 100% B from 41 to 46
min, 100% B to 65% B from 46 to 47 min, and re-equilibration with 65% B from 47 to 52
min. A 60:40 ethyl acetate:hexanes fraction afforded semipure material containing ω-
hydroxyemodin (6). The HPLC gradient system was 25 to 40% B from 0 to 40 min, 40% B
to 100%B from 40 to 41 min, 100% B from 41 to 46 min, 100% B to 25% B from 46 to 47
min, and re-equilibration with 25% B from 47 to 52 min. A 100% ethyl acetate fraction
afforded semi-pure material containing 2, ω- dihydroxyemodin (5). The HPLC gradient
system was 10 to 40% B from 0 to 40 min, 40% B to 100%B from 40 to 41 min, 100% B
from 41 to 46 min, 100% B to 10% B from 46 to 47 min, and re-equilibration with 10% B
from 47 to 52 min.
For the isolation of 9H-xanthen-9-one, 8-hydroxy-1-(hydroxymethyl)-3-methyl- (8) and
paeciloxanthone (9) LO3336 (mdpD ∆) was utilized as the strain. A 10:90 ethyl
acetate:hexanes fraction afforded semi-pure material containing 8 and 9. The HPLC
gradient system was 50 to 75% B from 0 to 40 min, 75% B to 100%B from 40 to 41 min,
100% B from 41 to 46 min, 100% B to 50% B from 46 to 47 min, and re-equilibration with
50% B from 47 to 52 min. Paeciloxanthone required further purification and was isolated
using preparative TLC with 40:60 ethyl acetate:hexanes as eluent.
46

The procedure for isolating variecoxanthone A (1) from LO3895 (xptA ∆) was identical to
that of isolating 1 from LO2026 (stcJ ∆).
For the isolation of monodictyphenone (16) and 1(3H)-isobenzofuranone, 3-(2,6
dihydroxyphenyl)-4-hydroxy-6-methyl- (17) LO4178 (xptB ∆) was utilized as the strain. A
100% ethyl acetate fraction afforded semi-pure material containing 16 and 17. The HPLC
gradient system was 10 to 40% B from 0 to 40 min, 40% B to 100% B from 40 to 41 min,
100% B from 41 to 46 min, 100% B to 10% B from 46 to 47 min, and reequilibration with
10% B from 47 to 52 min.
Structural Characterization
Variecoxanthone A (1): yellow powder; IR υmax
KBr
3517, 2919, 1644, 1599, 1470, 1234,
1105, 1062; The
1
H NMR and
13
C NMR of 1 is highly similar to those of emericellin,
except that resonances corresponding to the C-prenyl group are missing. DEPT, HMQC,
and HMBC spectroscopy corroborated our assignment of 1 as variecoxanthone A.
1
H NMR
(CDCl3): δ = 1.72 (3H, br s), 1.81 (3H, br s), 2.46 (3H, br s), 4.45 (2H, d, J=7.6 Hz), 5.09
(2H, br s), 5.61 (1H, br t, J=7.6 Hz), 6.78 (1H, br d, J=8.4 Hz), 6.91 (1H, br d, J=8.4 Hz),
7.30 (1H, br s), 7.59 (1H, br t, J=8.4 Hz), 12.66 (1H, s);
13
C NMR (CDCl3): δ = 18.0, 18.3,
26.1, 57.3, 72.5, 106.8, 109.4, 110.7, 118.2, 119.7, 119.8, 134.5, 137.1, 139.4, 143.1, 152.9,
154.3, 155.9, 162.1, 184.7.
Shamixanthone (2): yellow powder; IR υmax
KBr
3507, 2916, 1643, 1603, 1573, 1480, 1429,
1243;
1
H NMR and
13
C NMR data (CDCl3), in good agreement with the published data
(Marquez-Fernandez, et al., 2007). DEPT, HMQC, and HMBC spectroscopy further
confirmed 2 to be shamixanthone.
1
H NMR (CDCl3): δ = 1.75 (3H, s), 1.79 (3H, s), 1.84
47

(3H, s), 2.36 (3H, br s), 2.73 (1H, br d, J = 3.2 Hz), 3.50 (2H, t, J = 6.0 Hz), 4.35 (1H, dd, J
= 10.8 and 3.2 Hz), 4.42 (1H, ddd, J = 10.8, 3.2, and 1.2 Hz), 4.59 (1H, br s), 4.80 (1H, br
s), 5.07 (1H, d, J = 3.6 Hz), 5.31 (1H, m), 5.41 (1H, br t, J = 3.6 Hz), 6.73 (1H, d, J = 8.4
Hz), 7.29 (1H, s), 7.44 (1H, d, J = 8.4 Hz), 12.59 (1H, s);
13
C NMR (CDCl3): δ = 17.5,
17.9, 22.6, 25.8, 27.5, 45.0, 63.2, 64.6, 109.2, 109.7, 112.3, 116.9, 118.9, 119.3, 121.0,
121.7, 133.3, 136.5, 138.3, 142.6, 149.4, 152.2, 152.8, 159.7, 184.5.
Emericellin (3): yellow powder; IR υmax
KBr
3517, 2917, 1642, 1600, 1480, 1237, 1008;
1
H
NMR and
13
C NMR data (CDCl3), in good agreement with the published data (Marquez-
Fernandez, et al., 2007).
1
H NMR (CDCl3): δ = 1.72 (3H, s), 1.75 (3H, s), 1.79 (3H, s),
1.81 (3H, s), 2.47 (3H, s), 3.49 (2H, d, J = 7.6 Hz), 4.45 (2H, d, J = 7.6 Hz), 4.49 (2H, d, J
= 8.0 Hz), 5.08 (2H, d, J = 8.0 Hz), 5.31 (1H, br t, J = 7.6 Hz), 5.62 (1H, br t, J = 7.6 Hz),
6.74 (1H, d, J = 8.4 Hz), 7.34 (1H, s), 7.44 (1H, d, J = 8.4 Hz), 12.53 (1H, s);
13
C NMR
(CDCl3): δ = 17.8, 17.9, 18.1, 25.8, 25.9, 27.5, 57.1, 72.2, 109.1, 110.0, 118.0, 118.9,
119.5, 119.6, 121.6, 133.4, 134.2, 136.9, 139.1, 142.6, 152.6, 152.8, 154.0, 159.9, 184.7.
Epishamixanthone (4): yellow powder; IR υmax
KBr
3507, 2925, 1643, 1570, 1482, 1430,
1239;
1
H NMR (CDCl3), in good agreement with the published data (Ishida et al., 1976).
1
H NMR (CDCl3): δ = 1.75 (3H, br s), 1.79 (3H, s), 1.99 (3H, s), 2.38 (3H, s), 2.55 (1H, br
d, J = 11.6 Hz), 3.51 (2H, d, J = 7.2 Hz), 4.34 (1H, J = 11.6 and 10.4 Hz), 4.47 (1H, ddd, J
= 10.4, 3.6, and 1.6 Hz), 4.62 (1H, br d, J = 3.6 Hz), 4.78 (1H, br s), 5.06 (1H, d, J = 1.2
Hz), 5.32 (1H, m), 5.50 (1H, td, J = 3.6 and 1.2 Hz), 6.72 (1H, d, J = 8.4 Hz), 7.29 (1H, s),
7.42 (1H, d, J = 8.4 Hz), 12.57 (1H, s);
13
C NMR (CDCl3): δ = 17.8, 18.2, 22.8, 26.0, 27.7,
48

44.2, 63.5, 64.2, 109.4, 109.9, 111.7, 117.1, 119.1, 119.7, 121.8, 121.9, 133.6, 136.7, 138.7,
142.5, 149.5, 152.3, 153.0, 160.0, 184.6.
2, ω-dihydroxyemodin (5): brown powder; IR υmax
ZnSe
3401, 1682, 1620, 1382, 1209,
1140; The UV and NMR spectra are similar to ω-dihydroxyemodin (7), but a mass
difference of +16 Da and one fewer aromatic proton indicates that the anthraquinone is
tetraoxygenated. The methylene (CH2) resonance of 4.58 ppm in the
1
H NMR spectrum,
identical to the methylene resonance in dihydroxyemodin, indicates that the C-ring does not
bear another hydroxyl group. A
13
C NMR resonance of 181.3 is indicative 148 of a
carbonyl carbon that is not hydrogen-bonded to a hydroxyl group, ruling out the C-4
position. This leaves C-2 as the position of the fourth hydroxyl group.
1
H NMR (DMSO-
d6): 7.60 (1H, s), 7.21 (1H, s), 7.19 (1H, s), 4.58 (2H, s);
13
C NMR (DMSOd6): δ = 62.8,
110.1, 110.4, 115.1, 117.5, 120.9, 125.4, 134.0, 140.1, 152.2, 153.5, 181.3, 191.1.
ω-dihydroxyemodin (6): yellow powder; IR υmax
ZnSe
3369, 2917, 1675, 1627, 1475, 1387,
1257, 1204, 1173, 1024
1
H NMR and
13
C NMR data (DMSO-d6), in good agreement with
the published data (Bok et al., 2009; Morooka et al., 1990).
1
H NMR (DMSO-d6): δ = 4.58
(2H, s), 6.56 (1H, br s), 7.09 (1H, br s), 7.21 (1H, s), 7.60 (1H, s), 12.05 (1H, s);
13
C NMR
(DMSO-d6): δ = 62.7, 108.6, 109.5, 109.7, 11438, 117.7, 121.5, 133.6, 135.8, 153.5, 162.1,
165.2, 166.7, 182.1, 190.3.
Emodin (7): yellow powder; IR υmax
ZnSe
3373, 2947, 2835, 1676, 1450, 1115, 1028;
1
H
NMR and
13
C NMR data (DMSO-d6), in good agreement with the published data (Bok, et
al., 2009; Morooka, et al., 1990).
1
H NMR (DMSO-d6): δ = 2.36 (3H, br s), 6.54 (1H, J = 2
Hz), 7.05 (1H, J = 2 Hz), 7.09 (1H, br s), 7.40 (1H, br s), 11.96 (1H, br s), 12.02 (1H, br s);
49

13
C NMR (DMSO-d6): δ = 22.1, 108.6, 109.5, 109.5, 114.0, 121.1, 124.8, 133.4, 135.7,
148.9, 162.1, 165.1, 166.4, 181.9, 190.3.
9H-xanthen-9-one, 8-hydroxy-1-(hydroxymethyl)-3-methyl- (8): yellow powder; IR
υmax
ZnSe
3175, 2917, 1653, 1609, 1471, 1271, 1233, 1076
1
H NMR and
13
C NMR data
(CDCl3), in good agreement with the published data (Ayer and Taylor, 1976; Hein et al.,
1998).
1
H NMR (CDCl3): δ = 2.50 (3H, br s), 4.98 (2H, s), 6.80 (1H, br d, J = 8.4 Hz), 6.92
(1H, br d, J = 8.4 Hz), 7.15 (1H, br s), 7.60 (1H, t, J = 8.4 149 Hz), 12.65 (1H, s);
13
C NMR
(CDCl3): δ = 22.2, 65.4, 106.9, 109.6, 110.9, 116.8, 118.2, 127.5, 137.1, 142.8, 147.7,
155.9, 158.3, 162.2, 184.6.
Paeciloxanthone (9): yellow powder; IR υmax
KBr
3215, 2922, 1648, 1610, 1480, 1264, 944;
1
H NMR and
13
C NMR data (CDCl3), in good agreement with the published data (Wen et
al., 2008).
1
H NMR (CDCl3): δ = 1.76 (3H, s), 1.80 (3H, s), 2.51 (3H, br s), 3.51 (2H, d, J =
6.8 Hz), 4.43 (1H, t, J = 8.0 Hz), 4.95 (2H, d, J = 8.0 Hz), 5.25 (br t, J = 7.6 Hz), 6.76 (1H,
d, J = 8.8 Hz), 7.08 (1H, br s), 7.15 (1H, s), 7.23 (1H, s), 7.28 (1H, s), 7.46 (1H, d, J = 8.8
Hz), 12.54 (1H, s);
13
C NMR (CDCl3): δ = 18.2, 22.2, 26.0, 27.7, 65.5, 109.6, 110.4, 116.8,
118.2, 119.2, 121.9, 127.5, 133.6, 137.3, 142.8, 147.5, 153.1, 158.3, 160.2, 184.8.
Aloe-emodin (11): orange powder; IR υmax
ZnSe
3391, 2949, 2837, 1681, 1419, 1205, 1143,
1027;
1
H NMR and
13
C NMR data (DMSO-d6), in good agreement with the published data
(Kametani et al., 2007).
1
H NMR (DMSO-d6): δ = 4.62 (2H, d, J = 6 Hz), 5.60 (1H, t, J = 6
Hz), 7.29 (1H, s), 7.38 (1H, br d, J = 8.4 Hz), 7.72-7.69 (2H, m), 7.80 (1H, t, J = 8.4 Hz),
11.9 (2H, br s);.
13
C NMR (DMSO-d6): δ = 62.7, 115.2, 116.7, 117.8, 120.0, 121.4, 125.1,
133.9, 134.1, 138.0, 154.4, 162.0, 162.3, 182.2, 192.4.
50

Chrysophanol (12): yellow powder; IR υmax
KBr
2916, 1676, 1627, 1476, 1452, 1272,1212,
1163;
1
H NMR and
13
C NMR data (CDCl3), in good agreement with the published data
(Miethbauer et al., 2008). DEPT, HMQC, and HMBC spectroscopy further confirmed 12 to
be chrysophanol.
1
H NMR (CDCl3): δ = 2.47 (3H, s), 7.11 (1H, br s), 7.29 (1H, dd, J = 8.8
and 1.2 Hz), 7.69-7.65 (2H, m), 7.83 (1H, dd, J = 7.4, 1.2 150 Hz), 12.02 (1H, s), 12.13
(1H, s);
13
C NMR (CDCl3): 22.5, 114.0, 116.1, 120.2, 121.6, 124.6, 124.8, 133.5, 133.9,
137.2, 149.6, 162.7, 163.0, 182.3, 192.8.
Monodictyphenone (16): yellow powder; IR υmax
ZnSe
3209, 1684, 1628, 1455, 1206, 1031,
923;
1
H NMR and
13
C NMR data (acetone-d6), in good agreement with the published data
(Krick et al., 2007).
1
H NMR (acetone-d6): δ = 2.31 (1H, s), 6.33 (2H, d, J = 8.0 Hz), 6.97
(1H, br s), 7.20 (1H, t, J = 8.0 Hz), 7.33 (1H, br s);
13
C NMR (acetone-d6): δ = 20.5, 107.3,
111.9, 120.5, 121.6, 129.0, 131.3, 135.9, 138.7, 153.5, 162.2, 137.1, 201.6.
1(3H)-isobenzofuranone, 3-(2,6-dihydroxyphenyl)-4-hydroxy-6-methyl- (17): yellow
powder; IR υmax
ZnSe
3307, 2364, 2344, 1683, 1615, 1208, 1145; The
1
H NMR and
13
C NMR
of 17 is similar to those of monodictyphenone, except that the carbonyl resonance at 201.6
ppm is missing and a new proton resonance at 6.91 ppm and a carbon resonance at 73.9
ppm is observed, consistent with a (Ar)2CH-O-grouping. As 17 is 16 Da less than
monodictyphenone and that reduction of the carbonyl would add two protons, reduction
followed by dehydration (18 fewer Daltons) would account for the mass difference. The
only functional group in proximity capable of participating in dehydration is the carboxylic
acid group. DEPT, HMQC, and HMBC spectroscopy corroborated our assignment of 17 as
variecoxanthone A.
1
H NMR (acetone-d6): δ = 2.34 (3H, br s), 6.37 (2H, d, J=8.0 Hz), 6.91
51

(1H, s), 6.93 (1H, t, J=8.0 Hz), 7.01 (1H, br s), 7.08 (1H, br s);
13
C NMR (acetone-d6): δ =
20.5, 73.9, 107.3, 109.2, 115.5, 120.6, 129.9, 134.2, 140.1, 152.2, 157.6, 171.3.

52

Table 2-1. Gene designations of the text and corresponding annotations from two
websites
a
.

a. CADRE (http://www.cadre-genomes.org.uk) which uses the same designations as
the Aspergillus genome database (AspGD,http://www.aspgd.org) and the Broad Institute
Aspergillus Comparative Database
(http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html).
Putative functions are from BLAST searches performed previously (Bok et al., 2009) or for
this study.

53

Table 2-2. Primers used in this study (5’  3’)
AN10039
AN10039P1: GTA CAA CAC CGG CCT CTA GC
AN10039P2: ACC ACA CCC A TA CGC A TA CC
AN10039P3: CGA AGA GGG TGA AGA GCA TTG GAC A TG ACG ACA TGA T AC GG
AN10039P4: GCA TCA GTG CCT CCT CTC AGA CAG CTG CGT GAC CTT TCT TTT
CC
AN10039P5: TGG CAG CA T CTA AGG A TT GG
AN10039P6: GAA GCC A TC CCC ACT AA T CC

mdpA
mdpAP1: GTC ACC GAC CTG AAG TAC CC
mdpAP2: TGT CTT GTG AGT TGG GA T CG
mdpAP3: CGA AGA GGG TGA AGA GCA TTG CAA CCG A TA GAG CCT GAA CC mdpAP4:
GCA TCA GTG CCT CCT CTC AGA CAG GGA TAC AGT TCC GAA CAA GC
mdpAP5: TGA GGG ACT GAG GGT CTT CC
mdpAP6: GAC ACC A TG AGG GAC TGA GG

mdpB
mdpBP1: ACC TCA A TT CCA ACG TCA GC
mdpBP2: AAA GTT GCC CTT GTG ACT GG
mdpBP3: CGA AGA GGG TGA AGA GCA TTG AGT GTC TAG GAC GGG AAG ACC
mdpBP4: GCA TCA GTG CCT CCT CTC AGA CAG TAG TTT CTG CGT CGG AAT CG
mdpBP5: CCA GCC TCG ACA ACA GAT CC
mdpBP6: GGC GCT GAC CTA TAA TTT GG

mdpC
mdpCP1: CGC ACA GCT TCA TTC CTA CC
mdpCP2: CAA CAT GCC TCC AAT TAG CC
mdpCP3: CGA AGA GGG TGA AGA GCA TTG TGG AGA CA T TGG TGC TTT CC
mdpCP4: GCA TCA GTG CCT CCT CTC AGA CAG GTA AAA CCC GCC TTC A TA CG
mdpCP5: A TT CCG ACG CAG AAA CTA CC
mdpCP6: ATA TGC AGC CGA ACA TGA CC

mdpD
mdpDP1: TGT AAC CAG TGT TGG GAC ACC
mdpDP2: GAA AGT GGC AGT GCA AGT CC
mdpDP3: CGA AGA GGG TGA AGA GCA TTG TTG GGT AGG GTC A TT GAA GC
54

Table 2-2 (continued). Primers used in this study (5’  3’)
mdpDP4: GCA TCA GTG CCT CCT CTC AGA CAG CAG TCG CAA TGT GA T TGA GC
mdpDP5: CAA TAC CTC AAC CAG GAG TCG
mdpDP6: CAG TGT TGG AGG ACA TGA GG

mdpE
mdpEP1: CGA GGC AAC AGA CAA A TT CC
mdpEP2: TAT ACC ACC CCG AAC TCT GC
mdpEP3: CGA AGA GGG TGA AGA GCA TTG A TC AA T CGG GGG A TT ACA GC mdpEP4:
GCA TCA GTG CCT CCT CTC AGA CAG GAG TGG TCG GAG TCT TTT TCC
mdpEP5: ATG GAC CTT TGC GTG TTT CC
mdpEP6: GAG CA T GCG GTA GAA TTT CC

mdpF
mdpFP1: GGT TCT GCG AGA TCT CA T CC
mdpFP2: GCG AGA TCT CA T CCA CTA ACG
mdpFP3: CGA AGA GGG TGA AGA GCA TTG AGT CTA GCC GA T GCT TTT GC mdpFP4:
GCA TCA GTG CCT CCT CTC AGA CAG A TT GGA TGG AGT GAG GTT GG
mdpFP5: CCC A TT CGA CCG A TA ACT CC
mdpFP6: ACG GAG GAG AAG GAC TTT GC

mdpG
mdpGP1: TGG TGT GAA TTC AGC TTT CG
mdpGP2: ACA TAT GGT CA T GCG AGT GC
mdpGP3: CGA AGA GGG TGA AGA GCA TTG AAG TGA CAA GCG TCA GA T CG mdpGP4:
GCA TCA GTG CCT CCT CTC AGA CAG CCC A TC CTC ACT CA T CAA CC
mdpGP5: TTG ACT GAA CCC TGC TAG GC
mdpGP6: TAC TGG AAG CGC TGA TAT GC

mdpH
mdpHP1: AAC AAC CTC GTG GAC TAC GC
mdpHP2: CTG TCC ACG GAG AAG AGT GG
mdpHP3: CGA AGA GGG TGA AGA GCA TTG TGG TTG A TG AGT GAG GA T GG mdpHP4:
GCA TCA GTG CCT CCT CTC AGA CAG TAG AGT CGC TTC GGG ACA TCA ACC
mdpHP5: TCC CAG CGA GCA GAA GA T AGA AG
mdpHP6: CTG GGA TTG GAG AAC GTA GC

mdpI
mdpIP1: CAG CGA GA T CAA CCA TCA CC
mdpIP2: TTC TGC ATA TCA GCG CTT CC
mdpIP3: CGA AGA GGG TGA AGA GCA TTG GGG TTT CAG TGG AAC TGT CG
mdpIP4: GCA TCA GTG CCT CCT CTC AGA CAG AGA TGG A TT GTG TGC TGA
55

Table 2-2 (continued). Primers used in this study (5’  3’)
GG
mdpIP5: GGA GTT CAT CGA GCG TAT CG
mdpIP6: CGG GTA CCG TAG CCT AAA CC

mdpJ
mdpJP1: TAC AAT CCC AGG CCA TTA GG
mdpJP2: GGA AGA AA T GCC TGA GCA AGC
mdpJP3: CGA AGA GGG TGA AGA GCA TTG CGA T AC GCT CGA TGA ACT CC mdpJP4:
GCA TCA GTG CCT CCT CTC AGA CAG TCG GTG GCG TTA AGA A TA GC
mdpJP5: GTA GTC A TG ACG GGG AA T GG
mdpJP6: CTC CAG ACA TGG AGG GAA GG

mdpK
mdpKP1: TCC CGC AAC CTT CTT AAA CC
mdpKP2: CTC AAG GAC CCC A TC A TA CC
mdpKP3: CGA AGA GGG TGA AGA GCA TTG CGG GTA CCG TAG CCT AAA CC
mdpKP4: GCA TCA GTG CCT CCT CTC AGA CAG AGC CCT GA T CGA GGT TAA GG
mdpKP5: CA T CTC GGC AGT CTT TCT CG
mdpKP6: GCA CAG AGG TTT AGC A TC TCG

mdpL
mdpLP1: TGA TCC AGA A TC TGC TCT CG
mdpLP2: CGC CTA CTG TCG AAA CAA GC
mdpLP3: CGA AGA GGG TGA AGA GCA TTG GGT AGA TGG TTG GGT TTT GC mdpLP4:
GCA TCA GTG CCT CCT CTC AGA CAG GGG TCT TGG CCA TCT AGT ACG
mdpLP5: CTC GGT CTG ACC A TT CTT GC
mdpLP6: GTG TTT TGC TCT TGC ACA GG

AN0153
AN0153P1: GAG AAA GAC TGC CGA GAT GC
AN0153P2: GCC GAG ATG CTA AAC CTC TG
AN0153P3: CGA AGA GGG TGA AGA GCA TTG A TG A TG CTT CCA GGA TCA GC
AN0153P4: GCA TCA GTG CCT CCT CTC AGA CAG CCG TCA GTC AGT CAA AGT GG
AN0153P5: CTG CCT CCT TTA CCC GTC TCC
AN0153P6: AGC CTT GCT GCC TCC TTT ACC

xptA
xptAP1: GTC GCT CTC CAA CA T TGA CC
xptAP2: GCT GAA GGG A TA AAC AGT GG
xptAP3: CGA AGA GGG TGA AGA GCA TTG CCA A TT CCA CCC AAA GTA CG xptAP4:
GCA TCA GTG CCT CCT CTC AGA CAG GAC CAC AAC CGG ACA CTT CC
56

Table 2-2 (continued). Primers used in this study (5’  3’)
xptAP5: GTG TTC TTA TCC CGG TTT CC
xptAP6: CA T GGA AGC TCT GGA GAA GG

xptB
xptBP1: CTT TGC TGG CA T TGT AGT CG
xptBP2: GCG ACT CTG CAC TA T CTG A TT ACC
xptBP3: CGA AGA GGG TGA AGA GCA TTG CTG GTC AGT TTG CA T GAT GG xptBP4:
GCA TCA GTG CCT CCT CTC AGA CAG GGC AGA GTA CCG TCA CTT CG
xptBP5: GA T TA T A TG CCT GGG GAA GC
xptBP6: GTG TGG A TT CGT GGA ACT GG

xptC
xptCP1: GTG A TA CGG AGA TAG A TA CTG
xptCP2: GTA GA T CAA CCT TGT GAA GTC
xptCP3: CGA AGA GGG TGA AGA GCA TTG GCA GAA TTA CGG A TA GCT TGG xptCP4:
GCA TCA GTG CCT CCT CTC AGA CAG A TC TAT TAG ACC GCA GGC AG
xptCP5: TAC GAG CAG A TT CTC AAA AGC
xptCP6: TAC TCT CGA GCT CA T TCA GC

AN7999
AN7999P1: TGA GGT CGT CAA ACA TAC AGC
AN7999P2: TGG GAC AGA ACC TTC CAA GC
AN7999P3: CGA AGA GGG TGA AGA GCA TTG GTT TGG A TT TGG A TG A TG CTT G
AN7999P4: GCA TCA GTG CCT CCT CTC AGA CAG GAA GCT TCA GAG TAA GTC
CG
AN7999P5: CA T ACG CAA GCA TAC TCG AC
AN7999P6: CCA AGC GTA TGG ACT CTC AC

AN8514
AN8514P1: ACA CGA TGG CGA AGA TA T GG
AN8514P2: A TG GCA GTG GAC TAG A TT GG
AN8514P3: CGA AGA GGG TGA AGA GCA TTG GCA GCT TGT CCT TTT GTG ACC
AN8514P4: GCA TCA GTG CCT CCT CTC AGA CAG CA T TGG CTG TCG TTG A TT CG
AN8514P5: CCA GCG TCT CAG CTC TA T GG
AN8514P6: CCA TCC AGC TAC TCG ACA CG

AN10289
AN10289P1: CTG AAG GGA TGG TGG AAA GG
AN10289P2: CTT TTA CTA CGG GGC TCA CG
AN10289P3: CGA AGA GGG TGA AGA GCA TTG CGT A TC AAC TGG CTT TCA CAG G
57

Table 2-2 (continued). Primers used in this study (5’  3’)
AN10289P4: GCA TCA GTG CCT CCT CTC AGA CAG CCT TTC CCT TTC TCT CTT CC
AN10289P5: GTA TGC TGG TTG CCA GAG TCC
AN10289P6: TTT CCT GGC TGG TTC AGA GG

AN11080
AN11080P1: GCA GCA GAA TGA GAA CAG AGG
AN11080P2: AGA GAG CAG AAG AGG CAG AGC
AN11080P3: CGA AGA GGG TGA AGA GCA TTG ACG GTC GTG A TT TCC TTT GC
AN11080P4: GCA TCA GTG CCT CCT CTC AGA CAG AGT GCC TGA TAA CTC TGC TTC
G
AN11080P5: TTA TCC CTT CAC CTG CTT CC
AN11080P6: CTT TCG TAA GGG GAA CAA GG

AN11194
AN11194P1: ACG A TC TA T CA T GGG GTT CC
AN11194P2: TTG AA T CA T CGG TGC ACT CC
AN11194P3: CGA AGA GGG TGA AGA GCA TTG CAA CAA TCA CCA GGA CTA CTC G
AN11194P4: GCA TCA GTG CCT CCT CTC AGA CAG CTG ACA CTC CTG ACC CTT ACG
AN11194P5: GAG GTC CCA GCT GAA GAA GG
AN11194P6: CAG ACT TCT TCA GCC TTT GC

AN11202
AN11202P1: TA T CCT ACC GGG AGT CAA CC
AN11202P2: CGC CCC TTT GTC TGA A TA TG
AN11202P3: CGA AGA GGG TGA AGA GCA TTG GTC TGC TGT GCC TTA TGT ACT CC
AN11202P4: GCA TCA GTG CCT CCT CTC AGA CAG TAG CAG AAG TCC CGA GTC ACG
AN11202P5: CAG CAG GAC TTT TGG TTT GC
AN11202P6: GAC TTG GTC GTA TGC CTT GG

Blue and red sequences are tails that anneal to the A. fumigatus pyrG fragment (AfpyrG)
during fusion PCR.

58

Table 2-3. A. nidulans strains used in this study

Strain Genotype Source
stcJ ∆
LO2026
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB (Bok et al.,
2009)
mdpG ∆, cclA ∆
LO2149
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
cclA:AfpyroA; mdpG::AfpyrG
(Bok et al.,
2009)
AN0523 ∆,
cclA ∆
LO2154
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
cclA:AfpyroA; AN0523::AfpyrG
(Bok et al.,
2009)
AN1034 ∆,
cclA ∆
LO2159
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
cclA:AfpyroA; AN1034::AfpyrG
(Bok et al.,
2009)
AN2032 ∆,
cclA ∆
LO2165
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
cclA:AfpyroA; AN2032::AfpyrG
(Bok et al.,
2009)
AN3230 ∆,
cclA ∆
LO2169
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
cclA:AfpyroA; AN3230::AfpyrG
(Bok et al.,
2009)
AN3386 ∆,
cclA ∆
LO2174
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
cclA:AfpyroA; AN3386::AfpyrG
(Bok et al.,
2009)
AN6000 ∆,
cclA ∆
LO2179
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
cclA:AfpyroA; AN6000::AfpyrG
(Bok et al.,
2009)
AN6448 ∆,
cclA ∆
LO2184
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
cclA:AfpyroA; AN6448::AfpyrG
(Bok et al.,
2009)
AN7071 ∆,
cclA ∆
LO2189
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
cclA:AfpyroA; AN7071::AfpyrG
(Bok et al.,
2009)
AN7909 ∆,
cclA ∆
LO2194
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
cclA:AfpyroA; AN7909::AfpyrG
(Bok et al.,
2009)
AN10039
LO3366,
LO3367
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
AN10039::AfpyrG
This
study
mdpA ∆
LO3346-
LO3348
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpA::AfpyrG
This
study
mdpB ∆
LO3361,
LO3362
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpB::AfpyrG
This
study
59

Table 2-3 (continued). A. nidulans strains used in this study

mdpC ∆
LO3371-
LO3373
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpC::AfpyrG
This
study
mdpD ∆
LO3336-
LO3338
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpD::AfpyrG
This
study
mdpE ∆
LO3391,
LO3392
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpE::AfpyrG
This
study
mdpF ∆
LO3381-
LO3383
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpF::AfpyrG
This
study
mdpG ∆
LO3341,
LO3342
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpG::AfpyrG
This
study
mdpH ∆
LO3351-
LO3353
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpH::AfpyrG
This
study
mdpI ∆
LO3396,
LO3398
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpI::AfpyrG
This
study
mdpJ ∆
LO3401-
LO3403
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpJ::AfpyrG
This
study
mdpK ∆
LO3356-
LO3358
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpK::AfpyrG
This
study
mdpL ∆
LO3387,
LO3388
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
mdpL::AfpyrG
This
study
xptA ∆
LO3895-
LO3897
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
xptA::AfpyrG
This
study
xptB ∆
LO4178,
LO4179
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
xptB::AfpyrG
This
study
xptC ∆
LO4428,
LO4429
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
xptC::AfpyrG
This
study
AN7999 ∆
LO4433-
LO4435
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
AN7999::AfpyrG
This
study
AN8514 ∆
LO3900-
LO3902
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
AN8514::AfpyrG
This
study
60

Table 2-3 (continued). A. nidulans strains used in this study

AN10289 ∆
LO4168-
LO4170
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
AN10289::AfpyrG
This
study
AN11080 ∆
LO3915-
LO3917
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
AN11080::AfpyrG
This
study
AN11194 ∆
LO3920-
LO3922
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
AN11194::AfpyrG
This
study
AN11202 ∆
LO3925-
LO3927
pyrG89; pyroA4, nkuA::argB; riboB2, stcJ::AfriboB;
AN11202::AfpyrG
This
study
61

Figure 2-1. Prenyl xanthones and compounds that emerged from the study of targeted gene
deletions. The compounds are as follows: variecoxanthone A, 1; shamixanthone, 2; emericellin,
3; epishamixanthone, 4; 2, ω-dihydroxyemodin, 5; ω-hydroxyemodin, 6; emodin, 7; 9H-xanthen-
9-one, 8-hydroxy-1-(hydroxymethyl)-3-methyl-, 8; paeciloxanthone, 9; endocrocin, 10; aloe-
emodin, 11; chrysophanol, 12; cichorine, 13; austinol, 14; dehydroaustinol, 15;
monodictyphenone, 16; and 1(3H)-isobenzofuranone, 3-(2,6-dihydroxyphenyl)-4-hydroxy-6-
methyl-, 17.
62

Figure 2-2. HPLC profiles of extracts from stcJ Δ; stcJ Δ, cclA Δ; and stcJ Δ, cclA Δ, mdpG Δ
strains, as detected by UV absorbance at 254 nm. Numbering of peaks correspond to the
compounds in Figure 2-1. Shamixanthone 2 and emericellin 3 elute at the same retention time.

63

Figure 2-3. (Top) Organization of the genes surrounding the PKS mdpG involved in prenyl
xanthone biosynthesis. Each arrow indicates gene size and direction of transcription. On the
basis of a set of deletions we created and analyzed, genes shown in black are involved in prenyl
xanthone biosynthesis while those in gray are not. (Middle) Organization of the genes for
monodictyphenone biosynthesis in a cclA Δ background (Chiang et al., 2010). AN0147 and
AN0148 are circled to emphasize that AN0147 is unnecessary for monodictyphenone generation
but required for xanthone synthesis, whereas AN0148 is necessary for monodictyphenone
generation but not required for xanthone synthesis. (Bottom) Organization of the genes outside
of the mdpG cluster that are involved in prenyl xanthone synthesis. The genes that were
successfully deleted were AN10039 through mdpL, xptA, xptC, AN7999, and xptB.

64

Figure 2-4. HPLC extracts of strains in the cluster as detected by UV absorbance at 254 nm. ω-
Hydroxyemodin 6 and the unrelated metabolite austinol 14 elute at the same retention time.

65

Figure 2-5. (a) HPLC extracts of the prenyltransferase deletant strains xptA Δ and xptB Δ as
detected by UV absorption at 254 nm. (b) HPLC extract of xptC Δ. (c) Mass spectra of the major
LC/MS peak from (top) stcJ Δ control extract and (bottom) xptC Δ extract.

66

Figure 2-6. Proposed biosynthetic pathway of prenyl xanthones.

67

Figure 2-7. UV/Vis and ESI-MS of variecoxanthone A (1, positive mode), shamixanthone (2,
positive mode), emericellin (3, positive mode), epishamixanthone (4, positive mode), 2, ω
dihydroxyemodin (5, negative mode), ω- hydroxyemodin (6, negative mode).

68

Figure 2-8. UV/Vis and ESI-MS of emodin (7, negative mode), compound 8 (positive mode),
paeciloxanthone (9, positive mode), aloe-emodin (11, negative mode), chrysophanol (12,
negative mode), monodictyphenone (16, negative mode), compound 17 (negative mode).
69

Figure 2-9.
1
H and
13
C NMR spectra of variecoxanthone A 1.
70

Figure 2-10.
1
H and
13
C NMR spectra of shamixanthone 2.
71

Figure 2-11.
1
H and
13
C NMR spectra of emericellin 3.
72

Figure 2-12.
1
H and
13
C NMR spectra of epishamixanthone 4.
73

Figure 2-13.
1
H and
13
C NMR spectra of 2, ω-dihydroxyemodin 5.
74

Figure 2-14.
1
H and
13
C NMR spectra of ω-hydroxyemodin 6.
75

Figure 2-15.
1
H and
13
C NMR spectra of emodin 7.
76

Figure 2-16.
1
H and
13
C NMR spectra of compound 8.
77

Figure 2-17.
1
H and
13
C NMR spectra of paeciloxanthone 9.
78

Figure 2-18.
1
H and
13
C NMR spectra of aloe-emodin 11.
79

Figure 2-19.
1
H and
13
C NMR spectra of chrysophanol 12.
80

Figure 2-20.
1
H and
13
C NMR spectra of monodictyphenone 16.
81

Figure 2-21.
1
H and
13
C NMR spectra of compound 17.
82

CHAPTER III: Molecular genetic characterization of the biosynthesis cluster of a
prenylated isoindolinone alkaloid aspernidine A in Aspergillus nidulans
3.1 Abstract
Aspernidine A is a prenylated isoindolinone alkaloid isolated from the model fungus Aspergillus
nidulans. A genome-wide kinase knock out library of A. nidulans was examined and it was
found that a mitogen-activated protein kinase gene, mpkA, deletion strain produces aspernidine
A. Targeted gene deletions were performed in the kinase deletion background to identify the
gene cluster for aspernidine A biosynthesis. Intermediates were isolated from mutant strains
which provided information about the aspernidine A biosynthesis pathway.

83

3.2 Introduction
Secondary metabolites (SMs) that filamentous fungi produce have served as a valuable source of
low molecular weight molecules with a variety of biological activities. Many of the bioactive
SMs that are easily accessible under conventional laboratory conditions have already been
isolated and patented for drug development. However, advances in genome sequencing revealed
that there is an abundance of potential SM gene clusters that have yet to be associated with their
final metabolites.(Chiang et al., 2010b; Galagan et al., 2005; Sanchez et al., 2012b)
One major group of SMs is polyketides (PKs) whose core structure is furnished by polyketide
synthases (PKSs). Using a variety of genome mining methods, we and others have successfully
identified the immediate products of all 14 nonreducing (NR)-PKSs in the model fungus A.
nidulans (Ahuja et al., 2012; Brown et al., 1996a; Gerke et al., 2012; Nielsen et al., 2011;
Schroeckh et al., 2009; Watanabe et al., 1999). However, for several of these NR-PKSs in A.
nidulans, the final, downstream metabolites remain unknown. We are interested in using this
information to comprehensively link metabolites to genes and to elucidate their biosynthetic
pathways.
One such metabolite of interest is aspernidine A. Aspernidine A was discovered previously by
the Hertweck group by screening 45 different culture conditions using the A. nidulans AXB4A2
strain (see Supplementary Table 3-3 for genotype) and was isolated from 14 L of malt medium
cultured for 7 days (Scherlach et al., 2010a). The aromatic group in this compound suggested
that its biosynthesis is initiated by an NR-PKS. Prior to this study, our group had not been able to
detect the production of aspernidine A in the genome-sequenced strain A. nidulans FGSC A4 and
all other FGSC A4 background mutant strains. A chief requirement for the elucidation of the
84

genetic biosynthetic basis of SMs is the availability of a strain that expresses the required gene
cluster to yield enough material for chemical and biological characterization. We have recently
obtained and screened a genome-wide kinase knock-out (KO) library, a resource provided by the
Fungal Genetics Stock Center (FGSC) to the Aspergillus research community (Colin P. De
Souza, 2013), with the expectation that manipulating the expression of kinases, which are key
players in many aspects of regulation and signal transduction, would be a novel approach for
activating cryptic gene clusters. Herein we report that we have successfully found a kinase KO
strain that produces aspernidine A and we performed serial deletions of the surrounding genes to
identify the genes involved in its biosynthesis. We also isolated and characterized intermediates
from the deletion strains, which allowed us to propose a biosynthesis pathway.

85

3.3 Results and Discussion
Through screening of this library (a total of 98 kinase KO strains), we found one strain (mpkA ∆)
that consistently produced compounds 8 and 9 which were distinctly different from the
compounds produced by the control strain (1-7) (Figure 3-1). Compound 8 was isolated from this
strain from yeast agar glucose (YAG) medium at a titer of almost 15 mg/L and identified as
aspernidine A. MpkA is one of four mitogen-activated protein kinase (MAPK) genes that the A.
nidulans genome harbors (Atoui et al., 2008; Bayram et al., 2012; Furukawa et al., 2005;
Hagiwara et al., 2009; Jun et al., 2011; Kawasaki et al., 2002).

The kinase has been shown to
play a pivotal role in cell wall integrity signalling as well as in regulation of germination of
conidial spores and polarized growth (Bussink and Osmani, 1999; Fujioka et al., 2007). The
discovery of a strain of A. nidulans that produces aspernidine A (8) in high titer allowed us to
identify and analyze the gene cluster involved in its biosynthesis. Herein we report the
identification of the biosynthetic gene cluster for aspernidine A biosynthesis through a series of
targeted gene deletions in the mpkA Δ background. We isolated and characterized 3 novel related
compounds, aspernidine C-E (9-11), allowing us to propose a biosynthesis pathway for
aspernidine A.
For full characterization of compound 8, the mpkA Δ strain was cultivated at a larger scale and the
target compound was purified using flash chromatography and preparative HPLC.
1
H and
13
C
NMR analysis identified 8 as aspernidine A (Scherlach et al., 2010a). Additionally, a potentially
new metabolite 9 was isolated, and the similarity of UV spectra and MS fragmentation patterns
(Figure 3-6) suggested it was structurally related to 8. Compound 9 had a molecular formula of
C25H35NO4
(deduced from HRESI-MS), suggesting the presence of an additional methyl group.
This was confirmed by one- and two-dimensional NMR spectroscopy (F). Though several
86

isoindoline derivatives have been identified from fungi, compound 9 appears to be a new
compound, which we named aspernidine C.
The chemical structure of aspernidine A led us to hypothesize that it might be derived from
orsellinaldehyde, which was shown previously to be the product of the NR-PKS, PkfA.(Ahuja et
al., 2012) To test this hypothesis, we generated a pkfA deletion in the mpkA Δ background,
replacing pkfA with the A. fumigatus pyroA gene, a nutritional selection marker required for
pyridoxine biosynthesis. The deletion was verified by diagnostic PCR. We cultured this mpkA ∆,
pkfA ∆ double mutant and the mpkA Δ strain under the same conditions that yielded aspernidine A.
In the double mutant, aspernidine A production was eliminated (Figure 3-1). This confirmed that
PkfA, the PKS encoded by pkfA, is required for the biosynthesis of aspernidine A.
We next set out to identify additional genes involved in aspernidine A biosynthesis. Taking
advantage of the fact that secondary metabolism genes in A. nidulans are usually clustered, we
focused on the genes surrounding pkfA (Table 3-1, Figure 3-3). We performed gene deletions in
the mpkA Δ background, and all deletions were verified by diagnostic PCR. Again, all strains
were cultivated in the same culture conditions that yielded aspernidine A. LC/MS analysis of the
extracts from the gene deletion strains showed elimination of aspernidine A in deletants of genes
from AN3225 through AN3230 (Figure 3-4). The putative functions of the genes within the
cluster along with the genes immediately outside are shown in Table 3-1. Deletants of AN3224
and AN3231 continued to produce aspernidine A, indicating that we have identified the
boundaries of the gene cluster. We now designate the genes surrounding pkfA as pkfB- pkfF.
In silico analysis was carried out using the Aspergillus 16-way comparative database asp2_v7
provided through the Aspergillus Genome Database (AspGD, http://www.aspgd.org/) to compare
87

the surrounding genes of this proposed gene cluster with those of other fungal species.
Interestingly, this analysis shows that our proposed aspernidine A gene cluster is present as an
insertion of ~20kb in A. nidulans within a highly conserved region of the Aspergillus genome
(Supplementary Figure 3-13). Furthermore, Anderson et al. developed an algorithm to accurately
predict SM gene clusters based on an annotated genome sequence and a catalog of gene
expression (Andersen et al., 2013). Using this algorithm, they predicted the number of genes in
the cluster for pkfA to be six, which matches our findings from targeted gene deletions. These
data further suggest that we have correctly determined the extent of the gene cluster responsible
for the biosynthesis of aspernidine A.
The deletant strains in which aspernidine A production was eliminated were examined for
intermediates or shunt products that are part of the biosynthetic process for aspernidine A (Figure
3-4). Extracts from strains carrying deletions of pkfC, pkfD, pkfE, and pkfA showed no obvious
intermediates. Strains carrying deletions of pkfB and pkfF each displayed a significant new peak
in the chromatogram. Both strains were cultured at a larger scale and the metabolites were
isolated by flash chromatography and preparative HPLC. The structures of compounds 10 and
11, which we have named aspernidine D and E, respectively (Figure 3-2), were determined using
both one- and two-dimensional NMR spectroscopy (see Supporting Information for detailed
structural characterization, Figures 3-7 to 3-12).
Our gene deletion data and the intermediates we isolated and structurally characterized by
NMR allow us to propose a biosynthetic pathway for aspernidine A (Figure 3-5). The starting
point is the production of orsellinaldehyde by the NR-PKS PkfA as shown previously (Ahuja et
al., 2012). Although we were unable to identify the immediate products after polyketide
biosynthesis, isolation of compound 10 from the mpkA Δ, pkfB Δ strain led us to propose that
88

hydroxylation, methylation of one of the phenol groups, and prenylation, presumably catalyzed
by the gene product of pkfE, a prenyltransferase gene, would be needed to yield aspernidine D
(10). Subsequently, the gene product of the cytochrome P450 monooxygenase gene pkfB is
responsible for hydroxylation of aspernidine D (10) to yield aspernidine E (11). An aromatic
dialdehyde, asperugin A, was found as a metabolic product of A. rugulosus, a species closely
related to A. nidulans (Ballanti.Ja et al., 1965). This compound shows structural relation to the
aspernidines (Scherlach et al., 2010a), leading us to hypothesize that the choline dehydrogenase
gene pkfF may be responsible for further oxidation of aspernidine E (11), to form this dialdehyde
intermediate. Furthermore, this intermediate will need to be transformed in a series of steps,
some of which are enzyme-mediated, to generate aspernidine A (8). Although fungal SM
biosynthetic genes are often clustered in one region of the chromosome, we have identified two
examples in A. nidulans where the genes are located in at least two distinct genomic loci and
therefore we cannot exclude the possibility that additional genes in the genome are involved in
aspernidine A biosynthesis (Lo et al., 2012; Sanchez et al., 2011a).

89

3.4 Materials and Methods
Generation of fusion PCR fragments, A. nidulans protoplasting, and transformation
A. nidulans strains used in this study are listed in Table 3-3. The control strain and mpkA ∆ strain
were a part of an arrayed kinase knock-out library obtained from the Fungal Genetics Stock
Center (http://www.fgsc.net/Aspergillus/KO_Cassettes.htm). All other deletant strains were
generated by replacing each gene with a deletion construct harboring the Aspergillus fumigatus
pyroA gene in the recipient strain mpkA ∆ (FGSC1404). The construction of deletion constructs
by fusion PCR, protoplast production, and transformation were carried out as described
previously(Szewczyk et al., 2006). For the construction of deletion constructs by fusion PCR,
two ~1,000 base pair fragments upstream and downstream of every targeted gene were amplified
from A. nidulans genomic DNA by PCR. Primers used in this study are listed in Table 3-2. The
two amplified flanking sequences and the A. fumigatus pyroA selection marker cassette were
fused together into one construct by fusion PCR using nested primers. Diagnostic PCR of the
deletant strains was performed employing the external primers (P1 and P6) from the first round
of PCR. The difference in size between the gene replaced by the selection marker and the native
gene allowed us to determine whether the transformant carried the correct gene replacement. For
further verification, multiple diagnostic PCRs were carried out using external primers paired
with primers located within the selection marker gene in which case the deletants yielded PCR
products of the expected size whereas no product would be seen in the non-deletants. Genotypes
of all strains are given in Table 3-3.

90

Fermentation and LC/MS analysis
All strains were cultivated at 37°C on solid YAG (complete medium; 5 g yeast extract /L, 15 g of
agar /L, and 20 g of D-glucose /L supplemented with 1.0 mL/L of trace element solution) starting
with 1 x 10
7
spores per 100 x 15 mm Petri dishes (with 25 mL medium per plate). After four
days of cultivation at 37°C, the agar was chopped into small pieces and the material was
extracted with 50 mL of methanol (MeOH) followed by 50 mL of 1:1 CH2Cl2/MeOH, each with
1 hour of sonication. The extract was evaporated in vacuo to yield a residue, then subsequently
partitioned between H2O (25 mL) and ethyl acetate (EtOAc, 25 mL x 2). The combined EtOAc
layer was evaporated in vacuo, redissolved in 200 µL of DMSO, then diluted with 4 volumes of
MeOH. 10 µL of this dilute crude extract was injected for HPLC-DAD-MS analysis.
LC/MS was performed using a ThermoFinnigan LCQ advantage ion trap mass spectrometer with
a reverse-phase C18 column (Alltech Prevail; 2.1 x 100 mm with a 3 µm particle size) at a flow
rate of 125 µL/min. The solvent gradient for HPLC-DAD-MS was 95% acetonitrile
(MeCN)/H2O (solvent B) in 5% MeCN/H2O (solvent A) both containing 0.05% formic acid, as
follows: 0% solvent B from 0 to 5 min, 0 to 100% solvent B from 5 min to 35 min, 100% solvent
B from 35 to 40 min, 100 to 0% solvent B from 40 to 45 min, and re-equilibration with 0%
solvent B from 45 to 50 min.
Isolation and Identification of Secondary Metabolites
All strains were cultivated at 37°C for 4 days on ~40 YAG plates at 1 x 10
7
spores per 100 x 15
mm plate (~25 mL of YAG per plate). Similar to the method described above, the agar was
chopped and sonicated in MeOH, followed by 1:1 CH2Cl2/MeOH. The organic material was
91

evaporated and extracted twice with an equal volume of EtOAc. All EtOAc layers were
combined and evaporated in vacuo.
For isolation of mpkA ∆ strain metabolites, the crude extract in the EtOAc layer (397 mg) was
coated on 6 g SiO2 gel (Merck 230-400 mesh, ASTM) which was then suspended in CH2Cl2 and
applied to a SiO2 gel column (20 x 75 mm). The column was then eluted with CH2Cl2-MeOH
mixture of increasing polarity (fraction A, 1:0, 350 ml; fraction B, 49:1, 350 ml; fraction C, 19:1,
350 ml; fraction D, 9:1, 350 ml; and fraction E, 7:3, 350 ml). All fractions were analyzed by
HPLC-DAD-MS. Fraction B was subjected to semi-preparative reverse phase HPLC
(Phenomenex Luna 5 µm C18 (2), 250 x 10 mm) with flow rate of 5.0 ml/min and monitored by
a UV detector at 254 nm. The gradient system was MeCN (solvent B) in 5% MeCN/H2O
(Solvent A) both containing 0.05% TFA: 30 to 50% B from 0 to 7 min, 50 to 90% B from 7 to 12
min, maintained at 90% from 12 to 22 min, 90 to 100% B from 22 to 27 min, maintained at
100% from 27 to 32 min, 100 to 30% B from 32 to 37 min, and re-equilibration with 30% B
from 37 to 42 min. Compounds 8 (14.8 mg) and 9 (4.3 mg) were eluted at 20.10 and 25.0
minutes respectively.
For isolation of mpkA ∆, pkfB ∆ strain metabolites, after 2 x extraction with EtOAc, the aqueous
layer was acidified with 6M HCl to approximately pH 2.5. The two EtOAc extracts were
combined with the acidified EtOAc extract and evaporated in vacuo. The crude extract (214 mg)
was applied to a SiO2 gel column and eluted into five fractions using the same gradient system
used for the mpkA ∆ strain. Fraction A was subjected to further purification using semi-
preparative reverse phase HPLC. Compound 10 (9 mg) was eluted at 21.80 minutes.
92

For isolation of mpkA ∆, pkfF ∆ strain metabolites, the crude extract in EtOAc layer (241 mg) was
applied to a SiO2 gel column (20 x 75 mm) and eluted into five fractions using the same gradient
system used for the mpkA ∆ strain. Fraction B was subjected further purification using semi-
preparative reverse phase HPLC. Compound 11 (1.93 mg) was eluted at 23.64 minutes.
Compound structure identification with NMR analysis
For compound structure elucidation,
1
H and
13
C spectra were collected on a nuclear magnetic
resonance (NMR) Varian Mercury Plus 400 and Varian VNMRS-600 spectrometer. High
resolution electrospray ionization mass spectrum (HRESI-MS) was obtained with an Agilent
Technologies 1200 series high-resolution mass spectrometer.

93

3.5 Supplementary Information
Generation of fusion PCR fragments, A. nidulans protoplasting, and transformation
A. nidulans strains used in this study are listed in Table 3-3. The control strain and mpkA strain
were a part of an arrayed kinase knock-out library obtained from the Fungal Genetics Stock
Center (http://www.fgsc.net/Aspergillus/KO_Cassettes.htm). All other deletant strains were
generated by replacing each gene with a deletion construct harboring the Aspergillus fumigatus
pyroA gene in the recipient strain mpkA FGSC1404). The construction of deletion constructs
by fusion PCR, protoplast production, and transformation were carried out as described
previously.(Szewczyk et al., 2006) For the construction of deletion constructs by fusion PCR,
two ~1,000 base pair fragments upstream and downstream of every targeted gene were amplified
from A. nidulans genomic DNA by PCR. Primers used in this study are listed in Table 3-2. The
two amplified flanking sequences and the A. fumigatus pyroA selection marker cassette were
fused together into one construct by fusion PCR using nested primers. Diagnostic PCR of the
deletant strains was performed employing the external primers (P1 and P6) from the first round
of PCR. The difference in size between the gene replaced by the selection marker and the native
gene allowed us to determine whether the transformant carried the correct gene replacement. For
further verification, multiple diagnostic PCRs were carried out using external primers paired
with primers located within the selection marker gene in which case the deletants yielded PCR
products of the expected size whereas no product would be seen in the non-deletants. Genotypes
of all strains are given in Table 3-3.

94

Fermentation and LC/MS analysis
All strains were cultivated at 37°C on solid YAG (complete medium; 5 g yeast extract /L, 15 g of
agar /L, and 20 g of D-glucose /L supplemented with 1.0 mL/L of trace element solution) starting
with 1 x 10
7
spores per 100 x 15 mm Petri dishes (with 25 mL medium per plate). After four
days of cultivation at 37°C, the agar was chopped into small pieces and the material was
extracted with 50 mL of methanol (MeOH) followed by 50 mL of 1:1 CH2Cl2/MeOH, each with
1 hour of sonication. The extract was evaporated in vacuo to yield a residue, then subsequently
partitioned between H2O (25 mL) and ethyl acetate (EtOAc, 25 mL x 2). The combined EtOAc
layer was evaporated in vacuo, redissolved in 200 L of DMSO, then diluted with 4 volumes of
MeOH. 10 L of this dilute crude extract was injected for HPLC-DAD-MS analysis.
LC/MS was performed using a ThermoFinnigan LCQ advantage ion trap mass spectrometer with
a reverse-phase C18 column (Alltech Prevail; 2.1 x 100 mm with a 3 m particle size) at a flow
rate of 125 µL/min. The solvent gradient for HPLC-DAD-MS was 95% acetonitrile
(MeCN)/H2O (solvent B) in 5% MeCN/H2O (solvent A) both containing 0.05% formic acid, as
follows: 0% solvent B from 0 to 5 min, 0 to 100% solvent B from 5 min to 35 min, 100% solvent
B from 35 to 40 min, 100 to 0% solvent B from 40 to 45 min, and re-equilibration with 0%
solvent B from 45 to 50 min.
Isolation and Identification of Secondary Metabolites
All strains were cultivated at 37°C for 4 days on ~40 YAG plates at 1 x 10
7
spores per 100 x 15
mm plate (~25 mL of YAG per plate). Similar to the method described above, the agar was
chopped and sonicated in MeOH, followed by 1:1 CH2Cl2/MeOH. The organic material was
95

evaporated and extracted twice with an equal volume of EtOAc. All EtOAc layers were
combined and evaporated in vacuo.
For isolation of mpkA  strain metabolites, the crude extract in the EtOAc layer (397 mg) was
coated on 6 g SiO2 gel (Merck 230-400 mesh, ASTM) which was then suspended in CH2Cl2 and
applied to a SiO2 gel column (20 x 75 mm). The column was then eluted with CH2Cl2-MeOH
mixture of increasing polarity (fraction A, 1:0, 350 ml; fraction B, 49:1, 350 ml; fraction C, 19:1,
350 ml; fraction D, 9:1, 350 ml; and fraction E, 7:3, 350 ml). All fractions were analyzed by
HPLC-DAD-MS. Fraction B was subjected to semi-preparative reverse phase HPLC
(Phenomenex Luna 5 µm C18 (2), 250 x 10 mm) with flow rate of 5.0 ml/min and monitored by
a UV detector at 254 nm. The gradient system was MeCN (solvent B) in 5% MeCN/H2O
(Solvent A) both containing 0.05% TFA: 30 to 50% B from 0 to 7 min, 50 to 90% B from 7 to 12
min, maintained at 90% from 12 to 22 min, 90 to 100% B from 22 to 27 min, maintained at
100% from 27 to 32 min, 100 to 30% B from 32 to 37 min, and re-equilibration with 30% B
from 37 to 42 min. Compounds 8 (14.8 mg) and 9 (4.3 mg) were eluted at 20.10 and 25.0
minutes respectively.
For isolation of mpkA , pkfB  strain metabolites, after 2 x extraction with EtOAc, the aqueous
layer was acidified with 6M HCl to approximately pH 2.5. The two EtOAc extracts were
combined with the acidified EtOAc extract and evaporated in vacuo. The crude extract (214
mg) was applied to a SiO2 gel column and eluted into five fractions using the same gradient
system used for the mpkA strain. Fraction A was subjected to further purification using semi-
preparative reverse phase HPLC. Compound 10 (9 mg) was eluted at 21.80 minutes.
96

For isolation of mpkA , pkfF  strain metabolites, the crude extract in EtOAc layer (241 mg) was
applied to a SiO2 gel column (20 x 75 mm) and eluted into five fractions using the same gradient
system used for the mpkA strain. Fraction B was subjected further purification using semi-
preparative reverse phase HPLC. Compound 11 (1.93 mg) was eluted at 23.64 minutes.
Detailed Structural Characterization of Compounds
Aspernidine A (8): yellow powder;
1
H NMR (CDCl3), in good agreement with the published
data.(Scherlach et al., 2010b)
1
H NMR (CDCl3): δ = 1.55 (3H, s), 1.57 (3H, s), 1.62 (3H, s), 1.65
(3H, s) 1.95 (2H, m), 2.02 (2H, m), 2.02 (2H, m), 2.02 (2H, m), 3.89 (3H, s), 4.32 (2H, s), 4.64
(2H, d, J = 8.0 Hz), 5.03 (1H, m), 5.05 (1H, m), 5.47 (1H, br t, J = 8.0 Hz), 6.20 (1H, br s), 6.63
(1H, br s), 6.97 (1H, s). DEPT, HMQC, and HMBC spectroscopy corroborated our assignment
of 8 as aspernidine A;
1
H NMR and
13
C NMR data (DMSO-d6):
1
H NMR: δ = 1.53 (3H, s), 1.54
(3H, s), 1.56 (3H, s), 1.62 (3H, s) 1.92 (2H, m), 1.96 (2H, m), 1.98 (2H, m), 1.99 (2H, m), 3.81
(3H, s), 4.15 (2H, s), 4.49 (2H, d, J = 6.8 Hz), 5.04 (1H, m), 5.04 (1H, m), 5.48 (1H, t, J = 6.8
Hz), 6.75 (1H, br s), 6.75 (1H, br s), 6.97 (1H, s);
13
C NMR: δ = 15.73, 16.04, 17.57, 25.52,
25.95, 26.23, 39.16, 39.25, 42.42, 56.04, 68.38, 97.08, 120.45, 122.99, 124.49, 127.90, 130.70,
134.61, 137.48, 140.50, 146.16, 148.98, 154.07, 170.28. HRESI-MS, [M + H]
+
m/z found
400.2497 calc. for C24H33NO4:400.2482.
Aspernidine C (9): yellow powder; DEPT, HMQC, and HMBC spectroscopy corroborated our
assignment of 9 as aspernidine C;
1
H NMR (CDCl3): δ = 1.57 (3H, s), 1.57 (3H, s), 1.66 (3H, s),
1.66 (3H, s), 1.94 (2H, m), 2.04 (2H, m), 2.04 (2H, m), 2.04 (2H, s), 3.88 (3H, s), 3.98 (3H, s),
4.36 (2H, s), 4.57 (2H, d, J = 7.6), 5.06 (1H, m), 5.08 (1H, m), 5.55 (1H, t, J = 7.6 Hz), 6.78 (1H,
s);
13
C NMR (CDCl3): δ = 16.19, 16.58, 17.90, 25.91, 26.55, 26.92, 39.84, 39.90, 43.45, 56.51,
97

60.69, 70.10, 101.45, 119.87, 123.96, 124.50, 127.49, 128.31, 131.56, 135.58, 142.49, 143.68,
148.99, 155.47, 171.78. HRESI-MS, [M + H]
+
m/z found 414.2657 calc. for
C25H35NO4:414.2639.
Aspernidine D (10): yellow powder; DEPT, HMQC, and HMBC spectroscopy corroborated our
assignment of 10 as aspernidine D;
1
H NMR (CDCl3): δ = 1.56 (3H, s), 1.57 (3H, s), 1.64 (3H,
s), 1.65 (3H, s), 1.95 (2H, m), 1.95 (2H, m), 2.01 (2H, m), 2.01 (2H, m), 2.52 (3H, s), 3.89 (3H,
s), 4.54 (2H, d, J = 7.2 Hz), 5.06 (1H, br s), 5.06 (1H, br s), 5.54 (1H, t, J = 7.2 Hz), 6.27 (1H, s),
10.09 (1H, s), 12.14 (1H, s);
13
C NMR (CDCl3): δ = 16.14, 16.60, 17.87, 18.53, 25.89, 26.55,
26.90, 39.82, 39.88, 56.24, 69.36, 106.19, 114.24, 120.13, 124.08, 124.52, 131.51, 133.32,
135.41, 139.02, 142.16, 158.18, 159.70, 193.85. HRESI-MS, [M - H]
-
m/z found 385.2384 calc.
for C24H34O4:385.2384.
Aspernidine E (11): yellow powder; DEPT, HMQC, and HMBC spectroscopy corroborated our
assignment of 11 as aspernidine E;
1
H NMR (acetone-d6): δ = 1.59 (3H, s), 1.60 (3H, s), 1.65
(3H, s), 1.65 (3H, s), 1.96 (2H, m), 2.06 (2H, m), 2.06 (2H, m), 2.06 (2H, m), 3.84 (1H, s), 3.95
(3H, s), 4.53 (2H, d, J = 7.2 Hz), 4.92 (2H, s), 5.10 (1H, m), 5.12 (1H, m), 5.51 (1H, t, J = 7.2
Hz), 6.79 (1H, s), 10.29 (1H, s);
13
C NMR (acetone-d6): δ = 16.12, 16.49, 17.80, 25.92, 27.15,
27.49, 40.35, 40.52, 56.60, 62.24, 69.29, 105.01, 114.20, 121.65, 124.86, 125.22, 131.73, 134.76,
135.85, 141.66, 143.32, 158.91, 160.14, 195.45. HRESI-MS, [M - H]
-
m/z found 401.2336 calc.
for C24H34O5:401.2333.

98

Table 3-1. Putative function of genes within the aspernidine A cluster
Gene designation Putative function
AspGD
annotation
Broad annotation
pkfB Cytochrome p450 AN3225 ANID_03225.1
pkfC
Short chain
dehydrogenase
AN3226 ANID_03226.1
pkfD Hypothetical protein AN3227 ANID_03227.1
pkfE Prenyltransferase AN3228 ANID_03228.1
pkfF Choline dehydrogenase AN3229 ANID_03229.1
pkfA Polyketide synthase AN3230 ANID_03230.1

99

Table 3-2. Primers used in this study (5’  3’)

AN3224_P1 AAACCAGGCCATTCAGAGC
AN3224_P2 TTCAGAGCGGAAAACATCG
AN3224_P3 CGAAGAGGGTGAAGAGCATTGGCAAACACGGAAGAGGATG
AN3224_P4 ATCAGTGCCTCTCAGACAGACCTCGAACTACCGCACAAG
AN3224_P5 AGATGCGGTGCTACGAAAG
AN3224_P6 GTTTGTTGGAGGGCAACG

AN3225_P1 TCTGCGTGTGAAGTTGTGC
AN3225_P2 TGTATCAACGCGGCATTTC
AN3225_P3 CGAAGAGGGTGAAGAGCATTGAGATGTGCCAGATGCAACC
AN3225_P4 ATCAGTGCCTCTCAGACAGCATCTCCTGGGCTAGGTTGA
AN3225_P5 GGCTTCAGGTTAATGCTTGC
AN3225_P6 GCCGAGGTATTGGATTCTCA

AN3226_P1 GTGGGCGAACTCTGACAAAC
AN3226_P2 CGCAAGACGCTGTCAAAG
AN3226_P3 CGAAGAGGGTGAAGAGCATTGGGTTGCAAGTAGTCGAACAG
AN3226_P4 ATCAGTGCCTCTCAGACAGTCTGCGTGTGAAGTTGTGC
AN3226_P5 AGATGTGCCAGATGCAACC
AN3226_P6 TACCAGGATCTGCGAAAGG

AN3227_P1 TTTGTATGGGGAGTGCATTG
AN3227_P2 GTGCATTGCCTGATTCACC
AN3227_P3 CGAAGAGGGTGAAGAGCATTGTGCGACAGTTAGTGAGAGC
AN3227_P4 ATCAGTGCCTCTCAGACAGTGCAGACCGGAATGTTCTC
AN3227_P5 ATGCCAGATCCGTCCTTTC
AN3227_P6 TGCTGACAATGCCAGATCC

AN3228_P1 TGCGACAGTTAGTGAGAGCGT A
AN3228_P2 TCGTAAGGACGAGGACAGGA
AN3228_P3 CGAAGAGGGTGAAGAGCATTGTTCACCATTTCTTTGGTAGAGTAGGG
AN3228_P4 ATCAGTGCCTCTCAGACAGTGACAGGAAAGATGCACTGG
AN3228_P5 TTACCATCGGTGACGAGGTC
AN3228_P6 GCGAAATTACCATCGGTGAC

AN3229_P1 CCGCGAAGCTTTATTAGGG
AN3229_P2 TATTAGGGCCCGTTTGTCG
AN3229_P3 CGAAGAGGGTGAAGAGCATTGCCAGTGATCGGTTGTGGAG
AN3229_P4 ATCAGTGCCTCTCAGACAGTTCATTCGCTTCCCTTTCC
AN3229_P5 GGACTGGCTTGGAGTAGCC
AN3229_P6 GACCACTGGCCAACACAAG

AN3230_P1 GGTTGACGGTTGCATTGAC
AN3230_P2 GTCGTCTGACCTGGGATTTG
100

Table 3-2 (continued). Primers used in this study (5’  3’)

AN3230_P3 CGAAGAGGGTGAAGAGCATTGCCCAGTCACAGAGGGAGAAA
AN3230_P4 ATCAGTGCCTCTCAGACAGTCAACGACACAGGAATCACC
AN3230_P5 TATCTATCACCCGGCCTGAC
AN3230_P6 AGGGCGTAGACACAATGGAG

AN3231_P1 ATCGCCCTGGATAAGAGTGG
AN3231_P2 CTGCAGCACCATCTAACGAC
AN3231_P3 CGAAGAGGGTGAAGAGCATTGTGGGACTGCGACTAGTTGAG
AN3231_P4 ATCAGTGCCTCTCAGACAGCGCACTCAAGAGACCGACTA
AN3231_P5 TCCCTGGTATTGAGTCGGAC
AN3231_P6 ATCAAGACCGCACAGACTCT

Sequences in blue and red represent tails that anneal to the A.fumigatus pyroA fragment
(AfpyroA) during fusion PCR.

101

Table 3-3. A.nidulans strains used in this study

Strain Genotype Source
Control wA3; argB2; ΔnkuA::argB pyroA4; sE15 nirA14 chaA1 fwA1 FGSC,(Colin
P. De Souza,
2013)
mpkA ∆ pyrG89; wA3; argB2; ΔnkuA::argB pyroA4; sE15 nirA14
chaA1 fwA1 AN5666::AfpyrG
FGSC, (Colin
P. De Souza,
2013)
mpkA ∆,
AN3224 ∆
pyrG89; wA3; argB2; ΔnkuA::argB pyroA4; sE15 nirA14
chaA1 fwA1 mpkA::AfpyrG; AN3224::AfpyroA
This study
mpkA ∆,
pkfB ∆
pyrG89; wA3; argB2; ΔnkuA::argB pyroA4; sE15 nirA14
chaA1 fwA1 mpkA::AfpyrG; AN3225::AfpyroA
This study
mpkA ∆,
pkfC ∆
pyrG89; wA3; argB2; ΔnkuA::argB pyroA4; sE15 nirA14
chaA1 fwA1 mpkA::AfpyrG; AN3226::AfpyroA
This study
mpkA ∆,
pkfD ∆
pyrG89; wA3; argB2; ΔnkuA::argB pyroA4; sE15 nirA14
chaA1 fwA1 mpkA::AfpyrG; AN3227::AfpyroA
This study
mpkA ∆,
pkfE ∆
pyrG89; wA3; argB2; ΔnkuA::argB pyroA4; sE15 nirA14
chaA1 fwA1 mpkA::AfpyrG; AN3228::AfpyroA
This study
mpkA ∆,
pkfF ∆
pyrG89; wA3; argB2; ΔnkuA::argB pyroA4; sE15 nirA14
chaA1 fwA1 mpkA::AfpyrG; AN3229::AfpyroA
This study
mpkA ∆,
pkfA ∆
pyrG89; wA3; argB2; ΔnkuA::argB pyroA4; sE15 nirA14
chaA1 fwA1 mpkA::AfpyrG; AN3230::AfpyroA
This study
mpkA ∆,
AN3231 ∆
pyrG89; wA3; argB2; ΔnkuA::argB pyroA4; sE15 nirA14
chaA1 fwA1 mpkA::AfpyrG; AN3231::AfpyroA
This study
AXB4A2 pyrG89; pabaA1; bgaO; fwA1; argB2::pAXB4A (Scherlach et
al., 2010b;
Weidner et al.,
1998)

102

Figure 3-1. LC-DAD-MS analysis of a control strain and strains carrying mpkA Δ, and both mpkA Δ
and pkfA Δ. HPLC profiles of extracts of strains as detected by UV absorbance at 254 nm.

103

Figure 3-2. Structures of compounds elucidated throughout this study. The compounds are as
follows: asperthecin, 1; austinol, 2; dehydroaustinol, 3; sterigmatocystin, 4; emericellin, 5;
shamixanthone, 6; epishamixanthone, 7; aspernidine A, 8; aspernidine C, 9; aspernidine D, 10;
aspernidine E, 11.

104

Figure 3-3. Organization of genes surrounding the PKS pkfA involved in aspernidine A
biosynthesis in A. nidulans. Black open reading frames (ORFs) are genes involved in aspernidine
A biosynthesis while gray ORFs are genes not involved in aspernidine A biosynthesis.

105

Figure 3-4. Boundary of the aspernidine A biosynthesis gene cluster. HPLC profile of extracts of
strains in the cluster as detected by UV absorption at 254 nm. Numbers on peaks correspond to
the compounds shown in Figure 3-2.

106

Figure 3-5. Proposed biosynthesis pathway of aspernidine A

107

Figure 3-6. UV/Vis and ESI-MS of isolated compounds 8-11

108

Figure 3-7.
1
H NMR spectrum of compound 9

109

Figure 3-8.
13
C NMR spectrum of compound 9

110

Figure 3-9.
1
H NMR spectrum of compound 10

111

Figure 3-10.
13
C NMR spectrum of compound 10

112

Figure 3-11.
1
H NMR spectrum of compound 11

113

Figure 3-12.
13
C NMR spectrum of compound 11

114

Figure 3-13. Putative aspernidine A cluster embedded within a conserved syntenic region of
Aspergillus genome. Block arrows: gray, genes of the aspernidine A cluster; black, conserved
synteny block genes; white, putative genes that are not a part of the synteny block.

115

CHAPTER IV: Application of an efficient gene targeting system linking secondary
metabolites to their biosynthetic genes in Penicillium canescens
4.1 Abstract
Penicillium species are known to produce a variety of bioactive secondary metabolites. Many of
its secondary metabolite biosynthesis gene clusters remain silent under conventional laboratory
culture conditions, and to access and understand the complexity of their secondary metabolism, a
holistic approach is necessary. However, this is hindered by the lack of an efficient gene
targeting system in most fungal species. We first developed a gene targeting system in P.
canescens by generating a ku70-, pyrG- strain. We then took the genome-wide approach by
systematically deleting 29 polyketide synthase genes and 20 non-ribosomal peptide synthetase
genes. Wild-type strains were challenged on 10 different media and Czapek’s solid media was
chosen based on its secondary metabolite profile. Screening of the mutant library revealed the
genetic links to three polyketides and one non-ribosomal peptide. We believe that the efficient
gene targeting system we developed in P. canescens will be an indispensable tool to link
phenotype to genotype in this species. Furthermore, we expect that the library we generated will
be an important resource towards a systematic understanding of secondary metabolite
biosynthesis.

116

4.2 Introduction
Penicillium species are known producers of various natural products, some of which have
interesting bioactivities (Frisvad et al., 2004; Kozlovskii et al., 2013; Kozlovsky et al., 2011).
One of these Penicillium species, Penicillium canescens, is known to produce griseofulvin, an
orally administered anti-fungal drug, canescin, which has been shown to have antibiotic activity,
and Sch 642305, which inhibits the DNA primase in bacteria (Nicoletti et al., 2007). However,
no secondary metabolites have been linked to their biosynthetic genes in P. canescens to our
knowledge. This is due in large part to the lack of an efficient gene targeting system for genetic
manipulation in this fungal species. In this study, we established a gene targeting system in P.
canescens that would facilitate the rapid generation of a large number of gene deletions to
overcome this issue.
In fungal species, the availability of full genome sequences provides a means to find core
secondary metabolite (SM) genes such as polyketide synthase (PKS) genes and nonribosomal
synthetase (NRPS) genes (Khaldi et al., 2010; Medema et al., 2011). These core SM genes are
large and contain several conserved protein domains, making it possible for them to be identified
from genome sequences. Analysis of genome sequences of multiple species of filamentous fungi
have shown that these organisms harbor large numbers of core SM genes that far exceed the
number of SMs that have been identified(Bok et al., 2006; Galagan et al., 2005; Machida et al.,
2005; Nierman et al., 2005). However, predicting the exact product of these SM genes from its
sequence alone can be difficult, due in part to the iterative manner in which some of these
enzymes work (type I PKS) and also due to the tailoring enzymes that add modifications to the
core polyketide structure (Keller et al., 2005).
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There are two strategies to link SMs to their biosynthesis genes. The first strategy can be applied
if the target SM is structurally similar to compounds of which their biosynthesis genes have been
identified in other fungal species. BLAST searches can be performed to locate the homologue of
the known biosynthesis gene in the species of interest. The involvement of the homologous gene
in the biosynthesis of the target SM can be verified by gene knockouts. If the gene is indeed
involved, the production of the target SM will be eliminated. Genes of tailoring enzymes tend to
be found clustered around the SM core genes in filamentous fungi, which often facilitate the
search for genes involved in a single biosynthetic pathway. Additional gene deletions of
surrounding genes can be performed to find and verify all genes involved in the biosynthetic
pathway. This strategy will be further discussed and demonstrated in Chapter 5. The second
strategy is a genome-wide approach that allows for a rapid, high-throughput screening to link
SMs to core biosynthesis genes (Nielsen et al., 2011). A library of mutants carrying deletions of
core SM biosynthesis genes is generated. The deletant strains are then screened for any changes
in the SM profile. This global approach is unbiased and is especially effective for SMs whose
core structure biosynthesis have not been identified in other species. We will discuss and
demonstrate this strategy in this chapter.
The full genome sequence of P. canescens was made publicly available by the Joint Genome
Institute in 2013. Our analysis revealed that this species appears to contain as many as 29 PKS
genes and 20 NRPS genes (Table 4-1). We applied our gene targeting system to generate a
knockout library in P. canescens as a tool for linking these putative core SM genes to SMs.
Herein we report that by using this knockout library, we have successfully linked three PKs
griseofulvin (1), xantheopcin (2), and 15-deoxyoxalicine B (3) and one NRP amauromine (4) to
their core genes responsible for their biosynthesis (Figure 4-1).
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4.3 Results and discussion
Development of protoplasting, transformation, and regeneration protocol
The first task in developing an efficient gene targeting system is to establish an efficient protocol
for protoplasting and regeneration. The key to successful and efficient protoplasting is to find the
osmotic stabilizer concentration that works best for the species of interest (Maliszewska and
Zboinska, 1996). To find the best system for P. canescens, we tested 6 different osmotic
stabilizer concentrations. All steps required to isolate protoplasts were tested for each
concentration, and protoplasts were monitored microscopically after each step to ensure they
maintained good morphology. As a result, we determined that 1.2M MgSO4 was the best osmotic
stabilizer for this species. We also tested different enzyme combinations, and found that Vino
Taste Pro (Novozymes) worked well with this species and addition of Yatalase (TaKaRa) did not
result in any enhancement.
We next set out to find the best regeneration condition for this species. Again, the key was to
find the best osmotic stabilizer that would yield good growth and sporulation. We tested
regeneration with glucose minimal media (GMM) with various concentrations of KCl and
sorbitol. We chose these reagents because they are the two major osmotic stabilizers used in
fungal transformation methods and the two are fundamentally different: one is an ionic osmotic
stabilizer and the other is an organic stabilizer. Protoplasts were plated onto GMM containing
various concentrations of osmatic stabilizers and their growth and sporulation was monitored.
Protoplasts plated on GMM containing 0.6M KCl showed the most rapid growth and sporulation
was observed as early as 3 days after plating. 1.0M sorbitol showed good growth but no
sporulation was observed until 6 days after plating. From these results, we determined that 0.6M
KCl was the best osmotic stabilizer for P. canescens.
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Creation of ku70-, pyrG- double mutant to develop an efficient gene targeting system
To develop a system that would facilitate the rapid generation of a large number of individual
knock-out strains, we created a ku70-, pyrG- double mutant strain of P. canescens ATCC10419.
In living cells, DNA damage repair can occur either via non-homologous end joing (NHEJ) or
homologous recombination. Deletion of the homolog of ku70 eliminates a major component of
the NHEJ system, shifting all DNA damage repair to homologous recombination, thus improving
gene targeting efficiency (Krappmann et al., 2006; Nayak et al., 2006; Takahashi et al., 2006).
We first located the Ku70 homolog in P. canescens by performing BLAST analysis using the
sequence of NkuA, the Ku70 homolog in Aspergillus nidulans (Chiang et al., 2008). Protein ID
369793 had 84% sequence similarity to NkuA and high sequence similarity to Ku70 homologs in
other species, suggesting that this is the homolog of Ku70 in P. canescens. To create the ku70-
mutant, we replaced the ku70 gene of the wild type strain with a hygromycin resistance marker
(hph). The strategy is illustrated in Figure 4-2. To do this, we first amplified a 2kb region
upstream and downstream of the ku70 homolog gene. We then fused these fragments with the
hph construct amplified from pCB1003 to generate the ku70 KO construct. This fragment was
then transformed into P. canescens following a protocol established for A. niger with
modifications mentioned in the previous section. Transformants were selected on media
supplemented with 0.1mg/mL of hygromycin. This concentration was determined by testing
growth inhibition with media supplemented with different concentrations of hygromycin ranging
from 0.001mg/mL to 1mg/mL. Correct replacement of the gene with hph was confirmed by
diagnostic PCR, and Southern blotting was performed to confirm the lack of ectopic integration
of the resistance marker.
120

Next we created a pyrG auxotrophic mutant in the ku70- background (Figure 4-3). We located
the pyrG gene in P. canescens using the same method described above. The upstream and
downstream regions of the pyrG coding region were amplified by PCR, and the two fragments
were joined together using fusion PCR. Transformation with this construct should result in the
elimination of the pyrG coding region. Transformants were selected on media supplemented with
uracil and 1.5mg/mL 5-fluoorotic acid (FOA) (Gouka et al., 1995). The concentration of 5-FOA
was determined by growth inhibition tests with media supplemented with different
concentrations of 5-FOA ranging from 0.5mg/mL to 4mg/mL. In transformants with intact pyrG,
the non-toxic FOA is metabolized into a toxic fluouracil, rendering them non-viable on FOA
selection media. On the other hand, the pyrG- mutants survive because the FOA is not
metabolized. Deletion of pyrG was confirmed by diagnostic PCR.
pyrG complementation for use as selectable marker
In Aspergillus species such as A. nidulans, A. terreus, and A. oryzae, the pyrG gene from A.
fumigatus, AfpyrG, is used in the transformation construct as a selectable marker. We first
decided to follow these examples and tried using AfpyrG to complement the pyrG deletion in P.
canescens. We inserted Afpyrg into the original pyrG locus with a construct consisting of 1kb
flanking sequences corresponding to the upstream and downstream region of the pyrG locus and
the AfpyrG gene. The transformation yielded only one transformant, and its growth was
significantly slower than the wild type. This suggested that the AfpyrG gene was not able to
sufficiently complement the pyrG of P. canescens. We therefore switched to using the native P.
canescens pyrG gene (PcanpyrG). We repeated the test transformation with this gene, and
obtained >10 transformants that showed growth speed comparable to that of wild type. From
121

these results, we concluded that we would use PcanpyrG for all subsequent experiments as the
selectable marker.
Construction and initial characterization of a genome-wide PKS and NRPS library
To find putative PKS genes we performed bioinformatic analysis using the JGI database and
searched for genes harboring putative beta-ketoacyl synthase and acyl transferase domains. We
found a total of 29 genes, and further conserved domain structure analysis revealed within them
there are 12 nonreducing PKSs (NR-PKSs), 15 highly reducing PKSs (HR-PKSs), and 2 partially
reducing PKSs (PR-PKSs). We next looked for putative NRPS genes by searching for genes
harboring putative condensation domains and adenylation domains. From this search, we found
20 putative NRPS genes. The complete list of putative PKSs and NRPSs is shown in Table 4-1.
Using the information obtained from this analysis, knockout constructs for a total of 49 SM core
genes were assembled by fusion PCR and all targeted genes were replaced by PcanpyrG marker
in the ku70-, pyrG- background. Replacement of the targeted gene was confirmed by diagnostic
PCR. All 52 deletion mutant strains were viable and able to sporulate, indicating that none of
these genes are essential for either growth or conidiation.
For initial characterization of the library, we focused on homologs of core SM genes that have
been linked to metabolites in other fungal species and verified their links in P. canescens.
In A. nidulans, the wA gene is involved in the production of a polyketide, naphthopyrone YWA1,
which is a precursor of the green conidial pigment (Fujii et al., 2001; Watanabe et al., 1999). In
A. nidulans, deletion of the wA gene results in the formation of white conidiospores. In P.
canescens, one of the PKSs, Protein ID 366620, has 82% sequence similarity to wA. The wild
type P. canescens produces conidiospores with a green pigment. As expected, deletion of the
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gene for Protein ID 366620 resulted in the formation of white conidiospores (Figure 4-4). This
suggests Protein ID 366620 is involved in the formation of the green pigmentation in
conidiospores.
The gene cluster for the biosynthesis of griseofulvin (1) has been reported previously in P.
aethiopicum (Chooi et al., 2010). We performed a BLAST search for the homolog of the NR-
PKS gene GsfA in P. canescens and found that Protein ID 243077 has 90% sequence similarity.
Wild type P. canescens produced griseofulvin in small amounts when cultured on yeast extract
sucrose (YES) solid medium. We grew the strain carrying the gene deletion for Protein ID
243077 on YES solid medium and found that the production of griseofulvin was eliminated
(Figure 4-5). This confirms that the homolog of GsfA, Protein ID 243077, is the PKS involved in
griseofulvin biosynthesis in P. canescens.
Selecting media that support secondary metabolite production in P. canescens
Filamentous fungi are known to produce different SMs in different media and culture conditions
(Bode et al., 2002; Davis et al., 1966; Oxford et al., 1935; Pitt et al., 1983; Scherlach and
Hertweck, 2006). We grew wild type P.canescens on ten different types of solid media, CZA,
CYA, YAG, YES, MEA, MB,TYG, GMM, LMM, and LCMM and compared their SM profiles
using LC-MS. We needed to select one type of medium for our first screening of the knockout
library. We based our selection on the following criteria: 1) diversity of SMs produced and 2)
good separation of each metabolite on HPLC that would allow us to efficiently isolate and
characterize target metabolites. We compared SM profiles of all ten media, and based on our
criteria, chose CZA as the first medium to screen the knockout library.

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Protein ID 308305 is involved in the biosynthesis of xanthoepocin
P. canescens produces a compound on CZA that elutes at around 27.5 min. Neither the mass nor
UV spectrum of this compound matched any of the known metabolites of P. canescens. We
therefore cultivated the strain in large scale and the target compound was isolated by flash
chromatography and HPLC and characterized by NMR. The compound was determined to be
xanthoepocin (2), an antibiotic isolated previously from the culture broth of P. simplissimum
IFO5762 (Igarashi et al., 2000). Biological assays were performed and showed that xanthepocin
inhibited the growth of MRSA and Bacillus subtilis with the MICs of 0.78 – 1.56µg/mL with
weak cytotoxicity (35 µg/mL). Through the screening of our PKS knockout library on CZA, we
found that 2 completely disappeared in the mutant strain carrying the Protein ID 308305 gene
deletion (Figure 4-6). This suggests we have found the core PKS responsible for the biosynthesis
of 2.
Protein ID 371741 is involved in the biosynthesis of amauromine
Another compound we found that P. canescens produced on CZA was amauromine (4), also
known as nigrifortine (Figure 4-7). To our knowledge, this compound being has never been
reported as being produced by P. canescens, and its biosynthesis gene cluster and biosynthesis
pathway have yet to be elucidated. This compound was first found and isolated from
Amauroascus sp. No. 6237 and later found also in P. nigricans (Laws and Mantle, 1985; Takase
et al., 1984a; Takase et al., 1984b). Amauromine is a diketopiperazine antibiotic, and it shows
vasodilator activity by blocking the calcium ion channel. Its structural uniqueness and bioactivity
attracted the attention of synthetic chemists and its total synthesis has been explored by multiple
groups (Depew et al., 1999; Takase et al., 1985, 1986a; Takase et al., 1984c, 1986b).
124

We isolated this compound as described above from CZA solid media large-scale cultures of P.
canescens for structural characterization. NMR data was in good agreement with published data
(Takase et al., 1984a) and confirmed the compound as amauromine. The chemical structure of
amauromine shows that it is a diketopiperazine derived from two tryptophan molecules and
carries two reverse prenyl moieties (3’-(3’, 3’)- dimethylallyls (3’-DMAs)). Metabolites with
similar structural characteristics have been isolated and characterized from other fungal species.
These include acetylaszonalenin and its isomers from multiple Aspergillus species (Bhat et al.,
1990, 1993; Kimura et al., 1982; Rank et al., 2006; Yin et al., 2009), roquefortine C from P.
roquefortii (Barrow et al., 1979), and fructigenines A and B from P. fructigenum (Arai et al.,
1989). The biosynthetic gene cluster for acetylaszonalenin was reported in Neosartorya fischeri,
which revealed the putative involvement of an NRPS (AnaPS), an acetyltransferase (AnaAT),
and a prenyltransferase (AnaPT) in the biosynthesis pathway (Yin et al., 2009). We have
previously heterologously expressed this gene cluster in A. nidulans and confirmed that AnaPS
uses tryptophan and anthranilic acid as starter units and forms the cyclic dipeptide (R)-
benzodiazepinedione. The prenyltransferase AnaPT catalyzes the transfer of a dimethylallyl
moiety to the C3 position of the indole ring of the cyclic dipeptide, and also the formation of a
ring system between C2 of the indoline and and N atom of the diketopiperazine ring to yield
aszonalenin. Subsequent acetylation by AnaAT completes the formation of acetylaszonalenin.
In the screening of our NRPS knockout library in P. canescens, the production of 4 was
eliminated in the mutant strain carrying the deletion of ProteinID 371741 (Figure 4-7).
Conserved domain analysis of this NRPS revealed that it consists of one adenylation (A) domain,
two peptide carrier protein (PCP) domains, and two condensation (C) domains. Adjacent to the
NRPS gene, we found Protein ID 371742, a prenyltransferase with 48% sequence similarity to
125

AnaPT. From the genomic sequence data and chemical structure of amauromine, we propose that
for its biosynthesis, two tryptophan molecules are used as substrates for this NRPS to form a
cyclic dipeptide. It is then prenylated at the C3 position of each tryptophan molecule and ring
formation also occurs, catalyzed by the prenyltransferase. We propose that the two enzymes are
sufficient to complete the formation of amauromine.
Protein ID 400488 is involved in the biosynthesis of 15-deoxyoxalicine B
Another major SM of P. canescens was isolated from CZA solid media cultured in large scale
and identified as 15-deoxyoxalicine B (3). In our gene deletion library, the deletion of Protein ID
400488 resulted in the elimination of the production of this compound. This compound belongs
to a rare skeletal class of meroterpenoids whose molecular and genetic basis of biosynthesis has
not previously been elucidated (Zhang et al., 2003). This warranted a detailed investigation into
its biosynthesis gene cluster and pathway. We performed gene knockouts of genes surrounding
the Protein ID 400488 gene to identify the boundaries of the biosynthesis gene cluster. We also
isolated and identified intermediates from the gene deletion mutants which allowed us to propose
the biosynthesis pathway for 15-deoxyoxalicine B. This is discussed in detail in Chapter 5.

126

4.4 Materials and methods
Strains
The Penicillium canescens strain ATCC10419 was used as the parent strain for ku70 deletion.
The ku70- mutant was used as the control strain for SM analysis and also as the parent strain for
pyrG deletion. All SM core gene deletion strains were constructed in the P. canescens
ATCC10419 ku70-, pyrG- background.
Molecular manipulations
Primers used in this study are listed in Table 4-2. All targeted genes were individually replaced
by either the A. fumigatus pyrG gene (AfpyrG) or P. canescens pyrG gene (PcanpyrG).
Construction of double joint fusion PCR products, protoplast generation, and transformation
were carried out according to previous procedures (Guo et al., 2012) with modifications to the
osmotic stabilizer: 1.2M MgSO4 was used for protoplasting, and 0.6M KCl was used for
protoplast regeneration. Diagnostic PCR of the mutant strains was carried out using the external
primers fromt eh first round of PCR.
Media
CZA (Czapek’s) media was prepared by adding the following in 1L H2O: 3g NaNO3, 0.5g KCl,
0.5g MgSO4·7H2O, 0.01g FeSO4·7H2O, 1g K2HPO4, 30g sucrose, and 15g agar. YES media was
prepared by combining 20g of yeast extract, 10g of sucrose, 1mL of trace element solution, and
15g of agar in 1L H2O. For LC/MS screening, 1 x 10
7
spores of each gene deletant were
cultivated on 100 x 15mm petri dishes containing solid media for 5 days at 25˚C for 5 days. The
agar was chopped into ~2cm2 pieces, and the material was extracted using sonication with
methanol, followed by 1:1 methanol: dichloromethane. The organic solvents were removed in
127

vacuo, and the remaining material was partitioned between H2O and ethyl acetate. The ethyl
acetate layer was collected and evaporated, and the crude material was redissolved in 1mL of
20% DMSO in methanol.
LC/MS analysis
LC/MS was carried out using a ThermoFinnigan LCQ advantage ion trap mass spectrometer
with an RP C18 column (Alltech Prevail; 2.1 x 100mm with a 3µm particle size) at a slow rate of
125µL/ min and monitored by a UV detector at 254nm. The solvent gradient was 95% MeCN-
H2O (solvent B) in 5% MeCN- H2O (solvent A) both containing 0.05% formic acid: 0% B from
0 to 5min, 0 to 100% B from 5 to 35min, 100% B from 35 to 40min, 100% B to 0% B from 40 to
45min, and reequiliberation with 0%B from 45 to 50min.
Isolation and structure elucidation of secondary metabolites
For scale up, P. canescens ATCC10419 was cultivated at 25˚C on 100 x 15mm petri dishes
containing a total of 1L solid media for 5 days. Agar was chopped into small pieces and
extracted as described previously. The extract was subjected to silica gel column
chromatography, using methanol and dichloromethane as eluent. The materials were further
separated by preparative HPLC (Phenomenex Luna 5µm C18, 250 x 21.2mm) with a flow rate of
5.0mL/min and measured by a UV detector at 254nm.

128

Table 4-1. List of all putative PKS and NRPS genes in P. canescens

129

Table 4-2. List of primers (5’  3’)
ku70 deletion construct
ku70_P1 GTTGTGATCCCGAGGCTTG
ku70_P2 AGGCTTGGCAAGGTCAGAT
ku70_P3 TGACCTCCACTAGCTCCAGTGAAGCAGTGGGAGAGTGAA
ku70_p4 AATAGAGTAGATGCCGACCGTCCAGCACTTTGGCCATT
ku70_p5 AACTTTCACCCCGGCTTC
ku70_p6 CAAAGCGGCCCCTAACTT

hph gene from pCB1003 (outside EcoRI site)
hph Fw GTTGTAAAACGACGGCCAGT
hph Rev GCAGGTCGACTCTAGAGGATC

pyrG deletion construct
PyrG_P1 AAGACGGCCGAATTGACA
PyrG_P2 TTGACACCCGACGGAGTT
PyrG_P3 GCATACGGATCACCTACATGCGGGGTGAAGAAGTGGTG
PyrG_P4 CATGTAGGTGATCCGTATGC
PyrG_P5 GCGTCGTGGGAGTGTTTC
PyrG_P6 CGGTTCGTCGATTCATCC

P.can_PyrG_Fw CAATGCTCTTCACCCTCTTCG
P.can_PyrG_Rev CTGTCTGAGAGGAGGCACTG

PKS deletion constructs
243077-P1 ACTTTCGCCGGAGGAGAC
243077-P2 AGGGCTCTGTCGTGATGG
243077-P3 CGAAGAGGGTGAAGAGCATTGTCTTTGATCGCCCTCTGG
243077-P4 CAGTGCCTCCTCTCAGACAGATTTTGGCGGGCTTTTTG
243077-P5 GGGCATTCCAGCAAGATG
243077-P6 CAAAGGAGACGGCGAGTC

317201-P1 TCCCCTGGCTTCTCTGTG
317201-P2 CCTCCTGGCAAACGCTTA
317201-P3 CGAAGAGGGTGAAGAGCATTGCGAGCCTGGTGTCGTTCT
317201-P4 CAGTGCCTCCTCTCAGACAGCAAACCACTGCCCCACTC
317201-P5 CACACGCGAAGGACATTG
317201-P6 ACGCGACTTCCAGTCCAC

371551-P1 TTCGGATGATCGGCCATA
371551-P2 CGGCTGTGAGCTTGTCG
130

371551-P3 CGAAGAGGGTGAAGAGCATTGAGGCGCGTATTGATTCCA
371551-P4 CAGTGCCTCCTCTCAGACAGcccttagccttccgcact
371551-P5 aggcaaccaacccacctt
371551-P6 CGCGAGGGGTCAGAGTAA

400488-P1 AACGACCCGCATACTGGA
400488-P2 CTCAGGCCACGAATACGC
400488-P3 CGAAGAGGGTGAAGAGCATTGACGGAACTGGTGGGGAAC
400488-P4 CAGTGCCTCCTCTCAGACAGTCAAGCCCACTTCCAAGG
400488-P5 CCCAGAGTTGTCCGATGC
400488-P6 GGTTGTCCCATCGTCCAG

352123-P1 ACAATTTCGGGCATCTGG
352123-P2 GGCATCTGGGGAGGAAAC
352123-P3 CGAAGAGGGTGAAGAGCATTGTCGAGTGTGGTGGACGAG
352123-P4 CAGTGCCTCCTCTCAGACAGCCAGATCGTGCCCTCAGT
352123-P5 TGGCCAACACAGGCATC
352123-P6 GGCCCAGTCTCCGTCATA

308305-P1 AACGCGGCTGGTACTGAC
308305-P2 CGGCCCAGTCGTTCATAC
308305-P3 CGAAGAGGGTGAAGAGCATTGCGTTCCATTCCGTTTTCC
308305-P4 CAGTGCCTCCTCTCAGACAGTTAATCGCTTGGCGTTGG
308305-P5 GCCCATCTTTTGCATTCG
308305-P6 AACACGCCCATCTTTTGC

352316-P1 GCCGTCGTATTTGCCAAG
352316-P2 TCCGGTCTAGCGAAATGG
352316-P3 CGAAGAGGGTGAAGAGCATTGTGTGGAGCCCCAAATCTC
352316-P4 CAGTGCCTCCTCTCAGACAGTGCCCCCAATAGTTCGTG
352316-P5 GGCCACACCGTTCTTCTC
352316-P6 TGGTCCCTACGGAATTGG

378503-P1 TCAGAGGCACCGACAATG
378503-P2 CAGTGGCCGGGTGAATAG
378503-P3 CGAAGAGGGTGAAGAGCATTGGCGTGCTGATCCTTGGTC
378503-P4 CAGTGCCTCCTCTCAGACAGCCCACTTTCAGCCGTTGT
378503-P5 TGGGATCTTCAGCGAGGT
378503-P6 GGGTTTGGCTGACATGGA

366620-P1 TTCCGCAAGCCACAATCT
131

366620-P2 GCGTTCGGGAAAATCGTA
366620-P3 CGAAGAGGGTGAAGAGCATTGTGGCTCGGGTAATGCTGT
366620-P4 CAGTGCCTCCTCTCAGACAGTGCAGGTCCACACCACAC
366620-P5 TCAACGAAGCCTCCAAGC
366620-P6 CCCACAGACCCAGAAACG

368325-P1 ACAAAGGTGACCGCCAAG
368325-P2 CGAGCTGCCTCTGGAGAA
368325-P3 CGAAGAGGGTGAAGAGCATTGTTCGGCCCACATTAGCTC
368325-P4 CAGTGCCTCCTCTCAGACAGCGGTGGGCCTACAAAAGA
368325-P5 TGTCGGCAATTTGGGATT
368325-P6 CGCATACCGCTTTCCATC

395714-P1 ATATCCAAGGGGCGCATC
395714-P2 TTCTGCGTCCCGGTATCT
395714-P3 CGAAGAGGGTGAAGAGCATTGGGAAAGGTGAGCCCGAAT
395714-P4 GTGCCTCCTCTCAGACAGTCGCCTCTCACTCGCTTC
395714-P5 TCGATGCTTTTGCTTGTCC
395714-P6 GGTCGGCATCTTTCACGA

248590-P1 TATGTGACGCGTGGGATG
248590-P2 AGCCGACTATGCCCTTCC
248590-P3 CGAAGAGGGTGAAGAGCATTGCCGAAGCGTCTCCTTTGA
248590-P4 CAGTGCCTCCTCTCAGACAGTGTTTGCGACCTTTGTTGG
248590-P5 GGAGCTGCTGGTTTGGTG
248590-P6 AGACGGCGGACATGATTC

365444-P1 TGATTAGCGCCGAGAAGC
365444-P2 CTTTCGGGTGGTCGAGTG
365444-P3 CGAAGAGGGTGAAGAGCATTGCATCGGAGATTGGCCTTG
365444-P4 CAGTGCCTCCTCTCAGACAGCGACTTGGTTCGGATTGG
365444-P5 GCGAAGGACTGGTTGTGG
365444-P6 CCTCCGTCGCTTTTCTTG

328953-P1 AGTCTGTACTGCGGGTCCA
328953-P2 CTGCGGGTCCATTGACTT
328953-P3 CGAAGAGGGTGAAGAGCATTGTACGGCGTCCAAGGAGAC
328953-P4 CAGTGCCTCCTCTCAGACAGCGCCACCTCGTATTGGTG
328953-P5 AGAGCGGCATCCACTTTG
328953-P6 TGAAGGAGCGGTGTGTTG

132

406411-P1 GACCACCCCCTGAAGCTC
406411-P2 TTGCCTGGACCAACTTCC
406411-P3 CGAAGAGGGTGAAGAGCATTGGTAGGCTGGACGCCATTG
406411-P4 CAGTGCCTCCTCTCAGACAGTTCGTCGCCAACCCTAAG
406411-P5 GGTGCGTTGCTGTCGAG
406411-P6 TGTTGAGAGGCCACTTCG

427089-P1 ATGCGAAAGCTCGACAGC
427089-P2 CGACCTTTGATGCGTTCC
427089-P3 CGAAGAGGGTGAAGAGCATTGGGTCATACGCACGGCTTT
427089-P4 GTGCCTCCTCTCAGACAGCTTGTCCTCCCCCTCACA
427089-P5 GCGCAGAATCGCAGTAGC
427089-P6 GCGGCAATGCCACATTA

399280-P1 CTTGCAAGGTGCTTGCTG
399280-P2 GGTTACCAAGCCGTTGTCA
399280-P3 CGAAGAGGGTGAAGAGCATTGACGGTCCCCGATAGAGGT
399280-P4 GTGCCTCCTCTCAGACAGCATGGCTCAAGGGATACAGG
399280-P5 CCAGGGCTCACTAGACTTCC
399280-P6 TGTTTGGGTTGCTAACTTCG

435808-P1 GACCGAGGAATGGTGTGG
435808-P2 TCCGGGTCACACACATTG
435808-P3 CGAAGAGGGTGAAGAGCATTGTGGCGAGAACGAGAAAGG
435808-P4 GTGCCTCCTCTCAGACAGTGAAACACACCAGCTCCATC
435808-P5 AATCAACCAGCACACCTTCC
435808-P6 GCAGTGGCCATATCAGTGAC

320000-P1 CGCTGTTCGGTCTCATGG
320000-P2 CATGGCGCATTTGATCTG
320000-P3 CGAAGAGGGTGAAGAGCATTGAAGGCCAACAGGAGTTTGG
320000-P4 GTGCCTCCTCTCAGACAGTGCTCACGCAGCTCATTC
320000-P5 TGGGAGTCTCTGGGAACG
320000-P6 AGCTGCTGGGGTCAGATG

358616-P1 AGCGGGCCTATGAAGAGC
358616-P2 AATCGGCCATGCACTACG
358616-P3 CGAAGAGGGTGAAGAGCATTGGGTTGGTGCGTCGATCTC
358616-P4 GTGCCTCCTCTCAGACAGCTTGCTGCGCTTTGTATCC
358616-P5 GCAGGCCATCTTTGTTCC
358616-P6 GCGATTTCACGTTCTTTCG
133

385201-P1 AGCGCAAAGTGCAATCG
385201-P2 TCCGGCAAATGACTGTCC
385201-P3 CGAAGAGGGTGAAGAGCATTGGTATCTCGGGTCCGGTAGC
385201-P4 GTGCCTCCTCTCAGACAGTGAGCAACGGTAGTGACCTC
385201-P5 GAAGGCAGGGAGACAAGTGA
385201-P6 TGTGGAGTGAGATGGTCTGG

401759-P1 TCTGTGCGACGATTCACC
401759-P2 CATAGTCGGCGGGTTTCA
401759-P3 CGAAGAGGGTGAAGAGCATTGCGTTTCCGTTCCACAACC
401759-P4 GTGCCTCCTCTCAGACAGTTTGCCCCCTACATGTCC
401759-P5 CGTCATGGCGCTTTCTG
401759-P6 GTTTCAGAGCCCGTGACC

365434-P1 TGAATCTCCTGGCCCTTG
365434-P2 CTGGCTCGGCTGGTTAAG
365434-P3 CGAAGAGGGTGAAGAGCATTGGAAGCAGCGTCGGAAGAG
365434-P4 GTGCCTCCTCTCAGACAGCGCCTTGACCATTTGTCG
365434-P5 TTGGCCTGGTGATTTCG
365434-P6 GCCGAGGGTGTTAGAGACG

364328-P1 GCTTGCCATCTCCATTCG
364328-P2 TGCGATGATGACGAGTGG
364328-P3 CGAAGAGGGTGAAGAGCATTGTATGAGCGGGGTGAAACC
364328-P4 GTGCCTCCTCTCAGACAGCCGCGCTAGTCTTGCTTC
364328-P5 GCTGTGTTGCGCTCGATA
364328-P6 CCTCGTTGAAAGGCTCCA

443472-P1 TGTGCAGTTGACGTGTGC
443472-P2 TCACGACAGTGGCTTTGC
443472-P3 CGAAGAGGGTGAAGAGCATTGGGCTGGAAGAAAGGATGG
443472-P4 GTGCCTCCTCTCAGACAGTCAGGGATGGAAGACAGAGG
443472-P5 TCAGCTGAGGAGCAGAAAGC
443472-P6 CCTTCATTCCGCTTTATAGGC

434561-P1 GTACCCAGCCAGCTTTCG
434561-P2 TGATGAAGGCGTGACAGG
434561-P3 CGAAGAGGGTGAAGAGCATTGGGCAAGGAGGTGGTATCG
434561-P4 GTGCCTCCTCTCAGACAGATTTGGTTGCGGACATGG
434561-P5 AATCCTGCTCGCTGTTGC
134

434561-P6 CCACCGATCTTTCCAAGC

362850-P1 CTTGAGCTGCCAGAGTATCG
362850-P2 TCAGCATCAAGGCTGTCAAC
362850-P3 CGAAGAGGGTGAAGAGCATTGACCGCTACAGCATCAGTTCC
362850-P4 CAGTGCCTCCTCTCAGACAGATTTGGATTGCTCCATCAGG
362850-P5 GGTCGAGACTTCAAGGCAAC
362850-P6 CCGGACTGCTTGAAAGATTG

394144-P1 TGTGGCGTTGGATTGAAA
394144-P2 TGAAACGTCGCTCAGTGG
394144-P3 CGAAGAGGGTGAAGAGCATTGGTTGGAATCCCGTTGTCG
394144-P4 GTGCCTCCTCTCAGACAGTTGCTGCGTCTTCTTTGC
394144-P5 TGCTAAAGGCCGTCAACC
394144-P6 GGCTCAATGTTGGTGTTGG

391502-P1 TGGCGACACCATTGAGAA
391502-P2 TCTCATCCCAGGCTGTCC
391502-P3 CGAAGAGGGTGAAGAGCATTGTTTTGGCTGACGGAGAGG
391502-P4 GTGCCTCCTCTCAGACAGTCCAATGCGAACAACGTG
391502-P5 TGCTTGTCGTCCGTTGAA
391502-P6 CCGTTCCATTGGTTGGAG

NRPS deletion constructs
316552-P1 CCTTTGCTCCCAATGCTG
316552-P2 CAATGCTGGCGGAAAAAG
316552-P3 GAAGAGGGTGAAGAGCATTGGGCGTTCGACAATTGAGC
316552-P4 CAGTGCCTCCTCTCAGACAGCCGCCTTCCCTTATAGCC
316552-P5 TACTGCCACACGCTGGAA
316552-P6 TCACTGAGCTGCCGTCTG

315960-P1 GGCCCTCATGCTCAATGT
315960-P2 CCCTTTCCTTCCCTCGTC
315960-P3 CGAAGAGGGTGAAGAGCATTGCCTTGCGTGAATGTGCAA
315960-P4 CAGTGCCTCCTCTCAGACAGTGCGCATGTTCAGGTAGC
315960-P5 TACCATGCGGCAGTGATG
315960-P6 CGAGTCGGGAATTGTTGG

369610-P1 CCGGGACCATGTAAGCTG
369610-P2 GCTGGGGCATTCATCTTC
369610-P3 CGAAGAGGGTGAAGAGCATTGGAGTCGGCAGGTCGAGTG
135

369610-P4 CAGTGCCTCCTCTCAGACAGCTCGCCTGTAGCCAGACC
369610-P5 GGGTAGCGAGGTGATTGG
369610-P6 TTCGGCATTGCCTAGGAG

368421-P1 ACGTGGTTGGTGGAGACG
368421-P2 AGACGGGGATGGGTTTTC
368421-P3 CGAAGAGGGTGAAGAGCATTGCTAAGAACCGCCGAGGTG
368421-P4 CAGTGCCTCCTCTCAGACAGTGCCCGTTTCTGGTATCC
368421-P5 AGCGGGTCGTTAACATGC
368421-P6 GTCGGTGCCAAACAGGAG

344392-P1 ATCGCGCAGACAAAGGTC
344392-P2 TCCGTCGAGCTCAAGTCC
344392-P3 CGAAGAGGGTGAAGAGCATTGGGTGATGTCGCAGGTTCC
344392-P4 CAGTGCCTCCTCTCAGACAGATGAACCCGACCCAAAGC
344392-P5 TGCCCTCTTCCCTTGTTG
344392-P6 GCACTTATGACGGCAGCA

367730-P1 TGGCTTCGGCAAGAGAAC
367730-P2 AGCCACCCATCACGAGAC
367730-P3 CGAAGAGGGTGAAGAGCATTGGCAGCTGTGGCTGAAGGT
367730-P4 CAGTGCCTCCTCTCAGACAGCGGCGGCGTCTATAACTG
367730-P5 CGAACACCACCAGCACAG
367730-P6 CCTCGGTGCACTTGGTTC

371741-P1 CGACATGGGCGAAGAGAC
371741-P2 TGGCAAAGGTCACCGACT
371741-P3 CGAAGAGGGTGAAGAGCATTGTGCCGCAGTTGAATGAGA
371741-P4 CAGTGCCTCCTCTCAGACAGTTTCCCAGTCGATTGAACG
371741-P5 AGTCGGATGGGGGAGAAG
371741-P6 CACGGAATTGCCCAGAAC

372572-1 GGCGGCAAAGAGTCAATG
372572-2 TCCCGAGAGATGGATTGC
372572-3 CGAAGAGGGTGAAGAGCATTGAAACCCGGGAGTTGTATCG
372572-4 GTGCCTCCTCTCAGACAGATGGGACCTCCACTGCTG
372572-5 AGTCCGGCACGAAGTGTC
372572-6 ATCCACAAATGCGTGCTG

372228-1 GCCCCTCGGTGAAAACTC
372228-2 TTCACGTTGCACCTCAGC
136

372228-3 CGAAGAGGGTGAAGAGCATTGCACGGTTCTGTCGCATTG
372228-4 CAGTGCCTCCTCTCAGACAGCGCAGCCTTCTCAAGACC
372228-5 CCCTTTTGCTTGGTGCTG
372228-6 CTCATCGCCGCCATTATC

407019-P1 GACGGTTGCTCGAGGGTA
407019-P2 TGATGTGCTGGCCATGTT
407019-P3 CGAAGAGGGTGAAGAGCATTGGGGAAGCAGACGCAAAGA
407019-P4 CAGTGCCTCCTCTCAGACAGGGGCCTCCTTTTGTCGAT
407019-P5 TGGGTAAGTTCGGGCATAAG
407019-P6 GCGATCTTAGCTAGGGCTTTC

435720-P1 CGTCCAATCGGCTTATCG
435720-P2 TGCGGCAGATGCGTATAA
435720-P3 CGAAGAGGGTGAAGAGCATTGGAGCATGGTTGGGAATCG
435720-P4 CAGTGCCTCCTCTCAGACAGCCCGTCAAGGTTTCAAGG
435720-P5 ATTTGGACTTTCGGCAAGG
435720-P6 GACTTCGGTGGTCACATCG

439034-P1 TGGCTTTCCTCCGTGGTA
439034-P2 CGGCGTCCTTAGGGTTCT
439034-P3 CGAAGAGGGTGAAGAGCATTGTATCCCACGGCTCGGTTA
439034-P4 CAGTGCCTCCTCTCAGACAGaagccctgaagcgattatacc
439034-P5 CTCCAGTATGCTTGGGTTGG
439034-P6 CTACGCTATCTGGGCTCTCG

367729-P1 GCATTCCGGTCTTGCTATGT
367729-P2 AGTCTCGCCTCCTAGCACAA
367729-P3 CGAAGAGGGTGAAGAGCATTGGTGATCGTCGCTGCATTCTA
367729-P4 CAGTGCCTCCTCTCAGACAGATCCACCCTAACACGAGTGC
367729-P5 CTTAGGGCCCAAGATAATCG
367729-P6 TAAGCAGGCCTAACCCTTCC

336181-P1 GGTGCACGGATATTTGTCTG
336181-P2 GAGTTCTCTCGCCACCAAAC
336181-P3 CGAAGAGGGTGAAGAGCATTGAAGGAACGCATGAAATCTGG
336181-P4 CAGTGCCTCCTCTCAGACAGCTTCCTTCCACCCCTCTACC
336181-P5 GCCTTAATGTTGTCGTGCAG
336181-P6 AGGAAGGCAATCTGAGCATC

368433-P1 AGATGAAGGGCGTGATGC
137

368433-P2 CCTTGAGGACCCTTGCAG
368433-P3 CGAAGAGGGTGAAGAGCATTGTTTCCTGCGGTCGAAGAG
368433-P4 CAGTGCCTCCTCTCAGACAGTTTCCGTTTGGTCTTGATCC
368433-P5 CAGCCTTTCCAATCGTCTTC
368433-P6 TCTCAACCCCAATCTGCTTC

440327-P1 ctggctaggaggtcgaacag
440327-P2 gctcgaaaaaggtctgatgc
440327-P3 CGAAGAGGGTGAAGAGCATTGTGAAGACTTTAGCGGGCAGT
440327-P4 CAGTGCCTCCTCTCAGACAGAAGTTGGAAACCGTCAATGG
440327-P5 AGGGTCGTGGTACATGGAAT
440327-P6 TGGGCCACTTCTCACAATTA

369110-P1 TAGGCCGCAACAGAGACC
369110-P2 AGAGCAGGGATGCGTGAC
369110-P3 CGAAGAGGGTGAAGAGCATTGGGGACTGCCTGTCACCTG
369110-P4 CAGTGCCTCCTCTCAGACAGTTCCCACGGCGATTGTAT
369110-P5 CGCGATAGTTGGGTCCAC
369110-P6 GATCAGGGCGATGTTTGG

372566-P1 CACGGGATCCTTCCGATT
372566-P2 CACATGCATACCCGAGCA
372566-P3 CGAAGAGGGTGAAGAGCATTGATTGCCTGCGAAATCAGC
372566-P4 CAGTGCCTCCTCTCAGACAGAATAGAACGCCGGTCAACAG
372566-P5 GTCGTTGTCGATGGAAGGAT
372566-P6 ACTCATTGTTCCGAGCCAAA

369609-P1 CTGGCTCGTCAATCAAACAA
369609-P2 GCAGCGTCATACCAGTCAAC
369609-P3 CGAAGAGGGTGAAGAGCATTGATTCCCAAGAACCACTGTGC
369609-P4 CAGTGCCTCCTCTCAGACAGGTTTTCGCAACCCATTGACT
369609-P5 AAGTGCGACGACCTTGTACC
369609-P6 ATTCTGATTCCCTGGTGGTG

370383-1 ACTGCGAATCTCTTCGGCTA
370383-2 TCCGCTGAGTATCATTGACG
370383-3 CGAAGAGGGTGAAGAGCATTGCGCATCTGCATTGTATCCAC
370383-4 CAGTGCCTCCTCTCAGACAGTAGTGCATTGCTTGGTCAGC
370383-5 AGAGGATCCGCTCGTAAACA
370383-6 TCCAACTCCGTTTCAACTCC
138

Figure 4-1. Structures of compounds identified in this study. The compounds are as follows:
griseofulvin, 1; xanthoepocin, 2; 15-deoxyoxalicine B, 3; amauromine, 4.
139

Figure 4-2. Strategy for ku70 deletion in P. canescens using hygromycin resistance marker (hph)

140

Figure 4-3. Strategy for pyrG deletion

141

Figure 4-4. Morphological difference between control (top wells) and mutants carrying Protein
ID 366620 deletion (bottom wells).

142

Figure 4-5. HPLC profile of extracts from control strain and Protein ID 243077 deletant as
detected by (A) UV at 254 nm and (B) mass spectrometry in positive ion mode [M+H]
+
m/z =
353. EIC = extracted ion chromatogram.

143

Figure 4-6. HPLC profile of extracts from control strain and Protein ID 308305 deletant as
detected by (A) UV at 254 nm and (B) mass spectrometry in positive ion mode [M-H]
-
m/z =
605. EIC = extracted ion chromatogram.

144

Figure 4-7. HPLC profile of extracts from control strain and Protein ID 371741 deletant as
detected by (A) UV at 254 nm and (B) mass spectrometry in positive ion mode [M-H]
-
m/z =
510. EIC = extracted ion chromatogram.

145

Figure 4-8. HPLC profile of extracts from control strain and Protein ID 400488 deletant as
detected by (A) UV at 254 nm and (B) mass spectrometry in positive ion mode [M-H]
-
m/z =
504. EIC = extracted ion chromatogram.

B. EIC 504
A. UV 254 nm
146

Figure 4-9. UV-Vis and ESIMS spectra of compounds 1 to 4.

147

Figure 4-10.
1
H NMR spectrum of griseofulvin (1)

148

Figure 4-11.
1
H NMR spectrum of xanthoepocin (2)

149

Figure 4-12.
1
H NMR spectrum of 15-deoxyoxalicine B (3)

150

Figure 4-13.
1
H NMR spectrum of amauromine (4)

151

CHAPTER V: Molecular genetic analysis of a diterpenic meroterpenoid 15-deoxyoxalicine
B biosynthesis gene cluster in Penicillium canescens
5.1 Abstract
Meroterpenoids are a class of fungal metabolites that are produced from polyketide and
terpenoid precursors. 15-deoxyoxalicine B belongs to one structural group consisting of a unique
pyridinyl- α -pyrone polyketide subunit and a diterpenoid subunit connected through a
characteristic asymmetric spiro carbon atom. An understanding of the genes involved in the
biosynthesis of this class of compounds should provide a means to facilitate engineering of
second-generation molecules and increasing production of the first-generation compounds. We
found that the filamentous fungus Penicillium canescens produces 15-deoxyoxalicine B. Using
targeted gene deletions, we have identified a cluster of 12 contiguous genes that are involved in
the biosynthesis of 15-deoxyoxalicine B. This gene cluster included one non-reducing polyketide
synthase gene which we have designated olcA. Chemical analysis of wild-type and gene deletion
mutant extracts enabled us to isolate and characterize 7 additional natural products that are either
intermediates or shunt products of the biosynthesis pathway. Two of the compounds identified
have not been reported previously. Our data have allowed us to propose a biosynthesis pathway
for 15-deoxyoxalicine B.

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5.2 Introduction
Natural products have served as important sources of bioactive molecules for decades. Most
natural products are secondary metabolites (SMs) of microorganisms and plants, and their wide
range of bioactivities are derived from their tremendous structural diversity. Intriguingly, the
core structures of these natural products are assembled from simple precursors, and structural
diversity is the result of reactions catalyzed by different enzymes that are responsible for
numerous modifications in the course of the biosynthesis process.
Terpenoids and polyketides are two classes of SMs that are particularly rich in structural
diversity, each built upon core structures assembled by terpene cyclases and polyketide synthases
(PKS), respectively (Christianson, 2006; Fischbach and Walsh, 2006). Furthermore, these two
classes of enzymes can work in concert to produce a hybrid molecule between terpenoids and
polyketides called meroterpenoids. Meroterpenoids are found widely in plants, fungi, and
bacteria and show a broad range of bioactivities (Geris and Simpson, 2009). Examples of known
meroterpenoids with therapeutic uses include the acetylcholinesterase inhibitor territrem for the
potential treatment of Alzheimer’s disease (Chen et al., 1999; Kuno et al., 1996), the acyl-CoA
cholesterol acetyltransferase inhibitor pyripyropene for the treatment and prevention of
atherosclerosis (Omura et al., 1993; Sunazuka et al., 2008), and mycophenolic acid as an
immunosuppressive agent (Colombo et al., 1979; Colombo et al., 1982; Muth and Nash, 1975).
Although many meroterpenoids have been isolated and characterized, the genes involved in their
biosynthesis have been revealed in only a few instances in fungi (Guo et al., 2012; Holm et al.,
2014; Itoh et al., 2010; Lin et al., 2013; Lo et al., 2012; Matsuda et al., 2013).
Oxalicines A and B were first isolated from Penicillium oxalicum by Ubillas et al. in 1989
(Ubillas et al., 1989) and were the first of a rare skeletal class of meroterpenoids (Figure 5-1).
153

Since then, other related compounds such as 15-deoxyoxalicines A, B and decaturins A-F have
been isolated from P. thiersii and/ or P. decaturense, (Li et al., 2005; Zhang et al., 2003) and
many of them were shown to have antiinsectican activity against the fall armyworm (Spodoptera
frugiperda). To our knowledge, no molecular and genetic basis for the biosynthesis of oxalicines
or decaturins has been reported previously. These compounds form a structurally unique class of
natural products because their basic structure is composed of two subunits: a pyridinyl- α -
pyrone polyketide subunit (Figure 5-1, blue), and a diterpenoid subunit. This pyridinyl- α -
pyrone polyketide subunit itself is rare among natural products, having only been found in
anibine, a plant metabolite (Mors et al., 1957), and the pyripyropenes , a group of potent acyl-
CoA cholesterol acyltransferase inhibitors isolated from Aspergillus fumigatus. The biosynthesis
pathway of pyripyropene A has been determined previously (Itoh et al., 2010), and its early stage
of biosynthesis leading to the formation of the pyridinyl- α -pyrone polyketide subunit is most
likely highly similar to that of oxalicines and decaturins. However, pyripyropenes incorporate
sesquiterpenoid components in later steps rather than diterpenoid components. Furthermore, the
two subunits of the oxalicines and decaturins are connected through a unique and characteristic
asymmetric spiro carbon atom, a feature that is lacking in pyripyropenes.
In recent years, the Joint Genome Institute (JGI) has released a number of complete genome
sequences for various Penicillium species, making it feasible to perform in silico analysis of
potential SM gene clusters. In this study we attempted to identify the biosynthesis genes for
oxalicines and decaturins by performing a BLAST search using as reference the two genes that
are responsible for the formation of the pyridinyl- α -pyrone polyketide subunit in pyripyropene
A biosynthesis in A. fumigatus, CoA-ligase Pyr1 and NR-PKS Pyr2 (Itoh et al., 2010). We found
that P. canescens ATCC10419 harbors genes with high sequence similarity to each of the two
154

genes in close proximity to each other in the genome. P. canescens is known to produce
griseofulvin, canescin, curvulininc acid, Sch642305, and tryptophan-containing alkaloids (Brian
et al., 1953; Kozlovskii et al., 1997; Nicoletti et al., 2007). Genome sequence data analysis
shows that this species harbors many core SM biosynthesis genes, suggesting that it has the
potential to produce far more SMs than is currently known. Through screening of P. canescens
grown on 10 different media, we identified a condition that produces 15-deoxyoxalicine B (1) in
high titer. We next developed a targeted gene-deletion procedure in P. canescens ATCC10419.
For many fungal species, even if the complete genome sequence is available, linking SMs to
specific genes is often challenging. This is because for most of these organisms, gene targeting
systems are not available or are inefficient, and this was also the case for P. canescens. To
develop an efficient gene targeting system for P. canescens that would facilitate the rapid
generation of gene deletions, we created a ku70 ∆, pyrG ∆ double mutant strain of P. canescens
ATCC 10419. The ku70 gene was deleted to increase homologous recombination rates, thereby
improving targeting efficiency (Krappmann et al., 2006; Ninomiya et al., 2004). pyrG was then
deleted in the ku70 ∆ background to create an auxotrophic mutant (dEnfert, 1996). Using the
ku70 ∆, pyrG ∆ double mutant strain as the parent strain, we performed a series of targeted gene
deletions, and combined with metabolite analysis, we were able to identify and characterize a
gene cluster containing 12 contiguous genes that are involved in the biosynthesis of 1. Several of
the gene deletant strains accumulated chemically stable intermediates or shunt products in
sufficient amounts for full characterization. These strains were cultivated in large-scale and we
were able to isolate a total of 6 related compounds. Two of these compounds have not been
reported previously (Figure 5-2). Combined with further bioinformatic analysis, we have
proposed a biosynthetic pathway for 15-deoxyoxalicine B.
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5.3 Results
Isolation and Characterization of 15-deoxyoxalicine B
Fungal species are known to produce different secondary metabolites when cultivated on
different culture media (Bode et al., 2002; Davis et al., 1966; Nielsen et al., 2011; Oxford et al.,
1935; Pitt et al., 1983). We grew P. canescens on 10 different solid culture media and found that
compound 1 was the major product produced in Czapek’s medium (Figure 5-3). For full
characterization of compound 1, the strain was subjected to large-scale cultivation and the target
peak was purified using flash chromatography and subsequently by preparative HPLC. NMR and
mass spectometry characterization revealed the molecule to be 15-deoxyoxalicine B (Zhang et
al., 2003).
Identification of the Gene Cluster Responsible for 15-deoxyoxalicine B Biosynthesis
Bioinformatic analysis using the JGI database (http://genome.jgi-
psf.org/programs/fungi/index.jsf) was performed, and a total of 32 genes harboring putative beta-
ketoacyl synthase and acyl transferase domains were found. Out of the 32 genes, there are 12
nonreducing PKSs (NR-PKSs), 15 highly reducing PKSs (HR-PKSs), 2 partially reducing PKSs
(PR-PKSs) and 3 PKS- nonribosomal peptide synthetase (NRPS) hybrids.
The polyketide portion of 1 consists of an α-pyrone with an attached pyridine ring. This feature
is structurally highly similar to the distinct polyketide portion of pyripyropenes, a group of
fungal sesquiterpene meroterpenoids produced by Aspergillus fumigatus FO-1289 (Omura et al.,
1982; Simpson et al., 1982). The biosynthetic gene cluster and the biosynthetic pathway of
pyripyropene A in A. fumigatus have been reported previously by Itoh et al (Itoh et al., 2010). In
the biosynthesis of pyripyropene A, CoA ligase Pyr1 catalyzes the formation of nicotine-derived
156

coenzyme A (CoA), or nicotinyl-CoA. PKS Pyr2 uses this nicotinyl-CoA as a starter unit to
which it catalyzes the condensation of two malonyl-CoA molecules to form an α-pyrone moiety.
Because of their structural similarity, we hypothesized that the early stage of the biosynthetic
pathway of 1 would be highly similar to that of pyripyropene A and that the core biosynthesis
gene would be an NR-PKS accompanied by a CoA ligase in close proximity in the genome. We
therefore examined the NR-PKS genes in the P. canescens genome using BLAST analysis and
found that out of the 15 NR-PKSs, Protein ID 400488 had the highest corresponding gene
sequence similarity to pyr2, and the gene is in close proximity to a CoA ligase gene with high
gene sequence similarity to pyr1, Protein ID 437327 (Table 5-1). We therefore hypothesized that
this was the core NR-PKS gene responsible for the biosynthesis of 15-deoxyoxalicine B. To
confirm our hypothesis, we deleted the Protein ID 400488 gene in a P. canescens ATCC10419
strain with a kus70-, pyrG- background and cultivated the deletion strain under 15-
deoxyoxalicine B-producing conditions. The kus70 gene deletion is known to improve the gene-
targeting efficiency because it increases the rate of homologous recombination (Krappmann et
al., 2006; Ninomiya et al., 2004). Analysis of the resultant SMs in the crude organic extract using
LC-DAD-MS showed the complete elimination of compound 1 (Figure 5-5). This confirmed our
hypothesis that Protein ID 400488 is involved in the biosynthesis of 1, and we designated this
gene as olcA.
Next, we set out to identify additional genes involved in the biosynthesis of 1. This process is
facilitated by the fact that fungal secondary metabolite biosynthesis genes are usually clustered.
We individually deleted 13 genes surrounding olcA. The 13 deletants were cultivated under 15-
deoxyoxalicine B-producing conditions and their SM profiles were examined by LC/MS (Figure
5-5). Deletion of genes corresponding to Protein IDs 333321, 437321, 351329, 367480, 393266,
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410812. 437327, 333335, 367485, and 351342 resulted in complete elimination of 1. Deletion of
Protein ID 351326 greatly diminished production of 1. SM profiles remained unchanged after
deletion of Protein ID 410805 and 367486, indicating that these genes are not involved in the
biosynthesis of 1 and that we have established the border of the gene cluster. We now designate
the genes surrounding olcA that are involved in the biosynthesis of 1 as olcB-olcL (Table 5-1).
Identification, Purification, and Structural Determination of the Intermediates and Shunt
Products from Mutant Strains
The deletant strains in which production of 1 was eliminated or greatly reduced were examined
for the presence of new metabolites that may represent intermediates or shunt products of the
biosynthetic pathway of 1. Extracts from strains carrying deletions of olcC, olcE, olcH, and olcI
showed no obvious intermediates that would be a part of the biosynthesis pathway of 1. Seven
strains carrying gene deletions of seven genes, olcB, olcF, olcG, olcJ, olcK, and olcL
accumulated chemically stable intermediates or shunt products in sufficient amounts that allowed
us to isolate them and characterize them by using NMR (NMR data for all the intermediates and
shunt products are shown in Supporting Information). These strains were subjected to large-scale
cultivation and the target compounds were isolated by flash chromatography and semi-
preparatory HPLC. Among the isolated compounds were four decaturin analogs: decaturin A
(Zhang et al., 2003) (3) was isolated from olcB deletants, decaturin D (Li et al., 2005) (5) was
isolated from olcJ deletants, decaturin F (Wang et al., 2013) (6) was isolated from olcF deletants,
and decaturin C (Li et al., 2005) (4) was isolated from both olcK and olcL deletants. The olcK
and olcL deletants also yielded 15-deoxyoxalicine A (Li et al., 2005) (2). NMR analysis of all
five of these compounds is in agreement with published NMR data. Compounds 7 and 8 are
previously unreported compounds. Compound 7 was isolated from olcG deletant strains NMR
158

data showed that 7 has not yet undergone spiro cyclization. Compound 8 was isolated from olcJ
deletants. NMR analysis showed that this compound is an oxidized form of decaturin D (5)
which was also isolated from the same deletant strains. We designate these two compounds
predecaturin E (7) and decaturin G (8).

159

5.4 Discussion
We used a combination of genomics, efficient gene targeting, and natural product chemistry to
elucidate the biosynthesis pathway of the diterpenic meroterpenoid 15-deoxyoxalicine B (1).
Several oxalicine analogues (oxalicines A and B) and decaturin analogues (decaturins A-D) have
been isolated from Penicillium spp. such as P. oxalicum, P. thiersii, and P. decateurense (Abe et
al., 2007; Li et al., 2005; Ubillas et al., 1989; Wang et al., 2013; Zhang et al., 2003), but this
study is the first to our knowledge to give the molecular and genetic basis of the biosynthesis of
this class of compounds.
On the basis of the structure similarity of the polyketide subunit with pyripyropenes, we
performed bioinformatics analysis to find homologs of the responsible genes in Penicllium
species. Using the genome sequence database provided by JGI, we found that P. canescens
harbors homologs of the two genes: a CoA ligase with 71% similarity to Pyr1 and an NR-PKS
with 59% similarity to Pyr2. We then proceeded to find the optimal condition for the production
of 1 in P. canescens, and then performed a series of targeted gene deletions to identify the genes
involved in the biosynthesis pathway. We showed that the pathway involves at least 12 genes in
a single cluster. We have isolated and characterized 5 additional intermediates, 2 of which have
not been reported previously. Further bioinformatic analysis of the genes involved in the
biosynthetic gene cluster, along with comparison to the biosynthetic pathway of pyripyropene A
which shares a similar polyketide subunit, has allowed us to propose a biosynthetic pathway for
1 (Figure 5-6).
1 has a polyketide subunit as an α-pyrone with an attached pyridine ring, very similar to
pyripyropene A. This structural similarity has allowed us to propose the early steps of the
biosynthesis pathway of 1. Although strains carrying deletions of olcI and olcA showed no
160

obvious intermediates, these genes have high protein sequence similarity to genes involved in the
first steps of the biosynthesis of pyripyropene A, pyr1 and pyr2, respectively. Therefore we
propose that CoA ligase OlcI catalyzes the formation of nicotine-derived coenzyme A (CoA), or
nicotinyl-CoA. PKS OlcA then uses this nicotinyl-CoA as a starter unit to which it catalyzes the
condensation of two malonyl-CoA molecules to form an α-pyrone moiety, HPPO.
Unlike pyripyropene A in which a farnesyl pyrophosphate (FPP) is attached to HPPO in the
subsequent step, 1 is linked with geranylgeranyl pyrophosphate (GGPP). The deletion of olcC
significantly decreased the production of 1, suggesting that a large portion of the GGPP used
here seems to be generated by OlcC, however, the deletion did not completely eliminate the
production of 1. Bioinformatic analysis of the P. canescens genome showed 5 additional genes
encoding GGPP synthetases, which may provide the GGPP necessary to produce 1 in the olcC
deletant strain. This olcC gene is unique because, based on further bioinformatic analysis, no
other GGPP synthetases in P. canescens are located in sufficient proximity to a PKS to be a part
of a biosynthetic gene cluster. Furthermore, the pyripyropene A gene cluster in A. fumigatus is
located on a completely separate chromosome from the FPP synthetase.
In pyripyropene A biosynthesis, the attachment of FPP to HPPO to form farnesyl-HPPO is
catalyzed by the prenyltransferase Pyr6. The homolog of Pyr6 in P. canescens is OlcH (68%
protein sequence similarity), and this is most likely the enzyme that catalyzes the attachment of
GGPP to HPPO to form geranylgeranyl-HPPO.
The next steps consist of the epoxidation and cyclization of the terpenoid subunit. It was
demonstrated in the pyripyropene biosynthesis pathway that the FAD-dependent monooxygenase
(FMO) Pyr5 catalyzes the epoxidation step, and the integral membrane protein Pyr4 functions as
the terpene cyclase. The olc gene cluster also contains homologs of these two enzymes, OlcE
161

(57% similarity) and OlcD (74% similarity). From this, we propose that the FMO OlcE catalyzes
the epoxidation of geranyl-geranyl-HPPO and OlcD catalyzes the cyclization of the terpenoid
component, resulting in the formation of the three terpene rings seen in predacaturin E (7). The
similarities between the pyripyropene biosynthetic gene cluster and the putative 15-
deoxyoxalicine B biosynthetic gene clusters end at this point.
Deletion of olcG resulted in the accumulation of 7, suggesting that OlcG, a putative cytochrome
P450, is the next enzyme in the biosynthesis pathway. We propose that OlcG catalyzes the allylic
oxidation of compound 7, which is followed by double bond isomerization and spirocylization
with concomitant loss of water to form decaturin E. Although our deletion strains did not
produce decaturin E, we isolated and identified decaturin D (5) and decaturin G (8) from olcJ
deletion strains. This result suggests that in the absence of OlcJ, decaturin E may be shunted to a
pathway in which it is oxidized to a ketone, possibly by OlcF, to form 5, which undergoes further
allylic oxidation to yield 8.
The next steps involve the rearrangement of the diterpenic subunit leading to the formation of the
hemiacetal seen in decaturin C (4). Conserved domain analysis showed OlcF is a putative short
chain dehydrogenase. We propose that decaturin E is the substrate of OlcJ, and it is hydroxylated
to form decaturin F (6). 6 subsequently undergoes oxidation and lactolization catalyzed by OlcF,
resulting in the formation of the hemiacetal structure of 4.
Deletion of olcK and olcL both resulted in similar SM profiles, showing the accumulation of 4.
olcB deletants accumulated decaturin A (3), which has an added hydroxyl group to 4. These data
suggest that oxidation of 4 to form 3 requires both OlcK and OlcL. Conserved domain analysis
of OlcK showed that this enzyme belongs to the 2-oxoglutarate-Fe(II) oxygenase superfamily
and has a 54% protein sequence similarity to Fum3, a fumonisin C-5 hydroxylase in Fusarium
162

verticillioides (Ding et al., 2004). Interestingly, this enzyme superfamily includes peroxisomal
enzymes. OlcL, on the other hand, is a putative MFS transporter. Computer analysis by
TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) indicated that it is a highly hydrophobic
protein with 14 transmembrane helices. Analysis of Pex19 (peroxisome biogenesis factor 19)
binding sequences (http://www.peroxisomedb.org/) in the OlcL protein revealed one putative
Pex19 binding site between amino acids 132 and 143, within the 2
nd
transmembrane helix. This
finding suggests that OlcL may be inserted in the peroxisomal membrane via the import receptor
Pex19. Based on these analyses, although speculative, we hypothesize that OlcK may be a
peroxisomal enzyme that catalyzes the hydroxylation of 4 once it is shuttled into the peroxisome
by the MFS transporter OlcL. However, further localization studies will be necessary to test our
hypothesis.
In the final step of 15-deoxyoxalicine B biosynthesis, the hemiacetal group of 3 undergoes
oxidative cleavage followed by lactonization. This spirocyclization/ lactonization is catalyzed by
a predicted cytochrome P450, OlcB, to yield compound 1. In both olcK and olcL deletants, in
addition to the production of 4, we identified the production of 15-deoxyoxalicine A (2). On the
basis of the function of OlcB, it is most likely that in the absence of OlcK and/or OlcL, 4 is
accumulated and can be catalyzed by OlcB to yield 2 in a shunt pathway.

163

5.5 Materials and Methods
Strains and Molecular Genetic Manipulations. The P. canescens wild-type and mutant strains
used in this study are listed in Table S2. All DNA insertions into the P. canescens genome were
performed using protoplasts and standard PEG transformation. A strain of P. canescens
ATCC10419 was altered to improve gene targeting efficiency. First, the homolog of ku70 was
deleted by replacing it with the hygromycin resistance marker (hph). Hygromycin deletion
cassettes were generated using the double joint PCR technique. Two ~1,000 base pair fragments
upstream and downstream of the targeted gene were amplified from P. canescens genomic DNA
by PCR. The two amplified flanking sequences and the hygromycin phosphor-transferase gene
(hph) marker cassette amplified from pCB1003 (Fungal Genetics Stock Center) were fused
together into one construct by fusion PCR using nested primers. Next, we created an auxotrophic
mutant in the ku70- background by using a similar tactic, only this time we did not use a deletion
cassette. We fused together the upstream and downstream fragments of pyrG from P. canescens
to form the deletion construct. The mutation was selected by growth on media supplemented
with uracil and fluoroorotic acid (FOA). FOA is toxic to cells that still have a functioning pyrG
gene. Diagnostic PCR of the deletant strains was performed employing the external primers (P1
and P6) from the first round of PCR. The difference in size between the gene replaced by the
selection marker and the native gene allowed us to determine whether the transformant carried
the correct gene replacement. For transformants in which the size of the P1/P6 PCR products are
similar to that of the control, additional diagnostic PCRs were carried out using external primers
paired with primers located within the selection marker gene, in which case the deletants yielded
PCR products of the expected size whereas no product would be seen in the non-deletants. All
164

deletant strains were generated by replacing each targeted gene with the P. canescens pyrG gene
in the kus70-, pyrG- background strain of P. canescens.
Fermentation and LC-MS Analysis. Wild-type P. canescens ATCC 10419 and mutant strains
were cultivated at 26
o
C on Czapek’s agar plates (complete medium; 3 g NaNO3/L, 0.5 g KCl/L,
0.5 g MgSO4·7H2O/L, 0.01 g FeSO4·7H2O/L, 1 g K2HPO4/L, 30 g sucrose/L, and agar 15 g/L)
starting with 1 x 10
7
spores per Petri dish (D = 10 cm). After 5 days of cultivation, agar was
chopped into small pieces and extracted by 80 ml MeOH followed by 80 ml 1:1 CH2Cl2/MeOH,
each with 1 hour of sonication. The extract was evaporated in vacuo to yield a water residue,
which was suspended in 50 ml H2O and partitioned with 50 ml EtOAc. The EtOAc layer was
evaporated in vacuo, re-dissolved in 1 ml of 20% DMSO in MeOH and a portion (10 μl) was
examined by high performance liquid chromatography-photodiode array detection-mass
spectroscopy (HPLC-DAD-MS) analysis.
HPLC-MS was carried out using a ThermoFinnigan LCQ Advantage ion trap mass spectrometer
with a RP C18 column (Alltech Prevail C18 3 mm 2.1 x 100 mm) at a flow rate of 125 μl/min.
The solvent gradient for HPLC-DAD-MS was 95% MeCN/H2O (solvent B) in 5% MeCN/H2O
(solvent A) both containing 0.05% formic acid, as follows: 0% solvent B from 0 to 5 min, 0 to
100% solvent B from 5 min to 35 min, 100 to 0% solvent B from 40 to 45 min, and re-
equilibration with 0% solve B from 45 to 50 min.
Isolation and Characterization of Metabolites. For structure elucidation, the P. canescens
wild-type and mutant strains were cultivated on ~80 Czapek’s agar plates (~25 mL of medium
per plate, D = 10 cm) at 1 x 10
7
spores per plate at 26
o
C for 6 days. Extraction was performed in
the same manner as described above. The crude material was subjected to flash chromatography
and further separated via semi-preparative reverse phase HPLC (Phenomenex Luna 5 μm C18
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(2), 250 x 10 mm) with a flow rate of 5.0 ml/min and monitored by a UV detector at 235 nm. See
Supporting Information for more details about isolation. NMR spectra were collected on a
Varian VNMRS-600 spectrometer. . High-resolution electrospray ionization mass spectrum
(HRESI-MS) was obtained with an Agilent Technologies 1200 series high-resolution mass
spectrometer.

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5.6 Supplementary Information
Detailed Structural Characterization of Compounds

15-deoxyoxalicine B (1): white powder;
1
H NMR (CDCl3), in good agreement with published
data

(Zhang et al., 2003): δ = 0.92 (3H, s), 1.30 (1H, dt, J = 13, 3.6 Hz), 1.49 (1H, dt, J = 14.4,
3.6 Hz), 1.62 (1H, m), 1.70 (3H, s), 1.89 (3H, s), 2.05 (1H, td, J = 13, 4.2), 2.21 (2H, m), 2.30
(1H, m), 2.33 (1H, m), 2.43 (1H, m), 2.49 (1H, m), 2.67 (1H, dd, J = 11.4, 6), 2.97 (1H, d, J =
16.8), 3.10 (1H, d, J = 16.8), 4.37 (1H, d, J = 12.9), 4.45 (1H, d, J = 12.9), 5.07 (1H, s), 5.17 (1H,
s), 5.72 (1H, br s), 6.64 (1H, s), 7.39 (1H, dd, J = 8.4, 4.8), 8.11 (1H, dt, J = 8.4, 1.8), 8.66 (1H,
dd, J = 4.8, 1.8), 8.99 (1H, d, J = 1.8). HRESI-MS, [M + H]
+
m/z found 504.2374 calc. for
C30H34NO6: 504.2381.
Decaturin A (3): white powder,
1
H NMR (CDCl3), in good agreement with published data
(Zhang et al., 2003): δ = 0.87 (3H, s), 1.00 (3H, s), 1.07 (3H, s), 1.29 (1H, dt, J = 13.8, 3.6), 1.48
(1H, dt, J = 14.4, 3.6), 1.65 (1H, m), 1.67 (3H, s), 1.72 (1H, m), 1.84 (1H, td, J = 13.8, 3.6), 1.87
(1H, m), 1.93 (1H, td, J =12, 7.2), 2.06 (1H, m), 2.10 (1H, td, J = 14.4, 3.6), 2.22 (1H, m), 2.24
(1H, dd, J = 12.6, 4.8), 2.94 (1H, d, J = 16.2), 3.08 (1H, d, J = 16.2), 3.93 (1H, d, J = 9.6), 4.12
(1H, dd, J = 9.6, 2.4), 5.69 (1H, br d, J = 5.4), 6.61 (1H, s), 7.39 (1H, dd, 8.4, 4.8), 8.10 (1H, dt, J
= 8.4, 1.8), 8.67 (1H, dd, J = 4.8, 1.8), 9.00 (1H, d, J = 1.8). HRESI-MS, [M + H]
+
m/z found
506.2546 calc. for C30H35NO6: 506.2537.
Decaturin C (4): white powder,
1
H NMR (CDCl3), in good agreement with published data (Li
et al., 2005): δ = 0.87 (3H, s), 0.97 (3H, s), 1.03 (3H, s), 1.27 (1H, m), 1.29 (1H, m), 1.35 (1H,
m), 1.53 (1H, m), 1.56 (2H, m), 1.66 (3H, br s), 1.73 (1H, m), 1.80 (2H, m), 2.14 (1H, m), 2.16
(1H, m), 2.20 (1H, m), 2.92 (1H, d, J = 16.2), 3.08 (1H, d, J = 16.2), 3.89 (1H, dd, J = 9, 1.8),
167

4.20 (1H, dd, J = 9, 3), 5.70 (1H, br d, J = 5.4), 6.60 (1H, s), 7.39 (1H, dd, J = 8.4, 4.8), 8.12 (1H,
dt, J = 8.4, 1.8), 8.67 (1H, dd, J = 4.8, 1.8), 9.00 (1H, d, J = 1.8). HRESI-MS, [M + H]
+
m/z
found 490.2591 calc. for C30H35NO5: 490.2588.
Decaturin F (6): white powder,
1
H NMR (CDCl3), in good agreement with published data (Li et
al., 2005; Wang et al., 2013): δ = 0.68 (1H; 13.2, 3.0), 0.71 (3H; s), 0.78 (1H; dd; 13.8, 2.4), 0.89
(3H; s), 1.02 (3H; s), 1.35 (1H; dd; 12.6, 4.2), 1.42-1.46 (1H; m), 1.42-1.46 (2H; m), 1.56 (1H;
m), 1.58 (1H; m), 1.59 (1H; m), 1.61 (3H; s), 2.06 (1H; m),2.24 (1H; 13.2, 3.0), 2.63 (1H; m),
2.88 (1H; d; 16.2), 3.02 (1H; dd; 12, 4.8), 3.04 (1H; d; 16.2), 3.76 (1H; d; 12), 3.79 (1H; d; 12),
5.65 (1H; br d; 4.8), 7.36 (1H; s), 7.54 (1H; dd; 8.4, 4.8), 8.24 (1H; dt; 8.4, 1.8), 8.67 (1H; 4.8,
1.8), 9.07 (1H; d; 1.8)
Supplemental Methods
Cultivation and LC-MS analysis
P. canescens ATCC 10419 was cultivated at 26
o
C on solid Czapek’s medium (CZA; complete
medium; 3 g NaNO3/L, 0.5 g KCl/L, 0.5 g MgSO4 7H2O/L, 0.01 g FeSO4 7H2O/L, 1 g
K2HPO4/L, and 30 g sucrose/L) starting with 1 x 10
7
spores per Petri dish (D = 10 cm). After 5
days of cultivation, agar was chopped into small pieces and extracted by 80 ml MeOH followed
by 80 ml 1:1 CH2Cl2/MeOH, each with 1 hour of sonication. The extract was evaporated in
vacuo to yield a water residue, which was suspended in 50 ml H2O and partitioned with 50 ml
EtOAc. The EtOAc layer was evaporated in vacuo, re-dissolved in 1 ml of 20% DMSO in
MeOH and a portion (10 μl) was examined by high performance liquid chromatography-
photodiode array detection-mass spectroscopy (HPLC-DAD-MS) analysis.
168

HPLC-MS was carried out using a ThermoFinnigan LCQ Advantage ion trap mass spectrometer
with a RP C18 column (Alltech Prevail C18 3 mm 2.1 x 100 mm) at a flow rate of 125 μl/min.
The solvent gradient for HPLC-DAD-MS was 95% MeCN/H2O (solvent B) in 5% MeCN/H2O
(solvent A) both containing 0.05% formic acid, as follows: 0% solvent B from 0 to 5 min, 0 to
100% solvent B from 5 min to 35 min, 100 to 0% solvent B from 40 to 45 min, and re-
equilibration with 0% solve B from 45 to 50 min.
Isolation and identification of secondary metabolites
All strains were cultivated at 26
o
C for 6 days on ~80 CZA plates at 10 x 10
6
spores per 10 cm
plate. Similar to the method described above, the agar was chopped and sonicated in MeOH,
followed by 1:1 CH2Cl2/MeOH. The organic material was evaporated and extracted twice with
an equal volume of EtOAc. All EtOAc layers were combined and evaporated in vacuo.
For isolation of 15-deoxyoxalicine B and its biosynthetic intermediates, the crude extract in the
EtOAc layer (~150 mg) was coated on 2.3 g C18 silica gel (Cosomil 75C18-OPN, Nacalia
Tesque), which was then suspended in MeOH and applied to a silica gel column (32 x 50 mm).
After equilibrating the column to the starting solvent system of 1:9 MeOH-H2O, the extract was
eluted with MeOH-H2O mixtures of decreasing polarity (fraction A, 1:9, 150 mL; fraction B, 1:1,
150 mL; fraction C, 3:1, 150 mL; fraction D, 1:0, 150 mL). All fractions were analyzed by
HPLC-DAD-MS. Fraction C was subjected to semi-preparative reverse phase HPLC
(Phenomenex Luna 5 μm C18 (2), 250 x 10 mm) with a flow rate of 5.0 ml/min and monitored
by a UV detector at 235 nm. The gradient system was MeCN (solvent B) in 5% MeCN/H2O
(solvent A) both containing 0.05% TFA: 30 to 100% solvent B from 0 to 35 min, maintained at
169

100% from 35 to 38 min, 100 to 30% solvent B from 38 to 39 min, and re-equilibration with
30% solvent B from 39 to 43 min.

170

Table 5-1. Putative function of genes within the 15-deoxyoxalicine B cluster
gene
designation
protein
ID
A.fumigatus
homologs
(Afu6gxxxxx)
Similarity/
Identity
(%)
putative function
410805 cytoskeletal protein adducin
olcB 333321 cytochrome P450
CYP3/CYP5/CYP6/CYP9
subfamilies
olcC 351326 geranylgeranyl pyrophosphate
synthase/Polyprenyl synthetase
olcD 437321 13950(pyr4) 57/41 integral membrane protein (terpene
cyclase)
olcE 351329 13970(pyr5) 74/60 FAD-dependent monooxygenase
olcF 367480 short chain dehydrogenase
olcG 393266 cytochrome P450
CYP3/CYP5/CYP6/CYP9
subfamilies
olcH 410812 13980(pyr6) 68/52 prenyltransferase
olcA 400488 13930(pyr2) 59/42 PKS
olcI 437327 13920(pyr1) 71/58 CoA ligase
olcJ 333335 cytochrome P450
CYP3/CYP5/CYP6/CYP9
subfamilies
olcK 367485 hypothetical protein
olcL 351342 predicted transporter (major
facilitator superfamily)
367486 hypothetical protein

171

Table 5-2. Primers used in this study (5’  3’)
410805-P1 TTGGCTGGATCGGTGATT
410805-P2 AGCGGACGATTTTTGCTG
410805-P3 CGAAGAGGGTGAAGAGCATTGCAGCGCATAAACGCATTG
410805-P4 CAGTGCCTCCTCTCAGACAGCCTAAACACCACGCAAAGG
410805-P5 TGTGGCATCACAGCAAGG
410805-P6 ATCCTGGGCGATTGAGG

olcA deletion
400488-P1 AACGACCCGCATACTGGA
400488-P2 CTCAGGCCACGAATACGC
400488-P3 CGAAGAGGGTGAAGAGCATTGACGGAACTGGTGGGGAAC
400488-P4 CAGTGCCTCCTCTCAGACAGTCAAGCCCACTTCCAAGG
400488-P5 CCCAGAGTTGTCCGATGC
400488-P6 GGTTGTCCCATCGTCCAG

olcB deletion
333321-P1 CGCATGTGGCTGTACTCG
333321-P2 CGTTTATGCGCTGGCTTT
333321-P3 CGAAGAGGGTGAAGAGCATTGTCGGAGCCTGAAGTCGTC
333321-P4 CAGTGCCTCCTCTCAGACAGTATGGAACACCCCGCAGT
333321-P5 GTCCGACCGAGGAGGAAT
333321-P6 AAGGGTCAGGGCATGGAT

olcC deletion
351326-P1 GGATGGTTGGGTAGCTCGT
351326-P2 ACATTGTGGGCAAACATGG
351326-P3 CGAAGAGGGTGAAGAGCATTGAACTGCGATCCGCATCAT
351326-P4 CAGTGCCTCCTCTCAGACAGCCCATCCTTTGCATGGTC
351326-P5 CATTCCGCCCAGAGTCAG
351326-P6 GCATGAGTCCCGATACGC

olcD deletion
437321-P1 TTGGATTCCCGCTGTTTG
437321-P2 TCAGAATTGCTGCGGATG
437321-P3 CGAAGAGGGTGAAGAGCATTGAAGGTGAAGGGCCGACTC
437321-P4 CAGTGCCTCCTCTCAGACAGCGAGCTACCCAACCATCC
437321-P5 TCGCCACATTTCTGTTCG
437321-P6 GCACAGCAGCAGAAATGC

olcE deletion
351329-P1 TTCTGCACTGCGATTTGC
351329-P2 CTTGCTGTCGGGTTCTGG
351329-P3 CGAAGAGGGTGAAGAGCATTGGTAAAAGCCGGGTTGTGG
351329-P4 CAGTGCCTCCTCTCAGACAGACAGTTCGACGTAGCCTTGG
172

351329-P5 GGCAAGCATGGTTTGATAGG
351329-P6 CCGCAAGTAAGTCCATACCC

olcF deletion
367480-P1 CGATCTTGCGAGCTTTCC
367480-P2 CTCGGAAGGCAAGGACTG
367480-P3 CGAAGAGGGTGAAGAGCATTGCGGCTCGGATCGAAGTAG
367480-P4 CAGTGCCTCCTCTCAGACAGATCACCGCCCTGTTTGAC
367480-P5 AGGGCCAGGTTGGATCTC
367480-P6 ACCAAGCCTCACGTCTCG

olcG deletion
393266-P1 CTTTGATCGGAGGCCAAG
393266-P2 TGCCTCGTGATCGAATTG
393266-P3 CGAAGAGGGTGAAGAGCATTGGCCCGCAGGTGAAGTATG
393266-P4 CAGTGCCTCCTCTCAGACAGCCAGCACAGGGAAGAACC
393266-P5 ACCCAGTCGTTCCACACC
393266-P6 TGTACGCGCCACTTTGG

olcH deletion
410812-P1 CCTCGGCTAACCAGTGGA
410812-P2 GCCATTTACCCCGATCCT
410812-P3 CGAAGAGGGTGAAGAGCATTGTTGGCGGAGAATTGGAAA
410812-P4 CAGTGCCTCCTCTCAGACAGAAATGGGGATGGCTCGAT
410812-P5 CCAAGGCACCACATCCTT
410812-P6 CGCGTACTGGGGAGTGAA

olcI deletion
437327-P1 ACGCCATTTCTGGACACC
437327-P2 GGACTGGACCGCATCAAA
437327-P3 CGAAGAGGGTGAAGAGCATTGTGATCCGCTCAGCATGAA
437327-P4 CAGTGCCTCCTCTCAGACAGTCCGATTTTGGGGGAAAC
437327-P5 CGCCATTCACACAACGAC
437327-P6 CTACTGGGCGGTTCATCG

olcJ deletion
333335-P1 TCCATCCTCCCGTCTCTG
333335-P2 GCCGGGTGCGTAGTTATG
333335-P3 CGAAGAGGGTGAAGAGCATTGGGGCGCATCAATCATTTC
333335-P4 CAGTGCCTCCTCTCAGACAGTTGTTTCCCCCAAAATCG
333335-P5 AGCGATGCCAGAAGTTGC
333335-P6 AGTCCACCCTCCCTGTCC

olcK deletion
367485-P1 GTACGCCACAGCCATCG
367485-P2 GGCCACTATGGGGTGTAGG
173

367485-P3 CGAAGAGGGTGAAGAGCATTGTGGAGAAGCACGGAAAGG
367485-P4 CAGTGCCTCCTCTCAGACAGAACCTTGGTCACCCATCG
367485-P5 CCAACGTCTCCCAACGAG
367485-P6 TAGGGAGCTGGGTGTTGC

olcL deletion
351342-P1 AAGCTGTTCCGTGCCAAC
351342-P2 TTGATTCCGGCGAAAAAG
351342-P3 CGAAGAGGGTGAAGAGCATTGGGACGGATTGTCCTGTCG
351342-P4 CAGTGCCTCCTCTCAGACAGCAATTGGCCGGATACAGG
351342-P5 TGGCCCGAGTTATCTAGTCG
351342-P6 GCCCACATCCCCTTACC

367486-P1 AAGTGCCTCGCATCCTGC
367486-P2 TAAGGGGATGTGGGCGGA
367486-P3 CGAAGAGGGTGAAGAGCATTGGGGTCCGCTCAGGGTAGA
367486-P4 CAGTGCCTCCTCTCAGACAGCAGATGTTCGCCCGGGTT
367486-P5 CGAACCGTGCAGGTGGAA
367486-P6 GCCTCACACACGTGCTCA

174

Table 5-3.
1
H-NMR Data for Compounds 1-3 (600 MHz in CDCl
3
)

position
15-deoxyoxalicine B (1)
1
H
δ
H
(int.; mult.; J in Hz)
15-deoxyoxalicine A (2)
1
H
δ
H
(int.; mult.; J in Hz)
Decaturin A (3)
1
H
δ
H
(int.; mult.; J in Hz)
2
3
4
5
6
7
9
10
11
12
14
15

16
17
18

19
20
21

22

23
24
25

26

27
28
29

30
31
32
33

34
8.99 (1H; d; 1.8)

8.11 (1H; dt; 8.4, 1.8)
7.39 (1H; dd; 8.4, 4.8)
8.66 (1H; dd; 4.8, 1.8)

6.64 (1H; s)

2.97 (1H; d; 16.8)
3.10 (1H; d; 16.8)

5.72 (1H; br s)
2.21 (1H; m)

2.21 (1H; m)
2.67 (1H; dd; 11.4, 6)

1.49 (1H; dt; 14.4, 3.6)
2.30 (1H; m)
1.30 (1H; dt; 13, 3.6)
2.05 (1H; td; 13, 4.2)

1.62 (1H; m)
2.49 (1H; m)
2.33 (1H; m)
2.43 (1H; m)

4.37 (1H; d; 12.9)
4.45 (1H; d; 12.9)
1.70 (3H; s)
0.92 (3H; s)

5.07 (1H; s)
5.17 (1H; s)
1.89 (3H; s)
9.00 (1H; d; 1.8)

8.12 (1H; dt; 8.4, 1.8)
7.40 (1H; dd; 8.4, 4.8)
8.67 (1H; dd; 4.8, 1.8)

6.62 (1H; s)

2.96 (1H; d; 16.2)
3.09 (1H; d; 16.2)

5.72 (1H; br s)
2.25 (2H; m)

1.99 (1H; m)

1.56 (2H; m)

1.56 (1H; m)
1.76 (1H; m)
2.01 (1H; br s)

1.76 (1H; m)
2.25 (1H; m)
2.40 (1H; m)
2.44 (1H; m)

4.37 (1H; d; 12)
4.53 (1H; d; 12)
1.70 (3H; s)
0.95 (3H; s)

4.76 (1H; s)
4.96 (1H; s)
1.80 (3H; s)
9.00 (1H; d; 1.8)

8.10 (1H; dt; 8.4, 1.8)
7.39 (1H; dd; 8.4, 4.8)
8.67 (1H; dd; 4.8, 1.8)

6.61 (1H; s)

2.94 (1H; d; 16.2)
3.08 (1H; 16.2)

5.69 (1H; br d; 5.4)
1.87 (1H; m)
2.06 (1H; m)
2.24 (1H; dd; 12.6, 4.8)

1.29 (1H; dt; 13.8, 3.6)
1.84 (1H; td; 13.8, 3.6)
1.48 (1H; dt; 14.4, 3.6)
2.10 (1H; td; 14.4, 3.6)

1.72 (1H; m)
1.93 (1H; td; 12, 7.2)
1.65 (1H; m)
2.22 (1H; m)

3.93 (1H; d; 9.6)
4.12 (1H; dd; 9.6, 2.4)
1.67 (3H; s)
0.87 (3H; s)
1.00 (3H; s)
1.07 (3H; s)

175

Table 5-4.
1
H-NMR Data for Compounds 4 and 6 (600 MHz in CDCl
3
)

position
Decaturin C (4)
1
H (in CDCl
3
)
δ
H
(int; mult.; J in Hz)
Decaturin F (6)
1
H (in DMSO-d
6
)
δ
H
(int.; mult.; J in Hz)
2
3
4
5
6
7
9
10
11
12
14
15

16
17
18

19
20
21

22
23
24
25

26

27
28
29

30
31
32
33
9.00 (1H; d; 1.8)

8.12 (1H; dt; 8.4, 1.8)
7.39 (1H; dd; 8.4, 4.8)
8.67 (1H; dd; 4.8, 1.8)

6.60 (1H; s)

2.92 (1H; d; 16.2)
3.08 (1H; d; 16.2)

5.70 (1H; br d; 5.4)
1.80 (1H; m)
2.14 (1H; m)
1.80 (1H; m)

1.35 (1H; m)
1.53 (1H; m)
1.56 (2H; m)
1.29 (1H; m)

1.27 (1H; m)
2.16 (1H; m)
1.73 (1H; m)
2.20 (1H; m)

3.89 (1H; dd; 9, 1.8)
4.20 (1H; dd; 9, 3)
1.66 (3H; br s)
0.87 (3H; s)
0.97 (3H; s)
1.03 (3H; s)
9.07 (1H; d; 1.8)

8.24 (1H; dt; 8.4, 1.8)
7.54 (1H; dd; 8.4, 4.8)
8.67 (1H; 4.8, 1.8)

7.36 (1H; s)

2.88 (1H; d; 16.2)
3.04 (1H; d; 16.2)

5.65 (1H; br d; 4.8)
2.06 (1H; m)
2.63 (1H; m)
1.58 (1H; m)

1.35 (1H; dd; 12.6, 4.2)
1.59 (1H; m)
1.42-1.46 (2H; m)
0.78 (1H; dd; 13.8, 2.4)

0.68 (1H; 13.2, 3.0)
2.24 (1H; 13.2, 3.0)
1.42-1.46 (1H; m)
1.56 (1H; m)
3.02 (1H; dd; 12, 4.8)

3.79 (1H; d; 12)
3.76 (1H; d; 12)
1.61 (3H; s)
1.02 (3H; s)
0.71 (3H; s)
0.89 (3H; s)

176

Table 5-5.
1
H-NMR Data for Compound 7 (600 MHz in CDCl
3
)

predecaturin E (7)

a
These assignments may be interchanged

177

Table 5-6.
1
H-NMR Data for Compound 8 (600 MHz in CDCl
3
)

decaturin G (8)

a
These assignments may be interchanged

178

Figure 5-1. Structurally related fungal meroterpenoids. The polyketide part is shown in blue and
the terpenoid part in red.

179

Figure 5-2. Structures of novel compounds isolated and characterized from this study
180

Figure 5-3. HPLC profiles of extracts from control and olcA ∆ strains as detected by (A) UV
absorbance 254 nm and (B) mass spectrometry in positive ion mode [M+H]
+
m/z = 504. EIC =
extracted ion chromatogram.

A. UV 254 nm B. EIC 504
181

Figure 5-4. Orientation of the genes surrounding the PKS olcA involved in 15-deoxyxoalicine B
biosynthesis. Each arrow indicates gene size and direction of transcription. On the basis of a set
of deletions we created and analyzed, genes shown in gray are involved in 15-deoxyoxalicine B
while those in white are not.

182

Figure 5-5. HPLC extracts of strains in the cluster as detected by UV absorbance at 254 nm. A
peak labeled by * appears at the same retention time as 1 in deletion strains. This compound was
identified as griseofulvin. Peaks labeled with † denote compounds that could not be
characterized because of poor yield or instability.

183

Figure 5-6. Proposed biosynthesis pathway for 1. All of the natural products isolated in this
study are boxed.

184

Figure 5-7. UV-Vis and ESI-MS spectra of compounds isolated from this study.

185

Figure 5-7 (continued). UV-Vis and ESI-MS spectra of compounds isolated from this study.

186

Figure 5-8.
1
H NMR spectrum of decaturin A (3)

187

Figure 5-9.
1
H NMR spectrum of decaturin C (4)

188

Figure 5-10.
1
H NMR spectrum of decaturin F (6)

189

Figure 5-11.
1
H NMR spectrum of predecaturin E (7)

190

Figure 5-12.
1
H NMR spectrum of decaturin G (8)

191

Chapter VI. Conclusion and perspective
Throughout this dissertation genome-wide strategies in tandem with the powerful approach of
combining genomics, bioinformatics, efficient gene targeting, and natural product chemistry was
employed in discovering secondary metabolites and elucidating their biosynthesis in filamentous
fungi. We first applied and demonstrated the efficacy of this approach in the model fungal
species A. nidulans, which resulted in the elucidation of two (prenyl xanthone and aspernidine)
biosynthesis pathways. We then demonstrated that this approach can be applied to other fungal
species. We successfully developed an efficient gene-targeting system in Penicillium canescens
which allowed us to systematically delete all core SM biosynthesis genes in the genome of this
organism. This resulted in the elucidation of the biosynthesis pathway of 15-deoxyoxalicine B,
and also linked two metabolites (xanthoepocin and amauromine) to their core biosynthesis genes.
In this current chapter we will discuss the overall themes that have emerged in the course of
these studies and address this dissertation’s overall message.
Ever since the release of the first genome sequence of a fungal species, one thing has been very
clear: fungal species harbor potential SM genes that far outnumber the number of SMs that have
actually been discovered. Many of these SMs are not produced under standard laboratory
conditions. Unlike primary metabolites that are absolutely necessary to live, SMs are not
necessary for the fungi to be viable. Some SMs are thought to be produced for niche security or
to outcompete other organisms in growth. Perhaps, then, it is because everything is so controlled
in the laboratory—we make sure the media contains all necessary nutrients, the temperature is
controlled for optimal growth, and we use sterile techniques to ensure there is no contamination
with other fungal or bacterial species. It would make sense that the fungi grown in these
controlled environments would not produce these types of metabolites simply because they do
192

not need to because there is no competition. To access these silent SM gene clusters while
maintaining the controlled nature of the laboratory environment, one of the approaches that have
been taken is OSMAC (one strain many compounds). It has been demonstrated that changing
conditions such as temperature, pH, media composition, etc. can alter the SM profile of a fungal
species. In this dissertation this strategy was applied in the two fungal organsisms. In the prenyl
xanthone project in A. nidulans, we found that the xanthones were produced on Yeast Extract
Sucrose (YES) and Yeast Agar Glucose (YAG) and shredded wheat media. In P. canescens we
found that, 15-deoxyoxalicine B, xanthoepocin, and amauromine were produced on Czapek’s
solid media.
The OSMAC strategy has been used since the early 2000s and it is a tried-and-true approach.
However, we wanted to explore something different to try to activate silent SM gene clusters that
even the OSMAC strategy has not yet been able to give us access to. We therefore directed our
attention to kinases. Kinases are key players in regulation and signal transduction, conveying a
variety of exterior information to intracellular targets to regulate complex processes in cells.
Protein kinases are one of the largest groups of kinases and they act on and regulate protein
activity by transferring phosphate groups from a high-energy donor molecule such as ATP to
specific protein substrates. Their enormous diversity as well as their key roles in every aspect of
regulation and signal transduction has made them an object of study in many eukaryotic systems.
It is possible, then, that they are also involved in regulating the production of SMs. We
hypothesized that altering the expression of kinases would lead to changes in SM profiles and
possibly the expression of SMs that have never been detected in any other culture conditions. We
took advantage of the availability of a genome-wide kinase knock-out library of non-essential
kinase genes in A. nidulans to test this hypothesis. We screened all mutant strains of the kinase
193

knock-out library and found one strain carrying the mpkA deletion that produced aspernidine A,
which had not been detected in A. nidulans previously. Since A. nidulans is a model organism
with a well-established gene targeting system, we were able to perform additional gene
knockouts in the mpkA- background to identify the gene cluster involved in the biosynthesis of
aspernidine A. Furthermore, the mpkA- strain and some of the additional gene knockout strains
produced intermediates in sufficient amounts to allow us to isolate and identify intermediates,
enabling us to propose a biosynthesis pathway. Several other kinase knockout strains showed
changes to their SM profiles also, but since A. nidulans is such a well-studied organism and
many of its SMs have been already identified, none of the SMs turned out to be unknown
compounds. However, since kinases are highly conserved among different species, we believe
that the data we have obtained from the A. nidulans kinase KO library can be applied to other
fungal species. For example, if the knockout of kinase A resulted in an altered SM profile in A.
nidulans, we can find the homolog of kinase A in a different species and perform a knockout of
the corresponding gene with the hypothesis that this would result in an altered SM profile in this
species as well, and possibly, the emergence of a novel SM.
Our group has spent many years working with A. nidulans along with other Aspergillus species,
and we have been successful in linking SMs to their biosynthesis gene clusters using a well-
established system for performing targeted gene deletions and characterizing its metabolites.
This is something that should be applicable to filamentous fungi other than Aspergillus as well.
Using the techniques and knowledge from our experience with working with Aspergillus species,
we decided to embark on the research of Penicillium species. JGI’s fungal portal gave us access
to genome information that we needed, and out of the several Penicillium species that had
recently fully sequenced, we chose P. canescens to start off. The biggest challenge was
194

developing the protocol and transformation system that would allow gene targeting rapidly and
efficiently. Protoplasting and transformation protocols must be tweaked and catered for each
different fungal species. Selection markers that worked in Aspergillus species may not
necessarily work in other species. After overcoming these issues, we were able to develop a gene
targeting system in P. canescens with the rate of transformants carrying the correct gene
insertion at >80%.
We then used this gene targeting system in P. canescens and applied a genome-wide strategy to
link SMs to their biosynthesis genes. This strategy was applied in the xanthone project in A.
nidulans, in which we used a genome-wide PKS knockout library to identify the core
biosynthesis gene of prenyl xanthone. In P. canescens, we created a genome-wide PKS and
NRPS gene knockout library and screened the entire library for changes in SM profiles. This
screening successfully linked 15-deoxyoxalicine B, xanthoepocin, and amauromine to their core
SM genes. In the case of 15-deoxyoxalicine B, we performed additional gene deletions and
determined a complex gene cluster containing 12 genes that is responsible for its biosynthesis.
We were also able to isolate and identify intermediates which allowed us to propose a
biosynthesis pathway. Similar to the situation in A. nidulans, the only limitation to this genome-
wide gene knockout approach is that the SM needs to be produced at detectable levels in the
wild-type. This can be overcome by applying the OSMAC strategy or any of the other techniques
used in other species to activate silent gene clusters. P. canescens harbors at least 52 putative
core SM genes, suggesting a huge potential for the discovery of novel SMs, possibly with
bioactivities that are beneficial for human use. Prior to our studies, none of them had been linked
to SMs due to a lack of tools to accomplish this task. Through our study we have developed the
195

necessary tool in P. canescens and we have shown proof that this tool can be used to successfully
establish the connection between gene and metabolite.
The world of fungal secondary metabolism is complex, and novel approaches and tools are
necessary for the advancement of SM biosynthesis research. The work described in the
dissertation illustrated the application of genome-wide strategies in two fungal species, A.
nidulans and P. canescens. We also described the development of a novel gene targeting system
in P. canescens. Applying these approaches and tools along with modern bioinformatics
analysis, gene targeting, and natural product chemistry will contribute to further discovery of
natural product biosynthesis in filamentous fungi.

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Abstract (if available)

Abstract Genome projects of filamentous fungi have generated an unprecedented amount of information, and in a moment in time often referred to as the “post-genomic era”, it is up to us to find ways to provide meaning to this immense quantity of genome sequence data. Fungi have long been known to be prolific producers of bioactive secondary metabolites, but bioinformatic analysis has showed that these organisms harbor the potential to produce far more secondary metabolites than are currently known. The work herein describes genome-wide strategies to approach two main challenges we now face: 1) finding ways to access the hidden natural products and 2) developing and/ or using genetic systems in fungal species to link secondary metabolites to their biosynthesis genes and elucidate their biosynthesis pathways. We have demonstrated the power of combining bioinformatics, molecular gene targeting, and natural product chemistry in secondary metabolite biosynthesis research. ❧ We initally used A. nidulans as the target species and took advantage of its well-established gene targeting system. First, genome-based deletion analysis in A. nidulans led us to find genes involved in the biosynthesis of xanthones located in at least three distinct loci in the genome. This was particularly interesting because it contradicted the general notion that fungal secondary metabolites are clustered. This highlighted the utility of genomics combined with efficient gene targeting to identify these dispersed genes. Next, we hypothesized that altering the expression of kinases would have an effect on secondary metabolite production and may activate silent gene clusters. We used a genome-wide kinase knockout library and screened its secondary metabolite profile and found that the mpkA deletion strain produced aspernidine A, a compound not previously isolated from A. nidulans. We performed additional gene deletions and determined the border of the biosynthesis gene cluster and proposed the biosynthesis pathway for aspernidine A. ❧ We then took advantage of the recent advances in genome sequencing of Penicillium species and chose Penicillium canescens as the target species. This species is a prolific producer of secondary metabolites but no previous work has been reported to link their metabolites to biosynthesis genes. This is due in large part to the lack of an efficient gene targeting system in this organism. We therefore developed a genetic system in P. canescens that would allow us to perform targeted gene manipulations rapidly and efficiently. We then applied this system and generated a genome-wide deletion library of polyketide synthase (PKS) genes and nonribosomal peptide synthetase (NRPS) genes. Using this library, we successfully linked four secondary metabolites, griseofulvin, xanthoepocin, 15-deoxyoxalicine B, and amauromine, to their core secondary metabolite biosynthesis genes. Interestingly, 15-deoxyoxalicine B is a structurally unique diterpenic meroterpenoid, a class of fungal metabolites whose biosynthesis mechanism remains largely unknown. We performed additional gene deletions and determined the gene cluster involved in its biosynthesis. We were able to isolate and characterize intermediates from the gene deletants, which allowed us to propose a biosynthesis pathway. We believe the gene targeting system and genome-wide PKS and NRPS deletion libraries will be important resources towards a systematic understanding of secondary metabolite biosynthesis in P. canescens.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Yaegashi, Junko (author)

Core Title Application of genome-wide strategies for the mining of secondary metabolite biosynthesis pathways in filamentous fungi

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 04/20/2017

Defense Date 03/24/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Aspergillus,gene targeting,genome mining,OAI-PMH Harvest,Penicillium

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Wang, Clay C.C. (committee chair), Okamoto, Curtis Toshio (committee member), Olenyuk, Bogdan (committee member), Stiles, Bangyan L. (committee member), Zhang, Chao (committee member)

Creator Email junko.yaegashi@gmail.com,yaegashi@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-553827

Unique identifier UC11298769

Identifier etd-YaegashiJu-3341.pdf (filename),usctheses-c3-553827 (legacy record id)

Legacy Identifier etd-YaegashiJu-3341.pdf

Dmrecord 553827

Document Type Dissertation

Format application/pdf (imt)

Rights Yaegashi, Junko

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA