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Genome mining of natural product biosynthesis pathways in filamentous fungi for novel drug discovery and production

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Genome mining of natural product biosynthesis pathways in filamentous fungi for novel drug discovery and production

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Content i

GENOME MINING OF NATURAL PRODUCT BIOSYNTHESIS
PATHWAYS IN FILAMENTOUS FUNGI FOR NOVEL DRUG
DISCOVERY AND PRODUCTION

by
Wei-Wen Sun

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

May 2018

Copyright 2018 Weiwen Sun
ii
DEDICATION

I dedicate this work to my parents, Cheng-Bo Sun and Ming-Yu
Yang, for their priceless love and support.

iii
ACKNOWLEDGEMENTS

I am very grateful to my mentor, Dr. Clay C. C. Wang. Without his generous support and
professional guidance, none of this dissertation would have been possible. I believe it’s
my fate to become his student since I had planned to join his lab even before I came to
USC. He welcomed me to his lab in spring 2013 from the PIBBS program. Since then, he
raised me up by teaching me knowledge, training my skills, and supporting my career.
His encouragement, insight, enthusiasm and perseverance has helped me overcome the
difficulties in my PhD career, and will be forever appreciated.

I am also thankful to my committee members, Dr. Richard Roberts, Dr. Wei-Chiang Shen,
Dr. Curtis Okamoto, and Dr. Bangyan Stiles, for their valuable advice and strong support.
Besides, I would like to thank Dr. Weiming Yuan for admitting me to the USC PIBBS
program, and Dr. Wange Lu and Dr. Bogdan Olenyuk as I did rotations in their labs. I
also feel honored working with our collaborators, Dr. Kenneth Bruno and Dr. Berl
Oakley.

It was also a pleasure to work with such an amazing group. I want to particularly thank
Dr. Yi-Ming Chiang and Dr. Chun-Jun Guo, who trained me to be a natural product
chemist. I also thank Dr. James Sanchez, Dr. Shu-Lin Chang, Dr. Hsu-Hua Yeh, Dr. Ting
Liu, Dr. Junko Yaegashi, Mike Praseuth for their substantial help. I certainly cannot
forget the happy time spending with Kevin Lin, Jillian Romsdahl, Jan van Dijk, Michelle
Grau, Adriana Blachowicz, Steven Glen, John Gallagher, Shauna Warren, Turlough
Heffernan, Catherine Kozachenko, Iarlaith Doherty, Stephanie Loekman, and Yi-En Liao.

Furthermore, I would like to thank the Guangzhou Elite Project and USC School of
iv
Pharmacy for their financial support. Last but not least, I want to thank my family, my
friends, and everyone who helped me during my PhD journey.

Chapter 2 is incorporated from Guo, C. J., Sun, W. W., Bruno, K. S., & Wang, C. C.
(2014). Molecular genetic characterization of terreic acid pathway in Aspergillus
terreus. Organic letters, 16(20), 5250.

Chapter 3 is incorporated from Sun, W. W., Guo, C. J., & Wang, C. C. (2016).
Characterization of the product of a nonribosomal peptide synthetase-like (NRPS-like)
gene using the doxycycline dependent Tet-on system in Aspergillus terreus. Fungal
Genetics and Biology, 89, 84-88.

Chapter 4 is incorporated from Sun, W. W., & Wang, C. C. (2017). Genome-based
deletion analysis in Aspergillus terreus reveals the acetylaranotin bis-thiomethylation
gene. Submitted.

Chapter 5 is incorporated from Guo, C. J., Sun, W. W., Bruno, K. S., Oakley, B. R.,
Keller, N. P., & Wang, C. C. (2015). Spatial regulation of a common precursor from two
distinct genes generates metabolite diversity. Chemical Science, 6(10), 5913-5921.

Chapter 6 is incorporated from Oakley, C. E., Ahuja, M., Sun, W. W., Entwistle, R.,
Akashi, T., Yaegashi, J., Guo, C. J., Cerqueira, G. C., Russo Wortman, J., Wang, C. C. &
Chiang, Y . M. (2017). Discovery of McrA, a master regulator of Aspergillus secondary
metabolism. Molecular Microbiology, 103(2), 347-365.

The author has made the most contributions to the studies described in Chapter 2, 3 and 4,
v
and some contributions to the studies in chapter 5 and 6. For all of the above journal
articles, the author has permission to reincorporate them herein.

vi
Table of Contents
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF ABBREVIATIONS viii
LIST OF TABLES x
LIST OF FIGURES xi
ABSTRACT xiii
CHAPTER I: Introduction 1
1.1 The significance of fungal genetics and fungal natural products research 1
1.2 The enzymology of polyketide synthases and nonribosomal peptide synthetases 2
1.3 The strategies and tools developed for genome mining of natural products
biosynthesis in Aspergillus species 4
1.4 Recent advances in the genome mining of natural products biosynthesis in
Aspergillus terreus 7
CHAPTER II: Molecular genetic characterization of terreic acid pathway in
Aspergillus terreus 11
2.1 Abstract 11
2.2 Introduction 11
2.3 Results and Discussion 12
2.4 Materials and Methods 17
CHAPTER III: Characterization of the product of a nonribosomal peptide
synthetase-like (NRPS-like) gene using the doxycycline dependent Tet-on
system in Aspergillus terreus 43
3.1 Abstract 43
3.2 Introduction 43
3.3 Results and Discussion 45
vii
3.4 Materials and Methods 48
CHAPTER IV: Genome-based deletion analysis in Aspergillus terreus reveals
the acetylaranotin bis-thiomethylation gene 67
4.1 Abstract 67
4.2 Introduction 67
4.3 Results and Discussion 69
4.4 Materials and Methods 74
CHAPTER V: Spatial regulation of a common precursor from two distinct
genes generates metabolite diversity 95
5.1 Abstract 95
5.2 Introduction 96
5.3 Results and Discussion 97
5.4 Materials and Methods 107
CHAPTER VI: Discovery of McrA, a master regulator of Aspergillus
secondary metabolism 138
6.1 Abstract 138
6.2 Introduction 138
6.3 Results and Discussion 139
6.4 Experimental procedures 149
CHAPTER VII: Summary, perspectives and future work 166
BIBLIOGRAPHY 172

viii
LIST OF ABBREVIATIONS

5-FOA

5-fluoroorotic acid
6-MSA

6-methylsalicylic acid
A

Adenylation
ACP

Acyl carrier protein
AT

Acyltransferase
BGC

Biosynthetic gene clusters
BLAST

Basic local alignment search tool
Btk

Bruton’s tyrosine kinase
C

Condensation
CRISPR

Clustered regularly interspaced short palindromic
repeats
DAD

Photodiode array detection
DAQ

Dopaquinone
DCM

Dichloromethane
DH

Dehydratase
DHN

Dihydroxynaphthalene
DOPA

Di-hydroxyphenylalanin
Dox

Doxycycline
DR

Direct repeat
EA

Ethyl acetate
EIC

Extracted ion chromatogram
ER

Enoylreductase
ETP

Epipolythiodiketopiperazine
GMM

Glucose minimal medium
HDAC

Histone deacetylase
HE

Heterologous expression
HPLC

High performance liquid chromatography
HPP

Hydroxylphenylpyruvate
HRESIMS

High resolution electrospray ionization mass spectrum
JGI

Joint genome institute
KAS

Ketoacyl synthase
KR

Ketoreductase
KS

Ketosynthase
LMM

Lactose minimal medium
mcrA

Multicluster regulator A
MeCN

Acetonitrile
MeOH

Methanol

ix
MFS

Major facilitator superfamily
MS

Mass spectrometry
NCBI

National center for biotechnology information
NMR

Nuclear magnetic resonance
NRPS

Nonribosomal peptide synthatase
OSMAC

One strain, many compounds
PCR

Polymerase chain reaction
PKS

Polyketide synthases
PT

Prenyltransferase
ROS

Reactive oxygen species
SAHA

Suberoylanilide hydroxamic acid
SAM

S-adenosylmethionine
SM

Secondary metabolite
SMURF

Secondary metabolite unique regions finder
T

Thiolation
TA

Terreic acid
TE

Thioesterase
TFA

Trifluoroacetic acid
TH

Thioester hydrolase
WT

wild type
YAG

Yeast extract agar

x
LIST OF TABLES

Table 2-1. The at gene cluster and gene function prediction 21
Table 2-2. Primers used in this study 25
Table 2-3. A. terreus strains used in this study 28
Table 2-4.
1
H and
13
C NMR data for compound 1 29
Table 2-5.
1
H and
13
C NMR data for compound 2 29
Table 2-6.
1
H and
13
C NMR data for compound 3 30
Table 2-7.
1
H and
13
C NMR data for compound 4 30
Table 2-8. AtX (ATEG_06275.1) homologs with sequence identity over 40% 31
Table 3-1. NMR data for compound 1 52
Table 3-2. Primers used in this study 57
Table 3-3. Fungal strains used in this study 58
Table 4-1. The expected and isolated compounds from genetic engineering 79
Table 4-2. Primers used in this study 81
Table 4-3. A. terreus strains used in this study 82
Table 4-4.
1
H and
13
C NMR data for compound 9 83
Table 4-5.
1
H and
13
C NMR data for compound 10 84
Table 4-6.
1
H and
13
C NMR data for compound 11 85
Table 5-1. Primers used in this study 119
Table 5-2. Fungal strains used in this study 123
Table 5-3.
1
H and
13
C NMR data for compound 1 124
Table 5-4. NMR data for compound 5 125
Table 6-1. A. nidulans strains used in this study 152
Table 6-2. Primers used to create deletions of A. terreus and P . canescens mcrA
homologs 162

xi
LIST OF FIGURES

Figure 1-1. Fusion PCR strategy and efficient gene targeting system 10
Figure 2-1. A. Compounds derived from 6-MSA; B. Isolated intermediates or shunt
products of TA 22
Figure 2-2. (A) Organization of the TA gene cluster; (B) The DAD traces of extracts
from the wildtype and mutants as detected by UV 23
Figure 2-3. Proposed biosynthetic pathway for terreic acid 24
Figure 2-4. The DAD traces and EIC extraction profile from the wildtype, atXΔ and
atXΔ fed with 3-methylcatechol (5) 33
Figure 2-5. Schematic of the diagnostic PCR strategy 34
Figure 2-6. Results of diagnostic PCR for all the gene deletion strains 35
Figure 2-7. UV-Vis and ESIMS spectra of compounds 36
Figure 2-8.
1
H NMR and
13
C NMR spectrum of TA 1 37
Figure 2-9.
1
H NMR and
13
C NMR spectrum of compound 2 38
Figure 2-10.
1
H NMR and
13
C NMR spectrum of compound 3 39
Figure 2-11.
1
H NMR,
13
C NMR, gHMBC, gHMQC, gCOSY , NOESY spectrum of
compound 4 42
Figure 3-1. Schematic overview of the Dox-regulated gene expression system used in
this study 53
Figure 3-2. HPLC profiles of strain extracts as detected by UV 54
Figure 3-3. UV-Vis and ESIMS spectra of compound 1 55
Figure 3-4. Proposed biosynthetic pathway for compound 1 56
Figure 3-5. The schematic design of the pgnA gene native promoter replacement and
heterologous expression 59
Figure 3-6. Diagnostic PCR analysis schematic design and results 60
Figure 3-7. (A) The difference between NRPS and NRPS-like genes;
(B) The ORF codes of the pgnA gene 62
Figure 3-8. Alignment of amino acid sequence of PgnA, ApvA, AtmelA, AtqA, BtyA,
MicA, and TdiA 64
Figure 3-9.
1
H NMR and
13
C spectra of compound 1 66
Figure 4-1. Acetylaranotin related compounds isolated in this study 77
Figure 4-2. The DAD traces and EIC extraction profile from the wild type and ataSΔ
strains 78
Figure 4-3. Proposed biosynthetic pathway for acetylaranotin derivatives 79
Figure 4-4. The DAD traces of extracts from the mutant strains as detected by UV 80
Figure 4-5. Alignment of A. fumigatus GtmA/TmtA and A. terreus AtaS 85
Figure 4-6. The direct repeat strategy and marker recycling 86
Figure 4-7. Diagnostic PCR strategy and results 87
xii
Figure 4-8. UV-Vis and ESIMS spectra of identified compounds 88
Figure 4-9. Oxidation of two isomers of 11 by 3% H2O2 89
Figure 4-10.
1
H and
13
C NMR spectra of compound 9 90
Figure 4-11. 1D and 2D NMR spectra of compound 10 93
Figure 4-12.
1
H and
13
C NMR spectra of compound 11 94
Figure 5-1. The proposed biosynthesis strategy for aspulvinones and butyrolactones 112
Figure 5-2. (A) Compounds related to this study; (B) HPLC profiles of extracts of
HE strains 113
Figure 5-3. Phenotype, total extracts, conidial extracts and hyphal extracts of
A. terreus wild type and mutant strains 114
Figure 5-4. HPLC profiles of extracts of A. terreus PT genes deletants 116
Figure 5-5. (A) Phenotype of the strains; (B) Replacing atmelA and apvA with gfp 117
Figure 5-6. The schematic design of molecular genetic experiments in this study 126
Figure 5-7. Relative quantification analysis of gene expression levels in
WT-hyphae, WT-conidia, and CW6058.1-conidia 127
Figure 5-8. HPLC profiles of extracts of the strains 128
Figure 5-9. The gene apvA is inserted in a highly conserved region among
Aspergillus species that contains genes putatively encoding life-essential proteins 129
Figure 5-10. Homology analysis of 59 NRPS-like homologs obtained from the
Broad Institute Aspergillus Comparative Database 130
Figure 5-11. Proposed biosynthetic pathway for butyrolactones and aspulvinones 132
Figure 5-12. UV-Vis and ESIMS spectra of compounds 1, 5 133
Figure 5-13. Diagnostic PCR strategies 134
Figure 5-14.
1
H NMR and
13
C spectra of compound 1 136
Figure 5-15.
1
H NMR and
13
C spectra of compound 5 137
Figure 6-1. Phenotype of the strains 154
Figure 6-2. HPLC profile scans of parental strains and AN8694 deletion strains 155
Figure 6-3. Effects of alteration of mcrA and laeA expression on SM production 156
Figure 6-4. Reinsertion of mcrA returns secondary metabolite production to parental
levels 157
Figure 6-5. HPLC profile scans of parental strains and AN8694 deletion strains 159
Figure 6-6. Deletion of mcrA homologs alters secondary metabolite production in
A. terreus and P . canescens 160
Figure 6-7. UV-Vis and ESIMS (positive or negative mode) spectra of new and
unknown compounds identified in this study 165
Figure 7-1. The secondary metabolites involved in chapter VII 171
Figure 7-2. The DAD traces of extracts from the P . canescens wildtype and mutant 171
xiii
ABSTRACT

Filamentous fungi are well known producers of a wide variety of bioactive secondary
metabolites. Genome sequencing projects of filamentous fungi have opened the
post-genomic era of genome mining for natural products. Bioinformatic analysis has
shown that fungal species have the potential to produce far more secondary metabolites
than have already been isolated. The challenge lies in how to unlock this hidden power.
Combining bioinformatics, molecular gene targeting, and natural product chemistry in
secondary metabolite biosynthesis research, the work herein describes different aspects of
natural product discovery: 1) biosynthesis gene cluster characterization, 2) genetic tool
development for biosynthesis pathway activation, 3) structural modification of natural
product by genetic engineering, and 4) exploration of the complex natural product
regulation mechanisms. Using the gene loss-of-function analysis approach, we first
characterized the biosynthesis gene cluster for terreic acid, which can also be used as a
probe to uncover structurally similar compounds from other genome sequenced fungi.
Next, we adopted the Tet-on system as a genetic tool to Aspergillus terreus and
successfully activated a previously uncharacterized NRPS-like gene, pgnA, which is
responsible for production of phenguignardic acid. Furthermore, we discovered the
bis-thiomethylation gene for acetylaranotin, and engineered the acetylaranotin
biosynthesis pathway to generate more analogues. Finally, exploring regulation
mechanisms of natural product biosynthesis allowed us to discover the importance of
spatial regulation and a global regulator, McrA.

1
CHAPTER I: Introduction

1.1 The significance of fungal natural products and fungal genetics research

Natural products that are derived from natural sources such as plants, animals or
microorganisms have benefited human health for probably thousands of years, which can
be dated from prehistoric times (Cragg and Newman, 2001). An impressive number of
modern drugs have been elucidated or derived from natural products, many of which
were based on the traditional medicinal practices. Quinine (isolated from the bark of
Cinchona succirubra Pav. ex Klotsch), morphine (isolated from Papaver somniferum L.),
acetylsalicylic acid (derivative of salicin that isolated from Salix alba L.) and digitoxin
(isolated from Digitalis purpurea L.) are all representative natural product drugs from
plants, whereas penicillin (isolated from Penicillium notatum), cyclosporine (isolated
from Beauveria nivea) and griseofulvin (isolated from Penicillium griseofulvum) are
equally well known fungal natural product drugs (Dias et al., 2012). The continued
research into natural products is important because up to 50% of all drugs in current
clinical use are derived from natural products (Pan et al., 2013). Speaking of fungi, they
have been closely related to human life for a long history. Except the important medicinal
application, mushrooms have served as delicious food, and yeasts have been used to
make bread and alcoholic beverages worldwide. Because of their broad application and
massive economic value, a lot of attention has been drawn to fungal research.

Filamentous fungi feature the formation of hyphae, and are important sources of natural
products that have potential for pharmaceutical and agricultural purposes. They are
known to produce antibiotics (e.g., penicillin), antifungal drugs (e.g., griseofulvin)
immunosuppressants (e.g., cyclosporine), cholesterol-lowering agents (e.g., lovastatin)
2
and potent poisons (e.g., aflatoxins) (Keller et al., 2005). Filamentous fungi encompass
many genera of fungi including Aspergillus, Penicillium, Fusarium, Cladosporium,
Emericella, Eurotium, Paecilomyces (Pitt and Hocking, 2009), among which Aspergillus
is one of the most-extensively studied genera. Because of the medical and commercial
significance, the genomes of many members of this genus have been sequenced so far,
and high-throughput genome sequencing techniques have greatly facilitated the genome
mining of secondary metabolites (SMs) identified in Aspergillus species. Recent studies
have revealed that the genes responsible for biosynthesizing one secondary metabolite are
usually clustered within a chromosome, often being apart from each other by less than
2kb (Keller et al., 2005), which provides important clues for gene cluster characterization.
The core structural genes within gene clusters are usually responsible for the biosynthesis
of secondary metabolite backbones, while other genes encoding tailoring enzymes, like
methyltransferases, decarboxylases, oxidases, hydrolases, expoxidases, prenyltransferases
and cyclases, will further expand the chemical diversity by modifying the backbone. In
addition, genes encoding regulatory proteins or transporters can also be found within the
cluster. Typical examples include characterized gene clusters for the biosynthesis of
aflatoxins and sterigmatocystin (Brown et al., 1996; Yu et al., 2004a). The
characterization of gene structures and functions in fungi will help us explore the
unknown genes with homologous sequence. Based on understanding the sequence,
function and interaction of a significant number of genes, we can use them as basic
elements to design and construct novel artificial biological pathways, or to redesign the
existing natural biological pathways.

1.2 The enzymology of polyketide synthases and nonribosomal peptide synthetases

There are two major classes of enzymes involved in the construction of secondary
3
metabolite backbones: polyketide synthases (PKS) and nonribosomal peptide synthetases
(NRPS). They are responsible for the biosynthesis of secondary metabolites, polyketides
and non-ribosomal peptides. The structures of polyketides derive from simple building
blocks like malonyl-CoA and methylmalonyl-CoA assembled by PKSs, which is a similar
process to fatty acids synthesis. The major difference is that fatty acids have full
reduction of the beta-carbon, which is an optional event in polyketide synthesis (Keller et
al., 2005). There are three types of PKS. Type I PKS consist of very large multifunctional
proteins with individual functional domains, including bacterial modular (non-iterative)
type I PKS and fungal (also some bacterial) iterative type I PKS. Type II PKS consist of
individual proteins, and exclusively exist in bacteria. Type III PKS (chalcone and stilbene
synthases) are very simple β-ketoacyl synthase (KAS) proteins, which are distributed in
mainly plants, some bacteria and fungi (Cox, 2007; Hertweck, 2009). Most of our
research targets are the fungal iterative type I PKS, which use a single multidomain
module iteratively throughout the biosynthesis of the polyketides, while bacterial type I
PKS have separate modules for each methylmalonyl CoA addition. Their canonical core
domains include an acyltransferase (AT) domain, a ketosynthase (KS) domain and a
thiolation (T) domain (also referred to as ACP (acyl carrier protein) domain). The AT
domain selects either malonyl-CoA or methylmalonyl-CoA as substrates, and then
transfer the acyl group to the ACP domain, which provides a thiol group for the acyl
chain to covalently attach. The KS domain catalyzes the C-C bond formation during
chain elongation by decarboxylating the downstream acyl unit and adding it to the
upstream acyl chain. The ketoreductase (KR) domain, dehydratase (DH) and
enoylreductase (ER) domain are optional in the fungal type I PKS, and their presence
determines the extent of chain reduction. The final hydrolysis of the thioester to release
the polyketide chain is catalyzed by a thioesterase (TE) domain.

4
Non-ribosomal peptides derive from proteinogenic and non-proteinogenic amino acids
and other carboxylic acids assembled by the multimodular NRPS. A typical module of
fungal NRPS contains one adenylation (A) domain responsible for selecting amino acid
or other carboxylic acid monomer as substrate, one thiolation (T) domain (also referred to
as PCP (peptidyl carrier protein) domain) responsible for carrying the substrate, and one
condensation (C) domain responsible for catalyzing the peptide-bond formation and chain
elongation. The typical order of these domains is C-A-T. Compared to typical NRPS,
NRPS-like genes encode single module (A-T-TE) proteins missing the condensation (C)
domain (Balibar et al., 2007a; Fischbach and Walsh, 2006b). The A domain is responsible
for aryl acid substrate recognition and activation. The activated substrate is loaded onto
the thiolation (T) domain. The thioesterase (TE) domain is suggested to be involved in
the condensation and releasing of the final product. By examining the domain
arrangement of PKS and NRPS, we are able to predict the structure of their products, and
vice versa.

1.3 The strategies and tools developed for genome mining of natural products
biosynthesis in Aspergillus species

As previously mentioned in 1.1, the genomes of many members of Aspergillus species
have been sequenced so far, and bioinformatic analysis revealed that there are far more
secondary metabolite gene clusters than the secondary metabolites that have been isolated
before. This indicates that most gene clusters predicted by genome analysis don’t produce
any known secondary metabolites under normal laboratory conditions, and thus we call
them silent gene clusters. The obligation of genome mining for natural products is to
activate the silent gene clusters, understand their biosynthesis and regulation, and use the
knowledge for further natural products discovery and production. Several strategies have
5
been successfully developed to uncover the hidden secondary metabolites of silent gene
clusters in Aspergillus species. One strategy is defined as the OSMAC (one strain, many
compounds) approach, which uses different culture conditions (like medium recipe,
temperature, shaking speed) to generate metabolic diversity. Another broadly used
strategy is to manipulate the cluster-specific regulatory genes by deleting them or placing
them under the control of an inducible promoter. In addition to pathway-specific regulator,
global regulators also play important roles in silent gene cluster regulation, although the
result of their manipulation cannot be predictable without completely understanding the
complex regulatory network. LaeA is such a representative global regulator, and its
deletion and overexpression in A. nidulans allowed the characterization of the
terrequinone A gene cluster (Bok et al., 2006). The discovery of another equally
important global regulator, McrA, is described in Chapter 5. Epigenetic processes that
involve covalent modification of DNA and chromatin also regulate gene expression in
eukaryotic cells. Recent research has shown that suberoylanilide hydroxamic acid
(SAHA), which inhibits histone deacetylase (HDAC) and further affect chromatin
arrangement, generated the production of nygerone A (Henrikson et al., 2009) . The last
strategy that must be mentioned is refactoring and heterologous expression. This involves
replacement of the native promoter of each gene within the gene cluster of interest with a
constitutive or inducible promoter, and expression of them in a heterologous host.
Formation of the complete assembly line in the heterologous host will allow the
production of the final natural product, while the incomplete assembly line might produce
intermediates in the biosynthetic pathway. The same strategy can also be applied to
characterize a suspect gene cluster.

Once a secondary metabolite is found, we are interested in characterizing its biosynthetic
pathway and engineering second-generation analogs. There are several approaches for
6
characterizing a gene cluster: targeted gene deletion analysis in native organisms,
heterologous expression of suspected genes in an alternative host, and in vitro
characterization of expressed proteins. By deleting a gene, it can be learned whether that
gene was responsible for the formation of a particular natural product. Deletion of an
important PKS/NRPS gene should lead to the loss of the corresponding secondary
metabolite. Deletion of tailoring genes that contribute to the metabolite’s formation
should result in not only the secondary metabolite loss, but also the accumulation of
intermediates in the biosynthetic pathway in many cases. These intermediates may
provide important clues to the biosynthesis of the final product, and they may also have
interesting biological properties of their own. The implementation of this approach is
illustrated in Chapter 2. Similar to the heterologous expression approach described above,
the in vitro characterization of expressed proteins approach tries to form the biochemical
assembly line in vitro instead of in a heterologous host.

The development of the above approaches was facilitated by a fusion PCR technique and
the establishment of an efficient gene targeting system. (Figure 1-1) The fusion PCR
technique allows constructing DNA fragments for transformation by two-round of PCR
without ligation, which accelerates the gene deletion or promoter replacement process.
The typical primers are designed with a ~20 bp tail for combining the ~1 kb flanking
regions with the selectable marker, which can be a gene encoding an essential nutrient
that cannot be self-produced by the recipient strain (Yu et al., 2004b). (Figure 1-1a) The
efficient gene targeting system in Aspergillus species is established by deleting the human
Ku70 or Ku80 gene homologs (KusA), which are essential for nonhomologous enjoining
repair for DNA double-stranded breaks (Nayak et al., 2006; Ninomiya et al., 2004). In A.
nidulans, this system increased the frequency of desired homologous recombination from
∼13% to ∼90%. (Figure 1-1b) Additionally, a procedure for producing transformable
7
protoplasts from hyphae has been developed using readily available enzyme to remove
the cell wall, which makes the transformation of fusion PCR construct into fungal cells
feasible (Szewczyk et al., 2006).

Although the deletion of Ku70 or Ku80 homologues and development of selection
markers established the efficient gene targeting system, the introduction of such genetic
tools into fungi can be labor-intensive and time consuming, and it has been made only
available to a few model fungi so far. Most recently, gene targeting advance using the
CRISPR/Cas9 genome editing technique has been applied to filamentous fungi. As
accurate RNA guided mutagenesis can be achieved by transforming a target fungus with a
single plasmid, it has been proven to be a powerful and efficient tool for gene disruption
analysis. Its application has made the gene disruption available to fungal microorganisms
such as A. aculeatus, A. fumigatus, Beauveria bassiana, Pyricularia oryzae,
Saccharomyces cerevisiae, Trichoderma reesei, and Ustilago maydis (Arazoe et al., 2015;
Chen et al., 2017; Fuller et al., 2015; Liu et al., 2015; Mans et al., 2015; Nødvig et al.,
2015; Schuster et al., 2016; Zhang et al., 2016).

1.4 Recent advances in the genome mining of natural products biosynthesis in
Aspergillus terreus

Aspergillus terreus, as the main producer of lovastatin and a pathogen that causes
aspergillosis, was fully sequenced early in 2005, and its sequence is publicly available in
the Aspergillus Comparative Database. A Secondary Metabolite Unique Regions Finder
(SMURF) analysis of the A. terreus NIH 2624 genome identified 28 polyketide synthase
(PKS) genes, 20 nonribosomal peptide synthetase (NRPS) genes, one PKS-NRPS hybrid
gene, two PKS-like genes, and 14 NRPS-like genes (Khaldi et al., 2010); all of these
8
genes are responsible for the biosynthesis of secondary metabolite backbones,
polyketides and non-ribosomal peptides. In contrast to the diversity of backbone genes,
only a few secondary metabolites were identified that corresponded to their biosynthetic
gene cluster, including lovastatin (Campbell and Vederas, 2010), (+)-geodin (Nielsen et
al., 2013), isoflavipucine (Gressler et al., 2011a), dehydrocurvularin (Xu et al., 2013),
terretonin (Guo et al., 2012), acetylaranotin (Guo et al., 2013d), asperfuranone (Chiang et
al., 2013b), terrein (Zaehle et al., 2014), and citreoviridin (Lin et al., 2016). The gene
clusters involved in the biosynthesis of other secondary metabolites isolated from A.
terreus still remain to be identified, and most gene clusters predicted by genome analysis
don’t produce any known secondary metabolites under laboratory conditions. In order to
obtain more valuable natural products, an efficient method is desired to be developed in A.
terreus to activate silent gene clusters. Our long-term goal is to discover more medically
and commercially valuable secondary metabolites from filamentous fungi and decipher
their biosynthetic pathway and complex regulation mechanism. An objective of this
application is to use known gene clusters as probes to mine other genome-sequenced
microorganisms for structurally related secondary metabolites. By elucidating the
biosynthetic pathway of a compound, it also becomes possible to rationally modify the
compound towards a more potent drug through genetic manipulation.

In this dissertation, we report our efforts in accomplishing the above objectives in
genome mining for natural products in filamentous fungi using different strategies. We
characterized the biosynthesis gene cluster and pathway of terreic acid using the gene
deletion approach as described in Chapter 2. Chapter 3 describes the successful adoption
of the Tet-on system as an effective gene activation tool in A. terreus, which allowed us
to link an NRPS-like gene with its product. In Chapter 4, we made efforts to rationally
manipulate acetylaranotin biosynthesis genes to generate second-generation analogues.
9
Chapter 5 and Chapter 6 demonstrate two important secondary metabolites regulation
mechanisms, spatial regulation and global regulation, respectively.

10
a.

b.

Figure 1-1. a. T he tw o - r ou n d fu s i on PCR t ec hni q ue and g ene kn oc k - o ut s tr at eg y . b . T he kusA- , pyrG- ef fi c i ent g en e tar g eti n g s y s t e m f or A. terreus . kusA i s the n on- h om o logou s en d j oi n i n g r epa i r g ene, an d pyrG i s a s ele c ti on m a rk er .
11
CHAPTER II: Molecular genetic characterization of terreic
acid pathway in Aspergillus terreus

2.1 Abstract

Terreic acid is a natural product derived from 6-methylsalicylic acid (6-MSA). A compact
gene cluster for its biosynthesis was characterized. Isolation of the intermediates and
shunt products from the mutant strains, combined with bioinformatic analyses, allowed
for the proposition of a biosynthetic pathway for terreic acid.

2.2 Introduction

Filamentous fungi are well known producers of a wide variety of secondary metabolites
with interesting biological activity and important pharmaceutical potential. Terreic acid
(TA, 1) (Figure 2-1), a quinone epoxide, is one such compound isolated from Aspergillus
terreus. Prior studies have recognized the inhibitory effect of TA (1) against bacteria
(Yamamoto et al., 1980). TA (1) has attracted attention because it selectively inhibits the
catalytic activity of Bruton’s tyrosine kinase (Btk), the kinase that plays vital roles in
mast cell activation and B cell development (Kawakami et al., 1999). Because of its
inhibitory effect, TA (1) has been used as a chemical probe to examine the function of
Btk (Kawakami et al., 1999). Ibrutinib, also known as PCI-32765, is a selective inhibitor
of Btk and has been approved by the FDA recently in the treatment of mantle cell
lymphoma and chronic lymphocytic leukemia (Byrd et al., 2013). One of the research
interests in our lab is in the discovery of natural product inhibitors of Btk such as TA (1)
as potential cancer therapeutics.

12
Compared to its biological activity, knowledge of the biosynthesis pathway for TA (1) in
A. terreus is quite limited. Pioneering work using a radio labeled precursor approach
demonstrated that the carbon skeleton of TA (1) originated from 6-methylsalicylic acid
(6-MSA, 2) (Figure 2-1) (Read and Vining, 1968). The 6-MSA (2) precursor undergoes
decarboxylation and a series of oxidation steps to give TA (1) (Read and Vining, 1968;
Read et al., 1969). Recent genome sequencing of A. terreus enabled us to use a genome
mining approach to decipher the biosynthesis of TA (1) at the genetic level. We can use
the genes for TA (1) biosynthesis as probes to uncover structurally similar compounds
with potential Btk inhibitory activity from other genome sequenced fungi. A previous
study reported the cloning of the gene atX in A. terreus and its identification as a 6-MSA
synthase (6-MSAS) (Fujii et al., 1996). Although atX is the only 6-msas gene identified
in A. terreus, the genetic linkage between atX and TA (1) has not been confirmed
experimentally until this study.

2.3 Results and Discussion

In this study, we analyzed the A. terreus NIH 2624 genome and identified the at cluster
responsible for TA (1) biosynthesis. A. terreus was cultivated on yeast extract agar (YAG),
a medium on which TA (1) is consistently produced. The compound TA (1) was identified
in the ethyl acetate (EA) layer after extraction from the acidified water layer. The
structure of TA (1) was confirmed by comparing its
1
H and
13
C NMR spectra with
published data (Findlay and Radics, 1972). Next, we deleted the gene ATEG_06275.1
(atX) using an efficient gene targeting system we developed for A. terreus (Guo et al.,
2013a). As expected, examination of the secondary metabolite profile of the atXΔ strain
showed that the production of TA (1) was eliminated (Figure 2-2), providing direct
evidence that AtX synthesizes 6-MSA (2) that incorporates into the TA (1) pathway.
13

Secondary metabolite genes are often clustered in filamentous fungi (Keller et al., 2005).
Previous bioinformatic analyses suggested that the at cluster contains seven genes from
ATEG_06272.1 to ATEG_06278.1.1 but there was no experimental proof (Boruta and
Bizukojc, 2014a). To explicitly characterize the entire gene cluster, a total of 12 genes
from ATEG_06270.1 to ATEG_06282.1 were individually deleted. The LC/MS profiles
of the verified gene deletion mutants indicated that the production of TA (1) was impaired
in the strains ATEG_06272.1Δ (atAΔ), ATEG_06273.1Δ (atBΔ), ATEG_06274.1Δ (atCΔ),
ATEG_06276.1Δ (atDΔ), ATEG_06277.1Δ (atEΔ), ATEG_06278.1Δ (atFΔ) and
ATEG_06280.1Δ (atGΔ), suggesting that these genes are involved in the biosynthesis of
TA (1) (Figure 2-2). TA (1) was produced in the ATEG_06270.1Δ, ATEG_06271.1Δ,
ATEG_06281.1Δ and ATEG_06282.1Δ mutants indicating we have defined the left and
right borders of the entire gene cluster.

Among the genes involved in TA (1) production, the atAΔ, atCΔ, and atEΔ strains
produced intermediates or shunt products that were UV active (Figure 2-2). Compound 2
(Figure 2-1B) was purified from large-scale cultures of the atAΔ strain (Figure 2-2). The
structure of compound 2 was confirmed to be 6-MSA (2) by comparing the
1
H and
13
C
NMR spectra with those of the published data (Fujii et al., 1996). The atCΔ strain
accumulated chemically stable compound 3 (Figures 2-1B and 2-2). The structure of
compound 3 was identified as terremutin (3), a dihydroquinone epoxide intermediate
proposed to be involved in the TA (1) pathway (Read et al., 1969). The structure of
compound 4, purified from the atEΔ strain, was found to be
(2Z,4E)-2-methyl-2,4-hexadienedioic acid. Since no published NMR data are available
for compound 4, thorough 1D and 2D NMR analyses enabled us to determine its
structure. The NOE correlation between H-3 and the 2-methyl protons, as well as the
14
coupling constant (J = 15.2 Hz) between H-4 and H-5, led us to determine the
conformation of 4 to be 2Z, 4E. Interestingly, compound 4 no longer maintains the
six-carbon ring moiety as identified in other characterized intermediates (2 and 3),
suggesting that it might be a shunt product in the TA (1) pathway.

Elucidation of the above intermediates’ structures, combined with the sequence analyses
of the involved genes, allowed us to propose a biosynthetic pathway for TA (1) (Figure
2-3, Table 2-1). The first step of the pathway is the synthesis of 6-MSA (2) by the
6-MSAS AtX. The domain architecture of AtX includes β-ketoacyl synthase (KS),
acyltransferase (AT), thioester hydrolase (TH), ketoreductase (KR), and acyl carrier
protein (ACP) (Figure 2-3). The previous study reveals that the TH domain in AtX acts as
a product releasing domain which catalyzes a thioester hydrolysis to give 6-MSA (2),
representing a first example of a product-releasing domain embedded in the middle of a
PR-PKS (Moriguchi et al., 2010). In the biosynthesis of 6-MSA (2), AtX utilizes three
malonyl-CoAs as its substrates and catalyzes a series of programmed reactions including
Claisen condensation, dehydration, reduction and cyclization to yield compound 2
(Moriguchi et al., 2010).

A recent bioinformatic study by Boruta et al. predicted that AtA catalyzes the conversion
of 2 to 5 and AtE catalyzes hydroxylation of 5 to 6 (Figure 2-3). They predicted that AtC
is involved in the dehydrogenation of 6 to a quinone precursor of terreic acid (1). They
did not propose the enzyme responsible for the final epoxidation. The study also
suggested that the cluster contains a putative transporter gene (atB) and a regulatory gene
(atF) (Boruta and Bizukojc, 2014a).

In our study, only compound 2 (but no other intermediates) was accumulated in the atAΔ
15
strain (Figure 2-2). Conserved domain search of AtA indicated that the putative protein
contains a salicylate 1-monooxygenase domain. Salicylate 1-monooxygenase is an
enzyme that catalyzes the conversion of salicylate to catechol, and is FAD dependent
(Table 2-1) (Katagiri et al., 1965). AtA shares around 30% amino acid identity with a
characterized salicylate 1-monooxygenase gene, salA, in A. nidulans (Graminha et al.,
2004). This suggests that AtA might catalyze the decarboxylative hydroxylation of 2 to 5.
Previous literature reported the characterization of two FAD-dependent monooxygenases,
TropB and SorbC, that catalyze the hydroxylative dearomatization of their substrates (al
Fahad et al., 2014; Davison et al., 2012). In comparison, the aromaticity of compound 2
still remains after the decarboxylative hydroxylation by AtA.

The conversion of 3-methylcatechol (5) to terremutin (3) requires a series of oxidation
modifications such as hydroxylation and epoxidation (Figure 2-3). We were able to
isolate a shunt product (4) in the 2L scaled-up cultures (original scale: 25 ml) of the atEΔ
(Figure 2-2). We could identify compound 4 after feeding the atXΔ strain with compound
5 (Figure 2-4), suggesting that the accumulation of 4 is likely due to the decomposition of
5. This degradation is probably catalyzed by an unidentified catechol 1, 2-dioxygenase
(Sistrom and Stanier, 1954), the coding gene of which is not located in the terreic acid (1)
cluster. A similar cleavage and degradation of aromatic acids has also been identified in A.
terreus (Milstein et al., 1987). Homology analysis of AtE showed that the putative protein
has 80% similarity in amino acid sequence with CYP619C2 characterized in A. clavatus
(Artigot et al., 2009). CYP619C2 is involved in the biosynthesis of patulin (Figure 2-1A),
another natural product derived from 6-MSA (1). The study showed that CYP619C2 is
capable of catalyzing the p-hydroxylation of both m-cresol to toluquinol and
m-hydroxybenzyl alcohol to gentisyl alcohol (Artigot et al., 2009). Based on the
hydroxylation function of its homolog, we propose that AtE catalyzes the hydroxylation
16
of 5 to 6. Lastly, since the deletion of atC accumulated terremutin (3), we propose that
AtC is required for the oxidation of terremutin (3) to TA (1) (Figure 2-3).

The at cluster contains one gene, atF , that encodes a putative zinc family transcription
factor (Table 2-1). Deletion of atF eliminated the production of only TA (1) but not other
types of secondary metabolites in A. terreus, suggesting that AtF specifically regulates
the expression of genes in the at cluster. The cluster also contains one gene, atB, coding
for a putative major facilitator superfamily (MFS) tansporter (Table 2-1). These MFS
transporter genes, identified in several secondary metabolite gene clusters, have been
shown to be responsible for transporting the metabolites out of its producing organisms.
Tri12, for example, is involved in the transport of trichothecene in Fusarium species
(Alexander et al., 1999). However, there are cases in which deletion of the MFS
transporter gene in the pathway does not lead to significant loss of the metabolites (Lo et
al., 2012a). Production of TA (1) is greatly diminished in the atBΔ strain (Figure 2-2),
indicating that AtB might play a role in the transport of TA (1).

There are two genes (atD and atG) in the cluster to which we could not assign their
functions. Although they are involved in the biosynthesis of TA (1), we could not detect
any intermediates or shunt products accumulated in the mutant strains. A homologous
protein of AtD could be identified in the patulin cluster (PatJ) (Puel et al., 2010), but the
function of both putative enzymes is unknown. Conserved domain analysis of AtG
showed that it contains a cytochrome P450 monooxygenase domain, but no characterized
homolog can be identified in a BLASTp analysis. It is possible that AtG catalyzes one of
the oxidation steps occurred in the conversion of 6 to terremutin (3). Thus for AtD and
AtG, further effort is necessary to elucidate their specific functions in the biosynthesis of
TA (1).
17

In conclusion, we report the identification of a cluster of eight genes that are responsible
for the biosythesis of TA (1). The biosynthetic pathway is proposed based on the
bioinformatic analyses of the involved genes and the chemical analyses of the gene
deletion strains. The cluster contains a key gene atX that codes for a 6-MSAS. BLASTp
analysis of AtX showed that its homologs are wide-spread in Ascomycetes (Table 2-8),
but in most cases, only one 6-msas gene resides in the genome of each single fungus. All
6-MSASs have high similarity in amino acid sequence and domain architecture, strongly
suggesting that they share a common origin. The diversity of 6-MSA (2) derivatives is
enriched due to the tailoring proteins, with specific catalytic activities in each pathway
(Artigot et al., 2009; Holm et al., 2014).

Thus, understanding the function of these
tailoring proteins will expand our knowledge of this important class of natural products.

2.4 Materials and Methods

Strains and molecular manipulations
Primers used in this study are listed in Table 2-2. A. terreus strains used in this study are
listed in Table 2-3. All targeted genes were individually replaced by the A. fumigatus
pyrG gene (AfpyrG) in the A. terreus kusA-, pyrG- strain. The construction of double joint
fusion PCR products, protoplast generation, and transformation were carried out
according to previous procedures (Guo et al., 2012). Diagnostic PCR of the mutant
strains was carried out by using the external primers from the first round of fusion PCR.
The difference in size between the gene replaced by the resistant marker and the native
gene allowed us to determine if the transformants carried correct gene replacement. When
the targeted gene size was similar to the marker AfpyrG, an internal primer that
specifically binds to AfpyrG was used with an outside primer. When the targeted gene
18
was successfully replaced by AfpyrG, a PCR fragment of around 4 kb could be amplified
from the mutant strains (Figure 2-5 and 2-6).

Fermentation and LC-MS analysis
A. terreus NIH 2624 was cultivated at 30 °C on YAG plates at 10 × 10
6
spores per plate
(D = 10 cm). After 6 days, agar was chopped into small pieces and extracted by 80 ml 1:1
CH2Cl2/MeOH. The extract was evaporated in vacuo to yield a water residue, which was
suspended in 25 ml H2O and partitioned with ethyl acetate (EtOAc, 25 ml × 2). The
EtOAc layer was discarded. The pH of the water layer was then adjusted to around 2 by
6M HCl and partitioned with 25ml EtOAc. The EtOAc layer was evaporated in vacuo,
re-dissolved in 1 ml of 20% DMSO in MeOH and a portion (10μl) was examined by high
performance liquid chromatography–photodiode array detection–mass spectrometry
(HPLC–DAD–MS) analysis. HPLC–MS was carried out using a ThermoFinnigan LCQ
Advantage ion trap mass spectrometer with an RP C18 column (Alltech Prevail C18 3
mm 2.1 × 100 mm) at a flow rate of 125 μl/min. The condition for MS analysis was
carried out as previously described (Guo et al., 2012).

Isolation of secondary metabolites
For scale up, A. terreus wild type and knock-outs were cultivated at 30 °C on YAG plates
(D = 15 cm, ~50 ml per plate, total volume = 2 liter) for 6 days. Agar was chopped into
small pieces and then soaked in 2 L of 1:1 CH2Cl2/MeOH for 24 hrs. After filtration, the
crude extract was evaporated in vacuo to yield a residue, which was then suspended in
500 ml water and partitioned with EtOAc (500 ml) three times. The EtOAc layer was
discarded. The pH of the water layer was then adjusted to around 2 and extracted with
EtOAc (500 ml × 2). The combined EtOAc layer was evaporated in vacuo to a crude
extract.
19

Further purification was carried on by gradient HPLC on a C18 reverse phase column
(Phenomenex Luna 5μm C18, 250 × 10 mm) with a flow rate of 5.0 ml/min and
measured by a UV detector at 254 nm. The gradient system was MeCN (solvent B) and 5%
MeCN/H2O (solvent A) both containing 0.05% TFA.

The gradient condition for HPLC analysis of crude extract from WT was 0-2 min 100-80%
A, 2-7 min 80-66% A, 7-27 min 66-54.8% A, 27-29 min 54.8-0% A, 29-31 min 0-100%
A, 31-33 min 100% A. Compound 1 (28 mg/L) was eluted at about 5 min. The gradient
condition for HPLC analysis of crude extracts from ATEG_06272.1Δ and
ATEG_06274.1Δ was 0-5 min 100-80% A, 5-20 min 80-60% A, 20-25 min 60-0% A,
25-30 min 0-100% A, 30-35 min 100% A. Compounds 2 (16 mg/L) and 3 (5 mg/L) were
eluted at 16.0 min and 6.5 min, respectively. The gradient condition for HPLC analysis of
crude extract from ATEG_06277.1Δ was 0-5 min 100-80% A, 5-10 min 80% A, 10-12
min 80-0% A, 12-14 min 0% A, 14-16 min 0-100% A, 16-18 min 100% A. Compound 4
(7.5 mg/L) was eluted at 9.0 min.

Feeding compound 5 to atXΔ
The 3-methylcatechol (5) was bought from Sigma Aldrich. It was dissolved in DMSO
and added to YAG medium when the medium’s temperature dropped to around 50 °C
after autoclaving. The atXΔ mutant was cultivated at 30 °C on YAG containing 0.2 mg/ml
compound 5. The WT strain and the atXΔ mutant were also cultivated on ordinary YAG
as control. After 6 days cultivation, the followed extraction and LC-MS analysis were
similar as described above.

20
Spectral data of compounds
HRESIMS spectra were obtained on an Agilent Technologies 1200 series high-resolution
mass spectrometer. NMR spectra were collected on a Varian Mercury Plus 400
spectrometer. For UV-Vis and ESIMS spectra, see Figure 2-7; For NMR spectra, see
Table 2-4, 2-5, 2-6, 2-7, and Figure 2-8, 2-9, 2-10, 2-11. The molecular formula of
compound 4 was determined by HRESIMS to be C7H7O4 [M - H]
-
(m/z 155.0341, calcd
for C7H7O4 155.0350). The NMR data of compounds 1, 2, 3 were in good agreement with
the published data (Dewi et al., 2012; Findlay and Radics, 1972; Fujii et al., 1996)

21
Table 2-1. The at gene cluster and gene function prediction. The proteins of patJ and patL
are not characterized.
gene cofactors
(putative)
Blast homologs
(% identity)
putative function
72 (atA) FAD/
NAD(P)
salA (27%) 6-MSA
decarboxylase
73 (atB) unknown MFS transporter
74 (atC) FAD VBS (40%) GMC
oxidoreductase
75 (atX) - - 6-MSAS
76 (atD) unknown patJ (67%) Unknown
77 (atE) NADH/
NADPH
patI (64%)
patH (55%)
Cytochrome P450
monooxygenase
78 (atF) unknown patL (38%) Transcription
factor
80 (atG) NADH/
NADPH
unknown Cytochrome P450
monooxygenase

22

Figure 2-1. A. Compounds derived from 6-MSA; B. Intermediates or shunt products
isolated in this study.

23

Figure 2-2. (A) Organization of the 6-MSAS gene cluster involved in TA (1) biosynthesis
in A. terreus. Black open reading frames (ORFs) are involved in TA (1) biosynthesis
while grey ones are not. (B) The DAD traces of extracts from the wildtype and mutants as
detected by UV (200-600 nm). Numbers on peaks correspond to compounds in Figure 1.
*
This metabolite is not related to TA (1) (For more details, see Figure 2-7).
†
This
metabolite is related to TA (1) and its structure is similar to 4 according to UV absorption
spectra but not isolated due to low production yield.

WT
ATEG_06270.1 Δ
ATEG_06271.1 Δ
ATEG_06272.1 Δ
(atA Δ)
ATEG_06273.1 Δ
(atB Δ)
ATEG_06274.1 Δ
(atC Δ)
ATEG_06275.1 Δ
(atX Δ)
ATEG_06276.1 Δ
(atD Δ)
ATEG_06277.1 Δ
(atE Δ)
ATEG_06278.1 Δ
(atF Δ)
ATEG_06279.1 Δ
ATEG_06280.1 Δ
(atG Δ)
ATEG_06281.1 Δ
ATEG_06282.1 Δ
Time (min)
4 6 8 10 12 16 14
Time (min)
4 6 8 10 12 16 14
*
*
*
*
*
1
1
1
1
1
1
2
3
4
†
1
ATEG_0XXXX.1
6270
6271
6273
(atB)
6272
(atA)
6274
(atC)
6275
(atX)
6276
(atD)
6277
(atE)
6278
(atF) 6279
6280
(atG) 6281 6282
5 Kb
(A)
(B)
24

Figure 2-3. Proposed biosynthetic pathway for TA (1). Isolated and structurally
characterized compounds are boxed.

25
Table 2-2. Primers used in this study
primer Sequence (5′→3′)
ATEG_06270.1P1 GGC GAT TGG TCT TTA ATC TG
ATEG_06270.1P2 AGA CAT GGC GTC TAT CTC GT
ATEG_06270.1P3 CGA AGA GGG TGA AGA GCA TTG GGA TCA CAG TAT TCG CCA GT
ATEG_06270.1P4 CAT CAG TGC CTC CTC TCA GAC AGA CAA CAA CCA TCA GGT CGT G
ATEG_06270.1P5 GAT TCG ACG GTG AAG AAC TC
ATEG_06270.1P6 GCC TCT ATG GTC GAA CAA TC
ATEG_06271.1P1 CCA CCA CAG TGT GAA ACC TA
ATEG_06271.1P2 CAA ATG CAA GAC TCC TGT GA
ATEG_06271.1P3 CGA AGA GGG TGA AGA GCA TTG GGG TTG TTA TCA CTG CTG CT
ATEG_06271.1P4 CAT CAG TGC CTC CTC TCA GAC AGG CTA TGG CAG ACT ACA GAA C
ATEG_06271.1P5 GTC ATG ACC TAT CCC ATT GA
ATEG_06271.1P6 CCT TAT ACC GAT GGA TGT CG
ATEG_06272.1P1 CCA GAG TTA CGA CGA TTT C
ATEG_06272.1P2 GTC CTT CTC AAC CTG TGT AT
ATEG_06272.1P3 CGA AGA GGG TGA AGA GCA TTG TAG GTC ACA CAC AAG ACA CT
ATEG_06272.1P4 CAT CAG TGC CTC CTC TCA GAC AGC CAT AAA GCT TCC AGA AGA G
ATEG_06272.1P5 GGG ACA CTA CAC GAC TGT AT
ATEG_06272.1P6 GTA TGT CAC AGG GAA CAA TG
ATEG_06273.1P1 GGT CTC ATT CTC GAC TTT GC
ATEG_06273.1P2 ATT CTC GAC TTT GCT GAG GA
ATEG_06273.1P3 CGA AGA GGG TGA AGA GCA TTG ACA ATG ATA TCG CGA TCC TG
ATEG_06273.1P4 CAT CAG TGC CTC CTC TCA GAC AGC GTT ACG ACG CTC AAG TTA G
ATEG_06273.1P5 GGT ACC AAA CAG CTT CGT CT
ATEG_06273.1P6 CCG AAA GAG GTA CCA AAC AG
ATEG_06274.1P1 TGT ACC TCA TTC GTC CCA TT
ATEG_06274.1P2 CCA TTC CAA TAG GGT TGC TA
ATEG_06274.1P3 CGA AGA GGG TGA AGA GCA TTG TCC CAG TAT GGA GAA CTC GT
ATEG_06274.1P4 CAT CAG TGC CTC CTC TCA GAC AGA ACT TAT GGG GCT TTC TGA
ATEG_06274.1P5 ACA TCT GGT GGG ATG GTT T
ATEG_06274.1P6 ACC CAC ACT GAC CTG GAG TT
ATEG_06275.1P1 CGC CTA TCT CTC CCC TTA GT
ATEG_06275.1P2 CGG ATG TCT AAA AGG AGC AG
ATEG_06275.1P3 CGA AGA GGG TGA AGA GCA TTG ATG CCT GAA GAG GAG AGA TG
ATEG_06275.1P4 CAT CAG TGC CTC CTC TCA GAC AGG CTG ACG GTT TCT CCT TAT G
ATEG_06275.1P5 TTT CTG AGG TGA TGG GCT AC
ATEG_06275.1P6 CTG TTC GAT GAG TGT GGC TA
ATEG_06276.1P1 ATG CCT GAA GAG GAG AGA TG
26
ATEG_06276.1P2 GCG TCG CCT CTA CAT CTT AT
ATEG_06276.1P3 CGA AGA GGG TGA AGA GCA TTG AGG ATG GCC TAA GCA CGT A
ATEG_06276.1P4 CAT CAG TGC CTC CTC TCA GAC AGC CAA ATG GAT TGA GTG TGT G
ATEG_06276.1P5 GAA GTG GCT ATC AGC TTT CG
ATEG_06276.1P6 GTG CCT TGC ATA CAG GAA GT
ATEG_06277.1P1 CCA AAT GGA TTG AGT GTG TG
ATEG_06277.1P2 GTG TGT GTA TGC CTT CGA GA
ATEG_06277.1P3 CGA AGA GGG TGA AGA GCA TTG GTG CCT TGC ATA CAG GAA GT
ATEG_06277.1P4 CAT CAG TGC CTC CTC TCA GAC AGG AGT TGG GTG GAA GAT TGA G
ATEG_06277.1P5 GTC CTC CTG GAA CTG CTC TA
ATEG_06277.1P6 AGG AAA CGT CAG TCC CTT CT
ATEG_06278.1P1 AGA GTC ACC ACA AGC GAC AT
ATEG_06278.1P2 CCG ATT GGT TAA GAA TGG AC
ATEG_06278.1P3 CGA AGA GGG TGA AGA GCA TTG GGC TGG TTG AAC AAT AGC AG
ATEG_06278.1P4 CAT CAG TGC CTC CTC TCA GAC AGG TCG TTC TTA TCG CAA CCA T
ATEG_06278.1P5 CAT CTC CCG AAG CAC TCT AC
ATEG_06278.1P6 GCA AGC TGC CTT ACA TCA AT
ATEG_06279.1P1 TCT GCG TCA GCT ATG GT CTA
ATEG_06279.1P2 ATC CCT GGA AAA CTG TTG G
ATEG_06279.1P3 CGA AGA GGG TGA AGA GCA TTG GCA GTC AAT GAG TTG CAA TG
ATEG_06279.1P4 CAT CAG TGC CTC CTC TCA GAC AGA CGG CTG AAT TAG TTC GTG T
ATEG_06279.1P5 GAC TAG TGA TCG GCA GGA TG
ATEG_06279.1P6 AAG CCG AGG TAA TGA TGC T
ATEG_06280.1P1 TAT CGG CAA AGA CAA GAT CG
ATEG_06280.1P2 GTA CGA GGT CAT GTC TGG TG
ATEG_06280.1P3 CGA AGA GGG TGA AGA GCA TTG CCG CCA ATA CTC TGT CTT TC
ATEG_06280.1P4 CAT CAG TGC CTC CTC TCA GAC AGT AGA CGT GCC AAA CTG TGA C
ATEG_06280.1P5 CAG AGG GTA TCG GTC ATG TT
ATEG_06280.1P6 GAG CGG TGT CCA TTG TTA CT
ATEG_06281.1P1 AGC TCG TTT ACC CCT GTT GG
ATEG_06281.1P2 CAG GCA TCA AAA GCC GAA GG
ATEG_06281.1P3 CGA AGA GGG TGA AGA GCA TTG GTT GCA CAC GTC CTC TCT CA
ATEG_06281.1P4 GCA TCA GTG CCT CCT CTC AGA CAG CTG GTG CTA GCT GGC TTC TT
ATEG_06281.1P5 GGC TCG CCA AGG AGT AAT CA
ATEG_06281.1P6 ACT CTG TAC TCC GTC CCT CC
ATEG_06282.1P1 TGC ACA CCA ACA CTC CTG TA
ATEG_06282.1P2 ACG TAG GAC CAA CCC TGA TAG T
ATEG_06282.1P3 CGA AGA GGG TGA AGA GCA TTG GTG ACT GCG GAT GGA AGT GT
ATEG_06282.1P4 GCA TCA GTG CCT CCT CTC AGA CAG GAA CCA AAC GCT GTC ACT GG
27
ATEG_06282.1P5 CAG CCA TAA ACT CCA CGA AGC
ATEG_06282.1P6 CTC ACA ATC ATC GGC TCC CC
Internal primer used for diagnostic PCR
AfpyrGR CGG GAG CAG CGT AGA TGC C

28
Table 2-3. A. terreus strains used in this study
Fungal strain or
Transformants
Gene mutation(s) Genotype
Aspergillus terreus NIH2624 - wildtype
CW6061.1, CW6061.2, CW6061.3 ATEG_06270.1Δ nkuA:: hph; pyrG-, ATEG_06270.1:: AfpyrG
CW6062.1, CW6062.2 ATEG_06271.1Δ nkuA:: hph; pyrG-, ATEG_06271.1:: AfpyrG
CW6063.1, CW6063.2 ATEG_06272.1Δ nkuA:: hph; pyrG-, ATEG_06272.1:: AfpyrG
CW6064.1, CW6064.2, CW6064.3 ATEG_06273.1Δ nkuA:: hph; pyrG-, ATEG_06273.1:: AfpyrG
CW6065.1, CW6065.2, CW6065.3 ATEG_06274.1Δ nkuA:: hph; pyrG-, ATEG_06274.1:: AfpyrG
CW6066.1, CW6066.2 ATEG_06275.1Δ nkuA:: hph; pyrG-, ATEG_06275.1:: AfpyrG
CW6067.1, CW6067.2, CW6067.3 ATEG_06276.1Δ nkuA:: hph; pyrG-, ATEG_06276.1:: AfpyrG
CW6068.1, CW6068.2, CW6068.3 ATEG_06277.1Δ nkuA:: hph; pyrG-, ATEG_06277.1:: AfpyrG
CW6069.1, CW6069.2, CW6069.3 ATEG_06278.1Δ nkuA:: hph; pyrG-, ATEG_06278.1:: AfpyrG
CW6070.2, CW6070.2, CW6070.3 ATEG_06279.1Δ nkuA:: hph; pyrG-, ATEG_06279.1:: AfpyrG
CW6071.1, CW6071.2, CW6071.3 ATEG_06280.1Δ nkuA:: hph; pyrG-, ATEG_06280.1:: AfpyrG
CW6072.1, CW6072.2 ATEG_06281.1Δ nkuA:: hph; pyrG-, ATEG_06281.1:: AfpyrG
CW6073.1, CW6073.2, CW6073.3 ATEG_06282.1Δ nkuA:: hph; pyrG-, ATEG_06282.1:: AfpyrG

29

Table 2-4.
1
H and
13
C NMR data for compound 1.
(400 MHz and 100 MHz in acetone-d6)
Position δ H (J in Hz) δ C
1 188.7, C
2 119.9, C
3 154.4, C
4 192.2, C
5 3.97, d (4.0) 52.9, CH
6 3.87, d (4.0) 54.7, CH
7 1.83, s 8.7, CH3

Table 2-5.
1
H and
13
C NMR data for compound 2.
(400 MHz and 100 MHz in acetone-d6)
Position δ H (J in Hz) δ C
1 113.2, C
2 164.2, C
3 6.73, d (7.6) 116.1, CH
4 7.29, t (8.0) 135.0, CH
5 6.78, d (8.8) 123.5, CH
6 142.6, C
7 2.56, s 24.0, CH3
8 174.2, C
8-OH 11.2, br s

30

Table 2-6.
1
H and
13
C NMR data for compound 3
(400 MHz and 100 MHz in acetone-d6)
Position δ H (J in Hz) δ C
1 Not Identified
2 108.9, C
3 164.1, C
4 4.63, s 66.0, CH
5 3.66, dd (3.2, 1.2) 52.3, CH
6 3.37, dd (3.6, 1.2) 55.3, CH
7 1.64, s 7.5, CH3

Table 2-7.
1
H and
13
C NMR data for compound 4.
(400 MHz and 100 MHz in acetone-d6)
Position δ H (J in Hz) δ C HMBC
a
COSY NOESY
1 168.4, C
2 136.1, C
3 6.67, d (12.0) 136.3, CH 1, 5, 7 H-4, H-5 H-4, H3-7
4 8.22, dd (15.2, 11.6) 141.5, CH 3, 5, 6 H-3, H-5 H-3, H-5
5 6.06, d (15.6) 126.6, CH 2, 3, 4, 6 H-3, H-5 H-4
6 168.0, C
7 2.10, s 21.4, CH3 1, 2, 3 H-3
a
HMBC correlations are from proton(s) to the indicated carbon.

31
Table 2-8. AtX (ATEG_06275.1) homologs with sequence identity over 40%.
Seq ID Species Amino
acids
Identity/Sim
ilarity (%)
gb|AAK48943.1|AF3603
98_1
Byssochlamys nivea 1778 64/77
gb|ADF47133.1| Penicillium griseofulvum 1774 63/75
ref|XP_002564832.1| Penicillium chrysogenum
Wisconsin 54-1255
1776 63/75
gb|EOD48890.1| Neofusicoccum parvum
UCRNP2
1796 63/75
ref|XP_001273093.1| Aspergillus clavatus NRRL 1 1720 64/76
emb|CDM36380.1| Penicillium roqueforti 1776 62/76
gb|EKG18983.1| Macrophomina phaseolina
MS6
1754 63/75
gb|AAC23536.1| Aspergillus parasiticus 1766 60/75
gb|EIT77765.1| Aspergillus oryzae 3.042 1766 60/75
dbj|BAE65442.1| Aspergillus oryzae RIB40 1766 60/74
gb|EOD47055.1| Neofusicoccum parvum
UCRNP2
1779 57/73
gb|AFP89390.1| Cladosporium phlei 1792 57/72
ref|XP_002849666.1| Arthroderma otae CBS 113480 1773 56/72
gb|ERF77015.1| Endocarpon pusillum Z07020 1801 56/71
ref|XP_003015543.1| Arthroderma benhamiae CBS
112371
1776 55/71
ref|XP_003169413.1| Arthroderma gypseum CBS
118893
1776 55/71
ref|XP_003019112.1| Trichophyton verrucosum HKI
0517
1747 55/71
ref|XP_003233709.1| Trichophyton rubrum CBS
118892
1774 55/71
gb|EKG12695.1| Macrophomina phaseolina
MS6
1738 55/71
gb|AAX35547.1| Glarea lozoyensis 1791 55/70
gb|EYE91246.1| Aspergillus ruber CBS 135680 1782 55/70
gb|ETS75984.1| Pestalotiopsis fici W106-1 1796 54/71
ref|XP_001791162.1| Phaeosphaeria nodorum SN15 1720 54/71
gb|EGX43547.1| Arthrobotrys oligospora ATCC
24927
1761 53/69
32
gb|AAS98200.1| Aspergillus ochraceus 1766 50/67
ref|XP_001826575.2| Aspergillus oryzae RIB40 1701 59/74
ref|XP_001402408.2| Aspergillus niger CBS 513.88 1787 48/64
gb|EHA22196.1| Aspergillus niger ATCC 1015 1779 48/64
ref|XP_002560460.1| Penicillium chrysogenum
Wisconsin 54-1255
1783 47/63
gb|AAB49684.1| Penicillium griseofulvum 1783 47/64
gb|EKV11531.1| Penicillium digitatum PHI26 1785 47/63
gb|EGD94108.1| Trichophyton tonsurans CBS
112818
1685 55/72
gb|EGE06642.1| Trichophyton equinum CBS
127.97
1579 53/71
gb|AAR90279.1| Bipolaris maydis 1766 41/59
ref|WP_003879650.1| Mycobacterium fortuitum 1723 42/58
ref|YP_001071753.1| Mycobacterium sp. JLS 1704 43/57
ref|YP_640635.1| Mycobacterium sp. MCS 1704 42/57
ref|WP_003928522.1| Mycobacterium vaccae 1712 42/57
emb|CDO33136.1| Mycobacterium vulneris 1716 42/57
ref|YP_004522781.1| Mycobacterium sp. JDM601 1718 42/58
gb|ACN64831.1| Streptomyces
diastatochromogenes
1739 43/59
ref|WP_005628551.1| Mycobacterium hassiacum 1712 42/57
ref|YP_004076616.1| Mycobacterium gilvum Spyr1 1735 42/57
ref|YP_954639.1| Mycobacterium vanbaalenii
PYR-1
1744 43/57
ref|YP_001133955.1| Mycobacterium gilvum
PYR-GCK
1698 42/57
ref|YP_008908204.1| Mycobacterium neoaurum
VKM Ac-1815D
1721 42/57
ref|YP_005002033.1| Mycobacterium rhodesiae
NBB3
1725 41/57
ref|WP_003926867.1| Mycobacterium
thermoresistibile
1758 41/56
ref|WP_003086619.1| Amycolatopsis vancoresmycina 1742 42/57
gb|AAZ77673.1| Streptomyces antibioticus 1756 41/56

33

Figure 2-4. (A) The DAD traces of extracts from the wildtype, atXΔ and atXΔ fed with
3-methylcatechol (5) as detected by UV (200-600 nm). The peak labeled with “*”
corresponds to the compound labeled with “*” in Figure 2-2. (B) EIC extraction of 1, 4
and 5 (m/z=153, 155 and 123 [M-H]
-
) from the negative ESI-MS trace in the profiles of
the wildtype, atXΔ and atXΔ fed with 3-methylcatechol (5). Feeding the atXΔ mutant
with 0.2 mg/ml 3-methylcatechol (5) recovers TA (1) production and accumulates 4. This
suggests that 3-methylcatechol (5) is an intermediate of TA (1) and compound 5 could be
degraded to 4. The retention time of 3-methylcatechol (5) is 12.83 min, but it was not
detected probably due to complete decomposition.
Time (min)
4 6 8 10 12 16 14
1
1
*
4
WT
ATEG_06275.1 Δ
(atX Δ)
ATEG_06275.1 Δ
(atX Δ)+5
Time (min)
4 6 8 10 12 16 14
WT
ATEG_06275.1 Δ
(atX Δ)
ATEG_06275.1 Δ
(atX Δ)+5
1
4
1
(A) DAD traces (B) EIC extraction of 1, 4 and 5
34

Figure 2-5. Schematic of the diagnostic PCR strategy.
We used two redundant strategies to determine if the target gene had been deleted by
replacement with AfpyrG. In one strategy, DNA from transformants is amplified with two
primers, P1 from the chromosomal region just outside of the 5’ flank of the transforming
DNA fragment and P6 from just outside of the 3’ flank. If the target gene is different in
size from the AfpyrG gene, which was used as a selectable marker for transformation, the
PCR fragment amplified from a correct transformant (a) will be different in size from the
fragment amplified if the target gene is intact (b). In some instances the target gene and
the AfpyrG cassette will be of comparable size and a second strategy is applied. In the
second strategy, P1 or P6 are used with internal primers specific to the AfpyrG cassette.
For example, if the target gene has been replaced by the AfpyrG gene (c), P1 and
AfpyrGR will amplify a fragment of a predictable size. If the target gene has not been
replaced (d), the AfpyrGR primer will not anneal and there will be no specific
amplification.
35

Figure 2-6. Results of diagnostic PCR for all the gene deletion strains.
ATEG_06270.1
F1+R6: WT=2998 bp; KO=4066 bp
F1+R6

ATEG_06273.1
F1+R6: WT=4236 bp; KO=3983 bp
F1+R6

ATEG_06276.1
F1+R6: WT=3490 bp; KO=3983 bp
F1+R6

ATEG_06278.1
F1+PyrG-R: WT=blank; KO=2905 bp
F1+PyrG-R

ATEG_06271.1
F1+R6: WT=3021 bp; KO=3936 bp
F1+R6

ATEG_06273.1
F1+PyrG-R: WT=blank; KO=2944 bp
F1+PyrG-R

ATEG_06277.1
F1+R6: WT=3669 bp; KO=3960 bp
F1+R6

ATEG_06279.1
F1+R6: WT=3978 bp; KO=39262bp
F1+R6

ATEG_06272.1
F1+R6: WT=3591 bp; KO=3964 bp
F1+R6

ATEG_06274.1
F1+R6: WT=4423 bp; KO=4007 bp
F1+R6

ATEG_06277.1
F1+PyrG-R: WT=blank; KO=2916 bp
F1+PyrG-R

ATEG_06279.1
F1+PyrG-R: WT=blank; KO=2943 bp
F1+PyrG-R

ATEG_06272.1
F1+PyrG-R: WT=blank; KO=2913 bp
F1+PyrG-R

ATEG_06275.1
F1+R6: WT=7646 bp; KO=3959 bp
F1+R6

ATEG_06278.1
F1+R6: WT=4102 bp; KO=3924 bp
F1+R6

ATEG_06280.1
F1+R6: WT=2653 bp; KO=3998 bp
F1+R6

WT
6061.1
6061.2
6061.3
WT
6062.1
6062.2
WT
6063.1
6063.2
WT
6063.1
6063.2
WT
6064.1
6064.2
6064.3
WT
6064.1
6064.2
6064.3
WT
6065.1
6065.2
6065.3
WT
6066.1
6066.2
6066.3
WT
6067.1
6067.2
6067.3
WT
6068.1
6068.2
6068.3
WT
6068.1
6068.2
6068.3
WT
6069.1
6069.2
6069.3
WT
6069.1
6069.2
6069.3
WT
6070.1
6070.2
6070.3
WT
6070.1
6070.2
6070.3
WT
6071.1
6071.2
6071.3
ATEG_06281.1
F1+R6: WT= 3882bp; KO=4031 bp
F1+R6

ATEG_06282.1
F1+PyrG-R: WT=blank; KO=2887 bp
F1+PyrG-R

ATEG_06281.1
F1+PyrG-R: WT=blank; KO=2960 bp
F1+PyrG-R

ATEG_06283.1
F1+R6: WT=3254 bp; KO=3940 bp
F1+R6

ATEG_06282.1
F1+R6: WT=4078 bp; KO=3958 bp
F1+R6

ATEG_06282.1
F1+PyrG-R: WT=blank; KO=2887 bp
F1+PyrG-R

WT
KO_A
KO_B
KO_C
WT
KO_A
KO_B
KO_C
WT
6072.1
6072.2
6072.3
WT
6072.1
6072.2
6072.3
WT
6073.1
6073.2
6073.3
WT
6073.1
6073.2
6073.3
36

Figure 2-7. UV-Vis and ESIMS spectra of compounds 1 to 4 and unrelated compound (*).
The molecular formula of the compound labeled with “*” was determined by HRESIMS
to be C12H16NO5 [M - H]
-
(m/z 254.1035, calcd for C12H16NO5 254.1034). The compound
labeled with “*” is not terreic acid (1) pathway related based on two evidence: (1). It can
be detected in the atXΔ strain; (2). The HRESIMS predicted molecular formula suggests
that the compound contains one nitrogen, but there is no nitrogen-bearing group
identified in terreic acid (1) or any of the proposed intermediates in its pathway.

2 0 0 3 0 0 4 0 0 50 0 6 0 0
w aveleng th ( nm)
0
2 0
4 0
6 0
8 0
10 0
33 4 .0 0
2 3 7.0 0
2 0 0 3 0 0 4 0 0 50 0 6 0 0
w aveleng th ( nm)
0
2 0
4 0
6 0
8 0
10 0
24 2 .0 0
32 0 .0 0
2 0 0 3 0 0 4 0 0 50 0 6 0 0
m/z
0
2 0
4 0
6 0
8 0
10 0
[M-H ]ˉ
151.12
2 0 0 3 0 0 4 0 0 50 0 6 0 0
w aveleng th ( nm)
0
2 0
4 0
6 0
8 0
10 0
2 57.0 0
2 0 0 3 0 0 4 0 0 50 0 6 0 0
m/z
2 0
4 0
6 0
8 0
10 0
[M-H ]ˉ
155.12
2 0 0 3 0 0 4 0 0 50 0 6 0 0
w aveleng th ( nm)
0
2 0
4 0
6 0
8 0
10 0
2 53 .0 0
10 0 2 0 0 3 0 0 4 0 0 50 0 6 0 0
m/z
0
2 0
4 0
6 0
8 0
10 0
[M-H ]ˉ
155.0 0
2 0 0 3 0 0 4 0 0 50 0 6 0 0
w aveleng th ( nm)
0
2 0
4 0
6 0
8 0
10 0
33 0 .0 0
2 3 5.0 0
10 0 2 0 0 3 0 0 4 0 0 50 0 6 0 0
m/z
0
2 0
4 0
6 0
8 0
10 0
[M-H ]ˉ
2 53 .8 9
O
HO
O
O
OH O
OH
O
HO
OH
O
Compound labeled with “*”
Terreic acid (1)
6-methylsalicylic
acid (2)
Terremutin (3)
10 0
10 0
2 0 0 3 0 0 4 0 0 50 0 6 0 0
m/z
0
2 0
4 0
6 0
8 0
10 0
[M-H ]ˉ
153.10
10 0
37

a.
1
H NMR spectrum of TA 1, acetone-d6: 2.05 (m) ppm

b.
13
C NMR s pectrum of TA 1, acetone-d6: 206.6 (s), 29.92 (m) ppm
Figure 2-8.
1
H NMR and
13
C NMR spectrum of TA 1.
1.00
1.00
2.97
3.956
3.966
3.869
3.860
1.824
1.826
ppm 0 1 2 3 4 5 6 7 8 9

38

a.
1
H NMR spectrum of compound 2, acetone-d6: 2.05 (m) ppm

b.
13
C NMR spectrum of compound 2, acetone-d6: 207.1 (s), 29.92 (m) ppm
Figure 2-9.
1
H NMR and
13
C NMR spectrum of compound 2.
207.129
174.181
164.159
142.590
135.060
123.452
116.082
113.170
23.991
29.531
29.730
29.920
30.111
30.301
ppm 0 20 40 60 80 100 120 140 160 180 200 220
20131125_6272_HCL_YAG_cpd4
Sample Name:
20131125_6272_HCL_YAG_cpd4
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
20131125_6272_HCL_YAG_cpd4_20131125_01
FidFile: CARBON_01
Pulse Sequence: CARBON (s2pul)
Solvent: acetone
Data collected on: Nov 25 2013

1.45
1.00
2.13
3.43
11.153
7.286
7.306
7.266
6.786
6.764
6.738
6.719
2.560
2.085
2.083
2.072
2.070
2.067
2.062
2.059
2.056
2.053
2.050
2.045
2.039
ppm 0 2 4 6 8 10 12
20131125_6272_HCL_YAG_cpd4
Sample Name:
20131125_6272_HCL_YAG_cpd4
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
20131125_6272_HCL_YAG_cpd4_20131125_01
FidFile: PROTON_02
Pulse Sequence: PROTON (s2pul)
Solvent: acetone
Data collected on: Nov 25 2013

39

a.
1
H NMR spectrum of compound 3, acetone-d6: 2.05 (m) ppm

b.
13
C NMR spectrum of compound 3, acetone-d6: 207.2 (s), 29.92 (m) ppm
Figure 2-10.
1
H NMR and
13
C NMR spectrum of compound 3.
207.251
108.864
65.955
55.277
52.266
29.920
30.118
30.309
30.499
30.674
29.729
29.539
29.341
7.490
7.833
ppm 0 20 40 60 80 100 120 140 160 180 200 220
6274-Cpd5_170mg
Sample Name:
6274-Cpd5_170mg
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
6274-Cpd5_170mg_20140125_01
FidFile: CARBON_02
Pulse Sequence: CARBON (s2pul)
Solvent: acetone
Data collected on: Jan 25 2014

4.633
2.086
2.762
3.362
3.364
3.370
3.373
3.651
3.654
3.659
3.662
4.046
2.056
2.050
2.045
1.715
1.706
1.702
1.674
1.657
1.646
1.641
1.632
1.618
1.587
-0.024
ppm 0 1 2 3 4 5 6 7 8
6274-Cpd5_170mg
Sample Name:
6274-Cpd5_170mg
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
6274-Cpd5_170mg_20140125_01
FidFile: PROTON_01
Pulse Sequence: PROTON (s2pul)
Solvent: acetone
Data collected on: Jan 25 2014

40

a.
1
H NMR spectrum of compound 4, acetone-d6: 2.05 (m) ppm

b.
13
C NMR spectrum of compound 4, acetone-d6: 207.1 (s), 29.92 (m) ppm

0.83
1.00
1.10
1.81
8.228
8.257
8.219
8.190
6.683
6.653
6.078
6.039
2.092
2.096
3.317
2.084
2.077
2.070
2.066
2.060
2.050
1.971
1.967
1.850
1.847
1.195
ppm 0 2 4 6 8 10 12
20131122_6277_cpd3_ace
Sample Name:
20131122_6277_cpd3_ace
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
20131122_6277_cpd3_ace_20131122_01
FidFile: PROTON_02
Pulse Sequence: PROTON (s2pul)
Solvent: acetone
Data collected on: Nov 22 2013

206.542
206.206
167.977
168.434
141.522
136.309
136.149
126.637
29.920
30.110
30.164
30.301
30.362
30.499
30.667
29.729
29.531
29.340
21.444
ppm 0 20 40 60 80 100 120 140 160 180 200 220
20131122_6277_cpd3_ace
Sample Name:
20131122_6277_cpd3_ace
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
20131122_6277_cpd3_ace_20131122_01
FidFile: CARBON_01
Pulse Sequence: CARBON (s2pul)
Solvent: acetone
Data collected on: Nov 22 2013

41
F2 (ppm)
1 2 3 4 5 6 7 8
F1
(ppm)
20
40
60
80
100
120
140
160
180
200
F2 (ppm)
0 1 2 3 4 5 6 7 8
F1
(ppm)
20
40
60
80
100
120
140

c. gHMBC spectrum of compound 4

d. gHMQC spectrum of compound 4
42
F2 (ppm)
1 2 3 4 5 6 7 8
F1
(ppm)
2
3
4
5
6
7
8
F2 (ppm)
1 2 3 4 5 6 7 8
F1
(ppm)
2
3
4
5
6
7
8

e. gCOSY spectrum of compound 4

f. NOESY spectrum of compound 4
Figure 2-11.
1
H NMR,
13
C NMR, gHMBC, gHMQC, gCOSY , NOESY spectrum of 4.
43
CHAPTER III: Characterization of the product of a
nonribosomal peptide synthetase-like (NRPS-like) gene using
the doxycycline dependent Tet-on system in Aspergillus terreus

3.1 Abstract

Genome sequencing of the fungus Aspergillus terreus uncovered a number of silent core
structural biosynthetic genes encoding enzymes presumed to be involved in the
production of cryptic secondary metabolites. There are five nonribosomal peptide
synthetase (NRPS)-like genes with the predicted A-T-TE domain architecture within the
A. terreus genome. Among the five genes, only the product of pgnA remains unknown.
The Tet-on system is an inducible, tunable and metabolism-independent expression
system originally developed for Aspergillus niger. Here we report the adoption of the
Tet-on system as an effective gene activation tool in A. terreus. Application of this system
in A. terreus allowed us to uncover the product of the cryptic NRPS-like gene, pgnA.
Furthermore expression of pgnA in the heterologous A. nidulans host suggested that the
pgnA gene alone is necessary for phenguignardic acid (1) biosynthesis.

3.2 Introduction

Filamentous fungi are important sources of secondary metabolites (SMs) that have
potential for pharmaceutical and agricultural purposes. Recent whole genome-sequencing
projects have revealed that the number of core structural biosynthetic genes for putative
SMs far exceeds the number of isolated natural products, indicating that most fungal SM
biosynthetic pathways are silent under normal laboratory culture conditions (Chiang et al.,
2009a). Several approaches as mentioned in 1.3, have been developed to activate these
44
silent biosynthetic pathways using primarily the model organism Aspergillus nidulans.
Many of these approaches, such as overexpressing cluster-specific regulatory activators,
manipulation of the global regulator laeA expression, have hinged on the use of the
inducible alcA promoter system that encodes the alcohol dehydrogenase I (ADHI) gene
which is positively regulated by the alcR gene (Ahuja et al., 2012b; Bergmann et al.,
2007; Bok et al., 2009; Chiang et al., 2010; Chiang et al., 2009b; Waring et al., 1989; Yeh
et al., 2012b).

Among the filamentous fungi, Aspergillus terreus, the main producer of lovastatin,
represents a potential reservoir of cryptic SMs. Several labs, including our own, have
been interested in mining the A. terreus genome for novel SMs (Awakawa et al., 2009;
Boruta and Bizukojc, 2014b; Chiang et al., 2013a; Guo et al., 2015; Guo et al., 2012; Guo
et al., 2014; Guo et al., 2013b; Qiao et al., 2011; Wang et al., 2014b; Xu et al., 2013;
Zaehle et al., 2014). Several attempts using the constitutive gpdA promoter (Gressler et
al., 2011b) and/or the inducible alcA promoter to drive gene expression, have been
evaluated in the effort to turn on silent biosynthetic pathways present in A. terreus.
However, these attempts have not succeeded as envisioned, either because gpdA
activation is insufficient to yield a significant amount of the product although the target
genes were overexpressed (Gressler et al., 2011b), or due to the absence of the ethanol
catabolism regulatory gene alcR in A. terreus as predicted by bioinformatic analyses
(Flipphi et al., 2009).

The Tet-on system is an inducible, tunable and metabolism-independent expression
system functional in filamentous fungi of the genus Aspergillus, including A. niger, A.
fumigatus and A. nidulans (Dümig and Krappmann, 2015; Kalb et al., 2015; Kwon et al.,
2014; Macheleidt et al., 2015; Meyer et al., 2011; Ouedraogo et al., 2011; Richter et al.,
2014; Samantaray et al., 2013; Wartenberg et al., 2012). It was established by
45
constitutively expressing a doxycycline-dependent transcriptional activator rtTA under
the gpdA promoter and fusing the target gene behind a minimal promoter sequence (Pmin)
preceded by several operator (tetO7) sites that rtTA would bind to (Meyer et al., 2011)
(Figure 3-1). We were interested in adopting and employing the Tet-on system in A.
terreus as an important tool to mine its secondary metabolome. There are five
nonribosomal peptide synthetase (NRPS)-like genes with predicted A-T-TE domain
architecture within A. terreus genome, including pgnA, atmelA, atqA, apvA and btyA
(Guo and Wang, 2014). The difference between NRPS and NRPS-like genes is shown in
Figure 3-7A. Among the five genes, only the product of pgnA is unknown at the start of
this study, while the other four genes have been linked to their secondary metabolites
(Guo et al., 2015; Guo et al., 2013a). Here we report our efforts on activating the silent
NRPS-like gene ATEG_08899.1 (pgnA) via the Tet-on system. Application of this system
to activate pgnA allowed us to discover its product, phenguignardic acid (1, Figure 3-1), a
phytotoxic secondary metabolite produced by Guignardia bidwellii the causal agent of
black rot in grapes (Molitor et al., 2012). Additional heterologous expression of pgnA in
A. nidulans confirmed that the single gene pgnA is necessary to produce phenguignardic
acid.

3.3 Results and Discussion

Activation of the pgnA gene using an inducible Ptet-on stimulates the production of
phenguignardic acid.
Previous data have shown A. terreus lacks the alcR gene, therefore an alternative to the
alcA induction system needed to be developed (Flipphi et al., 2009). For this study, we
adopted the Tet-on system developed for A. niger to A. terreus for promoter replacement.
Our analysis identified five NRPS-like genes with A-T-TE domain architecture in A.
terreus. Since the products of the other four NRPS-like genes in A. terreus were known,
46
we focused on replacing the promoter of the pgnA gene (ATEG_08899.1), a gene whose
product was still unknown. We replaced the native promoter of pgnA with the Ptet-on
promoter cloned from an A. niger plasmid (Meyer et al., 2011). The replacement was
accomplished by using a kusA-, pyrG- mutant strain of A. terreus and fusion PCR. All
strains were verified by diagnostic PCR (Figure 3-6). We cultivated two separate
promoter exchange strains and a control strain in LCMM media and used doxycycline
(Dox) as an inducer. Metabolite profiles from each strain were analyzed by LC-DAD-MS,
and the Ptet-on_pgnA strains with Dox induction were able to produce a new metabolite
with m/z of 309 [M-H]
-
(compound 1, Figure 3-2A and Figure 3-3). The compound was
isolated from scaled-up cultivation of the Ptet-on_pgnA strain by semi-preparative HPLC
purification. The molecular formula was established to be C18H14O5 by its
13
C NMR,
DEPT and HRESIMS spectral data, and by comparison to the published NMR spectra of
phenguignardic acid (Molitor et al., 2012). The compound also exhibited an optical
rotation of [α]
21
D
= -53 (c = 0.4, CH2Cl2), indicating it is (-)-phenguignardic acid ((R)-1,
Figure 3-1) (Stoye et al., 2013).

Heterologous expression of pgnA in the heterologous host A. nidulans LO4389
demonstrates that only one gene is necessary for biosynthesis of compound 1.
Our data so far suggested that turning on the expression of a single gene pgnA in A.
terreus was necessary to elicit the compound 1 production. To confirm that indeed
overexpression of only one single gene pgnA without any other accessory genes in A.
terreus is sufficient for compound 1 biosynthesis, we expressed pgnA in a heterologous
host, A. nidulans LO4389 (Ahuja et al., 2012b). We have deleted the biosynthesis clusters
of the major UV active metabolites sterigmatocystin from the selected strain of A.
nidulans. The pgnA gene was fused with the alcA promoter by fusion PCR and
transformed into A. nidulans using our standard procedure (Figure 3-5, Chiang et al.,
2013). We cultivated the OE:pgnA A. nidulans strain in lactose minimal media (LMM)
47
and as a control we also cultivated the parental A. nidulans strain. We induced the alcA
promoter using 50 μg/ml cyclopentanone as inducer. Using HPLC-DAD-MS we detected
compound 1 in the OE:pgnA A. nidulans and not the A. nidulans parental control strain
demonstrating that only pgnA is necessary for compound 1 biosynthesis (Figure 3-2B).

The A. terreus genome contains fourteen NRPS-like genes and the SM products for the
majority of them are unknown (Khaldi et al., 2010). Five among the fourteen NRPS-like
genes have the predicted A-T-TE domain architectures, missing the condensation (C)
domain compared to typical NRPSs (Figure 3-7) (Fischbach and Walsh, 2006a; Guo and
Wang, 2014). The adenylation (A) domain recognizes and activates the aryl acid
substrates, and loads them onto the thiolation (T) domain. The thioesterase (TE) domain
shares the missing condensation (C) domain function, and is responsible for condensation
and final product release (Balibar et al., 2007b). The alignment of PgnA amino acid
sequence with other NRPS-like proteins is shown in Figure 3-8. BLASTP analysis of the
protein sequence of pgnA shows 56% similarity with TdiA, a characterized NRPS-like
protein involved in terrequinone biosynthesis. In the biosynthesis of the terrequinone core,
the A domain of TdiA is shown to activate and load indole-pyruvic acid as its substrate
(Balibar et al., 2007b). Based on the function of TdiA, we propose a speculative
biosynthetic pathway for 1 in Figure 3-4. It involves the activation of phenylpyruvic acid
(PPA), a precursor available in both A. terreus and A. nidulans, by the PgnA A domain to
AMP-PPA followed by loading the PPA unit to the T domain and eventually transferring
to the TE domain. Another PPA unit is loaded onto the T domain. The TE domain likely
promotes the enolate formation on the attached unit, followed by a nucleophilic attack on
the carbonyl to yield an ether linkage between the two units. Then the TE domain likely
catalyzes a similar reaction to give the cyclized dioxolanone core and releases compound
1 (Figure 3-4).

48
With the elucidation of the product of pgnA by utilizing the Tet-on system, product
characterization of all five NRPS-like genes with A-T-TE domain architecture in A.
terreus has been completed. Both activation of the single pgnA gene in native A. terreus
and the heterologous expression in A. nidulans led to the identical product, indicating that
indeed the single pgnA gene is necessary for compound 1 production. The identification
and structural characterization of phenguignardic acid (1) has demonstrated the
effectiveness of the Tet-on system for activating silent genes to discover natural products
in A. terreus. Once the genome information is accessible for Guignardia bidwellii, the
sequence of PgnA could facilitate the mining of the gene responsible for phenguignardic
acid biosynthesis in Guignardia bidwellii.

In conclusion, we report here the adoption of the Tet-on system as an effective gene
activation tool in A. terreus. Application of the Tet-on system in A. terreus allowed us to
discover the product of the NRPS-like gene, pgnA. With the heterologous expression of
pgnA in A. nidulans, we have demonstrated that pgnA gene alone is necessary for
phenguignardic acid (1) biosynthesis in A. terreus.

3.4 Materials and Methods

Strains and molecular genetic manipulations.
The plasmid pVG4.1 containing the Tet-on system was kindly provided by Dr. Vera
Meyer. The Tet-on system (Ptet-on) was amplified from pVG4.1 by PCR with primers F4
and R5 (Meyer et al., 2011). Replacement of endogenous promoters with Ptet-on was
carried out as shown in Figure 3-5. Primers used in this study are listed in Table 3-2. The
fungal strains used in this study are listed in Table 3-3. The scheme for heterologous
expression of the pgnA gene of A. terreus in A. nidulans strain LO4389 is shown in
Figure 3-5. The construction of fusion PCR products, protoplast generation, and
49
transformation were carried out as previously described (see 2.4). The scheme of
diagnostic PCR is shown in Figure 3-6A, and all transformants were verified by
diagnostic PCR.

Fermentation and LC-MS analysis.
A. terreus NIH2624 wild type strain and Ptet-on_pgnA mutant strains were cultivated at
37 °C in 25 ml LCMM liquid medium (6 g/l NaNO3, 0.52 g/l KCl, 0.52 g/l MgSO4.7H2O,
1.52 g/l KH2PO4, 20 g/l lactose, 10 g/l D-glucose supplemented with 1 ml/l of trace
element solution (EDTA disodium salt 50 g/l, ZnSO4·7H2O 22 g/l, H3BO3 11.4 g/l,
MnCl2·4H2O 5.06 g/l, CoCl2·6H2O 1.61 g/l, CuSO4·5H2O 1.57 g/l, (NH4)6Mo7O24·4H2O
1.1 g/l, FeSO4·7H2O 4.99 g/l)) at 1 × 10
6
spore/ml per 125 ml flask with shaking at 180
rpm. After 18 h of incubation, 25 μl sterile doxycycline (50 μg/μl) was added into the
culture of the Ptet-on_pgnA mutant strains, making the final concentration of doxycycline
at 50 μg/ml. The culture of A. terreus NIH2624 wild type strain and Ptet-on_pgnA mutant
strains without adding doxycycline were used as controls. Then the incubator temperature
was lowered to 30 °C and the culture medium was collected after 72 h. The medium was
filtered and extracted with equal volume of ethyl acetate (EtOAc). Then the pH of the
medium was adjusted to around 2 by addition of 6M HCl and extracted with equal
volume of EtOAc again. See 2.4 for LC-MS sample preparation and analysis.

A. nidulans strain LO4389 and the pgnA heterologous expression strains were cultivated
at 37 °C in 50 ml LMM liquid medium (6 g/l NaNO3, 0.52 g/l KCl, 0.52 g/l
MgSO4.7H2O, 1.52 g/l KH2PO4, 20 g/l lactose supplemented with 1 ml/l of trace element
solution) at 1 × 10
6
spore/ml per 125 ml flask with shaking at 180 rpm. The nutrients
pyridoxine and riboflavin were supplemented. After 18 h of incubation, 44 μl
cyclopentanone was added into the medium to make final concentration at 10 mM for
alcA induction as previously reported (Chiang et al., 2013a). Then the incubator
50
temperature was lowered to 30 °C and the culture medium was collected 72 h after
cyclopentanone induction. The medium was filtrated and extracted with equal volume of
EtOAc. See 2.4 for LC-MS sample preparation and analysis.

Isolation of secondary metabolites.
For structure elucidation, the Ptet-on_pgnA mutant strain was cultivated at 37 °C in a
total of 1.5 liter LCMM liquid medium (50 ml per 125 ml flask) at 1 × 10
6
spore/ml with
shaking at 180 rpm. To induce expression of the pgnA gene, sterile doxycycline at a final
concentration of 50 μg/μl was added into each flask after 18 h of incubation. Then the
incubator temperature was lowered to 30 °C and the culture medium was collected after
72 h induction. The medium was filtered and extracted with equal volume of ethyl acetate
(EtOAc). Then the pH of the medium was adjusted to around 2 by addition of 6M HCl
and extracted with equal volume of EtOAc again. The EtOAc layers were combined and
evaporated to a crude extract. Further purification was carried on by gradient HPLC on a
C18 reverse phase column [Phenomenex Luna 5μm C18, 250 × 10 mm] with a flow rate
of 5.0 ml/min and measured by a UV detector at 304 nm. The gradient system was MeCN
(solvent B) and 5% MeCN/H2O (solvent A) both containing 0.05% TFA. The gradient
condition for HPLC analysis of crude extract from the Ptet-on_pgnA mutant strain was
0-5 min 100-80% A, 5-35 min 80-40% A, 35-38 min 40-0% A, 38-40 min 0-0% A, 40-42
min 0-100% A. Compound 1 (2.1 mg/L) was eluted at about 32.5 min.

Compound identification.
NMR spectra were collected on a Varian Mercury Plus 400 spectrometer. High resolution
electrospray ionization mass spectrum (HRESIMS) of compound 1 was obtained on
Agilent 6210 time of flight LC–MS. Optical rotation was measured with a Jasco P-2000
polarimeter at a wavelength of 589 nm.

51
The compound phenguignardic acid (1) was isolated as an amorphous colorless solid.
The molecular formula was established to be C 18H14O5 by its
13
C NMR, DEPT and
HRESIMS spectral data (HRESIMS, m/z=309.0757, expected 309.0763 [M-H]
-
). For
UV-Vis and ESIMS spectra, see Figure 3-3; For NMR data (chloroform-d), see Table 3-1
and Figure 3-9.

52
Table 3-1. NMR data for compound 1 (400 and 100 MHz in CDCl3). The left two lanes
are experimental data, and the right two lanes are published data.
Position δH (J in Hz) δC δH (J in Hz) δC
2 106.1, C 105.9, C
4 135.7, C 135.5, C
5 163.1, C 162.9, C
6 168.6, C 168.7, C
7
3.51, d (16.0)
3.44, d (16.0)
40.9, CH2
3.51, d (14.7)
3.44, d (14.7)
41.0, CH2
8 6.22, s 109.3, CH 6.20, s 109.7, CH
1' 132.4, C 132.3, C
2'/6' 7.60, d (8.0) 130.0, C 7.59, m 130.0, CH
3'/5' 7.34, t (8.0) 128.8, CH 7.34, m 128.9, CH
4' 7.31, m 129.1 CH 7.31, m 129.2 CH
1'' 131.0, CH 130.7, CH
2''/6'' 7.22, m 131.2, CH 7.22, m 131.2, CH
3''/5'' 7.16, m 128.5, CH 7.16, m 128.6, CH
4'' 7.16, m 127.8, CH 7.16, m 127.9, CH

53

Figure 3-1. Schematic overview of the doxycycline-regulated gene expression system
(Ptet-on, gray part) used in this study. The constitutive promoter PgpdA drives the
expression of a doxycycline (Dox)-dependent transcriptional activator rtTA (TcgrA as a
terminator). In the absence of Dox, rtTA does not bind to the rtTA dependent tetO7-Pmin
promoter, so there is no transcription activation of the target gene. In contrast, in the
presence of Dox, rtTA binds to tetO7-Pmin and initiates the transcription of the target
gene. A. fumigatus pyrG is used as a selection marker for transformation.

54

Figure 3-2. (A) HPLC profiles of extracts from A. terreus wild type, Ptet-on_pgnA
mutant with and without doxycycline induction as detected by UV (200-600 nm). The
maximum uAU value for compound 1 peak is around 110000. (B) HPLC profiles of
extracts from A. nidulans LO4389 (control), and pgnA heterologous expression strain
(alcA_pgnA).

5 10 15 20 25 30 35 40
Time (min)
control
1
alcA_pgnA
WT
Ptet-on
Ptet-on+Dox
Dox
5 10 15 20 25 30 35 40
Time (min)
1
(A) A. terreus
(B) A. nidulans
55

Figure 3-3. UV-Vis and ESIMS spectra of compound 1.

56

Figure 3-4. Proposed biosynthetic pathway for compound 1.

57
Table 3-2. Primers used in this study

Primer Sequence (5′→3′)
Primers used in the promoter replacement experiment
ATEG_08899.1F1 GGC CAA AAG GGA CTT GGT TG
ATEG_08899.1F2 AAA CCA CCG GGT TCG ATC TG
ATEG_08899.1R3
CGA AGA GGG TGA AGA GCA TTG CCG TCC GAC AGA ACA AGT
GAA
ATEG_08899.1F4
GCA TCA GTG CCT CCT CTC AGA CAG CAA GCG CGC AAT TAA
CCC
ATEG_08899.1R5 GGT GTT TAA ACG GTG ATG TC
ATEG_08899.1F6
GACATC ACC GTT TAA ACA CCA TGA ATA AGA AGC TCA AGC
TTT
ATEG_08899.1R7 ATA CCG CAG GTT GCT GAG AC
ATEG_08899.1R8 CGA AGG CAG GCA CAA TTA C
Primers used in the heterologous expression experiment
ATEG8899.1HEF
CCA ATC CTA TCA CCT CGC CTC AAA ATG AAT AAG AAG CTC
AAG CT
ATEG8899.1HER
CGA AGA GGG TGA AGA GCA TTG ATG GGA CAG GCG ATA GAT
AA
58
Table 3-3. Fungal strains used in this study
Fungal strain or
transformants
Gene mutation(s) Genotype
NIH2624
(A. terreus)
- wild-type
CW9001.1, CW9001.2
(A. terreus)
Ptet-on_pgnA kusA:: hph; pyrG-, AfpyrG_Ptet-on_pgnA
LO4389
(A. nidulans)
- pyrG89; pyroA4; nkuA::argB; riboB2; stcA-W
CW6053.1, CW6053.2,
CW6053.3
(A. nidulans)
stcJΔ, alcA(p)_pgnA
pyrG89; pyroA4; nkuA::argB; riboB2; stcA-W
yA::alcA(p)_pgnA_AfpyrG

59

Figure 3-5. The schematic design of the pgnA gene native promoter replacement by
Ptet-on in A. terreus (A), and heterologous expression of the A. terreus pgnA gene in A.
nidulans (B). In (A), the fusion construct consists of a ~1 kb sequence upstream of the
target gene, a selectable A. fumigatus pyrG (AfpyrG) marker, the Tet-on system (Ptet-on),
and a portion (~1 kb) of the target gene. The native promoter of the target gene is about
200 bp. The fusion construct was transformed into the A. terreus protoplasts to replace
the native promoter by homologous recombination. In (B), the fusion construct consists
of a ~1 kb 5’-yA flanking region, the alcA promoter, the pgnA gene, the AfpyrG marker,
and a ~1 kb 3’-yA flanking region. The fusion construct was transformed into the A.
nidulans protoplasts to replace the yA gene by homologous recombination.
5 ’-yA-alcA AfpyrG-3 ’-yA
5 ’-yA yA 3’-yA
pgnA 5 ’-yA alcA AfpyrG 3 ’-yA
fusion PCR
5 ’-yA 3 ’-yA
alcA AfpyrG
pgnA
pgnA
Ptet-on AfpyrG
pgnA coding
sequence
fusion PCR
Ptet-on AfpyrG
native
promoter
upstream
sequence
upstream
sequence
pgnA coding
sequence
Ptet-on
AfpyrG
upstream
sequence
pgnA
A In A. terreus
B In A. nidulans
pgnA
60

Figure 3-6. Diagnostic PCR analysis schematic design (A) and results (B) of native
promoter replacement strains. Genomic DNA from transformants or WT is amplified
with two primers, F1 from the chromosomal region just outside of the upstream sequence
of the native promoter, and R8 from outside of part of the pgnA coding sequence. If the
native promoter is replaced by the single AfpyrG-Ptet-on fusion strand, the amplified
fragment from transformants will predictably be 3961 bp longer than that amplified from
WT, and the diagnostic PCR results proved this case.

CW9001.1
CW9001.2
WT
Ptet-on AfpyrG
native
promoter
upstream
sequence
pgnA coding
sequence
Ptet-on
AfpyrG
upstream
sequence
pgnA
pgnA
F1
R8
F1
R8
F1+R8
WT=2139bp
Mutant=6100bp
6.0
10.0
3.0
2.0
1.0
(kb)
A
B
61
A.

B.
ATGAATAAGAAGCTCAAGCTTTTCTCTATGCCAGGCGCACAAACATCTCAAATCGTGATCATGTTGTTCC
AAAGCCTACTGCATCTTCTCGAAGCTATCGCTTCGCGGGAGCCGACACGCTACATTATTACCTATTCTAT
TGGAAACACCCATACGCCGGAGATATTTTCATACTCCGACCTCTTACAATCAGCCAGAAAGGCCGCCGGA
GCTCTTCGTTTCAAATACCATGTCGTCCCCGGATCGGTTGTTCTTCTACACTTTAATGATCATTGGAATT
CGATGTTGTGGTTCTGGGCCACCCTCATAGCAGACTGTATCCCAGCTATGTCCACTCCATTCTCCAACAA
TCCCGAGACAAGATTACGCCATTTGAAACACCTATCAACCACTTTAAGGAGTCCGAAATGCCTGACCACG
GCTTCTCTGGCCGCGGAATTCGCTGGCCAAGAATACATTACACCAATCTGCGTGCAATCGCTTGATTACG
AAAACCTGGTACATTTACCGATCAAGGAGGGGGGCGACATTGCAGTGCTTATGTTTACGTCTGGGAGCTC
AGGCCACTGCAAGGTCGTGCCTTTGACTCACGAGCAGATATTGGCATCTCTTTCGGGAAAAGCATGGACC
TTCCCACTGCCAGACAACACGGCACAGCTGAATTGGGTCGGAATGAATCATGTCGCCTCTTTGGTCGAAG
TTCATCTCTTCAGCATCTATACCCACAGTGATCAAGTCCATATCCCAACAGTTGAAGTCTTGTCTCACGT
TACCCTGTTCCTTGATTTGATACATCGACACCGTGTGTCTCGAACTTTCGCACCTAACTTTTTCCTCGCA
AAACTACGCGCAGCATTGAGTGCGGATGATACCTTGGCCAAGTACACCGGGAGTCTCAGCAACCTGCGGT
ATATTGTCTCCGGTGGCGAGGCGAACGTTACCCAGACGATCAATGATTTGGCCCAGATGCTGAAGAAGTG
TGGAGCTGTCTCGAACGTAATTGTGCCTGCCTTCGGCATGACAGAGACCTGTGCGGGCGCCATTTACAAT
ACCTCGTTTCCCCAGTATGATGTCGAGCACGGACTTCCATTTGCTTCCGTGGGGTCCTGCATGCCAGGGA
TCCAAGTGAGAATTGTCCAGCTTAATGGAAACGGCAATAGTGTTCCTCCGGGCACAGTAGGTAATCTCGA
GATCTGCGGTCCGGTGGTTCTCAAAGGTTATTTCAACAATCCTGCTGCTACAAAGTCGACATTCACGAAC
GACAATTGGTTCAAAACCGGAGATTTAGCTTTCGTTGACGATAACGGAATGCTGGTACTTGCTGGACGTG
AAAAGGATAGCATCATTGTGAATGGGGCCAACTACAGTCCACACGATATCGAGTCCGCCATCGACGAAGC
AAACATCCCCGGCCTTATCTCTGGTTTCACTTGTTGTTTCTCCACGTTCCCGCCCAGCGCAGACACAGAG
GAGGTCATCATTGTTTATCTCCCAAATTACACACCAGCGGACACAGTTCGACGATCTGAAACTGCAGCCG
CGATCAGAAAGGTCGCCATGATGTCAGTCGGCGTGCGTGCCACAGTTCTCCCGCTCGACCGGACAATGCT
GGAGAAATCGACTCTGGGCAAGCTTGCCCGCGGCAAGATCAAGGCTGCTTATGAAAGGGGAGACTATAAA
AGTTATCAAGAAGCGAACGAACAGATGATGGCTCTACACCACAAAGTGTCGCATCATCAGCCGCGGTCTG
GTCTCGAACAGAGTCTACTCGGCGTCTTCACCCGCACTATACCCGAGAACTTGACGGAGGACTTCGACGT
GTTGACGTCAATATTTGATCTGGGAATCACATCCATCGAGCTCCTCAAGCTCAAGAGAGGTATCGAAGAT
CTGATAGGTCACGGACAGATTCCTCTCATCACCCTGATGACAAACCCCACTATCCGGACATTATCAGACG
CGCTGAAGCAGCACGCTCAGCAAAGAGACTGCAGCATATACAACCCTGTAGTCGTATTACAGAGTCAAGG
A
T
TE
A
T C
canonical NRPS
NRPS-like
62
CAAAAAACCACCCATCTGGCTTGTCCACCCAGTCGGCGGAGAAGTCATGATATTCATGAACCTAGCAAAG
TTTATCATCGATCGACCTGTGTATGGGCTGCGAGCACGCGGGTTCAACGACGGTGAGGATCCATTCCACA
CGTTCGAAGAAATAGTCAGCACATATCATGCAAGTATCAAGGAAAAGCAACCAAGTGGCCCGTACGCAAT
TGCCGGTTATTCCTACGGCGCGAAAGTTGCATTTGATATTGCCAAGGCCCTGGAGCACAATGGAGACGAG
GTCCGCTTTCTGGGTTTGCTTGATCTTCCACCTAGTCTGAATGGTACGCAGATGCGTGCAGTCGCCTGGA
AGGAAATGCTGCTTCATATATGCCGTATGGTTGGTGTGATTCGGGAGGAGGGCATCAAGAAAATATACCC
GAGGTTGGAACCAGAAAATATCTCTCCACGCCATGCGATAGAAACAGTGATGGGTGAGGCTGATGTCACG
CGATTGGCGGAACTAGGGTTGACAGCGTCCGCGTTGGAGAGGTGGGCTAATCTAACACATGCTCTGCAAC
GTTGTATTGTCGATCATAAGACGAATGGCTCCGTTGCAGGCGCTGACGCGTTTTACTGTGATCCAATGGC
TTCGATGGCGATATCGAACGAGCAATGGGCATGTGACTATATTGGGAAGTGGAGTGACCATACACGGTCA
CCACCGAGGTTCCATCACATTGCGGGGACGCATTACACCATACTGGATGCAGAAAATATCTTCTCATTTC
AGAAGACATTTCTCAGGGCGCTGAATGACCGTGGAATCTAA

Figure 3-7. (A) The difference between NRPS and NRPS-like genes. NRPS enzymes are
identified as enzymes with at least one module composed of an adenylation domain (A),
a thiolation domain (T) and a condensation domain (C). NRPS-like enzymes usually
consist of an A domain, a T domain, but lack a canonical C domain. (B) The ORF codes
of the pgnA gene.

63

64

Figure 3-8. Alignment of amino acid sequence of PgnA, ApvA, AtmelA, AtqA, BtyA,
MicA, and TdiA. NRPS abbreviations: PgnA, A. terreus phenguignardic acid synthetase
(accession id in GenBank: EAU31031.1; locus id: ATEG_08899); ApvA, A. terreus
aspulvinone synthetase (accession id in GenBank: EAU36966.1; locus id: ATEG_02004);
AtmelA, A. terreus melanin synthetase (accession id in GenBank: EAU36837.1; locus id:
ATEG_03563); AtqA, A. terreus asterriquinone synthetase (accession id in GenBank:
EAU39346.1; locus id: ATEG_00700); BtyA, A. terreus butyrolactone synthetase
(accession id in GenBank: EAU36089.1; locus id: ATEG_02815); MicA, A. nidulans
microperfuranone synthetase (accession id in GenBank: CBF82791.1; locus id:
ANIA_03396); TdiA, A. nidulans terrequinone synthetase (accession id in GenBank:
CBF80711.1; locus id: ANIA_08513). The conserved amino acids are highlighted. The
amino acids covering adenylation domain, thiolation domain, and thioesterase domain
(using PgnA as reference) are shown in blue, brown, and green, respectively.

65

a.
1
H NMR spectrum of compound 1
66

b.
13
C NMR spectrum of compound 1
Figure 3-9.
1
H NMR and
13
C spectra of compound 1.

67
CHAPTER IV: Genome-based deletion analysis in Aspergillus
terreus reveals the acetylaranotin bis-thiomethylation gene

4.1 Abstract

Acetylaranotin is an epipolythiodiketopiperazine (ETP) secondary metabolite with a
broad range of bioactivities. We demonstrated that ATEG_01465.1 located outside of
acetylaranotin gene cluster is responsible for catalyzing the S-methylation of its
biosynthetic pathway. Combining the previous characterization of acetylaranotin
biosynthetic gene cluster together with the identification of its S-methyltransferase
provides a means to obtain second-generation acetylaranotin derivatives previously
inaccessible. By permutations of targeted deletions of ATEG_01465.1, acetyltransferase
(AtaH), and benzoate hydroxylase (AtaY), three novel acetylaranotin derivatives were
produced by Aspergillus terreus.

4.2 Introduction

Epipolythiodiketopiperazines (ETPs) are an important class of fungal secondary
metabolites featuring the presence of a transannular disulfide bridge. Their broad range of
biological activities, including antiviral, antibacterial, antiallergic, antimalarial, anticancer,
and cytotoxic effects, are imparted by this unique disulfide bridge by at least two reported
mechanisms: 1) the generation of reactive oxygen species (ROS) via redox cycling
between the disulfide and dithiol forms and 2) the inactivation of free thiol-containing
proteins via disulfide exchange and formation of intermolecular disulfide bond (Boyer et
al., 2013; Gardiner et al., 2005; Nicolaou et al., 2012). So far, nearly twenty families of
distinct ETP fungal metabolites have been isolated and structurally characterized (Welch
and Williams, 2014), among which gliotoxin produced by Aspergillus fumigatus has been
68
examined most extensively (Dolan et al., 2017). Recent studies have shown that gliotoxin
can induce apoptosis in mammalian cells by activating the proapoptotic protein Bak, and
have immunosuppressive properties by inhibiting the transcription factor NF-κB through
inhibiting proteolysis of IKB (Kroll et al., 1999; Pardo et al., 2006). Acetylaranotin (1,
Figure 4-1) is an ETP secondary metabolite that was first isolated from Arachniotus
aureus (Nagarajan et al., 1968), and it exhibits a range of bioactivities such as antiviral
activity via inhibition of viral RNA polymerase (Neuss et al., 1968), antiproliferative
activity via induction of apoptosis against cancer cell lines (Choi et al., 2011) and
antifungal activity (Suzuki et al., 2000). Because of the intriguing bioactivities and
structural complexity, total synthetic efforts of acetylaranotin have been accomplished by
both the Reisman and Tokuyama groups (Codelli et al., 2012; Fujiwara et al., 2012).
Genes involved in the biosynthesis of acetylaranotin have been identified in Aspergillus
terreus using gene knock out approaches by our group, which revealed that the
biosynthesis pathway shares several homologues with the biosynthetic pathway of
gliotoxin (Guo et al., 2013d).

The A. terreus strain not only produces acetylaranotin and its ETP intermediate
acetylapoaranotin (3, Figure 4-1), but also produces their corresponding
bis-thiomethylated derivatives (Guo et al., 2013d). Since thiomethylation decreases the
biological activities of the ETPs (Boyer et al., 2013), we are interested in developing A.
terreus strains with decreased amounts of the inactive bis-thiomethylated derivatives in
order to increase the amount of the desired bioactive acetylaranotin compounds. Recently,
the Doyle and Hertweck groups independently identified the S-adenosylmethionine
(SAM)-dependent gliotoxin bis-thiomethyltransferase (GtmA/TmtA) responsible for the
thiomethylation. An important discovery from their study is the observation that the gene
for the bis-thiomethyltransferase lies outside of the gli gene cluster in A. fumigatus and
explains why we could not identify a bis-thiomethyltransferase gene in the acetylaranotin
69
gene cluster in A. terreus in our previous study (Dolan et al., 2014; Scharf et al., 2014).
Using the findings from the Doyle and Hertweck groups’ discovery in gliotoxin
biosynthesis, in this study we report the creation of an improved acetylaranotin and
acetylapoaranotin producer strain with the deletion of AtaS, the homologue of
GtmA/TmtA, from A. terreus. The ataS gene is also located outside of the previously
identified ata gene cluster in A. terreus, and the ataSΔ strain eliminated the
bis-thiomethylation of acetylaranotin and acetylapoaranotin. In addition in this study, we
created double and triple gene deletion mutants of the three genes in the acetylaranotin
pathway, ataS, ataH (acetyltransferase), and ataY (benzoate hydroxylase), with the
intention of producing additional ETP derivatives from the acetylaranotin biosynthetic
pathway.

4.3 Results and Discussion

The deletion of acetylaranotin pathway-specific bis-thiomethyltransferase AtaS
from A. terreus improved its ETP producing activity
The production of acetylaranotin (1) and acetylapoaranotin (3) from A. terreus wild type
strain is always accompanied by the appearance of their methylated forms 2 and 4 (Figure
4-2), which may lose most of their bioactivities because of the disulfide bridge disruption
by thiomethylation. In our initial discovery of the acetylaranotin biosynthesis pathway,
we proposed that the AtaM domain of AtaIMG is responsible for methylating the free
dithiol of acetylaranotin intermediates, resulting in their flux to the methylation pathway,
however we were unable to verify the initial hypothesis (Guo et al., 2013d). In light of
the recent report by both the Doyle and Hertweck groups that a separate SAM-dependent
bis-thiomethyltransferase (GtmA/TmtA) in the gliotoxin pathway lies outside of the gli
gene cluster in A. fumigatus (Dolan et al., 2014; Scharf et al., 2014), we hypothesized that
there exists a protein homologue of GtmA/TmtA in the A. terreus genome responsible for
catalyzing the bis-thiomethylation of acetylaranotin. Searching for homologues of the
70
GtmA/TmtA protein in A. terreus by BlastP shows that AtaS (ATEG_01465.1) has 64.5%
sequence similarity, and is also located outside of the ata gene cluster (Figure 4-5).

To confirm our hypothesis, we deleted the ataS gene in an A. terreus NIH2624 strain with
a kusA-, pyrG- background by replacing it with the pyrG marker through an efficient gene
targeting system developed for A. terreus (Guo et al., 2013a). The kusA gene deletion
impaired the nonhomologous end-joining process, and thus improved gene targeting
efficiency through increasing the homologous recombination rate (Ninomiya et al., 2004).
The ataSΔ mutant strain was cultivated under the acetylaranotin-producing conditions,
and analysis of its secondary metabolites profile by LC-DAD-MS showed that the
production of the two bis-thiomethylated analogues,
bisdethiobis(methylthio)-acetylaranotin (2) and
bisdethiobis(methylthio)-acetylapoaranotin (4) were greatly diminished compared to the
wild type (WT) control, while the yield of acetylaranotin (1) and acetylapoaranotin (3)
remained the same or increased (Figure 4-1 and 4-2). The elimination of the
bis-thiomethylation allowed us to demonstrate that AtaS encodes the
bis-thiomethyltransferase for the acetylaranotin biosynthetic pathway in A. terreus. Since
the deletion of ataS can prevent ETP compounds loss by thiomethylation, it provides us
with an improved strain with presumed better activity against pathogens in coculture. In
addition, the ataSΔ mutant strain also facilitates the purification of aetylaporanotin (3),
which was previously inseparable from bisdethiobis(methylthio)-acetylaranotin (2) by
HPLC in the WT strain chemical profile.

Multiple knockouts were performed based on ataSΔ to create strains producing ETP
intermediates of acetylaranotin
The discovery of AtaS as a thiomethyltransferase for acetylaranotin biosynthesis pathway
provides us with a possible solution to bypass the thiomethylation branch via its
71
disruption. Deletion of ataH and ataY from A. terreus WT strain led to the production of
S-methylated ETP intermediates without acetylation or dihydrooxepin moiety, and thus
provided verification of their bioinformatically predicted function as an acetyltransferase
and a benzoate hydroxylase, respectively (Guo et al., 2013d). Thus, we made
permutations of targeted deletions of AtaS, AtaH, and AtaY with the desire to obtain ETP
intermediates without acetylation or dihydrooxepin moiety or both, which are possible to
have interesting bioactivities. By analyzing the acetylaranotin biosynthetic pathway
(Figure 4-3), we deduced the results as following: (1) the ataSΔ&ataHΔ double mutant
strain should allow the production of compound 15 and 16; (2) the ataSΔ&ataYΔ double
mutant strain should allow the production of compound 17; (3) the
ataSΔ&ataHΔ&ataYΔ triple mutant strain should allow the production of compound 14
(Table 4-1). Because there is only one pyrG marker that can be utilized for A. terreus
kusA-, pyrG- strain, the direct repeat strategy was employed to recycle the marker and
carry out the multiple knockouts (Nielsen et al., 2007) (Figure 4-6). We first deleted ataS
using the direct repeat strategy, and then the mutant was selected on media containing
1.3mg/ml 5-FOA to recycle the pyrG marker. After acquisition of the ataSΔ, pyrG-
mutant, ataH and ataY were individually deleted using the same direct repeat strategy to
obtain the ataSΔ&ataHΔ and ataSΔ&ataYΔ double mutants. Based on the ataSΔ&ataHΔ
mutant with recycled pyrG marker, ataY was knocked out to create the
ataSΔ&ataHΔ&ataYΔ triple mutant strain. Then the mutant strains were cultivated under
the same acetylaranotin-producing conditions, and their chemical profiles were analyzed
by LC-DAD-MS and shown in Figure 4-4. Compared to ataSΔ, the three mutant strains
accumulated amounts of chemically stable intermediates or shunt products that were
sufficient for structure determination. Compounds 9, 10 and 11 are previously
unidentified compounds purified from scaled-up cultures by flash chromatography and
subsequently by preparative HPLC, and their structures were elucidated via NMR
spectroscopy and high-resolution electrospray ionization MS (HRESIMS) analysis (Table
72
4-4, 4-5, 4-6, and Figure 4-10. 4-11. 4-12). However, we were not able to fully infer the
stereochemical configuration of 10 and 11 from acetylaranotin (1), and the two HPLC
peaks of compound 11 are probably the enantiomers to each other. Adding D2O to the
NMR sample of compound 11 led to the disappearance of the thiol hydrogen peak, and
confirmed the existence of free thiols. Oxidation of 11 using 3% H2O2 likely allowed the
disulfide bond formation as detected by mass spectrometry, indicating the two thiol
groups point to the same direction (Figure 4-9).

Compared to ataHΔ (Guo et al., 2013d), the ataSΔ&ataHΔ mutant still produced
compound 6 and 7 instead of the expected compound 15 and 16, indicating the existence
of other methyltransferases in the genome functioning on the acetylaranotin pathway to
prevent the formation of ETP intermediates (Figure 4-4). We speculate that the toxicity of
15 and 16 is beyond the strain’s self-tolerance, since we observed that its growth had
been adversely affected. The production of shunt product 10 is probably the result of
self-detoxification as one thiol group is methylated and the other is replaced by
hydroxylation.

Similarly, removal of ataS from the ataYΔ mutant didn’t allow production of the expected
compound 17, but still produced its methylated form, compound 8, and the shunt product
compound 12 (Guo et al., 2013d), further suggesting the catalysis of thiomethylation by
redundant methyltransferases. Surprisingly, the double mutant strain produced an
unexpected compound 11, which is likely to be derived from compound 14 via disulfide
reduction. The gliotoxin reductase GliT is the homologue of AtaT domain, and can
catalyze the NADPH-mediated reduction of gliotoxin disulfide bond (Schrettl et al.,
2010), indicating AtaT domain is a promising candidate for catalyzing such a reduction
step for compound 14. However, we cannot exclude the alternative of cytosolic disulfide
reduction. The production of compound 11 indicates that compound 14 would be
73
detoxified via disulfide reduction at the meantime of being converted to compound 8 via
acetylation and methylation.

Instead of accumulating the expected compound 14, deletion of both the ataH and ataY
genes from ataSΔ strain led to the production of 9 and 11, which are the derivatives of
compound 14 as a result of S-methylation and disulfide reduction, respectively. The
production of 11 in ataSΔ&ataHΔ&ataY triple mutant indicates the same mechanism as
11 was accumulated in ataSΔ&ataY double mutant, while the accumulation of 9 provides
another clue for the existence of thiomethylation catalyzed by other methyltransferases
besides AtaS.

Microorganisms are powerful and efficient bioreactors for producing complicated
chemical compounds, namely natural products. Based on the extensive understanding of
natural products biosynthesis, the producer microorganism can be rationally modified
through genetic manipulation to improve the activity of producing certain natural product
or its derivatives. One of the efficacious strain engineering strategies proven by extensive
studies is via disruption of a particular gene that acts downstream in a biosynthesis
pathway as a tailoring enzyme (Ito et al., 2009; Ju et al., 2009; Mendes et al., 2001;
Remsing et al., 2003; Zhang et al., 2008). Our genetic modification of A. terreus
represents another effort of strain engineering. The gene disruptions were accomplished
based on the recent description of the biosynthetic gene cluster for acetylaranotin and the
thiomethyltransferase for gliotoxin. This effort allowed us to establish an improved A.
terreus ataSΔ mutant strain producing 1 and 2 with eliminated production of their
bis-thiomethylated forms. Nevertheless, our rational design didn’t allow us to obtain the
expected acetylaranotin derivatives from the multi-gene mutants. Instead, these mutant
strains continued to produce the methylated derivatives or reduced form of the expected
compounds. The production of methylated derivatives 6, 7, 8, 9, and 10 are probably due
74
to the existence of redundant methyltransferases since the pathway specific
methyltransferase has already been deleted. Similarly, there may be additional redundant
methyltransferases responsible for detoxifying ETP pathways in A. fumigatus in addition
to the major methyltransferase, GtmA/TmtA. By blasting the methyltransferase in A.
terreus didn’t identify any other homologue of GtmA/TmtA except AtaS, leaving the
additional methyltransferase/s still a mystery. The mutants also accumulated 11, which is
probably due to the function of AtaT domain or the cytosol redox process. Interestingly,
the compound 11 remained stable free-dithiol form, which is considered as an active and
toxic state. The irreversible thiomethylation and the reversible disulfide oxidoreduction
represent two major ways to prevent self-intoxication as clarified by gliotoxin and
holomycin biosynthesis (Li et al., 2012; Scharf et al., 2014; Schrettl et al., 2010; Smith et
al., 2016). This study provides further insight into how A. terreus protects itself during
ETP production when the major thiomethylation mechanism is damaged.

In conclusion, we have established an A. terreus ataSΔ mutant strain with improved ETP
producing activity. We demonstrated that ataS located outside of acetylaranotin gene
cluster is the thiomethyltransferase responsible for converting acetylaranotin and its
related intermediates to their methylated forms. Although three novel ETP derivatives
were obtained, further strain improvement by disruption of ataH or/and ataY by targeted
gene deletion from ataSΔ mutant strain still led to the accumulation of the methylated
forms of the desired ETP intermediates. These results point to the importance of
redundant methyltransferases, the discovery and functions of which await further
exploration.

4.4 Materials and Methods

Strains and Molecular Manipulations. The primers used in this study are listed in Table
75
4-2. The A. terreus wild-type and mutant strains used in this study are listed in Table 4-3.
The ataSΔ mutant was created by replacing the ataS gene with the A. fumigatus pyrG
gene (AfpyrG) in the KusA-, pyrG- background of A. terreus. To create the ataSΔ&ataHΔ
and ataSΔ&ataYΔ double mutant strains, the KusA-, pyrG-, ataS- background of A.
terreus was first created using the direct repeat strategy, and then ataHΔ and ataYΔ were
individually deleted also using the direct repeat strategy. The ataSΔ&ataHΔ&ataY
mutant was created by replacing ataY with the pyrG gene in the KusA-, pyrG-, ataS-, and
ataH- background. The construction of double joint-fusion PCR products, protoplast
generation, and the transformation were carried out according to previous procedures
(Guo et al., 2012). Diagnostic PCR of three randomly picked transformants for each gene
replacement was carried out using the external primer pairs F1 and R6 after pyrG marker
recycling if using direct repeat strategy. The difference in size between the native gene
and gene deletion with marker recycling (ataSΔ, ataSΔ&ataHΔ, ataSΔ&ataYΔ) or
between the native gene and the pyrG marker (ataSΔ&ataHΔ&ataYΔ), allowed us to
determine if the transformants carried correct gene mutation. At least two of the three
transformants were determined to be correct for each mutant (Figure 4-7).

Fermentation and LC-MS analysis. The A. terreus NIH 2624 wild type and mutant
strains were cultivated at 30 °C on Czapek’s agar plates (NaNO3, 3 g/L; KCl, 0.5 g/L;
MgSO4·7H2O, 0.50 g/L; K2HPO4, 1.0 g/L; FeSO4·7H2O, 0.01 g/L; sucrose, 30 g/L; agar,
15 g/L) at 10 × 10
6
per 10 cm plate. After 5 days, agar was chopped into small pieces and
extracted with 50 ml of 1:1 CH2Cl2/MeOH with sonication for 1.0 h. The extract was
evaporated in vacuo to yield a water residue, which was suspended in 25 ml of water and
partitioned with 25 ml of ethyl acetate (EtOAc). See 2.4 for HPLC-MS sample
preparation and analysis. The solvent gradient condition for HPLC-MS analysis was 0-5
min 100-80% A, 5-20 min 80-60% A, 20-45 min 60-40% A, 45-50 min 40-0% A, 50-55
min 0-100 % A, 55-60 min 100% A.
76

Isolation and Characterization of Secondary Metabolites. For structure elucidation,
the A. terreus wild-type and mutant strains were cultivated on forty Czapek’s agar plates
( ∼50 mL of medium per plate, D = 15 cm) at 30 °C for 5 days. The UV-active secondary
metabolites were isolated via flash chromatography and reverse-phase HPLC. A 10 μL
portion of each purified compound solution from the reverse-phase HPLC was then
examined using an Agilent Technologies 1200 series high-resolution mass spectrometer.
The UV-Vis and ESIMS spectra of the identified compounds are shown in Figure 4-8.
NMR spectra were collected on a Varian Mercury Plus 400 spectrometer.

77

Figure 4-1. Acetylaranotin related compounds isolated in this study.

78

Figure 4-2. (A) The DAD traces of extracts from the wild type and ataSΔ strains as
detected by UV (200-600nm). (B-E) EIC extraction of 1, 2, 3 and 4 from the positive
ESI-MS trace in the profiles of the wild type and ataSΔ strains. Peaks labeled with *
correspond to compounds not involved in 1 biosynthesis.

79

Figure 4-3. Proposed biosynthetic pathway for the three novel acetylaranotin derivatives
(in gray box): 9, 10, and 11. All of the natural products isolated in this study are boxed.

Table 4-1. The expected compounds and isolated compounds from genetic engineering.
Mutant Expected compounds Isolated compounds
ataSΔ & ataHΔ 15, 16 6，7 ，10
ataSΔ & ataY 17 8 ，11
ataSΔ & ataHΔ & ataYΔ 14 9 ，11

80

Figure 4-4. The DAD traces of extracts from the mutant strains as detected by UV
(200-600nm). Peaks labeled with * correspond to compounds not involved in 1
biosynthesis, or could not to be characterized due to poor yield or instability. Compound
11 has two isomers.

81
Table 4-2. Primers used in this study
primer Sequence (5′→3′)
ATEG_01465.1F1 TAG CAT CAC GGT TGT CTC GT
ATEG_01465.1F2 AGA AAA GCG TAC CAT CCT CCG
ATEG_01465.1R3 AAC CGG AGC TTT ACC TTG GGT CTC TGG TCC TGG AGA GTT GA
ATEG_01465.1DR-F CCC AAG GTA AAG CTC CGG TT
ATEG_01465.1DR-R CGA AGA GGG TGA AGA GCA TTG GAG AGA TGC TGG CTC ACT CG
ATEG_01465.1F4 GCA TCA GTG CCT CCT CTC AGA CAG CCC AAG GTA AAG CTC CGG TT
ATEG_01465.1R5 CGA TCT CAA CCA GCC AGA GT
ATEG_01465.1R6 GAT CCA TAG TGA CAG CCC CG
ATEG_03466.1F1 GGG ACT ATC CTC TTC CTC GT
ATEG_03466.1F2 CAC AGG TCG TAC TTG GTG TC
ATEG_03466.1R3 GTC TCC CAC CAA TAT TCT CGG TGT CCT TGT TGA CGG ACT T
ATEG_03466.1DR-F C GAG AAT ATT GGT GGG AGA C
ATEG_03466.1DR-R CGA AGA GGG TGA AGA GCA TTG CGT CAA GAT GAA AGT TGG CAC
ATEG_03466.1F4 CAT CAG TGC CTC CTC TCA GAC AGC GAG AAT ATT GGT GGG AGA C
ATEG_03466.1R5 GAT ATA AGG TCG TCG CTT GG
ATEG_03466.1R6 ATC CCG CGA TAT TAG CAA C
ATEG_03468.1 F1 ATG CAA CAG AGT GAC ACG AG
ATEG_03468.1 F2 CTC CAC TTA CCA GGA GTC CA
ATEG_03468.1 R3 GAA GTA AAG GCC TGA GAG GGT CGC ATT TGG GGT ACA TAT C
ATEG_03468.1DR-F C CCT CTC AGG CCT TTA CTT C
ATEG_03468.1DR-R CGA AGA GGG TGA AGA GCA TTG GTT CCC TCC GCG TAA ACT TC
ATEG_03468.1 F4 CAT CAG TGC CTC CTC TCA GAC AGC CCT CTC AGG CCT TTA CTT C
ATEG_03468.1 R5 GCG ACG AGC ATA TTT CAA GT
ATEG_03468.1 R6 CAG CTT CCG GAT TCT TTT C

82
Table 4-3. A. terreus strains used in this study
Fungal strain or
Transformants
Gene mutation(s) Genotype
Aspergillus terreus NIH2624 - wildtype
CW9002.1, CW9002.2, CW9002.3 ataSΔ nkuA::hph; pyrG-, tmtA::AfpyrG
CW9003.1, CW9003.2, CW9003.3 ataSΔ&ataHΔ nkuA::hph; pyrG-, tmtA-, ataH::AfpyrG
CW9004.1, CW9003.2, CW9003.3 ataSΔ&ataYΔ nkuA::hph; pyrG-, tmtA-, ataY::AfpyrG
CW9005.1, CW9005.2, CW9005.3 ataSΔ&ataHΔ&ataYΔ nkuA::hph; pyrG-, tmtA-, ataH-, ataY::AfpyrG

Table 4-4.
1
H and
13
C NMR data for compound 9
a
(400 and 100 MHz in acetone-d 6).
Position δ H (J in Hz) δ C
1 168.9
2 74.2
3 3.03, d (15.9)
3.15, d (15.7)
38.6
4 133.8
5 6.02, m 120.5
6 5.91, m 123.9
7 5.66, d (9.9) 131.5
8 4.78, m 75.1
9 4.78, m 69.6
-SCH 3 2.25, s 14.8
HRESIMS prediction: C 20H 22N 2O 4S 2
a
The absolute configuration was inferred from acetylaranotin (1).

83

Table 4-5.
1
H and
13
C NMR data for compound 10
a
(400 and 100 MHz in DMSO-d 6).
Position δ H (J in Hz) δ C HMBC
b
COSY NOESY
1 166.1
2 86.4
3 H a: 2.77, d (15.8)
H b: 2.96, d (15.0)
42.0 1, 2, 4, 5, 9 H b-3, H-5, H-9
H a-3, H-5, H-9
H b-3
H a-3
4 111.7
5 6.59, s 136.7 3, 4, 6, 9 H-3, H-9
6 6.28, dd (8.3, 2.4) 138.1 5, 7, 8 H-7, H-8 H-7
7 4.77, m 110.8 6, 8, 9 H-6 H-6, H-8
8 4.48, d (7.8) 71.2 H-6, H-9 H-7
9 4.77, m 63.5 4, 7, 8 H-3, H-5, H-8
1’ 164.3
2’ 71.0
3’ 2.77, d (15.8)
3.21, d (15.1)
41.1 1’, 2’, 4’, 5’, 9’ H b-3’, H-5’, H-9’
H a-3’, H-5’, H-9’
H b-3’, H 3-SCH 3
H a-3’
4’ 111.5
5’ 6.66, s 136.5 3’, 4’, 6’, 9’ H-3’, H-9’
6’ 6.28, dd (8.3, 2.4) 138.0 5’, 7’, 8’ H-7’, H-8’ H-7’
7’ 4.77, m 110.7 6’, 8’, 9’ H-6’ H-6’, H-8’
8’ 4.19, d (7.7) 71.0 H-6’, H-9’ H-7’
9’ 4.77, m 63.0 4’, 7’, 8’ H-3’, H-5’, H-8’
-SCH 3 2.28, s 13.0 2’ H a-3’
HRESIMS: C 19H 19N 2O 6S [M-OH]
+
(m/z=403.0946, calc m/z=403.0958)
a
The absolute configuration was inferred from acetylaranotin (1).
b
HMBC correlations are from proton(s) to the indicated carbon.

84

Table 4-6.
1
H and
13
C NMR data for compound 11
a
(400 and 100 MHz in DMSO-d 6).
Position δ H (J in Hz) δ C
1 167.8
2 69.4
3 2.94, d (16.4)
3.36 (covered)
44.0
4 141.3
5 5.78, m 116.1
6 5.86, m 124.2
7 5.66, d (9.5) 132.4
8 4.18, dd (15.33, 2.8) 57.5
9 4.43, d (15.2) 73.3
-SH 9.20, s
HRESIMS: C 18H 15N 2O 2S 2 [M –2H 2O+H]
+
(m/z=355.0563, calc m/z=355.0569)
a
The absolute configuration was inferred from acetylaranotin (1).

85

Figure 4-5. Alignment of A. fumigatus GtmA/TmtA and A. terreus AtaS. Query: GtmA /TmtA
(Afu2g11120); Subject: AtaS (ATEG_01465.1).

86

Figure 4-6. The direct repeat strategy and marker recycling. The replacement of target gene by
AfpyrG is carried out by transforming bipartite fusion PCR fragments 5’-flanking-DR-AfpyrG and
AfpyrG-DR-3’-flanking. The two fragments contain overlapping 5’ and 3’ truncations of AfpyrG.
After the target gene is replaced by the AfpyrG marker, the mutant will be selected on medium
containing 5-fluoroorotic acid (5-FOA). Because AfpyrG encodes an orotidine-5’-phosphate
decarboxylase which can convert 5-FOA into 5-fluorouracil, a toxic compound, only the mutant
with the direct repeat recombination, namely the deletion of AfpyrG, can survive.

B
5 ’-flanking DR 5 ’-AfpyrG 3 ’-flanking
fusion PCR
3 ’-AfpyrG
DR
5 ’-flanking-DR-AfpyrG
AfpyrG-DR-3 ’-flanking
5 ’-flanking target gene 3 ’-flanking
5 ’-flanking DR AfpyrG DR 3 ’-flanking
5-FOA selection
5 ’-flanking DR
AfpyrG
DR 3 ’-flanking
5 ’-flanking DR 3 ’-flanking
WT
atmelA
apvA
apvA ,
3 Days
87
（A ）

（B ）

Figure 4-7. Diagnostic PCR strategy (A) and results (B).
F1
R6
F1
R6
F1
R6
DR
native gene
AfpyrG
ATE G_01465.1 ATE G_01465.1 &ATE G_03466.1
ATE G_01465.1 &ATE G_03468.1 ATE G_01465.1 &
ATE G_03466.1 &ATE G_03468.1
CW9002.1
CW9002.2
WT
F1+R6
WT=2992bp
Mutant=1870bp
CW9002.3
CW9003.1
CW9003.2
WT
F1+R6
WT=3169bp
Mutant=2184bp
CW9003.3
CW9004.1
CW9004.2
WT
F1+R6
WT=2966bp
Mutant=2093bp
CW9004.3
CW9005.1
CW9005.2
WT
F1+R6
WT=2966bp
Mutant=3978bp
CW9005.3
88

Figure 4-8. UV-Vis and ESIMS spectra of identified compounds.
89

Figure 4-9. Oxidation of two isomers of 11 by 3% H 2O 2 for 1 hour likely allowed the disulfide
bond formation.

90

a.
1
H NMR spectra of compound 9.

b.
13
C NMR spectra of compound 9.
Figure 4-10.
1
H and
13
C NMR spectra of compound 9.
Triple KO-P10
Sample Name:
1465-3466-3468-Peak10_right
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
1465-3466-3468-Peak10_right_20150127_01
FidFile: PROTON_01
Pulse Sequence: PROTON (s2pul)
Solvent: acetone
Data collected on: Jan 27 2015
Temp. 25.0 C / 298.1 K
Operator: wang
Relax. delay 1.000 sec
Pulse 45.0 degrees
Acq. time 2.559 sec
Width 6402.0 Hz
64 repetitions
OBSERVE H1, 400.0992830 MHz
DATA PROCESSING
FT size 32768
Total time 3 min 54 sec
0.24
1.53
1.59
1.63
1.00
3.12
1.81
1.81
2.58
0.08
0.19
5.24
0.66
0.23
8.017
2.248
2.273
2.333
2.404
2.409
2.422
2.600
2.875
3.008
3.012
3.015
3.048
3.051
3.130
3.170
4.742
4.781
4.784
4.822
5.331
5.650
5.674
5.886
5.888
5.894
5.898
5.901
5.903
5.911
5.914
5.916
5.922
5.927
6.015
6.020
6.026
6.033
7.278
7.286
2.242
2.218
2.211
2.184
2.178
2.142
2.086
2.079
2.071
2.061
2.056
2.050
2.044
2.039
1.194
1.189
ppm 1 2 3 4 5 6 7 8

Triple KO-P10
Sample Name:
1465-3466-3468-Peak10_right
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
1465-3466-3468-Peak10_right_20150127_01
FidFile: CARBON_01
Pulse Sequence: CARBON (s2pul)
Solvent: acetone
Data collected on: Jan 27 2015
Temp. 25.0 C / 298.1 K
Operator: wang
Relax. delay 1.000 sec
Pulse 45.0 degrees
Acq. time 1.304 sec
Width 25125.6 Hz
6000 repetitions
OBSERVE C13, 100.6049495 MHz
DECOUPLE H1, 400.1013435 MHz
Power 35 dB
continuously on
WALTZ-16 modulated
DATA PROCESSING
Line broadening 0.5 Hz
FT size 65536
Total time 3 hr, 59 min
206.307
168.900
131.501
133.848
131.409
120.450
123.902
75.177
75.055
74.156
69.613
69.560
29.920
30.110
30.301
30.491
38.586
29.722
29.531
29.341
14.760
ppm 0 20 40 60 80 100 120 140 160 180 200 220

91

a.
1
H NMR spectra of compound 10.

b.
13
C NMR spectra of compound 10.
6mg
Sample Name:
1465-3466-doubleKO_Peak1_20141220
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
1465-3466-doubleKO_Peak1_20141220_20141220_01
FidFile: PROTON_02
Pulse Sequence: PROTON (s2pul)
Solvent: dmso
Data collected on: Dec 20 2014
Operator: wang
Relax. delay 1.000 sec
Pulse 45.0 degrees
Acq. time 2.559 sec
Width 6402.0 Hz
32 repetitions
OBSERVE H1, 400.0990993 MHz
DATA PROCESSING
FT size 32768
Total time 1 min 57 sec
1.00
0.97
2.08
1.82
4.39
1.00
0.99
0.98
1.12
2.07
2.34
3.12
0.09
0.03
6.288
6.303
6.309
6.596
6.659
6.664
6.282
6.267
6.261
4.755
4.771
4.776
4.782
4.799
4.803
5.254
5.378
4.751
4.737
4.485
4.467
4.203
4.187
3.351
3.226
3.188
2.989
2.984
2.947
2.791
2.752
2.505
2.500
2.496
2.277
2.199
2.142
1.562
ppm 1 2 3 4 5 6 7

6mg
Sample Name:
1465-3466-doubleKO_Peak1_20141220
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
1465-3466-doubleKO_Peak1_20141220_20141220_01
FidFile: CARBON_01
Pulse Sequence: CARBON (s2pul)
Solvent: dmso
Data collected on: Dec 20 2014
Operator: wang
Relax. delay 1.000 sec
Pulse 45.0 degrees
Acq. time 1.304 sec
Width 25125.6 Hz
4000 repetitions
OBSERVE C13, 100.6050427 MHz
DECOUPLE H1, 400.1011674 MHz
Power 35 dB
continuously on
WALTZ-16 modulated
DATA PROCESSING
Line broadening 0.5 Hz
FT size 65536
Total time 2 hr, 39 min
166.055
164.340
136.521
136.689
137.954
138.099
110.539
110.745
110.791
111.652
86.432
39.506
39.719
39.925
40.138
41.106
41.990
63.034
63.499
70.953
71.006
71.227
39.300
39.094
38.881
12.952
ppm 0 20 40 60 80 100 120 140 160 180 200 220

92

c. HMBC NMR spectra of compound 10.

d. COSY NMR spectra of compound 10.
6mg
Sample Name:
1465-3466-doubleKO_Peak1_20141220
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
1465-3466-doubleKO_Peak1_20141220_20141220_01
FidFile: gCOSY_01
Pulse Sequence: gCOSY
Solvent: dmso
Data collected on: Dec 21 2014
Operator: wang
Relax. delay 1.000 sec
Acq. time 0.150 sec
Width 3900.2 Hz
2D Width 3900.2 Hz
64 repetitions
128 increments
OBSERVE H1, 400.0991064 MHz
DATA PROCESSING
Sq. sine bell 0.075 sec
F1 DATA PROCESSING
Sq. sine bell 0.033 sec
FT size 2048 x 2048
Total time 2 hr, 49 min
F2 (ppm)
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
F1
(ppm)
2
3
4
5
6
7

93

e. NOESY NMR spectra of compound 10.
Figure 4-11. 1D and 2D NMR spectra of compound 10.

6mg
Sample Name:
1465-3466-doubleKO_Peak1_20141220
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
1465-3466-doubleKO_Peak1_20141220_20141220_01
FidFile: NOESY_01
Pulse Sequence: NOESY
Solvent: dmso
Data collected on: Dec 21 2014
Operator: wang
Relax. delay 1.000 sec
Acq. time 0.150 sec
Width 3900.2 Hz
2D Width 3900.2 Hz
32 repetitions
2 x 200 increments
OBSERVE H1, 400.0991064 MHz
DATA PROCESSING
Gauss apodization 0.069 sec
F1 DATA PROCESSING
Gauss apodization 0.047 sec
FT size 2048 x 2048
Total time 5 hr, 4 min
F2 (ppm)
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
F1
(ppm)
2
3
4
5
6
7

94

a.
1
H NMR spectra of compound 11.

b.
13
C NMR spectra of compound 11.
Figure 4-12.
1
H and
13
C NMR spectra of compound 11.
TripleKO-Peak3
Sample Name:
WWS-1465-3466-3468-Peak3
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
WWS-1465-3466-3468-Peak3_20150128_01
FidFile: PROTON_03
Pulse Sequence: PROTON (s2pul)
Solvent: dmso
Data collected on: Jan 29 2015
Temp. 25.0 C / 298.1 K
Operator: wang
Relax. delay 1.000 sec
Pulse 45.0 degrees
Acq. time 2.559 sec
Width 6402.0 Hz
64 repetitions
OBSERVE H1, 400.0990988 MHz
DATA PROCESSING
FT size 32768
Total time 3 min 54 sec
1.00
0.16
1.22
1.09
1.08
1.09
1.03
0.21
1.17
0.28
9.203
8.314
5.671
5.777
5.781
5.840
5.847
5.853
5.861
5.871
5.876
5.883
5.647
4.413
4.451
4.195
4.158
3.360
3.162
2.962
2.918
2.500
2.505
2.539
2.496
ppm 2 3 4 5 6 7 8 9

TripleKO-Peak3
Sample Name:
WWS-1465-3466-3468-Peak3
Data Collected on:
Agilent-NMR-mercury400
Archive directory:
/home/wang/vnmrsys/data
Sample directory:
WWS-1465-3466-3468-Peak3_20150128_01
FidFile: CARBON_01
Pulse Sequence: CARBON (s2pul)
Solvent: dmso
Data collected on: Jan 29 2015
Temp. 25.0 C / 298.1 K
Operator: wang
Relax. delay 1.000 sec
Pulse 45.0 degrees
Acq. time 1.304 sec
Width 25125.6 Hz
6000 repetitions
OBSERVE C13, 100.6050423 MHz
DECOUPLE H1, 400.1011674 MHz
Power 35 dB
continuously on
WALTZ-16 modulated
DATA PROCESSING
Line broadening 0.5 Hz
FT size 65536
Total time 3 hr, 59 min
167.805
141.297
132.402
124.201
116.122
79.196
73.319
69.387
39.510
39.716
39.921
40.135
44.037
57.543
39.296
39.091
38.877
ppm 0 20 40 60 80 100 120 140 160 180 200 220

95
CHAPTER V ：Spatial Regulation of a Common Precursor
from Two Distinct Genes Generates Metabolite Diversity

5.1 Abstract

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase
genes usually encode proteins that generate various backbone precursors. These
precursors are modified by other tailoring enzymes to yield a large variety of different
secondary metabolites. The number of core synthesis genes in a given species correlates,
therefore, with the number of types of secondary metabolites the organism can produce.
In our study, heterologous expression showed that two NRPS-like proteins of A. terreus,
encoded by atmelA and apvA, release the same natural product, aspulvinone E. In hyphae
this compound is converted to aspulvinones whereas in conidia it is converted to melanin.
The genes are expressed in different tissues and this spatial control is probably regulated
by their own specific promoters. Comparative genomics indicates that atmelA and apvA
might share a same ancestral gene and the gene apvA is inserted in a highly conserved
region in Aspergillus species that contains genes coding for life-essential proteins. Our
data reveal the first case in secondary metabolite biosynthesis in which the tissue specific
production of a single compound directs it into two separate pathways, producing distinct
compounds with different functions. Our data also reveal that a single
trans-prenyltransferase, AbpB, prenylates two substrates, aspulvinones and
butyrolactones, revealing that genes outside of contiguous secondary metabolism gene
clusters can modify more than one compound thereby expanding metabolite diversity.
Our study raises the possibility of incorporation of spatial, cell-type specificity in
expression of secondary metabolites of biological interest and provides new insight into
designing and reconstituting their biosynthetic pathways.
96

5.2 Introduction

Recently, application of an efficient gene targeting system enabled us to link two
NRPS-like genes apvA and btyA to their corresponding SMs, aspulvinones and
butyrolactones, respectively, in A. terreus NIH 2624 (Guo et al., 2013c). In that study, we
demonstrated that one NRPS-like gene, atmelA, is involved in the synthesis of a brown
conidial melanin in this fungus (Guo et al., 2013c). The A. terreus genome contains 14
NRPS-like genes with predicted A-T-TE or similar domain architecture and the SM
products for the majority of these are unknown. Here we report our efforts to
systematically characterize the products of these 14 NRPS-like genes. We heterologously
expressed each gene in A. nidulans under the control of the inducible alcA promoter.
Surprisingly, our study reveals that two NRPS-like genes, apvA and atmelA, are
responsible for the formation of the same intermediate, aspulvinone E (1, Figure 5-1A).
The aspulvinone E produced by AtmelA is further modified by a tyrosinase AtmelB and
incorporates into the brown melanin biosynthesis pathway in A. terreus (Figure 5-1A). In
parallel, the aspulvinone E synthesized by ApvA is further prenylated by a
trans-prenyltransferase (AbpB) to produce aspulvinones (Figure 5-1A). AbpB also
prenylates butyrolactones, and this reveals that modifying genes outside of specific
clusters can be responsible for modifying compounds from more than one SM gene
cluster, thus expanding the diversity of SMs produced by an organism (Figure 5-1B).

Our results suggest that the promoter of apvA drives expression in hyphae resulting in the
production of aspulvinone E (1), which is modified to produce aspulvinone variants. The
atmelA promoter drives expression in conidia, also resulting in the production of
aspulvinone E (1) but in this cell type it is converted to melanin. Further genetic analysis
97
of apvA and atmelA indicates that these two genes may share a common ancestral gene.
The gene apvA, which may result from duplication of the ancestral gene, is inserted in a
genomic region consisting of genes that codes for life-essential proteins. Our study
suggests an unprecedented pathway for conidial pigment biosynthesis in A. terreus that
incorporates an NRPS-like product aspulvinone E (1) as its substrate (Figure 5-1). Our
data also demonstrated how the SM diversity can be expanded by 1). allocating the same
natural product in different fungal compartments to produce molecules with different
functions; 2). encoding tailoring enzyme that is capable of chemically modifying more
than one type of SMs.

5.3 Results and Discussion

Heterologous expression of the NRPS-like genes apvA and atmelA in A. nidulans
both result in aspulvinone E production.
Using a recently reported efficient heterologous expression (HE) system in A. nidulans
(Chiang et al., 2013c), the individual NRPS-like genes identified in the A. terreus genome
were expressed at either the wA or yA locus of A. nidulans under regulation of the
inducible alcA promoter (Figure 3-5). In a previous study, targeted deletion of apvA
depleted production of aspulvinones in A. terreus indicating that this NRPS-like gene is
responsible for the biosynthesis of the aspulvinone core (Guo et al., 2013c). As expected,
heterologous expression of apvA results in accumulation of aspulvinone E (1) which is
speculated to be the first intermediate in the aspulvinone pathway (Figure 5-1and Figure
5-2A, B) (Guo et al., 2013c). Unexpectedly, our HE experiments revealed that AtmelA
produces the same compound (Figure 5-2A, B). The gene apvA is responsible for
aspulvinone biosynthesis while atmelA is involved in the synthesis of the brown conidial
pigment (Guo et al., 2013c). The brown conidial melanin is still produced in the apvA
98
deletant strain as shown in Figure 5-3A. In contrast, deletion of atmelA generates an
albino mutant which is still capable of synthesizing aspulvinones (Guo et al., 2013c).
Together, these data reveal that despite having the same activity (i.e. synthesis of
aspulvinone E), ApvA and AtmelA function in different roles in the fungus.

Aspulvinone E occurs as a precursor in both aspulvinone and melanin pathways.
To test our hypothesis that the aspulvinone E is an intermediate in two different pathways,
the aspulvinone pathway and melanin pathway, we wished to delete the first tailoring
gene in each pathway to accumulate the precursor produced by the gene responsible for
the backbone metabolite. Since we have established the genetic linkage between apvA
and aspulvinones (Guo et al., 2013c), we set out to identify the first tailoring enzyme,
which we presumed was responsible for prenylating 1 to give 4 (Figure 5-2A). However,
we could not locate a PT gene proximal to apvA (Guo et al., 2013c). We then targeted
each of the putative PT genes in the A. terreus genome for deletion (Figure 5-4). The
individual genes were knocked out using fragments created by fusion PCR (Guo et al.,
2013c). The SM profiles of the correct mutants were examined by LC-MS, and of the 11
different mutants, only the ATEG_01730.1Δ strain accumulated aspulvinone E (1) (Figure
5-4). Unexpectedly, removal of ATEG_01730.1 leads to the accumulation of
butyrolactone II (7) as well (Figure 5-4). Previous study showed tha the NRPS-like gene
btyA is responsible for the biosynthesis of butyrolactone core (Guo et al., 2013c). The
expression of three genes, apvA, btyA, and abpB were also analyzed using real-time
quantitative reverse transcription PCR (qRT-PCR) (Figure 5-7C). Our data showed that
these three genes are co-expressed under aspulvinone and butyrolactone producing
conditions. These pieces of evidence suggest that this single PT is responsible for the
prenylation of two different metabolites, aspulvinone E (1) and butyrolactone II (7). Thus,
we name the gene a (aspulvinone) b (butyrolactone) p (PT) B.
99

Definitively establishing the role of AtmelA in the synthesis of aspulvinone E (1) required
additional gene deletion experiments. We first deleted the gene apvA using the direct
repeat (DR) strategy (Nielsen et al., 2008) followed by the recycling of the AfpyrG
marker. This ensures that aspulvinone E (1) identified in the later mutants is not from
ApvA. However, limited information is available about the biosynthesis of brown
conidial melanin in A. terreus. Our study suggests that the melanin pathway originates
from an NRPS-like product aspulvinone E (1), which is unlike the two main precursors
[di-hydroxyphenylalanin (DOPA) and dihydroxynaphthalene (DHN)] of currently known
melanins.(Langfelder et al., 2003) The biosynthesis of DOPA melanin starts with a
tyrosine that is oxidized to either DOPA or dopaquinone (DAQ) by a tyrosinase
(Langfelder et al., 2003). Considering that aspulvinone E (1) shares the same phenol
moiety as tyrosine, we speculate that a similar tyrosinase might catalyze the
hydroxylation of 1 at the ortho position to give a dihydroxylated intermediate that
becomes incorporated into the brown melanin (Figure 5-1). Following the cluster
paradigm, we examined the genes surrounding atmelA and identified one gene
ATEG_03564.1 (atmelB) that encodes a putative tyrosinase. Removal of this gene in the
apvAΔ background changed the strain’s phenotype: the brown melanin is no longer
synthesized and the conidia turned into bright yellow (Figure 5-3A). As expected, the
yellow material is aspulvinone E (1), which accumulated in the metabolite profiles of the
apvA and atmelB double deletion strain (Figure 5-3B). We next deleted the gene atmelA
in the double apvAΔ, atmelBΔ strain. The LC-MS profile of this triple deletion mutant
shows the abrogation of the aspulvinone E (1) which had reappeared in the double mutant
(Figure 5-3B), in accord with our HE results that AtmelA is also capable of producing
Aspulvinone E (1).

100
Aspulvinone E (1) produced by ApvA and AtmelA accumulates in different fungal
tissues.
Our studies indicate that ApvA and AtmelA synthesize the same product aspulvinone E
(1). We next asked how the fungus is able to allocate the same chemical synthesized by
different proteins into their own specific pathway without cross-interference. Previous
literature reports have shown that the production of SMs and/or their precursors can be
specific to both cellular organelle and fungal tissue (Berthier et al., 2013; Lim et al.,
2014). We speculated that the aspulvinone E (1) from the two genes might be generated
in different fungal tissues. Since removal of atmelA or atmelB changes the phenotype of
A. terreus conidia (Figure 5-3A), it is likely that aspulvinone E (1) from AtmelA might be
produced in conidia. Likewise aspulvinones, derived from 1 that is produced by ApvA,
might be produced inside the hyphae and secreted into the medium.

To test this hypothesis, we performed tissue-specific extraction(Berthier et al., 2013; Lim
et al., 2014) to reinvestigate the SM profiles of the four strains (1. wild type; 2. abpAΔ; 3.
apvAΔ, atmelBΔ; 4. apvAΔ, atmelBΔ, atmelAΔ). Fungal cultures were divided into three
fractions: 1. Conidial (mostly conidia and minor conidiophore), 2. Top layer agar
(mixture of conidiophore, vegative hyphae, minor invasive hyphae), 3. Lower layer agar
(mostly invasive hyphae) (Figure 5-3C-D). Compared to the SM profiles of total extracts
(Figure 5-3B.), extraction of the conidia showed the accumulation of 1 only in the apvAΔ,
atmelBΔ strain (Figure 5-3C), not in strains carrying abpBΔ or atmelAΔ. This result
indicates that aspulvinone E (1) from AtmelA is produced in conidia. Extraction of the
hyphae (mostly invasive) fraction showed the production of 1 in the abpBΔ strain and
compound 4 in wild type, indicating that aspulvinone E (1) from ApvA and its derivative
4 is specifically produced in hyphae.

101
Exchanging atmelA with apvA, under control of the atmelA promoter, restores
melanin production in A. terreus.
Next, we asked about the molecular mechanism for regulating the tissue-specific
production of 1. We first examined the expression profiling of atmelA and apvA in
different tissues using Real-Time qRT-PCR (Figure 5-7). As expected, the gene atmelA is
specifically expressed in conidia compartment (Fig 5-7B) while the gene apvA is locally
expressed in hyphae (Fig 5-7C). We then probed this question by determining whether
ApvA could replace AtmelA in the brown melanin biosynthesis. We assumed that
tailoring enzymes like AtmelB could still recognize aspulvinone E (1) generated from
either ApvA or AtmelA. Another study suggests that the products of two tubulin genes,
benA and tubC, are functionally interchangeable. The method they used was to disrupt
benA and then put tubC under control of the benA promoter.(May, 1989; Oakley, 2004)
Herein we implemented a similar strategy by replacing the coding region of atmelA with
that of apvA, placing apvA under control of the atmelA promoter (atmelAp) in the apvAΔ
background (Figure 5-6 i). As shown in Figure 5-5A, the mutant stain (apvAΔ,
atmelA::apvA) produces brownish conidia indicating that the brown conidial pigment is
produced in the mutant strain (apvAΔ, atmelA::apvA). As anticipated, the production of
aspulvinones (1 or 4) were not detected (Figure 5-8). The pigment is not produced as
much as in the wild type (Figure 5-5A) probably because atmelAp, after all, is not the
native promoter of ApvA. Quantitative analysis of their expression using Real-Time
qRT-PCR suggests that the expression level of apvA, which is under the control of atmelA
promoter, is lower than that of atmelA (Figure 5-7B). This could be one of the reasons
that leads to less production of the conidial melanin in the mutant strain (apvAΔ,
atmelA::apvA). But more importantly, our experiment shows that compound 1 from apvA
can be incorporated into the melanin pathway when apvA is regulated by atmelAp. As
mentioned earlier, the production of 1 from atmelA is conidia-specific. Thus, this
102
experiment shows that the product of ApvA can also be produced inside conidia and
incorporated into melanin, suggesting that the tissue specific allocation of their products
may be due to cell-type specific expression of the genes atmelA and apvA under
regulation of their specific promoters.

Tissue specific expression of gfp can be identified when using the coding sequence of
gfp to replace atmelA and apvA.
To test our hypothesis, we generated two mutants (atmelAp-gfp, apvAp-gfp) in which the
coding regions of apvA and atmelA were replaced by the green fluorescent protein coding
sequence (gfp), placing gfp under control of their specific promoters (Figure 5-6 ii). Both
the melanin pathway and aspulvinones biosynthetic pathways are active when A. terreus
is cultivated on LCMM agar. Under this culture condition, we propose that atmelAp will
turn on the expression of gfp specifically inside the conidia while the gfp regulated by
apvAp will display green fluorescent signal in the hyphae. As expected, we were able to
visualize localization of GFP within the conidia only in the strain carrying atmelAp-gfp
but not in the parental strain (used as control) or the strain apvA-gfp (Figure 5-5B). In
comparison, hyphal localization of GFP fluorescence was observed only in strain
apvAp-gfp.(Figure 5-5B) Thus, the tissue specific accumulation of aspulvinone E (1) is
probably due to cell-type-specific expression of the two genes apvA and atmelA.

Our study suggests that metabolite diversity could be expanded by spatial regulation of
the same precursor biosynthesized by two distinct genes. Melanin plays an important role
in fungal pathogenesis as well as in the protection of the producing organisms from
ultraviolet radiation (Eisenman and Casadevall, 2012; Gao and Garcia-Pichel, 2011;
Langfelder et al., 2003). No definite structures for melanins have been elucidated due to
their large molecular weight, insolubility in aqueous or organic solvents, and
103
heterogeneity.(Langfelder et al., 2003) The characterized conidial melanin of most
Aspergillus species belongs to the dihydroxynaphthalene (DHN) melanin
class.(Langfelder et al., 2003) Our study reports a unique and unprecedented pathway of
conidial pigment biosynthesis in A. terreus that originates from the NRPS-like product
aspulvinone E (1) (Figure 5-1). We also identified one putative tyrosinase encoded by the
gene atmelB that is involved in a tailoring modification of aspulvinone E (1) to yield the
brown conidial pigment (Figure 5-1). We speculate that the function of the tyrosinase,
AtmelB, might resemble that of its homolog which participates in
dihydroxyphenylalanine (DOPA)-melanin biosynthesis (Langfelder et al., 2003) and
catalyzes the ortho hydroxylation of the phenol moiety in the aspulvinone E (1) core
(Figure 5-1). It is entirely possible that other genes are involved in the polymerization of
the dual hydroxylated aspulvinone E and more efforts will be needed to decipher the
whole biosynthetic pathway of the brown conidial melanin in A. terreus.

Compounds in the aspulvinone family are found to have anti-influenza A viral (H1N1)
activity and are potent inhibitors of firefly luciferase (Cruz et al., 2011; Gao et al., 2013b)
The production of aspulvinones, as revealed in a previous study, is a general stress
response of A. terreus.(Hanlon and O'Connor, 2006). The biological functions of
aspulvinones, and what role they play in the biology of A. terreus, remain elusive.
AtmelA shares 73% sequence similarity with ApvA. We noted that there is a 63 bp
sequence in atmelA, corresponding to the TE domain of AtmelA, which has strong
nucleotide identity with a portion of apvA, indicating that these two genes may share a
common origin (Figure 5-10C). Interestingly, homology analysis of apvA and its
surrounding genes showed that apvA is inserted into a conserved genome region
consisting of genes that are essential for fungal survival (Figure 5-9). It is possible that
the insertion of apvA is due to the duplication and translocation of its ancestral homolog.
104
The production of aspulvinone-type SMs by A. terreus, in response to general stress,
could be related to the chromosomal location of its producing gene apvA. However, more
experiments are definitely necessary to elucidate the exact role the aspulvinone-type
metabolites play in the growth, survival or metabolic processes of the fungus A. terreus.

Previous literature suggested that p-hydroxylphenylpyruvate (HPP), which originates
from the shikimate pathway, is the biosynthetic precursor of aspulvinones (Nitta et al.,
Chem. Pharm. Bull., 1983, 31, 1528; Dewick, Nat. Prod. Rep., 1984, 7, 25). The
shikimate pathway, highly conserved in bacteria, fungi, and plant species, generates
carbon skeletons for the aromatic amino acids including tryptophan, tyrosine, and
phenylalanine (Tohge et al., Front. Plant Sci., 2013, 4). HPP is proposed to be the
intermediate in the conversion of prephenate to L-tyrosine in the shikimate pathway.
Limited information has been reported regarding the tissue localization of HPP in fungus.
Our study suggests that the distribution of HPP is not limited to certain types of fungal
compartments since both the biosynthesis of brown conidial melanin and hyphae specific
aspulvinones requires the prescence of HPP. Our data indicate that the tissue-specific
expression of apvA and atmelA are due to the promoters of the two genes. The promoter
of apvA drives expression specifically in hyphae while the promoter of atmelA drives
expression in conidia. There are many examples of hyphal versus spore specific gene
regulation. For example, the expression of the melanin synthesis gene atmelA might be
regulated by spore specific transcriptional regulators as demonstrated in a recent study
showing that BrlA, the transcription factor required to initiate conidiophore development
in Aspergillus spp., is necessary for fumiquinazoline gene expression and product
production (Lim et al., 2014). The fact that atmelAp is capable of turning on the
expression of both apvA (SM gene) and the gfp (reporter gene), suggests that it might be a
useful tool for directing the expression of genes specifically inside of conidia.
105

Besides spatial regulation of the intermediate, the SM diversity can be further expanded,
as shown in our study, by encoding those tailoring enzymes which are capable of
modifying SMs with different chemical scaffolds. Early literature reported the enzymatic
characterization of aspulvinone dimethylallyltransferase in A. terreus. This enzyme is
capable of catalyzing the mono or dual prenylation of aspulvinone E (1) (Takahashi et al.,
1978). The substrate promiscuity of some PTs has also been tested in vitro by feeding
experiments (Yu et al., 2011). Our study reveals the in vivo versatility of AbpB, as it
accepts substrates with different chemical scaffolds. Interestingly, the three genes are
dispersed in the A. terreus genome (abpB on chromosome II; apvA on chromosome III;
btyA on chromosome IV), representing another deviation from the SM gene cluster
paradigm. We have discovered in A. nidulans that in some cases, the corresponding PT
genes are not located in the same cluster as the core PKS genes (Lo et al., 2012b; Sanchez
et al., 2011). Further comparison of the butyrolactones with aspulvinones shows that the
chemical modifications after prenylation, including epoxidation and dehydrogenation, are
very similar (Figure 5-11). Thus, these two natural product families may share the same
set of tailoring enzymes that specifically modifies the prenyl groups attached by AbpB.

Fungi are capable of producing a large variety of SMs with unique chemical scaffolds. It
is usually expected that core synthetic genes, in a given species, are behind the
biosynthesis of different core intermediates, as demonstrated in a study showing that all
individual NRPKS in A. nidulans generates a unique PKS product (Ahuja et al., 2012a).
Phylogenetic analysis, using the protein sequences of those NRPS-like homologs
obtained from the Broad Institute Aspergillus comparative database, revealed several
other characterized NRPS-like homologs including TdiA (terrequinone A biosynthesis in
A.nidulans) (Balibar et al., 2007a), RalA (ralfuranone biosynthesis in Ralstonia
106
solanacearum) (Wackler et al., 2011), MicA (microperfuranone biosynthesis in
A.nidulans) (Yeh et al., 2012a), AtpA (asterrequinone biosynthesis in A. terreus) (Guo et
al., 2013c), BtyA (butyrolactone biosynthesis in A. terreus) (Guo et al., 2013c), and EchA
(echosides biosynthesis in Streptomyces sp. LZ35) (Zhu et al., 2014) (Figure 5-10). These
NRPS-like enzymes, with A-T-TE domain architecture, fall within Clade I of the
phylogenetic tree (Figure 5-10). They are capable of synthesizing natural products with
various chemical scaffolds using similar substrates like phenylpyruvic acid. Previous
literature suggested that these pyruvic acid substrates could be produced via the
Shikimate pathway (Arai and Yamamoto, 1990; Knaggs, 2003; Nitta et al., 1983). The TE
domains are proposed to catalyze various condensation, cyclization and releasing
reactions to yield different chemical backbones (Balibar et al., 2007a). In comparison,
another characterized NRPS-like protein (encoded by ATEG_03630.1), with A-T-R
domain arrangement, belongs to Clade V (Figure 5-10) (Wang et al., 2014a). In this case,
the aryl acid substrate, generated by an adjacent PKS (encoded by ATEG_03629.1), is
loaded onto the A domain and is reduced to its aryl-aldehyde precursor by the R domain.
Thus, SM diversity is enriched from these disparate starting points. It is also common to
identify two highly homologous core synthetic genes, in different species, that produces
the same precursor. For example, in the biosynthesis of the meroterpenoids austinol (A.
nidulans) and terretonin (A. terreus), the two PKS genes ausA and trt4 synthesize the
same intermediate 3, 5-dimethylorsellinic acid (Guo et al., 2012).

Our work suggests that chemical diversity in SM biosynthesis can be expanded via
multiple dimensions. Prior to our work, the richness of the fungal SM pool was presumed
to be determined by the number of SM gene clusters while the clustered genes are
associated with the biosynthesis of a distinct type of SM, with some exceptions
(Wiemann et al., 2013). In our study, we have shown that A. terreus is deploying a
107
different strategy to enrich its natural product pool. Although the same precursor
aspulvinone E (1) is shared in two pathways (the aspulvinone pathway, a typical SM
pathway and the melanin pathway, producing a self-protection pigment that might also be
involved in the pathogenicity of this fungus), the tissue specific expression of their
biosynthetic genes results in the production of the same compound in different fungal
tissues and allows it to be modified into two different products that, we assume, confer
selective advantages in the specific tissues in which they are produced. The localization
of production is possibly regulated by their specific promoters, but it is entirely possible
that a more complex regulatory mechanism underlies this phenomenon. This expands our
insight into spatial regulation of SMs in fungi (Lim and Keller, 2014). More investigation
into these promoters might provide a means to the cell-type-directed biosynthesis of SMs
or manipulating the location of the expression of some genes responsible for products
with interesting biological activities. Finally, our data demonstrate that AbpB prenylates
compounds in two pathways revealing that two pathways may share the same tailoring
genes. It will be of interest to determine if this characteristic is common to, and specific
to, prenyl transferases.

5.4 Materials and Methods

Strains and molecular manipulations
Primers used in this study are listed in Table 5-1. The fungal strains used in this study are
listed in Table 5-2. The construction of fusion PCR products, protoplast generation, and
transformation were carried out as previously described.(Chiang et al., 2013c; Guo et al.,
2013c) The scheme of diagnostic PCR is shown in Figure 5-13.

For real time qRT-PCR, the A. terreus wild type strain and the mutant strain (apvAΔ,
108
atmelA::apvA) were cultivated on LCMM agar for mRNA extraction from conidia. The A.
terreus wild type strain was cultivated in LCMM liquid broth for mRNA extraction from
hyphae. Total mRNA was extracted by using the Qiagen RNeasy Plant Mini Kit. The
cDNA was made from the equal amount of mRNA. The expression of every gene was
analyzed by ABI 7900HT Fast Real-Time PCR system.

Fermentation and LC-MS analysis
1. A. nidulans strain LO4389 and other HE mutant strains were fermented and analyzed
according to 3.4.

2. For the prenyltransferase screening experiment, A. terreus NIH 2624 and the mutant
strains were point inoculated at 30°C on LCMM agar plates. After 5 days, the secondary
metabolite profile was analyzed according to 4.4.

3. For the extraction of different fungal tissues for aspulvinone production, the procedure
was similar to a previously reported procedure.(Berthier et al., 2013; Lim et al., 2014)
The plates containing 20 ml LCMM agar (1.5% agar) were overlaid with another 10 ml
LCMM agar (0.75% agar). Plates were point inoculated with wild type or the DR mutant
strains and grown at 30 °C for 4 days. Conidia were harvested by adding 7 ml salt
solution (8.5 g/l NaCl) followed by gently scraping with a sterile spreader. The conidial
solution was inspected under the microscope to be largely free of hyphae and
conidiophores. The conidial fraction was sonicated for 1 hr and extracted twice with an
equal volume of EtOAc. The top layer was then washed with 10 ml sterile water twice
and removed with a sterile spatula. The bottom agar layer was then chopped into small
pieces and extracted as previously described. Preparation of the HPLC-MS samples from
different tissues and the conditions for MS analysis were as previously described.(Guo et
109
al., 2013c)

Isolation of secondary metabolites
For scale up, A. nidulans HE strains were cultivated at 37 °C in 1 liter LMM liquid
medium (~100 ml per flask) at 1 × 10
6
spore/ml per flask with shaking at 180 rpm. The
nutrients uridine, uracil, pyridoxine and riboflavin were supplemented if necessary. To
induce expression, 88 μl cyclopentanone was added into the medium after 18 hrs of
incubation. The incubator temperature was then changed to 30 °C and the culture medium
was collected 72 hrs after cyclopentanone induction. The medium was filtrated and
extracted three times with equal volume of EtOAc. The combined EtOAc layers were
evaporated to a crude extract. Further purification of fractions with targeted compounds
was carried out by gradient HPLC on a C18 reverse phase column [Phenomenex Luna
5μm C18, 250 × 10 mm] with a flow rate of 5.0 ml/min and measured by a UV detector
at 254 nm. The gradient system was MeCN (solvent B) and 5% MeCN/H 2O (solvent A)
both containing 0.05% TFA. Compound 1 was identified in both the secondary
metabolites profiles of the alcA_apvA and alcA_atmelA mutant strains. The gradient
condition for semi-preparative HPLC analysis of the crude of the alcA_atmelA strain was
0-2 min 100%-70% A, 2-5 min 70% A, 15-17 min 70%-0% A, 17-19 min 0%-100% A,
19-21 min 100% A. Compounds 1 (76.17 mg/L of medium) was eluted at 13.1 min.

Fluorescence microscopy
The wild type and two mutant strains (atmelAp-gfp, apvAp-gfp) were cultivated on the
two-layer agar plates as mentioned. Plates were point inoculated with wild type or the
mutant strains and grown at 30 °C for 5 days. The conidia were collected as previously
described and the conidial solution was diluted 10 times with sterile water. A 10 µL
portion of the diluted solution was placed on a pre-cleaned microscope slide and covered
110
with a coverslip. For imaging of the hyphal GFP fluorescence signal, the top layer was
removed as previously described. A small portion of hyphae containing agar (<10 µL)
was placed on a pre-cleaned microscope slide and covered with a coverslip. All the
samples were examined under an optical microscope to confirm that the conidia and
hyphae could be clearly visualized. Images were taken with a Zeiss LSM 510 Meta NLO
(Thornwood, NY) confocal imaging system equipped with Argon, red HeNe, and green
HeNe lasers and a Coherent Chameleon Ti-Sapphire laser mounted on a vibration-free
table.

Real-Time qRT-PCR analysis of the expression of genes atmelA, apvA, abpB, and
btyA.
The A. terreus wild type and the mutant strain CW6058.1 (apvAΔ, atmelAp-apvA) were
cultivated on LCMM agar at 30 °C for 72 hours for extracting mRNA from spores. The A.
terreus wild type was cultured in LCMM broth (1M spores/ml of medium) at 37 °C,
180rpm, for 60 hours for hyphae mRNA extraction. The β-tubulin gene atbenA
(ATEG_00287.1, homologue of A. nidulans benA gene) was used as a control and
quantification standard. Total mRNA was extracted by using the Qiagen RNeasy Plant
Mini Kit. The total mRNA was digested by Recombinant DNase I (ambion by life
technologies) to remove DNA contamination. The cDNA library was made from the same
amount of mRNA by using TaqMan reverse transcription reagents (T04141) and the
random hexamers. The expression of every gene was analyzed by ABI 7900HT Fast
Real-Time PCR system by following KAPA SYBR FAST qPCR kit (KK4601) protocol.
The experiments are performed in triplicate manner and the result is shown in Figure 5-7.

111
Spectral data of Compounds
NMR spectra were collected on a Varian Mercury Plus 400 spectrometer. The spectral
data of compounds 2, 3 and 4 have been reported before.
Aspulvinone E (1).Yellowish amorphous solid; For UV-Vis and ESIMS spectra, see
Figure 5-12; For NMR spectra, see Table 5-3 and Figure 5-14. The NMR data were in
good agreement with the published data. Butyrolactone II (5). Colorless amorphous
solid; For UV-Vis and ESIMS spectra, see Figure 5-12; For NMR spectra, see Table 5-4
and Figure 5-15.

112

Figure 5-1. (A). AtmelA and ApvA may synthesize the identical natural product 1
accumulated in different fungal tissues. (B). A trans-prenyltransferase, AbpB, prenylates
two substrates, aspulvinones and butyrolactones.

113

Figure 5-2. (A) Compounds related to this study. (B) HPLC profiles of extracts of HE
strains as detected by total scan UV .

5 10 15 20 25 30 35 40
Time (min)
B
LO4389
alcA_apvA 1
1 alcA_atmelA
O
OH
O
HO
OH
O
OH
O
HO
OH
O
HO
HO
O
OH
O
O
R
O
HO
HO
O
OH
O
O
A
aspulvinone E (1) aspulvinone H (2)
butyrolactone II (5) R = butyrolactone III (3)
O
R = butyrolactone I (4)
114

Figure 5-3. (A) Phenotype of A. terreus wild type and other A. terreus mutant strains
growing on LCMM for 5 days. Morphological change of the fungal conidia can be
observed if the deleted genes are involved in the biosynthesis of the brown conidial
pigment. Total extracts (B), conidial extracts (C) and hyphal extracts (D) of A. terreus
wild type and mutant strains as detected by UV at 370 nm. Aspulvinone related natural
products are labeled in red. In Figures 5-3B, 3C, and 3D, the black box represents the top
layer agar as described in tissue-specific extraction. The green box represents the bottom
layer agar in tissue-specific extraction. The numbering of the peaks corresponds to the
natural products shown in Figure 5-2A.The aspulvinone E (1) and its related natural
products can be detected in LC/MS traces Bi, Bii, Biii, Ciii, Di, Dii, and Diii. Trace
amount of 1 identified in D iii is due to the diffusion of this compound synthesized in the
WT
apvAΔ
apvAΔ, atmelBΔ
A
20 25 30 35 40 45
Time (min)
i. WT
iii. apvAΔ, atmelBΔ
1
2
*
iv. apvAΔ, atmelBΔ, atmelAΔ
15
1
ii. abpBΔ
15 20 25 30 35 40 45
Time (min)
i. WT
ii. abpBΔ
iii. apvAΔ, atmelBΔ
1
iv. apvAΔ, atmelBΔ, atmelAΔ
15 20 25 30 35 40 45
Time (min)
i. WT
iii. apvAΔ, atmelBΔ
1
2
*
iv. apvAΔ, atmelBΔ, atmelAΔ
ii. abpBΔ
B
C
D
1
apvAΔ, atmelBΔ,
atmelAΔ
115
conidia of the mutant strain. *This metabolite is related with aspulvinones according to
its UV absorption and MS spectrum.

116

Figure 5-4. HPLC profiles of extracts of A. terreus prenyltransferase (PT) genes deletants
as detected by UV at 330nm and 370nm. The numbering of the peaks corresponds to the
natural products shown in Figure 5-2A. The “*” compound is an aspulvinone derivative
according to its UV absorption spectrum. ATXXXXX is abbreviated for
“ATEG_XXXXX.1”. Deletion of the gene ATEG_00702.1, a homolog of tdiB involved in
the asterriquinone biosynthesis, was not achieved due to the unsuccessful PCR
amplification of its flanking region.

117

Figure 5-5. (A) Phenotype of the A. terreus mutant strain apvAΔ, atmelA::apvA (the A.
terreus wild type is used as positive control that produces melanin, the atmelAΔ strain is
used as negative control). (B) Replacing atmelA and apvA with gfp. Top column: conidia.
WT atmelAΔ
apvAΔ,
atmelA::apvA
A
B Control atmelAp-gfp apvAp-gfp
Control atmelAp-gfp apvAp-gfp
Merged
DIC
GFP
20 µm
Merged
DIC
GFP
118
Bottom column: hyphae. Top row: GFP. Middle row: differential interference contrast
(DIC). Bottom row: Merged GFP.

119
Table 5-1. Primers used in this study
primer Sequence (5′→3′)
Primers used in the heterologous expression experiments
ATEG2004.1HEF CCA ATC CTA TCA CCT CGC CTC AAA ATG ACT TTG AAC AAC CTA CA
ATEG2004.1HER CGA AGA GGG TGA AGA GCA TTG CGC TTG ACT TTC AAT AGA CG
ATEG3563.1HEF CCA ATC CTA TCA CCT CGC CTC AAA ATG CAA CCA AGC CTT ATT CC
ATEG3563.1HER CGA AGA GGG TGA AGA GCA TTG TTC CTC GAG AGT TTG AGA A
Primer used in the prenyltransferase deletion experiments
ATEG_00702.1F1 GTT ATG TTG GCC TCG AGA TG
ATEG_00702.1F2 GGC CAT TTT GTA ATG CTG TC
ATEG_00702.1R3 CGA AGA GGG TGA AGA GCA TTG AAG GTC TCA TCG GAG AGG AT
ATEG_00702.1F4 CAT CAG TGC CTC CTC TCA GAC AGC ATA ATG ACC ATC CGC TTG
ATEG_00702.1R5 ATG AAG GTC GCT CGT GTT AC
ATEG_00702.1R6 TTC TTC CAT TCC TCA CCA TC
ATEG_00821.1F1 GTA AAG GCC AAT GAA GAT GG
ATEG_00821.1F2 TAG TCC GAA TCC TCC CAT AG
ATEG_00821.1R3 CGA AGA GGG TGA AGA GCA TTG GAG GAC AAA TAG CCA GAT CG
ATEG_00821.1F4 CAT CAG TGC CTC CTC TCA GAC AGT TTA CCG GGT ATT CCA TCT G
ATEG_00821.1R5 ATC TGT TGA AGC GGC ATA GT
ATEG_00821.1R6 AAA CGC CAG TAC GAA TCT GT
ATEG_01730.1F1 ATT CTG CAT TTG GTC CTA CG
ATEG_01730.1F2 TCT CCA AGT AAG GAG CCA GA
ATEG_01730.1R3 CGA AGA GGG TGA AGA GCA TTG GGA AGA AAC GAT TCT GAT GC
ATEG_01730.1F4 CAT CAG TGC CTC CTC TCA GAC AGA GTG CTC CTT CAT CAC GTC T
ATEG_01730.1R5 GGA CAT CGA TTG TCT CAA CC
ATEG_01730.1R6 CTT TGT GTA CCA AGG CCA AG
ATEG_02823.1F1 GGG TTG GCA TCA AAC TCA
ATEG_02823.1F2 GGG ATG TCA TTC CAC AGT TC
ATEG_02823.1R3 CGA AGA GGG TGA AGA GCA TTG CGT ATG ACC TGG AGG TGA AG
ATEG_02823.1F4 CAT CAG TGC CTC CTC TCA GAC AGA GAG ACC CCC ATT TCA ATT C
ATEG_02823.1R5 GTC ATT GAT CCG TGC AAA G
ATEG_02823.1R6 TGA ATC GTT GCA GTA GTT CG
ATEG_03092.1F1 AGA AGT TGC CAT CGA AGT TG
ATEG_03092.1F2 GGG TTT TTG TAC TTG GTG CT
ATEG_03092.1R3 CGA AGA GGG TGA AGA GCA TTG GGT GGT AGT CGG TGA TAA GC
ATEG_03092.1F4 CAT CAG TGC CTC CTC TCA GAC AGA TCA GGT TCT GCA GTT ACG G
ATEG_03092.1R5 ATT CGG CCG TGT TCT CAT AC
ATEG_03092.1R6 TCC AAC TCC TAC CTT CAT CG
ATEG_04218.1F1 GCC CTA CTC TGA TCC TGA CA
120
ATEG_04218.1F2 CAT GGC CAA AGA CAA AAG AC
ATEG_04218.1R3 CGA AGA GGG TGA AGA GCA TTG TAT GCT TGA TGG CAG GAT G
ATEG_04218.1F4 CAT CAG TGC CTC CTC TCA GAC AGG AGC AGT AGG TTT GCA GGA C
ATEG_04218.1R5 GTC GGG TTC TGA GGG TTA CT
ATEG_04218.1R6 ATG ATG ATT CCG TGC TGA C
ATEG_04999.1F1 TCA GTG TGG ATG CAG GAT AG
ATEG_04999.1F2 GGT TGC TTC CAT TAT GTC GT
ATEG_04999.1R3 CGA AGA GGG TGA AGA GCA TTG GAG TCG ATG GGA TGT CAA GT
ATEG_04999.1F4 CAT CAG TGC CTC CTC TCA GAC AGT GAC TCT TGT ACT GGG TTT CC
ATEG_04999.1R5 CAC ATC TCC AAC AAC CAT CA
ATEG_04999.1R6 ATC TCG CTC ACA TCT CCA AC
ATEG_06111.1F1 GCT TCC ATG TCG AAC TGT G
ATEG_06111.1F2 CGA GTA CAT CTG TTG GTA GGC
ATEG_06111.1R3 CGA AGA GGG TGA AGA GCA TTG GAG GAG GTA CTG CTG GAA AA
ATEG_06111.1F4 CAT CAG TGC CTC CTC TCA GAC AGG TGT TAT ACT GGA GCC ACT GC
ATEG_06111.1R5 CAG GGC TAA TGC GTT ATT GT
ATEG_06111.1R6 GAC AGA CTC GAT GGA TGG TT
ATEG_06825.1F1 ATT CAG CCT CTC ATT GAA GC
ATEG_06825.1F2 GTA TCA CGA GAC CCA AAA CC
ATEG_06825.1R3 CGA AGA GGG TGA AGA GCA TTG TAG AAT GCA TGT TCG TCG AG
ATEG_06825.1F4 CAT CAG TGC CTC CTC TCA GAC AGC ATA GAG CGC TGC AAA TGT A
ATEG_06825.1R5 TGC TAC TGA CGA AAG TGG TC
ATEG_06825.1R6 ATC CGC GAC TAT GCT ACT GA
ATEG_08428.1F1 AAT TCA CCG AGA CAA CAT CC
ATEG_08428.1F2 GTT GGG TGT ATC AGG GAA GA
ATEG_08428.1R3 CGA AGA GGG TGA AGA GCA TTG ATG CTG TGT AAC ACG GAT TG
ATEG_08428.1F4 CAT CAG TGC CTC CTC TCA GAC AGC CAA GAG CTC AGT CGT TCA
ATEG_08428.1R5 ATC GCA GAG CTT CAG TCA TT
ATEG_08428.1R6 GTA TCC AAT CGC AGA GCT TC
ATEG_09980.1F1 CTG AAA AAT GAG CGG AGA AG
ATEG_09980.1F2 GGC AAA TCT GCC TGT TAG AC
ATEG_09980.1R3 CGA AGA GGG TGA AGA GCA TTG TGG TCG AAT ATG GGA CTA GC
ATEG_09980.1F4 CAT CAG TGC CTC CTC TCA GAC AGG GTA TGG GTT GCC AGA TAG A
ATEG_09980.1R5 GGC GAG CTG TAC TTC ATC A
ATEG_09980.1R6 AGA GTC GTC GCT GTA GGT GT
ATEG_10306.1F1 CTC GTG CAG GTT TAA CGA AC
ATEG_10306.1F2 CGT TAA TGT TCC TTG GGT GA
ATEG_10306.1R3 CGA AGA GGG TGA AGA GCA TTG GTG GAA GGG GAA ATG GTT AT
ATEG_10306.1F4 CAT CAG TGC CTC CTC TCA GAC AGA AGA TGA ATC GTG GCA GTG T
121
ATEG_10306.1R5 AGG GCT TAC AAT GGA TGC TA
ATEG_10306.1R6 TGG CCA ATG TAG GTA GAA GC
Primer used in the direct repeat (DR) disruption experiments
ATEG2004.1DR_F1 ATT ATG TAG CAG CAC GCA AG
ATEG2004.1DR_F2 GGT ATG GAT CGT TTC GTG TT
ATEG2004.1DR_R3 GAC AAA TTC CCG AGA AAC AGG CTG ATC ATG AAG ATG CTT G
ATEG2004.1DR_F4 CTG TTT CTC GGG AAT TTG TC
ATEG2004.1DR_R5 CGA AGA GGG TGA AGA GCA TTG TTA CTG CTG TCG ACT TCG TG
ATEG2004.1DR_R6 GTG ATT GTC GGC CAG AAT AG
ATEG3564.1DR_F1 CCA TGG AGA AGA AGA CCA AG
ATEG3564.1DR_F2 GTT AAC AAG CAC CAT TCT ACC C
ATEG3564.1DR_R3 TAC TCT TTG TGG TTT ACC GGT CAC GCA GTG AAG TCA TCA T
ATEG3564.1DR_F4 CCG GTA AAC CAC AAA GAG TA
ATEG3564.1DR_R5 CGA AGA GGG TGA AGA GCA TTG TGA GAC TGA AGA CGC TGA AG
ATEG3564.1DR_F6 CAT CAG TGC CTC CTC TCA GAC AGA GTT CCC GGT AAA CCA CAA
ATEG3564.1DR_R7 GAT AGT GAA CAC AGC GAG GA
ATEG3564.1DR_R8 GGC TGA AGA GGA TAG TGA ACA
ATEG3563.1DR_F1 CGT CGC TCA AAT GAC TTA GA
ATEG3563.1DR_F2 AGA GTC TTC TCC GTG GTC TG
ATEG3563.1DR_R3 ATT ACC ACC CGT AGA GTC GAA CCC TGT ACA TCC TGG AAA A
ATEG3563.1DR_F4 TCG ACT CTA CGG GTG GTA AT
ATEG3563.1DR_R5 CGA AGA GGG TGA AGA GCA TTG ATC TGT GCT GTG CCA TGA TA
ATEG3563.1DR_R6 GAG ACT CGT CTC TCG AGC TT
AfpyrG_DR_F1 CGG CGG CTT CTA TTT TAG AA
AfpyrG_DR_R2 GGA AGA GAG GTT CAC ACC (M2 primer(Nielsen et al., 2008))
AfpyrG_DR_R3 CAG TGC CTC CTC TCA GAC AG
AfpyrG_DR_F4 TGA TAC AGG TCT CGG TCC (M3 primer(Nielsen et al., 2008))
Primers used in the gene swap experiments
ATEG3563.1_SWA_F1 ATEG3563.1DR_F1
ATEG3563.1_SWA_F2 ATEG3563.1DR_F2
ATEG3563.1_SWA_R3 TGT AGG TTG TTC AAA GTC ATG GTG TGA TGA AGA AAT CCC C
ATEG3563.1_SWA_F4 CAT CAG TGC CTC CTC TCA GAC AGT CGA CTC TAC GGG TGG TAA T
ATEG3563.1_SWA_R5 CTC GAG CTT ATC TTC CCT GT
ATEG3563.1_SWA_R6 GAG ACT CGT CTC TCG AGC TT
Primers used in the green fluorescent (GFP) experiments
ATEG3563_GFP_F1 GGG GAT TTC TTC ATC ACA CCA TGA GTA AAG GAG AAG AAC T
ATEG2004_GFP_F1 CCC TTA TTG CAA CTC GGA CCA AAA ATG AGT AAA GGA GAA G
GFP_R2 CGA AGA GGG TGA AGA GCA TTG TTT GAG GCG ACC GGT TTA TTT G
Primers used in the real-time qRT-PCR
122
ATEG0287_RT_F GAC TGA ACT GGG TGG TGG TG
ATEG0287_RT_R GAG GTC GGA GGA GCC ATT G
ATEG3563_RT_F CAT CTG GGT TTT GCG GAT GC
ATEG3563_RT_R TGC GGC TGT TTG GAT TTG AC
ATEG2004_RT_F GAT CAT GCT GAT GAC GCA CA
ATEG2004_RT_R GGT CAA CGA TAT ACT GGG CGA
ATEG1730_RT_F AAA CGG ATC TGG GTC TGC TG
ATEG1730_RT_R GAT CAT CCA TAG TGC CCC CG
ATEG2815_RT_F CAG CAC GGT AAG GAC GAA GT
ATEG2815_RT_R TTC TGG ATT GCT CTG GGC TG
Primer used in the diagnostic PCR
AfpyrG_R CGG GAG CAG CGT AGA TGC C

123
Table 5-2. Fungal strains used in this study
Fungal strain or
transformants
Gene mutation(s) Genotype
Aspergillus terreus NIH2624 - wild-type
LO4389 (A. nidulans) None pyrG89; pyroA4; nkuA::argB; riboB2; stcA-W
CW6050.1, CW6050.2, CW6050.3 stcJΔ, alcA(p)-apvA
pyrG89; pyroA4; nkuA::argB; riboB2; stcA-W
wA::alcA(p)-apvA-AfpyrG
CW6052.1, CW6052.2, CW6052.3
stcJΔ,
alcA(p)-atmelA
pyrG89; pyroA4; nkuA::argB; riboB2; stcA-W
wA::alcA(p)-atmelA-AfpyrG
CW6054.1, CW6054.2, CW6054.3 abpBΔ kusA:: hph; pyrG-, abpBΔ
CW6055.1, CW6055.2, CW6055.3 apvAΔ kusA:: hph; pyrG-, apvAΔ
CW6056.1, CW6056.2, CW6056.3 apvAΔ, atmelBΔ kusA:: hph; pyrG-, apvAΔ, atmelBΔ
CW6057.1, CW6057.2, CW6057.3
apvAΔ, atmelBΔ,
atmelAΔ
kusA:: hph; pyrG-, apvAΔ, atmelBΔ, atmelAΔ
CW6058.1, CW6058.2, CW6058.3
apvAΔ,
atmelAp-apvA
kusA:: hph; pyrG-, apvAΔ,
atmelAp-apvA-AfpyrG
CW6059.1, CW6059.2, CW6059.3 atmelAp-gfp kusA:: hph; pyrG-, atmelAp-gfp-AfpyrG
CW6060.1, CW6060.2, CW6060.3 apvAp-gfp kusA:: hph; pyrG-, apvAp-gfp-AfpyrG

124

Table 5-3.
1
H and
13
C NMR data for compound 1 (400 MHz and 100 MHz in DMSO-d6)
(Gao et al., 2013a)
Position δ H (J in Hz) δ C
1 168.5, C
2 1001., C
3 162.1, C
4 140.4, C
5 6.64, s 107.8, CH
6 124.2, C
7 7.61, d (7.6) 132.2, CH
8 6.88, d (8.0) 116.2, CH
9 158.5, C
10 6.88, d (8.0) 116.2, CH
11 7.61, d (7.6) 132.2, CH
12 121.0, C
13 7.81, d (8.0) 128.8, CH
14 6.86, d (8.8) 115.4, CH
15 156.7, C
16 6.86, d (8.8) 115.4, CH
17 7.81, d (8.0) 128.8, CH

125

Table 5-4. NMR data for compound 5 (400 and 100 MHz in DMSO-d6)
Position δH (J in Hz) δC
1 158.0, C
2 6.88, d (6.8) 115.9, CH
3 7.52, d (7.2) 128.8, CH
4 121.0, C
5 7.52, d (7.2) 128.8, CH
6 6.88, d (6.8) 115.9, CH
7 127.5, C
8 138.1, C
9 168.0, C
10 84.7, C
11 169.8, C
12 3.40, d (3.2) 38.0, CH2
13 123.2 C
14 6.58, d (6.8) 131.2, CH
15 6.51, d (6.8) 114.7, CH
16 — 156.3, C
17 6.51, d (6.8) 114.7, CH
18 6.58, d (6.8) 131.2, CH
11-OCH3 3.74, s 53.6, CH3
126

Figure 5-6. The schematic design of molecular genetic experiments in this study: i. the
gene atmelA is replaced by apvA under the control of the atmelA promoter; ii. the coding
regions of both atmelA and apvA are replaced by gfp under the control of their own
promoters.
C
atmelA
apvA
5’-atmelA 3’-atmelA
AfpyrG 5’-atmelA 3’-atmelA
atmelAp
atmelAp transforming
fragment
(i)
(ii)
chromosomal
locus
atmelA
gfp
5’-atmelA 3’-atmelA
AfpyrG 5’-atmelA 3’-atmelA
atmelAp
atmelAp transforming
fragment
chromosomal
locus
apvA
gfp
5’-apvA 3’-apvA
AfpyrG 5’-apvA 3’-apvA
apvAp
apvAp transforming
fragment
chromosomal
locus
127

Figure 5-7. Relative quantification analysis of gene expression levels in WT-hyphae,
WT-conidia, and CW6058.1-conidia. (A) The size of Real-Time PCR product of the
β-tubulin gene atbenA from cDNA was analyzed using the genomic DNA as control. The
mRNAs were extracted from WT-hyphae (A, lane 2), WT-conidia (A, lane 3), and
CW6058.1-conidia (A, lane 4). (B) The relative expression level of atmelA and apvA in
WT-conidia and CW6058.1-conidia. (C) The relative expression level of atmelA, apvA,
abpB, and btyA in WT-hyphae. The gene expression levels are normalized using the
atbenA in the corresponding cDNA sample. Relative expression levels were calculated
using the 2
−ΔΔCt
method.
128

Figure 5-8. HPLC profiles of extracts of wild type and the apvAΔ, atmelA::apvA strain as
detected by UV at 370nm. The “*” compound is same as shown in Figure 4.

15 20 25 30 35 40 45
Time (min)
* 4
WT
apvAΔ, atmelA::apvA
129

Figure 5-9. The gene apvA (red arrow) is inserted in a highly conserved region among
Aspergillus species that contains genes putatively encoding life-essential proteins.

1Kb
2009 2008 2007 2006 2005 2004 2003 2002
A. terreus NIH2624 (ATEG_0XXXX)
AN0044 AN0043 AN0042 AN0040 AN0039 AN0038
A. oryzae RIB40 (AO090120000XXX)
386 387 388 390 391 392 393
A. flavus NRRL 3557 (AFL2G_08XXX)
390 391 392 393 394 395 396
A. niger CBS 513.88 (An14g06XXX)
600 590 580 570 560 550 540
A. aculeatus ATCC16872 (Aacu16872_XXXXXX)
025887 026236 026244 041954 026172 025841 051463
A. clavatus NRRL 1 (ACLA_01XXXX)
5650 5660 5670 5680 5690 5700
N. fischeri NRRL 181 (NFIA_07XXXX)
4610 4600 4590 4580 4570 4560 4550
A. fumigatus A1163 (AFUB_060XXX)
260 270 280 290 300 310 320
A. nidulans FGSC A4
DNA excision repair protein RAD5
Squalene/phytoene synthase
CorA family metal ion transporter
E3 ubiquitin-protein ligase
DNA polymerase gamma
YvcK-like protein, carbon metabolism
Serine/threonine-protein kinase Tel1
130
131
Figure 5-10. (A) Homology analysis of 59 NRPS-like homologs obtained from the Broad
Institute Aspergillus Comparative Database. Phylogenetic analyses of the protein
sequences of all the NRPS-like genes identified in Aspergillus species. A Maximum
Likelihood phylogenetic tree is drawn to scale, with branch lengths in the same units as
those of the evolutionary distances used to infer the tree. The numbers on the branches
means the percentage of times this topology was reached in a bootstrap test of 1000
replicates. The characterized genes are shown in bold. Genes start with “ACLA” are from
the genome sequence of A.clavatus (“AFLA”, A. flavus; “Afu”, A. fumigatus; “AN”, A.
nidulans. “An”, A. niger; “ATEG”, A. terreus;) The percentage lower than 75% is
removed. Clade I is enlarged as shown in (B). The enlarged part of Clade I of the
phylogenetic tree including all the characterzied NRPS-like genes. These genes encode
proteins with A-T-TE domain architecture and usually the aryl acids are the subsrate of
their A domains. (C) The conserved nucleotide region identified within the two genes
atmelA and apvA.
132

Figure 5-11. Proposed biosynthetic pathway for butyrolactones and aspulvinones.

133

Figure 5-12. UV-Vis and ESIMS spectra of compounds 1, 5.
The UV-Vis and ESIMS spectra of compounds 2, 3, and 4 have been shown in the
previous paper. (Guo et al., 2013c)

134

Figure 5-13. Diagnostic PCR strategies.

(A) Diagnostic PCR for NRPS-like genes heterologous expression (1), PT gene deletions
(2), gene swap experiment (3), and gfp replacement experiment (4) (number corresponds
to the fragment that is inserted in each experiment). In one strategy, DNA from
transformants is amplified with two primers, F1 from the chromosomal region just
outside of the 5’ flank of the transforming DNA fragment and R6 from just outside of the
3’ flank. If the target gene is different in size from the inserted fragment, the PCR
fragment amplified from a correct transformant will be different in size from the fragment
amplified if the target gene is intact, as shown in the case of abpBΔ (A ii). In some
instances the target gene and the AfpyrG cassette will be of comparable size and a second
strategy is applied. In the second strategy, F1 or R6 are used with internal primers
specific to the AfpyrG cassette. For example, if the target gene has been replaced by the
AfpyrG gene, F1 and AfpyrGR will amplify a fragment of a predictable size. If the target
gene has not been replaced, the AfpyrGR primer will not anneal and there will be no
specific amplification, as shown in the case of mutant strain atmelAp-gfp-AfpyrG (A iii).
5’-flank
3’-flank
5’-flank 3’-flank Target gene
Inserted DNA fragment:
(1) alcA + NRPS-like gene + AfpyrG
(2) AfpyrG
(3) ATEG_02004.1 + AfpyrG
(4) gfp + AfpyrG
F1
R6
F1
R6
R
10000
8000
6000
5000
4000
3000
2500
2000
1500
750
1000
500
250
ii. abpBΔ
F1 + R6: WT = 3310 bp; KO = 3991 bp
WT
CW6054.2
CW6054.3
CW6054.1
F1 + R6
WT
CW6059.2
CW6059.3
CW6059.1
F1 + R6
iii. atmelAp-gfp-AfpyrG
F1 + R6: WT = 4918 bp; KO = 4753 bp;
F1 + AfpyrG_R: WT no band; KO = 3288 bp
WT
CW6059.2
CW6059.3
CW6059.1
F1 + AfpyrG_ R
(A)
i.
135

(B) Diagnostic PCR for direct repeat experiment
For direct repeat (DR) experiment, the diagnostic PCR experiments were performed after
the AfpyrG marker has been cut off via homologous recombination of the DR sequences
since the PCR experiments using F1 and R6 cannot be performed when there are two
copies of DR sequence integrated in the genome (C). An example of diagnostic PCR for
DR mutant strains is shown in B ii. Lane 1 shows PCR amplification from the wild type
strain. Lanes 5-10 show PCR amplification from the apvA DR deletion mutants when the
AfpryG marker has been excised.

1 2 3 4 5 6 7 8 9 10 11
DR 5’-flank 3’-flank
F1
R6
5’-flank 3’-flank Target gene
F1
R6
(B)
i.
ii. apvA DRΔ
F1 + R6: WT = 3850 bp; DR KO = 1975 bp
136

a.
1
H NMR spectrum of compound 1

b.
13
C NMR spectrum of compound 1
Figure 5-14.
1
H NMR and
13
C spectra of compound 1.
168.520
158.490
162.125
156.706
140.366
115.382
116.213
121.014
124.162
128.819
132.150
107.806
100.139
39.510
39.723
39.929
40.134
39.304
39.090
38.885
ppm 0 20 40 60 80 100 120 140 160 180 200 220
20131210_AT3563A1_cpd5
S ample Name:
20131210_AT3563A1_cpd5
Data Collected on:
Agilent-NMR -mercury400
Archive directory:
/home/wang/vnmrsys/data
S ample directory:
20131210_AT3563A1_cpd5_20131210_01
FidFile: CAR BON_01
Pulse S equence: CAR BON (s2pul)
S olvent: dmso
Data collected on: Dec 10 2013
O
OH
O
HO
OH
aspulvinone E
1.10
2.00
1.71
3.77
0.96
6.869
6.889
7.603
7.622
7.808
7.828
6.847
6.799
6.639
2.050
2.467
2.500
2.536
1.800
1.137
ppm 0 2 4 6 8 10 12
20131210_AT3563A1_cpd5
S ample Name:
20131210_AT3563A1_cpd5
Data Collected on:
Agilent-NMR -mercury400
Archive directory:
/home/wang/vnmrsys/data
S ample directory:
20131210_AT3563A1_cpd5_20131210_01
FidFile: PR OTON_02
Pulse S equence: PR OTON (s2pul)
S olvent: dmso
Data collected on: Dec 10 2013
O
OH
O
HO
OH
aspulvinone E
137

a.
1
H NMR spectrum of compound 5

b.
13
C NMR spectrum of compound 5
Figure 5-15.
1
H NMR and
13
C spectra of compound 5.
0.71
1.92
2.00
3.99
3.03
6.573
6.590
6.595
6.856
6.863
6.870
6.876
6.888
6.893
7.492
7.500
7.507
7.513
7.525
7.530
6.559
6.516
6.511
6.499
6.494
3.737
3.723
3.407
3.399
3.166
2.539
2.505
2.500
2.496
2.491
ppm 0 1 2 3 4 5 6 7 8 9 10 11 12
butyrolactone_II
S ample Name:
butyrolactone_II
Data Collected on:
Agilent-NMR -mercury400
Archive directory:
/home/wang/vnmrsys/data
S ample directory:
butyrolactone_II_20140224_01
FidFile: PR OTON_01
Pulse S equence: PR OTON (s2pul)
S olvent: dmso
Data collected on: Feb 24 2014
O
HO
HO
H
3
COOC
O
OH
butyrolactone II
169.831
167.986
157.972
156.341
115.901
121.030
123.187
127.485
128.834
131.197
138.133
114.651
84.744
39.510
39.716
39.921
40.135
53.556
39.296
39.091
38.877
38.016
ppm 0 20 40 60 80 100 120 140 160 180 200 220
butyrolactone_II
S ample Name:
butyrolactone_II
Data Collected on:
Agilent-NMR -mercury400
Archive directory:
/home/wang/vnmrsys/data
S ample directory:
butyrolactone_II_20140224_01
FidFile: CAR BON_01
Pulse S equence: CAR BON (s2pul)
S olvent: dmso
Data collected on: Feb 24 2014
O
HO
HO
H
3
COOC
O
OH
butyrolactone II
138

CHAPTER VI: Discovery of McrA, a master regulator of
Aspergillus secondary metabolism

6.1 Abstract

Using a new genetic screen, we have identified a conserved but uncharacterized gene,
designated mcrA, which negatively regulates secondary metabolite production in the
filamentous fungus Aspergillus nidulans. Deletion of mcrA stimulates SM production even
in strains carrying a deletion of the SM regulator laeA, and deletion of mcrA homologs in
Aspergillus terreus and Penicillum canescens alters the secondary metabolite profile of
these organisms. Deletions of this gene and its homologs may be valuable tools for
secondary metabolite discovery.

6.2 Introduction

The sequencing of fungal genomes has revealed that in most cases the number of SM
biosynthetic gene clusters (BGCs) in the genomes far outnumbers the number of SMs
known to be produced by the species. Given the importance of fungal SMs, the need for
activating fungal SM clusters to exploit the fungal secondary metabolome and the need to
prevent production of toxic fungal SMs such as aflatoxins, it is very important to
understand the regulation of fungal SM genes. A great deal of progress has been made in
understanding the veA/velB/laeA complex, a complex that is present in many fungi and is
involved in the regulation of development and the transcription of a large number of SM
gene clusters [reviewed in (Bayram and Braus, 2012)] and new components of this system
are still being discovered (Ramamoorthy et al., 2013).

139

In spite of these advances and other advances involving replacement of native promoters
with regulatable promoters, we still do not know how to activate the majority of the SM
clusters even in a well-studied fungus such as Aspergillus nidulans (Yaegashi et al., 2014).
There are clearly important aspects of fungal SM regulation that remain to be determined.
Much of the previous work in identifying regulators of fungal SM has been directed toward
identifying positive regulators, genes for which increased expression would lead to
activation of silent SM clusters or greater expression of weakly expressed clusters. There
is ample evidence that there are negative regulators of SM clusters, however, and that
inactivation of such genes can result in the activation of SM clusters (Bok et al., 2009;
Hoffmeister and Keller, 2007; Spröte and Brakhage, 2007; Tsitsigiannis and Keller, 2006).
Using a genetic screen for identifying negative regulators in A. nidulans, a predicted
zinc-finger transcription factor, AN8694, has been identified. We have deleted the gene
and found that the deletion upregulates the production of several secondary metabolites,
while inducing overexpression of the gene with a regulatable or strong constitutive
promoter results in suppression of SM production. This gene is, thus, a potent, negative
regulator of A. nidulans SM production. We assign the gene designation mcrA
(multicluster regulator A) to AN8694. Deletion of mcrA homologs in Aspergillus terreus
and Penicillium canescens alters SM production in these organisms, stimulating
production of compounds not produced by wild-type strains cultured under the same
conditions. These data suggest that deletions of mcrA homologs in fungi are promising
tools for compound discovery.

6. 3 Results and Discussion

AN8694 is highly conserved across many genera of ascomycetes
Using a genetic screen carried out by Oakley group for identifying negative regulators in A.
nidulans, a predicted but uncharacterized zinc-finger transcription factor, AN8694, has
140

been identified. The only mention of AN8694 that we could find in the literature is that its
transcription is downregulated by deletions of velB and vosA (Ahmed et al., 2013). The
gene model in the AspGD database predicts that its product is a 399 amino acid protein.
The product of AN8694 is predicted to be a zinc-finger transcription factor with a
GAL4-like Zn2Cys6 binuclear cluster DNA binding domain extending from amino acids
156 to 196. The predicted product of AN8694 also, notably, contains proline rich regions
including several polyproline regions. A BLAST search with AN8694 revealed that many
species of fungi have a single, strong homolog of AN8694. The homology was strong not
just among species of Aspergillus, but across many genera of ascomycetes revealing that
AN8694 encodes a protein that is highly conserved. Among species of Aspergillus the
identity generally extended over the entire molecule (>95% coverage). There were five hits
among Aspergillus species that showed less coverage. In some cases, the reduced coverage
may be due to errors in annotation. For example, the A. terreus homolog [designated
ATET_7219 in AspGD and ATEG_07219 in the fungal genome datablase (Fungidb.org)
and the National Center for Biotechnology information (http://www.ncbi.nlm.nih.gov/)]
has only 74% coverage because the product of this gene is annotated to lack the 93
N-terminal amino acids of the predicted product of AN8694. However, a TBLASTN
search of the A. terreus genome with the product of AN8694 reveals reading frames with
strong identity to aa 5-93 in the sequences upstream of the annotated ATET_7219 (using
the AspGD designation for consistency) start codon, suggesting that the start codon for the
A. terreus gene is misannotated. A number of species from other genera showed >90%
coverage. In a number of cases the homologous proteins in other species appeared to lack
the N-terminal 140 amino acids or so present in the predicted A. nidulans protein, but in
nearly all cases the region starting with the DNA binding domain and extending to the
C-terminus was very highly conserved.

141

AN8694 is a negative regulator of the production of nidulanins and other secondary
metabolites
From the genetic screen, we deduced that AN8694 is a negative regulator of expression of
AN1242. To determine if this was the case, we deleted AN8694 in strain LO7543, the
strain we originally mutated that carries AfriboB replacing the coding sequence of
AN1242. We designated the resulting strain LO8162. LO8162 grew on medium lacking
riboflavin, confirming that deletion of AN8694 activates expression of AN1242 and, thus,
that AN8694 is a negative regulator of transcription of AN1242 (Figure 6-1). To add an
additional layer of redundancy to the proof that AN8694 is a regulator of AN1242, we
reinserted AN8694 at the yA locus creating strains LO10255-10257. Deletion of AN8694,
thus, activates the AN1242 promoter and reinsertion of AN8694 at another locus in the
deletion strain inactivates the promoter.

While some regulators of fungal SM production are specific for particular clusters (e.g.
cluster-specific transcription factors that are associated with some SM gene clusters),
others (e.g. LaeA) regulate several or many gene clusters (Bok and Keller, 2004; Brakhage,
2013; Keller et al., 2006). Regulators that control the expression of multiple SM gene
clusters are often called global regulators. (This term seems to imply that the regulator gene
controls all SM gene clusters, but no regulator identified to date has been shown to regulate
more than a fraction of SM clusters.) Because AN8694 mutants were originally selected to
activate AN1242 (NRPS of nidulanin A) on glucose minimal medium (GMM) plates, we
expected that on the same medium AN8694Δ would activate expression of the entire
nidulanin A cluster resulting in nidulanin A production. We also wanted to determine if it
would activate production of other compounds as well. We deleted AN8694 in strain
LO1362 [=TN02A7 (Nayak et al., 2006)] in which all SM clusters are intact, creating strain
LO8158. We examined the effects of deletion of AN8694 as well as overexpression of this
gene on SM production in a variety of media. SMs were extracted from plates and media as
142

detailed in the Materials and Methods and analyzed by HPLC-diode array detector
(DAD)-MS. Previous efforts from our laboratories and others have resulted in the
purification and structural determination of many A. nidulans SMs (Yaegashi et al., 2014),
and we were able to identify these previously discovered compounds by comparing their
HPLC retention time, UV-Vis absorption, and mass spectra.

We first examined the metabolites produced in the AN8694Δ strain LO8158 relative to its
parental strain LO1362 cultivated on GMM plates (Figure 6-2, i and ii). A major
metabolite, sterigmatocystin (1), and a trace amount of terrequinone (2) were identified in
the parental strain LO1362. In addition to compounds 1 and 2, seven additional metabolites
(3 – 9) were detected in the AN8694Δ strain LO8158. Compounds 3 and 4 were identified
as shamixanthone and emericellin from the prenyl xanthone pathway by comparing the
spectral data previously reported by our groups (Sanchez et al., 2011). Compounds 5 – 9
were identified as nidulanin A and its derivatives, based on the extracted-ion
chromatogram (EIC) reported by Andersen et al. (Andersen et al., 2013), confirming that
AN8694 is a negative regulator of the nidulanin A biosynthesis pathway.

We next compared LO7543, the strain that was originally mutated, to LO8162, which is
identical except that it carries AN8694Δ (Figure 6-2, iii and iv). Since a key nidulanin A
biosynthesis gene is replaced in both strains and the entire sterigmatocystin cluster is
deleted in both strains, no nidulanins or sterigmatocystin were detected in either strain. As
seen for LO1362 and LO8158, shamixanthone (3) and emericellin (4) were upregulated by
AN8694Δ. Reinsertion of the AN8694 gene at the yA locus of LO8162 (creating strains
LO10255-LO10257 returned SM production to that seen with LO7543 (Figure 6-4),
demonstrating that the absence of AN8694 was the cause of enhanced production of SMs
in LO8162. Taken together, our data so far indicated that AN8694 is a negative regulator of
the biosynthesis of prenyl xanthones (3 and 4) and nidulanins (5 – 9).
143

Deletion of AN8694 results in the upregulation of several SMs under different culture
conditions
In many cases, microorganisms produce different SMs under different growth conditions
(Bode et al., 2002). We were consequently interested in determining if deletion of AN8694
affected SM production in a variety of culture conditions. When grown on lactose minimal
medium (LMM) plates, both the parental strain LO1362 and LO8158 (AN8694 deleted in
LO1362) produced austinol (15), dehydroaustinol (16), and intermediates from the
sterigmatocystin pathway (17 – 21) (Figure 6-5A i and ii) so AN8694 does not appear to
regulate production of these compounds on LMM plates. On the other hand, F9775B
(22), F9775A (23), shamixanthone (3), emericellin (4), and epishamixanthone (24), were
upregulated in AN8694Δ mutants, LO8158 and LO8162 (AN8694Δ in strain LO7543
which carries the AfriboB gene coding sequence replacing the coding sequence of
AN1242), on LMM plates (Figure 6-5A, i – iv). In yeast extract, agar, glucose (YAG)
plates, prenyl xanthones (3, 4, and 24), nidulanins (5 – 9), cichorine (10), asperthecin (27),
emodic acid (28), emodin (29), and an unknown (30) were identified (Figure 6-5B, i – iv).
However, there was no consistent pattern of upregulation or downregulation by AN8694Δ
on YAG plates.

Deletion of AN8694 in a genetic dereplication strain results in the discovery of novel
compounds
We have previously developed methods for deleting entire A. nidulans SM clusters
(Chiang et al., 2013c) and we have used these methods to engineer a strain (LO8030) in
which the SM clusters responsible for the biosynthesis of the following major SMs are
deleted: sterigmatocystin, the emericellamides, asperfuranone, monodictyphenone,
terrequinone, F9775A and B, asperthecin and both portions of the split SM cluster that
produces austinol and dehydroaustinol (Chiang et al., 2015). These deletions greatly
144

reduce the SM background allowing SMs from other clusters to be detected more easily. In
addition, deletion of major SM BGCs may free up CoA precursors to allow greater
production of low level SMs and we noted that nidulanins (5 – 9) were produced at low, but
detectable, levels in LO8030 (Figure 6-2, v). It also produced a trace amount of 11, a
compound we have discovered recently and designated aspercryptin (Chiang et al., 2015).

We deleted AN8694 in LO8030 creating strain LO8111. Compounds 5 – 11 were
upregulated in LO8111 (Figure 6-2, v and vi). AN8694 is, consequently, a negative
regulator of cichorine and aspercryptin as well as the nidulanins. In addition, two peaks (12
and 13) were consistently detected in LO8111 grown on GMM that were not detectable or
produced in low quantities in the LO8030 parent (Figure 6-2, v and vi). In order to
characterize the chemical structures of 12 and 13, we isolated both compounds from a
large-scale culture of LO8111. The structures of 12 and 13, elucidated by their
spectroscopic data, are new cichorines. Although not detected by HPLC-DAD-MS, a
minor compound, felinone A (14), a recently discovered antibiotic (Du et al., 2014), was
also isolated from large-scale cultures of LO8111.

In summary, our data indicate that AN8694 is involved in the regulation of the SM
biosynthetic pathways that produce nidulanins (5 – 9), sterigmatocystin (1), the prenylated
xanthones (3, 4 and 24), asperthecin (11), cichorines (10, 12 and 13), F9775A and B (23
and 22), the emericellamides, and possibly terrequinone (2) and asperthecin (27). In most,
but not all, cases, deletion of AN8694 increases compound production and overexpression
reduces compound production. In view of these findings we designate AN8694 mcrA
(multi-cluster regulator A).

145

Deletion of mcrA homologs in Aspergillus terreus and Penicillium canescens alters
production of secondary metabolites
Because mcrA is conserved in ascomycetes, we hypothesized that deletion of mcrA
homologs in species of fungi other than A. nidulans would lead to alterations of SM
profiles. As discussed above, we identified an mcrA homolog in A. terreus strain NIH2624
(ATET_7219) using a BLASTP search of the NCBI protein database. As discussed above,
we suspect that ATET_7219 is incorrectly annotated and that the product of ATET_7219 is
actually longer and aligns with the entire length of McrA. We also identified an mcrA
homolog in Penicillium canescens, CE25191_2989, in the Joint Genomes Institute
database. This gene, too, is shorter than mcrA as annotated with aa 103 of mcrA aligning
with aa 1 of P. canescens. However, the RNA reads in the JGI database suggest to us that
the P. canescens gene is longer than annotated. We deleted each of these genes and we
found that the deletions altered the metabolite profiles of each of these genes as anticipated
(Figure 6-6). As in A. nidulans, some metabolites were reduced whereas more metabolites
appeared or increased. These data indicate that deletions of mcrA homologs will be a useful
tool to elicit secondary metabolite production in ascomycetes.

Interactions with the veA/velB/laeA network
The veA/velB/laeA network is a very important regulator of secondary metabolism, and it is
still yielding secrets even though it has been studied extensively over many years. It is
likely that fully elucidating mcrA functions and interactions will require similar long term
efforts. We are curious, however, as to the relationship, if any, between mcrA and laeA.
LaeA is a positive regulator of secondary metabolism. Deletion of laeA reduces the
production of several secondary metabolites and overexpression increases SM production
(Bayram and Braus, 2012). We, therefore, created strains that carried mcrAΔ as well as
laeAΔ or laeA overexpressed (oelaeA) by replacing its native promoter with the inducible
146

and repressible alcA promoter (Felenbok, 1991; Waring et al., 1989) or the strong
constitutive gpdA promoter (Punt et al., 1991).

We examined the effects of mcrAΔ along with laeAΔ or overexpression of laeA on SM
production. These experiments were carried out on GMM plates under conditions identical
to those used to obtain the data shown in Figure 6-2. We first confirmed that SM
production of the parental strain L01362 is modest under these conditions (Figure 6-3i).
We also examined production of SMs in the alcA(p)mcrA strain LO8166. On GMM
alcA(p) is repressed by glucose resulting in downregulation of mcrA and, as anticipated,
SM production was boosted similar to the mcrA deletion (compare Figure 6-3ii to Figure
6-2ii). We deleted laeA in LO1362 creating the sister transformants LO10279-LO10281.
We confirmed that, as expected, the deletion abolished almost all SM production in each of
the three sister transformants and results for LO10279 are shown in Figure 6-3iii. Next we
created laeA, and mcrA double deletions strains by deleting laeA in the mcrAΔ strain,
LO8158, creating sister transformants LO10260-LO10262. Interestingly, we found that for
many SMs mcrAΔ overrode the inhibitory effects of laeAΔ. (All three strains gave similar
results. Results for LO10260 are shown in Figure 6-3iv). Austinol, dehydoroaustinol,
nidulanin A, nidulanin intermediates, terrequinone, shamixanthone and emericellin were
all produced in the laeAΔ, mcrAΔ strain. There was one notable exception.
Sterigmatocystin was produced in the double deletion strain, but at much lower levels than
in the mcrAΔ, laeA
+
strains (Figure 6-2ii) or mcrA repressed, laeA
+
strains (Figure 6-3ii).
This result suggests that laeA plays a particularly important role in sterigmatocystin
production that is not overridden by mcrAΔ. Surprisingly, two compounds, F9775A and
F9775B were produced in laeA, mcrA double deletion strains at much higher levels than in
the mcrAΔ, laeA
+
or mcrA repressed laeA
+
strain.

147

Overexpression of laeA results in enhanced SM production and putting laeA under control
of the strong constitutive promoter of the gpdA gene in the parental strain LO1362, creating
strains LO10285-LO10287, resulted in enhanced SM production as expected (Results for
LO10285 are shown in Figure 6-3v). Since both mcrAΔ and overexpression of laeA
produce SM production, we created strains carrying both mcrAΔ and gpdA(p)laeA by
putting laeA under control of gpdA(p) in LO8158 creating strains LO10269-10271. We
hoped that SM production would be doubly enhanced. SM production was strong in the
double mutant strains, but not dramatically greater than in the mcrAΔ parent (Figure 6-3vi).
However, one novel compound was produced (42) in these strains under these conditions
and it is possible that additional new compounds will be produced in this strain under other
conditions.

Using the genetic screen to isolate negative regulators of the nidulanin A cluster, we were
able to identify a previously unstudied negative regulator of secondary metabolism that we
designate mcrA. Our secondary metabolite profiles, obtained on a variety of media, reveal
that mcrA plays a role in the regulation of nidulanins, sterigmatocystin, the prenylated
xanthones, aspercryptin, cichorines, F9775A and B, the emericellamides, and possibly
terrequinone and microperfuranone. This list includes important bioactive SMs.
Sterigmatocystin, notably, is a precursor of aflatoxins (Brown et al., 1996) and both
sterigmatocystin and the aflatoxins are extremely toxic and carcinogenic and are major
public health and agricultural problems (Williams et al., 2004). It is noteworthy that the
effects of the mcrA deletion on production of secondary metabolites differ on different
growth media. This may indicate that McrA plays a role in connecting primary metabolism
with secondary metabolism. In addition, mcrAΔ allowed us to discover two new
compounds synthesized by the cichorine biosynthetic pathway and to discover that A.
nidulans produces felinone A.
148

Homology searches indicate that McrA, the protein product of mcrA, is likely a
transcription factor with a Gal4 DNA binding domain. Transcription profiling revealed
that mcrA is a master regulator, affecting the transcription of hundreds of genes. The mcrA
gene is conserved in ascomycetes, and there are only one or two strong homologs per
species, raising the possibility that mcrA homologs are conserved master regulators. It
follows that deletion of mcrA homologs in other fungi may activate silent secondary
metabolite BGCs and that deletions of mcrA homologs may be very valuable in
discovering compounds produced by heretofore-silent SM gene clusters. Our initial
experiments with A. terreus and P. canescens confirm that this is the case.

We also identified that mcrAΔ stimulates SM production regardless of whether laeA is
present, absent or overexpressed and this suggests that McrA, in general, acts
independently of LaeA. There are clearly connections to the veA/velB/laeA system,
however. Deletion of velB has been reported to downregulate AN8694, which we have
now designated mcrA (Ahmed et al., 2013) and this might be expected to stimulate SM
production. However, the velB deletion is not reported to stimulate SM production
(Bayram et al., 2008). Deletion of mcrA causes a modest upregulation of laeA and deletion
of laeA exacerbates the weak growth defect caused by mcrAΔ. mcrAΔ causes only a small
stimulation of sterigmatocystin production if laeA is deleted so for this metabolite laeAΔ is
epistatic to mcrAΔ. mcrAΔ and oelaeA collaborate to produce a new compound not found
in the single mutants and, finally, F9775 A and B are produced in mcrAΔ, laeAΔ strains but
not in either single mutant. These findings show that mcrA and the veA/velB/laeA system
are connected, but they are not easily integrated into a simple, linear signaling model.
Some of the findings are consistent with mcrA acting downstream of veA/velB/laeA. In this
case, mcrA would be proximal to SM production and deletion of mcrA would cause SM
production regardless of veA/velB/laeA signaling. This model does not, however, explain
the aforementioned effects of laeAΔ on growth of mcrAΔ strains, nor the epistatic effect of
149

laeAΔ on sterigmatocystin production nor the stimulation of F9775 A and B production in
the double deletion strain.

In this regard, we call mcrA a master SM regulatory gene because mutation or deletion of
this gene alters the production of a number of secondary metabolites. However, it may be
more appropriate to think of regulatory networks than master regulators. These networks
would integrate various signals (light, carbon source, nitrogen source, chemical
environment, physical contact with predatory or prey organisms, developmental stage and
perhaps many other things) and activate production of SMs as appropriate. Key regulators
such as LaeA and McrA would be at nodes of these networks and alteration of these
proteins would, thus, affect production of many SMs.

6.4 Experimental Procedures

Molecular Genetic Methods. The A. nidulans strains used in this study are listed in Table 6-1.
Replacement of the coding sequences of core SM biosynthetic genes was carried out as
previously described using fusion PCR to create transforming fragments (see 2.4). Correct
gene integration in each transformant was verified by at least three diagnostic PCR
amplifications using different primer pairs. Deletion of entire SM clusters was carried out
as previously described (Chiang et al., 2013b). Most clusters were deleted using the loop
out recombination procedure. Correct deletion of entire clusters was verified by diagnostic
PCR amplifications using primers outside of the ends of the clusters.

Molecular genetic manipulations in A. terreus and P. canescens including protoplasting
and gene targeting were carried out as previously described (Guo et al., 2014 ; Yaegashi et
al., 2015). Deletions were confirmed by diagnostic PCR. The diagnostic PCR strategy is
shown in Figure 2-5.
150

Fermentation and HPLC-DAD-MS analysis. For agar plate cultures, A. nidulans
strains were incubated at 37°C on GMM [as above except that Hutner’s trace element
solution (Hutner et al., 1950) was used at 1m L
-1
], LMM (15 g L
-1
D-lactose, 6 g L
-1

NaNO3, 0.52 g L
-1
KCl, 0.52 g L
-1
MgSO4·7H2O, 1.52 g L
-1
KH2PO4, 15 g L
-1
agar, and 1
ml L
-1
Hutner’s trace element solution), or YAG (5 g L
-1
yeast extract, 20 g L
-1
,
D-glucose, 15 g L
-1
agar, and 1 ml L
-1
Hutner’s trace element solution) plates were
supplemented with riboflavin (2.5 mg L
-1
), pyridoxine (0.5 mg L
-1
), or uracil (1 g L
-1
) and
uridine (10 mM) when necessary. Plates were inoculated with 1.0 X 10
7
spores per 10-cm
plate. After 5 days, three plugs (7-mm diameter) were cut out and transferred to a 7-ml
screw-cap vial. The material was extracted with 3 ml of methanol followed by 3 ml of 1:1
dichloromethane-methanol, each with a 1-hr sonication. The extract was transferred to a
clean vial and the solvent was evaporated to dryness by TurboVap LV (Caliper
LifeSciences). The residues were re-dissolved in 0.3 ml of DMSO:MeOH (1:4) and 10 μl
was injected for LC-DAD-MS analysis as described previously (Bok et al., 2009).
MS/MS was conducted with a normalized collision energy of 35 and isolation width of 2
m/z.

For liquid culture, 3  10
7
spores were grown in 30 ml GMM or LMM liquid medium
(recipes same as above except no agar added) in 125 ml flasks at 37ºC with shaking at
180 rpm. After 4 days, culture medium and hyphae were collected by filtration. Culture
medium was extracted with the same volume of EtOAc. In order to extract the most
acidic phenolic compounds, the water layer was extracted with the same volume of
EtOAc again after acidification (pH = 2). Both EtOAc extracts were then evaporated by
TurboVap LV. The residues were re-dissolved in 0.5 ml of DMSO:MeOH (1:4) and
analyzed by LC-DAD-MS as described above. The hyphae collected were soaked in 30
ml of methanol overnight to extract metabolites not secreted out to the liquid medium.
151

The methanol extract was then collected by filtration and the solvent was evaporated by
TurboVap LV. The residues were re-dissolved in 1 ml of DMSO:MeOH (1:4) and
analyzed by LC-DAD-MS as described above.

For alcA(p) induction, see 3.4. Culture medium and hyphae were collected 48 hr after
cyclopentanone induction by filtration, followed by extraction and HPLC-DAD-MS
analysis as described. Fermentation and LC-MS analysis for A. terreus and P. canescens
are listed in the captions to Figure 6-6.

Isolation of Secondary Metabolites
For scaling up to isolate compounds 12 and 13, 50 15-cm GMM plates (3 L of medium in
total) inoculated with A. nidulans strain LO8111 were grown for 5 days at 37°C. The agar
was chopped into pieces and extracted with 2.5 L of MeOH and then 2.5 L of 1:1
dichromethane-methanol as described in Materials and Methods. After removing the
solvent in vacuo, the total crude extract (~5.0 g) was applied to a reverse phase C18 gel
column (COSMOSIL 75C18-OPN, 35  150 mm) and eluted with MeOH-H2O mixtures
of decreasing polarity (fraction A, 1:9, 250 ml; fraction B, 3:7, 250 ml; fraction C, 7:3,
250 ml; fraction D, 1:0, 100 ml). Fraction B (~270 mg) containing compounds 12 and
13 was subjected to purification by semi-preparative reverse phase HPLC [Phenomenex
Luna 5 m C18 (2), 250  10 mm] and monitored by a PDA detector at 236 nm. The
gradient system (5 ml/min) was MeCN (solvent B) in 5% MeCN/H2O (solvent A) with
the following gradient conditions: 5 to 40% B from 0 to 20 min, 40 to 100% B from 20 to
21 min, maintained at 100% B from 21 to 22 min, 100 to 5% B from 22 to 23 min, and
re-equilibration with 5% B from 23 to 27 min. Compounds 12 (2.9 mg) and 13 (4.8 mg)
were eluted at 16.7 and 20.6 min, respectively. Cichorine (10, 6.7 mg) eluted at 12.8 min
was also isolated from this fraction. In addition, 3.0 mg of felinone A (14) eluted at 7.5
min was also isolated.

152

Table 6-1. A. nidulans strains used in this study. All strains carry veA1. Note that since
some of the molecular genetic manipulations are novel, there is no standard nomenclature
for them.
Strain Genotype
LO1362 pyroA4, riboB2, pyrG89, nkuA::argB
LO2804 pyroA4, riboB2, nkuA::argB (LO1362 transformed with a wild-type copy of
A. nidulans pyrG)
LO4389 pyroA4, riboB2, pyrG89, nkuA::argB, sterigmatocystin cluster
(AN7804-AN7825)∆
LO7543 pyroA4, riboB2, pyrG89, nkuA::argB, sterigmatocystin cluster
(AN7804-AN7825)∆, AN1242:: AfriboB_AfpyrG (AN1242 coding
sequence replaced)
LO7668 A riboflavin prototophic mutant of LO7543
LO8030 pyroA4, riboB2, pyrG89, nkuA::argB, sterigmatocystin cluster
(AN7804-AN7825)∆, emericellamide cluster (AN2545-AN2549)∆,
asperfuranone cluster (AN1036-AN1029)∆, monodictyphenone cluster
(AN10023-AN10021)∆, terrequinone cluster (AN8513-AN8520)∆, austinol
cluster part 1 (AN8379-AN8384)∆, austinol cluster part 2
(AN9246-AN9259)∆, F9775 cluster (AN7906-AN7915)∆, asperthecin
cluster (AN6000-AN6002)∆.
LO8111 AN8694 (mcrA)::AfpyroA in LO8030
LO8158-
LO8159
pyroA4, riboB2, pyrG89, nkuA::argB, AN8694 (mcrA)::AfpyroA
LO8162 pyroA4, riboB2, pyrG89, nkuA::argB, sterigmatocystin cluster ∆,
AN1242::AfriboB_AfpyrG (AN1242 coding sequence only replaced),
AN8694 (mcrA)::AfpyroA
LO8166-
LO8167
pyroA4, riboB2, pyrG89, nkuA::argB, AfpyroA-alcA(p)AN8694 [promoter
of AN8694 (mcrA) replaced with the alcA promoter]
LO8936 -LO8937 pyroA4, riboB2, pyrG89, nkuA::argB, AfpyrG-gpdA(p)AN8694 (mcrA)
[promoter of AN8694 (mcrA) replaced with the gpdA promoter]
LO9345 AN6448(pkbA = cicF)::AfpyrG in LO8111
LO10255-LO10257 pyroA4, riboB2, pyrG89, nkuA::argB, sterigmatocystin cluster ∆,
AN1242::AfriboB_AfpyrG (AN1242 coding sequence only replaced),
AN8694 (mcrA)::AfpyroA, yA::ptrA
r
-mcrA (mcrA deleted and reinserted at
the yA locus using pyrithiamine resistance as a selectable marker)
LO10260-LO10262 pyroA4, riboB2, pyrG89, nkuA::argB, AN8694 (mcrA)::AfpyroA,
laeA::AfpyrG (deletion of both mcrA and laeA)
LO10269-LO10271 pyroA4, riboB2, pyrG89, nkuA::argB, AN8694 (mcrA)::AfpyroA,
AtpyrG-gpdA(p)laeA (laeA promoter replaced with the gpdA promoter in a
mcrA deletion strain
153

LO10279-LO10281 pyroA4, riboB2, pyrG89, nkuA::argB, laeA::AtpyrG (laeA deletion)
LO10285-LO10287 pyroA4, riboB2, pyrG89, nkuA::argB, AtpyrG-gpdA(p)laeA (laeA driven by
the gpdA promoter)

154

Figure 6-1. Growth of a WT strain (FGSC4), LO7543, a strain carrying a replacement of
the coding region of AN1242 with the coding sequence of the AfriboB gene and LO8162,
which is a transformant of LO7543 in which AN8694 has been deleted. The strains are
growing on minimal medium supplemented or unsupplemented with riboflavin. In the
absence of riboflavin, LO7543 does not grow, but LO8162, carrying a deletion of
AN8694, grows, revealing that expression of AfriboB at the AN1242 locus has been
activated.

155

Figure 6-2. HPLC paired profile scans of parental strains and AN8694 deletion strains.
[UV-Vis total scan (200 – 600 nm)] (i) Parental strain LO1362, (ii) LO8158, the AN8694
deletion strain created from (i). (iii) parental strain LO7543, (iv) LO8162, the AN8694
deletion strain created from (iii), (v) parental strain LO8030, (vi) LO8111, the AN8694
deletion strain created from (v). All strains were grown on GMM plates. 1 is
sterigmatocystin, 2 is terrequinone, 3 is shamixanthone, 4 is emericellin, 5 is nidulanin A,
6 – 9 are nidulanin A derivatives (see Figure 6-7), 10 is cichorine, 11 is aspercryptin, 14 is
felinone A. Note that 2 and 5 were co-eluted at 31.6 min; 3 and 4 were co-eluted at 39.6
min.

156

Figure 6-3. Effects of alteration of mcrA and laeA expression on SM production. HPLC
UV-Vis total scan (200-600 nm). Growth was on GMM plates and extraction and HPLC
conditions were identical to those used for Figure 6-2. i. parental strain LO1362 (laeA
+
,
mcrA
+
). ii. LO8166 (laeA
+
, alcA(p)mcrA). The glucose in GMM plates represses the alcA
promoter resulting in repression of mcrA transcription and an SM profile similar to the
mcrA deletion strain AN8158 (Figure 6-2 ii). iii. LO10279 (laeAΔ, mcrA
+
). Deletion of
laeA eliminates most SM production. iv. LO10260 (laeAΔ, mcrAΔ). Deletion of mcrA
stimulates the production of SMs even if laeA is deleted. Compare to laeA
+
, mcrAΔ
strains (Figure 6-2 ii) or laeA
+
, mcrA repressed strains (ii). Sterigmatocystin (1) is a
notable exception. However, F9775 A and B (23 and 22) are produced in this strain
whereas they were not detected in laeA
+
, mcrAΔ strains under the same conditions. v.
LO10285 (gpdA(p)laeA, mcrA
+
). Upregulation of laeA driven by the strong constitutive
gpdA promoter results in enhanced SM production relative to the parental strain (i). vi.
L010269 (gpdA(p)laeA, mcrAΔ). Deletion of mcrA combined with overexpression of
laeA results in the production of a new compound (42). For traces iii-vi three separate
genetically identical strains (sister transformants listed in Table 6-1) were analyzed and in
each case the results were very similar for the three strains. Representative traces are
shown. 1 is sterigmatocystin, 2 is terrequinone, 3 is shamixanthone, 4 is emericellin, 5 is
nidulanin A, 6 – 9 are nidulanin A derivatives, 23 is F9775A, 22 is F9775B, 42 is an
unknown compound. Emericellamides were detected by mass spectroscopy in all samples
but they are not UV active and thus not visible in these traces.

157

Figure 6-4. Reinsertion of AN8694 (mcrA) returns secondary metabolite production to
parental levels. Traces are HPLC UV-Vis total scans (200-600 nm). Growth was on
GMM plates and extraction and HPLC conditions were identical to those used for Figure
6-2. i. The parental strain LO7543. ii. Strain LO8162 which carries a deletion of AN8694
in LO7543. Iii. Strain 10255 in which AN8694 has been reinserted at the yA locus of
strain LO8162. All three strains carry a deletion of the sterigmatocystin gene cluster and
replacement of AN1242 with AfriboB. Sterigmatocystin, nidulanin A and pathway
intermediates for these compounds are, thus, not produced. Deletion of AN8694 causes
an increase in production of austinol (16), dehydroaustinol (17), terrequinone (2),
shamixanthone (3) and emericellin (4) (the latter two form a single peak under these
conditions). Insertion of a functional copy of AN8694 at the yA locus returns SM
production to levels seen with the parental strain. The peak at 22 min is not yet identified,
but it does not correlate with AN8694 presence or absence.
158

A.

159

B.

Figure 6-5. HPLC paired profile scans of parental strains and AN8694 deletion strains. A.
HPLC UV-Vis total scan (200 – 600 nm) profiles of AN8694 deletion strains, LO8158
(ii), LO8162 (iv), LO8111 (vi), and their parental strains LO1362 (i), LO7543 (iii),
LO8030 (v) grown on LMM and B. YAG plates. 1 is sterigmatocystin, 2 is terrequinone,
3 is shamixanthone, 4 is emericellin, 5 is nidulanin A, 6 – 9 are nidulanins (see Figure
6-7), 10 is cichorine, 11 is aspercryptin, 12 is N-(4-carboxybutyl)cichorine, 13 is a
O-methyl-3-methylorsellinaldehyde dimer, 14 is felinone A, 15 is austinol, 16 is
dehydroaustinol, 17 – 21 are sterigmatocystin intermediates, 22 is F9775B, 23 is F9775A,
24 is epishamixanthone, 25 and 26 are unknowns, 27 is asperthecin, 28 is emodic acid, 29
is emodin, 30 is an unknown. Notice that 2, 5 and 19 co-eluted at 31.6 min; 3 and 4
co-eluted at 39.6 min. *: lumicrome.
160

Figure 6-6. Deletion of mcrA homologs alters secondary metabolite production in A.
terreus and P . canescens. In each case, deletion of the mcrA homolog alters the secondary
metabolite profile, causing the disappearance of a small number of peaks and the
appearance or enhancement of several peaks. Deletion of mcrA homologs, thus,
stimulates production of compounds not detected in wild-type strains and potentiates
discovery of additional secondary metabolites. All traces are UV-Vis scans 200-600 nm.
A. terreus strains were cultivated at 37 °C in LCMM liquid medium. After 18 h of
incubation, the temperature was switched to 30 °C and the culture medium was collected
after 72 h.
Penicillium canescens strains were cultivated at 26 °C on 10 cm diameter potato dextrose
agar plates inoculated at 1 × 10
7
spores per plate. After 6 days, agar was chopped into
small pieces and extracted with 80 ml 1:1 CH2Cl2/MeOH. The extract was evaporated in
161

vacuo to yield a water residue, which was suspended in 25 ml H2O and partitioned with
EtOAc.
Primers used to create deletions of A. terreus and P . canescens mcrA homologs are shown
in Table 6-2. The molecular genetic manipulations in A. terreus and P . canescens,
including protoplasting and gene targeting, were carried out as previously described (Guo
et al., 2014; Yaegashi et al., 2015).

Strains used for Figure 6-6
Fungal strain
mcrA homolog
deleted
Genotype
Aspergillus terreus NIH2624 - wildtype
CW9002.1, CW9002.2, CW9002.3 ATET_07219.1Δ nkuA:: hph; pyrG-, ATEG_06270.1:: AfpyrG
Penicillium canescens ATCC10419 - wildtype
CW9003.1, CW9003.2, CW9003.3 CE25191_2989Δ ku70::hph; pyrG-, CE25191_2989::PcanpyrG

162

Table 6-2. Primers used to create deletions of A. terreus and P . canescens mcrA homologs
ATET_07219.1P1
CAG AAT TTG GAG AGC AAA GG
ATET_07219.1P2 CCG AGG CTC TTT CTT TTC TC
ATET_07219.1P3
CGA AGA GGG TGA AGA GCA TTG GAT TGC TGG ACA TTG GGT AG
ATET_07219.1P4
CAT CAG TGC CTC CTC TCA GAC AGT TGT GCA TAC GCT ACA GAC C
ATET_07219.1P5
GTC CGA GAA AGA AGC ACA TC
ATET_07219.1P6
AAG GCG CAA TAC CAT AAC C
CE25191_2989P1
AGA CCT CGG GAT TGG CGA
CE25191_2989P2
GAT TGG CGA CGG TCC ACT
CE25191_2989P3
CGA AGA GGG TGA AGA GCA TTG CGC GTA TTG AGG CGG GTA
CE25191_2989P4
CAG TGC CTC CTC TCA GAC AGA GTA GAT CGT GTG CTG GCC AC
CE25191_2989P5
CTC CCT CGC AGG ATG CAG
CE25191_2989P6
CCT GAC GAC CAG CCC AAG

163

164

165

Figure 6-7. UV-Vis and ESIMS (positive or negative mode) spectra of new and unknown
compounds identified in this study. Felinone A (14) has very poor ionization in positive
or negative mode.

166

Chapter VII: Summary, perspectives and future work

In summary, my work represents different aspects of natural product drug discovery and
production from biosynthesis in filamentous fungi, especially Aspergillus species. The
first aspect is to characterize the biosynthesis gene cluster for a significant natural
product, and use its gene cluster as a probe to uncover structurally similar compounds
from other genome sequenced fungi. This is represented by the characterization of the
biosynthesis gene cluster and pathway of terreic acid using the gene deletion approach.
The second aspect is to activate silent gene clusters by promoter replacement to discover
new compounds as potential drug leads. My contribution lies in the adoption of the
Tet-on system as an effective gene activation tool in A. terreus, which allowed us to link
an NRPS-like gene with its product. The third aspect is to modify the target natural
product structure by genetic engineering. Following this aspect, we made efforts to
rationally manipulate acetylaranotin biosynthesis genes to generate second-generation
analogues. Another aspect is to expand the chemical diversity of natural product
biosynthesis by exploring pathway regulation mechanisms in filamentous fungi. My work
contributes to the demonstration of two important natural product biosynthesis regulation
mechanisms, spatial regulation and global regulation.

The natural product drug discovery field thrived after the discovery of the first true
antibiotic, penicillin, by Fleming in 1929, which opened the gate of “the Golden Age of
Antibiotics”. Intensive investigation of nature as a source of novel bioactive agents, in
particular novel drugs, has been promoted thereafter. A significant amount of structurally
diverse bioactive natural products has been contributed to the pharmaceutical industry by
microorganisms. However, in late 1980s, with the breakthroughs made by combinatorial
chemistry, the focus of drug discovery was shifted from natural product biosynthesis to
chemical synthesis. In the new millennium, with fast progress of genome sequencing, the
167

availability of microbial genome sequence information has brought the natural product
drug discovery back to the vision of scientific research, since microbial genome mining
accelerated natural products discovery. The introduction of robotics and automation to
this field also makes high-throughput screening available to add to the acceleration.
Along with the gradual characterization of gene structure and function and the
understanding of gene interaction and regulation, the amount of characterized genes and
gene networks will reach a certain threshold where the correct understanding of
unexplored gene function can be solely dependent on artificial intelligence, as machine
learning can use the experimentally characterized genes as big data to learn how to
identify the function of a new gene. At that time, we will completely unlock the power of
biosynthesis by assembling functional gene elements using computer-aided rational
design to not only discover and produce natural product drugs, but also take place of
chemical plants to produce organic chemicals in an environment-friendly way.

At this moment, we still need to make efforts to expand the knowledge of fungal genetics,
biology, and mechanisms of biosynthesis, because compared to the complexity of fungal
organisms and the rich diversity of fungal species, what we have understood is just a drop
in the sea. The 1000 fungal genome project supported by the Joint Genome Institute (JGI)
made a great effort to provide the scientific research community with a lot of information
to explore. The systematic studies of model fungal organisms like A. nidulans increased
the depth of our understanding of fungi and provide us with research templates. The
development of CRISPR/Cas9 genome editing technique and its introduction to fungal
research offers unparalleled potential for accurate and high throughput manipulations of
fungal genomes. The combination of above progresses starts a new chapter of fungal
biosynthesis research. What I can visualize in the following years is that the previous
hard-to-access fungal biosynthesis gene clusters, pathways and their regulations will be
explored by CRISPR or other newly emergent more efficient and high-throughput gene
168

targeting techniques. Based on the expanded knowledge, novel artificial biological
pathways will be designed and constructed, or the existing natural biological pathways
will be redesigned. The rapid progress of artificial intelligence and its gradual application
will also greatly facilitate the rational design using the design-build-test-analyze-learn
cycle.

Following my research, there are two projects with preliminary discoveries that can be
accomplished in the future. One project is characterization of an azaphilone biosynthesis
pathway using the doxycycline-dependent Tet-on system and CRISPR/Cas9 genome
targeting technique in Aspergillus terreus. Previously we made efforts to systematically
activate the silent PKS and NRPS gene clusters in A. terreus by overexpressing the
predicted cluster-specific transcription factors using the doxycycline-dependent Tet-on
system. We cultured the mutant strains in liquid LCMM under 37 ℃ for 18 hrs, and then
used the concentration of sterile doxycycline at 50 μg/ml as inducer as well as switching
the temperature to 30 ℃ in the meantime. After 72 hrs culture, secondary metabolites
profiles were analyzed by LC-MS using the ethyl acetate (EA) layers after extraction
from the unacidified and acidified LCMM media layer. We discovered that
overexpression of the transcription factor ATEG_03445 would allow the mutant strain to
produce a series of azaphilone secondary metabolites, one of which was isolated and its
structure was elucidated by NMR as compound 1 (Figure 7-1). We were not able to
identify other compounds from this series due to their instability or low yield. The
compound isolated from the single peak by HPLC would split into two or three peaks
which exist in equilibrium, and this might serve as a clue for identifying their structures.
Since ATEG_03445 is in proximity to the PKS ATEG_03446, and the PKS ATEG_03432
which is located not far away also produces a precursor (compound 2, Figure 7-1) of
Monascus azaphilones (Chiang et al., 2013b), we speculated that the gene cluster
containing both ATEG_03446 and ATEG_03432 was overexpressed. The recent
169

bioinformatic analysis studied the gene cluster containing the genes from ATEG_03432
to ATEG_03446, and predicted the biosynthesis pathway according to its homolog gene
cluster, azaA-L and R (EHA28230-28239, 28242-28244) in A. niger ATCC 1015 (Zabala
et al., 2012), which is responsible for azanigerone (another kind of azaphilone)
biosynthesis (Yin et al., 2016). Although the predicted pathway doesn’t include
compound 1, the bioinformatic prediction is still very reasonable, and verified our
speculation. Due to the difficulty of recycling the only available PyrG selection marker,
we were not able to carry out the gene disruption analysis. However, with the
introduction of CRISPR/Cas9 genome editing technique to A. terreus, the
characterization of the speculated gene cluster will become feasible soon.

The other project follows the discovery of the important global regulator, McrA. The
deletion of McrA homologue from P . canescens alters its secondary metabolites
production, stimulating production of compounds 3 and 4, which are not produced by the
wild-type strain cultured under the same conditions (Figure 7-2). By checking the
secondary metabolites list produced by Penicilium species, we figured out that
compounds 3 and 4 have the same mass as griseofulvin and decholorogriseofulvin,
respectively. By comparing compounds 3 and 4 with the standard compounds, we figured
out that they are indeed griseofulvin (3) and decholorogriseofulvin (4) (Figure 7-1).
Griseofulvin is an antifungal drug by inhibiting the function of mitotic spindle
microtubules in mitosis (Odds et al., 2003), and has been used for many years in medical
and veterinary applications. It also has newly discovered anticancer and antiviral
properties (Ho et al., 2001; Jin et al., 2008). The results indicate that McrA is a negative
regulator of the griseofulvin biosynthesis pathway in P . canescens. The gene cluster
containing the PKS gene gsfA 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 GsfA in P . canescens and found that Protein ID 243077 has 90% sequence
170

similarity. The gene deletion analysis performed by our group confirmed that Protein ID
243077 is the PKS responsible for the biosynthesis of griseofulvin in P . canescens since
its deletion led to the disappearance of griseofulvin production. To verify that McrA is a
negative regulator of the griseofulvin biosynthesis pathway, the expression level of
Penca1_243077 (gsfA homolog) was detected by RNA-seq, which shows that its
transcription in the mcrAΔ strain is about 30-fold higher than that in the WT strain. These
data suggest that deletion of mcrA homologs in griseofulvin producer strains is a
promising tool for griseofulvin production. Because griseofulvin is an antifungal drug,
the mcrAΔ griseofulvin producer strains can be used to coculture with pathogen fungi,
like candida ablicans, to test the strains’ bioactivity. In addition, the RNA-seq data of P .
canscens provided plenty of information about the genes regulated by McrA, which can
be explored later. The deletions of mcrA homologs can also be performed to elicit
secondary metabolite production in other ascomycetes.

171

Figure 7-1. The secondary metabolites involved in chapter VII. 3: griseofulvin; 4:
decholorogriseofulvin.

Figure 7-2. The DAD traces of extracts from the P . canescens wildtype and mutant as
detected by UV (200-600 nm).

172

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Abstract (if available)

Abstract Filamentous fungi are well known producers of a wide variety of bioactive secondary metabolites. Genome sequencing projects of filamentous fungi have opened the post-genomic era of genome mining for natural products. Bioinformatic analysis has shown that fungal species have the potential to produce far more secondary metabolites than have already been isolated. The challenge lies in how to unlock this hidden power. Combining bioinformatics, molecular gene targeting, and natural product chemistry in secondary metabolite biosynthesis research, the work herein describes different aspects of natural product discovery: 1) biosynthesis gene cluster characterization, 2) genetic tool development for biosynthesis pathway activation, 3) structural modification of natural product by genetic engineering, and 4) exploration of the complex natural product regulation mechanisms. Using the gene loss-of-function analysis approach, we first characterized the biosynthesis gene cluster for terreic acid, which can also be used as a probe to uncover structurally similar compounds from other genome sequenced fungi. Next, we adopted the Tet-on system as a genetic tool to Aspergillus terreus and successfully activated a previously uncharacterized NRPS-like gene, pgnA, which is responsible for production of phenguignardic acid. Furthermore, we discovered the bis-thiomethylation gene for acetylaranotin, and engineered the acetylaranotin biosynthesis pathway to generate more analogues. Finally, exploring regulation mechanisms of natural product biosynthesis allowed us to discover the importance of spatial regulation and a global regulator, McrA.

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Asset Metadata

Creator Sun, Weiwen (author)

Core Title Genome mining of natural product biosynthesis pathways in filamentous fungi for novel drug discovery and production

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 02/26/2019

Defense Date 05/22/2017

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

Tag acetylaranotin,Aspergillus nidulans,Aspergillus terreus,ataS,azaphilone,biosynthesis,epidithiodiketopiperazine (ETP),filamentous fungi,fungal genetics,gene clusters,genome mining,mcrA,natural products,nonribosomal peptide,OAI-PMH Harvest,Penicillium,pgnA,phenguignardic acid,polyketide,Secondary metabolites,spatial regulation,terreic acid,Tet-on,thiomethyltransferase

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Wang, Clay C.C. (committee chair), Roberts, Richard (committee member), Shen, Wei-Chiang (committee member)

Creator Email weiwensu@usc.edu,weiwensun1989@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-480002

Unique identifier UC11268062

Identifier etd-SunWeiwen-6067.pdf (filename),usctheses-c40-480002 (legacy record id)

Legacy Identifier etd-SunWeiwen-6067.pdf

Dmrecord 480002

Document Type Dissertation

Rights Sun, Weiwen

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