Genetic and chemical characterization of two highly-reducing polyketide synthase clusters from Aspergillus species

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Content i

GENETIC AND CHEMICAL CHARACTERIZATION OF TWO HIGHLY-REDUCING
POLYKETIDE SYNTHASE CLUSTERS FROM ASPERGILLUS SPECIES

by
Tzu-Shyang (Kevin) Lin

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTEHRN CALIFORNIA
In Partial Fulfilment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
MAY 2018
Copyright 2018 Tzu-Shyang (Kevin) Lin

ii

DEDICATION
Soli Deo Gloria

iii

ACKNOWLEDGEMENTS

I would like to thank my mentor and advisor Dr. Clay C. C. Wang for his support and guidance
in the past five years. From the moment I joined his lab he has continually expressed confidence
in me, which gave me confidence to face the challenges I encountered. He pushed me when I got
complacent. He pointed out my weaknesses frankly and motivated me to overcome them. He
taught me to value speed and efficiency. I have learned a lot from his charisma, experiences,
energy, generosity, and leadership style. I am forever grateful for his mentorship – this
dissertation would not have been possible without his constant encouragement, motivation, and
support. Acknowledgements

I would also like to thank the professors who served on my committee – Dr. Ian Haworth, Dr.
Bangyan Stiles, Dr. Pin Wang, Dr. Jianming Xie, and Dr. Tiger Zhang, for their expertise and
insights.

I would like to thank Dr. Yi-Ming Chiang for all his help and support. I am thankful for the
countless hours he spent personally tutoring me organic chemistry, sharpening my critical
thinking, imparting scientific vision, revising manuscripts, troubleshooting my experiments,
fixing instruments, and sharing his experiences and personal insights with me. I am thankful for
our friendship. I would also like to thank Dr. Shu-Lin Chang who trained me the first year and
for his patience and care for me, despite my stubbornness and all the mistakes I made.

iv

I am also thankful for all my colleagues - Jan Van Dijk, Jillian Romsdahl, Michelle Grau, Ada
Blachowicz, and Yien Liao. I respect every one of them very much for their diligence,
intelligence, perseverance, and work-life balance. I have learned a lot from their example, and I
am grateful for such a supportive and friendly work environment. I am also thankful for many
visiting students and volunteers in our lab – Catherine Kozachenko, Stephanie Loekman, Chi-
Ying Li, and Bethany Chen. It was a joy working with them. I am thankful for past members of
the lab, Dr. Chun-Jun Guo, Dr. Junko Yaegashi, Dr. Hsu-Hua Yeh, Dr. James Sanchez, and Dr.
John Gallagher. They are examples I look up to.

I would like to thank USC School of Pharmacy and the Graduate School for financial support.
Last but not least, I would like to thank my family and church family for their invaluable support
and prayers throughout this journey.

Chapter 2 is incorporated from Lin, T., Chiang, Y. and Wang, C. (2016). Biosynthetic Pathway
of the Reduced Polyketide Product Citreoviridin in Aspergillus terreus var. aureus Revealed by
Heterologous Expression in Aspergillus nidulans. Organic Letters, 18(6), pp.1366-1369.

Chapter 3 is incorporated from Lin T., Chen, B., Chiang, Y. and Wang, C. (2018). Discovery and
elucidation of the biosynthesis of aspernidgulenes, novel polyenes from Aspergillus nidulans,
using serial promoter replacement. Manuscript in preparation.

v

TABLE OF CONTENTS
DEDICATION............................................................................................................................... ii
ACKNOWLEDGEMENTS ........................................................................................................ iii
LIST OF TABLES ...................................................................................................................... vii
LIST OF FIGURES ..................................................................................................................... ix
ABSTRACT ................................................................................................................................ xiii
Chapter 1: Introduction ............................................................................................................... 1
1.1 The potential of natural products as a source of drug discovery ........................................... 1
1.2 The importance, role, genetics, and enzymology of fungal secondary metabolites .............. 7
1.3 Genome mining in Aspergillus ........................................................................................... 16
1.4 The development of an efficient gene targeting system and heterologous platform in
Aspergillus nidulans .................................................................................................................. 21
CHAPTER II: Biosynthetic Pathway of the Reduced Polyketide Product Citreoviridin in
Aspergillus terreus var. aureus Revealed by Heterologous Expression in Aspergillus
nidulans ........................................................................................................................................ 29
2.1 Abstract ............................................................................................................................... 29
2.2 Introduction ......................................................................................................................... 29
2.3 Results and Discussion ........................................................................................................ 31
2.4 Supplementary Information................................................................................................. 36

vi

CHAPTER III: Discovery and elucidation of the biosynthesis of aspernidgulenes, novel
polyenes from Aspergillus nidulans, using serial promoter replacement .............................. 70
3.1 Abstract ............................................................................................................................... 70
3.2 Introduction ......................................................................................................................... 70
3.3 Results and Discussion ........................................................................................................ 72
3.4 Supplementary Information................................................................................................. 77
CHAPTER IV: Conclusion and perspective .......................................................................... 161
BIBLIOGRAPHY ..................................................................................................................... 168

vii

LIST OF TABLES
Table 1-1. Mean values for molecular properties of natural, drug, and synthetic compounds. 28
Table 2-1. Citreoviridin (1) biosynthesis genes in A. terreus var. aureus, their homologs in other
species, and gene function predictions. 42
Table 2-S1. Primers used in this study. 44
Table 2-S2. A. nidulans strains used in this study. 46
Table 2-S3.
1
H and
13
C NMR data for compound 2 and 3. 48
Table 3-S1. Aspernidgulene biosynthesis genes in A. nidulans, their homologs in other species,
and gene function predictions. 87
Table 3-S2. Primers used in this study 88
Table 3-S3. Strains used in this study 89
Table 3-S4.
13
C chemical shift data of aspernidgulenes A1 (4) and A2 (5) and proguosene B in
CD3OD. 102
Table 3-S5.
1
H chemical shift data of aspernidgulenes A1 (4) and A2 (5) and proguosene B1 in
CD3OD, unless otherwise indicated. 103
Table 3-S6. NOESY correlation of aspernidgulenes A1 (4) and A2 (5) and proguosene B. 104
Table 3-S7.
1
H and
13
C NMR data for compound 1 105
Table 3-S8.
1
H and
13
C NMR data for compound 2 107
Table 3-S9.
1
H and
13
C NMR data for compound 4 in CD3OD 114
Table 3-S10.
1
H and
13
C NMR data for compound 4 in acetone-D6 124
Table 3-S11.
1
H and
13
C NMR data for compound 5 132
Table 3-S12.
1
H and
13
C NMR data for compound 6 140
Table 3-S13.
1
H and
13
C NMR data for compound 7 146
viii

Table 3-S14.
1
H and
13
C NMR data for compound 8 152
Table 3-S15.
1
H and
13
C NMR data for compound 9 154

ix

LIST OF FIGURES
Figure 2-1. Citreoviridin and other inhibitors of mitochondrial oxidative phosphorylation
produced by different fungal species. ........................................................................................... 40
Figure 2-2. (A) The citreoviridin (1) biosynthesis gene cluster. Black open reading frames
(ORFs) are involved in the biosynthesis while gray ones are not. (B) HPLC profile of metabolites
extracted from the culture media of A. nidulans strains expressing citreoviridin cluster genes
under the control of alcA(p). HPLC analysis was carried out using a C18 reverse phase column.
Detection was at 403 nm. “4x zoomed out” indicates that the y-axis scale is four times larger. . 41
Figure 2-3. Proposed biosynthetic pathway for citreoviridin (1). The more stable allylic
carbocation is shown in the solid-line. .......................................................................................... 43
Figure 2-S1. Production of citreoviridin by A. terreus var. aureus strain CBS 503.65. Detection is
at 403 nm. The identity of citreoviridin was verified by comparison with the retention time, UV-
Vis absorption, and mass spectra of the authentic standard.......................................................... 49
Figure 2-S2. UV-vis and ESIMS spectra of compounds 1 to 3. ................................................... 50
Figure 2-S3. Results of diagnostic PCR for all strains generated in the study. ............................ 51
Figure 2-S4.
1
H NMR spectrum of citreomontanin (2) in CDCl3. ................................................ 52
Figure 2-S5.
13
C NMR spectrum of citreomontanin (2) in CDCl3. .............................................. 52
Figure 2-S6.
1
H NMR spectrum of demethyl-citreomontanin (3) in DMSO-d6. .......................... 53
Figure 2-S7.
13
C NMR spectrum of demethyl-citreomontanin (3) in DMSO-d6. ......................... 54
Figure 3-1. Proposed biosynthetic pathway for asperniduglene A1 (4). ...................................... 84
Figure 3-2. (A) Organization of the aspernidgulene (1) biosynthesis gene cluster. (B) Total scan
HPLC profiles of culture media of A. nidulans strains expressing aspernidgulene biosynthesis
genes under the control of alcA(p)................................................................................................ 85
x

Figure 3-3. (A) New compounds isolated from the study. (B) Structures of representative
compounds containing the oxabicylo[2.2.1]heptane unit or their derivatives. ............................. 86
Figure 3-S1. Expanded total scan HPLC profiles of culture media of A. nidulans strains
expressing(i) sdgACF (ii) sdgADCF (iii) SdgADCF+An1784 under the control of alcA(p). ..... 90
Figure 3-S2. UV-vis and ESIMS spectra of compounds 10-12. ................................................... 91
Figure 3-S3. UV-vis and ESIMS spectra of compounds 13-15. ................................................... 92
Figure 3-S4. UV-vis and ESIMS spectra of compounds 16-18. ................................................... 93
Figure 3-S5. UV-vis and ESIMS spectra of compounds 19. ........................................................ 94
Figure 3-S6. Possible cyclization patterns of (14E, 16E) terminal di-ene after bis-epoxidation. . 95
Figure 3-S7. Possible cyclization patterns of (14E, 16Z) terminal di-ene after bis-epoxidation. . 96
Figure 3-S8. Possible cyclization patterns of (14Z, 16E) terminal di-ene after bis-epoxidation. . 97
Figure 3-S9. Possible cyclization patterns of (14Z,-16Z) terminal di-ene after bis-epoxidation. 98
Figure 3-S10. UV-vis and ESIMS spectra of compounds 1, 2, and 4. ......................................... 99
Figure 3-S11. UV-vis and ESIMS spectra of compounds 5-7. ................................................... 100
Figure 3-S12. UV-vis and ESIMS spectra of compounds 8 and 9. ............................................ 101
Figure 3-S13.
1
H NMR spectrum of compound 1 in CD3OD (400 MHz). ................................. 106
Figure 3-S14.
1
H NMR spectrum of compound 2 in CD3OD (400 MHz). ................................. 108
Figure 3-S15.
13
C NMR spectrum of compound 2 in CD3OD (400 MHz)................................. 109
Figure 3-S16. DEPT spectrum of compound 2 in CD3OD (400 MHz). ..................................... 110
Figure 3-S17. gHMQC spectrum of compound 2 in CD3OD (400 MHz). ................................. 111
Figure 3-S18. gHMBC spectrum of compound 2 in CD3OD (400 MHz). ................................. 112
Figure 3-S19. gCOSY spectrum of compound 2 in CD3OD (400 MHz). .................................. 113
Figure 3-S20.
1
H NMR spectrum of compound 4 in CD3OD (400 MHz). ................................. 115
xi

Figure 3-S21.
13
C NMR spectrum of compound 4 in CD3OD (400 MHz)................................. 116
Figure 3-S22. DEPT CH NMR spectrum of compound 4 in CD3OD (400 MHz). .................... 117
Figure 3-S23. DEPT CHn NMR spectrum of compound 4 in CD3OD (400 MHz). .................. 118
Figure 3-S24 HSQC spectrum of compound 4 in CD3OD (400 MHz). ..................................... 119
Figure 3-S25. gHMQC spectrum of compound 4 in CD3OD (400 MHz). ................................. 120
Figure 3-S26. HMBC spectrum of compound 4 in CD3OD (400 MHz). ................................... 121
Figure 3-S27. COSY spectrum of compound 4 in CD3OD (400 MHz). .................................... 122
Figure 3-S28. NOESY spectrum of compound 4 in CD3OD (400 MHz). .................................. 123
Figure 3-S29.
1
H NMR spectrum of compound 4 in acetone-D6 (400 MHz). ............................ 125
Figure 3-S30.
13
C NMR spectrum of compound 4 in acetone-D6 (400 MHz). .......................... 126
Figure 3-S31. HSQC spectrum of compound 4 in acetone-D6 (400 MHz). ............................... 127
Figure 3-S32. gHMQC spectrum of compound 4 in acetone-D6 (400 MHz). ............................ 128
Figure 3-S33. HMBC spectrum of compound 4 in acetone-D6 (400 MHz). .............................. 129
Figure 3-S34 COSY spectrum of compound 4 in acetone-D6 (400 MHz). ................................ 130
Figure 3-S35 NOESY spectrum of compound 4 in acetone-D6 (400 MHz). .............................. 131
Figure 3-S36.
1
H spectrum of compound 5 in CD3OD (600 MHz). ........................................... 133
Figure 3-S37.
13
C spectrum of compound 5 in CD3OD (600 MHz). .......................................... 134
Figure 3-S38. HSQC spectrum of compound 5 in CD3OD (600 MHz). .................................... 135
Figure 3-S39. gHMQC spectrum of compound 5 in CD3OD (600 MHz). ................................. 136
Figure 3-S40. HMBC spectrum of compound 5 in CD3OD (600 MHz). ................................... 137
Figure 3-S41. COSY spectrum of compound 5 in CD3OD (600 MHz). .................................... 138
Figure 3-S42. NOESY spectrum of compound 5 in CD3OD (600 MHz). .................................. 139
Figure 3-S43.
1
H spectrum of compound 6 in CD3OD (400 MHz). ........................................... 141
xii

Figure 3-S44.
13
C spectrum of compound 6 in CD3OD (400 MHz). .......................................... 142
Figure 3-S45. HSQC spectrum of compound 6 in CD3OD (400 MHz). .................................... 143
Figure 3-S46. HMBC spectrum of compound 6 in CD3OD (400 MHz). ................................... 144
Figure 3-S47. COSY spectrum of compound 6 in CD3OD (400 MHz). .................................... 145
Figure 3-S48.
1
H NMR spectrum of compound 7 in CD3OD (400 MHz). ................................. 147
Figure 3-S49.
13
C NMR spectrum of compound 7 in CD3OD (400 MHz)................................. 148
Figure 3-S50. HSQC NMR spectrum of compound 7 in CD3OD (400 MHz). .......................... 149
Figure 3-S51. HMBC NMR spectrum of compound 7 in CD3OD (400 MHz). ......................... 150
Figure 3-S52. COSY NMR spectrum of compound 7 in CD3OD (400 MHz). .......................... 151
Figure 3-S53.
1
H spectrum of compound 8 in CDCl3 (400 MHz). ............................................. 153
Figure 3-S54.
1
H spectrum of compound 9 in CDCl3 (600 MHz). ............................................. 155
Figure 3-S55.
13
C spectrum of compound 9 in CDCl3 (600 MHz). ............................................ 156
Figure 3-S56. HSQC spectrum of compound 9 in CDCl3 (600 MHz). ...................................... 157
Figure 3-S57. gHMQC spectrum of compound 9 in CDCl3 (600 MHz). ................................... 158
Figure 3-S58. HMBC spectrum of compound 9 in CDCl3 (600 MHz). ..................................... 159
Figure 3-S59. COSY spectrum of compound 9 in CDCl3 (600 MHz). ...................................... 160

xiii

ABSTRACT

Filamentous fungi have historically been a rich source of therapeutically relevant natural
products. Yet genome sequencing has revealed that the secondary metabolite capacity of
filamentous fungi remains largely untapped. Many more bioactive secondary metabolites await
discovery, with potentially valuable entities in the midst.

Two main goals of natural product research are the linking of known metabolites to their gene
clusters and the discovery of novel secondary metabolites. The first goal is important because
understanding the biosynthesis of complex organic molecules can inspire biomimetic synthesis
strategies. Furthermore, biosynthetic enzymes can act as useful biocatalysts that perform
challenging reactions in short, efficient routes. The second goal is important because all
secondary metabolites are inherently biologically relevant. Even if their function is currently
unknown, future discoveries can shed light on their utility, whether in medicine, agriculture, or
some other field. Moreover, the structures found in nature are often beyond the imagination of
medicinal chemists. Thus, they can inspire new scaffolds.

The work herein describes both the elucidation of the biosynthetic genes of a known mycotoxin
and the discovery of several new metabolites from A. nidulans. First, the biosynthetic genes of
citreoviridin, a potent mycotoxin that has caused significant health problems around the world,
were identified and verified experimentally by heterologous expression. The heterologous
expression platform used was a well-established A. nidulans system that has a clean background,
recyclable selection markers, and facile genetic amenability. Second, a silent gene cluster in A.
nidulans was turned on through serial promoter replacement, leading to the discovery of several
xiv

novel polyketide products, the aspernidgulenes. Their biosynthesis was proposed, representing
the first the genetic and enzymatic study for this class of compounds. Interestingly, while the
aspernidgulenes biosynthetic genes have significant homology with that of citreoviridin,
significant diversification of structure was achieved by the difference of two tailoring enzymes.

1

Chapter 1: Introduction

1.1 The potential of natural products as a source of drug discovery
Brief history of natural products
Ancient records indicate that humans have used natural products (NPs) as a source of medicine
for millennia. Records from Mesopotamia (2600 B.C.), Egypt (2900 B.C.), China (1100 B.C.),
India (1000 B.C.), Greece (100 A.D.) document the usage of medicinal herbs, oils, and
ointments.
1
The 1950’s and 1960’s was heralded as the Golden Age of natural product
discovery, with Nobel Prizes award to the scientists who discovered penicillin and
streptomycin.
2
The discovery of paclitaxel in 1971 was another success story for NPs.
3

Big Pharma’s decreased emphasis on natural product drug discovery
However, starting in the 1990’s and continuing through the early 2000’s, the many of the large
companies in the pharmaceutical industry have been shifting away from natural product (NP) as
a source of drugs leads.
4
Companies such as Bristol Myers, Merck, Johnson and Johnson, Pfizer,
Roche, Eli Lilly, and Bayer have either significantly downsized or completely decommissioned
their natural products program.
5
Instead, combinatory chemistry and diversity-oriented synthesis
were used to generate large compound libraries for high throughput screening (HTS).
6

There are several reasons why the industry moved away from natural products. These include the
incompatibility of whole crude extracts with HTS biochemical screening;
7

8

9
the time, cost, and
effort associated with fractionation of crude extracts and purification of leads;
10
the frequent re-
discovery of known structures;
11
problems with harvesting and physically accessing the source of
2

the natural products due to ecological, regulatory, geographical, and financial considerations;
1213

the minute quantities many promising lead compounds are made in;
14
the challenges involved in
the synthesis of low-quantity natural products that are structurally complex;
15
the shorter
timeline of high throughput screening of synthetic libraries;
16
and the perception that all the easy
NP discoveries have already been made.
17
One paper suggests that natural products do not fit
with the modern drug discovery paradigm in two fundamental ways: that are not compatible with
high-throughput screening and they are not easily amenable to de novo analogue synthesis.
18

The contribution of natural products in the last four decades
Despite marginalization by industry, NPs continued to be a significant pipeline for therapeutics.
In a survey of all approved small-molecule drugs between January 1, 1981 and December 31,
2014, it was found that 6% comes from unaltered natural product and 26% comes from natural
product derivatives.
19

The advantages of natural products
The continued contribution of NPs to drug discovery comes in the backdrop of unrealized
expectations for combinatory chemistry.
20

21

22

23
Prior to the 1990’s, pharmaceutical companies
employed functional whole-cell biological assays. With the advent of high throughput
biochemical assays, companies started to develop high-throughput synthesis
programs.
24
Combinatory chemistry allowed for the rapid assembly of large defined libraries of
structurally related compounds that can be screened simultaneously. This platform was in
contrast with natural products investigation, which required time-consuming screening of
extracts, bioassay-guided isolation, and purification and structural elucidation. The hope and
3

expectation was that these libraries of millions of combinatorial molecules would produce as
many or even more leads than traditional libraries.
25

However, the combinatorial design platform did not result in an increase in lead candidates or
drugs.
26
It was soon recognized that sheer numbers and raw speed are not enough; the quality of
screening libraries matters. Qualities such as diversity
27
and drug-likeness
28
need to be taken into
consideration in library design. As a result in the recent year there has been renewed interest in
natural product-based drug discovery.
29
The following section discusses the merits of NPs and
how NPs can contribute to the drug discovery process.

Inherently bioactive
One advantage of using natural products as a starting point for the discovery of drugs is that NPs,
by definition, have biological function. The genetic cost and metabolic energy associated with
NP production are only justified if the NPs confer some survival benefit to the organism, such as
defense against predators or signaling within its own community.
30
One review even renames
natural products as “naturally occurring ligands” to highlight their biological utility.
31
One
hypothesis states that all NPs have some receptor-binding activity.
32
While the biological function
of many NPs are not known, their inherent biological relevance makes them good starting points
for drug design.
33

To achieve their biological activities, many NPs are fine-tuned to interact with
biomacromolecules.
34
Some NPs have extremely high potency and high selectivity due to
evolutionary selection that have optimized their three-dimensional structures and functional
4

groups to bind to specific targets.
35
Since it is common for binding pockets or folds to be
conserved across species, a NP originally tended to target a microorganism can, in many
instances, bind to human proteins. An example is cyclosporin and rapamycin, which are likely
originally synthesized as chemical weapons, but have been utilized by humans as
immunosuppressants.
36

Another feature of natural products is that in many cases they are substrates for transporters.
37
In
fact, it is for this reason that natural products are exempt from the Lipinski’s rule of five,
38,39
which is used to guide the design of molecules with favorable physico-chemical properties.
40
A
review speculates that NPs have evolved to hitchhike on transporters to cross the cellular
membrane to reach their biological targets.
41
This feature of natural products is what, at least in
part, motivates projects to evaluate the metabolite-likeness of libraries.
42

The inherent biological relevance of NPs has several practical implications on drug discovery.

Firstly, since NPs fall into biologically relevant chemical space that has already been explored by
nature, the size of a NP library is significantly smaller than that of a synthetic library. An
analysis published in 2007 on the 126,140 unique compounds in the Dictionary of Natural
Products found that 60% do not violate Lipinski’s Rule of Five.
43
As of January 2018, there are
about 274,000 unique natural products in the Dictionary of Natural Products.
44
Assuming 60% of
them satisfy Lipinski’s rule of five, we are left with 164,400 bioactive compounds. That library
is smaller than a typical synthetic library, which can involve millions of compounds with no
guaranteed bioactivity
45
.
5

Secondly, because NPs are biologically validated structural entities, they have higher hit rates in
screening. An analysis of 35 Novartis screens that found natural products to have significantly
higher hit rates than synthetic or combinatorial libraries.
46
One review argues since proteins have
evolved to recognize NPs, the reason why HTS is able to find any hits at all is because the
libraries used are biased towards molecules that resemble NPs.
47
Thus, it has been suggested that
designing compound libraries to mimic the structures and properties of NP can potentially
increase hit rates.
48
A natural product-likeness score has been developed to assess a library’s
similarity to natural product space in order to help introduce NP-like features into libraries.
49

Occupy different chemical space
Besides occupying biologically relevant chemical space, natural products also occupy a different
and complementary chemical space than combinatorial libraries.
50
NPs offer an incredible
structural diversity unrivaled by the creativity of medicinal chemists.
51
In fact, it is generally
accepted that NPs are more structurally complex than synthetic molecules,
52
which are
constrained by factors that affect the efficiency of combinatory such as availability of reagents
and the suitability of reactions.
53

These synthetic limitations lead to certain structural differences between NPs and combinatorial
compounds. Several pharmaceutical companies, including Bayer, Roche, New Chem Entities,
and Signal Gene have conducted statistical comparison of natural products, drugs, and synthetic
compounds.
54555657
They drew overall conclusions that are remarkably similar. A review
summarized the computation results in Table 1-1.
58

6

For instance, due to the difficulties associated with chiral separation in combinatorial synthesis,
synthetic compounds have a much lower average number of chiral centers compared to NPs.
Other examples include the distribution of molecular weights (skewed towards the higher end for
NPs), the number of aromatic rings, and the ratio of nitrogen and oxygen (which affects average
lipophilicity).

It was concluded that certain properties of combinatorial libraries such as fewer chiral centers,
more rotatable bonds, and smaller molecular weight distribution as compared to NP is overall
unfavorable.
59
Since biological targets are usually stereospecific, chiral centers would confer
selectivity. Rigid ligands exhibit stronger binding due to lower entropic less, thus rotatable bonds
that confer flexibility are negatively impact binding. Thus, the review recommended that a
“natural-product-like” filter, much like a “drug-like” filter can be applied to combinatory
libraries. Similar recognition was made by Lee et al, who proposed that the novel scaffolds in
natural products should be incorporated into combinatorial libraries.
60
One aim, the review
suggested, would be to lower the overall nitrogen count of synthetic compounds, which is
significantly higher than that of natural products.

Lastly, Feher et al. conducted a principal component analysis to visualize how diverse
combinatorial compounds are compared to natural products based on these properties.
61
Their
findings are insightful. First, combinatorial compounds are significantly less diverse than natural
products and drugs, occupying less space on the plot. Second, much of the area covered by
7

natural products are not covered by synthetic compounds. Third, the areas where combinatorial
compounds are most diverse is not covered by natural products.

These statistical studies highlight the fact that natural products can contribute to drug discovery
in at least two ways: first, by guiding the design of combinatorial libraries to have property
distributions that result in greater diversity and higher biological relevance; second, by offering
novel chemical structures not found in combinatorial libraries.

1.2 The importance, role, genetics, and enzymology of fungal secondary metabolites
Fungal secondary metabolites are important pharmaceutically and agriculturally
Fungi are a rich source of natural products. It is known that the ability to produce secondary
metabolites is unevenly distributed among all groups of living organism. A survey of all known
antibiotics and bioactive compounds (22,500 at the time) showed that 17% came from
prokaryotic, unicellular bacteria, 45% came from filamentous actinomycetes, and 38% came
from fungi.
62
Fungi are the producers of many natural products that are important
pharmaceutically and agriculturally.
63

In terms of pharmaceutics, examples include the antibiotic penicillin
64
, the cholesterol-lowering
agent lovastatin,
65
the antifungal agent griseofulvin,
66
and the immunosuppressant agents
cyclosporine and mycophenolic acid.
67

68
Ergotamine has been used to treat acute migraine since
1926.
69
Fingolimod, a chemical derivative of the fungal NP myriocin, is the first approved oral
therapy for multiple sclerosis.
70
The echinocandins are a class of drugs approved for invasive
fungal infections.
71

8

In terms of agriculture, beauvericin is the active compound produced by entomopathogenic fungi
that are used to control pests.
7273
Fumagillin has been used to treat nosemosis in honey bees for
nearly 60 years.
74
Gibberellin, initially discovered as a fungal metabolite, was later identified to
be a plant hormone, and alternations of its signaling led to development of seedless fruits.
75

76

Furthermore, fungal natural products play an important role in the virulence and lifestyle of
phytopathogenic, which invade crops both pre- and post-harvest and cause severe economic
damage.
77
For example, the fungi Cochliobolus heterostrophus race T, which causes southern
corn leaf blight, requires T-toxin for its high virulence.
78
Additionally, the mycotoxins
themselves (for example, aflatoxin and citrinin) can affect the seed quality, germination, and
development of cereal grain, leading to loss of crops.
79

Natural products are secondary metabolites
All the natural products produced by fungi are classified as secondary metabolites (SMs).
Secondary metabolites contrast with primary metabolites, which are necessary for cellular
structure, maintenance, and reproduction. Primary metabolites are often ubiquitous in all living
organisms. Examples of primary metabolites include adenosine triphosphate, essential amino
acids, and sugars. Secondary metabolites, on the other hand, are not essential to the growth and
life of the producing organism. There is great variety among different organisms with regards to
the secondary metabolites made.
80
The differences between primary and secondary metabolites
are summarized well when Campbell defined primary metabolites as “ancient, successful, and
general solutions to global biological problems” and Bennette et al. said that secondary
9

metabolites “represent the splendid, idiosyncratic diversity of nature, endowing different species
with specific solutions to biological problems.”
81

82

The role of fungal secondary metabolites
Because fungi live in complex ecological communities consisting of many other organisms, SMs
confer a survival advantage to the producing fungi by providing protection, serving as signaling
molecules, or acting as virulence factors in plants and animals.
83
In many cases, the benefits SMs
confer are unknown. However, there are also many SMs with known functions. They include
antibiotics, insecticides, quorum sensing molecules, sporogenic factors, virulence factors, and
pigments that confer protection from exogeneous stresses such as radiation,
84
reactive oxygen
species,
85
and microbicidal peptides.
86

Classically, antibiotics are viewed as inter-microbial chemical weapons,
87
and this position is
supported by a recent study.
88
Penicillin is the most famous example of a fungal antibiotic. It has
been speculated that its production is coordinated with the production of the insecticides
aflatoxin and sterigmatocystin to help fungi secure a niche.
89
During fungal development,
penicillin is thought to be secreted into the environment to first eliminate fast-growing
prokaryotes. After the fungi has established itself, penicillin production stops and
sterigmatocystin is secreted to defend the fungi against eukaryotic predators.

However, an alternative hypothesis has emerged that stated that antibiotics are also signaling
agents that “regulate the homeostasis of microbial communities.”
90
This alternative hypothesis
was developed at least in part due to the observation that antibiotics at low concentrations
10

modulate bacterial transcription, triggering adaptive responses.
91
Since it is debatable whether in
ecological contexts antibiotics are produced in concentrations sufficient to exert their weapon
function,
92
antibiotics are proposed to meditate cooperation between diverse species.

Fungal secondary metabolites can also act as quorum sensing (QS) molecules or inhibitors of
quorum sensing. QS is a cell-cell communication system that was first discovered in bacteria. QS
molecules, also called autoinducers, are released by bacteria, and the concentration of these QS
molecules increases proportionally with bacterial population.
93
At a certain threshold stimulatory
concentration, the QS molecules modulate the gene expression of the entire community,
coordinating behaviors such as virulence, antibiotic production, bioluminescent, motility,
sporulation, and biofilm formation.
94

In fungi, QS molecules or molecule that have QS-like activities are known to influence
sporulation, development, and SM production. Psi factors, which are hydroxylated unsaturated C
fatty acids, are known to induce premature sexual sporulation in A. nidulans.
95
Researchers also
found that farnesol prevents mycelial development in Candida albicans.
96
A few years later
tyrosol was found to exert the opposite effect in stimulating germ-tube formation in C.
albicans.
97
Moreover, the mycotoxin zearalenone is known to enhance the formation of
perithecium in Fusarium graminaerum, and inhibition of its synthesis inhibited sexual
development.
98
Butyrolactone is an autoinducer and causes increased lovastatin production,
hyphal branching, and sporulation.
99

100

101

11

Other fungal SMs have the opposite function of inhibiting bacterial QS. A study screened 100
extracts from 50 Penicillium species and found that 33 had QS inhibitory activity.
102
From these
extracts, penicillic acid and patulin were isolated and the inhibitory activities of the purified
compounds were confirmed. The quorum quenching activities of these SM are thought to serve
as chemical defense for fungi, which do not have immune systems.

Lastly, fungal secondary metabolites are also virulence factors. For example, a studied showed
how DHN-melanin serves an important role in A. fumigatus pathogenicity.
103
It first inhibits
phagolysosomal acidification to increase the survival rate of the phagocytosed conidia. Then it
also inhibits phagocyte apoptosis to provide an intracellular niche for conidia survival. Another
molecule, siderophore, which is used for iron uptake and storage is also critical for virulence.
104

It is essential due to the limited iron availability in the host. Recently, a secreted peptide called
Qsp1 was identified and found to regulate the virulence of fungal pathogen Cryptococcus
neoformans.
105

Other than melanin and siderophores, the mechanisms of how other SMs contribute to fungal
virulence are unclear. However, experiments showing upregulation of certain SMs during
infection and experiments implicate these SMs in the fungus-host interaction process. These SMs
include destruxin,
106
beauvericin,
107
gliotoxin, fumitremorgin, fumagillin, and pseurotin.
108

The genetics of fungal secondary metabolites
Despite their remarkable structural diversity, most fungal SMs can be categorized into one of the
four classes: polyketides, non-ribosomal peptides, terpenes, and indole alkaloids.
109
The carbon
12

skeleton of SMs are made by backbone enzymes, and they are polyketide synthase (PKS) for
polyketides, non-ribosomal peptide synthetases (NRPSs) for non-ribosomal peptides and indole
alkaloids,
110
and terpene cyclases for terpenes. These backbone enzymes iteratively incorporate
monomeric units into a growing chain, and their enzymology is discussed later in this section.

Besides the backbone enzymes, other enzymes are involved in the biosynthesis of SMs.
Tailoring enzymes modify the carbon skeleton produced by the backbone enzyme. The
modifications they perform are diverse, including methylation, acecylation, prenylation,
halogenation, hydroxylation, epoxidation, as well as oxidative cleavage and rearrangement.
111

Other enzymes are responsible for the generation of the substrates of the backbone enzymes. In
the biosynthesis of ergotamine, tryptophan is prenylated by dimethylallyl tryptophan synthetase
(DMATS) and subsequently undergoes transformation into lysergic acid, which is then loaded
onto the NRPS for condensation.
112

Transporter and transcriptional regulator genes are also common in fungal biosynthesis.
Transporters can serve to rid the cell of toxin compounds, as in the case of the efflux pump
(GliA) in the gliotoxin gene cluster.
113
They can also transporter precursors to subcellular
destinations, as in the case of penicillin biosynthesis. A transporter PaaT putatively transports
side-chain precursors phenylacetic acid and pheoxyacetic acid to the peroxisome for the
biosynthesis of the antibiotic.
114

13

In terms of transcriptional regulators, there are both global and pathway-specific regulators of
SM biosynthesis. LaeA is called a “global” regulator of SM biosynthesis in Aspergillus genus
because it controls the synthesis of multiple SMs. Deletion of LaeA led to the loss of
sterigmatocystin, penicillin, and lovastatin production.
115
MrcA is another global regulator, but
its effect is negative – deletion of it led to the stimulation of the production of many SMs.
116
On
the other hand, an example of a pathway-specific regulator is AfoA, which activates the genes
involved in the asperfuranone pathway.
117

Still others genes are responsible for self-resistance against the SMs produced. Since many
fungal SMs are antifungals, how do the producing strains shield themselves from the toxicity of
the antifungals they produce? It turns out that self-defense genes are encoded in many fungal
biosynthetic genes clusters. These self-defense genes can confer self-resistance to the producing
strain by various means, including duplication of the target of the antifungal, enzymes that
chemically modify the antifungal to neutralize the effect, and transporters that export the
toxin.
118

For example, the antifungal activity of lovastatin comes from its ability to inhibit HMG-CoA
reductase and thus the biosynthesis of fungal sterol ergosterol, which is an essential cell-
membrane component.
119
To protect the producing strain, a second copy of HMG-CoA (lvrA) is
found in the lovastatin gene cluster of Aspergillus terreus, and that copy is thought to be resistant
to the toxin.
120
Other examples of putative resistance genes include methionine aminopeptidase-2
(target of fumagillin) and proteasome subunit (target of fellutamide B).
121

122
Another example of
self-resistance (F1-ATPase β subunit is the target of citreoviridin) is found in chapter 2.
14

One characteristic of fungal SMs biosynthesis is that the biosynthesis genes are clustered,
meaning they are located physically close to each other on the chromosome like bacterial
operons.
123
One possible reason for this feature is that SM clusters were originally transferred to
fungi ancestors from bacteria, and clustering has been maintained because it allows for
coordinated transcription regulation by chromatin-based mechanisms and thus minimizes the
number of regulatory steps needed.
124
An evidence that supports this hypothesis is the
observations that horizontal gene transfer events of entire clusters occurred for β-lactam
antibiotics and sterigmatocystin.
125

126
The significance of fungi gene clustering will be discussed
in the next section 1.3.

Enzymatic logic of polyketide synthases and nonribosomal peptide synthetases
Most fungal SMs are produced by either polyketide synthases (PKSs), nonribosomal peptide
synthetases (NRPSs), or hybrids of the two (PKS-NRPSs are most common; recently an NRPS-
PKS hybrid was discovered).
127
Both PKSs and NRPSs incorporate monomer units into a
growing oligomer chain in iterative elongation steps.
128
For PKSs, the monomer units are often
malonyl-CoA or methylmalonyl-CoA, and the chain start units can be acetyl-coA, propionyl-
CoA, or benzyl-CoA. For NRPSs, the monomer units include the 20 proteinogenic amino acids
and nonproteinogenic amino acids as well. In this discussion I will focus on the enzymology of
PKSs.

There are three subtypes of PKSs: types I, II, and III.
129
Type I PKSs are multidomain enzymes
with catalytic domains tethered together. On the other hand, type II and III PKSs are complexes
15

consisting of free-standing catalytic components. Type III PKSs are homodimers, and each
monomer consists of a single KS domain that catalyzes priming, extension, and cyclization
iteratively.
130
PKSs can also be classified as iterative or noniterative depending on whether a
catalytic domain is used during more than one round of iteration. To date, all fungal PKSs are
type I. Most of them are iterative, with a few exceptions such as the diketide synthases in
lovastatin (LovF) and compactin (MlcB) biosynthesis.
131

The enzymology of PKSs parallel that of fatty acid synthases, but differs in the existence of
incomplete reduction of the carbon chain.
132
The minimal domains are the ketosynthase (KS),
acyltransferase (AT), and acyl carrier protein (ACP) domains. First, a starter unit is loaded on the
KS domain. Subsequently, a malonyl-CoA starter unit is transferred to the phosphopantetheine
arm of the ACP domain by the AT domain. The KS domain then catalyzed Claisen condensation
of the malonyl extender unit to covalently link the starter and extender unit to produce a diketide.

Fungal type I PKSs are further divided into two subclasses: non-reducing (NR) PKSs and highly
reducing (HR) PKSs. These two subclasses differ by their domain architectures and the level of
reduction of their polyketide products. HR-PKSs, in addition to the minimal domains, contain
domains that modify the growing polyketide chain: the C-methyltransferase (CMet) domain, the
ketoreductase (KR), dehydratase (DH), and enoyl reductase (ER) domains. In the example of a
diketide, the CMet domain methylates the α-carbon of the diketide using S-adenosylmethionine
as a substrate. The KR domain would reduce the β-keto group to β-hydroxyl, which the DH
domain would reduce to α,β-unsaturated carbonyl, which the ER domain would reduce to α,β-
saturated carbonyl. These domains are not necessarily active in every elongation step in HR-
16

PKSs; instead, there seems to be some programming that allows each domain to selectively act
on certain steps. The rules and basis of this programming remains unknown.

NR-PKSs, on the other hand, contain the thioesterase/Claisen cyclase (TE/CLC) domain, the
starter unit-ACP transacylase (SAT) domain, and the product template (PT) domain. The
TE/CLC domain is a Claisen cyclase responsible for product release. The SAT domain is
responsible for selecting the acyl starter unit.
133
The PT domain is responsible for controlling the
reactivity of the poly-β-keto intermediates inside the reaction chamber to yield the correct aldol
cyclization or aromatization pattern.
134

1.3 Genome mining in Aspergillus
Genome mining vs. find and grind
Prior to the genomic age, SM discovery was driven largely by chance.
135
The process has been
called “grind and find.” The predominant methods were bioactivity-guided fractionation or by
chemical screening. The former involves several cycles of fractionation followed by bioassays to
identify and isolate the pure active component.
136
The latter involves isolating
chromatographically resolvable peaks with interesting spectroscopic properties. As the catalogue
of readily identified SMs grew, strategies were developed to minimize chance in the process.
These strategies included exploring new ecologies, judicious selection of genera, and developing
more sensitive analytical tools.
137

The development of genome mining, the process of translating biosynthetic genes to purified
molecules,
138
revolutionized SM discovery by potentially promising to eliminate the component
17

of chance. Whereas no structural information of the SMs produced could be known prior to
extraction in the pre-genomic era, genome sequences now provide clues about the compounds
produced by an organism even before it is cultivated. This ability minimizes the risk of re-
isolating known compounds.
139
Thus, the success of genome mining is determined by the degree
to which biosynthetic genes can be activated and correlated to an encoded structure.

The goals of genome mining
The goal of genome mining is three-fold. First, to be able to accurately predict SM structure
based on DNA sequence information. Second, to be able to accurately predict SM biosynthetic
enzyme function based on DNA sequence information. Third, to be able to rationally design
biosynthetic pathways of the desired molecule.
140
Currently, more experimental data are needed
to be able to reliably predict the SM structure or enzyme function based on sequence; more gene-
protein-SM connections need to be made.
141

Establishing the connection between SM genes and their encoded molecules have additional
benefits. They include inspiring more efficient total synthesis routes; facilitation metabolic
engineering to increase the yield of valuable compounds or engineering derivatives; discovering
potential green biocatalysts; discovering structurally-related compounds in other fungi; and
understanding the ecological functions of the SMs.
142

The SM potential of fungi based on genome sequencing
Genome mining in fungi is facilitated by the clustering feature of fungal SMs biosynthetic genes.
First, the clustering of genes allows researchers to quickly identify all the genes putatively
18

involved in the biosynthesis of a compound by first scanning for backbone enzymes and then
examining the functions of the nearby genes. The involvement of these putative genes can then
be verified by via knock out or heterologous expression experiments.

Second, gene clustering allows researchers to quickly estimate the SM production potential of a
given species by simply counting the number of backbone enzymes present. Indeed, shortly after
the genomes of the first Aspergillus species were sequenced and published, it became apparent
that the number of SM clusters vastly outnumbered the number of known SMs.
143
Clusters that
are not expressed or expressed at low levels under laboratory conditions are termed “silent” and
clusters not connected to a known metabolite are termed “orphan.”
144
The majority of SM
clusters in fungi currently fall into those two categories.
145

The discovery that the SM potential of fungal SM is largely untapped has spurred the
development of many strategies to “activate” or “awaken” those silent clusters in the hopes of
finding therapeutically relevant molecules. These strategies include the one strain-many-
compound approach, activation of in-cluster transcription factors, epigenetic manipulation,
chemical epigenetics, heterologous expression, and co-culturing.
146
In chapter 3 the activation of
a silent cluster, the aspernidgulenes cluster, via serial promoter replacement is discussed in
detail.

Furthermore, many strategies have also been developed to link orphan clusters to known
compounds. These include comparative metabolic profiling,
147
the genomisotopic approach,
chemical structure-based gene targeting, and the proteomic approach. In brief, In chapter 2 the
19

use of chemical structure-based gene targeting to link citreoviridin to its orphan cluster is
discussed in detail.

Advent of affordable genome sequencing
Large-scale, modern genome mining is made possible by the development of next-generation
sequencing, which sharply lowered the cost of whole-genome sequencing of fungi.
148
This
development allowed individual labs to sequence their own organisms of interest. Furthermore,
the undertaking of large-scale sequencing projects such as the 1000 Fungal Genomes Project
means that tens, if not hundreds, of thousands of orphan gene clusters will become available to
researchers in the upcoming years.
149
The potential of discovering new structures from fungal
genomes is thus largely untapped.

The Aspergillus genus
Aspergillus is one of the most studied genera of mold. It received its name came from Antonio
Micheli in 1729, who observed the resemblance of the spore-forming structure to the
aspergillum, an instrument used to disperse holy water by Catholic priests.
150

151
Members of this
genus are mainly saprophytes that grow on decomposing organic matter such as plant debris. The
Aspergilli have attracted the attention of biologists and chemical biologists for several reasons.
These include their ubiquity in nature, the ease with which they could be cultivated in the
laboratory, their industrial and agricultural significance, the pathogenicity of some species, and
their historic use in east Asian cuisine.
152

153

20

This genus has benefited humans immensely. A. niger is used as “cell factories” for the industrial
production of extracellular enzymes (e.g. amylases) and chemicals (e.g. citric acid and
gluconic).
154

155
A. oryzae, A. sojae, and A. kawachii have been used to ferment alcohol
beverages, miso, soy sauce for hundreds of years.
156
A. nidulans is an important eukaryotic model
for genetics and cell biology.
157

At the same time, the Aspergilli have caused much destruction. A. fumigatus is a deadly airborne
pathogen due to its production of aerosolized conidia (spores). Inhalation of conidia by
immunocompromised patients can lead to invasive aspergillosis, which has a mortality rate as
high as > 85%.
158

159

160
A. flavus is also a plant pathogen that infects peanuts, corn, and cotton. It
produces the potent carcinogen aflatoxin in the seeds of crops before harvest and in storage that
causes severe economic damage.
161
Between 2004-2005 there were more than 150 reported cases
of human death related to the consumption contaminated maize.
162

The Aspergillus genus also produces many important secondary metabolites, and thus have been
closely studied for their SM production potential. The first statin approved for human use,
lovastatin, was isolated from A. terreus.
163
Kojic acid, which is extensively used as a skin
lightening agent in cosmetic products in Asia, was originally isolated from A. oryzae.
164

Mycotoxins such as aflatoxins (A. flavus and A. parasiticus) and ochratoxin (A. ochraceus)
contaminate crops and much economic losses worldwide.
165
A database curated secondary
metabolites from the Aspergillus genus contains 807 unique non-redundant SMs from 675
species.
166
This section discusses the status SM discovery from two species in particular, A.
nidulans and A. terreus.
21

Status of secondary metabolite discovery in A. nidulans
Aspergillus nidulans has been used as a genetic model organism for many decades because of its
ability to utilize a wide range of nutrients, its amenability to genetic modification, and the
easiness phenotypes can be studied by growth testing.
167
Other advantages of this organism
include its ability to grow over a wide range of temperature (25-42 C), its abundant production of
darkly pigmented spores (conidia), and its ability to form heterokaryons, which allows for
genetic analysis through the sexual cycle.
168

The most recent analysis of the A. nidulans genome predicts the presence of 56 putative SM core
genes.
169
Of these, 27 are PKS genes, 2 are PKS-like genes, 11 are NRPS genes, 15 are NRPS-
like genes, and 1 is a hybrid NRPS-PKS gene. The current understanding of the products of these
56 clusters is summarized in the work of Yaegashi et al.
170
Advances that were made since the
publication of the work by Yaegashi include the discovery of fellutamide,
171
aspercryptin,
172
the
aspernidgulenes, the linking of viridicatin to its NRPS (AsqK, AN9226)
173
and siderophore to its
NRPS (SidD, AN6236), as well as the elucidation of the function of ivoA.
174
In chapter 3 the
discovery and the aspernidgulenes will be discussed.

1.4 The development of an efficient gene targeting system and heterologous platform in
Aspergillus nidulans
The role of gene targeting in studying fungal SMs
Gene targeting is a powerful tool to study the biosynthesis of fungal SMs. It involves the
homologous recombination of a transforming sequence into the genome, and can be used to for
22

systemic gene disruption, gene overexpression, and protein tagging.
175
Each of these applications
has been used to elucidate fungal SM biosynthesis on the genetic and protein level.

For example, Chiang et al. replaced the promoter of the transcriptional activator of a cryptic gene
cluster in Aspergillus nidulans with an alcohol inducible promoter.
176
Overexpression of this
transcription activator resulted in the production of the novel compound asperfuranone.
Subsequently, serial gene deletion was conducted to identify the boundary of the cluster. Some
deletion strains accumulated intermediates, and isolation of these intermediates allowed for the
proposal of a biosynthetic pathway of asperfuranone.

In another study, the coding regions of two NRPS-like proteins, atmelA and apvA, were replaced
with gfp.
177
Thus, gfp was placed under the control of either the promoter of atmelA or the
promoter of apvA. Both atmelA and apvA synthesize the same product, aspulvinone E. However,
the authors hypothesized that atmelA is expressed in the conidia where aspulvinone E is
converted to melanin, while apvA is expressed in the hyphae where aspulvinone E is converted to
apsulvinone variants. Their hypothesis was confirmed when the strain containing gfp under the
control of atmelA(p) showed fluorescence in the conidia while the strain containing gfp under the
control of apvA(p) showed fluorescence in the hyphae. This study showed that there is spatial
regulation of the expression of SM genes.

Gene targeting in Aspergillus nidulans
As outlined in section 1.5, the development of an efficient gene targeting system in Aspergillus
nidulans has led to the discovery to many compounds. Two technological advances have led to
23

the development of an efficient gene targeting system in this organism. The first is fusion PCR,
and the second is the deletion of nkuA, the protein responsible for nonhomologous end joining
DNA repair.

Prior to the development of fusion PCR, the construction of transforming cassettes involved
tedious and time-consuming sub-cloning steps.
178
Multiple sub-cloning steps were required
because filamentous fungi usually require long homologous sequences (at least 1 kb) for targeted
integration (in contrast, only about 50 bp is required for integration in yeast). The process can
take several days to several weeks, and is sometimes limited by the availability of restriction
enzyme sites.
179

The development of fusion PCR allowed for transformation cassettes to be generated in a way
that is rapid, simple, and relatively inexpensive. The process requires two steps. First, the
individual fragments, which usually minimally consists of the 5’ and 3’ homologous sequences
as well as a selectable marker, are amplified. The primers used have extensions that are
complementary to the ends of the other fragments to be fused. In the second step these fragments
are fused together via the regions of primer overlaps. The simplicity and speed of the procedure
made the first large-scale gene deletion, promoter swapping, and GFP tagging analyses feasible
in A. nidulans.
180
It has driven the first high-throughput genetic analyses of other fungi such as
Saccharomyces cerevisiae, Candida albicans, Cryptoccocus neoformans.
181

Even with the development of fusion PCR, however, gene targeting efficiency in filamentous
fungi was still limited by the rate of homologous recombination. To achieve 10% homologous
24

recombination efficiency, at least 1 kb of homologous DNA is required.
182
A study of gene
manipulation of filamentous fungi using fusion PCR showed that even with 3 kb flanking
sequence, the percentage of correct gene replacement ranged from 8 to 34%.
183
Other problems
associated with homologous recombination including integration into multiple sites and the
circularization of linear transformation cassettes. An even more efficient system needed to be
developed to complete genome-wide gene-targeting projects.

The development began with the discovery that deletion of the Neurospora crassa homologs of
KU70 and KU80, human proteins responsible for nonhomologous end joining DNA repair,
increased the homologous recombination efficiency in the fungi.
184
In strains containing the Ku
homolog deletions, correct integration success rate was 100% when ~2 kb of homologous
flanking sequences were used, as compared to the wild-type strain, which had 10-30% success
rate. Using the same idea, Nayak et al. deleted the KU70 homolog in A. nidulans (nkuA) and
increased the frequency of corrected gene targeting from ~13% to ~90% using a fragment with
2000 bp flanking DNA. The authors further showed that even with 500 bp flanking regions the
targeting efficiency was still ~90%. The nkuA knock out did not cause growth inhibition or
sensitivity to mutagens tested.
185

Implications/uses of the efficient gene targeting system in A. nidulans
These two technological advancements, coupled with the sequencing of the Aspergillus nidulans
genome, resulted in a powerful gene targeting system. A protocol detailing fusion PCR,
protoplasting, and transformation was published.
186
This protocol has been routinely used by our
lab to investigate SM pathways, including the pathways of emericellamide,
187

25

aspercethin,
188
asperfuranone,
189
orsellinic acid/F9775,
190
monodictyphenone,
191
prenyl-
xanthone,
192
microperfuranone,
193
cichorine,
194
austinol and dehydroaustinol,
195
aspernidine
A,
196
aspercryptin,
197
and fellutamide B.
198
In chapter 3, I describe my work on the discovery of
the aspernidgulenes by serial promoter replacement using this gene targeting system in A.
nidulans.

The development of Aspergillus nidulans as a heterologous expression host
Because most fungi do not have a genetic molecular system that allows for gene targeting,
heterologous expression is required to investigate the function of a biosynthetic gene. The gene
targeting system in A. nidulans has thus also been rewired to serve as a heterologous expression
platform. Two major advances were made in that facilitated the development: an efficient way to
delete entire SM gene clusters and a fast fusion-PCR based approach to place an unlimited
number of target genes under the control of regulatable promoters using marker recycling.
199

Deleting gene clusters to clean up SM background
There are two advantages to deleting active background gene clusters in a heterologous
expression host. First, reducing SM background facilitates the detection and purification of the
new compounds. Second, eliminating the production of background SMs may also free up
substrates such as malonyl-CoA and increase the yield of the desired product. Deleting an entire
cluster as opposed to only a key gene (for example the PKS) ensures that the remaining genes in
the cluster do not modify the products of the heterologously expressed cluster.

26

In the reported work, the authors developed a variant of the loop-out recombination procedure to
systematically delete gene clusters to create a cleaner SM background in A. nidulans. The
procedure involved replacing entire clusters (as large as 50 kb) with the selectable marker A.
fumgitus pyrG (AfpyrG), which encodes for orotidine 5’-monophosphate decarboxylase.
200
The
target strain contains the mutation pyrG89, which makes it a pyrimidines auxotroph. Targets
carrying the correct insertion can grow in the absence of pyrimidines.

To recycle the AfpyrG marker to additional rounds of cluster deletion, 5-fluoro-orotic acid (5-
FOA) is used. A strain expressing orotidine 5’-monophosphate decarboxylase converts 5-FOA to
the toxic 5-florouracil. Thus, 5-FOA selects for the loss of AfpyrG. The loss of AfpyrG happens
through a loop-out recombination technique adopted from A. oryzae and A. sojae (Figure x).
201

Using this procedure, the gene clusters encoding for sterigmatocystin, emericellamide, orsellinic
acid/F9775A, B, asperfuranone, monodictyphenone, and terrequinone are deleted.

Fusion PCR, alcA(p), and marker recycling
To heterologously express SM biosynthetic genes, each target gene is amplified from the
genomic DNA of the target fungus, placed under the control of the regulatable alcA promoter,
and transformed into A. nidulans. The final transformation cassette is generated through fusion
PCR as described previously. For large backbone genes (often the case with PKSs or NRPSs),
they are broken up into two or three small fragments with overlapping regions that will fuse by
homologous recombination during transformation. Doing so bypasses the difficulties associated
with amplifying long sequences using PCR. The target gene is placed under control of alcA
(alcohol dehydrogenase) promoter, which is inducible by alcohol.
202
The target gene can be
27

inserted into either the wA or yA locus. The wA gene encodes the a PKS responsible for green
pigment synthesis in the spores, and wA mutants have white spores.
203

204
The yA gene encodes
for conidial laccase, and yA mutants have yellow spores.
205

The selectable marker is placed at the 3’ end of the gene. Transformation of a second gene
removes the marker of the first gene and replaces it with a different marker (Figure x). Thus, the
markers can be recycled in theory indefinitely. In A. nidulans, three selectable markers have been
developed: AfpyrG, AfriboB, and AfpyroA. These markers complement pyrG89 (pyrimidine
auxotrophy), riboB2 (riboflavin auxotrophy), and pyroA4 (pyridoxine auxotrophy) respectively.

Applications of the A. nidulans heterologous expression system
This heterologous expression system was used to elucidate the SM genes of A. terreus, the
genetic system of which was developed around the same time.
206

207
The nine NR-PKSs in A.
terreus were heterologously expressed, and the products for all but two of them were obtained.
Furthermore, in the same study, the entire A. terreus asperfuranone pathway, which contains six
genes and is normally silent in A. terreus, was reconstituted. Expressing these six genes in
different combinations allowed for the porposed asperfuranone pathway to be refined.
208
In
another study, recombinant NRPS-like enzymes from A. terreus were heterologously expressed
in A. nidulans, providing insight into the NRPS programming.
209
In chapter II I will discuss the
heterologous expression of the citreoviridin gene cluster from A. terreus var. aureus using this
platform.

28

Table 1-1. Mean values for molecular properties of natural, drug, and synthetic compounds.
Natural Products Drugs Synthetics
Molecular weight (a.m.u) 360-414 340-356 393
LogP 2.4-2.9 2.1-2.2 4.3
Number of chiral centers 3.2-6.2 1.2-2.3 0.1-0.4
Number of N atoms 0.84 1.64 2.69
Number of O atoms 5.9 4.03 2.77
% of rings that are aromatic 31% 55% 80%

29

CHAPTER II: Biosynthetic Pathway of the Reduced Polyketide Product Citreoviridin in
Aspergillus terreus var. aureus Revealed by Heterologous Expression in Aspergillus
nidulans

2.1 Abstract
Citreoviridin (1) belongs to a class of F1-ATPase β-subunit inhibitors that are synthesized by
highly reducing polyketide synthases. These potent mycotoxins share an α-pyrone polyene
structure, and they include aurovertin, verrucosidin, and asteltoxin. The identification of the
citreoviridin biosynthetic gene cluster in Aspergillus terreus var. aureus and its reconstitution
using heterologous expression in Aspergillus nidulans are reported. Two intermediates were
isolated that allowed the proposal of the biosynthetic pathway of citreoviridin.

2.2 Introduction
Recent genome sequencing efforts of filamentous fungi have clearly demonstrated that the
number of secondary metabolites (SMs) that have been isolated and identified after decades of
intensive efforts is very much smaller than the number of SM biosynthetic gene clusters within
the genome.
210
The gene clusters that are not connected with any known compounds are called
orphan gene clusters, and they are a promising source of novel therapeutics from the
pharmacological point of view.
211
With the advent of new DNA sequencing technologies such as
next-generation sequencing, we are witnessing a rapid accumulation of fungal genomes in public
databases. Thus, natural product researchers are seizing this opportunity by intensely mining
these fungal genomes for orphan gene clusters that produce medicinally valuable compounds.
30

Citreoviridin (1) is a highly reduced polyketide product that belongs to a class of fungal
secondary metabolites that act as inhibitors of mitochondrial oxidative phosphorylation.
Members of this class of ATP synthase inhibitors contain a methylated α-pyrone, a polyene
linker, and either a tetrahydrofuran ring, as in the case of citreoviridin and verrucosidin, or a
dioxabicyclooctane moiety, as in the case of aurovertin and asteltoxin (Figure 2-1).
212

213

Citreoviridin binds to the β-subunit of F1-ATPase, thereby uncompetitively inhibiting ATP
hydrolysis and noncompetitively inhibiting ATP synthesis.
214

215
Recently, Chang et al.
216
used
citreoviridin to target ectopic ATP synthase in breast cancer. Other members of this class of ATP
synthase inhibitors have similarly been investigated as potential therapeutics against cancer.
217

Because of the potent biological activity of citreoviridin, investigators have been interested in the
biosynthesis of this mycotoxin since the 1980s. From 13C-labeled precursor studies, Steyn et
al.
218
found that the polyketide backbone of citreoviridin is derived from one acetyl-CoA starter
unit, eight malonyl-CoA extender units, and five methyl groups from S-adenosylmethionine
(SAM). The same group later proposed that tetrahydrofuran ring formation involved a
bisepoxidation step followed by epoxide hydrolysis.
219
Asai et al.
220
proposed that
citreomontanin (2) is likely the biogenic precursor of citreoviridin. Here we report our efforts to
identify the biosynthetic gene cluster for citreoviridin using an Aspergillus nidulans heterologous
expression system.
221
While our study was being completed, the biosynthesis gene cluster of a
similar highly reduced polyketide, aurovertin from Calcarisporium arbuscula, was reported.
222

31

2.3 Results and Discussion
Organisms sometimes achieve self-resistance against the metabolites they produce by harboring
duplicate or resistant targets within the metabolite biosynthetic gene cluster.
223
For instance,
Aspergillus terreus is thought to protect itself from the antifungal effects of lovastatin by
encoding a copy of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the target
of lovastatin, in the lovastatin biosynthetic locus. In fact, resistance-gene-driven genome mining
has led to the identification of the mycophenolic acid and thiotetronic acid gene clusters.
224

225

Bioinformatic analysis of A. terreus strain NIH2624 revealed that the organism contains an extra
copy of the F1-ATPase β-subunit of the known target of citreoviridin (ATEG_09616 and
ATEG_07609).
226
One gene, ATEG_09616, is located adjacent to a highly reducing poyketide
synthase (HR-PKS), ATEG_09617, and other putative tailoring enzymes required for the
synthesis of the tetrahydrofuran ring and methylated α-pyrone in citreoviridin. Specifically, a
methyltransferase (ATEG_09618), flavin-dependenent mono-oxygenase (ATEG_09620), and
hydrolase (ATEG_09619) were found in the locus. Moreover, genome database searches showed
that Metarhizium anisopliae, a known producer of the structurally similar toxin aurovertin,
227

also harbors a cluster containing a β-subunit of ATP synthase, and genes in the cluster have
homology to the locus we identified (Table 2-1).

Initial bioinformatics analysis was conducted on NIH2624 because it was the only A. terreus
strain with a publicly available genome at the time of the analysis. However, despite extensive
studies conducted on its secondary metabolites, NIH2624 has not been reported to produce
32

citreoviridin.
228
On the other hand, the A. terreus var. aureus strain CBS503.65 is a known
producer of the toxin.
229
When we cultured both strains, we detected the production of
citreoviridin only in A. terreus var. aureus CBS503.65 (Figure 2-S1). Furthermore, initial
attempts at the heterologous expression of ATEG_09617 were unsuccessful in obtaining the
production of the expected polyketide (data not shown). These results led us to investigate the
citreoviridin biosynthetic gene cluster in the A. terreus var. aureus CBS503.65 strain.

As no genome sequence of the A. terreus var. aureus CBS503.65 strain is publicly available, we
designed primers based on the sequence of the NIH2624 strain in an attempt to clone
homologues of the HR-PKS ATEG_09617 and its surrounding genes. At the onset we were
unsure whether the strategy would be successful. Gratifyingly, the putative citreoviridin
biosynthetic genes were cloned with relatively little difficulty. In total, five genes were cloned:
ctvA, ctvB, ctvC, ctvD, and ctvE (Figure 2-2A). Sequence analysis of the homologues revealed
that the two share 80–90% identity at the nucleotide level and 90–95% identity at the amino acid
level. On the basis of function prediction (Table 2-1), we hypothesized that ctvA to ctvD
constitute the core biosynthetic gene cluster while ctvE is the resistance gene.

Each gene in the putative citreoviridin gene cluster was fused to the alcohol-inducible alcA(p) by
fusion PCR and transformed into the host A. nidulans LO8030 according to the procedure
developed by us.
230
The major known metabolites of the heterologous host A. nidulans LO8030
were deleted to facilitate the detection of new compounds and to free up metabolite precursors
such as acetyl-CoA and malonyl-CoA.
231

232
Correct integration of the gene into the chromosome
was verified by diagnostic PCR (Figure 2-S3).
33

We first overexpressed the HR-PKS CtvA in an attempt to identify the highly reduced polyketide
backbone of 1. In the ctvA overexpression strain we detected the production of a new metabolite,
compound 3, compared with the LO8030 host control (Figure 2-2B, i and ii). Compound 3 is
bright yellow and has an ESI-MS peak at m/z 339 [M + H]+. It has a strong UV–vis absorption
around 420 nm (Figure 2-S2), consistent with the polyene chromophore of an ER-less HR-
PKS.
233

Scale-up and NMR characterization of the purified compound revealed the structure of
compound 3. The 1H and 13C NMR spectra of 3 were found to be almost identical to those of 2
except for the disappearance of the methoxy resonances (δH = 3.82 (3H, s), δC = 56.3; Table 2-
S3). Therefore, 3 is a demethylated form of the proposed citreoviridin biogenic intermediate 2
(Figure 2-3).

Since we did not know whether the methyltransferase or the monooxygenase would act next in
the biosynthetic pathway, we made two distinct mutant strains, ctvAB and ctvAC. We cultured
both strains and analyzed their metabolite profiles. The ctvAC strain had a metabolic profile
similar to that of the ctvA-only strain (Figure 2-2B, iv), while in the ctvAB strain we observed the
disappearance of compound 3 and the appearance of a new peak (Figure 2-2B, iii). The
corresponding ESI-MS peak was at m/z 353 [M + H]+, suggesting the addition of a methyl
group. Indeed, by comparison with published 1H and 13C NMR spectra (Figures 2-S4 and S5)
we confirmed the identity of the new compound as citreomontanin (2).

34

The formation of the tetrahydrofuran ring has been proposed to involve two steps: bisepoxidation
by a monooxygenase and epoxide ring opening by a regioselective hydrolase.
234
Since CtvC is
the only monooxygenase in the cluster, we first generated a triple overexpression ctvABC. We
observed the emergence of many peaks, including that of 1 (Figure 2-2B, v). Some of these
peaks correspond to species with molecular weight of 402 and similar UV–vis absorbance
patterns, suggesting that they might be stereoisomers of 1. Since bisepoxides are unstable and
reactive, spontaneous hydrolysis and degradation might be responsible for these peaks.
Citreoviridin 1 seems to be one of the major products, and it might be due to the greater stability
of the allylic carbocation intermediate compared with the other carbocation intermediates that
could form from epoxide opening (Figure 2-3). When ctvD was added to generate the ctvABCD
overexpression strain, the other peaks disappeared and only the peak for compound 1 remained
(Figure 2-2B, vi and vii). These results indicate that the four genes ctvA, ctvB, ctvC, and ctvD are
sufficient for citreoviridin biosynthesis.

On the basis of our results, we propose the biosynthetic pathway for citreoviridin shown in
Figure 2-3. The HR-PKS CtvA has a domain structure of KS-AT-DH-CMet-KR-ACP. It accepts
acetyl-CoA as the starter unit and catalyzes eight iterations of malonyl-CoA extension and four
iterations of SAM-dependent methylation at C4, C12, C14, and C16. The KR and DH domains
selectively act on the first six iterations to generate the hexaene chain. In the last three iterations,
the KR and DH domains terminate their functions to yield a β,δ-diketo ester moiety, which then
undergoes intramolecular cyclization to yield the α-pyrone in compound 3. Subsequently, CtvB
methylates the α-pyrone hydroxyl group of 3 to generate 2. In order to form the tetrahydrofuran
ring with the correct stereochemistry, the terminal alkenes of 2 need to undergo isomerization to
35

yield a (17Z)-hexaene,
235236
a step that could be catalyzed by CtvC. The (17Z)-hexaene then
undergoes bisepoxidation by CtvC to form a (17R,16R,15S,14R)-bisepoxide moiety. Lastly,
CtvD acts as a regioselective hydrolase to form the tetrahydrofuran ring with the substituents in
the correct absolute configuration, completing the biosynthesis of 1.

Comparison of the citreoviridin biosynthetic gene cluster with the aurovertin gene clusters in C.
arbuscula and M. anisopliae showed that there is 30–50% amino acid identity (Table 2-1). While
the C. arbuscula gene cluster lacks the putative resistance gene, in M. anisopliae the ATP
synthase β-subunit is highly conserved compared to that in A. terreus var. aureus.

CtvA selects acetyl-CoA as the starter unit, while AurA preferentially uses propionyl-CoA as the
primer for biosynthesis. Currently, the only other HR-PKS or HR-PKS module known to use
propionyl-CoA as a priming unit is the PKS-nonribosomal peptide synthase hybrid PsoA.
237
In
nonreducing polyketide synthases (NR-PKSs), the starter unit acyltransferase (SAT) domain is
responsible for starter unit selection,
238
yet the HR-PKSs do not contain SAT domains. These
results suggest that the KS domain might possess innate starter unit selectivity. Our identification
of CtvA, which has significant amino acid and domain architecture conservation with AurA
(69% amino acid identity between the KS domains as predicted by antiSMASH), paves the way
for future domain swapping experiments between the two closely related enzymes to verify the
model in which the KS domain possesses a programming role.
239

The findings of our study, in combination with the published studies done on the aurovertin
biosynthetic cluster, will allow us to identify the biosynthetic gene clusters of asteltoxin in A.
36

stellatus and verrucosidin and its derivatives in sponge-derived Penicillium aurantiogriseum
once their genomes become publicly available.
240

241
The elucidation of the citreoviridin
biosynthetic pathway strengthens the hypothesis that four genes are sufficient to generate the
remarkable diversity among these ATP synthase inhibitors.

In conclusion, we have reported the elucidation of the citreoviridin biosynthetic pathway in A.
terreus var. aureus by using A. nidulans as a heterologous expression host. We successfully
reconstituted the pathway and demonstrated that four genes, ctvA, ctvB, ctvC, and ctvD, are
sufficient for the production of the toxin.

2.4 Supplementary Information
Strains and molecular genetic manipulations.
The primers used to generate transforming fragments via fusion PCR are listed in Table S1. The
strains generated in this study are listed in Table S2. The procedures for fusion PCR,
protoplasting, and transformation are as previously described,1 with the exception that
VinoTaste Pro (Novo) was used at a final concentration of 100 mg/mL for protoplasting in place
of VinoFlow FCE. Correct insertion was verified by diagnostic PCR (Figure 2-S2). At least two
transformants with the correct inserts were used for each metabolite analysis.

Fermentation and HPLC analysis.
The A. terreus strain NIH2624 and A. terreus var. aureus strain CBS 503.65 were cultured in 45
mL liquid Czepek-Dox (30 g/L sucrose, 1 g/L K2HPO4, 5 g/L yeast extract, 3 g/L NaNO3, 0.5
g/L KCl, 0.5 g/L MgSO4·7 H2O, 0.01 g/L FeSO4·7 H2O) at 26 °C, shaking at 180 rpm for 4
37

days. For inoculation 4.5 × 107 spores were used. Culture media (3 mL) was collected by
filtration and extracted with equal volume ethyl acetate. The ethyl acetate layer was evaporated
by Turbo Vap LV (Caliper LifeSciences) and the crude extract was dissolved in 0.25 mL of
DMSO:MeOH (1:4) and 2 uL was injected for HPLC analysis. The gradient system was MeCN
(solvent B) and 5% MeCN/H2O (solvent A). The gradient condition for HPLC analysis was 0-25
min 90-45% A, 25-30 min 45-0% A, 30-33 min 0% A, 34-39 min 90% A. Authentic citreoviridin
(1 mg, 97% purity) was purchased from Enzo Life Sciences (Farmingdale, NY, USA) and was
used as standard. Citreoviridin was eluted at about 22 min. The yield of citreoviridin 1 in the
media extract is around 10.5 mg/L.

To cultivate A. nidulans, 3.0 × 107 spores were added to 30 mL liquid LMM (15 g/L lactose, 6
g/L NaNO3, 0.52 g/L KCl, 0.52 g/L MgSO4, ¬1.52 g/L KH2PO4, 1mL/L trace elements¬
(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)) in 125mL flasks at 37°C and shaking at 180 rpm. Pyridoxine (0.5 mg/mL) or uracil (1
mg/mL) and uridine (10 mM) were supplemented when necessary. After 42 hours, methyl ethyl
ketone (MEK), an inducer of alcA(p), was added to a final concentration of 50 mM.2 Culture
medium was collected by filtration 72 hours after MEK induction and then extracted with equal
volume of ethyl acetate. The ethyl acetate layer was evaporated by Turbo Vap LV (Caliper
LifeSciences) and the crude extract was dissolved in 0.25 mL of DMSO:MeOH (1:4) and 2 uL
was injected for high-performance liquid chromatography (HPLC) analysis. HPLC analysis was
carried out using a C18 reverse phase column (Agilent 5 µm, 4.6 x 150 mm) with a flow rate of
1.0 mL/min and measured by a UV detector at 403 nm. The gradient system was MeCN (solvent
38

B) and 5% MeCN/H2O (solvent A) both containing 0.05% TFA. The gradient was as described
above. The yield of citreoviridin 1 in the media extract of A. nidulans strain expressing ctvA,
ctvB, ctvC, and ctvD is around 2.8 mg/L.

Isolation of secondary metabolites.
To isolate compound 2, A. nidulans strain expressing ctvA and ctvB was cultivated in 45 mL
liquid LMM supplemented with uridine and uracil in 125 mL flasks (24 flasks, 1080 mL total).
The cultures were grown at 37 °C for 42 hours, and then induced with MEK to a final
concentration of 50 mM. The cultures were then grown for another 72 hours before being
harvested. Culture media was collected by filtration and extracted with ethyl acetate four times.
The ethyl acetate extract was evaporated in vacuo. The mycelium was soaked in 250 mL
methanol for at least 5 minutes and the methanol was evaporated in vacuo. The combined residue
from media and mycelium was 3.3 g.

The ethyl acetate extract contains a high amount of uridine and uracil. In order to remove the
residual uridine and uracil, the residual was dissolved in dichloromethane and passed through
filter paper. Uridine and uracil, which are poorly soluble in dichloromethane, were left behind.
The dichloromethane was evaporated in vacuo to yield a crude extract of 470 mg. The extract
was purified by a silica gel column (EMD®, 230 to 400 Mesh, ASTM, 1:19 20:0 ethyl
acetate:hexane gradient) to afford 37 fractions. Fraction 8 to 10 was combined, evaporated, and
purified again by a silica gel column (1:19 8:12 ethyl acetate:hexane gradient) to yield 15.5
mg of compound 2. The structure of 2 was determined by NMR (Table S3, Figures S4 and S5)
and the data is in good agreement with published data.3
39

To isolate compound 3, A. nidulans strain expressing ctvA was cultivated in 750 mL liquid LMM
supplemented with pyridoxine in 2 L flasks (4 flasks, 3 L total). The cultures were grown at 37
°C for 42 hours, and then induced with MEK to a final concentration of 50 mM. The cultures
were then grown for another 48 hours at 30 °C before they were harvested. The mycelium was
separated from the culture media and soaked in 200 mL methanol five times for at least 5
minutes. The methanol was evaporated in vacuo and the residue was redissolved in 250 mL
water, which was extracted three times with equal volume ethyl acetate. The ethyl acetate was
evaporated in vacuo and the crude was purified by C18 reverse phase column (Cosmosil 75C18-
OPN, Nacalai Tesque, Inc, 1:9 to 10:0 methanol:water gradient). The 100% MeOH fraction (276
mg) was subjected to purification by a Sephadex LH-20 column to yield demethyl-
citreomontanin 3 (30.0 mg). The structure of 3 was determined by NMR (Table 2-S3, Figures 2-
S6 and S7).

Compound identification.
NMR spectra were collected on a Varian Mercury Plus 400 spectrometer.
UV-vis and ESIMS spectra of compounds 2 and 3 (Figure 2-S2) was obtained by 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
included a papillary voltage 5.0 kV, a sheath gas flow rate at 50 arbitrary units, an auxiliary gas
flow rate at 0 arbitrary units, and the ion transfer capillary temperature at 275 °C.
40

Figure 2-1. Citreoviridin and other inhibitors of mitochondrial oxidative phosphorylation
produced by different fungal species.
41

Figure 2-2. (A) The citreoviridin (1) biosynthesis gene cluster. Black open reading frames
(ORFs) are involved in the biosynthesis while gray ones are not. (B) HPLC profile of metabolites
extracted from the culture media of A. nidulans strains expressing citreoviridin cluster genes
under the control of alcA(p). HPLC analysis was carried out using a C18 reverse phase column.
Detection was at 403 nm. “4x zoomed out” indicates that the y-axis scale is four times larger.
42

Table 2-1. Citreoviridin (1) biosynthesis genes in A. terreus var. aureus, their homologs in other
species, and gene function predictions.
gene putative function A. terreus NIH2624
homolog
aa (% identity, %
similarity)
gene (% identity)
C. arbuscula
homolog
aa (% identity, %
similarity)
M. anisopliae homolog
aa (% identity, %
similarity)
ctvA polyketide synthase XP_001218239.1 (89, 93)
ATEG_09617 (88)
AurA (46, 60) XP_007824472.2 (47, 63)
ctvB S-adenosylmethionine –
dependent
methyltransferase
XP_001218240.1 (94, 98)
ATEG_09618 (91)
AurB (53, 67) XP_007824471.1 (52, 67)
ctvC

Flavin-dependent
monooxygenase (FMO)
XP_001218242.1 (92, 96)
ATEG_09620 (87)
AurC (47, 67) XP_007824468.2 (45, 64)
ctvD hydrolase XP_001218241.1 (84, 88)
ATEG_09619 (85)
AurD (31, 47) XP_007824469.1 (33, 48)
ctvE ATP synthase β-chain XP_001218238.1 (96, 97)
ATEG_09616 (87)

None XP_007824466.2 (83, 89)

43

Figure 2-3. Proposed biosynthetic pathway for citreoviridin (1). The more stable allylic
carbocation is shown in the solid-line.
44

Table 2-S1. Primers used in this study
Name Sequence
ctvA_Af1_alcAEnd CCAATCCTATCACCTCGCCTCAAAATGGCACACATGGAACCGAT
ctvA_Br1_pyrGBeg CGAAGAGGGTGAAGAGCATTGGCAATCGCCACTATGTCTTAG
ctvA_Ar1 CTCCAGTGCGATCAGATAC
ctvA_Bf1 CATTGATGCATTCAGCGTAAAC
ctvA_Bf2 CGACATCTCGTCTCCGAACCA
ctvA_Bf3 AGGATGAGTGCATCATTGGC
ctvA_Br1_alcABeg CTATCACAATCAGCTTTTCAGGCAATCGCCACTATGTCTTAG
ctvB_Af1_alcAEnd CCAATCCTATCACCTCGCCTCAAAATGACCTTCTACCAGCTTTC
ctvB_Br1_pyrGBeg CGAAGAGGGTGAAGAGCATTGCGTGGATGCGTTACAATGAG
ctvB_Af1 ATGACCTTCTACCAGCTTTCCG
ctvB_Af2 ATCCACTTCTCTTGTGGTGG
ctvB_Br1_alcABeg CTATCACAATCAGCTTTTCAGCGTGGATGCGTTACAATGAG
ctvC_Af1_alcAEnd CCAATCCTATCACCTCGCCTCAAAATGGAAGGAAAGCACCCTCA
ctvC_Br1_pyrGBeg CGAAGAGGGTGAAGAGCATTGCTTAGGACTAGCGTCTGACC
ctvC_Af2 GCCAATACAGCCATTGAAGC
ctvC_Af3 GTCAAGTGACACCATTCACC
ctvC_Br1_alcABeg CTATCACAATCAGCTTTTCAGCTTAGGACTAGCGTCTGACC
ctvD_Af1_alcAEnd TCCTATCACCTCGCCTCAAAATGGCCCTTTCAGCCTACAT
ctvD_Br1_pyrGBeg CGAAGAGGGTGAAGAGCATTGGGCAAGCTCGAACTACGACC
yA_P1 TTCTTCCAGCTTCTGCTGCGT
yA_P2 CGACAACCAAGGGAAGTCAA
yA_P3 CGCATTCTAGAGAGAGTGTG
yA_p4 ATTGCGTCCCATCAAATGGG
yA_p5 CAAACTCCTTGACACCGT
yA_p6 GAGTCTGCAGCAAAGGCATTGA
45

alcA_Af1 CTGAAAAGCTGATTGTGATAG
alcA_Br1 TTTGAGGCGAGGTGATAGGATTGG
aclA_Br1.1 CTGAAAAGCTGATTGTGATAG
AfpyrG_Af1 CAATGCTCTTCACCCTCTTCG
AfpyrG_Br1 CTGAGAGGAGGCACTGATGC
yA_P4_pyrGEnd GCATCAGTGCCTCCTCTCAGACAGATTGCGTCCCATCAAATGGG
Green and purple sequences are the tail that anneal to the alcA promoter fragment during fusion
PCR. Red and blue sequences are the tail that anneal to the A. fumigatus pyrG (AfpyrG) fragment
during fusion PCR.

46

Table 2-S2. A. nidulans strains used in this study. Currently, there is no standard nomenclature
for the deletion of entire gene clusters. Note that 34 bp of the coding sequence of AN7804 in the
sterigmatocystin gene cluster was not deleted. All the genes in all the other clusters were deleted
in their entirety, however.
Strain Heterologously
expressed gene(s)
Genotype
LO8030 None pyroA4, riboB2, pyrG89, nkuA::argB,
sterigmatocystin cluster (AN7804-AN7825)Δ,
emericellamide cluster (AN2545-AN2549)Δ,
asperfuranone cluster (AN1039-AN1029)Δ,
monodictyphenone cluster (AN10023-AN10021)
Δ, terrequinone cluster (AN8512-8520)Δ, austinol
cluster part 1 (AN8379-AN8384)Δ, austinol cluster
part 2 (AN9246-9259)Δ, F9775 cluster (AN7906-
7915)Δ, asperthecin cluster (AN6000-AN6002)Δ.
CW7032, CW7034,
CW7035
ctvA pyrG89; pyroA4; nkuA::argB; riboB2; STΔ; easΔ;
afoΔ; mΔpΔ; TQΔ; ausΔ; F9775Δ; aptΔ
yA::AfriboB: alcA(p)-ctvA: AfpyrG
CW7051, CW7052,
CW7054
ctvAB pyrG89; pyroA4; nkuA::argB; riboB2; STΔ; easΔ;
afoΔ; mΔpΔ; TQΔ; ausΔ; F9775Δ; aptΔ
yA::AfriboB: alcA(p)-ctvA: alcA(p)-ctvB: AfpyroA
CW7061, CW7062 ctvAC pyrG89; pyroA4; nkuA::argB; riboB2; STΔ; easΔ;
afoΔ; mΔpΔ; TQΔ; ausΔ; F9775Δ; aptΔ
47

yA::AfriboB: alcA(p)-ctvA: alcA(p)-ctvC: AfpyroA
CW7091, CW7094,
CW7095
ctvABC pyrG89; pyroA4; nkuA::argB; riboB2; STΔ; easΔ;
afoΔ; mΔpΔ; TQΔ; ausΔ; F9775Δ; aptΔ
yA::AfriboB: alcA(p)-ctvA: alcA(p)-ctvB: alcA(p)-
ctvC: AfpyrG
CW7210, CW7211,
CW7212
ctvABCD pyrG89; pyroA4; nkuA::argB; riboB2; STΔ; easΔ;
afoΔ; mΔpΔ; TQΔ; ausΔ; F9775Δ; aptΔ
yA::AfriboB: alcA(p)-ctvA: alcA(p)-ctvB: alcA(p)-
ctvC: alcA(p)-ctvΔ: AfpyroA

48

Table 2-S3.
1
H and
13
C NMR data for compound 2 and 3
2
a
3
b

Position δH (multi, J in Hz) δC δH (multi, J in Hz) δC
1 ----- 164.0 ----- 162.2
2 5.48 (1H, s) 88.7 5.37 (1H, s) 89.8
3 ----- 170.8 ----- 169.9
4 ----- 107.7 ----- 108.1
5 ----- 154.9 ----- 154.7
6 6.30 (1H, d, 14.9) 118.4 6.62-6.44 (1H, m) 119.5
7 7.22 (1H, dd, 14.9, 11.3) 136.5 6.99 (1H, dd, 14.9, 11.2) 134.8
8 6.37 (1H, dd, 14.8, 11.2) 130.8 6.44-6.62 (1H, m) 131.2
9 6.57 (1H, dd, 14.6, 10.7) 139.3 6.69 (1H, dd, 14.7, 10.4) 138.5
10 6.31 (1H, dd, 14.9, 10.5) 127.2 6.38 (1H, dd, 15.1, 10.4) 127.5
11 6.45 (1H, d, 15.1) 142.5 6.62-6.44 (1H, m) 141.6
12 ----- 133.6 ----- 133.3
13 6.08 (1H, s) 140.0 6.12 (1H, s) 139.1
14 ----- 133.8 ----- 133.3
15 5.89 (1H, s) 136.7 5.91 (1H, s) 136.4
16 ----- 132.3 ----- 131.9
17 5.44-5.53 (1H, m) 126.2 5.48 (1H, q, 6.9) 126.0
18 1.72 (3H, d, 7) 14.2 1.68 (3H, d, 7.0) 13.8
19 1.78 (3H, s) 17.0 1.75 (3H, s) 16.6
20 1.98 (3H, s) 19.2 1.95 (3H, s) 18.8
21 1.97 (3H, s) 14.3 1.94 (3H, s) 14
22 1.96 (3H, s) 9.1 1.93 (3H, s) 8.8
OMe 3.82 (3H, s) 56.3 ----- -----

a
400 MHz and 100 MHz in CDCl3
b
400 MHz and 100 MHz in DMSO-d6

49

Figure 2-S1. Production of citreoviridin by A. terreus var. aureus strain CBS 503.65. Detection
is at 403 nm. The identity of citreoviridin was verified by comparison with the retention time,
UV-Vis absorption, and mass spectra of the authentic standard.
50

Figure 2-S2. UV-vis and ESIMS spectra of compounds 1 to 3.

51

Figure 2-S3. Results of diagnostic PCR for all strains generated in the study.
52

Figure 2-S4.
1
H NMR spectrum of citreomontanin (2) in CDCl3.

Figure 2-S5.
13
C NMR spectrum of citreomontanin (2) in CDCl3.
53

Figure 2-S6.
1
H NMR spectrum of demethyl-citreomontanin (3) in DMSO-d6.
54

Figure 2-S7.
13
C NMR spectrum of demethyl-citreomontanin (3) in DMSO-d6.

55

Supplemental Nucleotide and Protein Sequence Data
Green: start codon
Blue: predicted intron
Red: stop codon
Gene: ctvA
Size: 7527 bp
ATGGCACCCATGGAGCCGATTGCCATCGTTGGCACTGCCTGCCGATTTGCCGGCTCG
TCATCCACTCCTTCCAGGCTTTGGGAACTTCTCTTAAACCCCAAGGACGTGGCATCA
GAGCCACCCGCAGATCGATTCAATATCGATGCTTTCTATGACCCGGAAGGCTCCAAC
CCCATGGCGACCAATGCCCGCCAGGGGTATTTCCTTTCTGACAACGTCAAAGCCTTC
GATGCCCCGTTCTTCAATATCTCCGCAGCCGAAGCACTGGCACTCGACCCACAGCAG
CGGATGCTGCTGGAAGTCGTCTATGAATCACTGGAGACTGCTGGCCTGCGCTTAGAC
ACTCTCCGCGGCTCCTCGACGGGGGTCTACTGCGGTGTGATGAACTCCGACTGGGAG
GGCATATTCAGCGTCTCATGTGCAGCACCGCAGTATGGGAGTGTTGGGGTTGCCCGG
AATAACCTCGCTAACCGCATCTCCTACTTCTTCGACTGGCAAGGCCCGTCCATGTCC
ATCGATACCGCCTGCTCAGCGAGCATGGTAGCATTGCATGATGCCGTCTCCGCACTC
ACTCGCCACGACTGCGACATGGCTGCAGCTCTAGGTGCCAACCTCATGTTGTCTCCC
CAGATGTTCATCGCTGCATCCAATTTGCAGATGTTGTCCCCAACCAGCCGCAGCCGT
ATGTGGGATGCGCAGGCTGATGGTTATGCGCGTGGCGAGGGGGTCGCATCCGTGCT
CTTGAAACGGCTTTCAGATGCAGTGGCCGACGGCGACCCTATCGAATGTGTTATCCG
AGCTGTCGGCGTGAACCATGATGGCCGTAGCATGGGTTTCACCATGCCGTCGAGTGA
TGCACAAGTGCAACTGATCAGGTCTACTTATGCAAAAGCCGGATTGGATCCTCGCTG
CGCGGAAGATCGACCCCAATATGTCGAGGCCCATGGTACAGGCACGTTGGCGGGTG
56

ATCCCCAGGAAGCATCCGCCCTTCATCAGGCCTTCTTCAGTTCCTCGGACGAGGACA
CTGTACTGCATGTCGGTTCCATCAAGACAGTGGTAGGCCACGCGGAAGGGACTGCT
GGTCTCGCGGGTCTCATCAAGGCATCCCTGTGCATTCAGCATGGCATAATACCCCCG
AATCTTCTTTTCAATCGCTTGAACCCGGCTCTGGAGCCATATGCACGGCAATTGCGA
GTTCCAGTAGACGTGATCCCCTGGCCCTCCCTTCCTCCAGGCGTTCCCCGACGTGTTT
CAGTGAACTCCTTCGGCTTTGGTGGCACCAATGCTCATGTTATTCTGGAGAGCTATG
AACCTGCTAGAGACCTCACCAAGGACGGCTTCAATCAGAATGCGGTGCTTCCGTTTG
TCTTCTCTGCGGAGTCGGATTATAGTCTTGGGTCGGTTCTGGAGCAGTATTCCAGAT
ATCTCTCCAGATTTTCTGACGTGGACGTACACGATCTGGCATGGACGCTAATCGAGC
GCCGTTCCGCGCTGATGCACCGTGTCGCTTTTTGGGCGCCAGATATTGCACACCTCA
AAAGAAGGATCCAGGATGAGGTCGCCCTCCGGAAAGCAGGGACACCCTCGACAGTC
ATCTGCCGGCCACATGGCAAGACTAGGAAGCACATTCTGGGCGTCTTCACTGGTCAG
GGTGCCCAATGGGCGCAGATGGGACTTGAACTAATCACCGCGTCCACCATTGCGCG
AGGCTGGCTGGATGAGCTGCAACAGTCTCTCGATACTTTGCCGGAGGCGTATCGTCC
AGAGTTCTCGCTCTTTCAAGAGCTTGCTGCGGATCCGGCCGCATCACGACTATCGGA
GGCCCTTCTGTCGCAGACCCTCTGCACAGCAATGCAGATTATCTGGGTGAAGGTGCT
CTGGGCTCTGAACATCCACTTGGAAGCTGTGGTCGGTCACTCATCTGGCGAGATTGC
TGCGGCCTTTGCGGCTGGCTTTCTGACAGCTGAGGATGCCATTCGCATTGCCTACCTT
CGAGGTGTGTTTTGCTCGGCTTCAGGCAGCTCGGGGGAAGGTGCGATGCTGGCCGCT
GGTCTTTCGATGGACGAAGCGACTGCACTCTGTGACGACGTATCCTCGTCTGGGGGG
CGAATCAACGTGGCAGCGTCCAACTCGCCTGAAAGCGTCACGCTCTCTGGAGACCG
AGATGCAATTCTGCGAGCTGAGCAGCAGTTGAAGGATAGGGGAGTCTTTGCCCGTCT
ACTTCGTGTCAGTACCGCCTACCACTCCCATCACATGCAGCCATGTTCGCAGCCCTA
57

TCAGAACGCATTGAGTAGTTGCAACATTCAGATTCAGGCCCCGGTGCCCACCACCAC
CTGGTATTCAAGCGTCTATGCTGGGTGCCCCCTGGAGGAGCCTTCGGTCATAGAGAC
GCTCGGTACAGGAGAATACTGGGCGGAAAATCTAGTCAGTCCTGTGTTGTTCTCGCA
GGCACTAACGGCTGCCATATCCACCACAAACCCTTCCCTGGTCGTCGAAGTTGGACC
TCATCCAGCTCTGAAAGGACCTGCCTTACAGACGATCTCAGGAATAACGTCAGGGG
AGATCCCTTATATCGGGGTATCAGCCCGGAACAATTGTGCACTTGAGTCCATAGCCA
CAGCCATTGGATCTTTCTGGACGCATCTTGGTCCACAAGTCATCAATCCGCGAGGGT
ACCTGGCTCTTTTCCGGCCGAATGTGAGGTCTTCAGTTGTCCGTGGGCTGCCTTTGTA
TCCCTTTGACCATCGCCAAGAGCACGGTTATCAGACCCGCAAGGCTAATGGTTGGCT
GTACCGACGGTACACACCACACCCTCTGCTGGGTTCTCTGAGTGAAGACCTCGGGGA
GGGCGAGTTGCGGTGGAATCATTACCTCTCCCCCCGACGGCTCCCATGGCTCGATGG
CCACCGCGTCCAGGGCCAAATCGTGGTCCCTGCCACAGCTTATATCGTGATGGCTCT
CGAGGCCGCTCGCATACTGACCGCTGAGAAACAAAAGAGCTTGCATCTAATCCGTA
TAGACGACCTAGTCATCGGTCAAGCTATCTCCTTCCAGGATGAACGAGATGAGGTTG
AGACTCTGTTCCACCTCGCCCCTATGGTGGAGACCAAGGATGACAACACAGCAGTC
GGCCGGTTCCGCTGTCAGATGGCTGCTTCCGGGGGTCACGTCAAGACATGTGCGGAG
GGCATCCTCACGGTAACCTGGGGCTCGCCGCTGGATGATGTCCTCCCATACCCTAGG
TCTCCAGCGCCCGCAGGGCTAGCCCATGTAGCCGACATAGACGAGTACTATGCGTC
GCTCCGAAGCTTGGGTTACGAGTACACCGGCGCCTTCCAGGGAATTTTTTCTCTCTCC
CGGAAGATGGGTATCGCCACGGGCCAATTGTGTAACCCTGCATTAAATGGCTTTCTG
ATCCATCCAGCAGTTCTCGACACTGGATTACAGGGTCTTCTGGCCGCGGTGGGGGAG
GGACACCTCACGAGCCTACATGTTCCAACCCGCATTGATGCATTCAGCGTAAACCCT
GCAGCCTGTAGTAGCGGTTCGCTAGCCTTTGAGGCTGCCGTGACTCGGACAGGATTA
58

GACGGTCTCGTGGGCGACGTGGAGTTGTATACGGATACCAACGGCCCTGGTGCCGTC
TTCTTTGAAGGAGTGCACGTCTCCCCACTAGTGCCGCCATCCGCAGCGGATGATCCG
TCAGTATTTTGGGTGCAGCATTGGACACCCCTTAGCCTGGATGTCAACCGTTCCAAA
TCTCGACTGTCGCCGGAATGGATGGCCATGTTAGAAGGGTATGAGCGCCGGGCGTTC
CTTGCACTGAAGGACATCCTCCAGCAGGTCACACCAGAGCTTCGTGCCACTTTTGAC
TGGCATCGTGAAAGCGTTGTCAGTTGGATTGAGCACATTATGGAGGAAACCCGCGT
GGGTCGGCACGCCGTCTGCAAGCCTGAGTGGCTAGACCAAGAGCTAGAGAATCTCG
GACACATATGGGGGCGGCCAGACGCGCGCATTGAGGATCGAATGATGTATCGAGTT
TACCGGAACCTGCTACCCTTCCTCCGCGGGGAAGCGAAGATGCTAGATGCTCTTCGG
CAGGACGAATTGCTTACACAGTTCTATCGCGACGAGCACGAGCTGCGCGATATCAA
CCGTCGACTGGGTCAGTTGGTTGGTGACCTAGCCGTGCGCTTTCCACGTATGAAACT
CCTTGAAGTCGGCGCCGGGACAGGCTCTGCCACTCGAGAGGTACTCAAACATGTCG
GCCGGGCCTACCATTCCTACACGTTCACAGACATCTCGGTTGGCTTTTTTGAAGACA
TGTTGGAAACAATTCCCGAGCACGCGGACCGTCTGCTATTCCAGAAGCTCGATGTCG
GGCAAGACCCATTGCAGCAGGGCTTTGGTGAACACACTTACGATGTAATCATCGCCG
CTAACGTACTTCATGCCACACCGACGCTGCAAGAGACTCTGCGAAACGTGCGTCGTC
TACTCAAGCCAGGAGGGTATCTGATCGCTCTGGAGATCACTAACATTGATACAATCC
GCATCGGCTTCTTGATGTGTGCCTTTGACGGCTGGTGGCTTGGCCGGGAGGATGGCC
GTCCATGGGGTCCGGTGGTCTCTGCATCACAGTGGGATAGCCTACTCCGGGAGACGG
GATTCGGTGGCATAGACACTATCACTGATCGCGCCGCTGACCAGCTCACCATGTACT
CTGTCTTTGCCGCCCAAGCGGTGGACGACCAGATCACTCGATGTCGAGAACCTCTGA
CGCCGCTCCCTCCTCAACCTCCTTTCTGCCGGGGAGTGATCATCGGAGGCTCGCCTA
GTCTGGTGACAGGCATAAGAGTCATTATTCATCCTTTCTTCTCGACTGTTGAACATGT
59

TTCTACCATCGAGAACCTGACGGAGGGAGCACCAGCTGTTGTGTTGATGTTGGCTGA
CCTGAGCGACATCCCCTGCTTCGAAAATCTCACCGAGTCAAGACTGGCCGGACTCAA
AGCACTGGTGCAAATGGCCGAGAAGACGCTCTGGGTGACCACGGGCTCTGAAGCGG
ACAACCCTTATCTCTGCCTCAGCAAGGGCTTTCTCACTTCGATGAATTATGAACATCC
AGCTATCTTCCAATATCTGAACATCATCGACTCGGCTGACGTCCAACCCGTGGTCTT
GGCCGAGCATCTTCTGCGATTGGCCTATACCAACCAAAACAATGACTTCGCCCTCAC
GAATTGCGTCCACAGCACAGAGCTTGAGCTGCGTCTCTACCAGGGCGGGATTCTGAA
GTTCCCACGCATTAACGCGAGCGATGTCCTGAACAGTCGGTACGCGGCAGCTCGGC
GCCCAGTCACCCATTCTGTCACCAACATGCAGGACAGCGTGGTTGTACTTGACCAAA
GCCCAAGTGGGAAGCTTCGACTCGTGTTTGGGGAGGAGCTTGCAGGTGATCGCGCA
ACCGTCACCATTAACGTCCGATACTCGACCTCTCGTGCAATCCGCATCAATGGTGCT
GGATATCTGGTCCTTGTTCTCGGGCAGGATAAAGTTACCAAAGCGCGTCTGGTGGCT
CTGGCAGGTCAGTCTGCGAGCGTCGTCTCGTCCTCCTGTTATTGGGAGGTCCCAGCA
GATATCTTCGAGGAGCAGGAGCCCGCGTATCTGTACGCCACAGCAACAGCTTTGCTC
GCTGCCAGTTTGGTGCAGTCCAACGGCACCACAATCCTGGTACATGGCGCTGACATG
GTCCTACGCCATGCAATCGCCATAGAGGCCGCTTCACGGGTCATTCAGCCTATATTC
ACTACCACATCTCCCTCCGCAGCATCATCCGCGGGTCTTGGGAAGAGCATCCTCGTG
CATGAGAACGACACCCGGCGACAACTGGTTCATCTTCTCCCTCGATATTTCACAGCT
GCTGTGAATTTCGACCCTAGTGCCCGCCGACTCTTCGACCGAATGATGACAGTCGGT
CATCAATCGGGTGTCACAGAAGAACACCTTCTTACCACTTTGACAGCTGCCCTCCCT
CGTCCGTCAGCATCTCTGCTGCCGGCCCAGCCTCAGGCTGCCATGGACACTCTTCGC
AAAGCCTCATTGACTGCTTATCAGTTCACCGTCCAGTTGACAGCACCAGGACCCATC
ATCGCACCAATCGCCGACATCCAATCCTGTTCACAACAGTTAGCAGTCGTAGACTGG
60

AAACCATCTTGCGGCTCGGTTCCAGTACACCTCCAACCAGCCACTGAGCTGGTTCGT
CTCTCTGCTCAAAAGACATATCTCCTGGTGGGTATGACTGGTGCCCTCGGCCAATCC
ATCACGCAATGGCTGGTCACCCGCGGCGCTCGCAATATCGTCCTCACCAGCCGCAAG
CCATCAGTGGACCCCGCATGGATCGCAGAGATGCAGACCACAACAAGCGCGCGTGT
CCTCGTTACGCCAATGGATGTGACAAGCCGCGACTCGATCCTTGTGGTGGCACACGC
CCTGAAGGCCGACTGGCCGCCGCTCGGCGGCGTCGTCAACGGTGCCATGGTGCTCTG
GGACCGTCTCTTCGTCGACGCACCCCTGTCCGTTCTGACGGGACAGCTCGCCCCAAA
AGTCCAGGGGAGCCTTCTCCTCGATGAGATTTTTGGCCATGAACCGGGCCTTGATTT
CTTTATCCTCTTCGGTAGCGCTATCGCCACTATTGGAAATCTGGGTCAGTCTGCCTAC
ACAGCCGCCAGTAACTTCATGGTCGCGCTTGCGGCGCAACGCCGCGCCCGAGGGCT
TGTCGCAAGCGTCCTCCAGCCGGCGCAGGTCGCCGGTGCCATGGGTTATCTCAGGGA
TAAAGACGACAGCTTCTGGGCTCGGATGTTTGATATGATTGGGCGACATCTCGTCTC
CGAACCAGATCTGCACGAACTTTTGGCCCATGCTATCTTGTCGGGTCGTGGCCCTCC
AGCTGACGTTGGATACGGACCAGGCGAGGATGAGTGCATCATTGGCGGACTCCGCG
TCCAAGACCCTGCTGTATACCCAGATATCCTCTGGTTCCGTACGCCCAAAGTCTGGC
CATTCATCCACTATCACCACGAGGGAACTGGCCCTTCATCTGGGGCGGCTGGTTCGA
TATCGCTGGTCGATCAGCTGAAGTGTGCGACTAGCTTAGCCCAAGTTGGGGACATGG
TGGAAGCTGGCGTTGCGGCCAAACTGCACCATCGACTCCATCTCCCAGGCGAGGTTG
GAGGCGTCACTGGCGACACGCGTTTGACCGAGCTGGGGGTGGACTCGTTAATTGCG
GTGGACTTGCGTCGGTGGTTTGCGCAGGAGTTGGAGGTTGATATTCCCGTTCTGCAG
ATGCTGAGTGGGTGTTCAGTAAAGGAGCTGGCTGCTTCCGCGACGGCGTTGTTGCAT
CCGAAATTCTATCCGGAGGTGGTGGCCGATTCTGACGTGGGGAGTGAGAGGGATGG
61

TTCCTCGGACTCCCGTGGTGATACCTCTTCCTCCTCGTATCAGCTGATCACTCCGGAG
GAGGGGGACCATGACTGA

Protein: CtvA
Size: 2436 aa
MAPMEPIAIVGTACRFAGSSSTPSRLWELLLNPKDVASEPPADRFNIDAFYDPEGSNPMA
TNARQGVSCAAPQYGSVGVARNNLANRISYFFDWQGPSMSIDTACSASMVALHDAVSA
LTRHDCDMAAALGANLMLSPQMFIAASNLQMLSPTSRSRMWDAQADGYARGEGVAS
VLLKRLSDAVADGDPIECVIRAVGVNHDGRSMGFTMPSSDAQVQLIRSTYAKAGLDPR
CAEDRPQYVEAHGTGTLAGDPQEASALHQAFFSSSDEDTVLHVGSIKTVVGHAEGTAG
LAGLIKASLCIQHGIIPPNLLFNRLNPALEPYARQLRVPVDVIPWPSLPPGVPRRVSVNSFG
FGGTNAHVILESYEPARDLTKDGFNQNAVLPFVFSAESDYSLGSVLEQYSRYLSRFSDVD
VHDLAWTLIERRSALMHRVAFWAPDIAHLKRRIQDEVALRKAGTPSTVICRPHGKTRKH
ILGVFTGQGAQWAQMGLELITASTIARGWLDELQQSLDTLPEAYRPEFSLFQELAADPA
ASRLSEALLSQTLCTAMQIIWVKVLWALNIHLEAVVGHSSGEIAAAFAAGFLTAEDAIRI
AYLRGVFCSASGSSGEGAMLAAGLSMDEATALCDDVSSSGGRINVAASNSPESVTLSGD
RDAILRAEQQLKDRGVFARLLRVSTAYHSHHMQPCSQPYQNALSSCNIQIQAPVPTTTW
YSSVYAGCPLEEPSVIETLGTGEYWAENLVSPVLFSQALTAAISTTNPSLVVEVGPHPAL
KGPALQTISGITSGEIPYIGVSARNNCALESIATAIGSFWTHLGPQVINPRGYLALFRPNVR
SSVVRGLPLYPFDHRQEHGYQTRKANGWLYRRYTPHPLLGSLSEDLGEGELRWNHYLS
PRRLPWLDGHRVQGQIVVPATAYIVMALEAARILTAEKQKSLHLIRIDDLVIGQAISFQD
ERDEVETLFHLAPMVETKDDNTAVGRFRCQMAASGGHVKTCAEGILTVTWGSPLDDVL
PYPRSPAPAGLAHVADIDEYYASLRSLGYEYTGAFQGIFSLSRKMGIATGQLCNPALNGF
62

LIHPAVLDTGLQGLLAAVGEGHLTSLHVPTRIDAFSVNPAACSSGSLAFEAAVTRTGLDG
LVGDVELYTDTNGPGAVFFEGVHVSPLVPPSAADDPSVFWVQHWTPLSLDVNRSKSRL
SPEWMAMLEGYERRAFLALKDILQQVTPELRATFDWHRESVVSWIEHIMEETRVGRHA
VCKPEWLDQELENLGHIWGRPDARIEDRMMYRVYRNLLPFLRGEAKMLDALRQDELL
TQFYRDEHELRDINRRLGQLVGDLAVRFPRMKLLEVGAGTGSATREVLKHVGRAYHSY
TFTDISVGFFEDMLETIPEHADRLLFQKLDVGQDPLQQGFGEHTYDVIIAANVLHATPTL
QETLRNVRRLLKPGGYLIALEITNIDTIRIGFLMCAFDGWWLGREDGRPWGPVVSASQW
DSLLRETGFGGIDTITDRAADQLTMYSVFAAQAVDDQITRCREPLTPLPPQPPFCRGVIIG
GSPSLVTGIRVIIHPFFSTVEHVSTIENLTEGAPAVVLMLADLSDIPCFENLTESRLAGLKA
LVQMAEKTLWVTTGSEADNPYLCLSKGFLTSMNYEHPAIFQYLNIIDSADVQPVVLAEH
LLRLAYTNQNNDFALTNCVHSTELELRLYQGGILKFPRINASDVLNSRYAAARRPVTHS
VTNMQDSVVVLDQSPSGKLRLVFGEELAGDRATVTINVRYSTSRAIRINGAGYLVLVLG
QDKVTKARLVALAGQSASVVSSSCYWEVPADIFEEQEPAYLYATATALLAASLVQSNG
TTILVHGADMVLRHAIAIEAASRVIQPIFTTTSPSAASSAGLGKSILVHENDTRRQLVHLL
PRYFTAAVNFDPSARRLFDRMMTVGHQSGVTEEHLLTTLTAALPRPSASLLPAQPQAA
MDTLRKASLTAYQFTVQLTAPGPIIAPIADIQSCSQQLAVVDWKPSCGSVPVHLQPATEL
VRLSAQKTYLLVGMTGALGQSITQWLVTRGARNIVLTSRKPSVDPAWIAEMQTTTSAR
VLVTPMDVTSRDSILVVAHALKADWPPLGGVVNGAMVLWDRLFVDAPLSVLTGQLAP
KVQGSLLLDEIFGHEPGLDFFILFGSAIATIGNLGQSAYTAASNFMVALAAQRRARGLVA
SVLQPAQVAGAMGYLRDKDDSFWARMFDMIGRHLVSEPDLHELLAHAILSGRGPPADV
GYGPGEDECIIGGLRVQDPAVYPDILWFRTPKVWPFIHYHHEGTGPSSGAAGSISLVDQL
KCATSLAQVGDMVEAGVAAKLHHRLHLPGEVGGVTGDTRLTELGVDSLIAVDLRRWF
63

AQELEVDIPVLQMLSGCSVKELAASATALLHPKFYPEVVADSDVGSERDGSSDSRGDTS
SSSYQLITPEEGDH

Gene: ctvB
Size: 687 bp
ATGACCTCCTACCAGCTTTCCGATGCCGAAGGCGCCGACNACTACTACAATCCACTT
CTCTTGTGGTGGTACGACTTCTGGGTTCATTGGGTCAGCGCGCTCTTTGCCTGGAAGT
GTTCGTCTAAGGACATTCTTCTCCCTTTTTTCCTGTCCAACATTGGATCTCGGCACTG
TGATGTTGGCGTGGGCACGGGCTACTATCTCTCTGCTGTGCGGAAGCGTCGGCCGTC
CTGGCCGGAGAAGCTGACGCTAGTGGATTTTCACATCCGATGCCTGAGGAAGGCTG
CCAACCGCGTTGGAGTAGCGGATCGCACGGAGTGTGTGCTGGCCAATATCCTGGAG
CCCATCCCTATACAACCTGAACGGCAGTTCGACTCCATATCCCTTATGTACGTCCTG
CACTGTCTCCCGGGGACTTCTAAGGACAAAGGACGCGTGTTTGCCAACTTGAAGCCG
CTCCTCAAGGACAATGGCACTCTCTTCGGGTCTACGCTGCTGTGCCGTGGAGTTCGG
CAGAATTGGTTCAGTTGGCTCCTTCAGCGCATCTACAATGCTGTGGACATGTTTCAG
AATCGGTCAGACTATCCAGACGACTTTGTCCGTGCGCTGGAGGACGAGTTCGAGGA
GGTGGAGAGCGTGATTATTGGAACGGTCCTTATGTTCAAGGCGCGGAAGCCTCGTCG
CTAG

Protein: CtvB
Size: 228 aa
64

MTSYQLSDAEGADXYYNPLLLWWYDFWVHWVSALFAWKCSSKDILLPFFLSNIGSRHC
DVGVGTGYYLSAVRKRRPSWPEKLTLVDFHIRCLRKAANRVGVADRTECVLANILEPIP
IQPERQFDSISLMYVLHCLPGTSKDKGRVFANLKPLLKDNGTLFGSTLLCRGVRQNWFS
WLLQRIYNAVDMFQNRSDYPDDFVRALEDEFEEVESVIIGTVLMFKARKPRR

Gene: ctvC
Size: 1611 bp
ATGGAAGGAAAGCACCCTCAGTTCAAGGTCGTCATCGTCGGGGCATCCGTCACGGG
TCTGACCCTCGCCCACTGTCTGCATCGGGCCGGAATAGACTACGTGGTGCTGGAGAA
GCATCACGAAGTCCATCCGCCGATTGGCGCCGCGGTGGCCATCCTGCCGAATGGAG
GGCGGATCATGGAGCAGCTGGGGATCTTCCGGCACATTGAAGACCGCTGCCAGCCA
TTTCAAAGGGTGCATCTTTGCTTTCAGGATGGGTTCTACTATGATAGCCTTTCGCCTA
GTGTCGTCTTGAAGAGGTCGGTGCTTCCTTGGTCCCCGAGAGGGTGGAACAAAGTCG
TAAGAAGACGCTAACACGGCGGGTACGCAGGTTTGGCTTGAAATTCGCGTGCCTGG
AACGGACTCAGCTACTCGAGATCCTGTACGCCCATTTGCCTGACAAGTCCAGGGTCC
TCACGAGCAAAGGCGTCGTCCGGATCACTCCCCATGGCAGCAAGATGACGGTCACC
ACCGCAGATGGGGACGAGTTCCAGGGAGATCTGGTTGTCGGTGCGGACGGCGTGCA
CAGTGTGACGCGGCGAGAGATGTGGCGCATTGCTAATATAGAGCAGCCGGGGCTGA
TTCCGCTCAAAGAACAGGCCAGTATGTTGCTACACTCCTTATCGATTCCGTCCAATA
TTAATAGATGATGCTCTGCGGCTAGGCATGTCCGTCGAGTTCAGCTGCGTCTTCGGC
ATGTCCAACCCCATCCCCGGACGGAAGCGTTGGCAGCATGTGATCCGCATCGGGCC
CGGTTTCACGATCCTGATATTCCCTGCGACCGGGGAGAGCCTTTTCTGGGTGCTGAT
TGAAAAGCTACCACACAAGTATATCTACCCTGACGTTCCGCGATTCTCGCAGGAGGA
65

CGCCATTGCGCGCTGTGAGGCAGCCGCCAGCCAGCCTATCTGGGAGGAAGTACAAT
TCCGGGATATCTGGGCTCAACGCCGGGGATTCCGGATGGTTGCGTTGGAGGAGAAT
CTCTTCCGTACGTGGCATCATGGCCGGATCATCTGCATCGGGGATAGTATCAGCAAG
GTATGTCTTGCATGGAGCCTTTTTTTCCCCTTCATATACTGATAGCTGGGACTCTAGA
TGACCCCCAACATCGGTCAAGGCGCCAATACAGCCATTGAAGCGGCGGCTGGACTG
GCCAACGTGATATATGCGATTGCACAAAACCATGACCAGCCGTCAAGTGACACCAT
TCACCAGGCGCTCGCTGCCTTCAGCGAGAAAAACCGCAAACGACTGGATGCTATCC
ATCTAGAGTCGCGCTGGATCACGCGATTGGAGGCTTGCCAGGGACGGATGGTCACG
GCCTTCACGCGATATGTGGCGCCACACTGTGGCGATCTTTTTGCGTTGGGGGTGGTG
CGTAACTCATACAACGGTGAGGTACTGCAGTTCTTGCCGTTGACTGAACGTTCGGGG
AAGTACTGGCCGAAGTTGGAGTGGTGGAATACTTGGGGTCTGTCCAAGTGGCAGGA
ATTTGGGGAGAGGGTGATGTATTGCTCTGGACTTGTGATAGTTTTGTGGATTAGCTG
GGTGGTGTTCAATGTCAAGGAAGTATGA

Protein: CtvC
Size: 473 aa
MEGKHPQFKVVIVGASVTGLTLAHCLHRAGIDYVVLEKHHEVHPPIGAAVAILPNGGRI
MEQLGIFRHIEDRCQPFQRVHLCFQDGFYYDSLSPSVVLKRFGLKFACLERTQLLEILYA
HLPDKSRVLTSKGVVRITPHGSKMTVTTADGDEFQGDLVVGADGVHSVTRREMWRIA
NIEQPGLIPLKEQASMSVEFSCVFGMSNPIPGRKRWQHVIRIGPGFTILIFPATGESLFWVL
IEKLPHKYIYPDVPRFSQEDAIARCEAAASQPIWEEVQFRDIWAQRRGFRMVALEENLFR
TWHHGRIICIGDSISKMTPNIGQGANTAIEAAAGLANVIYAIAQNHDQPSSDTIHQALAAF
66

SEKNRKRLDAIHLESRWITRLEACQGRMVTAFTRYVAPHCGDLFALGVVRNSYNGEVL
QFLPLTERSGKYWPKLEWWNTWGLSKWQEFGERVMYCSGLVIVLWISWVVFNVKEV

Gene: ctvD
Size: 1132 bp
ATGGCCCTTTCAGCCTACATACTTCTCTGCTTGTCCGTACTTGGACTAGACGCCATCT
ATGGCTTCNGATTCCGGAATGGCTTCCTCGAGCTCATGGCCAACAGCTACCGAGAGC
GAAAGCTATCCGGCACGGCAGAGCCCTTGCAAGGCAACATCACCGGAACGGGATTT
GACGAACTCTTGGGAAATCTAATTGTCTTCTACTGGCCCGTGCTGGACGGACGCCAT
CCTGGTCTCAGTGTCCAGGCCTTTCACTTCTCTGGTGCAATCGTGGCCGTGTGGGTGG
CGATCCAGGTCCAGAGCTGGAGATCGCCAACCCGCACCAGCGTTCTGCTATCGTGA
GTGTCTCTACCCTCGTATCCCTATTCTTCATCTGTCGGCCATGTTCCGAACCAACCGT
CATAGTCCAACCCTTTTCGCCATGCTATCCCAGGTCGTGGCCATCGCCGTCGTCGTA
CCCCTCTGGTGCGCGATCCACATCTGGTCGTCGTCGTCGTCGTCGTCATCATCATCAT
CACGCCCAGTGGCGCGCGTTATCTCGGCATCAGCAGCTCATTCCATACGCCTCATCC
CCATCAGCATGATCCTGGGATTTGGAGTCCCGACTATCGCCATGGTCCTTCCGGAAT
CCGCGCACCAGGACTTGTTCGGCAAACAGATGGCCATTGCCGCATGGCAGCTCTGG
CCAATCTATGCAGCTCTGTGTCACTGGGGTCTACGGGCCTTTTTCCGACCAAGAGCG
AGCACGGGTATCTCCACGAGGAGTGCATGCCGGACCGCGTATACCTTTGCGTTTGTA
TGCGCGATTATCCCCCATGTAATCAGCTGGGGTCTTTCCCTGACCTTGGCTCCTACAA
ATCTCTTGGCGGATGTATCCCCTTGGCAGTTTGCTGGGGGTCGTACGGTCCAGGTTC
AATCCATGGCCCAAGGGGCGCTGTGGTTCCTACAGTGGGATCACCTCATTGGAATGG
GGAGCTTTCTCCTGTGGGCACTGCATATGCGGCGGACAGTTGAGAGGCAGTCGTCAT
67

TCACACAGACTTGCTATCTAGCGCTGAAAGTTGGAGGTCTTTGTCTGATCTCAGGGC
CCTGCGGTGCGGCTGTTTGGTTGCTGCGGGAGGAATCTCAATTCTGA

Protein: CtvD
Size: 354 aa
MALSAYILLCLSVLGLDAIYGFXFRNGFLELMANSYRERKLSGTAEPLQGNITGTGFDEL
LGNLIVFYWPVLDGRHPGLSVQAFHFSGAIVAVWVAIQVQSWRSPTRTSVLLSPTLFAM
LSQVVAIAVVVPLWCAIHIWSSSSSSSSSSSRPVARVISASAAHSIRLIPISMILGFGVPTIA
MVLPESAHQDLFGKQMAIAAWQLWPIYAALCHWGLRAFFRPRASTGISTRSACRTAYT
FAFVCAIIPHVISWGLSLTLAPTNLLADVSPWQFAGGRTVQVQSMAQGALWFLQWDHLI
GMGSFLLWALHMRRTVERQSSFTQTCYLALKVGGLCLISGPCGAAVWLLREESQF

Gene: ctvE
Size: 1733 bp
ATGTTCAGACTATCATCGTCAGGCCTTCTGAAAGGAGCCTGGGCCTCCCGGTCTCGT
GTGCCTCAGCTTGGTCGTTGTCTCTACAGCACCACTGCCTCTGCGGGCGCTGACAAG
ACCCAGGGAAAAATCCACACTGTGATCGGAGCAGGTAATCCGGAAGCCCCTGATGG
TCTCGTTTCAGACAAATCTGACCACGACTGATAGTTGTCGATGTTCAATTCAACCAT
GGTCGCCTTCCGCCCATTCTGAATGCCCTCGAAACCACCAACCAAGGCAAGAAACT
GGTCCTCGAGGTTGCGGTACTTCCCTCCTGTCCTCACTCGTCTACAGCCCCCCCAGAC
TGATCCTGTAACAGCAACATCTGGGCGAGCACACCGTTCGATGCATTGCCATGGATG
GTGAGTACCTCCAAGATATCCCTCACCCTCCAGTTAGAGACACACTCATGCAGTCAA
CAGGTACCGAGGGCCTCGTGCGAGGAACCTCCGTCACCGACACCGGAAATCCGATC
68

ATGGTACCCGTTGGACCAGCCACTCTGGGTCGCATCATGAACGTCACCGGCGATCCC
ATCGATGAGAGGGGCCCCATTGAAGGCGTTCGTCTGATGCCTATCCACACCGATCCT
CCTGCATACACCGAGCAATCCACCCATGCGGAAATCCTGGTCACCGGCATCAAAGT
GGTCGACCTCCTGGCCCCTTACGCTCGTGGTGGCAAGATCGGTCTGTTCGGAGGTGC
TGGAGTGGGAAAGACTGTCTTCATCCAAGAGCTGATTGTCTGAGTCCCTTCCCACTC
TCTCTATTTTGGTGCTGGCCCAGGCTAACGTTCTTTCGTAATAGAACAACATCGCGA
AAGCCCACGGTGGATACTCGGTGTTTACCGGCGTTGGAGAGAGAACTCGTGAGGGT
AATGACTTGTACCATGAGATGCAGGAGACAGGCGTCATCCAGCTCGATGGCGAGTC
CAAGGTCGCGCTGGTCTTTGGCCAGATGAACGAGCCCCCGGGTGCTCGCGCTCGCGT
TGCTCTTACCGGGTTGACAATTGCTGAATATTTCCGTGACGAAGGCCAGGATGGTAA
GACCAGCTCTCAATCCGTCCGGCTTCGCCCACAAAATACCTAACATATTCAATGCAG
TCCTTCTTTTCATCGACAACATCTTCCGGTTCACCCAGGCGGGGTCCGAAGTGTCCG
CGCTTCTTGGCCGGATTCCTTCTGCGGTGGGATATCAGCCCACGCTCGCCGTCGACA
TGGGAGCAATGCAGGAGCGTATCACTACGACTACCAAGGGCTCAATTACCTCCGTG
CAGGCCGTGTACGTGCCTGCTGACGATCTGACTGATCCTGCGCCCGCAACGACCTTT
ATCCATTTAGATGCGACGACCGAGCTCTCTCGTGGTATCTCCGAGCTTGGTATCTAC
CCTGCTGTGGACCCGCTGGGATCGAAGTCTCGACTGATGGACCCGCGTATCGTGGGC
GAGGAGCACTACGACACGGCGATGCGCGTGCAACGTACCCTCCAGGAGTACAAGTC
GCTGCAGGACATCATCGCTATCCTCGGTATGGATGAGCTCTCGGAAGCGGATAAGAT
AACTGTCGAGCGGGCTCGGAAGCTGCAGAAGTTCCTCAGTCAGCCATTCGCTGTTGC
GGAGGTGTTCACAGGTATCCCGGGACAGCTGGTGTCGCTCGAGGACACCATTCGGTC
TTTCAAGGCTGTTCTGGATGGACAGGGTGA

69

Protein: CtvE
Size: 468 aa
MFRLSSSGLLKGAWASRSRVPQLGRCLYSTTASAGADKTQGKIHTVIGAVVDVQFNHG
RLPPILNALETTNQGKKLVLEVAQHLGEHTVRCIAMDGGTEGLVRGTSVTDTGNPIMVP
VGPATLGRIMNVTGDPIDERGPIEGVRLMPIHTDPPAYTEQSTHAEILVTGIKVVDLLAPY
ARGGKIGLFGGAGNNIAKAHGGYSVFTGVGERTREGNDLYHEMQETGVIQLDGESKVA
LVFGQMNEPPGARARVALTGLTIAEYFRDEGQDVLLFIDNIFRFTQAGSEVSALLGRIPS
AVGYQPTLAVDMGAMQERITTTTKGSITSVQAVYVPADDLTDPAPATTFIHLDATTELS
RGISELGIYPAVDPLGSKSRLMDPRIVGEEHYDTAMRVQRTLQEYKSLQDIIAILGMDEL
SEADKITVERARKLQKFLSQPFAVAEVFTGIPGQLVSLEDTIRSFKAVLDGQG

70

CHAPTER III: Discovery and elucidation of the biosynthesis of aspernidgulenes, novel
polyenes from Aspergillus nidulans, using serial promoter replacement

3.1 Abstract
Through serial promoter exchanges, we isolated several novel polyenes, the aspernidgulenes, from
Aspergillus nidulans, and uncovered their biosynthetic pathway. A highly-reduced polyketide
synthase putatively produces a polyene pyrone, which putatively undergoes epoxidation, epoxide
opening, followed by another epoxidation and cyclization to assemble an oxabicylo[2.2.1]heptane
ring and a cyclopentanone moiety. Aspernidgulene A1 (4) is generated after hydrolytic cleavage
of the oxabicyclo[2.2.1]heptane ring.
3.2 Introduction
Natural products have historically been an important pipeline for therapeutics, and recently there
have been revived interests in natural product based drug discovery.
242
The rapid accumulation of
publically available genome data and advancements in molecular biology offer us unprecedented
opportunities to discovery novel chemical structures and to investigate the enzymatic basis of their
biosynthesis.
243
Still, more than a decade after the first filamentous fungi genomes were
published,
244

245

246
the vast majority of secondary metabolite gene clusters remain unelucidated.

For instance, a recent genome sequencing study of 9 members of the Penicillium species showed
that only 16% of the gene clusters containing either a polyketide synthase (PKS), non-ribosomal
peptide synthetase (NRPS), or PKs-NRPS hybrid in those genomes could be connected to a known
pathway (127/798).
247
For even a well-studied model fungus such as Aspergillus nidulans, less than
half of its secondary metabolite gene clusters have been linked to downstream products despite
71

intensive research.
248
The discrepancy between the number of known natural products and the
number of gene clusters suggests that the potential for secondary metabolites to be a source of
antibiotics and therapeutics is vast and still largely unexploited.
249

The genome of A. nidulans contains 27 PKSs, 15 of which are non-reducing polyketide synthases
(NR-PKSs) and 12 are high-reducing polyketide synthases (HR-PKSs).
250
Microarray
transcription data of Aspergillus nidulans indicates that one HR-PKS AN1784 is co-regulated with
three other genes in the putative cluster.
251

This four-gene cluster attracted our attention for two reasons. First, three of its four genes have
homology with the biosynthetic genes of aurovertin (aur) and citreoviridin (ctv) (Table 3-1), which
belong to a class of potent ATP synthase inhibitors made from HR-PKSs, leading us to speculate
that the AN1784 cluster might encode for a new member of this class.
252

253
These inhibitors have
been tested as therapeutic agents
254
and the conciseness of the enzymatic assemblies of their
unique ring systems have been noted.
255
Second, the four genes in the cluster are divergently
transcribed. AN1784 and 1785 are a pair of adjacent genes that have opposite orientations, and
AN1786 and 1787 form the other pair. Divergent transcription allows us to replace both promoters
in one transformation using one selectable marker as described previously.
256

257
Therefore, we can
quickly activate the entire cluster in two rounds of transformation.

Through serial promoter replacement, we discovered a series of new metabolites made by this
cluster, which we named the aspernidgulenes. Here we report our investigation of the
aspernidgulenes gene cluster (sdg).
72

Like the ctv cluster, the sdg cluster contains an HR-PKS and two enzymes putatively responsible
for cyclization of the polyketide product. The HR-PKS AN1784 is homologous to CtvA (Table 3-
1) and both enzymes share the same domain architecture of KS-AT-DH-CMet-KR-ACP
258,259
. We
thus named AN1784 SdgA. AN1785 is a putative membrane-associated hydrolase with homology
to CtvD and was named SdgD. AN1786 is a flavin-dependent mono-oxygenase with homology to
CtvC and was named SdgC. Previous work have demonstrated that CtvC and CtvD and its
homologs AurC and AurD are responsible for the assembly the tetrahydrofurane ring in
citreoviridin and 2,6-dioxabicyclo[3.2.1]-octane ring in aurovertin respectively.
260
Unlike the ctv
cluster, however, there is no methyl transferase (CtvB) or resistance gene (CtvE). Instead, AN1787
is a putative FAD-linked oxidase and is homologous with Sol5 of the solanapyrone biosynthesis
cluster.
261
We named it SdgF.

3.3 Results and Discussion
We first activated sdgA and sdgD together by replacing both promoters with the inducible alcA
promoter (alcA(p)) in a single transformation.
262
Under inducing conditions, three new peaks 1 –
3 not present in the host strain were detected in the media (Figure 3-1, i and ii). Next, we activated
the other divergently transcribed pair, sdgC and F to generate a strain expressing the entire cluster,
sdgADCF. In this strain three new peaks 4 – 6 emerged (Figure 3-1, iii). In addition, we also
activated AN1783, a putative dehydrogenase with homology to Sol3 from the solanopyrone
pathway (Table S1).
263
However, we did not detect any new peaks in the SdgADCF + AN1784
coexpressing strain, (Figure 3-1, iv), suggesting that AN1783 is not part of the sdg cluster as the
73

transcription data predicted.
5
Therefore, 4 – 6 are likely to be the final products of this four-gene
cluster.

We purified compounds 4 – 6 from large scale fermentation and elucidated their structures based
on spectroscopic data (see Supplementary Information for detailed structural elucidation of all
compounds). All three compounds contain a 2,3-dimethyl-γ-lactone substituted with a 1-
carboxyethyl group, a moiety that has been observed in other fungal natural products (Figure 3-
3B). In compound 4, the γ-lactone is linked to a trimethylpentanone by a triene (Figure 3-3A). We
named it aspernidgulene A1. Compound 5 is a 19-epimer of 4, so we named it aspernidgulene A2.
Compound 6 lacks the trimethylpentanone group and is a tetraene (Figure 3-3A). We named it
aspernidgulene B1.

Having identified the likely final products, we proceeded to investigate the functions of the
individual genes in the sdg cluster. First, we created two strains: SdgA and SdgADF. We found
that the metabolite profiles of both are identical to that of SdgAD expression strain (Figure 3-1, ii
and v, vi). These data indicate that SdgD and SdgF do not act immediately downstream of SdgA
and that the HR-PKS SdgA is alone is sufficient for the productions of peaks 1 - 3.

The purification of compound 1 – 3 was difficult due to the instability of the polyenes, which has
been noted by other chemists.
264
While we were unable to obtain high quality NMR spectral data
despite our best attempts, we were able to deduce the chemical structures of 1 and 2. They were
pyrone-containing hexaenoic acids, which we named preaspernidgulene A1 (1) and A2 (2) (Figure
3-3A). Since SdgA does not contain an oxidase domain, it is likely that the carboxylic acids on 1
74

and 2 are installed by unknown endogenous enzymes, as our lab has previously observed similar
oxidation of polyketides.
265

After concluding that SdgD and SdgF do not act immediately downstream of SdgA, we turned our
attention to SdgC. We created a SdgAC expression strain, which produced compounds 7 – 9
(Figure 3-1, vii). NMR characterization revealed their structures (Figure 3-3A). We named 7
aspernidgulene B2 due to its structural similarity to aspernidgulene B1 (6), and we named 8 and 9
preaspernidgulene A3 and A4 respectively. Compounds 7 – 9 all contain a secondary alcohol on
the side chain, suggesting that SdgC is responsible for the epoxidation of the side chain (Figure 3-
1) like CtvC
7
and AurC
6
in the citreoviridin and aurovertin biosynthesis pathways, respectively.
Epoxide ring opening provides the driving force for the cyclization to assemble the
oxabicyclo[2.2.1]heptane ring, which could then undergo hydrolytic cleavage to yield the 2,3-
dimethyl-γ-lactone moiety. It is fascinating that one enzyme is sufficient for the transformation of
the hexa-ene α-pyrone into the 2,3-dimethyl-γ-lactone moiety in 7.

Lastly, to establish the roles of the predicted hydrolase SdgD and the oxidase SdgF, we generated
and analyzed two strains SdgADC and SdgACF. The SdgADC strain produced 7 without
detectable amounts of 8 and 9 (Figure 3-1, viii). These data suggest that SdgD somehow facilitates
cyclization to form 7, eliminating the shunt products 8 and 9. The SdgACF strain produced 4 – 6
as its major products (Figure 3-1, ix), indicating that SdgF is involved in the formation of the
trimethylpentanone. Interestingly, the SdgACF strain also produced minor products such as 8 and
9 as well as 10, 13, 16-19 that were not found in the SdgADCf strain (Figure 3-S1). Based on UV
75

absorption and ionization patterns, it is possible that 10 and 13 are isomers of 4 and 5 (Figure 3-
S2, S3), suggesting that SdgD could play a role in the regioselective cyclization to form 4 – 6.

Based on our data, we propose the biosynthetic pathway of compound 4 (Figure 3-2). The carbon
backbone is synthesized by the HR-PKS SdgA, which accepts acetyl-CoA as the starter unit and
catalyzes malonyl-CoA extension, methylation, and

reduction. The resulting nonaketide offloads the HR-PKS via spontaneous lactonization, yielding
a hexa-ene α-pyrone. After the hexa-ene α-pyrone is produced, SdgC catalyzes epoxidation on the
penultimate double bond. The epoxide is then oxidized by SdgF to a ketone in a reaction involving
hydride shift. Subsequently, SdgC installs another epoxide on the last double bond. SdgD then
catalyzes epoxide opening, and the positive charge is carried down the polyene chain to C6, where
it is intercepted by the enol form of the β-keto lactone moiety, forming a the oxygenated
cyclopentanone ring and an oxabicylo[2.2.1]-heptane unit. Finally, SdgD catalyzes hydrolytic
cleavage of the bicyclic unit to form the γ-lactone moiety in compound 4.

The formation of 4 in strains not expressing SdgD (Figure 3-1, ix) likely resulted from spontaneous
formation and hydrolytic cleavage of the oxabicylo[2.2.1]-heptane. This hypothesis is supported
by the results of Miller et al., who obtained oxabicylo[2.2.1]-heptanes by treating β-ketolactone
with camphorsulfonic acid.
266
Moreover, treatment of prugosene A1 (Figure 3-3B) with diluted
NaOH led to the production of prugosene B1.
267
Thus, these reactions can happen spontaneously
under mild acidic or basic conditions.

76

The relative stereochemistry of the hydroxyl-trimethyl-cyclopentanone moieties in compounds 4
(14S*, 16R*, 17R*) and 5 (14S*, 16S*, 17R*) is likely determined by the configuration of the last
two double bonds and of the two epoxides installed on them. Given that each double bond could
either be E or Z and could be epoxidated in two different orientations, there are sixteen possible
combinations (Figure 3-S6-S9). Of them, four would result in the stereochemistry of 4 and another
four the stereochemistry of 5. The conformation depicted in Figure 1 is only one of the possibilities
for the formation of 4. The suppression of the production of compounds 10 and 13, which could
be epimers of 4 and 5 based on UV-vis absorption patterns and mass spectroscopy, suggests the
role of SdgD in stereo-control, but the timing and mechanism of double bond isomerization and
epoxidation are unclear.

Interestingly, NOESY data indicate that compounds 4, 5 (Table 3-S6) and wortmannilactone G are
(6R*), while prugosene B1 is (6S*). The stereochemistry at C6 is determined by how the β-keto
lactone intercepts the positive charge on C6 after epoxide opening. Since no C6 epimers
were isolated in all three studies,
268

269
this reaction could be enzymatically controlled by SdgD
and its homologs.

In our proposed pathway, compounds 6, 7, 8, and 9 are shunt products formed through spontaneous
epoxide opening. Reduction by possibly endogenous enzymes leads to the formation of 8 and 9.
Spontaneous cyclization and hydrolytic cleavage leads to accumulation of 7, and hydride shift,
possibly catalyzed by SdgF, leads to the accumulation of 6.

77

Using serial promoter replacement, we have unexpectedly discovered the aspernidgulenes. While
three genes in the cluster have homologs to the ctv and aur biosynthetic genes, the aspernigulenes
altogether fall into another structural class of molecules. Related natural products such as
shimalactone A, the prugosenes, wortmannilactones E-L
270
, and coccidiostatin A likely employ
homologous enzymes in their biosynthesis.

3.4 Supplementary Information
Strains and molecular genetic manipulations.
The primers used to generate transforming fragments via fusion PCR are listed in Table 3-2. The
strains generated in this study are listed in Table 3-2. The procedures for fusion PCR, protoplasting,
and transformation are as previously described,
1
with the exception that VinoTaste Pro (Novo)
was used at a final concentration of 100 mg/mL for protoplasting in place of VinoFlow FCE.

Fermentation and HPLC analysis.
To cultivate A. nidulans expressing genes of the aspernidgulene cluster, 3.0 × 10
7
spores were
added to 30 mL liquid LMM (15 g/L lactose, 6 g/L NaNO3, 0.52 g/L KCl, 0.52 g/L MgSO4, 1.52
g/L KH2PO4, 1mL/L trace elements (EDTA 10.0 g/L, ZnSO4·7H2O 4.4 g/L, MnCl2 1.01 g/L,
CoCl2·6H2O 0.32 g/L, CuSO4·5H2O 0.315 g/L, (NH4)6Mo7O24·4H2O 0.22 g/L, CaCl2·2H2O 1.47
g/L, FeSO4·7H2O 1.00 g/L)) in 125mL flasks at 37°C and shaking at 180 rpm. Pyridoxine (0.5
mg/mL), riboflavin (2.5 mg/L), and uracil (1 mg/mL) and uridine (10 mM) were supplemented
when necessary. After 42 hours, methyl ethyl ketone (MEK), an inducer of alcA(p), was added to
a final concentration of 50 mM.
2
Culture media was collected by filtration 72 hours after MEK
78

induction. The media was centrifuged for 10 minutes at 16,000 rcf and then 10 uL of the top layer
was injected for LC-DAD-MS analysis as described previously.
3

Isolation of secondary metabolites.
To isolate compound 4, A. nidulans strain expressing SdgADCB and AN1783 was cultivated in
750 mL liquid LMM as described above in 2000 mL flasks (4 flasks, 3L total). The cultures were
grown at 37 °C for 42 hours at ~180 rpm, and then induced with MEK to a final concentration of
50 mM. After another 72 hours of culturing the cultures were harvested. Culture media was
collected by filtration and extracted with ethyl acetate twice. The ethyl acetate extract was
evaporated in vacuo. The crude extract was purified by LiChroprep® RP-18 gel (40-64 µm) to
yield two fractions (6:4, 1:0 MeOH:H2O). The 60% MeOH fraction was evaporated and subjected
to purification by reversed-phase HPLC. The solvent system was MeCN with no TFA (solvent B)
and 95:5 H2O:MeCN with 0.005% TFA (solvent A). The gradient condition was 65% solvent B
from 0 to 4.0 minutes, 65% to 85% solvent B from 4.0 to 4.5 minutes, and 85% to 65% solvent B
from 4.5 to 5.0 minutes, and 65% solvent B from 5.0 to 9.0 minutes. Compound 4 was eluted
around 3.8 minutes. To neutralize the TFA, ~ 1 mL of 100 mM potassium phosphate (pH ~12)
was added to the collection tubes. MeCN was removed from the combined solution of eluent and
phosphate buffer by evaporation in vacuo. The remaining aqueous solution re-acdified with HCl
and extracted with EA, which was then washed with ddH2O to remove any residual phosphate.
The EA was then evaporated in vacuo, yielding 4.8 mg of compound 4.

To isolate compounds 5, A. nidulans strain expressing SdgADCB and AN1783 was cultivated in
750 mL liquid LMM as described above in 2000 mL flasks (2 flasks, 1500 mL total). The cultures
79

were grown at 37 °C for 42 hours at ~180 rpm, and then induced with MEK to a final concentration
of 50 mM. After another 72 hours of culturing the cultures were harvested. Culture media was
collected by filtration and extracted with ethyl acetate twice. The ethyl acetate extract was
evaporated in vacuo to yield ~124 mg of crude extract. The crude extract was purified by
LiChroprep® RP-18 gel (40-64 µm) to yield three fractions (3:7, 7:3, 1:0 MeOH:H2O). The 70%
MeOH fraction was evaporated and subjected to purification by reversed-phase HPLC. The solvent
system was MeCN with 0.05% TFA (solvent B) and 95:5 H2O:MeCN with 0.05% TFA (solvent
A). The gradient condition was 65% solvent B from 0 to 4.5 minutes, 65% to 100% solvent B from
4.5 to 5.0 minutes, and 100% to 65% solvent B from 5.0 to 5.5 minutes, and 65% solvent B from
5.5 to 9.5 minutes. Compound 4 was eluted around 3.8 minutes and 4.7 mg was purified.

To isolate compounds 6, A. nidulans strain expressing SdgADCB and AN1783 was cultivated in
50 mL liquid LMM as described above in 125 mL flasks (48 flasks, 2400 mL total). The cultures
were grown at 37 °C for 42 hours at ~180 rpm, and then induced with MEK to a final concentration
of 50 mM. After another 72 hours of culturing the cultures were harvested. Culture media was
collected by filtration and extracted with ethyl acetate once. The ethyl acetate extract was
evaporated in vacuo to yield ~150 mg of crude extract. The crude extract was purified by silica gel
to yield 30 fractions (3:97 10:90 MeOH:DCM). Fractions 11-18 was evaporated (41.8 mg) and
subjected to purification by reversed-phase HPLC. The solvent system was MeCN with 0.05%
TFA (solvent B) and 95:5 H2O:MeCN with 0.05% TFA (solvent A). The gradient condition was
75% solvent B from 0 to 4.5 minutes, 75% to 100% solvent B from 4.5 to 5.0 minutes, and 100%
to 75% solvent B from 5.0 to 5.5 minutes, and 75% solvent B from 5.5 to 9.0 minutes. Compound
6 began eluting around 5.7 minutes. To neutralize the TFA, ~ 1 mL of 20 mM potassium phosphate
80

dibasic was added to the collection tubes. MeCN was removed from the combined solution of
eluent and phosphate buffer by evaporation in vacuo. The remaining aqueous solution was
acidified with HCl and then extracted with EA. MgSO4 was added to remove water. The EA was
then evaporated in vacuo, yielding compound 6.

To isolate compound 7, A. nidulans strain expressing SdgADC was cultivated in 50 mL liquid
LMM as described above supplemented with pyridoxine (0.5 mg/L) in 125 mL flasks (25 flasks,
1250 mL total). The cultures were grown at 37 °C for 42 hours at ~180 rpm, and then induced with
MEK to a final concentration of 50 mM. After another 72 hours the cultures were harvested.
Culture media was collected by filtration and extracted with ethyl acetate twice. The ethyl acetate
extract was evaporated in vacuo to yield ~116 mg of crude extract. The crude extract was purified
by LiChroprep® RP-18 gel (40-64 µm) to yield two fractions (1:1 and 1:0 MeOH:H2O). The 100%
MeOH fraction (72.8 mg) was evaporated and subjected to purification by reversed-phase HPLC.
The solvent system was MeCN with no TFA (solvent B) and 95:5 H2O:MeCN with 0.005% TFA
(solvent A). The gradient condition was 75% solvent B from 0 to 3.5 minutes, 75% to 100% solvent
B from 3.5 to 4.0 minutes, and 100% to 75% solvent B from 4.0 to 4.5 minutes, and 75% solvent
B from 4.5 to 8.5 minutes. Compound 7 eluted around 4.3 minutes and 17.8 mg was purified.

Compounds 1 and 2 were purified from the culture medium of A. nidulans strain expressing SdgA.
Initially, the crude extract was applied to a silica gel (SiO2) column and eluted with 0% to 10 %
of MeOH/CH2Cl2. However, none of the fractions collected contained detectable 1 and 2,
indicating that 1 and 2 might not be stable in silica gel or in aprotic solvent. Therefore, the use of
silica gel and aprotic solvent were avoid in the purification step afterward. Specifically, 50 flasks
81

of 30 mL liquid LMM culture medium (~ 1500 ml total) were collected after 72 hr of induction.
The medium after filtration was directly applied to a flash reverse phase C18 gel column
(COSMOSIL 75C18-OPN, 30 100 mm). After flow through the culture medium to the C18
column, it was then eluted with MeOH-H2O mixtures of decreasing polarity (fraction A, 1:9, 300
ml; fraction B and C, 7:3, 200 ml each; fraction D, 1:0, 200 ml). Fraction B (~450 mg) containing
the metabolites of interest was further purified by semi-preparative reverse phase HPLC. The
solvent system was MeCN (solvent B) in 5% MeCN/H2O (solvent A) both containing 0.05% TFA.
The gradient condition was 40% to 75% solvent B from 0 to 15 minutes, 75% to 100% solvent B
from 15 to 16 minutes, maintained at 100 % B from 16 to 18 minutes, 100 to 40 % B from 18 to
19 minutes, and re-equilibration with 40 % B from 19 to 22 minutes. Compound 1 and 2 eluted at
12.2 min and 14.0 minutes, respectively, were collected and TFA was neutralized immediately
with 0.1 M of phosphate buffer (pH = 7). After evaporating the solvent in vacuo, the phosphate
salt was removed by gel filtration using Sephadex LH-20 (10 300 mm) eluted with MeOH.
After evaporating the MeOH, 7.3 mg of compound 1 with poor purity and 5.1 mg of compound 2
with good purity examined by 1H NMR was obtained (Figure S?). 1D and 2D NMR spectral data
of compound 2 were then collected immediately after good purity of 2 was obtained.

To isolate compounds 8 and 9, A. nidulans strain expressing SdgAC was cultivated in 750 mL
liquid LMM as described above in 2000 mL flasks (3 flasks, 2250 mL total). The cultures were
grown at 37 °C for 42 hours at ~180 rpm, and then induced with MEK to a final concentration of
50 mM. The temperature was lowered to 30 °C at the time of induction. Cultures were harvested
after 72 hours. Culture media was collected by filtration and extracted with ethyl acetate twice.
The ethyl acetate extract was evaporated in vacuo to yield ~158 mg of crude extract. The crude
82

extract was subject to silica gel to yield three fractions (6:4, 5:5, and 3:7 hexane:EA). The 60%
hexane fraction (53.3 mg) was evaporated and subjected to purification by reversed-phase HPLC.
The solvent system was MeCN with no TFA (solvent B) and 95:5 H2O:MeCN with 0.05% TFA
(solvent A). The gradient condition was 70% solvent B from 0 to 8.0 minutes, 70% to 100% solvent
B from 8.0 to 8.5 minutes, and 100% to 70% solvent B from 8.5 to 9.0 minutes, and 70% solvent
B from 9.0 to 13.0 minutes. Compound 7 began eluting around 5.7 minutes and compound 8 began
eluting around 7.5 minutes. To neutralize the TFA, ~ 1 mL of 100 mM potassium phosphate
dibasic was added to the collection tubes. MeCN was removed from the combined solution of
eluent and phosphate buffer by evaporation in vacuo. The remaining aqueous solution was
extracted with EA, which was then washed with ddH2O to remove any residual phosphate. The
EA was then evaporated in vacuo, yielding 15.0 mg of compound 6 and 15.5 mg of compound 8.

Compound identification.
NMR spectra were collected on a Varian Mercury Plus 400 spectrometer and Varian VNMRS-600
3-Channel spectrometer.

UV-vis and ESIMS spectra were obtained by 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 included a papillary voltage 5.0 kV , a sheath gas flow
rate at 50 arbitrary units, an auxiliary gas flow rate at 0 arbitrary units, and the ion transfer capillary
temperature at 275 °C.

83

HRESIMS spectra were obtained by Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap
mass spectrometer with an Eclipse XDB-C18 column (Agilent 5 um 4.6 ×150 mm) at a flow rate
of 125 µL/min. The condition for MS analysis included a sheath gas flow rate of 20, aux gas flow
rate 5, sweep gas flow rate 1, spray voltage 3.5 kV , capillary temperature 275 °C, s-lens RF level
55, aux gas heat temperature 325 °C, scan range 300-600 m/z, resolution 140,000, AGC target 3e6,
maximum injection time 200 ms.

84

Figure 3-1. Proposed biosynthetic pathway for asperniduglene A1 (4).

85

Figure 3-2. (A) Organization of the aspernidgulene (1) biosynthesis gene cluster. (B) Total scan
HPLC profiles of culture media of A. nidulans strains expressing aspernidgulene biosynthesis
genes under the control of alcA(p).

86

Figure 3-3. (A) New compounds isolated from the study. (B) Structures of representative
compounds containing the oxabicylo[2.2.1]heptane unit or their derivatives.

87

Table 3-S1. Aspernidgulene biosynthesis genes in A. nidulans, their homologs in other species,
and gene function predictions.
gene putative function Aspergillus terreus
var. aureus homolog
aa (% identity, %
similarity)

Calcarisporium
arbuscula
homolog
aa (% identity, %
similarity)
Alternaria solani
homolog
aa (% identity, %
similarity)
AN1783 dehydrogenase --- --- Sol3 (25, 45)
sdgA (AN1784) highly-reduced
polyketide synthase
CtvA (44, 59) AurA (45, 63) Sol1 (28, 44)
sdgD (AN1785) Hydrolase CtvD (24, 40) AurD (23, 41) ---
sdgC (AN1786)

flavin-dependent
monooxygenase (FMO)
CtvC (39, 56) AurC (37, 54) ---
sdgF (AN1787) FAD linked oxidase --- --- Sol5 (29, 44)
88

Table 3-S2. Primers used in this study.
SdgA_F1 TCCTATCACCTCGCCTCAAAATGGCATACGATAATACAGT
SdgA_R1 TGACTGAACCAACCAGGAGG
SdgA_R2 TCGTTGTCATCAGCAAAGGC
SdgA_R3 GTTCACGCTCACATCACACC
SdgD_F1 TCCTATCACCTCGCCTCAAAATGTCTCACCAATCTACAAT
SdgD_R1 TTAGTCCTCCTTCGCGATCC
SdgD_R2 CAACGTGCTCATCACAGTCC
SdgB_F1 TCCTATCACCTCGCCTCAAAATGGACAAGAGAAGTTTCAA
SdgB_R1 GAACAACTAGTCAGTGACGG
SdgB_R2 TGAGAAGGAGAGCGAGAACC
SdgC_F1 TCCTATCACCTCGCCTCAAAATGGCGACGGTCTCCGAACT
SdgC_R1 GCTGGACGATAGCAAAGAGC
SdgC_R2 GGTTGTTGAGGATGATGACG
SdgC_F2 CAGTGCCTCCTCTCAGACAGATGGCGACGGTCTCCGAACT
SdgB_F2 GAAGAGGGTGAAGAGCATTGATGGACAAGAGAAGTTTCAA
SdgD_F2 GCATCAGTGCCTCCTCTCAGATGTCTCACCAATCTACAAT
AN1783_P1 CTTTACGGACCCGATTGTCC
AN1783_P2 CTAGATATCTCCGACGCACG
AN1783_P3 GAAGAGGGTGAAGAGCATTGATTTCTTGGTTGCCGGCTCA
AN1783_P4 TCCTATCACCTCGCCTCAAAATGGCAACATCGTACGACGC
AN1783_P5 ATGGCAGTCCTGCAGATATG
AN1783_P6 TGAACATAGGCAAGAGCTGC

Green sequences are the tail that anneals to the alcA promoter fragment during fusion PCR. Blue
sequences are the tail that anneals to the selection markers A. fumigatus pyrG (AfpyrG), A.
fumgiatus riboB (AfriboB), or A. fumigatus pyroA (AfpyroA) fragment during fusion PCR.

89

Table 3-S3. A. nidulans strains used and generated in this study. STΔ indicates that the
sterigmatocystin gene cluster was deleted in its entirety
Strain Gene(s) with
promoters replaced
with alcA(p) turned on
Genotype
LO4389 None pyrG89; pyroA4; nkuA::argB; riboB2; STΔ
CW7382
CW7383
sdgAD pyrG89; pyroA4; nkuA::argB; riboB2; STΔ; AN1785-
AN1784::AfriboB-alcA(p)-AN1785-alcA(p)-AN1784
CW7390
CW7394.5
sdgADCB pyrG89; pyroA4; nkuA::argB; riboB2; STΔ; AN1785-
AN1784::AfriboB-alcA(p)-AN1785-alcA(p)-AN1784;
AN1788-AN1787::AfpyroA-alcA(p)-AN1788-alcA(p)-
AN1787
CW7415
CW7416
sdgADCB, AN1783 pyrG89; pyroA4; nkuA::argB; riboB2; STΔ; AN1785-
AN1784::AfriboB-alcA(p)-AN1785-alcA(p)-AN1784;
AN1788-AN1787::AfpyroA-alcA(p)-AN1788-alcA(p)-
AN1787; AN1783::alcA(p)-AN1783
CW7412
CW7414
sdgADB pyrG89; pyroA4; nkuA::argB; riboB2; STΔ; AN1785-
AN1784::AfriboB-alcA(p)-AN1785-alcA(p)-AN1784;
AN1788::AfpyrG-alcA(p)-AN1788
CW7424
CW7420
sdgADC pyrG89; pyroA4; nkuA::argB; riboB2; STΔ; AN1785-
AN1784::AfriboB-alcA(p)-AN1785-alcA(p)-AN1784;
AN1787::AfpyrG-alcA(p)-AN1787
CW7450
CW7451
sdgA pyrG89; pyroA4; nkuA::argB; riboB2; STΔ;
AN1784::AfriboB-alcA(p)-AN1784
CW7455.5
CW7459
sdgAC pyrG89; pyroA4; nkuA::argB; riboB2; STΔ;
AN1784::AfriboB-alcA(p)-AN1784; AN1787::AfpyrG-
alcA(p)-AN1787
CW7460
CW7460.3
sdgACB pyrG89; pyroA4; nkuA::argB; riboB2; STΔ;
AN1784::AfriboB-alcA(p)-AN1784; AN1788-
AN1787::AfpyroA-alcA(p)-AN1788-alcA(p)-AN1787

90

Figure 3-S1. Expanded total scan HPLC profiles of culture media of A. nidulans strains
expressing(i) sdgACF (ii) sdgADCF (iii) SdgADCF+An1784 under the control of alcA(p).

91

Figure 3-S2. UV-vis and ESIMS spectra of compounds 10-12.

92

Figure 3-S3. UV-vis and ESIMS spectra of compounds 13-15.

93

Figure 3-S4. UV-vis and ESIMS spectra of compounds 16-18.
94

Figure 3-S5. UV-vis and ESIMS spectra of compounds 19.

95

Figure 3-S6. Possible cyclization patterns of (14E, 16E) terminal di-ene after bis-epoxidation.

96

Figure 3-S7. Possible cyclization patterns of (14E, 16Z) terminal di-ene after bis-epoxidation.

97

Figure 3-S8. Possible cyclization patterns of (14Z, 16E) terminal di-ene after bis-epoxidation.

98

Figure 3-S9. Possible cyclization patterns of (14Z,-16Z) terminal di-ene after bis-epoxidation.

99

Figure 3-S10. UV-vis and ESIMS spectra of compounds 1, 2, and 4.
100

Figure 3-S11. UV-vis and ESIMS spectra of compounds 5-7.
101

Figure 3-S12. UV-vis and ESIMS spectra of compounds 8 and 9.

102

Table 3-S4.
13
C chemical shift data of aspernidgulenes A1 (4) and A2 (5) and proguosene B in
CD3OD.
position 4 5 6 7 Wortmanniloactone
G
Prugosene
B1
1
2
3
4
5
6
7
8
9
10
…
6-Me
4-Me
2-Me

179.6
48.3
178.5
43.3
87.9
49.5
137.0
132.7
132.7
130.3
…
12.2
14.7
7.8
179.5
48.2
177.5
42.7
87.5
49.6
136.6
132.9
132.4
130.5
…
12.1
14.6
7.8
179.4
48.1
177.8
42.8
87.5
49.7
139.2
132.6/137.6
132.6/137.6
130.1
…
12.1/12.3
14.6
7.8
179.5
48.2
177.6
42.7
87.5
49.5
136.6
132.9
132.7
130.9
…
12.2
14.6
7.8
179.1
47.9
175.9
42.4
87.2
49.5
136.7
132.6
135.1
132.6
…
11.9
14.3
7.6
179.5
48.4
177.7
43.3
88.9
49.8
130.5
135.2
134.2
134.9
…
20.8
13.8
8.7

103

Table 3-S5.
1
H chemical shift data of aspernidgulenes A1 (4) and A2 (5) and proguosene B1 in CD3OD, unless otherwise indicated.

position 4 4 (acetone-D6) 5 Prugosene B1
1
2
3
4
5
6
7
8
9
10
…
22/26
23/27
24/28

---
2.82 (1H, q, 7.1)
---
2.71 (1H, m)
4.42 (1H, d, 10.7)
---
5.78 (1H, d, 15.0)
6.32 (1H, dd, 15.0, 10.5)
6.24 (1H, dd. 14.4, 10.5)
6.59 (1H, dd, 14.4, 11.0)
…
1.06 (3H, s)
1.12 (3H, d, 7.03)
0.98 (3H, d, 7.20)
---
2.83 (1H, q, 7.2)
---
2.69 (1qd, 7.0, 10.7)
4.40 (1H, d, 10.7)
---
5.88 (1H, d, 15.2)
6.39 (1H, dd, 15.2, 10.5)
6.24 (1H, dd. 14.5, 10.5)
6.64 (1H, dd, 14.5, 11.1)
…
1.07 (3H, s)
1.10 (3H, d, 7.0)
0.95 (3H, d, 6.7)
---
2.81 (1H, q, 7.1)
---
2.66 (1H, qd, 7.0, 10.6)
4.41 (1H, d, 10.8)
---
5.79 (1H, d, 15.2)
6.33 (1H, dd, 15.2, 10.5)
6.25 (1H, dd, 14.5, 10.5)
6.59 (1H, dd, 14.5, 11.1)
…
1.06 (3H, s)
1.10 (3H, d, 7.1)
0.98 (3H, d, 7.1)
---
2.75 (1H, q, 7.2)
---
2.59 (1H, qd, 7.1, 10.5)
4.38 (1H, d, 10.6)
---
5.57 (1H, d, 14.2)
6.33 (1H, m)
6.25-6.4 (1H, m)
6.25-6.4 (1H, m)
…
1.41 (3H, s)
1.13 (3H, d, 7.0)
0.95 (3H, d, 7.2)

104

Table 3-S6. NOESY correlation of aspernidgulenes A1 (4) and A2 (5) and proguosene B.
position 4 (CD3OD) 4 (acetone-D6) 5 (CD3OD)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

---
5, 7, 24
---
5, 22, 23
2, 4, 7, 23
---
2, 5, 8, 9
7, 9, 10, 22
7,11
8
9, 13/14
---
11, 20, 21?
11, 20, 21?
---
---
13/14
13
17
13/14, 21
13/14, 20
4, 8, 24
4
2, 22
---
5, 7, 24
---
22, 23
2, 7
---
2, 5, 9
10, 22
7, 11
8, 21
9, 13, 14
---
11, 20, 21
17, 21
---
---
18, 19, 21
17, 19
17, 18
13
10, 13, 14, 17
4, 8, 24
4
2, 22

---
5, 7, 24
---
5, 22, 23
2, 7, 23
---
2,5,9,8
7,10,22
10,11
8,9,11,21
9, 10, 13, 21
---
11, 14, 18, 19
13, 17, 20, 21
---
---
13w, 14, 18, 21
13,17,21
4,8,13
NA
10,14,17
NA
2,13,14
2

105

Table 3-S7.
1
H and NMR data for compound 1
position δH (mult, J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
---
---
---

4.34 (1H, d, 9.7)
---
6.14 (1H, d, 11.1)
6.55 (1H, dd, 14.9, 11.1)
6.37 (1H, m)
6.42 (1H, m)
6.39 (1H, d, 14.6)
---
6.04 (1H, s)
---
5.91 (1H, s)
---
5.79 (1H, s)
---
2.20 (3H, s)
2.02 (3H, s)
1.99 (3H, s)
1.84 (3H, s)
1.04 (3H, d)
1.70 (3H, s)
106

Figure 3-S13.
1
H NMR spectrum of compound 1 in CD3OD (400 MHz).
107

Table 3-S8.
1
H and
13
C NMR data for compound 2.
position δC δH (mult, J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
171.7
97.4
175.7
41.3
89.6
133.2
132.9
129.1
140.2
130.6
136.4
137.6
137.5
139.8
134.8
154.7
119.9
170.5
19.9
19.9
14.6
15.2
22.3
9.4
20.8
---
---
---
---
4.59 (1H, s)
---
6.18 (1H, d, 11.1)
6.59 (1H, dd, 14.7, 11.1)
6.37 (1H, m)
6.47 (1H, m)
6.41 (1H, d, 8.6)
---
6.07 (1H, s)
---
5.96 (1H, s)
---
5.75 (1H, s)
---
2.26 (3H, s)
2.03 (3H, s)
2.01 (3H, s)
1.91 (3H, s)
1.15 (3H, s)
1.75 (3H, s)
1.10 (3H, s)
108

Figure 3-S14.
1
H NMR spectrum of compound 2 in CD3OD (400 MHz).
109

Figure 3-S15.
13
C NMR spectrum of compound 2 in CD3OD (400 MHz).
110

Figure 3-S16. DEPT spectrum of compound 2 in CD3OD (400 MHz).
111

Figure 3-S17. gHMQC spectrum of compound 2 in CD3OD (400 MHz).
112

Figure 3-S18. gHMBC spectrum of compound 2 in CD3OD (400 MHz).
113

Figure 3-S19. gCOSY spectrum of compound 2 in CD3OD (400 MHz).
114

Table 3-S9.
1
H and
13
C NMR data for compound 4 in CD3OD
position δC δH (mult, J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
179.5
48.2
177.5
42.7
87.5
49.6
136.6
132.9
132.4
130.5
130.0
138.6
59.5
47.2
220.4
77.1
44.9
10.3
21.5
13.5
12.2
12.1
14.6
7.8
---
2.82 (1H, q, 7.1)
---
2.71 (1H, m)
4.42 (1H, d, 10.7)
---
5.78 (1H, d, 15.0)
6.32 (1H, dd, 15.0, 10.5)
6.24 (1H, dd. 14.4, 10.5)
6.59 (1H, dd, 14.4, 11.0)
6.04 (1H, d, 11.1)
---
2.21 (1H, m)
2.21 (1H, m)
---
---
1.81 (1H, m)
0.92 (3H, d, 6.8)
1.23 (3H, s)
1.02 (3H, m)
1.74 (3H, s)
1.06 (3H, s)
1.12 (3H, d, 7.0)
0.98 (3H, d, 7.2)
115

Figure 3-S20.
1
H NMR spectrum of compound 4 in CD3OD (400 MHz).
116

Figure 3-S21.
13
C NMR spectrum of compound 4 in CD3OD (400 MHz).
117

Figure 3-S22. DEPT CH NMR spectrum of compound 4 in CD3OD (400 MHz).
118

Figure 3-S23. DEPT CHn NMR spectrum of compound 4 in CD3OD (400 MHz).
119

Figure 3-S24 HSQC spectrum of compound 4 in CD3OD (400 MHz).
120

Figure 3-S25. gHMQC spectrum of compound 4 in CD3OD (400 MHz).
121

Figure 3-S26. HMBC spectrum of compound 4 in CD3OD (400 MHz).
122

Figure 3-S27. COSY spectrum of compound 4 in CD3OD (400 MHz).
123

Figure 3-S28. NOESY spectrum of compound 4 in CD3OD (400 MHz).
124

Table 3-S10.
1
H and
13
C NMR data for compound 4 in acetone-D6
position δC δH (mult, J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
177.1
47.4
175.2
41.9
86.3
48.9
136.8
132.0
132.0
130.03
129.33
138.6
58.9
46.4
218.0
76.2
44.3
10.1
21.4
13.7
12.2
12.0
14.5
7.8
---
2.83 (1H, q, 7.2)
---
2.71 (1H, dq, 10.7, 7.0)
4.40 (1H, d, 10.7)
---
5.88 (1H, d, 15.2)
6.39 (1H, dd, 15.2, 10.4)
6.29 (1H, dd, 14.6, 10.4)
6.64 (1H, dd, 14.6, 11.1)
6.08 (1H, d, 11.1)
---
2.25 (1H, m)
2.20 (1H, m)
---
---
1.85 (1H, m)
0.92 (3H, d, 6.5)
1.21 (3H, s)
0.98 (3H, d, 6.5)
1.76 (1H, s)
1.07 (3H, s)
1.11 (3H, d, 7.0)
0.95 (3H, d, 6.8)
125

Figure 3-S29.
1
H NMR spectrum of compound 4 in acetone-D6 (400 MHz).
126

Figure 3-S30.
13
C NMR spectrum of compound 4 in acetone-D6 (400 MHz).
127

Figure 3-S31. HSQC spectrum of compound 4 in acetone-D6 (400 MHz).
128

Figure 3-S32. gHMQC spectrum of compound 4 in acetone-D6 (400 MHz).
129

Figure 3-S33. HMBC spectrum of compound 4 in acetone-D6 (400 MHz).
130

Figure 3-S34 COSY spectrum of compound 4 in acetone-D6 (400 MHz).
131

Figure 3-S35 NOESY spectrum of compound 4 in acetone-D6 (400 MHz).
132

Table 3-S11.
1
H and
13
C NMR data for compound 5
position δC δH (mult, J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
179.6
48.3
178.5
43.3
87.9
49.5
137.0
132.7
132.7
130.3
130.3
137.8
58.1
46.3
223.0
80.0
44.8
11.5
18.5
13.6
12.3
12.2
14.7
7.8
---
2.81 (1H, q, 7.1)
---
2.66 (1H, dt, 11.1, 7.1)
4.41 (1H, d, 10.8)
---
5.79 (1H, d, 15.2)
6.33 (1H, dd, 15.2, 10.5)
6.25 (1H, dd, 14.5, 10.5)
6.59 (1H, dd, 14.5, 11.1)
6.07 (1H, d, 11.1)
---
1.92 (1H, m)
2.23 (1H, m)
---
---
2.03 (1H, m)
0.94 (3H, s)
1.04 (3H, s)
1.00 (3H, d, 7.1)
1.76 (3H, s)
1.06 (3H, s)
1.10 (3H, d, 7.1)
0.98 (3H, d, 7.1)
133

Figure 3-S36.
1
H spectrum of compound 5 in CD3OD (600 MHz).
134

Figure 3-S37.
13
C spectrum of compound 5 in CD3OD (600 MHz).
135

Figure 3-S38. HSQC spectrum of compound 5 in CD3OD (600 MHz).
136

Figure 3-S39. gHMQC spectrum of compound 5 in CD3OD (600 MHz).
137

Figure 3-S40. HMBC spectrum of compound 5 in CD3OD (600 MHz).
138

Figure 3-S41. COSY spectrum of compound 5 in CD3OD (600 MHz).
139

Figure 3-S42. NOESY spectrum of compound 5 in CD3OD (600 MHz).
140

Table 3-S12.
1
H and
13
C NMR data for compound 6
position δC δH (mult, J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

18
19
20
21
22
23
24
179.4
48.1
177.8
42.8
87.5
49.7
139.2
132.6/137.6
132.6/137.6
130.1
136.7
136.1
144.6
136.1
209.4
14.9
28.7

12.1/12.3
18.3
14.00
17.2
12.1/12.3
14.6
7.8
---
2.84 (1H, q, 7.2)
---
2.70 (1H, dq, 13.1, 6.7)
4.44 (1H, d, 10.7)
---
5.92 (1H, d, 14.6)
6.43 (1H, m)
6.43 (1H, m)
6.69 (1H, dd, 13.8, 11.4)
6.47 (1H, d, 13.8)
---
7.16 (1H, s)
---
---
3.32 (1H, m)
1.40 (1H, m)
1.66 (1H, tq, 14.7, 7.4)
0.86 (3H, t, 7.4)
1.06 (3H, d, 6.7)
2.01 (3H, s)
2.09 (3H, s)
1.08 (3H, s)
1.11 (3H, d, 7.0)
0.99 (3H, d, 7.2)
141

Figure 3-S43.
1
H spectrum of compound 6 in CD3OD (400 MHz).
142

Figure 3-S44.
13
C spectrum of compound 6 in CD3OD (400 MHz).
143

Figure 3-S45. HSQC spectrum of compound 6 in CD3OD (400 MHz).
144

Figure 3-S46. HMBC spectrum of compound 6 in CD3OD (400 MHz).
145

Figure 3-S47. COSY spectrum of compound 6 in CD3OD (400 MHz).
146

Table 3-S13.
1
H and
13
C NMR data for compound 7
position δC δH (mult, J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
179.5
48.2
177.6
42.7
87.5
49.5
136.6
132.9
132.7
130.9
130.8
137.2
83.1
138.4
130.5
137.3
121.9
13.5
12.2
15.2
17.9
12.2
14.6
7.8
---
2.81 (1H, q, 7.2)
---
2.69 (1H, m)
4.41 (1H, d, 10.8)
---
5.79 (1H, d, 15.2)
6.34 (1H, 15.2, 10.5)
6.26 (1H, dd, 14.4, 10.5)
6.60 (1H,dd, 11.3, 10.5)
6.04 (1H, d, 11.3)
---
4.34 (1H, s)
---
6.09 (1H, s)
---
5.60 (1H, q, 6.8)
1.65 (3H, d, 6.8)
1.51 (3H, s)
1.69 (3H, s)
1.95 (3H, s)
1.06 (3H, s)
1.11 (3H, d, 7.1)
0.97 (3H, d, 7.2)
147

Figure 3-S48.
1
H NMR spectrum of compound 7 in CD3OD (400 MHz).
148

Figure 3-S49.
13
C NMR spectrum of compound 7 in CD3OD (400 MHz).
149

Figure 3-S50. HSQC NMR spectrum of compound 7 in CD3OD (400 MHz).
150

Figure 3-S51. HMBC NMR spectrum of compound 7 in CD3OD (400 MHz).
151

Figure 3-S52. COSY NMR spectrum of compound 7 in CD3OD (400 MHz).
152

Table 3-S14.
1
H NMR data for compound 8
position δH (mult, J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
---
3.53 (1H, q, 6.6)
---
2.50 (1H, m)
4.73 (1H, d, 11.1)
---
6.15 (1H, d, 10.4)
6.40 (1H, dd, 10.4, 3.9)
6.34 (1H, m)
6.23 (1H, m)
6.23 (1H, d, 9.0)
---
5.30 (1H, d, 9.8)
2.69 (1H, m)
3.79 (1H, d, 7.5)
---
5.41 (1H, q, 6.4)
1.55 (3H, d, 6.4)
1.51 (3H, s)
1.01 (3H, d, 6.7)
1.76 (3H, s)
1.81 (3H, s)
1.04 (3H, d, 7.1)
1.36 (3H, d, 6.5)
153

Figure 3-S53.
1
H spectrum of compound 8 in CDCl3 (400 MHz).
154

Table 3-S15.
1
H and
13
C NMR data for compound 9
position δC δH (mult, J in Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
169.8
49.3
207.4
48.4
86.4
129.1
131.5
126.5/126.7
135.9
126.5/126.7
139.6
133.3
137.1/137.2
37.1
82.1
137.1/137.2
122.0
13.3
11.8
16.9
12.7
15.3
22.3
8.6
19.4
---
3.62 (1H, q, 6.6)
---
---
4.92 (1H, s)
---
6.12 (1H, d, 10.9)
6.42 (1H, dd, 14.5, 10.9)
6.30 (1H, dd, 14.5, 9.0)
6.22 (1H, m)
6.22 (1H, d, 8.7)
---
5.29 (1H, d, 9.8)
2.68 (1H, m)
3.78 (1H, d, 7.6)
---
5.41 (1H, q, 6.7)
1.55 (3H, d, 6.7)
1.51 (3H, s)
1.01 (3H, d, 6.8)
1.76 (3H, s)
1.87 (3H, s)
1.12 (3H, s)
1.35 (3H, d, 6.6)
1.02 (3H, s)
155

Figure 3-S54.
1
H spectrum of compound 9 in CDCl3 (600 MHz).
156

Figure 3-S55.
13
C spectrum of compound 9 in CDCl3 (600 MHz).
157

Figure 3-S56. HSQC spectrum of compound 9 in CDCl3 (600 MHz).
158

Figure 3-S57. gHMQC spectrum of compound 9 in CDCl3 (600 MHz).
159

Figure 3-S58. HMBC spectrum of compound 9 in CDCl3 (600 MHz).
160

Figure 3-S59. COSY spectrum of compound 9 in CDCl3 (600 MHz).
161

CHAPTER IV: Conclusion and perspective

In this dissertation I have described the elucidation of the citreoviridin biosynthetic pathway
using heterologous expression and the discovery of the aspernidgulenes using serial promoter
replacement. Citreoviridin is a potent mycotoxin that selectively inhibits the β-subunit of ATP
synthase. Identifying its biosynthesis genes will allow the gene clusters of other structurally
similar ATP synthase inhibitors such as asteltoxin and verrucosidin to be probed. In fact, one
homolog of CtvA, the HR-PKS responsible for assembling the carbon backbone of citreoviridin,
was found in A. nidulans. The homolog is SdgA, and in its promixity are two other homologs of
the citreoviridin biosynthetic genes: SdgC and SdgD. However, when the promoters of the Sdg
genes were serially activated, to our surprise the resulting final product, aspernidgulen A1 and
A2, were remarkably structurally different from citreoviridin. In this chapter I will discuss the
lessons learned through the course of these two studies.

The first lesson is the importance of target selection. Given the availability and low cost of
genome sequencing, hundreds of thousands of fungal gene clusters will flood publicly available
database in the come years. The 1000 Fungal Genome Project, funded by the Department of
Energy, is an example of large-scale sequencing project fun. Moreover, it is becoming
increasingly feasible for individual labs to sequence their own strains of interest. In fact, in the
more developed world of bacterial SM research, there are ~1.1 million biosynthetic gene clusters
as of March 2018, as catalogued in the “Atlas of Biosynthetic Gene Clusters” by the Joint
Genome Institute.
271

162

The challenge facing natural products research is the problem of prioritization. Given that
genetic and chemical interrogation of a cluster is time consuming and laborious, researchers need
to focus their energy and time on the ones that are most likely to produce molecules with useful
bioactivities. How can this prediction be done?

One solution to the problem is resistance gene guided genome mining. The physical linkage of
the resistance gene to the rest of the biosynthetic machinery gives us information on the target of
the natural product without prior knowledge of the structure of the inhibitor. In the example of
citreoviridin, the presence of a duplicated copy of ATP synthase β-subunit in the cluster gave us
a substantial clue in linking the toxin to its biosynthetic genes. Thus, by looking for the presence
of duplicated ATP synthase β-subunit nearby backbone enzymes in other genomes, we can
identify other putative inhibitors.

In fact, resistance gene mining has been successfully applied in several instances. The cluster for
thiotetronic acid antibiotics, known inhibitors of fatty acid synthase, was identified by searching
for duplicated house-keeping genes in the genomes of 86 Salinispora.
272
Similarly, an orphan
cluster in A. nidulans was correlated with the proteasome inhibitor fellutamide B in part because
of the presence of a proteasome subunit in the cluster.
273
As the amount of publicly available
genome sequences rapidly increases in the next decade, comparative genomics can be a powerful
tool to efficiently identify candidate clusters for antifungals or antibiotics.

However, in both aforementioned cases orphan clusters were linked to a scaffold well-known to
inhibit its target. To my knowledge there has not yet been an example of using this approach to
163

discover a novel compound, a novel target, or an unknown inhibitory activity of a known
compound. It could be that known inhibitors with potent bioactivity and produced in sufficient
quantities are low-hanging fruits.

Validating a novel protein target requires biochemical characterization, which could pose a
challenge to many natural product labs. It can also be difficult assessing whether a duplicated
housekeeping gene is indeed involved in resistance or have other biological functions unrelated
to resistance. On the downstream side, if the target sequence is silent it needs to be either
heterologously expressed or turned on in the native host. Time will tell in the upcoming years as
to the fruitfulness of this approach in the discovery of novel inhibitors.

In the world of bacterial SM research, a public, web-based platform was recently developed to
automate the high-throughput screening of resistance genes. This web server is called Antibiotic
Resistance Target Seeker (ARTS).
274
It identifies putative resistance genes based on three
criteria: duplication, its physical location in the BGC, and evidence of horizontal gene transfer. It
also tells the user whether the identified resistance gene is a known resistance gene. Similar
platform for fungi awaits be developed.

The second lesson is to differentiate between silent clusters versus dead clusters. As discussed in
chapter 2, the citreoviridin cluster in A. terreus NIH 2624 seems to be nonfunctional presumably
due to some mutations in the HR-PKS that renders the enzyme incapable of producing the
polyketide precursor. It is conceivable that some of the backbone enzymes investigated have not
yielded product because of similar mutations. In those cases, the clusters are not so much silent
164

as they are “dead.” Thus, mutations in one or more of the biosynthetic genes is another barrier to
the elucidation of seemingly silent and orphan clusters, along with challenges such as compound
stability and detectability as well as the need for genes outside of the cluster.

An interesting follow-up experiment is to determine how of many the four citreoviridin
biosynthetic genes in A. terreus NIH 2624 have been inactive through mutation. Is it only CtvA,
or others as well? This question can be easily answered by mixing and matching citreoviridin
biosynthetic genes from NIH2624 and A. terreus var. aureus. If it can be shown that, for
example, CtvA and D have been inactivated and CtvB and C are not, there would be important
implications for secondary metabolite research.

Important implications arise because many tailoring enzymes serves as gateway keepers that
control whether a common precursor is converted to one final product versus another. For
example, it is likely that sclerotiorin and asperfuranone share the same precursor. Their
biosynthesis pathways diverge when AfoF introduces a hydroxyl group that prevents six-
membered ring cyclization. As the result, the five-membered ring in asperfuranone is formed.
Another example of enzymatic structural divergence is citreoviridin and the aspernidgulenes,
where methylation of the polyketide by CtvB ensures that the oxabicylo[2.2.1]heptane moiety
would not be formed.

Thus, nonsense or missense mutation on one or more key tailoring enzymes can cause a cluster
to accumulate interme some clusters incapable of producing the final product even if the right
environmental triggers are present. In this case, intermediates would be expected to accumulate
165

instead of the final product. An example of this scenario was reported in the works by Kato et
al.
275
The A. fumigatus strain Af293 was found to be incapable to producing fumitremorgin, a
potent, specific inhibitor of breast cancer resistant protein, but accumulated 6-
hydroxytryprostatin B, an intermediate in the pathway. The blockage of the fumitremorgin
pathway was traced back to a single mutation in ftmD, a methyltransferase, responsible for the
conversion of 6-hydroxytryprostatin B to tryprostatin A.

Fortunately, there is a simple solution to the problem of dead clusters. Expressing multiple
variants of the same cluster from closely related species circumvents the risk that we are working
with nonfunctional biosynthetic genes. The effectiveness of this strategy was demonstrated by
investigating the HR-PKS alleles of both A. terreus NIH2624 and A. terreus var. aureus
CBS503. The same approach could be taken to investigate other seemingly silent clusters.

Another potential solution is to use genome editing to restore enzyme function. In the work
published by Weber et al. CRISPR editing was used to reconstitute the production of trypacidin
by removing a single nucleotide insertion that led to a frameshift in the PKS tynC.
276
However,
such an approach would require knowledge of which mutations are silent and which mutations
are abolishing function.

The third lesson is the power of tailoring enzymes to generate structural diversity and confer
regioselectivity. The investigation of the aspernidgulenes gene cluster was motivated in part by
the hypothesis that it would encode for a novel ATP synthase β-subunit inhibitor due to the
cluster’s homology to the citreoviridin cluster. However, to our surprise the structure of
166

aspernidgulenes A1 and A2, which we believe are the final products of the cluster, are
remarkably different from that of citreoviridin. Citreoviridin contains an α-pyrone connected by
tetra-ene to a tetrahydrofuran. Aspernidgulenes A1 and A2, on the other hand, contain a 2,3-
dimethyl-γ-lactone connected by a tri-ene to a trimethylpentanone.

Though the polyketide precursors of the two compounds are structurally similarly, differing by
only two methyl groups, their biosynthesis almost begins to diverge immediately upon the first
modification. Methylation of the α-pyrone at the ctv biosynthetic pathway prevents the folding of
the pyrone to stabilize the positive charge generated by epoxide opening. Further downstream in
the biosynthesis, the presence of SdgE, which opens up the first epoxide to generate a keto group
restricts the number of possible rings formed and prepares for the assembly of the
oxabicylo[2.2.1]heptane moiety, which later undergoes hydrolytic cleavage to generate the 2,3-
dimethyl-γ-lactone moiety.

Thus, even though three sets of enzymes (CtvA and SdgA, CtvC and SdgC, and CtvD and SdgD)
have significant homology, the presence of CtvB in citreoviridin biosynthesis and SdgE in
aspernidgulenes biosynthesis led to different structues with different bioactivity. Even though the
bioactivity of the aspernidgulenes have not been tested, similar compounds showed various
activities: shimalactone A from Emericella variecolor (neuritogenic),
277
coccidiostatin A
(coccidiostat)
278
and prugosenes
279
from Penicillium rugulosum, wortmannilactones from
Talaromyces wortmannii (cathepsin B inhibitory and anthelmintic),
280

281
and ukulactones from
Penicillium sp. FKI-3389 (NADH-fumarate reductase inhibitory).
282

167

Lastly, the ability of tailoring enzymes to impart regioselectivity to biosynthesis is incredible.
Both CtvD and SdgD are putative membrane-bound hydrolases thought to catalyze epoxide ring
opening. In the absence of CtvD, an array of possible isomers with similar molecular weight and
UV absorption patterns were detected. Similarly, in the absence of SdgD, the number of possible
isomers and shunt products increased. Two other enzymes, CtvC and SdgC have possible roles in
the isomerization of the polyketide precursors. Remarkably, they can select from the four
possible conformations of the terminal di-enes and catalyze bis-epoxidation in a manner that
leads to the correct final stereochemistry. This observation highlights the efficiency of
biosynthesis to generate chiral molecules that pose a challenge for medicinal chemists.

The isolation of the epimers aspernidgulenes A1 and A2 may suggest that the basis for the
pentanone stereochemistry is non-enzymatic. However, the disappearance of two compounds, 10
and 13, with similar UV and mass as aspernidgulenes A1 and A2 upon the introduction of SdgD
may indicate that SdgD is still somehow responsible for regioselectivity. The fact that in the
studies of prugosene and wortmannilactone no C6 epimers were isolated also suggests an
enzymatic role of SdgD and its homologs. The precise function of this membrane-bound enzyme
is unclear from our data, and the isolation of peaks 10-20, especially 10 and 13 could shed on the
question. Yet given the chiral nature of the protein targets of SMs, it would not be surprising if
SdgD is later found to be another example of enzymatically-controlled regiostereochemistry.

168

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Asset Metadata

Creator Lin, Tzu-Shyang (Kevin) (author)

Core Title Genetic and chemical characterization of two highly-reducing polyketide synthase clusters from Aspergillus species

Contributor Electronically uploaded by the author (provenance)

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 05/02/2018

Defense Date 03/16/2018

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

Tag Aspergillus,aspernidgulenes,biosynthesis,citreoviridin,natural products,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Advisor Wang, Clay (committee chair), Xie, Jianming (committee member), Zhang, Yong (Tiger) (committee member)

Creator Email kevin.lin.ts@gmail.com,tzushyal@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-499283

Unique identifier UC11267370

Identifier etd-LinTzuShya-6305.pdf (filename),usctheses-c40-499283 (legacy record id)

Legacy Identifier etd-LinTzuShya-6305.pdf

Dmrecord 499283

Document Type Dissertation

Format application/pdf (imt)

Rights Lin, Tzu-Shyang (Kevin)

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

Abstract (if available)

Abstract Filamentous fungi have historically been a rich source of therapeutically relevant natural products. Yet genome sequencing has revealed that the secondary metabolite capacity of filamentous fungi remains largely untapped. Many more bioactive secondary metabolites await discovery, with potentially valuable entities in the midst. ❧ Two main goals of natural product research are the linking of known metabolites to their gene clusters and the discovery of novel secondary metabolites. The first goal is important because understanding the biosynthesis of complex organic molecules can inspire biomimetic synthesis strategies. Furthermore, biosynthetic enzymes can act as useful biocatalysts that perform challenging reactions in short, efficient routes. The second goal is important because all secondary metabolites are inherently biologically relevant. Even if their function is currently unknown, future discoveries can shed light on their utility, whether in medicine, agriculture, or some other field. Moreover, the structures found in nature are often beyond the imagination of medicinal chemists. Thus, they can inspire new scaffolds. ❧ The work herein describes both the elucidation of the biosynthetic genes of a known mycotoxin and the discovery of several new metabolites from A. nidulans. First, the biosynthetic genes of citreoviridin, a potent mycotoxin that has caused significant health problems around the world, were identified and verified experimentally by heterologous expression. The heterologous expression platform used was a well-established A. nidulans system that has a clean background, recyclable selection markers, and facile genetic amenability. Second, a silent gene cluster in A. nidulans was turned on through serial promoter replacement, leading to the discovery of several novel polyketide products, the aspernidgulenes. Their biosynthesis was proposed, representing the first the genetic and enzymatic study for this class of compounds. Interestingly, while the aspernidgulenes biosynthetic genes have significant homology with that of citreoviridin, significant diversification of structure was achieved by the difference of two tailoring enzymes.