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Secondary metabolites of Aspergillus nidulans

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Secondary metabolites of Aspergillus nidulans

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SECONDARY METABOLITES OF ASPERGILLUS NIDULANS

by

Hsien-Chun Lo

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

May 2008

Copyright 2008 Hsien-Chun Lo

ii
ACKNOWLEDGEMENTS

First and foremost, I would like to thank my advisor, Dr. Clay Wang, for his
guidance, encouragement, and mentorship during my time in his laboratory.
My appreciation also goes to my fellow lab mates, Dr. Yi-Ming Chiang and
James Sanchez, for giving me the greatest support and assistance on my experiments,
as well as academic studies, and to all the faculty members in the school of
Pharmacy for their academic instruction and inspiration. In particular, I want to show
my gratitude to Dr. Michael Kahn and Dr. Wei-Chiang Shen, who assisted me during
my research endeavors.
Lastly, I would like to thank Shiuan Wey, my beloved family, and dearest
friends who have given me all the support throughout my graduate studies.

iii
TABLE OF CONTENTS

Acknowledgements ................................................................................................. ii
List of Tables........................................................................................................... iv
List of Figures ......................................................................................................... v
Abbreviations .......................................................................................................... vii
Abstract ................................................................................................................... viii
CHAPTER ONE:
Introduction ..................................................................................................... 1
1.1 Secondary Metabolites .......................................................................... 1
1.2 Fungi as a Source of Natural Products .................................................. 1
1.2.1 Non-ribosomal Peptides (NRPS).................................................. 2
1.2.2 Polyketides (PKS) ........................................................................ 2
1.2.3 Terpenes and Indole Alkaloids..................................................... 6
1.3 Secondary Metabolites in Aspergillus nidulands .................................. 6

CHAPTER TWO:
Materials and Methods .................................................................................... 8
2.1 Chemicals and Media ............................................................................ 8
2.2 Discovery of a Gene Involved in Secondary Metabolite
Biosynthesis Pathway by Gene Deletion............................................... 8
2.2.1 Fusion PCR................................................................................... 10
2.3 Cultivation of Aspergillus nidulans Strains........................................... 12
2.4 Extraction .............................................................................................. 12
2.5 Liquid Chromatography/Mass Spectrometer (LC/MS)......................... 13
2.6 Purification ............................................................................................ 14
2.7 Structural identification......................................................................... 15
2.8 Stereochemical Determination of Amino Acid Residues...................... 15

CHAPTER THREE:
Results ............................................................................................................. 17
3.1 Biosynthesis of Emericellamides by AN 2545.3 Gene ......................... 17
3.2 Toward the Discovery of Genes for the Biosynthesis of Two
Terpenes, Austinol and Dehydroaustinol .............................................. 24
3.3 Characterization of novel emericellamides C-F.................................... 27

CHAPTER 4:
Discussion ....................................................................................................... 36
REFERENCES........................................................................................................ 40

iv
LIST OF TABLES
Table 1: Reagents and Their Sources ................................................................. 8
Table 2: The Formulation of Media.................................................................... 9
Table 3: The Genotype of All Strains in This Research..................................... 10
Table 4: Molecular Formula and Mass of Five Emericellamides....................... 19
Table 5:
1
H-NMR Data for Emericellamides A and C-F
(400 MHz in DMSO-d
6
)....................................................................... 30
Table 6:
13
C-NMR Data for Emericellamides A and C-F
(100MHz in DMSO-d
6
)........................................................................ 31

v
LIST OF FIGURES
Figure 1: Biosynthetic Mechanism of NRPS........................................................ 3
Figure 2: Biosynthetic Mechanism of PKS .......................................................... 5

Figure 3: Creation of a Single Fusion Fragment with an AfpyrG Marker
By PCR with Six Primers for the Gene Transformation ....................... 11

Figure 4: A schematic diagram of gene transformation........................................ 12
Figure 5: Total Ion Current (TIC) Trace of Wild Type A. Nidulans with
Six Mutants............................................................................................ 18

Figure 6: UV Trace of Wild Type A. Nidulans with Six Mutants ........................ 18

Figure 7: Chemical Structures of Emericellamide A, C-F.................................... 20
Figure 8: EIC Traces of Five Mutants and Wild Type with m/z of 610 ............... 21
Figure 9: EIC Traces of Five Mutants and Wild Type with m/z of 596 ............... 21
Figure 10: EIC Traces of Five mutants and Wild Type with m/z of 624 ............... 22
Figure 11: EIC traces of Five Mutants and Wild Type with m/z of 325 ................ 23
Figure 12: EIC Traces of Five Mutants and Wild Type with m/z of 491 ............... 24
Figure 13: Chemical Structure of Austinol and Dehydroaustinol........................... 25

Figure 14: UV Trace of Wild Type A. Nidulans, Four Mutants, Austinol, and
Dehydroaustinol .................................................................................... 25

Figure 15: TIC Trace of Wild Type A. nidulans, Four Mutants, Austinol, and
Dehydroaustinol. ................................................................................... 26

Figure 16: EIC Trace of Wild Type A. Nidulans, Four Mutants, and
Dehydroaustinol with m/z of 457 ......................................................... 27

Figure 17: EIC Trace of Wild Type A. Nidulans, Four Mutants, and Austinol
With m/z of 459 .................................................................................... 28

Figure 18: MS/MS Spectra of Emericellamide A, C-F........................................... 33
Figure 19: Assignment of MS/MS Fragmentation................................................. 34

vi
Figure 20: The Mechanism of Formation of FDAA-Amino Acid
Derivatives............................................................................................. 35
Figure 21: UV and EIC Traces of Standard FDAA-Amino Acids
And Hydrolysates .................................................................................. 35

Figure 22: A diagram of the Proposed Mechanism of Emericellamide
Biosynthetic Pathway in A. nidulans..................................................... 37

vii
ABBREVIATIONS
NRPS: non-ribosomal peptide synthase
PKS: polyketide synthase
DMSO: dimethyl sulfoxide
YAG: yeast extract glucose
GMM: glucose minimal medium
TIC: total ion current
EIC: extracted ion current
TQ: terrequinone
ST: sterigmatocystin
IHD: indices of hydrogen deficiency
GHMQC: Gradient-enhanced Heteronuclear Multiple-Quantum Correlation
DEPT: Distortionless enhancement by polarization transfer
HPLC: high performance liquid chromatography
LC/MS: liquid chromatography/mass spectrometer
HRFABMS: high resolution fast atom bombardment mass spectrometry

viii
ABSTRACT
The complete sequencing of genomes of several Aspergillus species,
including A. nidulans, revealed that these fungal species have a potential to produce
a surprising large range of natural products. The discovery of new natural products
and their biosynthetic pathway in A. nidulans has been facilitated by a rapid gene
modification technique by which the replacement of targeted genes, can be achieved
within a few days instead of months. The natural products emericellamides A, C-F, a
family of cyclic polyketide/nonribosomal peptides, biosynthesized by the mixed
PKS/NRPS cluster (AN2544-AN2547), have been identified with the application of
this rapid method. Once the emericellamide gene cluster was successfully elucidated,
it was undertaken to determine the genes responsible for the biosynthetic pathway of
two terpenes, austinol and dehydroaustinol, in the same species. Although the
preliminary data showed that the four putative terpene cyclase genes that were
studied are not responsible for the terpene biosynthesis pathway in A. nidulans,
additional candidate genes are currently being examined.

1
CHAPTER 1
INTRODUCTION
1.1 Secondary Metabolites
Secondary metabolites are natural products distinguished from primary
metabolites, which are small compounds of intermediary metabolism needed for
growth, development, and reproduction of a living organism. On the other hand,
secondary metabolites play nonessential roles (Vining, 1992), and are often used in
defense against predation and habitat encroachment, or even used as communication.
Therefore, these natural compounds endow the organisms that produce them,
including the filamentous fungus Aspergillus nidulans, survival advantage over
non-producing species.
1.2 Fungi as a Source of Natural Products
Secondary metabolites are largely found in bacteria, fungi, plants,
dinoflagellates, mollusk sponges, and insects. The fungal kingdom, encompassing
many species, is a rich source of natural products with important medicinal
properties. So far, 1,500 compounds in fungi have already been isolated, and more
than half of these natural products have antibacterial, antitumor, or antifungal agency
(Palaez, 2005). Many well-known human medicines have been isolated from a
variety of fungal species, such as penicillin, an antibiotic, lovastatin, a
cholesterol-lowering drug, and cyclosporine, an immunosuppressant (Dirk & Keller,
2007). Due to the structural diversity of these fungal secondary metabolites,
discovery of novel fungal natural products may lead to a variety of new medicines.
There are four major classes of fungal secondary metabolites, categorized by their

2
biosynthesis pathway: non-ribosomal peptides (NRP), polyketides (PK), terpenes,
and indole alkaloids (Keller, Turner, & Bennett, 2005).
1.2.1 Non-ribosomal peptides (NRPs). Rather than synthesized by the rRNA-
and tRNA-dependent ribosomal machinery, non-ribosomal peptides are
manufactured by nonribosomal peptide synthetases (NRPSs). NRPS contain a set of
repetitive catalytic units called modules (Schwarzer & Marahiel, 2001). Each module
in NRPS is responsible for incorporating one amino acid into the growing peptide
chain. A single cycle of peptide elongation is accomplished by three steps, substrate
recognition, acyl adenylation, and the formation of an amide bond between two
contiguous amino acids. The first step involves an adenylation (A) domain that
selects a specific amino acid and subsequently activates it as an amino acyl adenylate,
reacting with one molecule of ATP. This unstable adenylated amino acid
subsequently undergoes transport to the peptidyl carrier protein (PCP), or thiolation
(T) domain, and forms a thioester bond with the cysteamine thiol group of the T
domain-bound phosphopantetheine swinging arm. For the third step, the amide bond
between the amino acid bonded to the T domain and nascent peptide chain on the
preceding module will be formed at the condensation (C) domain. The growing
peptide chain can undergo some modification by other enzymes such as cyclase (Cy),
oxidase (Ox), epimerase (E) and N-methyltransferase (M). Finally,
termination/thioesterase (Te) domain releases the peptide (Figure 1).
1.2.2 Polyketides (PKs). The most abundant fungal natural products belong to
the polyketide family. Polyketide biosynthetic pathways are classified into type I,
type II and type III according to the catalytic activities of PKS (Shen, 2003). Type I

3
C
A
PCP Te
SH
NH
3
O
O
R
+
ATP
Mg
2+
-PPi
NH
3
O
O
R
AMP
A domain
PCP domain
NH
3
O
O
R
AMP
+
SH
H
N
O
R
2
SH
C domain
NH
3
O
R
1
SH
NH
2
O
R
2
SH
-AMP
-H
2
O
H
3
N
O
R
1
NH
3
O
R
SH
PCP
PCP
PCP
PCP
PCP
Te domain
H
3
N
R
H
N
O
n
O
S
Rn +1
H
2
O H
3
N
R
H
N
O
n
O
O
Rn +1
SH
PCP
+
PCP
NRPS: Reaction catalyzed by different functional domains

Figure 1. Biosynthetic mechanism of NRPS.
PKSs are multifunctional enzymes composed of several modules, or a single module,
which acts in an iterative fashion. The acyl transferase (AT) domain, acyl carrier
protein (ACP) domain, and ketosynthase (KS) domain are minimally required for
each module. So far only iterative type I PKS biosynthetic pathways have been

4
found in fungi (Dirk & Keller, 2007). All fungal polyketides are biosynthesized by
polyketide synthases (PKSs), which consist of several protein domains of specific
functions and catalyze repetitive Claisen condensations from acetyl CoA or malonyl
CoA. Selection, activation, and transfer of building blocks are achieved by the AT
domain in the first step of polyketide assembly. Subsequently, the formation of a
ketide bond between the growing ketide chain tethered to the ACP domain of the
upstream module and the activated unit in the downstream ACP is catalyzed by a
ketosynthase (KS) domain. In addition, optional modifications can be catalyzed by
the keto reductase (KR), dehydratase (DH), enoyl reductase (ER), cyclase (CYC),
and methyl transferase (MT) domains. Finally, the release of polyketides is catalyzed
by the termination/thioesterase (Te) domain (Figure 2) (Schwarzer & Marahiel,
2001). In the fungal PKS biosynthetic pathway, the growing polyketide chain
undergoes iterative biosynthetic reactions in the same module before the final
polyketide is released (Dirk & Keller, 2007). Distinct from type I PKSs, type II PKSs
are multienzyme complexes composed of several KS and ACP enzymes (Figure 2).
The selection and addition of an acyl group for the growing polyketide chains is
achieved by a KS domain in type II PKSs. Specially, the KS enzyme have two
subunits, KS
α
and KS
β
.

KS
α
performs similar function as the KS in type I PKSs.
However, KS
β
can catalyze the decarboxylation of malonyl-ACP to act as a chain
initiation factor. The type III PKSs are different from type II PKSs by the absence of
an ACP enzyme. Instead, KS domains of type III PKSs utilize acyl CoAs as
substrates directly. Each of the three types of PKSs can act iteratively with a single
module or complex.

5
AT ACP
AT domain
O
O O
S CoA
R
+
ACP
SH
-CoA
O
O O
S
R
ACP
KS domain
O
O O
S
R
ACP
S
R
O
-CO
2
O O
S
R
ACP
ACP
S
ACP
R
Te domain
R
OH
S
O O
ACP
R'
n
O
O
O
R'
n
ACP
SH
PKS: Reaction catalyzed by different functional domains
KS AT KR ACP
Loading
Module 1
PKS
Module 2
KS AT ACP Te
Releasing
Module N
SH
Type I PKS:
SH
SH
x
y
+ KSα KS
β ACP KS
n
Type II PKS: Type III PKS:

Figure 2. Biosynthesis mechanism of PKS.
In addition, mixed PKS/NRPS systems can be present to increase the
diversity of natural products. Emericellamide derivatives, the secondary metabolite

6
products that were identified and describe herein, are cyclic polyketide/nonribosomal
peptides.
1.2.3 Terpenes and indole alkaloids. Terpenes are composed of several
head-to-tail or tail-to-tail isoprene units (Eberhard, 2006). They are classified into
monoterpenes, sequiterpenes, diterpenes, and carotenoids, according to the number
of carbons in their backbone. The enzymes responsible for terpene biosynthesis may
vary. The defining fungal terpene biosynthetic enzyme is terpene cyclase (Dirk &
Keller, 2007). Another class of products are the indole alkaloids, tryptophan
derivatives whose biosynthesis uses this amino acid as a precursor. However, other
amino acids can also found to be the precursors in the indole alkaloid biosynthetic
pathway.
1.3 Secondary Metabolites in Aspergillus nidulans
In contrast to other eukaryotes, in which the genes involved in a particular
biosynthetic pathway are scattered over the genome, the genes of individual
secondary metabolite pathways in fungi, including A .nidulans, are almost always
clustered together, which potentially facilitates study and molecular genetic
manipulation (Dirk & and Keller, 2007; Brown et al., 1996). The sequencing of
genomes of Aspergillus nidulans , a filamentous fungus and the focus of this
research paper, revealed that there are more than 40 secondary metabolite pathways
in this species, but fewer than ten had been discovered at the time the genome was
completed (Galagan, 2004). Bioinformatic analysis of the genome of A. nidulans
suggested that A. nidulans contains 27 polyketide synthases, 14 nonribosomal
peptide synthetases, six fatty acid synthases, and one sequiterpene cyclase. In

7
addition to these findings, the development of breakthrough gene engineering
techniques such as fusion PCR enables us to identify new natural products and their
corresponding biosynthetic pathways more efficiently. Five emericellamide
derivatives, a family of cyclic polyketide/nonribosomal peptides, four of which were
novel, were identified in a project inspired by these developments.

8
CHAPTER 2
MATERIALS AND METHODS
2.1 Chemicals and Media
The chemicals and media used in this project are as follows (Tables 1 and 2):
Table 1. Reagents and Their Sources

Reagent Brand and Series Number
Agar EMD, AX0410-1
Yeast extract BD, 210933
Dextrose EMD, DX0145-5
Lactose EMD, LX0035.5
Sucrose EMD, 8550
Tween 80 Sigma, P1754-500ml
Pyridoxine EM Science, PX2070-1
NaCl VWR, VW6430-1
NaNO
3
EMD, SX0655-3
KCl EMD, 7300
KOH EMD, PX1480-11
ZnSO
4
·7H
2
O J.T. Baker 4382-04
H
3
BO
3
EMD, BX0865-11
MnCl
2
·4H
2
O J.T. Baker, 2540-04
FeSO
4
7H
2
0 J.T. Baker, 2074-01
CoCl
2
6H
2
0 EMD, EM-CX1800-3
CuSO
4
5H
2
O VWR, VW3312-2
EDTA Sigma, E-6758
Na
2
EDTA Sigma, ED2SS
(NH
4
)6Mo
7
O24 4H
2
O EMD, AX1310-3
FDAA, Marfey’s
reagent
Pierce, 48895

Solvent
Ethyl Acetate EMD, EX0241-1
Methanol BDH, BDH113504LG
Chloroform BDH, BDH1109-4LG
Acetonitrile Burdick & Jackson,
UN1648
Dichlromethane BDH, BDH1113-4LG

2.2 Discovery of a Gene Involved in Secondary Metabolite
Biosynthetic Pathway by Gene Deletion

The strategy used to discover a specific natural product and its biosynthetic
pathway is as follows: If a critical gene for the biosynthesis of a specific compound
is missing in the specific mutant, the mutant will no longer produce this

9
Table 2. The Formulation of Media
YAG Medium Plate
Yeast extract 5g
Dextrose 20g
Agar 15g
ddH
2
O 1L
Hutner’s trace
element
2ml

GMM Medium (pH of 6.5)
Dextrose 10g
20 x salts 50ml
ddH
2
O 1L
Hutner’s trace
element
1ml

Hutner’s Trace Elements
ZnSO
4
·7H
2
O 2.2g
H
3
BO
3
1.1g
MnCl
2
·4H
2
O 0.5g
FeSO
4
·7H
2
O 0.5
CoCl
2
·5H
2
O 0.16g
CuSO
4
·5H
2
O 0.16g
(NH
4
)
6
Mo
7
O
24
·4H
2
O 0.11g
Na
4
EDTA 5.0g

compound, whereas it will be observed from a wild type strain. In addition, other
genes responsible for the same biosynthetic pathway may be located adjacent to this
gene because of the fact that the genes responsible for the same secondary metabolite
biosynthetic pathway are usually contiguous. The production of a secondary
metabolite by the mutant strains can be compared with wild type strain using LC/MS
analysis.
In this project, six putative nonribosomal peptide synthetase (NRPS) genes
and four putative terpene cyclase genes were deleted individually from wild type A.
nidulans strain in order to discover and analyze nonribosonal peptide and terpene

10
biosynthetic pathways, respectively (Table 3). These putative NRPS genes are
AN2545.3, AN0607.3, AN1242.3, AN2621.3, AN8412.3, and AN9244.3. Putative
terpene cyclase genes were AN2611.3, AN2590.3, AN5359.3, and AN3277.3. All
Aspergillus nidulans mutant strains were created by the laboratory of Berl Oakley by
using fusion PCR techniques. The six primers for each deletion were designed by
using Primer3 software. Additionally, the prediction of targeted genes in the
Aspergillus nidulans gene database was from the Broad Institute website
(http://www.broad.mit.edu/annotation/genome/Aspergillus_group/MultiHome.html).
Table 3. The Genotype of All Strains in This Research
NRPS Knockout Strains
Strain Relevant genotype
AN2545.3
pyroA4, pyrG89, nku::argB, AN2545:: A. fumigatus
pyrG
AN0607.3
pyroA4, pyrG89, nku::argB, AN0607:: A. fumigatus
pyrG
AN1242.3
pyroA4, pyrG89, nku::argB, AN1242:: A. fumigatus
pyrG
AN2621.3
pyroA4, pyrG89, nku::argB, AN2621:: A. fumigatus
pyrG
AN8412.3
pyroA4, pyrG89, nku::argB, AN8412:: A. fumigatus
pyrG
AN9244.3
pyroA4, pyrG89, nku::argB, AN9244:: A. fumigatus
pyrG
Terpene Cyclase Knockout Strains
Strain Relevant Genotype
AN2611.3 ΔSTCJ::riboB, pyroA4, AN2611:: A. fumigatus pyrG
AN2590.3 ΔSTCJ::riboB, pyroA4, AN2590:: A. fumigatus pyrG
AN5359.3 ΔSTCJ::riboB, pyroA4, AN5359:: A. fumigatus pyrG
AN3277.3 ΔSTCJ::riboB, pyroA4, AN3277:: A. fumigatus pyrG

2.2.1 Fusion PCR. Fusion PCR is a rapid and efficient technique to facilitate
gene replacement, promoter replacement, gene knockout and gene tagging
(Szewcqyk et al., 2006). In this technique, the two 1,000 base pair fragments

11
flanking the targeted gene for deletion are first amplified by PCR. Two of the six
PCR primers, P3 and p4, have regions homologous to a selectable marker,
Aspergillus fumigatus pyrG (orotidine-5’phosphate decarboxylase gene).
Subsequently, the two ~1,000 base pair fragments upstream and downstream to the
targeted gene, together with the selectable marker gene, were amplified by PCR to
generate a single fusion fragment for the gene transformation (Figure 3). Followed
by protoplast generation, transformation with recipient nkuAΔ strain and selection,
the targeted gene deletion will be obtained (Figure 4). Primers 1 and 6 were used to
extend the fusion fragment, which may be degraded during PCR.

Figure 3. Creation of a single fusion fragment with an AfpyrG marker by PCR with
six primers for the gene transformation (Szewczyk et al., 2007).

12

Figure 4. A schematic diagram of gene transformation (Szewczyk et al., 2007).
2.3 Cultivation of Aspergillus nidulans Strains
A. nidulans wild type R153 and six NRPS mutant strains were cultivated in
GMM medium (1X 10
6
spores/ml) supplemented with addition of pyridoxine
(0.5μg/ml) and shaken at 100 rpm at 37
o
C for 4 days. Four terpene mutant strains
and a wild type strain were cultivated on YAG medium plate (d = 15 cm) at 37
o
C in
an incubator for 5 days.
2.4 Extraction
GMM culture medium was collected and filtrated, followed by extracting
twice with an equivalent volume of EtOAc (ethyl acetate). Subsequently, the EtOAc

13
layer was evaporated using the TurboVap Evaporation System (Caliper LifeSciences
Inc.) to obtain crude natural products, and this material was redissolved in methanol
(MeOH) at a concentration of 1mg/ml for LC/MS analysis.
When using YAG plates, the agar was sliced into pieces and soaked in 100%
MeOH for 1h with sonication (volume of MeOH is about 50ml/15cm plate), and the
chopped materials was extracted again with a mixture of 1:1 MeOH:CH
2
Cl
2
using
the same volume. The solvents were removed by rotary evaporation (BÜCHI In.),
and the dry material was dissolved in 50 ml water followed by extracting twice with
an equivalent volume of EtOAc. The EtOAc layer was evaporated in a Turbo Vap to
obtain crude natural products and this material was re-dissolved in MeOH at a
concentration of 1mg/ml for LC/MS analysis.
2.5 Liquid Chromatography/Mass
Spectrometry (LC/MS)
A ThermoFinnigan LCQ Advantage ion trap mass spectrometer with an RP
C
18
column (Alltech Prevail C18 3μm 2.1 X 100mm) was used to analyze the
secondary metabolites. The solvent system for HPLC was 95% MeCN/H
2
O (solvent
B) in 5% MeCN/H
2
O (solvent A), both containing 0.05% formic acid. The solvent
gradient was 0% B from 0 to 5 min, 0 to 100% B from 5 to 35 min, 100 to 0% from
35 to 40 min, and 100% B from 40 to 45 min for re-equilibration, at a flow rate of
125μL/min. Positive ion electrospray ionization (ESI) was used to detect the
analytes.

14
2.6 Purification
For structure elucidation of emericellamides, a 10 L GMM fermentation
volume was extracted with EtOAc in the same procedure to obtain 350 mg gray
powder. This crude extract then was separated by Si gel column (Merck 230-400
mesh, ASTM, 10 X 100 mm) and eluted with a CHCI
3
–MeOH solvent system of
increasing polarity (frac. A, 1:0, 300 ml; frac. B, 19:1, 300 ml; frac. C, 9:1, 300 ml;
frac. D, 7:3, 300 ml) to divide samples into four fractions. Fraction B, containing
emericellamides was evaporated by rotary evaporation and dissolved in CHCl
3
followed by the application of HPLC with a C
18
column [Phenomenex Luna 5 μm
C
18
, 250 X 21.2 mm] and measured by a UV detector at 200 nm wavelength to
obtain these five compounds. The solvent system was composed of MeCN (solvent
B) in 5 % MeCN/H
2
O (solvent A) both containing 0.05% formic acid. The solvent
gradient was 60 to 100% B from 0 to 15 min, 100% B from 15 to 20 min, and 60% B
from 20 to 22min for re-equilibration, at a flow rate of 10.0 ml/min. A mixture of
emericellamides C and D (6.6 mg, T
R
= 11.4 min), emericellamide A (1.2 mg, T
R
=
12.5 min), and a mixture of emericellamide E and F (5.6 mg, T
R
= 14.8 min) were
collected. The mixture of emericellamide C (2.8 mg, T
R
= 36.2 min) and D (2.5 mg,
T
R
= 37.8 min) and the mixture of emericellamide E (1.4 mg, T
R
= 47.0 min) and F
(2.6 mg, T
R
= 49.1 min) were further purified by the same HPLC with isocratic 50%
B solvent. Finally, the pure samples were dissolved in deuterated DMSO for NMR
analysis.
Previously, in the course of comparing the wild type strain with SUMO
knock out mutants, two terpenoids, austinol and dehydraustinol, were found in the

15
wild type strain. In order to identify the chemical structure of these two terpenoids,
20 YAG plates of wild type A. nidulans were extracted with EtOAc using the
procedure described above. Crude extract was separated by a Si gel column (Merck
230-400 mesh, ASTM) and eluted with a CHCI
3
–MeOH solvent system of
increasing polarity to divide the samples into four fractions (frac. A, 1:0, 300 ml; frac.
B, 19:1, 300 ml; frac. C, 9:1, 300 ml; frac. D, 7:3, 300 ml). Fraction B was collected
and further separated by normal phase HPLC [Phenomenex Luna 5 μm Si (2), 250 Χ
10 mm] and measured by a UV detector at 254 nm wavelength. Isocratic 1:1 ethyl
acetate/hexane was used as solvent system with a flow rate of 5.0 ml/min to obtain
austinol (16.2 mg R
T
= 9.4 min) and dehydroaustinol (24.7 mg, R
T
= 8.5 min).
Subsequently, the samples were dissolved in deuterated DMSO for NMR analysis.
2.7 Structural Identification
Varian Unity Plus 400 NMR with
1
H and
13
C spectra were used in the
structural identification of these two terpenes and five emericellamides.
Perkin–Elmer Spectrum BX IR was used to further confirm the structure of
the emericellamides. The emericellamide samples were dissolved in MeOH and
added on the plates of potassium bromide to be examined by IR spectroscopy.
2.8 Stereochemical Determination
of Amino Acid Residues
FDAA, Marfey’s reagent (1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide)
was used to determine the absolute configuration of the amino acids of
emericellamides C-F. Hydrolysis of the amino acid residues of emericellamides was
achieved by the addition of 1 mL of 6N HCl at 115 °C for 18 h. The HCl was

16
removed under a stream of N
2
. The hydrolysate was dissolved in 1 N NaHCO
3

(100μL) and treated with 1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide (L-Marfey’s
reagent) in acetone (10 mg/mL, 50μL). The reaction was heated at 40
o
C for 1 h in a
heating module followed by the neutralization with the addition of 2 N HCl (50μL),
and diluted with 50% aqueous CH
3
CN (300μL). The sample was then analyzed by
LC/MS with an RP C
18
column (Alltech Prevail C18 3μm 2.1 X 100mm).The solvent
system for HPLC was 95 % MeCN/H
2
O (solvent B) in 5 % MeCN/H
2
O (solvent A),
both containing 0.05 % formic acid. The solvent gradient was 0% B from 0 to 5min,
0 to 100% B from 5 to 35 min, 100 to 0 % from 35 to 40min, and 100% B from 40 to
45 min for re-equilibration, at a flow rate of 125μL/min. In comparison, the pure
alanine, valine, and leucine amino acids were used as standards under the same
treatment to determine absolute configurations of hydrolysates.

17
CHAPTER 3
RESULTS
3.1 Biosynthesis of Emericellamides
by AN2545.3 Gene
All strains were grown in the same conditions followed by the same
purification and analytical procedures. Therefore, the gene responsible for the
specific secondary metabolites can be found by comparing the MS profile of the
mutants and the wild type strains.
Total ion current (TIC) of the mutants and wild type revealed that
onlyΔAN2545.3 is missing five peaks between a retention time (R
T
) of 27.50 -31.30
min. Therefore, some secondary metabolites may not be present due to the deletion
of the AN2545.3 gene (Figure 5).
The UV trace (λ = 254nm) showed no difference in number of peaks for each
mutant because the missing metabolites have no UV absorption (λ= 254nm) (Figure
6). However, the consistency of UV traces of all strains showed that the other natural
products produced by all these strains were the same, as all strains were under the
same growth and purification conditions.
The five compounds produced by the wild type strain and absent in the
AN2545.3 deletion strain were studied further. Compound 1 has a molecular mass of
m/z = 609 (m/z = 610 [M+H]
+
). Compounds 2 and 3 have molecular masses of m/z =
595 (m/z = 596 [M+H]
+
), and Compounds 4 and 5 have molecular masses of m/z =
623 (m/z = 624 [M+H]
+
). The molecular formulas of these five compounds were

18

Figure 5. Total ion current (TIC) trace of wild type A. nidulans with six mutants.
TIC profile showed ΔAN2545.3 mutant was missing some secondary metabolites.

Figure 6. UV trace of wild type A. nidulans with six mutants. Missing metabolites
have no UV absorption (λ= 254nm).

19
obtained by analysis of these five compounds by HRFABMS, high resolution mass
spectrometry (Table 4).
Table 4. Molecular Formula and Mass of Five Emericellamides
Molecula
formula
Mass ([M+H]
+

m/z)
Emericellamide A (1) C
31
H
56
N
5
O
7
610.4154
Emericellamide C (2) C
30
H
54
N
5
O
7
596.4026
Emericellamide D (3) C
30
H
54
N
5
O
7
596.4016
Emericellamide E (4) C
32
H
58
N
5
O
7
624.4324
Emericellamide F (5) C
32
H
58
N
5
O
7
624.4312

The chemical structures of these five emericellamides have been further
identified. Emericellamide A, a mixed cyclic polyketide/nonribosomal peptide, is
also produced by a marine Emericella species (Emericella is Aspergillus in its sexual
stage) (Oh, Kauffman, Jensen, & Fenical, 2007). The four other compounds were
completely novel, and the structures were solved by our lab (Figure 7).
The positive mode extracted ion current (EIC) of m/z of 610, 596, and 624,
corresponding to the emericellamides, m/z of 325 corresponding to sterigmatocystin
(ST), and m/z of 489, corresponding to terrequinone (TQ), in all strains were
analyzed. EIC traces of emericellamides in ΔAN2545.3 showed that an m/z of 610,
596, and 624 was missing. Incidentally, the two compounds with m/z of 596 and two
compounds with m/z of 624 were closely spaced in the chromatogram (Figures 8, 9,
and 10). Therefore, the deletion of AN2545.3 gene destroyed the ability of the
mutant strain to produce these five emericellamides. The results suggested that the
AN2545.3 gene was crucial for the biosynthesis of emericellamides A, and C-F.

20

Figure 7. Chemical structures of emericellamides A, C-F.

21

Figure 8. EIC traces of five mutants and wild type with m/z of 610. Emericellamide
A was missing in AN2545.3 deletion strain.

Figure 9. EIC traces of five mutants and wild type with m/z of 596. Emericellamides
C and D were missing in ΔAN2545.3 mutant.

22

Figure 10. EIC traces of five mutants and wild type with m/z of 624.
Emericellamides E and F were missing in ΔAN2545.3 mutant.

The positive mode EIC m/z of 325, corresponding to sterigmatocystin (ST),
and m/z of 489, corresponding to terrequinone (TQ) in all strains were also analyzed.
ST and TQ appeared on the mass spectra of all strains because the biosynthetic gene
clusters for ST, AN7800.3-AN7830.3, and for TQ, AN8513.3-AN8526.3, still exist
in all strains (Figures 11 and 12). The results support the rationale that a mutant
strain will be missing only the natural products corresponding to the critical
biosynthesis genes that are missing, provided that the growth conditions of this
mutant are the same as in wild type strain.

23

Figure 11. EIC traces of five mutants and wild type with m/z of 325.
Sterigmatocystin (m/z = 325 [M+H]+) appeared on the mass spectra of all the strains
because biosynthetic gene clusters for ST still exist in all strains.

24

Figure 12. EIC traces of five mutants and wild type with m/z of 491. Terrequinone
(m/z = 491 [M+H]+) appeared on the mass spectra of all the strains.

3.2 Toward the Discovery of Genes for the
Biosynthesis of Two Terpenes, Austinol
and Dehydroaustinol

Because of the successful discovery of the AN2545.3 gene responsible for
the biosynthesis of the five emericellamides in A. nidulans, the same analytical
procedure was used to discover which genes are responsible for the biosynthesis of
two terpenes named austinol and dehydroaustinol, previously isolated from R153
wild type A. nidulans strain. In addition, the chemical structures of austinol and
dehydroaustinol have already been characterized (Marquez-Fernandez, et. al., 2007;
Simpson, et. al., 1982) (Figure 13).

25

Figure 13. Chemical structures of austinol and dehydroaustinol
The UV traces and TIC were analyzed in four putative terpene cyclase gene
knockout strains, wild type strain, and austinol and dehydroaustinol standards. The
results showed there is no difference in second metabolites among the four mutants
and wild type, as the mass spectrometry results are similar (Figures 14 and 15).

Figure 14. UV trace of wild type A. nidulans, four mutants, austinol, and
dehydroaustinol. There is no difference among these strains.

26

Figure 15. TIC trace of wild type A. nidulans ,four mutants, austinol, and
dehydroaustinol. There is no difference among these strains.

To further confirm the results, EIC of m/z of 457, corresponding to
dehydroaustinol, and EIC of m/z of 459, corresponding to austinol, in all the strains
were analyzed. The positive mode EIC in all the strains showed austinol and
dehydroaustinol were still produced by these four mutant strains, ΔAN2611.3,
ΔAN2590.3, ΔAN5359.3, and ΔAN3277.3 (Figures 16 and 17). Therefore, the
results indicated that these four genes may not be involved in the terpene
biosynthesis pathway, or there is parallel compensatory terpene biosynthesis
pathways.

27

Figure 16. EIC trace of wild type A. nidulans, four mutants, and dehydroaustinol. All
the strains produce dehydroaustinol with m/z of 457.

3.3 Characterization of novel emericellamides C-F

Emericellamides C-F isolated from A. nidulans were novel. These four
emericellamides were further examined to establish their chemical properties.
Emericellamide C was isolated as white powder. The molecular formula was
C
30
H
54
N
5
O
7
and

solved by HRFABMS and reconfirmed by
13
C NMR and DEPT
data.
The
1
H NMR spectrum in DMSO-d6 showed five amide NH signals (δ
H
7.53,
7.61, 7.82, 8.11, and 8.20), six α- amino protons [δ
H
4.38 (1H), 4.01-4.09 (3H), 3.91
(1H), and 3.58 (1H)], and one ester carbinol proton (δ
H
4.81), which means
emericellamide C is composed of five amino acids and one of them is glycine, the
only amino acid with two α- amino protons (Table 5).

28

Figure 17. EIC trace of wild type A. nidulans, four mutants, and austinol. All the
strains produce austinol with m/z of 459.

13
C NMR and DEPT spectra showed six amide or ester carbonyl groups
(δ
c
172.9, 171.6, 171.4, 171.2, 170.9, and 169.4) and one oxygenated carbon (δ
c
75.0),
which correlated with δ
H
4.81 in the gHMQC spectrum (Table 6). These results
consist with five amide NH signals and one ester carbinol proton of the
1
H NMR
spectrum. In addition, the correlation of oxygenated carbon with ester carbinol
proton in the gHMQC spectrum confirms there is an ester bond. These results,
together with seven indices of hydrogen deficiency (IHD) and the IR absorption band
at 3307 cm
-1
(amide N-H stretching), 1758 cm
-1
(ester C = O stretching), and 1636
cm
-1
(amide C = O stretching), suggested that emericellamide C is a monocyclic
peptide composed of five amino acids and one ester functional group. Compared
with the NMR spectrum of known emericellamide A,
1
H and
13
C NMR spectra of

29
these two emericellamide are very similar. The addition of a methyl group (δ
H
0.82;
δ
c
12.9, CH
3
-31) and the carbinol proton in emericellamide A [δ
H
4.81 (ddd, J = 9.2,
8.4, 3.6 Hz, H-22)] contribute to the differences in
1
H and
13
C NMR spectra.
Additionally, emericellamide C also contained one glycine, one valine, one leucine,
and two alanines.
Emericellamide D, a constitutional isomer of emericellamide C, also has
similar 1H and 13C NMR spectral features with emericellamide A (Tables 5 and 6).
Emericellamide A and D differ only in that CH3-30 in emericellamide A is replaced
by H in emericellamide D. This generated one methylene [δH 2.68 (1H, dd, J = 13.6,
9.6 Hz) and 2.18 (1H, d, J = 13.6 Hz); δC 30.8 (t), CH2-21] in emericellamide D
instead of one methine [δH 2.87 (1H, dq, J = 9.6, 6.8 Hz); δC 41.1 (d), CH-21] in
emericellamide A.
The position of the methyl group on emericellamides C-F can be further
confirmed by the analysis of MS/MS spectra of emericellamides A, C-F. The
intensityof dehydration fragments in compounds 3 (m/z of 295, 323, 408, 436, and
507) and 5(m/z of 323, 351, 436, 464, and 535) are stronger than compounds 2 and 4.
The results suggested the aliphatic side chains of compound 3 and 5 have two
carbonyl α-protons on C-21 that increase the probability of H
2
O elimination to
generate α,β-unsaturated carbonyl fragments (Figures 18 and 19).

30

Table 5.
1
H-NMR Data for Emericellamides A and C–F (400 MHz in DMSO-d
6
)

Position A C D E F
NRP
(L-Ala-1)
2 4.00–4.14
b
4.03–4.09
b
4.04–4.11
b
4.01–4.09
b
4.04–4.11
b

3 1.24 (d, 6.8) 1.22 (d, 6.8) 1.24 (d, 6.8) 1.22 (d, 6.8) 1.24 (d, 6.8)
2-NH 8.04 (br s) 7.82 (d, 4.4) 8.15 (d, 4.4) 7.85 (br s) 8.16 (d, 4.0)
NRP
(L-Ala-2)
5 4.00–4.14
b
4.03–4.09
b
4.09–4.17
b
4.01–4.09
b
4.09–4.17
b

6 1.21 (d, 6.8) 1.23 (d, 6.8) 1.22 (d, 6.8) 1.23 (d, 6.8) 1.22 (d, 6.8)
5-NH 7.44 (d, 7.2) 7.53 (d, 7.2) 7.42 (d, 7.6) 7.55 (d, 7.2) 7.45 (d, 7.2)
NRP
(L-Leu)
8 4.00–4.14
b
4.01–4.04
b
4.04–4.11
b
4.01–4.09
b
4.04–4.11
b

9 1.54–1.62
b
1.50–1.62
b
1.51–1.61
b
1.50–1.62
b
1.51–1.61
b

10 1.54–1.62
b
1.50–1.62
b
1.51–1.61
b
1.50–1.62
b
1.51–1.61
b

11 0.80 (d, 6.8) 0.82 (d, 6.8) 0.82 (d, 6.8) 0.82 (d, 6.8) 0.82 (d, 6.8)
12 0.89 (d, 6.8) 0.89 (d, 6.8) 0.90 (d, 6.8) 0.89 (d, 6.8) 0.90 (d, 6.8)
8-NH 8.29 (d, 8.4) 8.20 (d, 7.2) 8.19 (d, 8.4) 8.32 (d, 7.2) 8.31 (d, 7.6)
NRP
(L-Val)
14 3.98 (t, 7.6) 3.91 (t, 7.2) 3.93 (t, 7.6) 3.91 (t, 7.2) 3.93 (t, 7.6)
15 1.90 (m) 1.91 (m) 1.93 (m) 1.93 (m) 1.94 (m)
16 0.87 (d, 6.8) 0.89 (d, 6.8) 0.90 (d, 6.8) 0.89 (d, 6.8) 0.90 (d, 6.8)
17 0.88 (d, 6.8) 0.91 (d, 6.8) 0.88 (d, 6.8) 0.90 (d, 6.8) 0.88 (d, 6.8)
14-NH 8.22 (d, 8.4) 8.11 (d, 7.2) 8.10 (d, 7.6) 8.26 (d, 7.6) 8.25 (d, 7.6)
NRP (Gly) 19
3.62 (dd, 17.6,
2.4); 4.31 (dd,
17.6, 5.2)
3.58 (dd, 17.6,
2.0); 4.38 (dd,
17.6, 6.0)
3.75 (dd, 17.2,
4.0); 4.09–4.17
b

3.59 (dd, 17.6,
2.4); 4.35 (dd,
17.6, 6.0)
3.76 (dd, 17.2,
4.0); 4.09–4.17
b

19-NH 7.45 (br s)
7.61 (dd, 6.0,
2.0)
7.54 (t, 4.0) 7.56 (br, s) 7.51 (br s)
PK 21 2.87 (dq, 9.6, 6.8)
2.73 (dq, 9.2,
6.8)
2.18 (d, 13.6);
2.68 (dd, 13.6,
9.6)
2.72 (dq, 9.2,
6.8)
2.18 (d, 13.6);
2.68 (dd, 13.6,
9.6)
22 4.93 (br d, 9.6)
4.81 (ddd, 9.2,
8.4, 3.6)
5.04 (dd, 9.6,
3.6)
4.82 (ddd, 9.2,
8.4, 3.6)
5.05 (dd, 9.6,
2.4)
23 1.67 (m)
1.42 (m);
1.50–1.62
b

1.54–1.68
b

1.42 (m);
1.50–1.62
b

1.54–1.68
b

24 1.05–1.16
b
1.14–1.30
b

1.02 (m);
1.18–1.34
b

1.14–1.30
b

1.02 (m);
1.18–1.34
b

25 0.98–1.06
b
1.14–1.30
b
1.18–1.34
b
1.14–1.30
b
1.18–1.34
b

26~28 1.18–1.34
b
1.14–1.30
b
1.18–1.34
b
1.14–1.30
b
1.18–1.34
b

29 0.84 (t, 6.8) 0.85 (t, 6.8) 0.86 (t, 7.6) 1.14–1.30
b
1.18–1.34
b

30 0.90 (d, 6.8) 0.93 (d, 6.8) 0.83 (d, 7.6) 1.14–1.30
b
1.18–1.34
b

31 0.82 (d, 6.8) 0.85 (t, 6.8) 0.86 (t, 7.6)
32
0.93 (d, 6.8) 0.83 (d, 7.6)

31

Table 6:
13
C-NMR data for emericellamides A and C–F (100 MHz in DMSO-d
6
)
Position A C D E F
NRP (L-Ala-1) 1 171.4 (s) 171.6 (s) 171.8 (s) 171.4 (s) 171.9 (s)
2 48.2 (d) 48.2 (d) 48.4 (d) 48.3 (d) 48.4 (d)
3 16.3 (q) 16.2 (q) 16.1 (q) 16.2 (q) 16.1 (q)
NRP (L-Ala-2) 4 171.5 (s) 171.4 (s) 171.4 (s) 171.6 (s) 171.4 (s)
5 47.2 (d) 47.8 (d) 47.4 (d) 47.7 (d) 47.3 (d)
6 18.3 (q) 17.4 (q) 18.1 (q) 17.5 (q) 18.2 (q)
NRP (L-Leu) 7 170.9 (s) 170.9 (s) 170.8 (s) 171.0 (s) 170.9 (s)
8 51.7 (d) 51.8 (d) 51.6 (d) 51.8 (d) 51.6 (d)
9 39.4 (t) 38.9 (t) 39.4 (t) 39.0 (t) 39.5 (t)
10 24.5 (d) 24.5 (d) 24.4 (d) 24.5 (d) 24.4 (d)
11 20.7 (q) 20.7 (q) 20.7 (q) 20.7 (q) 20.7 (q)
12 23.2 (q) 23.3 (q) 23.2 (q) 23.2 (q) 23.2 (q)
NRP (L-Val) 13 171.2 (s) 171.2 (s) 171.2 (s) 171.2 (s) 171.3 (s)
14 60.2 (d) 60.2 (d) 60.2 (d) 60.3 (d) 60.3 (d)
15 30.1 (d) 29.8 (d) 29.8 (d) 29.8 (d) 29.8 (d)
16 18.8 (q) 18.7 (q) 18.6 (q) 18.7 (q) 18.7 (q)
17 19.1 (q) 19.0 (q) 19.1 (q) 19.0 (q) 19.1 (q)
NRP (Gly) 18 168.7 (s) 169.4 (s) 169.0 (s) 169.4 (s) 169.0 (s)
19 42.5 (t) 42.2 (t) 42.4 (t) 42.2 (t) 42.3 (t)
PK 20 172.9 (s) 172.9 (s) 169.3 (s) 172.8 (s) 169.3 (s)
21 41.1 (d) 42.7 (d) 38.0 (t) 42.8 (d) 38.1 (t)
22 76.6 (d) 75.0 (d) 74.4 (d) 75.0 (d) 74.4 (d)
23 33.2 (d) 28.8 (t) 36.4 (d) 28.8 (t) 36.5 (d)
24 33.5 (t) 31.1 (t) 32.0 (t) 31.3 (t) 32.0 (t)
25 26.6 (t) 23.5 (t) 26.3 (t) 23.5 (t) 26.4 (t)
26 28.9 (t) 28.6 (t) 28.9 (t) 28.9
b
(t) 29.2
b
(t)
27 31.2 (t) 30.9 (t) 31.2 (t) 28.8
b
(t) 28.7
b
(t)
28 22.1 (t) 22.1 (t) 22.1 (t) 28.7
b
(t) 28.9
b
(t)
29 14.0 (q) 14.0 (q) 14.0 (q) 30.9 (t) 31.3 (t)
30 14.3 (q) 14.4 (q) 14.7 (q) 22.1 (t) 22.1 (t)
31 12.9 (q) 14.0 (q) 14.0 (q)

32 14.4 (q) 14.7 (q)

32
Emericellamides E and F are a pair of constitutional isomers and have the
same molecular formula of C
32
H
58
N
5
O
7.
Analysis of
1
H and
13
C NMR spectra of
emericellamides C-F showed the spectra of emericellamide E and F are
superimposed with the spectra of emericellamide C and D, respectively. This result,
together with DEPT spectra suggested emericellamide E and F have two more CH
2

carbons extending on aliphatic side chains of emericellamide C and D, respectively.
The absolute configuration of amino acids in emericellamides C and D were
determined all L-isomers by Marfey’s reagent after hydrolysis and LC/MS analysis
(Oh et al., 2007). FDAA can react with amino acids to form FDAA derivatives with
D-amino acids or L-amino acids. FDAA derivatives of D-amino acids have stronger
intermolecular bonding, which reduces their polarity compared to FDAA derivatives
of L-amino acids (Jacobson, Sambandan, & Morgan, 1998) (Figure 20). Therefore,
the D-derivatives retain longer on reverse phase columns and elute later than L-
derivatives when separated by HPLC, as observed with a UV detector at 340 nm.
Given these properties of FDAA derivatives, the absolute configuration of amino
acids can be determined by comparing the LC/MS profile of the tested sample with
known amino acids.
By comparing the UV trace of emericellamide hydrolysate with standard
amino acids, valine, leucine, and alanines, all the amino acids of emericellamidess
possessed L-configuration (T
R
= 26.18 corresponding to L-alanine, T
R
=36.05
corresponding to L-valine, and T
R
=32.15 corresponding to L-leucine) (Figure 21).

33

Figure 18. MS/MS spectra of emericellamide A, C-F.
The configurations of C-21 and C-22 in emericellamides C and E are 21R
and 22R according to the large coupling constants (> 9.0 Hz). In addition, the
configuration of C-22 and C-23 in emericellamides D and F are 22S and 23S
according to the small coupling constants (< 4.0 Hz). All the absolute configurations
of emericellamides C-F have the same as emericellamide A (Oh, et. al., 2007).

34
NH
NH
NH
NH
NH O
+
O
O
O
O
O OH
R
1
R
2
NH
NH
NH
NH
NH O
+
O
O
O
O
O OH
NH
NH
NH
NH
NH O
+
O
O
O
O
O OH
R
1
R
2
1
3
4
6
7
11
12
13
16 17
18
19
20
21 23 25 27
29
a
5
(568)
b
4
(525)
a
3
(426)
b
2
(341)
a
2
(313)
Emericellamide C (2): R
1
= Me, R
2
= H
Emericellamide D (3): R
1
= H, R
2
= Me
b
3
(454)
1
3
4
6
7
11
12
13
16 17
18
19
20
21 23 25 27
29
30
a
5
(582)
b
4
(539)
a
3
(440)
b
2
(355)
a
2
(327)
b
3
(468)
31
Emericellamide E (4): R
1
= Me, R
2
= H
Emericellamide F (5): R
1
= H, R
2
= Me
Emericellamide A (1)
1
3
4
6
7
11
12
13
16 17
18
19
20
21 23 25 27
a
5
(596)
b
4
(553)
a
3
(454)
b
2
(369)
a
2
(341)
b
3
(482)
29
31
Emericellamides
1. protonation
2. ring opening

Figure 19. Assignment of MS/MS fragmentation.

35
NO
2
O 2 N
F
NH
O
NH
2
+
H
2
N CH C
R
OH
O
D or L amino acid 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA)
arylation in NaHCO
3
/acetone
40
o
C
NO
2
O
2
N
NH
O
NH
2
NO
2
O
2
N
NH
O
NH
2
NH
C
C
R H
O
OH
NH
C
C
H R
O
OH
LL LD

Figure 20: The mechanism of formation of FDAA-amino acid derivatives (Jacobson
et al., 1998).

Figure 21. UV and EIC traces of standard FDAA-amino acids and hydrolysates.

36
CHAPTER 4
DISCUSSION
The biosynthetic pathway for these five emericellamides can be proposed by
analyzing the genes surrounding AN2545.3 in a gene database. This gene cluster
contains an NRPS gene (AN2545.3 as mentioned), a PKS gene (AN2547.3), and a
transporter (AN2544.3).The gene cluster was designated as eas (emericellamide
synthesis) cluster and easA to AN2547.3, easB to AN2545.3 and easC to AN2544.3.
In addition, EasA, EasB, and EasC were designed for their gene products
respectively. The polyketide chain is first synthesized iteratively by EasA. EasA has
the ability to produce polyketides with different chain lengths and degrees of
methylation. Therefore, EasA may cause the structural variation observed in the
emericellamides. Subsequently, the growing polyketide chain is transferred to a
thiolation (T) domain in the first module of the NRPS EasB. EasB contains five
modules responsible for the arrangement of the five amino acids in the
emericellamides. Finally, the hydroxyl group on the polyketide chain attacks to form
the cyclic compound. From homologous comparison to genes of known function,
EasC may be a transporter which is involved in the export of the emericellamides
(Figure 22).
The selection and incorporation of a specific amino acid into the growing
peptide chain in NRPS is achieved by the A (adenylation) domain in each NRPS
module. Therefore, the amino acid sequence of the nonribosomal peptides can be
predicted by the arrangement of NRPS modules containing A domains. The
correlation between the sequence of the A domain and the amino acid that will be

37

Figure 22. A diagram of the proposed mechanism of emericellamide biosynthesis
pathway in A. nidulans.

selected for the growing peptide chain has been well-understood in bacteria (Torsten,
Henning, & Mohamed, 1999). However, less has been established with fungi. In
order to examine whether the sequence and amino acid correlations observed in
bacteria can be applied to A. nidulans, the sequence of the NRPS responsible for the
biosynthesis of emericellamides was entered into a software program that predicts
which amino acids will be incorporated, using a database of various NRPS sequences
(http://www.nii.res.in/nrps-pks.html). The results showed only the A domain for

38
glycine incorporation has a homologous sequence to other NRPS A domains.
Therefore, the correlation between the structure of a nonribosomal peptide and its
corresponding A domain in A. nidulans may be different from the correlation in
bacteria or other species in the database.
Regarding the attempted discovery of a terpene biosynthesis pathway in A.
nidulans, the results showed deletion of four putative terpene cyclase genes,
AN2611.3, AN2590.3, AN5359.3, and AN3277.3, did not eliminate the biosynthesis
of austinol and dehydroaustinol. Some factors may contribute these results. First,
these four genes may not be terpene cyclase genes. The protein sequence of the well-
characterized aristolochene cyclase from A. terreus was used in a BLAST similarity
search to predict terpene cyclase genes of A. nidulans. Even though these four
putative terpene cyclase genes are the four genes with highest similarity to the
aristolochene cyclase gene, it is still difficult to predict the genes that encode these
proteins due to the low similarity of primary sequences among terpene cyclases (Dirk
& Keller, 2007). Furthermore, terpene cyclases are structurally less homologous than
they are at the protein in sequence level.
Another reason is the possibility that these four putative terpene cyclase
genes were not successfully deleted. However, the same gene was deleted and the
constructs analyzed three separate times. In addition, the successful rate of gene
replacement is up to 90% according to previous research (Szewczyk et al., 2006).
Diagnosis PCR can be used in the future to directly detect whether the deletion
occurs on the targeted gene in the genome.

39
The cyclization step of austinol and dehydroaustinol may be also performed
by other cyclases in A. nidulans, which means that even though our putative terpene
cyclase has been deleted, another cyclase which has similar function can catalyze the
cyclization of terpenes. In this situation, these four strains, AN2611.3, AN2590.3,
AN5359.3, and AN3277.3, can be used as background strain to undergo further
deletion of other cyclases.
By analyzing the chemical structure of austinol and dehydroaustinol and the
AN3277.3 gene in the genome of A. nidulans, the polyketide portion in these two
terpenes may be biosynthesized by the gene AN3273.3, a PKS gene, in the same
cluster. The new mutant strain with the deletion of the AN3273.3 gene will be made
and analyzed in the future. If the deletion of the PKS gene can make cause this strain
to no longer produce austinol and dehydroaustinol, the gene cluster to which
AN3277.3 and AN3273.3 belong may be involved in the terpene biosynthesis
pathway, and the AN3277.3 gene may be the terpene cyclase for the biosynthesis of
austinol and dehydroaustinol.

40
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Adams, T. H., & Leonard, T. J. (1996). Twenty-five coregulated transcripts
define a sterigmatocystin gene cluster in Aspergillus nidulans. Proceedings of
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Dirk, H., & Keller, N. P. (2007). Natural products of filamentous fungi: enzymes,
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Eberhard, B. (2006). Terpenes Wiley-VCH.
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Jacobson, P. G., Sambandan, T. G., & Morgan, B. (1998). Determination of the
chirality of cysteines in somatostatin analogs. Journal of Chromatography A,
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Simpson, T. J., Stenzel, D. J., Bartlett, A. J., O'Brien, E., Holker, J. S. E. (1982).
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Abstract (if available)

Abstract The complete sequencing of genomes of several Aspergillus species, including A. nidulans, revealed that these fungal species have a potential to produce a surprising large range of natural products. The discovery of new natural products and their biosynthetic pathway in A. nidulans has been facilitated by a rapid gene modification technique by which the replacement of targeted genes, can be achieved within a few days instead of months. The natural products emericellamides A, C-F, a family of cyclic polyketide/nonribosomal peptides, biosynthesized by the mixed PKS/NRPS cluster (AN2544-AN2547), have been identified with the application of this rapid method. Once the emericellamide gene cluster was successfully elucidated, it was undertaken to determine the genes responsible for the biosynthetic pathway of two terpenes, austinol and dehydroaustinol, in the same species. Although the preliminary data showed that the four putative terpene cyclase genes that were studied are not responsible for the terpene biosynthesis pathway in A. nidulans, additional candidate genes are currently being examined.

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Asset Metadata

Creator Lo, Hsien-Chun (author)

Core Title Secondary metabolites of Aspergillus nidulans

School School of Pharmacy

Degree Master of Science

Degree Program Pharmacy / Pharmaceutical Sciences

Publication Date 04/25/2008

Defense Date 03/18/2008

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

Tag Aspergillus nidulans,OAI-PMH Harvest,Secondary metabolites

Language English

Advisor Wang, Clay C. C. (committee chair), Kahn, Michael (committee member), Shen, Wei-Chiang (committee member)

Creator Email hsienchl@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1129

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Document Type Thesis

Rights Lo, Hsien-Chun

Type texts

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

Repository Name Libraries, University of Southern California

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