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Uncovering the mechanism of the radiation tolerance of a Chernobyl isolated Cladosporium cladosporioides using genetic engineering

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Uncovering the mechanism of the radiation tolerance of a Chernobyl isolated Cladosporium cladosporioides using genetic engineering

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Uncovering the mechanism of the radiation tolerance of a Chernobyl isolated Cladosporium
cladosporioides using genetic engineering

by

Sujeung Lim

A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE
(Pharmaceutical Sciences)

May 2020

Copyright 2020 Sujeung Lim
ii

Acknowledgments
First and foremost, I would like to thank my advisor, Dr. Clay C. C. Wang, for his
guidance and his continuous support during my time in his laboratory. He taught me how to be
independent and how to think critically, which made me be a better scientist. His knowledge and
enthusiasm broadened my perspective of fungal natural products.
Next, I would like to thank my thesis committee: Dr. Jennica Zaro, and Dr. Martine Culty
for generously accepting to be on my thesis committee and providing me insightful comments.
I would like to thank my lab members- Dr. Yi-Ming Chiang, Dr. Ming-Shian Lee, Dr.
Swati Bijlani, Patrick Lehman, Chris Rabot, Bo Yuan, Ngan Pham, Jingyi Wang, and Gujie Xu-
for making the Wang lab a great and family-like research environment. I wish all the best for
their future.
I would also like to thank my previous lab members- Dr. Adriana Blachowicz and Dr.
Michelle Grau- for teaching me and guiding me with the necessary research techniques and
elemental concepts that I lack. Dr. Blachowicz and Dr. Grau provided me much guidance and
helped me to troubleshoot throughout my research. I am glad I met them during my time at USC.
I wish them the best at JPL and Zymergen.
Other than the people mentioned above, I am grateful to Daehee Kang for always being
there for me and for helping me to strive to be a better person. His encouragement helped me to
never give up on anything throughout my years of study.
Last but not least, I would like to send a big thank you to my family. Words cannot
express how grateful I am to my family. My family unconditionally supported me in every
possible way. Without them, I would not be able to complete my master’s degree successfully. I
love you!
iii

Table of Contents
Acknowledgments........................................................................................................................... ii
List of Tables ...................................................................................................................................v
List of Figures ................................................................................................................................ vi
Abbreviations ................................................................................................................................ vii
Abstract ............................................................................................................................................x
Chapter 1: Introduction ................................................................................................................1
1.1 Secondary metabolites ............................................................................................................1
1.2 Classes of secondary metabolites and biosynthetic gene clusters .........................................1
1.3 Fungal survival mechanisms ...................................................................................................2
Chapter 2: Pigment backbone gene knockout in Cladosporiaceae sp. IMV 00236 using
CRISPR-Cas9 .........................................................................................................................4
2.1 Introduction .....................................................................................................................4
2.2 Materials and Methods ....................................................................................................6
2.3 Results and Discussion ....................................................................................................9
Chapter 3: Gene knockout of ku70 in Cladosporiaceae sp. IMV 00236 ..................................16
3.1 Introduction ...................................................................................................................16
3.2 Material and Methods ....................................................................................................17
3.3 Results and Discussion ..................................................................................................17
Chapter 4: One Strain Many Compounds (OSMAC) approach to identify secondary
metabolites in Cladosporiaceae sp. IMV 00236 ..................................................................22
4.1 Introduction ...................................................................................................................22
iv

4.2 Materials and Methods ..................................................................................................22
4.3 Results and Discussion ..................................................................................................24
Chapter 5: Conclusions ...............................................................................................................27
Chapter 6: Supporting Information ...........................................................................................29
References .....................................................................................................................................33

v

List of Tables
Table 6.1. The formulation of the media .......................................................................................29
Table 6.2. The formulation of the Hutner’s trace element .............................................................30
Table 6.3. The raw data of UV-C sensitivity .................................................................................31

vi

List of Figures
Figure 2.1. The biosynthesis pathway of DHN-melanin and DOPA melanin ..............................11
Figure 2.2. Hygromycin resistance test of IMV 00236 ................................................................12
Figure 2.3. Sanger sequencing of backbone gene mutant .............................................................13
Figure 2.4. Growth of IMV 00236 wild type vs. IMV 00236 with disrupted backbone gene for
melanin biosynthesis on PDA at 26°C ...............................................................................14
Figure 2.5. UV-C sensitivity of IMV 00236 and IMV 00236 with disrupted backbone gene for
melanin biosynthesis ..........................................................................................................15
Figure 3.1. An agarose gel image of fragments amplification for Gibson assembly to generate
sgRNA construct for ku70 deletion....................................................................................18
Figure 3.2. An agarose gel image of colony PCR to confirm cloning of ku70 sgRNA in pFC334
............................................................................................................................................19
Figure 3.3. Sanger sequencing of ku70 mutants created by CRISPR-Cas9..................................20
Figure 3.4. Growth of IMV 00236 wild type vs. IMV 00236 ku70 strain on PDA at 26°C ......21
Figure 4.1. Morphology and growth of IMV 00236 using the OSMAC approach .....................24
Figure 4.2. Discovery of new compounds in Cladosporiaceae sp. IMV 00236 grown on GMM
agar, pH=4.5 in the presence of the light ...........................................................................25
Figure 4.3. Discovery of new compounds in Cladosporiaceae sp. IMV 00236 grown on GMM
agar, pH=7 in the presence of the light ..............................................................................26

vii

Abbreviations
A: adenylation
AntiSMASH: antibiotics and secondary metabolite analysis shell
ACP: acyl carrier protein
AT: acyltransferase
BGC: biosynthetic gene cluster
BLAST: basic local alignment search tool
C: condensation
CRISPR-Cas9: clustered regularly interspaced short palindromic repeats/CRISPR-associated
protein 9
CFU: colony forming unit
ChNPP: Chernobyl nuclear power plant
CYA: czapek yeast extract agar
CZA: czapek’s agar
DHN: dihydroxynaphthalene
DMSO: dimethyl sulfoxide
DNA: deoxyribonucleic acid
DOPA: dihydroxyphenylalanine
DSBs: double-stranded breaks
ESIMS: electronspray ionization mass spectrometry
GMM: glucose minimal media
HCl: hydrochloric acid
viii

HPLC-DAD-MS: high performance liquid chromatography-photodiode array detection-mass
spectroscopy
HR: homologous recombination
IMV: Institute for Microbiology and Virology, Academy of Sciences, Kiyv, Ukraine
KOH: potassium hydroxide
KS: ketoacyl synthase
LB: lysogeny broth
LCMM: lactose dextrose minimal media
LC/MS: liquid chromatography/mass spectrometry
LD50: lethal dose, 50%
LMM: lactose minimal media
MB: malt broth
MEA: malt extract agar
MeCN: acetonitrile
MeOH: methanol
NASA: National Aeronautics and Space Administration
NHEJ: nonhomologous end-joining
NRPS: nonribosomal peptide synthetase
ORF: open reading frame
OSMAC: one strain many compounds
PCP: peptidyl carrier protein
PEG: polyethylene glycol
PDA: potato dextrose agar
ix

PDB: potato dextrose broth
PKS: polyketide synthase
sgRNA: single chimeric guide RNA
SM: secondary metabolite
TYG: tryptone yeast extract glucose
UV: ultraviolet
UV-C: ultraviolet-C
WT: wild type
YAG: yeast extract agar glucose
YES: yeast extract sucrose

x

Abstract
Cladosporiaceae sp. IMV 00236 is a ubiquitous saprophytic fungus isolated from a wide
variety of sources, including air, soil, and textiles. It is a known plant pathogen and is also
commonly observed in the indoor mycobiome. Cladosporiaceae sp. IMV 00236 (Institute for
Microbiology and Virology, Academy of Sciences, Kiyv, Ukraine), was isolated from the
Chernobyl disaster site. It exhibited growth towards the radiation source, which was a previously
unknown phenomenon referred to as positive radiotropism. Due to its extreme isolation site and
unique characteristics, the IMV 00236 strain was selected for further characterization of the
ultraviolet (UV) irradiation resistance mechanism and its secondary metabolome.
In this thesis, chapter 1 is an introductory chapter that gives a broad overview of fungal
secondary metabolism. Chapter 2 deals with the role of one of the polyketide synthase (PKS)
gene clusters of Cladosporiaceae sp. IMV 00236 in radiation resistance. Chapter 3 describes the
utilization of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-
associated protein 9) for the construction of the ku70 mutant strain of Cladosporiaceae sp. IMV
00236. The deletion of ku70 results in the increase of the efficiency of homologous
recombination for further gene targeting. Chapter 4 describes the use of “one strain many
compounds” approach to discover silent gene clusters in Cladosporiaceae sp. IMV 00236 that
are activated only under certain specific growth conditions. Lastly, chapter 5 summarizes all the
results and their significance.
1

Chapter 1
Introduction
1.1 Secondary metabolites
Filamentous fungi are prolific producers of secondary metabolites (SMs). SMs are
structurally heterogeneous, low-molecular-mass, bioactive compounds, many of which are also
known as natural products. They are not directly involved in the growth of the organisms.
However, SMs act as chemical signals for communication and act as a defense mechanism for
fungal habitats by inhibiting the growth of other competitive organisms (Brakhage, 2013; Chiang
et al., 2009; Keller et al., 2005). Despite not being necessary for fungal growth, SMs are crucial
in fungal development and are consistently involved in their interaction with other organisms
(Keller, 2019). Many secondary metabolites are important clinically used drugs including
antibiotics (penicillin), cholesterol-reducing agents (lovastatin), and immunosuppressants
(cyclosporine) (Brakhage, 1998, 2013; Hoffmeister and Keller, 2007; Keller et al., 2005).
1.2 Classes of secondary metabolites and biosynthetic gene clusters
Fungi produce a variety of SMs, such as polyketides, non-ribosomal peptides, their
hybrids, and terpenes (Keller, 2019). The chemical class of the secondary metabolites is defined
by the backbone enzyme, which forms the basis of the chemical scaffold. Hence, specific
backbone enzymes are required to produce a particular class of SMs. (Keller, 2019; Wiemann
and Keller, 2014).
In filamentous fungi, the genes encoding the enzymatic activities to produce secondary
metabolites are cluster and forms a biosynthetic gene cluster (BGC) (Keller, 2019). BGCs are
composed of a chemically defining synthase or synthetase that uses primary metabolites to create
carbon backbones that are further modified by decorative, tailoring enzymes, which can change
2

the bioactivities of the metabolites (Chiang et al., 2009; Keller, 2019). Examples of the tailoring
enzymes are oxygenases, glycosyltransferases, methyltransferases, and cyclases (Brakhage,
2013; Walsh et al., 2001).
Polyketides are a diverse class of SMs that are most abundant in filamentous fungi.
Polyketides are synthesized by the backbone enzymes known as type 1 polyketide synthases
(PKSs) (Chiang et al., 2010). The enzyme system of PKSs is organized in a module, which
consists of a minimum of three domains- acyltransferase (AT), ketoacyl synthase (KS), and an
acyl carrier protein (ACP) that are needed for polyketide synthesis. The AT domain is
responsible for selecting extender unit to be added to the product and transfers them to the ACP.
The ACP domain loads the unit to extend the product, and the KS domain is responsible for
decarboxylative condensation of the extender unit with an acyl thioester (Brakhage, 2013;
Chiang et al., 2010). Non-ribosomal peptides are synthesized by its backbone enzyme known as
non-ribosomal peptide synthetases (NRPSs). A module of NRPSs consists of an adenylation (A),
a peptidyl carrier protein (PCP), and a condensation (C) domain. The amino acids are activated
by the A domain and then attached as a thioester to 4’ phosphopantetheine at the PCP domain.
Peptide bonds are formed by the C domain and released by the thioesterase (TE) domain
(Brakhage, 2013; Keller et al., 2005; Walsh et al., 2001). Terpenes are organic molecules
composed of several isoprene units and synthesized by terpene cyclases (TCs), which is crucial
for the production of different types of terpenes from different diphosphates (Brakhage, 2013;
Keller et al., 2005).
1.3 Fungal survival mechanisms
Over time, filamentous fungi have evolved to occupy a wide range of ecological niches.
It has been suggested that filamentous fungi have developed adaptation mechanisms allowing
them to survive and to endure extreme habitats, such as low and high temperatures (Kubicek and
3

Druzhinina, 2007; McKay et al., 2003), desiccation (Barnard et al., 2013; Kubicek and
Druzhinina, 2007), acidic and alkaline pH (Kubicek and Druzhinina, 2007), and radiation
(Blachowicz et al., 2019; Dadachova and Casadevall, 2008; Kubicek and Druzhinina, 2007;
Zhdanova et al., 2004). One of the adaptation mechanisms involves the production of SMs that
help the fungi to survive in the environment directly or indirectly. For instance, many fungi are
known to produce melanin, which is known to have the ability to shield the fungi against
radiation sources. One such example can be found from the melanized fungal strains that inhabit
in Arctic, Antarctic, and Chernobyl disaster sites. Such characteristics of fungi led the National
Aeronautics and Space Administration (NASA) to explore the Chernobyl isolated fungal species
(Blachowicz et al., 2019; Rummel et al., 2002). The Chernobyl isolated fungal strains colonized
the walls of a highly radioactive damaged reactor at Chernobyl and soil surrounding it
(Dadachova et al., 2007). Furthermore, 2000 species of fungi have been isolated from the
Chernobyl disaster sites within the last 30 years (Zhdanova et al., 2004). However, there are
significant gaps in the understanding of the molecular mechanisms of filamentous fungi’s
survival under extreme radiation conditions, which need to be explored in detail.

4

Chapter 2
2.1 Introduction
Cladosporiaceae sp. IMV 00236 is ubiquitous in nature, which is commonly isolated
from soil, food, paint, textiles, and other organic matters. It is a melanized fungus, which
produces several secondary metabolites of economic importance like viriditoxin - an anticancer
compound, cladosporin, and isocladosporin with known antifungal activities (Bensch et al.,
2012; Jacyno et al., 1993; Scott et al., 1971; Singh et al., 2017). Cladosporiaceae sp. IMV 00236
strain has been isolated from the wall surface of unit-4 of the Chernobyl nuclear power plant
(ChNPP) after the nuclear accident, which happened on April 25, 1986 (Blachowicz et al., 2019;
Dadachova et al., 2007). High levels of released radioactivity were detrimental to the
surrounding environment making it inhospitable (Blachowicz et al., 2019). However, melanized
fungal communities were the first to reappear and dominate the area within the failed reactor and
beyond in the exclusion zone (Dighton et al., 2008).
Cladosporiaceae sp. IMV 00236 exhibits positive radiotropism, which was a previously
unknown phenomenon of growing towards the radiation source (Blachowicz et al., 2019;
Dighton et al., 2008; Zhdanova et al., 2004). Some of the radiotropic fungi could grow into and
decompose hot particles, pieces of radioactive graphite from the reactor (Dadachova and
Casadevall, 2008; Zhdanova et al., 2004). Some reports showed that C. cladosporioides isolates
60 and 10 (from the 4
th
Block reactor room) showed statistically significant growth directed
towards
109
Cd source of radiation (Dadachova and Casadevall, 2008). Although not statistically
significant, a trend toward directional growth was seen for C. cladosporioides 396, which was
isolated from uncontaminated soils (Dadachova et al., 2007; Dadachova and Casadevall, 2008).
5

The term “melanin” has derived from melanos, a Greek word for black (Dadachova et al.,
2007). Melanin has high molecular weight and is insoluble in an aqueous or organic solvent
(Guo et al., 2015). Melanin has many beneficial properties, for instance, it protects against UV,
solar radiation and extreme temperatures (Dadachova et al., 2007). In fungi, the most important
types of melanin are the dihydroxynaphthalene (DHN) melanin and the dihydroxyphenylalanine
(DOPA) melanin (Langfelder et al., 2003). Many fungi synthesize melanin via the DHN-melanin
pathway (Figure 2.1) that is usually found in hyphae or spores (Dadachova et al., 2007;
Eisenman and Casadevall, 2012; Keller, 2019). In the DHN-melanin pathway, acetyl CoA and
malonyl CoA serve as precursors for a polyketide synthase (PKS) to form 1,3,6,8-THN. This is
followed by a series of reduction and dehydration reactions to produce the intermediates
scytalone, 1,3,8-trihydroxynaphthalene (THN), vermelone, and 1,8-dihydroxynaphthalene
(DHN). Finally, DHN is polymerized to form DHN-melanin (Eisenman and Casadevall, 2012;
Langfelder et al., 2003; Tsai et al., 2001). However, some fungi can synthesize melanin through
L-3,4-dihydroxyphenylalanine (L-DOPA) melanin pathway. L-DOPA or tyrosine are two
possible starting molecules. If tyrosine is the precursor, then it is first converted to L-DOPA. L-
DOPA is oxidized to dopaquinone by laccase. After several further enzymatic reactions,
dihydroxyindoles are produced that polymerize to form melanin (Eisenman and Casadevall,
2012).
Melanin is known to protect the fungi from radiations, therefore, we wanted to
investigate if melanin is responsible for imparting resistance to irradiation in Cladosporiaceae
sp. IMV 00236.
6

2.2 Material and Methods
Fungal strains and growth conditions
Cladosporiaceae sp. IMV 00236, originally isolated from the Chernobyl disaster site,
was used for the experiments. The fungal strains were grown on PDA (potato dextrose agar) at
26°C for 5 days. The spore suspensions were prepared in ST (0.85% NaCl, 0.1% Tween 80)
solution and spore count was enumerated using a hemocytometer. The spore suspension of
mutants was stored in 15% glycerol at -80°C.
Antibiotic sensitivity test
The antibiotic sensitivity of IMV00236 was tested against hygromycin. 10 mL of GMM
(glucose minimal media) with 1.5 % agar was transferred to 6 of 15 mL falcon tubes. Then
hygromycin with following concentrations: 0 μg/mL, 1 μg/mL, 10 μg/mL, 100 μg/mL, and 1
mg/mL was mixed with media in falcon tubes and poured to each well of the sterile, 6 wells
tissue culture plates (VWR, North America). 100 μL of the spore suspension was inoculated in
each well and spread evenly. The plate was then incubated at 26°C for 4 days. The effective
concentration range of hygromycin was then determined after the incubation. This was followed
by another round of antibiotic sensitivity test by varying the hygromycin concentration from 10
μg/mL - 100 μg/mL to determine the minimum inhibitory concentration.
Fungal transformation
The transformation in Cladosporiaceae sp. IMV 00236 was done using the standard
polyethylene glycol (PEG) transformation method as previously described (Bergmann et al.,
2009; Krappmann et al., 2006; Punt and van den Hondel, 1992) with some modifications. The
fungal culture was set up at a starting spore concentration of 1x10
8
sp/ml and incubated the
culture at 30°C, 110 rpm for 16 hours. After the incubation, the protoplasts were prepared by
digestion with the VinoTaste Pro enzyme (Novozymes) for 2 hours. The remaining protocol was
7

followed similar to that described previously (Bergmann et al., 2009; Krappmann et al., 2006;
Punt and van den Hondel, 1992).
Construction of backbone gene mutant of PKS gene cluster 24
The backbone gene mutant in IMV 00236 was created using the CRISPR-Cas9 system
developed by Mortensen lab (Nodvig et al., 2015). The 20 bp protospacer sequence 5’-TGA
GAC CGG CGA GAT TAT CC-3’, targeting the backbone gene was selected from the gene
sequence. The sgRNA (single chimeric guide RNA) targeting the backbone gene was
constructed as follows. The sgRNA backbone sequence was amplified in two fragments using
pFC334 as a template such that the protospacer sequence overlaps between the two fragments.
The protospacer sequence was incorporated in the reverse primer of 1
st
fragment and forward
primer of 2
nd
fragment. The two fragments were amplified using pFC334 as a template with
custom primers: Fw1 5’-TAG CTG TTT CCG CTG A-3’, Rev1 5’-GGA TAA TCT CGC CGG
TCT CAG ACG AGC TTA CTC GTT TCG TCC TCA CGG ACT CAT CAG TGA GAC CGG
TGA TGT CTG CTC AAG-3’, and Fw2 5’-TCG TCT GAG ACC GGC GAG ATT ATC CGT
TTT AGA GCT AGA AAT AGC AAG TTA AA-3’ and Rev2 5’-ATT CTG CTG TCT CGG
CTG- 3’. These fragments were then assembled in pFC332 (contains Cas9 and hygromycin
resistance gene for selection in fungi) using Gibson assembly mix (New England BioLabs
(NEB)) after linearizing the pFC332 plasmid with PacI (NEB).
The recombined plasmid was then transformed into chemically competent Escherichia
coli DH5α and the colonies were selected on lysogeny broth (LB) with ampicillin. The plasmid
was isolated from the colonies obtained using QIAprep Spin Miniprep Kit (Qiagen Hilden,
Germany) and cloning was confirmed by diagnostic PCR using primers that anneal outside the
cloning site. The purified plasmid was linearized, and 5 µg of linearized deoxyribonucleic acid
8

(DNA) with PvuI (NEB) was used for fungal transformation. The introduction of DNA was done
using protoplasts and standard PEG transformation. Correct transformants were selected based
on the loss of the pigment under selection conditions and confirmed by Sanger sequencing.
Ultraviolet-C (UV-C) exposure and survival evaluation
Strains were revived and grown at 26°C on the malt extract agar (MEA) for 4 days to
collect spores in the ST solution. To evaluate UV-C sensitivity of the mutant, approximately
1000 conidia suspended in 50 mL of MEA (half agar) cooled to 55°C and 5 mL of this
suspension (100 conidia per plate) was poured on top of 20 mL MEA in Petri plates (D = 10 cm).
Each strain was exposed to 0, 10, 15, 20, and 25 mJ/cm
2
doses of UV-C using Hoefer UVC 500
crosslinker (Amersham Biosciences, Little Chalfont, UK) in triplicates. UV-C exposed and
unexposed (control plates) were incubated at 26°C for 3 days. After 3 days, colony-forming units
(CFUs) were counted and percent survival was calculated using the formula: (N/N0) * 100%; the
number of CFUs obtained at a given dose/CFUs in the control plate *100%. Results from three
biological replicates were averaged and used for statistical analysis with Welch’s corrected t-test.
Lethal dose, 50% (LD50) values for IMV 00236 WT and its PKS backbone gene mutant were
calculated using LD50 calculator from AAT Bioquest (https://www.aatbio.com/tools/ld50-
calculator).

9

2.3 Results and Discussion
The secondary metabolite gene clusters in Cladosporiaceae sp. IMV 00236 were
predicted using the antibiotics and secondary metabolite analysis shell (antiSMASH) pipeline
(Medema et al., 2011). This analysis revealed a total of 31 putative secondary metabolite gene
clusters, including six terpene cyclase, six nonribosomal peptide synthetase (NRPS), seven
polyketide synthase (PKS), ten NRPS-like and two PKS-NRPS genes. More in-depth analysis of
the melanin molecular structure and the detailed annotation from antiSMASH led to the
hypothesis that gene cluster 24 may be responsible for melanin production in IMV 00236 strain.
To confirm our hypothesis, a CRISPR-Cas9 system was used to disrupt the backbone
gene of the identified gene cluster 24. The antibiotic sensitivity of Cladosporiaceae sp. IMV
00236 against hygromycin was first tested to proceed with fungal transformations where
hygromycin can be used as a selection marker. The effective hygromycin concentration was
determined using the IMV 00236 protoplast (spores with the digested cell wall, which are used
for downstream transformation) was determined to be 70 μg/mL (Figure 2.2). Upon
transformation with the CRISPR plasmid containing the protospacer for backbone gene deletion,
we observed the loss of the typical pigmentation in the IMV 00236 transformants. To confirm
the correct deletion within the targeted gene, genomic DNA was extracted from both the wild
type (WT) and selected mutant strains and it was sent for Sanger sequencing. The sequencing
revealed that 14 bp were deleted within the 20 bp targeted protospacer sequence in the IMV
00236 mutant strain (Figure 2.3). The sequencing analysis confirmed that CRISPR-Cas9 was
successfully used to disrupt melanin production in Cladosporiaceae sp. IMV 00236. Further,
both IMV 00236 WT and IMV 00236 mutant strain were point inoculated on the PDA plates to
10

confirm morphology and show the loss of pigmentation in the IMV 00236 mutant strain (Figure
2.4).
Upon confirming the correctness of the created genetic mutant of IMV 00236, both IMV
00236 mutant and WT were exposed to varying doses of UV-C (0, 10, 15, 20, 25 mJ/cm
2
) to
evaluate their sensitivity to UV. The analysis revealed that the backbone gene mutant was
significantly more UV-C sensitive compared to the WT strain IMV 00236, while both strains
exhibited dose-dependent UV-C sensitivity (Figure 2.5). In addition, LD50, the dose required to
kill 50% of populations, for IMV 00236 WT and its PKS backbone gene mutant was found to be
11.1 mJ/cm
2
and 8.2 mJ/cm
2
respectively. These results suggest that, as predicted, melanin plays
a role in protecting Cladosporiaceae sp. IMV 00236 from irradiation.

11

Figure 2.1. The biosynthesis pathway of A) DHN-melanin and B) DOPA-melanin. Pyroquilon
acts similar to tricyclazole (Tc, as shown in A), which inhibits both THN reductase reactions in
the DHN-melanin pathway (A) (Eisenman and Casadevall, 2012; Tsai et al., 2001).

A)
B)
12

Figure 2.2. Antibiotic sensitivity test of Cladosporiaceae sp. IMV 00236 strain against
hygromycin. (A) The effective concentration range of hygromycin was first determined using
IMV 00236 conidia and minimum inhibitory concentration was determined to lie between 10-
100 μg/mL. (B) The exact minimum inhibitory concentration - was further determined from the
range identified in (A) using protoplasts and was found to be 70 μg/mL.

100 μg/mL
1 μg/mL
10 μg/mL
Control
1 mg/mL
A)
Control 70 μg/mL
90 μg/mL
130 μg/mL
110 μg/mL
150 μg/mL
B)
13

Figure 2.3. Sanger sequencing result of the deletion within the targeted backbone gene
(BBmutant) as shown upon alignment with the parent strain (wt).

14

Figure 2.4. Growth of IMV 00236 WT compared to backbone mutant strain. The wild type
strain (A) and the mutant strain (B) on PDA after growth at 26°C for 3 days, showing colony
morphology and its pigmentation.

A) B)
15

Figure 2.5. UV-C sensitivity test of IMV 00236 WT (blue) and the mutant strains (red). The
histogram represents the percent viability following the exposure to varying doses of UV-C
radiation. The raw data are included in the supporting information section (Table 6.3). Statistical
significance was determined by Welch’s corrected t-test and is marked with * (p ≤ 0.05) (refer
Table 5.4).

16

Chapter 3
3.1 Introduction
There are two major repair pathways of DNA double-stranded breaks (DSBs) in all living
organisms (Carvalho et al., 2010; Mizutani et al., 2008)- homologous recombination (HR) and
the nonhomologous end-joining (NHEJ). The NHEJ does not need any sequence homology to
rejoin DSBs (Mizutani et al., 2008) and directly ligates the strand ends to rejoin the DSBs
(Mizutani et al., 2008). HR, on the other hand, requires intact a genetic sequence/material from
an undamaged homologous region as a template to repair DSBs (Mizutani et al., 2008). NHEJ
and HR pathways require the involvement of different proteins. One major protein required for
the NHEJ pathway is Ku protein (Krappmann, 2007; Mizutani et al., 2008).
Most filamentous fungi prefer/choose to repair DSBs by NHEJ (Mizutani et al., 2008).
Ku is a heterodimer of two proteins, Ku70 and Ku80, and is a major component of NHEJ. The
deletion of ku70 impairs NHEJ, thereby increasing the rate of HR for DNA repair (Carvalho et
al., 2010; Shrivastav et al., 2008). Several studies have shown that Ku-deficient mutants, for
example, in Aspergillus fumigatus, Aspergillus nidulans, Aspergillus, oryzae, Aspergillus sojae,
and Aspergillus niger, displayed improved gene targeting efficiency by HR (Carvalho et al.,
2010; Chiang et al., 2008; Krappmann, 2007; Krappmann et al., 2006; Meyer et al., 2007;
Mizutani et al., 2008; Nakamura et al., 2017).
Most of the gene clusters in Cladosporiaceae sp. IMV 00236 are still uncharacterized
that might be of economic importance and need to be investigated. Therefore, the main objective
of this study was to develop a strain that exhibits higher efficiency of HR that will facilitate gene
targeting. This was accomplished by creating a ku70Δ mutant strain of Cladosporiaceae sp. IMV
00236.
17

3.2 Material and Methods
Construction of C. cladosporioides ku70 mutant strain
The ku70Δ in IMV 00236 was created using an established CRISPR-Cas9 system as
previously described in the methods in chapter 2. The 20 bp protospacer sequence, 5’-CAA CCG
TCT CGA CAG GAA GT-3’, targeting ku70 was selected from the ku70 gene sequence. The
protospacer was introduced to the pFC332 using similar strategy as described in chapter 2 with
custom primers: Fw1 5’-TAG CTG TTT CCG CTG A-3’, Rev1 5’-ACT TCC TGT CGA GAC
GGT TGG ACG AGC TTA CTC GTT TCG TCC TCA CGG ACT CAT CAG CAA CCG CGG
TGA TGT CTG CTC AAG-3’, Fw2 5’-TCG TCC AAC CGT CTC GAC AGG AAG TGT TTT
AGA GCT AGA AAT AGC AAG TTA AA-3’ and Rev2 5’-ATT CTG CTG TCT CGG CTG-3’
(Figure 3.1).
The A. fumigatus transformation protocol from Dr. Sven Krappmann’s lab (Bergmann
et al., 2009; Krappmann et al., 2006; Punt and van den Hondel, 1992) in Germany was modified
and used for the transformation as described in chapter 2. The deletion in the transformants
obtained was confirmed by Sanger sequencing using the following primers: Fw 5’-CCT GTA
GGA ATG GAG CGG GA-3’ and Rev 5’- TGG CGT GCT ACT GTA CGG AA-3’.
3.3 Results and Discussion
The protein sequence of the ku70 gene in Cladosporiaceae sp. IMV 00236 was found by
searching for ATP-dependent DNA helicase II subunit 1 in antiSMASH. This protein sequence
was aligned against other fungal genomes using protein basic local alignment search tool
(BLAST). The protein BLAST result showed that there is a 76% sequence similarity between
Ku70 of Cladosporiaceae sp. and A. fumigatus; and high sequence similarity to Ku70 orthologs
in other species.
18

The cloning of sgRNA in pFC332 was confirmed by diagnostic PCR using primers
flanking the integration site (Figure 3.2). The generated construct was then linearized and 5 μg
was transformed into Cladosporiaceae sp. IMV 00236. Transformants were selected on GMM
media containing 70 μg/mL of hygromycin. The transformants obtained were verified using
Sanger sequencing (Figure 3.3). Two transformants were positive for a mutation in the ku70
gene, as analyzed by Sanger sequencing results. Both IMV 00236 WT and IMV 00236 ku70
mutant were point inoculated on the PDA plates to confirm morphology and pigmentation
(Figure 3.4). The ku70 mutant grows similar to the WT strain.

Figure 3.1. An agarose gel image of PCR amplified fragments 1 and 2 (436 bp and 585 bp
respectively) containing the protospacer for Gibson Assembly.

Fragment 1 Fragment 2
19

Figure 3.2. Colony PCR to confirm the cloning of sgRNA (containing the ku70 protospacer
sequence) in pFC332. All colonies gave the expected PCR product of 2 kb.

20

Figure 3.3. Sanger sequencing result of the disrupted ku70 gene. 250 bp upstream and
downstream from the targeted 20 bp were amplified, sequenced and sequences analyzed. The top
row is the sequence of the ku70 gene in WT, and the second row is the sequence of ku70 mutant
clones. A) 6 bp were deleted within 20 bp protospacer region. B) A total of 25 bp were deleted in
the second ku70 mutant.

A)
B)
21

Figure 3.4. Growth of IMV 00236 WT (A) and ku70 mutant strain (B) on PDA at 26°C after 3
days, showing colony morphology and its pigmentation.

A) B)
22

Chapter 4
4.1 Introduction
Fungi are well known for their ability to produce many natural products. However, gene
clusters responsible for the production of secondary metabolites often remain silent under
standard laboratory conditions. This result suggests that there are a lot of BGCs yet to be
explored. To activate these silent BGCs, conditions that trigger their expression need to be
identified. There are many methods to activate silent SM gene clusters, such as co-cultivation
with other organisms, transcriptional regulator modulation, heterologous expression in a different
host, and OSMAC (One Strain Many Compounds) (Lyu et al., 2020; Wiemann and Keller,
2014). OSMAC approach enables one to discover new compounds by culturing the organism
under different conditions, including different media compositions, pH, temperature, and light
(Bode et al., 2002; Chiang et al., 2011). These factors can induce or repress the synthesis of
natural products, for instance, penicillin and cephalosporin are synthesized from their respective
gene clusters, under specific growth conditions (Chiang et al., 2011; Wiemann and Keller, 2014).
In this study, an attempt was made to turn on the silent secondary metabolite BGCs and
to discover new secondary metabolites by using the OSMAC approach.
4.2 Material and Methods
Preparation of culture media
Eleven media (both agar and broth) with two different pH values, 4.5 and 7, were
prepared for the culture of Cladosporiaceae sp. IMV 00236. The media prepared were MEA,
GMM, LMM, lactose dextrose minimal media (LCMM), Czapek yeast extract agar (CYA)
media, Czapek’s agar (CZA) media, yeast extract sucrose (YES) media, yeast extract agar
glucose (YAG) media, tryptone yeast extract glucose (TYG) media, potato dextrose (PD), and
23

malt broth (MB). The formulation of all media is listed in chapter 6 section of this thesis (Table
6.1). The pH values for all the media were adjusted to 4.5 or 7 with 6 M hydrochloric acid (HCl)
or 5.5 M potassium hydroxide (KOH), respectively, using the pH meter. The agar plates were
prepared using sterile, six wells tissue culture plates (VWR, North America). 10 mL of each agar
media was poured to each well of 6 well tissue culture plates.
Fungal culture
2x10
8
spores were inoculated in 20 mL of each liquid media in 125 mL Erlenmeyer
flasks after harvesting the spores in the ST solution. These were cultured for a week at 28°C, 180
rpm. 2x10
8
spores were point inoculated on agar plates after harvesting the spores in the ST
solution. These were cultured for a week at room temperature.
Secondary metabolite (SM) extraction and analysis
For the liquid culture, each culture was filtered through Mira-cloth and then partitioned
with ethyl acetate. 3 mL of methanol was used to re-dissolve the extract. A 5 μL of the extract
was diluted into 95 μL of methanol and was analyzed using HPLC-DAD-MS.
For the agar culture, 3 mL of methanol was added to each well and allowed to soak for 1
hour. After 1 hour, the liquid was transferred to a glass test tube, and the solvent was evaporated
in vacuo. 3 mL of methanol and 3 mL of 1:1 methanol/dichloromethane were then added to each
well of the agar plate. All the liquids were transferred to the same glass test tube, and the solvent
was again evaporated in vacuo. The residue obtained was suspended in 3 mL of deionized water
and extracted with 3 mL of ethyl acetate. The ethyl acetate layer was evaporated in vacuo to
yield a residue that was suspended in 2 mL of MeOH. A concentration of 10 ppm of the
secondary metabolites extracted was prepared before injecting into LC/MS. The solvent gradient
for LC/MS was 95% acetonitrile/H2O (solvent B) in 5% acetonitrile/H2O (solvent A) both
24

containing 0.1% formic acid, as follows: 0 to 100% solvent B from 0 to 13 min, 100% solvent B
from 13 min to 40 min, and re-equilibration with 0% solvent B from 40 to 50 min.
4.3 Results and Discussion
The culture conditions resulted in the production of several unidentified secondary
metabolites. The morphology and the growth of Cladosporiaceae sp. IMV 00236 under different
conditions and in the presence of various carbon sources are presented below (Figure 4.1). From
this OSMAC approach, changes in secondary metabolite profiles were seen in the case of GMM
at pH 4.5, light, and pH 7.0 light. Three new peaks appeared when IMV 00236 was cultured in
the presence of light on GMM media at pH=4.5 (Figure 4.2). Two new peaks appeared when
IMV 00236 was grown in the light on GMM at pH=7 (Figure 4.3). In the case of all other
conditions tested, there were no differences observed in the secondary metabolite profile. The
same compound was expressed at varying amounts depending on the culture conditions. The
different peaks obtained from growth on GMM at pH=4.5 and GMM at pH=7 need to be further
characterized.

Figure 4.1. Morphology and growth of Cladosporiaceae sp. IMV 00236 under different light
and pH conditions on various media. Pictures were taken after 7 days of growth.

25

Figure 4.2. Secondary metabolites analysis of IMV 00236 on GMM, pH=4.5 grown in the
presence of light. Three new peaks (highlighted with blue) were observed under this condition.
Traces present PDA and ESIMS (positive mode) data for the unknown compounds 3-5 analyzed
by HPLC-DAD-MS.

3 4 5
26

Figure 4.3. Secondary metabolites analysis of IMV 00236 on GMM, pH=7 grown in the
presence of light. Two, new peaks (highlighted with yellow) are observed under this condition.
Traces present PDA and ESIMS (positive mode) data for the unknown compounds 6-7 analyzed
by HPLC-DAD-MS.

6 7
27

Chapter 5
Conclusions
The results obtained from this study identify an important aspect of fungal secondary
metabolism. The advanced bioinformatic tools and molecular biology techniques such as
CRISPR-Cas9 and OSMAC were used in this study.
Cladosporiaceae sp. IMV 00236 is a melanized fungi and since this strain was isolated
from a highly radiated environment, we wanted to study if melanin plays any role in protection
from radiation conditions in this fungus. In this study, we identified the putative gene cluster that
is responsible for melanin biosynthesis using bioinformatic tools. The genetic mutant of putative
melanin PKS backbone gene was constructed and when exposed to UV-C radiation, it was found
that the mutant is more sensitive to UV-C compared to the WT strain. This indicated that the
putative melanin gene does play a role in protection of this fungus under extreme radiation
mechanisms.
In addition to this, we wanted to create a strain that is genetically more amenable
compared to the wild type strain. Therefore, we created ku70 gene mutant in Cladosporiaceae
sp. IMV 00236. that is known to enhance the gene targeting efficiency by inhibiting non-
homologous end joining pathway. Furthermore, the ku70Δ mutant strain can be used to
characterize many biosynthetic gene clusters.
Finally, an attempt was made to discover silent gene clusters of Cladosporiaceae sp.
IMV 00236 using the OSMAC approach where changing the culture conditions produced a total
of five, unknown secondary metabolites. These five, unidentified secondary metabolites need to
be characterized in future.
28

Although there is a significant amount of discoveries made, there is still more to learn
about fungal secondary metabolism. The work described in this thesis can be used as a basis for
future studies.

29

Chapter 6
Supporting Information
Name Compositions

GMM
10 g/L glucose, 6 g/L NaNO3, 0.52 g/L KCl,
0.52 g/L MgSO4·7H2O, 1.52 g/L KH2PO4, 1
mL/L Hutner’s trace element

LCMM
20 g/L lactose, 10 g/L dextrose, 6 g/L
NaNO3, 0.52 g/L KCl, 0.52 g/L
MgSO4·7H2O, 1.52 g/L KH2PO4, 1 mL/L of
Hutner’s trace element

LMM
15 g/L lactose, 6 g/L NaNO3, 0.52 g/L KCl,
0.52 g/L MgSO4·7H2O, 1.52 g/L KH2PO4, 1
mL/L of Hutner’s trace element

CYA
100 mL/L Concentrated Czapek (NaNO3 30
g/L, KCl 5 g/L, MgSO4·7H2O, 0.1 g/L
FeSO4·7 H2O), 1 g/L K2HPO4, 5 g/L yeast
extract, 30 g/L sucrose

CZA
100 mL/L Concentrated Czapek (NaNO3 30
g/L, KCl 5 g/L, MgSO4·7H2O, 0.1 g/L
FeSO4·7 H2O), 1 g/L K2HPO4, 30 g/L sucrose
PDA 4 g/L potato extract, 20 g/L dextrose
MB 130 g/L malt extract

MEA
20 g/L malt extract, 1 g/L peptone, 20 g/L
glucose

TYG
3 g/L tryptone, 3 g/L yeast extract, 3 g/L
glucose, 1 g/L K2HPO4

YAG
5 g/L yeast extract, 20 g/L glucose, 1 mL/L
of Hutner’s trace element

YES
20 g/L yeast extract, 100 g/L sucrose, 1
mL/L of Hutner’s trace element
Table 6.1. The formulation of the media. These media were used in the OSMAC experiment.
15g/L agar was added to each media to prepare the agar plate. The formulation of potato
dextrose broth (PDB) is the same as PDA without agar added.
30

Name Compositions

Hutner’s trace element

2.2 g ZnSO4·7H2O
1.1 g H3BO3
0.5 g MnCl2·4H2O
0.5 g FeSO4· 7H2O
0.16 g CoCl2·6H2O
0.16 g CuSO4·5H2O
0.11 g (NH4)6Mo7O24·4H2O
5.0 g Na2EDTA
Table 6.2. The composition of Hutner’s trace element. Hutner’s trace element was prepared as
follows: add the solids in order to 80 ml of H2O and dissolve each completely before adding the
next. Heat the solution to boiling, cool to 60°C, adjust the pH to 6.5 with saturated KOH. Cool to
room temperature and adjust the volume to 100 ml with deionized water. Autoclave and store at
room temperature.

31

Dose [mJ/cm2] WT WT % Δ CFU Δ %
0 60 85.71 100 142.86
60 85.71 60 85.71
90 128.57 50 71.43
Average of first
triplicates 70 70
90 84.38 80 82.76
40 37.50 120 124.14
190 178.13 90 93.10
Average of second
triplicates 107 97
90 100.00 130 139.29
80 88.89 50 53.57
100 111.11 100 107.14
Average of third
triplicates 90 93
10 43 61.43 39 55.71
48 68.57 36 51.43
55 78.57 35 50.00
49 45.94 25 25.86
45 42.19 36 37.24
58 54.38 25 25.86
47 52.22 34 36.43
40 44.44 14 15.00
44 48.89 26 27.86
15 27 38.57 17 24.29
18 25.71 19 27.14
29 41.43 8 11.43
27 25.31 6 6.21
14 13.13 20 20.69
30 28.13 11 11.38
24 26.67 5 5.36
20 22.22 12 12.86
33 36.67 11 11.79
20 19 27.14 8 11.43
10 14.29 8 11.43
16 22.86 5 7.14
16 15.00 8 11.43
3 2.81 3 4.29
12 11.25 4 5.71
12 13.33 0 0.00
5 5.56 2 2.86
8 8.89 2 2.86
25 8 11.43 0 0.00
0 0.00 1 1.07
0 0.00 2 2.14
4 3.75 2 2.14
9 8.44 0 0.00
9 8.44 3 3.21
10 11.11 0 0.00
A)
32

4 4.44 0 0.00
2 2.22 0 0.00

multiple t-test WT vs mutant
Dose significance p value
0 No 0.363915
10 Yes 0.001184
15 Yes 0.000429
20 Yes 0.00762
25 Yes 0.008238

Table 6.3. The raw data of UV-C sensitivity. The raw data of UV-C sensitivity (A) and its
statistical significance determined by Welch’s corrected t-test (B) (p ≤ 0.05).

B)
33

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Abstract (if available)

Abstract Cladosporiaceae sp. IMV 00236 is a ubiquitous saprophytic fungus isolated from a wide variety of sources, including air, soil, and textiles. It is a known plant pathogen and is also commonly observed in the indoor mycobiome. Cladosporiaceae sp. IMV 00236 (Institute for Microbiology and Virology, Academy of Sciences, Kiyv, Ukraine), was isolated from the Chernobyl disaster site. It exhibited growth towards the radiation source, which was a previously unknown phenomenon referred to as positive radiotropism. Due to its extreme isolation site and unique characteristics, the IMV 00236 strain was selected for further characterization of the ultraviolet (UV) irradiation resistance mechanism and its secondary metabolome. ❧ In this thesis, chapter 1 is an introductory chapter that gives a broad overview of fungal secondary metabolism. Chapter 2 deals with the role of one of the polyketide synthase (PKS) gene clusters of Cladosporiaceae sp. IMV 00236 in radiation resistance. Chapter 3 describes the utilization of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for the construction of the ku70 mutant strain of Cladosporiaceae sp. IMV 00236. The deletion of ku70 results in the increase of the efficiency of homologous recombination for further gene targeting. Chapter 4 describes the use of “one strain many compounds” approach to discover silent gene clusters in Cladosporiaceae sp. IMV 00236 that are activated only under certain specific growth conditions. Lastly, chapter 5 summarizes all the results and their significance.

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Asset Metadata

Creator Lim, Sujeung (author)

Core Title Uncovering the mechanism of the radiation tolerance of a Chernobyl isolated Cladosporium cladosporioides using genetic engineering

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/20/2020

Defense Date 03/26/2020

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

Tag Chernobyl isolate,CRISPR-Cas9,filamentous fungi,fungal secondary metabolism,gene editing,genetic engineering,melanin,OAI-PMH Harvest,Secondary metabolites,UV-C

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Wang, Clay (committee chair), Culty, Martine (committee member), Zaro, Jennica (committee member)

Creator Email sjsue0412@gmail.com,sujeungl@usc.edu

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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