University of Southern California Dissertations and Theses

Genome mining of secondary metabolites in Scedosporium apiospermum and Paecilomyces variotii using CRISPR-Cas9 technology

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Genome mining of secondary metabolites in Scedosporium apiospermum and Paecilomyces variotii using CRISPR-Cas9 technology

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i

Genome Mining of Secondary Metabolites in Scedosporium apiospermum and
Paecilomyces variotii Using CRISPR-Cas9 Technology

by
Jingyi Wang

A Dissertation 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 Science)

May 2020

Copyright 2020 Jingyi Wang

ii

Acknowledgments

First and foremost, I would like to appreciate my advisor, Dr. Clay C. C. Wang, chair
and professor of the department of pharmacology and pharmaceutical sciences, for allowing
me to do the research and providing guidance throughout my research. He taught me how to
think critically in the research and how to be an excellent presenter. It was a great honor to
work and study in his lab.
My thesis committee member, Dr. Martine Culty, and Dr. Yong (Tiger) Zhang also
helped me to complete this project. Lectures given by them inspired me a lot.
My sincere gratitude also goes to my lab mates, Dr. Adriana Blachowicz and Dr. Swati
Bijlani, who taught me the methodology to carry out researches. Also, I would like to express
my gratitude to Dr. Yi-Ming Chiang, Dr. Simon Lee, Dr. Michelle Grau, and Bo (Eva) Yuan
for their generous help.
Last but not least, I thank my fellow Christian Râbot, Patrick Lehman, SuJeung Lim,
Ngan Pham, and Gujie Xu for stimulating discussion and the days we spend together in the
last two years.

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Table of Contents
Acknowledgments...................................................................................................................... ii
List of Tables ............................................................................................................................ iv
List of Figures ............................................................................................................................ v
Abstract ..................................................................................................................................... vi
Project 1: Biosynthetic Pathway of a Compound Isolated from Scedosporiumapiospermum .. 1
1.1 Introduction .............................................................................................................................. 1
1.2 Methods and materials ............................................................................................................ 3
1.3 Results and discussion ............................................................................................................... 6
Project 2: "One Strain Many Compounds" (OSMAC) of Scedosporium apiospermum .......... 13
2.1 Introduction ............................................................................................................................ 13
2.2 Methods and materials .......................................................................................................... 14
2.3 Results and discussion ............................................................................................................. 16
Project 3: Unraveling of usually silenced SMs by turning on a global transcription factor in
Paecilomyces variotii ......................................................................................................................... 19
3.1 Introduction ............................................................................................................................ 19
3.2 Methods and materials .......................................................................................................... 20
3.3 Results and discussion ............................................................................................................. 22
Project 4: Screening of antibiotic resistance of Chernobyl-isolated strains ............................... 32
4.1 Introduction ............................................................................................................................ 32
4.2 Methods and materials .......................................................................................................... 32
4.3 Results and discussion ............................................................................................................. 33
Conclusions and outlook ................................................................................................................... 38
References ........................................................................................................................................... 39

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List of Tables

Table 4.1. Number of strains tested against hygromycin B and phleomycin ............................. 34
Table 4.2. Strains which showed sensitivity against hygromycin B........................................ 35
Table 4.3. Strains which showed sensitivity against phleomycin ................................................ 37

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List of Figures

Figure 1.1. Metabolomic characterization of ISS (International Space Station)-grown
Scedosporium apiospermums..................................................................................................... 3
Figure 1.2. The S. apiospermum was grown under different culturing temperature. ................ 7
Figure 1.3. The S. apiospermum cultured on different agar medium ........................................ 7
Figure 1.4. Secondary metabolite production of S. apiospermum ............................................. 7
Figure 1.5. The S. apiospermum grown on different concentration of phleomycin. ................. 8
Figure 1.6. The sensitivity of IMV 00882 protoplasts against phleomycin............................... 8
Figure 1.7. Vector assembly of the plasmid which targets the gene of interest ...................... 10
Figure 2.1. S. apiospermum grow on 11 different medium ..................................................... 17
Figure 2.2. The LC/MS profile of S. apiospermum ................................................................. 18
Figure 3.1. The LC/MS of IMV 00236 grown on MEA plate (pH=4.5, with light) compare to
cheerios .................................................................................................................................... 23
Figure 3.2. Strucuture of the compounds isolated from cheerios. ........................................... 23
Figure 3.3.
1
H and
13
C NMR spectrum of ergosta-5-7-22-trienol (5) and pregn-7-dien-3,6,20
-trione (6) ............................................................................................................................ 25-36
Figure 3.4. The construction of the plasmid targeting the pyrG .............................................. 29
Figure 3.5. The knockout of pyrG using CRISPR-Cas9 ............................................................... 29
Figure 3.6. The vector which targets the mcrA. ....................................................................... 30
Figure 4.1. Example of the antibiotic resistance test ............................................................... 34

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Abstract

Both Scedosporium apiospermum and Paecilomyces variotii are Chernobyl-isolated
strains that have adapted to the high irradiation environment and shown unknown
phenomenon of growing towards the radiation source. The plasticity of fungi enables them to
protect themselves from the radiation by producing compounds to act as sunscreens.
Fungal secondary metabolites (SMs) are an underexploited source of bioactive molecules.
However, most of the gene clusters remain inactive under conventional laboratory conditions.
This thesis aimed to use cultivation based approaches and genetically modify tools to
stimulate the production of more SMs.
CRISPR-Cas9 system is a secure and versatile tool for genome editing. Here we were
able to use CRISPR-Cas9 to generate ku70 and pyrG double-disrupted mutant P. variotii and
further mutated the global regulator mcrA to reveal more SMs.

KEYWORDS: S. apiospermum, P. variotii, secondary metabolites (SMs), CRISPR-Cas9,
genome editing

1

Project 1: Biosynthetic Pathway of a Compound Isolated from
Scedosporium apiospermum
1.1 Introduction
Fungal secondary metabolites (SMs) are a rich source of medically useful compounds
due to their different biological activities. Up to date, the regulation of the biosynthesis of
many SM is still not fully understood. However, the rapid increase in the number of
sequenced fungal genomes and the development of versatile gene targeting methods enabled
the genetic manipulation of non-model filamentous fungi leading to the discovery of novel
SMs. SMs are not directly involved in fungal survival; however, they may confer
environmental advantage that enables adaptation to extreme habitats.
In the years after the Chernobyl nuclear accident took place, more than 2000 fungal
species were collected from the nuclear power plant (Zhdanova et al., 2004).
Chernobyl-isolated fungi are extremophiles that have adapted to the high irradiation
environment and shown unknown phenomenon of growing towards the radiation source
(Zhdanova et al., 2004; Dighton et al., 2008), referred as radiotropism. Scedosporium
apiospermum (IMV 00882) is one of the nuclear power plant accident-associated fungi that
were selected for SM characterization.
Scedosporium apiospermum is an emerging opportunistic fungal pathogen. The ecology
study showed that it is strongly related to human activities. The highest density of the species
is found in industrial areas or agricultural lands. It is also found in patients with cystic
fibrosis (CF), ranking second among the filamentous fungi in CF (Rougeron, A., 2018).
However, not a lot of attention was paid to them due to its rather low occurrence in the
ambient environment (Beguin, H. & Nolard, N., 1994). It turned out that the recovery of the
Scedosporium species is hampered by other extensively growing strains, such as
Cladosporium, Penicillium, and Aspergillus. The application of semi-selective culture media

2

facilitates the demonstration of the rapid occurrence of Scedosporium and draws more
scientists' attention to this species.
SM analysis of IMV 00882 via LC-MS showed a high production yield of orbuticin. It
was initially found in Penicillium verruculosum (Ito, M., et al., 1992). Orbuticin is a
macrocyclic polylactones, which indicates that they are derived from the polyketide pathway.
Polyketides exhibit a broad spectrum of biological activities. Many essential clinically and
industrially relevant compounds are polyketides. Abbanat, D. et al. (1998) also isolated
orbuticin from a marine fungus Hypoxylon oceanicum. Moreover, they found out that
orbuticin has antifungal activity by inhibiting cell wall formation. Schlingmann Gerhard et al.
(2002) concluded that orbuticin is the most potent compound among its similar lactides.
However, its biosynthetic pathway remains cryptic.
Organic extracts of IMV 00882 and Scedosporium apiospermum isolated from the
international space station were examined to test if the exposure to radiation alters the
production of secondary metabolites (Figure 1). Surprisingly, the yield of the orbuticin was
increased in the ISS-isolated S. apiospermum, which has been radiated (Adriana Blachowicz
et al., 2019).
Therefore, we are interested in the orbuticin and intend to elucidate its biosynthetic
pathway. To elucidate the unknown biosynthetic pathway, we are going to use the
CRISPR-Cas9 to knockout the hypothesized gene cluster responsible for the production of
the target compound.
CRISPR-Cas9 is an efficient genetic manipulation tool that can be widely applied to
plants and mammals. The system consists of the Cas9 nuclease and a single chimeric guide
RNA. The protospacer sequence in sgRNA is able to guide the Cas9 nuclease to the targeting
DNA sequence by base pairing. The CRISPR-Cas9 construction we take from Professor Uffe
H. Mortensen (2015) was initially adapt for Aspergillus, but can be extended to other

3

non-model filamentous fungi.
We first tried to knock out the gene cluster MSJF01000841.1, which is responsible for
the production of melanin, as revealed by the BLAST search of the NCBI database. Wild
type S. apiospermum is grey and yellowish. If the gene is successfully knock out, the strain
will turn white. Therefore, it is easy to select the successfully transformed colony.
A similar macrocyclic polylactone,menisporopsin A, was discovered in Menisporopsis
theobromae recently (Bunnak, W. et al., 2019). By comparing the annotation of gene clusters,
we hypothesized that gene cluster MSJF01000841.1 is responsible for the production of
orbuticin. Upon disrupting genes involved in the production of a compound of interest, we
will use HPLC and NMR to analyze the structure of intermediates and propose a biosynthetic
pathway.
Unraveling the biosynthetic pathway may lead to optimizing the production process of
this chemically interesting compound.

Figure 1.1. Metabolomic characterization of ISS (International Space Station)-grown
Scedosporium apiospermums. A significant increase in compound 1 (orbuticin) in space-growm
strain was observed.

4

1.2 Methods and materials
Culture condition optimization: The same amount of spores were inoculated on glucose
minimal medium (GMM), which has just enough ingredients to support the strain to grow.
The plates were placed under different temperatures. The radius of the colony was measured
on day 5 and day 7 to determine the best incubation temperature. The best agar medium was
selected using the same method. The same amount of spores were inoculated on different
agar mediums and placed in a 29°C incubator. The radius of the colony was measured on day
5.
CYA (Czapek Yeast Extract Agar): Concentrated Czapek 100 mL/L (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, and 15 g/L agar.
PDA (Potato Dextrose Agar): 4 g/L potato extract, 20 g/L dextrose, 15 g/L agar.
MB (Malt Broth): 130 g/L malt extract, 15 g/L agar.
YAG (Yeast Extract-Agar-Glucose): 5 g/L yeast extract, 20 g/L glucose, 15 g/L agar
supplemented with 1 mL/L of Hutner’s trace element solution.
YPDA (Yeast Extract-Peptone-Dextrose Agar): 5 g/L yeast extract, 10 g/L peptone, 20 g/L
dextrose, 20g/L agar).
YES (Yeast Extract Sucrose Medium): 20 g/L yeast extract, 100 g/L sucrose, 15 g/L agar
supplemented with 1 mL/L of Hutner’s trace element solution.
LC-MS/MS analysis: The strain was streaked on YES medium and grew at 29°C for 5 days.
The plate was then chopped into small pieces and sonicated for 1 hour with MeOH and
MeOH:DCM (1:1), respectively. The organic solvent was then evaporated, and the compound
was redissolved in double-distilled H2O and partitioned with the same volume of ethyl
acetate (EtOAc). The upper EtOAc layer was transferred to a new tube and air-dried. The
compound was redissolved in 500 μL of 10% DMSO/MeOH (vol/vol) and examined by

5

high-performance liquid chromatography-photodiode array detection-mass spectrometry
(HPLC-DAD-MS).
Antibiotic test: The sensitivity toward hygromycin and phleomycin was determined by
seeding 1 x10
5
spores in 10mL GMM top agar (7.5 g/L agar) with different concentrations of
antibiotic and plated in six-well plate. The growth was observed on day 4.
Genetic Manipulation:
Plasmid construction: The mutant strains were constructed using the CRISPR-Cas9 system
provided by Professor Uffe Mortensen (2015). The vector pFC333 contains the phleomycin
resistant cassette. Two fragments which have 25bp complementary sequence were amplified
using pFC334 as a template with FlashPrime
TM
High Fidelity DNA Polymerase (Initial
denaturation: 2 min at 98°C, denature: 30 s at 98°C, annealing: 30 s at 56°C, extension: 30 s
at 72°C, final extension: 10 min at 72°C). The fragment 1 includes the 20bp designed
protospacer and fragment 2 contains the 6bp hammerhead (inverted-repeat of the 5' end of the
protospacer). The 5' end of fragment 1 and 3' end of fragment 2 also have a ~20bp overlap to
the enzyme-specific cleavage site of pFC333.The pFC333 was linearized by Pacl (NEB).
Then the fragments were inserted into the pFC333 using Gibson assembly. The plasmid was
then propagated into E.coli-DH5α and purified using QIAprepSpin Miniprep Kit (Qiagen
Hilden, Germany). Diagnostic PCR was performed with a set of primers beyond the region of
insertion. The ethidium bromide-stained agarose gel showed the result of the Gibson
assembly. Correct insertion will give a 2kb band. The circular plasmid was again linearized
using PvuI.
Protoplast transformation: The protocol of PEG-mediated protoplasts was adapted from
Dr. Krappmann, S. (2015). The spores were harvested from the PDA plate and inoculated
into 200 mL liquid YES medium for overnight culture at 30°C and 110 rpm. The mycelium
was gathered by filtering. Then VinoTaste Pro enzyme (800mg 6-10 g/hl enzyme dissolved in

6

20 mL of citrate buffer(11.18 g/L KCl, 33.89 g/L NaCl, 14.70 g/L Na-citrate and the pH was
adjusted to 5.5 with citric acid)) was added to digest the mycelium for 2 hours and 15 min at
30°C and 100 rpm. The quality and quantity of the protoplasts were checked by microscopy.
Protoplasts were separated from remained mycelium by Mira cloth and washed twice with
STC1700 buffer (218 g/L sorbitol, 10 mL/L 1M Tris pH 5.5 stock-solution, 5.55 g/L CaCl2,
2 g/L NaCl). 5 μg of the linear or circular plasmid, which contains the sgRNA was added to
150 μl protoplasts (1x10
8
protoplasts/mL) and rest on ice for 30 min. 40% PEG (40%
polyethylene glycol 4000, 50 mM Tris-HCl [pH 8], 50 mM CaCl2) was added to the
protoplasts gradually, followed by 20 min incubation in the ice. The suspension was again
washed with STC1700 buffer and the precipitated. Then it was mixed with 12 mL top agar
(0.7% agar, supplemented with 1.2M sorbitol) and pour into a 10 cm petri dish. After
overnight incubation at 29°C, another 16 mL of top agar with phleomycin was added to the
top. After the colonies have grown to the top, they are repeatedly streaked on selective plates
to avoid heterokaryon formation.

1.3 Results and Discussion
The culture condition was first optimized (Figure 1.2, 1.3). The same amount of
spores was plated in the middle of the 10cm petri dish and incubated for 5 days. The radiuses
of the colonies were measured. We concluded that IMV 00882's growth is the best on the
YES medium, and the most suitable temperature is 29°C. The young colonies are cottony.
The color is light yellowish gray and develops darker with age. After the optimization, we
performed the LC/MS again to confirm the production of orbuticin (Figure 1.4).
The IMV 00882 was then tested for antibiotic sensitivity against hygromycin B (hph) and
phleomycin (ble) to determine the selection marker and associated plasmid for the
CRISPR-Cas9 system. The growth was not affected by hygromycin but inhibited by

7

Figure 1.2. The S. apiospermumwas grown under different culturing temperatures. The radius of
the colony was measured on day 5 and day 7. The best culture temperature was 29°C.

Figure 1.3. The S. apiospermumgrown on different agar medium. The radiuses of the colony were
measured on day 5. The best culture medium was YES.

Figure 1.4. Secondary metabolite production of S. apiospermum. Secondary metabolite profiles of
IMV 00882, when grown on YES in a 29°C incubator, to confirm the strain can still produce orbuticin
(1).

CYA PDA YAG YPDA YES

8

Figure1.5. TheS. apiospermumgrow on the various concentration of phleomycin. The strain
showed resistance against phleomycin. The lowest effective concentration was 20 µg/mL.

Figure 1.6. The sensitivity of IMV 00882 protoplasts against phleomycin. 1x10
5
protoplasts were
mixed with the PDA containing different concentrations of phleomycin (0-3µg/mL). As can be seen,
the lowest effective concentration dropped to 2 µg/mL.

9

phleomycin at 20 µg/mL (Figure 1.5). Thus we chose phleomycin as the selective marker
later in the transformation.
After protoplasting, we tested the antibiotic resistance again using the protoplasts (Figure
1.6). The growth of the strain was inhibited at the concentration of 2 µg/mL. It is much lower
compared to the conidia before the protoplasting because the protoplasts lost the cell wall.
We blasted all the 37 gene cluster against the NCBI database and found that gene cluster
MSJF01000841.1 is responsible for the conidial yellow pigment biosynthesis polyketide
synthase. A 20 bp long protospacer (5'-AGAATCCACAAAGAGAGCAT-3') adjacent to a
TGG PAM (Protospacer Adjacent Motif) was chosen from this gene cluster. The fragments
which contain the protospacer was amplified by Flash PCR. Figure 1.7(C) showed the PCR
used to diagnose the assembly of the plasmid. The plasmid was then propagated by the e.coli
and diagnosed by PCR to confirm the integration of protospacer contained inserts at the
correct locus.
The transformation was performed as described. However, the colony could barely grow
through to the top layer after three weeks of incubation. The transformants were then
restreaked on selective plates containing phleomycin for 4 rounds to obtain single colonies,
which are monokaryotic. The gDNA was extracted and sent for sanger sequencing. However,
no deletion was obtained.
Scedosporium apiospermum is a slow-growing pathogenic fungus (Ramsperger, M. et
al., 2014). It takes the strain for about 10 days to grow through the non-selective top agar.
Hence, when using selectable markers to select correct transformants, possibilities are the
conidia which did not take up the plasmid also grow on the debris of other dead spores. So
we tried to grow the strain on rich medium YES instead of GMM, but the growth rate was not
improved significantly.

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Figure 1.7. Vector assembly of the plasmid which targets the gene of interest.
(A) Confirmation of the correct size of the amplified pFC 333 vector from e.coli by gel
electrophoresis. (B) Fragments amplified by FlashPrime
TM
High Fidelity DNA Polymerase using
pFC334 as a template. Lane 1 is gene ruler. Lane 3 and 4 are insert 1, and lane 6 and 7 are insert 2. (C)
The inserts were assembled with the vector by Gibson assembly. Different vector:insert ratios were
tested, and the 1:1 was the best (lane 4). (D) The plasmid was then extracted from the E. coli.
Diagnostic PCR using a set of primers that give 2 kb was performed to check the correct insertion.

11

We came up with another hypothesis that the IMV 00882 is extremely sensitive to the
phleomycin after they lose the cell wall. So all the transformants died immediately. Only
wild-type conidia survived, but their growth was partially inhibited by phleomycin. Thus we
tried to optimize the protocolof transformation. The protoplasts were first inoculated into
liquid GMM and incubate at 30°C, 100 rpm for 1 hour to regenerate. Then the protoplasts
were mixed with top agar with no antibiotic. After the first layer is dry thoroughly, another 16
mL top agar contains 2 μg/mL phleomycin was added. At the same time, a positive control
(protoplasts take up the plasmid added to the plate without phleomycin) and negative control
(protoplasts without plasmid added to a plate with 2 μg/mL phleomycin) were made as
described. By day 8, the negative control was not growing at all. The transformants were able
to grow on selective plates at the same speed as non-selective plates. While the color of the
transformants was getting darker, the growth rate proves that the protoplast already took up
the plasmid, which contains the phleomycin resistance cassette. However, the targeted gene
was not mutated successfully.
We came up with several possibilities. The specificity of the Cas9 enzyme is believed to
be controlled by the PAM and protospacer. Firstly, the PAM can not successfully guide the
nuclease to the targeted protospacer. We used canonical PAM, which is a 5'-TGG-3' variant,
whereas different variants may have different efficiency. Another possibility is when the
protospacer mismatch with the target sequence, off-target genetic modifications may occur.
Lin, Y. et al. (2014) concluded that the off-target effects of CRISPR-Cas9 appear at a
frequency higher than 50%.
Unfortunately, we were not able to apply the CRISPR-Cas9 system to Scedosporium
apiospermum. This strain is not ideal enough due to its slow growth rate. It is also hard to use
the selectable marker to distinguish the correct transformation from background growth

12

easily.The selection marker we used was phleomycin. It can inhibit the growth of IMV 00882
at a very low concentration.
Moreover, phleomycin is mutagenic to the fungus by introducing double-strand breaks in
DNA ( Suzuki, H. et al., 1970). They can damage the DNA and attack cell walls (Burger, R.,
1998).The protoplasts are incredibly vulnerable and can be easily killed by this antibiotic.
We also grew the IMV 00882 on 5-FOA (5-fluoroorotic acid) plates. The 5-FOA can
inhibit the proliferation of cells by converting pyrimidine into the pyrimidine analog
fluoorotidine monophosphate, which can be further converted into fluorodeoxyuridine.
Fluorodeoxyuridine is a suicidal inhibitor of the thymidylate synthase and thus inhibit the
growth of the strain. However, the strain was not sufficiently inhibited by 5-FOA after
7-days-incubation. The 5-FOA can not form an ideal selection system either.
In conclusion, we were able to discover a bioactive compound in the
Chernobyl-associated strain IMV 00882 and hypothesized the gene cluster, which is
responsible for its production. But we lack an efficient tool to generate mutants in this strain.
Hopefully, new tools will be developed to employ genetic modification in this species to
exploit them thoroughly.

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Project 2: "One Strain Many Compounds" (OSMAC) of Scedosporium
apiospermum
2.1 Introduction
The whole-genome sequence of the S. apiospermum has revealed that the strain has 37
genomes and has the potential to produce a large variety of secondary metabolites. It has 11
PKSs (polyketide synthases) and 9 NRPSs (nonribosomal peptide synthetases). While only a
few compounds had been identified from this organism. Therefore we conducted one strain
many compounds (OSMAC) by cultivating the IMV 00882 in different fermentation
conditions. OSMAC is a cultivation-based technique designed based on the fact that a single
strain can produce different metabolites under different environmental conditions. Parameters
that affect the environmental condition include light regime, pH, temperature, carbon source,
and nitrogen source (Romano, S. et al., 2018).
The light regime is an environmental factor that can regulate the growth, stress resistance,
and pathogenicity of many fungal species. For some fungi, the light serves as the circadian
clock and brings about daily environmental fluctuations (Lombardi, L. & Brody, S., 2005).
The effect of light on fungi remains to be elucidated. Recently Fuller KK. et al. (2013)
focused on the effects of the light on a widely studied strain Aspergillus fumigatus. They
were able to find out that A. fumigatus responded to both blue and red light and revealed the
importance of the light on the physiology of fungus. Researches in N. crassa showed that
exposure to light affected nearly 6.0% of the whole genome. Yet, the response to the light
varies between fungal species. For some strains, the light may repress the germination of
spores, while other strains might be the opposite. Fungi, especially species isolated from
Chernobyl nuclear power plant, can protect themselves from solar radiation through a variety
of mechanisms. Some strains can produce melanin or melanin-like compounds to act as

14

sunscreens (Braga et al., 2015). Thus we compared the development of IMV 00882 under the
light and in the dark. Then the LC/MS was conducted to compare the compound produced.
The pH is also an environmental factor that can significantly change the fungi. The pH
we used in our study was mildly acidic (pH=4.5) and neutral (pH=7). Fungi can adapt to pH
stress (Piper, P., et al., 2001). Moreover, some fungi can actively modify the ambient pH by
secreting acids or alkali (Vylkova, S., 2017). Rougeron et al. (2015) found the S.
apiospermum abundant at the pH range of 6 to 8. So the pH=4.5 medium is giving pressure to
the strain. The yield of the secondary metabolites can be affected by it.

2.2 Methods and materials
Culture condition: Eleven kinds of different mediums and two types of cereals were used as
a source of nutrients. The pH was adjusted to 4.5 or 7 using HCl or KOH. For liquid medium,
1x10
8
spores were added to 5mL of medium and shook at 180 rpm in the dark for 7 days. For
the agar plate, different kinds of medium were plated in 6-well plates. Plates were either
placed in the dark or exposed to the light (36 watts fluorescent light). For cereals, 1x10
8

spores were added to 125 mL flasks containing 3 mL of PDB (potato dextrose broth) and 10
g of cheerios or brown rice. The flasks were placed in the 29℃ incubator for 10 days.
CYA (Czapek Yeast Extract Agar): Concentrated Czapek 100 mL/L (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,
and 15 g/L agar.
CZA (Czapek's Agar): Concentrated Czapek 100 mL/L, 1 g/L K2HPO4, 30 g/L sucrose, and
15 g/L agar.
GMM (Glucose Minimal Medium): 10 g/L glucose, 6 g/L NaNO3, 0.52 g/L KCl, 0.52 g/L
MgSO4·7H2O, 1.52 g/L KH2PO4, 15 g/L agar supplemented with 1 mL/L of Hutner’s trace
element solution.

15

LCMM (Lactose Glucose Minimal Medium): 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, 15 g/L agar supplemented with 1
mL/L of Hutner’s trace element solution.
LMM (Lactose Minimal Medium): 15 g/L lactose, 6 g/L NaNO3, 0.52 g/L KCl, 0.52 g/L
MgSO4·7H2O, 1.52 g/L KH2PO4, 15 g/L agar supplemented with 1 mL/L of Hutner’s trace
element solution.
PDA (Potato Dextrose Agar): 4 g/L potato extract, 20 g/L dextrose, 15 g/L agar.
MB (Malt Broth): 130 g/L malt extract, 15 g/L agar.
MEA (Malt Extract Agar): 20 g/L malt extract, 1 g/L peptone, 20 g/L glucose, 15 g/L agar.
TYG (Tryptone Yeast Extract Glucose Medium): 3 g/L tryptone, 3 g/L yeast extract, 3 g/L
glucose, 1 g/L K2HPO4, 15 g/L agar.
YAG (Yeast Extract-Agar-Glucose): 5 g/L yeast extract, 20 g/L glucose, 15 g/L agar
supplemented with 1 mL/L of Hutner’s trace element solution.
YES (Yeast Extract Sucrose Medium): 20 g/L yeast extract, 100 g/L sucrose, 15 g/L agar
supplemented with 1 mL/L of Hutner’s trace element solution.
LC-MS/MS analysis
Liquid culture: The medium was filtered through Mira-cloth and partitioned with EtOAc.
3 mL of MeOH was used to re-dissolve the extraction. And 5 μL of it was injected into 95 μL
of MeOH to analyze with high-performance liquid chromatography-photodiode array
detection-mass spectrometry (HPLC-DAD-MS).
Agar plate culture: 3 mL of DCM: MeOH(1:1, vol:vol) was added into each well and soaked
for 5 min. All the liquid was then transferred to a glass tube and evaporated. Then the crude
compound was suspended in 3 mL of water and partitioned with 3 mL of EtOAc. The EtOAc
layer was air-dried, re-dissolved in 3 mL of MeOH, and 60 μL was injected into the
HPLC-DAD-MS.

16

2.3 Results and Discussion
After 7 days of incubation, the S. apiospermum was able to grow better without light.The
light used was fluorescent light, which mostly emits light in the visible region of the spectrum
(approximately 400-700 nm in wavelength). A small amount of UV-A and UV-B radiation
will be emitted, while no UV-C (100–280 nm), which exists at Chernobyl nuclear power
plant, is emitted. Therefore the light we use is similar to solar UV radiation. UV radiation can
kill the conidia and hamper the conidial germination speed. Also, UV radiation can lead to
DNA damages. The result was aligned with the conclusion drew by Adriana Blachowicz
(2019) that the IMV 00882 showed a lower survival rate compared to other
Chernobyl-isolated strains.
The main SM orbuticin (1) can be found in most of the cultural conditions. Among them,
cheerios yielded the most abundant amount of orbuticin. Moreover, a few other new
compounds were found. Notable among these compounds, the S. apiospermum was able to
produce pseurotin A (compound 2) in liquid PDA (pH=4.5). Pseurotin A was found in a laeA
mutated Aspergillus fumigatus strain (Wiemann, P. et al., 2013). The biosynthesis genes of
both pseurotin and fumagillin are co-located in a supercluster. By checking the antibiotics
and secondary metabolite analysis shell (antiSMASH), S. apiospermum’s cluster 22
(MSJF01000596.1) shows 100% identity with both the pseurotin A and fumagillin
biosynthetic gene clusters in A.fumigates (Af293), respectively (BGC000103 and
BGC0001067). The genes are also intertwined. Curiously in our OSMAC experiments, only
pseurotin A was detected.
For other compounds, the yield was too low to scale up. Not enough compounds could be
separated and send for NMR. The LC/MS could only show the molecular weight of an
ionized fraction and its polarity. It is unlikely to solve the structure just base on the LC/MS
data.

17

In conclusion, different fermentation conditions have influenced the metabolome of IMV
00882. We can focus on the gene cluster MSJF01000596.1 and study the production of
fumagillin by gene modification using an inducible promoter to activate the fumagillin
production.

Figure 2.1. S. apiospermum grow on 11 different medium. S. apiospermum can grow better in the
dark. The size of the fungal population is not significantly affected by pH.

18

Figure 2.2. The LC/MS profile of S. apiospermum. The main secondary metabolites orbuticin (1)
can be detected in most cultural conditions.

19

Project 3: Unraveling of usually silenced SMs by turning on a global
transcription factor in Paecilomyces variotii
3.1 Introduction
Paecilomyces variotii (IMV 00236) normally produces only a few secondary metabolites.
One of the main products that can be detected under conventional laboratory conditions is
Viriditoxin. Viriditoxin has antibacterial activities, and its biosynthesis pathway has already
been revealed recently (Urquhart, A. et al., 2019). One PKS gene cluster is responsible for its
production. IMV 00236 has 6 NRPS and 7 PKS gene cluster. More potential interesting
compounds remained to be discovered. In this project, we were using different methods to
exploit these silent gene clusters.
Both cultivation based approaches and genetically modification tools can be applied to
stimulate the production of more SMs. Among these two methods, changing the nutrient
content is an easier and more efficient way to alter secondary metabolism (Romano, S. et al.,
2018). Therefore, OSMAC (one strain many compounds) was performed. Eleven different
media containing a variety of nutrient content, different pH, and the different light regime
were applied to culture the strain and thus allow the expression of the normally silenced gene
cluster in the laboratory condition. Though the OSMAC is very easy to perform, there is no
certainty that optimized conditions can stimulate the production of interesting compounds.
According to the OSMAC, the secondary metabolite profile of IMV 00236 is not greatly
altered by environmental conditions. The gene clusters remain cryptic in common laboratory
culture conditions. So we introduced the genetic approaches to the study.
SMs are under the control of the global epigenetic control mechanism.
The veA/velB/laeA complex is a representative of the global regulator. LaeA involves in the
chromatin modification, which is vital in the modulation of SMs. The deletion of laeA leads
to the silence of a relatively large number of gene clusters (Nancy P. Keller et al., 2005).

20

However, laeA is a positive regulator. If it is applied as a tool to explore more SMs, we need
to heterologously express the gene instead of using the CRISPR system to delete it directly.
On the other hand, the deletion of a negative regulator leads to the upregulation of secondary
metabolites. McrA is a negative regulator that encodes a zinc-finger transcription factor that
has previously been identified in A. nidulans (Oakley, C. et al., 2017). A BLAST search of
the A. nidulans mcrA showed that the gene is homologous across many different fungi
species. Thus it is a promising tool for compound discovery.
While this approach also has its limitations, Oakley, C (2017) noted that fewer than half
of the A. nidulans gene clusters are regulated by laeA and mcrA. So more compounds might
be omitted. Moreover, the production of the target compound will not be elevated. Therefore,
later heterologous expression of regulatable alcA promoter will be applied. In this project, we
tried to use the global regulator to eliciting the production of compounds.

3.2 Methods and materials
Strain culture: The strain was stocked at -80°C in 30% glycerol (v/v) and plated on potato
dextrose agar (PDA). Spores were collected on day 5. Then it was grown on different
mediums to conduct the OSMAC.
After transformation, protoplasts were grown on glucose minimum medium (GMM)
supplemented with 1.2M sorbitol. For pyrG auxotrophs, the growth medium was also
supplemented with 10 mM uridine and 1 mg/mL uracil, respectively.
Scale-up: 1x10
8
spores were added to 125 mL small flask containing 10g cheerios and 3 mL
of PDB and incubate at 26°C for 10 days. 75 mL of MeOH was added to the flask and
sonicated for 1 hour. Then the liquid was filtered through a mira-cloth and evaporated in
vacuo. The crude extract was then re-dissolved in ddH2O and partitioned with the same
amount of EtOAc. The EtOAc layer was evaporated in vacuo and subjected to a silica gel

21

columnelutedwith DCM: MeOH(30:1, v:v). The extract was divided into 5 fractions (Fraction
1-5) by monitoring the TLC. Each fraction was then analyzed with HPLC/MS in positive
mode using the Thermo Finnigan LCQ Advantage ion trap mass spectrometer with an RP C18
column (Alltech Prevail C18, particle size 3 μm, column 2.1 x 100 mm) at a flow rate of 2
mL/min. The solvent used was DCM:MeOH=40:1 (v:v).
Transformation Procedures and Construction of Transforming Molecules:
Plasmid construction: The mutant strains were constructed using the CRISPR-Cas9 system
provided by Professor Uffe Mortensen (2015). The pFC332 vector contains the Cas9
nuclease, a sgRNA insertion area with the PacI restriction site, the E.coli Ori (origin of
replication), a β-lactamase gene for selection against ampicillin and a hygromycin resistance
cassette. The vector was first linearized by PacⅠ. Then two fragments were obtained by using
pFC334 as a template. They were flanked by 25bp complementary sequences and contained
the designed protospacer and 6bp hammerhead. The fragments were then fused to the
linearized pFC332 using Gibson assembly. The plasmid was propagated into E.coli-DH5α
and purified using QIAprepSpin Miniprep Kit (Qiagen Hilden, Germany). A set of primers,
which is ~500bp upstream and downstream from the PacⅠ restriction site, was used to
diagnose the correct insertion of the fragments. The plasmid was linearized using the
restriction enzyme PvuⅠ.
Protoplast transformation: The PEG-mediated protoplasts were first achieved as previously
described (Dümig, M. & Krappmann, S. 2015). 1x10
5
spores were inoculated in MEA liquid
medium and shake for 17 hours at 110 rpm, 30℃. Then VinoTaste Pro enzyme was added to
digest the mycelium for 2 hours at 30°C and 100 rpm. After transformation, the protoplasts
were mixed with 20 mL top agar (0.7% agar, supplemented with 1.2M sorbitol) and
70 μg/mL hygromycin and pour into 15cm petri dish. After the agar is solidified, another 20
mL of top agar with 70 μg/mL hygromycin and 1 mg/mL 5-FOA was added to the top.

22

RNA-seq: Spores of transformants were collected from MEA plates. The genomic DNA was
extracted and dissolved in TE buffer. Then a diagnose PCR was conducted using the gDNA
as a template with a set of primers ~300bp upstream and downstream of the protospacer. The
PCR product was extracted after gel purification and send for Sanger sequencing.
Compound Identification:
1
H and
13
C nuclear magnetic resonance (NMR) spectra were
conducted on a Varian Mercury Plus 400 spectrometer. And the NMR data in CDCl3 were
aligned with published data.

3.3 Results and Discussion
The CRISPR-Cas9 gene modification is supported by homologous recombination
(HR), while the non-homologous end-joining pathway (NHEJ) tempers the HR. Ku70 is one
of the major eukaryotic NHEJ proteins. Other major eukaryotic NHEJ proteins include Lig1,
Lig4, Mre11, Rad50, etc. (Chiruvella, K.et al., 2013). The Ku heterodimer is composed
of Ku70 and Ku80. They bind to DNA double-strand break ends and participate in the DNA
repair. The knockout of the Ku70 can thus improve the efficiency of the Cas9 system by 4-5
folds (Van Trung Chu et al., 2015).
In this project, we have already knocked out the P. variotii Ku70. It greatly reduces the
frequency of homologous integration during the transformation and improves the
gene-targeting frequencies (Krappmann, S. et al., 2006).
The OSMAC was performed as described. In addition to the standard medium used in the
lab, cheerios and brown rice were used as a nutrition source. We found that the cheerios can
produce much more secondary metabolites other than the viriditoxin (3) and its derivatives.
Thus we scaled up the growth of IMV 00236 in cheerios. 1x10
8
spores were added to 125
mL small flask containing 20 g cheerios and 5 mL of PDB and incubate at 26°C for 10 days.
The flasks were washed by MeOH and then combined. After extracting with EtOAc, 3.24 g

23

Figure 3.1. The LC/MS of IMV 00236 grown on MEA plate (pH=4.5, with light) compared to
cheerios. On standard MEA plates, IMV 00236 can produce semiviriditoxin (3). While on cheerios, it
can produce a new compound (4) at a retention time of 25.89 min.

Figure 3.2. Strucuture of the compounds isolated from cheerios.

5 6

24

of the crude compound was fractionated by flash chromatography and then by preparative
HPLC, and sent for NMR. However, the main peaks were either steroids or fatty acids that
have no biological activity. Two compounds, ergosta-5-7-22-trienol (5) and
pregn-7-dien-3,6,20-trione (6) were separated and confirmed by NMR. Other peaks were too
small and close to each other, which are not able to be separated.
Ergosta-5,7,22-trienol (1). Colourless needles (CHCl3), m.p. 168-170 ºC; ESI-MS m/z 397
[M+H]+;
1
H-NMR (CDCl3) δ (ppm), 5.57 (1H, dd, J = 6.6, 2.5 Hz, H-6), 5.36 (1H, m, H-7),
5.24 (1H, dd, J = 15.9, 6.6 Hz, H-23), 5.17 (1H, dd, J = 15.9, 6.6 Hz, H-22), 3.60 (1H, m,
H-3), 1.02 (3H, d, J = 6.6 Hz), 0.95 (3H, s), 0.92 (3H, d, J = 6.8 Hz), 0.84 (3H, d, J = 6.6 Hz),
0.82 (3H, d, J = 6.7 Hz), 0.63 (3H, s);
13
C-NMR (CDCl3) δ (ppm), 38.5 (C-1), 32.0 (C-2), 70.6 (C-3), 41.0(C-4), 140.0 (C-5), 119.8
(C-6), 116.5 (C-7), 141.6 (C-8), 46.5 (C-9), 37.2 (C-10), 21.3 (C-11), 39.3 (C-12), 43.0
(C-13), 54.8 (C-14), 28.5 (C-15), 23.2 (C-16), 55.9 (C-17), 12.3 (C-18), 16.5 (C-19), 40.7
(C-20), 21.3 (C-21), 132.2 (C-22), 135.8 (C-23), 43.0 (C-24), 33.3 (C-25), 19.9 (C-26), 20.2
(C-27), 17.8 (C-28). The structure was confirmed by comparison with literature data(Li, X. et
al., 2007).
Pregn-7-dien-3,6,20-trione (2). Colourless needles (CHCl3), m.p. 168-170 ºC; ESI-MS m/z
329 [M+H]+;
1
H-NMR (CDCl3) δ (ppm), 5.80(1H, s, H-7), 2.66(H, dd, J = 12.6, 4.2 Hz, H-5),
2.60(1H, ddd, J = 16.1, 5.6, 2.1Hz, H-4), 2.54(1H, dd, J = 16.1, 12.1 Hz, H-4), 2.15(1H, ddd,
J = 12.6, 5.6, 2.1 Hz, H-1), 1.95(1H, m, H-11);
13
C-NMR (CDCl3) δ (ppm), 38.3 (C-1), 37.9 (C-2), 211.1 (C-3), 37.1 (C-4), 54.8 (C-5), 198.3
(C-6), 123.8 (C-7), 162.3 (C-8), 49.6 (C-9), 38.6 (C-10), 22.1 (C-11), 37.4 (C-12), 45.5
(C-13), 55.6 (C-14), 22.9 (C-15), 23.1 (C-16), 63.3 (C-17), 14.0 (C-18), 13.0 (C-19), 208.8
(C-20), 31.6 (C-21). The structure was confirmed by comparison with literature data (Pang, X.
et al., 2018).

25

1
HNMR spectrum of compound 1 in CDCl3

13
C NMR spectrum of compound 1 in CDCl3

26

1
H NMR spectrum of compound 2 in CDCl3

13
C NMR spectrum of compound 2 in CDCl3
Figure 3.3.
1
H and
13
C NMR spectrum of ergosta-5-7-22-trienol (5) and
pregn-7-dien-3,6,20-trione (6)

27

Ergosterol is a crucial component to form the fungal membranes. Since the Paecilomyces
variotii was isolated from the Chernobyl site, the strain has been exposed to radiation. UV
radiation is harmful to the fungus (Englander, L., Browning, M., & Tooley, P., 2006).
Conidia of most fungal species can be killed by exposure to radiation for a few hours (Braga,
G. et al., 2015). As Villarreal, P. et al. (2016) concluded, the ergosterol is playing a role in
minimizing the damages caused by UV radiation. The ultraviolet-C treatment can inhibit the
conversion of ergosterol to vitamin D2 (Xu, Z., Meenu, M., & Xu, B., 2020), which has an
antioxidant function in the oxidative stress responses and is resistant to the peroxidation of
membrane lipids (Wiseman, H., 1993). P. variotii showed survival at the level of 3.60% after
the exposure to the dose of 2000 J/m
2
UV-C (254 nm), much higher than the ~0.1% survival
rate of other Chernobyl isolated strains (Adriana Blachowicz et al., 2019). Thus, the survival
of Chernobyl-associated strains, including the P. variotii, may process mutagenesis, which
leads to enhanced protection from the radiation (Zavilgelsky, G. et al., 1998).
To further explore the SMs of this strain, we first generated the pyrG deletion strain to
broaden the selection marker. The PyrG is abidirectional marker that allows positive and
negative selection. It involves the complementation of the auxotrophic strain with its wild
type. The deletion of pyrG leads to the auxotrophic for uracil and uridine but are resistant to
5-FOA. In future studies, we will knock out the gene cluster which responsible for the
production of the target compound by either homologous recombination or CRISPR system.
Both of the methods can use pryG as the selection marker.
The designed plasmid contains a 20 bp protospacer sequence from IMV 00236. Then the
plasmid was introduced via protoplast transformation. The transformants were grown on
selective plates containing hygromycin (70 µg/mL) and 5-FOA (1mg/mL). They grew
through the top agar and formed single colonies. To make sure stable mutants were generated,
colonies were streaked on selective plates for two rounds. Then single colonies were picked

28

and plated on non-selective MEA medium to harvest spores. Growing on non-selective
medium led to the loss of the CRISPR-Cas9 plasmid and thus left monokaryotic strain. The
genomic DNA (gDNA) was then extracted. A set of primers, which was ~300 bp upstream
and downstream of the protospacer, was used to amplify the gDNA (total 595 bp). Then the
products were sent for Sanger sequencing and compared with the wild type IMV 00236. One
colony showed a successful deletion of 160 bp (Figure 3.4).
For some transformants, we were not able to amplify the region using the primer to
check the deletion. A possible reason is that there is a larger deletion which exceeds the
region of amplification.
In conclusion, we were able to obtain Ku70 and pyrG double mutant strain, which can be
applied to later genetic manipulation. If a valuable compound is found, the auxotrophic strain
can be applied to produce heterologous proteins.
Since we cannot find SMs other than sterol, we next tried to knock out the global
regulator mcrA. On account of the CRISPR-Cas9 system that has already been well adapted
to P. variotii, we still used Cas9 to mediate the mutation and selected the transformants with
hygromycin.
We performed a BLAST search of the genome of IMV 00236 and were able to find gene
cluster BS090_011480 from P. variotii is 62.17% identical to the mcrA of A. nidulans. And
both of them contain a GAL4-like Zn2Cys6 binuclear cluster DNA-binding domain.
The plasmid targeting the mcrA was constructed using the CRISPR-cas9 system provided
by Professor Uffe Mortensen. And the PEG-mediated transformation was performed as
previously described (Dümig, M., & Krappmann, S. 2015).

29

Figure 3.4. The construction of the plasmid targeting the pyrG. (A) Two fragments that have a 25
base pair flank were amplified by FlashPrime
TM
High Fidelity DNA Polymerase. Lane 2-4 is fragment
1. Lane 5-7 is fragment 2. (B) The fragments were inserted into pFC332 and transformed by e.coli.
The plasmid was extracted using the QIAprepSpin Miniprep Kit. The insertion was confirmed after
the miniprep. Correct insertion gives a 2 kb band (lane 2-4).

Figure 3.5. The knockout of pyrG using CRISPR-Cas9. The Sanger sequence of the mutant
revealed a 160bp deletion, including the protospacer marked in yellow.

30

Figure 3.6. The vector which targets the mcrA. (A) Fragments amplified by FlashPrime
TM
High
Fidelity DNA Polymerase. ((B) The inserts are assembled with the vector by Gibson assembly.
Different vector: insert ratios were tried. Lane 2 is 1:2 (vector:insert), lane 3 is 1:4, lane 4 is 1:5 and
lane 5 is 1:10. All of them give the correct assembly. (C) The size of the plasmids was confirmed
again after transformed in e.coli.

31

We successfully constructed the plasmid, which targets the mcrA. The transformation
was succeeded while no deletion occurs around the protospacer. During the CRISPR-Cas9
mediated transformation, we found out the efficiency of mutation is rather low. Despite the
transformation plate mentioned in pyrG knockout, which used both 5-FOA and hygromycin
as a selective marker, we also made plates only use hygromycin to select. However, 5
colonies survived the hygromycin selection showed no deletion at all. The rate of successful
gene modification is not ideal enough.
The efficiency and specificity of sgRNA in the CRISPR-Cas9 system is a significant
concern. The genome may consist of hundreds of NGG sites, which all can become the
potential cleavage site (Liang, G. et al., 2016). However, the efficiency of a certain candidate
guide RNA remains to be developed. And unlike animal cell lines has the screening libraries,
it takes fungi a longer time to regenerate. Thus the transgenic products can not be obtained
efficiently and used to screen the efficiency of sgRNAs.Since the multiple sgRNAs can exist
simultaneously and edit different loci at the same time (Xie, K. et al., 2015). We will next try
to use two sgRNAs in one transformation to make sure thatmcrAis mutated.
To conclude, we were able to identify and isolate two compounds ergosta-5-7-22-trienol
and pregn-7-dien-3,6,20-trionein IMV 00236. We also successfully generated the Ku70 and
pyrG double mutant strain. It provides a platform for future gene modification. We are still
working on the mcrA knockout. If the mcrAΔ is constructed and the production of new
compounds are stimulated, we can further confirm that mcrA is a global regulator. It will be a
useful tool that can be applied to other fungal species. And more essential SMs are likely
waiting to be discovered.

32

Project 4: Screening of antibiotic resistance of Chernobyl-isolated strains
4.1 Introduction
To carry out modern molecular biological research, the availability of a DNA-mediated
transformation system to generate genetically modified cells that have taken up DNA of
interest is of necessity. One of the significant barriers of gene modification is the availability
of selection pressures, used to distinguish transformants from the background of the recipient
host.
There are also disadvantages of using resistant markers. As mentioned, phleomycin can
introduce double-strand breaks when activated by iron ions and oxygen, which is mutagenic
to fungus (Suzuki, H. et al., 1970).
Selection markers can be divided into resistance markers and nutritional markers. The
CRISPR-Cas9 construct we inherited from Professor Uffe Mortensen included the
hygromycin (pFC332) or phleomycin (pFC333) resistance cassette. Therefore, we were
testing 128 strains in our lab against these two antibiotics.

4.2 Methods and materials
All the strains were grown on PDA plates, and conidia were collected day 5. The
concentrations of the spore suspension were measured. GMM medium (10 g/L glucose, 7.5
g/L agar, 6 g/L NaNO3, 0.52 g/L KCl, 0.52 g/L MgSO4·7H2O, 1.52 g/L KH2PO4, 1 ml/L
5.5M KOH, 15 g/L agar and 1 mL/L Hutner’s trace element solution) is mixed with different
concentration of hygromycin or phleomycin(0, 1, 10, 100μg/mL and 1mg/mL) and plated in
six-well plates. 10μl spore suspension was added on the top of the plates. The plates were
tapped to make the spore suspension is well spread. On days 3 and 5, the growth of the plate
was observed.

33

4.3 Results and discussion
Transformation selection markers enable us to pick up transformants in the parent
background. It facilitates later genetic manipulation of fungi. Therefore we were able to
distinguish strains that are suitable for the genome modification and set up an IMV library for
the lab as well.

34

Figure 4.1. Example of the antibiotic resistance test.IMV 00255 was placed on GMM with
different concentrations of hygromycin B. The lowest effective concentration to inhibit the growth of
this strain is 2 μg/mL.

Table 4.1. The number of strains tested against hygromycin B and phleomycin. A total of 145
strains were streaked on PDA plates. Thirty-three of the stock was either contaminated or not growing.
One hundred twelve of them were tested against the different concentrations of hygromycin B. 66
strains were able to be inhibited by hygromycin. If a strain was resistant to hygromycin B, it was
further tested against phleomycin. Twenty-six strains showed resistance to hygromycin but were
inhibited by phleomycin. Twenty strains were resistant to both antibiotics.

Hygromycin B sensitive 66
Phleomycin sensitive 26
Hygromycin B and Phleomycin
resistance
20
Glycerol stock contaminated 16
Not growing or slow-growing 17
Total strains tested 128

35

Table 4.2. Strains that showed sensitivity against hygromycin B.A Total of 66 strains revealed
sensitivity. Two rounds of the antibiotic test were conducted to figure out the lowest concentration of the
hygromycin to inhibit the strains.(“+”: growth, “-“: no growth.)

Strain ID
Spore
conc.
Control 1 µg/mL 10 µg/mL 100 µg/mL 1 mg/mL
Lowest effective
conc.
IMV 00134 2.4x10
4
+ + + Partial - -
800 µg/mL
IMV 00473A 5.2x10
5
+ + Pigment loss - -
800 µg/mL
IMV 00689A 7.7x10
5
+ + + Pigment loss -
800 µg/mL
IMV 01140 2.0x10
5
+ + + - -
800 µg/mL
M2 1.0x10
5
+ + + - - 80 µg/mL
M3 4.0x10
4
+ + - - -
40 µg/mL
M4 6.0x10
4
+ + Partial - - - 60 µg/mL
IMV 00255 2.5x10
4
+ + - - -
2 µg/mL
IMV 00268 3.0x10
4
+ + + - -
100 µg/mL
IMV 00330 2.4x10
5
+ + + Partial - -
200 µg/mL
IMV 00433 1.0x10
5
+ + + Partial - -
200 µg/mL
IMV 01220 5.5x10
5
+ Pigment loss Pigment loss Partial - -
600 µg/mL
IMV 01262 4.0x10
3
+ + + - - 60 µg/mL
IMV 00034 4.8x10
5
+ + Pigment loss Partial - -
600 µg/mL
IMV 00113 7.0x10
3
+ + Partial - - -
200 µg/mL
IMV 00738 3.4x10
5
+ + Partial - - -
200 µg/mL
IMV 00428 + + + Partial - -
200 µg/mL
IMV 01863 + + + Partial - -
200 µg/mL
IMV 00718 4.3x10
4
+ + + Partial - - 200 µg/mL
IMV 00094 8.2x10
5
+ + Pigment loss - -
80 µg/mL
IMV 00758 1.2x10
5
+ + + Pigment loss -
800 µg/mL
IMV 00609 5.5x10
5
+ + Pigment loss - -
100 µg/mL
IMV 00912 7.2x10
5
+ + + - - 200 µg/mL
IMV 01360 3.1x10
4
+ + + Partial - -
1mg/mL
IMV 01896 3.8x10
5
+ + + - - 80 µg/mL
IMV 01944 1.6x10
5
+ + Pigment loss - -
20 µg/mL
IMV 00769 1.8x10
5
+ + Pigment loss - - 20 µg/mL
IMV 00578 8.9x10
5
+ + + - -
20 µg/mL
IMV 00706 8.3x10
4
+ + Pigment loss - - 20 µg/mL
IMV 00190 5.7x10
5
+ + + - -
200 µg/mL
IMV 01960 1.7x10
4
+ + Pigment loss Pigment loss - 300 µg/mL
IMV 00371 7.0x10
5
+ + + - -
20 µg/mL
IMV 00856 8.4x10
5
+ + + Partial - - 400 µg/mL
IMV 00976 9.3x10
5
+ + Pigment loss - -
80 µg/mL
IMV 00316 6.0x10
5
+ + + - - 200 µg/mL
IMV 00277 1.0x10
5
+ + Pigment loss Partial - -
40 µg/mL
IMV 01154 6.0x10
5
+ + Pigment loss - - 10 µg/mL
IMV 01006 7.0x10
3
+ + Partially - - -
60 µg/mL
IMV 00413 5.8x10
5
+ + +
Pigment loss
- 200 µg/mL
IMV 00164 8.0x10
3
+ + Partially - - - 40 µg/mL
IMV 00334 7.3x10
5
+ +
Pigment loss
- - 200 µg/mL
IMV 00085 3.3x10
5
+ + + Partial - - 100 µg/mL
IMV 01738 1.8x10
5
+ + + Partial - - 600 µg/mL

36

Strain ID
Spore
conc.
Control 1 µg/mL 10 µg/mL 100 µg/mL 1 mg/mL
Lowest effective
conc.
IMV 01738 1.8x10
5
+ + + Partial - - 600 µg/mL
IMV 00100 3.3x10
4
+ + + - - 20 µg/mL
IMV 00119 9.0x10
4
+ +
Pigment loss Pigment loss
- 400 µg/mL
IMV 02017 1.0x10
5
+ + Partial - - - 40 µg/mL
IMV 01779 3.1x10
5
+ + + Partial - - 200 µg/mL
IMV 00543 3.6x10
4
+ + Partial - - - 20 µg/mL
IMV 00065 6.3x10
5
+ + + Partial - - 1 mg/mL
IMV 01272 8.0x10
3
+ + + + - 1 mg/mL
IMV 00212 2.0x10
5
+ +
Pigment loss
- - 20 µg/mL
IMV 01661 1.5x10
4
+ Partial - - - - 40 µg/mL
IMV 00282 1.0x10
3
+ + - - - 10 µg/mL
IMV 00791 6.7x10
4
+ + + - - 40 µg/mL
IMV 00804 1.0x10
5
+ + + - - 40 µg/mL
IMV 00013 3.0x10
5
+ +
Pigment loss
- - 20 µg/mL
IMV 00496 2.6x10
4
+ +
Pigment loss
- - 20 µg/mL
IMV 00182 2.4x10
5
+ +
Pigment loss
- - 20 µg/mL
IMV 01007 2.0x10
3
+ + + - - 20 µg/mL
IMV 02010 1.0x10
3
+ +
Pigment loss
- - 40 µg/mL
IMV 00768 + + + - - 100 µg/mL
IMV 00187 + + + - - 40 µg/mL
IMV 01651 + + + - - 100 µg/mL
IMV 00060 + + + + - 100 µg/mL
IMV 01762 + + + - - 100 µg/mL
IMV 00376 + - - - - 1 µg/mL

37

Table 4.3. Strains which showed sensitivity against phleomycin. Strains which are resistant to up
to 1 mg/mL hygromycin, are further tested against phleomycin. (“+”: growth, “-“: no growth.)

Strain ID
Spore
conc.
Control 1 µg/mL 10 µg/mL 100 µg/mL 1 mg/mL Effective conc.
IMV 01956 1.8x10
5
+ Pigment loss Pigment loss - -
40 µg/mL
IMV 00289 1.2x10
3
+ + + - -
100 ug/mL
IMV 00875 1.1x10
4
+ + + - -
40 µg/mL
IMV 01518 7.0x10
5
+ + + - N/A
100 µg/mL
IMV 01629 1.6x10
5
+ + + - -
60 µg/mL
IMV 01880 4.0x10
5
+ + - - N/A
6 µg/mL
IMV 00343 1.0x10
5
+ + - - N/A
8 µg/mL
IMV 01153 2.0x10
3
+ + - - N/A
8 µg/mL
IMV 01288 1.0x10
3
+ + - - N/A
20 µg/mL
IMV 00074 5.9x10
5
+ + Pigment loss - N/A
80 µg/mL
IMV 00262 1.52x10
6
+ + + - - 100 µg/mL
IMV 01762 + + + - - 100 µg/mL
IMV 00395 2.3x10
5
+ + + Partial - N/A
400 µg/mL
IMV 01038 6.9x10
5
+ + + Partial - -
400 µg/mL
IMV 00973 4.0x10
4
+ + + Partial - -
400 µg/mL
IMV 00188 5.5x10
5
+ + + + -
400 µg/mL
IMV 00899 8.1x10
5
+ Pigment loss Pigment loss Partial -

400 µg/mL
IMV 01463 1.8x10
5
+ + + + -
600 µg/mL
IMV 01255 8.0x10
5
+ + + Partial - -
600 µg/mL
IMV 00488 1.42x10
6
+ + + Partial - -
600 µg/mL
M7 5.7x10
5
+ + + Pigment loss -
>600 µg/mL
IMV 01851 4.1x10
5
+ Pigment loss Pigment loss Partial - -
1 mg/mL
IMV 01953 5.6x10
5
+ + Pigment loss Partial - -
1 mg/mL
IMV 01954 1.8x10
5
+ + Pigment loss Partial - -
1 mg/mL
IMV 01955 4.0x10
4
+ + + Partial - -
1 mg/mL
IMV 02033 7.0x10
4
+ + + Partial - -
1 mg/mL

38

Conclusions and outlook
In conclusion, we worked on the Chernobyl-associated strains to exploit the potential
of fungi’s ability to produce natural compounds that are hard to synthesize industrially. Both
IMV 00882 (Scedosporium apiospermum) and IMV 00236 (Paecilomyces variotii) showed
aquantitative change of metabolites after exposure to UV radiation. We, therefore, first
focused on these variations. We were then able to discover more SMs in both strains using
the OSMAC.
A great many fungal genomes have been sequenced and would constitute gene edits.
Genome modification also leads to the upscale production of the metabolites and can be
further applied in the industry. Our lab was able to sequence the whole genome and
performed genome annotation of several strains, including IMV 00882 and IMV 00236,
which provide an advance to the genome mining. We then constructed the ku70 and pyrG
double-disrupted mutant P. variotii to service for the CRISPR-Cas9 mediated genetic
manipulation. And use the CRISPR system to knock out the negative global regulator mcrA.
The experiment is still under progress. The success will allow us to discover more new
secondary metabolites. Furthermore, the application of mcrAΔ from A. nidulans to P. variotii
will demonstrate this global regulator is conserved in different fungal species and can be an
efficient tool for exploiting natural products.

39

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Abstract (if available)

Abstract Both Scedosporium apiospermum and Paecilomyces variotii are Chernobyl-isolated strains that have adapted to the high irradiation environment and shown unknown phenomenon of growing towards the radiation source. The plasticity of fungi enables them to protect themselves from the radiation by producing compounds to act as sunscreens. ❧ Fungal secondary metabolites (SMs) are an underexploited source of bioactive molecules. However, most of the gene clusters remain inactive under conventional laboratory conditions. This thesis aimed to use cultivation based approaches and genetically modify tools to stimulate the production of more SMs. ❧ CRISPR-Cas9 system is a secure and versatile tool for genome editing. Here we were able to use CRISPR-Cas9 to generate ku70 and pyrG double-disrupted mutant P. variotii and further mutated the global regulator mcrA to reveal more SMs.

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Asset Metadata

Creator Wang, Jingyi (author)

Core Title Genome mining of secondary metabolites in Scedosporium apiospermum and Paecilomyces variotii using CRISPR-Cas9 technology

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/30/2020

Defense Date 03/23/2020

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

Tag CRISPR-Cas9,genome editing,OAI-PMH Harvest,P. variotii,S. apiospermum,secondary metabolites (SMs)

Language English

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Advisor Wang, Clay (committee chair), Culty, Martine (committee member), Zhang, Yong (committee member)

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