Progress in Research Regarding Genetic Manipulation in

Aspergillus Oryzae

Qi Jin

1,3

, Hongliang Liu

, Kunhai Qin

, Yitong Shang

, Huanhuan Yan, Bin Zeng

1,2*

and Zhihong Hu

1, *

Jiangxi Key Laboratory of Bioprocess Engineering, College of Life Sciences, Jiangxi Science and

Technology Normal University, Nanchang 330013, China

College of Pharmacy, Shenzhen Technology University, Shenzhen518118, China

School of chemistry and chemical engineering, Jiangxi Science and Technology Normal University,

Nanchang 30013, China

Keywords: Aspergillus Oryzae, Selection Marker, Genetic Manipulation, Protoplast-Mediated Transformation,

Agrobacterium Tumefaciens-Mediated Transformation.

Abstract:

Aspergillus oryzae is a kind of important industrial microorganism, used in food fermentation, condiment

production, brewing, recombinant protein and enzyme production. Currently, genetic engineering

techniques are increasingly being used to improve fermentation performance and product yield of A. oryzae.

However, unlike other filamentous fungi, such as monascus ruber or Aspergillus niger, A. oryzae has

inherent resistance to the common antibiotics, which renders genetic manipulation difficult. In the past, the

genetic transformation of A. oryzae has mainly relied on protoplast-mediated transformation. The

Agrobacterium tumefaciens-mediated transformation system, developed in 2016, and the CRISPR/Cas9

gene editing system, has also been successfully applied in A. oryzae. In this review, we have summarized

the progress in research on genetic manipulation and gene editing methods in A. oryzae.

1 INTRODUCTION

Aspergillus oryzae, an important production strain

certified safe by FDA and WHO, has been used in

traditional food fermentation, flavoring production,

and brewing in Asia for a long time; in addition, its

importance in modern biotechnology industries,

such as those producing recombinant proteins and

enzymes, has increased (Wang, 2021). A. oryzae

genome was sequenced in 2005 (Machida M, 2005).

Currently, genetic engineering techniques are

increasingly being used to improve its fermentation

performance and product yield (Fleissner A, 2010).

However, unlike other filamentous fungi, such as

monascus ruber or Aspergillus niger, genetic

manipulation in A. oryzae was difficult as the

commonly used selection marker and transgenic

methods cannot be used in this organism. Recently,

multiple selection markers and Agrobacterium

tumefaciens-mediated transformation (ATMT) have

been developed, which have facilitated genetic

manipulation in A. oryzae. Gene editing refers to the

precise cutting, insertion or mutation of specific sites

in the genome of recipient cells, to realize the

specific modification of the genome. As a key

reverse genetics research method, gene editing

technology is an important approach for functional

genome research and genetic modification. It

significantly promotes the development of synthetic

biology and has important applications in fungal

genetics and breeding and it is a research hotspot of

fungal synthetic biology (Kumar A, 2021). The

known gene editing systems have been used in A.

oryzae. However, as A. oryzae possesses

multinucleate mycelia and conidia, homozygous

gene-edited strains are hard to be obtained using

traditional gene editing systems, which is unlike that

in monascus ruber and Aspergillus niger (Kitamoto

K, 2015). In this review, we have summarized the

progress in research regarding selection markers,

transgenic methods, and gene editing technology in

A. oryzae.

Jin, Q., Liu, H., Qin, K., Shang, Y., Yan, H., Zeng, B. and Hu, Z.

Progress in Research Regarding Genetic Manipulation in Aspergillus Oryzae.

DOI: 10.5220/0012001700003625

In Proceedings of the 1st International Conference on Food Science and Biotechnology (FSB 2022), pages 63-69

ISBN: 978-989-758-638-5

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

2 SELECTION MARKERS USED

FOR GENETIC ENGINEERING

OF A. ORYZAE

The use of suitable selection markers is important

for successful genetic manipulations, as markers

determine the feasibility of transformation, reduce

the probability of obtaining false-positive

transformants, and minimize the screening

workload. A. oryzae has inherent resistance to the

common antibiotics used as fungal transformation

selectors, such as hygromycin B, geneticin (G418),

phleomycin and bleomycin (Suzuki S, 2009).

Therefore, antibiotics are seldom used as selection

agents for A. oryzae genetic manipulation.

Auxotrophic and drug resistance-related genes

are most commonly used for A. oryzae genetic

manipulation. The fungal orotidine-5-

monophosphate (OMP) decarboxylase, a commonly

used auxotrophy marker that can convert orotidine

into uridine (the precursor of uracil), is encoded by

pyrG. Wild type A. oryzae cannot grow in a medium

containing 5-fluoroorotic acid (5-FOA), as OMP

decarboxylase can convert non-toxic 5-FOA into

toxic 5-fluorouracil, which inhibits growth. pyrG

mutants can grow in medium containing 5-FOA and

uridine/uracil (Jiang, 2013). Therefore, the pyrG

mutant can be selected using 5-FOA and

uridine/uracil supplementation in the medium, and

uridine/uracil auxotrophy can be used as the

selection marker for genetic transformation (Nguyen

KT, 2016). Other auxotrophic genes, such as argB

(encoding ornithine carbamoylase), niaD (encoding

nitrate reductase), adeA (encoding aminoimidazole

nucleotide synthetase), and adeB (encoding

phosphoramidyl carbazole carboxylase) have also

been used as selection markers (Table 1) for genetic

transformation of A. oryzae. Wild type A. oryzae is

sensitive to pyrithiamine (PT), the researchers

obtained the PT-resistance gene ptrA from a PT-

resistant A. oryzae mutant and used it as a selection

marker for transformation (Kubodera T, 2002). This

provides an effective resistance screening marker

that facilitates the screening of transformants and

promotes molecular biology research in A. oryzae.

3 TRANSGENIC METHODS

USED FOR A. ORYZAE

GENETIC ENGINEERING

The transformation method is the second-most

important factor affecting the outcomes of genetic

engineering. Usually, filamentous fungi are

transformed by two methods, protoplast-mediated

transformation (PMT) and Agrobacterium

tumefaciens-mediated transformation (ATMT). PMT

is usually mediated by polyethylene glycol (PEG)-

CaCl

, and involves the formation of particles

[which include PEG, divalent cations (Mg

, Ca

), and exogenous DNA] on the surface of

protoplasts; subsequently, the particles are absorbed

into the protoplast via endocytosis (Liu, 2012). The

preparation of highly efficient protoplasts plays a

pivotal role in PMT, as the state of protoplasts

considerably affects transformation efficiency. The

advantage of PMT is that introduction of exogenous

genes into protoplasts is relatively straightforward.

However, it also has several limitations, including

complicated procedures for preparing and cultivating

protoplasts and the low regeneration frequency of

the transformed protoplasts (Nguyen KT, 2016).

ATMT can not only transform plants, but can also

be used to transform bacteria, animals, and fungi. In

fungi, the first successful genetic manipulation via

ATMT was performed in yeast (Bundock P, 1995).

Currently, ATMT is being successfully used for

transforming A. nidulans, Neurospora crassa, A.

awamori, A. niger, Monascus sp., and other

filamentous fungi (Chen, 2011). ATMT is easier to

perform than PMT (Idnurm A, 2017), as it only

requires the co-cultivation of spores and

Agrobacterium tumefaciens harboring the target

gene, followed by selection in screening media. The

target gene is randomly inserted into the genome and

inherited stably by the newly divided cells.

Furthermore, the transformation efficiency is high.

However, attempts toward establishment of an

ATMT system in A. oryzae have been unsuccessful

until Nguyen et al. first established ATMT using

uracil auxotrophy as a screening marker in A. oryzae

in 2016 (Nguyen KT, 2016). We also developed a

dual selective marker ATMT system using

uridine/uracil auxotrophy and pt resistance genes as

selection markers (Sun, 2019). The establishment of

ATMT system considerably promoted the

identification of functional genes of A. oryzae. The

methods and selectable markers used for

transformation of A. oryzae are shown in Table 1.

FSB 2022 - The International Conference on Food Science and Biotechnology

4 GENE EDITING

TECHNOLOGY IN A. ORYZA

4.1 Homologous Recombination for

Gene Editing in A. oryzae

Early gene editing technology mainly takes

advantage of homologous recombination (HDR) to

replace the target genes. Therefore, adding

homologous arms on both sides of foreign DNA

sequences can realize the accurate integration of

foreign sequences (Komor A, 2017). However, in

eukaryotes, the frequency of homologous

recombination is very low, and foreign DNA

sequences are more likely to be randomly integrated

into other sites on the genome, resulting in off target

effect. Knock out genes required for non-homologous

end joining (NHEJ), such as ku70, ku80, kusA and

ligD, was very effective for increasing the HDR

efficiency (Jiang, 2013; Kwon MJ, 2019).

4.2 ZFNs, TALENs and CRISPR/cas9

System for Gene Editing in A. oryzae

Studies have found that the double strand breaks

(DSBs) that occur at specific DNA sites on the

genome can greatly improve the efficiency of

homologous recombination (Porteus, 2003).

Therefore, in recent years, researchers have

successively developed artificial specific

endonuclease to cleave double strand breaks at

genome-specific DNA sites. Currently, the most

widely used nucleases including: clustered regularly

interspaced short palindromic repeats (CRISPR),

transcription activator-like effector nuclease proteins

(TALENs) and zinc-finger nucleases (ZFNs) (Rajat,

2014). CRISPR/cas9 system is the most widely used

technology at present (Kitamoto K, 2015; Suzuki S,

2009). In the first two techniques, specific DNA-

binding proteins fused with endonucleases to cleave

the double-stranded DNA (dsDNA), resulting in

DNA DSB at specific sites (Rajat, 2014). However,

the CRISPR/Cas9 technique takes advantage of

single guide RNA (sgRNA) to guide Cas9

endonuclease to cleave the DNA double strand,

generating DSBs at the desired site (Zheng, 2017).

Then, the DSB is repaired via NHEJ or HDR. The

NHEJ error-prone features can be utilized to trigger

the mismatching of nucleobase pairs, resulting in the

non-deterministic editing of the target gene. For

HDR, the desired DNA sequence can act as the

template to replace the target gene, thus to realize

accurate gene editing.

ZFNs were first used in animal cells. However,

as the design of ZFNs is complex, the cost of ZFNs

is high, and the editing efficiency depends on the

sequences of target DNA, which limits the

development of this technology. Until now, ZFNs

have not been used in A. oryzae. TALENs is the

second generation gene editing technology and it has

been successfully applied in many species (Joung

JK, 2013). Recent research showed it can also work

in A. oryzae. For example, sC and ligD can be

successfully knocked out in A. oryzae by transient

expression of high-efficiency platinum-fungal

TALENs (PtFg TALENs) (Mizutani O, 2017).

CRISPR/Cas9 is considered a breakthrough in the

field of gene editing due to its versatility and

efficiency, as only the Cas9 endonuclease and the

corresponding gRNA have to be induced in vivo. In

many species, CRISPR/Cas9 system has been

successfully applied. In 2016, the CRISPR/Cas9

system was successfully used to edit the genes of A.

oryzae (Katayama T, 2016). The plasmids

expressing the gene encoding Cas9 (codon

optimized) nuclease and sgRNAs were transformed

into A. oryzae strain via PMT, and the target genes

were successfully knocked out by exploiting the

error-prone property of NHEJ. However, the

mutation efficiency of transformants was only 10%

to 20%, and the most common induced mutations

were 1 bp deletions or insertions (Katayama T,

2016). The low efficiency of traditional

CRISPR/Cas9 system in A. oryzae may due to its

multinucleate mycelia and conidia.

4.3 Optimization CRISPR/Cas9 System

in A. oryzae

Researchers have attempted to improve the

efficiency of the CRISPR/Cas9 system in A. oryzae.

The optimization mainly focused on three aspects:

first, using NHEJ related gene deletion strains

(Δku70, Δku80, ΔkusA and ΔligD) to increase HDR

for accurate gene editing. Sometimes, it needs to

replace, insert or delete the target gene by HDR.

Therefore, using CRISPR/Cas9 system in NHEJ

deficiency mutant strain can increase the HDR

efficiency. This is a useful strategy for many

species. However, it has not been reported in A.

oryzae, which may due to the reason that other

strategies were enough to realize accurate gene

editing by HDR in A. oryzae. The second strategy is

to increase the expression levels of Cas9 and gRNA

using different promoters or autonomous replication

plasmid. To increase the Cas9 and sgRNA levels,

strong promoters were selected for their expression.

Progress in Research Regarding Genetic Manipulation in Aspergillus Oryzae

For example, the A. oryzae amyB promoter was used

for Cas9 expression and the promoter of Aspergillus

Niger U6 RNA polymerase III (PU6) was used for a

heterologous expression of sgRNA (Katayama T,

2016). In addition, the use of the autonomous

replicating plasmid containing Aspergillus nidulans

AMA1 (allows for autonomous plasmid replication)

can also increase the expression of Cas9 and

sgRNA, thereby increasing the gene editing

efficiency (Katayama T, 2019). Another way of gene

editing involves transformation of the assembled

Cas9-CRISPR gRNA RNP complexes in vivo (Jie,

2019). Compared with the expression of Cas9 and

gRNA in vivo, this strategy has obvious advantages,

as the amount or rate of Cas9 translation or gRNA

transcription cannot limit the assembly of Cas9-

gRNA RNP, and it can protect gRNA from

degradation. Recently, this method was also

successfully used in A. oryzae. For example, Zou et

al. used vitro-assembled RNP in A. oryzae protoplast

using chemical reagents to improve the

transformation efficiency of CRISPR-Cas9 RNP

(Zou, 2020). Furthermore, they also added inositol

and benomyl to control the cell division and mitotic

cycles, respectively, which increased the formation

of mononuclear protoplasts. The mononuclear

transformation of A. oryzae increased significantly

increased from 0% to 40.0% with inositol and to

71.43% with benomyl (Zou, 2020). As using

autonomous replicating plasmid greatly improved

gene editing efficiency, multiple gene editing with

one plasmid was realized. For example, Takuya et

al. expressed two gRNA molecules from one gene

editing plasmid and edited two genes (Katayama T,

2019). In addition, this approach can be used to

replace or insert DNA sequences by co-transforming

a circular donor DNA (Katayama T, 2019; Nodvig

CS, 2018). Furthermore, a marker-free gene editing

Table 1: The selection markers and transformation method of A. oryzae.

Strains

Origin of

stain

Selection markers/selection mechanisms

Transgenic

methods

Ref.

niaD300

Mutagenesis

of RIB40

Nitrate reductase gene (niaD)/niaD-bearing strains only

grow in media with NO

as sole nitrogen; the transformants

can

row in media with NO

as sole nitro

en source.

PMT

(Unkles SE,

1989)

FN-16 ΔamdS

Mutagenesis

of FN-16

Acetamidase-encoding gene (amdS)/transformants can grow

in the presence of sucrose and CsCl, but the growth of

untransformed strains was restricted.

PMT (Gomi K, 1992)

NS4

mutagenesis

of niaD30

ATP sulfurylase gene (sC)/the sC mutants are SeO

resistant

and CrO

sensitive, and cannot use NO

and SO

as sole

nitro

en and sulfur sources.

PMT

(Yamada O,

1997)

PTR26

Mutagenesis

of HL1034

Pyrithiamine (PT) resistance gene (ptrA)/transformants can

row on media su

lemented with

rithiamine.

PMT

(Kubodera

T, 2002

)

SE29-70

HowB425

ΔpyrG

5-aminolevulinate synthase (hemA)/deletion of hemA

resulted in a lethal phenotype that could be rescued by the

supplementation of 5-aminolevulinic acid or hemA.

PMT

(Elrod SL,

2000)

NSR13/NSR1

mutagenesis

of NS4

Mutants of adenine genes (adeA/adeB)/AdeA/adeB failed to

grow without adenine; minimal medium supplemented with

adenine restored their

rowth.

PMT (Jin, 2004)

NSAR1

Gene

knocked out

of NSR13

The ornithine transcarbamylase (OTCase) (argB)/ArgB

deletion mutants did not grow in the absence of arginine;

growth was restored after complementation.

PMT

(Nguyen

KT, 2016)

Bm-resistance

mutant

RIB40 wild

type

Bleomycin (Bm)-resistance expression cassette

(BmR)/disruption of ligD with BmR replacement to enhance

the susce

tibilit

. or

zae to Bm.

PMT

(Suzuki S,

2009)

AUT1-PlD/

AS11, C2/

VS1ΔpyrG

RIB40/3.04

2/VS1

OMP decarboxylase gene (pyrG)/cells lacking pyrG are

uridine/uracil auxotrophic mutants and resistant to 5-FOA;

wild-type and pyrG transformants did not survive in the

resence of 5-FOA.

PMT (Zhu, 2013)

RIB40/ΔpyrG

Gene

knocked out

of RIB40

Cells lacking pyrG are uridine/uracil auxotrophic mutants

and resistant to 5-FOA.

ATMT (Jiang, 2013)

3.042/ΔpyrG

Gene

knocked out

of 3.042

Pyrithiamine (PT) resistance gene and OMP

decarboxylase gene/mechanisms are the same as

described above.

ATMT (Sun, 2019)

FSB 2022 - The International Conference on Food Science and Biotechnology

Table 2: Gene editing method in A. oryzae.

Strains

Selection

markers

Transgenic

methods

Editing

methods

Optimization methods Ref.

BCC7051/NID1/

NRRL2270ΔpyrG

pyrG/argB PMT CRISPR/Cas9

Optimized promoter (PU6,

PU3 and tRNA)

(Nodvig CS,

2018; Song,

2018

)

NSAR1

linear neo

cassette

PMT CRISPR/Cas9 Transcribed gRNA in vitro (Zheng, 2017)

RIB40/RIB128/RIB915 ptrA PMT CRISPR/Cas9

Construct including an

AMA1-based autonomously

replicating plasmi

(Katayama T,

2019)

NSAR1 pyrG PMT CRISPR/Cas9

Assembled Cas9-CRISPR

gRNA ribonucleoprotein

(

RNP

)

com

lexes in vitro

(Zou, 2020)

PFJo218 pyrG PMT CRISPR/Cas9

Used Single-stranded Oligo

nucleotides as Gene-

Targeting Substrates (GTS)

(Nodvig CS,

2018)

NSAR1 pyrG PMT CRISPR/Cas9

Chemical reagents were

adde

(Zou, 2020)

RIB40 ptrA/niaD PMT CRISPR/Cas9

Expressed two gRNAs from

a single genome-editing

lasmi

(Katayama T,

2019)

NID1 pyrG PMT CRISPR/Cas9

Transcripted two sgRNAs

from a sin

le tRNA-s

ace

(Nodvig CS,

2018

)

NSAR1

linear neo

cassette

PMT CRISPR/Cas9

Transcribed two sgRNAs in

vitro

(Zheng, 2017)

RIB40 ptrA/niaD PMT CRISPR/Cas9

Used conditional expression

of Aoace2 in plasmid to

force plasmid recover

(Joung JK,

2013)

system has also been developed in A. oryzae.

Takuya et al. used an inducible promoter to express

Aoace2, a gene causing cell lysis, in the gene editing

plasmid (Katayama T, 2019). Then, the transformed

plasmid can be removed by inducing Aoace2 after

the target gene edition finished. Thus, this method

can be used for the repeatable genetic engineering of

A. oryzae. The optimizations of gene editing

methods in A. oryzae are shown in Table 2.

5 CONCLUSIONS

A. oryzae genome was completely sequenced in

2005. So far, the genomes of 6 different A. oryzae

strains have been sequenced. Owing to the

development in molecular biology techniques and

increase in investigations regarding A. oryzae,

genetic transformation of this fungus for improving

traits or producing new products has received an

impetus. Agrobacterium tumefaciens-mediated

transgenic technology will further promote the

application of reverse and forward genetics in

functional gene research in A. oryzae. The gene

editing technology is a powerful tool for the

identification of functional genes. Nowadays, the

gene editing system in A. oryzae mainly depends on

CRISPR/Cas9. And the accurate gene editing main

depends on HDR as the NHEJ editing gene was

non-deterministic. However, the efficiency of HDR

was much lower than that of NHEJ, which limits the

application of this system. Recently, the single base

editor system was also developed based on

CRISPR/Cas9 (Komor A, 2016). This system allows

accurate and efficient mutation of cytosine (C) to

thymine (T) without generating dsDNA breaks. This

technique is more efficient than other gene editing

techniques, and will not produce side effects, such

as random insertion and deletion. Combination of

this method with the existing gene editing

technologies will promote the identification of

functional genes of A. oryzae and lay a theoretical

and technical foundation for genetic modification.

REFERENCES

Bundock P, Den DA, Beijersbergen A, Hooykaas PJ.

(1995) Trans-kingdom T-DNA transfer from

Agrobacterium tumefaciens to Saccharomyces

cerevisiae. Embo Journal. 14:3206-3214.

Progress in Research Regarding Genetic Manipulation in Aspergillus Oryzae

Chen X, Yang J, Peng Y. (2011) Large-scale insertional

mutagenesis in Magnaporthe oryzae by Agrobac-

terium tumefaciens-mediated transformation. Methods

in molecular biology (Clifton, NJ). 722:213-224.

Elrod SL, Jones A, Berka RM, Cherry JR. (2000) Cloning

of the Aspergillus oryzae 5-aminolevulinate synthase

gene and its use as a selectable marker. Current

Genetics. 38:291-298.

Fleissner A, Dersch P. (2010) Expression and export:

recombinant protein production systems for

Aspergillus. Applied microbiology and biotechnology.

87:1255-1270.

Gomi K, Kitamoto K, Kumagai C. (1992) Transformation

of the industrial strain of Aspergillus oryzae with the

homologous amdS gene as a dominant selectable

marker. Journal of Fermentation & Bioengineering.

74:389-391.

Idnurm A, Bailey A, Cairns T, Elliott C, Foster G, Ianiri

G, et al. (2017) A silver bullet in a golden age of

functional genomics: the impact of Agrobacterium-

mediated transformation of fungi. Fungal biology and

biotechnology. 4:6.

Jiang D, Zhu W, Wang Y, Sun C, Zhang KQ, Yang J.

(2013) Molecular tools for functional genomics in

filamentous fungi: recent advances and new strategies.

Biotechnol Adv. 31:1562-1574.

Joung JK, . Sander JD. (2013) TALENs: a widely

applicable technology for targeted genome editing.

Nature Reviews Molecular Cell Biology. 14:49-55.

Jie Q, Sun W, Lin S, Rong J, Ma L, Yi L. (2019)

Cytosolic delivery of CRISPR/Cas9 ribonucleo-

proteins for genome editing using chitosan-coated red

fluorescent protein. Chemical Communications. 55.

Jin FJ, Maruyama J, Juvvadi PR, Arioka M, Kitamoto K.

(2004) Adenine Auxotrophic Mutants of Aspergillus

oryzae: Development of a Novel Transformation

System with Triple Auxotrophic Hosts. Journal of the

Agricultural Chemical Society of Japan. 68:656-662.

Kumar A, Chadha S, Rath D. (2021) CRISPR-Cas9

system for functional genomics of filamentous fungi:

applications and challenges.

Kitamoto K. (2015) Cell biology of the Koji mold

Aspergillus oryzae. Bioscience, biotechnology, and

biochemistry. 79:863-869.

Kubodera T, Yamashita N, Nishimura A. (2002)

Transformation of Aspergillus sp. and Trichoderma

reesei using the pyrithiamine resistance gene (ptrA) of

Aspergillus oryzae. Bioscience Biotechnology &

Biochemistry. 66:404-406.

Komor A, Badran A, Liu D. (2017) CRISPR-Based

Technologies for the Manipulation of Eukaryotic

Genomes. Cell. 169:559.

Kwon MJ, Schutze T, Spohner S, Haefner S, Meyer V.

(2019) Practical guidance for the implementation of

the CRISPR genome editing tool in filamentous fungi.

Fungal Biol Biotechnol. 6:15.

Katayama T, Tanaka Y, Okabe T, Nakamura H, Fujii W,

Kitamoto K, et al. (2016) Development of a genome

editing technique using the CRISPR/Cas9 system in

the industrial filamentous fungus Aspergillus oryzae.

Biotechnol Lett. 38:637-642.

Katayama T, Nakamura H, Zhang Y, Pascal A, Fujii W,

Maruyama JI. (2019) Forced Recycling of an AMA1-

Based Genome-Editing Plasmid Allows for Efficient

Multiple Gene Deletion/Integration in the Industrial

Filamentous Fungus Aspergillus oryzae

. Appl Environ

Microbiol. 85.

Komor A, Kim Y, Packer M, Zuris J, Liu D. (2016)

Programmable editing of a target base in genomic

DNA without double-stranded DNA cleavage. Nature.

533:420-424.

Liu Z, Friesen T. (2012) Polyethylene glycol (PEG)-

mediated transformation in filamentous fungal

pathogens. Methods in molecular biology (Clifton,

NJ). 835:365-375.

Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai

G, et al. (2005) Genome sequencing and analysis of

Aspergillus oryzae. Nature. 438:1157-1161.

Mizutani O, Arazoe T, Toshida K, Hayashi R, Ohsato S,

Sakuma T, et al. (2017) Detailed analysis of targeted

gene mutations caused by the Platinum-Fungal

TALENs in Aspergillus oryzae RIB40 strain and a

ligD disruptant. J Biosci Bioeng. 123:287-293.

Nguyen KT, Ho QN, Pham TH, Phan TN, Tran VT.

(2016) The construction and use of versatile binary

vectors carrying pyrG auxotrophic marker and

fluorescent reporter genes for Agrobacterium-

mediated transformation of Aspergillus oryzae. World

J Microbiol Biotechnol. 32:204.

Nodvig CS, Hoof JB, Kogle ME, Jarczynska ZD,

Lehmbeck J, Klitgaard DK, et al. (2018) Efficient

oligo nucleotide mediated CRISPR-Cas9 gene editing

in Aspergilli. Fungal Genet Biol. 115:78-89.

Porteus, M. H. (2003) Chimeric Nucleases Stimulate Gene

Targeting in Human Cells. Science. 300:763-763.

Rajat, M., Gupta, Kiran, Musunuru. (2014) Expanding the

genetic editing tool kit: ZFNs, TALENs, and

CRISPR-Cas9. Journal of Clinical Investigation.

Suzuki S, Tada S, Fukuoka M, Taketani H, Tsukakoshi Y,

Matsushita M, et al. (2009) A novel transformation

system using a bleomycin resistance marker with

chemosensitizers for Aspergillus oryzae. Biochemical

& Biophysical Research Communications. 383:42-47.

Sun Y, Niu Y, He B, Ma L, Li G, Tran VT, et al. (2019) A

Dual Selection Marker Transformation System Using

Agrobacterium tumefaciens for the Industrial

Aspergillus oryzae 3.042. J Microbiol Biotechnol.

29:230-234.

Song L, Ouedraogo JP, Kolbusz M, Nguyen TTM, Tsang

A. (2018) Efficient genome editing using tRNA

promoter-driven CRISPR/Cas9 gRNA in Aspergillus

niger. PLoS One. 13:e0202868.

Unkles SE, Campbell EI, Ruiterjacobs YMJTD,

Broekhuijsen M, Macro JA, Carrez D, et al. (1989)

The development of a homologous transformation

system for Aspergillus oryzae based on the nitrate

assimilation pathway: A convenient and general

selection system for filamentous fungal

FSB 2022 - The International Conference on Food Science and Biotechnology

transformation. Molecular & General Genetics Mgg.

218:99-104.

Wang L, Hu T, Jiang Z, Yan Q, Yang S. (2021) Efficient

production of a novel alkaline cold-active

phospholipase C from Aspergillus oryzae by

molecular chaperon co-expression for crude oil

degumming. Food chemistry. 350:129212.

Yamada O, Lee BR, Gomi K. (1997) Transformation

System for Aspergillus oryzae with Double

Auxotrophic Mutations, niaD and sC. Journal of the

Agricultural Chemical Society of Japan. 61:1367-

1369.

Zheng YM, Lin FL, Gao H, Zou G, Zhang JW, Wang GQ,

et al. (2017) Development of a versatile and

conventional technique for gene disruption in

filamentous fungi based on CRISPR-Cas9 technology.

Sci Rep. 7:9250.

Zou G, Xiao M, Chai S, Zhu Z, Wang Y, Zhou Z. (2020)

Efficient genome editing in filamentous fungi via an

improved CRISPR-Cas9 ribonucleoprotein method

facilitated by chemical reagents. Microb Biotechnol.

Zhu L, Maruyama JI, Kitamoto K. (2013) Further

enhanced production of heterologous proteins by

double-gene disruption (ΔAosedD ΔAovps10 ) in a

hyper-producing mutant of Aspergillus oryzae.

Applied Microbiology & Biotechnology. 97:6347.

Progress in Research Regarding Genetic Manipulation in Aspergillus Oryzae