Construction of an Engineering Strain Producing High Yields

of -Transglucosidase via Agrobacterium tumefaciens-Mediated

Transformation of Asperillus niger
Ming LI, Liying ZHOU, Meng LIU, Yunyan HUANG, Xin SUN, and Fuping LU
y
Key Laboratory of Industrial Fermentation Microbiology, Education Ministry of China,
College of Biotechnology, Tianjin University of Science and Technology,
No. 29, 13 Main Street, Economic and Technological Development Zone, Tianjin, 300457, China
Received April 3, 2013; Accepted May 30, 2013; Online Publication, September 7, 2013
[doi:10.1271/bbb.130281]
In this study, Agrobacterium tumefaciens-mediated
transformation (ATMT) was used in breeding industrial
strains for the purpose of improving -transglucosidase
production. Firstly, an ecient ATMT system for
Asperillus niger was established by optimization of
several inuencing factors, in which transformation
eciency was improved up to 14-fold compared with the
initial conditions. Furthermore, binary vector pBI-Glu
containing an -transglucosidase expression cassette
was constructed and transferred into Agrobacterium
tumefaciens LBA4404 in order to infect A. niger. By the
ecient ATMT method, the gene for -transglucosi-
dase, driven by strong promoter P
glaA
(the glucoamylase
gene promoter), had a high expression level in A. niger
A-8 (25.02 U/mL). The optimized ATMT system was
found to be eective and suitable for A. niger, and
should be a useful tool for studying the function of
A. niger genes and for industrial breeding of this strain.
Key words: Agrobacterium tumefaciens-mediated trans-
formation; Aspergillus niger; optimization;
-transglucosidase
The lamentous fungus Aspergillus niger, widespread
in foods, plant products, and the soil, is one of the most
important microorganisms in biotechnology. Owing to
its high secretion capacity and being generally regarded
as safe (GRAS) status, A. niger has been in use for many
decades to produce a large number of enzymes (e.g.,
-transglucosidase, glucoamylase)
1)
and organic acids
(e.g., citric acid).
2,3)
The long history of using A. niger
on an industrial scale makes it well developed as a
transformation host to overexpress foreign proteins. In
recent years, a number of genes have been cloned and
expressed in the A. niger expression system.
46)
-Transglucosidase (EC 3.2.1.20) is a group of
enzymes that catalyze the hydrolysis of -glucosidic
linkages from the non-reducing terminal of substrates
releasing -glucose or transferring a glucosyl residue to
the 6-OH of the accepting glucose unit, yielding
isomaltooligosaccharide (IMO).
7,8)
It is widely distributed
in microorganisms, animals, and plants. Since an
-transglucosidase-producing strain was selected by
Hayashibara Biochemical Laboratories in 1982, it has
been widely used in the food industry and has attracted
much attention. In the past few years, studies of -
transglucosidase have focused mainly on the screening
of production strains and medium optimization,
911)
but
the low yield of -transglucosidase from wild-type
strains makes them uneconomical for extensive indus-
trial application, and thus, with the development of
genetic engineering techniques, constructing and using
a genetic engineered strain as host for large-scale
production is a new way to study the enzyme.
1215)
To
date, -transglucosidase gene has been cloned and
expressed in A. niger,
16)
E. coli,
12)
A. nidulans,
13,17)
and S. cerevisiae.
18)
These studies indicate that among
all of the hosts, the expression level of -transglucosi-
dase was the highest in A. niger. Nevertheless, the yield
obtained with the A. niger was still low and did not
reach the standard for industrial application. Hence it is
necessary to use A. niger as a host for the further study
to improve the production of -transglucosidase. In
order to characterize the -transglucosidase gene, and to
construct a prolic secretion strain of -transglucosidase
by recombinant DNA techniques, a highly ecient,
stable, and convenient transformation method for A.
niger must be established to make genetic manipulation
possible in this strain.
Agrobacterium tumefaciens, a plant pathogenic bac-
terium, transfers part of its Ti plasmid (T-DNA) to the
host cell and then inserts the T-DNA region into host
genome.
19,20)
Using this property, Agrobacterium tume-
faciens-mediated transformation (ATMT) is widely
applied as a transformation method for plants. It can
be used to achieve targeted gene disruption and random
insertional mutation. Since de Groot et al.
21)
reported
that ATMT can be used for the transformation of
lamentous fungi, it has been considered to be more
convenient, stable, and ecient than traditional trans-
formation methods used in fungi. Reports on ATMT
indicate that the transformation eciency of lamentous
fungi via A. tumefaciens is about 3009,000 transform-
ants per 10
7
spores, which was improved up to 600-fold
as compared to results for PEG transformation of
protoplasts.
19,22)
To date, a wide range of fungi species
y
To whom correspondence should be addressed. Fax: +86-22-60602298; E-mail: kdlimingyjs@sina.com
Abbreviations: ATMT, Agrobacterium tumefaciens-mediated transformation; As, acetosyringone; IMO, isomaltooligosaccharide; Rif, rifampicin;
Str, streptomycin; T-DNA, Ti plasmid
Biosci. Biotechnol. Biochem., 77 (9), 18601866, 2013
(e.g., Aspergillus fumigatus,
23)
Aspergillus awamori,
19)
Aspergillus japonicus,
24)
and Trichoderma viride
25)
)
have been established ATMT system, but the trans-
formation eciency of A. niger by ATMT was very
low, only ve transformants per 10
7
spores.
21)
Thus, it
is essential to establish an ecient ATMT method for
A. niger.
The transformation eciency of ATMT diers in
dierent fungal species. Several factors, including
spores freshness, the concentration of A. tumefaciens,
the spore concentration, co-culture temperature, and
time aect eciency to a great extent.
17,23,26)
In this
study, therefore, an ecient ATMT method for A. niger
was estabilished by optimization of these inuencing
factors. Then an A. niger genetic engineered strain
expressing -transglucosidase at a high level was
constructed and selected from a transformant library
by means of the improved transformation method.
Materials and Methods
Strains, media, and plasmids. A. niger TCCC41056, screened as a
strain producing glucoamylase and intracellular -transglucosidase by
our laboratory, was used as host strain for ATMT. A. niger UV-11,
27)
preserved in our laboratory, was used for amplication of the
glucoamylase gene promoter (P
glaA
). These strains were maintained
on PDA medium at 28

C. A. tumefaciens LBA4404, stored in our

laboratory, was used as a T-DNA donor for fungal transformation, and
grown on YEB medium (0.5% peptone, 0.5% yeast extract powder,
0.5% beef extract, 0.5% sucrose, 0.5% MgSO
4
.
7H
2
O, and 1.5% agar)
supplemented with rifampicin (Rif) and streptomycin (Str) or on IM
medium (2% NH
4
Cl, 0.6% MgSO
4
.
7H
2
O, 0.3% KCl, 0.5% glucose,
2 mmol Na
3
PO
4
, 50 mmol 2-(N-morpholino) ethanesulfonic acid
(MES), and 1.5% agar, pH 5.8) supplemented with 200 mmol/L of
acetosyringone (AS), at 28

C. E. coli DH5 was used for gene

cloning. It was grown in LB medium at 37

C. Fermentation medium
(3% soluble starch and 3% bran) was used to produce -trans-
glucosidase. The recombinant plasmids used in ATMT were con-
structed with the vectors pAN7-1,
28)
pBI121,
29)
and pT-Z, a covalently
closed circular pUCm-T by ligating open circular pUCm-T. Vector
pAN7-1 contains the Hygromycin B resistance gene (hph) driven by
the Aspergillus nidulans gpdA promoter and terminated by the
A. nidulans trpC terminator. Plasmid pBI121 possessing the T-DNA
border repeat sequence and the kan-resistance gene was used for
genetic transformation. Vector pT-Z containing the MCS region was
used in plasmid construction. All plasmids were preserved in our
laboratory.
Sensitivity of A. tumefaciens LBA4404 to cephamycin. Two
hundred microlitre of A. tumefaciens culture (OD
600
1:0) was spread
on YEB agar plates supplemented with various concentrations of
cephamycin (0, 20, 40, 60, 80, and 100 mg/mL) and this was incubated
at 28

C for 4 d. The growth of A. tumefaciens was observed every day.

Sensitivity of A. niger TCCC41056 to hygromycin B. A. niger
spores incubated for 4 d on PDA medium were harvested with sterile
water. The spore concentration was counted by hemocytometer and
diluted from 10
8
spores per milliliter to 10
5
spores per milliliter.
Two hundred microlitre of A. niger spore suspension (10
8
spores
per milliliter) was spread on PDA agar plates supplemented with
various concentrations of hygromycin B (0, 40, 60, 80, 100, and
120 mg/mL) and this was incubated at 28

C in dark for 4 d. The

growth of A. niger was recorded every day.
Construction of binary vector pBI-hph. The pBI121, EcoR I, and
Xba I sites lay between the left and the right border of T-DNA, and
hence the binary vector pBI-hph was constructed by insertion of a 2.6-
kb EcoR I/Xba I fragment from pAN7-1 containing the hph cassette
into the EcoR I/Xba I restricted pBI121 plasmid.
A. tumefaciens-mediated transformation. ATMT was developed to
introduce DNA into lamentous fungi. In this study, all the trans-
formation experiments were carried out according to the previously
described protocol unless otherwise noted.
21)
Initially, binary vector
pBI-hph, having the hph gene as selection marker, was introduced into
A. tumefaciens LBA4404 by electroporation.
30)
The transformants
were isolated on YEB agar plates supplemented with Rif (50 mg/mL),
Str (20 mg/mL), and Kan (50 mg/mL), and conrmed by PCR and
enzyme digestion. Positive A. tumefaciens transformants were cultured
in 5 mL of YEB on a rotatory shaker (180 r/min) at 28

C for 20 h.
A. tumefaciens cells were collected by centrifugation (5,000 r/min,
10 min) and resuspended in fresh YEB containing AS (200 mmol/L) at
a density of OD
600
0:15: After preincubation for 610 h at 28

C on a
shaker to an OD
600
of 0.4 to 1.0, 100 mL of the cell suspension was
mixed with an equal volume of spore suspension at a concentration
of 10
5
spores per milliliter to 10
8
spores per milliliter, and then
the mixture was spread onto lter paper placed on IM agar plate
(with 200 mmol/L AS). The plates were incubated at 20

C to 28

C in
the dark for 1 to 4 d. After co-cultivation, the lter paper was
transferred onto PDA plates containing hygromycin B and cephamycin
to inhibit the growth of A. tumefaciens and to select transformants.
After an additional incubation at 28

C for 4 d in the dark, the single

colony was transferred to new PDA agar plates and conrmed. All
the results presented below were obtained from three independent
experiments.
Extraction of genomic DNA and total RNA. A. niger spore
suspension was inoculated into PDA liquid medium at 28

C on a
rotary shaker (180 r/min). After 4 d, the mycelia were collected with
sterile gauze and washed with sterile water. Genomic DNA was
extracted by the CTAB method.
31)
Total RNA was extracted according
to the specications of the Trizol kit.
Analysis of transformants. The hph gene in the putative trans-
formants was conrmed by PCR using primers hphF and hphR with
genomic DNA as template. For Southern blot analysis, genomic DNA
from the transformants and the wild-type strain was digested with
Xba I overnight. The treated fragments were separated by electro-
phoresis on 0.8% agarose and transferred to a nylon membrane. Pre-
hybridization, hybridization, and washing of the membrane were
carried out at 68

C following the instructions in the DIG High Prime

DNA Labeling and Detection Start Kit I.
Mitotic stability of the transformants. To determine the stability of
the inserted T-DNA in transformants, hygromycin B resistant clones on
selection plates were selected randomly and cultured on PDA plates
without hygromycin B at 28

C for 10 successive generations. Spores

of the transformants were inoculated on a new PDA plate and cultured
4 d for one generation. Then the transformants were transferred to
PDA plates with 100 mg/mL of hygromycin B and this was incubated
at 28

C for 4 d.
Construction of binary vector pBI-Glu. Binary vector pBI-Glu was
constructed on the backbone of pBI-hph by inserting an -trans-
glucosidase expression cassette, as follows: Strong promoter P
glaA
was
amplied with sense primer PgF containing Kpn I and Hind III and
antisense primer PgR containing Nco I from the genomic DNA of
A. niger UV-11. An 820-bp PCR fragment was digested with Kpn I
and Nco I and ligated to pT-Z digested with the same restriction sites
to construct plasmid pT-P. The A. nidulans trpC terminator gene was
obtained by PCR, using plasmid pBI-hph as template and primers TrF
and TrR, which contained the BgI II and the Xba I restriction site
respectively. After digestion with BgI II and Xba I, this fragment was
inserted into pT-P treated with the same restriction sites, resulting in
pT-P-C. -Transglucosidase DNA was amplied with sense primer
TGF containing the glucoamylase signal peptide sequence and
antisense primer TGR by RT-PCR using the total RNA of A. niger
TCCC41056 as template. These primers were designed according to
the DNA sequence of the A. niger mRNA for a-glucosidase in NCBI
(no. AB285123.1), and contained the Nco I and the BgI II restriction
site respectively. The resulting 3.0-kb fragment was cloned into vector
pT-P-C between the glaA promoter and the trpC terminator. This
plasmid was named pT-P--C. Then, binary vector pBI-Glu was
Construction of a Strain Producing -Transglucosidase 1861
constructed by insertion of a 4.4-kb Hind III/Xba I fragment from
pT-P--C containing the -transglucosidase gene expression cassette
into the Hind III/Xba I restricted pBI-hph plasmid. The sequences of
primers are listed in Table 1.
Construction and selection of the engineered strain. Binary vector
pBI-Glu, including the hph gene and the -transglucosidase expression
cassette, was transferred into A. tumefaciens LBA4404 via electro-
poration,
30)
and the positive transformants were used to infect A. niger
TCCC41056 by ATMT, which was established and optimized in the
above experiments for random insertional mutagenesis.
A. niger transformants were identied by PCR with sense primer
PgF and antisense primer TrR. The copy number of the -trans-
glucosidase gene in the A. niger transformants was determined by
Southern blot analysis according to the instructions in DIG High Prime
DNA Labeling and Detection Start Kit I, in addition to digestion of
genomic DNA from the putative transformants with Hind III.
To determine the activity of -transglucosidase, the spores of each
positive transformant and the wild-type strain were inoculated into
50 mL fermentation medium in 250 mL asks and shaken (180 r/min)
at 28

C for 3 d. -Transglucosidase from wild-type A. niger is

intracellular enzyme. Hence, the fermentation broth of wild-type strain
must be disrupted by ultrasonication. After centrifugation, the super-
natant consisted of crude enzyme solutions. The -transglucosidase
from the A. niger engineered strain was secreted into the fermentation
broth in view of the role of the signal peptide, and so the crude enzyme
solution was obtained only by centrifugation of the fermentation broth.
The activity of -transglucosidase was measured by the national
standard method of China (QB 2525-2001) with methyl--d-glucopyr-
anoside as substrate.
32)
According to the national standard method,
dierent concentrations of standard glucose solution (100 mg/mL,
200 mg/mL, 300 mg/mL, 400 mg/mL, and 500 mg/mL) were rst
prepared. Standard (0.1 mL) solutions were then mixed with 3 mL of
color reagent 4-aminoantipyrin in tubes. The tubes were incubated at
40 0:5

C for 20 min, and the absorbance of these mixtures was

measured at a wavelength of 500 nm with sterile water as control. A
standard curve was made with the concentrations of standard glucose
solution and the absorbance values as horizontal and vertical
coordinates respectively.
Methyl--D-glucopyranoside (1 mL) was mixed with 1 mL of
0.02 mol/L of acetic acid-sodium acetate buer and this was incubated
at 40 0:5

C for 10 min in a tube. Then, an appropriate concentration

of crude enzyme solution was added and mixed intensely, and this was
incubated at 40 0:5

C for 60 min. The tube was immediately

transferred to boiling water for 5 min to end the reaction. Finally, the
amount of resulting glucose was measured according to the standard
curve, as described above.
One unit (U) of the activity was dened as the amount of enzyme
catalyzes the production of 1 mmol of glucose per min.
Results and Discussion
Determination of antibiotic sensitivity
During the selection phase of ATMT, cephamycin
was used to inhibit the growth of A. tumefaciens. The
sensitivities of A. tumefaciens LBA4404 were tested,
and the results showed that the inhibition by cephamycin
of the growth of A. tumefaciens was gradually strength-
ened with increasing concentrations, and that growth
was completely inhibited when the concentration of
cephamycin reached 20 mg/mL. Hence, cephamycin at
20 mg/mL was chosen and used in the following
transformation experiments.
Hygromycin B, a very convenient dominant selection
marker, is widely used in fungal genetic transformation.
In this study, the sensitivity of A. niger was tested in
PDA medium with various concentrations of hygrom-
ycin B. The growth of A. niger was aected by the
increase in hygromycin B and completely inhibited in
medium when the concentration reached 100 mg/mL.
These results suggest that hygromycin B is a suitable
marker for A. niger, and that 100 mg/mL of hygromycin
B is an eective concentration for the selection of
transformants.
Construction of binary vector pBI-hph
In order to establish a transformation system for
A. niger mediated by A. tumefaciens, binary vector pBI-
hph was constructed and identied by PCR and enzyme
digestion analysis. Then the recombinant plasmid was
transferred into A. tumefaciens LBA4404 and positive
clones were used during ATMT.
Eect of spore freshness on transformation frequency
To analyze the eect of spore freshness on trans-
formation eciency, a spore suspension was prepared
from A. niger stored at 4

C refrigerator about two

weeks and a fresh strain cultivated at 28

C for 4 d. Then
the spore suspension (10
7
spores per milliliter) was
mixed with an equal volume of A. tumefaciens culture
(OD
600
0:6) and co-cultivated at 20

C for 1 d. Under
the same experimental conditions, it was obvious that
transformation eciency was higher for the fresh strain
used as host for ATMT (Table 2). Currently, there is
no research in this regard, and the main reason for
the dierence in transformation eciency is not very
clear. Perhaps the reason is the fresh spores can adapt
to the environment quickly and their growth cycle is
shorter.
Table 1. Sequences of Primers Used in This Study
Primer names Primer sequence (5
0
3
0
) Restriction enzyme
PgF GGGGGTACCAAGCTTGGATCCGAACTCCAACCGGGG Kpn I, Hind III
PgR CCGGCCATGGCTGAGGTGTAATGATGCTGGGG Nco I
TrF GAAGATCTGGGCCCATGTCAACAAGAATA BgI II
TrR GGGTCTAGAAAGAAGGATTACCTCTAAACAAGTG Xba I
TGF TTAACCATGGCGTTCCGATCTCTACTCGCCCTGAGCGGCCTCGTCTGCACA- Nco I
GGGTTGGCATCCACCACTGCCCCTTCGCA
TGR GAAGATCTCTACCATTCCAATACCCAGTTTTCCGCCC BgI II
hphF CATGCCATGGCTGAACTCACCGCGACGTC
hphR CGGGATCCCGCGGTCGGCATCTACTCTAT
Table 2. The Eect of Spore Freshness on Transformation Frequency
Spore freshness Number of transformants
Spores saved for 2 weeks 6 2
Fresh spores 26 3
Each value was the mean SD of triplicate experiments.
1862 M. LI et al.
Eect of the A. tumefaciens concentration on trans-
formation eciency
The eect of the A. tumefacien concentration on
transformation eciency was tested as follows: Four
dierent concentrations (OD
600
0:4, 0.6, 0.8, and 1.0)
were chosen and co-cultivated with fresh spore suspen-
sion. As shown in Fig. 1A, transformation eciency
improved with increases in the A. tumefaciens cell
concentration from 0.4 to 0.8. However, when the
concentration was above 0.8, the number of transform-
ants decreased. The increased bacterial concentration,
resulting in an overgrowth of A. tumefaciens, might
have led to fungal growth limitation. In addition,
massive growth of A. tumefaciens also gives rise to
diculty at the selection stage. Similar results have been
reported for Coleophoma empetri (OD
600
0:6)
33)
and
A. awamori (OD
600
0:8).
19)
These studies indicated
that a higher concentration of cells can improve trans-
formation eciency.
Eect of the spore concentration on transformation
eciency
The eect of the spore concentration was tested.
Transformation eciency consistently increased with
the increases in the spore concentration within a certain
range of concentration (Fig. 1B). But there was no
statistically signicant dierence in transformation
eciency when the spore concentration was at 10
7
spores per milliliter (55 transformants) or 10
8
spores per
milliliter (59 transformants). Thus, 10
7
spores per
milliliter was the optimal spore concentration in this
experiment. This result agrees with other ATMT results
for fungi. In Trichoderma atroviride, more transforma-
tions were obtained when the spore concentration was at
10
7
spores per milliliter.
34)
In the ATMT of A. japoni-
cus, the optimal spore concentration was 10
8
spores per
milliliter. Guo et al. found that transformation eciency
began to decrease at very high spore concentrations,
which may lead to nutrient limitation.
24)
Eect of co-culture temperature on transformation
eciency
Co-cultivation is a critical step in transformation. In
order to analyze the eect of co-culture temperature on
transformation eciency, three temperatures (20

C,
24

C, and 28

C) were tested. The results indicated that

the optimal temperature for this ATMT system was
24

C (Fig. 1C). The dierent eciencies among these

temperatures suggested that a relatively low temperature
during co-culture is benecial for A. tumefaciens to
transfer its T-DNA to the host, consistently with
Hebeloma cylindrosporum,
35)
Leptosphaeria macu-
lans,
36)
A. awamori.
19)
In the experiments of Michielse
et al., four temperatures (20

C, 22.5

C, 25

C, and
28

C) were tested. The results indicated that co-

cultivation at 20

C resulted in variable transformation

frequencies, which hampered meaningful statistical
analysis, while at 28

C, excessive growth of A. tume-

faciens resulted in a decrease in transformation e-
ciency.
19)
These studies indicate dierent temperatures
signicantly inuence the number of transformants and
background growth. Although 28

C was the optimal

temperature for the growth of A. tumefaciens, it was not
appropriate for the transformation mechanism. This
0
10
20
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40
50
60
70
80
90
100
T
h
e

n
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b
e
r

o
f

t
r
a
n
s
f
o
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m
a
n
t
s
Concentration of A. tumefaciens /OD
600
A
0
10
20
30
40
50
60
70
80
90
100
10
8
10
7
10
6
B
T
h
e

n
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m
b
e
r

o
f

t
r
a
n
s
f
o
r
m
a
n
t
s
Spore concentration of A. niger (spores/mL)
10
5 0.4 0.6 0.8 1.0
20 24 28
0
10
20
30
40
50
60
70
80
90
100
C
T
h
e

n
u
m
b
e
r

o
f

t
r
a
n
s
f
o
r
m
a
n
t
s
Co-culture temperature (C)
1 2 3 4
0
10
20
30
40
50
60
70
80
90
100
D
T
h
e

n
u
m
b
e
r

o
f

t
r
a
n
s
f
o
r
m
a
n
t
s
Co-culture time (d)
Fig. 1. Eects of Four Factors on Transformation Eciency.
Each value is the mean SD of triplicate experiments. A, A. tumefaciens concentration. One hundred microlitre of A. tumefaciens cell
suspension (OD
600
0:4, 0.6, 0.8, and 1.0) was mixed with 100 mL of fresh spore suspension (10
7
spores/mL) and then incubated on an IM agar
plate containing 200 mmol/L of AS at 20

C for 1 d. B, Spore concentration. One hundred microlitre of A. tumefaciens cell suspension

(OD
600
0:8) was mixed with 100 mL of fresh spore suspension (10
5
, 10
6
, 10
7
, and 10
8
spores/mL) and then incubated on an IM agar plate
containing 200 mmol/L of AS at 20

C for 1 d. C, Co-culture temperature. One hundred microlitre of A. tumefaciens cell suspension (OD
600
0:8)
was mixed with 100 mL of fresh spore suspension (10
7
spores/mL) and then incubated on an IM agar plate containing 200 mmol/L of AS at
20

C, 24

C, and 28

C for 1 d. D, Co-culture time. One hundred microlitre of A. tumefaciens cell suspension (OD
600
0:8) was mixed with
100 mL of fresh spore suspension (10
7
spores/mL) and then incubated on an IM agar plate containing 200 mmol/L of AS at 24

C for 1 to 4 d.
Construction of a Strain Producing -Transglucosidase 1863
exorbitant temperature might induce loss of the trans-
formation function of A. tumefaciens and thus lead to
the decreasing of transformation eciency.
Eect of co-culture time on transformation eciency
Transformation eciency is closely related with co-
culture time. In this study, optimal transformation
frequency was attained when the mixture was co-
cultivated for 2 d, and extending the incubation time
led to reduced transformation eciency (Fig. 1D).
Perhaps a decreased co-culture time goes against the
transfer of T-DNA, and increased co-culture time might
lead to Agrobacterium background growth. However, for
dierent fungi, the co-culture time in ATMT made a
signicant dierence. For Colletotrichum trifolii, in-
creased transformation frequency was obtained with a
shorter co-culture time (48 h),
37)
while in ATMT of
Laccaria bicolor, the optimal co-culture time was 108 h.
38)
Analysis of transformants
To determine whether the hph cassette was integrated
in the genome, ve putative transformants were selected
randomly and detected by PCR and Southern blot. The
PCR results revealed that 1-kb fragments were present in
all ve transformants, but not in the wild-type strain
(Fig. 2A). Southern blot was performed using the hph
gene as probe. As shown in Fig. 2B, all the resistant
strains harbored single hybridizing fragment signals
at dierent sites, with no signal at any position in the
wild-type strain. These results indicate that hygromycin
B resistant clones were obtained by ATMT.
Mitotic stability of the transformants
In order to conrm the stability of the transformants,
20 hygromycin B resistant clones obtained by ATMT
were selected randomly and grown in PDA (hygromycin
B-free) medium for 10 successive generations. Then
they were transferred to resistant plate (PDA medium
containing 100 mg/mL of hygromycin B) and cultured
for 4 d. The genomic DNA of these transformants was
extracted and tested by PCR. The results showed that the
hph resistances of all the transformants were heritable
and stable (date not shown).
Establishment of ATMT for A. niger
Based on the above described results, a transforma-
tion system for A. niger mediated by A. tumefaciens was
established and optimized. One hundred microlitre of
fresh spore suspension (10
7
spores per milliliter) was
mixed with an equal volume of A. tumefaciens cell
suspension (OD
600
0:8) and spread onto lter paper
placed on IM plates (200 mmol/L AS added). After co-
cultivation at 24

C for 2 d, the lter paper was trans-

ferred onto PDA agar plates containing hygromycin B
(100 mg/mL) and cephamycin (20 mg/mL) to select
transformants. Transformation eciency reached 83
transformants per 10
7
spores by the established ATMT
for A. niger in this study, which improved by 16 times as
compared with that previously reported.
21)
Construction of binary vector pBI-Glu
According to Materials and Methods, P
glaA
, -
transglucosidase, and the trpC terminator gene were
cloned and sequenced respectively. The sequencing
results showed that the cloned sequences were consistent
with the reported sequences, excepting that the -
transglucosidase gene did not contain signal sequence.
Then binary vector pBI-Glu was constructed by cloning
P
glaA
, -transglucosidase, and the trpC terminator gene
into pBI-hph.
In improving the production of heterologous gene
products in lamentous fungi, the promoter, an essential
part of the expression vector in genetic engineering, has
a great inuence on the expression levels of heterolo-
gous proteins. The P
glaA
cloned from glucoamylase-
producing A. niger UV-11, which can drive the ex-
pression of foreign genes eectively, is frequently used
to construct expression vectors. It can be used to
construct industrial strains, too. Hence, P
glaA
was cloned
into pBI-Glu to drive the expression of -transglucosi-
dase gene to a high level.
-Transglucosidase derived from A. niger is an intra-
cellular enzyme, and it is dicult to extract. In this
research, the glucomylase signal peptide was used to
guide the intracellular enzyme to the extracellular
matrix. To some extent, this might reduce costs in
industrial production. Thus the glucoamylase signal
peptide sequence was fused to the N-terminus of the
-transglucosidase gene.
Construction of the engineered strain
A. tumefaciens LBA4404, harboring binary vector
pBI-Glu, was used to infect A. niger TCCC41056 by the
optimized transformation method. A. niger transform-
ants, obtained by ATMT, were selected and conrmed
by PCR. The results revealed that 4.4-kb fragments
A B
Fig. 2. Identication and Analysis of A. niger Transformants.
A, PCR identication of A. niger transformants. 1, DNA marker; 2, wild type; 37, transformants. B, Southern blot analysis of A. niger
transformants. 13, 56, transformants; 4, wild type.
1864 M. LI et al.
(including the sequences of P
glaA
, -transglucosidase
and terminator of trpC) were present in all transform-
ants, but not in the wild-type strain (data not shown).
Then, 400 positive transformants, conrmed by PCR,
were randomly selected to analyze mitotic stability, and
230 mitotically stable transformants were obtained
(about 57%). This suggested that the -transglucosidase
gene in most of the transformants was heritable and
stable.
Expression of -transglucosidase in the engineered
strain
All stable transformants and the wild-type strain were
cultured in fermentation medium for 3 d, and then the
enzyme activity was measured (data not shown). Among
the transformants, the productivity of -transglucosidase
of 13 transformants was higher than that of the wild-type
strain (Fig. 3). This indicates that genetic engineered
strains with high enzyme activity can be obtained by
ATMT. As shown in Fig. 3, it was obvious that highest
activity reached 25.02 U/mL, about 12.1-fold of the
control strain. One of the reasons for the high expression
level of -transglucosidase in the engineered strains
might be the multiplication of a region in the DNA with
a strong promoter. The enzyme assay showed that -
transglucosidase was secreted into the extracellular
matrix by the glucoamylase signal peptide, conrming
that the glucoamylase signal peptide contributed to the
secretion of -transglucosidase in the A. niger engi-
neered strain.
In previous studies, the -transglucosidase gene was
cloned and expressed in dierent hosts. Nakamura et al.
cloned the -transglucosidase gene from A. niger GN-3
and introduced it into A. nidulans, but the expression
level in the transformants (highest activity 18 mU/mg)
was lower than in A. niger GN-3 (21 mU/mg).
17)
The
activity was still lower when the same gene was inserted
into Emericella nidulans JVM10259, at only 0.962
mU/mg.
13)
When the -transglucosidase gene from A.
niger GN-3 was cloned into the same A. niger strain, the
activity of the recombinant -transglucosidase rose
240 mU/mg.
16)
Based on previous research results, there
is no doubt that enzyme activity is higher with A. niger
used as host. Therefore, in order to compare enzyme
activities, the activity of -transglucosidase from A.
niger A-8 was determined by the method of Lee et al.
16)
The result, that the enzyme activity was 57 U/mg,
suggests that A. niger engineered strain A-8 is poten-
tially valuable for producing high yields of -trans-
glucosidase.
The 13 transformants were then tested by Southern
blot using the -transglucosidase expression cassette
(including the sequences of P
glaA
, -transglucosidase,
and the terminator of trpC) as probe, and the wild-type
strain was used as control. The genome from each strain
was hybridized with the entire -transglucosidase
expression cassette gene. As shown in Fig. 4, all the
transformations had 12 -transglucosidase fragment
signals at various positions, without any signal at any
position in the wild-type strain. Twelve transformants
were single copies and only one transformant was a
double copy. A-1, A-2, A-6, and A-9 produced hybrid-
izing fragments of approximately the same size.
Based on the above results, the highest enzyme
activity was obtained from double-copy transformant
A-8 at 25.02 U/mL. It can be seen that the insertion of
foreign gene can aect the expression mechanism to a
certain extent. In fact, many studies have found that the
expression is inuenced by gene copy number and insert
locus. Gene dosage has an important eect on the
expression of heterologous proteins. Some experiments
found that one copy of the foreign protein expression
cassette was sucient for expression, and others that an
increase in copy number had a positive eect on
expression.
39,40)
Sreekrishna et al. integrated 20 copies
of the tnf gene into the chromosome of yeast, and the
expression of recombinants was 200 times that of the
single-copy strain.
41)
When Pichia pastori was used as
host for the expression of Bovine lysozyme, the
expression level decreased with increasing of copy
numbers, from 0.46 g/L (one copy) to 0.25 g/L (three
copies).
42)
Therefore, there is no denite conclusion as
to whether the inuence of gene dosage on the
expression of a heterologous protein is a positive eect
or a negative one, but in this study, the copy number
of -transglucosidase gene proved benecial to the
expression.
In addition, single-copy transformants A-1, A-2, A-6,
and A-9 showed almost the same activity (12.26 U/mL,
11.82 U/mL, 11.75 U/mL, and 11.09 U/mL respec-
tively), whereas the activity of -transglucosidase from
the other single-copy transformants was dierent. These
dierences suggest that the integration site of the foreign
gene also aected the expression level. The insert locus
of the foreign gene in the fungal genome is so random
that any chromosome can be used as an integration site
and the sites are dierent in the same chromosome.
When a foreign gene was transferred to another species
and inserted into the chromosome, the change in the
structure of host genome aected the expression of the
host and foreign genes.
43)
Kong and Liu concluded that
A A-1 A-2 A-3 A-4 A-5 A-6 A-7 A-8 A-9 A-10 A-11 A-12 A-13
0
5
10
15
20
25
30
T
h
e

a
c
t
i
v
i
t
y

o
f

-
t
r
a
n
s
g
l
u
c
o
s
i
d
a
s
e

(
U
/
m
L
)
Fig. 3. Determination of -Transglucosidase Activity.
Each value represents the mean SD of triplicate experiments. A,
wild type; A-1A-13, transformants.
Fig. 4. Southern Blot Analysis of A. tumefaciens Wild-Type and
Transformants.
A-1A-13, transformants; A, wild type.
Construction of a Strain Producing -Transglucosidase 1865
the chromosomal microenvironment near the insert
locus inuences the activity of the promoter of foreign
gene, thereby aecting the expression level.
44)
On the
basis of the above results, dierent insertion sites lead to
dierence in enzyme activity, but the reason for these
dierences needs future research on the molecular level.
Conclusions
In this study, we established an ecient ATMT
system for A. niger. The factors aecting transformation
eciency were optimized, and the highest transforma-
tion eciency was obtained (83 transformants per 10
7
spores) when a fresh spore suspension (10
7
spores per
milliliter) was co-cultivated with an equal volume of
A. tumefaciens (OD
600
0:8) in IM medium at 24

C
for 2 d. By this transformation method, -transglucosi-
dase-producing engineered strains were constructed. The
enzyme activity of mutant A-8 was 12.1-fold of the
wild-type strain. Thus the ATMT method is not only an
eective method for the transformation of lamentous
fungi, but also an ecient tool for random integration in
A. niger. Besides, ATMT provides a favorable reference
for gene engineered strain breeding. More importantly,
the A. niger engineered strain is potentially useful for
producing high yields of -transglucosidase.
Acknowledgments
This work was supported by the Tianjin Research
Program of the Application Foundation and Advanced
Technology Program (no. 11JCYBJC 09600), the Key
Technology Research and Development Program of
Tianjin, China (no. 11ZCKFSY00900), the Program for
Changjiang Scholars and the Innovative Research Team
in University (IRT1166), the National Natural Science
Foundation of China (no. 21176190), and the Chinese
National Program for High Technology Research and
Development (2013AA102106).
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