Functional evaluation of ToxA promoter in Trichoderma reesei Rut-
C30
Ortiz E.Gastón1*, Albertó Edgardo1, Blasco Martín2
1Instituto de Investigaciones Biotecnológicas, Instituto Tecnológico Chascomús (IIB–INTECH, UNSAM–
CONICET), Universidad de San Martín, Av. 25 de Mayo y Francia, Campus UNSAM, San Martín, 1650 Buenos
Aires, (ARGENTINA)
2Centro de Investigación y Desarrollo en Biotecnología Industrial, Instituto Nacional de Tecnología Industrial,
Av. General Paz 5445, Ediûcio 51, San Martín, Buenos Aires, (ARGENTINA)
E-mail: gas.ortiz@gmail.com
FULL PAPER
ABSTRACT
In order to expand the collection of promoters studied in Trichoderma
reesei Rut-C30, a functional study of the ToxA promoter was carried out by
analysis of GFP expression in T. reesei Rut-C30. For this purpose, the
binary pCBCT expression vector was employed to transform T. reesei Rut-
C30. The transformants obtained were evaluated by means of fluorescence
microscopy, fluorometry, dot blot and Western blot analysis. The low levels
of cytoplasmic GFP protein in fungal hyphae suggest that ToxA promoter
works as a weak constitutive promoter which can drive successfully the
expression of heterologous proteins in T. reesei Rut-C30
 2016 Trade Science Inc. - INDIA
KEYWORDS
TrichodermareeseiRut-C30;
Recombinant protein expres-
sion;
ToxA promoter;
Weak promoter.
INTRODUCTION
The genus Trichoderma is composed of several
species of filamentous fungi whose natural habitat
is the soil. Of all species included in this genus,T.
reesei is the species with major industrial impor-
tance[1]. It has been successfully employed for de-
cades in the cellulolytic enzyme production and it is
currently used as a host to recombinant proteins pro-
duction[2]. T. reesei is an interesting expression sys-
tem because it has the capacityto grown in cheap
fermentation systems and culture media with the par-
ticular ability to generate and secrete large amounts
of recombinant protein with conventional eukary-
otic post-translational modifications[3, 4]. Particularly,
the mutant T. reesei Rut-C30 has been used success-
fully in the production of several recombinant pro-
teins of different origins, for example
glucoamylase[5], endochitinase[6], â-glucosidase[7],
laccase[8], xylanases [9] fromfungalorigin;
bacterialxylanases; vegetable endopeptidases;
bovinechymosin and human erythropoietin[10]. How-
ever, vectors used for the production of these pro-
teins are based on the use of strong promoters such
as cbh1, cbh2, or their modifications. This small set
of promoters available, restricted the plasticity of
the expression system in T. reesei[11, 13]. In order to
overcome this drawback, numerous efforts have been
BTAIJ, 12(1), 2016 [039-044]
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An Indian Journal
Volume 12 Issue 1ISSN : 0974 - 7435
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40 Functional evaluation of ToxA promoter in Trichoderma reesei Rut-C30
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BTAIJ, 12(1) 2016
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focused upon the search fornew functional promot-
ers in T. reesei[10].
The ToxA promoter controls the expression of
toxin A in the fungus Pyrenophoratritici-repentis,
this promoter has been successfully used for consti-
tutive expression of recombinant proteins in vari-
ous fungi such as Colletotrichum magna, P. tritici-
repentis, Sclerotiniasclerotiorum,
Colletotrichumtrifolii, Verticilliumdahliae,
Alternariaalternata, Botrytis cinerea,
Cochliobolussativus, Fusarium sambucinum[14].
Taking into account the need to find new func-
tional promoters in T. reesei Rut-C30 and consider-
ing that ToxA promoter has shown functionality in a
broad host range; it was decided to study it func-
tionality inT. reesei Rut-C30. Due to this, the aim of
this work is to study the functionality of ToxA in T.
reesie Rut-C30 and expand the repertoire of pro-
moters studied in this strain.
MATERIALS AND METHODS
Expression vector
The binary plasmid pCBCT[15] was used forT.
reesei Rut-C30 transformation, mediated by
Agrobacteiumtumafaciens. This plasmid contains the
following elements: hygromycin B resistance gene
(hph) under control of the AspergillusnidulanstrpC
promoter, the gene of green fluorescent protein
(GFP) under control of ToxA promoter from
Pyrenophoratritici-repentis, the left (LB) and right
(RB) borders of T-DNA from A. tumefaciens, the
origin of replication (RK2 oriV), the neomycin re-
sistance gene (nptIII) and the backbone of mini bi-
nary vector pCB301. The pCBCT vector was am-
plified in Escherichia coli DH5á and isolated from
it using the plasmid DNA preparation described by
Sambrook[16].
Microorganisms and culture conditions
Forconidiation, T. reesei Rut-C30 was grown at
28°C in agarose medium potato glucose. The conidia
were collected using a solution of 80% Tween 0.05%
and counted in a Neubauer chamber. For expression
assays, Mandels[17] liquid medium supplemented with
2 g/L glucose was inoculated with T. reesei Rut-
C30 and T. reesei Rut-C30-GFP to a final concen-
tration of 1x106 spores/mL and incubated in an or-
bital shaker at 28°C, 250 rpm for 48 hours. The E.
coli DH5áwere grown in Luria-Bertani medium con-
taining 0.1 mg/mLampicillin for 24 h at 37°C and
250 rpm. Agrobacterium tumefaciensLBA1100
were grown in minimal media according to proto-
col described by Pardoand coworkers[18].
Trichoderma transformation and selection
Conidia were resuspended in 0.08% Tween 80,
counted and immediately employed for the transfor-
mation following the protocol described by
Michielse[19].
Protein extracts preparation and quantification
The mycelia were recovered from the liquid cul-
tures by filtration. Then, 2 g of that wet biomass
was milled using liquid nitrogen and mortar. Finally,
the resulting powder was resuspended in 1 mL of
distilled water and stored until use at -20°C. Total
protein was quantified using the Bradford method.
SDS-PAGE and Western blot:
Protein extracts were diluted in loading buffer
and 20 µg of total protein per lane was loaded on a
gel 10% SDS-PAGE, the electrophoresis was per-
formed at 160 V for 1 h. Subsequently, the proteins
were transferred to a nitrocellulose membrane and
the transfer verified by Ponceau red staining. The
membrane was washed with water, blocked with 1%
skim milk in TBS-0.1% Tween 20 during 60 min at
room temperature and incubated with primary anti-
GFP (rabbit) and secondary (IRDye® 800CW anti-
rabbit, LI-COR Biosciences) antibody diluted
1:1000 and 1:10000 in blocking buffer with 0.05%
Tween 20 respectively. For immunodetection the
membrane was analyzed using infrared scanner (LI-
COR Biosciences)
Fluorescence microscopy
A portion of mycelium was hydrated with dis-
tilled water and analyzed by microscopy using a fluo-
rescence microscope Nikon Eclipse E600 equipped
with SPOT-RT camera. The merged images were
obtained by superposition of corresponding chan-
nels using the ImageJ v1.44 software.

Ortiz E.Gastón et al. 41
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BTAIJ, 12(1) 2016
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Measurement of green fluorescent protein by
fluorometry
For measurement of green protein,T. reesei Rut-
C30 strains were culture and processed according
the protocol described by Dandan Lv[13]. The myce-
lia suspension was analyzed by fluorometry for GFP
expression using a microplate reader (Filtermax F5;
Molecular Device, LLC.).
Dot Blot analysis
Protein extracts and purified GFP protein were
diluted in phosphate saline buffer and 2 µL of each
sample were dropped in nitrocellulosic membrane
(Bio-Rad). The membrane was dried at room tem-
perature and blocked with 1% skim milk in TBS-
0.1% Tween 20 during 60 min at room temperature.
Finally, the membrane was incubated with primary
anti-GFP (rabbit) and secondary (IRDye® 800CW
anti-rabbit, LI-COR Biosciences) antibody diluted
1:1000 and 1:10000 in blocking buffer with 0.05%
Tween 20 respectively. For immunodetection the
membrane was analyzed using infrared scanner (LI-
COR Biosciences)
RESULTS AND DISCUSSION
Previous reports have shown the correct expres-
sion of GFP gene under control of different promot-
ers in the filamentous fungus T. reeseiRut-C30[11].
To assess the ability of ToxA promoter to drive ex-
pression of the green fluorescent protein, the binary
pCBCT vector was transformed into T. reeseiRut-
C30. After transformation, the positive transformants
of T. reeseiRut-C30 were selected for their ability
to grow in the presence of hygromycin in the growth
medium. One of these positive transformants was
selected toGFP expression confirmation byblue light
test. As shown in Figure 1A, the morphology shown
by thetransformant is similar to wild-type
strainsuggesting that the integration of the T-DNA
cassette in the fungus genome does not disrupt genes
essential for fungus viability. Fluorescence micros-
copy studies were carried out to evaluate the cellu-
lar localization and distributionof GFPprotein in
selectedT. reesieRut-C30transformants. The fungal
transformants showed a GFPcytoplasmatic localiza-
tion with a regular fluorescent signal along all
hyphaestructure,but some of this hyphae display a
higher expression of GFP in growing hypha tips Fig-
ure 1B. These results indicate that transformants
were able to express the GFP gene under ToxA pro-
moter.
To confirm the presence of GFP in T. reeseiRut-
C30 transformants Western blot analysis was per-
formed. For this purposeintracellular fungal extracts
were electrophoresed and electroblotted as de-
scribed above (see materials and methods)
andthePonceau staining was performed to verify the
Figure 1 : Fluorescence analysis for GFP expression under the ToxA promoter regulation. A: Blue light test, right
panels T. reeseiRut-C30 transformed withpCBCT in left panels T. reeseiRutC-30 untransformed. B: Fluorescence
microscopy, upper panels T. reeseiRut-C30 transformed withpCBCT in lower panels T. reeseiRut-C30
untransformed

42 Functional evaluation of ToxA promoter in Trichoderma reesei Rut-C30
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transfer of total intracellular proteins into the nitro-
cellulose membrane prior to antibodies hybridiza-
tion Figure 2B. After Western blotting the membrane
was infrared scanned and the resulting signal showed
the presence of an unique band of 26.9 kDa consis-
tent with the GFP molecular weight in the fungal
transformant extract Figure 2A. It is important to note
thepresence of dimer aggregates (53.8 kDa)in re-
combinant GFP purified from E. coli extract Figure
2. These results indicate that GFP protein is pro-
duced in theT. reeseiRut-C30 as a single in
tracellularly protein without presence of aggregates.
To assess promoter strength, a fluorometric quan-
titative assay was performed. For this purpose, the
wild type strain and the recombinant strain were
grown and processed under conditions previously
reported byDandan Lv[13]. The GFP fluorescence was
measuredby means of fluorometric assay and the in-
tensities obtained were compared with data previ-
ously reported for recombinant T. reeseiRut-C30
expressing DsRed2 under control of CBHI strong
promoter[13]. As shown in TABLE 1 the strength of
CHBI promoter results 6.5 fold higher than ToxA pro-
moter in T. reesie Rut-C30. In discordance with
previouslyreported for ToxA promoter strength in
other fungus genus, this promoter work as a weak
promoter in T.reeseiRut-C30[20]. To confirm the
strength of ToxA promoter, a quantitative dot blot
Figure 2 : Expression analysis of GFP by Western blot. A: Western blot using anti-GFP. B:Ponceau red staining. In
both cases, lane 1- Recombinant GFP expressed in E. coli, lane 2- protein extract from T. reeseiRut-C30
untransformed, lane 3- protein extract from T. reeseiRut-C30 transformed with pCBCT
Figure 3 : Quantification of intracellular GFP content. A: Dot blots using specific antibody (anti-GFP). Lane 1: 6
mg/L of standard purified GFP (Std) and undiluted sample (S). Lane 2: Std (5 mg/L) and sample diluted 1/5. Lane 3:
Std (4 mg/L) and sample dilution 1/10. Lane 4: Std (3 mg/L) and sample dilution 1/5. Lane 5: Std (2 mg/L) and
sample dilution 1/10. Lane 6: Std (1 mg/L). C: Calibration curve plotted from the intensities values obtained by
quantification of signal for each standard spot. D: The chart shows the values obtained for the sample and their
percentage in the proteome

Ortiz E.Gastón et al. 43
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analysis of GFP level expression was conducted.
As shown in Figure 3, GFP content is 0.1% of total
intra cellular protein. In T. reeseiRut-C30 the extra
cellular protin is 8% of total proteomeand CBHI
protein represent 60% of extracellular protein[21, 22].
From these values, it is possible to estimate that
CBHI protein amount is around 4.8% of proteome,
this value is significantly higher than the values ob-
tained for GFP expression under control of ToxA pro-
moter.
CONCLUSION
From the results presented, is possible to con-
clude thatToxA promoter is functional in T. reesei
Rut-C30. In addition, the study of the expression
level suggesting that ToxA promoter is working in
this strain as a weak promoter and can be employed
as good promoter for co-expression of accessory
proteins as chaperon proteins. The results presented
here extend the repertoire of functional promoters
described forT. reesei Rut-C30.
ACKNOWLEDGMENTS
This work was funded by grant2010 SJ10/
31from theUniversidad Nacional de San
Martin(issued to M. Blasco) and PICT Start Up
2010-1312 grant from the National Agency for Sci-
ence and Technology Promotion from the National
Ministry of Science and Technology of Argentina
(issued to Dr. E. Albertó). We are grateful to Dr.
Pardo (University of Quilmes, Argentina) for kindly
providing the pCBCT vector. We are grateful to
M.Sc. Fernanda who edited the final version of the
manuscript.
T. reesei
strain
Intracellularfluorescent
(fluorescentunits) Fluorescent ratio (Transformant / wild type) Reference
RutC30 55
RutC30-GFP 5342
97 In thiswork
RutC30 66
F1* 42061
637 13
TABLE 1 : The comparative study of total intracellular protein production by the parent strain T. reeseiRut-C30
and the recombinant strain
*F1 is a T. reesei RutC30 transformed with pWEF31-red vector
REFERENCES
[1] I.S.Druzhinina, Komoñ M.Zelazowska,
L.Atanasova, V.Seidl, C.P.Kubicek; Evolution and
ecophysiology of the industrial producer Hypocrea
jecorina (Anamorph Trichoderma reesei) and a new
sympatric agamospecies related to it, PLoS One, 5,
e9191 (2010).
[2] M.Onnela; Production of recombinant proteins in
the filamentous fungus trichoderma reesei sirkka ker
inen and merja penttil I, Biotechnology, 534–537
(1995).
[3] A.Schuster, M.Schmoll; Biology and biotechnology
of Trichoderma, Appl.Microbiol.Biotechnol., 87,
787–99 (2010).
[4] F.Meng, D.Wei, W.Wang; Heterologous protein ex-
pression in Trichoderma reesei using the cbhII
promoter.Plasmid, 70, 272–6 (2013).
[5] V.V.Joutsjoki, T.K.Torkkeli, K.M.Nevalainen;
Transformation of Trichoderma reesei with the
Hormoconis resinae glucoamylase P (gamP) gene:
production of a heterologous glucoamylase by Tri-
choderma reesei, Curr.Genet., 24, 223–8 (1993).
[6] Margolles E.Clark, C.K.Hayes, G.E.Harman,
M.Penttilä; Improved production of Trichoderma
harzianum endochitinase by expression in Tricho-
derma reesei, Appl.Environ.Microbiol., 62, 2145–
51 (1996).
[7] P.;Murray et al.; Expression in Trichoderma reesei
and characterisation of a thermostable family 3 beta-
glucosidase from the moderately thermophilic fun-
gus Talaromyces emersonii, Protein Expr.Purif., 38,
248–57 (2004).
[8] L.L.Kiiskinen et al.; Expression of melanocarpus
albomyces laccase in trichoderma reesei and char-
acterization of the purified enzyme, Microbiology,
150, 3065–74 (2004).
[9] B.C.Salles et al.; Identification of two novel xylanase-
encoding genes (xyn5 and xyn6) from

44 Functional evaluation of ToxA promoter in Trichoderma reesei Rut-C30
FULL PAPER
BTAIJ, 12(1) 2016
BioTechnology
An Indian Journal
Acrophialophora nainiana and heterologous expres-
sion of xyn6 in Trichoderma reesei, Biotechnol.Lett.,
29, 1195–201 (2007).
[10] V.G.Gupta et al.; Biotechnology and Biology of Tri-
choderma, Elsevier B.V., (2014).
[11] G.Zou et al.; Construction of a cellulase hyper-ex-
pression system in Trichoderma reesei by promoter
and enzyme engineering, Microb.Cell Fact., 11, 21
(2012).
[12] W.Wang, F.Meng, P.Liu, S.Yang, D.Wei; Construc-
tion of a promoter collection for genes co-expres-
sion in filamentous fungus Trichoderma reesei,
J.Ind.Microbiol.Biotechnol., 41, 1709–18 (2014).
[13] D.Lv, W.Wang, D.Wei; Plasmid construction of two
vectors for gene expression in trichoderma reesei,
Plasmid, 67, 67–71 (2012).
[14] J.A.Rollins et al.; Fungal biology MINIREVIEW
green fluorescent protein is lighting up fungal biol-
ogy, doi:10.1128/AEM.67.5.1987, (2001).
[15] M.Gorfer, S.Klaubauf, D.Bandian, J.Strauss;
Cadophora finlandia and Phialocephala fortinii:
Agrobacterium-mediated transformation and func-
tional GFP expression, Mycol.Res., 111, 850–5
(2007).
[16] J.Sambrook, D.W.Russell; Molecular cloning: A labo-
ratory manual (fourth edition), Book 2344 (CSHL
Press, 2001), at <http://www.amazon.com/Molecu-
lar-Cloning-Laboratory-Edition-Three/dp/
1936113422>
[17] M.Mandels, J.Weber, R.Parizek; Enhanced cellu-
lase production by a mutant of Trichoderma viride,
Appl.Microbiol., 21, 152–4 (1971).
[18] A.G.Pardo, M.Hanif, M.Raudaskoski, M.Gorfer;
Genetic transformation of ectomycorrhizal fungi
mediated by Agrobacterium tumefaciens,
Mycol.Res., 106, 132–137 (2002).
[19] C.B.Michielse, P.J.J.Hooykaas, Van Den Hondel,
C.a M.J.J., A.F.J.Ram; Agrobacterium-mediated
transformation of the filamentous fungus Aspergil-
lus awamori, Nat.Protoc., 3, 1671–8 (2008).
[20] R.M.Andrie, J.P.Martinez, L.M.Ciuffetti; Develop-
ment of ToxA and ToxB promoter-driven fluores-
cent protein expression vectors for use in filamen-
tous ascomycetes, Mycologia, 97, 1152–61 (2005).
[21] T.M.Pakula et al.; Monitoring the kinetics of glyco-
protein synthesis and secretion in the filamentous
fungus Trichoderma reesei: cellobiohydrolase I
(CBHI) as a model protein, Microbiology, 146(Pt
1), 223–32 (2000).
[22] T.M.Pakula, K.Salonen, J.Uusitalo, M.Penttilä; The
effect of specific growth rate on protein synthesis
and secretion in the filamentous fungus Trichoderma
reesei, Microbiology, 151, 135–43 (2005).