ISSN : 0974 - 7435
Volume 12 Issue 1
BioTechnology An Indian Journal FULL PAPER BTAIJ, 12(1), 2016 [039-044]
Functional evaluation of ToxA promoter in Trichoderma reesei RutC30
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
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 RutC30. 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 importance[1]. It has been successfully employed for decades in the cellulolytic enzyme production and it is currently used as a host to recombinant proteins production[2]. T. reesei is an interesting expression system because it has the capacityto grown in cheap fermentation systems and culture media with the particular 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 successfully in the production of several recombinant proteins 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]. However, vectors used for the production of these proteins 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
40
Functional evaluation of ToxA promoter in Trichoderma reesei Rut-C30
BTAIJ, 12(1) 2016
FULL PAPER
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
C30 and T. reesei Rut-C30-GFP to a final concentration of 1x106 spores/mL and incubated in an orbital shaker at 28°C, 250 rpm for 48 hours. The E. coli DH5áwere grown in Luria-Bertani medium containing 0.1 mg/mLampicillin for 24 h at 37°C and 250 rpm. Agrobacterium tumefaciensLBA1100 were grown in minimal media according to protocol described by Pardoand coworkers[18].
Trichoderma transformation and selection
Conidia were resuspended in 0.08% Tween 80, counted and immediately employed for the transformation following the protocol described by Michielse[19].
Protein extracts preparation and quantification
The mycelia were recovered from the liquid cultures 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.
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 resistance gene (nptIII) and the backbone of mini binary vector pCB301. The pCBCT vector was amplified 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-
BioTechnology An Indian Journal
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 performed 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 antiGFP (rabbit) and secondary (IRDye® 800CW antirabbit, 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 (LICOR Biosciences)
Fluorescence microscopy
A portion of mycelium was hydrated with distilled water and analyzed by microscopy using a fluorescence microscope Nikon Eclipse E600 equipped with SPOT-RT camera. The merged images were obtained by superposition of corresponding channels using the ImageJ v1.44 software.
BTAIJ, 12(1) 2016
Ortiz E.Gastón et al.
41
FULL PAPER
Measurement of green fluorescent protein by fluorometry
For measurement of green protein,T. reesei RutC30 strains were culture and processed according the protocol described by Dandan Lv[13]. The mycelia 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 temperature and blocked with 1% skim milk in TBS0.1% 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 (LICOR Biosciences)
RESULTS AND DISCUSSION
Previous reports have shown the correct expression of GFP gene under control of different promoters in the filamentous fungus T. reeseiRut-C30[11].
To assess the ability of ToxA promoter to drive expression of the green fluorescent protein, the binary pCBCT vector was transformed into T. reeseiRutC30. 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 microscopy studies were carried out to evaluate the cellular localization and distributionof GFPprotein in selectedT. reesieRut-C30transformants. The fungal transformants showed a GFPcytoplasmatic localization with a regular fluorescent signal along all hyphaestructure,but some of this hyphae display a higher expression of GFP in growing hypha tips Figure 1B. These results indicate that transformants were able to express the GFP gene under ToxA promoter.
To confirm the presence of GFP in T. reeseiRutC30 transformants Western blot analysis was performed. For this purposeintracellular fungal extracts were electrophoresed and electroblotted as described 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
BioTechnology An Indian Journal
42
FULL PAPER
Functional evaluation of ToxA promoter in Trichoderma reesei Rut-C30
BTAIJ, 12(1) 2016
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
transfer of total intracellular proteins into the nitrocellulose membrane prior to antibodies hybridization 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 consistent 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 recombinant GFP purified from E. coli extract Figure 2. These results indicate that GFP protein is produced in theT. reeseiRut-C30 as a single in tracellularly protein without presence of aggregates.
To assess promoter strength, a fluorometric quantitative 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 intensities obtained were compared with data previously 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 promoter 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
BioTechnology An Indian Journal
BTAIJ, 12(1) 2016
Ortiz E.Gastón et al.
43
FULL PAPER
TABLE 1 : The comparative study of total intracellular protein production by the parent strain T. reeseiRut-C30 and the recombinant strain
T. reesei strain RutC30
RutC30-GFP RutC30 F1*
Intracellularfluorescent (fluorescentunits) 55 5342 66 42061
Fluorescent ratio (Transformant / wild type) 97 637
Reference In thiswork
13
*F1 is a T. reesei RutC30 transformed with pWEF31-red vector
analysis of GFP level expression was conducted.
As shown in Figure 3, GFP content is 0.1% of total
REFERENCES
intra cellular protein. In T. reeseiRut-C30 the extra
cellular protin is 8% of total proteomeand CBHI [1] I.S.Druzhinina, Komoñ M.Zelazowska,
protein represent 60% of extracellular protein[21, 22].
L.Atanasova, V.Seidl, C.P.Kubicek; Evolution and
From these values, it is possible to estimate that
ecophysiology of the industrial producer Hypocrea
CBHI protein amount is around 4.8% of proteome,
jecorina (Anamorph Trichoderma reesei) and a new
this value is significantly higher than the values obtained for GFP expression under control of ToxA pro-
[2]
sympatric agamospecies related to it, PLoS One, 5, e9191 (2010). M.Onnela; Production of recombinant proteins in
moter.
the filamentous fungus trichoderma reesei sirkka ker
CONCLUSION
inen and merja penttil I, Biotechnology, 534–537 (1995).
[3] A.Schuster, M.Schmoll; Biology and biotechnology
From the results presented, is possible to con-
of Trichoderma, Appl.Microbiol.Biotechnol., 87,
clude thatToxA promoter is functional in T. reesei
787–99 (2010).
Rut-C30. In addition, the study of the expression [4] F.Meng, D.Wei, W.Wang; Heterologous protein ex-
level suggesting that ToxA promoter is working in
pression in Trichoderma reesei using the cbhII
this strain as a weak promoter and can be employed as good promoter for co-expression of accessory
[5]
promoter.Plasmid, 70, 272–6 (2013). V.V.Joutsjoki, T.K.Torkkeli, K.M.Nevalainen; Transformation of Trichoderma reesei with the
proteins as chaperon proteins. The results presented
Hormoconis resinae glucoamylase P (gamP) gene:
here extend the repertoire of functional promoters
production of a heterologous glucoamylase by Tri-
described forT. reesei Rut-C30.
choderma reesei, Curr.Genet., 24, 223–8 (1993).
[6] Margolles E.Clark, C.K.Hayes, G.E.Harman,
ACKNOWLEDGMENTS
M.Penttilä; Improved production of Trichoderma harzianum endochitinase by expression in Tricho-
This work was funded by grant2010 SJ10/
derma reesei, Appl.Environ.Microbiol., 62, 2145–
31from theUniversidad Nacional de San
51 (1996).
Martin(issued to M. Blasco) and PICT Start Up
[7]
P.;Murray et al.; Expression in Trichoderma reesei and characterisation of a thermostable family 3 beta-
2010-1312 grant from the National Agency for Sci-
glucosidase from the moderately thermophilic fun-
ence and Technology Promotion from the National
gus Talaromyces emersonii, Protein Expr.Purif., 38,
Ministry of Science and Technology of Argentina
248–57 (2004).
(issued to Dr. E. Albertó). We are grateful to Dr. [8] L.L.Kiiskinen et al.; Expression of melanocarpus
Pardo (University of Quilmes, Argentina) for kindly
albomyces laccase in trichoderma reesei and char-
providing the pCBCT vector. We are grateful to
acterization of the purified enzyme, Microbiology,
M.Sc. Fernanda who edited the final version of the
150, 3065–74 (2004).
manuscript.
[9] B.C.Salles et al.; Identification of two novel xylanaseencoding genes (xyn5 and xyn6) from
BioTechnology An Indian Journal
44
Functional evaluation of ToxA promoter in Trichoderma reesei Rut-C30
BTAIJ, 12(1) 2016
FULL PAPER
Acrophialophora nainiana and heterologous expression of xyn6 in Trichoderma reesei, Biotechnol.Lett., 29, 1195–201 (2007). [10] V.G.Gupta et al.; Biotechnology and Biology of Trichoderma, Elsevier B.V., (2014). [11] G.Zou et al.; Construction of a cellulase hyper-expression 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; Construction of a promoter collection for genes co-expression 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 biology, 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 functional GFP expression, Mycol.Res., 111, 850–5 (2007). [16] J.Sambrook, D.W.Russell; Molecular cloning:Alaboratory manual (fourth edition), Book 2344 (CSHL Press, 2001), at <http://www.amazon.com/Molecular-Cloning-Laboratory-Edition-Three/dp/ 1936113422>
[17] M.Mandels, J.Weber, R.Parizek; Enhanced cellulase 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 Aspergillus awamori, Nat.Protoc., 3, 1671–8 (2008).
[20] R.M.Andrie, J.P.Martinez, L.M.Ciuffetti; Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes, Mycologia, 97, 1152–61 (2005).
[21] T.M.Pakula et al.; Monitoring the kinetics of glycoprotein 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).
BioTechnology An Indian Journal
Ver+/-
