Cloning and Expression of Endoglucanase genes from Trichoderma

Journal of Applied Sciences Research, 4(11): 1546-1556, 2008
© 2008, INSInet Publication
Cloning and Expression of Endoglucanase genes from
Trichoderma species in Saccharomyces cerevisiae
1
Mala C. Ganiger, 1Dr. Sumangala Bhat, 2Pranav Chettri, and 3M.S. Kuruvinashetti
Institute of Agri-Biotechnology, University of Agricultural Sciences,
Krishinagar, Dharwad -580005, Karnataka, India.
Abstract: The present study was conducted to isolate and clone the endoglucanase gene from Trichoderma
harzianum and to study the expression of endoglucanase genes cloned from different species in
Saccharomyces cerevisiae (INVSc1 strain). Using specific primers, gene encoding β-1, 6 endoglucanase
(1.3 kb) from T. harzianum was cloned into pTZ57R/T vector. The clone was confirmed through PCR
and restriction analysis. The clone was sequenced and analyzed for the homology at the nucleotide and
at the protein level for domain analysis. Gene encoding β-1, 6 endoglucanase has shown 97% with
reported sequence both at the nucleotide and protein level. Endoglucanase genes cloned from different
species were expressed in Saccharomyces cerevisiae using pYES2/CT vector. The recombinant clones of
T. reesei has shown three times more, T. harzianum and T. virens has shown twice the activity when
compared to control INVSc1 having pYES2/CT vector. Further, the SDS-PAGE analysis of the
recombinant clone β-1, 6 endoglucanase from T. harzianum has shown the presence of corresponding to
β-1, 6 endoglucanase.
Key words: T. harzianum, β-1, 6 endoglucanase, Saccharomyces cerevisiae, INVSc1, SDS-PAGE
INTRODUCTION
Intensified use of fungicides has resulted in the
accumulation of toxic compounds potentially hazardous
to human and environment [8 ] and also in the buildup of
resistance of the pathogens [1 4 ]. In order to tackle these
national and global problems, effective alternatives to
chemical control are being investigated and the use of
antagonistic microbes seems to be one of the promising
approaches[9 ]. Antagonism may be accomplished by
competition, parasitism, antibiotics, or by a combination
of these modes of action [1 1 ,3 3 ]. Parasitism involves the
production of several hydrolytic enzymes that degrade
cell walls of pathogenic fungi[1 9 ]. Some species of
Trichoderma
have
been described as biological
control agents against several fungal plant pathogens [2 8 ].
T he d egradatio n and further assimila tion of
phytopathogenic fungi, namely, mycoparasitism, has
been proposed as the major mechanism accounting for
the antagonistic activity of Trichoderma species against
fungal pathogens [6 ]. From recent work, it appears that
Trichoderma mycoparasitism is a complex process
involving several successive steps [7 ]. Initially, the
mycoparasite grows directly towards its host and often
coils around it or attaches to it by forming hook-like
structures and ap resso ria [ 1 7 ] . F o llo wing these
interactions, Trichoderma spp. sometimes penetrates the
host mycelium, apparently by partially degrading its
cell walls [1 8 ]. Finally, it is assumed that Trichoderma
spp. utilize the intracellular contents of the host[1 8 ].
Chitin and β-1, 3-glucan are the main structural
components of fungal cells walls, except those from
members of the class Oomycetes, which contain β-1, 3glucan and cellulose [4 ]. Thus, chitinases (EC 3.2.1.14)
and β-1, 3-glucanases (EC 3.2.1.39), proteins secreted
by Trichoderma spp., have been suggested as the
key enzymes in the lysis of phytopathogenic fungal
cell
walls during mycoparasitic action [1 2 ,1 5 ,6 ,3 1 ].
However, other cell wall-degrading enzymes, including
those hydrolyzing minor polymers (proteins, β-1, 6glucans, α-1, 3-glucans, etc.), may be involved in the
effective and complete degradation of mycelial or
c o nid ia l w a lls of phytopa thoge nic fungi by
Trichoderma spp. A subtilisin-type serine proteinase
induced by chitin has already been described in a
mycoparasitic strain of Trichoderma harzianum [2 0 ].
Also, β-1, 6- glucanases (EC 3.2.1.75) have been
shown to lyse yeast and fungal cell walls in
filamentous fungi [3 2 ,3 4 ] and bacteria [2 9 ] and the recent
studies has shown the expression of β-1, 6- glucanases
from T. harzianum in yeast[1 3 ]. These enzymes have
been shown to be produced by several fungi and
bacteria and may be an important factor in biological
control[1 ,2 7 ].
W ith this background information an effort was
made to clone β-1, 6 endoglucanase namely pSGH
Corresponding Author: Mala C. Ganiger, Institute of Agri-Biotechnology, University of Agricultural Sciences, Krishinagar,
Dharwad -580005, Karnataka, India.
E-mail: [email protected]
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J. Appl. Sci. Res., 4(11): 1546-1556, 2008
fro m T. h a r zia n u m . Furthermo re, the yeast
Saccharomyces cerevisiae has been used as the host for
the expression and comparison of the fungus
endoglucanase genes from different species, with yeast
vector pYES2/CT.
M ATERIALS AND M ETHODS
M ethods: Cloning of β-1, 6 endoglucanase. Genomic
DNA was prepared from T. harzianum using the
protocol of Hedgedas and Khazhatourians (1996). The
β-1, 6 endoglucanase gene was cloned from T.
harzianum using the PCR method with the gene
specific primers (Forward primer, 5’GCAAGCTTCCA
TCAAGATGAAGTACTC3’, and Reverse primer,
5’G CG CG G CCG CCAAT CACTCGTGAT TTACC3’).
The PCR conditions were denaturing at 94 °C for 5
minutes, annealing at 54°C for 1 minute 30 seconds
and extension at 72°C for 1 minute with final
extension at 72°C for 45 minutes. The purified PCR
product of 1.3 kb (50 ng/µl) were ligated to pTZ57R/T
vector (2886 bp) as described in InsT/A clone™ PCR
product cloning kit (#k1214) from MBI, Fermentas
USA. The clone was named as pSGH. Further, the
clones were confirmed through PCR and restriction
analysis (Figure 1).
Sequencing: The nucleotide sequence of β-1, 6
endoglucanase (pSGH) cloned in pTZ57R/T was
determined using M13 primers employing primer
walking technique at Bangalore Genei, Private Ltd.,
Bangalore. The sequences were subjected to analysis
using
BLAST
algorithm
available at
http://www.ncbi.nim.gov.
Expression of the Recombinant Protein in Brewer’s
yeast (Saccharom yces cerevisiae): The glucanase genes
were cloned into the vector pYES2/CT (Invitrogen
Catsbad, CA) for inducible expression in Brewer’s
yeast. Yeast expression plasmids were constructed as
follows. The β-1, 4 endoglucanase from T. reesei
(already av ailab le, 8 9 4 b p ) an d the β-1, 6
endoglucanase from T. harzianum cloned in this study
were digested with Hind III and Not I and β-1, 6
endoglucanase from T. virens (already available,
1.3 kb) was digested with Bam HI and Not I restriction
enzymes, present in pTZ57R/T cloning vector.
Accordingly, expression vector pYES2/ CT was also
digested with the same restriction enzymes to generate
compatible cohesive ends. T he glucanase gene
fragments were ligated with pYES2/CT vector at 16°C
for 24 hrs and transformed into E. coli DH5α by CaCl2
method. The construct map of all the three
recombinants is shown in Figure 2. Plasmids from E.
coli transformants were isolated following the method
of Brinboin and Doly [5 ]. Further, the recombinants were
analysed for the presence of the insert with the
respective restriction enzymes and PCR confirmation
(Figure 3).
The positive clones obtained were used for
endoglucanase expression in INVSc1 Brewer’s yeast
cells (MATα his 3∆1 leu2 trp1-289 ura3-52/MATα his
3∆ leu2 trp1-289 ura3-52/ura3-52 Invitrogen). These
clones were transformed into Saccharomyces cerevisiae
INVSc1 using Li acetate method [2 1 ]. As a control, yeast
cells were also transformed with the vector pYES2/CT.
The positive clone were isolated and maintained on
SC-U medium with 2% glucose.
Isolation of the Glucanase Recombinant Plasmid
from B rewer’s Yeast: Two loops full of the Brewer’s
yeast transformant cells growing on yeast peptone
dextrose
medium were suspended in 200 µl of
breaking buffer (10 mM Tris-HCl, pH8.0, 1mM
Na 2 EDTA, 100 mM NaCl, and 0.1% [w/v] SDS). Glass
beads were added, and the cells were lysed by
vortexing for 5 minutes. An equal volume of phenol:
chloroform: isoamyl alcohol (25:24:1, v/v) was
added and mixed by vortexing for 60 seconds. The
mixture was centrifuged and the aqueous layer
was extracted with an equal volume of chloroform.
The plasmid in the aqueous layer was extracted. The
plasmid was analysed by PCR confirmation (Figure 4).
Induction for Expression by Galactose: Briefly, a
single colony of INVSc1 containing recombinant
pYES2/CT (pYESH, pYESR and pYESV) along with
control (pYES2/CT without gene) was inoculated into
50ml of appropriate SC selective medium containing
2% glucose. It was grown overnight at 30°C with
shaking
at 200 rpm. Next day, the cells were
harvested at 4°C at the rate of 13,000 rpm for 1
minute. The supernatant was discarded and the cells
were resuspended in 200 ml of SC-U liquid medium
containing 2 per cent raffinose along with 10mg/lit
adenine. The mixture was kept at 30°C with shaking at
the rate of 200 rpm for three days. After three days of
shaking the cells were centrifuged at 13,000 rpm for 1
minute at 4°C. The supernatant was discarded and the
cells were resuspended in 50 ml of induction medium
having 2 per cent galactose. The mixture was kept at
30°C with shaking at the rate of 200 rpm for three
days. The samples were centrifuged at 4°C for 1
minute at the rate of 13,000 rpm. The supernatant and
the cell pellets were stored at -20°C for further use.
Biochemical M ethods: Glucanase activity was
determined by using supernatant and cell lysates. A
general protocol for small-scale preparation of cell
lysates using acid-washed glassbeads was followed [2 ].
To prepare the cell lysates frozen cells were used.
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J. Appl. Sci. Res., 4(11): 1546-1556, 2008
Fig. 1: PCR and restriction Confirmation of β- 1, 6-endoglucanase (pSGH) from T. harzianum in pTZ57R/T
Lane - 1 Plasmid, Lane - 2-5 Amplification and Lane - 6 Restriction
M - Lambda DNA / Eco R I / Hind III Double Digest Bangalore Genei Cat no. # 105694
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J. Appl. Sci. Res., 4(11): 1546-1556, 2008
Fig. 2: Construct map of (a) pYESH containing full length β-1, 6-endoglucanase gene from T. harzianum (b)
pYESR containing full length β-1, 4-endoglucanase gene from T. reseei and (c) pYESV containing full
length β-1, 6-endoglucanase gene from T. virens in pYES2/CT yeast vector
The frozen cell pellet was resuspended in 500 µl of
breaking buffer (10 mM Tris-HCl, pH8.0, 1mM
Na 2 EDTA, 100 mM NaCl, and 0.1% [w/v] SDS).
The cells were centrifuged at 13,000 rpm for 5
minutes at 4°C. The supernatant was discarded and the
cells were resuspended in 200 µl of breaking buffer.
An equal volume of acid washed glass beads were
added. The mixture was vortexed for 30 seconds,
followed by 30 seconds on ice. This was repeated six
times for a total of six minutes to lyse the cells.
The mixture was centrifuged for 10 minutes at 13,000
rpm at 4°C. The supernatant was removed and
transferred to a fresh centrifuge tube.
Protein determination was done by the method
described by the Lowry. 50 µl each of supernatant and
lysed product from induced cells of all the clones
(pYESH, pYESR, pYESV and pYES2/CT) was taken
and total protein content was determined using
spectrophotometer at 660nm.
The endoglucanase activity was assayed as
laminarase and CMCase activity with laminarin and
CMC as substrate in 50mM acetate buffer (pH=4.8) at
45°C for 30 minutes. The amount of released sugar
was measured by 3’, 5’- dinitrosalicylic acid method
described by Katatny et al.,[2 4 ]. One unit of enzyme
activity was defined as the amount of enzyme that
released 1µmol of glucose equivalent per minute.
Electrophoresis: Sodium dodecyl sulfate-polyacrylmide
gel electrophoresis was carried out following the
procedure outlined in Sambrook and Russell[3 0 ]. SDSPAGE was done using Hoefer system with 4%
acrylmide stacking and 12% acrylmide separating
gels in accordance with the manufacturer instructions.
The gels were stained with Coomassie brilliant blue to
visualize the proteins.
RESULTS AND DISCUSSION
C loning and Sequence analysis of β-1, 6endoglucanase: U nder the experimental conditions
described, a specific band of 1284bp was successfully
amplified from T. harzianum chromosomal DNA,
which was confirmed by sequencing. This band
was subcloned into pTZ57R/T cloning vector, clones
were
confirmed
through
PCR and restriction
analysis. Finally, the clone was checked by sequencing.
The DNA fragment was identified as β-1, 6endoglucanase, since the nucleotide sequence was
found to have 97 per cent homology with known
sequences in database. The full length nucleotide
sequence of β-1, 6-endoglucanase gene was deposited
at NCBI database and has accession number
EU747838.
In cloned gene, 1197 bp corresponds to the unique
open reading frame (Figure 5) which codes for 399
amino acids with a predicted molecular mass of 44
kDa. The conserved domain search at NCBI has
indicated that the cloned β-1, 6 endoglucanase from
T. harzianum belongs to Glycosyl hydrolase family 55
(Figure 6). Also the amino acid alignment of pSGH
with the already availabled clone β-1, 6- endoglucanase
from T. virens was done which is shown in
Figure 7. The remarkable similarity of these amino acid
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J. Appl. Sci. Res., 4(11): 1546-1556, 2008
Fig. 3a: Lane-1 Restriction, Lane-2 control, Lane-3 Amplified gene, Lane-4-5 PCR Amplification M - 1Kb DNA
ladder Bangalore Genei Cat no. # 105678
Fig. 3b(I): Lane- 1-4 PCR Amplification M - Lambda DNA / Eco R I / Hind III Double Digest Bangalore Genei
Cat no. # 105694
Fig. 3b(ii): Lane- 1 Restriction M - Lambda DNA / Eco R I / Hind III Double Digest Bangalore Genei Cat no.
# 105694
Fig. 3c(I): Lane - 1-12 PCR Amplification M- Lambda DNA / Eco R I / Hind III Double Digest Bangalore
Genei Cat no. # 105694
Fig. 3c(ii): Lane - 1 Restriction, Lane 2- Control M - 1Kb DNA ladder Bangalore Genei Cat no. # 105678
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J. Appl. Sci. Res., 4(11): 1546-1556, 2008
Fig. 4:
PCR Confirmation of the Mobilised Recombinant pYES2/CT in Yeast Host INVSc1 (a) pYESH (b)
pYESR and (c) pYESV
Fig. 5: Open Reading Frame of T. harzianum β -1, 6-endoglucanase (pSGH)
Fig. 6: Conserved Domain of T. harzianum β -1, 6 endoglucanase(pSGH)
sequences indicated the similar substrate affinities of
this enzyme. In addition, the multiple alignment of β-1,
6-endoglucanase from T. harzianum with the other
organisms viz., plant pathogenic fungi, bacteria and
plants has shown the conserved regions of M -S-S-K-W D-S-G, D-D-H-N-Y-I-G-A, K-I-R-G-V-N-G-G-W , N-TA-R-G-K-R (Figure 8). These regions might be
important for β-glucanases of Glycosyl hydrolase
family 55 belonging to β-1, 3-glucanases, β-1, 6glucanase, cellulases and so on [2 3 ,1 0 ]. The marked boxes
show the conserved regions.
Expression of the Cloned Endoglucanase Genes in
Yeast: T he recombinant strains obtained showed the
high quantity of reducing sugar production, indicating
that the Trichoderma enzyme was secreted in active
forms by the yeasts. The control strain, S. cerevisiae
INVSc1 transformed with vector pYES2/CT, showed
less endoglucanase activity comparatively (Table1
and 2). Though the total crude protein was high in
pYESH, amount of reducing sugars released was
relatively low. This may be because of different
proteins like chitinase, glucanase and many others
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Table 1:
Total protein content in 50µl of supernatant and lysed product of different Trichoderm a species
Total Protein (µg/m l)
Species
---------------------------------------------------------------------------------------------Supernatant
Lysate
INVSc1 with pY ES2/CT
61
23
-----------------------------------------------------------------------------------------------------------------------------------------------------------pY ESH -Y
75
34
-----------------------------------------------------------------------------------------------------------------------------------------------------------pY ESR-Y
67
23
-----------------------------------------------------------------------------------------------------------------------------------------------------------pY ESV-Y
47
25
Table 2: Reducing sugar released per 50µl of protein by different clones
Reducing Sugar (µg /m l)
-------------------------------------------------------------------------------------------------------------------Lam inarin
Carboxym ethyl Cellulase
Species
-----------------------------------------------------------------------------------------------Supernatant
Lysate
Supernatant
Lysate
INVSc1 with pY ES2/CT
360
161
-----------------------------------------------------------------------------------------------------------------------------------------------------------pY ESH -Y
687
630
-----------------------------------------------------------------------------------------------------------------------------------------------------------pY ESR-Y
908
803
-----------------------------------------------------------------------------------------------------------------------------------------------------------pY ESV-Y
520
169
-
Fig. 7: Amino acid sequence alignment of β-1, 6-endoglucanase gene from T. harzianum and T. virens.
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Fig. 8: Multiple alignment of amino acid sequence of the
sequences present in the database.
β-1, 6-endoglucanase from T. harzianum with the
Lane 1-11: β- 1, 6-endoglucanase (pYESH) from T. harzianum, Lane 13-14 Control (pYES2/CT) in INVSc1
MPM W M Medium Range Bangalore Genei Cat no. # 105979
contributing to total protein content, percentage of
glucanase may be high in the total crude protein
from pYESR. Similar observations were made
earlier[2 6 ]. It can be concluded that, as different species
may differ in their potential to degrade glucan, the
glucanase enzyme secreted by T. reesei may be having
more potential to degrade carboxymethyl cellulose
compared to glucanase enzyme secreted by other
Trichoderma species. Therefore, it may be necessary to
purify glucanase from these culture filtrates in future to
have clear information on glucan degrading potentiality
of these species.
Glucanase activity was observed even in the
control (yeast with pYES2/CT). This is because S.
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cerevisiae contains a wide range of endo- and exo-1,
3-β-glucanases and β-1, 6-glucanases and about 15
genes are known to encode polypeptides with glucanase
or related enzymatic activities [3 ]. Some of these
glucanases have roles during cell separation, while
others exhibit transglycosylase activity and may be
involved in extending and rearranging 1, 3-β-glucan
and 1, 6-β-glucan chains, and cross linking these
polymers to other wall components.
Finally, the concentrated lysed culture was
analyzed for β-1, 6- endoglucanase protein from T.
harzianum by SDS-PAGE. The apparent molecular
masses of β-1, 6- endoglucanase produced by the
yeasts were approximately 44 kDa in S. cerevisiae
indicating expression of the cloned gene (Figure 9).
At the present time, we have cloned and expressed
the gene corresponding to β -1, 6-glucanase, named
bgn 16.2 [2 5 ] and that corresponds to the 44-kDa
glucanase from T. harzianum. So, the introduction of
these genes into a biological antagonist strain which
strongly expressed them could be useful in the
development of a transgenic T. harzianum with
enhanced mycoparasitic activity.
8.
9.
10.
11.
12.
13.
14.
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