differential involvement of -glucosidases from hypocrea jecorina
TRANSCRIPT
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Differential involvement of β-glucosidases from Hypocrea jecorina 1
in rapid induction of cellulase genes by cellulose and cellobiose 2
3
Qingxin Zhou1, 2, Jintao Xu1, Yanbo Kou, Xinxing Lv, Xi Zhang, Guolei Zhao, Weixin Zhang, 4
Guanjun Chen, Weifeng Liu 5
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No.27 Shanda South Road, State Key Laboratory of Microbial Technology, School of Life 7
Science, Shandong University, Jinan 250100, Shandong, P. R. China 8
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Running title: β-Glucosidases in cellulase gene expression 13
Keywords: β-glucosidase; cellulase; Hypocrea jecorina; transcription induction; 14
cellobiohydrolase 15
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1 These authors contribute equally to this work 18
2 Present address: Institute of Agro-Food Science & Technology, Shandong Academy of 19
Agricultural Sciences, Jinan 250100, China 20
Correspondence should be addressed to Weifeng Liu. Tel.:+86 531 88364324; Fax: +86 531 21
88565610; e-mail: [email protected] (W. Liu) 22
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00170-12 EC Accepts, published online ahead of print on 21 September 2012
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Summary 23
Appropriate perception of cellulose outside the cell by transforming it into an 24
intracellular signal ensures the rapid production of cellulases by cellulolytic 25
Hypocrea jecorina. The major extracellular β-glucosidase BglⅠ(CEL3a) has been 26
shown to contribute to the efficient induction of cellulase genes. Multiple 27
β-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, 28
exist in H. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an 29
intracellular β-glucosidase displaying in vitro transglycosylation activity. We then 30
presented evidence that these two major intracellular β-glucosidases were involved 31
in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a 32
and cel1b significantly compromised the efficient gene expression of the major 33
cellulase gene, cbh1. Simultaneous absence of BglⅠ, CEL1a and CEL1b further 34
deteriorated the cellulase gene induction by cellulose. The induction defect, 35
however, was not observed with cellobiose. The absence of the three 36
β-glucosidases rather facilitated the induced cellulase synthesis on cellobiose. 37
Furthermore, addition of cellobiose restored the productive induction in the 38
deletion strains on cellulose. The results indicate that the three β-glucosidases may 39
not participate in transforming cellobiose beyond hydrolysis to provoke cellulase 40
formation in H. jecorina. They may otherwise contribute to the accumulation of 41
cellobiose from cellulose as inducing signals. 42
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Introduction 45
Cellulose is a linear polymer of β-1, 4-linked glucose molecules piled up into 46
highly ordered fibrillar structures. As the most abundant biomass from plant cell 47
wall, its microbial decomposition not only plays a key role in the carbon cycle in 48
nature, but also provides great potentials for a number of applications, most 49
notably biofuel production (17, 18). Since its initial isolation as a decomposer of 50
cellulosic materials, Hypocrea jecorina (anamorph Trichoderma reesei) has been 51
developed into one of the most prolific cellulase producer in industry. The 52
cellulase mixture of H. jecorina has been shown to consist of at least three types 53
of enzymes that act upon the insoluble substrate to achieve the efficient 54
conversion of native crystalline cellulose to glucose (18). Among others, it has 55
been widely accepted that endoglucanases (EC 3.2.1.4) and exoglucanases (EC 56
3.2.1.91) act synergistically upon cellulose to produce mainly cellobiose. This 57
disaccharide and other cello-oligosaccharides are further hydrolyzed to glucose by 58
β-glucosidases (EC 3.2.1.21). 59
Although the cellulases have been extensively characterized in H. jecorina, the 60
regulation of cellulase production in H. jecorina is still insufficiently understood. 61
For rapid launching of the cellulase machinery, H. jecorina has either to sense the 62
presence of the insoluble cellulose outside the cell or to detect the substrate by 63
uptake of the degradation products (14-16, 20). Although the precise nature of the 64
true ‘inducer’ has been elusive, several lines of evidence have been presented 65
pointing to a role for β-glucosidases in the rapid induction of the cellulase genes. 66
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First, slow feeding of cellobiose as the sole carbon source or inhibition of 67
extracellular hydrolysis of cellobiose by β-glucosidase leads to cellulase formation 68
(7, 15, 35). Second, it has been shown that the absence of the extracellular 69
β-glucosidase (BglⅠ) results in induction delay of the cellulase genes (6), while 70
recombinant strains bearing multicopies of the bgl1 gene display enhanced 71
cellulase induction not only by cellulose but also by sophorose, a potent inducer 72
potentially formed by the plasma-membrane-bound β-glucosidase activity from 73
cellulose degradation products via transglycosylation reactions (20, 33). Both 74
extracellular and intracellular β-glucosidases have been reported to exist in H. 75
jecorina (3, 10, 20). While BglⅠ belonging to glycosyl hydrolase family 3 (GH3) 76
has been considered to account for the majority of extracellular and 77
cell-wall-bound activities, BglⅡ (CEL1a) belonging to GH family 1 (GH1) has 78
been shown to be intracellularly localized (27). Moreover, five other β-glucosidase 79
sequences have been identified (5). Like bgl1, cel1a and cel1b are among the 80
highly induced transcripts upon growth on cellulose or sophorose (5). However, 81
their significance for cellulase gene regulation has not yet been investigated. 82
We report here some enzymatic properties and cellular localization of a second 83
GH1 β-glucosidase (CEL1b). We further report disruptions of the major 84
extracellular and intracellular β-glucosidase gene loci in H. jecorina. The 85
corresponding knockout strains were used to investigate the contribution of these 86
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β-glucosidase activities to the induction of cellulase genes by cellulose and 87
cellobiose. 88
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Materials and Methods 90
Strains, plasmids and medium 91
Hypocrea jecorina QM9414(ATCC 26921)and its uridine-auxotrophic derivative 92
TU-6 with a mutant pyr4 (ATCC MYA-256; (8)) were maintained on malt extract 93
agar (Sigma) supplemented with 10 mM uridine when necessary. Strains were 94
grown in 1 L Erlenmeyer flasks on a rotary shaker (200 rpm) at 30℃ in the 95
medium as described by Mandels and Andreotti (21). Carbon sources were used at 96
a final concentration of 10 g l-1. Escherichia coli DH 5α was used for routine gene 97
cloning and vector construction. 98
For expression of CEL1b and its mutant derivatives in Escherichia coli, the 99
coding sequence of cel1b was amplified from the cDNA of H. jecorina with 100
primers harboring EcoRI and HindIII sites and ligated into pET32a(+) after it was 101
digested with the same enzymes to obtain pET32acel1b. All the relative mutants 102
were obtained by oligonucleotide-mediated mutagenesis of the cel1b gene using a 103
two-step fusion PCR with pET32acel1b as the template. The mutated sites were 104
verified by sequencing before being subcloned into pET32a. For expression of 105
CEL1b-EGFP in H. jecorina to determine the subcellular localization of CEL1b, 106
the cel1b gene was inserted into the NcoI site of pIG1783 and fused in frame with 107
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the egfp coding sequence to obtain pIGcel1b (26). Oligonucleotides including 108
gene-specific primers used in this study for plasmid constructions, gene deletion 109
or probe preparation are listed in Table S1. 110
For the transcript and secreted protein analysis, strains were pregrown on 111
glycerol (1% v/v) for 48 h. Mycelia were harvested by filtration and washed twice 112
with medium without carbon source. Equal amounts of mycelia were transferred 113
to a fresh medium containing the respective carbon sources including Avicel or 114
cellobiose without peptone, and incubation was continued for the indicated time 115
period. For resting cell cultivations, H. jecorina was pregrown on glycerol 116
medium, and then washed extensively with the minimal medium lacking carbon 117
source, resuspended in the replacement medium lacking nitrogen (and therefore 118
enabling no growth) as previously described except that sophorose was used at a 119
final concentration of 1 mM (29). 120
Production of recombinant CEL1b in E. coli 121
For purification of CEL1b, E. coli strain with the cel1b expression construct was 122
grown at 37°C until OD600 reached 0.5~0.6. IPTG (isopropyl-β-D-thio 123
galactopyranoside) of a final concentration of 100 µM was added and the 124
incubation was continued for 16 h at 20°C. The CEl1b protein was purified with 125
Ni-nitrilotriacetic acid-agarose (Qiagen) essentially according to the instructions 126
of the manufacturer (Qiagen). 127
Disruption of the cel1a, cel1b and bgl1 genes of H. jecorina 128
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The 2.7 kb pyr4 fragment released from pFGI with either XbaI/SalI or SpeI/SalI 129
was ligated into the same sites of pUC19 to obtain pUCpyr4 or pUCpyr4-1. The 130
two 2 kb fragments upstream to the ATG codon and downstream to the stop codon 131
of cel1a or cel1b respectively, were amplified from H. jecorina chromosomal 132
DNA and ligated into the corresponding sites to yield the disruption vector 133
pUCcel1apyr4 or pUCcel1bpyr4. The 6.7 kb fragments containing the complete 134
pyr4 gene plus the 5’ and 3’ regulatory sequences of cel1a or cel1b were released 135
from pUCcel1apyr4 or pUCcel1bpyr4 with EcoRI/Hind III or XbaI/Hind III, and 136
were used to transform H. jecorina TU6. 137
To construct the Δcel1aΔcel1b deletion strain, a 3.0 kb amdS fragment released 138
from pALK424 by SpeI and SalI digestion was introduced into pUC19 to generate 139
pUCamdS (12). The 2.0 kb of 5’ and 3’ regulatory fragments of the cel1b gene 140
were inserted into pUCamdS, respectively, resulting in the cel1b deletion vector 141
pUCcel1bamdS. The deletion cassette was further released from pUCcel1bamdS 142
by XbaI and HindIII digestion and used to transform the Δcel1a strain in the same 143
way as did for TU6 except that the transformants were selected on plates 144
containing acetamide as sole nitrogen source. Simultaneous deletions of Bgl I 145
encoding gene cel3a were made by further disrupting cel3a in the Δcel1aΔcel1b 146
strain. A hygromycin resistance cassette containing the gpd 147
(glyceraldehyde-3-phosphate dehydrogenase) promoter from H. jecorina and 148
hygromycin resistance gene from pRLMex30 (19) was ligated into the NotI and 149
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SpeI sites of pUC19 to obtain pUChph. The 2.0 kb of 5’ and 3’ flanking sequences 150
of the cel3a gene were successively inserted into pUChph, resulting in 151
pUCcel3ahph. After linearization with SacI, the disruption vector was used to 152
transform the Δcel1aΔcel1b strain. Transformants were selected on minimal 153
medium containing 100 μg/ml hygromycin. To complement the Δcel1aΔcel1b 154
strain with CEL1b (I174C), the cel1b disruption vector was digested with SpeI and 155
StuI to remove the pyr4 gene followed by ligating with the hygromycin resistance 156
cassette digested with the same enzymes to create pUCcel1bhph. This plasmid 157
was digested with SpeI, dephosphorylated using shrimp alkaline phosphatase 158
(Takara), and further ligated with the coding region for Cel1b (I174C) amplified 159
from pET32acel1b (I174C). After linearization with HindIII, the fragment was 160
transformed into the Δcel1aΔcel1b strain. 161
Transformation of H. jecorina was carried out essentially as described by 162
Penttila (25). Transformants were selected on minimal medium either for uridine 163
prototroph or for resistance to hygromycin and acetamide. 164
Fluorescence microscopy 165
For visualization of CEL1b-EGFP, spores of recombinant strains harboring the 166
chromosome-integrated pIGcel1b were inoculated in the minimal medium and 167
grown for 20 h at 30°C. Mycelia were used directly for microscopic observation. 168
To simultaneously stain the nuclei, 100 μg ml-1 of DAPI (4’, 169
6-Diamidino-2-phenylindol Dihydrochlorid) solution in 50% glycerol was added. 170
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Fluorescence was detected with a Nikon Eclipse 80i fluorescence microscope and 171
images were captured and processed with the NIS-ELEMENTS AR software. 172
Nucleic acid isolation and hybridization 173
Fungal mycelia were harvested by filtration, washed with tap water, and frozen in 174
liquid nitrogen. Fungal genomic DNA was isolated according to the instructions of 175
E.Z.N.A.™ Fungal DNA Miniprep Kit (Omega Biotech, Doraville, USA). Total 176
RNA was isolated with the Trizol reagent (Invitrogen) according to the 177
manufacturer’s protocol. Southern hybridization and Northern analysis were 178
performed with the digoxigenin nonradioactive system from Roche Applied 179
Science as described previously (9). Relative transcription levels were analyzed 180
semiquantitatively by densitometry using the software ImageJ 181
(http:///rsb.info.nih.gov/ij). The values were normalized by densitometry of the 182
cbh1 signal to that of the 18S RNA control. 183
Quantitative RT-PCR 184
The total RNA was further purified with TURBO DNA-free kit (Ambion) 185
according to the manufacturer’s instructions. Reverse transcription was carried out 186
using the PrimeScript RT reagent Kit (Takara) according to the instructions. 187
Quantitative PCR were performed using a Bio-Rad myIQ2 thermocycler (Bio-Rad) 188
and the SYBR Green Supermix (Takara). Reactions were performed in triplicate 189
with a total reaction volume of 20 μL including 300 nM each of forward and 190
reverse primers and 100 ng template cDNA. Data analysis was performed using 191
the Relative Quantitation/Comparative CT (ΔΔCT) method and was normalized to 192
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the endogenous control actin with expression on glycerol as the reference sample. 193
Enzyme activity measurements 194
β-glucosidase activity was determined by measuring the amount of p-nitrophenol 195
released from p-nitrophenyl-b-D-glucopyranoside (pNPG) (Sigma) used as 196
substrates. The transglycosylation activity of CEL1b was measured as described 197
previously (27). CBH activity in the culture supernatants were measured using 198
p-nitrophenol-D-cellobioside (pNPC, Sigma) as a substrate (4). Cellular extracts 199
used for assay of intracellular β-glucosidase activity were prepared as follows: H. 200
jecorina strains were grown on Mandels-Andreotti medium with Avicel or 201
glycerol as the carbon source. The mycelia were harvested and washed twice with 202
0.9% NaCl. Lysates were prepared by grinding the mycelia into fine powder under 203
liquid nitrogen, which were then suspended into 50 mM Na-phosphate buffer (pH 204
7.0) with protease inhibitors. Cell debris was removed by centrifugation at 14, 000 205
g for 10 min. Determination of total Avicel hydrolysis activity was performed at 206
50℃ with 1% Avicel in 50 mM sodium acetate (pH 5.0) with equal amount of 207
extracellular culture filtrates relative to biomass. Culture broth supernatant was 208
buffer exchanged using a 10 kDa molecular weight cut off centrifugal filter to 209
remove any soluble sugars prior to initiating hydrolysis experiments. Sugars 210
released were determined by the dinitrosalicylic acid (DNS) method of Miller et al. 211
with D-glucose as standard (23). 212
HPLC analysis 213
Analysis of in vitro transglycosylation activity by HPLC was performed as follows: 214
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Samples were desalted by mixing with bed resin TMD-8 (Sigma, USA) and 215
vortexing for 1 min. Salt-free supernatants were applied onto a Bio-Rad Aminex 216
HPX-42A carbohydrate column and analyzed by LC-10AD HPLC (Shimadzu, 217
Japan), equipped with a RID-10A refractive index detector. The column was 218
maintained at 75℃ and eluted with double distilled water at a flow rate of 0.4 219
ml/min. 220
Protein analysis 221
SDS-PAGE and Western blotting were performed according to standard protocols 222
(28). Total secreted and intracellular proteins were determined using the method 223
of Bradford protein assay. Detection of cellobiohydrolase CBH1 was performed 224
by immunoblot using a polyclonal antibody raised against amino acids (426-446) 225
of CBH1 (1) 226
227
Results 228
CEL1b is an intracellular β-glucosidase with transglycosylation activities 229
CEL1a and CEL1b are members of glycosyl hydrolase family 1, both of which are 230
among the highly induced β-glucosidase genes in H. jecorina growing on cellulose 231
or on sophorose (5). CEL1a has been shown to be an intracellular enzyme 232
displaying not only hydrolytic properties but also transglycosylation activity in 233
vitro (27). Amino acid sequence comparison of CEL1b with CEL1a and two other 234
characterized GH1 β-glucosidases from Phanerochaete chrysosporium revealed 235
relatively high sequence identity (53% between CEL1a and CEL1b) (Fig. S1). 236
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Both CEL1a and CEL1b as well as BglⅠalso showed significant homology to the 237
recently identified GH1-1, GH3-3 and GH3-4 of Neurospora crassa, respectively 238
(Fig. S3 and S4). The modeled structure of CEL1b with the determined structure 239
of CEL1a as template showed that the overall structure of CEL1b was very similar 240
to that of CEL1a forming a classical (β/α)8 barrel with the active site being located 241
at the bottom of the pocket (Fig. S2) (11). Specifically, while the surrounding 242
residues at the glycone binding site are highly conserved, residues around the 243
aglycone binding site are highly divergent, which have been proposed to be the 244
basis of substrate specificity (24). Residues with smaller side chains at the 245
aglycone site have been also observed in BGL1B from P. chrysosporium which is 246
more efficient than BGL1A in hydrolysis of cellobiose (32). In order to 247
characterize the role of amino acids around the aglycone binding site on enzymatic 248
activity of CEL1b, several mutants with directed change of these divergent amino 249
acids either to residues with smaller side chains (I174 to C, H265 to A) or to the 250
corresponding residues in CEL1a or BGL1B (Y187 to L, D242 to H, and S442 to 251
A), were made and purified from soluble extracts of E. coli Origami B (DE3) (Fig. 252
1A). All of the purified proteins exhibited hydrolytic activity toward 253
p-nitrophenyl-β-D-pyranoside (pNPG), though there were apparent differences in 254
the kinetic parameters, as summarized in Table 1. Among others, change of Ile 174 255
to Cys significantly increased the hydrolytic efficiency probably due to a wider 256
entrance of the catalytic site resulted from the smaller side chain of cysteine. 257
These results suggest that residues around the entrance of catalytic site of CEL1b 258
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may not only be involved in determining the substrate specificity, but also play a 259
role in affecting the overall hydrolytic efficiency. Similar to CEL1a, recombinant 260
CEL1b exhibited significant transglycosylation activity when incubated with 261
relatively high concentrations of glucose and cellobiose (Fig. 1B and Fig. 1C). A 262
maximal yield of 189.6 mg l-1 was achieved for cellotriose when CEL1b was 263
incubated with 20% cellobiose. None of the mutants displayed higher activity of 264
transglycosylation than WT (data not shown). Note that we cannot exclude the 265
possibility of the existence of sophorose in the transglycosylation products. To 266
determine the subcellular localization of CEL1b, CEL1b-EGFP fusion protein was 267
expressed from a constitutive gpd promoter in H. jecorina QM9414 strain. 268
CEL1b-EGFP fluorescence was readily observed to be dispersed throughout the 269
cytosol (Fig. 1D). Taken together, these results indicate that CEL1b is mainly an 270
intracellular β-glucosidase with in vitro transglycosylation activities. 271
Disruption of cel1a and cel1b compromises induced production of cellulases 272
To probe further into the in vivo function of CEL1a and CEL1b, mutants of H. 273
jecorina lacking the coding sequences of cel1a or cel1b were obtained by targeted 274
gene replacement (Fig. 2A). A Δcel1aΔcel1b strain that lacks cel1a and cel1b was 275
also constructed. Anchored PCR and Southern blot analysis confirmed that each 276
deletion event had occurred as predicted, integrating only once within the H. 277
jecorina genome resulting in the removal of the expected coding sequences while 278
leaving the flanking sequences intact (Fig. 2B). Analysis of the intracellular 279
β-glucosidase activity demonstrated that, while the parent and Δcel1b strains 280
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exhibited only a slight difference in pNPG hydrolytic activity, the absence of 281
CEL1a resulted in a dramatic decrease in the intracellular β-glucosidase activities 282
in Δcel1a and Δcel1aΔcel1b strains to about 25% that of the wild-type strain 283
when cultured on cellulose (Fig. 3A). Simultaneous absence of CEL1a and CEL1b 284
also led to a significant upregulation of the extracellular β-glucosidase activities 285
over a longer period of induction (12 h). To further test the effect of the absence of 286
intracellular CEL1a and CEL1b on cell growth on different carbon sources, WT 287
and mutant strains were incubated on plates containing glucose or cellobiose. 288
While there was hardly any difference in growth between the parent and mutant 289
strains on glucose, disruption of cel1a and cel1b apparently affect the growth of H. 290
jecorina on cellobiose (Fig. 3B). Total extracellular protein production and 291
cellobiohydrolase (CBH) activity were further measured with the parent and the Δ292
cel1aΔcel1b strains with Avicel as the carbon source. Similar to results observed 293
for bgl1-deleted strain (6), the initial rate of both extracellular protein production 294
and exoglucanase activity was lower for the mutant strain relative to the parental 295
strain (Fig. 3C and 3D). Together, these results indicate that intracellular CEL1a 296
and CEL1b not only participate in the metabolism of cellulose degradation 297
products including cellobiose, but may also be involved in efficient induction of 298
the cellulases. 299
The absence of CEL1a and CEL1b results in a delay in the induction of cellulase 300
gene transcription by cellulose 301
To test whether CEL1a and CEL1b exert their effect on cellulase production at the 302
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transcriptional level, we examined the endogenous cbh1 mRNA by Northern blot 303
(Fig. 4). This analysis showed that, in comparison to the rapid induction in WT 304
strain which occurred as early as 3 h upon induction by cellulose, productive 305
activation of transcription of cbh1 was delayed by about 3 h and 21 h in Δcel1b 306
and Δcel1a strains, respectively (Fig. 4A-C). This lag in gene expression was 307
further extended to 36 h in the Δcel1aΔcel1b strain although the final level of 308
transcription was almost the same (Fig. 4D). Correspondingly, secretion of CBH1 309
into the culture supernatant was also delayed in strains with deletions of cel1a and 310
cel1b as assayed by Western blot (Fig. 4E). Complementation of the Δcel1aΔ311
cel1b strains with the CEL1b (I174C) significantly increased the intracellular 312
pNPG hydrolytic activity and partially improved the induction kinetics to a degree 313
more rapid than that of the Δcel1a strain (Fig. 4F). Notably, the observed delay in 314
transcription induction cannot be attributed to the differential growth on cellulose 315
caused by the potentially compromised metabolism of cellulose degradation 316
products in mutants because a similar delay in the gene expression was observed 317
in a resting-cell inducing system (Fig. 5A and 5B). Surprisingly, analysis of cbh1 318
transcription on induction with lactose revealed that a significant lag in cbh1 319
transcription also existed in the absence of cel1a as compared with the wild type 320
strain (Fig. 5C and 5D). 321
The disaccharide sophorose has been assumed as a putative natural inducer for 322
cellulose-mediated induction which has been detected in culture fluids of H. 323
jecorina (13, 34). To ask whether intracellular CEL1a and CEL1b would 324
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otherwise participate in the formation of the inducer, we investigated the effect of 325
sophorose on the efficacy of cbh1 induction (Fig. 5E and 5F). Similarly efficient 326
cbh1 transcription occurred both in the wild type and theΔcel1aΔcel1b strains in 327
response to sophorose. The findings suggest that, intracellular CEL1a and CEL1b 328
contribute to the induction of cellulase genes probably through participating in the 329
formation of cellulase inducer. It has been reported that extracellular β-glucosidase 330
BglⅠmay also be involved in the formation of such an inducer (6, 20) (Table S2). 331
Deletion of bgl1 in a cel1a and cel1b mutant strain further deteriorates the 332
induction on cellulose 333
To probe further into the relationship between BglⅠand the intracellular 334
β-glucosidases in modulating cellulase induction, we deleted bgl1 in the Δcel1aΔ335
cel1b strain ( Δ triβG) on the assumption that the absence of these three 336
β-glucosidases would not only significantly slow down the hydrolysis of 337
cellobiose, but also eliminate any putatively associated activities transforming 338
cellobiose. Analysis of the β-glucosidase activity demonstrated that the ΔtriβG 339
strain displayed a dramatic decrease in the extracellular β-glucosidase activities as 340
compared with that of wild-type and Δcel1aΔcel1b strains though its growth on 341
cellobiose was only similarly retarded as did the cel1a and cel1b mutants (Fig. 3A 342
and 3B). Analysis of secreted CBH1 in the presence of cellulose revealed that 343
productive formation of CBH1 was further compromised in the ΔtriβG strain than 344
in the Δcel1aΔcel1b strain with a slower kinetics and a lower level of CBH1 345
secretion (Fig. 6A). Quantitative RT-PCR analysis of cbh1 mRNA showed that a 346
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similar lag in cbh1 expression occurred in ΔtriβG strain (Fig. 6B). This result was 347
further corroborated by both the significantly lower cellobiohydrolase activities as 348
measured by pNPC hydrolysis and lower amount of secreted protein in the ΔtriβG 349
cultures over the period of induction by Avicel (Fig. 3C and 3D). These results 350
indicate that, the major extracellular and intracellular β-glucosidases act together 351
to ensure the efficient production of cellulases on Avicel. 352
Cellulase gene induction by cellobiose is not compromised in the absence of 353
β-glucosidases 354
As the major end-product of cellulose hydrolysis by the synergistic action of 355
endoglucanases and cellobiohydrolases, cellobiose has been shown to be able to 356
induce cellulase production. This has been achieved by simultaneous addition of 357
inhibitors of β-glucosidase or by slow feeding of cellobiose (7, 30). It has been 358
assumed that cellobiose is transformed to sophorose by the membrane-bound 359
β-glucosidase and endoglucanases via transglycosylation (27, 33, 34). To further 360
test whether the induction defect as observed in the β-glucosidase-deleted strains 361
was due to their inability to transform cellobiose, we investigated the effect of 362
absence of the major β-glucosidases on the efficacy of cbh1 induction by 363
cellobiose (Fig. 6C). While cellobiose plus δ-gluconolactone was usually used to 364
provoke cellulase induction in H. jecorina, cellobiose alone was found to be 365
capable of inducing cellulase formation in both WT and mutant strains though the 366
level of secreted proteins was lower than those induced by Avicel in WT (data not 367
shown). However, in comparison with WT, induced production of cellulases as 368
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represented by CBH1 occurred more efficiently in the presence of 0.25% 369
cellobiose in the cel1a and cel1b deletion strain (Fig. 6C). Similar to the Δcel1aΔ370
cel1b strain, simultaneous absence of BglⅠwas also capable of provoking efficient 371
formation of cellulases at relatively lower concentrations of cellobiose as 372
compared with WT. Therefore, in accordance with previous results, lowering the 373
degree of cellobiose hydrolysis may facilitate the stimulation of cellulase synthesis. 374
These results also suggest that further metabolism of cellobiose beyond hydrolysis 375
by major extracellular and intracellular β-glucosidases may not be involved in the 376
efficient induction of cellulase genes. 377
Addition of cellobiose restores the efficient induction on Avicel in β-glucosidase 378
disrupted strains 379
The above results implicate that the productive induction defect as observed in the 380
absence of the major β-glucosidases may to a larger extent be caused by the 381
insufficient cellobiose initially available for triggering the induction cascade. We 382
therefore asked whether the addition of cellobiose would rescue the induction 383
defect on Avicel as displayed in Δcel1aΔcel1b and Δ triβG strains. After 384
induction with 1% Avicel plus 0.25% cellobiose, there was a slight decrease in the 385
amount of secreted protein and Avicel hydrolysis activities in the wild-type strain 386
(Fig. 7A and 7C). In contrast, the Δcel1aΔcel1b and ΔtriβG cultures produced a 387
comparable amount of protein including CBH1 to that produced by WT during the 388
early induction on Avicel (Fig. 7A and 7B). In addition, the induced Δcel1aΔ389
cel1b and ΔtriβG cultures showed a significant increase in hydrolytic activity 390
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toward Avicel over this same period of induction (Fig. 7C). These data indicate 391
that β-glucosidases may influence the productive transcription of cellulase genes 392
on Avicel by assuring an appropriate level of cellobiose available for triggering the 393
induction cascade. 394
395
Discussion 396
Successful perception of the existence of insoluble cellulose outside the cell is 397
critical for cellulolytic H. jecorina to initiate the rapid production of the enzymatic 398
machinery needed to sustain its growth on the breakdown products. In this study, 399
we demonstrate that the absence of intracellular β-glucosidases CEL1a and CEL1b 400
significantly delays the cbh1 gene expression on crystalline cellulose. As well, we 401
show that simultaneous absence of the major extracellular β-glucosidase BglⅠ 402
builds on this defect by displaying a further delay in initiating the induction 403
process. However, we further demonstrate that deletion of these major 404
β-glucosidases has no effect on the productive induction of cellulase genes by 405
cellobiose, and that addition of cellobiose rescues the induction defect of the 406
mutant strains on Avicel. 407
It is reasonably believed that one mode of perception of the presence of 408
cellulose outside the cell would be sensing its degradation products (14, 16, 22). 409
Several lines of evidence have been indeed obtained that cellobiose or its close 410
relatives are capable of efficiently inducting the cellulases in H. jecorina (7, 30). 411
In all cases, β-glucosidase capable of both hydrolyzing and transglycosylating 412
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cellobiose has been thought to play an important role within the cascade regulating 413
cellulase formation. A balance regarding the β-glucosidase-mediated metabolism 414
of cellobiose has thus been suggested to exist which significantly influences 415
cellobiose’s role in provoking cellulase formation (7, 20). In this work, our initial 416
observations that the absence of three major β-glucosidases compromised the 417
efficient induction of cellulases, and that the productive transcription was largely 418
restored by sophorose, indicate a potential involvement of these intracellular 419
β-glucosidases in the rapid induction of cellulases by forming a cellulase inducer. 420
However, our further result that the efficacy of induction on cellobiose was not 421
compromised in these mutants argues against the previous surmise that the 422
induction defect on Avicel results from the inability to process cellobiose into a 423
cellulase inducer. To the contrary, the absence of these major β-glucosidases rather 424
seemed to facilitate the induction on cellobiose by displaying a more productive 425
response. The response of β-glucosidase-deleted H. jecorina strains to cellobiose 426
described here is consistent with that recently reported in Neurospora crassa (36), 427
and could be ascribed to sparing more cellobiose for induction while in the 428
meantime unmasking the inducing effect from the glucose-mediated catabolite 429
repression. Together with the findings that transglycosylation activities of 430
β-glucosidases are only observed in vitro with extremely high concentrations of 431
sugars (27, 33), the above results imply that participation of transglycosylation 432
activities from these β-glucosidases in forming cellulase inducers, if any, is not 433
relevant and therefore may not constitute a major part of mechanism for their 434
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involvement in efficient induction of cellulases. Note that we cannot at present 435
draw the conclusion that cellobiose itself is the physiological inducer since 436
possibility exists that proteins other than β-glucosidases under study may still 437
participate in modifying cellobiose to another true inducer. It still remains to be 438
established for the physiological role of transglycosylation activity associated with 439
β-glucosidase in cellulase induction. 440
Given the comparable induction of a filter paper hydrolyzing activity to that 441
induced by cellulose when cellobiose was fed at a continuous low level (35), we 442
predict that the absence of the bulk of β-glucosidase in H. jecorina would also 443
make cellobiose alone an easily manipulated potent inducer for enzyme 444
production and regulation as reported recently in N. crassa (36). In this respect, a 445
possible interpretation for the defective induction in the β-glucosidase-deleted 446
strains on Avicel would be that the initial release of cellobiose from Avicel may be 447
less efficient probably due to a transient feedback inhibition of exoglucanases by 448
cellobiose whose hydrolysis is slowed down in the absence of β-glucosidases. The 449
insufficiently released cellobiose thus fails to efficiently initiate the induction 450
cascade when the mutants were incubated with Avicel. Possibility also exists that 451
cellooligosaccharides, released from cellulose may in the absence of 452
β-glucosidases not be cleaved at a sufficient rate to build up the required 453
concentration of cellobiose for induction. Our results that increasing the 454
intracellular β-glucosidase activity by expressing CEL1b (I174C) or addition of an 455
appropriate amount of cellobiose improved the induction defect in Δcel1aΔcel1b 456
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and ΔtriβG strains do point to such a possibility. Therefore, we hypothesize that a 457
subtle balance between cellobiose production and metabolism exists during Avicel 458
hydrolysis, which is tightly controlled by extracellular and intracellular 459
β-glucosidases in influencing their release from cellulose for signal induction (Fig. 460
8). Our results thus indicate that requirement for the three major β-glucosidases in 461
the rapid induction of cellulase genes on Avicel may lie in their ability to ensure an 462
appropriate amount of cellobiose from Avicel for efficiently initiating the signal 463
cascade in H. jecorina. 464
465
Acknowledgements 466
This work is supported by grants from the National Basic Research Program of 467
China (2011CB707402), New Century Excellent Talents in University 468
(NCET-10-0546), Shandong Provincial Funds for Distinguished Young Scientists 469
(JQ201108), and Independent Innovation Foundation of Shandong University, 470
IIFSDU. 471
472
473
474
475
476
477
478
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479
480
481
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30. Szakmary, K., A. Wottawa, and C. P. Kubicek. 1991. Origin of 582
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33. Umile, C., and C. P. Kubicek. 1986. A constitutive, plasma-membrane 595
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34. Vaheri, M., M. Leisola, and V. Kauppinen. 1979. Transglycosylation 598
products of cellulase system of Trichoderma reesei. Biotech Letters 599
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35. Vaheri, M. P., M. E. O. Vaheri, and V. S. Kauppinen. 1979. Formation 601
and release of cellulolytic enzymes during growth of Trichoderma reesei 602
on cellobiose and glycerol. Applied Microbiology and Biotechnology 603
8:73-80. 604
36. Znameroski, E. A., S. T. Coradetti, C. M. Roche, J. C. Tsai, A. T. 605
Iavarone, J. H. Cate, and N. L. Glass. 2012. Induction of 606
lignocellulose-degrading enzymes in Neurospora crassa by cellodextrins. 607
Proc Natl Acad Sci 109:6012-6017. 608
609
610
611
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612
613
614
FIGURE LEGEND 615
Figure 1 Enzymatic characterization and subcellular localization of CEL1b. (A) 616
SDS-PAGE analysis of purified CEL1b and its mutants produced in E. coli. M, 617
molecular mass standard; Lane WT,WT CEL1b; Lane 1,CEL1b I174C; Lane 2,618
CEL1b Y178L; Lane 3,CEL1b D242H; Lane 4, CEL1b H265A; Lane 5, CEL1b 619
S442A. (B) and (C) Recombinant CEL1b exhibited transglycosylation activity. 620
Purified CEL1b was incubated with glucose (20% and 40%, w/v) or cellobiose 621
(10% and 20%, w/v). Reaction products were analyzed by HPLC. G1: Glucose; 622
G2: Disaccharide; G3: Trisaccharide. G4: Tetrasaccharide. (D) CEL1b was mainly 623
localized in the cytoplasm. EGFP was used as a reporter to visualize subcellular 624
localization of CEL1b. Panels from left to right: phase-contrast image of mycelia; 625
fluorescent image of GFP-fused CEL1b; nuclei visualized by DAPI staining. 626
Figure 2 Targeted disruptions of cel1a, cel1b and cel3a. (A) Schematic illustration 627
of the homologous integration of the H. jecorina pyrG gene at the cel1a or cel1b 628
gene locus resulting in the deletion of the coding sequences. (B) Southern blot 629
analysis of Δcel1a, Δcel1b, Δcel1aΔcel1b and ΔtriβG strains. Genomic DNA 630
was digested with SacⅠ(Δcel1a), PstⅠ(Δcel1b andΔcel1aΔcel1b) and NcoI (ΔtriβG) 631
prior to electrophoresis and probed with a 2 kb DNA fragment upstream the ATG 632
codon of cel1a,cel1b, and cel3a respectively. Bands of 3.1 kb (lane 2), 3.5 kb 633
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(lane 4), 4.1 kb (lane 6), and 3.6 kb (lane 8) corresponding to the size expected for 634
the disrupted genes in Δcel1a, Δcel1b,Δcel1aΔcel1b, and ΔtriβG replaced the 635
parental 6.9 kb (lane 1), 6.6 kb (lane 3 and lane 5) and 2.6 kb (lane 7), 636
respectively. 637
Figure 3 Disruption of cel1a, cel1b and cel3a affected growth of H. jecorina on 638
cellobiose as well as cellulase production on Avicel. (A) Intracellular and 639
extracellular β-glucosidase activity in the parental and disruption strains. H. 640
jecorina strains were pre-cultured on glycerol for 48 h and then transferred to 641
Avicel. Intracellular and extracellular β-glucosidase activity was measured using 642
soluble cellular extract and extracellular proteins of culture filtrates, repectively, 643
with pNPG as substrate after 6 h and 12 h induction. Data shown are the means of 644
three independent experiments. (B) Disruption of cel1a, cel1b or cel3a resulted in 645
slower growth on cellobiose. Strains were incubated on plates containing either 646
glucose or cellobiose as the carbon source. (C) and (D) Extracellular protein 647
production and exoglucanase activity from culture supernatant of WT and mutant 648
H. jecorina strains grown in the presence of 1% (w/v) Avicel. Data shown are the 649
means of three independent experiments. 650
Figure 4 The absence of ce11a and cel1b resulted in delayed transcription of cbh1 651
gene on cellulose induction. H. jecorina strains including WT (A), Δcel1a (B), Δ652
cel1b (C) and Δcel1aΔcel1b (D) were pre-cultured on glycerol for 48 h and then 653
transferred to the same medium containing 1% (w/v) Avicel instead of glycerol. 654
One microgram of total RNA was electrophoresed and blotted onto Hybond N+ 655
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nylon membrane (Amsersham). cbh1 mRNA and 18S RNA was probed at 656
different times after Avicel induction. The values below the panels indicate the 657
ratio of the intensity of the cbh1 signal as measured by densitometry to that of the 658
18S RNA control. (E) Western blot analysis of CBHⅠ secreted into the culture 659
supernatant of WT and deletion mutant strains on 1% Avicel. Equal amount of 660
culture supernatant relative to biomass was loaded for all strains. (F) Northern blot 661
analysis of cbh1 mRNA and 18S RNA from Δcel1aΔcel1b complemented with 662
CEL1b (I174C) on 1% (w/v) Avicel. Quantitation was performed as in (A-D) 663
Figure 5 Productive transcription was compromised in a resting-cell inducing 664
system, but was restored by sophorose. Northern blot analysis of cbh1 mRNA and 665
18S RNA from WT (A) and Δcel1aΔcel1b strain (B) was performed in a 666
resting-cell inducing system with Avicel (1%, w/v) as the carbon source. Northern 667
blot analysis of cbh1 mRNA and 18S RNA was also performed for WT (C) and Δ668
cel1a (D) strains after the pre-cultured mycelia were transferred to 1% (w/v) 669
lactose. (E) and (F) Northern blot analysis of cbh1 mRNA from WT and Δcel1aΔ670
cel1b strains. H. jecorina strains were pre-cultured on glycerol for 48 h, and then 671
washed extensively with the minimal medium lacking carbon source, resuspended 672
the same medium with 1 mM sophorose, and incubation was continued for the 673
indicated time period before RNA was extracted for analysis. The values below 674
the panels indicate the ratio of the intensity of the cbh1 signal as quantitated in Fig. 675
4. 676
Figure 6 Simultaneous absence of Bgl I further compromised CBH1 expression,677
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and cellobiose efficiently induced cellulase production in β-glucosidase deletion 678
strains. (A)CBH1 in culture filtrates of WT, Δcel1aΔcel1b and ΔtriβG strains 679
after induction with Avicel (1%, w/v) was analyzed by western blot. Right panel, 680
CBH1 was quantitated by scanning densitometry of the developed membranes.681
(B)Gene expression of cbh1 as analyzed by quantitative RT-PCR after induction 682
with Avicel for different time periods. Relative gene expression levels were 683
normalized to 1 when incubated with glycerol. Expression levels of actin were 684
used as an endogenous control in all samples. Error bars indicate 1 SD. (C) 685
Western blot analysis CBHⅠ secreted in culture filtrates of WT, Δcel1aΔcel1b and 686
ΔtriβG strains after induction with cellobiose (0.25%, w/v). Equal amount of 687
culture filtrates relative to biomass were loaded for all strains. Right panel, CBH1 688
was quantitated by scanning densitometry of the developed membranes. 689
Figure 7 Cellobiose rescued the induction defect by Avicel in β-glucosidase 690
deletion strains. (A) Western blot analysis of CBHⅠ secreted in culture filtrates of 691
WT, Δcel1aΔcel1b and ΔtriβG strains after induction with 1% Avicel plus 692
cellobiose (0.25%, w/v). (B) Extracellular protein production and (C) cellulase 693
activity of culture supernatant of WT, Δcel1aΔcel1b and ΔtriβG strains from A 694
after 24 h of induction toward Avicel. Data shown are the means of three 695
independent experiments. 696
Figure 8 Model of β-glucosidases’ role in cellulase induction with cellulose versus 697
cellobiose in H. jecorina. Upon induction with cellulose, cellobiose is released 698
from cellulose by the synergistic action of exoglucanases and endoglucanases. 699
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Released cellobiose is then either transported intracellularly to initiate the 700
transcriptional induction or hydrolyzed by extracellular and intracellular 701
β-glucosidases (solid line). The absence of β-glucosidases, however, compromises 702
the initial release of cellobiose from Avicel and thus the efficient initiation of the 703
following signaling cascade though hydrolysis of cellobiose may also be slowed 704
down (dashed line). Upon incubation with cellobiose, the downstream pathway 705
from cellobiose for transcriptional induction is strengthened while that for 706
repression is weakened in the β-glucosidase deletion strains. The question mark 707
denotes putative cellobiose derivatives. 708
709
710
711
712
713
714
715
716
717
718
719
720
721
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722
723
Table 1 Kinetic parameters of different β-glucosidases and CEL1b mutants 724
Enzyme Vmax
(nmol/(L·min))
Km
(mmol/L) Kcat (s
-1) Kcat /Km (s-1 mmol-1 L) Reference
CEL1b 35.5 0.44 0.57 1.31 This study
CEL1b (I174C) 2903.73 1.88 31.55 16.82 This study
CEL1b (Y178L) 28.29 0.14 0.38 2.65 This study
CEL1b (D242H) 106.27 0.59 1.57 2.68 This study
CEL1b (H265A) 13.84 0.10 0.20 1.91 This study
CEL1b (S442A) 22.59 0.23 0.46 1.98 This study
CEL1a - 2.22 34.8 - (31)
CEL3a (BglⅠ) - 0.38 87.9 - (2)
725
726
727
728
729
730
731
732
733
734
735
736
737
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