Differential involvement of -glucosidases from Hypocrea jecorina

1

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

6

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

89

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

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33. Umile, C., and C. P. Kubicek. 1986. A constitutive, plasma-membrane 595

bound β-glucosidase in Trichoderma reesei. FEMS Microbiology Letters 596

34:291-295. 597

34. Vaheri, M., M. Leisola, and V. Kauppinen. 1979. Transglycosylation 598

products of cellulase system of Trichoderma reesei. Biotech Letters 599

1:41-46. 600

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

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612

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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

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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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Differential involvement of -glucosidases from Hypocrea jecorina

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