Mutants of Trichoderma reesei are defective in cellulose induction, but not basal expression of cellulase-encoding genes

Gene, 173 (1996) 1999203 0 1996 Elsevier Science B.V. All rights reserved.

199

0378-1119/96/$15.00

GENE 09816

Mutants of Trichoderma reesei are defective in cellulose induction, but not basal expression of cellulase-encoding genes (Endoglucanase

activity; coordinate expression; genomic analysis; hygromycin-B; promoter; transcript expression)

E. Torigoi, F. Henrique-Silva, and S. El-Gogary

J. Escobar-Vera,

J.C. Carle-Urioste,

0. Crivellaro,

H. El-Dorry

Department of Biochemistry, Institute of Chemistry, University of Sdo Paulo, S&o Paula, 05508-900 SP, Brazil Received by J.R. Kinghorn:

26 November

1995; Revised/Accepted:

27 December/28

December

1995; Received at publishers:

7 March

1996

SUMMARY

Four mutants of Trichoderma reesei defective in cellulose utilization were characterized at the molecular level. Genomic analysis of the cellulase-encoding genes (eel) and transcript induction using two well-established inducers of the ccl system ~ the insoluble polymer, cellulose and the soluble inducer, sophorose, - revealed that these mutants are defective in the transcription of ccl genes. The results also indicate that the ccl genes are coordinately expressed and most probably are regulated by the same mechanism. Using a heterologous gene construct, in which the hygromycin-B-resistanceencoding gene was placed under the control of the promoter of the major ccl gene, cbhl, we showed that the mutants synthesize basic levels of cellulase, but are defective in the ccl induction.

INTRODUCTION

Cellulose, the major constituent of lignocellulosic materials, is a linear polymer of glucose units joined by 1,4-B-glycosidic bonds. Cycling of this plant polysaccharide is brought about mostly by ubiquitous and abundant cellulolytic microorganisms (Coughlan and Ljungdahl, 1988). The utilization of cellulose as an energy carbon source by those microorganisms is achieved by an extracellular cellulase system that catalyzes the hydrolysis of cellulose to glucose (Montenecourt, 1983). Cellulose-degrading enzymes (Cel) of the filamentous Correspondence

to: Dr. S. El-Gogary,

Department

of Biochemistry,

Institute of Chemistry, University of SLo Paulo, Avenida Prof. Lineu Prestes 748, SBo Paulo, 05508-900 SP, Brazil. Tel. (55-11) 818-3848; Fax (55-11) 815-5579; e-mail: [email protected] Abbreviations:

Ab, antibody(ies);

lase; cbh, gene encoding

bp, base pair(s); CBH, cellobiohydro-

CBH; Cel, cellulase;

ccl, gene (DNA,

RNA)

encoding Cel; EG, endoglucanase; egl, gene encoding EG; Hph, hygromycin-B phosphotransferase; hph, gene encoding Hph; Hy, hygromycinB; kb, kilobase or 1000 bp; nt, nucleotide(s); Tr, Trichoderma reesei. PII SO378-1 119(96)00219-3

R, resistance/resistant;

fungus Trichoderma reesei (Tr) are among the best characterized; this cellulolytic system consists of three major classes of Cel: cellobiohydrolases (CBH), which cleave cellobiosyl units from the nonreducing ends of cellulose chains; endoglucanases (EG), which cleave internal glycosidic bonds; and B-glucosidases, which cleave cellooligosaccharides to produce glucose (for a review, refer to Beguin, 1990). The genes coding for CBHI, CBHII, EGI, EGII, and b-glucosidase have been isolated and characterized (Shoemaker et al., 1983; Penttila et al., 1986; Chen et al., 1987; Teeri et al., 1987; Van Arsdell et al., 1987; Saloheimo et al., 1988; Barnett et al., 1991). The ability of Tr to utilize different carbon sources, such as cellulose or glucose, is tightly regulated. Growth on cellulose as the sole carbon source results in induction of at least 1200-fold of the ccl transcripts (J.C.C.-U., F. H.-S., S. E.-G., O.C. and H. E.-D., unpublished), whereas glucose strongly represses the expression of those transcripts (Abrahao-Neto et al., 1995). Compelling evidences have implicated basal expression of the Cel activity in triggering the induction of the cellulase transcripts; this basal expression seems necessary to catalyze the forma-

200

tion of soluble inducer from cellulose (El-Gogary et al., 1989; Kubicek, 1987). A potential soluble inducer of the Cel activity (Mandels et al., 1962) is the disaccharide sophorose (2-O-B-glucopyranosyl-D-glucose). This inducer was shown to be one of the products catalyzed by the Tr Cel system and was detected during growth of TV on cellobiose (Mandels and Reese, 1960; Mandels et al., 1962; Vaheri et al., 1979). The aim of this report was to characterize Tr mutants defective in Cel activity. We found that these mutations affect cellulose-induced expression, but not basal expression, of those genes.

A

1

I

o

20

30

42

52

Cdf

0

0

20

49

75

&I-

O

0

0

0

0

(h)

B Sophorose

(h)

CelldOSe

4

-----

6

19

21

24

.

Cd+ Cd-

1 EXPERIMENTAL

cbhl

AND DISCUSSION

(a) Induction of the ccl activity by cellulose

The Tr expresses an extracellular cellulolytic system when grown on cellulose. The activity of this cellulolytic system was measured in the growth media filtrate during induction with cellulose (Fig. 1A). No activity was detectable in the growth medium of the four mutants (QM9136, 9977, 9978, and 9979) after more than 50 h induction. Under the same condition, substantial amount of the cellulolytic activity was measured in the growth medium of Tr QM9414; this activity was detectable after 20 h of induction.

Fig. 1. (A) Effect of cellulose activity.

QM9414,

American

The Tr expresses at least two cellobiohydrolases, CBHI and CBHII (Shoemaker et al., 1983; Chen et al., 1987) two endoglucanases, EGI and EGII (Penttila et al., 1986; Saloheimo et al., 1988; Van Arsdell et al., 1987) and a P-glucosidase (Barnett et al., 1991); this enzymatic system catalyzes, synergistically, the hydrolysis of crystalline cellulose to soluble sugars (Henrissat et al., 1985). The expression of the transcripts of this system was analyzed after induction of Tr cells with two known inducers of the Cel system: cellulose, the insoluble inducer and sophorose, a soluble disaccharide. Fig. 1B shows that the expression of the ccl transcripts, cbhl, cbh2, egll and egl2, were detected 19 h after the addition of cellulose to Tr QM9414 cells; the transcripts were also expressed 4 h after induction with sophorose. However, no transcripts were detected when the mutant cells were exposed to either cellulose or sophorose. Similar results were obtained for @-glucosidase (E.T.; F.H.-S; J.E.-V.; J.C.C.-U.; O.C.; H.E.-D. and S.E.-G., unpublished). It is important to note that, in Tr QM9414, the time course of the induction of the ccl transcripts by cellulose or sophorose appears to be the same. Therefore, it appears that the Cel system genes of the Tr QM9414 strain are expressed coordinately. The results also indicate that the

Type Culture

Collection

mutants

QM9977,

obtained

from the US Department

Inoculum

preparation

(El-Gogary incubated

twice with

on a rotary

26921);

The

All cultures

negative

mutants

to Hybond-N

previously

were incubated

1982) and aliquots

electrophoretically

membranes

(refer to Abrahao-Neto

was

cellulose)

et al., 1989). (B) of the ccl

strain

cellulase-

9414 and

by cellulose

time. RNAs were isolated containing

in 1.2% agarose

and hybridized

on a

on the expression

9136, 9977, 9978 and 9979, were induced (3 mM) for the indicated

and

in culture

time. Cel activity

(El-Gogary

cellulose and sophorose,

and Botstein,

were fractionated

buffer (pH 6.0)

media using Avicel (microcrystalline

Tr cells, cellulase-positive

( 1%) or sophorose

described

spores were centri-

100 mM K.phosphate

as described

mRNAs.

IL, USA.

shaker for 2 h. Mycelia were suspended

in the culture

Effect of inducers,

cellulase-negative Peoria,

from germinated

was added.

Cel from

et al., 1971) were

of Agriculture,

shaker (200 rpm) at 28°C for the indicated

as a substrate,

(Carlson

(ATCC

was obtained

and culture medium were as previously

media and 1% cellulose rotary

of the extracellular

Tr strain,

9978, 9979 and 9136 (Mandels

et al., 1989). Mycelia

fuged, washed

measured

(b) Expression of the cef transcripts

on the induction

a cellulase-positive

10 ug of RNA gel, transferred

to labeled probes as indicated

et al., 1995, for labeled probes).

inability of the mutants to utilize cellulose is not due to posttranscriptional events, such as translation or secretion processes. In addition, the failure of soluble inducer to induce cellulase transcripts in the mutants, indicates that the lack of induction is not due to the inability of the mutants to form the soluble inducer from cellulose. (c) Restriction analysis of the ccl genes

Since the defect in the expression of the ccl genes in these mutants is neither at translation nor at secretion processes, we therefore examined if these mutations are due to deletion in the structural genes of the cellulase or in the regulatory elements controlling the expression of those genes. To that end, we performed restriction analyses of the cellulase genes, cbhl, egll, cbh2 and egl2 (Fig. 2). No differences in the restriction pattern were

201

TAA

ATG 0.72

TAA

ATG

kb

kb

BamHl

4.4

w

kb

HindIll

I

I

“A

h

I y TAA

Af G

I

1.3 kb I

I I

I

ATG

I

I

kb

Nae I

TAA 1.1 kb

Hind III/Xho

2.6 I

1.8

kb 2.2 1.6

Fig. 2. Restriction restriction

enzymes,

analysis

of the ccl genes. DNAs

as indicated,

and fractionated

from the Tr Gel’ strain electrophoretically

labeled probes. The restriction map for each gene is presented hybridization. (A) and (B), cbhl. (C) and (D), egll.

above

observed between the Gel+ strain QM9414 and the four Cell mutants. It is impoitant to note that the probes and the restriction sites used in the analyses of each gene were chosen to produce a DNA pattern covering the entire structural gene and at least 1.5 kb of the 5’ flanking region of the gene. Moreover, no difference was observed in the restriction map analyzed for the B-glucosidase gene in QM9414 strain and the four mutants (data not shown). Therefore, it seems that the inability of those mutants to express the ccl transcripts is not due to deletion of the structural genes or of the promoters controlling the expression of those transcripts. (d) Basal expression of the ceCgenes

The insoluble polymer cellulose is the natural inducer of the eel transcripts. Low basal level of the Cel activity is implicated in triggering the expression of the ccl tran-

9414 and Gel-

in 0.8% agarose the blot; arrow

mutants

gel, transferred below

9136, 9977, 9978 and 9979 were digested to Hybond-N

each restriction

membranes

map presents

and hybridized the probes

with to

used for

scripts; this basal level is needed to generate from cellulose a soluble inducer such as sophorose (Mandels and Reese, 1960; Vaheri et al., 1979). This conclusion was reached from experiments in which the induction of the cbhl transcript by cellulose, but not sophorose, was impaired when antibodies (Ab) to the major members of the cellulase system were added to the culture medium (El-Gogary et al., 1989). To examine whether the basic expression of the eel genes is defective in the examined mutants, we analyzed the expression of a heterologous bacterial gene, the HyR gene (Gritz and Davies, 1983) placed under the control of the cbhl promoter. The vectors pCBHl-Hph-2.2 and pCBHlR-Hph-2.2 contain a 2.2-kb DNA fragment of the 5’ region of chhl fused to the hph gene in the correct and the opposite orientation respectively (J.C.C.-U., F.H.S., S.E.-G., O.C. and H.E.-D., unpublished). The plasmids

202 Transforming

Hygromycin

Plasmids

B

pBluescript

TAT.4

pCBHl-Hph-2.2

+

cbh

TVpCBH1 R-Hph-2.2

-2.2

_K

1

>I hph

kbl

cbh 1

1hph

TATA

ACKNOWLEDGMENTS

This study was supported by grants from PADCTCNPq (62.0622/91.1) and FAPESP (92/3558-4).

Mph Fig. 3. Expression

from the Pcbhl

Hy resistance

Tr eel-

to

pCBHlR-Hph-2.2

contain

in cbhl::hph fusion confers

Plasmids

F.H.S.,

transformation,

cells were plated

pCBHl-Hph-2.2

and the opposite

S.E.-G., O.C. and H.E.-D., on minimal

orientation,

medium

was 0.3 mg/ml, and cells were grown for 48 h. A 2.5cm the top of minimal days. A Bluescript

medium plasmid

used also as controls. presented;

similar

was removed containing

results

essentially

circle (containing

construct

with mutant

with the other

(pHph) QM9978

REFERENCES

as

of MgSO,

200 ug Hy/ml and grown for 4

obtained

were obtained

After

from each plate and placed on

and a promoter-less

The results

respec-

unpublished).

et al., 1988) except that the concentration

at least 15 transformants)

and

of the 5’ region of the cbhl

a 2.2-kb fragment

tively (J.C.C-U.,

(Penttila

promoter

mutants.

gene fused to hph in the correct

described

induce expression of four cellulase and the l%glucosidase transcripts, and that a point mutation or small deletion(s) in the elements controlling these genes is unlikely to occur simultaneously in the promoters of all examined genes. (3) The mutants are defective in the induction process but not in the basal expression of the ccl genes.

were are

three mutants

and the ccl+ strain QM9414.

Abrahao-Neto,

J., Rossini,

Crivellaro,

C.H.B.,

0. and El-Dorry,

cellulase

El-Gogary,

S., Henrique-Silva,

H.: Mitochondrial

Barnett, of

in Trichoderma reesei. Biochemistry

gene expression

CC., the

Berka,

gene

R.M. and Fowler,

encoding

an

Trichoderma reesei: evidence of cellulosic

Carlson,

(e) Conclusions (I) The data suggest that the mutants QM9136, 9977, 9978 and QM9979 are most probably defective in transcription factor(s) required for induction and/or the uptake of the soluble inducer formed from cellulose. Recently it was shown that one of these mutants, QM9979, is indeed defective in P-diglucoside permease (Kubicek et al., 1993). (2) The results also indicate that the ccl transcripts are coordinately expressed and most probably are regulated by the same transcriptional factor(s). This conclusion is based on the fact that all examined mutants failed to

F.,

mediate 34

(1995) 10456610462.

substrates.

T.: Cloning

extracellular for improved

Bio/Technology

M. and Botstein,

Chen, C.M., Gritzali, deduced

primary

9 (1991) 562-567.

reesei. Bio/Technology Coughlan, fungal Btguin,

P. and

Cellulose El-Gogary,

Millet,

J. (Eds.), Academic

by which

of hygromycin

Biochemistry

triggers

Pranz,

Natl.

Saccharomyces

H., Viet, C. and Schulein,

B-glucosidase

by cellulose

(1987) 1481-1487. Kubicek, C.P., Messner, E.M.:

of J.-P.,

Genetics

D. and El-Dorry, Acad.

hygromycin

Bio/Technology 3 (1985) 7222726. Kubicek, C.P.: Involvement of a conidial synthesis

and

cellobiohydrolase

B phosphotransferase and

In: Aubert,

of

Sci. USA 86

B resistance:

25

M.: Synergism

of

of cellulose. and a plasma-

of endoglucanase

in Trichoderma reesei. J. Gen. Microbial. R., Gruber,

F., Mandels,

the

cereoisiae. Gene

endoglucanase

in the induction

H.:

I gene

gene and its expres-

from Trichoderma reesei in the degradation

membrane-bound

and

Biochemistry

systems.

O., Eveleigh,

cellulose

sion in Escherichia coli

cellulases

sequence

Press, New York, 1988, pp. 11-30.

in Trichoderma reesei. Proc.

(1983) 179-188. Henrissat, B., Driguez,

of yeast

II from Trichoderma

Comparative

enzyme

(1989) 613886141. Gritz, L. and Davies, J.: Plasmid-encoded sequence

mRNAs with forms

D.W.: Nucleotide

L.G.:

cellulolytic

S., Leite, A., Crivellaro,

expression

Rev.

5 (1987) 274-278.

Degradation.

Mechanism

Annu.

regulated

of cellobiohidrolase

M.P. and Ljungdahl, and bacterial

degradation.

and intracellular

M. and Stafford, structure

from

rates of saccharification

D.: Two differentially

different 5’ ends encode secreted invertase. Cell 28 (1982) 145-154.

and amplification

beta-glucosidase

Beguin, P.: Molecular biology of cellulose Microbial. 44 (1990) 219-248.

were transformed into mutants cells and transformants were tested for their ability to grow on a minimal medium containing Hy. Fig. 3 shows that the cbhl promoter fused to hph gene in the correct but not the opposite orientation conferred HyR to mutants cells. The same result was obtained in at least five independent transformations. In addition, no HyR transformants were obtained using the cbhl promoter placed in the opposite orientation or with the promoter-less constructs, while between 100 to 200 transformants per ug of plasmid DNA were obtained with the construct having the cbhl promoter in the correct orientation. The results indicate that those mutants are not defective in the basal expression of the cellulase genes.

functions

133

M. and Kubicek-

Triggering

involvement of a inhibited P-diglucoside

of cellulase biosynthesis by cellulose: sophorose-inducible, glucoseconstitutive, permease.

J.

Biol.

Chem

268

(1993)

19364419368. Mandels, M. and Reese, E.T.: Induction of cellulase in fungi by cellobiose. J. Bacterial. 79 (1960) 816-826. Mandels, M., Parrish, F.W. and Reese, E. T.: Sophorose as an inducer of cellulase in Trichoderma uiride. J. Bacterial. 83 (1962) 400-408.

203 Mandels,

M., Weber, J. and Parizek,

R. Enhanced

cellulase

of Trichoderma uiride. Appl.

by a mutant

Shoemaker,

production

Microbial.

21 (1971)

152-154. B.S.: Regulation

in fungi. Trends

Bio/Technol.

M., Lehtovaara,

Knowles,

of cellulase

nucleotide

biosynthesis

and secretion

1 (1983) 156-161.

P., Nevalainen,

J.: Homology

reesei: complete

M., Nevalainen,

A versatile fungus

and

between

cellulase

sequence

genes

R. and

of Trichoderma

of the endoglucanase

I gene.

M., Salminen,

system

E. and Knowles,

for the cellulolytic

J.:

filamentous

Trichoderma reesei. Gene 61 (1988) 1555164.

Saloheimo,

M., Lehtovaara,

Johansson, Knowles,

G., Pettersson, J.K.C.:

EGIII,

reesei: the characterization (1988) 11-22.

P., Pentilla,

G., Claeyssens,

M., Tomme,

a new endoglucanase of both

gene

and

from

J.,

P. and

Trichodermu

enzyme.

Gene

63

M.:

M., Gelfand, Molecular

D., Kwok,

cloning

of

S., exo-

I derived from Trichoderma reesei strain L27. Bio/

1 (1983) 691-696. P., Kauppinen,

domains

gene

and

sequence

S., Salovuori,

I. and Knowles,

in Trichoderma reesei cellulolytic

expression

of cellobiohydrolase

enzymes:

II. Gene

51

(1987) 43-52. M., Leisola,

M. and Kauppinen,

V.: Transglycosylation

ducts of the cellulase system of Trichoderma reesei. Biotechnol. 1 (1979) 41-46. Van Arsdell, J.N., Kwok,

M., Teeri, T.T., Stahlberg,

V., Ladner,

Innis,

J.: Homologous

Vaheri,

H., Ratto,

transformation

Technology

Teeri, T.T., Lehtovaara,

H., Bhikhabhai,

Gene 45 (1986) 253-263. Penttila,

K.

cellobiohydrolase

Montenecourt, Penttila,

S., Schweickart,

Myambo,

and

Innis.

M.:

Saccharomyces

S., Schweickart,

Cloning,

V., Ladner,

characterization,

cereuisiae of endoglucanase

reesei. Bio/Technology

5 (1987) 60-64.

proLett.

M., Gelfand,

D.

expression

in

and I from

Trichoderma