Trichoderma reesei. - Springer Link

Mol Gen Genet (1993) 241:523-530 © Springer-Verlag 1993

High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes Pirkko L. Suominen 1, Arja L. Miintyl~i 1, Taina Karhunen 2, Satu Hakola 1, Helena Nevalainen 1,, 1Research Laboratories, Alko Ltd, P.O.B. 350, SF-00101 Helsinki, Finland 2 Fazer Chocolates Ltd, P.O.B. 4, SF-00941 Helsinki, Finland Received: 17 February 1993/Accepted: 16 June 1993

Abstract. Four cellulase genes of Trichoderma reesei, cbhl, cbh2, egll and egl2, have been replaced by the amdS marker gene. When linear D N A fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egll or egl2 slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2 gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose. Key words: Trichoderma reesei Gene replacement - Homologous recombination - Cellulases - Regulation

Introduction Transformation techniques have been developed for a wide range of filamentous fungi (reviewed by Finkelstein 1992). With few exceptions, filamentous fungi do not contain autonomous plasmids and D N A introduced into the nucleus is integrated into the genome. Both homologous and non-homologous recombination events occur frequently (reviewed by Esser and Mohr 1986). The frequency of homologous integration and gene replacement seems to vary according to the organism, the recipient strain, the target locus and the amount of homology Communicated by C. van den Hondel * Present address: Macquarie University, School of Biological Sciences, Sydney, N.S.W. 2109, Australia Correspondence to: P.L. Suominen

between the transforming D N A and the genome. However targeted recombination of the incoming DNA would be very advantageous. It allows the introduction of precise changes, made in vitro, into the structure of endogenous genes in order to study structure-function relationships both of proteins and of promoters. The biological function of a cloned gene can be elucidated by inactivation or complete removal of that gene by targeted transformation. The ability to modulate protein production by removal of chosen genes or by replacing them by others is an important tool in the construction of industrial production strains (Berka et al. 1990; Harkki et al. 1991; Suominen et al. 1992). It has been shown that homologous recombination of transforming DNA occurs in Trichoderma and that it can be used to alter cellulase production profiles (Harkki et al. 1991; Suominen et al. 1992; Seiboth et al. 1992). However, the frequency of homologous recombination was very low in both instances in which it was estimated (Harkki et al. 1991 ; Seiboth et al. 1992). Trichoderma reesei is known to be a good cellulase producer. It produces a complete set of cellulases which act synergistically to hydrolyse crystalline cellulose to glucose. Two different cellobiohydrolases and at least three different endoglucanases are produced; the two cellobiohydrolases have been suggested to differ in their stereospecificity and to hydrolyse different configurations of the nonreducing end groups in cellulose and thus have distinct physiological roles (Woodward 1989). Genes for both cellobiohydrolases have been cloned and characterized by sequencing (Shoemaker et al. 1983; Teeri et al. 1983 ; Teeri et al. 1987). Also the genes for the two main endoglucanases have been cloned and sequenced (Penttilfi et al. 1986; van Arsdell et al. 1987; Saloheimo et al. 1988). Studies of the behaviour of different Trichoderma mutant strains that are cellulase negative (Nevalainen and Palva 1978) suggest that there is an overall control system for coordinate expression of the enzymes participating in the hydrolysis of lignocellulosic material. In this paper, we report high frequency, onestep gene replacement at four different loci of T. reesei

524 and derive guidelines on how to target recombination in this filamentous fungus. We have used the gene replacement technique to study the effects of deletion of various cellulase genes on the expression of the remaining cellulase genes and on production of cellulolytic activities. Materials and methods

Bacterial and fungal strains and plasmids. Plasmids were propagated in Escherichia eoli K-12 strain XL1-Blue (Bullock et al. 1987). The plasmids were constructed from pUC19 as a basic vector and using standard recombinant DNA techniques. T. reesei strain VTT-D-79125 (Bailey and Nevalainen 1981), a mutant producing high cellulase levels, was used as an initial host in all transformations. Plasmid p3SR2 (Kelly and Hynes 1985) was kindly provided by Dr. M. Hynes. The plasmid carries the Aspergillus nidulans amdS gene for aeetamidase cloned into pBR322. DNA manipulations. T. reesei chromosomal DNA was isolated according to Raeder and Broda (1985). Plasmids were isolated from E. coli as described by Birnboim and Doly (1979) or by Holmes and Quigley (1981). DNA manipulations and Southern blot analyses were performed by standard techniques (Maniatis et al. 1982). Transformation of T. reesei. Transformation of T. reesei and selection of AmdS + transformants were carried out essentially as described by Penttilfi et al. (1987b) with the modifications described in Karhunen et al. (1993). Transformants were purified by selection of conidia on selective medium. Immunological methods. For Western blot analysis, purified transformants were grown in 96-well millititre filtration plates (Millipore) at 30°C for 7 days. Cellulase proteins in the culture supernatant were subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Specific proteins were detected by Western blotting and immunostaining using polyclonal or monoclonal antibodies and the ProtoBlot Western blot AP system (Promega) according to the recommendations of the manufacturer. For detection of cellobiohydrolase I (CBHI) a polyclonal antibody was used and for CBHII and endoglucanase I (EGI) monoclonal antibodies CII-8 and EI-2, respectively (Aho et al. 1991). Quantitation of secreted EGI was carried out by double antibody sandwich ELISA (enzyme-linked immunosorbent assay; Bfihler 1991) using the monoclonal anti-EGI antibody EI-2 (Aho et al. 1991) as capture antibody. For quantitation of CBHI and CBHII, ELISAs of the same format have been developed (R. Bfihler, unpublished), using the monoclonal antibodies CI-258 and CII-8, respectively (Aho et al. 1991), as capture antibodies. Media and culture conditions. All liquid cultures o f T. reesei were started from conidiospores grown on potato dextrose agar. The liquid cultivation medium contained 4% whey, 1.5% complex nitrogen source derived from

grain, 1.5% KHzPO4 and 0.5% (NH4)2SO 4. Cultures were maintained at 30 ° C and 250 rpm for 7 days. Enzyme assays. All assays were performed upon the culture supernatants after removing the mycelia. Activity against hydroxyethylcellulose (HEC, middle viscosity 1, Fluka AG) was measured as described by Bailey and Nevalainen (1981). For measurements of [~-glucanase activity, HEC was replaced by barley [3-glucan (Biocon). Activity against filter paper (FPU) was determined by the assay developed by Mandels et al. (1976). Xylanase activity was measured using birch xylan as substrate in the method described by Bailey et al. (1992). Soluble protein was assayed by the method of Lowry et al. (1951) using bovine serum albumin as standard. Construction of a cosmid library of T. reesei. A genomic bank was constructed from T. reesei QM6a DNA. Total genomic DNA was partially digested with Sau3A and ligated into the BamHI site of the cosmid vector p3030 (B. Hohn and A. Hinnen, unpublished). The resulting cosmid library consisted of about 36 000 independent colonies. Construction of pALK425, pALK429, pALK432, pALK454 and pALK471. For the construction of the plasmid used for cbhl replacement, pALK425, 5' and 3' regions of the cbhl locus were isolated using plasmid rescue from the strain ALKO2466 (Harkki et al. 1991). The restriction maps of the rescued regions and the wild-type cbhl locus were compared by Southern blotting and found to be similar. The 1.9 kb ScaI-EcoRI fragment from the 5' region ofcbhl and the 1.7 kb BamHIEeoRI fragment from the 3' region were subcloned into pUC19, together with the amdS gene on a 3.2 kb SphI-XbaI fragment from p3SR2 (Kelly and Hynes 1985). From the resulting plasmid pALK425, the 6.8 kb fragment shown in Fig. 1 can be isolated as a EeoRI fragment for transformation. The location of the 5' and 3' fragments on pALK425 with respect to the protein coding region of cbhl is shown in Fig. 1. Plasmid pALK432 was constructed to allow replacement of the cbh2 gene with the amdS gene. Regions both 5' and 3' to the ebh2 gene were subcloned from the k clone originally isolated by Teeri et al. (1987). A restriction map of the locus was constructed; the 3.4 kb PvuII fragment from the 5' region and the 2.1 kb PvuII-XbaI fragment from the 3' region were further subcloned into a derivative of pUC19. The location of these fragments with respect to the coding region of cbh2 is shown in Fig. 1. The amdS gene, as a 3.2 kb SphI-XbaI fragment from p3SR2, was subcloned between these fragments to give plasmid pALK432. From this plasmid, the 8.7 kb fragment shown in Fig. 1 can be isolated as a XhoI fragment for transformation. Two plasmids were constructed for replacement of the egll gene. The plasmid pALK429 contains the 0.9 kb XhoI-HindIII fragment from pTTcll (Penttil~i et al. 1987a). This fragment from the 3' region also contains part of the coding region of the egH gene. The 5' region of the egll gene present on pALK429 is a 1.0 kb XhoI-

525 pALK425: I

cbh'l 5'

amdS

1.9kb /

3.2kb

cbhl 3'

\1.7 kb

I cbh 1 2.3kb

pALK432: I

cbh2 5'

1.65kb

/ 1.4kb

""1 1./-,k b

amdS

cbh 2 3'

3.2 k b

2.1 kb

t

3.4. kb

~

[

cbh2 1.7kb

)

k clone, originally called egl3, isolated by Saloheimo et al. (1988). The locus was restriction mapped, and the 1.6 kb XhoI fragment from the 5' region and the 1.65 kb BalI-SmaI fragment from the 3' region were subcloned into pUC19. The locations of these fragments with respect to the coding region ofegl2 are shown in Fig. 1. The 3.2 kb fragment containing the amdS gene was subcloned between these fragments from egI2 locus to give the plasmid pALK454. The 6.45 kb fragment shown in Fig. 1 can be isolated as an EcoRI fragment from pALK454.

t 0.5kb

Results pALK429:

egll 5'

amdS

egll 3'

1.0kb \

3.2kb ./ \0.85kb ~ ' - ,,

0.9kb

1

I

't

[--~t~_ _ .,, g.55kb

pALK471:

pALK454:

I

I

egl. 1 5'

amdS

eg[1 3'

1.8 kb

3.2 kb

1.6 kb

eg[2 5' '1.6 kb

/ 2.2 kb

emdS

egt2 3'

3.2kb

1.65kb 1.4. kb

0.1kb

Fig. 1. Schematic representation of fragments used in transformations, pALK425, pALK432, pALK429, pALK471 and pALK454 are the plasmids from which each of the fragments used for transformation (upper line in each case) were isolated. The lower line in each case illustrates the part of the wild-type loci replaced by the amdS gene on the corresponding fragments. The open arrows represent the protein coding part of each gene

SphI fragment from the original X clone (Penttil/i et al. 1986). The fragment containing the amdS gene is the same as in pALK425. From the plasmid pALK429 the 5.1 kb fragment shown in Fig. 1 can be isolated as an EcoRI fragment for transformation. The egll replacement plasmid pALK471 contains longer flanking regions of the egll gene. The egll locus was isolated from the cosmid bank of T. reesei QM6a DNA using the egll cDNA (from pTTcl 1 ; Penttil/i et al. 1987a) as a probe. A restriction map of the flanking areas was constructed and the 1.8 kb ScaI-StuI fragment from the 5' region and the 1.6 kb ScaI-XhoI fragment from the Y region were subcloned into pUC19. The locations of these areas with respect to the coding region of egll are shown in Fig. 1. The 3.2 kb SphI-XbaI fragment bearing the amdS gene from p3SR2 was added to the plasmid containing the egll flanking regions. From the resulting plasmid pALK471 the 6.6 kb fragment shown in Fig. 1 can be isolated as an EcoRI fragment for transformation. For construction of the plasmid pALK454, regions both 5' and 3' to the egl2 gene were subcloned from the

Replacement of cbhl, cbh2, egll and eg12 loci with amdS In order to replace each of the four cellulase genes, cbhl, cbh2, egH and e912, plasmids pALK425, pALK432, pALK429, and pALK454 were constructed as described in the Materials and methods. The D N A fragments shown in Fig. 1 were used in transformations. Each fragment contains the amdS marker gene flanked by long 5' and 3' regions of the target locus. Linear D N A was used because we had previously shown that transformation with circular D N A will not lead to efficient replacement of the target locus (Karhunen et al. 1993). T. reesei strain VTT-D-79125 was transformed with the 6.8 kb fragment (Fig. 1) from the plasmid pALK425; 1.7 kb of the 3' and 1.9 kb of the 5' flanking region was included to facilitate homologous recombination at the cbhl locus. These regions were chosen in such a way that integration at the cbhl locus replaces the entire coding region, the promoter (2.3 kb) and 1.4 kb of the Y region (Fig. 1). Transformants were purified through selection of conidia on selective media. AmdS + transformants were then grown on microtitre plates for 7 days. The culture supernatants were analysed by Western blotting with CBHI-specific antibody to identify tansformants not secreting CBHI protein (not shown). Lack of CBHI secretion by transformants that continue to secrete other proteins indicates replacement of the cbhl locus. Several transformants from independent transformations were further analysed by Southern blotting (not shown) to verify lack of the cbhl coding region. These results were in agreement with the Western blot results. Of 44 transformants tested from the first transformation 16 did not produce CBHI, indicating a replacement frequency of 36 %. In other transformations the frequencies have been even higher, 44-47% (16/36, 28/59 and 16/36 from three independent transforlnations). Similar results have been obtained in all other transformations with the pALK425 fragment and also in other recipient strains. To find out if the high replacement frequency of the ebhl locus by the pALK425 fragment was locus dependent, we constructed plasmids from which similar fragments could be isolated for replacement of the ebh2, egll and egl2 loci (Fig. 1). The pALK432 fragment was transformed into strain VTT-D-79125 to replace the entire coding region, promoter (1.4 kb) and 0.5 kb of the 3' region of the cbh2 locus (Fig. 1). Transformants were

526

purified and tested for secretion of CBHII as described above for CBHI. The replacement frequencies for the cbh2 locus varied from 40 to 49 % (22/45 or 6/15 from two independent transformations). When the fragment of pALK429 was used to replace 0.85 kb of the promoter and 0.65 kb of the coding region of egll (Fig. 1), transformants not secreting EGI were more rarely found than in the previous cases with cbhl and cbh2. In several independent transformations their frequency was less than 10% (for example, 5/59) and in each case at least 20 transformants were tested. The best replacement frequency with the pALK429 fragment was 16% (3/19). The pALK429 (egll) fragment differs from the pALK425 (cbhl) and pALK432 (cbh2) fragments by having shorter flanking regions for direct homologous recombination - only 1.0 kb and 0.9 kb in comparison with 1.9 kb and 1.7 kb in pALK425 and 3.4 kb and 2.1 kb in pALK432. To test whether the low replacement frequency was due to the egll locus or to the shorter flanking regions plasmid pALK471 was constructed (Fig. 1). The pALK471 fragment carries 1.8 kb and 1.6 kb of the 5' and 3' flanking regions, respectively. When VTT-D-79125 was transformed with this fragment, the replacement frequencies of the egll locus varied from 24 to 32% (14/59 and 6/19 from two independent transformations). Thus the longer flanking regions of egll did increase the targeting frequency of the transforming D N A to the egll locus, but not to the level of 40-50% found with the cbhl and cbh2 loci (Table 1). The pALK454 fragment (Fig. 1) was used to replace the egl2 locus of VTT-D-79125 with the amdS gene. D N A was isolated from five purified AmdS + transformants. Southern blotting of these DNAs showed that only one had the wild-type egl2 locus and in four transformants the locus was replaced by the pALK454 fragment, giving a replacement frequency of 80%. However one of the four replacement strains also had copies of the pALK454 fragment elsewhere in the genome. Screening by Southern blotting for the correct replacement of the egl2 locus was necessary because of the lack of an EGIIspecific monoclonal or polyclonal antibody. Moreover, the small number of transformants tested may lead to over-estimation of the replacement frequency. When the pALK454 fragment was transformed into ALKO2721, a derivative of VTT-D-79125, the targeting frequency was 33%. Twelve randomly chosen AmdS + transformants were tested by Southern blotting, of which eight the wild-type e912 locus. In three transformants replacement of the resident egl2 locus with the pALK454 fragment had occurred and in one the fragment had integrated at the egl2 locus; however, the integration did not result in simple replacement but in more complicated changes at the egl2 locus (not shown), which were not further studied. By using linear D N A fragments, containing the amdS marker for selection of transformants flanked by long (1.6-3.4 kb) pieces of the 5' and 3' regions of a specific target locus, we have been able to replace four independent cellulase loci of T. reesei with the amdS marker with very high replacement frequencies, ranging from 32 to 50% (Table 1).

Table 1. Replacement frequencies of different cellulase loci

Locus

Replacing fragment

Length of homologous pieces (5' and Y)

Replacement frequences

cbhl cbh2 egll egll egl2

pALK425 pALK432 pALK429 pALK471 pALK454

1.9 kb 3.4 kb 1.0 kb 1.8 kb 1.6 kb

36-47% 40-49% 8-16% 24-32% 33%

and and and and and

1.7 kb 2.1 kb 0.9 kb 1.6 kb 1.6 kb

Southern blot analysis of the different replacement strains To obtain a set of strains, each lacking one of the cellulase genes, cbhl, cbh2, egll or e912, several transformants from each of the above described transformations were analysed by Southern blotting. In each case one blot was hybridized with the corresponding coding region. The Southern hybridization analyses correlated with the previous Western blot results: no signal was obtained from D N A from strains lacking the corresponding protein (not shown). A few strains from each transformation were studied more carefully: the Southern blots of the strains chosen for expression studies are shown in Fig. 2. Total D N A was digested with XhoI and hybridized with the transforming fragment used in each case. From the wild-type cbhl locus of the host strain VTT-D-79125, two bands, 9.2 kb and 1.4 kb, were seen as expected (Fig. 2A). If a replacement of the cbhl locus by the pALK425 fragment occurred, the 9.2 kb band was replaced by a 7.05 kb band and the 1.4 kb was unchanged. Bands of the correct sizes were observed when D N A from ALKO2862 was hybridized (Fig. 2A). From the egll locus of the host, bands of 2.75 kb and 2.5 kb were expected and observed. In ALKO2874 these bands were replaced by one larger band of 6.9 kb, as expected, if the locus were replaced by the pALK429 fragment (Fig. 2B). In the case of the ebb2 locus, the 5.0 kb and 10 kb bands of the host were replaced by a 14.5 kb band in ALKO3067, as expected (Fig. 2C). In ALKO3128 the 6.0 kb and the 2.55 kb bands have replaced the 4.5 kb, 2.6 kb and 1.6 kb bands of the egl2 locus of the host. In each case the results were confirmed by using several other restriction enzymes (not shown). The T. reesei strains ALKO2862, ALKO2874, ALKO3067 and ALKO3128 have the same genetic background (VTT-D-79125) and the only difference between the strains is the lack of one cellulase gene and its promoter. In the strains ALKO2682 the cbhl locus, in ALKO2874 the egll locus, in ALKO3067 the cbh2 locus and in ALKO3128 the egl2 locus is replaced by the amdS gene.

Effects of replacement of single cellulase 9enes on production of cellulolytic activities The strains ALKO2862, ALKO2874, ALKO3067 and ALKO3128, together with the parent strain VTT-D-

527 A

B V'i3"-D- ALKO 7,H 79125 2862

~,H

MTT-D- ALKO ~,H+E 79125 2874

C

D

VTT-D- ALKO ~,Bst ~,H+E 79125 3067

VTT-D- ALKO LH+E 79125 3128

14.5 10 --9.2

~iliji;ii;~!~iii~¸ i : ? ;

-- 6.9

- - 7.05

--6

5.0

--

~

4.5

iiiii!ii:~i¸

-- 2.75

--

-- 2.5

-

2.6

2.55

1.4

--

Fig. 2. Southern analysis of the replacement strains ALKO2862, ALKO2874, ALKO3067 and ALKO3128. Genomic DNA was digested with XhoI. Hybridization of the blot was done with the fragment used for transformation in each case. The probes were

1.6

labelled with digoxigenin according to the procedure of BoehringerMannheim. Molecular weight markers )~HindIII, )~EcoRI-HindIII and )~BstEII were used. Sizes of the hydridizing fragments are shown on the right

Table 2. Production of cellulases by the host strain Trichoderma reesei VTT-D-79125 and the transformants representing different replacement strains Strain

Replaced gene

Secreted protein (mg/ml)

Endoglucanase (HEC; (nkat/ml)

13-Glucanase (nkat/ml)

FPU/ml

13-Gluco- CBHI sidase (mg/1) (nkat/ml)

CBHII (mg/ml)

EGI (mg/ml)

VTT-D-79125 ALKO2862 ALKO3067 ALKO2874 ALKO3128

cbhl cbh2 e9ll egl2

7.4 4.5 7.2 7.6 7.5

1000 1300 940 760 460

6100 11000 6000 4800 3100

3.3 0.96 2.2 3.7 2.9

36 i 10 53 34 35

0.37 0.92 0.48 0.58

0.27 0.26 0.25 0.30

2.0 2.0 2.2 2.3

HEC, Hydroxyethylcellulose; FPU, filter paper hydrolysing activity; CBH, cellobiohydrolase; EGI, endoglucanase I Strains were grown for 7 days on cellulase inducing medium. Total secreted protein, enzyme activities and the amounts of CBHI,

CBHII and EGI were measured from culture supernatants by enzyme-linked immunosorbent assay. All results are the average of two independent experiments and each using three parallel flasks of each strain

79125, were grown in cellulase-inducing medium in flasks for 7 days. Lactose instead of cellulose was used as main carbon source to avoid possible effects on growth of the strains lacking cellulases. In this medium, all the strains seem to grow equally well, although we could not measure the dry weight of the mycelia produced because the medium contained particles. After removing the mycelia by centrifugation, the various cellulolytic activities and amounts o f total secreted protein were measured in the culture supernatant (Table 2). C B H I is the m a j o r cellulase produced by T. reesei and reduction of the a m o u n t of total protein (40%, Table 2) secreted by the cbhl replacement strain A L K O 2 8 6 2 occurred as expected. The filter paper-degrading activity produced by this strain was also decreased by 70%. Lack of the cbhl gene

and its p r o m o t e r in A L K O 2 8 6 2 increased H E C hydrolysing activity in the culture supernatant (Table 2), which m a y indicate enhanced production o f endoglucanases. Production of [3-glucanase activity was increased 1.8fold. Replacement of the cbh2 gene and its promoter, ALKO3067 had only minimal effects, if any, on the total a m o u n t of protein secreted and on activities against H E C and 13-glucan in the culture supernatant (Table 2). Filter paper-degrading activity produced by A L K O 3 0 6 7 was almost 35% lower (2.2 F P U / m l ) than that produced by the parent strain VTT-D-79125 (3.3 FPU/ml, Table 2). Activities against H E C and 13-glucan in the culture supernatant of the strain A L K O 2 8 7 4 (egll replacement) were decreased by 25 % and 21% respectively (Table 2), because of the lack of E G I production. There was a slight

528 increase of about 10 % in the filter paper-degrading activity produced by this strain (Table 2). Replacement of the egl2 gene, in ALKO3128, had the opposite effect on the filter paper-degrading activity it was decreased by 12% (2.9 FPU/ml compared with 3.3 FPU/ml, Table 2). Both ALKO2874 and ALKO3128 secreted as much 13-glucosidase as the parent strain (Table 2). Activities against HEC and 13-glucan produced by ALKO3128 (EGII negative) were even lower than those produced by the EGInegative strain ALKO2874 (Table 2) and were 55% (HEC) and 50 % ([3-glucan) lower than those produced by the parent. However egl2 replacement did not affect the total amount of protein secreted.

Effects of replacements of each cellulase gene on production of other cellulases The concentrations of CBHI, CBHII and EGI were measured in the same culture supernatants as the enzyme acitivities, using specific ELISA assays (Table 2). The amount of CBHI secreted by all the strains containing cbhl was fairly constant, about 2 mg/ml. In these growth conditions, deletion of cbh2 (ALKO3067) did not alter production of CBHI at all (2.0 mg/ml, Table 2). Also in the strains lacking egll (ALKO2874, 2.2 mg/ml) or egl2 (ALKO3128, 2.3 mg/ml) the amount of CBHI in the culture supernatant was essentially unaltered. The amount of EGI secreted by ALKO2862 (cbhl), ALKO3067 (cbh2), ALKO3128 (egl2) was approximately the same (0.27 mg/ml) as the amount secreted by the parent strain VTT-D-79125 (see Table 2). Production of CBHII was clearly affected by deletions of the other cellulase genes. Deletion of egll had the slightest effect, increasing secretion of CBHII by 30% (ALKO2874, Table 2). Deletion of egl2 increased secretion of CBHII by 55% and deletion of cbhl enhances CBHII by 150% (ALKO3128 and ALKO2862, Table 2). Discussion

In this report we have describe the successful replacement of four different loci in the filamentous fungus T. reesei; the replacement frequencies were high in each case, ranging from 32 to 50%. We have previously shown that homologous integration occurs frequently (37-63%) at the ebhl locus (Karhunen et al. 1993) and here we have shown that the high replacement frequencies are not limited to the ebhl locus, since all the other loci tested gave similarly high frequencies. However the replacement frequency was affected by structural features of the integrating DNA. The transforming D N A should be linear (Karhunen et al. 1993) and the 5' and 3' regions from the target locus flanking the selection marker should each be more than 1 kb in size, preferably over 1.4 kb. This was studied using the plasmids pALK429 and pALK471 for replacement of the egll locus by the amdS gene (Fig. 1). The main difference between these plasmids is the length of the homologous regions, which in pALK429 are 1.0 kb and 0.9 kb and in pALK471

1.8 kb and 1.6 kb. This increase in the size of the homology region raised the replacement frequency from about 10% to about 30%. However the frequency was never as high for the egll locus as for the cbh loci (40-50%). This may be due to the loci themselves or to the even longer homology region in the cbh plasmids (pALK425 1.7 kb and 1.9 kb; pALK432 3.4 kb and 2.1 kb). The technique of high frequency gene replacement described in this paper is not limited to the amdS marker gene or the host strain used here. Similar results have been achieved also with other T. reesei host strains and other markers (unpublished data from our laboratory). It is worth noticing that the replacement frequencies presented here are so high that screening for homologous integration events can easily be done by Southern blotting, as was done for egl2 in this study. This raises the possibility of investigating the function of newly isolated unknown genes by disrupting or replacing them or constructing mutations in vitro and studying them after integration in their natural location in the T. reesei genome. In this study, we have constructed a set of T. reesei strains in which the genes for the major cellulases CBHI, CBHII, EGI and EGII as well as their promoters were replaced by the amdS marker gene. Otherwise the strains ALKO2862, ALKO2874, ALKO3067 and ALKO3128 are isogenic. The effect of each deletion on expression of the other cellulases was studied in cellulase-inducing conditions using lactose as main carbon source. Cellulases seem to be coordinately regulated (Nevalainen and Palva 1978) and thus it was expected that each deletion might affect expression of the other cellulase genes; however, suprisingly small effects were noted. When the gene and promoter for the major cellulase CBHI was deleted, secretion of CBHII was doubled (Table 2). Lack of the CBHI protein, not deletion of the strong cbhl promoter, provides the signal to increase CBHII production, because the same effect was seen on CBHII production when the cbhl gene was disrupted not deleted (unpublished data from our laboratory). No effect on secretion of EGI could be detected (Table 2). However activities were increased against [~-glucan (1.8-fold, Table 2) and HEC (1.3-fold, Table 2), indicating possible increases in the amounts of EGII and/or other endoglucanases. The increase in ~3-glucanase activity is highly likely to be at least partly caused by the increase (2.5-fold, Table 2) in the amount of CBHII protein, since colonies of Saccharomyces cerevisiae expressing the T. reesei cbh2 gene have been shown degrade barley 13-glucan on plates (Penttilfi et al. 1988). Production of CBHII was increased not only in response to cbhl deletion but also when egll or egl2 were replaced (Table 2). This may indicate that the ebh2 promoter or the CBHII protein plays a key role in cellulase regulation. On the other hand, although expression of cbh2 seems to respond when there is any change in the production of other cellulases, the deletion of cbh2 itself did not affect production of CBHI or EGI (strain ALKO3067, Table 2). This is in accordance with the finding of Seiboth et al. (1992) that in a ebh2 disruption strain the final cellulase levels were not affected. In the

529 study of Seiboth et al. (1992), endoglucanase activity was measured and no reduction in the induced level of endoglucanase production was detected. In accordance with this finding, only a very small decrease in the activity against H E C was seen with the cbh2 deletion strain ALKO3067 (Table 2). However C B H I I is very i m p o r t a n t for efficient hydrolysis of filter paper; filter paperdegrading activity is reduced by 35% when C B H I I is deleted (ALKO3067, Table 2), C B H I production remaining at the original level. Thus this strain is not as efficient in total hydrolysis of cellulose as its C B H I I + counterpart and it is not able to compensate for the loss of C B H I I . Suprisingly there was hardly any change in the amounts of the other cellulases. Lack of E G I I production (ALKO3128) reduced the endoglucanase activity (HEC) in the culture supernatant by as much as 55%, whereas lack of E G I reduced it by only 25% (Table 2). Deletion of egl2 did not lower production of E G I protein (Table 2). Thus E G I I seems to account for m o s t of the endoglucanase activity produced by the host strain (VTT-D-79125). Another possibility is that for efficient hydrolysis of H E C , E G I needs the action of E G I I but E G I I can work more independently, and therefore the lack o f E G I only has a small effect. However we have shown that a 7-fold increase in the a m o u n t of E G I increases the activity against H E C only 2.6-fold (Karhunen et al. 1993), which would be in accordance with 25% o f the host strain's activity being due to E G I (25% reduction on egll deletion) and by independent action of both endoglucanases on H E C . However we cannot rule out the possibility that E G I I is limiting in the enzyme mixture produced by the E G I overproducer if the two enzymes work synergistically. These questions cannot be unambiguously answered before we can measure also the a m o u n t of E G I I in the enzyme mixtures produced by the different strains. The deletions of egll and egl2 have opposite effects on the production o f activity against filter paper; lack of E G I increases and lack of E G I I decreases this activity. The small increase in filter paper-hydrolysing activity produced by the egll deletion strain A L K O 2 8 7 4 can be explained by the small increase in production of both cellobiohydrolases (Table 2). Conversely the production of b o t h cellobiohydrolases was even higher in the egl2 deletion strain ALKO3128 (Table 2) and yet the filter paper-hydrolysing activity was clearly lower (3.7 compared with 2.9). Both strains secreted equal amounts of [3-glucosidase (Table 2), which might otherwise have affected the results. These findings strongly suggest that E G I I has a m u c h more i m p o r t a n t role than E G I in the total hydrolysis of cellulose.

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