Gene, 61 (1987) 155-164

Elsevier

155

GEN 02230

A versatile transformation system for the cellulolytic filamentous fungus Z’riclroderma reesei

(Recombinant DNA; cotransformation; lignocellulose; integration; heterologous expression)

Merja Penttilsl=, Helena Nevalainen b*, Marjaana Rltt(ie, Elina Salminenb and Jonathan Knowles”

a VTT, Biotechnical Laboratory, SF-02150 Espoo (Finland) and b Department of Cell Biology, University of Jyviiskyli, Vapaudenkatu 4, SF-40100 Jyviiskylii (Finland) Tel. (941)-29211

Received 16 July 1987

Revised 8 September 1987

Accepted 14 September 1987

SUMMARY

An eficient transformation system for the cellulolytic tilamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per pg of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia colt’ /I-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter.

INTRODUCTION

Despite the dramatic technological development that has occurred in the biological sciences recently, the genetics and molecular biology of many in- dustrially important frlamentous fungi is still poorly understood. This is in part due to the lack of a sexual reproduction cycle and the lack of a transformation

Correspondence to: Dr. M. Penttill, VTT Biotechnical Labo-

ratory, Tietotie 2, SF-02150 Espoo (Finland) Tel. (90)-4561.

* Present address: Research Laboratory, Oy Alko Ab, PL 350,

SF-00101 Helsinki (Finland), Tel. (90) 13311.

Abbreviations: /?Gal, /?-galactosidase; gpd, gene coding for

glyceraldehydephosphate dehydrogenase; MM, minimal medi-

um; PD, potato dextrose; PEG, polyethyleneglycol; XGal,

5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside.

system. Following the development of transforma- tion systems for the genetically well-studied fungi Neurospora crussa (Case et al., 1979) and A. niduluns (Ballance et al., 1983; Tilbum et al., 1983; Yelton et al., 1984), such systems have recently been developed for filamentous fungi of economical importance such as A. niger (e.g., Buxton et al., 1985; Kelly and Hynes, 1985; Hartingsveldt et al., 1987) or the antibiotic producers Penicillium chryso- genum (Cantoral et al., 1987) and Cephalosporium acremonium (Pefialva et al., 1985).

Filamentous fungi play an important role in the enzymatic hydrolysis and modification of ligno- cellulose. The mesophilic imperfect fungus T. reesei produces all the enzymes required for extensive hydrolysis of crystalline cellulose and is probably the most widely investigated of all cellulase-producing

0378-l 119/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

156

organisms. Intensive strain development using the direct approach of mutation and screening has pro- duced a number of T. reesei mutant strains secreting very large amounts of protein into the medium (reviewed by Montenecourt, 1983). Four of the T. reesei cellulase genes, cbhl (Shoemaker et al., 1983; Teeri et al., 1983), cbh2 (Teeri et al., 1987; Chen et al., 1987), egll (Penttila et al., 1986; Van Arsdell et al., 1987) and eg13 (Saloheimo et al., 1988) have been cloned and characterized.

We have developed an efficient transformation system for T. reesei which now permits the study of the expression of both homologous and heterologous genes in this organism. Transformation is based on complementation of a mutation in a T. reesei arginine auxotroph by the A. nidulans argB plasmid pSa143 (Berse et al., 1983) and on a dominant selection system based on the expression of A. nidulans amdS (Hynes et al., 1983) gene in T. reesei.

MATERIALS AND METHODS

(a) Strains and plasmids

T. reesei strains VTT-D-79125 (Bailey and Nevalainen, 1981) and QM 9414 (Mandels et al., 1971) were used as transformation hosts and strain VTT-D-79125 in preparation of auxotrophic mutants. The plasmid pSa143 (Berse et al., 1983) containing the A. nidulans argB gene was obtained from Dr. M. Skrzypek (Department of Genetics, University of Warsaw, Poland) and the amdS plas- mid p3SR2 (Hynes et al., 1983) was kindly donated by Dr. M. Hynes. The plasmid pAN5-41B (Van Gorcom et al., 1986) was kindly supplied by Dr. C.A.M.J.J. van den Hondel of the Medical Biologi- cal Laboratory TNO, Rijswijk (The Netherlands).

(b) Fungal growth media

Fungal strains were maintained on PD agar (Difco) slants under a daylight bulb. The Trichoderma MM contained (mg/ml): glucose 20, (NH,)SO, 5, KH,PO, 15, MgSO, 0.6, CaCl, 0.6, FeSO, * 7H,O 0.005, MnSO, . H,O 0.0016, ZnSO, - 7H,O 0.0014, and CoCI, 0.002. The pH was adjusted to 5.5. In all plates, 2% agar was used

as the solidifying agent. Colony growth was restricted by adding 0.1% Triton X- 100 to the medium.

For regeneration, transformed protoplasts were plated in 3 % selective top agar (or agarose) contain- ing 1 M sorbitol as osmotic stabilizer. The selective medium for amdS expression was MM glucose containing 10 mM acetamide as the sole nitrogen source instead of (NH&SO, and 12.5 mM CsCl. AmdS + transformants, cotransformed with the plasmid pAN5-41B, were screened for /?Gal expres- sion on MM glucose (pH 7) plates containing acetamide and 40 pg/ml of the colour reagent XGal (Sigma).

(c) Protoplast preparation and transformation

Cellophane disks on PD agar were inoculated with spore suspension and the fungus was grown for 20 h at 28 ‘C. The mycelium from five cellophanes was suspended into 15 ml of 1.2 M sorbitol - 100 mM potassium phosphate, pH 5.6 - solution containing 5 mg/ml of Novozym 234 (Novo) and incubated at 28 “C for approx. 1.5 h. Protoplasts were separated from undigested mycelial debris by filtering through sintered glass (porosity 1) and washed twice in 1.2 M sorbitol - 10 mM Tris . HCl, pH 7.5.

For better purification, protoplasts were prepared as described above but using 1.2 M MgSO, - 10 mM sodium phosphate buffer, pH 5.8, in protoplasting. Protoplasts were overlayed with an equal volume of 0.6 M sorbitol - 0.1 M Tris. HCl, pH 7.0, and centrifuged at 4000 x g for 15 min (Tilburn et al., 1983). Protoplasts were collected from interphase and were washed twice in 1 M sorbitol - 10 mM Tris * HCl, pH 7.5, and resuspended in 1 M sorbitol - 10 mM CaCl, - 10 mM Tris * HCl, pH 7.5, at a concentration of between 5 x 10’ - 5 x 108/ml. These protoplasts were either used immediately or stored at -70’ C until transformation. The storage of protoplasts at -70°C reduced the transformation frequencies about 50 2.

Transformation was carried out essentially as described by Ballance et al. (1983). Plasmid DNAs used for transformation were purified by centrifuga- tion once or twice in CsCl gradient. Two to live pg of transforming DNA (5 10 pg in cotransforma- tions) in 20 ~1 was mixed with 200 ~1 of protoplast suspension. 50 ~1 of 25% PEG 6000 (Fluka) - 50 mM CaCl, - 10 mM Tris * HCl, pH 7.5, was added

and the mixture was incubated on ice for 20 min. Incubation was continued at room temperature for 5 min after addition of 2 ml of the above mentioned PEG solution. Four ml of 1 M sorbitol - 10 mM CaCl, - 10 mM Tris * HCl, pH 7.5, was added. Aliquots of 100 1.11 and 500 ~1 were plated in agar overlay onto selective plates containing 1 M sorbitol. Plates were incubated at 28°C.

For determination of regeration frequencies, protoplasts were collected before and after addition of PEG and plated in agar overlay onto non-selective minimal media. Although over 90% of Trichoderma protoplasts can be regenerated if 1.0-1.2 M sorbitol is used, PEG treatment reduces the regeneration frequency to 12-35%. Sucrose (1.2 M) was also tested as a stabilizer on selection plates, but this gave two to four times lower transformation frequencies than sorbitol.

RESULTS AND DISCUSSION

(a) Isolation of arginine auxotrophs

Filtration enrichment was used for the isolation of arginine requiring auxotrophs of T. reesei from UV-mutagenized conidia. When grown in MM

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glucose medium, over 99% of the inoculated conidia were germinated and were then removed by succes- sive filtrations. The ungerminated conidia were plated on MM-arginine. 46% of the colonies germinating on this medium required a supplement of ornithine, citrulline or arginine (Table I). The remaining 54 y0 may have been arginine prototrophic conidia suffering from delayed germination. Both uracil- and tryptophan-requiring mutants were also isolated from similar enrichment experiments, although these were obtained with a lower frequency than arginine auxotrophs.

The argB gene codes for omithine carbamoyl transferase (OTCase, EC 2.1.3.3) which catalyzes the formation of citrulline from omithine and carbamoyl phosphate. Mutants defective in argB should therefore be recognizable as citrulline- requirers (Table I, Class III). Mutants of type IV should have either a block between citrulline and arginine (defective in argininosuccinate synthetase or argininosuccinase) or may possibly carry multiple mutations. In mutant types I and II the mutation was linked to formation of omithine. In addition, type II mutants seemed to be unable to use exogenous citrulline. These results are consistent with those obtained in isolation ofA. niger kg- mutants when a similar approach was used (Buxton et al., 1985).

TABLE I

Characterization of arginine requiring auxotrophs a

Mutant type” Number d Supplements b

No supplement Ornithine Citrulline Arginine

I 54 _ + + +

II 70 + +

III 90 + +

IV 383 - +

+ , growth; - , no growth.

a Auxotrophs requiring arginine were isolated after UV treatment and filtration enrichment of conidial suspensions as described by

Nevalainen (1985). A conidial suspension of T. reesei VTT-D-79125 (approx. 106/ml) was irradiated with UV-light (254 nm) for 15 min.

After this treatment the survival level was approx. 15%. After mutagenesis, conidia were grown in liquid minimal medium under constant

agitation (250 rev./min) at 28°C. The filtration enrichment method (Fries, 1947) was used to separate prototrophic growth from the

medium. Cultures were filtrated twice during the four- or five-day incubation period. The remaining conidia were pelleted, resuspended,

and plated on MM medium supplemented with arginine (100 pg/ml).

b Colonies growing on MM-arginine medium were analyzed in more detail by streaking on MM glucose plates containing ornithine (100

ng/ml), citrulline (200 fig/ml) or arginine, and grown for five to seven days at 28°C.

c The mutants were grouped into four groups (I-IV) according to their ability to grow on these plates.

d Number of mutants with the indicated growth characteristics.

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Two mutant strains, VTT-D-87305 and VTT-D-

87307, were chosen for transformation experiments.

(b} Traasformation of Trichwierma reesei with

Aspergiliw nidulans urgB plasmid

The argB - character of the two isolated Arg- mutants, VTT-D-87305 and VTT-D-87307, was confrmed by complementation of the mutation by tr~sfo~ation with the plasmid pSa143 (Fig. 1) containing the A. nidulans argB gene. Transforma- tion frequency with pSa143 was between 150-4OO/c(g of DNA. No background growth was detected and the tr~sfo~~t colonies were all roughly of equal size and sporulated reasonably well.

The Southern-blot analysis of a number of ArgB + tr~sfo~~ts is shown in Fig. 2. It can be seen that in each case, multiple tandem copies of pSa143 are integrated into the genome. The copy number of the argB gene and the sites of inte~ation appear to be different in each transformant. The difference in copy number is more clearly seen in dot hybrid- izations of total undigested DNA (Fig. 3). The results of both Southern-blot and dot-blot hybridiza- tion show that in the transformants analyzed, the copy number of argB apparently varies at least 20-fold.

pBR327

Fig. 1. The plasmid pSal43 containing the A. nidulans argB gene. Restriction sites for EcuRI (E), Sal1 (S) and BamHI (Et) are shown. Single line, pBR 327 sequences; open box, A. nidulans sequence carrying the a@ gene; striped box, S. cereviPiae LEU2 gene; stippled box, S. cerevisiae DNA. The construction of the plasmid is described by Berse et al. (1983).

(c) Transformation of Trichoderma reesei with Asprgillus niduians acetamidase gene amdS

The A. nidulans acetamidase gene nmd,S (Hynes et al., 1983) can be used as a marker in transforma- tion when tr~sfo~~ts are selected on plates containing acetamide as a sole nitrogen source and

u-l cdl v) u3 v)

5m&m~m&m5m ----b-N,--

tra 1 tra 2 tra3 tra 4 control

B‘-

E-S e

E-S -

E-S 3=f:

E-S --c

Fig. 2. Southern-blot analysis of argg ~ansform~ts of T. reesei.

Each lane contains 3 pg of total DNA from four transformants (tra l-4) and from the untransformed T. reesei control strain digested with BumHI (B) or EcoRI + Sat1 (E + S). Plasmid pSa143 was used as a 32P probe. The arrows mark the positions

of pSa143 (Fig. i), after digestion with the same enzymes (B, BumHI fragments; E-S, EcoRI-SalI fragments). DNA was isolated from T. reesei mycelia grown in liquid cultures in minimal medium supplemented with 0.2% peptone or in selective minimal medium. The method of Raeder and Broda (1985) was used, except that samples were treated with RNAse A before phenol- chloroform extraction. DNAs were run on 0.7% agarose gels, transferred to Gene Screen Plus membranes (New England Nuclear) and hybridized according to man~acturer’s instruc- tions. Probe was nick-translated (Mauiatis et al., 1982) with Klenow polymerase (Boehringer-Mannheim) using [a-32P]dCTP (Amersham). The filters were exposed on Kodak XARS film at -70°C using intensi~ng screens.

1.59

tta 1

tra2

tra4

control Fig. 3. Copy number analysis of the A. nidulans argB gene of T. reesei transformants. Indicated amounts of total undigested DNA from transformants (trai, cr&!, @al) and from the untrans- formed T. reesei (control) were used for dot hybridization. The approx. f&kb SulI fragment from pSaf43 (Fig. 1) was used as a probe. The DNA was boiled for 10 min in 0.3 M NaOH, cooled on ice, and transferred to nitroceffufose filter (Schfeicher & Schueff, BA85) using vacuum in 0.1 M Tris * HCf pH 7.5 - 1 M NH,. acetate. Hybridization was carried out essentially as de- scribed by Maniatis et al. (1982). It can be seen that the copy number of the a@ gene of the transformant tru4 is at least four times greater than that of the transformant traf, and in trans- formant rra2 at least 4 times greater than in the transformant trul. Thus, the copy number difference between the transfor- mants trul and tra2 is at feast 16-fold.

amdS, transfarmants

CsCl for reduction of background growth (Tilbum et al., 1983 ; Kelly and Hynes, 1985). Trichoderma

grows poorly on acetamide as a sole nitrogen source. This background growth can be reduced by including CsCl in the medium. Replacing agar with agarose reduces the growth of Trichoderma even further, suggesting that the residual growth is at least partly caused by impurities in agar.

T. reesei protoplasts, osmotically stabilized in 1.0 M sorbitol, were transformed with the plasmid p3SR2 (Hynes et al., 1983; Tilbum et al., 1983) which consists of the 5-kb EcoRI-Sal1 fragment containing the A. nidul~~ amdS gene and the large EcoRI-S’ufI fragment of pBR322. Tr~sform~ts of varying size were obtained (Fig. 4). Large AmdS + colonies (diam. > 5 mm) were obtained with a frequency of 50-lOO/pg of plasmid DNA used, and the smaller ones (diam. l-5 mm) with a frequency of 100-400/~g.

The observation that transformed colonies of varying size are obtained in amdS transformation has also been reported for A. nidulans (Tilbum et al., 1983; Wemars et al., 1985) and A. niger (Kelly and

control

Fig. 4. T. reesei amdS transformants on acetamide-CsCl-plates. Colonies classified as small are shown by arrows. The control plate shows the background growth on acetamide-CsCf when no amdS DNA is added to protopfasts. Transformation was carried out as described in MATERIALS AND METHODS, section C, using 4 pg of p3SR2 DNA. 100 ~1 of protoplasts were plated in selective top agar (see MATERIALS AND METHODS, section b).

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Hynes, 1985). The smaller colonies do not usually grow further after transfer to fresh selective medium and have therefore been termed as abortive trans- formants (Tilburn et al., 1983; Kelly and Hynes, 1985).

All large Trichoderma AmdS + colonies grew well when restreaked on acetamideCsC1 plates. Transfer of the small AmdS + colonies to new selective plates gave variable results. Approximately 15% of the 60 small colonies tested showed vigorous growth all over the plate, 35% grew moderately and the rest 50% only slightly better than the untransformed control strain. As Trikhoderma sporulates very poorly on acetamide, the small AmdS + colonies were streaked on PD plates and the spores obtained tested for AmdS + phenotype. From ten original small colonies, three gave spores with variable fre- quencies of AmdS + phenotype (94%) 44% and 5 %). The other seven clones gave only spores unable to grow on acetamide. Interestingly, all AmdS + spores obtained showed vigorous growth on acetamide plates, characteristic of large colony transformants.

These results are similar to those obtained by Wernars et al. (1985) with A. nidulans amdS trans- formants. They also noticed the conversion of small colonies to those showing continuous growth, and the same kind of conidial heterogeneity of AmdS + phenotype. Thus, it seems that at least a number of the small colonies became true transformants, the initial low growth rate perhaps caused by the delayed integration of transforming DNA into the chromo- some. As suggested by Wernars et al. (1985) the restoration of growth by reculturing might give more time for integration and thus result in stabilization of the AmdS + phenotype.

The presence of vector p3SR2 DNA in AmdS + clones was verified by Southern-blot analysis. Thirteen large and seven small colonies were purified on acetamide-CsC1 plates, sporulated on rich medium and grown for DNA isolation in liquid minimal medium containing acetamide as a sole nitrogen source.

Vector DNA could be detected in all large trans- formants and in two of the seven small AmdS’ colonies tested (Fig. 5). In the non-digested samples, the hybridization signal is visible in the region of chromosomal DNA, there is no indication for presence of free plasmids. Thus, it seems that, as in

tra a trab trac trad trae

A k/yI----

-21

5.0 kb - * -5.0 -4.3

3.7 kb - -3.5

3.7 kb -

Fig. 5. Southern-blot analysis of amdS transformants of 7”. reesei. Each lane contains 3 ng of DNA from big (tra a-c) and small (tra d-e) colony transformants undigested or digested with EcoRI + MI (E + S). Panel A was hybridized with the S-kb amdS-specific EcoRI + Sal1 fragment of p3SR2 and panel B with the 3.7 kb pBR322 specific EcoRI-Sal1 fragment. The arrows mark the positions ofp3SR2, digested with EcoRI + SalI. M, markers are shown on the right margin. See Fig. 2 for experimental details.

many other filamentous fungi, transformation occurs through integration into the Trichodemza genome. The hybridization pattern of the digested samples shows that several copies of p3SR2 are integrated at different locations in the genome, and that integra- tion has happened either through pBR322 or amdS sequences. From digestions with BamHI, cutting only once in p3SR2, it was concluded that integration of multiple copies occurred in tandem array (not shown). However, only one copy of intact amdS is sufftcient to result in the big colony type transfor- mant. The transformant tra-a (Fig. 5) contains one

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copy of p3SR2 integrated through pBR322 se- quences. Consequently, there would appear to be no direct correlation between the colony size of the transformants and the number of amdS genes integrated.

All seven small colony transformants tested were able to grow in selective liquid medium, although slowly, whereas the non-transformed Trichoderma control did not grow. Thus, it is possible that some amdS sequences are present but not yet in stabilized form. The reason for the lack of detectable trans- forming DNA in Southern hybridization in five out of seven of the small-colony transformants analyzed is not clear.

The extent of homology between the amdS gene and the T. reesei genome is not known. In the case of A. niger, where no homology was detected, the transforming amdS sequences were integrated as several tandem copies at different locations in the genome (Kelly and Hynes, 1985) as is shown here for Trichoderma. However, also in A. nidulans, where homology exists, integration occurs at random loca- tions in some strains studied (Wernars et al., 1985), although a preference for homologous recombination has also been reported (Tilburn et al., 1983; Wemars et al., 1985).

(d) Cotransformation with amdS and argB plasmids

Cotransformation provides a convenient means to introduce unselectable genetic material into many organisms. For this reason cotransformation of Trichoderma with a non-selectable plasmid was studied with the arginine auxotrophic mutant.

The argB - strain was transformed with equal molar amounts of A. nidulans plasmids pSa143 (argB) and p3SR2 (amdS), transformants were selected for Arg + phenotype and tested for acquisi- tion of amdS by streaking on acetamide-CsCl plates. Of the argB transformants, 86% were also AmdS + . Double selection on minimal acetamide-CsCl medium resulted in approx. 100 big Amd + Arg+ transformants per pg of DNA and a number of small colonies, characteristic of amdS transformation. Southern-blot analysis of cotransformants gave a very complex pattern which suggested that both plasmids had become integrated in variable numbers of copies at several different locations in the genome (not shown).

It is possible that the presence of homologous E. coli sequences on both plasmids used for cotrans- formation might increase the possibilities of homolo- gous recombination between the two plasmids and so lead to increased transformation frequencies. However, this was not observed, since the same

number of Arg’ transformants was obtained when the argB plasmid pSa143 was used alone or with the amdS plasmid p3SR2 in transformation.

(e) Stability of the Trichoderma reesei transfor-

mants

The argB transformants were phenotypically 100% stable through at least three generations. This was tested by successive platings of conidia from five transformants onto complete medium and thereafter testing the Arg+ phenotype on minimal medium (50-80 colonies/transformant).

In contrast, the amdS transformants showed a certain degree of mitotic instability. Ten large trans- formants of varying size were subcultured on acetamide-CsCl plates, sporulated on PD and replated on PD. To exclude heterogeneity caused by heterokaryosis, individual colonies arising from one spore were tested for AmdS + phenotype. One posi- tive clone from each original transformant was sub- jected to successive platings on non-selective PD-agar. After one cycle on non-selective medium six of the ten transformants gave 100% AmdS + conidia, three gave 4%85% and one transformant gave no AmdS + conidia at all. The result was the same after five non-selective growth cycles.

When the stability of the ArgB + phenotype of the argB amdS cotransformants was tested, three out of five proved 100% stable, whereas of the other two, one had lost the ArgB + character in 7% and the other in 80% of the progeny analysed. Interestingly,

the same individual colonies had also lost the AmdS + phenotype. Whether this instability of both markers is caused by the cointegration of argB and amdS into the same chromosomal location in these transformants is not known.

(f) Expression of Escherichia coli j?-galactosidase in

Trichoderma reesei

Van Gorcom et al. (1985; 1986) have shown that the E. coli j_?Gal can be produced in A. nidulans in

162

enzymatically active form when the 1acZ gene is coupled to either the trpC or the gpd promoter of A. nidulans. The colonies producing j?Gal can be distinguished by their deep blue colour on plates containing the chromogenic substrate XGal.

The plasmid pAN5-41B (Van Gorcom et al., 1986) which contains the E. coli 1acZ gene coupled in phase to the promoter and N-terminal protein coding region of the A. nidulans gpd gene was used in transformation of Trichoderma. Prototrophic T. reesei (QM9414) was cotransformed with the plas- mid p3SR2 (amdS) and pAN5-41B, transformants were selected for Amd + phenotype and screened for /IGal expression on acetamide-CsCl plates contain- ing XGal. No endogenous T. reesei t?Gal activity can be detected when glucose is present in the medium and pH of the XGal plates is neutral.

Certain transformed colonies developed a blue colour after between one and four days of growth. When 1.0/0.7 (p3SR2/pAN5-41B) molar ratio of the plasmids was used in transformation, 13 % of the big and 6% of the small Amd + clones showed jIGal activity. With a molar ratio of 1.0/2.6, 39% big and

7% small colonies showed the XGal+ phenotype. The presence of both plasmids in Amd’ transfor- mants showing blue colour was verified by Southern hybridization (not shown).

The apparent lower cotransformation frequency obtained with the plasmid pAN5-41B compared with the argB/amdS cotransformation (see RESULTS AND DISCUSSION, section d) may be due to the low expression level of the 1acZ gene. The colour reaction, although clearly detectable in some trans- formants, is not intense, and even lower levels of BGa.l activity would not have been detected. The intensity of the colour is not affected when transfor- mants are grown on glucose or glycerol as carbon source, or acetarnide as sole carbon and nitrogen source. This suggests that the Aspergillus gpd pro- moter is not regulated in Trichoderma.

(g) Conclusions

We have developed an efficient transformation system for the imperfect filamentous fungus T. reesei, by selection for the expression of two A. nidulans genes in this organism. The acetamidase gene amdS can be used as a dominant selectable marker in transformation of any Trichoderma strain,

and the argB gene in complementation of T. reesei argB - auxotrophs. Relatively high transformation frequencies were obtained with both selection sys- tems (loo-6OO/pg).

The characteristics of Trichoderma transformation are in general similar to those described for other lilamentous fungi, e.g., Neurospora and Aspergillus. Transformation occurs by integration of the trans- forming DNA into the recipient genome. A variable number of tandemly repeated vector sequences are integrated at a number of locations in the genome, indicating that non-homologous recombination occurs at high frequency. The frequency of cotrans- formation is also high allowing the stable integration of non-selectable DNAs into the Trichoderma chro- mosome. Moreover, the E. coli #?Gal is expressed in T. reesei in enzymatically active form.

In this work, three A. nidulans promoter se- quences, amdS, argB and gpd, were shown to give rise to gene expression in T. reesei. For amdS and argB, only one or two copies of the gene are sufficient to bring about a selectable phenotype. Whether the Trichoderma transcription system recognises the regulatory sequences in these promoters is not known. Nevertheless, there is an increasing amount of evidence to support the idea that filamentous fungal genes are often expressed at least to some extent in heterologous tilamentous fungal hosts (reviewed by Rarnbosek and Leach, 1987). It would seem that the introns present in the amdS gene (Corrick et al., 1987) and in the N-terminal coding region of the gpd gene (C.A.M.J.J. van den Hondel, pers. commun.) are processed by T. reesei. The level of expression of the gpd promoter, however, seems to be low compared to its activity in Aspergillus. Reciprocally, the T. reesei cellulase gene cbhl, containing two introns, is expressed in A. nidulans, although this too at a low level (M.P., J. Ballance, G. Turner and J.K., in preparation). However, it is probable that the chromosomal site of integration may also affect the level of gene expression and therefore the reasons for low level of expression of highly expressed genes in heterologous hosts needs further study.

In conclusion, the development of an efficient transformation system for T. reesei as described here makes possible the study of both homologous and heterologous gene expression in T. reesei. The in- formation so obtained will add to our knowledge of

163

the molecular biology of filamentous fungi and should permit the further development of these com- mercially important organisms.

ACKNOWLEDGEMENTS

We thank Dr. G. Turner and Dr. J. Ballance (Bristol University, U.K.) for much good advice and Dr. C.A.M.J.J. van den Hondel and Dr. P. Punt (Medical Biological Laboratory TNO, Rijswijk, The Netherlands) for plasmid pAN5-41B. The skilled technical assistance of Tuula Sinisalo, Seija Nordberg and Risto Poij%rvi is greatly acknowl- edged.

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Communicated by C.A.M.J.J. van den Handel.