Journal of Biotechnology, 17 (1991) 35-50
35
Elsevier BIOTEC 00573
Enzyme production by recombinant Trichoderma reesei strains Jaana M. Uusitalo 1, K.M. Helena Nevalainen z, Anu M. Harkki Jonathan K.C. Knowles ~ and Merja E. Penttil~i
1,.,
1 VTT, BiotechnicalLaboratory, Espoo, Finlandand 2 Research Laboratories, Alko Ltd., Helsinki, Finland
(Received 17 April 1990; revision accepted 20 June 1990)
Summary The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail. Cellulase; Chymosin; Filamentous fungi; Heterologous gene expression; Trichoderma reesei
Introduction
The industrially important filamentous fungus Trichoderma reesei is an efficient producer of hydrolases, especially of different cellulolytic enzymes which can Correspondence to: J. Uusitalo, VT-I', Biotechnical Laboratory, P.O. Box 202, SF-02151 Espoo, Finland. * Present address: Cultor Ltd., Technology Center, SF-02460 Kantvik, Finland. Abbreviations: ELISA, enzyme linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis;
PMSF, phenylmethylsulfonyi fluoride; SDS, sodium dodecyl sulphate; TCA, trichloroacetic acid. 0168-1656/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
36 degrade native crystalline cellulose to glucose. As a result of systematic improvement of production properties by mutagenesis and screening, the best strains today produce up to 40 g 1-1 of extracellular protein most of which consists of cellulases (Durand et al., 1988a,b). The main cellobiohydrolase, cellobiohydrolase I (CBHI) forms the major part, about 60% of the total secreted protein (Gritzali and Brown, 1979; Niku-Paavola, 1983). The proportion of cellobiohydrolase II (CBHII) is around 20% (Gritzali and Brown, 1979; Niku-Paavola, 1983) and endoglucanases, most of which is endoglucanase I (EGI), comprise 10% (Gritzali and Brown, 1979; Niku-Paavola, 1983). /3-Glucosidases account for only 1% of the total secreted protein (Niku-Paavola, 1983). In addition to enzymes for cellulose hydrolysis, hemicellulases and proteases are also produced in reasonably high amounts. Even though the amount of secreted protein has been improved in mutated strains, the proportion of the different cellulase components has remained more or less constant compared to the original strains. Mutagenesis and screening has only in few cases resulted in the isolation of T. reesei strains in which the relative amount of, for instance endoglucanase is increased (Sheir-Neiss and Montenecourt, 1984; Shoemaker et al., 1981). In addition, Tiraby (personal communication) has reported isolation of a T. reesei mutant lacking exoglucanase (exo-/3-1,4-glucanase) activity but still producing endoglucanase activity on cellulose medium in the presence of glucose. However, this strain was isolated when screening for cellulase-negative mutants. Also in this fine, most of the mutants represent classes in which both endoglucanase and exogiucanase activities are lacking and the amount of secreted protein is generally low. Thus, screening of hundreds of thousands of mutant colonies on plates without being able to specifically select changes in expression of a separate endoglucanase or exoglucanase component has not offered realistic possibilities for preparation of tailor-made enzyme mixtures for various biotechnical processes. Although knowledge about the genetic system of Trichoderma is limited, genetic engineering has provided important molecular biological data concerning Trichoderma. Genes coding for the four major cellulases of T. reesei, the two cellobiohydrolases CBHI (Shoemaker et al., 1983; Teeri et al., 1983) and CBHII (Chen et al., 1987; Teeri et al., 1987) and the two endoglucanases EGI (Penttil~i et al., 1986; van Arsdell et al., 1987) and EGII (previously EGIII) (Saloheimo et al., 1988) have been cloned and characterized, and the pgk gene coding for phosphoglycero kinase (Vanhanen et al., 1989). Transformation methods, based on both dominant and auxotrophic markers, have been developed (Penttila et al., 1987b; Durand et al., 1988a; Berges et al., 1989; Gruber et al., 1989) as well as methods for gene inactivation (Harkki et al., 1990) and targeted integration. This information now enables new approaches to the improvement and modification of the cellulolytic system of Trichoderma. The production of large amounts of cellulases by Trichoderma demonstrates that this fungus has an excellent secretion capacity. It is also noteworthy that there is only one copy of each of the major cellulase genes in the fungal genome indicating that the cellulase promoters in general, and especially that of the cbhl gene are very strong. Expression of cellulases is regulated at transcriptional level (E1-Gogary et al.,
37 1989; A. Jokinen and M. Penttil~i, in preparation). For example, the amount of cbhl specific m R N A increases at least 1000-fold in induced cultures compared to glucose repressed cultures (A. Jokinen, M.-L. Onnela and M. Penttil~i, in preparation). For these reasons, we are studying the use of the cbhl promoter to express and secrete also foreign proteins with Trichoderma. This paper reviews the use of genetic engineering to modify the cellulase profiles of Trichoderma and the development of Trichoderma as a host for production of foreign proteins. Expression and secretion of active calf chymosin in different growth conditions is described in detail.
Novel Trichoderma strains producing altered mixtures of ceilulases The present Trichoderma strains produce the separate individual cellulase components in industrially relevant amounts. For example, in a strain producing 20 g 1of extracellular protein, roughly 12 g of it would be CBHI, 4 g of CBHII, and 2 g of endoglucanases. The individual cellulases, however, differ in their specificity and activity towards different cellulosic substrates, and thus enzyme preparations enriched in or lacking a particular enzyme component would be useful for instance for the optimization of processes developed for lignocellulose modification. Table 1 lists several novel Trichoderma strains producing altered mixtures of cellulases, constructed by genetic engineering. Cellulase profiles have been changed for instance by insertional inactivation of the gene responsible for the synthesis of CBHI (VTT-D-87312; Table 1; Fig. 1; Harkki et al., 1990; Nevalainen et al.,
TABLE 1 T. reesei strains producing altered mixtures of ceilulases
Special feature
Reference
VTT-D-88358
Improved endoglucanase I production
Harkki et al., 1990 Nevalainen et al., 1990a,b
VTT-D-87312
No cellobiohydrolase I (gene inactivation)
Harkki et al., 1990 Nevalainen et al., 1990a,b
Alko 2466
Improved endoglucanase I production, no cellobiohydrolase I (gene inactivation)
M~intyl~iet al., 1989a,b Harkki et al., 1990 Nevalainen et al., 1990a,b
Alko 2654
Improved endoglucanase I production, no cellobiohydrolase I (gene replacement)
Karhunen, 1990
Alko 2566
No cellobiohydrolaseII (gene replacement)
Suominen, M~intyl~iand Nevalainen, unpublished data
T. reesei
strain
38
14 '7
12
03 " - " 1C ._
£ 8 O_ "(3 E 0 ~- 4 0 (/3 2 1
2
3
Fig. 1. Proportion of EGI of the total secreted protein produced by (1) cellulase producing mutant VTT-D-79125 (Bailey and Nevalainen, 1981), (2) VTT-D-87312, cellobiohydrolase I negative derivative of VTT-D-79125, (3) Alko 2466, EGI transformant of VTT-D-87312. (t3), Total secreted protein; (¢~), EGI protein measured by double sandwich ELISA assay (Harkki et al., 1990).
1990a,b) and by replacement of the gene coding for CBHII. The cbh2 gene has been replaced with the Aspergillus nidulans argB or the trpC gene in a T. reesei argB or tprC mutant (Alko 2566), respectively (Table 1; P. Suominen, A. M~intyRi and H. Nevalainen, unpublished data). The production of EGI by Trichoderrna has been improved by transforming a mutant strain already producing high amounts of all cellulases with a plasmid carrying the egll cDNA inserted into an expression cassette between the promoter and terminator sequences of the strongly expressed cbhl gene (VTl'-D-88358) (Table 1; Harkki et al., 1990; Nevalainen et al., 1990a,b). Using this strain the increase in EGI production was about two-fold. Further variation in the relative amounts of different cellulases has been obtained by using a cbhl negative strain as a transformation host for enhanced EGI production under the control of the cbhl promoter (Alko 2466; Table 1; Fig. 1; MantyRi et al., 1989a,b; Harkki et al., 1990; Nevalainen et al., 1990a,b), or by direct replacement of the chromosomal cbhl gene with the egll cDNA in the cbhl expression cassette with amdS as the selection marker (Alko 2654; Table 1; Karhunen, 1990). In the direct replacement studies, targeted integration of the linearized transforming vector was quite efficient, 37% of the transformants had the expression cassette integrated into the cbhl locus. Transformants show a variation in EGI production and are under more detailed investigation. The best endoglucanase producing transformants obtained by cotransformation have two- to four-fold increased EGI levels. Southern analysis of the transformants revealed integration of one copy of the expression cassette into the cbhl locus through the terminator sequences (Suominen et al., 1989; Harkki et al., 1990). This demonstrates that the cbhl promoter can be successfully used to enhance expression of other cellulases and it also suggests that for optimal expression the cassette should be integrated into the cbhl locus.
39 The T. reesei strain producing no CBHI has proven to be suitable for full scale operations. The enzyme mixture produced can be used in certain applications requiring specific modification of lignocellulose. The enzyme preparations, enriched with endoglucanase are used, for example in processes in which hydrolysis of fl-glucan in raw material is desired.
Secretion of active calf chymosin Chymosin (rennin) is an aspartyl proteinase, which is used in cheese making processes. At present it is isolated from the stomachs of young calves and more economical ways of production are desirable. Chymosin is naturally synthesized as preprochymosin from which a 16 amino acid long signal sequence is cleaved off during secretion. The resulting 365 amino acid long prochymosin form is inactive but can be autocatalytically activated to a 323 amino acid long chymosin in acidic conditions of pH 4.5 (Foltmann et al., 1977; Harris et al., 1982; Moir et al., 1982). If the pH is even lower (pH 2.0), prochymosin is cleaved to another active form, pseudochymosin (338 amino acids; Pederson et al., 1979). The fact that this post-translational processing is required for the formation of active mature chymosin, allows one to study the effect of different protein fusions on the expression and secretion of prochymosin since the final protein product is always the same. To produce chymosin in T. reesei, several different expression plasmids were constructed in which the prochymosin cDNA was differently fused to the promoter, signal sequence and the terminator of the cbhl gene, and these were transformed into T. reesei (Harkki et al., 1989). It is known that CBHI is induced in media containing cellulose, sophorose, cellobiose or lactose although the natural inducer of cellulases is not known (Enari, 1983; Bisaria and Mishra, 1989). In addition, cellulase induction is subjected to catabolite repression by readily metabolized carbon sources such as glucose. To find optimal conditions for protein production directed by the cbhl promoter, the T. reesei host strain Rut-C30 (Montenecourt and Eveleigh, 1979) and a chymosin producing clone VTF-D-88360 (pAMH102/3; Harkki et al., 1989) which had been transformed with a plasmid carrying the cbhl promoter and signal sequence fused to the prochymosin cDNA, were cultivated in different carbon and nitrogen sources in shake flasks (Table 2). All the components in the media except cellulose were soluble in order to avoid possible binding of chymosin to solid particles. Production of cellulases was measured as filter paper activity (FPA) at different time points of growth (Fig. 2). Even though the FPA-values differ slightly between the host and the transformant strain, induction of cellulase expression seems to occur similarly in these two strains. Highest final activity is obtained in SF-medium, although in LSG-medium cellulase production is observed earlier (Fig. 2). The production seems to reflect growth rate: in some of the media Trichoderma grows better and also produces more cellulases. If the cellulase activities are correlated with the biomass produced,
40 T ABLE 2 Different carbon and nitrogen source combinations used in optimization of protein production under the cbhl promoter and milk coagulation activities obtained Medium a
(SF) (WP) (CP) (LSG) (LP) (BP) (GSG) (GP)
Milk coagulation activities (mg 1-1 ) b Cultivation time (h)
Cellulose Whey extract Cellobiose Lactose Lactose Bran extract Glucose Glucose
+ peptone +peptone + peptone + spent grain extract + peptone + peptone + spent grain extract + peptone
48
96
120
0.3 -
0.8 0.7 -
0.3 0.3 -
a Cellulose used was crystalline Solka floc. The concentration of carbon source was 10 g 1-~, that of proteose peptone (Difco) 2 g 1-1 and of spent grain extract 5 g 1-1. The other components in the medium were, in g 1-1: KH2PO 4 15, (NH4)2SO 4 5, M g S O a - 7 H 2 0 0.6, CaC12.2H20 0.6, F e S O 4 . 7 H 2 0 0.005, MnSO4-H20 0.0016, Z n S O 4 . 7 H 2 0 0.0014, COC12-6H20 0.0037. pH of the me di um was adjusted to 4.8. 50 ml of the medium was inoculated with 5 × 106 spores and the fungus was cultivated in shake flasks 200 rpm at 28°C. b Milk coagulation activities were measured according to Bailey and Siika-aho (1988). The strain used was a chymosin producing transformant VTT-D-88360.
no clear difference in the inducing effect of the different growth conditions was noticed. As expected, cellulase production is low in GP-medium and probably occurs only after glucose has been consumed by the fungus below the repressing
A
B
/
lOO
100
/
It- 50
24
5(2
48
I 72
I 96
Cultivation time (h)
t 120
24
48
72
916
120
Cultivation time (h)
Fig. 2. Relative filter paper activities (FPA) (IUPAC, 1987) obtained in SF- ( x ), WP- (o), CP- (e), LSG(a), LP- (A), BP- (*), GSG- (E3) and GP- (m) media. Relative FPA values were calculated from glucose (rag) produced using SF cultures (120 h) as reference (FPA = 100, corresponds to 0.24 FPU m l - l ) . (A) host strain T. reesei Rut-C30, (B) chymosin producing strain VTT-D-88360.
41 SF
WP
proc hyrnosin chyrnosin
CP
GP
LSG
_ --
C
48
96
120
48
96
120
48
96
120
48
96
120
48
96
120
H
Fig. 3. Western blot analysis of chymosin produced in different media in shake flask cultivation with transformant VTT-D-88360. Symbols used above are the same as used in Table 2. Growth time in h is indicated under the lane. H = host strain T. reesei Rut-C30, C = purified calf chymosin. Proteins were separated on a 7-15% gradient SDS-PAGE (Laemmli, 1970), electroblotted onto nitrocellulose filters, treated with chymosin specific antibodies (kindly obtained from M. Hansen, NOVO A/S) and with alkaline phosphatase-coupled a-antibody (Promega Biotec), and stained by the ProtoBlot Immunoblotting System (Promega Biotec). Proteins were concentrated 15-20-fold depending on growth by TCA precipitation and diluted into 2 M NaOH-10 mM Tris, pH 8.5.
concentration. Reasonable amounts of cellulase were produced in SF- and WPmedium. Similar results have been obtained earlier by Bailey and Oksanen (1984). Extracellular chymosin activity produced by strain VTT-D-88360 was measured using a milk coagulation assay (Bailey and Siika-aho, 1988). This assay cannot detect small amounts of chymosin. Nevertheless, chymosin expression from the cbhl promoter seems to be induced in a similar way as cellulase expression in these different media (Table 2). Unexpectedly, no chymosin activity could be observed in SF-medium. However, chymosin is clearly produced (see Fig. 3), and thus might be bound to cellulose and consequently not be detectable with the activity assay. Secretion of chymosin was also studied by Western blotting using chymosinspecific antibodies (Fig. 3). After 48 h of growth in SF- and WP-medium both prochymosin and chymosin can be detected in the medium, whereas in LSG- and CP-medium, the processed chymosin form is clearly prominent. This can be explained by the effect of culture pH on the autocatalytic processing of prochymosin. In SF- and WP-medium, the pH value of the culture is still over 4.5 while in both LSG- (due to rapid growth) and in CP-medium, pH has already dropped to a value of 3.2. After 96 h of growth, the protein produced has been cleaved to the mature chymosin form also in the SF- and WP-medium. The highest amounts of active extracellular chymosin were detected after 96 h of growth (Table 2). After that the amount begins to decrease and in CP- and LSG-medium also degradation products can be seen (Fig. 3). This might indicate autodegradation of chymosin under these conditions or the action of host proteases.
42
C
~C
°
_~a_
/2/ 5
"~ 4£- g ~-' 4 3£~- ~ "~ 3
,,'-. . . . .
.~ 20
24
I
I
48
72
96
120
Cultivation time Ch]
70-
7~-
B
C 12
8 ~ 4c
C
~
8
ac ~
6
4
t~ 1 c - c o
1
# =>
oo
2
i 24
48
72
96
120
144
168
Cultivation time (h)
Fig. 4. Bioreactor cultivation (10 1) of chymosin producing transformants; (A) VTT-D-88360 (B) VTT-D-88361. The medium contained, in g l - l : whey extract 60, proteose peptone (Difco) 10, yeast extract (Difco) 5, (NH4)2SO 4 5, KH2PO 4 5, MgSO4.7H20 0.5, CaC12-2H20 0.5. Temperature (33°C for 48 h, subsequently 29°C), pH (>3.5, controlled by addition of NH4OH ) and pO 2 (_>_30%, controlled by agitation speed at a constant aeration rate of 5 1 min-1) were automatically controlled. The inoculum was grown in half-strength medium (buffered with 15 g 1 1 KH2PO4 ) in two stages of 50 ml for 3 d and after that 5 × 200 ml for 1 d. (zx) Reducing sugars, g I i (Miller, 1959) (e) Dry weight, g 1-1 (o) Milk coagulation activity, mg 1-1 (Bailey and Siika-aho, 1988) (t~) Soluble protein, g 1-1 (Lowry et al., 1951) (. . . . . . ) FPU, U m1-1 (IUPAC, 1987).
43 WP-medium was chosen to be used in bioreactor cultivations of two chymosin producing strains, strain VT-F-D-88360 described above and strain VTT-D-88361 (pAMH 104/15; Harkki et al., 1989). Like strain VTT-D-88360, strain VTT-D-88361 had also been transformed with the prochymosin cDNA fused to the promoter and signal sequence of the cbhl gene, but in this strain, the plasmid used for transformation additionally carried the selectable marker amdS (Harkki et al., 1989). Highest amounts of chymosin were obtained after 72 h of growth in the bioreactor (Fig. 4). After this time point, the amount of active chymosin began to decrease as previously noticed in shake flask cultivations. Also inactive prochymosin appeared to be degraded as judged by Western blots (Fig. 5A, C). The amount of intracellular chymosin at different time points of growth follows the profile of extracellular chymosin. However, the peak in chymosin levels seems to occur slightly earlier inside the cell than in the medium (Fig. 5 B, D). Intracellular chymosin is present in both prochymosin and chymosin forms indicating that autocatalytic processing partly occurs already inside the cell but is completed only in the culture medium where pH might be more optimal for processing. Transformant VTT-D-88361 produced about four times less cellulases, measured as FP-activity, in the bioreactor compared to transformant VTT-D-88360. Most probably due to the very large quantities of cellulases in the culture medium, the chymosin specific antibodies also detect cellulase specific bands in Western analysis (see Fig. 5A). The culture medium of strain VTT-D-88361 lacks, however, the major cellulase band (Fig. 5C) indicating that CBHI is not produced. This has been confirmed by Northern, and Southern analysis (Harkki et al., 1989) which also shows that in this strain, one copy of the chymosin expression cassette has been integrated into the cbhl locus and has inactivated the endogenous cbhl gene. The yields of active chymosin obtained with strains VTT-D-88360 and VTT-D88361 in WP-medium in the bioreactor were 7.3 mg 1 i and 13 mg 1-1, respectively. In SF-medium, in the bioreactor, the amounts of active chymosin were even higher, 17 mg 1-1 and 40 mg 1-1, respectively (Harkki et al., 1989). The amount of chymosin decreases only slightly if at all during prolonged cultivation in SF-medium (M. Bailey, unpublished). The yields obtained with these chymosin producing T. reesei transformants are comparable, or even higher than the amounts described for chymosin expressing Aspergillus nidulans strains (Cullen et al., 1987). It is obvious however, that chymosin is not produced with the same efficiency as CBHI by Trichoderma, even when the expression cassette is integrated into the cbhl locus. The amount of chymosin specific mRNA is only 1-5% of that of the cbhl mRNA in the transformant VTT-D-88360 (Harkki et al., 1989). Whether this is caused by instability of the chymosin mRNA or a lower transcription rate of the hybrid gene is under investigation. Chymosin expression could be improved in several ways. Longer fusions to the CBHI protein than described in this article could be advantageous. When prochymosin is fused to the signal sequence and in addition includes 20 N-terminal amino acids of the mature CBHI protein higher yields are obtained than using other DNA constructs carrying shorter CBHI ~equences (Harkki et al., 1989). The yields
44
A 69--
46-_ prochymosin chymosin
30--
22
31
44
54
75
97
117
C u l t i v a t i o n time (h)
B 69
46
prochymosin chymosin
30
C
22
31
44
54
Cultivation time
75
97
117
(h)
Fig. 5. Western analysis of chymosin-specific polypeptides produced in bioreactor cultivations (Fig. 4A, B). (A) strain VTT-D-88360, culture supernatant, (B) strain VTT-D-88360, mycelial extract, (C) strain VTT-D-88361, culture supernatant, (D) strain VTT-D-88361, mycelial extract. Growth time in h is indicated under the lane. The sizes of the molecular weight markers (kDa) used are shown on the left. C = purified calf chymosin (20 ng). SDS-PAGE and electroblotting were carried out as described in Fig. 3. Filters were treated with chymosin-specific antibodies and 125I-labelled protein A (Batteiger et al., 1982). Mycelial proteins were concentrated 30-fold by T C A precipitation and diluted into 2 M NaOH-10 m M Tris, pH 8.5. The intracellular proteins were extracted by grinding the freeze-dried mycelium in liquid nitrogen using a mortar and pestle. 0.05 M sodium citrate buffer, pH 6.0 containing 2% SDS and 1 m M PMSF was added, mixed thoroughly and incubated at room temperature for 30 min. The lysate was centrifuged to remove the cell debris.
45
C 69--
46-_
prochymosin
-- chymosin
30--
19
28
43
53
Cultivation
67
77
96
1 2 0 167
time(h)
D 69--
46-_
prochymosin
-- chymosin
30--
C
19
28
43
53
67
77
96
120
167
Cultivation time (h)
Fig. 5 (continued).
of chymosin would also be increased by screening for more efficient producers after mutagenesis of the strain, using a protease negative strain as a host, or possibly by increasing the copy number of the chymosin expression cassette in the strain.
46 Conclusions
Cellulases are presently used in starch processing, animal feed applications, grain alcohol production, malting and brewing, and in extraction of vegetable juices where the enzyme should be effective in hydrolysis of the raw material. In other types of applications such as wood and textile processing, controlled modification of lignocellulose or fabric must be carried out without too much hydrolysis of the processable material. Thus cellulase preparations meet different requirements in different applications. The application of genetic engineering to Trichoderma has made it possible to modulate cellulase production in such a way that new T. reesei strains producing novel cellulase profiles of commercial potential are now available. Several different combinations have been made without changing the viability of the organism. More detailed knowledge of the activity and substrate specificity of the individual cellulase enzymes would enable further improvements in application of cellulases in biotechnology. Now that the three dimensional structure of the catalytic subunit of C B H I I (Bergfors et al., 1989; Rouvinen et al., 1989, 1990) and of the terminal tail domain of CBHI (Kraulis et al., 1989), as well as expression systems for both Trichoderrna and yeast (Penttil~i et al., 1987a, 1988; Zurbriggen et al., 1990a,b) are available, a detailed investigation of the structure-function relationship of these enzymes is possible. The combination of genetic and protein engineering is likely to lead to novel developments in utilization of cellulosic materials in future. The production of calf chymosin with Trichoderma shows that this fungus is a useful organism not only for production of cellulases but also for proteins of heterologous origin. Chymosin was efficiently secreted and processed to the correct active form and appears to be non-glycosylated. As shown by Ward (1989) the yields of a foreign protein can be greatly improved by further mutagenesis and screening. Although some proteins might be difficult to express in heterologous hosts, such as the heme requiring lignin peroxidase of Phlebia radiata in Trichoderma (Saloheimo et al., 1989), filamentous fungi have proven promising alternatives to the other existing production organisms. To investigate further the capability of Trichoderma for heterologous protein production we are analysing the properties of the recombinant chymosin in detail and studying the expression and secretion of several other eukaryotic proteins in Trichoderma.
References
Bailey, M.J. and Nevalainen, K.M.H. (1981) Induction, isolation and testing of stable Trichoderma reesei mutants with improved production of solubilizing cellulase. Enzyme Microb. Technol. 3, 153-157. Bailey, M.J. and Oksanen, J. (1984) Cellulase production by mutant strains of Trichoderma reesei on non-cellulosic media. In: Proceedings of the 3rd European Congress in Biotechnology, vol II, Verlag Chemie GmbH, Mtinchen, pp. 157-162. Bailey, M.J. and Siika-aho, M. (1988) Production of microbial rennin. Biotechnol. Lett. 10, 161-166. Batteiger, B., Newhall, W.J. and Jones, R.B. (1982) The use of Tween as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods 55, 297-307.
47 Berges, T., Barreau, C. and Begueret, J. (1989) Development of transformation systems for the cellulolytic fungus Trichoderma reesei. Tricel 89, Int. Symp. Trichoderma Cellulases, Vienna, Austria, 14.-16.9.1989, Abstract no. P17, p. 21. Bergfors, T., Rouvinen, J., Lehtovaara, P., Caldentey, X., Tomme, P., Claeyssens, M., Pettersson, G., Teeri, T.T., Knowles, J. and Jones, T.A. (1989) Crystallization of the core protein of cellobiohydrolase II from Trichoderma reesei. J. Mol. Biol. 209, 167-169. Bisaria, V.S. and Mishra, S. (1989) Regulatory aspects of cellulase biosynthesis and secretion. CRC Crit. Rev. Biotechnol. 9, 61-103. Chen, C.M., Gritzali, M. and Stafford, D.W. (1987) Nucleotide sequence and deduced primary structure of cellobiohydrolase II of Trichoderma reesei. Bio/Technology 5, 274-278. Cullen, D., Gray, G.L., Wilson, L.J., Hayenga, K.J., Lamsa, M.H., Rey, M.W., Norton, S. and Berka, R.M. (1987) Controlled expression and secretion of bovine chymosin in Aspergillus nidulans. Bio/Technology 5, 369-376. Durand, H., Baron, M., Calmels, T. and Tiraby, G. (1988a) Classical and molecular genetics applied to Trichoderma reesei for the selection of improved cellulolytic industrial strains. In: Aubert, J.-P., Beguin, P. and Millet, J. (Eds.) Biochemistry and Genetics of Cellulose Degradation, New York, Academic Press, pp. 135-151. Durand, H., Clanet, M. and Tiraby, G. (1988b) Genetic improvement of Trichoderma reesei for large scale enzyme production. Enzyme Microb. Technol. 10, 341-345. El-Gogary, S., Leite, A., Crivellaro, O., Eveleigh, D.E. and E1-Dorry, H. (1989) Mechanism by which cellulose triggers cellobiohydrolase I gene expression in Trichoderma reesei. Proc. Natl. Acad. Sci. USA 86, 6138-6141. Enari, T.-M. (1983) Microbial Cellulases. In: Fogarty, W.M. (Ed.), Microbial Enzymes and Biotechnology, Applied Science Publishers, London and New York, pp. 183-223. Foltmann, B., Pedersen, V.B., Jacobsen, H., Kauffman, D. and Wybrandt, G. (1977) The complete amino acid sequence of procbymosin. Proc. Natl. Acad. Sci. USA 74, 2321-2324. Gritzali, M. and Brown, R.D., Jr. (1979) The cellulase system of Trichoderma. The relationship between purified extracellular enzymes from induced or cellulose-grown cells. Adv. Chem. Ser. 81, 237-260. Gruber, F., de Graaff, L.H., Visser, J. and Kubicek, C.P. (1989) Isolation of an OMP-decarboxylase deficient mutant strain of Trichoderma reesei QM 9414 and its complementation with the corresponding genes of Aspergillus niger and Neurospora crassa. Tricel 89, Int. Symp. Trichoderrna Cellulases, Vienna, Austria, 14.-16.9.1989, Abstract no P16, p. 21. Harkki, A., Uusitalo, J., Bailey, M., Penttil~i, M. and Knowles, J.K.C. (1989) A novel fungal expression system: secretion of active calf chymosin from the filamentous fungus Trichoderma reesei. Bio/Technology 7, 596-603. Harkki, A., Mantyl~, A., Penttila, M., Muttilainen, S., Biihler, R., Suominen, P., Knowles, J. and Nevalainen, H. (1990) Genetic engineering of Trichoderma to produce strains with novel cellulase profiles. Enzyme Microb. Technol. In press. Harris, T.J.R., Lowe, P.A., Lyons, A., Thomas, P.G., Eaton, M.A.W., Millican, T.A., Patel, T.P., Bose, C.C., Carey, N.H. and Doel, M.T. (1982) Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin. Nucl. Acids Res. 10, 2177-2187. IUPAC (International Union of Pure and Applied Chemistry) (1987) Measurement of cellulase activities. Pure and Appl. Chem. 59, 257-268. Karhunen, T. (1990) Trichoderma reesei -homeen endoglukanaasi I : n tuoton tehostaminen geeniteknisesti. MSc. thesis, University of Helsinki. Kraulis, P.J., Clore, G.M., Nilges, M., Jones, T.A., Pettersson, G., Knowles, J. and Gronenborn, A.M. (1989) Determination of the three dimensional structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. Biochemistry 28, 7241-7257. Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265-275. Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426-428.
48 Moir, D., Mao, J., Schumm, J.W., Vovis, G.F., Alford, B.L. and Taunton-Rigby, A. (1982) Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin. Gene 19, 127-138. Montenecourt, B.S. and Eveleigh, D.E. (1979) Selective screening methods for the isolation of high yielding mutants of Trichoderma reesei. Adv. Chem. Ser. 181,289-301. M~intyla, A., Harkki, A., Penttil~i, M., Muttilainen, S., Btihler, R., Vaara, T., Knowles, J. and Nevalainen, H. (1989a) Construction of Trichoderma reesei strains with new cellulase profiles. In: EMBO-Alko Workshop Molecular Biology of Filamentous Fungi, Espoo, Finland, 2.-7.7.1989, Abstract no. P43, p. 40. Mantyla, A., Harkki, A., Penttila, M., Muttilainen, S., Suominen, P., Biihler, R., Vaara, T., Knowles, J. and Nevalainen, H. (1989b) Recombinant Trichoderma reesei strains producing new cellulase mixtures. Tricel 89, Int. Symp. Trichoderma Cellulases, Vienna, Austria, 14.-16.9.1989, Abstract no P14, p. 20. Nevalainen, H., Harkki, A., Penttil~i, M., Saloheimo, M., Teeri, T. and Knowles, J. (1990a) Trichoderma reesei as a production organism for enzymes for the pulp and paper industry. In: Biotechnology of the Pulp and Paper Manufacture, Butterworth Publishers, Boston, pp. 577 583. Nevalainen, H., Penttil~i, M., Harkki, A., Teeri, T. and Knowles, J. (1990b) The molecular biology of Trichoderrna and its application to the expression of both homologous and heterologous genes. In: Leong, S.A. and Berka, R. (Eds), Molecular Industrial Mycology, Marcel Dekker Inc., New York. In press. Niku-Paavola, M.-L. (1983) Biochemistry of Trichoderma reesei VTT-D-80133 cellulases. In: Proceedings of the Soviet Union - Finland Seminar on Bioconversion of Plant Raw Materials by Micro-organisms. Tashkent, pp. 26-31. Pederson, V.B., Christensen, K.A. and Foltmann, B. (1979) Investigations on the activation of bovine prochymosin. Eur. J. Biochem. 94, 573-580. Penttil~i, M., Lehtovaara, P., Nevalainen, H., Bhikhabhai, R. and Knowles, J. (1986) Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene. Gene 45, 253-263. Penttil~i, M.E., Andrr, L., Saloheimo, M., Lehtovaara, P. and Knowles, J.K.C. (1987a) Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae. Yeast 3, 175 185. Penttil~i, M., Nevalainen, H., R~ittiS, M., Salminen, E. and Knowles, J. (1987b) A versatile transformation system for the cellulolytic filamentous fungus Triehoderma reesei. Gene 61, 155-164. Penttil~i, M.E., Andrr, L., Lehtovaara, P., Bailey, M., Teeri, T.T. and Knowles, J.K.C. (1988) Efficient secretion of two fungal cellobiohydrolases in Saccharomyces cerevisiae. Gene 63, 103-112. Rouvinen, J., Bergfors, T., Pettersson, G., Knowles, J. and Jones, T.A. (1989) Crystallographic studies on the core protein of cellobiohydrolase II from Trichoderma reesei. First European Workshop on Crystallography of Biological Macromolecules, Como, Italy, 15.-19.5.1989. Rouvinen, J., Bergfors, T., Teeri, T.T., Knowles, J.K.C. and Jones, T.A. (1990) Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei. Science 249, 380-386. Saloheimo, M., Lehtovaara, P., Penttila, M., Teeri, T.T., St~ihlberg, J., Johansson, G., Pettersson, G., Claeyssens, M., Tomme, P. and Knowles, J.K.C. (1988) EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme. Gene 63, 11-21. Saloheimo, M., Barajas, V., Niku-Paavola, M.-L. and Knowles, J.K.C. (1989) A lignin peroxidase-encoding cDNA from the white-rot fungus Phlebia radiata: characterization and expression in Trichoderma reesei. Gene 85, 343-351. Sheir-Neiss, G. and Montenecourt, B.S. (1984) Characterization of the secreted cellulases of Trichoderma reesei wild type and mutants during controlled fermentations. Appl. Microbiol. Biotechnol. 20, 46-53. Shoemaker, S.P., Raymond, J.C. and Brunet, R. (1981) Cellulases: diversity amongst improved Trichoderma strains. In: Hollander, A.E. (Ed.), Trends in the Biology of Fermentations for Fuels and Chemicals, Plenum Press, New York, pp. 89-109. Shoemaker, S., Schweickart, V., Ladner, M., Gelfand, D., Kwok, S., Myambo, K. and Innis, M. (1983) Molecular cloning of exocellobiohydrolase from Trichoderma reesei strain L27. Bio/Technology 1, 691-696.
49 Suominen, P., M~intyl~i,A. and Nevalainen, H. (1989) DNA-analysis of a recombinant Trichoderma reesei strain with altered ceUulase production. In: EMBO Alko Workshop Molecular Biology of Filamentous Fungi, Espoo, Finland, 2.-7.7. 1989, Abstract no. P44, p. 41. Teed, T., Salovuori, I. and Knowles, J. (1983) The molecular cloning of the major cellulase gene from Trichoderma reesei. Bio/Technology 1,696-699. Teed, T.T., Lehtovaara, P., Kauppinen, S., Salovuori, I. and Knowles, J. (1987) Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II. Gene 51, 43-52. Van Arsdell, J.N., Kwok, S., Schweikart, V.L., Ladner, M.B., Gelfand, D.H. and Innis, M.A. (1987) Cloning, characterization, and expression in Saccharomyces cerevisiae of endoglucanase I from Trichoderrna reesei. Bio/Technology 5, 60-64. Vanhanen, S., Penttila, M., Lehtovaara, P. and Knowles, J. (1989) Isolation and characterization of the 3-phosphoglycerate kinase gene ( p g k ) from filamentous fungus Trichoderma reesei. Curr. Genet. 15, 181-186. Ward, M. (1989) Heterologous gene expression in Aspergillus. In: Nevalainen, H. and Penttil~i, M. (Eds), Proceedings of the EMBO-Alko Workshop on Molecular Biology of Filamentous Fungi, Foundation for Biotechnical and Industrial Fermentation Research 6, pp. 119-128. Zurbdggen, B., Bailey, M.J., Penttil~i, M.E., Poutanen, K. and Linko, M. (1990) Pilot scale production of a heterologous Trichoderma reesei cellulase by Saccharomyces cerevisiae. J. Biotechnol. 13, 267-278. Zurbdggen, B.D., Penttil~i, M.E., Viikari, L. and Bailey, M.J. (1990b) Pilot scale production of a Trichoderma reesi endo-fl-glucanase by brewer's yeast. J. Biotechnol. In press.
35
Elsevier BIOTEC 00573
Enzyme production by recombinant Trichoderma reesei strains Jaana M. Uusitalo 1, K.M. Helena Nevalainen z, Anu M. Harkki Jonathan K.C. Knowles ~ and Merja E. Penttil~i
1,.,
1 VTT, BiotechnicalLaboratory, Espoo, Finlandand 2 Research Laboratories, Alko Ltd., Helsinki, Finland
(Received 17 April 1990; revision accepted 20 June 1990)
Summary The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail. Cellulase; Chymosin; Filamentous fungi; Heterologous gene expression; Trichoderma reesei
Introduction
The industrially important filamentous fungus Trichoderma reesei is an efficient producer of hydrolases, especially of different cellulolytic enzymes which can Correspondence to: J. Uusitalo, VT-I', Biotechnical Laboratory, P.O. Box 202, SF-02151 Espoo, Finland. * Present address: Cultor Ltd., Technology Center, SF-02460 Kantvik, Finland. Abbreviations: ELISA, enzyme linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis;
PMSF, phenylmethylsulfonyi fluoride; SDS, sodium dodecyl sulphate; TCA, trichloroacetic acid. 0168-1656/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
36 degrade native crystalline cellulose to glucose. As a result of systematic improvement of production properties by mutagenesis and screening, the best strains today produce up to 40 g 1-1 of extracellular protein most of which consists of cellulases (Durand et al., 1988a,b). The main cellobiohydrolase, cellobiohydrolase I (CBHI) forms the major part, about 60% of the total secreted protein (Gritzali and Brown, 1979; Niku-Paavola, 1983). The proportion of cellobiohydrolase II (CBHII) is around 20% (Gritzali and Brown, 1979; Niku-Paavola, 1983) and endoglucanases, most of which is endoglucanase I (EGI), comprise 10% (Gritzali and Brown, 1979; Niku-Paavola, 1983). /3-Glucosidases account for only 1% of the total secreted protein (Niku-Paavola, 1983). In addition to enzymes for cellulose hydrolysis, hemicellulases and proteases are also produced in reasonably high amounts. Even though the amount of secreted protein has been improved in mutated strains, the proportion of the different cellulase components has remained more or less constant compared to the original strains. Mutagenesis and screening has only in few cases resulted in the isolation of T. reesei strains in which the relative amount of, for instance endoglucanase is increased (Sheir-Neiss and Montenecourt, 1984; Shoemaker et al., 1981). In addition, Tiraby (personal communication) has reported isolation of a T. reesei mutant lacking exoglucanase (exo-/3-1,4-glucanase) activity but still producing endoglucanase activity on cellulose medium in the presence of glucose. However, this strain was isolated when screening for cellulase-negative mutants. Also in this fine, most of the mutants represent classes in which both endoglucanase and exogiucanase activities are lacking and the amount of secreted protein is generally low. Thus, screening of hundreds of thousands of mutant colonies on plates without being able to specifically select changes in expression of a separate endoglucanase or exoglucanase component has not offered realistic possibilities for preparation of tailor-made enzyme mixtures for various biotechnical processes. Although knowledge about the genetic system of Trichoderma is limited, genetic engineering has provided important molecular biological data concerning Trichoderma. Genes coding for the four major cellulases of T. reesei, the two cellobiohydrolases CBHI (Shoemaker et al., 1983; Teeri et al., 1983) and CBHII (Chen et al., 1987; Teeri et al., 1987) and the two endoglucanases EGI (Penttil~i et al., 1986; van Arsdell et al., 1987) and EGII (previously EGIII) (Saloheimo et al., 1988) have been cloned and characterized, and the pgk gene coding for phosphoglycero kinase (Vanhanen et al., 1989). Transformation methods, based on both dominant and auxotrophic markers, have been developed (Penttila et al., 1987b; Durand et al., 1988a; Berges et al., 1989; Gruber et al., 1989) as well as methods for gene inactivation (Harkki et al., 1990) and targeted integration. This information now enables new approaches to the improvement and modification of the cellulolytic system of Trichoderma. The production of large amounts of cellulases by Trichoderma demonstrates that this fungus has an excellent secretion capacity. It is also noteworthy that there is only one copy of each of the major cellulase genes in the fungal genome indicating that the cellulase promoters in general, and especially that of the cbhl gene are very strong. Expression of cellulases is regulated at transcriptional level (E1-Gogary et al.,
37 1989; A. Jokinen and M. Penttil~i, in preparation). For example, the amount of cbhl specific m R N A increases at least 1000-fold in induced cultures compared to glucose repressed cultures (A. Jokinen, M.-L. Onnela and M. Penttil~i, in preparation). For these reasons, we are studying the use of the cbhl promoter to express and secrete also foreign proteins with Trichoderma. This paper reviews the use of genetic engineering to modify the cellulase profiles of Trichoderma and the development of Trichoderma as a host for production of foreign proteins. Expression and secretion of active calf chymosin in different growth conditions is described in detail.
Novel Trichoderma strains producing altered mixtures of ceilulases The present Trichoderma strains produce the separate individual cellulase components in industrially relevant amounts. For example, in a strain producing 20 g 1of extracellular protein, roughly 12 g of it would be CBHI, 4 g of CBHII, and 2 g of endoglucanases. The individual cellulases, however, differ in their specificity and activity towards different cellulosic substrates, and thus enzyme preparations enriched in or lacking a particular enzyme component would be useful for instance for the optimization of processes developed for lignocellulose modification. Table 1 lists several novel Trichoderma strains producing altered mixtures of cellulases, constructed by genetic engineering. Cellulase profiles have been changed for instance by insertional inactivation of the gene responsible for the synthesis of CBHI (VTT-D-87312; Table 1; Fig. 1; Harkki et al., 1990; Nevalainen et al.,
TABLE 1 T. reesei strains producing altered mixtures of ceilulases
Special feature
Reference
VTT-D-88358
Improved endoglucanase I production
Harkki et al., 1990 Nevalainen et al., 1990a,b
VTT-D-87312
No cellobiohydrolase I (gene inactivation)
Harkki et al., 1990 Nevalainen et al., 1990a,b
Alko 2466
Improved endoglucanase I production, no cellobiohydrolase I (gene inactivation)
M~intyl~iet al., 1989a,b Harkki et al., 1990 Nevalainen et al., 1990a,b
Alko 2654
Improved endoglucanase I production, no cellobiohydrolase I (gene replacement)
Karhunen, 1990
Alko 2566
No cellobiohydrolaseII (gene replacement)
Suominen, M~intyl~iand Nevalainen, unpublished data
T. reesei
strain
38
14 '7
12
03 " - " 1C ._
£ 8 O_ "(3 E 0 ~- 4 0 (/3 2 1
2
3
Fig. 1. Proportion of EGI of the total secreted protein produced by (1) cellulase producing mutant VTT-D-79125 (Bailey and Nevalainen, 1981), (2) VTT-D-87312, cellobiohydrolase I negative derivative of VTT-D-79125, (3) Alko 2466, EGI transformant of VTT-D-87312. (t3), Total secreted protein; (¢~), EGI protein measured by double sandwich ELISA assay (Harkki et al., 1990).
1990a,b) and by replacement of the gene coding for CBHII. The cbh2 gene has been replaced with the Aspergillus nidulans argB or the trpC gene in a T. reesei argB or tprC mutant (Alko 2566), respectively (Table 1; P. Suominen, A. M~intyRi and H. Nevalainen, unpublished data). The production of EGI by Trichoderrna has been improved by transforming a mutant strain already producing high amounts of all cellulases with a plasmid carrying the egll cDNA inserted into an expression cassette between the promoter and terminator sequences of the strongly expressed cbhl gene (VTl'-D-88358) (Table 1; Harkki et al., 1990; Nevalainen et al., 1990a,b). Using this strain the increase in EGI production was about two-fold. Further variation in the relative amounts of different cellulases has been obtained by using a cbhl negative strain as a transformation host for enhanced EGI production under the control of the cbhl promoter (Alko 2466; Table 1; Fig. 1; MantyRi et al., 1989a,b; Harkki et al., 1990; Nevalainen et al., 1990a,b), or by direct replacement of the chromosomal cbhl gene with the egll cDNA in the cbhl expression cassette with amdS as the selection marker (Alko 2654; Table 1; Karhunen, 1990). In the direct replacement studies, targeted integration of the linearized transforming vector was quite efficient, 37% of the transformants had the expression cassette integrated into the cbhl locus. Transformants show a variation in EGI production and are under more detailed investigation. The best endoglucanase producing transformants obtained by cotransformation have two- to four-fold increased EGI levels. Southern analysis of the transformants revealed integration of one copy of the expression cassette into the cbhl locus through the terminator sequences (Suominen et al., 1989; Harkki et al., 1990). This demonstrates that the cbhl promoter can be successfully used to enhance expression of other cellulases and it also suggests that for optimal expression the cassette should be integrated into the cbhl locus.
39 The T. reesei strain producing no CBHI has proven to be suitable for full scale operations. The enzyme mixture produced can be used in certain applications requiring specific modification of lignocellulose. The enzyme preparations, enriched with endoglucanase are used, for example in processes in which hydrolysis of fl-glucan in raw material is desired.
Secretion of active calf chymosin Chymosin (rennin) is an aspartyl proteinase, which is used in cheese making processes. At present it is isolated from the stomachs of young calves and more economical ways of production are desirable. Chymosin is naturally synthesized as preprochymosin from which a 16 amino acid long signal sequence is cleaved off during secretion. The resulting 365 amino acid long prochymosin form is inactive but can be autocatalytically activated to a 323 amino acid long chymosin in acidic conditions of pH 4.5 (Foltmann et al., 1977; Harris et al., 1982; Moir et al., 1982). If the pH is even lower (pH 2.0), prochymosin is cleaved to another active form, pseudochymosin (338 amino acids; Pederson et al., 1979). The fact that this post-translational processing is required for the formation of active mature chymosin, allows one to study the effect of different protein fusions on the expression and secretion of prochymosin since the final protein product is always the same. To produce chymosin in T. reesei, several different expression plasmids were constructed in which the prochymosin cDNA was differently fused to the promoter, signal sequence and the terminator of the cbhl gene, and these were transformed into T. reesei (Harkki et al., 1989). It is known that CBHI is induced in media containing cellulose, sophorose, cellobiose or lactose although the natural inducer of cellulases is not known (Enari, 1983; Bisaria and Mishra, 1989). In addition, cellulase induction is subjected to catabolite repression by readily metabolized carbon sources such as glucose. To find optimal conditions for protein production directed by the cbhl promoter, the T. reesei host strain Rut-C30 (Montenecourt and Eveleigh, 1979) and a chymosin producing clone VTF-D-88360 (pAMH102/3; Harkki et al., 1989) which had been transformed with a plasmid carrying the cbhl promoter and signal sequence fused to the prochymosin cDNA, were cultivated in different carbon and nitrogen sources in shake flasks (Table 2). All the components in the media except cellulose were soluble in order to avoid possible binding of chymosin to solid particles. Production of cellulases was measured as filter paper activity (FPA) at different time points of growth (Fig. 2). Even though the FPA-values differ slightly between the host and the transformant strain, induction of cellulase expression seems to occur similarly in these two strains. Highest final activity is obtained in SF-medium, although in LSG-medium cellulase production is observed earlier (Fig. 2). The production seems to reflect growth rate: in some of the media Trichoderma grows better and also produces more cellulases. If the cellulase activities are correlated with the biomass produced,
40 T ABLE 2 Different carbon and nitrogen source combinations used in optimization of protein production under the cbhl promoter and milk coagulation activities obtained Medium a
(SF) (WP) (CP) (LSG) (LP) (BP) (GSG) (GP)
Milk coagulation activities (mg 1-1 ) b Cultivation time (h)
Cellulose Whey extract Cellobiose Lactose Lactose Bran extract Glucose Glucose
+ peptone +peptone + peptone + spent grain extract + peptone + peptone + spent grain extract + peptone
48
96
120
0.3 -
0.8 0.7 -
0.3 0.3 -
a Cellulose used was crystalline Solka floc. The concentration of carbon source was 10 g 1-~, that of proteose peptone (Difco) 2 g 1-1 and of spent grain extract 5 g 1-1. The other components in the medium were, in g 1-1: KH2PO 4 15, (NH4)2SO 4 5, M g S O a - 7 H 2 0 0.6, CaC12.2H20 0.6, F e S O 4 . 7 H 2 0 0.005, MnSO4-H20 0.0016, Z n S O 4 . 7 H 2 0 0.0014, COC12-6H20 0.0037. pH of the me di um was adjusted to 4.8. 50 ml of the medium was inoculated with 5 × 106 spores and the fungus was cultivated in shake flasks 200 rpm at 28°C. b Milk coagulation activities were measured according to Bailey and Siika-aho (1988). The strain used was a chymosin producing transformant VTT-D-88360.
no clear difference in the inducing effect of the different growth conditions was noticed. As expected, cellulase production is low in GP-medium and probably occurs only after glucose has been consumed by the fungus below the repressing
A
B
/
lOO
100
/
It- 50
24
5(2
48
I 72
I 96
Cultivation time (h)
t 120
24
48
72
916
120
Cultivation time (h)
Fig. 2. Relative filter paper activities (FPA) (IUPAC, 1987) obtained in SF- ( x ), WP- (o), CP- (e), LSG(a), LP- (A), BP- (*), GSG- (E3) and GP- (m) media. Relative FPA values were calculated from glucose (rag) produced using SF cultures (120 h) as reference (FPA = 100, corresponds to 0.24 FPU m l - l ) . (A) host strain T. reesei Rut-C30, (B) chymosin producing strain VTT-D-88360.
41 SF
WP
proc hyrnosin chyrnosin
CP
GP
LSG
_ --
C
48
96
120
48
96
120
48
96
120
48
96
120
48
96
120
H
Fig. 3. Western blot analysis of chymosin produced in different media in shake flask cultivation with transformant VTT-D-88360. Symbols used above are the same as used in Table 2. Growth time in h is indicated under the lane. H = host strain T. reesei Rut-C30, C = purified calf chymosin. Proteins were separated on a 7-15% gradient SDS-PAGE (Laemmli, 1970), electroblotted onto nitrocellulose filters, treated with chymosin specific antibodies (kindly obtained from M. Hansen, NOVO A/S) and with alkaline phosphatase-coupled a-antibody (Promega Biotec), and stained by the ProtoBlot Immunoblotting System (Promega Biotec). Proteins were concentrated 15-20-fold depending on growth by TCA precipitation and diluted into 2 M NaOH-10 mM Tris, pH 8.5.
concentration. Reasonable amounts of cellulase were produced in SF- and WPmedium. Similar results have been obtained earlier by Bailey and Oksanen (1984). Extracellular chymosin activity produced by strain VTT-D-88360 was measured using a milk coagulation assay (Bailey and Siika-aho, 1988). This assay cannot detect small amounts of chymosin. Nevertheless, chymosin expression from the cbhl promoter seems to be induced in a similar way as cellulase expression in these different media (Table 2). Unexpectedly, no chymosin activity could be observed in SF-medium. However, chymosin is clearly produced (see Fig. 3), and thus might be bound to cellulose and consequently not be detectable with the activity assay. Secretion of chymosin was also studied by Western blotting using chymosinspecific antibodies (Fig. 3). After 48 h of growth in SF- and WP-medium both prochymosin and chymosin can be detected in the medium, whereas in LSG- and CP-medium, the processed chymosin form is clearly prominent. This can be explained by the effect of culture pH on the autocatalytic processing of prochymosin. In SF- and WP-medium, the pH value of the culture is still over 4.5 while in both LSG- (due to rapid growth) and in CP-medium, pH has already dropped to a value of 3.2. After 96 h of growth, the protein produced has been cleaved to the mature chymosin form also in the SF- and WP-medium. The highest amounts of active extracellular chymosin were detected after 96 h of growth (Table 2). After that the amount begins to decrease and in CP- and LSG-medium also degradation products can be seen (Fig. 3). This might indicate autodegradation of chymosin under these conditions or the action of host proteases.
42
C
~C
°
_~a_
/2/ 5
"~ 4£- g ~-' 4 3£~- ~ "~ 3
,,'-. . . . .
.~ 20
24
I
I
48
72
96
120
Cultivation time Ch]
70-
7~-
B
C 12
8 ~ 4c
C
~
8
ac ~
6
4
t~ 1 c - c o
1
# =>
oo
2
i 24
48
72
96
120
144
168
Cultivation time (h)
Fig. 4. Bioreactor cultivation (10 1) of chymosin producing transformants; (A) VTT-D-88360 (B) VTT-D-88361. The medium contained, in g l - l : whey extract 60, proteose peptone (Difco) 10, yeast extract (Difco) 5, (NH4)2SO 4 5, KH2PO 4 5, MgSO4.7H20 0.5, CaC12-2H20 0.5. Temperature (33°C for 48 h, subsequently 29°C), pH (>3.5, controlled by addition of NH4OH ) and pO 2 (_>_30%, controlled by agitation speed at a constant aeration rate of 5 1 min-1) were automatically controlled. The inoculum was grown in half-strength medium (buffered with 15 g 1 1 KH2PO4 ) in two stages of 50 ml for 3 d and after that 5 × 200 ml for 1 d. (zx) Reducing sugars, g I i (Miller, 1959) (e) Dry weight, g 1-1 (o) Milk coagulation activity, mg 1-1 (Bailey and Siika-aho, 1988) (t~) Soluble protein, g 1-1 (Lowry et al., 1951) (. . . . . . ) FPU, U m1-1 (IUPAC, 1987).
43 WP-medium was chosen to be used in bioreactor cultivations of two chymosin producing strains, strain VT-F-D-88360 described above and strain VTT-D-88361 (pAMH 104/15; Harkki et al., 1989). Like strain VTT-D-88360, strain VTT-D-88361 had also been transformed with the prochymosin cDNA fused to the promoter and signal sequence of the cbhl gene, but in this strain, the plasmid used for transformation additionally carried the selectable marker amdS (Harkki et al., 1989). Highest amounts of chymosin were obtained after 72 h of growth in the bioreactor (Fig. 4). After this time point, the amount of active chymosin began to decrease as previously noticed in shake flask cultivations. Also inactive prochymosin appeared to be degraded as judged by Western blots (Fig. 5A, C). The amount of intracellular chymosin at different time points of growth follows the profile of extracellular chymosin. However, the peak in chymosin levels seems to occur slightly earlier inside the cell than in the medium (Fig. 5 B, D). Intracellular chymosin is present in both prochymosin and chymosin forms indicating that autocatalytic processing partly occurs already inside the cell but is completed only in the culture medium where pH might be more optimal for processing. Transformant VTT-D-88361 produced about four times less cellulases, measured as FP-activity, in the bioreactor compared to transformant VTT-D-88360. Most probably due to the very large quantities of cellulases in the culture medium, the chymosin specific antibodies also detect cellulase specific bands in Western analysis (see Fig. 5A). The culture medium of strain VTT-D-88361 lacks, however, the major cellulase band (Fig. 5C) indicating that CBHI is not produced. This has been confirmed by Northern, and Southern analysis (Harkki et al., 1989) which also shows that in this strain, one copy of the chymosin expression cassette has been integrated into the cbhl locus and has inactivated the endogenous cbhl gene. The yields of active chymosin obtained with strains VTT-D-88360 and VTT-D88361 in WP-medium in the bioreactor were 7.3 mg 1 i and 13 mg 1-1, respectively. In SF-medium, in the bioreactor, the amounts of active chymosin were even higher, 17 mg 1-1 and 40 mg 1-1, respectively (Harkki et al., 1989). The amount of chymosin decreases only slightly if at all during prolonged cultivation in SF-medium (M. Bailey, unpublished). The yields obtained with these chymosin producing T. reesei transformants are comparable, or even higher than the amounts described for chymosin expressing Aspergillus nidulans strains (Cullen et al., 1987). It is obvious however, that chymosin is not produced with the same efficiency as CBHI by Trichoderma, even when the expression cassette is integrated into the cbhl locus. The amount of chymosin specific mRNA is only 1-5% of that of the cbhl mRNA in the transformant VTT-D-88360 (Harkki et al., 1989). Whether this is caused by instability of the chymosin mRNA or a lower transcription rate of the hybrid gene is under investigation. Chymosin expression could be improved in several ways. Longer fusions to the CBHI protein than described in this article could be advantageous. When prochymosin is fused to the signal sequence and in addition includes 20 N-terminal amino acids of the mature CBHI protein higher yields are obtained than using other DNA constructs carrying shorter CBHI ~equences (Harkki et al., 1989). The yields
44
A 69--
46-_ prochymosin chymosin
30--
22
31
44
54
75
97
117
C u l t i v a t i o n time (h)
B 69
46
prochymosin chymosin
30
C
22
31
44
54
Cultivation time
75
97
117
(h)
Fig. 5. Western analysis of chymosin-specific polypeptides produced in bioreactor cultivations (Fig. 4A, B). (A) strain VTT-D-88360, culture supernatant, (B) strain VTT-D-88360, mycelial extract, (C) strain VTT-D-88361, culture supernatant, (D) strain VTT-D-88361, mycelial extract. Growth time in h is indicated under the lane. The sizes of the molecular weight markers (kDa) used are shown on the left. C = purified calf chymosin (20 ng). SDS-PAGE and electroblotting were carried out as described in Fig. 3. Filters were treated with chymosin-specific antibodies and 125I-labelled protein A (Batteiger et al., 1982). Mycelial proteins were concentrated 30-fold by T C A precipitation and diluted into 2 M NaOH-10 m M Tris, pH 8.5. The intracellular proteins were extracted by grinding the freeze-dried mycelium in liquid nitrogen using a mortar and pestle. 0.05 M sodium citrate buffer, pH 6.0 containing 2% SDS and 1 m M PMSF was added, mixed thoroughly and incubated at room temperature for 30 min. The lysate was centrifuged to remove the cell debris.
45
C 69--
46-_
prochymosin
-- chymosin
30--
19
28
43
53
Cultivation
67
77
96
1 2 0 167
time(h)
D 69--
46-_
prochymosin
-- chymosin
30--
C
19
28
43
53
67
77
96
120
167
Cultivation time (h)
Fig. 5 (continued).
of chymosin would also be increased by screening for more efficient producers after mutagenesis of the strain, using a protease negative strain as a host, or possibly by increasing the copy number of the chymosin expression cassette in the strain.
46 Conclusions
Cellulases are presently used in starch processing, animal feed applications, grain alcohol production, malting and brewing, and in extraction of vegetable juices where the enzyme should be effective in hydrolysis of the raw material. In other types of applications such as wood and textile processing, controlled modification of lignocellulose or fabric must be carried out without too much hydrolysis of the processable material. Thus cellulase preparations meet different requirements in different applications. The application of genetic engineering to Trichoderma has made it possible to modulate cellulase production in such a way that new T. reesei strains producing novel cellulase profiles of commercial potential are now available. Several different combinations have been made without changing the viability of the organism. More detailed knowledge of the activity and substrate specificity of the individual cellulase enzymes would enable further improvements in application of cellulases in biotechnology. Now that the three dimensional structure of the catalytic subunit of C B H I I (Bergfors et al., 1989; Rouvinen et al., 1989, 1990) and of the terminal tail domain of CBHI (Kraulis et al., 1989), as well as expression systems for both Trichoderrna and yeast (Penttil~i et al., 1987a, 1988; Zurbriggen et al., 1990a,b) are available, a detailed investigation of the structure-function relationship of these enzymes is possible. The combination of genetic and protein engineering is likely to lead to novel developments in utilization of cellulosic materials in future. The production of calf chymosin with Trichoderma shows that this fungus is a useful organism not only for production of cellulases but also for proteins of heterologous origin. Chymosin was efficiently secreted and processed to the correct active form and appears to be non-glycosylated. As shown by Ward (1989) the yields of a foreign protein can be greatly improved by further mutagenesis and screening. Although some proteins might be difficult to express in heterologous hosts, such as the heme requiring lignin peroxidase of Phlebia radiata in Trichoderma (Saloheimo et al., 1989), filamentous fungi have proven promising alternatives to the other existing production organisms. To investigate further the capability of Trichoderma for heterologous protein production we are analysing the properties of the recombinant chymosin in detail and studying the expression and secretion of several other eukaryotic proteins in Trichoderma.
References
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