Patent Application: US-2975598-A

Abstract:
the present invention relates to recombinant - dna - technology , and particularly to genes involved in the control of basic metabolic processes in fungi . the invention specifically provides a mutated form of the native glucose repressor gene cre of filamentous fungi , wherein the mutation is situated in the c - terminal domain , the n - terminal first zinc finger being intact and the c - terminal region including the second zinc finger being mutated so that the viability of a strain carrying said mutated gene is maintained and the glucose repression is relieved .

Description:
in order to address the role of crea in other filamentous fungi and especially in relation to cellulase expression , we have isolated the crea equivalent cre1 from the filamentous fungus trichoderma reesei which is one of the most extensively studied cellulolytic organisms ( reviewed e . g . by nevalainen and penttilä , 1995 ). the production of cellulases is dependent on the carbon source available , and glucose repression is very tight . the expression level of the major cellobiohydrolase 1 ( cbh1 ) is up to several thousand fold higher on media containing inducing carbon sources such as cellulose or sophorose compared with glucose containing media ( penttilä et al ., 1993 ). deletion analysis of the cbh1 promoter has suggested that a crea equivalent would be involved in glucose repression of cellulase expression in t . reesei ( penttilä et al ., 1993 ). in addition to the cre1 gene of t . reesei , we isolated the same gene from the related species t . harzianum . the expression studies of the t . reesei cre1 gene revealed unexpected indication of autoregulation . furthermore , a hypercellulolytic t . reesei strain was found to express a truncated form of the cre1 gene with an unexpected structure and properties . the truncated cre1 gene of the strain t . reesei rut - c30 was named cre1 - 1 . the present invention thus provides a mutated fungal glucose repressor gene which , when transformed to a fungal strain maintains said strain fully viable and renders said strain capable of producing elevated amounts of secreted proteins , even in the presence of glucose . furthermore , this strain has an altered glucose uptake , as well as altered growth characteristics on certain nitrogen sources . consequently , a further object of this invention is the use of said gene in a process for enhancing the production of secreted proteins in a fungal host , which process comprises transforming the truncated cre1 gene according to the invention into a suitable fungal host , and replacing the existing cre1 gene , cultivating the transformed host in a suitable growth medium comprising glucose , and recovering the protein produced . according to the present invention it is also possible to control the growth rate of a fungus by regulating the type of nitrogen source and the amount of glucose in the medium . fungal strains transformed with the truncated cre1 gene are also provided . unexpected results were obtained when studying the expression of the t . reesei cre1 gene . if glucose repression mediated through crei was regulated at the level of cre1 transcription , one would expect increase in transcription in the presence of glucose . however , high expression of cre1 does not correlate positively with glucose repression , in fact when glucose is present in repressing amounts the level of cre1 transcript is relatively low , and higher on the neutral carbon sources sorbitol and glycerol , and on cellulose . furthermore , addition of glucose to cultivations carried out on the neutral carbon sources reduced the level of cre1 transcript , which is indicative of autoregulation . further indication for autoregulation was obtained when studying expression of the mutated form of the glucose repressor gene cre , i . e . cre1 - 1 of the hypercellulolytic t . reesei strain rut - c30 . nearly 80 % of the protein coding region , including one of the two zinc fingers , is deleted as compared to the “ native ” cre1 gene of t . reesei qm9414 . the t . reesei cre1 - 1 mutation could suggest that the n - terminal zinc finger could be sufficient for dna - binding and thus retaining most of the viability of the fungus . however , the strain rut - c30 produces relatively high levels of cre1 - 1 mrna also on glucose medium compared with the strain qm9414 . assuming that crei down regulates its own expression when growing on glucose medium , the abundancy of cre1 - 1 mrna may be explained by the inability of the crei - 1 protein to bind the target sequences in its own promoter , or by the inability of the truncated form of the protein to confer repression of the gene . this should hold true also for other genes regulated by cre1 , which is here shown by derepression of the cellulase gene cbh1 . the high cellulase cbh1 mrna levels detected in the presence of glucose in combination with the mutation resulting in the truncated form of cre1 , support true carbon catabolite derepression in this strain . the results obtained with rut - c30 now further support the suggestion that cellulase expression is under control of carbon catabolite repression mediated at least partially by crei . it is likely that a similar situation holds true for other filamentous fungi , and furthermore that also other hydrolytic enzymes involved in utilization of plant polymeric substrates could be regulated by crei / crea proteins . by replacing the cre1 gene in t . reesei qm9414 with the mutated gene cre1 - 1 it is possible to construct a strain expressing the cre1 - 1 and transfer the glucose derepressed phenotype of rut - c30 into another trichoderma strain . a strain constructed in this way is fully viable , and is further able to produce secreted proteins in the presence of glucose . the properties are extremely useful in biotechnical processes when producing secreted proteins . we have also transformed the native full length cre1 gene into the rut - c30 strain . the characteristics of the new strain obtained indicated that the mutated gene cre1 - 1 of rut - c30 has a function partly allowing transcription of a cellulase gene suggesting competition between the products of cre1 and cre1 - 1 genes . alternatively , an additional mutation in rut - c30 is partly taking part in glucose derepression in addition to cre1 . other highly unexpected results are also described . it is for the first time shown that glucose consumption from the medium is under cre control and particularly so that some residual glucose can remain completely unutilized in a strain mutated in cre . this result shows that several mutant forms of cre can now be generated and transferred to fungal strains , which will result in changed glucose consumption of the strain . it is noteworthy that glucose containing cultures and for instance fed - batch cultivations with the necessity of careful control of glucose amount are generally used in biotechnical processes for protein production . using strains harboring cre mutations allows now a new type of process control . another unexpected result shown is that growth rate modulation is under cre control in a way dependent on the type of nitrogen source provided for the fungus . this allows the growth rate , however retaining the strain fully viable , to be controlled in biotechnical processes . fig1 alignment of the amino acid sequences of three t . reesei crei proteins ( rut - c30 ( seq id no : 7 ), vtt - d - 80133 ( seq id no : 5 ) and qm9414 ) ( seq id no : 5 ) as well as of t . harzianum t3 ( seq id no : 2 ). the domain containing the two zinc fingers is underlined in the t3 sequence . fig2 expression of cre1 in t . reesei strain qm9414 cultivated on minimal medium supplemented with either 2 % sorbitol , 5 % glucose or 3 % solka floc cellulose at the cultivation times indicated . 2 μg of total rna was loaded on gel . prior to blotting and hybridization the gel was stained with acridine orange ( ao ) to visualize the amount of rna loaded . hybridization probe was a 600 bp long internal pcr fragment of the cre1 ( see experimental ). fig3 effect of different carbon sources on the expression of cre1 and cbh1 of t . reesei qm9414 . cultivations were carried out for 72 - 92 h as indicated on minimal medium supplemented with either 2 % sorbitol or 2 % glycerol . at 72 h , glucose was added to 2 % into certain sorbitol and glycerol cultures and incubation was continued for 1 h ( total cultivation time 73 h ) or 15 h ( total cultivation time 87 h ). similarly , 1 mm sophorose was added into glycerol cultures twice , at 72 h and 82 h , and the incubation was continued for 15 h ( total 87 b ) or 20 h ( total 90 h ). northern blot was prepared and hybridized as described in the legend to fig2 . fig4 comparison of expression of cre1 and cbh1 in the t . reesei strains rut - c30 and qm9414 cultivated for 72 h on 5 % glucose or 2 % solca floc cellulose medium . northern analysis was performed as described in the legend to fig2 . the cre1 probe used was the 294 bp long fragment specific for the nucleotides from − 158 -+ 136 of the cre1 gene . fig5 construction of the vector for replacement of qm9414 cre1 by cre1 - 1 of rut - c30 . relevant restriction enzyme recognition sites used in the cloning steps are indicated with stars (*). the pcr primers are 5 ′ ggggaattcatagatggatagaaagagttgg ( sense ) ( seq id no : 8 ) and 5 ′ ggggaattcctcactatagggagaccggcctcgagttaattaagctt ( antisense ) ( seq id no : 9 ) fig6 production of cbhi into the culture medium by trichoderma reesei transformants . 40 μl and 200 μl aliquots of culture medium were dot blotted onto a nitrocellulose membrane after 3 days of cultivation in trichoderma minimal medium containing initially 5 % glucose and 0 . 2 % proteose peptone . monoclonal antibody ci - 258 raised against cbhi was used to detect cbhi . transformants 41 - 53a and 41 - 112a , 41 - 108a , 41 - 19a and 41 - 21a are amds + and cre1 + , 41 - 53a and 41 - 9a are amds + . the host strain rut - c30 and the strain qm9414 are shown as controls . fig7 trichoderma strains rut - c30 ( left ), rut - c30 transformed with pmi - 41 ( middle ), and qm9414 grown on trichoderma minimal plates supplemented with 2 % glycerol ( top row ), 2 % glycerol + 0 . 2 % peptone ( 2nd row ), 2 % glucose + 0 . 2 % peptone ( 3rd row ), and 2 % glucose ( bottom row ). fig8 northern analysis on expression of cbh1 and cre1 mrna by cre1 transformant 41 - 66a during cultivation in trichoderma minimal medium containing initially 2 % glucose and 0 . 2 % proteose peptone . total rna was isolated from mycelia grown for 1 , 2 and 3 days and 2 μg of total rna was loaded on gel . the host strain rut - c30 and the strain qm9414 are shown as controls . t . reesei strain qm9414 ( vtt - d - 74075 , atcc 26921 ) ( mandels et al . 1971 ) was used for gene expression studies , isolation of rna , and as a source of dna for preparation of pcr fragments used as a probe . dna of t . reesei strain vtt - d - 80133 ( bailey & amp ; nevalainen 1981 ), a mutant strain of qm9414 with increased cellulolytic activity , was used in the construction of the chromosomal gene library . t . reesei rut - c30 ( atcc 56765 ), a hypercellulolytic mutant strain ( montenecourt and eveleigh 1979 ) was cultivated for isolation of dna and rna . dna extracted from the t . harzianum isolate t3 ( wolffhechel , h ., 1989 ) was used for construction of a genomic lambda library . for rna isolation , 50 ml of each cultivation media in 250 ml conical flasks were inoculated with 10 7 spores and incubated in a rotary shaker at 200 rpm at 28 ° c . trichoderma minimal medium ( tmm ) contained kh 2 po 4 15 g / l , ( nh 4 ) 2 so 4 5 g / l , feso 4 × 7h 2 o 5 mg / l , mnso 4 × h 2 o 1 . 6 mg / l , znso 4 × h 2 o 1 . 4 mg / l , cocl 2 × 6h 2 o 3 . 7 mg / l , mgso 4 0 . 6 g / l , cacd 2 0 . 6 g / l , ph was adjusted to 4 . 8 . proteose peptone 2 g / l was added to certain culture media as indicated in text . this medium was used throughout the study supplemented with the appropriate carbon source , either 20 g / l ( sorbitol , glycerol , glucose ) or 50 g / l ( glucose ). in certain sorbitol or glycerol cultivations glucose was added into final concentration of 2 % by adding 4 ml of 25 % glucose into 50 ml of cultivation media after 72 h of growth . similarly , 1 mm α - sophorose ( serva ) was added into the culture medium twice , at 72 and 82 hours of growth . cellulose medium was tmm supplemented with solka floc cellulose 30 g / l and distiller &# 39 ; s spent grain 15 g / l and the ph was adjusted to 5 . 0 . mycelia was harvested from culture medium by filtration through gf / b glass microfibre filters ( whatman ), washed with sterile water and stored at − 70 ° c . for dna isolation , the cultivation was as described above except that tmm was supplemented with 2 % glucose and 0 . 2 % proteose peptone ( difco ) and mycelia was freeze - dried after harvesting and stored at − 20 ° c . for preparation of solid media , the ph of tmm was adjusted to 5 . 5 and 2 % agar was added . 0 . 1 % triton x - 100 was used to restrict spreading of the colonies . to clone the cre1 genes of trichoderma several redundant oligodeoxyribonucleotides were designed based on aspergillus crea protein sequences , synthesized and used as primers with trichoderma chromosomal dna as a template in a pcr reaction . the functional primer pair used for t . reesei was 5 ′ ggcggatcct ( c , t ) tggngt ( g , a ) tcngg ( antisense ) ( seq id no : 10 ) and 5 ′ ggcggatccacnca ( c , t ) acnggnga ( a , g ) aa ( a , g ) cc ( sense ) ( seq id no : 11 ) and for t . harzianum 5 ′ ggcggatcct ( c , t ) tggngt ( g , a ) tcngg ( antisense ( seq id no : 12 ) and 5 ′ ggcggatccttngg ( g , a ) tt ( g , a ) ta ( a , g ) ta ( t , g ) ttngg ( sense ) ( seq id no : 13 ). bamhi cleavage sites were included at the 5 ′ ends of the primers to facilitate cloning of the amplified fragment . pcr cycling used in both reactions was as follows : four cycles of repeated denaturation at 96 ° c . 1 min — annealing at 37 ° c . 30 sec — polymerization at 72 ° c . 1 min followed by 25 cycles of repeated denaturation at 96 ° c . 1 min — annealing at 55 ° c . 1 min — polymerization at 72 ° c . 1 min . the pcr products of expected size , around 600 - 700 bp , were cut with bamhi and ligated to bamhi linearized puc19 vector , and transformed into e . coli dh5α . the resulting plasmids were isolated using standard methods and sequenced to demonstrate cloning of trichoderma cre1 fragments . for hybridization , the cre1 fragments ( 600 bp for t . reesei , 660 bp for t . harzianum ) were released from the plasmids by bamhi digestion , purified from agarose gel by phenol extraction and labelled using random primed dna . labeling kit ( boehringer mannheim ) and α - 32 p - dctp ( amersham ) to a specific activity of 10 8 cpm / μg . the probe fragment corresponding to the full length protein coding region of the t . reesei cre1 gene was prepared by pcr using the t . reesei cre1 cdna clone as a template . the primers used , 5 ′ ggcggatccatgcaacgagcacagtcigcc ( sense ) ( seq id no : 14 ), and 5 ′ ggcggatccctacatggcatccatgaggtc ( antisense ) ( seq id no : 15 ), were complementary to the 5 ′ and 3 ′ ends of the protein coding region of the cre1 gene and contained bamhi cleavage sites at the ends to facilitate cloning . pcr cycling was repeated 25 times as follows : 94 ° c . 45 sec — 55 ° c . 30 sec .— 72 ° c . 2 min . the 294 bp long probe fragment corresponding to the nucleotides from − 158 to + 136 of the t . reesei cre1 was synthesized by pcr using the cdna clone as a template and the primers 5atcagcagtctctcctc ( sense ) ( seq id no : 16 ), and 5 ′ actgtgticctggaatg ( antisense ) ( seq id no : 17 ). the pcr cycling was repeated 25 times the following way : 95 ° c . 45 sec — 42 ° c . 30 sec — 72 ° c . 45 sec . dynazyme ™ dna - polymerase was used in each pcr reaction described above in the reaction conditions recommended by the manufacturer ( finnzymes oy , espoo , finland ). full length cbh1 cdna was obtained as an ecori - hindiii fragment from pttc01 ( penttilä et al . 1988 ) and the probe was prepared as described above . in order to clone the t . reesei chromosomal cre1 gene , the genomic cosmid library of t . reesei vtt - d - 80133 ( mäntylä et al . 1992 ) was plated out on ampicillin plates and after overnight incubation bacterial colonies were transferred to nitrocellulose membranes and screened with the 600 bp long t . reesei cre1 probe using colony hybridization . colonies giving a positive hybridization signal were purified and cosmid dna was prepared using standard plasmid isolation procedures . cosmid dna carrying the cre1 gene was partially restriction mapped and analyzed by southern hybridization . a 6 . 4 kb sali - hindiii restriction fragment containing the entire protein coding region with flanking regions was subcloned for sequencing into psp73 plasmid vector ( promega ) digested with sali - hindiii . the plasmid was designated pmi - 41 . in order to clone the cre1 gene of the t . harzianum isolate t3 , chromosomal dna was prepared according to raeder & amp ; broda ( 1985 ), partially digested with sau3a and size fractionated by sucrose gradient centrifugation ( sambrook et al . 1989 ), and dna fragments of about 22 kb were ligated to bamhi digested , phosphatase treated lambda dash ™ vector arms and the ligation mixture was packaged into lambda particles using gigapack ii gold packaging extract ( stratagene ). the lambda particles were used to infect appropriate e . coli host cells , and the library was screened using the 660 bp long t . harzianum cre1 fragment as a probe , and positive clones were isolated and purified according to manufacturer &# 39 ; s instructions ( stratagene ). bacteriophages were purified using a method based on the general procedure described in sambrook et al . ( 1989 ) with the following modifications : dnasei treatment was omitted and phage particles released from lysed host cells were precipitated using peg6000 , dissolved in sm and extracted with chloroform , pelleted by centrifugation at 25 000 rpm in kontron tst41 . 14 rotor for 2 h , and again dissolved in sm . ilunbda dna was isolated by digesting the phage particles with proteinase k followed by phenol extraction and ethanol precipitation . dna of the lambda clones containing e . harzianum cre1 gene , were partially restriction mapped and analysed by southern hybridization . two ecorv restriction fragments , one of 3 kb containing the 5 ′ end and the other of 4 . 9 kb containing the 3 ′ end with flanking regions , both giving a positive hybridization signal with cre1 probe , were subcloned into ecorv cut , phosphatase treated plasmid vector psp73 ( promega ) for sequencing . in order to isolate cre1 cdna clones , the cdna library of the t . reesei strain qm9414 constructed into lambda unizap xr vector ( nakari et al . 1993 ) was screened with the 600 bp long t . reesei cre1 probe prepared as described above . the cdna library of the t . reesei strain rut - c30 ( stalbrand et al ., 1995 ) was screened using the full length cre1 protein coding region as a probe . bacteriophage dna of positive clones were in vivo excised and converted into plasmid form according to the instructions of stratagene . for colony hybridization , bacterial colonies or lambda plaques were transferred from agar plates onto nitrocellulose membranes ( schleicher & amp ; schuell b a 85 ) by placing the membrane on the plate for 1 min . the membrane was placed on filter paper soaked in denaturing solution ( 0 . 5m naoh — 1 . 5m naca ) for 7 min and then on filter paper soaked in neutralizing solution ( 1 . 5 m nacl — 0 . 5 m tris - hcl ph 7 . 5 ) for 2 × 3 min , whereafter the membranes were soaked in 2 × ssc , and dna was fixed by baking the membranes at 80 ° c . for 2 h . to remove excess bacterial debris the membranes were washed prior to hybridization in 50 mm tris ph 8 — 1m nacl — 1 mm edta — 0 . 1 % sds at 42 ° c . for 1 h . hybridization was carried out in 50 % formamide — 5 × denhardt &# 39 ; s - 5 × sspe — 0 . 1 % sds — 100 μg / ml herring sperm dna - 1 μg / ml polya dna at 42 ° c . overnight with 10 6 cpm of probe per ml of hybridization solution . after hybridization the membranes were washed in 2 × ssc — 0 . 1 % sds at room temperature followed by a wash in 1 × ssc — 0 . 1 % sds at 68 ° c . for 1 h , and exposed to kodak xar - 5 x - ray film at − 70 ° c . for southern analysis 2 μg of trichoderma chromosomal dna was completely digested with restriction enzymes ( boehringer mannheim ), the resulting dna fragments were separated by electrophoresis in 0 . 8 % agarose gel and dna was capillary blotted onto hybond n nylon membrane . conditions for hybridization were as described for colony hybridization , except that the post hybridization washes were performed in 2 × ssc at room temperature 2 × 5 min followed by a wash in 1 × ssc — 0 . 1 % sds at 68 ° c . for 60 min . for dna dot blot hybridization , 1 μg of chromosomal dna was denatured in 0 . 4 m naoh for 10 min and neutralized by addition of an equal volume of 2 m ammonium acetate ph7 , and dot blotted onto a hybond n nylon membrane . hybridization and washing were done as described for southern analysis . total fungal rna was isolated according to chirgwin et al . ( 1979 ), glyoxylated and electrophoresed in 1 % agarose gel in 10 mm na - phosphate buffer ph 7 . 0 according to maniatis et al . ( 1982 ). rna ladder ( brl ) containing rna molecules of known length were used as molecular weight markers . the gel was stained with acridine orange ( 15 μg / ml ) for 15 min in 10 mm phosphate buffer to visualize rna and de - stained for 3 h in 10 mm phosphate buffer . thereafter rna was blotted onto hybond ™ n nylon membranes ( amersham ) by capillary blotting in 20 × ssc . hybridization was carried out in 50 % formamide — 10 % dextran sulphate — 1 % sds — 1m nacl — 125 μg / ml denatured herring sperm dna at 42 ° c . overnight using 10 6 cpm of probe per ml of hybridization solution . the membranes were washed at 42 ° c . in 5 × sspe , twice in 1 × sspe — 0 . 1 % sds , twice in 0 . 1 × sspe — 0 . 1 % sds , each wash lasting 15 min , and exposed to kodak xar - 5 x - ray film at − 70 ° c . for replacement of cre1 of t . reesei qm9414 by the mutated gene cre1 - 1 of t . reesei rut - c30 an expression vector pmi - 62 was constructed ( see also fig5 ) in the plasmid pbluescript sk − . pmi - 62 was constructed as follows : 5 ′- sequences of cre1 were obtained from pmi - 41 as the 3 kb ecori - bsteii fragment that was joined in frame to the bsteii - ecori fragment of cre1 - 1 cdna from plasmid pmi - 42 resulting in plasmid pmi - 60 . a 1 . 4 kb fragment of cre1 terminator , beginning 50 bp downstream of the translation stop codon and ending at the naturally occurring hindiii site , was amplified by pcr using pmi - 41 as a template and sequence specific primers that were tailed with ecori sites to facilitate cloning of fragment . the pcr primer sequences corresponding to nucleotides starting from 50 bp downstream of cre1 protein coding region and to vector sequences surrounding the single hindiii site were 5 ′ ggggaattcatagatggatagaaagagttgg ( seq id no : 18 ) and 5 ′ ggggaattcctcactatagggagaccggcctcgagttaattaagctt ( seq id no : 19 ), respectively . the pcr amplified cre1 terminator fragment was ecori cut and cloned to plasmid p3sr2 linearised using ecori , resulting in the plasmid pmi - 61 . pmi - 60 was cut using xhoi and pmi - 61 using sali - xhoi and the fragment containing the amds gene and cre1 terminator was ligated to xhoi linearized pmi - 60 resulting in plasmid pmi - 62 . trichoderma reesei was transformed according to penttilä et al . ( 1987 ). production of cbhi into culture medium was detected using antibodies . 200 μl and 40 μl aliquots of culture medium were dot blotted onto a nitrocellulose membrane . the membrane was incubated in 3 % non fat dried milk — tbs ( 10 mm tris - hcl ph8 — 150 mm nacl ) at 37 ° c . for 10 min , washed in tbs 3 × 10 min . the membrane was incubated in tbs containing monoclonal anti - cbhi - antibody ci - 258 ( aho et al . 1991 ) for 1 h at room temperature and washed in tbs . then the membrane was transferred to tbs containing anti - mouse polyvalent immunoglobulins conjugated with alkaline phosphatase ( sigma a - 0162 ) and incubated for 1 h at room temperature . the bound antibody was detected using the nbt and bcip reagents ( promega ) in a solution containing 100 mm tris - hcl ph 9 . 5 — 100 mm nacl — 5 mm mgcl 2 . the genomic cre1 genes were sequenced from both strands using sanger &# 39 ; s dideoxynucleotide method , sequence specific primers and sequenase version 2 polymerase ( usb ). cre1 cdna clones were sequenced from one strand . all other techniques not described in detail were carried out using standard methods ( e . g . sambrook et al . 1989 ). pc / gene nucleic acid and protein sequence analysis software release 6 . 80 ( intelligenetics inc .) was used for sequence manipulations . isolation and characterization of the t . reesei and t . harzianum cre1 genes fragments of the cre1 genes of t . reesei and t . harzianum were amplified from chromosomal dnas by polymerase chain reaction ( pcr ) using degenerate oligodeoxyribonucleotide primers designed on the basis of the aspergillus crea sequence information . primers , which gave rise to inserts with similarity to the a . nidulans crea gene were complementary at the 5 ′ end with the zinc finger region or the linker region connecting the two fingers , and at the 3 ′ end with the proline rich region showing similarity to the rgr1 protein of yeast ( see later ). the cre1 fragments were used as hybridization probes to isolate chromosomal copies of the cre1 genes of the t . reesei strain vtt - d - 80133 and t . harzianum t3 . the clones were restriction mapped and sequenced from subcloned fragments . the t . reesei cre1 gene contains an open reading frame ( orf ) of 1206 nucleotides ( seq id no : 3 ) which can be translated to a 402 amino acids long protein ( seq id no : 5 ), and the t . harzianum gene ( seq . id . no : 1 ) an orf of 1227 bp , encoding 409 amino acids . the nucleotide sequences of these two trichoderma cre1 genes show 89 % identity within the predicted protein coding region . in addition to the chromosomal copy , which was isolated from the t . reesei strain vtt - d - 80133 ( seq . id . no . 3 ), a cdna copy was isolated from the t . reesei strain qm9414 ( seq . id . no . 4 ). the only difference in cre1 nucleotide sequence observed between these two strains was at position 140 , where c is found in qm9414 instead of t in vtt - d - 80133 which changes amino acid no 47 from threonine ( qm9414 ) to isoleucine ( vtt - d - 80133 ). in the t . harzianum crei , threonine is found at this position . sequencing of the cdna copy confirmed that there are no introns in the t . reesei cre1 gene . sequencing of the 3 ′ ends of nine t . reesei cre1 cdna clones showed that the mrna was equally often cleaved at two different regions separated by approximately 170 bp at the 3 ′ end of the cre1 transcript . there is some heterogeneity in the cleavage sites , the shorter forms of the cdnas end 281 - 288 bp , and the longer ones 450 - 454 bp after the protein coding region . sequences of the chromosomal copies show several putative polyadenylation signals , aaatat ( seq id no : 20 ) or taatat ( seq id no : 21 ), which are located in the t . reesei cre1 gene at 263 , 494 and 665 bp , in the t . harzianum cre1 at 234 , 445 and 735 bp after the protein coding region . the codon usage of the trichoderma cre1 genes is biased against a in the third position , which is typical for filamentous fungi ( unkles 1992 ). the codon preference is more biased than that of cellulase genes of t . reesei but not as biased as for instance that of the translation elongation factor tef1 ( nakari et al . 1993 ) or the phosphoglycerate kinase gene pgk1 ( vanhanen et al . 1989 ). the deduced crei amino acid sequences of t . reesei and t . harzianum show 95 % similarity ( 92 . 5 % identity ) ( fig1 ) ( seq id nos : 5and 2respectively ). when compared with the crea protein sequences of a . nidulans and a . niger 72 % overall similarity ( 46 % identity ) is obtained . one of the two best conserved regions between trichoderma and aspergillus localizes in the dna - binding motif , the c 2 h 2 type zinc finger which is almost perfectly conserved throughout the 57 aa long region . interestingly , the amino acid sequences surrounding the zinc finger domain differ between the aspergillus and trichoderma proteins . on the amino terminal side of the finger , 13 amino acids are missing in both of the trichoderma proteins compared with the aspergillus sequences . on the carboxy terminal side an 8 - 9 aa long alanine stretch found in the aspergillus crea proteins is replaced by histidine and glutamine residues in trichoderma . the other equally well conserved region is found further downstream . within this region 39 out of 41 amino acids are identical between the trichoderma and aspergillus crei / crea proteins . characteristic to this part of the crei / crea proteins is the content of about 25 % proline , 25 % serine and 12 % threonine . the sequence shows similarity to the glucose repressor protein rgr1 of yeast ( sakai et al . 1990 ). expression of t . reesei cre1 is dependent on the carbon source available northern analysis shows that t . reesei qm9414 produces two major cre1 transcripts of 1 . 85 kb and 2 kb and a faint one of 2 . 6 kb , visible only after a prolonged exposure ( fig2 ). these were detected in all the cultivation conditions studied with approximately the same relative abundancy . these features are in good correlation with the observed size difference of 170 bp of the cdna clones analysed ( see above ) and demonstrate together with the southern analysis that both the two major transcripts are products of a single gene . because the t . reesei cellulase genes are subject to glucose repression , it was of interest to study expression of cre1 in conditions relevant for cellulase expression . the cultivations were carried out in shake flasks on media containing either glucose , solka floc cellulose , which is an efficient inducer , or on sorbitol which is a neutral carbon source in respect to cellulase expression ( penttila et al ., 1993 ). northern analysis revealed that the steady state levels of cre1 transcript show clear variation in these different cultivation conditions in repeated experiments . in contrast to expectation , cre1 mrna levels were surprisingly high on the inducing carbon source cellulose . moreover , they were lower on glucose than on cellulose or sorbitol medium ( fig2 ). thus , the expression of cre1 is regulated by the carbon source available . consequently , the effect of glucose on cre1 transcription was studied in more detail and a new series of cultivations was carried out . the fungus was grown for 72 h on a medium containing either sorbitol or glycerol , both neutral carbon sources in respect to cellulase expression , whereafter glucose was added to 2 %. at this point of cultivation the fungus has not yet utilized all sorbitol or glycerol . mycelia were collected for rna isolation 1 h and 15 h after the addition of glucose . in control mycelia grown on glycerol or sorbitol medium without glucose addition , cre1 mrna was equally abundant at different time points during the cultivation ( fig3 ). the levels of cre1 mrna remained unchanged also one hour after glucose addition to the sorbitol and glycerol cultures . however , after overnight incubation in the presence of glucose , cre1 mrna levels were significantly decreased compared with the sorbitol and glycerol cultures where glucose was not added . for comparison , the addition of sophorose , an efficient inducer of cellulase expression , into sorbitol or glycerol cultivations did not affect cre1 transcript levels at the time points studied ( fig3 ). the truncated form of cre1 expressed by the hypercellulolytic t . reesei strain rut - c30 northern analysis carried out for rut - c30 using the pcr - amplified 600 bp long probe specific for the central part of the cre1 gene gave no expression . when southern analysis of genomic dna cut with restriction enzymes ecori , sali , acci , pvuii and sphi was carried out using the complete protein coding region of cre1 as a probe it revealed weak hybridization to the probe and a pattern differing from that of the strain qm9414 ( data not shown ). subsequently the full length cre1 probe was used to isolate cre1 cdna of strain rut - c30 . sequence analysis of the rut - c30 cdna clones showed that the 5 ′ end of cre1 was identical to the previously sequenced cre1 of t . reesei up to nucleotide 261 , corresponding to the first cysteine , cys 87 , of the second zinc finger ( see fig1 ), whereafter the two sequences differed . the reading frame of the truncated cre1 ( hereafter referred to as cre1 - 1 ) continues after cys 87 , encoding eight additional amino acids ( tsitcfff ) followed by a stop codon . a representative plasmid containing a 1 . 3 kb cre1 - 1 cdna insert was designated pmi - 42 , and was deposited to deutsche sammlung von mikro - organismen und zellkulturen , braunschweig , germany , with dsm - accession number 10190 , and was also stored in the vtt culture collection , espoo , finland with accession number vtt - f - 95055 . the dna sequence of cre1 - 1 is given in seq . id . no . 6 . the cre1 - 1 mrnas are short and appear as a double band ( 1 . 1 and 1 . 3 kb ) in northern analysis ( see fig4 ). this is due to addition of the poly - a tail at two different regions as demonstrated by sequencing of four cdna clones . the 3 ′ end of the cre1 - 1 is dissimilar to all known trichoderma gene sequences . to study expression of the truncated form of cre1 and to compare that with expression of cre1 of strain qm9414 , northern analysis was carried out using a probe specific for the 5 ′- end sequences of the cre1 transcripts present in both the wild type cre1 and the cre1 - 1 of rut - c30 thus establishing similar hybridization conditions . cre1 - 1 mrna of rut - c30 seems to be more abundant than the cre1 mrna of strain qm9414 , especially on glucose medium where the expression level of cre1 - 1 is markedly elevated ( fig4 ). the rut - c30 strain was originally isolated as a hypercellulolytic strain by screening for carbon catabolite derepression ( montenecourt and eveleigh 1979 ) and it is consequently of interest to study the extent of possible glucose derepression of cellulase expression in this strain . the t . reesei strains rut - c30 and qm9414 were cultivated on glucose and in inducing conditions on solka floc cellulose . expression of the major cellulase , cellobiohydrolase i ( cbh1 ), was studied ( fig4 middle panel ). cbh1 mrna was produced by both of the strains on cellulose containing medium as expected . on glucose medium expression of cbh1 was repressed in qm9414 as shown previously ( penttilä et al . 1993 ), whereas rut - c30 produced relatively high levels of cbh1 mrna indicative of glucose derepression . transformation of the mutated gene cre1 - 1 of rut - c30 into the strain qm9414 to replace cre1 with cre1 - 1 the endogenous cre1 gene of trichoderma reesei qm9414 was replaced by the mutated gene cre1 - 1 of rut - c30 . for this purpose an expression vector pmi - 62 was constructed ( fig5 ) in the plasmid pbluescript sk − consisting of 5 ′- sequences of cre1 joined in frame to the cre1 - 1 cdna followed by the amds gene and cre1 terminator . this construct was transformed to the strain qm9414 and transformants were selected based on utilization of acetamide as the sole nitrogen source . transformant dna were analysed by southern hybridization to find those in which homologous recombination had taken place and the endogenous cre1 locus was replaced by the transformed construct . the transformant is viable and grows well , the growth rate being dependant on the growth medium used . furthermore , the transformant produces cellulases in the presence of glucose in the medium . in this way it was possible to construct a strain expressing the mutated gene cre1 - 1 and transfer the glucose derepressed phenotype of rut - c30 into another trichoderma strain . transformation of the trichoderma reesei strain rut - c30 with full length cre1 gene the cosmid clone pcos11 carrying the cre1 gene was isolated from a cosmid gene library of chromosomal t . reesei dna of the strain vtt - d - 80133 . from a positive clone ( pcos11 ) a 6 . 4 kb sali - hindiii restriction fragment containing the protein coding region with flanking regions of cre1 gene was isolated and cloned to a sali - hindiii cut vector psp73 resulting in plasmid pmi - 41 . pmi - 41 with plasmid p3sr2 ( hynes et al ., 1983 ), that carries acetamidase gene as a selection marker providing the transformants the ability to use acetamide as a sole nitrogen source , were co - transformed to the strain rut - c30 according to penttilä et al . 1987 . the integration of cre1 gene in the genome of acetamide utilizing transformants was verified by dot blot hybridization of chromosomal dna to the 600 bp long cre1 probe lacking sequences found in the endogenous mutant cre1 - 1 in rut - c30 . to study production of cellulases in cre1 transformant colonies , the host strain rut - c30 and the strain qm9414 were cultivated in microtiter plate wells in minimal medium containing 5 % glucose for 3 days . at this point of cultivation glucose was still detectable in the medium ( in repressing amounts ). culture medium was dot blotted onto nitrocellulose membrane . cellulases produced into the medium were detected immunologically using antibodies ( fig6 ). monoclonal antibody ci - 258 raised against the major cellulase cbhi was used ( aho et al ., 1991 ). the analysis showed that the strain qm9414 did not produce any detectable cbhi , indicative of glucose repression of cellulase expression , but rut - c30 and the cre1 transformants did , indicative of derepression . this shows that the mutated gene cre1 - 1 of rut - c30 has a function that dominates ( or codominates ) in the transformants over the transformed cre1 , or an additional mutation in rut - c30 is partly taking part in glucose derepression in addition to cre1 . spores of cre1 transformants and for comparison of transformants harbouring only the selection marker were plated on solid media to obtain colonies derived from a single spore . the medium used was potato dextrose agar ( difco ) supplemented with 0 . 1 % triton x - 100 to restrict spreading of the colonies . after 4 - 5 days of incubation at 28 ° c . a difference in growth was seen between cre1 transformants and the ones having only the selection marker , cre1 transformants formed bigger colonies than the others indicative of positive effect of cre1 on the growth / viability of the fungus . when transformant colonies were cultivated on solid trichoderma minimal medium containing 0 . 2 % peptone supplemented with different carbon sources such as 2 % glucose , 2 % glycerol or 2 % solka floc cellulose and 0 . 1 % triton x - 100 , a difference in the colony morphology and diameter were seen between the host strain and the cre1 transformants in each case , the transformants being bigger ( fig7 ). for comparison , when peptone was omitted from the medium , no notable differences between transformants and the host strain were seen . in addition in the colonies grown on potato dextrose agar medium ( difco ), the difference in colony growth was also seen . we conclude that cre1 affects the growth of the colony in a manner that is dependent on cultivation conditions especially on the presence of an organic nitrogen source . transformant colonies pmi - 41 - 66a and pmi - 41 - 112a and the host strain rut - c30 were cultivated in liquid trichoderma minimal medium containing initially glucose 20 g / l and 0 . 2 % proteose peptone ( difco ). after 1 , 2 and 3 days of cultivation , mycelia were collected and total rna was isolated . northern analysis of cbh1 mrna coding for the major cellulase cellobiohydrolase i ( cbhi ) showed that the strain rut - c30 produced abundant cbh1 mrna whereas in the transformants hybridization to cbh1 probe was significantly reduced ( fig8 ). northern analysis of the same samples with cre1 probe showed that the transformed cre1 gene was expressed in the transformants . we conclude that the transformed cre1 was able to regulate cbh1 expression in the host strain . 6 . 3 . effect of transformed cre1 on the utilization of glucose from cultivation medium rut - c30 was transformed with pmi - 41 containing the cre1 gene as described earlier . two independent transformant colonies pmi - 41 - 66a and pmi - 41 - 112a were cultivated in trichoderma minimal medium containing initially glucose 20 g / l as the sole carbon source and ammonium sulphate as the only nitrogen source in parallel with rut - c30 , the host strain for transformation in a rotary shaker at 28 ° c . for six days . four parallel shake flasks for each strain were inoculated . the amount of glucose in the medium was monitored daily using the god - perid method ( boehringer mannheim ) ( table 1 ). the amount of glucose in growth media of the untransformed rut - c30 decreased from the initial 20 g / l to 3 - 4 g / l during the first 3 days of cultivation and remained at that level till day 6 . the amount of glucose in the cultivation medium of cre1 transformants decreased below the level that was detected in the growth media of the strain rut - c30 when measured at day 4 and day 6 , in some flasks no glucose was detectable . in addition in the culture medium of the strain qm9414 cultivated in parallel , glucose was no more detectable at day 6 . based on these results we conclude that cre1 gene promotes consumption of glucose from culture medium especially when low level of glucose , below 3 g / l , is present . montenecourt , b . s ., and eveleigh , d . e . 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( 1992 ) gene organization in industrial filamentous fungi . in applied molecular genetics of filamentous fungi . kinghom j . r ., and turner g . ( eds ). glasgow : blackie academic & amp ; professional , pp . 28 - 53 . vanhanen , s ., penttilä , m ., lehtovaara , p ., and knowles , j . ( 1989 ) isolation and characterization of the 3 - phosphoglycerate kinase gene ( pgk ) from the filamentous fungus trichoderma reesei . curr genet 15 : 181 - 186 . wolffhechel , h . ( 1989 ) fungal antagonists of pythium ultimum isolated from a disease suppressive sphagnum peat . växtskyddsnotiser 53 : 7 - 11 . tttggctgcc gctcgatcac atg caa cga gct cag tct gcc gtg gat ttt tcc 833 aat cta ctt aat cca act tcg gca gca gga cag gac agc gac gcc gag 881 caa ggc agc gga gcc atg tct acc gct gcg gtt acc gtc atc aag ccc 929 aat ggg cct atc cca gga gca cag tca acg gag gct gcc aac gag ctt 977 cct cgt ccc tac aag tgc cct ctt tgt gaa aag gcc ttc cac cgt ctg 1025 gag cac cag acc agg cac atc cgc act cac acg ggc gag aag ccc cat 1073 gcc tgc cag ttc cct ggc tgc agc aag aag ttc tct cgt tcc gat gag 1121 ala cys gln phe pro gly cys ser lys lys phe ser arg ser asp glu ttg acc agg cac tcg agg ata cac agc aac ccc aac tcc agg cgc ggc 1169 cct cac cac atg cac gtc gat ggc atg atg ccc cct ccg gtg cca aag 1265 gcc atc cgc tct gct ccc acc tcg act ctg gtc tcg cct aac gtc tcg 1313 cct ccc cac tct tac tcc tcg ttt gtc atg ccc cag acc ccc atg gct 1361 cac tac aac cgt ggc aac gat atc aca atg ctg gca aag gct gca aac 1409 his tyr asn arg gly asn asp ile thr met leu ala lys ala ala asn cag atc gag cgg gaa act ctc tct ggc ggc ccg tct aac cac aac tca 1457 gln ile glu arg glu thr leu ser gly gly pro ser asn his asn ser agg cat cat ccc tac ttc ggc cag ggc ttg ccg aac tct cga ggc cac 1505 ccg cct tcc ctt tcc tcg tac cac atg gcg aga tct cac tcc aat gac 1553 gat gat gat cat tac agc agc atg agg cac gcc aag agg tcg agg cct 1601 aac tcg ccc aac tcc acg gct ccc tct tct ccc acc ttt tcc cac gac 1649 tct ctg tct ccc acc ccg gat cac act ccc atc gca act ccc gct cac 1697 tcc cct cga ctc cgc ccc ttt tcg ggc tat gag ctg ccg agt ctg aga 1745 aac ctg tct ctg cag cac aac acg act ccg gcg ctg gcc ccc atg gag 1793 ccc cac ctg gat gct ccc cag ttc ccc cct cag ctg cag gca aac aac 1841 aac cgc agc ccc ggc atg tcg ctt acc gac atc atc agc cgc ccc gac 1889 ggg agc cac agg aag ctc cct gtt cct cag gtt ccc aag gtg gcg gtg 1937 cag gac ctc ctc tca gac ggt gta ttc cct aac agc ggc aga agt tca 1985 act gca ggc agt ctt gca ggt ggc gac ctc atg gat cgg atg 2027 met gln arg ala gln ser ala val asp phe ser asn leu leu asn pro gly ala gln ser thr glu ala ala asn glu leu pro arg pro tyr lys cys pro leu cys glu lys ala phe his arg leu glu his gln thr arg his ile arg thr his thr gly glu lys pro his ala cys gln phe pro gly cys ser lys lys phe ser arg ser asp glu leu thr arg his ser ser ser phe val met pro gln thr pro met ala his tyr asn arg gly asn asp ile thr met leu ala lys ala ala asn gln ile glu arg glu met ser leu thr asp ile ile ser arg pro asp gly ser his arg lys asp gly val phe pro asn ser gly arg ser ser thr ala gly ser leu gca cag tct gcc gtg gat ttt tcc aac ctc ctg aat cca acg tcg gca 345 gca gga cag gac agc ggc gcc atg tct acc gcc gcg gtc acc gtc atc 393 aag ccc aat ggg ccc att cca gga aca cag tcg acc gag act gcc aac 441 gag ctg cct cgt ccc tac aag tgc cct ctt tgc gac aag gct ttc cac 489 glu leu pro arg pro tyr lys cys pro leu cys asp lys ala phe his cgc ctg gag cac cag acc agg cac att cgc acc cac acg ggc gag aag 537 ccc cat gcc tgc cag ttc cct ggc tgc agc aag aag ttc tcc cgc tcc 585 gat gag ctg acg agg cac tcg agg ata cac agc aac ccc aac tca agg 633 cct cac ccc atg cac gtc gat ggc ttg atg cac cct ccc gcc gcg cca 729 aag gcc atc cgc tct gcg ccc cct tcg acc ctc gtc tct ccc aac gtc 777 tcg cct ccg cac tcc tac tcg tcc ttt gtc atg cct cac ggt ccc att 825 tct cac tat ggc cgt ggc aac gac att acg atg ctt gcc aag gcg gca 873 ser his tyr gly arg gly asn asp ile thr met leu ala lys ala ala aat cag att gag cgc gag acg ctt tct ggc ggg ccg tcc aac cac aac 921 asn gln ile glu arg glu thr leu ser gly gly pro ser asn his asn tcg agg cac cac cct tac ttt ggc cag ggt gtt ccg ggt tct cga ggc 969 cac ccc tcg ctt tct tcg tac cac atg gcg aga gct cac tcc aac gac 1017 gag gat gac cac tac cat ggc agc ttg agg cac gcc aag agg tca agg 1065 ccc aac tcg ccc aac tct acg gct cct tct tct ccc acc ttt tcg cac 1113 gac tct ctg tcc ccc acc ccg gat cac act ccc atc gca act ccc gct 1161 cac tcc ccc cgt ctc cgt ccc ttt tcg ggc tac gag ctg ccg agt ctg 1209 aga aac ctg tcc ctg cag cac aat acg act ccg gcg ctg gcc ccc atg 1257 gag cct cac ctg gac gct ccg cag ttc cac cct cag ctg cag gca aac 1305 acc acc cgc agc ccc ggc atg tca ctt acc gac atc att agc cgc ccc 1353 gac ggc agt cag agg aaa ctg cct gtc cct cag gtc ccc aag gtg gcg 1401 gtt cag gac ctt ctc tcc gac ggc gtc ttc ccc aac agc ggc aga agt 1449 tcc acc acg ggc agt ctt gcc ggt ggc gac ctc atg gat cgg atg tag 1497 met gln arg ala gln ser ala val asp phe ser asn leu leu asn pro thr ala asn glu leu pro arg pro tyr lys cys pro leu cys asp lys gly pro ile ser his tyr gly arg gly asn asp ile thr met leu ala lys ala ala asn gln ile glu arg glu thr leu ser gly gly pro ser ser asn asp glu asp asp his tyr his gly ser leu arg his ala lys thr pro ala his ser pro arg leu arg pro phe ser gly tyr glu leu gln ala asn thr thr arg ser pro gly met ser leu thr asp ile ile lys val ala val gln asp leu leu ser asp gly val phe pro asn ser ctgccgctcg atcac atg caa cga gca cag tct gcc gtg gat ttt tcc aac 351 ctc ctg aat cca acg tcg gca gca gga cag gac agc ggc gcc atg tct 399 acc gcc gcg gtc acc gtc atc aag ccc aat ggg ccc att cca gga aca 447 cag tcg acc gag act gcc aac gag ctg cct cgt ccc tac aag tgc cct 495 gln ser thr glu thr ala asn glu leu pro arg pro tyr lys cys pro ctt tgc gac aag gct ttc cac cgc ctg gag cac cag acc agg cac att 543 leu cys asp lys ala phe his arg leu glu his gln thr arg his ile cgc acc cac acg ggc gag aag ccc cat gcc tgc acc tcc att acc tgc 591 arg thr his thr gly glu lys pro his ala cys thr ser ile thr cys met gln arg ala gln ser ala val asp phe ser asn leu leu asn pro thr ala asn glu leu pro arg pro tyr lys cys pro leu cys asp lys gly glu lys pro his ala cys thr ser ile thr cys phe phe phe 665 bp and t . harzianum cre1 gene at 234 , 445 and 735 after the