WO1998023764A1 - Promoteur de cbh i, tronque et obtenu a partir de trichoderma reesei, et utilisation de celui-ci - Google Patents

Promoteur de cbh i, tronque et obtenu a partir de trichoderma reesei, et utilisation de celui-ci Download PDF

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WO1998023764A1
WO1998023764A1 PCT/FI1997/000742 FI9700742W WO9823764A1 WO 1998023764 A1 WO1998023764 A1 WO 1998023764A1 FI 9700742 W FI9700742 W FI 9700742W WO 9823764 A1 WO9823764 A1 WO 9823764A1
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promoter
protein
sequence
cbhl
sequences
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PCT/FI1997/000742
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English (en)
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Marja ILMÉN
Maija-Leena Onnela
Merja Penttilä
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Röhm Enzyme Finland OY
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Priority to AU51234/98A priority Critical patent/AU5123498A/en
Priority to EP97945898A priority patent/EP0939825A1/fr
Publication of WO1998023764A1 publication Critical patent/WO1998023764A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi

Definitions

  • Trichoderma reesei is a filamentous fungus able to carry out efficient hydrolysis of crystalline cellulose to glucose through the action of a number of secreted cellulase enzymes.
  • the major secreted protein is cellobiohydrolase I (CBHI) that may constitute up to 60 % of the secreted proteins. This amount, derived from the single copy cbhl gene, corresponds to roughly 25% of all protein synthesized by the fungus in cellulase-inducing growth conditions.
  • Availability of the carbon source regulates production of CBHI (reviewed in Bisaria, V.S. et al, CRC Crit. Rev. Biotechnol.
  • Sophorose consists of two glucose units linked by a ⁇ -l,2-glycosidic bond. Sophorose has also been considered to be a possible natural inducer formed from ⁇ -l,4-linked cello-oligosaccharides such as cellobiose by transglycosylation (Vaheri, M. et al, . Biotechnol Lett T. 41-46 (1979); Gritzali, M. et al, Adv. Chem. Ser. 181: 237-260 (1979); Loewenberg, J.R., Arch. Microbiol 137: 53-57 (1984)). Sophorose is known to induce the cbhl promoter.
  • Promoter sequence comparisons carried out for fungal cellulase promoters have revealed little similarities and the functional significance of this similarity, if any, is unknown.
  • the analysis of the promoter of the cellulase gene cbh2 of 7 reesei by a preliminary gel shift analysis showed that cellular proteins bind the promoter sequences (Stangl, H. et al, Curr. Genet. 23: 115-122 (1993)).
  • the cbhl promoter is contained on a long 2.2 kb piece of T. reesei genomic DNA flanked on the 5' end by an EcoRl site and on the 3' end by the start of the CBHI coding sequence.
  • the ability to design recombinant constructs and vectors for the production of a desired sequence under the control of this promoter would be more efficient if it were possible to shorten the promoter but yet retain the ability of the promoter to respond to inducers such as sophorose. This is especially desirable when the vector is a shuttle vector that is to be maintained in a bacterial or yeast host.
  • such shortened forms of the cbhl promoter are unknown.
  • This invention is first directed to shortened sophorose inducible cellulase and hemicellulase promoters, and their use to express operably linked proteins in fungal hosts, especially filamentous fungi.
  • the invention is further directed to DNA sequences or vectors containing such promoters, useful for the transformation of a desired host cell.
  • the invention is further directed to recombinant hosts transformed with such DNA or vectors, and especially filamentous fungal hosts.
  • the invention is further directed to recombinant hosts transformed with such DNA or vectors, and with DNA sequences encoding proteins mediating transcriptional regulation through the promoter sequences of this invention.
  • the invention is further directed to a process for producing a desired protein or antisense RNA of interest and expressing the desired protein or antisense RNA that is operably linked to the shortened promoters of the invention.
  • FIG. 2 Schematic representation of the ⁇ -galactosidase expression vector pMLOl ⁇ and the derivatives containing mutant cbhl promoters.
  • pMLOl ⁇ consists of the T. reesei wild type cbhl promoter (hashed boxes), E. coli lacZ (black boxes labeled "lacZ" in the coding sequence) and the T. reesei cbhl terminator (open boxes) in the vector pBR322 ( — ).
  • the putative TATA- box is situated at -132 and the major transcription start points (tsp) at -83 and -93 upstream of the initiator ATG.
  • the linkers (LI - L3) contain the following restriction enzyme cleavage sites, LI : BaR, Bsml, BstETL, Xbal, Kpnl, Pstl, Xhol; L2: BaR, Bsml, Bst ⁇ ll, Xbal; L3: Beauty, Bsml, BstE ⁇ l. Other relevant restriction enzyme cleavage sites are also shown. See Example 1 and Table 1 for details of the vector constructions.
  • Figure 3 (A and B).
  • the expression cassette del7(5) lacks promoter sequences between nucleotides -1497 to -210 upstream of the translation initiation codon and del7(5)* lacks all sequences upstream of -210.
  • del5(l 1) lacks sequences between -1497 and -390 bp. In pMLOl ⁇ the wild type cbhl promoter is joined to lacZ.
  • del5(l 1) and pMLOl ⁇ have the expression cassettes integrated as a single copy at the cbhl locus and lack the endogenous cbhl.
  • B Northern analysis on lacZ expression under the shortest deletion derivatives of the cbhl promoter. pMI-33, pMI- 34 and pMI- 35 lack all sequences upstream of -184, -161 and -140, respectively. 5 ⁇ g RNA was loaded on both gels and the blots were hybridized with the probes indicated. AO, acridine orange stained gel.
  • Figure 4 DNA mobility shift assay visualising proteins from total protein lysates binding to the cbhl promoter fragment from -134 to -173.
  • Lanes 1-7 total protein lysate from a glucose grown culture. Lanes 2-4, 5, 50, lOOx amounts of specific competitor DNA (same as the labelled promoter fragment). Lanes 5-7, 5,10, lOOx amounts of unspecific competitor DNA. Lanes 8-14, total protein lysate from Avicel cellulose grown culture. Lanes 9- 11 same as lanes 2-4 and lanes 12-14 same as lanes 5-7.
  • Plasmid pAS34 is also called VTT-F-97077 and was deposited as VTT-F-97077 at the DSMZ (Deutche Sammlung von Mikroorganismem und Zelkulturen GmbH), Mascheroder Weg lb, D-38124 Braunschweig, F.R.G. in E. coli on March 7, 1997 and assigned accession number DSM 11451.
  • DSMZ Deutche Sammlung von Mikroorganismem und Zelkulturen GmbH
  • Mascheroder Weg lb D-38124 Braunschweig
  • F.R.G. in E. coli on March 7, 1997 and assigned accession number DSM 11451.
  • Plasmid pAS33 is also called VTT-F-97078 and was deposited as VTT-F-97078 at the DSMZ in E. coli on March 7, 1997 and assigned accession number DSM 11452.
  • Plasmid pAS28 is also called VTT-F-97079 and was deposited as VTT-F-97079 at the DSMZ in E. coli on March 7, 1997 and assigned accession number DSM 11453.
  • Plasmid pAS26 is also called VTT-F-97080 and was deposited as VTT-F-97080 at the DSMZ in E. coli on March 7, 1997 and assigned accession number DSM 11454.
  • This invention provides shortened sophorose inducible cellulase and hemicellulase promoters, methods for creating such shortened promoters and methods for their use. Surprisingly, it has been found that cellulase and hemicellulase promoters, such as the cbhl promoter, can be greatly shortened and still retain the ability to be induced by sophorose.
  • a “shortened” promoter is meant a promoter that is truncated on its 5' end (relative to the direction of transcription) when compared to the length of the native promoter.
  • a promoter that is shortened to remove at least one regulatory element of the native promoter is thus a shortened promoter within the invention.
  • a shortened T. reesei cbhl promoter of the invention that is smaller than the "native" T.
  • reesei cbhl promoter is one smaller than the 1497 bp sequence (bases -1497 to -1) that is found immediately 5' to the start of the CBHI coding sequence and which is flanked on its 3' end by the CBHI protein coding region.
  • the sequence of the native T. reesei cbhl promoter is known ( Figure 1 [SEQ ID No. 1] and Nakari et al, WO94/04673).
  • the shortened sophorose inducible promoters of the invention retain the ability to be induced by sophorose.
  • the level of expression of a desired protein of interest can be regulated by the presence or absence of sophorose in the medium.
  • inducers of the shortened cbhl promoters of the invention include cellobiose or other inducers naturally present in industrial (complex) growth media.
  • the shortened forms of the cellulase and hemicellulase promoters of the invention lack the glucose repression sites; therefore, in a preferred embodiment, the shortened promoters of the invention are also not repressed by the presence of glucose in the medium.
  • the native cellulase and hemicellulase promoters can be greatly shortened and retain the ability to express an operably linked gene, even in the absence of their ability to be induced by sophorose.
  • the cbhl promoter can be shortened to include only 140 bases and still functions to direct the expression of an operably linked gene, albeit at a low level of expression. Therefore, while such promoters are not optimally responsive to sophorose, and may show no response to sophorose, it has been found that the desired cellulase and hemicellulase promoter may be shortened to a length of 140 bases and still be functional.
  • the shortened promoters are of a length such that the strength of the full-length promoter is retained, cbhl promoters that retain at least 161 bases, or even 184 bases of the sequence that is proximal to the 5' end of the coding sequence, have a higher level of expression than those that contain 140 bases of the sequence that is proximal to the 5' end of the coding sequence, and not only can direct transcription of operably linked sequences in a relatively efficient and strong manner that is not repressed by glucose but also are inducible with sophorose. Longer (but shortened relative to the full- length) promoters are thus possible too.
  • the 5' end of the shortened promoters of the invention may be at any position that is further upstream of position - 133 (which is the first position upstream of the TATA site).
  • Especially useful 5' end points for the shortened promoters of the invention include promoters that end at positions -210, -340, -390, and -500.
  • Sequences mediating sophorose induction lie within the 161 bp sequence immediately upstream of (5' to) the initiator ATG in the cbhl promoter (i.e., proximal to the 5' end of the coding sequence).
  • the DNA element(s) responsible for sophorose induction is present in the sequence of bases that are found within the sequence at positions -161 to -1 in the native T. reesei cbhl promoter.
  • the sequences mediating sophorose induction are either in the 30 bp region 5' of TATA, or in the region downstream of the TATA- box. These sequences seem to be also responsible for the expression observed on cellobiose medium in the plate assays.
  • Position -133 is the first nucleotide upstream of the TATA sequence.
  • the sequence of the region between -161 and -133 is: 5'TGAGC TAGTA GGCAA AGTCA GCGAA TGTG [SEQ ID No. 2]. It is this sequence, or a fragment of this sequence that is thought to be responsible for sophorose induction of the cbhl promoter.
  • the sophorose responsive shortened promoter region can be inserted in multiple copies in a shortened cbhl promoter so as to maximally enhance induction of gene expression from this promoter.
  • this element can be inserted into other fungal promoters in one or multiple copies to bring these promoters under regulation mediated through this element.
  • promoters may be natural (native) promotors or engineered ones, for instance promoters that lack binding sites for a glucose repressor or some other transcription factor(s).
  • a transcription activating protein like DNA-binding activator protein recognizing this element can be expressed in the host cell leading to enhanced production of a desired protein product.
  • the organism from which the DNA binding protein and the corresponding target sequence originate can be different from the production host organism.
  • Sequences needed for glucose repression are found between -500 and -740 in the cbhl promoter.Removing sequences above (that is, 5' to,) -500 and preferably above -210 in the T. reesei cbhl promoter results in a modified promoter that can been induced by sophorose but not repressed by glucose. Modifications that result in glucose derepression are described in Nakari et al. , WO94/04673. If desired, to eliminate glucose repression, it is necessary to delete or to mutate the glucose repressor binding sites in the promoter. In the T.
  • reesei cbhl promoter there are such sites: six have the hexanucleotide sequences 5'-(C/G)TGGGG. The sites are found -691, -699, -725, -1006, - 1154 and -1510 bases upstream of the protein coding region (i.e., positions - 691, -699, -725, -1006, -1154 and -1510 of the cbhl promoter).
  • the 5' end of the chbl promoter is truncated anywhere between approximately position -500 to position -210 from the start of the coding sequence, and the resulting promoter still retains the ability to be induced by sophorose.
  • Shortened forms of other cellulase or hemicellulase promoters that are responsive to sophorose may be made and used as described for the shortened cbhl promoter exemplified herein, either by shortening the native promoter or by shortening and/or modifying the native promoter to include SEQ ID No. 2. Therefore, the invention includes cellulase or hemicellulase promoters that are shortened according to the guidance provided for the exemplified cbhl promoter, and thus that retain the ability to be induced by sophorose.
  • the shortened promoter is a shortened cellulase promoter, and in a highly preferred embodiment, it is a shortened cbhl, cbh2, egll, egl2 or egl5 promoter. In an especially preferred embodiment, the shortened promoter is a shortened cbhl promoter. In a further preferred embodiment, the shortened cellulase or hemicellulase sophorose inducible promoter of the invention is a shortened cellulase or hemicellulase promoter of any member of the Trichoderma species, and in particular, promoters of T. reesei, T. harzianum, T.
  • the species from which the shortened promoter is derived is T. reesei.
  • the promoter from which the shortened promoter is derived is the native T. reesei cbhl, cbh2, egll, eg!2 or egl5 promoter.
  • examples of cbhl and cbh2 promoters from Trichoderma that are known and could be shortened according to the invention include the T. koningii cbhl promoter (Wey, T.T. et al, Curr. Microbiol.
  • T reesei cbh2 promoter Stangl, H. et al, Curr. Genet. 25:115-122 (1993)
  • T. viride cbhl promoter Choeng, C. et al, Nucl. Acids Res. 75: 5559 (1990)
  • a method for cloning genes activating expression through a specific promoter can be based on expression of a complete cDNA library from the desired organism, in a second host, for example, in the yeast 5 * . cerevisiae.
  • the second host for example, the yeast strain, is first transformed with a reporter construct in which expression of a reporter gene is under the control of (operably linked to) a desired heterologous (or homologous) promoter thought to contain a binding site of (or is at least responsive to) the transcriptional regulatory protein in question.
  • This could be a promoter for which regulatory features (such as inducers, repressors. growth conditions that turn it on and off, etc) are known but for which the actual regulatory proteins are not known or at least the corresponding genes are not cloned.
  • this could be a promoter for which no known inducers or regulatory mechanisms have yet been identified.
  • the second strain such as, for example, the yeast strain discussed above, is then transformed with a sample from a cDNA bank that is to be screened for the presence of genes capable of expressing proteins that activate the promoter that is operably linked to the reporter gene.
  • a cDNA bank that is from the same organism as that of the promoter or from a different organism.
  • the clones in the cDN A bank are in the form of an expression library wherein expression of proteins encoded by the clones is provided in a constitutive or inducible manner.
  • the design of the expression library should be such that promoters operably linked to the cDNA constructs are capable of functioning in the organism.
  • the second host described above contains both a clone (from the expression library) that expresses a transcriptional activator that is capable of regulating the promoter that is operably linked to the reporter construct, and also the host contains the reporter construct
  • expression of the reporter should be such that induction of the reporter's promoter's expression occurs only when activators of the gene are present.
  • the presence of the activator can be identified as an increase in the expression of the reporter gene over a base level that is found in the absense of the activator.
  • a useful reporter sequence to identify transcriptional activator proteins when S. cerevisiae is the host is the H1S3 gene.
  • yeast cerevisiae host strains (his3- minus) are available where the HIS3 gene has been deleted or otherwise inactivated in a way that they cannot grow without added histidine unless the yeast has been transformed with a functional HIS3 gene. Consequently, by transforming such hosts with a HIS3 DNA construct to which a desired promoter has been operably linked and also transforming such hosts with the gene bank from which activator genes are to be identified, yeast clones harboring the desired activator gene can be found based on their ability to grow without histidine addition to the medium. Using yeast genetic methods, the ability of the activator to activate only in the presence of the specific promoter can be confirmed.
  • Possibly leakiness of the reporter construct can be avoided, when necessary, by placing a stuffer fragment in between the upstream vector sequences and the promoter, or alternatively, for example, by using appropriate amounts of the competitive inhibitor of the HIS3 gene product, aminotriatzole (for example, 1 - 100 mM), in the medium.
  • a stuffer fragment in between the upstream vector sequences and the promoter, or alternatively, for example, by using appropriate amounts of the competitive inhibitor of the HIS3 gene product, aminotriatzole (for example, 1 - 100 mM), in the medium.
  • the TATA region on the reporter gene's promoter can be provided from the desired reporter gene, for example, the HIS3 gene, or alternatively from the promoter of question.
  • the reporter gene can be also any other gene for which the desired result, activation or repression, can be detected in a similar manner.
  • it can. for instance, encode beta-galactosidase as described for many reporter systems, or it can be, for example, CUP1 or an antibiotic resistance marker such as G418 and its activity detected based on the copper or antibiotics resistance, respectively, that it confers to the yeast harboring the activator clone.
  • the advantage of the method is that no previous knowledge of the identity or presence of the transcriptional regulatory protein, such as the activator, or of the protein ' s binding site is needed.
  • large promoter fragments can be operably linked to the reporter gene.
  • the method works also for smaller fragments of the promoter, and once the activator has been cloned, its binding sites in the promoter can be mapped by replacing the whole promoter by overlapping smaller fragments of the same.
  • the method can also be used to test whether promoters or promoter fragments contain binding sites for certain activators.
  • a yeast-based system is especially useful for cloning of fungal activator genes regulating genes encoding filamentous fungal extracellular enzymes since the yeast S. cerevisiae does not generally produce such enzymes or transcriptional regulatory proteins responsible for their production, S. cerevisiae being an exception amongst yeasts and filamentous fungi. Thus it is unlikely that proteins native to the yeast host would activate the reporter construct causing background.
  • transcriptional activator proteins that regulate the transcription of themselves or of other transcriptional activator proteins can be identified, using the same reporter system as described above.
  • the host cell would be provided with at least three constructs: a first construct containing the reporter gene operably linked to a promoter capable of being activated by a known transcriptional activator protein; a second construct that contains the gene of the known transcriptional activator protein under its native promoter; and a third construct that is the representative of the cDNA bank that is being screened for the identification of a protein that will activate transcription of the known transcriptional activator.
  • the new transcriptional activator will effectively activate transcription of the known transcriptional activator, which, in turn, activates transcription of the reporter gene. This can be achieved also by operably linking the the promoter of the activator gene directly to the reporter gene.
  • the method is also useful to identify not just regulatory proteins that regulate the transcription of other regulatory proteins, but also, to identify those transcriptional regulatory proteins that interact in an ancillary manner with another protein required for transcription so as to alter its ability to enhance or repress transcription, but that may not bind the promoter.
  • This method is not limited by the type of host and would be useful to identify any transcriptional regulatory protein for any host and in any host as long as the basic transcription machinery of such host would be expected to bind to the promoter operably linked to the reporter gene and to the transcriptional regulatory protein, as provided by the cDNA bank.
  • activator proteins in bacterial hosts could be identified by using a promoter capable of functioning in such host in the presence of the activator.
  • transcriptional activator proteins that regulate transcription in filamentous fungi can be identified.
  • the identified proteins were called ACEI and ACEII and are capable of activating the promoter of the cellulase gene cbhl that encodes the major cellulase cellobiohydrolase I (CBHI) protein.
  • the ace 2 cDNA sequence is deposited in plasmid pAS26 and the corresponding gene in plasmid pAS33 at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) and assigned accession numbers DMS 1 1454 and 11452, respectively.
  • Is is possible to modulate the expression of ACEI and ACEII, and overexpress them under any inducible or constitutive promoter in Trichoderma or Aspergillus singly or together in various repressing, neutral or induced conditions in respect to cellulase production such as on glucose containing media or on media containing sorbitol, on cellulose or its derivatives cellobiose or sophorose, on xylan. lactose, or whey.
  • Transforming a fungal host with clones capable of expressing ACEI and/or ACEII either under their own promoters or under the control of a desired heterologous promoter enhances the levels of these proteins in the host cell and allows the maximal transcriptional expression of fungal proteins that are the natural targets for these proteins. Especially, such modulation is used to improve or modify expression of hydrolytic enzyme genes under their own, modified or heterologous promoters.
  • any desired protein can be placed under the control of a promoter that is known to respond to the ACEI or ACEII protein, for example, the T. reesei cbhl promoter, and expression of such protein can thereby be regulated or enhanced in a desired host cell. If such host cell naturally produced ACEI and/or ACEII then it may not be necessary to transform such a host with additional copies of the genes encoding these proteins. However, if the host cell does not naturally produce ACEI or ACEII.
  • the host cell may be transformed with additional copies of the genes encoding one or both ACEI and ACEII as necessary and as provided according to the invention. If production is desired in conditions were the activators are not naturally produced, they can be overexpressed under a promoter functional in all conditions, eg. the fungal glycerol phosphate dehydrogenase A (gpdA) promoter or cDNAl promoter of T. reesei.
  • gpdA fungal glycerol phosphate dehydrogenase A
  • the protein which expression is enhanced by producing the activators can be any homologous protein of Trichoderma or Aspergillus, or it can be any heterologous protein like the ⁇ -lactamase encoded by the lacZ gene of E. coli shown here.
  • Another way to enhance production of proteins is to modify the promoters in such a way that they contain additional copies of the ACEI and/or ACEII binding sites. Also promoters not normally under the regulation of the activators can be modified to contain one or more binding sites. By combining these methods the fungus can be manipulated to produce enzyme mixtures specifically tailored for each application.
  • the promoters and elements described herein are useful for expression of a desired coding or antisense RNA sequence in a fungal host.
  • the process for genetically engineering such coding or antisense sequences, for expression under a promoter of the invention is facilitated through the isolation and partial sequencing of pure protein encoding an enzyme of interest or by the cloning of genetic sequences which are capable of encoding such protein with polymerase chain reaction technologies; and through the expression of such genetic sequences.
  • the term "genetic sequences" is intended to refer to a nucleic acid molecule (preferably DNA). Genetic sequences that are capable of encoding a protein are derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof.
  • the preferred source of genomic DNA is a fungal genomic bank.
  • the preferred source of the cDNA is a cDNA bank prepared from fungal mRNA grown in conditions known to induce expression of the desired gene to produce mRNA or protein.
  • a coding sequence from any host including prokaryotic (bacterial) hosts, and any eukaryotic host plants, mammals, insects, yeast, and any cultured cell populations would be expected to function (encode the desired protein).
  • Genomic DNA may or may not include naturally occurring introns.
  • such genomic DNA may be obtained in association with the 5' promoter region of the gene sequences and/or with the 3' transcriptional termination region. According to the invention however, the native promoter region would be replaced with a promoter of the invention.
  • Such genomic DNA may also be obtained in association with the genetic sequences which encode the 5' non-translated region of the mRNA and/or with the genetic sequences which encode the 3' non-translated region.
  • a host cell can recognize the transcriptional and/or translational regulatory signals associated with the expression of the mRNA and protein, then the 5' and/or 3' non-transcribed regions of the native gene, and/or, the 5' and/or 3' non-translated regions of the mRNA may be retained and employed for transcriptional and translational regulation.
  • Genomic DNA can be extracted and purified from any host cell, especially a fungal host cell, which naturally expresses the desired protein by means well known in the art.
  • a genomic DNA sequence may be shortened by means known in the art to isolate a desired gene from a chromosomal region that otherwise would contain more information than necessary for the utilization of this gene in the hosts of the invention.
  • restriction digestion may be utilized to cleave the full-length sequence at a desired location.
  • nucleases that cleave from the 3 '-end of a DNA molecule may be used to digest a certain sequence to a shortened form, the desired length then being identified and purified by gel electrophoresis and DNA sequencing.
  • Such nucleases include, for example, Exonuclease III and Ba ⁇ 1. Other nucleases are well known in the art.
  • DNA preparations are randomly sheared or enzymatically cleaved, respectively, and ligated into appropriate vectors to form a recombinant gene (either genomic or cDNA) bank.
  • a DNA sequence encoding a desired protein or its functional derivatives may be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T., (Maniatis, T.
  • Libraries containing sequences coding for the desired gene may be screened and the desired gene sequence identified by any means which specifically selects for a sequence coding for such gene or protein such as, for example, a) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of this protein, or b) by hybridization-selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized, or, c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated protein product produced by the host containing the clone.
  • Oligonucleotide probes specific for a certain protein which can be used to identify clones to this protein can be designed from the knowledge of the amino acid sequence of the protein or from the knowledge of the nucleic acid sequence of the DNA encoding such protein or a related protein.
  • antibodies may be raised against purified forms of the protein and used to identify the presence of unique protein determinants in transformants that express the desired cloned protein.
  • Peptide fragments can be analyzed to identify sequences of amino acids that may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon.
  • amino acid sequence may be encoded by only a single oligonucleotide sequence
  • amino acid sequence may be encoded by any of a set of similar oligonucleotides.
  • all of the members of this set contain oligonucleotide sequences which are capable of encoding the same peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment
  • only one member of the set contains the nucleotide sequence that is identical to the exon coding sequence of the gene.
  • this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.
  • the genetic code one or more different oligonucleotides can be identified from the amino acid sequence, each of which would be capable of encoding the desired protein.
  • the probability that a particular oligonucleotide will, in fact, constitute the actual protein encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells.
  • oligonucleotide sequence a single oligonucleotide sequence, or a set of oligonucleotide sequences, that contain a theoretical "most probable" nucleotide sequence capable of encoding the protein sequences is identified.
  • the suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of a certain gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) may be synthesized by means well known in the art (see, for example, Oligonucleotides and Analogues, A Practical Approach, F. Eckstein, ed., 1992, IRL Press, New
  • the above-described DNA probe is labeled with a detectable group.
  • detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of nucleic acid hybridization and in general most any label useful in such methods can be applied to the present invention. Particularly useful are radioactive labels, such as 32 P, 3 H, 14 C, 35 S, ,25 I, or the like.
  • any radioactive label may be employed which provides for an adequate signal and has a sufficient half-life. If single stranded, the oligonucleotide may be radioactively labelled using kinase reactions. Alternatively, polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
  • a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
  • the elucidation of a partial protein sequence permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide.
  • an oligonucleotide complementary to this theoretical sequence or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides
  • a DNA molecule or set of DNA molecules
  • a bank is prepared using an expression vector, by cloning DNA or, more preferably cDNA prepared from a cell capable of expressing the protein into an expression vector.
  • the bank is then screened for members which express the desired protein, for example, by screening the bank with antibodies to the protein.
  • the above discussed methods are, therefore, capable of identifying genetic sequences that are capable of encoding a protein or biologically active or antigenic fragments of this protein.
  • the desired coding sequence may be further characterized by demonstrating its ability to encode a protein having the ability to bind antibody in a specific manner, the ability to elicit the production of antibody which are capable of binding to the native, non- recombinant protein, the ability to provide a enzymatic activity to a cell that is a property of the protein, and the ability to provide a non-enzymatic (but specific) function to a recipient cell, among others.
  • the coding sequence and the operably linked promoter of the invention are introduced into a recipient eukaryotic cell (preferably a fungal host cell) as a non-replicating DNA (or RNA), non-integrating molecule, the expression of the encoded protein may occur through the transient (nonstable) expression of the introduced sequence.
  • the coding sequence is introduced on a DNA molecule, such as a closed circular or linear molecule that is incapable of autonomous replication,
  • a DNA molecule such as a closed circular or linear molecule that is incapable of autonomous replication
  • the DNA molecule is a linear molecule that integrates into the host chromosome.
  • Genetically stable transformants may be constructed with vector systems, or transformation systems, whereby a desired DNA is integrated into the host chromosome. Such integration may occur de novo within the cell or, be assisted by transformation with a vector which functionally inserts itself into the host chromosome.
  • the gene encoding the desired protein operably linked to the promoter of the invention may be placed with a transformation marker gene in one plasmid construction and introduced into the host cells by transformation, or, the marker gene may be on a separate construct for co-transformation with the coding sequence construct into the host cell.
  • the nature of the vector will depend on the host organism. In the practical realization of the invention the filamentous fungus Trichoderma has been employed as a model. Thus, for Trichoderma and especially for T. reesei, vectors incorporating DNA that provides for integration of the expression cassette (the coding sequence operably linked to its transcriptional and translational regulatory elements) into the host's chromosome are preferred. It is not necessary to target the chromosomal insertion to a specific site. However, targeting the integration to a specific locus may be achieved by providing specific coding or flanking sequences on the recombinant construct, in an amount sufficient to direct integration to this locus at a relevant frequency.
  • Cells that have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector in the chromosome, for example the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co- transformation.
  • a genetic marker especially for the transformation of the hosts of the invention is amdS, encoding acetamidase and thus enabling Trichoderma to grow on acetamide as the only nitrogen source.
  • Selectable markers for use in transforming filamentous fungi include, for example, acetamidase (the amdS gene), benomyl resistance, oligomycin resistance, hygromycin resistance, aminoglycoside resistance, bleomycin resistance; and, with auxotrophic mutants, ornithine carbamoyltransferase (OCTase or the argB gene).
  • acetamidase the amdS gene
  • benomyl resistance oligomycin resistance
  • hygromycin resistance aminoglycoside resistance
  • bleomycin resistance bleomycin resistance
  • auxotrophic mutants ornithine carbamoyltransferase (OCTase or the argB gene).
  • OCTase or the argB gene auxotrophic mutants
  • the use of such markers is also reviewed in Finkelstein, D.B. in: Biotechnology of Filamentous Fungi: Technology and Products, Chapter 6, Finkelstein, D.B.
  • the cloned coding sequences obtained through the methods described above, and preferably in a double-stranded form, may be operably linked to sequences controlling transcriptional expression in an expression vector, and introduced into a host cell, either prokaryote or eukaryote, to produce recombinant protein or a functional derivative thereof.
  • a host cell either prokaryote or eukaryote
  • antisense RNA or a functional derivative thereof it is also possible to express antisense RNA or a functional derivative thereof.
  • the present invention encompasses the expression of the protein or a functional derivative thereof, in eukaryotic cells, and especially in fungus.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are
  • An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence.
  • a coding sequence and a promoter region sequence linked to the 5' end of the coding sequence are said to be operably linked if induction of promoter function results in the transcription of RNA encoding the desired protein (or antisense RNA) and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the protein (or antisense RNA), or (3) interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operably linked to a DNA sequence if the promoter was capable of effecting transcription of that DNA sequence.
  • the precise nature of the regulatory regions needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences involved with initiation of transcription and translation respectively, such as the TATA box, capping sequence, CAAT sequence, and the like, with those elements necessary for the promoter sequence being provided by the promoters of the invention.
  • Such transcriptional control sequences may also include enhancer sequences or upstream activator sequences, as desired.
  • telomeres are associated in their native state with a particular gene which is capable of a high level of expression in the host cell.
  • control regions may or may not provide an initiator methionine (AUG) codon, depending on whether the cloned sequence contains such a methionine.
  • a promoter of the invention sufficient to direct the initiation of the synthesis of the desired RNA in the host cell.
  • a fusion product that contains a partial coding sequence (usually at the amino terminal end) of a protein and a second coding sequence (partial or complete) of a second protein.
  • the first coding sequence may or may not function as a signal sequence for secretion of the protein from the host cell.
  • the sequence coding for desired protein may be linked to a signal sequence which will allow secretion of the protein from, or the compartmentalization of the protein in, a particular host.
  • Such fusion protein sequences may be designed with or without specific protease sites such that a desired peptide sequence is amenable to subsequent removal.
  • the native signal sequence of a fungal protein is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the peptide that is operably linked to it.
  • Secretion signals for the filamentous fungi will generally function outside of their native host. For example, secretion signals from one type of Trichoderma, such as T. reesei, will function in another type of Trichoderma and in other filamentous fungi. Aspergillus leader/secretion signal elements also function in Trichoderma.
  • the non-transcribed and/or non-translated regions 3' to the sequence coding for a desired protein can be obtained by the above-described cloning methods.
  • the 3 '-non-transcribed region may be retained for its transcriptional termination regulatory sequence elements, or for those elements which direct polyadenylation in eukaryotic cells. Where the native expression control sequences signals do not function satisfactorily in a host cell, then sequences functional in the host cell may be substituted.
  • the vectors of the invention may further comprise other operably linked regulatory elements such as DNA sequences to target insertion at a desired site in the chromosome.
  • the vectors may also contain sequences that confer antibiotic resistance, or origins of replication for maintenance of the vector in one or more host cells, especially if the vector is designed to be capable of being maintained in a bacterial host.
  • the introduced sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. In Bacillus hosts, integration of the desired DNA may be necessary.
  • Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • preferred S. cerevisiae yeast plasmids include those containing the 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Bot- stein, D.. et al, Miami Wntr. Symp.
  • the DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, including transformation.
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. If the gene of interest is being expressed under the control of one of the glucose derepressed promoters of the invention, and if this medium includes glucose, expression of the cloned gene sequence(s) results in the production of the desired protein that is operably linked to the glucose derepressed promoter of the invention.
  • This expression can take place in a continuous manner in the transformed cells (for example, if the promoter expresses in the absence of an inducer like sophorose), or in a controlled manner, for example, by induction of expression by inclusion of inducing amounts of sophorose in the medium.
  • Useful sophorose concentrations range from about 0.1 mM to about 20 mM.
  • Useful glucose concentrations range from about 0.5 % to 5%.
  • Fungal transformation is carried out also accordingly to techniques known in the art. for example, using, for example, homologous recombination to stably insert a gene into the fungal host and/or to destroy the ability of the host cell to express a certain protein.
  • Fungi useful as recombinant hosts for the purpose of the invention include, e.g. Trichoderma, Aspergillus, Claviceps purpurea, Penicillium chrysogenum, Magnaporthe grisea, Neurospora, Mycosphaerella spp.
  • Collectotrichum trifolii the dimorphic fungus Histoplasmia capsulatum, Nectia haematococca (an&vaov ⁇ r.Fusarium solani f. sp. phaseoli and f. sp. pisi), Ustilago violacea, Ustilago maydis, Cephalosporium acremonium,
  • Trichoderma genus Trichoderma are classified on the basis of morphological evidence of similarity. T. reesei was formerly known as T. viride Pers. or T. koningii Oudem; sometimes it was classified as a distinct species of the T. longibrachiatum group. The entire genus Trichoderma, in general, is characterized by rapidly growing colonies bearing tufted or pustulate, repeatedly branched conidiophores with lageniform phialides and hyaline or green conidia borne in slimy heads (Bissett, J., Can. J. Bot. (52:924-931 (1984)).
  • T. reesei The fungus called T. reesei is clearly defined as a genetic family originating from the strain QM6a, that is, a family of strains possessing a common genetic background originating from a single nucleus of the particular isolate QM6a. Only those strains are called T. reesei.
  • Trichoderma harzianum which acts as a biocontrol agent against plant pathogens.
  • a transformation system has also been developed for this Trichoderma species (Herrera-Estrella, A. et al, Molec. Microbiol. 4:839-843 (1990)) that is essentially the same as that taught for T. reesei (EP 244,234).
  • Trichoderma harzianum is not assigned to the section
  • T. reesei is available from a variety of sources, such as, for example, ATCC26921 (T. reesei strain QM9414).
  • recipient cells After the introduction of the vector and selection of the transformant, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
  • Expression of the cloned gene sequence(s) under control of the promoter of the invention results in the synthesis and secretion of the desired heterologous or homologous protein, or in the production of a fragment of this protein, into the medium of the host cell.
  • the coding sequence is the sequence of an enzyme that is capable of hydrolysing lignocellulose.
  • examples of such sequences include a DNA sequence encoding cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII), endoglucanase I (EGI), endoglucanase II (EGII), endoglucanase III (EGIII), ⁇ - glucosidases, xylanases (including endoxylanases and ⁇ -xylosidase), side- group cleaving activities, (for example, ⁇ -arabinosidase, -D-glucuronidase, and acetyl esterase), mannanases, pectinases (for example, endo- polygalacturonase, exo-polygalacturonase, pectinesterase, or, pec
  • the expressed protein may be purified or isolated, as desired, from the medium of the host in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.
  • the cells may be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose. polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or im- munoprecipitation.
  • the protein that is expressed under direction of the promoters of the invention can also be useful in an enzyme preparation.
  • enzyme preparation is meant a composition containing enzymes.
  • the enzymes have been extracted from (either partially or completely purified from) a microbe or the medium used to grow such microbe.
  • Extracted from means that the desired enzymes are separated from the cellular mass. This can be performed by any method that achieves this goal, including breaking cells and also simply removing the culture medium from spent cells. Therefore, the term “enzyme preparation” includes compositions containing medium previously used to culture a desired microbe(s) and any enzymes that have been released from the microbial cells into such medium during the culture or downstream processing steps.
  • the host used to express the desired protein may also be substantially incapable of synthesizing one or more enzymes or proteins native to that host.
  • a host that is "substantially incapable" of synthesizing one or more enzymes is meant a host in which the activity of one or more of the listed enzymes is repressed, deficient, or absent when compared to the wild-type.
  • the T. reesei strain QM9414 (Mandels, M. et al, Appl Microbiol.
  • VTT-D-74075 was used throughout the study. It was grown on potato dextrose agar (Difco) to obtain spores which were suspended into 0.8% NaCl - 0.025% Tween 80 - 20% glycerol and stored at -70°C.
  • Trichoderma minimal medium pH 4.8
  • Trichoderma minimal medium pH 4.8
  • sorbitol 5% glucose
  • 50 ml of growth media in 250 ml conical shake flasks were inoculated with 10 7 spores and Trichoderma was grown shaking at 200 rpm at 28 °C for 48 to 87 h depending on the growth.
  • sorbitol-based cultures -sophorose Serva was added to 1 mM concentration twice, at 72 and 82 h, to ensure prolonged high level of induction of cellulase genes.
  • Mycelia were always collected 15 h after the first addition of sophorose which is representative of cellulase mRNA peak levels. Northern analyses also included mycelia grown for the same time without sophorose addition as well as mycelia grown for 15 h shorter time period. Since the latter two cultivations always gave the same result, only results from the longer cultivation without sophorose addition are shown. The amount of glucose in the medium was measured daily using the GOD-Perid system (Boehringer Mannheim). Mycelia were harvested by filtration through GF/B glass microfibre filters (Whatman), washed with sterile water or 0.7% NaCl and stored at -70 °C.
  • E. coli DH5 ⁇ was used as a host for transformations and maintenance of the plasmids. All ligation joints and PCR amplified DNA fragments were sequenced from double stranded vectors using the dideoxy termination method, sequence specific primers, and the SequenaseTM version 2 DNA polymerase (USB). The VentTM polymerase (NEB) was used for PCR amplification.
  • the expression construct pMLOl ⁇ (Fig.2) consists of the wild type T. reesei cbhl promoter (Nakari, T. et al, WO 94/04673) beginning at an Eco RI site located 2.2 kb upstream of the protein coding region (Teeri. T. et al, Bio/Technology 1 : 696-699 ( 1983)), a 3.1 kb E. coli lacZ fragment of the plasmid pAN924-21 (van Gorcom, R.F.M. et al, Gene 40: 99-106 (1985)), and a cbhl terminator as a 1.6 kb Avall-BamUl fragment (Shoemaker, S.
  • a linker containing a Sail restriction site was cloned into the Eco RI site at the 5' end of the cbhl promoter and a linker containing a Sphl site at the 3' end of the cbhl terminator (Fig. 2).
  • Other lacZ expression vectors described below contain modified cbhl promoters and are derivatives of pMLOl ⁇ (Fig. 2). Sequences of oligonucleotides used as PCR primers in construction of mutant cbhl promoters are listed in Table 1.
  • pMI-33 primers 2 and 1 (Table 1) were used. The amplified fragment was cut with BstEll-Kspl and ligated with BstE ⁇ -Kspl cut pMLOl ⁇ . pMI-34 and pMI-35 were constructed in the same way using primer pairs 3 and 1, and 4 and 1, respectively.
  • Trichoderma reesei strain QM9414 was transformed according to Penttila, M. et al, Gene 61: 155-164 (1987)). Prior to transformations expression cassettes consisting of the cbhl promoter, lacZ, and cbhl terminator were released from the vector sequences with Sail and Sphl cutting at the 5' end of the promoter and at the 3' end of the terminator, respectively, if not otherwise stated in the text. This DNA (20 ⁇ g) was purified by phenol extraction and co- transformed with 3 ⁇ g of the plasmid p3SR2 (Hynes, M.J. et al, Mol Cell.
  • ⁇ Gal activity was assayed by adding 10 ⁇ l of 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside (X-gal, 10 mg/ml) on top of the colonies, incubating the plates at room temperature and following the formation of blue colour as an indication of ⁇ Gal activity. The plates were photographed 3-5 h after addition of X-gal.
  • X-gal 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside
  • Anti-CBHI antibodies were detected using alkaline- phosphatase conjugated anti-mouse polyvalent antibodies (Sigma), and the complex was further detected using the ProtoBlot reagents (Promega).
  • Blots were hybridized with probes specific for the cbhl promoter, lacZ and cbhl cDNA. Hybridization was done in 50% formamide - 5 x Denhardt's - 5xSSPE - 0.1% SDS - 100 ⁇ g/ml denatured herring sperm DNA - 1 ⁇ g/ml poly A DNA at 42 °C overnight and washed twice for 5 min in 2xSSC at room temperature, and for 30 min in 2xSSC - 0.1% SDS at 65 °C.
  • Northern hybridizations were done at 42 °C in 50% formamide - 10% dextran sulphate - 1% SDS - 1 M NaCl - 125 ⁇ g/ml denatured herring sperm DNA overnight and washed at 42 °C in 5xSSPE, twice in lxSSPE-0.1% SDS and twice in 0.1% SSPE - 0.1% SDS, for 15 min each wash.
  • Probes were the complete T. reesei cbhl, cbh2 (Penttila, M.E. et al, Gene 63: 103-112 (1988)) and egll cDNAs (Penttila. M.E. et al, Yeast 3: 175-185 (1987)), the 2.2 kb EcoBl- BamHl fragment of the cbhl promoter obtained from pMLOl ⁇ , and the 3.1 kb long E. coli lacZ gene from pMLOl ⁇ .
  • the T. reesei actin probe covering the fifth exon of the gene was amplified from chromosomal DNA using sequence specific primers (Matheucci, E. et al, Gene 161: 103-106 (1995)). Probes were labelled using the Random Primed DNA Labeling Kit (Boehringer Mannheim) and [ - 2 P]dCTP (Amersham).
  • the basic expression vector constructed, pMLOl ⁇ consists of 2.2 kb of the wild type cbhl promoter linked to the 3.1 kb protein encoding region of lacZ, followed by a 1.6 kb cbhl terminator (Fig. 2).
  • a polylinker was inserted into the cbhl promoter region of both plasmids at the Xbal site at 1497 bp upstream of the protein coding region to enable generation of mutant promoters.
  • the expression cassettes (see later) were co- transformed into the T.
  • reesei which has a lower pH optimum.
  • the fungi were grown for 1 or 2 days, whereafter X- gal was added on top of the colonies and the formation of blue colour as an indication of ⁇ Gal activity was followed over several hours.
  • Initial screening for ⁇ Gal-producing transformants was done on cellobiose containing medium that allows for lacZ expression from the cbhl promoter. On the average, 70- 80% of the transformants were ⁇ Gal + .
  • the plate assay circumvents the use of more laborious analyses of ⁇ Gal activities of cellular extracts as an initial selection screen of the transformants to be studied in detail. lacZ was expressed from the wild type cbhl promoter in the same carbon source-dependent fashion as endogenous cbhl.
  • the expression constructs need to be targeted to a specific locus in the genome to avoid copy number and position effects in gene regulation studies. All the constructs were designed to be targeted into the cbhl locus via homologous recombination through the promoter and terminator regions thus avoiding any extra vector sequences which might affect promoter function (our unpublished observations). To achieve this, the expression cassette was released prior to transformation from the vector sequences using restriction enzymes cutting 5' of the cbhl promoter and 3' of the terminator (see Fig.2).
  • a series of lacZ expression vectors with deletion derivatives of the cbhl promoter were generated starting from the polylinker at -1497 in pMLOl ⁇ and proceeding towards the protein coding region.
  • the deletion variants (Fig. 2) were transformed into Trichoderma.
  • the transformants were ⁇ Gal + in the X- gal plate assay when grown on cellobiose medium.
  • deletion constructs including del5(l 1) and del7(5), and pMLOl ⁇ , were digested with restriction enzymes cutting at the polylinker at -1497 and at the 3' end of the cbhl terminator thus generating expression cassettes without the sequences upstream of -1497 (see Fig. 2).
  • Comparison of random transformants with and without the upstream sequences using the X-gal plate assay gave similar results in each case suggesting that the sequences upstream of -1497 did not affect the regulatory properties of the promoter.
  • the constructs were pMI-33, pMI-34 and pMI-35 covering sequences from -1 to -184, to -161, and to -140. respectively (Fig. 2).
  • the host was transformed with BstEll-Sphl cut DNA producing expression cassettes lacking sequences upstream of -1497.
  • Expression of lacZ mRNA was studied by Northern analyses (Fig. 2). lacZ expression under the pMI-33 and pMI-34 promoters was occurring on sorbitol medium and was still clearly induced by the addition of sophorose. The same result was seen in the plate assay.
  • lacZ expression in the pMI-35 transformants was more variable both in the plate assay and in the Northern analysis (Fig. 3).
  • lacZ was expressed on sorbitol medium at the level comparable with that of pMI-33 and pMI-34 and was induced by sophorose, whereas the remaining seven clones produced lacZ at a very low level and in five of these clones a very weak sophorose induction might have occurred (results of four representative clones shown in Fig. 3).
  • Sequences mediating sophorose induction lie within the 161 bp situated upstream of the initiator ATG in the cbhl promoter, either in the 30 bp region 5' of TATA, or in the region downstream of the TATA-box. These sequences seem to be also responsible for the expression observed on cellobiose medium in the plate assays.
  • the 6-bp nucleotide sequence 5'GGC(T/A)AA is repeated 15 times in cbhl promoter of T. reesei.
  • One of the repeats is situated between nucleotides -161 and -146 upstream of initiator ATG, within the 29-bp region that is sufficient for sophorose induction in T. reesei.
  • the repeats are found in both upper and lower strands.
  • the same sequence is found also in cbh2 (3x), egll (2x), xyll (3x), egl5 (3x) promoters of T. reesei.
  • sequences are found in cellulase and hemicellulase promoters in other filamentous fungi. These include Aspergillus tubigiensis xlnA, Aspergillus nidulans xlnC, Aspergillus niger xynB, Aspergillus aculeatus endoglucanase (FI- CMCase),and Aspergillus kawachii xynC. The sequence is not found in the crel promoter of T. reesei, or in the glucoamylase (glaA) promoters of Aspergillus niger or Aspergillus oryzae.
  • Aspergillus tubigiensis xlnA Aspergillus nidulans xlnC
  • Aspergillus niger xynB Aspergillus aculeatus endoglucanase
  • FI- CMCase Asper
  • 5'CGAAT is found in glucoamylase (glaA) promoters of Aspergillus niger and Aspergillus oryzae this sequence was shown to be a part of a region which is responsible for high expression and starch induction (reviwed in MacKenzie, D.A., et al, J. Gen. Microbiol. 139:2295-2207 (1993)).
  • pRS315 (Sikorski, R.S., and Hieter, P., Genetics 722:19-27 (1989)), the yeast single-copy vector containing the LEU2 marker, was digested with the restriction enzymes BamHI and Sail.
  • GGATCC is a BamHI site and GTCGAC is a Sail site.
  • the PCR fragment was digested with the above mentioned enzymes and ligated to the vector followed by sequencing of the PCR fragment.
  • the HIS3 gene of the resulting pASl plasmid contains a minimal promoter, 55 bp upstream from the ATG, which is not able to support growth in a medium lacking histidine.
  • a 1.4 kb Sacl-fragment from a non-relevant cDNA (5 ' end of a glutamate receptor cDNA from rat) was ligated in front of the HIS3 gene.
  • This plasmid was used as a negative control containing no promoter elements, since the polylinker region present in the vector pASl caused leakage of the HIS3 gene.
  • GAC TTT GCC TAC TAG CTC ACA CAT TCG CTG ACT TTG CCT ACT AGC TCA CAC ATT CGC TGA CTT TGC CTA CTA GCT CAG (SEQ ID 9), covering sequences from -161 to -133 in three copies and BamHI compatible ends (underlined), are synthesized, annealed and ligated to the BamHI cut vector.
  • Oligonucleotides having a random sequence of similar size and overall nucleotide composition 5 'GAT CCT GAA GAA TGG GAA GCA TTG CTA AGC GGT GTG AAG AAT GGG AAG CAT TGC TAA GCG GTG TGA AGA ATG GGA AGC ATT GCT AAG CGG TGG(SEQ ID 10) and 5 'GAT CCC ACC GCT TAG CAA TGC TTC CCA TTC TTC ACA CCG CTT AGC AAT GCT TCC CAT TCT TCA CAC CYGC TTA GCA ATG CTT CCC ATT CTT CAG (SEQ ID 11) are made as controls and cloned into the same vector.
  • the transformants carrying the reporter plasmids do not grow on media lacking histidine.
  • the reporter yeast is transformed with a cDNA library of T. reesei, transformant colonies are selected on SC-LEU-URA plates and subsequently screened for HIS + phenotype.
  • T. reesei QM9414 was grown on minimal medium (Penttila, M. et al, Gene 61: 155-164 (1987) supplemented with different carbon sources.
  • sorbitol sorbitol+sophorose, sorbitol+mannobiose, sorbitol+xylobiose, sorbitol+cellobiose, cellobiose, glycerol, glycerol+marmobiose, glycerol+xylobiose, mannose, xylose, xylitol, arabinose, arabitol, galactose, lactose, Lenzing xylan, methylglucuronoxylan, oat spelts xylan.
  • RNA was isolated as described by Chirgwin, J.M. et al., Biochem. J. 18:5294-5299 (1979) and analysed by Northern blotting and hybridized. The following genes were used as probes: cbhl, egl5, bgll, xyll, xyl2, bxll, abfl, girl, axel, manl, agll, agl2, agl3.
  • DNA mobility shift assays were performed to study if cellular proteins bind to the DNA sequence found immediately upstream of the TATA box of the cbhl promoter.
  • the fragment to be labelled was made by annealing two oligonucleotides containing the following sequences 5' aattcATTAA
  • the unspesific DNA used for competition in the binding reaction was prepared by annealing two oligonucleotides containing the sequences 5' AATTCGATAA AGATAGCCTC ATTAAACGGA ATGAGCTAGT T 3' (SEQ ID NO.14) and 5' CTAGAACTAG CTCATTCCGT TTAATGAGGC TATCTTTATC G 3 '(SEQ ID NO. 15).
  • the mycelial total protein lysates were prepared from T.reesei grown in shake flasks on minimal media containing 2% glucose or 1% Avicel cellulose for 20h and 5d respectively.
  • Mycelia were harvested from the culture media by filtration trough GF/B glass-microfiber filters (Whatman), washed with buffer A ( 20 mM Hepes, pH 7.9, lOOmMKCl, 2mM EDTA, lOmM DTT and 2mM PMSF) and grounded under liquid nitrogen. The ground mycelia was dissolved in buffer A and incubated on ice for 10 min. The cell debris was spinned down and the supernatant was collected and used for mobility shift assays.
  • buffer A 20 mM Hepes, pH 7.9, lOOmMKCl, 2mM EDTA, lOmM DTT and 2mM PMSF
  • the total protein lysate (10 ⁇ g) was incubated with the labelled DNA fragment ( 1 ng, 20 000 CPM) for 30 minutes at 25°C in a 20 ⁇ l total volume in a reaction mixture containing 25mM Hepes, 50mM NaCl, 10% glycerol,
  • ORGANISM Trichoderma reesei
  • AATAATTGTA CAATCAAGTG GCTAAACGTA CCGTAATTTG CCAACGGCTT GTGGGGTTGC 1500
  • CTCATTAAAC GGAATGAGCT AGTAGGCAAA GTCAGCGAAT GTGTATATAT AAAGGTTCGA 2100
  • ORGANISM Trichoderma reesei
  • ORGANISM Trichoderma reesei
  • STRAIN QM9414
  • ORGANISM Trichoderma reesei
  • CAGGAGACTT GTACACCATC TTTTGAGGCA CAGAAACCCA ATAGTCAACC GCGGACTGCG 180 CATC 184
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Trichoderma reesei
  • STRAIN QM9414

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Abstract

L'invention concerne un promoteur de CBH I (cellobiohydrolase I), tronqué et obtenu à partir de Trichoderma reesei. Ce promoteur a la capacité d'être induit par la sophorose. L'invention concerne en outre des vecteurs contenant de tels promoteurs, des hôtes transformés à l'aide de ces vecteurs, ainsi que des procédés d'utilisation de tels promoteurs pour l'expression de séquences qui lui sont liées de manière fonctionnelle.
PCT/FI1997/000742 1996-11-29 1997-12-01 Promoteur de cbh i, tronque et obtenu a partir de trichoderma reesei, et utilisation de celui-ci WO1998023764A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU51234/98A AU5123498A (en) 1996-11-29 1997-12-01 Truncated cbh i promoter from trichoderma reesei and use thereof
EP97945898A EP0939825A1 (fr) 1996-11-29 1997-12-01 Promoteur de cbh i, tronque et obtenu a partir de trichoderma reesei, et utilisation de celui-ci

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US3215696P 1996-11-29 1996-11-29
US60/032,156 1996-11-29
US3295996P 1996-12-13 1996-12-13
US60/032,959 1996-12-13
US4014097P 1997-03-10 1997-03-10
US60/040,140 1997-03-10

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US09/066,597 Continuation-In-Part US6001595A (en) 1996-11-29 1998-04-27 Promoters and uses thereof

Publications (1)

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WO1998023764A1 true WO1998023764A1 (fr) 1998-06-04

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Application Number Title Priority Date Filing Date
PCT/FI1997/000742 WO1998023764A1 (fr) 1996-11-29 1997-12-01 Promoteur de cbh i, tronque et obtenu a partir de trichoderma reesei, et utilisation de celui-ci
PCT/FI1997/000743 WO1998023642A1 (fr) 1996-11-29 1997-12-01 Genes codant des proteines de regulation transcriptionnelle, obtenus a partir de trichoderma reesei, et utilisations de ceux-ci

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/FI1997/000743 WO1998023642A1 (fr) 1996-11-29 1997-12-01 Genes codant des proteines de regulation transcriptionnelle, obtenus a partir de trichoderma reesei, et utilisations de ceux-ci

Country Status (3)

Country Link
EP (2) EP0950064A1 (fr)
AU (2) AU5123498A (fr)
WO (2) WO1998023764A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6001595A (en) * 1996-11-29 1999-12-14 Rohm Enzyme GmbH Promoters and uses thereof
WO2002092757A2 (fr) * 2000-11-15 2002-11-21 Kemin Industries, Inc. Enzyme surproduisant des micro-organismes transgeniques
EP2397491A1 (fr) 2010-06-21 2011-12-21 Technische Universität Wien LeaA de Trichoderma reesei
EP2708553A1 (fr) 2012-09-18 2014-03-19 Technische Universität Wien Cellule fongique modifiée
CN112961788A (zh) * 2021-02-24 2021-06-15 江南大学 一种在里氏木霉中高产木聚糖酶的方法及其应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002240882A1 (en) * 2000-12-29 2002-07-16 Rhein Biotech Gesellschaft Fur Neue Biotechnologische Prozesse Und Produkte Mbh Method for producing heterologous proteins in a homothallic fungus of the sordariaceae family
FI120310B (fi) * 2001-02-13 2009-09-15 Valtion Teknillinen Parannettu menetelmä erittyvien proteiinien tuottamiseksi sienissä
WO2021007630A1 (fr) 2019-07-16 2021-01-21 Centro Nacional De Pesquisa Em Energia E Materiais Lignée de champignon trichoderma modifiée pour la production d'un cocktail enzymatique

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004673A1 (fr) * 1992-08-19 1994-03-03 Alko Group Ltd. Promoteurs fongiques actifs en presence du glucose

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004673A1 (fr) * 1992-08-19 1994-03-03 Alko Group Ltd. Promoteurs fongiques actifs en presence du glucose

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 228, 1996, F. HENRIQUE-SILVA et al., "Two Regulatory Regions Controlling Basal and Cellulose-Induced Expression of the Gene Encoding Cellohydrolase I of Trichoderma Reesei are Adjacent to Its TATA Box", pages 229-237. *
DIALOG INFORMATION SERVICES, File 154, MEDLINE, Dialog Accession No. 08945369, Medline Accession No. 97156936, ILMEN M. et al., "Functional Analysis of the Cellobiohydrolase I Promoter of the Filamentous Fungus Trichoderma Reesei"; & MOL. GEN. GENET., (GERMANY), 13 Dec. 1996, 253 (3), pages 303-14. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6001595A (en) * 1996-11-29 1999-12-14 Rohm Enzyme GmbH Promoters and uses thereof
WO2002092757A2 (fr) * 2000-11-15 2002-11-21 Kemin Industries, Inc. Enzyme surproduisant des micro-organismes transgeniques
WO2002092757A3 (fr) * 2000-11-15 2003-03-13 Kemin Ind Inc Enzyme surproduisant des micro-organismes transgeniques
EP2397491A1 (fr) 2010-06-21 2011-12-21 Technische Universität Wien LeaA de Trichoderma reesei
WO2011161063A1 (fr) 2010-06-21 2011-12-29 Technische Universität Wien Leaa de trichoderma reesei
EP2708553A1 (fr) 2012-09-18 2014-03-19 Technische Universität Wien Cellule fongique modifiée
WO2014044640A1 (fr) 2012-09-18 2014-03-27 Technische Universität Wien Cellule fongique modifiée
CN112961788A (zh) * 2021-02-24 2021-06-15 江南大学 一种在里氏木霉中高产木聚糖酶的方法及其应用

Also Published As

Publication number Publication date
WO1998023642A1 (fr) 1998-06-04
EP0950064A1 (fr) 1999-10-20
AU5123598A (en) 1998-06-22
AU5123498A (en) 1998-06-22
EP0939825A1 (fr) 1999-09-08

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