WO1998023642A1 - Genes codant des proteines de regulation transcriptionnelle, obtenus a partir de trichoderma reesei, et utilisations de ceux-ci - Google Patents

Genes codant des proteines de regulation transcriptionnelle, obtenus a partir de trichoderma reesei, et utilisations de ceux-ci Download PDF

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WO1998023642A1
WO1998023642A1 PCT/FI1997/000743 FI9700743W WO9823642A1 WO 1998023642 A1 WO1998023642 A1 WO 1998023642A1 FI 9700743 W FI9700743 W FI 9700743W WO 9823642 A1 WO9823642 A1 WO 9823642A1
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nucleic acid
acid molecule
sequence
seq
polypeptide
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PCT/FI1997/000743
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Anu Saloheimo
Nina Aro
Marja ILMÉN
Merja Penttilä
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Röhm Enzyme Finland OY
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Priority to AU51235/98A priority Critical patent/AU5123598A/en
Priority to EP97945899A priority patent/EP0950064A1/fr
Publication of WO1998023642A1 publication Critical patent/WO1998023642A1/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

  • the invention is in the field of transcriptional regulation of fungal gene expression.
  • Trichoderma reesei is widely used in production of hydrolytic enzymes for industrial applications.
  • the promoters used especially the cbhl promoter of the gene encoding the major cellulase of this fungus, are strong and lead to high expression levels of most of the products. There is, however, a need for even more efficient expression systems for both homologous and heterologous products.
  • This kind of limitation can be overcome by overexpressing an activator protein that is capable of inducing the production promoter.
  • an activator protein that is capable of inducing the production promoter.
  • One example of this is the overexpression of the alcR activator of Aspergillus which activates the alcA promoter.
  • Another example is the S. cerevisiae GAL4 activator, or the corresponding protein LAC9 of K. lactis, which activates the GALl promoter of S. cerevisiae.
  • the CREI of Trichoderma is the first protein shown to regulate cellulase expression (Ilmen, M., et al, Mol. Gen. Genet. 255:303-314 (1996) and Mol. Gen. Genet. 257:451-460 (1996)). From Aspergillus, XYLR activating the gene expression of the xylanase genes is characterized (WO 97/00962). No positively acting regulatory proteins involved in the induction of cellulase promoters of any fungi are, however, known.
  • Another option to increase (stimulate) promoter function is to modify the promoter so that it contains multiple binding sites for the activator(s) thus sequestering the activator so that it binds in a larger amount to the desired promoter and further activates the promoter's expression.
  • the fungus could be tailored to produce an optimal combination of enzymes for each industrial application.
  • Cloning of activator genes can be attempted by analyzing all proteins binding to previously characterized binding sites, and then making antibodies to the proteins or making nucleic acid probes based on the N-terminal amino acid sequences of the proteins, and using conventional cloning methods to obtain the activator gene. Also, if the binding site is known, the so-called one-hybrid system can be used (Clontech). Also, regulatory mutant complementation, or alternatively heterologous hybridization with a DNA encoding the desired activator from an other organism can be used and the cloned genes analyzed.
  • This invention is first directed to a method for cloning transcriptional regulatory elements (binding sites) and transcriptional regulatory proteins, in particular, transcriptional activator elements and transcriptional activator proteins, in a manner that does not depend upon the availability of mutants or data therefrom, and does not require that the DNA binding site of such proteins be known in advance.
  • the invention is further directed to the identification of the T. reesei acel and ace2 transcriptional activator proteins using such method, and their cloning.
  • the invention is further directed to nucleic acid sequences encoding T. reesei acel and ace2, including expression cassettes from which the proteins encoded by these genes can be expressed, including vectors providing the same.
  • the invention is further directed to hosts transformed with such nucleic acid sequences.
  • the invention is further directed to a method of stimulating gene expression in hosts transformed with sequences encoding acel and ace2 by providing to such host a DNA construct in which the gene of interest is operably linked to a promoter that further contains one or more binding sites for the acel and/or ace2 transcriptional activator proteins that are heterologous to the native promoter structure.
  • the invention is further directed to a method for enhancing expression of a desired gene in cells capable of expressing ACEI and/or ACEII by inserting into the promoter of the gene a binding site for ACEI and/or ACEII, or multiple copies of such sites.
  • FIG. 1 Plasmid pAS3.
  • FIG. 2 Plasmid pMS95.
  • FIG. 3 Plasmid pAJ401.
  • FIG. 4 The acel cDNA sequence (SEQ ID NO. 5).
  • FIG. 5a to e The acel chromosomal sequence and the deduced protein sequence of ACEI (SEQ ID NO. 6 and 7).
  • FIG. 6 The ace2 cDNA sequence (SEQ ID NO. 8).
  • FIG. 7a to c The ace2 chromosomal sequence and the deduced protein sequence of ACEII (SEQ ID NO. 9 and 10).
  • FIG. 8 Plasmid p -66
  • FIG. 9 Northern analysis of T. reesei QM9414 host (lane 1) and five transformants (lanes 2-6) that overproduce the ACEII protein on Solka floe cellulose or medium containing glucose.
  • FIG. 10. The expression vector pIvfl-69.
  • FIG. 11. The expression vector pARO3.
  • FIG. 12. Plasmid pALK1062NB .
  • Plasmid pAS34 is also called VTT-F-97077 and was deposited as VTT-F-
  • 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
  • Plasmid pAS28 is also called VTT-F-97079 and was deposited as VTT-F-
  • Plasmid pAS26 is also called VTT-F-97080 and was deposited as VTT-F-
  • the invention describes a method for identification and isolation of transcriptional regulatory protein-encoding genes, and especially genes encoding transcriptional activator proteins.
  • the advantage of the method of the invention is that no information about the DNA binding site (sequence) that is recognized by the transcriptional regulatory protein is needed to practice the method because the method of the invention allows for the identification of the sites where specific activators bind.
  • the 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 S. 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.
  • 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 cDNA 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.
  • the methods of the invention are also useful for identifying transcriptional repressor proteins.
  • a useful reporter sequence to identify transcriptional activator proteins when S. cerevisiae is the host is the HIS 3 gene.
  • S. 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.
  • 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.
  • aminotriatzole for example, 1 - 100 mM
  • 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, CUPl 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 of the invention 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 of the invention 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 promoter of the activator gene directly to the reporter gene.
  • the method of the invention 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 to 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.
  • ACEI and ACEII transcriptional activator proteins that regulate transcription in filamentous fungi. These proteins are called ACEI and ACEII and are capable of activating the promoter of the cellulase gene cbhl that encodes the major cellulase cellobiohydrolase I
  • CBHI CBHI protein.
  • ace2 genes were obtained by screening of a T. reesei gene bank that had been induced to maximally express a variety of T. reesei extracellular enzymes including cellulases as well as xylanases and other hemicellulases.
  • the encoded proteins contain DNA binding regions but show no other obvious amino acid similarity to any other protein known, not even when compared against the data base containing the complete yeast genome.
  • ACEI (Example 3 and 4) has an open reading frame of 733 amino acids (SEQ ID No. 7). It contains the C 2 H 2 zinc finger domain that characterizes one major class of DNA binding domains present in a variety of regulatory proteins from other species.
  • the acel cDNA sequence is shown in SEQ ID No. 5 and the sequence of the acel gene containing three introns is shown in SEQ ID No. 6.
  • ACEII (Example 3 and 5) has an open reading frame of 341 amino acids (SEQ ID No.10).
  • the ace2 cDNA sequence is shown in SEQ ID No. 8 and the ace2 gene sequence, which contains no introns, is shown in SEQ ID No. 9.
  • the DNA binding domain of ACEII is a binuclear zinc cluster typical of many activators of fungal origin only.
  • ACEI and ACEII it 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.
  • 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.
  • 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.
  • 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 AC ⁇ I 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 process for genetically engineering a coding sequence, for expression under an ACEI or ACEII-sensitive promoter, 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, for example, by cDNA cloning or 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.
  • genomic DNA may be obtained in association with the 5' promoter region of the gene sequences and/or with the 3' transcriptional termination region.
  • the native promoter region would be replaced with an ACEI and/or ACEII responsive promoter.
  • 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 either genomic DNA or cDNA
  • suitable 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 Iigation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and
  • 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.
  • 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
  • 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. Although occasionally an amino acid sequence can be encoded by only a single oligonucleotide sequence, frequently the amino acid sequence are encoded by any of a set of similar oligonucleotides.
  • 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. Because 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.
  • 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.
  • codon usage rules 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 can be synthesized by means well known in the art (see, for example, Oligonucleotides and Analogues, A Practical Approach, F. Eckstein, ed., 1992, LRL Press, New York) and employed as a probe to identify and isolate a clone to such gene by techniques known in the art. Those members of the above-described gene bank that are found to be capable of such hybridization are then analyzed to determine the extent and nature of coding sequences which they contain.
  • 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 2 P, H, l4 C, 35 S, 125 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.
  • 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.
  • oligonucleotide complementary to this theoretical sequence or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides, one obtains a DNA molecule (or set of DNA molecules), capable of functioning as a probe(s) for the identification and isolation of clones containing a gene.
  • 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), or non-integrating molecule, the expression of the encoded protein may occur through the transient
  • the coding sequence is introduced as a DNA molecule, such as a closed circular or linear DNA molecule that is incapable of autonomous replication and most preferably, a linear molecule that integrates into the host chromosome.
  • Genetically stable fungal 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.
  • ACEII inducible promoter of the invention may be provided along 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 ACEI and ACEII regulated embodiments 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.
  • 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 that allow for selection of host cells that 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 intro- quizzed into the same cell by co-transformation.
  • marker genes should not to be confused with the marker gene that is used to identify transcriptional regulatory proteins according to that method of the invention (although such markers could be used in fungal hosts in that regard). Rather, the discussion here regards the use of these markers as markers for the transformation event.
  • 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 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. et al, eds., Butterworth
  • 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 “operably linked” to the nucleotide sequence which encodes the polypeptide.
  • 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.
  • Two DNA sequences (such as 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 mRNA encoding the desired protein 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 proteins, 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.
  • Expression of a protein in eukaryotic hosts such as fungus requires the use of regulatory regions functional in such hosts, and preferably fungal regulatory systems.
  • a wide variety of transcriptional and translational regulatory sequences can be employed, depending upon the nature of the host.
  • these regulatory signals are associated in their native state with a particular gene that 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.
  • AUG initiator methionine
  • Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis in the host cell. Promoters from filamentous fungal genes which encode a mRNA product capable of translation are preferred, and especially, strong promoters can be employed provided they also function as promoters 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 that 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.
  • Aspergillus leader/secretion signal elements also function in Trichoderma.
  • the non-transcribed and/or non-translated regions 3 1 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 that 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 may further comprise other operably linked regulatory elements such as DNA elements that confer antibiotic resistance, or origins of replication for maintenance of the vector in one or more host cells.
  • the introduced sequence is incor- porated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • 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 that do not contain the vector; the number of copies of the vector that 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. 7P:265-274 (1982); Broach, J.R., in: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, J.R., Cell 25:203-204 (1982); Bollon, D.P., et al, J. Clin. Hematol. Oncol. 70:39-48 (1980); Maniatis, T., In: Cell Biology: A Comprehensive
  • 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 this medium includes glucose, expression of the cloned gene sequence(s) results in the production of the desired protein, or in the production of a fragment of this protein as desired. This expression can take place in a continuous manner in the transformed cells, or in a controlled manner, for example, by induction of expression.
  • 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., CoUectotrichum trifoli the dimorphic fungus Histoplasmia capsulatum, Nectria haematococca solani f. sp. phaseoli and f. sp. pisi),
  • Ustilago violacea Ustilago maydis, Cephalosporium acremonium, Schizophyllum commune, Podospora anserina, Sordaria macrospora, Mucor circinelloides, Humicola, Melanocarpus, Myceliophthora, Chaetomium and CoUectotrichum capsici. Transformation and selection techniques for each of these fungi have been described (reviewed in Finkelstein, D.B. in: Biotechnology of Filamentous Fungi: Technology and Products, Chapter 6, Finkelstein, D.B. et al, eds., Butterworth-Heinemann, publishers, Stoneham, MA, (1992), pp. 113- 156).
  • Trichoderma reesei Trichoderma reesei, T. harzianum, T. longibrachiatum, T. viride, T. koningii, Aspergillus nidulans, A. niger, A. terreus, A.ficum, A. oryzae, A. awamori and N. crassa.
  • the hosts of the invention are meant to include all Trichoderma.
  • 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. 62:924-931 (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 species For example, Cheng, C, et al, Nucl. Acids. Res. 18:5559 (1990), discloses the nucleotide sequence of T. viride cbhl. The gene was isolated using a probe based on the T reesei sequence. The authors note that there is a 95% homology between the amino acid sequences of the T viride and T. reesei gene. Goldman, G.H. et al, Nuc Acids Res. 18:6717 (1990), discloses the nucleotide sequence of phosphoglycerate kinases from T.
  • T. viride and notes that the deduced amino acid sequence is 81% homologous with the phosphoglycerate kinase gene from T. reesei. Thus, the species classified to T. viride and T. reesei must genetically be very close to each other.
  • Trichoderma can be found in the formerly discussed Trichoderma section Longbrachiatum, there are some other species of Trichoderma that are not assigned to this section. Such a species is, for example, Trichoderma harzianum, which acts as a biocontrol agent against plant pathogens. A transformation system has also been developed for this Trichoderma species (Herrera-Estrella,
  • Trichoderma species are available from a wide variety of resource centers that contain fungal culture collections.
  • Trichoderma species are catalogued in various databases. These resources and databases are summarized by O'Donnell, K. et al, in Biochemistry of Filamentous Fungi: Technology and Products, D.B. Fingelstein et al, eds.,
  • 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) 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 EH (EGUI), ⁇ -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, pectin and pectin acid lyase), and enzymes of lig
  • the gene for the native cellobiohydrolase CBHI sequence has been cloned by Shoemaker et al. (Shoemaker, S., et al, Bio/Technology 7:691-695 (1983)) and Teeri et al. (Teeri, T., et al. , Bio/Technology 1 :696-699 ( 1983)) and the entire nucleotide sequence of the gene is known (Shoemaker, S., et al, Bio/Technology 7:691-695 (1983)). From T.
  • the expressed protein may be isolated and purified 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 DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, hydroxy- apatite or by electrophoresis or immunoprecipitation.
  • the reporter plasmid pAS3 ( Figure 1) was constructed as follows. 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. The HIS3 reporter gene of S. cerevisiae was cloned from cosmid p3030 (Hohn and Hinnen, unpublished; see Penttila, M. ⁇ ., et al, Mol. Gen. Genet.
  • 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.
  • pASl was digested with the restriction enzymes Sacl and Xbal.
  • Sacl and Xbal A 1.15 kb promoter fragment upstream from the TATA box of the T. reesei cbhl promoter was cloned from the plasmid pMLO16 (Ilmen, M., eta , Mol. Gen. Genet.
  • TCTAGA is a Xbal site.
  • the promoter fragment was digested with the restriction enzymes Sacl and Xbal and ligated in front of the HIS3 gene in the pASl vector.
  • Plasmid pMS95 ( Figure 2), used as a negative control plasmid, was constructed as follows. pAS 1 plasmid was digested with the restriction enzyme
  • Sacl 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. Plasmids pAS3 and pMS95 were transformed into the yeast strain
  • DBY746 (ATCC 44773, his3- ⁇ leu2-3 leu2- ⁇ ⁇ 2 ura3-52 trpl-2 9 cyH cir; Dr. D. Bothstein, Massachusetts Institute of Technology, Cambridge,MA) by electroporation according to the manufacturer ' s instructions (Bio-Rad). Transformants were plated on synthetic complete plates (Sherman, F., Meth. Enzymol. 194:3-21 (1991)) supplemented with 1M sorbitol and all other amino acids except leusine (SC-Leu). Colonies were streaked on SC-Leu and SC-Leu- His -plates.
  • the reporter strain DBY746-pAS3 and the control strain DBY746- pMS95 could not grow on media lacking histidine showing that the Trichoderma promoter or the non-relevant DNA fragment could not drive the expression of the reporter gene in yeast.
  • a cDNA library of Trichoderma reesei grown in hydrolase-inducing conditions Solka floe cellulose, spent grain, locust bean gum, lactose, acetyl glucuronoxylan, arabinoxylan
  • the expression library could be prepared using an inducible yeast promoter such as GALl (West et al., 1984).
  • GALl West et al., 1984.
  • the growth conditions and preparation of mRNA from T. reesei Rut-C30 strain have been described (Stalbrand, H., et al, Appl. Environ. Microbiol. (57:1090-1097 (1995)).
  • cDNA synthesized by the ZAP-cDNA synthesis kit (Stratagene), was ligated to the EcoRI-XhoI cut plasmid pAJ401.
  • the DBY746-pAS3 reporter yeast strain was transformed with the library plasmid stock by electroporation according to the manufacturer's instructions (Bio-Rad). Electroporation with the total of 40 ⁇ g of the plasmid pool gave a library of 10 6 yeast cells growing on SC-Leu-Ura plates supplemented with 1M sorbitol. After 7d of growth at 30 °C the colonies were scraped from the plates. In order to screen HIS + colonies the library was plated on SC-Leu-Ura-His plates to a density of 10 6 cells per plate and grown at 30°C for 5d.
  • the library is plated on a medium containing galactose instead of glucose as a carbon source. 0.004% of the yeast cells could grow in the selection conditions. Plasmid DNA was isolated from 48 growing colonies and transformed to the DBY746 yeast strain with and without the reporter constructs. 75% of the plasmids supported growth of all the strains on media lacking histidine. The existance of the Trichoderma reesei his3 gene in these clones was verified by sequencing of the cDNA from the 5' end followed by homology comparison of the open reading frame (ORF) against the yeast and Neurospora crassa hi 3 genes.
  • ORF open reading frame
  • pAS27 contained a 1.9 kb cDNA and was named acel (activator of cellulase expression).
  • the other four plasmids each contained an identical cDNA of 1.4 kb that was different from the ⁇ ce7 DNA.
  • pAS26 was studied further and the gene was named ace2.
  • a total of 8 x 10 5 plaques were plated to NZY plates to a density of 5 x 10 4 per 150 mm diameter plate. Two replicas were taken from each plate to nitrocellulose filters.
  • Hybridization was carried out overnight at 42°C in a solution containing 50% formamide, 5x Denhardt's, 5xSSPE, 0.1% SDS, 100 ⁇ g/ml herring sperm DNA, 1 ⁇ g/ml polyA DNA and
  • RNAs The ORF of 733 aa starting from the first ATG codon of the cDNA maintains the frame of the original ORF and contains 242 additional amino acids. There is a rather long (over 600 bp) non-translated 5' leader sequence in the cDNA but the existance of three in-frame stop codons before the first ATG in the cDNA confirms that the plasmid contains at least the whole coding sequence of the acel gene ( Figure 4; SEQ ID No. 5).
  • Hybridization conditions were as in the plaque hybridization except that the amount of the 2 P-labelled full-length cDNA probes was 10 6 cpm/ml.
  • the filters were washed twice at room temperature in 2xSSC - 0.1% SDS for 5 min followed by a wash at 68°C in O.lxSSC - 0.1% SDS for 60 min.
  • the strains used were QM9414 (ATCC 26921,Mandels, M., et al, Appl. Microbiol.
  • VTT-D-79125 and VTT-D-80133 cellulase overproducing strains,Bailey, M.J. and Nevalainen, K.M.H., Enzyme Microb. Technol 5:153- 157 (1981)
  • Rut-C30 and the cellulase negative strains VTT-D-81152, VTT-D- 81153, VTT-D-81155, VTT-D-81158 and VTT-D-81168 (el- 18, cel-7, cel-1, cel-22 and cel-25 in Nevalainen, K.M.H. and Palva, E.T. Appl. Environ.
  • the filters were prewashed at 42°C in a solution containing 50mM Tris pH 8, 1M NaCl, lrnM EDTA and 0.1% SDS. Hybridization conditions and washes were the same as those used in Southern hybridization except that the amount of the probes was 5 x 10 5 cpm/ml. Two cosmids giving strong signals were purified and found to contain the acel chromosomal gene. The gene was subcloned as a 7 kb HindHI fragment into the pZErO-1 vector (Invitrogen) resulting in plasmid pAS34. Sequencing of the chromosomal gene revealed three introns, one of which interrupts the long 5' non-coding sequence present in the cDNA.
  • a chromosomal lambda library of T reesei strain QM9414 (Vanhanen, S., et al, Curr. Genet. 75:181-186 (1989)) was screened in plaque hybridization using PCR fragments of the coding sequence of ace2 gene as a probe.
  • a total of 4 x 10 4 plaques were plated to NZY plates to a density of 10 'per 150 mm diameter plate. Two replicas were taken from each plate. The filters were denatured, neutralized and fixed. Hybridization conditions and washes were as in the screening of the cosmid library. Three plaques giving strong signals with the ace2 probe were purified.
  • Lambda DNA was isolated from the purified clones and the chromosomal ace2 gene was subcloned as a 6 kb EcoRI-Hindlll fragment into the pZErO-1 vector resulting in plasmid pAS33. Sequencing of the pAS33 plasmid revealed no introns. In the promoter regions of the ace genes, several sequences similar to the consensus binding sites for the glucose repressor proteins CREI of T. reesei (Ilmen, M., et al, Mol. Gen.Genet. 257:451-460 (1996)) and CREA of A. nidulans (Kulmburg, P., et al, Mol. Microbiol. 7:847-857 (1993)) can be found. This suggests that the ace genes are under glucose repression mediated by the CREI repressor protein.
  • the acel cDNA sequence (SEQ ID NO. 5) is shown in Figure 4, the chromosomal sequence (SEQ ED NO. 6) and the deduced protein sequence (SEQ ED NO. 7) is shown in Figure 5.
  • Amino acids 387-403 form a putative bipartite nuclear targeting signal RRKKNATPEDVAPKKCR (SEQ ID NO. 25) (basic residues are shown in bold) fitting well to the consensus (at least two basic residues separated by a spacer often residues from a block of at least three basic residues within the next five amino acids, Dingwall, C, and Laskey, R.A., Trends Biochem. Sci. 7(5:478-481 (1991)).
  • Sequencing of the ace2 cDNA from the pAS26 plasmid revealed a 1373 bp cDNA with an ORF of 341 aa starting from the first ATG codon only 6 bp after the start of the cDNA.
  • Northern hybridization using the cDNA as a probe gave 2-3 signals of about the size of the cDNA and a weaker signal of about 1.6 - 1.8 kb.
  • the last codon preceding the start of the cDNA is a stop codon in the frame of the ORF.
  • the acel cDNA sequence (SEQ ID NO. 8) is shown in Figure 6, the chromosomal sequence (SEQ ED NO. 9) and the deduced protein sequence (SEQ ID NO. 10) is shown in Figure 7.
  • At the N-terminus of the deduced protein there is a sequence ACDRCHDKKLRCPRISGSPCCSRCAKANVAC ( SEQ ID 26) fitting well to the consensus of a fungal Zn 2 C 6 binuclear cluster domain [G/A/S/T V]-C-x(2)-C-[R/K/H]-x(2)-[R/K/H]-x(2)-C-x(5 to 9)-C-x(2)-C-x(6 to 8)-C (conserved residues are shown in bold, Pan, T., and Coleman, J.E.
  • This area in bicoid protein is called the PRD repeat (Berleth et al, EMBOJ. 7:1749-1756 (1988)).
  • this histidine-rich area is followed by a glutamine and proline-rich stretch (EQQQEQQQGQPQHPPPPVQ) (SEQ ED 28).
  • EQQQEQQQGQPQHPPPPVQ glutamine and proline-rich stretch
  • This area gave also similarity to some regulatory proteins of Drosophila in the search.
  • the ACEII sequence showed no significant similarity to known regulatory proteins except for the short regions mentioned above most probably corresponding to general features of regulatory proteins.
  • Trichoderma was grown in 250 ml conical flasks in 50 ml of the minimal medium containing 2% of carbon source indicated, 1.5% KH 2 PO 4 , 0.5%
  • RNA concentrations were measured spectrophotometrically and further controlled by running 2 ⁇ g samples in an agarose gel followed by an acridine orange staining.
  • RNAs were glyoxylated according to Sambrook, J., et al, Molecular Cloning. A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (1989)), run in a 1 % agarose gel and capillary blotted to the Hybond N nylon filter.
  • the filters were hybridized with 2 P-labelled cDNA probes overnight at 42°C in 10-15 ml of the RNA hybridization solution containing 50% formamide, 1M NaCl, 1% SDS, 10% dextran sulphate, 100 ⁇ g/ml herring sperm DNA and 4-8 x 10 5 cpm/ml of the probe.
  • the filters were washed for 20 min once at room temperature in lxSSC - 0.1% SDS and three times at 68 C in 0.2xSSC - 0.1%
  • pMI-66 Trichoderma expression vector was as follows. The coding region of the acel gene (working name S26) was first cloned from plasmid pAS28 by PCR using the 5 'primer GCT CTA GAG CCG GAT CCA TCC TTT TCG AAC CCC CGC (SEQ ED NO. 11) and the 3 'primer TCC CCC CGG GGG GAG GAT CCT TAC TCT TGA AAC CCC TGG T (SEQ LD
  • the underlined GGATCC is a BamHI site.
  • the PCR fragment was ligated to the pGEM-t vector purchased from Promega.
  • the resulting plasmid was digested with the restriction enzyme BamHI and the isolated acel fragment was ligated to a similarily-cut pAN52-lNotI fungal expression vector (Punt, P.J. et al, Gene (59:49-57 (1988)) under the constitutive gpdA promoter of A. nidulans that supports high expression levels also in Trichoderma.
  • the resulting pMI-66 plasmid (FIG. 8) was linearized and transformed to T. reesei. Three different T. reesei host strains were used. The effect of the activator on cellulase production was studied in the strain QM9414 (ATCC 26921, Mandels, M., et al,
  • pMLO16-67A is QM9414 where the native cbhl gene is replaced by an expression casette where E. coli LacZ gene is under the T. reesei cbhl promoter.
  • T. reesei Construction of Trichoderma strains producing ACEI under the control of the cDNAl promoter of T. reesei Construction of the pMI-69 expression vector was as follows. Plasmid pAS28 containing the full-length acel cDNA in the Bluescript SK- vector was digested from the 5 'end of the cDNA with the restriction enzyme Spel. The strong cDNAl promoter of T reesei (Nakari-Setala, T. and Penttila, M., Appl Environ. Microbiol. 61 :3650-3655 (1995)) was first cloned by PCR using the
  • the underlined ATCGAT is a Clal site
  • the underlined ACTAGT is a Spel site
  • the underlined GCTAGC is a Nhel site.
  • the PCR fragment was digested with the Clal enzyme and ligated to the similarily digested pSP72 resulting in plasmid pMI-36.
  • the promoter fragment was then cloned from the plasmid pMI-36 by digestion with the restriction enzymes Spel and Clal and ligated to the Spel-Clal cut pAS28 in front of the acel cDNA.
  • the expression vector pMI-69 ( Figure 10) is linearized with the restriction enzyme Notl and transformed to the same T. reesei strains as in Example 7. Transformants are analysed as in Example 7.
  • pARO3 Trichoderma expression vector was as follows. The coding region of the ace2 gene (working name S5) was first cloned from plasmid pAS26 by PCR using the 5 'primer ATC TGT CTA GAC AGG ATC CCC GGC AAG CAT GTG ATC GAT (SEQ ED NO. 15) and the 3 'primer TAC GTC CCG GGC TGG ATC CTC ACT TCA GCA GTC TGG CTC (SEQ ID NO. 15) and the 3 'primer TAC GTC CCG GGC TGG ATC CTC ACT TCA GCA GTC TGG CTC (SEQ
  • the underlined GGATCC is a BamHI site.
  • the PCR fragment was ligated to the pGEM-t vector resulting in plasmid pARO2.
  • pARO2 was digested with the restriction enzyme BamHI and the isolated ace2 coding region was ligated to a similarily-cut pAN52-1 Notl vector.
  • the resulting pARO3 plasmid (FIG. 11) was linearized and protoplast transformation of T. reesei QM9414 was performed as in Example 7. Cotransformation of the T.
  • reesei reporter strains was done using the plasmid pAN8-l containing the phleomycin resistance marker according to Nyyss ⁇ nen, E., et al, Bio/Technology 77:591-595 (1993)). Transformants having different copy numbers (1-4) were screened by
  • the plasmid pMI-66 containing the ⁇ ce7 production cassette was transformed into three different T. reesei strains earlier transformed with the ace2 cassette (Example 9). Among the transformants from each of the host strains
  • Example 7 Hydrolase production of the strains overproducing the activators singly or both of them on glucose-containing medium, on sorbitol- containing medium, or on inducing conditions on cellobiose, whey or Solka floe cellulose or a combination of plant materials, as described in Example 1, was analysed. Expression can be detected by enzyme activity measurements or by
  • the expression vector pMI-73 was first prepared.
  • the expression cassette including the gpdA promoter, the acel coding region and the trpC terminator was digested from plasmid pARO3 ( Figure 11) with the restriction enzymes Notl and Hind ⁇ l and blunt-ended by using the T4
  • the plasmid pALK1062 ⁇ B ( Figure 12), containing the 5 'and 3' flanking regions of the egll gene of T reesei and the hph expression cassette coding for the marker for selection of the transformants (HmB phosphotransferase conferring resistance to hygromycin B), was digested with BamHI, similarily blunt-ended and the acel expression cassette was ligated into the vector.
  • the egll 5 '-flanking fragment is the 1.4 kb Xhol - Sacl about 2.2 kb upstream of the egll gene and the 3 ' -flanking fragment is the 1.6 kb AvrR - Smal about 0.2 kb from the end of the egll gene.
  • Both of the flanking fragments have been isolated from T. reesei VTT-D-80133 (Bailey and Nevalainen, Enzyme Microb. Technol. 3: 153-157 (1981)) and they have been subcloned from a lambda clone, originally called egl3, isolated by Saloheimo et al. (Gene 63: 11-21 (1988)).
  • the hph gene is from E. coli and it is expressed from T. reesei pki (pyruvate kinase) promoter. T. reesei cbh2 terminator is ligated after the hph gene to terminate transcription.
  • the hph expression cassette originates from the plasmid pRLMex30 (Mach et al, Curr. Genet. 25: 567-570 (1994)).
  • pMI-73 was digested with the restriction enzyme Bam ⁇ l and the released ace2 coding region was replaced with the acel coding region similarily digested from the plasmid pMI- 66.
  • the plasmids pMI-73 and pMI-76 were digested with the restriction enzyme Asp718, the production cassettes were separated from the vector parts by agarose gel electrophoresis and purified from the gel by using the QIAquick gel extraction kit (Qiagen).
  • the T. reesei strain ALKO2221 (7th generation low protease mutant from T. reesei QM9414/ATCC26921) was transformed with the expression cassette of the pMI-76 plasmid.
  • the transformants were selected by their ability to grow on minimal medium plates containing lOO ⁇ g/ml hygromycin B. About 30 transformants were purified and grown in shake flasks on a complex medium containing whey and a complex nitrogen source derived from cereal grain (Suominen, P.L., et al, Mol. Gen. Genet. 241:523-530 (1993)) for 7 days.
  • the total protein secreted to the medium was TCA-precipitated and analysed by
  • the total MUL activity representing the activities of the CBHI and EGI enzymes was first measured (van Tilbeurgh et al, Methods in Enzymology 7(50:45-59 (1988)). In this measurement the activity of the ⁇ -glucosidase enzyme was inhibited by the presence of 100 mM glucose. The portion of the EGI activity was measured by inhibiting the action of CBHI by the presense of 5 mM cellobiose, and this value was subtracted from the total MUL activity.
  • the production of total secreted protein and CBHI activity by the selected transformants is shown in Table 1. The values have been normalised so that the level produced by the host strain (the medium of five parallel bottles) is given the value 1. The chosen transformants produced 1.1 - 1.2 times more MUL activity compared to the host strain ALKO2221.
  • the T. reesei strain ALKO2221 was transformed with the expression cassette of the pMI-73 plasmid, transformants were purified, grown in shake flasks and analysed similarily as in Example 12.
  • the production of total secreted protein and CBHI activity by the selected transformants is shown in Table 2. The values have been normalised so that the level produced by the host strain is given the value 1. About 10 % increase in MUL activity was observed in the culture supernatants of the chosen transformants.
  • the T. reesei strain ALKO3625 (ALKO2221/pALK807/20) was transformed with the expression cassette of the pMI-76 plasmid, transformants were purified and grown in shake flasks similarily as in Example 12.
  • ALKO3625 strain has been constructed as described in WO93/24621, Example 4. This strain contains 4-5 copies of the cbhl promoter - xylanase 1 production cassettes integrated at unknown positions in the genome (estimated from Southern blot and by scanning of a dot blot filter). The production of xylanase I was measured using standard xylanase activity measurement (Bailey M.J. et al, J. Biotechnol.
  • the T. reesei strain ALKO3625 was transformed with the expression cassette of the pMI-73 plasmid, transformants were purified and grown in shake flasks similarily as in Example 12.
  • the production of total secreted protein and xylanase activity by the selected transformants is shown in Table 4. The values have been normalised so that the level produced by the host strain is given the value 1.
  • the increase in xylanase activity in the culture supernatants of chosen transformants was 1.2 - 1.6 times compared to the host.
  • Fragments of 30- 200 bp of cbhl promoter are synthesized by PCR and cloned into the negative control reporter plasmid using the same strategy as described in the previous example and transformed into the yeast strain DBY746. Possible leakiness of the constructs is avoided, when necessary, by using different amounts (1-100 mM) of aminotriazole in the medium.
  • the reporter yeast strain is transformed with plasmids pAS26 and pAS27 encoding ACEII and ACEI. respectively. Transformants are selected and subsequently screened for HIS + phenotype as described in Example 1. Transformants that are able to grow only in the presence of both plasmids are obtained. In these colonies the reporter plasmid carries a specific nucleotide sequence which is recognized and bound by either ACEI or ACEII leading to activation of HIS3 gene expression.
  • 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 ere 7 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
  • 5'CGAAT Another sequence element of interest found within the above mentioned region is 5'CGAAT, which is found in glucoamylase (glaA) promoters of
  • cbhl promoter sequence between and including the nucleotides -184 and -1 is shown as SEQID NO. 18
  • cbhl promoter sequence between and including the nucleotides -161 and -1 is shown as SEQID NO.19
  • cbhl promoter sequence between and including the nucleotides -140 and -1 is shown as SEQID NO.20.
  • 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 23) 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 24) 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 as described in Example 1. Plasmids originating from the cDNA library that support growth only in the presence of the reporter plasmid, but not alone or with the negative control plasmid, are obtained. These plasmids carry genes that code for proteins which activate transcription through binding to the cbhl promoter sequences present in the reporter construct.
  • the gene encoding xylanase I of Trichoderma reesei is fused to the cbhl promoter of T. reesei.
  • the fusion can be made in such a way that the TATA box of the gpdA gene of Aspergillus nidulans is inserted between the promoter and the xylanase gene to ensure the binding of the basic transcription machinery to the construct.
  • the 29 bp promoter fragment mentioned in Examples 16 and 17, or the 6 bp part of it, 5' GGC(T/A)AA, mentioned in Example 16 can be multiplied in front of the TATA box instead of the promoter.
  • the construct is transformed to Aspergillus nidulans strains.
  • T. reesei QM9414 was grown on minimal medium
  • Example 6 supplemented with different carbon sources. These were sorbitol, sorbitol+sophorose, sorbitol+mannobiose, sorbitol+xylobiose, sorbitol+cellobiose, cellobiose, glycerol, glycerol+mannobiose, glycerol+xylobiose. mannose, xylose, xylitol, arabinose, arabitol, galactose, lactose, Lenzing xylan, methylglucuronoxylan.
  • RNA was isolated as described by Chirgwin, J.M. et al, Biochem. J. 75:5294-5299 (1979) and analysed by Northern blotting and hybridized as described in Example 6. The following genes were used as probes: cbhl, egl5, bgll. xyll, xyl2, bxll, abfl, girl, axel, manl, agll, agl2, agl3.
  • Labelling of the DNA fragment containing the acel or acel gene or part of the gene is done by random priming using a labelling kit (Pharmacia) and 32 P- dATP according to the manufacturer ' s insturctions.
  • the radioactively labelled DNA is denatured by incubation for three to five minutes at 100° C and is kept single stranded by rapid chilling on ice, before addition to a hybridization buffer containing 6xSSC, 5x Denhardf s solution, 0.1% sodium pyrophosphate and 100 ⁇ g/ml heat denatured herring sperm DNA.
  • Screening of the genomic libraries for acel or acel related genes is performed by hybridization.
  • the sample is first prehybridized in 6xSSC, 0.1 % SDS, 0.05%) sodium pyrophosphate and 100 ⁇ g/ml denatured herring sperm DNA at 60°C for 3 - 5 hours, followed by hybridization in 6xSSC, 0.1% SDS, 0.05% sodium pyrophosphate and 100 ⁇ g/ml denatured herring sperm DNA at 57 °C for
  • Labelling of synthetic oligonucleotides is done by using gamma- 32 P ATP.
  • the reaction mixture contains, in a final volume of 50 ⁇ l: 37 pmol oligonucleotide. 66 mM Tris-HCl pH 7.6, 1 mM ATP, 1 mM spermidine, 10 mM MgCL. 15 mM dithiothreitol, 200 ⁇ g/ml BSA, 34 pmol gamma 32 P-ATP (New England Nuclear, 6000 Ci/mmol) and 30 units T4-polynucleotide kinase.
  • the reaction mixture is incubated for 60 min at 37°C, after which the reaction is terminated by the addition of 4 ⁇ l 0.5 M EDTA, pH 8.0.
  • the genomic libraries are screened using oligonucleotide probes in the following way.
  • the filters are wetted and washed for 60 min at room temperature in 3xSSC according to Maniatis, T. et al, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, second edition, 1988).
  • Prehybridization is for two hours at 65 °C in 6xSSC, 0.5% SDS, lOxDenhardt's, 100 ⁇ g.ml heat denatured herring sperm DNA.
  • Hybridization is done by adding the 32 P-labelled oligo (see above) into the hybridization buffer (which is the same buffer as the prehybridization except that no herring DNA is added).
  • Hybridization is performed for 18 hours at a final temperature of 38 ° C, achieved by slow, controlled cooling from the initial temperature of 65 °C. After hybridization, the filters are washed in 2xSSC, after which the filters are washed in prewarmed hybridization buffer at 38 °C. Finally the filters are washed for 30 min at 38 °C in 6xSSC, and 0.05%) sodium pyrophosphate. Hybridizing plaques are identified by exposure of Kodak XAR X-ray film for 72 hours at -70 °C using an intensifying screen.
  • ORGANISM Trichoderma reesei
  • TTCCACTCCC TCGAGTGTCT CAGTTGCTCG TCTGGCTCTC GTCTGCCCAC CCCCCGTCGG 240 TGCCGAGTCC TTGCGTTCAT ATATCCAATT TACGCCCCGT CTTCTGTCAA GGCAGTTTTT 300
  • TTCACTCTCC TACATCTCCC AGCGCTTCAT CTGCTGCCGG CGACTTCGTC CCTCCTACTC 780
  • AAGTTCGCCC CGGCGACCGG CCTTACACTA ACGGATACTT TATCGACCTG AAGGAGCAAA 1500
  • CTGCAGCTTC CTCGTACGAG CAGTACCCTC CCTACCAGAA CGGTTCCACC TTCATCATCA 2400
  • MOLECULE TYPE DNA (genomic) (71) ORIGINAL SOURCE:
  • ORGANISM Trichoderma reesei
  • ACATCTCTTC TATCGACAAG GGAAGCTAAC CGCTTGCCGC TGCACTGTTA GGACTCGTGT 1020
  • ACATCTCCCA GCGCTTCATC TGCTGCCGGC GACTTCGTCC
  • AGCACCACCG CGATGCTTCC CAGCCTCAGC CACCGCGCTG TCAACCGCAT CCGCGAACAC 1680 ACTCTCCGCC CTCTGCTGGA GAAGCCCACG TTGAAGGAAT TCGAGCCCAT CGTCCTAGAC 1740
  • GTCATGTCCA CCGACTTCCC CATGTATCCG GCTGATGACG ATTGGCTCGC TACCTACGGC 2880 GCGCAGCCCA ACACCATCGA CGCCATGGAC CTGGGTCTCG AGAACCTTTC CCCTGCCTCT 2940
  • ORGANISM Trichoderma reesei
  • B STRAIN: Rut-C-30
  • AGTCCGCCAT CGAGGCCATT TCGCCCTCAC GAGCCCTTGA ACCACAGCCA TGAACACAGT 180 CATAGTCACA GTCACAATCA TAACGGGGTA GGCGTCAGCT TTGACTGGCT CGATCTCATG 240
  • ORGANISM Trichoderma reesei
  • CATCCCTCGA CATGCATCAC GTCTCAAGGC AGCAGCTGAG AGAGTACGCC GACACCGTGG 1080 GAACCGGCTT CGACCTGCAG TCCACCCTCG ACAGCCTCCT CCACCACGCC CAGGATCTCC 1140
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE:

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Abstract

Cette molécule d'acide nucléique purifié code une protéine possédant la capacité de réguler de façon transcriptionnelle des promoteurs. L'invention concerne notamment des activateurs transcriptionnels du promoteur de CBH I (cellobiohydrolase I), obtenu à partir de Trichoderma reesei, l'ADN codant de telles protéines, ainsi que des procédés d'utilisation associés.
PCT/FI1997/000743 1996-11-29 1997-12-01 Genes codant des proteines de regulation transcriptionnelle, obtenus a partir de trichoderma reesei, et utilisations de ceux-ci WO1998023642A1 (fr)

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AU51235/98A AU5123598A (en) 1996-11-29 1997-12-01 Genes encoding transcriptional regulatory proteins from trichoderma reesei and uses thereof
EP97945899A EP0950064A1 (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

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US3215696P 1996-11-29 1996-11-29
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US3295996P 1996-12-13 1996-12-13
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US4014097P 1997-03-10 1997-03-10
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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

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Cited By (4)

* 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
WO2002053758A2 (fr) * 2000-12-29 2002-07-11 Rhein Biotech Gesellschaft für neue Biotechnologische Prozesse und Produkte mbH Procede de fabrication de proteines heterologues dans un champignon homothallique de la famille sordariaceae
WO2002064624A3 (fr) * 2001-02-13 2002-11-21 Valtion Teknillinen Tutkimuskeskus Procede ameliore de production de proteines secretees dans les champignons
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

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Publication number Priority date Publication date Assignee Title
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
CN112961788B (zh) * 2021-02-24 2023-06-13 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

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AU4712193A (en) * 1992-08-19 1994-03-15 Alko Group Limited Fungal promoters active in the presence of glucose

Non-Patent Citations (3)

* 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 Cellobiohydrolase I of Trichoderma Reesei are Adjacent to Its TATA Box", page 236. *
DIALOG INFORMATION SERVICES, File 154, MEDLINE, Dialog Accession No. 09081041, Medline Accession No. 97223688, ESCOBAR-VERA J. et al., "Transcriptional Control of the Cellulase Genes in Trichoderma Reesei"; & BRAZ. J. MED. BIOL. RES. (BRAZIL), July 1996, 29 (7), pages 905-9. *
DIALOG INFORMATION SERVICES, File 154, MEDLINE, Dialog Accession No. 8791479, Medline Accession No. 96411785, ZEILINGER S. et al., "Different Inducibility of Expression of the Two Xylanase Genes Xyn1 and Xyn2 in Trichoderma Reesei"; & J. BIOL. CHEM. (UNITED STATES), 11 Oct. 1996, 271 (41), pages 25624-9. *

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
WO2002053758A2 (fr) * 2000-12-29 2002-07-11 Rhein Biotech Gesellschaft für neue Biotechnologische Prozesse und Produkte mbH Procede de fabrication de proteines heterologues dans un champignon homothallique de la famille sordariaceae
WO2002053758A3 (fr) * 2000-12-29 2002-12-05 Rhein Biotech Proz & Prod Gmbh Procede de fabrication de proteines heterologues dans un champignon homothallique de la famille sordariaceae
WO2002064624A3 (fr) * 2001-02-13 2002-11-21 Valtion Teknillinen Tutkimuskeskus Procede ameliore de production de proteines secretees dans les champignons
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

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WO1998023764A1 (fr) 1998-06-04
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EP0939825A1 (fr) 1999-09-08
EP0950064A1 (fr) 1999-10-20

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