WO2000050567A1 - Cellules fongiques contenant un systeme de reparation des mesappariements de l'adn inactive - Google Patents

Cellules fongiques contenant un systeme de reparation des mesappariements de l'adn inactive Download PDF

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WO2000050567A1
WO2000050567A1 PCT/DK2000/000063 DK0000063W WO0050567A1 WO 2000050567 A1 WO2000050567 A1 WO 2000050567A1 DK 0000063 W DK0000063 W DK 0000063W WO 0050567 A1 WO0050567 A1 WO 0050567A1
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polypeptide
interest
filamentous fungal
mismatch repair
fungal cell
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PCT/DK2000/000063
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English (en)
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Torben Vedel Borchert
Lars Christiansen
Jesper Vind
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Novozymes A/S
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Priority to AU25364/00A priority Critical patent/AU2536400A/en
Priority to EP00903565A priority patent/EP1157095A1/fr
Priority to JP2000601131A priority patent/JP2002536990A/ja
Publication of WO2000050567A1 publication Critical patent/WO2000050567A1/fr

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    • 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
    • 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
    • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus

Definitions

  • a process for making DNA libraries in filamentous fungal cells using a novel cloned gene involved in the mismatch repair system of filamentous fungal cells is described.
  • the mismatch repair system is a system within cells which recognises mismatches in newly synthesised duplex DNA sequences .
  • the mismatch repair system then either corrects the mismatches which are seen as errors by e . g. using the methylated ""old 11 strain as template or alternatively it may mediate degradation of the duplex DNA sequences which comprise the mismatches .
  • mismatch repair system' ' will limit the ""diversity ' ' within a cell, diversity being represented as duplex DNA sequences which comprise mismatches .
  • duplex DNA sequence which comprises a single mismatch represents a diversity of two different DNA sequences within the cell. If the mismatch repair system corrects the mismatch there will only be a diversity of one within the cell.
  • mismatch repair system mediates the degradation of such a duplex DNA sequence the diversity will be lost. See figure 1 for a graphic illustration on how the mismatch repair system may work within a cell.
  • duplex DNA sequences comprising mismatches represent a DNA library of interest, then the diversity of this library may be limited when transformed (placed) into cells with an active mismatch repair system.
  • EP 449923 describes bacterial cells wherein the mismatch system is inactivated.
  • WO 97/37011 describes yeast cells wherein the mismatch system is inactivated. See the working examples of this document .
  • WO 97/05268 describes mice cells wherein the mismatch system is inactivated. See the working examples of this document .
  • the problem to be solved by the present invention is to provide an improved strategy for making DNA libraries in filamentous fungal cells.
  • a filamentous fungal cell population comprising such a DNA library may then be used to select a polypeptide of interest.
  • polynucleotide sequences with particular properties might be selected, such as promoters, terminators and other regulatory elements with changed/improved properties .
  • the solution is based on that the present inventors have cloned a NOVEL gene involved in the mismatch repair system of a filamentous fungal cell. Further, this gene is the first gene cloned which is involved in the mismatch repair system of a filamentous fungal cell.
  • the gene comprises a very characterising DNA sequence encoding the polypeptide sequence shown in positions 683-758 of SEQ ID NO 2.
  • This DNA has been used to clone the full length gene encoding the polypeptide sequence shown in positions 1-940 of SEQ ID NO 2. See working examples herein (vide infra) .
  • the gene cloned as described in working examples herein is a gene cloned from an Aspergillus oryzae filamentous fungal cell.
  • the present invention relates to a filamentous fungal cell, wherein a gene involved in the mismatch repair system has been inactivated and in which the gene involved in the mismatch repair system comprises: (a) a DNA sequence encoding the polypeptide sequence shown in positions 683-758 of SEQ ID NO 2; or (b) a DNA sequence encoding a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 683-758 of SEQ ID NO 2 ; and in a second aspect the present invention relates to a filamentous fungal cell, wherein a gene involved in the mismatch repair system has been inactivated and in which the gene involved in the mismatch repair system comprises : (a) a DNA sequence encoding the polypeptide sequence shown in positions 1-940 of SEQ ID NO 2; or (b) a DNA sequence encoding a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1-940 of SEQ ID NO 2.
  • filamentous fungal cell of the first or second aspect of the invention is very suitable for making a DNA library of interest in filamentous fungal cells.
  • the present invention relates to a process for preparing a filamentous fungal cell population wherein individual cells in the population comprise individually different DNA sequences of interest representing a
  • DNA library of interest comprising following steps:
  • FIG 1 One of the advantages of allowing an individual mismatch repair inactivated filamentous fungal cell duplicated DNA of interest at least once as descried under step (b) of the third aspect is illustrated in figure 1.
  • the process of the third aspect using a filamentous fungal mismatch repair inactivated cell as described herein allows preparation of a DNA library wherein eventual hetero duplex DNA mismatches are not corrected. This gives a DNA library with a higher diversity as compared to a DNA library made in a fila- mentous fungal cell NOT having an inactivated mismatch repair system (see figure 1) .
  • Duplication of DNA sequence of interest means that the two strands are replicated such that two separate sets of double stranded DNA are generated, each being based on a separate one of the two original strands .
  • a filamentous fungal cell population wherein individual cells in the population comprise a DNA library of interest as described above may be used to select a polypeptide of interest .
  • the present invention relates to a process for production of a polypeptide of interest comprising the steps of the third aspect and wherein the DNA sequences of interest encode a polypeptide of interest and which further comprises following step:
  • step (c) selecting from the resultant population of filamentous fungal cells of step (b) of the third aspect a desired polypeptide of interest .
  • an advantage of the process of the fourth aspect may be that the polypeptide of interest is selected from a filamentous fungal cell expressing the polypeptide. Consequently, it is directly known that the polypeptide can be expressed from a filamentous fungal cell, which is usefully if it is subsequently required to produce the polypeptide in large scale in a filamentous fungal cell. This may be of particular interest when the DNA library encodes polypeptides of interest which are derived from filamentous fungal cells, since it is known that filamentous fungal polypeptides preferably are produced in industrial relevant high yields in filamentous fungal cells.
  • a gene ' ' denotes herein a gene (a DNA sequence) will is capable of being expressed into a polypeptide within said cell. Accordingly, said gene sequence will be defined as an open reading frame starting from a start codon (normally ""ATG”, “"GTG”, or “"TTG") and ending at a stop codon (normally ""TAA”, TAG” or ""TGA” ) .
  • Such standard elements may include a promoter, a ribosomal binding site, a termination sequence, and may be others elements as known in the art.
  • mismatch repair system 11 shall herein be understood according to the art, as a system within cells which recognises mismatches in duplex DNA sequences. See e.g. WO 97/37011, page 1, line 21-28)
  • the mismatch repair system then either corrects the mismatches which are seen as errors by e . g. using the methylated ""old 11 strain as template or alternatively it may mediate degradation of the duplex DNA sequences which comprise the mismatches.
  • the ""mismatch repair system 1 ' will limit the ""diversity 11 within the cell represented by such duplex DNA sequences which comprise mismatches. 6
  • a duplex DNA sequence which comprises a single mismatch represents a diversity of two different DNA sequences within the cell. If the mismatch repair system corrects the mismatch their will only be a diversity of one within the cell. Alternatively, if the mismatch repair system mediates the degradation of such a duplex DNA sequence this diversity will be lost.
  • a polypeptide encoded by a gene involved in the mismatch repair system recognises a mismatch by a mechanism involving binding to the mismatch.
  • a suitable assay to test whether or not a filamentous fungal cell as described herein is inactivated in its mismatch repair system is to use a ""gel shift assay' 1 or alternatively termed a ""gel retardation assay 1 '.
  • This is a standard assay used in the art. See WO 97/05268, page 16,17 and 25.
  • cell extracts are prepared of both (a) a filamentous fungal cell wherein the gene, as described herein, involved in the mismatch repair system is inactivated; and (b) the corresponding filamentous fungal cell wherein the gene is NOT inactivated. These extracts are then bound/mixed to oligonucleotides containing the base- pair mismatched G:T; G:A; G:G; A:C, and an extrahelical TG dinucleotide and run on a nondenaturing gel.
  • the gel shift assay demonstrates that the control filamentous fungal cell wherein the gene is NOT inactivated comprises any protein (s) which binds to any of above mentioned oligonucleotides and these binding protein (s) are NOT seen in the filamentous fungal cell wherein the gene, as described herein, involved in the mismatch repair system is inactivated then it is a confirmation that the mismatch repair system in the latter is inactivated.
  • DNA sequence encoding a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 683-758 of SEQ ID NO 2 ' ' and ""a DNA sequence encodincr a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1-940 of SEQ ID NO 2 ' ' ; is determined as the degree of identity between two sequences indicating a derivation of the first sequence from the second.
  • the identity may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B.
  • the polypeptide encoded by an analogous DNA sequence of the invention exhibits a degree of identity preferably of at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and especially at least 97% with amino acid sequence shown in positions 683-758 of SEQ ID NO 2, according to the first aspect of the invention; or with amino acid sequence shown in positions 1-940 of SEQ ID NO 2, according to the second aspect of the invention.
  • DNA library' denotes herein a library of at least two different DNA sequences.
  • the DNA library preferably comprises at least 1000 different DNA sequences, more preferably at least 10000 different DNA sequences, and even more preferably at least 100000 different DNA sequences.
  • step (a) in the process of the third aspect of the invention shall herein be understood broadly in the sense that it is NOT identical DNA sequences of interest which are placed in the filamentous fungal cell population.
  • the term should preferably denotes a situation wherein a cell within the filamentous fungal cell population comprises at least two different DNA sequences of interest which are so partially homologous that they are capable of hybridising/recombining to each other within the cell. It is within the skilled persons general knowledge to determine how partially homologous such sequences have to be in order to obtain said recombination within the cell.
  • a practical example may be that single stranded oligonucleotide sequences partially homologous to chromosomal DNA sequence are placed within the cell or duplex DNA sequences comprising mismatches (e.g. comprised within a vector) are placed within the cell. See below for further description of such examples .
  • the specific experimental way of placing these DNA sequences within a filamentous cell may be done according to any of the many suitable techniques, such as transformation techniques .
  • step (b) of the third aspect of the invention denotes that after an individual cell has duplicated itself at least once then the mismatch repair system may be activated again without loosing the advantage of the process.
  • FIG 1. In this example a duplex DNA sequence comprising a single mismatch is placed in filamentous cell.
  • This figure illustrates an example wherein a duplex DNA sequence comprising a single mismatch is placed in filamentous cell.
  • the two individually different single stranded DNA sequences within the duplex DNA have individually been duplicated providing two different duplex sequences, one in each duplicated cell, without any mismatches.
  • the mismatch repair system is active, a mismatch within a duplex is corrected.
  • This figure shows three partial Aspergillus oryzae polypeptide sequences: "msh2 ' Ao-collO/13/15 ; derived from cloned PCR fragments.
  • the three partial polypeptide sequences are aligned with two other partial polypeptide sequences of known mismatch repair proteins: a human mismatch repair protein, msh2 -human . p; and a fungal Saccharomyces cerevisiae mismatch repair protein, S.c. msh2.
  • the underlined sequences in the figure derive from the construction of the PCR fragments.
  • This figure shows an alignment of the proposed polypeptide sequence of the putative Aspergillus oryzae mismatch repair protein (Ao.MSH2) with the polypeptide sequences of three known mismatch repair proteins from human (msh2 -human .p) , mouse (msh2-mus .p) , and yeast (S.c. msh2).
  • a filamentous fungal cell as described herein, wherein a gene, as described herein, involved in the mismatch repair system has been inactivated.
  • the NOVEL gene, as described herein, involved in the mismatch repair system may be inactivated by any of the numerous known techniques known to the skilled person.
  • An embodiment of the invention relates to a filamentous fungal cell as described herein, wherein the gene involved in the mismatch repair is defective.
  • Numerous methods are known to the skilled person to make a gene defective when the DNA sequence is KNOWN. These methods includes deleting part of the DNA sequence of the gene; introducing frame-shift mutations by deleting or inserting nucleotides; introducing stop codons etc.
  • a preferred embodiment of the invention relates to a filamentous fungal cell as described herein, wherein the gene involved in the mismatch repair has been inactivated transitorily.
  • a number of methods are known to the skilled person for doing this, including insertion of a regulable promoter upstream of the gene or e.g. permanently deleting part of the gene on the chromosome followed by inserting a vector (e.g. a plasmid) into the cell which comprises the gene.
  • the plasmid may then comprise a regulable promoter up-steam of the gene or it may be that the plasmid can be removed from the cell when the mismatch repair system shall be inactivated transitorily and then re-inserted into the cell when the mismatch repair system shall be re-activated.
  • a preferred way to make a filamentous fungal cell which is capable of transitorily inactivate the mismatch repair system as described herein is first to permanently inactive the mismatch repair gene described herein on the chromosome of the cell followed by inserting a plasmid into the cell which comprises the gene, wherein the plasmid is characterised by that it comprises a suitable replication initiating sequence and a suitable selectable marker.
  • the suitable replication initiating sequence is AMA1 (Gems, D., et al . (1991, Gene 98:61-67).
  • AMA1 Gams, D., et al . (1991, Gene 98:61-67).
  • a more detailed description of suitable replication initiating sequences and suitable selectable markers is provided immediately below and in working example 4 herein is provided an example of this strategy using a plasmid comprising AMAl as replication initiating sequence and AmdS as selectable marker .
  • the term "fungal replication initiating sequence” is defined as a nucleic acid sequence which is capable of supporting autonomous replication of an extrachromosomal molecule, e.g., a plasmid or a DNA vector, in a fungal host cell, normally without structural rearrangement of the plasmid or integration into the host cell genome.
  • the replication ini- tiating sequence may be of any origin as long as it is capable of mediating replication initiating activity in a fungal cell.
  • the replication initiating sequence is obtained from a filamentous fungal cell, more preferably a strain of Aspergill us, Fusari um or Al ternaria , and even more preferably, a strain of A . nidulans , A . oryzae , A . niger, F. oxysporum or Al ternaria al tena ta .
  • a replication initiating sequence may be identified by methods well-known in the art. For instance, the sequence may be identified among genomic fragments derived from the organism in question as a sequence capable of sustaining autonomous replication in yeast, (Ballance and Turner, Gene, 36 (1985), 321- 331), an indication of a capability of autonomous replication in filamentous fungal cells.
  • the replication initiating activity in fungi of a given sequence may also be determined by transforming fungi with contemplated plasmid replicators and selecting for colonies having an irregular morphology, indicating loss of a sectorial plasmid which in turn would lead to lack of growth on selective medium when selecting for a gene found on the plasmid (Gems et al, Gene, 98 (1991) 61-67) .
  • AMAl was isolated in this way.
  • An alternative way to isolate a replication initiating sequence is to isolate natural occurring plasmids (e.g. as disclosed by Tsuge et al . , Genetics 146 (1997) 111-120 for Al ternaria a t erna ta ) .
  • replication initiating sequences include, but are not limited to, the ANSI and AMAl sequences of Aspergill us nidulans , e.g., as described, respectively, by Cullen, D., et al. (1987, Nucleic Acids Res. 15:9163-9175) and Gems, D., et al. (1991, Gene 98:61-67) .
  • replication initiating activity is used herein in its conventional meaning, i.e. to indicate that the sequence is capable of supporting autonomous replication of an ex- trachromosomal molecule, such as a plasmid or a DNA vector in a fungal cell .
  • selective pressure is defined herein as culturing a filamentous fungal cell, containing a DNA vector containing a fungal selective marker gene operably linked to a polynucleo- tide sequence of interest, in the presence of an effective amount or the absence of an appropriate selective agent.
  • the effective amount of the selective agent is defined herein as an amount sufficient for allowing the selection of cells containing the selection marker from cells which do not contain the selection marker.
  • the fungal selective marker is selected from the group of genes which encodes a product capa- ble of providing resistance to biocide or viral toxicity, resistance to heavy metal toxicity, or prototrophy to auxotrophs.
  • the prototrophy is obtained from an enzyme selected from the group of metabolic pathways consisting of nucleotide synthesis, cofactor synthesis, amino acid synthesis, acetamide metabolism, proline metabolism, sul- fate metabolism, and nitrate metabolism.
  • the fungal selective marker is a gene selected from the group consisting of argB (ornithine carbamoyl- transferase) , amdS (acetamidase) , bar (phosphinothricin acetyl- transferase) , hemA ( 5-aminolevulinate synthase), hemB (porpho- bilinogen synthase), hygB (hygromycin phosphotransferase) , niaD (nitrate reductase), prn (proline permease), pyrG (orotidine- 5' -phosphate decarboxylase) , pyroA, riboB, sC (sulfate adenyl- transferase) , and trpC (anthranilate synthase) .
  • argB ornithine carbamoyl- transferase
  • amdS acetamidase
  • the fungal cell is cultivated in a suitable medium and under suitable conditions for screening or selecting for transfor- mants harbouring the variant polynucleotide sequence of interest having or encoding the desired characteristic.
  • the cultivation may be performed in accordance with methods well-known in the art for screening of polynucleotide variant libraries.
  • the filamentous fungal cell The filamentous fungal cell
  • the filamentous fungal cell as described herein includes all filamentous forms of the subdivision Eumycota and Oomycota.
  • the filamentous fungi are characterised by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides .
  • Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.
  • vegetative growth by yeasts such as Sa ccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal cell is a cell of a species of, but is not limited to, Acremonium, Aspergill us , Fusarium, Humicola , Mucor, Mycel ioph thora , Neurospora , Penicill ium, Scytal idium, Thiela via , Tolypocladi um, and Trichoderma .
  • filamentous fungal cells of use in the present invention include an Aspergill us cell, an Acremonium cell, a Fusarium cell, a Humicola cell, a Mucor cell, a Myceliophthora cell, a Neurospora cell, a Penicillium cell, a Thielavia cell, a Tolypocladium cell, and a Trichoderma cell. 14
  • the filamentous fungal cell is an Asper ⁇ gill us a wamori , Aspergill us foetidus, Aspergill us j aponicus, Aspergillus nidulans , Aspergillus niger, or Aspergillus oryzae cell ; a Fusari um bactridioides , Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
  • Trichoderma harzianum Trichoderma koningii , Trichoderma longibrachia tum, Trichoderma reesei , or Trichoderma viride cell .
  • step (a) of the process of the third aspect of the invention Placing individually different DNA sequences of interest in a filamentous fungal cell population according to step (a) of the process of the third aspect of the invention:
  • a practical example may be that single stranded oligonucleotide sequences partially homologous to chromosomal DNA sequence are placed within the cell. See Calissano et al . (Fungal genetic newsletter 43:15-16 (1995) for further description of this.
  • duplex DNA sequences comprising mismatches are placed within the cell.
  • the different DNA sequences of interest is comprised in a plasmid wherein the plasmid is characterised by that it comprises a suitable replication initiating sequence and a suitable selectable marker as described above.
  • the suitable replication initiating sequence is AMAl (Gems, D., et al . (1991, Gene 98:61-67).
  • step (a) Growing the population of step (a) for a period of time allowing an individual DNA sequence of interest in the population to be duplicated at least once under conditions wherein the mismatch repair system gene, as describe herein has been inactivated, according to step (b) of the third aspect of the invention.
  • Growing of the population may be done in any of the numerous suitable known media for growing filamentous fungal cells. It is within the skilled persons general knowledge to choose such a suitable media.
  • Said cells may of course be allowed to be duplicated more than once under conditions wherein the mismatch repair system gene has been inactivated.
  • mismatch repair system under step (b) has been inactivated transitorily.
  • the mismatch repair system is then re-activated in order to avoid these lethal mutations in the filamentous fungal cell as such.
  • the strategy for this transitorily inactivation may be any of the strategies described above.
  • Another strategy to limit introduction of mutations on the chromosome is to transitorily stop the chromosomal replication while replicating the extra-chromosomal element under mismatch repair deficient conditions. This can be achieved by introducing mutations in elements being solely necessary for the chromosomal replication.
  • a preferred strategy is to use a filamentous fungal cell wherein the gene involved in the mismatch repair system as described herein is permanently inactivated on the chromosome of the cell followed by inserting a plasmid into the cell which comprises the gene, wherein the plasmid is characterised by that it comprises a suitable replication initiating sequence and a suitable selectable marker. See above for a further explanation of this strategy.
  • the suitable replication initiating sequence is AMAl (Gems, D., et al . (1991, Gene 98:61-67).
  • a further embodiment relates to the process of the third aspect of the invention, wherein the mismatch repair system under step (b) is defective.
  • the invention relates to a process as described herein, wherein, under step (b) of the third aspect of the invention, there is an in vivo intergenic recombination of partially homologous DNA sequences of interest .
  • a process for production of a polypeptide of interest comprising the steps of the third aspect of the invention and wherein the DNA sequences of interest encode a polypeptide of interest, according to the fourth aspect of the invention:
  • the desired polypeptide of interest may be any polypeptide comprising an desired technical feature, such as improved stability; a desired specific activity; a desired pH optimum; an improved wash performance in a detergent; etc.
  • the specific strategy for selecting this desired polypeptide of interest may be any of the numerous selecting strategies known to the skilled person, such as plate screening assays, micro-titer plate based assays, etc.
  • An embodiment of the invention relates to a process of the fourth aspect of the invention, which further comprises following steps:
  • step (e) placing the DNA sequence encoding the polypeptide of interest of step (c) of the fourth aspect or the modified polypeptide of interest of step (d) into a filamentous fungal cell which is suitable for large scale production of the polypeptide of interest ;
  • step (f) cultivating the filamentous fungal cell of step (e) in a fermentor of at least 10000 m3 under conditions permitting expression of the polypeptide of interest; and (g) isolating the polypeptide of interest.
  • step (d) relates to an industrial very relevant process, wherein the selected polypeptide of interest is produced in large scale.
  • step (d) relates to a situation wherein e.g. the desired polypeptide of interest is selected in order to e.g. identify a polypeptide with improved wash performance in a detergent according to step (c) of the third aspect of the invention.
  • This polypeptide while having improved wash performance in a detergent may not be sufficiently stable for a commercial product. Accordingly, it may be required to make some further amino acid substitutions in this selected polypeptide, such as e.g. suitable Proline substitutions on order to obtain sufficient stability to commercialising this polypeptide .
  • a further embodiment relates to a process of the embodiment immediately above, wherein the filamentous fungal cell which is suitable for large-scale production of the polypeptide of interest of step (e) said embodiment is another filamentous fungal cell as compared to the filamentous fungal cell of step (a) of the third aspect of the invention.
  • This embodiment relates to a situation wherein the filamentous fungal cell used to select the polypeptide of interest is different from the one which is used for large scale production.
  • a further embodiment relates to a process as described herein, wherein the polypeptide of interest is a polypeptide derived from a filamentous fungal cell.
  • the term derived from a filamentous fungal cell should be understood in the sense that the information in the amino acid sequence of the polypeptide of interest is derived from a polypeptide obtained from a filamentous fungal cell.
  • filamentous fungal polypeptide may be a variant of a wild-type filamentous fungal polypeptide and/or may be a polypeptide which is a result of a recombination/shuffling of two or more different filamentous fungal polypeptides.
  • the invention relates to a process as described herein, wherein the polypeptide of inter- est is an enzyme, such as an amylase, a protease, a cellulase, a lipase, a xylanase; a phospholipase .
  • an enzyme such as an amylase, a protease, a cellulase, a lipase, a xylanase; a phospholipase .
  • Chemicals used as buffers and substrates were commercial products of at least reagent grade.
  • a gel shift assay suitable for determining if a filamentous fungal cell as described herein is inactivated in the mismatch repair system The principle in this gel shift assay is that cell extracts are prepared of both (a) a filamentous fungal cell wherein the gene, as described herein, involved in the mismatch repair system is inactivated; and (b) the corresponding filamentous fungal cell wherein the gene is NOT inactivated. These extracts are then bound/mixed to oligonucleotides containing the base-pair mismatched G:T; G:A; G:G; A:C, and an extrahelical TG dinucleotide and run on a nondenaturing gel.
  • control filamentous fungal cell wherein the gene is NOT inactivated comprises any protein (s) which binds to any of above mentioned oligonucleotides and these binding protein (s) is NOT seen in the filamentous fungal cell wherein the gene, as described herein, involved in the mismatch repair system is inactivated then it is a confirmation that the mismatch repair system in the latter is inactivated.
  • oligonucleotide U is radiolabelled and annealed with any of the unlabelled oligonucleotides L-G.T, L-G.A, L-G.C, L-A.C, L- T.G., or L-HOM.
  • Oligonucleotide sequences are derived from
  • L-G.T 5'-GAGCATAGGAGGCTGACATTGGGGCCTGGCAGCTTCCC-3 ' (SEQ ID NO:
  • L-G.A. 5'-GAGCATAGGAGGCTGACAATGGGGCCTGGCAGCTTCCC-3' (SEQ ID NO
  • L-A.C. 5'-GAGCATAGGAGGCTGACACCGGGGCCTGGCAGCTTCCC-3' (SEQ ID NO 7) (resulting in a A.C mismatch);
  • L-HOM 5'-GAGCATAGGAGGCTGACACCGGGGCCTGGCAGCTTCCC-3' (SEQ ID NO
  • EXAMPLE 2 Cloning of a gene involved in the mismatch repair system of an
  • SEQ ID NO 1 DNA sequence
  • SEQ ID NO 2 the translated amino acid sequence
  • the numbers indicated are reference numbers from the public available GenBank database. Based on the C-terminal homology between known mismatch repair proteins, a set of degenerate primers were designed, aiming at amplification of a partial sequence of the Aspergillus oryzae homolog:
  • Pr 117858 (SEQ ID NO 10) : P-GGCNCARATHGGNTGYTTYGTNCC Pr 117859 (SEQ ID NO 11) : P-GCCCANGCNARNCCRAANCC
  • PCR-cycle profile [96°C; 2 min - 30 cycles of (94°C; 15s - 50°C; 15s - 72°C; 30s) - 72°C,7 min - 4°C; hold].
  • the 230bp PCR fragment was blunt end ligated into filled in BamHl site of pUC19.
  • pUC19 was BamHl cleaved in presence of calf intestine alkaline phosphatase, followed by filling in the sticky ends by klenow polymerase and dNTP .
  • Three individual plasmids harbouring the insert were isolated from E.coli XL1 transformants of above ligation, and sequenced. Alignment of polypeptides derived by translation of the cloned PCR fragments, revealed a strong homolgy to known mismatch repair protein sequences (see Figure 2) .
  • the three Aspergillus sequences of Figure 2 are equal to the sequence shown in SEQ ID NO 2 from positions 683-758, except from position 685 which in the final cloned sequence is a Thr (T) in sted of an lie (I) as indicated above. This is due to the sequence in above mentioned consensus primers.
  • a radiolabeled probe of the cloned fragment was generated by PCR, using 0.5 mg pUC19 ' msh2 ' -13 (see above) as template in a 100 ml reaction with Taq polymerase, 30 pmol pUC forward and reverse primers and o.2 mM of dG- , dC-, dTTP and 0.2mM dATP + 32 P-dATP.
  • the generated radiolabeled probe was liberated from pUC19 sequences by EcoRI- Hind3 digestion followed by gel purification of the resulting 293 bp fragment.
  • the probe was hybridized to a membrane gridded cosmid library of genomic DNA from A . oryzae strain A1560 (the father of JaL142) (W096/29391) .
  • a positive clone was identified on the filter when analyzed in a phosphoimager, and the clone was identified as ⁇ 31A2.
  • the ⁇ 31A2 cosmid DNA was propagated and used for southern analysis, using the same radiolabeled primer as above.
  • the insert was sequentially sequenced, starting with primers pointing out from the previously determined sequence, followed by primers based on the sequences determined in the last run:
  • the 3825 bp sequence hereby determined (SEQ ID NO 1) was translated in the frame previously determined in the PCR fragment.
  • the resulting protein (SEQ ID NO 2) called Ao.MSH2 was aligned to the protein sequences of known mismatch repair proteins in Figure 3. From the alignment in shown in Figure 3 the cloned and sequenced DNA clearly encompasses the coding sequence for a homolog of yeast, man and mouse mismatch repair proteins, with one intron in the N-terminal part. The position of the intron was deduced by the standard splice rules, and constitutes the only possibility.
  • msh.2 CDS was deleted from pUC19msh2P (see example 2) by PCR, introducing a Notl site instead. This was done by the primers:
  • the resulting PCR product of appr. 8.9 Kb was isolated, ligated into pUC19, and transformed into E. coli XL1. From the resulting transformants pMsh2 ⁇ was isolated, and the correctness of the new junction and its surroundings verified by sequencing [primer 138149 (SEQ ID NO 26) : CCTTTCCACTTTAATCCTAAGC] . (Xll/pMsh2 ⁇ : Lac3073) .
  • the chromosomal gene is deleted in an Aspergillus oryzae cell according to standard techniques known in the art for crossing in such a deleted gene on the chromosome by homologous recombination (Miller, B.L., et al . , 1985 Mol. and Cell. Biol. 5:1714-1721).
  • the plasmid constructed as described below is highly suitable for making a filamentous fungal cell wherein the mismatch repair system may be transitorily inactivated, wherein this plasmid may be inserted into a mismatch disrupted strain of example 3 when the mismatch repair system shall be activated and deleted from the strain when the mismatch repair system shall be inactivated.
  • mismatch repair gene may cause the accumulation of new chromosomal mutations, thus such a strain might be genetically unstable. Consequently it was decided to perform the chromosomal disruption in a strain where mismatch repair gene was expressed from an extra chromosomal element readily lost when the ⁇ mismatch repair phenotype was wanted.
  • the extra-chromosomal element was here a plasmid comprising AMAl as replication initiating sequence and AmdS as selectable marker.
  • the mismatch repair gene (SEQ ID NO 1) was cloned into an autonomously replicating construct harbouring one AMAl repeat .
  • [ ] * indicates that the site has been filled in by Klenow-polymerase and dNTP
  • pMT1505DHS was isolated (LaC 3212), and the mismatch repair expression cassette was introduced as a BamHl - Muni fragment in the corresponding 25 sites in pMT1505DHS, resulting in the plasmid pAma-msh2 (LaC 3216) .
  • EXAMPLE 5 Construction of plasmid pMT1505 used in example 4: Plasmids pMT1505: constructed as described below in Example 5 pHelpl : contains the pyrG gene from A . oryzae as a selective marker and the AMAl sequences which enable autonomous replication in A . nidulans as described by Gems, D., et al . (1991. Gene 98: 61-67) pToC68: as described in EP 0 531 372 (Novo Nordisk A/S)
  • pMT1466 was constructed by inserting an Sphl/Narl fragment from pHelpl into pToC68.
  • pMT1489 was constructed by digesting pMT1466 with Sphl and Stul, then religating.
  • pMTl500 was constructed by digesting pMT1489 with Aatll and Narl and ligating a linker.
  • pMT1504 was constructed by digesting pMT1500 with Nhel and religating.
  • pMT1505 was constructed by inserting a 2.7 kb Xbal fragment containing the amdS encoding gene from A . nidulans genomic DNA (Corrick, C ., et al . 1987, Gene 53:63-71) into pMT1504 which had been cut with Nhel .
  • EXAMPLE 6 EXAMPLE 6 :
  • the plasmid p418MsHII (from lac3159) is cut with Sail and Xhol and treated with calf-intestmal phosphatase. In this manner part of the msHI I gene is cut out.
  • the large band (6400 bp) containing the vector and most of the msHI I gene is isolated from a 1% agarose gel.
  • the plasmid pJal554 was constructed by ligating a Spel/SspBI cut fragment (5330 bp) from pS02 with a Asp718/Nhel cut fragment (316 bp) from pS02. Plasmid pJal554 is cut with Sail and a 2350 bp band which contains the pyrG gene is isolated on a 1% agarose gel. The 2350bp band with pyrG is ligated with the cut p418MsHII plasmid and transformed into E . col l . The right E . col i transformant is identified by restriction analysis and a plasmid preparation is made from this transformant.
  • the plasmid thus prepared is cut with £coRI m order to linearize the plasmid before it is transformed into for example Aspergill us oryzae Jal250. Transformants are selected on mini- mal plates .
  • a transformant where a double crossover event has taken place is identified by making an Aspergillus chromosomal DNA prep followed by a PCR screen for full-length mshll gene using appropriate primers.
  • a Southern blot is made using chromosomal DNA which is randomly fragmented with appropriate enzymes as well as appropriate probes for the deleted msHI I fragment (which is not there any longer) as well as a positive control probe .
  • a screen for mutations in the niaD gene is made. This is done by growing the parent strain Aspergill us Jal250 and the msHI I inactivated strain on plates .
  • the strain with no expression of the MsHII protein will have a higher rate of niaD mutations (more chlorate resistant clones), than the control strain.
  • a per fragment is made using the oligo's: 000120j 2 (SEQ ID NO 27) : TCTGCGAATCGCTTGGATCCCGAACGCGACAACAC, 000120J (SEQ ID NO 28): GAGCTCAGATCTCTTAGGTTCTGGACGAGAAGA, and pUC19msh2P as template.
  • This PCR fragment contains the 5 "end of the msHII gene including the presumed part of 5' msHII mRNA.
  • Another PCR fragment is made using the oligo's: 000120J3 (SEQ ID NO 29) : GTTGTCGCGTTCGGGATCCAAGCGATTCGCAGAAG, 1298-TAKA (SEQ ID NO 30) : GCAAGCGCGCGCAATACATGGTGTTTTGATCAT, and pENI1298 as template (PCT DK99/00552) .
  • Both PCR reactions are done using PWO polymerase according to the manufacturers manual (Boehringer-Mannheim) .
  • the PCR fragments are purified using the Qiagen PCR purification kit (Qiagen) .
  • Qiagen Qiagen PCR purification kit
  • the two PCR fragments are mixed and a third PCR reaction is done with primer 1298-TAKA and 000120J4. In this manner the two PCR fragments are assembled.
  • the assembled PCR fragment is cut with BssHII and Bglll, and purified from a 1.5 % agarose gel and ligated with pENI1298 which was cut with BssHII and Bgl II (purified from 1 % agarose gel) .
  • the ligation mixture is transformed into E . col l .
  • a DNA- prep is made of each of the resulting E . col i transformants .
  • the assembled PCR fragment is sequenced to confirm that no unwanted mutations are introduced during the procedure.
  • the cor- rect construct contains the TAKA promoter, which drives the transcription of the msHII anti-sense mRNA.
  • the resulting plasmid is transformed into for example Aspergil l us oryzae Jal250 along with pENI1298 as control, and transformant are selected on minimal plates.
  • the resulting transformants are isolated on minimal plates and incubated at 37°C until they sporulate.
  • a screen for mutations in the niaD gene is made.
  • a spore-suspension is prepared of the control transformants (pEN ⁇ l298) and of the msHII Anti-sense RNA transformants, and equal amounts of spores are plated on to chlorate-containing plate as described by Unkles et al . (S. E. Unkles, E.C.Campbell. Y.M.J.T. de Ruite Jacobs, M. Broekhui- sen, J.A. Macro, D.Carrez, R.
  • the strain with no or low expression of the MsHII protein will have a higher rate of niaD mutations (more chlorate resistant clones), than the control strain.

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Abstract

L'invention concerne un procédé permettant d'établir des bibliothèques d'ADN dans des cellules fongiques filamenteuses à l'aide d'un gène cloné nouveau intervenant dans le système de réparation des mésappariements de cellules fongiques filamenteuses.
PCT/DK2000/000063 1999-02-24 2000-02-17 Cellules fongiques contenant un systeme de reparation des mesappariements de l'adn inactive WO2000050567A1 (fr)

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AU25364/00A AU2536400A (en) 1999-02-24 2000-02-17 Fungal cells with inactivated dna mismatch repair system
EP00903565A EP1157095A1 (fr) 1999-02-24 2000-02-17 Cellules fongiques contenant un systeme de reparation des mesappariements de l'adn inactive
JP2000601131A JP2002536990A (ja) 1999-02-24 2000-02-17 不活性化されたｄｎａミスマッチ修復系を有する菌類細胞

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002046396A1 (fr) * 2000-12-06 2002-06-13 Novozymes A/S Methode pour la production d'une bibliotheque de polynucleotides
WO2002059331A1 (fr) * 2001-01-24 2002-08-01 Novozymes A/S Mutation in vivo et recombinaison dans des champignons filamenteux
US6783941B2 (en) 2000-12-06 2004-08-31 Novozymes A/S Method for producing a polynucleotide library in vitro by mismatch repair of heteroduplexes
US7122330B2 (en) 2000-04-13 2006-10-17 Mark Aaron Emalfarb High-throughput screening of expressed DNA libraries in filamentous fungi
EP2527448A1 (fr) 2011-05-23 2012-11-28 Novozymes A/S Intégrations simultanées spécifiques au site de copies de plusieurs gènes dans des champignons filamenteux
EP2527432A1 (fr) 2011-05-23 2012-11-28 Novozymes A/S Marqueur de sélection bidirectionnelle codant pour la cytosine désaminase
US8551751B2 (en) 2007-09-07 2013-10-08 Dyadic International, Inc. BX11 enzymes having xylosidase activity
US8673618B2 (en) 1996-10-10 2014-03-18 Dyadic International (Usa), Inc. Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US8680252B2 (en) 2006-12-10 2014-03-25 Dyadic International (Usa), Inc. Expression and high-throughput screening of complex expressed DNA libraries in filamentous fungi

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3433358B1 (fr) 2016-03-24 2022-07-06 Novozymes A/S Variants de cellobiohydrolase et polynucléotides codant pour ces derniers

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WO1990007576A1 (fr) * 1988-12-26 1990-07-12 Setratech Procede de recombinaison in vivo de sequences d'adn partiellement homologues
WO1997005268A1 (fr) * 1995-07-26 1997-02-13 Setratech Recombinaison homologue dans des cellules eucaryotes a systeme de reparation des mesappariements inactive
WO1997037011A1 (fr) * 1996-04-01 1997-10-09 Setratech S.A.R.L. Recombinaison meiotique in vivo de sequences d'adn partiellement homologues
WO1998031837A1 (fr) * 1997-01-17 1998-07-23 Maxygen, Inc. Evolution de cellules entieres et d'organismes par recombinaison recursive de sequences

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WO1990007576A1 (fr) * 1988-12-26 1990-07-12 Setratech Procede de recombinaison in vivo de sequences d'adn partiellement homologues
WO1997005268A1 (fr) * 1995-07-26 1997-02-13 Setratech Recombinaison homologue dans des cellules eucaryotes a systeme de reparation des mesappariements inactive
WO1997037011A1 (fr) * 1996-04-01 1997-10-09 Setratech S.A.R.L. Recombinaison meiotique in vivo de sequences d'adn partiellement homologues
WO1998031837A1 (fr) * 1997-01-17 1998-07-23 Maxygen, Inc. Evolution de cellules entieres et d'organismes par recombinaison recursive de sequences

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8916363B2 (en) 1996-10-10 2014-12-23 Dyadic International (Usa), Inc. Construction of Highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US8673618B2 (en) 1996-10-10 2014-03-18 Dyadic International (Usa), Inc. Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US7122330B2 (en) 2000-04-13 2006-10-17 Mark Aaron Emalfarb High-throughput screening of expressed DNA libraries in filamentous fungi
US7794962B2 (en) 2000-04-13 2010-09-14 Dyadic International (Usa), Inc. High-throughput screening of expressed DNA libraries in filamentous fungi
WO2002046396A1 (fr) * 2000-12-06 2002-06-13 Novozymes A/S Methode pour la production d'une bibliotheque de polynucleotides
US6783941B2 (en) 2000-12-06 2004-08-31 Novozymes A/S Method for producing a polynucleotide library in vitro by mismatch repair of heteroduplexes
WO2002059331A1 (fr) * 2001-01-24 2002-08-01 Novozymes A/S Mutation in vivo et recombinaison dans des champignons filamenteux
US8680252B2 (en) 2006-12-10 2014-03-25 Dyadic International (Usa), Inc. Expression and high-throughput screening of complex expressed DNA libraries in filamentous fungi
US8551751B2 (en) 2007-09-07 2013-10-08 Dyadic International, Inc. BX11 enzymes having xylosidase activity
WO2012160097A1 (fr) 2011-05-23 2012-11-29 Novozymes A/S Marqueur de sélection bidirectionnel codant pour une cytosine désaminase
WO2012160093A1 (fr) 2011-05-23 2012-11-29 Novozymes A/S Intégrations simultanées spécifiques d'un site de multiples copies géniques dans un champignon filamenteux
EP2527432A1 (fr) 2011-05-23 2012-11-28 Novozymes A/S Marqueur de sélection bidirectionnelle codant pour la cytosine désaminase
EP2527448A1 (fr) 2011-05-23 2012-11-28 Novozymes A/S Intégrations simultanées spécifiques au site de copies de plusieurs gènes dans des champignons filamenteux

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