EP1157095A1 - 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

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Publication number
EP1157095A1
EP1157095A1 EP00903565A EP00903565A EP1157095A1 EP 1157095 A1 EP1157095 A1 EP 1157095A1 EP 00903565 A EP00903565 A EP 00903565A EP 00903565 A EP00903565 A EP 00903565A EP 1157095 A1 EP1157095 A1 EP 1157095A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
interest
filamentous fungal
mismatch repair
fungal cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00903565A
Other languages
German (de)
English (en)
Inventor
Torben Vedel Borchert
Lars Christiansen
Jesper Vind
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novozymes AS
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Filing date
Publication date
Application filed by Novozymes AS filed Critical Novozymes AS
Publication of EP1157095A1 publication Critical patent/EP1157095A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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 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 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.
  • 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:
  • 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 .
  • step (c) selecting from the resultant population of filamentous fungal cells of step (b) of the third aspect a desired polypeptide of interest .
  • 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 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.
  • 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.
  • 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 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 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 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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-A.C. 5'-GAGCATAGGAGGCTGACACCGGGGCCTGGCAGCTTCCC-3' (SEQ ID NO 7) (resulting in a A.C mismatch);
  • L-HOM 5'-GAGCATAGGAGGCTGACACCGGGGCCTGGCAGCTTCCC-3' (SEQ ID NO
  • 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 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.
  • 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 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.
  • 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 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 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) .

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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.
EP00903565A 1999-02-24 2000-02-17 Cellules fongiques contenant un systeme de reparation des mesappariements de l'adn inactive Withdrawn EP1157095A1 (fr)

Applications Claiming Priority (3)

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DKPA199900253 1999-02-24
DK990253 1999-02-24
PCT/DK2000/000063 WO2000050567A1 (fr) 1999-02-24 2000-02-17 Cellules fongiques contenant un systeme de reparation des mesappariements de l'adn inactive

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EP1157095A1 true EP1157095A1 (fr) 2001-11-28

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EP (1) EP1157095A1 (fr)
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CN (1) CN1341146A (fr)
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2017165760A1 (fr) 2016-03-24 2017-09-28 Novozymes A/S Variants de cellobiohydrolase et polynucléotides codant pour ces derniers

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US7883872B2 (en) 1996-10-10 2011-02-08 Dyadic International (Usa), Inc. Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
KR20020026456A (ko) 2000-04-13 2002-04-10 에말파브 마크 아론 사상균에서 발현된 ｄｎａ 라이브러리의 고산출량 스크리닝
US6783941B2 (en) 2000-12-06 2004-08-31 Novozymes A/S Method for producing a polynucleotide library in vitro by mismatch repair of heteroduplexes
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
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
CA2736661A1 (fr) 2007-09-07 2009-03-12 Dyadic International, Inc. Enzymes fongiques inedites
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

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FR2641793B1 (fr) * 1988-12-26 1993-10-01 Setratech Procede de recombinaison in vivo de sequences d'adn presentant des mesappariements de bases
CA2263958C (fr) * 1995-07-26 2012-06-19 Mixis France Recombinaison homologue dans des cellules eucaryotes a systeme de reparation des mesappariements inactive
US7115413B2 (en) * 1996-04-01 2006-10-03 Mixis France S.A. Meiotic recombination in vivo of partially homologous DNA sequences
AU743305C (en) * 1997-01-17 2006-03-30 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017165760A1 (fr) 2016-03-24 2017-09-28 Novozymes A/S Variants de cellobiohydrolase et polynucléotides codant pour ces derniers

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WO2000050567A1 (fr) 2000-08-31

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