WO2016050917A1 - Procédés de modification génomique - Google Patents

Procédés de modification génomique Download PDF

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WO2016050917A1
WO2016050917A1 PCT/EP2015/072710 EP2015072710W WO2016050917A1 WO 2016050917 A1 WO2016050917 A1 WO 2016050917A1 EP 2015072710 W EP2015072710 W EP 2015072710W WO 2016050917 A1 WO2016050917 A1 WO 2016050917A1
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target locus
nuclease
recognition site
site
host cell
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PCT/EP2015/072710
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Adrianus Wilhelmus Hermanus Vollebregt
VAN DE Peter Jozef Ida VONDERVOORT
Bernard Meijrink
Johannes Andries Roubos
Hesselien Touw-Riel
Van Den Marco Alexander Berg
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Dsm Ip Assets B.V.
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Publication of WO2016050917A1 publication Critical patent/WO2016050917A1/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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the present invention relates to a method for the modification of the genome of a fungal host cell at a target locus.
  • mammalian cell lines are used for antibody production; fungal cells are preferred organisms for production of polypeptides and secondary metabolites; bacterial cells are preferred for small metabolite and antibiotic production; and plant cells are preferred for taste and flavor compounds.
  • Recombinant techniques are widely employed for optimization of the productivity of such cells and/or the processes in which they are used. This can involve a multitude of options, including, but not limited to over expression of a gene of interest, deletion or inactivation of competing pathways, changing compartmentalization of enzymes, increasing protein or metabolite secretion, increasing organelle content and the like.
  • target sequences are altered in vitro to create mutant alleles with inserted sequences encoding antibiotic resistance markers.
  • regulatory authorities in most countries object to the use of antibiotic resistance markers in view of the potential risks of spreading resistance genes to the biosphere from large-scale use of production strains carrying such genes.
  • selectable marker genes may need to be removed so that production strains may be used commercially and/or so that the same marker gene may be recycled for use in sequential strain modification.
  • An efficient method to remove marker genes is represented by the Cre-/ox recombination system.
  • the method generally requires several steps including construction and introduction of a Cre-expression vector in the cell, expression of the Cre-protein intracellularly, followed by the Cre-mediated recombination event between two lox sites at both termini of the marker to be excised.
  • Cre protein could be directly introduced into Aspergillus oryzae protoplasts, polyethylene glycol and carrier DNA, such as for example single stranded or double stranded DNA. Authors found that no recombination events took place when carrier DNA was dispensed with.
  • Zhang et al. disclose a Cre-/ox recombination system in the filamentous fungus C. parasitica wherein Cre recombinase is provided to a cell by anastomosis with a second, Cre-expressing, donor cell.
  • the present invention is based on a method which enables a significant shortening of the time it takes to construct marker free isolates of fungal strains, such as filamentous fungi and yeast.
  • a nuclease enzyme such as Cre recombinase
  • Cre recombinase is introduced directly into the cell, rather than expressed within the cell via a nucleic acid construct. This has the advantage of not requiring a step of introducing nucleic acid constructs encoding a nuclease into the cell.
  • the invention relates to a method for the modification of the genome of a fungal host cell at a target locus, the method comprising:
  • a fungal host cell comprising in its chromosome a target locus, the target locus comprising a recognition site for a nuclease; and (b) introducing into said cell the nuclease protein or mRNA encoding a nuclease protein which cleaves DNA at the recognition site such that recombination occurs at the target locus, thereby to modify the genome of the host cell at the target locus.
  • FIG. 1A sets out a schematic diagram of plasmid pEBA513 for transient expression of Cre recombinase in fungi.
  • pEBA513 is a pAMPF21 derived vector containing the AMA1 region and the CAT chloramphenicol resistance gene. Depicted are the Cre recombinase gene (Cre) expression cassette, containing the A. niger glaA promoter (Pgla), Cre recombinase coding region, and niaD terminator.
  • the hygromycin resistance cassette consisting of the A. nidulans gpdA promoter (PgpdA), hygB coding region and the P. chrysogenum penDE terminator (TpenDE) is indicated.
  • FIG. 1 B sets out a map of pGBTOPEBA205 for expression of T. emersonii CBHI in T. emersonii. Depicted is EBA205 expressed from the glucoamylase promoter (PglaA). In addition, the glucoamylase flank (3'glaA) of the expression cassette is depicted.
  • PglaA glucoamylase promoter
  • Figure 1 C sets out a schematic diagram of plasmid pDEL_PdxA-2, which is the basis for a replacement cassette to inactivate the pdxA gene in A. niger.
  • the replacement cassette comprises the pdxA flanking regions, the ble marker cassette, mutant loxP sites and E. coli DNA.
  • WO2013135729A1 For a further description see WO2013135729A1 .
  • Figure 2 sets out the detection of pGBTOPEBA-205 expression plasmid in the R. emersonii genome by PCR. Genomic DNA was isolated and analysed by PCR from transformant A-A4 (lanes 2-4) and the empty strain (lanes 5-7). Plasmid DNA was used as control template for the PCR reactions: pGBTOPEBA-4 (lane 8), pGBTOPEBA-8 (lane 9) and pGBTOPEBA-205 (lane 10).
  • Lanes 1 and 1 1 contain a molecular weight marker.
  • Figure 3 sets out pCHYhph, the hygromycin B resistance marker containing plasmid used for integration in GG799 for production of chymosin.
  • Figure 4 sets out pCHYnat, the nourceothricin resistance marker containing plasmid used for integration in GG799H for production of chymosin.
  • Figure 5 sets out pCHYkanMX, the hygromycin B resistance marker containing plasmid used for integration in GG799HN for production of chymosin.
  • SEQ ID NO: 1 sets out the nucleic acid sequence of the Ble-For primer.
  • SEQ ID NO: 2 sets out the nucleic acid sequence of the Ble-Rev primer.
  • SEQ ID NO: 3 sets out the nucleic acid sequence of the EBA205-For primer.
  • SEQ ID NO: 4 sets out the nucleic acid sequence of the EBA205-Rev primer.
  • SEQ ID NO: 5 sets out the nucleic acid sequence of the EBA4-For primer.
  • SEQ ID NO: 6 sets out the nucleic acid sequence of the EBA4-Rev primer.
  • SEQ ID NO: 7 sets out the nucleic acid sequence of the EBA8-For primer.
  • SEQ ID NO: 8 sets out the nucleic acid sequence of the EBA8-Rev primer.
  • SEQ ID NO: 9 sets out the nucleic acid sequence of the kanMX gene under S.c. tef control, flanked by lox66 and lox71 .
  • SEQ ID NO: 10 sets out the nucleic acid sequence of the hph gene under S.c. tef control, flanked by lox66 and lox71 .
  • SEQ ID NO: 1 1 sets out the nucleic acid sequence of the natl gene under S.c. tef control, flanked by lox66 and lox71 .
  • the articles “a” and “an” are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article.
  • an element may mean one element or more than one element.
  • the invention concerns a method for carrying out modification at a target locus, or target loci, typically within a target genome, although the invention may be used to modify an extra-chromosomal target locus.
  • the modification method of the invention results in alteration of the target locus, for example the insertion of nucleic acid sequence at the target locus.
  • the method may be carried out such that insertion of new sequence at the target locus is accompanied by removal of existing sequence from the target locus. That is to say, the method may be used to substitute a sequence at the target locus with an alternative sequence.
  • the invention may be used to delete sequence from a target locus, for example a sequence encoding a marker gene, or may be used to carry out substitution of one or more nucleic acids at the target locus.
  • the method of the invention may be carried out in vitro, ex vivo or in vivo.
  • the method of the invention may conveniently be carried out in vivo in a fungal host cell.
  • the method of the invention is not carried out on a human or animal cell. That is to say, the method of the invention is not typically carried out in the form of a method of treatment.
  • the method of the invention may be carried out in an ex vivo or in vitro format.
  • ex vivo or in vitro should be understood to encompass methods carried out on microorganisms (both on whole living cells or on non-cellular material), but to exclude methods carried out on humans or animals.
  • the method of the invention may be carried out to achieve alteration of, the sequence of, the target locus.
  • Such alteration may be, for example addition of new sequence, replacement of existing sequence and/or deletion/removal of existing sequence.
  • the invention is carried out in vivo in a fungal host cell.
  • the fungal host cell may, preferably, be one which produces a compound of interest.
  • the fungal host cell may be capable of producing the compound of interest prior to application of the method of the invention.
  • the method of the invention may be used to modify the target locus so that production of the compound of interest by the fungal host cell is altered, for example production may be increased.
  • the fungal host cell may be one which produces the compound of interest as a result of application of the method of the invention.
  • the invention may be used, for example, in the optimization of the productivity of a fungal host cell and/or the processes in which they are used.
  • the invention may be used, for example, to introduce novel nucleic acids such that the fungal host cell is rendered capable of producing a new compound of interest.
  • the invention may be used sequentially, such that a plurality of novel nucleic acid sequences is introduced into the host cell, resulting in the introduction of an entirely new metabolic pathway.
  • a target locus may be any nucleic sequence which is to be modified.
  • the target locus may be a sequence within a genome (the complete genetic material of an organism), for example a locus on a chromosome.
  • a chromosome could be a linear or a circular chromosome.
  • the target locus could be extrachromosomal for example a locus on a plasmid, a minichromosome or artificial chromosome.
  • the target locus may be located on a plasmid, a phage, or any other nucleic acid sequence which is able to replicate or be replicated in vitro or in a host cell.
  • the invention relates to a method for the modification of a fungal host cell at a target locus, for example in the genome of the fungal host cell, the method comprising:
  • the method of the invention is carried out, wherein introduction of the nuclease protein or mRNA encoding the nuclease protein takes place in the absence of carrier DNA.
  • the method may be carried out in the presence of a carrier, such as nucleic acid, for example DNA or RNA.
  • Introduction of the nuclease protein in the context of this invention is intended to mean introduction of protein per se: not a DNA sequence encoding the nuclease.
  • nuclease protein may occur by contacting the fungal host cell with a composition comprising isolated nuclease protein.
  • the composition is enriched in such protein.
  • the composition may comprise the nuclease protein as the major or sole enzymatic component, e.g., a mono-component composition.
  • the composition may comprise multiple enzymatic activities.
  • isolated nuclease protein as used herein means a nuclease protein that is removed from at least one component, e.g. other polypeptide or protein material, with which it is naturally associated.
  • an isolated nuclease protein may contain at most 10%, at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, even more preferably at most 1 % and most preferably at most 0,5% as determined by SDS-PAGE of other polypeptide or protein material with which it is natively associated.
  • the isolated protein may be substantially free of any other impurities.
  • the isolated nuclease protein may be at least 50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 80% pure, at least 90% pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% as determined by SDS-PAGE or any other analytical method suitable for this purpose and known to the person skilled in the art.
  • nuclease protein may occur preferably by contacting the fungal host cell with the composition comprising isolated nuclease protein under suitable conditions to allow nuclease mediated DNA cleavage at the recognition site and recombination at the target locus.
  • suitable conditions comprise contacting a fungal protoplast with the composition comprising isolated nuclease protein and optionally PEG in sufficient amount to allow nuclease mediated DNA cleavage at the recognition site and recombination at the target locus.
  • amount of nuclease protein e.g. Cre recombinase protein is more than 5U Cre recombinase / 1 x10 5 protoplasts.
  • at least 10U, 20U, 30U, 40U, 50U Cre recombinase are used per 1 x10 5 protoplasts.
  • At most 1000 U Cre recombinase are used per 1 x10 5 protoplasts.
  • the composition does not comprise PEG. Cre recombinase units (U) are defined in the examples.
  • suitable conditions to allow nuclease mediated DNA cleavage at the recognition site and recombination at the target locus comprise contacting fungal cells with the composition comprising isolated nuclease protein and electroporating the mixture comprising the composition and the cells.
  • suitable conditions to allow nuclease mediated DNA cleavage at the recognition site and recombination at the target locus comprise contacting fungal cells with the composition comprising isolated nuclease protein and a Lithium salt, such as e.g. lithium acetate or lithium chloride.
  • a Lithium salt such as e.g. lithium acetate or lithium chloride.
  • the lithium salt may be added in any form suitable, for example as a solution or as a powder lithium salt.
  • compositions comprising nuclease with the fungal host cell may be used, which are typically applied in genetic transformation or transfection of host cells.
  • the person skilled in the art is aware of such methods which include but are not limited to particle bombardment or microprojectile bombardment, the above mentioned protoplast methods and Agrobacterium mediated transformation (AMT).
  • AMT Agrobacterium mediated transformation
  • the protoplast method is used for filamentous fungi. Procedures for transformation are inter alia described by J.R.S. Fincham, Transformation in fungi. 1989, Microbiological reviews. 53, 148-170. Transformation may involve a process consisting of protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se.
  • a suitable method of transforming Fusarium species is described by Malardier et al., 1989, Gene 78:147156 or in WO 96/00787.
  • Other methods can be applied such as a method using biolistic transformation as described in: Christiansen et al., Biolistic transformation of the obligate plant pathogenic fungus, Erysiphe graminis f.sp. hordei. 1995, Curr Genet. 29:100-102.
  • Yeast may be transformed using any method known in the art such as the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, 1983; Hinnen et al., 1978, and Gietz RD, Woods RA. 2002.
  • the mRNA may be one which has been produced by in vitro translation.
  • the nuclease used in a method of the invention may cause a single- or double- stranded cleavage at the target locus.
  • the host cell may comprise in its chromosome two or more target loci, for example three, four, five or more target loci, each of which comprises a recognition site for a nuclease.
  • the method is carried out so that two or more target loci comprise a recognition site for a nuclease
  • at least two of the target loci may comprise a recognition site for different nucleases.
  • the nuclease may be a site-specific recombinase or an endodeoxyribonuclease characterized by a large recognition site, for example a meganuclease.
  • recombinase or "site-specific recombinase” or the like refers to enzymes or recombinases that recognize and bind to a short nucleic acid site or "site- specific recombinase site", i.e., a recombinase recognition site, and catalyze the recombination of nucleic acid in relation to these sites.
  • site-specific recombinase site i.e., a recombinase recognition site, and catalyze the recombination of nucleic acid in relation to these sites.
  • enzymes include recombinases, transposases and integrases.
  • the site-specific recombinase for use in the invention may be the ere gene of bacteriophage P1 , the Cre recombinase or the FLP recombinase.
  • the endodeoxyribonuclease characterized by a large recognition site may be a meganuclease.
  • the meganuclease may be a homing endonuclease, such as for example a member of the LAGLI DADG family of homing endonucleases, such as I- Crel or l-Scel.
  • the nuclease used in the invention may be a naturally-occurring nuclease, such as one of those described above, or a modified non-naturally occurring variant thereof.
  • each target locus may comprise:
  • a a pair of recognition sequences for a site-specific recombinase; or b. a recognition site for an endodeoxyribonuclease characterized by a large recognition site.
  • site-specific recombinase site refers to short nucleic acid sites or sequences, i.e., recombinase recognition sites, which are recognized by a sequence- or site-specific recombinase and which become the crossover regions during a site- specific recombination event.
  • sequence-specific recombinase target sites include, but are not limited to, lox sites, att sites, dif sites and frt sites.
  • lox site refers to a nucleotide sequence at which the product of the cre gene of bacteriophage P1 , the Cre recombinase, can catalyze a site- specific recombination event.
  • lox sites are known in the art, including the naturally occurring loxP, loxB, loxL and loxR, as well as a number of mutant, or variant, lox sites, such as ⁇ 66, ⁇ 71 , ⁇ 51 1 , ⁇ 514, ⁇ 86, ⁇ 1 17, loxC2, ⁇ 2, loxP3 and lox P23.
  • fuse site refers to a nucleotide sequence at which the product of the FLP gene of the yeast 2 micron plasmid, FLP recombinase, can catalyze site-specific recombination.
  • the site-specific recombination sites and recombinase are selected such that the recombinase may target the site-specific recombination sites leading to out- recombination of sequence located between the recombination sites.
  • the site-specific recombination sites may be such that out-recombination following recombinase action gives rise to a single mutant site-specific recombination site at the target locus which is not recognized by the recombinase.
  • the lox sites may be lox66 and lox 71 (Albert, H., Dale, E. C, Lee, E., & Ow, D. W. (1995). Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant Journal, 7(4), 649-659).
  • Recognition sites for an endodeoxyribonculease characterized by a large recognition site are from about 15 to about 40 base pairs. As a result of this size, such sites occurs rarely in any given genome and therefore meganucleases are very specific.
  • the recognition site is selected based on the specific endodeoxyribonculease being used. For example, the l-Scel meganuclase recognizes an 18-base pair sequence.
  • the modification to the genome of the fungal host cell may be any desired modification.
  • the modification may be any desired modification.
  • the modification may be any desired modification.
  • the modification may be any desired modification.
  • the modification may be any desired modification.
  • the modification may be any desired modification.
  • the modification may be any desired modification.
  • the modification may be any desired modification.
  • the modification may be any desired modification.
  • the modification may be any desired modification.
  • the modification may be
  • nucleic acid sequence such as removal of a nucleic acid sequence encoding a marker at the target locus
  • the fungal host cell may be a yeast cell or a filamentous fungus cell.
  • a yeast cell may be a cell such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain. More preferably from Kluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowia lipolytica and Pichia pastoris.
  • a filamentous fungal cell may be any filamentous form of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
  • the filamentous fungi are characterized 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.
  • Filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Rasamsonia, Talaromyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.
  • Preferred filamentous fungal cells belong to a species of an Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Rasamsonia, Talaromyces, Thielavia, Fusarium or Trichoderma genus, and most preferably a species of Aspergillus niger, Acremonium alabamense, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Rasamsonia Talaromyces emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum, Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris or Penicillium chrysogenum.
  • a more preferred host cell belongs to the genus Aspergillus, more preferably the host cell belongs to the species Aspergillus niger.
  • the host cell according to the invention is an Aspergillus niger host cell, the host cell preferably is CBS 513.88, CBS124.903 or a derivative thereof.
  • Useful strains in the context of the present invention may be Aspergillus niger CBS 513.88, CBS124.903, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 101 1 , CBS205.89, ATCC 9576, ATCC14488-14491 , ATCC 1 1601 , ATCC12892, P. chrysogenum CBS 455.95, P.
  • a method for the modification of the genome of a fungal host cell at a target locus comprising:
  • the host cell comprises in its chromosome two or more target loci, each of which comprises a recognition site for a nuclease.
  • nuclease is a site-specific recombinase or an endodeoxyribonuclease characterized by a large recognition site, preferably a site-specific recombinase, more preferably a Cre recombinase.
  • nuclease is a naturally- occurring nuclease or a modified non-naturally occurring variant thereof.
  • each target locus comprises:
  • nucleic acid sequence such as removal of a nucleic acid sequence encoding a marker at the target locus
  • the fungal host cell is a yeast cell or a filamentous fungus cell.
  • each target locus comprises a pair of recognition sequences for a site-specific recombinase which are lox sites and wherein the site-specific recombinase is Cre recombinase. 12.
  • introduction of the nuclease protein occurs by contacting the fungal host cell with a composition comprising isolated nuclease protein under suitable conditions to allow nuclease mediated DNA cleavage at the recognition site and recombination at the target locus.
  • a method according to embodiment 12 wherein suitable conditions to allow nuclease mediated DNA cleavage at the recognition site and recombination at the target locus comprise contacting a fungal protoplast with the composition comprising isolated nuclease protein in sufficient amount to allow nuclease- mediated DNA cleavage at the recognition site and recombination at the target locus.
  • suitable conditions to allow nuclease mediated DNA cleavage at the recognition site and recombination at the target locus comprise contacting the fungal cell with the composition comprising isolated nuclease protein and electroporating the mixture comprising the composition and the cells
  • suitable conditions to allow nuclease mediated DNA cleavage at the recognition site and recombination at the target locus comprise contacting the fungal cell with the composition comprising isolated nuclease protein and a lithium salt.
  • the Rasamsonia emersonii (R. emersonii) strains used herein are derived from ATCC16479, which is used as wild-type strain. ATCC16479 was formerly also known as Talaromyces emersonii and Penicillium geosmithia emersonii. Upon the use of the name Rasamsonia emersonii also Talaromyces emersonii is meant. Other strain designations of R. emersonii MCC 16479 are CBS393.64, IF031232 and IM1 1 16815.
  • TEC-142 Rasamsonia (Talaromyces) emersonii strain TEC-142 is deposited at CENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands on 1 st July 2009 having the Accession Number CBS 124902.
  • TEC-142S is a single isolate of TEC-142.
  • Cre recombinase was obtained from New England Biolabs, catalog no M0298L. Cre recombinase from New England BioLabs catalogue noM0298L is a Type I topoisomerase from bacteriophage P1 that catalyses the site-specific recombination of DNA between loxP sites.
  • One unit is defined as the amount of enzyme necessary to produce maximal site- specific recombination of 0.25 ⁇ g pLox2+ control DNA in 30 minutes at 37°C in a total reaction volume of 50 ⁇ . Maximal recombination is determined by agarose gel analysis and by transformation of reactions followed by selection on ampicillin plates. Reaction conditions: 1 X Cre Recombinase Reaction Buffer, incubate at 37°C. 1 X Cre Recombinase Reaction Buffer: 33mM NaCI, 50 mM Tris-HCI, 10mM MgCI2, pH 7.5 at 25°C.
  • Strains were grown from stocks on Rasamsonia agar medium in 10 cm diameter Petri dishes for 5-7 days at 40°C. For MTP fermentations, strains were grown in 96-well plates containing Rasamsonia agar medium. Strain stocks were stored at -80°C in 10% glycerol. Chromosomal DNA isolation
  • Strains were grown in YGG medium (per liter: 8 g KCI , 16 g glucose. H 2 0 , 20 ml of 10% yeast extract, 10 ml of 100x pen/strep, 6.66 g YNB+amino acids, 1 .5 g citric acid, and 6 g K 2 HP0 4 ). for 16 hours at 42°C, 250 rpm, and chromosomal DNA was isolated using the DNeasy plant mini kit (Qiagen, Hilden, Germany).
  • pEBA513 was constructed by DNA2.0 (Menlo Park, USA) and contains the following components: expression cassette consisting of the A. niger glaA promoter, ORF encoding Cre-recombinase (AAY56380) and A. nidulans niaD terminator; expression cassette consisting of the A. nidulans gpdA promoter, ORF encoding hygromycin B resistance protein and P. chrysogenum penDE terminator (Genbank: M31454.1 , nucleotides 1750-2219); pAMPF21 derived vector containing the AMA 1 region and the CAT chloramphenicol resistance gene.
  • Figure 1A represents a map of pEBA513.
  • R. emersonii was transformed to obtain a multicopy Cbhl strain.
  • Plasmid pGBTOPEBA205 ( Figure 1 B), described in WO201 1/054899, encoding R. emersonii Cbhl driven by the A. niger glucoamylase promoter was used in the transformation.
  • R. emersonii transformation was performed according to the protocol described in WO201 1/054899. R.
  • pDEL_PdxA-2 plasmid was determined using primer Ble-For (SEQ ID NO: 1 ) and Ble- Rev (SEQ ID NO: 2) and of pGBTOPEBA205 with primer EBA205-For (SEQ ID NO: 3) and EBA205-Rev (SEQ ID NO: 4).
  • Primers directed against pGBTOPEBA4 (SEQ ID NO: 5 and 6) and pGBTOPEBA8 (SEQ ID NO: 7 and 8) were used as a control.
  • Ble-Rev (SEQ ID NO: 2): 5'-CACGAAGTGCACGCAGTTG-3'.
  • EBA205-For (SEQ ID NO: 3): 5'-CTTCTGCTGAGCAGCTCTGCC-3'; and EBA205-Rev (SEQ ID NO: 4): 5'-GTTCAGACCGCAAGGAAGGTTG -3'.
  • EBA4-For (SEQ ID NO: 5): 5'-CGAGAACCTGGCCTACTCTCC-3'
  • EBA4-Rev (SEQ ID NO: 6): 5'-CAGAGTTGTAGTCGGTGTCACG-3'
  • EBA8-For (SEQ ID NO: 7): 5'-GAAGGGTATCAAGAAGCGTGCC-3'
  • EBA8-Rev (SEQ ID NO: 8): 5'-GCCGAAGTTGTGAGGGTCAATG-3'
  • PCR conditions for the reactions 50 ⁇ reaction mix with 5 ⁇ of template DNA, 20 pmol of each primer, 0.2 mM of dNTPs, 1 x Phusion HF buffer and 1 U of Phusion DNA- Polymerase, according to Phusion High-Fidelity DNA Polymerase Manual (Finnzymes, Espoo, Finland), 30 s denaturation at 98°C, amplification in 30 cycles (10 s 98°C, 10 s 55°C, 15 s 72°C), and a final incubation of 10 min at 72°C.
  • Transformant A-A4 is a co-transformant that contains one or more copies of pGBTOPEBA205.
  • the expected 452 bp PCR product of pGBTOPEBA-205 bp was observed in the transformant ( Figure 2, lane 4), which is detected in the control PCR in which pGBTOPEBA205 was used as a template (lane 10), but not in the empty strain (lane 7).
  • the EBA4 and EBA8 PCR reactions no specific bands were observed in the transformants, but the expected PCR products of 256 bp and 306 bp, respectively, were generated when plasmid DNA was used as template (lanes 8 and 9).
  • Table 1 overview of different experimental conditions for testing the effect of incubation of protoplasts with purified Cre recombinase vs the actual method of transformation with pEBA513 vector for generation of marker free isolates. Trafo nr Host strain 'Cre' condition # +/- PEG Incubation protoplasts time
  • the phleomycin resistant R. emersonii transformant A-A4 was transformed with AMA plasmid pEBA513 carrying the Cre recombinase gene and was aimed for selection of phleomycin-sensitive transformants. Cre recombinase was transiently expressed in R. emersonii transformant A-A4 to remove the lox-flanked phleomycin resistance gene by recombination over the lox66 and lox71 site.
  • the transformant was transformed with milliQ water (control) or with 1 .8 and 3.6 ⁇ g of pEBA513 carrying a Cre recombinase and hygromycin expression cassette.
  • pEBA513 transformed protoplasts were plated in overlay on regeneration medium containing 100 ⁇ g ml of hygromycin B. Hygromycin-resistant transformants were grown on PDA containing 100 ⁇ g ml of hygromycin B to allow expression of the Cre recombinase. Removal of the ble marker was tested phenotypically by growing the transformants on media with and without 10 ⁇ g ml of phleomycin. The majority (>80%) of the transformants after transformation with pEBA513 (with the Cre recombinase) were phleomycin sensitive, indicating that Cre recombinase works very efficiently in R. emersonii and that transformants lost the (ble) marker upon introduction and expression of the recombinase.
  • Regeneration agar plates were incubated at 42 ° C for 14 days until sporulation of mycelium had occurred.
  • Spores were scraped from the agar plates by pipetting 5 ml of 10% glycerol on top of the fully grown agar plates and by scraping with sterile 'hockey sticks' from VWR. Spore suspensions were serially diluted and dilution series were plated on PDA agar plates. PDA agar plates were incubated for 4 days at 42 ° C and plates containing single colonies were used for screening for isolates with loss of phleomycin resistance gene.
  • Table 2 overview of results on frequencies of marker free isolates obtained using different experimental conditions as described in Table 1 .
  • the transformant still contained multiple R. emersonii Cbhl copies; this was confirmed with PCR using EBA205 specific oligonucleotides.
  • Cre-recombinase mediated marker removal can successfully occur in filamentous fungi and in the absence of carrier DNA when protoplasts are treated with a sufficient amount of Cre-recombinase.
  • the experiments also show that the latter may occur either in the presence or in the absence of polyethylene glycol.
  • the amount of Cre-recombinase to be used in the absence of carrier DNA was found to be above 5U Cre recombinase / 1 x10 5 protoplasts. Successful results were obtained with 50U Cre recombinase / 2x10 5 protoplasts.
  • GG799 This Kluyveromyces lactis strain is used as a wild-type strain. This strain is obtained from New England BioLabs, Ipswich, Massachusetts, USA. Cre recombinase was obtained from New England BioLabs, catalogue no M0298L.
  • the K. lactis strain was grown in 100 mL YEPD (10 g/l yeast extract, 20 g/l peptone, 20 g/l D-glucose) at 30°C at 250 rpm in a shake flask to an OD600 of 2.0.
  • the culture was harvested by centrifugation at 4000g for 10 minutes and the cells were resuspended in 1 ml TE (10 mM Tris-HCI pH 7.5 and 1 mM EDTA). Again, the cells were harvested by centrifugation the pellet is resuspended in 1 ml TEL (TE + 0.2 M lithium acetate). After one hour at room temperature without shaking, the cells were pelleted (harvested) again (by centrifugation).
  • the pellet was resuspended in 1 ml TEL.
  • 80 ⁇ of cells were used. After adding max 20 ⁇ DNA, milliQ or Cre recombinase, the cells were mixed and 600 ⁇ of PTEL (TEL + 40% PEG 4000) was added. After thorough mixing, the mixture was incubated for 30 minutes at 30°C while shaking 200 rpm. Then 70 ⁇ DMSO was added and after mixing by inversion, the mixture was incubated for 15 minutes at 42°C without shaking. After this, the cells were harvested by centrifugation and are plated on selective and non-selective agar plates. Construction of the expression vectors
  • Plasmid pCHYkanMX contains the kanMX marker under control of the Saccharomyces cerevisiae Tef promoter and terminator, flanked with lox66 and lox71, conferring resistance to G418 when integrated in the K. lactis genome (SEQ ID NO: 9).
  • an expression cassette of chymosin composed of an one kb (1000 base pairs) K. lactis derived PGK1 promoter, mating factor alpha leader fused to the chyB coding gene as described in WO2013164481 and a 300 bp K. lactis derived PGK1 terminator.
  • the marker and expression cassette were flanked with 1 kb LAC4 5' and 3' fragments as depicted in Figure 5.
  • Plasmid pCHYhph contains the hph marker under control of the Saccharomyces cerevisiae Tef promoter and terminator, flanked with lox66 and lox71 , conferring resistance to hygromycin B when integrated in the K. lactis genome (SEQ ID NO: 10).
  • SEQ ID NO: 10 Next to this resistance marker lies an expression cassette of chymosin as described above. The marker and expression cassette were flanked with 1 kb PRC1 5' and 3' fragments as depicted in Figure 3.
  • Plasmid pCHYnat contained the natl marker under control of the Saccharomyces cerevisiae Tef promoter and terminator, flanked with lox66 and lox71, conferring resistance to nourseotricin when integrated in the K. lactis genome (SEQ ID NO: 1 1 ).
  • SEQ ID NO: 1 1 Next to this resistance marker lies an expression cassette of chymosin as described above. The marker and expression cassette were flanked with 1 kb BAR1 5' and 3' fragments as depicted in Figure 4.
  • Plasmid pCHYhph was linearized by digestion with Notl, the fragment containing the expression cassette and the resistance marker was purified and transformed to Kluyveromyces lactis strain GG799 by electroporation. Electroporation is described in WO2007060247. Transformants were selected on G418 containing YEPD agar plates. After each transformation, 30 random transformants were tested for increased chymosin productivity compared to the wild-type parent strain GG799. For this, milk clotting activity was measured after cultivation in 500 mL Erlenmeyer flask containing 100 mL YEP2D/MES medium, both cultivation and milk clotting assay as described in WO2013164481 . From the pCHYhph transformants with detectable chymosin productivity, single copy transformants in the PRC1 locus were selected by PCR. One of the found single copy transformants was denoted GG799H.
  • Plasmid pCHYnat was linearized by digestion with Notl, the fragment containing the expression cassette and the resistance marker was purified and transformed to Kluyveromyces lactis strain GG799H by electroporation. Transformants were selected on nourseothricin containing YEPD agar plates. After each transformation, 30 random transformants were tested for increased chymosin productivity compared to the parent strain. For this, milk clotting activity was measured after cultivation in 500 ml_ Erlenmeyer flask containing 100 ml. YEP2D/MES medium, both cultivation and milk clotting assay as described in WO2013164481 . From the pCHYnat transformants with increased chymosin productivity, single copy transformants in the BAR1 locus were selected by PCR. One of the found single copy transformants was denoted GG799HN.
  • Plasmid pCHYkanMX was linearized by digestion with Notl, the fragment containing the expression cassette and the resistance marker was purified and transformed to Kluyveromyces lactis strain GG799HN by electroporation. Transformants were selected on nourseothricin containing YEPD agar plates. After each transformation, 30 random transformants were tested for increased chymosin productivity compared to the parent strain. For this, milk clotting activity was measured after cultivation in 500 ml. Erlenmeyer flasks containing 100 ml. YEP2D/MES medium, both cultivation and milk clotting assay as described in WO2013164481 . From the pCHYkanMX transformants with increased chymosin productivity, single copy transformants in the BAR1 locus were selected by PCR. One of the found single copy transformants was denoted GG799HNK.
  • the Cre enzyme was administered to strain GG799HNK by electroporation.
  • To a volume of 40 ⁇ of competent cells, used for the electroporation we added 0, 1 or 10 Units of the Cre recombinase.
  • cells were plated on non-selective YEPD agar plates. From each treatment, ten random colonies were transferred to a YEPD agar containing MTP, next to control strains GG799 and GG799HNK. After incubation for 3 days at 30°C the colonies were replicated on medium containing either G418 or nourseothricin or hygromycin B or no antibiotic.
  • the pSH65 plasmid was used, using methods of transformant selection and induction of Cre-recombinase induction as described by Guldener et al. 1996 (A New Efficient Gene Disruption Cassette for Repeated Use in Budding Yeast. Nucleic acids research 24; 2519-2524) and Guldener et al 2002 (A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Research 30 (6): e23. doi: 10.1093/nar/30.6.e23). Of ten tested pSH65 transformants, all cells had lost their resistance markers. These results are summarized in Table 3.
  • Table 3 Percentage of 10 tested colonies that are sensitive to the tested antibiotic- Application of Cre recombinase was by electroporation.
  • Cre enzyme by transformation was also tested using Lithium acetate transformation.
  • Different quantities of Cre recombinase were administered to strain GG799HNK by Lithium acetate transformation.
  • a volume of 80 ⁇ of competent cells, used for the electroporation contained 10 Units of the Cre recombinase.
  • cells were plated on non-selective YEPD agar plates. From each treatment, ten random colonies were transferred to a YEPD agar containing MTP, next to control strains GG799 and GG799HNK.

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Abstract

Cette invention concerne un procédé de modification du génome d'une cellule hôte fongique sur un locus cible, le procédé comprenant : (a) l'utilisation d'une cellule hôte fongique comprenant dans son chromosome un locus cible, le locus cible comprenant un site de reconnaissance pour une nucléase ; et (b) l'introduction dans ladite cellule de la protéine nucléase ou de l'ARNm codant pour une protéine nucléase qui clive l'ADN sur le site de reconnaissance pour qu'une recombinaison se produise sur le locus cible, pour modifier ainsi le génome de la cellule hôte sur le locus cible.
PCT/EP2015/072710 2014-10-01 2015-10-01 Procédés de modification génomique WO2016050917A1 (fr)

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