WO1995017513A1 - Retransformation de champignons filamenteux - Google Patents

Retransformation de champignons filamenteux Download PDF

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Publication number
WO1995017513A1
WO1995017513A1 PCT/DK1994/000488 DK9400488W WO9517513A1 WO 1995017513 A1 WO1995017513 A1 WO 1995017513A1 DK 9400488 W DK9400488 W DK 9400488W WO 9517513 A1 WO9517513 A1 WO 9517513A1
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protein
dna
gene
niger
dna sequence
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PCT/DK1994/000488
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English (en)
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Solvejg Reeh
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Novo Nordisk A/S
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Priority to PCT/DK1994/000488 priority Critical patent/WO1995017513A1/fr
Priority to AU12726/95A priority patent/AU1272695A/en
Publication of WO1995017513A1 publication Critical patent/WO1995017513A1/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
    • C12N15/67—General methods for enhancing the expression
    • C12N15/69—Increasing the copy number of the vector
    • 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
    • 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)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • the present invention relates to process for producing a pro ⁇ tein in filamentous fungi in improved yields.
  • a gene amplification process is described in WO 88/06623 (Gist- Brocades) .
  • a prokaryotic, especially a Bacil- lus host is transformed with a vector carrying a gene of inter ⁇ est, which gene generally is present in the chromosome of the host, and a marker gene.
  • the DNA is integrated in the host chromosome by homologous or illegitimate recombination and am ⁇ plified by growth under selection pressure.
  • the gene of inter- est will typically be amplified in a tandem arrangement.
  • retransformation according to the present method has the advantage over the amplification procedure described in WO 88/06623 that the copies of the gene introduced into the fungus are integrated at different sites on the host chromosome due to the presence of different DNA sequences homologous to the host chromosome on the vector with which the host is trans ⁇ formed. This reduces the risk that the gene will be deleted from the cell as a result of recombination, as may often happen when genes are amplified by the procedure described above.
  • "scatter- ed" gene transformants i.e. transformants in which the gene is integrated at different sites in the chromosome
  • Correct transformants are then iden ⁇ tified by selection.
  • the retransformation process of the pre ⁇ sent invention is different in that it does not require gene amplification under selection pressure, selection for "scatter ⁇ ed" genes in a mixture of cells also containing tandemly arran- ged genes, or let alone fusion of host cells.
  • the process of the present in ⁇ vention differs from that described by Farman and Oliver in that recombinant DNA constructs are integrated into the fungal chromosome at different sites of the chromosome and are prac ⁇ tically not recombined out of the cell. Furthermore, the pre ⁇ sent process makes it possible to integrate said DNA constructs at sites on the host chromosome which result in improved expres ⁇ sion and hence in improved yields.
  • the present invention relates to a process for producing a protein in a filamentous fungus in improved yields, the process comprising
  • step (b) retransforming the transformant of step (a) with a second recombinant DNA construct which comprises a DNA sequence enco- ding a protein preceded by a promoter sequence operably con ⁇ nected thereto, as well as with a second DNA sequence coding for a suitable marker for the selection of transformants,
  • step (c) culturing the retransformed filamentous fungus of step (b) in a suitable culture medium under conditions permitting the production of the protein.
  • step (a) and/or step (b) said first or second DNA construct, and/or said first or second DNA sequence comprises at least one stretch of DNA homologous to the host.
  • a still further embodiment comprises the process as above, wherein the DNA sequence encoding a protein in step (b) encodes a protein which is the same or different from the protein enco ⁇ ded for in step (a) .
  • the protein(s) may be homologous or heterologous to the host organism.
  • the term "homologous” is intended to indicate that the protein in question is one which is produced by the host organism in nature, while the term “heterologous” is intended to indicate proteins which are not, in nature, produced by the host organism in question.
  • Heterologous proteins may be derived from prokaryotic or eukaryotic organisms, such as fungi or higher eukaryotes such as plants or mammals.
  • the protein may be an enzyme.
  • homologous is meant to indicate DNA sequences which are not identical, but suffi ⁇ ciently similar to be recognised and recombined by recombina ⁇ tion enzymes in the cell, or which would hybridize to the same DNA probe.
  • the term “homologous” may also be defined as intend ⁇ ed to indicate a sequence identity at the DNA level of at least 70% (after aligning the sequences) .
  • filamentous fungus is intended to include, but not being limited to all fungi belonging to Eumycota, such as the groups Phycomycetes, Zygomycetes, Ascomycetes, Basidiomycetes, Deuteromycetes, or fungi imperfecti, including Hyphomycetes, such as the genera Aspergillus, Trichoderma, Penicillium, Fu ⁇ arium or Humicola .
  • Fig. 1 shows the construction of plasmid p960.
  • Fig. 2 shows the construction of plasmid pSRe423.
  • Fig. 3 shows the construction of plasmid pSRe424.
  • Fig. 4 shows the construction of plasmid pSRe426.
  • Fig. 5 shows the construction of plasmid pSRe427.
  • Fig. 6 shows the construction of plasmid pSRe428.
  • Fig. 7 shows the construction of plasmid pSRe433.
  • Fig. 8 shows the construction of plasmid pStal4.
  • Fig. 9 shows the construction of plasmid pSRe418.
  • Fig. 10 shows the construction of plasmid pSRe419.
  • Fig. 11 shows the construction of plasmid pMEM1231.
  • Fig. 12 shows the construction of plasmid pSRe408.
  • Fig. 13 shows the construction of plasmid pHLL.
  • Fig. 14 shows the construction of plasmid p960.
  • Fig. 15 shows a comparison of yields of lipase secretion from before and after transformation and retransformation according to the invention.
  • Fig. 16 shows a comparison of yields of lipase normalised to the yield of amylase secretion before and after retransforma- tion according to the invention.
  • Fig. 17 shows a Southern blot of a deletion transformant.
  • the first and second DNA constructs as well as the first and second DNA sequences encoding the se ⁇ lection marker(s) may conveniently be inserted into a recombi- nant expression vector for transformation of the host cell.
  • the recombinant expression.vector may be any vector which may rea ⁇ dily be subjected to recombinant DNA procedures.
  • the vec- tor will typically be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been inte ⁇ grated.
  • the transformation of a filamentous fungus host cell the vector will be integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated, due to the presence of at least one stretch of DNA homologous to the host.
  • the integration can proceed in any way, such as through a Campbell type or a double reciprocal type recombination.
  • Each DNA construct comprising the protein coding sequence and the promoter may be inserted in the same vector as that con ⁇ taining the DNA sequence encoding the selection marker, or it may be inserted in a separate vector for introduction into the host cell.
  • the vector or vectors may be linear or closed cir- cular molecules.
  • the host cell is transformed, in each transfor ⁇ mation step, with more than one vector, of which one carries the DNA sequence coding for the selection marker, and another carries the DNA construct comprising the protein-coding sequen- ce and the promoter.
  • the DNAs with which the cell is transformed integrate into the chromosome.
  • transformants coding for an increased number of integrated genes of interest can be isolated from a relatively small number of transformants. If only one round of transforma ⁇ tion is done a large number of transformants have to be screened to obtain a transformant with the same number of integrated genes of interest.
  • one purpose of doing retransformations is to increase the number of integrated genes of interest in the chromosomes.
  • Another purpose of doing retransformations is to direct the integrations of the genes of interest into many differently located parts of the chromosomes, thereby decreasing the pro ⁇ bability of deletions of a substantial part of the integrated genes of interest.
  • a still further object of the invention is a means of avoiding expression of harmful proteins by provoking a disruption/dele ⁇ tion of the genes coding for said harmful proteins. If said proteins are proteases this means that the resulting strain is more suitable for production of proteins susceptible to diges ⁇ tion by proteases.
  • harmful protein is meant to indicate a protein that is harmful to either the desired gene product or to the host itself. Another meaning of harmful in this context is a protein which contaminates the final product, or is difficult to separate from the desired end product.
  • An example hereof is the excessive production of the TAKA amylase during production of e.g. a lipase product.
  • the invention resides in the possibility of an unstable plasmid integration into the gene including regulatory sequences encoding the "harmful" protein, through recombinoge- nic sequences leading to cells with unstable chromosomes, which are stabilised by deleting the integrated plasmids together with parts of the chromosomal gene, thereby reducing or avoid ⁇ ing the expression of the harmful protein.
  • the two plasmids are sometimes integrated separately, but also sometimes together in the chromosome. Du- ring the transformation the two different plasmids are able to recombine under the formation of a plasmid complex, if the vec ⁇ tor comprises parts that are identical or homologous.
  • this plasmid complex is therefore dependent on the DNA sequences of the gene of interest, the selection gene, and the vector proper.
  • the direction of integration of this plasmid complex may there ⁇ fore be directed to different genes or sites in the chromosomes by either changing the selection gene, the vector part, or by inserting a pool of random DNA fragments in the complex.
  • the promoter sequence which may be preceded by upstream activa ⁇ ting sequences and enhancer sequences as known in the art may be any DNA sequence exhibiting a strong transcriptional activi ⁇ ty in filamentous fungi and may be derived from a gene encoding an extracellular or intracellular protein such as an amylase, a glucoamylase, a protease, a lipase, a cellulase or a glyco- lytic enzyme.
  • suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic pro- teinase, A. niger neutral ⁇ -amylase, A . niger acid stable ⁇ - amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease or A. oryzae triose phosphate isome- rase.
  • the filamentous fungus host organism may conveniently be one which has previously been used as a host for producing recom ⁇ binant proteins, e.g. a strain of Aspergillus sp. , such as A. niger, A. nidulans or A. oryzae .
  • a strain of Aspergillus sp. such as A. niger, A. nidulans or A. oryzae .
  • the use of A . oryzae in the production of recombinant proteins is extensively described in, e.g. EP 238 023.
  • a preferred promoter for use in the process of the present invention is the A. oryzae TAKA amylase promoter as it exhibits a strong tran ⁇ scriptional activity in A. oryzae .
  • the sequence of the TAKA amylase promoter appears from EP 238 023.
  • the DNA sequence encoding the protein is expressed from different promoters on the first and second DNA constructs whereby it may i.a. be possible to obtain expres ⁇ sion of the gene of interest during different growth phases.
  • the DNA construct may also com ⁇ prise other sequences required for gene expression, such as a transcription initiation site and termination and polyadeny- lation sequences. These may suitably be derived from the same sources as the promoter.
  • the techniques used to transform a fungal host cell may suitab ⁇ ly be adapted from the methods of transforming A. nidulans de ⁇ scribed in, for instance, Yelton et al., Proc. Natl . Acad . Sci . USA 81, 1984, 1470-1474, or EP 215 594, or from the methods of transforming A. niger described in, for instance Buxton et al.. Gene 37, 1985, 207-215 or US 4,885,249, or from the method of transforming A. oryzae described in EP 238 023.
  • the host cell is transformed with a first DNA sequence coding for a selection marker which is ca ⁇ pable of being incorporated in the genome of the host organism on transformation, but which is either not expressed by the host before transformation or expressed in amounts which are not sufficient to permit growth under selective conditions. Transformants may then be selected and isolated from nontrans- formants on the basis of the incorporated selection marker.
  • the host cell is subsequently transformed with a second DNA sequen ⁇ ce typically coding for a different selection marker in order to distinguish between transformants from each transformation step.
  • a DNA sequence enco- ding an inactive marker gene (e.g. not provided with a promoter sequence) should be integrated in the host chromosome in the locus of the corresponding gene by Campbell recombination.
  • a DNA se ⁇ quence encoding the same inactive marker gene should be inte ⁇ grated in the chromosome as before by homologous recombination, resulting in a host which only contains one active marker gene.
  • the host may also be mutated so as to be deficient in the mar ⁇ ker gene product, and may subsequently be retransformed once more with a DNA sequence encoding said marker gene provided with a promoter for its expression. In this way, selection for only one phenotype is required.
  • Suitable selection markers may be derived from the A. nidulans or A. niger arqB gene, the A. nidulans trpC gene, the A. nidu ⁇ lans amdS gene, the Neurospora crassa pyr4 or DHFR genes, or the A. niger or A. oryzae niaD or pyrG genes, or from genes whose products confer antibiotic resistance to the host, e.g. hygromycin, ornithine or phleomycin resistance.
  • Preferred selection markers for use in the present invention are derived from the A. nidulans or A. niger amdS or arqB genes.
  • Wild-type A . oryzae strains are usually ArgB + (which means that the arqB gene is expressed in A. oryzae) .
  • an ArgB " mutant strain of A. oryzae which does not express the AraB " gene
  • the amdS gene may be used as the selection marker in wild type A. oryzae strains which do not express this gene in sufficient amounts to permit growth under selective conditions.
  • the first DNA sequence coding for a selection marker may be derived from the A . ni ⁇ dulans or A. niger amdS gene
  • the second DNA sequence coding for the selection marker may be derived from the A. nidulans, niger or oryzae niaD gene.
  • the first and second DNA constructs may additionally comprise a signal sequence per ⁇ mitting secretion of the expressed protein into the culture me ⁇ dium, by ensuring efficient direction of the expressed product into the secretory pathway of the host cell.
  • the signal sequen ⁇ ce may code for a naturally occurring signal peptide or a functional part thereof or it may be a synthetic sequence pro ⁇ viding secretion of the protein from the cell.
  • the signal sequence may be derived from a gene coding for a secreted pro ⁇ tein derived from any source.
  • the signal sequence may be derived from a gene encoding an Aspergillus sp. amylase, protease, or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease, a gene encoding a Humicola insolens cellulase, or a gene encoding a Coprinus sp. peroxidase.
  • the signal sequence is preferably derived from the gene en ⁇ coding A. oryzae TAKA amylase, A. niger neutral ⁇ -amylase, A. niger acid-stable ⁇ -amylase, A. niger glucoamylase, or a Co ⁇ prinus macrorhizu ⁇ or cinereus peroxidase.
  • the protein produced by the present process is preferably an enzyme.
  • suitable enzymes are Upa ⁇ ses, cellulases, oxidoreductases (e.g. a peroxidase) , proteases or amylases.
  • the medium used to culture the transformed host cells may be any conventional medium suitable for growing filamentous fungi.
  • the transformants are usually stable and may be cultured in the absence of selection pressure.
  • the mature protein secreted from the host cells may convenient ⁇ ly be recovered from the culture medium by well-known procedu ⁇ res including separating the cells from the medium by centrifu- gation or filtration, and precipitating proteinaceous compo ⁇ nents of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • a salt such as ammonium sulphate
  • the first DNA construct may also comprise a DNA fragment homologous to part of the genome of the host filamentous fungus
  • the second DNA construct may comprise a DNA fragment homologous to part of the genome of the host filamentous fungus
  • both the first and the second DNA constructs may comprise a DNA fragment homologous to part of the genome of the host filamentous fun ⁇ gus.
  • both the first and second DNA constructs in ⁇ clude such homologous DNA fragments, in which case the homo ⁇ logous DNA fragment present on the first DNA construct may differ from the homologous DNA fragment present on the second DNA construct so as to ensure integration of the vectors at different sites on the host genome, thus reducing the risk of loss of the DNA sequence encoding the protein present in either DNA construct.
  • the homologous DNA fragment should preferably have a length of at least 1 kb.
  • said DNA fragments may be smaller, such as a length of 0.2, 0.4, 0.6, or 0.8 kb.
  • the homologous DNA fragment is preferably a random fragment of the genome of the host filamentous fungus. It is, however, pre ⁇ ferable to select homologous DNA fragments which do not cause the DNA construct to be introduced into the host chromosome at a site where it would interfere with vital functions of the cell.
  • the filamentous fungus may be retransformed more than once, in which case the host cell is each time transformed with a DNA sequence coding for a selection marker not expressed by the host cell.
  • this may be accomplished either by inserting a gene encoding a different selection marker on each subsequent vector, by inactivating the marker gene introduced into the host cell by mutation after each (re)transformation and reusing the same selection marker.
  • This inactivation can be obtained in different ways, such as through deletion or mutation.
  • telomeres may also be possible to estab- lish a pool of vectors for (re)transformation of a host cell by introducing on separate vectors carrying a DNA construct as de ⁇ scribed above DNA fragments homologous to different parts of the host chromosome.
  • Suitable DNA fragments may, for instance, be prepared by partial restriction of the host DNA.
  • the isolated pools of DNA fragments restricted with enzyme A are ligated in a diluted DNA solution, so that the ends of the DNA fragments ligate with themselves creating circularised fragments.
  • the circularised fragments have to be cut with one or two restriction enzymes, B or B plus C which are different from the first used restriction enzyme A.
  • DNA fragments are dephosphorylated and ligated with other DNA fragments that are restricted with the same enzymes B or B and C and code for the gene of interest, the selection gene, a vector, or combinations hereof.
  • This DNA construct may make up a vector which is replicable in a selected host, such as a Bacillus, Escherichia or yeast strain, for propagation of plasmids.
  • the vector may be a derivative of an E. coli vector as pBR322 and pUC19 or of the E. coli Lambda and M13 phage
  • the vector may also be derivatives of Bacillus phages and Bacillus vectors such as pU B110, pE194, pC194, pAMbetal and pVWOl.
  • the vector may also be a derivative of a yeast vector, such as a 2 micron ( ⁇ m) derived plasmid vector.
  • E. coli plasmids Propagation of E. coli plasmids was done in strain MT 172 (E. coli K12, MC 1000, hsdR(r ⁇ -m ⁇ +) ) .
  • Media for liquid cultures were either YP, 2% maltose medium according to Maniatis et al . or of the minimal MdU-2 medium containing 4.3% maltose, 111 mM urea, 4 mM MgS0 4 , 5.7 mM K 2 S0 4 , 17 mM NaCl, 7.3 mM NaH z P0 4 and trace elements. pH was adjusted to 5.0.
  • the chlorate re ⁇ sistant mutants with the ability to grow on nitrite and hypo- xanthine and with no or only weak growth on nitrate were tested for complementation by the niaD encoding plasmid pStal4 isola- ted by S.E. Unkles et al . 1989.
  • the frequency of niaD mutants were 1 per 10 7 viable conidia.
  • Irradiated co ⁇ nidia were spread on Cove minimal agarose plates containing 1% glucose, 10 mM urea, 0.030 mg/ml D-methionine, 0.055 mM sul ⁇ phate and from 10 to 250 mM selenate.
  • the frequency of selenate resistant mutants were 1 per 10 4 .
  • the selenate resistant mu ⁇ tants, unable to grow on 1 mM sulphate containing Cove minimal agar plates, were tested for complementation by the sC gene encoding plasmid pAndyl described in Bailey, A. M.
  • Protoplasts were prepared and transformed according to T. Chris- tensen et a ⁇ . (1988) . 15-50 ⁇ g of DNA were incubated with 5xl0 7 protoplasts per transformation. Transformants were selected on Cove minimal agar plates containing 1 M sucrose as the carbon source and osmotic stabiliser, plus the selective nitrogen source such as 10 mM acetamide or 10 mM nitrate for the selec- tion of respectively acetamidase and nitrate reductase trans ⁇ formants.
  • the ATP sulphurulase transformants were selected on the same selection plates except that agarose was used instead of agar. Analysis of the secretion of lipase of the isolated transfor ⁇ mants
  • the transformants were purified twice through conidial spores, cultured in shake flasks containing MdU-2 minimal medium, where- after the supernatants were analyzed for the secreted amount of lipase and amylase by rocket immune electrophoresis (RIE) .
  • RIE rocket immune electrophoresis
  • the lipase activity was determined by an esterase assay, the enzy ⁇ matic cleavage of p-nitro phenyl butyrate or by the enzymatic cleavage of triglycerides.
  • Chromosomal A . oryzae DNA was prepared from 3 days old mycelial by the method of T. Christensen et al . (1988). Southern and Dot blot analysis was done according to the methods of Maniatis et al . 1982 as all the other DNA methods except when specified in methods.
  • the restriction enzymes were from the company Biolabs, Inc. and were used in accordance with the manufacturer's in ⁇ structions.
  • the Humicola lanigunosa lipase gene was isolated as a cDNA clo- ned by E. Boel et al. (1988) .
  • the lipase plasmid p960 is descri ⁇ bed in EP patent application no. 88307980.8 and shown in Fig. 1, and was used to express the lipase gene.
  • This pUC derived plasmid, p960 encodes the promoter of the TAKA amylase, the lipase gene with its signal peptide and polyadenylation site and the terminator of amyloglucosidase of A. niger.
  • p960 Two derivatives of p960 were constructed by extending the TAKA promoter region of p960 with 50 base pairs (pSRe424) or 400 base pairs (pSRe423) of the upstream region the TAKA promoter.
  • the plasmid constructions are shown in Figs. 2 and 3.
  • pSRe427 and pSRe428 Two derivatives of p960 were constructed by extending the TAKA promoter region of p960 with 50 base pairs (pSRe424) or 400 base pairs (pSRe423) of the upstream region the TAKA promoter.
  • the plasmid constructions are shown in Figs. 2 and 3.
  • pSRe427 and pSRe428 Two derivatives of p960 were constructed by extending the TAKA promoter region of p960 with 50 base pairs (pSRe424) or 400 base pairs (pSRe423) of the upstream region the TAKA promoter.
  • the triose phosphate isomerase promoter is a strong promoter and active during exponential growth, while the TAKA promoter is activated, when the cells are entering the stationary pha- se.
  • the Triose phosphate isomerase gene of A. oryzae was iso ⁇ lated by M. Trier Hansen, Novo Nordisk A/S as a genomic clone.
  • a BamHl site was inserted in front of the translation initia ⁇ tion site of the triose phosphate isomerase gene, TPI and the TPI promoter fragment EcoRl-BamHl was inserted in p960 deleted of the TAKA promoter fragment EcoRl-BamHl.
  • the construction is shown in Fig. 7.
  • NiaD nitrate reductase encoding plasmid pStal4 isolated by S. E. Unkless et al . (1989) was used as the selection plasmid for the isolation of nitrate reductase, NiaD, transformants. Also the NiaD plasmids, pSRe418 and pSRe419 encoding the 4 kb Bgl2 frag ⁇ ment of pStal4, were used as selection plasmids; these two plas ⁇ mids are different from the other NiaD plasmids in that they lack the NiaD promoter. The NiaD plasmids, pStal4, pSRe418 and pSRe419 are shown in Figs. 8, 9, and 10.
  • ATP sulphurulase (sC) gene encoding plasmid pANDYl was used as the selection plasmid for the isolation of ATP sulphurulase transformants.
  • sC gene plasmid pMEM1231, encoding the 3.1 kb Bgl2-EcoRV sC gene fragment of pANDYl in pUC19 was used.
  • the construction of plasmid pMEM1231 is shown in Fig. 11.
  • This plasmid was constructed from plasmid pToC68, described in EP 238 023, wherein a Sail 0.9 kb fragment from a genomic Sau3A clone coding for the alkaline protease from A. oryzae was in ⁇ serted.
  • a cDNA sequence of this protease has been published by Tatsumi et al . Mol . Gen . Genet . 219 (1989) 33-38.
  • the A. oryzae wild-type strain A1560 was transformed with the lipase plasmid p960 and the AmdS plasmid p35R2 and transfor- mants were isolated and analyzed for lipase secretion as de ⁇ scribed in the experimental protocol.
  • a lipase transformant secreting high amounts of lipase was chosen for a second transformation.
  • a spontaneous NiaD mutant of this transformant was retransformed in several ways.
  • HLL Humicola lanuginosa lipase
  • G G G G G G G one of which is complementary to HLL mRNA in the region coding for Phe-Asn-Gln-Phe-Asn was synthesized on an Applied Biosy- stems, Inc. DNA synthesizer and purified by polyacrylamide gel electrophoresis. Approximately 10,000 E. coli recombinants from the Humicola lanuginosa cDNA library were transferred to What ⁇ man 540 paper filters. The colonies were lysed and immobilized as described by Gergen et al . (Nucleic Acids Res. 7, 2115-2135, 1979) . The filters were hybridized with the 32 P-labelled HLL- specific pentadecamer mixture as described by Boel et al . (EMBO J.
  • DNA sequences containing unique restriction sites were added to the 5' and the 3' ends of the cDNA as follows. pHLL 702,3 was di ⁇ gested with Sau961 which digests HLL cDNA in the 3' untransla ⁇ ted region and the resulting ends were filled in with E. coli DNA polymerase (Klenow fragment) and the four dNTPs. This DNA was subsequentially digested with Sacl which cuts the HLL cDNA once just 3' to the initiating methionine codon. The resulting 0.9 kb HLL cDNA fragment was purified by agarose gel electropho- resis, electroeluted and made ready for ligation reactions.
  • a 5' adaptor two oligonucleotides, 927 and 928, were synthesi- zed.
  • the sequence of the adaptor is shown in Fig. 13.
  • This adap ⁇ tor was designed to add a Hindlll and a BamHI site just 5' to the initiating Met codon of HLL cDNA.
  • the two oligos were kinased with ATP and T 4 polynucleotide kinase, annealed to each other and ligated to the purified 0.9 kb HLL cDNA sequence in a pUC19 vector that had been digested with Hindlll and Hindi and purified on a 0.7% agarose gel.
  • the resulting plasmid pHLL carried the HLL cDNA as a portable 0.9 kb BamHI fragment.
  • the construction of pHLL is shown in Fig. 13.
  • HLL cDNA is under transcriptional control of the TAKA promoter from Aspergillus oryzae and the AMG terminator from Aspergillus niger.
  • the con- struction of p960 is shown in Fig. 14.
  • p775 contains the TAKA promoter and AMG terminator and has a unique BamHI site as a cloning site.
  • HLL is synthesized as a 291 amino acid residue long precursor with a signal peptide of 17 residues, and a short propeptide of 5 residues.
  • the NiaD selection plasmid was pStal4.
  • Fig. 15 In Table 1 and in Fig. 15 is shown the lipase secretion of an isolated lipase retransformants and of the parent strain after culturing the strains in shake bottles.
  • the numbers at the top of Fig. 15 indicate the "Example” re ⁇ ferred to, and the "23" at the bottom identifies the parent strain or grand parent strain HL23.
  • the yield is given in arbi ⁇ trary units.
  • the lipase transformant HL23NiaD was retransformed with the li ⁇ pase plasmids, pSRe424 and pSRe423 coding for a larger fragment of the TAKA promoter.
  • the selection plasmids were pStal4, pSRe418 or pSRe419.
  • Fig. 15 and Table 1 show the shake bottles fermentations of some of these retransformants.
  • the lipase se- cretion was increased approximately twofold.
  • the lipase transformant HL23NiaD was retransformed with the pACYC177 derived lipase plasmids pSRe426, pSRe427 and pSRe428 together with the pUC derived selection plasmids pStal4, pSRe418, and pSRe419.
  • Fig. 15 and Table 1 show that the lipase secretion of some of these retransformants has been increased approximately twofold.
  • the lipase transformant HL23NiaD was retransformed with the li ⁇ pase plasmid pSRe433, which codes for a TPI driven expression of the lipase.
  • the NiaD selection plasmid was pSRe419.
  • Fig. 15 and Table 1 is seen that the lipase secretion of one of the retransformants increased by a factor of about two.
  • sC mutant of one of the NiaD retransformants, SRe37g, secre ⁇ ting lipase in high amount was isolated and this sC mutant was once more retransformed, but with the lipase plasmid pSRe424 and the sC coding selection plasmid pMEM1231 shown in Fig. 11.
  • Fig. 16 is shown the lipase secretion normalised to the TAKA amylase secretion of some of the isolated sC retransformants (C) and of the parent (B) and the grandparent (A) strain.
  • the ratio of lipase secretion normalised to TAKA amylase secretion has increased a factor of approximately 6 after two rounds of retransformations. Table 1.
  • the deletion of the structural part of the TAKA amylase gene may be explained by a recombination between the TAKA promoter on the chromosome and the plasmid, and between the TAKA termi ⁇ nator on the chromosome and the glucoamylase terminator on the plasmid.
  • the simultaneous deletion of two out of the three TAKA amylase genes located on different chromosomes may indicate that recom- binogenic sequences or direct repats are present in the TAKA promoter and in the glucoamylase terminator.
  • chromosomal genes e.g. the alkaline protease and retransformation with protease sensitive genes, e.a. Chymosin
  • the wild-type strain A1560 was transformed with the plasmid pSRe408, coding for the TAKA amylase promoter, the internal 900 base pair Sall-Kpnl-Sall fragment of the A ⁇ pergillu ⁇ oryzae al ⁇ kaline protease gene and the A. niger amyloglucosidase termina ⁇ tor.
  • the construction of pSRe408 is shown in Fig. 12. Trans- formants were tested for secretion of the alkaline protease and one turned out to have less protease than detectible.
  • a southern analysis of the transformant and the parent strain showed an insertion having the size of the plasmid pSRe408.
  • a spontaneous NiaD mutant of this alkaline protease negative strain was retransformed with several genes as for example Chy- mosin encoded by the plasmid pToC56 described in EP Bl 0 238 023.
  • the chymosin secretion of transformants of the alkaline protease negative strain and of wild-type strain were compared after SDS gel and Western Blot analysis.
  • the highest secreting chymosin transformant of the alkaline protease negative strain secreted 2-3 fold more chymosin than the chymosin transformants of the wild type A1560.

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Abstract

L'invention concerne un procédé de production d'une protéine dans un champignon filamenteux permettant d'améliorer les rendements. Ledit procédé comprend: (a) la transformation d'un champignon filamenteux approprié au moyen d'un premier produit de synthèse d'ADN recombinant comprenant une séquence d'ADN codant ladite protéine, ainsi qu'au moyen d'une première séquence d'ADN codant pour un marqueur approprié de sélection de transformateurs, (b) la retransformation dudit transformateur au moyen d'un deuxième produit de synthèse d'ADN recombinant comprenant une séquence d'ADN codant ladite protéine ou une autre protéine, ainsi qu'au moyen d'une deuxième séquence d'ADN codant pour un marqueur approprié de sélection de transformateurs, (c) la culture du champignon filamenteux retransformé dans un milieu de culture approprié dans des conditions permettant de produire la protéine. Le premier ou le deuxième produit de synthèse d'ADN et/ou la première ou la deuxième séquence d'ADN peuvent comprendre une extension de l'homologue d'ADN vers l'hôte. L'invention est mise en application dans la production de différentes protéines au moyen de différents champignons filamenteux jouant le rôle d'hôtes.
PCT/DK1994/000488 1993-12-23 1994-12-22 Retransformation de champignons filamenteux WO1995017513A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/DK1994/000488 WO1995017513A1 (fr) 1993-12-23 1994-12-22 Retransformation de champignons filamenteux
AU12726/95A AU1272695A (en) 1993-12-23 1994-12-22 Retransformation of filamentous fungi

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DK1437/93 1993-12-23
DK173793 1993-12-23
PCT/DK1994/000488 WO1995017513A1 (fr) 1993-12-23 1994-12-22 Retransformation de champignons filamenteux

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5821350A (en) * 1995-11-01 1998-10-13 Nexia Biotechnologies, Inc. Aspergillus niger beta-galactosidase gene
WO1998046772A3 (fr) * 1997-04-11 1999-02-25 Gist Brocades Bv Transformation genetique comme outil pour la construction de champignons filamenteux industriels de recombinaison
EP1137808A1 (fr) * 1998-12-03 2001-10-04 Sunol Molecular Corporation Methodes de fabrication de cellules recombinees
WO2009046978A1 (fr) * 2007-10-12 2009-04-16 F. Hoffmann-La Roche Ag Expression de protéine à partir d'acides nucléiques multiples
WO2009076709A1 (fr) * 2007-12-19 2009-06-25 Applimex Systems Pty Ltd Plateforme multi-promoteur pour production de protéines
WO2018162517A1 (fr) 2017-03-10 2018-09-13 F. Hoffmann-La Roche Ag Procédé de production d'anticorps multispécifiques
US10227523B2 (en) 2013-11-26 2019-03-12 Advanced Enzyme Systems, Llc Glycosyl hydrolase enzymes in high temperature industrial processes
CN113061538A (zh) * 2021-03-23 2021-07-02 江南大学 一种构巢曲霉自诱导型表达系统及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989010959A1 (fr) * 1988-05-06 1989-11-16 Codon Super-transformants pour taux eleves d'expression dans des cellules eucaryotiques
EP0357127A1 (fr) * 1988-08-16 1990-03-07 Gist-Brocades N.V. Remplacement de gènes comme règle de construction de souches d'aspergillus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989010959A1 (fr) * 1988-05-06 1989-11-16 Codon Super-transformants pour taux eleves d'expression dans des cellules eucaryotiques
EP0357127A1 (fr) * 1988-08-16 1990-03-07 Gist-Brocades N.V. Remplacement de gènes comme règle de construction de souches d'aspergillus

Non-Patent Citations (1)

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Title
DIALOG INFORMATION SERVICE, File 155, Medline, Dialog Accession No. 08002363, Medline Accession No. 92140363, FARMAN M.L. et al., "Transformation Frequencies Are Enhanced and Vector DNA is Targeted During Retransformation of Leptosphaeria Maculans, a Fungal Plant Pathogen"; & MOL. GEN. GENET. (GERMANY), Jan. 1992, 231 (2), pages *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5821350A (en) * 1995-11-01 1998-10-13 Nexia Biotechnologies, Inc. Aspergillus niger beta-galactosidase gene
WO1998046772A3 (fr) * 1997-04-11 1999-02-25 Gist Brocades Bv Transformation genetique comme outil pour la construction de champignons filamenteux industriels de recombinaison
JP2001518798A (ja) * 1997-04-11 2001-10-16 デーエスエム ナムローゼ フェンノートシャップ 工業的組み換え生物を溝築するための手段としての遺伝子変換
US6432672B1 (en) 1997-04-11 2002-08-13 Gerardus Cornelis Maria Selten Gene conversion as a tool for the construction of recombinant industrial filamentous fungi
EP2298913A1 (fr) * 1997-04-11 2011-03-23 DSM IP Assets B.V. Conversion de gène en tant qu'outil pour la construction de champignon filamenteux industriel recombinant
EP1137808A1 (fr) * 1998-12-03 2001-10-04 Sunol Molecular Corporation Methodes de fabrication de cellules recombinees
EP1137808A4 (fr) * 1998-12-03 2002-10-02 Sunol Molecular Corp Methodes de fabrication de cellules recombinees
JP2010540583A (ja) * 2007-10-12 2010-12-24 エフ．ホフマン−ラ ロシュ アーゲー 複数の核酸からのタンパク質発現
WO2009046978A1 (fr) * 2007-10-12 2009-04-16 F. Hoffmann-La Roche Ag Expression de protéine à partir d'acides nucléiques multiples
EP2592148A1 (fr) * 2007-10-12 2013-05-15 F. Hoffmann-La Roche AG Expression protéinique pour plusieurs acides nucléiques
EP2592147A1 (fr) * 2007-10-12 2013-05-15 F. Hoffmann-La Roche AG Expression protéinique pour plusieurs acides nucléiques
AU2008309934B2 (en) * 2007-10-12 2014-03-06 F. Hoffmann-La Roche Ag Protein expression from multiple nucleic acids
US8771988B2 (en) 2007-10-12 2014-07-08 Hoffmann-La Roche Inc. Protein expression from multiple nucleic acids
US9428766B2 (en) 2007-10-12 2016-08-30 Hoffmann-La Roche Inc. Protein expression from multiple nucleic acids
WO2009076709A1 (fr) * 2007-12-19 2009-06-25 Applimex Systems Pty Ltd Plateforme multi-promoteur pour production de protéines
US10227523B2 (en) 2013-11-26 2019-03-12 Advanced Enzyme Systems, Llc Glycosyl hydrolase enzymes in high temperature industrial processes
US11401457B2 (en) 2013-11-26 2022-08-02 Advanced Enzyme Systems, Llc Glycosyl hydrolase enzymes in high temperature industrial processes
WO2018162517A1 (fr) 2017-03-10 2018-09-13 F. Hoffmann-La Roche Ag Procédé de production d'anticorps multispécifiques
CN113061538A (zh) * 2021-03-23 2021-07-02 江南大学 一种构巢曲霉自诱导型表达系统及其应用

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