WO1999051756A2 - Promoteur et produits de synthese destines a l'expression de proteines de recombinaison dans des champignons filamenteux - Google Patents

Promoteur et produits de synthese destines a l'expression de proteines de recombinaison dans des champignons filamenteux Download PDF

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WO1999051756A2
WO1999051756A2 PCT/EP1999/002243 EP9902243W WO9951756A2 WO 1999051756 A2 WO1999051756 A2 WO 1999051756A2 EP 9902243 W EP9902243 W EP 9902243W WO 9951756 A2 WO9951756 A2 WO 9951756A2
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aspergillus
dna
promoter
fungus
protein
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PCT/EP1999/002243
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WO1999051756A3 (fr
WO1999051756A9 (fr
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Heidi Sisniega Barroso
Francisco Javier Casqueiro Blanco
Francisco José MORALEJO LORENZO
Juan Francisco Martin Martin
Santiago Gutierrez Martin
María José HIJARRUBIA IBRAHIM
José Luis DEL RIO PERICACHO
Ignacio Faus Santasusana
Rosa Elena Cardoza Silva
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Urquima, S.A.
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Priority to JP2000542467A priority Critical patent/JP2002510497A/ja
Priority to AU42575/99A priority patent/AU4257599A/en
Priority to EP99944133A priority patent/EP1084262A1/fr
Priority to CA002325571A priority patent/CA2325571A1/fr
Publication of WO1999051756A2 publication Critical patent/WO1999051756A2/fr
Publication of WO1999051756A9 publication Critical patent/WO1999051756A9/fr
Publication of WO1999051756A3 publication Critical patent/WO1999051756A3/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C07K14/43—Sweetening agents, e.g. thaumatin, monellin
    • 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
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • This invention relates to improvements in the expression of proteins, particularly of fusion proteins, by recombinant DNA technology, using filamentous fungi as the host. These improvements refer mainly to the use of a new promoter and new DNA constructions containing it.
  • Filamentous fungi are known to produce in nature a wide range of homologous proteins in large amounts. For this reason, filamentous fungi have been regarded as attractive hosts for the expression of recombinant proteins. For instance, Aspergillus awamori has been used for the production of recombinant proteins such as bovine chymosin and human lactoferrin.
  • thaumatins are proteins with a very sweet taste and the ability to increase the palatability of food. In industry they are currently extracted from the arils of the fruit of the plant Thaumatoccocus daniellii Benth (M.Witty, J.D. Higginbotham, Thaumatin ,1994, CRC Press, Boca Raton, Florida). Thaumatins can be isolated from these arils in at least five different forms (I, II, III, b and c) , thaumatins I and II being the most abundant types in the arils.
  • the present invention provides a new expression system that makes use of the promoter from the glutamate dehydrogenase (gdh) gene from filamentous fungi of the genus Aspergillus, particularly from Aspergillus awamori .
  • gdh glutamate dehydrogenase
  • One of the objects of the present invention is a new promoter for the expression of recombinant proteins in filamentous fungi that comprises a nucleotide sequence - or a complementary strand thereof - selected from the group consisting of: (a) the nucleotide sequence numbered 1-740 in the enclosed SEQ ID No. 1; (b) a nucleotide sequence that is analogous to that defined in (a) ; and (c) a nucleotide sequence that hybridizes under stringent conditions to that defined in (a) .
  • the promoter comprising the sequence defined in (a), i.e. the nucleotide sequence numbered as 1-740 in SEQ ID No. 1, which corresponds to the gdhA promoter of the glutamate dehydrogenase A gene from Aspergillus awamori .
  • glutamate dehydrogenase A disclosed herein is the first glutamate dehydrogenase identified and described in the filamentous fungus Aspergillus awamori , there may exist other glutamate dehydrogenases in Aspergillus awamori .
  • Aspergillus awamori can be used as a probe for the identification and isolation of other homologous promoters/genes of glutamate dehydrogenases in Aspergillus awamori as well as in other organisms, preferably in filamentous fungi, more preferably in fungi of the genus Aspergillus , still more preferably in Aspergillus awamori and Aspergillus niger , and specially in Aspergillus awamori , following the teachings of the present invention.
  • the present invention is not limited to the specific gdhA promoter from Aspergillus awamori disclosed herein but also relates to the promoter of any glutamate dehydrogenase gene from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans .
  • Aspergilli include Aspergillus awamori , Aspergillus niger, Aspergillus oryzae and Aspergillus soiae
  • the invention relates to a promoter of a glutamate dehydrogenase gene from Aspergillus awamori or Aspergillus niger.
  • the invention relates to a promoter of a glutamate dehydrogenase gene from Aspergillus awamori .
  • the use of the novel nucleotide sequence shown in SEQ ID No. 1 or a portion thereof as probe is also a object of the present invention.
  • the term "a portion thereof” denotes any part of the nucleotide sequence of SEQ ID No .1 that is functional as a probe.
  • Another object of the present invention is a new DNA sequence, purified and isolated, that encodes a glutamate dehydrogenase protein and that comprises a nucleotide sequence - or a complementary strand thereof - selected from the group consisting of: (a) the nucleotide sequence numbered 741-2245 in the enclosed SEQ ID No. 1; (b) a nucleotide sequence that is analogous to that defined in
  • nucleotide sequence encoding a glutamate dehydrogenase is the sequence defined in (a), i.e. the nucleotide sequence numbered as 741-2245 in SEQ ID No. 1.
  • the present invention is not limited, however, to the specific gdhA gene from Aspergillus awamori disclosed herein but also relates to any glutamate dehydrogenase gene from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans .
  • the invention relates to the DNA sequences encoding glutamate dehydrogenase from Aspergillus awamori or Aspergillus niger . In a more preferred embodiment, the invention relates to the DNA sequences encoding glutamate dehydrogenase from Aspergillus awamori .
  • Another object of the invention are the novel proteins encoded by any of the DNA sequences defined above .
  • this protein has the amino acid sequence shown in the enclosed SEQ ID No. 2.
  • glutamate dehydrogenase from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans , more preferably a glutamate dehydrogenase from Aspergillus awamori or Aspergillus niger. and still more preferably a glutamate dehydrogenase from Aspergillus awamori .
  • the invention further relates to the use of the glutamate dehydrogenase promoters above described for the expression of recombinant proteins in filamentous fungi .
  • glutamate dehydrogenases from several microorganisms are already known and their genes have been disclosed, in particular the glutamate dehydrogenase A (gdhA) gene from Aspergillus nidulans (A.R. Hawkins et al . , Mol. Gen. Genet. 1989, 218(1), pp. 105-111).
  • gdhA glutamate dehydrogenase A
  • the glutamate dehydrogenase promoter from Aspergillus awamori has proven to be very strong in promoting transcription of heterologous genes. Therefore, this promoter as well as related ghd promoters from Aspergilli are expected to drive high-level transcription of genes and thus are expected to be of use in the expression of recombinant proteins in filamentous fungi .
  • the gdh promoter is from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans .
  • desired proteins include, but are not limited to, enzymes, hormones, cytokines, growth factors, structural proteins, plasma proteins and others.
  • a non- limiting list of examples of proteins that can be expressed includes human proteins such as interferons, interleukins, tissue plasminogen activator, serum albumin, growth hormone, and growth factors .
  • Other proteins can be of non-human origin such as lipases of both fungal and non-fungal origin, proteases, thaumatins, bovine chymosin, etc.
  • Polypeptides which can be of human and non-human origin, include calcitonin, glucagon, insulin, nerve growth factor, epidermal growth factor, the anticoagulant Hirudin and analogs such as R3-hirulog.
  • a further object of the present invention are the DNA constructions that comprise: a) a promoter from a glutamate dehydrogenase gene from a fungus of the genus Aspergillus ; b) a DNA sequence encoding a protein normally expressed from a filamentous fungus or a portion thereof; c) a DNA sequence encoding a cleavable linker peptide; and d) a DNA sequence encoding a desired protein.
  • the promoter under a) comprises a gdh promoter from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans , more preferably it is from Aspergillus awamori or Aspergillus niger, still more preferably it is from Aspergillus awamori , yet more preferably it comprises any of the new promoters described above, and more particularly it comprises the nucleotide sequence 1-740 in SEQ ID No . 1.
  • the DNA sequence under b) encodes a protein normally expressed from a filamentous fungus or a portion thereof that is functional, i.e.
  • Such protein under b) include glucoamylase, oc- amylase and aspartyl proteases from Aspergillus awamori , Aspergillus niger, Aspergillus oryzae and Aspergillus soiae, cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase III from Trichoderma species, glucoamylase from Neurospora and Humicola species, the protein B2 from Acremonium chrysogenum and a glutamate dehydrogenase from a filamentous fungi.
  • the DNA sequence under b) encodes a protein or portion thereof selected from the group consisting of: i) glucoamylase from Aspergillus awamori , Aspergillus niger, Aspergillus orvzae or Aspergillus soiae; ii) B2 from Acremonium chrvsogenum; and iii) a glutamate dehydrogenase from a filamentous fungi; more preferably, the DNA sequence under b) encodes a protein or portion thereof selected from the group consisting of: i) glucoamylase from Aspergillus awamori , Aspergillus niger, Aspergillus orvzae or Aspergillus soiae; ii) B2 from Acremonium chrvsogenum,- and iii) a glutamate dehydrogenase from Aspergillus awamori or Aspergillus
  • the DNA sequence under c) encodes a cleavable linker peptide; as used herein, cleavable linker peptide means a peptide sequence which under certain circumstances allows the separation of the sequences bordering the cleavable linker, for example sequences that are recognized and cleaved by a protease or cleaved after exposure to certain chemicals .
  • the DNA sequence under c) contains a KEX2 processing sequence.
  • the desired protein under d) can be in principle any recombinant protein.
  • the DNA sequence under d) encodes thaumatin; particularly preferred constructions for the preparation of thaumatin include those wherein the DNA sequence encoding thaumatin under d) is the synthetic gene encoding thaumatin II coming from plasmid pThlX, which is disclosed in EP 684312.
  • the gdh promoters when expressing a desired protein, it is also possible to use a gdh promoter to express directly a desired protein. Therefore, it is a further object of the present invention the new DNA constructions that comprise a gdh promoter from a fungus of the genus Aspergillus operatively linked to a DNA sequence encoding the protein that it is desired to express.
  • the gdh promoter is from a fungus of the genus Aspergillus with the proviso that it is not from Aspergillus nidulans , more preferably it is from Aspergillus awamori or Aspergillus niger, still more preferably it is from Aspergillus awamori, yet more preferably it is one of the new promoters described above, and more particularly it comprises the nucleotide sequence 1-740 in SEQ ID No. 1.
  • all the above DNA constuctions may additionally contain other elements which include, but are not limited to, signal sequences, termination sequences, polyadenylation sequences, selection sequences, sequences that allow the replication of the DNA, etc.
  • signal sequences termination sequences
  • polyadenylation sequences selection sequences
  • sequences that allow the replication of the DNA etc.
  • signal sequences from glucoamylase, -amylase and aspartyl proteases from Aspergillus awamori , Aspergillus niger, Aspergillus orvzae and Aspergillus soiae signal sequences from cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase III from Trichoderma species, signal sequences from glucoamylase from Neurospora and Humicola species and the signal sequence from the protein B2 from Acremonium chrvsogenum.
  • a termination sequence is a nucleotide sequence which is recognized- by the expression host to terminate transcription. Examples include the terminators from the A. nidulans trpC gene, the A ⁇ awamori , A. niger, A. orvzae or A. soiae glucoamylase gene, the A ⁇ awamori , A. niger , A.
  • a selection sequence is a sequence useful as selection marker to allow the selection of transformed host cells.
  • any known selection marker for the filamentous fungus that is intended to be used as host can be employed. Examples of such selection markers include genes confering resistance to a drug such as an antibiotic (e.g. hygromycin or phleomycin) as well as auxotrophic markers such as argB, trpC, niaD and pyrG.
  • a polyadenylation sequence is a nucleotide sequence which when transcribed is recognized by the expression host to add polyadenosine residues to transcribed mKNA.
  • Examples include the polyadenylation sequences from the A__ nidulans trpC gene, the ⁇ awamori , A. niger, A. orvzae or A ⁇ . soiae glucoamylase genes and the Mucor miehei carboxyl protease gene.
  • the present invention also relates to the filamentous fungus cultures capable of producing a recombinant protein that have been transformed with plasmids that contain any of the DNA constructions mentioned above.
  • filamentous fungi that may be used as expression hosts include the following genera: Aspergillus , Trichoderma, Neurospora , Penicillium, Acremonium, Cephalosporium, Achlva , Phanerochaete , Podospora, Endothia , Mucor, Fusarium, Humicola, Cochliobolus , Rhizopus and Pyricularia.
  • the filamentous fungus is selected from a fungus of the genus Aspergillus , and more preferably it is selected from Aspergillus awamori, Aspergillus niger , Aspergillus oryzae, Aspergillus nidulans or Aspergillus soiae.
  • the recombinant protein produced is thaumatin.
  • a further object of the present invention is to provide a process for producing a recombinant protein in a filamentous fungus that comprises the following steps: a) preparation of an expression plasmid that contains a DNA construction as defined above; b) transformation of a strain of filamentous fungus with said expression plasmid; c) culture of the transformed strain under appropriate nutrient conditions to produce the desired protein, either intracellularly, extracellularly or in both ways simultaneously; and d) depending on each case, separation and purification of the desired protein from the fermentation broth.
  • Preferred is the process wherein the recombinant protein produced is thaumatin..
  • the accompanying examples describe the identification and isolation of the glutamate dehydrogenase A gene and its promoter region from Aspergillus awamori . This was achieved using a probe from Neurospora crassa. The selection of a suitable DNA fragment from the glutamate dehydrogenase gene in Neurospora crassa to be used as a probe to get the homologous gene in Aspergillus awamori is not, however, straightforward. In this case, there were no clear homology sequences that could be detected, and therefore what was used was a 2.6 kb BamHI fragment that contained the Neurospora crassa gdh gene. This is a large fragment of DNA, and is certainly not the optimal size fragment.
  • the expression system of the present invention offers several advantages over the prior art systems. On the one hand, it allows to reach concentrations of expressed protein of about 100 mg/1, which are one order of magnitude higher than the best described (for example, using the process described in EP 684312, concentrations of about 5-10 mg/1 are attained; see I. Faus et al . , Appi . Microbiol . Biotechnol . , 1998, vol. 49, pp. 393-398) .
  • the use of the promoter of the present invention leads to higher concentrations of expressed protein. And last but not least, with the constructions of the present invention it is possible to use a more economical nitrogen source (ammonium sulfate) than the one that is commonly used (asparagine) .
  • promoter means a DNA sequence operative in a filamentous fungus capable of promoting transcription of a coding region when operatively associated therewith.
  • recombinant protein means a protein that is not expressed under standard normal conditions by the host, and that is only expressed by the host as a result of the introduction into said host of a DNA sequence that allows for the expression of said recombinant protein.
  • This recombinant protein can be fungal or non-fungal, and it can even be found in the non-recombinant host .
  • FIG. 1 parts A, B and C. Schematic representation of the steps involved in the construction of the B2KEX expression cassette.
  • Figure 4 parts A and B. Alignment of the deduced amino acid sequences of NADP-specific glutamate dehydrogenases of A. awamori, I _ nidulans (Genebank accession number P18819), N. crassa (P00369), £ _ cerevisiae (P07262), £L occidentalis (P29507), . bisporus (P54387) , S ⁇ _ tvphimurium (P15111) , E ⁇ coli (P00370) and C _ glutamicum (P31026) . Identical amino acids are shadowed. Motifs a-i with several consecutive conserved residues are overlined.
  • Figure 5 Complementation of the gdhA mutation in two strains of A ⁇ . nidulans with the gdhA gene of A ⁇ awamori .
  • Part A 1, . nidulans A686 mutant; 2, transformant A686-4; 3, transformant A686-6; 4, transformant A686-7.
  • Part B 1, A. nidulans A699 mutant; 2, transfomant A699-2; 3, transformant A699-3; and 4, transformant A699-4.
  • Figure 7 Northern blot analysis of the transcripts of the gdhA and . ⁇ - ' actin genes.
  • A hybridization with a probe internal to the gdhA gene (0.694 kb PvuII fragment).
  • B hybridization with the ⁇ -actin gene of A ⁇ nidulans as control .
  • Figure 8. Slot Blot analysis of the trancript of the A. awamori gdhA gene, during the course of a fermentation in MDFA medium with 1% glucose and 10 mM ammonium sulfate (part A) .
  • Part B relative level of the expression of the gdhA to the ⁇ -actin gene.
  • Part C NADP-dependent glutamate dehydrogenase activity in the same cultures from where the mRNAs were extracted.
  • Figure 9 Slot Blot analysis of the transcript of the A. awamori gdhA gene during the course of a fermentation in MDFA medium with different nitrogen sources (part A) .
  • the medium contained ammonium sulfate 10 mM as a control and glutamic acid, glutamine, sodium nitrite, sodium nitrate and asparagine as nitrogen source, all of them at a concentration of 10 mM.
  • the transcript of the ⁇ -actin gene was also studied for comparative purposes.
  • Part B Relative level of expression of the gdhA to the ⁇ -actin gene.
  • FIG. 10 parts A, B and C. Schematic representation of the steps involved in the construction of the GDH expression cassette.
  • FIG. 1 parts A and B. Schematic representation of the steps involved in the construction of the GPD expression cassette.
  • FIG. 12 Production (expressed as concentratin CT of secreted protein in mg/1) of thaumatin from A ⁇ _ awamori strains TB2bl-44 and TGDTh-4 in fermentor studies.
  • the medium used was MDFA supplemented with the components described below.
  • Empty squares Strain TB2bl-44; 6.0% sucrose, pH 6.2, fedbatch with asparagine.
  • Empty circles TB2 -44, 6.0% sucrose, pH 6.2, fedbatch with ammonium sulfate.
  • Filled triangles Strain TGDTh-4; 6.0 % sucrose, pH 6.2, fed-batch with ammonium sulfate.
  • This plasmid contains: (i) a sulfanilamide resistance marker
  • a DNA sequence which encodes a fusion protein comprising in his turn (a) the synthetic gene encoding thaumatin II, (b) a spacer sequence which in turn contains a KEX2 processing sequence, and (c) the complete glucoamylase gene (genomic) of Aspergillus niger; (iii) the signal sequence ("pre") and the "pro” sequence of the glucoamylase gene (glaA) of Aspergillus niger, and finally (iv) the promoter region sequence of the glucoamylase gene (glaA) of Aspergillus niger.
  • three new expression cassettes were prepared, which contained: (i) a drug resistance marker (most of the times it was a phleomycin resistance marker) ; (ii) a DNA sequence which encodes a fusion protein comprising in his turn (a) the synthetic gene of thaumatin II, (b) a spacer sequence which in turn contains a KEX2 processing sequence, and (c) a cDNA sequence that encodes most of the B2 protein (except sequences in the COOH end) from Acremonium chrvsogenum; (iii) the signal sequence of the B2 gene of Acremonium chrvsogenum and (iv) three different promoter regions.
  • Escherichia coli DH5a served as the recipient strain for high-frequency plasmid transformation.
  • E. coli WK6 was used as host for obtaining single-stranded DNA from pBluescript plasmids for sequencing purposes .
  • Protein B2 is an extracellular protease produced by the filamentous fungus Acremonium chrvsogenum. This protein is expressed and secreted in the late stages of growth of Acremonium chrvsogenum (between 120 and 144 hours after the start of growth) .
  • Plasmid pJElA (Laboratory of Prof. Juan-Francisco Martin, Universidad de Leon, Leon, Spain) contains the promoter region, leader peptide (including the signal sequence) and coding region of the B2 gene from Acremonium chrvsogenum.
  • the gene itself has 1298 base pairs and two introns . These two introns are not present in the sequence that has been subcloned in pJElA, since these subcloned sequences were obtained from a cDNA. Upstream from the ATG start point of translation there is a promoter region of 477 base pairs.
  • Acremonium chrvsogenum is grown in a defined medium which contains sucrose and glucose as carbon sources and asparagine as nitrogen source, the gene is expressed at its highest levels between 72 and 96 hours of growth.
  • Plasmid pJElA was digested sequentially with BamHI and Ncol, releasing a 560 bp fragment that was purified from a 0.8% agarose gel. This fragment contains most of the coding region of the B2 gene, but excludes the active center of the protein.
  • plasmid pJL43b J.L. Barredo, Ph.D. Thesis, Universidad de Leon, Leon, Spain
  • Plasmid p43bB2CT was digested with ⁇ col, treated with the Klenow fragment of D ⁇ A polymerase I (in order to obtain blunt ends) and then digested with Stul, yielding a fragment of 3874. bp that was also purified from a 0.8% agarose gel.
  • the single-stranded oligonucleotides ThSl and ThS2 (sequences shown below) where used, using plasmid pThlX as a template, to amplify by polymerase chain reaction (PCR) the KEX2-like and thaumatin sequences present in pThlX.
  • the first 18 nucleotides present in ThSl correspond to the KEX2-like sequence .
  • ThSl 5 ' - CGA ATG AAA AGG AAA AGG ATGGCCACCTTCGAG - 3 ' Arg Met Lys Arg Lys Arg
  • ThS2 5 ' - TTA TTA GGC GGT GGG GCA
  • a 655 bp D ⁇ A fragment was obtained by PCR using plasmid pThTX as the template and ThSl and ThS2 oligonucleotides as primers.
  • This D ⁇ A fragment was ligated with the previously obtained fragment from p43bB2CT, yielding plasmid p43bB2CTTh.
  • This plasmid (aprox. 4530 bp) contains part of the B2 protein gene fused to a KEX-2 sequence and to the synthetic gene encoding thaumatin II.
  • the transcription termination signal present in this construct is the terminator sequence from the cycl gene of Saccharomvces cerevisiae.
  • Plasmid p43bB2CTTh was digested with BamHI, treated with calf intestinal - alkaline phosphatase (CIP) and purified from a 0.8% agarose gel.
  • a 900 bp BamKE-BamHI fragment from pJElA was also isolated. Subsequent ligation of these two D ⁇ A fragments generated plasmid pB2KEX (5430 bp) .
  • the 900 bp BamHI-BamHI fragment from pJElA contains the B2 gene promoter sequence (477 bp) , the leader peptide sequence (318 bp) and 107 bp of the amino terminal sequence of the B2 gene.
  • Plasmid pB2KEX was then digested with Xbal, treated with the Klenow fragment of DNA polymerase I (in order to obtain blunt ends) and then digested with Sail, yielding a fragment of 2400 bp that was purified in a 0.8% agarose gel.
  • Plasmid pJL43b was digested with Hindlll, also treated with the Klenow fragment of DNA polymerase I, and then digested with Xhol. A fragment of 4500 bp was purified as before. Finally, the two gel-purified fragments described above were ligated, generating plasmid pB2KTh (6900 bp; Fig. 1C) .
  • Plasmid pJL43bl is a derivative of plasmid pJL43b, where the promoter that drives expression of the phleomycin resistance gene
  • This plasmid contains a cassette to express thaumatin that comprises: (i) a phleomycin resistance marker; (ii) a DNA sequence which encodes a fusion protein comprising in his turn (a) the synthetic gene of thaumatin II, (b) a spacer sequence which in turn contains a KEX2 processing sequence, and (c) a cDNA sequence that encodes most of the B2 protein (except sequences in the COOH end) from Acremonium chrysoqenum; (iii) the signal sequence of the B2 gene of Acremonium chrvsogenum and (iv) the promoter region of the B2 gene of Acremonium chrvsogenum.
  • phleomycin resistance gene (phleo) is driven by the promoter of the glyceraldehyde-3- phosphate dehydrogenase gene from Aspergillus nidulans .
  • A. awamori ATCC 22342 was used as the source of DNA and RNA.
  • . nidulans mutants A686 (gdhAl, yA2 , methH2 , galAl) and . nidulans A699 (gdhAl, biAl) (J.R. Kinghorn, J.A. Pateman, J. Gen. Microbiol . 1973, vol. 78, pp. 39-46) were obtained from the Fungal Genetics Stock Center, and were used for complementation studies with the gdhA gene from awamori .
  • Filamentous fungi were routinely maintained on solid Power sporulation medium (F. Fierro et al . , Appi. Microbiol. Biotechnol . 1996, vol. 43, pp. 597-604) at 30 2 C for 3 days.
  • A. awamori and A ⁇ _ nidulans seed cultures in CM medium containing 20 g/1 malt extract; 5 g/1 yeast extract; 5 g/1 glucose
  • CM medium containing 20 g/1 malt extract; 5 g/1 yeast extract; 5 g/1 glucose
  • a genomic library of total DNA of A ⁇ awamori ATCC 22342 was constructed in a Lambda GEM12 phage vector. Total DNA was extracted and partially digested with Sau3AI to obtain DNA fragments of between 17 and 23 kb. This DNA was purified by sucrose-gradient centrifugation, ligated to Lambda GEM12 phage arms, and packaged in vitro using a Gigapack III Gold packaging system (Stratagene) resulting in a total of 8x10 4 recombinant phages .
  • a Gigapack III Gold packaging system Stratagene
  • BamHI fragments of 1.7, 5.5 and 10 kb were subcloned in pBluescript KS+ plasmid, giving rise to plasmids pBl.7, pB5.5 and pBlO, as shown in Figure 2. They were then sequenced by generating ordered sets of deletions with the Erase-a-base system (Promega Co., Wis.) by digestion with exonuclease III from appropriate ends, followed by removal of single-stranded DNA with SI exonuclease. Sequencing of fragments of the gdhA gene was performed by the dideoxynucleotide chain termination method.
  • a 2.1 kb Xbal-Xbal fragment corresponding to the right end of plasmid pB5.5 was subcloned in pBluescript SK+ plasmid, creating plasmid pBSGh. More specifically, this 2.1 Kb Xbal- Xbal fragment was generated by digesting pB5.5 at an internal Xbal site and at a second Xbal site in the polylinker of pBSKS ⁇ (and close to the BamHI site shown in Fig. 3) .
  • ORFl contained two putative introns at positions 785-850 and 1414-1471 (following the numbering in SEQ ID No. 1) that showed lariat and 5 ' and 3 ' splicing sequences similar to those of other fungal introns (D.J. Ballance, Yeast 1986, vol. 2, pp. 229-236) .
  • the presence of the two introns was confirmed by sequencing the DNA regions corresponding to introns I and II obtained by PCR from a A,, awamori cDNA library using as primers oligonucleotides I A and I B for intron I, and II A and II B for intron II (sequences shown below) .
  • cDNA for these experiments was obtained from total RNA extracted as described above, from mycelia grown for 48 h in MDFA medium. The first and second cDNA strands were synthetized using a cDNA synthesis kit from Stratagene (La Jolla, Ca) . This cDNA was used for PCR amplification of the fragments containing the exon-exon junctions by the following program: 1 cycle at 94 2 C for 5 min, 50 2 C for 1 min, 72 2 C for 1 min followed by 30 cycles at 94 2 C for 1 min, 50 2 C for 1 min, 72 S C for 1 min and finally one cycle at 72 2 C for 8 min.
  • ORFl encodes a putative NADP-dependent glutamate dehydrogenase
  • ORFl encoded a protein of 460 amino acids (see SEQ ID No . 2) with a deduced molecular mass of 49.4 kDa and a pi value of 5.62. Comparison of the protein encoded by ORFl with other proteins in the SWISS-PROT data base showed that the encoded protein has a high homology with NADP-dependent glutamate dehydrogenases of ⁇ _ nidulans (84.7% of identical amino acids), IL. crassa (74.4% identity), Saccharomvces cerevisiae (66.5% identity) and Schwanniomvces occidentalis (66.9% identity), as shown in Figure 4. The homology is extensive throughout the entire protein.
  • All these proteins are ⁇ ADP-dependent glutamate dehydrogenases that catalyze the reductive amination of ⁇ -ketoglutarate, in the presence of ATP, to form L-glutamate.
  • the protein encoded by ORFl contains nine conserved motifs when compared with other fungal and yeast glutamate dehydrogenases .
  • One of the conserved domains corresponds to a region implicated in the catalytic mechanism of the enzyme. The consensus sequence of this region is [LIV] -X (2) -G-G- [SAG]-K-X-[GV]-X(3)-[D ⁇ S]-[PL] (PROSITE PS00074) .
  • the lysine residue K 114 located in the glycine-rich region GGGK 11 GG corresponds to the lysine in the active center of Glu/Leu/Phe/Val (GLFV) dehydrogenases. Therefore, following standard fungal gene nomenclature, the gene encoded by ORFl was named gdhA. A.2.1.3. The cloned gene complements A. nidulans gdhA mutants
  • A. nidulans A686 and A699 strains were transformed by a known method (M.M. Yelton et al . Proc. Natl. Acad. Sci. USA 1984, vol. 81, pp. 1470-4) with plasmid pGDHaw (7.1 kb) , which contains the j_ awamori gdhA gene in a 2570 bp Xbal-Xbal fragment .
  • This fragment contains also an upstream promoter region of 740 bp and a 322 bp region downstream from ORFl (gdhA gene) .
  • the 2570 bp Xbal-XBal fragment was inserted into the Xbal 'site of the fungal vector p43gdh, which contains the phleomycin resistance marker under control of the A. awamori gdhA promoter as shown later in this patent application.
  • nidulans mutants A686 and A699 grow very poorly in plates with 100 mM ammonium sulfate, whereas three randomly selected transformants grow very well in this medium.
  • the residual growth of A ⁇ . nidulans gdhA mutants A686 and A699 in ammonium sulfate as nitrogen source is known (J.R. Kinghorn, J.A. Pateman, Heredity 1973, vol. 31, pp. 427) and is due to the presence of a second glutamate dehydrogenase activity that allows partial growth of these mutants .
  • Nicotinamide adenine dinucleotide phosphate (NADP) -specific glutamate dehydrogenase (NADP-GDH) activity was assayed by following the reductive amination of ⁇ -ketoglutarate in the presence of ammonium and NADPH and expressed as units of enzyme activity per mg protein.
  • the initial reaction velocity was estimated from the change in optical density at 340 nm in a Hitachi U-2001 spectrophotometer.
  • One unit of glutamate dehydrogenase was defined as the activity that catalyzes the oxydation of one nanomol of NADPH per minute.
  • the NADP-dependent glutamate dehydrogenase activity was measured in the A. nidulans gdhA mutants A686 and A699, and in three randomly selected transformants complemented with the A ⁇ awamori gdhA gene. Results are shown in Table 1 and they clearly indicated that while the glutamate dehydrogenase activity in strains A686 and A699 was clearly below the detection levels, significant levels of glutamate dehydrogenase activity were obtained in the transformants with the A ⁇ awamori gdhA gene, particularly at 24 and 48 h of growth. Some of the transformants, like A699-4, showed relatively high levels of glutamate dehydrogenase activity, perhaps due to integration of more than one copy of the gdhA gene in the genome of this transformant .
  • Table 1 NADP-dependent glutamate dehydrogenase activity (U/mg of protein) , in the A ⁇ nidulans gdhA mutants A686 and A699, and in three transformants of each of these mutants with the A ⁇ . awamori gdhA gene.
  • Primer extension analysis using as primer the oligonucleotide "Pe” 5 ' -GGGGTTCTTCTGGAAGAGGGT-3 ' revealed a single band in the extension reaction (Fig. 8).
  • the 5 ' -end of the mRNA corresponds to a thymine (T) located 86 bp upstream of the ATG initiation codon.
  • the gdhA gene is transcribed as a monocistronic transcript of 1.7 kb, and its expression is regulated by nitrogen.
  • RNA of A. awamori was obtained by the phenol-SDS method from mycelia grown for 12, 24, 48, 60 or 72 h in MDFA medium with 55.5 mM glucose and 10 mM ammonium sulfate as carbon and nitrogen sources, respectively.
  • MDFA base medium without ammonium sulfate was supplemented with glutamic acid, L-glutamine, sodium nitrite, sodium nitrate and L-asparagine at 10 mM final concentrations.
  • RNA was fixed by UV irradiation using an UV- Stratalinker 2400 lamp (Stratagene, La Jolla, Calif.).
  • RNA 5 ⁇ g was loaded on a filter (NYTRAN. 0.45) by vacuum in a Bio-Dot SF Microfiltration apparatus.'. (Slot Blotting, Bio-Rad) .
  • the R ⁇ A was fixed by UV irradiation as above .
  • the filters were pre-hybridized for 3 h at 42 2 C in 50% formamide, 5 x Denhardt's solution, 5 x SSPE, 0.1% SDS, 500 ⁇ g of denatured salmon-sperm D ⁇ A per ml, and hybridized in the same buffer containing 100 ⁇ g of denatured salmon-sperm D ⁇ A per ml at 42 2 C for 18 h, using as probe an internal D ⁇ A fragment (0.694 kb PvuII) of the A ⁇ . awamori gdhA gene.
  • the filters were washed once in 2 x SSC, 0.1% SDS at 42 2 C for 15 min, once in 0.1 x SSC, 0.1% SDS at 42 2 C for 15 min, and once more in 0.1 x SSC, 0.1% SDS at 55 2 C for 20 min and then autoradiographed with Amersham X-ray film.
  • mR ⁇ A was purified from total R ⁇ A by using the Poly(A) Quick mR ⁇ A isolation kit (Stratagene, La Jolla, Calif.).
  • gdhA steady state transcript ' levels in the cell are higher than those of the ⁇ - actin gene (arrows) indicating that the glutamate dehydrogenase A is expressed from a very efficient promoter.
  • gdhA hybridizing RNA was compared to ⁇ -actin hybridizing RNA in MDFA medium with ammonium sulfate ( Figure 8A) and expressed as the ratio of counts in the gdhA-hybridizing band to the ⁇ - actin hybridizing counts ( Figure 8B) .
  • Results indicate that expression of both genes (gdhA and ⁇ -actin) is associated with the growth of Aj . awamori but whereas low steady state levels of ⁇ -actin mRNA remained in the cells until 96 hours of growth, the levels of glutamate dehydrogenase mRNA decreased ⁇ drastically after 48 hours.
  • the glutamate dehydrogenase enzymatic activity detected when A. awamori is grown in MDFA medium with ammonium sulfate (10 mM) as nitrogen source at different times of the culture is shown in Fig. 8C .
  • glutamate dehydrogenase plays a central role in nitrogen utilization by A., awamori , it was also of interest to study if expression of gdhA was regulated by different nitrogen sources.
  • very high gdhA transcript (mRNA) levels were obtained in media containing NH 4 + , or asparagine as sole nitrogen sources.
  • Glutamic acid repressed transcription of the gdhA gene, whereas intermediate levels of expression (normalized with respect to the ⁇ -actin gene) were observed in media that contained nitrate, glutamine or nitrite as nitrogen source.
  • the results were expressed per mg of protein in the cell extracts the highest specific activity was observed in MDFA medium with nitrate as the sole nitrogen source. This is due to the fact that in the presence of nitrate, A___ awamori grows very slowly. The lowest activity was observed in MDFA medium with glutamate as nitrogen source, confirming the results observed previously at the transcription level .
  • Table 2 NADP-dependent glutamate dehydrogenase activity in A ⁇ awamori cultures grown for 24 h in MDFA medium supplemented with different nitrogen sources.
  • Plasmid pBSGh was used as a template to obtain a 750 bp DNA fragment corresponding to the promoter region of the gdhA gene. This fragment was obtained by DNA amplification using the oligonucleotides gdhl and gdh2 and the Pfu enzyme (Stratagene) .
  • gdhl 5 ' - TTTT GTCGAC TTG CGA CGG CGT ATT GCT - 3 '
  • This amplified DNA fragment was digested with Sail and Ncol and purified in a 0.8% agarose gel.
  • Plasmid pJL43 (a derivative of pJL43b, Dr. Jose Luis Barredo, Ph.D. Thesis, Universidad de Leon, Leon, Spain) was digested with Sail and Ncol and a large fragment (3740 bp) was purified in a 0.8% agarose gel. This DNA fragment was then ligated with the Sall-Ncol fragment previously amplified, yielding plasmid p43gdh (4500 bp) , where the pcbC promoter from Penicillium chrvsogenum has been replaced by the gdhA promoter from Aspergillus awamori .
  • plasmid p43gdh was digested with Ncol, treated first with the Klenow fragment of DNA polymerase I and then with calf-intestinal phosphatase (CIP) .
  • CIP calf-intestinal phosphatase
  • a fragment of 1140 bp containing the B2 protein gene was amplified via the PCR technique, using plasmid pJElA as the template and oligonucleotides NTB2b and CTB2b as primers (sequences given below) .
  • This 1140 bp fragment was digested with BamHI and then treated with the Klenow fragment of DNA polymerase I.
  • pB2KEX was sequentially digested with Xbal, treated with the Klenow fragment from DNA polymerase I and finally digested with BamHI. A fragment of 4637 bp was purified from a 0.8% agarose gel.
  • plasmid p43gdhB2 was sequentially digested with Sail, treated with the Klenow fragment from DNA polymerase I and finally digested with BamHI. A fragment of 1173 bp was purified from a 0.8% agarose gel. The ligation of these two fragments yielded plasmid pGDHTh (5810 bp) , where a new Sail site was created. This allows for the excision of the complete GDHTh cassette as a 2670 bp Sall-Sall fragment.
  • Plasmid pJL43 was linearized by digestion with Sail and ligated to a 2170 bp Sall-Dral fragment from pGDHTh (see Fig. 10, part B) .
  • plasmid pGD71 was constructed as follows: plasmid PAN7-1 (P.J. Punt et al . , J. Biotecnol . 1990, vol. 17, pp. 19-34) was sequentially digested with Xbal, treated with the Klenow fragment from DNA polymerase I, and finally digested with Hindlll, and purified from a 0.8% agarose gel. In parallell, plasmid pGDHTh was digested with Ecll36ll (or Sad*, a variant of Sad from Fermentas that recognizes the standard Sad restriction site but leaves a blunt end), Hindlll and Dral. A fragment of 2175 bp was purified from an agarose gel . Ligation of these two fragments yielded plasmid pGD71 (see Fig. 10, part C) .
  • Plasmids p43GDTh and pGD71 contain a cassette to express thaumatin that comprises: (i) a DNA sequence which encodes a fusion protein comprising in his turn (a) the synthetic gene of thaumatin II, (b) a spacer sequence which in turn contains a KEX2 processing sequence, and (c) a cDNA sequence that encodes most of the B2 protein (except sequences in the COOH end) from Acremonium chrvsogenum; (ii) the signal sequence of the B2 gene of Acremonium chrvsogenum, (iii) the promoter region from the Aspergillus awamori glutamate dehydrogenase A gene, and (iv) a drug resistance gene that can be used as a transformation marker.
  • Plasmid p43GDTh has the phleomycin resistance gene (phleo) driven by the the pcbC promoter from Penicillium chrvsogenum.
  • Plasmid pGD71 contains the hygromyci ⁇ B resistance gene driven by the glyceraldehyde-3- pho ⁇ phate dehydrogenase promoter from Aspergillus nidulans .
  • the expression cassette GPDTh is similar to the expression cassette B2KEX, except that the B2 promoter from Acremonium chrvsogenum has been replaced by the promoter from the glyceraldehyde-3-phosphate dehydrogenase (named "gpd” from now on) gene from Aspergillus nidulans.
  • the complete promoter region of the gpd gene is present in plasmid pAN52-l (P.J. Punt et al . , J. Biotecnol. 1990, vol. 17, pp. 19-34).
  • a Sacl-Ncol fragment (880 bp) from pAN52-l has been subcloned, generating pJL43bl.
  • Plasmid pJL43bl was digested with ⁇ col and treated first with the Klenow fragment of D ⁇ A polymerase I and then with calf-intestinal phosphatase (CIP) , as shown in Figure 11.
  • CIP calf-intestinal phosphatase
  • a 1140 bp fragment of D ⁇ A was obtained by D ⁇ A amplification using the PCR technique, using pJElA as template and oligonucleotides ⁇ TB2b and CTB2b as primers.
  • This fragment of DNA was digested with BamHI and treated with the Klenow fragment from DNA polymerase I, yielding a fragment of 425 bp that was purified from a 0.8% agarose gel.
  • NTB2b 5 ' - ATG CGT GCT GCT ACT CTC - 3 '
  • CTB2b 5' - CTG GCC GTT GTT GAT GAG - 3'
  • the next step in the construction of a complete expression cassette was the addition of the second portion of the B2 gene, the KEX2 sequence and the synthetic thaumatin II gene.
  • plasmid pB2KEX was once again used.
  • pB2KEX was sequentially digested with Xbal, treated with the Klenow fragment from DNA polymerase I and finally digested with BamHI. A fragment of 4637 bp was purified from a 0.8% agarose gel.
  • plasmid pblB2 was sequentially digested with BamHI and Ecll36II (or Sad*) (leaves blunt ends), and a 1300 bp fragment was purified from a 0.8% agarose gel. The ligation of these two fragments yielded plasmid pGPDTh (5800 bp) .
  • the GPDTh cassette was isolated from pGPDTh by digestion with Ecll36ll (or Sad*) , Hindlll and Dral, yielding a DNA fragment 2800 bp long.
  • plasmid pB2KThbl was sequentially digested with BamHI, treated with the Klenow fragment from DNA polymerase I and finally digested with Hindlll.
  • a 4500 bp fragment was isolated from a 0.8% agarose gel.
  • the plasmid resulting from the ligation of these two fragments was named pGPThbl.
  • This plasmid contains a cassette for the expression of thaumatin that is identical to the expression cassette B2KEX except that the promoter from the B2 gene of Acremonium chrvsogenum has been replaced by the promoter from the gpd gene from Aspergillus nidulans .
  • Aspergillus awamori strain NRRL312 was obtained from the American Type Culture Collection (ATCC) . Using standard mutagenesis techniques with nitrosoguanidine (NTG) , a derivative of this strain was obtained, and was named LpR66. This mutant strain secretes into the growth medium an inactive exoprotease aspergillopepsin A (named "pepA” from now on) . In all of the transformation experiments that are described below the strain that was used was Aspergillus awamori strain LpR66.
  • the antibiotic phleomycin was used as the selection marker.
  • Strain LpR66 can grow in plates that contain 20 ⁇ g/ml of phleomycin. Therefore, all transformants were selected in plates with 25 ⁇ g/ml of the antibiotic .
  • the regeneration medium that was used is TSAS, which contains 30 g/1 of Triptone-Soja (Difco) , 103 g/1 of sucrose and 1.5% agar (Difco) .
  • the transformation protocol was similar to the one described by Yelton (see above) with some modifications.
  • a plate containing Power medium was inoculated with 10 7 spores. This plate was incubated for 72 hours at 30 2 C, at which point the spores were scraped from the plate and were inoculated in 100 ml of CM medium (500 ml shake flask) . Incubation was for 16- 18 hours at 250 rpm and 28 a C.
  • the mycelium obtained from this growth was filtered through a 30 ⁇ m nylon filter (Nytal) and washed with 10 mM sodium phosphate buffer (pH 5.8) which also contained 0.6 M magnesium sulfate.
  • protoplast buffer (10 mM sodium phosphate buffer (pH 5.8) which also contained 1.2 M magnesium sulfate) .
  • An equal volume of buffer containing the enzyme "Lysing” (Sigma) was added, yielding a final concentration of 3 mg/ml of the enzyme.
  • the mycelium solution was left to incubate for 3-4 hours at 100 rpm and 30 2 C until protoplasts were formed. Protoplast formation was monitored by visual inspection using a light microscope. Protoplasts were filtered, washed and finally resuspended in STC solution, to a final concentration of 10 8 protoplasts/ml.
  • Transformants were analyzed by PCR to detect whether the thaumatin II gene had been incorporated into their genome essentially as described (cf . EP 684312) . Those transformants that were positive were then further analyzed for expression of thaumatin by immunoblot analysis and ELISA (enzyme-linked immunoassay) also as described (cf. EP 684312).
  • C Recombinant strains that produce thaumatin
  • CM medium malt extract, 5 g/1; yeast extract, 5 g/1; glucose, 5 g/1.
  • SMM medium 8% sodium citrate; 1.5% (NH 4 ) 2 S0 4 ; 0.13%
  • glucose sucrose, maltose, etc.
  • the pH of the medium is adjusted to 6.2 with H 2 S0 4 .
  • MDFA medium 1.2% L-asparagine; 0.8% of salt solution I [2% Fe(NH 4 ) 2 (S0 4 ) 2 .6H 2 0] ; and 14.4% of salt solution II [10.4% K 2 HP0 4 ; 10.2% KH 2 P0 4 ; 1.15% Na 2 CuS0 4 .5H 2 0 ; 0.2%MgS0 4 .7H 2 0; 0.02% ZnS0 4 .7H 2 0; 0.005% CuS0 4 .5H 2 0; 0.05% Cad-,.2110] .
  • the carbon source used was either maltose (usually 6.5%) or a mix of sucrose (3.6%) and glucose (2.7%) . Other amounts of carbon source are indicated in each experiment that is described.
  • the initial pH of this medium is 6.5.
  • Dry weight was determined by passing a sample through a pre- filter (Nucleopore, Cat .No. 211114) .
  • the biological material retained in the pre-filter was washed with 40 ml of pure ethanol and 50 ml of distilled water. It was then incubated at 90°C until a constant weight could be recorded. The filtrate was aliquoted and frozen for further analysis.
  • Thaumatin concentration in the culture broth was determined by an enzyme-linked immunoassay (ELISA) and by immunoblotting
  • Glucose levels were determined using a SIGMA DIAGNOSTICS kit (Procedure number 510).
  • Maltose concentration in the culture broth was determined as follows: 250 ⁇ l of sample filtrate were placed in a test-tube that had been previously chilled; 1.250 ml of anthrone solution (prepared by dissolving 2 g anthrone in 50 ml absolute ethanol and then adding 950 ml of 75% H 2 S0 4 ) were then added, and the sample was kept chilled for five minutes. The sample was then transferred to a boiling water bath, and incubated for 10 minutes. Finally the samples were once again chilled .and the absorbance read at 625 nm. Maltose concentrations were determined by comparison to a calibration curve generated by measuring the absorbance of maltose solutions of known concentrations (range: 0 - 0.2 g/1).
  • This strain is a derivative of Lpr66 that was obtained by transformation of the aforementioned LpR66 strain with the expression plasmid pB2_KTh-bl .
  • This expression cassette contains the synthetic thaumatin II gene under the control of the promoter of the B2 protein from Acremonium chrvsogenum. In shake-flask cultures with MDFA medium this strain secretes 6-8 mg thaumatin/1.
  • Lpr66 It is a derivative of Lpr66 which was obtained by transformation of the aforementioned LpR66 strain with the expression cassette p43GDTh.
  • This expression cassette contains the synthetic thaumatin II gene under the control of the promoter of the gdhA gene from Aspergillus awamori. In shake-flask cultures with MDFA medium (with 6.0% sucrose) this strain secretes 6-8 mg thaumatin/1.
  • This strain is a derivative of Lpr66 which was obtained by transformation of the aforementioned LpR66 strain with the expression cassette pGPThbl .
  • This expression cassette contains the synthetic thaumatin II gene under the control of the promoter of the gpd gene from Aspergillus nidulans . In shake-flask cultures with MDFA medium this strain secretes 9- 10 mg thaumatin/liter .
  • Strains TB2 -44 and TGP-3 were re-transformed with expression plasmid pGD71, which contains the thaumatin gene under control of the glutamate dehydrogenase promoter from A. awamori and a hygromycin B resistance gene as a selection marker for transformation experiments.
  • a battery of different transformants (see Table 3) was analyzed in shake flask experiments. It was shown that re-transformation of strain TGP-3 did not result in better producing strains. However, re-transformation of TB2bl-44 did result in better producing strains when cultured in shake-flasks under the standard conditions mentioned before.
  • Table 3 Production of thaumatin in shake flasks by retransformed strains grown in MDFA medium for 96 h. Quantification by ELISA. All strains were retransformed using hygromicin B resistance as selection marker.
  • Biomass was removed by filtration through filter paper. The filtrate was collected in a filtering flask that was submerged in ice. The clarified broth was then centrifugated at 6000 rpm for 15 minutes at 4 2 C.
  • the clarified fermentation broth was further concentrated by ultrafiltration using a ProFluxTM Ml2 Tangential Filtration System.
  • the system configuration was: base unit, level switch, 2.5 1 reservoir, cooling coil, inlet and oulet pressure transducers, secondary pump, one Spiral-wound membrane cartridges S1Y3 (Molecular weight cut-off 3,000 Daltons) .
  • the system was operated as follows: (1) Calibration of the pressure sensors. (2) Adjustment of alarm set points: low inlet pressure 3.0 Bars, high inlet pressure 3.5 Bars, differential pressure 0.3 Bars. (3) Washing of the system and the cartridges with deoinized, distilled water (4) Fill- up of the reservoir with process solution; the solution is kept at 8-10 2 C by recirculating cold water (HAAKE, DC1-K20 refrigerated circulator) through the cooling coil. (5) Setting of the level switch at the desired concentration volume (1/4 to 1/5 of the initial volume) . (6) Operation of the recirculation pump at 75 %. (7) Adjustment of the Back Pressure Valve to obtain a 3.0 Bar inlet pressure. If necessary, back pressure was reduced during operation.
  • the solution was diafiltered in order to remove low molecular weight solutes (Salts, sugars, etc.).
  • the system configuration allows the operation in the "pumped diafiltration with automatic safety stop” mode.
  • the dialysate (five volumes of deionized water) was transferred by the secondary pump in steps as directed by the level switch. Once the dialysate supply is exhausted, the system and the secondary pump will shut off automatically.
  • the diafiltered solution is drained from the system, sterilized by filtration (Stericup, 0.22 ⁇ m, Millipore) and stored at 4 2 C.
  • Recombinant thaumatin was purified to homogeneity using a four step purification scheme that is detailed in Table 4.
  • the starting point for the particular purification protocol that is described here are 500 ml of fermentation broth obtained from the growth of strain TGDTh-4, with thaumatin present at a concentration of 50 mg/1.
  • Proteins from this broth were precipitated with ammonium sulfate (20-50% range) .
  • the precipitate was then re-suspended in 25 mM phosphate buffer, pH 7.0.

Abstract

L'invention concerne de nouveaux promoteurs des gènes de glutamate déshydrogénase (gdh) provenant d'Aspergillus awamori et d'aspergilli qui lui sont associés, ainsi que de nouvelles séquences d'ADN codant des glutamate déshydrogénases provenant d'Aspergillus awamori. L'invention concerne également l'utilisation des promoteurs des gènes de gdh provenant d'un champignon du genre Aspergillus pour l'expression de protéines de recombinaison dans des champignons filamenteux.
PCT/EP1999/002243 1998-04-02 1999-04-01 Promoteur et produits de synthese destines a l'expression de proteines de recombinaison dans des champignons filamenteux WO1999051756A2 (fr)

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AU42575/99A AU4257599A (en) 1998-04-02 1999-04-01 Promoter and constructions for expression of recombinant proteins in filamentousfungi
EP99944133A EP1084262A1 (fr) 1998-04-02 1999-04-01 Promoteur et produits de synthese destines a l'expression de proteines de recombinaison dans des champignons filamenteux
CA002325571A CA2325571A1 (fr) 1998-04-02 1999-04-01 Promoteur et produits de synthese destines a l'expression de proteines de recombinaison dans des champignons filamenteux

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EP1231272A2 (fr) * 2001-02-08 2002-08-14 Laboratorios Ovejero S.A. Procédé d'obtention de chymosine bovine (rennine)
WO2003070957A2 (fr) * 2002-02-20 2003-08-28 Novozymes A/S Production de polypeptides de plantes
WO2007014726A2 (fr) * 2005-07-30 2007-02-08 Maltagen Forschung Gmbh Thaumatine obtenue a partir d'orge transgenique
US8268585B2 (en) 1998-10-06 2012-09-18 Dyadic International (Usa), Inc. Transformation system in the field of filamentous fungal hosts
EP2505651A2 (fr) 2006-12-10 2012-10-03 Dyadic International, Inc. Isolat de champignon avec activité protéase réduite
US8551751B2 (en) 2007-09-07 2013-10-08 Dyadic International, Inc. BX11 enzymes having xylosidase activity
US8673618B2 (en) 1996-10-10 2014-03-18 Dyadic International (Usa), Inc. Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US20140206086A1 (en) * 2011-08-24 2014-07-24 Novozymes, Inc. Methods for Obtaining Positive Transformants of a Filamentous Fungal Host Cell
US20140212977A1 (en) * 2011-08-24 2014-07-31 Debbie Yaver Methods For Producing Multiple Recombinant Polypeptides In A Filamentous Fungal Host Cell

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EP0249350A1 (fr) * 1986-06-06 1987-12-16 Panlabs, Inc. Systèmes d'expression de champignons filamenteux
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US8673618B2 (en) 1996-10-10 2014-03-18 Dyadic International (Usa), Inc. Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US8916363B2 (en) 1996-10-10 2014-12-23 Dyadic International (Usa), Inc. Construction of Highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US8268585B2 (en) 1998-10-06 2012-09-18 Dyadic International (Usa), Inc. Transformation system in the field of filamentous fungal hosts
WO2002002776A1 (fr) * 2000-07-06 2002-01-10 Institute Of Microbiology, Chinese Academy Of Sciences Procede permettant d'augmenter le taux d'assimilation de l'azote par les plantes
EP1231272A2 (fr) * 2001-02-08 2002-08-14 Laboratorios Ovejero S.A. Procédé d'obtention de chymosine bovine (rennine)
EP1231272A3 (fr) * 2001-02-08 2002-11-13 Laboratorios Ovejero S.A. Procédé d'obtention de chymosine bovine (rennine)
WO2003070957A2 (fr) * 2002-02-20 2003-08-28 Novozymes A/S Production de polypeptides de plantes
WO2003070957A3 (fr) * 2002-02-20 2003-12-24 Novozymes As Production de polypeptides de plantes
WO2007014726A2 (fr) * 2005-07-30 2007-02-08 Maltagen Forschung Gmbh Thaumatine obtenue a partir d'orge transgenique
WO2007014726A3 (fr) * 2005-07-30 2007-06-07 Maltagen Forschung Gmbh Thaumatine obtenue a partir d'orge transgenique
EP2505651A2 (fr) 2006-12-10 2012-10-03 Dyadic International, Inc. Isolat de champignon avec activité protéase réduite
US8551751B2 (en) 2007-09-07 2013-10-08 Dyadic International, Inc. BX11 enzymes having xylosidase activity
US20140206086A1 (en) * 2011-08-24 2014-07-24 Novozymes, Inc. Methods for Obtaining Positive Transformants of a Filamentous Fungal Host Cell
US20140212977A1 (en) * 2011-08-24 2014-07-31 Debbie Yaver Methods For Producing Multiple Recombinant Polypeptides In A Filamentous Fungal Host Cell
US9404101B2 (en) * 2011-08-24 2016-08-02 Novozymes, Inc. Methods for obtaining positive transformants of a filamentous fungal host cell
US9493790B2 (en) * 2011-08-24 2016-11-15 Novozymes, Inc. Methods for producing multiple recombinant polypeptides in a filamentous fungal host cell
US10822601B2 (en) 2011-08-24 2020-11-03 Novozymes, Inc. Methods for producing multiple recombinant polypeptides in a filamentous fungal host cell

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