Patents Available Fungal Expression

Our product and project portfolio represents a huge diversity of different enzymes screened from a variety of microorganisms in nature or from molecular evolution in the laboratory. Our fungal expres-sion systems include the host strains Aspergillus oryzae, Aspergillus niger and Fusarium venenatum. Most of these species have been used in industrial processes for more that 30 years.

The ability to express enzymes of any class from any source is the key for unlocking the practical use of enzymes for industry. We produce the enzyme in yields often 1000 fold higher than what is achieved by the indigenous organism.

Our technologies within the fungal expression area covers for example a process for expression of a protein product in Aspergillus oryzae, enabling industrial production of many different polypeptides and proteins. Another example relates to fungi, which do not produce intracellular proteases. This is useful for the production of proteins susceptible for proteolytic degradation.  

• Promoters for use in Aspergillus

• Host cell expressing reduced levels of a metalloprotease and methods using the host cell in protein production

Title of Technology:  Promoters for use in Aspergillus

Abstract: The technology concerns s process for expression of a protein prod-uct in Aspergillus. The process comprises transforming an Aspergillus strain with a vector system comprising DNA-sequences encoding a promoter including upstream activating sequences derived from an A. niger amylase, a suitable marker for selection of transformants, and a DNA-sequence encoding the desired protein product. The process enables industrial production of many different polypeptides and pro-teins in Aspergillus, preferably A. niger. Examples of such products are chymosin or prochymosin and other rennets, proteases, lipases and amylases. Also disclosed is an effective promoter for expression of a protein in Aspergillus, preferably Aspergillus niger being derived from a gene encoding an A. niger amylase. The A. niger amylases are the neutral and acid stable .alpha.-amylases and a new amylase not so far described and designated XA amylase. Also disclosed is the novel amylase from A. niger XA amylase.

Detailed Description:  The technology relates to a process for expression of protein products in Aspergillus, recombinant DNA vectors, a promoter for Aspergillus and transformed fungi. The technology is also directed to a new amy-lase from A. niger

The transformation technique used was a method adapted from the methods for transformation of A. nidulans (Ballance et al. Biochem. Biophys. Res. Commun., 112 (1983), 284-289; Tilburn et al., Gene 26 (1983), 205-221, Yelton et al. Proc. Natl. Acad. Sci. U.S.A., 81 (1984), 1470-1474) and similar to the method of Buxton et al. (Gene 37 (1985), 207-214) for transformation of A. niger. In the process the chosen Aspergillus strain is transformed with a vector system contain-ing a selection marker which is capable of being incorporated into the genome of the host strain, but which is not harboured in the host strain before the transformation. Transformants can then be selected and isolated from nontransformants on the basis of the incorporated selection marker.

Preferred selection markers are the argB (A. nidulans or A. niger), trpC (A. nidulans), amdS (A. nidulans), or pyr4 (Neurospora crassa) genes, or the DHFR (dihydrofolate reductase or mutants hereof) ge-ne. More preferred selection markers are the argB or the amdS gene.

Besides promoter and upstream activating sequences the vectors will normally contain further DNA-sequences encoding functions facilitat-ing gene expression such as transcription terminators and polyade-nylation signals.

The expressed product may be accumulated within the cells requiring disruption of the cells to isolate the product. To avoid this further pro-cess step and also to minimize the amount of possible degradation of the expressed product within the cells it is preferred that the product is secreted from the cells. For this purpose the gene for the desired pro-duct is provided with a preregion ensuring effective direction of the expressed product into the secretory pathway of the cell. This preregion which might be a naturally occurring signal or leader pep-tide or functional parts thereof or a synthetic sequence providing se-cretion is generally cleaved from the desired product during secretion leaving the mature product ready for isolation from the culture broth.

The preregion may be derived from genes for secreted proteins from any source of organism.

The gene for the desired product functionally linked to the promoter and terminator sequences may be incorporated in a vector containing the selection marker or may be placed on a separate vector or plas-mid capable of being integrated into the genome of the host strain. As used herein the expression "vector system" includes a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA-information to be inte grated into the host genome. Vectors or plasmids may be linear or closed circular molecules. According to a preferred embodiment of the present technology A. niger is cotrans-formed with two vectors, one including the selection marker and the other comprising the remaining foreign DNA to be introduced in the host strain, including promoter, the gene for the desired product and transcription terminator and polyadenylation sequences.

Normally the A. niger transformants are stable and can be cultured in the absence of a selection pressure. If the transformants turn out to be unstable the selection marker may be used for selection during cultivation. The transformed cells are then cultured under a selection pressure corresponding to the marker in question.

The technology provides for a method for production of high yields of many different polypeptide or protein products in Aspergillus, espe-cially A. niger. A. niger strains have for years been used in commer-cial scale for the production of for instance amyloglucosidase and other extracellular enzymes and accordingly fermentation technology for these microorganisms is well developed and the microorganisms are approved for use in the food industry. The technology offers the possibility of using A. niger in the industrial production of high amounts of in principle any polypeptide or protein product. Examples of such products are chymosin or prochymosin and other rennets, proteases, amyloglucosidases, acid stable amylases from Aspergillus, fungal lipases or prokaryotic lipases, and thermostable bacterial and fungal amylases.

Glossary of Terms  

 Patent References

Title of Technology:  Host cell expressing reduced levels of a metalloprotease and meth-ods using the host cell in protein production

Abstract: The present technology relates to novel host cells and to methods of producing proteins. More specifically the technology relates to a host cell useful for the expression of heterologous proteins, which host cell has been genetically modified in order to express significantly re-duced levels of a metalloprotease. Moreover the technology relates to a method of producing a heterologous protein, which method com-prises cultivating the host cell in a suitable growth medium, followed by recovery of the desired protein.

By the method, the proteolytic action arising from metalloproteases have been significantly reduced, thereby improving the stability of the protein obtained by the method. Moreover, the protein obtained by the method can be obtained as a precursor protein, i.e. a zymogen, a hy-brid protein, a protein obtained as a pro sequence or pre-pro se-quence, or in unmaturated form.

Detailed Description:  Host Cells

The present technology provides a host cell useful for the expression of heterologous proteins, which cell, when compared to the parental cell, has been genetically modified in order to express significantly reduced levels of a metalloprotease.

The parental cell is the source of said host cell. It may be a wild-type cell. Alternatively, besides a decrease in metalloprotease level, it may be genetically altered in another respect.

In order to produce the desired protein, the host cell obviously must hold structural (i.e. regions comprising the coding nucleotide se-quences) and regulatory (i.e. regions comprising nucleotide se-quences necessary or e.g. transcription , translation and termination) genetic regions necessary for the expression of the desired product. The nature of such structural and regulatory regions greatly depends on the product and the host cell in question.

Preferably, the host cell is modified by methods known in the art for introduction of an appropriate cloning vehicle, i.e. a plasmid or a vec-tor, comprising a DNA fragment encoding the desired product. The cloning vehicle may be introduced into the host cell either as an auto-nomously replicating plasmid or integrated into the chromosome. Preferably the cloning vehicle comprises one or more structural re-gions operably linked to one or more appropriate regulatory regions.

The cloning vehicle may also comprise a selectable marker, e.g. a gene, the product of which complements a defect in the host cell, or one which confers antibiotic resistance, such as ampicillin, kanamy-cin, chloramphenicol or tetracycline resistance.

The procedures used to ligate the DNA construct, the promoter, ter-minator and other elements, respectively, and to insert them into suit-able cloning vehicles containing the information necessary for replica-tion, are well known to persons skilled in the art ¢vide e.g. Sambrook et al.; Molecular Cloning, Cold Spring Harbor, N.Y., 1989!.

The host cell may be any host cell conventionally used for heterolo-gous expression of proteins. Preferably, the host cell is a yeast or a filamentous fungus capable of producing a desired protein.

Products

The desired end product, i.e. the heterologous protein expressed by the host cell, may be any eubacterial or eucaryotic protein.

In a more specific embodiment, the product is an eucaryotic enzyme, such as insulin, growth hormone, glucagon, somatostatin, interferon, PDGF, factor VII, factor VIII, urokinase, EPO, chymosin, tissue plas-minogen activator, or serum albumin.

In another preferred embodiment, the product is an enzyme of fungal, of yeast, or of bacterial origin. Preferably the enzyme is a glycosidase enzyme, e.g. an amylase, in particular an .alpha.-amylase (EC 3.2.1.1), a .beta.-amylase (EC 3.2.1.2), a glucan 1,4-.alpha.-glucosidase (EC 3.2.1.3), a cellulase (EC 3.2.1.4), an endo-1,3(4)-.beta.-glucanase (EC 3.2.1.6), an endo-1,4-.beta.-glucanase (EC 3.2.1.8), a polygalacturonase (EC 3.2.1.15), an .alpha.-glucosidase (EC 3.2.1.20), a .beta.-glucosidase (EC 3.2.1.21), an .alpha.-galactosidase (EC 3.2.1.22), a .beta.-galactosidase (EC 3.2.1.23), a xylan-endo-1,3-.beta.-xylosidase (EC 3.2.1.32), an endo-1,3-.beta.-glucanase (EC 3.2.1.39), an endo-1,3-.alpha.-glucanase (EC 3.2.1.59), an endo-1,2-.beta.-glucanase (EC 3.2.1.71), an endo-1,6-.beta.-glucanase (EC 3.2.1.75), a cellulose-1,4-.beta.-cellobiosidase (EC 3.2.1.91, also known as cellobiohydrolases).

In another preferred embodiment the enzyme is a lipolytic enzyme, in particular a lipase, an esterase, a phospholipase, or a lysophospholi-pase.

In a third preferred embodiment the enzyme is a phytase, in particular a 3-phytase (EC 3.1.3.8) or a 6-phytase (EC 3.1.3.26).

In a fifth preferred embodiment the enzyme is an oxidoreductase, such as a peroxidase or a laccase, a pectinase, or a cutinase.

Preferred hybrid polypeptides are prochymosin and pro-trypsin-like proteases.

Metalloproteases

In a preferred embodiment, the metalloprotease is a Fusarium metal-loprotease, preferably a Fusarium oxysporum metalloprotease. In a most preferred embodiment, the metalloprotease is a Fusarium ox-ysporum p45 metalloprotease.

In another preferred embodiment, the metalloprotease is a neutral metalloprotease, which is a metalloprotease possessing optimal pro-teolytic activity in the neutral pH region, i.e. in the range of about pH 6-8, preferably the range of about pH 6.5-7.5, around pH 7.

More particularly, the metalloprotease is a neutral Aspergillus metal-loprotease of group NpI or NpII.

Genetic Modifications

The host cell, genetically modified in order to express significantly reduced levels of a metalloprotease, may be modified using standard recombinant DNA technology. The gene sequence responsible for the production of metalloprotease may be inactivated or eliminated en-tirely.

Owing to genetic modification, the host cell expresses significantly reduced levels of metalloproteases. In a preferred embodiment the level of metalloprotease expressed by the host cell is reduced more than about 50%, preferably more than about 85%, more preferred more than about 90%, most preferred more than about 95%. In a most preferred embodiment, the product expressed by the host cell is essentially free of any metalloprotease activity.

Methods of Producing Proteins

The technology provides a method of producing proteins (i.e. polypep-tides and/or proteins), which method comprises cultivating the host cell in a suitable growth medium, followed by recovery of the desired product.

By the method of the technology, the proteolytic action of metallopro-teases have been significantly reduced, thereby improving the stability of the product obtained. Moreover, owing to the absence of metallo-protease, the heterologous protein expressed by the host cell may be obtained as a precursor protein, i.e. a zymogen, a hybrid protein, a protein obtained as a pro sequence or pre-pro sequence, or in un-maturated form.

The broth or medium used for culturing may be any conventional me-dium suitable for growing the host cell in question, and may be com-posed according to the principles of the prior art. The medium pref-erably contain carbon and nitrogen sources and other inorganic salts. Suitable media, e.g. minimal or complex media, are available from commercial suppliers, or may be prepared according to published re-ceipts, e.g. the American Type Culture Collection (ATCC) Catalogue of strains.

After cultivation, the protein is recovered by conventional method for isolation and purification proteins from a culture broth. Well-known purification procedures include separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, and chromatographic methods such as e.g. ion exchange chromatogra-phy, gel filtration chromatography, affinity ch romatography, etc.

Glossary of Terms

Patent References