Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-02-28
2003-04-22
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S455000, C435S320100, C536S023100
Reexamination Certificate
active
06551797
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to an expression system for production of proteins in fungi of the genera Trametes or Polyporus, to its preparation and to its use.
2. The Prior Art
Various prokaryotic and eukaryotic expression systems are known for protein production. Examples of prokaryotic expression systems are
Escherichia coli
and
Bacillus subtilis
. The methods for genetic manipulation of these organisms are well established. Specific disadvantages of these expression systems are the frequently disappointingly low production rate in particular of eukaryotic proteins, the folding of the produced proteins in such a way that they are often not in active form, and, in particular, the absence of the post-translational modification of the expressed proteins. As example of the absence of post-translational modification, mention may be made of the absence of incorporation of prosthetic groups or the absence of glycosylation of the protein to be expressed.
These disadvantages of prokaryotic expression systems can be avoided by using eukaryotic systems.
Widespread eukaryotic expression systems which are widely used include cell culture systems both of mammalian cells and insect cells, and eukaryotic microorganisms such as yeasts or filamentous fungi. Whereas the protein to be expressed is usually produced in active form with these expression systems, the production rate is in many cases too low, especially on expression of heterologous proteins. Expression in the yeast
Saccharomyces cerevisiae
or in filamentous fungi from the ascomycetes class may serve as example thereof.
High production rates in filamentous fungi such as Aspergillus have been described in particular for the expression of homologous proteins or proteins of filamentous fungi of the ascomycetes class. Expression of heterologous proteins often takes place with only low or moderate yields. WO 96/00290, for example, describes heterologous expression of the laccase LCC1 from the filamentous fungus of the basidiomycetes class,
Polyporus pinsitus
. On expression in Aspergillus, a filamentous fungus of the ascomycetes class, yields of up to 0.35 g/l are obtained in the fermentation. This is a comparatively small increase in yield compared with the production of 0.1-0.2 g/l by a comparable wild-type strain in the fermentation.
There is an increasing interest in industrial applications especially for enzymes from the basidiomycetes class and therein especially the white rot fungi. Examples which may be mentioned are hydrolytic enzymes such as cellulases, hemicellulases or lipases, or else oxidoreductases such as lignin peroxidases, manganese peroxidases, laccases, cellobiose-quinone oxidoreductase or cellobiose oxidase. Potential applications for these enzymes exist, for example, in wood and pulp processing.
One class of enzymes occurring among others in basidiomycetes and of great interest for industrial applications is the class of laccase enzymes (p-hydroxyphenol oxidase, EC 1.10.3.2.). Laccases belong to the protein family called the “blue copper proteins” and usually contain four copper ions which are arranged in three copper centers referred to as type 1 to type 3. Laccases are further distinguished by generally being secreted proteins and possibly containing a glycosylation content of up to 10 to 45% of the molecular weight. Beside the depolymerization of macromolecular compounds such as lignin, laccases are also able to catalyze the polymerization in particular of aromatic compounds. An example thereof is lignin biosynthesis in plants, in which the laccases present in plants are involved. Possible industrial applications of laccases are in paper manufacture for the delignification of pulp, in polymerization reactions of all types, for example in waste water treatment. The use of laccases in organic chemical synthesis is also known, for example in coupling reactions or the side-chain oxidation of aromatic compounds. However, a precondition for industrial application of all these processes is that the laccase enzyme can be provided at reasonable cost and in relatively large amounts.
DNA vectors said to be suitable for transformation and selection of transformants have been described for various filamentous fungi from the basidiomycetes class. A method for homologous transformation of the basidiomycete
Phanerochaete chrysosporium
has been described (M. Alic et al. (1991) Curr. Genet. 19, 491-494). DNA constructs for transformation of the basidiomycete
Pleurotus ostreatus
has been described (K. Yanai et al. (1996) Biosci. Biotech. Biochem. 60, 472-475). U.S. Pat No. 5,362,640 describes DNA vectors for the transformation of the basidiomycete
Coriolus hirsutus
. Likewise, a DNA vector for transformation of the basidiomycete
Coriolus versicolor
has been described (Y. Iimura et al. (1992) 5th International Conference on Biotechnology in the Pulp and Paper Industry, 427-431). It has not been disclosed for any of these expression systems from the basidiomycetes class that a significant increase in the expression rate for homologous or heterologous proteins has been achieved.
Known expression vectors containing genetic regulatory elements for expression in filamentous fungi of the ascomycetes class cannot be efficiently expressed in filamentous fungi of the basidiomycetes class. Thus, on transformation of filamentous fungi of the basidiomycetes class they do not allow the selection of positive transformants on the basis of, for example, acquired antibiotic resistance or the expression of a color-forming indicator protein or on the basis of the complementation of an auxotrophic gene defect.
SUMMARY OF THE INVENTION
The present invention relates to an expression system for the production of a protein in a filamentous fungi consisting of
a) a host organism selected from the genera Trametes and Polyporus and
b) a DNA vector which comprises a selection marker gene which codes for a protein which, after transformation of the host organism, allows selection of positive transformants and is selected from the group of antibiotic resistance genes which code for proteins which abolish the growth-inhibiting effect of antibiotics to which the host organism is not resistant, of genes which encode proteins which are capable of a color-forming reaction, and of genes which complement a genetic defect in the host organism (auxotrophy), where expression of the selection marker gene is controlled by at least one genetic regulatory element which is active in the host organism, and
c) a DNA vector which comprises a gene which codes for the protein to be produced, where expression of this gene and, where appropriate, also secretion of the protein thus produced is controlled by a genetic regulatory element which is active in the host organism, where the DNA vector which comprises a selection marker gene, and the DNA vector which comprises the gene which codes for the protein to be produced may also be present as a DNA vector.
Suitable and preferred as antibiotic resistance genes are genes which confer resistance to an antibiotic from the group of hygromycin, bialaphos, kanamycin, geneticin, bleomycin, oligomycin, G418, zeocin, benomyl and phleomycin.
It is possible and preferred to use further selection marker genes which code for proteins which are capable of a color-forming reaction, for example the glucuronidase gene or the gene for green fluorescent protein (GFP).
Selection marker genes able to complement a genetic defect in the host organism (auxotrophy) are particularly suitable.
Host organisms preferred for the expression system according to the invention are monokaryotic strains from the genera Trametes and Polyporus.
Host organisms of the species
Trametes versicolor
are particularly preferred.
The host organism in the expression system according to the invention is preferably distinguished by also having a genetic defect in metabolism (auxotrophy), on the basis of which one or more metabolites essential for growth can no longer be synthesized, and the host organism is no longer able
Hessing Johanna
Pfaller Rupert
van den Hondel Cornelis
van Gorcom Robertus
Collard & Roe P.C.
Consortium für Electrochemische Industrie GmbH
Katcheves Tina
Yucel Remy
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