Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-02
2002-08-20
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S069800, C435S455000, C435S320100, C435S471000, C435S484000, C435S477000, C435S478000, C435S254110, C435S254300, C435S254400, C435S254500, C435S254600, C435S254700, C435S254800, C435S254900, C536S023100, C536S023200, C536S023700, C536S024100
Reexamination Certificate
active
06436643
ABSTRACT:
The invention relates to a process for site-directed integration of multiple copies of a gene in a mould, to a transformed mould obtainable by such process, to a process for culturing such transformed mould, and to a process for producing and optionally secreting a desired protein by culturing such transformed mould. In particular, the invention provides a process for preparing a protein by a mould transformed by multicopy integration of at least one expressible gene comprising a structural gene encoding a desired protein into the genome of a mould, especially of moulds belonging to the genus Aspergillus.
In this specification the expression “expressible gene” means a structural gene encoding a protein, either homologous or heterologous to the host organism, in combination with DNA sequences for proper transcription and translation of the structural gene, and optionally with secretion signal DNA sequences, which DNA sequences should be functional in the host mould. Further, in this specification the expressions “mould” and “filamentous fungus” are considered as synonyms.
BACKGROUND OF THE INVENTION AND PRIOR ART
1. Filamentous fungi and especially species such as
Aspergillus awamori, Aspergillus niger, Trichoderma reesei
and
Fusarium graminearum
have shown to be attractive hosts for large scale production of homologous and heterologous proteins. They have the capacity to secrete substantial amounts of protein into the medium, large scale fermentation is generally well established and most of them they have a GRAS (Generally Recognized As Safe) status, which makes it possible to use these species in the food and food-processing industry. Moreover, the mould
Fusarium graminearum
A 3/5, the Quorn
R
myco-protein fungus, has also been used as a commercial human food source in the UK for over 10 years (Royer et al.; Bio/Technology 13 (1995) 1479-1483).
The production of fungal proteins, of either homologous or heterologous origin, by filamentous fungi is usually very efficient and production levels of grams per liter were reached. However, compared to this the production levels of heterologous proteins of mammalian, bacterial or plant origin in moulds are relatively low. In order to improve the production of both homologous and heterologous proteins several strategies have been developed. The basic strategy that is commonly applied to achieve higher protein production in moulds is the introduction of multiple copies of the gene encoding the desired protein.
2. Whereas moulds have been successfully used for the production of enzymes, antibody fragments and peptides at laboratory and commercial scale (xylanase, pectinase, etc), the acceptance of products from these genetically modified organisms (GMO) in the market has experienced some unexpected difficulties in the past few years.
(a) In general there is a growing concern about the use of antibiotic resistance genes in genetically modified organisms. The main reason for this concern is the possibility that such a gene might be transferred into and expressed in gut micro-organisms, which would thereby become antibiotic resistant (“Report on the use of antibiotic resistance markers in genetically modified food organisms” published by the Advisory Committee on Novel Foods and Processes, Ministry of Agriculture, Fisheries and Food, England, 1994).
(b) Further, the presence of other foreign DNA such as remnants of vector DNA used in cloning is also undesired.
(c) Another concern is the fact that in general the genetically modified strains contain randomly integrated genetic material. In the perception of some consumer organisations this would constitute an unpredictable safety risk, and could mean a barrier to the acceptation of derived products.
3. Therefore, the recombinant mould should ideally contain multiple copies of the gene encoding the desired protein integrated at only a predetermined locus in the genome and no other foreign DNA should be present in order to produce proteins in moulds in both an economically attractive manner and in a way that deals with the concerns about genetically modified organisms as described above.
The generation of mould strains that meet these criteria has not been reported in literature.
The commonly applied system for integration of single or multiple copies of a gene into the genome of moulds, e.g. Aspergillus, Trichoderma and
Fusarium graminearum
makes use of plasmids which in addition to the gene encoding a desired protein contain bacterial marker genes encoding resistance to antibiotics (e.g. Ampicillin) and other vector sequences. Therefore, genetically modified moulds will usually contain antibiotic resistance genes and other vector DNA.
Whereas, targeted integrations of a single gene copy have been described regularly (e.g. Timberlake, “Gene Cloning and Analysis” (Chapter 3) in the book “More Gene Manipulations in Fungi” (1991) 51-85, edited by Bennett and Lasure; Gouka et al. Applied and Environmental Microbiology 62 (1996) 1951-1957) it has been proven to be very difficult to obtain mould strains that contain multiple gene copies integrated at a predetermined locus in the genome. Gouka et al. (Curr. Genet. 27 (1995) 536-540) reported the selection of targeted multi-copy integrations at the pyrG locus in
A. awamori
, but the recombinant strains were obtained from transformations in which DNA was used containing vector sequences and no information was presented on the number of gene copies that were integrated at the pyrG locus. For
Aspergillus nidulans
a similar observation on targeted tandem integration at the argB locus was published (Van den Hondel and Punt, “Gene transfer systems and vector development” (Chapter 1) in the book “Applied Molecular Genetics” (1991) 1-28, edited by Peberdy et al.).
Several other publications indicate that site-directed integration of multiple gene copies could not be obtained, although it was desired for scientific or commercial purposes, (Kubicek-Pranz et al. J. of Biotech. 20 (1991) 83-94; Van den Hondel et al. Antonie van Leeuwenhoek 61 (1992) 153-160; Verdoes et al. Transgenic Research 2 (1993) 84-92; Archer et al. Antonie van Leeuwenhoek 65 (1994) 245-250; Van Gemeren et al. Applied Microbiology and Biotechnology 45 (1996) 755-763; Van Gemeren, “Expression and secretion of defined cutinase variants by
Aspergillus awamori
” (Chapter 5) Thesis University of Utrecht (1997) ISBN 90-393-1229-X).
4. Previously, two processes have been described in literature that, in principle, might allow the generation of mould strains that contain multiple copies of a gene that are integrated at a predetermined locus in the genome without the presence of other foreign DNA.
The first process describes the preparation of a protein by a fungus transformed by site-directed multicopy integration of an expression vector in the ribosomal DNA locus of the fungal genome as described in International PCT patent application WO-A-91/00920; Unilever, published Jan. 24, 1991. Although the Examples were carried out with yeasts, it was envisaged that such process is also applicable to moulds. Thus such process could make it possible to construct a mould strain in which multiple copies of a gene are integrated at a predetermined locus of the genome, without the presence of other foreign DNA.
However, transformation of moulds follows a somewhat different pattern than the transformation of yeasts. Whereas in the yeast
Saccharomyces cerevisiae
transforming DNA is integrated into the genome of the cell via homologous recombination at the corresponding homologous site, in filamentous fungi such as the mould
Aspergillus awamori
DNA integrates mainly via illegitimate recombination at random sites in the genome (Finkelstein, “Transformation” Chapter 6 in the book “Biotechnology of Filamentous Fungi” (1992) 113-156, edited by Finkelstein and Ball). For instance, for the mould
A. awamori
Gouka et al. (Curr. Genet. 17 (1995) 536-540) performed an analysis on a large number of transformants and showed that DNA integrated via homologous recombination in approximately 10% of the trans
Beijersbergen Alida Godelieve Maria
de Groot Marcellus Johannes Augustinus
Musters Wouter
Guzo David
Morgan & Lewis & Bockius, LLP
Unilever Patent Holdings BV
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