Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1992-06-11
1999-07-20
LeGuyader, John L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 691, 4351723, 435183, 43525411, 4352543, 4352545, 4353201, 536 231, 536 232, 536 237, C12Q 168, C12N 1580
Patent
active
059255153
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a transformant selection system, particularly for a strain of fungus, more particularly Penicillium chrysogenum.
Furthermore, this invention relates to a transformant selection marker, viz. the acetyl-CoA synthetase (facA) gene, isolated from Penicillium chrysogenum.
The filamentous fungus Penicillium chrysogenum is the most applied producer of the .beta.-lactam compounds penicillin G and penicillin V. Penicillins G/V are used as antibiotics themselves or they are chemically converted into semi-synthetic .beta.-lactams. P. chrysogenum has a long record of industrial application. Since the second world war it has been the microorganism of choice for large scale production of penicillins all over the world. Over the years significant improvements have been made in the yield of the penicillin production process, both by strain improvement and by process development. Strain improvement has been pursued by the application of random mutagenesis by chemical and physical means as well as by targeted mutagenesis of key enzymes in metabolic pathways connected to penicillin biosynthesis, followed by extensive selections for strains with increased penicillin titers. For reviews see for example Hersbach, G. J. M., Van der Beek, C. P. and Van Dijck, P. W. M., "The Penicillins: Properties, Biosynthesis and Fermentation" in Biotechnology of Industrial Antibiotics, E. van Damme (ed), Marcel Dekker (NY), 1984 and Rowlands, R. T., Enzyme Microb. Technol. 6, 1984, 3-10 and 290-300.
A novel approach to strain improvement has become possible with the development of recombinant DNA technology. Prerequisites for the application of recombinant DNA techniques to any organism or cell-line, are the availability of a gene transfer and a selection or detection system, which permits the identification of the usually small number of recombinant DNA containing transformed cells, in a vast majority of non-transformed cells. The development of efficient gene transfer and selection systems for .beta.-lactam producing industrial strains, particularly for P. chrysogenum is very difficult for two major reasons. First of all, it is a common observation that .beta.-lactam producing industrial strains are by far more difficult to transform than wild type or laboratory strains (Ingolia & Queener, Med. Res. Rev. 9, 1989, 245-264). The feature of impaired transformation of industrial strains might be related to the extensive mutagenesis of these strains. For example, mutations affecting the composition and/or assembly of the cell membrane or the cell wall are likely to accumulate in industrial strains leading to changes in morphology in the course of a strain improvement program. (Lein, in: Overproduction of Microbial Metabolites, Vanek and Hostalek (eds), 1986, Butterworth Publishers, 105-139). These morphological changes might for instance affect the generation of protoplasts from mycelium, the stability of protoplasts, their capacity for uptake of DNA, the regeneration of protoplasts into mycelium etc. Secondly, it is an additional requirement that the gene transfer and selection procedure should not affect the level of penicillin production.
The difficulties encountered in the development of transformant selection systems are also related to the limited knowledge of the genetic system of P. chrysogenum, which is difficult to study (e.g. because of the multinucleate nature of the filamentous mycelium and the absence of a sexual cycle, which only permits parasexual analysis (Pontecorvo et al., Adv. Genet. 5, 1953, 141-238)), the physical barriers which hinder the uptake of exogenous DNA (Peberdy, in: Biochemistry of Cell Walls and Membranes in Fungi; Kuhn, P. J., Trinci, A. P. J., Jung, M. J., Gossey, M. W., Copping, L. G. (eds) Springer-Verlag, Berlin 1989, 5-30) and the lack of DNA elements which allow for stable extra-chromosomal replication of the transforming DNA, which consequently results in very low transformation frequencies because the transforming DNA has to integrate into the genome of the host.
At this
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Bovenberg Roelof Ary Lans
Gouka R. J.
Van Gorcom Robert Franciscus Maria
Van Hartingsveldt Willem
Gist-Brocades B.V.
LeGuyader John L.
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