Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Patent
1990-11-09
1994-04-12
Schwartz, Richard A.
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
435 691, 4351723, 4353201, 43525411, C12N 115, C12N 1580, C12P 2100
Patent
active
053025270
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the novel use of a marker system in the transformation of filamentous fungi. In particular it relates to the use of the nitrate reductase gene as a selection marker in transformations of the antibiotic-producing species Penicillium chrysogenum and Acremonium chrysogenum.
The development and exploitation of recombinant DNA technology in filamentous fungi has been slow, in part because of the lack of efficient and industrially usable systems for readily selecting those cells which have been transformed with the desired vector DNA. Transformation and hence stable inclusion of the desired DNA into the DNA sequence of the fungal cell is a rare event and thus such a selection system for determining those few cells, perhaps among millions of untransformed cells, which have acquired the desired DNA is a necessity. Various systems have been proposed using the presence of a marker gene in the vector along with the desired DNA, by which the transformed cells can be recognised and selected.
Thus selection of successfully transformed cells of filamentous fungi has previously been demonstrated with, for example, the acetamidase (amdS) gene which confers upon transformants the ability to utilise acetamide as the sole source of nitrogen [see for instance Kelly & Hynes, EMBO J, 4, p475 (1985)] and hence provides a method of identifying such transformants. A characteristic of filamentous fungal transformation is a low efficiency of transformation, since exogenous DNA is not readily and stably integrated into the host genome. This results in a high background level of non-transformants and, in the case of the amdS selection marker system, it has been found that some of these non-transformants will also grow on acetamide as the sole nitrogen source, even though they are deficient in the expression of the acetamidase gene. This makes the amdS marker selection system difficult to operate, particularly where there is a low efficiency of transformation, since it does not clearly distinguish those cells which have undergone transformation.
This problem has been to some extent overcome by the development of antibiotic selection as a marker method. For example, transformation with a vector which includes a phosphotransferase gene as a marker confers upon the transformants resistance to the aminoglycosidic antibiotics G418 and hygromycin B [see for instance Punt et al, Gene, 56, p117 (1987)]. Transformed cells may then be recognised by their ability to grow on a medium containing one or other of these antibiotics. Although this system provides a clear-cut method of selecting transformants, the use of the resultant antibiotic resistant fungal strains on a large industrial scale is undesirable since it requires a high degree of containment making industrial exploitation both difficult and expensive. Furthermore, it is considered desirable in the use of such strains to maintain a selection pressure by the presence of antibiotic in the fermentation medium, in order to prevent growth of revertants where the integrated genes are not stably maintained; such continuous use of antibiotic in the fermentation medium is, of course, disadvantageous.
Other marker systems for filamentous fungi have been described, such as the reversions of arginine auxotrophy using the argB gene [Buxton et al, Gene, 37, p207 (1985)], tryptophan C auxotrophy using the trpC gene [Sanchez et al, Gene 51, p97 (1987)] or uracil auxotrophy using the pyr-4 or pyrG genes [Cantoral et al, Biotechnology, 5, p494 (1987)]. These techniques, however, require complicated and lengthy procedures involving mutation and selection to obtain suitable auxotrophic mutants. Also such procedures often lead to a loss of antibiotic productivity in antibiotic producing strains of filamentous fungi.
There is a need, therefore, to develop a marker system for the identification of transformed filamentous fungi where isolation of suitable mutants which are deficient in the expression of the marker gene is straightforward and selection procedures provide clear and
REFERENCES:
Arst et al., 1979, Mol-Gen. Genet., 174:89-100.
Fu et al., 1987, PNAS, 84:8243-8247.
Timberlake et al., Science, 244, 1313-1317, 1989.
Unkles et al., Molecular and General Genetics, 99, 99-104, 1989.
Daboussi et al., Current Genetics, 15, 453-456, 1989.
Batt et al., Canadian Journal of Microbiology, 28, 1206-1209, 1982.
Unkles, Molecular and Genetic Aspects of Nitrate Assimilation, chapter 22, (1989 Symposium Report), Ed. J. L. Ray, J. R. Kinghorn, Oxford University Press.
Birkett John A.
Kinghorn James R.
Glaxo Group Limited
Schwartz Richard A.
Vogel Nancy T.
LandOfFree
Nitrate reductase as marker for filamentous fungi does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nitrate reductase as marker for filamentous fungi, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nitrate reductase as marker for filamentous fungi will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2098199