Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1997-04-03
2003-04-15
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S320100, C435S254110, C435S252300, C435S069100, C536S023200
Reexamination Certificate
active
06548285
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of molecular biology, in particular the invention is concerned with selectable marker genes to be used in transformation of organisms.
BACKGROUND OF THE INVENTION
The
Aspergillus nidulans
amdS gene is probably the most frequently used selectable marker for the transformation of filamentous fungi and has been applied in most of the industrially important filamentous fungi such as e.g.
Aspergillus niger
(Kelly and Hynes 1985, EMBO J. 4: 475-479),
Penicillium chrysogenum
(Beri and Turner 1987, Curr. Genet. 11: 639-641),
Trichoderma reesei
(Pentillä et al. 1987, Gene 61: 155-164),
Aspergillus oryzae
(Christensen et al. 1988, Bio/technology 6: 1419-1422) and
Trichoderma harzianum
(Pe'er et al. 1991, Soil Biol. Biochem. 23: 1043-1046).
The popularity of the amdS gene as a selectable marker is most likely a result of the fact that it is the only available non-antibiotic marker gene which can be used as a dominant selectable marker in the transformation of fungi. Dominant selectable markers provide the advantage that they can be used directly in any strain without the requirement for mutant recipient strains. The antibiotic-resistance genes are, however, not preferred for use in industrial strains because the regulatory authorities in most countries object to the use of antibiotic markers in view of the potential risks of spread of antibiotic-resistance genes in the biosphere upon large-scale use of production strains carrying such genes.
The amdS gene has been used as a dominant marker even in fungi known to contain an endogenous amdS gene, i.e.
A. nidulans
(Tilburn et al. 1983, Gene 26: 205-221) and
A. oryzae
(Gomi et al. 1991, Gene 108: 91-98). In these cases the background of non-transformants can be suppressed by the inclusion of CsCl in the selection medium. In addition, high-copynumber transformants are provided with a growth advantage over the non-transformants (when acetamide is the sole nitrogen-source) because of the higher gene dosage.
Apart from the
A. nidulans
and
A. oryzae
amdS genes, by coincidence a sequence was found in the genome of the yeast
Saccharomyces cerevisiae
, which shows homology to the
A. nidulans
amdS gene (Chang and Abelson 1990, Nucleic Acids Res. 18:7180). The yeast amdS-like sequence was shown not to be essential in yeast. It is, however, not known whether the yeast amdS-like gene actually encodes a protein with amidase activity which might allow to use the gene as selectable marker. amdS genes have not been found in other fungi, despite attempts to detect such genes with heterologous hybridization using the
A. nidulans
amdS gene as probe (see e.g. Kelly and Hynes 1985 EMBO J. 4: 475-479). This is also in line with the observation that, in contrast to
A. nidulans
and
A. oryzae
, most fungi grow very poor, if at all, on acetamide (see e.g. Beri and Turner 1987, Curr. Genet. 11: 639-641; Pentillä et al. 1987, Gene 61: 155-164). The cloning and sequencing of two bacterial acetamidase genes has been reported, i.e. those of
Pseudomonas aeruginosa
(Brammar et al. 1987, FEBS Lett. 215: 291-294) and of
Mycobacterium smegatis
(Mahenthiralingam et al. 1993, J. Gen. Microbiol. 139: 575-583). However, these bacterial acetamidases appear to be unrelated to the above mentioned fungal acetamidases since no sequence similarities can be detected and the bacterial acetamidases are also much smaller than their fungal counterparts. No reports of the use of these bacterial acetamidases as selectable markers have appeared.
In addition to its dominant character, the amdS selectable marker provides the advantage of being a bidirectional marker. This means that, apart from the positive selection for the presence of the amdS gene using acetamide as sole carbon- or nitrogen-source, a counterselection can be applied using fluoracetamide to select against the presence of the amdS gene (Hynes and Pateman 1970, Mol. Gen. Genet. 108, 107-106). The fluoracetamide counterselection has been applied to cure genetically engineered strains from recombinant constructs carrying the amdS gene (e.g. Ward et al. 1993, Appl. Microbiol. Biotechnol. 39, 738-743).
A disadvantage of the amdS marker is the fact that the
A. nidulans
amdS gene is a heterologous gene in industrial fungi such as
A. niger, A. oryzae, T. reesei
and
P. chrysogenum
. Even though this may seem trivial to most molecular biologists, regulatory authorities often object that production strains containing the heterologous
A. nidulans
amdS gene posses a new (the gene being heterologous) and unnecessary (the marker gene not being necessary once the transformant strain is obtained) property, the risks of which cannot be foreseen. Unfortunately, the only industrial filamentous fungus for which an homologous amdS gene is available is
A. oryzae.
We have previously addressed this problem by developing a method to obtain recombinant fungal production strains that are free of selectable markers (EP-A-0 635 574). In this method the bidirectionality of the amdS marker is used to remove the marker from specially constructed expression cassettes once they have been introduced in the fungal genome. The method is, however, less compatible with the high copy numbers which are often necessary in industrial production strains. For these situations, a homologous and dominant selectable marker would still be required.
SUMMARY OF THE INVENTION
The present invention discloses novel DNA sequences encoding acetamidase genes from fungi other than
Aspergillus nidulans, Aspergillus oryzae
and
Saccharomyces cerevisiae.
Preferably, these DNA sequences encode acetamidases which comprise an internal consensus fragment, the amino acid positional identity of which is less than 100% when compared with each of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, whereas this amino acid positional identity is more than 30% when compared with one of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
The invention also discloses recombinant DNA constructs comprising the DNA sequences encoding the acetamidases of the invention, as well as recombinant cells containing these constructs.
The invention further discloses recombinant cells in which an endogenous copy of the gene encoding the acetamidase of the invention has been inactivated.
In a further embodiment, the invention discloses a process in which the recombinant cells of the invention are cultured in order to obtain a product of interest.
Finally, the invention discloses methods for obtaining the acetamidase genes of the invention, as well as methods for the inactivation of endogenous copies of these acetamidase genes.
REFERENCES:
patent: 0 635 574 (1995-01-01), None
Chang, T. et al., Nucl. Acids Res., vol. 18, No. 23, p. 7180, 1990.*
Gomi, K. et al., Gene, vol. 108, No. 1, pp. 91-98, 1990.*
Corrick, C. et al., Gene, vol. 53, pp. 63-71, Jul. 13, 1987.*
Kuwahara, M. et al., J. Ferment. Technol., vol. 59, No. 6, pp. 573-577, 1980.*
Van der Walt, J. et al., System. Appl. Microbiol., vol. 16, pp. 330-332, 1993.*
Price, V. et al., Meth. Enzymol., vol. 185, pp. 308-318, 1990.*
Lee, C.C. et al., Science, vol. 239, No. 239, pp. 1288-1291, 1988.*
Masuda, Y. et al., Curr. Gen., vol. 25, pp. 412-417, 1994.*
Hashimoto, Y. et al., Bioch. Biophys. Acta, vol. 1088, pp. 225-233, 1991.*
Beri, R.K., et al., “Transformation ofPenicillium chrysogenumusing theAspergillus nidulansamdS gene as a dominant selective marker,”Curr Genet(1987) 11:639-641.
Brammar, W.J., et al., “The nucleotide sequence of the amiE gene ofPseudomonas aeruginosa,”FEBS Letters(1987) 215(2):291-294.
Christensen, T., et al., “High Level Expression of Recombinant Genes inAspergillus oryzae,”Bio/Technology(1988) 6:1419-1422.
Hynes, M.J., et al., “The Genetic Analysis of Regulation of Amidase Synthesis inAspergillus nidulans,”Molec Gen Genetics(1970) 108:107-116.
Katsuya, G., et al., “Cloning and molecular characterization of the acetamidase-encoding gene(amdS) fromAspergillus oryzae
Bakhuis Janna G.
Bovenberg Roelof A. L.
Selten Gerardus C. M.
Swinkels Bart W.
Vollebregt Adrianus W. H.
DSM N.V.
Morrison & Foerster / LLP
Prouty Rebecca E.
Steadman David J
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