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
1998-11-23
2001-11-13
McKelvey, Terry (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...
C435S243000, C435S254210, C435S254300, C435S254600, C435S320100, C435S325000, C435S410000, C536S023740
Reexamination Certificate
active
06316220
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a transcription factor found in filamentous fungi, especially in Aspergillii, DNA sequences coding for said factor, its transformation into and expression in fungal host organisms, and the use of said factor in such hosts for increasing the expression of a polypeptide of interest being produced by said host.
BACKGROUND OF THE INVENTION
Transcription factors are well known proteins involved in the initiation of transcription. They have been studied intensively in many different organisms and have also been described in fungi. Dhawale and Lane (NAR (1993) 21 5537-5546) have recently compiled the transcription factors from fungi, including the filamentous fungi.
Many of the transcription factors are regulatory proteins; they bind to the promoter DNA and either activate or repress transcription as a response to stimuli to the cell.
The expression of the &agr;-amylase gene in
A. oryzae
is regulated in response to the available carbon source. The gene is expressed at its maximum when the organism is grown on starch or maltose (Lachmund et al. (1993)
Current Microbiology
26 47-51; Tada et al. (1991)
Mol. Gen. Genet.
229 301-306). The expression of &agr;-amylase is regulated at the transcriptional level as shown by Lachmund et al. (supra), which strongly suggests that transcription factors are involved in the regulation, but so far no gene for such a factor has been identified.
The promoter of the &agr;-amylase gene has been studied by deletion analysis (Tada et al. (1991)
Agric. Biol. Chem.
55 1939-1941; Tsuchiya et al. (1992)
Biosci. Biotech. Biochem.
56 1849-1853; Nagata et al. (1993)
Mol. Gen. Genet.
237 251-260). The authors of these papers propose that a specific sequence of the promoter is responsible for the maltose induction. Nagata et al. (supra) is used this sequence as a probe in a gel shift experiment to see whether any proteins from
A. nidulans
nuclear extracts were able to bind to the promoter sequence. Three such proteins were found, but no involvement of these proteins in expression was shown. None of the proteins have been purified or identified by other means. Their genes likewise remain unknown.
SUMMARY OF THE INVENTION
The present invention relates to a transcription factor regulating the expression of the x-amylase promoter in filamentous fungi.
Accordingly, in a first aspect the invention relates to a DNA construct comprising a DNA sequence encoding a transcription factor of the invention, which DNA sequence comprises
a) the transcription factor encoding part of the DNA sequence cloned into plasmid pToC320 present in
E. coli
ToC1058, DSM 10666, or
b) an analogue of the DNA sequence defined in a), which
i) is at least 60% homologous with the DNA sequence defined in a), or
ii) hybridizes with the same nucleotide probe as the DNA sequence defined in a), or
iii) encodes a transcription factor which is at least 500homologous with the transcription factor encoded by a DNA sequence comprising the DNA sequence defined in a), or
iv) encodes a transcription factor which is immunologically reactive with an antibody raised against the purified transcription factor encoded by the DNA sequence defined in a), or
v) complements the mutation in ToC879, i.e. enables ToC879 to grow on cyclodextrin and produce lipase when transformed with said DNA sequence.
The full length genomic DNA sequence encoding a transcription factor has been derived from a strain of the filamentous fungus
Aspergillus oryzae
and has been cloned into plasmid pToC320 present in
E. coli
ToC1058, DSM 10666.
Said transcription factor encoding DNA sequence harboured in pToC320, DSM 10666, is believed to have the same sequence as that presented in SEQ ID NO: 1 and SEQ ID NO: 2. Accordingly, whenever reference is made to the transcription factor encoding part of the DNA sequence cloned into plasmid pToC320 present in DSM 10666 such reference is also intended to include the transcription factor encoding part of the DNA sequence presented in SEQ ID NO: 1 and SEQ ID NO: 2.
Accordingly, the terms “the transcription factor encoding part of the DNA sequence cloned into plasmid pToC320 present in DSM 10666” and “the transcription factor encoding part of the DNA sequence presented in SEQ ID NO: 1 and SEQ ID NO: 2” may be used interchangeably.
In further aspects the invention provides an expression vector harbouring the DNA construct of the invention, a cell comprising said DNA construct or said expression vector and a method of producing a peptide exhibiting transcription factor activity, which method comprises culturing said cell under conditions permitting the production of the transcription factor.
Such a transcription factor of the invention will typically originate from a filamentous fungus.
The term “filamentous fungus” is intended to include the groups Phycomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and fungi imperfecti, including Hyphomycetes such as the genera Aspergillus, Penicillium, Trichoderma, Fusarium and Humicola.
The invention also relates to a method of producing a filamentous fungal host cell comprising the introduction of a DNA fragment coding for any such factor into a filamentous fungus wherein an &agr;-amylase promoter or a co-regulated promoter regulates the expression of a polypeptide of interest in a manner whereby said factor will be expressed in said fungus.
In a further aspect the invention relates to a method of producing a polypeptide of interest, the expression of which is regulated by an &agr;-amylase promoter or a co-regulated promoter, comprising growing a filamentous fungal host cell as described above under conditions conducive to the production of said factor and said polypeptide of interest, and recovering said polypeptide of interest.
Finally the invention relates to the use of said factor for regulating the expression of a polypeptide of interest in a filamentous fungus.
In this context, regulation means to change the conditions under which the factor of the invention is active. This could mean different pH, substrate, etc. regimes, whereby the resulting effect is an improved regulation of the expression of the protein of interest.
Furthermore, regulation also comprises events occurring in the growth phase of the fungus during which the transcription factor is active. Depending on the circumstances, both advancing and/or postponing the phase wherein the factor is active may enhance the expression and thus the yield.
In addition, using standard procedures known in the art, the specific DNA sequences involved in the binding of a transcription factor may be identified, thereby making it possible to insert such sequences into other promoters not normally regulated by the factor and enabling those promoters to be under the regulation of said factor.
REFERENCES:
Berendsen, Science, vol. 282, pp. 642-643, Oct. 1998.*
Gene 1994, 145(2), 179-187, Verdoes, Jan C. et al., “The Effect of Multiple Copies of the Upstream Region on Expression of theAspergillus NigerGlucoamylase Encoding Gene”.
Appl. Microbiol Biotechnol (1995) 43: 195-205, Verdoes JC et al: “Molecular-Genetic Strain Improvement For the Overproduction of Fungal Proteins by Filamentous Fungi”.
Mol. Gen. Genet (Germany) Feb. 1993, 237 (1-2) pp. 251-260, Nagata O. et al.:Aspergillus NidulansNuclear Proteins Bind To A CCAAT Element and the Adjacent Upstream Sequence in the Promoter Region of the Starch-Inducible Taka-Amylase A Gene.
Nucleic Acids Research, 1993, vol. 21, No. 24, 5537-5546, Dhawale, Shree S. et al.: “Complilation of Sequence-Specific DNA-Binding Proteins Implicated in Transcriptional Control in Fungi”.
Mol. Gen Genet (1991), 229: 301-306, Tada, Setsuzo et al., “Construction of a Fusion Gene Comprising the Taka-Amylase A Promoter and theEscherichia Coli&bgr;-Glucuronidase Gene and Analysis of its Expression inAspergillus Oryzae”.
Current Microbiology vol. 26 (1993), pp. 47-51, Lachmund, Astrid et al., Regulation of &agr;-Amylase Formation inAspergillus OryzaeandAspergillus NidulansTransformants.
Journal of Bacteriology, Jan. 1992, vol.
Garbell Jason T.
Lambiris Elias J.
McKelvey Terry
Novozymes A/S
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