Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-03-02
2001-12-04
Cochrane Carlson, Karen (Department: 1653)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023700, C536S024320, C536S023200, C530S350000, C530S324000, C530S300000, C435S069100, C435S252300, C435S320100, C435S006120, C435S173300, C435S069800, C435S254110, C435S007100
Reexamination Certificate
active
06326477
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to recombinant-DNA-technology, and particularly to genes involved in the control of basic metabolic processes in fungi. The invention specifically provides mutated glucose repressor genes to be transformed to fungal strains in order to render the strains capable of producing elevated amounts of secreted proteins without reducing the viability of the strain.
BACKGROUND OF THE INVENTION
Carbon catabolite repression is a major mechanism controlling metabolic processes of both prokaryotic and eukaryotic microbes. In the presence of readily metabolizable carbon sources such as glucose the expression of structural genes required for utilization of other alternative carbon sources is decreased. Cellulases and other secreted hydrolase enzymes are proteins whose production is repressed by glucose. In general, glucose repression is mediated through the action of specific proteins that regulate transcription by binding promoter sequences of the target genes (Trumbly, 1992).
The creA gene of the filamentous fungus
Aspergillus nidulans
is a well characterized regulatory gene mediating carbon catabolite repression (Arst and Bailey, 1977). The central role of the CREA protein of
A. nidulans
in carbon catabolite repression has been verified extensively by genetic (Arst et al., 1990) and molecular analysis (Mathieu and Felenbok, 1994). Mutations of the creA gene are recessive to the wild type gene. Some of the mutants have abnormal morphology (Arst et al., 1990), and haploid
A. nidulans
creA
−
strains constructed by genetic engineering, lacking the entire protein coding region of the creA gene (Dowzer and Kelly, 1991) have extremely reduced viability. Additionally, the carbon metabolism in the mutant strains is altered in different ways. The most extreme of the in vivo isolated creA mutants with respect to growth impairment is the creA30 mutation that additionally results in formation of an abnormally compact colony. Molecular analysis of the creA30 gene sequence revealed that the gene is truncated at the 3′ end corresponding to a protein having two intact zinc fingers but lacking the sequences C-terminal to the DNA-binding region (Dowzer and Kelly, 1991). This is the most extreme mutation of creA described so far.
The CREA protein has two zinc fingers of C
2
H
2
type involved in base recognition. The CREA fingers are strikingly similar to the ones of the glucose repressor MIG1 of
Saccharomyces cerevisiae
. In addition, the C-terminal one of the two fingers has significant similarities with the fingers found in mammalian early growth response proteins involved in developmental regulation. Obvious sequence similarities in other parts of the proteins are, however, missing Nehlin and Ronne, 1990). Another yeast gene RGR1, is also involved in glucose repression. The phenotypic effects of the rgr1-1 mutation somewhat resemble those of crea mutant alleles, and a RGR1
−
strain is not viable (Sakai et al., 1990).
Similarities in carbon catabolite repression have thus been found between yeast and filamentous fungi, and some of the basic features of glucose repression are probably universal in eukaryotic microorganisms. Considering that crea has such a central role in Aspergillus as a regulator of different sets of genes that are subject to carbon catabolite repression, it is likely that similar mechanisms mediating glucose repression are found in related organisms. Besides
A. nidulans
, the creA gene has so far been isolated only from the closely related species,
A. niger
(Drysdale et al., 1993).
DESCRIPTION OF THE INVENTION
In order to address the role of creA in other filamentous fungi and especially in relation to cellulase expression, we have isolated the creA equivalent cre1 from the filamentous fungus
Trichoderma reesei
which is one of the most extensively studied cellulolytic organisms (reviewed e.g. by Nevalainen and Penttilä, 1995). The production of cellulases is dependent on the carbon source available, and glucose repression is very tight. The expression level of the major cellobiohydrolase 1 (cbh1) is up to several thousand fold higher on media containing inducing carbon sources such as cellulose or sophorose compared with glucose containing media (Penttilä et al., 1993). Deletion analysis of the cbh1 promoter has suggested that a crea equivalent would be involved in glucose repression of cellulase expression in
T. reesei
(Penttilä et al., 1993). In addition to the cre1 gene of
T. reesei
, we isolated the same gene from the related species
T. harzianum
. The expression studies of the
T. reesei
cre1 gene revealed unexpected indication of autoregulation. Furthermore, a hypercellulolytic
T. reesei
strain was found to express a truncated form of the cre1 gene with an unexpected structure and properties. The truncated cre1 gene of the strain
T. reesei
Rut-C30 was named cre1-1.
The present invention thus provides a mutated fungal glucose repressor gene which, when transformed to a fungal strain maintains said strain fully viable and renders said strain capable of producing elevated amounts of secreted proteins, even in the presence of glucose. Furthermore, this strain has an altered glucose uptake, as well as altered growth characteristics on certain nitrogen sources.
Consequently, a further object of this invention is the use of said gene in a process for enhancing the production of secreted proteins in a fungal host, which process comprises transforming the truncated cre1 gene according to the invention into a suitable fungal host, and replacing the existing cre1 gene, cultivating the transformed host in a suitable growth medium comprising glucose, and recovering the protein produced.
According to the present invention it is also possible to control the growth rate of a fungus by regulating the type of nitrogen source and the amount of glucose in the medium.
Fungal strains transformed with the truncated cre1 gene are also provided.
Unexpected results were obtained when studying the expression of the
T. reesei
cre1 gene. If glucose repression mediated through CREI was regulated at the level of cre1 transcription, one would expect increase in transcription in the presence of glucose. However, high expression of cre1 does not correlate positively with glucose repression, in fact when glucose is present in repressing amounts the level of cre1 transcript is relatively low, and higher on the neutral carbon sources sorbitol and glycerol, and on cellulose. Furthermore, addition of glucose to cultivations carried out on the neutral carbon sources reduced the level of cre1 transcript, which is indicative of autoregulation.
Further indication for autoregulation was obtained when studying expression of the mutated form of the glucose repressor gene cre, i.e. cre1-1 of the hypercellulolytic
T. reesei
strain Rut-C30. Nearly 80% of the protein coding region, including one of the two zinc fingers, is deleted as compared to the “native” cre1 gene of
T. reesei
QM9414. The
T. reesei
cre1-1 mutation could suggest that the N-terminal zinc finger could be sufficient for DNA-binding and thus retaining most of the viability of the fungus. However, the strain Rut-C30 produces relatively high levels of cre1-1 mRNA also on glucose medium compared with the strain QM9414. Assuming that CREI down regulates its own expression when growing on glucose medium, the abundancy of cre1-1 mRNA may be explained by the inability of the CREI-1 protein to bind the target sequences in its own promoter, or by the inability of the truncated form of the protein to confer repression of the gene. This should hold true also for other genes regulated by cre1, which is here shown by derepression of the cellulase gene cbh1.
The high cellulase cbh1 mRNA levels detected in the presence of glucose in combination with the mutation resulting in the truncated form of cre1, support true carbon catabolite derepression in this strain. The results obtained with Rut-C30 now further support the suggestion that cellulase expression is under control of car
Ilmen Marja
Penttila Merja
Söderlund Hans
Carlson Karen Cochrane
Crowell & Moring LLP
Robinson Hope
Valtion teknillinen tutkimuskeskus
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