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
1999-05-14
2001-10-09
Schwartzman, Robert A. (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...
C435S252300, C435S252330, C435S254110, C435S254210, C435S254300, C435S254500, C435S320100, C435S471000, C435S483000, C435S484000, C530S350000, C536S023740, C536S024100, C536S024500
Reexamination Certificate
active
06300095
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the technical field of the expression of the gdh and hex genes of
Penicillium chrysogenum
and of the act gene also of
P. chrysogenum
and of
Acremonium chrysogenum
. From analysis of the nucleotide sequence of said genes the existence of a promoter region which includes the translation initiation site, and which can be used to construct powerful expression and secretion vectors that are useful both for
P. chrysogenum
and for
A. chrysogenum
and related species, is deduced. In addition, these promoters can be used to block gene expression by means of antisense constructs. The expression of other genes in filamentous fungi can be directed under the control of the aforesaid promoters, with the production of antibiotics and/or proteins inherent therein being increased.
PRIOR ART
P. chrysogenum
and
A. chrysogenum
are filamentous fungi which are of industrial interest because of their ability to produce penicillin and cephalosporin, respectively. During the last decade there has been considerable development of genetic manipulation techniques applicable in both microorganisms. The techniques for genetic manipulation of
P. chrysogenum
and
A. chrysogenum
include the transformation of protoplasts with vectors which use the phleomycin resistance gene (hereinafter called ble
R
gene) (Kolar, M. et al. (1988), Gene 62, 127-134) as a selection marker, as well as the expression of additional intact copies of genes of interest and the replacement of the promoter of the gene in question by another promoter which is able to improve its expression. The expression of homologous genes in fungi such as
P. chrysogenum
or
A. chrysogenum
can be negatively regulated, whereas in the case of heterologous genes it is possible that their promoter may not be efficiently recognized by the said fungi. With the aim of avoiding these problems, genes were identified and cloned which are expressed constitutively and in which the said expression preferably does not show negative catabolic regulation, called hereinafter strong promoters. In general it is considered that the high-expression genes have signals in the promoter region which facilitate high transcription levels and which play a fundamental role in functions implicated in primary cellular metabolism. These genes include: the genes which code for NADP-dependent glutamate dehydrogenase (EC.1.4.1.4) (hereinafter called gdh gene), &bgr;-N-acetylhexosaminidase (EC.3.2.1.52) (hereinafter called hex gene) and &ggr;-actin (hereinafter called act gene).
There are earlier references to the gdh, hex and act genes from microorganisms other than those which are used in the present invention. The most relevant bibliography includes: (I) the nucleotide sequence of the gdh gene of the fungus
Neurospora crassa
(Kinnaird, J. H. and Fincham, J. R. S. (1983), Gene 26, 253-260) as well as the regulation of the expression of the gdhA gene of
Aspergillus nidulans
(Hawkins, A. R. et al. (1989), Mol. Gen. Genet. 418, 105-111), (II) the cloning and expression of the hexl gene of
Candida albicans
(Cannon, R. D. et al. (1994), J. Bacteriol. 2640-2647) and (III) the characterization of the act gene of
A. nidulans
(Fidel, S. et al. (1988), Gene 70, 283-293). The expression of heterologous genes in
P. chrysogenum
using the promoters of the pcbC or penDE genes was described by Cantwell, C. A. et al. in 1992 (Proc. R. Soc. London Ser. B 248, 283-289). In addition, the expression of heterologous genes in
A. chrysogenum
using the promoters of the &bgr;-isopropyl malate dehydrogenase gene (Japanese Patent Laid Open Publication No. 80295/1989) and glyceraldehyde 3-phosphate dehydrogenase gene (European Patent Application 0376226A1/1989) has also been described.
The inactivation of gene expression in industrial strains is sometimes necessary for the elimination of undesirable enzyme activities. Owing to the fact that the level of ploidy of many industrial strains makes it difficult in most cases to block expression by direct gene disruption, it is necessary to use systems for inactivation of expression which are independent of the level of ploidy. The development of antisense constructs expressed under the control of strong promoters makes interruption of gene expression possible. Constructs of this type are especially useful in industrial strains owing to the fact that their levels of ploidy (Künkel et al. (1992) Appl. Microbiol. Biotech. 36, 499-502) make it difficult to obtain complete gene inactivation. The use of antisense constructs for blocking enzyme activities has been described in yeasts (Atkins, D. et al. (1994), Biol. Chem. H-S 375, 721-729) and plants (Hamada, T. (1996), Transgenic Research 5, 115-121; John, M. E. (1996) Plant Mol. Biol. 30, 297-306). The hex promoter has the special feature of coding for an extracellular enzyme, which allows it to be used for the expression of extracellular proteins.
There are no citations in the prior art, however, which describe either the gene sequences of the filamentous fungi used in the present invention or those of the enzymes synthesized by the expression thereof. Nor is there any description in said Prior Art of the use of the strong promoters of the genes of the fungi described in the present invention for the expression, secretion or inactivation of gene expression.
DETAILED DESCRIPTION OF THE INVENTION
The use of strong promoters to overexpress certain genes can lead to improvement in the production of penicillin or cephalosporin, and also to the synthesis of new antibiotics derived from the latter.
This invention describes a new process for obtaining strains of
P. chrysogenum
and
A. chrysogenum
with the ability to express homologous or heterologous genes under the control of strong promoters. The characterization and subsequent use of the promoters corresponding to the genes which code for NADP-dependent glutamate dehydrogenase (EC.1.4.1.4)—gdh gene—of
P. chrysogenum
, &bgr;-N-acetylhexosaminidase (EC.3.2.1.52)—hex gene—of
P. chrysogenum
and &ggr;-actin —act gene—of
P. chrysogenum
and
A. chrysogenum
are described. The use of said promoters to overexpress genes related to the biosynthesis of penicillin and/or cephalosporin in the above-mentioned strains is one of the aims of the present invention. These promoters can also be used to block gene expression by means of antisense constructs.
The present invention is based on
P. chrysogenum
and
A. chrysogenum
as nucleic acid donors. Once the genomic DNA had been purified, DNA libraries of both microorganisms were constructed as described in Examples 1 and 4, and they were screened with: (I) synthetic oligonucleotides corresponding to the gdh gene of
N. crassa
in order to clone the homologous gene of
P. chrysogenum
, (II) combinations of oligonucleotides synthesized on the basis of the amino terminal sequence of the enzyme &bgr;-N-acetylhexosaminidase in order to clone the hex gene of
P. chrysogenum
and (III) a fragment of the act gene of
A. nidulans
in order to clone the homologous genes of
P. chrysogenum
and
A. chrysogenum
. The clones purified by virtue of their ability to generate positive hybridization with the corresponding probe were subsequently analysed, the presence of the genes sought being determined.
The gdh gene of
P. chrysogenum
was identified in a 7.2 kb EcoRI fragment and in two BamHI fragments of 2.9 and 1.5 kb respectively. The restriction map of the DNA region which includes it is shown in FIG.
1
. The 2,816 nucleotide sequence (SEQ ID NO:1) was then determined, which includes an open reading frame (ORF) with a very marked preferential codon usage pattern, the ATG translation initiation codon of which was found in position 922 and the TAA translation termination codon in position 2,522. The presence of 2 introns of 159 bp and 56 bp was also determined between positions 971-1130 and 1262-1318 respectively. Said ORF codes for a protein of 49,837 Da, with an isoelectric point of 6.18, the 461 amino acid sequence of which (SEQ ID NO:5) has 72.4% identity with the ami
Barredo Fuente José Luis
Collados De La Vieja Alfonso J.
Diez Garcia Bruno
Moreno Valle Migeul Angel
Rodriguez Saiz Marta
Antibioticos S.A.
Ladas & Parry
Schwartzman Robert A.
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