Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reissue Patent
2000-08-16
2001-11-13
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S254200, C435S471000
Reissue Patent
active
RE037447
ABSTRACT:
The present invention relates to new genetically modified yeasts belonging to the genus Kluyveromyces and to their use to produce advantageously recombinant proteins.
The advances accomplished in the field of molecular biology have made it possible to modify microorganisms in order to make them produce proteins of interest and for example heterologous proteins (mammalian proteins, artificial proteins, chimetic proteins, and the like). In particular, numerous genetic studies have been performed on the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae. More recently, genetic tools have been developed so as to use the yeast Kluyveromyces as host cell for the production of recombinant proteins. The discovery of the plasmid pKD1, derived from K.drosophilarum (EP 241 435), has made it possible to develop a particularly advantageous host vector system for the secretion of recombinant proteins (EP 361 991, EP 413 622).
However, the application of this system of production is still limited, in particular by the problems of the efficacy of gene expression in these recombinant microorganisms, by the problems of stability of the plasmids and also by the problems of degradation of the recombinant products by the cells in which they are synthesized. A proteolysis phenomenon can indeed manifest itself during transit of the protein of interest in the secretory pathway of the recombinant yeast, or by the existence of secreted proteases or proteases present in the culture medium following an undesirable cell lysis which occurs during fermentation.
The applicant has now shown that it is possible to improve the levels of production of the said recombinant proteins, that is to say in their integral form, in Kluyveromyces yeasts, by modifying at least one gene encoding a cellular protease, and especially a protease transiting through the secretory pathway. Surprisingly, the applicant has furthermore shown that such modifications are particularly advantageous since they make it possible to increase the levels of production of recombinant proteins, and this is all the more advantageous since the said modification is without apparent effect on the growth rate and the viability of the modified cells under industrial fermentation conditions. Still surprisingly, the applicant has also shown that the said modifications do not affect the stability of the transformant yeasts, which makes it possible to use the said yeasts in a particularly advantageous manner to produce recombinant proteins.
The subject of the present invention is therefore yeasts of the genus Kluyveromyces having one or more genetic modifications of at least one gene encoding a protease, modifying the proteolytic activity of the said yeasts. Preferably, the genetic modification(s) render the said gene partially or totally incapable of encoding the natural protease. In another preferred embodiment of the invention, the gene(s) thus genetically modified encode a non-functional protease, or a mutant having a modified proteolytic activity spectrum. In another preferred embodiment of the invention, the gene(s) encoding the said proteases are placed under the control of a regulated promoter.
The yeasts of the genus Kluyveromyces according to the invention comprise the yeasts as defined by van der Walt [in: The Yeasts (1987) N. J. W. Kregervan Rij (ed): Elsevier: p.224], and preferably the yeasts K.marxianus var.lactis (K.lactis), K.marxianus var. marxianus (K.fragilis), K.marxianus var. drosophilarum (K.drosophilarum), K.waltii, and the like.
Genetic modification should be understood to mean more particularly any suppression, substitution, deletion or addition of one or more bases in the gene(s) considered. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or alternatively by exposing the said yeasts to a treatment by means of mutagenic agents. As mutagenic agents, there my be mentioned for example physical agents such as energetic radiation (X, g, ultra violet rays and the like), or chemical agents capable of reacting with various functional groups of the bases of DNA, and for example alkylating agents [ethyl methanesulphonate(EMS), N-methyl-N′-nitro-nitrosoguanidine, N-nitroquinoline 1-oxide (NQO)], bialkylating agents, intercalating agents and the like. Deletion is understood to mean any suppression of the gene considered. It may relate in particular to a part of the region encoding the said proteases and/or of all or part of the transcriptional promoter region. The genetic modifications can also be obtained by gens disruption, for example according to the procedure initially described by Rothstein [Meth. Enzymol. 101 (1983) 202]. In this case, the entire coding sequence will be preferably disrupted so as to allow the replacement, by homologous recombination, of the wild-type genomic sequence by a non-functional or mutant sequence.
The said genetic modification(s) may be located in the gene encoding the said proteases, or outside the region encoding the said proteases, for example in the regions responsible for the transcriptional expression and/or regulation of the said genes. The inability of the said genes to encode the natural proteases can manifest itself either by the production of a protein which is inactive because of structural or conformational modifications, or by the absence of production, or by thy production of a protease having a modified enzymatic activity, or alternatively by the production of the natural protease at an attenuated level or according to a desired mode of regulation.
Moreover, certain modifications such as point mutations are by nature capable of being corrected or attenuated by cellular mechanisms, for example during the replication of DNA preceding cell division. Such genetic modifications are thereby of limited interest at the industrial level since the phenotypic properties resulting therefrom are not perfectly stable. The applicant has now developed a process which makes it possible to prepare Kluyveromyces yeasts having one or more genetic modifications of at least one gene encoding a protease, the said modification(s) being segregationally stable and/or non-reversible. The yeasts having such modifications are particularly advantageous as cellular host for the production of recombinant proteins. The invention also makes it possible to produce yeasts in which the modification(s) made render the gene(s) considered totally or only partially incapable of producing a functional protease.
Preferably the yeasts according to the invention have one or more segregationally stable genetic modifications. Still according to a preferred embodiment, the genetic modification(s) its non-reversible. Still according to a preferred embodiment of the invention, the genetic modification(s) leave(s) no residual activity for the gens considered.
Preferably, the gene(s) encoding one or more proteases are chosen from the genes encoding proteases transiting through the secretorypathway of Kluyveromyces. Such proteases may be located in the endoplasmic reticulum, the compartment of the Golgi apparatus, the post-Golgi compartment, and for example the cellular vacuoles, the vesicles of the endosome, the secretion vesicles, or the extracellular medium.
As example of such genes, there may be mentioned the Kluyveromyces genes encoding a protease chosen from the families comprising protease A, protease B, or a carboxypeptidase (and for example carboxypeptidase Y or carboxypeptidase S), or alternatively endopeptidase KEX1 of K. lactis or a protease with similar activity, and for example protease YAP3 [Egel-Mitani et al., Yeast 6 (1990) 127], or more generally any other protease involved in the maturation of certain secreted proteins.
In a preferred embodiment of the invention, the considered gene(s) encode proteases which are not involved in the cleavage of the signal peptide of the recombinant proteins expressed in the form of preproteins. There may be mentioned by way of examples of particularly
Fleer Reinhard
Fournier Alain
Yeh Patrice
Aventis Pharma S.A.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Priebe Scott D.
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