Modified Kluyveromyces yeasts, their preparation and use

Details Modified Kluyveromyces yeasts, their preparation and use Modified Kluyveromyces yeasts, their preparation and use

1995-02-06

1997-10-21

Chambers, Jasemine C.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

4351723, 4352542, C12P 2100, C12N 1509, C12N 119, C12N 1581

Patent

active

056795440

DESCRIPTION:

BRIEF SUMMARY
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 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 group

REFERENCES:
Fleer et al., "Stable multicopy vectors for high level secretion of recombinant human serum albumin by Kluyveromyces yeasts", Bio/Technology 9:968-975 Oct. 1991.
Sturley et al., "Secretion and lipid association of human apolipoprotein E in Saccharomyces cerevisiae requires a host mutation in sterol esterification uptake", J. Biol. Chem. 266:16273-16276 Sep. 1991.
Chem. Abstracts Abstract 120893j 99(15):520, 1983, Grieve Kitchen Dulley Bartley, Partial Characterization of Cheese-Ripening Proteinases Produced by the Yeast Kluyveromyces lactis.
FEBS Letters 234(2):464-70, 1988, Tanguy-Rougeau Wesolowski-Lou Fukuhara, The Kluyveromyces lactis KEX1 Gene Encodes a Subtilisin-Type Serine Proteinase.
Cell 48:887-97, 1987, Valls Hunter Rothman Stevens, Protein Sorting in Yeast: The Localization Determinant of Yeast Vacuolar Carboxypeptidase Y Resides in.
Molec. Cell Biol. 7(12):4390-99, 1987 Moehle Tizard Lemmon Smart Jones, Protease B of the Lysosomelike Vacuole of the Yeast Saccharomyces cerevisiae Is Homologous to the Subtilisin.
Nucleic Acids Res. 17(4): 1779, 1989, Lott Page Boiron Benson Reiss, Nucleotide Sequence of the Candida albicans Aspartyl Proteinase Gene.
EP 336056, filing date Jan. 24, 1989, Publ Date Oct. 11, 1989, Yamamoto, Protease.

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