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
2002-06-25
Ketter, James (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...
C435S069300, C435S069400, C435S069510, C435S069520, C435S069600, C435S183000, C435S254200, C435S254210, C435S255300, C435S320100, C435S440000
Reissue Patent
active
RE037767
ABSTRACT:
The present invention relates to the field of biotechnology, and more particularly to that of industrial fermentations by recombinant microorganisms.
Still more particularly, it relates to a host/vector pair which is highly stable in a complex medium, its preparation and its use in industrial fermentation.
The advances accomplished in the field of molecular biology have made it possible to modify microorganisms in order to make them produce specific recombinant proteins, preferably heterologous proteins. In particular, numerous genetic studies have been performed on the bacterium E. coli. More recently, yeasts such as Saccharomyces, Kluyveromyces, Pichia, or even Hansenula, have emerged as promising host organisms for this mode of protein production.
However, the industrial application of these new modes of production is still limited, especially by the problems of the efficacy of gene expression in these recombinant microorganisms, and by the difficulty of obtaining recombinant cells which are stable under industrial fermentation conditions. One of the essential operational constraints is indeed linked to the segregational stability of an expression vector inside the host used. At the industrial level, a vector should possess a high stability over at least 25 successive generations, which approximately represent the number of generations required to go as far as the end of a 200-m
3
fed batch-type industrial fermenter (Principles of Fermentation Technology, Stanburry and Whitaker, Pergamon Press, Oxford, 1984). The stability of the vector must be even higher in the case of continuous fermentation where it must reach not less than about one hundred generations.
In bacteria, the most common solution used in the laboratory consists of inserting a gene for resistance to an antibiotic into the plasmid used, which endows the bacteria with the capacity to survive and grow in a selective medium containing said antibiotic. However, because of security and regulatory constraints in the field of biotechnology, it is essential to be able to avoid the use of antibiotic resistance genes at the industrial level. In yeasts, the most commonly used method consists of culturing cells with a defective pathway for the biosynthesis of amino acids (Trp, Leu, His) or of purine (adenine) or pyrimidine (uracil) bases, said cells being transformed by a vector containing a gene which is capable of complementing this defect. However, this approach requires the use of media lacking the amino acid or the base for which the host strain is auxotrophic. The use of such synthetic media has numerous disadvantages. In particular, these media are expensive, which is incompatible with an industrial use, and furthermore, they lead to slower growth of the cells and to a smaller biomass.
A solution has been proposed to avoid the use of a synthetic medium or of antibiotic resistance genes, which consists of (i) mutating a gene which is essential for survival in a complex medium in the host cell and (ii) introducing an intact copy of said gene into the expansion plasmid used. This system, the principle of which is to force the host cell to retain its plasmid, has enabled the stability of the host/vector pair to be increased. This system has, in particular, been described for E. coli, for the dapD gene which encodes tetrahydropicolinate-N-succinyl transferase (EP 258 118), for the va1S gene whose product is an enzyme which is required for protein synthesis (Skogman and Nilsson, Gene 31 (1984) 117), and for the ssb gene whose product is essential for DNA replication and for the survival of the cell (Porter et al., Bio/technology vol. 8 (1990) 47). Ferrari et al. have also described the use of the racemase alanine gene for stabilising a plasmid inside a B. subtilis mutant in which this gene was not functional (Bio/technology vol. 3 (1985) 1003). Application WO 86/01224 describes a similar selection system which is suitable for the yeast S. cerevisiae. This system uses the yeast S. cerevisiae which has a mutation in 2 genes which are involved in the biosynthesis of uracil. It consists of (i) inactivating one of the genes for the synthesis of uracil, (ii) transforming said cell with a vector carrying the active gene, and (iii) blocking the other metabolic pathway by mutagenesis.
Another approach for obtaining expression systems which are stable in complex media consists of using vectors which are integrated into the genome of the host cell. However, this system enables only a small number of copies of the vector to be obtained per recombinant cell, and furthermore, the transformation frequency is low. Under these conditions, the levels of expression of heterologous genes are not always satisfactory. A method enabling amplification of a gene which is integrated into the genome has, moreover, been developed in S. cerevisiae, by directing integration towards the genes encoding ribosomal proteins, said genes being present in multiple copies in the genome. However, this system proves to be unstable when the integrated genes are expressed at high levels, whether they are homologous or heterologous genes.
Currently, the use of the new yeasts, different from S. cerevisiae, for the production of recombinant proteins requires the development of tools which are adapted to these microorganisms, in order to resolve in particular the problems of stability of expression vectors for heterologous genes. More specifically, yeasts which are taxonomically related to the Kluyveromyces genus appear to possess a particularly advantageous capacity for secreting recombinant proteins. This has been observed in particular in the case of the yeast K. lactis, for the production of chymosin (EP 96430), IL-1&bgr; or human serum albumin (EP 361991). However, no sufficiently stable multicopy expression vectors exist in this organism to permit its use in industrial processes. In particular, no vectors exist which are stable in complex media, enabling large-scale processes, especially continuous processes, to be envisaged using this organism. Indeed, although certain vectors which are stable in K. lactis have been described (EP 361991), the introduction of a heterologous gene expression cassette into these vectors produces a substantial destabilising effect, especially under conditions for inducing production.
A particularly efficient means for stabilising host/vector pairs in which the host is a yeast of the Kluyveromyces genus, has now been found.
One embodiment of the invention consists of a host/vector pair which is highly stable in a complex medium, characterised in that the host is a yeast of the Kluyveromyces genus in which a gene which is essential for its growth in said medium is nonfunctional, and in that the vector carries a functional copy of said gene.
Within the context of the present invention, complex medium is understood to mean any medium for industrial fermentation which is compatible with the economic constraints of a large-scale operation. In particular, it relates to media containing industrial-type raw materials: maize soluble extract, yeast extract, molasses or “distillers”, for example, as opposed to defined synthetic media which are supplemented (for example with antibiotics). However, it is understood that the present invention may also be used on synthetic media, although this embodiment is less advantageous.
Moreover, it is understood that the functional gene which is present in the vector may be a homologous or heterologous gene.
Genes which are essential for the growth of the host cell in a complex medium include genes which are involved in the metabolism of a carbon source present in the medium (galactose, lactose, glucose and the like), and genes participating in cellular division, in membrane synthesis, in protein synthesis or DNA replication or transcription.
More preferably, the invention consists of a host/vector pair which is highly stable in a complex medium, characterised in that the host is a yeast of the Kluyveromyces genus in which a gene which is involved in glycolysis is nonfunctional, and in that the vector car
Fleer Reinhard
Fournier Alain
Yeh Patrice
Aventis Pharma S.A.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Ketter James
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