Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reissue Patent
2001-05-09
2003-11-25
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S069100, C435S061000, C435S086000, C435S183000, C435S189000, C435S190000, C435S252300, C435S253300
Reissue Patent
active
RE038331
ABSTRACT:
This application is a 371 of PCT/FR93/01202 Dec. 7, 1993.
The present invention relates to cells modified with respect to the catabolism of betaine, to their preparation and their use, especially for the improved production of metabolites and/or enzymes. The invention also relates to DNA fragments carrying genes for the catabolism of betaine.
Glycine betaine (N,N,N-trimethylglycine) is generally known for its osmoprotective properties, which confer on bacteria tolerance to osmotic stress (Csonka, 1989). To explain the origin of this property, it has been proposed that the molecular effects of glycine betaine on the activity of water and on the osmotic pressure of the cytoplasm in Escherichia coli were more important than those of the solutes which it replaces (Cayley et al, 1992). Furthermore, in addition to its osmoprotective potentials, it has been described that glycine betaine could also promote the production of enzymes (JP 8,260,709) or of metabolites, such as amino acids (Patent JP 202703); antibiotics (Patent AU 825513) and vitamins (White et al, 1971). However, most bacteria except cyanobacteria and other CO
2
-fixing prokaryotes do not synthesize glycine betaine which is mainly synthesized by plants. It should therefore be added to production media in fermenters, which generates an additional cost in an industrial process. The present invention provides a solution to this problem.
The applicant has indeed demonstrated that it is possible, by modifying the catabolism of betaine of cells, especially by genetic means, to potentiate the effect of this compound on the production of enzymes or of metabolites without affecting the rate of growth of cells, their viability and the like, under industrial fermentation conditions. The applicant has also identified, isolated and characterized DNA fragments containing genes involved in the catabolism of betaine, which make it possible in particular to prepare cells specifically modified with respect to the catabolism of betaine, and whose modifications are segregationally stable and nonreversible. These fragments also make it possible to stimulate the catabolism of betaine by amplification of the appropriate enzymatic activities. The present invention therefore makes it possible to potentiate the effects of betaine and, thereby, to use this compound economically in industrial fermentation processes.
A first subject of the invention therefore relates to a modified cell exhibiting at least one modification with respect to a gene involved in the catabolism of betaine.
In a first embodiment, the term modified cell designates more particularly any cell having a substitution and/or a deletion and/or an insertion of one or more bases in the considered gene(s) and degrading betaine less rapidly. Such modifications can be obtained in vitro (on isolated DNA fragments carrying genes for the catabolism of betaine) or in situ, for example, by means of genetic engineering techniques, or alternatively by exposing the said cells to a treatment by means of mutagenic agents.
As mutagenic agents, there may be mentioned for example physical agents such as energetic radiation (X-, g- or ultraviolet rays end the like) or chemical agents capable of reacting with various functional groups of the bases of DNA, and for example alkylating agents [ethyl methane-sulphonate (EMS), N-methyl-N′-nitro-N-nitrosoguanidine, N-nitroquinoline 1-oxide (NQO)], dialkylating agents, intercalating agents and the like.
Deletion is understood to mean the removal of all or part of the gene considered. This may especially be a portion of the coding region and/or of all or part of the promoter region for transcription.
The genetic modifications can also be obtained by gene disruption, for example according to the procedure initially described by Rothstein (1983). In this case, all or part of the gene is preferably perturbed so as to allow the replacement, by homologous recombination, of the wild-type genomic sequence by a nonfunctional or mutant sequence prepared in vitro.
The said modification(s) may be located in the coding portion of the gene or in the regions responsible for the expression end/or transcriptional regulation of the said genes. The (total or partial) incapacity of the said cells to degrade betaine can manifest itself either by the production of inactive enzymes because of structural or conformational modifications, or by the absence of production, or by the production of enzymes having an impaired activity, or alternatively by the production of natural enzymes at en 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, in this case, of limited interest at the industrial level since the phenotypic properties resulting therefrom are not perfectly stable. According to the present invention, it is now possible, by virtue of the identification of DNA fragments carrying genes for the catabolism of betaine, to prepare modified cells in which the said modification(s) are segregationally stable and/or nonreversible. The cells exhibiting such modifications are particularly advantageous as cellular host for the production of metabolites end/or enzymes.
In another particular embodiment, the modified cells of the invention are cells in which at least one gene involved in the catabolism of betaine is amplified, and which as a result degrade betaine more rapidly.
The amplification can be obtained by introducing a DNA fragment carrying a gene for the catabolism of betaine into the cell. This fragment is preferably part of a vector, which my be an autonomously replicating vector or an integrative vector. Moreover, the DNA fragment may be homologous or heterologous in relation to the modified cell, that is to say that the amplified gene(s) may be genes from the said cell or genes obtained from other cellular sources and encoding an activity of the same type. The choice of the vector and of the origin of the amplified fragment depends on the cells considered and the applications envisaged. The DNA fragment may be introduced into the cells by any method allowing the introduction of a foreign DNA into a cell. This may be in particular transformation, electroporation, conjugation, protoplast fusion, or any other techniques known to persons skilled in the art.
According to studies carried out essentially in Rizobium meliloti, the degradation of glycine betaine is performed on media of low osmolarity, by three successive demethylations (see FIG.
1
). The first stage is catalysed by betaine homocysteine methyltransferase E.C. 2.1.1.5. and leads to dimethylglycine; the second is catalysed by dimethylglycine dehydrogenase E.C. 1.5.99.2 and generates momomethylglycine or sarcosine; finally the third is catalysed by sarcosine dehydrogenase E.C. 1.5.99.1 and the product of the reaction is glycine (Smith et al, 1988).
Preferably, the modifications exhibited by the cells of the invention affect one of the fist two stages of the catabolism of betaine, or optionally the two first stages simultaneously.
Preferably, the cells of the invention are cells which product metabolites and/or enzymes. In this respect, they may also be recombinant cells which product metabolites and/or enzymes, that is to say cells modified by recombinant DNA techniques so as to improve their production capacity (cf. especially WO 91/11518, EP 346000). Still more preferably, the cells of the invention are chosen from cells of the genus Pseudomonas, Streptomyces, actinomycetes, Propionibacterium, Corynabacterium, Bacillus, Escherichia, Salmonella, Rhizobium, Agrobacterium, Rhodopseudomonas, Xanthomonas, Clostridium end Methanobacterium.
Another aspect of the invention relates to a process for preparing cells exhibiting a modification of at least one gene involved in the catabolism of betaine which are capable of being used under industrial fermentation conditions.
T
Cameron Beatrice
Crouzet Joel
Achutamurthy Ponnathapu
Finnegan Henderson Farabow Garrett & Dunner LLP
Rao Manjunath N.
Rhone-Poulenc Biochimie
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