Cells with altered betaine catabolism and their use in producing

Details Cells with altered betaine catabolism and their use in producing Cells with altered betaine catabolism and their use in producing

1995-05-18

1997-11-25

Wax, Robert A.

Chemistry: molecular biology and microbiology

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

435 86, 435183, 4352533, C12P 104, C12P 1942, C12N 900, C12N 121

Patent

active

056911637

DESCRIPTION:

BRIEF SUMMARY
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.sub.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 (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

REFERENCES:
White, R.F. et al. (1973) "Betaine-homocysteine transmethylase in Pseudomonas denitrificans, a vitamin B.sub.12 overproducer" J. Bact. 113(1):218-223.
Kawahara, Y. et al. (1990) "Effect of glycine betaine, an osmoprotective compound on the growth of Brevibacterium lactofermentum" Appl. Microbiol. Biotechnol. 33:574-577.
Lago, B.D. et al. (1969) "Alternate requirement for vitamin B12 or methionine in mutants of Pseudomonas denitrificans, a vitamin B12-producing bacterium" J. Bact. 99(1):347-349, Jul. 1969.

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