Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1997-09-30
2001-03-06
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S476000, C435S252300, C435S252330, C435S488000
Reexamination Certificate
active
06197590
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a new method for the amplification and stabilization of the copy number of chosen genes in a microorganism.
The invention is illustrated by means of examples given hereinafter in which the sequence coding for an enzyme, beta-galactosidase, and the genes coding for three enzymes involved in the biosynthesis of L-threonine are transferred inside the chromosome of a microorganism.
These genes have been shown to be stably fixed in the chromosome. The resulting strains are therefore better producers of, in the first instance, the corresponding enzymes or, secondly, of L-threonine amino acids, than are the parental strains.
The bacteria are capable of synthesizing useful active principles such as enzymes and amino acids. The production by fermentation involves complex biochemical procedures controlled by multiple genes and gene products. The products of the fermentation are obtained in relatively small quantities. In order to improve this production by fermentation, it would be useful to increase the number of copies of the genes involved, in a manner such that the number of these copies could be controlled and stabilized in the chromosome or the episomes of the microorganism.
Several methods have been described in the scientific literature in which the copy number of a gene can be amplified in a microorganism, in particular by means of circular plasmid vectors. However, the number of copies obtained by these methods is extremely variable and depends on the particular vector, the length of the chosen gene and the particular physiological conditions of the microorganism. None of these methods are concerned with the amplification of the gene to a chosen level, avoiding an unnecessary excess of copies, and this with a stable fixation of these copies in the chromosome.
Various documents suggest the use of transposons as vectors in bacteria or other organisms, in particular:
U.S. Pat. No. 4,670,388,
EP-A-0,200,708
EP-A-0,091,723
GENE, vol. 42 (1986), pages 185-192
THE EMBO JOURNAL, vol. .4, No. 4, 1985, pages 891-898, and 5 BIOTECHNOLOGY, vol. 4, No. 5, May 1986, pages 446-449, New York, US.
However, none of these documents describes a process by which it is possible to obtain a stable integration of a specific DNA sequence with a defined copy number.
SUMMARY OF THE INVENTION
One aim of the invention is to describe a method for amplifying and stably fixing the copies of chosen genes in a microorganism with more flexibility and more reliability than was hitherto possible. This aim as well as other objects of the invention, such as are envisaged hereafter, have been accomplished, for example, by providing the
Escherichia coli
bacterium with a transposable element having a wide host range and derived from the Mu phage, and in which the genes of the transposase cannot be expressed or are deleted. Such a transposon is a defective transposon. Under particular reversible genetic conditions which are easy to reproduce for organisms of the same genus or of a different genus, this transposon and the genes which can be introduced into it by genetic engineering technology can be made to multiply inside the chromosome of the microorganism. When the particular genetic conditions are removed, the various copies of the transposon remain fixed in a stable manner inside the chromosome.
The present invention relates to a process for integration of a chosen gene or of a specific DNA sequence (inside a bacterium) in a DNA sequence such as a chromosome or an episome, wherein:
a) the said chosen gene or the chosen DNA sequence is cloned inside a defective transposon outside the essential parts of the transposon,
b) the said transposon is integrated in the DNA sequence inside the bacterium.
More particularly, the process according to the invention is used to amplify the chosen copy number of genes in order to obtain a specific number of copies. In this case the process according to the invention is a process for integration of a specific number of copies of a chosen gene or of a specific DNA sequence in a DNA sequence such as the chromosome or episome of a bacterium, wherein:
a) the said chosen gene or the chosen DNA sequence is cloned inside a transposon outside the essential parts of the transposon, the said transposon being defective,
b) the said transposon is integrated in the DNA sequence such as the chromosome or the episome of the said bacterium,
c) the said defective transposon is complemented so as to be able to transpose several times and the complementation is then stopped after a specific number of transpositions.
According to the invention any transposon can be used which transposes inside any site in the bacterial DNA.
In the present text the term “transposons” is intended to mean both “true transposons” and also phages having the capacity to transpose.
Preferred transposons are Tn3, Tn5, Tn7, TnlO, Tn903, TnHoHo, IS 1, IS 10, IS 50 and the MudI and MudII phages. The MudI and MudII phages are particularly preferred.
The use of defective transposons, which are transposons incapable of transposing by themselves, makes it possible to obtain a stable integration. For such a use the transposons used have no genes for the transposase or these genes are not active in the said bacterium. A transposon is very particularly preferred which lacks genes coding for the transposase, but it is also possible to use a transposon in which the trans-acting transposases are repressed or inactive under normal conditions.
According to the invention it is possible to use any bacterium in which the transposons described hereinabove can transpose.
The preferred bacteria are members of the Enterobacteriaceae family, for example
Escherichia coli, Citrobacter freundii, Erwinia herbicola
or
E. chrysanthemi, Salmonella thyphimurium, Shiaella soneii, Enterobacter cloacae, Serratia marcescens
or members of the Rhizobiaceae family, such as
Agrobacterium tumefaciens
or members of the Pseudomonas family, such as Pseudomonas putida for example.
By the term chosen gene there is understood a gene which is not normally present in the said transposon or which is not present in the host bacterium.
As chosen gene there may be used a cloned gene or a hybrid gene, a gene segment or a synthesized gene. The gene can be expressed in the host bacterium and may be of animal, vegetable or microbial origin. Several genes can be inserted in the transposon.
The chosen gene can be under the control of a promoter which is itself inside the transposon and which ensures the expression of the said gene in the said bacterium.
The cloned genes are inserted by known genetic engineering techniques or according to the process described hereinbelow.
For convenience sake, it is simpler to start from a plasmid containing the defective transposon. The transposon is made defective by deletion or mutation.
The plasmid containing the transposon is isolated from the bacterium and cleaved with appropriate restriction enzymes. The chosen genes are also extracted from their host and then purified after cleavage by means of restriction enzymes. The cloning of a marker gene such as an antibiotic-resistant gene (not for example) or another “reporter” gene (lacz for example) at the same time as the inserted genes is preferable for the selection of the transposition events. The number of transpositions is evaluated by determination of the expression of the marker gene. The said expression of the marker gene can be determined by the resistance to an antibiotic when the marker gene is an antibiotic-resistant gene; the expression of the marker gene can also be determined by the, coloration of a substituent when the marker gene codes for an enzymatic activity which can be evaluated by a colorimetric assay. The various fragments are recombined by ligation in vitro and transformed in the appropriate recipient bacterium. The transformants are selected and then analyzed in order to establish that the structure is suitable. The resulting composite defective transposon can be placed in other strains by transformation
Beyou Anne
Jarry Bruno
Kurahashi Osamu
Richaud Francois
Takinami Koichi
Cooley & Godward LLP
Eurolysine
Yucel Remy
LandOfFree
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