Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-05-08
1999-10-12
Guzo, David
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
43525232, 4353201, 435471, 435476, 435487, 536 241, C12P 2100, C12N 121, C12N 1577, C07H 2104
Patent
active
059653914
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention relates to a DNA which regulates gene expression in coryneform bacteria.
BACKGROUND OF THE INVENTION
Each organism is required, in the course of growth to synthesize new cell substances. There are numerous cell components, like, for example, amino acids and porphyrins which are to be newly formed starting from metabolites of the citrate cycle. This means that the metabolites involved in the citrate cycle must be newly synthesized. In the growth of microorganisms on acetate, ethanol or fatty acids, metabolites of the acetate cycle are newly synthesized by a reaction sequence which has been called the glyoxylate cycle (Kornberg, Biochemical Journal 99 (1966) 1-11), key enzymes for the glyoxylate cycle being the enzymes isocitrate lyase and malate synthase. Since the named enzymes in many organisms can grow exclusively on acetate, ethanol or fatty acids, but not on carbohydrates, the activity or the new synthesis of the two enzymes is mainly regulated by the carbon source of the medium.
Because of their club-like configuration, Corynebacterium glutamicum and the closely associated C. melassocolae, B. flavum and B. lactofermentum are counted as coryneform bacteria. These types of bacteria also belong to the known class of "glutamic acid bacteria" since they are capable under certain growth conditions of liberating large amounts of glutamate in the medium. The named microorganisms are of considerable industrial interest since they can be used for the production of amino acids, purines and proteins. For C. glutamicum, C. melassecolae, B. flavum and B. lactofermentum, growth upon acetate or ethanol is already known and it has been found that they are involved in a glyoxylate cycle, i.e. also utilize the enzymes isocitrate lyase and malate synthase (for an overview see Kinoshita, Amino acids, in Biology of Industrial Organisms, 1985, pages 115-142, Benjamin/Cummings Publishing).
In spite of long term industrial use of these organisms only recently have molecular biological methods been developed with the aid of which coryneform bacteria can be genetically modified for certain specific purposes. As a rule, the gene to be cloned is cloned under the control of its own promoters on vectors which are available in higher copy numbers in coryneform bacteria. It has been found in many cases that a strong overexpression of individual genes is a drawback to the growth of coryneform bacteria and thus has an effect on the production of desired products. This has its origin in an overproduction of the corresponding gene products to toxic effects within the metabolism of the cell and gives rise to a reduction in the growth of these cell. An example of such a case is the homologous overexpression of mutated genes which code for deregulated enzymes, i.e. such enzymes whose activity no longer has end product blocking, for instance, the homologous overexpression of the HOM 1 gene which codes for a deregulated homoserine dehydrogenase (Reinscheid et al., Applied the Environmental Microbiology 60 (1994), 126-132). There are however, also known cases in which the overexpression of nonmutated genes is detrimental in a homologous system for the growth of C. glutamicum (for example Eikmanns et al., Microbiology 140 (1994) 1817-1828). In addition, there are significant problems when genes, which do not stem from coryneform bacteria, should be overexpressed in them. In order to express a desired gene in coryneform bacteria, without having to take into consideration a growth blockage by the corresponding gene product, there are various possibilities: a desired gene can be integrated in a single copy number in the chromosome of coryneform bacteria. Since one copy of these genes is provided in the organism, as a rule, no toxic effects arise from the corresponding gene product. A weakness of this process is found in the work-intensive methodology to achieve the desired goal. ln addition, with a single copy number of the inserted gene, seldom is a sufficient quantity of a desired material formed.
An alte
REFERENCES:
Biochemical Journal, 99 (1966), pp. 1-11, Kornberg, H.L.
Biology of Industrial Microorganisms, 1985, pp. 115 to 142, Kinoshita, S.
Applied and Environmental Microbiology, 60, (1994), pp. 126 to 132, Reinscheid et al.
Microbiology, 140 (1994), 1817 to 1828, Eikmanns et al.
Bio/Technology 6 (1988) pp. 428 to 431, Tsuchiya et al.
"Malate synthase from C. glutamicum: sequence analysis of the gene and biochmical characterization of the enzyme" by Reinscheid et al., published in Microbiology, vol. 140, 1994.
Eikmanns Bernhard
Reinscheid Dieter
Sahm Hermann
Dubno Herbert
Forschungszentrum Julich GmbH
Guzo David
Myers Jonathan
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