Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing nitrogen-containing organic compound
Patent
1996-07-02
1998-06-02
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing nitrogen-containing organic compound
4352523, 43525233, 43525234, 4353201, 536 231, 536 232, 536 237, C12P 1300, C12N 120, C07H 2104
Patent
active
057598240
DESCRIPTION:
BRIEF SUMMARY
This is a national stage application of PCT/EP94/03317 filed on Oct. 7, 1994.
The present invention relates to recombinant genetic material for expression of the genes of butyrobetaine/crotonobetaine-L-carnitine, microorganisms which contain this recombinant genetic material, and the use of such microorganisms in a biotechnological process for the production of L-carnitine.
L-carnitine is a natural, vitamin-like substance of great importance in human metabolism. In the utilization of fatty acids, L-carnitine is essential as a transmitter substance of the mitochondrial membrane and, thus, as a transporter of metabolic energy. If L-carnitine is synthesized in inadequate amounts by the body, it must be added to the diet to avoid deficiency symptoms. Especially in the diet of infants, who are still unable to biosynthesize their own L-carnitine, L-carnitine is an essential nutrient.
L-carnitine preparations are employed as active constituents of pharmaceutical products. Supplementation with L-carnitine is indicated in the case of carnitine deficiency and other therapeutic indications, especially in cardiac disorders, etc.
The biosynthesis of L-carnitine in higher organisms is known; further functions in and importance for metabolism are objects for intensive research activity. In addition to a metabolic pathway which is described for microorganisms, especially of the genus Pseudomonas (.gamma.-butyrobetainehydroxylase catalysis, Lindstedt et al., Biochemistry 16, 2181-2188, 1977), L-carnitine is formed as a metabolic intermediate of certain microorganisms, e.g. of Agrobacterium/Rhizobium sp..
EP-A-0 158 194 discloses a process for the microbiological production of L-carnitine, starting from, for example, .gamma.-butyrobetaine, in which an L-carnityl dehydrogenase negative production mutant is obtained by means of traditional microbiological selection processes, using which relatively good yields of L-carnitine are already obtained within a reaction time of 20 to 30 hours. A further optimization of this process with respect to the volume/time yields, however, is not possible using this classical microbiological method.
It is, therefore, the object of the present invention to provide a more economical biotechnological process for the production of L-carnitine, in which L-carnitine is obtained in a significantly shorter reaction time with even better yields.
Investigations on .gamma.-butyrobetaine/crotonobetaine metabolism led to the identification of five genes, bcoA/B, bcoC, bcoD, bcoE and bcoT, which code for enzymes of the .gamma.-butyrobetaine/crotonobetaine metabolic pathway and are contained, inter alia, in an operon, the so-called butyrobetaine-L-carnitine operon (bco). In this context the abbreviations have the following meanings synthetase (bcoB) gene, i.e. a unique gene which codes for an enzyme product which has both .gamma.-butyrobetaine-CoA synthetase activity and crotonobetaine-CoA synthetase activity.
It was found that the gene products of the genes bcoA/B, bcoc and bcoD are responsible for the biosynthesis of L-carnitine, while bcoE encodes a carnitine dehydrogenase which causes the degradation of the metabolic intermediate L-carnityl-CoA to betaine. bcoT is presumably a gene which codes for a transport protein of a transport system assigned to butyrobetaine metabolism. This gene is not essential for L-carnitine biosynthesis.
The present invention accordingly relates to DNA fragments and vectors which comprise one or more of the genes bcoc, bcoA/B and bcoD coding for the enzymes for the biosynthesis of L-carnitine in .gamma.-butyrobetaine/crotonobetaine metabolism and, optionally, additionally the potential transport gene bcoT.
The invention further relates to microorganisms which contain these DNA fragments and/or vectors. The invention further relates to a biotechnological process for the production of L-carnitine using the microorganisms according to the invention.
The designations bcoA/B, bcoc, bcoD and bcoT, as are used here in the description and the claims, comprise, as defined, both the ge
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Werlen Josef
Zimmermann Thomas
Lonza A.G.
Nashed Nashaat T.
Wax Robert A.
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