Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Patent
1997-03-20
2000-08-22
Elliott, George C.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
43525232, 43525233, 4353201, 536 232, 536 237, C12P 1306, C12N 121, C12N 1531, C12N 1563
Patent
active
061070637
DESCRIPTION:
BRIEF SUMMARY
This application is a National Phase filing of PCT/DE95/00017, filed Jan. 9, 1995.
The invention relates to a method for the microbial production of L-isoleucine wherein a threonine dehydratase gene is altered so as to deregulate the feed back inhibition of L-isoleucine by threonine dehydratase. The invention also relates to a gene structure and vector corresponding to the altered threonine dehydratase gene. The invention also relates to a transformed cell which contains the aforementioned gene structure.
The amino acid L-isoleucine is essential for man and animals. It is widely used in dietetic foods and as a component of various nutrient mixtures for medicinal purposes. L-Isoleucine is also used as an additive or as a reagent in the pharmaceutical and chemical industries.
Microorganisms, which secrete L-isoleucine into a fermentation medium, are used to obtain this amino acid, which is formed by the biosynthetic path shown in FIG. 1. As is also demonstrated in FIG. 1, there are key enzymes in the biosynthesis of the L-isoleucine, namely aspartic acid kinase, homoserine dehydrogenase and threonine dehydratase. The activity of the aspartic acid kinase and of the homoserine dehydrogenase is inhibited by the feedback of the amino acid, L-threonine, which is also formed within the scope of the biosynthesis of the L-isoleucine, while the key enzyme, threonine dehydratase, which is specific for the synthesis of L-isoleucine, is inhibited by the feedback of the end product of the biosynthetic chain, L-isoleucine.
In the past, attempts have been made time and again to increase the formation of L-isoleucine by obtaining mutants of L-isoleucine producers which, relative to the wild types, form more L-isoleucine.
To obtain such mutants, mutagenesis was carried out exclusively in vivo, that is, a mutagen was allowed to act on the whole of the genome. Mutated microorganisms, the above-mentioned key enzymes of which no longer are subject to a feed-back inhibition, were selected on the basis of the resistance to amino acid analogs.
For example, a mutation, which leads to a resistance to .alpha.-aminobutyrate or isoleucine hydroxamate, is described in U.S. Pat. No. 4,329,427. According to this example, microorganisms admittedly produce increased amounts of isoleucine; however, the question as to which enzymes are no longer inhibited by feedback, is not clarified.
The U.S. Pat. No. 4,442,208 and 4,601,983 disclose that, after an in vivo mutagenesis, it was possible to isolate a DNA fragment, which is not defined more precisely and which brings about resistance to .alpha.-aminohydroxyvaleric acid. After this fragment is transferred to a Corynebacterium or a Brevibacterium strain, the latter produce increasing amounts of L-isoleucine.
Methods have also been described, for which, by means of oversynthesis of feedback-regulated key enzymes, L-isoleucine can be formed increasingly (see, for example, the German Offenlegungsschrift 3,942,947 and the European publication 0 137 348).
Overall, all previously described methods for increasing the L-isoleucine production have the common feature that mutations were more likely to lead randomly to deregulated key enzymes, which are no longer subject to feedback inhibition and that the amino acid synthesis was increased by these means.
It is an object of the invention to provide a method for the microbial production of L-isoleucine, by means of which defined mutations of the key enzyme of the L-isoleucine biosynthesis, the threonine dehydratase, is selectively changed so that feedback inhibition by L-isoleucine no longer takes place.
The objective, on which the invention is based, is accomplished owing to the fact that, in a gene of a threonine dehydratase, present in vitro, one or several bases are exchanged in such a manner by mutation in the region of the gene coding for the allosteric domains of the enzyme, that at least one amino acid in the amino acid sequence of the allosteric domains of the enzyme is replaced by a different one in such a manner, that the enzyme is no longer inhib
REFERENCES:
Gavrilova et al., Chemical Abstracts 108(144395d): 176 (1988).
Moeckel et al., Chemical Abstracts 121(199457z): 554(1994).
Cordes et al. (1992) Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum. Gene 112:113-116, 1992.
Komatsubara et al. (1980) Transductional construction of an isoleucine-producing strain of Serratia marcescens. J. Gen. Microbiol. 119:51-61, Jul. 1980.
Eggeling Lothar
Moeckel Bettina
Sahm Hermann
Elliott George C.
Forschungszentrum Juelich GmbH
Schwartzman Robert
LandOfFree
Production of L-isoleucine by means of recombinant microorganism does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Production of L-isoleucine by means of recombinant microorganism, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Production of L-isoleucine by means of recombinant microorganism will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-579266