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
1994-07-01
1997-08-26
Hendricks, Keith D.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
435194, 43525233, 536 232, C12N 912, C12N 1554, C12P 1308
Patent
active
056610122
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/JP93/01640, filed Nov. 10, 1993.
TECHNICAL FIELD
The present invention relates to the microbiological industry, and describes a method for the production of L-threonine by fermentation. L-threonine is an essential amino acid, and it is used as a component of various nutritional mixtures for medical purposes. Furthermore, it is used as a feed additive for animals, as a reagent in the pharmaceutical and chemical industries, and as a growth factor for the production of amino acids such as lysine and homoserine by microorganisms.
BACKGROUND ART
In the past, bacteria separated from the natural environment or artificial mutants of such bacteria have been used as microorganisms for the production of L-threonine by fermentation. Many L-threonine-producing artificial mutants are known, the majority of which are resistant to .alpha.-amino-.beta.-hydroxyvaleric acid and belong to the genus Escherichia, Serratia, Brevibacterium or Corynebacterium. Regarding Escherichia, in JPA 55-131397, JPA 59-31691, JPA 56-15696 and JPW 3-501682 are there described methods for the production of L-threonine by bacteria transformed by recombinant plasmids containing threonine operons.
Also, of the heretofore known threonine-producing bacteria, Escherichia coli BKIIM (VKPM) B-3996 strain has exhibited the most superior level of threonine production and coefficient of consumption (JPW 3-501682). According to the present invention, "coefficient of consumption" refers to the number of grams of sugar required for the production of one gram of threonine. If this bacterium is cultured in an experimental fermenter, adding sugar-ammonia to the culture medium in response to the signals from the pH sensor, then the maximum degree of biosynthesis of threonine is 85 g/l, and the coefficient of consumption is 2 g of sugar per one gram of threonine.
Aspartokinase (hereunder abbreviated to AK) is an enzyme which converts aspartic acid to .beta.-phosphoaspartic acid, and it is the main regulatory site of the biosynthesis pathway of aspartic acid and its derivative amino acids. As shown in FIG. 1, there are three types of AK from E. coli (AK I, AK II, AK III) and the first two of these are bifunctional enzymes having homoserine dehydrogenase (hereunder sometimes abbreviated to HD) activity. One of these is AK I-HD I which is coded for by the thrA gene, and the other is AK II-HD II which is coded for the metL(M) gene.
Only AK III is a monofunctional enzyme, and it is the product of a gene named lysC, and is known to undergo repression and feedback inhibition by lysine. On the other hand, AK I undergoes concerted repression by threonine and isoleucine, and inhibition by threonine, while AK II undergoes repression by methionine. The proportion of the intracellular activities thereof is AK I:AK II:AK III=approximately 5:1:4.
The lysC gene of E. coli has already been cloned, and its base sequence has been determined (Cassan, M., Parsot, C., Cohen, G. N. and Patte. J. C., J. Biol. Chem., 261, 1052, 1986). Also, the production of L-threonine by fermentation using strains of E. coli whose AK III activity has been reinforced, is described by Mizukami, et al. (Mizukami, T. et al., Agric. Biol. Chem., 50, 1015, 1986). However, sufficient release of the lysine-dependent feedback inhibition on the AK III reported by Mizukami, et al. has not yet been achieved.
Thus, the subject matter of the present invention is obtaining AK III with sufficient release of the feedback inhibition due to lysine, and providing a method for the production of L-threonine by fermentation which is much improved over the prior art.
DISCLOSURE OF INVENTION
The inventors of the present invention, as a result of diligent research carried out to overcome the above mentioned problem, have succeeded in obtaining a gene (mutant lysC, or lysC*) which codes for AK III in E. coli and which sufficiently releases the feedback inhibition due to lysine, and have discovered that by introducing this gene into threonine-producing bacteria, L-threonine is efficiently
REFERENCES:
Cassan et al., J. Biol. Chem., 261(3):1052-1057, Jan. 25, 1986.
Mizukami et al., Agric. Biol. Chem. 50(4):1015-1018 1986.
Kojima Hiroyuki
Ogawa Yuri
Sano Konosuke
Ajinomoto Co. Inc.
Hendricks Keith D.
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