Method of producing L-lysine

Details Method of producing L-lysine Method of producing L-lysine

1996-10-08

1998-06-16

Wax, Robert A.

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

4351721, 435190, 4352523, 536 232, C12N 120, C12N 904

Patent

active

057669252

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

The present invention relates to microbial industry, and in particular relates to a method of producing L-lysine by fermentation, and coryneform bacteria preferable for use in this production method.
L-lysine has been hitherto produced by fermentation using L-lysine-producing bacteria belonging to the genus Brevibacterium, Corynebacterium, Bacillus or Escherichia, which is synthesized in a biosynthesis system of any of these microorganisms from oxaloacetate through aspartate, aspartate .beta.-aldehyde and so on. Various enzymes such as phosphoenol pyruvate carboxylase, aspartokinase and dihydrodipicolinate synthase participate in such an L-lysine biosynthesis pathway, however, many of these enzymes undergo feedback inhibition by L-lysine as a final product or by aspartic acid as an intermediate product. Thus when L-lysine is produced by fermentation, in order to improve the productivity, many mutant strains which do not undergo such inhibition are used.
For example, it is known that aspartokinase (hereinafter referred to as "AK") undergoes concerted inhibition by L-lysine and L-threonine synthesized in a branched pathway from the L-lysine synthesis pathway in coryneform bacteria belonging to the genera such as Brevibacterium and Corynebacterium. A mutant strain harboring AK which does not undergo the inhibition is used for L-lysine production (J. Gen. Appl. Microbiol., 16, 373-391 (1970)).
A mutant strain, which lacks homoserine dehydrogenase (hereinafter referred to as "HD") considered to be an enzyme having the greatest influence on L-lysine productivity, is also used for production of L-lysine by fermentation. This is attributed to the fact that L-threonine is not synthesized due to deficiency in HD to catalyze a reaction for producing L-homoserine from aspartate .beta.-semialdehyde as a first reaction in a synthesis pathway inherent to L-threonine branching from the L-lysine synthesis pathway through aspartate .beta.-semialdehyde, resulting in progress of the L-lysine synthesis reaction without inhibition of the AK activity. As such an HD deficient strain, an HD completely deficient strain of Corynebacterium glutamicum is known (Nakayama, K. et al.; J. Gen. Appl. Microbiol. 7(3), 145-154 (1961)).
In addition to the HD completely deficient strain as described above, a mutant strain harboring so-called leaky type HD is considered to be effective for L-lysine production, as well. The HD completely deficient strain cannot synthesize L-threonine and L-methionine, and thus it cannot grow unless these amino acids are present in a medium. On the contrary, if an HD leaky type strain can be obtained that harbors a leaky type HD which does not substantially exhibit activity so much to suppress L-lysine production but has HD activity a little, it becomes possible to make growth without addition of L-threonine and L-methionine to a medium, and it becomes convenient to prepare the medium.
Additionally, the leaky type HD has small affinity to aspartate .beta.-semialdehyde as its substrate. Therefore, the HD leaky type strain synthesizes a considerable amount of aspartate .beta.-semialdehyde for synthesizing L-threonine, L-methionine and L-isoleucine required for the growth. Aspartate .beta.-semialdehyde synthesized in a considerable amount is consequently converted into L-lysine.
On the other hand, the HD completely deficient strain is considered to be still useful in that it completely suppresses production of L-threonine in amount, however, the deficiency in HD as a result of mutation has a possibility to restore the activity due to reverse mutation. Thus an HD deficient strain, in which such a possibility is extremely low with a destroyed HD gene, is considered to be more useful. A nucleotide sequence of an HD gene has been reported by Peoples et al. for Corynebacterium glutamicum (Peoples, O. P. et al., Molecular Microbiology, 2(1), 63-72 (1988)).
Since the HD leaky type strain and the HD deficient strain do not produce L-threonine, AK does not undergo feedback inhibition. Accordingly

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Katinka et al. (1980) Nucleotide sequence of the thrA gene of Escherichi coli. Proc. Natl. Acad. Sci. USA 77 (10): 5730-5733, Oct. 1980.
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Mateos et al. (1987) Nucleotide sequence of the homoserine dehydrogenase (thrA) of Brevibacterium lactofermentum. Nucleic Acids Research 15 (24): 10598, Dec. 1987.

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