PRODUCTION DE MICROORGANISMES PRODUCTEURS DE L-LYSINE

PRODUCTION OF MICROORGANISMS PRODUCING L-LYSINE

Abrégé anglais

Described is a method for the preparation of microorganisms which have an
elevated L-lysine production capability by
mutation of microorganisms of the genuses Corynebacterium and Brevibacterium
with known mutagens in a known manner,
characterized in that strains of said genuses are mutated, selecting those
wich are resistant to regenerative inhib~~on by 2-azido-.epsilon.-
caprolactam.

Revendications

3
WHAT IS CLAIMED IS:
1. A process for producing microorganisms which
have increased L-lysine productivity comprising:
- mutating strains of mircroorganisms of a
genus selected from the group consisting of Corynebacterium
and Brevibacterium;
- and selecting the strains that are resistant
to feedback inhibition by 2-azido-.epsilon.-caprolactam.
2. A process according to claim 1, wherein the
step of mutating is performed with N-methyl-N'-nitro-N-
nitrosoguanidine or by U.V. radiation.
3. A process according to claim 1 or 2, wherein
the microorganisms are selected from the group consisting
of B. amoniagenis, B. divaricatum, B. flavum, B. keto-
glutamicum, B. lactofermentum, B. linens, B. sp.,
C. acetoacidophilum, C. acetoglutamicum, C. glutamicum,
C. lilium and C. sp.
4. A process according to claim 3, wherein the
microorganisms are B. flavum or C. glutamicum.
5. A process according to claim 4, wherein the
microorganisms are B. flavum having ATCC deposit number
21.474 or C. glutamicum having ATCC deposit number 21526.

Description

CA 02075044 2000-08-28
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The production of microorq_anisms producincr L-lysine
The present invention relates to a process for
producing L-lysine and microorganisms therefor.
L-lysine is an essential amino acid and is widely
used as additive to human and animal food. It is also
employed in medicine as component of infusion solutions.
L-lysine is obtained by hydrolysis of proteins
with acid, by synthesis of D,L-lysine and subsequent
resolution of the racemate and by synthesis with.the aid
of microorganisms. Microbiological processes for pre-
paring.L-lysine are described, for example, in Trends in
Biotechnology 1 (1983) 70-74.
We have found an improved process for producing
microorganisms which produce L-lysine.
The present invention relates to a process for
producing microorganisms which have increased L-lysine
productivity comprising:
- mutating strains of mircroorganisms of a genus
selected from the group consisting of Corynebacterium and
Brevibacterium; and
- selecting the strains that are resistant to
feedback inhibition by 2-azido-E-caprolactam.
Surprisingly, 2-azido-e-caprolactam has con-
siderably higher efficiency in the selection of mutants
after mutagenesis than the compounds described in
JP 51-19 186 and EP 175 309, such as fluoro- or chloro-
caprolactam.
It is thus possible by selection with azidocapro-
lactam to increase the lysine productivity of strains by
more than 10$.
The mutants according to the invention can be
produced by conven°cional mutagenesis, eg. with N-methyl
N'-nitro-N-nitrosoguanidine or by U.V. radiation.


CA 02075044 2000-08-28
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Examples of suitable microorganisms of the genera
Corynebacterium (C) and Brevibacterium (B) are the
following:
B. amoniagenis, 8. divaricatum, B. flavum, B.
ketoglutamicum, B. lactofermentum, B. linens, B. sp., C.

~o~~o~~
- 2 - O.Z. 0050/41780
acetoacidophilum, C. acetoglutamicum, C. glutamicum, C.
lilium and C. sp. Preference is given to B, flavum and C.
glutamicum, especially B. flavum ATCC 21.474 and C.
glutamicum ATCC 21526. The latter has the special advan-
Cage that it is homoserine-dependent and, moreover, is
resistant to S-(2-aminoethyl)-L-cysteine.
As already indicated, it is beneficial for the
strains to be homoserine-dependent. It is also useful for
the strains to be resistant to S-(2-aminoethyl)-L-cys-
teine. If the strains do not possess this resistance it
can be introduced as described in US 3,707,441 by
treating the strains with N-methyl-N'-vitro-N-nitroso-
guanidine and subsequent selection.
Example
Corynebacterium glutamicum ATCC 21 526 was
treated with 250 ~g/ml N-methyl-N'-vitro-N-nitrosoguani-
dine in tris/maleic acid buffer, pH 6.0, at 30°C for 30
min. The cells were then washed with 0.1 M tris buffer,
pH 7.2, plated on minimal agar plates and then incubated
at 28°C for from 4 to 14 days.
The minimal agar had the following composition:
20 g/1 agar 0.1 g/1 MnS04-H20
2 g/1 ( NH4 ) ZSO4 100 ~gf 1 biotin
0.5 g/1 RHZPO4 30 mg/1 each Met, Thr, Leu
0.5 g/1,K2HpO4 4 g/1 lactate
0 .4 g/1 MgS04 ~ 7H20
0. O1 g/1 FeSO, ~ 7H20 pH = 7 . 0
The colonies- producing lysine amongst those
growing on the agar plates after incubation were identi-
fied. The strains which produced 10% more lysine than the
initial strain were isolated.