KR100442036B1 - Method for Culturing Artificially Mutated L-Lysine-Producing Microorganism and process for Producing L-Lysine - Google Patents

Abstract

The present invention is characterized by adding a fermentation broth or an intermediate culture fermentation broth of an artificial mutated L-lysine producing strain to a fermenter containing an artificial mutated L-lysine producing strain and an L-lysine production medium. It relates to a method for preparing L-lysine. In addition, the present invention is characterized in that the seed culture fermentation broth of the strain L- lysine producing strain or the intermediate culture fermentation broth is added to the seed culture medium to prepare a seed culture of the artificial strain treated L- lysine production strain It is about a method. In addition, the present invention is characterized in that the artificial mutant-treated L- lysine producing L- lysine-producing strain or intermediate culture fermentation broth is added to the culture medium of the artificial mutated L- lysine producing strain characterized in that It relates to a method of culturing the production strain.

Description

Method for culturing L-lysine producing strain treated with artificial mutants and producing method of L-lysine {Method for Culturing Artificially Mutated L-Lysine-Producing Microorganism and process for Producing L-Lysine}

The present invention relates to a method for culturing L-lysine producing variant strains and a method for preparing L-lysine.

Conventional methods for producing L-lysine include enzymatic conversion using DL-α-amino-ε-caprolactam as a substrate, direct fermentation by amino acid nutrition, drug-resistant mutant strains, and amino acid non-constitutive cell fusion strains. The production of L-lysine mainly by the latter is the main axis. Patents by microorganisms include Republic of Korea Patents 44119, 48428 and 73609, Republic of Korea Patent Publication 92-14933 and Publication 92-18228, Japan Patent Publication Hei 4-356194, Hei 4-116092, Hei 5-111386 Hei 5-30985, Hei 6-7182, Pyeong 7-155184, Pyeong 10-113183, Pyeong 10-165180, etc., which include improved fertility by the fermentation process, development of artificially mutated high-mutant strains by mutagens, recombinant strains or DNAs associated with them. Intercepts, etc. Among these patents, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) is used as a development method of high titer mutant strains because it has a stronger effect on microbial mutation than any other material. However, when several strains with improved fermentation concentrations or yields are applied to fermenters due to several times of artificial mutations, they often suffer from unexpected results, which are delayed due to lack of trace metabolites. If it is. It is believed that this is because characteristic changes occur in addition to the desired site of the strain.

The present inventors studied L-lysine high titer mutant strains by artificial mutants, but the L-fermentation concentration and yield in flask fermentation were high, but in particular, in order to solve the fact that the culture time was significantly delayed compared to the parent strain in the seed culture stage. As a result of the addition of a portion at the beginning of the culture, the seed culture time was 20% or more, and the culture time in the production medium was shortened by 5% or more, thus completing the present invention.

In general, the present invention is characterized by adding a culture fermentation broth of the strain L- lysine producing strain or an intermediate culture fermentation broth to the seed culture medium to prepare a seed culture of the artificial strain treated L- lysine production strain Provide a way to.

In addition, the present invention is characterized in that the artificial mutant-treated L- lysine producing L- lysine-producing strain or intermediate culture fermentation broth is added to the culture medium of the artificial mutated L- lysine producing strain characterized in that Provided is a method of culturing a production strain.

In addition, the present invention is characterized in that the fermentation tank containing the artificial mutated L- lysine producing strain and L- lysine production medium is fermented by adding the fermentation broth or intermediate culture fermentation broth of the artificial mutated L- lysine producing strain. This provides a method for producing L-lysine.

The present invention is a method for stabilizing the fermentation process by shortening the seed culture time and production culture time in L-lysine fermentation using microorganisms, mainly N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter, NTG It is useful to apply the mutagenic high titer strain to the production site in a short time.

Therefore, the present invention specifically relates to adding L-lysine producing high titer mutant strains obtained by treating NTG to L-lysine producing strains or adding intermediate fermentation broths thereof to the seed culture medium. Provided are methods for preparing a species culture of lysine producing variant strains.

In addition, the present invention is added to the culture medium of the L- lysine production strain treated with the artificial mutant strain culture fermentation broth or its intermediate culture fermentation broth obtained by treating NTG to L- lysine producing strains. Characterized in that it provides a method for culturing the artificial mutated L-lysine producing strain.

In addition, the present invention is the seed culture fermentation broth of the L- lysine producing strain treated with the artificial mutant in the fermenter containing L- lysine producing high titer strain and L- lysine production medium obtained by treating NTG to L- lysine producing strain or It provides a method for producing L-lysine, characterized in that the fermentation broth by addition of the intermediate culture.

The present invention is explained in detail by the following examples. Corynebacterium glutamicum KFCC 11043, a L-lysine producing strain used as a test strain, was obtained from α-amino-β-hydroxy valeric acid, S- (β -Aminoethyl) -L-cysteine, methyl lysine and the like and L- leucine and guanine leak (leaky), acetic acid non-magnetic properties. The medium used for the cultivation of the strains is shown in each example, L-lysine concentration was high performance liquid chromatography (HPLC), the equivalent was quantified using the Bertrand method if necessary.

(Example 1)

First, KFCC 11043, the experimental strain, was grown in a Luria bertani (LB) solid medium at 30 ° C incubator for 20 to 24 hours. g, urea 5g, biotin 50 ㎍, process water 1L (pH 7.0, before sterilization)] was inoculated with one platinum and incubated at 30 ℃, shaking culture incubator for 220 rpm, 20 hours and centrifuged at 10 ℃, 3,000 rpm. Only the supernatant from which the cells were removed was stored in a 4 ° C. refrigerator and tested in the same medium (volume 100 ml) and culture conditions in a 500 ml shake flask for each concentration. Cells were suspended in sterile physiological saline to maintain the same inoculation, and the residue and cell growth (Optical density) of each culture time was measured and the results are shown in Table 1 below.

TABLE 1

Effect of Species Culture Fermented Supernatant on Cell Growth of Corynebacterium Glutamicum KFCC11043

Experimental results showed that when the culture fermentation supernatant was added more than 0.5%, the incubation time could be reduced by 20% compared to the case without addition. Therefore, KFCC11043, a high titer L-lysine producing strain produced by NTG treatment, showed a trace amount of metabolite leakage. It is considered to have a leaky property.

(Example 2)

The effect was tested in the L-lysine production medium of the 7L fermenter, using the fact that the incubation time was shortened when more than 0.5% of the medium volume (0.5 ml supernatant was added to the 100 ml medium volume) was shortened. The culture method was carried out by fed-batch fermentation method in which additional sugars and additional products were added during the cultivation. The medium components and culture conditions used were as follows and the results are shown in Table 2 below.

7L fermentation tank medium components: molasses (reduced sugar) 280g / l, corn steep liquor 100ml / l, (NH4) 2SO4 35g / l, KH2PO41.0g / l, MgSO47H2O 0.5g / l, FeSO4 7H2O 10mg / l, biotin 300㎍ / l, thiamine hydrochloride 500 μg / l, antifoam 3 ml / l)

Culture conditions: temperature 30 ℃, pH 7.2, stirring speed 600 rpm, aeration rate 1vvm (air volume / medium / minute)

TABLE 2

Nutritional Supplementation of L-lysine Fermentation of Corynebacterium glutamicum KFCC11043 by Addition of Species Culture Supernatant (7L Fermentation Tank)

* PCV: Packed Cell Volume

As a result of the experiment, when 0.5% or more of the culture supernatant was added, the incubation time could be reduced by about 5% from 65 hours to 61.5 hours without changing the fermentation concentration and yield. This method can be applied to any amino acid-producing bacteria culture as well as to the production plant.

The method of the present invention will be very useful industrially by increasing the production yield of L-lysine as well as providing stabilization of the fermentation process by shortening the seed culture time and production culture time in fermentation of L-lysine using microorganisms. .

Claims (9)

In the method for producing a seed culture of the artificial mutated L-lysine-producing strain, the artificial mutant, characterized in that the addition of the supernatant from the seed culture fermentation broth of the strain or the intermediate culture fermentation broth additionally added to the seed culture medium A method of preparing a seed culture of the treated L-lysine producing strain. The method of claim 1, wherein the L-lysine producing strain has been mutated by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. The method of claim 1 or 2, wherein the strain is Corynebacterium glutamicum KFCC 11043. In the method of culturing the artificial mutated L-lysine producing strain, L-lysine treated with artificial mutant, characterized in that the supernatant from which the cells were removed from the strain culture fermentation broth or intermediate culture fermentation broth is added to the culture medium of the strain. Cultivation method of lysine producing strain. The method of claim 4, wherein the L-lysine producing strain has been mutated by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The method of claim 4 or 5, wherein the strain is Corynebacterium glutamicum KFCC 11043. In the method for producing L-lysine using the artificial mutated L-lysine producing strain, L- lysine characterized in that the addition of the supernatant from which the bacteria were removed from the seed culture fermentation broth of the strain or the intermediate culture fermentation broth. Lysine manufacturing method. 8. The method of claim 7, wherein the L-lysine producing strain has been mutated by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The method of claim 7 or 8, wherein the strain is Corynebacterium glutamicum KFCC 11043.