KR100187607B1 - Process for producing l-lysine by fermentation employing a microorganism resistant to acyl-lysine or methylated acyl - Google Patents

Abstract

The present invention relates to a method for producing L-lysine by fermentation. More specifically, the present invention relates to a method for producing L-lysine in high yield by culturing microorganisms belonging to the genus Corynebacterium.

Description

Method for preparing L-lysine by fermentation

The present invention relates to a method for producing L-lysine by fermentation. More specifically, the present invention relates to a method for producing L-lysine in high yield by culturing microorganisms belonging to the genus Corynebacterium.

L-lysine is an important amino acid that is used as a feed additive for poultry and swine because it is lacking in feed crops such as corn.

Commonly known methods of preparing L-lysine by fermentation yield L-lysine productivity, such as homoserine nutrition, S- (2-aminoethyl) -L-cysteine resistance, α-chlorocaprolactam resistance, and the like. Imparting the necessary properties to the microorganisms (hereinafter referred to as native strains) belonging to the genus Brevibacterium or Corynebacterium, and culturing the microorganisms in a medium containing a carbon source and a nitrogen source And collecting L-lysine produced and accumulated in a culture broth by a resin adsorption method or the like. In addition, methods of using improved strains of these L-lysine producing microorganisms, such as L-alanine nutrients, fluoropyruvate sensitive strains, L-leucine homologue resistant strains, and the like, have also been studied. The yield of L-lysine has not been satisfactory yet. Therefore, there is a need for a method for producing L-lysine more efficiently using L-lysine producing microorganisms or mutants thereof having more improved L-lysine productivity and improved fermentation yields at lower cost than conventional methods.

It is an object of the present invention to further improve the L-lysine productivity of L-lysine-producing microorganisms or improved mutants thereof and to enhance the fermentation yield, thereby efficiently producing L-lysine at a lower cost than conventional methods. To provide.

In order to further improve the L-lysine productivity of L-lysine-producing microorganisms or improved mutants thereof and to enhance fermentation yield, intensive studies by the inventors have shown that they are resistant to acyl-lysine and / or methylated acyl-lysine. It was found that this obtained L-lysine-producing mutant was suitable for this purpose, and based on this finding, the present invention was completed.

That is, the present invention provides a step of culturing microorganisms belonging to the genus Brevibacterium or Corynebacterium which is resistant to acyl-lysine and / or methylated acyl-lysine and capable of producing L-lysine and produced in culture broth. It provides a method of producing L- lysine by fermentation, comprising the step of collecting the accumulated L- lysine.

Acyl-lysine used in the present invention refers to a compound comprising a fatty acid residue having 5 to 20 carbon atoms bonded to an amino group via an acyl bond at the α-position and / or ε-position of L-lysine or DL-lysine. Examples of such compounds include N ^α -stearoyl-L-lysine, N ^ε -decanoyl-L-lysine, N ^ε -myristoyl-L-lysine, N ^ε -palmitoyl-L-lysine, N ^α , N ^ε -octanoyl-L-lysine, N ^α , N ^{ε -dilauroyl} -L-lysine and the like. Methylated acyl-lysine represents acyl-lysine methylated at an amino group in the α-position. Examples of such compounds include N ^α -methyl-N ^{ε -stearoyl} -L-lysine, N ^α , N ^α -dimethyl-N ^ε -decanoyl-DL-lysine, N ^α , N ^α -dimethyl-N ^ε − Palmitoyl-DL-lysine, N ^α , N ^α , N ^α -trimethyl-N ^ε -myristoyl-DL-lysine, N ^α , N ^α , N ^α -trimethyl-N ^ε -palmitoyl-DL-lysine This includes.

The mutants used herein belong to the genus Brevibacterium or Corynebacterium and are resistant to acyl-lysine and / or methylated acyl-lysine, ie even in the presence of acyl-lysine at a concentration of 25 mg / l. And / or even grow well in the presence of methylated acyl-lysine at a concentration of 25 mg / l and homoserine trophic, S- (2-aminoethyl) -L-cysteine resistance, α-chlorocaprolactam resistance or their And known properties which are essential for obtaining L-lysine productivity, such as mixed properties and the like. The well-growing properties herein indicate that when growth in the absence of acyl-lysine and methylated acyl-lysine is 100, the relative growth is at least 50.

Specific examples of mutants are as follows:

Brevibacterium lactofermentum AJ 12592 (FERM BP-3239),

Brevibacterium lactofermentium AJ 12593 (FERM BP-3240),

Corynebacterium glutamicum AJ 12596 (FERM BP-3242).

The above strains were deposited on January 24, 1991, to the Institute of Microbiological Industry and Technology (FRI) of the Institute of Industrial Technology and Technology, located in the 1st and 3rd half of Higashi 1-chome, Tsukuba City, 305, under the Treaty of Budapest.

As parent strains used to obtain these mutants, microorganisms belonging to the genus Brevibacterium or Corynebacterium already known as producing L-lysine are used. In addition, the native strains shown below can also be used as parent strains.

Brevibacterium lactofermentum ATCC 13869,

Brevibacterium flavum ATCC 14067,

Brevibacterium divaricatum ATCC 14020,

Corynebacterium glutamicum ATCC 13032,

Corynebacterium acetoacidophilum ATCC 13870,

Corynebacterium acetoglutamicum ATCC 15806.

After giving L-lysine productivity to these native strains, resistance to acyl-lysine and / or methylated acyl-lysine can be conferred. Alternatively, resistance to acyl-lysine and / or methylated acyl-lysine may be imparted to these autologous strains, followed by L-lysine productivity.

In mutating the parent strain, conventional methods such as contact with N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter abbreviated NG) can be applied. In screening acyl-lysine and / or methylated acyl-lysine resistant strains from such mutants, in a medium containing acyl-lysine or methylated acyl-lysine at a concentration that can significantly inhibit the growth of the parent strain. A method may be employed that includes collecting mutants that grow well. Specific methods of collecting resistant strains can be illustrated as follows:

Brevibacterium lactofermentum AJ 12435 (FERM BP-2294) live cells, L-lysine-producing microorganisms resistant to S- (2-aminoethyl) -L-cysteine, at 30 ° C. Treat for minutes. Cells showing a 1.0% survival rate are seeded onto agar plate medium, the minimum medium shown in Table 1 supplemented with 50 mg / l of N ^α , N ^ε -dioctanoyl-L-lysine, one of the acyl-lysines. After incubation at 30 ° C. for one week, growth colonies are collected. The concentration of acyl-lysine or methylated acyl-lysine supplemented to the medium is appropriately selected according to the minimum growth inhibitory concentration of the parent strain selected and used to effectively collect the desired resistant strain. One of the resistant strains is Brevibacterium lactofermentum AJ 12592 (FERM BP-3239), which will be further provided in the following experiments. Similarly, Brevibacterium lactofermentum AJ 12593 (FERM BP-3240) is obtained as N ^α , N ^α , N ^α -trimethyl-N ^ε -palmitoyl-DL-lysine-resistant strains.

ingredient density Glucose Ammonium Sulfate KH ₂ PO ₄ MgSO ₄ .7H ₂ OFeSO ₄ .7H ₂ OMnSO ₄ .4H ₂ OBiotinthiamine hydrochlorideurea7Agar 20g / l10g / l1g / l0.4g / l10mg / l10mg / l50μg / l100μg / l2g / l20g / l

In addition, Corynebacterium glutamicum AJ 3463 (FERM BP-1987) was used as the parent strain, and Corynebacterium glutamicum AJ 12596 (FERM BP-3242) was used as N ^α , N ^α , N ^α − Obtained as trimethyl-N ^ε -palmitoyl-DL-lysine resistant strain.

The resistance of each resistant strain to acyl-lysine or methylated acyl-lysine thus obtained is measured as follows and compared with the resistance of the parent strain. Acyl-lysine or methylated acyl-lysine is added to the media shown in Table 2 at respective concentrations and 4 ml of media is separately charged into small test tubes. After sterilization, the test strain is incubated on the medium. After the culture is shaken at 30 ° C. for 24 hours, the turbidity is measured at a wavelength of 562 nm and the relative growth rate is calculated based on the growth rate in medium without the addition of acyl-lysine or methylated acyl-lysine. . The results are shown in Table 3. Mutants of the present invention exhibit good relative growth rates of greater than 50 in the presence of acyl-lysine or methylated acyl-lysine at a concentration of 25 mg / l, while the parent strain exhibits very poor relative growth rates of less than 10. As mentioned above, the mutants of the present invention are clearly distinguished from the parent strain because they are resistant to acyl-lysine or methylated acyl-lysine.

ingredient density Sucrose ammonium sulphate KH ₂ PO ₄ MgSO ₄ .7H ₂ OFeSO ₄ .7H ₂ OMnSO ₄ .4H ₂ O biotinthiamine hydrochloride nicotinamide protein hydrolyzate urea 20g / l5g / l1g / l0.4g / l10mg / l10mg / l100μg / l200μg / l5mg / l1g / l (calculated as nitrogen) 3g / l

Test strain reagent Reagent concentration Relative growth rate Brevibacterium lactofermentum AJ 12435 N ^α , N ^ε -dioctanoyl-L-lysine 0 (mg / l) 10029865 Brevibacterium lactofermentum AJ 12592 N ^α , N ^ε -dioctanoyl-L-lysine 0 (mg / l) 10092811410 Brevibacterium lactofermentum AJ 12435 N ^α , N ^α , N ^α -trimethyl-N ^ε -palmitoyl-DL-lysine 0 (mg / l) 10048985 Brevibacterium lactofermentum AJ 12593 N ^α , N ^α , N ^α -trimethyl-N ^ε , palmitoyl-DL-lysine 0 (mg / l) 100100531311 Corynebacterium glutamicum AJ 3463 N ^α , N ^ε -dioctanoyl-L-lysine 0 (mg / l) 10038765 Corynebacterium glutamicum AJ 12596 N ^α , N ^ε -dioctanoyl-L-lysine 0 (mg / l) 100100934524

Using such resistant strains, L-lysine can be produced fermented in conventional nutrient media containing carbon sources, nitrogen sources, inorganic ions, growth factors and nutrients used in conventional methods. As the carbon source, sugars (e.g., glucose, sucrose, molasses, starch hydrolysates, etc.), organic acids (e.g., acetic acid, propionic acid, etc.), alcohols (e.g., ethanol, propanol, etc.) can be used. As the nitrogen source, ammonium sulfate, ammonium nitrate, ammonium chloride, urea, ammonia or the like can be used.

As conditions for culturing the strain, the aerobic culture is carried out at a fermentation temperature of 24 to 37 ℃, preferably 30 to 34 ℃. The number of days required to ferment is generally 2 to 7 days. The pH at the start of fermentation or during fermentation is in the range of 5.0 to 8.5, preferably 6.0 to 7.5. In order to adjust the pH, inorganic or organic acidic or alkaline substances may be used, in addition, urea, calcium carbonate, ammonia gas and the like may be used.

After fermentation, L-lysine is collected from the culture broth using known methods using ion exchange resins, crystallization and the like and mixing methods thereof.

Example

Next, the present invention will be described in more detail with reference to the following examples.

Example 1

Glucose 36g / l, ammonium chloride, _{20g / l, KH 2 PO 4} g / l, MgSO 4 .7H 2 O 400mg / l, FeSO 4 .7H 2 O 10mg / l, MnSO 4 .4H 2 O 8mg / l, soybean Medium (pH: 7.0) containing 3 mg / l of protein hydrolysate (calculated by nitrogen), 0.1 mg / l of thiamin hydrochloride and 0.3 mg / l of biotin is separately charged into a 500 ml shake flask in a volume of 20 ml each. . After sterilization by heating at 115 ° C. for 10 minutes, 1 g of pre-dry heat sterilized calcium carbonate is added to the flask. Each strain was inoculated onto the medium and then incubated for 48 hours at 31.5 ° C. The amount of L-lysine accumulated in the culture broth is quantitatively determined by acid-copper ninhydrin colorimetric method. The results are shown in Table 4. For all resistant strains, the accumulation of L-lysine was significantly increased compared to the corresponding parent strain.

Strain Accumulated L-Lysine (g / l) % Yield based on sugar Brevibacterium lactofermentum AJ 12435 (parent strain) 9.5 26.4 Brevibacterium lactofermentum AJ 12592 (resistant strain) 11.8 32.8 Corynebacterium glutamicum AJ 3463 (parent strain) 7.0 19.5 Corynebacterium glutamicum AJ 12596 (resistant strain) 10.7 29.7

Example 2

Black strap molasses (blackstrap) 80g / l (calculated as per), ammonium sulfate, _{50g / l, KH 2 PO 4} 1g / l, MgSO 4 .7H 2 O 1g / l, soybean hydrolyzate 10mg / l (calculated as Nitrogen ), A medium (pH: 7.0) consisting of 0.1 mg / l thiamine hydrochloride and 0.3 mg / l biotin is separately charged into a 500 ml shake flask in a volume of 20 ml each. After sterilization by heating at 120 ° C. for 10 minutes, 1 g of pre-dry heat-sterilized calcium carbonate at 180 ° C. is added to the flask. Each strain is inoculated onto the medium and then shake-cultured at 31.5 ° C. for 72 hours. The amount of L-lysine accumulated in the fermentation broth is assayed. The results are shown in Table 5. In resistant strains, the accumulation of L-lysine was significantly increased compared to the parent strain.

Strain Accumulated L-Lysine (g / l) % Yield based on sugar Brevibacterium lactofermentum AJ 12435 (parent strain) 20.4 25.5 Brevibacterium lactofermentum AJ 12593 (resistant strain) 24.8 31.0

According to the present invention, L-lysine-producing microorganisms resistant to acyl-lysine and / or methylated acyl-lysine can be used to produce L-lysine in high yields at high yields, and the cost of industrial production of L-lysine Can reduce the cost.

Claims (1)

Culturing a microorganism belonging to the genus Corynebacterium that is resistant to acyl-lysine and / or methylated acyl-lysine and capable of producing L-lysine; and A method for producing L-lysine by fermentation, comprising collecting L-lysine produced and accumulated in a culture broth.