KR950012899B1 - Novel hybrid microorganism - Google Patents

Abstract

The fermentation of demethyl tetracycline (I) with hybrid of Streptomyces aureofaciens (II) CKD 3562 (KCTC 8526P) (III) in the media of starch 5.5%, cotton seed powder 2%, calcium carbonate 0.6%, yeast extract 0.13%, vegetable oil 0.5%, casein 0.1%, ammonium chloride 0.2%, trace elements at pH 7.0 yields 3750 mg/l. Treated (II) ATCC 12551 with UV, his requiring mutant and arg requiring mutant are selected, and the protoplasts of each mutant are fused by PEG method. (III) can grow without inhibition by chloride and copper used for conversing (I) into minocycline in antibiotic.

Description

Novel cell fusion strain and preparation of demethyl tetracycline using the same

1 is a result of analysis of DMT and DMCT by high-performance liquid chromatography according to Example 2.

The present invention is a demethyltetracycline (DMT) which is a precursor capable of increasing the chemical synthesis yield of minocycline [compound having R ₁ = N (CH ₃ ) ₂ , R ₂ = H of the general formula (1)] , R ₁ = H, R ₂ = OH in the formula (1)] relates to a method for producing a novel microbial fusion strain.

More specifically, the present invention is directed to a nutrient medium containing carbon sources such as starch and hydrolyzate thereof, and organic nitrogen sources such as corn steep liquor and cotton seed meal. Cultured cell fusion strains) to minimize the accumulation of demethyl chlortetracycline (DMCT), a compound of R ₁ = Cl, R ₂ = OH of formula (1) in the culture medium. It is characterized by not only facilitating recovery of methyltetracycline (DMT) but also chemical conversion to minocycline.

Demethylchlortetracycline (DMCT) has been used as a synthetic starting material for minocycline, which is currently produced industrially. A study on this has been carried out to isolate demethylchlortetracycline (DMCT) from mutant strains of Streptomyces aureofaciens ATCC 12551, which is produced by Tetracycline (see J. Am. Chem. Soc. 79, 4561, 1957: USP 2,808,289), and there have been many studies on the minocycline synthesis method using demethylchlortetracycline (DMCT) as reference material (USP 3,526,629; USP 4,038,315; GB 1,469,384: USP). 3,226,436: USP 3,148,212: USP 3,901,942).

The inventors have discovered that Streptomyces aueofaciens ATCC 12551, a known strain producing demethylchlortetracycline (DMCT), produces a small amount of demethyltetracycline (DMT) in culture. It was confirmed that the minocycline was more useful than the conventional precursor demethylchlortetracycline (DMCT).

Accordingly, the present inventors use the known strain Streptomyces aureofaciens ATCC 12551 as a mutant, UV, EMS (ethylmethan sulfonate) for the purpose of developing a strain accumulating a large amount of demethyltetracycline (DMT). Treated by a known method to obtain an auxotrophic mutant to be used as a marker for identifying the cell fusion strain, and a series of cells such as protoplast formation, cell fusion, regeneration as indicated in Example (1) Numerous cell fusions were obtained by fusion method (Ref .: Okanishi et al. J. Gen. Microbiol 80,389, 1974). Among the cell fusions, a candidate strain with a large degree of proliferation inhibition of Bacillus subtilis, which is the first to be tested, was selected, and a large amount of demethyltetracycline (DMT), which is a target product, was accumulated in the culture medium through liquid culture. Methylchlortetracycline (DMCT) completed the present invention by selecting a small amount of cell fusion strain, Streptomyces Oreopathians CKD 3562.

In the present invention, the obtained high titer cell fusion strain Streptomyces Oreopaciens CKD 3562 (KCTC 8526P) has the following characteristics compared with the known parent strain Streptomyces Oreopasions ATCC 12551.

1) Morphological Properties

As shown in Table 1, the end of the aerial mycelium of the two strains has a unique spiral spiral, but the cell fusion strains have a slightly shorter length of the mycelia, and the sporulation ability is significantly improved compared to the intimate strains having a weak sporulation ability. It became. In particular, cell fusion strains form greyish brown spores in oatmeal agar medium.

TABLE 1

Microscopic Observation of the Proton strain ATCC 12551 and High Potency Cell Fusion Cell CKD 3562

2) Cultural Properties

end. Influence of chloride ions

Table 2 shows the growth and production of demethyltetracycline with different chloride ion concentrations while incubating the CKD 3562 of the parent strain and the cell fusion strain CKD 3562 in a carbon source with starch and a nitrogen source with corn leachate and cottonseed meal, respectively.

That is, as shown in Table 2, the cell growth of cell fusion strains did not show a significant difference as the chloride ion concentration increased, but the growth rate of the cell strains of the fusion strains was increased, and the production capacity of demethyltetracycline (DMT) was increased in the culture medium. While the number of chloride ions decreases, the cell fusion strain shows little change, indicating that the cell fusion strain is a mutant with a difference in affinity (Ka value) or metabolism itself.

TABLE 2

Effect of Chloride Ion Concentration in Culture of Probiotics and Cell Fusion

I. Promoting Chloride Addition Reactions and Inhibitors

Effects of copper ions (Cu ++), known as promoters of biosynthetic chloride addition reactions, and dimercapto chiadiazole (2,5-dimercapto-1,3,4-thiadiazole, DMTD) as inhibitors on probiotics and cell fusion strains As shown in Tables 3-1 and 3-2, the parental strains were expected to vary with the concentration of copper ions and dimecaptothiadiazole (DMTD), that is, the former increased as the concentration increased. Decreased concentration of dimethyl tetracycline (DMCT) decreased with decreasing concentration of demethylchlor tetracycline (DMCT), but cell fusion strain showed no physiologically different strains because it did not respond to the above two substances. have.

Table 3-1

Differences in the Chloride Addition Reaction of the Probiotic ATCC 1255l and Cell Fusion CKD 3562

* Chloride addition reaction accelerator

Table 3-2

Differences in the Chloride Addition Reaction between the Proton strain ATCC 12551 and the Cell Fusion Line CKD 3562 (Dimercapto Tooth Diazole * Effect)

* Chloride addition reaction inhibitors

In addition to the above characteristics, 1) selected cell fusion strains can be adjusted physiologically without the addition of alkalis or acids during cultivation. 2) the variation of fermentation performance according to the concentration of corn leachate (cornsteep liquor) in the raw material is low, and 3) the formation of spores to preserve the strain, It has advantages such as advantages in development and the like.

The present invention is described in more detail in the following examples, and the cell fusion strain Streptomyces Oreopathians CKD 3562 specified in the Examples was deposited as KCTC 8256P on September 24, 1992 by KCTC (Korea Institute of Science and Technology).

Example 1

Species culture of histidine-retained strain (His-) and arginine-retained strain (Arg-) among amino acid extracts obtained under conditions where the mortality was 99.996 by irradiating UV spores at 240 μw / cm ² for 90 seconds to spore suspensions Was inoculated in S medium supplemented with 1% glycine (glucose 10g, peptone 4g, Yeast extract 4g, MgSO ₄ 0.5g, KH ₂ PO ₄ 2g, K ₂ HPO ₄ 4g, distilled water 1000ml) to 28 After incubation, the cells were collected and suspended in P3 buffer (NaC1 4.1g, MgCl ₂ · 6H ₂ O 1.0g, CaCl ₂ · 2H ₂ O 0.75g, Sucrose 137g, 0.25M TES buffer 100ml, H ₂ O 850ml). 2 mg / ml was added to form protoplasts.

Each protoplast formed was collected and PWP buffer (NaCl 4.1g, MgCl ₂ · 6H ₂ O 2.05g, CaCl ₂ · 2H ₂ O 2.95g, Sucrose 137g, 0.25M TES buffer 100ml, H ₂ O 850ml) 1.0 Suspended together in ml, 0.9 ml of 50% PEG 6000 solution was added to induce fusion, followed by R ₂ regeneration (Sucrose 103g, Glucose 10g, K ₂ SO ₄ 0.25g, MgCl ₂ · 6H ₂ O 10.2g, KH ₂ PO ₄ 0.05g, CaCl ₂ · 2H ₂ O 2.95g, L-proline 3.0g, 0.2M-TES buffer 100ml, Agar 22g, H ₂ O 850ml) was reached to select the fusion strain.

Example 2

Strains used: Streptomyces Oreopathians ATCC 12551 (Probiotics)

Streptomyces Oreofasion CKD 3562

Spawn culture medium: corn leachate 4%, ammonium sulfate 0.2%, calcium carbonate 1.2%, rice bran oil 0.1%, pH 7.0 ± 0.2

Presentation medium: starch 5.5%, cottonseed powder 2%, calcium carbonate 0.6%, yeast extract 0.13%, animal and vegetable oil 0.5%, casein 0.1%, ammonium chloride 0.2%, magnesium sulfate, appropriate amount of trace elements, pH 7.0 ± 0.2

Analysis method: The target product DMT and by-product DMCT were analyzed by the following high performance liquid chromatography method, and the analysis example is shown in FIG.

Column: Bondapak C ₁ 8 (39 × 300mm)

Mobile phase: EDTA 0.34g, citric acid 1.58g, sodium citrate 2.94g, DMF 250ml, HNO ₃ 7.5ml H ₂ O 750ml (pH 2.5)

Sample injection volume: 10 μl

Temperature: Room temperature

Mobile phase flow rate: 1.0ml / min

Cultivation method: 80 ml of spawn culture medium was put into a 500 ml round flask and sterilized under pressure at 121 ° C. for 20 minutes, and the two strains were inoculated and incubated in a reciprocating shaker at a temperature of 28 ° C. and 100 rpm for about 30 hours. The fermentation broth was put into a 500 ml Erlenmeyer flask and sterilized as the seed medium condition, and then inoculated with 5% of the seed broth and incubated at 25 ° C. at 220 rpm for 7 days.

Results: As can be seen in Table 4, the cell fusion strain DMT biosynthesis ability was clearly improved (about 5 times, 3289/675) compared to the parent strain.

TABLE 4

Comparison of Antibiotic Production Ability between Probiotics and Cell Fusion

* DMT: Demethyltetracycline (Purpose)

DMCT: Demethylchlortetracycline (by-product)

Example 3

The strain and the medium composition of the present embodiment are the same as in Example 2, and 800ml of medium was put into a 7L round flask and sterilized under pressure at 121 ° C for 40 minutes, and then inoculated with the strain, followed by reciprocating shaking for about 30 hours at 28 ° C and 100rpm. Incubate in phase. This seed culture is inoculated 5% in a 19L fermentation medium filled with 10L fermentation medium and incubated for 7 days under conditions of 25 ℃-28 ℃, 500-900rpm, 0.5-1.0vvm (air flow rate). During the cultivation was maintained in a natural state without adjusting the pH and the experimental results are shown in Table 5.

TABLE 5

Comparison of Antibiotic Production Ability between Probiotics and Cell Fusion

Example 4

The strain and medium composition of this Example were the same as in Example 2, and the seed culture was incubated for 25 hours at 19 ℃ fermenter at 28 ° C and 500 rpm for 25 hours, and then inoculated with 6.5L in a 250L fermenter filled with 130L fermentation broth at 28 ° C, 150 After 7 days of incubation at -250rpm and 0.5-1.0vvm, the concentration of DMT, the target product, was nearly 90% in the case of cell fusion strain, and its absolute concentration was increased by 10 times compared to that of the probiotic strain. Compared with the results of Examples 2 and 3 shows that the production capacity of the target product DMT is increased as the culture vessel is increased, which has advantages in industrial production.

TABLE 6

Comparison of Antibiotic Production Ability between Probiotics and Cell Fusion

Claims (2)

Streptomyces aureofaciens forms gray-brown spores in oatmeal agar medium and produces relatively large amounts of demethylchlortetracycline (DMCT) in the culture medium, but produces a relatively large amount of target product demethyltetracycline (DMT). ) Fusion mutant strain CKD-3562 KCTC 8526P. Streptomyes aureofaciens cell fusion CKD 3562 KCTC 8526P of claim 1 was cultured in a medium containing a carbon source, an energy source, and a nitrogen source to form demethyltetracycline (DMT) and demethylchlortetracycline (DMCT). A method for producing demethyltetracycline, characterized in that fractionation of demethyltetracycline is produced after accumulation and accumulation.