JPS6117474B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to a method for producing L-serine by fermentation. As a method for producing L-serine by a fermentation method, a method for producing L-serine from glycine using microorganisms such as Corynebacterium and Pseudomonas is known. The present inventors conducted research to develop a more efficient method for producing L-serine. As a result, many temperature-sensitive mutant strains belonging to the genus Pseudomonas produced L-serine in a higher yield than glycine. It has been found that this species has the ability to produce and accumulate in the culture medium. That is, the microorganism used in the present invention belongs to the genus Pseudomonas and is a mutant strain that is temperature sensitive and has the ability to produce L-serine from glycine. A temperature-sensitive mutant strain is one that has a lower growth rate at high temperatures than its parent strain, or
Alternatively, it refers to a mutant strain that cannot grow at all at the high temperatures at which the parent strain can grow. The method for collecting such mutant strains is to collect N-methyl-
After subjecting the parent strain to a mutation treatment such as treatment with N'-nitro-N-nitrosoguanidine, a mutant strain that can grow at lower temperatures but not at higher temperatures compared to the parent strain may be selected. Examples of mutant strains used in the present invention include the following. Pseudomonas rhodomethylofaciens AJ11263 (FEFRM-P4533) Pseudomonas megarubescens AJ11268 (FERM-P4538) Specific collection methods for these exemplary mutant strains and experiments to demonstrate the degree of temperature sensitivity Experimental example with data shown below 1 Pseudomonas megalbetscens AJ11262 or Pseudomonas rhodomethylofaciens
AJ11261 was cultured in a broth liquid medium containing 2.0 ml/dl methanol at 30°C for 24 hours, and the resulting bacterial cells were
Washed with 50mM phosphate buffer. This bacterial cell was added to 50mM phosphate buffer (PH7.0) containing N-methyl-N'-nitro-N-nitrosoguanidine.
The suspension was suspended in water, shaken at 30°C for 15 minutes, and then washed. The mutagenized cells were then spread on a broth agar plate and cultured at 30°C for 4 to 5 days to form colonies. The grown colonies were replicated onto the following synthetic agar plate medium using methanol as the carbon source, and incubated at 37°C.
After culturing for 3 to 4 days, unviable colonies were collected from the original methanol-containing broth agar plate. Each mutant strain obtained in this way was applied to two methanol synthetic agar plates and cultured for 2 to 3 days at 30°C and 37°C to examine growth. This strain was designated as a temperature-sensitive strain that loses its ability to assimilate methanol at high temperatures. Composition of synthetic agar medium: KH ₂ PO ₄ 0.6g/ (NH ₄ ) ₂ HPO ₄ 2.0g/ MgSO ₄・7H ₂ O 0.2g/ NaCl 0.5g/ FeSO ₄・7H ₂ O 10mg/ CaCl ₂・2H ₂ O 10mg / MnSO ₄・4H ₂ O 5mg / Yeast extract 0.2g / Agar 20g / Methanol (separately sterilized) 15ml / PH 7.2 A typical mutant strain obtained in this way is Pseudomonas megabetscens AJ.
AJ 11268 mutated from 11262, and Pseudomonas rhodomethylofaciens AJ 11261
An example of this is AJ 11263, which is more mutagenized. Experimental Example 2 Experimental data showing the temperature sensitivity of the mutant strains obtained as described above are shown in Table 1.

【table】

[Table] Science
AJ 11261 + + + + −
AJ 11263 + + + − −
Experimental method: Each strain was inoculated into a methanol synthetic liquid medium containing 15 ml of methanol/1 g of yeast extract (the agar was removed from the methanol synthetic agar medium and the amount of yeast extract was increased to 1 g/). Shaking culture was carried out for 48 hours at room temperature. Pseudomonas rhodomethylofaciens AJ
11261 (FERM-P4531) and Pseudomonas
Megalbetscens AJ 11262 (FERM-P4532) is a new bacterial species, and its mycological properties are as follows. AJ 11261 (a) Morphology 1 Cell shape and size: large rod, 1.0
~1.5×2-5 microns 2 Presence or absence of cell pleomorphism: Yes, granules of poly-β-hydroxybutyric acid are formed within the bacterial cells 3 Presence or absence of motility, epiphytic status of flagella: Yes, polar flagella 4 Spores Presence, shape, size, location: None 5 Gram staining: Negative 6 Acid-fastness: Negative (b) Growth status in each medium 1 Broth agar plate culture: Moderate growth, circular, whole-rimmed, semi-lenticular, homogeneous , yellow-orange, opaque 2 Broth agar slant culture: Moderate growth, filamentous, red-orange, opaque, no water-soluble pigments produced. 3 Meat juice liquid culture: Moderate growth, ring-shaped 4 Meat juice gelatin puncture culture: Growth on top, aerobic, non-liquefaction. 5 Litmus milk: No change (c) Physiological properties 1 Nitrate reduction: Positive 2 Denitrification reaction: Negative 3 MR test: Negative 4 VP test: Negative 5 Indole production: Negative 6 Hydrogen sulfide production: Negative 7 Starch Hydrolysis: Negative 8 Use of citric acid: Koser medium, slightly used Christensen medium, used slightly 9 Use of inorganic nitrogen sources: Methanol medium Nitrate, used Ammonium salt, used 10 Pigment production: not produced 11 Urease: Negative 12 Oxidase: Positive 13 Catalase: Positive 14 Growth range: Positive Temperature 10-38℃ Cannot grow at 41℃ PH 6-8 15 Attitude toward oxygen: Aerobic 16 Presence or absence of acid and gas production from sugars Acid production Gas Production of L-arabinose + - D-xylose + - D-glucose + - D-mannose + - D-fructose + - D-galactose + - Maltose - - Sugar - - Lactose - - D-sorbitol - - D-mannite - - Glycerin + - Starch - - 17 Accumulation of poly-β-cedroxybutyric acid: + 18 Auxotrophy: None 19 Assimilation of the following compounds (according to Stanier's medium) D-glucose ± trehalose - 2- Ketogluconic acid - L-valine - β-alanine - DL-arginine - Satucarose + Propionic acid - Propylene glycol + Ethanol + D-xylo - D-ribose - Levulinic acid - Citraconic acid - Mesotartrate + D(-)-Tartaric acid - Sorbitol - Erythritol - 2,3-butylene glycol - Metabenzoic acid - Para-benzoic acid - DL-lactic acid - Malonic acid + Testosterone - Cellobiose - DL-β-hydroxybutyrate
+ L-histidine - pantothenic acid - acetic acid + succinic acid + citric acid ± L-ornithine - 5-keto-gluconic acid - L-lysine - L-alanine ± dulcitrate - formic acid + 20 Other properties L-serine hydroxy Methyltransferase activity: Positive Hydroxypyruvate reductase activity:
Positive As mentioned above, this bacterial species is a Gram-negative bacillus, has a polar monoflagellate, is motile, and is aerobic. It also has the property of producing acid rather than sugar, and according to the 8th edition of Barge's Manual, it belongs to the genus Pseudomonas. In addition, a new bacterial species that differs from known bacterial species has characteristics such as lack of auxotrophy, accumulation of poly-β-hydroxybutyric acid within the bacterial body, lack of ability to assimilate arginine, and red pigmentation. Therefore, we named it Pseudomonas rhodomethylofaciens. AJ11262 (a) Morphology 1 Cell shape and size: large rod, 1.0
~1.5 x 2-5 microns 2 Presence or absence of cell pleomorphism: Yes, granules of poly-β-hydroxybutyric acid are accumulated within the bacterial cells. 3 Presence or absence of motility, epiphytic status of flagella: Yes, polar flagella 4 Presence or absence of spores, shape, size, site: None 5 Gram staining: Negative 6 Acid-fastness: Negative (b) Growth status in each medium 1 Juice Agar plate culture: Moderate growth, round, semi-lenticular, homogeneous, red-orange, opaque, no water-soluble pigments produced. 2 Meat juice agar slant culture: Moderate growth, filamentous, red-orange, opaque 3 Meat liquid liquid culture: Moderate growth, ring-shaped 4 Meat juice gelatin puncture culture: Growth on top, aerobic, non-liquefied. 5 Litmus milk: No change (c) Physiological properties 1 Nitrate reduction: Positive 2 Denitrification reaction: Negative 3 MR test: Negative 4 VP test: Negative 5 Indole production: Negative 6 Hydrogen sulfide production: Negative 7 Starch Hydrolysis: Negative 8 Use of citric acid: Koser medium Used Christensen medium Used 9 Use of inorganic nitrogen source: (Methanol medium) Nitrate Used Ammonium salt Used 10 Pigment production: Not produced 11 Urease: Positive 12 Oxidase: Positive 13 Catalase: Positive 14 Growth range: Temperature 10-38℃ Cannot grow at 41℃ PH 6-8 15 Attitude toward oxygen: Aerobic 16 Presence or absence of acid and gas production from sugars Acid production Gas production L -arabinose ± - D-xylose - - D-glucose - - D-mannose ± - D-fructose - - D-galactose - - malt sugar - - sugar - - lactose - - D-sorbitol - - D- Mannite - - Glycerin + - Starch - - 17 Accumulation of poly-β-hydroxybutyric acid: + 18 Auxotrophy: None 19 Assimilation of the following compounds (according to Stanier's medium) D-glucose + trehalose - 2-keto- Gluconic acid ± L-valine - β-alanine + DL-arginine - Satucarose + Propionic acid - Propylene glycol + Ethanol + D-xylose + D-ribose + Levulinic acid + Citraconic acid + Mesotartrate - D(-)-Tartaric acid - Sorbitol - Erythritol - 2,3-butylene glycol - Metabenzoic acid - Parabenzoic acid - DL-lactic acid - Malonic acid + Testosterone - Cellobiose - DL-β-Hydroxybutyrate + L-Histidine - Pantothenic acid ± Acetic acid - Succinic acid + Citric acid + L-ornithine - 5-keto-gluconic acid ± L-lysine - L-alanine + dulcitrate ± formic acid - 20 Other properties L-serine hydroxymethyltransferase activity: positive hydroxypyruvate reductase activity :
Positive AJ11262 is a Gram-negative bacillus, has a polar monoflagellate, is motile, and is aerobic as described above. This property belongs to the genus Pseudomonas according to the 8th edition of Purging Aids Manual. None of the bacterial species described as belonging to the genus Pseudomonas has properties similar to this species, and it has properties that differ in many respects from the new fungus Pseudomonas rhodomethylofaciens. ing. That is, acid production from sugars is weak, β-alanine, D-xylose, D-
It has the ability to assimilate ribose, levulinic acid, and citraconic acid, and does not have the ability to assimilate mesotartaric acid, acetic acid, and formic acid. Therefore, this bacterial species is a new bacterial species belonging to the genus Pseudomonas, and was named Pseudomonas megalbetscens. Microorganisms that utilize carbon-containing compounds such as methanol are described in Bergey's Manual of Determinative, 7th edition.
Bacterioolgy 7th ed.), it is classified into the genus Protaminobacter or Methanomonas, and AJ 11261 and AJ 11262 of the present invention should be classified into the genus Protaminobacter according to the Burgess Manual, 7th edition. be. However, in the 8th edition of the Virgies Manual, the genus Protaminobacter is not recognized as a genus, and its representative species, Protaminobacter spp.
Louver is considered to be a microorganism related to methanol-assimilating microorganisms belonging to the genus Bacillus, Vibrio, or Pseudomonas. In addition, recent research by Urakami and Komagata et al. revealed that Protaminobacter ruber has a similar cell morphology, coenzyme Q type, and bacterial cell fatty acid composition to methanol-assimilating bacteria that form red colonies of the genus Pseudomonas. According to the recent classification system, it is appropriate that Protaminobacter ruber and its related microorganisms belong to the genus Pseudomonas. It is thought that there is. Accordingly, the microorganisms belonging to the genus Pseudomonas used in the present invention include Protaminobacter ruba and its closely related microorganisms as described in the Burgess Manual, 7th edition. The medium for culturing these mutant strains contains, in addition to glycine, a carbon source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients such as vitamins and amino acids. Glycine may be added to the medium from the beginning of culture, but it may also be added after the mutant has grown to a certain extent to reduce the growth inhibition of the mutant by glycine. Alternatively, small amounts of glycine may be added in portions so that the concentration of glycine in the medium is low enough not to inhibit the growth of the mutant strain. Methanol is most suitable as a carbon source, but alcohols such as ethanol and propanol, organic acids such as formic acid and acetic acid, carbohydrates such as glucose, etc. can also be used if an appropriate strain is selected. As the nitrogen source, ammonia water, ammonia gas, ammonium salts, nitrates, amino acids, and other common nitrogen sources can be used. As an inorganic ion,
Potassium ions, magnesium ions, iron ions, manganese ions, calcium ions, chloride ions, sulfate ions, phosphate ions, and others are appropriately added to the medium as necessary. As organic micronutrients such as vitamins and amino acids, corn staple liquor, yeast extract, meat extract, etc. containing these may be added. Cultivation is preferably carried out under aerobic conditions, and the most favorable results can be obtained by adjusting the pH to an appropriate range of 5 to 9. In addition, the culture temperature is adjusted to an appropriate temperature in the range of 25 to 34°C in the early stage of cultivation so that the mutant strain can proliferate, and in the latter stage of cultivation it is adjusted to an appropriate temperature in the range of 33 to 40°C.
It is desirable to carry out the test while controlling the temperature to a level that suppresses the growth of mutant strains within the range of . Thus, if the culture is continued for 2 to 8 days, a significant amount of L-serine will be produced and accumulated in the culture solution.L-serine can be collected from the culture solution using normal methods such as using ion exchange resin. It can be carried out. Example 1 20 ml of a liquid medium having the following composition was placed in a 500 ml shaking flask and sterilized. Add 20ml of methanol/
One platinum loop of Pseudomonas megabetscens AJ11268, which had been cultured at 30°C for 48 hours in a broth slant containing P.
After 24 hours, 10 ml of methanol was added to the medium and cultured for a further 24 hours, and then 8 g of glycine and 20 ml of methanol were added. Then change the culture temperature
As a result of further culturing at 42° C. for 48 hours, 4.1 g/L-serine was produced. When the parent strain Pseudomonas megabetscens AJ11262 was cultured in the same manner, the amount of L-serine produced was 0.8 g/
It was nothing more than a simple thing. Liquid medium composition KH ₂ PO ₄ 0.5g/ (NH ₄ ) ₂ HPO ₄ 5.0g/ MgSO ₄・_{4H 2} O 0.4g/ NaCl 0.5g/ FeSO ₄・7H ₂ O 10mg/ MnSO ₄・4H ₂ O 5.0mg/ Yeast extract 2.0g / Methanol (separately sterilized) 20ml / CaCO ₃ (separately sterilized) 50g / PH 6.8 Example 2 Pseudomonas rhodomethylofaciens AJ 11263 was cultured for 48 hours in the same manner as in Example 1.
Similarly, glycine 8g/methanol 2.0%
After adding (v/v), 3.8 g/L-serine was produced as a result of culturing at a culture temperature of 42° C. for 48 hours. In the parent strain Pseudomonas rhodomethylofaciens AJ 11261, only 0.9 g/L-serine was produced in the same manner. Example 3 Pseudomonas megabetscens AJ11268 and Pseudomonas rhodomethylofaciens AJ11263 were cultured at 30°C for 48 hours in the same manner as in Example 1, and 8 g/g of glycine and 2.0% methanol (v/
After adding v), the culture temperature was changed to 30℃, 34℃, 38℃, 42℃.
The cells were cultured for an additional 48 hours at different temperatures. As a result, L-
The amount of serine produced and the yield of L-serine to consumed glycine (yield to consumed glycine) were as shown in Table 2.

【table】

Claims (1)

[Claims] 1 A mutant strain belonging to the genus Pseudomonas that is temperature sensitive and has the ability to produce L-serine from glycine is cultured in a medium containing glycine, and L-serine produced and accumulated in the medium is collected. A method for producing L-serine.