JPH0525475B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-threonine by fermentation. Conventional fermentation methods for producing L-threonine include methods using microorganisms belonging to the genus Brevibacterium (see Japanese Patent Publication No. 45-26708) and methods using recombinant microorganisms belonging to the genus Escherichia (Japanese Unexamined Patent Publication No. 55-1982). 131397) etc. are known. In these methods, breeding is carried out with the aim of obtaining strains with high L-threonine productivity, but on the other hand, efforts are being made to improve L-threonine productivity by examining methods of culturing L-threonine-producing strains. There are also attempts. For example, Agric.Biol.Chem.
Volume 43, pages 2087-2092 (1979) describes the influence of oxygen supply on L-threonine fermentation. According to this, in L-threonine production using microorganisms belonging to the genus Brevibacterium, when the dissolved oxygen partial pressure in the liquid medium is 0.01 atm or more, L-
It has been confirmed that while maximum accumulation of threonine is obtained, productivity decreases when the dissolved oxygen partial pressure becomes zero. The present inventors conducted various studies to improve the production method of L-threonine using conventional fermentation methods, and found that
Microorganisms belonging to the genus Escherichia and capable of producing L-threonine are
It has been found that L-threonine can be produced at an extremely high yield by culturing with oxygen supply conditions set in the range of 0.07 atm or more and 0.17 atm or less. That is, the present invention involves culturing a microorganism belonging to the genus Escherichia and having the ability to produce L-threonine while controlling the oxygen supply conditions so that the dissolved oxygen partial pressure in the liquid medium is in the range of 0.07 atm to 0.17 atm. This is a characteristic method for producing L-threonine. The microorganisms capable of producing L-threonine used in the present invention include α-
Amino-β-hydroxyvaleric acid resistant and isoleucine auxotrophic mutant strain (Japanese Patent Publication No. 45-26709), an L-threonine-producing bacterium containing plasmid pBR322 containing a gene involved in L-threonine biosynthesis of the genus Escherichia ( JP-A-55-131397, JP-A-55-156591), etc. When culturing these microorganisms, the culture is performed under oxygen supply conditions such that the partial pressure of dissolved oxygen in the liquid medium is 0.07 atm or more and 0.17 atm or less. A conventional method may be used to measure the dissolved oxygen partial pressure of the culture solution. For example, measurement may be carried out by attaching a commercially available oxygen membrane electrode for measuring dissolved oxygen partial pressure to the fermenter. The period during which the oxygen supply conditions are set so that the dissolved oxygen partial pressure is between 0.07 atm and 0.17 atm is the period after microbial growth has started and when the dissolved oxygen partial pressure to be controlled is reached, or when bacterial cell growth is at L. - From the time when the amount of bacterial cells necessary for threonine production is reached until the production of L-threonine is substantially completed. In addition,
Temporarily, deviation from the above oxygen supply conditions will not cause much harm. Further, the medium for culturing the L-threonine producing bacteria is not particularly different from the conventional method. That is, various sugars, organic acids, etc. can be used as a carbon source, and ammonia gas, aqueous ammonia, ammonium salts, etc. can be used as a nitrogen source. The carbon source and nitrogen source may be added at once or may be added over time over several times during culture. In addition, inorganic ions such as phosphate ions, iron ions, magnesium ions, potassium ions, etc. are added to the medium as appropriate. Furthermore, organic micronutrients such as vitamins and amino acids are used if necessary. Oxygen supply conditions for culture are dissolved oxygen partial pressure of 0.07 atm.
During cultivation, the culture medium pH is kept in the range of 5 to 8 and the temperature is kept in the range of 27 to 37℃.
The most desirable results can be obtained by adjusting the range. Thus, if the culture is continued for 1 to 5 days, L.
- Threonine is produced in high yield. The L-threonine produced and accumulated in the culture medium may be collected by any conventional method. Examples are shown below. Example 1 50 ml of the seed culture medium shown in Table 1 was placed in a 500 ml flask and sterilized under pressure at 115°C for 15 minutes. This is combined with Esierihiakori AJ11335 (FERM BP−
1484) (Japanese Patent Publication No. 55-131397, Esierhia coli
AJ11332 (FERM BP-1640) was inoculated with 1 to 3 platinum loops of L-threonine producing bacteria (containing plasmid pBR322 containing genes involved in L-threonine biosynthesis) and shaken at 31.5°C for 24 hours. A seed culture solution was obtained by culturing. Next, 240 ml of the culture medium prepared so that the final concentration after inoculating the seed culture solution has the medium composition shown in Table 1 was placed in a 1-volume jar fermenter, and the medium was sterilized at 115°C for 15 minutes. Obtained. Seed culture solution 60
ml was inoculated and culture was started at 31.5°C with aeration rate of 1/3 VVm, stirring number of 1000 rpm, and pH 6.0. The dissolved oxygen partial pressure in the culture solution was measured with an oxygen membrane electrode, and from the time when the dissolved oxygen concentration shown in Table 2 was reached, the aeration amount or stirring rate was changed based on this value until the end of the culture. This value was maintained for 72 hours. In addition, during the cultivation, 300g of glucose/ammonium sulfate was added.
A medium prepared by previously pressurizing 100 g/100 g of medium at 115° C. for 15 minutes was added three times in 30 ml portions. Table 2 shows the amount of L-threonine accumulated in the culture solution after the completion of the culture.

[Table] Table 2 Dissolved oxygen partial pressure L-threonine (atm) Accumulation amount (g/) 0.05 1.59 0.07 3.26 0.10 3.75 0.15 4.20 0.17 3.63 0.20 2.84 Example 2 Escherichia spp.・Koli AJ11332 (FERM P-4898) from 1 to
Three platinum loops were inoculated to obtain a seed culture solution. Next, in the same manner as in Example 1, the cells were cultured at 31.5° C. for 72 hours at the dissolved oxygen partial pressure shown in Table 3, and the amount of L-threonine accumulated in the culture solution is shown in Table 3.
However, a medium containing 300 g of glucose and 100 g of ammonium sulfate was added once during the culture. Table 3 Dissolved oxygen partial pressure L-Threonine (ATM) Accumulation amount (g/) 0.05 0.38 0.07 0.93 0.10 1.04 0.15 1.37 0.17 1.15 0.20 0.80

Claims (1)

[Claims] 1 A microorganism belonging to the genus Escherichia and capable of producing L-threonine was cultured in a liquid medium with aeration and stirring.
- When producing threonine, culture is carried out while controlling the oxygen supply conditions so that the dissolved oxygen partial pressure in the liquid medium is in the range of 0.07 to 0.17 atm by changing the aeration amount and/or the number of stirrings. A method for producing L-threonine using a characteristic fermentation method.