Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing nitrogen-containing organic compound
Reexamination Certificate
2000-02-28
2002-01-29
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing nitrogen-containing organic compound
C435S252100, C435S252300
Reexamination Certificate
active
06342377
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a microorganism which produces and accumulates 5-aminolevulinic acid at a high concentration, and to a process for producing 5-aminolevulinic acid using the same.
BACKGROUND ART
5-Aminolevulinic acid is a compound which is broadly present in the biosphere as a precursor of tetrapyrrole compounds and takes important roles in vivo. 5-Aminolevulinic acid is a natural compound which shows excellent functions as, for example, herbicides, insecticides, plant growth regulators, plant photosynthesis reinforcing agents, and the like, and also has advantageous characteristics such as no toxicity upon humans and animals and no residual property in the environment due to its high decomposability (e.g., see JP-A-61-502814, JP-A-2-138201 etc.).
However, 5-aminolevulinic acid has a problem in that it lacks in practicability for use in the above uses because of its high production cost (
CHEMICAL WEEK
, Oct., 29 (1984)).
Accordingly, many chemical synthesis processes have been examined (e.g., see JP-A-2-76841), but satisfactory processes have not been developed yet.
On the other hand, other processes for producing 5-aminolevulinic acid have also been examined using microorganisms belonging to the genus Rhodobacterium, the genus Propionibacterium, the genus Methanobacterium, the genus Methanosarcina, and the like. However, the processes using microorganisms belonging to the genus Propionibacterium, the genus Methanobacterium, the genus Methanosarcina and the like (e.g., see JP-A-5-184376) are not satisfactory because the production yield is very low.
It is known that purple non-sulfur bacteria, such as microorganisms belonging to the genus Rhodobacterium, and the genus Rhodopseudomonas, and the like, have high ability to synthesize tetrapyrrole compounds using 5-aminolevulinic acid as a metabolic intermediate. However, the use of these microorganisms has a problem in that the produced 5-aminolevulinic acid is metabolized into tetrapyrrole compounds so that a desired amount of 5-aminolevulinic acid is not accumulated.
Thus, since biosynthesis of 5-aminolevulinic acid is regulated in a complicated manner in vivo, it is not easy to obtain a strain capable of accumulating 5-aminolevulinic acid at a high concentration.
On the other hand, a method using a mutant strain of the genus Rhodobacterium capable of accumulating 5-aminolevulinic acid at a maximum amount of 14.3 mM in which glucose is used as a carbon source has been proposed (JP-A-8-168391). However, this method cannot be considered as an industrially advantageous method because it is necessary to keep a dissolved oxygen concentration at a low level during culturing so that aeration in the culture vessel must be carried out by feeding a mixture of air and a nitrogen gas.
Also, this method requires oxygen when 5-aminolevulinic acid is aerobically accumulated using glucose as the carbon source. As a consequence, there is a possibility that 5-aminolevulinic acid is produced efficiently even under conditions that oxygen is present sufficiently because the dissolved oxygen concentration is reduced due to consumption of oxygen by the microorganism. However, it is not possible to produce 5-aminolevulinic acid efficiently under relatively high dissolved oxygen concentration conditions.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a microorganism which can produce 5-aminolevulinic acid efficiently even under conditions that a dissolved oxygen concentration is relatively high without requiring feeding of a nitrogen gas or the like. Another object of the present invention is to provide a process for producing 5-aminolevulinic acid using the microorganism.
Under the above conditions, the inventors of the present invention have conducted intensive studies with the aim of obtaining a microorganism capable of producing 5-aminolevulinic acid efficiently and, as a result, found a process for selecting 5-aminolevulinic acid accumulating microorganisms having high productivity and then, using this method, found a microorganism capable of accumulating 5-aminolevulinic acid at a high concentration even with a relatively high dissolved oxygen concentration. Thus the present invention has been accomplished.
Also, the present inventors have developed a markedly efficient process for selecting of 5-aminolevulinic acid accumulating microorganisms which can evaluate a large number of mutant strains by appropriately changing saccharides, glycine, levulinic acid, and the like, and a repeated mutant treatment has become possible. Thus, the present inventors have succeeded in breeding a strain capable of accumulating 5-aminolevulinic acid at a high concentration. Thereafter, the present invention was completed by establishing a culturing process in which saccharides, glycine, levulinic acid, iron components, and the like are appropriately added, and dissolved oxygen and oxidation-reduction potential in the culture broth are controlled.
That is, the present invention provides a 5-aminolevulinic acid-producing microorganism having a 5-aminolevulinate synthase activity of from 2 to 7 (nmol/min/mg protein) under aerobic culturing conditions at a dissolved oxygen concentration of from 0.70 to 6.60 ppm.
Also, the present invention provides a process for producing 5-aminolevulinic acid, comprising culturing at least one of such 5-aminolevulinic acid producing microorganisms and recovering 5-aminolevulinic acid from the resulting culture.
Furthermore, the present invention provides a process for producing 5-aminolevulinic acid, comprising culturing a 5-aminolevulinic acid-producing microorganism in a medium containing an iron component at an amount of from 5 to 500 &mgr;M and recovering 5-aminolevulinic acid from the resulting culture mixture.
Moreover, the present invention provides a process for culturing a 5-aminolevulinic acid-producing microorganism, comprising culturing a 5-aminolevulinic acid producing microorganism in a medium containing an iron component at an amount of from 5 to 500 &mgr;M.
REFERENCES:
patent: 5733770 (1998-03-01), Watanabe et al.
patent: 5763235 (1998-06-01), Watanabe et al.
patent: 5-95782 (1993-04-01), None
patent: 6-169758 (1994-06-01), None
patent: 8-168391 (1996-07-01), None
Ken Sasaki et al, Influence of Iron on the Excretion of 5-Aminolevulinic Acid by a Photosynthetic BacteriumRhodobacter sphaeroides, 1989, vol. 68, No. 5, pp. 378-381.
Ellen L. Neidle et al, 5-Aminolevulinic Acid Availability and control of Spectral Complex Formation in HemA and HemT Mutants ofRhodobacter sphaeroides, Apr. 1993, vol. 175, pp. 2304-2313.
Hotta Yasushi
Kaminaga Tomomi
Miyachi Nobuya
Nishikawa Seiji
Tanaka Tohru
Cosmo Research Intitute
Lilling Herbert J.
Sughrue & Mion, PLLC
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