Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
2000-03-21
2002-08-20
Ketter, James (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S064000, C435S252500, C435S248000, C435S170000
Reexamination Certificate
active
06436664
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for producing shikimic acid. Shikimic acid is useful for intermediates of aromatic compounds synthesis such as phenylalanine, tyrosine, tryptophane, p-aminobenzoic acid or p-hydroxybenzoic acid. Shikimic acid is also used as component of waste disposal material and the like.
BACKGROUND ART
Shikimic acid that is an aromatic intermediate is synthesized in four enzymatic reactions from phosphoenol pyruvate and erythrose-4-phosphate. These four enzymes are encoded by aroA, aroB, aroC, and aroD genes in
Bacillus subtilis
. Shikimic is converted into chorismic acid by enzymatic reactions by aroI, aroE and aroF. The pathway from phosphoenol pyruvate and erythrose-4-phosphate to chorismic acid is called as shikimic acid pathway. Shikimic acid pathway is also known as a common pathway for biosynthesis of aromatic amino acids L-tryptophane, L-phenylalanine and L-tyrosine.
Shikimic acid is obtained from plants heretofore, and it has not been produced by direct fermentation using microorganism.
Bacillus subtilis
1-118 (aroI116, amy4), which has been known as amylase-deficient strain, is known to have a mutation in aroI gene (Yuki, S., Japan.
J. Genetics,
50(2), 155-157 (1975)). However, it is not known that the strain 1-118 produces shikimic acid.
Although, it also has been known that
Bacillus subtilis
SB130 (aroE130, hisH32) has a mutation in a gene coding for 5-enolpyruvylshikimate-3-phosphate synthase (EC:2.5.1.19), it is not known that the strain produces shikimic acid.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a method for producing shikimic acid by direct fermentation and a microorganism that is used in the method.
As a result of diligent and repeated investigation in order to achieve the object described above, the present inventors determined that strains of
B. subtilis
, carrying defective shikimate kinase enzyme accumulated shikimate. Further, the present inventor succeeded in improving shikimic acid productivity of the bacterium by enhancing an activity of shikimate dehydrogenase and an activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase (sometimes referred to as “DAHP synthase”).
Further, the present inventors found that the shikimic acid productivity of
B. subtilis
strain can be increased by depletion of aroE gene product, 5-enolpyruvylshikimate-3-phosphate, and enhancing the activity of shikimate dehydrogenase synthase, even if the strain possesses active shikimate kinase.
That is, aspects of the present invention are as follows:
(1) A method for producing shikimic acid, comprising the steps of cultivating a bacterium belonging to the genus Bacillus which is deficient in shikimate kinase activity and has shikimic acid productivity in a medium, producing and accumulating shikimic acid in the medium, and collecting shikimic acid from the medium;
(2) The method of (1), wherein the bacterium is
Bacillus subtilis;
(3) The method of (1), wherein shikimate dehydrogenase activity in the cell of the bacterium is enhanced;
(4) The method of (1), wherein shikimate dehydrogenase activity is enhanced by increasing copy number of a gene encoding shikimate dehydrogenase, enhancing expression regulation sequence of the gene or integrating the gene into chromosomal DNA of the bacterium;
(5) The method of (4), wherein the shikimate dehydrogenase gene, in which its inherent promoter is displaced by a promoter of other gene or to which a promoter of other gene is added, is integrated into the chromosomal DNA;
(6) The method of (5), wherein activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase in the cell of the bacterium is further enhanced;
(7) The method of (6), wherein activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase is enhanced by increasing copy number of a gene encoding 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase, enhancing expression regulation sequence of the gene or integrating the gene into chromosomal DNA of the bacterium;
(8) A bacterium belonging to the genus Bacillus having shikimic acid productivity, wherein the bacterium is deficient in shikimate kinase activity, and shikimate dehydrogenase activity in the cell of the bacterium is enhanced; and
(9) The bacterium of (8), wherein activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphatesynthase in its cell is enhanced.
(10) A method for producing shikimic acid, comprising the steps of cultivating a bacterium belonging to the genus Bacillus which is deficient in 5-enolpyruvylshikimate-3-phosphate synthase activity and has shikimic acid productivity in a medium, producing and accumulating shikimic acid in the medium, and collecting shikimic acid from the medium.
(11) The method of (10), wherein said bacterium is
Bacillus subtilis.
(12) The method of (10), wherein shikimate dehydrogenase activity in the cell of said bacterium is enhanced.
(13) The method of (12), wherein shikimate dehydrogenase activity is enhanced by increasing copy number of a gene encoding shikimate dehydrogenase, enhancing expression regulation sequence of said gene or integrating said gene into chromosomal DNA of said bacterium.
(14) A bacterium belonging to the genus Bacillus having shikimic acid productivity, wherein said bacterium is deficient in 5-enolpyruvylshikimate-3-phosphate synthase activity, and shikimate dehydrogenase activity in the cell of said bacterium is enhanced.
In the present invention, the term “having shikimic acid productivity” refers to an activity to accumulate shikimic acid in a medium, when the bacterium of the present invention is cultivated in the medium.
The present invention will be explained in detail below.
A bacterium belonging to the genus Bacillus of the first embodiment of the present invention is deficient in shikimate kinase activity. As the bacterium belonging to the genus Bacillus, there are exemplified
Bacillus amyloliquefaciens, Bacillus subtilis
and Bacillus.
A bacterium belonging to the genus Bacillus deficient in shikimate kinase activity is exemplified by a mutant which substantially completely loses shikimate kinase activity and a mutant of which shikimate kinase activity is significantly lower than that of wild type strain. Alternatively, a bacterium belonging to the genus Bacillus may be a strain in which a shikimate kinase gene (aroI) on its chromosome is disrupted by homologous recombination.
Depletion of shikimate kinase activity of the bacterium belonging to the genus Bacillus may be achieved by the conventionally known mutation treatment and selection of mutant strain which has a mutation in its gene coding for the enzyme (aroI). The mutation treatment includes a method for treating the bacterium belonging to the genus Bacillus with ultraviolet irradiation or a mutating agent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for the mutation treatment.
An aroI mutant of the bacterium belonging to the genus Bacillus may be also obtained by gene conversion method using aroI mutant of a bacterium belonging to the genus Bacillus. That is, a wild type aroI gene on a plasmid is converted to a mutant aroI gene by gene conversion method using the plasmid carrying a cloned aroI gene and a mutant strain having a mutation in its aroI gene. Then a wild type aroI gene in a bacterium belonging to the genus Bacillus is converted to the mutant aroI gene.
Concretely, a bacterium belonging to the genus Bacillus is exemplified by
Bacillus subtilis
1-118 (aroI116, amy4) and I-116 which was derived from the strain 1-118 (see after-mentioned Example 1) or the like. The strain 1-118 is described in Yuki, S., Japan
J. Genetics,
50 (2), 155-157 (1975).
A bacterium belonging to the genus Bacillus of the present invention may be preferably enhanced in shikimate dehydrogenase activity. Furthermore, it is preferable that the bacterium is further enhanced in DAHP synthase. Shikimate dehydrogenase activity or DAHP synthase activity in a bacterial cell can be enhanced by, for example, increasing the copy number of genes coding
Abalakina Elena Georgievna
Bachina Tatyana Aleksandrovna
Iomantas Yurgis Antanas Vladovich
Kozlov Yuri Ivanovich
Polanuer Boris Mironovich
Ajinomoto Co. Inc.
Ketter James
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Schnizer Richard
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