Methods for producing L-valine and L-leucine

Details Methods for producing L-valine and L-leucine Methods for producing L-valine and L-leucine

1999-01-14

2001-04-10

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing alpha or beta amino acid or substituted amino acid...

C435S116000, C435S252330, C435S252800, C435S320100

Reexamination Certificate

active

06214591

ABSTRACT:

TECHNICAL FIELD
This invention relates to a microorganism belonging to the genus Escherichia having the capability of producing L-valine or L-leucine and, more particularly, a microorganism whose capability of producing L-valine or L-leucine is enhanced.
BACKGROUND ART
In the past, L-valine and L-leucine have been produced by a method of fermentation primarily using a microorganism belonging to the genus Brevibacterium, Corynebacterium or Serratia or a mutant thereof which produces L-valine or L-leucine (Amino acid fermentation, JAPAN SCIENTIFIC SOCIETY'S PRESS, pp.397-422, 1986). Although the conventional methods have considerably enhanced the productivity of these amino acids, the development of a more efficient, cost-effective technique is required in order to meet increasing demand for L-valine and L-leucine in the future.
On the other hand, a microorganism belonging to the genus Escherichia is potentially utilized as a potent L-valine or L-leucine-producing microorganism due to its rapid growth rate, progress in genetic analysis and plentiful genetic materials. However, there are few reports documenting the production of these amino acids with from Escherichia microorganisms, and as for L-branched chain amino acids, only a few reports deal with the production of L-isoleucine (Japanese Patent Application Laid-Open No. 5-304969(1993) and Japanese Patent Application Laid-Open No. 5-130882(1993).
DISCLOSURE OF THE INVENTION
The object of the present invention, in view of the aforementioned points, is to provide an efficient and cost-effective method for producing L-valine and L-leucine by enhancing the capability of producing L-valine or L-leucine of a microorganism belonging to the genus Escherichia.
As a result of a wholeheartedly conducted study of the production of L-valine and L-leucine by mutants of microorganisms belonging to the genus Escherichia, the present inventors have found that a mutation, whereby lipoic acid is required for growth and/or H
+
-ATPase is deficient, enhances the capability of producing L-valine or L-leucine of a L-valine or L-leucine-producing microorganism.
Thus, a first microorganism of the present invention is a microorganism belonging to the genus Escherichia and having the capability of producing L-valine or L-leucine, which requires lipoic acid for growth. A second microorganism of the present invention is a microorganism belonging to the genus Escherichia and having the capability of producing L-valine or L-leucine, which is deficient in H
+
-ATPase. Furthermore, a third microorganism of the present invention is a microorganism belonging to the genus Escherichia and having the capability of producing L-valine or L-leucine, which requires lipoic acid for growth and is deficient in H
+
-ATPase.
The present invention also provides a method for producing L-valine or L-leucine comprising culturing the aforementioned microorganism in a liquid medium to allow the L-valine or L-leucine to be produced and accumulated in the medium and collecting it.
In the specification, the phrase “H
+
-ATPase deficient” means that cells do not substantially express H
+
-ATPase activity, and includes both of that an H
+
-ATPase gene does not express due to entire or partial deletion of an atp operon coding for eight subunits of H
+
-ATPase or split of the atp operon and that the H
+
-ATPase gene has substitution, insertion or deletion of one or more nucleotides therein so that the H
+
-ATPase protein which is produced by expression of the gene does not have H
+
-ATPase activity. The ilvGMEDA operon means a operon including each of ilvG, ilvM, ilvE and ilvD genes, and the operon may additionally include ilvA gene, which expresses inactivated threonine deaminase, or may not include ilvA gene substantially.
The invention will be described in detail as follows:
<1> Microorganism of the Present Invention
A microorganism of the invention is one which belongs to the genus Escherichia and has the capability of producing L-valine or L-leucine and has any one of the following properties:
1. Lipoic acid is required for growth.
2. H
+
-ATPase is deficient.
3. Lipoic acid is required for growth and H
+
-ATPase activity is deficient.
In the present invention, the microorganism may possess any one of the aforementioned properties 1 to 3, and preferably possess property 3.
A microorganism having such properties can be obtained by giving the capability of producing L-valine or L-leucine to a microorganism belonging to the genus Escherichia, which is mutated so that it requires lipoic acid for growth and/or is deficient in H
+
-ATPase, or by enhancing the capability of producing L-valine or L-leucine in the aforementioned mutant. The microorganism of the present invention can be also obtained by inducing a mutation whereby lipoic acid is required for growth and/or a mutation whereby H
+
-ATPase is deficient in a microorganism belonging to the genus Escherichia.
The microorganism to be used in obtaining the aforementioned microorganisms can include a strain, which belongs to the genus Escherichia such as
Escherichia coli
(hereinafter, also referred to as
E. coli
) and exhibits no pathogenicity. For example, the following strains can be used.
Escherichia coli
K-12 (ATCC10798)
Escherichia coli
W3110 (ATCC27325)
Escherichia coli
W1485 (ATCC12435)
In order to introduce a mutation whereby lipoic acid is required for growth and/or a mutation whereby H
+
-ATPase is deficient into these microorganisms belonging to the genus Escherichia, the usual methods for introducing mutation, such as irradiation with X-ray or ultraviolet rays, or contact with mutagens including N-methyl-N′-nitro-N-nitrosoguanidine (hereinafter abbreviated as NG) and nitrous acid, can be applied. Additionally, the introduction of a mutation into a microorganism belonging to the genus Escherichia can be carried out by other genetic technique such as gene recombination, transduction, cell fusion and the like.
An example of the means for obtaining a mutant is as follows:
A mutant which requires lipoic acid for growth (hereinafter, referred to as a lipoic-acid-requiring strain) is obtained by culturing mutagenized bacterial cells on an agar plate, and by isolating colonies which exhibit lipoic acid requirement (A. A. Herbert and J. R. Guest: J. Gen. Microbiol., 53, 363-381 (1968)). As a lipoic acid requiring strain, specifically,
E. coli
W1485lip2 (ATCC25645) can be used.
A mutant which is deficient in H
+
-ATPase (hereinafter, referred to as a H
+
-ATPase-deficient strain) is obtained by selecting mutants which cannot grow on an agar plate containing citric acid as the sole carbon source and can grow on an agar plate containing glucose as the sole carbon source from mutagenized bacterial cells, and by further selecting, from these mutants, strains which do not exhibit H
+
-ATPase activity. As a H
+
-ATPase-deficient strain, specifically,
E coli
AN718 (
E. coli
Genetic Stock Center, Yale University, Department of Biology) can be used.
H
+
-ATPase is a membrane-binding enzyme with approximately 500,000 KD in molecular weight, in which 8 types of subunits complicatedly associate, and functions to pump H
+
outside of cytoplasm through changes in the free energy caused by ATP hydrolyzation and to synthesize ATP utilizing a H
+
-concentration gradient between the inside and outside of cytoplasmic membrane caused by intracellular respiration. Additionally, this enzyme is divided into an f0 fraction, which is localized on the inner membrane and exhibits H
+
-transport activity, and an F1 fraction, which is localized in the membrane surface and catalyzes the decomposition and synthesis of ATP, and the F0 is composed of 3 types of subunits a, b and c, while the F1 is composed of 5 types of subunits &agr;, &bgr;, &ggr;, &dgr;, &egr;. A strain which has a mutation in any of these subunits can be used as a H
+
-ATPase-deficient strain. The mutation of the H
+
-ATPase deficiency may in

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