Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
2000-07-10
2002-06-11
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S252300, C435S320100, C435S252330, C435S232000, C536S023200
Reexamination Certificate
active
06403342
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to DNA coding for a mutant a-isopropylmalate synthase. Also, the present invention relates to an L-leucine-producing microorganism having the mutant a-isopropylmalate synthase, and a method for producing L-leucine by using the microorganism. L-Leucine is an essential amino acid which can be used as a nutritious additive for food or feed, reagents or materials for medical treatment, pharmaceutical or chemical industry, or a growth factor used for production of other amino acids, such as lysine.
Description of the Background
In the past, L-leucine has been produced by a method of fermentation primarily using microorganisms belonging to the genus Brevibacterium, Corynebacterium or Serratia or mutants thereof which produce L-leucine (Amino acid fermentation, JAPAN SCIENTIFIC SOCIETY'S PRESS, pp.397-422, 1986).
The highest level of L-leucine accumulation was obtained when using Brevibacterium flavum VKPM B-2736. This strain produces L-leucine at a concentration up to 26 g/L on sucrose-containing media for 72 h of fermentation in a laboratory fermenter (USSR Author Certificate 1394711). Moreover,
Brevibacterium lactofermentum
34 is known to produce L-leucine up to 34 g/L on a medium with glucose (Appl. Environ. Microbiol. 51, p.1024 (1986)).
Although the productivity of L-leucine has been improved to some extent, the development of a more efficient and cost-effective method for producing L-leucine is necessary in order to meet the increasing demand for L-leucine.
On the other hand, microorganisms belonging to the genus Escherichia might be potentially utilized as a potent L-leucine-producing bacteria due to their rapid growth rate, prominent data obtained from genetic analysis and plentiful genetic materials. However, there are few reports which disclose the production of L-leucine using bacteria belonging to the genus Escherichia.
As L-leucine-producing bacterial strains of the genus Escherichia, a strain which is resistant to &bgr;-thienylalanine, &bgr;-hydroxyleucine (Japanese Patent Publication No. 62-34397 (1987) and a strain which is resistant to 4-azaleucine or 5,5,5-trifluoroleucine (Japanese Patent Application Laid-Open No. 8-70879 (1996)) are known.
However, neither L-leucine-resistant bacteria belonging to the genus Escherichia nor any relation between L-leucine resistance and productivity of L-leucine is known.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to improve productivity of L-leucine of bacteria belonging to the genus Escherichia and to provide an efficient and cost-effective method for producing L-leucine.
It is, in particular, an object of the present invention to provide a DNA coding for a protein of the following (A) or (B):
(A) a protein having an amino acid sequence shown in SEQ ID NO:2 which has a substitution selected from the following (a) to (e):
(a) a substitution of another amino acid residue for a threonine residue at position 482,
(b) a substitution of another amino acid residue for a glutamic acid residue at position 386,
(c) a substitution of another amino acid residue for a proline residue at position 428,
(d) a substitution of another amino acid residue for a glycine residue at position 479, and
(e) a substitution of another amino acid residue for a glycine residue at position 462,
(B) a protein having the amino acid sequence of the protein of (A), which sequence has deletion, substitution, insertion or addition of one or a few amino acid residues, the protein of (B) having &agr;-isopropylmalate synthase activity and feedback inhibition of the activity by L-leucine being desensitized equivalently to that of the protein of (A).
It is, moreover, also an object of the present invention to provide a microorganism transformed with any of the the DNA defined above.
It is also an object of the present invention to provide a method of producing L- leucine.
The above objects and others are provided by a microorganism transformed with the DNA as defined above in (A)(a)-(e) and/or (B).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present inventors have surprisingly discovered that desensitization of feedback inhibition by L-leucine of &agr;-isopropylmalate synthase (hereinafter abbreviated as IPMS) contributes to the production of L-leucine. The present invention is predicated upon this discovery.
Thus, the present invention provides a DNA coding for a protein of the following (A) or (B) (hereinafter also referred to as DNA of the present invention):
(A) a protein having an amino acid sequence shown in SEQ ID NO: 2 which has a substitution selected from the following (a) to (e):
(a) a substitution of another amino acid residue for a threonine residue at position 482,
(b) a substitution of another amino acid residue for a glutamic acid residue at position 386,
(c) a substitution of another amino acid residue for a proline residue at position 428,
(d) a substitution of another amino acid residue for a glycine residue at position 479, and
(e) a substitution of another amino acid residue for a glycine residue at position 462,
(B) a protein having the amino acid sequence of the protein of (A), which sequence has deletion, substitution, insertion or addition of one or a few amino acid residues, said protein of (B) having &agr;-isopropylmalate synthase activity and feedback inhibition of the activity by L-leucine being desensitized equivalently to that of the protein of (A).
The DNA of the present invention is preferably one in which the substitution is a substitution selected from the following (a′) to (e′):
(a′) a substitution of an isoleucine residue for a threonine residue at position 482,
(b′) a substitution of a lysine residue for a glutamic acid residue at position 386,
(c′) a substitution of a leucine residue for a proline residue at position 428,
(d′) a substitution of a cysteine residue for a glycine residue at position 479, and
(e′) a substitution of an aspartic acid residue for a glycine residue at position 462.
Specific examples of the DNA of the present invention include ones which has a nucleotide sequence shown in SEQ ID NO: 1, which sequence has a mutation selected from the following (i) to (v):
(i) a mutation of cytosine at position 1445 to thymine,
(ii) a mutation of guanine at position 1156 to adenine,
(iii) a mutation of cytosine at position 1283 to thymine,
(iv) a mutation of guanine at position 1435 to thymine, and
(v) a mutation of guanine at position 1385 to adenine.
The present invention also provides a microorganism which is transformed with the DNA of the present invention, and has an ability to produce L-leucine (hereinafter, also referred to as “microorganism of the present invention”). The microorganism of the present invention preferably belongs to the genus Escherichia. The microorganism of the present invention is more preferably Escherichia coli.
The present invention further provides a method for producing L-leucine, which entails:
culturing any one or more bacteria of the present invention in a culture medium to produce and accumulate L-leucine in the medium, and
recovering L-leucine from the medium.
The present invention will be further explained below.
A. DNA of the present invention
The DNA of the present invention has a mutation to desensitize feedback inhibition by L-leucine of IPMS encoded by the DNA, in a DNA coding for a wild type IPMS.
The phrase “feedback inhibition by L-lysine is desensitized” means that the degree of the feedback inhibition is lowered. The lowering of the degree of feedback inhibition can be determined by measuring lowering of the IPMS activity by L-leucine and comparing it with that of wild strain or a parent strain.
IPMS is exemplified by those originating from bacteria belonging to the genus Escherichia, especially IPMS originating from
E. coli.
The mutation of IPS to desesitize feedback inhibition by L-leucine is exemplified by the following substitutions (a) to (e) in the amino acid sequence shown SEQ ID NO: 2:
(a) a sub
Gusyatiner Mikhail Markovich
Ivanovskaya Lirina Valerievna
Kozlov Yuly Ivanovich
Lunts Maria Grigorievna
Voroshilova Elvira Borisovna
Anjinomoto Co., Inc.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Saidha Tekchand
LandOfFree
DNA coding for mutant isopropylmalate synthase... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with DNA coding for mutant isopropylmalate synthase..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and DNA coding for mutant isopropylmalate synthase... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2929952