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
2001-01-18
2004-05-18
Achutamurthy, Ponnathapu (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...
C536S023200, C536S023700, C435S252100, C435S252300
Reexamination Certificate
active
06737255
ABSTRACT:
TECHNICAL FIELD
This invention relates to a method for producing L-valine by fermentation, particularly, a DNA coding for acetohydroxy acid synthase isozyme III which is free from feedback inhibition by L-valine, a microorganism which harbors the DNA, and a method for producing L-valine using the bacterium.
BACKGROUND ART
In the past, L-valine has 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.
As bacteria other than above-mentioned bacteria used for producion of L-valine, it is exemplified by L-valine producer belonging to the genus Escherichia which requires lipoic acid for growth and/or which is deficient in H
+
-ATPase activity, and a bacterium belonging to the genus Escherichia which has preceding charasteristics and which is introduced an ilvGMEDA operon expressing ilvG, ilvM, ilvE and ilvD genes and not expressing threonine deaminase (WO96/06926).
The final step of L-valine biosynthesis is carried out by a group of ilvGMEDA operon-encoded enzymes. The ilvGMEDA operon includes each of ilvG, ilvM, ilvE, ilvD and ilvA genes, which encodes a large subunit and a small subunit of isozyme II of acetohydroxy-acid synthase, transaminase, dihydroxy-acid dehydratase and threonine deaminase, respectively. Of these enzymes, acetohydroxy-acid synthase, transaminase and dihydroxy-acid dehydratase catalyze the synthetic pathways from pyruvic acid to L-valine and from 2-ketobutyric acid to L-isoleucine, and threonine deaminase catalyzes the deamination from L-threonine to 2-ketobutyric acid, which is a rate-limiting step of L-isoleucine biosynthesis. Incidentally, the expression of ilvGMEDA operon suffers control (attenuation) by L-valine and/or L-isoleucine and/or L-leucine.
As acetohydroxy acid synthase concerning L-valine biosynthesis, isozyme III (hereinafter, also referred to as AHAS III) is known, apart from isozyme II (hereinafter, also referred to as AHAS II). AHAS III is coded by ilvIH operon which consists of ilvI coding for a large subunit (catalytic subunit) and ilvH coding for a small subunit (control subunit). AHAS III suffers feedback inhibition by L-valine.
Incidentally, it has been reported that the mutant ilvH gene cloned from the mutant
Escherichia coli
resistant to L-valine had an amino acid substitution of
14
gly with asp (Vyazmensky, M. et al., Biochemistry, 35, 10339-10346 (1996)). Further, ilvH612 has been known as the AHAS III mutation (De Felice et al.,
J. Bacteriol.,
120, 1058-1067(1974)). The ilvH gene in the ilvIH operon of
Esherichia coli
MI262 (Guardiola et al.,
J. Bacteriol.,
120, 536-538 (1974); De Felice et al.,
J. Bacteriol.,
120, 1068-1077(1974)) contains the ilvH612 double mutation by which
29
Asn is substituted with Lys and
92
Gln is substituted with a termination codon(TAG), respectively.
As described above, a DNA coding for AHAS II has been utilized for breeding of L-valine producer, however, for AHAS III no case has been reported.
DISCLOSURE OF THE INVENTION
The object of the present invention, in view of the aforementioned points, is to provide a DNA coding for AHAS III which is free from a feedback inhibition by L-valine, a microorganism which harbors the DNA, and a method for producing L-valine using the bacterium.
As a result of diligent investigation in order to achieve the object described above, the present inventors found that L-valine productivity is increased when a DNA coding for valine resistant AHAS III isolated from an L-valine resistant mutant is introduced into
Escherichia coli
. Thus the present invention has been completed.
That is, aspects of the present invention are as follows:
(1) A DNA coding for a small subunit of acetohydroxy acid synthase isozyme III originating from
Escherichia coli
which has a mutation to replace an amino acid residue corresponding to serine residue at the amino acid number 17 with another amino acid residue in SEQ ID NO: 2, or both of a mutation to replace an amino acid residue corresponding to serine residue at the amino acid number 17 and a mutation to replace an amino acid residue corresponding to glycine residue at the amino acid number 14 with another amino acid residue in SEQ ID NO: 2;
(2) The DNA of (1), wherein the mutation of the amino acid residue corresponding to serine residue at the amino acid number 17 is replacement of the serine residue with phenylalanie residue and the mutation of the amino acid residue corresponding to glycine residue at the amino acid number 14 is replacement of the glycine residue with aspartic acid residue;
(3) A DNA coding for acetohydroxy acid synthase isozyme III originating from
Escherichia coli
which is free from an inhibition by L-valine and has an activity to catalyze two reactions to generate &agr;-acetolactate from pyruvate and &agr;-aceto-&agr;-hydroxybutyrate from &agr;-ketobutyrate and pyruvate;
(4) The DNA of (3), wherein the DNA codes for a large subunit and a small subunit of acetohydroxy acid synthase isozyme III, the small subunit having a mutation to replace an amino acid residue corresponding to serine residue at the amino acid number 17 with another amino acid residue, or a mutation to replace an amino acid residue corresponding to asparagine residue at the amino acid number 29 with another amino acid residue, or a mutation to delete a C-terminal region from the amino acid number 91 downwards, in SEQ ID NO: 2, or a combination of two or more mutations selected from the group consisting of aforementioned mutations and a mutation to replace an amino acid residue corresponding to glycine residue at the amino acid number 14 with another amino acid residue in SEQ ID NO: 2.
(5) The DNA of (4), wherein the mutation of the amino acid residue corresponding to serine residue at the amino acid number 17 is replacement of the serine residue with phenylalanine residue, the mutation of the amino acid residue corresponding to asparagine residue at the amino acid number 29 is replacement of the asparagine residue with lysine residue or tyrosine residue, and the mutation of the amino acid residue corresponding to glycine residue at the amino acid number 14 is replacement of the glycine residue with aspartic acid residue.
(6) A bacterium which harbors the DNA according to claims
1
or
3
on chromosomal DNA or plasmid in the bacterium and has an ability to produce L-valine;
(7) The bacterium of (6), wherein expression of the DNA is enhanced;
(8) The bacterium of (7), wherein the expression is enhanced by locating the DNA under the control of a potent promoter or amplifying a copy number of the DNA;
(9) A method for producing L-valine comprising the steps of cultivating the bacterium according to claim
6
in a culture medium, producing and accumulating L-valine in the culture medium, and collecting L-valine from the culture medium.
The present invention will be explained in detail below.
The first DNA of the present invention is a DNA encoding a small subunit of AHAS III which exhibits acetohydroxy synthase activity without suffering a feedback inhibition by L-valine along with a large subunit. Acetohydroxy synthase activity herein refers to an activity to catalyze two reactions to generate &agr;-acetolactate from pyruvate, and &agr;-aceto-&agr;-hydroxybutyrate from &agr;-ketobutyrate and pyruvate. AHAS III small subunit of
Escherichia coli
has an amino acid sequence depicted in SEQ ID NO: 2 in Sequence Listing.
Aforementioned mutation is selected from a mutation to replace an amino acid residue corresponding to serine residue at the amino acid number 17 with another amino acid residue in SEQ ID NO: 2, or both of a mutation to replace an amino acid residue cor
Akhverdian Valery Zavenovich
Belaryeva Alla Valentinovna
Doroshenko Vera Georgievna
Gorshkova Nataliya Vasilyevna
Gusyatiner Mikhail Markovich
Achutamurthy Ponnathapu
Ajinomoto Co. Inc.
Kerr Kathleen
Oblon, Spivak, McClelland, Maier & Neustadt PC
LandOfFree
Mutant ilvH gene and method for producing L-valine does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Mutant ilvH gene and method for producing L-valine, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mutant ilvH gene and method for producing L-valine will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3264658