Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2001-06-22
2004-09-14
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C536S023200
Reexamination Certificate
active
06790647
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to microbiological industry, to the method of L-arginine production and concerns the using of new feedback-resistant mutant enzymes in arginine biosynthesis pathway of
E. coli
arginine-producer strains.
2. Description of the Related Art
The biosynthesis of arginine from glutamate in
E. coli
cells is carried out by a series of reactions initiated by the acetylation of glutamate by N-acetylglutamate synthase (NAGS) encoded by argA. This process is regulated via transcription repression of the arg regulon and by feedback inhibition of NAGS by arginine [Cunin R., et al.,
Microbiol. Rev., vol.
50, p.314-352, 1986]. L-Arginine represses argA expression with a ratio greater than 250 and inhibits NAGS activity (Ki=0.02 mM) [Leisinger T., Haas D.,
J. Biol. Chem., vol.
250, p.1690-1693, 1975]. For enhanced biosynthesis of arginine in
E. coli
, the feedback-resistant (may be referred to as “fbr”) NAGS enzymes are required.
The feedback-resistant mutants of enzymes can be obtained by spontaneous, chemical or site-directed mutagenesis.
Some argA fbr mutants were isolated and studied. The
Serratia marcescens
cells carrying the chromosomal fbr argA mutations were unstable and gave rise to argA mutants with reduced activity or with altered affinity for glutamate [Takagi T., et al.,
J. Biochem. vol.
99, p.357-364 1986].
The fbr argA genes from the five
E. coli
strains with fbr NAGS were cloned and different single-base substitutions in argA genes were found in each of the fbr NAGS strains and it was revealed that the substitutions cause replacing His-15 with Tyr, Tyr-19 with Cys, Ser-54 with Asn, Arg-58 with His, Gly-287 with Ser and Gln-432 with Arg (Rajagopal B. S. et al.,
Appl. Environ. Microbiol.,
1998, vol.64, No.5, p. 1805-1811).
As a rule, the fbr phenotype of enzyme arises as a result of the replacing the amino acid residue with another in a single or in a few sites of protein sequence and these replacements lead to reducing the activity of enzyme. For example, the replacing of natural Met-256 with 19 other amino acid residues in
E. coli
serine acetyltransferase (SAT) (cysE gene) leads in most cases to fbr phenotype but the mutant SAT proteins do not restore the level of activity of natural SAT (Nakamori S. et al.,
AEM,
64(5):1607-11, 1998).
So, the disadvantage of the mutant enzymes, obtained by these methods, is a reduce in the activity of mutant enzymes as compared to wild type enzymes.
SUMMARY OF THE INVENTION
An object of the present invention is to provide mutant feedback resistant and high active enzymes which play a key role in biosynthesis of arginine by
E. coli.
In present invention the novel procedure for synthesis a large set of mutant argA genes is proposed by using the full randomization of fragment of argA gene. The simultaneous substitutions of some amino acid residues in fragment of protein sequence, in which the fbr mutation can be localized, can be able to give a mutant proteins with the level of its activity restored near to natural due to more correct restored three dimensional structure of enzyme. Thus the present invention described below has been accomplished.
That is the present invention provides:
(1) A mutant N-acetylglutamate synthase wherein the amino acid sequence corresponding to positions from 15 to 19 in a wild type N-acetylglutamate synthase is replaced with any one of amino acid sequences of SEQ ID NOS: 1 to 4, and feedback inhibition by L-arginine is desensitized;
(2) The mutant N-acetylglutamate synthase according to (1), wherein a wild type N-acetylglutamate synthase is that of
Escherichia coli.
(3) The mutant N-acetylglutamate synthase according to (1), which includes deletion, substitution, insertion, or addition of one or several amino acids at one or a plurality of positions other than positions from 15 to 19, wherein feedback inhibition by L-arginine is desensitized;
(4) A DNA coding for the mutant N-acetylglutamate synthase as defined in any one of (1) to (3);
(5) A bacterium belonging to the genus Escherichia which is transformed with the DNA as defined in (4) and has an activity to produce L-arginine; and
(6) A method for producing L-arginine comprising the steps of cultivating the bacterium as defined in (5) in a medium to produce and accumulate L-arginine in the medium and collecting L-arginine from the medium.
The NAGS having any of fbr mutation as described above may be referred to as “the mutant NAGS”, a DNA coding for the mutant NAGS may be referred to as “the mutant argA gene”, and a NAGS without mutation may be referred to as “a wild type NAGS”.
Hereafter, the present invention will be explained in detail.
<1> Mutant NAGSs and Mutant argA Genes
The mutant NAGSs and the mutant argA genes coding the same were obtained by randomized fragment-directed mutagenesis. To obtain the numerous mutations in argA gene the full randomization of 15-nucleotide fragment of argA gene which codes the region from 15-th to 19-th amino acid residues in protein sequence was carried out. The full randomized 15-nucleotide fragment gives 4
15
or near 10
9
different DNA sequences which can code 20
5
different amino acid residues in 5-mer peptide. The likelihood of in frame non-introducing the stop codons in this sequences is equal of about 0.95
5
or 78%. So, the full randomization of the argA gene fragment coded the peptide from 15-th to 19-th amino acid residues must give approximately 2.5 million different protein sequences with diversity in this peptide fragment of NAGS structure. Subsequent selection and screening of recombinant clones carrying mutant argA genes cloned into expression vector allows to choose the fbr variants of mutant NAGS with different level of its biological activity up to level of activity of derepressed wild-type (wt) NAGS. In the selection, the inventors considered that the strain harboring the mutant argA gene would be obtained by using argD
−
, and prob
−
or proA
−
strain, because such a strain cannot produce L-proline due to inhibition of NAGS thereby cannot grow if excess amount of L-arginine exists in a culture medium, but the strain harboring fbr NAGS can grow in a minimal medium because glutamate-semialdehyde, a precursor of L-proline, can be supplied by acetylornithine deacetylase (the argE product) from N-acetylglutamate-semialdehyde, a precursor of L-arginine (Eckhardt T., Leisinger T.,
Mol. Gen. Genet
., vol. 138, p.225-232, 1975). However, the inventors found that it is difficult to obtain fbr NAGS having high activity by the above method as described in the abter-mentioned following Example, and that fbr NAGS having high activity can be obtained by introducing the mutant argA into a wild type strain and selection of a strain which shows delay of cell growth.
The amino acid sequences of the mutant NAGS suitable for fbr phenotype of NAGS were defined by the present invention. Therefore, the mutant NAGS can be obtained based on the sequences by introducing mutations into a wild type argA using ordinary methods. As a wild type argA gene, the argA gene of
E. coli
can be mentioned (GenBank Accession Y00492; the DNA sequence appears as SEQ ID NO: 15 and the corresponding protein sequence appears as SEQ ID NO:16).
The amino acid sequence of positions from 15 to 19 in the mutant NAGS of the present invention is any one of the sequnece of SEQ ID NOS: 1 to 4. The corresponding amino acid sequence of known mutant NAGS, in which tyr at a position 19 is replaced with Cys, and the wild type NAGS of
E. coli
are illustrated in SEQ ID NOS: 5 and 6, respectively. Examples of nucleotide sequence encoding these amino acid sequences are shown in SEQ ID NOS: 7 to 12. Table 1 shows these sequence.
TABLE 1
SEQ
SEQ
Amino acid sequence
ID NO:
Nucleotide sequence
ID NO:
Val Val Trp Arg Ala
1
GTAGTATGGCGGGCA
7
Leu Phe Gly Leu His
2
TTGTTCGGATTGCAC
8
Ser Arg Arg Ser Arg
3
TCGCGGCGGTCCAGA
9
Gly Trp Pro Cys Val
4
GGGTGGCCATGCGTG
10
His Ser Val Pro Cys
5
CATTCGGTTCCCTGT
11
His
Altman Irina Borisovna
Gusyatiner Mikhail Markovich
Leonova Tatyana Viktorovna
Ptitsyn Leonid Romanovich
Rostova Yulia Georgievna
LandOfFree
Mutant N-acetylglutamate synthase and method for L-arginine... 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 N-acetylglutamate synthase and method for L-arginine..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mutant N-acetylglutamate synthase and method for L-arginine... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3256539