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
1999-09-15
2003-07-01
Prouty, Rebecca E. (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...
C435S252320, C435S106000, C435S089000, C435S041000, C435S233000, C435S477000, C435S487000, C536S023200
Reexamination Certificate
active
06586214
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides nucleotide sequences from coryneform bacteria coding for the pgi gene and a process for increasing metabolic flux through the pentose phosphate cycle by attenuating the pgi gene.
2. Background Information
Nucleotides, vitamins and in particular L-amino acids, very particularly lysine and tryptophan, are used in the foodstuffs industry, in animal nutrition, human medicine and the pharmaceuticals industry.
It is known that these substances are produced by fermentation using strains of coryneform bacteria, in particular
Corynebacterium glutamicum
. Due to its great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up of the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.
The performance characteristics of these microorganisms are improved using methods of mutagenesis, selection and mutant selection. In this manner, strains are obtained which are resistant to antimetabolites or are auxotrophic for regulatorily significant intermediates and produce nucleotides, vitamins or amino acids.
For some years, methods of recombinant DNA technology have also been used to improve strains of Corynebacterium which produce nucleotides, vitamins and L-amino acids.
A typical raw material for the production of these compounds is glucose, which is usually used in the form of starch hydrolysate. Sucrose is also used as a raw material.
On cellular absorption, glucose is phosphorylated with consumption of phosphoenolpyruvate (phosphotransferase system) (Malin & Bourd, Journal of Applied Bacteriology 71, 517-523 (1991)) and is then available to the cell as glucose-6-phosphate. Sucrose is converted into fructose and glucose-6-phosphate by a phosphotransferase system (Shio et al., Agricultural and Biological Chemistry 54, 1513-1519 (1990)) and invertase reaction (Yamamoto et al., Journal of Fermentation Technology 64, 285-291 (1986)).
During glucose catabolism, the enzymes glucose-6-phosphate dehydrogenase (EC 1.1.14.9) and glucose-6-phosphate isomerase (EC 5.3.1.9) compete for the substrate glucose-6-phosphate. The enzyme glucose-6-phosphate isomerase catalyses the first reaction step of the Embden-Meyerhof-Parnas pathway or glycolysis, namely conversion into fructose-6-phosphate. The enzyme glucose-6-phosphate dehydrogenase catalyses the first reaction step of the oxidative portion of the pentose phosphate cycle, namely conversion into 6-phosphogluconolactone.
In the oxidative portion of the pentose phosphate cycle, glucose-6-phosphate is converted into ribulose-5-phosphate, so producing reduction equivalents in the form of NADPH. As the pentose phosphate cycle proceeds further, pentose phosphates, hexose phosphates and triose phosphates are interconverted. Pentose phosphates, such as for example 5-phosphoribosyl-1-pyrophosphate are required, for example, in nucleotide biosynthesis. 5-Phosphoribosyl-1-pyrophosphate is moreover a precursor for aromatic amino acids and the amino acid L-histidine. NADPH acts as a reduction equivalent in numerous anabolic biosyntheses. Four molecules of NADPH are thus consumed for the biosynthesis of one molecule of L-lysine from oxalacetic acid.
The significance of the pentose phosphate cycle to biosynthesis and the production of amino acids, in particular L-lysine, by coryneform bacteria is known and has been the focus of much special interest.
Oishi & Aida (Agricultural and Biological Chemistry 29, 83-89 (1965)) have accordingly reported the “hexose monophosphate shunt” of
Brevibacterium ammoniagenes
. Investigations using
13
C isotope methods by Ishino et al. (Journal of General and Applied Microbiology 37, 157-165 (1991)) into glucose metabolism during glutamic acid and lysine fermentation indicate a correlation between lysine production and metabolic flux through the pentose phosphate pathway.
SUMMARY OF THE INVENTION
Object of the Invention
The inventors set themselves the object of providing a process for increasing metabolic flux through the pentose phosphate cycle.
Description of the Invention
Nucleotides, vitamins and in particular L-amino acids, very particularly L-lysine and L-tryptophan, are used in the foodstuffs industry, in animal nutrition, human medicine and the pharmaceuticals industry. There is accordingly general interest in providing improved processes for the production of these products.
The present invention provides an isolated polynucleotide containing a polynucleotide sequence selected from the group
a) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide containing the amino acid sequence of SEQ ID no. 2,
b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID no. 2,
c) polynucleotide which is complementary to the polynucleotides of a) or b), and
d) polynucleotide containing at least 100 successive bases of the polynucleotide sequence of a), b) or c).
The present invention also provides the polynucleotide as claimed in claim 1, wherein it preferably comprises replicable DNA containing:
(i) the nucleotide sequence shown in SEQ ID no. 1 or
(ii) at least one sequence which matches the sequence (i) within the degeneration range of the genetic code, or
(iii) at least one sequence which hybridises with the complementary sequence to sequence (i) or (ii) and optionally
(iv) functionally neutral sense mutations in (i).
The present invention also provides
a polynucleotide as claimed in claim 2, containing the nucleotide sequence as shown in SEQ ID no. 1,
a polynucleotide as claimed in claim 2 which codes for a polypeptide which contains the amino acid sequence as shown in SEQ ID no. 2,
a vector containing the polynucleotide as claimed in claim 1, indent d, in particular pMC1, deposited in
E. coli
DSM 12969.
and coryneform bacteria acting as host cells which contain the vector as claimed in claim 6.
“Isolated” means separated from its natural environment. “Polynucleotide” generally relates to polyribonucleotides and polydeoxyribonucleotides, wherein the RNA or DNA may be unmodified or modified.
“Polypeptides” are taken to mean peptides or proteins which contain two or more amino acids connected by peptide bonds.
The polypeptides according to the invention include the polypeptide according to SEQ ID no. 2, in particular those having the biological activity of glucose-6-phosphate isomerase and also those which are at least 70% identical to the polypeptide according to SEQ ID no. 2, preferably being at least 80% and particularly preferably at least 90% to 95% identical to the polypeptide according to SEQ ID no. 2 and having the stated activity.
This invention furthermore relates to a process for the fermentative production of nucleotides, vitamins and in particular L-amino acids, very particularly lysine and tryptophan, using coryneform bacteria which in particular already produce the stated substances and in which the nucleotide sequences coding for the pgi gene are attenuated, in particular expressed at a low level.
In this connection, the term “attenuation” describes the reduction in or switching off of the intracellular activity of one or more enzymes (proteins) in a microorganism, which enzymes are coded by the corresponding DNA, for example by using a weak promoter or a gene or allele which codes for a corresponding enzyme having low activity or inactivates the corresponding enzyme (protein) and optionally by combining these measures.
The microorganisms provided by the present invention are capable of producing nucleotides, vitamins and in particular L-amino acids, very particularly lysine and tryptophan, from glucose, sucrose, lactose, fructose, maltose, molasses, s
Burke Kevin
Dunican L. K.
Marx Achim
McCormack Ashling
Mockel Bettina
Degussa - AG
Pillsbury & Winthrop LLP
Prouty Rebecca E.
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