Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-12-16
2002-05-21
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
Reexamination Certificate
active
06391603
ABSTRACT:
The present invention relates to genes of purine biosynthesis from
Ashbya gossypii
and to the use thereof in riboflavin synthesis.
Vitamin B2, also called riboflavin, is essential for humans and animals. Vitamin B2 deficiency is associated with inflammations of the mucous membranes of the mouth and throat, itching and inflammations in the skin folds and similar cutaneous lesions, conjunctival inflammations, reduced visual accuracy and clouding of the cornea. Babies and children may experience cessation of growth and loss of weight. Vitamin B2 therefore has economic importance, especially as vitamin supplement in cases of vitamin deficiency and as supplement to animal feed. It is also employed for coloring foodstuffs, for example in mayonnaise, icecream, blancmange etc.
Vitamin B2 is prepared either chemically or microbially (see, for example, Kurth et al. (1996) riboflavin, in: Ullmann's Encyclopedia of industrial chemistry, VCH Weinheim). In the chemical preparation process, riboflavin is, as a rule, obtained as pure final product in multistage processes, it being necessary to employ relatively costly starting materials such as, for example, D-ribose. An alternative to the chemical synthesis of riboflavin is the preparation of this substance by microorganisms. The starting materials used in this case are renewable raw materials such as sugars or vegetable oils. The preparation of riboflavin by fermentation of fungi such as
Eremothecium ashbyii
or
Ashbya gossypii
is known (The Merck Index, Windholz et al., eds. Merck & Co., page 1183, 1983), but yeasts such as, for example, Candida, Pichia and Saccharomyces, or bacteria such as, for example, Bacillus, clostridia or corynebacteria, have also been described as riboflavin producers.
EP 405370 describes riboflavin-overproducing bacterial strains obtained by transformation of the riboflavin biosynthesis genes from
Bacillus subtilis.
These genes described therein, and other genes involved in vitamin B2 biosynthesis from prokaryotes are unsuitable for a recombinant riboflavin preparation process using eukaryotes such as, for example,
Saccharomyces cerevisiae
or
Ashbya gossypii.
DE 44 20 785 describes six riboflavin biosynthesis genes from
Ashbya gossypii,
and microorganisms transformed with these genes, and the use of such microorganisms for riboflavin synthesis.
It is possible with these processes to generate producer strains for microbial riboflavin synthesis. However, these producer strains often have metabolic limitations which cannot be eliminated by the inserted biosynthesis genes or are sometimes induced thereby. Such producer strains are sometimes unable to provide sufficient substrate for saturating some steps in the biosynthesis, so that the biosynthetic capacity of some segments of metabolism cannot be fully exploited.
It is therefore desirable to enhance further sections of metabolic pathways, thereby to eliminate metabolic bottlenecks and thus further optimize the microorganism employed for the microbial riboflavin synthesis (producer strains) in respect of their ability for riboflavin synthesis. It is desirable to identify the enhancing sections of the complex metabolism and to enhance these in a suitable way.
The present invention relates to novel proteins of purine biosynthesis, the genes therefor and the use thereof for microbial riboflavin synthesis.
Purine metabolism (for a review, see, for example, Voet, D. and Voet, J. G., 1994, Biochemie, VCH Weinheim, pages 743-771; Zalkin, H. and Dixon, J. E., 1992, De novo purine nucleotide biosynthesis, in: Progress in nucleic acid research and molecular biology, Vol. 42, pages 259-287, Academic Press) is a part of the metabolism which is essential for all life forms. Faulty purine metabolism may in humans lead to serious diseases (e.g. gout). Purine metabolism is moreover an important target for treating oncoses and viral infections. Numerous publications have appeared describing substances which intervene in purine metabolism for these indications (as review, for example Christopherson, R. I. and Lyons, S. D., 1990, Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents, Med. Res. Reviews 10, pages 505-548).
Investigations on the enzymes involved in purine metabolism (Smith, J. L., Enzymes in nucleotide synthesis, 1995, Curr. Opinion Struct. Biol. 5, 752-757) aim to develop novel immunosuppressives, antiparasitic or antiproliferative medicines (Biochem. Soc. Transact. 23, pages 877-902, 1995).
These medicines are normally not naturally occurring purines, pyrimidines or compounds derived therefrom.
The present invention relates to a protein having the polypeptide sequence depicted in SEQ ID NO:2 or a polypeptide sequence obtainable from SEQ ID NO:2 by substitution, insertion or deletion of up to 15% of the amino acids, and having the enzymatic activity of a phosphoribosyl-pyrophosphate synthetase.
The sequence depicted in SEQ ID NO:2 is the gene product of the KPR1 gene (SEQ ID No:1) obtained from
Ashbya gossypii.
The invention further relates to a protein having the polypeptide sequence depicted in SEQ ID NO:5 or a polypeptide sequence obtainable from SEQ ID NO:5 by substitution, insertion or deletion of up to 10% of the amino acids, and having the enzymatic activity of a glutamine-phosphoribosyl-pyrophosphate amidotransferase.
The sequence depicted in SEQ ID NO:5 is the gene product of the ADE4 gene (SEQ ID NO:3) obtained from
Ashbya gossypii.
The invention further relates to a protein having the polypeptide sequence depicted in SEQ ID NO:8 or a polypeptide sequence obtainable from SEQ ID NO:8 by substitution, insertion or deletion of up to 20% of the amino acids, and having the enzymatic activity of an IMP dehydrogenase.
The sequence depicted in SEQ ID NO:8 and 9 is the gene product of the GUA1 gene (SEQ ID NO:7) obtained from
Ashbya gossypii.
The invention further relates to a protein having the polypeptide sequence depicted in SEQ ID NO:11 or a polypeptide sequence obtainable from SEQ ID NO:11 by substitution, insertion or deletion of up to 10% of the amino acids, and having the enzymatic activity of a GMP synthetase.
The sequence depicted in SEQ ID NO:11 is the gene product of the GUA2 gene (SEQ ID NO:10) obtained from
Ashbya gossypii.
The invention further relates to a protein having the polypeptide sequence depicted in SEQ ID NO:13 or a polypeptide sequence obtainable from SEQ ID NO:13 by substitution, insertion or deletion of up to 10% of the amino acids, and having the enzymatic activity of a phosphoribosyl-pyrophosphate synthetase.
The sequence depicted in SEQ ID NO:13 is the gene product of the KPR2 gene (SEQ ID NO:12) obtained from
Ashbya gossypii.
These gene products mentioned can be modified by conventional methods of gene technology, such as site-directed mutagenesis, so that particular amino acids are replaced, additionally inserted or deleted. Amino acid residues are normally (but not exclusively) replaced by those of similar volume, charge or hydrophilicity/hydrophobicity in order not to lose the enzymatic properties of the gene products. In particular, modifications of the amino acid sequence in the active center frequently results in a drastic alteration in the enzymatic activities. However, modifications of the amino acid sequence and other, less essential sites are often tolerated.
It is possible with the novel proteins
1. for up to 15, preferably up to 10 and particularly preferably up to 5, % of the amino acids to be modified, by comparison with sequences depicted in the sequence listing, in the case of the gene product of the AgKPR1 gene;
2. for up to 10 and particularly preferably up to 5% of the amino acids to be modified, by comparison with the sequences depicted in the sequence listing, in the case of the gene product of the AgADE4 gene;
3. for up to 20, preferably up to 15, particularly preferably up to 10 and especially preferably up to 5, % of the amino acids to be modified, by comparison with the sequences depicted in the sequence listing, in the case of the gene product of t
Doval Jose Luis Revuelta
Garcia Maria Angeles Santos
Höffken Hans Wolfgang
Jimenez Alberto
Pompejus Markus
Achutamurthy Ponnathapu
BASF - Aktiengesellschaft
Keil & Weinkauf
Steadman David J.
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