Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Reexamination Certificate
2000-07-24
2002-03-12
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
C435S254200, C536S023200
Reexamination Certificate
active
06355468
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates inter alia to a Rhodotorula phenylalanine lyase polypeptide, to polynucleotides encoding the polypeptide, and to methods of obtaining and using these products.
BACKGROUND OF THE INVENTION
Phenylalanine ammonia lyase (PAL; EC 4.3.1.5.) is an enzyme that is found in several plants, yeast, and Streptomyces. PAL catalyzes the nonoxidative deamination of L-phenylalanine to trans-cinnamic acid. The enzyme has a potential role in the treatment and diagnosis of phenylketonuria (Ambrus et al.,
Science
, 201, 837-839 (1978)) and cancer, and is commercially useful for the manufacture of L-phenylalanine from ammonia and t-cinnamate.
Many references describe PAL-producing yeast strains that are useful in fermentation cultures for producing phenylalanine.
Rhodotorula glutinis
can be employed to obtain PAL activity in the presence of inducer, but the activity reaches a maximum after about six hours of induction and then diminishes thereafter. PAL similarly is rapidly degraded in the absence of the inducer during fermentation and has a half-life of approximately 2-5 hours during fermentations of most
Rhodotorula rubra
strains.
U.S. Pat. No. 4,598,047 describes mutant strains of
Rhodotorula rubra
(GX 5902, GX 5903, GX 5904 specifically) that are useful for PAL production.
Rhodotorula graminis
wild-type strain KGX 39 (also known as GX 5007) is a soil isolate that similarly has PAL activity (Durham et al.,
J. Bact
., 160, 771-777 (1984)). KGX 39 has several advantages over other production strains of
Rhodotorola rubra
. It grows 15-20% faster and requires less yeast extract, has no L-methionine requirement during induction, and its PAL half-life during fermentation is about 8 to 9 hours.
R. graminis
KGX 39, however, is undesirable as a production strain due to low PAL titers obtained during fermentation.
An over-producing PAL mutant also has been obtained by mutagenesis of strain KGX 39, as described in U.S. Pat. No. 4,757,015. This mutagenized strain (deposited as ATCC 20804) has high PAL specific activity and titer, high PAL specific productivity, high stability, and lower fermentation times to maximum PAL concentration than any of the previously-available PAL-producing yeast strains.
The use of yeast-derived PAL to produce a variety of optically-active unnatural amino acids having phenylalanine-like structures as chiral synthons for synthesis recently has been described (see, U.S. Pat. No. 5,981,239, incorporated by reference in its entirety herein). According to this reference, the stereospecific introduction of ammonia is accomplished with use of microorganism cells (i.e., cells of the yeast strain
Rhodotorula graminis
ATCC 20804) as the biocatalyst for the stereospecific conversion. Phenylalanine ammonia lyase from
R. graminis
ATCC 20804 was found to demonstrate broad substrate specificity for introduction of a molecule of ammonia stereoselectively onto the double bond of a 3-substituted acrylic acid. This newly discovered activity of
R. graminis
PAL should prove useful commercially.
In particular, phenylalanine and its derivatives also have been used as essential building blocks in the construction of various types of biologically active molecules. For instance, protease inhibitors employed in the treatment of human immunodeficiency virus and human cytomegalovirus infections contain a phenylalanine-like architecture as their pharmacophores. Presently there is a need for a general process of preparing a variety of optically active unnatural amino acids (i.e., amino acids that are not found in nature) having phenylalanine-like structures as chiral synthons for synthesis of these drug candidates. Based on the broad substrate specificity of
R. graminis
, it would be useful to obtain the polypeptide and nucleic acid sequences of its PAL, e.g., amongst other things, for optimization of its enzymatic activities in these synthesis reactions.
Accordingly, while polynucleotides encoding phenylalanine ammonia lyase have been isolated from the yeasts
Rhodosporidium toruloides
(PCT WO 88/02824) and
Rhodotorula rubra
(Filpula et al.,
Nucleic Acids Research
, 16, 11381 (1988), it would be useful to obtain the polynucleotide sequence of still other species. There is a need for strains that can be employed for the production of phenylalanine, phenylalanine analogs, and other optically active unnatural amino acids having phenylalanine-like structures. The present invention thus is directed, amongst other things, to methods, vectors, sequences, and compositions to meet that need. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein. The description and examples are provided to enhance the understanding of the invention, but are not intended to in any way limit the scope of the invention.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a Rhodotorula phenylalanine lyase polypeptide, polynucleotides encoding the polypeptide, and methods of obtaining and using these products.
REFERENCES:
patent: 4436813 (1984-03-01), Wood et al.
patent: 4574117 (1986-03-01), Vollmer et al.
patent: 4584273 (1986-04-01), Finkelman et al.
patent: 4598047 (1986-07-01), McGuire
patent: 4600692 (1986-07-01), Wood et al.
patent: 4636466 (1987-01-01), McGuire et al.
patent: 4728611 (1988-03-01), Wood et al.
patent: 4732851 (1988-03-01), Wood et al.
patent: 4757015 (1988-07-01), Orndorff et al.
patent: 5981239 (1999-11-01), Liu
patent: 0 167 411 (1986-08-01), None
patent: WO 88/02024 (1988-03-01), None
patent: WO 93/07279 (1993-04-01), None
Ambrus K. et al, Phenylalanine Depletion for the Management of Phenylketonuria: Use of Enzyme Reactors with Immobilized Enzymes, Science, (1978); 201; 837-839.*
Durham D. R. et al, Dissimilation of Aromatic Compounds in Rhodotorula Graminis: Biochemical Characterization of Pleiotropically Negative Mutants, J. Bacteriol. (1984); 160, 771-777.*
John G. Anson, et al., “Complete nucleotide sequence of theRhodosporidium toruloidesgene coding for phenylalanine ammonia-lyase,”Gene, 58:189-199 (1987).
Godwin B. D'Cunha, et al., “Stabilization of phenylalanine ammonia lyase containingRhodotorula glutiniscells for the continuous synthesis of L-phenylalanine methyl ester/96/,”Enzyme and Microbial Technology, 19:421-427, 1996.
Christopher T. Evans, et al., “BioConversion of Trans-Cinnamic Acid to L-Phenylalanine in an Immobilized Whole Cell Reactor,”Biotechnology and Bioengineering, 30:1067-1072 (1987).
James D.B. Faulkner, et al., “High-level expression of the phenylalanine ammonia lyase-encoding gene fromRhodosporidium toruloidesinSaccharomyces cerevisiaeandEscherichia coliusing a bifunctional expression system,”Gene, 143(1):13-20 (1994).
David Filpula, et al., “Nucleotide sequence of gene for phenylalanine ammonia-lyase forRhodotorula rubra,”Nucleic Acids Research, 16:11381 (1988).
Andreas Gloge, et al., “Phenylalanine Ammonia-Lyase: The Use of Its Broad Substrate Specificity for Mechanistic Investigations and Biocatalysis—Synthesis of L-Arylalanines,”Chem. Eur. J.6:18, 3386-3390 (2000).
Daniel S. Hodgins, “Yeast Phenylalanine Ammonia-lyase,”The Journal of Biological Chemistry, 246 (9):2977-2983 (1971).
Katsuhiko Nakamichi, et al., “Induction and Stabilization of L-Phenylalanine Ammonia-Lyase Activity inRhodotorula glutinis,”Eur. J. Appl. Microbiol Biotechnol, 18:158-162 (1983).
Derwent Abstract 85-095789 (Abstract to JP 83-151415).
JAPIO Abstract 81-026197 (Abstract to JP 56-26197).
JAPIO Abstract 88-148992 (Abstract to JP 63-148992).
GenBank Accession No. x13094 and x13095.
GenBank Accession No. x51513.
Kootstra Anna B.
Yoshida Roberta K.
Marshall Gerstein & Borun
Nashed Nashaat T.
PCBU Services, Inc.
Walicka M
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