Ketoreductase gene and protein from yeast

Details Ketoreductase gene and protein from yeast Ketoreductase gene and protein from yeast

1999-10-08

2003-03-11

Martinell, James (Department: 1633)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S320100, C435S252330, C435S069100, C435S254100, C435S254200, C435S254210, C536S023740, C536S023700, C536S023200

Reexamination Certificate

active

06531308

ABSTRACT:

FIELD OF INVENTION
This invention relates to recombinant DNA technology. In particular the invention pertains to the generation and use of recombinant hosts harboring genetic mutations which provide for increased expression levels of a fungal ketoreductase polypeptide useful in bioenzymatic processes for the stereospecific reduction of ketones.
BACKGROUND OF THE INVENTION
2,3 Benzodiazepine derivatives are potent antagonists of the AMPA (&agr;-amino-3-hydroxy-5 methylisoxazole-4-propionic acid) class of receptors in the mammalian central nervous system (See I. Tarnawa et al. In
Amino Acids: Chemistry, Biology and Medicine
, Eds. Lubec and Rosenthal, Leiden, 1990). These derivative compounds have potentially widespread applications as neuroprotective agents, particularly as anti-convulsants. One series of 2,3 benzodiazepines is considered particularly advantageous for such use, and this series of compounds has the following general formula:
Wherein R is hydrogen or C
1
-C
10
alkyl; and
X is hydrogen, C
1
-C
10
alkyl, acyl, aryl, amido or carboxyl, or a substituted derivative thereof.
The clinical potential for these compounds has led to interest in developing more efficient synthetic methods. Biologically-based methods in which a ketoreductase enzyme provides a stereospecific reduction in a whole-cell process using fungal cells have been described in U.S. patent application Ser. No. 08/413,036. U.S. patent application Ser. No. 09/182,985 described the cloning of a ketoreductase gene from
Z.rouxii
, recombinant host cells that express the ketoreductase gene, and methods for stereospecifically reducing a ketone using either the recombinantly expressed ketoreductase or host cells expressing the cloned ketoreductase.
Maximizing expression levels of commercially important recombinantly expressed proteins is a common objective in the realm of biotechnology. Traditionally, protein properties have been modified by engineering changes in the DNA, mRNA, or polypeptide sequence based on a structural and/or functional understanding of that particular molecule, and testing for an improvement in the property which is being optimized. However, attempts to rationally alter one property of a recombinantly expressed polypeptide are often tedious, information intensive efforts. Additionally, these pursuits of a optimally expressed polypeptide often result in alterations which improve a targeted property at the expense of other important previously existing characteristics.
Recently, molecular “breeding” and directed evolution techniques have gained favor as methods of optimizing a particular protein's characteristics (Ness et al., (1999), DNA shuffling of subgenomic sequences of subtilisin,
Nat. Biotechnol.,
17 (9):893). Directed evolution procedures are typically used to make desired changes in the characteristics of a protein absent specific knowledge of the genetic sequence or protein structure which confers that characteristic to the molecule. Changes in enzyme specific activity, substrate specificity, thermal stability, chemical stability, and other properties have been successfully modified by this approach (see Ness, et al.). The procedure typically involves incorporating random mutations into a genetic sequence, and/or inducing random recombination between similar coding nucleotide sequences, and screening or selecting for altered but desirable phenotypes in hosts expressing the resulting mutations.
Subsequent to the selection of hosts exhibiting the desired or improved upon properties, sequence analysis often reveals that the mutated coding sequences contain a combination of missense and silent mutations. Backcrossing against the unmated wild type sequence is generally employed to remove mutations that do not contribute to the desired property of the protein and could potentially have detrimental effects on untargeted but desired properties of the original molecule. In at least one relevant example the expression level of an enzyme increased measurably as a result of mutations in the promoter region of a targeted gene (Stemmer et al., (1994), Rapid evolution of a protein in vitro by DNA shuffling, Nature 370:389-391). Back-crossing resulted in a protein having the same property as the best mutant after all four silent mutations had been reverted back to the wild type sequence. Likewise, Ness et. al. (J. E. Ness, et al., DNA shuffling of subgenomic sequences of subtilisin, Nature Biotechnology 17: 893-896, 1999) reported directed evolution experiments resulted in hosts having altered expression levels. Ness et al. suggested that this phenotype was likely attributable to recombination within the coding sequence which affected secretion or maturation of the expressed protein product.
The present invention provides a method of increasing the ability of a recombinant host cell to express an hetereologous polypeptide which is more efficient, and less labor and knowledge intensive than current methods. The present invention also provides for mutated ketoreductase encoding polynucleotides which are expressed in recombinant host cells at much greater levels than the wild-type polynucleotides.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to increased expression of polypeptides. The present invention is based on the discovery that silent mutations within the coding region of a polynucleotide can have unexpected and profound effects upon the expression level of the polypeptide encoded thereby. Ketoreductase encoding polynucleotide sequences are disclosed which exhibit increased levels of expression of ketoreductase.
Accordingly, the present invention relates to an isolated DNA molecule encoding a ketoreductase enzyme from
Z. rouxii
, said DNA molecule comprising a nucleotide sequence identified as SEQ ID NO:1.
In particularly preferred embodiments the invention is directed to isolated DNA molecules encoding a ketoreductase protein, wherein said DNA molecules comprise any one of the polynucleotide sequences identified in SEQ ID NO:16.
Having the cloned ketoreductase encoding nucleic acid molecules enables the production of recombinant ketoreductase proteins, and the production of recombinant host cells expressing said proteins, wherein said recombinant cells can be used in a stereospecific reduction of ketones.
The invention also provides the protein products of said nucleic acids, in substantially purified form. Also provided are methods for the formation of chiral alcohols using a purified ketoreductase enzyme, or recombinant host cells that express said nucleic acid molecules.
In another embodiment the present invention relates to a substantially purified ketoreductase protein molecule from
Z. rouxii.
In another embodiment the present invention relates to a ketoreductase protein molecule from
Z. rouxii
, wherein said protein molecule comprises the sequence identified as SEQ ID NO:2.
In particularly preferred embodiments the invention is directed to a ketoreductase protein molecule wherein said protein molecule comprises any one of the sequences identified in SEQ ID NO:17.
In a further embodiment the present invention relates to a ribonucleic acid molecule encoding ketoreductase protein, said ribonucleic acid molecule comprising the sequence identified as SEQ ID NO:3.
In particularly preferred embodiments the invention is directed to a ribonucleic acid molecule encoding ketoreductase protein, said ribonucleic acid molecule arising from the transcription of any one of the polynucleotide sequences identified in SEQ ID NO:16.
In yet another embodiment, the present invention relates to a recombinant DNA vector that incorporates a ketoreductase encoding polynucleotide of the present invention in operable-linkage to gene expression sequences, enabling said polynucleotide to be transcribed and translated in a host cell.
In still another embodiment the present invention relates to host cells that have been transformed or transfected with a recombinant DNA vector of the present invention such that said ketoreductase gene is expressed in the host cell.
In a still further embo

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