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AssigneePharmacia Corporation
InventorsBolten L. Suzanne, Robert A. Clayton, Alan M. Easton, Leslie C. Engel, Dean M. Messing, John S. Ng, Beverly Reitz, Mark C. Walker, Ping T. Wang
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The present invention relates to a novel cytochrome P450-like enzyme (Aspergillus ochraceus 11 alpha hydroxylase) and an oxidoreductase (Aspergillus ochraceus oxidoreductase) isolated from cDNA library generated from the mRNA of Aspergillus ochraceus spores. When the cDNA encoding the 11 alpha hydroxylase was co-expressed in Spodoptera frugiperda (Sf-9) insect cells with the cDNA encoding human oxidoreductase as an electron donor, it successfully catalyzed the conversion of the steroid substrate 4-androstene-3,17-dione (AD) to 11 alpha-hydroxy-AD as determined by HPLC analysis. The invention also relates to nucleic acid molecules associated with or derived from these cDNAs including complements, homologues and fragments thereof, and methods of using these nucleic acid molecules, to generate, for example, polypeptides and fragments thereof. The invention also relates to the generation of antibodies that recognizes the A. ochraceus 11 alpha hydroxylase and oxidoreductase and methods of using these antibodies to detect the presence of these native and recombinant polypeptides within unmodified and transformed host cells, respectively. The invention also provides methods of expressing the Aspergillus 11 alpha hydroxylase gene separately, or in combination with human or Aspergillus oxidoreductase, in heterologous host cells, to facilitate the bioconversion of steroid substrates to their 11 alpha hydroxy-counterparts.
An isolated and purified nucleic acid, encoding an Aspergillus ochraceus 11 alpha hydroxylase.
An isolated and purified Aspergillus ochraceus 11 alpha hydroxylase.
An isolated and purified nucleic acid, encoding an Aspergillus ochraceus oxidoreductase.
An isolated and purified Aspergillus ochraceus oxidoreductase.
An isolated and purified nucleic acid encoding an enzyme that can catalyze the 11 alpha hydroxylation of a compound selected from the group consisting of:
A method of expressing a protein that can catalyze the 11 alpha hydroxylation of a compound selected form the group consisting of:
25 The method of claim 21 wherein said 11 alpha hydroxylase is Aspergillus ochraceus 11 alpha hydroxylase and said electron donor protein is Aspergillus ochraceus oxidoreductase.
26 The method of claim 22 wherein said 11 alpha hydroxylase is Aspergillus ochraceus 11 alpha hydroxylase and said electron donor protein is Aspergillus ochraceus oxidoreductase
An isolated and purified polypeptide that can catalyze the 11 alpha hydroxylation of a compound selected from the group consisting of:
An expression cassette comprising a heterologous DNA encoding an enzyme from the metabolic pathway for the synthesis of sitosterol to eplerenone wherein said enzyme catalyzes at least one conversion selected from the group consisting of:
A method of determining the specific activity of a cloned 11 alpha hydroxylase comprising the steps of;
A composition comprising from about 0.5-500 g/L molasses, 0.5-50 g/L cornsteep liquid, 0.5-50 g/L KH2PO4, 2.5-250 g/L NaCl, 2.5-250 g/L glucose, and 0.04-4 g/L progesterone, pH 3.5-7.
Dependencies are inferred from the claim text.