NEOPINONE ISOMERASE AND METHODS OF USING
Disclosed are methods for converting a first morphinan alkaloid compound into a second morphinan alkaloid compound in the presence of a neopinone isomerase enzyme under reaction conditions permitting the conversion of the first alkaloid compound into the second alkaloid compound. The first alkaloid compound can be neopinone or neomorphinone. The second alkaloid compound can be codeinone or morphinone. Related compositions are also disclosed.
1 - 68 . (canceled)
69 . A substantially pure nucleic acid comprising one or more of the nucleic acid sequences selected from the group consisting of:
(i) SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17;
(ii) a nucleic acid sequence that is substantially identical to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17;
(iii) a nucleic acid sequence that is substantially identical to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 but for the degeneration of the genetic code;
(iv) a nucleic acid sequence that is complementary to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17;
(v) a nucleic acid sequence encoding a polypeptide comprising one or more of the amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21;
(vi) a nucleic acid sequence that encodes a functional variant of a polypeptide comprising one or more of the amino acid sequences set forth in in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21; and
(vii) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (i), (ii), (iii), (iv), (v) or (vi).
70 . The nucleic acid according to claim 69 , wherein the nucleic acid sequence that is substantially identical to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO; 16 or SEQ ID NO: 17 is at least 70% identical to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO; 16 or SEQ ID NO: 17.
71 . The nucleic acid according to claim 69 , wherein the nucleic acid sequence encodes a neopinone isomerase and is selected from SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 12 and SEQ ID NO: 53 or a nucleic acid sequence substantially identical thereto.
72 . The nucleic acid according to claim 69 , wherein the polypeptide comprising one or more amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21 is selected from SEQ ID NO:2 and SEQ ID NO:54.
73 . A polypeptide comprising one or more of the amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21 or functional variants thereof.
74 . The polypeptide of claim 73 , wherein the polypeptide comprises an amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:54.
75 . A chimeric nucleic acid sequence comprising as operably linked components:
(a) a nucleic acid sequence according to claim 69 encoding a neopinone isomerase; and
(b) a nucleic acid sequence capable of controlling expression of neopinone isomerase in a host cell.
76 . The chimeric nucleic acid sequence of claim 75 , wherein the nucleic acid sequence capable of controlling expression of neopinone isomerase comprises a promoter, a transcriptional terminator, a transcriptional enhancer, or combinations thereof.
77 . A recombinant expression vector comprising as operably linked components:
(a) a nucleic acid sequence capable of controlling expression in a host cell; and
(b) a nucleic acid sequence according to claim 69 encoding a neopinone isomerase;
wherein the expression vector is suitable for expression in a host cell.
78 . The recombinant expression vector of claim 77 , wherein the nucleic acid sequence capable of controlling expression in a host cell comprises a promoter, a transcriptional terminator, a transcriptional enhancer, or combinations thereof.
79 . The recombinant expression vector of claim 77 , further comprising one or more nucleic acid sequences encoding marker genes, one or more origins of replication, genetic elements required for the integration of the vector or a portion thereof into the host cell’s genome, or combinations thereof.
80 . A host cell comprising a recombinant polynucleotide comprising a nucleic acid sequence according to claim 69 .
81 . The host cell of claim 80 , wherein the host cell is Escherichia coli .
82 . A method of making neopinone isomerase, the method comprising:
(a) providing a chimeric nucleic acid sequence according to claim 75 and introducing the chimeric nucleic acid sequence into a host cell and growing the host cell to produce the neopinone isomerase.