TITLE: NEOPINONE ISOMERASE AND METHODS OF USING

RELATED APPLICATION

[0001] This Patent Cooperation Treaty Application claims the benefit under 35 USC § 119 (e] from U.S. Provisional Patent Application No 62/594,854, filed on December 05, 2017, and from U.S. Provisional Patent Application No 62/686,337, filed on June 18, 2017, which are both incorporated by reference herein in their entirety.

FIELD OF THE DISCLOSURE

[0002] The methods and systems disclosed herein relate to a class of chemical compounds known as alkaloids and methods of making alkaloids. In particular, the methods and systems disclosed herein relate to alkaloid morphinan compounds.

BACKGROUND OF THE DISCLOSURE

[0003] The following paragraphs are provided by way of background to the present disclosure. Except where expressly stated, they are not however an admission that anything discussed therein is prior art or part of the knowledge of persons skilled in the art.

[0004] Alkaloids are a class of nitrogen containing organic chemical compounds that are naturally produced by opium poppy ( Papaver somniferum), and a range of other plant species belonging to the Papaveraceae family of plants, as well as other plant families including, for example the Lauraceae, Annonaceae, Euphorbiaceae and the Moraceae. The interest of the art in alkaloid compounds is well established and can be explained by the pharmacological properties of these compounds, as well as their utility as feedstock materials in the manufacture of pharmaceutical compounds.

[0005] The manufacture of a class of compounds known as alkaloid morphinan compounds, for example, codeine and morphine, can involve the conversion of precursor alkaloid compounds into one or more intermediary alkaloid compounds to yield the desired morphinan alkaloid compound. In biosynthetic production systems, enzymes can catalyze the conversion reaction of precursor compounds into intermediate compounds, or into a desired product. However in many biosynthetic production systems, alkaloid compounds are not efficiently converted into the desired products, for example, due to substrate inhibition, or they can be converted into products other than the desired alkaloids products, each of which results into low alkaloid product yields. [0006] Thus, for example, in a biosynthetic production system in which it is desired that morphine or codeine is produced, wherein the system involves the alkaloid morphinan compounds neopinone or neomorphinone as a precursor compound, the reaction can be inefficient. In particular, it has been observed that substantial quantities of neopine and neomorphine can accumulate in such systems, at the expense of morphine and codeine (see: Nature Chemical Biology, 2014, 10: 837] The accumulation of neopine and neomorphine is believed to occur as the precursor compounds neopinone and neomorphine are converted to neopine and neomorphine, respectively, rather than to the desired intermediate reaction compounds, namely codeinone and morphinone.

[0007] It is noted that in biosynthetic systems for the production of morphine or codeine, the undesirable conversion of neopinone and neomorphine to neopine and neomorphine, respectively, is believed by the prior art to be catalyzed by codeinone reductase, while the desired reaction from neopinone and neomorphine to codeinone and morphinone, respectively, is believed by the prior art to the prior art to proceed spontaneously (see: Tetrahedron Letters, 1993, 34: 5703]

[0008] There exists therefore a need in the art for improved processes to produce morphinan alkaloid compounds. In particular, there exists a need in the art for production systems in which the alkaloid compounds neopinone or neomorphinone are efficiently converted to codeinone and morphinone, respectively.

SUMMARY OF THE DISCLOSURE

[0009] The following paragraphs are intended to introduce the reader to the more detailed description, not to define or limit the claimed subject matter of the present disclosure.

[00010] In one aspect, the present disclosure relates to morphinan alkaloid compounds.

[00011] In another aspect, the present disclosure relates to enzymes useful in the synthesis of morphinan alkaloid compounds.

[00012] Accordingly, in one aspect, the present disclosure provides, in at least one embodiment, a method of making a second morphinan compound having a saturated carbon bond at position CS-CM and a mono-unsaturated bond at position C 7- C8, the method comprising: (i) providing a first morphinan compound having a mono- unsaturated carbon bond at position CS-CM and a saturated carbon bond at position CyCs; and

(ii) contacting the first morphinan compound with neopinone

isomerase under reaction conditions permitting the conversion of the first morphinan compound into the second morphinan compound.

[00013] In some embodiments, the first and second morphinan compounds can possess a bridging oxygen atom between carbon atoms C ₄ and Cs, forming a tetrahydrofuranyl ring within the morphinan chemical structure.

[00014] In some embodiments, the first morphinan compound can be a chemical compound having the chemical structure (I):

(I); and the second morphinan compound can be a chemical compound having the chemical structure (II):

wherein Ri is either a hydroxyl group, or a methoxy group.

[00015] In some embodiments, the method can further include a step c) comprising isolating the second morphinan compound.

[00016] In some embodiments, Ri can be a methoxy group, and the method can further include reacting the second morphinan compound in the presence of codeinone reductase to form a third morphinan compound having the chemical structure (III]:

[00017] In some embodiments, the method can further include a step c] comprising isolating the third morphinan compound having chemical structure (III] [00018] In some embodiments, additionally a fourth morphinan having the chemical structure (IV] :

can be formed, wherein the quantity of compound (IV] upon completion of the reaction does constitute no more than about 20% (w/w] of all morphinan compounds.

[00019] In some embodiments, compound (IV] upon completion of the reaction can constitute no more than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of 1% (w/w] of all morphinan compounds.

[00020] In some embodiments, Ri can be a methoxy group and the method further includes reacting the second morphinan compound in the presence of codeinone reductase to form a third morphinan compound having the chemical structure (III]:

the method further includes reacting the third morphinan compound in the presence of codeinone-O-demethylase to form a fourth morphinan compound having the chemical structure (V] :

[00021] In some embodiments, the method can further include a step c] comprising isolating the fourth morphinan compound having chemical structure (V] [00022] In some embodiments, Ri can be a methoxy group and the method further includes reacting the second morphinan compound in the presence of morphinone reductase B to form a third morphinan compound selected from the morphinan compounds having the chemical structure (XIV]:

(XVI]:

[00023] In some embodiments, the method can further include a step c] comprising isolating the third morphinan compound having chemical structure (XIV]; (XV] or (XVI]

[00024] In some embodiments, Ri can be a hydroxyl group and the method further includes reacting the second morphinan compound in the presence of codeinone reductase to form a third morphinan compound having the chemical structure (V]

[00025] In some embodiments, additionally a fourth morphinan compound having the chemical structure (VI] :

can be formed, wherein compound the quantity of compound (VI] upon completion of the reaction does constitute no more than about 20% (w/w] of all morphinan compounds.

[00026] In some embodiments, compound (VI] upon completion of the reaction can constitute no more than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of 1% (w/w] of all morphinan compounds.

[00027] In some embodiments, Ri can be a hydroxyl group and the method further includes reacting the second morphinan compound in the presence of morphinone reductase B to form a third morphinan compound selected from the morphinan compounds having the chemical structure (XVII]:

(XVIII] :

[00028] In some embodiments, the method can further include a step c] comprising isolating the third morphinan compound having chemical structure (XVII] or (XVIII]

[00029] In some embodiments, the first morphinan compound can be formed in a reaction comprising providing a precursor morphinan compound and converting the precursor morphinan compound to form the first morphinan compound.

[00030] In some embodiments, the precursor morphinan compound can be a morphinan compound having the chemical structure (VII]:

[00031] In some embodiments, the precursor morphinan compound can be a compound having chemical structure (VII] and the precursor compound is reacted in the presence of T6-0-demethylase to form the first morphinan compound, wherein in the first morphinan compound Ri is a methoxy group.

[00032] In some embodiments, the morphinan precursor compound can be a morphinan compound having chemical structure (VIII]:

and the precursor morphinan compound is reacted in the presence of T6 -0- demethylase, to form the first morphinan compound, wherein in the first morphinan compound Ri is a hydroxyl group.

[00033] In some embodiments, the precursor compound can be a compound having chemical structure (VII] and the precursor compound is reacted in the presence of codeine-O-demethylase to form a further precursor compound having the chemical structure (VIII], and the further precursor compound is reacted in the presence of T6- O-demethylase, to form the first morphinan compound, wherein in the first morphinan compound Ri is a hydroxyl group.

[00034] In some embodiments, the reaction conditions can be in vitro reaction conditions. [00035] In some embodiments, the reaction conditions can be in vivo reaction conditions.

[00036] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence comprising one or more nucleic acid sequences selected from:

(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 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].

[00037] In some embodiments, 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 can be at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or 100% identical to SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 or SEQ.ID NO: 17.

[00038] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence comprising SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 and SEQ.ID NO: 17. [00039] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence selected from SEQ.ID NO: 1, SEQ.ID NO: 9, SEQ.ID NO: 12 and SEQ.ID NO: 53.

[00040] In some embodiments, the 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 can be selected from SEQ.ID NO: 2 and SEQ.ID NO: 54.

[00041] In another aspect, the present disclosure provides, in at least one embodiment, a method for preparing a second morphinan compound comprising:

(A] providing a chimeric nucleic acid sequence comprising as operably linked components:

(a] a nucleic acid sequence encoding a neopinone isomerase polypeptide 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]; and (b] one or more nucleic acid sequences capable of controlling expression in a host cell;

(B] introducing the chimeric nucleic acid sequence into a host cell capable of producing a first morphinan compound having the chemical structure (!]:

(C] growing the cell to produce a second morphinan having the chemical structure (II]:

wherein Ri is either a hydroxyl group, or a methoxy group.

[00042] In some embodiments, 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 can be at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or 100% identical to SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 or SEQ.ID NO: 17.

[00043] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence comprising SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 and SEQ.ID NO: 17.

[00044] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence selected from SEQ.ID NO: 1, SEQ.ID NO: 9, SEQ.ID NO: 12 and SEQ.ID NO: 53. [00045] In some embodiments, the 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 can be selected from SEQ.ID NO: 2 and SEQ.ID NO: 54.

[00046] In some embodiments, the method can further include a step c] comprising isolating the second morphinan compound.

[00047] In some embodiments, Ri can be a methoxy group, and the cell further includes a polynucleotide encoding a codeinone reductase capable of catalyzing a reaction, permitting the conversion of the second morphinan compound to form a third morphinan compound having the chemical structure (III]:

[00048] In some embodiments, the nucleic acid encoding the codeinone reductase can be operably linked to the nucleic acid comprising a nucleic acid sequence selected from (i]; (ii]; (iii]; (iv]; (v]; and (vi]

[00049] In some embodiments, the method can further include a step c] comprising isolating the third morphinan compound having chemical structure (III] [00050] In some embodiments, additionally a fourth morphinan having the chemical structure (IV] :

can be formed, wherein the quantity of compound (IV) upon completion of the reaction does constitute no more than about 20% (w/w) of all morphinan compounds.

[00051] In some embodiments, compound (IV) upon completion of the reaction can constitute no more than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of 1% (w/w) of all morphinan compounds.

[00052] In some embodiments, Ri can be a methoxy group and the cell further includes a polynucleotide encoding a codeinone reductase capable of catalyzing a reaction permitting the conversion of the second morphinan compound in the presence of codeinone reductase to form a third morphinan compound having the chemical structure (III):

the cell further includes a polynucleotide encoding a codeine-O-demethylase capable of catalyzing a reaction permitting the conversion of the third morphinan compound in the presence of codeine-O-demethylase to form a fourth morphinan compound having the chemical structure (V] :

[00053] In some embodiments, the nucleic acid encoding the codeinone reductase or the nucleic acid encoding the codeine-O-demethylase can be operably linked to the nucleic acid comprising a nucleic acid sequence selected from (i]; (if); (iii); (iv); (v); and (vi). [00054] In some embodiments, the method can further include a step c] comprising isolating the fourth morphinan compound having chemical structure (V] [00055] In some embodiments, Ri can be a hydroxyl group and the cell further includes a nucleic acid encoding a codeinone reductase capable of catalyzing a reaction permitting the conversion of the second morphinan in the presence of codeinone reductase to form a third morphinan compound having the chemical structure (V] [00056] In some embodiments, additionally a fourth morphinan having the chemical structure (VI] :

is formed, wherein compound the quantity of compound (VI] upon completion of the reaction does constitute no more than about 20% (w/w] of all morphinan compounds.

[00057] In some embodiments, compound (VI] upon completion of the reaction can constitute no more than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of 1% (w/w] of all morphinan compounds.

[00058] In some embodiments, the first morphinan can be formed in a reaction in the cell, the reaction comprising converting a precursor morphinan compound to form the first morphinan.

[00059] In some embodiments, the precursor compound can be a compound having the chemical structure (VII]:

[00060] In some embodiments, the precursor compound can be a compound having chemical structure (VII], the cell can comprise T6-0-demethylase, and wherein the first morphinan compound Ri can be a methoxy group.

[00061] In some embodiments, the precursor compound can be a compound having chemical structure (VIII]:

wherein the cell can comprise T6-0-demethylase, and wherein the first morphinan Ri can be a hydroxyl group.

[00062] In some embodiments, the precursor compound can be a compound having chemical structure (VIII], the cell can comprise T6-0-demethylase, and in the first morphinan compound Ri can be a hydroxyl group.

[00063] In some embodiments, the precursor compound can be a compound having chemical structure (VII], the cell comprises codeine-O-demethylase catalyzing a reaction to form a further precursor compound having the chemical structure (VIII] from precursor compound (VII], and the cell further comprises T6-0-demethylase, to form the first morphinan compound from the precursor compound having the chemical structure (VIII], wherein in the first morphinan compound Ri is a hydroxyl group.

[00064] In another aspect, the present disclosure provides, in at least one embodiment, 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;

(iv] 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], (hi], (iv], (v] or (vi]

[00065] In some embodiments, 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 can be at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or 100% identical to SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 or SEQ.ID NO: 17.

[00066] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence comprising SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 and SEQ.ID NO: 17.

[00067] In some embodiments, the nucleic acid sequence encodes a neopine isomerase and can be selected from SEQ.ID NO: 1, SEQ.ID NO: 9, SEQ.ID NO: 12 and SEQ.ID NO: 53.

[00068] In some embodiments the 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 can be selected from SEQ.ID NO: 2 and SEQ.ID NO: 54.

[00069] In another aspect, the present disclosure provides, in at least one embodiment, a substantially pure protein comprising:

(i] 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 (ii] a functional variant of 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.

[00070] In some embodiments, SEQ.ID NO: 18, SEQ.ID NO: 19, SEQ.ID NO: 20 or SEQ.ID NO: 21 can comprise an amino acid sequence that is at least 90% identical, at least91% identical, atleast92% identical, atleast 93% identical, atleast94% identical atleast95%, atleast96%, atleast97%, atleast98%, or atleast99% identical to SEQ.ID NO: 18; SEQ.ID NO: 19; SEQ.ID NO: 20; or SEQ.ID NO: 21, respectively.

[00071] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence comprising SEQ.ID NO: 18, SEQ.ID NO: 19, SEQ.ID NO; 20 and SEQ.ID NO: 21.

[00072] In some embodiments, the substantially pure protein can comprise SEQ.ID NO: 2, or SEQ.ID NO: 54.

[00073] In another aspect, the present disclosure provides, in at least one embodiment, a chimeric nucleic acid sequence comprising as operably linked components:

(a] a nucleic acid sequence encoding a neopinone isomerase, the nucleic acid sequence comprising one or more 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), (h], (iii], (iv), (v] or (vf); and

(b] a nucleic acid sequence capable of controlling expression of neopinone isomerase in a host cell.

[00074] In some embodiments, 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 can be at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or 100% identical to SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 or SEQ.ID NO: 17.

[00075] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence comprising SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 and SEQ.ID NO: 17.

[00076] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence selected from SEQ.ID NO: 1, SEQ.ID NO: 9, SEQ.ID NO: 12 and SEQ.ID NO: 53.

[00077] In some embodiments, the 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 can be selected from SEQ.ID NO: 2 and SEQ.ID NO: 54.

[00078] In another aspect, the present disclosure provides, in at least one embodiment, 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 encoding a neopinone isomerase, the nucleic acid sequence comprising one or more 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), (h], (hi], (iv], (v] or (vi]; and

wherein the expression vector is suitable for expression in a host cell.

[00079] In some embodiments, 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 can be at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or 100% identical to SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 or SEQ.ID NO: 17.

[00080] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence comprising SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 and SEQ.ID NO: 17.

[00081] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence selected from SEQ.ID NO: 1, SEQ.ID NO: 9, SEQ.ID NO: 12 and SEQ.ID NO: 53.

[00082] In some embodiments, the 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 can be selected from SEQ.ID NO: 2 and SEQ.ID NO: 54. [00083] In another aspect, the present disclosure provides, in at least one embodiment, a host cell comprising a recombinant polynucleotide comprising a nucleic acid sequence 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]; and

[00084] In some embodiments, 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 can be at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or 100% identical to SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 or SEQ.ID NO: 17.

[00085] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence comprising SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 and SEQ.ID NO: 17.

[00086] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence selected from SEQ.ID NO: 1, SEQ.ID NO: 9, SEQ.ID NO: 12 and SEQ.ID NO: 53. [00087] In some embodiments, the 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 can be selected from SEQ.ID NO: 2 and SEQ.ID NO: 54.

[00088] In another aspect, the present disclosure provides, a method of making neopinone isomerase, the method comprising:

(a] providing a chimeric nucleic acid sequence comprising as operably linked components:

(I] a nucleic acid sequence encoding a neopinone isomerase 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]; and (II] one or more nucleic acid sequences capable of controlling expression in a host cell;

(b] introducing the chimeric nucleic acid sequence into a host cell and growing the host cell to produce the neopinone isomerase; and

(c] recovering the neopinone isomerase from the host cell.

[00089] In some embodiments, 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 can be at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or 100% identical to SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 or SEQ.ID NO: 17.

[00090] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence comprising SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 and SEQ.ID NO: 17.

[00091] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence selected from SEQ.ID NO: 1, SEQ.ID NO: 9, SEQ.ID NO: 12 and SEQ.ID NO: 53.

[00092] In some embodiments, the 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 can be selected from SEQ.ID NO: 2 and SEQ.ID NO: 54.

[00093] In another aspect, the present disclosure provides, in at least one embodiment, a use of a neopinone isomerase as a catalytic agent in a reaction to make a second morphinan compound having a saturated carbon bond at position CS-CM and a mono-unsaturated carbon bond at position CyCs, using a first morphinan compound having a mono-unsaturated carbon bond at position Cs-Ci4 and a saturated carbon bond at position CyCs as a substrate.

[00094] In some embodiments, the first and second morphinan compound can possess a bridging oxygen atom between carbon atoms C4 and C5, forming a tetrahydrofuranyl ring within the morphinan chemical structure.

[00095] In some embodiments, the first morphinan compound can be a chemical compound having the chemical structure (!]: (!]; and the second morphinan compound can be a chemical compound having the chemical structure (II]:

wherein Ri is either a hydroxyl group, or a methoxy group.

[00096] In another aspect, the present disclosure provides, in at least one embodiment, a pharmaceutical composition comprising a morphinan compound prepared in accordance with any one of the methods of the present disclosure.

[00097] In another aspect, the present disclosure provides, in at least one embodiment, a use of a morphinan compound prepared in accordance with any one of the methods of the present disclosure to prepare a pharmaceutical composition comprising the morphinan compound.

[00098] In another aspect, the present disclosure provides, in at least one embodiment, a method for treating a patient with a morphinan compound prepared according to the methods of the present disclosure, the method comprising administering to the patient a pharmaceutical composition comprising a morphinan compound, wherein the pharmaceutical composition is administered in an amount sufficient to ameliorate a medical condition in the patient.

[00099] Other features and advantages will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating preferred implementations of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those of skill in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[000100] The disclosure is in the hereinafter provided paragraphs described, by way of example, in relation to the attached figures. The figures provided herein are provided for a better understanding of the example embodiments and to show more clearly how the various embodiments may be carried into effect. The figures are not intended to limit the present disclosure.

[000101] FIG. 1 depicts a prototype chemical structure of a morphinan (FIG. 1A) and furanyl morphinan (FIG. IB). Various atoms within the chemical structures have been numbered for ease of reference.

[000102] FIG. 2 depicts the chemical structures of certain morphinan compounds: thebaine (FIG. 2A); neopinone (FIG. 2B); neopine (FIG. 2C); codeinone (FIG. 2D); oripavine (FIG. 2E) neomorphinone (FIG. 2F); neomorphine (FIG. 2G); and morphinone (FIG. 2H).

[000103] FIG. 3 depicts the chemical structures of certain further morphinan compounds: codeine (FIG. 3A); morphine (FIG. 3B); hydrocodone (FIG. 3C); 14- hydroxycodeinone (FIG. 3D); oxycodone (FIG. 3E); 14-hydroxymorphinone (FIG. 3F); and oxymorphinone (FIG. 3G).

[000104] FIG. 4 depicts certain chemical example reactions involving the conversion of various alkaloid morphinan compounds to other alkaloid morphinan compounds.

[000105] FIG. 5 depicts certain other chemical example reactions involving the conversion of various alkaloid morphinan compounds to other alkaloid morphinan compounds.

[000106] FIG. 6 depicts certain experimental results, notably a photograph of an SDS PAGE gel electrophoresis experiment showing the expression of neopinone isomerase in Escherichia coli.

[000107] FIG. 7 depicts certain experimental results, notably LC-MS/MS trace obtained in the performance of a neopinone isomerase in vitro assay.

[000108] FIG. 8 depicts certain experimental results, notably LC-MS/MS trace obtained in the performance of another a neopinone isomerase in vitro assay [000109] FIG. 9 depicts certain experimental results, notably a bar graph obtained in the performance of a neopinone isomerase in vivo assay.

[000110] FIG. 10 depicts certain experimental results, notably bar graphs obtained in the performance of a neopine isomerase gene silencing experiment. Shown are the production of NISO transcript (FIG. 10A], thebaine (FIG. 10B], codeinone (FIG. 10C], neopine (FIG. 10D], codeine (FIG. 10E] and morphine (FIG. 10F], in each case in the presence of P. somniferum tissue transformed with empty vector (pTRV2], or with a vector designed to silence NISO expression (pNISO]

[000111] FIG. 11 depicts an amino acid sequence alignment of NISO (SEQ.ID NO: 2] and PR-polypeptides including notably: PR10-8 (SEQ.ID NO: 38]; PR10-9 (SEQ.ID NO: 40]; PR10-10 (SEQ.ID NO: 42]; PR10-5 (SEQ.ID NO: 34]; PR10-4 (SEQ.ID NO: 32], PR10-11 (SEQ.ID NO: 44]; PR10-12 (SEQ.ID NO: 46]; MLP15 (SEQ.ID NO: 30]; MLP1 (SEQ.ID NO: 56], MLP2 (SEQ.ID NO: 24]; MLP3 (SEQ.ID NO: 26]; MLP4 (SEQ.ID NO: 28]; PR10-14 (SEQ.ID NO: 50]; PR10-15 (SEQ.ID NO: 30]; and a thebaine synthase polypeptide (SEQ.ID NO: 58] Various Domains are indicated, notably Domain 1 (1] (including Subdomain la (la], and Subdomain lb (lb]]; Domain 2 (2] (including Subdomain 2a (2a], and Subdomain 2b (2b]]; Domain 3 (3]; and Domain 4 (4] Identical amino acid residues are highlighted in black for ease of comparison. It is noted that an interspersed sequence that is unique to PR10-14, starting at amino acid 121 and ending at amino acid 214 is shown highlighted in grey. The interspersed sequence is shown separately in order to permit alignment between PR10-14 and the other sequences shown.

[000112] FIG. 12 depicts a schematic representation of a NISO polypeptide (FIG. 12 A] and certain experimental results obtained in the performance of an experiment to evaluate the in vitro activity of NISO mutants (FIG. 12B] FIG. 12A further shows the location of Domain 1 (1] (including Subdomain la (la], and Subdomain lb (lb]]; Domain 2 (2] (including Subdomain 2a (2a], and Subdomain 2b (2b]]; Domain 3 (3]; and Domain 4 (4] within the NISO polypeptide. Vertical bars in FIG. 12B represent neopine produced as a percentage of total product. All assays contained COR. Shown are results from assays comprising no NISO (-], and the inclusion of NISO (NISO], a Domain 1 mutant (D1], a Domain 2 mutant (D2], a Domain 3 mutant (D3], a Domain 4 mutant (D4], a Domain la mutant (Ala], a Domain lb mutant (Alb], a Domain 2 mutant (A2b] and a Domain 2 mutant (A2b]

[000113] FIG. 13 depicts a schematic representation of a PR10-8 polypeptide (FIG. 13 A] and certain experimental results obtained in the performance of an experiment to evaluate the activity of PR10-8 mutants (FIG. 13B] FIG. 13A further shows the location of Domain 1 (1], Domain 2 (2], and Domain 4 (4] within the PR10- 8 polypeptide. Vertical bars in FIG. 13B represent neopine produced as a percentage of total product. All assays contained COR. Shown are results from assays comprising no NISO and no PR10-8 (-], and the inclusion of NISO (NISO], PR10-8 (PR10-8], a PR10- 8 Domain 1 mutant (PR10-8A1; containing Domain 1 from NISO], a PR10-8 Domain 2 mutant (PR10-8A2; containing Domain 2 from NISO], a PR10-8 Domain 4 mutant (PR10-8A4; containing Domain 4 from NISO]

[000114] FIG. 14 depicts certain experimental results, notably bar graphs obtained in the performance of an experiment evaluating morphinan alkaloid compound production in assays using MorB alone (MorB], or a combination of NISO and MorB (MorB + NISO] The morphinan alkaloid evaluated is hydrocodone.

[000115] FIG. 15 depicts certain experimental results, notably bar graphs obtained in the performance of an experiment evaluating morphinan alkaloid compound production in assays using no enzyme; T60DM, COR and CODM ; or T60DM, COR, CODM and NISO. The morphinan alkaloids evaluated are: neopine, codeinone, codeine, neomorphine and morphine, as indicated by the legend in FIG. 15.

[000116] FIG. 16 depicts certain experimental results, notably bar graphs obtained in the performance of an experiment evaluating morphinan alkaloid compound production in assays using COR alone, or COR together with NISO. The substrate morphinan alkaloids evaluated are: codeine, neopine, morphine, neomorphine codeinone, and morphinone, and the product alkaloid morphinans evaluated are codeine and morphine as indicated by the legend in FIG. 16. Substrate conversion (FIG. 16A] and product formation (FIG. 16B] are shown.

[000117] The figures together with the following detailed description make apparent to those skilled in the art how the disclosure may be implemented in practice.

DESCRIPTION

[000118] Various compositions, systems or processes will be described below to provide an example of an embodiment of each claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover processes, compositions or systems that differ from those described below. The claimed subject matter is not limited to compositions, processes or systems having all of the features of any one composition, system or process described below or to features common to multiple or all of the compositions, systems or processes described below. It is possible that a composition, system or process described below is not an embodiment of any claimed subject matter. Any subject matter disclosed in a composition, system or process described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicant(s), inventor(s) or owner(s) do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.

[000119] As used herein and in the claims, the singular forms, such "a”, "an” and "the” include the plural reference and vice versa unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, "comprise,” "comprises” and "comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.

[000120] The term "or” is inclusive unless modified, for example, by "either”.

[000121] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and sub combinations of ranges and specific embodiments therein are intended to be included. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about.” The term "about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range, as will be readily recognized by context. Furthermore any range of values described herein is intended to specifically include the limiting values of the range, and any intermediate value or sub -range within the given range, and all such intermediate values and sub -ranges are individually and specifically disclosed ( e.g . a range of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5] Similarly, other terms of degree such as "substantially" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of the modified term if this deviation would not negate the meaning of the term it modifies.

[000122] Unless otherwise defined, scientific and technical terms used in connection with the formulations described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[000123] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Terms and Definitions

[000124] The terms“morphinan”,“morphinan alkaloid compound”,“morphinan” alkaloid”, or "morphinan compound”, as may be used interchangeably herein, refer to a class of molecules having the prototype chemical structure shown in FIG. 1A, and includes compounds having the prototype chemical structure shown in FIG. IB. Certain specific carbon and nitrogen atoms can be referred herein by reference to their position within the morphinan chemical structure e.g. Ci, C ₂ , N17 etc. Various modifications to the prototype chemical structures are possible, and morphinans include, for example, compounds wherein the C3 carbon atom comprises a hydroxyl side group, or a methoxy side group, the C ₆ carbon atom comprises a hydroxyl group or an oxo group, and wherein the nitrogen atom N17 comprises a methyl side group.

[000125] The term "furanyl morphinan”, as used herein, refers to a class of chemical compounds having the prototype chemical structure as shown in FIG. IB. Furanyl morphinans can be derived from morphinan compounds having the chemical structure shown in FIG. 1A by the formation of a tetrahydrofuranyl ring chemical structure established by a bridging oxygen atom between C4 and C5. It is noted that the tetrahydrofuranyl ring can also be referred to as a dihydrofuranyl chemical structure due to benzene resonance.

[000126] The term "thebaine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2A.

[000127] The term "neopinone”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2B.

[000128] The term "neopine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2C.

[000129] The term "codeinone”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2D.

[000130] The term "oripavine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2E.

[000131] The term "neomorphinone”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2F.

[000132] The term "neomorphine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2G.

[000133] The term "morphinone”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 2H.

[000134] The term "codeine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3 A.

[000135] The term "morphine”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3B.

[000136] The term "hydrocodone”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3C.

[000137] The term "14-hydroxycodeinone”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3D.

[000138] The term "oxycodone”, as used herein, refers to a chemical compound having the chemical structure set forth in FIG. 3E.

[000139] The term "14-hydroxymorpinone”, as used herein, refers to a chemical compound having the structure set forth in FIG. 3F.

[000140] The term "oxymorphone”, as used herein, refers to a chemical compound having the structure set forth in FIG. 3G. [000141] The terms "neopinone isomerase” or "NISO”, as may be used interchangeably herein, refer to any and all proteins comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any neopinone isomerase polypeptide set forth herein, including, for example, SEQ.ID NO: 2, and SEQ.ID NO: 54 or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any neopinone isomerase polypeptide set forth herein, but for the use of synonymous codons, provided however that, neopinone isomerases, exclude any and all PR-Proteins, and further include all neopinone isomerases set forth herein.

[000142] The terms "codeinone reductase” or "COR”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any codeinone reductase polypeptide set forth herein, including, for example, SEQ.ID NO: 4, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any codeinone reductase polypeptide set forth herein, but for the use of synonymous codons.

[000143] The terms "T6-0-demethylase” or "T60DM”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any T6-0-demethylase polypeptide set forth herein, including, for example, SEQ.ID NO: 6, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any T6-0-demethylase polypeptide set forth herein, but for the use of synonymous codons.

[000144] The terms "codeine-O-demethylase” or "CODM”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any codeine-O-demethylase polypeptide set forth herein, including, for example, SEQ.ID NO: 8, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any codeine-O-demethylase polypeptide set forth herein, but for the use of synonymous codons. [000145] The terms "morphinone reductase B” or "MorB”, as may be used interchangeably herein, refer to any and all enzymes comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any morphinone reductase B polypeptide set forth herein, including, for example, SEQ.ID NO: 22, or (if) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any morphinone reductase B polypeptide set forth herein, but for the use of synonymous codons.

[000146] The term "PR-protein” refers to any and all proteins comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequences constituting any PR protein polypeptide set forth herein, including, for example, SEQ.ID NO: 38, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any PR-protein polypeptide set forth herein, but for the use of synonymous codons, provided however that, PR-proteins, exclude any and all neopine isomerases, and further include all PR-proteins set forth herein.

[000147] The terms "nucleic acid sequence encoding neopinone isomerase”, and "nucleic acid sequence encoding a neopinone isomerase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a neopinone isomerase polypeptide, including, for example, SEQ.ID NO: 1, SEQ.ID NO: 9, SEQ.ID NO: 12, and SEQ.ID NO: 53. Nucleic acid sequences encoding a neopinone isomerase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the neopinone isomerase polypeptide sequences set forth herein; or (ii] hybridize to any neopinone isomerase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.

[000148] The terms "nucleic acid sequence encoding codeinone reductase”, and "nucleic acid sequence encoding a codeinone reductase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a codeinone reductase polypeptide, including, for example, SEQ.ID NO: 3. Nucleic acid sequences encoding a codeinone reductase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the codeinone reductase polypeptide sequences set forth herein; or (ii] hybridize to any codeinone reductase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.

[000149] The terms "nucleic acid sequence encoding T6-0-demethylase”, and "nucleic acid sequence encoding a T6-0-demethylase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a T6-0- demethylase polypeptide, including, for example, SEQ.ID NO: 5. Nucleic acid sequences encoding a T6-0-demethylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the T6-0- demethylase polypeptide sequences set forth herein; or (ii] hybridize to any T6-0- demethylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.

[000150] The terms "nucleic acid sequence encoding codeine-O-demethylase”, and "nucleic acid sequence encoding a codeine-O-demethylase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a codeine-O-demethylase polypeptide, including, for example, SEQ.ID NO: 7. Nucleic acid sequences encoding a codeine-O-demethylase polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the codeine-O-demethylase polypeptide sequences set forth herein; or (ii] hybridize to any codeine-O-demethylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.

[000151] The terms "nucleic acid sequence encoding morphinone reductase B”, and "nucleic acid sequence encoding a morphinone reductase B polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a morphinone reductase B polypeptide, including, for example, SEQ.ID NO: 13. Nucleic acid sequences encoding a morphinone reductase B polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to the morphinone reductase B polypeptide sequences set forth herein; or (ii] hybridize to any morphinone reductase B nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.

[000152] The terms "nucleic acid sequence encoding a PR-protein, and "nucleic acid sequence encoding a "PR-polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a PR-polypeptide, including, for example, SEQ.ID NO: 37. Nucleic acid sequences encoding a PR-polypeptide further include any and all nucleic acid sequences which (i] encode polypeptides that are substantially identical to PR-polypeptide sequences set forth herein; or (ii] hybridize to any nucleic PR-polypeptide encoding nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons, provided however that, nucleic acid sequences encoding PR-polypeptides, exclude any and all nucleic acid sequences encoding neopine isomerases, and further include all nucleic acid sequences encoding PR-polypeptides set forth herein.

[000153] The terms "polynucleotide”, "nucleic acid” or "nucleic acid sequence” as used herein, refer to a sequence of nucleoside or nucleotide monomers, consisting of naturally occurring bases, sugars and intersugar (backbone] linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present disclosure may be deoxyribonucleic polynucleotides (DNA] or ribonucleic acid polynucleotides (RNA] and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The nucleic acid sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil, and xanthine and hypoxanthine. A sequence of nucleotide or nucleoside monomers may be referred to as a polynucleotide sequence, nucleic acid sequence, a nucleotide sequence or a nucleoside sequence.

[000154] The term "polypeptide”, as used herein in conjunction with a reference SEQ.ID NO, refers to any and all polypeptides comprising a sequence of amino acid residues which is (i] substantially identical to the amino acid sequence constituting the polypeptide having such reference SEQ.ID NO, or (ii] encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding the polypeptide having such reference SEQ.ID NO, but for the use of synonymous codons. A sequence of amino acid residues may be referred to as an amino acid sequence, or polypeptide sequence.

[000155] The terms "nucleic acid sequence encoding a polypeptide” or "nucleic acid encoding a polypeptide”, as used herein in conjunction with a reference SEQ.ID NO, refers to any and all nucleic acid sequences encoding a polypeptide having such reference SEQ.ID NO. Nucleic acid sequences encoding a polypeptide, in conjunction with a reference SEQ.ID NO, further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the polypeptide having such reference SEQ.ID NO; or (ii) hybridize to any nucleic acid sequences encoding polypeptides having such reference SEQ.ID NO under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.

[000156] By the term "substantially identical” it is meant that two amino acid sequences preferably are at least 70% identical, and more preferably are at least 85% identical and most preferably at least 95% identical, for example 96%, 97%, 98% or 99% identical. In order to determine the percentage of identity between two amino acid sequences the amino acid sequences of such two sequences are aligned, using for example the alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48:1073) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, computer programs will be employed for such calculations. Computer programs that may be used in this regard include, but are not limited to, GCG (Devereux et ah, Nucleic Acids Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et al, J. Mol. Biol., 1990:215:403). A particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994, Nucleic Acid Res 22(22): 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10 and a gap extension penalty of 0.1, so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.

[000157] By "at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 ( e.g . 20, 25, 30, 40 or 50] nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm=81.5° C.-16.6 (LoglO [Na+]]+0.41(% (G+C]-600/l], or similar equation]. Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1° C. decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5° C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride/sodium citrate (SSC]/5xDenhardt's solution/1.0% SDS at Tm (based on the above equation] -5° C, followed by a wash of 0.2xSSC/0.1% SDS at 60° C. Moderately stringent hybridization conditions include a washing step in 3xSSC at 42° C. It is understood however that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions maybe found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1.-6.3.6 and in: Sambrook et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.

[000158] The term "functional variant”, as used herein in reference to polynucleotides or polypeptides, refers to polynucleotides or polypeptides capable of performing the same function as a noted reference polynucleotide or polypeptide. Thus, for example, a functional variant of the polypeptide set forth in SEQ.ID NO: 2, refers to a polypeptide capable of performing the same function as the polypeptide set forth in SEQ.ID NO: 2. Functional variants include modified a polypeptide wherein, relative to a noted reference polypeptide, the modification includes a substitution, deletion or addition of one or more amino acids. In some embodiments, substitutions are those that result in a replacement of one amino acid with an amino acid having similar characteristics. Such substitutions include, without limitation (i] glutamic acid and aspartic acid; (i] alanine, serine, and threonine; (iii] isoleucine, leucine and valine, (iv] asparagine and glutamine, and (v] tryptophan, tyrosine and phenylalanine. Functional variants further include polypeptides having retained or exhibiting an enhanced alkaloid biosynthetic bioactivity.

[000159] The term "chimeric”, as used herein in the context of polynucleotides, refers to at least two linked polynucleotides which are not naturally linked. Chimeric polynucleotides include linked polynucleotides of different natural origins. For example, a polynucleotide constituting a microbial promoter linked to a polynucleotide encoding a plant polypeptide is considered chimeric. Chimeric polynucleotides also may comprise polynucleotides of the same natural origin, provided they are not naturally linked. For example a polynucleotide constituting a promoter obtained from a particular cell-type may be linked to a polynucleotide encoding a polypeptide obtained from that same cell-type, but not normally linked to the polynucleotide constituting the promoter. Chimeric polynucleotides also include polynucleotides comprising any naturally occurring polynucleotides linked to any non-naturally occurring polynucleotides.

[000160] The term "in vivo", as used herein to describe methods of making morphinan compounds, refers to contacting a first morphinan compound with a polypeptide capable of mediating conversion of a first morphinan compound within a cell, including, for example, a microbial cell or a plant cell, to form a second morphinan compound.

[000161] The term "in vitro" as used herein to describe methods of making morphinan compounds refers to contacting a first morphinan with a polypeptide capable of mediating a conversion of the first morphinan in an environment outside a cell, including, without limitation, for example, in a microwell plate, a tube, a flask, a beaker, a tank, a reactor and the like, to form a second morphinan.

[000162] The terms "substantially pure” and "isolated”, as may be used interchangeably herein describe a compound, e.g., an alkaloid, polynucleotide or a polypeptide, which has been separated from components that naturally accompany it. Typically, a compound is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, 95%, 96%, 97%, or 98%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction] in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides, by chromatography, gel electrophoresis or HPLC analysis.

[000163] The term "recovered” as used herein in association with an enzyme or protein or morphinan, refers to a more or less pure form of the enzyme or protein or morphinan.

General Implementation

[000164] As hereinbefore mentioned, the present disclosure relates to alkaloids. The current disclosure further relates to certain polynucleotides and polypeptides. The herein provided methods and compositions are useful in that they facilitate a novel and efficient means of making certain alkaloids, notably certain morphinan alkaloids, including codeinone, codeine, morphine and morphinone. The methods and compositions can avoid the synthesis of certain side products, notably neopine and neomorphine, which in the methods known to the art can be produced at the expense of desired products, such as the aforementioned codeinone, codeine, morphine and morphinone. The current disclosure further provides cells and organisms not normally capable of synthesizing these morphinan alkaloid compounds. Such cells and organisms may be used as a source whence these morphinan alkaloids can economically be extracted. The morphinan alkaloids produced in accordance with the present disclosure are useful inter alia in the manufacture of pharmaceutical compositions.

[000165] Accordingly, the present disclosure provides, in at least one aspect, and in at least one embodiment, a method of making a second morphinan compound having a saturated carbon bond at position CS-CM and a mono-unsaturated carbon bond at position CyCs, the method comprising:

(i] providing a first morphinan compound having a mono- unsaturated carbon bond at position CS-CM and a saturated carbon bond at position CyCs; and

(ii] contacting the first morphinan compound with neopinone isomerase (NISO) under reaction conditions permitting the conversion of the first morphinan compound into the second morphinan compound.

[000166] In preferred embodiments, the first morphinan and the second morphinan each possess a bridging oxygen atom between carbon atoms C ₄ and Cs forming a tetrahydrofuranyl ring within the morphinan chemical structure, thus having the prototype chemical structure shown in FIG. IB.

[000167] In at least one aspect, the present disclosure provides, in an embodiment, a method of making a second morphinan compound, the method comprising:

(a] providing a first morphinan compound; and

(b) contacting the first morphinan compound with a neopinone isomerase under conditions permitting the conversion of the first morphinan into the second morphinan;

wherein the first morphinan is a chemical compound having the chemical structure (I):

(I); and the second morphinan is a chemical compound having the chemical structure

(II):

wherein Ri is either a hydroxyl group, or a methoxy group. [000168] In what follows, various example embodiments are provided to make morphinan alkaloids, including codeinone, morphinone, codeine and morphine. The alkaloid compounds that may be used and made, and the methods are further illustrated with reference to FIG. 4.

Synthesis of Codeinone

[000169] As shown in FIG. 4, in one embodiment, in the first and second morphinan compound, Ri is a methoxy group, and the method comprises:

(a] providing a first morphinan compound;

(b] contacting the first morphinan compound with a neopinone isomerase under conditions permitting the conversion of the first morphinan compound into the second morphinan;

wherein the first morphinan compound is a chemical compound having the chemical structure (IX]:

(IX]; and the second morphinan compound is a chemical compound having the chemical structure (X] :

It is noted that compound (IX] and (X] are known as neopinone and codeinone, respectively.

Synthesis of Morphinone [000170] As shown in FIG. 4, in one embodiment, in the first and second morphinan compound, Ri is a hydroxyl group, and the method comprises:

(a] providing a first morphinan compound;

(b] contacting the first morphinan compound with a neopinone isomerase under conditions permitting the conversion of the first morphinan compound into the second morphinan compound;

wherein the first morphinan compound is a chemical compound having the chemical structure (XI]:

the second morphinan is a chemical compound having the chemical structure

(XII]:

It is noted that compound (XI] and (XII] are known as neomorphinone and morphinone, respectively.

Synthesis of Codeine

[000171] As shown in FIG. 4, in one embodiment, in the first and second morphinan compound, Ri is a methoxy group, and the method comprises:

(a] providing a first morphinan compound;

(b] contacting the first morphinan compound with a neopinone isomerase under conditions permitting the conversion of the first morphinan compound into the second morphinan; wherein the first morphinan compound is a chemical compound having the chemical structure (IX]:

the second morphinan compound is a chemical compound having the chemical structure (X] :

(X]; and

the method further comprising contacting the second morphinan compound with codeinone reductase under reaction conditions permitting the second morphinan compound into a third morphinan compound, wherein the third morphinan compound has the chemical structure (III]:

It is noted that compound (IX], (X] and (III] are known as neopinone, codeinone and codeine, respectively. [000172] In some embodiments, a reaction mixture can be prepared to include the first morphinan compound and both neopinone isomerase and codeinone reductase.

[000173] In some embodiments, a reaction mixture can be prepared to include the first morphinan compound and neopinone isomerase, and only upon completion of the reaction, codeinone reductase can be added to the reaction mixture.

[000174] In some embodiments, certain quantities of the second morphinan compound may in the presence of codeinone reductase be converted into a fourth morphinan compound, the fourth morphinan having the chemical structure (IV]:

It is noted that compound (IV] is known as neopine.

[000175] In general upon completion of the reaction, the reaction mixture constitutes no more than about 20% (w/w] of compound (IV] of all morphinan compounds.

[000176] In some embodiments, compound (IV] upon completion of the reaction constitute no more than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of 1% (w/w] of all morphinan compounds.

Synthesis of Morphine

[000177] As shown in FIG. 4, in one embodiment, in the first and second morphinan compound, Ri is a hydroxyl group, and the method comprises:

(a] providing a first morphinan compound;

(b] contacting the first morphinan compound with a neopinone isomerase under conditions permitting the conversion of the first morphinan compound into the second morphinan compound;

wherein the first morphinan compound is a chemical compound having the chemical structure (XII]: the second morphinan compound is a chemical compound having the chemical structure (XIII]:

(XIII]; and the method further comprising contacting the second morphinan compound with codeinone reductase under reaction conditions permitting the conversion of the second morphinan compound into a third morphinan, wherein the third morphinan has the chemical structure (V] :

It is noted that compound (XII], (XIII] and (V] are known as neomorphinone, morphinone and morphine, respectively.

[000178] As shown in FIG. 4, in one embodiment, in the first and second morphinan compound, Ri is a hydroxyl group, and the method comprises:

(a] providing a first morphinan compound; (b] contacting the first morphinan compound with a neopinone isomerase under conditions permitting the conversion of the first morphinan compound into the second morphinan compound; wherein the first morphinan compound is a chemical compound having the chemical structure (XII]:

the second morphinan compound is a chemical compound having the chemical structure (XIII]:

(XIII]; and the method further comprising contacting the second morphinan compound with codeinone reductase under reaction conditions permitting the second morphinan compound into a third morphinan, wherein the third morphinan compound has the chemical structure (V] :

It is noted that compound (XII], (XIII] and (V] are known as neomorphinone, morphinone and morphine, respectively.

[000179] In some embodiments, a reaction mixture can be prepared to include the first morphinan compound and both neopinone isomerase and codeinone reductase.

[000180] In some embodiments a reaction mixture can be prepared to include the first morphinan compound and neopinone isomerase, and only upon completion of the reaction, codeinone reductase can be added to the reaction mixture.

[000181] In some embodiments, certain quantities of the second morphinan may in the presence of codeinone reductase be converted into a fourth morphinan, the fourth morphinan having the chemical structure (VI]:

It is noted that compound (VI] is also known as neomorphine.

[000182] In general upon completion of the reaction, the reaction mixture constitutes no more than about 20% (w/w] of compound (VI] of all morphinan compounds.

[000183] In some embodiments, compound (VI] upon completion of the reaction constitutes no more than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of 1% (w/w] of all morphinan compounds.

[000184] As further shown in FIG. 4, in one embodiment, in the first and second morphinan compound, Ri is a methoxy group, and the method comprises:

(a] providing a first morphinan compound;

(b] contacting the first morphinan compound with a neopinone isomerase under conditions permitting the conversion of the first morphinan compound into the second morphinan compound;

wherein the first morphinan compound is a chemical compound having the chemical structure (IX]:

the second morphinan compound is a chemical compound having the chemical structure (X] :

(X);

the method further comprising contacting the second morphinan compound with codeinone reductase under reaction conditions permitting the second morphinan compound into a third morphinan compound wherein the third morphinan compound has the chemical structure (III]:

the method further comprising contacting the third morphinan compound with codeine-O-demethylase under reaction conditions permitting the conversion of the third morphinan compound into a fourth morphinan compound, wherein the fourth morphinan compound has the chemical structure (V]:

It is noted that compound (IX], (X], (III] and (V] are known as neopinone, codeinone, codeine and morphine, respectively.

[000185] In some embodiments, a reaction mixture can be prepared to include the first morphinan compound and each neopinone isomerase, codeinone reductase and codeine-O-demethylase.

[000186] In some embodiments, a reaction mixture can be prepared to include the first morphinan compound and neopinone isomerase, and only upon completion of the reaction, codeinone reductase and codeinone-O-demethylase can be added to the reaction mixture, either together or sequentially.

[000187] In some embodiments, the reaction mixture can include 2 -oxoglutarate to facilitate the demethylation reaction catalyzed by codeine-O-demethylase.

Synthesis of 14-Hydroxymorphinone and Oxymorphone

[000188] As shown in FIG.4 and FIG. 5, in one embodiment, in the first and second morphinan compound, Ri is a hydroxyl group, and the method comprises:

(a] providing a first morphinan compound;

(b] contacting the first morphinan compound with a neopinone isomerase under conditions permitting the conversion of the first morphinan compound into the second morphinan compound; wherein the first morphinan compound is a chemical compound having the chemical structure (XII]:

(XII]; and the second morphinan compound is a chemical compound having the chemical structure (XIII]:

(XIII]; and the method further comprising contacting the second morphinan compound with morphinone reductase B under reaction conditions permitting the second morphinan compound into a third morphinan compound, wherein the third morphinan compound has the chemical structure (XVII]:

wherein the third morphinan has the chemical structure (XVIII]:

[000189] In some embodiments a mixture of compound (XVII] and (XVIII] is formed. It is noted that compound (XII], (XIII], (VII] and (XVIII] are known as neomorphinone, morphinone, 14-hydroxymorphinone and oxymorphone, respectively. [000190] In some embodiments, a reaction mixture can be prepared to include the first morphinan compound and both neopinone isomerase and morphinone reductase B.

[000191] In some embodiments a reaction mixture can be prepared to include the first morphinan compound and neopinone isomerase, and only upon completion of the reaction, morphinone reductase B can be added to the reaction mixture.

[000192] In some embodiments, certain quantities of the second morphinan may in the presence of codeinone reductase be converted into a fourth morphinan, the fourth morphinan having the chemical structure (VI]:

It is noted that compound (VI] is also known as neomorphine.

[000193] In general upon completion of the reaction, the reaction mixture constitutes no more than about 20% (w/w] of compound (VI] of all morphinan compounds.

[000194] In some embodiments, compound (VI] upon completion of the reaction constitute no more than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of 1% (w/w] of all morphinan compounds.

Synthesis of 14-Hvdroxycodeinone. Oxycodone and Hvdrocodone

[000195] As shown in FIG.4 and FIG. 5, in one embodiment, in the first and second morphinan compound, Ri is a methoxy group, and the method comprises:

(c] (a] providing a first morphinan compound;

(d] (b] contacting the first morphinan compound with a neopinone isomerase under conditions permitting the conversion of the first morphinan compound into the second morphinan;

wherein the first morphinan compound is a chemical compound having the chemical structure (IX]: the second morphinan compound is a chemical compound having the chemical structure (X] :

(X]; and

the method further comprising contacting the second morphinan compound with morphinone reductase B under reaction conditions permitting the second morphinan compound into a third morphinan compound, wherein the third morphinan compound has the chemical structure: (XIV]:

or wherein the third morphinan compound has the chemical structure (XV]:

or wherein the third morphinan compound has the chemical structure (XVI]:

[000196] In some embodiments, a mixture comprising two or more of compounds

(XIV], (XV] and (XVI] are formed. It is noted that compound (IX], (X], (XIV], (XV] and

(XV] are known as neopinone, codeinone, 14-hydroxycodeinone, oxycodone and hydrocodone, respectively.

[000197] In some embodiments, a reaction mixture can be prepared to include the first morphinan compound and both neopinone isomerase and morphinone reductase

B.

[000198] In some embodiments, a reaction mixture can be prepared to include the first morphinan compound and neopinone isomerase, and only upon completion of the reaction, morphinone reductase B can be added to the reaction mixture.

[000199] In some embodiments, certain quantities of the second morphinan compound may in the presence of codeinone reductase be converted into a fourth morphinan compound, the fourth morphinan having the chemical structure (IV]:

It is noted that compound (IV] is known as neopine.

[000200] In general, upon completion of the reaction, the reaction mixture constitutes no more than about 20% (w/w] of compound (IV] of all morphinan compounds.

[000201] In some embodiments, compound (IV] upon completion of the reaction constitute no more than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of 1% (w/w] of all morphinan compounds.

[000202]

In vitro synthesis

[000203] In accordance with certain aspects of the present disclosure, a first morphinan compound is brought in contact with a neopinone isomerase, generally in catalytic quantities, under reaction conditions permitting an enzyme catalyzed chemical conversion of the first morphinan compound to form a second morphinan compound under in vitro reaction conditions. Under such in vitro reaction conditions, the initial reaction constituents can be provided in more or less pure form and can contacted with each other and mixed under conditions that permit the requisite chemical reactions, upon enzyme catalysis, to substantially proceed. Substantially pure forms of the first morphinan compound can be chemically synthesized, or isolated from natural sources, including from poppy plants, including Papaver somniferum. Other plant species that may be used in accordance herewith to obtain an alkaloid substrate include, without limitation, plant species belonging to the plant families of Eupteleaceae, Lardizabalaceae, Circaeasteraceae, Menispermaceae, Berberidaceae, Ranunculaceae, and Papaveraceae (including those belonging to the subfamilies of Pteridophylloideae, Papaveroideae and Fumarioideae], and further include plants belonging to the genus Argemone, including Argemone mexicana (Mexican Prickly Poppy], plants belonging to the genus Berberis, including Berberis thunbergii (Japanese Barberry], plants belonging to the genus Chelidonium, including Chelidonium majus (Greater Celandine], plants belonging to the genus Cissampelos, including Cissampelos mucronata (Abuta], plants belonging to the genus Cocculus, including Cocculus trilobus (Korean Moonseed], plants belonging to the genus Corydalis, including Corydalis chelanthifolia (Ferny Fumewort], Corydalis cava; Corydalis ochotenis; Corydalis ophiocarpa; Corydalis platycarpa; Corydalis tuberosa; and Cordyalis bulbosa, plants belonging to the genus Eschscholzia, including Eschscholzia californica (California Poppy], plants belonging to the genus Glaucium, including Glaucium flavum (Yellowhorn Poppy], plants belonging to the genus Hydrastis, including Hydrastis canadensis (Goldenseal], plants belonging to the genus Jeffersonia, including Jeffersonia diphylla (Rheumatism Root], plants belonging to the genus Mahonia, including Mahonia aquifolium (Oregon Grape], plants belonging to the genus Menispermum, including Men isperm um canadense (Canadian Moonseed], plants belonging to the genus Nandina, including Nandina domestica (Sacred Bamboo], plants belonging to the genus Nigella, including Nigella sativa (Black Cumin], plants belonging to the genus Papaver, including Papaver bracteatum (Persian Poppy], Papaver somniferum, Papaver cylindricum, Papaver decaisnei, Papaver fugax, Papaver nudicale, Papaver oreophyllum , Papaver orientale, Papaver paeonifolium, Papaver persicum, Papaver pseudo-orientale, Papaver rhoeas, Papaver rhopalothece, Papaver armeniacum, Papaver setigerum, Papaver tauricolum , and Papaver triniaefolium, plants belonging to the genus Sanguinaria, including Sanguinaria canadensis (Bloodroot], plants belonging to the genus Stylophorum, including Stylophorum diphyllum (Celandine Poppy], plants belonging to the genus Thalictrum, including Thalictrum flavum (Meadow Rue], plants belonging to the genus Tinospora, including Tinospora cordifolia (Heartleaf Moonseed], plants belonging to the genus Xanthoriza, including Xanthoriza simplicissima (Yellowroot] and plants belonging to the genus Romeria including Romeria carica.

[000204] Referring again to FIG. 4, in some embodiments, the first morphinan compound can be formed in a reaction comprising providing a precursor morphinan compound and converting the precursor morphinan compound to form the first morphinan.

[000205] In some embodiments, the precursor compound can be a compound having the chemical structure (VII]:

[000206] In some embodiments, the precursor compound can be a compound having chemical structure (VII] and the precursor compound is reacted in the presence of T6-0-demethylase to form the first morphinan compound, wherein in the first morphinan compound, Ri is a methoxy group. The foregoing reaction can be performed in-vivo or in-vitro.

[000207] In some embodiments, the precursor compound can be a compound having chemical structure (VIII]:

and the precursor compound is reacted in the presence of T6-0-demethylase, to form the first morphinan compound, wherein in the first morphinan compound, Ri is a hydroxyl group.

[000208] In some embodiments, the precursor compound can be a compound having chemical structure (VII] and the precursor compound is reacted in the presence of codeine-O-demethylase to form a further precursor compound having the chemical structure (VIII], and the further precursor compound is reacted in the presence of T6- O-demethylase, to form the first morphinan compound, wherein in the first morphinan compound, Ri is a hydroxyl group. The foregoing reactions can be performed in-vivo or in-vitro.

[000209] In accordance herewith, more or less pure forms of a neopinone isomerase enzyme may be isolated from natural sources, including microbial species, and any of the hereinbefore mentioned plant species, or the enzyme may be prepared recombinantly. Thus provided in here further is a method of making, neopinone isomerase, the method comprising:

(a] providing a chimeric nucleic acid sequence comprising as operably linked components:

(i] a nucleic acid sequence encoding a neopinone isomerase; and

(ii] one or more nucleic acid sequences capable of controlling expression in a host cell;

(b] introducing the chimeric nucleic acid sequence into a host cell and growing the host cell to produce the neopinone isomerase; and

(c] recovering the neopinone isomerase from the host cell.

[000210] In some embodiments, the neopinone isomerase is an enzyme encoded by a nucleic acid sequence comprising one or more 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]

[000211] In some embodiments, 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 can be at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or 100% identical to SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 or SEQ.ID NO: 17.

[000212] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence selected from SEQ.ID NO: 1, SEQ.ID NO: 9, SEQ.ID NO: 12 and SEQ.ID NO: 53

[000213] In some embodiments, the 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 is selected from SEQ.ID NO: 2 and SEQ.ID NO: 54.

[000214] In some embodiments, two or more, or three or more nucleic acid sequences from the group consisting of SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO: 16 or SEQ.ID NO: 17 are selected.

[000215] Growth of the host cells leads to production of the neopinone isomerase. The neopinone isomerase polypeptide subsequently can be recovered, isolated and separated from other host cell components by a variety of different protein purification techniques including, e.g. ion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, reverse phase chromatography, gel filtration, etc. Further general guidance with respect to protein purification may for example be found in: Cutler, P. Protein Purification Protocols, Humana Press, 2004, Second Ed. Thus substantially pure preparations of the neopinone isomerase polypeptides may be obtained.

[000216] In accordance herewith, a first morphinan compound is brought in contact with neopinone isomerase under reaction conditions permitting an enzyme catalyzed chemical conversion of the first morphinan compound to form the second morphinan compound.

[000217] In some embodiments, the agents are brought in contact with each other and mixed to form a mixture. In some embodiments, the mixture is an aqueous mixture comprising water and further optionally additional agents to facilitate enzyme catalysis, including buffering agents, salts, pH modifying agents, cofactors, or other enzymes, including, in certain embodiments, as herein described, codeinone reductase or codeine-O-demethylase, or as also herein described, in certain embodiments. Reactions may be performed at a range of different temperatures. In preferred embodiments, a reaction is performed at a temperature between about 18 °C and 60 °C, or between about 37 °C and 55 °C, or at around 50 °C.

[000218] Reactions further may be conducted at different pH’s, for example, in certain embodiments at a pH between 7 and 9. The pH may be controlled by the addition of buffering agents to the reaction mixtures. In embodiments, in which a reaction mixture includes both neopinone isomerase and codeinone reductase, a pH between about 5.0 and 9.0, and more preferably about 7.5. It is noted that in reactions involving both neopinone isomerase and codeinone reductase the operable pH range may differ from the operable pH range of reactions involving the use codeinone reductase alone, for example, in some embodiments the operable pH range may be greater in reactions involving both neopinone isomerase and codeinone reductase, ranging from about 5.0 to about 9.0.

[000219] In embodiments hereof, wherein a reaction mixture includes the enzyme codeinone reductase, the reaction mixture preferably further includes the cofactor nicotineamide adenine dinucleotide phosphate (NADP ⁺ ]· In the performance of the reaction NADP ⁺ can be converted to NADPH. It is noted that the NADPH formed can be used as a cofactor in reactions involving neopinone or neomorphinone to form neopine and neomorphine, respectively.

[000220] In embodiments hereof, wherein a reaction mixture includes both codeinone reductase and neopinone isomerase, in preferred embodiments, the quantities of neopinone isomerase included in the reaction mixture exceed the quantities of codeinone reductase. Thus, for example, the quantities of neopinone isomerase may exceed the quantities of codeinone reductase by a factor of at least about 5X, 10X, 15X, 20X or 25X on a weight basis. In preferred embodiments, the quantities of neopinone isomerase, exceed the quantities of codeine reductase by a factor of about 17X, 18X, 19X, 20X, 21X, 22X or 23X on a weight basis. At these quantities, the formation of neopine and neomorphine at the expense of codeinone and morphinone, respectively, can be significantly reduced, in particular, when compared to reaction mixtures not including neopinone isomerase. In preferred embodiments, upon completion of the reaction, neopine or neomorphine constitute no more than about 20% (w/w] of all morphinan compounds present in the reaction mixture. In further preferred embodiments, neopine or neomorphine constitute no more than about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of 1% (w/w] of all morphinan compounds present in the reaction mixture.

[000221] Upon completion of the in vitro reaction the second morphinan compound, or morphinan compounds derived therefrom, including codeine and morphine, may be obtained in more or less pure form.

In vivo synthesis

[000222] In accordance with certain aspects of the present disclosure, a first morphinan compound is brought in contact with a neopinone isomerase enzyme, generally in catalytic quantities, under reaction conditions permitting an enzyme catalyzed chemical conversion of the first morphinan compound to form a second morphinan compound under in vivo reaction conditions. Under such in vivo reaction conditions, living cells are modified in such a manner that they produce the second morphinan. In certain embodiments, the living cells can be microorganisms, including bacterial cells, including Escherichia coli cells, for example and fimgal cells, including, yeast cells, Saccharomyces cerevisiae cells and Yarrowia lipolytica cells, for example. In other embodiments, the living cells are multicellular organisms, including plants.

[000223] In some embodiments, the living cells can be selected to be host cells capable of producing the first morphinan compound, but not the second morphinan compound. In some embodiments, the living cells can be selected to be host cells capable of producing a first morphinan compound having formula (I], but not second morphinan compound having formula (II]. Such cells include, without limitation, bacteria, yeast, other fimgal cells, plant cells, or animal cells. Thus, by way of example only, a host cell can be a yeast host cell capable of producing a first morphinan having formula (I], but not a second morphinan having formula (II] In order to modulate such host cells in such a manner that they produce the second morphinan compound, a neopinone isomerase in accordance herewith can be heterologously introduced and expressed in the host cells.

[000224] In some embodiments, the living cells naturally produce the second morphinan compound, however the living cells are modulated in such a manner that the levels of the second morphinan compound produced by the cell are modulated, relative to the levels produced by the cell without heterologous introduction of the neopinone isomerase in such living cells. [000225] In order to produce the second morphinan compound, provided herein is further a method for preparing a second morphinan compound comprising:

(A] providing a chimeric nucleic acid sequence comprising as operably linked components:

(a] a nucleic acid sequence encoding a neopinone isomerase polypeptide comprising a polypeptide sequence encoded by a polynucleotide having a nucleic acid sequence comprising one or more 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]; and

(b] one or more nucleic acid sequences capable of controlling expression in a host cell;

(B] introducing the chimeric nucleic acid sequence into a host cell capable of producing a first morphinan having the chemical structure (!]: (!]; and

(C] growing the cell to produce a second morphinan having the chemical structure (II]:

wherein Ri is either a hydroxyl group, or a methoxy group.

[000226] In some embodiments, 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 can be at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or 100% identical to SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 or SEQ.ID NO: 17.

[000227] In some embodiments, the neopine isomerase can be a polypeptide encoded by a nucleic acid sequence selected from SEQ.ID NO: 1, SEQ.ID NO: 9, SEQ.ID NO: 12 and SEQ.ID NO: 53

[000228] In some embodiments, the 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 is selected from SEQ.ID NO: 2 and SEQ.ID NO: 54.

[000229] In some embodiments, two, three or four nucleic acid sequences from the group consisting of SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO: 16 or SEQ.ID NO: 17 are selected.

[000230] In some embodiments, the sequences SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO: 16 and SEQ.ID NO: 17 are selected, and SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO: 16 or SEQ.ID NO: 17 are linked in a 5’ to 3’ direction in the order: SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO: 16, SEQ.ID NO: 17.

[000231] In some embodiments, the nucleic acid sequence further comprises an interspersing sequence between SEQ.ID NO: 14 and 15 no longer than 39 nucleic acid residues.

[000232] In some embodiments, the nucleic acid sequence further comprises an interspersing sequence between SEQ.ID NO: 15 and 16 no longer than 6 nucleic acid residues.

[000233] In some embodiments, the nucleic acid sequence further comprises an interspersing sequence between SEQ.ID NO: 16 and 17 no longer than 66 nucleic acid residues.

[000234] In some embodiments, the nucleic acid sequence further comprises an interspersing sequence between SEQ.ID NO: 14 and 15 comprising or consisting of a nucleic acid sequence encoding SEQ.ID NO: 59.

[000235] In some embodiments, the nucleic acid sequence further comprises an interspersing sequence between SEQ.ID NO: 14 and 15 comprising a nucleic acid sequence encoding an amino acid sequence substantially identical to SEQ.ID NO: 59.

[000236] In some embodiments, the nucleic acid sequence further comprises an interspersing sequence between SEQ.ID NO: 15 and 16 comprising or consisting of a nucleic acid sequence encoding SEQ.ID NO: 60.

[000237] In some embodiments, the nucleic acid sequence further comprises an interspersing sequence between SEQ.ID NO: 15 and 16 comprising a nucleic acid sequence encoding an amino acid sequence substantially identical to SEQ.ID NO: 60.

[000238] In some embodiments, the nucleic acid sequence further comprises an interspersing sequence between SEQ.ID NO: 16 and 17 comprising or consisting of a nucleic acid sequence encoding SEQ.ID NO: 61.

[000239] In some embodiments, the nucleic acid sequence further comprises an interspersing sequence between SEQ.ID NO: 16 and 17 comprising a nucleic acid encoding an amino acid sequence substantially identical to SEQ.ID NO: 61.

[000240] In some embodiments, the method can further include a step c] comprising isolating the second morphinan.

[000241] In some embodiments, the nucleic acid sequences can be isolated from any of the hereinbefore mentioned plant species. Thus the present disclosure further includes a substantially pure polynucleotide comprising a nucleic acid sequence comprising one or more 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]

[000242] In some embodiments, 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 can be at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or 100% identical to SEQ.ID NO: 14, SEQ.ID NO: 15, SEQ.ID NO; 16 or SEQ.ID NO: 17.

[000243] In some embodiments, the nucleic acid sequence encodes a neopine isomerase and is selected from SEQ.ID NO: 1, SEQ.ID NO: 9, SEQ.ID NO: 12 and SEQ.ID NO: 53

[000244] In some embodiments the 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 is selected from SEQ.ID NO: 2 and SEQ.ID NO: 54. [000245] In accordance herewith, the nucleic acid sequence encoding neopinone isomerase can be linked to a nucleic acid sequence capable of controlling expression of neopinone isomerase in a host cell. The present disclosure also provides, a nucleic acid sequence encoding a neopinone isomerase linked to a promoter capable of controlling expression in a host cell. Accordingly, the present disclosure provides in one embodiment, a chimeric polynucleotide comprising a polynucleotide comprising a nucleic acid sequence comprising as operably linked components:

(a] a polynucleotide comprising a nucleic acid sequence encoding a neopinone isomerase; and

(b] a polynucleotide comprising a nucleic acid sequence capable of controlling expression of neopinone isomerase in a host cell.

[000246] Nucleic acid sequences capable of controlling expression in host cells that may be used herein include any transcriptional promoter capable of controlling expression of polypeptides in host cells. Generally, promoters obtained from bacterial cells are used when a bacterial host is selected in accordance herewith, while a fungal promoter will be used when a fimgal host is selected, a plant promoter will be used when a plant cell is selected, and so on. Further nucleic acid elements capable elements of controlling expression in a host cell include transcriptional terminators, enhancers and the like, all of which may be included in the chimeric nucleic acid sequences of the present disclosure.

[000247] In accordance with the present disclosure, the chimeric nucleic acid sequences comprising a promoter capable of controlling expression in host cell linked to a nucleic acid sequence encoding a neopinone isomerase, can be integrated into a recombinant expression vector which ensures good expression in the host cell. Accordingly, the present disclosure includes a recombinant expression vector comprising as operably linked components:

(a] a polynucleotide comprising a nucleic acid sequence capable of controlling expression in a host cell; and

(b] a polynucleotide comprising nucleic acid sequence encoding a neopinone isomerase,

wherein the expression vector is suitable for expression in a host cell. The term "suitable for expression in a host cell” means that the recombinant expression vector comprises the chimeric nucleic acid sequence of the present disclosure linked to genetic elements required to achieve expression in a host cell. Genetic elements that may be included in the expression vector in this regard include a transcriptional termination region, one or more nucleic acid sequences encoding marker genes, one or more origins of replication and the like. In preferred embodiments, the expression vector further comprises genetic elements required for the integration of the vector or a portion thereof in the host cell's genome, for example if a plant host cell is used the T-DNA left and right border sequences which facilitate the integration into the plant's nuclear genome.

[000248] Pursuant to the present disclosure, the expression vector may further contain a marker gene. Marker genes that may be used in accordance with the present disclosure include all genes that allow the distinction of transformed cells from non- transformed cells, including all selectable and screenable marker genes. A marker gene may be a resistance marker such as an antibiotic resistance marker against, for example, kanamycin or ampicillin. Screenable markers that may be employed to identify transformants through visual inspection include b-glucuronidase (GUS] (U.S. Pat. Nos. 5,268,463 and 5,599,670] and green fluorescent protein (GFP] (Niedz et ah, 1995, Plant Cell Rep., 14: 403]

[000249] One host cell that particularly conveniently may be used is Escherichia coli. The preparation of the E. coli vectors may be accomplished using commonly known techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing, the Polymerase Chain Reaction (PCR] and other methodologies. A wide variety of cloning vectors is available to perform the necessary steps required to prepare a recombinant expression vector. Among the vectors with a replication system functional in E. coli, are vectors such as pBR322, the pUC series ofvectors, the M13 mp series of vectors, pBluescript etc. Typically, these cloning vectors contain a marker allowing selection of transformed cells. Nucleic acid sequences may be introduced in these vectors, and the vectors may be introduced in £. coli by preparing competent cells, electroporation or using other well known methodologies to a person of skill in the art. £. coli may be grown in an appropriate medium, such as Luria-Broth medium and harvested. Recombinant expression vectors may readily be recovered from cells upon harvesting and lysing of the cells. Further, general guidance with respect to the preparation of recombinant vectors and growth of recombinant organisms may be found in, for example: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001, Third Ed.

[000250] Further included in the present disclosure are a host cell wherein the host cell comprises a chimeric nucleic acid sequence comprising in the 5’ to 3’ direction of transcription as operably linked components one or more polynucleotides comprising nucleic acid sequences encoding a neopinone isomerase. As hereinbefore mentioned the host cell is preferably a host cell capable of producing a morphinan alkaloid having chemical structure (I], not a morphinan alkaloid having chemical structure (II], but for the introduction of the chimeric nucleic acid sequences of the present disclosure.

[000251] As hereinbefore mentioned, in other embodiments, the host cells naturally produce a morphinan compound having chemical structure (II], however the host cells are modulated in such a manner that the levels of the morphinan compound having chemical structure (II] produced in the cells is modulated, relative to levels of such morphinan compound produced by the cell without heterologous introduction of the herein enzymes in such host cells. Such modulations may be achieved by a variety of modification techniques, including, but not limited to, the modulation of the enzymatic activity of an neopinone isomerase, for example by modulating the native nucleic acid sequences encoding the neopinone isomerase, for example by gene silencing methodologies, such as antisense methodologies; or by the use of modification techniques resulting in modulation of activity of the enzymes using for example site directed mutagenesis, targeted mutagenesis, random mutagenesis, virus- induced gene silencing, the addition of organic solvents, gene shuffling or a combination of these and other techniques known to those of skill in the art, each methodology designed to alter the activity of the enzymes of the neopinone isomerase, in such a manner that level of product morphinan compound in the host cells increases.

[000252] In some embodiments, the hereinbefore mentioned methods to modulate expression levels of the polynucleotides encoding neopinone isomerase of the present disclosure may result in modulations in the levels of plant alkaloids, including without limitation in the levels of morphine, codeine and codeinone. Thus, the present disclosure includes the use of the methodologies to modify the levels of plant morphinan alkaloids, in a plant naturally capable of producing plant morphinan alkaloid compounds. [000253] In some embodiments the plant belongs to the plant family of Papaveraceae.

[000254] In some embodiments, the plant belongs to the plant species Papaver somniferum, Papaver bracteatum, Papaver nudicale, Papaver orientale or Papaver rhoeas.

[000255] In another aspect of the present disclosure, the polynucleotides encoding a neopinone isomerase may be used to examine the presence of the polynucleotide in a cell, or a cell extract, such as a polynucleotide containing extract. Accordingly, in some embodiments the polynucleotides encoding neopinone isomerase may be labeled and used as a probe to examine the presence of the polynucleotide in a cell, or a cell extract.

[000256] In another aspect of the present disclosure, the polynucleotides encoding neopinone isomerase of the present disclosure may be used to genotype plants.

[000257] In some embodiments, the plant belongs to the plant family of Papaveraceae.

[000258] In some embodiments, the plant belongs to the species Papaver somniferum, Papaver bracteatum , Papaver nudicale, Papaver orientale or Papaver rhoeas.

[000259] In general, genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to identify segregants in subsequent generations of a plant population. Molecular marker methodologies can be used for phylogenetic studies, characterizing genetic relationships among plant varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance. See, e.g., Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed., Springer-Verlag, Berlin (1997] For molecular marker methodologies, see generally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2] in: Genome Mapping in Plants (ed. Andrew H. Paterson] by Academic Press/R. G. Landis Company, Austin, Tex., pp.7-21. The particular method of genotyping in accordance with the present disclosure may involve the employment of any molecular marker analytic technique including, but not limited to, restriction fragment length polymorphisms (RFLPs] RFLPs reflect allelic differences between DNA restriction fragments caused by nucleic acid sequence variability. As is known to those of skill in the art, RFLPs are typically detected by extraction of plant genomic DNA and digestion of the genomic DNA with one or more restriction enzymes. Typically, the resulting fragments are separated according to size and hybridized with a nucleic acid probe. Restriction fragments from homologous chromosomes are revealed. Differences in fragment size among alleles represent an RFLP. Thus, the present disclosure further provides a means to follow segregation of a portion or genomic DNA encoding a polynucleotide of the present disclosure, as well as chromosomal nucleic acid sequences genetically linked to these polynucleotides using such techniques as RFLP analysis. Linked chromosomal nucleic sequences are within 50 centiMorgans (cM), often within 40 or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2, or 1 cM of a genomic nucleic acid sequence encoding a polypeptide of the present disclosure. Thus, in accordance with the present disclosure the polynucleotides of the present disclosure may be used as markers to evaluate in a plant population the segregation of polynucleotides genetically linked thereto. In some embodiments, the plant population comprises or consists of plants belonging to the plant families Papaveraceae. In other embodiments, the plant population comprises or consists of plants belonging to the plants species Papaver somniferum, Papaver bracteatum Papaver nudicale, Papaver orientale or Papaver rhoeas.

[000260] In accordance with the present disclosure, the polynucleotide probes employed for molecular marker mapping of plant nuclear genomes selectively hybridize, under selective hybridization conditions, to a genomic sequence encoding a polypeptide of the present disclosure, including, in specific embodiments polypeptides comprising the amino acid sequence set forth in SEQ.ID NO: 2, or SEQ.ID NO: 54.

[000261] Typically, these probes are cDNA probes. Typically, these probes are at least 15 bases in length, more preferably at least 20, 25, 30, 35, 40, or 50 bases in length. Generally, however, the probes are less than about 1 kilobase in length. Preferably, the probes are single copy probes that hybridize to a unique locus in a haploid plant chromosome complement. Some exemplary restriction enzymes employed in RFLP mapping are EcoRI, EcoRv, and Sstl. As used herein the term "restriction enzyme” includes reference to a composition that recognizes and, alone or in conjunction with another composition, cleaves a polynucleotide at a specific nucleic acid sequence. [000262] Other methods of differentiating polymorphic (allelic] variants of the nucleic acid sequences of the present disclosure can be used by utilizing molecular marker techniques well known to those of skill in the art, including, without limitation: 1] single stranded conformation analysis (SSCP]; 2] denaturing gradient gel electrophoresis (DGGE]; 3] RNase protection assays; 4] allele-specific oligonucleotides (ASOs]; 5] the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and 6] allele-specific PCR. Other approaches based on the detection of mismatches between the two complementary DNA strands include, without limitation, clamped denaturing gel electrophoresis (CDGE]; heteroduplex analysis (HA], and chemical mismatch cleavage (CMC]

[000263] Thus, the present disclosure further provides a method of genotyping comprising the steps of contacting, under stringent hybridization conditions, a sample suspected of comprising a nucleic acid encoding a polypeptide of the present disclosure, including, in specific embodiments polypeptides comprising the amino acid sequence set forth in SEQ.ID NO: 2, or SEQ.ID NO: 54 with a nucleic acid probe capable of hybridizing to a polynucleotide sequence encoding the foregoing. Generally, the sample is a plant sample, and in some embodiments, a sample suspected of comprising a Papaver somniferum nucleic acid sequence encoding polynucleotides of the present disclosure. The polynucleotide probe selectively hybridizes, under stringent conditions, to a subsequence of the nucleic acid sequence encoding the polypeptide comprising a polymorphic marker. Selective hybridization of the polynucleotide probe to the polymorphic marker nucleic acid sequence yields a hybridization complex. Detection of the hybridization complex indicates the presence of that polymorphic marker in the sample. In preferred embodiments, the polynucleotide probe comprises a portion of a nucleic acid sequence encoding polypeptide of the present disclosure.

Uses of Morphinan Compounds

[000264] It will be clear form the foregoing that the neopinone isomerase according to the present disclosure may be used in a variety of processes and In another aspect, the present disclosure provides, in at least one embodiment, a use of a neopinone isomerase as a catalytic agent in a reaction to make a second morphinan product having a saturated carbon bond at position CS-CM and a mono-unsaturated carbon bond at position CyCs, using a first morphinan having a mono-unsaturated carbon bond at position Cs-Cwand a saturated carbon bond at position CyCs as a substrate.

[000265] In some embodiments, the first and second morphinan can possess a bridging oxygen atom between carbon atoms C ₄ and Cs, forming a tetrahydrofuranyl ring within the morphinan chemical structure.

[000266] In some embodiments, the first morphinan can be a chemical compound having the chemical structure (I) :

the second morphinan can be a chemical compound having the chemical structure (II):

wherein Ri is either a hydroxyl group, or a methoxy group.

[000267] The morphinan alkaloid compounds obtained in accordance with the present disclosure may be formulated for use as a pharmaceutical drug, therapeutic agent or medicinal agent. Thus the present disclosure further includes a pharmaceutical composition comprising a morphinan compound prepared in accordance with the methods of the present disclosure. Pharmaceutical drug preparations comprising a morphinan product in accordance with the present disclosure can comprise vehicles, excipients and auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like. These vehicles, excipients and auxiliary substances are generally pharmaceutical agents that may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, benzoates, and the like. It is also preferred, although not required, that the preparation will contain a pharmaceutically acceptable excipient that serves as a stabilizer. Examples of suitable carriers that also act as stabilizers for peptides include, without limitation, pharmaceutical grades of dextrose, sucrose, lactose, sorbitol, inositol, dextran, and the like. Other suitable carriers include, again without limitation, starch, cellulose, sodium or calcium phosphates, citric acid, glycine, polyethylene glycols (PEGs], and combinations thereof. The pharmaceutical composition may be formulated for oral and intravenous administration and other routes of administration as desired. Dosing may vary and may be optimized using routine experimentation.

[000268] In another aspect, the present disclosure further provides, in an embodiment, a use of a morphinan compound prepared in accordance with any one of the methods of the present disclosure to prepare a pharmaceutical composition comprising the morphinan compound.

[000269] The morphinan compounds of the present disclosure further may be used as precursor or feedstock material for the production of derivative morphinan compounds. Thus, for example, as has been described herein, codeinone made in accordance with the present disclosure can be used as a precursor to make codeine, codeine can be used as a precursor to make morphine, and morphinone can be used as a precursor compound to make morphine. It will be clear to those of skill in the art that the morphinone compounds made in accordance with the present disclosure can be used to make a wide variety of derivative morphinan compounds. Upon finishing synthesis the morphinan compounds can be used to formulate pharmaceutical drugs, as hereinbefore described.

[000270] In further embodiments, the present disclosure provides methods for treating a patient with a pharmaceutical composition comprising a morphinan compound prepared in accordance with the present disclosure. Accordingly, the present disclosure further provides a method for treating a patient with a morphinan compound prepared according to the methods of the present disclosure, the method comprising administering to the patient a pharmaceutical composition comprising a morphinan compound, wherein the pharmaceutical composition is administered in an amount sufficient to ameliorate a medical condition in the patient.

SUMMARY OF SEQUENCES

[000271] SEQ.ID NO: 1 sets forth a polynucleotide sequence encoding a neopinone isomerase polypeptide.

[000272] SEQ.ID NO: 2 sets forth a deduced amino acid sequence of a neopinone isomerase polypeptide.

[000273] SEQ.ID NO: 3 sets forth a polynucleotide sequence encoding a codeinone reductase polypeptide.

[000274] SEQ.ID NO: 4 sets forth a deduced amino acid sequence of a codeinone reductase polypeptide.

[000275] SEQ.ID NO: 5 sets forth a polynucleotide sequence encoding a T6-0- demethylase polypeptide.

[000276] SEQ. ID NO: 6 sets forth a deduced amino acid sequence of a T-6-0- polypeptide.

[000277] SEQ.ID NO: 7 sets forth a polynucleotide sequence encoding a codeine- O-demethylase polypeptide.

[000278] SEQ. ID NO: 8 sets forth a deduced amino acid sequence of a codeine-6- O-polypeptide.

[000279] SEQ.ID NO: 9 sets forth a polynucleotide sequence encoding a neopinone isomerase polypeptide.

[000280] SEQ.ID NO: 10 sets forth a polynucleotide sequence encoding a PR polypeptide (PR10-8]

[000281] SEQ.ID NO: 11 sets forth a deduced amino acid sequence encoding a PR- polypeptide (PR10-8]

[000282] SEQ.ID NO: 12 sets forth a polynucleotide sequence encoding a neopinone isomerase polypeptide (codon optimized]

[000283] SEQ.ID NO: 13 sets forth a polynucleotide sequence encoding a morphinone reductase polypeptide.

[000284] SEQ.ID NO: 14 sets forth a polynucleotide sequence encoding a portion of the neopinone isomerase polypeptide set forth in SEQ.ID NO: 1. [000285] SEQ.ID NO: 15 sets forth a polynucleotide sequence encoding a portion of the neopinone isomerase polypeptide set forth in SEQ.ID NO: 1.

[000286] SEQ.ID NO: 16 sets forth a polynucleotide sequence encoding a portion of a the neopinone isomerase polypeptide set forth in SEQ.ID NO: 1.

[000287] SEQ.ID NO: 17 sets forth a polynucleotide sequence encoding a portion of the neopinone isomerase polypeptide set forth in SEQ.ID NO: 1.

[000288] SEQ.ID NO: 18 sets forth a deduced amino acid sequence of a portion of the neopinone isomerase polypeptide set forth in SEQ.ID NO: 2.

[000289] SEQ.ID NO: 19 sets forth a deduced amino acid sequence of a portion of the neopinone isomerase polypeptide set forth in SEQ.ID NO: 2.

[000290] SEQ.ID NO: 20 sets forth a deduced amino acid sequence of a portion of the neopinone isomerase polypeptide set forth in SEQ.ID NO: 2.

[000291] SEQ.ID NO: 21 sets forth a deduced amino acid sequence of a portion of the neopinone isomerase polypeptide set forth in SEQ.ID NO: 2.

[000292] SEQ.ID NO: 22 sets forth a deduced amino acid sequence of a morphinone reductase B polypeptide.

[000293] SEQ.ID NO: 23 sets forth a polynucleotide sequence of a PR-polypeptide (MLP-2]

[000294] SEQ.ID NO: 24 sets forth a deduced amino acid sequence of a PR- polypeptide (MLP-2]

[000295] SEQ.ID NO: 25 sets forth a polynucleotide sequence of a PR-polypeptide (MLP-3]

[000296] SEQ.ID NO: 26 sets forth a deduced amino acid sequence of a PR- polypeptide (MLP-3]

[000297] SEQ.ID NO: 27 sets forth a polynucleotide sequence of a PR-polypeptide

(MLP-4]

[000298] SEQ.ID NO: 28 sets forth a deduced amino acid sequence of a PR- polypeptide (MLP-4]

[000299] SEQ.ID NO: 29 sets forth a polynucleotide sequence of a PR-polypeptide (MLP-15]

[000300] SEQ.ID NO: 30 sets forth a deduced amino acid sequence of a PR- polypeptide (MLP-15] [000301] SEQ.ID NO: 31 sets forth a polynucleotide sequence of a PR-polypeptide (PR10-4).

[000302] SEQ.ID NO: 32 sets forth a deduced amino acid sequence of a PR- polypeptide (PR10-4]

[000303] SEQ.ID NO: 33 sets forth a polynucleotide sequence of a PR-polypeptide

(PR10-5).

[000304] SEQ.ID NO: 34 sets forth a deduced amino acid sequence of a PR- polypeptide (PR10-5]

[000305] SEQ.ID NO: 35 sets forth a polynucleotide sequence of a PR-polypeptide (PR10-7).

[000306] SEQ.ID NO: 36 sets forth a deduced amino acid sequence of a PR- polypeptide (PR10-7]

[000307] SEQ.ID NO: 37 sets forth a polynucleotide sequence of a PR-polypeptide (PR10-8).

[000308] SEQ.ID NO: 38 sets forth a deduced amino acid sequence of a PR- polypeptide (PR10-8]

[000309] SEQ.ID NO: 39 sets forth a polynucleotide sequence of a PR-polypeptide (PR10-9).

[000310] SEQ.ID NO: 40 sets forth a deduced amino acid sequence of a PR- polypeptide (PR10-9]

[000311] SEQ.ID NO: 41 sets forth a polynucleotide sequence of a PR-polypeptide (PR10-10).

[000312] SEQ.ID NO: 42 sets forth a deduced amino acid sequence of a PR- polypeptide (PRIO-IO]

[000313] SEQ.ID NO: 43 sets forth a polynucleotide sequence of a PR-polypeptide

(PR10-11).

[000314] SEQ.ID NO: 44 sets forth a deduced amino acid sequence of a PR- polypeptide (PRlO-ll]

[000315] SEQ.ID NO: 45 sets forth a polynucleotide sequence of a PR-polypeptide (PR10-12).

[000316] SEQ.ID NO: 46 sets forth a deduced amino acid sequence of a PR- polypeptide (PR10-12] [000317] SEQ.ID NO: 47 sets forth a polynucleotide sequence of a PR-polypeptide (PR10-13]

[000318] SEQ.ID NO: 48 sets forth a deduced amino acid sequence of a PR- polypeptide (PR10-13]

[000319] SEQ.ID NO: 49 sets forth a polynucleotide sequence of a PR-polypeptide

(PR10-14]

[000320] SEQ.ID NO: 50 sets forth a deduced amino acid sequence of a PR- polypeptide (PR10-14]

[000321] SEQ.ID NO: 51 sets forth a polynucleotide sequence of a PR-polypeptide (PR10-15]

[000322] SEQ.ID NO: 52 sets forth a deduced amino acid sequence of a PR- polypeptide (PR10-15]

[000323] SEQ.ID NO: 53 sets forth a polynucleotide sequence encoding a neopinone isomerase polypeptide (truncated]

[000324] SEQ.ID NO: 54 sets forth a deduced amino acid sequence of a neopinone isomerase polypeptide (truncated]

[000325] SEQ.ID NO: 55 sets forth a polynucleotide sequence of a PR-polypeptide (MLP-1]

[000326] SEQ.ID NO: 56 sets forth a deduced amino acid sequence of a PR- polypeptide (MLP-1]

[000327] SEQ.ID NO: 57 sets forth a polynucleotide sequence of a thebaine synthase polypeptide.

[000328] SEQ.ID NO: 58 sets forth a deduced amino acid sequence of a thebaine synthase polypeptide.

EXAMPLES

[000329] Hereinafter are provided examples of specific implementations for performing the methods of the present disclosure, as well as implementations representing the compositions of the present disclosure. The examples are provided for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.

Example 1 - Cloning and sequencing of gene encoding neopinone isomerase

[000330] The Papaver somniferum neopinone isomerase gene was cloned and sequenced. The neopinone isomerase gene was identified within a subset of genes encoding pathogenesis-related 10 (PR10] proteins, based on gene expression and protein abundance profiles in various opium poppy ( Papaver somniferum ] organs, tissues and cell types and cellular fractions. Nucleic acid sequences are set forth in SEQ.ID NO: 1 (including some 5’ and 3’ upstream and downstream sequences and SEQ.ID NO: 9 (coding sequence only]. The amino acid sequence is set forth in SEQ.ID NO: 2.

Example 2 - Expression of neopinone isomerase in E. coli

[000331] A codon-optimized PR10-3 gene (SEQ.ID NO: 12], encoding neopinone isomerase (NISO] was expressed in Escherichia coli with a His6-tag on the N-terminus, and cloned into the pACE vector or pET 19b vector. Expression vectors were transformed into E. coli stain Rosetta (DE3] pLysS (EMD Chemicals], which were subsequently induced overnight using 0.2 mM isopropyl b-D-thiogalactoside (IPTG] at 16°C. Cells were harvested by centrifugation and sonicated in 50 mM sodium phosphate, pH 7.0, 300 mM NaCl and 10% (v/v] glycerol. After centrifugation at 20,000g for 10 min, the supernatant was loaded onto Talon (Clontech] cobalt-affinity resin. Purification was performed according to the manufacturer’s instructions. Purified, recombinant proteins were desalted using PD 10 column (GE healthcase], and stored in 50 mM sodium phosphate, pH 7.0, 50 mM NaCl and 10% (v/v] glycerol. Protein concentration was determined by the Bradford assay (Bio-Rad] using bovine serum albumin as the standard. Chemical crosslinking was performed to demonstrate that neopinone isomerase functions as a homodimer. Briefly, the cross-linking reagent bis[sulfosuccinimidyl] suberate (BS3] was prepared at a concentration of 10 mM in PBS buffer (10 mM sodium phosphate buffer, pH 7.4, 137 mM NaCl, 2.7 mM KC1] The BS3 solution (2 pL] was added to 20 pL of purified protein (1 pg/pL] The reaction was incubated on ice for 1 h followed by the addition of 5 pL of 10% (w/v] sodium dodecyl sulfate to quench the reaction. Protein samples were then boiled for 5 min and subjected to SDS-PAGE and immunoblot analysis. SDS-PAGE results are shown in FIG. 6

Example 3 - Expression of neopinone isomerase in veast

[000332] Yeast strains with chromosome-integrated T60DM and C0R1.3 genes were constructed using a USER cloning system. USER (uracil -specific excision reaction] -based cloning has been used for the integration of multiple genes into the yeast genome owing to its relatively straightforward application and independence from the enzyme-based ligation of DNA fragments. Multiple PCR products of BIA biosynthetic genes and Gall/GallO promoter regions were simultaneous cloned to the USER cloning vectors initially nicked with As/SI and Nb.Bsml and then transformed into yeast cells using the LiAc/PEG/single-stranded carrier DNA (ssDNA] transformation method. The high-copy number pESC-Ura (or, alternatively, pESC-Leu or pESC-His] vector was used to express neopinone isomerase (NISO] gene candidates using the GallO promoter. PCR-amplified candidate genes from cDNA using primers flanked with Spel and Notl restriction sites were ligated to the pESC-Ura vector to generate transient expressing constructs. Transient expression constructs were individually transformed to the platform yeast strains with chromosome-integrated BIA biosynthetic genes using the LiAc/PEG/single-stranded carrier DNA (ssDNA] transformation method. Each yeast strain transiently expressing a candidate gene was inoculated in SD-drop out medium overnight. The overnight cultures were then diluted into a SD-drop out medium containing 2% (w/v] galactose and 200 mM of the BIA suitable for conversion by the baseline yeast strain and/or the transient expression construct. Yeast cultures were grown for 24 h.

Example 4 - Neopinone isomerase in vitro activity

NISO PRIO-type protein-coupled T60DM-C0R assays with thebaine as substrate

[000333] A 50 pL -reaction mixture containing purified recombinant T60DM (~9 pg], purified recombinant C0R1.3 (~l-6 pg], 100 pM thebaine, 800 pM NADPH, 8 mM a-ketoglutaric acid, 800 pM ferrous sulfate, and 8 mM Na-ascorbate in 50 mM Bis-tris propane (pH 7.0 or 6.0] buffer was incubated at 30°C for 1 hr. When indicated, assays also included NISO (~0.5 pg]. All assays were quenched with 200 pL of acetonitrile and centrifuged at 17,000 g for 40 min. The supernatant was analyzed by LC-MS/MS. Results are shown in FIG. 7.

NISO PRIO-tvpe protein-coupled COR activity assays using codeinone or morphinone as substrate

[000334] A 50 pL -reaction mixture containing individual purified recombinant NISO (~5-10 pg], purified recombinant C0R1.3 (~l-6 pg], 80 pM codeinone or morphinone, 800 pM NADPH in 50 mM Bis-tris propane (pH 7.0 or 6.0] buffer was incubated at 30°C for 1 hr. When indicated, assays also included NISO (~0.6 pg]. All assays were quenched with 200 pL of acetonitrile and centrifuged at 17,000 g for 40 min. The supernatant was analyzed by LC-MS/MS. Results are shown in FIG. 8.

NISO PRIO-type protein-coupled T6QDM-COR CODM assays with thebaine as substrate - detection of morphine

[000335] A 50 pL -reaction mixture containing purified recombinant T60DM (~9 pg], purified recombinant C0R1.3 (~l-6 pg], purified CODM, 100 mM thebaine, 800 mM NADPH, 8 mM a-ketoglutaric acid, 800 mM ferrous sulfate, and 8 mM Na-ascorbate in 50 mM Bis-tris propane (pH 7.0 or 6.0] buffer was incubated at 30°C for 1 hr. When indicated, assays also included NISO (~0.5 pg]. All assays were quenched with 200 pL of acetonitrile and centrifuged at 17,000 g for 40 min. The supernatant was analyzed by LC-MS/MS. Results are shown in FIG. 15. As can be seen in FIG. 15 in the presence of NISO codeine production increases substantially. Furthermore, morphine production increases as well. By contrast neopine production is reduced.

Example 5 - Neopinone isomerase in vivo activity

[000336] Yeast cells expressing neopinone isomerase were prepared as described in Example 3. Yeast cells were removed from their culture medium by centrifugation and 5 pL of supernatant, containing alkaloids secreted by the yeast cells into the culture medium, was subjected to high-resolution mass spectrometry (MS] analysis. Alkaloids produced in yeast were characterized by high-resolution MS ⁿ analysis. For MS ⁿ experiments, alkaloids were injected by HPLC for electrospray ionization (ESI] prior to analysis by LTQ-Orbitrap-XL. Operation was conducted using LTQ Tune Plus v. 2.5.5 SP1 and Xcalibur v. 2.1.0.1140, with additional analyses using QualBrowser feature of Xcalibur. Internal calibration, external calibration, tuning, and general operations were performed using routine procedures optimized for alkaloid detection, with the exception that ESI, rather than heated HESI, was employed with reduced flow rates to minimize heat-induced degradation of analytes during ionization. Data acquisition for MS ² was performed using a single scan event, involving CID conducted in linear ion trap (IT] with normalized collision energy (NCE] of 35%, and detection by FTMS with resolution of 60,000 FWHM and scan range m/z 90-340. Data acquisition for MS ³ was performed by first identifying the most intense ions in MS ² , optimizing NCE for CID-based fragmentation of these ions (15-35%], and adjusting FTMS scan ranges for detection of MS ³ ions. To ensure sufficient MS ³ detection by FTMS, individual runs comprised of only one scan event were conducted for each MS ² ion subjected to CID analysis. Error was maintained at < 2 ppm to allow prediction of elemental formulae for all ions. Compound identity was based on comparisons with authentic standards and MS ⁿ assignments available in the literature. Results are shown in FIG. 9.

Example 6 - Gene-silencing of neopinone isomerase

[000337] Virus induced gene silencing (VIGS) of a nucleic acid sequence encoding neopine isomerase was performed using the tobacco rattle virus (TRV) vector system, which is based on the pYL156 plasmid, as described previously (Hagel and Facchini, 2010). The vector used to silence NISO expression, labeled pPR10-3, targeted a 187 bp region of the coding sequence. Agrobacterium tumefaciens strains harboring (i) pTRVl and (ii) either pPR10-3 or the empty pTRV2 vector were co-inoculated at a 1:1 ratio into the cotyledons and apical meristem of 2 to 3-week-old opium poppy, Bea’s choice variety, seedlings and leaves using a needleless syringe. Infiltrated plants were harvested after 8 to 12 weeks of growth. Tissue was harvested for RNA extraction as described above, from a 2 cm section of stem located approximately 1 cm below the flower bud.

[000338] Plants were screened for infection by RT-PCR to identify samples containing sequences specific to pYL156 plasmid (GAPDH). Samples included 10 and 12 plants containing pPR10-3 and pTRV2 constructs, respectively. Stem cDNA from selected plants was analyzed by qRT-PCR to determine relative transcript abundance. Latex was collected from flower buds for alkaloid analysis. Latex samples were freeze- dried for 24 h and resuspended in a solution of methanol and acetonitrile (1: 1) to a final concentration of 25 pg (dry weight)/pL. Samples were incubated on a shaker at room temperature and 200 rpm for 4 h. Extracts were centrifuged at 21,000 ^c g for 20 min at 4°C and the supernatant prepared for LC-MS analysis. Major alkaloids, thebaine, noscapine and papaverine were analyzed by 10 pL injection of a 1:50 dilution, using full-scan mode. For targeted analysis of codeinone, neopine, codeine, morphinone, neomorphine and morphine, 2 pL of undiluted sample was analyzed by MRM to identify trace amounts of metabolites.

[000339] Suppression of NISO transcript abundance in opium poppy by VIGS lead to a significant drop in morphine (FIG. 10F) accumulated in the latex, and an increase in upstream metabolites including thebaine (FIG. 10B), codeinone (FIG. 10C), neopine (FIG. 10D] and codeine (FIG. 10E] The greatest change in metabolite levels was recorded for neopine, which increased 10-fold, from 6.7 to 69 pg g ¹ dry weight. (FIG. 10] NISO transcript levels were significantly suppressed ( pPR10-3 ] (p = 0.0003] compared with the control (empty vector, pTRV2 ] (FIG. 10A] Alkaloid content (pg g ¹ dry weight] is shown for various metabolites. Values represent the mean ± standard deviation of 10 and 12 biological replicates from pPRIO- 3 and p77?V2-infected plants, respectively. The asterisk marks statistically significant differences as determined using an unpaired, two-tailed Student t test (significant p values noted on graph].

Example 7 - Identification of NISO functional domains

[000340] Several proteins with similarity to NISO (SEQ.ID NO: 2] were identified in the Papaver somniferum latex transcriptome including the following proteins PR10- 8 (SEQ.ID NO: 38 (nucleic acid sequence: SEQ.ID NO: 37]; PR10-9 (SEQ.ID NO: 40 (nucleic acid sequence: SEQ.ID NO: 39]; PR10-10 (SEQ.ID NO: 42 (nucleic acid sequence: SEQ.ID NO: 37]; PR10-5 (SEQ.ID NO: 34 (nucleic acid sequence: SEQ.ID NO: 33]; PR10-4 (SEQ.ID NO: 32 (nucleic acid sequence: SEQ.ID NO: 31], PR10-11 (SEQ.ID NO: 44 (nucleic acid sequence: SEQ.ID NO: 43]; PR10-12 (SEQ.ID NO: 46 (nucleic acid sequence: SEQ.ID NO: 45]: MLP15 (SEQ.ID NO: 30 (nucleic acid sequence SEQ.ID NO: 29]; MLP1 (SEQ.ID NO: 56 (nucleic acid sequence: SEQ.ID NO: 55], MLP2 (SEQ.ID NO: 24 (nucleic acid sequence: SEQ.ID NO:23]; MLP3 (SEQ.ID NO: 26 (nucleic acid sequence: SEQ.ID NO: 25]; MLP4 (SEQ.ID NO: 28 (nucleic acid sequence: SEQ.ID NO: 27]; PR10-14 (SEQ.ID NO: 50 (nucleic acid sequence: SEQ.ID NO: 49]; PR10-15 (SEQ.ID NO: 30 (nucleic acid sequence: SEQ.ID NO: 29]; and a thebaine synthase polypeptide (SEQ.ID NO: 58 (nucleic acid sequence: SEQ.ID NO: 57] Sequences of all of the foregoing were aligned as shown in FIG. 11. The following domains showing significant sequence similarity were identified: Domain 1 (SEQ.ID NO: 14] (comprising Subdomain la, and lb], Domain 2 (SEQ.ID NO: 15] (comprising Subdomains 2a and 2b], Domain 3 (SEQ.ID NO: 16] and Domain 4 (SEQ.ID NO: 17] (as shown in FIG. 11] Mutant proteins D1, D2, D3, D4, Ala, Alb A2a, A2b with altered Domains 1, 2, 3, 4, la, lb, 2a and 2b, respectively, were prepared and neopine formation in the presence of each of the mutant proteins and COR was assessed and compared to a positive control (intact NISO] and a negative control (no NISO] The results are shown in FIG. 12. The impact of altering Domain 1, Domain 2 and Domain 3 is particularly significant, with mutant proteins D1, Ala, Alb, D2, A2a, Nib and D3, showing neopine formation levels very similar to those achieved in the absence of NISO. The effect on neopine formation using the D4 mutant was less pronounced, but nevertheless significant in all cases. Thus all four Domains 1, 2, 3, and 4 are contributing to NISO activity.

[000341] In a further experiment, PR10-8 mutant proteins were prepared, notably RM0-8D1, RM0-8D2, and PR10-8A4, which contained independent replacement of the indicated Domain in PR10-8 with the corresponding Domain from NISO. Neopine formation in the presence of the mutant proteins and COR was assessed, and compared to neopine formation using COR and NISOAl, NIS0A2, and NIS0A3. Results are shown in FIG. 13. In the absence of NISO, neopine constitutes about 25% of the reaction product. Neopine formation can be reduced to 5% or less in the presence of NISO. Consistent with the previous experiment, in the presence of NISOAl, NIS0A2, or NIS0A3 an increase in neopine is observed, relative to wildtype NISO. By contrast, PR10-8, as well as the PR10-8Al, PR10-8A2, and PR10-8A4 mutants were unable to reduce neopine production. Thus the relatively modest sequence differences between Domains D1, D2, D3, D4 of NISO and PR10-8 nevertheless are significant with respect to the ability to suppress neopine production in the presence of COR. In this respect, related polypeptides exhibiting significant sequence similarity, for example 95%, 96%, 97%, 98%, or 99% to the sequence of Domains D1, D2, D3, or D4 of NISO are likely to be particularly effective in suppressing neopine formation in the presence of COR.

Example 8 - Production of hydrocodone

[000342] Yeast strains with chromosomally integrated T60DM were transformed (as described in Example 3] with NISO, or with NISO and further a morphine reductase form Pseudomonas putida M10 (SEQ.ID NO: 13 (nucleic acid sequence]; SEQ.ID NO: 22 (amino acid sequence], referred to as MorB. The production of hydrocone was assayed and the results are shown in FIG. 14. As can be seen in FIG. 14, in the presence of MorB alone trace amounts of hydrocone are produced. By contrast, in the presence of both NISO and MorB significant quantities of hydrocone are produced. Thus it is inferred that NISO is able to reduce neopine production and hydrocone can be produced in the presence of NISO at the expense of neopine.

Example 9 - Production of codeine and morphine [000343] Assay mixtures containing purified recombinant COR-B (~0.1 pg] alone, or purified recombinant COR-B (~0.1 pg] together with purified recombinant NISO (0.196 pg] (1:4 molar ratio], Na-ascorbate cofactor, and ImM NADPH, were incubated for 20 mins using one of the following substrates: codeine, neopine, morphine, neopmorphine, codeinone and morphinone. The percentages substrate conversion and products formed were determined. Results are shown in FIG. 16. As can be seen in FIG. 16, in the presence of NISO, codeinone substrate turnover increased almost 2- fold. The products of assays using codeinone and morphinone as a substrate, resulted in a substantial increase in product in the presence of COR-B and NISO together, i.e. codeine and morphine, respectively, when compared to assay mixtures including COR-

B alone.

SEQUENCE LISTING

[000344] SEQ.ID NO: 1 AT GT ACAGCT CAT AAT GT CACAAT AT CAGCT GATT CTTTTT CT AT AT AAACT CG TT AT ACCAACAT GGACT CAGT AT CAGCT GCT CT AGT ATTT CAT AGTT CCAT AT A CTT GT GT GCAATGGCT CAT CAT GGT GTTT CAGGT CT AGTT GGGAAAATT GT AA CT GAATT GGAGGT GAATT GT AAT GCCGACGAATTTT AT AAGATTTT GAAGCGC GAT GAAGAT GTT CCACGGGCAGTTT CT GAT CTTTT CCCT CCCGT CAAAATT GC CAAAGGAGAT GGACTT GTTT CT GGTT GT AT CAAGGAAT GGGACT GT GTT CTT GAT GGT AAGGCGAT GAGCGGCAAGGAGGAAACAACACACAACGAT GAAACG AGGACTTT GCGT CACCGT GAATTGGAAGGAGACTT GAT GAAGGATT ACAAGA AGTTT GATT CCAT AATT GAAGTT AAT CCAAAACCAAAT GGACATGGAAGCATT GT GACGT GGT CAATT GAGT AT GAGAAAAT GAACGAAGATT CT CCGGCT CCCT TT GCTT AT CT AGCTT CCTT CCAT CAGAACGTT GT GGAAGTT GATT CT CACCT C T GCCTTT CT GAAT AAGAT GCAAGT ACAT GAACACGACTTT AGT GTT CGAT GT A CGT CAGT AT AT GTT GTTT AAAT GTT CTT CTT GCGGT AT GCCT AT GT CT ACGT G AT CAAGTT CAGT GTT CGT ACACGT GAGCTTT GT GGTTTT GT GGTT ACCT AT [000345] SEQ.ID NO: 2

MDSVSAALVFHSSIYLCAMAHHGVSGLVGKIVTELEVNCNADEFYKILKRDEDVP RAVSDLFPPVKIAKGDGLVSGCIKEWDCVLDGKAMSGKEETTHNDETRTLRHRE LEGDLMKDYKKFDSIIEVNPKPNGHGSIVTWSIEYEKMNEDSPAPFAYLASFHQN VVEVDSHLCLSE

[000346] SEQ.ID NO: 3

AT GGAGAGT AAT GGT GT ACCT AT GAT CACT CT CAGTT CCGGCATT CGGAT GC CT GCTTT AGGT AT GGGAACAGCT GAAACAAT GGT AAAAGGAACAGAAAGAGA GAAATT GGCGTTTTT GAAAGCGAT AGAGGT CGGTT ACAGACACTT CGAT ACA GCT GCT GCAT ACCAAAGT GAAGAGT GT CTT GGT GAAGCT AT AGCT GAAGCAC TT CAACTT GGT CT AAT AAAAT CT CGAGAT GAACT CTT CAT CACTT CCAAGCT C TGGT GCGCT GAT GCT CACGCT GAT CTT GT CCT CCCT GCT CTT CAGAATT CT C T GAGGAAT CTT AAATT GGACT AT CTT GAT CT AT ATTT GAT ACACCAT CCGGT A AGCTT GAAGCCAGGGAAGTTT GTT AACGAAAT ACCAAAGGAT CAT AT CCTT C CAAT GGACT ACAAAT CT GT AT GGGCAGCCAT GGAAGAGT GT CAGACCCTT GG CTT CACT AGGGCAAT CGGGGT CT GT AATTT CT CAT GCAAAAAGCTT CAAGAG TT GAT GGCAGCAGCCAAGAT CCCT CCAGTT GT GAAT CAAGT GGAGAT GAGCC CGACTTT ACAT CAAAAAAAT CT GAGGGAAT ATT GCAAGGCCAAT AAT AT CAT G AT CACT GCACACT CGGTTTT GGGAGCCAT AT GT GCT CCAT GGGGCAGCAAT G CAGTT AT GGATT CT AAGGT GCTT CACCAGATT GCT GT GGCAAGAGGAAAAT C T GTT GCCCAGGTT AGT AT GAGAT GGGTTT ACCAGCAAGGCGCGAGT CT AGT G GT GAAAAGTTT CAAT GAAGGGAGGAT GAAGGAAAACCTT AAGAT ATTT GATT G GGAACT AACGGCAGAGAAT AT GGAAAAGAT CAGT GAGATT CCGCAAT CT AGA ACAAGCT CT GCT GATTT CTT GTT AT CACCGACT GGACCTTT CAAAACT GAAGA AGAGTT CT GGGAT GAGAAGGATT GA [000347] SEQ.ID NO: 4

MESNGVPMITLSSGIRMPALGMGTAETMVKGTEREKLAFLKAIEVGYRHFDTAA AYQSEECLGEAIAEALQLGLIKSRDELFITSKLWCADAHADLVLPALQNSLRNLKL DYLDLYLIHHPVSLKPGKFVNEIPKDHI LPMDYKSVWAAMEECQTLGFTRAIGVC NFSCKKLQELMAAAKIPPVVNQVEMSPTLHQKNLREYCKANNIMITAHSVLGAIC APWGS NAVM DS KVLH Q I AVARG KS VAQVS M RWVYQQGAS LVVKS F N E G R M KE NLKIFDWELTAENMEKISEIPQSRTSSADFLLSPTGPFKTEEEFWDEKD

[000348] SEQ.ID NO: 5

AT GGAGAAAGCAAAACTT AT GAAGCT AGGT AAT GGT AT GGAAAT ACCAAGT G TT CAAGAATT GGCT AAACT CACGCTT GCCGAAATT CCAT CT CGAT ACGT AT GC GCCAAT GAAAACCTTTT GTT GCCT AT GGGT GCAT CT GT CAT AAAT GAT CAT GA AACCATT CCTGT CAT CGAT AT AG AAAATTT ATT AT CT CCAG AACCAAT AAT CG GAAAGTT AGAATT AGAT AGGCTT CATTTT GCTT GCAAAGAAT GGGGTTTTTTT CAGGT AGT GAACCAT GGAGT CGACGCTT CATT GGT GGAT AGT GT AAAAT CAG AAATT CAAGGTTT CTTT AACCTTT CT AT GGAT GAGAAAACT AAAT AT GAACAG GAAGAT GGAGAT GT GGAAGGATTT GGACAAGGCTTT ATT GAAT CAGAGGACC AAACACTT GATT GGGCAGAT AT ATTT AT GAT GTT CACT CTT CCACT CCATTT AA GGAAGCCT CACTT ATTTT CAAAACT CCCAGT GCCT CT CAGGGAGACAAT CGA AT CCT ACT CAT CAG AAAT G AAAAAGTT AT CCAT G GTTCTCTTT AAT AAG AT G G AAAAAGCT CT ACAAGT ACAAGCAGCCGAGATT AAGGGT AT GT CAGAGGT GTT T AT AGAT GGGACACAAGCAAT GAGGAT GAACT ATT AT CCCCCTT GT CCT CAA CCAAAT CT CGCCAT CGGT CTT ACGT CGCACT CGGATTTT GGCGGTTT GACAA T CCT CCTT CAAAT CAACGAAGT GGAAGGATT ACAGAT AAAAAGAGAGGGGAC AT GG ATTT CAGT CAAACCT CT ACCT AAT GCGTT CGT AGT GAAT GTT GGAGAT A TTTT GGAGAT AAT GACT AAT GGAATTT ACCAT AGT GT CGAT CACCGGGCAGT A GT AAACT CAACAAAT GAGAGGCT CT CAAT CGCAACATTT CAT GACCCT AGT CT AGAGT CGGT AAT AGGCCCAAT AT CAAGCTT GATT ACT CCAGAGACACCT GCT TT GTTT AAAAGT GGAT CT ACAT AT GGGGAT CTT GT GGAGGAAT GT AAAACAAG GAAGCT CGAT GG AAAAT CATTT CTT GACT CCAT GAGG ATTT G A

[000349] SEQ.ID NO: 6 MEKAKLMKLGNGMEIPSVQELAKLTLAEI PSRYVCANENLLLPMGASVI NDHETI P VIDIENLLSPEPIIGKLELDRLHFACKEWGFFQVVNHGVDASLVDSVKSEIQGFFN LSMDEKTKYEQEDGDVEGFGQGFIESEDQTLDWADIFMMFTLPLHLRKPHLFSK LPVPLRETIESYSSEMKKLSMVLFNKMEKALQVQAAEI KGMSEVFIDGTQAMRM NYYPPCPQPNLAIGLTSHSDFGGLTILLQINEVEGLQIKREGTWISVKPLPNAFVV NVGDILEIMTNGIYHSVDHRAWNSTNERLSIATFHDPSLESVIGPISSLITPETPAL

FKSGSTYGDLVEECKTRKLDGKSFLDSMRI

[000350] SEQ.ID NO: 7

AT GGAGACACCAAT ACTT AT CAAGCT AGGCAAT GGTTT GT CAAT ACCAAGT GT T CAGGAATT GGCT AAACT CACGCTT GCAGAAATT CCAT CT CGAT ACACAT GCA CCGGT GAAAGCCCGTT GAAT AAT ATT GGTGCGT CT GT AACAGAT GAT GAAAC AGTT CCT GT CAT CGATTT GCAAAATTT ACT AT CT CCAGAACCCGT AGTT GGAA AGTT AGAATT GGAT AAGCTT CATT CT GCTT GCAAAGAAT GGGGTTT CTTT CAG CT GGTT AACCAT GGAGT CGACGCTTT ACT GAT GGACAAT AT AAAAT CAGAAAT T AAAGGTTT CTTT AACCTT CCAAT GAAT GAGAAAACT AAAT ACGGACAGCAAG AT GGAGATTTT GAAGGATTT GGACAACCCT AT ATT GAAT CGGAGGACCAAAG ACTT GATT GGACT GAAGT GTTT AGCAT GTT AAGT CTT CCT CT CCATTT AAGGA AGCCT CATTT GTTT CCAGAACT CCCT CT GCCTTT CAGGGAGACACT GGAAT C CT ACCT AT CAAAAAT G AAAAAACT AT CAACGGTT GT CTTT G AGAT GTT GG AAA AAT CT CT ACAATT AGTT GAGATT AAAGGT AT GACAGACTT ATTT GAAGAT GGG TT GCAAACAAT GAGGAT GAACT ATT AT CCT CCTT GT CCT CGACCAGAGCTT GT ACTT GGT CTT ACGT CACACT CGGATTTT AGCGGTTT GACAATT CT CCTT CAAC TT AAT GAAGTT GAAGGATT ACAAAT AAGAAAAGAAGAGAGGT GGATTT CAAT C AAACCT CT ACCT GATGCGTT CAT AGT GAAT GTT GGAGACATTTTGGAGAT AAT GACT AAT GGGATTT ACCGT AGCGT CGAGCACCGGGCAGT AGT AAACT CAACA AAGGAGAGGCT CT CAAT CGCGACATTT CAT GACT CT AAACT AGAGT CAGAAA T AGGCCCAATTT CGAGCTT GGT CACACCAGAGACACCT GCTTT GTT CAAAAG AGGT AGGT AT GAGGAT ATTTT GAAGGAAAAT CTTT CAAGGAAGCTT GAT GGA AAAT CATTT CT CGACT ACAT GAGGAT GT GA

[000351] SEQ.ID NO: 8 METPILIKLGNGLSI PSVQELAKLTLAEIPSRYTCTGESPLNNIGASVTDDETVPVI D LQNLLSPEPWGKLELDKLHSACKEWGFFQLVNHGVDALLMDNIKSEIKGFFNLP MNEKTKYGQQDGDFEGFGQPYIESEDQRLDWTEVFSMLSLPLHLRKPHLFPEL PLPFRETLESYLSKMKKLSTWFEMLEKSLQLVEIKGMTDLFEDGLQTMRMNYY PPCPRPELVLGLTSHSDFSGLTI LLQLNEVEGLQIRKEERWISIKPLPDAFIVNVGD ILEIMTNGIYRSVEHRAVVNSTKERLSIATFHDSKLESEIGPISSLVTPETPALFKRG RYEDILKENLSRKLDGKSFLDYMRM

[000352] SEQ.ID NO: 9 AT GGACT CAGT AT CAGCT GCT CT AGT ATTT CAT AGTT CCAT AT ACTT GT GT GC AAT GGCT CAT CAT GGT GTTT CAGGT CT AGTT GGGAAAATT GT AACT GAATT GG AGGT GAATT GT AAT GCCGACGAATTTT AT AAGATTTT GAAGCGCGAT GAAGAT GTT CCACGGGCAGTTT CT GAT CTTTT CCCT CCCGT CAAAATT GCCAAAGGAG AT GGACTT GTTT CT GGTT GT AT CAAGGAAT GGGACT GT GTT CTT GATGGT AAG GCGATGAGCGGCAAGGAGGAAACAACACACAACGATGAAACGAGGACTTTG CGT CACCGT GAATT GGAAGGAGACTT GAT GAAGGATT ACAAGAAGTTT GATT CCAT AATT GAAGTT AAT CCAAAACCAAAT GGACAT GGAAGCATT GT GACGT G GT CAATT GAGT AT GAGAAAAT GAACGAAGATT CT CCGGCT CCCTTT GCTT AT C T AGCTT CCTT CCAT CAGAACGTT GT GGAAGTT GATT CT CACCT CT GCCTTT CT GAATAA

[000353] SEQ.ID NO: 10

AT GGCT CAT CACGGT GTTT CAGGT CT AGTT GGGAAACTT GT AACT CAATT AGA GGT CAATT GT GAT GCT GACGAATTTT AT AAAATTT GGAAGCACCAT GAAGAAG TT CCAAAGGCAGTTT CT CATTTTTT CCCT GCCGT CAAAGTT GT CAAAGGAGAT GGACTT GTTT CT GGTT GT AT CAAGGAAT GGCACT AT AT CCT CGAGGGT AAGG CGAT GAGCGCAAT GGAGGAAACGACACACAAT GAT GAAACAAGGACTTT ACA T CACCAGGT AGTT GAAGGAGAAGT GAT GAAGGATT ACAAGGCGATT GCTT CC AT AATT CAAGTT AAT CCAAAT CCAAAT GGACAT GGAAGCATT GT GACGTGGT C AATT GAGT AT GAGAAAAT GAACGAAGATT CT CCAACT CCCTTT GCTT AT CTT G AATT CTT CCAT CAGAACAT AAT CGAT AT GAATT CT CACCT CT ACGT AGGCT CT GATT CT CACCT CCACGTT GAT GAAT AA

[000354] SEQ.ID NO: 11

MAHHGVSGLVGKLVTQLEVNCDADEFYKIWKHHEEVPKAVSHFFPAVKWKGD

GLVSGCIKEWHYILEGKAMSAMEETTHNDETRTLHHQWEGEVMKDYKAIASIIQ

VNPNPNGHGSIVTWSIEYEKMNEDSPTPFAYLEFFHQNII DMNSHLYVGSDSHLH

VDE

[000355] SEQ.ID NO: 12

AT GGCGCACCACGGT GT GAGCGGCCT GGTT GGCAAGAT CGT GACCGAGCT GGAAGTT AACT GCAACGCGGAT GAGTT CT AT AAGATT CT GAAACGT GACGAA GAT GTT CCGCGT GCGGT GAGCGACCT GTTT CCGCCGGTT AAGAT CGCGAAA GGT GAT GGCCT GGT GAGCGGCT GCATT AAAGAGT GGGACT GCGT GCT GGAT GGCAAGGCGAT GAGCGGT AAAGAGGAAACCACCCACAACGACGAAACCCGT ACCCT GCGT CACCGT GAGCT GGAAGGT GACCT GAT GAAGGATT ACAAGAAAT T CGAT AGCAT CATT GAGGTT AACCCGAAACCGAACGGT CACGGCAGCAT CGT GACCT GGAGCATT GAGT ACGAAAAGAT GAACGAAGACAGCCCGGCGCCGTT CGCGT AT CT GGCGAGCTTT CACCAGAACGT GGTT GAGGTT GAT AGCCACCT GTGCCTGAGCGAATAA

[000356] SEQ.ID NO: 13

CGAGGAAGGCCT CCACGAAGGCGAT CAGGT CGAGGTT GGAAT CCCGGGAG ACGGTGGGGAAGCCCACCAGGCGGT CGAGCAT CT CGGCGGCGGT CAT GGC GCGCT CCTT GGT CACGTTT CGGGAAAT CCT AT CACGCCCGCGGGGCAGGCC GGCGGCCT GGGCGT CAT AGCCCCAGACT AT CCGGT CGAT GCCGT CACT CTT TTT GACAGCAGT CATT AACAGGCTT AAGAT GT CGGGCGACGACGGAT ACCCG T CCATT CACT CTT CAT CT GGAGGCGT ATT GATGCCGGAT ACAT CCTT CT CCAA CCCCGGGCT CTT CACCCCGCT GCAGCT GGGCAGCCT CAGCCT GCCCAACC GCGT GAT CAT GGCGCCGCT GACCCGCT CGCGCACGCCGGACAGCGTT CCC GGCAGGTT GCAGCAGAT CT ACT AT GGCCAGCGCGCCAGCGCCGGGCT GAT CAT CAGCGAGGCCACCAAT AT CT CGCCCACCGCCCGCGGCT ACGT CT ACAC GCCGGGGAT CT GGACCGACGCGCAGGAAGCCGGCT GGAAGGGCGT CGT CG AGGCGGT GCAT GCCAAGGGCGGGCGCAT CGCCCT GCAGCT GT GGCACGT C GGCCGT GT CT CCCACGAGCT GGT GCAGCCCGACGGCCAGCAGCCCGT GGC ACCGAGCGCCCT CAAGGCCGAGGGGGCGGAAT GCTT CGT CGAGTT CGAGG ACGGCACGGCGGGGCT GCACCCCACCAGCACGCCGCGGGCGCTT GAGACC GACGAGAT CCCCGGCAT CGT CGAGGACT ACCGCCAGGCT GCGCAGCGCGC CAAGCGT GCCGGCTT CGACAT GGT CGAGGT CCACGCCGCCAACGCCT GCCT GCCCAACCAGTT CCT CGCCACCGGCACCAACCGGCGCACCGACCAGT ACG GCGGCT CCAT CGAGAACCGGGCGCGCTT CCCGCT GGAGGT GGT CGACGCC GT GGCCGAGGT GTT CGGGCCCGAGCGGGT CGGCAT CCGCCT GACCCCCTT CCT CGAGCT CTT CGGCCT CACCGACGACGAGCCCGAGGCGAT GGCCTT CT A CCT GGCCGGCGAGCT CGACCGCCGCGGCCT GGCCT ACCT CCACTT CAACG AGCCCGACT GGAT CGGT GGCGAT AT CACCT ACCCCGAAGGCTT CCGGGAGC AGAT GCGCCAGCGCTT CAAGGGT GGGCT GAT CT ACT GCGGCAACT ACGAT G CCGGGCGCGCCCAGGCCCGCCT GGAT GACAACACCGCCGACGCCGT GGCC TT CGGCCGCCCCTT CAT CGCCAACCCCGAT CT GCCCGAGCGCTT CCGCCT G GGGGCCGCCCT CAACGAGCCCGACCCCAGCACCTT CT ACGGCGGCGCCGA GGT CGGCT ACACCGACT ACCCCTT CCT CGACAACGGCCACGACCGGCT CGG CT GAGT CAGCGT CCGCCCCT GGAAGCAT CAGCAAGCCCGGCCAGCGT GCC GGGCTT GT GGCGT GAT GGGGAGGGT GGT GCGGGGCAT CGT CGGT GATTGG CGCACAT CAACACCGCGGCGT CAGAT CCACAGAACGCATT CCGAGGGACCG CC

[000357] SEQ.ID NO: 14

TT GAAGCGCGAT GAAGAT GTT CCACGGGCAGTTT CT GAT CTTTT CCCT CCCG T CAAAATT GCCA

[000358] SEQ.ID NO: 15

T GGGACT GT GTT CTT GATGGT AAGGCGAT GAGCGGCAAGGAGGAAACAACA CACAACGAT GAAACGAGGACTTT GCGT CACCGT GAATT G

[000359] SEQ.ID NO: 16

GACTT GAT GAAGGATT ACAAGAAGTTT GATT CCAT AATT GAAGTT AAT CCAAA ACC [000360] SEQ.ID NO: 17

GCT CCCTTT GCTT AT CT AGCTT CCTT CCAT CAGAACGTT GT GGAAGTT GATT C T CACCT CT GCCTTT CT GAAT AA

[000361] SEQ.ID NO: 18

LKRDEDVPRAVSDLFPPVKIA [000362] SEQ.ID NO: 19

DCVLDGKAMSGKEETTHNDETRTLRHREL

[000363] SEQ.ID NO: 20

DLMKDYKKFDSIIEVNPK

[000364] SEQ.ID NO: 21 APFAYLASFHQNWEVDSHLCLSE

[000365] SEQ.ID NO: 22

MPDTSFSNPGLFTPLQLGSLSLPNRVIMAPLTRSRTPDSVPGRLQQIYYGQRAS AGLIISEATNISPTARGYVYTPGIWTDAQEAGWKGVVEAVHAKGGRIALQLWHV GRVSHELVQPDGQQPVAPSALKAEGAECFVEFEDGTAGLHPTSTPRALETDEIP GIVEDYRQAAQRAKRAGFDMVEVHAANACLPNQFLATGTNRRTDQYGGSIENR ARFPLEWDAVAEVFGPERVGIRLTPFLELFGLTDDEPEAMAFYLAGELDRRGLA YLHFNEPDWIGGDITYPEGFREQMRQRFKGGLIYCGNYDAGRAQARLDDNTAD AVAFGRPFIANPDLPERFRLGAALNEPDPSTFYGGAEVGYTDYPFLDNGHDRLG

[000366] SEQ.ID NO: 23

T GTTT CGT CAT CAAAGGT CGTTT GCT CCTT AACAAT CAT CCT AAT ACCCAGCT AAGACAAT AAT CAGT AT CAACT AT CAGAAAT GGCT CAT GCT CAT GGT ATTT CA GGT CT AGTTGGGAAACTT ATT ACT GAAT CGGAGGTT AACT GCAACGCT GACA AGTTTT ACCAAAT GTTT AAGCACGAT GAAAAT ATT ACAAAT AT AATT CCT CAT A T CT AT ACT AGTTT CAAGGTT GT CGAGGGAGATGGACTT ATTT CT GGTT GT ACC AAGGAAT GGGGCT AT CTTT CT GAGGGCAAAGCAAGGATT GTT AAGGAGCAAA CGACCTTT GAT GACGAAACAAGGACGAT ACAT CATT GCGCAAAAGCAGGAGA CAT GAT GAAT GATT ACAAGAAGTT CGTT CT AACACTT GT AGTT AAT CCAAAGG CT CAT G G AC AAG G AAG C AC AGT C AAGT G GATT AT AG ATT AT G AG AAG AT AAA T GAGGATT CT CCAGTT CCTTTT GCTT AT CT AT CT CT GT GCATT AAGAT CACT G AAGGT CT GAACT CT CACAT CT ACGCTT CCGAAT AGGTT AT CAAT GGAT AT GT C CACCGAT AT GTTT GT GT AT CGGCGAAT AT CAGGACT CAGT AT AT AT GGT GT GT GCT AAT GGAGTTT CT ACT AGAT CT CCT AT GAT CGACCT AAT AAAT GCGT ACGT ACTT GCAT GT AT GT GT GGT GT GTTT CATTT CGTTT CGTTTTT CAT CT ACTTT CT GTAATTTCTA

[000367] SEQ.ID NO: 24

MAHAHGISGLVGKLITESEVNCNADKFYQMFKHDENITNIIPHIYTSFKVVEGDGLI SGCTKEWGYLSEGKARIVKEQTTFDDETRTIHHCAKAGDMMNDYKKFVLTLVVN PKAHGQGSTVKWIIDYEKINEDSPVPFAYLSLCI KITEGLNSHIYASE

[000368] SEQ.ID NO: 25

T GT GCGAGGAT ACAT AATTT CT AT AT AAACT CATT CAACT AGCT AAT AATT CAT CAAAATT CAATT GAATTT AT AT GT GT CATT GTTTTT CAAACTT ACTT AT AT AT CA AGGAAAACAAT CAAT AT CAATT ACCAAAAAT GGCT CAACAT CAT ACCATTT CA GGT CTT ATT GGGAAGCTT GT GACCGAAT CAGAAGTT AATT GCGATGCT GAAA AAT ATT ACAAAAT AATT AAGCACCACGAAGAT GT ACCT AAT GCAACCCCTT AT GTTT CCGAT GT CAAAGTT ACT GAAGGACAT GGT ACCACTT CGGGTT GT GT CA AGCAAT GGAACTTT GTT GTT GCGGGT CGAAACGAAT AT GT CCTT GAAAAAAC AACAT ACAAT GAT GAAACAAGGACAAT AT GT CACAGT GACTTT GAAGGAGAC CT GAT GAAGAAAT ACAAGAAGTTT GAT GCAAT CCTT GT AGTT AAGCCAAAGGA T AATGGACAT GGT AGT AAT GT GAGAT GGACT ATT GAAT AT GAGAAGAAT AACG AGGATT CT CCGGTT CCAATT GATT AT CT AGGTTT CTT CCAAT CGTT AAT CGAT G ACTT GAACT CT CAT CTTT GCTCCT CTT AAT AATTT GG ATT GAT GAT ACGT AT C AACACCTT CT ACGT ACAGTT CGAT CGCTT AT GT GGGT AT GT ATTT GT GT GAT A AT AAAT AGT AT GT GGATTTTT CACAAT AT AT ACAAT AAT GT GCAT ACAT GCACG T GT GATTT GT CTT ATTT ATTTT CATT AT CATTTTTT GT CAT GTTTT AAGGCGT AT AATATG

[000369] SEQ.ID NO: 26

MAQHHTISGLIGKLVTESEVNCDAEKYYKIIKHHEDVPNATPYVSDVKVTEGHGT TSGCVKQWNFVVAGRNEYVLEKTTYNDETRTICHSDFEGDLMKKYKKFDAILVV KPKDNGHGSNVRWTIEYEKNNEDSPVPIDYLGFFQSLIDDLNSHLCSS

[000370] SEQ.ID NO: 27

CTT CCACACACT CTT GT GCAAACTT CT AT ACT ACAT AT CT ACAAT CAACAACT A T AT CAAT GGCT AGTT AT GATT AT GGT CTTT CCGGT CT AATT GGGAAATTT AT AA TT CAATT G GAGAT CAAT AG CGAT GCT GACAATTTTT AT GAAAT CT AT AAG CATT GCAAAGAT GTT CCT AAGGCAGTT CCT CAT CTTTT CACT GGT GTT AAAGTT ACC AAAGGAGAT GAACT CGTTT CT GGTT GT AT CAAGGAAT GGAACT AT GTT CTT GA GGGT AAGGCGAT GACCGCT GT G GAG GAAACAAC AATT GACGAT GCAACAAG GACCTT GACACACCACGT AATT GAAGGAGACGT GAT GAAGGATT ACAAGAAG TT CGAT GT GATT ATT GAAGCT AAT CCGAAGCCT AGT GGACAAGGAACCATT G GAGGAAGCATT GT GACT GT GT CT ATT GT AT AT GACAGAAT GAATGCGAAGT C T CCAGCT CCCTT CGATT ATT ACAAATT CT ATT AT CAGAACAT AGT AGAT AT GGA TGCT CACAT CT CCACTT CTT AGT AAACT AT CTT AAT CT CCGT GTT GGGT GTGC GTAT GCAT GT GCAT AT GT ACGT CAGT ACT CGTT GAT CAATTT GTAT GCGTT AC TT CACGAGAT CT ATT GCAT CT CT AT AACT AT GT AT CATTTT AAAT AAAT GGAGT AAGTT ATTT AAAAT AAAAAAAA

[000371] SEQ.ID NO: 28

MASYDYGLSGLIGKFIIQLEINSDADNFYEIYKHCKDVPKAVPHLFTGVKVTKGDE LVSGCIKEWNYVLEGKAMTAVEETTI DDATRTLTHHVI EGDVMKDYKKFDVIIEAN P KPSGQGTI GGS IVTVS I VYDR M N AKS PAPFDYYKFYYQN I VDM DAH I STS

[000372] SEQ.ID NO: 29

ATT GAT GAT GGTT GAT ACT GGTT CATT CACAAT CAT CCT AAT ATT AATT AGTT A AGGCAAGAACCAGT AT CAACCAT CAT CAAT GGCCCAT CAACAT ACAATTT CAG GT CTT GT GGGAAAACTT ATT ACT GAAT CGGAGGTT AACT GCAAT GCCGACAA GT ATT ACCAAAT ATTT AAGCACCAT GAAGACCTT CCAAGCGCAAT CCCT CAT A TTT ACACT AGCGT CAAAGCT GT CGAGGGACAT GGAACT ACTT CT GGAT GT GT CAAGGAGT GGT GCT AT ATT CTT GAGGGGAAACCACTT ACAGTT AAGGAGAAA ACAACGT ACAAT GAT GAAACAAG AACGAT AAAT CAT AAT GG AAT AG AAGGAG GCAT GAT GACT GATT ACAAGAAGTT CGTT GCAACACTT GT AGTT AAGCCAAAA GCT AAT GGGCAAGGAAGCAT CGT GACAT GGAT AGT GGATT AT GAGAAGATT A AT GAGGATT CT CCAGTT CCTTT CGACT AT CT AGCTTT CTT CCAACAAAACAT C GAAGACTT GAACT CT CACCT CT GT GCTT CT GATT AAATT AT CAAT GGGT AT GT CCAT AT GCAACGAT GAACAT CAGT GTT CT CT GT AT GAT AAT AAAGT CT AT AT G TGGA

[000373] SEQ.ID NO: 30

MAHQHTISGLVGKLITESEVNCNADKYYQIFKHHEDLPSAIPHIYTSVKAVEGHGT

TSGCVKEWCYILEGKPLTVKEKTTYNDETRTINHNGI EGGMMTDYKKFVATLVVK

PKANGQGSIVTWIVDYEKINEDSPVPFDYLAFFQQNIEDLNSHLCASD

[000374] SEQ.ID NO: 31

GCTTT ATTT AT AT GCCGGCCCT CAAT AACCAAAGACACT CGAT GCAT CTGTGC AGT ACAT AT AT AT ACGT ACT GCATT ATT AAGACACACAAACCAACAGCTT CATT T GT CT CT CGAGT AGT AACAAT CAAT AT CAT CAATTTT CAT CCAT GGCT CAT CC CCAT CCT ATTT CAGGT CT AGTT GGGAAACT AGT GACT GAATT GGAGGTT AACT GCGACGCT GACAAGT ATT ACAAAATTTTT AAGCACCAT GAAGAT GTT CCAAAA GCAGT ACCT CAT AT GT ACACT AGCGT CAAAGTT GT CGAGGGACAT GGAATT A CTT CT GGTT GT GT CAAGGAAT GGGGTT AT CTT CTT GAGGGAAAAGAACT GAT T GT CAAGGAAACAACAACAT ACACT GAT GAAACAAGGACGAT ACAT CAT AGC GCAGT AGGAGGACACAT GACGAAGATTT ACAAGAAG TTT GAT GCAACGCTT G T AGT CAAT CCAAAGCCT AGT GGCCAT GGAAGCACGGT GAGTT GGACT ATT GA TT AT GAGAAAATT AACGAGGATT CT CCCGTT CCT ATT CCAT AT CT AGCTTT CTT CCAT AAGCT CAT CGAGGACTT GAACT CT CACCT CT GCGCTT CT GATT AAAGAA ATT ATT GATTT ATT GTT CT CGAT GGACAATTT CAGCT GTT GGTTT GT GT GT GTT AAT AAT GCAGT ACGT AT AT AT AT GT ACT GCACAGAT GCAT CGAGT GCTTTT GG TT ATT GAGGGCCGGCAT AT AAAT AAAGCCACT CCT ACT CAAGT ATTT

[000375] SEQ.ID NO: 32

MAHPHPISGLVGKLVTELEVNCDADKYYKIFKHHEDVPKAVPHMYTSVKVVEGH GITSGCVKEWGYLLEGKELIVKETTTYTDETRTI HHSAVGGHMTKIYKKFDATLW NPKPSGHGSTVSWTIDYEKI NEDSPVPIPYLAFFHKLIEDLNSHLCASD

[000376] SEQ.ID NO: 33

TTT GACCGGCAGTTT AT GAT AAT CT CACCAGCAGT AGAT ACTT AT GAT AGGT A ATT GGCCACT AAACAAAT GCTT GTTTTT CT AT AT AAACT AGAT CAACTTT CATT GAAATTT AT CAT CAACT GCT CCAGCAATT AT AGTT CT CT ACAAACTT CAAT AT A T AGGGCAACAAT CAT CAACCAT CT AT GGCGCAT CAT GGT GTTT CAGGT CT AG TT GGGAAACTT GT AACT GAATTGGAGGT CCATT GCAAT GCT GACGCAT ACT AT AAAAT CTTT AAGCACCAAGAAGAT GT ACCAAAGGCAAT GCCT CAT CTTT ACAC T GGCGGGAAAGTT AT CAGT GGAGAT GCAACCCGTT CT GGTT GT AT CAAGGAA T GGAACT ACATT CTT GAGGGT AAGGCGCT GAT CGCAGT GGAGGAAACAACA CAT GACGAT GAAACAAGGACCTT AACACACCGCAT AACT GGAGGAGACTT GA CAAAGGATT ACAAAAAGTT CGTT AAGAT CGTT GAAGTT AAT CCAAAGCCT AAT GGACAT GGAAGCATT GT GACT GT AT CCCTT GT GT AT GAGAAAAT GAACGAGG GTT CT CCAACT CCCTTT AATT AT CT ACAATTT GT CCAT CAGACCATT GT AGGCT T GAATT CT CACAT CTGCGCTT CTT AGT AAAAT ACAT CCGAACTT CAGCGTTGG GTTT AAGT AT GCACGT ACGAT CGT CGGT ACTT GTT GTTT AATT AGTT GT ACT G T ACGTT ATT CCT ACACACT GCACT AT CAT GCCT AT GT AT GTTT GATT AAAT AAG ACT AT GGAACT AT GGGATTT AT CAT AT GCGAT GAT CCTTTT GAAT AAAT CAAAT AAGT CATTT AAAAT GT GTTTTTTTTTTT CT CTTTT CT

[000377] SEQ.ID NO: 34

MAHHGVSGLVGKLVTELEVHCNADAYYKIFKHQEDVPKAMPHLYTGGKVISGDA

TRSGCIKEWNYI LEGKALIAVEETTHDDETRTLTHRITGGDLTKDYKKFVKIVEVN

PKPNGHGSIVTVSLVYEKMNEGSPTPFNYLQFVHQTIVGLNSHICAS

[000378] SEQ.ID NO: 35

GCAT CACAAATT AAACCAACGAGAT CAGCGACT ACACT AT AAT AT ACT GCAAT T ATT AGGAAAAT GAGGT ACGAGTTT AT AAACGAGTTT GAT GCACAT GCAT CAG CAGACGAT GTTT GGGGAGGAAT CT AT GGCT CCATT GATT ACCCT AAACT AGT GGTT CAATT ACTT CCAACT GT CCT CGAAAAGAAGGAAAT CTT GGAAGGCGAT GGT CAT AAT GTT GGT ACT GTT CT GCAT GTT GT GT ACCTT CCAGGATTT GTT CC GCGGACGT ACAACGAGAAGATT GT AACGAT GGAT CACAAAAAACGTT ACAAG GAAGT ACAAAT GGTT GAAGGAGGAT ACTT GGAT AT GGGATTT ACAT AT GT CAT GGT AATT CAT GAAGT ACT AGCAAAAGAAT GT AATT CTT GT AT CATT AGAT CAAT T GTT AAGT GT GAAGT CAAGGAT GAATTT GCT GCAAAT GTTT CT AAT ATT CGCA ACACCTTT GAT GGAT AT GT CGCCTT AGCCCGAGCCGTT CCGGAAT AT ATT GC GAAGCAGCACGCAACAT CAGCAGCT AATT AACTT GCT GCCGCAGTT AAT AAA T GGATTTT CGAT GGT CT AAAT AAT AT GGAACT GGAT AAAGT ACCT AGGACT GA GATT ACT GTTT CCTT CCT AT GTT ATT CCT CTT GT GAT CTT CTTTT CT CT CTTT CT AT GTTTTT GT GCTTT AT CTTTT

[000379] SEQ.ID NO: 36

MRYEFINEFDAHASADDVWGGIYGSIDYPKLWQLLPTVLEKKEILEGDGHNVGT

VLHWYLPGFVPRTYNEKIVTMDHKKRYKEVQMVEGGYLDMGFTYVMVIHEVLA

KECNSCIIRSIVKCEVKDEFAANVSNIRNTFDGYVALARAVPEYIAKQHATSAAN

[000380] SEQ.ID NO: 37

T AGAAAATTT AGGGGGGGCT AGT GACCCCACT GACCCCAGT GT AGATT CGC CACT GAT AT CAGTT GCT CT AAT CTT CCAT ACT GCCAT AT ATTT CT GT GCAAATT T CAGGAT AACAAT CTT GAACT GTT GAT GGCT CAT CACGGT GTTT CAGGT CT AG TT GGGAAACTT GT AACT CAATT AGAGGT CAATT GT GAT GCT GACGAATTTT AT AAAATTT GGAAGCACCAT GAAGAAGTT CCAAAGGCAGTTT CT CATTTTTT CCC T GCCGT CAAAGTT GT CAAAGGAGAT GGACTT GTTT CT GGTT GT AT CAAGGAA T GGCACT AT AT CCT CGAGGGT AAGGCGAT GAGCGCAAT GGAGGAAACGACA CACAAT GAT GAAACAAGGACTTT ACAT CACCAGGT AGTT GAAGGAGAAGT GA T GAAGGATT ACAAGGCGATT GCTT CCAT AATT CAAGTT AAT CCAAAT CCAAAT GGACAT GGAAGCATT GT GACGT GGT CAATT GAGT AT GAGAAAAT GAACGAAG ATT CT CCAACT CCCTTT GCTT AT CTT GAATT CTT CCAT CAGAACAT AAT CGAT A T GAATT CT CACCT CT ACGT AGGCT CT GATT CT CACCT CCACGTT GAT GAAT AA AAT GT CATT ACCGT AAGT ACAT GAACGCGGCTTT AGT GTTT GAT GT ACGT CAG T AT GT GCT GTTT GAATT GAT CAGTTT CCT GT GTT ATT CTT ACTT GAAT CAGTT G CTT AT GCT AGT CTT GCAGT ATGCCT GT GT CT ACGT GCCT GT GTTT CAT AAT AA T AAAGGCT AAGAGCACTT GCAAGTT AT AATT CT CTT CTTT AT AT CCCTTTT CCT AT GGT GT ATT CT GTTT AAT CAAGTT CT GTTTT CT CT AGCACAAGGGTTT CCAC AAATT AT CT CAGTT ACCCT GAATT ATTTTTT CTT AATT GCAAAT GT AAAAGGT A CT AAAAGG AG AATT ACT AGT ACCT AGT AGT CGT AACCCAAT CAATT G AGCCAA ATTT GATGCCT AT AAT AT GCGAT AAT GT AGCT AAGAAAGCTTT CT GAAT CAAC AGT AT AT AT AT ATT GTT GCGGT GT CAACT CCT ACTT CTTTT ATT AGAGTT AGTT T ATT ACCTT ATT ATTT GTTTT CCGT ACGT ACTT AACATT CAGTTT CCT AGTTT AT AAGATTT CTT CAGT GAGTT GCTT GCTT ACCAAGTTT ATT CAGCT AT AT AT AGCT CGAT CCT AGCTT GT AACAGGACAAATT AT CAAT AT AAGAAGTT

[000381] SEQ.ID NO: 38

MAHHGVSGLVGKLVTQLEVNCDADEFYKIWKHHEEVPKAVSHFFPAVKWKGD

GLVSGCIKEWHYILEGKAMSAMEETTHNDETRTLHHQWEGEVMKDYKAIASIIQ

VNPNPNGHGSIVTWSIEYEKMNEDSPTPFAYLEFFHQNII DMNSHLYVGSDSHLH

VDE

[000382] SEQ.ID NO: 39 ACT GT AACGT GCAAGT CCGCAT AGT CTT ACTT ATT CAAACATTT AT AT AAACC CAT AGCCCT AAGCAT AT AGaAT CAaT aT CAACT GCT AAGGT CTT CCAAAATT CT AT AT ACTTTTT CAGCAACAAACT GTT AAT GGCT CAT CAT GGCGTTT CT GGTTT A GTT GGGAAACTT GT AACT CAATT GGAGGT CAATT GT GAT GCT GAT AAATT GT A T AAAAT CT AT AAGCACCAT GAAGAT GTT CCAAAGGCAATTT CT CAT CTTTT CAC CGGT GT AAAAGTT CT CGAAGGACAT GGACTT CGTT CT GGCT GT AT CAAGGAA T GGAAAT AT ATT ATT GAT GGT AAGGCGTT GACT GCT GT GGAGGAAACAACCC AT GGCGAT GAAACAAGGACTTT AAAACAT CGCGT CATT GAT GGAGACTT GAT GAAGGATT ACAAGAAGTT CGACAAGAT CATT GAAGCT AAT CCAAAGCCAAAT GGACAT GGAAGCATT GT GACT GT CT CT CTTTT GT AT GAGAAGAT AAAT GAGG ACT CT CCAGCT CCGTTT GAT CAT CT CAAATT CTT CCAT CAAAACAT AGAAGAT AT GAATT CT CACAT CT GCGCTT CAGAGT AAAAT AT CT CAT CTT CATT GTT GGG T GT ACGT AT GCGTT CAGT AAGT CAGT GCTT GAGAAATT AGTT GT GT GCGTT AT T CCAGT CAGT GTTTT GT GT AAGT AGTT GGAAT GTT GGAT GCGTT ATT CCT ACA GTGTGCTAT AT GCTT AGGGCT AT GGGTTT AT AT AAAT GTTT GAAT AAAAGT AA AAAAACT AAAAAGAGACT AGCCAAAGGCACACAGGGGAT AGN AACAAAT AAA TTTAAA

[000383] SEQ.ID NO: 40

MAHHGVSGLVGKLVTQLEVNCDADKLYKIYKHHEDVPKAISHLFTGVKVLEGHG

LRSGCIKEWKYII DGKALTAVEETTHGDETRTLKHRVIDGDLMKDYKKFDKII EAN

PKPNGHGSIVTVSLLYEKINEDSPAPFDHLKFFHQNIEDMNSHICASE

[000384] SEQ.ID NO: 41

AGT ATTT CAT AGTT CCAT AT ACTT GT GT GCAAT GGCT CAT CAT GGT GTTT CAG GT CT AGTT GGGAAACTT GTT ACT CAGCT GGAGGT CAATT GCGAT GCAGACAT ATTTT AT AAAAT CGTT AAGCACCAT GAAGAAGTT CCAAACGT AATT CCT CATTT TTT CACCGGCGTT CAAGT GACCAAAGGAGATGGACTT GTTT CT GGTT GT AT C AAGGAAT GGAACT AT GTT CTT GAGGGT AAGGCGAT GACCGCT GT GGAGGAA ACAACCCACGCCGAT GAAACAAGGACCCT AACACACCACAT AACT GAAGGAG ACGCG AT GAAAGATT ACAAG AAGTTT GAT GT GAT CGTT G AAACT AAT CCAAAG CCT AAT GGACAT GGAAGCGTT GT GACAT ATT CT ATT GT GT AT GAGAAAAT CAA T GAGGATT CT CCAGCT CCCTTT GATT AT CT AAAATT CTT CCAT CAGAACAT AG T AGACAT GAGT GCT CACAT CT GCT CTT CTGCAT AAT AT ACCAAT GAACTT CAG T GTT GTT GCGT GGACGT ATT CACGT GAAAAT GAACGT CGGT GCTT GCT GTT C AATTT GT GT GCGTT ATT CCTT CACT AT GAT GAT GT CT AT GGAT GTTT GGTT AAA T AAGACTT GT GT GT GGACT AT CGGAT CT ATT GCAT CT CT GCT GAT CTTTTT AA AT AAAACAT ACAGT AT AAAAT ATTT AATT AGTT GCGCCTT GTT AGT CT GT GACT CCCAT AT CCAAAAT CT ATT ATT GT GATTT AAAACTT GCGAACT GAT CAAAAAT C T AT ATT GT GCCAAAAATTT AAT ACTT ATT GGAAAC

[000385] SEQ.ID NO: 42

MAHHGVSGLVGKLVTQLEVNCDADI FYKIVKHHEEVPNVIPHFFTGVQVTKGDGL

VSGCIKEWNYVLEGKAMTAVEETTHADETRTLTHHITEGDAMKDYKKFDVIVETN

PKPNGHGSWTYSIVYEKINEDSPAPFDYLKFFHQNIVDMSAHICSSA

[000386] SEQ.ID NO: 43 TT GCCCT AAAT ACAGCT CATT AT CCAGT CACCACCCTTT AT CATT CCT GT AGT TT CT GGTT GTTT CT AT AT AAACT CGTT CAGCT AAGACT AATTTT CAT CGCAAT C ACACT CAT CCT AAT ATT CAGCT AAGGAAACCGT AAGT AT CAACTTTT AGCAAT GGCT CAT ACT CGT GGT ATTT CAGGT CT AGTT GGGAAACTT GTT AT GGAAACG GAGGTT AACT GCAACGCT GACAAGT ATT ACCAAAT AT AT AAGCACCAT GAAGA T CTT CCAAGCGCAAT CCCT CAT ATT GT CACT AGCGCCAAAGCT GTT GAGGGA CAT GGAACT ACTT CTGGTT GCGT CAAGGAGT GGGGCT AT AT GCAT GAGGGT A AAACACTT ACTT GCAAGGAGAAAACT ACCT AT AACGAT GAAACAAGGACGAT A T GT CAT AGCAT AT CT GAAGGAGACTT GAT GAAT GATT ACAAGAAGTT CGAT GC AACACTT GT CGTT GAT CCAAAGGAT AATGGACAT GGAAGCATT GT GAAGT AT A TTTT AGATT AT GAGAAGAT AAAT GAGGATT CT CCGGTT CCT ATT CATT AT CT AG CT CT GT GCAAT CAAGCCACCGAAGACTT GAACACTT ACCTTT GT GCTT CT GT C T AAGTT AT CAAT GGAT AT CT CCGCCGAAT AAAT AT GCAAGT AT GAAT ACCACT GTT CT ACTT CT AT CAGT GGTATCT AAT AAT AAAGT CT AT AT GT GGAATTT CCAC T AGACCT ATT GT CT AT AAT AAAT GCTT CCAT ACTT GT ACGT ACCT GTT GTTTT C TT CATTT CTTTTT GTT AT GGAGT ACT GTTTTT CGT CT ACT AT CTTTT ATTTTT AC T GAAAAT CAAGGCGT AAT AAT A

[000387] SEQ.ID NO: 44

MAHTRGISGLVGKLVMETEVNCNADKYYQIYKHHEDLPSAIPHIVTSAKAVEGHG

TTSGCVKEWGYMHEGKTLTCKEKTTYNDETRTICHSISEGDLMNDYKKFDATLV

VDPKDNGHGSIVKYILDYEKINEDSPVPIHYLALCNQATEDLNTYLCASV

[000388] SEQ.ID NO: 45

GTT CGAT AT AT AT ACTT CT GTT CAT ACTT CAGGGCAACAAT CGT CAACT GT CA AT GGCT CGT CACGGAGGTT CAGGT CT AGT AGGGAAACTT GT AACT GAACTGG AGGT CT ACTGCGAT GCT GACAAAT ATT AT AAAAT CT GGAAGCACCACGAAGA T GTT CCGAAGGCAAT GCCT CAT AT GTT CACT GGT GT CCAACCT AT CAAAGGA GAT GGAAT CT GTT CCGGCAGCAT CAAGGAAT GGAACT AT AT CATT GAAGGT A AGGCAAT GAGAGCT AT GGAGGAAT CAACACAT AACGAT GAAACGAGAACAAT AAGT CACCGT GTT GT AGAAGGAGACCT GCT GAAGGATT ACAAGAAGTTT GAA T CGAT AAAT GAAAT CAAT CCT AAGCCT AACGGAAAT GGAT GCGT CGT GACAT GGACT ATT GCAT AT GAGAAAAT CAAT GAGGATT CT CCAACT CCCTTT GCAT AT AT ACCTTT CGT CCAT CAGGCCATT GAAGACACGAACAAACAT CTT GCT GGTT C CGAGT AAAT GGT CT ACGCCGT CT AT ACAT GAAT AACCCGATT CT CCGT CGGG GGT ACGT AT GCT CAT GCACGT ACATTT ATT AAT CAGTT GAAGTTT AT GT GGGT T ATT GTT GCAGT AT AT GCCT AAATGGCCATTT CGGCCT AT ATTT GTT GT CATT GTT CT GT CAGT AACT ACCT AGTTT GGT GT GT ACT CT CATT AGAGAGAAACT AA AT GT ACCAACT ATTT GAT GATTT G AATTTT CTTT CCT GAT AAAAAAA

[000389] SEQ.ID NO: 46

MARHGGSGLVGKLVTELEVYCDADKYYKIWKHHEDVPKAMPHMFTGVQPIKGD GICSGSIKEWNYIIEGKAMRAMEESTHNDETRTISHRWEGDLLKDYKKFESINEI NPKPNGNGCVVTWTIAYEKINEDSPTPFAYIPFVHQAI EDTNKHLAGSE

[000390] SEQ.ID NO: 47 CTT CAAT AAT CT CCAAT CT ATT GAGCAAAAAT CCT CAACT ACTT GAT GGCT CAT CAT GGT GTTT CGGGTTT AGT CGGGAAAGTT GT AACT GAATT GGAGCT CAATT GCGATGCT GACGAAT ACT AT AAAGT CT AT AAGCACCAT CAACT AGT ACCAAAT GAGGCAGTTT CT CAT CTTTT CACT GGT GTT AAAGCT CTT GAAGGAGGAGACG GCCT CAGT CCCGTT CAT AT CAAGGAAT GGAGCT AT ATT CTT GAGGGAAAGAC AAT GACCGCCGTGGAAGAAT CAACAT AT GACGAT GAAACAAGGACCAT AT CG CACCGCAT CGTT GAAGGAGAT GTT AT GAAGGATT ACAAGAAGTTT GAT GAGA T CGTT GT AGCT AAACCAAAGCCT GAT GGACAT GGAAGCATT GT AT CCAT AT CT AT AAT GT AT GAG AAAAT AAACG AGG ATT CT CCAACT CCATTT G ACAT CCT GAA AACTTT CCAT CAGAACATT CT AGACCT AAGT GCT CACAT CT GT GCTT CCGAGT AAAAT AT CT CT CAAGT GTTT GGGTGTT ACTT GTTGT CATTT ATGTGT GCGTT AT T CAT GCAT GGACT AT GCAT GGCTTT GT AACCGCAGTTT AT CGCTT CTTT GAT C AT CTTTTTTT CTTTTTTT AT ACTT CTTTTTT AAAGAAGTTTT GGCT CT AT GT CCG T CCCTT GCT ATTTT AATTTTTT GTT CTTT GAT CAGT AAAT ATTTTT GCTT AAAAA AAAAACAAT CAT GAGCT ACGT CT AT GT

[000391] SEQ.ID NO: 48

MAHHGVSGLVGKVVTELELNCDADEYYKVYKHHQLVPNEAVSHLFTGVKALEG

GDGLSPVHIKEWSYILEGKTMTAVEESTYDDETRTISHRIVEGDVMKDYKKFDEI

VVAKPKPDGHGSIVSISIMYEKINEDSPTPFDILKTFHQNI LDLSAHICASE

[000392] SEQ.ID NO: 49

CAACAACT AT CAGCT GGT CCAGCT GACCACT GTTT CT GCT GAT GAACCTT AA GGAACAAATTT CAGCAGAGAGAT ACAAGCT GAGGGT GTT AT CAAAT ATT CGA CGT AGGAAGCGCT AT CAAACCT GACTTT CAT CCTT GAAATT AGAAGTT CAAGT CT CCGCTTT AAG AAT AATT AGT GAT CCATT CAAAACTT CAACCGT GTCT CCAA AT GT CGCATT GT AT GT GT CACCACAGT GAATTT CAGGT AAAATTTT CAAAT AA GACT GTT GAT AGGATT CT CCT AAAAGGGGATT CAGAAAGT AT GACT GTT AGAT CAAGAAATTT CCAAAAT AGAAAGGT CAACTT AT AATTT CT AGT GGTT AT AT CTT AT CGCAAT ACT AGT GGT GGAAAGGT CAACTT AT AATTT CTGGGT GGAT CT GG TT GAACGAT CGTT GCCGCCAATT ACAGCT CACT ATT GAGT CAT CAGT AGTT CA GCACAATTT CAT CAATT CATT CCT GT AGTT GCAGGTT GTTT AATT CT AT AT AAG CT CAT GAAAT AT CAGT ATT CAT CT AAG GC AAG AAT CAGT AT CAACT AT CAGCA AT GGCACAT CACT ATT CCACTT CCGGT CT AGTT GGGAAACTT GTT ACT GAAAT GGAGGTT AACT GCAACGCCGAAAACT ATT ACCAAAT ATTT AAGCAGCAT GAA GGCGTT CCAAAAGCAAT ACCT CAT ATTTTT ACGAGCAT GAAAGTT CTT GAGGG ACAT GGACTT ACTT CCGGTT GT AT CAAGGAAT GGCACT AT CTT CAT GAGGGA AAAGCACT CAAATT CAAGGAGACCACGACAT AT AACGAT GAAGAAAGGACGA T AT GT CACAGCGTT AT AGGAGGT GACTT GTT GAAT GATT ACAAGAACTT CAGT GCGACACTT CT GGTT AAGGTT AAGCCT ATGGGT CAT GGAACT ACGT ACCT GG CT CCGCCAGT GCAGCCAGCT CCCAAGCAACATTTT AGCCAACCAGCCCAGC CGGCAT CCAAGCAT CAT CATTTT AGCCTT CAT AGGCCT CATTT AAACCAACCA GCACAGCCAGATT CCAAGCAT CAT CTT AGT CTT CAT AGGCCT CATTT AAACCT TT GCAAGACCATTT CACACT GCCCACT GACCGGCCGT GT CTT GGGT GT GCAA GATT CTT CCCCACCT GCT CCT ACCT ACGTGGCT CCGCCAGTT CCT ACAT ACG TGGCT CCGCCCAT GCAT GGAAGCACT GT GAT GT GGATT AT AGATT AT GAGAA GAT CAAT AAGGATT CT CCAAT CCCCGTT CCTT AT CT GGCTTT CTT CCAT CAGA T CATT GT AGACTT GAACT CT CACTT CT CCGCTT CTT ATT AAATT AT GGAT AGAT AT GCAT GCCCACGGATTT AT AT AT AT GT AT GCAACGACAAT CACAGT GT CTT G T GT ACGAT AT AT GT GT GGAAAT AAAAAACT AT AT AT AAACGT GGT CAT GCCAA AAAT CT ATTTT CGGCCT AAT CAAGGCTTT ACTT ATT GCAT GT GT AT GAGT GGT TT GTTT CAT AAT ACT AGGGCAT AT GAAT AAGT GAAT GAATT AT GT AAGT CATTT GGATT GATT CTTT CAT GTT AT AAT AAGAT AAAAAACAAACATT CAGAAT AAT GT CGGCAT CGT AT GGGCCGCGACAGT GCAT CACT AAG

[000393] SEQ.ID NO: 50

MAHHYSTSGLVGKLVTEMEVNCNAENYYQIFKQHEGVPKAIPHIFTSMKVLEGH GLTSGCIKEWHYLHEGKALKFKETTTYNDEERTICHSVIGGDLLNDYKNFSATLL VKVKPMGHGTTYLAPPVQPAPKQHFSQPAQPASKHHHFSLHRPHLNQPAQPDS KHHLSLHRPHLNLCKTISHCPLTGRVLGVQDSSPPAPTYVAPPVPTYVAPPMHG STVMWI I DYEKI N KDSPI PVPYLAFFHQI I VDLNS HFSASY

[000394] SEQ.ID NO: 51

CGAT CAT CCAAAT ATTT AGCT AAGCCAACAATT AT AATT CAGT AT CAAT CACT A T AT CACT AT CAGCAAT GGCT CAGCCT CAAT GT ATTT CAGGT CT AT CT GGGAAA CTT GT GACT AAAT CAAAT GTT AACT GCGGT GCCAACGATTTTT ACACAATTTTT AAGCAGCAT GT AGAT GTT CCGAAAGCGAT ACCT CAAATTT ACAAGT GCGT GA AAGTT GTT GAGGGAGAT GGAACT ACTT CCGGTT GT AT CAAGGAAT GGGGAT A CCATT GT GAGGGT AAGGAACT GATT GT CAAGGAGAAAACGACAT ACACCGAT GAAACAAGGACGAT AT GT CACT GCGT AGT AGGAGGAGACAT AGCAAAT GAGT ACAAGAAATTTT AT GCAATT CTT GT GGTT AAT CCAAAGCCTT GT GGT AAT GGA AGCATT GT GAGTT GGACT GTT GATT ACGAGAAGATT AAT AAGGATT CT CCAAT T CCT ATT CCTT AT AT AGCT CT GTT CGCT CGGGT CATT GAAGGCTT GGACT CCT ACCT CT GCGCTT AT GCTT AAATT AT CGAT GGGTTT GCAT AT AT AT CAT GGGAG AAT CCT AGGGT CCT GCAGAT GGT AAT AAAACT AT AAAT AAGCGT GGACTT GC CAAGATT CT ACCTT CT ACCTT AT CAT CAAGGCTT ACAT ATT GCAT GT CT GCGT GGTTT GTTT CGT ATT AAAT GAAT AAGTT AATT AT CCAAGT CGTTT GGAGT GTTT CT ACT GTTT AT AAT GAACAAGT GATTT AT CT AAGT CGTTT GAATT GTTT GTTTT T GTTT ATT AAATT CT CAT CATT AAC

[000395] SEQ.ID NO: 52

MAQPQCISGLSGKLVTKSNVNCGANDFYTIFKQHVDVPKAIPQIYKCVKWEGDG TTSGCIKEWGYHCEGKELIVKEKTTYTDETRTICHCWGGDIANEYKKFYAILVVN PKPCGNGSIVSWTVDYEKINKDSPIPIPYIALFARVIEGLDSYLCAYA

[000396] SEQ.ID NO: 53

AT GGCT CAT CAT GGT GTTT CAGGT CT AGTT GGGAAAATT GT AACT GAATT GGA GGT GAATT GT AAT GCCGACGAATTTT AT AAGATTTT GAAGCGCGAT GAAGAT G TT CCACGGGCAGTTT CT GAT CTTTT CCCT CCCGT CAAAATT GCCAAAGGAGAT GGACTT GTTT CT GGTT GT AT CAAGGAAT GGGACT GT GTT CTT GAT GGT AAGG CGAT GAGCGGCAAGGAGGAAACAACACACAACGAT GAAACGAGGACTTT GC GT CACCGT GAATT GGAAGGAGACTT GAT GAAGGATT ACAAGAAGTTT GATT C CAT AATT GAAGTT AAT CCAAAACCAAAT GGACAT GGAAGCATT GT GACGT GGT CAATT GAGT AT GAGAAAAT GAACGAAGATT CT CCGGCT CCCTTT GCTT AT CT A GCTT CCTT CCAT CAGAACGTT GT GGAAGTT GATT CT CACCT CT GCCTTT CT GA ATAA [000397] SEQ.ID NO: 54

MAHHGVSGLVGKIVTELEVNCNADEFYKILKRDEDVPRAVSDLFPPVKIAKGDGL

VSGCIKEWDCVLDGKAMSGKEETTHNDETRTLRHRELEGDLMKDYKKFDSI IEV

NPKPNGHGSIVTWSIEYEKMNEDSPAPFAYLASFHQNVVEVDSHLCLSE

[000398] SEQ.ID NO: 55

CTT CAAT AAT CT CCAAT CT ATT GAGCAAAAAT CCT CAACT ACTT GAT GGCT CAT CAT GGT GTTT CGGGTTT AGT CGGGAAAGTT GT AACT GAATT GGAGCT CAATT GCGATGCT GACGAAT ACT AT AAAGT CT AT AAGCACCAT CAACT AGT ACCAAAT GAGGCAGTTT CT CAT CTTTT CACT GGT GTT AAAGCT CTT GAAGGAGGAGACG GCCT CAGT CCCGTT CAT AT CAAGGAAT GGAGCT AT ATT CTT GAGGGAAAGAC AAT GACCGCCGTGGAAGAAT CAACAT AT GACGAT GAAACAAGGACCAT AT CG CACCGCAT CGTT GAAGGAGAT GTT AT GAAGGATT ACAAGAAGTTT GAT GAGA T CGTT GT AGCT AAACCAAAGCCT GAT GGACAT GGAAGCATT GT AT CCAT AT CT AT AAT GT AT GAGAAAAT AAACG AGG ATT CT CCAACT CCATTT G ACAT CCT GAA AACTTT CCAT CAGAACATT CT AGACCT AAGT GCT CACAT CT GT GCTT CCGAGT AAAAT AT CT CT CAAGT GTTT GGGTGTT ACTT GTTGT CATTT ATGTGT GCGTT AT T CAT GCAT GGACT AT GCAT GGCTTT GT AACCGCAGTTT AT CGCTT CTTT GAT C AT CTTTTTTT CTTTTTTT AT ACTT CTTTTTT AAAGAAGTTTT GGCT CT AT GT CCG T CCCTT GCT ATTTT AATTTTTT GTT CTTT GAT CAGT AAAT ATTTTT GCTT AAAAA AAAAACAAT CAT GAGCT ACGT CT AT GT [000399] SEQ.ID NO: 56

MMAHHGVSGLVGKWTELELNCDADEYYKVYKHHQLVPNEAVSHLFTGVKALE GGDGLSPVHIKEWSYILEGKTMTAVEESTYDDETRTISHRIVEGDVMKDYKKFDE IVVAKPKPDGHGSIVSISIMYEKINEDSPTPFDILKTFHQNILDLSAHICASEYPYDV PDYA

[000400] SEQ.ID NO: 57

AT GG ATT CT ATT AATT CTT CCAT AT ACTT CT GT GCAT ATTTT AGAG AACT AAT C AT CAAATT GTT GAT GGCT CCT CTT GGT GTTT CAGGTTT AGT CGGGAAACTTT C AACT GAATT GGAGGT CGATT GCGATGCT GAAAAAT ATT AT AACAT GT AT AAGC ACGGAGAAGAT GT GCAAAAGGCAGTT CCT CAT CTTT GCGTT GACGT CAAAGT T AT CAGT GGAGAT CCGACCAGTT CAGGTT GT AT CAAGGAAT GGAAT GTT AAC ATT GAT GGT AAGACGATT CGCT CAGT AGAGGAAACAACACACAAT GAT GAAA CGAAAACGTT GCGT CACCGT GT ATTT GAAGGAGACAT GAT GAAGGATTT CAA GAAGTTT GAT ACGAT AAT GGT AGT CAAT CCAAAGCCGGAT GGAAAT GGAT GT GTT GT GACACGGT CAATT GAGT AT GAGAAAACCAACGAGAATT CT CCGACT C CCTTT GATT AT CT ACAATT CGGCCAT CAGGCCATT GAAGACAT GAACAAAT AC TT ACGCGATT CT GAAT AA [000401] SEQ.ID NO: 58

MDSINSSIYFCAYFRELIIKLLMAPLGVSGLVGKLSTELEVDCDAEKYYNMYKHGE DVQKAVPHLCVDVKVISGDPTSSGCIKEWNVNIDGKTIRSVEETTHNDETKTLRH RVFEGDMMKDFKKFDTIMVVNPKPDGNGCWTRSIEYEKTNENSPTPFDYLQFG HQAIEDMNKYLRDSE

[000402] SEQ.ID NO: 59

KGDGLVSGCIKEW

[000403] SEQ.ID NO: 60

EG

[000404] SEQ.ID NO: 61

PNGHGSIVTWSIEYEKMNEDSP