Compositions and methods for making noscapine and synthesis intermediates thereof

1. A method of making noscapine comprising:

(a) providing a noscapine pathway precursor selected from a canadine derivative and a papaveroxine derivative; wherein

the canadine derivative is selected from the group of canadine derivatives consisting of each of (S)—N-methylcanadine; 1-hydroxy-N-methylcanadine; 1,13-dihydroxy-N-methylcanadine, 1-hydroxy-13-O-acetyl-N-methylcanadine; and 1,8-dihydroxy-13-O-acetyl-N-methyl canadine; and

the papaveroxine derivative is papaveroxine;

(b) contacting the noscapine pathway precursor with the enzymes listed below (i) in vitro or (ii) in a microorganism heterologously provided with the noscapine pathway precursor under reaction conditions permitting the catalysis of the noscapine pathway precursor to form noscapine;

wherein

(i) when the canadine derivative is (S)—N-methylcanadine, contacting the (S)—N-methylcanadine with each of the enzymes (i) CYP82Y1; (ii) CYP82X2; (iii) AT1; (iv) CYP82X1; (v) OMT; (vi) CXE1; and (vii) NOS;

(ii) when the canadine derivative is 1-hydroxy-N-methylcanadine, contacting the 1-hydroxy-N-methylcanadine with each of the enzymes (i) CYP82X2; (ii) AT1; (iii) CYP82X1; (iv) OMT; (v) CXE1; and (vi) NOS;

(iii) when the canadine derivative is 1,13-dihydroxy-N-methylcanadine, contacting the 1,13-dihydroxy-N-methylcanadine with each of the enzymes (i) AT1; (ii) CYP82X1; (iii) OMT; (iv) CXE1; and (v) NOS;

(iv) when the canadine derivative is 1-hydroxy-13-O-acetyl-N-methylcanadine, contacting the 1-hydroxy-13-O-acetyl-N-methylcanadine with each of the enzymes (i) CYP82X1; (ii) OMT; (iii) CXE1; and (iv) NOS;

(v) when the canadine derivative is 1,8-dihydroxy-13-O-acetyl-N-methylcanadine, contacting the 1,8-dihydroxy-13-O-acetyl-N-methylcanadine with each of the enzymes (i) OMT; and (ii) CXE1; and (iii) NOS;

(vi) when the papaveroxine derivative is papaveroxine, contacting the papaveroxine solely with the enzyme NOS; and

wherein

NOS comprises the amino acid sequence of SEQ.ID NO: 14; or a sequence at least 90% identical thereto;

CYP82Y1 comprises the amino acid sequence of SEQ.ID NO: 2, or a sequence at least 90% identical thereto;

CYP82X2 comprises the amino acid sequence of SEQ.ID NO: 4, or a sequence at least 90% identical thereto;

AT1 comprises the amino acid sequence of SEQ.ID NO: 6 or a sequence at least 90% identical thereto;

CYP82X1 comprises the amino acid sequence of SEQ.ID NO: 8, or a sequence at least 90% identical thereto;

OMT comprises the amino acid sequence of SEQ.ID NO: 10, or a sequence at least 90% identical thereto; and

CXE1 comprises the amino acid sequence of SEQ.ID NO: 12, or a sequence at least 90% identical thereto.

2. A method for producing noscapine from a pathway precursor selected from a canadine derivative and a papaveroxine derivative; wherein

the canadine derivative is selected from the group of canadine derivatives consisting of (S)—N-methylcanadine; 1-hydroxy-N-methylcanadine; 1,13-dihydroxy-N-methylcanadine, 1-hydroxy-13-O-acetyl-N-methylcanadine; and 1,8-dihydroxy-13-O-acetyl-N-methylcanadine heterologously provided to a host cell; and

the papaveroxine derivative is papaveroxine heterologously provided to a host cell;

the method comprising:

(a) providing a chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of transcription as operably linked components:

(i) one or more nucleic acid sequences encoding one or more of the polypeptides selected from the group of polypeptides listed below; and

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

(b) introducing the chimeric nucleic acid sequence into the host cell, and growing the host cell to produce the polypeptide and noscapine;

wherein:

(i) when the canadine derivative is (S)—N-methylcanadine, the enzymes are each of (i) CYP82Y1; (ii) CYP82X2; (iii) AT1; (iv) CYP82X1; (v) OMT; (vi) CXE1; and (vii) NOS;

(ii) when the canadine derivative is 1-hydroxy-N-methylcanadine, the enzymes are each of (i) CYP82X2; (ii) AT1; (iii) CYP82X1; (iv) OMT; (v) CXE1; and (vi) NOS;

(iii) when the canadine derivative is 1,13-dihydroxy-N-methylcanadine, the enzymes are each of (i) AT1; (ii) CYP82X1; (iii) OMT; (iv) CXE1; and (v) NOS;

(iv) when the canadine derivative is 1-hydroxy-13-O-acetyl-N-methylcanadine, the enzymes are each of (i) CYP82X1; (ii) OMT; (iii) CXE1; and (iv) NOS;

(v) when the canadine derivative is 1,8-dihydroxy-13-O-acetyl-N-methylcanadine, the enzymes are each of (i) OMT; (ii) CXE1; and (iii) NOS;

(vi) when the papaveroxine derivative is papaveroxine, the enzyme is solely NOS; and

wherein

NOS comprises the amino acid sequence of SEQ.ID NO: 14, or a sequence at least 90% identical thereto;

CYP82Y1 comprises the amino acid sequence of SEQ.ID NO: 2, or a sequence at least 90% identical thereto;

CYP82X2 comprises the amino acid sequence of SEQ.ID NO: 4, or a sequence at least 90% identical thereto;

AT1 comprises the amino acid sequence of SEQ.ID NO: 6, or a sequence at least 90% identical thereto;

CYP82X1 comprises the amino acid sequence of SEQ.ID NO: 8, or a sequence at least 90% identical thereto;

OMT comprises the amino acid sequence of SEQ.ID NO: 10, or a sequence at least 90% identical thereto; and

CXE1 comprises the amino acid sequence of SEQ.ID NO: 12, or a sequence at least 90% identical thereto.

3. The method according to claim 2 , wherein the method further includes step (c) comprising recovering noscapine.

4. The method according to claim 1 wherein:

CYP82Y1 comprises the amino acid sequence of SEQ.ID NO: 2, or a sequence at least 95% identical thereto;

CYP82X2 comprises the amino acid sequence of SEQ.ID NO: 4, or a sequence at least 95% identical thereto;

AT1 comprises the amino acid sequence of SEQ.ID NO: 6, or a sequence at least 95% identical thereto;

CYP82X1 comprises the amino acid sequence of SEQ.ID NO: 8, or a sequence at least 95% identical thereto;

OMT comprises the amino acid sequence of SEQ.ID NO: 10, or a sequence at least 95% identical thereto;

CXE1 comprises the amino acid sequence of SEQ.ID NO: 12, or a sequence at least 95% identical thereto; and

NOS comprises the amino acid sequence of SEQ.ID NO: 14, or a sequence at least 95% identical thereto.

5. The method according to claim 1 wherein:

CYP82Y1 comprises the amino acid sequence of SEQ.ID NO: 2;

CYP82X2 comprises the amino acid sequence of SEQ.ID NO: 4;

AT1 comprises the amino acid sequence of SEQ.ID NO: 6;

CYP82X1 comprises the amino acid sequence of SEQ.ID NO: 8;

OMT comprises the amino acid sequence of SEQ.ID NO: 10;

CXE1 comprises the amino acid sequence of SEQ.ID NO: 12; and

NOS comprises the amino acid sequence of SEQ.ID NO: 14.

6. The method according to claim 1 , wherein the reaction conditions are in vitro reaction conditions.

7. The method according to claim 1 , wherein the microorganism is a bacterial cell or a fungal cell.

8. The method according to claim 7 , wherein the bacterial cell is an E. coli cell.

9. The method according to claim 7 , wherein the fungal cell is a yeast cell.

10. The method according to claim 2 wherein:

CYP82Y1 comprises the amino acid sequence of SEQ.ID NO: 2, or a sequence at least 95% identical thereto;

CYP82X2 comprises the amino acid sequence of SEQ.ID NO: 4, or a sequence at least 95% identical thereto;

AT1 comprises the amino acid sequence of SEQ.ID NO: 6, or a sequence at least 95% identical thereto;

CYP82X1 comprises the amino acid sequence of SEQ.ID NO: 8, or a sequence at least 95% identical thereto;

OMT comprises the amino acid sequence of SEQ.ID NO: 10, or a sequence at least 95% identical thereto;

CXE1 comprises the amino acid sequence of SEQ.ID NO: 12, or a sequence at least 95% identical thereto; and

NOS comprises the amino acid sequence of SEQ.ID NO: 14, or a sequence at least 95% identical thereto.

11. The method according to claim 2 wherein:

CYP82Y1 comprises the amino acid sequence of SEQ.ID NO: 2;

CYP82X2 comprises the amino acid sequence of SEQ.ID NO: 4;

AT1 comprises the amino acid sequence of SEQ.ID NO: 6;

CYP82X1 comprises the amino acid sequence of SEQ.ID NO: 8;

OMT comprises the amino acid sequence of SEQ.ID NO: 10;

CXE1 comprises the amino acid sequence of SEQ.ID NO: 12; and

NOS comprises the amino acid sequence of SEQ.ID NO: 14.

12. The method according to claim 2 wherein the cell is a bacterial cell, a fungal cell, or a plant cell.

13. The method according to claim 11 , wherein the bacterial cell is an E. coli cell.

14. The method according to claim 11 wherein the fungal cell is a yeast cell.