Nucleic acid encoding n-methylputrescine oxidase and uses thereof
The gene encoding N-methylputrescine oxidase (MPO) and constructs comprising such DNA are provided, including methods of regulating MPO expression independently or with other alkaloid biosynthesis genes to modulate alkaloid production in plants and host cells. MPO genes or fragments thereof are useful for reducing pyrrolidine or tropane alkaloid production in plants, for increasing pyrrolidine or tropane alkaloid production in plants, and for producing an MPO enzyme in host cells.
1. A method of producing N-methylpyrrolinium, comprising incubating N-methylputrescine with a recombinant MPO enzyme.
2. The method of claim 1 , wherein the recombinant MPO enzyme has the amino acid sequence set forth in SEQ ID NO: 3, or a variant thereof, and the MPO enzyme is fused to a polyhistidine tag.
3. The method of claim 1 , wherein the recombinant MPO enzyme is produced by:
(1) genetically engineering a cell with a nucleic acid construct comprising an isolated nucleic acid molecule selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 1;
(b) the nucleotide sequence set forth in SEQ ID NO: 2;
(c) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3;
(d) a nucleotide sequence that is at least 95% identical to the full length nucleotide sequences of (a), (b), or (c) and encodes an N-methylputrescine oxidase (MPO) enzyme;
(e) a nucleotide sequence that hybridizes under high stringent conditions to the full length nucleotide sequences of (a), (b), (c), or (d) and encodes an MPO enzyme; and
(f) a nucleotide sequence that differs from the nucleic acid sequence of (a) or (b) due to the degeneracy of the genetic code and encodes an MPO enzyme; and
(2) growing the engineered cell under conditions such that MPO is produced.
4. The method of claim 3 , wherein the genetically engineered cell is selected from the group consisting of bacteria, yeast, filamentous fungi, algae, green plants, insect, and mammalian cells.
5. The method of claim 3 , wherein the isolated nucleic acid molecule is the nucleotide sequence set forth in SEQ ID NO: 1.
6. The method of claim 3 , wherein the isolated nucleic acid molecule is the nucleotide sequence set forth in SEQ ID NO: 2.
7. The method of claim 3 , wherein the isolated nucleic acid molecule is the nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3.
8. The method of claim 5 , wherein the nucleotide sequence set forth in SEQ ID NO: 1 is operably linked to a heterologous promoter sequence in the nucleic acid construct.
9. The method of claim 6 , wherein the nucleotide sequence set forth in SEQ ID NO: 2 is operably linked to a heterologous promoter sequence in the nucleic acid construct.
10. The method of claim 7 , wherein the nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3 is operably linked to a heterologous promoter sequence in the nucleic acid construct.
11. The method of claim 8 , wherein the genetically engineered cell is a bacterial cell.
12. The method of claim 9 , wherein the genetically engineered cell is a bacterial cell.
13. The method of claim 10 , wherein the genetically engineered cell is a bacterial cell.
14. The method of claim 11 , wherein the bacterial cell is Escherichia coli.
15. The method of claim 12 , wherein the bacterial cell is E. coli.
16. The method of claim 13 , wherein the bacterial cell is E. coli.
17. A method of producing 1-methyl-2-cyanopyrrolidine, comprising:
(a) incubating N-methylputrescine with a recombinant MPO enzyme; and
(b) incubating the reaction product of (a) with KCN.
18. The method of claim 17 , wherein the recombinant MPO enzyme has the amino acid sequence set forth in SEQ ID NO: 3, or a variant thereof, wherein the MPO enzyme is fused to a polyhistidine tag.