Specialized (iso)eugenol-4-O-methyltransferases (s-IEMTs) and methods of making and using the same
Specialized (iso)eugenol 4-O-methyltransferase (s-IEMT) enzymes having increased capacity for methylation of monolignols are disclosed. The s-IEMTs have unique activity favoring methylation of coniferyl alcohol versus sinapyl alcohol. Various s-IEMTs methylate ferulic acid. Means for producing the various s-IEMTs are provided. The s-IEMTs are useful for modification of lignin content and production of aromatic compounds.
1. A specialized (iso)eugenol 4-O-methyltransferase (s-IEMT) enzyme comprising amino acid substitutions at each of residues 133, 165, 175, 166, 169, 26, 30 and 33, where the amino acid residues correspond to those of SEQ ID NO. 1.
2. The s-IEMT of claim 1 further comprising an amino acid substitution at residue 319 of SEQ ID NO. 1.
3. The s-IEMT enzyme of claim 2 selected from the group consisting of s-IEMTs comprising the amino acid substitutions
T133L/E165I/F175I/F166W/H169F/M26H/S30R/V33S;
T133L/E165I/F1751/F166W/H169F/M26S/S30F/V33Y;
T133L/E165I/F175I/F166W/H169F/M26S/S30F/V33G; and
T133L/E165I/F175I/F166W/H169F/M26H/S30R/V33S/T319M.
4. An isolated nucleic acid sequence comprising a sequence encoding a specialized (iso)eugenol 4-O-methyltransferase enzyme having amino acid substitutions at each of residues 133, 165, 175, 166, 169, 26, 30 and 33 of SEQ ID NO. 1.
5. The isolated nucleic acid sequence of claim 4 further comprising an amino acid substitution at residue 319 of SEQ ID NO: 1.
6. The isolated nucleic acid sequence of claim 5 selected from the group consisting of sequences encoding an s-IEMT having the amino acid substitutions
T133L/E165I/F175I/F166W/H169F/M26H/S30R/V33S;
T133L/E165I/F1751/F166W/H169F/M26S/S30F/V33Y;
T133L/E165I/F175I/F166W/H169F/M26S/S30F/V33G; and
T133L/E165I/F175I/F166W/H169F/M26H/S30R/V33S/T319M.
7. A plant transformed with an expression vector adapted for expression in plants of the specialized IEMT enzyme encoded by the nucleic acid sequence of claim 4 or claim 5 .
8. The plant of claim 7 selected from the group consisting of Arabidopsis , corn, switchgrass, poplar, angiosperms and gymnosperms.
9. An expression vector adapted for expression in cultured cells of the specialized IEMT enzyme encoded by the nucleic acid sequence of claim 4 or claim 5 .
10. The expression vector of claim 9 wherein the cultured cells are selected from the group consisting of cultured bacterial, yeast, plant, insect and mammalian cells.
11. An isolated purified (iso)eugenol 4-O-methyltransferase (s-IEMT) obtained from the cultured cells of claim 10 .
12. An isolated purified (iso)eugenol 4-O-methyltransferase (s-IEMT) obtained from the plant claim 7 .