Monolignol 4-O-methyltransferases and uses thereof
Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.
1. A modified (iso)eugenol 4-O-methyltransferase (IEMT) enzyme having three amino acid substitutions selected from the group consisting of enzymes having amino acid substitutions T133L/E165I/F175I and T133L/L139Q/E165F, wherein the modified amino acid residues correspond to those of SEQ ID NO: 1.
2. An isolated nucleic acid molecule encoding a modified (iso)eugenol 4-O-methyltransferase enzyme having three amino acid substitutions selected from the group consisting of sequences encoding amino acid substitutions T133L/E165I/F175I and T133L/L139Q/E165F, wherein the modified amino acid residues correspond to those of SEQ ID NO: 1.
3. An expression vector adapted for expression in plants of a modified IEMT enzyme comprising the nucleic acid sequence of claim 2 .
4. A plant containing the expression vector of claim 3 .
5. The plant of claim 4 selected from the group consisting of Arabidopsis , poplar, corn, rice and switchgrass.
6. A modified (iso)eugenol 4-O-methyltransferase enzyme having four or five amino acid substitutions selected from the group consisting of enzymes having amino acid substitutions T133L/E165I/F175I/F166W, T133L/E165I/F175I/H169F, T133L/E165I/F175I/F166W/H169W, T133L/E165I/F175I/F166W/H169F, T133I/E165I/F175I/Y326F/N327V and T133L/E165I/F175I/F166W/T135N, wherein the modified amino acid residues correspond to those of SEQ ID NO: 1.
7. An isolated nucleic acid sequence encoding the modified (iso)eugenol 4-O-methyltransferase enzyme of claim 6 .
8. An expression vector adapted for expression in plants specifically designed to express the modified IEMT enzyme sequence of claim 7 .
9. A plant containing the expression vector of claim 8 .
10. The plant of claim 9 selected from the group consisting of Arabidopsis , poplar, corn and switchgrass.
11. A modified (iso)eugenol 4-O-methyltransferase (m-IEMT) having an increased activity, compared to wild type (iso)eugenol 4-O-methyltransferase (wt-IEMT), for 4-O-methylation of coniferyl alcohol or sinapyl alcohol or both, said m-IEMT having three, four or five amino acid substitutions selected from the group consisting of T133L/E165I/F175I, T133L/L139Q/E165F, T133L/E165I/F175I/F166W, T133L/E165I/F175H/H169F, T133L/E165I/F175I/F166W/H169W, and T133L/E165I/F175I/F166W/H169F, wherein the modified amino acid residues correspond to those of SEQ ID NO: 1.