Compositions and methods for modulating the sensitivity of cells to AHAS inhibitors
Methods and materials useful for modulating the sensitivity of cells to an inhibitor of acetohydroxyacid synthase (AHAS) are disclosed. For example, nucleic acid molecules encoding AHAS large subunits are disclosed as well as methods for using such nucleic acid molecules to transform microbial cells and plant cells, and to confer modulated sensitivity to AHAS-inhibiting compounds onto such cells. Further provided are materials and methods useful for modulating growth, development, activity, and characteristics of host cells and organisms.
1. An isolated nucleic acid molecule comprising
a nucleic acid sequence encoding an acetohydroxyacid synthase (AHAS) exhibiting 90% or greater identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5;
wherein the AHAS has at least one amino acid substitution at an amino acid position selected from the group consisting of R9, G35, A36, L38, I56, G114, A116, A122, I171, K173, I186, N194, A234, M272, D296, D297, P319, K337, S348, P352, F361, E364, V367, V491, W494, F498, D500, P567, and G568 of SEQ ID NO: 1, 3, or 5;
and further wherein said AHAS has reduced sensitivity to an AHAS inhibitor compared to the AHASs encoded by SEQ ID NO 1, 3 or 5.
2. An isolated polypeptide encoded by a nucleic acid molecule according to claim 1 .
3. A nucleic acid molecule according to claim 1 , wherein said at least one amino acid substitution is at an amino acid position selected from the group consisting of A36, G114, A122, D297, and W494,of SEQ ID NO: 1, 3, or 5.
4. A recombinant nucleic acid construct comprising a nucleic acid molecule according to claim 1 operably linked to a heterologous nucleic acid sequence.
5. The recombinant nucleic acid construct of claim 4 , wherein said heterologous nucleic acid sequence is a heterologous promoter.
6. The recombinant nucleic acid construct of claim 4 , wherein said heterologous nucleic acid sequence encodes a transit peptide.
7. A host cell comprising a nucleic acid molecule according to claim 1 .
8. The host cell according to claim 7 , wherein said nucleic acid molecule is integrated into the genome of said host cell via homologous recombination or ectopic insertion.
9. The host cell according to claim 7 , wherein said nucleic acid molecule is integrated into a chloroplast genome or nuclear genome of said host cell.
10. The host cell according to claim 7 , wherein said host cell is an algal cell, a bacterial cell, a fungal cell, or a plant cell.
11. The host cell according to claim 7 , wherein said host cell is a Nannochloropsis cell.
12. The host cell according to claim 7 , wherein said nucleic acid molecule further comprises a second heterologous nucleic acid sequence to be expressed by said host cell, said second heterologous nucleic acid sequence encoding a reporter protein.
13. A host organism comprising the host cell of claim 7 .
14. A biological sample, biomass, or progeny derived from a host organism according to claim 13 .
15. A method for modulating sensitivity of a cell to an AHAS inhibitor, said method comprising introducing into said cell a nucleic acid molecule according to claim 1 , wherein said nucleic acid molecule confers reduced sensitivity of said cell to said AHAS inhibitor as compared to a control cell.
16. The method of claim 15 , said method further comprising a step of selecting cells that have been successfully transformed with said nucleic acid molecule by culturing said cells in a growth medium containing at least one AHAS inhibitor that is inhibitory to the growth of untransformed cells.
17. The method of claim 15 , said method further comprising a step of regenerating from said transformed cell a transformed organism.
18. The method of claim 15 , wherein said nucleic acid molecule is integrated into the genome of said host cell via homologous recombination or ectopic insertion.
19. The method of claim 15 , wherein said nucleic acid molecule is integrated into a chloroplast genome or nuclear genome of said host cell.
20. The method of claim 15 , wherein said host cell is an algal cell, a bacterial cell, a fungal cell, or a plant cell.
21. The method of claim 15 , wherein said host cell is a Nannochloropsis cell.
22. The method of claim 15 , wherein said nucleic acid molecule further comprises a second heterologous nucleic acid sequence to be expressed by said host cell said second heterologous nucleic acid sequence encoding a reporter protein.
23. The nucleic acid molecule of claim 3 , wherein the amino acid substitution is selected from the group consisting of: A36T, A36S, A122V, and D297N.