Mutant polypeptides and uses thereof
AssigneeInvista North America S.A.R.L.
InventorsEric ALTHOFF, Nadia KADI, Mihai AZOITEI, Yih-En A. BAN, Daniela GRABS-RÖTHLISBERGER, Alexander PISARCHIK, Alexandre ZANGHELLINI, Adriana L. BOTES
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Abstract
The present disclosure provides novel polypeptides with 3-buten-2-ol dehydratase activity, polypeptides with catalytic activity in the conversion of 3-methyl-3-buten-2-ol to isoprene, and crystal structure data for one of such polypeptides. Methods of making and using the polypeptides and their related crystal structure data are also provided.
Claims10
-20. (canceled)
A composition comprising 1,3-butadiene and a polypeptide comprising an amino acid sequence with at least 90% amino acid sequence homology to SEQ ID NO:
- 1, wherein said amino acid sequence comprises one to five mutations at the following X positions of SEQ ID NO:
- 1:
- R1-95X96R97-98X99R100-122X123R124-186X187R188-203
- X204R205-211X212R213-272X273X274X275R276-323
- X324R325-327X328R329-359X360R361-365X366R367-381
- X382R383-398,
- wherein:
- X96 is mutated to a different amino acid selected from L, I, M, A, G, and V;
- X99 is mutated to a different amino acid selected from L, I, M, A, G, and V;
- X123 is mutated to a different amino acid selected from L, I, M, A, and G;
- X187 is mutated to a different amino acid selected from L, M, A, G, and V;
- X204 is mutated to a different amino acid selected from L, I, M, A, and G;
- X212 is mutated to a different amino acid selected from F, Y, and W;
- X273 is mutated to a different amino acid selected from C, M, S, and T;
- X274 is mutated to a different amino acid selected from F, Y, and W;
- X275 is mutated to a different amino acid selected from L, I, M, A, and G;
- X324 is mutated to a different amino acid selected from L, I, M, G, V, E, D, N, and Q;
- X328 is mutated to a different amino acid selected from I, M, A, G, and V;
- X360 is mutated to a different amino acid selected from F, Y, and W;
- X366 is mutated to a different amino acid selected from L, I, M, A, G, V, C, and T;
- X382 is mutated to a different amino acid selected from Y and W;
- and each R is the same as the corresponding amino acid in SEQ ID NO:
- 1, with or without an N-terminal signal peptide, and with or without an N-terminal methionine,
- wherein the polypeptide has an activity in converting 3-buten-2-ol to 1,3-butadiene and/or in converting 3-methyl-3-buten-2-ol to isoprene,
- and provided that the mutations chosen from one or more of positions X96, X99, X123, and X187 are not the only mutations of SEQ ID NO:
- 1.
The composition of claim 1, wherein the amino acid sequence of the polypeptide has at least 95% amino acid sequence homology to SEQ ID NO:
- 1.
The composition of claim 1, wherein the amino acid sequence of the polypeptide comprises one to three mutations at the following X positions of SEQ ID NO:
- 1:
- R1-95X96R97-98X99R100-122X123R124-186X187R188-203
- X204R205-211X212R213-272X273X274X275R276-323X324
- R325-327X328R329-359X360R361-365X366R367-381X382
- R383-398
- wherein:
- X96 is mutated to a different amino acid selected from L, I, M, A, G, and V;
- X99 is mutated to a different amino acid selected from L, I, M, A, G, and V;
- X123 is mutated to a different amino acid selected from L, I, M, A, and G;
- X187 is mutated to a different amino acid selected from L, M, A, G, and V;
- X204 is mutated to a different amino acid selected from L, I, M, A, and G;
- X212 is mutated to a different amino acid selected from F, Y, and W;
- X273 is mutated to a different amino acid selected from C, M, S, and T;
- X274 is mutated to a different amino acid selected from F, Y, and W;
- X275 is mutated to a different amino acid selected from L, I, M, A, and G;
- X324 is mutated to a different amino acid selected from L, I, M, G, V, E, D, N, and Q;
- X328 is mutated to a different amino acid selected from I, M, A, G, and V;
- X360 is mutated to a different amino acid selected from F, Y, and W;
- X366 is mutated to a different amino acid selected from L, I, M, A, G, V, C, and T;
- X382 is mutated to a different amino acid selected from Y and W;
- and each R is the same as the corresponding amino acid in SEQ ID NO:
- 1, with or without an N-terminal signal peptide, and with or without an N-terminal methionine,
- wherein the polypeptide is capable of converting 3-buten-2-ol to 1,3-butadiene and/or converting 3-methyl-3-buten-2-ol to isoprene,
- and provided that the mutations chosen from one or more of positions X96, X99, X123, and X187 are not the only mutations in SEQ ID NO:
- 1.
The composition of claim 1, wherein the polypeptide is mutated at X187 to a different amino acid selected from L, M, A, G, and V.
The composition of claim 1, wherein the polypeptide is mutated at X328 to a different amino acid selected from I, M, A, G, and V.
The composition of claim 1, wherein the polypeptide is mutated at X382 to a different amino acid selected from Y and W.
The composition of claim 1, wherein the polypeptide comprises the following two mutations:
- L328V and F382W.
The composition of claim 1, wherein the polypeptide comprises the following three mutations:
- I187M, L328V, and F382W.
The composition of claim 1, wherein the polypeptide has an activity in the catalysis of the dehydration of 3-buten-2-ol to 1,3-butadiene that is at least 80% of that of a polypeptide consisting of SEQ ID NO:
- 1, 4, 5, 7, or 8, wherein said activity is observed in at least one activity assay.
Dependencies are inferred from the claim text.