Mutant polypeptides and uses thereof

Patent metadata

Field	Value
Publication number	US-10214736-B2
Application number	US-201715593395-A
Country	US
Kind code	B2
Filing date	May 12, 2017
Priority date	Feb 27, 2015
Publication date	Feb 26, 2019
Grant date	Feb 26, 2019

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.

First claim

We claim:

1. 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:
R 1-95 X 96 R 97-98 X 99 R 100-122 X 123 R 124-186 X 187 R 188-203 X 204 R 205-211 X 212 R 213-272 X 273 X 274 X 275 R 276-323 X 324 R 325-327 X 328 R 329-359 X 360 R 361-365 X 366 R 367-381 X 382 R 383-398 , 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.

2. The polypeptide of claim 1 , wherein said amino acid sequence has at least 95% amino acid sequence homology to SEQ ID NO: 1.

3. The polypeptide of claim 1 , wherein said amino acid sequence comprises one to five mutations at the following X positions of SEQ ID NO: 1: R 1-95 X 96 R 97-98 X 99 R 100-122 X 123 R 124-186 X 187 R 188-203 X 204 R 205-211 X 212 R 213-272 X 273 X 274 X 275 R 276-323 X 324 R 325-327 X 328 R 329-359 X 360 R 361-365 X 366 R 367-381 X 382 R 383-398 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.

4. The polypeptide of claim 1 , wherein X 187 is mutated to a different amino acid selected from M and equivalent amino acids.

5. The polypeptide of claim 1 , wherein X 328 is mutated to a different amino acid selected from V and equivalent amino acids.

6. The polypeptide of claim 1 , wherein X 382 is mutated to a different amino acid selected from W and equivalent amino acids.

7. The polypeptide of claim 1 , comprising the following two mutations: L328V and F382W.

8. The polypeptide of claim 1 , comprising the following three mutations: I187M, L328V, and F382W.

9. The polypeptide 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.

10. The polypeptide of claim 1 , wherein the polypeptide has an activity in the catalysis of the dehydration of 3-methyl-3-buten-2-ol to isoprene 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.

11. A polynucleotide comprising a nucleic acid sequence encoding the polypeptide of claim 1 .

12. A vector comprising the polynucleotide of claim 11 .

13. A host cell transformed or transduced with the polynucleotide of claim 11 .

14. The host cell of claim 13 , wherein the host cell is a bacterium or a fungus.

15. A composition comprising the polypeptide of claim 1 and 3-buten-2-ol, 3-methyl-3-buten-2-ol, or both.

16. A method for producing 1,3-butadiene by the dehydration of 3-buten-2-ol to 1,3-butadiene in the presence of the polypeptide of claim 1 .

17. A method for producing 1 isoprene by the dehydration of 3-methyl-3-buten-2-ol to isoprene in the presence of the polypeptide of claim 1 .

Assignees

• Invista North America Sarl

Inventors

• Althoff Eric
• Kadi Nadia
• Azoitei Mihai Luchian
• Ban Yih-En A
• Grabs-Röthlisberger Daniela
• Pisarchik Alexander
• Zanghellini Alexandre
• Botes Adriana L

Classifications

• G01N33/573

  for enzymes or isoenzymes · CPC title

• C07C11/167

  1, 3-Butadiene · CPC title

• C07K2299/00

  Coordinates from 3D structures of peptides, e.g. proteins or enzymes · CPC title

• C08F36/22

  the radical having three or more carbon-to-carbon double bonds · CPC title

• C12P5/007

  containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00) · CPC title