NOVEL NADH-DEPENDENT ENZYME MUTANTS TO CONVERT ACETONE INTO ISOPROPANOL

• US 20190309266A1
• Filed: 04/08/2019
• Published: 10/10/2019
• Est. Priority Date: 04/06/2018
• Status: Active Grant

• Current Assignee: Braskem SA
• Original Assignee: Braskem SA
• Inventors: QUEIROZ, Veronica Leite, PARIZZI, Lucas Pedersen, GOUVEA, Iuri Estrada, OKAMOTO, Debora Noma, GUIDO, Rafael Victorio Carvalho, NASCIMENTO, Alessandro Silva, POLIKARPOV, Igor
• Granted Patent: US 11,530,391 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Y 101/01001 Alcohol dehydrogenase (1.1.1.1)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present disclosure relates to biological processes and systems for the production of isopropanol and/or acetone utilizing modified alcohol dehydrogenases that exhibit increased activity with NADH as a cofactor. The disclosure further relates to polynucleotides and polypeptides of the modified alcohol dehydrogenases, and host cells containing the polynucleotides and expressing the polypeptides.

Claims

1. A nucleic acid construct comprising a polynucleotide sequence encoding a modified alcohol dehydrogenase, wherein the alcohol dehydrogenase exhibits activity with cofactor NADH as compared to an unmodified alcohol dehydrogenase;

and wherein the polynucleotide sequence shares at least 85% sequence identity with SEQ ID NO;

1.

2. A nucleic acid construct of claim 1, wherein the modified alcohol dehydrogenase exhibits a cofactor preference for NADH over NADPH, as compared to an unmodified alcohol dehydrogenase.

3. The nucleic acid construct of claim 1, wherein the modified alcohol dehydrogenase exhibits an increased activity for reduction of acetone to isopropanol with NADH, as compared to an unmodified alcohol dehydrogenase.

4. The nucleic acid construct of claim 1, wherein the modified alcohol dehydrogenase exhibits an increased activity for oxidation of isopropanol to acetone, as compared to an unmodified alcohol dehydrogenase.

5. A polypeptide sequence comprising a modified alcohol dehydrogenase that exhibits activity with cofactor NADH as compared to an unmodified alcohol dehydrogenase;

and wherein the modified alcohol dehydrogenase shares at least 85% sequence identity with SEQ ID NO;

2.

6. The polypeptide sequence of claim 5, wherein the modified alcohol dehydrogenase exhibits a cofactor preference for NADH over NADPH, as compared to an unmodified alcohol dehydrogenase.

7. The polypeptide sequence of claim 5, wherein the modified alcohol dehydrogenase exhibits an increased activity for reduction of acetone to isopropanol with NADH, as compared to an unmodified alcohol dehydrogenase.

8. The polypeptide sequence of claim 5, wherein the modified alcohol dehydrogenase exhibits an increased activity for oxidation of isopropanol to acetone, as compared to an unmodified alcohol dehydrogenase.

9. The polypeptide sequence of claims 5, wherein the modified alcohol dehydrogenase exhibits at least a 10 fold increase in activity with cofactor NADH as compared to an unmodified alcohol dehydrogenase.

10. The polypeptide sequence of claim 5, wherein the modified alcohol dehydrogenase sequence is selected from SEQ ID NO:

3-27.

11. The polypeptide sequence of claim 5, wherein the modified alcohol dehydrogenase sequence comprises an amino acid substitution at one or more of the following residues:

T38, G198, S199, Y218, I175, I173, R200, P201, C203, G244, E247, T248, K219, G243, Q251, Y267, L294, C295, K340, and K342.

13. The polypeptide sequence of claim 5, wherein the modified alcohol dehydrogenase sequence comprises an amino acid substitution at residue G198 with a D, E, G, K, N, R, S, A, or V amino acid residue.

14. The polypeptide sequence of claim 5, wherein the modified alcohol dehydrogenase sequence comprises one or more of the following amino acid substitutions:

G198E, E247Q, E247D, E247N, Y218F, G198D, I173C, G198L, S199D, Y218P, or Y218A.

15. The polypeptide sequence of claim 5, wherein the modified alcohol dehydrogenase sequence comprises a G198E amino acid substitution.

16. A method of producing a recombinant microorganism that produces a modified alcohol dehydrogenase that exhibits activity with cofactor NADH as compared to an unmodified alcohol dehydrogenase, the method comprising introducing a polynucleotide sequence encoding the polypeptide sequence of claim 5 into a microorganism.

17. The method of claim 16, wherein the microorganism is a bacterium or a fungus.

18. The method of claim 16, wherein the modified alcohol dehydrogenase exhibits a cofactor preference for NADH over NADPH, as compared to an unmodified alcohol dehydrogenase.

19. A recombinant microorganism expressing a polypeptide sequence comprising a modified alcohol dehydrogenase that exhibits activity with cofactor NADH as compared to an unmodified alcohol dehydrogenase;

and wherein the modified alcohol dehydrogenase shares at least 85% sequence identity with SEQ ID NO;

2.

20. The recombinant microorganism of claim 19, wherein the modified alcohol dehydrogenase exhibits a cofactor preference for NADH over NADPH, as compared to an unmodified alcohol dehydrogenase.