Enzyme used in biosynthesis of 1, 4-BDO and screening method of the same

Patent metadata

Field	Value
Publication number	US-9493746-B2
Application number	US-201414504279-A
Country	US
Kind code	B2
Filing date	Oct 1, 2014
Priority date	Jul 30, 2012
Publication date	Nov 15, 2016
Grant date	Nov 15, 2016

Abstract

Recombinant butyraldehyde dehydrogenases (Blds) with improved production of 1,4-BDO, as well as recombinant microorganisms comprising polynucleotides encoding the recombinant Blds, and methods of producing 1,4-BDO by using the recombinant microorganisms.

First claim

What is claimed is:

1. A butyraldehyde dehydrogenase (bld) mutant polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7′ and wherein the mutant polypeptide has butyraldehyde dehydrogenase activity“.

2. A polynucleotide that encodes the bld mutant of claim 1 .

3. A microorganism that produces 1,4-butanediol, the microorganism comprising the bld mutant polypeptide of claim 1 .

4. The microorganism of claim 3 , wherein the microorganism comprises an exogenous polynucleotide encoding the bld mutant polypeptide of claim 1 .

5. The microorganism of claim 3 , further comprising a butanol dehydrogenase enzyme that catalyzes the conversion of 4-hydroxybutyraldehyde into 1,4-butanediol.

6. The microorganism of claim 5 , wherein the microorganism comprises a gene encoding the butanol dehydrogenase enzyme.

7. The microorganism of claim 6 , wherein the butanol dehydrogenase enzyme is from Clostridium saccharoperbutylacetonicum.

8. The microorganism of claim 3 , further comprising
a gene encoding succinyl-CoA:coenzyme A transferase that converts succinate into succinyl-CoA,
a gene encoding CoA-dependent succinate semialdehyde dehydrogenase that converts succinyl-CoA into succinate semialdehyde,
a gene encoding 4-hydroxybutyrate dehydrogenase that converts succinate semialdehyde into 4-hydroxybutyrate, and
a gene encoding 4-hydroxybutyryl-CoA:acetyl-CoA transferase that converts 4-hydroxybutyrate into 4-hydroxybutyryl-CoA.

9. The microorganism of claim 3 , wherein the microorganism is Escherichia coli.

10. A method of producing 4-hydroxybutyaldehyde, the method comprising:
contacting 4-hydroxybutyryl-CoA with the bld mutant polypeptide of claim 1 , whereby 4-hydroxybutyaldehyde is produced.

11. A method of producing 1,4-BDO, the method comprising:
contacting 4-hydroxybutyryl-CoA with the bld mutant polypeptide of claim 1 ; and
contacting the obtained reaction product with butanol dehydrogenase, whereby 1,4-BDO is produced.

12. A method of producing 1,4-BDO, the method comprising:
incubating a microorganism comprising a polynucleotide encoding the bld mutant of claim 1 and a polynucleotide encoding butanol dehydrogenase (bdh) with a carbon source, whereby the microorganism produces 1,4-BDO; and
separating 1,4-BDO from the incubation product.

13. The method of claim 12 , further comprising introducing the polynucleotide encoding the bld mutant of claim 1 and the polynucleotide encoding butanol dehydrogenase into the microorganism prior to incubating the microorganism with a carbon source.

14. The method of claim 12 , wherein the microorganism further comprises
a gene encoding succinyl-CoA:coenzyme A transferase that converts succinate into succinyl-CoA,
a gene encoding CoA-dependent succinate semialdehyde dehydrogenate that converts succinyl-CoA into succinate semialdehyde,
a gene encoding 4-hydroxybutyrate dehydrogenase that converts succinate semialdehyde into 4-hydroxybutyrate, and
a gene encoding 4-hydroxybutyryl-CoA:acetyl-CoA transferase that converts 4-hydroxybutyrate into 4-hydroxybutyryl-CoA.

15. The mutant polypeptide of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

16. The mutant polypeptide of claim 1 , wherein the polypeptide comprises amino acid sequence of SEQ ID NO: 3.

17. The mutant polypeptide of claim 1 , wherein the polypeptide comprises amino acid sequence of SEQ ID NO: 4.

18. The mutant polypeptide of claim 1 , wherein the polypeptide comprises amino acid sequence of SEQ ID NO: 5.

19. The mutant polypeptide of claim 1 , wherein the polypeptide comprises amino acid sequence of SEQ ID NO: 6.

20. The mutant polypeptide of claim 1 , wherein the polypeptide comprises amino acid sequence of SEQ ID NO: 7.

Assignees

• Samsung Electronics Co Ltd
• Ajou Univ Industry-Academic Coop Found

Inventors

• Park Jinhwan
• Lee Pyungcheon
• Park Jaechan
• Lee Youngmin
• Lee Wooyong
• Park Jinwoo
• Cho Kwangmyung
• Hwang Heejin

Classifications

• C12P7/24

  containing a carbonyl group · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12P7/18

  polyhydric · CPC title

• C12Y102/01057

  Butanal dehydrogenase (1.2.1.57) · CPC title

• C12N9/0008 Primary

  acting on the aldehyde or oxo group of donors (1.2) · CPC title