3-hydroxypropionic acid production by recombinant yeasts expressing an insect aspartate 1-decarboxylase

Patent metadata

Field	Value
Publication number	US-9845484-B2
Application number	US-201414908730-A
Country	US
Kind code	B2
Filing date	Jul 31, 2014
Priority date	Jul 31, 2013
Publication date	Dec 19, 2017
Grant date	Dec 19, 2017

Abstract

Provided herein are recombinant yeast cells having an active 3-Hydroxypropionic Acid (3-HP) pathway and further comprising a heterologous polynucleotide encoding an aspartate 1-decarboxylase (ADC) of the Class Insecta, Bivalvia, Branchioporia, Gastropoda, or Leptocardii. Also described are methods of using the recombinant yeast cells to produce 3-HP and acrylic acid.

First claim

What is claimed is:

1. A recombinant yeast cell comprising a heterologous polynucleotide encoding an aspartate 1-decarboxylase, wherein the aspartate 1-decarboxylase has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 162, and wherein the cell is capable of producing 3-hydroxypropionic acid (3-HP).

2. The recombinant yeast cell of claim 1 , wherein the aspartate 1-decarboxylase comprises a glutamine at a residue corresponding to position 377 of the amino acid sequence of SEQ ID NO: 162.

3. The recombinant yeast cell of claim 1 , wherein the aspartate 1-decarboxylase comprises a F 1 aa 19 S 2 aaY 3 motif or an Y 1 aa 19 S 2 aaY 3 motif, wherein F 1 is phenylalanine, Y 1 is tyrosine, aa 19 is an intervening sequence of nineteen amino acids, S 2 is serine, aa is a single amino acid, and Y 3 is tyrosine.

4. The recombinant yeast cell of claim 1 , wherein the heterologous polynucleotide encoding the aspartate 1-decarboxylase comprises a promoter foreign to the polynucleotide encoding the aspartate 1-decarboxylase.

5. The recombinant yeast cell of claim 1 , wherein the cell produces a greater amount of 3-HP compared to the cell without the heterologous polynucleotide encoding the aspartate 1-decarboxylase, when cultivated under identical conditions.

6. The recombinant yeast cell of claim 1 , wherein the cell belongs to a genus selected from Candida, Issatchenkia, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Torulaspora , and Zygosaccharomyces.

7. The recombinant yeast cell of claim 6 , wherein the cell is selected from I. orientalis, C. lambica , and S. bulderi.

8. The recombinant yeast cell of claim 1 , wherein the cell is capable of growing at a pH of less than 4 in media containing 75 g/L or greater 3-HP.

9. A method of producing 3-HP, comprising:
(a) cultivating the recombinant yeast cell of claim 1 in a fermentable medium under suitable conditions to produce 3-HP; and
(b) recovering the 3-HP.

10. The method of claim 9 , wherein the fermentable medium is at a pH of less than 5.

11. A method of producing acrylic acid or a salt thereof, comprising:
(a) cultivating the recombinant yeast cell of claim 1 in a fermentable medium under suitable conditions to produce 3-HP;
(b) recovering the 3-HP;
(c) dehydrating the 3-HP under suitable conditions to produce acrylic acid or a salt thereof; and
(d) recovering the acrylic acid or salt thereof.

12. The recombinant yeast cell of claim 1 , wherein the aspartate 1-decarboxylase has an amino acid sequence comprising or consisting of SEQ ID NO: 162.

13. The recombinant yeast cell of claim 1 , wherein the aspartate 1-decarboxylase has at least 97%, sequence identity to the amino acid sequence of SEQ ID NO: 162.

14. The recombinant yeast cell of claim 1 , wherein the aspartate 1-decarboxylase has at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 162.

15. The method of claim 9 , wherein the aspartate 1-decarboxylase has at least 97%, sequence identity to the amino acid sequence of SEQ ID NO: 162.

16. The method of claim 9 , wherein the aspartate 1-decarboxylase has at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 162.

17. The method of claim 9 , wherein the aspartate 1-decarboxylase has an amino acid sequence comprising or consisting of SEQ ID NO: 162.

18. The method of claim 11 , wherein the aspartate 1-decarboxylase has at least 97%, sequence identity to the amino acid sequence of SEQ ID NO: 162.

19. The method of claim 11 , wherein the aspartate 1-decarboxylase has at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 162.

20. The method of claim 11 , wherein the aspartate 1-decarboxylase has an amino acid sequence comprising or consisting of SEQ ID NO: 162.

Assignees

• Novozymes As

Inventors

• Barnhart Michelle
• Negrete-Raymond Ana
• Frias Janice
• Barbier Gui
• Catlett Michael

Classifications

• C12Y401/01011

  Aspartate 1-decarboxylase (4.1.1.11) · CPC title

• C12P7/42 Primary

  Hydroxy-carboxylic acids · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12N1/16

  Yeasts; Culture media therefor · CPC title

• C12Y401/01001

  Pyruvate decarboxylase (4.1.1.1) · CPC title