Microorganisms for C4-dicarboxylic acid production

Patent metadata

Field	Value
Publication number	US-9447440-B2
Application number	US-201414159786-A
Country	US
Kind code	B2
Filing date	Jan 21, 2014
Priority date	Feb 28, 2011
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

Abstract

The present invention relates to isolated polypeptides having bicarbonate transporter activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, and methods of producing C4-dicarboxylic acids, such as malic acid.

First claim

What is claimed is:

1. A recombinant fungal or prokaryotic host cell comprising a heterologous polynucleotide encoding a bicarbonate transporter, wherein the heterologous polynucleotide:
(a) encodes a bicarbonate transporter having at least 90% sequence identity to SEQ ID NO: 4; or
(b) has at least 90% sequence identity to SEQ ID NO: 3, or the cDNA sequence thereof;
wherein the host cell is capable of producing a greater amount of a C4-dicarboxylic acid compared to the host cell without the heterologous polynucleotide when cultivated under the same conditions.

2. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide encodes a bicarbonate transporter having at least 90% sequence identity to SEQ ID NO: 4.

3. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide encodes a bicarbonate transporter having at least 95% sequence identity to SEQ ID NO: 4.

4. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide has at least 90% sequence identity to SEQ ID NO: 3, or the cDNA sequence thereof.

5. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide has at least 95% sequence identity to SEQ ID NO: 3, or the cDNA sequence thereof.

6. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide encodes a polypeptide that comprises or consists of SEQ ID NO: 4.

7. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide encoding the bicarbonate transporter is operably linked to a promoter foreign to the polynucleotide.

8. The recombinant host cell of claim 1 , further comprising a heterologous polynucleotide encoding a C4-dicarboxylic acid transporter.

9. The recombinant host cell of claim 8 , wherein the heterologous polynucleotide encoding a C4-dicarboxylic acid transporter is operably linked to a promoter foreign to the polynucleotide.

10. The recombinant host cell of claim 1 , further comprising a heterologous polynucleotide encoding a malate dehydrogenase.

11. The recombinant host cell of claim 10 , wherein the heterologous polynucleotide encoding a malate dehydrogenase is operably linked to a promoter foreign to the polynucleotide.

12. The recombinant host cell of claim 1 , further comprising a heterologous polynucleotide encoding a pyruvate carboxylase.

13. The recombinant host cell of claim 12 , wherein the heterologous polynucleotide encoding a pyruvate carboxylase is operably linked to a promoter foreign to the polynucleotide.

14. The recombinant host cell of claim 13 , wherein the host cell is a filamentous fungal host cell.

15. The recombinant host cell of claim 14 , wherein the host cell is selected from the group consisting of an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus, Rhizopus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , and Trichoderma.

16. The recombinant host cell of claim 14 , wherein the host cell is an Aspergillus host cell.

17. The recombinant host cell of claim 16 , wherein the host cell is an Aspergillus oryzae host cell.

18. The recombinant host cell of claim 16 , wherein the host cell is an Aspergillus niger host cell.

19. The recombinant host cell of claim 1 , wherein the C4-dicarboxylic acid is malic acid.

20. The recombinant host cell of claim 1 , wherein the host cell is capable of producing a greater amount of the C4-dicarboxylic acid by at least 25% compared to the host cell without the heterologous polynucleotide that encodes the bicarbonate transporter, when cultivated under the same conditions.

21. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide encodes a bicarbonate transporter having at least 97% sequence identity to SEQ ID NO: 4.

22. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide encodes a bicarbonate transporter having at least 98% sequence identity to SEQ ID NO: 4.

23. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide encodes a bicarbonate transporter having at least 99% sequence identity to SEQ ID NO: 4.

24. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide has at least 97% sequence identity to SEQ ID NO: 3, or the cDNA sequence thereof.

25. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide has at least 98% sequence identity to SEQ ID NO: 3, or the cDNA sequence thereof.

26. The recombinant host cell of claim 1 , wherein the heterologous polynucleotide has at least 99% sequence identity to SEQ ID NO: 3, or the cDNA sequence thereof.

27. A recombinant Aspergillus oryzae host cell comprising a heterologous polynucleotide that encodes a bicarbonate transporter, wherein:
the heterologous polynucleotide is operably linked to a promoter foreign to the polynucleotide;
the heterologous polynucleotide encodes a bicarbonate transporter having at least 95% sequence identity to SEQ ID NO: 4; and
the host cell is capable of producing a greater amount of malic acid compared to the host cell without the heterologous polynucleotide when cultivated under the same conditions.

28. The recombinant host cell of claim 27 , wherein the heterologous polynucleotide encodes a bicarbonate transporter that comprises or consists of SEQ ID NO: 4.

29. A method of producing a C4-dicarboxylic acid, comprising:
(a) cultivating the recombinant host cell of claim 1 in a medium under suitable conditions to produce the C4-dicarboxylic acid; and
(b) recovering the C4-dicarboxylic acid.

30. The method of claim 29 , wherein the C4-dicarboxylic acid is malic acid.

Assignees

• Novozymes Inc

Inventors

• Fischer Amanda
• Brown Stephen
• Luttringer Sheryl

Classifications

• C07K14/38

  from Aspergillus · CPC title

• C12P7/46 Primary

  Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid · CPC title

• C12N1/14

  Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor · CPC title