Production of organic acids from Aspergillus cis-aconitic acid decarboxylase (cadA) deletion strains

Patent metadata

Field	Value
Publication number	US-10947548-B2
Application number	US-201916393149-A
Country	US
Kind code	B2
Filing date	Apr 24, 2019
Priority date	Apr 24, 2018
Publication date	Mar 16, 2021
Grant date	Mar 16, 2021

Abstract

This application provides recombinant Aspergillus fungi having an endogenous cis-aconitic acid decarboxylase (cadA) gene genetically inactivated, which allows aconitic acid production by the recombinant fungi. Such recombinant fungi can further include an exogenous nucleic acid molecule encoding aspartate decarboxylase (panD), an exogenous nucleic acid molecule encoding β-alanine-pyruvate aminotransferase (BAPAT), and an exogenous nucleic acid molecule encoding 3-hydroxypropionate dehydrogenase (HPDH). Kits including these fungi, and methods of using these fungi to produce aconitic acid and 3-hydroxypropionic acid (3-HP) are also provided.

First claim

We claim:

1. An isolated recombinant Aspergillus fungus capable of producing 3-hydroxypropionic acid (3-HP), comprising:
a genetic inactivation of an endogenous cis-aconitic acid decarboxylase (cadA) gene,
an exogenous nucleic acid molecule encoding aspartate 1-decarboxylase (panD),
an exogenous nucleic acid molecule encoding β-alanine-pyruvate aminotransferase (BAPAT), and
an exogenous nucleic acid molecule encoding 3-hydroxypropionate dehydrogenase (HPDH).

2. The isolated recombinant Aspergillus fungus of claim 1 , wherein the Aspergillus fungus is Aspergillus pseudoterreus.

3. The isolated recombinant Aspergillus fungus of claim 1 , wherein the Aspergillus fungus is Aspergillus terreus.

4. The isolated recombinant Aspergillus fungus of claim 1 , wherein the endogenous cadA gene is genetically inactivated by complete or partial deletion mutation or by insertional mutation.

5. The isolated recombinant Aspergillus fungus of claim 1 , wherein the cadA gene prior to its genetic inactivation encodes a cis-aconitic acid decarboxylase with an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 50 or 52.

6. The isolated recombinant Aspergillus fungus of claim 1 , wherein the cadA gene prior to its genetic inactivation comprises a coding sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:49, 51, 59 or 92.

7. The isolated recombinant Aspergillus fungus of claim 1 , wherein the exogenous nucleic acid molecule encoding panD
comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 53 or 65, and/or
encodes an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 54.

8. The isolated recombinant Aspergillus fungus of claim 1 , wherein the exogenous nucleic acid molecule encoding BAPAT
comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 55 or 71, and/or
encodes an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 56.

9. The isolated recombinant Aspergillus fungus of claim 1 , wherein the exogenous nucleic acid molecule encoding HPDH
comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 57 or 80, and/or
encodes an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 58.

10. The isolated recombinant Aspergillus fungus of claim 1 , wherein the exogenous nucleic acid molecule encoding panD, the exogenous nucleic acid molecule encoding BAPAT, and the exogenous nucleic acid molecule encoding HPDH are part of a single exogenous nucleic acid molecule.

11. A composition comprising the isolated recombinant Aspergillus fungus of claim 1 .

12. A kit, comprising:
the isolated recombinant Aspergillus fungus of claim 1 ; and
a medium for culturing the fungus.

13. A method of making 3-hydroxypropionic acid (3-HP), comprising:
culturing the isolated Aspergillus fungus of claim 1 in a culture media under conditions that permit the isolated Aspergillus fungus to make 3-HP; thereby making 3-HP.

14. The method of claim 13 , wherein the isolated Aspergillus fungus is cultured in Riscaldati medium or modified Riscaldati medium comprising 20× trace elements.

15. The method of claim 13 , further comprising isolating the 3-HP from the culture media or from the isolated Aspergillus fungus.

Assignees

• Battelle Memorial Institute

Inventors

• Deng Shuang
• Dai Ziyu
• Magnuson Jon K

Classifications

• C12N15/80 Primary

  for fungi · CPC title

• C12Y101/01059

  3-Hydroxypropionate dehydrogenase (1.1.1.59) · CPC title

• C12Y401/01011

  Aspartate 1-decarboxylase (4.1.1.11) · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title

• C12P7/48

  Tricarboxylic acids, e.g. citric acid · CPC title