Method for increasing citric acid production by Aspergillus niger fermentation

Patent metadata

Field	Value
Publication number	US-10612007-B2
Application number	US-201815867836-A
Country	US
Kind code	B2
Filing date	Jan 11, 2018
Priority date	Jan 12, 2017
Publication date	Apr 7, 2020
Grant date	Apr 7, 2020

Abstract

The invention discloses a method for improving citric acid production by Aspergillus niger fermentation, which integrates Aspergillus niger GABA pathway succinate semialdehyde dehydrogenase SSD gene into Aspergillus niger genome to obtain recombinant Aspergillus niger strain, and uses recombinant black The Aspergillus strain ferments to produce citric acid; the expression of the succinate semialdehyde dehydrogenase SSD gene is regulated by the Pgas promoter. The method of the invention realizes the expression of succinate semialdehyde dehydrogenase SSD in Aspergillus niger to enhance the GABA pathway so as to strengthen the TCA cycle and promote the synthesis of citric acid.

First claim

What is claimed is:

1. A recombinant Aspergillus niger strain, comprising:
a genome comprising an inserted Aspergillus niger gamma-aminobutyric acid (GABA) pathway succinate semialdehyde dehydrogenase (SSD) gene, wherein the GABA pathway SSD gene is under control of a Pgas promoter,
wherein the GABA pathway SSD gene is set forth in SEQ ID NO:1,
wherein the Pgas promoter sequence is SEQ ID NO:3,
wherein the A. niger strain produces a higher amount of citrate as compared with a wild type A. niger strain grown under identical conditions.

2. The recombinant A. niger strain of claim 1 , wherein the SSD gene produces a protein having an amino acid sequence set forth in SEQ ID NO: 2.

3. The recombinant A. niger strain of claim 1 , wherein the Pgas promoter is induced by a low pH of between pH 2.0 and pH 3.5.

4. The recombinant Aspergillus niger strain of claim 1 , wherein the gene sequence of GABA pathway SSD gene and the Pgas promoter are encoded onto an expression cassette transformed into the A. niger strain.

5. The recombinant Aspergillus niger strain of claim 4 , wherein the expression cassette further comprises at least one trp terminator sequence of SEQ ID NO:6.

6. The recombinant Aspergillus niger strain of claim 4 , wherein the expression cassette further comprises a gpdA promoter sequence of SEQ ID NO:4.

7. The recombinant Aspergillus niger strain of claim 4 , wherein the expression cassette further comprises a hygromycin resistance (hph) gene sequence of SEQ ID NO:5.

8. The recombinant Aspergillus niger strain of claim 4 , wherein the expression cassette encodes a trp terminator sequence of SEQ ID NO:6, a gpdA promoter sequence of SEQ ID NO:4, and an hgh gene sequence of SEQ ID NO:5.

9. The recombinant Aspergillus niger strain of claim 8 , wherein the expression cassette sequences are in the order of gpdA-hph-trp and Pgas-SSD-trp.

10. The recombinant Aspergillus niger strain of claim 1 , wherein the A. niger strain is derived from A. niger strain H915-1.

11. The recombinant Aspergillus niger strain of claim 1 , wherein the amount of citrate produced by the A. niger strain is at least 10% higher under identical conditions than a corresponding wild type Aspergillus niger strain.

12. The recombinant Aspergillus niger strain of claim 1 , wherein the amount of citrate produced by the A. niger strain is at least 45.2% higher under identical conditions than a corresponding wild type Aspergillus niger strain.

13. An expression cassette of succinate semialdehyde dehydrogenase SSD, comprising a promoter Pgas, a succinate semialdehyde dehydrogenase SSD gene and a terminator trp in order of Pgas-LGT1-trp.

14. The expression cassette in claim 13 , wherein a sequence of the promoter Pgas is set forth in SEQ ID NO: 3, an amino acid sequence of succinate semialdehyde dehydrogenase SSD is set forth in SEQ ID NO: 2, and a sequence of terminator trp is set forth in SEQ ID NO: 6.

15. A method for the reconstruction of reconstructed A. niger mentioned in claim 1 contains the following steps:
(1) constructing an expression cassette of succinate semialdehyde dehydrogenase with Pgas-SSD-trp;
(2) constructing a resistant gene expression cassette gpdA-hph-trp;
(3) inserting expression cassettes in step (1) and (2) into A. niger , screening resistant strains and confirming reconstructed strains with PCR.

16. The method in claim 15 , wherein a sequence of gpdA promoter in resistant gene cassette is set forth in SEQ ID NO: 4.

17. The method in claim 15 , wherein a sequence of resistant gene hph in resistant gene cassette is set forth in SEQ ID NO: 5.

18. A method of expressing citric acid from Aspergillus niger , which comprises:
incubating an A. niger strain in growth medium comprising malt extract and tryptone at 35° C. for seven days to generate spores, wherein the A. niger strain comprises a genome comprising a gamma-aminobutyric acid (GABA) pathway succinate semialdehyde dehydrogenase (SSD) gene, wherein the SSD gene is under the control of a Pgas promoter, wherein the SSD gene sequence is SEQ ID NO:1, and wherein the Pgas promoter sequence is SEQ ID NO:3,
harvesting the spores,
inoculating seed culture medium with the harvested spores at a density of 10 6 spores per mL to generate a seed culture, wherein the seed medium comprises corn starch medium comprising a total sugar concentration of 10% and a total nitrogen concentration of 0.2%,
growing the spores in the seed culture medium at 37° C. for 24 hours at pH 3.5,
inoculating a fermentation medium with the seed culture at 1/10 volume,
incubating the fermentation medium for 72 hours at 35° C. at pH 2.0, and
centrifuging the fermentation medium and discarding bacteria to obtain citric acid.

19. The method of claim 18 , wherein the A. niger strain produces a higher amount of citrate as compared with a wild type A. niger strain grown under identical conditions.

20. The method of claim 18 , wherein the A. niger strain produces between 10% and 45.2% more citrate as compared with a wild type A. niger strain under identical conditions.

Assignees

• Univ Jiangnan
• Jiangsu Guoxin Union Energy Co Ltd

Inventors

• Liu Long
• Chen Jian
• Du Guocheng
• Li Jianghua
• Yin Xian
• Hu Zhijie
• Jiang Jianwei
• Sun Fuxin
• Jin Sai
• Zhang Cheng
• Jiang Xiaodong

Classifications

• C12N9/0008 Primary

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

• C12P7/48 Primary

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

• C12Y102/01076

  Succinate-semialdehyde dehydrogenase (acetylating) (1.2.1.76) · CPC title

• C12N15/80

  for fungi · CPC title