CN-119570641-B - Recombinant aspergillus niger strain for high production of trans-aconitic acid and application thereof

Abstract

The invention provides a recombinant aspergillus niger strain for high production of trans-aconitic acid, belonging to the technical field of genetic engineering. The recombinant Aspergillus niger strain can express aconitase and aconitase isomerase simultaneously. The genetically engineered aspergillus niger strain provided by the invention has the advantage that the fermentation yield of trans-aconitic acid is generally higher than that of an original strain, wherein the strain with the highest yield can reach 51g/L on the shake flask level, and the genetically engineered aspergillus niger strain has a very high application value.

Inventors

• LV XUEFENG
• WANG MIN
• HUANG XUENIAN
• Guo laixian
• GU MENG
• ZHANG YUNPENG

Assignees

• 中国科学院青岛生物能源与过程研究所

Dates

Publication Date

20260512

Application Date

20230905

Claims (8)

1. 1. A recombinant aspergillus niger strain for producing trans-aconitic acid, characterized in that the recombinant aspergillus niger strain heterologously expresses aconitase and aconitic acid isomerase; The aconitase is Aco, and the aconitase is TbrA or Adi1; the amino acid sequence of aconitase Aco is shown as SEQ ID NO. 1; the amino acid sequence of aconitate isomerase TbrA is shown as SEQ ID NO. 2; The amino acid sequence of aconitate isomerase Adi1 is shown in SEQ ID NO. 3.
2. 2. The recombinant Aspergillus niger strain of claim 1, wherein the recombinant Aspergillus niger strain is one or more of Aspergillus niger MEFC1501, aspergillus niger ATCC 1015, aspergillus niger NRRL 41873, aspergillus niger NRRL 27809, aspergillus niger NRRL 31821, aspergillus niger NRRL 6276, aspergillus niger Co827, aspergillus niger 5016, aspergillus niger ATCC 9142, aspergillus niger MTCC 282, and wherein the Aspergillus niger MEFC1501 is deposited with China general microbiological culture Collection center with a strain deposit number of CGMCC No.40614.
3. 3. The recombinant aspergillus niger strain according to claim 1, wherein the recombinant aspergillus niger strain is obtained by introducing an expression cassette containing aconitase gene and an expression cassette containing aconitase gene.
4. 4. A recombinant aspergillus niger strain according to claim 3, characterized in that the expression cassette comprises a promoter selected from one or more of the group consisting of pgla promoter, pgpdAt promoter, pgpdAn promoter, pacoA promoter, pcitA promoter, picdA promoter, pcadA promoter, pmfsA promoter, pgasA promoter.
5. 5. A recombinant aspergillus niger strain according to claim 3, wherein the expression cassette comprises a terminator selected from TtrpC terminator or Tpgk terminator.
6. 6. A whole cell catalyst comprising the recombinant aspergillus niger strain according to any of claims 1-5.
7. 7. Use of a recombinant strain of aspergillus niger according to any of claims 1-5 or of a whole-cell catalyst according to claim 6 for the production of trans aconitic acid.
8. 8. A method for producing trans aconitic acid, which is characterized by comprising the following steps: Step one, constructing and obtaining the recombinant aspergillus niger strain according to any of claims 1-5; and step two, inoculating the recombinant aspergillus niger strain into a fermentation medium for fermentation to obtain trans-aconitic acid.

Description

Recombinant aspergillus niger strain for high production of trans-aconitic acid and application thereof Technical Field The invention belongs to the field of genetic engineering, and in particular relates to a recombinant Aspergillus niger strain for high-yield trans-aconitic acid, a method for producing trans-aconitic acid by using the recombinant Aspergillus niger strain and application of the recombinant Aspergillus niger strain. Background Trans aconitic acid (trans-Aconitic acid, CAS: 4023-65-8) is an unsaturated tricarboxylic acid. Because of containing unsaturated double bond and three carboxyl groups, the polymer can be used as a monomer compound for preparing a polymer material, can also be used as a synthesis precursor of other compounds, and is listed as one of the platform compounds with the most application value by the United states department of energy. The trans-aconitate prepared by taking trans-aconitic acid as a raw material is a new-generation plasticizer for PVC, PLA and other plastic industries with safety, environmental protection and excellent performance, and has wide application prospect. Trans aconitic acid also has good nematicide activity, is a novel biopesticide for preventing and treating nematodes, and can be used as a food additive for enhancing the flavor of foods, beverages and the like. In addition, trans aconitic acid is used as a stereoisomer of cis aconitic acid (cis-Aconitic acid, CAS: 585-84-2) which is a key intermediate of tricarboxylic acid cycle, has a certain inhibition effect on aconitase which is a key enzyme in tricarboxylic acid cycle, can interfere with tricarboxylic acid cycle, thereby affecting vital activity and exhibiting a certain biological activity. At present, trans-aconitic acid is mainly produced by a chemical synthesis method, and has the advantages of complex process, more byproducts and high cost, so that large-scale production is not formed. In order to develop a more green and efficient trans-aconitic acid production process, early researchers obtain an aspergillus terreus engineering strain (CN 112011469A, CN 112029671A) capable of producing aconitic acid by blocking a cis-aconitic acid decarboxylase gene (cadA) catalyzing cis-aconitic acid decarboxylation to produce itaconic acid in aspergillus terreus and overexpressing aconitic acid isomerase CICC00358, but the yield is still lower and cannot meet the requirement of large-scale production. In addition to aspergillus terreus, many fungi have the capability of efficiently synthesizing organic acid, and in common industrial chassis strains, aspergillus niger (Aspergillus niger) is an excellent citric acid producing strain, and the produced citric acid is a direct precursor of cis-aconitic acid, and the aspergillus niger does not synthesize itaconic acid, so that the synthesis of itaconic acid is not required to be blocked, and the path for synthesizing trans-aconitic acid can be simplified compared with aspergillus terreus. In addition, aspergillus niger is a traditional fermentation strain widely used in the food industry, is recognized by the American FDA and world health organization as a generally safe, reliable and nontoxic (GENERAL REGARDED AS SAFE, GRAS) microorganism, and can be used in a large number of production fields of proteins, organic acids, antigens and the like as a cell factory. Aspergillus niger is thus a potential strain for large-scale production of trans-aconitic acid. At present, no report exists on the production of trans-aconitic acid by adopting Aspergillus niger. In addition, aspergillus tubingensis, aspergillus date, aspergillus usamii, candida and the like can also be used as potential hosts for producing trans aconitic acid. Disclosure of Invention In order to solve the defects of high cost, low yield, complex process and the like existing in the existing plant extraction or chemical synthesis production of trans-aconitic acid, the invention provides aconitase capable of efficiently catalyzing synthesis of cis-aconitic acid, aconitic acid isomerase capable of efficiently catalyzing conversion of cis-aconitic acid into trans-aconitic acid, an Aspergillus niger genetic engineering strain capable of synthesizing trans-aconitic acid, and a method for producing trans-aconitic acid by fermentation of Aspergillus niger, so as to realize a one-step method for producing trans-aconitic acid, namely producing trans-aconitic acid by one-step fermentation of a carbon source in microbial cells. Alternatively, the microorganism having trans aconitic acid synthesis ability includes, but is not limited to, the aspergillus niger (Aspergillus niger). In one aspect, the application provides a recombinant Aspergillus niger strain capable of producing trans-aconitic acid in high yield, wherein the recombinant Aspergillus niger strain can express aconitase and aconitate isomerase simultaneously. In one embodiment, the aconitase is Aco and the aconitate isomerase is selected from TbrA