Recombinant aspergillus niger with high yield of trans-aconitic acid as well as construction method and application of recombinant aspergillus niger

Abstract

The invention provides recombinant aspergillus niger capable of producing trans-aconitic acid at high yield as well as a construction method and application of the recombinant aspergillus niger, and relates to the technical field of genetic engineering. Specifically, the recombinant aspergillus niger is used for heterologous expression of aconitic acid isomerase TbrA and / or Adi1. A new engineering strain is provided for efficient production of trans-aconitic acid, the source dilemma that plant extraction or chemical synthesis is needed in traditional production of trans-aconitic acid is solved, and the yield of trans-aconitic acid is greatly increased.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a recombinant Aspergillus niger with high trans-aconitic acid production, a construction method thereof, and an application thereof. Background Art

[0002] Trans-Aconitic acid (CAS: 4023-65-8) is an unsaturated tricarboxylic acid. Because it contains unsaturated double bonds and three carboxyl groups, it can be used as a monomer compound for preparing polymer materials, and can also be used as a synthetic precursor for other compounds. It is listed as one of the most valuable platform compounds by the U.S. Department of Energy. Trans-aconitic acid esters prepared with trans-aconitic acid as raw material are a new generation of safe, environmentally friendly, and excellent plasticizers for PVC, PLA and other plastic industries, and have broad application prospects. Trans-aconitic acid also has good nematicidal activity and is a new type of biological pesticide for nematode control. In addition, trans-aconitic acid, as a stereoisomer of cis-Aconitic acid (CAS: 585-84-2), a key intermediate of the tricarboxylic acid cycle, has a certain inhibitory effect on aconitase, a key enzyme in the tricarboxylic acid cycle, can interfere with the tricarboxylic acid cycle, thereby affecting life activities and showing certain biological activity. In addition, trans-aconitic acid can also be used as a food additive to enhance the flavor of food, beverages, etc.

[0003] Trans-aconitic acid is mainly produced by plant extraction or chemical synthesis. The content in plants is extremely low, while the chemical synthesis process is complex, with many by-products, high cost, and no large-scale production. In order to develop a greener and more efficient trans-aconitic acid production process, the researchers used the industrial production strain of itaconic acid, Aspergillus terreus, as a cell factory. They first blocked the aconitate decarboxylase gene (cadA) in Aspergillus terreus that catalyzes the decarboxylation of aconitate to produce itaconic acid. On this basis, they used the promoter PcadA of the aconitate decarboxylase gene to overexpress aconitate isomerase, and obtained an Aspergillus terreus engineered strain that can produce aconitate (CN 112011469A, CN112029671A), but the yield and production efficiency of trans-aconitate still need to be improved. Summary of the invention

[0004] It is found in the present invention that, except Aspergillus terreus, many fungi have the ability to efficiently synthesize organic acids. In the commonly used industrial chassis strains, Aspergillus niger is an excellent citric acid producer, and the citric acid produced by it is the direct precursor of cis-aconitic acid, and Aspergillus niger does not synthesize itaconic acid, so there is no need to block the synthesis of itaconic acid, and compared with Aspergillus terreus, the path of synthesizing trans-aconitic acid can be simplified. In addition, Aspergillus niger is a traditional fermentation strain widely used in the food industry, and is identified as a generally safe, reliable and non-toxic (Generally regarded as safe, GRAS) microorganism by the U.S. FDA and the World Health Organization, and can be used as a cell factory in a large number of production fields such as proteins, organic acids, antigens, etc. Therefore, Aspergillus niger is a potential strain for large-scale production of trans-aconitic acid. And there is no relevant report of using Aspergillus niger to produce trans-aconitic acid.

[0005] In addition, Aspergillus tubingensis, Aspergillus date palm, Aspergillus usami, Candida albicans, etc. can also be used as potential hosts for the production of trans-aconitic acid.

[0006] The purpose of the present invention is to provide a recombinant Aspergillus niger that can efficiently catalyze the conversion of cis-aconitic acid into trans-aconitic acid, and a method for producing trans-aconitic acid by fermentation using the above-mentioned recombinant Aspergillus niger, aiming to solve the defects of the existing plant extraction or chemical synthesis production of trans-aconitic acid, such as high cost, low yield, and complex process.

[0007] In one aspect, the present application provides a recombinant Aspergillus niger that produces high levels of trans-aconitic acid, wherein aconitate isomerase is heterologously expressed in the recombinant Aspergillus niger;

[0008] The aconitate isomerase comprises TbrA and / or Adi1.

[0009] This application is the first to use Aspergillus niger to produce trans-aconitic acid. Since the citric acid produced in Aspergillus niger is the direct precursor of cis-aconitic acid, and Aspergillus niger does not synthesize itaconic acid, compared with the existing trans-aconitic acid engineering bacteria Aspergillus terreus, there is no need to block the synthesis of itaconic acid in Aspergillus niger, which simplifies the synthesis of trans-aconitic acid, saves the cost of engineering bacteria production, and improves the production efficiency of trans-aconitic acid.

[0010] In addition, since the aconitase Aco of Aspergillus niger itself is subject to strict feedback inhibition regulation, it is unable to accumulate cis-aconitic acid in large quantities. In the present application, aconitate isomerase is introduced into Aspergillus niger to convert the cis-aconitic acid produced in Aspergillus niger into trans-aconitic acid, thereby avoiding feedback inhibition regulation in the synthesis process of cis-aconitic acid.

[0011] Further, the recombinant Aspergillus niger is selected from one or more of Aspergillus niger MEFC1501, Aspergillus niger ATCC 1015, Aspergillus niger NRRL41873, Aspergillus niger NRRL 27809, Aspergillus niger NRRL 31821, Aspergillus niger NRRL 6276, Aspergillus niger Co827, Aspergillus niger 5016, Aspergillus niger 3008, Aspergillus niger GCB75, Aspergillus niger ATCC 9142, and Aspergillus niger MTCC 282.

[0012] Furthermore, the recombinant Aspergillus niger also includes a promoter, which promotes the expression of aconitate isomerase; preferably, the promoter is selected from one or more of PglaA promoter, PgpdAt promoter, PgpdAn promoter, PacoA promoter, PcitA promoter, PicdA promoter, PcadA promoter, PmfsA promoter, and Pgas promoter; more preferably, the promoter is PglaA promoter.

[0013] More preferably, the PglaA promoter sequence is SEQ ID NO.3.

[0014] More preferably, the PgpdAt promoter sequence is shown as SEQ ID NO.4, the PgpdAn promoter sequence is shown as SEQ ID NO.5, the PacoA promoter sequence is shown as SEQ ID NO.6, the PcitA promoter sequence is shown as SEQ ID NO.7, the PicdA promoter sequence is shown as SEQ ID NO.8, the PcadA promoter sequence is shown as SEQ ID NO.9, the PmfsA promoter sequence is shown as SEQ ID NO.10, and the Pgas promoter sequence is shown as SEQ ID NO.11.

[0015] Furthermore, the amino acid sequence of TbrA includes the sequence shown in SEQ ID No.1 or an amino acid sequence having at least 60% sequence homology with the sequence of SEQ ID NO.1; preferably, the aconitate isomerase gene encoding TbrA includes the sequence shown in SEQ ID No.14 or a nucleotide sequence having at least 60% sequence homology with the sequence of SEQ ID NO.14.

[0016] Furthermore, the amino acid sequence of Adi1 includes an amino acid sequence as shown in SEQ ID No.2 or an amino acid sequence having at least 60% sequence homology with the SEQ ID NO.2 sequence; preferably, the aconitate isomerase gene encoding Adi1 includes a sequence as shown in SEQ ID No.15 or a nucleotide sequence having at least 60% sequence homology with the SEQ ID NO.15 sequence.

[0017] The aconitate isomerase TbrA is derived from Bacillus thuringiensis, and the aconitate isomerase Adi1 is derived from Ustilago maydis.

[0018] Preferably, the amino acid sequence of TbrA includes the sequence shown in SEQ ID No.1; the aconitate isomerase gene encoding the TbrA includes the sequence shown in SEQ ID No.14; the amino acid sequence of Adi1 includes the sequence shown in SEQ ID No.2; the aconitate isomerase gene encoding the Adi1 includes the sequence shown in SEQ ID No.15.

[0019] Furthermore, the recombinant Aspergillus niger also includes a terminator, and the terminator terminates the expression of aconitate isomerase; preferably, the terminator includes a TtrpC terminator and / or a Tpgk terminator.

[0020] In a preferred embodiment, the TtrpC terminator sequence is shown as SEQ ID NO.12.

[0021] In a preferred embodiment, the Tpgk terminator sequence is shown as SEQ ID NO.13.

[0022] The terminator for terminating the expression of the aconitate isomerase in the present application can be a common terminator for fungi or a terminator specific to the host bacteria. Those skilled in the art can make an adaptive selection according to the actual situation. The present application does not impose a mandatory limitation on the sequence of the terminator.

[0023] On the other hand, the present application also provides a biomaterial, wherein the biomaterial is any one of A1) to A3):

[0024] A1) an expression cassette, the expression cassette comprising a promoter, an aconitate isomerase gene and a terminator; the aconitate isomerase comprises TbrA and / or Adi1;

[0025] A2) a recombinant vector, wherein the recombinant vector contains the expression cassette described in A1);

[0026] A3) A whole-cell catalyst, wherein the whole-cell catalyst contains the recombinant vector described in A2) or the above-mentioned recombinant Aspergillus niger.

[0027] In a preferred embodiment, the aconitate isomerase expression cassette is located on a recombinant vector or is introduced into a recombinant microorganism using a recombinant vector, and the recombinant vector can be pUC57.

[0028] A person skilled in the art can select a known plasmid to construct a recombinant vector according to actual conditions and perform conventional optimization and modification on the constructed recombinant vector. The present application does not impose mandatory restrictions on the type of plasmid.

[0029] Those skilled in the art can imagine that other commonly used expression elements can be adaptively added to the expression cassette described in the present application to assist the expression of aconitate isomerase in Aspergillus niger, such as adding tags, fluorescent protein markers, resistance screening markers, etc.

[0030] In a preferred embodiment, the recombinant vector and the recombinant microorganism contain a resistance screening marker gene, which can be selected by those skilled in the art according to actual conditions, and the present application does not impose mandatory limitations on the resistance screening marker gene.

[0031] On the other hand, the present application also provides a method for constructing the above-mentioned recombinant Aspergillus niger with high trans-aconitate production, the method comprising: introducing an aconitate isomerase gene into Aspergillus niger to obtain a recombinant Aspergillus niger with high trans-aconitate production; the aconitate isomerase comprises TbrA and / or Adi1.

[0032] On the other hand, the present application also provides the use of the above-mentioned recombinant Aspergillus niger or the above-mentioned biological material in the production of trans-aconitic acid.

[0033] On the other hand, the present application also provides a method for producing trans-aconitic acid, the method comprising:

[0034] Step 1: constructing the above-mentioned recombinant Aspergillus niger or the above-mentioned biological material;

[0035] Step 2: inoculating the recombinant Aspergillus niger into a fermentation medium for fermentation to obtain trans-aconitic acid.

[0036] Preferably, the fermentation medium uses monosaccharides, polysaccharides or mixtures thereof that can be utilized by Aspergillus niger as carbon sources, and the carbon sources include but are not limited to glucose, fructose, sucrose, molasses, corn flour, potato starch powder, starch hydrolyzate and the like.

[0037] In a preferred embodiment, the fermentation medium comprises: 160 g L -1 Glucose, 2 g L -1 NH4NO3, 0.2 g L -1 (NH4)2HPO4, 20mg L -1 FeSO4, 0.4 g L -1 MgSO4, 40 mg L -1 ZnSO4, 40mg L -1 CuSO4, pH 3.5.

[0038] In a preferred embodiment, the method for producing trans-aconitic acid comprises the following steps:

[0039] The recombinant Aspergillus niger heterologously expressing aconitate isomerase was inoculated into the Aspergillus niger spore production medium and cultured at 32° C. for 6 days to obtain mature spores. The mature spores were then inoculated into the fermentation medium and fermented at 34° C. and 220 rpm for 135 hours.

[0040] The recombinant Aspergillus niger comprises a promoter, an aconitate isomerase gene and a terminator, wherein the promoter is a PglaA promoter, the aconitate isomerase encoded by the aconitate isomerase gene is TbrA or Adi1, and the terminator is a TtrpC terminator or a Tpgk terminator.

[0041] The trans-aconitic acid production of the modified recombinant Aspergillus niger strain in the present application is generally higher than that of the starting strain, among which the strain with the highest production reaches 35 g / L at the shake flask level, and has strong application value.

[0042] On the other hand, the present application also provides trans-aconitic acid produced by the above method.

[0043] The present invention has the following beneficial effects:

[0044] 1. The process of synthesizing trans-aconitic acid using the recombinant Aspergillus niger is safe, reliable and non-toxic; Aspergillus niger is a microorganism recognized as generally safe, reliable and non-toxic (GRAS) by the U.S. FDA and the World Health Organization, and is widely used in traditional fermentation strains in the food industry. This application uses Aspergillus niger as a starting strain, heterologously expresses aconitate isomerase, and then obtains a recombinant Aspergillus niger that produces trans-aconitic acid, providing a highly safe engineering strain for the efficient production of trans-aconitic acid;

[0045] 2. The fermentation production of trans-aconitic acid using the recombinant Aspergillus niger constructed in the present application solves the source dilemma of trans-aconitic acid, which requires plant extraction or chemical synthesis in traditional production, and also avoids the side effect of itaconic acid produced in existing trans-aconitic acid engineering strains (such as Aspergillus terreus). The recombinant Aspergillus niger described in the present application greatly increases the production of trans-aconitic acid, among which the strain with the highest yield reaches 35 g / L at the shake flask level, which has a strong application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0047] Figure 1 The statistical diagram of the synthesis of cis-aconitic acid and trans-aconitic acid by shake flask fermentation of recombinant Aspergillus niger strains using different promoters to construct isomerase TbrA expression cassettes;

[0048] Figure 2 The statistical diagram of the synthesis of cis-aconitic acid and trans-aconitic acid by shake flask fermentation of recombinant Aspergillus niger strains using different promoters to construct isomerase Adi1 expression cassettes;

[0049] Figure 3 This is a statistical diagram of the synthesis of cis-aconitic acid and trans-aconitic acid by shake flask fermentation of a recombinant Aspergillus niger strain using pyrithiamine ptrA as a screening marker.

[0050] Strain deposit information:

[0051] Aspergillus niger MEFC1501 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the culture deposit number: CGMCC NO.40614, the deposit date: April 27, 2023, and the deposit location is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0052] Technical terms:

[0053] Aconitate isomerase (AI): Aconitate isomerase is an enzyme protein that can convert cis-aconitate into trans-aconitate.

[0054] Expression: The term "expression" includes any step involved in the production of aconitate isomerase or a mutant thereof including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0055] Homology: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules in molecular evolution research.

[0056] Recombination: Broadly speaking, any gene exchange process that causes genotype changes is called recombination.

[0057] One-step cloning: One-step cloning method homologous recombination is a technology that uses the principle of homologous recombination to perform seamless cloning.

[0058] Expression cassette: An expression cassette is a group of DNA sequences consisting of a promoter, target gene and reporter gene, which can be expressed in specific tissues and easily detected.

[0059] Expression vector: An expression vector is a vector that adds expression elements (such as promoter, RBS, terminator, etc.) to the basic skeleton of a cloning vector to enable the expression of the target gene.

[0060] Whole-cell catalyst: Whole-cell biocatalysis refers to the process of using a complete biological organism (i.e., whole cell, tissue, or even individual) as a catalyst for chemical transformation. The corresponding complete biological organism involved in the catalytic process is the whole-cell catalyst.

[0061] Engineered strain: A fungal cell strain that uses genetic engineering methods to efficiently express exogenous genes.

[0062] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0063] Introduction: The process of integrating exogenous genes into the genome of target cells for stable expression, or the process of inserting exogenous genes into vectors and introducing them into host cells for expression, as well as other forms of introducing exogenous genes into host cells for expression are called gene introduction.

[0064] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0065] Unless otherwise specified, in the following embodiments, reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market. If no specific conditions are indicated in the examples, conventional conditions or conditions recommended by the manufacturer are followed.

[0066] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are performed in accordance with the kit instructions.

[0067] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields, and can be specifically performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0068] In the present invention, the plasmid extraction adopts the Plasmid Mini KitI kit (D6943-02) of OMEGA Company, the DNA fragment recovery adopts the Cycle-Pure Kit kit (D6492-02) of OMEGA Company, and the gel recovery adopts the Gel Extraction Kit kit (D2500-01) of OMEGA Company.

[0069] Aspergillus niger solid medium PDA: 3.9 g L -1 Potato dextrose agar medium (Difco™ Potato Dextrose Agar, BD, LOT: 1165825) was sterilized and plate preparation was performed.

[0070] Aspergillus niger liquid medium PDB: 2.4 g L -1 Potato dextrose medium (Difco™ Potato Dextrose Broth, BD, LOT: 2095227).

[0071] Aspergillus niger spore production (slant) medium: 10 g L -1 Glucose, 2 g L -1 NaNO3, 0.2 g L -1 KH2PO4, 20 mg L - 1 FeSO4, 5 g L -1 MgSO4, 0.5 g L -1 NaCl, 40 mg L -1 ZnSO4, 40mg L -1 CuSO4, 1.5% agar, sterilize at 115℃ for 15min, dispense into test tubes, and prepare slant.

[0072] Regeneration screening medium plate PDA-SH: 3.9 g L -1 Potato dextrose agar medium (DifcoTM Potato Dextrose Agar, BD, LOT: 1165825) and 1.2 M sorbitol were sterilized and cooled to about 55°C, and hygromycin B (Solarbio, Catalog No.: M419099) was added to a final concentration of 100 μg / mL to prepare a plate.

[0073] Regeneration screening medium top layer PDBS: 2.4 g L -1 Potato dextrose broth (Difco™ Potato Dextrose Broth, BD, LOT: 2095227), 1.2 M sorbitol and 0.5% agarose were sterilized to prepare top agar.

[0074] Regeneration screening medium plate CD-SPt: 10 g L -1 Glucose, 3 g L -1 NaNO3, 2 g L -1 KCl, 1 g L - 1 KH2PO4, 0.5 g L -1 MgSO4, 20 mg L -1 FeSO4, 1.5 g L -1 Agar, and 1.2 M sorbitol, the balance being water, are sterilized and cooled to about 55°C, and pyrithiamine (Sigma, Catalog No.: P0256) is added to a final concentration of 100 μg / L to prepare a plate.

[0075] Screening medium plate CD-Pt: 10 g L -1 Glucose, 3 g L -1 NaNO3, 2 g L -1 KCl, 1 g L -1 KH2PO4, 0.5gL -1 MgSO4, 20 mg L -1 FeSO4, 1.5 g L -1 Agar, with the balance being water, was sterilized and cooled to about 55°C, and pyrithiamine (Sigma, Catalog No.: P0256) was added to a final concentration of 100 μg / L to prepare a plate.

[0076] Aspergillus niger fermentation medium: 160 g L -1 Glucose, 2 g L -1 NH4NO3, 0.2 g L -1 (NH4)2HPO4, 20mg L - 1 FeSO4, 0.4 g L -1 MgSO4, 40 mg L -1 ZnSO4, 40mg L -1 CuSO4, pH 3.5.

[0077] Plasmid pSGF957 was obtained from Seoul National University. The plasmid is described in Kim, JG, Choi, YD, Chang, YJ, Kim, SU, Genetic transformation of Monascus purpureus DSM1379, Biotechnology Letters, 2003, 25, 1509-1514.

[0078] Aspergillus niger MEFC1501 is deposited in the China General Microbiological Culture Collection Center, the culture collection number is: CGMCC NO.40614, the preservation time is: April 27, 2023, and the preservation location is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0079] Aspergillus niger Co827 was purchased from China Industrial Microbiology Culture Collection Center.

[0080] Aspergillus niger NRRL 27809, NRRL 31821, NRRL 41873, and NRRL 6276 were purchased from the ARS Culture Collection.

[0081] Aspergillus terreus CICC 40205 was purchased from China Industrial Microbiological Culture Collection Center.

[0082] Plasmid pSGF957 (obtained from Seoul National University, the plasmid is described in Kim, JG, Choi, YD, Chang, YJ, Kim, SU, Genetic transformation of Monascus purpureus DSM1379, Biotechnology Letters, 2003, 25, 1509-1514).

[0083] Plasmid pAN52-4 (obtained from TNO Medical Biological Laboratory, the plasmid is described in Punt P.J., Zegers ND, Busscher M., Pouwels PH, van den Hondel CA, Journal of Biotechnology, 1991, 7, 19-33).

[0084] Plasmid pmWM23 is described in patent publication number CN111944706A.

[0085] Plasmid pUC57-Kan was purchased from Jinweizhi Biotechnology Co., Ltd.

[0086] The amino acid sequence of the following TbrA is shown in SEQ ID No.1; the amino acid sequence of Adi1 is shown in SEQ ID No.2; the PgpdAt promoter sequence is shown in SEQ ID NO.4; the PgpdAn promoter sequence is shown in SEQ ID NO.5; the PacoA promoter sequence is shown in SEQ ID NO.6; the PcitA promoter sequence is shown in SEQ ID NO.7; the PicdA promoter sequence is shown in SEQ ID NO.8; the PcadA promoter sequence is shown in SEQ ID NO.9; the PmfsA promoter sequence is shown in SEQ ID NO.10; the Pgas promoter sequence is shown in SEQ ID NO.11; the TtrpC terminator sequence is shown in SEQ ID NO.12; and the Tpgk terminator sequence is shown in SEQ ID NO.13.

[0087] The invention uses Aspergillus niger as a starting strain, expresses heterologous aconitate isomerase in the genome of the starting strain through genetic engineering, obtains a recombinant Aspergillus niger strain, and realizes the synthesis of trans-aconitate in Aspergillus niger, wherein the starting strain is a currently known Aspergillus niger capable of synthesizing citric acid, such as Aspergillus niger MEFC1501, Aspergillus niger Co827, Aspergillus niger NRRL 27809, Aspergillus niger NRRL 31821, Aspergillus niger NRRL 41873, Aspergillus niger NRRL 6276 or other genetically engineered bacteria or wild strains capable of synthesizing citric acid, and Aspergillus niger MEFC1501 is taken as an example to specifically describe the construction method of the recombinant Aspergillus niger strain of the invention.

[0088] Determination method of trans-aconitic acid, citric acid, cis-aconitic acid, etc.: The content and purity of each organic acid in the fermentation broth are detected by high performance liquid chromatography.

[0089] The present invention is the first to use Aspergillus niger to construct an engineered bacterium for producing trans-aconitic acid. Figure 1As shown, Aspergillus niger can efficiently synthesize citric acid, and citric acid can be converted into cis-aconitic acid under the action of aconitase Aco. Cis-aconitic acid can be partially converted into trans-aconitic acid in a natural state. In addition, there is an aconitate isomerase in microorganisms such as corn smut, Bacillus thuringiensis, and some plants, which can catalyze the isomerization of cis-aconitic acid to generate trans-aconitic acid (Geiser E. et al., Ustilagomayd is produces itaconic acid via the unusual intermediate trans-aconitate, Microbial Biotechnology, 2016, 9 (1): 116-126). The present invention uses Aspergillus niger as a starting strain, and heterologously expresses aconitate isomerase derived from corn smut or aconitate isomerase derived from Bacillus thuringiensis in Aspergillus niger, and obtains an Aspergillus niger engineered strain that produces trans-aconitic acid, which can solve the source dilemma of trans-aconitic acid that traditionally requires plant extraction or chemical synthesis by fermentation production.

[0090] Example 1 Construction of recombinant Aspergillus niger heterologously expressing aconitate isomerase TbrA from Bacillus thuringiensis

[0091] 1. Construction of the expression cassette for aconitate isomerase TbrA

[0092] The aconitate isomerase TbrA gene was artificially synthesized and codon optimized for Aspergillus niger expression. The optimized sequence was shown in SEQ ID NO.14 and cloned into the pUC57-Kan vector to construct the plasmid puc57-TbrA.

[0093] 1) Construction of the expression cassette of aconitate isomerase TbrA using the PgpdAt promoter

[0094] PCR amplification was performed using fuPgpdAt-TbrA-F (5'-ctcatcaatcatcacaacatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0095] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and fuPgpdAt-TbrA-R (5'-cgaagcaggggatcttcatgttgtgatgattgatgag-3') as primer pairs and plasmid pMWM23 as template. The product was purified and recovered to obtain the PgpdAt-TtrpC fragment.

[0096] The two fragments were cloned in one step, and PCR amplification was performed using PgpdAt-F (5'-ttacactctgggaggatccaggtac-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgpdAt-Tbr A-TtrpC expression cassette fragment.

[0097] 2) Construction of the expression cassette of aconitate isomerase TbrA using the PgpdAn promoter

[0098] PCR amplification was performed using fuPgpdAn-TbrA-F (5'-ccgcttgagcagacatcaccatgaagatcccctgcttcgt-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0099] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and fuPgpdAn-TbrA-R (5'-acgaagcaggggatcttcatggtgatgtctgctcaagcgg-3') as primer pairs and plasmid pAN52-4 as template, and the product was purified and recovered to obtain the fragment of PgpdAn-TtrpC. PgpdAn is the promoter of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene, and TtrpC is the terminator of the Aspergillus nidulans tryptophan synthase.

[0100] The two fragments were cloned in one step, and PCR amplification was performed using PgpdAn-F (5'-ttgatcgagacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgpdAn-TbrA-TtrpC expression cassette fragment.

[0101] 3) Construction of the expression cassette of aconitate isomerase TbrA using the PglaA promoter

[0102] PCR amplification was performed using fuPglaA-TbrA-F (5'-gcatcattacacctcagcaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0103] PCR amplification was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and PglaA-R (5'-tgctgaggtgtaatgatgctgggg at-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the PglaA promoter.

[0104] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0105] The three fragments were cloned in one step, and PCR amplification was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PglaA-TbrA-TtrpC expression cassette fragment.

[0106] 4) Construction of the expression cassette of aconitate isomerase TbrA using the PcadA promoter

[0107] PCR amplification was performed using fuPcadA-TbrA-F (5'-cctcttaaattgaccatgaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0108] PCR amplification was performed using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and PcadA-R (5'-tcatggtcaatttaagagg-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain the fragment of PcadA promoter.

[0109] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0110] The three fragments were cloned in one step, and PCR amplification was performed using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PcadA-Tbr A-TtrpC expression cassette fragment.

[0111] 5) Construction of the expression cassette of aconitate isomerase TbrA using the PmfsA promoter

[0112] PCR amplification was performed using fuPmfsA-TbrA-F (5'-accgacttctcatccatcttcaaaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0113] PCR amplification was performed using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the PmfsA promoter.

[0114] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0115] The three fragments were cloned in one step, and PCR amplification was performed using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PmfsA-TbrA-TtrpC expression cassette fragment.

[0116] 6) Construction of the expression cassette of aconitate isomerase TbrA using the PacoA promoter

[0117] PCR amplification was performed using fuPacoA-TbrA-F (5'-cctcgcatagagagcttccatcatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0118] PCR amplification was performed using PacoA-F (5'-tggcaccggtccgcggga-3') and PacoA-R (5'-gatggaagctctctatgcgagg-3') as a primer pair and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain a fragment of the PacoA promoter.

[0119] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0120] The three fragments were cloned in one step, and PCR amplification was performed using PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PacoA-TbrA-TtrpC expression cassette fragment.

[0121] 7) Construction of the expression cassette of aconitate isomerase TbrA using the PcitA promoter

[0122] PCR amplification was performed using fuPcitA-TbrA-F (5'-cttttttagactcttgttggattcaaaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0123] PCR amplification was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and PcitA-R (5'-ttgaatccaacaagagtctaaaaaag-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain the fragment of Pcit A promoter.

[0124] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0125] The three fragments were cloned in one step, and PCR amplification was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-at tacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PcitA-TbrA-TtrpC expression cassette fragment.

[0126] 8) Construction of the expression cassette of aconitate isomerase TbrA using the PicdA promoter

[0127] PCR amplification was performed using fuPicdA-TbrA-F (5'-cgcaggccacgcttcactgtcgaaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0128] PCR amplification was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and PicdA-R (5'-ttcgacagtgaagcgtggcctgcg-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the PicdA promoter.

[0129] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0130] The three fragments were cloned in one step, and PCR amplification was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PicdA-TbrA-TtrpC expression cassette fragment.

[0131] 9) Construction of the expression cassette of aconitate isomerase TbrA using the PgasA promoter

[0132] PCR amplification was performed using fuPgasA-TbrA-F (5'-gtcttctttcgttcacctcctcacatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0133] PCR amplification was performed using PgasA-F (5'-ctgctctctctctgctctctctttct-3') and PgasA-R (5'-gtgaggaggtgaacgaaagaagac-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as template. The product was purified and recovered to obtain the fragment of the PgasA promoter.

[0134] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0135] The three fragments were cloned in one step, and PCR amplification was performed using PgasA-F (5'-ctgctctctctctgctctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgasA-TbrA-TtrpC expression cassette fragment.

[0136] 2. Construction of the expression cassette of the hygromycin B selection marker gene hph:

[0137] Using hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') as primer pair and plasmid pSGF957 as template, PCR amplification of PtrpC-hph-TtrpC fragment was performed, PtrpC is the promoter of Aspergillus nidulans tryptophan synthase gene, hph is the hygromycin phosphotransferase gene, and TtrpC is the terminator of Aspergillus nidulans tryptophan synthase; after purification and recovery of the amplified product, the hygromycin B resistance hph gene expression cassette PtrpC-hph-TtrpC was obtained.

[0138] 3. Transform Aspergillus niger protoplasts with the aconitate isomerase TbrA expression cassette to obtain a recombinant Aspergillus niger strain:

[0139] 1) Preparation of Aspergillus niger protoplasts

[0140] The spore suspension of Aspergillus niger MEFC1501 was inoculated into 50 mL of liquid medium PDB, with a spore concentration of about 10 7 / mL, culture at 200rmp, 33℃ for 12-18h. Collect the grown mycelium by filtration with a sterile single-layer 100-mesh nylon cloth, rinse three times with a sterilized 0.6M MgSO4 solution, squeeze dry and place in a sterile 50mL conical flask; weigh 1g of mycelium, add 10mL of enzymatic solution, and treat at 30℃, 120rpm for 1-2h. The enzymatic solution contains: 1% cellulase (Sigma, Catalog No.: C1184), 1% lyase (Sigma, Catalog No.: L1412), 1% snail enzyme (Shanghai Shenggong, Catalog No.: SB0870), 0.6M MgSO4, and filter and sterilize through a 0.22μm sterile filter.

[0141] The mixture after enzymatic hydrolysis was filtered through a 300-mesh nylon cloth to collect the filtrate. The protoplasts were collected by centrifugation at 4°C, washed once with a precooled 1.0 M sorbitol solution, and then washed once with a precooled STC (1.0 M sorbitol, 50 mM Tris·HCl-pH8.0, 50 mM CaCl2). Finally, the protoplasts were resuspended in 150 μL precooled STC and the protoplast concentration was adjusted to 5 × 10 7 / mL to obtain a protoplast suspension.

[0142] 2) Co-transformation of Aconitate Isomerase TbrA Expression Cassette and Resistance Gene hph into Aspergillus niger protoplasts:

[0143] Add about 5 μg of aconitate isomerase TbrA expression cassette DNA and 1 μg of hygromycin B resistance hph gene expression cassette DNA to the above protoplast suspension, then add 50 μL of PSTC (40% PEG4000, 50Mm Tris-HCl pH8.0, 50mM CaCl2), gently mix, and place on ice for 30 minutes. Add 1.5mL of PSTC, mix, and place at room temperature for 20 minutes; then mix with the upper agar layer and pour into the regeneration screening medium plate PDA-SH, and culture at 30°C in the dark for 3-4 days.

[0144] The transformants were transferred from the plate to the screening plate PDA-H (4 g of potato dextrose agar medium was weighed and dissolved in 100 mL of distilled water, sterilized, and hygromycin was added to a final concentration of 100 μg / mL when cooled to about 55°C to prepare the plate), and cultured at 30°C for 3-5 days to obtain transformants.

[0145] In the preparation process of the recombinant Aspergillus niger strain containing the aconitate isomerase TbrA expression cassette, the aconitate isomerase TbrA expression cassette and the hygromycin B resistance hph gene expression cassette can also be connected into one expression cassette, and then transformed into protoplasts, which does not affect the screening of positive transformants and the identification of genotypes.

[0146] 3) Verification of the genotype of the recombinant Aspergillus niger strain:

[0147] The above-obtained recombinant Aspergillus niger transformant was selected and inoculated on a PDA-H plate to culture spores, and then the spores were inoculated in a liquid culture medium for culture, and the mycelia was collected to extract the genome, and PgpdAt-F743 (5'-ttacactctgggaggat ccaggtact-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primer pairs for PCR to amplify the PgpdAt-TbrA-TtrpC inserted into the genome; hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcga acccagg-3') were used as primer pairs to amplify the PtrpC-hph-TtrpC fragment inserted into the recombinant bacteria, and the PCR product was analyzed by 0.8% agarose gel electrophoresis. The starting strain Aspergillus niger MEFC1501 genomic DNA template was used as a negative control.

[0148] Similarly, PCR was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primers to amplify PglaA-TbrA-TtrpC inserted into the genome; PCR was performed using PgpdAn-F (5'-ttgatcga gacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primers to amplify PgpdAn-TbrA-TtrpC inserted into the genome; and PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacc tctaaacaagtgtac-3') as primer pair to amplify PcadA-TbrA-TtrpC inserted into the genome; PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair to amplify PmfsA-TbrA-TtrpC inserted into the genome; PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair to amplify PmfsA-TbrA-TtrpC inserted into the genome PCR was performed using primers PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify the PacoA-TbrA-TtrpC inserted into the genome; PCR was performed using primers PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify the PcitA-TbrA-TtrpC inserted into the genome; and PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and Ttr PCR was performed using pC-R (5'-attacctctaaacaagtgtac-3') as the primer pair to amplify PicdA-TbrA-Ttrp C inserted into the genome; PCR was performed using PgasA-F (5'-ctgctctctctctgctctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as the primer pair to amplify PgasA-TbrA-TtrpC inserted into the genome.

[0149] The transformants with correct genotype verification were transferred to PDA-H plates for subculture for 3 times. Then, the spores were collected and diluted appropriately with physiological saline, and 100 μL was spread on the PDA-H plate to grow independent single colonies, which was single spore isolation. Spores were taken from the single colony for single spore isolation again, and a total of 4 single spore isolation subcultures were performed. The recombinant strains after single spore isolation were again identified for genotyping and verified, and some of the recombinant strains with correct verification were selected for shake flask fermentation screening. The specific shake flask screening method and results are shown in Figure 1 .

[0150] Example 2 Construction of recombinant Aspergillus niger heterologously expressing aconitate isomerase Adi1 from Ustilago maydis

[0151] 1. Construction of the expression cassette for aconitate isomerase Adi1

[0152] The aconitate isomerase Adi1 gene was artificially synthesized and codon optimized for Aspergillus niger expression. The optimized sequence was shown in SEQ ID NO.15 and cloned into the pUC57-Kan vector to construct the plasmid puc57-Adi1.

[0153] 1) Construction of the expression cassette of aconitate isomerase Adi1 using the PgpdAt promoter

[0154] PCR amplification was performed using fuPgpdAt-Adi1-F (5'-ctcatcaatcatcacaacatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0155] Using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and fuPgpdAt-Adi1-R (5'-tggtgtcgatggggtgcagcatgttgtgatgattgatgag-3') as primer pair, PCR amplification was performed with plasmid pMWM23 as template, and the product was purified and recovered to obtain the PgpdAt-TtrpC fragment.

[0156] The two fragments were cloned in one step, and PCR amplification was performed using PgpdAt-F (5'-ttacactctgggaggatccaggtac-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgpdAt-Adi1-TtrpC expression cassette fragment.

[0157] 2) Construction of the expression cassette of aconitate isomerase Adi1 using the PgpdAn promoter

[0158] PCR amplification was performed using fuPgpdAn-Adi1-F (5'-ccgcttgagcagacatcaccatgctgcaccccatcgacaccat-3') and fuAdi1-Ttrp CR (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0159] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and fuPgpdAn-Adi1-R (5'-tggtgtcgatggggtgcagcatggtgatgtctgctcaagcgg-3') as primer pairs and plasmid pAN52-4 as template, and the product was purified and recovered to obtain the fragment of PgpdAn-TtrpC. PgpdAn is the promoter of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene, and TtrpC is the terminator of the Aspergillus nidulans tryptophan synthase.

[0160] The two fragments were cloned in one step, and PCR amplification was performed using PgpdAn-F (5'-ttgatcgagacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgpdAn-Adi1-Ttrp C expression cassette fragment.

[0161] 3) Construction of the expression cassette of aconitate isomerase Adi1 using the PglaA promoter

[0162] PCR amplification was performed using fuPglaA-Adi1-F (5'-gcatcattacacctcagcaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0163] PCR amplification was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and PglaA-R (5'-tgctgaggtgtaatgatgctgggg at-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the PglaA promoter.

[0164] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0165] The three fragments were cloned in one step, and PCR amplification was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PglaA-Adi1-TtrpC expression cassette fragment.

[0166] 4) Construction of the expression cassette of aconitate isomerase Adi1 using the PcadA promoter

[0167] PCR amplification was performed using fuPcadA-Adi1-F (5'-cctcttaaattgaccatgaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0168] PCR amplification was performed using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and PcadA-R (5'-tcatggtcaatttaagagg-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain the fragment of PcadA promoter.

[0169] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0170] The three fragments were cloned in one step, and PCR amplification was performed using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primers. The product was purified and recovered to obtain PcadA-Adi1

[0171] -TtrpC expression cassette fragment.

[0172] 5) Construction of the expression cassette of aconitate isomerase Adi1 using the PmfsA promoter

[0173] PCR amplification was performed using fuPmfsA-Adi1-F (5'-accgacttctcatccatcttcaaaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0174] PCR amplification was performed using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the PmfsA promoter.

[0175] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0176] The above three fragments were cloned in one step, and PCR amplification was performed using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PmfsA-Adi1-TtrpC expression cassette fragment.

[0177] 6) Construction of the expression cassette of aconitate isomerase Adi1 using the PacoA promoter

[0178] PCR amplification was performed using fuPacoA-Adi1-F (5'-cctcgcatagagagcttccatcatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0179] PCR amplification was performed using PacoA-F (5'-tggcaccggtccgcggga-3') and PacoA-R (5'-gatggaagctctctatgcgagg-3') as a primer pair and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain a fragment of the PacoA promoter.

[0180] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0181] The three fragments were cloned in one step, and PCR amplification was performed using PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PacoA-Adi1-TtrpC expression cassette fragment.

[0182] 7) Construction of the expression cassette of aconitate isomerase Adi1 using the PcitA promoter

[0183] PCR amplification was performed using fuPcitA-Adi1-F (5'-cttttttagactcttgttggattcaaaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0184] PCR amplification was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and PcitA-R (5'-ttgaatccaacaagagtctaaaaaag-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain the fragment of Pcit A promoter.

[0185] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0186] The three fragments were cloned in one step, and PCR amplification was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-at tacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PcitA-Adi1-TtrpC expression cassette fragment.

[0187] 8) Construction of the expression cassette of aconitate isomerase Adi1 using the PicdA promoter

[0188] PCR amplification was performed using fuPicdA-Adi1-F (5'-cgcaggccacgcttcactgtcgaaatgctgcaccccatcgacacca-3') and fuAdi1-Ttrp CR (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0189] PCR amplification was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and PicdA-R (5'-ttcgacagtgaagcgtggcctgcg-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the PicdA promoter.

[0190] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0191] The three fragments were cloned in one step, and PCR amplification was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PicdA-Adi1-TtrpC expression cassette fragment.

[0192] 9) Construction of the expression cassette of aconitate isomerase Adi1 using the PgasA promoter

[0193] PCR amplification was performed using fuPgasA-Adi1-F (5'-gtcttctttcgttcacctcctcacatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0194] PCR amplification was performed using PgasA-F (5'-ctgctctctctctgctctctctttct-3') and PgasA-R (5'-gtgaggaggtgaacgaaagaagac-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as template. The product was purified and recovered to obtain the fragment of the PgasA promoter.

[0195] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0196] The above three fragments were cloned in one step, and PCR amplification was performed using PgasA-F (5'-ctgctctctctctgctctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the product, the PgasA-Adi1-TtrpC expression cassette fragment was obtained.

[0197] 2. Construction of the expression cassette of the hygromycin B selection marker gene hph:

[0198] As described in Example 1.

[0199] 3. Transform Aspergillus niger protoplasts with the aconitate isomerase Adi1 expression cassette to obtain a recombinant Aspergillus niger strain:

[0200] 1) Preparation of Aspergillus niger protoplasts

[0201] As described in Example 1.

[0202] 2) Co-transformation of Aconitate Isomerase Adi1 Expression Cassette and Resistance Gene hph into Aspergillus niger protoplasts:

[0203] Add about 5 μg of aconitate isomerase Adi1 expression cassette DNA and 1 μg of hygromycin B resistance hph gene expression cassette DNA to the above protoplast suspension, then add 50 μL of PSTC (40% PEG4000, 50 mM Tris-HCl pH8.0, 50 mM CaCl2), gently mix, and place on ice for 30 minutes. Add 1.5 mL of PSTC, mix, and place at room temperature for 20 minutes; then mix with the upper agar layer and pour into the regeneration screening medium plate PDA-SH, and culture at 30°C in the dark for 3-4 days.

[0204] The transformants were transferred from the plate to the screening plate PDA-H (4 g of potato dextrose agar medium was weighed and dissolved in 100 mL of distilled water, sterilized, and hygromycin was added to a final concentration of 100 μg / mL when cooled to about 55°C to prepare the plate), and cultured at 30°C for 3-5 days to obtain transformants.

[0205] In the preparation process of the recombinant Aspergillus niger strain containing the aconitate isomerase Adi1 expression cassette, the aconitate isomerase Adi1 expression cassette and the hygromycin B resistance hph gene expression cassette can also be connected into one expression cassette, and then transformed into protoplasts, which does not affect the screening of positive transformants and the identification of genotypes.

[0206] 3) Verification of the genotype of the recombinant Aspergillus niger strain:

[0207] The above-obtained recombinant Aspergillus niger transformant was selected and inoculated on a PDA-H plate to culture spores, and then the spores were inoculated in a liquid culture medium for culture, and the mycelia was collected to extract the genome, and PgpdAt-F743 (5'-ttacactctgggaggat ccaggtact-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primer pairs for PCR to amplify the PgpdAt-Adi1-TtrpC inserted into the genome; hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaa cccagg-3') were used as primer pairs to amplify the PtrpC-hph-TtrpC fragment inserted into the recombinant bacteria, and the PCR product was analyzed by 0.8% agarose gel electrophoresis. The starting strain Aspergillus niger MEFC1501 genomic DNA template was used as a negative control.

[0208] Similarly, PCR was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primers to amplify PglaA-Adi1-TtrpC inserted into the genome; PCR was performed using PgpdAn-F (5'-ttgatcga gacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primers to amplify PgpdAn-Adi1-TtrpC inserted into the genome; PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacc tctaaacaagtgtac-3') as primer pair to amplify PcadA-Adi1-TtrpC inserted into the genome; PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair to amplify PmfsA-Adi1-TtrpC inserted into the genome; PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-a ttacctctaaacaagtgtac-3') as primer pair to amplify PacoA-Adi1-TtrpC inserted into the genome; PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair to amplify PcitA-Adi1-TtrpC inserted into the genome; PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and Ttrp PCR was performed using the primer pair CR (5'-attacctctaaacaagtgtac-3') to amplify PicdA-Adi1-TtrpC inserted into the genome; PCR was performed using the primer pair PgasA-F (5'-ctgctctctctctgctctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify PgasA-Adi1-TtrpC inserted into the genome.

[0209] The transformants with correct genotype verification were transferred to PDA-H plates for subculture, and subcultured for 3 times. Then, the spores were collected separately and diluted appropriately with physiological saline, and 100 μL was spread on the PDA-H plate to grow independent single colonies, which is single spore isolation. Spores were taken from the single colony and single spore isolation was performed again, and a total of 4 single spore isolation subcultures were performed. The recombinant strains after single spore isolation were again genotyped for verification. Some of the recombinant strains with correct verification were selected for shake flask fermentation screening. The specific shake flask screening method and results are shown in Example 4. Figure 2 .

[0210] Example 3 Construction of a recombinant Aspergillus niger strain using pyrithiamine ptrA as a selection marker

[0211] Example 2 was repeated. The difference from Example 2 was that the selection marker gene used in the recombinant Aspergillus niger strain in this example was the pyrithiamine selection marker ptrA gene. The specific method is described as follows:

[0212] 1. Construction of expression cassettes of aconitate isomerase Adi1 with different promoters, see Example 2.

[0213] 2. Construction of recombinant Aspergillus niger strain using the pyrithiamine resistance gene ptrA as a selection marker

[0214] 1) Construction of the expression cassette of the pyrithiamine resistance gene ptrA

[0215] PCR amplification was performed using ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atggggtgacgatgagccgc-3') as primer pairs and plasmid pMWM23 as template. After the amplified product was purified and recovered, the pyrithiamine resistance gene ptrA expression cassette was obtained.

[0216] Prepare Aspergillus niger MEFC1501 protoplasts according to the preparation method in Example 1. Add about 5 μg of aconitase Adi1 expression cassette DNA and 1 μg of screening marker ptrA fragment to the protoplast suspension, then add 50 μL PSTC (40% PEG4000, 50Mm Tris-HCl pH8.0, 50mM CaCl2), mix gently, and ice bath for 30 minutes. Add 1.5mL PSTC, mix well and place at room temperature for 20 minutes; then mix with the upper agar and pour into the regeneration screening medium plate CD-SPt, and culture at 33°C in the dark for 3-4 days. Transfer the transformant from the plate to the screening plate CD-Pt, and culture at 33°C for 3-5 days to obtain the transformant.

[0217] The prepared recombinant Aspergillus terreus transformants were selected and inoculated on CD-Pt plates to culture spores, and then the spores were inoculated in PDB liquid culture medium for culture, the mycelia were collected to extract the genome, and PgpdAt-F743 (5'-ttacactctgggaggatc caggtact-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primer pairs for PCR to amplify PgpdAt-Adi1-TtrpC inserted into the genome; ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atggggtgacgatgagccg c-3') were used as primer pairs to amplify the ptrA fragment inserted into the recombinant bacteria, and the PCR products were analyzed by 0.8% agarose gel electrophoresis. The genomic DNA template of the starting strain Aspergillus niger MEFC1501 was used as a negative control.

[0218] Similarly, PCR was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primers to amplify PglaA-Adi1-TtrpC inserted into the genome; PCR was performed using PgpdAn-F (5'-ttgatcga gacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primers to amplify PgpdAn-Adi1-TtrpC inserted into the genome; PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacc tctaaacaagtgtac-3') as primer pair to amplify PcadA-Adi1-TtrpC inserted into the genome; PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair to amplify PmfsA-Adi1-TtrpC inserted into the genome; PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-a ttacctctaaacaagtgtac-3') as primer pair to amplify PacoA-Adi1-TtrpC inserted into the genome; PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair to amplify PcitA-Adi1-TtrpC inserted into the genome; PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and Ttrp PCR was performed using the primer pair CR (5'-attacctctaaacaagtgtac-3') to amplify PicdA-Adi1-TtrpC inserted into the genome; PCR was performed using the primer pair PgasA-F (5'-ctgctctctctctgctctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify PgasA-Adi1-TtrpC inserted into the genome.

[0219] The verification and separation and purification of positive transformants refer to Example 1 or Example 2, and the specific shake flask screening method and results are shown in Example 4. Figure 3 .

[0220] Example 4 Recombinant Aspergillus niger fermentation to produce trans-aconitic acid

[0221] 1. Shake flask screening of recombinant Aspergillus niger strains producing trans-aconitic acid

[0222] The recombinant Aspergillus niger strains obtained in Example 1, Example 2 and Example 3 were inoculated into the Aspergillus niger spore production medium and cultured at 32°C for 6 days to obtain mature spores. The spores cultured to maturity were then inoculated into the trans-aconitic acid fermentation medium, and three bottles of each strain were made in parallel, and fermented at 34°C and 220rpm for 135h. The mycelium in the fermentation broth was filtered out, and the fermentation supernatant was appropriately diluted and analyzed by high performance liquid chromatography (HPLC).

[0223] 2. Analysis of aconitic acid content in fermentation broth

[0224] The fermentation supernatant of the recombinant strain and the starting strain Aspergillus niger MEFC1501 was diluted and analyzed by high performance liquid chromatography. The standard curve was prepared with different concentrations of cis-aconitic acid and trans-aconitic acid standards to analyze and compare the content and purity of cis-aconitic acid and trans-aconitic acid in the fermentation broth. The chromatographic conditions are as follows: chromatographic column: Aminex HPX-87H Organic Acid Analysis Column, 300mm×7.8mm (Bio-rad, Cat No.1250140); mobile phase: 5mmol / L sulfuric acid; flow rate: 0.5mL / min; column temperature: 30℃; detection temperature: 30℃; UV detector (210nm).

[0225] The fermentation of trans-aconitic acid by the recombinant strains obtained in Example 1, Example 2 and Example 3 is as follows: Figure 1-3 shown.

[0226] Depend on Figure 1-3 The analysis results showed that the content of cis-aconitic acid and trans-aconitic acid in the fermentation broth of the recombinant strain was significantly higher than that of the wild-type strain at the shake flask fermentation level. The effects of the two isomerases TbrA and Adi1 were not much different, but the gene expression driven by different promoters showed obvious differences, and the content of trans-aconitic acid could reach 34g / L. There was no significant difference between different terminators such as TtrpC or Tp gk, and there was no significant difference between different screening tags such as hygromycin or pyrithiamine. Different transformation methods, such as co-transformation with the screening tag or constructing the expression cassette of the target gene and the expression cassette of the screening tag together for transformation, all obtained similar results.

[0227] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A recombinant Aspergillus niger with high trans-aconitic acid production, characterized in that: Aconitate isomerase is heterologously expressed in the recombinant Aspergillus niger; The aconitate isomerase comprises TbrA and / or Adi1.

2. The recombinant Aspergillus niger according to claim 1, characterized in that The recombinant Aspergillus niger is selected from 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 3008, Aspergillus niger GCB75, Aspergillus niger ATCC9142, and Aspergillus niger MTCC 282.

3. The recombinant Aspergillus niger according to claim 1, characterized in that The recombinant Aspergillus niger also includes a promoter, which promotes the expression of aconitate isomerase; preferably, the promoter is selected from one or more of PglaA promoter, PgpdAt promoter, PgpdAn promoter, PacoA promoter, PcitA promoter, PicdA promoter, PcadA promoter, PmfsA promoter, and Pgas promoter; more preferably, the promoter is PglaA promoter.

4. The recombinant Aspergillus niger according to claim 1, characterized in that The amino acid sequence of TbrA includes the sequence shown in SEQ ID No.1 or an amino acid sequence having at least 60% sequence homology with the sequence of SEQ ID NO.1; preferably, the aconitate isomerase gene encoding the TbrA includes the sequence shown in SEQ ID No.14 or a nucleotide sequence having at least 60% sequence homology with the sequence of SEQ ID NO.

14.

5. The recombinant Aspergillus niger according to claim 1, characterized in that The amino acid sequence of Adi1 includes an amino acid sequence as shown in SEQ ID No.2 or an amino acid sequence having at least 60% sequence homology with the SEQ ID NO.2 sequence; preferably, the aconitate isomerase gene encoding the Adi1 includes a sequence as shown in SEQ ID No.15 or a nucleotide sequence having at least 60% sequence homology with the SEQ ID NO.15 sequence.

6. The recombinant Aspergillus niger according to claim 1, characterized in that The recombinant Aspergillus niger also includes a terminator, which terminates the expression of aconitate isomerase; preferably, the terminator includes a TtrpC terminator and / or a Tpgk terminator.

7. Biomaterial, characterized in that The biological material is any one of A1)-A3): A1) an expression cassette, the expression cassette comprising a promoter, an aconitate isomerase gene and a terminator; the aconitate isomerase comprises TbrA and / or Adi1; A2) a recombinant vector, wherein the recombinant vector contains the expression cassette described in A1); A3) A whole-cell catalyst, which contains the expression cassette as described in A1) or the recombinant vector as described in A2) or the recombinant Aspergillus niger as described in any one of claims 1-6.

8. The method for constructing a recombinant Aspergillus niger with high trans-aconitic acid production according to claim 1, characterized in that: The aconitate isomerase gene is introduced into Aspergillus niger to obtain a recombinant Aspergillus niger with high trans-aconitate production; the aconitate isomerase includes TbrA or Adi1.

9. Use of the recombinant Aspergillus niger according to any one of claims 1 to 6 or the biological material according to claim 7 in the production of trans-aconitic acid.

10. A method for producing trans-aconitic acid, characterized in that: The method comprises: Step 1, constructing the recombinant Aspergillus niger according to any one of claims 1 to 6 or the biomaterial according to claim 7; Step 2: inoculating the recombinant Aspergillus niger into a fermentation medium for fermentation to obtain trans-aconitic acid.

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