A recombinant aspergillus niger strain with high trans-aconitic acid yield and application thereof

Abstract

The application provides a recombinant Aspergillus niger strain with high trans-aconitic acid yield, and belongs to the technical field of genetic engineering. The recombinant Aspergillus niger strain can simultaneously express aconitase and aconitate isomerase. The genetically engineered Aspergillus niger strain provided by the application has a generally higher trans-aconitic acid yield than a starting strain, and the strain with the highest yield can reach 51 g / L at a shake flask level, and has a very strong application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a recombinant Aspergillus niger strain that produces high levels of trans-aconitine, a method for producing trans-aconitine using this recombinant Aspergillus niger strain, and its applications. Background Technology

[0002] trans-aconitic acid ( trans Trans-aconitic acid (CAS: 4023-65-8) is an unsaturated tricarboxylic acid. Due to its unsaturated double bonds and three carboxyl groups, it can be used as a monomer compound for preparing polymer materials and as a precursor for the synthesis of other compounds. It is listed by the U.S. Department of Energy as one of the most valuable platform compounds. Trans-aconitic esters, obtained from trans-aconitic acid, are a new generation of safe, environmentally friendly, and high-performance plasticizers for the PVC, PLA, and other plastics industries, with broad application prospects. Trans-aconitic acid also has excellent nematicidal activity, making it a novel biological pesticide for nematode control; it can also be used as a food additive to enhance the flavor of food and beverages. Furthermore, trans-aconitic acid is a key intermediate in the tricarboxylic acid cycle, specifically cis-aconitic acid (…). cis The stereoisomer of aconitic acid (CAS: 585-84-2) has a certain inhibitory effect on aconitase, a key enzyme in the tricarboxylic acid cycle, which can interfere with the tricarboxylic acid cycle and thus affect life activities, exhibiting certain biological activity.

[0003] Currently, trans-aconitic acid is mainly produced through chemical synthesis, which is complex, produces numerous byproducts, and is costly, thus hindering large-scale production. To develop a greener and more efficient trans-aconitic acid production process, previous researchers have blocked the cis-aconitic acid decarboxylase gene, which catalyzes the decarboxylation of cis-aconitic acid to itaconic acid, in Aspergillus terrestris. cadA They overexpressed aconitine isomerase CICC00358 and obtained engineered strains of Aspergillus terrestris (CN112011469A, CN112029671A) that can produce aconitine, but the yield is still relatively low and cannot meet the needs of large-scale production.

[0004] Besides Aspergillus terreus, many fungi possess the ability to synthesize organic acids efficiently. Among commonly used industrial chassis strains, Aspergillus niger (… Aspergillus nigerAspergillus niger is an excellent citric acid-producing bacterium, and the citric acid it produces is a direct precursor to cis-aconitic acid. Furthermore, Aspergillus niger does not synthesize itaconic acid, thus eliminating the need to block itaconic acid synthesis. Compared to Aspergillus terreus, this simplifies the pathway for trans-aconitic acid synthesis. In addition, Aspergillus niger is a traditional fermentation strain widely used in the food industry and is recognized by the US FDA and the World Health Organization as a General Regarded As Safe (GRAS) microorganism, making it suitable for large-scale production of proteins, organic acids, and antigens. Therefore, Aspergillus niger is a potential strain for large-scale trans-aconitic acid production. However, there are currently no reports on the use of Aspergillus niger for trans-aconitic acid production.

[0005] In addition, Aspergillus tabingensis, Aspergillus jujuba, Aspergillus umbellatus, and Candida albicans can also serve as potential hosts for the production of trans-aconitine. Summary of the Invention

[0006] To address the shortcomings of existing plant extraction or chemical synthesis methods for producing trans-aconic acid, such as high cost, low yield, and complex processes, this invention provides an aconitase that efficiently catalyzes the synthesis of cis-aconic acid, an aconitase that efficiently catalyzes the conversion of cis-aconic acid to trans-aconic acid, a genetically engineered strain of *Aspergillus niger* capable of synthesizing trans-aconic acid, and a method for producing trans-aconic acid through fermentation using *Aspergillus niger*. This achieves a one-step production of trans-aconic acid, specifically, the one-step fermentation of trans-aconic acid in microbial cells using a carbon source. Optionally, the microorganisms capable of trans-aconic acid synthesis include, but are not limited to, *Aspergillus niger*. Aspergillus niger ).

[0007] On the one hand, this application provides a recombinant Aspergillus niger strain that produces high levels of trans-aconitine, wherein the recombinant Aspergillus niger strain can simultaneously express aconitase and aconitine isomerase.

[0008] In one embodiment, the aconitase is Aco, and the aconitase isomerase is selected from TbrA or Adi1.

[0009] Preferably, the aconitase is derived from Aspergillus terrestris (…). Aspergillus terreus Aco(ATEG_03325) of ); the aconitine isomerase is selected from Bacillus thuringiensis (Bacillus thuringiensis) Bacillus thuringiensis TbrA from smut fungus (Maize smut) or derived from smut fungus (Maize smut) Ustilago maydis Corola ) of Adi1.

[0010] In one embodiment, the aconitase Aco contains an amino acid sequence as shown in SEQ ID NO. 1 or having at least 60% homology with the amino acid sequence shown in SEQ ID NO. 1; and / or,

[0011] The amino acid sequence of the aconitine isomerase TbrA comprises an amino acid sequence as shown in SEQ ID NO.2 or having at least 60% homology with the amino acid sequence shown in SEQ ID NO.2; and / or,

[0012] The amino acid sequence of the aconitine isomerase Adi1 comprises an amino acid sequence as shown in SEQ ID NO.3 or having at least 60% homology with the amino acid sequence shown in SEQ ID NO.3.

[0013] In one embodiment, the gene encoding the aconitase Aco comprises a nucleotide sequence as shown in SEQ ID NO. 17 or having at least 60% homology with the sequence shown in SEQ ID NO. 17;

[0014] The gene encoding the aconitine isomerase TbrA comprises a nucleotide sequence as shown in SEQ ID NO.15 or having at least 60% homology with the sequence shown in SEQ ID NO.15;

[0015] The gene encoding the aconitine isomerase Adi1 comprises a nucleotide sequence as shown in SEQ ID NO.16 or having at least 60% homology with the sequence shown in SEQ ID NO.16.

[0016] Preferably, the sequences with at least 60% homology described in this application are sequences with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homology.

[0017] In one embodiment, the host cell of the recombinant Aspergillus niger strain is selected from one or more of the following: Aspergillus niger MEFC1501, Aspergillus niger ATCC 1015, Aspergillus niger NRRL 41873, Aspergillus niger NRRL 27809, Aspergillus niger NRRL 31821, Aspergillus niger NRRL6276, Aspergillus niger Co827, Aspergillus niger 5016, Aspergillus niger 3008, Aspergillus niger GCB75, Aspergillus niger ATCC 9142, and Aspergillus niger MTCC282.

[0018] Preferably, the host cell of the recombinant Aspergillus niger strain is Aspergillus niger MEFC1501.

[0019] In one embodiment, the recombinant Aspergillus niger strain is obtained by introducing an expression cassette containing an aconitase gene and an expression cassette containing an aconitase isomerase gene.

[0020] In one embodiment, the promoter of the aconitase gene expression cassette is selected from one or more of the following promoters: PglaA promoter, PgpdAt promoter, PgpdAn promoter, PacoA promoter, PcitA promoter, PicdA promoter, PcadA promoter, PmfsA promoter, and PgasA promoter.

[0021] Optionally, the PglaA promoter sequence is shown in SEQ ID NO.4.

[0022] Optionally, the PgpdAt promoter sequence is shown in SEQ ID NO.5.

[0023] Optionally, the PgpdAn promoter sequence is shown in SEQ ID NO.6.

[0024] Optionally, the PacoA promoter sequence is shown in SEQ ID NO.7.

[0025] Optionally, the PcitA promoter sequence is shown in SEQ ID NO.8.

[0026] Optionally, the PicdA promoter sequence is shown in SEQ ID NO.9.

[0027] Optionally, the PcadA promoter sequence is shown in SEQ ID NO.10.

[0028] Optionally, the PmfsA promoter sequence is shown in SEQ ID NO.11.

[0029] Optionally, the PgasA promoter sequence is shown in SEQ ID NO.12.

[0030] Preferably, the promoter is a PmfsA promoter or a PgasA promoter; more preferably, the nucleotide sequence of the PmfsA promoter is as shown in SEQ ID NO.11 or a nucleotide sequence having at least 60% sequence homology with the sequence of SEQ ID NO.11; even more preferably, the nucleotide sequence of the PgasA promoter is as shown in SEQ ID NO.12 or a nucleotide sequence having at least 60% sequence homology with the sequence of SEQ ID NO.12.

[0031] In one embodiment, the expression box further includes a terminator selected from the TtrpC terminator or the Tpgk terminator.

[0032] Optionally, the TtrpC terminator sequence is as shown in SEQ ID NO.13.

[0033] Optionally, the Tpgk termination sequence is as shown in SEQ ID NO.14.

[0034] On the other hand, this application also provides a biological material selected from any one of A1 to A3 below:

[0035] A1. An expression cassette, comprising a promoter, a target gene, and a terminator, wherein the target gene comprises a nucleotide sequence as shown in SEQ ID NO. 17 or having at least 60% homology with the sequence shown in SEQ ID NO. 17, and contains a nucleotide sequence as shown in SEQ ID NO. 15 or having at least 60% homology with the sequence shown in SEQ ID NO. 15, or a nucleotide sequence as shown in SEQ ID NO. 16 or having at least 60% homology with the sequence shown in SEQ ID NO. 16;

[0036] A2. A recombinant vector containing the expression cassette as described in A1;

[0037] A3, a whole-cell catalyst containing an expression cassette as described in A1, or a recombinant vector as described in A2, or the recombinant Aspergillus niger strain described therein.

[0038] On the other hand, this application also provides the use of the recombinant Aspergillus niger strain or the biomaterial in the production of trans-aconitine or products containing trans-aconitine or trans-aconitine structural units.

[0039] On the other hand, this application also provides a method for producing trans-aconitic acid, comprising:

[0040] Step 1: Construct the recombinant Aspergillus niger strain or the biomaterial described above;

[0041] Step 2: Inoculate the recombinant Aspergillus niger strain into a fermentation medium for fermentation to obtain trans-aconitine.

[0042] In one embodiment, the fermentation medium uses monosaccharides, polysaccharides, or mixtures thereof available to Aspergillus niger as carbon sources, including but not limited to glucose, fructose, sucrose, molasses, corn flour, potato starch, and starch hydrolysate.

[0043] In one embodiment, the fermentation medium contains: 160 g / L glucose, 2 g / L NH4NO3, 0.2 g / L (NH4)2HPO4, 20 mg / L FeSO4, 0.4 g / L MgSO4, 40 mg / L ZnSO4, 40 mg / L CuSO4, and pH 3.5.

[0044] The beneficial effects of this application include at least the following:

[0045] 1. The recombinant Aspergillus niger strain provided in this application is based on Aspergillus niger (… Aspergillus niger Using Aspergillus terreus as the starting strain, Aspergillus terreus Aconitase Aco and maize smut fungus (from which aconitase Aco and maize smut fungus are derived) Ustilago maydis Corola Aconitine isomerase Adi1 or Bacillus thuringiensis (Bt) are sources of this enzyme. Bacillus thuringiensis The aconitine isomerase TbrA from Aspergillus niger was heterologously expressed in Aspergillus niger to obtain an engineered strain of Aspergillus niger that produces trans-aconitine. This can solve the source dilemma of trans-aconitine, which traditionally requires plant extraction or chemical synthesis, through fermentation production, and can also utilize Aspergillus niger to synthesize trans-aconitine.

[0046] 2. Aspergillus niger is a traditional fermentation strain widely used in the food industry. It is recognized by the US FDA and the World Health Organization as a generally regarded as safe (GRAS) microorganism and can be used extensively as a cell factory in the production of proteins, organic acids, antigens, etc. This invention provides a safe, non-toxic cell factory for the efficient production of trans-aconitine.

[0047] 3. Experiments have shown that the trans-aconitine fermentation yield of the genetically engineered Aspergillus niger strain provided by this invention is generally higher than that of the original strain. The strain with the highest yield can reach 51 g / L at the shake flask level, which has strong application value. Attached Figure Description

[0048] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0049] Figure 1 The present invention provides an example of the combination of PglaA-driven aconitine isomerase TbrA and aconitine enzyme ATEG_03325 driven by different promoters, which measures the content of trans-aconitine produced by shake-flask fermentation of recombinant Aspergillus niger strain.

[0050] Figure 2 The present invention provides an example of the combination of PgpdAn-driven aconitine isomerase Adi1 and aconitine enzyme ATEG_03325 driven by different promoters to measure the trans-aconitine content produced by shake-flask fermentation of recombinant Aspergillus niger strain.

[0051] Information on strain preservation:

[0052] Aspergillus niger MEFC1501 ( Aspergillus nigerMEFC1501 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.40614, on April 27, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0053] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0054] Unless otherwise specified, the materials, reagents, instruments, and methods used in the following examples are all conventional materials, reagents, instruments, and methods in the art, and are all commercially available. Unless otherwise specified, the methods described are conventional methods in the field of molecular biology or are performed in accordance with the relevant product instructions.

[0055] Technical terms:

[0056] Aconitate hydratase (ACO): Aconitate hydratase is an enzyme protein that is normally synthesized in living organisms. It can remove one molecule of H2O from citric acid to obtain cis-aconitate.

[0057] Aconitate isomerase (AI): Aconitate isomerase is an enzyme protein that can convert cis-aconitate into trans-aconitate.

[0058] Expression: The term “expression” includes any step involving the production of aconitase or aconitase isomerase or mutants thereof, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0059] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0060] Importation: The process of integrating a foreign gene into the genome of a target cell to achieve stable expression, or the process of inserting a foreign gene into a vector and introducing it into a host cell for expression, as well as other forms of introducing foreign genes into host cells for expression, are called gene importation.

[0061] In the following embodiments:

[0062] Plasmid extraction was performed using the OMEGA Plasmid Mini Kit I (D6943-02), DNA fragment recovery was performed using the OMEGA Cycle-Pure Kit (D6492-02), and gel recovery was performed using the OMEGA Gel Extraction Kit (D2500-01).

[0063] Aspergillus niger solid culture medium PDA: 3.9 g·L -1 Potato dextrose agar medium (Difco) TM Potato Dextrose Agar (BD, LOT: 1165825), sterilized and then plated.

[0064] Aspergillus niger liquid culture medium PDB: 2.4 g·L -1 Potato Dextrose Agar (Difco) TM Potato Dextrose Broth, BD, LOT: 2095227).

[0065] Aspergillus niger sporulation slant culture 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, 40 mg·L -1 CuSO4, 1.5% agar, sterilized at 115℃ for 15 min, dispensed into test tubes, and used to prepare slant agar.

[0066] Regeneration screening medium plate PDA-SH: 3.9 g·L -1 Potato dextrose agar medium (Difco) TM Potato Dextrose Agar (BD, LOT:1165825) and 1.2M sorbitol were sterilized and cooled to about 55°C. Hygromycin B (Solarbio, Catalog No.: M419099) was added to a final concentration of 100 μg / mL to prepare plates.

[0067] Top layer of regeneration screening medium: PDBS 2.4 g·L -1 Potato Dextrose Agar (Difco) TMPotatoDextrose Broth (BD, LOT: 2095227), 1.2M sorbitol, and 0.5% agarose were sterilized to prepare the top agar.

[0068] Regeneration and screening medium plate CD-SPt: 10 g·L -1 Glucose, 3g·L -1 NaNO3, 2g·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 1.2M sorbitol, with the remainder being water, were sterilized and cooled to approximately 55°C. Then, pyrithione (Sigma, Catalog No.: P0256) was added to a final concentration of 100 μg / L to prepare plates.

[0069] Screening medium plate CD-Pt: 10 g·L -1 Glucose, 3 g / L -1 NaNO3, 2g·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, with the remainder being water, was sterilized and cooled to approximately 55°C. Pyrithionein (Sigma, Catalog No.: P0256) was added to a final concentration of 100 μg / L to prepare plates.

[0070] Aspergillus niger fermentation medium: 160 g·L -1 Glucose, 2 g / L -1 NH4NO3, 0.2 g·L -1 (NH4)2HPO4, 20 mg·L -1 FeSO4, 0.4 g·L -1 MgSO4, 40 mg·L -1 ZnSO4, 40 mg·L -1 CuSO4, pH 3.5.

[0071] The plasmid pSGF957 was obtained from Seoul National University and is documented in Kim, JG, Choi, YD, Chang, YJ, Kim, SU, Genetic transformation of Monascus purpureus DSM1379, Biotechnology Letters, 2003, 25, 1509-1514.

[0072] The plasmid pUC57-Kan was purchased from Genewiz Biotechnology Co., Ltd.

[0073] Aspergillus niger MEFC1501 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 40614, deposited on April 27, 2023, at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0074] Aspergillus niger Co827 was purchased from the China Industrial Microbial Culture Collection Center.

[0075] Aspergillus niger NRRL 27809, NRRL 31821, NRRL 41873, and NRRL 6276 were purchased from the Agricultural Culture Collection (ARS).

[0076] Aspergillus terreus CICC 40205 was purchased from the China Industrial Microbial Culture Collection Center.

[0077] Plasmid pAN52-4 (obtained by 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).

[0078] The plasmid pmWM23 is described in the patent with publication number CN111944706A.

[0079] This invention uses *Aspergillus niger* as the starting strain. Through genetic engineering, heterologous aconitase and aconitase are expressed in the genome of the starting strain to obtain a recombinant *Aspergillus niger* strain, realizing the synthesis of trans-aconitine from *Aspergillus niger*. The starting strain is a known *Aspergillus niger* strain 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* NRRL6276, or other genetically engineered or wild-type strains capable of synthesizing citric acid. The following describes the construction method of the recombinant *Aspergillus niger* strain of this invention using *Aspergillus niger* MEFC1501 as an example.

[0080] Detection method:

[0081] Determination of trans-aconitic acid, citric acid, cis-aconitic acid, etc.: The content and purity of each organic acid in the fermentation broth were determined by high performance liquid chromatography.

[0082] Example 1: Construction of recombinant Aspergillus niger heterologous expression of aconitase Aco from Aspergillus terreus and aconitase TbrA from Bacillus thuringiensis.

[0083] 1. Constructing an expression cassette for the aconitase gene ATEG_03325

[0084] 1) Construct an expression cassette for the aconitase gene ATEG_03325 using the PgpdAt promoter.

[0085] Using fuPgpdAt-03325-F (5'-aacaactcatcaatcatcacaacatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus terreus CICC 40205 as a template. After purification and recovery of the product, the fragment of the ATEG_03325 gene was obtained.

[0086] Using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and fuPgpdAt-03325-R (5'-atgcgcgcaaggcgggtggagatcatgttgtgatgattgatgagttgtt-3') as primer pairs and plasmid pmWM23 as a template, PCR amplification was performed. After purification and recovery, the PgpdAt-TtrpC fragment was obtained. PgpdAt is the promoter of Aspergillus terrestris glyceraldehyde-3-phosphate dehydrogenase gene, and TtrpC is the terminator of Aspergillus nidulans tryptophan synthase.

[0087] The two fragments were cloned in one step using PgpdAt-F (5'-ttacactctgggaggatccaggtac-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the product, the PgpdAt-ATEG_03325-TtrpC expression cassette fragment was obtained.

[0088] 2) Construct an expression cassette for the aconitase gene ATEG_03325 using the PgpdAn promoter.

[0089] Using fuPgpdAn-03325-F (5'-ccgcttgagcagacatcaccatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus terreus CICC 40205 as a template. After purification and recovery of the product, the fragment of the ATEG_03325 gene was obtained.

[0090] Using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and fuPgpdAn-03325-R (5'-atgcgcgcaaggcgggtggagatcatggtgatgtctgctcaagcgg-3') as primer pairs and plasmid pAN52-4 as a template, PCR amplification was performed. After purification and recovery, the PgpdAn-TtrpC fragment was obtained. PgpdAn is the promoter of Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene, and TtrpC is the terminator of Aspergillus nidulans tryptophan synthase.

[0091] The two fragments were cloned in one step using PgpdAn-F (5'-ttgatcgagacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the product, the PgpdAn-ATEG_03325-TtrpC expression cassette fragment was obtained.

[0092] 3) Construct an expression cassette for the aconitase gene ATEG_03325 using the PglaA promoter.

[0093] Using fuPglaA-03325-F (5'-cagcatcattacacctcagcaatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus terreus CICC 40205 as a template. After purification and recovery of the product, the fragment of the ATEG_03325 gene was obtained.

[0094] Using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and PglaA-R (5'-tgctgaggtgtaatgatgctggggat-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PglaA promoter were obtained.

[0095] Using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0096] The three fragments were cloned in one step using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PglaA-ATEG_03325-TtrpC expression cassette fragment was obtained.

[0097] 4) Construct an expression cassette for the aconitase gene ATEG_03325 using the PcadA promoter.

[0098] Using fuPcadA-03325-F (5'-cctcttaaattgaccatgaatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus terreus CICC 40205 as a template. After purification and recovery of the product, the fragment of the ATEG_ATEG_03325 gene was obtained.

[0099] Using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and PcadA-R (5'-ggtcaatttaagaggacgatcttcg-3') as primer pairs, and the genome of Aspergillus terrestris CICC 40205 as a template, PCR amplification was performed. After purification and recovery of the product, fragments of the PcadA promoter were obtained.

[0100] Using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0101] The three fragments were cloned in one step using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PcadA-ATEG_03325-TtrpC expression cassette fragment was obtained.

[0102] 5) Construct an expression cassette for the aconitase gene ATEG_03325 using the PmfsA promoter.

[0103] Using fuPmfsA-03325-F (5'-accgacttctcatccatcttcaaaatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus terreus CICC 40205 as a template. After purification and recovery of the product, the fragment of the ATEG_03325 gene was obtained.

[0104] Using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PmfsA promoter were obtained.

[0105] Using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0106] The three fragments were cloned in one step using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PmfsA-ATEG_03325-TtrpC expression cassette fragment was obtained.

[0107] 6) Construct an expression cassette for the aconitase gene ATEG_03325 using the PacoA promoter.

[0108] Using fuPacoA-03325-F (5'-cctcgcatagagagcttccatcatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus terreus CICC 40205 as a template. After purification and recovery of the product, the fragment of the ATEG_03325 gene was obtained.

[0109] Using PacoA-F (5'-tggcaccggtccgcggga-3') and PacoA-R (5'-gatggaagctctctatgcgagg-3') as primer pairs, and the genome of Aspergillus terreus CICC40205 as a template, PCR amplification was performed, and the PacoA promoter fragment was obtained after purification and recovery of the product.

[0110] Using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0111] The three fragments were cloned in one step using PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PacoA-ATEG_03325-TtrpC expression cassette fragment was obtained.

[0112] 7) Construct an expression cassette for the aconitase gene ATEG_03325 using the PcitA promoter.

[0113] Using fuPcitA-03325-F (5'-cttttttagactcttgttggattcaaaatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus terreus CICC 40205 as a template. After purification and recovery of the product, the fragment of the ATEG_03325 gene was obtained.

[0114] Using PcitA-F (5'-caaccaaggaccgcgatg-3') and PcitA-R (5'-ttgaatccaacaagagtctaaaaaag-3') as primer pairs, and the genome of Aspergillus terreus CICC40205 as a template, PCR amplification was performed. After purification and recovery of the product, the fragment of the PcitA promoter was obtained.

[0115] Using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as a template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0116] The three fragments were cloned in one step using PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PcitA-ATEG_03325-TtrpC expression cassette fragment was obtained.

[0117] 8) Construct an expression cassette for the aconitase gene ATEG_03325 using the PicdA promoter.

[0118] Using fuPicdA-03325-F (5'-cgcaggccacgcttcactgtcgaaatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus terreus CICC 40205 as a template. After purification and recovery of the product, the fragment of the ATEG_03325 gene was obtained.

[0119] Using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and PicdA-R (5'-ttcgacagtgaagcgtggcctgcg-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PicdA promoter were obtained.

[0120] Using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0121] The three fragments were cloned in one step using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PicdA-ATEG_03325-TtrpC expression cassette fragment was obtained.

[0122] 9) Construct an expression cassette for the aconitase gene ATEG_03325 using the PgasA promoter.

[0123] Using fuPgasA-03325-F (5'-gtcttctttcgttcacctcctcacatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus terreus CICC 40205 as a template. After purification and recovery of the product, the fragment of the ATEG_03325 gene was obtained.

[0124] Using PgasA-F (5'-ctgctctctctctgctctctttct-3') and PgasA-R (5'-gtgaggaggtgaacgaaagaagac-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PgasA promoter were obtained.

[0125] Using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as a template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0126] The three fragments were cloned in one step using PgasA-F (5'-ctgctctctctctgctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PgasA-ATEG_03325-TtrpC expression cassette fragment was obtained.

[0127] 2. Constructing the targeting element for aconitine isomerase TbrA

[0128] The aconitine isomerase TbrA gene was artificially synthesized, and its codons were optimized for expression in Aspergillus niger. The gene was then cloned into the pUC57-Kan vector to construct the plasmid puc57-TbrA.

[0129] 1) Construct an expression cassette for aconitine isomerase TbrA using the PgpdAt promoter.

[0130] Using fuPgpdAt-TbrA-F (5'-ctcatcaatcatcacaacatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template, PCR amplification was performed. After purification and recovery of the product, the TbrA gene fragment was obtained.

[0131] Using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and fuPgpdAt-TbrA-R (5'-cgaagcaggggatcttcatgttgtgatgattgatgag-3') as primer pairs and plasmid pmWM23 as a template, PCR amplification was performed. After purification and recovery of the product, the PgpdAt-TtrpC fragment was obtained.

[0132] The two fragments were cloned in one step using PgpdAt-F (5'-ttacactctgggaggatccaggtac-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the product, the PgpdAt-TbrA-TtrpC expression cassette fragment was obtained.

[0133] 2) Construct an expression cassette for aconitine isomerase TbrA using the PgpdAn promoter.

[0134] Using fuPgpdAn-TbrA-F (5'-ccgcttgagcagacatcaccatgaagatcccctgcttcgt-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template, PCR amplification was performed, and the TbrA gene fragment was obtained after purification and recovery of the product.

[0135] Using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and fuPgpdAn-TbrA-R (5'-acgaagcaggggatcttcatggtgatgtctgctcaagcgg-3') as primer pairs and plasmid pAN52-4 as a template, PCR amplification was performed. After purification and recovery, the PgpdAn-TtrpC fragment was obtained. PgpdAn is the promoter of Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene, and TtrpC is the terminator of Aspergillus nidulans tryptophan synthase.

[0136] The two fragments were cloned in one step using PgpdAn-F (5'-ttgatcgagacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PgpdAn-TbrA-TtrpC expression cassette fragment was obtained.

[0137] 3) Construct an expression cassette for aconitine isomerase TbrA using the PglaA promoter.

[0138] Using fuPglaA-TbrA-F (5'-gcatcattacacctcagcaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template, PCR amplification was performed. After purification and recovery of the product, the TbrA gene fragment was obtained.

[0139] Using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and PglaA-R (5'-tgctgaggtgtaatgatgctggggat-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PglaA promoter were obtained.

[0140] Using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0141] The three fragments were cloned in one step using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PglaA-TbrA-TtrpC expression cassette fragment was obtained.

[0142] 4) Construct an expression cassette for aconitine isomerase TbrA using the PcadA promoter.

[0143] Using fuPcadA-TbrA-F (5'-cctcttaaattgaccatgaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template, PCR amplification was performed. After purification and recovery of the product, the TbrA gene fragment was obtained.

[0144] Using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and PcadA-R (5'-tcatggtcaatttaagagg-3') as primer pairs, and the genome of Aspergillus terreus CICC40205 as a template, PCR amplification was performed. After purification and recovery of the product, fragments of the PcadA promoter were obtained.

[0145] Using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0146] The three fragments were cloned in one step using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PcadA-TbrA-TtrpC expression cassette fragment was obtained.

[0147] 5) Construct an expression cassette for aconitine isomerase TbrA using the PmfsA promoter.

[0148] Using fuPmfsA-TbrA-F (5'-accgacttctcatccatcttcaaaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template, PCR amplification was performed. After purification and recovery of the product, the TbrA gene fragment was obtained.

[0149] Using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PmfsA promoter were obtained.

[0150] Using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0151] The three fragments were cloned in one step using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PmfsA-TbrA-TtrpC expression cassette fragment was obtained.

[0152] 6) Construct an expression cassette for aconitine isomerase TbrA using the PacoA promoter.

[0153] Using fuPacoA-TbrA-F (5'-cctcgcatagagagcttccatcatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template, PCR amplification was performed. After purification and recovery of the product, the TbrA gene fragment was obtained.

[0154] Using PacoA-F (5'-tggcaccggtccgcggga-3') and PacoA-R (5'-gatggaagctctctatgcgagg-3') as primer pairs, and the genome of Aspergillus terreus CICC 40205 as a template, PCR amplification was performed, and the PacoA promoter fragment was obtained after purification and recovery of the product.

[0155] Using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0156] The three fragments were cloned in one step using PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The PacoA-TbrA-TtrpC expression cassette fragment was obtained after purification and recovery of the product.

[0157] 7) Construct an expression cassette for aconitine isomerase TbrA using the PcitA promoter.

[0158] Using fuPcitA-TbrA-F (5'-cttttttagactcttgttggattcaaaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template, PCR amplification was performed, and the TbrA gene fragment was obtained after purification and recovery of the product.

[0159] 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, PCR amplification was performed. After purification and recovery of the product, the fragment of the PcitA promoter was obtained.

[0160] Using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0161] The three fragments were cloned in one step using PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The products were purified and recovered to obtain the PcitA-TbrA-TtrpC expression cassette fragment.

[0162] 8) Construct an expression cassette for aconitine isomerase TbrA using the PicdA promoter.

[0163] Using fuPicdA-TbrA-F (5'-cgcaggccacgcttcactgtcgaaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template, PCR amplification was performed. After purification and recovery of the product, the TbrA gene fragment was obtained.

[0164] Using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and PicdA-R (5'-ttcgacagtgaagcgtggcctgcg-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PicdA promoter were obtained.

[0165] Using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0166] The three fragments were cloned in one step using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PicdA-TbrA-TtrpC expression cassette fragment was obtained.

[0167] 9) Construct an expression cassette for aconitine isomerase TbrA using the PgasA promoter.

[0168] Using fuPgasA-TbrA-F (5'-gtcttctttcgttcacctcctcacatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template, PCR amplification was performed. After purification and recovery of the product, the TbrA gene fragment was obtained.

[0169] Using PgasA-F (5'-ctgctctctctctgctctctttct-3') and PgasA-R (5'-gtgaggaggtgaacgaaagaagac-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PgasA promoter were obtained.

[0170] Using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0171] The three fragments were cloned in one step using PgasA-F (5'-ctgctctctctctgctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PgasA-TbrA-TtrpC expression cassette fragment was obtained.

[0172] 3. Construction of the hph expression cassette for the hygromycin B selection marker gene:

[0173] Using hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') as primer pairs and plasmid pSGF957 as a template, PCR amplification of the PtrpC-hph-TtrpC fragment was performed. PtrpC is the promoter of the Aspergillus nidulans tryptophan synthase gene, hph is the hygromycin phosphotransferase gene, and TtrpC is the terminator of the Aspergillus nidulans tryptophan synthase gene. After purification and recovery of the amplification product, the hygromycin B resistance hph gene expression cassette PtrpC-hph-TtrpC was obtained.

[0174] 4. The aconitase gene ATEG_03325 expression cassette and the aconitase isomerase TbrA expression cassette were co-transformed into Aspergillus niger protoplasts to obtain recombinant Aspergillus niger strains:

[0175] 1) Preparation of Aspergillus niger protoplasts

[0176] A spore suspension of Aspergillus niger MEFC1501 was inoculated into 50 mL of liquid PDB medium, resulting in a spore concentration of approximately 10. 7The mycelia were cultured at 200 rpm and 33 °C for 12-18 h. The grown mycelia were collected by filtration through a sterile 100-mesh nylon cloth and rinsed three times with sterile 0.6 M MgSO4 solution. After pressing dry, the mycelia were placed in a sterile 50 ml Erlenmeyer flask. 1 g of mycelia were weighed and added to 10 mL of enzymatic hydrolysate, and the mixture was treated at 30 °C and 120 rpm for 1-2 h. The enzymatic hydrolysate consisted of 1% cellulase (Sigma, Catalog No.: C1184), 1% lyase (Sigma, Catalog No.: L1412), 1% snailase (Shanghai Sangon Biotech, Catalog No.: SB0870), and 0.6 M MgSO4, and was sterilized by filtration through a 0.22 μm sterile filter.

[0177] The enzymatically hydrolyzed mixture was filtered through a 300-mesh nylon cloth, and the filtrate was collected. Protoplasts were collected by centrifugation at 4 °C, washed once with pre-cooled 1.0 M sorbitol solution, and then washed once with pre-cooled STC (1.0 M sorbitol, 50 mM Tris·HCl-pH 8.0, 50 mM CaCl2). Finally, the protoplasts were resuspended in 150 μl of pre-cooled STC, and the protoplast concentration was adjusted to 5 × 10⁻⁶ using STC. 7 Protoplast suspension was obtained by 1 / mL.

[0178] 2) Co-transformation of Aspergillus niger protoplasts with the aconitase gene ATEG_03325 expression cassette, the aconitase isomerase TbrA expression cassette, and the resistance gene hph:

[0179] Add approximately 5 μg of aconitase gene ATEG_03325 expression cassette DNA, approximately 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, 50 Mm Tris-HCl pH 8.0, 50 mM CaCl2), mix gently, and incubate on ice for 30 min. Add 1.5 mL of PSTC, mix well, and let stand at room temperature for 20 min. Then mix with the upper agar and pour onto PDA-SH regeneration selection medium plates. Incubate at 30°C in the dark for 3-4 days.

[0180] Transformants were transferred from the plate to screening plates PDA-H (4g of potato dextrose agar medium was dissolved in 100ml of distilled water, sterilized, and cooled to about 55°C. Hygromycin was added to a final concentration of 100μg / mL to prepare the plate). The plate was then incubated at 30°C for 3-5 days to obtain transformants.

[0181] In the preparation of the recombinant Aspergillus niger strains containing the ATEG_03325 expression cassette and TbrA expression cassette driven by each promoter, the ATEG_03325 expression cassette can also be linked with the hygromycin B resistance hph gene expression cassette to form a single expression cassette, or the TbrA expression cassette can be linked with the hygromycin B resistance hph gene expression cassette to form a single expression cassette. Then, the protoplasts can be transformed without affecting the screening of positive transformants or the identification of genotypes.

[0182] 3) Verification of the genotype of the recombinant Aspergillus niger strain:

[0183] The recombinant Aspergillus niger transformants obtained above were inoculated onto PDA-H plates to culture spores. The spores were then cultured in liquid medium, and the hyphae were collected to extract the genome. PCR was performed using PgpdAt-F743 (5'-ttacactctgggaggatccaggtact-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs to amplify the PgpdAt-ATEG_03325-TtrpC element and / or PgpdAt-TbrA-TtrpC inserted into the genome; PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R were used as primer pairs. PCR was performed using primers (5'-attacctctaaacaagtgtac-3') to amplify the PglaA-ATEG_03325-TtrpC element and / or PglaA-TbrA-TtrpC inserted into the genome; PCR was performed using primers PgpdAn-F (5'-ttgatcgagacctaatacagc-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') to amplify the PgpdAn-ATEG_03325-TtrpC element and / or PgpdAn-TbrA-TtrpC inserted into the genome; PCR was performed using primers PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') to amplify the PgpA-ATEG_03325-TtrpC element and / or PgpdAn-TbrA-TtrpC inserted into the genome. PCR was performed using primers (5'-gtacagtggccatgaaatccaatc-3') to amplify the PcadA-ATEG_03325-TtrpC element and / or PcadA-TbrA-TtrpC inserted into the genome; PCR was performed using primers (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify the PcadA-ATEG_03325-TtrpC element and / or PcadA-TbrA-TtrpC inserted into the genome; PCR was performed using primers (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-gtacagtggccatgaaatccaatc-3') to amplify the PcadA-ATEG_03325-TtrpC element and / or PcadA-TbrA-TtrpC inserted into the genome. PCR was performed using primers (5'-attacctctaaacaagtgtac-3') to amplify PacoA-ATEG_03325-TtrpC elements and / or PacoA-TbrA-TtrpC inserted into the genome.PCR was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pairs to amplify the PcitA-ATEG_03325-TtrpC element and / or PcitA-TbrA-TtrpC inserted into the genome; PCR was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pairs to amplify the PicdA-ATEG_03325-TtrpC element and / or PicdA-TbrA-TtrpC inserted into the genome; PCR was performed using PgasA-F (5'-ctgctctctctgctctctttct-3') and TtrpC-R primer pairs to amplify the PcitA-ATEG_03325-TtrpC element and / or PicdA-TbrA-TtrpC inserted into the genome. PCR was performed using primers (5'-attacctctaaacaagtgtac-3') to amplify the PgasA-ATEG_03325-TtrpC element and / or PgasA-TbrA-TtrpC inserted into the genome.

[0184] Using hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') as primer pairs, the PtrpC-hph-TtrpC fragment inserted into the recombinant bacteria was amplified, and the PCR products were analyzed by 0.8% agarose gel electrophoresis.

[0185] The genomic DNA template of the originating strain Aspergillus niger MEFC1501 was used as a negative control.

[0186] Transformants with verified genotypes were transferred to PDA-H plates and passaged three times. Spores were then collected and serially diluted with physiological saline. 100 μL of each diluted spore was spread onto PDA-H plates to allow for the growth of independent single colonies, thus achieving single-spore isolation. Spores were then collected from these single colonies for further single-spore isolation, for a total of four passages. The recombinant strains isolated from single spores underwent genotype identification for verification. A selection of correctly identified recombinant strains were then screened using shake-flask fermentation.

[0187] Example 2. Construction of recombinant Aspergillus niger heterologously expressing aconitase Aco and aconitase Adi1

[0188] 1. Construct the expression cassette of aconitase gene ATEG_03325, as described in Example 1.

[0189] 2. Construction of an expression cassette for aconitine isomerase Adi1

[0190] The aconitine isomerase Adi1 gene was artificially synthesized, and its codons were optimized for expression in Aspergillus niger. It was then cloned into the pUC57-Kan vector to construct the plasmid puc57-Adi1.

[0191] 1) Construct an expression cassette for aconitine isomerase Adi1 using the PgpdAt promoter.

[0192] Using fuPgpdAt-Adi1-F (5'-ctcatcaatcatcacaacatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs, PCR amplification was performed using puc57-Adi1 plasmid as a template. After purification and recovery of the product, the fragment of the Adi1 gene was obtained.

[0193] Using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga -3') and fuPgpdAt-Adi1-R (5'-tggtgtcgatggggtgcagcatgttgtgatgattgatgag -3') as primer pairs and plasmid pmWM23 as template, PCR amplification was performed. After purification and recovery of the product, the PgpdAt-TtrpC fragment was obtained.

[0194] The two fragments were cloned in one step using PgpdAt-F (5'-ttacactctgggaggatccaggtac-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the product, the PgpdAt-Adi1-TtrpC expression cassette fragment was obtained.

[0195] 2) Construct an expression cassette for aconitine isomerase Adi1 using the PgpdAn promoter.

[0196] Using fuPgpdAn-Adi1-F (5'-ccgcttgagcagacatcaccatgctgcaccccatcgacaccat-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs, PCR amplification was performed using puc57-Adi1 plasmid as a template. After purification and recovery of the product, the fragment of the Adi1 gene was obtained.

[0197] Using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and fuPgpdAn-Adi1-R (5'-tggtgtcgatggggtgcagcatggtgatgtctgctcaagcgg-3') as primer pairs and plasmid pAN52-4 as a template, PCR amplification was performed. After purification and recovery, the PgpdAn-TtrpC fragment was obtained. PgpdAn is the promoter of Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene, and TtrpC is the terminator of Aspergillus nidulans tryptophan synthase.

[0198] The two fragments were cloned in one step using PgpdAn-F (5'-ttgatcgagacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the product, the PgpdAn-Adi1-TtrpC expression cassette fragment was obtained.

[0199] 3) Construct an expression cassette for aconitine isomerase Adi1 using the PglaA promoter.

[0200] Using fuPglaA-Adi1-F (5'-gcatcattacacctcagcaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template, PCR amplification was performed. After purification and recovery of the product, the fragment of the Adi1 gene was obtained.

[0201] Using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and PglaA-R (5'-tgctgaggtgtaatgatgctggggat-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PglaA promoter were obtained.

[0202] Using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0203] The three fragments were cloned in one step using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The products were purified and recovered to obtain the PglaA-Adi1-TtrpC expression cassette fragment.

[0204] 4) Construct an expression cassette for aconitine isomerase Adi1 using the PcadA promoter.

[0205] Using fuPcadA-Adi1-F (5'-cctcttaaattgaccatgaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs, and puc57-Adi1 plasmid as template, PCR amplification was performed. After purification and recovery of the product, the fragment of the Adi1 gene was obtained.

[0206] 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, PCR amplification was performed. After purification and recovery of the product, fragments of the PcadA promoter were obtained.

[0207] Using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0208] The three fragments were cloned in one step using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The products were purified and recovered to obtain the PcadA-Adi1-TtrpC expression cassette fragment.

[0209] 5) Construct an expression cassette for aconitine isomerase Adi1 using the PmfsA promoter.

[0210] Using fuPmfsA-Adi1-F (5'-accgacttctcatccatcttcaaaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs, and puc57-Adi1 plasmid as template, PCR amplification was performed. After purification and recovery of the product, the fragment of the Adi1 gene was obtained.

[0211] Using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PmfsA promoter were obtained.

[0212] Using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0213] The three fragments were cloned in one step using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PmfsA-Adi1-TtrpC expression cassette fragment was obtained.

[0214] 6) Construct an expression cassette for aconitine isomerase Adi1 using the PacoA promoter.

[0215] Using fuPacoA-Adi1-F (5'-cctcgcatagagagcttccatcatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs, and puc57-Adi1 plasmid as template, PCR amplification was performed. After purification and recovery of the product, the fragment of the Adi1 gene was obtained.

[0216] Using PacoA-F (5'-tggcaccggtccgcggga-3') and PacoA-R (5'-gatggaagctctctatgcgagg-3') as primer pairs, and the genome of Aspergillus terreus CICC 40205 as a template, PCR amplification was performed. After purification and recovery of the product, fragments of the PacoA promoter were obtained.

[0217] Using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0218] The three fragments were cloned in one step using PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PacoA-Adi1-TtrpC expression cassette fragment was obtained.

[0219] 7) Construct an expression cassette for aconitine isomerase Adi1 using the PcitA promoter.

[0220] Using fuPcitA-Adi1-F (5'-cttttttagactcttgttggattcaaaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template, PCR amplification was performed, and the product was purified and recovered to obtain the fragment of the Adi1 gene.

[0221] 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, PCR amplification was performed. After purification and recovery of the product, the fragment of the PcitA promoter was obtained.

[0222] Using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0223] The three fragments were cloned in one step using PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PcitA-Adi1-TtrpC expression cassette fragment was obtained.

[0224] 8) Construct an expression cassette for aconitine isomerase Adi1 using the PicdA promoter.

[0225] Using fuPicdA-Adi1-F (5'-cgcaggccacgcttcactgtcgaaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template, PCR amplification was performed. After purification and recovery of the product, the fragment of the Adi1 gene was obtained.

[0226] Using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and PicdA-R (5'-ttcgacagtgaagcgtggcctgcg-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PicdA promoter were obtained.

[0227] Using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0228] The three fragments were cloned in one step using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PicdA-Adi1-TtrpC expression cassette fragment was obtained.

[0229] 9) Construct an expression cassette for aconitine isomerase Adi1 using the PgasA promoter.

[0230] Using fuPgasA-Adi1-F (5'-gtcttctttcgttcacctcctcacatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template, PCR amplification was performed. After purification and recovery of the product, the fragment of the Adi1 gene was obtained.

[0231] Using PgasA-F (5'-ctgctctctctctgctctctttct-3') and PgasA-R (5'-gtgaggaggtgaacgaaagaagac-3') as primer pairs, PCR amplification was performed using the genome of Aspergillus niger MEFC1501 as a template. After purification and recovery of the product, fragments of the PgasA promoter were obtained.

[0232] Using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template, PCR amplification was performed. After purification and recovery of the product, the TtrpC fragment was obtained.

[0233] The three fragments were cloned in one step using PgasA-F (5'-ctgctctctctctgctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the products, the PgasA-Adi1-TtrpC expression cassette fragment was obtained.

[0234] 3. Construction of the hph expression cassette for the hygromycin B selection marker gene, as described in Example 1.

[0235] 4. The aconitase gene ATEG_03325 expression cassette and the aconitase isomerase Adi1 expression cassette were co-transformed into Aspergillus niger protoplasts to obtain recombinant Aspergillus niger strains:

[0236] 1) Preparation of Aspergillus niger protoplasts, refer to Example 1.

[0237] 2) Aconitase gene ATEG_03325 expression cassette, aconitase Adi1 expression cassette, and resistance gene hph Co-transformation of Aspergillus niger protoplasts:

[0238] Approximately 5 μg of aconitase gene ATEG_03325 expression cassette DNA, approximately 5 μg of aconitase Adi1 expression cassette DNA, and 1 μg of hygromycin B resistance were added to the above protoplast suspension. hph Add the gene expression cassette DNA, then add 50 μL of PSTC (40% PEG4000, 50 Mm Tris-HCl pH 8.0, 50 mM CaCl2), mix gently, and incubate on ice for 30 min. Add 1.5 mL of PSTC, mix well, and let stand at room temperature for 20 min; then mix with the upper agar and pour onto PDA-SH regeneration selection medium plates, and incubate at 30°C in the dark for 3-4 days.

[0239] Transformants were transferred from the plate to screening plates PDA-H (4g of potato dextrose agar medium was dissolved in 100ml of distilled water, sterilized, and cooled to about 55°C. Hygromycin was added to a final concentration of 100μg / mL to prepare the plate). The plate was then incubated at 30°C for 3-5 days to obtain transformants.

[0240] In the preparation of the recombinant Aspergillus niger strains containing the ATEG_03325 expression cassette and Adi1 expression cassette driven by each promoter, the ATEG_03325 expression cassette can also be linked with the hygromycin B resistance hph gene expression cassette to form a single expression cassette, or the Adi1 expression cassette can be linked with the hygromycin B resistance hph gene expression cassette to form a single expression cassette. Then, the protoplasts can be transformed without affecting the screening of positive transformants or the identification of genotypes.

[0241] 3) Verification of the genotype of the recombinant Aspergillus niger strain:

[0242] The recombinant Aspergillus niger transformants obtained above were inoculated onto PDA-H plates to culture spores. The spores were then cultured in liquid medium, and the hyphae were collected to extract the genome. PCR was performed using PgpdAt-F743 (5'-ttacactctgggaggatccaggtact-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs to amplify the PgpdAt-ATEG_03325-TtrpC element and / or PgpdAt-Adi1-TtrpC inserted into the genome. PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R were used as primer pairs to amplify the genome. PCR was performed using primers (5'-attacctctaaacaagtgtac-3') to amplify the PglaA-ATEG_03325-TtrpC element and / or PglaA-Adi1-TtrpC inserted into the genome; PCR was performed using primers PgpdAn-F (5'-ttgatcgagacctaatacagc-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') to amplify the PgpdAn-ATEG_03325-TtrpC element and / or PgpdAn-Adi1-TtrpC inserted into the genome; PCR was performed using primers PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'- attacctctaaacaagtgtac-3') to amplify the PgpA-ATEG_03325-TtrpC element and / or PgpdAn-Adi1-TtrpC inserted into the genome. PCR was performed using primers (5'-gtacagtggccatgaaatccaatc-3') to amplify the PcadA-ATEG_03325-TtrpC element and / or PcadA-Adi1-TtrpC inserted into the genome; PCR was performed using primers (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify the PcadA-ATEG_03325-TtrpC element and / or PcadA-Adi1-TtrpC inserted into the genome; PCR was performed using primers (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-gtacagtggccatgaaatccaatc-3') to amplify the PcadA-ATEG_03325-TtrpC element and / or PcadA-Adi1-TtrpC inserted into the genome; PCR was performed using primers (5'-attacctctaaacaagtgtac-3') to amplify PacoA-ATEG_03325-TtrpC elements and / or PacoA-Adi1-TtrpC inserted into the genome.PCR was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pairs to amplify the PcitA-ATEG_03325-TtrpC element and / or PcitA-Adi1-TtrpC inserted into the genome; PCR was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pairs to amplify the PicdA-ATEG_03325-TtrpC element and / or PicdA-Adi1-TtrpC inserted into the genome; PCR was performed using PgasA-F (5'-ctgctctctctgctctctttct-3') and TtrpC-R primer pairs to amplify the PcitA-ATEG_03325-TtrpC element and / or PicdA-Adi1-TtrpC inserted into the genome. PCR was performed using primers (5'-attacctctaaacaagtgtac-3') to amplify the PgasA-ATEG_03325-TtrpC element and / or PgasA-Adi1-TtrpC inserted into the genome.

[0243] Using hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') as primer pairs, the PtrpC-hph-TtrpC fragment inserted into the recombinant bacteria was amplified, and the PCR products were analyzed by 0.8% agarose gel electrophoresis. The genomic DNA template of the originating strain *Aspergillus niger* MEFC1501 was used as a negative control.

[0244] Transformants with verified genotypes were transferred to PDA-H plates and passaged three times. Spores were then collected and serially diluted with physiological saline. 100 μL of each diluted spore was spread onto PDA-H plates to allow for the growth of independent single colonies, thus achieving single-spore isolation. Spores were then collected from these single colonies for further single-spore isolation, for a total of four passages. The recombinant strains isolated from single spores underwent genotype identification for verification. A selection of correctly identified recombinant strains were then screened using shake-flask fermentation.

[0245] Example 3: Construction of recombinant Aspergillus niger strain using pyridine thiamine ptrA as a selection marker

[0246] Example 2 was repeated, except that the selection marker gene used in the recombinant Aspergillus niger strain in this example was the pyridine thiamine selection marker ptrA gene. The specific method is described below:

[0247] 1. Construction of different promoter expression cassettes for aconitase ATEG_03325, as described in Example 1.

[0248] 2. Construction of different promoter expression cassettes for aconitine isomerase Adi1, as described in Example 2.

[0249] 3. Recombinant Aspergillus niger strains were constructed using the pyridine thiamine resistance gene ptrA as a selection marker.

[0250] 1) Construction of the pyridine thiamine resistance gene ptrA expression cassette

[0251] Using ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atggggtgacgatgagccgc-3') as primer pairs and plasmid pmWM23 as template, PCR amplification was performed. After purification and recovery of the amplification product, the ptrA expression cassette of pyridine thiamine resistance gene was obtained.

[0252] Aspergillus niger MEFC1501 protoplasts were prepared according to the preparation method in Example 1. Approximately 5 μg of aconitase gene ATEG_03325 expression cassette DNA, approximately 5 μg of aconitase Adi1 expression cassette DNA, and 1 μg of the selection marker ptrA fragment were added to the protoplast suspension. Then, 50 μL of PSTC (40% PEG4000, 50 Mm Tris-HCl pH 8.0, 50 mM CaCl2) was added, and the mixture was gently mixed and incubated on ice for 30 min. 1.5 mL of PSTC was added, mixed, and incubated at room temperature for 20 min. The mixture was then combined with the upper agar and poured onto regeneration selection medium plates CD-SPt. The plates were incubated at 33°C in the dark for 3–4 days. Transformants were transferred from the plates to selection plates CD-Pt and incubated at 33°C for 3–5 days to obtain transformants.

[0253] The prepared recombinant Aspergillus niger transformants were picked and inoculated onto CD-Pt plates to culture spores. Then, the spores were inoculated into PDB liquid medium for culture. The hyphae were collected and the genome was extracted. The PmfsA-ATEG_03325-TtrpC element or PgpdAn-Adi1-TtrpC inserted into the genome was amplified. Using ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atggggtgacgatgagccgc-3') as primer pairs, the ptrA fragment inserted into the recombinant bacteria was amplified. The PCR products were analyzed by 0.8% agarose gel electrophoresis.

[0254] The genomic DNA template of the originating strain Aspergillus niger MEFC1501 was used as a negative control.

[0255] The verification and purification of positive transformants were carried out in accordance with Examples 1 and 2.

[0256] Example 4: Fermentation of trans-aconitic acid by recombinant Aspergillus niger

[0257] 1. Shake-flask screening of trans-aconitine produced by recombinant Aspergillus niger strains

[0258] Stable recombinant Aspergillus niger strains obtained from Examples 1, 2, and 3 were inoculated into Aspergillus niger sporulation medium and cultured at 32°C for 6 days to obtain mature spores. Each mature spore was then inoculated into trans-aconitine fermentation medium, with three replicates per strain, and fermented at 34°C and 220 rpm. Mycelia were removed from the fermentation broth by filtration, and the fermentation supernatant was appropriately diluted and analyzed by high-performance liquid chromatography (HPLC).

[0259] 2. Analysis of the content and purity of trans-aconitic acid in the fermentation broth

[0260] The fermentation supernatants of the recombinant strain and the starting strain *Aspergillus niger* MEFC1501 were diluted and analyzed by high-performance liquid chromatography (HPLC). Standards of different concentrations of trans-aconitic acid were used as a standard curve to analyze and compare the content and purity of trans-aconitic acid in the fermentation broth. The chromatographic conditions were as follows: column: Aminex HPX-87H Organic Acid Analysis Column, 300 mm × 7.8 mm (Bio-rad, Cat No. 1250140); mobile phase: 5 mmol / L sulfuric acid; flow rate: 0.5 mL / min; column temperature: 30℃; detection temperature: 30℃; UV detector (210 nm).

[0261] The results are as follows Figure 1 and Figure 2 As shown.

[0262] The results showed that, at the shake-flask fermentation level, the contents of cis-aconitic acid and trans-aconitic acid in the fermentation broth of the recombinant strain were significantly higher than those of the wild-type strain. The effects of the two isomerases, TbrA and Adi1, were not significantly different, but gene expression driven by different promoters showed significant differences, with trans-aconitic acid content reaching the highest at 51.11 g / L. There were no significant differences between different terminators such as TtrpC or Tpgk, nor between different selection tags such as hygromycin or pyrithiamine. Different transformation methods, such as co-transformation with selection tags or transformation by combining the expression cassette of the target gene with the expression cassette of the selection tag, all yielded similar results.

[0263] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A recombinant Aspergillus niger strain producing trans-aconitic acid, characterized in that, The recombinant Aspergillus niger strain heterologously expresses aconitase and aconitase isomerase; The aconitase is Aco, and the aconitase is selected from TbrA or Adi1. The amino acid sequence of the aconitase Aco is shown in SEQ ID NO.1; The amino acid sequence of the aconitine isomerase TbrA is shown in SEQ ID NO.2; The amino acid sequence of the aconitine isomerase Adi1 is shown in SEQ ID NO.

3.

2. The recombinant Aspergillus niger strain according to claim 1, characterized in that, The recombinant Aspergillus niger strain is one or more of the following: 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, and Aspergillus niger MTCC 282; the Aspergillus niger MEFC1501 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC NO.40614.

3. The recombinant Aspergillus niger strain according to claim 1, characterized in that, The recombinant Aspergillus niger strain was obtained by introducing an expression cassette containing an aconitase gene and an expression cassette containing an aconitase isomerase gene.

4. The recombinant Aspergillus niger strain according to claim 3, characterized in that, The expression box includes a promoter selected from one or more of the following: PglaA promoter, PgpdAt promoter, PgpdAn promoter, PacoA promoter, PcitA promoter, PicdA promoter, PcadA promoter, PmfsA promoter, and PgasA promoter.

5. The recombinant Aspergillus niger strain according to claim 3, characterized in that, The expression box includes a terminator, which is selected from the TtrpC terminator or the Tpgk terminator.

6. A whole-cell catalyst, characterized in that, The whole-cell catalyst contains the recombinant Aspergillus niger strain as described in any one of claims 1-5.

7. The use of the recombinant Aspergillus niger strain as described in any one of claims 1-5 or the whole-cell catalyst as described in claim 6 in the production of trans-aconitine.

8. A method for producing trans-aconitic acid, characterized in that, include: Step 1: Construct the recombinant Aspergillus niger strain as described in any one of claims 1-5; Step 2: Inoculate the recombinant Aspergillus niger strain into a fermentation medium for fermentation to obtain trans-aconitine.

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