A recombinant aspergillus niger with high yield of trans-aconitic acid, and a construction method and application thereof

Abstract

The application provides a recombinant Aspergillus niger with high trans-aconitic acid yield and a construction method and application thereof, and relates to the technical field of genetic engineering. Specifically, the recombinant Aspergillus niger heterogeneously expresses aconitate isomerase TbrA and / or Adi1. The application provides a new engineering strain for efficient production of trans-aconitic acid, solves the source predicament of needing plant extraction or needing chemical synthesis in traditional production of trans-aconitic acid, and greatly improves the yield of trans-aconitic acid.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a recombinant Aspergillus niger with high yield of trans-aconitic acid as well as a construction method and application thereof. BACKGROUND

[0002] Trans-aconitic acid (CAS: 4023-65-8) is an unsaturated tricarboxylic acid. Because it contains an unsaturated double bond and three carboxyl groups, it can be used as a monomer compound for the preparation of polymeric materials, and also as a synthetic precursor of other compounds, and is listed by the US Department of Energy as one of the most valuable platform compounds. Trans-aconitate, which is prepared from trans-aconitic acid, is a new generation of safe and environmentally friendly plasticizer for the PVC, PLA and other plastic industries, and has a broad application prospect. Trans-aconitic acid also has good nematicidal activity and is a new type of biological pesticide for nematicide. In addition, as a stereoisomer of cis-aconitic acid (CAS: 585-84-2), a key intermediate of the tricarboxylic acid cycle, trans-aconitic acid has a certain inhibitory effect on aconitase, a key enzyme in the tricarboxylic acid cycle, can interfere with the tricarboxylic acid cycle, and thus affect life activities, showing certain biological activity. In addition, trans-aconitic acid can also be used as a food additive for flavoring of food, beverages and the like.

[0003] Trans-aconitic acid is mainly produced by plant extraction or chemical synthesis. The content of trans-aconitic acid in plants is extremely low, and the chemical synthesis process is complex, has many by-products, and is high in cost. Moreover, no large-scale production has been formed. In order to develop a more green and efficient production process of trans-aconitic acid, researchers used Aspergillus versicolor, an industrial production strain of itaconic acid, as a cell factory. First, the cadA gene of Aspergillus versicolor, which catalyzes the decarboxylation of cis-aconitic acid to generate itaconic acid, was blocked. On this basis, the aconitate isomerase was overexpressed using the promoter PcadA of the cadA gene, and an Aspergillus versicolor engineering strain capable of producing aconitic acid was obtained (CN 112011469A, CN 112029671A). However, the yield and production efficiency of trans-aconitic acid still need to be improved. SUMMARY

[0004] It is found in the present application that many fungi have the ability to efficiently synthesize organic acids in addition to Aspergillus terreus. Among commonly used industrial chassis strains, Aspergillus niger is an excellent citric acid producing strain, and the citric acid produced by Aspergillus niger is a 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, 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 recognized by the US FDA and the World Health Organization as a Generally regarded as safe (GRAS) microorganism, which can be used as a cell factory for large-scale production of proteins, organic acids, antigens and other fields. Therefore, Aspergillus niger is a potential strain for large-scale production of trans-aconitic acid. However, there is no related report on the production of trans-aconitic acid by Aspergillus niger at present.

[0005] In addition, Aspergillus tubigensis, Aspergillus daoxianensis, Aspergillus usamii, Candida and the like can also be used as potential hosts for producing trans-aconitic acid.

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

[0007] In one aspect, the present application provides a recombinant Aspergillus niger with high yield of trans-aconitic acid, wherein aaconitate isomerases are heterologously expressed in the recombinant Aspergillus niger.

[0008] The aconitate isomerases comprise TbrA and / or Adi1.

[0009] In the present application, Aspergillus niger is used for the first time to produce trans-aconitic acid. Since the citric acid produced by Aspergillus niger is a direct precursor of cis-aconitic acid, and Aspergillus niger does not synthesize itaconic acid, compared with the existing trans-aconitic acid engineering strain Aspergillus terreus, the synthesis of itaconic acid does not need to be blocked in Aspergillus niger, which simplifies the path of synthesizing trans-aconitic acid, saves the cost of engineering bacteria, and improves the production efficiency of trans-aconitic acid.

[0010] In addition, since the aconitase Aco of Aspergillus niger is strictly regulated by feedback inhibition, it cannot accumulate cis-aconitic acid in large quantities, and in the present application, the cis-aconitic acid produced in Aspergillus niger is converted to trans-aconitic acid by introducing aconitate isomerases into Aspergillus niger, thereby avoiding the feedback inhibition regulation in the synthesis process of cis-aconitic acid.

[0011] Further, the recombinant A. niger is selected from one or more of A. niger MEFC1501, A. niger ATCC 1015, A. niger NRRL 41873, A. niger NRRL 27809, A. niger NRRL 31821, A. niger NRRL 6276, A. niger Co827, A. niger 5016, A. niger 3008, A. niger GCB75, A. niger ATCC 9142, A. niger MTCC 282.

[0012] Further, the recombinant A. niger further comprises a promoter which initiates the expression of the 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, 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 as shown in SEQ ID NO. 4, the PgpdAn promoter sequence is as shown in SEQ ID NO. 5, the PacoA promoter sequence is as shown in SEQ ID NO. 6, the PcitA promoter sequence is as shown in SEQ ID NO. 7, the PicdA promoter sequence is as shown in SEQ ID NO. 8, the PcadA promoter sequence is as shown in SEQ ID NO. 9, the PmfsA promoter sequence is as shown in SEQ ID NO. 10, and the Pgas promoter sequence is as shown in SEQ ID NO. 11.

[0015] Further, the amino acid sequence of the TbrA comprises a sequence as 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 comprises a sequence as 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] Further, the amino acid sequence of the Adi1 comprises a sequence as shown in SEQ ID No. 2 or an amino acid sequence having at least 60% sequence homology with the sequence of SEQ ID NO. 2; preferably, the aconitate isomerase gene encoding Adi1 comprises a sequence as shown in SEQ ID No. 15 or a nucleotide sequence having at least 60% sequence homology with the sequence of SEQ ID NO. 15.

[0017] The TbrA aconitase is derived from Bacillus thuringiensis, and the Adil aconitase is derived from Ustilago maydis.

[0018] Preferably, the amino acid sequence of the TbrA comprises the sequence shown in SEQ ID No. 1; the aconitase gene encoding the TbrA comprises the sequence shown in SEQ ID No. 14; the amino acid sequence of the Adil comprises the sequence shown in SEQ ID No. 2; and the aconitase gene encoding the Adil comprises the sequence shown in SEQ ID No. 15.

[0019] Further, the recombinant Aspergillus niger further comprises a terminator which terminates the expression of the aconitase; preferably, the terminator comprises a TtrpC terminator and / or a Tpgk terminator.

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

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

[0022] The terminator which terminates the expression of the aconitase in the present application can be a general terminator for fungi, or a specific terminator for the host fungus, and those skilled in the art can make adaptive selection according to the actual situation, and the sequence of the terminator is not compulsorily limited in the present application.

[0023] In another aspect, the present application also provides a biological material, which is any one of A1) to A3):

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

[0025] A2) a recombinant vector containing the expression cassette of A1);

[0026] A3) a whole-cell catalyst containing the recombinant vector of A2) or the recombinant Aspergillus niger described above.

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

[0028] Those skilled in the art can select known plasmids for the construction of the recombinant vector according to the actual situation, and make routine optimization and modification on the constructed recombinant vector, and the type of the plasmid is not compulsorily limited in the present application.

[0029] The person skilled in the art can conceive that other commonly used expression elements can be added to the expression cassette described in the present application to assist the expression of aconitate isomerase in A. niger, such as 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 the person skilled in the art according to the actual situation, and the resistance screening marker gene is not compulsorily limited in the present application.

[0031] In another aspect, the present application also provides a method for constructing the recombinant A. niger with high yield of trans-aconitate, which comprises: introducing aconitate isomerase gene into A. niger to obtain a recombinant A. niger with high yield of trans-aconitate; and the aconitate isomerase comprises TbrA and / or Adil.

[0032] In another aspect, the present application also provides the use of the recombinant A. niger or the biological material in the production of trans-aconitate.

[0033] In another aspect, the present application also provides a method for producing trans-aconitate, which comprises:

[0034] Step one, constructing the recombinant A. niger or the biological material as described above;

[0035] Step two, inoculating the recombinant A. niger into a fermentation medium to perform fermentation to obtain trans-aconitate.

[0036] Preferably, the fermentation medium uses monosaccharides, polysaccharides or mixtures thereof that can be utilized by A. niger as a carbon source, which includes but is not limited to glucose, fructose, sucrose, molasses, corn flour, potato powder, starch hydrolysate, etc.

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

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

[0039] The recombinant Aspergillus niger expressing aconitate isomerase is inoculated into an Aspergillus niger sporulation medium, and mature spores are obtained after 6 days of culture at 32 DEG C, and then the culture to maturity is inoculated into a fermentation medium, and fermentation is carried out at 34 DEG C and 220 rpm for 135 hours.

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

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

[0042] In another aspect, the application also provides trans-aconitic acid produced by the above method.

[0043] The application has the following beneficial effects:

[0044] 1. The process for synthesizing trans-aconitic acid by using the recombinant Aspergillus niger is safe, reliable and non-toxic; Aspergillus niger is a microorganism generally regarded as safe (GRAS) by the US FDA and the World Health Organization, and is widely used as a traditional fermentation strain in the food industry; in the application, Aspergillus niger is used as a starting strain, aconitate isomerase is expressed heterologously, and then a recombinant Aspergillus niger producing trans-aconitic acid is obtained, thereby providing an engineering strain with high safety for efficient production of trans-aconitic acid.

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

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

[0047] Figure 1 A statistical diagram of the synthesis amount of cis-aconitic acid and trans-aconitic acid synthesized by shake flask fermentation of the recombinant Aspergillus niger strains constructed by using different promoters to express the isomerase TbrA;

[0048] Figure 2 Figure 1 shows the statistical chart of the synthesis amount of cis-aconitic acid and trans-aconitic acid synthesized by shake flask fermentation of the recombinant Aspergillus niger strain constructed with the isomerase Adi1 expression cassette using different promoters;

[0049] Figure 3 Figure 1 shows the statistical chart of the synthesis amount of cis-aconitic acid and trans-aconitic acid synthesized by shake flask fermentation of the recombinant Aspergillus niger strain constructed with the isomerase Adi1 expression cassette using different promoters;

[0050] Strain preservation information:

[0051] Aspergillus niger MEFC1501 is preserved in the China General Microbiological Culture Collection Center, and the strain preservation 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. 1, Beichen West Road, Beijing City, Chaoyang District, China. DETAILED DESCRIPTION

[0052] Technical terms:

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

[0054] Expression: The term "expression" includes any step related to the production of aconitate isomerase or its mutants, 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 the amino acid sequences of two protein molecules in molecular evolution studies.

[0056] Recombination: In a broad sense, any process of gene exchange that causes changes in genotype is called recombination.

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

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

[0059] Expression vector: An expression vector is a vector that increases expression elements (such as promoters, RBS, terminators, etc.) on the basis of the basic skeleton of a cloning vector, so that the target gene can be expressed.

[0060] Whole cell catalyst: Whole cell biocatalysis refers to a process of chemical transformation using an intact organism (i.e. whole cell, tissue or even individual) as catalyst, and the intact organism involved in the catalytic process is called whole cell catalyst.

[0061] Engineered strain: A strain of bacteria in which a foreign gene is highly expressed by genetic engineering.

[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 application. The term "host cell" encompasses any progeny of the parent cell that is not identical to the parent cell (due to mutations that occur during replication).

[0063] Introduction: The process of integrating a foreign gene into the genome of a target cell to achieve stable expression, or inserting a foreign gene into a vector to express in a host cell, and other forms of introducing a foreign gene into a host cell for expression, is referred to as the introduction of a gene.

[0064] In order to more clearly illustrate the overall concept of the present application, the following embodiments will be described in detail with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without one or more of these specific details. In other instances, well-known features have not been described in detail to avoid obscuring the present application.

[0065] In the following examples, the reagents or instruments used are not specified by the manufacturer, but are conventional products that can be purchased on the market, unless otherwise specified. In the examples, the specific conditions are not specified, but are carried out according to the conventional conditions or the conditions recommended by the manufacturer.

[0066] In the following examples, the plasmids, endonucleases, PCR enzymes, column DNA extraction kits and DNA gel recovery kits used are commercial products, and the specific operations are carried out according to the kit instructions.

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

[0068] Plasmid Mini Kit I (D6943-02) from OMEGA, Cycle-Pure Kit (D6492-02) from OMEGA and Gel Extraction Kit (D2500-01) from OMEGA were used for the extraction of plasmid, DNA fragment and gel respectively.

[0069] Aspergillus niger solid medium PDA: 3.9 g / L -1 Potato Dextrose Agar (Difco™ Potato Dextrose Agar, BD, LOT: 1165825) was prepared in plates after sterilization.

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

[0071] Aspergillus niger sporulation (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, 40 mg / L -1 CuSO4, 1.5% agar, sterilized at 115°C for 15 min, aliquoted into test tubes and prepared into slants.

[0072] Regeneration selection medium plate PDA-SH: 3.9 g / L -1 Potato Dextrose Agar (Difco™ Potato Dextrose Agar, BD, LOT: 1165825) and 1.2 M sorbitol were prepared into plates after sterilization and cooled to about 55°C, then hygromycin B (Solarbio, Catalog No.: M419099) was added to a final concentration of 100 μg / mL.

[0073] Regeneration selection 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 prepared into top layer agar after sterilization.

[0074] Regeneration selection 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 rest being water, sterilized and cooled to about 55°C when pyridoxal phosphate (Sigma, Catalog No.: P0256) is added to a final concentration of 100 μg / L, to prepare the plate.

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

[0076] 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.

[0077] Plasmid pSGF957, obtained from Seoul National university, the plasmid is described in Kim, J.G., Choi, Y.D., Chang, Y.J., Kim, S.U., Genetic transformation of Monascus purpureus DSM 1379, Biotechnology Letters, 2003, 25, 1509-1514.

[0078] Aspergillus niger MEFC1501 was deposited in China General Microbiological Culture Collection Center, and the strain preservation number was CGMCC NO. 40614, the preservation time was April 27, 2023, and the preservation place was Institute of Microbiology, Chinese Academy of Sciences, No. 1, Yanchun West Road, Beijing City, China.

[0079] Aspergillus niger Co827 was purchased from China General Microbiological Culture Collection Center.

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

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

[0082] Plasmid pSGF957 (obtained from Seoul National University, the plasmid is described in Kim, J.G., Choi, Y.D., Chang, Y.J., Kim, S.U., Genetic transformation of Monascus purpureus DSM 1379, 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 N.D., Busscher M., Pouwels P.H., van den Hondel C.A., Journal of Biotechnology, 1991, 7, 19-33).

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

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

[0086] The amino acid sequence of TbrA is shown as SEQ ID No. 1; the amino acid sequence of Adil is shown as SEQ ID No. 2; the PgpdAt promoter sequence is shown as SEQ ID NO. 4; the PgpdAn promoter sequence is shown as SEQ ID NO. 5; the PcoA 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; the Pgas promoter sequence is shown as SEQ ID NO. 11; the TtrpC terminator sequence is shown as SEQ ID NO. 12; and the Tpgk terminator sequence is shown as SEQ ID NO. 13.

[0087] The present application uses Aspergillus niger as a starting strain, expresses a 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-aconitic acid in Aspergillus niger. The starting strain is currently known Aspergillus niger that can synthesize 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 strains or wild strains that can synthesize citric acid. The following takes Aspergillus niger MEFC1501 as an example to specifically describe the construction method of the recombinant Aspergillus niger strain.

[0088] The determination method of trans-aconitic acid, citric acid, cis-aconitic acid, etc. is to detect the content and purity of each organic acid in the fermentation broth by high performance liquid chromatography.

[0089] In the present application, Aspergillus niger is used for the first time to construct an engineered strain for producing trans-aconitic acid. Figure 1As shown, Aspergillus niger can efficiently synthesize citric acid, which can be converted into cis-aconitic acid under the action of aconitate Aco. Cis-aconitic acid can be partially converted into trans-aconitic acid in the natural state, in addition, there is an aconitate isomerase in microorganisms such as Ustilago maydis and Bacillus thuringiensis and some plants, which can catalyze the isomerization of cis-aconitic acid to generate trans-aconitic acid (Geiser E. et al., Ustilago maydis produces itaconic acid via the unusual intermediate trans-aconitate, Microbial Biotechnology, 2016, 9(1): 116-126). In the present application, Aspergillus niger is used as the starting strain, and the aconitate isomerase from Ustilago maydis or the aconitate isomerase from Bacillus thuringiensis is heterologously expressed in Aspergillus niger, and an engineered Aspergillus niger strain producing trans-aconitic acid is obtained, which can solve the source dilemma of trans-aconitic acid traditionally requiring plant extraction or chemical synthesis through fermentation production.

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

[0091] 1. Construction of an 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 is shown in SEQ ID NO. 14. It was cloned in the pUC57-Kan vector to construct the plasmid puc57-TbrA.

[0093] 1) Construction of an expression cassette for aconitate isomerase TbrA with PgpdAt promoter

[0094] PCR amplification was performed with fuPgpdAt-TbrA-F (5'-ctcatcaatcatcacaacatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs, and puc57-TbrA plasmid as template. The TbrA gene fragment was obtained after purification and recovery of the product.

[0095] The fragment of PgpdAt-TtrpC was obtained by PCR amplification using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and fuPgpdAt-TbrA-R (5'-cgaagcaggggatcttcatgttgtgatgattgatgag-3') as primer pair and plasmid pmWM23 as template.

[0096] The two fragments were cloned in one step to obtain the expression cassette fragment of PgpdAt-TbrA-TtrpC by PCR amplification using PgpdAt-F (5'-ttacactctgggaggatccaggtac-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair.

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

[0098] The fragment of TbrA gene was obtained by PCR amplification using fuPgpdAn-TbrA-F (5'-ccgcttgagcagacatcaccatgaagatcccctgcttcgt-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pair and puc57-TbrA plasmid as template.

[0099] The fragment of PgpdAn-TtrpC was obtained by PCR amplification using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and fuPgpdAn-TbrA-R (5'-acgaagcaggggatcttcatggtgatgtctgctcaagcgg-3') as primer pair and plasmid pAN52-4 as template. PgpdAn is the promoter of triosephosphate dehydrogenase gene of Aspergillus nidulans and TtrpC is the terminator of tryptophan synthetase of Aspergillus nidulans.

[0100] The two fragments above were cloned in one step, and PgpdAn-F (5'- ttgatcgagacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primer pairs for PCR amplification. 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 with PglaA promoter

[0102] fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primer pairs, and puc57-TbrA plasmid was used as the template for PCR amplification. The product was purified and recovered to obtain the TbrA gene fragment.

[0103] PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and PglaA-R (5'-tgctgaggtgtaatgatgctggggat-3') were used as primer pairs, and the genome of Aspergillus niger MEFC1501 was used as the template for PCR amplification. The product was purified and recovered to obtain the PglaA promoter fragment.

[0104] fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primer pairs, and plasmid pAN52-4 was used as the template for PCR amplification. The product was purified and recovered to obtain the TtrpC fragment.

[0105] The three fragments above were cloned in one step, and PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primer pairs for PCR amplification. 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 with PcadA promoter

[0107] The TbrA gene fragment was obtained by PCR amplification using puc57-TbrA plasmid as template, fuPcadA-TbrA-F (5'-cctcttaaattgaccatgaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pair, and then the product was purified and recovered.

[0108] The PcadA promoter fragment was obtained by PCR amplification using the genome of Aspergillus terreus CICC 40205 as template, PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and PcadA-R (5'-tcatggtcaatttaagagg-3') as primer pair, and then the product was purified and recovered.

[0109] The TtrpC fragment was obtained by PCR amplification using plasmid pAN52-4 as template, fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair, and then the product was purified and recovered.

[0110] The PcadA-TbrA-TtrpC expression cassette fragment was obtained by PCR amplification using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair, and then the product was purified and recovered.

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

[0112] The TbrA gene fragment was obtained by PCR amplification using puc57-TbrA plasmid as template, fuPcadA-TbrA-F (5'-cctcttaaattgaccatgaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pair, and then the product was purified and recovered.

[0113] The genomic DNA of A. niger MEFC1501 was used as a template for PCR amplification with the primer pair of PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3'), and the PmfsA promoter fragment was obtained after purification and recovery of the PCR product.

[0114] The plasmid pAN52-4 was used as a template for PCR amplification with the primer pair of fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'), and the TtrpC fragment was obtained after purification and recovery of the PCR product.

[0115] The three fragments were subjected to one-step cloning, and the PmfsA-TbrA-TtrpC expression cassette fragment was obtained after PCR amplification with the primer pair of PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3').

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

[0117] The plasmid puc57-TbrA was used as a template for PCR amplification with the primer pair of fuPacoA-TbrA-F (5'-cctcgcatagagagcttccatcatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3'), and the TbrA gene fragment was obtained after purification and recovery of the PCR product.

[0118] The genomic DNA of A. terreus CICC 40205 was used as a template for PCR amplification with the primer pair of PacoA-F (5'-tggcaccggtccgcggga-3') and PacoA-R (5'-gatggaagctctctatgcgagg-3'), and the PacoA promoter fragment was obtained after purification and recovery of the PCR product.

[0119] The TtrpC fragment was obtained by PCR amplification using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as a primer pair and plasmid pAN52-4 as a template.

[0120] The PacoA-TbrA-TtrpC expression cassette fragment was obtained by PCR amplification using PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as a primer pair and the three fragments above as a template.

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

[0122] The TbrA gene fragment was obtained by PCR amplification using fuPcitA-TbrA-F (5'-cttttttagactcttgttggattcaaaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as a primer pair and puc57-TbrA plasmid as a template.

[0123] The PcitA promoter fragment was obtained by PCR amplification using PcitA-F (5'-caaccaaggaccgcgatg-3') and PcitA-R (5'-ttgaatccaacaagagtctaaaaaag-3') as a primer pair and the genome of Aspergillus terreus CICC 40205 as a template.

[0124] The TtrpC fragment was obtained by PCR amplification using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as a primer pair and plasmid pAN52-4 as a template.

[0125] The three fragments were cloned in one step, and the PicdA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pairs were used for PCR amplification. 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 with the PicdA promoter

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

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

[0129] The fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pairs were used for PCR amplification with the plasmid pAN52-4 as the template, and the product was purified and recovered to obtain the TtrpC fragment.

[0130] The three fragments were cloned in one step, and the PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pairs were used for PCR amplification. 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 with the PgasA promoter

[0132] The TbrA gene fragment was obtained by PCR amplification using puc57-TbrA plasmid as template, fuPgasA-TbrA-F (5'-gtcttctttcgttcacctcctcacatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pair.

[0133] The PgasA promoter fragment was obtained by PCR amplification using the genome of Aspergillus niger MEFC1501 as template, PgasA-F (5'-ctgctctctctctgctctctttct-3') and PgasA-R (5'-gtgaggaggtgaacgaaagaagac-3') as primer pair.

[0134] The TtrpC fragment was obtained by PCR amplification using plasmid pAN52-4 as template, fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair.

[0135] The PgasA-TbrA-TtrpC expression cassette fragment was obtained by PCR amplification using PgasA-F (5'-ctgctctctctctgctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair.

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

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

[0138] 3. Transforming Aspergillus niger protoplasts with the aconitate isomerase TbrA expression cassette to obtain recombinant Aspergillus niger strains:

[0139] 1) Preparation of Aspergillus niger protoplasts

[0140] Spore suspension of Aspergillus niger MEFC1501 was inoculated into 50 mL liquid medium PDB, with a spore concentration of about 10 7 individuals / mL, and cultured at 200 rpm and 33°C for 12-18 h. The mycelium that grew was collected by filtering with sterile single-layer 100-mesh nylon cloth, and rinsed with sterilized 0.6 M MgSO4solution three times, and then dried by pressing and placed in a sterile 50-mL flask. 1 g of the mycelium was weighed, 10 mL of enzyme solution was added, and the mixture was treated at 30°C and 120 rpm for 1-2 h. The enzyme solution was composed of 1% cellulase (Sigma, Catalog No.: C1184), 1% lyticase (Sigma, Catalog No.: L1412), 1% snailase (Shanghai Sangon, Catalog No.: SB0870), and 0.6 M MgSO4. The solution was sterilized by filtering through a 0.22-μm sterile filter.

[0141] The mixture after enzyme treatment was filtered with 300-mesh nylon cloth, and the filtrate was collected. The 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-pH8.0, 50 mM CaCl2), and finally resuspended in 150 μL of pre-cooled STC. The concentration of the protoplasts was adjusted to 5×10 7 individuals / mL with STC, and a protoplast suspension was obtained.

[0142] 2) Co-transformation of Aspergillus niger protoplasts with the aconitate isomerase TbrA expression cassette and the hph resistance gene:

[0143] About 5 μg of the aconitate isomerase TbrA expression cassette DNA and 1 μg of the hygromycin B resistance hph gene expression cassette DNA were added to the protoplast suspension, and 50 μL of PSTC (40% PEG4000, 50 mM Tris-HCl pH8.0, 50 mM CaCl2) was added, and the mixture was mixed gently and incubated in an ice bath for 30 min. 1.5 mL of PSTC was added, the mixture was mixed and incubated at room temperature for 20 min, and then mixed with the upper agar and poured into a regeneration and selection medium plate PDA-SH, and incubated at 30°C in the dark for 3-4 days.

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

[0145] In the preparation of the recombinant Aspergillus niger strain containing the TbrA expression cassette, the TbrA expression cassette can also be linked to the hph gene expression cassette of the hygromycin B resistance to form an expression cassette, and then the protoplast is transformed, without affecting the screening of positive transformants and the identification of the genotype.

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

[0147] The recombinant Aspergillus niger transformants obtained above were inoculated on PDA-H plates to culture spores, and then the spores were inoculated in liquid culture medium for culture, and the mycelium was collected to extract the genome. PCR was performed with a primer pair of PgpdAt-F743 (5'-ttacactctgggaggat ccaggtact-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify PgpdAt-TbrA-TtrpC inserted into the genome, and a primer pair of hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcga acccagg-3') to amplify PtrpC-hph-TtrpC inserted into the recombinant strain. 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.

[0148] Similarly, PglaA-TbrA-TtrpC inserted into the genome was amplified by PCR with primer pair of PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PgpdAn-TbrA-TtrpC inserted into the genome was amplified by PCR with primer pair of PgpdAn-F (5'-ttgatcga gacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PcadA-TbrA-TtrpC inserted into the genome was amplified by PCR with primer pair of PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PmfsA-TbrA-TtrpC inserted into the genome was amplified by PCR with primer pair of PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PacoA-TbrA-TtrpC inserted into the genome was amplified by PCR with primer pair of PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PcitA-TbrA-TtrpC inserted into the genome was amplified by PCR with primer pair of PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PicdA-TbrA-TtrpC inserted into the genome was amplified by PCR with primer pair of PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); and PgasA-TbrA-TtrpC inserted into the genome was amplified by PCR with primer pair of PgasA-F (5'-ctgctctctctctgctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3').

[0149] The transformants with correct genotypes were transferred to PDA-H plates for subculture, and subcultured for 3 times. Then spores were collected and diluted with physiological saline, and 100 μL of the diluted spores were inoculated on PDA-H plates to grow into independent single colonies, i.e. single spore isolation. Spores were collected from the single colonies and subjected to single spore isolation again, and the process was repeated for 4 times. The recombinant strains after single spore isolation were subjected to genotype identification again, and some of the recombinant strains with correct genotypes were selected for shake flask fermentation screening. The shake flask screening method and results are shown in Table 2. Figure 1 .

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

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

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

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

[0154] PCR amplification was performed using the plasmid puc57-Adi1 as a template, with fuPgpdAt-Adi1-F (5'-ctcatcaatcatcacaacatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as a primer pair. The Adi1 gene fragment was obtained after purification and recovery of the product.

[0155] PCR amplification was performed using the plasmid pmWM23 as a template, with fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and fuPgpdAt-Adi1-R (5'-tggtgtcgatggggtgcagcatgttgtgatgattgatgag-3') as a primer pair. The PgpdAt-TtrpC fragment was obtained after purification and recovery of the product.

[0156] The two fragments were cloned in one step, and the expression cassette fragment of PgpdAt-Adil-TtrpC was obtained by PCR amplification using PgpdAt-F (5'- ttacactctgggaggatccaggtac-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs.

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

[0158] The Adil gene fragment was obtained by PCR amplification using fuPgpdAn-Adil-F (5'- ccgcttgagcagacatcaccatgctgcaccccatcgacaccat-3') and fuAdil-TtrpC-R (5'- tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adil plasmid as a template.

[0159] The PgpdAn-TtrpC fragment was obtained by PCR amplification using fuAdil-TtrpC-F (5'- cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and fuPgpdAn-Adil-R (5'- tggtgtcgatggggtgcagcatggtgatgtctgctcaagcgg-3') as primer pairs and plasmid pAN52-4 as a template.

[0160] The two fragments were cloned in one step, and the expression cassette fragment of PgpdAn-Adil-TtrpC was obtained by PCR amplification using PgpdAn-F (5'- ttgatcgagacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs.

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

[0162] The Adil gene fragment was obtained by PCR amplification using fuPcadA-Adil-F (5'-cctcttaaattgaccatgaatgctgcaccccatcgacacca-3') and fuAdil-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as a primer pair and puc57-Adil plasmid as a template.

[0163] The PglaA promoter fragment was obtained by PCR amplification using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and PglaA-R (5'-tgctgaggtgtaatgatgctggggat-3') as a primer pair and the genome of Aspergillus niger MEFC1501 as a template.

[0164] The TtrpC fragment was obtained by PCR amplification using fuAdil-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as a primer pair and plasmid pAN52-4 as a template.

[0165] The PglaA-Adil-TtrpC expression cassette fragment was obtained by PCR amplification using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as a primer pair.

[0166] 4) Construction of an expression cassette of coniferyl acid isomerase Adil using PcadA promoter

[0167] The Adil gene fragment was obtained by PCR amplification using fuPcadA-Adil-F (5'-cctcttaaattgaccatgaatgctgcaccccatcgacacca-3') and fuAdil-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as a primer pair and puc57-Adil plasmid as a template.

[0168] The genomic DNA of A. niger MEFC1501 was used as template for PCR amplification with primer pair PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3'), and the fragment of PmfsA promoter was obtained after purification and recovery of the PCR product.

[0169] The genomic DNA of A. niger MEFC1501 was used as template for PCR amplification with primer pair PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3'), and the fragment of PmfsA promoter was obtained after purification and recovery of the PCR product.

[0170] The three fragments were cloned in one step, and the PcadA-Adi1-TtrpC expression cassette fragment was obtained after PCR amplification with primer pair PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3').

[0171] The three fragments were cloned in one step, and the PcadA-Adi1-TtrpC expression cassette fragment was obtained after PCR amplification with primer pair PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3').

[0172] 5) Construction of an expression cassette of aconitate isomerase Adi1 with PmfsA promoter

[0173] The genomic DNA of A. niger MEFC1501 was used as template for PCR amplification with primer pair PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3'), and the fragment of PmfsA promoter was obtained after purification and recovery of the PCR product.

[0174] The genomic DNA of A. niger MEFC1501 was used as template for PCR amplification with primer pair PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3'), and the fragment of PmfsA promoter was obtained after purification and recovery of the PCR product.

[0175] The fuAdil-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pair was used to perform PCR amplification with plasmid pAN52-4 as the template, and the TtrpC fragment was obtained after purification and recovery of the product.

[0176] The three fragments were cloned in one step, and the PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pair was used to perform PCR amplification, and the PmfsA-Adil-TtrpC expression cassette fragment was obtained after purification and recovery of the product.

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

[0178] The fuPacoA-Adil-F (5'-cctcgcatagagagcttccatcatgctgcaccccatcgacacca-3') and fuAdil-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') primer pair was used to perform PCR amplification with the puc57-Adil plasmid as the template, and the Adil gene fragment was obtained after purification and recovery of the product.

[0179] The PacoA-F (5'-tggcaccggtccgcggga-3') and PacoA-R (5'-gatggaagctctctatgcgagg-3') primer pair was used to perform PCR amplification with the genome of Aspergillus terreus CICC 40205 as the template, and the PacoA promoter fragment was obtained after purification and recovery of the product.

[0180] The fuAdil-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pair was used to perform PCR amplification with plasmid pAN52-4 as the template, and the TtrpC fragment was obtained after purification and recovery of the product.

[0181] The three fragments were cloned in one step, and the PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pairs were used for PCR amplification. The PacoA-Adi1-TtrpC expression cassette fragment was obtained after purification and recovery of the product.

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

[0183] The fuPcitA-Adi1-F (5'-cttttttagactcttgttggattcaaaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') primer pairs were used for PCR amplification with the puc57-Adi1 plasmid as the template. The Adi1 gene fragment was obtained after purification and recovery of the product.

[0184] The PcitA-F (5'-caaccaaggaccgcgatg-3') and PcitA-R (5'-ttgaatccaacaagagtctaaaaaag-3') primer pairs were used for PCR amplification with the genome of Aspergillus terreus CICC 40205 as the template. The PcitA promoter fragment was obtained after purification and recovery of the product.

[0185] The fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pairs were used for PCR amplification with the plasmid pAN52-4 as the template. The TtrpC fragment was obtained after purification and recovery of the product.

[0186] The three fragments were cloned in one step, and the PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') primer pairs were used for PCR amplification. The PcitA-Adi1-TtrpC expression cassette fragment was obtained after purification and recovery of the product.

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

[0188] The Adil gene fragment was obtained by PCR amplification using puc57-Adil plasmid as template, fuPicdA-Adil-F (5'-cgcaggccacgcttcactgtcgaaatgctgcaccccatcgacacca-3') and fuAdil-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pair, and then purifying and recovering the product.

[0189] The PicdA promoter fragment was obtained by PCR amplification using the genome of Aspergillus niger MEFC1501 as template, PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and PicdA-R (5'-ttcgacagtgaagcgtggcctgcg-3') as primer pair, and then purifying and recovering the product.

[0190] The TtrpC fragment was obtained by PCR amplification using plasmid pAN52-4 as template, fuAdil-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair, and then purifying and recovering the product.

[0191] The PicdA-Adil-TtrpC expression cassette fragment was obtained by one-step cloning of the above three fragments, using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair, and then purifying and recovering the product.

[0192] 9) Construction of an expression cassette of aconitate isomerase Adil using PgasA promoter

[0193] The Adil gene fragment was obtained by PCR amplification using puc57-Adil plasmid as template, fuPgasA-Adil-F (5'-gtcttctttcgttcacctcctcacatgctgcaccccatcgacacca-3') and fuAdil-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pair, and then purifying and recovering the product.

[0194] The genomic DNA of A. niger MEFC1501 was used as template for PCR amplification with primer pair PgasA-F (5'-ctgctctctctctgctctctttct-3') and PgasA-R (5'-gtgaggaggtgaacgaaagaagac-3'). The fragment of PgasA promoter was obtained after purification and recovery of the PCR product.

[0195] The plasmid pAN52-4 was used as template for PCR amplification with primer pair fuAdil-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'). The fragment of TtrpC was obtained after purification and recovery of the PCR product.

[0196] The three fragments above were used for one-step cloning. The expression cassette of PgasA-Adil-TtrpC was obtained by PCR amplification with primer pair PgasA-F (5'-ctgctctctctctgctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3').

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

[0198] The same as described in Example 1.

[0199] 3. Transformation of A. niger protoplasts with the expression cassette of aconitate isomerase Adil:

[0200] 1) Preparation of A. niger protoplasts

[0201] The same as described in Example 1.

[0202] 2) Co-transformation of A. niger protoplasts with the expression cassette of aconitate isomerase Adil and the resistance gene hph:

[0203] About 5 μg of the expression cassette of aconitate isomerase Adil and 1 μg of the expression cassette of the resistance gene hph were added to the protoplast suspension above, followed by 50 μL of PSTC (40% PEG4000, 50 mM Tris-HCl pH 8.0, 50 mM CaCl2), and mixed gently. The mixture was incubated in ice bath for 30 min. Then 1.5 mL of PSTC was added, mixed and incubated at room temperature for 20 min. The mixture was then mixed with the top agar and poured into the regeneration and selection medium PDA-SH, and incubated at 30°C in dark for 3-4 days.

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

[0205] In the preparation of the recombinant Aspergillus niger strain containing the aconitate isomerase Adil expression cassette, the aconitate isomerase Adil expression cassette can also be linked to the hygromycin B resistance hph gene expression cassette to form an expression cassette, and then the protoplast is transformed, without affecting the screening of positive transformants and the identification of the genotype.

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

[0207] The recombinant Aspergillus niger transformants obtained above were inoculated on PDA-H plates to culture spores, and then the spores were inoculated in liquid culture medium for culture, and the mycelium was collected to extract the genome. PCR was performed with a primer pair of PgpdAt-F743 (5'-ttacactctgggaggat ccaggtact-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify PgpdAt-Adil-TtrpC inserted into the genome; and a primer pair of hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaa cccagg-3') to amplify the PtrpC-hph-TtrpC fragment inserted into the recombinant strain. 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.

[0208] Similarly, PglaA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PgpdAn-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PgpdAn-F (5'-ttgatcga gacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PcadA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PmfsA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PacoA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PcitA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PicdA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); and PgasA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PgasA-F (5'-ctgctctctctctgctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3').

[0209] The transformants with correct genotypes were transferred to PDA-H plates for subculture, and subcultured for 3 times. Then spores were collected and diluted with physiological saline, and 100 μL of the diluted spores were spread on PDA-H plates to grow into independent single colonies, which were single spore isolates. The spores from the single colonies were collected again for single spore isolation, and the subculture was performed for 4 times. The recombinant strains after single spore isolation were identified again. Some of the recombinant strains with correct genotypes were selected for shake flask fermentation screening. The shake flask screening method and results are shown in Example 4. Figure 2 .

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

[0211] Example 2 was repeated, except that in this example, the screening marker gene used in the recombinant Aspergillus niger strains was pyrithiamine screening marker ptrA gene. The specific method is described as follows:

[0212] 1. Construction of expression cassettes of aconitate isomerase Adil under different promoters, referring to Example 2.

[0213] 2. Construction of recombinant Aspergillus niger strains using pyrithiamine resistance gene ptrA as a screening marker

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

[0215] The pyrithiamine resistance gene ptrA expression cassette was obtained by PCR amplification using plasmid pmWM23 as a template, with ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atggggtgacgatgagccgc-3') as primers. The amplified product was purified and recovered.

[0216] Aspergillus niger MEFC1501 protoplasts were prepared according to the preparation method in Example 1. About 5 μg of aconitate isomerase Adil expression cassette DNA and 1 μg of screening marker ptrA fragment were added to the protoplast suspension, and 50 μL of PSTC (40% PEG4000, 50 mM Tris-HCl pH 8.0, 50 mM CaCl2) was added, mixed gently, and placed in an ice bath for 30 min. 1.5 mL of PSTC was added, mixed, and then placed at room temperature for 20 min. Then mixed with the upper agar and poured into the regeneration and screening medium plate CD-SPt, and cultured at 33°C in the dark for 3-4 days. The transformants were transferred from the plate to the screening plate CD-Pt, and cultured at 33°C for 3-5 days to obtain the transformants.

[0217] The prepared recombinant A. terreus transformant was inoculated on CD-Pt plate to culture spores, and then the spores were inoculated in PDB liquid medium for culture, and the mycelium was collected to extract the genome. PgpdAt-F743 (5'-ttacactctgggaggatccaggtact-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as a primer pair to perform PCR to amplify PgpdAt-Adi1-TtrpC inserted into the genome; ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atggggtgacgatgagccgc-3') were used as a primer pair to amplify the ptrA fragment inserted into the recombinant strain, and the PCR product was analyzed by 0.8% agarose gel electrophoresis. The genome DNA template of the starting strain A. niger MEFC1501 was used as a negative control.

[0218] Similarly, PglaA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PgpdAn-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PgpdAn-F (5'-ttgatcga gacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PcadA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PmfsA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PacoA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PcitA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); PicdA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3'); and PgasA-Adil-TtrpC inserted into the genome was amplified by PCR using primers pair of PgasA-F (5'-ctgctctctctctgctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3').

[0219] Verification and isolation of positive transformants Figure 3 .

[0220] Example 4: Fermentation of trans-aconitic acid by recombinant Aspergillus niger

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

[0222] 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 for 135 h. Mycelia were removed from the fermentation broth by filtration, 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 supernatants of the recombinant strain and the starting strain *Aspergillus niger* MEFC1501 were diluted and analyzed by high-performance liquid chromatography (HPLC). Standard curves were constructed using standards of different concentrations of cis-aconitic acid and trans-aconitic acid to analyze and compare the content and purity of cis-aconitic acid and 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).

[0225] The fermentation production of trans-aconitic acid by the recombinant strains obtained in Examples 1, 2, and 3 are as follows: Figures 1-3 As shown.

[0226] Depend on Figures 1-3 The analysis 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 increased compared with 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 34 g / L. Different terminators, such as TtrpC or Tpgk, showed no significant differences. Different selection tags, such as hygromycin or pyrithionein, also showed no significant differences. 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.

[0227] The above merely provides an example of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application should be included in the scope of claims of the present application.

Claims

1. A recombinant Aspergillus niger with high trans-aconitine production, characterized in that, The recombinant Aspergillus niger heterologous expression of aconitic acid isomerase; The aconitine isomerase is TbrA and / or Adi1, the amino acid sequence of TbrA is shown in SEQ ID No. 1, and the amino acid sequence of Adi1 is shown in SEQ ID No.

2.

2. The recombinant Aspergillus niger according to claim 1, characterized in that, The recombinant Aspergillus niger 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 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 that initiates the expression of aconitine isomerase.

4. The recombinant Aspergillus niger according to claim 3, characterized in that, The promoter is 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 Pgas promoter.

5. The recombinant Aspergillus niger according to claim 4, characterized in that, The promoter is the PglaA promoter.

6. The recombinant Aspergillus niger according to claim 1, characterized in that, The aconitine isomerase gene encoding the TbrA is shown in SEQ ID No.

14.

7. The recombinant Aspergillus niger according to claim 1, characterized in that, The aconitine isomerase gene encoding Adi1 is shown in SEQ ID No.

15.

8. The recombinant Aspergillus niger according to claim 1, characterized in that, The recombinant Aspergillus niger also includes a terminator that terminates the expression of aconitine isomerase.

9. The recombinant Aspergillus niger according to claim 8, characterized in that, The terminator is either the TtrpC terminator or the Tpgk terminator.

10. A whole-cell catalyst, wherein the whole-cell catalyst contains recombinant Aspergillus niger as described in any one of claims 1-9.

11. The method for constructing high-yield trans-aconitine recombinant Aspergillus niger as described in claim 1, characterized in that: Aconitine isomerase gene was introduced into Aspergillus niger to obtain a recombinant Aspergillus niger that produces high levels of trans-aconitine; the aconitine isomerase includes TbrA or Adi1.

12. The use of the recombinant Aspergillus niger as described in any one of claims 1-9 or the whole-cell catalyst as described in claim 10 in the production of trans-aconitine.

13. A method for producing trans-aconitic acid, characterized in that, The method includes: Step 1: Construct the recombinant Aspergillus niger as described in any one of claims 1-9; Step 2: Inoculate the recombinant Aspergillus niger into a fermentation medium for fermentation to obtain trans-aconitine.

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