Recombinant Aspergillus niger strain with high yield of trans-aconitic acid and application of recombinant Aspergillus niger strain

Abstract

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

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to a recombinant Aspergillus niger strain with high trans-aconitic acid production, a method for producing trans-aconitic acid by using the recombinant Aspergillus niger strain, and application thereof. Background Art

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

[0003] At present, trans-aconitic acid is mainly produced by chemical synthesis, which has complex processes, many by-products and high costs, so large-scale production has not been formed. In order to develop a greener and more efficient trans-aconitic acid production process, researchers previously blocked the aconitate decarboxylase gene (cadA) that catalyzes the decarboxylation of aconitate to itaconic acid in Aspergillus terreus and overexpressed aconitate isomerase CICC00358 to obtain an Aspergillus terreus engineered strain that can produce aconitate (CN112011469A, CN112029671A), but the yield is still relatively low and cannot meet the needs of large-scale production.

[0004] In addition to Aspergillus terreus, many fungi have the ability to efficiently synthesize organic acids. Among the commonly used industrial chassis strains, Aspergillus niger is an excellent citric acid producer. The citric acid it produces 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 synthesis path of trans-aconitic acid can be simplified. In addition, Aspergillus niger is a traditional fermentation strain widely used in the food industry. It is recognized as a generally safe, reliable and non-toxic (General Regarded As Safe, GRAS) microorganism by the US FDA and the World Health Organization. It can be used as a cell factory in large quantities in the production of proteins, organic acids, antigens, etc. Therefore, Aspergillus niger is a potential strain for large-scale production of trans-aconitic acid. However, there are currently no reports on the use of Aspergillus niger to produce trans-aconitic acid.

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

[0006] In order to solve the defects of existing plant extraction or chemical synthesis of trans-aconitic acid, such as high cost, low yield, and complex process, the present invention provides an aconitase that can efficiently catalyze the synthesis of cis-aconitic acid and an aconitase that efficiently catalyzes the conversion of cis-aconitic acid into trans-aconitic acid, and a genetically engineered strain of Aspergillus niger capable of synthesizing trans-aconitic acid, and a method for producing trans-aconitic acid by fermentation using Aspergillus niger, so as to realize the one-step production of trans-aconitic acid, that is, in microbial cells, trans-aconitic acid is produced by one-step fermentation using a carbon source. Optionally, the microorganism with the ability to synthesize trans-aconitic acid includes but is not limited to the Aspergillus niger.

[0007] On the one hand, the present application provides a recombinant Aspergillus niger strain with high production of trans-aconitic acid, wherein the recombinant Aspergillus niger strain can simultaneously express aconitase and aconitate isomerase.

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

[0009] Preferably, the aconitase is Aco (ATEG_03325) derived from Aspergillus terreus; the aconitate isomerase is selected from TbrA derived from Bacillus thuringiensis, or Adi1 derived from Ustilago maydis Corola.

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

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

[0012] The amino acid sequence of the aconitate isomerase Adi1 comprises an amino acid sequence as shown in SEQ ID NO.3 or an amino acid sequence 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 a nucleotide sequence having at least 60% homology to the sequence shown in SEQ ID NO.17;

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

[0015] The gene encoding the aconitate isomerase Adi1 includes a nucleotide sequence as shown in SEQ ID NO.16 or a nucleotide sequence 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 the present application are preferably sequences with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% homology.

[0017] In one embodiment, the host cell of the recombinant Aspergillus niger strain is selected from one or more of Aspergillus niger MEFC1501, Aspergillus niger ATCC 1015, Aspergillus niger NRRL 41873, Aspergillus niger NRRL 27809, Aspergillus niger NRRL 31821, Aspergillus niger NR RL6276, 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 aconitate isomerase gene.

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

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

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

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

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

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

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

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

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

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

[0030] Preferably, the promoter is PmfsA promoter or 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 SEQ ID NO.11 sequence; 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 SEQ ID NO.12 sequence.

[0031] In one embodiment, the expression cassette further comprises a terminator, and the terminator is selected from TtrpC terminator or Tpgk terminator.

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

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

[0034] On the other hand, the present application also provides a biomaterial selected from any one of the following A1 to A3:

[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 to the sequence as shown in SEQ ID NO.17, and contains a nucleotide sequence as shown in SEQ ID NO.15 or having at least 60% homology to the sequence as shown in SEQ ID NO.15, or a nucleotide sequence as shown in SEQ ID NO.16 or having at least 60% homology to the sequence as shown in SEQ ID NO.16;

[0036] A2, a recombinant vector containing the expression cassette described in A1;

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

[0038] On the other hand, the present application also provides the use of the recombinant Aspergillus niger strain or the biological material in the production of trans-aconitic acid or products containing trans-aconitic acid or containing trans-aconitic acid structural units.

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

[0040] Step 1, constructing the recombinant Aspergillus niger strain or the biological material;

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

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

[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, pH 3.5.

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

[0045] 1. The recombinant Aspergillus niger strain provided in the present application uses Aspergillus niger as the starting strain, and heterologously expresses aconitase Aco from Aspergillus terreus and aconitase Adi1 from Ustilago maydisCorola or aconitase TbrA from Bacillus thuringiensis in Aspergillus niger to obtain an engineered Aspergillus niger strain producing trans-aconitic acid. The source dilemma of trans-aconitic acid, which traditionally requires plant extraction or chemical synthesis, can be solved by fermentation production, and trans-aconitic acid can be synthesized using Aspergillus niger.

[0046] 2. Aspergillus niger is a traditional fermentation strain widely used in the food industry. It is recognized as a generally safe, reliable and non-toxic (GRAS) microorganism by the US FDA and the World Health Organization. It can be used as a cell factory in large quantities in the production of proteins, organic acids, antigens, etc. The present invention provides a safe and non-toxic cell factory that can efficiently produce trans-aconitic acid.

[0047] 3. Experiments have shown that the trans-aconitic acid fermentation yield of the genetically engineered Aspergillus niger strain provided by the present invention is generally higher than that of the starting strain, among which the strain with the highest yield can reach 51 g / L at the shake flask level, and has strong application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

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

[0050] Figure 2 The present invention provides an example of a combination of aconitase Adi1 driven by PgpdAn and aconitase ATEG_03325 driven by different promoters, and the content of trans-aconitate produced by shake flask fermentation of the recombinant Aspergillus niger strain.

[0051] Strain deposit information:

[0052] Aspergillus niger MEFC1501 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the culture deposit number: CGMCC No.40614, the deposit date: April 27, 2023, and the deposit location is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0053] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0054] The materials, reagents, instruments and methods used in the following examples are conventional materials, reagents, instruments and methods in the art unless otherwise specified, and can be obtained through commercial channels. The methods described are conventional methods in the field of molecular biology technology or are performed with reference to the corresponding product instructions unless otherwise specified.

[0055] Technical terms:

[0056] Aconitase (ACO): Aconitase is an enzyme protein normally synthesized in organisms, which can remove a molecule of H2O from citric acid to obtain cis-aconitic acid.

[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 involved in the production of aconitase or aconitate isomerase or a mutant thereof including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0059] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0060] Introduction: The process of integrating exogenous genes into the genome of target cells for stable expression, or the process of inserting exogenous genes into vectors and introducing them into host cells for expression, as well as other forms of introducing exogenous genes into host cells for expression are called gene introduction.

[0061] In the following embodiments:

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

[0063] Aspergillus niger solid medium PDA: 3.9 g·L -1 Potato dextrose agar medium (Difco TM Potato Dextrose Agar, BD, LOT: 1165825), and the plates were prepared after sterilization.

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

[0065] Aspergillus niger spore-forming slant medium: 10 g·L -1 Glucose, 2 g L -1 NaNO3, 0.2 g·L -1 KH2PO4, 20mg·L -1 FeSO4, 5 g·L -1 MgSO4, 0.5 g·L -1 NaCl, 40 mg L -1 ZnSO4, 40mg·L -1 CuSO4, 1.5% agar, sterilize at 115℃ for 15min, dispense into test tubes, and prepare slant.

[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 1.2 M sorbitol were sterilized and cooled to about 55°C, and hygromycin B (Solarbio, Catalog No.: M419099) was added to a final concentration of 100 μg / mL to prepare a plate.

[0067] Regeneration screening medium top layer PDBS: 2.4 g·L -1 Potato dextrose medium (Difco TM Potato Dextrose Broth, BD, LOT: 2095227), 1.2 M sorbitol and 0.5% agarose were sterilized to make top agar.

[0068] Regeneration screening medium plate CD-SPt: 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, 20mg·L -1 FeSO4, 1.5 g·L -1 Agar, and 1.2 M sorbitol, the balance being water, are sterilized and cooled to about 55°C, and pyrithiamine (Sigma, Catalog No.: P0256) is added to a final concentration of 100 μg / L to prepare a plate.

[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, 20mg·L -1 FeSO4, 1.5 g·L -1 Agar, with the balance being water, was sterilized and cooled to about 55°C, and pyrithiamine (Sigma, Catalog No.: P0256) was added to a final concentration of 100 μg / L to prepare a plate.

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

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

[0072] Plasmid pUC57-Kan was purchased from Jinweizhi Biotechnology Co., Ltd.

[0073] Aspergillus niger MEFC1501 is deposited in the China General Microbiological Culture Collection, the culture collection number is: CGMCC NO.: 40614, the preservation time is: April 27, 2023, and the preservation location is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

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

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

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

[0077] Plasmid pAN52-4 (obtained from TNO Medical Biological Laboratory, the plasmid is described in Punt P.J., Zegers ND, Busscher M., Pouwels PH, van den Hondel CA, Journal of Biotechnology, 1991, 7, 19-33).

[0078] Plasmid pmWM23 is described in patent publication number CN111944706A.

[0079] The invention uses Aspergillus niger as a starting strain, expresses heterologous aconitase and aconitate isomerase in the genome of the starting strain through genetic engineering, obtains a recombinant Aspergillus niger strain, and realizes the synthesis of trans-aconitate in Aspergillus niger, wherein the starting strain is a currently known Aspergillus niger capable of synthesizing citric acid, such as Aspergillus niger MEFC1501, Aspergillus niger Co827, Aspergillus niger NRRL 27809, Aspergillus niger NRRL 31821, Aspergillus niger NRRL 41873, Aspergillus niger NRRL6276 or other genetically engineered bacteria or wild strains capable of synthesizing citric acid, and Aspergillus niger MEFC1501 is taken as an example to specifically describe the construction method of the recombinant Aspergillus niger strain of the invention.

[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 detected by high performance liquid chromatography.

[0082] Example 1 Construction of recombinant Aspergillus niger heterologously expressing aconitase Aco from Aspergillus terreus and aconitate isomerase TbrA from Bacillus thuringiensis

[0083] 1. Construction of the expression cassette of aconitase gene ATEG_03325

[0084] 1) Construction of the expression cassette of aconitase gene ATEG_03325 using the PgpdAt promoter

[0085] PCR amplification was performed using fuPgpdAt-03325-F (5'-aacaactcatcaatcatcacaacatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain the fragment of the ATEG_03325 gene.

[0086] PCR amplification was performed using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and fuPgpdAt-03325-R (5'-atgcgcgcaaggcgggtggagatcatgttgtgatgattgatgagttgtt-3') as primer pairs, and plasmid pMWM23 as a template. The product was purified and recovered to obtain the fragment of PgpdAt-TtrpC. PgpdAt is the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene of Aspergillus terreus, and TtrpC is the terminator of the tryptophan synthase of Aspergillus nidulans.

[0087] The two fragments were cloned in one step, and PCR amplification was performed using PgpdAt-F (5'-ttacactctgggaggatccaggtac-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgpdAt-ATE G_03325-TtrpC expression cassette fragment.

[0088] 2) Construction of the expression cassette of aconitase gene ATEG_03325 using the PgpdAn promoter

[0089] PCR amplification was performed using fuPgpdAn-03325-F (5'-ccgcttgagcagacatcaccatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain the fragment of the ATEG_03325 gene.

[0090] PCR amplification was performed using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and fuPgpdAn-03325-R (5'-atgcgcgcaaggcgggtggagatcatggtgatgtctgctcaagcgg-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the fragment of PgpdAn-TtrpC. PgpdAn is the promoter of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene, and TtrpC is the terminator of the Aspergillus nidulans tryptophan synthase.

[0091] The above two fragments were cloned in one step, and PCR amplification was performed using PgpdAn-F (5'-ttgatcgagacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgpdAn-ATEG_03325-TtrpC expression cassette fragment.

[0092] 3) Construction of the expression cassette of aconitase gene ATEG_03325 using the PglaA promoter

[0093] PCR amplification was performed using fuPglaA-03325-F (5'-cagcatcattacacctcagcaatgatctccacccgccttgcgcgcat-3') and fu03325-Ttr pC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as template. The product was purified and recovered to obtain the fragment of the ATEG_03325 gene.

[0094] PCR amplification was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and PglaA-R (5'-tgctgaggtgtaatgatgctggggat-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the Pgl aA promoter.

[0095] PCR amplification was performed using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0096] The above three fragments were cloned in one step, and PCR amplification was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After the product was purified and recovered, the PglaA-ATEG_03325-TtrpC expression cassette fragment was obtained.

[0097] 4) Construction of the expression cassette of aconitase gene ATEG_03325 using the PcadA promoter

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

[0099] PCR amplification was performed using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and PcadA-R (5'-ggtcaatttaagaggacgatcttcg-3') as primer pair and the genome of Aspergillus terreus CICC 40205 as template. The product was purified and recovered to obtain the fragment of PcadA promoter.

[0100] PCR amplification was performed using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0101] The three fragments were cloned in one step, and PCR amplification was performed using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PcadA-ATEG_03325-TtrpC expression cassette fragment.

[0102] 5) Construction of the expression cassette of aconitase gene ATEG_03325 using the PmfsA promoter

[0103] PCR amplification was performed using fuPmfsA-03325-F (5'-accgacttctcatccatcttcaaaatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as template. The product was purified and recovered to obtain the fragment of the ATEG_03325 gene.

[0104] PCR amplification was performed using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the PmfsA promoter.

[0105] PCR amplification was performed using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0106] The above three fragments were cloned in one step, and PCR amplification was performed using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After the product was purified and recovered, the PmfsA-ATEG_03325-TtrpC expression cassette fragment was obtained.

[0107] 6) Construction of the expression cassette of aconitase gene ATEG_03325 using PacoA promoter

[0108] PCR amplification was performed using fuPacoA-03325-F (5'-cctcgcatagagagcttccatcatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as template. The product was purified and recovered to obtain the fragment of the ATEG_03325 gene.

[0109] PCR amplification was performed using PacoA-F (5'-tggcaccggtccgcggga-3') and PacoA-R (5'-gatggaagctctctatgcgagg-3') as a primer pair and the genome of Aspergillus terreus CICC40205 as a template. The product was purified and recovered to obtain a fragment of the PacoA promoter.

[0110] PCR amplification was performed using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0111] The above three fragments were cloned in one step, and PCR amplification was performed using PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PacoA-ATEG_03325-TtrpC expression cassette fragment.

[0112] 7) Construction of the expression cassette of aconitase gene ATEG_03325 using PcitA promoter

[0113] PCR amplification was performed using fuPcitA-03325-F (5'-cttttttagactcttgttggattcaaaatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as template. The product was purified and recovered to obtain the fragment of the ATEG_03325 gene.

[0114] PCR amplification was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and PcitA-R (5'-ttgaatccaacaagagtctaaaaaag-3') as primer pairs and the genome of Aspergillus terreus CICC40205 as a template. The product was purified and recovered to obtain the fragment of the PcitA promoter.

[0115] PCR amplification was performed using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0116] The above three fragments were cloned in one step, and PCR amplification was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-at tacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PcitA-ATEG_03325-TtrpC expression cassette fragment.

[0117] 8) Construction of the expression cassette of aconitase gene ATEG_03325 using PicdA promoter

[0118] PCR amplification was performed using fuPicdA-03325-F (5'-cgcaggccacgcttcactgtcgaaatgatctccacccgccttgcgcgcat-3') and fu03325-TtrpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as template. The product was purified and recovered to obtain the fragment of the ATEG_03325 gene.

[0119] PCR amplification was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and PicdA-R (5'-ttcgacagtgaagcgtggcctgcg-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of Picd A promoter.

[0120] PCR amplification was performed using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0121] The three fragments were cloned in one step, and PCR amplification was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PicdA-ATE G_03325-TtrpC expression cassette fragment.

[0122] 9) Construction of the expression cassette of aconitase gene ATEG_03325 using PgasA promoter

[0123] PCR amplification was performed using fuPgasA-03325-F (5'-gtcttctttcgttcacctcctcacatgatctccacccgccttgcgcgcat-3') and fu03325-Tt rpC-R (5'-ttcagtaacgttaagtggatccttagttgctagcagccttgcgggc-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as template. The product was purified and recovered to obtain the fragment of the ATEG_03325 gene.

[0124] PCR amplification was performed using PgasA-F (5'-ctgctctctctctgctctctctttct-3') and PgasA-R (5'-gtgaggaggtgaacgaaagaagac-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as template. The product was purified and recovered to obtain the fragment of the PgasA promoter.

[0125] PCR amplification was performed using fu03325-TtrpC-F (5'-gcccgcaaggctgctagcaactaaggatccacttaacgttactgaa-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

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

[0127] 2. Construction of targeting element for aconitate isomerase TbrA

[0128] The aconitate isomerase TbrA gene was artificially synthesized, codon optimized for Aspergillus niger expression, and cloned into the pUC57-Kan vector to construct the plasmid puc57-TbrA.

[0129] 1) Construction of the expression cassette of aconitate isomerase TbrA using the PgpdAt promoter

[0130] PCR amplification was performed using fuPgpdAt-TbrA-F (5'-ctcatcaatcatcacaacatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0131] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and fuPgpdAt-TbrA-R (5'-cgaagcaggggatcttcatgttgtgatgattgatgag-3') as primer pairs and plasmid pMWM23 as template. The product was purified and recovered to obtain the PgpdAt-TtrpC fragment.

[0132] The two fragments were cloned in one step, and PCR amplification was performed using PgpdAt-F (5'-ttacactctgggaggatccaggtac-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgpdAt-Tbr A-TtrpC expression cassette fragment.

[0133] 2) Construction of the expression cassette of aconitate isomerase TbrA using the PgpdAn promoter

[0134] PCR amplification was performed using fuPgpdAn-TbrA-F (5'-ccgcttgagcagacatcaccatgaagatcccctgcttcgt-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0135] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and fuPgpdAn-TbrA-R (5'-acgaagcaggggatcttcatggtgatgtctgctcaagcgg-3') as primer pairs and plasmid pAN52-4 as template, and the product was purified and recovered to obtain the fragment of PgpdAn-TtrpC. PgpdAn is the promoter of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene, and TtrpC is the terminator of the Aspergillus nidulans tryptophan synthase.

[0136] The two fragments were cloned in one step, and PCR amplification was performed using PgpdAn-F (5'-ttgatcgagacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgpdAn-TbrA-TtrpC expression cassette fragment.

[0137] 3) Construction of the expression cassette of aconitate isomerase TbrA using the PglaA promoter

[0138] PCR amplification was performed using fuPglaA-TbrA-F (5'-gcatcattacacctcagcaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0139] PCR amplification was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and PglaA-R (5'-tgctgaggtgtaatgatgctggggat-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the Pgl aA promoter.

[0140] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacct ctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0141] The three fragments were cloned in one step, and PCR amplification was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PglaA-TbrA-TtrpC expression cassette fragment.

[0142] 4) Construction of the expression cassette of aconitate isomerase TbrA using the PcadA promoter

[0143] PCR amplification was performed using fuPcadA-TbrA-F (5'-cctcttaaattgaccatgaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0144] PCR amplification was performed using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and PcadA-R (5'-tcatggtcaatttaagagg-3') as primer pairs and the genome of Aspergillus terreus CICC40205 as a template. The product was purified and recovered to obtain a fragment of the PcadA promoter.

[0145] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacct ctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0146] The three fragments were cloned in one step, and PCR amplification was performed using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PcadA-TbrA-TtrpC expression cassette fragment.

[0147] 5) Construction of the expression cassette of aconitate isomerase TbrA using the PmfsA promoter

[0148] Using fuPmfsA-TbrA-F (5'-accgacttctcatccatcttcaaaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pair, puc57-TbrA plasmid as template for PCR amplification, and the product was purified and recovered to obtain the TbrA gene fragment.

[0149] PCR amplification was performed using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the PmfsA promoter.

[0150] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctc taaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0151] The three fragments were cloned in one step, and PCR amplification was performed using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PmfsA-TbrA-TtrpC expression cassette fragment.

[0152] 6) Construction of the expression cassette of aconitate isomerase TbrA using the PacoA promoter

[0153] PCR amplification was performed using fuPacoA-TbrA-F (5'-cctcgcatagagagcttccatcatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0154] PCR amplification was performed using PacoA-F (5'-tggcaccggtccgcggga-3') and PacoA-R (5'-gatggaagctctctatgcgagg-3') as a primer pair and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain a fragment of the PacoA promoter.

[0155] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacct ctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

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

[0157] 7) Construction of the expression cassette of aconitate isomerase TbrA using the PcitA promoter

[0158] Using fuPcitA-TbrA-F (5'-cttttttagactcttgttggattcaaaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pair, puc57-TbrA plasmid as template for PCR amplification, and the product was purified and recovered to obtain the TbrA gene fragment.

[0159] PCR amplification was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and PcitA-R (5'-ttgaatccaacaagagtctaaaaaag-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain the fragment of the PcitA promoter.

[0160] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctc taaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0161] The three fragments were cloned in one step, and PCR amplification was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-at tacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PcitA-TbrA-TtrpC expression cassette fragment.

[0162] 8) Construction of the expression cassette of aconitate isomerase TbrA using the PicdA promoter

[0163] PCR amplification was performed using fuPicdA-TbrA-F (5'-cgcaggccacgcttcactgtcgaaatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pairs and puc57-TbrA plasmid as template. The product was purified and recovered to obtain the TbrA gene fragment.

[0164] PCR amplification was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and PicdA-R (5'-ttcgacagtgaagcgtggcctgcg-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of Picd A promoter.

[0165] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacct ctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0166] The three fragments were cloned in one step, and PCR amplification was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PicdA-TbrA-TtrpC expression cassette fragment.

[0167] 9) Construction of the expression cassette of aconitate isomerase TbrA using the PgasA promoter

[0168] Using fuPgasA-TbrA-F (5'-gtcttctttcgttcacctcctcacatgaagatcccctgcttcg-3') and fuTbrA-TtrpC-R (5'-tcagtaacgttaagtggatccttaggggatgatcagctcg-3') as primer pair, puc57-TbrA plasmid as template for PCR amplification, and the product was purified and recovered to obtain the TbrA gene fragment.

[0169] PCR amplification was performed using PgasA-F (5'-ctgctctctctctgctctctctttct-3') and PgasA-R (5'-gtgaggaggtgaacgaaagaagac-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as template. The product was purified and recovered to obtain the fragment of the PgasA promoter.

[0170] PCR amplification was performed using fuTbrA-TtrpC-F (5'-cgagctgatcatcccctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attacctc taaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0171] The three fragments were cloned in one step, and PCR amplification was performed using PgasA-F (5'-ctgctctctctctgctctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgasA-TbrA-TtrpC expression cassette fragment.

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

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

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

[0175] 1) Preparation of Aspergillus niger protoplasts

[0176] The spore suspension of Aspergillus niger MEFC1501 was inoculated into 50 mL of liquid medium PDB, with a spore concentration of about 10 7 / mL, culture at 200rmp, 33℃ for 12-18h. Collect the grown mycelium by filtration with a sterile single-layer 100-mesh nylon cloth, rinse three times with a sterilized 0.6M MgSO4 solution, squeeze dry and place in a sterile 50ml conical flask; weigh 1g of mycelium, add 10mL of enzymatic solution, and treat at 30℃, 120rpm for 1-2h. The enzymatic solution contains: 1% cellulase (Sigma, Catalog No.: C1184), 1% lyase (Sigma, Catalog No.: L1412), 1% snail enzyme (Shanghai Shenggong, Catalog No.: SB0870), 0.6M MgSO4, and filter and sterilize through a 0.22μm sterile filter.

[0177] The mixture after enzymatic hydrolysis was filtered through a 300-mesh nylon cloth to collect the filtrate. The protoplasts were collected by centrifugation at 4°C, washed once with a precooled 1.0 M sorbitol solution, and then washed once with a precooled STC (1.0 M sorbitol, 50 mM Tris·HCl-pH8.0, 50 mM CaCl2). Finally, the protoplasts were resuspended in 150 μl of precooled STC and the protoplast concentration was adjusted to 5 × 10 7 / mL to obtain a protoplast suspension.

[0178] 2) The expression cassette of aconitase gene ATEG_03325, the expression cassette of aconitase isomerase TbrA and the resistance gene hph are co-transformed into Aspergillus niger protoplasts:

[0179] Add about 5 μg of aconitase gene ATEG_03325 expression cassette DNA, about 5 μg of aconitase isomerase TbrA expression cassette DNA and 1 μg of hygromycin B resistance hph gene expression cassette DNA to the above protoplast suspension, then add 50 μL of PSTC (40% PEG4000, 50Mm Tris-HCl pH8.0, 50mM CaCl2), gently mix, and ice bath for 30 minutes. Add 1.5mL of PSTC, mix well, and place at room temperature for 20 minutes; then mix with the upper agar and pour into the regeneration screening medium plate PDA-SH, and culture at 30°C in the dark for 3-4 days.

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

[0181] In the process of preparing the recombinant Aspergillus niger strain containing the ATEG_03325 expression cassette and TbrA expression cassette driven by each promoter, the ATEG_03325 expression cassette and the hygromycin B resistance hph gene expression cassette can also be connected to form an expression cassette, or the TbrA expression cassette and the hygromycin B resistance hph gene expression cassette can be connected to form an expression cassette, and then the protoplasts are transformed, which does not affect the screening of positive transformants and 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 picked and inoculated on PDA-H plates to culture spores, and then the spores were inoculated into liquid culture medium for culture, the mycelium was collected to extract the genome, and PCR was performed using PgpdAt-F743 (5'-ttacactctgggaggatcc aggtact-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'-ggattgcctgaacattg PCR was performed using primers PgpdAn-F (5'-ttgatcgagacctaaacaagtgtac-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify the PglaA-ATEG_03325-TtrpC element and / or PglaA-TbrA-TtrpC inserted into the genome; tacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primers for PCR to amplify the PgpdAn-ATEG_03325-TtrpC element and / or PgpdAn-TbrA-TtrpC inserted into the genome; PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primers for PCR to amplify the PcadA-ATEG_03325-TtrpC element and / or PcadA-TbrA-TtrpC inserted into the genome; PmfsA-F (5'-gtacagtggccatga PCR was performed using primers 5'-aatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify the PmfsA-ATEG_03325-TtrpC element and / or PmfsA-TbrA-TtrpC inserted into the genome; PCR was performed using primers PacoA-F (5'-tggcaccggtccgc ggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify the PacoA-ATEG_03325-TtrpC element and / or PacoA-TbrA-TtrpC inserted into the genome;PCR was performed using primers PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify the PcitA-ATEG_03325-TtrpC element and / or PcitA-TbrA-TtrpC inserted into the genome; and primers PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify the PcitA-ATEG_03325-TtrpC element and / or PcitA-TbrA-TtrpC inserted into the genome. ac-3') as primer pair to perform PCR to amplify PicdA-ATEG_03325-TtrpC element and / or PicdA-TbrA-TtrpC inserted into the genome; PgasA-F (5'-ctgctctctctctgctctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pair to perform PCR to amplify PgasA-ATEG_03325-TtrpC element and / or PgasA-TbrA-TtrpC inserted into the genome. ;

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

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

[0186] The transformants with correct genotype verification were transferred to PDA-H plates for subculture for 3 times. Then, the spores were collected and diluted appropriately with physiological saline, and 100 μL was spread on the PDA-H plate to grow independent single colonies, which was single spore isolation. Spores were taken from the single colony for single spore isolation again, and a total of 4 single spore isolation subcultures were performed. The recombinant strains after single spore isolation were again genotyped for verification. Some of the recombinant strains with correct verification were selected for shake flask fermentation screening.

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

[0188] 1. Construct an expression cassette of aconitase gene ATEG_03325, referring to Example 1.

[0189] 2. Construction of the expression cassette of aconitate isomerase Adi1

[0190] The aconitate isomerase Adi1 gene was artificially synthesized, codon optimized for Aspergillus niger expression, and cloned into the pUC57-Kan vector to construct the plasmid puc57-Adi1.

[0191] 1) Construction of the expression cassette of aconitate isomerase Adi1 using the PgpdAt promoter

[0192] PCR amplification was performed using fuPgpdAt-Adi1-F (5'-ctcatcaatcatcacaacatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0193] Using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and fuPgpdAt-Adi1-R (5'-tggtgtcgatggggtgcagcatgttgtgatgattgatgag-3') as primer pair, PCR amplification was performed with plasmid pMWM23 as template, and the product was purified and recovered to obtain the PgpdAt-TtrpC fragment.

[0194] The two fragments were cloned in one step, and PCR amplification was performed using PgpdAt-F (5'-ttacactctgggaggatccaggtac-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgpdAt-Adi1-TtrpC expression cassette fragment.

[0195] 2) Construction of the expression cassette of aconitate isomerase Adi1 using the PgpdAn promoter

[0196] PCR amplification was performed using fuPgpdAn-Adi1-F (5'-ccgcttgagcagacatcaccatgctgcaccccatcgacaccat-3') and fuAdi1-Ttrp CR (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0197] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and fuPgpdAn-Adi1-R (5'-tggtgtcgatggggtgcagcatggtgatgtctgctcaagcgg-3') as primer pairs and plasmid pAN52-4 as template, and the product was purified and recovered to obtain the fragment of PgpdAn-TtrpC. PgpdAn is the promoter of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene, and TtrpC is the terminator of the Aspergillus nidulans tryptophan synthase.

[0198] The two fragments were cloned in one step, and PCR amplification was performed using PgpdAn-F (5'-ttgatcgagacctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PgpdAn-Adi1-Ttrp C expression cassette fragment.

[0199] 3) Construction of the expression cassette of aconitate isomerase Adi1 using the PglaA promoter

[0200] PCR amplification was performed using fuPglaA-Adi1-F (5'-gcatcattacacctcagcaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0201] PCR amplification was performed using PglaA-F (5'-ggattgcctgaacattgacattcgg-3') and PglaA-R (5'-tgctgaggtgtaatgatgctgggg at-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the PglaA promoter.

[0202] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-atta cctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

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

[0204] 4) Construction of the expression cassette of aconitate isomerase Adi1 using the PcadA promoter

[0205] PCR amplification was performed using fuPcadA-Adi1-F (5'-cctcttaaattgaccatgaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0206] PCR amplification was performed using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and PcadA-R (5'-tcatggtcaatttaagagg-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain the fragment of Pcad A promoter.

[0207] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0208] The three fragments were cloned in one step, and PCR amplification was performed using PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primers. The product was purified and recovered to obtain PcadA-Adi1

[0209] -TtrpC expression cassette fragment.

[0210] 5) Construction of the expression cassette of aconitate isomerase Adi1 using the PmfsA promoter

[0211] PCR amplification was performed using fuPmfsA-Adi1-F (5'-accgacttctcatccatcttcaaaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0212] PCR amplification was performed using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and PmfsA-R (5'-tttgaagatggatgagaagtcggt-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the PmfsA promoter.

[0213] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0214] The above three fragments were cloned in one step, and PCR amplification was performed using PmfsA-F (5'-gtacagtggccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PmfsA-Adi1-TtrpC expression cassette fragment.

[0215] 6) Construction of the expression cassette of aconitate isomerase Adi1 using the PacoA promoter

[0216] PCR amplification was performed using fuPacoA-Adi1-F (5'-cctcgcatagagagcttccatcatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0217] PCR amplification was performed using PacoA-F (5'-tggcaccggtccgcggga-3') and PacoA-R (5'-gatggaagctctctatgcgagg-3') as a primer pair and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain a fragment of the PacoA promoter.

[0218] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-atta cctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0219] The three fragments were cloned in one step, and PCR amplification was performed using PacoA-F (5'-tggcaccggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PacoA-Adi1-TtrpC expression cassette fragment.

[0220] 7) Construction of the expression cassette of aconitate isomerase Adi1 using the PcitA promoter

[0221] PCR amplification was performed using fuPcitA-Adi1-F (5'-cttttttagactcttgttggattcaaaatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0222] PCR amplification was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and PcitA-R (5'-ttgaatccaacaagagtctaaaaaag-3') as primer pairs and the genome of Aspergillus terreus CICC 40205 as a template. The product was purified and recovered to obtain the fragment of Pcit A promoter.

[0223] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0224] The three fragments were cloned in one step, and PCR amplification was performed using PcitA-F (5'-caaccaaggaccgcgatg-3') and TtrpC-R (5'-at tacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PcitA-Adi1-TtrpC expression cassette fragment.

[0225] 8) Construction of the expression cassette of aconitate isomerase Adi1 using the PicdA promoter

[0226] PCR amplification was performed using fuPicdA-Adi1-F (5'-cgcaggccacgcttcactgtcgaaatgctgcaccccatcgacacca-3') and fuAdi1-Ttrp CR (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0227] PCR amplification was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and PicdA-R (5'-ttcgacagtgaagcgtggcctgcg-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as a template. The product was purified and recovered to obtain the fragment of the PicdA promoter.

[0228] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-attac ctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0229] The three fragments were cloned in one step, and PCR amplification was performed using PicdA-F (5'-ctttaacgttgcagatacagggatgcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. The product was purified and recovered to obtain the PicdA-Adi1-TtrpC expression cassette fragment.

[0230] 9) Construction of the expression cassette of aconitate isomerase Adi1 using the PgasA promoter

[0231] PCR amplification was performed using fuPgasA-Adi1-F (5'-gtcttctttcgttcacctcctcacatgctgcaccccatcgacacca-3') and fuAdi1-TtrpC-R (5'-tcagtaacgttaagtggatccttaggacaggctacggtcg-3') as primer pairs and puc57-Adi1 plasmid as template. The product was purified and recovered to obtain the Adi1 gene fragment.

[0232] PCR amplification was performed using PgasA-F (5'-ctgctctctctctgctctctctttct-3') and PgasA-R (5'-gtgaggaggtgaacgaaagaagac-3') as primer pairs and the genome of Aspergillus niger MEFC1501 as template. The product was purified and recovered to obtain the fragment of the PgasA promoter.

[0233] PCR amplification was performed using fuAdi1-TtrpC-F (5'-cgaccgtagcctgtcctaaggatccacttaacgttactga-3') and TtrpC-R (5'-atta cctctaaacaagtgtac-3') as primer pairs and plasmid pAN52-4 as template. The product was purified and recovered to obtain the TtrpC fragment.

[0234] The above three fragments were cloned in one step, and PCR amplification was performed using PgasA-F (5'-ctgctctctctctgctctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs. After purification and recovery of the product, the PgasA-Adi1-TtrpC expression cassette fragment was obtained.

[0235] 3. Construction of the hygromycin B selection marker gene hph expression cassette, refer to Example 1.

[0236] 4. The expression cassette of aconitase gene ATEG_03325 and aconitase isomerase Adi1 were co-transformed into Aspergillus niger protoplasts to obtain a recombinant Aspergillus niger strain:

[0237] 1) Preparation of Aspergillus niger protoplasts, see Example 1.

[0238] 2) The expression cassette of aconitase gene ATEG_03325, the expression cassette of aconitase isomerase Adi1 and the resistance gene hph are co-transformed into Aspergillus niger protoplasts:

[0239] Add about 5 μg of aconitase gene ATEG_03325 expression cassette DNA, about 5 μg of aconitase isomerase Adi1 expression cassette DNA and 1 μg of hygromycin B resistance hph gene expression cassette DNA to the above protoplast suspension, then add 50 μL of PSTC (40% PEG4000, 50Mm Tris-HCl pH8.0, 50mM CaCl2), gently mix, and ice bath for 30 minutes. Add 1.5mL of PSTC, mix well, and place at room temperature for 20 minutes; then mix with the upper agar and pour into the regeneration screening medium plate PDA-SH, and culture at 30°C in the dark for 3-4 days.

[0240] The transformants were transferred from the plate to the screening plate PDA-H (4 g of potato dextrose agar medium was weighed and dissolved in 100 ml of distilled water, sterilized, and hygromycin was added to a final concentration of 100 μg / mL when cooled to about 55°C to prepare the plate), and cultured at 30°C for 3-5 days to obtain transformants.

[0241] In the process of preparing the recombinant Aspergillus niger strain containing the ATEG_03325 expression cassette and the Adi1 expression cassette driven by each promoter, the ATEG_03325 expression cassette and the hygromycin B resistance hph gene expression cassette can also be connected to form an expression cassette, or the Adi1 expression cassette and the hygromycin B resistance hph gene expression cassette can be connected to form an expression cassette, and then the protoplasts are transformed, which does not affect the screening of positive transformants and the identification of genotypes.

[0242] 3) Verification of the genotype of the recombinant Aspergillus niger strain:

[0243] The recombinant Aspergillus niger transformants obtained above were picked and inoculated on PDA-H plates to culture spores, and then the spores were inoculated into liquid culture medium for culture, the mycelium was collected to extract the genome, and PCR was performed using PgpdAt-F743 (5'-ttacactctgggaggat ccaggtact-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'-ggattgcctgaac PCR was performed using primers PgpdAn-F (5'-ttgatcgag acctaatacagc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primers for PCR to amplify the PgpdAn-ATEG_03325-TtrpC element and / or PgpdAn-Adi1-TtrpC inserted into the genome; PcadA-F (5'-ctaccaacagtctcgcggtgaatag-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primers for PCR to amplify the PcadA-ATEG_03325-TtrpC element and / or PcadA-Adi1-TtrpC inserted into the genome; PmfsA-F (5'-gtacagtg gccatgaaatccaatc-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primer pairs for PCR to amplify the PmfsA-ATEG_03325-TtrpC element and / or PmfsA-Adi1-TtrpC inserted into the genome; PacoA-F (5'-tggcacc ggtccgcggga-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') were used as primer pairs for PCR to amplify the PacoA-ATEG_03325-TtrpC element and / or PacoA-Adi1-TtrpC inserted into the genome;PCR was performed using the primer pair PcitA-F (5'-caaccaaggaccg cgatg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify the PcitA-ATEG_03325-TtrpC element and / or PcitA-Adi1-TtrpC inserted into the genome; PCR was performed using the primer pair PicdA-F (5'-ctttaacgttgcagatacagggat gcg-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') to amplify the Pi cdA-ATEG_03325-TtrpC element and / or PicdA-Adi1-TtrpC; PCR was performed using PgasA-F (5'-ctgctctctctctgctctctctttct-3') and TtrpC-R (5'-attacctctaaacaagtgtac-3') as primer pairs to amplify PgasA-ATEG_03325-TtrpC element and / or PgasA-Adi1-TtrpC inserted into the genome. ;

[0244] The primer pair hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') were used to amplify the PtrpC-hph-TtrpC fragment inserted into the recombinant bacteria, and the PCR product was analyzed by 0.8% agarose gel electrophoresis. The genomic DNA template of the starting strain Aspergillus niger MEFC1501 was used as a negative control.

[0245] The transformants with correct genotype verification were transferred to PDA-H plates for subculture for 3 times. Then, the spores were collected and diluted appropriately with physiological saline, and 100 μL was spread on the PDA-H plate to grow independent single colonies, which was single spore isolation. Spores were taken from the single colony for single spore isolation again, and a total of 4 single spore isolation subcultures were performed. The recombinant strains after single spore isolation were again genotyped for verification. Some of the recombinant strains with correct verification were selected for shake flask fermentation screening.

[0246] Example 3 Construction of a recombinant Aspergillus niger strain using pyrithiamine ptrA as a selection marker

[0247] Example 2 was repeated. The difference from Example 2 was that the selection marker gene used in the recombinant Aspergillus niger strain in this example was the pyrithiamine selection marker ptrA gene. The specific method is described as follows:

[0248] 1. Construction of expression cassettes of different promoters of aconitase ATEG_03325, refer to Example 1.

[0249] 2. Construction of expression cassettes of aconitate isomerase Adi1 with different promoters, see Example 2.

[0250] 3. Construction of recombinant Aspergillus niger strain using the pyrithiamine resistance gene ptrA as a selection marker

[0251] 1) Construction of the expression cassette of the pyrithiamine resistance gene ptrA

[0252] PCR amplification was performed using ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atggggtgacgatgagccgc-3') as primers and plasmid pMWM23 as template. After the amplified product was purified and recovered, the pyrithiamine resistance gene ptr A expression cassette was obtained.

[0253] Prepare Aspergillus niger MEFC1501 protoplasts according to the preparation method in Example 1. Add about 5 μg of aconitase gene ATEG_03325 expression cassette DNA, about 5 μg of aconitase isomerase Adi1 expression cassette DNA and 1 μg of screening marker ptrA fragment to the protoplast suspension, then add 50 μL PSTC (40% PEG4000, 50Mm Tris-HCl pH8.0, 50mM CaCl2), mix gently, and ice bath for 30 minutes. Add 1.5mL PSTC, mix well and place at room temperature for 20 minutes; then mix with the upper agar and pour into the regeneration screening medium plate CD-SPt, and culture at 33°C in the dark for 3 to 4 days. Transfer the transformant from the plate to the screening plate CD-Pt, and culture at 33°C for 3 to 5 days to obtain the transformant.

[0254] The prepared recombinant Aspergillus niger transformants were picked and inoculated on CD-Pt plates to culture spores, and then the spores were inoculated into PDB liquid culture medium for culture, the mycelium was collected to extract the genome, and the PmfsA-ATEG_03325-TtrpC element or PgpdAn-Adi1-TtrpC inserted into the genome was amplified; ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atgg ggtgacgatgagccgc-3') were used as primer pairs to amplify the ptrA fragment inserted into the recombinant bacteria, and the PCR products were analyzed by 0.8% agarose gel electrophoresis.

[0255] The genomic DNA template of the starting strain Aspergillus niger MEFC1501 was used as a negative control.

[0256] The verification and separation and purification of positive transformants were carried out as described in Examples 1 and 2.

[0257] Example 4 Recombinant Aspergillus niger fermentation to produce trans-aconitic acid

[0258] 1. Shake flask screening of recombinant Aspergillus niger strains producing trans-aconitic acid

[0259] The recombinant Aspergillus niger strains obtained in Example 1, Example 2 and Example 3 were inoculated into the Aspergillus niger spore production medium and cultured at 32° C. for 6 days to obtain mature spores. The spores cultured to maturity were then inoculated into the trans-aconitic acid fermentation medium, and each strain was fermented in three bottles at 34° C. and 220 rpm. The mycelium in the fermentation broth was filtered out, and the fermentation supernatant was appropriately diluted and analyzed by high performance liquid chromatography.

[0260] 2. Analysis of the content and purity of trans-aconitic acid in the fermentation broth

[0261] The fermentation supernatant of the recombinant strain and the starting strain Aspergillus niger MEFC1501 was diluted and analyzed by high performance liquid chromatography. The standard of trans-aconitic acid with different concentrations was used as the standard curve to analyze and compare the content and purity of trans-aconitic acid in the fermentation broth. The chromatographic conditions are as follows: chromatographic column: Aminex HPX-87H Organic Acid Analysis Column, 300mm×7.8mm (Bio-rad, Cat No.1250140); mobile phase: 5mmol / L sulfuric acid; flow rate: 0.5mL / min; column temperature: 30℃; detection temperature: 30℃; UV detector (210nm).

[0262] The results are as follows Figure 1 and Figure 2 shown.

[0263] The results showed that the content of cis-aconitic acid and trans-aconitic acid in the fermentation broth of the recombinant strain was significantly higher than that of the wild-type strain at the shake flask fermentation level. The effects of the two isomerases TbrA and Adi1 were not much different, but the gene expression driven by different promoters showed obvious differences, among which the content of trans-aconitic acid could reach up to 51.11 g / L. There was no significant difference between different terminators such as TtrpC or Tpgk, and there was no significant difference between different screening tags such as hygromycin or pyrithiamine. Different transformation methods, such as co-transformation with the screening tag or constructing the expression cassette of the target gene and the expression cassette of the screening tag together for transformation, all gave similar results.

[0264] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A recombinant Aspergillus niger strain with high trans-aconitic acid production, characterized in that: The recombinant Aspergillus niger strain can simultaneously express aconitase and aconitate isomerase.

2. The recombinant Aspergillus niger strain according to claim 1, characterized in that The aconitase is Aco and the aconitate isomerase is selected from TbrA or Adi1.

3. The recombinant Aspergillus niger strain according to claim 1, characterized in that The amino acid sequence of the aconitase Aco comprises an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO.1; and / or, The amino acid sequence of the aconitate isomerase TbrA comprises an amino acid sequence as shown in SEQ ID NO.2 or an amino acid sequence having at least 60% homology with the amino acid sequence as shown in SEQ ID NO.2; and / or, The amino acid sequence of the aconitate isomerase Adi1 comprises an amino acid sequence as shown in SEQ ID NO.3 or an amino acid sequence having at least 60% homology with the amino acid sequence as shown in SEQ ID NO.

3.

4. The recombinant Aspergillus niger strain according to claim 1, characterized in that The host cell of the recombinant Aspergillus niger strain is selected from one or more of Aspergillus niger MEFC1501, Aspergillus niger ATCC 1015, Aspergillus niger NRRL 41873, Aspergillus niger NRRL 27809, Aspergillus niger NRRL 31821, Aspergillus niger NRRL 6276, Aspergillus niger Co827, Aspergillus niger 5016, Aspergillus niger 3008, Aspergillus niger GCB75, Aspergillus niger ATCC 9142, and Aspergillus niger MTCC 282, preferably Aspergillus niger MEFC1501.

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

6. The recombinant Aspergillus niger strain according to claim 5, characterized in that The promoter of the expression cassette of the aconitase gene is selected from one or more of the PglaA promoter, PgpdAt promoter, PgpdAn promoter, Pa coA promoter, PcitA promoter, PicdA promoter, PcadA promoter, PmfsA promoter and Pgas promoter.

7. The recombinant Aspergillus niger strain according to claim 5, characterized in that The expression cassette further comprises a terminator, and the terminator is selected from the group consisting of a TtrpC terminator or a Tpgk terminator.

8. Biomaterial, characterized in that Select any one of the following A1 to A3: 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 to 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 to 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 to the sequence shown in SEQ ID NO.16; A2, a recombinant vector containing the expression cassette described in A1; A3, a whole-cell catalyst, comprising the expression cassette as described in A1, or the recombinant vector as described in A2, or the recombinant Aspergillus niger strain as described in any one of claims 1-7.

9. Use of the recombinant Aspergillus niger strain according to any one of claims 1 to 7 or the biomaterial according to claim 8 in the production of trans-aconitic acid or a product containing trans-aconitic acid or a trans-aconitic acid structural unit.

10. A method for producing trans-aconitic acid, characterized in that: include: Step 1, constructing a recombinant Aspergillus niger strain as described in any one of claims 1 to 7 or a biological material as described in claim 8; Step 2: inoculating the recombinant Aspergillus niger strain into a fermentation medium for fermentation to obtain trans-aconitic acid.

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