CN112011469B - A recombinant Aspergillus terreus strain producing trans-aconitic acid and its construction method and application - Google Patents

Abstract

A recombinant aspergillus terreus strain for producing trans-aconitic acid and a construction method and application thereof belong to the technical field of genetic engineering. In order to improve the yield of trans-aconitic acid synthesized by a biological method, the invention provides a recombinant Aspergillus terreus strain for producing trans-aconitic acid, which is obtained by taking Aspergillus terreus (Aspergillus terreus) as an initial strain, knocking out a aconitate decarboxylase CadA gene in the initial strain and over-expressing aconitate isomerase Adi 1. The amino acid sequence of the aconitic acid isomerase Adi1 is shown as SEQ ID NO. 2; the nucleotide sequence is shown as SEQ ID NO. 3. According to the invention, the Adi1 is overexpressed on the basis of knockout of CadA, so that cis-aconitic acid is converted into trans-aconitic acid, and thus higher trans-aconitic acid yield is obtained, and the method can be used for producing the trans-aconitic acid.

Description

A recombinant Aspergillus terreus strain producing trans-aconitic acid and its construction method and application

technical field

The invention belongs to the field of genetic engineering, and in particular relates to a recombinant Aspergillus terreus strain with high-yield trans-aconitic acid and a construction method and application thereof.

Background technique

Trans-Aconitic acid (CAS: 4023-65-8) is an unsaturated tricarboxylic acid. Because it contains unsaturated double bonds and abundant hydroxyl groups, it can be used as a monomer compound for the preparation of polymeric materials, and can also be used as a synthetic precursor for other compounds, such as trimethyl trans-aconitic acid, etc. In addition, trans-aconitic acid, as a stereoisomer of cis-Aconitic acid (CAS: 585-84-2), a key intermediate in the TCA cycle, is a key enzyme in the TCA cycle. Acidase has a certain inhibitory effect, which can interfere with the tricarboxylic acid cycle, thereby affecting life activities and showing certain biological activities. A number of studies have shown that trans-aconitic acid has a good effect on nematode control and other aspects, so it has a good potential in the development of biological pesticides.

At present, trans-aconitic acid is mainly produced by chemical synthesis. Its production process is complicated, with many by-products, high cost, and there is no large-scale production, which will be unfavorable for the downstream application of trans-aconitic acid and product quality. development. In order to develop a greener and more efficient process for the production of trans-aconitic acid, researchers obtained a strain by blocking the cis-aconitic acid decarboxylase gene (cadA) that catalyzes the decarboxylation of cis-aconitic acid to itaconic acid in Aspergillus terreus. Aspergillus terreus engineered strain At-ΔcadA capable of producing aconitic acid (A in Figure 1). According to the analysis of biosynthetic pathway and fermentation results, it is speculated that the direct product of cadA knockout accumulation is cis-aconitic acid. However, cis-aconitic acid, as an intermediate of the tricarboxylic acid cycle, will be quickly converted into isocitrate and further metabolized.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the existing synthesis process of trans-aconitic acid and improve the yield of trans-aconitic acid, the present invention provides a recombinant Aspergillus terreus strain with high-yield trans-aconitic acid. ( Aspergillus terreus ) is the starting strain, which is obtained by knocking out the cis-aconitic acid decarboxylase CadA gene and overexpressing the aconitic acid isomerase Adi1 in the starting strain.

In one embodiment of the present invention, the starting strain is Aspergillus terreus CICC40205, Aspergillus terreus NRRL1960, Aspergillus terreus DSM23081, Aspergillus terreus TN484 or Aspergillus terreus TN484-M1.

The CadA in the present invention refers to the cis-aconitic acid decarboxylase in Aspergillus terreus, and the sequence of the gene is not limited in the present invention; in a preferred embodiment, the amino acid sequence of the cadA is as shown in SEQ ID No. 1; in other embodiments, the cadA also includes an amino acid sequence with a high degree of homology to the sequence shown, preferably, the high degree of homology includes at least 99% homology with the target sequence , 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60% of the sequence (ie more than 60% homology).

In an embodiment of the present invention, the amino acid sequence of the aconitate isomerase Adi1 is shown in SEQ ID NO.2.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the aconitate isomerase Adi1 is shown in SEQ ID NO.3.

The present invention also provides a method for constructing the above-mentioned recombinant Aspergillus terreus, using the Aspergillus terreus as a starting strain, knocking out the CadA gene of cis-aconitic acid decarboxylase, and introducing the expression element of the aconitic acid isomerase Adi1 into the recombinant Aspergillus terreus. Among the above-mentioned starting strains, a recombinant Aspergillus terreus strain was constructed.

In one embodiment of the present invention, the promoter used for the expression element of aconitate isomerase Adi1 is the P cadA promoter or the P gpdAt promoter.

The present invention also provides the application of the above-mentioned recombinant Aspergillus terreus in the production of trans-aconitic acid.

In one embodiment of the present invention, the application refers to the production of trans-aconitic acid after the recombinant Aspergillus terreus is fermented and cultured, and the fermentation conditions are 37° C., 220 rpm for 72 h to 168 h.

In one embodiment of the present invention, the medium formula used in the fermentation is: 100 g L ^-1 glucose, 2 g L ^-1 NH ₄ NO ₃ , 0.2 g L ^-1 (NH ₄ ) ₂ HPO ₄ , 20 mg L ^-1 FeSO ₄ , 0.4 g L ^-1 MgSO ₄ , 40 mg L ^-1 ZnSO ₄ , 40 mg L ^-1 CuSO ₄ , the balance being water.

To be further defined, the DNA repair-related protein ku80 Genbank accession number is EAU32303.1, and the cis-aconitic acid decarboxylase cadA Genbank accession number is MK026070.1.

In one embodiment of the present invention, the blocking of cadA is to completely knock out the cadA gene, and no fragments of the cadA gene remain in the genome of the engineered strain.

In one example, the cadA -knocked out Aspergillus terreus At-∆cadA was used as the starting strain, and the P gpdAt promoter was used to express adi1 in a site-directed manner to obtain a pure-bred transformant integrating the adi1 expression element, denoted as At-∆cadA-ku80 ::PgpdAt-adi1; In another embodiment, the construction of knockout cadA overexpression adi1 gene targeting element and the construction of heterologous expression adi1 Aspergillus terreus engineering strain are carried out to obtain a pure-bred transformant integrating the adi1 expression element, Denoted as At-∆cadA::adi1.

The method for producing trans-aconitic acid using the genetically engineered strain provided by the present invention includes the step of inoculating the strain into a medium for fermentation. After the fermentation broth was treated, the production of trans-aconitic acid, the production ratio of trans-aconitic acid and cis-aconitic acid, and the trans-aconitic acid production ratio of the two pure-bred transformants and the control group were compared by high performance liquid chromatography. Changes in the ratio of cephalic acid to cis-aconitic acid.

beneficial effect

As shown in B in Figure 1, there is an aconitic acid isomerase Adi1 in Ustilago maydis , 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). The present invention takes Aspergillus terreus which does not express cis-aconitic acid decarboxylase as the starting strain, and obtains a more efficient adi1 gene by heterologously expressing the adi1 gene in Aspergillus terreus at different genomic sites and using different promoters. The trans-aconitic acid-producing Aspergillus terreus engineered strain (A in Figure 1) circumvented the defects of the chemical synthesis of trans-aconitic acid, and developed a more efficient green process for the production of trans-aconitic acid through microbial fermentation.

Experiments have shown that the genetically engineered strains At-∆cadA::adi1 and At-∆cadA-ku80::PgpdAt-adi1 of the present invention have higher trans-aconitic acid levels than the control strain At-∆cadA. yield and lower cis-aconitic acid production, and the trans-aconitic acid production of strain At-∆cadA::adi1 was higher than that of strain At-∆cadA-ku80::PgpdAt-adi1. In addition, the genetically engineered strain of the present invention can realize the conversion of cis-aconitic acid to trans-aconitic acid in 72h in the middle stage of fermentation, while the control strain At-∆cadA realizes the same ratio of cis-aconitic acid to trans-aconitic acid. Transformation takes 168 h. Therefore, the introduction of the adi1 gene under the drive of the P cadA promoter or the P gpdAt promoter shortened the fermentation time of trans-aconitic acid to a certain extent.

Description of drawings

Figure 1.A is a schematic diagram of the construction of a high-yielding trans-aconitic acid Aspergillus terreus engineered strain, and B is a schematic diagram of the action of aconitic acid isomerase Adi1;

Figure 2. Schematic diagram of the construction strategy of the recombinant strain of Aspergillus terreus expressing aconitic acid isomerase Adi1, A is PgpdAt as the adi1 heterologous expression promoter integrated at ku80 site; B is the knockout of cadA and the use of PcadA promoter to drive adi1 heterologous expression;

Figure 3. Genome PCR verification results of the constructed At-ΔcadA-ku80::PgpdAt-adi1 engineering strain, A is the result of PCR verification with primer U-ku80-F/adi-R(TtrpC), WT is the starting strain At -ΔcadA; B is the result verified with primer adi-F/D-ku80-R, WT is the starting strain At-Δku80-ΔpyrG;

Figure 4. Genome PCR verification results of the constructed At-ΔcadA::adi1 engineering strain;

Figure 5. HPLC analysis of organic acids produced by strain fermentation, CA71-1: (the 1st bottle of 3 parallel experiments of transformant No. 7 of At-ΔcadA::adi1 strain); GA11-2: (At-ΔcadA- ku80::PgpdAt-adi1 strain No. 1 transformant, the second bottle of 3 parallel experiments); Δcad-1: (the first bottle of 3 parallel experiments of At-ΔcadA strain);

Figure 6. The analysis results of trans-aconitic acid produced by engineering strain shake flask fermentation, A is the output of cis- and trans-aconitic acid in 72h, the left side is trans-aconitic acid, and the right side is cis-aconitic acid , B is the change of the ratio of trans-aconitic acid to cis-aconitic acid in different transformants at 72h (left), 112h (middle) and 168h (right). Three transformants of At-ΔcadA-ku80::PgpdAt-adi1 engineered strain: GA11, GA31 and GA51, three transformants of At-ΔcadA::adi1 engineered strain: CA21, CA71 and CA101, starting strain At-ΔcadA engineering One transformant of the strain: Δcad; SEM of the three parallel experiments represented by the Error bar in Figure A, and the bar height in Figure B is the average of the ratio of aconitic acid to cis-aconitic acid in the three parallel experiments.

Detailed ways

The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, and the protection content of the present invention is not limited to the following embodiments. Variations and advantages that can occur to those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the appended claims are the scope of protection. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common knowledge in the field, and the present invention has no special limited contents. Conditions were as described in Sambrook et al., Molecular Cloning, Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as suggested by the manufacturer.

Prepare the medium for use according to the following medium formula:

Regeneration selection medium plate PDA-SH: 3.9 g L ^-1 Potato Dextrose Agar (Difco ^TM PotatoDextrose Agar, BD, LOT: 1165825), and 1.2 M sorbitol, sterilized and cooled to about 55 °C Plates were prepared by adding hygromycin B (Solarbio, Catalog No.: M419099) to a final concentration of 100 μg/mL.

PDAS: 3.9 g L ^-1 Potato Dextrose Agar Medium (Difco ^™ Potato Dextrose Agar, BD, LOT: 1165825) and 1.2 M sorbitol, sterilized to prepare plates.

PDBS: 2.4 g L ^-1 potato dextrose medium (Difco ^™ Potato Dextrose Broth, BD, LOT: 9239568), 1.2 M sorbitol and 0.5% agarose, sterilized to make top agar.

Aspergillus terreus sporulation medium: 10 g L ^-1 glucose, 2 g L ^-1 NaNO ₃ , 0.2 g L ^-1 KH ₂ PO ₄ , 5 g L ^-1 MgSO ₄ , 0.02 g L ^-1 FeSO ₄ , 0.5 g L ^-1 NaCl, 0.04 g L ^-1 ZnSO ₄ , 0.04 g L ^-1 CuSO ₄ , 0.5% bran, 1.5% agar powder, sterilize at 115 ℃ for 25 min, and prepare a plate.

Organic acid fermentation medium IPM: 100 g L ^-1 glucose, 2 g L ^-1 NH ₄ NO ₃ , 0.2 g L ^-1 (NH ₄ ) ₂ HPO ₄ , 20 mg L ^-1 FeSO ₄ , 0.4 g L ^-1 MgSO ₄ , 40 mg L ^-1 ZnSO ₄ , 40 mg L ^-1 CuSO ₄ , adjust the pH to 3.5 with sulfuric acid, and sterilize at 115 °C for 30 min.

Aspergillus terreus sporulation medium: 10 g L ^-1 glucose, 2 g L ^-1 NaNO ₃ , 0.2 g L ^-1 , KH ₂ PO ₄ , 20 mg L ^-1 FeSO ₄ , 5 g L ^-1 MgSO ₄ , 0.5 g L ^-1 NaCl, 40 mg L ^-1 ZnSO ₄ , 40 mg L ^-1 CuSO ₄ , 0.5% bran, 1.5% agar, sterilized at 115 ℃ for 15 min, then divided into test tubes, sterilized at 115 ℃ for 25 min, Prepare bevels.

The Aspergillus terreus used in the present invention is obtained from the China Industrial Microorganism Culture Collection and Management Center, and the deposit number is CICC 40205.

Aspergillus terreus engineered strain At-∆cadA (recorded in: Lv Xuefeng et al., Aconitic acid-containing Aspergillus terreus strain and its construction method and application, CN 201910649851.1, available to the public through Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences.)

In the present invention, plasmid extraction adopts OMEGA's Plasmid Mini KitI kit (D6943-02), DNA fragment recovery adopts OMEGA's Cycle-Pure Kit (D6492-02), and gel recovery adopts OMEGA's GelExtraction Kit (D2500- 01).

Plasmid pSGF957 (obtained by Seoul National university, the plasmid is recorded in Kim, J.G., Choi, Y.D., Chang, Y.J., Kim, S.U., Genetic transformation of Monascus purpureusDSM1379, Biotechnology Letters, 2003, 25, 1509-1514).

Plasmid pXH-106 (recorded in: Lu Xuefeng et al., Aconitic acid-containing Aspergillus terreus strain and its construction method and application, CN 201910649851.1, available to the public through the Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences).

Plasmid pXH2-1 (recorded in: Huang X et al. Cloning, characterization and application of a native glyceraldehyde-3-phosphate dehydrogenase promoter for Aspergillus terreus . J Ind Microbiol Biotechnol 2014, 41:585–592. Publicly available through Qingdao Biotechnology, Chinese Academy of Sciences obtained by the Institute of Energy and Processes)

The DNA Marker used in the present invention was purchased from Beijing Quanshijin Biology, the product name is: 1Kb DNALadder, and the product catalog number is: BM201-01.

The recombinant Aspergillus terreus described in Example 1 and Example 2 uses Aspergillus terreus as the starting strain, and the starting strain is to knock out the cis-aconitic acid decarboxylase CadA gene and overexpress aconitic acid isomerase obtained from Adi1. The starting strain is the commonly used Aspergillus terreus, such as Aspergillus terreus CICC40205, Aspergillus terreus NRRL1960, Aspergillus terreus DSM23081, Aspergillus terreus TN484, Aspergillus terreus TN484-M1 or other genetically engineered bacteria or wild strains capable of producing itaconic acid, etc. , the starting strain used in this example is Aspergillus terreus CICC40205.

Embodiment 1, with P gpdAt promoter site-specific expression adi1

1) Construction of targeting element expressing adi1 gene

ku80 site: This site is a DNA repair-related protein, Genbank accession number EAU32303.1

SEQ ID NO. 2 is the amino acid sequence of the aconitate isomerase Adi1 of A. zeae, optimized according to the codon preference of Aspergillus terreus, and the nucleotide fragment encoding Adi1 is synthesized as SEQ ID NO. 3.

The following primers were designed and synthesized according to the gene sequence information:

U-ku80-F: 5’-cgcgggtttctagaagtcacatcagc-3’;

U-ku80-R(PgpdAt): 5’-gtacctggatcctcccagagtgtaaggtggatctggagcagagg-3’;

PgpdAt-F743: 5'-ttacactctgggaggatccaggta-3';

PgpdAt-R(adi1) : 5'-gtgtcgatggggtgcagcattgtgatgattgatgagttg-3';

adi1-F : 5'-atgctgcaccccatcgacac-3';

adi1-R(TtrpC): 5'-cagtaacgttaagtggatccttaggacaggctacggtcgctag-3';

TtrpC-F: 5'-ggatccacttaacgttactg-3';

hph-R(-TtrpC) : 5'-cggtcggcatctactctattcc-3';

D-ku80-F(hph-TtrpC): 5’-ggaatagagtagatgccgaccgtagcccggagttaggtagatag-3’;

D-ku80-R : 5'-catcaccgaccctacgctgt-3';

C-ku80-F: 5'-ggtggtttctctctatcatgg-3';

C-ku80-R : 5'-gcgaaggcgaaaagtagtctc-3';

Using Aspergillus terreus At-∆cadA genomic DNA as template, pfu DNA polymerase (full gold, catalog number: AS231-01) was used for PCR amplification, and primers U-ku80-F/U-ku80-R (PgpdAt ) can amplify the upstream homology arm U-ku80 of the ku80 gene with a size of about 1.0 kb, and use the primers D-ku80-F(hph-TtrpC)/D-ku80-R to amplify the ku80 with a size of 1.0 kb Gene downstream homology arm D-ku80.

Using plasmid pXH2-1 as a template, PCR amplification was performed with primers PgpdAt-F743/PgpdAt-R(adi1) to obtain the Aspergillus terreus constitutive promoter P gpdAt , namely the PgpdAt fragment.

Similarly, using plasmid pXH2-1 as a template, PCR amplification of terminator TtrpC and hygromycin resistance gene hph was performed with primer TtrpC-F/hph-R(-TtrpC) to obtain TtrpC-hph fragment.

The Adi1 fragment was obtained by using the plasmid where the adi1 gene was synthesized as a template and PCR amplification with primers adi1-F/adi1-R (TtrpC).

All PCR products were detected by 1.0% agarose gel electrophoresis and recovered and purified by gel tapping. U-ku80 fragment, PgpdAt fragment, Adi1 fragment, TtrpC-hph fragment and D-ku80 fragment are fused by the method of fusion PCR, and the product of this fusion PCR is used as a template, and C-ku80-F/C-ku80- R was used as a primer to amplify the gene targeting element ku80::PgpdAt-adi1-hph fragment with a size of about 7.0 kb, which can be used for the work of expressing Adi1 with the PgpdAt promoter at the ku80 site.

2) Construction of an engineered strain of Aspergillus terreus heterologously expressing adi1 at the ku80 locus

Aspergillus terreus At-∆cadA is an aconitic acid-producing engineered strain obtained by knocking out the cadA gene. The spores of the engineered strain At-∆cadA were inoculated into 50 mL of IPM liquid medium to make the spore concentration about 10 ⁷ /mL, and cultured at 200 rpm and 32 °C for 12-18 h. The grown mycelia were collected by filtration with a sterile single-layer 500-mesh nylon cloth, rinsed three times with sterile 0.6 M MgSO ₄ solution, pressed dry and placed in a sterile 50 mL conical flask, and an appropriate amount of enzyme was added according to the weight of the mycelium. The solution (10 mL of enzymatic solution was added to each 1 g of mycelium) was treated at 30 °C and 60 rpm for 1-3 h. The mixed solution after enzymolysis was filtered with 300-mesh nylon cloth or lens paper, and the filtrate was collected. Protoplasts were collected by centrifugation at 4 °C, 4000 rpm, washed once with pre-cooled 1.0 M sorbitol solution, and then washed with pre-cooled STC (STC composition: 1.0 M sorbitol, 50 mM Tris-HCl (pH 8.0), 50 mM CaCl ₂ ) Wash once, and finally resuspend the protoplasts in pre-cooled STC, and adjust the protoplast concentration to 5×10 ⁷ /mL with STC to obtain a protoplast suspension. To 150 μL of this protoplast suspension was added 10 μL of the DNA fragment (about 2 μg) of the targeting element ku80::PgpdAt-adi1-hph. Then add 50 μL of PSTC in ice bath (PSTC composition: 40% PEG4000, 1.2 M sorbitol, 50 mM Tris-HCl (pH 8.0), 50 mM CaCl ₂ ), mix gently, and ice bath for 30 min. Add 1 mL of PSTC at room temperature, mix well and place at room temperature for 20 min. Then, it was mixed with 30 mL of PDBS top agar and poured onto 10 PDA-SH plates for regeneration screening culture, and cultured at 30 °C in the dark for 5-7 days.

The transformants with good growth conditions were picked from the transformation screening plate and transferred to PDAH (PDA plate supplemented with 100 mg/L hygromycin B) plate, and cultured at 30 °C for 5 days for passage purification. The spores of the stable passage transformants were inoculated in IPM liquid medium, cultured at 30 °C and 200 rpm for 48 h, and the hyphae were collected to extract genomic DNA. The primers U-ku80-F/adi-R (TtrpC) and adi-F/ The two pairs of primers D-ku80-R were verified by PCR, and the genome of the At-∆cadA strain was used as a control. The positive transformants could amplify the bands with sizes of about 3.0 kb and 5.5 kb, respectively, while the control could not amplify the bands. 5 positive transformants were selected for single spore isolation and purification, and 3 single spores were verified for each transformant, and the primers U-ku80-F/adi-R (TtrpC) and adi-F/D-ku80-R were used for genome again. PCR verification, as shown in Figure 3, obtained a pure-bred transformant integrating the adi1 expression element at the ku80 site, that is, a genetically engineered strain for high-yield trans-aconitic acid, denoted as At-∆cadA-ku80::PgpdAt- adi1.

Example 2. Site-directed expression of Adi1 at the cadA site using the P cadA promoter

1) Construction of knockout cadA overexpression adi1 gene targeting element

The following primers were designed and synthesized according to the genome information of Aspergillus terreus and the gene sequence information of SEQ ID NO.3:

U-cadA-F: 5’-gcgataaatgttgaacgaggc-3’;

U-cadA-R(adi1) : 5'-gtgtcgatggggtgcagcattggtcaatttaataggacaattttc-3';

adi1-F: 5'-atgctgcaccccatcgacac-3';

adi1-R(TtrpC): 5'-cagtaacgttaagtggatccttaggacaggctacggtcgctag-3';

TtrpC-F: 5'-ggatccacttaacgttactg-3';

TtrpC-R: 5'-aagaaggttacctctaaacaag-3';

pyrG/loxp-F(TtrpC): 5'-cttgtttagaggtaaccttctttaagggagatggtgattgaactag-3';

pyrGAn-R(PgpdAtF743): 5'-gcatcaaatcgtcgtaccgca-3';

D-cadA-F(pyrGAn): 5'-tgcggtacgacgatttgatgctaaatgggaagcgatatggaaac-3';

D-cadA-R: 5’-cattgcagggaagtatatgcttc-3’;

C-cadA-F: 5'-tgtggttcctaccaaggtggc-3';

C-cadA-R: 5’-cgactatagctggattgatcac-3’;

Using Aspergillus terreus At-∆ku80 (Aspergillus terreus At-∆ku80 is an engineered strain with ku80 gene knockout for Aspergillus terreus CICC40205) (Lu Xuefeng et al., A method and application for improving the application efficiency of gene targeting technology in Aspergillus terreus, ZL201510275491 .5) The genomic DNA is used as the template, and pfu DNA polymerase (Fermentas, catalog number: EP0501) is used for PCR amplification, and the primer U-cadA-F/U-cadA-R (adi1) can be used for amplification to obtain a size of about The upstream homology arm U-cadA of the 1.2 kb cadA gene can be amplified with primers D-cadA-F(pyrGAn)/D-cadA-R to obtain the 1.3 kb downstream homology arm D-cadA of the cadA gene.

Using plasmid pXH2-1 as a template, PCR amplification of terminator TtrpC was performed with primers TtrpC-F/TtrpC-R to obtain TtrpC fragment.

Using plasmid pXH-106 as a template, PCR amplification was performed with primers pyrG/loxp-F(TtrpC)/pyrGAn-R(PgpdAtF743) to amplify the Aspergillus niger pyrG _An gene expression element as a selection marker, that is, a pyrG _An fragment.

The Adi1 fragment was obtained using the plasmid where the adi1 gene was synthesized as a template, and the primers adi1-F/adi1-R (TtrpC) were used.

All PCR products were detected by 1.0% agarose gel electrophoresis and recovered and purified by gel tapping. U-cadA fragment, adi1 fragment, TtrpC fragment, pyrG _An fragment and D-cadA fragment were fused by fusion PCR method, and the product of the fusion PCR was used as a template, and C-cadA-F/C-cadA-R was used as a template. The ΔcadA::adi1 gene targeting element with a size of about 5.7 kb was obtained as primer amplification, which can be used to express Adi1 at the cadA site using the PcadA promoter.

2) Construction of an engineered strain of Aspergillus terreus heterologously expressing adi1 at the cadA site

At-∆ku80-∆pyrG is a uracil auxotrophic engineered strain obtained by knocking out the pyrG gene on the basis of the At-∆ku80 strain (Lu Xuefeng et al., A method and application for improving the application efficiency of gene targeting technology in Aspergillus terreus) , ZL201510275491.5). The spores of the engineered strain At-∆ku80-∆pyrG were inoculated into 50 mL of IPM-FU (IPM supplemented with 1 g L ^-1 5-fluoroorotic acid and 10 mM uridine) liquid medium to make the spores. The concentration was about 10 ⁷ cells/mL, and cultured at 200 rpm and 32 °C for 12-18 h. The grown mycelia were collected by filtration with a sterile single-layer 500-mesh nylon cloth, rinsed three times with sterile 0.6 M MgSO ₄ solution, pressed dry and placed in a sterile 50 mL conical flask, and an appropriate amount of enzyme was added according to the weight of the mycelium. The solution (10 mL of enzymatic solution was added to each 1 g of mycelium) was treated at 30 °C and 60 rpm for 1-3 h. The mixed solution after enzymolysis was filtered with 300-mesh nylon cloth or lens paper, and the filtrate was collected. Protoplasts were collected by centrifugation at 4°C, 4000 rpm, washed once with pre-chilled 1.0 M sorbitol solution, and then with pre-chilled STC (STC composition: 1.0 M sorbitol, 50 mM Tris-HCl (pH 8.0), 50 mM CaCl ₂ ) was washed once, and finally the protoplasts were resuspended in pre-cooled STC, and the protoplast concentration was adjusted to 5×10 ⁷ /mL with STC to obtain a protoplast suspension. To 150 μL of this protoplast suspension was added 10 μL of the DNA fragment (approximately 2 μg) of the targeting element ∆cadA::adi1. Then add 50 μL of PSTC in ice bath (PSTC composition: 40% PEG4000, 1.2 M sorbitol, 50 mM Tris-HCl (pH 8.0), 50 mM CaCl ₂ ), mix gently, and ice bath for 30 min. Add 1 mL of PSTC at room temperature, mix well and place at room temperature for 20 min. Then, it was mixed with 30 mL of PDBS top agar and poured onto 10 PDAS plates for regeneration screening and cultured at 30 °C in the dark for 5-7 days.

The transformants with good growth conditions were picked from the transformation screening plate and transferred to the PDA plate, and cultured at 30 °C for 5 days for passage purification. The spores of the stable passage transformants were inoculated in IPM liquid medium, cultured at 30 °C and 200 rpm for 48 h, and the hyphae were collected to extract genomic DNA, which was verified by PCR with primers U-cadA-F/D-cadA-R. The genome of the At-∆ku80-∆pyrG strain served as a control. Positive transformants can amplify a band of about 6.0 kb in size, and the control can amplify a band of about 4.5 kb in size. 5 positive transformants were selected for single spore isolation and purification, each transformant was verified for 3 single spores, and the primers U-cadA-F/D-cadA-R were used again for genomic PCR verification, as shown in Figure 4, to obtain cadA The pure-bred transformants with the adi1 expression element integrated into the site, that is, the genetically engineered strain for high production of trans-aconitic acid, were recorded as At-∆cadA::adi1.

Example 3. Shake flask analysis of aconitic acid produced by fermentation of heterologous expression of adi1 Aspergillus terreus engineering strain

Three positive transformants of At-∆cadA-ku80::PgpdAt-adi1 (Example 1) and At-∆cadA::adi1 (Example 2) were selected respectively, and At-∆cadA was used as a control strain to inoculate respectively To Aspergillus terreus sporulation slant medium, cultivated at 32 °C for 7 days to obtain mature spores. Then inoculate a spore on the slant into a bottle of IPM (55 mL medium in a 500 mL conical flask), the inoculum volume is about the final concentration of 2×10 ⁵ spores/mL, and each transformant is set with 3 shakers. Flasks were fermented for 70-170 h at 37 °C, 220 rpm as parallel.

The supernatant was filtered through a 0.45 μm filter and then appropriately diluted, and the content of organic acids was detected by high performance liquid chromatography (High Performance Liquid Chromatography, HPLC). Column: Bio-rad AminexHPX-87H, 300 mm x 7.8 mm; mobile phase: 5 mM sulfuric acid; flow rate: 0.5 mL/min; column temperature: 55 °C; inspection temperature: 35 °C; UV detector (210 nm). The results are shown in Figures 5 and 6, HPLC chromatograms and fermentation yields of cis/trans aconitic acid of strains At-∆cadA, At-∆cadA-ku80::PgpdAt-adi1 and At-∆cadA::adi1 Statistical comparative analysis showed that in the middle 72 h of shake flask fermentation: the trans-aconitic acid production and peak profile of At-∆cadA::adi1 transformant CA71 (15.34 g/L) were much higher than those of At-∆cadA-ku80::PgpdAt -adi1 transformant GA11 (9.46g/L) and the starting strain At-∆cadA transformant ∆cad (5.52g/L), while the yield of cis-aconitic acid CA71 (1.48g/L) GA11 (2.53g/L) L) and peaks were lower than the original strain At-∆cadA transformant ∆cad (4.12g/L). Among them, the transformant CA71 of At-∆cadA::adi1 produced the best trans-aconitic acid. The ratio of aconitic acid/cis-aconitic acid production can be clearly found in At-∆cadA::adi1 transformants CA101 (9:1), CA71 (10:1) and CA21 (10:1) At-∆cadA::adi1 in the middle of 72 h fermentation. The -∆pgpdAt::adi1 transformants GA11 (7:2), GA31 (4:1) and GA51 (2:1) are all much larger than the starting strain At-∆cadA transformant ∆cad (4:3), and are shown in the figure As shown in A in 6, comparing the production ratio of trans-aconitic acid/cis-aconitic acid at three time points of fermentation at 72 h, 112 h and 168 h, it can be clearly found that the transformant CA71 was fermented at 72 h. The ratio of trans-aconitic acid/cis-aconitic acid production was the highest. Based on the above results of trans-aconitic acid production and trans-aconitic acid/cis-aconitic acid production ratio, it is proved that the introduction of isomerase Adi1 can realize the conversion of cis-aconitic acid to trans-aconitic acid in 72 h in the middle stage of fermentation, Among them, the Adi1 isomerase guided by the P cadA promoter is the best, and the yield can be increased while the ratio is increased, and the Adi1 isomerase guided by the P gpdAt promoter is the second. Adi1 isomerase can increase the production of trans-aconitic acid and shorten the fermentation time.

The above descriptions are merely examples of the present application, and are not intended to limit the present application. Various modifications and variations of this application are possible for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the scope of the claims of this application.

Gene and amino acid sequence involved in the present invention:

SEQ ID NO.1 Cisaconitic acid decarboxylase CadA

MTKQSADSNAKSGVTSEICHWASNLATDDIPSDVLERAKYLILDGIACAWVGARVPWSEKYVQATMSFEPPGACRVIGYGQKLGPVAAAMTNSAFIQATELDDYHSEAPLHSASIVLPAVFAASEVLAEQGKTISGIDVILAAIVGFESGPRIGKAIYGSDLLNNGWHCGAVYGAPAGALATGKLLGLTPDSMEDALGIACTQACGLMSAQYGGMVKRVQHGFAARNGLLGGLLAHGGYEAMKGVLERSYGGFLKMFTKGNGREPPYKEEEVVAGLGSFWHTFTIRIKLYACCGLVHGPVEAIENLQGRYPELLNRANLSNIRHVHVQLSTASNSHCGWIPEERPISSIAGQMSVAYILAVQLVDQQCLLSQFSEFDDNLERPEVWDLARKVTSSQSEEFDQDGNCLSAGRVRIEFNDGSSITESVEKPLGVKEPMPNERILHKYRTLAGSVTDESRVKEIEDLVLGLDRLTDISPLLELLNCPVKSPLV

SEQ ID NO.2

Sequence features:

Length: 443aa

Molecular Type: Amino Acid Sequence

Original Source: Ustilago maydis

Specificity name: aconitate isomerase Adi1

MLHPIDTTIYRAGTSRGLYFLASDLPAEPSERDAALISIMGSGHPLQIDGMGGGNSLTSKVAIVSASTQRSEFDVDYLFCQVGITERFVDTAPNCGNLMSGVAAFAIERGLVQPHPSDTTCLVRIFNLNSRQASELVIPVYNGRVHYDDIDDMHMQRPSARVGLRFLDTVGSCTGKLLPTGNASDWIDGLKVSIIDSAVPVVFIRQHDVGITGSEAPATLNANTALLDRLERVRLEAGRRMGLGDVSGSVVPKLSLIGPGTETTTFTARYFTPKACHNAHAVTGAICTAGAAYIDGSVVCEILSSRASACSASQRRISIEHPSGVLEVGLVPPENAAQSLVDVAVVERSIALIAHARVYYTTPDRRRSYDSPLTSPSTPADTHNLFDAAYRPVIQPSDTDVEAPHMLALENKEQCVSRCDTALHHIVASYGASDAHASDRSLS

SEQ ID NO.3

Sequence features:

Length: 1332bp

Molecular Type: DNA Sequence

Original Source: Ustilago maydis

Specificity name: aconitate isomerase adi1 gene

ATGCTGCACCCCATCGACACCACCATCTACCGCGCCGGCACCAGCCGCGGTCTGTACTTCCTGGCCTCCGACCTGCCCGCCGAGCCTTCTGAGCGCGACGCTGCTCTGATCTCCATCATGGGCTCCGGCCACCCCCTGCAAATCGACGGCATGGGCGGCGGCAACTCCCTGACCTCCAAGGTCGCCATCGTCTCCGCCTCCACCCAGCGCAGCGAGTTCGACGTCGACTACCTGTTCTGCCAGGTCGGCATCACCGAGCGCTTCGTCGACACCGCCCCCAACTGCGGCAACCTGATGTCCGGCGTCGCCGCCTTCGCCATCGAGCGTGGTCTGGTCCAGCCCCACCCCTCCGACACCACCTGCCTGGTCCGCATCTTCAACCTGAACTCCCGCCAGGCCTCCGAGCTGGTCATCCCCGTCTACAACGGCCGCGTCCACTACGACGACATCGACGACATGCACATGCAGCGCCCCAGCGCCCGCGTCGGTTTGCGTTTCCTGGACACCGTCGGCAGCTGCACCGGCAAGCTGCTGCCCACCGGCAACGCCAGCGACTGGATCGACGGCCTGAAGGTCAGCATCATCGACTCCGCCGTCCCCGTCGTCTTCATCCGCCAGCACGACGTCGGCATCACGGGCAGCGAGGCCCCTGCTACCCTGAACGCCAACACCGCCCTGCTGGACCGCCTGGAGCGCGTTCGTCTGGAGGCCGGTCGCCGTATGGGCCTGGGTGACGTCTCCGGCAGCGTCGTCCCCAAGCTGAGCCTGATCGGCCCCGGCACCGAGACCACCACCTTCACCGCCCGCTACTTCACCCCCAAGGCCTGCCACAACGCCCACGCCGTCACCGGTGCCATCTGCACCGCCGGTGCCGCTTACATCGACGGCAGCGTCGTGTGCGAGATCCTGTCCAGCCGCGCCTCCGCCTGCTCCGCTTCTCAGCGTCGCATCAGCATCGAGCACCCCAGCGGCGTCCTGGAGGTCGGTCTGGTCCCCCCTG AGAACGCCGCCCAGTCCCTGGTCGACGTCGCCGTTGTCGAGCGCAGCATCGCCCTGATCGCCCACGCCCGTGTCTACTACACCACCCCCGACCGCCGCCGCTCCTACGATAGCCCTCTGACCTCCCCCTCCACCCCCGCTGACACCCACAACCTGTTCGACGCCGCCTACCGCCCCGTCATCCAGCCTAGCGACACCGACGTCGAGGCCCCCCACATGCTGGCCCTGGAGAACAAGGAGCAGTGCGTCTCCCGCTGCGACACCGCCCTGCACCACATCGTCGCCTCCTACGGCGCCAGCGACGCCCATGCTAGCGACCGTAGCCTGTCCTAA

SEQ ID NO.4 Gene encoding cis-aconitic acid decarboxylase CadA

ATGACCAAACAATCTGCGGACAGCAACGCAAAGTCAGGAGTTACGTCCGAAATATGTCATTGGGCATCCAACCTGGCCACTGACGACATCCCTTCGGACGTATTAGAAAGAGCAAAATACCTTATTCTCGACGGTATTGCATGTGCCTGGGTTGGTGCAAGAGTGCCTTGGTCAGAGAAGTATGTTCAGGCAACGATGAGCTTTGAGCCGCCGGGGGCCTGCAGGGTGATTGGATATGGACAGGTAAATTTTATTCACTCTAGACGGTCCACAAAGTATACTGACGATCCTTCGTATAGAAACTGGGGCCTGTTGCAGCAGCCATGACCAATTCCGCTTTCATACAGGCTACGGAGCTTGACGACTACCACAGCGAAGCCCCCCTACACTCTGCAAGCATTGTCCTTCCTGCGGTCTTTGCAGCAAGTGAGGTCTTAGCCGAGCAGGGCAAAACAATTTCCGGTATAGATGTTATTCTAGCCGCCATTGTGGGGTTTGAATCTGGCCCACGGATCGGCAAAGCAATCTACGGATCGGACCTCTTGAACAACGGCTGGCATTGTGGAGCTGTGTATGGCGCTCCAGCCGGTGCGCTGGCCACAGGAAAGCTCCTCGGTCTAACTCCAGACTCCATGGAAGATGCTCTCGGAATTGCGTGCACGCAAGCCTGTGGTTTAATGTCGGCGCAATACGGAGGCATGGTAAAGCGTGTGCAACACGGATTCGCAGCGCGTAATGGTCTTCTTGGGGGACTGTTGGCCCATGGTGGGTACGAGGCAATGAAAGGTGTCCTGGAGAGATCTTACGGCGGTTTCCTCAAGATGTTCACCAAGGGCAACGGCAGAGAGCCTCCCTACAAAGAGGAGGAAGTGGTGGCTGGTCTCGGTTCATTCTGGCATACCTTTACTATTCGCATCAAGCTCTATGCCTGCTGCGGACTTGTCCATGGTCCAGTCGAGGCTATCGAAAACCTTCAGGGGAGATACCCCGAGCTCTTGAA TAGAGCCAACCTCAGCAACATTCGCCATGTTCATGTACAGCTTTCAACGGCCTCGAACAGTCACTGTGGATGGATACCAGAGGAGAGACCCATCAGTTCAATCGCAGGGCAGATGAGTGTCGCATACATTCTCGCCGTCCAGCTGGTCGACCAGCAATGTCTTTTGTCCCAGTTTTCTGAGTTTGATGACAACCTGGAGAGGCCAGAAGTTTGGGATCTGGCCAGGAAGGTTACTTCATCTCAAAGCGAAGAGTTTGATCAAGACGGCAACTGTCTCAGTGCGGGTCGCGTGAGGATTGAGTTCAACGATGGTTCTTCTATTACGGAAAGTGTCGAGAAGCCTCTTGGTGTCAAAGAGCCCATGCCAAACGAACGGATTCTCCACAAATACCGAACCCTTGCTGGTAGCGTGACGGACGAATCCCGGGTGAAAGAGATTGAGGATCTTGTCCTCGGCCTGGACAGGCTCACCGACATTAGCCCATTGCTGGAGCTGCTGAATTGCCCCGTGAAATCGCCACTGGTATAA

SEQUENCE LISTING

<110> Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences

Shandong Lukang Sherile Pharmaceutical Co., Ltd.

<120> A recombinant Aspergillus terreus strain with high-yield trans-aconitic acid and its construction method and application

<130>

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 490

<212> PRT

<213> Cisaconitic acid decarboxylase CadA

<400> 1

Met Thr Lys Gln Ser Ala Asp Ser Asn Ala Lys Ser Gly Val Thr Ser

1 5 10 15

Glu Ile Cys His Trp Ala Ser Asn Leu Ala Thr Asp Asp Ile Pro Ser

20 25 30

Asp Val Leu Glu Arg Ala Lys Tyr Leu Ile Leu Asp Gly Ile Ala Cys

35 40 45

Ala Trp Val Gly Ala Arg Val Pro Trp Ser Glu Lys Tyr Val Gln Ala

50 55 60

Thr Met Ser Phe Glu Pro Pro Gly Ala Cys Arg Val Ile Gly Tyr Gly

65 70 75 80

Gln Lys Leu Gly Pro Val Ala Ala Ala Met Thr Asn Ser Ala Phe Ile

85 90 95

Gln Ala Thr Glu Leu Asp Asp Tyr His Ser Glu Ala Pro Leu His Ser

100 105 110

Ala Ser Ile Val Leu Pro Ala Val Phe Ala Ala Ser Glu Val Leu Ala

115 120 125

Glu Gln Gly Lys Thr Ile Ser Gly Ile Asp Val Ile Leu Ala Ala Ile

130 135 140

Val Gly Phe Glu Ser Gly Pro Arg Ile Gly Lys Ala Ile Tyr Gly Ser

145 150 155 160

Asp Leu Leu Asn Asn Gly Trp His Cys Gly Ala Val Tyr Gly Ala Pro

165 170 175

Ala Gly Ala Leu Ala Thr Gly Lys Leu Leu Gly Leu Thr Pro Asp Ser

180 185 190

Met Glu Asp Ala Leu Gly Ile Ala Cys Thr Gln Ala Cys Gly Leu Met

195 200 205

Ser Ala Gln Tyr Gly Gly Met Val Lys Arg Val Gln His Gly Phe Ala

210 215 220

Ala Arg Asn Gly Leu Leu Gly Gly Leu Leu Ala His Gly Gly Tyr Glu

225 230 235 240

Ala Met Lys Gly Val Leu Glu Arg Ser Tyr Gly Gly Phe Leu Lys Met

245 250 255

Phe Thr Lys Gly Asn Gly Arg Glu Pro Pro Tyr Lys Glu Glu Glu Val

260 265 270

Val Ala Gly Leu Gly Ser Phe Trp His Thr Phe Thr Ile Arg Ile Lys

275 280 285

Leu Tyr Ala Cys Cys Gly Leu Val His Gly Pro Val Glu Ala Ile Glu

290 295 300

Asn Leu Gln Gly Arg Tyr Pro Glu Leu Leu Asn Arg Ala Asn Leu Ser

305 310 315 320

Asn Ile Arg His Val His Val Gln Leu Ser Thr Ala Ser Asn Ser His

325 330 335

Cys Gly Trp Ile Pro Glu Glu Arg Pro Ile Ser Ser Ile Ala Gly Gln

340 345 350

Met Ser Val Ala Tyr Ile Leu Ala Val Gln Leu Val Asp Gln Gln Cys

355 360 365

Leu Leu Ser Gln Phe Ser Glu Phe Asp Asp Asn Leu Glu Arg Pro Glu

370 375 380

Val Trp Asp Leu Ala Arg Lys Val Thr Ser Ser Gln Ser Glu Glu Phe

385 390 395 400

Asp Gln Asp Gly Asn Cys Leu Ser Ala Gly Arg Val Arg Ile Glu Phe

405 410 415

Asn Asp Gly Ser Ser Ile Thr Glu Ser Val Glu Lys Pro Leu Gly Val

420 425 430

Lys Glu Pro Met Pro Asn Glu Arg Ile Leu His Lys Tyr Arg Thr Leu

435 440 445

Ala Gly Ser Val Thr Asp Glu Ser Arg Val Lys Glu Ile Glu Asp Leu

450 455 460

Val Leu Gly Leu Asp Arg Leu Thr Asp Ile Ser Pro Leu Leu Glu Leu

465 470 475 480

Leu Asn Cys Pro Val Lys Ser Pro Leu Val

485 490

<210> 2

<211> 443

<212> PRT

<213> aconitate isomerase Adi1

<400> 2

Met Leu His Pro Ile Asp Thr Thr Ile Tyr Arg Ala Gly Thr Ser Arg

1 5 10 15

Gly Leu Tyr Phe Leu Ala Ser Asp Leu Pro Ala Glu Pro Ser Glu Arg

20 25 30

Asp Ala Ala Leu Ile Ser Ile Met Gly Ser Gly His Pro Leu Gln Ile

35 40 45

Asp Gly Met Gly Gly Gly Asn Ser Leu Thr Ser Lys Val Ala Ile Val

50 55 60

Ser Ala Ser Thr Gln Arg Ser Glu Phe Asp Val Asp Tyr Leu Phe Cys

65 70 75 80

Gln Val Gly Ile Thr Glu Arg Phe Val Asp Thr Ala Pro Asn Cys Gly

85 90 95

Asn Leu Met Ser Gly Val Ala Ala Phe Ala Ile Glu Arg Gly Leu Val

100 105 110

Gln Pro His Pro Ser Asp Thr Thr Cys Leu Val Arg Ile Phe Asn Leu

115 120 125

Asn Ser Arg Gln Ala Ser Glu Leu Val Ile Pro Val Tyr Asn Gly Arg

130 135 140

Val His Tyr Asp Asp Ile Asp Asp Met His Met Gln Arg Pro Ser Ala

145 150 155 160

Arg Val Gly Leu Arg Phe Leu Asp Thr Val Gly Ser Cys Thr Gly Lys

165 170 175

Leu Leu Pro Thr Gly Asn Ala Ser Asp Trp Ile Asp Gly Leu Lys Val

180 185 190

Ser Ile Ile Asp Ser Ala Val Pro Val Val Phe Ile Arg Gln His Asp

195 200 205

Val Gly Ile Thr Gly Ser Glu Ala Pro Ala Thr Leu Asn Ala Asn Thr

210 215 220

Ala Leu Leu Asp Arg Leu Glu Arg Val Arg Leu Glu Ala Gly Arg Arg

225 230 235 240

Met Gly Leu Gly Asp Val Ser Gly Ser Val Val Pro Lys Leu Ser Leu

245 250 255

Ile Gly Pro Gly Thr Glu Thr Thr Thr Phe Thr Ala Arg Tyr Phe Thr

260 265 270

Pro Lys Ala Cys His Asn Ala His Ala Val Thr Gly Ala Ile Cys Thr

275 280 285

Ala Gly Ala Ala Tyr Ile Asp Gly Ser Val Val Cys Glu Ile Leu Ser

290 295 300

Ser Arg Ala Ser Ala Cys Ser Ala Ser Gln Arg Arg Ile Ser Ile Glu

305 310 315 320

His Pro Ser Gly Val Leu Glu Val Gly Leu Val Pro Pro Glu Asn Ala

325 330 335

Ala Gln Ser Leu Val Asp Val Ala Val Val Glu Arg Ser Ile Ala Leu

340 345 350

Ile Ala His Ala Arg Val Tyr Tyr Thr Thr Pro Asp Arg Arg Arg Ser

355 360 365

Tyr Asp Ser Pro Leu Thr Ser Pro Ser Thr Pro Ala Asp Thr His Asn

370 375 380

Leu Phe Asp Ala Ala Tyr Arg Pro Val Ile Gln Pro Ser Asp Thr Asp

385 390 395 400

Val Glu Ala Pro His Met Leu Ala Leu Glu Asn Lys Glu Gln Cys Val

405 410 415

Ser Arg Cys Asp Thr Ala Leu His His Ile Val Ala Ser Tyr Gly Ala

420 425 430

Ser Asp Ala His Ala Ser Asp Arg Ser Leu Ser

435 440

<210> 3

<211> 1332

<212> DNA

<213> aconitate isomerase adi1 gene

<400> 3

atgctgcacc ccatcgacac caccatctac cgcgccggca ccagccgcgg tctgtacttc 60

ctggcctccg acctgcccgc cgagccttct gagcgcgacg ctgctctgat ctccatcatg 120

ggctccggcc accccctgca aatcgacggc atgggcggcg gcaactccct gacctccaag 180

gtcgccatcg tctccgcctc cacccagcgc agcgagttcg acgtcgacta cctgttctgc 240

caggtcggca tcaccgagcg cttcgtcgac accgccccca actgcggcaa cctgatgtcc 300

ggcgtcgccg ccttcgccat cgagcgtggt ctggtccagc cccacccctc cgacaccacc 360

tgcctggtcc gcatcttcaa cctgaactcc cgccaggcct ccgagctggt catccccgtc 420

tacaacggcc gcgtccacta cgacgacatc gacgacatgc acatgcagcg ccccagcgcc 480

cgcgtcggtt tgcgtttcct ggacaccgtc ggcagctgca ccggcaagct gctgcccacc 540

ggcaacgcca gcgactggat cgacggcctg aaggtcagca tcatcgactc cgccgtcccc 600

gtcgtcttca tccgccagca cgacgtcggc atcacgggca gcgaggcccc tgctaccctg 660

aacgccaaca ccgccctgct ggaccgcctg gagcgcgttc gtctggaggc cggtcgccgt 720

atgggcctgg gtgacgtctc cggcagcgtc gtccccaagc tgagcctgat cggccccggc 780

accgagacca ccaccttcac cgcccgctac ttcaccccca aggcctgcca caacgcccac 840

gccgtcaccg gtgccatctg caccgccggt gccgcttaca tcgacggcag cgtcgtgtgc 900

gagatcctgt ccagccgcgc ctccgcctgc tccgcttctc agcgtcgcat cagcatcgag 960

caccccagcg gcgtcctgga ggtcggtctg gtcccccctg agaacgccgc ccagtccctg 1020

gtcgacgtcg ccgttgtcga gcgcagcatc gccctgatcg cccacgcccg tgtctactac 1080

accacccccg accgccgccg ctcctacgat agccctctga cctccccctc cacccccgct 1140

gacacccaca acctgttcga cgccgcctac cgccccgtca tccagcctag cgacaccgac 1200

gtcgaggccc cccacatgct ggccctggag aacaaggagc agtgcgtctc ccgctgcgac 1260

accgccctgc accacatcgt cgcctcctac ggcgccagcg acgcccatgc tagcgaccgt 1320

agcctgtcct aa 1332

<210> 4

<211> 1529

<212> DNA

<213> Gene encoding cis-aconitic acid decarboxylase CadA

<400> 4

atgaccaaac aatctgcgga cagcaacgca aagtcaggag ttacgtccga aatatgtcat 60

tgggcatcca acctggccac tgacgacatc ccttcggacg tattagaaag agcaaaatac 120

cttattctcg acggtattgc atgtgcctgg gttggtgcaa gagtgccttg gtcagagaag 180

tatgttcagg caacgatgag ctttgagccg ccgggggcct gcagggtgat tggatatgga 240

caggtaaatt ttattcactc tagacggtcc acaaagtata ctgacgatcc ttcgtataga 300

aactggggcc tgttgcagca gccatgacca attccgcttt catacaggct acggagcttg 360

acgactacca cagcgaagcc cccctacact ctgcaagcat tgtccttcct gcggtctttg 420

cagcaagtga ggtcttagcc gagcagggca aaacaatttc cggtatagat gttattctag 480

ccgccattgt ggggtttgaa tctggcccac ggatcggcaa agcaatctac ggatcggacc 540

tcttgaacaa cggctggcat tgtggagctg tgtatggcgc tccagccggt gcgctggcca 600

caggaaagct cctcggtcta actccagact ccatggaaga tgctctcgga attgcgtgca 660

cgcaagcctg tggtttaatg tcggcgcaat acggaggcat ggtaaagcgt gtgcaacacg 720

gattcgcagc gcgtaatggt cttcttgggg gactgttggc ccatggtggg tacgaggcaa 780

tgaaaggtgt cctggagaga tcttacggcg gtttcctcaa gatgttcacc aagggcaacg 840

gcagagagcc tccctacaaa gaggaggaag tggtggctgg tctcggttca ttctggcata 900

cctttactat tcgcatcaag ctctatgcct gctgcggact tgtccatggt ccagtcgagg 960

ctatcgaaaa ccttcagggg agataccccg agctcttgaa tagagccaac ctcagcaaca 1020

ttcgccatgt tcatgtacag ctttcaacgg cctcgaacag tcactgtgga tggataccag 1080

aggagagacc catcagttca atcgcagggc agatgagtgt cgcatacatt ctcgccgtcc 1140

agctggtcga ccagcaatgt cttttgtccc agttttctga gtttgatgac aacctggaga 1200

ggccagaagt ttgggatctg gccaggaagg ttacttcatc tcaaagcgaa gagtttgatc 1260

aagacggcaa ctgtctcagt gcgggtcgcg tgaggattga gttcaacgat ggttcttcta 1320

ttacggaaag tgtcgagaag cctcttggtg tcaaagagcc catgccaaac gaacggattc 1380

tccacaaata ccgaaccctt gctggtagcg tgacggacga atcccgggtg aaagagattg 1440

aggatcttgt cctcggcctg gacaggctca ccgacattag cccattgctg gagctgctga 1500

attgccccgt gaaatcgcca ctggtataa 1529

Claims (7)

1.A recombinant Aspergillus terreus strain for producing trans-aconitic acid is characterized in that the recombinant Aspergillus terreus is obtained by taking Aspergillus terreus (Aspergillus terreus) as an initial strain, knocking out a aconitate decarboxylase cadA gene in the initial strain and overexpressing aconitate isomerase Adi 1; the amino acid sequence of the aconitate isomerase Adi1 is shown as SEQ ID NO.2, and the nucleotide sequence of the gene coding the aconitate isomerase Adi1 is shown as SEQ ID NO. 3; the promoter used by the expression element of the aconitate isomerase Adi1 is Pcad A promoter or PgpdAt promoter.

2. The recombinant Aspergillus terreus strain of claim 1, wherein the starting strain is constructed as Aspergillus terreus CICC40205, Aspergillus terreus NRRL1960, Aspergillus terreus DSM23081, Aspergillus terreus TN484 or Aspergillus terreus TN 484-M1.

3. The recombinant aspergillus terreus strain of claim 1, wherein the aconitate decarboxylase CadA has the amino acid sequence shown in SEQ ID No. 1.

4. The method for constructing a recombinant Aspergillus terreus strain according to any one of claims 1 to 3, characterized in that an Aspergillus terreus is used as a starting strain, the aconitate decarboxylase cadA gene is knocked out, and the expression element of the aconitate isomerase Adi1 is introduced into the starting strain to construct a recombinant Aspergillus terreus strain.

5. Use of a recombinant Aspergillus terreus strain according to any one of claims 1 to 3 for the production of trans-aconitic acid.

6. The use according to claim 5, wherein the use is to produce trans-aconitic acid by fermentation culture of the recombinant aspergillus terreus strain under the following fermentation conditions: the fermentation time at 37 ℃ and 220rpm is 72-168 h.

7. The use according to claim 6, wherein the fermentation is carried out using a medium formulation comprising: 100 g L^-1Glucose, 2g 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 CuSO₄And the balance being water.

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