CN112029671B - Recombinant aspergillus terreus strain for producing trans-aconitic acid and preparation method and application thereof - Google Patents

Abstract

A recombinant aspergillus terreus strain for producing trans-aconitic acid and a preparation 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 for producing the trans-aconitic acid, which takes Aspergillus terreus (Aspergillus terreus) as an initial strain, knocks out a aconitic acid decarboxylase cadA gene in the initial strain, and overexpresses aconitic acid isomerase Tbra. The amino acid sequence of the aconitic acid isomerase TbrA is shown as SEQ ID NO.2, and the nucleotide sequence is shown as SEQ ID NO. 3. On the basis of knocking out cadA, the invention obtains a more efficient trans-aconitic acid-producing aspergillus terreus engineering strain which can be used for producing trans-aconitic acid by heterologously expressing tbrA gene in aspergillus terreus at different genomic sites and by using different promoters.

Description

A kind of recombinant Aspergillus terreus strain producing trans-aconitic acid and its preparation method and application

technical field

The invention belongs to the field of genetic engineering, in particular to a trans-aconitic acid-producing recombinant Aspergillus terreus and a preparation 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.

Trans-aconitic acid is mainly produced by chemical synthesis, the process is complicated, the by-products are many, the cost is high, and there is no large-scale production. 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 the biosynthetic pathway and fermentation results, it can be speculated that the direct product of cadA knockout accumulation is cis-aconitic acid, and cis-aconitic acid, as an intermediate of the tricarboxylic acid cycle, will be quickly converted into isocitrate and further metabolized. Therefore, converting cis-aconitic acid to more stable trans-aconitic acid at the first time is beneficial to achieve higher trans-aconitic acid production.

SUMMARY OF THE INVENTION

In order to improve the yield of biosynthesized trans-aconitic acid, the present invention provides a recombinant Aspergillus terreus strain with high-yield trans-aconitic acid. The strain was obtained by mutating the cis-aconitic acid decarboxylase CadA gene and overexpressing the aconitic acid isomerase TbrA.

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 TbrA is shown in SEQ ID NO.2.

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

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

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

The invention also provides the application of the above recombinant Aspergillus terreus strain 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 strain is fermented and cultured, and the fermentation conditions are 37° C., 220 rpm for 72h-168h.

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.

beneficial effect

As shown in B in Figure 1, there is an aconitic acid isomerase TbrA in Bacillus thuringiensis , which can catalyze the isomerization of cis-aconitic acid to generate trans-aconitic acid (Du C., et al. , Genetic and Biochemical Characterization of a Gene Operon for trans -Aconitic Acid, a Novel Nematicide from Bacillus thuringiensis , The Journal of Biological Chemistry, 2017, 292(8):3517–3530). On the basis of knocking out cadA , the present invention obtains a more efficient trans-aconitic acid-producing A. terreus engineering strain by heterologously expressing the tbrA gene in Aspergillus terreus at different genomic sites and using different promoters (A in Figure 1 ). ).

Description of drawings

Figure 1. Schematic diagram of the construction of trans-aconitic acid-producing recombinant Aspergillus terreus strains; A is the schematic diagram of the construction of trans-aconitic acid-producing A. terreus engineered strains; B is the catalytic principle of aconitic acid isomerase TbrA;

Figure 2. Schematic diagram of the construction strategy of the Aspergillus terreus strain for site-directed expression of aconitate isomerase TbrA; A: PgpdAt was used as the tbrA heterologous expression promoter for site-directed integration at the ku80 site; B: cadA was knocked out and driven by the PcadA promoter Heterologous expression of tbrA ;

Figure 3. Genome PCR verification results of the constructed At-∆cadA-ku80::PgpdAt-tbrA engineering strain, M is the DNA molecular weight marker; A: PCR verification with primer U-ku80-F/tbrA-R(TtrpC) Result, WT is the control strain At-∆cadA; B: The result verified with primer PgpdAt-F743-F/D-ku80-R, WT is the control strain At-∆ku80-∆pyrG;

Figure 4. Genome PCR verification results of the constructed At-∆cadA::tbrA engineered strain; M is the DNA molecular weight marker, WT is the control strain At-∆cadA;

Figure 5. HPLC chromatograms of cis and trans aconitic acid produced by engineered strains fermented for 72 h, CT81-2 (long dashed line): (3 parallel experiments of the fermentation of transformant CT81 of At-∆cadA::tbrA strain 8 The 2nd bottle in the 1 (solid line): (flask 1 of 3 parallel experiments of the At-∆cadA strain); CAA is cis-aconitic acid and TAA is trans-aconitic acid.

Figure 6. Analysis results of the yield and proportion of trans-aconitic acid produced by engineering strains in shake flasks; A: The yield of cis- and trans-aconitic acid at 72h Head acid, B: The ratio of trans-aconitic acid to cis-aconitic acid in different transformants fermentation at 72h (left white), 112h (middle gray) and 168h (right twill); At-∆cadA-ku80 ::Three transformants of PgpdAt-tbrA engineered strain: GT41, GT71 and GT81, At-∆cadA::Three transformants of tbrA engineered strain: CT41, CT71 and CT81, one of the starting strain At-ΔcadA engineered strain Transformant: ∆cad; SEM of the three parallel experiments represented by the Error bar in panel A, and the bar height in panel 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. As described in Sambrook et al., Molecular Cloning, Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

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).

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.

Aspergillus terreus CICC40205, purchased from China Industrial Microorganism Culture Collection and Management Center.

Aspergillus terreus NRRL1960 was purchased from the American Agricultural Culture Collection (ARS Culture Collection).

Aspergillus terreus DSM23081 was purchased from the German Collection of Microorganisms DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen).

Aspergillus terreus TN484, described in Yahiro, K., Takahama, T., Park, YS, Okabe, M., Breeding of Aspergillus terreus Mutant Tn-484 for Itaconic Acid Production with High-Yield, Journal of Fermentation and Bioengineering, 1995, 79 (5): 506-508.

Aspergillus terreus TN484-M1, described in Dwiarti, L., Yamane, K., Yamatani, H., Kahar, P., Okabe, M., Purification and characterization of cis-aconitic aciddecarboxylase from Aspergillus terreus TN484-M1, J Biosci Bioeng, 2002, 94(1): 29-33.

Plasmid pXH2-1, described 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. Public available through Qingdao Biotechnology, Chinese Academy of Sciences Obtained from the Institute of Energy and Process Research.

Plasmid pXH-106: described in: Lu Xuefeng et al., Aconitic acid-containing Aspergillus terreus strain and construction method and application thereof, CN 201910649851.1.

Aspergillus terreus At-∆cadA, recorded in Lv Xuefeng et al., Aconitic acid-containing Aspergillus terreus strain and its construction method and application, CN 201910649851.1, the above materials are publicly available through the Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences .

Aspergillus terreus At-∆ku80 is described in Lv Xuefeng et al., A method and application for improving the application efficiency of gene targeting technology in Aspergillus terreus, ZL201510275491.5, available to the public through the Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences.

Example 1. Construction of a recombinant Aspergillus terreus strain with high trans-aconitic acid production.

For the recombinant Aspergillus terreus with high trans-aconitic acid production described in this example, Aspergillus terreus was used as the starting strain, the cis-aconitic acid decarboxylase CadA gene was knocked out in the starting strain, and aconitic acid was overexpressed It is obtained by isomerase TbrA, and 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 genes capable of producing itaconic acid Engineering bacteria or wild strains can be used, and the starting strain used in this embodiment is Aspergillus terreus CICC40205, and the specific construction method is described as follows:

1. Expression of tbrA using the P gpdAt promoter at the ku80 site

1) Construction of tbrA gene targeting element expressed at ku80 site (GenBank accession number EAU32303.1)

SEQ ID NO. 2 is the amino acid sequence of aconitic acid isomerase TbrA of Bacillus thuringiensis, optimized according to the codon preference of Aspergillus terreus, and the nucleotide fragment encoding TbrA 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(tbrA) : 5’-gacgaagcaggggatcttcattgtgatgattgatgagttgt-3’;

tbrA-F : 5'-atgaagatcccctgcttcgtc-3';

tbrA-R(TtrpC) : 5’-cagtaacgttaagtggatccttaggggatgatcagctcgccct-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 ( Aspergillus terreus ) At-∆cadA genomic DNA as a template, pfu DNA polymerase (Fermentas, catalog number: EP0501) was used for PCR amplification, and primers U-ku80-F/U-ku80-R (PgpdAt ), the upstream homology arm U-ku80 of the ku80 gene with a size of about 1.0 kb can be obtained by amplification, and ku80 with a size of 1.0 kb can be obtained by amplification with primers D-ku80-F(hph-TtrpC)/D- ku80 -R Gene downstream homology arm D-ku80.

Using plasmid pXH2-1 as a template, PCR amplification was performed with primers PgpdAt-F743/PgpdAt-R(tbrA) to obtain the constitutive promoter PgpdAt of Aspergillus terreus, that is, 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 TbrA fragment was obtained by using the plasmid containing the synthetic tbrA gene as a template and PCR amplification with primers tbrA-F/tbrA-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, tbrA fragment, TtrpC-hph fragment and D-ku80 fragment were fused by the method of fusion PCR, and the product of this fusion PCR was used as a template, and C-ku80-F and C-ku80- R was used as a primer to amplify the gene targeting element ku80::PgpdAt-tbrA-hph fragment with a size of about 6.8 kb, which could be used for the work of expressing TbrA with the PgpdAt promoter at the ku80 site.

2) Construction of an engineered strain of Aspergillus terreus heterologously expressing tbrA at the ku80 site

Aspergillus terreus At-∆cadA is obtained by knocking out the cadA gene (the gene of cis-aconitic acid decarboxylase cadA, GenBank accession number MK026070.1, the amino acid sequence encoded by the cadA gene described in the present invention is shown in SEQ ID NO.1) The obtained aconitic acid-producing engineering strain (Lu Xuefeng et al., an aconitic acid-producing Aspergillus terreus strain and its construction method and application, CN201910649851.1). The spores of the engineered strain At-∆cadA were inoculated into 50 mL of IPM liquid medium so that the spore concentration was 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), and 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 washed 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 the protoplast suspension, add 10 μL of the DNA fragment (about 2 μg) of the targeting element ku80::PgpdAt-tbrA-hph, and then add 50 μL of PSTC in an ice bath (PSTC composition: 40% PEG4000, 1.2 M Yamanashi) alcohol, 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 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 2.8 kb and 6.0 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 primers U-ku80-F/tbra-R (TtrpC) and PgpdAt-F743-F/D-ku80-R were used again. Perform genomic PCR validation, numbered 1-1 to 1-3, 2-1 to 2-3, 3-1 to 3-3, 4-1 to 4-3, 5-1 to 5-3, as in As shown in Figure 3, a pure transformant with the tbrA expression element integrated at the ku80 site was obtained, which was denoted as At-∆cadA-ku80::PgpdAt-tbrA.

2. Expression of tbrA using the PcadA promoter at the cadA site

1) Construction of knockout cadA overexpression tbrA gene targeting element

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

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

U-cadA-R(tbrA): 5’-gacgaagcaggggatcttcattggtcaatttaataggacaattttc-3’;

tbrA-F: 5’-atgaagatcccctgcttcgtc-3’;

tbrA-R(TtrpC) : 5’-cagtaacgttaagtggatccttaggggatgatcagctcgccct-3’;

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

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

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

pyrGAn-R(F743): 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 ( Aspergillus terreus ) At-∆ku80 genomic DNA as a template, pfu DNA polymerase (Fermentas, catalog number: EP0501) was used for PCR amplification, and primers U-cadA-F/U-cadA-R (tbrA ), the upstream homology arm U-cadA of the cadA gene with a size of about 1.2 kb can be obtained by amplification, and the downstream homology arm of the cadA gene with a size of 1.3 kb can be obtained by using primers D-cadA-F(pyrGAn)/D-cadA-R to amplify Homology arm D-cadA.

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

Using plasmid pXH-106 as template, primers pyrG/loxp-F (TtrpC) and pyrGAn-R (F743) were used to amplify the Aspergillus niger pyrG _An gene expression element as a selection marker, namely pyrG _An fragment.

The TbrA fragment was obtained by using the plasmid where the tbrA gene was synthesized as a template and amplified with primers tbrA-F and tbrA-R (TtrpC).

All PCR products were detected by 1.0% agarose gel electrophoresis and recovered and purified by gel tapping. The U-cadA fragment, tbrA fragment, TtrpC fragment, pyrG _An fragment and D-cadA fragment were fused by fusion PCR, and the product of the fusion PCR was used as a template, and C-cadA-F and C-cadA-R were used as templates. The ΔcadA::tbrA gene targeting element with a size of about 5.5 kb was obtained as primer amplification, which can be used to express TbrA with the PcadA promoter at the cadA site.

2) Construction of an engineered strain of Aspergillus terreus that heterologously expresses tbrA at the cadA site

Aspergillus terreus At-∆ku80 is an engineered strain that knocks out the ku80 gene (GenBank accession number: EAU32303.1) against A. terreus CICC40205, and At-∆ku80-∆pyrG knocks out the pyrG gene on the basis of the At-∆ku80 strain The obtained uracil auxotrophic engineering 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-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 ) 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::tbrA. 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 5.8 kb in size, and the control can amplify a band of about 4.0 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, numbered 1-1 to 1- 3, 2-1 to 2-3, 3-1 to 3-3, 4-1 to 4-3, 5-1 to 5-3, as shown in Figure 4, to obtain cadA sites with integrated tbrA expression The pure-bred transformants of the element were denoted At-∆cadA::tbrA.

Example 2. Shake flask analysis of aconitic acid produced by fermentation of an engineered strain of Aspergillus terreus heterologously expressing tbrA .

Three positive transformant strains of At-∆cadA-ku80::PgpdAt-tbrA and At-∆cadA::tbrA were selected respectively, and At-∆cadA was used as a control strain, and were inoculated into Aspergillus terreus sporulation slant medium respectively, 32 Cultivated 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 inoculation amount is about the final concentration of 2×10 ⁶ spores/mL, and each transformant is set with 3 shakers. Flasks were used as parallel, and were fermented for 72 h, 112 h and 168 h at 37 °C and 220 rpm, respectively.

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-tbrA and At-∆cadA::tbrA Statistical comparative analysis showed that in the middle 72h of shake flask fermentation: the trans-aconitic acid production and peak profile of At-∆cadA::tbrA transformant CT81 (19.05 g/L) were much higher than those of At-∆cadA-ku80::PgpdAt- The tbrA transformant GT41 (1.62g/L) and the starting strain At-∆cadA transformant ∆cad (5.52g/L), while the CT81 cis-aconitic acid yield (2.36g/L) and peak graph were lower than The transformant ∆cad (2.53g/L) of the starting strain At-∆cadA0, among which, the transformant CT81 of At-∆cadA::tbrA improved the production of trans-aconitic acid by 245% at 72h; The ratio of cephalic acid/cis-aconitic acid production in the middle stage of 72h fermentation can be clearly found that the At-∆cadA::tbrA transformants CT41 (9:1), CT71 (8:1) and CT81 (8:1) are much larger than The starting strain At-∆cadA transformant ∆cad (4:3), while the isomerase introduced by the P gpdAt promoter TbrA strain At-∆cadA-ku80::PgpdAt-tbrA transformant GT41 (2:1), GT71 ( 5:1) and GT81 (7:1) also achieved different degrees of increase in the ratio of trans-aconitic acid/cis-aconitic acid, and the target product trans-aconitic acid yield (1.6g/L, 2.5g/L) L, 1.3g/L) was lower than the starting strain At-∆cadA transformant ∆cad (5.52g/L); 112h fermentation, At-∆cadA::tbrA transformant CT81 trans-aconitic acid yield was 23.13g/L L, the production of ∆cad trans-aconitic acid in the transformant of the starting strain At-∆cadA was 12.36g/L, and the production of At-∆cadA::tbrA trans-aconitic acid increased by 87.1% in 112h; after 168h of fermentation, At-∆cadA ::tbrA transformant CT81 trans-aconitic acid yield was 24.65g/L, the starting strain At-∆cadA transformant ∆cad trans-aconitic acid yield was 13.19g/L, 168h At-∆cadA::tbrA trans-aconitic acid yield The yield of aconitic acid increased by 86.8%. Compared with the increase of 245% at 72h, the yield of At-∆cadA::tbrA in the later stage did not increase much. From the perspective of the fermentation acid production rate of a certain fermentation period, the 72h fermentation At- The -∆cadA::tbrA transformant CT81 was optimal.

In summary, the results show that the introduction of the isomerase TbrA under the guidance of both promoters can achieve the conversion of cis-aconitic acid to trans-aconitic acid in the middle of fermentation for 72h, and the TbrA isomerase under the guidance of the P cadA promoter can achieve In the end, the production of trans-aconitic acid was greatly improved and the fermentation time was shortened.

Amino acid and nucleotide sequences of the present invention:

SEQ ID NO.1 Cisaconitic acid decarboxylase CadA

MTKQSADSNAKSGVTSEICHWASNLATDDIPSDVLERAKYLILDGIACAWVGARVPWSEKYVQATMSFEPPGACRVIGYGQKLGPVAAAMTNSAFIQATELDDYHSEAPLHSASIVLPAVFAASEVLAEQGKTISGIDVILAAIVGFESGPRIGKAIYGSDLLNNGWHCGAVYGAPAGALATGKLLGLTPDSMEDALGIACTQACGLMSAQYGGMVKRVQHGFAARNGLLGGLLAHGGYEAMKGVLERSYGGFLKMFTKGNGREPPYKEEEVVAGLGSFWHTFTIRIKLYACCGLVHGPVEAIENLQGRYPELLNRANLSNIRHVHVQLSTASNSHCGWIPEERPISSIAGQMSVAYILAVQLVDQQCLLSQFSEFDDNLERPEVWDLARKVTSSQSEEFDQDGNCLSAGRVRIEFNDGSSITESVEKPLGVKEPMPNERILHKYRTLAGSVTDESRVKEIEDLVLGLDRLTDISPLLELLNCPVKSPLV

SEQ ID NO.2

Sequence features:

Length: 357 aa

Molecular Type: Amino Acid Sequence

Original Source: Bacillus thuringiensis

Specificity name: aconitate isomerase TbrA

MKIPCFVMRGGTSKGLFFLDKHLPSNKSIRDEVILKALGAGNARGVDGMGTLDPLSNKIAIIRISTTPGIDIDYLFLQADLKRRILDDSVNCGNIIAAVAPYAVESGLIKVGSGKETITIRNLNTNVIVESTIATKNGNVVYNGDIKIDGVPGTGAPIDLNFKNSIGSVTGKLFPTGAKTDVINGINVSCVDVSVPLIIIRASELGIIGNESPDVLNANKTFLHDIDLIRKKVACLANLGDVSNKVIPKIAVISKPRESGTITSRYFIPHQCHSTHAVTGSLALSAAIKINGTTAYHVAKNNDLNKMKKLNQIIIEHPAGKIQTESVIEESSNGYVIKKSSITRTARLLFKGELIIP

SEQ ID NO.3

Sequence features:

Length: 1074 bp

Molecular Type: DNA Sequence

Original Source: Bacillus thuringiensis

Specificity name: aconitate isomerase gene tbrA

ATGAAGATCCCCTGCTTCGTCATGCGCGGCGGCACCAGCAAGGGCCTGTTCTTCCTGGACAAGCACCTGCCCTCCAACAAGTCCATCCGCGACGAGGTCATCCTGAAGGCCCTGGGCGCCGGCAACGCCCGTGGTGTCGATGGTATGGGCACCCTGGACCCCCTGAGCAACAAGATCGCCATCATCCGCATCAGCACCACCCCCGGCATCGACATCGACTACCTGTTCCTGCAAGCCGACCTGAAGCGCCGCATCCTGGACGACAGCGTCAACTGCGGCAACATCATCGCCGCCGTCGCCCCCTACGCCGTCGAATCTGGCCTGATCAAGGTCGGCAGCGGCAAGGAGACCATCACCATCCGCAACCTGAACACCAACGTCATCGTCGAGAGCACCATCGCCACCAAGAACGGCAACGTCGTCTACAACGGCGACATCAAGATCGACGGCGTCCCCGGCACCGGCGCTCCTATTGACCTGAACTTCAAGAACAGCATCGGCAGCGTCACCGGCAAGCTGTTCCCCACCGGCGCCAAGACCGACGTCATCAACGGCATCAACGTCTCCTGCGTCGACGTCAGCGTCCCCCTGATCATCATCCGCGCCTCCGAGCTGGGCATCATCGGCAACGAGAGCCCCGACGTCCTGAACGCCAACAAGACCTTCCTGCACGACATCGACCTGATCCGCAAGAAGGTCGCCTGCCTGGCCAACCTGGGCGACGTCTCCAACAAGGTCATCCCCAAGATCGCCGTCATCAGCAAGCCCCGCGAGTCCGGCACCATCACCTCCCGTTACTTCATCCCCCACCAGTGCCACTCCACCCACGCCGTCACCGGCAGCCTGGCTCTGTCCGCTGCCATCAAGATCAACGGCACCACCGCCTACCACGTCGCCAAGAACAACGACCTGAACAAGATGAAGAAGCTGAACCAGATCATCATCGAGCACCCCGCCGGCAAGATCCAGACCGAGTCCGTCATCGAGGAGTCCTCCAACG GCTACGTCATCAAGAAGAGCTCCATCACCCGCACCGCCCGCCTGCTGTTCAAGGGCGAGCTGATCATCCCCTAA

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 Sheriler Pharmaceutical Co., Ltd.

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

<130>

<160> 28

<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> 357

<212> PRT

<213> aconitate isomerase TbrA

<400> 2

Met Lys Ile Pro Cys Phe Val Met Arg Gly Gly Thr Ser Lys Gly Leu

1 5 10 15

Phe Phe Leu Asp Lys His Leu Pro Ser Asn Lys Ser Ile Arg Asp Glu

20 25 30

Val Ile Leu Lys Ala Leu Gly Ala Gly Asn Ala Arg Gly Val Asp Gly

35 40 45

Met Gly Thr Leu Asp Pro Leu Ser Asn Lys Ile Ala Ile Ile Arg Ile

50 55 60

Ser Thr Thr Pro Gly Ile Asp Ile Asp Tyr Leu Phe Leu Gln Ala Asp

65 70 75 80

Leu Lys Arg Arg Ile Leu Asp Asp Ser Val Asn Cys Gly Asn Ile Ile

85 90 95

Ala Ala Val Ala Pro Tyr Ala Val Glu Ser Gly Leu Ile Lys Val Gly

100 105 110

Ser Gly Lys Glu Thr Ile Thr Ile Arg Asn Leu Asn Thr Asn Val Ile

115 120 125

Val Glu Ser Thr Ile Ala Thr Lys Asn Gly Asn Val Val Tyr Asn Gly

130 135 140

Asp Ile Lys Ile Asp Gly Val Pro Gly Thr Gly Ala Pro Ile Asp Leu

145 150 155 160

Asn Phe Lys Asn Ser Ile Gly Ser Val Thr Gly Lys Leu Phe Pro Thr

165 170 175

Gly Ala Lys Thr Asp Val Ile Asn Gly Ile Asn Val Ser Cys Val Asp

180 185 190

Val Ser Val Pro Leu Ile Ile Ile Arg Ala Ser Glu Leu Gly Ile Ile

195 200 205

Gly Asn Glu Ser Pro Asp Val Leu Asn Ala Asn Lys Thr Phe Leu His

210 215 220

Asp Ile Asp Leu Ile Arg Lys Lys Val Ala Cys Leu Ala Asn Leu Gly

225 230 235 240

Asp Val Ser Asn Lys Val Ile Pro Lys Ile Ala Val Ile Ser Lys Pro

245 250 255

Arg Glu Ser Gly Thr Ile Thr Ser Arg Tyr Phe Ile Pro His Gln Cys

260 265 270

His Ser Thr His Ala Val Thr Gly Ser Leu Ala Leu Ser Ala Ala Ile

275 280 285

Lys Ile Asn Gly Thr Thr Ala Tyr His Val Ala Lys Asn Asn Asp Leu

290 295 300

Asn Lys Met Lys Lys Leu Asn Gln Ile Ile Ile Glu His Pro Ala Gly

305 310 315 320

Lys Ile Gln Thr Glu Ser Val Ile Glu Glu Ser Ser Asn Gly Tyr Val

325 330 335

Ile Lys Lys Ser Ser Ile Thr Arg Thr Ala Arg Leu Leu Phe Lys Gly

340 345 350

Glu Leu Ile Ile Pro

355

<210> 3

<211> 1074

<212> DNA

<213> aconitate isomerase gene tbrA

<400> 3

atgaagatcc cctgcttcgt catgcgcggc ggcaccagca agggcctgtt cttcctggac 60

aagcacctgc cctccaacaa gtccatccgc gacgaggtca tcctgaaggc cctgggcgcc 120

ggcaacgccc gtggtgtcga tggtatgggc accctggacc ccctgagcaa caagatcgcc 180

atcatccgca tcagcaccac ccccggcatc gacatcgact acctgttcct gcaagccgac 240

ctgaagcgcc gcatcctgga cgacagcgtc aactgcggca acatcatcgc cgccgtcgcc 300

ccctacgccg tcgaatctgg cctgatcaag gtcggcagcg gcaaggagac catcaccatc 360

cgcaacctga acaccaacgt catcgtcgag agcaccatcg ccaccaagaa cggcaacgtc 420

gtctacaacg gcgacatcaa gatcgacggc gtccccggca ccggcgctcc tattgacctg 480

aacttcaaga acagcatcgg cagcgtcacc ggcaagctgt tccccaccgg cgccaagacc 540

gacgtcatca acggcatcaa cgtctcctgc gtcgacgtca gcgtccccct gatcatcatc 600

cgcgcctccg agctgggcat catcggcaac gagagccccg acgtcctgaa cgccaacaag 660

accttcctgc acgacatcga cctgatccgc aagaaggtcg cctgcctggc caacctgggc 720

gacgtctcca acaaggtcat ccccaagatc gccgtcatca gcaagccccg cgagtccggc 780

accatcacct ccgttactt catcccccac cagtgccact ccacccacgc cgtcaccggc 840

agcctggctc tgtccgctgc catcaagatc aacggcacca ccgcctacca cgtcgccaag 900

aacaacgacc tgaacaagat gaagaagctg aaccagatca tcatcgagca ccccgccggc 960

aagatccaga ccgagtccgt catcgaggag tcctccaacg gctacgtcat caagaagagc 1020

tccatcaccc gcaccgcccg cctgctgttc aagggcgagc tgatcatccc ctaa 1074

<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

<210> 5

<211> 21

<212> DNA

<213> U-cadA-F

<400> 5

gcgataaatg ttgaacgagg c 21

<210> 6

<211> 46

<212> DNA

<213> U-cadA-R(tbrA)

<400> 6

gacgaagcag gggatcttca ttggtcaatt taataggaca attttc 46

<210> 7

<211> 21

<212> DNA

<213> tbrA-F

<400> 7

atgaagatcc cctgcttcgt c 21

<210> 8

<211> 43

<212> DNA

<213> tbrA-R(TtrpC)

<400> 8

cagtaacgtt aagtggatcc ttaggggatg atcagctcgc cct 43

<210> 9

<211> 20

<212> DNA

<213> TtrpC-F

<400> 9

ggatccactt aacgttactg 20

<210> 10

<211> 22

<212> DNA

<213> TtrpC-R

<400> 10

aagaaggtta cctctaaaca ag 22

<210> 11

<211> 46

<212> DNA

<213> pyrG/loxp-F(TtrpC)

<400> 11

cttgtttaga ggtaaccttc tttaagggag atggtgattg aactag 46

<210> 12

<211> 21

<212> DNA

<213> pyrGAn-R(F743)

<400> 12

gcatcaaatc gtcgtaccgc a 21

<210> 13

<211> 44

<212> DNA

<213> D-cadA-F(pyrGAn)

<400> 13

tgcggtacga cgatttgatg ctaaatggga agcgatatgg aaac 44

<210> 14

<211> 23

<212> DNA

<213> D-cadA-R

<400> 14

cattgcaggg aagtatatgc ttc 23

<210> 15

<211> 21

<212> DNA

<213> C-cadA-F

<400> 15

tgtggttcct accaaggtgg c 21

<210> 16

<211> 22

<212> DNA

<213> C-cadA-R

<400> 16

cgactatagc tggattgatc ac 22

<210> 17

<211> 26

<212> DNA

<213> U-ku80-F

<400> 17

cgcgggtttc tagaagtcac atcagc 26

<210> 18

<211> 44

<212> DNA

<213> U-ku80-R(PgpdAt)

<400> 18

gtacctggat cctcccagag tgtaaggtgg atctggagca gagg 44

<210> 19

<211> 24

<212> DNA

<213> PgpdAt-F743

<400> 19

ttacactctg ggaggatcca ggta 24

<210> 20

<211> 41

<212> DNA

<213> PgpdAt-R(tbrA)

<400> 20

gacgaagcag gggatcttca ttgtgatgat tgatgagttg t 41

<210> 21

<211> 21

<212> DNA

<213> tbrA-F

<400> 21

atgaagatcc cctgcttcgt c 21

<210> 22

<211> 43

<212> DNA

<213> tbrA-R(TtrpC)

<400> 22

cagtaacgtt aagtggatcc ttaggggatg atcagctcgc cct 43

<210> 23

<211> 20

<212> DNA

<213> TtrpC-F

<400> 23

ggatccactt aacgttactg 20

<210> 24

<211> 22

<212> DNA

<213> hph-R(-TtrpC)

<400> 24

cggtcggcat ctactctatt cc 22

<210> 25

<211> 44

<212> DNA

<213> D-ku80-F(hph-TtrpC)

<400> 25

ggaatagagt agatgccgac cgtagcccgg agttaggtag atag 44

<210> 26

<211> 20

<212> DNA

<213> D-ku80-R

<400> 26

catcaccgac cctacgctgt 20

<210> 27

<211> 21

<212> DNA

<213> C-ku80-F

<400> 27

ggtggtttct ctctatcatg g 21

<210> 28

<211> 21

<212> DNA

<213> C-ku80-R

<400> 28

gcgaaggcga aaagtagtct c 21

Claims (7)

1. a recombinant aspergillus terreus strain producing trans-aconitic acid, is characterized in that, described recombinant aspergillus terreus is the starting bacterial strain with aspergillus terreus (Aspergillus terreus), is to knock out cis-aconitic acid decarboxylase CadA in the starting bacterial strain gene, and obtained by overexpressing aconitate isomerase TbrA; the amino acid sequence of the aconitate isomerase TbrA is shown in SEQ ID NO. 2, and the gene core encoding the aconitate isomerase TbrA The nucleotide sequence is shown in SEQ ID NO. 3; the promoter used for the expression element of the aconitate isomerase TbrA is the PcadA promoter. 2. The recombinant Aspergillus terreus strain according to claim 1, wherein the starting strain is Aspergillus terreus CICC40205, Aspergillus terreus NRRL1960, Aspergillus terreus DSM23081, Aspergillus terreus TN484 or Aspergillus terreus TN484-M1. 3. The recombinant Aspergillus terreus strain according to claim 1, wherein the amino acid sequence of the aconitic acid decarboxylase CadA is as shown in SEQ ID No.1. 4. the construction method of the recombinant Aspergillus terreus strain described in any one of claim 1-3, it is characterized in that, with described Aspergillus terreus as starting bacterial strain, knock out cis-aconitic acid decarboxylase CadA gene and described aspergillus terreus The expression element of head acid isomerase TbrA was introduced into the starting strain to construct a recombinant Aspergillus terreus strain. 5. The application of the recombinant Aspergillus terreus strain described in any one of claims 1-3 in the production of trans-aconitic acid. 6. The application according to claim 5, characterized in that, the application refers to the production of trans-aconitic acid after the recombinant Aspergillus terreus strain is fermented and cultured, and the fermentation conditions are 37° C., 220rpm for 72h- 168h. 7. application according to claim 6, is characterized in that, the used culture medium formula of described 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 , and the balance was water.

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