Trichoderma reesei engineering strain for producing trans-aconitic acid as well as construction method and application of Trichoderma reesei engineering strain

Abstract

The invention discloses a Trichoderma reesei engineering strain for producing trans-aconitic acid as well as a construction method and application of the Trichoderma reesei engineering strain, and relates to the technical field of biological engineering. The construction method comprises the following steps: transforming a coding gene of mitochondrial tricarboxylic acid transport protein and a coding gene of aconitic acid isomerase into a trichoderma reesei host bacterium, and carrying out gene recombination construction to obtain the trichoderma reesei engineering strain, the amino acid sequence of the mitochondrial tricarboxylic acid transporter is as shown in SEQ ID NO. 1; the amino acid sequence of the aconitic acid isomerase is as shown in SEQ ID NO. 2 or SEQ ID NO. 3. A new production strain is provided for microbial fermentation production of trans-aconitic acid, a large amount of trans-aconitic acid can be produced through direct fermentation by using common carbon sources such as glucose or cellulose, and the highest yield can reach 26.5 g / L.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to an engineered strain of Trichoderma reesei for producing trans-aconitic acid, a construction method thereof and an application thereof. Background Art

[0002] Trans-aconitic acid (TAA) is a small C6 organic acid containing three carboxyl groups and an unsaturated double bond. It is a stereoisomer of cis-aconitic acid, an intermediate in the tricarboxylic acid cycle. Its unique chemical structure offers potential for diverse applications, including significant effectiveness in nematode control and the development of new biopesticides. Field trials have shown that 560 grams per mu of trans-aconitic acid is as effective as 180 grams per mu of the chemical pesticide thiazolyl, but at a cost less than 20% and with superior environmental friendliness.

[0003] Traditional production methods for trans-aconitic acid (such as plant extraction and chemical synthesis) suffer from low efficiency, high cost, and numerous byproducts, making large-scale production impossible. This leads to insufficient raw material supply and severely restricts the development of downstream applications. Currently, research progress has been made in the production of trans-aconitic acid using genetically engineered microorganisms. For example, the Qingdao Institute of Bioenergy and Process Technology, Chinese Academy of Sciences, in collaboration with Shandong Lukang Pharmaceutical Co., Ltd., has achieved green microbial production of trans-aconitic acid through synthetic biology strategies, increasing the trans-aconitic acid content from 57% to 90%, and achieving a shake flask fermentation yield of 20g / L.

[0004] Trichoderma reesei, a GRAS (Generally Regarded as Safe) strain, is an important industrial production strain and has been widely used in fermentation industries such as food and feed. However, since these strains cannot synthesize trans-aconitic acid, this study proposes to construct an engineered strain of T. reesei through metabolic engineering for the fermentation production of trans-aconitic acid, hoping to provide technical support for breakthroughs in microbial green manufacturing of trans-aconitic acid. Summary of the Invention

[0005] The present invention aims to provide an engineered strain of Trichoderma reesei for producing trans-aconitic acid, as well as its construction method and application, to address the aforementioned problems of the prior art. The present invention provides a novel strain for the microbial fermentation of trans-aconitic acid. This strain can directly ferment and produce large amounts of trans-aconitic acid using common carbon sources such as glucose or cellulose, with a maximum yield of up to 26.5 g / L.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for constructing an engineered strain of Trichoderma reesei that produces trans-aconitic acid, comprising the steps of transforming a gene encoding a mitochondrial tricarboxylic acid transporter and a gene encoding an aconitate isomerase into a Trichoderma reesei host bacterium, and constructing the engineered strain of Trichoderma reesei by genetic recombination;

[0008] The amino acid sequence of the mitochondrial tricarboxylic acid transporter is shown in SEQ ID NO.1;

[0009] The amino acid sequence of the aconitate isomerase is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0010] Preferably, the amino acid sequence of the aconitate isomerase is shown as SEQ ID NO.2.

[0011] Furthermore, the nucleotide sequence of the gene encoding the mitochondrial tricarboxylic acid transporter is shown in SEQ ID NO.4;

[0012] When the amino acid sequence of the aconitate isomerase is shown as SEQ ID NO.2, the nucleotide sequence of its encoding gene is shown as SEQ ID NO.5; when the amino acid sequence of the aconitate isomerase is shown as SEQ ID NO.3, the nucleotide sequence of its encoding gene is shown as SEQ ID NO.6.

[0013] Furthermore, the Trichoderma reesei host strain is Trichoderma reesei QM6a, Rut-C30 or PC-3-7.

[0014] Furthermore, the gene encoding the mitochondrial tricarboxylic acid transporter and the gene encoding the aconitate isomerase are transformed into the Trichoderma reesei host bacteria in the form of a co-expression plasmid;

[0015] The backbone plasmid of the co-expression plasmid is LML2.0a plasmid.

[0016] Furthermore, the co-expression plasmid includes a mitochondrial tricarboxylic acid transporter encoding gene expression cassette and an aconitate isomerase encoding gene expression cassette;

[0017] The mitochondrial tricarboxylic acid transporter encoding gene expression cassette comprises a first promoter, the mitochondrial tricarboxylic acid transporter encoding gene and a first terminator connected in sequence;

[0018] The aconitate isomerase encoding gene expression cassette comprises a second promoter, the aconitate isomerase encoding gene and a second terminator connected in sequence.

[0019] The present invention also provides an engineered strain of Trichoderma reesei for producing trans-aconitic acid, which is constructed according to the above construction method.

[0020] The present invention also provides the use of the above-mentioned engineered strain of Trichoderma reesei in the fermentation production of trans-aconitic acid.

[0021] The present invention also provides a method for producing trans-aconitic acid by fermentation, comprising the steps of fermenting and culturing the above-mentioned engineered strain of Trichoderma reesei to obtain the trans-aconitic acid.

[0022] Furthermore, the fermentation medium used in the fermentation culture includes calcium carbonate; and / or

[0023] The concentration of the calcium carbonate is 20-80 g / L.

[0024] The present invention discloses the following technical effects:

[0025] The present invention uses a Trichoderma reesei strain as a host strain and genetically modifies it to express a mitochondrial tricarboxylic acid transporter and aconitate isomerase. This transforms the previously incapable Trichoderma reesei strain into one that can efficiently synthesize and secrete trans-aconitate. The engineered strain obtained in the present invention can directly ferment and produce large amounts of trans-aconitate using common carbon sources such as glucose or cellulose, with a maximum yield of up to 26.5 g / L in shake flask fermentation. This invention provides a new method for the production of trans-aconitate from microorganisms and can be applied to the industrial production of trans-aconitate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 Flowchart for constructing expression plasmids in the present invention; A: single expression plasmid Ppdc-Tcbh2; B: dual expression plasmid pEno-Pdc;

[0028] Figure 2 Flow chart of the construction of co-expression plasmids in the present invention; A: intermediate plasmid pmttA-Pdc; B: co-expression plasmid pmttA-TbrA; C: co-expression plasmid pmttA-Adi1;

[0029] Figure 3 This is a statistical chart of the trans-aconitic acid production of each engineered strain when glucose is used as the carbon source. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The LML2.0a plasmid used in the following examples has been disclosed in the document “Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Scientific Reports. 2016, 6: 20761”.

[0036] The trans-aconitic acid production assay is as follows: 1 mL of fermentation broth is placed in a centrifuge tube and centrifuged at 14,000 × g for 30 minutes. The supernatant is then filtered through a 0.22 μm aqueous filter and 100 μL is transferred to a disposable plastic vial to prepare the processed sample. The processed sample is then analyzed for trans-aconitic acid content using high-performance liquid chromatography (HPLC). The column used is a Bio-Rad AmineX HPX-87H (300 mm × 7.8 mm); the mobile phase is 5 mM H₂SO₄; the flow rate is 0.5 mL / min; the column temperature is 30°C; the injection volume is 10 μL; and the detectors are differential detectors and UV detectors, with a UV detection wavelength of 210 nm.

[0037] Example 1 Obtaining the genes encoding mitochondrial tricarboxylic acid transporter and aconitate isomerase

[0038] The method for obtaining the coding genes for the mitochondrial tricarboxylic acid transporter and aconitate isomerase is to provide the amino acid sequences (SEQ ID NOs. 1-3) and entrust a conventional gene company to synthesize the corresponding nucleotide coding sequences. The synthesis rules can be based on the codon preference of the host. Due to differences in hosts, codon preferences also vary. Based on the codon preference of Trichoderma reesei, the final synthesized DNA sequences are shown in SEQ ID NOs. 4-6. Because codon preference is a range, other DNA sequences similar to the sequences shown in SEQ ID NOs. 4-6 that can encode the same amino acid sequence can also be used.

[0039] The amino acid sequence of the mitochondrial tricarboxylic acid transporter mttA from Aspergillus terreus is shown in SEQ ID NO.1; the amino acid sequence of the aconitate isomerase TbrA from Bacillus thuringiensis is shown in SEQ ID NO.2; and the amino acid sequence of the aconitate isomerase Adi1 from Ustilago maydis is shown in SEQ ID NO.3.

[0040] The nucleotide sequence of the gene encoding the mitochondrial tricarboxylic acid transporter mttA from Aspergillus terreus is shown in SEQ ID NO.4; the nucleotide sequence of the gene encoding the aconitate isomerase TbrA from Bacillus thuringiensis is shown in SEQ ID NO.5; and the nucleotide sequence of the gene encoding the aconitate isomerase Adi1 from Ustilago maydis is shown in SEQ ID NO.6.

[0041] Example 2 Construction of a mitochondrial tricarboxylic acid transporter and aconitate isomerase co-expression plasmid

[0042] 1. Construction of a single expression plasmid (Ppdc-Tcbh2)

[0043] 1) Using primers Ppdc-F and Ppdc-R, amplify the Ppdc sequence using the Trichoderma reesei genome as a template

[0044] Ppdc-F: 5'-ACTAGTGAGTCATTTATGAAAGGAGGGAGCATTCTTCGA-3' (SEQ ID NO. 7);

[0045] Ppdc-R: 5'-ATTTAAATCATGATTGTGCTGTAGCTGCGC-3' (SEQ ID NO. 8).

[0046] Amplification reaction system: 10× PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; primers (upstream and downstream primers are both 10μM) 1.5μL; genomic template (200ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0047] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 45 sec, for 30 cycles; 68°C for 5 min.

[0048] 2) Using primers Tcbh2-1 and Tcbh2-2, amplify the Tcbh2 sequence using the Trichoderma reesei genome as a template

[0049] Tcbh2-1: 5'-AATCATGATTTAAATGGCTTTCGTGACCGGGCTT-3' (SEQ ID NO. 9);

[0050] Tcbh2-2: 5'-AGTGCCAAGCTTATTTTGGGTATGGTTTCCACGTGCA-3' (SEQ ID NO. 10).

[0051] Amplification reaction system: 10× PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; primers (upstream and downstream primers are both 10μM) 1.5μL; genomic template (200ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0052] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 15 sec, for 30 cycles; 68°C for 5 min.

[0053] 3) Using the LML2.0a plasmid as a backbone, a single expression vector was constructed. The restriction endonuclease SwaI on the existing plasmid LML2.0a was single-digested. The Ppdc fragment in step 1) and the Tcbh2 fragment in step 2) were homologously recombined with the single-digested LML2.0a using the Vazyme One Step Clone Kit to construct a Ppdc-Tcbh2 single expression plasmid ( Figure 1 Middle A).

[0054] The nucleotide sequence of the Ppdc fragment is shown in SEQ ID NO.11, and the nucleotide sequence of the Tcbh2 fragment is shown in SEQ ID NO.12.

[0055] 2. Construction of dual expression plasmid (pEno-Pdc)

[0056] 1) Using primers Peno-F and Peno-R, the Peno sequence was amplified using the Trichoderma reesei genome as a template.

[0057] Peno-F: 5'-ATTACGAATTCTTAATGCCAACTCCTTGACGCCAA-3' (SEQ ID NO. 13);

[0058] Peno-R: 5'-TTAATTAAACATTTTGAAGCTATTTCAGGT-3' (SEQ ID NO. 14).

[0059] Amplification reaction system: 10× PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; primers (upstream and downstream primers are both 10μM) 1.5μL; genomic template (200ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0060] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 45 sec, for 30 cycles; 68°C for 5 min.

[0061] 2) Using primers Tcbh1-1 and Tcbh1-2, the Tcbh1 sequence was amplified using the Trichoderma reesei genome as a template.

[0062] Tcbh1-1: 5'-TCAAAATGTTAATTAAGCTCCCGTGGCGAAAGCC-3' (SEQ ID NO. 15);

[0063] Tcbh1-2: 5'-TTATACGAAGTTATTCTAGAATTTCCACTGTTGCTATTATGCTGT-3' (SEQ ID NO. 16).

[0064] Amplification reaction system: 10× PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; primers (upstream and downstream primers are both 10μM) 1.5μL; genomic template (200ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0065] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 15 sec, for 30 cycles; 68°C for 5 min.

[0066] 3) Using the single expression plasmid Ppdc-Tcbh2 in step 1 as the backbone to construct a co-expression vector, the restriction endonuclease PacI / XbaI on the existing plasmid Ppdc-Tcbh2 was double-digested, and the amplified Peno fragment and Tcbh1 fragment were homologously recombined with the double-digested Ppdc-Tcbh2 using the Vazyme One Step Clone Kit to construct the pEno-Pdc dual expression plasmid ( Figure 1 Middle B).

[0067] The nucleotide sequence of the Peno fragment is shown in SEQ ID NO.17; the nucleotide sequence of the Tcbh1 fragment is shown in SEQ ID NO.18.

[0068] 3. Construction of co-expression plasmid

[0069] 1) Using primers mttA-F1 and mttA-F2, the nucleotide fragment mttA-DNA of the mitochondrial tricarboxylic acid transporter was amplified using SEQ ID NO. 4 as a template.

[0070] mttA-F1: 5'-TGAAATAGCTTCAAAATGGACTCGAAGATCCAGACGAACGTCC-3' (SEQ ID NO. 19);

[0071] mttA-F2: 5'-TTTCGCCACGGAGCTTTAGTTGGGCTGGGTCAGGAACTTGTAGAC-3' (SEQ ID NO. 20).

[0072] Amplification reaction system: 10× PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; 10μM primers HEWL-F1 / HEWL-F2 1.5μL each; genomic template (200ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0073] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 30 sec, for 30 cycles; 68°C for 5 min.

[0074] The expression plasmid was constructed using the Trichoderma reesei dual expression plasmid pEno-Pdc as the backbone. The restriction endonuclease PacI on the existing plasmid pEno-Pdc was single-digested and seamlessly connected using the Vazyme One Step Clone Kit. The nucleotide fragment mttA-DNA of the mitochondrial tricarboxylic acid transporter amplified above was connected to form the intermediate plasmid pmttA-Pdc ( Figure 2 Middle A).

[0075] 2) Using primers TbrA-F1 and TbrA-F2, the nucleotide fragment TbrA-DNA of aconitate isomerase from Bacillus thuringiensis was amplified using SEQ ID NO. 5 as a template.

[0076] TbrA-F1: 5'-AGCTACAGCACAATCATGAAGATCCCCTGCTTCGTCA-3' (SEQ ID NO. 21); TbrA-F2: 5'-CCGGTCACGAAAGCCTTAGGGGATGATCAGCTCGCC-3' (SEQ ID NO. 22).

[0077] Amplification reaction system: 10× PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; 10μM primers HEWL-F1 / HEWL-F2 1.5μL each; genomic template (200ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0078] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 30 sec, for 30 cycles; 68°C for 5 min.

[0079] The intermediate plasmid pmttA-Pdc was used as the backbone to construct an expression plasmid. The restriction endonuclease SwaI on the existing plasmid pmttA-Pdc was digested and seamlessly connected using the Vazyme One Step Clone Kit. The nucleotide fragment TbrA-DNA encoding the aconitate isomerase gene amplified from Bacillus thuringiensis was connected to form the co-expression plasmid pmttA-TbrA ( Figure 2 Middle B).

[0080] 3) Using primers Adi1-F1 and Adi1-F2, the nucleotide fragment Adi1-DNA of aconitate isomerase from Ustilago maydis was amplified using SEQ ID NO. 6 as a template.

[0081] Adi1-F1: 5'-AGCTACAGCACAATCATGCTGCACCCCATCGACA-3' (SEQ ID NO. 23);

[0082] Adi1-F2: 5'-CCGGTCACGAAAGCCTTAGCTGAGGCTGCGGTCC-3' (SEQ ID NO. 24).

[0083] Amplification reaction system: 10× PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; 10μM primers HEWL-F1 / HEWL-F2 1.5μL each; genomic template (200ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0084] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 40 sec, for 30 cycles; 68°C for 5 min.

[0085] The intermediate plasmid pmttA-Pdc was used as the backbone to construct an expression plasmid. The restriction endonuclease SwaI on the existing plasmid pmttA-Pdc was digested and seamlessly connected using the Vazyme One Step Clone Kit. The nucleotide fragment Adi1-DNA of the aconitate isomerase encoding gene amplified from Ustilago maydis was connected to form the co-expression plasmid pmttA-Adi1( Figure 2 Middle C).

[0086] Example 3 Construction of Trichoderma reesei engineered strains

[0087] The co-expression plasmids pmttA-TbrA and pmttA-Adi1 prepared in Example 2 were introduced into Trichoderma reesei, respectively, to obtain two engineered strains of Trichoderma reesei. The present invention refers to the introduction of plasmids into Trichoderma reesei by Agrobacterium-mediated transformation of Trichoderma reesei and clonal screening, randomly integrating the heterologous gene expression cassette into the Trichoderma reesei genome to form engineered and optimized strains, thereby achieving heterologous gene expression. The transformation method of the present invention is Agrobacterium tumefaciens-mediated conjugative transfer.

[0088] 1) The co-expression plasmids pmttA-TbrA and pmttA-Adi1 were electroporated into Agrobacterium, respectively. The plasmid-containing Agrobacterium was then co-cultured with the Trichoderma reesei host strain QM6a (ATCC 13631) on IM plates (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105(3): 259-264) for Agrobacterium tumefaciens-mediated conjugative transfer. After two days of co-cultivation, the transformants were transferred to PDA plates containing cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) for screening until the transformants grew hyphae and spores. The transformants were then screened and verified to obtain the Trichoderma reesei genetically engineered strains QM6a::mttA-TbrA and QM6a::mttA-Adi1.

[0089] 2) The co-expression plasmid pmttA-TbrA was electroporated into Agrobacterium, and then the Agrobacterium containing the plasmid was co-cultured with the Trichoderma reesei host strains Rut-C30 (ATCC 56765) and PC-3-7 (ATCC 66589) on IM plates (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105(3):259-264), respectively, for Agrobacterium tumefaciens-mediated conjugative transfer. After two days of co-cultivation, the transformants were transferred to PDA plates containing cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) for screening until the transformants grew hyphae and spores, and then screened and verified to obtain the Trichoderma reesei engineered strains RutC30::mttA-TbrA and PC37::mttA-TbrA.

[0090] Example 4

[0091] The host strains of Trichoderma reesei QM6a, Rut-C30 and PC-3-7, as well as the four engineered strains QM6a::mttA-TbrA, QM6a::mttA-Adi1, RutC30::mttA-TbrA and PC37::mttA-TbrA constructed in Example 3 were inoculated into glucose carbon source fermentation medium (see Table 1), respectively. The medium loading volume in the 250 mL Erlenmeyer flask was 50 mL, and the inoculum size was 10 8 The culture medium was incubated at 28°C and 220 rpm, and samples were taken on the 8th day to determine the trans-aconitic acid content.

[0092] Table 1 Glucose carbon source fermentation medium

[0093]

[0094] Note: a Trace element formula (1000 mL): 1.6 g MnSO4·4H2O, 5 g FeSO4·7H2O, 2 g CoCl2·6H2O, 1.4 g ZnSO4·7H2O, dissolved in water and diluted to 1000 mL.

[0095] The fermentation results showed that the host strains QM6a, Rut-C30 and PC-3-7 of Trichoderma reesei could not produce trans-aconitic acid ( Figure 3 ). The genes encoding the mitochondrial tricarboxylic acid transporter and aconitate isomerase were introduced into Trichoderma reesei and expressed in the engineered strains. Trans-aconitate was significantly detected in the fermentation broth. Among them, the engineered strains with higher yields were QM6a::mttA-TbrA, RutC30::mttA-TbrA, and PC37::mttA-TbrA, indicating that the co-expression plasmid pmttA-TbrA was superior to the co-expression plasmid pmttA-Adi1 in producing trans-aconitate during fermentation. Figure 3 ).

[0096] Experimental Example 1

[0097] The calcium carbonate concentration in the glucose carbon source fermentation medium shown in Table 1 was adjusted to 0-80 g / L, while other components remained unchanged. Engineered strains QM6a::mttA-TbrA, RutC30::mttA-TbrA, and PC37::mttA-TbrA were fermented using glucose carbon source fermentation medium with varying calcium carbonate concentrations, using the same method as in Example 4. Samples were taken on day 8 to determine the trans-aconitic acid content. The results are shown in Table 2.

[0098] Fermentation results showed that calcium carbonate concentration significantly affected trans-aconitic acid production. Without calcium carbonate, trans-aconitic acid production was very low. However, at a calcium carbonate concentration of 60 g / L, trans-aconitic acid production reached its highest level, reaching 14.8 g / L (Table 2).

[0099] Table 2 Effect of calcium carbonate concentration on trans-aconitic acid production

[0100]

[0101]

[0102] Note: a The fermentation was carried out in shake flasks with 100 g / L glucose as the carbon source.

[0103] Example 5 Construction of optimized strains of Trichoderma reesei

[0104] 1. The hygromycin B selection marker of the engineered strain is missing;

[0105] Engineered strains QM6a::mttA-TbrA, RutC30::mttA-TbrA, and PC37::mttA-TbrA were selected for marker deletion screening. This marker deletion screening method was performed according to the literature protocol (Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Scientific Reports. 2016, 6:20761). In summary, the engineered strain was inoculated into xylose PDA liquid medium (containing 20 g / L xylose, 100 g / L potato water, and no agar) to induce resistance gene deletion. After incubation at 28°C and 200 rpm in a shaker for 48 hours, a small amount of mycelium was spotted onto xylose PDA solid medium plates (containing 20 g / L xylose, 100 g / L potato water, and 20 g / L agar). The plates were incubated at 28°C for 5-7 days, and spores were collected. The spores were then diluted according to a concentration gradient and plated onto xylose PDA solid medium plates. The plates were incubated at 28°C for 48 hours, and the appropriate spore concentration was selected to allow the T. reesei strain to grow as a single colony from the xylose PDA solid medium plates. Pick an agar block with a monoclonal strain of Trichoderma reesei and place it on a glucose PDA solid medium (containing 20g / L glucose, 100g / L potato water, 20g / L agar) plate, and culture it at 28°C for 24h to allow the fungal hyphae in the agar block to spread to the plate. Then pick this agar block to a resistance PDA well plate (containing 20g / L glucose, 100g / L potato water, 20g / L agar, 150μg / mL hygromycin B and 150μg / mL cephalosporin), and culture it at 28°C for 48h to verify whether the resistance loss is successful: if it cannot grow in the resistance PDA well plate, it means that the resistance has been lost. Pick the PDA plate after the agar block and continue to culture it at 28°C for 4-6 days to allow the spread hyphae to grow fully and produce spores, and collect the spores of the resistance-deficient strain. The strains are the engineered strains QM6a::mttA-TbrA, RutC30::mttA-TbrA and PC37::mttA-TbrA without resistance markers.

[0106] Elimination of the hygromycin B resistance marker did not affect trans-aconitic acid production. After elimination of the hygromycin B resistance marker, the next round of gene introduction can be performed.

[0107] 2. Construction of optimized strains of Trichoderma reesei;

[0108] The co-expression plasmid pmttA-TbrA was introduced into the engineered strain of Trichoderma reesei multiple times to obtain an optimized strain of Trichoderma reesei with high production of trans-aconitic acid.

[0109] 1) The electroporated Agrobacterium containing the pmttA-TbrA plasmid was co-cultured with the non-resistance marker engineered Trichoderma reesei QM6a::mttA-TbrA, RutC30::mttA-TbrA and PC37::mttA-TbrA on IM plates for Agrobacterium tumefaciens-mediated conjugative transfer. After two days of co-cultivation, the transformants were transferred to PDA plates containing cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) for screening until the transformants grew hyphae and spores. They were then screened and verified, and the hygromycin B selection marker was deleted to obtain the optimized Trichoderma reesei strains QM6a::mttA-TbrA+, RutC30::mttA-TbrA+ and PC37::mttA-TbrA+.

[0110] 2) The Agrobacterium containing the pmttA-TbrA plasmid was again co-cultured with the optimized Trichoderma reesei strains QM6a::mttA-TbrA+, RutC30::mttA-TbrA+, and PC37::mttA-TbrA+ on an IM plate for Agrobacterium tumefaciens-mediated conjugative transfer; then, screening and verification were performed using the same protocol as in step 1), and hygromycin B was used to screen for marker deletion, to obtain the optimized Trichoderma reesei strains QM6a::mttA-TbrA++, RutC30::mttA-TbrA++, and PC37::mttA-TbrA++.

[0111] Example 6

[0112] The optimized Trichoderma reesei strains QM6a::mttA-TbrA+, RutC30::mttA-TbrA+, PC37::mttA-TbrA+, QM6a::mttA-TbrA++, RutC30::mttA-TbrA++ and PC37::mttA-TbrA++ constructed in Example 5 were inoculated into glucose carbon source fermentation medium (based on Table 1, the calcium carbonate concentration was adjusted to 60 g / L) and fermented using the same method as in Example 4. Samples were taken on the 8th day to determine the trans-aconitic acid content. The results are shown in Table 4.

[0113] The optimized Trichoderma reesei strains QM6a::mttA-TbrA+, RutC30::mttA-TbrA+, PC37::mttA-TbrA+, QM6a::mttA-TbrA++, RutC30::mttA-TbrA++ and PC37::mttA-TbrA++ constructed in Example 5 were inoculated into cellulose carbon source fermentation medium (Table 3) and fermented using the same method as in Example 4. Samples were taken on the 12th day to determine the trans-aconitic acid content. The results are shown in Table 4.

[0114] Table 3 Cellulose carbon source fermentation medium

[0115]

[0116] Note: a Trace element formula (1000mL): Dissolve 1.6g MnSO4·4H2O, 5g FeSO4·7H2O, 2g CoCl2·6H2O, and 1.4g ZnSO4·7H2O in water and adjust the volume to 1000mL. b. Preparation of ball-milled cellulose suspension: In a 500mL Erlenmeyer flask, add 20g microcrystalline cellulose, 190mL deionized water, and glass beads with a diameter of 0.5-1cm (ideally, flattening the bottom). Sterilize (121°C, 20min). Secure the flask with a rubber bag to prevent evaporation. Place in a shaker at 200rpm for 10 days, remove, and sterilize again (108°C, 20min). This creates a stable ball-milled cellulose suspension with a concentration of 10%.

[0117] Fermentation results showed that the optimized strains of T. reesei produced significantly higher trans-aconitic acid yields (Table 4). Furthermore, trans-aconitic acid production gradually increased with increasing incorporation of the mitochondrial tricarboxylic acid transporter and aconitate isomerase (Table 4). The optimized strain QM6a::mttA-TbrA++ exhibited the highest yield, reaching 26.5 g / L using glucose as the carbon source (Table 4).

[0118] The optimized strains RutC30::mttA-TbrA++ and PC37::mttA-TbrA++ could efficiently synthesize trans-aconitic acid using cellulose as a carbon source, with the highest yield reaching 16.5 g / L (Table 4).

[0119] Table 4 Trans-aconitic acid production of optimized Trichoderma reesei strains

[0120]

[0121] In summary, the present invention successfully produced trans-aconitic acid by fermentation through genetic modification of a Trichoderma reesei host strain. This research demonstrates for the first time that, although a Trichoderma reesei host strain cannot synthesize trans-aconitic acid, it can, through genetic engineering, ferment and produce trans-aconitic acid using glucose or cellulose as a carbon source. Furthermore, experiments have confirmed the potential of optimized Trichoderma reesei strains to produce trans-aconitic acid using cellulose fermentation, providing an excellent strain for the industrial production of trans-aconitic acid.

[0122] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for constructing an engineered strain of Trichoderma reesei for producing trans-aconitic acid, characterized in that: The method comprises the steps of transforming the coding gene of the mitochondrial tricarboxylic acid transporter and the coding gene of aconitate isomerase into the host bacteria of Trichoderma reesei, and constructing the engineered strain of Trichoderma reesei by gene recombination; The amino acid sequence of the mitochondrial tricarboxylic acid transporter is shown in SEQ ID NO.1; The amino acid sequence of the aconitate isomerase is shown in SEQ ID NO.2 or SEQ ID NO.

3.

2. The construction method according to claim 1, characterized in that The amino acid sequence of the aconitate isomerase is shown in SEQ ID NO.

2.

3. The construction method according to claim 1, characterized in that The nucleotide sequence of the gene encoding the mitochondrial tricarboxylic acid transporter is shown in SEQ ID NO.4; When the amino acid sequence of the aconitate isomerase is shown as SEQ ID NO.2, the nucleotide sequence of its encoding gene is shown as SEQ ID NO.5; when the amino acid sequence of the aconitate isomerase is shown as SEQ ID NO.3, the nucleotide sequence of its encoding gene is shown as SEQ ID NO.

6.

4. The construction method according to claim 1, characterized in that The Trichoderma reesei host strain is Trichoderma reesei QM6a, Rut-C30 or PC-3-7.

5. The construction method according to claim 1, characterized in that The coding gene of the mitochondrial tricarboxylic acid transporter and the coding gene of the aconitate isomerase are transformed into the Trichoderma reesei host bacteria in the form of a co-expression plasmid; The backbone plasmid of the co-expression plasmid is LML2.0a plasmid.

6. The construction method according to claim 5, characterized in that: The co-expression plasmid includes a mitochondrial tricarboxylic acid transporter encoding gene expression cassette and an aconitate isomerase encoding gene expression cassette; The mitochondrial tricarboxylic acid transporter encoding gene expression cassette comprises a first promoter, the mitochondrial tricarboxylic acid transporter encoding gene and a first terminator connected in sequence; The aconitate isomerase encoding gene expression cassette comprises a second promoter, the aconitate isomerase encoding gene and a second terminator connected in sequence.

7. An engineered strain of Trichoderma reesei producing trans-aconitic acid constructed according to the construction method according to any one of claims 1 to 6.

8. Use of the engineered strain of Trichoderma reesei according to claim 7 in the fermentative production of trans-aconitic acid.

9. A method for producing trans-aconitic acid by fermentation, characterized in that: The method comprises the step of fermenting and culturing the engineered strain of Trichoderma reesei according to claim 7 to obtain the trans-aconitic acid.

10. The method according to claim 9, characterized in that The fermentation medium used in the fermentation culture includes calcium carbonate; and / or The concentration of the calcium carbonate is 20-80 g / L.

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