CN116004677B - Construction method and application of Trichoderma reesei engineering bacteria for producing itaconic acid - Google Patents

Abstract

The invention discloses a construction method and application of an engineering strain of Trichoderma reesei that produces itaconic acid, and belongs to the field of bioengineering. The above-mentioned construction method includes the following steps: (1) Using the filamentous fungus Trichoderma reesei that does not produce itaconic acid or a derivative derived from Trichoderma reesei as a starting strain, introduce encoding cis-aconitic acid decarboxylase and mitochondrial tricarboxylic acid The exogenous gene of the acid transporter was used to obtain strain I; (2) the endogenous membrane transporter of strain I was overexpressed to obtain the itaconic acid-producing Trichoderma reesei engineering strain. The present invention obtains engineering bacteria of Trichoderma reesei through genetic modification. This engineering bacteria can use common substances such as glucose, glycerol, liquefied starch, cellulose, and cellulose hydrolyzates as carbon sources to directly ferment and produce a large amount of itaconic acid. The maximum output can reach 60g/L. It can be seen that the present invention provides a new method for the production of itaconic acid derived from microorganisms, and can be applied to the industrial production of itaconic acid.

Description

Construction method and application of an engineering strain of Trichoderma reesei that produces itaconic acid

Technical field

The invention relates to the field of bioengineering, and in particular to a construction method and application of an engineering strain of Trichoderma reesei that produces itaconic acid.

Background technique

Itaconic acid, also known as methylenesuccinic acid or methylenesuccinic acid, is an important unsaturated dicarboxylic acid that is widely used in the production of high-value chemicals such as latex, plastics, and resins. Itaconic acid has antibacterial, anti-inflammatory, anti-tumor, anti-viral and other properties, and has good application prospects in medicine and animal husbandry. In addition, itaconic acid is also an intermediate in the production of potential biofuel 3-methyltetrahydrofuran, which has great potential to replace petroleum-based methacrylic acid and acrylic acid.

Industrial production of itaconic acid is achieved through microbial fermentation, and the main strain of industrial production is Aspergillus terreus. In the fermentation tank, the yield of itaconic acid fermented by Aspergillus terreus has reached 130-160g/L. Ustilago maysa is a basidiomycete fungus that also has the ability to produce itaconic acid.

Advantages of Trichoderma reesei: As an important industrial production strain, Trichoderma reesei is a GRAS (Generally Regarded as Safe) strain, which is very suitable for industrial scale-up production and has been widely used in fermentation industries such as food and feed. However, since the Trichoderma reesei strain cannot produce itaconic acid, the present invention uses genetic modification technology to construct an engineering strain of Trichoderma reesei for the fermentation production of itaconic acid.

Contents of the invention

The purpose of the present invention is to provide a construction method and application of a Trichoderma reesei engineering bacterium that produces itaconic acid, so as to solve the problems existing in the above-mentioned prior art. The Trichoderma reesei engineering bacterium can produce a large amount of itaconic acid, Provides a new approach for microbially derived itaconic acid.

In order to achieve the above objects, the present invention provides the following solutions:

The invention provides a method for constructing engineering bacteria of Trichoderma reesei, which includes the following steps:

(1) Using the non-itaconic acid-producing filamentous fungus Trichoderma reesei or its derivatives derived from Trichoderma reesei as the starting strain, introduce foreign genes encoding cis-aconitic acid decarboxylase and mitochondrial tricarboxylic acid transporter , obtain strain I;

(2) Overexpress the endogenous membrane transport protein of the strain I to obtain an itaconic acid-producing Trichoderma reesei engineering strain.

Preferably, the starting strains include Trichoderma reesei QM6a, QM9414, Rut-C30, RL-P37, NG14 and PC-3-7.

Preferably, the mitochondrial tricarboxylic acid transporter and cis-aconitate decarboxylase are derived from a protein-coding gene annotated by Aspergillus terreus that has the function of expressing the mitochondrial tricarboxylic acid transporter and cis-aconitate decarboxylase.

The amino acid sequence of the cis-aconitate decarboxylase is shown in SEQ ID NO: 1, and the gene sequence encoding the cis-aconitate decarboxylase is shown in SEQ ID NO.2; the mitochondrial tricarboxylic acid transporter The amino acid sequence of the protein is shown in SEQ ID NO.5, and the gene sequence encoding the mitochondrial tricarboxylic acid transporter is shown in SEQ ID NO.6.

Preferably, the endogenous membrane transport protein is an mfs superfamily transport protein; the genes encoding the endogenous membrane transport proteins include mfs1, mfs2, mfs3, mfs4, and mfs5, and their nucleotide sequences are as shown in SEQ ID NO. 10. Shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14.

The invention also provides an engineering strain of Trichoderma reesei, which is constructed by the construction method.

The present invention also provides a method for producing itaconic acid, which utilizes the Trichoderma reesei engineering bacteria to ferment, collects the fermentation liquid, and obtains itaconic acid.

Preferably, the fermentation conditions are: the inoculum amount is 10 ⁸ spores/50 mL liquid culture medium, the fermentation temperature is 28-30°C, and the rotation speed is 200 rpm.

Preferably, the liquid culture medium includes the following concentration components: carbon source 50-100g/L, peptone 1-6g/L, KH ₂ PO ₄ 0.15g/L, K ₂ HPO ₄ 0.15g/L, CaCl ₂ ·2H ₂ O 0.10g/L, MgSO ₄ ·7H ₂ O 0.10g/L, calcium carbonate 20-40g/L, NaCl 0.05g/L and 1mL/L trace element solution.

Preferably, the carbon source includes any one of glucose, glycerol, liquefied starch, cellulose, and cellulose hydrolyzate;

The trace elements include the following weight components: 1.6g MnSO ₄ ·4H ₂ O, 5g FeSO ₄ ·7H ₂ O, 2g CoCl ₂ ·6H ₂ O, 1.4g ZnSO ₄ ·7H ₂ O, dissolved in water and diluted to volume 1L.

The present invention also provides the construction method, or the application of the Trichoderma reesei engineering bacteria in the production of itaconic acid.

The invention discloses the following technical effects:

The present invention uses Trichoderma reesei as the starting strain and introduces exogenous genes through genetic modification, so that the modified strain can express mitochondrial tricarboxylic acid transporter and cis-aconitate decarboxylase, so that bacteria that originally did not have the ability to produce itaconic acid can Trichoderma reesei has acquired the ability to synthesize itaconic acid; further, by overexpressing the endogenous membrane transporter of Trichoderma reesei, the production of itaconic acid is increased, and Trichoderma reesei is transformed to be able to efficiently synthesize secreted itaconic acid. Engineered strains of acid.

Experiments have proven that the engineering strain of Trichoderma reesei obtained by the present invention can directly ferment and produce a large amount of itaconic acid using common substances such as glucose, glycerol, liquefied starch, cellulose, cellulose hydrolyzate, etc. as carbon sources, and its maximum shake flask output can Up to 60g/L. It can be seen that the present invention provides a new method for the production of itaconic acid derived from microorganisms, and can be applied to the industrial production of itaconic acid.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the drawings of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 1 is a flow chart of expression plasmid construction in the present invention; A: mttA-cad1 dual-gene expression vector; B: different strains and engineering strains constructed based on genetic modification to ferment glucose and produce itaconic acid;

Figure 2 is a flow chart for expression plasmid construction in the present invention; A: mfs expression vector; B: engineering strain TrIA02, etc., fermenting glucose to produce itaconic acid;

Figure 3 shows the itaconic acid production of the genetically engineered strain TrIA02 when glycerol, liquefied starch, cellulose, and cellulose hydrolyzate are used as carbon sources in the present invention.

Detailed ways

Various exemplary embodiments of the invention will now be described in detail. This detailed description should not be construed as limitations of the invention, but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It should be understood that the terms used in the present invention are only used to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that every intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or value intermediate within a stated range and any other stated value or value intermediate within a stated range is also included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents relate. In the event of conflict with any incorporated document, the contents of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to the skilled person from the description of the invention. The specification and examples are intended to be illustrative only.

The words "includes", "includes", "has", "contains", etc. used in this article are all open terms, which mean including but not limited to.

Strains involved in the following examples: Trichoderma reesei strain QM6a (purchased from American Type Culture Collection ATCC 13631), QM9414 (purchased from American Type Culture Collection ATCC 26921), Rut-C30 (purchased from American Type Culture Collection ATCC 26921) ATCC 56765), RL-P37 (NRRL 15709), NG14 (purchased from American Type Culture Collection ATCC 56767), PC-3-7 (purchased from American Type Culture Collection) Collection library ATCC66589).

Example 1 Fermentation production of itaconic acid using genetically engineered bacteria

1. Preparation of ball-milled cellulose suspension

In a 500mL Erlenmeyer flask, add 20g corn straw powder (can pass through a 50-mesh sieve), 200mL deionized water, add glass beads with a diameter of 0.5-1cm (it is appropriate to flatten the bottom), and sterilize (121°C, 20min ). Tie the mouth of the bottle tightly with a rubber bag to prevent water from evaporating, then place it in a shaker, shake at 200 rpm for 10-15 days (preferably 12 days), take it out, and sterilize again (115°C, 20 min). A 10% ball-milled cellulose suspension was prepared (calculated based on the initial corn straw solid dry matter dosage).

2. Prepare saccharified starch solution

Dissolve 0.5g calcium chloride in 1kg hot water at 60°C, then add 1kg corn starch, and then add 0.5g high-temperature ɑ-amylase (commercial enzyme, Ningxia Xiasheng Industrial Group Co., Ltd.) to make a batter, and The pH of this batter is adjusted to 6.0. Then keep it at 97-98°C for 1.5h, then cool to 60-62°C (preferably 60°C), adjust the pH to 4.0, add 0.8mL of glucoamylase (commercial enzyme, Ningxia Xiasheng Industrial Group Co., Ltd.), Keep at 60-62°C for 27-28h ((preferably 60°C), 28h). Add distilled water to adjust the volume to 2.5L, and then sterilize with wet heat at 115°C for 30 minutes to obtain a saccharified starch solution with a sugar content of approximately 400g/L.

3. Preparation of cellulose hydrolyzate

Alkali-pretreated dry corn stover contains 62.6% cellulose, 21.4% hemicellulose and 8.2% lignin. The cellulose hydrolysis experiment can be carried out in a shake flask, using pretreated corn straw as the substrate, and the loading capacity of the substrate is 15% (150g straw dry matter/total reaction volume of 1L). Two enzymes are added to hydrolyze cellulose: cellulase and beta-glucosidase. The loading capacity of cellulase (commercial enzyme, Ningxia Xiasheng Industrial Group Co., Ltd.) is 20FPU/g dry biomass; the loading capacity of β-glucosidase (commercial enzyme, Ningxia Xiasheng Industrial Group Co., Ltd.) is 40CBU/g Dry biomass. The reaction shake flask was placed at 50°C (100 rpm), pH 5.0, and reacted for 96 hours. After the reaction, insoluble matter was removed by centrifugation, the supernatant hydrolyzate was diluted to 1.5L, and then sterilized by moist heat at 115°C for 30 minutes. A 10% cellulose hydrolyzate was prepared (calculated based on the initial alkali pretreated dry corn straw solid matter dosage).

4. Fermentation of genetically engineered bacteria to produce itaconic acid

Genetically engineered bacteria were inoculated into 50 mL of culture medium containing glucose, glycerol, liquefied starch, cellulose, and cellulose hydrolyzate as carbon sources in a 250 mL Erlenmeyer flask (formula: carbon source 50-100 g/L, peptone 6 g/L , KH ₂ PO ₄ 0.15g/L, K ₂ HPO ₄ 0.15g/L, CaCl ₂ ·2H ₂ O 0.10g/L, MgSO ₄ ·7H ₂ O 0.10g/L, calcium carbonate 20-40g/L, NaCl0 .05g/L, 1mL/L trace element liquid. Trace element liquid formula (1000mL: 1.6g MnSO ₄ ·4H ₂ O, 5g FeSO ₄ ·7H ₂ O, 2g CoCl ₂ ·6H ₂ O, 1.4g ZnSO ₄ ·7H ₂ O, dissolved in water, diluted to 1L), the inoculation amount is 10 ⁸ spores/50mL culture medium, culture at 28°C, 220rpm, and sample itaconic acid content on the eighth day. When glucose and liquefied starch are used as carbon sources , the final carbon source concentration is 100g/L (calculated based on glucose molecule content), and the calcium carbonate concentration is 40g/L; when using glycerol as the carbon source, the final carbon source concentration is 80g/L, and the calcium carbonate concentration is 40g/L; Cellulose and cellulose hydrolyzate are used as carbon sources. The final carbon source concentration is 50g/L (calculated based on the initial corn straw solid dry matter input), and the calcium carbonate concentration is 20g/L.

Embodiment 2 Determination method of itaconic acid content

Take the fermentation broth fermented according to Example 1 in a centrifuge tube, add 1 times the volume of 2mol/LH ₂ SO ₄ , put it into a water bath shaker at 80°C and 100rpm and shake it for 30 minutes. After the calcium carbonate in the fermentation broth is completely dissolved, After mixing the fermentation broth and the water beads on the tube wall, take 1 mL of the liquid into a 1.5 mL centrifuge tube, centrifuge at 14,000 × g for 30 min, absorb the supernatant, and measure the itaconic acid content with high-performance liquid chromatography (HPLC).

Determination of itaconic acid content by high performance liquid chromatography (HPLC): mobile phase: 5mM H ₂ SO ₄ ; flow rate: 0.5mL/min; column temperature: 30°C; detector: UV detector; wavelength: 210nm; column: AmineX HPX -87X, (300mm×7.8mm).

Example 3 Simultaneous expression of mitochondrial tricarboxylic acid transporter encoding gene mttA and cis-aconitate decarboxylase encoding gene cad1 derived from Aspergillus terreus in Trichoderma reesei

1. Construction of cad1 single gene expression vector (pOEcad1)

1) Use primers Ppdc-F and Ppdc-R to amplify the Ppdc sequence using the Trichoderma reesei genome as a template.

Ppdc-F: 5’-ACTAGTGAGTCATTTATGAAAGGAGGGAGCATTCTTCGA-3’;

Ppdc-R: 5’-CATGATTGTGCTGTAGCTGCGC-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; primer (10μM each) 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL) 1μL.

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

2) Use primers cad1-1 and cad1-2 to amplify the cad1 sequence using the codon-optimized cad1 plasmid as a template.

cad1-1: 5’-AGCTACAGCACAATCATGACGAAGCAGAGCGCCG-3’;

cad1-2: 5’-CCGGTCACGAAAGCCTCAGACGAGGGGGCTCTTGACG-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; primers (10μM each) 1.5μL; synthetic plasmid template (10ng) 1μL; KOD-Plus-Neo (1U/μL )1μL.

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

3) Use primers Tcbh2-1 and Tcbh2-2 to amplify the Tcbh2 sequence using the Trichoderma reesei genome as a template;

Tcbh2-1: 5’-GGCTTTCGTGACCGGGCTT-3’;

Tcbh2-2: 5’-AGTGCCAAGCTTATTTTGGGTATGGTTTCCACGTGCA-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; primer (10μM each) 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL) 1μL.

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

4) Use LML2.0a (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) as the skeleton to construct an expression vector. The restriction endonuclease SwaI on the existing plasmid LML2.0a was digested with a single enzyme, and the Vazyme One StepClone Kit was used to perform homologous recombination to construct the Ppdc-cad1-Tcbh2 expression cassette and obtain the cad1 single gene expression vector pOEcad1 (Figure 1A).

Among them, the amino acid sequence of cad1 is shown in SEQ ID NO.1, the nucleotide sequence of cad1 is shown in SEQ ID NO.2, the nucleotide sequence of Ppdc is shown in SEQ ID NO.3, and the nucleotide sequence of Tcbh2 As shown in SEQ IDNO.4.

2. Construction of mttA-cad1 dual gene expression vector (pOEmttA-cad1).

1) Use primers Peno-F and Peno-R to amplify the Peno sequence using the Trichoderma reesei genome as a template.

Peno-F: 5’-GATTACGAATTCTTAATTAATGCCAACTCCTTGACGCCAA-3’;

Peno-R: 5’-CATTTTTGAAGCTATTTCAGGT-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; primer (10μM each) 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL) 1μL.

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

2) Use primers mttA-1 and mttA-2 to amplify the mttA sequence using the codon-optimized mttA plasmid as a template. mttA-1: 5’-TGAAATAGCTTCAAAATGTCTAAGAAGCACATTGTTATCATTG-3’;

mttA-2: 5’-TTTCGCCACGGAGCTTTCAGATGACGTTGGAGTTGTGG-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; primers (10μM each) 1.5μL; synthetic plasmid template (10ng) 1μL; KOD-Plus-Neo (1U/μL )1μL.

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

3) Use primers Tcbh1-1 and Tcbh1-2 to amplify the Tcbh1 sequence using the Trichoderma reesei genome as a template.

Tcbh1-1: 5’-AGCTCCGTGGCGAAAGCC-3’;

Tcbh1-2: 5’-CATTATACGAAGTTATTCTAGAATTTCCACTGTTGCTATTATGCTGT-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; primer (10μM each) 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL) 1μL.

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

4) Use the cad1 single gene expression vector pOEcad1 (Figure 1A) as the backbone to construct an expression vector. Double digestion was carried out with the restriction endonuclease PacI/XbaI on the existing plasmid pOEcad1, and homologous recombination was performed using Vazyme One Step Clone Kit to construct the Peno-mttA-Tcbh1 expression cassette and obtain the mttA-cad1 dual gene expression vector pOEmttA- cad1 (Fig. 1A).

Among them, the amino acid sequence of mttA is shown in SEQ ID NO.5, the nucleotide sequence of mttA is shown in SEQ ID NO.6, the nucleotide sequence of Peno is shown in SEQ ID NO.7, and the nucleotide sequence of Tcbh1 As shown in SEQ IDNO.8.

3. Introduce the mttA-cad1 dual-gene expression vector pOEmttA-cad1 into Trichoderma reesei to obtain the genetically engineered strain TrIA01

The expression or heterologous expression involved in the present invention is Agrobacterium-mediated transformation and cloning screening of Trichoderma reesei, and the relevant genes are integrated into the Trichoderma reesei genome for expression. The transformation method involved in the present invention is Agrobacterium tumefaciens-mediated conjugative transfer.

1) Electroporate the plasmid pOEmttA-cad1 into Agrobacterium, and then combine the Agrobacterium containing the plasmid pOEmttA-cad1 with the Trichoderma reesei host strain QM6a (ATCC 13631), QM9414 (ATCC 26921), Rut-C30 (ATCC56765), RL- P37 (NRRL 15709), NG14 (ATCC 56767), PC-3-7 (ATCC 66589) in IM plate (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105(3):259-264). Culture, perform Agrobacterium tumefaciens-mediated conjugation transfer, and after two days of co-culture, transformants are transferred to PDA plates containing cefotaxime (300 μg/mL) and hygromycin B (75 μg/mL) for screening until transformation The seeds grow mycelium and spores, which are then screened and verified.

2) Inoculate the above-verified transformants into 50 mL of culture medium with glucose as the carbon source (see Example 1) in a 250 mL Erlenmeyer flask. The inoculation amount is ¹⁰ spores/50 mL of culture medium, and cultured at 28°C and 220 rpm. On the 8th day, samples were taken to determine the itaconic acid content.

3) When mttA-cad1 is co-expressed in Trichoderma reesei strains, itaconic acid can be significantly fermented and produced. The strain with the highest yield is the QM6a transformant, named TrIA01. When glucose is used as the carbon source, itaconic acid production can reach 42.5g/L (Figure 1B).

Example 4 Overexpression of own membrane transporter encoding gene in Trichoderma reesei

Using the amino acid sequence (SEQ ID NO. 9) of the major facilitator superfamily protein mfsA derived from Aspergillus terreus as a template, five endogenous membrane transport proteins of Trichoderma reesei were identified through homology alignment. The coding genes are named mfs1, mfs2, mfs3, mfs4, and mfs5, and their gene sequences are shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14 respectively. .

1. Construction of endogenous membrane transporter encoding gene overexpression vector pOEmfs1

1) Use primers mfs1-F1 and mfs1-F2 to amplify the upstream sequence mae1-F of mfs1 using the Trichoderma reesei genome as a template (the nucleotide sequence is shown in SEQ ID NO. 15).

mfs1-F1: 5’-ATTACGAATTCTTAATTAACTGTTGGCAAATGGCTGGATGA-3’;

mfs1-F2: 5’-CATTATACGAAGTTATTCTAGACGGCAGCACAGGACGATGAA-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; 10μM primer mfs1-F1/mfs1-F2 each 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL)1μL.

Reaction program: 94°C 2min; 98°C 10sec, 58°C 30sec, 68°C 25sec, 30 cycles; 68°C 5min.

2) Using the primers pdc-F and pdc-R and the amplification conditions in Example 3, use the Trichoderma reesei genome as a template to amplify the promoter Ppdc sequence (the nucleotide sequence is shown in SEQ ID NO. 3).

3) Use primers mfs1-1 and rmfs1-2 to amplify the mfs1 partial gene sequence mfs1-O (the nucleotide sequence is shown in SEQ ID NO. 16) using the Trichoderma reesei genome as a template.

mfs1-1: 5’-AGCTACAGCACAATCATGAAGCAAGATGAAGCGATACAGC-3’;

mfs1-2: 5’-AGTGCCAAGCTTATTTGCGACGAAGATGGCACAGAAC-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; 10μM primer mfs1-1/mfs1-2 each 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL)1μL.

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

4) Use lcNG (Wu et al. An efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei. J. Gen. Appl. Microbiol., 2019, 65: 301-307.) as the backbone to construct an expression vector on the existing plasmid lcNG The restriction endonuclease PacI/XbaI was used for double digestion, and the Vazyme One Step Clone Kit was used for seamless connection, and the upstream sequence mfs1-F was connected; SwaI was used for single digestion, and the Vazyme One Step Clone Kit was used for seamless connection. , connect the promoter Ppdc and part of the gene sequence mfs1-O; construct the mfs1 overexpression plasmid pOEmfs1 (Figure 2A).

2. Construction of endogenous membrane transporter encoding gene overexpression vector pOEmfs2

1) Use primers mfs2-F1 and mfs2-F2 to amplify the upstream sequence mfs2-F of mfs2 using the Trichoderma reesei genome as a template (the nucleotide sequence is shown in SEQ ID NO. 17);

mfs2-F1: 5’-ATTACGAATTCTTAATTAACTGTTGGCAAATGGCTGGATGA-3’;

mfs2-F2: 5’-CATTATACGAAGTTATTCTAGACGGCAGCACAGGACGATGAA-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; 10μM primer mfs2-F1/mfs2-F2 each 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL)1μL.

Reaction program: 94°C 2min; 98°C 10sec, 58°C 30sec, 68°C 25sec, 30 cycles; 68°C 5min.

2) Using the primers pdc-F and pdc-R and the amplification conditions in Example 3, use the Trichoderma reesei genome as a template to amplify the promoter Ppdc sequence (the nucleotide sequence is shown in SEQ ID NO. 3);

3) Use primers mfs2-1 and mfs2-2 to amplify the mfs2 partial gene sequence mfs2-O (the nucleotide sequence is shown in SEQ ID NO. 18) using the Trichoderma reesei genome as a template.

mfs2-1: 5’-AGCTACAGCACAATCATGAAGCAAGATGAAGCGATACAGC-3’;

mfs2-2: 5’-AGTGCCAAGCTTATTTGCGACGAAGATGGCACAGAAC-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; 10μM primer mfs2-1/mfs2-2 each 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL)1μL.

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

4) Use lcNG (Wu et al. An efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei. J. Gen. Appl. Microbiol., 2019, 65: 301-307.) as the backbone to construct an expression vector on the existing plasmid lcNG The restriction endonuclease PacI/XbaI was used for double digestion, and Vazyme One Step Clone Kit was used for seamless connection, and the upstream sequence mfs2-F was connected; SwaI was used for single digestion, and Vazyme One Step Clone Kit was used for seamless connection. , connect the promoter Ppdc and part of the gene sequence mfs2-O; construct the mfs2 overexpression plasmid pOEmfs2 (Figure 2A).

3. Construction of endogenous membrane transporter encoding gene overexpression vector pOEmfs3

1) Use primers mfs3-F1 and mfs3-F2 to amplify the upstream sequence mfs3-F of mfs3 using the Trichoderma reesei genome as a template (the nucleotide sequence is shown in SEQ ID NO: 19).

mfs3-F1: 5’-ATTACGAATTCTTAATTAAGAAGCGTCTCCAGGACATTCC-3’;

mfs3-F2: 5’-CATTATACGAAGTTATTCTAGAGGGCGAATCTGAAAGCGGATG-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; 10μM primer mfs3-F1/mfs3-F2 each 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL)1μL.

Reaction program: 94°C 2min; 98°C 10sec, 58°C 30sec, 68°C 25sec, 30 cycles; 68°C 5min.

2) Using the primers pdc-F and pdc-R and the amplification conditions in Example 3, use the Trichoderma reesei genome as a template to amplify the promoter Ppdc sequence (the nucleotide sequence is shown in SEQ ID NO. 3).

3) Use primers mfs3-1 and mfs3-2 to amplify the mfs3 partial gene sequence mfs3-O using the Trichoderma reesei genome as a template (the nucleotide sequence is shown in SEQ ID NO. 20).

mfs3-1: 5’-AGCTACAGCACAATCATGTCATCAAAATTTGACAATGAACAAGA-3’;

mfs3-2: 5’-AGTGCCAAGCTTATTTCGGAACGGACCAAGCACATC-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; 10μM primer mfs3-1/mfs3-2 each 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL)1μL.

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

5) Use lcNG (Wu et al. An efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei. J. Gen. Appl. Microbiol., 2019, 65: 301-307.) as the backbone to construct an expression vector on the existing plasmid lcNG The restriction endonuclease PacI/XbaI was used for double digestion, and Vazyme One Step Clone Kit was used for seamless connection, and the upstream sequence mfs3-F was connected; SwaI was used for single digestion, and Vazyme One Step Clone Kit was used for seamless connection. , connect the promoter Ppdc and part of the gene sequence mfs3-O; construct the mfs3 overexpression plasmid pOEmfs3 (Figure 2A).

4. Construction of endogenous membrane transporter encoding gene overexpression vector pOEmfs4

1) Use primers mfs4-F1 and mfs4-F2 to amplify the upstream sequence mfs4-F of mfs4 using the Trichoderma reesei genome as a template (the nucleotide sequence is shown in SEQ ID NO. 21).

mfs4-F1: 5’-ATTACGAATTCTTAATTAAGCTGTGGAGTTGCCGATAAGG-3’;

mfs4-F2: 5’-CATTATACGAAGTTATTCTAGACAGACGACGGCGGTATCAT-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; 10μM primer mfs4-F1/mfs4-F2 each 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL)1μL.

Reaction program: 94°C 2min; 98°C 10sec, 58°C 30sec, 68°C 25sec, 30 cycles; 68°C 5min.

2) Using the primers pdc-F and pdc-R and the amplification conditions in Example 3, use the Trichoderma reesei genome as a template to amplify the promoter Ppdc sequence (the nucleotide sequence is shown in SEQ ID NO: 3).

3) Use primers mfs4-1 and mfs4-2 to amplify the mfs4 partial gene sequence mfs4-O (the nucleotide sequence is shown in SEQ ID NO. 22) using the Trichoderma reesei genome as a template.

mfs4-1: 5’-AGCTACAGCACAATCATGGGCGCCTCAGAAGACAC-3’;

mfs4-2: 5’-AGTGCCAAGCTTATTTGCATCTGCCATCCTGACCTGTA-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; 10μM primer mfs4-1/mfs4-2 each 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL)1μL.

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

4) Use lcNG (Wu et al. An efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei. J. Gen. Appl. Microbiol., 2019, 65: 301-307.) as the backbone to construct an expression vector on the existing plasmid lcNG The restriction endonuclease PacI/XbaI was used for double digestion, and Vazyme One Step Clone Kit was used for seamless connection, and the upstream sequence mfs4-F was connected; SwaI was used for single digestion, and Vazyme One Step Clone Kit was used for seamless connection. , connect the promoter Ppdc and part of the gene sequence mfs4-O; construct the mfs4 overexpression plasmid pOEmfs4 (Figure 2A).

5. Construction of endogenous membrane transporter encoding gene overexpression vector pOEmfs5

1) Use primers mfs5-F1 and mfs5-F2 to amplify the upstream sequence mfs5-F of mfs5 using the Trichoderma reesei genome as a template (the nucleotide sequence is shown in SEQ ID NO. 23).

mfs5-F1: 5’-ATTACGAATTCTTAATTAACCTCCGAGTTGGCTATCACTGA-3’;

mfs5-F2: 5’-CATTATACGAAGTTATTCTAGAGGGTCCTGGATGTATGCGATGA-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; 10μM primer mfs5-F1/mfs5-F2 each 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL)1μL.

Reaction program: 94°C 2min; 98°C 10sec, 58°C 30sec, 68°C 25sec, 30 cycles; 68°C 5min.

2) Using the primers pdc-F and pdc-R and the amplification conditions in Example 3, use the Trichoderma reesei genome as a template to amplify the promoter Ppdc sequence (the nucleotide sequence is shown in SEQ ID NO. 3).

3) Use primers mfs5-1 and mfs5-2 to amplify the mfs5 partial gene sequence mfs5-O (the nucleotide sequence is shown in SEQ ID NO. 24) using the Trichoderma reesei genome as a template.

mfs5-1: 5’-AGCTACAGCACAATCATGTCGTCAACACCCGACCC-3’;

mfs5-2: 5’-AGTGCCAAGCTTATTTGAGGATGCGGAAGACGATGA-3’.

Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mMMgSO ₄ 3μL; 10μM primer mfs5-1/mfs5-2 each 1.5μL; genome template (200ng) 1μL; KOD-Plus-Neo (1U/μL)1μL.

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

4) Use lcNG (Wu et al. An efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei. J. Gen. Appl. Microbiol., 2019, 65: 301-307.) as the backbone to construct an expression vector on the existing plasmid lcNG The restriction endonuclease PacI/XbaI was used for double digestion, and Vazyme One Step Clone Kit was used for seamless connection, and the upstream sequence mfs5-F was connected; SwaI was used for single digestion, and Vazyme One Step Clone Kit was used for seamless connection. , connected to the promoter Ppdc and part of the gene sequence mfs5-O; the overexpression plasmid pOEmfs5 of mfs1 was constructed (Figure 2A).

Example 5

The expression vectors pOEmfs1 to pOEmfs5 constructed in the above Example 4 were respectively introduced into the Trichoderma reesei engineering strain TrIA01 to obtain the engineering strain TrIA02 with improved itaconic acid production.

The expression in the present invention is Agrobacterium-mediated transformation and cloning screening of Trichoderma reesei, and the endogenous genes of Trichoderma reesei are integrated into the Trichoderma reesei genome for expression. The transformation method of the present invention is Agrobacterium tumefaciens-mediated conjugation transfer.

1) The expression plasmids pOEmfs1 ~ pOEmfs5 were electroporated into Agrobacterium respectively, and then the Agrobacterium containing the expression plasmids were co-cultured with the Trichoderma reesei host strain TrIA01 (culture conditions reference: Covert etal. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol.Res.105(3):259-264), Agrobacterium tumefaciens-mediated conjugative transfer was performed, and after two days of co-culture, the transformants were transferred to cells containing cefotaxime (300 μg/mL) and G418 (400 μg/mL). Screen on PDA plates until the transformants grow hyphae and spores, and then verify.

2) Inoculate the above transformants into 50 mL of fermentation medium with glucose as the carbon source (see Example 1) in a 250 mL Erlenmeyer flask. The inoculation amount is ¹⁰ spores/50 mL of medium at 28-30°C (preferably 30 ℃), culture at 200rpm, and take samples to measure the itaconic acid content on the 8th day to verify its biological function.

3) Membrane transport proteins can transport itaconic acid out of cells. Therefore, after membrane transport proteins are expressed in large quantities in the Trichoderma reesei engineering strain TrIA01, the strain can significantly accumulate more itaconic acid in the fermentation broth (except for mfs4). Among them, the transformant with the highest yield is the mfs1 gene transformant, named TrIA02. When glucose is used as the carbon source, itaconic acid production can reach 60g/L (Figure 2B).

4) The engineering strain TrIA02 can directly ferment and produce large amounts of itaconic acid using common carbon sources such as glycerol, liquefied starch, cellulose, and cellulose hydrolyzate (Figure 3). Experiments show that Trichoderma reesei can ferment itaconic acid with a variety of carbon sources after genetic modification.

It can be seen from the results of the above examples that the present invention successfully fermented and produced itaconic acid after genetically modifying Trichoderma reesei. The research results of the present invention are the first to show that although the original strain of Trichoderma reesei cannot produce itaconic acid, after genetic engineering modification, it can use common carbon sources such as glucose, glycerol, liquefied starch, cellulose, and cellulose hydrolyzate as substrates. Fermentation produces itaconic acid. And it was confirmed through experiments that the potential of the Trichoderma reesei engineered strain to ferment itaconic acid in shake flasks provides an excellent strain for the industrial large-scale fermentation production of itaconic acid.

The above-described embodiments only describe the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. All deformations and improvements shall fall within the protection scope determined by the claims of the present invention.

Claims (7)

1. A method for constructing Trichoderma reesei engineering bacteria, which is characterized by comprising the following steps: (1) Using the non-itaconic acid-producing filamentous fungus Trichoderma reesei or its derivatives derived from Trichoderma reesei as the starting strain, introduce foreign genes encoding cis-aconitic acid decarboxylase and mitochondrial tricarboxylic acid transporter , obtain strain I; (2) Overexpress the endogenous membrane transport protein of strain I to obtain an itaconic acid-producing Trichoderma reesei engineering strain; The starting strain is Trichoderma reesei QM6a; The amino acid sequence of the cis-aconitate decarboxylase is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the cis-aconitate decarboxylase is shown in SEQ ID NO.2; the mitochondria The amino acid sequence of the tricarboxylic acid transporter is shown in SEQ ID NO.5, and the nucleotide sequence of the gene encoding the mitochondrial tricarboxylic acid transporter is shown in SEQ ID NO.6; The endogenous membrane transport protein is an mfs superfamily transport protein; the gene encoding the endogenous membrane transport protein is mfs1, mfs2, mfs3 or mfs5, and their nucleotide sequences are as SEQ ID NO. 10 and SEQ ID NO. 11. Shown as SEQ ID NO.12 or SEQ ID NO.14. 2. An engineering strain of Trichoderma reesei, characterized in that it is constructed by the construction method of claim 1. 3. A method for producing itaconic acid, which is characterized by utilizing the Trichoderma reesei engineering bacteria for fermentation according to claim 2, collecting the fermentation liquid, and obtaining itaconic acid. 4. The method of claim 3, wherein the fermentation conditions are: the inoculum amount is 10 ⁸ spores/50 mL liquid culture medium, the fermentation temperature is 28-30°C, and the rotation speed is 200 rpm. 5. The method of claim 4, wherein the liquid culture medium includes the following concentration components: carbon source 50-100 g/L, peptone 1-6 g/L, KH ₂ PO ₄ 0.15 g/ L, K ₂ HPO ₄ 0.15 g/L, CaCl ₂ ·2H ₂ O 0.10 g/L, MgSO ₄ ·7H ₂ O 0.10 g/L, calcium carbonate 20-40 g/L, NaCl 0.05 g/L and 1 mL /L trace element liquid. 6. The method of claim 5, wherein the carbon source includes any one of glucose, glycerol, cellulose and cellulose hydrolyzate; The trace element liquid includes the following weight components: 1.6 g MnSO ₄ ·4H ₂ O, 5 g FeSO ₄ ·7H ₂ O, 2g CoCl ₂ ·6H ₂ O and 1.4 g ZnSO ₄ ·7H ₂ O, dissolved in water and determined Capacity to 1 L. 7. The construction method according to claim 1, or the application of the Trichoderma reesei engineering bacteria according to claim 2 in the production of itaconic acid.