CN116640792A - Method for improving xylanase yield in Trichoderma reesei and application thereof - Google Patents
Method for improving xylanase yield in Trichoderma reesei and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The present invention relates to a method for increasing xylanase yield in trichoderma reesei. The invention takes Trichoderma reesei endogenous protein Xyn2 as a fusion expression element, connects coding genes of the fusion expression element Xyn2 and xylanase Xyn10A together to form a fusion protein gene, and then heterologously expresses the fusion protein to synthesize xylanase. The nucleotide sequence of the fusion expression element Xyn2 is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 1. The xylanase enzyme activity obtained by the method is obviously higher than that of xylanase Xyn10A directly expressed, the enzyme activity of xylanase produced by recombinant Trichoderma reesei Xyn2-Xyn10A with glucose as a carbon source reaches 170U/mL, and the enzyme activity of xylanase produced by the strain with cellulose as a carbon source reaches 270U/mL, which is obviously higher than that of a strain using common fusion expression elements CBHI signal peptide and CBHII-CBM, is more than 2 times of that of a starting strain, and the enzyme activity level of xylanase in an extracellular medium of Trichoderma reesei strain is obviously improved.
Description
Technical Field
The invention relates to a method for improving xylanase yield in trichoderma reesei and application thereof, belonging to the technical field of bioengineering.
Background
The lignocellulose substance is a main component of biomass resources, and has important strategic significance for the development and utilization of the biomass resources in China, such as economic development, environmental protection and other social core problems. Trichoderma reesei, an industrial filamentous fungus, can produce various cellulases and hemicellulases, and plays an important role in degrading lignocellulose substances. Xylanase is a main component of a hemicellulase system, so that products such as xylo-oligosaccharide, xylose and the like can be produced by degrading xylan, and the overall high-efficiency degradation of lignocellulose can be promoted by synergistic action with cellulase. Compared with cellulase, the expression level and the enzyme activity level of xylanase in Trichoderma reesei are lower, so that the efficient heterologous xylanase introduced into Trichoderma reesei can promote effective improvement of xylanase activity, and further the integral degradation capacity of xylanase to lignocellulose can be improved.
Trichoderma reesei has strong protein synthesis and secretion capacity, high safety, availability of cheap raw materials and other advantages, and is also developed as a chassis cell for expression and secretion of heterologous proteins. The secretory production of the protein is beneficial to the subsequent purification and scale-up of the target protein. At present, the signal peptide (17 amino acids at the N-terminal) of the CBHI of the main cellulase is often used for guiding the expression and secretion of the heterologous protein, but the problem of low production level of the heterologous recombinant protein is often encountered. In response to this problem, scientists have found that fusion of the N-terminal catalytic domain of CBHI (about 480 amino acids) to the N-terminal of a heterologous protein is effective in promoting enhanced secretion yield of synthesis. This is because CBHI as a fusion expression element can enhance the structural stability of mRNA of a heterologous protein, promote efficient entry of the heterologous protein into the secretory pathway, and efficient folding, transport, etc. However, because of the large molecular weight of CBHI, the difficulty of genetic manipulation and subsequent protein purification is increased, so that researchers develop a CBM domain (about 108 amino acids) with a short N-terminal of another cellulase CBH II as a fusion expression element, and the fusion expression element has a good secretagogue effect on heterologous proteins. In addition, the related research on the fusion expression element of trichoderma reesei in the prior art is less, and the short and small efficient fusion expression element still needs to be further excavated for efficient secretion production of heterologous proteins so as to promote the development and upgrading of trichoderma reesei chassis cells and realize low-cost production of more protein products.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for improving xylanase yield in Trichoderma reesei and application thereof.
The technical scheme of the invention is as follows:
a method for increasing xylanase yield in trichoderma reesei by directing expression of heterologous xylanases using endogenous protein Xyn2 as a fusion expression element.
According to the invention, the nucleotide sequence of the fusion expression element Xyn2 is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 1.
Preferably according to the invention, the heterologous xylanase is xylanase Xyn10A. The nucleotide sequence of the xylanase Xyn10A has been disclosed in the doctor graduation paper of Shandong university, functional analysis of the thermophilic fungus Emersen basket glycoside hydrolase (An Jianlu, 2021).
A recombinant Trichoderma reesei is prepared through linking the endogenous protein Xyn2 gene and xylanase Xyn10A gene to skeleton plasmid, and transforming the recombinant plasmid to Trichoderma reesei.
According to the preferred method of the invention, the construction method of the recombinant trichoderma reesei comprises the following specific steps:
(1) The Trichoderma reesei QM9414 genome DNA is used as a template, and a fusion expression element Xyn2 sequence is obtained through PCR amplification;
(2) Taking plasmid pPICZ alpha A-Xyn10A as a template, and obtaining xylanase Xyn10A sequence through PCR amplification;
(3) Connecting coding genes of a fusion expression element Xyn2 and xylanase Xyn10A together through overlap extension PCR, and obtaining a fusion fragment Xyn2-Xyn10A through PCR amplification by taking the fragment as a template;
(4) The method comprises the steps of taking Trichoderma reesei QM9414 genome DNA as a template, carrying out PCR amplification to obtain an upstream and downstream homologous arm sequence of pyr4 gene, connecting the upstream and downstream homologous arm sequence into a vector pUC19-hph containing a hygromycin resistance gene hph expression cassette, and then sequentially introducing a promoter tcu and a terminator TrpC between the upstream homologous arm and the hph expression cassette to obtain a recombinant expression vector pUC19-Ptcu1-pyr4site;
(5) Connecting the fusion fragment xyn2-xyn10A between a promoter and a terminator of the recombinant expression vector pUC19-Ptcu1-pyr4site to obtain a recombinant expression vector Ptcu1-xyn2-xyn10A;
(6) Transferring the recombinant expression vector Ptcu1-xyn2-xyn10A into Trichoderma reesei QM 9414-delta pyr4 protoplast, and obtaining recombinant Trichoderma reesei xyn2-xyn10A through regeneration, screening and verification.
According to a preferred embodiment of the present invention, in step (1), the primer sequences used for the PCR amplification are as follows:
xyn2F:5’-ACGACCTGGTTGATACGACAGGCGCGCCATGGTCTCCTTCACCTCCCTC-3’；
xyn2R:5’-TCTGGTTCGTATCGACGTAGGTGTCAGAACCTCCAGAACCTCCGCTGACGGTGATGGAAGCA-3’。
according to a preferred embodiment of the present invention, in step (2), the primer sequences used for the PCR amplification are as follows:
xyn10F:5’-TGCTTCCATCACCGTCAGCGGAGGTTCTGGAGGTTCTGACACCTACGTCGATACGAACCAG-3’；
xyn10R1:5’-TTAGTGATGGTGATGGTGGTGGACCGTCTGTCCAAGCC-3’。
according to a preferred embodiment of the present invention, in step (3), the primer sequences used for the PCR amplification are as follows:
xyn2F:5’-ACGACCTGGTTGATACGACAGGCGCGCCATGGTCTCCTTCACCTCCCTC-3’；
xyn10R2:5’-TGATTTCAGTAACGTTAAGTCTTAAGTTAGTGATGGTGATGGTGGTGGACC-3’。
according to a preferred embodiment of the present invention, in step (4), the sequence of the PCR amplification primer of the upstream homology arm is as follows:
upF:5’-GAATTCGAGCTCGGTACCCGGGATGATAATGGACTGGACCG-3’；
upR:5’-ATTCTTATAATCTCTAGAGGATCCTACCCGATATTGCGACTTT-3’；
the PCR amplification primer sequence of the downstream homology arm is as follows:
downF:5’-AGACTAGTCCAATCGTCGACGGGAGGGAAGGGAAGAAAGAAGTAA-3’；
downR:5’-ACCATGATTACGCCAAGCTTCTCGTAGGCGCAGTGGGCTT-3’。
the application of the recombinant trichoderma reesei in the production of xylanase.
A method for increasing xylanase yield in trichoderma reesei, comprising the following steps:
inoculating the recombinant Trichoderma reesei xyn2-xyn10A into a MA liquid culture medium which takes 1% (v/v) glycerol as a carbon source for preculture, culturing for 30-40 h at 30+/-2 ℃ and 180-220 rpm, and collecting hyphae; then transferring into MA liquid culture medium which takes 2% (w/v, g/mL) glucose as carbon source, and continuously culturing at 30+/-2 ℃ and 180-220 rpm to produce xylanase.
Further preferably, the MA liquid medium formula is: na was added to 1L of the medium 2 HPO 4 ·12H 2 O 17.907g、(NH 4 ) 2 SO 4 1.4 g、KH 2 PO 4 2.0g, 0.3g urea, tween-800.5mL, and adjusting pH to 5.0 with anhydrous citric acid; before use, mgSO with final concentration of 0.6g/L is added 4 Final concentration of 0.4g/L CaCl 2 And adding trace elements (final concentration 1×, mother liquor 1000×), 10mM uridine;
the 1000 times trace element mother liquor formula comprises: feSO 4 ·7H 2 O 0.5g，MnSO 4 ·H 2 O 0.16g，ZnSO 4 ·7H 2 O 0.14g，CoC l2 ·2H 2 O0.2 g with ddH 2 O is fixed to volume of 100mL, and filtering and sterilizing are carried out.
The invention has the beneficial effects that:
1. the invention takes Trichoderma reesei endogenous protein Xyn2 as a fusion expression element, connects coding genes of the fusion expression element Xyn2 and xylanase Xyn10A together to form a fusion protein gene, and then heterologously expresses the fusion protein to synthesize xylanase. The xylanase yield obtained by the method is obviously higher than that of xylanase Xyn10A directly expressed, the effect of promoting expression is obviously better than that of the common expression element CBHI signal peptide and CBH II-CBM, and the secretion production level of xylanase is obviously improved.
2. The xylanase produced by the recombinant Trichoderma reesei xyn2-xyn10A taking glucose as a carbon source has the enzyme activity reaching 170U/mL, and the xylanase produced by the strain taking cellulose as a carbon source has the enzyme activity reaching 270U/mL, which is obviously higher than that of a strain using a common fusion expression element CBHI signal peptide and CBHII-CBM, is more than 2 times of that of a starting strain, and obviously improves the enzyme activity level of the xylanase in an extracellular culture medium of the Trichoderma reesei strain.
Drawings
FIG. 1 is a schematic diagram of the construction of an expression strain in which Xyn2 is used as a fusion expression element to direct the heterologous xylanase Xyn10A.
In the figure, A is a schematic diagram of construction of an expression strain in which Xyn2 is used as a fusion expression element to guide heterologous xylanase Xyn10A; panel B and C are schematic diagrams of the construction of CBHI signal peptide and CBHII-CBM-directed Xyn10A expression strain, respectively, as controls for the beneficial effects of the methods shown in panel A.
FIG. 2 is an agarose gel electrophoresis chart verifying the successfully constructed PCR products of each recombinant Trichoderma reesei.
FIG. 3 shows SDS-PAGE analysis of recombinant Trichoderma reesei extracellular fermentation broth under glucose culture conditions.
In the figure, A is the analysis of fermentation broth after 48h of culture; panel B shows the fermentation broth analysis after 72h of incubation.
FIG. 4 shows Western blot analysis of recombinant Trichoderma reesei extracellular fermentation broth under glucose culture conditions.
In the figure, A is the analysis of fermentation broth after 48h of culture; panel B shows the fermentation broth analysis after 72h of incubation.
FIG. 5 shows the xylanase enzyme activity assay of recombinant Trichoderma reesei extracellular fermentation broth under glucose culture conditions.
FIG. 6 shows Western blot analysis of recombinant Trichoderma reesei extracellular fermentation broth under cellulose culture conditions.
In the figure, A is the analysis of fermentation broth after 48h of culture; panel B shows the fermentation broth analysis after 72h of incubation.
FIG. 7 shows the xylanase enzyme activity assay of recombinant Trichoderma reesei extracellular fermentation broth under cellulose culture conditions.
Detailed Description
The present invention will be further described with reference to examples and drawings, but the scope of the present invention is not limited thereto. The medicines and reagents related to the examples are common commercial products unless specified; the experimental procedures referred to in the examples, unless otherwise specified, are conventional in the art. The primers in the examples were all synthesized by Beijing qing Biotechnology Co.
The starting strain Trichoderma reesei (Trichoderma reesei) QM9414 in the examples is an existing strain available from the American type culture Collection under accession number ATCC 26921.
Trichoderma reesei QM 9414-. DELTA.pyr4 was obtained by knocking out pyr4 gene in Trichoderma reesei QM9414, and the construction method can be referred to in: a novel transcriptional regulator RXE1modulates the essentialtransactivator XYR1and cellulase gene expression in Trichoderma reesei.
The plasmid pPICZ alpha A-Xyn10A is an existing plasmid, and the construction process can be seen in the doctor graduation paper of Shandong university, functional analysis of thermophilic fungus Emersen basket glycoside hydrolase (An Jianlu, 2021).
Example 1: construction of Xyn2 as fusion expression element for guiding expression vector of heteroxylanase Xyn10A
1. Construction of Xyn2 as fusion expression element for guiding expression vector of heteroxylanase Xyn10A
(1) A fungal DNA extraction kit (E.Z.N.A) was used. TM Fungal DNA Mini Kit, OMEGA) extracting Trichoderma reesei QM9414 genome DNA, taking the genome DNA as a template, designing a primer Xyn2F/Xyn2R, and performing PCR amplification to obtain an Xyn2 complete gene fragment, wherein the fragment contains a sequence of a fusion expression element Xyn2, the nucleotide sequence of the fusion expression element Xyn2 is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 1; the primer Xyn2R contains a segment of nucleotide for encoding GGSGGS sequence, thereby realizing the introduction of a segment of lin between Xyn2 and Xyn10AA ker sequence.
(2) The primer Xyn10F/Xyn10R1 is designed, and a cDNA coding segment Xyn10A (coding catalytic domain Ala23-Thr 342) of the xylanase Xyn10A derived from Emerson basket bacteria is obtained by PCR amplification by taking a plasmid pPICZ alpha A-Xyn10A as a template. The Xyn2 and Xyn10A were ligated together using conventional overlap extension PCR to form a fusion fragment of about 1.7kb; and then taking the fusion fragment as a template, and carrying out PCR amplification by using a primer xyn2F/xyn10R2 to obtain a fusion fragment xyn2-xyn10A. Because the primer xyn10R2 has a coding sequence of 6 histidines, the tail end of the synthesized xyn10A protein is loaded with a corresponding histidine tag sequence.
Wherein, the sequence of the primer is as follows:
xyn2F:5’-ACGACCTGGTTGATACGACAGGCGCGCCATGGTCTCCTTCACCTCCCTC-3’；
xyn2R:5’-TCTGGTTCGTATCGACGTAGGTGTCAGAACCTCCAGAACCTCCGCTGACGGTGATGGAAGCA-3’；
xyn10F:5’-TGCTTCCATCACCGTCAGCGGAGGTTCTGGAGGTTCTGACACCTACGTCGATACGAACCAG-3’；
xyn10R1:5’-TTAGTGATGGTGATGGTGGTGGACCGTCTGTCCAAGCC-3’；
xyn10R2:5’-TGATTTCAGTAACGTTAAGTCTTAAGTTAGTGATGGTGATGGTGGTGGACC-3’；
the PCR amplification system is as follows: 10. Mu.M upstream primer 2. Mu.L, 10. Mu.M downstream primer 2. Mu.L, 10ng of genomic DNA, 5. Mu.L of 2.5mM dNTP mixture, 10. Mu.L of 5X TransStart FastPfu buffer, 1. Mu.L of TransStartFastPfiDNA polymerase, ddH 2 O is added to 50 mu L;
the PCR amplification procedure was as follows: pre-denaturation at 98 ℃ for 5min; denaturation at 95℃for 30sec, annealing at 58℃for 30sec, extension at 72℃for 3min,30 cycles; final extension at 72℃for 10min; after completion of PCR, 1% agarose gel electrophoresis was performed, and a DNA fragment was recovered.
(3) Primers upF/upR and downF/downR were designed and PCR amplified to yield homologous arm fragments downstream of the Trichoderma reesei pyr4 gene of about 2kb and 1.5kb, respectively. Respectively connecting the upstream and downstream homology arm fragments of pyr4 gene into a vector pUC19-hph containing a hygromycin resistance gene hph expression cassette, wherein the upstream and downstream homology arm fragments are respectively positioned at two sides of the hph expression cassette; and then, a trichoderma reesei constitutive promoter tcu and a terminator TrpC are sequentially introduced between the homologous arm and the hph expression cassette, so that a vector pUC19-Ptcu1-pyr4site which is successfully driven by the promoter tcu 1and subjected to pyr4 site-specific recombination is constructed. Finally, the fusion fragment Xyn2-Xyn10A is connected into the vector and is positioned between a promoter and a terminator, so that the recombinant expression vector Ptcu1-Xyn2-Xyn10A guided by the Xyn2 as a fusion expression element is obtained, and a schematic diagram of the recombinant expression vector is shown in FIG. 1A.
Wherein, the sequence of the primer is as follows:
upF:5’-GAATTCGAGCTCGGTACCCGGGATGATAATGGACTGGACCG-3’；
upR:5’-ATTCTTATAATCTCTAGAGGATCCTACCCGATATTGCGACTTT-3’；
downF:5’-AGACTAGTCCAATCGTCGACGGGAGGGAAGGGAAGAAAGAAGTAA-3’；
downR:5’-ACCATGATTACGCCAAGCTTCTCGTAGGCGCAGTGGGCTT-3’.
the PCR amplification system and the amplification procedure were as described above.
2. Construction of a CBHI Signal peptide and a CBH II-CBM respectively guide a heterologous xylanase Xyn10A expression vector
When expressing heterologous proteins in Trichoderma reesei, it is often guided by the signal peptide (the first 17 amino acids at the N-terminus) of the endogenous protein CBHI (NCBI accession number XP-006969224.1). In addition, the N-terminal CBM domain of Trichoderma reesei CBH II (the first 108 amino acids of the N-terminal, NCBI accession number XP_006962580.1 of CBH II) can be used as a fusion expression element with short sequence, and can promote the efficient expression and secretion of heterologous proteins. Thus, we used CBHI signal peptide and CBH II-CBM to direct expression secretion of heterologous xylanase Xyn10A, respectively, as a reference to measure the beneficial effect of Xyn2 as a fusion expression element to promote expression secretion of heterologous proteins as described above.
The plasmid pPICZαA-Xyn10A is used as a template, nested PCR is adopted, a corresponding fragment of the Xyn10A is amplified by using a primer cbh1SPF3/Xyn10R1, then a second round of amplification is carried out by using a product as a template and a primer cbh1SPF2/Xyn10R2, and then a third round of amplification is carried out by using a second round of product as a template and a primer cbh1SPF1/Xyn10R2, so that the fragment cbh1SP-Xyn10A is obtained.
Designing a primer CBMF/CBMR, and obtaining a coding sequence of an N-terminal CBM structural domain (108 amino acids) of CBH II by PCR amplification by taking Trichoderma reesei QM9414 genome as a template; designing a primer Xyn 10-F/Xyn 10R2, using a pPICZ alpha A-Xyn10A plasmid as a template, amplifying a catalytic domain coding sequence cbh2CBM of Xyn10A, and connecting the cbh2CBM and Xyn10A by adopting an overlap extension PCR method to obtain a fusion fragment cbh2CBM-Xyn10A of about 1.3kb. Because the primer Xyn10R2 has a coding sequence of 6 histidines, the tail end of the synthesized Xyn10A protein is loaded with a corresponding histidine tag sequence.
The sequences of the primers are as follows:
cbh1SPF1:5’-ACGACCTGGTTGATACGACAGGCGCGCCATGTATCGGAAGTTGGCCGTCATCT-3’；
cbh1SPF2:5’-CGGAAGTTGGCCGTCATCTCGGCCTTCTTGGCCACAGCTCGTGCT-3’；
cbh1SPF3:5’-CCTTCTTGGCCACAGCTCGTGCTGCAGGCCTCAACACAGC-3’；
CBMF:5’-ACGACCTGGTTGATACGACAGGCGCGCCATGATTGTCGGCATTCTCACCACG-3’；
CBMR:5’-GCTGTGTTGAGGCCTGCTCCCGATCCGACTGGAGGTACTCTG-3’；
xyn2F*：5’-AGAGTACCTCCAGTCGGATCGGGAGCAGGCCTCAACACAGC-3’；
the obtained DNA fragments CBH1SP-Xyn10A and CBH2CBM-Xyn10A are respectively connected into the pyr4 site-directed recombinant vector pUC19-Ptcu1-pyr4site and are positioned between a promoter and a terminator to respectively obtain recombinant expression vectors Ptcu1-CBH1SP-Xyn10A and Ptcu1-CBH2CBM-Xyn10A of CBHI signal peptide guided Xyn10A, and the schematic diagrams of the two vectors are shown in FIG. 1B and FIG. 1C.
Example 2: construction of heterologous xylanase Xyn10A expression Strain guided by Xyn2 as fusion expression element
1. Preparation of Trichoderma reesei protoplast
Preparation of a concentration of 5X 10 7 ～5×10 8 1mL of Trichoderma reesei QM 9414-delta pyr4 spore suspension is inoculated into 50mL of MM liquid culture medium, and cultured at 28 ℃ and 160rpm for 16 hours to obtain mycelium culture; centrifuging mycelium culture at 4000rpm for 5min, discarding supernatant, adding cell wall lyase solution, re-suspending,and hydrolyzing at 30deg.C and 60rpm for 2.5-3 hr, observing with microscope, filtering with G2 funnel when a large amount of free protoplast appears, centrifuging at 4deg.C and 3500rpm for 5min to collect protoplast, washing the collected protoplast twice with 15mL pre-cooled STC solution, and re-suspending with pre-cooled STC solution to give protoplast concentration of 5×10 7 ～5×10 8 individual/mL; all of the above operations were performed on ice.
The MM liquid culture medium comprises the following components (mass percent): 1% glucose, 0.5% (NH) 4 ) 2 SO 4 、1.5％ KH 2 PO 4 0.1% peptone, adjusting pH to 5.0-5.5 with NaOH, and adding MgSO to a final concentration of 0.6g/L before use 4 Final concentration of 0.4g/LCaCl 2 And adding trace elements (final concentration 1×, mother liquor 1000×), 10mM uridine;
the 1000 times trace element mother liquor formula comprises: feSO 4 ·7H 2 O 0.5g，MnSO 4 ·H 2 O 0.16g，ZnSO 4 ·7H 2 O 0.14g，CoC l2 ·2H 2 O0.2 g with ddH 2 O is fixed to 100mL, and filtering and sterilizing are carried out;
the cell wall lyase liquid comprises the following components: 0.24g of cell wall lyase, dissolved in 5.7mL of NaCl solution with the concentration of 0.7M, filtered and sterilized, and added with 0.3mL of PBS buffer with the pH of 5.8;
the STC solution had the following composition: 0.7. 0.7M D sorbitol, 0.05M CaCl 2 ，10mMTris-HCl，pH 7.5。
2. Transformation of recombinant expression vectors and transformant regeneration
Taking 150 mu L of recombinant expression vectors Ptcu1-xyn2-xyn10A, ptcu1-cbh1SP-xyn10A and Ptcu1-cbh2CBM-xyn10A obtained in the example 1 respectively, carrying out tangential linearization by using Mss I restriction enzyme, purifying and recycling fragments, uniformly mixing with 150 mu L of Trichoderma reesei QM 9414-delta pyr4 protoplast prepared in the step 1, adding the fragments into a 10mL centrifuge tube, dropwise adding 1.5mL of precooled PTC solution after ice bath for 20min, standing at room temperature for 20min, adding 2mL of STC solution, and uniformly mixing to obtain a conversion solution; the transformation solution is coated on a regeneration culture medium which does not contain uridine and is cultured for 5 to 7 days at the temperature of 28 ℃.
Wherein, the PTC solution comprises the following components: the mass-to-volume ratio (w/v) was 60% PEG 4000 (polyethylene glycol 4000), 10mM Tris-HCl,10mM CaCl 2 ，pH 7.5；
The regeneration medium comprises the following components (mass percent): 1% glucose, 0.5% (NH) 4 ) 2 SO 4 、1.5％ KH 2 PO 4 1, M D-sorbitol, 1.5% agar powder, adjusting pH to 5.0-5.5 with NaOH, and adding MgSO with final concentration of 0.6g/L before use 4 Final concentration of 0.4g/LCaCl 2 And trace elements (final concentration 1×, mother liquor 1000×) were added. 1000 times trace element mother liquor has the same formula.
Transferring the colony growing on the regeneration culture medium to a screening culture medium without uridine, and culturing at 28 ℃ for 5-7 days to obtain the transformant.
Wherein, the components of the screening culture medium are as follows (mass percent): 1% glucose, 0.5% (NH) 4 ) 2 SO 4 、1.5％KH 2 PO 4 Regulating pH to 5.0-5.5 with NaOH, and adding MgSO with final concentration of 0.6g/L before use 4 Final concentration of 0.4g/L CaCl 2 And trace elements (final concentration 1×, mother liquor 1000×) were added. 1000 times trace element mother liquor has the same formula.
After the recombinant expression vectors Ptcu1-xyn2-xyn10A, ptcu1-cbh1SP-xyn10A and Ptcu1-cbh2CBM-xyn10A are respectively transformed into Trichoderma reesei protoplast, the upstream homologous arm sequence and the downstream homologous arm sequence of pyr4 gene are carried on the recombinant expression vectors, homologous recombination occurs between the upstream homologous arm and the downstream homologous arm sequence and the upstream and downstream regions of pyr4 gene in genome, and an expression cassette and a screening marker hph are integrated on a site between the upstream and the downstream homologous arms of pyr4, so that the purpose of obtaining corresponding expression strains is achieved.
3. Verification and acquisition of Trichoderma reesei expression Strain
The genome DNA of the obtained transformant is extracted, the genome DNA is taken as a template, and the PCR technology is adopted to carry out the anchoring verification of the upstream homology arm sequence, wherein the upstream homology arm sequence anchoring verification primers are AnUp-F and An-R, and the length of a target band is about 2.5kb; the downstream homology arm sequence anchors and verifies primer An-F/AnUp-R, the length of the destination band is about 2.5kb;
the primer sequences are as follows:
AnUp-F:5’-TCAAGGGTTCACGAAAGACG-3’，
An-R:5’-CTCACGCCTCTGTCTGTAAT-3’；
An-F:5’-GAAACCGACGCCCCAGCACT-3’，
AnDown-R:5’-CCGTAGCCGTGGCGGTCATT-3’。
the transformant with correctly anchored upstream and downstream homology arms is purified by adopting a monospore separation method, the internal gene verification between the upstream and downstream homology arms of pyr4 is carried out by adopting a PCR technology and In-F and In-R primers, no target bands exist, namely the internal gene is successfully replaced by an expression cassette and a screening marker hph, the construction of the expression strain is successful, and the verification result is shown In figure 2.
The primer sequences are as follows:
In-F:5’-GACCTCGCCGGGACGTGCAA-3’，
In-R:5’-TCTTTAGCTTTGACCGGTGAGCCGG-3’
the above-mentioned verification PCR reaction systems were 20. Mu.L, and specific reaction systems and reaction conditions were described in GenStar2×Taq PCR Starmix (available from Wohawave technologies Co., ltd.).
As can be seen from FIG. 2, xyn 2-directed expression strains of the heterologous xylanase Xyn10A as well as CBHI signal peptide and CBH II-CBM-directed expression strains, respectively, used as references were successfully constructed. In the upstream and downstream homology arm sequence anchoring verification, the original strain Trichoderma reesei QM 9414-delta pyr4 has no target band, the recombinant Trichoderma reesei has a bright target band in the upstream and downstream homology arm sequence verification, the upstream homology arm band size is about 2.5kb, and the downstream homology arm band size is about 1.5kb, and the downstream homology arm band sizes are consistent with the expected band sizes; in the verification of the internal genes, the starting strain Trichoderma QM 9414-. DELTA.pyr4 had a bright target band of about 1.5kb in size, and the absence of this band in recombinant Trichoderma reesei indicated successful acquisition of recombinant Trichoderma reesei xyn2-xyn10A, cbh1SP-xyn10A and cbh2CBM-xyn10A.
Example 3: analysis of expression and secretion levels of heteroxylanase Xyn10A under glucose culture conditions
Spores of the recombinant Trichoderma reesei xyn2-xyn10A, cbh1SP-xyn10A, cbh CBM-xyn10A obtained in example 2 and Trichoderma reesei QM 9414-. DELTA.pyr4 as a starting strain were inoculated into MA liquid medium containing 0.2% peptone and 1% glycerol, respectively, placed in a shaker at 30℃and 200rpm for 36 hours, and an equal amount of wet weight mycelium (about 2 g) was transferred into MA liquid medium containing 2% glucose, and an extracellular fermentation broth sample was collected at fixed time.
1. SDS-PAGE and Western blot analysis of extracellular fermentation broths
20. Mu.L of the fermentation broth was taken and analyzed by conventional SDS-PAGE, and subjected to conventional Coomassie brilliant blue staining and decolorization, and the results are shown in FIG. 3.
As can be seen from FIG. 3, in comparison with the starting strain Trichoderma reesei QM 9414-. DELTA.pyr4, clear protein bands were present in the extracellular fermentation broths of the recombinant Trichoderma reesei Xyn2-Xyn10A, cbh SP-Xyn10A and cbh2CBM-Xyn10A constructed in example 2, the sizes of the bands were consistent with the expected molecular weights, indicating that three recombinant Trichoderma reesei strains successfully expressed the secretory xylanase Xyn10A under glucose fermentation culture conditions. The content of xylanase Xyn10A in the fermentation liquid of the recombinant Trichoderma reesei Xyn2-Xyn10A is obviously more than that of xylanase Xyn10A in the fermentation liquid of the recombinant Trichoderma reesei cbh1SP-Xyn10A and cbh2CBM-Xyn10A, which shows that the effect of promoting the expression and secretion of xylanase Xyn10A by using Xyn2 as a fusion expression element is optimal.
In order to further verify the results of SDS-PAGE, sensitive Western blot was used to quantify the secretion levels of xylanase Xyn10A in the extracellular fermentation broth of each recombinant Trichoderma reesei, as well as the starting strain. As described in example 1, xyn10A carries 6 His tags at the ends, so Western blot analysis was performed using anti-His antibodies, and the results are shown in FIG. 4.
As can be seen from FIG. 4, the protein hybridization bands of xylanase Xyn10A showed molecular weights consistent with those of SDS-PAGE, and the brightness analysis of the hybridization bands showed that the content of xylanase Xyn10A in the recombinant Trichoderma reesei Xyn2-Xyn10A fermentation broth was significantly higher than the content of xylanase Xyn10A in the recombinant Trichoderma reesei CBH1SP-Xyn10A and CBH2CBM-Xyn10A fermentation broth, further demonstrating that the expression secretion level of xylanase Xyn10A guided by fusion expression element Xyn2 was significantly higher than that guided by the existing fusion expression elements CBHI signal peptide and CBH II-CBM.
2. Xylanase enzyme activity analysis in fermentation liquor
The xylanase enzyme activity analysis method comprises the following steps: 20. Mu.L of the 10-fold diluted fermentation broth and 100. Mu.L of xylan substrate (0.5% w/v) dissolved in acetic acid-sodium acetate buffer (pH 4.0) were mixed uniformly and reacted at 60℃for 30min, and then 120. Mu.L of LDNS (3, 5-dinnitrosalicylic acid) was added and mixed uniformly and boiled for 10min to terminate the reaction. After cooling, the reaction solution was taken and the absorbance of the product was read at 550nm, and the result is shown in FIG. 5.
One enzyme activity unit of xylanase is defined as the amount of enzyme required to produce 1. Mu. Mol of reducing sugar per minute.
As can be seen from FIG. 5, the xylanase enzyme activity was not detected in the fermentation broth since the endogenous (hemi) cellulase gene of the starting strain Trichoderma reesei QM 9414-. DELTA.pyr4 was in a transcription repressed state under glucose conditions. And obvious xylanase enzyme activities can be detected in extracellular fermentation liquid of recombinant Trichoderma reesei Xyn2-Xyn10A, cbh1SP-Xyn10A and cbh2CBM-Xyn10A, which indicates that the recombinant Trichoderma reesei Xyn2-Xyn10A and the cbh2CBM-Xyn10A can be successfully expressed and secreted. Wherein the level of Xyn 2-directed xylanase enzyme activity is significantly higher than the corresponding levels of CBHI signal peptide and CBH II-CBM-directed enzyme activity, about three times the latter two, the recombinant Trichoderma reesei Xyn2-Xyn10A reaches about 170U/mL when cultured for 96 hours, and the recombinant Trichoderma reesei CBH1SP-Xyn10A and CBH2CBM-Xyn10A are both less than 55U/mL.
Example 4: analysis of expression and secretion levels of heteroxylanase Xyn10A under cellulose culture conditions
Spores of the recombinant Trichoderma reesei xyn2-xyn10A, cbh1SP-xyn10A, cbh CBM-xyn10A obtained in example 2 and Trichoderma reesei QM 9414-. DELTA.pyr4 as a starting strain were inoculated into MA liquid medium containing 0.2% peptone and 1% glycerol, respectively, placed in a shaker at 30℃and 200rpm for 36 hours, and an equal amount of wet weight mycelium (about 2 g) was transferred into MA liquid medium containing 1% microcrystalline cellulose, and a fermentation broth sample was taken at fixed time to preserve the cellulase enzyme activity to be measured. Wherein the MA liquid medium formulation is as shown in example 3.
1. Western blot analysis of extracellular fermentation broths
Because the Trichoderma reesei strain has more endogenous secretase components under the condition of cellulose, the xylanase xyn10A band cannot be intuitively analyzed by SDS-PAGE, so that Western blot analysis is directly performed on extracellular fermentation broth, and the result is shown in FIG. 6.
As can be seen from FIG. 6, compared with the starting strain Trichoderma reesei QM 9414-. DELTA.pyr4, the recombinant Trichoderma reesei Xyn2-Xyn10A, cbh SP-Xyn10A constructed in example 2 and the expression strain extracellular fermentation broth of cbh2CBM-Xyn10A both showed distinct protein hybridization bands, the sizes of which were consistent with the expected molecular weights, indicating successful expression and secretion of xylanase Xyn10A under the conditions of cellulose culture. The brightness analysis of the hybridized strip shows that the expression level of the xylanase Xyn10A guided by Xyn2 in the recombinant Trichoderma reesei Xyn2-Xyn10A is obviously higher than that of the xylanase Xyn10A guided by CBHI signal peptide and CBH II-CBM, namely the expression and secretion promoting effect of the xylanase Xyn10A by taking Xyn2 as a fusion expression element is optimal.
2. Xylanase enzyme activity analysis in fermentation liquor
Xylanase enzyme activity assay method is shown in example 3.
Under the condition of cellulose, the Trichoderma reesei strain can generate a certain level of endogenous xylanase, so that the measured xylanase enzyme activity is the total enzyme activity caused by the expression of the endogenous enzyme and Xyn10A, and the result is shown in FIG. 7.
As can be seen from FIG. 7, the level of xylanase total enzyme activity in the extracellular fermentation broth of Xyn 10A-expressing strain induced by Xyn2 in recombinant Trichoderma reesei Xyn2-Xyn10A was significantly higher than the corresponding xylanase Xyn10A total enzyme activity level of CBHI signal peptide and CBH II-CBM-induced strain, reaching about 270U/mL, whereas the xylanase Xyn10A total enzyme activities of recombinant Trichoderma reesei CBH1SP-Xyn10A and CBH2CBM-Xyn10A were less than 180U/mL. The result further shows that under the condition that Xyn2 is used for guiding the efficient expression and secretion of the heterologous xylan Xyn10A, the xylanase expression and secretion average of the Trichoderma reesei strain under the conditions of glucose and cellulose is remarkably improved. Under the condition of high-yield cellulose of endogenous cellulase, compared with xylanase enzyme activity expressed by Trichoderma reesei QM 9414-delta pyr4 of an original strain, the enzyme activity is improved by more than 2 times, and the enzyme activity is obviously higher than that of a strain guided by the existing fusion expression element CBHI signal peptide and CBH II-CBM, so that a high-efficiency cellulase-xylanase production system is obtained.
Claims (10)
1. A method for increasing xylanase yield in trichoderma reesei, characterized in that the method employs endogenous protein Xyn2 as a fusion expression element for directing expression of heterologous xylanases.
2. The method of claim 1, wherein the fusion expression element Xyn2 has a nucleotide sequence shown in SEQ ID No.2 and an amino acid sequence shown in SEQ ID No. 1.
3. The method of claim 1, wherein the heterologous xylanase is xylanase Xyn10A.
4. A recombinant Trichoderma reesei is characterized in that an endogenous protein Xyn2 gene of claim 2 and a xylanase Xyn10A gene of claim 3 are respectively connected to a skeleton plasmid, and then the recombinant plasmid is transformed into the Trichoderma reesei to obtain the recombinant Trichoderma reesei.
5. The method for constructing recombinant trichoderma reesei of claim 4, comprising the specific steps of:
(1) The Trichoderma reesei QM9414 genome DNA is used as a template, and a fusion expression element Xyn2 sequence is obtained through PCR amplification;
(2) Taking plasmid pPICZ alpha A-Xyn10A as a template, and obtaining xylanase Xyn10A sequence through PCR amplification;
(3) Connecting coding genes of a fusion expression element Xyn2 and xylanase Xyn10A together through overlap extension PCR, and obtaining a fusion fragment Xyn2-Xyn10A through PCR amplification by taking the fragment as a template;
(4) The method comprises the steps of taking Trichoderma reesei QM9414 genome DNA as a template, carrying out PCR amplification to obtain an upstream and downstream homologous arm sequence of pyr4 gene, connecting the upstream and downstream homologous arm sequence into a vector pUC19-hph containing a hygromycin resistance gene hph expression cassette, and then sequentially introducing a promoter tcu and a terminator TrpC between the upstream homologous arm and the hph expression cassette to obtain a recombinant expression vector pUC19-Ptcu1-pyr4site;
(5) Connecting the fusion fragment xyn2-xyn10A between a promoter and a terminator of the recombinant expression vector pUC19-Ptcu1-pyr4site to obtain a recombinant expression vector Ptcu1-xyn2-xyn10A;
(6) Transferring the recombinant expression vector Ptcu1-xyn2-xyn10A into Trichoderma reesei QM 9414-delta pyr4 protoplast, and obtaining recombinant Trichoderma reesei xyn2-xyn10A through regeneration, screening and verification.
6. The method of claim 5, wherein in step (1), the primer sequences used for the PCR amplification are as follows:
xyn2F:5’-ACGACCTGGTTGATACGACAGGCGCGCCATGGTCTCCTTCACCTCCCTC-3’；
xyn2R:5’-TCTGGTTCGTATCGACGTAGGTGTCAGAACCTCCAGAACCTCCGCTGACG GTGATGGAAGCA-3’；
in the step (2), the primer sequences used for the PCR amplification are as follows:
xyn10F:5’-TGCTTCCATCACCGTCAGCGGAGGTTCTGGAGGTTCTGACACCTACGTCG ATACGAACCAG-3’；
xyn10R1:5’-TTAGTGATGGTGATGGTGGTGGACCGTCTGTCCAAGCC-3’。
7. the method of claim 5, wherein in step (3), the primer sequences used for the PCR amplification are as follows:
xyn2F:5’-ACGACCTGGTTGATACGACAGGCGCGCCATGGTCTCCTTCACCTCCCTC-3’；
xyn10R2:5’-TGATTTCAGTAACGTTAAGTCTTAAGTTAGTGATGGTGATGGTGGTGGA CC-3’；
in step (4), the PCR amplification primer sequences of the upstream homology arms are as follows:
upF:5’-GAATTCGAGCTCGGTACCCGGGATGATAATGGACTGGACCG-3’；
upR:5’-ATTCTTATAATCTCTAGAGGATCCTACCCGATATTGCGACTTT-3’；
the PCR amplification primer sequence of the downstream homology arm is as follows:
downF:5’-AGACTAGTCCAATCGTCGACGGGAGGGAAGGGAAGAAAGAAGTAA-3’；
downR:5’-ACCATGATTACGCCAAGCTTCTCGTAGGCGCAGTGGGCTT-3’。
8. use of the recombinant trichoderma reesei of claim 5 for the production of xylanase.
9. A method for increasing xylanase yield in trichoderma reesei, comprising the specific steps of:
inoculating the recombinant Trichoderma reesei xyn2-xyn10A according to claim 5 into a MA liquid culture medium with 1% (v/v) glycerol as a carbon source for preculture, culturing at 30+/-2 ℃ for 30-40 h at 180-220 rpm, and collecting hyphae; then transferring into MA liquid culture medium which takes 2% (w/v, g/mL) glucose as carbon source, and continuously culturing at 30+/-2 ℃ and 180-220 rpm to produce xylanase.
10. The method of claim 9, wherein the MA broth formulation is: na was added to 1L of the medium 2 HPO 4 ·12H 2 O 17.907g、(NH 4 ) 2 SO 4 1.4 g、KH 2 PO 4 2.0g, 0.3g urea, tween-800.5mL, and adjusting pH to 5.0 with anhydrous citric acid; before use, mgSO with final concentration of 0.6g/L is added 4 Final concentration of 0.4g/LCaCl 2 And adding trace elements (final concentration 1×, mother liquor 1000×), 10mM uridine;
the 1000 times trace element mother liquor formula comprises: feSO 4 ·7H 2 O 0.5g，MnSO 4 ·H 2 O 0.16g，ZnSO 4 ·7H 2 O 0.14g，CoC l2 ·2H 2 O0.2 g with ddH 2 O is fixed to volume of 100mL, and filtering and sterilizing are carried out.
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| CN117343950B (en) * | 2023-12-06 | 2024-02-06 | 中国农业科学院北京畜牧兽医研究所 | Method for improving expression level of trichoderma reesei expression exogenous protein |
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