CN116426507A - Trichoderma reesei CBH І signal peptide mutant and application thereof in promoting efficient expression and secretion of heterologous protein - Google Patents
Trichoderma reesei CBH І signal peptide mutant and application thereof in promoting efficient expression and secretion of heterologous protein Download PDFInfo
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Abstract
The invention relates to a Trichoderma reesei CBHI signal peptide mutant and application thereof in promoting efficient expression and secretion of heterologous proteins. Wherein the amino acid sequence of the Trichoderma reesei CBHI signal peptide mutant SPM1 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO. 2. The invention is based on Trichoderma reesei CBHI signal peptide, and two leucine are added after the fourth lysine to obtain Trichoderma reesei CBHI signal peptide mutant SPM1. The mutant enhances the guiding capability of the heterologous protein and can effectively promote the expression and secretion of the heterologous protein XynST11. After the Trichoderma reesei CBHI signal peptide mutant SPM1 is introduced in the expression and secretion process of the heterologous protein XynST11, the secretion amount of XynST11 in an extracellular medium and the xylanase enzyme activity level generated by XynST11 in fermentation broth are greatly improved to 25U/mL which is far higher than that of the CBHI signal peptide, so that the Trichoderma reesei CBHI signal peptide mutant SPM1 has great application value in promoting the expression and secretion of the heterologous protein and preparing xylanase.
Description
Technical Field
The invention relates to a Trichoderma reesei CBHI signal peptide mutant and application thereof in promoting efficient expression and secretion of heterologous proteins, and belongs 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, a filamentous fungus, is an excellent representation of efficient degradation of cellulose in nature. Trichoderma reesei has strong cellulase synthesis and secretion capacity, and the content of exonuclease CBHI in an extracellular cellulase system is extremely high and accounts for more than 60% of the total secreted protein. Because Trichoderma reesei has the advantages of high-efficiency protein synthesis and secretion capacity, high safety, availability of cheap raw materials and the like, the Trichoderma reesei is also developed as a chassis cell for expression and secretion of heterologous proteins.
Secretory production of the protein is beneficial to downstream purification of target protein products and effective amplification of production scale. In the process of protein expression and secretion of eukaryotes, a signal peptide sequence at the N-terminal end of the protein plays an essential important role. The signal peptide mediates that the neosecretory protein smoothly enters the endoplasmic reticulum, namely, endoplasmic reticulum translocation is completed, and the neosecretory protein can be folded, processed and modified in the endoplasmic reticulum and enters a subsequent transportation link. Thus, a highly efficient signal peptide sequence is effective in promoting the production level of secreted proteins. Because CBHI is the highest secreted endogenous protein in Trichoderma reesei, the secretory synthesis of heterologous proteins is currently often guided by the CBHI's high-efficiency signal peptide sequence (17 amino acids at the N-terminus). However, the problem of low secretion production level of the heterologous recombinant protein is often encountered, so that a more efficient mutant sequence form needs to be developed on the basis of a CBHI efficient signal peptide sequence so as to further promote the efficient secretion production of the heterologous protein and promote the development and upgrading of trichoderma reesei chassis cells, and no corresponding technology exists at present.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a Trichoderma reesei CBHI signal peptide mutant and application thereof in promoting efficient expression and secretion of heterologous proteins.
The technical scheme of the invention is as follows:
a trichoderma reesei CBHI signal peptide mutant SPM1 having an amino acid sequence that is mutated with respect to the amino acid sequence of the trichoderma reesei CBHI signal peptide only as follows: two leucine additions were made after lysine four;
the amino acid sequence of the Trichoderma reesei CBHI signal peptide is as follows: MYRKLAVISAFLATARA.
According to the invention, the amino acid sequence of the Trichoderma reesei CBHI signal peptide mutant SPM1 is shown in SEQ ID NO. 1.
According to the invention, the nucleotide sequence of the Trichoderma reesei CBHI signal peptide mutant SPM1 is shown in SEQ ID NO. 2.
In a second aspect of the present invention, there are provided an expression cassette and a recombinant vector containing the gene encoding the trichoderma reesei CBHI signal peptide mutant SPM1. The recombinant vector is not particularly limited as long as it can replicate in a host, and may be any vector known in the art. For example, the vectors include, but are not limited to, plasmids, phages. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases integrate into the genome itself.
According to the invention, preferably, the recombinant vector is obtained by connecting a coding gene of the Trichoderma reesei CBHI signal peptide mutant SPM1 with an expression vector, and the expression vector is a plasmid.
Further preferably, the plasmid is pUC19-Ptcu1-pyr4site.
In a third aspect of the present invention, there is provided a recombinant host cell comprising a gene encoding said trichoderma reesei CBHI signal peptide mutant SPM1. Wherein the "host cell" is a host cell having a meaning generally understood in the art capable of introducing a gene encoding a mutant of the present invention, which is referred to as a recombinant host cell after introduction. The strain of the invention may be Trichoderma reesei (Trichoderma reesei) QM9414.
In a fourth aspect of the invention, there is provided an application of the trichoderma reesei CBHI signal peptide mutant SPM1 in promoting efficient expression and secretion of a heterologous protein.
Preferably, according to the invention, the heterologous protein is a heterologous xylanase.
Experimental procedures not described in detail in the present invention may be performed according to conventional experimental procedures in the art.
Advantageous effects
The invention is based on Trichoderma reesei CBHI signal peptide, and two leucine are added after the fourth lysine to obtain Trichoderma reesei CBHI signal peptide mutant SPM1. The mutant enhances the guiding capability of the heterologous protein and can effectively promote the expression and secretion of the heterologous protein XynST11. After the Trichoderma reesei CBHI signal peptide mutant SPM1 is introduced in the expression and secretion process of the heterologous protein XynST11, the secretion amount of XynST11 in an extracellular medium and the xylanase enzyme activity level generated by XynST11 in fermentation broth are greatly improved to 25U/mL which is far higher than that of the CBHI signal peptide, so that the Trichoderma reesei CBHI signal peptide mutant SPM1 has great application value in promoting the expression and secretion of the heterologous protein and preparing xylanase.
Drawings
FIG. 1 is a schematic diagram showing the alignment of the original sequence of CBHI signal peptide and the SPM1 sequence of CBHI signal peptide mutant.
FIG. 2 is a schematic diagram of the construction of a strain using CBHI signal peptide and CBHI signal peptide mutant SPM1 to direct expression and secretion of heterologous xylanase XynST11, respectively.
FIG. 3 is an agarose gel electrophoresis chart verifying that CBHI signal peptide and CBHI signal peptide mutant SPM1 respectively guide successful construction of heterologous xylanase XynST11 expression strain.
FIG. 4 is a graph showing comparison of expression and secretion levels of CBHI signal peptide and CBHI signal peptide mutant SPM1, respectively, directing the expression and secretion of heterologous xylanase XynST11.
Wherein, the A graph shows the enzyme activity level analysis of xylanase produced by XynST11 in the extracellular fermentation broth; panel B shows Western blot analysis of XynST11 in extracellular fermentation broth.
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 RXE1 modulates the essentialtransactivator XYR1and cellulase gene expression in Trichoderma reesei.
Example 1: carrier construction for CBHI signal peptide and CBHI signal peptide mutant SPM1 guiding expression and secretion of heterologous protein XynST11
1. Carrier construction for CBHI signal peptide guiding expression and secretion of heteroxylanase XynST11
(1) The primers xynST11F/xynST11R were designed and a cDNA coding fragment of the xylanase xynST11 (Asp 42-Ser331, excluding the own signal peptide coding sequence and stop codon) was obtained by PCR amplification using a plasmid containing the full-length sequence of the Streptomyces-derived xylanase gene xynST11 (see, e.g., article Biochemical Characterization of Xylanases from Streptomyces sp.B6 and Their Application in the Xylooligosaccharide Production from Viscose Fiber Production Waste. Liu L, xu M, cao Y, wang H, shao J, xu M, zhang Y, wang Y, zhang W, meng X, liu W.J Agric Food chem.2020Mar 11;68 (10): 3184-3194.) as a template, and approximately 0.7kb in length. Meanwhile, a primer xynST11-GFPF/xynST11-GFPR is designed, a cDNA coding sequence of the full length of the green fluorescent protein sfGFP is obtained through amplification, and two fragments are connected by adopting a conventional overlap extension PCR method, so that the xynST11-sfGFP fragment is obtained, and the length of the fragment is about 1.6kb. The primer cbh1-SPF1/cbh1-SPF2 was further designed, and the coding sequence (ATGTATCGGAAGTTGGCCGTCATCTCGGCCTTCTTGGCCACAGCTCGTGCT) of the CBHI signal peptide was loaded to the 5' end of the aforementioned xynST11-sfGFP fragment by nested PCR, to generate a cbh1SP-xynST11-sfGFP fragment sequence.
Wherein, the sequence of the primer is as follows:
xynST11F:5’-CCTTCTTGGCCACAGCTCGTGCTGACACCTACGTCGATACGAACCAGA T-3’；
xynST11R:5’-GTATCGACGTAGGTGTCAGCGGTTCCCGATCC-3’；
xynST11-GFPF:5’-CTCGTGCTCGACCTCGGGTGGAGGTGGAGGTTCTATGGTGAGCA AGGGCGA-3’；
xynST11-GFPR:5’-TGATTTCAGTAACGTTAAGTCTTAAGTTACTTGTACAGCTCGTCCA TGCCG-3’；
cbh1-SPF1:5’-ACGACCTGGTTGATACGACAGGCGCGCCATGTATCGGAAGTTGGCCG TCATCT-3’；
cbh1-SPF2:5’-CGGAAGTTGGCCGTCATCTCGGCCTTCTTGGCCACAGCTCGTGCT-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.
(2) 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 recombinant expression vector pUC19-Ptcu1-pyr4site which is successfully driven by the promoter tcu 1and fixed by pyr4 is constructed. Finally, the amplified fragment cbh1SP-xynST11-sfGFP is connected into the recombinant expression vector and is positioned between a promoter and a terminator, so that the recombinant expression vector Ptcu1-cbh1SP-xynST11 is obtained.
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 vector for CBHI signal peptide mutant SPM1 to guide expression and secretion of heteroxylanase XynST11
Designing a primer SPM1F, and introducing two codon sequences for encoding lysine between the codon sequences corresponding to the fifth and sixth amino acids of the CBHI signal peptide to obtain a Trichoderma reesei CBHI signal peptide mutant SPM1. The amino acid sequence of the Trichoderma reesei CBHI signal peptide mutant SPM1 is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2. Then, the obtained cbh1SP-xynST11-sfGFP fragment is used as a template by taking SPM1F/xynST11-GFPR as a primer, and the fragment SPM1-xynST11-sfGFP is obtained through PCR amplification.
The sequences of the primers are as follows:
SPM1F:5’-ACGACCTGGTTGATACGACAGGCGCGCCATGTATCGGAAGTTGTTGTTGG CCG-3’；
xynST11-GFPR:5’-TGATTTCAGTAACGTTAAGTCTTAAGTTACTTGTACAGCTCGTCCA TGCCG-3’。
and connecting the constructed SPM1-xynST11-sfGFP fragment into a recombinant expression vector pUC19-Ptcu1-pyr4site, and constructing a recombinant expression vector Ptcu1-SPM1-xynST11.
The pairs of the original sequence of the CBHI signal peptide and the SPM1 amino acid sequence of the CBHI signal peptide mutant in this example are shown in FIG. 1.
Example 2: construction of a strain in which CBHI Signal peptide and CBHI Signal peptide mutant SPM1 direct expression and secretion of heterologous xylanase XynST11
1. Preparation of Trichoderma reesei protoplast
Preparation of a concentration of 5X 10 7 ～5×10 8 individual/mL of Trichoderma reesei QM 9414-. DELTA.pyr4 spore suspension was taken1mL is inoculated in 50mL of MM liquid culture medium, and is cultivated for 16h at 28 ℃ and 160rpm to obtain mycelium culture; centrifuging mycelium culture at 4000rpm for 5min, discarding supernatant, adding cell wall lyase solution, re-suspending, performing enzymolysis at 30deg.C and 60rpm for 2.5-3 hr, filtering with G2 funnel when large amount of free protoplast appears, centrifuging filtrate at 4deg.C and 3500rpm for 5min to collect protoplast, washing the collected protoplast twice with 15mL of 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-cbh1SP-xynST11 and Ptcu1-SPM1-xynST11 obtained in the example 1 respectively, linearizing the vectors by using restriction enzyme Mss I, purifying and recovering fragments, uniformly mixing with 150 mu L of Trichoderma reesei QM 9414-delta pyr4 protoplast prepared in the step 1, adding the mixture into the bottom of a 10mL centrifuge tube, dropwise adding 1.5mL of precooled PTC solution after ice bath for 20min, standing for 20min at room temperature, adding 2mL of STC solution, and uniformly mixing to obtain a conversion solution; the transformation solution is spread on a regeneration medium plate without uridine and cultured for 5-7 days at 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-cbh1SP-xynST11 and Ptcu1-SPM1-xynST11 are respectively transformed into Trichoderma reesei protoplast, because the recombinant expression vectors carry an upstream homology arm sequence and a downstream homology arm sequence of pyr4 gene, the upstream homology arm and the downstream homology arm undergo homologous recombination with the upstream and downstream regions of pyr4 gene in genome, and a XynST11 expression cassette guided by a signal peptide and a screening marker hph are integrated at a site between the upstream homology arm and the downstream homology arm of pyr4, thereby achieving the purpose of obtaining the corresponding expression strain.
In this example, the construction of the strain in which CBHI signal peptide and CBHI signal peptide mutant SPM1 respectively guide expression and secretion of heteroxylanase XynST11 is schematically shown in FIG. 2.
3. Verification and acquisition of recombinant Trichoderma reesei 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 1.5kb; 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 3.
Wherein, the primer sequence is 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. 3, in the upstream and downstream homology arm sequence anchoring verification, the original strain Trichoderma QM 9414-. DELTA.pyr4 had no target band, and the recombinant protein expression strain had a bright target band in the upstream and downstream homology arm sequence verification, the upstream homology arm band was about 2.5kb in size, and the downstream homology arm band was about 1.5kb in size, which were all consistent with the expected band size; 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, whereas the recombinant strain was free of the band, indicating successful acquisition of the target recombinant strains cbh1SP-xynST11 and SPM1-xynST11. It was demonstrated that both CBHI signal peptide-directed XynST11 expression strain and CBHI mutant signal peptide SPM 1-directed XynST11 expression strain were successfully constructed and named recombinant Trichoderma reesei cbh1SP-xynST11 and SPM1-xynST11, respectively.
Example 3: CBHI signal peptide and signal peptide mutant SPM 1-guided analysis of expression and secretion levels of heterologous xylanase XynST11
Spores of the recombinant Trichoderma reesei cbh1SP-xynST11, SPM1-xynST11 and the starting strain QM 9414-. DELTA.pyr4 obtained in example 2 were inoculated into MA liquid medium containing 0.2% peptone and 1% glycerol, respectively, and cultured in a shaker at 30℃and 200rpm for 36 hours, and an equal amount of wet weight mycelium (about 2 g) was transferred to MA liquid medium containing 2% glucose, and extracellular fermentation broths were taken at 48 hours, 72 hours and 96 hours, respectively.
Wherein, the formula of the MA liquid culture medium is as follows: na was added to 1L of the medium 2 HPO 4 ·12H 2 O 17.907g、(NH 4 ) 2 SO 4 1.4g、KH 2 PO 4 2.0g of urea, 0.3g of Tween-800.5mL, and the pH was adjusted 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; 1000 x trace element mother liquor formula: feSO 4 ·7H 2 O 0.5g，MnSO 4 ·H 2 O 0.16g，ZnSO 4 ·7H 2 O0.14g，CoC l2 ·2H 2 O0.2 g with ddH 2 O is fixed to volume of 100mL, and filtering and sterilizing are carried out.
1. 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 6) were mixed uniformly, reacted at 70℃for 30min, then 120. Mu.L of LDNS (3, 5-dinnitrosalicylic acid) was added, and the mixture was 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. 4A.
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. 4A, the CBHI signal peptide guides the fermentation broth of recombinant Trichoderma reesei cbh1SP-xynST11 expressed by XynST11 to have no obvious xylanase XynST11 enzyme activity, which indicates that the expression and secretion level of xylanase XynST11 is extremely low; meanwhile, the fermentation liquor of the starting strain QM 9414-delta pyr4 also has no obvious xylanase XynST11 enzyme activity. In the fermentation broth of the recombinant Trichoderma reesei SPM1-xynST11 guided by the CBHI signal peptide mutant SPM1, the enzyme activity level of the xylanase XynST11 is remarkably improved, and the xylanase XynST11 reaches about 25U/mL when fermented for 96 hours, which indicates that the expression secretion amount of the xylanase XynST11 is greatly promoted under the guidance of the CBHI signal peptide mutant SPM1.
2. Western blot analysis of extracellular fermentation broths
In order to further verify the results of the above enzyme activity assay, sensitive Western blot was used to quantify the secretion levels of xylanase Xyn11ST in the extracellular fermentation broths of recombinant Trichoderma reesei cbh1SP-xynST11, SPM1-xynST11 and the starting strain QM9414- Δpyr4. As described in example 1, the ends of xylanase XynST11 were tagged with sfGFP, and therefore Western blot analysis was performed using GFP antibodies, and the results are shown in FIG. 4B.
As can be seen from FIG. 4B, the molecular weight of the protein shown by the hybridization band is consistent with the predicted molecular weight (53.8 kDa), and the brightness analysis of the hybridization band shows that the expression and secretion level of xylanase XynST11 guided by CBHI signal peptide mutant SPM1 is obviously higher than that of CBHI signal peptide, further verifies that the CBHI signal peptide mutant SPM1 greatly promotes the expression and secretion level of heterologous xylanase XynST11, and has great application value in promoting the expression and secretion of the heterologous protein and preparing xylanase.
Claims (7)
1. A trichoderma reesei CBHI signal peptide mutant SPM1, characterized in that the amino acid sequence thereof has only the following mutations with respect to the amino acid sequence of the trichoderma reesei CBHI signal peptide: two leucine additions were made after lysine four;
the amino acid sequence of the Trichoderma reesei CBHI signal peptide is as follows: MYRKLAVISAFLATARA.
2. The trichoderma reesei CBHI signal peptide mutant SPM1 encoding gene of claim 1.
3. The coding gene of claim 2, wherein the nucleotide sequence is shown in SEQ ID No. 2.
4. A recombinant vector comprising the gene encoding the trichoderma reesei CBHI signal peptide mutant SPM1 of claim 2.
5. A recombinant host cell comprising the gene encoding the trichoderma reesei CBHI signal peptide mutant SPM1 of claim 2.
6. Use of the trichoderma reesei CBHI signal peptide mutant SPM1 of claim 1 or the trichoderma reesei CBHI signal peptide mutant SPM1 gene of claim 2 for promoting efficient expression and secretion of a heterologous protein.
7. The use according to claim 6, wherein the heterologous protein is a heterologous xylanase.
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