CN114107360B - Method for improving cellulase expression of trichoderma reesei by interfering phosphatase gene - Google Patents

Method for improving cellulase expression of trichoderma reesei by interfering phosphatase gene Download PDF

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CN114107360B
CN114107360B CN202210104206.3A CN202210104206A CN114107360B CN 114107360 B CN114107360 B CN 114107360B CN 202210104206 A CN202210104206 A CN 202210104206A CN 114107360 B CN114107360 B CN 114107360B
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trichoderma reesei
ymr1
interfering
gene
expression
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CN114107360A (en
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苏小运
孙先花
姚斌
罗会颖
王晓璐
秦星
王苑
涂涛
张�杰
柏映国
于会民
黄火清
张红莲
王亚茹
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Institute of Animal Science of CAAS
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)

Abstract

The invention relates to the field of genetic engineering, in particular to a method for improving the expression of trichoderma reesei cellulase by interfering phosphatase gene. The invention relates to phosphatase gene related to trichoderma reesei participating in intracellular protein degradationymr1RNAi mediated gene silencing is carried out, and the operation improves the protein expression quantity and the cellulase activity of the trichoderma reesei.

Description

Method for improving cellulase expression of trichoderma reesei by interfering phosphatase gene
Technical Field
The invention relates to the field of genetic engineering, in particular to a method for improving the expression of trichoderma reesei cellulase by interfering phosphatase gene.
Background
The filamentous fungus trichoderma reesei is widely used as an important industrial production strain for industrial products such as: production of feed enzymes, organic acids, antibiotics, etc. It has the characteristics of high expression quantity, strong secretion capacity and the like, and simultaneously has a plurality of post-translational processing modes similar to those of eukaryotes, such as glycosylation, protease cleavage, disulfide bond formation and the like. In the mixed fermentation liquor, the expression of cellobiohydrolase accounts for more than 50% of the total extracellular secreted protein. However, the high cost of cellulase is still one of the major bottlenecks in cellulose biorefinery, so that new methods need to be developed continuously to improve the expression of cellulase and reduce the application cost.
In trichoderma reesei, cellulase expression is affected by a number of regulatory pathways. The main regulation is the coordinated regulation of transcription factors such as main activating factors and inhibiting factors, which occur at the transcription level. Transcription and translation can be upgraded to higher levels due to the combined use of these multiple strategies, but this may cause new problems for protein secretion, and some proteins that cannot be folded in time may be degraded by the ERAD pathway introduced into the endoplasmic reticulum, forming a bottleneck limiting protein expression. In theory, however, the ERAD pathway, like other cellular processes, may not be perfect in itself, which can lead to "accidental injury" of the correctly folded protein. However, the ERAD degradation pathway of endoplasmic reticulum is not clearly understood at present, and the research of improving protein expression by modifying the pathway through genetic engineering is not many. In trichoderma reesei, glucosidase II is part of the endoplasmic reticulum quality control system. In Trichoderma reesei Rut C-30 mutant strains, it was found by genomic sequencinggls2αThe gene is subjected to frame shift mutation and is over-expressedgls2αThe gene results in reduced RutC-30 protein secretion and reduced cellulase activity (Carvalho et al 2011). Therefore, it is important to further explore novel regulatory genes of the ERAD pathway.
Disclosure of Invention
The invention aims to provide a method for improving the expression of Trichoderma reesei cellulase by interfering a phosphatase gene.
The method for improving the expression of the Trichoderma reesei cellulase by interfering with the phosphatase gene comprises introducing a regulatory gene into Trichoderma reeseiymr1The step of interfering with the sequence of (1), the regulatory geneymr1The nucleotide sequence of (a) is shown as SEQ ID NO: 1 is shown.
The method for improving the expression of the Trichoderma reesei cellulase by interfering the phosphatase gene comprises the following steps of amplifying the following primers to obtain the regulatory geneymr1The interfering sequence of (a):
ymr1iF: 5’GACAATGACCAGCAGACCATCGAG3’
ymr1iR: 5’TCGCTCTGATAATGGCCAGGGGAG3’。
the method for improving the expression of the Trichoderma reesei cellulase by interfering with phosphatase gene comprises the step of constructing a regulatory geneymr1The step of recombining the vector with the interference sequence of (1).
The method for improving the expression of the Trichoderma reesei cellulase by interfering with the phosphatase gene according to the invention, whereintef1Promoter, and regulatory geneymr1The interfering sequence of,pkiThe promoters are spliced in series in a seamless splicing mode to obtain the recombinant vector.
The invention relates to phosphatase gene related to trichoderma reesei participating in intracellular protein degradationymr1RNAi mediated gene silencing is carried out, and the operation improves the protein expression quantity and the cellulase activity of the trichoderma reesei.
Drawings
FIG. 1 is a plasmid map of pPtef1-ymr1-Ppki for improving cellulase expression;
FIG. 2 showsymr1iComparing the strain with the extracellular protein of SUS4-1 strain;
FIG. 3 showsymr1iComparing the cellulase activity of the strain with that of SUS4-1 strain;
FIG. 4 showsymr1iThe transcript abundance of the strain-associated cellulase self-gene.
Detailed Description
Example 1 construction of plasmid pPtef1-ymr1-Ppki
Construction of pPtef1-ymr1-Ppki plasmid:tef1a promoter,ymr1Interference gene 413 bp,pkiA promoter and an ampr + ori moiety. Amplifying the ampr + ori part by using an APA-GOD plasmid as a template; taking a trichoderma reesei genome as a template,tef1a promoter,ymr1Interference gene 413 bp (ymr 1iF: GACAATGACCAGCAGACCATCGAG)
(ii) a ymr1iR: CTTTTCGCTCTGATAATGGCCAGG) andpkithe 4 fragments obtained by the promoter, electrophoresis and recovery are connected by a homologous recombination method. Escherichia coli Trans1-T1 competent cells were transformed, colony PCR was performed on coliform colonies growing on the plate, coliform colonies identified as positive by PCR were sequenced, and the correctly sequenced plasmid was designated as pPtef1-ymr 1-Ppkin plasmid, as shown in FIG. 1.
Example 2 introduction of pPtef1-ymr1-Ppki plasmid
(1) Transformation of plasmid pPtef1-ymr1-Ppki into Trichoderma reesei SUS4-1
Inoculating Trichoderma reesei SUS4-1 on a potato culture medium (PDA) plate, standing and culturing at 28 ℃ for 7 d until spores are produced, scraping the spores, inoculating the spores into a 100mg/ml PDB culture medium containing uracil, and shaking and culturing at 28 ℃ and 160 rpm overnight. And filtering the germinated hyphae by a 200-mesh sieve, and adding 10 mg/ml cellulase for digestion at 30 ℃ for 2-3 hours. After collection of protoplasts, pPtef1-ymr1-Ppki was used as a plasmidSspI, enzyme digestion and transformation of the Trichoderma reesei host cell.
(2) PCR verification of the introduction of pPtef1-ymr 1-Ppkin plasmid into the genome of Trichoderma reesei
Individual transformants were picked, inoculated into 24-well plates containing MM-glucose medium, and cultured at 28 ℃ for 5 to 7 days. Genomic DNA was extracted to verify the introduction of pPtef1-ymr1-Ppki plasmid into the genome of Trichoderma reesei. PDA sporulation was performed on transformants that amplified a PCR band that matched the expected PCR band.
Example 3.pPtef1-ymr1-PpkiInfluence on protein expression
(1) Interferenceymr1Shake flask induction of transformants of (1)
Interferenceymr1The transformant and the starting strain of (2X 10) were inoculated separately7Spores were cultured in 50 ml of MM-glucose medium at 28 ℃ and 160 rpm for 2 days. Was transferred to 50 ml of MM +2% Avicel medium at an inoculum size of 10% to induce expression of cellulase. Samples were taken every 24 h starting on day 3 and continued for up to 7 days.
(2) Interferenceymr1Protein concentration of transformant of gene, determination of cellulase
Protein quantification is carried out by a Coomassie brilliant blue method, after adding 250 mul of 1 × dye reagent dye and 10 mul of protein standard, reaction is carried out for 10 minutes at room temperature, and then the light absorption value at 595 nm is measured, and the result is shown in figure 2. It can be seen thatymr1The protein concentration of the transformant after gene interference is higher than that of the original strain during the whole culture period.
Cellulase determination was performed using sodium carboxymethylcellulose (1.5% CMC-Na) as substrate. Prepared with citric acid-disodium hydrogen phosphate buffer (0.05M, pH 5.0.0). Adding 100 mul of enzyme solution which is diluted properly into 900 mul of CMC-Na substrate, oscillating and mixing uniformly, preserving heat for 30 min in a water bath at 50 ℃, adding 1.5 ml of DNS reagent into each test tube when the reaction is ended, boiling for 5 min in boiling water, rapidly cooling, and measuring the absorbance at 540 nm. The amount of enzyme required to hydrolyze sodium carboxymethylcellulose per hour at 50 ℃ and pH 5.0 with 1 ml of liquid enzyme to produce 1. mu. mol of reducing sugar (in terms of glucose) was defined as one enzyme activity unit (U), and the results are shown in FIG. 3. Visible pairymr1The enzyme activity of the endo-cellulase of the transformant after gene interference is higher than that of the original strain in the whole culture stage. After SUS4-1 is cultured in MM-Avicel for 6 days, the enzyme activity of endo-cellulase is 28.0U/ml, and the enzyme activity of pPtef1-ymr1-Ppki strain cellulase is 43.1U/ml, which is improved by 0.54 times compared with the original strain. Therefore, the temperature of the molten metal is controlled,ymr1the reduction of the expression level of the gene plays an important role in improving the cellulase.
Example 4 transcriptional abundance of pPtef1-ymr1-Ppki Strain-related cellulases and self genes
Extracting RNA of pPtef1-ymr1-Ppki strain and SUS4-1 strain cultured in microcrystalline cellulose Avicel for 24 h, and determining related cellulase and self-matrixTranscript abundance of genes (see FIG. 4). Can see, it is rightymr1After the RNAi interference is performed,cbh1、cbh2、egl1andegl2the transcription level of the genes is significantly higher than that of the control strainymr1The transcription level of the gene was lower than that of the control strain. In the detection experiment of the related gene expression level, each sample is provided with three samples for parallel induction culture, each sample is subjected to 3 repeated fluorescence quantification technologies, the actin is selected as the reference gene, and a method for calculating the transcription level change of each gene is adopted 2-ΔΔCtA method.
The above embodiments are only used to explain the technical solutions of the present application, and do not limit the protection scope of the present application.
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> a method for improving the expression of Trichoderma reesei cellulase by interfering with phosphatase gene
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2349
<212> DNA
<213> Trichoderma reesei
<400> 1
atggaagcca agaagatcat cgaacatgtc caggccatca acggcggcca gacggccacc 60
ggcacgctga cactgaccaa cttccacatg atctttaccg ccatcatcga acagccacct 120
caagacggcc aagagtcgac gcctccgccg aagaagcgcg agaaatggat cacgtacccg 180
atgctctccc attgcacatt ccggccgacg cctccgtcgt cccgccaggc accgtcaata 240
cgcatccaat gccgggactt taccttcatc accttcaact tccccaccac cgaggtggcg 300
cgtgaggctt ttgaccatat caagtgtcgc acttgcaagc ttggcaccgt ccagaaccta 360
tttgccttta gccacaagcc gcccaagatg gagaaggagg tcaacggctg gaacatctat 420
gaccccagag cggagtttcg tcgccagggc atcagcgaga agctgccgga caagggctgg 480
aggatcacca acatcaacca cgactacagt ttttgcgaca cctatccggc cgtcctcgtc 540
gtgccgtcca agatctccga taatgttctc aaatacgcca aggagttccg gtccaggaat 600
cggatccccg ccctctctta catccatccg gtcaataact gcaccatcac gcggagttct 660
cagccgttgg caggcatcac caggaggact aatgtccagg acgaaaagct ggtcgctgct 720
tctttttcgg cctcgaccgg tagtggtggc tcggaaagtt cctcgccaat gtcgagtcag 780
gcagaggtgt ccaacggcag cgtcatgctg gaaactcaac tcacggaaac ggagcggtac 840
gaagacgagc tcatttctag atctgctgct ctctacgatg acaagaccgg caagcggctc 900
atctatggcg ctcaacagaa caacctcatc gtggatgctc gtccgacgat caatgccatg 960
gtgaatcagg tgcaggggat gggctcagag ccgatggaca agtacaagtt tgcacagaag 1020
gttttcctca acattgacaa catccacatc atgcgaaact ccctgctcaa ggtggtggag 1080
gcgctcaagg atgccgactt gtctcctctg ccccccaacc gcgagctcct gcaccagagc 1140
aactggctaa ggcacattca cgatatcctt gatgggtcag ccctcattgc ccgtcaggtc 1200
ggcatcaagc attcgcacgt tctgatccac tgctcagatg gctgggatcg caccagccag 1260
ctgagtgccc tggcccaggt catgctggac ccctactatc gcaccattga cggcttcatc 1320
gtcctcgtag aaaaagactg gctgtctttt ggccacatgt tcaggttacg ctccggccac 1380
ctcaattccg aagactggtt caccgtacaa aaggacgcgt tcgccggtct caaggtgcag 1440
ccgggagaga gtgacggacg gaccgacgcc ttccaaaacg tcattagcgg cgccaaacgc 1500
ttcttcagct cgaacaaaga agacgccgac cttgcttcca tgggcgagac tgccagcggt 1560
caggtagtgg acgaggaagc caccgtgcca aagatgatca gcccggtttt ccaccagttc 1620
ctcgactgca tgtaccagct gctgcggcag aacaagacgc gcttcgagta caatgaacga 1680
ttcctccggc gtctgatgta tcactcgtac tcgtgtcagt acggaacctt tctgtacaac 1740
tcagagaagc agcgaaaaga tgcccgcgta gcagagagga cgtcatcagt gtgggactac 1800
ttcctctgcc ggagagctga attcaccaac cctgactacg acccgacaat cgatgatcat 1860
gtcaagggaa aggaacgcat cattctgccg cgtcttgacg agatccgttg gtggcatcag 1920
ctgtttggcc gcaccgagga cgaaatgaac ggtgcgctca acgccgccgc cattgcggaa 1980
agcgaccggc aggctgccgt ctcgagcctc cactatccca gcgtggcccg agcagaagag 2040
ggagcgccag agacatcggc atcgtcggcc ccgagtccac ggccgcccag cctgtcaacc 2100
agccagtcgg tgctcacgtc ggtcgagacc gcgcacggca tcttgacgcc cgaagacagg 2160
cacgccactc tacaacacag cgtgtctgca gacgggatgg gcgcctttgc ggcccttcgc 2220
gatggcatcg ctggcctcaa cattggcaag gccgtcatgt ccaactttgg ccgtgccgcc 2280
gagcccgctg ctgaaggctc gacggcagcc cccatttctc gcgatcagga gatgcgggag 2340
atgacgtag 2349

Claims (4)

1. A method for improving the expression of Trichoderma reesei cellulase by interfering with phosphatase gene, which comprises introducing regulatory gene into Trichoderma reeseiymr1The step of interfering with the sequence of (1), the regulatory geneymr1The nucleotide sequence of (a) is shown as SEQ ID NO: 1 is shown.
2. The method of claim 1, wherein the regulatory gene is obtained by amplifying the following primersymr1The interfering sequence of (a):
ymr1iF: 5’GACAATGACCAGCAGACCATCGAG3’,
ymr1iR: 5’TCGCTCTGATAATGGCCAGGGGAG3’。
3. the method of claim 1, wherein the method comprises constructing a vector comprising the regulatory geneymr1The step of recombining the vector with the interference sequence of (1).
4. The method of claim 3, wherein the expression of the Trichoderma reesei cellulase is increased by interfering with a phosphatase genetef1Promoter, and regulatory geneymr1The interfering sequence of,pkiThe promoter is seamlessly spliced to obtain the recombinant vector.
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