WO2020020003A1 - 用于快速筛选重组菌株的重组表达载体及应用 - Google Patents

用于快速筛选重组菌株的重组表达载体及应用 Download PDF

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WO2020020003A1
WO2020020003A1 PCT/CN2019/095900 CN2019095900W WO2020020003A1 WO 2020020003 A1 WO2020020003 A1 WO 2020020003A1 CN 2019095900 W CN2019095900 W CN 2019095900W WO 2020020003 A1 WO2020020003 A1 WO 2020020003A1
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gene
recombinant
trichoderma reesei
cellulase
enzyme activity
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PCT/CN2019/095900
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French (fr)
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姚斌
苏小运
高飞
罗会颖
黄火清
柏映国
王苑
涂涛
王亚茹
孟昆
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中国农业科学院饲料研究所
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Priority to US17/263,562 priority Critical patent/US20210388411A1/en
Priority to EP19841706.5A priority patent/EP3831948B1/en
Priority to JP2021504371A priority patent/JP7334997B2/ja
Publication of WO2020020003A1 publication Critical patent/WO2020020003A1/zh

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    • 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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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
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    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/885—Trichoderma
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    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
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    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91097—Hexosyltransferases (general) (2.4.1)
    • G01N2333/91102—Hexosyltransferases (general) (2.4.1) with definite EC number (2.4.1.-)
    • G01N2333/91114—Cellulose synthases (2.4.1.12)
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G01N2333/926—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
    • G01N2333/928—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
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    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention belongs to the field of agricultural biotechnology, and particularly relates to a recombinant expression vector for rapid screening of recombinant strains and applications.
  • Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin.
  • cellulose as a main component for constructing a cell wall, is a natural linear macromolecular polymer formed by connecting 8000 to 10,000 glucose through ⁇ -1,4-glycosidic bonds, and cellobiose is a basic unit thereof.
  • Degradation of cellulosic polysaccharides in plant cell walls into fermentable simple oligosaccharides and monosaccharides such as glucose requires complex cellulase enzyme system synergy.
  • Trichoderma reesei As one of the major filamentous fungi, Trichoderma reesei has a strong ability to secrete cellulases. Industrially, the modified Trichoderma reesei can produce more than 100g / L of cellulase, so it has important application value in degrading plant cell wall polysaccharides and related applications.
  • the cellulase system of Trichoderma reesei includes two exo-cellulases, five endo-cellulases, one beta-glucosidase, and three lytic polysaccharides monooxygenases (LPMOs, ie Former GH61 family member).
  • Trichoderma reesei With the continuous optimization of the genetic manipulation system of Trichoderma reesei, its transformation efficiency can basically meet the needs of industry, but when a large number of transformants are obtained, how to screen for transformants that efficiently express the target protein has become the key to the problem.
  • Trichoderma reesei is a filamentous fungus, which is different from the common single pure line colonies.
  • Trichoderma reesei is a multinucleated colony with a multi-nucleated mycelium entanglement structure.
  • Flow cytometry is a technique for rapid quantitative analysis and sorting of single cells or other biological particles in the flow.
  • Flow cytometry can analyze tens of thousands of cells at high speed, and measure multiple cell characteristic parameters from one cell at the same time for qualitative or quantitative analysis. It has the characteristics of fast speed, high precision, and good accuracy.
  • the existing flow detection technology can only detect changes in the regulatory factors acting on the transcription stage, but cannot detect changes in the regulatory factors in the later stages of the transcription stage.
  • Trichoderma reesei cellulase is secreted and not expressed intracellularly through multiple key links including transcription, translation, transport, and glycosylation. Therefore, the existing flow detection technology, which can only detect changes in transcription levels, cannot meet the requirements for screening cellulases with improved enzyme activity.
  • the present application provides a recombinant expression vector for rapid screening of recombinant strains and a rapid screening method and application.
  • the present invention can quickly The screening of Trichoderma reesei cellulase-producing strains greatly shortens the screening time, improves the screening work efficiency, and obtains high-yield pure line transformants to meet the needs of actual production.
  • the object of the present invention is to provide a recombinant expression vector for rapid screening of highly expressing strains.
  • Another object of the present invention is to provide a recombinant strain containing the above-mentioned recombinant expression vector.
  • Yet another object of the present invention is to provide a method for quickly screening Trichoderma reesei with high expression of cellulase.
  • Yet another object of the present invention is to provide a method for quickly screening Trichoderma reesei with high cellulase expression and high enzyme activity.
  • a recombinant expression vector for rapid screening of highly expressing strains in which the gene expression cassette of the recombinant expression vector includes elements in order from upstream to downstream: cbh1 promoter, red fluorescent protein gene DsRed, and cell surface
  • the protein anchors the signal peptide genes AfMp1 and cbh1 terminators.
  • the nucleotide sequence of the DsRed gene is shown in SEQ ID NO.2
  • the nucleotide sequence of the AfMp1 gene is shown in SEQ ID NO.3.
  • nucleotide sequence of the cbh1 promoter is shown in SEQ ID NO.1:
  • the nucleotide sequence of the DsRed gene is shown in SEQ ID NO.2:
  • the nucleotide sequence of the AfMp1 gene is shown in SEQ ID NO.3:
  • nucleotide sequence of the cbh1 terminator is shown in SEQ ID NO.4:
  • the present invention also provides a recombinant strain for quickly screening a recombinant expression vector of a high expression strain, and the recombinant strain is preferably Trichoderma reesei.
  • the invention also provides a method for rapid screening of recombinant Trichoderma reesei, which method comprises the following steps:
  • the gene expression cassette containing the element cbh1 promoter, the red fluorescent protein gene DsRed, the cell surface protein anchor signal peptide gene AfMp1, and the cbh1 terminator is introduced into a plasmid to obtain a recombinant expression vector, in which the nucleotides of the DsRed gene The sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the AfMp1 gene is shown in SEQ ID NO.3;
  • the recombinant strains exhibiting red fluorescence on the surface are screened with a flow cytometer to obtain the recombinant strains.
  • the invention also provides a method for quickly screening Trichoderma reesei with high cellulase expression and high enzyme activity, which is characterized in that the method includes the following steps:
  • the gene expression cassette containing the element cbh1 promoter, the red fluorescent protein gene DsRed, the cell surface protein anchor signal peptide gene AfMp1, and the cbh1 terminator is introduced into a plasmid to obtain a recombinant expression vector, in which the nucleotides of the DsRed gene The sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the AfMp1 gene is shown in SEQ ID NO.3;
  • the cellulase enzyme activity of the recombinant Trichoderma reesei is screened in the determination step (4) to obtain the recombinant Trichoderma reesei with improved cellulase enzyme activity.
  • the genes related to protein secretion pathways include bip1 gene, hac1 gene, ftt1 gene, sso2 gene, sar1 gene, ypt1 gene.
  • the nucleotide sequence of the bip1 gene is shown in SEQ ID NO.5:
  • the nucleotide sequence of the hac1 gene is shown in SEQ ID NO.6:
  • step (3) of the method for rapidly screening strains highly expressing genes related to the protein secretion pathway a recombinant strain mediated by Agrobacterium tumefaciens is used to construct a gene mutant library of the recombinant strain.
  • the screening recombinant vector uses the Ti plasmid as a backbone plasmid, and the p plasmid is linked to the pry4 gene.
  • the nucleotide sequence of the pry4 gene is shown in SEQ ID NO 7:
  • the selection marker gene is pyr4, which is a gene for a nutritionally selective marker gene orotidine-5'-monophosphate decarboxylase.
  • the invention also provides a method for quickly screening genes related to protein secretion pathways that are highly associated with increasing cellulase enzyme activity, including the following steps:
  • step (3) introducing the target gene to be screened into the recombinant strain obtained in step (2), or randomly inserting a vector or gene into the recombinant strain obtained in step (2) to obtain a mutant library of the recombinant strain;
  • step (4) determining the cellulase enzyme activity of the recombinant strains exhibiting red fluorescence on the surface selected in step (4), and obtaining the recombinant strains with improved cellulase enzyme activity;
  • the invention constructs a recombinant expression vector containing a fusion gene expression cassette, and the expression cassette is sequentially connected from the upstream to the downstream of the cbh1 promoter, the cbh1 gene signal peptide sequence, the red fluorescent protein gene, the MP1 anchor protein signal peptide sequence derived from Aspergillus fumigatus, and cbh1 Gene terminator.
  • This recombinant expression vector can induce fluorescence display on the surface of the strain, thereby facilitating rapid screening.
  • the invention provides a method for quickly screening recombinant Trichoderma reesei with high expression of cellulase, and the identification of cellulase expression ability and red fluorescence intensity according to the red fluorescence intensity of recombinant Trichoderma reesei expressing cellulase.
  • the present invention places the DsRed gene under the control of a cellulase promoter.
  • CBH1 is the highest expressed cellulase in Trichoderma reesei, accounting for 50-60% of secreted proteins. Therefore, the DsRed gene was linked to the downstream of the strongly-inducible CBH1 promoter, and the depth of red was positively correlated with the degree of induction of the cbh1 promoter. Then, DsRed was coupled with a flow cytometer to screen T. reesei cellulose for high-throughput Enzyme-producing strains.
  • the red fluorescent protein with signal peptide can be effectively anchored to the cell wall, and the higher the red fluorescence intensity of the recombinant Trichoderma reesei is, the higher the cellulase expression ability is.
  • the fluorescence intensity of red fluorescent protein can well characterize the expression of cellulase, and the fluorescence intensity of red fluorescent protein on the cell wall is positively correlated with the amount of cellulase secreted in the fermentation broth.
  • Figure 1 is a Trichoderma reesei colony, the left is SUS2: the starting strain, and the right is SUS4: a positive transformant transformed into the pDsRed-AfMP1 plasmid;
  • Figure 2 shows Trichoderma reesei hyphae under a fluorescent microscope, the left picture shows Trichoderma reesei hyphae showing red fluorescent protein on the surface; the right picture shows Trichoderma reesei hyphae observed under a bright field fluorescence microscope;
  • FIG. 3 is a flow cytometry sorting diagram of a plasmid transformant related to a secretory pathway by a flow cytometer
  • Figure 4 shows the colony PCR results of secretion pathway-related transformants screened by flow cytometry.
  • 4-1 is hac1 colony PCR
  • 4-2 is bip1 colony PCR
  • 4-3 is ypt1 colony PCR
  • 4-4 is sso2 colony PCR.
  • 4-5 is sar1 colony PCR
  • 4-6 is ftt1 colony PCR;
  • FIG. 5 is a graph showing the results of measuring EG enzyme activity in a shake flask fermentation broth of a starting strain and a transformant-related gene
  • FIG. 6 is a copy number identification diagram of a transformant related to a modified secretory pathway
  • FIG. 7 shows the results of colony PCR verifying the Ti-pyr4 plasmid transformed into Agrobacterium
  • FIG. 8 is a flow cytometry sorting diagram of a randomly inserted mutant library of Agrobacterium
  • FIG. 9 is a PCR verification result of randomly inserted transformant colonies of Agrobacterium screened by convection screening
  • FIG. 10 shows the results of measuring the enzyme activity of transformants randomly inserted into Agrobacterium.
  • the components of the MM medium described in the following examples include: (NH 4 ) 2 SO 4 , 5.0 g / L; KH 2 PO 4 , 15.0 g / L; MgSO 4 ⁇ 7H 2 O, 0.6 g / L; CaCl 2 ⁇ 2H 2 O, 0.6g / L; CoCl 2 ⁇ 6H 2 O, 0.0037g / L; FeSO 4 ⁇ 7H 2 O, 0.005g / L; ZnSO 4 ⁇ 7H 2 O, 0.0014g / L; MnSO 4 ⁇ H 2 O, 0.0016 g / L; glucose (or carbon source such as Avicel), 20 g / L; the solvent used was water.
  • the cbh1 promoter and terminator were amplified from the Trichoderma reesei Tu6 genome to construct the cbh1p-DsRed-AfMp-cbh1t expression cassette.
  • the pAPA plasmid was linearized with the cbh1p-DsRed-AfMp-cbh1t expression cassette. The method is to perform homologous recombination in vivo, then transform E. coli, select positive transformants, and extract plasmids.
  • Trichoderma reesei SUS2 was inoculated on a potato culture medium (PDA) plate and allowed to stand for 7 days at 30 ° C for spore production. The spores were scraped off and inoculated into 100 mL of PDB medium containing uridine, and shaken at 30 ° C and 180 rpm Incubate overnight. The germinated hyphae were collected by filtration on 12 layers of gauze, 10 mg / mL yeast wall-breaking enzyme was added, and digested at 30 ° C for 1-2 hours. Protoplasts were collected, and the pDsRed-AfMp1 plasmid was transformed into the Trichoderma reesei SUS2 strain by PEG-mediated protoplast transformation.
  • PDA potato culture medium
  • Transformants were grown and selected on MM-glucose agar medium containing 1M sorbitol. A single clone was picked, inoculated on MM-lactose agar medium, cultured in a 30 ° C incubator for 5 days, and the color change was observed. It can express the red color that is visible to the naked eye. As shown in Figure 1, the surface of the positive transformant that successfully transformed and expressed the pDsRed-AfMp1 plasmid displayed red fluorescent protein, and the transformant was named SUS4.
  • the present invention obtains a transformant capable of expressing red fluorescent protein on the cell surface by expressing the red fluorescent protein displayed on the cell surface in Trichoderma reesei, and the transformant is cultured in MM-Avicel for 24 hours, as shown in FIG. 2,
  • the expression of red fluorescent protein can be observed under a confocal microscope.
  • T. reesei-derived protein secretion pathway-related genes in T. reesei SUS4
  • T. reesei-derived protein secretion pathway Six gene fragments related to the T. reesei-derived protein secretion pathway were selected: bip1 gene, hac1 gene, ftt1 gene, sso2 gene, sar1 gene, and ypt1 gene, respectively, and connected with appropriate promoters and terminators to construct them
  • the expression cassettes are: eno1p-bip1-eno1t, eno1p-hac1-eno1t, pdc1p-ftt1-pdc1t, pdc1p-sso2-pdc1t, gpd1p-sar1-gpd1t, gpd1p-ypt1-gpd1t.
  • the pAPA plasmid was linearized and ligated with the above six gene fragments by a seamless splicing method at a molar ratio of 1: 2, respectively, to construct six secretory pathway-related plasmids: pAPA-eno1-bip1, pAPA-eno1-hac1, and pAPA-pdc1. -sso2, pAPA-pdc1-ftt1, pAPA-gpd1-sar1, and pAPA-gpd1-ypt1.
  • the recombinant plasmid was transformed into E. coli T competent cells, and colony PCR was performed on the E. coli colonies to identify whether the six fragments have been ligated to pAPA.
  • the coliform colonies identified as positive by PCR were picked and inoculated on 1 mL of LB (containing 100 ⁇ g / mL ampicillin) medium, cultured at 37 ° C. with shaking at 220 rpm overnight, and the plasmid was extracted for preparation for transformation.
  • the above six secretion pathway related plasmids were mixed in equal volumes, and then transformed into Trichoderma reesei SUS4 strain by PEG-mediated protoplast transformation.
  • the transformant fungus pieces are picked and cultured on a new screening medium PDA, and 4 transformants can be picked on each plate and cultured at 28 ° C to produce spores.
  • spore concentration at 106 to 108 spores.
  • the analysis of transformant spores was performed using a flow cytometer.
  • the sample pressure was set to 1000 EPS (1,000 signal particles were analyzed per second).
  • the forward angle scattered light area and width map were used to remove adhesion and miscellaneous signals.
  • the fluorescence signal was excited using a 488 nm laser.
  • the negative control strain expressing RFP was used to set the voltage to distinguish the expressed spore clusters. According to the signal difference of negative positive spores, the gate was used to delimit the positive spore area, and all samples were analyzed by the same method.
  • the spores with the strongest fluorescent signal were directly sorted into 6-well plates containing PDA, and a total of 36 transformants were sorted and cultured to produce spores.
  • the flow sorting diagram is shown in Figure 3.
  • the selected transformant spores were inoculated into 25 mL of liquid MM-glucose, and cultured at 30 ° C. with shaking at 180 rpm for 1 day. Genomic DNA was extracted, and PCR was used to verify whether the six recombinant plasmids related to protein secretion have been successfully transformed into Trichoderma reesei cells.
  • the secretion pathway-related transformants selected by flow cytometry were subjected to colony PCR using 6 pairs of secretion pathway-related primers, respectively, to determine which secretion pathway-related plasmids the selected red fluorescence enhanced transformants contained.
  • the primers used are shown in the following table.
  • the PCR fragment size is about 200bp.
  • the electrophoresis results are shown in Figure 4.
  • the enzyme activity of 31 transformants obtained by PCR of the starting strain SUS4 and the colonies was determined.
  • the 3-6d fermentation broth was selected and the endocellulase enzyme activity (CMC enzyme activity) was measured.
  • Endo-cellulase enzyme activity assay The determination was performed using sodium carboxymethyl cellulose (CMC) as a substrate. Take 1000 mg of sodium carboxymethylcellulose and dilute to 50 mL with a citric acid-disodium hydrogen phosphate buffer solution (0.05M, pH 5.0) to obtain a 2% sodium carboxymethyl cellulose solution.
  • CMC sodium carboxymethyl cellulose
  • Fig. 5 The results of the enzyme activity measurement are shown in Fig. 5. There were 7 transformants with improved enzyme activity, of which three were transformants containing the hac1 gene and named SUS4-H7, SUS4-H28, SUS4-H43, and four were bip Transformants of the genes were named SUS4-B3, SUS4-B5, SUS4-B26, and SUS4-B31.
  • RTQactF (5'-TGAGAGCGGTGGTATCCACG-3 ')
  • RTQactR (5'-GGTACCACCAGACATGACAATGTTG-3 ')
  • RTQhacF (5'-ACAACGTCCTGCAGTGTCAA-3 ')
  • RTQhacR (5'-TAGCGATCTGCATCAAGGGC-3 ')
  • RTQbipF (5'-AAGAAGGTTACCCACGCCG-3 ')
  • RTQbipR (5'-ATCAAAGGTACCACCACCACCGAG-3 ')
  • the copy number of Bgl3P1 gene was relatively quantified by 2- ⁇ Ct method. Transformant copy number identification results are shown in Figures 6A and 6B. Quantitative PCR analysis showed that one or two copies of the HAC1 and BIP1 genes were integrated in the chromosome.
  • Example 3 Construction of a library of gene mutants to screen transformants with high cellulase expression and high enzyme activity
  • the pyr4 gene was transformed by Trichoderma reesei to construct the pTi-pyr4 plasmid of Agrobacterium.
  • the constructed pTi-pyr4 plasmid was transformed into AGL1 competent AGL1.
  • This plasmid contains the Trichoderma transformation screening marker gene pyr4, and the left arm (LB) and right arm (RB) used for random insertion of Agrobacterium.
  • the transformant colony grows, pick a single colony for verification, and the colony PCR verifies the target gene pry4.
  • the amplified target fragment of 500bp is consistent with the size of the pre-verified fragment and sent for sequencing and compared correctly.
  • Agrobacterium (3 ⁇ L), which had been transferred into the target plasmid pTi-pyr4, was inoculated into 3 mL of LB liquid (containing 50 ⁇ g / mL kanamycin and 25 ⁇ g / mL rifampicin) and cultured in a 28 ° C. shaker at 220 rpm.
  • a SUS4 Trichoderma reesei spore suspension was prepared, coated on a CM flat plate covered with cellophane, and pregerminated at 24 ° C for about 3 hours.
  • 100 ⁇ l of the above-mentioned Agrobacterium bacterium solution was coated on a CM plate pre-germinated with Trichoderma reesei fungus hyphae, and co-cultured at 25 ° C. in a dark environment to obtain a library of Agrobacterium tumefaciens-mediated T. reesei mutants.
  • Agrobacterium-mediated transformation was used to transform the pTi-pyr4 plasmid into SUS4 strain protoplasts, and a library of Agrobacterium-transformed mutants was successfully constructed, and the transformants were copied onto a PDA plate containing cephalosporin through a transmembrane, as shown in Figure 8 As shown, Trichoderma reesei strains were screened by flow cytometry after sporulation, and the surface of the flow cytometer showed red fluorescence.

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Abstract

提供了一种用于快速筛选高表达菌株的重组表达载体和快速筛选高表达菌株的方法。所述方法将一个外源的红色荧光蛋白（DsRed）和烟曲霉细胞表面蛋白定位信号肽（AfMP1）融合表达，将该融合基因(DsRed-AfMP1)整合到里氏木霉基因组中，从而构建里氏木霉表面展示红色荧光蛋白的菌株。将表面展示红色荧光蛋白的里氏木霉菌株通过流式细胞仪分选，可以快速分离出有益于纤维素酶活提高的基因变化。

Description

用于快速筛选重组菌株的重组表达载体及应用 技术领域
本发明属于农业生物技术领域，具体涉及一种用于快速筛选重组菌株的重组表达载体及应用。
背景技术
植物细胞壁主要由纤维素(cellulose)、半纤维素(hemicellulose)和木质素(lignin)组成。其中，纤维素作为构建细胞壁的主要成分，是一种由8000～10000个葡萄糖通过β-l,4-糖苷键连接而形成的天然线性大分子聚合物，以纤维二糖为其基本单元。将植物细胞壁中的纤维素多糖降解为可发酵的简单寡糖和单糖如葡萄糖需要复杂的纤维素酶酶系协同作用。
里氏木霉作为主要的丝状真菌之一，具有强大的分泌纤维素酶的能力。工业上，经改造的里氏木霉，其产纤维素酶的产量可达到100g/L以上，因此，在降解植物细胞壁多糖以及相关应用上具有重要的应用价值。
里氏木霉的纤维素酶系包括两个外切纤维素酶、五个内切纤维素酶、一个β-葡萄糖苷酶和三个裂解性多糖单加氧酶(lytic polysaccharides monooxygenases,LPMOs，即原GH61家族成员)。
随着里氏木霉遗传操作体系的不断优化，其转化效率已经可以基本满足工业的需要，但当得到大量的转化子时，如何筛选高效表达目的蛋白的转化子成了问题的关键。里氏木霉为丝状真菌，不同于常见的单一纯系菌落，里氏木霉为多核的菌落为多核菌丝相互缠绕形成的菌团结构。为了在高假阳性背景的转化平板上获得纯系转化子，只能通过繁琐的单孢分离过程分离单核孢子，导致遗传改造周期过长，进而导致筛选高表达量的菌株更加费时费力。
流式细胞仪技术(Flow cytometry)是一种对处在液流中的单个细胞或其它生物颗粒等进行快速定量分析和分选的技术。流式细胞仪可以高速分析上万个细胞，同时从一个细胞中测得多个细胞特征参数，进行定性或定量分析，具有速度快、精度高、准确性好等特点。但是现有的流式检测技术只能检测到作用于转录阶段的调控因子变化，而无法检测转录阶段后期的调控因子变化。里氏木霉纤维素酶通过转录、翻译、转运和糖基化修饰等多个关键环节后进行分泌表达，而非胞内 表达。因此，现有的只能检测转录水平变化的流式检测技术不能满足筛选酶活提高的纤维素酶的要求。
发明内容
为了解决现有技术中存在的无法快速筛选酶活提高的里氏木霉转化子的问题，本申请提供一种用于快速筛选重组菌株的重组表达载体和快速筛选方法及应用，本发明能够快速筛选里氏木霉纤维素酶高产菌株，大大缩短了筛选时间，提高了筛选的工作效率，获得高产量纯系转化子，以适应实际生产的需要。
本发明的目的在于提供一种用于快速筛选高表达菌株的重组表达载体。
本发明的再一目的在于提供一种含有上述重组表达载体的重组菌株。
本发明的再一目的在于提供快速筛选高表达纤维素酶的里氏木霉的方法。
本发明的再一目的在于提供一种快速筛选高表达纤维素酶且高酶活的里氏木霉的方法。
根据本发明具体实施方式，用于快速筛选高表达菌株的重组表达载体，在所述重组表达载体的基因表达盒中由上游到下游依次包括元件：cbh1启动子、红色荧光蛋白基因DsRed、细胞表面蛋白锚定信号肽基因AfMp1及cbh1终止子，其中，DsRed基因的核苷酸序列如SEQ ID NO.2所示，AfMp1基因的核苷酸序列如SEQ ID NO.3所示。
cbh1启动子的核苷酸序列如SEQ ID NO.1所示：
Figure PCTCN2019095900-appb-000001
Figure PCTCN2019095900-appb-000002
DsRed基因的核苷酸序列如SEQ ID NO.2所示：
Figure PCTCN2019095900-appb-000003
AfMp1基因的核苷酸序列如SEQ ID NO.3所示：
Figure PCTCN2019095900-appb-000004
cbh1终止子的核苷酸序列如SEQ ID NO.4所示：
Figure PCTCN2019095900-appb-000005
本发明还提供了用于快速筛选高表达菌株的重组表达载体的重组菌株，所述重组菌株优选为里氏木霉。
本发明还提供了快速筛选重组里氏木霉的方法，所述方法包括以下步骤：
(1)将含有元件cbh1启动子、红色荧光蛋白基因DsRed、细胞表面蛋白锚定信号肽基因AfMp1及cbh1终止子的基因表达盒导入质粒中，得到重组表达载体，其中，DsRed基因的核苷酸序列如SEQ ID NO.2所示，AfMp1基因的核苷 酸序列如SEQ ID NO.3所示；
(2)用步骤(1)构建的重组表达载体转化宿主细胞，得到重组菌株；
(3)培养重组菌株，诱导红色荧光蛋白表达于重组菌株的表面；
(4)筛选展示红色荧光的重组菌株。
根据本发明的具体实施方式，用流式细胞仪筛选表面展示红色荧光的重组菌株，得到重组菌株。
本发明还提供了快速筛选高表达纤维素酶且高酶活的里氏木霉的方法，其特征在于，所述方法包括以下步骤：
(1)将含有元件cbh1启动子、红色荧光蛋白基因DsRed、细胞表面蛋白锚定信号肽基因AfMp1及cbh1终止子的基因表达盒导入质粒中，得到重组表达载体，其中，DsRed基因的核苷酸序列如SEQ ID NO.2所示，AfMp1基因的核苷酸序列如SEQ ID NO.3所示；
(2)用步骤(1)构建的重组表达载体转化宿主细胞，得到重组里氏木霉；
(3)向步骤(2)得到的所述重组里氏木霉中导入蛋白分泌途径相关的基因，或者向步骤(2)得到的重组里氏木霉中随机插入载体或基因，获得重组里氏木霉的突变体库；
(4)用流式细胞仪筛选表面展示强烈红色荧光的重组里氏木霉；
(5)测定步骤(4)中筛选重组里氏木霉的纤维素酶酶活，获得纤维素酶酶活提高的重组里氏木霉。
根据本发明的具体实施方式，快速筛选与提高纤维素酶酶活相关的蛋白分泌途径相关基因的方法中，步骤(3)中，与蛋白分泌途径相关基因包括bip1基因、hac1基因、ftt1基因、sso2基因、sar1基因、ypt1基因。
bip1基因的核苷酸序列如SEQ ID NO.5所示：
Figure PCTCN2019095900-appb-000006
Figure PCTCN2019095900-appb-000007
hac1基因的核苷酸序列如SEQ ID NO.6所示：
Figure PCTCN2019095900-appb-000008
Figure PCTCN2019095900-appb-000009
根据本发明的具体实施方式，快速筛选高表达蛋白分泌途径相关基因的菌株的方法的步骤(3)中，用根瘤农杆菌介导重组菌株，构建所述重组菌株的基因突变体库。
根据本发明具体实施方式，步骤(3)中，筛选重组载体以Ti质粒为骨架质粒，Ti质粒上连接有pry4基因，pry4基因的核苷酸序列如SEQ ID NO 7所示：
Figure PCTCN2019095900-appb-000010
Figure PCTCN2019095900-appb-000011
所述筛选标记基因为pyr4，是营养选择性标记基因乳清酸核苷-5'-单磷酸脱羧酶的基因。
本发明还提供快速筛选高与提高纤维素酶酶活相关的蛋白分泌途径相关基因的方法，包括以下步骤：
(1)构建含有红色荧光蛋白基因和细胞表面蛋白锚定信号肽基因的DsRed-AfMP1融合基因的重组表达载体，其中，DsRed基因的核苷酸序列如SEQ ID NO.2所示，AfMp1基因的核苷酸序列如SEQ ID NO.3所示；
(2)用步骤(1)构建的重组表达载体转化菌株，得到重组菌株；
(3)向步骤(2)得到的所述重组菌株中导入待筛选的目的基因，或者向步骤(2)得到的重组菌株中随机插入载体或基因，获得重组菌株的突变体库；
(4)用流式细胞仪筛选表面展示红色荧光的重组菌株；
(5)测定步骤(4)中筛选出的表面展示红色荧光的重组菌株的纤维素酶酶活，获得纤维素酶酶活提高的重组菌株；
(6)确定纤维素酶酶活提高的重组菌株中所表达的外源目的基因，或被干扰的内源基因，从而获得与提高纤维素酶酶活相关的蛋白分泌途径相关基因。
本发明的有益效果：
本发明构建了含有融合基因表达盒的重组表达载体，表达盒由上游至下游依次连接cbh1启动子、cbh1基因信号肽序列、红色荧光蛋白基因、烟曲霉来源的MP1锚定蛋白信号肽序列和cbh1基因终止子。此重组表达载体可诱导菌株表面展示荧光，从而利于快速筛选。
本发明提供了一种快速筛选高表达纤维素酶的重组里氏木霉的方法，根据表达纤维素酶的重组里氏木霉的红色荧光强度的高低鉴定纤维素酶的表达能力，红色荧光强度越高的重组里氏木霉表达纤维素酶能力越强。为了将DsRed的表达与T.reesei纤维素酶的表达相关联，本发明将DsRed基因置于纤维素酶启动子的 控制下。CBH1是里氏木霉中表达量最高的纤维素酶，占分泌蛋白的50～60％。因此，将DsRed基因与强诱导CBH1启动子的下游连接，其红色的深度与cbh1启动子的诱导程度正相关，再将DsRed和流氏细胞仪相耦联高通量筛选里氏木霉纤维素酶高产菌株。实验证明，具有信号肽的红色荧光蛋白可以有效锚定到细胞壁上，红色荧光强度越高的重组里氏木霉的纤维素酶表达能力越高。红色荧光蛋白的荧光强度可以很好地表征纤维素酶的表达量，细胞壁上红色荧光蛋白的荧光强度和发酵液中纤维素酶的分泌量呈正相关。
附图说明
图1为里氏木霉菌落，左图为SUS2：出发菌株，右图为SUS4：转入pDsRed-AfMP1质粒的阳性转化子；
图2显示荧光显微镜下里氏木霉菌丝，左图为表面展示红色荧光蛋白的里氏木霉菌丝；右图为明场下荧光显微镜中观察到的里氏木霉菌丝；
图3为流式细胞仪对分泌途径相关质粒转化子的流式分选图，；
图4显示流式筛选的分泌途径相关转化子的菌落PCR结果，其中，4-1为hac1菌落PCR，4-2为bip1菌落PCR，4-3为ypt1菌落PCR，4-4为sso2菌落PCR，4-5为sar1菌落PCR，4-6为ftt1菌落PCR；
图5为出发菌株及分泌途径相关基因转化子摇瓶发酵液的EG酶活测定结果图；
图6为改造分泌途径相关基因转化子的拷贝数鉴定图；
图7显示验证转化入农杆菌的Ti-pyr4质粒的菌落PCR结果；
图8为流式细胞仪对农杆菌随机插入突变体库的流式分选图；
图9为对流式筛选的农杆菌随机插入转化子菌落的PCR验证结果；
图10为农杆菌随机插入转化子酶活的测定结果。
具体实施方式
下面结合附图对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中所述MM培养基的成分包括：(NH 4) 2SO 4，5.0g/L；KH 2PO 4，15.0g/L；MgSO 4·7H 2O，0.6g/L；CaCl 2·2H 2O，0.6g/L；CoCl 2·6H 2O，0.0037g/L；FeSO 4·7H 2O，0.005g/L；ZnSO 4·7H 2O，0.0014g/L；MnSO 4·H 2O，0.0016g/L；葡萄糖(或Avicel等碳源)，20g/L；所用溶剂为水。
实施例1在里氏木霉细胞表面展示红色荧光蛋白
1.构建表达红色荧光蛋白和烟曲霉细胞表面蛋白锚定信号肽融合基因(DsRed-AfMP1)的重组质粒pDsRed-AfMP1
从含DsRed的质粒中扩增DsRed基因片段，以及从含AfMP1的质粒中扩增AfMP1基因片段，其中DsRed基因F端引物含有51bp的cbh1基因信号肽序列，将这两个片段通过overlap PCR连接起来。
从里氏木霉Tu6基因组中扩增cbh1启动子与终止子，构建cbh1p-DsRed-AfMp-cbh1t表达盒，同时将pAPA质粒线性化后与cbh1p-DsRed-AfMp-cbh1t表达盒采用无缝拼接的方式在体内做同源重组，然后转化大肠杆菌，挑选阳性转化子，并提取质粒。
2.pDsRed-AfMp1融合基因在里氏木霉菌株SUS2中的表达
将里氏木霉SUS2接种于土豆培养基(PDA)平板上，30℃静置培养7d待其产孢，将孢子刮下并接种于100mL含有uridine的PDB培养基中，30℃、180rpm振摇培养过夜。在12层纱布上过滤收集萌发的菌丝，加入10mg/mL的酵母破壁酶，在30℃消化1-2小时。收集原生质体，将pDsRed-AfMp1质粒以PEG介导的原生质体转化法转入里氏木霉SUS2菌株。
转化子在含1M山梨醇的MM-glucose琼脂培养基上生长、选择。挑取单个克隆，接种于MM-lactose琼脂培养基上，30℃培养箱中培养5d，观察颜色变化。能表达肉眼可见的红色，如图1所示，成功转化并表达pDsRed-AfMp1质粒的阳性转化子的表面展示红色荧光蛋白，该转化子命名为SUS4。
本发明通过在里氏木霉中表达于细胞表面展示的红色荧光蛋白，获得了能够在细胞表面表达红色荧光蛋白的转化子，转化子在MM-Avicel中培养24h后，如图2所示，在共聚焦显微镜下可以观察到红色荧光蛋白的表达。
实施例2.筛选高表达纤维素酶且纤维素酶酶活高的转化子
1.在里氏木霉SUS4中表达里氏木霉来源的蛋白分泌途径相关基因
选取6个表达里氏木霉来源的蛋白分泌途径相关基因片段：分别为bip1基因、hac1基因、ftt1基因、sso2基因、sar1基因、ypt1基因，并与适当的启动子和终止子连接，分别构建表达盒为：eno1p-bip1-eno1t、eno1p-hac1-eno1t、pdc1p-ftt1-pdc1t、pdc1p-sso2-pdc1t、gpd1p-sar1-gpd1t、gpd1p-ypt1-gpd1t。
将pAPA质粒线性化，分别和上述6个基因片段按1:2的摩尔比用无缝拼接法连接，构建6中分泌途径相关质粒：pAPA-eno1-bip1、pAPA-eno1-hac1、pAPA-pdc1-sso2、pAPA-pdc1-ftt1、pAPA-gpd1-sar1和pAPA-gpd1-ypt1。将重组质粒转化大肠杆菌T感受态细胞，对大肠杆菌菌落做菌落PCR，以鉴定6个片段是否分别已经连接到pAPA上。将PCR鉴定为阳性的大肠菌落挑取并接种于1mL LB(含100μg/mL氨苄)培养基上，37℃、220rpm振摇培养过夜，提取质粒，备用于转化。将上述六种分泌途径相关质粒等体积混合后，以PEG介导的原生质体转化法转入里氏木霉SUS4菌株。
将转化子菌块挑至新的筛选培养基PDA上进行培养，每个平板可挑4个转化子，28℃培养至产孢。用8ml无菌水冲洗平板上的孢子(所有转化子的孢子)，重悬混匀，200目筛过滤至无菌尖底离心管中，即为流式细胞仪分析及分选的孢子悬液，孢子浓度控制在10 6至10 8个孢子。
转化子孢子的分析使用流式细胞仪进行，样品压力设为1000EPS(每秒分析1000个信号颗粒)，利用前向角散射光面积和宽度图，去除粘连和杂信号，荧光信号使用488nm激光激发，利用RFP表达的阴性对照菌株设定电压以分辨表达的孢子集群。根据阴阳性孢子的信号差别，设门圈定阳性孢子区域，所有样品的分析在相同的方法下进行。将荧光信号最强的孢子直接分选到含PDA的6孔板中，共分选了36个转化子，培养至产孢，流式分选图如图3所示。
将筛选出的转化子孢子接种于25mL液体MM-glucose中，30℃、180rpm振摇培养1d。提取基因组DNA，通过PCR验证6个表达蛋白分泌相关的重组质粒是否已成功转化进入里氏木霉细胞。
将过流式细胞仪筛选出的分泌途径相关的转化子分别用6对分泌途径相关引物进行菌落PCR，以确定筛选出的红色荧光增强的转化子是含有何种分泌途径相关质粒。所用引物如下表所示，PCR片段大小约200bp，电泳结果如图4所示。
表1引物序列
Figure PCTCN2019095900-appb-000012
如图4所示，流式细胞仪筛选出的36个转化子中共菌落PCR出31个转化子，其中22个为含hac1基因的转化子，9个为含bip1基因的转化子，且经测序为这两个转化子序列，其他四个分泌途径相关质粒序列均未扩增出。实验结果说明，hac1基因和bip1基因对DsRed-AfMP1转化子菌株的纤维素酶产生能力有增强作用，而另外4种分泌相关基因的过表达在当前菌株和培养条件下不能起到促进纤维素酶分泌表达的作用。
2.测定分泌相关的重组质粒转化子纤维素酶活
(1)诱导培养分选出的转化子
将出发菌株的孢子和流式细胞仪分选出的孢子，接种1×10 7个于100mL MM-glucose培养基中。30℃、180rpm振摇培养1d。将菌丝用12层纱布过滤，收集菌丝并用大量无菌水冲洗，以去除残余的葡萄糖。
称取等量(500mg)的菌丝，接种于100mL MM-Avicel液体培养基中，出发菌株和每个转化子均做3个平行。30℃、180rpm振摇培养168h以诱导纤维素酶的生产。从第72h开始，每24h收集发酵液，储存于4℃冰箱备用。
(2)纤维素酶酶活测定
1)纤维素酶的提取
将每个时间段收集的2mL发酵液8000rpm离心取上清液。
2)纤维素酶酶活测定
对出发菌株SUS4和菌落PCR出的31个转化子的酶活进行测定。选取3-6d的发酵液，分别测定内切纤维素酶酶活(CMC酶活)。
内切纤维素酶酶活测定：采用羧甲基纤维素钠(CMC)作为底物进行测定。 取1000mg羧甲基纤维素钠，用柠檬酸-磷酸氢二钠缓冲液(0.05M、pH 5.0)定容至50mL，得到2％的羧甲基纤维素钠溶液。
取酶液100μL，加入到10mL柠檬酸-磷酸氢二钠缓冲液(0.05M、pH 5.0)中，得到稀释101倍的酶液。在各试管中加入2％的羧甲基纤维素钠溶液和柠檬酸-磷酸氢二钠缓冲液(0.05M、pH 5.0)各0.45mL，50℃水浴平衡后，加入已经稀释的酶液0.1mL(空白先不加)，振荡混合均匀。50℃水浴保温30min，迅速冷却。向各试管中加入1.5mL DNS试剂，再向空白中加酶液0.1mL，混合均匀。在沸水中煮10min，迅速冷却。以0号管为参比，测定540nm的吸光度。1mL液体酶，在50℃、pH 5.0的条件下，每小时水解羧甲基纤维素钠，产生1μmol还原糖(以葡萄糖计)所需要的酶量定义为一个酶活力单位(U)。
酶活测定结果如图5所示，酶活提高的转化子一共7个，其中三个为含有hac1基因的转化子，命名为SUS4-H7，SUS4-H28，SUS4-H43，四个为含有bip基因的转化子，命名为SUS4-B3，SUS4-B5，SUS4-B26，SUS4-B31。
3.转化子的拷贝数的鉴定
转化子拷贝数的鉴定：转化子拷贝数的鉴定采用qRT-PCR的方法，选用7个转化子的基因组为模板，actin基因作为内参基因；将各基因组稀释5倍，取1μl为模板。引物如下：
RTQactF(5'-TGAGAGCGGTGGTATCCACG-3')
RTQactR(5'-GGTACCACCAGACATGACAATGTTG-3')
RTQhacF(5'-ACAACGTCCTGCAGTGTCAA-3')
RTQhacR(5'-TAGCGATCTGCATCAAGGGC-3')
RTQbipF(5'-AAGAAGGTTACCCACGCCG-3')
RTQbipR(5'-ATCAAAGGTACCACCACCGAG-3')
Bgl3P1基因的拷贝数用2 -△△Ct法相对定量。转化子拷贝数的鉴定结果如图6A和图6B所示，定量PCR分析表明，在染色体中整合了1至2个拷贝的HAC1和BIP1基因。
实施例3.构建基因突变体库以筛选高表达纤维素酶且高酶活的转化子
1.农杆菌pTi-pyr4质粒的构建
扩增里氏木霉转化pyr4基因，构建农杆菌pTi-pyr4质粒。
将构建好的pTi-pyr4质粒转化根癌农杆菌感受态AGL1中。该质粒含有木霉转化筛选标记基因pyr4，以及用以农杆菌随机插入的左臂(LB)及右臂(RB)。
待转化子菌落长出后，挑取单菌落验证，菌落PCR验证目的基因pry4，如图7所示，扩增出的目的片段500bp，与预验证片段大小相吻合，送测序后比对正确。
2.构建农杆菌pTi-pyr4随机插入里氏木霉突变体库
将已转入目的质粒pTi-pyr4的农杆菌(3μL)接种于3mL LB液体(含50μg/mL卡那霉素和25μg/mL利福平)于28℃摇床中220rpm培养。
制备SUS4里氏木霉孢子悬液，涂布至铺有玻璃纸的CM平板，24℃预萌发3h左右。将上述农杆菌菌液100μl涂布于已预萌发有里氏木霉真菌菌丝的CM平板，25℃黑暗环境下共培养，获得根癌农杆菌介导的里氏木霉突变体库。
在不含尿嘧啶和头孢霉素的MM平板上经初筛、复筛获得可正常生长的菌落,视为疑似转化子，抽提基因组DNA，并通过PCR验证转化子。
3.农杆菌pTi-pyr4随机插入突变体库流式细胞仪分选
利用农杆菌介导的转化将pTi-pyr4质粒转化入SUS4菌株原生质体，成功构建了农杆菌转化突变体库，并将转化子通过转膜影印于含有头孢霉素的PDA平板上，如图8所示,产孢后过流式筛选，流式分选表面展示红色荧光的里氏木霉菌株。
用引物验证过流式筛选得到的转化子中是否含有质粒pTi-pry4。如图9所示，验证结果表明转化SUS4后筛选得到4个含有该质粒，分别为SUS4-T7、SUS4-T33、SUS4-T37、SUS4-T47。
4.过流式筛选得到的农杆菌pTi-pyr4随机插入转化子酶活分析
以SUS4为对照菌株，对过流式筛选得到的转化子进行摇瓶发酵分析。MM+Avicel诱导后，取样测定上清中CMC-Na酶活。如图10所示，通过摇瓶发酵发现四株转化子的内切葡聚糖酶活性(26.3～33.3U/mL)分别高于亲本菌株(11.1U/mL)。

Claims (11)

  1. 一种用于快速筛选高表达纤维素酶的里氏木霉的重组表达载体，其特征在于，在所述重组表达载体的基因表达盒中由上游到下游依次包括元件：cbh1启动子、红色荧光蛋白基因DsRed、细胞表面蛋白锚定信号肽基因AfMp1及cbh1终止子，其中，DsRed基因的核苷酸序列如SEQ ID NO.2所示，AfMp1基因的核苷酸序列如SEQ ID NO.3所示。
  2. 含有权利要求1所述的用于快速筛选高表达纤维素酶的里氏木霉的重组表达载体的重组里氏木霉。
  3. 权利要求1所述的用于快速筛选高表达纤维素酶的里氏木霉的重组表达载体的应用。
  4. 快速筛选重组里氏木霉的方法，其特征在于，所述方法包括以下步骤：
    (1)将含有元件cbh1启动子、红色荧光蛋白基因DsRed、细胞表面蛋白锚定信号肽基因AfMp1及cbh1终止子的基因表达盒导入质粒中，得到重组表达载体，其中，DsRed基因的核苷酸序列如SEQ ID NO.2所示，AfMp1基因的核苷酸序列如SEQ ID NO.3所示；
    (2)用步骤(1)构建的重组表达载体转化宿主细胞，得到重组菌株；
    (3)培养重组菌株，诱导红色荧光蛋白表达于重组菌株的表面；
    (4)筛选展示红色荧光的重组菌株。
  5. 根据权利要求4所述的快速筛选重组里氏木霉的方法，其特征在于，在步骤(4)中，用流式细胞仪筛选表面展示红色荧光的重组菌株。
  6. 快速筛选高表达纤维素酶且高酶活的里氏木霉的方法，其特征在于，所述方法包括以下步骤：
    (1)将含有元件cbh1启动子、红色荧光蛋白基因DsRed、细胞表面蛋白锚定信号肽基因AfMp1及cbh1终止子的基因表达盒导入质粒中，得到重组表达载体，其中，DsRed基因的核苷酸序列如SEQ ID NO.2所示，AfMp1基因的核苷酸序列如SEQ ID NO.3所示；
    (2)用步骤(1)构建的重组表达载体转化宿主细胞，得到重组里氏木霉；
    (3)向步骤(2)得到的所述重组里氏木霉中导入蛋白分泌途径相关的基因，或者向步骤(2)得到的重组里氏木霉中随机插入载体或基因，获得重组里氏木霉的突变体库；
    (4)用流式细胞仪筛选表面展示强烈红色荧光的重组里氏木霉；
    (5)测定步骤(4)中得到的重组里氏木霉的纤维素酶酶活，获得纤维素酶酶活提高的重组里氏木霉。
  7. 根据权利要求6所述的快速筛选高表达纤维素酶且高酶活的里氏木霉的方法，其特征在于，步骤(3)中，与蛋白分泌途径相关基因包括bip1基因、hac1基因、ftt1基因、sso2基因、sar1基因、ypt1基因。
  8. 根据权利要求6所述的快速筛选高表达纤维素酶且高酶活的里氏木霉的方法，其特征在于，步骤(3)中，用根瘤农杆菌介导里氏木霉转化，得到里氏木霉突变体库。
  9. 快速筛选高与提高纤维素酶酶活相关的蛋白分泌途径相关基因的方法，其特征在于，所述方法包括以下步骤：
    (1)构建含有红色荧光蛋白基因和细胞表面蛋白锚定信号肽基因的DsRed-AfMP1融合基因的重组表达载体，其中，DsRed基因的核苷酸序列如SEQ ID NO.2所示，AfMp1基因的核苷酸序列如SEQ ID NO.3所示；
    (2)用步骤(1)构建的重组表达载体转化菌株，得到重组菌株；
    (3)向步骤(2)得到的所述重组菌株中导入待筛选的目的基因，或者向步骤(2)得到的重组菌株中随机插入载体或基因，获得重组菌株的突变体库；
    (4)用流式细胞仪筛选表面展示红色荧光的重组菌株；
    (5)测定步骤(4)中筛选出的表面展示红色荧光的重组菌株的纤维素酶酶活，获得纤维素酶酶活提高的重组菌株；
    (6)确定纤维素酶酶活提高的重组菌株中所表达的外源目的基因，或被干扰的内源基因，从而获得与提高纤维素酶酶活相关的蛋白分泌途径相关基因。
  10. 根据权利要求9所述的快速筛选高与提高纤维素酶酶活相关的蛋白分泌途径相关基因的方法，其特征在于，所述重组菌株为里氏木霉，
  11. 根据权利要求9所述的快速筛选高与提高纤维素酶酶活相关的蛋白分泌途径相关基因的方法，其特征在于，与蛋白分泌途径相关的基因包括为bip1基因、hac1基因。
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