WO2021143696A1 - 调控里氏木霉蛋白表达效率的因子、调控方法及应用 - Google Patents

调控里氏木霉蛋白表达效率的因子、调控方法及应用 Download PDF

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WO2021143696A1
WO2021143696A1 PCT/CN2021/071349 CN2021071349W WO2021143696A1 WO 2021143696 A1 WO2021143696 A1 WO 2021143696A1 CN 2021071349 W CN2021071349 W CN 2021071349W WO 2021143696 A1 WO2021143696 A1 WO 2021143696A1
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protein
trichoderma reesei
gene
target gene
target
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French (fr)
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周志华
邹根
刘睿
柴顺星
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中国科学院分子植物科学卓越创新中心
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    • C12N9/2405—Glucanases
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    • C12Y302/01021—Beta-glucosidase (3.2.1.21)

Definitions

  • the present invention belongs to the field of biotechnology. More specifically, the present invention relates to factors, methods and applications for regulating the expression efficiency of Trichoderma reesei protein.
  • Trichoderma reesei is the most used strain in the cellulase industry.
  • Trichoderma reesei strain Qm6a is the starting strain of each cellulase-producing strain. In order to obtain new cellulase-producing strains, Qm6a has undergone two rounds of linear accelerator mutagenesis and screening to obtain a more efficient cellulase-producing mutant strain Qm9414 , But it is still blocked by carbon metabolism, the enzyme production status will increase with the fermentation time, the glucose concentration will increase, and the synthesis of cellulase will be gradually inhibited.
  • Another mutant strain RL-P37 was obtained by UV mutagenesis on the basis of NG14.
  • Another cellulase high-producing strain CL-847 which is widely used in industry, was mutagenized by Qm9414. Because of its ability to produce high cellulase, it is widely used in textile, papermaking, pulping and bioenergy fields in industry. At present, the highest fermentation level of this bacterium can reach 100g/L of protein production. It has strong protein secretion ability and is safe for humans and animals. It has been developed as a good fungal host for expressing homologous and heterologous proteins. In the industrial application of cellulase, the further improvement of the enzyme production and protein secretion ability of Trichoderma reesei will help to further open the cellulase application market.
  • Trichoderma reesei the true induction mechanism of cellulase production by Trichoderma reesei has not yet been discovered.
  • the strong protein secretion ability of the T. reesei mutant strain has not been resolved. Only by clarifying these mechanisms can we transform Trichoderma reesei from the root to have higher cellulase production and protein secretion capacity.
  • Trichoderma reesei it is necessary to carry out more in-depth transformation of Trichoderma reesei to improve its enzyme production capacity and protein secretion capacity, reduce the industrial production cost of cellulase, and obtain useful industrial strains.
  • the purpose of the present invention is to provide factors, methods and applications for regulating the expression efficiency of Trichoderma reesei protein.
  • a method for improving the protein expression efficiency of Trichoderma reesei comprising: down-regulating a target gene in the genome of Trichoderma reesei, the target gene selected from: genes encoding ORF4 protein, encoding The gene for ⁇ -glucosidase.
  • the protein is an endogenous protein or a heterologous protein from Trichoderma reesei.
  • the endogenous protein includes (but is not limited to): cellulase, hemicellulase, ⁇ -glucosidase, carbohydrate permeable enzyme, and protein synthesis secretion pathway related enzymes.
  • the heterologous protein includes a non-cellulase secretory protein; preferably, it includes (but is not limited to): heterologous glucosidase, heterologous ferulic acid esterase FEA, structural protein (such as spider silk protein, silk protein, etc.), functional protein (such as immunomodulatory protein, serum albumin, etc.).
  • a non-cellulase secretory protein preferably, it includes (but is not limited to): heterologous glucosidase, heterologous ferulic acid esterase FEA, structural protein (such as spider silk protein, silk protein, etc.), functional protein (such as immunomodulatory protein, serum albumin, etc.).
  • the gene encoding the heterologous protein is inserted (replaced) into the Trichoderma reesei genome; or, the gene encoding the heterologous protein is introduced into the Trichoderma reesei cell by an expression vector .
  • the gene encoding the heterologous protein is inserted (replaced) into the cbh1 position in the Trichoderma reesei genome.
  • the starting strain of the Trichoderma reesei strain is Rut-C30, Qm9414, RC30-8.
  • down-regulating the target gene in the genome of Trichoderma reesei includes: inhibiting, knocking out, silencing or mutating the target gene, or inhibiting the expression or activity of the protein encoded by the target gene; or
  • the down-regulating agent that down-regulates the target gene is transferred into Trichoderma reesei (preferably includes an agent that inhibits or knocks out the target gene or suppresses the expression or activity of the protein encoded by the target gene); or Regulate the upstream signal pathway or upstream gene of the target gene, and down-regulate the target gene.
  • gene editing is performed by using CRISPR/Cas technology to knock out the target gene; or by homologous recombination technology, the segment where the target gene is located is deleted or inserted to knock out the target gene.
  • the target gene or interfere with the expression of the target gene through an interfering molecule; the interfering molecule is a dsRNA, antisense nucleic acid, small interfering RNA, microRNA, or capable of expressing the target gene or its transcript as the target of inhibition or silencing Or form a construct of the dsRNA, antisense nucleic acid, small interfering RNA, and microRNA.
  • the ORF4 protein has the amino acid sequence shown in SEQ ID NO: 2, and also includes its homologues (homologous sequences).
  • the ⁇ -glucosidase has the amino acid sequence shown in SEQ ID NO: 15, and also includes its homologs (homologous sequences).
  • the gene encoding the ORF4 protein is homologously replaced by introducing the ferulic acid esterase FEA expression cassette, thereby knocking out the gene encoding the ORF4 protein, and introducing the ferulic acid esterase FEA expression cassette at the same time.
  • the gene encoding the ORF4 protein has the nucleotide sequence shown in SEQ ID NO:1, and based on the sequence, it is targeted at positions 257-277, so that the target position can be spliced. Disrupt gene function.
  • the gene encoding ⁇ -glucosidase has the nucleotide sequence shown in SEQ ID NO: 3, and based on this sequence, it is targeted at positions 2607-2626 of the target position. Cut, destroy gene function.
  • the use of a modulator of a target gene is provided for improving the protein expression efficiency of Trichoderma reesei; the modulator includes a down-regulator; the down-regulator is targeted to the ORF4 protein or its code Genes or down-regulators targeting endogenous glucosidase or its encoding genes.
  • the downregulator includes: a CRISPR/Cas gene editing reagent that targets and knocks out the target gene (sgRNA as shown in SEQ ID NO: 14); or deletes the target gene through homologous recombination Or insert operation to knock out the target gene reagent (such as the homology arm for the target gene); or interference molecule, which includes the target gene or its transcript as the target of suppression or silencing dsRNA, antisense Nucleic acid, small interfering RNA, microRNA, or a construct capable of expressing or forming the dsRNA, antisense nucleic acid, small interfering RNA, or microRNA.
  • sgRNA CRISPR/Cas gene editing reagent that targets and knocks out the target gene
  • interference molecule which includes the target gene or its transcript as the target of suppression or silencing dsRNA, antisense Nucleic acid, small interfering RNA, microRNA, or a construct capable of expressing or forming the dsRNA, anti
  • a recombinant Trichoderma reesei strain or its spores, mycelia, or protoplasts the target genes in the genome of which are down-regulated; wherein the down-regulated target genes are selected from: encoding ORF4 protein The gene encoding endogenous ⁇ -glucosidase.
  • the target gene in the Trichoderma reesei strain or its spores, mycelium, or protoplasts, the target gene is inhibited, knocked out, silenced or mutated, or the expression of the protein encoded by the target gene or The activity is inhibited; or the Trichoderma reesei strain or its spores, mycelia, or protoplasts are transformed into the target gene down-regulator; preferably, the target gene down-regulator includes: suppressor or An agent that knocks out the target gene, or inhibits the expression or activity of the protein encoded by the target gene.
  • the Trichoderma reesei strain or its spores, mycelia, or protoplasts further include a gene encoding a heterologous protein; preferably, the gene encoding the heterologous protein is expressed by The vector is introduced into the Trichoderma reesei cell, or inserted (homologous replacement) into the Trichoderma reesei genome, such as (but not limited to) inserted into the cbh1 position.
  • Trichoderma reesei strains or spores, mycelia, or protoplasts thereof is provided for the expression of Trichoderma reesei endogenous protein or heterologous protein.
  • a method for recombinant expression of a heterologous protein comprising: inserting a gene encoding the heterologous protein into any of the recombinant Trichoderma reesei strains or spores, mycelia, or protoplasts Culture the Trichoderma reesei strain or its spores, mycelia, or protoplasts to express heterologous protein; preferably, the encoding gene of the heterologous protein is inserted (homologous substitution) into it The cbh1 position in the genome of Trichoderma spp. or its spore, mycelium, or protoplast.
  • a method for recombinantly expressing the endogenous protein of Trichoderma reesei comprising: culturing the recombinant Trichoderma reesei strain or its spores, mycelia, or protoplasts, thereby recombinantly expressing the Endogenous protein (such as cellulase) from Trichoderma spp.
  • kits for protein expression which comprises: any of the aforementioned recombinant Trichoderma reesei strains or spores, mycelia, or protoplasts; or target genes Downregulators (such as plasmids for knocking out target genes, or RNA interference reagents), the target genes are selected from the group consisting of genes encoding ORF4 protein and genes encoding endogenous ⁇ -glucosidase.
  • the endogenous protein includes (but is not limited to): cellulase, hemicellulase, ⁇ -glucosidase, carbohydrate permeating enzyme, protein synthesis secretion pathway related enzymes.
  • the heterologous protein includes a non-cellulase secretory protein; preferably, it includes (but is not limited to): heterologous glucosidase, heterologous ferulic acid esterase FEA, structural protein (such as spider silk protein, silk protein, etc.), functional protein (such as immunomodulatory protein, serum albumin, etc.).
  • a non-cellulase secretory protein preferably, it includes (but is not limited to): heterologous glucosidase, heterologous ferulic acid esterase FEA, structural protein (such as spider silk protein, silk protein, etc.), functional protein (such as immunomodulatory protein, serum albumin, etc.).
  • Figure 1 Use the yeast two-hybrid system to verify the protein interaction between orf4 and ACE1.
  • the positive control is Lae1 and Vel1, which have been reported to have protein interactions
  • the experimental group is AD-orf4 and BD-ACE1
  • the negative control is AD-orf4 and BD empty vector
  • the blue color indicates that there is protein interaction.
  • Trichoderma reesei is the main microorganism producing commercial cellulase. Although the common strains currently used in industry have improved the cellulase production compared with wild strains, there is still room for improvement. In addition, the induction mechanism of cellulase produced by Trichoderma reesei has not been truly resolved, so it is particularly important to find new enzyme production and key factors related to protein analysis. After in-depth research, the inventors found that some target genes in the Trichoderma reesei genome are closely related to the strain’s endogenous or exogenous protein production and/or activity. Down-regulating these target genes can significantly increase the strain’s performance. Endogenous or exogenous protein production and/or activity.
  • the target gene or the substance and method for regulating the target gene can be applied to realize the improvement of Trichoderma reesei, increase the protein expression efficiency of Trichoderma reesei, and enhance the output of endogenous or exogenous protein expressed and/ Or activity.
  • ORF4 polypeptide protein refers to a polypeptide having the sequence of SEQ ID NO: 2, and also includes variant forms or homologs of the sequence of SEQ ID NO: 2 that have the same function as the ORF4 polypeptide.
  • variant forms include (but are not limited to): several (such as 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, and more preferably 1- 8, 1-5) amino acid deletion, insertion and/or substitution, and addition or deletion of one or several (usually within 20, preferably within 10) at the C-terminus and/or N-terminus, and more Preferably within 5) amino acids.
  • ⁇ -glucosidase refers to a polypeptide having the sequence of SEQ ID NO: 15, and also includes variant forms or homologues of the sequence of SEQ ID NO: 15 that have the same function as the ⁇ -glucosidase polypeptide .
  • variant forms include (but are not limited to): several (such as 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and more preferably 1- 8, 1-5) amino acid deletion, insertion and/or substitution, and addition or deletion of one or several (usually within 20, preferably within 10) at the C-terminus and/or N-terminus, and more Preferably within 5) amino acids.
  • any high homology with the ORF4 polypeptide or ⁇ -glucosidase for example, the homology with the sequence shown in SEQ ID NO: 2 or 15 is 85% or higher; preferably, the homology is 90 % Or higher; more preferably, the homology is 95% or higher, such as 98% or 99% homology
  • a protein with the same function as ORF4 polypeptide or ⁇ -glucosidase is also included in the present invention .
  • ORF4 polypeptides or ⁇ -glucosidase enzymes are listed in the specific examples of the present invention, it should be understood that due to the existence of some different varieties of Trichoderma reesei, the sequences between them are highly conserved.
  • Homologous polypeptides or polynucleotides should also be included in the present invention, and methods of regulating these homologous polypeptides or polynucleotides (homologs) similar or identical to those of the present invention should also be included in the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.
  • the inventors gradually knocked out the genome of Trichoderma reesei and found that the protein secretion ability of the knockout strain was increased. After screening this sequence of single genes, it was finally found that the methyltransferase in this region is related to the regulation of cellulase production. Experiments have verified that the methyltransferase enzyme can regulate the cellulase inhibitor ACE1. To regulate the secretion of cellulase. And genes at other sites are also related to cellulase production. Knockout of the methyltransferase can increase the FPU enzyme activity of Trichoderma reesei Rut-C30 by more than 25%.
  • the present invention determines for the first time that some target genes in the genome of Trichoderma reesei are closely related to the endogenous or exogenous protein production and/or activity of the strain. Based on this new discovery, the present invention provides a method for improving the protein expression efficiency of Trichoderma reesei, the method comprising: down-regulating a target gene in the genome of Trichoderma reesei; wherein the down-regulated target gene is selected from: Gene encoding ORF4 protein, gene encoding endogenous ⁇ -glucosidase.
  • a variety of methods well known to those skilled in the art can be used to regulate (down-regulate or mutate) them. Including but not limited to inhibiting, knocking out, silencing or mutating the target gene, or inhibiting the expression or activity of the protein encoded by the target gene; or transferring the down-regulator that down-regulates the target gene into Trichoderma reesei ; Or through site-directed mutagenesis reagents for targeted mutations.
  • Trichoderma reesei strain including gene silencing, gene blockade, gene knockout, gene suppression, and the like. These methods are all included in the present invention.
  • the target gene in the genome can be modified by gene blocking technology based on gene insertion or deletion based on homologous recombination, so that the target gene is blocked; it is also possible to design interfering RNA or antisense nucleotides for the target gene. Suppress or silence target gene expression.
  • a method for down-regulating target genes is gene blocking technology.
  • a target gene blocking plasmid is constructed in vitro, and other unrelated target genes are inserted into the chromosomal target genes of the Trichoderma reesei strain through homologous recombination. Element, so that the target gene on the chromosome can no longer encode an active protein.
  • the selection of irrelevant elements is easy to choose by those skilled in the art, for example, the application of some resistance genes.
  • a method of gene blocking (knockout) can be found in Genetic Manipulation of Streptomyces: a Laboratory Manual, for example.
  • a resistance screening gene When designing a construct for gene block or knockout, it is preferable to include a resistance screening gene at the same time, so as to facilitate subsequent screening of strains whose genes have been blocked or knocked out.
  • the present invention also provides Trichoderma reesei or its spores, mycelia, or protoplasts that down-regulate the target gene, and more particularly, Trichoderma reesei or its spores, bacteria obtained by CRISPR/Cas technology deletion of the target gene Filaments, protoplasts, the strain or its spores, mycelium, or protoplasts do not express the target gene or the expression level is significantly reduced.
  • the invention also relates to the use of the strain or its spores, mycelium or protoplasts to improve the protein expression efficiency of Trichoderma reesei.
  • the present invention also provides a kit for protein expression, which comprises: the genetically engineered (recombined) Trichoderma reesei or its spores, mycelia, and protoplasts of the present invention.
  • the present invention also provides a kit for protein expression, which contains: a down-regulator of the target gene, such as a plasmid for knocking out the target gene, or an RNA interference reagent, or a site-directed mutagenesis reagent.
  • a down-regulator of the target gene such as a plasmid for knocking out the target gene
  • an RNA interference reagent such as a RNA interference reagent
  • a site-directed mutagenesis reagent such as a site-directed mutagenesis reagent.
  • the kit for protein expression may also include other reagents applied to the production process of Trichoderma reesei, such as the basic medium of Trichoderma reesei.
  • the kit for protein expression may also include instructions for use, explaining the method of culturing the Trichoderma reesei, or the method of using the downregulator or site-directed mutagenesis reagent to down-regulate the target gene in Trichoderma reesei .
  • the inventors knocked out the ORF in SEQ ID NO:1 of the Rut-C30 genome and found that this sequence is related to the cellulase production and protein secretion ability of Trichoderma reesei.
  • the present inventors also expressed heterologous genes in this region by homologous recombination and obtained higher yields than other sites. On this basis, further knocking out a gene SEQ ID NO: 3 predicted to be ⁇ -glucosidase can further Increase production. Therefore, the genes and non-coding polynucleotide sequences involved in the present invention have good industrial application prospects for Trichoderma reesei strains.
  • the enzyme activity of the cellulase produced by its spores is higher than 15IU/mL (U/mL); It is preferably higher than 18IU/mL.
  • the ferulic acid esterase FEA expression box (SEQ ID NO: 5) in the Trichoderma reesei Rut-C30 strain is expressed by homologous recombination at the cbh1 site, and then SEQ ID is further knocked out
  • the enzyme activity of the cellulase produced by the spores is higher than 200 IU/mL; preferably higher than 220 IU/mL.
  • the polynucleotide shown in SEQ ID NO: 3 is further knocked out in the high-yielding heterologous ⁇ -glucosidase strain U10 in which the Trichoderma reesei has deleted SEQ ID NO: 1 ,
  • the ⁇ -glucosidase enzyme activity (pNPGase) produced by its spores is higher than 2000IU/ml; preferably higher than 2500IU/ml.
  • High-yield strains are obtained through the transformation of the present invention, and the carbon source for fermentation culture of transformants is simple and easy-to-obtain agricultural wastes such as bran, which has a very obvious effect in improving the hydrolysis efficiency of cellulase preparations, such as application in food In the production of other industries, its production costs will also be greatly reduced, and it has a wider potential for industrial applications.
  • the active site of the present invention can also be used for the recombinant expression of heterologous genes.
  • the heterologous gene expression cassette is introduced into the genome through Agrobacterium-mediated transformation, PEG-mediated protoplast transformation, based on ku70/ku80, CRSIPR/Cas9, etc., into the genome, preferably into the cbh1 position, replacing part or all of the sequence Recombinant expression of the sequence is carried out, and further improvement is carried out to obtain derivative strains with higher yield or more optimized enzyme system or engineering strains of other recombinant proteins.
  • These engineered strains can also be further established for gene manipulation without selection markers, and have huge application prospects in industry.
  • the strain of the present invention can be used as the basis for further optimizing derivative strains or engineering strains of other recombinant proteins. After constructing a marker-free screening system, it can be used as the object of molecular manipulation, and genetic manipulations such as knock-in and knock-out genes can be performed. Purposeful genetic modification, or over-expression of cellulase genes that are under-expressed by Trichoderma reesei, such as over-expression of cellulase activator Xyr1, etc., or knock-out of cellulase repressor Cre1, etc., to obtain further high yields Engineering strains.
  • the modified strain of the present invention is a living cell. Once the spores, hyphae, protoplasts and related culture mixture of living cells of the strain of the present invention are obtained, it can be obtained in large quantities by means of inoculation, passage, regeneration, etc. Invented strain. This is usually done by inoculating it into a solid plate medium or a liquid medium to expand the strain to obtain the living cells of the present invention. The obtained living cells can be further subjected to laboratory domestication, genetic breeding and molecular genetic manipulation to obtain mutants and transformants. The present invention can also be used as a host cell for heterologous expression.
  • Methods well known to those skilled in the art can be used to mutagenize the living cells of the present invention, resulting in changes in the gene coding, enzyme activity, and morphology of the living cells.
  • These methods include the use of physical methods such as radiation, particles, lasers, and ultraviolet light, and the use of chemical mutagenesis methods such as alkylating agents, base analogs, hydroxylamine, and acridine pigments.
  • the mutagenesis can be multiple generations of the above method or multiple methods, and is not limited to these methods.
  • Based on the strain provided by the present invention it can be further bred by physical and chemical methods, and new cellulase genes and related regulatory genes can be introduced, and the enzyme production performance of the obtained mutants and transformants can be further improved.
  • the method is one or a combination of the above.
  • the methods well known to those skilled in the art use the polynucleotide sequences of the present invention to construct expression constructs (vectors) and to further modify host cells.
  • expression constructs vectors
  • the signal pathways, signal pathways and proteins involved in cellulase production that have been discovered or newly discovered in strains are further improved (such as increasing the expression of beneficial factors and reducing the expression of harmful factors).
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art. The steps used are well known in the art. Such as Agrobacterium-mediated fungal transformation methods, protoplast transformation methods, electroporation transformation methods, CRISPR/Cas9 genome editing methods, gene bombardment methods, etc., and are not limited to these methods.
  • the optimized strain of Trichoderma reesei obtained by the modification of the present invention can use lignocellulose-containing industrial and agricultural production wastes, such as cheap raw materials such as bran, corn steep liquor, soybean meal, etc., to produce cellulase through liquid or solid fermentation, and its advantages
  • the cellulase produced has high activity, reasonable composition, and strong ability to hydrolyze lignocellulose.
  • the invention provides a new strain resource for solving the problems of low enzyme activity, unreasonable enzyme components, high production cost, low saccharification efficiency and the like in the utilization of cellulose resources.
  • the engineered strain of Trichoderma reesei obtained by introducing the FEA expression box homologously replacing cbh1 and then further knocking out SEQ ID NO:1 can utilize industrial and agricultural production wastes containing lignocellulose, such as bran, corn milk, and soybean meal.
  • lignocellulose such as bran, corn milk, and soybean meal.
  • the advantages of producing cellulase through liquid or solid-state fermentation, such as cheap raw materials, are that the produced cellulase has high activity, reasonable components, and strong ability to hydrolyze lignocellulose. Under the same fermentation conditions, this strain has twice the yield of the FEA expression cassette introduced randomly.
  • the invention provides a new strain resource for solving the problems of low enzyme activity, unreasonable enzyme components, high production cost, low saccharification efficiency and the like in the utilization of cellulose resources.
  • the optimized strain of Trichoderma reesei obtained by knocking out SEQ ID NO: 3 of the present invention can utilize lignocellulose-containing industrial and agricultural production wastes, such as cheap raw materials such as bran, corn steep liquor, and soybean meal powder, to be produced through liquid or solid fermentation
  • lignocellulose-containing industrial and agricultural production wastes such as cheap raw materials such as bran, corn steep liquor, and soybean meal powder
  • Cellulase has the advantages of high cellulase activity, reasonable composition, and strong ability to hydrolyze lignocellulose.
  • the invention provides a new strain resource for solving the problems of low enzyme activity, unreasonable enzyme components, high production cost, low saccharification efficiency and the like in the utilization of cellulose resources.
  • the optimized Trichoderma reesei strain obtained by the method of the present invention can utilize lignocellulose-containing industrial and agricultural production wastes, such as cheap raw materials such as soybean meal powder, to produce cellulase through liquid or solid fermentation. Its advantage lies in the production of cellulase. Decreased significantly.
  • the knock-out strain of the present invention can provide an expression host for expressing non-cellulase secreted proteins.
  • the fermentation method includes: (1) A slope or a flat plate made by planting Trichoderma reesei on a potato culture medium After activation, a spore suspension with a concentration of 10 6 ⁇ 10 8 mL -1 was prepared, and 10% of the inoculum was added to Sandcastle's medium (seed medium: 1% yeast extract, 1% peptone, 4% glucose ), 28 °C, 200 rpm shaking culture to obtain seed liquid, and then the seed liquid was inserted into the liquid fermentation medium with 10% inoculum, the initial pH was 5.0, the filling volume was 10 mL in a 50 mL Erlenmeyer flask, at 28 °C, 200 rpm Culture in a shaker for 5-7 days; (2) Centrifuge the fermentation broth obtained in step (1), and take the supernatant as the crude enzyme solution; the induced fermentation medium contains 5% inducer
  • the culture medium and culture method applied to cultivating the strain of the present invention are not limited to those disclosed above, and other culture media and culture methods conventionally applied to culturing Trichoderma reesei can also be applied to the present invention.
  • the fermentation system can be scaled up for industrial production. Depending on the size of the system, those skilled in the art can make appropriate adjustments based on their general knowledge to facilitate the growth or production of strains.
  • the liquid fermentation crude enzyme solution can be used to obtain relatively pure cellulase, enzyme powder or other heterologous recombinant proteins through methods such as ultrafiltration, salting out or organic solvent precipitation.
  • FPA filter paper enzyme activity
  • CMC enzyme activity CMC enzyme activity
  • ⁇ -glucosidase enzyme activity ⁇ -glucosidase enzyme activity and other recombinant protein activities of the produced cellulase.
  • Trichoderma reesei Rut-C30 strain (purchased from ATCC, strain number: ATCC 56765) was inoculated on a plate made of potato culture medium and incubated at 28°C for 7 days to make a concentration of 10 6 to 10 8 /mL of spore suspension, add 10% (v/v) inoculum to Sandburg's medium (seed medium (v/v): 1% yeast extract, 1% peptone, 4% glucose), 28 °C, 200 rpm shaking culture, to obtain the bacterial solution.
  • seed medium v/v
  • the PCR product was purified and digested with XbaI, and the digested DNA fragment was recovered using the Axygen PCR product column recovery kit.
  • the DNA fragment and the recovered vector pHDt/sk (containing Kan and hyg selection markers) were purchased from Sigma) after dephosphorylation, ligated with T4 DNA ligase at 16°C overnight to obtain the vector pHDt/sk-full. And verify the correctness of the sequence by sequencing.
  • SEQ ID NO:1 (orf4) nucleic acid sequence:
  • the artificially optimized Cas9 gene (SEQ ID NO: 4; positions 4117-4137 are the nucleus entry signal) as the template, and the primer Cas9F (ATGGACAAGAAGTACAGCATTGG (SEQ ID NO: 8)) and Cas9R (TTAGACCTTGCGCTTCTTCTTGGG (SEQ ID NO: 9)) PCR to obtain the sequence.
  • the primer Cas9F ATGGACAAGAAGTACAGCATTGG (SEQ ID NO: 8)
  • Cas9R TTAGACCTTGCGCTTCTTCTTGGG (SEQ ID NO: 9)
  • Trichoderma reesei genomic DNA as template, using forward primer: 5'ACGACGGCCAGTGCCAAGCTTAGGACTTCCAGGGCTACTTG 3'(SEQ ID NO: 10) and reverse primer: 5'GATTGTGCTGTAGCTGCGCTGCTTTGATCGTTTTGAGGTGC 3'(SEQ ID NO: 11) to obtain Ppdc for amplification
  • forward primer 5'ACGACGGCCAGTGCCAAGCTTAGGACTTCCAGGGCTACTTG 3'(SEQ ID NO: 10)
  • reverse primer 5'GATTGTGCTGTAGCTGCGCTGCTTTGATCGTTTTGAGGTGC 3'(SEQ ID NO: 11)
  • Trichoderma reesei genomic DNA as a template, using forward primer 5'AGAAGAAGAGGAAGGTGTGACCCGGCATGAAGTCTGACCG 3'(SEQ ID NO: 16) and reverse primer 5'TAATTGCGCGG ATCCTCTAGATGGACGCCTCGATGTCTTCC 3'(SEQ ID NO: 17) to amplify Tpdc as a terminator.
  • the vector was constructed by a one-step cloning method, and the recombinant vector pDHt/sk-ppdc-Cas9-tpdc containing an inducible promoter was successfully constructed by verifying the sequence.
  • the above constructed plasmid was transformed into Agrobacterium tumefaciens AGL1, and transformed into Trichoderma reesei Rut-C30 under the mediation of Agrobacterium tumefaciens.
  • Hygromycin was used as the selection pressure to inoculate the grown transformants
  • DNA was extracted as a template after 2 days, and the forward primer 5'TACGTCGGCCCCCTGGCC 3'(SEQ ID NO: 12) was designed through the promoter on the vector and the reverse primer of the Cas9 gene was 5'GAGGTTGTCAAACTTGCGCTGCG 3' (SEQ ID NO: 13) PCR to identify positive transformants.
  • the obtained transformants were named C30-pc.
  • pMD18T purchased from Takara, Amp resistant
  • pMD18T purchased from Takara, Amp resistant
  • the obtained in vitro transcription template is extracted and purified with phenol: chloroform: isoamyl alcohol (25:24:1), and finally used Nuclease free water dissolves DNA and can be used for in vitro transcription of gRNA.
  • C30-pc spores were inoculated on a PDA plate, cultured at 28°C for 7 days, and washed with 0.85% NaCl+0.02% Tween 80. Use sterilized forceps to cover a piece of cellophane on the PDA plate, flatten it, add about 150ul of spore suspension on the cellophane, spread evenly with a glass rod, do 6 same treatments, incubate at 28 degrees for about 14 hours.
  • Lysing the cell wall with a lysing enzyme remove the cell wall debris to obtain a protoplast suspension, mix with purified gRNA fragments and transform with PEG-mediated protoplast transformation, spread on the screening plate, set 28 Cultivation at °C, the growth of hyphae can be observed in about 5 days. After the transformant grows, the corresponding sgRNA knockout can be obtained after verification.
  • a lysing enzyme Sigma#L1412 Lysing Enzymes
  • step (2) Centrifuge the fermentation broth obtained in step (1), and take the supernatant as the crude enzyme solution; the fermentation medium contains 5% inducer (3% microcrystalline cellulose and 2% bran) Inorganic salt culture medium (0.4% KH2PO4, 0.28% (NH 4 ) 2 SO 4 , 0.06% MgSO 4 ⁇ 7H 2 O, 0.05% CaCl 2 , 0.06% urea, 0.3% peptone, 0.1% Tween 80, 0.5% CaCO 3 , 0.001% FeSO 4 ⁇ 7H 2 O, 0.00032% MnSO 4 ⁇ H 2 O, 0.00028% ZnSO 4 ⁇ 7H 2 O, 0.0004% CoCl 2 ),
  • Example 3 Interaction protein screening of the protein encoded by SEQ ID NO:1
  • the yeast two-hybrid vector pGADT7 AD Vector and pGBKT7 DNA-BD Vector purchased from Takara were used to construct orf4 (ie SEQ ID NO:1) and other known transcriptional regulatory factors including: ACE1 (GenBank accession number AF190793), Vel1 ( GenBank accession number XM_006966084), Hda1 (GenBank accession number XP_006968036), Cre1 (GenBank accession number AAB01677), Lae1 (GenBank accession number AFX86442, etc., get AD-orf4, AD-ACE1, AD-Vel1, AD-Hda1, AD-Cre1 , AD-Lae1, AD-Cre1 and BD-orf4, BD-ACE1, BD-Vib1, BD-Hda1, BD-Cre1, BD-Lae1, BD-Cre1, etc.
  • ACE1 GenBank accession number AF190793
  • Vel1 GenBank accession number XM_0069
  • AH109 yeast transformed with different combinations of AD and BD vectors Strains purchased from Takara
  • blank controls were respectively spread on the screening plate containing SD+Dropout+Ade+His medium. After culturing at 30°C for 2 to 3 days, pick a few colonies to SD+Dropout+3- AT+X- ⁇ -GAL auxotrophic medium, cultured at 30°C for 2-3 days, observing that the yeast can grow and turn blue at the same time, it indicates that there is an interaction between the two proteins.
  • ACE1 is a cellulase inhibitor.
  • orf4 modifies certain sites of ACE1 to activate its function of inhibiting cellulase transcription.
  • the ferulic acid esterase FEA (nucleotide shown in SEQ ID NO: 5) was homologously substituted for the cbh1 position of Trichoderma reesei using CRISPR/Cas9 technology, and then Knock out SEQ ID NO:1, and the obtained transformants are named U10FEA1 ⁇ U10FEA5, and only homologously replace the cbh1 position of Trichoderma reesei without knocking out SEQ ID NO:1 as a control (FEA ⁇ 1).
  • the transformant and C30-pc were inoculated on a plate made of potato culture medium and placed at 28°C for 7 days to prepare a spore suspension with a concentration of 10 6 ⁇ 10 8 spores/mL, and press 10% (v/v ) Inoculated into Sandcastle's medium (seed medium (v/v): 1% yeast extract, 1% peptone, 4% glucose), cultured with shaking at 28°C and 200 rpm to obtain the seed liquid, and then the seeds
  • seed medium (v/v) 1% yeast extract, 1% peptone, 4% glucose
  • the inoculum was 10% (v/v) into the liquid fermentation medium, the initial pH was 5.0, and the filling volume was 10 mL in a 50 mL Erlenmeyer flask, and cultured in a shaker at 28°C and 200 rpm for 5-7 days; the obtained fermentation
  • the supernatant was centrifuged and the supernatant was taken as the crude enzyme solution;
  • Example 5 Increasing the level of extracellular secreted protein by operating endogenous ⁇ -glucosidase
  • Example 1 Using the Trichoderma reesei strain Qm9414 (purchased from ATCC 26921) as the starting strain, the method in Example 1 was used to knock out the ⁇ -glucosidase encoding gene (nucleotide shown in SEQ ID NO: 3) to design sgRNA : 5'TAATACGACTCACTATA GGCAAGTATCCGTATCCCGA GTTTTAGAGCTAGAAATAGC 3'(SEQ ID NO: 19), the 2607-2626 of the nucleotide shown in SEQ ID NO: 3 is cut to destroy its function, and transformants ⁇ bgl1 to ⁇ bgl3 are obtained.
  • ⁇ -glucosidase gene (SEQ ID NO: 3):
  • ⁇ -glucosidase protein (SEQ ID NO: 15):
  • the transformant and Qm9414 were inoculated on a plate made of potato culture medium and cultured at 28°C for 7 days to prepare a spore suspension with a concentration of 10 6 ⁇ 10 8 spores/mL at a rate of 10% (v/v)
  • the amount of inoculum was connected to Sandcastle's medium (seed medium (v/v): 1% yeast extract, 1% peptone, 4% glucose), cultured with shaking at 28°C and 200 rpm to obtain the seed liquid, and then the seed liquid
  • seed medium (v/v): 1% yeast extract, 1% peptone, 4% glucose) cultured with shaking at 28°C and 200 rpm to obtain the seed liquid, and then the seed liquid
  • the 10% (v/v) inoculum volume is connected to the liquid fermentation medium, the initial pH is 5.0, and the filling volume is 10 mL in a 50 mL Erlenmeyer flask.
  • the obtained fermentation broth is centrifuged Separate and take the supernatant as the crude enzyme solution; the fermentation medium is an inorganic salt culture solution (0.4% KH 2 PO 4 , 0.28) containing 5% inducer (3% microcrystalline cellulose and 2% bran) %(NH 4 ) 2 SO 4 , 0.06% MgSO 4 ⁇ 7H 2 O, 0.05% CaCl 2 , 0.06% urea, 0.3% peptone, 0.1% Tween 80, 0.5% CaCO 3 , 0.001% FeSO 4 ⁇ 7H 2 O, 0.00032% MnSO 4 ⁇ H 2 O, 0.00028% ZnSO 4 ⁇ 7H 2 O, 0.0004% CoCl 2 ).
  • the enzyme activity detection is specifically as described in Table 3.
  • Example 6 To further increase the output of exogenous protein by operating endogenous ⁇ -glucosidase
  • Trichoderma reesei strain (patent number 201210258194.6, with RC30-8 as the starting strain) overexpressing the exogenous ⁇ -glucosidase trbgls was used to knock out the SEQ ID NO:1 sequence using the method in Example 1 to obtain U10.
  • the transformant and U10 were inoculated on a plate made of potato culture medium and cultured at 28°C for 7 days to prepare a spore suspension with a concentration of 10 6 ⁇ 10 8 spores/mL at a rate of 10% (v/v)
  • the amount of inoculum was connected to Sandcastle's medium (seed medium (v/v): 1% yeast extract, 1% peptone, 4% glucose), cultured with shaking at 28°C and 200 rpm to obtain the seed liquid, and then the seed liquid
  • seed medium (v/v) 1% yeast extract, 1% peptone, 4% glucose
  • the 10% (v/v) inoculum volume is connected to the liquid fermentation medium, the initial pH is 5.0, and the filling volume is 10 mL in a 50 mL Erlenmeyer flask.
  • the obtained fermentation broth is centrifuged Separate and take the supernatant as the crude enzyme solution; the fermentation medium is an inorganic salt culture solution (0.4% KH 2 PO 4 , 0.28) containing 5% inducer (3% microcrystalline cellulose and 2% bran) %(NH 4 ) 2 SO 4 , 0.06% MgSO 4 ⁇ 7H 2 O, 0.05% CaCl 2 , 0.06% urea, 0.3% peptone, 0.1% Tween 80, 0.5% CaCO 3 , 0.001% FeSO 4 ⁇ 7H 2 O, 0.00032% MnSO 4 ⁇ H 2 O, 0.00028% ZnSO 4 ⁇ 7H 2 O, 0.0004% CoCl 2 ).
  • the enzyme activity detection is as follows:
  • pNPG p-nitrophenyl- ⁇ -D-glucoside
  • the pNP concentration is 10 mg/ml.

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Abstract

提供调控里氏木霉蛋白表达效率的因子、调控方法及应用。所述的调控里氏木霉蛋白表达效率的因子包括影响里氏木霉表达、合成或分泌蛋白质效率的非转录调控因子。还提供了它们在里氏木霉表达、合成与分泌蛋白质中的应用以及构建的重组的里氏木霉菌株。

Description

调控里氏木霉蛋白表达效率的因子、调控方法及应用 技术领域
本发明属于生物技术领域，更具体地，本发明涉及调控里氏木霉蛋白表达效率的因子、调控方法及应用。
背景技术
里氏木霉(Trichoderma reesei)是纤维素酶工业中利用最多的菌株。里氏木霉菌株Qm6a为各个高产纤维素酶菌株的出发株，为了获取高产纤维素酶的新菌株，Qm6a经过两轮直线加速器的诱变和筛选得到了更高效的产纤维素酶突变菌株Qm9414，但是仍然受到碳代谢阻遏，产酶状况会随着发酵时间增加，葡萄糖浓度升高，纤维素酶的合成逐渐受到抑制。为了获得更高的纤维素酶产量，对Qm6a重新进行育种，通过三轮诱变和筛选得到了抗碳代谢阻遏的高产菌株Rut-C30：第一轮通过紫外诱变和抗代谢阻遏筛选获得了菌株M7；第二轮对M7通过N-硝基胍诱变得到突变株NG14，相比Qm9414，NG14胞外蛋白产量和滤纸酶活分别提高了1倍和4倍；第三轮在NG14的基础上经过紫外诱变和抗代谢阻遏筛选的得到了更高效的产纤维素酶菌株Rut-C30。此外，在NG14的基础上紫外诱变获取了另一突变株RL-P37，另一工业上广泛应用的纤维素酶高产菌株CL-847是由Qm9414诱变而来。由于其高产纤维素酶的能力，在工业上被广泛用于纺织、造纸、制浆及生物能源等领域。该菌目前最高发酵水平可达100g/L的蛋白产量，具有很强的蛋白分泌能力，同时对人畜安全，已经被开发成表达同源蛋白和异源蛋白的良好真菌宿主。在纤维素酶工业应用中，里氏木霉的产酶和蛋白分泌能力的进一步提高有助于纤维素酶应用市场的进一步打开。
虽然传统的诱变育种可以获得不少优良的工业菌株，但Kubicek等认为，从1978年到1991年单纯通过传统诱变方法获得里氏木霉的突变株，在产酶能力上已经没有大的改观。近年来，随着分子生物学的进一步发展，人们已经逐渐从常规的诱变育种和改善发酵条件等传统生物学转向利用比较基因组学、蛋白质组学、转录组学和代谢组学等系统生物学，以此来分析了解一些高产菌株重要突变基因的功能和相关分子机制，并利用这些机制进行进一步遗传改造以获取更符合工业需求的生产菌株。然而，里氏木霉产纤维素酶的真正诱导机制还并未发现。此外里氏木霉突变株强大蛋白分泌能力也未被解析。只有弄清楚这些机理，才能从根源上改造里氏木霉，使其具有更高的纤维素酶产量以及蛋白分泌能力。
因此，有必要对里氏木霉进行更加深入的改造，提高其产酶能力和蛋白分泌能力，降低纤维素酶的工业生产成本，进而获得有用的工业菌株。
发明内容
本发明的目的在于提供调控里氏木霉蛋白表达效率的因子、调控方法及应用。
在本发明的第一方面，提供一种提高里氏木霉的蛋白表达效率的方法，包括：下调里氏木霉基因组中的靶基因，所述靶基因选自：编码ORF4蛋白的基因，编码β-葡萄糖苷酶的基因。
在一个优选例中，所述的蛋白是里氏木霉内源蛋白或异源蛋白。
在另一优选例中，所述的内源蛋白包括(但不限于)：纤维素酶，半纤维素酶，β葡萄糖苷酶，糖类透过酶，蛋白合成分泌途径相关酶。
在另一优选例中，所述的异源蛋白包括非纤维素酶分泌蛋白；较佳地，包括(但不限于)：异源葡萄糖苷酶，异源阿魏酸酯酶FEA，结构蛋白(如蜘蛛丝蛋白、蚕丝蛋白等)，功能蛋白(如免疫调节蛋白、血清白蛋白等)。
在另一优选例中，所述的异源蛋白的编码基因插入(替换)到里氏木霉基因组中；或，所述的异源蛋白的编码基因由表达载体引入到里氏木霉细胞内。
在另一优选例中，所述的异源蛋白的编码基因插入(替换)到里氏木霉基因组中cbh1位置。
在另一优选例中，所述的里氏木霉菌株的出发菌株为Rut-C30，Qm9414，RC30-8。
在另一优选例中，下调里氏木霉基因组中的靶基因包括：抑制、敲除、沉默或突变所述的靶基因，或抑制所述的靶基因编码的蛋白的表达或活性；或将下调所述靶基因的下调剂转入里氏木霉中(较佳地包括抑制或敲除所述的靶基因或抑制所述的靶基因编码的蛋白的表达或活性的作用的试剂)；或调节所述靶基因的上游信号通路或上游基因，下调所述靶基因。
在另一优选例中，通过采用CRISPR/Cas技术进行基因编辑以敲除所述的靶基因；或通过同源重组技术，对靶基因所在区段进行缺失或插入的操作以敲除所述的靶基因；或通过干扰分子干扰靶基因的表达；所述的干扰分子是以所述靶基因或其转录本为抑制或沉默靶标的dsRNA、反义核酸、小干扰RNA、微小RNA，或能表达或形成所述dsRNA、反义核酸、小干扰RNA、微小RNA的构建物。
在另一优选例中，所述的ORF4蛋白具有SEQ ID NO:2所示的氨基酸序列，还包括其同源物(同源序列)。
在另一优选例中，所述β-葡萄糖苷酶具有SEQ ID NO:15所示的氨基酸序列，还包括其同源物(同源序列)。
在另一优选例中，通过引入阿魏酸酯酶FEA表达框同源替换编码ORF4蛋白的基因，从而敲除该编码ORF4蛋白的基因，同时引入阿魏酸酯酶FEA表达框。
在另一优选例中，所述编码ORF4蛋白的基因具有SEQ ID NO:1所示的核苷酸 序列，基于该序列，靶向于其第257-277位，使该靶位置发生剪切，破坏基因功能。
在另一优选例中，所述编码β-葡萄糖苷酶的基因具有SEQ ID NO:3所示的核苷酸序列，基于该序列，靶向于其第2607-2626位，使该靶位置发生剪切，破坏基因功能。
在本发明的另一方面，提供靶基因的调节剂的用途，用于提高里氏木霉的蛋白表达效率；所述调节剂包括下调剂；所述下调剂为靶向于ORF4蛋白或其编码基因或靶向于内源葡萄糖苷酶或其编码基因的下调剂。
在一个优选例中，所述的下调剂包括：靶向且敲除靶基因的CRISPR/Cas基因编辑试剂(如SEQ ID NO:14所示的sgRNA)；或通过同源重组对靶基因进行缺失或插入操作、从而敲除所述的靶基因的试剂(如针对靶基因的同源臂)；或干扰分子，其包括以所述靶基因或其转录本为抑制或沉默靶标的dsRNA、反义核酸、小干扰RNA、微小RNA，或能表达或形成所述dsRNA、反义核酸、小干扰RNA、微小RNA的构建物。
在本发明的另一方面，提供一种重组的里氏木霉菌株或其孢子、菌丝体、原生质体，其基因组中的靶基因被下调；其中，下调的靶基因选自：编码ORF4蛋白的基因，编码内源β-葡萄糖苷酶的基因。
在一个优选例中，所述的里氏木霉菌株或其孢子、菌丝体、原生质体中，所述靶基因被抑制、敲除、沉默或突变，或该靶基因编码的蛋白的表达或活性被抑制；或所述里氏木霉菌株或其孢子、菌丝体、原生质体中，转入了所述靶基因的下调剂；较佳地，所述靶基因的下调剂包括：抑制或敲除所述的靶基因，或抑制所述的靶基因编码的蛋白的表达或活性的作用的试剂。
在另一优选例中，所述的里氏木霉菌株或其孢子、菌丝体、原生质体，其中还包括异源蛋白的编码基因；较佳地，所述异源蛋白的编码基因由表达载体引入到里氏木霉细胞内，或插入(同源替换)到里氏木霉基因组中，如(但不限于)插入到的cbh1位置。
在本发明的另一方面，提供前面任一所述的里氏木霉菌株或其孢子、菌丝体、原生质体的用途，用于表达里氏木霉内源蛋白或异源蛋白。
在本发明的另一方面，提供一种重组表达异源蛋白方法，包括：将异源蛋白的编码基因插入到任一所述的重组的里氏木霉菌株或其孢子、菌丝体、原生质体的基因组中，培养该里氏木霉菌株或其孢子、菌丝体、原生质体，从而表达异源蛋白；较佳地，所述的异源蛋白的编码基因插入(同源替换)到里氏木霉或其孢子、菌丝体、原生质体基因组中的cbh1位置。
在本发明的另一方面，提供一种重组表达里氏木霉内源蛋白方法，包括：培养所述的重组的里氏木霉菌株或其孢子、菌丝体、原生质体，从而重组表达里氏木霉内源蛋白(如纤维素酶)。
在本发明的另一方面，提供一种用于蛋白表达的试剂盒，其中包含：前面任一所 述的重组的里氏木霉菌株或其孢子、菌丝体、原生质体；或靶基因的下调剂(如用于敲除靶基因的质粒，或RNA干扰试剂)，所述靶基因选自：编码ORF4蛋白的基因，编码内源β-葡萄糖苷酶的基因。
在一个优选例中，所述的内源蛋白包括(但不限于)：纤维素酶，半纤维素酶，β葡萄糖苷酶，糖类透过酶，蛋白合成分泌途径相关酶。
在另一优选例中，所述的异源蛋白包括非纤维素酶分泌蛋白；较佳地，包括(但不限于)：异源葡萄糖苷酶，异源阿魏酸酯酶FEA，结构蛋白(如蜘蛛丝蛋白、蚕丝蛋白等)，功能蛋白(如免疫调节蛋白、血清白蛋白等)。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、利用酵母双杂系统验证orf4和ACE1存在蛋白互作。阳性对照为已经报道存在蛋白互作的Lae1和Vel1，实验组为AD-orf4和BD-ACE1，阴性对照为AD-orf4和BD空载体，以及AD空载体和BD-ACE1。显蓝色说明存在蛋白互作。
具体实施方式
里氏木霉是商业纤维素酶的主要生产微生物，目前工业上应用的常见菌株虽然相比野生株纤维素酶产量已经有了一定的提高，但是还有一定的上升空间。加上里氏木霉产纤维素酶的诱导机制还未被真正解析，因此寻找新的产酶以及蛋白分析相关的关键因子显得尤为重要。本发明人经过深入的研究，发现里氏木霉基因组中的一些靶基因与该菌株的内源或外源蛋白产量和/或活性密切相关，下调这些靶基因，可以极为显著地提高该菌株的内源或外源蛋白产量和/或活性。因此，所述靶基因或调节所述靶基因的物质和方法可应用于实现里氏木霉的改良，提高里氏木霉的蛋白表达效率，增强其表达的内源或外源蛋白产量和/或活性。
在本发明中，“ORF4多肽(蛋白)”指具有SEQ ID NO:2序列的多肽，还包括具有与ORF4多肽相同功能的、SEQ ID NO:2序列的变异形式或同源物。这些变异形式包括(但并不限于)：若干个(如1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个，还更佳如1-8个、1-5个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。
在本发明中，“β-葡萄糖苷酶”指具有SEQ ID NO:15序列的多肽，还包括具有与β-葡萄糖苷酶多肽相同功能的、SEQ ID NO:15序列的变异形式或同源物。这些变异形式包括(但并不限于)：若干个(如1-50个，较佳地1-30个，更佳地1-20个， 最佳地1-10个，还更佳如1-8个、1-5个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。
任何与所述的ORF4多肽或β-葡萄糖苷酶同源性高(比如与SEQ ID NO:2或15所示的序列的同源性为85％或更高；优选的，同源性为90％或更高；更优选的，同源性为95％或更高，如同源性98％或99％)的、且具ORF4多肽或β-葡萄糖苷酶相同功能的蛋白也包括在本发明内。
尽管本发明的具体实施例中，列举了具体的ORF4多肽或β-葡萄糖苷酶，但是应理解，由于里氏木霉存在一些不同的变种，它们之间序列高度保守，来自于这些菌中的同源多肽或多核苷酸也应被包含在本发明中，对这些同源多肽或多核苷酸(同源物)的如本发明类似或相同的调控方法也应被包含在本发明中。比对序列相同性的方法和工具也是本领域周知的，例如BLAST。
本发明人通过里氏木霉基因组逐步敲除，发现敲除株的蛋白分泌能力有所增加。再对这段序列进行单基因的筛选工作，最终发现该区域的甲基转移酶和产纤维素酶的调控有关，通过实验验证，该甲基转移酶酶可通过调控纤维素酶的抑制因子ACE1来调控纤维素酶的分泌。而且其他位点的基因也和纤维素酶的产量有关。该甲基转移酶的敲除可以使里氏木霉Rut-C30的FPU酶活提高25％以上。因此是一个产纤维素酶的关键因子，可用于里氏木霉工业菌株改造，非常具有应用价值。另一方面，本发明人还进一步对一个预测为β-葡萄糖苷酶的基因进行敲除，发现敲除株的蛋白分泌能力还能进一步提高。通过实验发现，该β-葡萄糖苷酶对里氏木霉诱导产酶的最强诱导物槐糖有较强的水解能力，可能导致胞内诱导物下降，和上述甲基转移酶都是诱导产纤维素酶的关键因子。
因此，本发明首次确定里氏木霉基因组中的一些靶基因与该菌株的内源或外源蛋白产量和/或活性密切相关。基于该新发现，本发明提供了一种提高里氏木霉的蛋白表达效率的方法，所述方法包括：下调里氏木霉基因组中的靶基因；其中，所述下调的靶基因选自：编码ORF4蛋白的基因，编码内源β-葡萄糖苷酶的基因。
在得知了所述的靶基因后，可以采用本领域人员熟知的多种方法来调节(下调或变异)它们。包括但不限于抑制、敲除、沉默或突变所述的靶基因，或抑制所述的靶基因编码的蛋白的表达或活性；或将下调所述靶基因的下调剂转入里氏木霉中；或通过定点突变试剂来进行靶向突变。
可以采用多种本领域已知的方法在里氏木霉株中下调所述靶基因，包括基因沉默、基因阻断、基因敲除、基因抑制等。这些方法均被包含在本发明中。
例如，可以通过基于同源重组的基因插入或缺失的基因阻断技术来改造基因组中的靶基因，从而使得靶基因被阻断；也可以针对靶基因设计干扰性RNA或反义核苷酸 来使靶基因表达抑制或沉默。
一种下调靶基因的方法是基因阻断技术，在本发明的优选实施方式中，体外构建靶基因阻断质粒，通过同源重组的方法，在里氏木霉株染色体靶基因中插入其他无关元件，从而使得染色体上的靶基因不再能够编码活性的蛋白质。当进行基因阻断时，无关元件的选择是本领域技术人员易于选择到的，例如应用一些抗性基因。一种基因阻断(敲除)的方法例如可参见Genetic Manipulation of Streptomyces:a Laboratory Manual中所记载的。
在设计用于进行基因阻断或敲除的构建物时，同时包含抗性筛选基因是优选的，从而有利于后续筛选出发生基因被阻断或敲除的菌株。
本发明还提供了下调所述靶基因的里氏木霉或其孢子、菌丝体、原生质体，更特别的是采用CRISPR/Cas技术缺失靶基因后获得的里氏木霉或其孢子、菌丝体、原生质体，该菌株或其孢子、菌丝体、原生质体不表达所述靶基因或表达量显著性降低。本发明还涉及所述菌株或其孢子、菌丝体、原生质体的用途，用于提高里氏木霉的蛋白表达效率。
基于本发明人的工作，本发明还提供了一种用于蛋白表达的试剂盒，其中包含：本发明的遗传工程化(重组)的里氏木霉或其孢子、菌丝体、原生质体。
本发明还提供了一种用于蛋白表达的试剂盒，其中包含：所述靶基因的下调剂，如用于敲除靶基因的质粒，或RNA干扰试剂，或定点突变试剂等。
所述的用于蛋白表达的试剂盒中，还可以包括其它应用于里氏木霉生产过程的试剂，例如里氏木霉的基础培养基。
所述的用于蛋白表达的试剂盒中，还可以包括使用说明书，说明培养所述里氏木霉的方法，或说明利用所述下调剂或定点突变试剂下调里氏木霉中靶基因的方法。
在本发明实施例中，本发明人通过对Rut-C30的基因组SEQ ID NO:1中的ORF进行敲除发现，这段序列和里氏木霉纤维素酶产量及蛋白分泌能力相关。本发明人还在该区域同源重组表达异源基因获得了比其他位点表达更高的产量，在此基础上进一步敲除一个预测为β-葡萄糖苷酶的基因SEQ ID NO:3可以进一步提高产量。因此，本发明涉及的基因及非编码多核苷酸序列对里氏木霉菌株具有很好的工业应用前景。
在本发明的具体实施例中，在里氏木霉Rut-C30菌株中敲除ORF4的多核苷酸序列后，其孢子生产的纤维素酶的酶活高于15IU/mL(U/mL)；较佳地高于18IU/mL。在本发明的另一实施例中，在里氏木霉Rut-C30菌株中阿魏酸酯酶FEA表达框(SEQ ID NO:5)在cbh1位点同源重组表达后，进一步敲除SEQ ID NO:1所示的多核苷酸序列后，其孢子生产的纤维素酶的酶活高于200IU/mL；较佳地高于220IU/mL。
在本发明的另一具体实施例中，在里氏木霉已经缺失SEQ ID NO:1的高产异源β-葡萄糖苷酶菌株U10中进一步还敲除SEQ ID NO:3所示的多核苷酸，其孢子生产的 β-葡萄糖苷酶酶活(pNPGase)高于2000IU/ml；较佳地高于2500IU/mL。
通过本发明的改造获得高产菌株，加上转化子发酵培养的碳源是麸皮等简单易得的农用废弃物，在提高纤维素酶制剂的水解效率方面有着十分明显的作用，如应用在食品其他行业生产中，也将大大降低其生产成本，有着更为广泛的工业应用潜力。
进一步地，本发明的活性位点，还可以用于异源基因的重组表达。通过农杆菌介导的转化、PEG介导的原生质体转化、基于ku70/ku80、CRSIPR/Cas9等手段将异源基因表达框引入到基因组中，优选引入到cbh1位置，取代该序列的部分或全部序列进行重组表达，进行进一步改良而获得产量更高或酶系更为优化的衍生菌株或者其他重组蛋白的工程菌株。这些工程菌株还可以进一步建立无筛选标记的基因操作，在工业上有着巨大的应用前景。
更进一步的，本发明的菌株可以作为进一步优化衍生菌株或者其他重组蛋白的工程菌株的基础，构建无标记筛选系统后，可作为分子操作的对象，可以敲入和敲除基因等遗传操作，进行有目的性的遗传改造，或过表达里氏木霉表达量过少的纤维素酶基因，如过表达纤维素酶激活因子Xyr1等，或敲除纤维素酶阻遏因子Cre1等，获得进一步高产的工程菌株。
本发明的改造菌株是活体细胞，一旦获得了本发明菌株的孢子、菌丝、原生质体及其相关的还有活体细胞的培养混合物，就可以用接种传代、再生等手段来大批量地获得本发明的菌株。这通常是将其接种到固体平板培养基或液体培养基中进行菌株的扩大培养而获得本发明的活体细胞。而获得的活体细胞可进一步进行实验室驯化、遗传育种和分子遗传操作等来获得突变体和转化子。也可利用本发明作为异源表达的宿主细胞。
本领域的技术人员熟知的方法能用于诱变本发明的活体细胞，而造成活体细胞的基因编码改变、酶活特性和形态学上的改变。这些方法包括利用射线、粒子、激光、紫外光等物理方法，利用烷化剂、碱基类似物(base analog)、羟胺(hydroxylamine)、吖啶色素等化学诱变方法。诱变可是以上一种方法或多种方法的多代诱变，且不限于这些方法。基于本发明提供的菌株，可以进一步进行物理化学等方式进行育种，也可以导入新的纤维素酶基因和相关调控基因，获得的突变体和转化子产酶性能可获得进一步提高，所述的育种方法为上述的一种或一种以上相结合。
本领域的技术人员熟知的方法将本发明的多核苷酸序列用于构建表达构建物(载体)和进一步改造宿主细胞。例如，对于菌株中已发现或新发现的与纤维素酶生产相关的信号途径、信号通路及其中涉及的蛋白进行进一步的改良(例如增加有益因子的表达，减少有害因子的表达)。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。所用的步骤在本领域众所周知。如农杆菌介导的真菌转化方法，原生质体转化方法、电击转化 方法、CRISPR/Cas9基因组编辑法、基因枪法等，且不限于这些方法。
本发明通过改造获得的里氏木霉优化菌株能够利用含有木质纤维素的工农业生产废弃物，如麸皮、玉米浆、豆饼粉等廉价原料，通过液态或固态发酵生产纤维素酶，其优势在于所产纤维素酶活力高，组分合理，水解木质纤维素能力强。本发明为解决纤维素资源利用中酶活力不高、酶组分不合理导致的生产成本高，糖化效率低等问题提供了新的菌种资源。
本发明通过引入FEA表达框同源替换cbh1后进一步敲除SEQ ID NO:1获得的里氏木霉工程菌株能够利用含有木质纤维素的工农业生产废弃物，如麸皮、玉米浆、豆饼粉等廉价原料，通过液态或固态发酵生产纤维素酶，其优势在于所产纤维素酶活力高，组分合理，水解木质纤维素能力强。相同发酵条件下，该菌株是随机引入FEA表达框产量的2倍。本发明为解决纤维素资源利用中酶活力不高、酶组分不合理导致的生产成本高，糖化效率低等问题提供了新的菌种资源。
本发明通过敲除SEQ ID NO:3获得的里氏木霉优化菌株能够利用含有木质纤维素的工农业生产废弃物，如麸皮、玉米浆、豆饼粉等廉价原料，通过液态或固态发酵生产纤维素酶，其优势在于所产纤维素酶活力高，组分合理，水解木质纤维素能力强。本发明为解决纤维素资源利用中酶活力不高、酶组分不合理导致的生产成本高，糖化效率低等问题提供了新的菌种资源。
本发明的方法获得的里氏木霉优化菌株能够利用含有木质纤维素的工农业生产废弃物，如豆饼粉等廉价原料，通过液态或固态发酵生产纤维素酶，其优势在于所产纤维素酶显著下降。本发明的敲除菌株可为表达非纤维素酶分泌蛋白提供表达宿主。
本发明在制备纤维素酶和异源蛋白方面的应用可以通过液态发酵实现，作为一个优选实施例方式，发酵方法包括：(1)将里氏木霉菌种在土豆培养基制成的斜面或平板活化后，制成浓度为10 6～10 8mL -1的孢子悬液，按10％的接种量接入沙堡氏培养基(种子培养基：1％酵母膏，1％蛋白胨，4％葡萄糖)中，28℃，200rpm振荡培养，得种子液，然后将种子液以10％接种量接入液体发酵培养基中，初始pH 5.0，装液量为10mL于50mL三角瓶，于28℃，200rpm摇床中培养5-7天；(2)将步骤(1)获得的发酵液离心分离，取上清液作为粗酶液；所述的诱导型发酵培养基是含有5％诱导物(3％微晶纤维素和2％麸皮)的无机盐培养液(0.4％KH 2PO 4，0.28％(NH 4) 2SO 4，0.06％MgSO 4·7H 2O，0.05％CaCl 2，0.06％urea，0.3％tryptone，0.1％Tween 80，0.5％CaCO 3，0.001％FeSO 4·7H 2O，0.00032％MnSO 4·H 2O，0.00028％ZnSO 4·7H 2O，0.0004％CoCl 2)。
应用于培养本发明的菌株的培养基和培养方法并不限于以上公布的那些，其它常规应用于培养里氏木霉的培养基和培养方法也可以应用于本发明中。
如上所述发酵体系可以进行体系放大进行工业生产，根据体系的大小不同，本领域技术人员根据所掌握的一般知识可以进行适当的调整以有利于菌株的生长或生产。
如上所述液体发酵粗酶液，可经超滤、盐析或有机溶剂沉淀等方法获得较纯的纤维素酶、酶粉或其他异源重组蛋白。通过发酵并测定所产纤维素酶的滤纸酶活(FPA)、CMC酶活、β-葡萄糖苷酶酶活及其他重组蛋白的活性。应理解，分离和纯化纤维素酶、重组蛋白的方法不受限于本发明中所提供的那些，其它本领域技术人员已知的方法也可应用于本发明中。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、SEQ ID NO:1多核苷酸的获得
1、培养里氏木霉菌株
将里氏木霉Rut-C30菌种(购自ATCC，菌株编号：ATCC 56765)接种在土豆培养基制成的平板上，置于28℃培养7天后，制成浓度为10 6～10 8个/mL的孢子悬液，按10％(v/v)的接种量接入沙堡氏培养基(种子培养基(v/v)：1％酵母膏，1％蛋白胨，4％葡萄糖)中，28℃，200rpm振荡培养，得菌液。
2、抽提里氏木霉基因组DNA
收集菌液到离心管，12000g离心获得菌丝。弃去上清液，加入0.5ml酚:氯仿:异戊醇(v:v:v＝25:24:1)，再加入0.5ml DES(0.1M Tris-HCl，pH 8.0，1.0％SDS，2％Triton X-100，10mM EDTA，0.1M NaCl)。加入0.1g beads后，放入Fastprep，6M/S，破碎30s，间隔5分钟后重复一次。裂解后的菌液12000g，4℃，离心10min。吸取上清0.4ml，加入等体积的异丙醇，-20℃沉淀30分钟。12000g，4℃，离心10min，弃上清。沉淀用75％乙醇洗涤后，12000g，4℃，离心5min，弃上清，晾干5min，之后加入200μl无菌水。
3、orf4多核苷酸(SEQ ID NO:1)的克隆
从上述分离到的里氏木霉基因组DNA，以正向引物为：5’ATGTCCCAATACGACTCCATCGG 3’(SEQ ID NO:6)，其5’端添加XbaI识别位点：TCTAGA；反向引物为5’TTACAACCGCAACTAAAACAC 3’(SEQ ID NO:7)，其5’端添加XbaI识别位点。
将PCR产物纯化后用XbaI酶切，应用AxygenPCR产物柱回收试剂盒回收酶切的DNA片段，将该DNA片段和经同样酶切的回收的载体pHDt/sk(含有Kan和hyg 筛选标记，购自Sigma公司)去磷酸化处理后，用T4DNA连接酶在16℃下连接过夜，得到载体pHDt/sk-full。并通过测序验证序列正确与否。
SEQ ID NO:1(orf4)核酸序列：
Figure PCTCN2021071349-appb-000001
SEQ ID NO:2(orf4)多肽序列：
Figure PCTCN2021071349-appb-000002
实施例2、orf4功能研究
1、里氏木霉CRISPR/Cas9底盘细胞的构建
通过对酿脓链球菌(Streptococcus pyogenes)的Cas9编码基因的密码子进行分析，以人工优化的Cas9基因(SEQ ID NO:4；其中第4117-4137位为入核信号)为模板，通过引物Cas9F(ATGGACAAGAAGTACAGCATTGG(SEQ ID NO:8))和Cas9R(TTAGACCTTGCGCTTCTTCTTGGG(SEQ ID NO:9))PCR分离获得该序列。以pDHt/sk载体为骨架，构建密码子优化的Cas9基因表达载体。
以里氏木霉基因组DNA为模板，利用正向引物：5’ACGACGGCCAGTGCCAAGCTTAGGACTTCCAGGGCTACTTG 3’(SEQ ID NO:10)和反向引物：5’GATTGTGCTGTAGCTGCGCTGCTTTGATCGTTTTGAGGTGC 3’(SEQ ID NO:11)扩增获得Ppdc作为优化的Cas9基因表达的启动子。
以里氏木霉基因组DNA为模板，利用正向引物5’AGAAGAAGAGGAAGGTGTGACCCGGCATGAAGTCTGACCG 3’(SEQ ID NO:16)和反向引物5’TAATTGCGCGG ATCCTCTAGATGGACGCCTCGATGTCTTCC 3’(SEQ ID NO:17)扩增Tpdc作为终止子。
采用一步法克隆的方法构建载体，通过验证序列成功构建包含诱导型启动子的重组载体pDHt/sk-ppdc-Cas9-tpdc。
将上述构建好的质粒转化入根癌农杆菌AGL1中，在根癌农杆菌的介导下转化入里氏木霉Rut-C30中，用潮霉素作为筛选压力，将长出的转化子接种于SDB培养液中，2天后抽提DNA为模板，通过载体上的启动子处设计正向引物5’TACGTCGGCCCCCTGGCC 3’(SEQ ID NO:12)和Cas9基因的反向引物为5’GAGGTTGTCAAACTTGCGCTGCG 3’(SEQ ID NO:13)PCR鉴定阳性转化子。获得的转化子分别命名为C30-pc。
2.orf4的功能性敲除
建立sgRNA：5’TAATACGACTCACTATA GGGAAAGGCACAATTCGT GTGTTTTAGAGCTAGAAATAGC 3’(SEQ ID NO:14)，其靶向于SEQ ID NO:1(在基因的第257-277位发生剪切破坏其基因功能)。
将构建好的sgRNA连入pMD18T(购自Takara公司，Amp抗性)，以此为模板，获得的体外转录模板用苯酚：氯仿：异戊醇(25:24:1)抽提纯化，最后用Nuclease free water溶解DNA，即可用于gRNA的体外转录。
gRNA的体外转录，具体方法参考试剂盒说明书。将C30-pc孢子接种于PDA平板，28℃培养7d，使用0.85％NaCl+0.02％吐温80洗下孢子。用灭菌的镊子在PDA平板上盖一张玻璃纸，将其铺平，在玻璃纸上加入150ul左右的孢子悬液，用玻棒涂布均匀，做6个同样的处理，28度培养约14h。用裂解酶(Sigma#L1412 lysing enzymes)裂解细胞壁，除去细胞壁碎片后即可获得原生质体悬液，与纯化gRNA片段混合后用PEG介导的原生质转化法转化，涂布于的筛选平板，置28℃培养，5天左右可观察到菌丝长出。待转化子长出后，验证后即可获得对应sgRNA的敲除子。
3.敲除子酶活筛选
(1)将里氏木霉菌种接种在土豆培养基制成的平板上置于28℃培养7天后，制成浓度为10 6～10 8个/mL的孢子悬液，按10％(v/v)的接种量接入沙堡氏培养基(种子培养基(v/v)：1％酵母膏，1％蛋白胨，4％葡萄糖)中，28℃，200rpm振荡培养，得种子液，然后将种子液以10％(v/v)接种量接入液体发酵培养基中，初始pH 5.0，装液量为10mL于50mL三角瓶，于28℃，200rpm摇床中培养5-7天；
(2)将步骤(1)获得的发酵液离心分离，取上清液作为粗酶液；所述的发酵培养基是含有5％诱导物(3％微晶纤维素和2％麸皮)的无机盐培养液(0.4％KH2PO4，0.28％(NH 4) 2SO 4，0.06％MgSO 4·7H 2O，0.05％CaCl 2，0.06％尿素，0.3％蛋白胨，0.1％Tween 80，0.5％CaCO 3，0.001％FeSO 4·7H 2O，0.00032％MnSO 4·H 2O，0.00028％ZnSO 4·7H 2O，0.0004％CoCl 2)，
(3)在0.05mol/L pH5.0的醋酸-醋酸钠或柠檬酸-柠檬酸钠缓冲液中，将里氏木霉出发株及其敲除子粗酶液加入到滤纸中，50～60℃水解60min，测定滤纸上的纤维素酶的酶活(单位：Filter Paper Unit，FPU)。测得酶活如表1。
表1、里氏木霉转化子及其敲除菌株的滤纸酶活
Figure PCTCN2021071349-appb-000003
由表1结果可见，orf4敲除使得纤维素酶的酶活显著性升高，因此其与纤维素酶的产酶相关，是关键基因。
实施例3、SEQ ID NO:1编码的蛋白的互作蛋白筛选
利用酵母双杂交载体pGADT7 AD Vector，pGBKT7 DNA-BD Vector(购于Takara公司)分别构建orf4(即SEQ ID NO:1)和其他已知转录调控因子包括：ACE1(GenBank登录号AF190793)，Vel1(GenBank登录号XM_006966084)，Hda1(GenBank登录号XP_006968036)，Cre1(GenBank登录号AAB01677)，Lae1(GenBank登录号AFX86442等，得到AD-orf4，AD-ACE1，AD-Vel1，AD-Hda1，AD-Cre1，AD-Lae1，AD-Cre1以及BD-orf4，BD-ACE1，BD-Vib1，BD-Hda1，BD-Cre1，BD-Lae1，BD-Cre1等。将不同组合的AD和BD载体转化的AH109酵母菌株(购自Takara)，并分别做好空白对照，分别涂布于含有SD+Dropout+Ade+His培养基筛选平板。30℃培养2～3天后，挑取菌落少许至SD+Dropout+3-AT+X-α-GAL营养缺陷培养基，30℃培养2-3天，观察酵母菌能生长同时变蓝色的说明两个蛋白之间存在互作。
结果发现，共转化AD-orf4和BD-ACE1的转化子能够生长并显蓝色，如图1所示，说明orf4和ACE1存在互作。
ACE1是一个纤维素酶抑制因子，该结果提示orf4通过对ACE1某些位点进行修饰，进而激活其抑制纤维素酶转录的功能。
实施例4、利用orf4表达异源蛋白
以实施例2获得的C30-pc菌株为出发株，利用CRISPR/Cas9技术将阿魏酸酯酶FEA(SEQ ID NO:5所示的核苷酸)同源替换里氏木霉cbh1位置，继而敲除SEQ ID NO:1，获得的转化子命名为U10FEA1～U10FEA5，仅同源替换里氏木霉cbh1位置而不敲除SEQ ID NO:1的菌株作为对照(FEAΔ1)。将转化子和C30-pc接种到在土豆培养基制成的平板上置于28℃培养7天后，制成浓度为10 6～10 8个/mL的孢子悬液，按10％(v/v)的接种量接入沙堡氏培养基(种子培养基(v/v)：1％酵母膏，1％蛋白胨，4％葡萄糖)中，28℃，200rpm振荡培养，得种子液，然后将种子液以10％(v/v)接种量接入液体发酵培养基中，初始pH 5.0，装液量为10mL于50mL三角瓶，于28℃，200rpm摇床中培养5-7天；获得的发酵液离心分离，取上清液作为粗酶液；所述的发酵培养 基是含有5％诱导物(3％微晶纤维素和2％麸皮)的无机盐培养液(0.4％KH 2PO 4，0.28％(NH 4) 2SO 4，0.06％MgSO 4·7H 2O，0.05％CaCl 2，0.06％尿素，0.3％蛋白胨，0.1％Tween 80，0.5％CaCO 3，0.001％FeSO 4·7H 2O，0.00032％MnSO 4·H 2O，0.00028％ZnSO 4·7H 2O，0.0004％CoCl 2)。将发酵液用0.05mol/L pH6.0的醋酸-醋酸钠缓冲液稀释，取50ul加入到0.625mM pNPF(先用DMSO配成10mM贮存液，取0.5ml溶于7.5ml含有5％Triton X-100的0.05mol/L pH6.0的醋酸-醋酸钠缓冲液中，pNPF的制备按照文献Analytical Biochemistry 387(2009)128-129所述制备)中，37℃水解10min，离心后取上清液于OD405读数。结果测得阿魏酸酯酶FEA酶活如表2所示。
表2、C30-pc及其转化子的pNPF酶活
Figure PCTCN2021071349-appb-000004
由表2结果可见，替换cbh1后进一步敲除orf4(SEQ ID NO:1)可以获得比仅替换cbh1位点更高的异源蛋白表达量。敲除orf4是提高异源蛋白表达产量的一个优选策略。
实施例5、通过对内源β-葡萄糖苷酶的操作提高胞外分泌蛋白水平
以里氏木霉菌株Qm9414(购自ATCC 26921)为出发株，用实施例1中的方法，敲除β-葡萄糖苷酶编码基因(SEQ ID NO:3所示的核苷酸)，设计sgRNA：5’TAATACGACTCACTATA GGCAAGTATCCGTATCCCGAGTTTTAGAGCTAGAAATAGC 3’(SEQ ID NO:19)，对SEQ ID NO:3所示的核苷酸的第2607-2626位进行剪切，破坏其功能，获得转化子Δbgl1～Δbgl3。
β-葡萄糖苷酶的基因(SEQ ID NO:3)：
Figure PCTCN2021071349-appb-000005
Figure PCTCN2021071349-appb-000006
β-葡萄糖苷酶的蛋白(SEQ ID NO:15)：
Figure PCTCN2021071349-appb-000007
将转化子和Qm9414接种到在土豆培养基制成的平板上置于28℃培养7天后，制成浓度为10 6～10 8个/mL的孢子悬液，按10％(v/v)的接种量接入沙堡氏培养基(种子培养基(v/v)：1％酵母膏，1％蛋白胨，4％葡萄糖)中，28℃，200rpm振荡培养，得种子液，然后将种子液以10％(v/v)接种量接入液体发酵培养基中，初始pH 5.0，装液量为10mL于50mL三角瓶，于28℃，200rpm摇床中培养5-7天；获得的发酵液离心分离，取上清液作为粗酶液；所述的发酵培养基是含有5％诱导物(3％微晶纤维素和2％麸皮)的无机盐培养液(0.4％KH 2PO 4，0.28％(NH 4) 2SO 4，0.06％MgSO 4·7H 2O，0.05％CaCl 2，0.06％尿素，0.3％蛋白胨，0.1％Tween 80，0.5％CaCO 3，0.001％FeSO 4·7H 2O，0.00032％MnSO 4·H 2O，0.00028％ZnSO 4·7H 2O，0.0004％CoCl 2)。酶活检测具体如表3所述。
表3、Qm9414及其转化子的发酵液的纤维素酶酶活
Figure PCTCN2021071349-appb-000008
Figure PCTCN2021071349-appb-000009
由表3的结果可见，敲除内源β-葡萄糖苷酶可显著提高内源纤维素酶的产量。
实施例6、通过对内源β-葡萄糖苷酶的操作进一步提高外源蛋白产量
以过表达外源β葡萄糖苷酶trbgls的里氏木霉菌株(专利号201210258194.6，以RC30-8为出发株)，用实施例1中的方法进行对SEQ ID NO:1序列进行敲除，获得U10。以U10为出发株，将内源β-葡萄糖苷酶编码基因SEQ ID NO:3所示的核苷酸敲除(sgRNA：5’TAATACGACTCACTATA GGCAAGTATCCGTATCCCGAGTTTTAGAGCTAGAAATAGC 3’(SEQ ID NO:18)；剪切位置：SEQ ID NO:3所示的核苷酸的第2607-2626位)，获得蛋白功能被破坏的菌株，获得的转化子命名为U10ΔB1。
将转化子和U10接种到在土豆培养基制成的平板上置于28℃培养7天后，制成浓度为10 6～10 8个/mL的孢子悬液，按10％(v/v)的接种量接入沙堡氏培养基(种子培养基(v/v)：1％酵母膏，1％蛋白胨，4％葡萄糖)中，28℃，200rpm振荡培养，得种子液，然后将种子液以10％(v/v)接种量接入液体发酵培养基中，初始pH 5.0，装液量为10mL于50mL三角瓶，于28℃，200rpm摇床中培养5-7天；获得的发酵液离心分离，取上清液作为粗酶液；所述的发酵培养基是含有5％诱导物(3％微晶纤维素和2％麸皮)的无机盐培养液(0.4％KH 2PO 4，0.28％(NH 4) 2SO 4，0.06％MgSO 4·7H 2O，0.05％CaCl 2，0.06％尿素，0.3％蛋白胨，0.1％Tween 80，0.5％CaCO 3，0.001％FeSO 4·7H 2O，0.00032％MnSO 4·H 2O，0.00028％ZnSO 4·7H 2O，0.0004％CoCl 2)。酶活检测具体如下所述：
以对硝基苯-β-D-葡萄糖苷(pNPG)为底物检测异源葡萄糖苷酶的酶活及理化特性，具体操作如下：
(1)对硝基苯酚(pNP)标准曲线制备
取8*3排列的PCR板，按表4加入溶液，共8组，每组3个平行重复。
表4
Figure PCTCN2021071349-appb-000010
pNP浓度为10mg/ml。上表每份标样加100μl 1M NaCO 3，终止反应并显色，每管吸取100μl于酶标板中，用酶标仪测405nm光吸收，标样编号0为空白对照。各样品值减空白后制备标线。
(2)异源葡萄糖苷酶的标准酶活测定
在100μl反应体系中，加入50μl浓度为4mM的pNPG，然后加入浓度为0.1M的 NaAc/HAc(pH5.0)缓冲液稀释至一定稀释度的酶液50μl，60℃反应10分钟，再加入100μl 1M NaCO 3终止反应并显色(对照为在上述反应体系中先加入100μl 1M NaCO 3后再加酶液)，用酶标仪测405nm光吸收值，样品测定值减去对照后利用标准曲线计算酶活单位(U)。结果如表5。
酶活单位(U)定义：1U为每分钟催化pNPG产生1μmol pNP所需的酶量。
表5、U10及其转化子的发酵液的pNPG酶活
Figure PCTCN2021071349-appb-000011
由表5的结果可见，敲除内源β-葡萄糖苷酶可以进一步提高外源蛋白(异源葡萄糖苷酶)产量。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (18)

  1. 一种提高里氏木霉的蛋白表达效率的方法，包括：下调里氏木霉基因组中的靶基因，所述靶基因选自：编码ORF4蛋白的基因，编码β-葡萄糖苷酶的基因。
  2. 如权利要求1所述的方法，其特征在于，所述的蛋白是里氏木霉内源蛋白或异源蛋白。
  3. 如权利要求2所述的方法，其特征在于，所述的内源蛋白包括：纤维素酶，半纤维素酶，β葡萄糖苷酶，糖类透过酶，蛋白合成分泌途径相关酶；或
    所述的异源蛋白包括非纤维素酶分泌蛋白；较佳地，包括：异源葡萄糖苷酶，异源阿魏酸酯酶FEA，结构蛋白，功能蛋白。
  4. 如权利要求2所述的方法，其特征在于，所述的异源蛋白的编码基因插入到里氏木霉基因组中；或，所述的异源蛋白的编码基因由表达载体引入到里氏木霉细胞内。
  5. 如权利要求1所述的方法，其特征在于，所述的异源蛋白的编码基因插入到里氏木霉基因组中cbh1位置。
  6. 如权利要求1所述的方法，其特征在于，下调里氏木霉基因组中的靶基因包括：
    抑制、敲除、沉默或突变所述的靶基因，或抑制所述的靶基因编码的蛋白的表达或活性；或将下调所述靶基因的下调剂转入里氏木霉中；或调节所述靶基因的上游信号通路或上游基因，下调所述靶基因。
  7. 如权利要求6所述的方法，其特征在于，通过采用CRISPR/Cas技术进行基因编辑以敲除所述的靶基因；或通过同源重组技术，对靶基因所在区段进行缺失或插入的操作以敲除所述的靶基因；或通过干扰分子干扰靶基因的表达；所述的干扰分子是以所述靶基因或其转录本为抑制或沉默靶标的dsRNA、反义核酸、小干扰RNA、微小RNA，或能表达或形成所述dsRNA、反义核酸、小干扰RNA、微小RNA的构建物。
  8. 如权利要求1所述的方法，其特征在于，通过引入阿魏酸酯酶FEA表达框同源替换编码ORF4蛋白的基因，从而敲除该编码ORF4蛋白的基因，同时引入阿魏酸 酯酶FEA表达框；或
    所述编码ORF4蛋白的基因具有SEQ ID NO:1所示的核苷酸序列，基于该序列，靶向于其第257-277位，使该靶位置发生剪切，破坏基因功能；或
    所述编码β-葡萄糖苷酶的基因具有SEQ ID NO:3所示的核苷酸序列，基于该序列，靶向于其第2607-2626位，使该靶位置发生剪切，破坏基因功能。
  9. 靶基因的调节剂的用途，用于提高里氏木霉的蛋白表达效率；所述调节剂包括下调剂；所述下调剂为靶向于ORF4蛋白或其编码基因或靶向于内源葡萄糖苷酶或其编码基因的下调剂。
  10. 如权利要求9所述的用途，其特征在于，所述的下调剂包括：靶向且敲除靶基因的CRISPR/Cas基因编辑试剂；或通过同源重组对靶基因进行缺失或插入操作、从而敲除所述的靶基因的试剂；或干扰分子，其包括以所述靶基因或其转录本为抑制或沉默靶标的dsRNA、反义核酸、小干扰RNA、微小RNA，或能表达或形成所述dsRNA、反义核酸、小干扰RNA、微小RNA的构建物。
  11. 一种重组的里氏木霉菌株或其孢子、菌丝体、原生质体，其特征在于，其基因组中的靶基因被下调；其中，下调的靶基因选自：编码ORF4蛋白的基因，编码内源β-葡萄糖苷酶的基因。
  12. 如权利要求11所述的里氏木霉菌株或其孢子、菌丝体、原生质体，其特征在于，所述的里氏木霉菌株或其孢子、菌丝体、原生质体中，所述靶基因被抑制、敲除、沉默或突变，或该靶基因编码的蛋白的表达或活性被抑制；或所述里氏木霉菌株或其孢子、菌丝体、原生质体中，转入了所述靶基因的下调剂；较佳地，所述靶基因的下调剂包括：抑制或敲除所述的靶基因，或抑制所述的靶基因编码的蛋白的表达或活性的作用的试剂。
  13. 如权利要求11所述的里氏木霉菌株或其孢子、菌丝体、原生质体，其特征在于，其中还包括异源蛋白的编码基因；较佳地，所述异源蛋白的编码基因由表达载体引入到里氏木霉细胞内，或插入到里氏木霉基因组中，如插入到的cbh1位置。
  14. 权利要求11～13任一所述的里氏木霉菌株或其孢子、菌丝体、原生质体的用途，用于表达里氏木霉内源蛋白或异源蛋白。
  15. 一种重组表达异源蛋白方法，其特征在于，所述方法包括：将异源蛋白的编码基因插入到权利要求11～13任一所述的重组的里氏木霉菌株或其孢子、菌丝体、原生质体的基因组中，培养该里氏木霉菌株或其孢子、菌丝体、原生质体，从而表达异源蛋白；较佳地，所述的异源蛋白的编码基因插入到里氏木霉或其孢子、菌丝体、原生质体基因组中的cbh1位置。
  16. 一种重组表达里氏木霉内源蛋白方法，其特征在于，所述方法包括：培养权利要求11～13任一所述的重组的里氏木霉菌株或其孢子、菌丝体、原生质体，从而重组表达里氏木霉内源蛋白。
  17. 一种用于蛋白表达的试剂盒，其特征在于，其中包含：
    权利要求11～13任一所述的重组的里氏木霉菌株或其孢子、菌丝体、原生质体；或
    靶基因的下调剂，所述靶基因选自：编码ORF4蛋白的基因，编码内源β-葡萄糖苷酶的基因。
  18. 如权利要求14或16所述，其特征在于，所述的内源蛋白包括：纤维素酶，半纤维素酶，β葡萄糖苷酶，糖类透过酶，蛋白合成分泌途径相关酶；或
    所述的异源蛋白包括非纤维素酶分泌蛋白；较佳地，包括：异源葡萄糖苷酶，异源阿魏酸酯酶FEA，结构蛋白，功能蛋白。
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