Construction method for improving gene editing efficiency of Trichoderma reesei engineering strain

Abstract

The invention discloses a construction method for improving gene editing efficiency of a recombinant trichoderma reesei engineering strain, and belongs to the technical field of gene editing. In the research, the T.reesei CJ2095 is taken as an original strain, the gene ku80 is knocked out, and the gene editing efficiency is improved to 7.14% from 1.04% of that of a control strain. In order to realize the recycling of the screening marker of the orotidine-5 '-phosphate decarboxylase gene pyr4, the gene pyr4 is knocked out. In addition, homologous recombinase rad51 and rad52 genes of a T.reesei endogenesis are respectively overexpressed, and the overexpressed gene rad52, namely the homologous recombination efficiency of the engineering strain T.reesei CJ2095 delta ku80 delta pyr4: rad52, can be improved to 43.75%, which is 42 times of that of an original strain.

Description

Technical Field

[0001] The present invention relates to a construction method for improving the gene editing efficiency of Trichoderma reesei engineering strains, belonging to the technical field of gene editing. Background Art

[0002] Trichoderma reesei is a safe filamentous fungus with high cellulase production. Due to its strong protein expression ability, T. reesei has become the main strain for industrial production. It is reported that the highest titer of cellulase production exceeds 100 g·L on an industrial scale. -1 Currently, T. reesei strains have produced industrial enzymes such as cellulase, xylanase, and phytase, and are widely used in fields such as food additives, feeds, and energy.

[0003] At present, there are two most commonly used transformation methods for T. reesei - protoplast transformation method and Agrobacterium tumefaciens - mediated transformation method. The protoplast transformation method has a relatively high transformation efficiency and a wide range of applications, including some plant cells and fungal cell types that are difficult to transform. However, factors such as the complex process of protoplast preparation and low regeneration efficiency limit its application. The Agrobacterium tumefaciens - mediated transformation method has good genetic stability, simple operation and low cost. Therefore, the Agrobacterium tumefaciens - mediated transformation method is more widely used in T. reesei. The repair mode of T. reesei is mainly non - homologous end joining, supplemented by homologous recombination. Therefore, the homologous recombination efficiency in T. reesei is extremely low. In experiments, when knocking out genes by homologous recombination, a large amount of time and effort need to be invested in screening transformants. With the development of gene editing technology, the gene editing efficiency of different species of microorganisms is constantly improving. In Saccharomyces cerevisiae, homologous recombination can be achieved with a homologous arm of only about 50 bp. However, for T. reesei, even when the homologous arm reaches 1200 bp, its homologous recombination efficiency is still not high. When the ku70 gene is knocked out in T. reesei, the homologous recombination rate increases from 5% - 10% to over 95% when the homologous arm is 1 kb (published in the paper "Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina"). This is because the ku protein participates in non - homologous end joining repair. When the ku protein forms a complex with DNA, other functional factors involved in non - homologous end joining repair will bind to the complex and act together at the DNA break, thus enabling non - homologous end joining repair to proceed. However, compared with traditional model organisms such as yeast and Escherichia coli, the homologous recombination efficiency of T. reesei is still very low. To solve the problem of low homologous recombination efficiency in T. reesei, the present invention knocks out the key gene ku80 for non - homologous end joining repair and strengthens the key gene rad52 for homologous recombination in T. reesei endogenous, so as to greatly improve the homologous recombination efficiency of T. reesei. Summary of the Invention

[0004] Aiming at the deficiencies of the above - mentioned prior art, the purpose of the present invention is to systematically construct and improve the gene editing efficiency of Trichoderma reesei strains by genetic engineering means, and increase the application potential of subsequent Trichoderma reesei strains in the field of bioengineering.

[0005] The first technical solution provided by the present invention is the application of gene ku80 and gene rad52 in improving the gene editing efficiency of Trichoderma reesei, and the application is to knock out the DNA repair protein gene ku80 of Trichoderma reesei and over - express the homologous recombination enzyme gene rad52.

[0006] In some embodiments, the nucleotide sequence of the gene ku80 has the NCBI accession number NW_006711151.1; the nucleotide sequence of the gene rad52 has the NCBI accession number NW_006711159.1.

[0007] The second technical solution provided by the present invention is a Trichoderma reesei engineering strain with high gene editing efficiency. The engineering strain uses Trichoderma reesei as the starting strain, knocks out the DNA repair protein gene ku80 in the starting strain, and overexpresses the homologous recombinase gene rad52.

[0008] In some embodiments, the nucleotide sequence of the gene ku80 has the NCBI accession number NW_006711151.1; the nucleotide sequence of the gene rad52 has the NCBI accession number NW_006711159.1.

[0009] In some embodiments, Trichoderma reesei CJ2095 is used as the starting strain for transformation. Trichoderma reesei CJ2095 is disclosed in the patent with the publication number CN117487675A, corresponding to Trichoderma reesei CJ2095 in the patent with the publication number CN117487675A.

[0010] The third technical solution provided by the present invention is a method for constructing the Trichoderma reesei engineering strain described in the second technical solution. The method is to knock out the gene ku80 of Trichoderma reesei CJ2095 and at the same time overexpress the homologous recombinase gene rad52 in Trichoderma reesei CJ2095.

[0011] In some embodiments, the method includes the following steps:

[0012] (1) Insert the gene ku80 knockout cassette into the vector backbone to obtain the gene ku80 knockout recombinant plasmid. The structure of the gene ku80 knockout cassette is "upstream homologous arm of gene ku80 - hygromycin resistance gene expression cassette - downstream homologous arm of gene ku80";

[0013] (2) Insert the gene rad52 expression cassette into the vector backbone to obtain the gene rad52 expression recombinant plasmid. The structure of the gene rad52 expression cassette is "gda410 - pyr4 cassette - Pgpd1 - gene rad52 and its transcription terminator";

[0014] (3) Transform the gene ku80 knockout recombinant plasmid in step (1) and the gene rad52 expression recombinant plasmid in step (2) into Agrobacterium tumefaciens respectively. Pick the transformants and co - culture and transform them with Trichoderma reesei CJ2095 to screen for the Trichoderma reesei engineering strain with high gene editing efficiency.

[0015] In some embodiments, in step (1), all amplified fragments of the gene ku80 knockout cassette are ligated to the vector backbone obtained by PCR amplification by a one-step ligation method.

[0016] In some embodiments, in step (2), all amplified fragments of the gene rad52 expression cassette except the pyr4 cassette are ligated to the vector backbone obtained by PCR amplification by a one-step ligation method. Then, pCAMBIA1303-LB-RB-gda410-Pgpd1-rad52-Trad52 and the pyr4 cassette obtained by PCR amplification are ligated by double digestion.

[0017] In some embodiments, the nucleotide sequence of the upstream homologous arm of the DNA repair protein gene ku80 is as shown in SEQ ID NO.3, the nucleotide sequence of the downstream homologous arm of the DNA repair protein gene ku80 is as shown in SEQ ID NO.4, the nucleotide sequence of the hygromycin resistance gene expression cassette is as shown in SEQ ID NO.11, the nucleotide sequence of the pyr4 cassette is as shown in SEQ ID NO.7, and the nucleotide sequence of the gene rad52 and its transcription terminator is as shown in SEQ ID NO.9.

[0018] In some embodiments, the promoter is Pgpd1, and its nucleotide sequence is as shown in SEQ ID NO.10.

[0019] In some embodiments, the Agrobacterium tumefaciens is AGL1.

[0020] In some embodiments, the pCAMBIA1303-LB-RB plasmid is used as the vector backbone to construct the gene expression cassette and the gene knockout cassette.

[0021] The fourth technical solution provided by the present invention is a method for gene editing of Trichoderma reesei, which is to knockout or knock in a target gene using the Trichoderma reesei engineering strain described in the second technical solution as the chassis cell.

[0022] In some embodiments, the gene editing is to knockout the pyr4 gene.

[0023] In some embodiments, the specific steps of knocking out the pyr4 gene include the following:

[0024] S1, construct a pyr4 gene knockout cassette, and the structure of the pyr4 gene knockout cassette is "upstream homologous arm of the pyr4 gene - downstream homologous arm of the pyr4 gene";

[0025] S2. Transform the pyr4 gene knockout cassette of S1 into Agrobacterium tumefaciens, pick the transformants and co-culture them with the Trichoderma reesei engineering strain described in the second technical solution, and screen to obtain Trichoderma reesei with the pyr4 gene knocked out.

[0026] Furthermore, the NCBI number of the nucleotide sequence of the pyr4 gene is NW_006711148.1.

[0027] In some embodiments, the nucleotide sequence of the upstream homologous arm of the pyr4 gene is as shown in SEQ ID NO.5, and the nucleotide sequence of the downstream homologous arm of the pyr4 gene is as shown in SEQ ID NO.6.

[0028] The present invention also screened suitable working concentrations of 5-fluoroorotic acid (5-FOA) and hygromycin for screening positive transformants of Trichoderma reesei CJ2095.

[0029] Compared with the prior art, the technical effects of the present invention are as follows:

[0030] (1) The DNA repair protein gene ku80 was knocked out to obtain the recombinant strain T. reesei CJ2095Δku80 with significantly improved gene editing efficiency. Its gene editing efficiency was increased from 1.04% of the control strain to 7.14%.

[0031] (2) In order to realize the recycling of the orotidine-5'-phosphate decarboxylase gene pyr4 screening marker, the gene pyr4 on the genome of the recombinant strain T. reesei CJ2095Δku80 was knocked out to obtain the recombinant strain T. reesei CJ2095Δku80Δpyr4.

[0032] (3) The effects of overexpressing the endogenous homologous recombination enzymes rad51 and rad52 genes of T. reesei on the homologous recombination efficiency were investigated. The results showed that by overexpressing the gene rad52, the homologous recombination efficiency of the engineering strain T. reesei CJ2095Δku80Δpyr4::rad52 could be increased to 43.75%, which was 42 times that of the starting strain. Description of the Drawings

[0033] Figure 1 Optimization of the hygromycin working concentration.

[0034] Figure 2 Construction of the recombinant plasmid for knocking out the ku80 gene and verification of positive clones. a: Schematic diagram of the construction of the ku80 gene knockout plasmid; b: Schematic diagram of the ku80 gene knockout; c: Gel diagram of nucleic acid verification of the ku80 gene knockout.

[0035] Figure 3 Optimization of the working concentration of 5 - FOA.

[0036] Figure 4 Effect of ku80 gene knockout on the gene editing efficiency of T. reesei. a: Schematic diagram of the construction of the pyr4 gene knockout plasmid; b: Gel diagram of nucleic acid verification for pyr4 gene knockout, and the red arrow indicates the lane where pyr4 was correctly knocked out; c: Effect of ku80 gene knockout on the pyr4 gene knockout efficiency.

[0037] Figure 5 Phenotypic verification of the recombinant strain T. reesei CJ2095Δku80Δpyr4.

[0038] Figure 6 Effect of overexpressing the key enzyme genes rad51 or rad52 on the gene editing efficiency of T. reesei. a: Schematic diagram of the construction of plasmids for overexpressing rad51 gene and rad52 gene; b: Verification of the excision of the pyr4 selection marker; c: Excision efficiency of the pyr4 selection marker; d: Schematic diagram of the excision of the pyr4 selection marker. Specific implementation manners

[0039] The following are descriptions of the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0040] Culture media:

[0041] LB medium (1L): 10 g peptone, 10 g NaCl, 5 g yeast extract.

[0042] PDA medium (1L): 37 g potato dextrose agar medium.

[0043] Malt extract agar medium (1L): 30 g malt extract, 20 g agar powder.

[0044] MM medium (1L): 10 g (NH4)2SO4, 20 g glucose, 6.7 g YNB.

[0045] IM medium (1L): 1.45 g KH2PO4, 2.05 g K2HPO4, 0.5 g (NH4)2SO4, 0.5 g MgSO4·7H2O, 0.15 g NaCl, 5 g glycerol, 0.06 g CaCl2, 0.0025 g FeSO4·7H2O, 1.8 g glucose, 8.54 g 2 - morpholinoethanesulfonic acid, 200 μM acetosyringone.

[0046] E. coli and A. tumefaciens were cultured in LB medium. T. reesei was cultured in PDA medium. Malt extract agar medium was used to culture T. reesei for spore production. The co-culture medium (IM) was used for the transformation of T. reesei. When the above media needed to be configured into solid media, 15 g·L -1 agar powder was added, and 1 g·L -1 uracil was added when culturing uracil auxotrophic strains.

[0047] Transformation of T. reesei:

[0048] T. reesei was genetically edited by Agrobacterium tumefaciens-mediated transformation. Agrobacterium tumefaciens containing the recombinant plasmid was inoculated into an LB test tube containing kanamycin and rifampicin and cultured for 24 - 36 h. 400 μL of the bacterial cells were collected and centrifuged, the supernatant was discarded, and the bacterial cell pellet was resuspended in 5 mL of an IM test tube containing MES and AS, and pre-cultured at 30 °C and 200 rpm until the OD 600 reached between 0.6 and 0.8, protected from light. An equal volume of T. reesei spores and the pre-cultured Agrobacterium tumefaciens were spread on an IM plate containing cellophane and cultured at 24 °C in the dark for 48 h. After the culture was completed, the cellophane was transferred to a screening plate and cultured at 30 °C for 2 - 7 d waiting for the transformants to grow. The transformants were picked and cultured on a re-screening plate for 1 - 2 d, and then the hyphae were picked and cultured in a 2 mL centrifuge tube containing MM medium until the hyphae grew out. The genomic DNA of the strains with growing hyphae was extracted and verified. The strains with correct verification were streaked on a PDA plate containing 0.1% (v / v) Triton-X100 to isolate single spores, and the single spores were selected and cultured on PDA medium for spore production, and finally stored in 40% glycerol.

[0049] Extraction of the T. reesei genome

[0050] The method for extracting the T. reesei genome refers to the paper by Meng Qingshan. Mycelia or spores were inoculated into the seed medium and cultured at 30 °C and 200 rpm for 2 - 3 d. Then, centrifuged at 12,000×g for 3 min, the supernatant was discarded, 500 μL of chromosomal extract was added, and an appropriate amount of glass beads was added, followed by shaking for 20 min. Then, placed in a water bath at 65 °C for 30 min. Next, a phenol-chloroform-isoamyl alcohol mixture (25:24:1) was added, shaken for 10 min, and centrifuged at 12,000×g for 3 min. The supernatant was transferred to a new 2 mL centrifuge tube, 50 μL of 3 M sodium acetate and 600 μL of isopropanol were added, mixed, and ice-bathed for 15 min. Finally, centrifuged at 4 °C and 12,000×g for 10 min, the supernatant was discarded, the DNA was washed with 75% ethanol, and after the ethanol evaporated, it was dissolved in an appropriate amount of sterile water for subsequent PCR verification operations.

[0051] In the following examples, the experimental methods not specifically noted can be completed according to conventional methods or according to the usage instructions of the manufacturers of the products used. The materials, reagents, etc. used, unless otherwise specified, can be obtained through commercial channels.

[0052] The primer sequences involved in the following examples are shown in Table 1.

[0053] Table 1 Primers used in this study

[0054]

[0055]

[0056] Note: The underlines represent the corresponding restriction enzyme sites.

[0057] The strains involved in the following examples are shown in Table 2:

[0058] Table 2 Strains used and constructed in the study

[0059]

[0060]

[0061] The following examples involve plasmids:

[0062] pCAMBIA1303-TrpC-Hygro-gpd1-GFP is stored in this laboratory. (A plasmid commonly used in Trichoderma transformation, which can be purchased from Hunan Fenghui Biotechnology Co., Ltd., Shanghai Bohu Biotechnology Co., Ltd., and Shanghai Newp Biotechnology Co., Ltd., etc., with the catalog numbers BR513, PJZ314, and V013008 respectively;

[0063] Example 1 Construction of recombinant plasmid

[0064] 1. Construction of recombinant plasmid for knocking out gene ku80 and recombinant plasmid for knocking out gene ku70

[0065] Among them, the nucleotide sequence of the upstream homologous arm of the DNA repair protein gene ku70 is shown as SEQ ID NO.1, the nucleotide sequence of the downstream homologous arm of the DNA repair protein gene ku70 is shown as SEQ ID NO.2, the nucleotide sequence of the upstream homologous arm of the DNA repair protein gene ku80 is shown as SEQ ID NO.3, the nucleotide sequence of the downstream homologous arm of the DNA repair protein gene ku80 is shown as SEQ ID NO.4, and the nucleotide sequence of the hygromycin resistance gene expression cassette (hyg cassette) is shown as SEQ ID NO.11.

[0066] Taking the construction of the plasmid pCAMBIA1303-LB-RB-UPku80-hyg casssette-DOWNku80 for knocking out gene ku80 as an example, its construction process is as follows: Using the T. reesei CJ2095 genome as a template, and using Δku80-up-FW / RS and Δku80-down-FW / RS as primers respectively, the upstream and downstream homologous arms UPku80 (1.2 kb) and DOWNku80 (1.2 kb) of the gene ku80 were obtained by PCR amplification. The conditions for PCR amplification were: pre-denaturation at 95 °C for 5 min, denaturation at 94 °C for 30 s; annealing at 55 °C for 30 s, extension at 72 °C (extension for 1 min according to 1 kb for the fragment length), 30 cycles; extension at 72 °C for 10 min. Using the plasmid pCAMBIA1303-TrpC-Hygro-gpd1-GFP (preserved in this laboratory) as a template, and hyg(Δku80)-FW and hyg(Δku80)-RS as primers, the hygromycin resistance gene expression cassette hyg cassette was obtained by PCR amplification. Using the plasmid pCAMBIA1303-TrpC-Hygro-gpd1-GFP as a template, and using backbone-FW and backbone-RS as primers, the gene fragment pCAMBIA1303-LB-RB amplified by PCR was used as the vector backbone. All the PCR amplified gene fragments UPku80, DOWNku80, hyg cassette, pCAMBIA1303-LB-RB were ligated using the ClonExpress MultiS One Step Cloning Kit, and the ligation solution was transformed into E. coli JM109 competent cells, and in the presence of a final concentration of 25 μg·mL -1On solid LB medium containing kanamycin, culture at 37°C for 10 h. Perform PCR amplification verification using Δuniversal validation 1-FW and Δuniversal validation 4-RS as primers, and perform gene sequencing verification to screen for positive clones. Finally, obtain the recombinant plasmid pCAMBIA1303-LB-RB-UPku80-hyg casssette-DOWNku80 using the EndoFree Plasmid MiniKit. The construction process of the plasmid pCAMBIA1303-LB-RB-UPku70-hyg casssette-DOWNku70 with the ku70 gene knocked out is the same as above.

[0067] 2. Construction of the knockout plasmid for the pyr4 gene

[0068] The nucleotide sequence of the upstream homologous arm of the pyr4 gene is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream homologous arm of the pyr4 gene is shown in SEQ ID NO.6.

[0069] Using the T. reesei CJ2095 genome as a template, and using Δpyr4-up-FW / RS and Δpyr4-down-FW / RS as primers respectively, PCR amplify to obtain the upstream and downstream homologous arms UPpyr4 (1.2 kb) and DOWNpyr4 (1.2 kb) of the pyr4 gene. The conditions for PCR amplification are: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s; annealing at 55°C for 30 s, extension at 72°C (extend for 1 min according to 1 kb for the fragment length), cycle 30 times; extension at 72°C for 10 min. Using the plasmid pCAMBIA1303-TrpC-Hygro-gpd1-GFP as a template, and using backbone-FW and backbone-RS as primers, PCR amplify the gene fragment pCAMBIA1303-LB-RB as the vector backbone. All the PCR-amplified gene fragments UPpyr4, DOWNpyr4, pCAMBIA1303-LB-RB are ligated using the ClonExpress MultiS One Step Cloning Kit, and the ligation solution is transformed into E. coli JM109 competent cells, in the presence of a final concentration of 25 μg·mL -1Cultivate on solid LB medium containing kanamycin at 37 °C for 10 h. Conduct PCR amplification verification using Δuniversal validation 1-FW and Δuniversal validation 4-RS as primers, and conduct gene sequencing verification to screen for positive clones. Finally, obtain the recombinant plasmid pCAMBIA1303-LB-RB-UPpyr4-DOWNpyr4 using the EndoFree Plasmid Mini Kit.

[0070] 3. Construction of recombinant plasmids expressing rad51 gene and rad52 gene

[0071] The nucleotide sequence of the pyr4 cassette is shown in SEQ ID NO.7, the nucleotide sequence of the homologous recombination gene rad51 and its transcription terminator is shown in SEQ ID NO.8, and the nucleotide sequence of the homologous recombination gene rad52 and its transcription terminator is shown in SEQ ID NO.9.

[0072] For overexpression of the rad51 gene and the rad52 gene, since the pyr4 selection marker cassette contains the homologous fragment gad410, the recombinant plasmids for overexpression of the rad51 gene and the rad52 gene need to be constructed by using both the one-step cloning kit and restriction enzyme digestion and ligation methods. Using the plasmid pCAMBIA1303-TrpC-Hygro-gpd1-GFP as a template, and Pgpd1(rad51)-FW and Pgpd1(rad51)-RS as primers, the promoter Pgpd1 of the gene gpd1 was obtained by PCR amplification. Using the T. reesei CJ2095 genome as a template, and rad51-FW and rad51-RS as primers, the gene rad51 and its terminator fragment Trad51 were obtained by PCR amplification. Using the T. reesei CJ2095 genome as a template, and pyr4-FW and pyr4-RS as primers, the pyr4 selection marker cassette (pyr4 cassette) was obtained by PCR amplification. Using the T. reesei CJ2095 genome as a template, and gda410-FW and gda410-RS as primers, the homologous fragment gda410 derived from the pyr4 selection marker cassette was obtained by PCR amplification for the excision of the pyr4 selection marker cassette. The conditions for PCR amplification were: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s; annealing at 55°C for 30 s, extension at 72°C (extension for 1 min according to 1 kb of fragment length), 30 cycles; extension at 72°C for 10 min. Using the plasmid pCAMBIA1303-TrpC-Hygro-gpd1-GFP as a template, and backbone-FW and backbone-RS as primers, the gene fragment pCAMBIA1303-LB-RB amplified by PCR was used as the vector backbone. The above homologous fragment gda410 and the gene fragment pCAMBIA1303-LB-RB were ligated by the one-step cloning kit ClonExpressMultiS One Step Cloning Kit. Then, the recombinant plasmid of the positive transformant after correct verification was extracted, and using gda410(EcoRI)-FW and gda410(XbaI)-RS as primers, the vector fragment pCAMBIA1303-LB-RB-gda410 containing EcoRI and XbaI was obtained by whole plasmid PCR amplification. In addition, the above pyr4 selection marker cassette, Pgpd1, the gene rad51 and its terminator fragment Trad51 and the gene fragment pCAMBIA1303-LB-RB were ligated by the one-step cloning kit ClonExpressMultiS One Step Cloning Kit.Next, extract the recombinant plasmid of the positively transformed clones after correct verification. Using rad51(XbaI)-FW and rad51(EcoRI)-RS as primers, PCR amplification was performed to obtain the gene fragment pyr4 cassette-rad51-Trad51 containing EcoRI and XbaI. Finally, the vector fragment pCAMBIA1303-LB-RB-gda410 was ligated with the gene fragment pyr4 cassette-rad51-Trad51 by double digestion with EcoRI and XbaI, and the ligation solution was transformed into competent E. coli JM109 cells and cultured on solid LB medium containing kanamycin at a final concentration of 25 μg·mL. -1 Cultivate at 37 °C for 10 h. Using rad51 validation-FW and rad51 validation-RS as primers, PCR amplification verification was carried out, and gene sequencing verification was also carried out to screen positive clones. The recombinant plasmid pCAMBIA1303-LB-RB-gda410-pyr4 cassette-Pgpd1-rad51-Trad51 was obtained through the EndoFree Plasmid Mini Kit. The construction process of the recombinant plasmid overexpressing the gene rad52 was the same as above. However, due to the restriction of enzyme digestion sites, when constructing the recombinant plasmid overexpressing the gene rad52 using enzyme digestion and ligation, EcoRI and HindIII were selected as the enzyme digestion sites for ligation.

[0073] The construction of the pyr4 recovery plasmid was similar to the above construction process, and only the vector fragment pCAMBIA1303-LB-RB-gda410 was ligated with the pyr4 cassette by enzyme digestion to obtain pCAMBIA1303-LB-RB-gda410-pyr4 cassette. The primers used in this example are shown in Table 1.

[0074] Example 2 Construction of Recombinant A. tumefaciens

[0075] The recombinant plasmids pCAMBIA1303-LB-RB-UPku80-hyg casssette-DOWNku80, pCAMBIA1303-LB-RB-UPku70-hyg casssette-DOWNku70, pCAMBIA1303-LB-RB-UPpyr4-DOWNpyr4, pCAMBIA1303-LB-RB-gda410-pyr4cassette-Pgpd1-rad51-Trad51, pCAMBIA1303-LB-RB-gda410-pyr4cassette-Pgpd1-rad52-Trad52, and pCAMBIA1303-LB-RB-gda410-pyr4 cassette in Example 1 were transformed into A. tumefaciens. The transformation method was carried out according to the steps in the master's thesis "Construction of an antibiotic-free Aspergillus niger expression system and expression of lipase" by Si-Yuan Zhu, and then A. tumefaciens containing the recombinant plasmids constructed in Example 1 was obtained.

[0076] Selection of the working concentration of hygromycin in Example 3

[0077] During the construction of the recombinant strain, hygromycin was used as a selection marker, and thus it was necessary to select an appropriate working concentration of the antibiotic. Experiments were carried out according to the reported working concentrations of hygromycin, and three different concentrations of hygromycin, 0 μg·mL -1 , 20 μg·mL -1 , and 40 μg·mL -1 , were studied. When the working concentration of hygromycin was 20 μg·mL -1 , the starting strain T. reesei CJ2095 could no longer grow ( Figure 1 ). Therefore, the working concentration of hygromycin in subsequent experiments was selected as 20 μg·mL -1 .

[0078] Construction of engineering strains T. reesei CJ2095Δku70 and T. reesei CJ2095Δku80 in Example 4

[0079] A. tumefaciens containing the recombinant plasmids pCAMBIA1303-LB-RB-UPku80-hyg casssette-DOWNku80 and pCAMBIA1303-LB-RB-UPpyr4-DOWNpyr4 was co-cultured with T. reesei on solid IM medium covered with cellophane at 24 °C in the dark for 48 h. The cellophane was transferred to a new PDA solid screening medium (containing 20 mg·L -1hygromycin and 200 mg·L -1 cefotaxime sodium), and cultured at 30 °C for 2 - 7 d. Pick the grown monoclonal colonies onto the rescreening solid medium and culture for 2 - 3 d. Pick the hyphae into an EP tube containing 1 mL of seed medium, culture for 2 d, extract its genomic DNA, and verify it. Streak the positive clones on the PDA solid medium containing 0.1% (v / v) Triton-X 100 and culture for 2 - 3 d to isolate single spores, inoculate them into the PDA medium for spore production culture for 5 - 7 d, and preserve the strains. Finally, obtain the engineered strains Trichoderma reesei CJ2095Δku70 and Trichoderma reesei CJ2095Δku80.

[0080] After PCR verification, Trichoderma reesei CJ2095Δku80 was successfully constructed ( Figure 2 -c). However, after repeated experiments, the correct Trichoderma reesei CJ2095Δku70 positive transformants were not obtained. Therefore, finally, the recombinant strain Trichoderma reesei CJ2095Δku80 was selected for subsequent experimental operations.

[0081] Example 5 Selection of the working concentration of 5-FOA

[0082] To verify the effect of knocking out the DNA repair protein ku80 gene on the gene editing efficiency of Trichoderma reesei, the orotidine-5'-phosphate decarboxylase pyr4 gene in Trichoderma reesei was further knocked out in the study. The pyr4 gene itself serves as a selection marker. When the pyr4 gene is absent in Trichoderma reesei, it will lead to the inactivation of orotidine-5′-phosphate decarboxylase. The engineered Trichoderma reesei strains with this gene knocked out must be supplemented with uracil additionally to grow normally during subsequent growth. Additionally, if the medium contains 5-FOA, the wild-type strains containing the pyr4 gene cannot grow, while the recombinant Trichoderma reesei strains lacking the pyr4 gene can grow. Therefore, when constructing the pyr4 gene knockout cassette, the hygromycin gene is not required as a selection marker. When screening for positive transformants, only 5-FOA needs to be added to the screening medium as a selection pressure. At the same time, as a uracil auxotrophic strain, uracil needs to be added additionally during culture. The optimal working concentration of 5-FOA was optimized in the study. When the working concentration of 5-FOA in the PDA solid medium is 250 mg·L -1 , the growth of Trichoderma reesei CJ2095 has been inhibited; when the concentration is 500 mg·L -1 , Trichoderma reesei CJ2095 cannot grow at all ( Figure 3 ). Therefore, the working concentration of 5-FOA was selected as 500 mg·L -1For subsequent experiments.

[0083] Example 6 Knockout of the DNA repair protein gene ku80 and its effect on gene editing efficiency

[0084] Agrobacterium tumefaciens containing the recombinant plasmid pCAMBIA1303-LB-RB-UPpyr4-DOWNpyr4 was co-cultured with Trichoderma reesei CJ2095 and Trichoderma reesei CJ2095Δku80 on MM medium (containing 500 mg·L -1 of 5-FOA and 1 g·L -1 of uracil). The grown monoclonal colonies were picked and cultured on a solid medium for rescreening for 2 - 3 days. The mycelia were picked and transferred into an EP tube containing 1 mL of seed medium and cultured for 2 days. Their genomic DNA was extracted and verified. The positive clones were streaked on PDA solid medium containing 0.1% (v / v) Triton-X 100 and cultured for 2 - 3 days to isolate single spores, which were then inoculated into PDA medium for spore production culture for 5 - 7 days, and the strains were preserved. Finally, the engineered strains Trichoderma reesei CJ2095Δpyr4 and Trichoderma reesei CJ2095Δku80Δpyr4 were obtained.

[0085] As Figure 4 shown, in this example, the gene pyr4 was knocked out in the strains Trichoderma reesei CJ2095 and Trichoderma reesei CJ2095Δku80 respectively to verify the effect of knocking out the ku80 gene on the gene editing efficiency of Trichoderma reesei ( Figure 4 -a). As Figure 4 -c shown, the knockout efficiency of the gene pyr4 in Trichoderma reesei CJ2095 was very low, and its positive knockout rate was only 1.04%. When the gene ku80 was knocked out and the gene pyr4 was knocked out again in the recombinant strain Trichoderma reesei CJ2095Δku80, the positive knockout rate increased from 1.04% of the control strain Trichoderma reesei CJ2095 to 7.14%.

[0086] In the phenotypic verification, it was found that the recombinant strain Trichoderma reesei CJ2095Δku80Δpyr4 could not grow on MM medium, but could grow normally on MM + 1 g·L -1 uracil medium and MM + 1 g·L -1 uracil + 500 mg·L -1 5-FOA medium. The strain Trichoderma reesei CJ2095Δku80Δpyr4 after knockout of the pyr4 gene could not grow in MM medium and could only grow in the medium containing 1 g·L -1 uracil (Figure 5 )。These phenomena are consistent with the reports. When the pyr4 gene is knocked out in the strain, the recombinant strain becomes uracil auxotrophic.

[0087] Example 7 Construction of Repeated Selection Markers

[0088] As Figure 6 shown, in order to be able to recycle the pyr4 selection marker and compare whether the homologous recombination efficiency of the recombinant strain increases after overexpression of the rad51 and rad52 genes, a homologous region gene gda410 with a size of 410 bp was added in front of the pyr4 gene when constructing the overexpression plasmid ( Figure 6 a). When homologous recombination occurs, gda410 and the pyr4 gene undergo homologous recombination, thereby removing the pyr4 gene and only leaving the gda410 gene fragment, achieving the recycling of the pyr4 selection marker ( Figure 6 d). Therefore, in subsequent experiments, when a selection marker is needed, a gene fragment containing "gda410 - pyr4 cassette" is constructed on the recombinant plasmid to achieve the recycling of the pyr4 selection marker.

[0089] Example 8 Effect of Expressing Key Enzyme Genes rad51 or rad52 on Gene Editing Efficiency

[0090] A. tumefaciens containing the recombinant plasmid pCAMBIA1303-LB-RB-gda410-pyr4 cassette constructed in Example 1 was co-cultured with T. reesei CJ2095Δku80Δpyr4 to construct the strain T. reesei CJ2095Δku80*. A. tumefaciens containing the recombinant plasmids pCAMBIA1303-LB-RB-gda410-pyr4 cassette-Pgpd1-rad51-Trad51 and pCAMBIA1303-LB-RB-gda410-pyr4 cassette-Pgpd1-rad52-Trad52 constructed in Example 1 were respectively spread on MM medium with the strain T. reesei CJ2095Δku80Δpyr4 to obtain the strains T. reesei CJ2095Δku80Δpyr4::rad51 and T. reesei CJ2095Δku80Δpyr4::rad52. Spores of the recombinant strains T. reesei CJ2095Δku80*, T. reesei CJ2095Δku80Δpyr4::rad51 and T. reesei CJ2095Δku80Δpyr4::rad52 were spread on MM solid medium containing 5-FOA and uracil, and the grown strains were further inoculated onto a rescreening plate (MM solid medium containing 5-FOA and uracil), and the genome was extracted for verification. Strains with the pyr4 gene correctly excised were obtained by PCR verification. When the pyr4 gene was correctly excised, Primer1 and Primer3 could not amplify the target band; while Primer2 and Primer3 could amplify a gene fragment of 2076 bp ( Figure 6 b and 6d). When the pyr4 gene of the recombinant strain T. reesei CJ2095Δku80* was correctly excised, Primer4 and Primer5 could not amplify the target band ( Figure 6 b and 6d).

[0091] The excision efficiency of the pyr4 gene of the two recombinant strains T. reesei CJ2095Δku80Δpyr4::rad51 and T. reesei CJ2095Δku80Δpyr4::rad52 was counted. 6 positive transformants were selected from 48 transformants of the recombinant strain T. reesei CJ2095Δku80Δpyr4::rad51, and the excision rate of the pyr4 gene was 12.5%; while the excision rate of the pyr4 gene of the recombinant strain T. reesei CJ2095Δku80Δpyr4::rad52 was 43.75% ( Figure 6c). The pop - out rate of the screening marker gene in the recombinant strain T. reesei CJ2095Δku80* was only 8.33% ( Figure 6 -c). The results showed that the pyr4 gene pop - out efficiency of the recombinant strain T. reesei CJ2095Δku80Δpyr4::rad52 was significantly higher than that of the recombinant strain T. reesei CJ2095Δku80Δpyr4::rad51, indicating that after overexpressing the rad52 gene, the homologous recombination ability of T. reesei was significantly improved.

[0092] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Application of gene ku80 and gene rad52 in improving the gene editing efficiency of Trichoderma reesei, characterized in that, The application is to knockout the DNA repair protein gene ku80 of Trichoderma reesei and overexpress the homologous recombinase gene rad52. The NCBI accession number of the nucleotide sequence of the gene ku80 is NW_006711151.1; the NCBI accession number of the nucleotide sequence of the gene rad52 is NW_006711159.

1.

2. Trichoderma reesei engineering strain with high gene editing efficiency, characterized in that, The engineering strain uses Trichoderma reesei as the starting strain, knocks out the DNA repair protein gene ku80 in the starting strain, and overexpresses the homologous recombinase gene rad52. The NCBI accession number of the nucleotide sequence of the gene ku80 is NW_006711151.1; the NCBI accession number of the nucleotide sequence of the gene rad52 is NW_006711159.

1.

3. The Trichoderma reesei engineering strain according to claim 2, wherein, Use Trichoderma reesei CJ2095 as the starting strain.

4. A method for constructing the Trichoderma reesei engineering strain according to claim 2 or 3, characterized in that, The method is to knockout the gene ku80 of Trichoderma reesei CJ2095 and at the same time overexpress the homologous recombinase gene rad52 in Trichoderma reesei CJ2095.

5. The method according to claim 4, wherein The method includes the following steps: (1) Insert the gene ku80 knockout cassette into the vector backbone to obtain the gene ku80 knockout recombinant plasmid. The structure of the gene ku80 knockout cassette is "upstream homologous arm of gene ku80 - hygromycin resistance gene expression cassette - downstream homologous arm of gene ku80"; (2) Insert the gene rad52 expression cassette into the vector backbone to obtain the gene rad52 expression recombinant plasmid. The structure of the gene rad52 expression cassette is "gda410 - pyr4 cassette - Pgpd1 - gene rad52 and its transcription terminator"; (3) Transform the gene ku80 knockout recombinant plasmid in step (1) and the gene rad52 expression recombinant plasmid in step (2) into Agrobacterium tumefaciens respectively. Pick the transformants and co - culture and transform them with Trichoderma reesei CJ2095, and screen to obtain the Trichoderma reesei engineering strain with high gene editing efficiency.

6. The method according to claim 5, wherein The nucleotide sequence of the upstream homologous arm of the DNA repair protein gene ku80 is as shown in SEQ ID NO.3, the nucleotide sequence of the downstream homologous arm of the DNA repair protein gene ku80 is as shown in SEQ ID NO.4, the nucleotide sequence of the hygromycin resistance gene expression cassette is as shown in SEQ ID NO.11, the nucleotide sequence of the pyr4 cassette is as shown in SEQ ID NO.7, the nucleotide sequence of the gene rad52 and its transcription terminator is as shown in SEQ ID NO.9, and the nucleotide sequence of the promoter Pgpd1 is as shown in SEQ ID NO.

10.

7. The method according to claim 5, characterized in that Use the pCAMBIA1303 - LB - RB plasmid as the vector backbone.

8. A method for gene editing of Trichoderma reesei, characterized in that, The method is to use the Trichoderma reesei engineering strain described in claim 2 or 3 as the chassis cell to knockout or knockin the target gene.

9. The method according to claim 8, characterized in that The gene editing is to knockout the pyr4 gene; Optionally, the specific steps for knocking out the pyr4 gene include the following: S1. Construct a pyr4 gene knockout cassette, and the structure of the pyr4 gene knockout cassette is "upstream homologous arm of the pyr4 gene - downstream homologous arm of the pyr4 gene". S2. Transform the pyr4 gene knockout cassette of S1 into Agrobacterium tumefaciens, pick transformants and co - culture them with the Trichoderma reesei engineering strain described in claim 2 or 3, and screen to obtain Trichoderma reesei with the pyr4 gene knocked out.

10. The method according to claim 9, wherein The NCBI accession number of the nucleotide sequence of the pyr4 gene is NW_006711148.

1. The nucleotide sequence of the upstream homologous arm of the pyr4 gene is as shown in SEQ ID NO.5, and the nucleotide sequence of the downstream homologous arm of the pyr4 gene is as shown in SEQ ID NO.6.

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