CN114591849A - Method for constructing myceliophthora thermophila strain with high homologous recombination efficiency and application thereof - Google Patents
Method for constructing myceliophthora thermophila strain with high homologous recombination efficiency and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a method for constructing myceliophthora thermophila strain with high homologous recombination efficiency and application thereof. The mutant strain delta Ku80 obtained by the invention can improve the homologous recombination efficiency of a myceliophthora thermophila CRISPR-Cas9 gene editing system from 57% of a wild type to 86%, reduce the workload of myceliophthora thermophila genetic transformation and improve the homologous recombination efficiency.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for constructing a myceliophthora thermophila strain with high homologous recombination efficiency and application thereof.
Background
Myceliophthora thermophila is a myceliophthora thermophila capable of quickly degrading cellulose and has higher cellulase expression and secretion capacity. Compared with the production of the filamentous fungi trichoderma reesei and penicillium decumbens by industrial cellulase, the myceliophthora thermophila has high enzyme activity and strong thermal stability, and has wide application prospect in industrial production. Meanwhile, the glycan structure produced by myceliophthora thermophila is more similar to the human glycan form than most other non-mammalian production hosts, and shows great application potential in the field of medicine. Therefore, the development of myceliophthora thermophila into novel filamentous fungus underpan cells through genetic engineering is of great significance for the production of industrial cellulases, therapeutic proteins, vaccines and primary and secondary metabolites.
At present, the development of a filamentous fungus genetic system and a genome editing technology is relatively slow, and the extremely low genetic transformation efficiency and homologous recombination efficiency severely restrict the use of filamentous fungi as underpant cells, so that the basic research, development and utilization of the filamentous fungi are hindered. In filamentous fungi, DNA double-strand breaks are mainly repaired by two mechanisms of non-homologous end connection and homologous recombination, but the non-homologous end connection efficiency in the filamentous fungi is usually obviously higher than the homologous recombination efficiency, exogenous DNA is mostly integrated into genome chromosomes in a random insertion mode, so that the screening workload is large, the site-specific knockout efficiency is low, and further, the relevant functional gene research and the targeted modification of strain gene engineering are affected extremely adversely. Therefore, the myceliophthora thermophila strain with high homologous recombination efficiency has great application prospect and practicability in the genetic engineering modification.
In recent years, although some studies report that the deletion of a key gene on a non-homologous end-joining pathway can improve the homologous recombination efficiency, the deletion of the same functional gene has distinct effects among different eukaryotic host cells, for example, a saccharomyces cerevisiae strain with the deletion of Ku70 has the effect of increasing the efficiency of site-specific integration of a DNA sequence into its genome, while the efficiency of site-specific integration of a mammalian cell with the deletion of Ku70 into a target gene is not improved. Furthermore, considering the low efficiency of genetic transformation in filamentous fungi, it is not clear whether knocking out a key gene in the non-homologous end joining pathway of filamentous fungi will indirectly affect its genetic transformation.
Disclosure of Invention
The invention aims to provide a method for constructing a myceliophthora thermophila strain with high homologous recombination efficiency.
Still another objective of the invention is to provide a method for improving the homologous recombination efficiency of the myceliophthora thermophila CRISPR-Cas9 gene editing system.
The method for constructing the myceliophthora thermophila strain with high homologous recombination efficiency comprises the step of knocking out a Ku80 gene of the myceliophthora thermophila, wherein the nucleotide sequence of the Ku80 gene is shown as SEQ ID NO. 1.
The method for constructing the myceliophthora thermophila strain with high homologous recombination efficiency according to the invention comprises the following steps:
preparing protoplast by taking myceliophthora thermophila as an original strain;
preparing a Ku80 gene knockout box;
and (3) introducing the knockout box into the protoplast by adopting a CRISPR-Cas9 gene editing system, and culturing and screening on a culture medium.
The method for constructing the myceliophthora thermophila strain with high homologous recombination efficiency according to the invention, wherein the myceliophthora thermophila is the strain ATCC 4246.
A method of increasing the efficiency of homologous recombination of a myceliophthora thermophila CRISPR-Cas9 gene editing system, the method comprising the steps of:
knocking out a Ku80 gene of myceliophthora thermophila, wherein the nucleotide sequence of the Ku80 gene is shown as SEQ ID NO. 1;
preparing protoplasts of the myceliophthora thermophila strain lacking the Ku80 gene;
the target gene knockout cassette, Cas9 and sgRNA were introduced into protoplasts of the myceliophthora thermophila strain lacking Ku80 gene, and cultured and selected on a medium containing antibiotics.
The invention has the following beneficial effects:
the mutant strain delta Ku80 obtained by the invention can improve the homologous recombination efficiency of a myceliophthora thermophila CRISPR-Cas9 gene editing system from 57% of a wild type to 86%, reduce the workload of myceliophthora thermophila genetic transformation and improve the homologous recombination efficiency.
Drawings
FIG. 1 shows the results of genomic PCR validation of mutant strain Δ Ku80 obtained by knocking out the Ku80 gene;
FIG. 2 shows the results of genomic PCR validation of mutant strain Δ Ku70 obtained by knocking out the Ku70 gene;
fig. 3 shows the results of genomic PCR validation of mutant Δ alp1 obtained by knocking out alp1 gene from wild type, mutant Δ Ku80 and mutant Δ Ku70, a: verifying the result of the wild host transformant; b: verification results of host transformants of the mutant strain delta Ku 80; c: verification of the mutant strain Δ Ku70 host transformants.
Detailed Description
The materials, reagents, apparatus and methods used in the following examples, which are not specifically illustrated, are all conventional in the art and are commercially available. The construction of the gene knockout box of the invention adopts the Beijing all-purpose gold biotechnology, Inc. pEASY-Blunt Cloning Kit and pEASY-Uni Seamless Cloning and Assembly Kit. PDA was purchased from BD corporation, usa. Lywallases were purchased from Sigma-Aldrich, usa;
STC solution: 50 mM calcium chloride, 1M sorbitol, 10 mM Tris-HCl pH 7.5;
PEG solution: 25% PEG 6000, 50 mM calcium chloride, 10 mM Tris-HCl pH 7.5;
upper medium: 2% sucrose, 20 mL/L50 XVogel's salt solution, 1M sorbitol, 0.75% agarose;
the lower layer culture medium: 2% sucrose, 20 mL/L50 XVogel's salt solution, 1M sorbitol, 1.5% agar;
myceliophthora thermophila ATCC42464 is purchased from American type culture Collection.
Example 1 construction of a Ku80 Gene knock-out myceliophthora thermophila Gene engineering Strain Δ Ku80
1. Construction of the Ku80 Gene knockout cassette
The Ku80 knock-out cassette was constructed with the primers shown below:
Ku80-F1（SEQ IDNO：4），Ku80-R1（SEQ IDNO：5）；
Ku80-F2（SEQ IDNO：6），Ku80-R2（SEQ IDNO：7）；
G418-F1（SEQ IDNO：8），G418-R1（SEQ IDNO：9）。
taking myceliophthora thermophila genome DNA as a template, adopting a primer Ku80-F1/Ku80-R1 to amplify a Ku80 gene fragment, recovering an amplification product, connecting the recovered amplification product with a pEASY-Blunt vector, transforming a Trans I cloning host, and carrying out sequencing verification to obtain an intermediate vector pEASY-Blunt-Ku 80; secondly, pEASY-Blunt-Ku80 is used as a template, primers Ku80-F2/Ku80-R2 are adopted to amplify the upstream and downstream of a Ku80 gene fragment, an amplification product is connected with a geneticin G418 resistance gene through a homologous recombination method after being recovered, a Trans I cloning host is transformed, and sequencing verification is carried out to obtain the Ku80 gene knockout box. Ku80 gene sequence SEQ ID NO:1 is shown.
2. Preparation of Ku80 knockout Strain
And (3) coating the myceliophthora thermophila spore suspension on a PDA (personal digital Assistant) plate paved with cellophane, culturing for 15 h in a constant-temperature incubator at 37 ℃, and collecting tender hyphae. Cell walls were lysed with 0.5% lywallzyme, 30 ℃ enzymeAfter 2 h of decomposition, filtering by double-layer mirror wiping paper, and centrifuging at 2000 rpm and 4 ℃ to collect the protoplast. Washing with pre-cooled STC solution for 1 time, re-suspending the protoplasts in the pre-cooled STC solution, and adjusting the protoplast concentration to 5 × 10 with the STC solution7And (4) obtaining a protoplast suspension.
To 200. mu.L of the above protoplast suspension were added 10. mu.L of linearized fragment (Ku 80 knock-out cassette, Cas9, sgRNA) and 50. mu.L of PEG solution, gently mixed, and ice-cooled for 20 min. Add 2 mL PEG solution, mix gently, room temperature for 5 min. Then 4 mL of STC solution was added, mixed with 5 mL of the upper medium containing geneticin, poured onto the lower medium containing geneticin, and cultured at 37 ℃ for 3 days to obtain a transformant.
Transformants having geneticin resistance were transferred from the plates to PDA plates, cultured in an incubator at 45 ℃ for 3 days, genome was extracted, and PCR results were analyzed by agarose gel electrophoresis, as shown in FIG. 1, and verified by PCR using Ku80-F1/Ku80-R1 as primers, the wild type strain amplified a band of about 2.0 kb in size, and the mutant strain Δ Ku80 amplified a band of about 3.0 kb in size and did not have a band of the same size as the wild type strain, indicating that homologous recombination occurred at the Ku80 gene site in the knockout cassette.
Example 2 construction of a Ku70 Gene knock-out myceliophthora thermophila Gene engineering Strain Δ Ku70
1. Construction of the Ku70 Gene knockout cassette
Primers used to construct the Ku70 knockout cassette are as follows:
Ku70-F1 （SEQ IDNO：10），Ku70-R1（SEQ IDNO：11）；
Ku70-F2（SEQ IDNO：12），Ku70-R2（SEQ IDNO：13）；
G418-F1（SEQ IDNO：14），G418-R1（SEQ IDNO：15）。
taking myceliophthora thermophila genome DNA as a template, adopting a primer Ku70-F1/Ku70-R1 to amplify a Ku70 gene fragment, recovering an amplification product, connecting the recovered amplification product with a pEASY-Blunt vector, transforming a Trans I cloning host, and carrying out sequencing verification to obtain an intermediate vector pEASY-Blunt-Ku 70; secondly, pEASY-Blunt-Ku70 is used as a template, primers Ku70-F2/Ku70-R2 are adopted to amplify the upstream and downstream of a Ku70 gene fragment, an amplification product is connected with a geneticin G418 resistance gene through a homologous recombination method after being recovered, a Trans I cloning host is transformed, and sequencing verification is carried out to obtain the Ku70 gene knockout box. The sequence of the Ku70 gene is shown in SEQ ID NO. 2.
2. Acquisition procedure for preparation of Ku70 knockout Strain
And (3) coating the myceliophthora thermophila spore suspension on a PDA (personal digital Assistant) plate paved with cellophane, culturing for 15 h in a constant-temperature incubator at 37 ℃, and collecting tender hyphae. Dissolving cell wall with 0.5% muramidase, performing enzymolysis at 30 deg.C for 2 hr, filtering with double-layer mirror paper, centrifuging at 2000 rpm and 4 deg.C, and collecting protoplast. Washing with pre-cooled STC solution for 1 time, resuspending the protoplasts in the pre-cooled STC solution, and adjusting the protoplast concentration to 5X10 with the STC solution7And (4) obtaining a protoplast suspension.
To 200. mu.L of the above protoplast suspension were added 10. mu.L of linearized fragment (Ku 70 knock-out cassette, Cas9, sgRNA) and 50. mu.L of PEG solution, gently mixed, and ice-cooled for 20 min. Add 2 mL PEG solution, mix gently, room temperature for 5 min. Then 4 mL of STC solution was added, mixed with 5 mL of the upper medium containing geneticin, poured onto the lower medium containing geneticin, and cultured at 37 ℃ for 3 days to obtain a transformant.
Transformants having geneticin resistance were transferred from the plates to PDA plates, cultured in an incubator at 45 ℃ for 3 days, genome was extracted, and PCR results were analyzed by agarose gel electrophoresis, as shown in FIG. 2, and verified by PCR using Ku70-F1/Ku70-R1 as primers, the wild type strain amplified a band of about 2.0 kb in size, and the mutant strain Δ Ku70 amplified a band of about 3.0 kb in size and did not have a band of the same size as the wild type strain, indicating that homologous recombination occurred at the Ku70 gene site in the knockout cassette.
Example 3 verification of the efficiency of homologous recombination in the Geotrichum thermophilum genetic engineering strains delta Ku80 and delta Ku70
1. Construction of alp1 Gene knockout cassette
The alp1 gene knockout cassette primers were constructed as follows:
alp1-F1（SEQ IDNO：16），alp1-R1（SEQ IDNO：17）；
alp1-F2（SEQ IDNO：18），alp1-R2（SEQ IDNO：19）；
HygR-F1（SEQ IDNO：20），HygR-R1（SEQ IDNO：21）。
taking myceliophthora thermophila genome DNA as a template, adopting a primer alp1-F1/alp1-R1 to amplify an alp1 gene fragment, recovering an amplification product, connecting the recovered amplification product with a pEASY-Blunt vector, transforming a Trans I cloning host, and performing sequencing verification to obtain an intermediate vector pEASY-Blunt-alp 1; secondly, using pEASY-Blunt-alp1 as a template, adopting primers alp1-F2/alp1-R2 to amplify the upstream and downstream of an alp1 gene fragment, connecting the amplified product with a hygromycin HygR resistance gene through a homologous recombination method after recovering the amplified product, transforming a Trans I cloning host, and carrying out sequencing verification to obtain the alp1 gene knockout box. The alp1 gene sequence is shown as SEQ ID NO: 3, respectively.
Protoplast transformation and screening validation
Respectively coating the myceliophthora thermophila wild type, the mutant strain delta Ku80 and the mutant strain delta Ku70 spore suspension on a PDA flat plate paved with cellophane, culturing for 15 h in a constant-temperature incubator at 37 ℃, and collecting young hyphae. Dissolving cell wall with 0.5% muramidase, performing enzymolysis at 30 deg.C for 2 hr, filtering with double-layer mirror paper, centrifuging at 2000 rpm and 4 deg.C, and collecting protoplast. Washing with pre-cooled STC solution for 1 time, resuspending the protoplasts in the pre-cooled STC solution, and adjusting the protoplast concentration to 5X10 with the STC solution7And each mL, obtaining the protoplast suspension of the myceliophthora thermophila wild type, the mutant strain delta Ku80 and the mutant strain delta Ku 70.
To 200. mu.L of the above protoplast suspension were added 10. mu.L of linearized fragment (alp 1 knock-out cassette, Cas9, sgRNA) and 50. mu.L of PEG solution, gently mixed, and ice-cooled for 20 min. Add 2 mL PEG solution, mix gently, room temperature for 5 min. Then 4 mL of STC solution was added, mixed with 5 mL of hygromycin-containing upper medium, poured onto hygromycin-containing lower medium, and cultured at 37 ℃ for 3 days to obtain transformants.
Transformants with hygromycin resistance are transferred to a PDA (personal digital assistant) plate from the plate, the plate is cultured in a constant temperature incubator at 45 ℃ for 3 days, a genome is extracted, agarose gel electrophoresis is used for analyzing a PCR result, as shown in figure 3, the primer alp1-F1/alp-R1 is used for PCR verification, 8 transformants in a wild-type host are amplified to form a band with the size of about 3.0 kb and do not have a band with the size of 1.5 kb as that of a wild-type strain, and the knockout box in the transformants is integrated to an alp1 gene site in a homologous recombination mode, wherein the homologous recombination probability is 8/14, namely 57%; the 12 transformants in the host of the knockout strain delta Ku80 amplified a band with the size of about 3.0 kb and did not have a band with the size of 1.5 kb as that of the wild strain, which indicates that the knockout cassette in the transformants is integrated into the alp1 gene site in a homologous recombination mode, and the homologous recombination probability is 12/14, namely 86%; the number of transformants in the host of the knockout strain delta Ku70 is sharply reduced, only 6 transformants are obtained, wherein 4 transformants amplify a band with the size of about 3.0 kb and do not have a band with the size of 1.5 kb of the wild strain, which indicates that the knockout cassette in the transformants is integrated to the alp1 gene site in a homologous recombination mode, the homologous recombination probability is 4/6, namely 67%, but the improvement effect of the homologous recombination probability is not obvious.
The above examples are only for explaining the technical solutions of the present application, and do not limit the scope of protection of the present application.
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> a method for constructing myceliophthora thermophila strain with high homologous recombination efficiency and application thereof
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cagcacagat ctcgagggtg ggaaagagag gggggggagg ggcgatgctg gggcgatgct 180
gcttggagcc gcatccgggg agggggccct gctgttcatc catatccagg atgatgcgag 240
attgaagcaa gataaataac acggcttccc cctccccttt cgatccggac cagaccatcg 300
tctccaacac cccaaagtcg atccgacaag tcccaatcca ccccgcccgc ccctccctcc 360
gtcgccgtcc cggtcttccg atttcgtcaa gatgcacttc tccaccgctc tcctggcctt 420
cctgcccgcc gccctcgcgg cccctactgc cgagaccctc gacaagcgcg ccccgatcct 480
gactgctcgc gctggccagg tcgtcccggg caagtacatc atcaagctcc gcgacggagc 540
cagcgacgat gtccttgagg ccgccatcgg caagctccgc tccaaggccg accacgtcta 600
ccgcggcaag ttcaggggct ttgccggcaa gctcgaggat gacgtccttg acgccatccg 660
tcttctcccc gaagtgagtc cgcgtcccgg aaagaaatag agcgagcggg ggagagagtg 720
aagggcgaaa agagccgtgt tttgttaacc gcttgtcttt tctttctctc ttgcaatagg 780
tcgagtacgt cgaggaggag gccatcttca ccatcaacgc gtacacctcg cagtccaacg 840
ccccctgggg ccttgcgcgc ctctcgtcca agaccgcggg ctccaccacc tacacctacg 900
acaccagcgc cggcgagggc acctgtgcct atgtgatcga cacgggcatc tacactagcc 960
actccgtatg tctcgcggtt acctcccctt tcggaagaag gggcatccat atgctgaccc 1020
ctcctgatca caggacttcg gcggccgtgc cactttcgcc gccaacttcg tcgacagctc 1080
taacaccgat ggcaacggcc acggcaccca cgtcgccggc accatcggcg gcaccacgta 1140
cggtgttgcc aagaagacca agctctacgc cgtcaaggtt ctcggctccg acggctctgg 1200
caccacgtat gcctcgcacc cgcgcacccg cacacccgcc cggccgttat cttctgactg 1260
acattcctct ttctcctctc tagttctggt gtcattgctg gcatcaactt cgtcgctgac 1320
gacgcgccca agcgcagctg ccccaagggc gtcgtcgcca acatgtcgct cggcggtagc 1380
tactcggcct ccatcaacaa cgccgccgcc gccctcgtca ggtcgggcgt cttcctggcc 1440
gtcgccgccg gcaacgagaa ccagaacgcc gccaac 1476
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cggcggagct aaccttgtca gtg 23
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cgcgcatgac ccggagattc t 21
<210> 6
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
catgcatgtt gcatgatgat gccgaagacg tgcgcagcta tc 42
<210> 7
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgctccttc aatatcagat acgcctgtct cgcccatgtt gat 43
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atctgatatt gaaggagc 18
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atcatcatgc aacatgcatg 20
<210> 10
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
acgggaacga caaggctgcg aaatc 25
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gcggggccga gagagcagca 20
<210> 12
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
catgcatgtt gcatgatgat cgccgacgag ctcaccgaca g 41
<210> 13
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atgctccttc aatatcagat gcgcggagaa gacacgggtg ga 42
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atctgatatt gaaggagc 18
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
atcatcatgc aacatgcatg 20
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ctagtcctcg cagcaaactc 20
<210> 17
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gttggcggcg ttctggttct c 21
<210> 18
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gtaaagataa tgctaaatca caccacctac acctacgaca c 41
<210> 19
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ttataggtta atgtcatgat ctgaacttgc cgcggtagac 40
<210> 20
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
atcatgacat taacctataa aaatagg 27
<210> 21
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tgatttagca ttatctttac atcttg 26
Claims (6)
1. A method for constructing a myceliophthora thermophila strain with high homologous recombination efficiency is characterized by comprising the step of knocking out a Ku80 gene of the myceliophthora thermophila, wherein the nucleotide sequence of the Ku80 gene is shown as SEQ ID NO. 1.
2. The method for constructing a myceliophthora thermophila strain with high homologous recombination efficiency according to claim 1, characterized in that it comprises the step of constructing the Ku80 gene knockout cassette.
3. The method for constructing myceliophthora thermophila strain with high homologous recombination efficiency according to claim 2, characterized in that it comprises the following steps:
preparing protoplast by taking myceliophthora thermophila as an original strain;
preparing a Ku80 gene knockout box;
introducing a Ku80 gene knockout cassette into the prepared protoplast;
culturing and screening are carried out on the medium.
4. The method for constructing myceliophthora thermophila strain with high homologous recombination efficiency according to claim 3, wherein the Ku80 gene knockout cassette is introduced into protoplasts by CRISPR-Cas9 gene editing system.
5. The method for constructing a myceliophthora thermophila strain with high homologous recombination efficiency according to claim 1, wherein the myceliophthora thermophila is strain ATCC 4246.
6. A method for improving the homologous recombination efficiency of a myceliophthora thermophila CRISPR-Cas9 gene editing system, which is characterized by comprising the following steps:
knocking out a Ku80 gene of myceliophthora thermophila, wherein the nucleotide sequence of the Ku80 gene is shown as SEQ ID NO. 1;
preparing protoplasts of the myceliophthora thermophila strain lacking the Ku80 gene;
the target gene knockout cassette, Cas9 and sgRNA were introduced into protoplasts of the myceliophthora thermophila strain lacking Ku80 gene, and cultured and selected on a medium containing antibiotics.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717727A (en) * | 2009-12-23 | 2010-06-02 | 贵州大学 | Myceliophthora thermophilia strain and application thereof in aspect of producing keratinase |
| CN102352320A (en) * | 2011-09-29 | 2012-02-15 | 北京可力美施生物科技有限公司 | Novel myceliophthora thermophila strain and application thereof |
-
2022
- 2022-05-10 CN CN202210501195.2A patent/CN114591849A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717727A (en) * | 2009-12-23 | 2010-06-02 | 贵州大学 | Myceliophthora thermophilia strain and application thereof in aspect of producing keratinase |
| CN102352320A (en) * | 2011-09-29 | 2012-02-15 | 北京可力美施生物科技有限公司 | Novel myceliophthora thermophila strain and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| GENBANK: CP003004.1: "Myceliophthora thermophila ATCC 42464 chromosome 3, complete sequence", 《GENBANK: CP003004.1》 * |
| MIN JIN KWON ET AL: "Practical guidance for the implementation of the CRISPR genome editing tool in filamentous fungi", 《FUNGAL BIOL BIOTECHNOL》 * |
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