CN115109735B - Trichoderma reesei engineering bacterium for producing alpha-L-arabinofuranosidase as well as construction method and application thereof - Google Patents
Trichoderma reesei engineering bacterium for producing alpha-L-arabinofuranosidase as well as construction method and application thereof Download PDFInfo
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- CN115109735B CN115109735B CN202110299855.9A CN202110299855A CN115109735B CN 115109735 B CN115109735 B CN 115109735B CN 202110299855 A CN202110299855 A CN 202110299855A CN 115109735 B CN115109735 B CN 115109735B
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Classifications
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention relates to trichoderma reesei engineering bacteria for high yield of alpha-L-arabinofuranosidase, a construction method and application thereof. In the invention, four cellulase genes cel5a, cel7b, cel6a and cel7a in Trichoderma reesei engineering bacteria with high yield of alpha-L-arabinofuranosidase are knocked out and inactivated, and a transcription activator coding gene xyr1 is inserted; the transcriptional activator encoding gene xyr1 is expressed under the drive of the strong promoter P tcu1. The Trichoderma reesei engineering bacteria constructed by the invention can be stably passaged, can obviously improve the yield of Trichoderma reesei alpha-L-arabinofuranosidase, has the activity of the alpha-L-arabinofuranosidase of the Trichoderma reesei engineering bacteria about 8-9 times of that of an original strain, and is beneficial to the application of Trichoderma reesei in degrading hemicellulose substances rich in arabinose residues. In addition, the Trichoderma reesei engineering bacteria constructed by the invention can also be used for producing xyloglucanase and cellulose endonuclease.
Description
Technical Field
The invention relates to trichoderma reesei engineering bacteria for high yield of alpha-L-arabinofuranosidase, and a construction method and application thereof, and belongs to the technical field of bioengineering.
Background
The degradable cellulose and hemicellulose substances are main components of biomass resources, and the development and the utilization of the degradable cellulose and hemicellulose substances have important strategic significance on the social core problems of economic development, environmental protection and the like in China. Some microorganisms exist in nature and can use cellulose and hemicellulose as carbon sources and energy sources to play an important role in the carbon circulation process. Wherein the representative strain Trichoderma reesei (Trichoderma reesei) can produce various cellulases and hemicellulases, and is widely applied to the fields of energy, textile, pulp and paper making and the like.
Xylan and arabinan are main components of plant hemicellulose, and efficient degradation and utilization of the xylan and the arabinan have important economic value. Degradation of xylan and arabinan requires the involvement of a variety of degrading enzymes, of which α -L-arabinofuranosidase (α -L-arabinofuranosidase, EC3.2.1.55) is capable of hydrolysing the arabinose monomers or oligomeric arabinose side-branches linked to the xylan/arabinan backbone in α -1,3, α -L,2 or α -1,5 linkages, which are necessary for complete hydrolysis of xylan/arabinan. Besides the energy field, the alpha-L-arabinofuranosidase is widely applied to the food and feed processing field, and has larger market demand.
The Trichoderma reesei genome contains four alpha-L-arabinofuranosidase coding genes which are named abf1, abf2, abf3 and bxl1 respectively, wherein abf and abf code for two main alpha-L-arabinofuranosidases. Studies have shown that abf and abf2 are capable of expression at the transcriptional level under sophorose or lactose carbon source conditions, but no significant enzyme activity can be detected in extracellular medium due to the lower levels. To date, trichoderma reesei producing strains having high levels of exocytosis yield of alpha-L-arabinofuranosidase have not been obtained, which greatly limits the use of Trichoderma reesei for efficient degradation of hemicellulose-containing arabinose residues.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a Trichoderma reesei engineering bacterium with high yield of alpha-L-arabinofuranosidase, a construction method and application thereof, and the extracellular alpha-L-arabinofuranosidase activity of the Trichoderma reesei engineering bacterium is 8-9 times that of a starting strain.
The technical scheme of the invention is as follows:
A Trichoderma reesei engineering bacterium for high yield of alpha-L-arabinofuranosidase, wherein four cellulase genes cel5a, cel7b, cel6a and cel7a in the Trichoderma reesei engineering bacterium are knocked out and inactivated, and a transcription activator coding gene xyr1 is inserted.
Preferably, according to the present invention, the transcriptional activator encoding gene xyr1 is expressed under the drive of a strong promoter; preferably, the strong promoter is P tcu1.
According to the invention, the alpha-L-arabinofuranosidase activity of the Trichoderma reesei engineering bacteria is 8-9 times of that of the original strain.
The related genes are all genes with known sequences, wherein the JGI protein ID of the gene cel5a encoded protein is Tr120312, the JGI protein ID of the gene cel7b encoded protein is Tr122081, the JGI protein ID of the gene cel6a encoded protein is Tr72567, the JGI protein ID of the gene cel7a encoded protein is Tr123989, and the JGI protein ID of the gene xyr1 encoded protein is Tr122208.
The construction method of the Trichoderma reesei engineering strain for high yield of alpha-L-arabinofuranosidase is characterized in that Trichoderma reesei QM9414 is taken as an original strain, a homologous recombination technology is adopted, four cellulase genes cel5a, cel7b, cel6a and cel7a in the original strain are sequentially knocked out, so that the genes are inactivated, then a transcription activator coding gene xyr1 is inserted, xyr1 gene is expressed under the drive of a strong promoter P tcu1, xyr gene is over-expressed, pyr4 gene is taken as a screening marker gene, and screening and verification are carried out to obtain the Trichoderma reesei engineering strain.
In the present invention, there is no particular requirement for the sequence of knockout of the four cellulase genes cel5a, cel7b, cel6a, cel7 a.
The construction method of the Trichoderma reesei engineering bacteria for high yield of the alpha-L-arabinofuranosidase comprises the following steps:
(1) Taking Trichoderma reesei QM9414 genome DNA as a template, performing PCR amplification to obtain upstream and downstream homology arms of a cel5a gene, respectively inserting the homology arms into pDonorpyr vectors, constructing a knockout vector pDonorcel a on two sides of a pyr4 gene expression cassette in the pDonorpyr vectors, transforming the knockout vector into Trichoderma reesei QM 9414-delta pyr4, screening after homologous recombination to obtain a cel5a gene knockout strain, and marking the cel5a gene knockout strain as Trichoderma reesei delta cel5a;
(2) Taking Trichoderma reesei QM9414 genome DNA as a template, performing PCR amplification to obtain upstream and downstream homology arms of a cel7b gene, respectively inserting the upstream and downstream homology arms into pDonorpyr vectors, constructing a knockout vector pDonorcel b on two sides of a pyr4 gene expression cassette in the pDonorpyr vectors, transforming the knockout vector into Trichoderma reesei Deltacel 5a in the step (1), screening after homologous recombination to obtain cel5a and cel7b gene knockout strains, and marking the cell 5a and the cel7b gene knockout strains as Trichoderma reesei Deltacel 5a-cel7b;
(3) Taking Trichoderma reesei QM9414 genome DNA as a template, performing PCR amplification to obtain upstream and downstream homology arms of a cel6a gene, respectively inserting the homology arms into pMDpyr vectors, constructing knockout vectors pMDcel a on two sides of pyr4 gene expression cassettes in pMDpyr vectors, transforming the knockout vectors into Trichoderma reesei Deltacel 5a-cel7b in the step (2), screening after homologous recombination to obtain cel5a, cel7b and cel6a gene knockout strains, and marking the cell 5a, cel7b and cel6a gene knockout strains as Trichoderma reesei Deltacel 5a-cel7b-cel6a;
(4) Constructing a knockout vector pUCcbh pyr4 of a cel7a gene, transforming the knockout vector into trichoderma reesei delta cel5a-cel7b-cel6a in the step (3), and screening after homologous recombination to obtain cel5a, cel7b, cel6a and cel7a gene knockout strains, namely trichoderma reesei delta 4cel;
(5) The Trichoderma reesei QM9414 genome DNA is used as a template, xyr genes are obtained through PCR amplification, inserted into a vector pMDP tcu1-TtrpC and positioned between a promoter tcu fragment and a terminator TrpC fragment, an over-expression vector pMDOExyr1 is constructed, the over-expression vector is transformed into the Trichoderma reesei delta 4cel in the step (4), after transformation, the cell 5a, the cell 7b, the cell 6a and the cell 7a gene knockout and xyr1 gene over-expression strains are obtained through screening, and the Trichoderma reesei engineering strain is the Trichoderma reesei delta 4celOExyr with high yield of alpha-L-arabinofuranosidase.
Preferably, according to the invention, the transformation is performed using PEG mediated protoplast transformation.
In the invention, pyr4 gene is taken as a screening marker gene for bacterial strain screening, uridine is added into a culture medium during screening, bacterial strains containing pyr4 gene can survive, and bacterial strains not containing pyr4 gene can not survive; the pyr4 gene is deleted by the action of 5' fluoroorotic acid, allowing the selectable marker to be reused.
In the preparation method, the construction methods of pDonorpyr vectors, trichoderma reesei QM 9414-delta pyr4 and pMDpyr vectors, knockout vector pUCcbh1pyr4 and vector pMDP tcu1-TtrpC are all the prior art.
The application of the Trichoderma reesei engineering bacteria in producing alpha-L-arabinofuranosidase, xyloglucanase and/or cellulose endonuclease.
According to a preferred embodiment of the invention, the application comprises the following steps:
Inoculating the Trichoderma reesei engineering bacteria into MA liquid culture medium containing 1% glycerol and 0.2% peptone, culturing at 180-200rpm at 28+ -2deg.C for 24-36 hr, collecting mycelium, transferring into MA liquid culture medium containing 2% lactose, and continuously culturing for 96-120 hr to obtain fermentation broth.
Further preferably, the MA liquid medium comprises the following components: adding Na2HPO4·12H2O 17.907g、(NH4)2SO41.4 g、KH2PO42.0 g、 urea 0.3g and Tween-80.5 mL into each 1L culture medium, regulating pH to 5.0 with anhydrous citric acid, and adding MgSO 4 with final concentration of 0.6g/L, caCl 2 with final concentration of 0.4g/L and microelements before use;
Wherein the trace element (1000X) is composed of 5.0mg/L FeSO4·7H2O,1.6mg/L MnSO4·H2O,1.4mg/L ZnSO4·7H2O,2.0mg/L CoCl2·2H2O.
The application of the Trichoderma reesei engineering bacteria in degrading hemicellulose substances rich in arabinose residues.
The invention has the technical characteristics and beneficial effects that:
1. According to the invention, trichoderma reesei QM9414 is taken as an initial strain, four main cellulase genes cel5a (JGI protein ID Tr 120312), cel7b (JGI protein ID Tr 122081), cel6a (JGI protein ID Tr 72567) and cel7a (JGI protein ID Tr 123989) in the genome of trichoderma reesei QM9414 are knocked out, and simultaneously, transcription activating factor coding genes xyr (JGI protein ID Tr 122208) of cellulase and hemicellulase genes are overexpressed at a high level, so that trichoderma reesei delta 4celOExyr engineering bacteria with high yield of alpha-L-arabinofuranosidase is constructed. The experiment results of the invention can infer that the transcription activator XYR1 has positive regulation effect on the transcription expression of the alpha-L-arabinofuranosidase encoding gene, and the deletion of four main cellulase genes ensures that the regulation of the transcription activator XYR1 acts on the alpha-L-arabinofuranosidase encoding gene more, so that the transcription expression level of the alpha-L-arabinofuranosidase is improved, and the aim of high yield of the alpha-L-arabinofuranosidase is achieved.
2. The engineering bacteria of Trichoderma reesei delta 4celOExyr1 constructed by the invention can be stably passaged, the yield of Trichoderma reesei alpha-L-arabinofuranosidase can be obviously improved, the enzyme activity of the alpha-L-arabinofuranosidase of the Trichoderma reesei delta 4celOExyr1 engineering bacteria is about 8-9 times of that of a starting strain, and the application of Trichoderma reesei in degrading hemicellulose substances rich in arabinose residues is facilitated. In addition, the engineering bacteria of Trichoderma reesei delta 4celOExyr1 constructed by the invention can also be used for producing xyloglucanase and cellulose endoenzyme.
Drawings
FIG. 1 is a schematic diagram of cel5a gene knockout vector pDonorcel a;
FIG. 2 is a schematic diagram of cel7b gene knockout vector pDonorcel b;
FIG. 3 is a schematic diagram of a cel6a gene knockout vector pMDcel a;
FIG. 4 is a schematic diagram of the cel7a knockout vector pUCcbh pyr 4;
FIG. 5 is a schematic diagram of xyr gene overexpression vector pMDOExyr;
FIG. 6 shows the relative transcript levels of genes cel5a (panel A), cel7B (panel B), cel6a (panel C), cel7a (panel D) and xyr1 (panel E) in the Δ4celOExyr1 strain and the starting strain QM 9414;
FIG. 7 is a SDS-PAGE electrophoresis of the fermentation broths of strain Delta4 celOExyr1 and of the starting strain QM 9414;
FIG. 8 is a graph showing the enzymatic activity analysis of alpha-L-arabinofuranosidase of fermentation broths of strain Delta4 celOExyr and of the starting strain QM 9414;
FIG. 9 shows the relative transcript levels of the a-L-arabinofuranosidase encoding gene abf of the Δ4celOExyr1 strain and of the starting strain QM 9414.
Detailed Description
The present invention will be further described with reference to examples and drawings, but the scope of the present invention is not limited thereto. The medicines and reagents related to the examples are common commercial products unless specified; the experimental procedures referred to in the examples, unless otherwise specified, are conventional in the art. The primers in the examples were all synthesized by Beijing qing Biotechnology Co.
The starting strain Trichoderma reesei (Trichoderma reesei) QM9414 was purchased from the American type culture Collection under the accession number ATCC 26921.
EXAMPLE 1 Ace15a Strain construction
1) Construction of cel5a gene knockout vector
Extracting Trichoderma reesei QM9414 genome DNA by using a fungus DNA extraction kit (E.Z.N.A. TM Fungal DNA MINI KIT, OMEGA), designing primers cel5a-upF/cel5a-upR by using the genome DNA as a template, and obtaining a cel5a gene upstream homology arm fragment by PCR amplification of about 1.5kb; designing a primer cel5a-downF/cel5a-downR, and obtaining a cell 5a gene downstream homologous arm fragment of about 1.5kb through PCR amplification; and a section of about 0.5kb fragment (named cel5 a-DR) at the 3' -end of the upstream homology arm fragment is obtained by PCR amplification using cel5aDR-F/cel5aDR-R as an upstream primer and a downstream primer.
Wherein, the sequence of the primer is as follows:
cel5a-upF：5’-TAGGGATAACAGGGTAATTCAACCAGACTGTCCCTTCTACTTT-3’，
cel5a-upR：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTAATGTCGATGACGGGGAGATATTATCG-3’；
cel5a-downF：5’-CACTCTGAGCTGAATGCAGAAGCCT-3’，
cel5a-downR：5’-ATTACCCTGTTATCCCTATAATCATTCGGATCAGCCGTACCTG-3’；
cel5aDR-F：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTAGTTGTGCAAACAGTAACTCAGGGAA-3’，
cel5aDR-R：5’-AGGCTTCTGCATTCAGCTCAGAGTGTGTCGATGACGGGGAGATATTATCG-3’；
The PCR amplification system is as follows: 10. Mu.M upstream primer 2. Mu.L, 10. Mu.M downstream primer 2. Mu.L, genomic DNA 10ng,2.5mM dNTP mix 5. Mu.L, 5X TRANSSTART FASTPFU buffer 10. Mu.L, transStartFastPfuDNA polymerase 1. Mu.L, ddH 2 O up to 50. Mu.L;
The PCR amplification procedure was as follows: pre-denaturation at 98 ℃ for 5min; denaturation at 95℃for 30sec, annealing at 58℃for 30sec, extension at 72℃for 3min,30 cycles; final extension at 72℃for 10min; after completion of PCR, 1% agarose gel electrophoresis was performed, and a DNA fragment was recovered.
And (3) fusing and connecting the cel5a-DR fragment and the downstream homology arm fragment of the cel5a gene by adopting a fusion PCR method, respectively connecting the fused fragment and the upstream homology arm fragment of the cel5a gene into pDonorpyr vectors under the action of BP cloning enzyme, and respectively locating at two sides of pyr4 gene expression cassettes in the pDonorpyr vectors to construct a cel5a gene knockout vector pDonorcel a. Pyr4 is a commonly used uridine-deficient screening tag.
Wherein, the construction method of pDonorpyr4 vector refers to literature Two major facilitator superfamily sugar transporters from Trichoderma reesei and their roles in induction of cellulase biosynthesis.Zhang W,Kou Y,Xu J,Cao Y,Zhao G,Shao J,Wang H,Wang Z,Bao X,Chen G,Liu W.J Biol Chem.2013Nov 15;288(46):32861-72;
The fusion PCR amplification system comprises: 20ng of cel5a-DR fragment, 20ng of downstream homology arm fragment, 8. Mu.L of 2.5mM dNTP mixture, 20. Mu.L of 5X TRANSSTART FASTPFU buffer, 1. Mu.L of TRANSSTART FASTPFU DNA polymerase, 10. Mu.M upstream primer cel5 aDR-F5. Mu.L, 10. Mu.M downstream primer cel5 a-downR. Mu.L, ddH 2 O make up 100. Mu.L;
the fusion PCR amplification procedure was as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30sec, annealing at 53℃for 6min, extension at 72℃for 4min,28 cycles; final extension at 72℃for 10min; after completion of PCR, 1% agarose gel electrophoresis was performed, and a DNA fragment was recovered.
2) Preparation of Trichoderma reesei protoplast
Preparing Trichoderma reesei QM 9414-delta pyr4 spore suspension with the concentration of 5X 10 7～5×108/mL, taking 1mL, inoculating in 50mL of mycelium growth medium, and culturing at 28 ℃ and 160rpm for 16h to obtain mycelium culture; centrifuging mycelium culture at 4000rpm for 5min, washing the precipitate with 0.7M NaCl solution three times, adding cell wall lyase solution each time for 25mL, re-suspending, and carrying out enzymolysis at 30 ℃ and 60rpm for 2.5-3 h, when a large amount of free protoplasts appear, filtering by using a G2 funnel, centrifuging filtrate at 4 ℃ and 3800rpm for 5min to collect protoplasts, washing the collected protoplasts twice with 15mL of precooled STC solution, and then adding precooled STC solution for re-suspending to ensure that the protoplast concentration is 5 multiplied by 10 7～5×108 per mL; all of the above operations were performed on ice.
Wherein, trichoderma reesei QM 9414-Deltapyr 4 is obtained by knocking out pyr4 gene in Trichoderma reesei QM9414, and the method for constructing the strain is described in literature A novel transcriptional regulator RXE1 modulates the essential transactivator XYR1and cellulase gene expression in Trichoderma reesei.Wang L,Lv X,Cao Y,Zheng F,Meng X,Shen Y,Chen G,Liu W,Zhang W.Appl Microbiol Biotechnol.2019Jun;103(11):4511-4523.
The mycelium growth medium comprises the following components (mass percent): 2% glucose, 0.5% (NH 4)2SO4、1.5％KH2PO4, 1% peptone, pH adjusted to 5.5 with NaOH), adding MgSO 4 with final concentration of 0.6g/L and CaCl 2 with final concentration of 0.4g/L before use, adding trace elements (final concentration 1×, mother liquor 1000×), 10mM uridine, fixing volume of 1000×trace element mother liquor formula ：FeSO4·7H2O 0.5g,MnSO4·H2O 0.16g,ZnSO4·7H2O 0.14g,CoCl2·2H2O 0.2g, to 100mL with ddH 2 O, filtering, and sterilizing;
the cell wall lyase liquid comprises the following components: 0.24g of cell wall lyase dissolved in 5.7mL of 0.7M NaCl solution, filtered and sterilized, and added with 0.3mL of PBS buffer with pH of 5.8;
The STC solution had the following composition: 0.7M D-sorbitol, 0.05M CaCl 2, 10mM Tris-HCl, pH 7.5.
3) Transformation of knockout vector pDonorcel a, transformant regeneration, screening verification and selectable marker removal
Taking 150 mu L of the knockout carrier pDonorcel a obtained in the step 1), carrying out tangential digestion by using I-SceI enzyme, purifying and recovering fragments, uniformly mixing with 150 mu L of Trichoderma reesei QM 9414-Deltapyr 4 protoplast prepared in the step 2), adding the mixture into the bottom of a 10mL centrifuge tube, dropwise adding 1.5mL of precooled PTC solution after ice bath for 20min, standing at room temperature for 20min, adding 2mL of STC solution, and uniformly mixing to obtain a conversion solution; the transformation solution is spread on a regeneration medium plate without uridine and cultured for 5-7d at 28 ℃.
Wherein, the PTC solution comprises the following components: the mass-to-volume ratio (g/mL) is 60% PEG 4000 (polyethylene glycol 4000), 10mM Tris-HCl,10mM CaCl 2, pH 7.5;
The regeneration medium comprises the following components (mass percent): 2% glucose, 0.5% (NH 4)2SO4、1.5％KH2PO4, 1% peptone, 1M D-sorbitol, 1.5% agar powder, pH was adjusted to 5.5 with NaOH, and before use, 0.6g/L MgSO 4 and 0.4g/LCaCl 2 were added, and microelements (final concentration 1×, mother liquor 1000×) 1000×microelements mother liquor formulation was the same.
Transferring the colonies growing on the regeneration medium plate to a screening medium plate without uridine, and culturing at 28 ℃ for 5-7d to obtain transformants.
Wherein, the components of the screening culture medium are as follows (mass percent): 2% glucose, 0.5% (NH 4)2SO4、1.5％KH2PO4, 1.5% agar powder, pH was adjusted to 5.5 with NaOH), mgSO 4 at a final concentration of 0.6g/L and CaCl 2 at a final concentration of 0.4g/L were added before use, and trace elements (final concentration 1X, mother liquor 1000X) 1000X trace element mother liquor was formulated as above.
The genome DNA of the obtained transformant is extracted, the genome DNA is taken as a template, and the PCR technology is adopted to carry out the anchoring verification of the upstream homology arm sequence, wherein the upstream homology arm sequence anchoring verification primers are cel5a-YZ-F1 and An-R, and the length of a target strip is about 1.5kb; the downstream homology arm sequence anchoring verification primer is An-F/cel5a-YZ-R2, and the length of a target band is about 1.5kb; the primer sequences were as follows:
cel5a-YZ-F1:5’-AAACGAATTCTAGGTTTATCGAGTC-3’，
An-R:5’-TGAAGCAGAAATAGGGTGAATGATA-3’；
An-F:5’-TATTTTTGATTTGGCATTTGCTTTT-3’，
cel5a-YZ-R2:5’-GCTGAAGACGGTCTTGCGCGAAGTA-3’。
Purifying the transformant with correctly anchored upstream and downstream homology arms by adopting a single spore separation method, and carrying out internal gene verification of the cel5a by adopting a PCR technology and using the cel5a-YZ-F3 and the cel5a-YZ-R3 primers, wherein no target band exists, namely the cel5a gene is successfully knocked out, and the Deltacel 5a strain is successfully constructed.
Wherein, the primer sequence is as follows:
cel5a-YZ-F3:5’-GTACCCTTGTTTCCTGGTGTTGCT-3’，
cel5a-YZ-R3:5’-TGTTATTTGTAGTACATTCGGCGTGA-3’。
the above-mentioned verification PCR reaction systems were 20. Mu.L, and specific reaction systems and reaction conditions were described in GenStar X Taq PCR StarMix (available from Wohan Homox technologies Co., ltd.).
The schematic diagram of the cel5a gene knockout vector pDonorcel a is shown in fig. 1, the knockout vector pDonorcel a contains an upstream homology arm sequence and a downstream homology arm sequence of the cel5a gene, after the cell 5a gene knockout vector is transformed into trichoderma reesei QM 9414-delta pyr4 protoplast, homologous recombination occurs between the upstream homology arm and the downstream region of the cel5a gene in the genome, and a uridine defect screening tag pyr4 in the knockout vector pDonorcel a is integrated on the trichoderma reesei genome to replace the cel5a gene, so that the purpose of knockout of the cel5a gene is achieved.
Spores of the Deltacel 5a strain were spread on a screening medium plate containing 5-fluoroorotic acid (5-Fluoroorotic acid, 5-FOA) at a final concentration of 1.2mg/mL for reverse screening, and the screening tag pyr4 was recombined by Trichoderma reesei's own recombinase, and transformants capable of growing on the plate containing 5-FOA were successfully removed, thereby obtaining uridine utilization deficient strain Deltacel 5a.
Example 2: construction of Deltacel 5a-cel7b Strain
1) Construction of cel7b gene knockout vector
Similar to the method of example 1, using Trichoderma reesei QM9414 genomic DNA as a template, designing a primer cel7b-upF/cel7b-upR, and performing PCR amplification to obtain a cel7b gene upstream homology arm fragment of about 1.5kb; designing a primer cel7b-downF/cel7b-downR, and obtaining a cell 7b gene downstream homologous arm fragment of about 1.5kb through PCR amplification; and a section of about 0.5kb fragment (named cel7 b-DR) at the 3' -end of the upstream homology arm fragment is obtained by PCR amplification using cel7bDR-F/cel7bDR-R as an upstream primer and a downstream primer.
Wherein, the sequence of the primer is as follows:
cel7b-upF：5’-TAGGGATAACAGGGTAATGGGAGGGAAATGGAATACGACAACT-3’，
cel7b-upR：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTAATTTGGGACAACAAGAAGGACTAAGA-3’；
cel7b-downF：5’-AGCGTTGACTTGCCTCTGGTCTGTC-3’，
cel7b-downR：5’-ATTACCCTGTTATCCCTAGCTCATCTCCGTGTCACTGGTCAAT-3’；
cel7bDR-F：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTACCTCGTCCTTTGTCCGTTGTCTAAT-3’，
cel7bDR-R：5’-GACAGACCAGAGGCAAGTCAACGCTTTTGGGACAACAAGAAGGACTAAGA-3’。
and (3) fusing and connecting the cel7b-DR fragment and the downstream homology arm fragment of the cel7b gene by adopting a fusion PCR method, respectively connecting the fused fragment and the upstream homology arm fragment of the cel7a gene into pDonorpyr vectors under the action of BP cloning enzyme, respectively positioning the fused fragment and the upstream homology arm fragment of the cel7a gene at two sides of a pDonorpyr vector pyr4 gene expression cassette, and constructing a cel7b gene knockout vector pDonorcel b.
The amplification system and the amplification procedure of the PCR and the fusion PCR are the same as those of example 1; wherein the upstream primer in the fusion PCR amplification system is cel7bDR-F, and the downstream primer is cel7b-downR.
2) Preparation of Trichoderma reesei Δcel5a protoplast
The uridine utilization deficient strain Δcel5a prepared in example 1 was used to prepare trichoderma reesei Δcel5a protoplasts by the method of preparing protoplasts in step 2) of example 1.
3) Transformation of knockout vector pDonorcel b, regeneration of transformants, screening verification and removal of screening tag
150. Mu.L of the knock-out vector pDonorcel b obtained in step 1) was taken, the fragment was recovered by I-SceI enzyme tangential digestion, and was uniformly mixed with 150. Mu.L of Trichoderma reesei Δcel5a protoplast prepared in step 2), and transformants were prepared and verified by screening according to the method of transformation of the knock-out vector pDonorcel a and regeneration of the transformants in step 3) of example 1.
The genome DNA of the obtained transformant is extracted, the genome DNA is taken as a template, and the PCR technology is adopted to carry out the anchoring verification of the upstream homology arm sequence, wherein the upstream homology arm sequence anchoring verification primers are cel7b-YZ-F1 and An-R, and the length of a target strip is about 1.5kb; the downstream homology arm sequence anchoring verification primers are An-F and cel7b-YZ-R2, and the length of the target band is about 1.5kb; the primer sequences were as follows:
cel7b-YZ-F1:5’-CCGCTCATCACACTGCTACGCCTAC-3’，
An-R:5’-TGAAGCAGAAATAGGGTGAATGATA-3’；
An-F:5’-TATTTTTGATTTGGCATTTGCTTTT-3’，
cel7b-YZ-R2:5’-TCTTCACGCTGCTCAAGGCTTTCAT-3’。
Purifying the transformant with correctly anchored upstream and downstream homology arms by adopting a single spore separation method, and carrying out internal gene verification of the cel7b by using the cel7b-YZ-F3 and the cel7b-YZ-R3 primers, wherein no target strip exists, namely the cel7b gene is successfully knocked out, and the Deltacel 5a-cel7b strain is successfully constructed.
Wherein, the primer sequence is as follows:
cel7b-YZ-F3:5’-CTCGGCTGTATCTCCTGGACTCTGA-3’，
cel7b-YZ-R3:5’-CTGGCTGTTGTCGTTCCAAATGCT-3’。
The above-identified PCR reaction system was 20. Mu.L, and specific reaction system and reaction conditions were described in GenStar X Taq PCR StarMix (available from Wohan Homox technologies Co., ltd.).
The schematic diagram of the cel7b gene knockout vector pDonorcel b is shown in fig. 2, the knockout vector pDonorcel b contains an upstream homology arm sequence and a downstream homology arm sequence of the cel7b gene, after the homologous recombination is carried out on the upstream homology arm and the downstream homology arm with the upstream and downstream regions of the cel7b gene in the trichoderma reesei Δcel5a genome after the homologous recombination is carried out, and a uridine defect type screening tag pyr4 in the knockout vector pDonorcel b is integrated on the trichoderma reesei Δcel5a genome to replace the cel7b gene, so that the purpose of knockout of the cel7b gene is achieved.
Spores of the Deltacel 5a-cel7b strain are coated on a 5-FOA screening medium plate with a final concentration of 1.2mg/mL for reverse screening, screening tag pyr4 is recombined by using the self recombinase of Trichoderma reesei, transformants growing on the plate with 5-FOA can be obtained, and the screening tag is successfully removed, so that uridine utilization defective strain Deltacel 5a-cel7b is obtained.
Example 3: construction of Deltacel 5a-cel7b-cel6a Strain
1) Construction of cel6a gene knockout vector
Designing primers cel6a-upF/cel6a-upR by taking Trichoderma reesei QM9414 genome DNA as a template, and obtaining a cel6a gene upstream homology arm fragment of about 2kb through PCR amplification; designing a primer cel6a-downF/cel6a-downR, and obtaining a cell 6a gene downstream homologous arm fragment of about 1.8kb through PCR amplification; and a fragment of about 0.4kb at the 5' end of the pyr4 gene expression cassette (designated cel6 a-DR) was obtained by PCR amplification using cel6aDR-F/cel6aDR-R as the upstream and downstream primers.
Wherein, the sequence of the primer is as follows:
cel6a-upF：5’-ATAAGAATGCGGCCGCTGGAGTAGATGCTGGAGTCTGGTGT-3’，
cel6a-upR：5’-GGACTAGT GGTGCAATACACAGAGGGTGATCTT-3’；
cel6a-downF：5’-AGGCGCGCCGGCTTTCGTGACCGGGCTTCAAACA-3’，
cel6a-downR：5’-AGCTTTGTTTAAACAGTTAGGTGATGGGTACAACTGCTT-3’；
cel6aDR-F：5’-CGCGGATCCGCGGTCGACAACTGCATCCAAACCATCC-3’，
cel6aDR-R：5’-CCCAAGCTTGGGGGAAGAAAGAAGTAAAGAAAGGCAT-3’；
the amplification system and the amplification procedure of the PCR were the same as those of example 1.
The pyr4 gene expression cassette is connected to a pMD19T vector (Takara), a pMDpyr vector is constructed, an upstream homology arm fragment and a cel6a-DR are connected to the upstream and downstream of the pyr4 gene expression cassette in the pMDpyr vector in sequence, and finally a downstream homology arm fragment is connected to the downstream of the cel6a-DR, and a cel6a gene knockout vector pMDcel a is constructed.
2) Preparation of protoplast of Deltacel 5a-cel7b Strain
The uridine prepared in example 2 was used to prepare Trichoderma reesei Deltace 5a-cel7b protoplasts using the preparation method of the protoplasts in example 1 step 2) using the defective strain Deltace 5a-cel7 b.
3) Transformation of knockout vector pMDcel a, transformant regeneration, screening verification and screening tag removal
Taking 150 mu L of the knockdown vector pMDcel a obtained in the step 1), carrying out enzyme tangentially, purifying and recovering fragments, uniformly mixing with 150 mu L of Trichoderma reesei Deltace 5a-cel7b protoplast prepared in the step 2), preparing transformants according to the method of transforming the knockdown vector pDonorcel a in the step 3) of example 1 and regenerating the transformants, and screening and verifying.
The genome DNA of the obtained transformant is extracted, the genome DNA is taken as a template, and the PCR technology is adopted to carry out the anchoring verification of upstream and downstream homology arm sequences, wherein the upstream homology arm sequence anchoring verification primers are cel6a-YZ-F1 and cel6a-YZ-R1, and the length of a target band is about 2kb; the downstream homology arm sequence anchoring verification primer is cel6a-YZ-F2/cel6a-YZ-R2, and the length of the target band is about 1.8kb; the primer sequences were as follows:
cel6a-YZ-F1:5’-CAAGTGGCTACAACGACAATAAGT-3’，
cel6a-YZ-R1:5’-TACCTACCTTGGCTGGTTTGCTTG-3’；
cel6a-YZ-F2:5’-GGAGGCGAACTGCACATTGGAAGG-3’，
cel6a-YZ-R2:5’-AATTCGACAACGTGGAACAACCTG-3’。
Purifying the transformant with correctly anchored upstream and downstream homology arms by adopting a single spore separation method, and carrying out internal gene verification of the cel6a by adopting a PCR technology and using the cel6a-YZ-F3 and the cel6a-YZ-R3 primers, wherein no target strip exists, namely the cel6a gene is successfully knocked out, and the Deltacel 5a-cel7b-cel6a strain is successfully constructed.
Wherein, the primer sequence is as follows:
cel6a-YZ-F3:5’-ACGACTATTACTCCCAGTGTCTTCCC-3’，
cel6a-YZ-R3:5’-CAGCGTCCAAATACATCGCAACAT-3’。
The above-identified PCR reaction system was 20. Mu.L, and specific reaction system and reaction conditions were described in GenStar X Taq PCR StarMix (available from Wohan Homox technologies Co., ltd.).
The schematic diagram of the cel6a gene knockout vector pMDcel a is shown in fig. 3, the knockout vector pMDcel a contains an upstream homology arm sequence and a downstream homology arm sequence of the cel6a gene, after the homologous recombination is carried out on the upstream homology arm and the downstream homology arm with an upstream region and a downstream region of the cel6a gene in the trichoderma reesei Δcel5a-cel7b genome after the homologous recombination is carried out, and a uridine defect type screening tag pyr4 in the knockout vector pMDcel a is integrated on the trichoderma reesei Δcel5a-cel7b genome to replace the cel6a gene, so that the purpose of knockout of the cel6a gene is achieved.
Spores of the Deltacel 5a-cel7b-cel6a strain are coated on a 5-FOA screening medium plate with a final concentration of 1.2mg/mL for reverse screening, screening tag pyr4 is recombined by using the self recombinase of Trichoderma reesei, transformants growing on the plate with 5-FOA can be successfully removed, and thus uridine utilization defective strain Deltacel 5a-cel7b-cel6a is obtained.
Example 4: construction of Deltacel 5a-cel7b-cel6a-cel7a (Delta4 cel) Strain
1) Construction of cel7a gene knockout vector
The construction of the cel7a gene knockout vector pUCcbh for pyr4 is described in reference Deciphering the effect of the different N-glycosylation sites on the secretion,activity,and stability of cellobiohydrolase I from Trichoderma reesei.Qi F,Zhang W,Zhang F,Chen G,Liu W.Appl Environ Microbiol.2014Jul;80(13):3962-71,, wherein the cbh1 gene in the reference is the same gene as the cel7a gene of the present invention.
2) Preparation of protoplast of Deltacel 5a-cel7b-cel6a Strain
Uridine prepared in example 3 was prepared into Trichoderma reesei Deltacel 5a-cel7b-cel6a protoplasts using the preparation method of the protoplast of example 1 step 2) using the defective strain Deltacel 5a-cel7b-cel6 a.
3) Transformation of knockout vector pUCcbh pyr4, transformant regeneration, screening verification and screening tag removal
150. Mu.L of the knock-out vector pUCcbh a from step 1) was taken, linearized, purified and the fragment recovered, and mixed with 150. Mu.L of Trichoderma reesei Deltace 5a-cel7b-cel6a protoplast prepared in step 2), transformants were prepared and verified by screening according to the method of transformation of knock-out vector pDonorcel a and regeneration of transformants in step 3) of example 1.
The genome DNA of the obtained transformant is extracted, the genome DNA is taken as a template, and the PCR technology is adopted to carry out the anchoring verification of the upstream homology arm sequence, wherein the upstream homology arm sequence anchoring verification primers are cel7a-YZ-F1 and An-R, and the length of a target strip is about 1.5kb; the upstream homology arm sequence anchoring verification primers are An-F and cel7a-YZ-R2, and the length of the target band is about 1.5kb; the primer sequences were as follows:
cel7a-YZ-F1:5’-TGTTTCGGCTACGGTGAAGAACTG-3’，
An-R:5’-TGAAGCAGAAATAGGGTGAATGATA-3’；
An-F:5’-TATTTTTGATTTGGCATTTGCTTTT-3’，
cel7a-YZ-R2:5’-CTTATTGAGACCATGCGTGCTAAT-3’。
Purifying the transformant with correctly anchored upstream and downstream homology arms by adopting a single spore separation method, and carrying out internal gene verification of the cel7a by adopting a PCR technology and using the cel7a-YZ-F3 and the cel7a-YZ-R3 primers, wherein no target strip exists, namely the cel7a gene is successfully knocked out, and the Deltacel 5a-cel7b-cel6a-cel7a strain is successfully constructed.
Wherein, the primer sequence is as follows:
cel7a-YZ-F3:5’-ACTCAACAGACAGGCTCCGTGGTC-3’，
cel7a-YZ-R3:5’-AGATTCGACCTGAGCAGGGACACC-3’。
The above-identified PCR reaction system was 20. Mu.L, and specific reaction system and reaction conditions were described in GenStar X Taq PCR StarMix (available from Wohan Homox technologies Co., ltd.).
The schematic diagram of the cel7a gene knockout vector pUCcbh a 1pyr4 is shown in fig. 4, the knockout vector pUCcbh pyr4 contains an upstream homology arm sequence and a downstream homology arm sequence of the cel7a gene, after the homologous recombination is carried out on the upstream homology arm and the downstream homology arm of the cel7a gene in the trichoderma reesei Δcel5a-cel7b-cel6a genome after the homologous recombination is carried out on the upstream homology arm and the downstream homology arm sequence are transformed into the trichoderma reesei Δcel5a-cel7b-cel6a protoplast, and a uridine defect screening tag pyr4 in the knockout vector pUCcbh pyr4 is integrated on the trichoderma reesei Δcel5a-cel7b-cel6a genome to replace the cel7a gene, so that the purpose of knockout of the cel7a gene is achieved.
Spores of the Deltacel 5a-cel7b-cel6a-cel7a strain are coated on a screening medium plate containing 5-FOA with a final concentration of 1.2mg/mL for reverse screening, screening tag pyr4 is recombined by using the self recombinase of Trichoderma reesei, transformants growing on the plate containing 5-FOA can be successfully removed, and thus uridine utilization defective strain Deltacel 5a-cel7b-cel6a-cel7a, abbreviated as Delta4 cel, is obtained.
Example 5: construction of Δ4celOExyr1 Strain
1) Construction of transcriptional activator xyr1 over-expression vector
The plasmid pMDhph is used as a template, primers hphF/hphR are designed, a hph gene expression cassette is obtained through PCR amplification, the hph gene expression cassette is connected to a plasmid pMDP tcu1-TtrpC and positioned at the 3' end of a TrpC sequence, and a vector pMDP tcu1-TtrpC -hph is constructed.
The genome DNA of Trichoderma reesei QM9414 is used as a template, primers xyr-upF/xyr 1-upR are designed, a xyr gene segment is obtained through PCR amplification, the xyr gene segment is connected into a vector pMDP tcu1-TtrpC -hph and positioned between a promoter tcu segment and a terminator TrpC segment, and a xyr gene overexpression vector pMDOExyr1 is constructed.
Wherein, the sequence of the primer is as follows:
hphF:5’-CCACTCCACATCTCCACTCGACGTGGCAGCACGAGATAACG-3’，
hphR:5’-TTACGCCAAGTTTGCACGCC ACGAGCTTGTGCTGCGGAATC-3’；
xyr1-upF:5’-TTGGCGCGCC ATGTTGTCCAATCCTCTCCGTCGCT-3’，
xyr1-upR:5’-GGACTAGT TTAGAGGGCCAGACCGGTTCCGTTA-3’；
Construction of plasmid pMDhph Two major facilitator superfamily sugar transporters from Trichoderma reesei and their roles in induction of cellulase biosynthesis.Zhang W,Kou Y,Xu J,Cao Y,Zhao G,Shao J,Wang H,Wang Z,Bao X,Chen G,Liu W.J Biol Chem.2013Nov 15;288(46):32861-72.
Construction of plasmid pMDP tcu1-TtrpC Characterization of a copper responsive promoter and its mediated overexpression of the xylanase regulator 1results in an induction-independent production of cellulases in Trichoderma reesei.Lv X,Zheng F,Li C,Zhang W,Chen G,Liu W.Biotechnol Biofuels.2015Apr 14;8:67.
2) Preparation of protoplast of Delta4 cel Strain
The uridine utilization deficient strain Δ4cel prepared in example 4 was used to prepare trichoderma reesei Δ4cel protoplasts by the method of preparing protoplasts in step 2) of example 1.
3) Transformation of over-expression vector pMDOExyr, regeneration of transformant and screening verification
Taking 150 mu L of the over-expression vector pMDOExyr a obtained in the step 1), linearizing, purifying and recovering fragments, uniformly mixing with 150 mu L of Trichoderma reesei delta 4cel protoplast prepared in the step 2), and obtaining a transformant according to the method of transforming the knockout vector pDonorcel a and regenerating the transformant in the step 3) of the example 1, wherein the difference is that: coating the transformation solution on a regeneration culture medium plate which does not contain uridine but contains hygromycin with the final concentration of 120 mug/mL, and culturing for 5-7d at 28 ℃; colonies grown on the regeneration medium plate were transferred to a screening medium plate containing no uridine but a final concentration of 120. Mu.g/mL hygromycin, and cultured at 28℃for 5-7d to obtain transformants.
The genome DNA of the obtained transformant is extracted, the genome DNA is taken as a template, the inserted xyr gene expression cassette is verified by adopting a PCR technology, the verification primer is YZxyr-F/YZxyr 1-R, and the length of a target band is about 3.0kb; the primer sequences were as follows:
YZxyr1-F:5’-CCACAAGAGCCTACTGCCAAATC-3’，
YZxyr1-R:5’-TTAGAGGGCCAGACCGGTTCCGT-3’；
And (3) separating and purifying the transformant by adopting a single spore separation method to obtain the Trichoderma reesei delta 4celOExyr1 engineering bacteria successfully inserted with xyr gene.
The above-identified PCR reaction system was 20. Mu.L, and specific reaction system and reaction conditions were described in GenStar X Taq PCR StarMix (available from Wohan Homox technologies Co., ltd.).
A schematic diagram of xyr gene over-expression vector pMDOExyr1 is shown in FIG. 5, and xyr gene is driven by constitutive strong promoter tcu1 in over-expression vector pMDOExyr1 to realize high-level xyr1 transcriptional expression.
EXAMPLE 6 analysis of the levels of the four cellulase-encoding genes ce15a, ce17b, ce16a, cea of the A4 cellOExyr1 Strain and of the starting Strain QM9414
Spores of Trichoderma reesei Delta4 celOExyr1 engineering bacteria constructed in example 5 and Trichoderma reesei QM9414 serving as a control strain are respectively inoculated into MA liquid culture medium containing 1% of glycerol, and are cultured for 24 hours at 28 ℃;3 sets of parallel experiments were set up.
Wherein, the formula of the MA liquid culture medium is as follows: Na2HPO4·12H2O 17.907g、(NH4)2SO41.4g、KH2PO42.0g、 urea 0.3g and Tween-800.5mL were added to each 1L of medium, and the pH was adjusted to 5.0 with anhydrous citric acid. Adding 1% glycerol as a carbon source and adding 2g/L peptone during preculture; 1% Avicel (microcrystalline cellulose) was added as a carbon source at the time of induction. Before use, adding MgSO 4 with a final concentration of 0.6g/L and CaCl 2 with a final concentration of 0.4g/L, and adding microelements (1×withfinal concentration, 1000×withmother liquor) and 10mM uridine; 1000 Xmicroelement mother liquor formulation ：FeSO4·7H2O 0.5g,MnSO4·H2O 0.16g,ZnSO4·7H2O 0.14g,CoCl2·2H2O 0.2g, was sterilized by filtration with ddH 2 O to a volume of 100 mL.
Total RNA in the sample was extracted using TRizol reagent (Invitrogen Co.) after the completion of the culture. RNA was reverse transcribed using TaKaRa PRIMESCRIPT TM RT REAGENT KIT WITH GDNA ERASER kit, 20. Mu.L of the reaction system, and the method was described in the specification.
1 Μl of the reverse transcription product was used for subsequent qRT-PCR amplification, and qRT-PCR analysis was performed according to TaKaRa SYBR Premix Ex Taq TM kit, with an amplification system of 20 μl, actin gene as an internal reference gene, and experimental procedures were described in the specification.
Wherein, qRT-PCR primers of the internal reference gene actin are as follows:
actin-F：5’-TGAGAGCGGTGGTATCCACG-3’，
actin-R：5’-GGTACCACCAGACATGACAATGTTG-3’，
the qRT-PCR primer of the cellulase coding gene cel5a is as follows:
cbh1-F：5’-CTTGGCAACGAGTTCTCTT-3’，
cbh1-R：5’-TGTTGGTGGGATACTTGCT-3’，
the qRT-PCR primer of the cellulase coding gene cel7b is as follows:
cbh2-F：5’-CTGTCGCAAAGGTTCCCTCT-3’，
cbh2-R：5’-TCCGGCAAGTCATACACCAC-3’，
the qRT-PCR primer of the cellulase coding gene cel6a is as follows:
eg1-F：5’-CGGCTACAAAAGCTACTACG-3’，
eg1-R：5’-CTGGTACTTGCGGGTGAT-3’，
The qRT-PCR primer of the cellulase coding gene cel7a is as follows:
eg2-F：5’-GCTGTACCACAGATGGCACT-3’，
eg2-R：5’-CATCCGACAGGTAAGCGGAA-3’，
The qRT-PCR primer of gene xyr encoding the transcriptional activator is as follows:
xyr1-F：5’-CCATCAACCTTCTAGACGAC-3’，
xyr1-R：5’-AACCCTGCAGGAGATAGAC-3’，
The qRT-PCR reaction system is as follows: 1. Mu.L of reverse transcription product, 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 10. Mu.L of 2 XSYBR Green q RT-PCR Mix, and 20. Mu.L in total of ddH 2 O8. Mu.L;
the qRT-PCR reaction procedure was as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30sec, annealing at 58℃for 30sec, elongation at 72℃for 1min for 30sec, 30 cycles; finally, the extension is carried out for 5min at 72 ℃.
As shown in fig. 6, the transcription level of the four main cellulase coding genes cel5a, cel7b, cel6a and cel7a in the trichoderma reesei Δ4celOExyr1 engineering bacteria is obviously reduced compared with that of the trichoderma reesei QM9414 of the control strain, and almost no transcription expression exists, which is consistent with the knocked out result of the four corresponding genes; the expression level of xyr gene of delta 4celOExyr1 engineering bacteria is obviously higher than that of trichoderma reesei QM9414 in a control strain, which shows that xyr gene in the strain realizes high-level expression under the drive of tcu promoter.
Example 7: analysis of extracellular fermentation broth of Δ4celOExyr1 Strain
Equal spores of Trichoderma reesei Delta4 CelOExyr1 engineering bacteria constructed in example 5 and Trichoderma reesei QM9414 as a control strain are inoculated into MA liquid culture medium containing 1% glycerol and 0.2% peptone respectively, and after culturing for 24 hours at 28 ℃ and 180rpm, mycelia are collected by filtration. Transferring the collected mycelium into MA liquid culture medium containing 2% lactose, and culturing at 28deg.C and 180rpm for 96 hr to obtain fermentation broth. The MA liquid medium was formulated as described in example 6.
1) SDS-PAGE and mass spectrometry of extracellular fermentation broth
20. Mu.L of the fermentation broth was analyzed by conventional SDS-PAGE, and subjected to conventional Coomassie brilliant blue staining and decolorization. As a result, as shown in FIG. 7, there were more bands in the fermentation broth of engineering bacteria of Trichoderma reesei Delta4 CelOExyr1 than in the extracellular fermentation broth of Trichoderma reesei QM9414 as a control strain. The main three strips in the gel are cut off and sent to Beijing Hua big gene company for mass spectrum analysis, and the mass spectrum results show that the strips 1,2 and 3 are xyloglucanase, alpha-L-arabinofuranosidase ABF1 and cellulose endoenzyme EG3 respectively.
2) Enzyme activity analysis of alpha-L-arabinofuranosidase in fermentation broth
Taking a proper amount of fermentation liquor, and carrying out enzyme activity analysis on the fermentation liquor by using alpha-L-arabinofuranosidase, wherein the analysis method comprises the following steps: after 50. Mu.L of the properly diluted fermentation broth and 150. Mu.L of pNP-. Alpha. -L-arabinofuranoside substrate in pH 4.8 acetic acid-sodium acetate buffer were uniformly mixed, the reaction was stopped by adding 50. Mu.L of 10% sodium carbonate after 30min reaction at 50 ℃. The absorbance of the product was read at a wavelength of 420 nm.
One enzyme activity unit of α -L-arabinofuranosidase is defined as the amount of enzyme that releases 1 μmol 4-nitropheny per minute under standard conditions.
As shown in FIG. 8, compared with the Trichoderma reesei QM9414, the enzyme activity of the arabinofuranosidase in the Trichoderma reesei Delta4 CelOExyr1 engineering bacteria fermentation broth is obviously increased, and the enzyme activity reaches 8.06U/mL after 96 hours of culture, which is 8-9 times that of the control strain. This is in agreement with the upregulation of the expression of alpha-L-arabinofuranosidase ABF1 in the above mass spectrometry results.
Example 8: analysis of the transcriptional level of the alpha-L-arabinofuranosidase encoding Gene abf1 in the Delta4 CelOExyr1 engineering bacterium
The culture, total RNA extraction and reverse transcription of Trichoderma reesei Delta4 celOExyr1 engineering and control strains Trichoderma reesei QM9414 were the same as in example 6.
1 Μl of the reverse transcription product was used for subsequent qRT-PCR amplification, and qRT-PCR analysis was performed according to TaKaRa SYBR Premix Ex Taq TM kit, with an amplification system of 20 μl, actin gene as an internal reference gene, and experimental procedures were described in the specification.
Wherein, qRT-PCR primers of the internal reference gene actin are as follows:
actin-F：5’-TGAGAGCGGTGGTATCCACG-3’，
actin-R：5’-GGTACCACCAGACATGACAATGTTG-3’，
the qRT-PCR primer of the alpha-L-arabinofuranosidase encoding gene abf is as follows:
abf1-F：5’-GCCAGTCCATACGACCAACA-3’，
abf1-R：5’-GATGATGGGCACGACGAGAT-3’，
The qRT-PCR reaction system is as follows: 1. Mu.L of reverse transcription product, 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 10. Mu.L of 2 XSYBR Green q RT-PCR Mix, and 20. Mu.L in total of ddH 2 O8. Mu.L;
the qRT-PCR reaction procedure was as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30sec, annealing at 58℃for 30sec, elongation at 72℃for 1min for 30sec, 30 cycles; finally, the extension is carried out for 5min at 72 ℃.
As shown in FIG. 9, the expression level of abf gene of Trichoderma reesei Delta4 celOExyr1 engineering bacteria is higher than that of Trichoderma reesei QM9414, which shows that the expression deletion of four main cellulases and the over-expression of transcription activator in Trichoderma reesei Delta4 celOExyr engineering bacteria realize the high-level expression of abf gene. This is consistent with the result of a significant increase in the arabinofuranosidase ABF1 protein content and enzyme activity level in the extracellular fermentation broth.
Claims (7)
1. The Trichoderma reesei engineering bacterium for producing alpha-L-arabinofuranosidase is characterized in that four cellulase genes cel5a, cel7b, cel6a and cel7a in the Trichoderma reesei engineering bacterium are knocked out and inactivated, and a transcription activator coding gene xyr1 is inserted, and the transcription activator coding gene xyr is expressed under the drive of a strong promoter P tcu1; the original strain of the Trichoderma reesei engineering bacteria is Trichoderma reesei QM9414 with a deposit number of ATCC 26921.
2. The construction method of Trichoderma reesei engineering bacteria producing alpha-L-arabinofuranosidase as claimed in claim 1, characterized in that Trichoderma reesei QM9414 is used as an original strain, homologous recombination technology is adopted to sequentially knock out four cellulase genes cel5a, cel7b, cel6a and cel7a in the original strain, so as to inactivate the genes, then a transcription activator coding gene xyr1 is inserted, xyr1 gene is expressed under the drive of a strong promoter P tcu1, so that xyr1 gene is overexpressed, pyr4 gene is used as a screening marker gene, and screening and verification are carried out to obtain the recombinant strain.
3. The construction method of Trichoderma reesei engineering bacteria producing alpha-L-arabinofuranosidase as claimed in claim 2, which is characterized by comprising the following steps:
(1) Taking Trichoderma reesei QM9414 genome DNA as a template, performing PCR amplification to obtain upstream and downstream homology arms of a cel5a gene, respectively inserting the homology arms into pDonorpyr vectors, constructing a knockout vector pDonorcel a on two sides of a pyr4 gene expression cassette in the pDonorpyr vectors, transforming the knockout vector into Trichoderma reesei QM 9414-delta pyr4, screening after homologous recombination to obtain a cel5a gene knockout strain, and marking the cel5a gene knockout strain as Trichoderma reesei delta cel5a;
(2) Taking Trichoderma reesei QM9414 genome DNA as a template, performing PCR amplification to obtain upstream and downstream homology arms of a cel7b gene, respectively inserting the upstream and downstream homology arms into pDonorpyr vectors, constructing a knockout vector pDonorcel b on two sides of a pyr4 gene expression cassette in the pDonorpyr vectors, transforming the knockout vector into Trichoderma reesei Deltacel 5a in the step (1), screening after homologous recombination to obtain cel5a and cel7b gene knockout strains, and marking the cell 5a and the cel7b gene knockout strains as Trichoderma reesei Deltacel 5a-cel7b;
(3) Taking Trichoderma reesei QM9414 genome DNA as a template, performing PCR amplification to obtain upstream and downstream homology arms of a cel6a gene, respectively inserting the homology arms into pMDpyr vectors, constructing knockout vectors pMDcel a on two sides of pyr4 gene expression cassettes in pMDpyr vectors, transforming the knockout vectors into Trichoderma reesei Deltacel 5a-cel7b in the step (2), screening after homologous recombination to obtain cel5a, cel7b and cel6a gene knockout strains, and marking the cell 5a, cel7b and cel6a gene knockout strains as Trichoderma reesei Deltacel 5a-cel7b-cel6a;
(4) Constructing a knockout vector pUCcbh pyr4 of a cel7a gene, transforming the knockout vector into trichoderma reesei delta cel5a-cel7b-cel6a in the step (3), and screening after homologous recombination to obtain cel5a, cel7b, cel6a and cel7a gene knockout strains, namely trichoderma reesei delta 4cel;
(5) The Trichoderma reesei QM9414 genome DNA is used as a template, xyr genes are obtained through PCR amplification, inserted into a vector pMDP tcu1-TtrpC and positioned between a promoter tcu fragment and a terminator TrpC fragment, an over-expression vector pMDOExyr1 is constructed, the over-expression vector is transformed into Trichoderma reesei delta 4cel in the step (4), after transformation, cell 5a, cell 7b, cell 6a and cell 7a gene knockout and xyr1 gene over-expression strains are obtained through screening, and the Trichoderma reesei strain is the Trichoderma reesei engineering strain for producing alpha-L-arabinofuranosidase and is named Trichoderma reesei delta 4celOExyr1.
4. The method of claim 3, wherein said transformation employs PEG-mediated protoplast transformation.
5. Use of the trichoderma reesei engineering bacteria according to claim 1 for producing alpha-L-arabinofuranosidase, xyloglucanase and/or endo-cellulose.
6. The use according to claim 5, comprising the steps of:
Inoculating the Trichoderma reesei engineering bacteria of claim 1 to MA liquid culture medium containing 1% glycerol and 0.2% peptone, culturing at 28+ -2deg.C and 180-200rpm for 24-36h, collecting mycelium, transferring to MA liquid culture medium containing 2% lactose, and continuously culturing for 96-120h to obtain fermentation broth.
7. The use according to claim 6, wherein the MA liquid medium comprises the following components: adding Na2HPO4·12H2O 17.907g、(NH4)2SO41.4 g、KH2PO42.0 g、 urea 0.3g and Tween-80.5 mL into each 1L culture medium, regulating pH to 5.0 with anhydrous citric acid, and adding MgSO 4 with final concentration of 0.6g/L, caCl 2 with final concentration of 0.4g/L and microelements before use;
Wherein 1000 times of trace elements are 5.0mg/L FeSO4·7H2O,1.6mg/L MnSO4·H2O,1.4mg/L ZnSO4·7H2O,2.0mg/L CoCl2·2H2O.
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Citations (4)
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|---|---|---|---|---|
| CN104561002A (en) * | 2015-01-08 | 2015-04-29 | 山东大学 | Copper ion permease gene promoter Ptcu1 in trichoderma reesei and application |
| CA2981966A1 (en) * | 2015-04-23 | 2016-10-27 | IFP Energies Nouvelles | Inducible trichoderma reesei promoters |
| JP2017201902A (en) * | 2016-05-10 | 2017-11-16 | 国立大学法人信州大学 | Method for producing cellulosic biomass-degrading enzyme |
| CN111117986A (en) * | 2020-01-16 | 2020-05-08 | 南京林业大学 | Encoding gene of calcium-dependent heat-resistant α -L-arabinofuranosidase, preparation technology and application |
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| CN104561002A (en) * | 2015-01-08 | 2015-04-29 | 山东大学 | Copper ion permease gene promoter Ptcu1 in trichoderma reesei and application |
| CA2981966A1 (en) * | 2015-04-23 | 2016-10-27 | IFP Energies Nouvelles | Inducible trichoderma reesei promoters |
| JP2017201902A (en) * | 2016-05-10 | 2017-11-16 | 国立大学法人信州大学 | Method for producing cellulosic biomass-degrading enzyme |
| CN111117986A (en) * | 2020-01-16 | 2020-05-08 | 南京林业大学 | Encoding gene of calcium-dependent heat-resistant α -L-arabinofuranosidase, preparation technology and application |
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| Towards an Understanding of Oxidative Damage in an α-L-Arabinofuranosidase of Trichoderma reesei: a Molecular Dynamics Approach;Jesus D Castaño;Appl Biochem Biotechnol;第193卷(第10期);全文 * |
| 阿拉伯呋喃糖苷酶的重组表达及其发酵工艺优化;李鸿雁;食品与发酵工业;第46卷(第15期);全文 * |
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