CN115109735A

CN115109735A - Trichoderma reesei engineering bacterium capable of producing alpha-L-arabinofuranosidase at high yield and construction method and application thereof

Info

Publication number: CN115109735A
Application number: CN202110299855.9A
Authority: CN
Inventors: 刘巍峰; 张伟欣; 郭俊琪; 李春艳; 孟祥锋
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2021-03-22
Filing date: 2021-03-22
Publication date: 2022-09-27
Anticipated expiration: 2041-03-22
Also published as: CN115109735B

Abstract

The invention relates to a trichoderma reesei engineering bacterium capable of highly producing alpha-L-arabinofuranosidase and a construction method and application thereof. Four cellulase genes cel5a, cel7b, cel6a and cel7a in the trichoderma reesei engineering bacteria for high yield of the alpha-L-arabinofuranosidase are knocked out and inactivated, and a transcription activation factor coding gene xyr1 is inserted; transcription activator coding gene xyr1 in strong promoter P _tcu1 Driving the expression. The Trichoderma reesei engineering bacteria constructed by the invention can be stably passaged, and can obviously improve Trichoderma reesei alpha-L-arabinofuranoseThe yield of glycosidase and the enzyme activity of the alpha-L-arabinofuranosidase of the trichoderma reesei engineering bacteria are about 8-9 times of those of the original strain, and the application of the trichoderma reesei in degrading hemicellulose substances rich in arabinose residues is facilitated. In addition, the Trichoderma reesei engineering bacteria constructed by the invention can also be used for producing xyloglucanase and cellulose endonuclease.

Description

Trichoderma reesei engineering bacterium capable of highly producing alpha-L-arabinofuranosidase and construction method and application thereof

Technical Field

The invention relates to a trichoderma reesei engineering bacterium capable of highly producing alpha-L-arabinofuranosidase and a construction method and application thereof, belonging to the technical field of biological engineering.

Background

The degradable cellulose and hemicellulose substances are main components of biomass resources, and the development and 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 the nature, can utilize cellulose and hemicellulose as a carbon source and an energy source, and play an important role in the carbon circulation process. The representative strain Trichoderma reesei (Trichoderma reesei) can produce various cellulases and hemicellulases, and is widely applied to the fields of energy, textile, paper pulp and papermaking and the like.

Xylan and araban are main components of plant hemicellulose, and the high-efficiency degradation and utilization of the xylan and araban have important economic value. The degradation of xylan and arabinan requires the involvement of a number of degrading enzymes, among which alpha-L-arabinofuranosidase (alpha-L-arabinanosidase, EC3.2.1.55), capable of hydrolyzing the monomeric or oligomeric arabinose side branches linked to the xylan/arabinan backbone in alpha-1, 3, alpha-L, 2 or alpha-1, 5 linkages, is necessary for the complete hydrolysis of xylan/arabinan. Besides the energy field, the alpha-L-arabinofuranosidase is widely applied to the food and feed processing field, and the market demand is large.

The genome of trichoderma reesei contains four alpha-L-arabinofuranosidase encoding genes which are respectively named as abf1, abf2, abf3 and bxl1, wherein abf1 and abf2 encode two main alpha-L-arabinofuranosidases. Studies have shown that abf1 and abf2 are expressed at the transcriptional level under the carbon source of sophorose or lactose, but no significant enzyme activity could be detected in the extracellular medium due to the low levels. To date, Trichoderma reesei-producing strains of alpha-L-arabinofuranosidase having high levels of extracellular secretion have not been obtained, which greatly limits the use of Trichoderma reesei in the efficient degradation of hemicellulose-like materials containing arabinose residues.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a trichoderma reesei engineering bacterium for high yield of alpha-L-arabinofuranosidase, a construction method and application thereof, wherein the extracellular alpha-L-arabinofuranosidase enzyme activity of the trichoderma reesei engineering bacterium is 8-9 times of that of an original strain.

The technical scheme of the invention is as follows:

the Trichoderma reesei engineering strain capable of highly producing alpha-L-arabinofuranosidase is characterized in that four cellulase genes cel5a, cel7b, cel6a and cel7a in the Trichoderma reesei engineering strain are knocked out and inactivated, and a transcription activation factor coding gene xyr1 is inserted.

Preferably, according to the 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 optimization of the invention, the enzyme activity of the alpha-L-arabinofuranosidase of the Trichoderma reesei engineering strain is 8-9 times that of the original strain.

The related genes are known sequence genes, wherein the JGI protein ID of the protein coded by the gene cel5a is Tr120312, the JGI protein ID of the protein coded by the gene cel7b is Tr122081, the JGI protein ID of the protein coded by the gene cel6a is Tr72567, the JGI protein ID of the protein coded by the gene cel7a is Tr123989, and the JGI protein ID of the protein coded by the gene xyr1 is Tr 122208.

The construction method of the trichoderma reesei engineering bacteria for high yield of the alpha-L-arabinofuranosidase comprises the steps of sequentially knocking out four cellulase genes cel5a, cel7b, cel6a and cel7a in a starting strain by using trichoderma reesei QM9414 as the starting strain and adopting a homologous recombination technology, inactivating the genes, inserting a transcription activation factor coding gene xyr1, inserting a xyr1 gene in a strong promoter P _tcu1 The expression is driven to cause xyr1 gene to be over-expressed, and pyr4 gene is used as a screening marker gene to obtain the gene after screening and verification.

In the invention, the knocking-out sequence of the four cellulase genes cel5a, cel7b, cel6a and cel7a is not specially required.

The construction method of the trichoderma reesei engineering bacteria for high yield of the alpha-L-arabinofuranosidase specifically comprises the following steps:

(1) performing PCR amplification by taking Trichoderma reesei QM9414 genome DNA as a template to obtain upstream and downstream homologous arms of a cel5a gene, respectively inserting the upstream and downstream homologous arms into a pDOORpyr 4 vector, locating at two sides of a pyr4 gene expression box in a pDOORpyr 4 vector, constructing a knockout vector pDOORcel 5a, transforming the knockout vector into Trichoderma reesei QM 9414-delta pyr4, and screening after homologous recombination to obtain a cel5a gene knockout strain, which is recorded as Trichoderma reesei delta cel5 a;

(2) performing PCR amplification by taking Trichoderma reesei QM9414 genomic DNA as a template to obtain upstream and downstream homologous arms of a cel7b gene, respectively inserting the upstream and downstream homologous arms into a pDOORpyr 4 vector, locating at two sides of a pyr4 gene expression box in a pDOORpyr 4 vector, constructing a knockout vector pDOORcel 7b, transforming the knockout vector into Trichoderma reesei delta cel5a in the step (1), and screening to obtain cel5a and cel7b gene knockout strains which are marked as Trichoderma reesei delta cel5a-cel7b after homologous recombination;

(3) performing PCR amplification by taking Trichoderma reesei QM9414 genomic DNA as a template to obtain upstream and downstream homologous arms of the cel6a gene, respectively inserting the upstream and downstream homologous arms into a pMDpyr4 vector, locating at two sides of a pyr4 gene expression cassette in a pMDpyr4 vector, constructing a knockout vector pMDcel6a, transforming the knockout vector into Trichoderma reesei delta cel5a-cel7b in the step (2), and screening to obtain cel5a, cel7b and cel6a gene knockout strains after homologous recombination, wherein the strains are marked as Trichoderma reesei delta cel5a-cel7b-cel6 a;

(4) constructing a knockout vector pUCbh 1pyr4 of the cel7a gene, transforming the knockout vector into the trichoderma reesei delta cel5a-cel7b-cel6a in the step (3), and screening to obtain cel5a, cel7b, cel6a and cel7a gene knockout strains after homologous recombination, wherein the strains are marked as trichoderma reesei delta 4 cel;

(5) using Trichoderma reesei QM9414 genome DNA as a template, carrying out PCR amplification to obtain xyr1 gene, and inserting a vector pMDP _tcu1 -T _trpC And (3) constructing an over-expression vector pMDOExyr1 between a promoter tcu1 fragment and a terminator TrpC fragment, transforming the over-expression vector into the Trichoderma reesei delta 4cel obtained in the step (4), and screening after transformation to obtain cel5a, cel7b, cel6a and cel7a gene knockout and xyr1 gene over-expression strains, namely Trichoderma reesei engineering bacteria for high yield of alpha-L-arabinofuranosidase, which is recorded as Trichoderma reesei delta 4 OEcelxyr 1.

Preferably, according to the invention, the transformation is carried out by PEG-mediated protoplast transformation.

In the invention, the pyr4 gene is used as a screening marker gene to screen strains, uridine is added into a culture medium during screening, the strains containing the pyr4 gene can survive, and the strains without the pyr4 gene cannot survive; the pyr4 gene was deleted by 5' fluoroorotic acid, allowing the selection marker to be reused.

In the preparation method, the related pDOnorpyr4 vector, Trichoderma reesei QM 9414-delta pyr4 vector, pMDpyr4 vector, knockout vector pUCbh 1pyr4 vector and vector pMDP _tcu1 -T _trpC The construction methods of (A) are all the prior art.

The Trichoderma reesei engineering bacteria are applied to the production of alpha-L-arabinofuranosidase, xyloglucanase and/or cellulose endonuclease.

According to a preferred embodiment of the present invention, the application comprises the following steps:

inoculating the Trichoderma reesei engineering bacteria to an MA liquid culture medium containing 1% of glycerol and 0.2% of peptone, culturing at the temperature of 28 +/-2 ℃ and the rotation speed of 180-200rpm for 24-36h, collecting mycelia, transferring the mycelia into the MA liquid culture medium containing 2% of lactose, and continuously culturing for 96-120h to obtain fermentation liquor.

Further preferably, the MA liquid medium has the following composition: adding Na into each 1L of culture medium ₂ HPO ₄ ·12H ₂ O 17.907g、(NH ₄ ) ₂ SO ₄ 1.4g、KH ₂ PO ₄ 2.0g, urea 0.3g, Tween-800.5 mL, adjusting pH to 5.0 with anhydrous citric acid, and adding MgSO with final concentration of 0.6g/L before use ₄ The final concentration of CaCl is 0.4g/L ₂ And trace elements;

wherein the microelement (1000X) component is 5.0mg/L FeSO ₄ ·7H ₂ O，1.6mg/L MnSO ₄ ·H ₂ O，1.4mg/L ZnSO ₄ ·7H ₂ O，2.0mg/L CoCl ₂ ·2H ₂ O。

The trichoderma reesei engineering bacteria are applied to degradation of hemicellulose substances rich in arabinose residues.

The invention has the technical characteristics and beneficial effects that:

1. the invention takes Trichoderma reesei QM9414 as an original strain, knockouts four main cellulase genes cel5a (JGI protein ID Tr120312), cel7b (JGI protein ID Tr122081), cel6a (JGI protein ID Tr72567) and cel7a (JGI protein ID Tr123989) in a Trichoderma reesei QM9414 genome, and simultaneously over-expresses a transcription activation factor coding gene xyr1(JGI protein ID Tr122208) of cellulase and hemicellulase genes at a high level to construct the Trichoderma reesei delta 4 OEcelxyr 1 engineering bacteria with high alpha-L-arabinofuranosidase yield. The transcriptional activator XYR1 regulates most cellulase coding genes and hemicellulase coding genes of trichoderma reesei, and the experimental result of the invention can conjecture that the transcriptional activator XYR1 has a positive regulation effect on the transcriptional expression of the alpha-L-arabinofuranosidase coding genes, and the deletion of the four main cellulase genes enables the regulation of the transcriptional activator XYR1 to act on the alpha-L-arabinofuranosidase coding genes more, so that the transcriptional expression level of the alpha-L-arabinofuranosidase is improved, and the aim of high yield of the alpha-L-arabinofuranosidase is fulfilled.

2. The Trichoderma reesei delta 4celOExyr1 engineering bacteria 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 Trichoderma reesei delta 4celOExyr1 alpha-L-arabinofuranosidase of the engineering bacteria is about 8-9 times of that of the original strain, and the application of Trichoderma reesei in degrading hemicellulose substances rich in arabinose residues is facilitated. In addition, the Trichoderma reesei delta 4celOExyr1 engineering bacteria constructed by the invention can also be used for producing xyloglucanase and cellulose endonuclease.

Drawings

FIG. 1 is a schematic diagram of cel5a gene knockout vector pDOORcel 5 a;

FIG. 2 is a schematic diagram of cel7b gene knock-out vector pDOORcel 7 b;

FIG. 3 is a schematic representation of cel6a knockout vector pMDcel6 a;

FIG. 4 is a schematic representation of cel7a knock-out vector pUCbh 1pyr 4;

FIG. 5 is a schematic view of xyr1 gene overexpression vector pMDOExyr 1;

FIG. 6 shows relative transcription levels of genes cel5a (FIG. A), cel7B (FIG. B), cel6a (FIG. C), cel7a (FIG. D) and xyr1 (FIG. E) in the Δ 4cel OExyr1 strain and the starting strain QM 9414;

FIG. 7 is a SDS-PAGE electrophoresis chart of the fermentation liquor of the strain Δ 4celOExyr1 and the starting strain QM 9414;

FIG. 8 is a graph showing the enzyme activity analysis of the α -L-arabinofuranosidase in fermentation broth of Δ 4celOExyr1 strain and the starting strain QM 9414;

FIG. 9 shows relative transcription levels of α -L-arabinofuranosidase encoding gene abf1 from Δ 4celOExyr1 strain and starting strain QM 9414.

Detailed Description

The invention will be further illustrated with reference to the following examples and figures, without however restricting the scope of the invention thereto. The drugs and reagents mentioned in the examples are all common commercial products unless otherwise specified; the experimental procedures referred to in the examples are those conventional in the art unless otherwise specified. The primers in the examples were synthesized by Biotechnology, Inc., of Okagaku, Beijing.

The starting strain Trichoderma reesei (Trichoderma reesei) QM9414 was purchased from American type culture Collection and deposited under accession number ATCC 26921.

Example 1: delta cel5a strain construction

1) construction of cel5a gene knockout vector

Fungal DNA extraction kit (e.z.n.a) was used. ^TM Fungal DNA Mini Kit, OMEGA) to extract genome DNA of Trichoderma reesei QM9414, taking the genome DNA as a template, designing primers cel5a-upF/cel5a-upR, and obtaining an upstream homologous arm fragment of cel5a gene with about 1.5kb through PCR amplification; designing a primer cel5a-downF/cel5a-downR, and obtaining a cel5a gene downstream homology arm fragment with about 1.5kb through PCR amplification; and using cel5aDR-F/cel5aDR-R as an upstream and downstream primer to carry out PCR amplification to obtain a fragment (named cel5a-DR) with about 0.5kb at the 3' end of the upstream homology arm fragment.

Wherein, the sequences of the primers are 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 μ M of the forward primer 2 μ L, 10 μ M of the reverse primer 2 μ L, genomic DNA 10ng, 2.5mM dNTP mix 5 μ L, 5 XStart Fastpfu buffer 10 μ L, TransStartFastpfu DNA polymerase 1 μ L, ddH ₂ O is complemented to 50 mu L;

the PCR amplification procedure was as follows: pre-denaturation at 98 ℃ for 5 min; denaturation at 95 ℃ for 30sec, annealing at 58 ℃ for 30sec, extension at 72 ℃ for 3min, and 30 cycles; final extension at 72 deg.C for 10 min; after completion of PCR, electrophoresis was performed on a 1% agarose gel, and the DNA fragment was recovered.

A cel5a-DR fragment and a cel5a gene downstream homologous arm fragment are connected in a fusion mode through a fusion PCR method, the fusion fragment and the cel5a gene upstream homologous arm fragment are respectively connected to a pDroppyr 4 vector under the action of BP clonase and are respectively positioned on two sides of a pyr4 gene expression box in the pDroppyr 4 vector, and a cel5a gene knockout vector pDropdorcel 5a is constructed. Among them, pyr4 is a commonly used uridine auxotrophy selection tag.

Wherein, the construction method of pDOnorpyr4 vector is described in Two major surface transmitters from Trichoderma reesei and the role in indexing of cell biosyntheses.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 is as follows: cel5a-DR fragment 20ng, downstream homology arm fragment 20ng, 2.5mM dNTP mix 8. mu.L, 5 XStart Fastpfu buffer 20. mu.L, Transstart Fastpfu DNA polymerase 1. mu.L, 10. mu.M upstream primer cel5 aDR-F5. mu.L, 10. mu.M downstream primer cel5a-downR 5. mu.L, ddH ₂ O is complemented to 100 mu L;

the fusion PCR amplification procedure was as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30sec, annealing at 53 ℃ for 6min, extension at 72 ℃ for 4min, 28 cycles; final extension at 72 deg.C for 10 min; after completion of PCR, 1% agarose gel electrophoresis was performed, and the DNA fragment was recovered.

2) Preparation of Trichoderma reesei protoplast

Prepared at a concentration of 5X 10 ⁷ ～5×10 ⁸ Inoculating 1mL of spore suspension of Trichoderma reesei QM 9414-delta pyr4 into 50mL of hypha growth culture medium, and culturing at 28 ℃ and 160rpm for 16h to obtain hypha culture; centrifuging the mycelium culture at 4000rpm for 5min, washing and precipitating for three times with 0.7M NaCl solution, adding a cell wall lyase solution 25mL each time, resuspending, carrying out enzymolysis at 30 ℃ and 60rpm for 2.5-3 h, observing by a microscope, filtering by using a G2 funnel when a large amount of free protoplasts appear, centrifuging the filtrate at 4 ℃ and 3800rpm for 5min to collect the protoplasts, washing the collected protoplasts twice with 15mL of precooled STC solution, and then adding the precooled STC solution for resuspension to ensure that the concentration of the protoplasts is 5 multiplied by 10 ⁷ ～5×10 ⁸ Per mL; all the above operations were performed on ice.

Wherein, the Trichoderma reesei QM 9414-delta pyr4 is to knock out pyr4 gene in Trichoderma reesei QM9414, the strain construction method refers to the documents A novel transcriptional regulator RXE1 models the expression vector promoter XYR1 and cell gene expression in Trichoderma reesei 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 components of the hypha growth culture medium are as follows (all in percentage by mass): 2% glucose, 0.5% (NH) ₄ ) ₂ SO ₄ 、1.5％KH ₂ PO ₄ 1% peptone, adjusted to pH 5.5 with NaOH, added to final concentration 0.6g/L MgSO ₄ The final concentration of CaCl is 0.4g/L ₂ And trace elements (final concentration 1 ×, mother liquor 1000 ×) and 10mM uridine were added; 1000 times microelement mother liquor formula: FeSO ₄ ·7H ₂ O 0.5g，MnSO ₄ ·H ₂ O 0.16g，ZnSO ₄ ·7H ₂ O 0.14g，CoC _l2 ·2H ₂ O0.2 g from ddH ₂ O, fixing the volume to 100mL, and filtering and sterilizing;

the components of the cell wall lytic enzyme solution are as follows: 0.24g cell wall lyase dissolved in 5.7mL 0.7M NaCl solution, filter sterilization, adding 0.3mL pH 5.8 PBS buffer solution;

the composition of the STC solution is as follows: 0.7M D-sorbitol, 0.05M CaCl ₂ ，10mM Tris-HCl，pH 7.5。

3) Transformation of knock-out vector pDONORCEL5a, regeneration of transformants, screening verification and removal of screening marker

Taking 150 mu L of the knockout vector pDONORCEL5a obtained in the step 1), carrying out enzyme digestion linearization by using I-SceI, purifying and recovering fragments, uniformly mixing with 150 mu L of the trichoderma reesei QM 9414-delta pyr4 protoplast prepared in the step 2), adding the mixture into the bottom of a 10mL centrifuge tube, carrying out ice bath for 20min, dropwise adding 1.5mL of precooled PTC solution, carrying out 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 was spread on a regeneration medium plate containing no uridine and cultured at 28 ℃ for 5-7 days.

Wherein, the PTC solution comprises the following components: the mass to volume ratio (g/mL) was 60% PEG 4000 (polyethylene glycol 4000), 10mM Tris-HCl, 10mM CaCl ₂ ，pH 7.5；

The regeneration medium comprises the following components (all in percentage by mass): 2% glucose, 0.5% (NH) ₄ ) ₂ SO ₄ 、1.5％KH ₂ PO ₄ 1% peptone, 1M D-sorbitol, 1.5% agar powder, adjusted to pH 5.5 with NaOH, added to MgSO 0.6g/L final concentration before use ₄ The final concentration was 0.4g/LCaCl ₂ And trace elements (final concentration 1 ×, mother liquor 1000 ×) were added. The formula of 1000 times of the microelement mother liquor is the same as the formula.

Transferring the colony growing on the regeneration medium plate to a screening medium plate without uridine, and culturing at 28 ℃ for 5-7 days to obtain a transformant.

Wherein the screening culture medium comprises the following components (in percentage by mass): 2% glucose, 0.5% (NH) ₄ ) ₂ SO ₄ 、1.5％KH ₂ PO ₄ 1.5% agar powder, adjusting pH to 5.5 with NaOH, adding MgSO 0.6g/L to give final concentration before use ₄ The final concentration of CaCl is 0.4g/L ₂ And trace elements (final concentration 1 ×, mother liquor 1000 ×) were added. The formula of 1000 times of the microelement mother liquor is the same as the formula.

Extracting the obtained transformant genome DNA, taking the genome DNA as a template, and performing upstream and downstream homologous arm sequence anchoring verification by adopting a PCR technology, wherein upstream homologous arm sequence anchoring verification primers are cel5a-YZ-F1 and An-R, and the length of a target band is about 1.5 kb; the verification primer of the downstream homology arm sequence anchoring is An-F/cel5a-YZ-R2, and the length of a target band is about 1.5 kb; the primer sequences are 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 transformants with correctly anchored upstream and downstream homology arms by adopting a single spore isolation method, and carrying out gene verification in cel5a by adopting a PCR (polymerase chain reaction) technology and using cel5a-YZ-F3 and cel5a-YZ-R3 primers, wherein no target band exists, namely the cel5a gene is successfully knocked out, and the delta cel5a strain is successfully constructed.

Wherein the primer sequences are as follows:

cel5a-YZ-F3:5’-GTACCCTTGTTTCCTGGTGTTGCT-3’，

cel5a-YZ-R3:5’-TGTTATTTGTAGTACATTCGGCGTGA-3’。

the above-mentioned verified PCR reaction systems were 20. mu.L, and the details of the reaction systems and reaction conditions were described in GenStar2 XTaq PCR StarMix (available from Wuhan Feiyu science Co., Ltd.).

The schematic diagram of the cel5a gene knockout vector pDOORcel 5a is shown in figure 1, after the upstream homology arm sequence and the downstream homology arm sequence of the cel5a gene are contained in the knockout vector pDOORcel 5a and are transformed into trichoderma reesei QM 9414-delta pyr4 protoplasts, the upstream homology arm and the downstream homology arm are subjected to homologous recombination with the upstream and downstream regions of the cel5a gene in a genome, a uridine-deficient screening tag pyr4 in the knockout vector pDOORcel 5a is integrated on the trichoderma reesei genome to replace the cel5a gene, and thus the aim of knocking out the cel5a gene is achieved.

Spores of the Δ cel5a strain were plated on a screening medium plate containing 5-Fluoroorotic acid (5-fluorogenic acid,5-FOA) at a final concentration of 1.2mg/mL to perform reverse screening, the screening tag pyr4 was recombined with a recombinase of Trichoderma reesei itself, and a transformant that could grow on the plate containing 5-FOA was successfully removed by screening, whereby a uridine utilization defective strain Δ cel5a was obtained.

Example 2: construction of a.DELTA.cel 5a-cel7b Strain

1) construction of cel7b gene knockout vector

Similar to the method of example 1, the primer cel7b-upF/cel7b-upR is designed by using Trichoderma reesei QM9414 genome DNA as a template, and the upstream homologous arm fragment of the cel7b gene is obtained by PCR amplification, and is about 1.5 kb; designing a primer cel7b-downF/cel7b-downR, and obtaining a cel7b gene downstream homology arm fragment with about 1.5kb through PCR amplification; and using cel7bDR-F/cel7bDR-R as an upstream primer and a downstream primer to obtain a fragment of about 0.5kb at the 3' end of the upstream homology arm fragment (named cel7 b-DR).

Wherein, the sequences of the primers are 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’。

a cel7b-DR fragment and a cel7b gene downstream homologous arm fragment are connected in a fusion mode through a fusion PCR method, the fusion fragment and the cel7a gene upstream homologous arm fragment are respectively connected to a pDroppyr 4 vector under the action of BP clonase and are respectively positioned on two sides of a pDroppyr 4 vector pyr4 gene expression box, and a cel7b gene knockout vector pDropdorcel 7b is constructed.

The amplification system and the amplification procedure of the above PCR and fusion PCR were the same as in example 1; wherein the upstream primer in the fusion PCR amplification system is cel7bDR-F, and the downstream primer is cel7 b-downR.

2) Preparation of Trichoderma reesei delta cel5a protoplast

The uridine prepared in example 1 was used to prepare a trichoderma reesei Δ cel5a protoplast using the deficient strain Δ cel5a by the method for preparing protoplasts according to step 2) of example 1.

3) Transformation of knock-out vector pDONORCEL7b, regeneration of transformant, screening verification and removal of screening tag

Taking 150 mu L of the knockout vector pDOORcel 7b obtained in the step 1), carrying out enzyme digestion linearization by using I-SceI, purifying and recovering fragments, mixing with 150 mu L of the Trichoderma reesei delta cel5a protoplast prepared in the step 2), preparing a transformant according to the methods of transformation of the knockout vector pDOORcel 5a and regeneration of the transformant in the step 3) of the embodiment 1, and carrying out screening verification.

Extracting the obtained transformant genome DNA, taking the genome DNA as a template, and performing upstream and downstream homology arm sequence anchoring verification by adopting a PCR (polymerase chain reaction) technology, wherein upstream homology arm sequence anchoring verification primers are cel7b-YZ-F1 and An-R, and the length of a target strip is about 1.5 kb; the verification primers of the downstream homology arm sequence anchor are An-F and cel7b-YZ-R2, and the length of a target band is about 1.5 kb; the primer sequences are as follows:

cel7b-YZ-F1:5’-CCGCTCATCACACTGCTACGCCTAC-3’，

An-R:5’-TGAAGCAGAAATAGGGTGAATGATA-3’；

An-F:5’-TATTTTTGATTTGGCATTTGCTTTT-3’，

cel7b-YZ-R2:5’-TCTTCACGCTGCTCAAGGCTTTCAT-3’。

a transformant with correctly anchored upstream and downstream homologous arms is purified by adopting a single spore separation method, and cel7b internal gene verification is carried out by using cel7b-YZ-F3 and cel7b-YZ-R3 primers, so that no target band exists, namely the cel7b gene is successfully knocked out, and the delta cel5a-cel7b strain is successfully constructed.

Wherein the primer sequences are as follows:

cel7b-YZ-F3:5’-CTCGGCTGTATCTCCTGGACTCTGA-3’，

cel7b-YZ-R3:5’-CTGGCTGTTGTCGTTCCAAATGCT-3’。

the above-mentioned verified PCR reaction system is 20. mu.L, and the specific reaction system and reaction conditions are described in GenStar2 XTaq PCR StarMix (available from Wuhan Feiyu technology Co., Ltd.).

A schematic diagram of a cel7b gene knockout vector pDONORCEL7b is shown in figure 2, the knockout vector pDORCEL 7b contains an upstream homology arm sequence and a downstream homology arm sequence of a cel7b gene, after the vector is transformed into a trichoderma reesei delta cel5a protoplast, the upstream and downstream homology arms and an upstream and downstream region of a cel7b gene in a trichoderma reesei delta cel5a genome undergo homologous recombination, a uridine-deficient screening tag pyr4 in the knockout vector pDORCEL 7b is integrated onto the trichoderma reesei delta cel5a genome to replace the cel7b gene, and therefore the purpose of knocking out the cel7b gene is achieved.

Spores of the delta cel5a-cel7b strain are coated on a 5-FOA screening medium plate with the final concentration of 1.2mg/mL for reverse screening, a screening tag pyr4 is recombined by using recombinase of trichoderma reesei, transformants which can grow on the plate with the 5-FOA are screened and successfully removed, and accordingly the uridine utilization defective strain delta cel5a-cel7b is obtained.

Example 3: construction of delta cel5a-cel7b-cel6a Strain

1) construction of cel6a gene knockout vector

The method comprises the steps of (1) designing primers cel6a-upF/cel6a-upR by using Trichoderma reesei QM9414 genome DNA as a template, and obtaining an upstream homologous arm fragment of cel6a gene with about 2kb through PCR amplification; designing a primer cel6a-downF/cel6a-downR, and obtaining a cel6a gene downstream homology arm fragment with about 1.8kb through PCR amplification; and using cel6aDR-F/cel6aDR-R as an upstream primer and a downstream primer to carry out PCR amplification to obtain a fragment (named cel6a-DR) of about 0.4kb at the 5' end of the pyr4 gene expression cassette.

Wherein, the sequences of the primers are 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 PCR amplification system and the PCR amplification procedure were the same as in example 1.

The expression cassette of the pyr4 gene is connected into a pMD19T vector (Takara) to construct a pMDpyr4 vector, the upstream homologous arm segment and the cel6a-DR are sequentially connected into the upstream and downstream of the pyr4 gene expression cassette in the pMDpyr4 vector, and finally the downstream homologous arm segment is connected into the downstream of the cel6a-DR to construct a cel6a gene knockout vector pMDcel6 a.

2) Preparation of protoplast of strain delta cel5a-cel7b

The uridine prepared in example 2 was used to prepare a trichoderma reesei Δ cel5a-cel7b protoplast using the deficient strain Δ cel5a-cel7b by the method for preparing the protoplast according to step 2) of example 1.

3) Transformation of knockout vector pMDcel6a, regeneration of transformant, screening verification and removal of screening tag

Taking 150 mu L of the knockout vector pMDcel6a obtained in the step 1), carrying out enzyme digestion linearization, purifying and recovering fragments, mixing the fragments with 150 mu L of the trichoderma reesei delta cel5a-cel7b protoplast prepared in the step 2), preparing a transformant according to the methods of transformation of the knockout vector pDOORcel 5a and regeneration of the transformant in the step 3) of the embodiment 1, and carrying out screening verification.

Extracting the obtained transformant genome DNA, taking the genome DNA as a template, and performing upstream and downstream homology arm sequence anchoring verification by adopting a PCR (polymerase chain reaction) technology, wherein upstream homology arm sequence anchoring verification primers are cel6a-YZ-F1 and cel6a-YZ-R1, and the length of a target strip is about 2 kb; the verification primer of the downstream homology arm sequence anchor is cel6a-YZ-F2/cel6a-YZ-R2, and the length of a target band is about 1.8 kb; the primer sequences are 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 transformants with correctly anchored upstream and downstream homology arms by adopting a single spore isolation method, carrying out cel6a internal gene verification by adopting a PCR (polymerase chain reaction) technology and using cel6a-YZ-F3 and cel6a-YZ-R3 primers, wherein no target band exists, namely the cel6a gene is successfully knocked out, and the delta cel5a-cel7b-cel6a strain is successfully constructed.

Wherein the primer sequences are as follows:

cel6a-YZ-F3:5’-ACGACTATTACTCCCAGTGTCTTCCC-3’，

cel6a-YZ-R3:5’-CAGCGTCCAAATACATCGCAACAT-3’。

The schematic diagram of the cel6a gene knockout vector pMDcel6a is shown in FIG. 3, the knockout vector pMDcel6a contains an upstream homology arm sequence and a downstream homology arm sequence of a cel6a gene, after the upstream homology arm sequence and the downstream homology arm sequence are transformed into a Trichoderma reesei delta cel5a-cel7b protoplast, the upstream and downstream homology arms and an upstream and downstream region of the cel6a gene in a Trichoderma reesei delta cel5a-cel7b genome are subjected to homologous recombination, a uridine-deficient screening tag pyr4 in the knockout vector pMDcel6a is integrated onto a Trichoderma reesei delta cel5a-cel7b genome to replace a cel6a gene, and thus the aim of knocking out the cel6a gene is achieved.

Spores of the delta cel5a-cel7b-cel6a strain are coated on a 5-FOA screening medium plate with the final concentration of 1.2mg/mL for reverse screening, a screening tag pyr4 is recombined by using recombinase of trichoderma reesei, a transformant capable of growing on the plate containing the 5-FOA is screened and successfully removed, and thus the uridine utilization defective strain delta cel5a-cel7b-cel6a is obtained.

Example 4: construction of a.DELTA.cel 5a-cel7b-cel6a-cel7a (.DELTA.4cel) strain

1) construction of cel7a gene knockout vector

The construction method of the knockout vector pUCCbh1pyr4 of cel7a gene refers to literature, Deciting the effect of the differentiation sites on the differentiation, activity, and stability of cellular hydrolases I from Trichoderma reesei, Qi, Zhang W, Zhang F, Chen G, Liu W, applied Environ Microl.2014Jul; 3962-71, wherein the cbh1 gene in the reference and the cel7a gene in the invention are the same gene.

2) Preparation of protoplast of delta cel5a-cel7b-cel6a strain

The uridine prepared in example 3 was used to prepare a trichoderma reesei delta cel5a-cel7b-cel6a protoplast using the deficient strain delta cel5a-cel7b-cel6a by the method for preparing the protoplast according to step 2) of example 1.

3) Transformation of knockout vector pUCbh 1pyr4, regeneration of transformant, screening verification and removal of screening tag

Taking 150 mu L of the knockout vector pUCbh 1pyr4 obtained in the step 1), linearizing the vector, purifying and recovering fragments, uniformly mixing the fragment with 150 mu L of the trichoderma reesei delta cel5a-cel7b-cel6a protoplast prepared in the step 2), preparing a transformant according to the methods of transformation of the knockout vector pDOORcel 5a in the step 3) of the example 1 and regeneration of the transformant, and carrying out screening and verification.

Extracting the obtained transformant genome DNA, taking the genome DNA as a template, and performing upstream and downstream homologous arm sequence anchoring verification by adopting a PCR technology, wherein upstream homologous arm sequence anchoring verification primers are cel7a-YZ-F1 and An-R, and the length of a target strip is about 1.5 kb; the upstream homology arm sequence anchor verification primers are An-F and cel7a-YZ-R2, and the length of a target band is about 1.5 kb; the primer sequences are 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 transformants with correctly anchored upstream and downstream homology arms by adopting a single spore isolation method, and carrying out gene verification in cel7a by adopting a PCR technology and using cel7a-YZ-F3 and cel7a-YZ-R3 primers, wherein no target band exists, namely the cel7a gene is successfully knocked out, and the delta cel5a-cel7b-cel6a-cel7a strain is successfully constructed.

Wherein the primer sequences are as follows:

cel7a-YZ-F3:5’-ACTCAACAGACAGGCTCCGTGGTC-3’，

cel7a-YZ-R3:5’-AGATTCGACCTGAGCAGGGACACC-3’。

A schematic diagram of a cel7a gene knockout vector pUCbh 1pyr4 is shown in FIG. 4, after the knockout vector pUCbh 1pyr4 contains an upstream homology arm sequence and a downstream homology arm sequence of a cel7a gene, the upstream homology arm sequence and the downstream homology arm sequence are transformed into a Trichoderma reesei delta cel5a-cel7b-cel6a protoplast, homologous recombination occurs between the upstream homology arm and the downstream region of the cel7a gene in a Trichoderma reesei delta cel5a-cel7b-cel6a genome, a uridine-deficient screening tag pyr4 in the knockout vector pUCbh 1pyr4 is integrated on a Trichoderma reesei delta cel5a-cel7b-cel6a genome, and the cel7a gene is replaced, so that the goal of knockout of the cel7a gene is achieved.

Spores of the delta cel5a-cel7b-cel6a-cel7a strain are coated on a screening medium plate containing 5-FOA with the final concentration of 1.2mg/mL for reverse screening, a screening tag pyr4 is recombined by using recombinase of trichoderma reesei, a transformant capable of growing on the plate containing the 5-FOA is successfully removed by screening, and accordingly the uridine utilization defective strain delta cel5a-cel7b-cel6a-cel7a, which is abbreviated as delta 4cel, is obtained.

Example 5: construction of the.DELTA.4ceOExyr 1 Strain

1) Transcriptional activator xyr1 overexpression vector construction

Using plasmid pMDhph as template, designing primer hphF/hphR, obtaining hph gene expression box by PCR amplification, connecting the hph gene expression box to plasmid pMDP _tcu1 -T _trpC In the sequence 3' of TrpC, construct the vector pMDP _tcu1 -T _trpC -hph。

The genome DNA of Trichoderma reesei QM9414 is used as a template, primers xyr1-upF/xyr1-upR are designed, a xyr1 gene fragment is obtained by PCR amplification, and a xyr1 gene fragment is connected to a vector pMDP _tcu1 -T _trpC In hph, located at promoter tcu1Between the fragment and the terminator TrpC fragment, xyr1 gene overexpression vector pMDOExyr1 was constructed.

Wherein, the sequences of the primers are 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 the plasmid pMDhph is described in the literature Two major defect transmitters from Trichoderma reesei and the role in indexing of cellulose biosyntheses.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.2013 Nov15; 288(46):32861-72.

Plasmid pMDP _tcu1 -T _trpC See the Characterisation of a compressor responsive promoter and its medium expressed overexpression of the xylase regulator 1 responses in an index-index production of cells in Trichoderma reesei Lv X, Zheng F, Li C, Zhang W, Chen G, Liu W.Biotechnol biofuels.2015r 14; 8:67.

2) Preparation of protoplast of delta 4cel strain

The uridine prepared in example 4 was used to prepare a trichoderma reesei Δ 4cel protoplast using the deficient strain Δ 4cel by the method for preparing the protoplast according to step 2) of example 1.

3) Transformation of over-expression vector pMDOExyr1, regeneration of transformant and screening verification

Taking 150 mu L of the overexpression vector pMDOExyr1 obtained in the step 1), linearizing, purifying and recovering fragments, mixing with 150 mu L of the Trichoderma reesei delta 4cel protoplast prepared in the step 2), and obtaining a transformant according to the method for transforming the knock-out vector pDOORcel 5a and regenerating the transformant in the step 3) of the example 1, wherein the difference is that: spreading the transformation solution on a regeneration 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 growing on the regeneration medium plate were transferred to a selection medium plate not containing uridine but containing hygromycin at a final concentration of 120. mu.g/mL, and cultured at 28 ℃ for 5-7d to obtain transformants.

Extracting the obtained transformant genome DNA, taking the genome DNA as a template, verifying the inserted xyr1 gene expression cassette by adopting a PCR (polymerase chain reaction) technology, and verifying that the primer is YZxyr1-F/YZxyr1-R, wherein the length of a target band is about 3.0 kb; the primer sequences are as follows:

YZxyr1-F:5’-CCACAAGAGCCTACTGCCAAATC-3’，

YZxyr1-R:5’-TTAGAGGGCCAGACCGGTTCCGT-3’；

and (3) separating and purifying transformants by adopting a single spore separation method to obtain the trichoderma reesei delta 4celOExyr1 engineering bacteria in which the xyr1 gene is successfully inserted.

xyr1 Gene overexpression vector pMDOExyr1 is shown in figure 5, in overexpression vector pMDOExyr1, xyr1 gene is driven by constitutive strong promoter tcu1, achieving high level xyr1 transcription expression.

Example 6: relative transcription level analysis of delta 4celOExyr1 strain and starting strain QM9414 four cellulase coding genes cel5a, cel7b, cel6a, cel7a and transcriptional activator coding gene xyr1

Respectively inoculating spores of the trichoderma reesei delta 4celOExyr1 engineering bacteria and the control strain trichoderma reesei QM9414 constructed in the embodiment 5 into MA liquid culture media containing 1% of glycerol, and culturing for 24h at 28 ℃; set up 3 parallel experiments.

Wherein, the formula of the MA liquid culture medium is as follows: adding Na into each 1L of culture medium ₂ HPO ₄ ·12H ₂ O 17.907g、(NH ₄ ) ₂ SO ₄ 1.4g、KH ₂ PO ₄ 2.0g, urea 0.3g, Tween-800.5 mL, and the pH was adjusted to 5.0 with anhydrous citric acid. Adding 1% glycerol as a carbon source during pre-culture, and adding 2g/L peptone; during induction, 1% Avicel (microcrystalline cellulose) was added as a carbon source. Before use, 0.6g/L MgSO (MgSO) needs to be added to the final concentration ₄ And final concentration of 0.4g/L CaCl ₂ And trace elements (final concentration 1 ×, mother liquor 1000 ×) and 10mM uridine were added; 1000 times microelement mother liquor formula: FeSO ₄ ·7H ₂ O 0.5g，MnSO ₄ ·H ₂ O0.16g，ZnSO ₄ ·7H ₂ O 0.14g，CoC _l2 ·2H ₂ O0.2 g from ddH ₂ And (4) metering the volume of O to 100mL, and filtering and sterilizing.

After the incubation, total RNA was extracted from the sample using TRizol reagent (Invitrogen). PrimeScript using TaKaRa ^TM The RT reagent Kit with gDNA Eraser Kit carries out reverse transcription on RNA, the reaction system is 20 mu L, and the method refers to the instruction.

mu.L of the reverse transcription product was used for subsequent qRT-PCR amplification, SYBR Premix Ex Taq according to TaKaRa ^TM The kit carries out qRT-PCR analysis, the amplification system is 20 mu L, the actin gene is used as an internal reference gene, and the experimental program refers to the instruction.

Wherein the qRT-PCR primers of the reference gene actin are as follows:

actin-F：5’-TGAGAGCGGTGGTATCCACG-3’，

actin-R：5’-GGTACCACCAGACATGACAATGTTG-3’，

qRT-PCR primers for the cellulase encoding gene cel5a were as follows:

cbh1-F：5’-CTTGGCAACGAGTTCTCTT-3’，

cbh1-R：5’-TGTTGGTGGGATACTTGCT-3’，

qRT-PCR primers for the cellulase encoding gene cel7b were as follows:

cbh2-F：5’-CTGTCGCAAAGGTTCCCTCT-3’，

cbh2-R：5’-TCCGGCAAGTCATACACCAC-3’，

qRT-PCR primers for the cellulase encoding gene cel6a were as follows:

eg1-F：5’-CGGCTACAAAAGCTACTACG-3’，

eg1-R：5’-CTGGTACTTGCGGGTGAT-3’，

qRT-PCR primers for the cellulase encoding gene cel7a were as follows:

eg2-F：5’-GCTGTACCACAGATGGCACT-3’，

eg2-R：5’-CATCCGACAGGTAAGCGGAA-3’，

qRT-PCR primers for the transcriptional activator-encoding gene xyr1 were as follows:

xyr1-F：5’-CCATCAACCTTCTAGACGAC-3’，

xyr1-R：5’-AACCCTGCAGGAGATAGAC-3’，

the qRT-PCR reaction system is as follows: reverse transcription product 1. mu.L, upstream primer 0.5. mu.L, downstream primer 0.5. mu.L, 2 XSSYBR Green q RT-PCR Mix 10. mu.L, ddH ₂ O8. mu.L, 20. mu.L in total;

the qRT-PCR reaction procedure was as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30sec, annealing at 58 ℃ for 30sec, extension at 72 ℃ for 1min30 sec, 30 cycles; final extension at 72 ℃ for 5 min.

The result is shown in fig. 6, the transcription levels of four main cellulase coding genes cel5a, cel7b, cel6a and cel7a in trichoderma reesei Δ 4celOExyr1 engineering bacteria are obviously reduced compared with the transcription level of a control strain trichoderma reesei QM9414, and almost no transcription expression exists, which is consistent with the knock-out result of four corresponding genes; the expression level of the xyr1 gene of the delta 4celOExyr1 engineering strain is obviously higher than that of a control strain Trichoderma reesei QM9414, and the result shows that the xyr1 gene in the strain realizes high-level expression under the drive of a tcu1 promoter.

Example 7: analysis of extracellular fermentation liquid of delta 4cel OExyr1 strain

The spores of Trichoderma reesei DELTA 4CelOExyr1 engineering strain and Trichoderma reesei QM9414 strain, which were constructed in example 5, were inoculated in the same amount to MA liquid medium containing 1% glycerol and 0.2% peptone, respectively, and cultured at 28 ℃ and 180rpm for 24 hours, followed by filtration to collect mycelia. Transferring the collected mycelia into MA liquid culture medium containing 2% lactose, and culturing at 28 deg.C and 180rpm for 96 hr to obtain fermentation broth. The MA liquid medium was prepared as described in example 6.

1) SDS-PAGE and mass spectrum of extracellular fermentation liquid

20 μ L of the fermentation broth was taken and subjected to conventional SDS-PAGE analysis and conventional Coomassie Brilliant blue staining and destaining. The result is shown in fig. 7, compared with the extracellular fermentation solution of the control strain trichoderma reesei QM9414, a plurality of strips are increased in the fermentation solution of trichoderma reesei Δ 4CelOExyr1 engineered bacteria. The main three bands in the gel were cut off and sent to Beijing Huada Gene company for mass spectrometry, and the mass spectrometry results show that the

bands

1, 2 and 3 are xyloglucanase, alpha-L-arabinofuranosidase ABF1 and cellulose endonuclease EG3 respectively.

2) Analysis of enzyme activity 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 appropriately diluted fermentation broth and 150. mu.L of pNP-alpha-L-arabinofanoside substrate dissolved in acetic acid-sodium acetate buffer solution of pH 4.8 were mixed uniformly, the reaction was carried out at 50 ℃ for 30min, and then 50. mu.L of 10% sodium carbonate was added to terminate the reaction. The absorbance of the product was read at a wavelength of 420 nm.

One unit of enzyme activity of α -L-arabinofuranosidase is defined as the amount of enzyme that releases 1 μmol4-nitropheny per minute under standard conditions.

The result is shown in fig. 8, compared with a control strain trichoderma reesei QM9414, the enzymatic activity of the arabinofuranosidase in the trichoderma reesei delta 4CelOExyr1 engineering bacteria fermentation liquor is remarkably improved, and the enzymatic activity reaches 8.06U/mL after 96 hours of culture and is 8-9 times that of the control strain. This is consistent with the upregulation of α -L-arabinofuranosidase ABF1 expression in the mass spectrometry results described above.

Example 8: transcript level analysis of alpha-L-arabinofuranosidase encoding gene abf1 in delta 4CelOExyr1 engineering bacteria

The culture, total RNA extraction and reverse transcription of the engineered Trichoderma reesei Δ 4celOExyr1 strain and the control strain Trichoderma reesei QM9414 were the same as in example 6.

Wherein the qRT-PCR primers of the reference gene actin are as follows:

actin-F：5’-TGAGAGCGGTGGTATCCACG-3’，

actin-R：5’-GGTACCACCAGACATGACAATGTTG-3’，

qRT-PCR primers for the alpha-L-arabinofuranosidase encoding gene abf1 were as follows:

abf1-F：5’-GCCAGTCCATACGACCAACA-3’，

abf1-R：5’-GATGATGGGCACGACGAGAT-3’，

The result is shown in fig. 9, in the fermentation process, the expression amount of abf1 genes of the trichoderma reesei Δ 4 ceoexyr 1 engineering bacteria is higher than that of the control strain trichoderma reesei QM9414, which shows that the expression of four main cellulases in the trichoderma reesei Δ 4 ceoexyr 1 engineering bacteria is absent and the overexpression of the transcriptional activator, so that the high-level expression of abf1 genes is realized. The result is consistent with the result that the protein content and the enzyme activity level of the arabinofuranosidase ABF1 in the extracellular fermentation liquid are obviously improved.

Claims

1. The trichoderma reesei engineering bacterium for high yield of 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 activation factor coding gene xyr1 is inserted.

2. The trichoderma reesei engineering bacterium capable of highly producing α -L-arabinofuranosidase according to claim 1, wherein the transcription activator encoding gene xyr1 is expressed under the drive of a strong promoter; preferably, the strong promoter is P _tcu1 。

3. The trichoderma reesei engineering bacterium capable of highly producing alpha-L-arabinofuranosidase according to claim 1, wherein the enzyme activity of the alpha-L-arabinofuranosidase of the trichoderma reesei engineering bacterium is 8-9 times that of an original strain.

4. The method for constructing the trichoderma reesei engineering bacteria for high yield of alpha-L-arabinofuranosidase according to claim 1, wherein trichoderma reesei QM9414 is used as an initial strain, homologous recombination technology is adopted to sequentially knock out four cellulase genes cel5a, cel7b, cel6a and cel7a in the initial strain to inactivate the genes, and then transcription activation factor encoding genes xyr1 and xyr1 genes are inserted into a strong promoter P _tcu1 Driven to express, xyr1 gene is over expressed, pyr4 gene is used as screening marker gene, and screening and verification are carried out to obtain the gene.

5. The method for constructing the trichoderma reesei engineering bacterium with high alpha-L-arabinofuranosidase yield according to claim 4, which is characterized by comprising the following steps:

6. The method of claim 5, wherein the transformation is performed by PEG-mediated protoplast transformation.

7. Use of the engineered Trichoderma reesei strain of claim 1 in the production of α -L-arabinofuranosidase, xyloglucanase, and/or endo-cellulose.

8. Use according to claim 7, characterized in that it comprises the following steps:

the Trichoderma reesei engineering bacteria as claimed in claim 1 is inoculated in MA liquid culture medium containing 1% glycerol and 0.2% peptone, cultured at 28 + -2 deg.C and 180-200rpm for 24-36h, the mycelium is collected, transferred into MA liquid culture medium containing 2% lactose, and cultured for 96-120h to obtain fermentation broth.

9. The use according to claim 8, wherein the MA liquid medium has the following composition: adding Na to 1L of culture medium ₂ HPO ₄ ·12H ₂ O 17.907g、(NH ₄ ) ₂ SO ₄ 1.4 g、KH ₂ PO ₄ 2.0g, 0.3g of urea and Tween-800.5 mL, adjusting the pH to 5.0 with anhydrous citric acid, and adding MgSO 0.6g/L until the final concentration is reached ₄ The final concentration of CaCl is 0.4g/L ₂ And trace elements;

10. The use of the engineered trichoderma reesei strain of claim 1 for degrading hemicellulose-like substances rich in arabinose residues.