CN114875059B - Construction and application of novel trichoderma reesei heterologous protein expression system - Google Patents

Abstract

The invention discloses construction and application of a novel trichoderma reesei heterologous protein expression system, and belongs to the technical field of biology. The invention constructs a DCL expression system suitable for the expression of the trichoderma reesei heterologous gene by using the promoter and the signal peptide of the trichoderma reesei gene DCL. The recombinant trichoderma reesei containing the system is cultured in the MM basic culture medium containing corncob, bran or bran leaching solution, so that the high-efficiency expression of the protein in cells or outside cells can be realized. Compared with the existing CBH expression system of Trichoderma reesei, the protein expression level can be improved by 1.38-20 times. The culture medium used by the dcl expression system provided by the invention has low raw materials and is easy to obtain, and the target protein can be directly secreted to the outside of cells under the optimized culture medium condition by the fermented heterologous protein, so that the separation and purification of the heterologous protein are simplified, and the industrialized production of the heterologous protein is facilitated.

Description

Construction and application of novel trichoderma reesei heterologous protein expression system

Technical Field

The invention belongs to the technical field of biology, and particularly relates to construction and application of a novel trichoderma reesei heterologous protein expression system.

Background

Trichoderma reesei (Trichoderma reesei) is a Wen Fusheng-philic filamentous fungus, and cellulase produced by the Trichoderma reesei accounts for more than 90% of the market share of the world, and is widely applied to the fields of food, textile, feed, bioenergy and the like. As safe strains authenticated by FDA, trichoderma reesei has strong protein synthesis and secretion capacity, outstanding industrial fermentation performance and similar higher eukaryote translation modification system, so that the Trichoderma reesei becomes an ideal cell factory for producing recombinant proteins.

The regulation and control of protein production firstly occurs at the transcription level, the utilization of a strong promoter to drive gene expression is one of main strategies for improving the yield of heterologous proteins, and meanwhile, the establishment of culture conditions suitable for the regulation and control is a key for realizing the high yield of the heterologous proteins. The promoters available for recombinant protein expression in Trichoderma reesei are very few, cellobiohydrolase CBHI, encoded by a single copy gene, account for at least 50% of extracellular protein secretion, pcbh1 thus being the most commonly used strong promoter. The intensity of Pcbh1 is greatly influenced by the type of carbon source, the pH value and the like, and has great limitation in practical application, and the yield of proteins, particularly non-fungal source proteins, is often lower than that of endogenous proteins and even cannot be expressed or secreted outside cells. At present, the potential of producing heterologous proteins in trichoderma cell factories is not really developed, and development of new expression elements and optimal fermentation conditions thereof are very necessary to make up for the defects of the existing expression systems.

Disclosure of Invention

In the pre-transcriptome sequencing of Trichoderma reesei, the inventors found that one gene (designated dcl) was transcribed at higher or comparable levels to cbh1 under different carbon sources (e.g.cellulose, glucose, lactose), and that Pdcl was very likely a strong promoter comparable to Pcbh 1. In the invention, the inventor constructs a trichoderma reesei heterologous protein expression system by using a promoter Pdcl, takes red fluorescent protein RFP which is difficult to secrete and is actually positioned in cytoplasm as a report system, establishes a culture condition suitable for efficient secretion of the red fluorescent protein RFP, discovers that the production of intracellular and extracellular proteins can reach better effects under various carbon source culture conditions, and provides a novel method and means for efficiently producing heterologous proteins by using trichoderma reesei.

The first object of the present invention is to provide a trichoderma reesei heterologous protein expression system consisting of a dcl promoter, a dcl signal peptide, a cbh2 terminator and a heterologous protein.

In one embodiment, the sequences of the dcl promoter, the dcl signal peptide and the cbh2 terminator are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively.

In one embodiment, the signal peptide of dcl is linked before the gene encoding the heterologous protein and the promoter of dcl is used to initiate expression of the heterologous protein.

It is a second object of the present invention to provide a plasmid containing the trichoderma reesei heterologous protein expression system.

In one embodiment, the promoter of dcl, the signal peptide of dcl, the heterologous protein encoding gene, and the cbh2 terminator are integrated onto the starting plasmid in that order to initiate and terminate expression of the heterologous protein.

In one embodiment, a recombinant plasmid is constructed using the pEASY-block T simple plasmid as the starting plasmid.

It is a third object of the present invention to provide a recombinant microbial cell containing said plasmid.

In one embodiment, trichoderma reesei TU6 is used as the starting strain.

A fourth object of the present invention is to provide a method for producing a heterologous protein using the trichoderma reesei heterologous protein expression system, the method comprising the steps of:

(1) Constructing a recombinant plasmid containing the trichoderma reesei heterologous protein expression system;

(2) The recombinant plasmid is guided into Trichoderma reesei to construct recombinant Trichoderma reesei;

(3) Culturing the recombinant trichoderma reesei in a reaction system of an MM basal medium containing glucose, wherein the reaction system also contains cellulose, corncob, bran or bran extract.

In one embodiment, the heterologous protein includes, but is not limited to, a red fluorescent protein.

In one embodiment, the uracil-deficient strain Trichoderma reesei TU6 is the starting strain.

In one embodiment, the recombinant plasmid further expresses the pyr4 gene and a positive transformant is obtained using pyr4 gene as a selectable marker.

In one embodiment, the nucleotide sequence of the pyr4 gene is set forth in SEQ ID NO. 4.

In one embodiment, the glucose content in the MM basal medium is 2% (w/v).

Preferably, the reaction system contains corncob, bran or bran extract.

More preferably, the corncob is present in an amount of 5% (w/v), the bran is present in an amount of 3% (w/v), and the bran extract is present in a concentration of 3%.

In one embodiment, the recombinant trichoderma reesei is inoculated into an MM medium containing glucose and pre-cultured for 40-50 hours at 25-32 ℃ at 150-250 rpm, the obtained culture solution is transferred into an MM medium containing corncob, bran or bran extract, and fermented for 120-144 hours at 25-32 ℃ at 150-250 rpm.

Preferably, the pH of the MM medium is 5.1 to 5.3.

The fifth object of the present invention is to provide the use of the trichoderma reesei expression system or the recombinant plasmid containing trichoderma reesei in expressing a heterologous protein.

In one embodiment, the heterologous protein includes, but is not limited to, a red fluorescent protein, a green fluorescent protein, and the like, capable of being expressed in trichoderma reesei.

The invention has the beneficial effects that:

the invention constructs the recombinant strain suitable for the expression of the trichoderma reesei heterologous protein by using the promoter and the signal peptide of the trichoderma reesei gene DCL, and realizes the efficient expression of the protein. The strain is cultured in MM basal medium+5% corncob, the expression intensity of extracellular protein is 1.38 times of that of CBH expression system, and the MM basal medium+3% bran (pH 5.1-5.3) or MM basal medium+3% bran leaching solution (pH 5.1-5.3) can make DCL expression system express heterologous protein RFP in the cell and the MM basal medium+3% bran leaching solution (pH 5.1-5.3) is full liquid medium, so that the strain is more suitable for large-scale production, and simultaneously can directly secrete protein out of cell, simplify the separation and purification of heterologous protein, and is favorable for industrialized production of heterologous protein.

Drawings

FIG. 1 is a backbone plasmid map of a dcl system vector that does not contain the target gene.

FIG. 2 is a plasmid map of the backbone of the cbh1 system vector that does not contain the target gene.

FIG. 3 is a schematic diagram of construction of RFP expression plasmids in two systems.

FIG. 4 is a PCR identification electrophoretogram of DCL-RFP positive transformants; 1: DCL-RFP-1,2: DCL-RFP-2,3: DCL-RFP-3,4: DCL-RFP-4.

FIG. 5 is a PCR identification of CBH1-RFP positive transformants; 1: CBH1-RFP-1;2: CBH1-RFP-2;3: CBH1-RFP-3;4: CBH1-RFP-4;5: CBH1-RFP-5.

FIG. 6 is SDS-PAGE analysis of extracellular proteins of DCL-RFP positive transformants under cellulose induction; 1 DCL-RFP-1,2: DCL-RFP-2,3: DCL-RFP-3,4: DCL-RFP-4,5: the starting strain TU6.

FIG. 7 shows SDS-PAGE analysis of extracellular proteins of CBH1-RFP positive transformants under cellulose induction; 1: starting strain TU6,2: CBH1-RFP-1,3: CBH1-RFP-2,4: CBH1-RFP-3,5: CBH1-RFP-4,6: CBH1-RFP-5.

FIG. 8 shows SDS-PAGE analysis and RFP relative fluorescence intensity of fermentation results of two recombinant strains under different medium conditions; 1: DCL-RFP-4,2: CBH1-RFP-1,3: the starting strain TU6.

FIG. 9 shows SDS-PAGE analysis of fermentation results under MM basal medium+3% bran extract and relative fluorescence intensity of RFP; 1: DCL-RFP-4,2: CBH1-RFP-1,3: the starting strain TU6.

Detailed Description

1. Strains and plasmids

Coli Escherichia coli strain Trans-T1 (purchased from Beijing full gold Biotechnology Co., ltd.) was used as a recombinant plasmid.

Trichoderma reesei TU-6 strain is uracil-deficient strain (disclosed in Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene, 2012) and expresses heterologous proteins as a host.

High fidelity Fastpfu was purchased from Beijing full gold biotechnology Co.

The plasmid pEASY-block T simple is purchased from Beijing full-type gold biotechnology Co., ltd, and the dcl expression system plasmid Pdcl-SSdcl-RFP and the cbh1 expression system plasmid Pcbh1-SScbh1-RFP are respectively constructed by taking the plasmid pEASY-block T simple as a framework.

The red fluorescent protein gene (GenBank: AB 830533.1) was synthesized by Nanjing Jinsri Biotechnology Co.

The seamless splice kit was purchased from the biological technology company limited of nanking nuozhen.

2×Master Mix (Dye Plus) was purchased from Nanjinouzan Biotech Inc.

2. Culture medium

(1) PDA culture medium

Glucose	20g
		Potato	200g
Agar powder	20g

Deionized water is fixed to 1L, the natural pH is carried out, the temperature is 115 ℃, and the high-pressure steam sterilization is carried out for 20 min.

(2) LB medium

Peptone	1％
		Sodium chloride	1％
Yeast extract	0.5％

Natural pH,121 ℃, and high pressure steam sterilization for 20 min.

( 3) Basic culture medium of trichoderma reesei (abbreviation: MM medium )

The pH was adjusted to 5.3, and the mixture was autoclaved at 121℃for 20 min.

(4) Fermentation medium

a) MM basal medium+1% (w/v) cellulose, pH 5.1-5.3;

b) MM basal medium+5% (w/v) corncob, pH 5.1-5.3;

c) MM basal medium+3% (w/v) bran, pH 5.1-5.3;

d) MM basal medium+1% (w/v) cellulose+5% (w/v) corncob+3% (w/v) bran, pH 5.1-5.3;

e) MM basic culture medium+3% (v/v) bran leaching liquor, pH is 5.1-5.3; wherein, 30g of bran is weighed and added with 1L of ddH ₂ O, boiling for 20min, filtering with double-layer gauze, and collecting the lower filtrate as 3% testa Tritici leaching solution; MM basal medium was dissolved in 3% branAnd (3) leaching the extract.

3. Main instrument and reagent

CTAB plant genomic DNA rapid extraction kit (brand: aidlab/Edley, cat# DN 1402) was used to extract the genome of Trichoderma reesei.

A multifunctional fluorescent plate reader (brand: synergy H4) was used to detect the fluorescence intensity of RFP.

4. Electrotransformation of Trichoderma reesei

(1) A rapid plasmid miniprep kit (purchased from Tiangen Biochemical technology (Beijing)) is used for extracting a large amount of CBH1-RFP plasmids and DCL-RFP, and then a vacuum centrifugal concentrator is used for concentrating the plasmids to ug/ul level, and the plasmids are preserved at the temperature of minus 20 ℃ for standby;

(2) Trichoderma reesei TU6 spores (2-3 dishes per transformation) on freshly cultured dishes (d=35 mm), prepared as spore suspension by washing the spores with 1.1M sorbitol (Amresco), and filtered through a 200 mesh cell sieve (d=50 mm) to remove residual mycelia;

(3) Transferring the spore liquid into a sterile centrifuge tube, centrifuging for 5min at 4 ℃ and discarding the supernatant;

(4) Spores were washed with 1.1M pre-chilled sorbitol, 3000g centrifuged at 4℃for 5min, and the supernatant was discarded;

(5) Repeating the step (4) twice;

(6) Resuspension spores with 100ul 1.1m pre-chilled sorbitol, and standing on ice for 30min;

(7) Opening the electric converter, and setting electric conversion parameters as follows: 1.8kV,800 Ω,25uF;

(8) Adding the plasmid into the spore liquid (the volume of the plasmid sample is not more than 20 ul), gently mixing, and transferring the spore liquid into a precooled electrorotating cup for electric shock;

(9) Immediately after the electric shock is finished, 900ul of precooled 1.1M sorbitol solution is added, gently mixed and placed on ice;

(10) 10 XYEPD was diluted 10-fold with 1.1M sorbitol solution, 5ml of diluted YEPD (1X) was added to the electrotransformed spore liquid and incubated overnight at 30 ℃;

(11) A proper amount of spore liquid is coated on a MM+2% glucose+1.1M sorbitol+0.1wt% TritonX-100 plate, and cultured at 28 ℃.

5. Identification and fermentation of transformants

After the transformants developed, the genome of the transformants was crude extracted using a university pall tissue DNA extraction buffer (purchased from the scientific company, of enokikai, beijing) as follows:

(1) Selecting a small amount of mycelium in 25ul of extraction buffer solution, and swirling for 5s;

(2) Heating at 95-98 ℃ for 10min;

(3) Vortex again for 5s and centrifuge for 10s;

(4) Preserving at-20 ℃ for standby.

Using the supernatant from the crude extract as a template, PCR was used to determine whether the RFP expression cassette was successfully integrated into the genome using the primer pair of Table 3, using 2X of the enzymeMaster Mix (Dye Plus) (available from Nanjinouzan Biotech Co., ltd.) under amplification conditions of: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 1min,30 cycles; finally, the extension is carried out for 5min at 72 ℃.

Selecting positive transformants, transferring the positive transformants to a PDA plate, culturing for 7-9 days, scraping a small amount of spores after the spores are mature, inoculating the spores to MM medium and 2% glucose, pre-culturing for 48 hours at 28 ℃ and 200rpm, transferring the spores to MM basal medium and 1% cellulose (pH 5.1-5.3), culturing for 5-6 days at 28 ℃ and 200rpm, and analyzing intracellular and extracellular protein expression conditions by SDS-PAGE.

TABLE 1 primers required for construction of dcl System vector backbone plasmid

TABLE 2 primer required for construction of the backbone plasmid of the cbh1 System vector

TABLE 3 primers used for identification of transformants

Embodiment one: construction of expression vectors

1. Construction of dcl expression System

Specifically, the construction of an expression plasmid containing the dcl promoter, the signal peptide, the cbh2 terminator, pyr4 gene and other elements is as follows:

(1) Preparation of Trichoderma reesei TU6 genomic DNA

a) Inoculating Trichoderma reesei TU6 spore liquid into MM basal medium and 2% glucose, culturing at 30deg.C for 48 hr, collecting mycelium, centrifuging at maximum rotation speed in 2ml centrifuge tube for 10min, and pouring out supernatant;

b) Fully mashing hypha by using a mortar until the hypha is uniformly and evenly pulped;

c) Extracting genome of Trichoderma reesei TU6 strain by using CTAB plant genome DNA rapid extraction kit;

d) The extracted genome DNA is preserved at-20 deg.c for further use.

(2) Respectively amplifying a dcl promoter, a signal peptide, a cbh2 terminator and a screening gene pyr4 sequence by using the Trichoderma reesei TU6 genome as a template through a primer pair in the table 1; using pEASY-block T simple plasmid as template to amplify expression vector skeleton, using high-fidelity Fastpfu as enzyme, and the amplification condition is: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 72℃for 2min,30 cycles; finally, the extension is carried out for 10min at 72 ℃. The dcl promoter signal peptide, cbh2 terminator, screening gene pyr4 were ligated to the pEASY-block T simple backbone fragment by overlapping between the different fragments (underlined in Table 1) using a seamless splice kit to obtain a dcl expression vector backbone plasmid not containing a heterologous gene (FIG. 1).

2. construction of cbh1 expression System

Specifically, the preparation of vector backbone plasmid containing cbh1 promoter signal peptide and cbh2 terminator and pyr4 gene and other elements:

amplifying the cbh1 promoter signal peptide by using the Trichoderma reesei TU6 genome as a template and the dcl expression backbone plasmid constructed as above as a template through a primer pair in Table 2, and amplifying a carrier backbone fragment by using high-fidelity Fastpfu as an enzyme under the amplification conditions that: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 72℃for 4min,30 cycles; finally, the extension is carried out for 10min at 72 ℃. The dcl promoter signal peptide in the dcl expression backbone plasmid was replaced with the cbh1 promoter signal peptide by overlap between fragments (underlined) using a seamless splice kit to obtain a cbh1 system vector backbone plasmid that did not contain the target gene (fig. 2).

3. Construction of RFP expression plasmid based on two expression systems

The synthesized RFP gene was inserted into the DCL expression backbone plasmid behind the DCL promoter signal peptide and the CBH1 expression backbone plasmid behind the CBH1 promoter signal peptide, respectively, to obtain the RFP expression plasmid DCL-RFP in the DCL expression system (FIG. 3B) and the RFP expression plasmid CH1-RFP in the CBH1 expression system (FIG. 3A).

Embodiment two: construction of recombinant strains and fermentation of proteins

1. The expression plasmids DCL-RFP and CH1-RFP obtained in the example were transformed into Trichoderma reesei, and after the transformants grew, the genome of the transformants was roughly extracted by using a Universall tissue DNA extraction buffer (purchased from Beijing Inocover technologies Co., ltd.) as follows:

(1) Selecting a small amount of mycelium in 25ul of extraction buffer solution, and swirling for 5s;

(2) Heating at 95-98 ℃ for 10min;

(3) Swirling for 5s again, centrifuging for 10s, and taking supernatant;

(4) Preserving at-20 ℃ for standby.

Using the supernatant from the crude extract as a template, PCR was used to determine whether the RFP expression cassette was successfully integrated into the genome using the primer pair of Table 3, using 2X of the enzymeMaster Mix (Dye Plus), amplification conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58 ℃30s,72℃extension 1min,30 cycles; finally, the extension is carried out for 5min at 72 ℃.

For the DCL expression system, 23 transformants were obtained in total by electrotransformation, of which 4 transformants had correct expression cassette sequences (fig. 4), named: DCL-RFP-1-4.

For the CBH1 expression system, 55 transformants were obtained in total by electrotransformation, and RFP expression cassettes were identified by PCR cloning and sequencing, and as a result, it was found that the expression cassette sequences of 5 transformants in total were correct (fig. 5), and they were named: CBH 1-RFP-1-5.

2. Intracellular and extracellular protein expression was analyzed by SDS-PAGE:

for the DCL expression system, inoculating the identified 4 positive transformants to MM medium plus 2% glucose, pre-culturing for 48 hours at 28 ℃ and 200rpm, transferring to MM basal medium plus 1% cellulose (pH5.1-5.3), culturing for 5-6 days at 28 ℃ and 200rpm, centrifuging extracellular fermentation liquor, taking supernatant to obtain extracellular proteins, crushing hyphae to obtain intracellular proteins, and carrying out SDS-PAGE protein analysis on the extracellular proteins. The results are shown in FIG. 6, in which DCL-RFP-1,2,4 was successfully fermented to RFP (arrow). As is clear from SDS-PAGE gel analysis, the amount of target protein fermented by the transformant DCL-RFP-4 was maximized, and thus, the following experiment was mainly conducted using the transformant DCL-RFP-4.

5 positive transformants obtained by the CBH1 expression system are inoculated in 50mL of MM medium taking glucose (2% w/v) as a unique carbon source, pre-cultured for 48h at 28 ℃ and 200rpm, transferred to MM medium (pH 5.1-5.3) taking cellulose (1% w/v) as a unique carbon source, fermented for 5-6 days at 28 ℃ and 200rpm, and the supernatant is obtained by centrifuging an extracellular fermentation liquid, so that extracellular proteins are obtained after fermentation mycelium is broken, and SDS-PAGE protein analysis is carried out on the extracellular proteins respectively. As a result (FIG. 7), it was found that RFP was well expressed in the cell of the CBH1 expression system (arrow point), but was not secreted extracellularly. As is clear from SDS-PAGE gel analysis, the fermentation result of CBH1-RFP-1 transformant was best, and thus CBH1-RFP-1 was mainly used in the following experiments.

Fermentation results under cellulose induction conditions show that compared with a CBH1 expression system, the DCL expression system can also successfully express RFP, and the expression intensity is equivalent to that of the CBH1 expression system. Therefore, the DCL expression system can also be used as an alternative to expressing heterologous genes in Trichoderma reesei.

Embodiment III: selection of optimal culture conditions

According to the fermentation result of the MM basal medium plus 1% cellulose (pH 5.1-5.3), two strains of CBH1-RFP-1 and DCL-RFP-4 are selected, wherein the MM basal medium plus 1% cellulose and the pH 5.1-5.3 are respectively used; MM basal medium+5% corncob, pH is 5.1-5.3; MM basic culture medium is added with 3 percent of bran, and the pH is 5.1 to 5.3; MM basal culture medium, cellulose, corncob, bran and pH 5.1-5.3, wherein the MM basal culture medium, the cellulose, the corncob and the bran are respectively 1% and 5%; MM basic culture medium+3% bran leaching solution, pH is 5.1-5.3; fermenting culture medium with 1.5% corn steep liquor and 1% cellulose and pH of 5.1-5.3, and selecting optimal culture medium suitable for DCL expression system according to the expression level of extracellular protein and relative fluorescence intensity of RFP.

In order to explore the fermentation conditions most suitable for a DCL expression system, two recombinant strains of DCL-RFP-4 and CBH1-RFP-1 are inoculated into 50mL of MM medium taking glucose (2% w/v) as the sole carbon source, after being pre-cultured for 48 hours at 28 ℃, the two recombinant strains are respectively transferred into MM basal medium+1% cellulose, MM basal medium+5% corncob, MM basal medium+3% bran, MM basal medium+1% cellulose+5% corncob+3% bran, MM basal medium+3% bran extract and 1.5% corn steep liquor+1% cellulose medium (pH is 5.1-5.3), fermented for 120 hours at 200rpm at 28 ℃, and the intracellular and extracellular protein expression conditions of the two recombinant strains are analyzed by SDS-PAGE, and meanwhile, the fluorescence intensity of RFP is detected.

The results are shown in FIG. 8:

in the MM basal medium plus 5% corncob, the extracellular RFP fluorescence intensity of the DCL expression system is 1.38 times of that of the CBH expression system, so that the DCL expression system is suitable for extracellular expression of target proteins;

in MM basal medium+3% bran, the extracellular RFP fluorescence intensity of DCL expression system is 7.39 times of that of CBH expression system, and the intracellular RFP fluorescence intensity is 4.12 times of that of CBH expression system;

under the conditions of MM basal medium and 3% bran extract (full liquid medium), the DCL expression system can express target protein in and out of the cell well, the fluorescence intensity of the extracellular RFP is 19.96 times of that of the CBH expression system, and the fluorescence intensity of the intracellular RFP is 16.65 times of that of the CBH expression system.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Shenzhen Cork Biotechnology Co., ltd

<120> construction of novel Trichoderma reesei heterologous protein expression system and application thereof

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gcacgtggaa accataccca aattgcctac agctgcggag catgagccta tggcgatcag 540

tctggtcatg ttaaccagcc tgtgctctga cgttaatgca gaatagaaag ccgcggttgc 600

aatgcaaatg atgatgcctt tgcagaaatg gcttgctcgc tgactgatac cagtaacaac 660

tttgcttggc cgtctagcgc tgttgattgt attcatcaca acctcgtctc cctcctttgg 720

gttgagctct ttggatggct ttccaaacgt taatagcgcg tttttctcca caaagtattc 780

gtatggacgc gcttttgcgt gtattgcgtg agctaccagc agcccaattg gcgaagtctt 840

gagccgcatc gcatagaata attgattgcg catttgatgc gatttttgag cggctgtttc 900

aggcgacatt tcgcccgccc ttatttgctc cattatatca tcgacggcat gtccaatagc 960

ccggtgatag tcttgtcgaa tatggctgtc gtggataacc catcggcagc agatgataat 1020

gattccgc 1028

<210> 4

<211> 2389

<212> DNA

<213> artificial sequence

<400> 4

ctggcagact tgtgtgtatc attcacccta tttctgcttc atagtacatg tactgtacct 60

gaacggctca accgctattt acgactctta tttttttgtg gcgttggtca cgtttgccag 120

ctgttgtccg tctttctagg gctcctcaaa cttgacctga ccgagctccc tttctggacc 180

cggtgggctt cacttccagc tgctgagcga cctgagccga acatcctcag tccttgtcca 240

gcgcaattca ttttctttcc ttttcttttt ttttattcct ttctttactt ttattctctc 300

tttttctcct cttcctcttc ttcttctttc tcctcctcct ccatatcctc actctcgtct 360

ccctcattac taccctctcg gctcctcagg tccaccaacc ctcccgcacc caaacctctg 420

ccgctgaaac ccattcggtg gtcgccgttt tttttttttt ttttttctca cccccaaagt 480

cgcaatatcg ggtatcgccg ccggcattga atcgccttct ccgctagcat cgactactgc 540

tgctctgctc tcgttgccag cgctgctccc tagaattttg accaggggac gagcccgaca 600

ttaaagcaac tccctcgcct cgagacgact cggatcgcac gaaattctcc caatcgccga 660

cagttcctac tcctcttcct cccgcacggc tgtcgcgctt ccaacgtcat tcgcacagca 720

gaattgtgcc atctctctct tttttttccc cccctctaaa ccgccacaac ggcaccctaa 780

gggttaaact atccaaccag ccgcagcctc agcctctctc agcctcatca gccatggcac 840

cacacccgac gctcaaggcc accttcgcgg ccaggagcga gacggcgacg cacccgctga 900

cggcttacct gttcaagctc atggacctca aggcgtccaa cctgtgcctg agcgccgacg 960

tgccgacagc gcgcgagctg ctgtacctgg ccgacaagat tggcccgtcg attgtcgtgc 1020

tcaagacgca ctacgacatg gtctcgggct gggacttcca cccggagacg ggcacgggag 1080

cccagctggc gtcgctggcg cgcaagcacg gcttcctcat cttcgaggac cgcaagtttg 1140

gcgacattgg ccacaccgtc gagctgcagt acacgggcgg gtcggcgcgc atcatcgact 1200

gggcgcacat tgtcaacgtc aacatggtgc ccggcaaggc gtcggtggcc tcgctggccc 1260

agggcgccaa gcgctggctc gagcgctacc cctgcgaggt caagacgtcc gtcaccgtcg 1320

gcacgcccac catggactcg tttgacgacg acgccgactc cagggacgcc gagcccgccg 1380

gcgccgtcaa cggcatgggc tccattggcg tcctggacaa gcccatctac tcgaaccggt 1440

ccggcgacgg ccgcaagggc agcatcgtct ccatcaccac cgtcacccag cagtacgagt 1500

ccgtctcctc gccccggtta acaaaggcca tcgccgaggg cgacgagtcg ctcttcccgg 1560

gcatcgagga ggcgccgctg agccgcggcc tcctgatcct cgcccaaatg tccagccagg 1620

gcaacttcat gaacaaggag tacacgcagg cctgcgtcga ggccgcccgg gagcacaagg 1680

actttgtcat gggcttcatc tcgcaggaga cgctcaacac cgagcccgac gatgccttta 1740

tccacatgac gcccggctgc cagctgcccc ccgaagacga ggaccagcag accaacggat 1800

cggtcggtgg agacggccag ggccagcagt acaacacgcc gcacaagctg attggcatcg 1860

ccggcagcga cattgccatt gtgggccggg gcatcctcaa ggcctcagac cccgtagagg 1920

aggcagagcg gtaccgatca gcagcgtgga aagcctacac cgagaggctg ctgcgatagg 1980

ggagggaagg gaagaaagaa gtaaagaaag gcatttagca agaaggggga aaagggaggg 2040

aggacaaacg gagctgagaa agagctcttg tccaaagccc ggcatcatag aatgcagctg 2100

tatttaggcg acctcttttt ccatcttgtc gatttttgtt atgacgtacc agttgggatg 2160

atggatgatt gtaccccagc tgcgattgat gtgtatcttt gcatgcaaca acacgcgatg 2220

gcggaggcga actgcacatt ggaaggttca tatatggtcc tgacatatct ggtggatctg 2280

gaagcatgga attgtatttt tgatttggca tttgcttttg cgcgtggagg gaacatatca 2340

ccctcgggca tttttcattt ggtaggatgg tttggatgca gttgtcgac 2389

Claims (9)

1. A trichoderma reesei heterologous protein expression system, which is characterized by comprising a dcl promoter, a dcl signal peptide, a cbh2 terminator and a heterologous protein; the nucleotide sequence of the dcl promoter is shown as SEQ ID NO.2, and the sequence of the signal peptide for encoding the dcl is shown as SEQ ID NO. 2; connecting a signal peptide of dcl in front of a coding gene of the heterologous protein and utilizing a promoter of the dcl to start expression of the heterologous protein; the sequence of the cbh2 terminator is shown in SEQ ID NO. 3.

2. A recombinant plasmid comprising the trichoderma reesei heterologous protein expression system of claim 1.

3. The recombinant plasmid according to claim 2, wherein the promoter of dcl, the signal peptide of dcl, the heterologous protein encoding gene, and the cbh2 terminator are integrated into the starting plasmid in that order to initiate and terminate expression of the heterologous protein.

4. The recombinant plasmid according to claim 3, wherein the plasmid pEASY-block T simple plasmid is used as a starting plasmid to construct the recombinant plasmid.

5. Trichoderma reesei comprising the heterologous protein expression system of Trichoderma reesei of claim 1 or the recombinant plasmid of any one of claims 2 to 4.

6. Trichoderma reesei according to claim 5, characterized in that Trichoderma reesei TU6 is used as starting strain.

7. A method for producing a heterologous protein, characterized in that the heterologous protein is produced by using the trichoderma reesei heterologous protein expression system of claim 1 or the trichoderma reesei of any one of claims 5 to 6, the method comprising:

(1) Constructing a recombinant plasmid containing the trichoderma reesei heterologous protein expression system;

(2) The recombinant plasmid is guided into Trichoderma reesei to construct recombinant Trichoderma reesei;

(3) Inoculating the recombinant trichoderma reesei into an MM culture medium containing glucose, pre-culturing for 40-50 h at 25-32 ℃ at 150-250 rpm, transferring the obtained culture solution into an MM culture medium containing one or more of cellulose, corncob, bran or bran leaching solution, and fermenting for 120-144 h at 25-32 ℃ at 150-250 rpm.

8. The method of claim 7, wherein the MM medium has a pH of 5.1-5.3.

9. Use of trichoderma reesei heterologous protein expression system according to claim 1 or 2, or recombinant plasmid according to any one of claims 2-4, or trichoderma reesei according to claim 5 or 6, for the production of heterologous proteins.