CN116814628A - Myceliophthora thermophila small molecule heat shock protein constitutive strong promoter Phsp and application thereof - Google Patents

Abstract

The application relates to the technical field of agricultural biology, in particular to a myceliophthora thermophila micromolecular heat shock protein constitutive strong promoter Phsp、And applications thereof. The constitutive strong promoter P of the present applicationhspCan start the high-efficiency expression of the gene in myceliophthora thermophila without an inducer. The application provides a promoter P containing constitutive stronghspThe myceliophthora thermophila overexpression vector can be used for high-efficiency expression of endogenous or exogenous genes, and provides a favorable tool for engineering modification of myceliophthora thermophila.

Description

Myceliophthora thermophila small molecule heat shock protein constitutive strong promoter Phsp、And applications thereof

Technical Field

The application relates to the technical field of agricultural biology, in particular to a myceliophthora thermophila micromolecular heat shock protein constitutive strong promoter Phsp、And applications thereof.

Background

Myceliophthora thermophila is a thermophilic fungus capable of rapidly degrading cellulose, and has higher cellulase expression and secretion capacity. Compared with the industrial cellulase production strains Trichoderma reesei and Penicillium decumbens, the myceliophthora thermophila has high enzyme activity and high thermal stability, and has wide application prospect in industrial production. Meanwhile, compared with most other non-mammalian production hosts, the myceliophthora thermophila produces glycan structures more similar to human glycan forms, and has great application potential in the field of medicine. Therefore, the myceliophthora thermophila is developed into a filamentous fungus chassis cell through genetic engineering, and has important significance for the production of industrial enzyme preparations, therapeutic proteins and vaccines.

At present, the reported constitutive strong promoters in myceliophthora thermophila are not numerous, mainly the transcription elongation factor EF-1 is startedSon Ptef-1And pyruvate decarboxylase promoter PpdcTwo kinds. When multiple proteins are required to be simultaneously over-expressed in myceliophthora thermophila, the available constitutive strong promoters for gene expression are very limited, so that the basic research and development and utilization of myceliophthora thermophila are seriously hampered, and the development of myceliophthora thermophila into industrial chassis cells is limited. Therefore, the novel constitutive strong promoter has a great application prospect and practicability for engineering transformation of myceliophthora thermophila.

Disclosure of Invention

The application aims to provide a myceliophthora thermophila small molecule heat shock protein constitutive strong promoter.

It is still another object of the present application to provide the use of the above-described myceliophthora thermophila small molecule heat shock protein constitutive strong promoter.

It is still another object of the present application to provide an expression vector comprising the above-mentioned myceliophthora thermophila small molecule heat shock protein constitutive strong promoter.

It is still another object of the present application to provide a method for expressing a gene of interest in myceliophthora thermophila.

The myceliophthora thermophila small molecule heat shock protein constitutive strong promoter PhspThe nucleotide sequence is shown as SEQ ID NO. 1.

The application provides a high promoter P containing the myceliophthora thermophila micromolecular heat shock protein constitutivehspMyceliophthora thermophila overexpression vector of (a).

The application provides a method for realizing the efficient expression of a target gene by using the myceliophthora thermophila overexpression vector.

The method for expressing a target gene in myceliophthora thermophila according to the present application comprises the steps of:

construction of the myceliophthora thermophila small molecule heat shock protein constitutive strong promoter PhspAnd an expression vector for the gene of interest;

the obtained expression vector is introduced into myceliophthora thermophila and expressed.

The application has the following beneficial effects:

the constitutive strong promoter P of the present applicationhspCan be used forIn myceliophthora thermophila, efficient expression of genes can be initiated without an inducer. The application provides a promoter P containing constitutive stronghspThe myceliophthora thermophila overexpression vector can be used for high-efficiency expression of endogenous or exogenous genes, and provides a favorable tool for engineering modification of myceliophthora thermophila.

Drawings

FIG. 1 shows overexpressionMtlpmoGenome PCR verification results of myceliophthora thermophila transformants obtained by the genes; FIG. 2 shows SDS-PAGE results of supernatant of the fermentation broth of the recombinant myceliophthora thermophila expression strain at day 4;

FIG. 3 shows recombinant proteins after separation and purificationMtSDS-PAGE results of LPMO;

FIG. 4 shows recombinant proteins after separation and purificationMtProtein mass spectrum identification results of LPMO;

FIG. 5 shows different pairs of promoters for recombinant proteinsMtEffect of Lac expression results.

Description of the embodiments

The materials, reagents, instruments and methods used in the examples below, without any particular description, are conventional in the art and are commercially available.

The gene over-expression vector construction of the application adopts high-fidelity DNA polymerase 2xPhanta Max Master Mix,Trans1-T1 clone competent cells, pEASY-Blunt Cloning Kit and pEASY-Uni Seamless Cloning and Assembly Kit kit. Myceliophthora thermophila ATCC 42464 was purchased from the American type culture Collection. Lywallases are purchased.

STC solution: 50 mM calcium chloride, 1M sorbitol, 10 mM Tris-HCl pH 7.5;

PEG solution: 25% PEG 6000, 50 mM calcium chloride, 10 mM Tris-HCl pH 7.5;

upper medium: 2% sucrose, 20 mL/L50 XVogel's saline, 1M sorbitol, 0.75% agarose; lower medium: 2% sucrose, 20 mL/L50 XVogel's saline, 1M sorbitol, 1.5% agar;

mycelium lysate: 0.05 M sodium hydroxide, 1 mM ethylenediamine tetraacetic acid, 1% triton X-100;

fermentation medium: 40 g/L glucose, 10 g/L yeast extract, 0.15 g/L potassium dihydrogen phosphate, 0.15 g/L dipotassium hydrogen phosphate, 0.10 g/L magnesium sulfate heptahydrate, 0. 0.10 g/L calcium chloride, 0.1 mg/L biotin, 1 mL/L50 XVogel's salt solution.

EXAMPLE 1 obtaining myceliophthora thermophila small molecule Heat shock protein promoter PhspSequence(s)

Primer P was designed based on the myceliophthora thermophila ATCC 42464 genome data published in the United states department of energy, genome research institute databasehsp-F (5'-CATGCATGTTGCATGATGATAAGGACTGGCCGATGCAGTC-3') and PhspR (5'-GTGGTGAGGGTGAAGGACTTCATGTTGCGTTGCTGTTG-3') PCR amplification was performed using the myceliophthora thermophila ATCC 42464 genome as template and high fidelity DNA polymerase 2X Phanta Max Master Mix. Amplification product PhspThe band gel with the size of 1624 and bp was recovered by 1% agarose gel electrophoresis, then ligated into pEASY-Blunt vector and sequenced to verify.

Myceliophthora thermophila small molecule heat shock protein promoter PhspThe sequence is shown as SEQ ID NO. 1.

EXAMPLE 2 construction of promoter P containing small molecule heat shock proteinhspA kind of electronic deviceMtlpmoGene overexpression vector

Designing primersMtlpmo-F (5'-AAGTCCTTCACCCTCACCAC-3') andMtlpmor (5'-GCCTAGGAACTTAGTGGTGGTGGTGGTGGTGGACGCACTGCGAGTAGTAG-3') PCR amplification with myceliophthora thermophila ATCC 42464 genome as template and high fidelity DNA polymeraseMtlpmoRecovery was detected by 1% agarose gel electrophoresis. Design of primer TMtlpmo-F (5'-CCACCACTAAGTTCCTAGGCAGGGCACCT-3') and TMtlpmoR (5'-CCTCTAAACAAGTGTACCTGAAAGAGCTTGCGGACAAGG-3') PCR amplification with myceliophthora thermophila ATCC 42464 genome as template and high fidelity DNA polymerase, amplification product TMtlpmoRecovery was detected by 1% agarose gel electrophoresis. Then, the primer is usedMtlpmo-F and TMtlpmoR to amplify the productMtlpmoAnd TMtlpmoPCR amplification using high fidelity DNA polymerase as template, amplified productMtlpmo-TMtlpmoRecovery was detected by 1% agarose gel electrophoresis.

Primers Vec-F (5'-CAGGTACACTTGTTTAGAGG-3') and Neo-T-R (5'-ATCATCATGCAACATGCATG-3') are designed, the plasmid pEASY-Blunt-PtrpC-Neo-T is used as a template, high-fidelity DNA polymerase is adopted for PCR amplification, and the amplified product Vec is detected and recovered by 1% agarose gel electrophoresis.

Amplification product P Using pEASY-Uni Seamless Cloning and Assembly Kit kithsp、Mtlpmo-TMtlpmoSeamless splicing with Vec, and transferring the spliced product into E.coli clone competent cellsTrans1-T1, selecting positive clone, and performing sequencing verification to obtain myceliophthora thermophilaMtlpmoGene overexpression vector pEASY-Blunt-PtrpC-neo-T-Phsp-Mtlpmo-TMtlpmo。

EXAMPLE 3 transformation, screening and recombinant proteins of myceliophthora thermophilaMtLPMO purification and identification

The myceliophthora thermophila spore suspension is coated on a potato dextrose agar plate paved with cellophane, and mycelium is collected after culturing for 15 hours at 37 ℃. Then, mycelium cell walls were digested with 0.5% lywallase, and after 2 hours of digestion at 30 ℃, protoplasts were collected by centrifugation with double-layered paper-towel filtration. The protoplasts were washed and resuspended with pre-chilled STC solution to adjust the protoplast concentration to 5X10 ⁷ And (3) obtaining protoplast suspension by using the volume per mL. 200. Mu.L of the protoplast suspension was taken and 10. Mu.L of the overexpression vector pEASY-Blunt-PtrpC-neo-T-P was addedhsp-Mtlpmo-TMtlpmoAnd 50. Mu.L of PEG solution, gently mixed, and ice-bathed for 20 minutes. Then, 2 mL of PEG solution was added and left at room temperature for 5 minutes. 4 mL of STC solution was added, and after mixing with 100. Mu.g/mL of geneticin-containing upper medium, the mixture was poured onto 100. Mu.g/mL of geneticin-containing lower medium, and after 3 days of culture at 37℃myceliophthora thermophila transformants were grown.

Transferring transformant with geneticin resistance from plate to PDA plate, culturing in 45 deg.C constant temperature incubator for 3 days, extracting genome with mycelium lysate, and collecting genome with Phsp-F2 (5'-CGATCAAAGCCACGGACATC-3') and TMtlpmoPCR verification with primers-c-R (5'-AGAATGCTTGGAGTGTCGAG-3'), as shown in FIG. 1, part of the transformant amplified a band of interest with a size of about 1.5 kb, the wild type strain without the corresponding band, indicating successful acquisition of the DNA fragment containing the DNA fragmentMtlpmoRecombinant myceliophthora thermophila expression strain of the gene expression cassette.

Randomly selecting recombinant myceliophthora thermophila expression strain according to 10 ⁶ The fermentation medium was inoculated at a concentration of individual/mL spore suspension and SDS-PAGE analysis was performed on the fermentation broth supernatant, as shown in FIG. 2, with a significant increase in extracellular protein content of recombinant myceliophthora thermophila expression strain T7 compared to the wild-type strain. Recombinant myceliophthora thermophila expression strain T7 is subjected to recombinant protein by collecting fermentation liquor on day 4 and utilizing a nickel affinity chromatographic columnMtPurification of LPMO, as shown in FIG. 3, yields an electrophoretically pure eluted fraction, the molecular weight of the protein and the recombinant protein of interestMtLPMO is equivalent; further carrying out protein mass spectrum identification on the eluted component, as shown in figure 4, totally identifying 11 specific peptide fragments matched with target recombinant proteinMtLPMO demonstrated that the eluted fraction was indeed recombinant proteinMtLPMO. These results indicate that myceliophthora thermophila small molecule heat shock protein promoter PhspHas promoter activity.

Designing primersMtlac-F (5'-ATGAAGTCCTTCATCAGCGC-3') andMtlacr (5'-CCCTCGCTCAGTGGTGGTGGTGGTGGTGCGCCTTGACCAGCCACTC-3') PCR amplification with myceliophthora thermophila ATCC 42464 genome as template and high fidelity DNA polymeraseMtlacRecovery was detected by 1% agarose gel electrophoresis.

Design of primer TMtlac-F (5'-CCACCACCACTGAGCGAGGGAGGAAAAAGGAAAC-3') and TMtlacR (5'-CCTCTAAACAAGTGTACCTGGCAGTCCTTGCAGATACTGC-3') PCR amplification with myceliophthora thermophila ATCC 42464 genome as template and high fidelity DNA polymerase, amplification product TMtlacRecovery was detected by 1% agarose gel electrophoresis.

Then, the primer is usedMtlac-F and TMtlacR to amplify the productMtlacAnd TMtlacPCR amplification using high fidelity DNA polymerase as template, amplified productMtlac-TMtlacRecovery was detected by 1% agarose gel electrophoresis.

Primers Vec-F (5'-CAGGTACACTTGTTTAGAGG-3') and P were designedhsp-lcc-R（5'-GCGCTGATGAAGGACTTCATGTTGCGTTGCTGTTGTTTTTTAAGCTGTTGCCGAGTTGCTG-3') was expressed as plasmid pEASY-Blunt-PtrpC-neo-T-Phsp-Mtlpmo-TMtlpmoAs a template, PCR amplification is carried out by adopting high-fidelity DNA polymerase, and the amplified product Vec-1 is detected and recovered by 1% agarose gel electrophoresis. Amplification product pairs Using the pEASY-Uni Seamless Cloning and Assembly Kit kitMtlac-TMtlacSeamless splicing with Vec-1, and transferring the spliced product into E.coli clone competent cellsTrans1-T1, selecting positive clone, and performing sequencing verification to obtain myceliophthora thermophilaMtlacGene overexpression vector pEASY-Blunt-PtrpC-neo-T-Phsp-Mtlac-TMtlac。

Primer P was designed based on the myceliophthora thermophila ATCC 42464 genome data published in the United states department of energy, genome research institute databasetef-1-F (5'-CATGCATGTTGCATGATGATGTAATGGCAGCACCTTGACG-3') and Ptef-1R (5'-GCGCTGATGAAGGACTTCATTTTGACGGTATTTGTGTTCTGAAGAACGAAACTGGC-3') PCR amplification was performed using the myceliophthora thermophila ATCC 42464 genome as template and high fidelity DNA polymerase 2X Phanta Max Master Mix. Amplification product Ptef-1The band gel with the size of about 1818 and bp was recovered by 1% agarose gel electrophoresis, then ligated into pEASY-Blunt vector and sequenced to verify. Myceliophthora thermophila transcription elongation factor EF-1 promoter Ptef-1The sequence is as follows: (SEQ ID NO: 2)

Primer P was designed based on the myceliophthora thermophila ATCC 42464 genome data published in the United states department of energy, genome research institute databasegpd-F (5'-CATGCATGTTGCATGATGATGATGGAGGACTACCATCGTC-3') and PgpdR (5'-GCGCTGATGAAGGACTTCATTTTGATTTCTGGAGGGATGAGGGAGCTTTG-3') PCR amplification was performed using the myceliophthora thermophila ATCC 42464 genome as template and high fidelity DNA polymerase 2X Phanta Max Master Mix. Amplification product PgpdThe band gel with the size of 1428 and bp was recovered by 1% agarose gel electrophoresis, then ligated into pEASY-Blunt vector and sequenced to verify. Myceliophthora thermophila glyceraldehyde-3-phosphate dehydrogenase promoter PgpdThe sequence is shown as SEQ ID NO. 3.

Designing primersMtlacF (5'-ATGAAGTCCTTCATCAGCGC-3') and Neo-T-R (5'-ATCATCATGCAACATGCATG-3') as plasmids pEASY-Blunt-PtrpC-neo-T-Phsp-Mtlac-TMtlacAs a template, PCR amplification is carried out by adopting high-fidelity DNA polymerase, and the amplified product Vec-2 is detected and recovered by 1% agarose gel electrophoresis. Amplification product P Using pEASY-Uni Seamless Cloning and Assembly Kit kittef-1Or PgpdSeamless splicing with Vec-2, and transferring the spliced product into E.coli clone competent cellsTrans1-T1, selecting positive clone, and performing sequencing verification to obtain myceliophthora thermophilaMtlacGene overexpression vector pEASY-Blunt-PtrpC-neo-T-Ptef-1-Mtlac-TMtlacAnd pEASY-Blunt-PtrpC-neo-T-Pgpd-Mtlac-TMtlac。

Then, the overexpression vector pEASY-Blunt-PtrpC-neo-T-P was usedtef-1-Mtlac-TMtlac、pEASY-Blunt-PtrpC-neo-T-Pgpd-Mtlac-TMtlacTransformation of myceliophthora thermophila protoplast to obtain a myceliophthora thermophila protoplast containing different promotersMtlacRecombinant myceliophthora thermophila expression strain of gene expression cassette according to 10 ⁶ Inoculating the fermentation medium at a concentration of each mL of spore suspension, and carrying out laccase enzyme activity determination on the supernatant protein of the fermentation liquid. As shown in FIG. 5, the different promoters are specific for myceliophthora thermophila recombinant proteinMtThe expression influence difference of Lac is obvious, wherein the Lac contains small molecule heat shock protein promoter PhspRecombinant of (2)MtThe myceliophthora thermophila expression strain has the highest enzyme activity (775+/-120U/L), and then contains a transcription elongation factor EF-1 promoter Ptef-1Recombinant of (2)MtMyceliophthora thermophila expression strain (605+/-30U/L) containing glyceraldehyde-3-phosphate dehydrogenase promoter PgpdRecombinant of (2)MtThe myceliophthora thermophila expression strain has no enzyme activity.

The above embodiments are only for understanding the technical solution of the present application, and do not limit the protection scope of the present application.

Claims (5)

1. Myceliophthora thermophila small molecule heat shock protein constitutive strong promoter PhspCharacterized in that the myceliophthora thermophila micromolecular heat shock protein constitutive strong promoter PhspThe nucleotide sequence of (2) is shown as SEQ ID NO. 1.

2. Comprising the following claims1A strong promoter P for the small molecule heat shock protein composition of myceliophthora thermophilahspMyceliophthora thermophila overexpression vector of (a).

3. The myceliophthora thermophila small molecule heat shock protein constitutive strong promoter P of claim 1hspIs used in the application of (a).

4. The use according to claim 3, wherein said myceliophthora thermophila small molecule heat shock protein constitutive strong promoter PhspIs used for expressing the target gene in myceliophthora thermophila.

5. A method for expressing a gene of interest in myceliophthora thermophila, comprising the steps of:

construction of a constitutive strong promoter P comprising the myceliophthora thermophila small molecule heat shock protein according to claim 1hspAnd an expression vector for the gene of interest;

the obtained expression vector is introduced into myceliophthora thermophila and expressed.