CN113186190B

CN113186190B - Blue light mediated regulation plasmid and construction method and application thereof

Info

Publication number: CN113186190B
Application number: CN202110571089.7A
Authority: CN
Inventors: 张后今; 闫云君; 王志乾; 龚梦瑶; 韩娟; 张婷
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2021-05-25
Filing date: 2021-05-25
Publication date: 2022-05-03
Anticipated expiration: 2041-05-25
Also published as: CN113186190A

Abstract

The invention discloses a blue light regulation promoter PC120 capable of being identified and combined by blue light protein SV40-VP-EL, belonging to the technical field of microorganisms. The invention also discloses a blue light mediated regulation plasmid containing the blue light regulation promoter PC120, and the plasmid also comprises a fusion gene PGAP-SV40-VP-EL for transcribing and translating the blue light protein. The invention further discloses a preparation method of the plasmid and application of the plasmid and/or the construction method in pichia pastoris regulation. The blue light mediated regulation plasmid provides a non-invasive blue light regulation mode with regulation reversibility and high space-time resolution for the induction expression of pichia pastoris, and is beneficial to widening the regulation method of pichia pastoris in production and life.

Description

Blue light mediated regulation plasmid and construction method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a blue-light mediated regulation plasmid and a construction method and application thereof.

Background

Most of the traditional regulation methods of gene expression process are the artificial control of gene expression by combining exogenous chemical inducer with soluble transcription factor, and the method is one of the main driving forces for promoting the rapid development of biotechnology, synthetic biology and medical research in the last century. While these traditional regulation approaches can initially achieve time and expression control of gene expression, their limited reversibility and their inducer-based transport regulation mechanisms have presented limitations and drawbacks, such as: its potential off-target effects, delayed transport processes and toxicity. Under ideal conditions, the biological regulation and control element can be rapidly and accurately turned on or off randomly, the capability of analyzing and controlling a complex biological gene network is effectively improved, and the time regulation precision of a light regulation mode can reach the millisecond range.

The EL222 Light-sensitive protein derived from rhodobacter vitis (Erythrobacter litoralis) can effectively realize Light regulation operation, the EL222 is composed of 222 amino acids, the N-terminal is a Light-oxidation regulatory domain (LOV), the C-terminal is a helix-turn-helix (HTH) DNA binding domain, and the C-terminal of the LOV is a J α helix connecting the LOV and the HTH. The core of LOV is the Per-Arnt-sim (pas) domain, which binds to Flavin Mononucleotide (FMN). Belongs to a system for photo-induced homodimerization. Under blue light illumination, the combination of FMN and LOV causes the J α helix to wobble from PAS, releasing the HTH, allowing EL222 to dimerize and bind to DNA. In the dark, EL222 spontaneously reverses when the N-terminal LOV domain inhibits the DNA-binding C-terminal HTH domain, rapidly inactivating EL 222. At present, the EL222 protein has been preliminarily verified in Escherichia coli and Saccharomyces cerevisiae, but the applicability thereof has not been reported in Pichia pastoris.

Pichia pastoris, as an eukaryote, has many of the advantages of eukaryotic expression systems, such as protein processing, folding, post-translational modifications, etc., and is easier to handle. Compared with eukaryotic expression systems such as mammalian cell culture and baculovirus, the pichia pastoris expression system has the advantages of rapidness, simplicity, cheapness, high expression level and the like. The pichia pastoris has similar molecular and genetic operation advantages as the saccharomyces cerevisiae, and the expression level of the foreign protein is ten times or even hundreds times of that of the saccharomyces cerevisiae. The advantages make pichia pastoris the first choice for eukaryotic protein expression systems. However, the induction regulation mode of pichia pastoris is still in the stage of exogenous chemical inducer, and the inducer used most often is strong in methanol toxicity and flammable, so that the application of pichia pastoris in the fields of food, medicine, agricultural product processing and the like is not facilitated. Therefore, the development of a novel light regulation mode of pichia pastoris can effectively solve the defects of toxicity, instability, transport delay and the like of the traditional chemical inducer, and expands a method for regulating and controlling pichia pastoris expression, which becomes a technical problem to be urgently solved in the field.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a blue light regulation promoter PC120 which can quickly and accurately realize blue light induced expression of a target gene.

The invention also aims to provide a blue-light mediated regulation plasmid, a construction method and application thereof, which can be used for blue-light induced expression of pichia pastoris, and the regulation mode is non-toxic and harmless, quick in response, simple and convenient to operate, good in reversibility and high in space-time resolution.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a blue light regulation promoter PC120, and the nucleic acid sequence of the promoter is shown in SEQ ID No. 1.

Preferably, the promoter is recognized and bound by the blue light protein SV 40-VP-EL.

Preferably, the amino acid sequence of the blue light protein SV40-VP-EL is shown as SEQ ID NO. 2.

Preferably, the nucleic acid sequence of the blue light protein SV40-VP-EL is shown as SEQ ID NO. 3.

The invention also provides a blue light mediated regulation plasmid, which comprises the blue light regulated promoter PC120 and a fusion gene PGAP-SV40-VP-EL of transcription and translation blue light protein.

Preferably, the nucleic acid sequence of the fusion gene PGAP-SV40-VP-EL is shown as SEQ ID NO. 4.

The invention also provides a construction method of the blue light mediated regulation plasmid, which comprises the following construction steps:

replacing sequences of pAO815 plasmid AOX1promoter, AOX1promoter and AOX1terminator by fusion gene PGAP-SV40-VP-EL by using pAO815 plasmid as a skeleton to obtain plasmid pGSVEA; and replacing the CGTTCGTTTGTGC sequence of the plasmid pGSVEA by another fusion gene containing the promoter PC 120.

Preferably, the fusion gene comprising the promoter PC120 further comprises a reporter gene and/or a target gene.

Preferably, the fusion gene is site-directed assembled on the pAO815 plasmid by means of Gibsonassambly.

The invention also provides the application of the blue light mediated regulation plasmid and/or the construction method of the blue light mediated regulation plasmid in pichia pastoris induced expression.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the invention provides a novel blue light induction regulation mode for an induction expression method of pichia pastoris. The method adopts a light regulation mode to carry out induction expression on the pichia pastoris, has the advantages of no toxicity, no harm, quick response, simple and convenient operation, good reversibility and high space-time resolution, and promotes the wide application of the pichia pastoris in the fields of food, medicine, agricultural product processing and the like.

The invention can realize blue light induced expression of target genes only by one plasmid, and expands a method for regulating and controlling expression of pichia pastoris.

Drawings

FIG. 1 is a schematic diagram of the mechanism of blue light regulatory plasmid.

FIG. 2 is a diagram of pGSVEACA vector, in which PpHIS4 is Pichia integration site and selection marker, AmpR is antibiotic resistance, ori is replication initiation site, GAP promoter is Pichia GAP promoter, SV40 is nuclear localization signal, VP16 is transcriptional activation domain, EL222 is blue light response protein, AOX1terminator is yeast AOX1terminator, PC120 is blue light regulation promoter, GFP is green fluorescent protein, ADH 1terminator is Saccharomyces cerevisiae ADH1 terminator.

FIG. 3 shows the cloning of Pichia pastoris transformed with pGSVEACA vector on MD plates.

FIG. 4 shows the positive clone of blue light regulation engineering bacteria genome PCR verification.

FIG. 5 shows an MD plate with blue light-regulating engineering bacteria induced for 12h under blue light condition and dark condition, the left is dark condition and the right is blue light condition.

FIG. 6 shows MD shake flask cultures induced by blue Light regulated engineering bacteria under blue Light and Dark conditions, respectively, (A) fluorescence intensities of experimental group Full Light and control group Full Dark 1h after blue Light induction; (B) fluorescence intensity of experimental group Full Light and control group Full Dark after 4h of blue Light induction; (C) fluorescence intensity of experimental group Full Light and control group Full Dark after 7h of blue Light induction; (D) fluorescence intensity of experimental group Full Light and control group Full Dark after 10h of blue Light induction; (E) trend plots of the mean fluorescence intensity values of hourly samples taken during blue Light induction for the experimental group Full Light and the control group Full Dark.

Detailed Description

The invention provides a blue light regulation promoter PC120, and the nucleic acid sequence of the promoter is shown in SEQ ID No. 1. The promoter PC120 can be identified and combined by blue light protein SV40-VP-EL, so that blue light induced expression of target genes can be rapidly and accurately realized.

The amino acid sequence of the blue light protein SV40-VP-EL is shown as SEQ ID NO.2, and the nucleic acid sequence is shown as SEQ ID NO. 3. The nucleic acid sequence of the blue light protein SV40-VP-EL of the invention may also be a variant of the remaining nucleic acid sequence of the protein SV 40-VP-EL.

The invention provides a blue light mediated regulation plasmid, which comprises the blue light regulated promoter PC120 and a fusion gene PGAP-SV40-VP-EL of transcription and translation blue light protein. PGAP in the fusion gene PGAP-SV40-VP-EL represents Pichia pastoris GAP promoter, SV40 represents nuclear localization signal SV40, VP represents transcription activation domain VP16, and EL represents blue light response protein EL 222.

In the present invention, the nucleic acid sequence of PGAP is the GAP 1promoter sequence of Pichia pastoris GS115 ATCC:20864 (GenBank accession No: CP014716REGION:809514.. 809990).

The nucleic acid sequence of the fusion gene PGAP-SV40-VP-EL is shown in SEQ ID NO. 4.

The invention also provides a construction method of the blue light mediated regulation plasmid, which comprises the following steps: replacing sequences of pAO815 plasmid AOX1promoter, AOX1promoter and AOX1terminator by fusion gene PGAP-SV40-VP-EL by using pAO815 plasmid as a skeleton to obtain plasmid pGSVEA; and replacing the CGTTCGTTTGTGC sequence of the plasmid pGSVEA by another fusion gene containing the promoter PC120 to obtain the blue light regulation plasmid pGSVEACA. The pGSVEACA plasmid constructed by the present invention is shown in FIG. 2.

The plasmid pGSVEA obtained by the invention can be transcribed and translated to obtain the blue light protein SV40-VP-EL after being introduced into pichia pastoris, and preferably, the pichia pastoris is GS 115.

The blue light regulation plasmid pGSVEACA obtained by the invention can be transcribed and translated to obtain blue light protein SV40-VP-EL, under the irradiation of blue light, the N end (photooxidation regulation domain) and the C end (helix-turn-helix DNA binding domain) of the photosensitive protein EL222 in the blue light protein SV40-VP-EL interact with each other, so that the EL222 is dimerized and combined with the promoter PC120, and the transcription of another fusion gene containing the promoter PC120 is started.

In the present invention, another fusion gene containing the promoter PC120 is PC120-GFP-TADH1, wherein PC120 represents the blue light-regulated promoter PC120, GFP represents green fluorescent protein GFP, TADH1 represents yeast ADH 1terminator ADH 1terminator, and the nucleic acid sequence of TADH1 is ADH 1terminator sequence of Saccharomyces cerevisiae Y169 (GenBank accession No: CP033484REGION:156786.. 157080). In the present invention, the green fluorescent protein GFP can be replaced by other reporter genes, and can also be replaced by other target genes.

The nucleic acid sequence of the fusion gene PC120-GFP-TADH1 is shown in SEQ ID NO. 5.

In the invention, two fusion genes can be assembled on the pAO815 plasmid in a site-specific manner by means of Gibsonassambly. The present invention is not limited to a specific assembly method.

The invention also provides application of the construction method of the blue light mediated regulatory plasmid and/or the blue light mediated regulatory plasmid in pichia pastoris induced expression. According to the invention, the blue light regulation plasmid pGSVEACA is transformed and introduced into the pichia pastoris, so that blue light induced expression of a target gene can be realized, a method for regulating and controlling expression of the pichia pastoris is expanded, and the wide application of the pichia pastoris in the fields of food, medicine, agricultural product processing and the like is promoted.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

This example optimizes the gene sequence.

VP16 and EL222 gene sequences and protein sequences retrieved from NCBI are optimized according to pichia pastoris codon preference, and DNA sequences are synthesized again.

The specific optimization method comprises the following steps: optimumGene according to gene synthesis company Kinsery codon optimization software^TMOptimizing the gene, mainly referring to the codon preference of pichia pastoris, and finally completing the optimization of the gene codon by combining a filter element and balancing the GC content of the gene to obtain an SV40-VP-EL nucleic acid sequence shown as SEQ ID No. 3; a PGAP-SV40-VP-EL nucleic acid sequence shown as SEQ ID NO. 4; the PC120-GFP-TADH1 nucleic acid sequence is shown as SEQ ID NO. 5.

Example 2

In this example, a blue light regulation plasmid pGSVEACA was designed and constructed with the plasmid pAO815 commonly used in Pichia pastoris as a backbone.

The specific construction method comprises the following steps:

the PGAP-SV40-VP-EL gene sequence obtained by optimization in example 1 is completely synthesized, and then a Gibson assembly technology is used for replacing pAO815 plasmid AOX1promoter and the sequence between AOX1promoter and AOX1terminator to obtain plasmid pGSVEA;

the PC120-GFP-TADH1 gene sequence obtained through optimization in example 1 is subjected to total synthesis, and then the CGTTCGTTTGTGC sequence of the plasmid pGSVEA is replaced by a Gibson assembly technology, so that the blue light regulation plasmid pGSVEACA is obtained.

In the embodiment, the PGAP-SV40-VP-EL gene sequence does not contain a terminator sequence, so that an AOX1terminator on the pAO815 plasmid skeleton is needed to be used to construct a complete SV40-VP-EL open reading frame, and the constitutive expression of SV40-VP-EL is realized.

In this example, in order to achieve the effect of blue light induction by a single plasmid, the PC120-GFP-TADH1 gene sequence needs to be inserted into the downstream of PGAP-SV40-VP-EL on the basis of the constructed plasmid pGSVEA, so the PC120-GFP-TADH1 gene sequence is used to replace the CGTTCGTTTGTGC sequence of the plasmid pGSVEA.

Example 3

This example describes transformation, inducible expression and analysis of the blue light-modulating plasmid pGSVEACA.

(1) Extraction and quantitative detection of plasmid

1) 10mL of overnight culture of the cells were collected, and the resulting mixture was centrifuged at 13400g for 1min to discard the supernatant and collect the precipitate.

2) Adding 500 mu L of solution I, solution II and solution III in sequence, immediately turning the mixture gently up and down for 6-8 times, standing the mixture for 5min, and centrifuging the mixture for 10min at 13400 g.

Wherein solution i had a composition of 25mM Tris-HCl (pH 8.0), 10mM EDTA, 50mM glucose;

the composition of solution II was 250mM NaOH, 1% (W/V) SDS;

the composition of solution III was 3M potassium acetate, 5M acetic acid.

3) Adding the supernatant collected in the last step into a filter column, centrifuging at 13400g for 1min, adding 450 mu L of isopropanol, and mixing uniformly. Then adding the mixture into an adsorption column, centrifuging the mixture for 1min at 13400g, and then discarding waste liquid.

4) Adding 700 μ L of rinsing solution into adsorption column, centrifuging at 13400g for 1min, and discarding waste liquid.

5) After the rinsing liquid is removed by the adsorption column, 150 mu L of deionized water is dripped into the center of the adsorption membrane, and the adsorption membrane is kept stand for 2min and then centrifuged at 13400g for 2 min.

6) The tube was collected and the plasmid pGSVEACA concentration was determined and stored at-20 ℃ until use.

(2) Linearization and recovery of plasmid pGSVEACA

1) To the sample was added 1/10 volumes of 3M sodium acetate solution and 2.5 volumes of absolute ethanol, mixed well, and left at-20 ℃ for 1 h. Then, the mixture was centrifuged at 12000rpm for 10min, and the supernatant was discarded.

2)2.5 times volume of pre-cooled 75% ethanol heavy suspension precipitation, 12000rpm centrifugation for 10min, abandon the supernatant. And blowing on a clean bench for 15 min.

3) 25 μ L of deionized water was added dropwise to the bottom of the tube to resuspend and measure the plasmid concentration, and the mixture was stored at-20 ℃ for further use.

(3) Electrotransformation of Pichia pastoris GS115

1) 5-10 mu g of linearized plasmid pGSVEACA is added into the prepared pichia pastoris GS115 competent cells and mixed evenly, and the mixture is transferred into a new precooled electric rotating cup (the specification of 0.2 cm) and ice-bathed for 5 min.

2) The electric beaker was quickly removed and wiped dry, shocked under voltage 1.5kV, capacitance 25 μ F, resistance 200 Ω, 1mL of pre-cooled 1M sorbitol was immediately added, the liquid was aspirated and transferred to a new 1.5mL sterile centrifuge tube, incubated at 30 ℃ for 1.5 h.

3) Most of the supernatant was discarded after centrifugation at 12000rpm for 20s, and the cells were resuspended and plated on MD selection plates and incubated at 28 ℃ for 2 days until single colonies visible to the naked eye appeared. The results of yeast transformation are shown in FIG. 3.

(4) Induction expression and fluorescence intensity qualitative analysis of pichia pastoris transformant

Transformed yeast transformants were randomly picked and inoculated into 5mL MD liquid medium and shake-cultured for 19h at 28 ℃. Preserving bacteria, extracting genome DNA, designing gene positive detection primer for PCR amplification to obtain positive strain. The primers used in the PCR of the present invention are shown in Table 1.

TABLE 1 PCR primers

In Table 1, the GAP-F sequence is shown as SEQ ID NO.6, the ADH-R sequence is shown as SEQ ID NO.7, the AOX-R sequence is shown as SEQ ID NO.8, and the AOX-F sequence is shown as SEQ ID NO. 9.

The PCR amplification results are shown in FIG. 4, which indicates that the yeast transformants cultured by picking contain the plasmid of interest.

And selecting 3-5 positive clones according to the PCR detection result, streaking the positive clones on an MD culture medium, culturing the positive clones in a dark environment at the temperature of 28 ℃ for 24 hours, and then carrying out blue light induction incubation on the experimental group.

After the blue light is induced for 24 hours, the experimental group and the control group are placed under excitation light of 475nm for qualitative comparison of fluorescence intensity, the comparison effect is shown in fig. 5, it can be seen that the strain after the blue light induction emits green fluorescence under the excitation light of 475nm, which indicates that green fluorescent protein is obtained through transcription and translation in pichia pastoris cells after the blue light induction, and the PC120 promoter can successfully recognize and combine with the blue light protein SV40-VP-EL, so that the transcription and translation of the reporter gene is accurately started.

(5) Flow cytometry analysis of Pichia transformants

Selecting 3-4 positive clones according to a PCR detection result, transferring 50-100 mu L of culture solution into a 50mLMD liquid culture medium respectively, performing shake culture at 28 ℃ in a dark environment until the OD 600nm of the thallus reaches 0.8-1.2, and performing blue light induction expression. Blue light induction the experimental group and the control group were sampled every 1h for a total of 10h induction.

The method comprises the following steps of pretreating a sample to be analyzed by flow cytometry, wherein the method comprises the following specific operations: and centrifuging the sample bacterium liquid at 4 ℃ at 5000rpm for 1min, discarding the supernatant, then resuspending the sample bacterium liquid by using PBS and diluting the sample bacterium liquid to a state invisible to the naked eye, filtering the sample diluted liquid with a proper volume by using a 300-mesh filter screen, and storing the sample diluted liquid in a flow analysis tube for flow cytometry analysis.

The relationship between the fluorescence intensity and the induction time obtained after the flow cytometry analysis of the samples of the experimental group and the control group is shown in table 2 and fig. 6.

TABLE 2 fluorescence intensity of the cells under blue light induction and dark conditions for different periods of time

As can be seen from Table 2 and FIG. 6, the fluorescence intensity of GFP reached the highest after 8 hours of continuous irradiation with blue light, which was 16.4 times higher than that of the contemporary control group. A single and uniform peak is shown in the histogram, indicating that blue light can achieve a uniform induction effect on GS115 cells carrying pGSVEACA plasmid. And the cross overlapping parts of the histograms of the experimental group and the control group are not much, which indicates that the induction intensity is relatively good. Overall, the flow cytometry results indicated that pGSVEACA plasmids could efficiently achieve efficient and uniform response of GS115 cells to blue light.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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actacttttg gttacggtgt tcaatgtttc gctagatatc ctgatcatat gaagcaacac 480

gatttcttta agtctgctat gccagaaggt tacgttcaag agagaactat tttctttaag 540

gatgatggta actacaagac tagagctgag gttaagtttg agggagatac tttggttaac 600

agaatcgaat tgaagggtat tgatttcaag gaggatggta acatcttggg tcataagttg 660

gaatacaact acaactctca caacgtttac attatggctg ataagcaaaa gaacggtatt 720

aaggttaact tcaagatcag acataacatc gaggatggtt ctgttcaatt ggctgatcac 780

taccaacaaa acactcctat tggagatggt cctgttttgt tgccagataa tcactatttg 840

tctactcaat ctgctttgtc taaagatcca aacgaaaaga gagatcatat ggttttgttg 900

gagtttgtta ctgctgctgg tattactcac ggtatggatg aattgtataa ataaggtacc 960

gaacaaaaac tcatctcaga agaggatctg aatagcggcg gccgccatca tcatcatcat 1020

cattgagttt tagccttaga catgactgtt caagttgggc acttacgaga agtaaataag 1080

ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat 1140

tcttgttctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta tagcatgagg 1200

tcgctcttat tgaccacacc tctaccggca tgccgagcaa atgcctgcaa atcgctcccc 1260

atttcaccca attgtagata tgctaactcc agcaatgagt tgatgaatct cggtgtgtat 1320

tttatgtcct cagaggacaa cacctgttgt aatcgttctt ccacacg 1367

<210> 6

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gatttttgta gaaatgtctt ggtgtcctc 29

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgtgtggaag aacgattaca acag 24

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggctagcgaa gatctcccg 19

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cgggagatct tcgctagcc 19

Claims

1. A blue light mediated regulatory plasmid comprising a blue light regulated promoter PC120 and a fusion gene PGAP-SV40-VP-EL for transcription and translation of a blue light protein;

the nucleic acid sequence of the fusion gene PGAP-SV40-VP-EL is shown in SEQ ID NO. 4;

the nucleic acid sequence of the promoter is shown as SEQ ID NO. 1.

2. The blue-light mediated regulatory plasmid of claim 1, wherein the promoter is recognized and bound by the blue-light protein SV 40-VP-EL.

3. The blue light-mediated regulatory plasmid of claim 2, wherein the amino acid sequence of the blue light protein SV40-VP-EL is shown as SEQ ID No. 2.

4. The blue light-mediated regulatory plasmid of claim 2, wherein the nucleic acid sequence of the blue light protein SV40-VP-EL is shown as SEQ ID No. 3.

5. The method for constructing the blue-light mediated regulatory plasmid according to any one of claims 1 to 4, wherein the construction step comprises:

replacing the pAO815 plasmid AOX1promoter and the sequence between the AOX1promoter and the AOX1terminator by the fusion gene PGAP-SV40-VP-EL by taking the pAO815 plasmid as a framework to obtain a plasmid pGSVEA; and replacing the CGTTCGTTTGTGC sequence of the plasmid pGSVEA by another fusion gene containing the promoter PC 120.

6. The method according to claim 5, wherein the fusion gene comprising the promoter PC120 further comprises a reporter gene and/or a target gene.

7. The method of claim 5, wherein the fusion gene is site-specifically assembled on the pAO815 plasmid by means of Gibsonassambly.

8. The use of the blue-light mediated regulatory plasmid according to any one of claims 1 to 4 and/or the method of constructing the blue-light mediated regulatory plasmid according to any one of claims 5 to 7 in pichia pastoris inducible expression.