CN110616162A

CN110616162A - Pichia pastoris for expressing flavone synthase

Info

Publication number: CN110616162A
Application number: CN201910923991.3A
Authority: CN
Inventors: 周金林; 周志华; 叶德晓; 王平平; 严兴
Original assignee: Foshan Huiteng Biotechnology Co Ltd
Current assignee: Foshan Huiteng Biotechnology Co Ltd
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2019-12-27
Anticipated expiration: 2039-09-27
Also published as: CN110616162B

Abstract

The invention provides pichia pastoris for expressing flavone synthase, which contains an expression vector, wherein the expression vector contains polynucleotide shown as SEQ ID NO. 3 or SEQ ID NO. 4. The invention discloses a method for expressing FNS I (flavone synthase) by pichia pastoris, which has the advantages of stable expression and high efficiency and can be used for constructing and optimizing flavone compound cell factories.

Description

Pichia pastoris for expressing flavone synthase

Technical Field

The invention relates to the technical field of biological enzymes, and particularly relates to pichia pastoris for expressing flavone synthase.

Background

Flavonoid compoundsThe compounds are important plant natural compounds with high structural diversity, 9000 flavonoid compounds with different structures are separated at present, and can be divided into flavones (flavanones), flavanones (flavanones) and isoflavones (isoflavone) according to the structuresisoflavone) Flavanols, flavonols and anthocyanidins. Among them, flavones are the largest type among flavonoids. The flavone not only has wide application to the physiology, ecology and agriculture of plants, but also has remarkable treatment in the aspects of preventing and treating diseases such as cancer, cardiovascular diseases and the like. For example, apigenin (apigenin) is a flavonoid that is distributed in some warm tropical vegetables and fruits, especially in high celery content. Apigenin has multiple biological activities such as anti-tumor, anti-inflammatory and antioxidant effects, has excellent anti-tumor activity, can inhibit tumor cell proliferation, induce tumor cell apoptosis, inhibit tumor invasion and metastasis, and improve chemotherapy drug sensitivity and antioxidant effect (Chengting, etc., Chinese modern application pharmacy, 2019). Luteolin (luteolin) is also a flavonoid compound present in various vegetables and medicinal plants, and has various pharmaceutical activities including anti-tumor, anti-oxidation, anti-inflammatory, protecting nervous system, etc. (wangnao et al, life science, 2013). Chrysin (chrysin) is a flavonoid compound with wide pharmacological activity, can be extracted from oroxylum indicum of bignoniaceae, is one of main effective components of propolis, and has various pharmacological effects of resisting tumors, preventing and treating cardiovascular and cerebrovascular diseases and the like (ginger, golden ginger and the like, Chinese herbal medicines, 2011). Flavone has been increasingly regarded as important in drug development and design due to its excellent pharmacological activity (Stefan Martens et al, Molecules of Interest, 2005). Therefore, how to prepare these flavone compounds in large quantities is receiving more and more attention.

The chemical synthesis method of flavone mainly has two modes, one is a chemical total synthesis method, and the industrial synthesis method uses 2, 4-dimethoxy-6-hydroxyacetophenone as a raw material and anisic aldehyde to obtain apigenin through condensation, oxidation of aureole and high-temperature demethylation; secondly, a chemical semi-synthesis method is adopted, naringenin is obtained by hydrolyzing naringin serving as a raw material, and then the naringenin is dehydrogenated by pyridine and iodine under an alkaline condition to obtain apigenin; or subjecting naringin as raw material to oxidative dehydrogenation with iodine under alkaline condition to obtain apigenin, extracting to obtain apigenin, and hydrolyzing with acid. Chemical synthesis of other flavone compounds was carried out using a similar strategy.

The biosynthesis of flavones comprises the steps of firstly synthesizing chalcone derivatives from three molecules of malonyl-CoA and one molecule of p-coumaroyl-CoA or one molecule of cinnamoyl-CoA or one molecule of caffeoyl-CoA, then converting the chalcone derivatives into flavanones, and finally generating flavones from the flavanones under the catalysis of flavone synthase (FNS). In the biosynthetic pathway of flavones, flavone synthase is the rate-limiting enzyme among them. It has now been found that there are two distinct FNSs in plants, one of which is FNS I, which is a soluble dioxygenase, and the other is FNS II, which is a membrane-bound cytochrome P450 enzyme. Many of the low cost flavanone compounds can be converted to more pharmaceutically valuable flavone compounds by the catalytic action of FNS, for example, naringenin to apigenin, eriodictyol (eriodicytol) to luteolin and pinocembrin (pinocembrin) to luteolin. Through comparative analysis of documents, the catalytic efficiency of most FNS I is higher than that of FNSII (Effect Leonard, APPLID AND ENVIRONMENTAL MICROBIOLOGY, 2005), but the conversion efficiency of the FNS I of the existing type cannot meet the requirements of production AND scientific research.

Disclosure of Invention

The technology discloses that pichia pastoris is used for realizing high expression of FNS I, so that the existing production and scientific research requirements are met.

Pichia pastoris for expressing flavone synthase is integrated with an expression vector, and the expression vector contains polynucleotide shown as SEQ ID NO. 3 or SEQ ID NO. 4.

Preferably, the pichia pastoris is pichia pastoris GS115, and the expression vector is pPIC9K-DcFNS or pPIC 9K-AgFNS; the pPIC9K-DcFNS contains a polynucleotide shown as SEQ ID NO. 3; the pPIC9K-AgFNS contains the polynucleotide shown in SEQ ID NO. 4. The recombinant Pichia pastoris GS115-pPIC9K-DcFNS is an expression strain containing an expression vector pPIC9K-DcFNS, and can express flavone synthase of an amino acid sequence shown in SEQ ID NO. 1 through induction. The recombinant Pichia pastoris GS115-pPIC9K-AgFNS is an expression strain containing an expression vector pPIC9K-AgFNS, and can express flavone synthase of an amino acid sequence shown in SEQ ID NO. 2 through induction.

The technology simultaneously discloses construction methods of the two pichia pastoris, which respectively comprise the following steps:

recombinant pichia pastoris GS115-pPIC 9K-DcFNS:

1) constructing a recombinant plasmid pPIC 9K-DcFNS: adding Sal I and Not I restriction sites at both ends of the polynucleotide shown as SEQ ID NO. 3, and connecting the polynucleotide into a pPIC9K vector subjected to double restriction by Sal I and Not I to obtain a recombinant plasmid pPIC 9K-DcFNS;

2) cloning of the recombinant plasmid pPIC 9K-DcFNS: transforming the recombinant plasmid pPIC9K-DcFNS into escherichia coli Top10 to construct recombinant escherichia coli Top10-pPIC9K-DcFNS, and extracting the recombinant plasmid pPIC9K-DcFNS after amplifying and culturing the recombinant escherichia coli Top10-pPIC 9K-DcFNS;

3) constructing the recombinant pichia pastoris: and (3) carrying out enzyme digestion linearization on the recombinant plasmid pPIC9K-DcFNS obtained in the step (2) by Sal I, then carrying out electric transformation on the linearized plasmid into pichia pastoris GS115, and screening positive clones to obtain the recombinant pichia pastoris GS115-pPIC 9K-DcFNS.

And the number of the first and second groups,

recombinant pichia pastoris GS115-pPIC 9K-AgFNS:

I) constructing a recombinant plasmid pPIC 9K-AgFNS: adding Sal I and Not I restriction sites at both ends of the polynucleotide shown as SEQ ID NO. 4, and connecting the polynucleotide into a pPIC9K vector subjected to double restriction by Sal I and Not I to obtain a recombinant plasmid pPIC 9K-AgFNS;

II) cloning of the recombinant plasmid pPIC 9K-AgFNS: transforming the recombinant plasmid pPIC9K-AgFNS into escherichia coli Top10 to construct recombinant escherichia coli Top10-pPIC9K-AgFNS, and extracting the recombinant plasmid pPIC9K-AgFNS after the recombinant escherichia coli Top10-pPIC9K-AgFNS is subjected to amplification culture;

III) construction of recombinant Pichia pastoris: and (3) carrying out enzyme digestion linearization on the recombinant plasmid pPIC9K-AgFNS obtained in the step (2) by Sal I, then carrying out electric transformation on the linearized plasmid into pichia pastoris GS115, and screening positive clones to obtain the recombinant pichia pastoris GS115-pPIC 9K-AgFNS.

The recombinant pichia pastoris GS115-pPIC9K-DcFNS and GS115-pPIC9K-AgFNS have the same expression method, and specifically comprise the following steps: activating the pichia pastoris by using a YPD liquid culture medium for 20 hours, inoculating the pichia pastoris in a BMGY culture medium according to the inoculation amount of 1%, and culturing the pichia pastoris at the temperature of 30 ℃ at 200r/min until the OD600 is 2-6; centrifuging at room temperature for 5min, collecting thallus, and suspending thallus with BMMY culture medium with pH of 6.0; inducing expression at 30 ℃ at 200r/min, adding methanol into the culture medium every 24h, maintaining the final concentration of methanol at 0.5% inducing condition, and inducing expression for 5 day; and centrifuging at 4 ℃ to collect thalli, wherein the supernatant is the crude enzyme solution.

The invention discloses a method for expressing FNS I (flavone synthase) by pichia pastoris, which has the advantages of stable expression and high efficiency and can be used for constructing and optimizing flavone compound cell factories.

Drawings

FIG. 1 is a photograph of agarose gel electrophoresis of flavone synthase DcFNS and AgFNS;

FIG. 2 is a western blot of flavone synthases DcFNS and AgFNS;

FIG. 3 is a western blot comparison of flavone synthases DcFNS and AgFNS expressed in different hosts;

FIG. 4 is a graph comparing the catalytic naringenin efficiencies of flavone synthases DcFNS and AgFNS expressed in different hosts.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. It will be understood by those skilled in the art that the following examples are illustrative of the present invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Nucleotide candidate sequences of flavone synthase, DcFNS (SEQ ID NO:3) and AgFNS (SEQ ID NO:4), were obtained from genomic and transcriptomic data of various plants by data mining analysis, respectively. The sequence is expressed in escherichia coli to prepare crude enzyme liquid containing the expression product of the sequence, and the catalytic activity of the crude enzyme liquid on naringenin is detected by in vitro catalysis with alpha ketoglutaric acid as a cofactor. The invention discovers that expression products of DcFNS and AgFNS can efficiently catalyze naringenin to synthesize apigenin with high added value and multiple important physiological activities. And verifying the expression effect of DcFNS or AgFNS by using different expression bacteria, and determining that the pichia pastoris is a better choice.

Example 1 cloning of flavone synthase DcFNS and its expression in E.coli

Two primers of SEQ ID NO. 5 and SEQ ID NO. 6 in the sequence Listing were synthesized, and PCR was performed using the above primers using cDNA reverse transcribed from RNA extracted from plants as a template. The DNA polymerase is KOD DNA polymerase with high fidelity from BAO bioengineering GmbH. The PCR amplification procedure was: 94 ℃ for 2 min; 15s at 94 ℃, 30s at 58 ℃ and 2min at 68 ℃ for 35 cycles; the temperature is reduced to 10 ℃ in 10min at 68 ℃. The PCR product was detected by agarose gel electrophoresis, and the results are shown in FIG. 1.

The target DNA band is cut off by irradiating under ultraviolet. Then, the DNA, i.e., the DNA fragment of the amplified flavone synthase gene, was recovered from the agarose Gel using the Axygen Gel Extraction Kit (AEYGEN). The recovered PCR product was cloned into a PMDT vector using PMD18-T cloning kit from Takara, Inc., Boehringer Bio Inc., and the constructed vector was named PMDT-DcFNS. The gene sequence of DcFNS is obtained by sequencing.

The DcFNS gene has the nucleotide sequence of SEQ ID NO. 3. The 1 st to 1074 th nucleotides from the 5 ' end of SEQ ID NO:3 are the Open Reading Frame (ORF) of DcFNS, the 1 st to 3 rd nucleotides from the 5 ' end of SEQ ID NO:3 are the initiation codon ATG of the DcFNS gene, and the 1072 nd and 1074 th nucleotides from the 5 ' end of SEQ ID NO:3 are the termination codon TAG of the DcFNS gene. The flavone synthase DcFNS codes a protein DcFNS containing 357 amino acids, has an amino acid residue sequence of SEQ ID NO:1, and has a theoretical molecular weight of 40.3kDa and an isoelectric point pI of 5.73 as predicted by software.

Synthesizing two primers of SEQ ID NO. 7 and SEQ ID NO. 8 in the sequence table, adding two enzyme cutting sites of BamH I and Xho I at two ends of the synthesized primers, and carrying out PCR by using plasmid PMDT-DcFNS as a template. The PCR amplification procedure was as above. The PCR product was separated by agarose gel electrophoresis, and the recovered PCR product was ligated into pET28a vector (Invitrogen) digested with BamH I and Xho I using One-step mutiscolone kit from Vazyme. The obtained recombinant plasmid was designated pET28 a-DcFNS.

The recombinant plasmid pET28a-DcFNS and the empty vector pET28a are transformed into escherichia coli BL21(DE3) to construct recombinant escherichia coli BL21-pET28a-DcFNS and BL21-pET28 a.

Respectively inoculating BL21-pET28a-DcFNS and BL21-pET28a into LB culture medium, culturing at 37 ℃ and 200rpm to OD₆₀₀About 0.6-0.8, cooling the bacterial liquid to 4 ℃, adding IPTG with the final concentration of 50 mu M, and inducing expression for 15h at 18 ℃ and 200 rpm. The cells were collected by centrifugation at 4 ℃ and resuspended by adding lysis buffer (50mM phosphate buffer, 1mM EDTA, 1mM DTT, pH 7.4), cells were disrupted by sonication, cell lysate supernatant was collected by centrifugation at 12000g at 4 ℃ and samples were subjected to SDS-PAGE electrophoresis and Western blot analysis, the results are shown in FIG. 2.

Example 2 cloning of flavone synthase AgFNS and expression thereof in E.coli

Two primers of SEQ ID NO. 9 and SEQ ID NO. 10 in the sequence Listing were synthesized, and PCR was performed using the above primers using cDNA reverse transcribed from RNA extracted from plants as a template. The DNA polymerase is KOD DNA polymerase with high fidelity from BAO bioengineering GmbH. The PCR amplification procedure was: 94 ℃ for 2 min; 15s at 94 ℃, 30s at 58 ℃ and 2min at 68 ℃ for 35 cycles; the temperature is reduced to 10 ℃ in 10min at 68 ℃. The PCR product was detected by agarose gel electrophoresis, and the results are shown in FIG. 1.

The target DNA band is cut off by irradiating under ultraviolet. Then, the DNA, i.e., the DNA fragment of the amplified flavone synthase gene, was recovered from the agarose Gel using the Axygen Gel Extraction Kit (AEYGEN). The recovered PCR product was cloned into a PMDT vector using PMD18-T cloning kit of Takara, Inc., Boehringer Bio Inc., and the constructed vector was named PMDT-AgFNS. And obtaining the gene sequence of AgFNS through sequencing.

The AgFNS gene has a nucleotide sequence of SEQ ID NO. 4. The 1 st to 1068 th nucleotides from the 5 ' end of SEQ ID NO. 4 are open reading frames of AgFNS, the 1 st to 3 th nucleotides from the 5 ' end of SEQ ID NO. 4 are the initiation codon ATG of AgFNS gene, and the 1066 th and 1068 th nucleotides from the 5 ' end of SEQ ID NO. 4 are the termination codon TGA of AgFNS gene. The flavone synthase AgFNS codes a protein AgFNS containing 355 amino acids, has an amino acid residue sequence of SEQ ID NO:1, and has a theoretical molecular weight of 40.0kDa and an isoelectric point pI of 6.20 according to prediction by software.

Synthesizing two primers of SEQ ID NO. 11 and SEQ ID NO. 12 in the sequence table, adding two enzyme cutting sites of BamH I and Xho I at two ends of the synthesized primers respectively, and carrying out PCR by using plasmid PMDT-AgFNS as a template. The PCR amplification procedure was as above. The PCR product was separated by agarose gel electrophoresis, and the recovered PCR product was ligated into a BamH I and Xho I double-digested pET28a vector using One-step mutisclone kit from Vazyme. The obtained recombinant plasmid was designated pET28 a-AgFNS.

The recombinant plasmid pET28a-AgFNS is transformed into Escherichia coli BL21(DE3) to construct recombinant Escherichia coli BL21-pET28 a-AgFNS.

BL21-pET28a-AgFNS and BL21-pET28a (example 1) were inoculated into LB medium and cultured at 37 ℃ and 200rpm to OD₆₀₀About 0.6-0.8, cooling the bacterial liquid to 4 ℃, adding IPTG with the final concentration of 50 mu M, and inducing expression for 15h at 18 ℃ and 200 rpm. The cells were collected by centrifugation at 4 ℃ and resuspended by adding lysis buffer (50mM phosphate buffer, 1mM EDTA, 1mM DTT, pH 7.4), cells were disrupted by sonication, cell lysate supernatant was collected by centrifugation at 12000g at 4 ℃ and samples were subjected to SDS-PAGE electrophoresis and Western blot analysis, the results are shown in FIG. 2.

Example 3 expression of flavone synthase DcFNS in Pichia pastoris

Two primers of SEQ ID NO. 13 and SEQ ID NO. 14 in the sequence list were synthesized, two ends of the synthesized primers were added with two restriction sites Sal I and Not I, respectively, and PCR was performed using the plasmid PMDT-DcFNS prepared in example 1 as a template. The PCR amplification procedure was as above. The PCR product was separated by agarose gel electrophoresis, and the recovered PCR product was ligated into the Sal I and Not I double-digested pPIC9K vector using One-step mutis clone kit from Vazyme. The obtained recombinant plasmid was named pPIC 9K-DcFNS.

The recombinant plasmid pPIC9K-DcFNS is transformed into the Escherichia coli Top10 to construct recombinant Escherichia coli Top10-pPIC 9K-DcFNS.

Extracting recombinant plasmid pPIC9K-DcFNS from Top10-pPIC9K-DcFNS, adopting Sal I to linearize the recombinant plasmid pPIC9K-DcFNS and the no-load plasmid pPIC9K, adopting an electric transformation method to transfer the recombinant plasmid pPIC9K-DcFNS and the no-load plasmid pPIC9K into Pichia pastoris GS115 cells, screening positive clones, and respectively naming GS115-pPIC9K-DcFNS and GS115-pPIC 9K.

Respectively inoculating GS115-pPIC9K-DcFNS and GS115-pPIC9K to YPD liquid culture medium for 20h, inoculating to 50mL BMGY culture medium at 1%, and culturing OD at 30 deg.C and 200r/min₆₀₀To 2-6. Centrifuging at room temperature for 5min at 3000g, collecting thallus, re-suspending the thallus with 5ml BMMY (pH 6.0), inducing expression at 30 deg.C at 200r/min, adding methanol with final concentration of 0.5% into culture medium every 24 hr, and maintaining the inducing condition. Inducing expression of 5day, centrifuging at 4 deg.C and 12000g, collecting thallus, collecting supernatant as crude enzyme solution, and performing Western blot analysis on the sample, with the results shown in figure 3 and figure 4. The result shows that the enzyme activity of DcFNS expressed by the pichia pastoris is higher than that expressed by escherichia coli, and the enzyme yield is more.

Example 4 expression of flavone synthase AgFNS in Pichia pastoris

Two primers of SEQ ID NO. 15 and SEQ ID NO. 16 in the sequence list were synthesized, two ends of the synthesized primers were added with two restriction sites Sal I and Not I, respectively, and PCR was performed using the plasmid PMDT-AgFNS prepared in example 2 as a template. The PCR amplification procedure was as above. The PCR product was separated by agarose gel electrophoresis, and the recovered PCR product was ligated into the Sal I and Not I double-digested pPIC9K vector using One-step mutis clone kit from Vazyme. The obtained recombinant plasmid was named pPIC 9K-AgFNS.

The recombinant plasmid pPIC9K-AgFNS is transformed into the Escherichia coli Top10 to construct recombinant Escherichia coli Top10-pPIC 9K-AgFNS.

Extracting a recombinant plasmid pPIC9K-AgFNS from Top10-pPIC9K-AgFNS, linearizing the pPIC9K-AgFNS by Sal I, transferring the pPIC9K-AgFNS into Pichia pastoris GS115 cells by an electrical transformation method, screening positive clones, and respectively naming the positive clones as GS115-pPIC 9K-AgFNS.

Are respectively provided withGS115-pPIC9K-AgFNS and GS115-pPIC9K (example 9) were inoculated into YPD liquid medium for 20 hours, respectively, and inoculated into 50mL BMGY medium at 1% inoculum size, and OD was cultured at 30 ℃ and 200r/min₆₀₀To 2-6. Centrifuging at room temperature for 5min at 3000g, collecting thallus, re-suspending the thallus with 5ml BMMY (pH 6.0), inducing expression at 30 deg.C at 200r/min, adding methanol with final concentration of 0.5% into culture medium every 24 hr, and maintaining the inducing condition. Inducing expression of 5day, centrifuging at 4 deg.C and 12000g, collecting thallus, collecting supernatant as crude enzyme solution, and performing Western blot analysis on the sample, with the results shown in figure 3 and figure 4. The result shows that AgFNS enzyme activity expressed by the pichia pastoris is higher than that expressed by escherichia coli, and the enzyme yield is more.

SEQUENCE LISTING

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gatcatggtc attatttgag caatggaagg ttcaggaatg ctgaccatca agcagtagtg 840

aattcaactt ccaccagatt gtcaattgca actttccaga acccggctca gaatgcgata 900

gtatatccgt taaagatcag ggagggagag aaggcaattc tggatgaggc catcacctac 960

gctgaaatgt ataagaaaaa catgactaaa catattgcgg tggctaccca gaagaaattg 1020

gccaaggaga aaaggttgca agatgagaag gccaagatga agatatga 1068

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence

<400> 5

atggctccaa caactattac tgc 23

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence

<400> 6

ctaaaatttc gtctccagct tggc 24

<210> 7

<211> 41

<212> DNA

<213> Artificial sequence

<400> 7

agcaaatggg tcgcggatcc atggctccaa caactattac t 41

<210> 8

<211> 41

<212> DNA

<213> Artificial sequence

<400> 8

tggtggtggt ggtgctcgag aaatttcgtc tccagcttgg c 41

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence

<400> 9

atggctccat caactataac tgc 23

<210> 10

<211> 24

<212> DNA

<213> Artificial sequence

<400> 10

tcatatcttc atcttggcct tctc 24

<210> 11

<211> 41

<212> DNA

<213> Artificial sequence

<400> 11

agcaaatggg tcgcggatcc atggctccat caactataac t 41

<210> 12

<211> 41

<212> DNA

<213> Artificial sequence

<400> 12

tggtggtggt ggtgctcgag tatcttcatc ttggccttct c 41

<210> 13

<211> 41

<212> DNA

<213> Artificial sequence

<400> 13

ctgaagctta cgtagaattc atggctccaa caactattac t 41

<210> 14

<211> 41

<212> DNA

<213> Artificial sequence

<400> 14

gcgaattaat tcgcggccgc aaatttcgtc tccagcttgg c 41

<210> 15

<211> 41

<212> DNA

<213> Artificial sequence

<400> 15

ctgaagctta cgtagaattc atggctccat caactataac t 41

<210> 16

<211> 41

<212> DNA

<213> Artificial sequence

<400> 16

gcgaattaat tcgcggccgc tatcttcatc ttggccttct c 41

Claims

1. Pichia pastoris for expressing flavone synthases, characterized in that an expression vector is integrated, and the expression vector contains polynucleotide as shown in SEQ ID NO. 3 or SEQ ID NO. 4.

2. The pichia pastoris expressing flavone synthase according to claim 1, wherein the pichia pastoris is pichia pastoris GS 115.

3. The pichia pastoris expressing flavone synthase according to claim 2, wherein the expression vector is pPIC9K-DcFNS or pPIC 9K-AgFNS; the pPIC9K-DcFNS contains a polynucleotide shown as SEQ ID NO. 3; the pPIC9K-AgFNS contains the polynucleotide shown in SEQ ID NO. 4.

4. The method for constructing pichia pastoris according to any one of claims 1 to 3, comprising the steps of:

5. The method for constructing pichia pastoris according to any one of claims 1 to 3, wherein: I) constructing a recombinant plasmid pPIC 9K-AgFNS: adding Sal I and Not I restriction sites at both ends of the polynucleotide shown as SEQ ID NO. 4, and connecting the polynucleotide into a pPIC9K vector subjected to double restriction by Sal I and Not I to obtain a recombinant plasmid pPIC 9K-AgFNS;

6. The method for expressing flavone synthase by pichia pastoris according to any one of claims 1 to 3, wherein: activating the pichia pastoris by using a YPD liquid culture medium for 20 hours, inoculating the pichia pastoris in a BMGY culture medium according to the inoculation amount of 1%, and culturing the pichia pastoris at the temperature of 30 ℃ at 200r/min until the OD600 is 2-6; centrifuging at room temperature for 5min, collecting thallus, and suspending thallus with BMMY culture medium with pH of 6.0; inducing expression at 30 ℃ at 200r/min, adding methanol into the culture medium every 24h, maintaining the final concentration of methanol at 0.5% inducing condition, and inducing expression for 5 day; and centrifuging at 4 ℃ to collect thalli, wherein the supernatant is the crude enzyme solution.