Core promoter optimized pichia pastoris protein expression system and application thereof

Abstract

The invention relates to a pichia pastoris protein expression system optimized by a core promoter and application of the pichia pastoris protein expression system. Specifically, the efficient promoter is designed and obtained on the basis of an upstream 300bp region of an initiation codon of a trichoderma reesei high-expression gene, and the promoters are used as a core promoter 1 of a synthetic expression system (SES) in fungal cells. When the SES system containing the promoter screened by the inventor is used for target protein expression in fungal host cells, the expression efficiency of the SES system is higher than that of SES-A in the existing technical system. On the basis, the invention provides an optimized synthetic expression system, a fungal host cell containing the optimized synthetic expression system and application of the fungal host cell in expression of target protein.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial genetic engineering, and specifically relates to a core promoter-optimized Pichia pastoris protein expression system and application thereof. BACKGROUND

[0002] Pichia pastoris has shown good application effects in academic research and industrial production due to its high growth density, strong protein expression, good genetic stability and mild glycosylation modification, and has become the preferred chassis cell for recombinant protein expression. Common promoters such as PAOX1, PGAP, PTEF1 and the like are mostly endogenous promoters, and their expression is affected by the complex regulation network of host cells, resulting in unstable expression efficiency and limited regulation accuracy. Moreover, as a widely used PAOX1, it depends on the methanol induction characteristics, which poses certain risks in terms of industrial facility safety and food application safety.

[0003] The synthetic expression system (SES) is a general functional protein expression system developed by the laboratory of Dominik Mojzita of the National Technology Research Center of Finland for various fungal hosts. The SES system mainly includes two modules. One is to drive the expression of an artificial transcription factor (Bm3R1-NLS-VP16) by a core promoter 1, so that the artificial transcription factor can accumulate. The other module is a combination of multiple binding sites of the artificial transcription factor and a core promoter element 2, which promotes the expression of the downstream target protein under the action of the artificial transcription factor. SES-A is a high-efficiency protein expression system constructed based on SES, in which the core promoter elements An008CP and An201CP derived from Aspergillus niger are applied to drive the expression of the artificial transcription factor and the target protein, respectively. As a powerful and multifunctional protein expression platform, the practicability of the SES-A system has been verified in various yeasts, including Saccharomyces cerevisiae, Pichia pastoris, Pichia kudriavzevii, Yarrowia lipolytica, Pachysolen tannophilus, Rhodotorula mucilaginosa and Candida utilis, and has also been successfully applied in two filamentous fungi, i.e., Trichoderma reesei and Aspergillus niger, covering multiple fungi with industrial value. The SES-A system can achieve an expression level of the target protein that reaches or exceeds the effect of a strong endogenous promoter or a traditional gene expression tool. However, the effect of the SES-A system in Pichia pastoris cells is still limited, and it is necessary to further screen and optimize the core promoter elements of the SES-A system, further improve the protein expression efficiency of the SES system, enrich the protein expression tools of Pichia pastoris, and improve the application potential of Pichia pastoris in industrialization. SUMMARY

[0004] To further optimize the application of SES in fungal host cells, the inventors designed and obtained the highly efficient promoters of this invention based on the 300bp upstream region of the highly expressed genes in *Trichoderma reesei*, and used these promoters as the core promoter 1 of SES. When the SES system containing the promoters selected by the inventors was used for target protein expression in fungal host cells, it showed higher expression efficiency than the existing technology system SES-A.

[0005] In this regard, the present invention includes, but is not limited to, the following:

[0006] In one aspect, the present invention provides an optimized synthetic expression system comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises, from 5' to 3', a first core promoter, an artificial transcription factor coding region and a first transcription terminator element, and the second polynucleotide comprises, from 5' to 3', an artificial transcription factor binding site, a second core promoter, a target protein coding region and a second transcription terminator element.

[0007] The first core promoter drives the expression of an artificial transcription factor in its coding region. The artificial transcription factor binds to its binding site on a second polynucleotide to act on the second core promoter, which in turn promotes the expression of the target protein gene in the target protein coding region.

[0008] The first core promoter is selected from SEQ ID NO:4-57.

[0009] In one aspect, the first core promoter of the present invention is selected from SEQ ID NO:4-44. In one aspect, the first core promoter of the present invention is selected from SEQ ID NO:4-12. In one aspect, the first core promoter of the present invention is selected from SEQ ID NO:4-13. In one aspect, the first core promoter of the present invention is selected from SEQ ID NO:4, 5, 6, 8, 10 and 13. In one aspect, the first core promoter of the present invention is selected from SEQ ID NO:5.

[0010] In one aspect, the target protein of the present invention is selected from fluorescent proteins, enzymes, antibodies, and fusion proteins; preferably, the target protein is selected from fluorescent proteins, enzymes, and fusion proteins.

[0011] In one aspect, the target protein of the present invention is a fluorescent protein, preferably mCherry;

[0012] And / or, the target protein is an enzyme, preferably alkaline proteinase K (Pro K);

[0013] And / or, the target protein is a fusion protein, preferably a fusion protein of human lysozyme and mCherry.

[0014] In one aspect, the target protein of the present invention is a fluorescent protein. In another aspect, the fluorescent protein of the present invention is mCherry.

[0015] In one aspect, the target protein of the present invention is an enzyme. In another aspect, the enzyme of the present invention is alkaline proteinase K (Pro K).

[0016] In one aspect, the target protein of the present invention is a fusion protein. In another aspect, the fusion protein of the present invention is a fusion protein of human lysozyme and mCherry (shLYZ-mCherry).

[0017] In one aspect, the amino acid sequence of the mCherry described in this invention is SEQ ID NO:61;

[0018] And / or, the amino acid sequence of the Pro K is SEQ ID NO:64;

[0019] And / or, the amino acid sequence of the human lysozyme and mCherry fusion protein is shown in SEQ ID NO:59.

[0020] In one aspect, the amino acid sequence of the mCherry described in this invention is shown in SEQ ID NO:61.

[0021] In one aspect, the amino acid sequence of Pro K according to the present invention is shown in SEQ ID NO:64.

[0022] In one aspect, the amino acid sequence of the human lysozyme and mCherry fusion protein of the present invention is shown in SEQ ID NO:59.

[0023] In one aspect, the target protein coding region of the present invention comprises a single copy of the target protein gene.

[0024] In one aspect, the target protein coding region of the present invention contains multiple copies of the target protein gene, preferably, the multiple copies are 2 or more, more preferably, the multiple copies are 2-10, and most preferably, the multiple copies are 2 or 3.

[0025] In one aspect, the target protein coding region of the present invention contains 2 or 3 copies of the target protein gene.

[0026] In one aspect, the target protein coding region of the present invention comprises two copies of the target protein gene. In another aspect, the target protein coding region of the present invention comprises three copies of the target protein gene.

[0027] In one aspect, the amino acid sequence of the artificial transcription factor encoded by the artificial transcription factor coding region of the present application is shown as SEQ ID NO: 66.

[0028] And / or, the first transcription terminator element is Ttefl, preferably, the nucleotide sequence of Ttefl is SEQ ID NO: 69.

[0029] And / or, the artificial transcription factor binding site is 8 artificial transcription factor binding sites in series, preferably, the nucleotide sequence of the 8 artificial transcription factor binding sites in series is shown as SEQ ID NO: 68.

[0030] And / or, the second core promoter is selected from An201CP, preferably, the nucleotide sequence of An201CP is shown as SEQ ID NO: 71.

[0031] And / or, the second transcription terminator element is Tpdc1, preferably, the nucleotide sequence of Tpdc1 is shown as SEQ ID NO: 70.

[0032] In one aspect, the amino acid sequence of the artificial transcription factor encoded by the artificial transcription factor coding region of the present application is shown as SEQ ID NO: 66. In one aspect of the present application, the framework of the synthetic expression system (SES) of the present application is constructed according to the report of the article (Rantasalo A et al., A universal gene expression system for fungi. Nucleic Acids Res. 2018 Oct 12; 46(18): e111. doi: 10.1093 / nar / gky558. PMID: 29924368; PMCID: PMC6182139), the main difference is to replace the core promoter 1 therein to expand the screening range.

[0033] In one aspect, the nucleotide sequence of the artificial transcription factor coding region of the present application is shown as SEQ ID NO: 67.

[0034] In one aspect, the first transcription terminator element of the present application is Ttefl. In one aspect, the nucleotide sequence of Ttefl of the present application is shown as SEQ ID NO: 69. In the present application, the Ttefl transcription terminator sequence is derived from the gene tef1 of Trichoderma reesei, which terminates transcription during transcription. Transcription terminators derived from different genes can be selected, in theory, as long as the terminator can play the role of transcription termination, for example, Tpdc1 in the present application can also be used here as the first transcription terminator.

[0035] In one aspect, the artificial transcription factor binding site of the present application is eight artificial transcription factor binding sites in tandem. In one aspect, the nucleotide sequence of the eight artificial transcription factor binding sites in tandem of the present application is set forth in SEQ ID NO: 68.

[0036] In one aspect, the second core promoter of the present application is selected from An201CP. In one aspect, the nucleotide sequence of the An201CP of the present application is set forth in SEQ ID NO: 71.

[0037] In one aspect, the second transcription terminator element of the present application is selected from Tpdc1 or ADH1, preferably Tpdc1. In one aspect, the nucleotide sequence of the Tpdc1 of the present application is SEQ ID NO: 70. In the present application, the Tpdc1 transcription terminator sequence is derived from the pdc1 gene of Trichoderma reesei, which terminates transcription during transcription. Transcription terminators derived from different genes can be selected, and theoretically, any terminator that can play a role in terminating transcription can be substituted for each other. In one aspect of the present application, ADH1 is derived from Saccharomyces cerevisiae.

[0038] In another aspect, the present application provides a fungal host cell comprising the synthetic expression system according to the present application; preferably, the first polynucleotide and the second polynucleotide of the synthetic expression system are integrated into the genome of the fungal host cell as expression cassettes.

[0039] In one aspect, the first polynucleotide and the second polynucleotide of the synthetic expression system of the present application are integrated into the genome of the fungal host cell by different expression cassettes, respectively.

[0040] In one aspect, the first polynucleotide and the second polynucleotide of the synthetic expression system of the present application are integrated into the genome of the fungal host cell as a polynucleotide in tandem.

[0041] In one aspect, the fungal host cell of the present application is selected from Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Trichophaea longibrachiatum, Rhodotorula mucilaginosa, Candida, Trichoderma reesei, and Aspergillus niger; preferably, the host cell is selected from Pichia pastoris, Trichoderma reesei, and Aspergillus niger.

[0042] In one aspect, the fungal host cell according to the present application is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Trichoderma reesei and Aspergillus niger. In addition to Pichia pastoris used in the examples of the present application, the document Rantasalo A et al., A universal gene expression system for fungi. Nucleic Acids Res. 2018 Oct 12; 46(18): el l l (doi: 10.1093 / nar / gky558. PMID: 29924368; PMCID: PMC6182139) reports that the SES system can also express target proteins in Saccharomyces cerevisiae, Trichoderma reesei and Aspergillus niger.

[0043] In one aspect, the fungal host cell according to the present application is Pichia pastoris. Preferably, the fungal host cell according to the present application is Pichia pastoris PpHR3.

[0044] In yet another aspect, the present application provides the use of the synthetic expression system according to the present application and the host cell according to the present application for expressing a target protein, preferably, the target protein is selected from the group consisting of fluorescent proteins, enzymes, antibodies and fusion proteins.

[0045] In yet another aspect, the present application provides a method for producing a target protein, comprising the following steps:

[0046] (1) culturing the fungal host cell according to the present application under conditions suitable for expression of the target protein,

[0047] (2) recovering the target protein.

[0048] In the present application, the conditions suitable for expression of the target protein can be routinely determined by the skilled person in the art depending on the type of the target protein to be expressed.

[0049] In one aspect of the present application, by analyzing the transcriptome data of Trichoderma reesei, promoter sequences driving high expression of their genes in Trichoderma reesei are screened as a library of heterologous core promoter elements for driving the expression of artificial transcription factors, realizing the optimization of the SES system. The present application provides heterologous core promoter sequences with better test results, consisting of base sequences shown in SEQ ID NO. 4 to SEQ ID NO. 57. Specifically, in one aspect, the first core promoter according to the present application is designed from the 300 bp region upstream of the start codon of the high expression gene of Trichoderma reesei. In the present application, the optimized SES of the first core promoter shows much higher expression efficiency than the existing technical system SES-A in the expression of fluorescent proteins (mCherry) and alkaline protease (Pro K).

[0050] In an aspect of the present application, the first nucleotide and the second nucleotide described in the present application can be integrated into a specific site in the genome of the Pichia pastoris host as an expression cassette, and multiple copy site-specific integration can be achieved by means such as CRISPR / Cas9.

[0051] In an aspect of the present application, the Pichia pastoris strain PpHR3 can be used, the genome of which is integrated into a Cas9 gene and three genes related to homologous recombination (RAD52, RAD59 and MRE11), and only an sgRNA vector and a polynucleotide with a 50 bp short homology arm need to be introduced to achieve site-specific integration of the target gene, and the system has the ability to integrate multiple fragments at the same time, and can quickly achieve site-specific integration of the SES expression module. For example, the Pichia pastoris strain PpHR3 of the present application can be obtained by referring to the following literature: Gao J et al., Enhancing Homologous Recombination Efficiency in Pichia pastoris for Multiplex Genome Integration Using Short Homology Arms. ACS Synth Biol. 2022 Feb 18; 11(2): 547-553. doi: 10.1021 / acssynbio.1c00366. Epub 2022 Jan 21. PMID: 35061355.

[0052] In an aspect, the present application provides the use of the optimized SES system to express heterologous alkaline protease or fusion protein in Pichia pastoris.

[0053] Compared with the prior art, the present application has at least the following beneficial effects:

[0054] The SES system described in the present application effectively avoids the influence of endogenous regulation in the host on the expression level by using a heterologous core promoter; the protein expression efficiency of the optimized SES system is significantly higher than that of the control system SES-A; the SES system is suitable for high-efficiency expression of heterologous proteins with industrial value in Pichia pastoris; and the multi-copy strategy can be flexibly applied to further enhance the expression efficiency of the target protein in the SES system. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 A schematic diagram of the composition of the protein expression system SES

[0056] Figure 2 A graph showing the efficiency of mCherry expressed by the SES system optimized with different heterologous promoter elements

[0057] Figure 3Activity determination results of Pro K expressed by different SES systems in Pichia pastoris

[0058] Figure 4 Activity determination results of Pro K expressed by SES-CP32 in Pichia pastoris using multi-copy strategy

[0059] Figure 5 Efficiency results chart of fusion protein shLYZ-mCherry expressed by SES systems optimized by different heterologous promoter elements DETAILED DESCRIPTION

[0060] In order to make the technical personnel in the art better understand the present application scheme, the technical solutions of the present application are described clearly and completely in combination with specific examples below. It should be pointed out that the following detailed description is exemplary and is only a part of the embodiments of the present application, but not all the embodiments.

[0061] Based on the embodiments in the present application, all other embodiments obtained by the ordinary skilled in the art without making creative efforts shall belong to the scope of protection of the present application.

[0062] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by the ordinary skilled in the art to which the present application belongs. The experimental materials used in the embodiments of the present application are all conventional experimental materials in the art, and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out according to the conventional experimental methods or according to the operation instructions recommended by the suppliers.

[0063] Example 1 Plasmid construction of SES-A expression module

[0064] The DNA fragment of SES-A plasmid was synthesized by Hangzhou Qikexing Biotechnology Co., Ltd. Figure 1), and the company was commissioned to connect by Gibson assembly method (Novagen ClonExpress Ultra One Step Cloning Kit V2, C116), respectively, to construct artificial transcription factor modules (which use An008CP as core promoter 1) and target protein mCherry, Pro K and shLYZ-mCherry expression modules (which use An201CP as core promoter 2) to pUC57-Kana and pUC57-amp. The vector sequences of the constructed plasmids pUC57-sTF-Kana, pUC57-mCherry-Amp, pUC57-Pro K-Amp and pUC57-shLYZ-mCherry-Amp are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:65, respectively.

[0065] Example 2 for optimizing SES heterologous core promoter screening and design

[0066] The transcriptome of T. reesei under different carbon source conditions was analyzed from public RNA-Seq data, the NCBI accession number of the transcriptome data is PRJNA232512, and the genes highly expressed under three conditions were selected as candidates, and the 300bp sequence upstream of the start codon ATG was obtained as the core promoter, and the sequence is shown in SEQ ID NO:4 to SEQ ID NO:57.

[0067] The core promoter sequence was synthesized by Hangzhou Qikexi Biotechnology Co., Ltd. and constructed between the restriction enzyme sites Mlu I and Pac I in the pUC57-sTF-Kana plasmid, that is, the core promoter 1 An008CP of the artificial transcription factor module in the SES-A system was replaced by the sequence shown in any one of SEQ ID NO:4 to SEQ ID NO:57. The target protein expression module in the optimized SES system and the SES-A system is the same.

[0068] Example 3 Site-specific integration of the SES system in Pichia pastoris

[0069] For Pichia pastoris transformation, only the region covered by CorePromoterl-Bm3R1-NLS-VP16-Ttefl and 8xBS-CorePromoter2-mCherry-Tpdc1 expression cassettes needs to be amplified. The KeyPo high-fidelity enzyme (2xKeyPo Master Mix, Vazyme) is used to amplify the Donor-DNA, which is purified by the gel recovery kit (Omega) and used for Pichia pastoris transformation. When evaluating the effectiveness of the screened promoter, the Intl-sTFF and Intl-sTFR primers are used to amplify the artificial transcription factor expression cassette, and the Int6-ProK / mCherry / shLYZF and Int6-ProK / mCherry / shLYZR primers are used to amplify the target protein mCherry expression cassette. With the help of the HGP-sgRNA-Intl-Int6 sgRNA plasmid (construction method reference Fang H et al, Engineering Pichia pastoris for Efficient De Novo Synthesis of 2'-Fucosyllactose, Journal of Agricultural and Food Chemistry 2025 73(14), 8555-8566. DOI: 10.1021 / acs.jafc.5c00598), the artificial transcription factor and target protein expression cassette Donor-DNA are integrated into the Intl and Int6 sites on the genome of Pichia pastoris strain PpHR3 by homologous recombination through electroporation. When expressing alkaline protease using the optimized protein expression system, the Intl-sTFF and Intl-sTFR primers are used to amplify the artificial transcription factor expression cassette, and the Int6-ProK / mCherry / shLYZF and Int6-ProK / mCherry / shLYZR, Int15-ProKF and Int15-ProKR, Int18-ProKF and Int18-ProKR primers are used to amplify the alkaline protease Pro K expression cassette. With the help of the HGP-sgRNA-Intl-Int6 and HHP-sgRNA-Intl8-Intl5 sgRNA plasmids, respectively, the corresponding expression cassettes (artificial transcription factor expression cassette and 1-3 Pro K expression cassettes) are integrated into the Intl, Int6, Intl5 and Intl8 sites on the genome of Pichia pastoris strain PpHR3 by electroporation.

[0070] In the expression of alkaline protease using the optimized protein expression system, the artificial transcription factor expression cassette was amplified using Int1-sTFF and Int1-sTFR primers, and the shLYZ-mCherry expression cassette was amplified using Int6-ProK / mCherry / / shLYZF and Int6-ProK / mCherry / shLYZR primers, and the corresponding expression cassettes were integrated into the Int1 and Int6 sites of the Pichia pastoris strain PpHR3 genome by electroporation with the help of the HGP-sgRNA-Int1-Int6 sgRNA plasmid.

[0071] The primers used in this example are shown in Table 1, wherein the underlined part represents the homologous arm sequence corresponding to the Int1, Int6, Int15 and Int18 integration sites. For the source of the Pichia pastoris strain PpHR3 and the method of integrating the target protein expression cassette into the strain, please refer to the literature Gao J et al., Enhancing Homologous Recombination Efficiency in Pichia pastoris for Multiplex Genome Integration Using Short Homology Arms. ACS Synth Biol. 2022 Feb 18; 11(2): 547-553. doi: 10.1021 / acssynbio.1c00366. Epub 2022 Jan 21. PMID: 35061355. For specific information about these integration sites, sgRNA sequences, and homologous arm sequences on the primers used, please refer to Gao J et al., Synthetic Biology Toolkit for Marker-Less Integration of Multigene Pathways into Pichia pastoris via CRISPR / Cas9, ACS Synthetic Biology 2022 11(2), 623-633. DOI: 10.1021 / acssynbio.1c00307.

[0072] Table 1 Primers used for amplifying Donor-DNA for transformation of Pichia pastoris

[0073]

[0074]

[0075] The primers were synthesized by Hangzhou Qikexi Biological Technology Co., Ltd.

[0076] The specific operation steps of Pichia pastoris electrotransformation are as follows:

[0077] (1) Fresh Pichia pastoris single colony was inoculated into 5 mL of YPD medium (the composition of YPD medium: 1% yeast extract, 2% peptone, 2% glucose), and cultured at 30°C overnight.

[0078] (2) The bacterial solution was transferred to a 50 mL YPD flask, and the initial OD600 was controlled at 0.15-0.2, and cultured at 30°C for 4-5 h.

[0079] (3) When OD600 reached 0.8-1.0, Pichia pastoris competent cells were prepared from the bacterial solution. The bacterial solution was transferred to a 50 mL sterile centrifuge tube and centrifuged at 4000 rpm for 5 min.

[0080] (4) The supernatant was discarded, 8.55 mL Beds (10 mM N-diglycine, 3% (v / v) ethylene glycol, 1 M sorbitol, adjusted to pH 8.3 with sodium hydroxide), 1 mL of 1 M DTT, and 450 μL of dimethyl sulfoxide (DMSO) were added, and the precipitate was gently blown and resuspended.

[0081] (5) The resuspended liquid was incubated at room temperature at 100 rpm for 5 min, and then centrifuged at 4000 rpm for 5 min.

[0082] (6) The supernatant was discarded, 950 μL of Beds and 50 μL of DMSO were added, and the precipitate was gently blown and resuspended. Centrifugation at 4000 rpm for 30 seconds.

[0083] (7) The supernatant was discarded, 190 μL of Beds and 10 μL of DMSO were added, and the precipitate was gently blown and resuspended. The resuspended cells were Pichia pastoris competent cells, which could be stored in an ultra-low temperature refrigerator.

[0084] (8) 40 μL of Pichia pastoris competent cells were taken, 500 ng of sgRNA plasmid and 1 μg of Donor-DNA were added. After mixing, it was transferred to a pre-cooled electrotransformation cup (with a distance of 2.0 mm) and placed on ice for 2 min.

[0085] (9) The parameters of the electrotransformation instrument were set as follows: voltage 1500 V, resistance 400 Ω, capacitance 25 μF.

[0086] (10) After electrotransformation, 1 mL of pre-cooled incubation solution was quickly added. If the transformant was screened by deficiency, the incubation solution was 1 M sorbitol; if the transformant was screened by antibiotic, the incubation solution was a mixture of YPD and 1 M sorbitol at a ratio of 1:1.

[0087] (11) Transfer the mixture to 1.5 mL pre-cooled sterile EP tube, and place in a 30°C shaking incubator for 2-4 h. Take an appropriate amount of bacterial liquid (reference transformation efficiency) and spread on the plate containing the selection of antibiotics or auxotrophic. Place the plate in a 30°C biochemical incubator for 2-3 days.

[0088] (12) Use the primers in Table 1 to screen positive clones by colony PCR, and after sequencing the PCR products, the construction of the strain is completed. Int1-F1 / Int1-R1, Int6-F1 / Int6-R1, Int15-F1 / Int15-R1, Int18-F1 / Int18-R1 are used for colony PCR to detect the integration of expression cassette at Int1, Int6, Int15 and Int18 sites, respectively. When using HGP-sgRNA-Int1-Int6 and HHP-sgRNA-Int18-Int15 sgRNA plasmids, 0.2 g / L G418 and 0.2 g / L hygromycin B are added to the YPD plate (the composition of YPD plate medium is: 1% yeast extract, 2% peptone, 2% glucose, 2% agar powder).

[0089] Example 4 Detection of expression of target protein

[0090] The mCherry protein was selected to evaluate the expression efficiency of different SES systems. In order to directly observe the expression of mCherry, Pichia pastoris cells were cultured on YPD plates at 30°C, and observed in the dark using a hand-held 520-550 nm excitation light source and a 550 nm filter. Positive clones that emit fluorescence were selected. In order to quantitatively measure the expression of mCherry, a single colony was inoculated into 5 mL of YPD medium and cultured at 30°C and 220 rpm / min for 48 hours, then centrifuged and resuspended in sterile water. 200 μL of the suspension was transferred to a black 96-well enzyme plate, and the mCherry fluorescence was measured at 578-615 nm using a multifunctional reader (BioTek, Synergy H1), which was normalized according to the cell density (OD600). The quantitative detection method of the expression level of the fusion protein shLYZ-mCherry of human lysozyme and mCherry is consistent with that of mCherry.

[0091] The recombinant Pichia pastoris strains carrying the Pro K gene were inoculated into glass test tubes containing 5 mL of YPD medium and cultured at 30°C and 220 rpm / min for about 12 hours. After adjusting the OD600 values of different recombinant Pichia pastoris strains to the same level, 1 mL of cell suspension was transferred to a flask containing 50 mL of YPD medium and cultured at 30°C and 220 rpm / min for 72 hours. 5 mL of 20% glucose was added every 24 hours. The fermentation supernatant was collected every 12 hours and the OD600 and enzyme activity were detected. In order to measure the activity of protease K, Suc-phe-pNA was selected as the chromogenic substrate of serine protease Pro K. After the enzyme cut Suc-phe-pNA, p-nitroaniline (pNA) was released, and the activity of serine protease could be quantitatively determined by colorimetric detection at 405 nm. The total volume of the reaction solution was 200 μL, which contained 2 mM Suc-phe-pNA substrate, 50 mM Tris-HCl (pH 8.0) and 10 μL of crude enzyme solution. The activity of Pro K was determined by recording the absorbance at 405 nm, which was determined after about 1.5 hours, and 1 unit was defined as an increase of 1 μmol pNA per minute at 37°C.

[0092] The experimental results show that the 41 heterologous core promoter optimized SES systems are significantly better than SES-A in expressing mCherry, with the highest expression efficiency being 5.4 times that of SES-A. Figure 2 The top 9 optimized SES systems were used to express alkaline protease Pro K in Pichia pastoris. After 72 hours of fermentation in a flask, the expression efficiency of Pro K by SES-CP32 was the highest, which was 1.6 times that of SES-A. Figure 3 Figure 4 When the sTF was maintained at 1 copy, the enzyme activity of Pro K was significantly improved as the copy number of Pro K increased from 1 to 3. After 72 hours of fermentation, the enzyme activity in the fermentation supernatant of 1*sTF+3*Pro K was 4.6 times that of 1*sTF+1*Pro K. Figure 4 The top 10 optimized SES systems were used to express the fusion protein of human lysozyme and red fluorescent protein (shLYZ-mCherry) in Pichia pastoris. The detection results showed that SES-CP64, SES-CP97, SES-CP32, SES-CP121, SES-CP47 and SES-CP60 all had better protein expression efficiency of shLYZ-mCherry than SES-A, and the protein expression efficiency of shLYZ-mCherry by the best SES-CP64 was 3.52 times that of SES-A.​Figure 5 ).

[0093] SEQUENCE LIST

[0094] SEQ ID NO. 1: Capital letters part is An008CP, underlined part is artificial transcription factor coding sequence, shaded part is Ttefl base sequence.

[0095]

[0096] SEQ ID NO. 2: Capital letters part is 8 artificial transcription factor binding sites in series, underlined capital letters part is An201CP, shaded part is mCherry coding sequence, underlined shaded part is Tpdc1 base sequence.

[0097]

[0098]

[0099] SEQ ID NO. 3: Capital letters part is 8 artificial transcription factor binding sites in series, underlined capital letters part is An201CP, shaded part is alpha-factor signal peptide sequence, underlined shaded part is Pro K coding sequence, wavy line part is Tpdc1.

[0100]

[0101] SEQ ID NO. 4: CP60

[0102] agtacgtaccagcaggctggtgccggtgatggatcaaaatactcccggatagcgttccgcggtcacgtggagccgctttgccactcgcaaatttgtgcctctagggccgcaaaccctatcggagtttttgcgaacgtctcaatccaccctcgacttttcggtgctgtcacattatcctacccaaaccgttgtcgccatattagcataaaccggtacgtccatcaccaaacctgccgtcaccccaaaccaccaacctcctcgatcggccaattcccctcgtactgacacgacgaagcaaac

[0103] SEQ ID NO. 5: CP32

[0104] cctcagaagcgcgggattggccaaattcaccccacactgcgataaaagcgcgacaactgcttatcacggtcatgccgcgaaagcacgtggggcttatcgcacgtgattgtaaatgcacaattcaattcgggtgcgggctgcgcaaatcacaatcgggcttagggctgtgctgccctctcgcctagcaattttccggggcagcagcaagtgccaaagccgccggccgcaaaaacatcaccacccactttcaagtccttcgacgcaacacatcggagaagcgacaacaccacaaccgtcgcc

[0105] SEQ ID NO. 6: CP97

[0106] ggctgaatgtcatcaggtcgcggtttgcttaggtaatgagtaactcggcagctctgtctgccagacaactcaacgctagccatatcacgtgccaggtcacaacctgtcgtctcgagcctttgccgccccaccccgcgcacccacaacggctcaatttcggctgcacaactctttgcgactcacgtggagcgagccctagaacttttcctgcccacacgatttcgcccaaacaattcgtgaccgccgacggacgactccagataccctcaacgccccgacaaaccccaagtcacagccacc

[0107] SEQ ID NO. 7: CP146

[0108] cagtgagcagagtacctattaaagggagatgtcctgctcctcccccccgaggcgcgaagctccctttccctctcttctctctccttctttccccccagcttcgatctctatttaagtctctcgctcagctcggaacgcatcgacactcgttttagtgctttttgcaactaacaacaacatcgtcaacacttcagcggctactgtattcacttactcgcgtctttgctgtcttacacgcaaaagaacaaaacaagctcaactcgctcacgccttttttgaatcacacccaaaaccgtcaca

[0109] SEQ ID NO. 8: CP64

[0110] aggattctaccgacttacaccagcagacggccccgatatccgacagtgaaaaggatgtgctgactcagctggcggattgctcgaggatgagataaggaaaaaaaagtggcatcacgtgcacaggcgagagggtatcatcacgtggattagacgggcgcccaggcccacgccactcccacgcaaaaattagggctggcaattcgacactccaccgcgaaaaaggcgttgctctcccccccaaattaacaatctgtgaatcgtcatccccagcttcacgcctaaccaccaccgaccgccaag

[0111] SEQ ID NO. 9: CP213

[0112] agttgtggttttggtctcgatttgggggtatataaggcgtgaggattcccggttgatggaatttggatttttctgtcttctcttcagcgagaaaaatcgagggttgctgagatactgtttcccgcttgctctataacttcttcttttttttttgctcttttggcctttaacgttcttgaaggcgttggtt

[0113] SEQ ID NO. 10: CP47

[0114] agcgagcggtacctagccttaccgcacattttcgcagcgcaaggcccgtacccgcgagggcgtgcacgcgcctggcctcgctcgcgccaccccttcctgctccaggccaaatcagtaatccaccggccgccaacgcgtgccagctacaaaagcccagctcgtccccaagaacgtcgttcccgcacccattcacttttcccatcatcacccgctgccagtccgcccttctgacatcttgcatcatcaacactcaaccttttcagtctctgaaatctatttgaaatcaacgttattttcaaa

[0115] SEQ ID NO. 11: CP25

[0116] acctgataccagccggtacatccttacacagtattgcacagtgcccatctgactttctcctgccatagcgccgcccacgggtgagaatctcaagtgcggctgcttccagctgggggatacataaacctcgtcgcccgtggcatcttggacgcctcgatctctcttctctccttctacttgagcataatatcccaacactctgtttgaattgcattcattcgcaaacaacacctttttctatcgcgtcgttttcctctcttcctctcttttcccacatcaccacacacacaaccgcccatc

[0117] SEQ ID NO. 12: CP96

[0118] taactggccttcccagctcaatggctgtccgtggtggggtccggacaaggggggctgcatgcgccacacctcccgagaagttctgtggctggtgcggacttcttgcggcccaacaaacgaggcagcaatcctgactcacccgagcaagccacactggttatcgaagccccaatcaggcatagcgctgaccctatcgcccacaaagcgagaaatcaccaaactctcccaaatttcgctttgcctttgggaactttgcatcaccaaaccactcaagtaacgacaccgcacaccgtcgtcaaa

[0119] SEQ ID NO. 13: CP121

[0120] ccccttcgtatgtgtcctgccgaggtcgagcagaccgctcaggtgcttggctttttgaatcacgtgatcagattatccttgggtttgcgcgcaggcattggccacatttgattggctgaaagagtcctttatcccaaaaaggctagcctgtggacgcacgggccgcacaatctgggaacctcaggtctcgcttaaaatttcgcacacaaactattttcccggaagcccttttcacttttctgcttcgtgcaggcttcgtcaatttttcataccaacctcatcaccacacaacagcccaaa

[0121] SEQ ID NO. 14: CP190

[0122] GAAAAAAAAGATCGTAGGAGGAGCTGAGATCGATAAAGCGAAAACTCGGTTCCCGCCAGGCTCAGGCTCAGGCTCCGGCTGCTGCGGGTCCAGCCTCGTTGGTGC GTGTGTGCCCTG GTGGCCC GTGC GCGCA AAAAATGCCCCTAACGCTTGCACACTCGGTTGCCTTGATTT CGGCAAATCCCCGTTATCCTCAATCTCTACTACCACCTGTTCCGCGC AC GACGCAAAAAACCCCCGTCAC T AATTCTTCACACATCAATTCAATCTTCGAGGATCGCCCGACAAGAAC AAC AATCAAG

[0123] SEQ ID NO. 15: CP67

[0124] TACAGCGGCGAGAGAGATTGC GATGAGCCCTCTCCCTACCTACAGACGGCTGACAATGTCCGTATACCACCAGCCAACGTGATGAAAACAAGGACATGAGGAACAGCCTGCGAGAGCTGGAAGATGAAGAGGGCCAGAAAAAAAAGTATAAAGAAGACCTCGATTCCCGCCATCCAACAATCTTTTCCATCCTCATCAGCACACTCATCTACAACC ATCACCACATTC ACTCAACTCCTCTTTCTCAACTCTCCAAACACAAACATTCTTTGTTGAATACCAACC ATCACCACCTTTCAAG

[0125] SEQ ID NO. 16: CP75

[0126] acagcccattgaagaggaaccggaatatgcgatccgacccaatctttttcctgcttcaactaacaaaagtaatgctactagcacaatctcacgaaacgggcttctttttcgcatacccgagaatccgccgatagccatcatcgtgccatcacgtgatacacctttagcgggattaacatcagaccatacacttgctcgatccatgccgagattctccattttcgctattccaagtccccatcagacagcacaacgactctcgcagcttcaaacacagcttcttaaagctcctcactcacg

[0127] SEQ ID NO.17:CP132

[0128] tgacgacatatcatcgagagccagatcgaatcgaacgtggtaccgttgagccggaagtggaacccagcaataagccaatcagatgtctcaaatggatatgccgggaaatatgggctccgctgactcactcgccgcaaaccagcagaggcaaactcggagcctaggattatccggcagggcagtccccgctcacgtgacggattcctcttgcccacaatcatttggcgaaatcccccaaaaatctcaccgcgccactgcagattcagcgtccacacatcatcgccaaatcaatctatcacc

[0129] SEQ ID NO.18:CP214

[0130] attccagtttgcggatagcgtggctcaggagagcgaacacgaaattataaaagaggccatggcgagctccctggggagattctgctctgtatcacaacccaccaacatttccaaagtttacaacctccttgaacaccctttcccttgtcaatcgac

[0131] SEQ ID NO.19:CP66

[0132] tccgtatccgtaccgccgtccctttctgctgcttgtctctggtctctgctgctctgctctgctcccaaaaacccccaccaatgggcggcatgagcaaagtcacgtgcgctacccacttgccgagattttggcgcgcacaaacgcctgccctaggcccggcttagtcattttgaccacacagcgaccaattcgtgaaattgaacctatcccagcgacccggctgctcaccatcatttttctgctcaaacgccaaatcccgacgatacaccttgtcgccaacaacccacacaacgcggcaag

[0133] SEQ ID NO.20:CP34

[0134] ttcctttctttgcctccctcctctttcatcctcatcgtcgtcttctcatcaacactcatcaagacctaaacactcttcaacaacaactgctttgcgactgtgattaacttcatcagtcgcctcacttggtatgttgtgttctcgctccttttgctctctcgattcccttttttgctatctgagcacactcttcagacactaataaccgggcatcatcagaacatcaactctcgtgacactctccaaccaaacctccatttccatcaacaagacatcaccaccaccaccaccatcatcatc

[0135] SEQ ID NO.21:CP151

[0136] aattccctccaagctctagaaaagttcgtggatttttggttttctactgagctaccaagaattgctcggcggggcagggctatcgcgcgcgcggccaatgagcgcagacttgccgttttggtaagccctaaagcaccggggtttaagcggaccacatcggaacattgactcagaaacacttttgctttgaaatcaaccaggcggtttttctgaagtggcgcccgggtctccttgctgttggaacaatcttcccccaaacatcatcaacaccccaagaagcaacagccaaacaccgtcaaa

[0137] SEQ ID NO. 22: CP1

[0138] acggaggaagagcgctgccgttctagggcaaaatctcggagatccaccgtccggagctcaactgatcacgtgatacaggcattgcgagtgccaagccattactcatcagagccctcgcttagcccactctttgaaagttgtgctaaaattccgggtctagtgcacgtgaacccacaaactttcagcagaactgctaccggccgctggaaattctagcacttcgtaaaccctcccccccccaaaacttcatccaacctcatcgccatcgagggccggccatcaataccgaagttattcaag

[0139] SEQ ID NO. 23: CP168

[0140] ccaaaccatcgttttcccttgcctttcacctgctggcaacatgacgacaccgttgcttttcacaatctctactagctccgcgcagctgttttcccacgccgttgctccccttctcatctctgacacgatactaaaacaacagcctttctagcccctagaaaaccacagctctcgacctctcttttccgccgcccgtatcacaacttgcgcgaccgacaaagcccctcccacagccagaagaaagccctcttctaccaccgaattttctcgacgacagaaccaattcaaaaccagagcatc

[0141] SEQ ID NO.24:CP11

[0142] tcctttcgagacaagtttatatagccggtggcgcacccagccccagctcgcagctctcaaaccatatatactagagccctttcattacctgaagctttccaatctacttcacactatccaacaccctcgcctcatcgtgagcaaaaagctacctgccgacaacaaccgccaccaccaccaccaccagctacagaaatccagcactcaatctttctcttcacgcggtcgcttcgccatactatctcaatacacggaaccgcattcaccctccagcaaccacattactatcagttcacaaaa

[0143] SEQ ID NO.25:CP23

[0144] ccagtcgcggactcgcagtttctagcgcaatggagctgctcaagctcgggccaatcacgcagaaccagatctgcgtcacgtgccggtgggcaggcgaaccctatcacgctatcctgcttcgggaggaaaatttccagtggccagcaacgaagtcgccccctcagtgcaccgaacttttcctcgcttaactccctcctactctccagcttgcgagccttcaaatccgtcgccagatttcgtcaagtactcgaccctcgcaagacactccacggcaaattttcatcgacacaatcagacaaa

[0145] SEQ ID NO.26:CP173

[0146] catggccaatcgcccgccgtcgaccgccacccaatccgagcaacacaagagtgcccgcggtcagtggtggaaaggtacgagagtactttgcgtttaccagctgtcacgtgacacaatttgtctcgttatcgataagcgcactttggaagaaaattgtggtggggcttcgcttggtccatcgctcacgtgcgtgccctaaggaaaatcacaactccagcttcccagcacattttcgcgagagcagcctccaaccgaagtcaagcaaccaaacatccccatccctaccacaaatccgtcaag

[0147] SEQ ID NO.27:CP152

[0148] taagaccgcaaatggcttggcccgtcattaccccatacatgtaggtactaattttctggcatcgcctgcctgcttactaagttcagcctccaacaaccgtccaggggtcgtggtcatgtgattcgtccccaacccggctgaccgcggtgtgggaactaggcgaagcggacggcgcacgaaattttagggcttcacaaaatagcccaccgggaatctcgcccttctctatcctcatcttcgaattttccacccaaagtttgaggaccaaaagccagccatcgccaggccaacaccgtcaag

[0149] SEQ ID NO. 28: CP2

[0150] tatcgctgctgcacagcacctgcctcccgcacgtattttttattttctttatccccctccaccaaccccaaaacgcgccaaaaaaaagagagcaagagacaacgcacggtccaccgcgcaatcccagatcagccgccaccgcagcctggcgctaggctcaactattaaagctcctgccccccgcccgcaagtccggaatttctcctttctgcatcgcccatctttttctcgcccttcttccccaccacagtttcatcactacaaacaacaacaccaaacaatcacacatcatcattcaca

[0151] SEQ ID NO. 29: CP3

[0152] ctctaggtctcgcagccctaagcaccaccgaccagtgagcccttcctcgcctgccccggaaccagcccatgcccaatcgggcaagcgggcacagcgattttcttccgctccttggtatcgcgaccacttccctccacaaccctcccagggaacgactaacccagccaaaccaaagcaaacacacagccagccagatttctttcgccgcttcgaggtcggaggaaatttgtgtgttttcttttctttcatcgttccttctttctatttcttttccataccccccgaaaagaatccgaaacg

[0153] SEQ ID NO.30:CP7

[0154] gcccacgataacctcgcctgcttgaagcctgcgtgcgacattcaaccggcgcatcacgtgcagataagtgcagctgattactgccttatcggatggaattgagcatcacaccagccacgtcacagccacaagaccccactccgatatccttgcttcagcttgcttcaccagccaccgcggggcactgactgcatcccctcattagaaaaaaaaaaattcctctatcgccccgttcttcttctcttcccttcttctcccaagctcaaacaactcttgtcaagcatcacgacatcagtcaaa

[0155] SEQ ID NO.31:CP117

[0156] agaggaatcttagagtttgggggaaaatggctgcgatgacaagcggaaactttgtcatgtgaggtcgcggcctgcgccacaccagctggaggcgctagagcagacgttatcagagcccgcttatctacacgtgatgtcagccacaacgcacactttcgagccaccccacttcgctaggttggaatttcatgtagggcttccgcccacaaaagttatccaattcgagatctctggttgtacgcctgtccgcaaacaccgtcaactttccaacatcgaccctccgagcagcaaaccgccacg

[0157] SEQ ID NO. 32: CP192

[0158] ataataaggcaggtacctaccaaggtagcgcgattgccccctttgcagctgggctgggctggaccgggcaagagccatcccgcttccgtacaagcaacgaccccttcccgttcttacgcaatgcccctcttgcaagaatcccttttgcgcgatagataatcccatcctctcctccattctcccgcccttggccaactttctctcctctgcttgcttctcccgctattctatcccaaatctctcctctgagctccctcctctcttattttgccctctacaaaacctctacaaaccgccata

[0159] SEQ ID NO. 33: CP98

[0160] tcacccacgaggcgttgagttaaagtgttcccccaagcctctcttcgtgcggtgtaaaccctccccaaacactttttcttctctcttccttatcatcctcaactctccccctactattttcccctaataataataattcctcgccctttcagcccgcctacctacaggttcaactgaaggattttcccgcggcatctctacctacaacccttccgctctcaattccatcccttcctaatcagccaacgccaaaccctatccacgtcacaaagaaaggagttcgtctcataattcttcgcc

[0161] SEQ ID NO.34:CP108

[0162] agcttgagattgctgactcatgctaggattctgggaagatgaggtacgtaccccactttccatctttgttcgacaaattgaatcgagctctcaccgcttgcccgaacgaggctagcagagcacatgccctagccgttcgacacacattttctcactggccgcaaaaaccgcaaaaacgggctcggcctcttctcttttcctcttttcgcgatacctcaagacctcgagtcaattaaaaatccccatacctctaggatatctttttgcccttaaataacctgccttcagaaataagaagat

[0163] SEQ ID NO.35:CP45

[0164] tatcctcattcattgccctgtcagtgtgttggctcacgtctccaatcctccgcccctcctcctgcaaagtaaataccttctcaaaacacgtctggaatcctgcaagtctccatcacaaggagcttcttcatcaaccaccttatacgagcaacatcatttgcatcatcgttgatccacatctcctcgcgcctcagagtgtcgtcaccagtataaataaccgcatcaagctctcgtccttcttcgttccacaatccaagaagcacctcaaaacgatcaaagcagcgcagctacagcacaatc

[0165] SEQ ID NO.36:CP154

[0166] gtgccaggcggctggctagctggctaggcctcaaactccactgcaggccggcgcgctcatcacctggcagtgggctcagcgctcccaagctccgccccgagagccccccaaaccccaaaaaccaattcaaatctcaatccacaatcaaaatataaacactcttctcgccccatcctcaaccgcatcccacctcagacacccgcatccatcctctgcctctgccacccgccatcgccgcacaactcccgccgccttcccgccctctccgtcttccaaaacagcatcaccgacgccgtcgaa

[0167] SEQ ID NO.37:CP83

[0168] aatcatccacgtgcgccacgtttcgacaatttttttcgtcgtcaaggcggggtgaagcgctggcttttaggcagttgatgcctcaggccgctacccctctactagggctcccgcagtggtggggggggctgccttgcatcaatactgcgcagttccatctggtgcaatctttttttttcttgtgactcctctccagtagatacccaactcttccagctctcttcaaccagacgacctctctctctccatatcctttcttctctcgccctcgatttcacacacacacacacaatcgcagcc

[0169] SEQ ID NO. 38: CP161

[0170] aatcatccacgtgcgccacgtttcgacaatttttttcgtcgtcaaggcggggtgaagcgctggcttttaggcagttgatgcctcaggccgctacccctctactagggctcccgcagtggtggggggggctgccttgcatcaatactgcgcagttccatctggtgcaatctttttttttcttgtgactcctctccagtagatacccaactcttccagctctcttcaaccagacgacctctctctctccatatcctttcttctctcgccctcgatttcacacacacacacacaatcgcagcc

[0171] SEQ ID NO. 39: CP29

[0172] footagtgggggcctatgtgcatcgcccggtccaggtccatttctggagccagtcacgtgcattgactcagaaggcatcgagggaatcagcatcacgtgccgatagcgcgcccgccccattgtttggtcgcctaatcagccaatctaaattttgcaagccctaaggtgggaaagtggcccgaaaaaagtgcgccgttgccctactctccatcctgtgcaaaaccatctaacatcgtcttcccaccctctacgatacgccctcagttacggtacctctccggagcaggataattcaag

[0173] SEQ ID NO.40: CP162

[0174] ttttttttttattagggaagggaactcgcctttcattcctcaaccttcctttttttttttcccacaagggggggcaattacagaaagacgcgccctttttctacttcccagtctctcgcttcgaattctcggtttcactctttttttgacggtctttccggtcctgcgcgcctccttccgcacacatcgtttcgcatcctcccttgaggattcacgctcgtttttctctctacctgaaaaaaaaataagggagataaaaaaaagaaagacaccaacaaatcgtcgccatc

[0175] SEQ ID NO.41: CP180

[0176] tgctccagggcgccgcttgaaaggagcagacctcttttcgcatctttcttttttgcttttgcaacttaattcatcagtcctttttgacatcgtttttttttgagggcggccgcctcgcacagttctggcctttcagtcactccttaagacaaacaaccatcatttacattctatatcgttccttgacgcctttttgaatctcttcgtcgcctgaccgagcacgagaagcacacgtccaatcgctacagcatcaactcaagaaccgcaagtttcacgactactttcaccagaaccgccaag

[0177] SEQ ID NO.42:CP187

[0178] ctctctccattccgcgttagcctcgtcgagcccaggaaaattgttaaggcggcgtcggcttccctcccagcaaaaccgcgcaagtggccgtgattggcccgcgaggcaacccgagcccccacccttggtgctcacagactctgctctctccatccgccctcctcccgaaaatctccattttgctcttcctgcaaaaggcagcaagcatcaacaaactcctctcaactcctccaccccccaattgcacgcccgctgcaattgctcccctcgttgctgaggccttcgtatttcgccctcaag

[0179] SEQ ID NO.43:CP106

[0180] GCGTGTGGAAGCGAATGTGCTTAGATAATCCACGGATTTTTTTTTCCC GC CGACGATAGGGCAGGATTGTGGGTTTGGGGGG AACGGCACATGTTGCCC GGCTTAGGGCTCGTGGCTAGAAGGTCACGTGCA TGGTTTCCAGACGC CAAATTT CCTATCCACACAACCTTGACGATTCCTCGAGATCACCCGCGC AAAAGTTCACCAGCAGCCACCAC TTAGGAAGCAAGTCAAGTGAGTATCTCATAT ACTCAATTCTTACCCGTCCGAGCC AT A ACC TCTGACAATTCTACAGA

[0181] SEQ ID NO. 44: CP116

[0182] TGGGC GTGT TTCGTGGGGTCGTT CGAATTGAGTTTAGTTTCGTTTGAGAGGC GAAACCGGAAGAACCAACGGCATTGGCTGGTCAATACATCAAGTTCGTT CCTTTCACGTGACTTCTCCAGCACCCTCGCGGGTCTTGCCTAGCGGTA AGCCTTAGGGCAAGAAAGCCCCACAATTGGCGCGATCGAAGCGAGGA GGGAAAAAAAAAGGTGTGCCATCAATTTAACTTTCTGTGCCTTATTC CACGCTCAACCTCTTCGTCGTTCCTATCACCCTCGAGAAAACAGCCAG TCCTCACC

[0183] SEQ ID NO. 45: CP155

[0184] gctcctccggctgactctggctcctcctgctctgcgctctgcctggacggacggacggactcgctcgctggtcccggcgtcacgacaataaaaaggccggcctgcctcccgcccccctctgctccttctcctgtctcgcctcgtcggtctctgtctcacgcaacaaagagtcgttccgttcactcggcccgggtctcgtctttcttcctgccatccccagccagcatcttcactcccacgcgtcaagtcaacaagagtccccctgaacaaaaaaagacaagcccagcgtcgaccgcagcc

[0185] SEQ ID NO.46:CP102

[0186] cttcatgatgacgtcgcacagttgccccgcctaaacacccctcaatcaagctggtgcttctcctgggtgttgcgatactaaattgcgcggctgcctcccgccagctgcagtgaagccacgatccatataaattgccacgcttgtgctcgtcatcggcttgcatcatcatcctattccattctcactcctggcaacacaacctcaaacaaagaaaaacatctgcttccaaacatctacgacactttcaacacacccacccacatacacacacacacacacaaaccatccacaaccaacaag

[0187] SEQ ID NO.47:CP33

[0188] CGGTGTGTGTTGCTTGATGTGTGAATGCTGCTGGAGCTTTGATCCCCTGAGACGGTGGGGATGATGGTGGCTGAAATGTCTCGCTCTGAACGAGGTGCTGGGAGTTGGGAGGGCGGTATGAAGCGAGGAGGAGGAGGAAGGACTACTATAAGAAGAGTCGAGAATCATCATTGTCTCAGACCTTTTCCTCTTCAGCAAGTCACACAGTTTATGACCCTTCACCTCAACTACTTACTTACACTCTTCAGAAGACATCGAAATCACCAATCGTCACCACTACCATCACCACCATCGCCCATC

[0189] SEQ ID NO. 48: CP92

[0190] TGGCGGATTTGGTTCTCGATACCCTCCTCGAGAAAAAATGCAGGCCTTTTCACGTGACATCAACC GCACCTGCCACTCTCGTGCCTTAGCGCGGACGAGGGCCAAAGAGGGCTGGAGACTTGTTGAGCA GGAAGATTTATCCCACAGAATTAGCGGAAAAATATATGCCCCGGTACCTTTCTGGTTTGGCTCG CTAGACTCAGGTTGATGCACACAACAATTGAAATTCTTCCAACCCCTCCACCAACTTCTTAGGA CCCTTTTGATTCCCAAGACCTCGTACGCCGGTAAGGAATC

[0191] SEQ ID NO. 49: CP40

[0192] GCTGTAAGCTGCAACTGAACCTGGAACCTGAAGTACCCTCGCTGTTGCCGTGGCAGGTGGAAGGC GGTCCAAGCCCAAAGCCTGCAGCGCTAGTGCAGCCGGAAAGAGTCGCTCTGAAAAAGTGTCCCCT TC GAGACCCGCTGCAATTTTTTTCCCTCCCCCCCCAGAAAAGCCATTCGCCCGCTCGGTGCAGAAT TTATTTCCCTCGAGTCCCCGATTGTCCTCCTCTTCCTCCTTTCATCTCCATCTCCCCTCAGTTCT TTCTTTGTTTCTGGGAAAGCGTTGCACCTCAGTCAA

[0193] SEQ ID NO. 50: CP21

[0194] GCTGTAAGCTGCAACTGAACCTGGAACCTGAAGTACCCTCGCTGTTGCCGTGGCAGGTGGAAGGC GGTCCAAGCCCAAAGCCTGCAGCGCTAGTGCAGCCGGAAAGAGTCGCTCTGAAAAAGTGTCCCCT TC GAGACCCGCTGCAATTTTTTTCCCTCCCCCCCCAGAAAAGCCATTCGCCCGCTCGGTGCAGAAT TTATTTCCCTCGAGTCCCCGATTGTCCTCCTCTTCCTCCTTTCATCTCCATCTCCCCTCAGTTCT TTCTTTGTTTCTGGGAAAGCGTTGCACCTCAGTCAA

[0195] SEQ ID NO. 51: CP143

[0196] aagccaatcgcacgatcccaacaaagcgttcatgttcgtcatttgcccctccttttcggcctttgacttgcccccccctttagtacctaaaagtaggtagccagtggtaaaccaagtccagttgctattgccccgacttagcttcagctttaggcgttaccccgccggccgagaccagcgagcttattactaggcagtttgtagtacttgtaggtatgaagcttttctttccctcctccctttcttctaccatacctacctaaatacctgacatattcagaacaacatcgacaagtcatc

[0197] SEQ ID NO. 52: CP178

[0198] cgtgcccctctgtgctccggggggcgtgtggctactcgttaggccttgtgtgccagatcgctcagggaactgatctctccggacacctcgagctttcgccgcttgcaaccttctctcctccatatcaccgtcttcccgcgatttccaccttcataaaccaacgctccctctccgtcctctgcagaaccgtgttgaggaggaagcatccgcatctttctttttgttcagattcccatcgactccggccgcgggcttgctctcaccccacttccatcgtgaacacagctcccagtcgacatc

[0199] SEQ ID NO. 53: CP27

[0200] ctggtcacacaggcccaagctcgccatttctcgccctccaccccctggccgctcttattttcctgcccagtcctcgtccgcggcaaagcaaataagtagccctggccacccccttctgctaccgactcttcttcttcttcctcctcccactttctttcttcactcacgcttcaccagcgcccgttttccccatacaccatccaaaaggctttttctggatccttctttttttttaatacccccccctttgtttgttgtgataccccccgcgctttaaagatacccttttttctatccaga

[0201] SEQ ID NO. 54: CP30

[0202] gttttaggctgcacagggtgtggttcagggactcaactgggcccggctgaaccagcgcggcagccagatgtggccaaagggacacttgttcaattgaagtgagtgcgtctaagtcatcaaggccactgcaagtacaaaacctcgcccgtccatccgtctttcgtgcaactgcattttgcacttccgcatcaccacttttttttcttcttcttctttttctattttctttttcttctcgaatcctccctctcccccgtccatctgtgatacccgcttgataccccaatcacaaccgtcgcc

[0203] SEQ ID NO. 55: CP128

[0204] Ccttacgcccgtgtgccaaaacggtactaaactgctgtaaaccgcgtcccaccagcgcccaagcaccatttgccgccagggccaatcacacaggcgaaaaaaaaattcaggtaacgcaccttttctcccccctccgtcctctttcctgctccttctcctcccaccttctcgatttctcctctccacatccttccagcactccactgcttttttcttttatctgccgcgcagactctctctccctcttcgcttcttctcttccgcttcacgacaagcccgatttttgcgcaatccatcaca

[0205] SEQ ID NO. 56: CP36

[0206] Ccttacgcccgtgtgccaaaacggtactaaactgctgtaaaccgcgtcccaccagcgcccaagcaccatttgccgccagggccaatcacacaggcgaaaaaaaaattcaggtaacgcaccttttctcccccctccgtcctctttcctgctccttctcctcccaccttctcgatttctcctctccacatccttccagcactccactgcttttttcttttatctgccgcgcagactctctctccctcttcgcttcttctcttccgcttcacgacaagcccgatttttgcgcaatccatcaca

[0207] SEQ ID NO. 57: CP46

[0208] gcacgcccccccgcaccctcaaattgtcgcaattgttttgctggaacacgtccatttccccaacaggcctagcgccgcaacagctgcgagagggagggtgattgctgatctcgcgcggcgagcaccaaaaagtacttaactttggagccc cccgccaaatccaaacctctgcatttacactgtttctctcaactctccacgctgcacgattaccattctgacatttgccgcatctcgcgcatctcgcatcaacactttacgttactttttccacgttaaccaaaaacccccaacttcaaa

[0209] SEQ ID NO:58: ShLYZ-mCherry base sequence, where the underlined portion encodes shLYZ and the ununderlined portion encodes mCherry. ATGAAAGCTTTGATTGTTTTGGGTTTGGTTTTGTTGTCTGTTACTGTTCAAGGTAAAG TTTTTGAAAGATGTGAATTGGCTAGAACTTTGAAAAGATTGGGTATGGATGGTTATAGAGGTATTTCTTTGGCTAAT TGGATGTGTTTGGCTAAATGGGAATCTGGTTATAATACTAGAGCTACTAACTATAATGCTGGTGACAGATCAACTGA TTATGGAATCTTTCAAATTAATTCTAGATATTGGTGTAATGATGGTAAAACTCCTGGTGCTGTTAATGCTTGTCATT TGTCTTGTTCTGCTTTGTTGCAAGATAATATTGCTGATGCTGTTGCTTGTGCTAAAAGAGTTGTTAGAGATCCACAA GGTATTAGAGCTTGGGTTGCTTGGAGAAATAGATGTCAAAATAGAGATGTTAGACAATATGTTCAAGGTTGTGGTGT TGTCTCCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTCCACATGGAGGGCTCCGTCAACGGCCACGAGTTCGAAATCGAGGGCGAGGGTGAGGGCCGCCCGTACGAGGGCACGCAGACCGCCAAGCTCAAGGTCACCAAGGGCGGCCCCCTCCCCTTCGCCTGGGACATCCTCTCCCCGCAGTTCATGTACGGCTCCAAGGCCTACGTCAAGCACCCCGCCGACATCCCCGACTACCTCAAGCTCTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTCATGAACTTCGAGGACGGCGGCGTCGTCACCGTCACCCAAGACTCCTCCCTCCAAGACGGCGAGTTCATCTACAAGGTCAAGCTCCGCGGCACCAACTTCCCCTCCGACGGCCCCGTCATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGCATGTACCCCGAGGACGGCGCCCTCAAGGGCGAGATCAAGCAGCGCCTCAAGCTCAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTCCCCGGCGCCTACAACGTCAACATCAAGCTCGACATCACCTCCCACAACGAGGACTACACCATCGTCGAGCAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTCTACAAGTAA

[0210] SEQ ID NO: 59: ShLYZ-mCherry amino acid sequence, with shLYZ underlined and mCherry not underlined MKALIVLGLVLLSVTVQGKVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGY NTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRN RCQNRDVRQYVQGCGVVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0211] SEQ ID NO: 60: mCherry base sequence ATGGTCTCCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTCCACATGGAGGGCTCCGTCAACGGCCACGAGTTCGAAATCGAGGGCGAGGGTGAGGGCCGCCCGTACGAGGGCACGCAGACCGCCAAGCTCAAGGTCACCAAGGGCGGCCCCCTCCCCTTCGCCTGGGACATCCTCTCCCCGCAGTTCATGTACGGCTCCAAGGCCTACGTCAAGCACCCCGCCGACATCCCCGACTACCTCAAGCTCTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTCATGAACTTCGAGGACGGCGGCGTCGTCACCGTCACCCAAGACTCCTCCCTCCAAGACGGCGAGTTCATCTACAAGGTCAAGCTCCGCGGCACCAACTTCCCCTCCGACGGCCCCGTCATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGCATGTACCCCGAGGACGGCGCCCTCAAGGGCGAGATCAAGCAGCGCCTCAAGCTCAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTCCCCGGCGCCTACAACGTCAACATCAAGCTCGACATCACCTCCCACAACGAGGACTACACCATCGTCGAGCAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTCTACAAGTAA

[0212] SEQ ID NO: 61: mCherry amino acid sequence MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0213] SEQ ID NO: 62: a-factor-Pro K coding sequence, with underlined portion being a-factor signal peptide (for secretion of Pro K outside of cell) coding sequence, and shaded portion being Pro K coding sequence.

[0214]

[0215] SEQ ID NO: 63: a-factor-Pro K amino acid sequence, with underlined portion being a-factor signal peptide for secretion of Pro K outside of cell, and shaded portion being Pro K amino acid sequence.

[0216]

[0217] SEQ ID NO: 64: Pro K amino acid sequence APAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDAAMEKISGKPDHVYKNVFSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYASSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDHNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGATDRYDRRSSFSNYGSVLDIFAPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGRTTAANACRYIADTANKGDLSNIPFGTVNLLAYNNYQAHHHHHH

[0218] SEQ ID NO: 65: pUC-57-shLYZ-mCherry-Amp, wherein the upper case part is the binding site of 8 tandem artificial transcription factors, the underlined upper case part is An201CP, the shaded part is the human lysozyme coding sequence, wherein the bold part is the signal peptide coding sequence of the human lysozyme itself, the underlined shaded part is the mCherry coding sequence, and the wavy line part is Tpdc1.

[0219]

[0220]

[0221] SEQ ID NO: 66: Amino acid sequence of transcription factor Bm3R1-NLS-VP16 MESTPTKQKAIFSASLLLFAERGFDATTMPMIAENAKVGAGTIYRYFKNKESLVNELFQQHVNEFLQCIESGLANERDGYRDGFHHIFEGMVTFTKNHPRALGFIKTHSQGTFLTEESRLAYQKLVEFVCTFFREGQKQGVIRNLPENALIAILFGSFMEVYEMIENDYLSLTDELLTGVEESLWAALSRQSSRADPKKKRKVSTAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG*

[0222] SEQ ID NO: 67: Coding sequence for transcription factor Bm3R1-NLS-VP16 ATGGAGAGCACCCCCACCAAGCAGAAGGCCATCTTCAGCGCCTCCCTCCTGCTCTTCGCTGAACGGGGCTTCGACGCCACGACGATGCCGATGATTGCTGAGAACGCCAAGGTCGGAGCCGGCACCATCTATAGATACTTTAAGAACAAGGAGTCGCTCGTCAACGAGCTCTTTCAGCAGCACGTCAATGAGTTCCTTCAGTGCATCGAGAGCGGCCTCGCCAACGAGCGCGACGGCTACCGCGACGGCTTCCACCACATCTTCGAGGGCATGGTCACCTTCACCAAGAACCACCCCCGCGCCCTCGGCTTCATCAAGACCCACTCCCAGGGCACCTTCCTGACCGAGGAGAGCCGTCTGGCCTACCAGAAGCTCGTCGAGTTCGTCTGCACCTTCTTCCGCGAGGGCCAGAAGCAGGGCGTCATCCGAAACCTCCCCGAGAACGCCCTGATCGCCATCCTCTTCGGCTCGTTCATGGAGGTCTACGAGATGATCGAGAACGACTACCTCTCCCTGACCGACGAGCTGCTCACCGGCGTCGAGGAGTCCCTCTGGGCCGCCCTGTCCCGCCAGAGCAGCCGCGCCGACCCCAAGAAGAAGCGCAAGGTCAGCACCGCCCCCCCCACCGACGTCAGCCTCGGCGACGAGCTGCACCTCGACGGCGAGGACGTCGCCATGGCCCACGCCGACGCCCTGGACGACTTCGACCTGGACATGCTCGGCGACGGCGACTCGCCCGGCCCCGGCTTCACCCCCCACGACTCCGCCCCCTACGGCGCCCTCGACATGGCCGACTTCGAGTTCGAGCAGATGTTCACCGACGCCCTCGGCATCGACGAGTACGGCGGTTAA

[0223] SEQ ID NO: 68: Nucleotide sequence of 8 concatenated binding sites for an artificial transcription factor ATCGATTCAAATGAGCTAGGGACCTGGCGGAATGAACATTCATTCCGAGCATAACGGAATGAAGGTTCATTCCGAGGGGACCTGGCGGAATGAACTTTCATTCCGAGCATAACGGAATGAACATTCATTCCGAGGGGACCTGGCGGAATGAAGGTTCATTCCGAGCATAACGGAATGAACTTTCATTCCGAGGGGACCTGGCGGAATGAACATTCATTCCGAGCATAACGGAATGAAGGTTCATTCCGAGGGATGATAATGCGATTGCTAGC

[0224]

[0225]

[0226] SEQ ID NO: 71: An201CP core promoter element TTCTCTTTTCTTAAGAATATGTTCAAAGACTAGGATGGATAAATGGGGTATATAAAGCACCCTGACTCCCTTCCTCCAAGTTCTATCTAACCAGCCATCCTACACTCTACATATCCACACCAATCTACTACAATTA

Claims

1. An optimized synthetic expression system, characterized in that, It includes a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises, from 5' to 3', a first core promoter, an artificial transcription factor coding region and a first transcription terminator element, and the second polynucleotide comprises, from 5' to 3', an artificial transcription factor binding site, a second core promoter, a target protein coding region and a second transcription terminator element; The first core promoter drives the expression of an artificial transcription factor in its coding region. The artificial transcription factor binds to its binding site on a second polynucleotide to act on the second core promoter, which in turn promotes the expression of the target protein gene in the target protein coding region. The first core promoter is selected from SEQ ID NO:4-57.

2. The synthetic expression system according to claim 1, characterized in that, The target protein is selected from fluorescent proteins, enzymes, antibodies, and fusion proteins; preferably, the target protein is selected from fluorescent proteins, enzymes, and fusion proteins.

3. The synthetic expression system according to claim 1, characterized in that, The target protein is a fluorescent protein, preferably mCherry; And / or, the target protein is an enzyme, preferably alkaline proteinase K; And / or, the target protein is a fusion protein, preferably a fusion protein of human lysozyme and mCherry.

4. The synthetic expression system according to claim 3, characterized in that, The amino acid sequence of the mCherry is SEQ ID NO:61; And / or, the amino acid sequence of the alkaline proteinase K is SEQ ID NO:64; And / or, the amino acid sequence of the human lysozyme and mCherry fusion protein is selected from SEQ ID NO:

59.

5. The synthetic expression system according to claim 1, characterized in that, The target protein coding region contains multiple copies of the target protein gene. Preferably, the multiple copies are 2 or more copies. More preferably, the multiple copies are 2-10 copies. Most preferably, the multiple copies are 2 or 3 copies.

6. The synthetic expression system according to claim 1, characterized in that, The amino acid sequence of the artificial transcription factor encoded by the coding region of the artificial transcription factor is shown in SEQ ID NO:66; And / or, the first transcription terminator element is Ttef1, preferably, the nucleotide sequence of Ttef1 is as shown in SEQ ID NO:69; And / or, the artificial transcription factor binding site is a binding site for eight tandem artificial transcription factors, preferably, the nucleotide sequence of the binding site for the eight tandem artificial transcription factors is shown in SEQ ID NO:68; And / or, the second core promoter is selected from An201CP, preferably, the An201CP nucleotide sequence is shown in SEQ ID NO:71; And / or, the second transcription terminator element is Tpdc1, preferably, the nucleotide sequence of Tpdc1 is SEQ ID NO:

70.

7. A fungal host cell, characterized in that, The invention comprises a synthetic expression system according to any one of claims 1-6; preferably, the first and second polynucleotides in the synthetic expression system are integrated into the genome of a fungal host cell as expression cassettes.

8. The fungal host cell according to claim 7, characterized in that, The fungal host cells are selected from Saccharomyces cerevisiae, Pichia pastoris, Yersinia lipolytica, Saccharomyces brevis, Rhodotorula rubra, Candida albicans, Trichoderma reesei, and Aspergillus niger; preferably, the host cells are selected from Pichia pastoris, Trichoderma reesei, and Aspergillus niger.

9. The application of the synthetic expression system according to any one of claims 1-6 and the fungal host cell according to claim 7 in expressing a target protein, preferably, the target protein is selected from fluorescent proteins, enzymes, antibodies and fusion proteins.

10. A method for producing a target protein, characterized in that, Includes the following steps: (1) Culture the fungal host cells according to claim 7 under conditions suitable for the expression of the target protein. (2) The target protein is recovered.

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