Core promoter optimized pichia pastoris protein expression system and application thereof

Abstract

This invention relates to a Pichia pastoris protein expression system with optimized core promoters and its applications. Specifically, this invention designs highly efficient promoters based on the 300bp upstream region of the start codon of a highly expressed gene in *Trichoderma reesei*, and uses these promoters as the core promoter 1 of a synthetic expression system (SES) in fungal cells. When the SES system containing the inventor's selected promoters is used for target protein expression in fungal host cells, it exhibits higher expression efficiency than the existing SES-A system. Based on this, this invention provides an optimized synthetic expression system, a fungal host cell containing this optimized synthetic expression system, and its application in expressing target proteins.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering technology. Specifically, this invention relates to a Pichia pastoris protein expression system with optimized core promoter and its application. Background Technology

[0002] Pichia pastoris, due to its high growth density, strong protein expression, good genetic stability, and mild glycosylation modification, has demonstrated excellent application results in academic research and industrial production, and has become the preferred chassis cell for recombinant protein expression. Commonly used promoters such as PAOX1, PGAP, and PTEF1 are mostly endogenous promoters, whose expression is affected by the complex regulatory network of the host cell, leading to unstable expression efficiency and limited regulatory precision. Furthermore, PAOX1, as a widely used promoter, relies on methanol-induced properties, posing certain risks to industrial facility safety and food application safety.

[0003] The Synthetic Expression System (SES) is a universal functional protein expression system developed by the Dominik Mojzita laboratory at the Technical Research Centre of Finland for various fungal hosts. The SES system mainly consists of two modules: one is the expression of an artificial transcription factor (Bm3R1-NLS-VP16) driven by a core promoter 1, which allows the artificial transcription factor to accumulate; the other module consists of multiple binding sites of the artificial transcription factor tandemly with a core promoter element 2, which promotes the expression of downstream target proteins under the action of the artificial transcription factor. SES-A is a highly efficient protein expression system constructed based on SES, using core promoter elements An008CP and An201CP derived from Aspergillus niger to drive the expression of the artificial transcription factor and the target protein, respectively. The SES-A system, a powerful and multifunctional protein expression platform, has been validated in various yeasts, including *Saccharomyces cerevisiae*, *Pichia pastoris*, *Pichia kudriozwee*, *Yarrowia lipolytica*, *Saccharomyces longipes*, *Rhodotorula rubiginosa*, and *Candida*. It has also been successfully applied in *Trichoderma reesei* and *Aspergillus niger*, covering several industrially valuable fungi. The SES-A system can achieve target protein expression levels that meet or exceed those of potent endogenous promoters or traditional gene expression tools. However, the effectiveness of the SES-A system in *Pichia pastoris* cells remains relatively limited. Further screening and optimization of its core promoter elements are necessary to improve the protein expression efficiency of the SES system, enrich the protein expression tools available for *Pichia pastoris*, and enhance the industrial application potential of *Pichia pastoris*. Summary of the Invention

[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 coding region of the artificial transcription factor of the present invention is shown in SEQ ID NO:66;

[0028] And / or, the first transcription terminator element is Ttef1, preferably, the nucleotide sequence of Ttef1 is SEQ ID NO:69;

[0029] 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;

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

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

[0032] In one aspect, the amino acid sequence of the artificial transcription factor encoded by the coding region of the artificial transcription factor described in this invention is shown in SEQ ID NO:66. In another aspect, the framework of the synthetic expression system (SES) of this invention is constructed with reference to 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 being the replacement of the core promoter 1 to broaden the screening scope.

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

[0034] In one aspect, the first transcription terminator element of the present invention is Ttef1. In another aspect, the nucleotide sequence of Ttef1 described in the present invention is shown in SEQ ID NO:69. In the present invention, the Ttef1 transcription terminator sequence is derived from the *Trichoderma reesei* *tef1* gene and terminates transcription during transcription. Transcription terminators derived from different genes can be selected; theoretically, any terminator that can perform transcription termination can be substituted for one another. For example, Tpdc1 in the present invention can also be used here as the first transcription terminator.

[0035] In one aspect, the artificial transcription factor binding site of the present invention is a binding site for eight tandem artificial transcription factors. In one aspect, the nucleotide sequences of the eight tandem artificial transcription factor binding sites of the present invention are shown in SEQ ID NO:68.

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

[0037] In one aspect, the second transcription terminator element of the present invention is selected from Tpdc1 or ADH1, preferably Tpdc1. In one aspect, the nucleotide sequence of Tpdc1 in the present invention is SEQ ID NO:70. In the present invention, the Tpdc1 transcription terminator sequence originates from the *Trichoderma reesei* pdc1 gene and terminates transcription during transcription. Transcription terminators derived from different genes can be selected; theoretically, any terminator that can perform transcription termination can be substituted for one another. In one aspect of the present invention, ADH1 is derived from *Saccharomyces cerevisiae*.

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

[0039] In one aspect, the first and second polynucleotides in the synthetic expression system of the present invention are integrated into the genome of a fungal host cell via different expression cassettes.

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

[0041] In one aspect, the fungal host cell of the present invention is selected from Saccharomyces cerevisiae, Pichia pastoris, Yersinia lipolytica, Saccharomyces simulans, Rhodotorula rubra, Candida albicans, 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 cells described in this invention are selected from Pichia pastoris, Saccharomyces cerevisiae, Trichoderma reesei, and Aspergillus niger. Besides Pichia pastoris used in the embodiments of this application, the literature 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) reported that the SES system can also express the target protein in Saccharomyces cerevisiae, Trichoderma reesei, and Aspergillus niger.

[0043] In one aspect, the fungal host cell of the present invention is Pichia pastoris. Preferably, the fungal host cell of the present invention is Pichia pastoris PpHR3.

[0044] In another aspect, the present invention provides a synthetic expression system according to the invention and the use of a host cell according to the invention in expressing a target protein, preferably, the target protein being selected from fluorescent proteins, enzymes, antibodies and fusion proteins.

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

[0046] (1) Culture the fungal host cells according to the present invention under conditions suitable for the expression of the target protein.

[0047] (2) The target protein is recovered.

[0048] In this invention, those skilled in the art can routinely determine the conditions suitable for the expression of the target protein based on the type of the target protein to be expressed.

[0049] In one aspect of this invention, by analyzing the transcriptome data of *Trichoderma reesei*, promoter sequences driving high gene expression in *Trichoderma reesei* are screened as a heterologous core promoter element library to drive the expression of artificial transcription factors, thereby optimizing the SES system. This invention provides heterologous core promoter sequences with good testing results, composed of the base sequences shown in SEQ ID NO. 4 to SEQ ID NO. 57. Specifically, in one aspect, the first core promoter of this invention is designed from the 300bp upstream region of the start codon of the highly expressed gene in *Trichoderma reesei*. In this invention, the SES system with the optimized first core promoter exhibits significantly higher expression efficiencies in both mCherry (luciferase) and ProK (alkaline protease) compared to the existing SES-A system.

[0050] In one aspect of the present invention, the first and second nucleotides of the present invention can be used as expression cassettes to be integrated into specific sites within the Pichia pastoris host genome, and multi-copy site-specific integration can be achieved through CRISPR / Cas9 or other means.

[0051] In one aspect of the present invention, the Pichia pastoris strain PpHR3 can be used. The genome of this strain is integrated with the Cas9 gene and three genes related to enhancing homologous recombination (RAD52, RAD59, and MRE11). Only an sgRNA vector and a polynucleotide with a 50bp short homologous arm are needed to achieve site-directed integration of the target gene. Furthermore, this system has the ability to integrate multiple fragments simultaneously, enabling rapid site-directed integration of the SES expression module. For example, the Pichia pastoris strain PpHR3 of the present invention can be obtained from the following literature: Gao J et al., Enhancing Homologous Recombination Efficiency in Pichiapastoris for Multiplex Genome Integration Using Short Homology Arms. ACS SynthBiol. 2022 Feb 18; 11(2):547-553. doi:10.1021 / acssynbio.1c00366.Epub 2022 Jan21.PMID:35061355.

[0052] In one aspect, the present invention provides the application of an optimized SES system in the expression of heterologous alkaline proteases or fusion proteins in Pichia pastoris.

[0053] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0054] The SES system described in this invention effectively avoids the influence of host endogenous regulation on expression levels 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; it is suitable for efficient expression of industrially valuable heterologous proteins in Pichia pastoris using SES; and a multi-copy strategy can be flexibly applied to further enhance the expression efficiency of target proteins in the SES system. Attached Figure Description

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

[0056] Figure 2 Efficiency results of mCherry expression in SES systems optimized for different heterogeneous promoter elements.

[0057] Figure 3Results of Pro K activity assay in Pichia pastoris using different SES systems

[0058] Figure 4 To improve the Pro K activity assay results of SES-CP32 secreted expression in Pichia pastoris using a multicopy strategy.

[0059] Figure 5 Figure showing the efficiency results of expressing the fusion protein shLYZ-mCherry using a SES system optimized for different heteroprogenitor elements. Detailed Implementation

[0060] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0061] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0062] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

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

[0064] The DNA fragment of the SES-A plasmid was synthesized by Hangzhou Qingke Biotechnology Co., Ltd. Figure 1The company was commissioned to use the Gibson assembly method (Novizan ClonExpress Ultra One Step Cloning Kit V2, C116) to connect artificial transcription factor modules (using An008CP as the core promoter 1) and target protein mCherry, Pro K and shLYZ-mCherry expression modules (using An201CP as the core promoter 2) into pUC57-Kana and pUC57-amp, respectively. 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 is used to optimize the screening and design of heterogeneous core promoters in SES.

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

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

[0068] Example 3: Site-directed integration of the SES system into Pichia pastoris

[0069] For Pichia pastoris transformation, the Donor-DNA only needs to amplify the regions covered by the CorePromoter1-Bm3R1-NLS-VP16-Ttef1 and 8×BS-CorePromoter2-mCherry-Tpdc1 expression cassettes. The Donor-DNA is amplified using KeyPo high-fidelity enzyme (2×KeyPo Master Mix, Vazyme), purified by a gel extraction kit (Omega), and then used for Pichia pastoris transformation. To evaluate the effectiveness of the screened promoters, artificial transcription factor expression cassettes were amplified using Int1-sTFF and Int1-sTFR primers, and the target protein mCherry expression cassette was amplified using Int6-ProK / mCherry / shLYZF and Int6-ProK / mCherry / shLYZR primers. With the assistance of the HGP-sgRNA-Int1-Int6 sgRNA plasmid (construction method referred to 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 the target protein expression cassette Donor-DNA were integrated into the Int1 and Int6 sites of the genome of Pichia pastoris strain PpHR3 via homologous recombination. When expressing alkaline proteases using the optimized protein expression system, artificial transcription factor expression cassettes were amplified using Int1-sTFF and Int1-sTFR primers, and alkaline protease Pro K expression cassettes were amplified using Int6-ProK / mCherry / / shLYZF and Int6-ProK / mCherry / shLYZR, Int15-ProKF and Int15-ProKR, and Int18-ProKF and Int18-ProKR primers. With the assistance of HGP-sgRNA-Int1-Int6 and HHP-sgRNA-Int18-Int15sgRNA plasmids, the corresponding expression cassettes (artificial transcription factor expression cassettes and 1-3 Pro K expression cassettes) were integrated into the Int1, Int6, Int15, and Int18 sites of the Pichia pastoris strain PpHR3 genome via electroporation.

[0070] When expressing alkaline proteases using the optimized protein expression system, artificial transcription factor expression cassettes were amplified using Int1-sTFF and Int1-sTFR primers, and shLYZ-mCherry expression cassettes were amplified using Int6-ProK / mCherry / / shLYZF and Int6-ProK / mCherry / shLYZR primers. With the assistance of the HGP-sgRNA-Int1-Int6 sgRNA plasmid, the corresponding expression cassettes were integrated into the Int1 and Int6 sites of the Pichia pastoris strain PpHR3 genome via electroporation.

[0071] The primers used in this embodiment are shown in Table 1. The underlined parts represent the homologous arm sequences corresponding to the integration sites Int1, Int6, Int15, and Int18. For information on the source of PpHR3 in Pichia pastoris strain 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 on 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 transforming Pichia pastoris.

[0073]

[0074]

[0075] The primers were synthesized by Hangzhou Qingke Biotechnology Co., Ltd.

[0076] The specific steps for electroporation of Pichia pastoris are as follows:

[0077] (1) Inoculate a single fresh Pichia pastoris colony into 5 mL of YPD medium (YPD medium consists of 1% yeast extract, 2% peptone, and 2% glucose) and incubate overnight at 30°C.

[0078] (2) Transfer the bacterial culture to a 50mL YPD shake flask, control the initial OD600 at 0.15-0.2, and incubate at 30℃ for 4-5h.

[0079] (3) When the OD600 reaches 0.8-1.0, take the bacterial culture to prepare Pichia pastoris competent cells. Transfer the bacterial culture to a 50mL sterile centrifuge tube and centrifuge at 4000rpm for 5min.

[0080] (4) Discard the supernatant, add 8.55 mL Beds (10 mM N-diglycine, 3% (v / v) ethylene glycol, 1 M sorbitol, pH adjusted to 8.3 with sodium hydroxide), 1 mL of 1 M DTT, and 450 μL of dimethyl sulfoxide (DMSO), and gently resuspend the precipitate by blowing.

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

[0082] (6) Discard the supernatant, add 950 μL of Beds and 50 μL of DMSO, and gently resuspend the precipitate by pipetting. Centrifuge at 4000 rpm for 30 seconds.

[0083] (7) Discard the supernatant, add 190 μL of Beds and 10 μL of DMSO, and gently resuspend the precipitate by pipetting. The cells obtained by resuspending are Pichia pastoris competent cells, which can be stored in an ultra-low temperature freezer.

[0084] (8) Take 40 μL of Pichia pastoris competent cells, add 500 ng sgRNA plasmid and 1 μg Donor-DNA. Mix well and transfer to a pre-cooled electroporation vessel (2.0 mm spacing) and place on ice for 2 min.

[0085] (9) The parameters of the electric converter are set as follows: voltage 1500V, resistance 400Ω, and capacitance 25μF.

[0086] (10) After electroporation, quickly add 1 mL of pre-cooled incubation solution. If the transformant is selected for defective screening, the incubation solution is 1 M sorbitol; if the transformant is selected for antibiotic screening, the incubation solution is a 1:1 mixture of YPD and 1 M sorbitol.

[0087] (11) Transfer the mixture to a 1.5 mL pre-cooled sterile EP tube and incubate in a shaking incubator at 30°C for 2-4 h. Take an appropriate amount of bacterial culture (refer to the transformation efficiency) and spread it on a plate containing the screening antibiotic or defective strain. Incubate the plate in a biochemical incubator at 30°C for 2-3 days.

[0088] (12) Positive clones were screened by colony PCR using the primers in Table 1. After sequencing the PCR products, the strain was constructed. Int1-F1 / Int1-R1, Int6-F1 / Int6-R1, Int15-F1 / Int15-R1, and Int18-F1 / Int18-R1 were used for colony PCR detection of expression cassettes integrated 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 of G418 and 0.2 g / L of hygromycin B were added to YPD plates (YPD plate medium consists of 1% yeast extract, 2% peptone, 2% glucose, and 2% agar powder), respectively.

[0089] Example 4: Detection of target protein expression

[0090] mCherry protein was selected to evaluate the expression efficiency of different SES systems. To directly observe mCherry expression, Pichia pastoris cells were cultured on YPD plates at 30°C and observed in the dark using a handheld 520-550 nm excitation light source and a 550 nm filter. Positive clones emitting fluorescence were selected. To quantitatively measure mCherry expression, single colonies were inoculated into 5 mL of YPD medium and cultured at 30°C and 220 rpm / min for 48 hours, followed by centrifugation and resuspending in sterile water. 200 μL of the suspension was transferred to a black 96-well microplate, and mCherry fluorescence was measured at 578-615 nm using a multi-functional reader (BioTek, Synergy H1). This value was normalized according to cell density (OD600). The quantitative detection method for the expression level of the human lysozyme-mCherry fusion protein shLYZ-mCherry was consistent with that for mCherry.

[0091] Recombinant Pichia pastoris strains carrying the Pro K gene were inoculated into glass tubes containing 5 mL of YPD medium and cultured at 30 °C and 220 rpm / min for approximately 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 shake flask containing 50 mL of YPD medium and cultured for another 72 hours at 30 °C and 220 rpm / min. 5 mL of 20% glucose was added every 24 hours. The fermentation supernatant was collected every 12 hours, and OD600 and enzyme activity were measured. To measure proteinase K activity, Suc-phe-pNA was selected as the chromogenic substrate for the serine protease Pro K. After digestion of Suc-phe-pNA, p-nitroaniline (pNA) was released, and the activity of the serine protease could be quantitatively determined by colorimetric detection at 405 nm. The total volume of the reaction solution was 200 μL, containing 2 mM Suc-phe-pNA substrate, 50 mM Tris-HCl (pH 8.0), and 10 μL of crude enzyme solution. Pro K activity was determined by recording absorbance at 405 nm, approximately 1.5 hours later. One unit was defined as the increase of 1 μmol pNA per minute at 37 °C.

[0092] Experimental results show that the SES system optimized with 41 heterogeneous core promoters significantly outperforms SES-A in expressing mCherry. Figure 2 The expression efficiency was improved by up to 5.4 times. Using the optimized first 9 SES system to secrete alkaline protease Pro K in Pichia pastoris, SES-CP32 showed the highest Pro K expression efficiency at 72 h after shake-flask fermentation, increasing by 1.6 times compared to SES-A. Figure 3 Furthermore, the use of a multi-copy strategy significantly improved the efficiency of SES-CP32 in expressing Pro K in Pichia pastoris. Figure 4 When the sTF count remained at 1 copy, the enzyme activity of Pro K significantly increased as the Pro K copy number increased from 1 to 3. After 72 hours of fermentation, the enzyme activity in the supernatant of 1*sTF + 3*Pro K fermentation reached 4.6 times that of 1*sTF + 1*Pro K. Figure 4 Using the optimized top 10 SES systems, a fusion protein of human lysozyme and red fluorescent protein (shLYZ-mCherry) was secreted and expressed in Pichia pastoris. Results showed that SES-CP64, SES-CP97, SES-CP32, SES-CP121, SES-CP47, and SES-CP60 all exhibited better shLYZ-mCherry protein expression efficiency than SES-A, with the optimal SES-CP64 demonstrating 3.52 times the shLYZ-mCherry protein expression efficiency of SES-A.Figure 5 ).

[0093] sequence list

[0094] SEQ ID NO.1: The uppercase part is An008CP, the underlined part is the coding sequence of the artificial transcription factor, and the shaded part is the Ttef1 base sequence.

[0095]

[0096] SEQ ID NO.2: The uppercase letters represent the binding sites of 8 tandem artificial transcription factors, the underlined uppercase letters represent An201CP, the shaded part represents the coding sequence of mCherry, and the underlined shaded part represents the base sequence of Tpdc1.

[0097]

[0098]

[0099] SEQ ID NO.3: The uppercase part represents the binding sites of 8 tandem artificial transcription factors, the underlined uppercase part is An201CP, the shaded part is the α-factor signal peptide sequence, the underlined shaded part is the Pro K coding sequence, and the wavy part is Tpdc1.

[0100]

[0101] SEQ ID NO.4: CP60

[0102] agtacgtaccagcaggctggtgccggtgatggatcaaaatactcccggatagcgttccgcggtcacgtggagccgctttgccactcgcaaatttgtgcctctagggccgcaaaccctatcggagtttttgcgaacgtctcaatccaccct cgacttttcggtgctgtcacattatcctacccaaaccgttgtcgccatattagcataaaccggtacgtccatcaccaaacctgccgtcaccccaaaccaccaacctcctcgatcggccaattcccctcgtactgacacgacgaagcaaac

[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] gaaaaaaaagatcgtaggaggagctgagatcgataaagcgaaaactcggttcccgccaggctcaggctcaggctccggctgctgcgggtccagcctcgttggtgcgttgtgtgccctggtggcccgtgcgcgcaaaaatgccctaacgcttgcacactcggttgccttgatttcggcaaatccccgttatcctcaatctctactaccacctgttccgcgcacgacgcaaaaacccccgtcactaattcttcacacatcaattcaatcttcgaggatcgcccgacaagaacaacaatcaag

[0123] SEQ ID NO.15:CP67

[0124] tacagcggcagagagattgcgatgagccctctccctacctacagacggctgacaatgtccgtataccaccagccaacgtgatgaaaacaaggacatgaggaacagcctgcgagagctggaagatgaagagggccagaaaaaaaagtataaagaagacctcgattcccgccatccaacaatcttttccatcctcatcagcacactcatctacaaccatcaccacattcactcaactcctctttctcaactctccaaacacaaacattctttgttgaataccaaccatcaccacctttcaag

[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] agaggaatcttagagtttgggggaaaatggctgcgatgacaagcggaaactttgtcatgtgaggcgcggcctgcccacaccagctggaggcgctagacgacgttatcagagcccgcttatctacacgtgatgtcagccacaacgca cactttcgagccaccccactcgctaggttggaatttcatgtagggcttcgcccacaaaagttatccaattcgagatctctgtgtcgcctgtcgcaaacccgtcaactttccacatcgaccctcgagcagcaaaccg

[0157] SEQ ID NO.32:CP192

[0158] fatheraggcaggtacctaccaaggtagcgcgattgccccctttgcagctgggctgggctgacgggcaagagccatcccgcttcgtacaagcaacgaccccttcccgttcgcgcaatgccccttgc gatagataccatcctctctctctctccgccttggccaactttctctctctgcttgctctccccgtattctccaaatctctctctcctcctctctttattttgccctctacaaaacctctacaaaccgccata

[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] gaagacttccgctcttcttcaagcccacccaattgcaccactggaaggtcatcaaagtcctctagcagataacgggttgattgccaagatttccacccttagtcagccgcactggccaatcgcatcgctcggccctaatttttgcgcccacacaaaaaattcacaatttcaaccagccccaacctcgagagctttacatcctggtgctgaccttttgcagaagagaagtggtcttcacagctaaatttctctttcctcacccatctcgaaactctttgcgcagaggcgaggctgtaccca

[0171] SEQ ID NO.39:CP29

[0172] footagtgggggcctatgtgcatcgcccggtccaggtccatttctggagccagtcacgtgcattgactcagaaggcatcgagggaatcagcatcacgtgccgatagcgcgcccgccccattgtttggtcgcctaatcagccaatctaaattttgcaagccctaaggtgggaaagtggcccgaaaaaagtgcgccgttgccctactctccatcctgtgcaaaaccatctaacatcgtcttcccaccctctacgatacgccctcagttacggtacctctccggagcaggataattcaag

[0173] SEQ ID NO.40: CP162

[0174] ttttttttttattagggaagggaactcgcctttcattcctcaaccttcctttttttttttcccacaagggggggcaattacagaaagacgcgccctttttctacttcccagtctctcgcttcgaattctcggtttcactctttttttgacggtctttccggtcctgcgcgcctccttccgcacacatcgtttcgcatcctcccttgaggattcacgctcgtttttctctctacctgaaaaaaaaataaagggagataaaaaaaagaaagacaccaacaaatcgtcgccatc

[0175] SEQ ID NO.41: CP180

[0176] tgctccagggcgccgcttgaaaggagcagacctcttttcgcatctttcttttttgcttttgcaacttaattcatcagtcctttttgacatcgtttttttttgagggcggccgcctcgcacagttctggcctttcagtcactccttaagacaaacaaccatcatttacattctatatcgttccttgacgcctttttgaatctcttcgtcgcctgaccgagcacgagaagcacacgtccaatcgctacagcatcaactcaagaaccgcaagtttcacgactactttcaccagaaccgccaag

[0177] SEQ ID NO.42:CP187

[0178] ctctctccattccgcgttagcctcgtcgagcccaggaaaattgttaaggcggcgtcggcttccctcccagcaaaaccgcgcaagtggccgtgattggcccgcgaggcaacccgagcccccacccttggtgctcacagactctgctctctccatccgccctcctcccgaaaatctccattttgctcttcctgcaaaaggcagcaagcatcaacaaactcctctcaactcctccaccccccaattgcacgcccgctgcaattgctcccctcgttgctgaggccttcgtatttcgccctcaag

[0179] SEQ ID NO.43:CP106

[0180] gcgtgtggaagcgaatgtgcttagataatccacggatttttttttcccgccgcgacgatagggcaggattgtgggttttggggggaacggcacatgttgcccggcttagggctcgtggctagaaggtcacgtgcatggtttccagacgccaaatttcctatccacacaaccttgacgattcctcgagatcacccgcgcaaaagttcaccagcagccaccactttaggaagcaagtcaagtgagtatctcatatactcaattcttacccgtccgagccataacctctgacaattctacaga

[0181] SEQ ID NO.44:CP116

[0182] tgggcgtgttccgtggggtcggtcgaattgagttagttcgtttgagaggcggaaccggaagaaccacggcattggctggtcaatacatcaagttcgttcctttcacgtgacttctccagcacttctcgcgggtcttgccaagcggtaagccttagggcaagaaagcccccacaattggcgcgatcgaagcgaggagggaaaaaaaaaaggtgtgccatcaatttaacatttctgtgccttattccacgctcaacctcttcgtcgtttccatcaccgtcgagaaaacagccagtcgtcacc

[0183] SEQ ID NO.45:CP155

[0184] gctcctccggctgactctggctcctcctgctctgcgctctgcctggacggacggacggactcgctcgctggtcccggcgtcacgacaataaaaaggccggcctgcctcccgcccccctctgctccttctcctgtctcgcctcgtcggtctctgtctcacgcaacaaagagtcgttccgttcactcggcccgggtctcgtctttcttcctgccatccccagccagcatcttcactcccacgcgtcaagtcaacaagagtccccctgaacaaaaaaagacaagcccagcgtcgaccgcagcc

[0185] SEQ ID NO.46:CP102

[0186] cttcatgatgacgtcgcacagttgccccgcctaaacacccctcaatcaagctggtgcttctcctgggtgttgcgatactaaattgcgcggctgcctcccgccagctgcagtgaagccacgatccatataaattgccacgcttgtgctcgtcatcggcttgcatcatcatcctattccattctcactcctggcaacacaacctcaaacaaagaaaaacatctgcttccaaacatctacgacactttcaacacacccacccacatacacacacacacacacaaaccatccacaaccaacaag

[0187] SEQ ID NO.47:CP33

[0188] cggtgtcgttgcttgatgtgtgaatgctgctggagctttgatcccctgagacggtggggatgatggtggctgaaatgtctcgctctgaacgaggtgctgggagttgggagggcggtatgaagcgaggaggaggaggaaggactactataagaagagtcgagaatcatcattgtctcagaccttttcctcttcagcaagtcacacagtttatgacccttcacctcaactacttacttacactcttcagaagacatcgaaatcaccaaatcgtcaccaccaccatcaccatcaccgcccatc

[0189] SEQ ID NO.48:CP92

[0190] tggcggatttggttctcgataccgtcactcgagaaaaaatgcaggcgtgtttcacgtgacatcaaccgcacttgccactctcgtgccttagcgcggacgagggccaaagagggctggagacttgtggagcaggaaaattattcccacagaattagcggaaaaatatatgccccggtacctttctggtttggctcgcttagactcaggttgatgcacacaacaattgaaattcttccaacccctccaccaacttcttaggaccctttcgattcccaagacctcgtacgccggtaaggaatc

[0191] SEQ ID NO.49:CP40

[0192] gctgtagcgctgcaactgaacctggaacttgaagtaccctcgctgttgccgtggcaggtggaaggcggtccaagcccaaagcctgcagcgctagtgcggccggaaagagtcgctctgaaaaagtgtccccttcgagacccgctgcaatttttttccctccccccccagaaaagccattcgcccgctcggtgcagaatttatttccctcgagtcccccgattgtcctcctcttcctcctttcatctccatctcccctcagttctttctttgtttctgggaaagcgttgcacctcagtcaaa

[0193] SEQ ID NO.50:CP21

[0194] tgtactacctagatactactactactagccggaaagacacggagaccgaccagtcgaactctcaacaggcagctgctggccccgtagaaacctctcagcctcttgcagaatgggggggaaactggcaagatggggtcaattggcccaacctagacggctagcggctgcacgtgacccggaagaaagctcttaccggagtggtcgctttcccgtcctaaactgctcattttgaaaacttctcccggcctttaccacctcattcgcatatccatcgctgattgctctcggtcatctgccaag

[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] gtgcccaaccgcgaaaatgctgtgaagacgcaccagaggaggaagaaaaggcggggccgtggttctctttagccccggccccactcccgccaatcacagcggacgtctcgagacctcctccgccagcccgcgcgataagcgctatcttcacgtgcaccacacaaactcgcttctcgtagggtttctcacccccacagattgcatgaggaaaatttggccctgaaaagcttcagcttcacgacggcaatccgcgactttggcacgtttcccacgaattaacgccccaacccctcagacaca

[0207] SEQ ID NO.57:CP46

[0208] gcacgccccccgcaccctcaaattgtcgcaattgttttgctggaacacgtccatttccccaacaggcctagcgccgcaacagctgcgagagggagggtgattgctgatctcgcgcggcgagcaccaaaaagtacttaactttggagccc 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: Amino acid sequence of ShLYZ-mCherry, where the underlined part is shLYZ and the non-underlined part is mCherry MKALIVLGLVLLSVTVQGKVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGY NTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRN RCQNRDVRQYVQGCGVVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK*

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

[0212] SEQ ID NO:61: mCherry amino acid sequence MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK*

[0213] SEQ ID NO:62: α-factor-Pro K coding sequence, where the underlined part is the coding sequence for the α-factor signal peptide (used to secrete Pro K into the extracellular space), and the shaded part is the coding sequence for Pro K.

[0214]

[0215] SEQ ID NO:63: α-factor-Pro K amino acid sequence, where the underlined red part is the α-factor signal peptide used to secrete ProK outside the cell, and the shaded part is the ProK amino acid sequence.

[0216]

[0217] SEQ ID NO:64:Pro K amino acid sequence APAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDAAMEKISGKPDHVYKNVFSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYASSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKV LDDNGSGQYSTIIAGMDFVASDHNRNRCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNADARNYSPASEPSVCTVGATDRYDRRSSFSNYGSVLDIFAPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGRTTAANACRYIADTANKGDLSNIPFGTVNLLAYNNYQAHHHHHH*

[0218] SEQ ID NO:65: pUC-57-shLYZ-mCherry-Amp, where the uppercase part represents the binding sites of 8 tandem artificial transcription factors, the underlined uppercase part is An201CP, the shaded part is the human lysozyme coding sequence, 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 part is Tpdc1.

[0219]

[0220]

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

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

[0223] SEQ ID NO:68: Nucleotide sequence of binding sites for 8 tandem artificial transcription factors ATCGATTCAAATGAGCTAGGGACCTGGCGGAATGAACATTCATTCCGAGCATAACGGAATGAAGGTTCATTCCGAGGGGACCTGGCGGAATGAACTTTCATTCCGAGCATAACGGAATGAACATTCATTCCGAGGGGACCTGGCGGAATGAAGGTTCATTCCGAGCATAACGGAATGAACATTCATTCCGAGGGGACCTGGCGGAATGAACATTCATTCCGAGCATAACGGAATGAAGGTTCATTCCGAGGGATGATAATGCGATTGCTAGC

[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: 5 or 8. The second core promoter is selected from An201CP, and its nucleotide sequence is shown in SEQ ID NO:

71. 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. The artificial transcription factor binding sites are binding sites for eight artificial transcription factors in tandem, and their nucleotide sequences are shown in SEQ ID NO:

68.

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.

3. The synthetic expression system according to claim 2, characterized in that, The target protein is selected from fluorescent proteins, enzymes, and fusion proteins.

4. The synthetic expression system according to claim 3, characterized in that, The target protein is a fluorescent protein.

5. The synthetic expression system according to claim 4, characterized in that, The fluorescent protein is mCherry.

6. The synthetic expression system according to claim 3, characterized in that, The target protein is an enzyme.

7. The synthetic expression system according to claim 6, characterized in that, The enzyme is alkaline proteinase K.

8. The synthetic expression system according to claim 3, characterized in that, The target protein is a fusion protein.

9. The synthetic expression system according to claim 8, characterized in that, The fusion protein is a fusion protein of human lysozyme and mCherry.

10. The synthetic expression system according to claim 5, characterized in that, The amino acid sequence of the mCherry is SEQ ID NO:

61.

11. The synthetic expression system according to claim 7, characterized in that, The amino acid sequence of the alkaline proteinase K is SEQ ID NO:

64.

12. The synthetic expression system according to claim 9, characterized in that, The amino acid sequence of the human lysozyme and mCherry fusion protein is selected from SEQ ID NO:

59.

13. The synthetic expression system according to claim 1, characterized in that, The target protein coding region contains multiple copies of the target protein gene.

14. The synthetic expression system according to claim 13, characterized in that, The term "multiple copies" refers to two or more copies.

15. The synthetic expression system according to claim 14, characterized in that, The multiple copies refer to 2-10 copies.

16. The synthetic expression system according to claim 15, characterized in that, The multiple copies refer to 2 or 3 copies.

17. The synthetic expression system according to claim 1, characterized in that, The first transcription terminator element is Ttef1; And / or, the second transcription terminator element is Tpdc1.

18. The synthetic expression system according to claim 17, characterized in that, The nucleotide sequence of Ttef1 is shown in SEQ ID NO: 69; And / or, the nucleotide sequence of Tpdc1 is SEQ ID NO:

70.

19. A fungal host cell, characterized in that, The synthetic expression system includes any one of claims 1-18.

20. The fungal host cell according to claim 19, characterized in that, The first and second polynucleotides in the synthetic expression system are integrated into the genome of the fungal host cell as expression cassettes.

21. The fungal host cell according to claim 19 or 20, characterized in that, The fungal host cells are selected from Saccharomyces cerevisiae, Pichia pastoris, Yersinia lipolytica, Saccharomyces simulans, Rhodotorula rubra, Candida albicans, Trichoderma reesei, and Aspergillus niger.

22. The fungal host cell according to claim 21, characterized in that, The fungal host cells were selected from Pichia pastoris, Trichoderma reesei, and Aspergillus niger.

23. The use of the synthetic expression system according to any one of claims 1-18 and the fungal host cell according to any one of claims 19-22 in expressing the target protein.

24. The application according to claim 23, characterized in that, The target protein is selected from fluorescent proteins, enzymes, antibodies, and fusion proteins.

25. A method for producing a target protein, characterized in that, Includes the following steps: (1) Culture the fungal host cells according to any one of claims 19-22 under conditions suitable for the expression of the target protein. (2) The target protein is recovered.

Images