High-efficiency protein expression system and construction method and application thereof

Abstract

This invention discloses a high-efficiency protein expression system, its construction method, and its applications. The system includes a heterozygous promoter activator and a heterozygous promoter. The heterozygous promoter activator consists of the DNA-binding protein TetR, a linker peptide, and the transcription activator Mit1-Δ60-96. The heterozygous promoter consists of the protein-binding sequence TetO, a spacer sequence, and a basal promoter. The linker peptide is (GGGGS)×2, the spacer sequence is TCGCGCGC, and the basal promoter is P. AOX1 The core promoter of this invention. In the absence of a heterozygous promoter activator, the target protein gene initiated by the heterozygous promoter is expressed very weakly; in the presence of a heterozygous promoter activator, the target protein gene is expressed efficiently. This invention's system has good applicability and scalability, and the expression intensity of various target proteins, including xylanase, phytase, and glucosidase, is significantly higher than that of commonly used P. AOX1 Promoter.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a high-efficiency protein expression system, its construction method, and its application. Background Technology

[0002] Pichia pastoris ( Pichia pastoris As a eukaryotic microbial expression system, Pichia pastoris has become an important platform for recombinant protein production due to its advantages such as protein folding and modification capabilities (e.g., glycosylation), rapid growth, and ease of large-scale cultivation. However, existing Pichia pastoris protein expression systems still suffer from core defects such as methanol dependence, limited expression efficiency, insufficient universality, and narrow carbon source adaptability, making it difficult to meet the demands of industrial production for safety, efficiency, and flexibility.

[0003] (1) Inherent limitations of methanol-induced systems

[0004] Currently, the most commonly used Pichia pastoris is the methanol-induced Pichia pastoris. AOX1 Startup Subsystem - P AOX1 It is the promoter of the endogenous methanol oxidase gene in Pichia pastoris, which is strongly induced by methanol and can drive high-level expression of the target protein. However, this system has the following problems: ① Safety risks: Methanol is toxic, flammable, and volatile, requiring special protection and storage conditions for production, increasing operational complexity; ② Carbon source limitation: P AOX1 The promoter is inhibited by the "glucose effect"—when glucose is used as the carbon source, the promoter activity is significantly shut down, resulting in a sharp drop in the expression level of the target protein.

[0005] (2) The existing system lacks sufficient expressive efficiency and universality.

[0006] ①P AOX1 ① Promoters exhibit significant differences in expression effects for different target proteins and lack systematic optimization; ② Activation efficiency needs improvement: Existing transcriptional activators typically retain redundant domains that do not participate in P... AOX1 Regulation occupies protein space, thus reducing activation efficiency; ③ Limited universality: For different proteins such as xylanase, phytase, and glucosidase, P AOX1 The expression intensity fluctuates greatly, making it difficult to adapt to the production needs of diverse proteins.

[0007] (3) Defects in the integration of exogenous regulation systems

[0008] To overcome methanol dependence, researchers attempted to integrate a tetracycline-regulated system with Pichia pastoris, but existing solutions have some shortcomings: ① Inappropriate promoter selection: some systems still use P... AOX1① Methanol-regulated promoters drive heterozygous promoter activators, failing to completely resolve methanol dependence; ② Lack of element optimization: the selection of linker peptides lacks specificity, leading to unreasonable conformation of fusion proteins; ③ Sequence competition interference: if there are sequence repetitions between heterozygous promoters and activators, it will trigger competition for transcriptional resources and reduce the overall expression level.

[0009] It is evident that existing systems are ill-suited for non-methanol-induced scenarios, exhibiting low carbon source compatibility. Furthermore, some target proteins (such as secretory enzymes) are sensitive to the "strict induction" characteristics of promoters, resulting in relatively low protein secretion expression efficiency. Therefore, developing a novel expression system that is methanol-independent, highly efficient, universal, and compatible with multiple carbon sources has become one of the urgent technical challenges to be addressed in this field. Summary of the Invention

[0010] The purpose of this invention is to provide a high-efficiency protein expression system, its construction method, and its application.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] A highly efficient protein expression system, comprising a heterozygous promoter activator and a heterozygous promoter (such as...) Figure 1 As shown in the figure, the heterozygous promoter activator is composed of DNA-binding protein TetR, linker peptide, transcription activator Mit1-Δ60-96, and the heterozygous promoter is composed of protein-binding sequence TetO, spacer sequence, and basic promoter.

[0013] The DNA-binding protein TetR sequence is shown in SEQ ID NO: 1, the linker peptide is (GGGGS) × 2, the transcription activator Mit1-△60-96 sequence is shown in SEQ ID NO: 4, the protein-binding sequence TetO sequence is shown in SEQ ID NO: 7, the spacer sequence is TCGCGCGC, and the basal promoter is derived from P. AOX1 The promoter sequence is shown in SEQ ID NO: 8.

[0014] Furthermore, the protein-binding sequence TetO is a 6-copy TetO sequence.

[0015] Furthermore, the promoter expressed by the hybrid promoter activator is the pGAP promoter.

[0016] Furthermore, the sequence of the heterozygous promoter is shown in SEQ ID NO: 9.

[0017] The method for constructing the high-efficiency protein expression system of the present invention includes the following steps:

[0018] (1) Construct the recombinant backbone plasmid pPICZA-TetR-Int18 expressing the heterozygous promoter activator, and then use the recombinant backbone plasmid pPICZA-TetR-Int18 as a template to construct the recombinant plasmid pPICZA-TetR-Int18 expressing the heterozygous promoter activator without the linker peptide. The recombinant plasmid pPICZA-TetR-(GGGGS)2-Mit1-△60-96-Int18 was constructed using the recombinant plasmid pPICZA-TetR-(GGGGS)2-Mit1-△60-96-Int18 as a template. Finally, the recombinant plasmid pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 was constructed using the recombinant plasmid pPICZA-TetR-(GGGGS)2-Mit1-△60-96-Int18 as a template.

[0019] (2) Construct a recombinant backbone plasmid 9K-p1TocP carrying a heterozygous promoter. Then, using the recombinant backbone plasmid 9K-p1TOcP as a template, construct a recombinant plasmid 9K-p6TOcP containing a heterozygous promoter with 6 copies of TetO. Finally, construct a recombinant plasmid 9K-p6TOcP containing a heterozygous promoter-target protein gene expression cassette-target protein gene fragment.

[0020] (3) The recombinant plasmid pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 and the recombinant plasmid 9K-p6TOcP-target protein gene fragment were introduced into Pichia pastoris GS115 competent cells.

[0021] The present invention provides a recombinant plasmid comprising the recombinant plasmid pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 and the recombinant plasmid 9K-p6TOcP-target protein gene fragment.

[0022] The present invention also provides a recombinant engineered strain, which is obtained by electroporating the above-mentioned recombinant plasmid into Pichia pastoris GS115 competent cells.

[0023] The high-efficiency protein expression system described in this invention can be used for protein production, wherein the protein includes, but is not limited to, xylanase, glucosidase, and phytase.

[0024] Compared with the prior art, the outstanding effect of the present invention is as follows:

[0025] (1) This invention integrates the tetracycline regulation system with the P of Pichia pastoris AOX1The promoter constructs a novel, highly efficient protein expression system. In the absence of a heterozygous promoter activator, the target protein gene initiated by the heterozygous promoter is expressed very weakly; however, in the presence of a heterozygous promoter activator, the target protein gene is expressed efficiently.

[0026] (2) The high-efficiency protein expression system of the present invention has good applicability and scalability. The expression intensity of various target proteins, including xylanase, phytase and glucosidase, is much higher than that of commonly used P. AOX1 Promoter. Depending on the culture conditions, the increase can range from 2-12 times or 90-600 times.

[0027] (3) The high-efficiency protein expression system of the present invention achieves high-efficiency expression of the target protein without methanol induction. It can also achieve high-efficiency expression of the target protein using glucose as a carbon source, which greatly expands the application range of Pichia pastoris protein expression system and achieves unexpected beneficial effects.

[0028] The high-efficiency protein expression system, its construction method, and its applications described in this invention will be further explained below with reference to the accompanying drawings and specific embodiments.

[0029] It should be understood that the following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, experimental methods in the following examples are generally performed under conventional conditions (such as those described in "J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002") or as recommended by the manufacturer. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the high-efficiency protein expression system of the present invention.

[0031] Figure 2 The Zn2Cys6 binuclear cluster DNA-binding domain is located at positions 60-96 of the Mit1 protein.

[0032] Figure 3 The results show the comparative effects of different heterozygous promoter activators on the growth of chassis strains.

[0033] Figure 4 This study compares the effects of hybrid promoter activators containing different transcription activators and linker peptides.

[0034] Figure 5 To express the effect of promoter type on the overall protein expression level of the system by expressing the heterozygous promoter activator.

[0035] Figure 6 The effect of the copy number of the protein-binding sequence TetO in a heterozygous promoter on the expression level of a protein expression system.

[0036] Figure 7 The results of expressing different target proteins using the high-efficiency protein expression system of the present invention are shown, wherein (a) is the effect of application in glucosidase and (b) is the effect of application in phytase. Detailed Implementation

[0037] The primer sequences (SEQ ID NO:21-55) used in the experiments of this invention are shown in Table 1 below.

[0038] Table 1

[0039]

[0040]

[0041] Example 1: Construction of the recombinant backbone plasmid pPICZA-TetR-Int18 expressing a heterozygous promoter activator

[0042] The heterozygous promoter activator consists of a DNA-binding protein, a linker peptide, and a transcription activator. The activator also includes a promoter and a terminator at its head and tail, respectively, to enable successful expression. In this invention, the DNA-binding protein is TetR, derived from the *E. coli* Tn10 tetracycline resistance operon system, and is a protein containing the amino acid sequence shown in SEQ ID NO: 1 (NCBI GenBank: CAA25291.1). To facilitate comparison of the effects of different linkers and transcription activators on the entire system and to achieve successful and convenient expression of the activator, it is typically constructed into a plasmid vector. Based on the above objectives, this invention also provides a recombinant backbone plasmid pPICZA-TetR-Int18 for expressing the heterozygous promoter activator.

[0043] The entire TetR gene was synthesized, and the synthesized gene was optimized according to the codon preference of Pichia pastoris. The synthesized nucleotide fragment (SEQ ID NO: 2) was inserted between the EcoRI and XhoI restriction sites of the pPICZA plasmid, thereby obtaining the recombinant plasmid pPICZA-TetR.

[0044] The recombinant plasmid pPICZA-TetR was digested with the restriction endonuclease BamHI, and the linearized fragment "pPICZA-TetR-line" was obtained after gel purification. Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primers "Int18-1" and "Int18-2" (as shown in SEQ ID NO:21-22), and the integration site gene fragment "Int18" was obtained after gel purification. The Int18 gene fragment and "pPICZA-TetR-line" were ligated using a homologous recombination kit (Beijing TransGen Biotech Co., Ltd., CU101-01). The ligation product was transformed into Escherichia coli Top10 competent cells (Beijing Zhuangmeng International Biotechnology Co., Ltd., ZC104), and then plated on LBLZ bleomycin-resistant plates for positive transformant selection. Afterwards, transformants that were correctly identified by colony PCR were inoculated into LB medium and plasmids were extracted. The recombinant backbone plasmid pPICZA-TetR-Int18 was obtained after sequencing verification by Shanghai Sangon Biotech Co., Ltd.

[0045] Example 2: Construction of recombinant plasmids expressing activators of different heterozygous promoters

[0046] (1) Discovery of transcription activator Mit1-Δ60-96

[0047] Mit1 is a methanol-inducible transcription factor 1 derived from Pichia pastoris, which regulates P... AOX1 It plays an important role in promoter activity and is a protein containing the amino acid sequence shown in SEQ ID NO: 3 (NCBI GenBank: XP_002493066.1). Analysis of Mit1 using the CD-Search online server shows that the protein contains a Zn2Cys6 binuclear cluster DNA-binding domain belonging to the GAL4 class, located at positions 60-96 of the protein. Figure 2 The primary function of this domain is to guide Mit1 to P by forming two helices aligned with the Zn2Cys6 motif and binding to a specific DNA sequence. AOX1 Upstream of the promoter, but in regulating P AOX1 This domain does not play a role in promoter activity. Therefore, the inventors hypothesize that deleting this domain will not affect the transcriptional activation of Mit1, and may even enhance activation due to the removal of unnecessary redundant sequences. The transcriptional activator with the amino acid sequence deleted from the 60-96 region of the Mit1 protein is named Mit1-△60-96, which is a protein containing the amino acid sequence shown in SEQ ID NO: 4.

[0048] (2) Construction of recombinant plasmid pPICZA-TetR-Mit1-△60-96-Int18 expressing a heterozygous promoter activator without linker peptides

[0049] Using the recombinant backbone plasmid pPICZA-TetR-Int18 as a template, PCR amplification was performed using primers "pPICZA-1" and "TetR-2" (as shown in SEQ ID NO:23-24). After gel purification, the linearized plasmid fragment "pPICZA-TetR-Int18-line" was obtained. Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs "Mit1-1 and Mit1-59-2" (as shown in SEQ ID NO:25-26) and "Mit1-97-1 and Mit1-2" (as shown in SEQ ID NO:27-28), respectively. After gel purification, the gene fragments encoding the first 59 amino acids of Mit1, "Mit1(1-59)" and "Mit1(97-888)" encoding amino acids 97-888 of Mit1, were obtained, respectively. The gene fragments Mit1 (1-59) and Mit1 (97-888) were ligated to the plasmid "pPICZA-TetR-Int18-line" using a homologous recombination kit (Beijing TransGen Biotech Co., Ltd., CU101-01). The ligation product was transformed into E. coli Top10 competent cells (Beijing Zhuangmeng International Biotechnology Co., Ltd., ZC104), and then plated on LBLZ bleomycin-resistant plates for positive transformant selection. Transformants that were correctly identified by colony PCR were then inoculated into LB medium, and plasmids were extracted. After sequencing verification by Shanghai Sangon Biotech Co., Ltd., the recombinant plasmid pPICZA-TetR-Mit1-△60-96-Int18 expressing a heterozygous promoter activator without the linker peptide was obtained.

[0050] (3) Construction of recombinant plasmids expressing hybrid promoter activators containing different linker peptides

[0051] Whether there is a linker peptide between the DNA-binding protein and the transcription activator, as well as the total number and length of the linker peptides, will have a significant impact on the size and structure of the fusion protein, and thus affect the activation effect of the transcription activator on the heterozygous promoter. Therefore, this invention constructs a heterozygous promoter activator containing a variety of different linker peptides, specifically including: no linker peptide, 1 set of flexible linker peptides GGGGS, 2 sets of flexible linker peptides (GGGGS) × 2, 1 set of rigid linker peptides EAAAK, and 2 sets of rigid linker peptides (EAAAK) × 2.

[0052] Using the recombinant plasmid pPICZA-TetR-Mit1-△60-96-Int18 as a template, PCR amplification was performed on “Mit1-GGGGS-1 and Mit1-2” (as shown in SEQ ID NO: 29 and 28). After gel extraction and purification, the gene fragment “GGGGS-Mit1-△60-96” encoding Mit1-△60-96 with the added linker peptide GGGGS was obtained. The gene fragment “GGGGS-Mit1-△60-96” was ligated to “pPICZA-TetR-Int18-line” using a homologous recombination kit (Beijing TransGen Biotech Co., Ltd., CU101-01). The ligation product was transformed into E. coli Top10 competent cells (Beijing Zhuangmeng International Biotechnology Co., Ltd., ZC104) and then plated on LBLZ bleomycin-resistant plates for positive transformant screening. Afterwards, transformants that were correctly identified by colony PCR were inoculated into LB medium and plasmids were extracted. After sequencing verification by Shanghai Sangon Biotech Co., Ltd., the recombinant plasmid pPICZA-TetR-GGGGS-Mit1-△60-96-Int18 was obtained, which expresses a heterozygous promoter activator containing a set of flexible linker peptides GGGGS.

[0053] Similarly, using the recombinant plasmid pPICZA-TetR-Mit1-△60-96-Int18 as a template, the corresponding gene fragments were obtained by PCR amplification using primer pairs “Mit1-EAAAK-1 and Mit1-2” (as shown in SEQ ID NO:30 and 28). The recombinant plasmid pPICZA-TetR-EAAAK-Mit1-△60-96-Int18 could be obtained by performing the same operation.

[0054] Using the recombinant plasmid pPICZA-TetR-Mit1-△60-96-Int18 as a template, PCR amplification was performed using primers “Mit1-F” and “TetR-2” (as shown in SEQ ID NO:31, 24). After gel purification, the linearized plasmid fragment “pPICZA-TetR-Mit1-△60-96-Int18-line” was obtained. Annealing ligation was performed using primer pairs “(GGGGS)2-1” and “(GGGGS)2-2” (as shown in SEQ ID NO:32-33) to obtain two sets of flexible linker peptides (GGGGS) × 2 corresponding to the gene fragment “(GGGGS)2”. The gene fragments “pPICZA-TetR-Mit1-△60-96-Int18-line” and “(GGGGS)2” were ligated using a homologous recombination kit to obtain the recombinant plasmid pPICZA-TetR-(GGGGS)2-Mit1-△60-96-Int18, which expresses a heterozygous promoter activator containing two sets of flexible linker peptides GGGGS. Similarly, the gene fragments “(EAAAK)2-1” and “(EAAAK)2-2” (as shown in SEQ ID NO:34-35) were annealed to obtain the corresponding gene fragment “(EAAAK)2”, and the recombinant plasmid pPICZA-TetR-(EAAAK)2-Mit1-△60-96-Int18 was obtained through the same operation.

[0055] (4) Construction of recombinant plasmids expressing heterozygous promoter activators of transcription activators published in existing cases.

[0056] CN115322998A discloses the transcription activator Mit1AD. Sequence analysis of the disclosed information in this patent shows that the transcription activator Mit1AD is the full-length Mit1.

[0057] Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs “Mit1-1 and Mit1-2” (as shown in SEQ ID NO:25 and 28) to obtain the gene fragment “Mit1” corresponding to the full-length Mit1 protein. The gene fragment “Mit1” was ligated with “pPICZA-TetR-Int18-line” to obtain the recombinant plasmid pPICZA-TetR-Mit1-Int18.

[0058] Using the recombinant plasmid pPICZA-TetR-Mit1-Int18 as a template, PCR amplification was performed on “Mit1-GGGGS-1 and Mit1-2” (as shown in SEQ ID NO:29 and 28) and “Mit1-EAAAK-1 and Mit1-2” (as shown in SEQ ID NO:30 and 28), respectively, yielding gene fragments “GGGGS-Mit1” encoding Mit1 with the linker peptide GGGGS and “EAAAK-Mit1” encoding Mit1 with the linker peptide EAAAK, respectively. The gene fragments “GGGGS-Mit1” and “EAAAK-Mit1” were then ligated to “pPICZA-TetR-Int18-line” to obtain the recombinant plasmids pPICZA-TetR-GGGGS-Mit1-Int18 and pPICZA-TetR-EAAAK-Mit1-Int18, respectively.

[0059] Using the recombinant plasmid pPICZA-TetR-Mit1-Int18 as a template, PCR amplification was performed using primers “Mit1-F” and “TetR-2” (as shown in SEQ ID NO: 31, 24). After gel purification, the linearized plasmid fragment “pPICZA-TetR-Mit1-Int18-line” was obtained. The gene fragments “(GGGGS)2” and “(EAAAK)2” were ligated to “pPICZA-TetR-Mit1-Int18-line” to obtain the recombinant plasmids pPICZA-TetR-(GGGGS)2-Mit1-Int18 and pPICZA-TetR-(EAAAK)2-Mit1-Int18, respectively.

[0060] CN116113643A discloses the transcription activator Mxr1_TAD and its corresponding amino acid and DNA sequences, as shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The gene fragment "Mxr1_TAD" was synthesized according to SEQ ID NO: 6. Using the gene fragment "Mxr1_TAD" as a template, PCR amplification was performed on "Mxr1_TAD-1 and Mxr1_TAD-2" (as shown in SEQ ID NO: 36-37) to obtain the gene fragment "Mxr1_TAD-ligator" for ligation construction. The gene fragment "Mxr1_TAD-ligator" was ligated to "pPICZA-TetR-Int18-line" to obtain the recombinant plasmid pPICZA-TetR-Mxr1_TAD-Int18.

[0061] Using recombinant plasmid pPICZA-TetR-Mxr1_TAD-Int18 as a template, PCR amplification was performed using primer pairs “Mxr1_TAD-GGGGS-1 and Mxr1_TAD-2” (as shown in SEQ ID NO:38 and 37) and “Mxr1_TAD-EAAAK-1 and Mxr1_TAD-2” (as shown in SEQ ID NO:39 and 37), respectively, to obtain gene fragments “GGGGS-Mxr1_TAD” encoding Mxr1_TAD with the linker peptide GGGGS added and “EAAAK-Mxr1_TAD” encoding Mxr1_TAD with the linker peptide EAAAK added, respectively. Gene fragments “GGGGS-Mxr1_TAD” and “EAAAK-Mxr1_TAD” were ligated with “pPICZA-TetR-Int18-line” to obtain recombinant plasmids pPICZA-TetR-GGGGS-Mxr1_TAD-Int18 and pPICZA-TetR-EAAAK-Mxr1_TAD-Int18, respectively.

[0062] Using the recombinant plasmid pPICZA-TetR-Mxr1_TAD-Int18 as a template, PCR amplification was performed using primers “Mxr1_TAD-F” and “TetR-2” (as shown in SEQ ID NO:40, 28). After gel purification, the linearized plasmid fragment “pPICZA-TetR-Mxr1_TAD-Int18-line” was obtained. Annealing ligation was performed using primer pairs “(GGGGS)2-1” and “(GGGGS)2-Mxr1_TAD-2” (as shown in SEQ ID NO:32, 41) to obtain two sets of flexible linker peptides (GGGGS) × 2 corresponding to the gene fragment “(GGGGS)2-Mxr1_TAD”. The linearized plasmid fragment “pPICZA-TetR-Mxr1_TAD-Int18-line” was ligated with “(GGGGS)2-Mxr1_TAD” to obtain the recombinant plasmid pPICZA-TetR-(GGGGS)2-Mxr1_TAD-Int18, which expresses a heterozygous promoter activator containing two sets of flexible linker peptides GGGGS. Similarly, the primer pairs “(EAAAK)2-1” and “(EAAAK)2-Mxr1_TAD-2” (as shown in SEQ ID NO:34, 42) were annealed to obtain the corresponding gene fragment “(EAAAK)2-Mxr1_TAD”, and the recombinant plasmid pPICZA-TetR-(EAAAK)2-Mxr1_TAD-Int18 could be obtained through the same operation.

[0063] A total of 15 recombinant plasmids expressing different heterozygous promoter activators were obtained, which are summarized in Table 2 below.

[0064] Table 2

[0065]

[0066] Example 3: Comparison of the effects of different heterozygous promoter activators on the growth of chassis strains

[0067] The 15 recombinant plasmids from Example 2 were linearized using the restriction endonuclease Kpn2I. The 15 linearized vectors were purified and recovered, and then electroporated into *Pichia pastoris* GS115 competent cells (Invitrogen Biotech, Inc., C18100, USA). Positive transformants were screened on bleomycin-containing resistant plates to obtain recombinant strains expressing different heterozygous promoter activators: GS115 / pPICZA-TetR-Mit1-△60-96-Int18, GS115 / pPICZA-TetR-GGGGS-Mit1-△60-96-Int18, and GS115 / p PICZA-TetR-EAAAK-Mit1-△60-96-Int18, GS115 / pPICZA-TetR-(GGGGS)2-Mit1-△60-96-Int18, GS115 / pPICZA-TetR-(EAAAK)2-Mit1-△60-96-Int18, GS115 / pPICZA-TetR-Mit1-Int18, GS115 / pPICZA-TetR- GGGGS-Mit1-Int18, GS115 / pPICZA-TetR-EAAAK-Mit1-Int18, GS115 / pPICZA-TetR-(GGGGS)2-M it1-Int18, GS115 / pPICZA-TetR-(EAAAK)2-Mit1-Int18, GS115 / pPICZA-TetR-Mxr1_TAD-Int18, GS115 / pPICZA-TetR-GGGGS-Mxr1_TAD-Int18, GS115 / pPICZA-TetR-EAAAK-Mxr1_TAD-Int18, GS1 15 / pPICZA-TetR-(GGGGS)2-Mxr1_TAD-Int18, GS115 / pPICZA-TetR-(EAAAK)2-Mxr1_TAD-Int18.

[0068] The above 15 recombinant strains and GS115 were inoculated into BMGY medium (1% (w / v) yeast extract, 2% (w / v) tryptone, 1.34% (w / v) amino-free yeast nitrogen source, 10% (v / v) 1 M potassium phosphate buffer (pH 6.0), 1% (v / v) glycerol, autoclaved at 115℃ for 20 min), and cultured at 30℃ and 250 rpm for 20 h. Cells were collected by centrifugation at 6000 rpm and 4℃ for 5 min. The collected cells were then resuspended in BMMY medium (1% (w / v) yeast extract, 2% (w / v) tryptone, 1.34% (w / v) amino-free yeast nitrogen source, 10% (v / v) 1 M potassium phosphate buffer (pH 6.0), autoclaved at 115℃ for 20 min, and then 1% (v / v) methanol was added) until the initial OD600 was close to 0.5. The cells were then cultured at 30℃ with shaking at 250 rpm. OD600 was measured daily and 1% methanol was added to the final concentration.

[0069] The results are as follows Figure 3 As shown, heterozygous promoter activators expressing the Mit1-△60-96 or Mxr1_TAD transcription activator series did not affect the growth of the strains, and the OD600 reached approximately 36 after 120 h of methanol-induced fermentation. However, the recombinant strains expressing the Mit1 series of heterozygous promoter activators showed poor growth, which worsened with increasing linker peptide length. The representative strains GS115 / pPICZA-TetR-Mit1-Int18, GS115 / pPICZA-TetR-GGGGS-Mit1-Int18, and GS115 / pPICZA-TetR-(GGGGS)2-Mit1-Int18 had OD600 values ​​of 32.6, 30.6, and 27.8 after 120 h of methanol-induced fermentation, respectively, which were 10.9%, 16.4%, and 24.0% lower than the control strain GS115's 36.6.

[0070] Example 4 Construction of recombinant plasmid carrying a heterozygous promoter

[0071] (1) Construction of recombinant backbone plasmid 9K-p1TOcP carrying a heterozygous promoter

[0072] The heterozygous promoter consists of a protein-binding sequence, a spacer sequence, and a basic promoter, and is used to initiate the expression of downstream target genes. To facilitate comparison of the effect of protein-binding sequence copy number on heterozygous promoter activity, and to facilitate the replacement of downstream target genes thereby improving the flexibility of the system, this invention also provides a recombinant backbone plasmid 9K-p1TOcP carrying a heterozygous promoter.

[0073] In this invention, the protein-binding sequence is TetO, which is the DNA sequence shown in SEQ ID NO: 7, the spacer sequence is TCGCGCGC, and the basic promoter is derived from P. AOX1 The promoter is the DNA sequence shown in SEQ ID NO: 8. Sequentially linking SEQ ID NO: 7, the spacer sequence, and SEQ ID NO: 8 yields the heterozygous promoter p1TOcP containing 1 copy of TetO, the sequence of which is shown in SEQ ID NO: 9.

[0074] Using plasmid pPIC9K as a template, PCR amplification was performed on "pPIC9K-1 and pPIC9K-2" (as shown in SEQ ID NO:43-44) and "p1TOcP-1 and p1TOcP-2" (as shown in SEQ ID NO:45-46) using primer pairs, respectively, to obtain the linearized plasmid fragment "pPIC9K-line" and the heterozygous promoter p1TOcP gene fragment "p1TOcP-fragment". The "pPIC9K-line" and "p1TOcP-fragment" were then ligated to obtain the recombinant backbone plasmid 9K-p1TOcP.

[0075] (2) Construction of recombinant plasmid 9K-p1TOcP-PuXyn containing a heterozygous promoter-xylanase expression cassette

[0076] The recombinant backbone plasmid 9K-p1TOcP and plasmid pPIC9K-PuXyn (disclosed in CN120173917A) were digested with restriction endonucleases EcoRI and NotI, respectively, to obtain the backbone plasmid fragment "9K-p1TOcP-fragment" and the xylanase PuXyn gene fragment "PuXyn-fragment". The two DNA fragments were then ligated to obtain the recombinant plasmid 9K-p1TOcP-PuXyn containing the heterozygous promoter-xylanase PuXyn expression cassette.

[0077] Example 5: Comparison of the application effects of different hybrid promoter activators

[0078] The plasmids 9K-p1TOcP-PuXyn and pPIC9K-PuXyn were linearized using the restriction endonuclease SalI. The linearized vectors were purified, recovered, and electroporated into Pichia pastoris GS115 competent cells. Positive transformants were screened on histidine auxotrophic plates to obtain recombinant strains GS115 / 9K-p1TOcP-PuXyn containing the heterozygous promoter-xylanase PuXyn expression cassette, as well as strains containing normal P... AOX1 The recombinant strain GS115 / pPIC9K-PuXyn, which is a promoter-xylanase PuXyn expression cassette.

[0079] The 15 linearized vectors from Example 3 were electroporated into GS115 / 9K-p1TOcP-PuXyn competent cells, and positive transformants were screened on bleomycin-containing resistant plates to obtain recombinant strains that simultaneously expressed a heterozygous promoter activator and contained a heterozygous promoter-xylanase PuXyn expression cassette.

[0080] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-Mit1-△60-96-Int18,

[0081] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-GGGGS-Mit1-△60-96-Int18,

[0082] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-EAAAK-Mit1-△60-96-Int18,

[0083] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-(GGGGS)2-Mit1-△60-96-Int18,

[0084] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-(EAAAK)2-Mit1-△60-96-Int18,

[0085] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-Mit1-Int18,

[0086] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR- GGGGS-Mit1-Int18,

[0087] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-EAAAK-Mit1-Int18,

[0088] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-(GGGGS)2-Mit1-Int18,

[0089] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-(EAAAK)2-Mit1-Int18,

[0090] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-Mxr1_TAD-Int18,

[0091] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-GGGGS-Mxr1_TAD-Int18,

[0092] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-EAAAK-Mxr1_TAD-Int18,

[0093] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-(GGGGS)2-Mxr1_TAD-Int18,

[0094] GS115 / 9K-p1TOcP-PuXyn / pPICZA-TetR-(EAAAK)2-Mxr1_TAD-Int18.

[0095] The 15 recombinant strains mentioned above, as well as GS115 / 9K-p1TOcP-PuXyn and GS115 / pPIC9K-PuXyn, were inoculated into BMGY medium and cultured at 30°C and 250 rpm for 20 h. Cells were then collected by centrifugation at 6000 rpm and 4°C for 5 min. The collected cells were resuspended in BMGY medium until the initial OD600 approached 0.5, and cultured with shaking at 30°C and 250 rpm, with 1% methanol added daily. After five days of fermentation, the fermentation supernatant of different strains was recovered by centrifugation at 6000 rpm and 4°C for 5 min, and xylanase activity was measured. To simplify the description, the names of the 17 recombinant strains have been simplified; the specific correspondences are shown in Table 3 below.

[0096] Table 3

[0097]

[0098] Xylanase activity assay: Under pH 5.0 and 50℃ conditions, 100 μL of 1% beech xylan substrate and 100 μL of appropriately diluted enzyme solution were mixed in a water bath and reacted for 30 min. The reaction was terminated by adding 600 μL of DNS solution and then incubated in a boiling water bath for 5 min. After cooling, the absorbance at 540 nm (A540) was measured, and the reducing sugar content was read from the standard curve. One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under given conditions.

[0099] The results are as follows Figure 4As shown: (1) The background expression activity of the heterozygous promoter is extremely low. The recombinant strain p1TOcP-Xyn containing only the heterozygous promoter-xylanase PuXyn expression cassette is basically unable to express the target protein. The specific enzyme activity of the fermentation supernatant is only 0.55 U / mL; (2) The protein expression system of the present invention can achieve efficient expression of the target protein. When the heterozygous promoter expression cassette and the heterozygous promoter activator are present at the same time, the overall protein expression ability of the system is greatly improved. The activation abilities of the three transcription activators, from strongest to weakest, were Mit1-△60-96 > Mit1 > Mxr1_TAD. In strains simultaneously containing a heterozygous promoter expression cassette and transcription activators Mit1-△60-96, Mit1, or Mxr1_TAD, the specific enzyme activities in the fermentation supernatant reached 66.9-100.5 U / mL, 55.7-83.8 U / mL, and 33.2-49.9 U / mL, respectively, representing increases in protein expression levels of 121-182 times, 101-152 times, and 60-90 times compared to the recombinant strain p1TOcP-Xyn. Furthermore, expression of transcription activators Mit1-△60-96 or Mit1 could restore protein expression levels to those of the native P. AOX1 The promoter exhibits even higher protein expression levels. Natural P AOX1 The specific enzyme activity of the supernatant of the recombinant strain pAOX1-Xyn expressing xylanase was 53.9 U / mL, while the specific enzyme activities of the supernatant of the recombinant strains p1TOcP-Xyn / TetR-(GGGGS)2-Mit1-△60-96 and p1TOcP-Xyn / TetR-(GGGGS)2-Mit1 were 100.5 U / mL and 83.8 U / mL, respectively, which were 1.86 times and 1.55 times that of strain pAOX1-Xyn; (3) The situation of the linker peptide is more complicated and varies depending on the transcription activator factor it is linked to. For the transcription activators Mit1-△60-96, Mit1 and Mxr1_TAD, the optimal linker peptides are (GGGGS)×2, (GGGGS)×2 and (EAAAK)×2, respectively.

[0100] In summary, the optimal heterozygous promoter activator was determined to be TetR-(GGGGS)2-Mit1-Δ60-96, where the DNA-binding protein is TetR, the linker peptide is (GGGGS)×2, and the transcription activator is Mit1-Δ60-96. Under optimal conditions, the protein expression level of the protein expression system can reach 100.5 U / mL, which is 182 times that of recombinant strains containing only the heterozygous promoter-xylanase PuXyn expression cassette, and is higher than that of natural P AOX1 The promoter expression of xylanase in recombinant strains was 1.86 times that of recombinant strains.

[0101] Example 6: Optimization of promoters expressing hybrid promoter activators

[0102] Existing hybrid promoter activators are for P AOX1 Expression under promoter control requires methanol induction, and due to the sequence repetition with the heterozygous promoter, there may be competition for transcriptional elements, thereby interfering with the protein expression ability of the entire system. Therefore, this invention also optimizes the promoter for expressing the heterozygous promoter activator.

[0103] Using the recombinant plasmid pPICZA-TetR-(GGGGS)2-Mit1-△60-96-Int18 as a template, PCR amplification was performed using primer pairs “pPICZA-TetR-1 and pPICZA-2” (as shown in SEQ ID NO:47-48) and “pTEF-1 and pTEF-2” (as shown in SEQ ID NO:49-50), respectively, to obtain the linearized plasmid fragment “pPICZA-TetR-(GGGGS)2-Mit1-△60-96-Int18-line” and the promoter pTEF gene fragment “pTEF-fragment”. Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primer pairs “pGAP-1 and pGAP-2” (as shown in SEQ ID NO:51-52) and “pGCW14-1 and pGCW14-2” (as shown in SEQ ID NO:53-54), respectively, to obtain the promoter pGAP gene fragment “pGAP-fragment” and the promoter pGCW14 gene fragment “pGCW14-fragment”. The plasmid fragments “pPICZA-TetR-(GGGGS)2-Mit1-△60-96-Int18-line” were ligated with “pTEF-fragment”, “pGAP-fragment”, and “pGCW14-fragment”, respectively, to obtain the recombinant plasmids pTEF-TetR-(GGGGS)2-Mit1-△60-96-Int18, pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18, and pGCW14-TetR-(GGGGS)2-Mit1-△60-96-Int18. The promoters driving the expression of the heterozygous promoter activator in these recombinant plasmids are expressed by P… AOX1 The bootloaders were replaced with pTEF, pGAP, and pGCW14 bootloaders, respectively.

[0104] The three recombinant plasmids were linearized with restriction endonuclease Kpn2I and electroporated into GS115 / 9K-p1TOcP-PuXyn competent cells, respectively. Positive transformants were screened on bleomycin-containing resistant plates to obtain the corresponding recombinant strains.

[0105] GS115 / 9K-p1TOcP-PuXyn / pTEF-TetR-(GGGGS)2-Mit1-△60-96-Int18 (abbreviated as p1TOcP-Xyn / pTEF-TetR-(GGGGS)2-Mit1-△60-96),

[0106] GS115 / 9K-p1TOcP-PuXyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 (abbreviated as p1TOcP-Xyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96),

[0107] GS115 / 9K-p1TOcP-PuXyn / pGCW14-TetR-(GGGGS)2-Mit1-△60-96-Int18 (abbreviated as p1TOcP-Xyn / pGCW14-TetR-(GGGGS)2-Mit1-△60-96).

[0108] The three recombinant strains mentioned above, as well as strains p1TOcP-Xyn / TetR-(GGGGS)2-Mit1-△60-96 and pAOX1-Xyn, were inoculated into BMGY medium and cultured at 30°C and 250 rpm for 20 h. Cells were then collected by centrifugation at 6000 rpm and 4°C for 5 min. The collected cells were then resuspended in either BMMY or BMDY medium (1% methanol in BMMY medium was replaced with 2% glucose, all other parameters remained unchanged) until the initial OD600 was approximately 0.5. The cells were then cultured at 30°C and 250 rpm with shaking. Strains cultured in BMMY medium were supplemented daily with a final concentration of 1% methanol, while strains cultured in BMDY medium were supplemented daily with a final concentration of 1% glucose. After five days of fermentation, the fermentation supernatants of different strains were recovered by centrifugation at 6000 rpm and 4°C for 5 min, and xylanase activity was measured.

[0109] The results are as follows Figure 5 As shown, the type of promoter expressing the heterozygous promoter activator has a significant impact on the overall protein expression level of the system, with the pGAP promoter being the optimal choice. Under BMMY medium conditions, the xylanase expression level of the protein expression system using the pGAP promoter to express the heterozygous promoter activator was 127 U / mL, which is the optimal level for P… AOX1 The promoter corresponds to a 1.26-fold increase in xylanase expression levels. However, under BMDY medium conditions, the advantage of expressing the heterozygous promoter activator using the pGAP promoter is even more pronounced. In the presence of glucose, P... AOX1 Promoter activity was significantly inhibited, with P AOX1Directly driving xylanase expression or driving the expression of a hybrid promoter activator with the promoter only yielded low protein expression levels, with specific enzyme activities of 1.5 U / mL and 6.1 U / mL, respectively. However, the pGAP promoter is a constitutive promoter, and its activity is not inhibited by glucose. Therefore, the corresponding protein expression system can still achieve high levels of target protein expression, with a specific enzyme activity of xylanase reaching 117.5 U / mL. AOX1 The promoter directly drives xylanase expression levels by 78.3 times, which is P AOX1 The xylanase expression level in the promoter-driven heterozygous promoter activator expression system was 19.3 times higher. These results demonstrate that the Pichia pastoris protein expression system constructed in this invention can not only achieve high-level expression of the target protein in a conventional methanol-induced system, but is also unaffected by inhibition from carbon sources such as glucose, enabling efficient expression of the target protein under non-methanol conditions. This significantly expands the application range of the Pichia pastoris protein expression system and achieves unexpected beneficial effects.

[0110] Example 7 Optimization of protein binding sequence copy number in heterozygous promoters

[0111] (1) Construction of recombinant backbone plasmids carrying heterozygous promoters with different copy numbers of TetO

[0112] The whole genome was synthesized according to the DNA sequences shown in SEQ ID NO: 10-18 to obtain gene fragments containing 2-10 copies of the TetO sequence, which were named 2TetO, 3TetO, 4TetO, and so on up to 10TetO. Using the recombinant backbone plasmid 9K-p1TOcP as a template, PCR amplification was performed using primer pairs “9K-p1TOcP-1 and pPIC9K-2” (as shown in SEQ ID NO: 55 and 44) ​​to obtain the corresponding linearized plasmid fragment “9K-p1TOcP-line”. “9K-p1TOcP-line” was ligated to gene fragments 2TetO-10TetO to obtain recombinant plasmids containing heterozygous promoters of 2-10 copies of TetO, which were named 9K-p2TOcP, 9K-p3TOcP, 9K-p4TOcP, and so on up to 9K-p10TOcP.

[0113] (2) Construction of recombinant plasmids containing different heterozygous promoter-xylanase expression cassettes

[0114] Referring to step (2) of Example 4, the acquisition and ligation of the backbone plasmid fragment and the xylanase PuXyn gene fragment "PuXyn-fragment" were completed, and nine recombinant plasmids 9K-p2TOcP-PuXyn, 9K-p3TOcP-PuXyn, 9K-p4TOcP-PuXyn, 9K-p5TOcP-PuXyn, 9K-p6TOcP-PuXyn, 9K-p7TOcP-PuXyn, 9K-p8TOcP-PuXyn, 9K-p9TOcP-PuXyn, and 9K-p10TOcP-PuXyn were obtained in sequence.

[0115] (3) Construction of recombinant strains containing different heterozygous promoter-xylanase expression cassettes and analysis of xylanase expression

[0116] The recombinant plasmid pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 from Example 6 was linearized with restriction endonuclease Kpn2I and electroporated into GS115 competent cells. Positive transformants were screened on bleomycin-resistant plates to obtain the recombinant strain GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18.

[0117] The nine recombinant plasmids from step (2) were linearized using the restriction endonuclease SalI. The linearized vectors were purified and recovered, and then electroporated into competent cells of recombinant strain GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18. Positive transformants were screened on histidine auxotrophic plates to obtain recombinant strains containing 2-10 copies of the heterozygous promoter-xylanase PuXyn expression cassette.

[0118] GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 / 9K-p2TOcP-PuXyn (abbreviated as p2TOcP-Xyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96),

[0119] GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 / 9K-p3TOcP-PuXyn (abbreviated as p3TOcP-Xyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96),

[0120] GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 / 9K-p4TOcP-PuXyn (abbreviated as p4TOcP-Xyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96),

[0121] GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 / 9K-p5TOcP-PuXyn (abbreviated as p5TOcP-Xyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96),

[0122] GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 / 9K-p6TOcP-PuXyn (abbreviated as p6TOcP-Xyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96),

[0123] GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 / 9K-p7TOcP-PuXyn (abbreviated as p7TOcP-Xyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96),

[0124] GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 / 9K-p8TOcP-PuXyn (abbreviated as p8TOcP-Xyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96),

[0125] GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 / 9K-p9TOcP-PuXyn (abbreviated as p9TOcP-Xyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96),

[0126] GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 / 9K-p10TOcP-PuXyn (abbreviated as p10TOcP-Xyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96).

[0127] The nine recombinant strains and strain p1TOcP-Xyn / pGAP-TetR-(GGGGS)2-Mit1-△60-96 were inoculated into BMGY medium and cultured at 30°C and 250 rpm for 20 h. Cells were then collected by centrifugation at 6000 rpm and 4°C for 5 min. The collected cells were resuspended in either BMMY or BMDY medium until the initial OD600 was approximately 0.5, and cultured with shaking at 30°C and 250 rpm. Strains cultured in BMMY medium were supplemented daily with a final concentration of 1% methanol, while strains cultured in BMDY medium were supplemented daily with a final concentration of 1% glucose. After five days of fermentation, the fermentation supernatant from each strain was recovered by centrifugation at 6000 rpm and 4°C for 5 min, and xylanase activity was measured.

[0128] The results are as follows Figure 6 As shown, the copy number of the protein-binding sequence TetO in the heterozygous promoter has a significant impact on the expression level of the protein expression system, exhibiting a "first increase, then decrease" trend. Specifically, within the TetO copy number range of 1-6, protein expression levels increase with increasing TetO copy number; however, when the TetO copy number exceeds 6, protein expression levels decrease. At a TetO copy number of 6, protein expression levels reached their highest under both culture medium conditions. Specifically, under BMMY medium, the xylan-specific enzyme activity reached 532.6 U / mL, and under BMDY medium, the xylan-specific enzyme activity reached 516.7 U / mL. Under the same culture medium conditions, P... AOX1 The promoter-driven xylanase expression levels were only 53.9 U / mL and 1.5 U / mL, respectively (data in Examples 5-6). Using the protein expression system of this invention, the protein expression levels were increased by 9.88 and 344 times, respectively, achieving significant beneficial effects. Furthermore, under different TetO copy number conditions, the protein expression levels of the protein expression system of this invention were quite similar in glucose-containing BMDY medium and methanol-induced BMMY medium, further demonstrating that the Pichia pastoris protein expression system constructed in this invention is not inhibited by carbon sources such as glucose and can achieve efficient expression of the target protein under non-methanol conditions.

[0129] Example 8: Expression of different target proteins using the optimized protein expression system

[0130] After the above multi-step optimization for different components, the optimal protein expression system is now determined as follows: (1) The optimal hybrid promoter activator is TetR-(GGGGS)2-Mit1-△60-96, in which the DNA binding protein is TetR, the linker peptide is (GGGGS)×2, the transcription activator is Mit1-△60-96, and the optimal promoter for initiating the expression of the hybrid promoter activator is the pGAP promoter; (2) The optimal hybrid promoter is p6TOcP, which contains 6 copies of the TetO sequence, the spacer sequence is TCGCGCGC, and the basic promoter is derived from P AOX1 The promoter is the DNA sequence shown in SEQ ID NO: 8.

[0131] (1) Construction of recombinant plasmids containing p6TOcP-expression cassettes of different target proteins

[0132] To further verify the universality of this system, the optimized protein expression system was used to express two other different target proteins, namely glucosidase and phytase.

[0133] The recombinant backbone plasmid 9K-p6TOcP and plasmid pPIC9K-BaBgl(WT) (disclosed in CN118530972A) were digested with restriction endonucleases EcoRI and NotI, respectively, to obtain the backbone plasmid fragment "9K-p6TOcP-fragment" and the glucosidase BaBgl gene fragment "BaBgl-fragment". The two DNA fragments were then ligated to obtain the recombinant plasmid 9K-p6TOcP-BaBgl containing the heterozygous promoter-glucosidase expression cassette.

[0134] The phytase EcPhy in this application is derived from Escherichia coli and is a protein containing the amino acid sequence shown in SEQ ID NO: 19 (NCBI GenBank: AAA72086.1). The entire genome was synthesized by a gene synthesis company based on this protein sequence. The synthesized gene was optimized according to the codon preference of Pichia pastoris, and the synthesized DNA fragment (SEQ ID NO: 20) was inserted between the EcoRI and NotI restriction sites of the pPIC9K plasmid and the 9K-p6TOcP plasmid, respectively, to obtain phytase EcPhy. AOX1 The plasmid vector pPIC9K-EcPhy, which directly drives phytase expression, and the plasmid vector 9K-p6TOcP-EcPhy, which drives phytase expression with the heterozygous promoter p6TOcP.

[0135] (2) Construction of recombinant strains containing p6TOcP-expression cassettes of different target proteins

[0136] Recombinant plasmids pPIC9K-BaBgl(WT), 9K-p6TOcP-BaBgl, pPIC9K-EcPhy, and 9K-p6TOcP-EcPhy were linearized using the restriction endonuclease SalI, and the linearized vectors were purified and recovered. Two linearized vectors from the pPIC9K series were electroporated into competent cells of strain GS115, and two linearized vectors from the 9K-p6TOcP series were electroporated into competent cells of recombinant strain GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18. Positive transformants were screened on histidine auxotrophic plates, yielding the following four recombinant strains:

[0137] GS115 / pPIC9K-BaBgl(WT) (abbreviated as pAOX1-BaBgl),

[0138] GS115 / pPIC9K-EcPhy (abbreviated as pAOX1-EcPhy)

[0139] GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 / 9K-p6TOcP-BaBgl(WT) (abbreviated as p6TOcP-BaBgl / pGAP-TetR-(GGGGS)2-Mit1-△60-96),

[0140] GS115 / pGAP-TetR-(GGGGS)2-Mit1-△60-96-Int18 / 9K-p6TOcP-EcPhy (abbreviated as p6TOcP-EcPhy / pGAP-TetR-(GGGGS)2-Mit1-△60-96).

[0141] (3) Enzyme production analysis of recombinant strains

[0142] The four recombinant strains were inoculated into BMGY medium and cultured at 30°C and 250 rpm for 20 h. Cells were then collected by centrifugation at 6000 rpm and 4°C for 5 min. The collected cells were resuspended in either BMMY or BMDY medium until the initial OD600 approached 0.5, and cultured with shaking at 30°C and 250 rpm. Strains cultured in BMMY medium were supplemented daily with a final concentration of 1% methanol, while strains cultured in BMDY medium were supplemented daily with a final concentration of 1% glucose. After five days of fermentation, the fermentation supernatants of different strains were recovered by centrifugation at 6000 rpm and 4°C for 5 min, and the corresponding enzyme activities were measured.

[0143] Method for determining glucosidase activity: 150 µL of 5 mM pNPG (prepared to a 20 mM stock solution concentration using 100 mM sodium phosphate buffer at pH 5.5) was incubated in a 50°C water bath for 10 min. Then, 50 µL of diluted enzyme solution was added, and the reaction was continued at 50°C for 15 min. The reaction was then terminated by adding 200 µL of 1 M Na₂CO₃ to ice. The absorbance was measured at 405 nm, and the amount of p-nitrobenzene (pNP) released was recorded using a standard curve. One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol of pNP per minute under given conditions.

[0144] Method for determining phytase activity: Add an appropriate amount of sodium acetate buffer (0.25 mol / L, pH 5.50) to the supernatant of the fermentation broth, dilute appropriately to a final volume of 1 mL, and preheat at 37℃ for 5 min. Add 2 mL of sodium phytate solution (0.00761 mol / L, pH 5.50), mix, and react at 37℃ for 30 min. Add 2 mL of freshly prepared colorimetric / stop solution (2.35 g / L ammonium vanadate solution: 100 g / L ammonium molybdate solution: 30% nitric acid = 1:1:2), let stand at room temperature for 10 min, and then measure the absorbance at 415 nm. Definition of phytase activity: 1 unit (U) of enzyme activity is defined as the amount of enzyme that releases 1 μmol of inorganic phosphorus per minute by hydrolyzing sodium phytate solution at 37℃ and pH 5.5.

[0145] The results are as follows Figure 7 As shown: The protein expression system of the present invention has broad applicability and scalability, and has good application effects in different target proteins. (a) Figure shows the application effect in glucosidase, with P AOX1 The promoter-driven expression levels of glucosidase were 74.5 U / L and 2 U / L in BMMY and BMDY media, respectively, while the glucosidase expression levels obtained using the protein expression system of this invention under the same conditions were 197.3 U / L and 183.0 U / L, respectively. AOX1 The promoter conditions were 2.65 times and 91.5 times. When applied to phytase, this invention yielded even more significant beneficial effects, as shown in Figure (b), with P... AOX1 The promoter-driven phytase expression levels were 63.7 U / mL and 1.4 U / mL in BMMY and BMDY media, respectively, while the expression levels obtained using the protein expression system of this invention under the same conditions were 715.2 U / mL and 804.4 U / mL, respectively. AOX1 The increase in promoter conditions is 11.2 times and 574.6 times, which is a huge improvement.

[0146] In summary, this invention provides a highly efficient protein expression system for Pichia pastoris, consisting of two parts: a heterozygous promoter activator and a heterozygous promoter. Through gradual optimization of each key component of the system, this invention achieves excellent results: in the absence of the heterozygous promoter activator, the target protein gene initiated by the heterozygous promoter is expressed very weakly; while in the presence of the heterozygous promoter activator, the target protein gene is expressed efficiently. This system has good applicability and scalability, and the expression intensity of various target proteins, including xylanase, phytase, and glucosidase, is significantly higher than that of commonly used Pichia pastoris systems. AOX1 Promoter. Depending on the culture conditions, the increase can range from 2-12 times or 90-600 times. Furthermore, this system achieves efficient expression of the target protein without methanol induction; glucose can also be used as a carbon source to achieve efficient expression of the target protein, greatly expanding the application range of the Pichia pastoris protein expression system and achieving unexpected beneficial results.

[0147] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A high-efficiency protein expression system, characterized in that: It includes a heterozygous promoter activator and a heterozygous promoter. The heterozygous promoter activator is composed of a DNA-binding protein TetR, a linker peptide, and a transcription activator Mit1-Δ60-96. The heterozygous promoter is composed of 6 copies of a protein-binding sequence TetO, a spacer sequence, and a basic promoter. The DNA-binding protein TetR sequence is shown in SEQ ID NO: 1, the linker peptide is (GGGGS) × 2, the transcription activator Mit1-△60-96 sequence is shown in SEQ ID NO: 4, the protein-binding sequence TetO sequence is shown in SEQ ID NO: 7, the spacer sequence is TCGCGCGC, and the basal promoter is derived from P. AOX1 The promoter sequence is shown in SEQ ID NO:

8.

2. The high-efficiency protein expression system according to claim 1, characterized in that: The promoter expressed by the hybrid promoter activator is the pGAP promoter.

3. The high-efficiency protein expression system according to claim 1, characterized in that: The sequence of the heterozygous promoter is shown in SEQ ID NO:

9.

4. The application of the high-efficiency protein expression system according to any one of claims 1-3 in protein production.

5. The application according to claim 4, characterized in that: The protein includes xylanase, glucosidase, and phytase.

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