Glucose-induced pichia pastoris promoter and application thereof

Abstract

The invention discloses a glucose-induced pichia pastoris promoter and application thereof, and belongs to the technical field of bioengineering. According to the invention, a glucose response activation cis-element is screened and identified, and is used for constructing a hybrid promoter containing GRACE. The promoter PAOX1PM6-3d constructed by the invention can respond to glucose-induced high-efficiency expression of target protein, and the titers of xylanase, TGase enzyme and snow flea antifreeze protein are respectively 3.98 times, 1.65 times and 3.33 times of those of a methanol-induced promoter, so that the promoter PAOX1PM6-3d has great industrial application potential.

Description

Technical Field

[0001] This invention relates to a glucose-induced Pichia pastoris promoter and its application, belonging to the field of bioengineering technology. Background Technology

[0002] Inducible promoters are indispensable in synthetic systems, enabling precise regulation of gene expression, optimization of enzyme levels, and reprogramming of cellular function through synthetic genetic circuits in engineered metabolic pathways. However, unlike prokaryotic promoters, the sequence-function relationships of eukaryotic promoters remain poorly understood, making their engineering more challenging. This complexity stems from their long promoter sequences, which contain multiple cis-regulatory elements, typically 4–10 bp in length. These elements, along with trans-acting factors, are dynamically regulated by inducer-derived signals that reshape intracellular cues, prompting transcription factors (TFs) to selectively bind to activation or repression sites, thereby regulating gene expression according to cellular state. The complex regulatory characteristics of yeast limit the design of custom promoters in synthetic biology, which often relies on a well-defined set of promoters. Pichia pastoris ( Komagataella phaffii Original name Pichia pastoris With its high-density growth, efficient secretion, genetic stability, and post-translational modification capabilities, *Pichia pastoris* has become an important host for both academia and industry. Although *Pichia pastoris* possesses a powerful genetic toolkit, it lacks an efficient induction system other than the alcohol oxidase I promoter (PAox1). PAox1 is widely used for recombinant protein expression, but its activation is strictly dependent on methanol. Methanol poses significant safety and toxicity risks, including serious health hazards from inhalation, ingestion, or exposure, such as visual impairment and systemic organ damage. Furthermore, methanol-driven fermentation, which relies on alcohol oxidases, requires high oxygen levels and generates substantial amounts of heat, complicating process control. Additionally, methanol concentrations exceeding 1% increase cell lysis, leading to a final biomass reduction of up to 50%. While *Pichia pastoris* is a recognized and commonly used strain in recombinant protein production, its dependence on methanol-inducible systems limits its wider application, particularly in the field of food biotechnology, where a non-toxic, food-grade expression platform is urgently needed.

[0003] In addition to methanol toxicity, naturally inducible promoter systems are further restricted by carbon metabolite repression (CCR), which reduces their responsiveness to alternative carbon sources and complicates dynamic regulation. In Pichia pastoris (… K. phaffiiAmong these, the PAox1 promoter is particularly affected because methanol induction is strongly inhibited by preferred carbon sources such as glucose or glycerol. Other methanol-free promoters include the ethanol-regulated PADH, the rhamnose-induced PRHAx, and the glucose-responsive promoter of the high-affinity glucose transporter Gth1 (P... GTH1 Both have limited induction efficiency. It is worth noting that P... GTH1 While strict regulation can be achieved, it is inhibited when glucose concentration exceeds 0.2%, posing a challenge to large-scale fermentation. Efforts to develop methanol-free systems in Pichia pastoris include: overexpressing the positive regulator Mit1 while knocking out the glucose repressors Nrg1, Mig1, and Mig2; replacing the natural promoters of Mxr1 and Mit1 with derepressed Cat1 or Aox2 promoters; and activating the Das1 promoter by overexpressing KpTrm1 in glucose medium. However, these methods only achieve 40%-60% of the PAox1 methanol-inducible strength. Therefore, there is an urgent need to develop a novel promoter in Pichia pastoris that meets food safety requirements. Summary of the Invention

[0004] The present invention provides a glucose-responsive cis-acting element GRACE having the nucleotide sequence shown in SEQ ID NO.3.

[0005] The present invention also provides a promoter containing the sequence shown in SEQ ID NO.3.

[0006] In one embodiment, the promoter includes, but is not limited to, the promoter PAox1P3 shown in SEQ ID NO.6, the promoter PAox1PM6 shown in SEQ ID NO.8, the promoter PAox1PM6-3 shown in SEQ ID NO.9, or the promoter PAox1PM6-3d shown in SEQ ID NO.13.

[0007] In one embodiment, the promoter is PAox1PM6-3d, the nucleotide sequence of which is shown in SEQ ID NO.13.

[0008] The present invention also provides a gene expression cassette or recombinant plasmid containing the promoter.

[0009] In one embodiment, the plasmid includes, but is not limited to, the pPIC sequence plasmid.

[0010] In one embodiment, the plasmid is pPIC3.5k.

[0011] The present invention also provides recombinant microorganisms, using the recombinant plasmid as a vector, to express the target protein.

[0012] In one embodiment, the target protein includes, but is not limited to, xylanase, transglutaminase, and snow flea antifreeze protein.

[0013] In one embodiment, the nucleotide sequence of the gene XynA encoding xylanase is shown in SEQ ID NO.14; the nucleotide sequence of the gene TGase encoding glutamine transaminase is shown in SEQ ID NO.15; and the nucleotide sequence of the gene SfAFP encoding snow flea antifreeze protein is shown in SEQ ID NO.16.

[0014] In one embodiment, the recombinant microorganism uses GS115Δku70 as the host and PAox1PM6-3d to regulate the expression of the xylanase gene XynA.

[0015] In one embodiment, the recombinant microorganism uses GS115Δku70 as a host and PAox1PM6-3d to regulate the expression of the TGase gene of glutamine transaminase.

[0016] In one embodiment, the recombinant microorganism uses GS115Δku70 as the host and PAox1PM6-3d to regulate the expression of the SfAFP gene, which is an antifreeze protein of the snow flea.

[0017] The present invention also provides the application of the promoter in regulating the expression of target proteins.

[0018] In one embodiment, the application involves linking the target protein downstream of the promoter, culturing recombinant bacteria expressing the target protein in a culture medium, and inducing protein expression with glucose.

[0019] In one embodiment, the target protein includes, but is not limited to, xylanase, transglutaminase, and snow flea antifreeze protein.

[0020] In one embodiment, the concentration of glucose in the culture medium is 1~10 g / L.

[0021] The present invention also provides the application of the promoter or the recombinant microorganism in the field of fermentation.

[0022] Beneficial effects: 1. This invention combines P Aox1 and P GTH1 By targeting the deletion of regulatory fragments and motifs, a short glucose-responsive cis-element (GRACE) was identified. Gradual replacement of the natural cis-element with GRACE alleviated glucose inhibition and transformed the traditionally glucose-inhibited promoter into an inducible system. Furthermore, the amplification of the methanol-specific Mit1-activated cis-module significantly improved promoter strength, resulting in a 190% increase in regulated EGFP activity.

[0023] 2. This invention also constructs a promoter containing GRACE that responds to glucose induction and achieves efficient expression of the target protein, solving the problem of induction dependence on non-food-safe components such as methanol in traditional Pichia pastoris expression systems. The promoter P... AOX1PM6-3d By regulating the expression of target proteins, the titer of xylanase can be 3.98 times that of the methanol-inducible promoter, the titer of TGase can be 1.65 times that of the methanol-inducible promoter, and the expression level of snow flea antifreeze protein can be 3.33 times that of the methanol-inducible promoter.

[0024] 3. The present invention also verified the effect of the promoter in inducing xylanase expression in a 5L bioreactor. The yield reached 2939 U / L after 70 hours, which has great potential for industrial application. Compared with the titer of 1126 U / L regulated by PAox1 at the same time point, it increased by 1.6 times, and the overall improvement was 123%. Attached Figure Description

[0025] Figure 1 Promoter fragment design and construction for fragment hybridization promoter library assembly.

[0026] Figure 2 For the construction and screening of heterozygous promoter variants; wherein, (A) a five-step single-copy integration process: (i) electroporation of 0.1 µg linear library DNA, (ii) using (iii) Liquid screening under low antibiotic pressure, (iv) Replica culture on a G418 concentration gradient, (v) Two rounds of consecutive flow cytometry sorting (FACS); (B) Screening of gate-2 isolates in 24-well deep plates using the same de-inhibition and inhibition conditions as during sorting.

[0027] Figure 3 A sliding window deletion analysis was performed to identify key glucose response elements; a systematic 260 bp PGTH1 deletion analysis was conducted on PAox1P3 to identify key regulatory subregions. The schematic diagram on the left shows 12 consecutive 30 bp deletions (ΔRE1 to ΔRE12), each overlapping by 10 bp in the 5' to 3' direction. The table lists the nucleotide coordinates of each deletion relative to the ATG start codon. The bar chart on the right shows the effect of each deletion on promoter activity tested under 0.2% glucose conditions, with PAox1P3 serving as a reference control (set as 100%).

[0028] Figure 4To evaluate derepression capability, PGTH1 regulatory fragments were inserted into PAox1 guided by heterozygous promoter structures. (A) The best-performing variants in each group, namely PAG1-PAG3 (glucose-restricted), PAG31-PAG33 (partially repressed), and PAG79-PAG81 (strongly repressed), were recloned into pPIC3.5k-EGFP for sequence analysis. (B) Promoter variants based on the native PAox1 sequence, labeled with key regulatory elements (right side). (C) Promoter activity was evaluated in shake flasks under glucose concentration gradients. PGTH1 activity at 0.2% glucose concentration was used as a reference.

[0029] Figure 5 Modifications targeting cis-regulatory elements for glucose response activation and methanol-induced recovery; (A) Six PAox1P-derived promoters in which the native Mxr1 site was sequentially replaced with GRACE activator motifs (positions 1-3-5, then 2-4-6), producing 1-6 copies of GRACE. Annotated regulators are shown on the left. (B) Shake-flask activity at glucose gradients (0.2-2.0% w / v). (C) Additional constructs using a repeat series of Mit1 sites in the PAox1PM6 backbone. (D) Promoter activity was assessed using the same method as in (B).

[0030] Figure 6 Functional characterization of wild-type and engineered promoters; among them, shake-flask culture analysis of (AC) wild-type PAox1 (induced with 1% methanol) and engineered PAox1PM6-3d (induced with 1% glucose) shows the yield and OD600 of (A) XynA (Aspergillus niger xylanase), (B) TGase (Streptomyces moharawata glutamine transaminase), and (C) SfAFP (Snowflake antifreeze protein). SDS-PAGE analysis depicted the seven-day expression profile of each protein in shake-flask culture. (D) 5 L fed-batch fermentation of XynA under PAox1PM6-3d and PAox1 promoters. Detailed Implementation

[0031] (a) Sequence Description The sequences of PAox1 are shown in SEQ ID NO. 1; the sequence of EGFP is shown in SEQ ID NO. 2; the sequence of the glucose-responsive activated cis-element (GRACE) is shown in SEQ ID NO. 3; the sequence of the glucose-responsive promoter PAox1P is shown in SEQ ID NO. 4; the 260 bp of PGTH1 is shown in SEQ ID NO. 5; the sequence of the glucose-responsive promoter PAox1P3 is shown in SEQ ID NO. 6; MXR1 is shown in SEQ ID NO. 7; the sequence of the glucose-responsive promoter PAox1PM6 is shown in SEQ ID NO. 8; the sequence of the glucose-responsive promoter PAox1PM6-3 is shown in SEQ ID NO. 9; the sequence of Mit-W2A is shown in SEQ ID NO. 10; the sequence of Mit-W2B is shown in SEQ ID NO. 11; the sequence of Mit-W3A is shown in SEQ ID NO. 12; and the sequence of the promoter PAox1PM6-3d is shown in SEQ ID NO. 10. The nucleotide sequence of the xylanase gene (XynA) is shown in SEQ ID NO. 13; the nucleotide sequence of the Streptomyces morigensis glutamine transaminase gene TGase is shown in SEQ ID NO. 15; and the nucleotide sequence of the snow flea antifreeze protein gene SfAFP is shown in SEQ ID NO. 16.

[0032] (a) Materials and reagents *E. coli* JM109 was used as the cloning host, and *Pichia pastoris* GS115 was used as the expression host. Chemical reagents and culture media were used for PCR with 2×Phanta Max Master Mix (Nanjing Vazyme) or PrimeSTAR®Max DNA polymerase (Takara). Genomic DNA, plasmids, and gel fragments were purified using kits from Sangon Biotech or Thermo Fisher Scientific; oligonucleotides were synthesized by Sangon Biotech and sequenced using Sanger sequencing. Bacterial peptone and yeast extracts were purchased from Oxoid; antibiotics, methanol, and other remaining reagents were purchased from Maclean's, Sigma-Aldrich, or Sangon Biotech.

[0033] Detailed information on the strains is shown in Table 1. Primer sequences used in the examples are shown in Table 2.

[0034] Table 1. Important strains constructed

[0035] Table 2. Main primer sequences

[0036] (ii) Culture medium Inorganic Salt Medium (BSM): Solution A (900mL) contains 16.6g magnesium sulfate heptahydrate, 1.0g calcium sulfate dihydrate, 40g glycerol, 27.3g potassium sulfate, and 11.1g ammonium sulfate; Solution B (100mL) contains 12g disodium hydrogen phosphate hexahydrate. Solutions A and B are sterilized separately to prevent salt precipitation. The composition (g / L) of PTM1 trace element solution is as follows: 0.09g potassium iodide, 0.02g boric acid, 0.2g biotin, 0.5g cobalt chloride, 20g zinc chloride, 0.2g sodium molybdate dihydrate, 65g ferrous sulfate heptahydrate, 6g copper sulfate pentahydrate, 3g manganese sulfate monohydrate, and 5.0mL / L 98% sulfuric acid.

[0037] Glycerol-buffered basal medium (BMGY): 20 g / L peptone, 10 g / L yeast extract, 1.34% YNB, 100 mM potassium phosphate buffer pH 6.0, 4 × 10⁻⁵% biotin, 10 g / L glycerol.

[0038] Glucose + Buffered Basal Medium (BMDY): 20 g / L peptone, 10 g / L yeast extract, 1.34% YNB, 100 mM potassium phosphate buffer pH 6.0, 4 × 10⁻⁵% biotin, 10 g / L glucose.

[0039] Methanol + buffered basal medium (BMMY): 20 g / L peptone, 10 g / L yeast extract, 1.34% YNB, 100 mM potassium phosphate buffer pH 6.0, 4 × 10⁻⁵% biotin, 10 g / L methanol.

[0040] LB medium: tryptone 15 g / L, yeast extract 10 g / L, NaCl 5 g / L, ddH2O 1L, pH 7.2.

[0041] YPD medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L.

[0042] YPD-G418: Yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, 100 μg / mL G418, 1L to a final volume.

[0043] (III) Detection Methods Fluorescence intensity detection: The fluorescence intensity of cell cultures in the well plates was detected using a Synergy H1 microplate reader (BioTek, 488 / 520 nm).

[0044] Glutamine transaminase (TGase) activity assay: The isohydroxamic acid method was used. The sample (60 µL) was mixed with 150 µL of substrate (30 mM CBZ-glutamine-glycine, 100 mM hydroxylamine, 10 mM reduced glutathione, 50 mM MES, pH 6.0), incubated at 37°C for 10 min, and then 60 µL of trichloroacetic acid / ferric chloride / hydrochloric acid was added to terminate the reaction. The absorbance was measured at 525 nm using a BioTek Cytation 5; one unit (U) was defined as the amount of enzyme producing 1 μmol of isohydroxamic acid per minute at 37°C.

[0045] Xylanase (XynA) activity assay: The DNS method was used for determination. A diluted enzyme solution (50 µL) was added to 450 µL of a 10 g / L beech xylan solution, incubated at 50°C for 10 min, and the reaction was terminated by adding 1.5 mL of DNS. The mixture was boiled for 5 min, diluted with 3 mL of water, and the absorbance was measured at 545 nm. 1 U is defined as the amount of enzyme that produces 1 µmol of reducing sugar per minute.

[0046] SfAFP assay: SfAFP in the culture supernatant was quantified using the BCA total protein assay, followed by 12% SDS-PAGE. The abundance of the target protein was quantified using ImageJ (National Institutes of Health, NIH). SfAFP titer = total protein × (SfAFP band / total protein in lane). For protein analysis, yeast culture supernatant (30 μL) was mixed with 10 μL of 4×NuPAGE LDS buffer (Thermo Fisher Scientific) containing reducing agent and heated at 90°C for 10 minutes. Then, 15 μL of the mixture was loaded into a 12% NuPAGE Bis-Tris gel (MES buffer, pH≈7). The gel was stained with Coomassie Brilliant Blue R-250, and the band intensity was quantified using ImageLab software (Bio-Rad Laboratories).

[0047] Example 1: Discovery of glucose-responsive cis-elements (GRACE) A combinatorial library of approximately 4000 PAox1-PGTH1 hybrid promoters was constructed in the pPIC3.5K vector, as shown below. Figure 1As shown, to avoid disrupting regulatory sites, sites A, B, C, and D were set in the promoter PAOX1 (936 bp in total, as shown in SEQ ID NO.1), with A located at -936, B at -729, C at -520, and D at -00 (i.e., the 3' end of the promoter). The regulatory fragment of the promoter PGTH1 (-459 to -199, as shown in SEQ ID NO.4) is rich in transcription factor binding sites (TFBS). Sites A, B, C, and D were set at -459, -369, -279, and -199 of the full-length sequence of the promoter PGTH1 (Gene ID: 8197946). A hybrid promoter library was constructed according to the following steps, and the EGFP sequence shown in SEQ ID NO.2 was synthesized. The promoter-free pPIC3.5K vector backbone was amplified by PCR using primers PLSpeI / PLXbaI to obtain the SpeI-XbaI terminus. The full-length PAox1 and the 260 bp PGTH1 regulatory regions were designed into four positional patterns, each containing SpeI, SacI, EcoRI, and XbaI restriction sites. Exchanges or repetitions at positions B and C in PAox1 resulted in four patterns: ABCD, ACBD, ABBD, and ACCD. The PGTH1 fragment was digested into subfractions: AB (90 bp), BC (90 bp), CD (80 bp), AC (180 bp), BD (170 bp), and the full-length AD, BD, and CD (260 bp), thus enabling various recombination configurations, such as... Figure 1 As shown. The amplified fragments and pPIC3.5K vector were digested with paired enzymes, purified by gel electrophoresis, and then mixed in equimolar amounts. The mixture was ligated overnight at 16°C using T4 ligase to construct recombinant plasmids regulating GFP proteins with different promoters. The ligation products were transformed into *E. coli* JM109, and colonies were randomly selected for Sanger sequencing. After extensive plasmid extraction, the plasmids were linearized with SalI, and 0.1 μg was introduced into strain GS115Δku70 (published in *Deletion of the Pichia pastoris KU70 HomologueFacilitates Platform Strain Generation for Gene Expression and Synthetic Biology*) via electroporation.

[0048] The recombinant bacteria constructed above were cultured overnight at 30°C in YPG medium containing 100 μg / ml ampicillin. Cells were collected, washed, and resuspended in medium to OD600=1.0. They were then incubated for 24 h at 30°C in BMDY medium containing 2 g / L glucose (derepressed) and 10 g / L glucose (repressed), respectively. Cells were then collected, washed with PBS (pH 7.4), and diluted to OD600=0.3 for two rounds of flow cytometry (FACS) screening to construct libraries. The first round excluded the top 1% to remove possible multicopy anomalies. The second round analyzed promoter variants by inducing for 48 h at 30°C in BMDY medium containing 2 g / L glucose (limited glucose conditions) or 10 g / L glucose (excess glucose conditions), respectively. Thirty clones were isolated from the top 3% of fluorescence intensity under limited glucose conditions, 48 ​​clones from the top 3% of fluorescence intensity under excess glucose conditions, and 20 clones from the bottom 1% of fluorescence intensity under excess glucose conditions. All selected clones were re-validated in 24-well plates under their respective induction conditions. Of the 30 clones (PAG1–PAG30) selected under limited glucose conditions, 13 showed more than 2-fold higher EGFP yield than PGTH1 (Figure 2). Under excess glucose conditions, no clones exhibiting enhanced fluorescence were selected from the 48 clones. The promoter sequences of the selected clones were confirmed by colony PCR sequencing. The selected recombinant bacteria were inoculated onto YPD-G418 (100 μg / ml) and incubated at 30°C for 48 h. The sorted clones were then re-selected for 48 h in 24-well deep-well plates (10 ml basal medium, 30°C, 500 rpm) under both derepression and repression conditions. Promoters with different combinations of PAG1, PAG2, etc. were obtained, and the results are as follows: Figure 2 As shown.

[0049] To identify key regulatory subregions, a systematic 260 bp PGTH1 regulatory fragment deletion analysis was performed on PAox1P3 (shown in SEQ ID NO.6). The 260 bp was divided into 12 consecutive 30 bp deletions (ΔRE1 to ΔRE12), with each deletion overlapping by 10 bp in the 5' to 3' direction. Figure 4 The nucleotide coordinates of each deleted fragment relative to the ATG start codon are marked. Recombinant Pichia pastoris containing fluorescent proteins regulated by the above promoters was cultured in BMDY medium containing 2 g / L at 30°C for 48 h. The effect of each deleted sequence on promoter activity was examined, and the results are as follows: Figure 3As shown, response elements are present in fragments RE8 and RE9. Through computer TFBS analysis of the 260bp sequence in PGTH1, a glucose response activation cis element containing 26bp was confirmed, and its nucleotide sequence is shown in SEQ ID NO.3.

[0050] Example 2 Construction of Hybrid Promoters Based on the PAox1 promoter, the transcriptional repressor binding sites ΔNrg1, ΔMig1, and ΔCre-1 were targeted and deleted to obtain PAox1P (nucleotide sequence shown in SEQ ID NO.4). Then, a 260 bp PGTH1 regulatory fragment (nucleotide sequence shown in SEQ ID NO.5) was inserted into three different positions (-729 bp, -520 bp, -182 bp) within PAox1 to construct a heterozygous promoter. Following the method in Example 1, the constructed promoter regulated EGFP expression and was transformed into GS115Δku70. The recombinant bacteria were cultured in LMDY medium with glucose concentrations of 2 g / L, 4 g / L, 6 g / L, 10 g / L, and 20 g / L at 30°C for 48 h, and fluorescence intensity was detected. The results showed that the promoter PAox1P3 (SEQ ID NO.6), with the regulatory fragment inserted into PAox1 at -182 bp, exhibited the best effect.

[0051] Recombinant plasmids containing the promoter PAox1P3 were constructed and applied to pPIC 3.5kJ / P3400. Aox1p3 Based on the plasmid, the selected glucose-responsive activation cis-elements (GRACE) were superimposed to replace the binding sites (-847~-831, -800~-781, -302~-283, -267~-248, -586~-559, -630~-611) of transcription factor Mxr1 (nucleotide sequence shown in SEQ ID NO.7), resulting in PAox1PM1, PAox1PM2, PAox1PM3, PAox1PM4, PAox1PM5, and PAox1PM6, respectively. The EGFP gene was constructed downstream of the promoter, and the promoter regulated the expression of the fluorescent protein. The obtained recombinant bacteria were cultured in BMDY medium containing different concentrations of glucose at 30℃ and 220 rpm / min for 48 h, and the expression of fluorescence intensity was detected. The results showed that ( Figure 5 The PAOX1PM6 promoter (SEQ ID NO.8) showed the best induction effect at a glucose concentration of 10 g / L.

[0052] GeneXplain analyzed the transcription factor binding sites (TFBS) in the PAox1PM6 promoter using the TRANSFAC database. The binding sites for Mit-W2A, Mit-W2B, and Mit1-W3A were obtained, with sequences SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, respectively. Based on the PAox1PM6 promoter, the binding sites for Mit-W2A, Mit-W2B, and Mit1-W3A were increased by one copy, resulting in PAox1PM6-1, PAox1PM6-2, and PAox1PM6-3 (shown in SEQ ID NO. 12). Further deletion of the complex framework (60 bp) of Hap2345p and ABaA in PAox1PM6-3 yielded the promoter PAox1PM6-3d (shown in SEQ ID NO. 13). Recombinant bacteria using pPIC3.5k as the vector and regulated by the promoter PAox1PM6-3d to express EGFP were constructed according to the aforementioned method. These bacteria were cultured in BMDY medium containing different glucose concentrations at 30℃ and 220 rpm / min for 48 h. The results showed that… Figure 5 (D) PAox1PM6-3d is not only active under glucose induction at a concentration of 10 g / L, but also has a fluorescence intensity 2.8 times that of the wild-type promoter PGTH1, showing good glucose responsiveness.

[0053] Example 3: Expression of xylanase (XynA) in Pichia pastoris under glucose induction. The gene sequence encoding xylanase shown in SEQ ID NO. 14 was synthesized. The EGFP position of the pPIC3.5k-EGFP plasmid was replaced, and transcription was initiated by the PAox1PM6-3d promoter to construct the recombinant plasmid pPIC3.5K-PAox1PM6-3d-XynA. The recombinant plasmid was transformed into GS115Δku70 to obtain recombinant strain G1. Following the same method, the recombinant plasmid pPIC3.5K-PAox1-XynA, whose gene expression is regulated by the PAox1 promoter, was constructed. The pPIC3.5K-PAox1-XynA plasmid was linearized and transformed into Pichia pastoris GS115Δku70. ku Recombinant bacteria P1 was obtained from 70.

[0054] The recombinant strain G1 was cultured in YPD medium at 30°C and 220 rpm / min until the OD600 reached 0.5. Then, glucose was added to a final concentration of 2 g / L, and the culture was induced at 30°C and 220 rpm / min for 12 h. Subsequently, the glucose concentration was increased by 2 g / L every 6 h until the glucose concentration in the fermentation system reached 10 g / L. The culture was continued at 30°C and 220 rpm / min for a total of 168 h. The control strain P1 was cultured in YPD medium at 30°C until the OD600 reached 6, and then induced with methanol at a final concentration of 10 g / L at 30°C and 220 rpm / min for a total of 168 h.

[0055] The results show that ( Figure 6 A) The titer of xylanase (XynA) produced by PAox1PM6-3d regulation was 462.3 U / mL, which is 3.98 times that of xylanase regulated by PAox1 (116 U / mL).

[0056] Example 4: Expression of Streptomyces mogulata glutamine transaminase (TGase) in Pichia pastoris under glucose induction. The TGase enzyme shown in SEQ ID NO. 15 was synthesized and constructed into the pPIC3.5k-EGFP plasmid according to the method in Example 3. The EGFP gene was replaced, and the expression of TGase was regulated by the promoter PAox1PM6-3d to obtain the recombinant plasmid pPIC3.5K-PAox1PM6-3d-TGase. This plasmid was then transformed into GS115Δku70 to obtain the recombinant strain G2. Following the same method, the recombinant plasmid pPIC3.5K-PAox1-TGase, whose gene expression is regulated by the promoter PAox1, was constructed. The pPIC3.5K-PAox1-TGase plasmid was linearized and transformed into Pichia pastoris GS115Δku70. ku Recombinant bacteria P2 was obtained from 70.

[0057] The engineered strain obtained was cultured in YPD medium at 30°C and 220 rpm / min until the OD600 reached 0.5. Then, it was induced with glucose at a final concentration of 2 g / L at 30°C and 220 rpm / min for 12 h. After that, the glucose concentration was increased by 2 g / L every 6 h until the glucose concentration in the fermentation system reached 10 g / L. The fermentation was continued at 30°C and 220 rpm / min for a total of 168 h.

[0058] The control strain P2 was cultured in YPD medium at 30℃ and 220 rpm / min until the OD600 reached 6. Then, it was induced with methanol at a final concentration of 10 g / L at 30℃ and 220 rpm / min for 168 h.

[0059] The results show that ( Figure 6 B), the glutamine transaminase (TGase) titer regulated by the PAox1PM6-3d promoter was 4.12 U / mL, which is 1.65 times the TGase titer regulated by the PAox1 promoter (2.5 U / mL).

[0060] Example 5: Expression of snowflake antifreeze protein (SfAFP) in Pichia pastoris under glucose induction. The antifreeze protein of *Salvia hyrax* shown in SEQ ID NO. 16 was synthesized and constructed into the pPIC3.5k-EGFP plasmid according to the method in Example 3. The EGFP gene was replaced, and the expression of SfAFP was regulated by the promoter PAox1PM6-3d, resulting in the recombinant plasmid pPIC3.5k-PAox1PM6-3d-SfAFP. This plasmid was transformed into GS115Δku70 to obtain recombinant strain G3. Following the same method, the recombinant plasmid pPIC3.5k-PAox1-SfAFP, with gene expression regulated by the promoter PAox1, was constructed. The pPIC3.5k-PAox1-SfAFP plasmid was linearized and transformed into *Pichia pastoris* GS115Δku70. ku Recombinant bacteria P3 was obtained from 70.

[0061] The engineered strain obtained was cultured in YPD medium at 30°C and 220 rpm / min until the OD600 reached 0.5. It was then induced with glucose at a final concentration of 2 g / L at 30°C for 12 h. The glucose concentration was then increased by 2 g / L every 6 h until the glucose concentration in the fermentation system reached 10 g / L. The fermentation was continued at 30°C and 220 rpm / min for a total of 168 h.

[0062] The control strain P3 was cultured in YPD medium at 30℃ and 220 rpm / min until the OD600 reached 6. Then, it was induced with methanol at a final concentration of 10 g / L at 30℃ and 220 rpm / min for 168 h.

[0063] The results show that ( Figure 6 C), the titer of snow flea antifreeze protein (SfAFP) regulated by the promoter PAox1PM6-3d was 0.2 g / L, which was 3.33 times that of PAox1-induced SfAFP (0.06 g / L).

[0064] The results of Examples 3-5 show that this fermentation strategy enables the target protein titer to reach its peak on day 3 or 4, exceeding the total yield of proteins fermented for 7 days under PAox1 promoter regulation. This demonstrates that the PAox1PM6-3d promoter can achieve high expression levels in a shorter culture time, confirming its ability to rapidly and efficiently produce proteins.

[0065] Example 6: Preparation of xylanase by fermentation in a 5L bioreactor The recombinant strain G1 constructed in Example 3 was used for fermentation using a glucose-restricted fed-batch fermentation method. The specific steps were as follows: a single colony was picked and inoculated into 30 ml of YPD medium and cultured at 30°C and 220 rpm for 24 h. A 1% amine inoculum was transferred to a 1 L shake flask containing 200 ml of YPD medium and cultured at 30°C and 220 rpm for 24 h to obtain the seed culture. The seed culture was then inoculated into an inorganic salt medium to achieve an OD600 of 0.2. A two-stage glucose-fed fermentation strategy was employed to maintain promoter induction and minimize activity loss. The first stage (from inoculation to 15 h of fermentation) involved adding 10 g / L glycerol as a carbon source and a 20 g / L glucose solution containing 12 ml / L PTM1, fed linearly at a rate of 9.1 mg / h / L. Phase 2: From hour 16 to 24, the feed rate was adjusted to maintain the glucose concentration at 1 g / L to 5 g / L. The glucose concentration was then increased to 10 g / L and maintained at this concentration until the end of the fermentation process. The fermentation temperature was controlled at 30℃ for 120 hours. Glucose levels were monitored every 3 hours; enzyme activity was measured every 12 hours. Results showed that xylanase regulated by the PAox1PM6-3d promoter reached a titer of 2939.0 U / L within 70 hours.

[0066] Comparative Example 1: Methanol-based fed-batch fermentation for xylanase expression The recombinant strain P1 constructed in Example 3 was used for fermentation. Fermentation was carried out using methanol-fed batch fermentation, with fermentation parameters similar to those in Example 6, except that the OD600 value at inoculation was 1. The methanol induction method was as follows: The culture was first carried out in an inorganic salt medium containing 10 g / L glycerol at 30°C for 48 h. Subsequently, dissolved oxygen (DO)-triggered glycerol-fed batch culture was performed using glycerol at a concentration of 500 g / L (w / v) containing 12 ml / L PTM1 trace element. The feeding rate started at 9.1 mL / h / L and increased to 18.6 mL / h within 2-3 h. Then, methanol was fed in continuously to maintain a methanol concentration of 10 g / L. Glucose levels were monitored every 3 h; enzyme activity was measured every 12 h. The results showed that xylanase activity reached 1126 U / L after 70 h of fermentation and 1036.7 U / L after 7 days of fermentation.

[0067] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A glucose-responsive cis-acting element GRACE, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

3.

2. A promoter containing the sequence shown in SEQ ID NO.

3.

3. The promoter according to claim 3, characterized in that, The promoter is PAox1PM6-3d, and its nucleotide sequence is shown in SEQ ID NO.

13.

4. A polynucleotide containing the promoter of claim 2 or 3, characterized in that, The polynucleotide is a gene expression cassette or a recombinant plasmid.

5. The polynucleotide according to claim 4, characterized in that, The plasmids include, but are not limited to, pPIC sequence plasmids.

6. Recombinant microorganisms, characterized in that, It contains the polynucleotide as described in claim 4 or 5.

7. The recombinant microorganism according to claim 6, characterized in that, Using Pichia pastoris as a host, the promoter is applied to regulate the expression of the target protein; the target protein includes, but is not limited to, xylanase, transglutaminase, and snow flea antifreeze protein.

8. A method for regulating the expression of a target protein, characterized in that, The gene encoding the target protein is linked downstream of the promoter described in claim 2 or 3, the recombinant bacteria expressing the target protein are cultured in a culture medium, and protein expression is induced with glucose.

9. The method according to claim 8, characterized in that, The target protein includes, but is not limited to, xylanase, transglutaminase, and snow flea antifreeze protein; the concentration of glucose in the culture medium is 1~10 g / L.

10. The application of the promoter of claim 1 or 2 or the recombinant microorganism of claim 6 or 7 in the field of fermentation.

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