A high-yield brazzein sweet protein pichia pastoris engineering strain and a construction method and application thereof

Abstract

The application discloses a Pichia pastoris engineering strain for high-yield Brazzein sweet protein and a construction method and application thereof, and belongs to the technical field of biotechnology. The application discloses a Pichia pastoris engineering strain for high-yield Brazzein sweet protein and a construction method and application thereof, and belongs to the technical field of biotechnology. The application constructs a recombinant yeast strain containing a Brazzein gene, wherein the Brazzein gene is derived from Pentadiplandra brazzeana The amino acid sequence of the protein coded by the Brazzein gene is SEQ ID No: 1. The Brazzein gene is integrated at multiple copies and multiple sites, and the promoter and signal peptide element are optimized and combined, and Pp PDI , Pp BIP and Pp ERO1 chaperone proteins are co-expressed to promote disulfide bond formation and correct folding, thereby effectively improving the secretion efficiency and yield of the target protein, and having an industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Pichia pastoris engineered strain that produces high levels of Brazzein sweet protein, its construction method, and its application. Background Technology

[0002] Brazzein is a plant from Africa Pentadiplandra brazzeana Brazzein, a naturally occurring sweet protein isolated from plants, exists in two forms: 53 and 54 amino acids, with a molecular weight of approximately 6.5 kDa. Structurally, it consists of an α-helix and three β-sheets, and contains four disulfide bonds, contributing to its stable structure and thus exhibiting strong protein stability. Furthermore, brazzein is approximately 2000 times sweeter than sucrose and possesses advantages such as good flavor, high water solubility, non-cariogenicity, and no glycemic reaction, making it a highly promising novel natural sweetener. However, brazzein is present in extremely low amounts in plants, and extraction costs are high, hindering large-scale application. Therefore, heterologous production of brazzein using microbial cell factories has become a research hotspot.

[0003] Pichia pastoris, as a methanol-enabled eukaryotic expression system, is widely recognized as one of the most mature and efficient eukaryotic exogenous protein expression platforms. It possesses advantages such as protein processing and modification capabilities, high-density fermentation, and high expression levels. Furthermore, Pichia pastoris can efficiently secrete recombinant proteins into extracellular culture media, which not only simplifies downstream purification processes but also avoids degradation of the target protein by intracellular proteases. Therefore, it has been widely used in the industrial production of exogenous proteins.

[0004] Despite the advantages mentioned above, the Pichia pastoris expression system still faces a series of technical challenges when expressing Brazzein using this system, such as low yield and lower sweetness compared to natural proteins. Therefore, effectively increasing yield, improving the correct folding and secretion efficiency of disulfide-rich Brazzein proteins, and constructing stable, high-yield engineered strains of Brazzein with high sweetness are of significant economic and industrial importance. Summary of the Invention

[0005] The main problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a Pichia pastoris engineered strain that produces high levels of Brazzein protein, its construction method and application.

[0006] To address the aforementioned problems, this invention provides a recombinant yeast strain containing the Brazzein gene, which is derived from... Pentadiplandra brazzeana .

[0007] Furthermore, the Brazzein gene encodes a Brazzein protein, the amino acid sequence of which is SEQ ID No:1.

[0008] To facilitate the purification or detection of Brazzein protein, a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No:1 in the sequence listing.

[0009] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0010] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0011] Those skilled in the art can readily mutate the nucleotide sequence encoding the Brazzein protein of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that possess 75% or more of the nucleotide sequence identity with the Brazzein protein isolated in this invention, provided they encode and function as Brazzein, are derived from and equivalent to the nucleotide sequence of this invention.

[0012] Furthermore, the recombinant yeast strain may contain 3, 6, or 9 copies of the Brazzein gene.

[0013] The present invention also provides biomaterials related to the Brazzein protein described above, wherein the biomaterials may be any of the following: c1) The nucleic acid molecule that encodes the Brazzein protein described above; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3).

[0014] In a specific embodiment, the nucleotide sequence of the Brazzein gene is SEQ ID No:2, SEQ ID No:3, or SEQ ID No:4.

[0015] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, they may be vectors such as pZQC-PNSI-2-3Brazzein, pZQC-PNSⅡ-5-3Brazzein, pZQC-PNSⅣ-9-3Brazzein, pZQC-PNSⅢ-10-ERO1, pZQC-PNSⅡ-4-PDI, and pZQC-PNSI-14-BIP.

[0016] In one specific embodiment, the Brazzein gene can be expressed by any of the following promoters: 1) P with nucleotide sequence SEQ ID No:5 AOX1 promoter; 2) P with nucleotide sequence SEQ ID No:6 DAS2 promoter; 3) P with nucleotide sequence SEQ ID No:7 DAS1 Promoter.

[0017] Furthermore, the Brazzein gene is also fused with any of the following signal peptides: 1) The α-mating factor signal peptide (α-peptide) with the nucleotide sequence SEQ ID No:8; 2) The nucleotide sequence of the SP0030 peptide is SEQ ID No:9.

[0018] The expression box described in this article can be as follows: I:P AOX1 α-Mating factor signal peptide – Braz opt1 (SEQ ID NO:2) T AOX1 II: P DAS2 α-Mating factor signal peptide – Braz opt2 (SEQ ID NO:3) T CYC1 III: P DAS1 SP0030 signal peptide – Braz opt3 (SEQ ID NO:4) T TEF1 .

[0019] The recombinant microorganisms described in this article may be recombinant Pichia pastoris.

[0020] To reduce the degradation of the target protein and improve its secretion efficiency, co-expression of chaperone proteins containing accessory protein folds (such as Pp) can also be used. PDI Pp BIP Pp ERO1 (etc.) to relieve endoplasmic reticulum stress.

[0021] In this article, the recombinant yeast strain also contains a chaperone protein gene that helps with protein folding; the chaperone protein gene may be any of the following: Pp PDI ,Pp BIP Pp ERP1 Pp AHA1 Pp GPX1 ,Pp HAC1 ,Pp IRE1 ,Pp SEC53 ,Pp YPT6 ,Pp HRD1 ,Pp UBC1 ,Pp BRF2 ,Pp BMH2 ,Pp SED4 ,Pp SLY1 ,Pp SSA1 ,Pp SSA4 ,Pp SSO2 ,Pp WSC4 ,Pp YDJ1 .

[0022] In one specific embodiment, P can be selected. pPDI Pp BIP Pp ERO1 Genes were co-expressed to co-express the Pichia pastoris endogenous chaperone protein Pp. PDI ,Pp BIP and Pp ERO1 It helps Brazzein form the correct disulfide bonds (Brazzein contains 4 disulfide bonds) to increase the production of Brazzein protein and improve secretion efficiency.

[0023] The Pp PDI The nucleotide's GenBank number is XM_002494247; update date PLN 17-FEB-2023; the Pp BIP The nucleotide's GenBank number is AY965684; update date PLN 12-SEP-2005; the Pp ERO1 The nucleotide's GenBank number is XM_002489600; updated 17-FEB-2023.

[0024] The Pp PDI Pp BIP Pp ERO1 Genes can be generated by P AOX1 P GAP P FLD1 Expression is driven by promoters or gene promoters themselves.

[0025] The Pp PDI Pp BIP Pp ERO1 The gene expression vector can be: pZQC-P AOX1 -Pp PDI pZQC-P GAP -Pp PDI pZQC-P FLD1 -Pp PDI pZQC-P PDI -Pp PDI ;pZQC-P AOX1 -Pp BIP pZQC-P GAP -Pp BIP pZQC-P FLD1 -Pp BIP pZQC-P BIP -Pp BIP ;pZQC-P AOX1 1-Pp ERO1 pZQC-P GAP -Pp ERO1 pZQC-P FLD1 -Pp ERO1 pZQC-P PDI -Pp ERO1 .

[0026] The terminator can be: T AOX1 T CYC1 or T TEF1 .

[0027] The Brazzein protein and chaperone protein genes are integrated into the recipient yeast strain at the following sites: PNSI-2, PNSI-9, PNSI-13, PNSI-14, PNSⅡ-4, PNSⅡ-5, PNSⅡ-6, PNSⅣ-9, PNSⅢ-4, or PNSⅢ-10.

[0028] Preferably, the integration sites of the Brazzein protein are neutral sites such as PNSI-2, PNSⅡ-5, and PNSⅣ-9.

[0029] Preferably, the integration site of the chaperone protein is a neutral site of PNSⅡ-4, PNSI-14, or PNSⅢ-10.

[0030] In this study, Ura3-deficient yeast monoclonal strains were used as recipient strains for multiple rounds of gene modification. Through optimized combinations of multiple copies, multiple sites, and multiple regulatory elements (different promoters, signal peptides, and terminators), the recombinant yeast strains were obtained.

[0031] This invention also provides the application of the recombinant yeast strain described above in the production of Brazzein.

[0032] The present invention also provides a method for producing Brazzein protein, comprising fermentation using the recombinant yeast strain described above to obtain the Brazzein protein.

[0033] The fermentation process conditions can be as follows: 1) The initial culture medium was BSM (phosphate buffer system) with a glycerol concentration of 40 g / L. The recombinant strain was inoculated at 10% concentration, at 30°C, pH 5.5 (adjusted with ammonia), and dissolved oxygen (DO) was maintained at 20% through stirring and aeration. 30%; 2) After glycerol is depleted, the glycerol feeding stage begins (50% glycerol, containing PTM1), and the flow rate is adjusted according to DO feedback; 3) Once the cell wet weight reaches 180 g / L, stop feeding, starve the cells for 30 minutes, and then begin methanol induction (100% methanol, containing PTM1), controlling the methanol concentration at 0.5%. 1.0% (methanol electrode online monitoring). Induction ended after 96 hours.

[0034] This invention integrates the Brazzein gene through multiple copies and at multiple sites, and optimizes the combination of promoter and signal peptide elements, while co-expressing Pp. PDI ,Pp BIP and Pp ERO1 Chaperone proteins promote disulfide bond formation and proper folding, thereby effectively improving the secretion efficiency and yield of the target protein. This invention also provides a fed-batch methanol-induced fermentation process based on this engineered strain and its application in the production of Brazzein protein.

[0035] The present invention has the following beneficial effects: 1) Significantly increased expression levels: Through the optimized combination of multiple copies, multiple sites, and multiple regulatory elements (different promoters, signal peptides, and terminators), the instability caused by homogeneous repetitive sequences was avoided, and the expression level of Brazzein was significantly increased.

[0036] 2) Genetic stability of the strain: Site-directed integration using neutral sites avoids the positional effects and copy number loss of random integration.

[0037] 3) Improved protein folding efficiency: through co-expression of chaperone protein (Pp) PDI Pp BIP Pp ERO1 (etc.), which relieved endoplasmic reticulum stress, reduced the degradation of target proteins, and improved secretion efficiency.

[0038] 4) The process can be scaled up: The developed 5 L tank fermentation process lays the foundation for industrial production. Attached Figure Description

[0039] Figure 1 A schematic diagram of the constructed vector pZQC-PNSI-2-3Brazzein spectrum.

[0040] Figure 2 A schematic diagram of the constructed vector pZQC-PNSⅡ-5-3Brazzein spectrum.

[0041] Figure 3 A schematic diagram of the constructed vector pZQC-PNSⅣ-9-3Brazzein spectrum.

[0042] Figure 4 The vector pZQC-P was constructed AOX1 -Pp PDI A schematic diagram of the spectrum.

[0043] Figure 5 The vector pZQC-P was constructed AOX1 -Pp BIP A schematic diagram of the spectrum.

[0044] Figure 6 The constructed vector pZQC-P AOX1 -Pp ERO1 A schematic diagram of the spectrum.

[0045] Figure 7 The results of colony PCR verification for 3-copy strains show Brazzein integration at neutral sites 4-9. Lanes 1-16 show clones selected for colony PCR verification.

[0046] Figure 8 The results of colony PCR verification for 1-2 neutral sites integrating Brazzein are shown. Lanes 1-24 are clones selected for colony PCR verification.

[0047] Figure 9 The results of colony PCR verification for 3-copy strains show Brazzein integration at 2-5 neutral sites. Lanes 1-24 show clones selected for colony PCR verification.

[0048] Figure 10A comparison of protein expression levels for strains with 3, 6, and 9 copies.

[0049] Figure 11 Image showing the SDS-PAGE results of Brazzein-induced expression in engineered strain (96 h).

[0050] Figure 12 The expression levels of the overexpressed chaperone protein in the 9-copy strain were compared with those in the 6-copy strain.

[0051] Figure 13 The results of Brazzein proteomic analysis are shown, where A represents peptide coverage and B represents raw mass spectrometry data.

[0052] Figure 14 This is a BSA protein detection kit. The linear relationship between BSA protein concentration and absorbance is shown. Detailed Implementation

[0053] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0055] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0056] Pichia pastoris in the following examples ( Pichia pastoris GS115 was purchased from Baosai Biotechnology (product number: YC243).

[0057] The plasmid pUC19 used in the following examples is a product from the Novizan ClonExpress Ultra One Step Cloning Kit V3 kit, catalog number C117-01 / 02.

[0058] For details on the preparation of the culture medium and the culture of yeast cells in the following examples, please refer to the Invitrogen™ User Guide: Pichia Expression Kit (Source: https: / / www.thermofisher.cn / order / catalog / product / K171001# / K171001).

[0059] The primer information used in the following examples is shown in Table 1 below.

[0060] Table 1. Primer information used in this invention

[0061] The following examples use statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0062] Example 1: Codon optimization and expression cassette construction of the Brazzein gene 1. Brazzein three-copy expression cassette gene sequence According to Pichia pastoris ( Pichia pastoris Using the codon frequency table of GS115, three different codon translation optimizations were performed on the brazzein gene without altering the amino acid sequence (SEQ ID No:1). This resulted in codon-optimized sequence A (SEQ ID No:2), codon-optimized sequence B (SEQ ID No:3), and codon-optimized sequence C (SEQ ID No:4). All three sequences were obtained through whole-genome synthesis.

[0063] The brazein gene expression cassette (nucleotide sequence SEQ ID No: 10) was obtained through gene synthesis: P AOX1 – α-Mating factor signal peptide – Braz opt1– T AOX1 P DAS2 – α-Mating factor signal peptide – Braz opt2–T CYC1 P DAS1 – SP0030 signal peptide – Braz opt3– T TEF1The frame was then integrated into the pPICZA vector (nucleotide sequence SEQ ID No:38).

[0064] 2. Obtain DNA fragments from the 5' and 3' homologous arms of the neutral sites PNSI-2, PNSⅡ-5, and PNSⅣ-9. Using the genome of GS115 WT strain as a template, the 5' homologous arm sequences and 3' homologous arm sequences of neutral sites PNSI-2, PNSⅡ-5, and PNSⅣ-9 were amplified. The above fragments were separated by 1% (w / v) agarose gel electrophoresis and purified using a gel DNA mini-recovery kit (Tiangen).

[0065] The primers used to amplify the neutral sites PNSI-2, PNSⅡ-5, and PNSⅣ-9 are: The PNSI-2 5' homologous arm sequence was amplified using Neu1-2 5'TYB and Neu 1-2 5'TYB overlap AOX1 R; The PNSI-23' homologous arm was amplified using the Neu 1-2 3' TYB overlap TEF terminator and Neu 1-2 3 TYB R. The 5' homologous arm of PNSⅡ-5 was amplified using Neu 2-5 5'TYB overlap ori F and Neu 2-5 5'TYB overlap PAOX1 R; The 3' homologous arm of PNSⅡ-5 was amplified using Neu 2-5 3'TYB overlap T TEFF and Neu 2-5 3'TYB overlap T TEFF R. The PNSⅣ-95' homologous arm was amplified using Neu 4-9 5'TYB overlap ori F and 4-9 5'TYBoverlap PAOX1 R; The PNSⅣ-9 3' homologous arm was amplified using 4-9 3' TYB overlap TEEF and Neu 4-9 overlap PTEF1.

[0066] 3. Obtain the autonomous replication sequence of E. coli plasmid and the bleomycin resistance DNA fragment. Plasmid P was extracted using a plasmid extraction kit (Tiangen). UC19 ( Seamless Cloning Kit, Novizan, catalog number C117-01 / 02 includes a built-in plasmid. ）Using this as a template, the E. coli autonomous replication sequence (PUC ori) and the bleomycin (BleoR) expression cassette sequence (nucleotide sequence SEQ ID No: 10) were amplified using the high-fidelity enzyme 2xphanta mix (Novizan P515). The above fragments were separated by 1% (w / v) agarose gel electrophoresis and purified using a gel DNA mini-recovery kit.

[0067] 4. Obtain URA3 DNA fragments of genes Using the genome of GS115 WT strain as a template and Ura3 F / Ura3 R as primers, amplification was performed separately. URA3 The DNA fragments of the gene were separated by 1% (w / v) agarose gel electrophoresis and purified using a gel DNA mini-recovery kit (Tiangen).

[0068] 5. Assemble the pZQC-Brazzein vector Using a seamless cloning kit (Novazia, catalog number C117-01 / 02), the above-mentioned homologous arm fragments at different neutral sites and the three-copy Brazzein expression frame fragments were assembled and ligated to the autonomous replication sequence of the E. coli plasmid and the bleomycin-resistant DNA fragments, respectively, to form the vector pZQC-PNSI-2-3Brazzein (… Figure 1 ), pZQC-PNSⅡ-5-3Brazzein ( Figure 2 ), pZQC-PNSⅣ-9-3Brazzein ( Figure 3 ).

[0069] The recombinant vector pZQC-PNSI-2-3Brazzein is described as follows: It is a recombinant vector with an autonomously replicating sequence (PUC ori, SEQ ID No:25) and bleomycin resistance (BleoR, SEQ ID No:26) as the backbone, with PNSI-2 3' homologous arm (SEQ ID No:28) and PNSI-2 5' homologous arm (SEQ ID No:29) connected to both sides respectively, and a sequence expression cassette sequence (DNA fragment of SEQ ID No:10), URA3 sequence (SEQ ID No:27), and a repeated PNSI-2 5' homologous arm sequence inserted between the two homologous arm sequences, and seamlessly cloned and ligated.

[0070] The structure of the recombinant vector pZQC-PNSⅡ-5-3Brazzein is described as follows: It is a recombinant vector obtained by seamless cloning and ligation of an autonomously replicating sequence (PUCori, SEQ ID No:25) and bleomycin resistance (BleoR, SEQ ID No:26) as a backbone, consisting of 5'-BleoR-3' (SEQ ID No:26) and 5'-PUCori-3' fragments (SEQ ID No:25), respectively. The 3' homologous arm (SEQ ID No:30) and 5' homologous arm (SEQ ID No:31) of PNSⅡ-5 are connected to the two sides, respectively. The sequence expression cassette sequence (DNA fragment of SEQ ID No:10), the URA3 sequence (SEQ ID No:27), and the repeated PNSI-25' homologous arm sequence are inserted between the two homologous arm sequences.

[0071] The structure of the recombinant vector pZQC-PNSⅣ-9-3Brazzein is described as follows: It is a recombinant vector obtained by seamless cloning and ligation of an autonomously replicating sequence (PUCori, SEQ ID No:25) and bleomycin resistance (BleoR, SEQ ID No:26) as a backbone, with the 3' homologous arm (SEQ ID No:32) and 5' homologous arm (SEQ ID No:33) of PNSⅣ-9 attached to both sides, and a sequence expression cassette sequence (DNA fragment of SEQ ID No:10), URA3 sequence (SEQ ID No:27), and a repeated 5' homologous arm sequence of PNSI-2 inserted between the two homologous arm sequences.

[0072] 6. Construction of Brazzein engineered strains and acquisition of homologous repair fragments. Using the plasmids pZQC-PNSI-2-3Brazzein, pZQC-PNSⅡ-5-3Brazzein, and pZQC-PNSⅣ-9-3Brazzein obtained in step 5 as templates, and employing the high-fidelity enzyme 2×phanta mix (Novizan P515), homologous recombination fragments targeting the PNSI-2 site (5' homologous arm - 3-copy Brazzein expression cassette - PNSI-2 3' homologous arm), the PNSⅡ-5 site (5' homologous arm - 3-copy Brazzein expression cassette - PNSⅡ-5 3' homologous arm), and the PNSⅣ-9 site (5' homologous arm - 3-copy Brazzein expression cassette - PNSⅣ-9 3' homologous arm) were amplified using primers with the 5' and 3' homologous arms of the neutral sites PNSI-2, PNSⅡ-5, and PNSⅣ-9, respectively. These fragments were used to construct a 9-copy Brazzein expression strain.

[0073] 7. Construction of multiple copy strains of Pichia pastoris Brazzein Ura 3 - Preparation of defective yeast: URA3 was knocked out via homologous recombination. The upstream fragment of URA3 was amplified using primers PP-26 and PP-29; the downstream homologous arm of URA3 was amplified using PP-19 and PP-25; and the knockout fragment (nucleotide sequence SEQ ID No: 34) was amplified by fusion PCR using primers PP-25 and PP-26. The fusion fragment was then transformed into Pichia pastoris GS115 via electroporation. Positive clones were screened using 5FOA plates (YNB 0.17%, ammonium sulfate 0.5%, D-(+)-glucose 2%, casein amino acids 0.5%, agar 2%, 5-fluoroorotic acid (5-FOA) 0.1%, uracil stock solution (0.5 mg / mL)). Positive clones were further screened using PP-23 and PP-24. Finally, the target Ura was obtained. 3 - Clones.

[0074] With Ura 3 - The defective yeast monoclonal strain is a basal species. The steps for constructing a Brazzein triple-copy strain are as follows: (1) Pick fresh and healthy Ura from the plate 3 - Defective yeast clones were inoculated into 50 mL of YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) and cultured overnight at 220 rpm and 30°C. (2) To be cultured to OD 600 When the bacterial culture reaches 1-2, transfer the bacterial culture to a sterile 50 mL centrifuge tube, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, and collect the bacterial cells; (3) Add 30 mL of lithium acetate sorbitol Tris mixture (LST buffer) to the collected bacterial cells for resuspending, and let stand at 30°C for 30 minutes; centrifuge the bacterial solution at 5000 rpm for 5 minutes after standing, and remove the supernatant; (4) The precipitated bacterial cells were resuspended in 10 mL of pre-cooled sorbitol solution, centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded; step (4) was repeated twice. (5) Add about 400 µL of pre-cooled sorbitol solution to the last collected cells, carefully resuspend, and complete the preparation of Pichia pastoris competent cells. Dispense 80 µL / tube into sterile pre-cooled EP tubes for later use (make and use immediately). (6) Add 2 µg of homologous recombination fragments (total volume not exceeding 20 µl) obtained from step 6 above at neutral sites PNSI-2, PNSⅡ-5, and PNSⅣ-9 to 80 µL of Pichia pastoris competent cells, gently blow them evenly with a pipette, and incubate on ice for 5 minutes. (7) Add competent cells after mixing recombinant fragments into pre-cooled electrode cups (0.1 cm apart); set the voltage of the electroporator to 780 V, place the electrode cups into the electroporator, and prepare 1 mL of pre-cooled sorbitol solution in advance; (8) Click the start button of the electric transducer and record the voltage and electric shock time; (9) Remove the electrode cup, add 1 mL of the pre-cooled sorbitol solution prepared in advance, and mix thoroughly by blowing and stirring. (10) Place the mixed bacterial solution in a 30℃ incubator and let it stand for 1.5 hours; The above bacterial cultures were spread in different gradients onto SC-Ura agar plates (0.67% YNB, 2% glucose, 0.077% CSM-Ura (or 0.77 g / L), 2% agar) without uracil, and incubated at 30°C for 2-3 days. After single colonies grew, PCR verification was performed using primer Neu4-9 3'TYB outer R2 and primer TEF terminator internal F2 to screen for positive clones (e.g., Figure 7 The clones that obtained the target band in the PCR results were positive clones. They integrated the Brazzein gene expression cassette with three copies at the neutral site PNSⅣ-9, and finally obtained the Brazzein 3-copy strain.

[0075] (11) Inoculate the positive clones that were verified in the previous step into YPD and culture them overnight.

[0076] (12) The overnight culture was spread on SC+Ura medium (SC-Ura base, with uracil added to 50 mg / L and 5-FOA to 1 mg / mL) containing 5-FOA (5-fluoroorotic acid) and uracil and cultured. Clones were picked to obtain the marker-recovered strains for the next step of strain construction.

[0077] (13) Following the same procedure as above, based on the three-copy strain overexpressed at the neutral site PNSⅣ-9, three copies of the Brazzein expression cassette were further overexpressed at the neutral site PNSI-2. PCR verification was performed using primer Neu1-2 3'TYB upstream R2 and primer TEF terminator internal F2 to screen for positive clones. The selected clones were incubated at 30℃ for 2-3 days. After single clones grew, genotyping was performed to obtain a 6-copy strain (e.g., ...). Figure 8 Clones that produce the target band in the PCR results are considered positive clones.

[0078] (14) Following the same procedure as above, based on the 6-copy strain with neutral site PNSⅣ-9 and PNSI-2, overexpress a 3-copy Brazzein expression cassette at the neutral site PNSⅡ-5. PCR verification was performed using primer Neu2-5 3' external homologous arm R2 and primer TEF terminator internal F2. Positive clones were screened, and the selected clones were incubated at 30℃ for 2-3 days. After single clones grew, genotyping was performed to obtain a 9-copy strain (e.g., ...). Figure 9 Clones that produce the target band in the PCR results are considered positive clones.

[0079] Example 2: Detection of Brazzein 3, 6, and 9 copy strain protein expression and SDS-PAGE Tested strains: Brazzein 3, 6, 9 copies, positive strain Brazzein 1-46 (2 copies), negative strain GS115.

[0080] 1. High-throughput expression induced by deep-well plates (1) Pre-culture: From freshly activated YPD plates, pick uniform single colonies with a sterile toothpick or 10 μL pipette tip, and inoculate them into 6 mL of BMGY medium (1% yeast extract, 2% peptone, 1% glycerol, 1.34% yeast basal nitrogen source (YNB), 100 mM potassium phosphate buffer (pH 6.0), sterilize at 115℃ for 25 min, and add 4 × 10⁻⁶ filtered sterile medium before use. -5 24-well deep-well plates (one clone per well) containing % biotin were used. The plates were covered with a breathable sealing membrane and incubated at 30 °C with shaking at 250 rpm for 18 days. 20 h, to OD 600 Reaching 4-6.

[0081] (2) Collection and resuspension of bacterial cells: Centrifuge the deep well plate at 5000 rpm for 5 min and discard the supernatant. Add 6 mL of sterile deionized water to each well, wash the bacterial cells by pipetting up and down 10 times, centrifuge again at 5000 rpm for 5 min and discard the supernatant.

[0082] (3) Transfer to BMMY induction medium: Add 6 mL of BMMY medium (1% yeast extract, 2% peptone, 1.34% YNB, 100 mM potassium phosphate buffer (pH 6.0)) to each well of the precipitate, sterilize at 115°C for 25 min, and add 4 × 10⁻⁶ sterile saturated medium before use. -5 Add 0.5% biotin and 0.5% methanol (preheated to 30 °C), and gently agitate to completely resuspend the cells. Seal the deep-well plate with sealing film.

[0083] (4) Methanol induction: The deep-well plate was placed in a shaker at 30 ℃ and continuously incubated at 250 rpm. Every 24 h, the sealing film was removed in a clean bench and sterile methanol was added to each well to a final concentration of 0.5% (v / v). After adding methanol, the plate was resealed and induction continued.

[0084] (5) Sampling: At 96 h and 120 h after induction, 200 μL of bacterial culture was taken into 1.5 mL centrifuge tubes in a clean bench, centrifuged at 12,000 rpm for 3 min, and the supernatant was separated (for SDS). PAGE analysis).

[0085] 2. SDS PAGE detection of secreted proteins (1) Sample preparation: Take 400 μL of fermentation supernatant at each time point (96 h, 120 h), add 100 μL of 5× protein loading buffer (containing 100 mM DTT), mix well, boil for 10 min, centrifuge at 12,000 rpm for 2 min, and take the supernatant for later use.

[0086] (2) Electrophoresis: Prepare a 16% separating gel and a 5% stacking gel (Brazzein has a molecular weight of about 6.5 kDa, so a high-concentration separating gel is chosen to ensure good separation of small molecule proteins). Load 20 μL of sample into each well and electrophore at a constant voltage of 80 V until the bromophenol blue front reaches the bottom of the separating gel.

[0087] (3) Staining and destaining: After electrophoresis, the gel was placed in Coomassie Brilliant Blue R Stain with 250 staining solution (0.25% Coomassie Brilliant Blue, 45% methanol, 10% glacial acetic acid) for 1 hour with shaking, then transfer to destaining solution (10% methanol, 10% glacial acetic acid) until the background is clear.

[0088] (4) Image acquisition and analysis: Images were captured using a gel imaging system, and the target band (at approximately 6.5 kDa) was scanned in grayscale using ImageJ software.

[0089] The results are as follows Figure 10 As shown: comparing strains with 3, 6, and 9 copies, the expression level of Brazzein increased sequentially with the increase of copy number, proving that the high copy strategy can effectively enhance the expression of Brazzein.

[0090] Example 3: Construction of strains co-expressing chaperone proteins 1. Obtaining homologous recombination fragments Referring to the homologous recombination method in Example 1, using the genome of GS115 WT strain as a template, the fragment of the 5' homologous arm at the PNSI-14 position was amplified with 1 / 14 UPF and 1 / 14 UPR primers; the fragment of the 3' homologous arm at the PNSI-14 position was amplified with 1 / 14 down FP and 1 / 14 DOWN RP primers. The fragment of the 5' homologous arm of PNSⅢ-10 was amplified using 3 / 10 UPF and 3 / 10 UPR primers; the fragment of the 3' homologous arm of PNSⅢ-10 was amplified using 3 / 10 DOWN FP and 3 / 10 down RP primers, respectively. The fragment at the 5' homologous arm of PNSII-4 was amplified using 2 / 4 up FP and 2 / 4 up RP primers; the fragment at the 3' homologous arm of PNSII-4 was amplified using 2 / 4 down FP and 2 / 4 down RP primers. The above fragments were separated by 1% (w / v) agarose gel electrophoresis and purified using a gel DNA mini-recovery kit (Tiangen).

[0091] 2. Constructing a chaperone protein overexpression vector To construct combinations of chaperone protein overexpression with different promoter strengths, four different types of promoters were selected: P AOX1 P GAP P FLD1 And the native promoters of each overexpressed gene.

[0092] Promoters of different strengths and Pp PDI ,Pp BIP ,Pp ERO1 Combined and connected; different types of combined fragments are obtained: 1) First, using the genome of GS115 WT strain as a template, Pp was amplified. PDI Gene ORF+ terminator fragment (290 bp after ORF) (SEQ ID No: 11); amplification of Pp BIPGene ORF+ terminator fragment (199 bp after ORF) (SEQ ID No: 12); amplification of Pp ERO1 Gene ORF+ terminator fragment (300bp after ORF) (SEQ ID No: 13); 2) Using the genome of GS115 WT strain as a template, P was amplified separately. AOX1 (The nucleotide sequence is SEQ ID No:5, P) GAP (nucleotide sequence is SEQ ID No:14), P FLD1 (nucleotide sequence is SEQ ID No:15) and the native promoters (first 500 bp of ORF) of each overexpressed gene, native promoter P PDI The sequences are No:16, native promoter P. BIP The sequences are No:17, native promoter P. ERO1 The sequence number is No:18.

[0093] 3) Using a seamless cloning method, Pp PDI Pp BIP Pp ERO1 The gene ORF+ terminator fragment sequence is associated with the 5' and 3' homologous arms of the chromosomal neutral integration sites PNSⅡ-4, PNSI-14, and PNSⅢ-10 (corresponding to SEQ ID No: 19-24, respectively) and with P... AOX1 P GAP P FLD1 Various chaperone protein overexpression vectors were constructed by combining the native promoters of each overexpressed gene: pZQC-P AOX1 -Pp PDI pZQC-P GAP -Pp PDI pZQC-P FLD1 -Pp PDI pZQC-P PDI -Pp PDI pZQC-P AOX1 -Pp BIP pZQC-P GAP -Pp BIP pZQC-P FLD1 -Pp BIP pZQC-P BIP -Pp BIP pZQC-P AOX1 1-Pp ERO1 pZQC-P GAP -Pp ERO1 pZQC-P FLD1 -Pp ERO1 pZQC-PERO1 -Pp ERO1 .

[0094] Recombinant vector pZQC-P AOX1 -Pp PDI The structure is described as follows: It uses an autonomously replicating sequence (PUC ori, SEQ ID No:25) and bleomycin resistance (BleoR, SEQ ID No:26) as its backbone, with PNSⅡ-4 5' homologous arm (SEQ ID No:19) and PNSⅡ-4 3' homologous arm (SEQ ID No:20) connected to each side, and sequence P inserted between the two homologous arm sequences. AOX1 The promoter sequence (SEQ ID No:5) and PpPDI expression cassette sequence (SEQ ID No:35), the URA3 sequence (SEQ ID No:27), the repeated PNSI-2 5' homologous arm sequence, and the recombinant vector obtained by seamless cloning and ligation are detailed in the following documents. Figure 4 .

[0095] Recombinant vector pZQC-P GAP -Pp PDI pZQC-P FLD1 -Pp PDI pZQC-P PDI -Pp PDI and Recombinant vector pZQC-P AOX1 -Pp PDI The only difference is that the DNA fragment in SEQ ID No:5 is replaced with the DNA fragments in SEQ ID No:14, 15, and 16.

[0096] Recombinant vector pZQC-P AOX1 -Pp BIP The structure is described as follows: it uses an autonomously replicating sequence (PUC ori, SEQ ID No:25) and bleomycin resistance (BleoR, SEQ ID No:26) as a backbone, with PNSI-14 5' homologous arm (SEQ ID No:21) and PNSI-14 3' homologous arm (SEQ ID No:22) connected to each side, and P inserted between the two homologous arm sequences. AOX1 The promoter sequence (SEQ ID No:5) and PpBIP expression cassette sequence (SEQ ID No:36), URA3 sequence (SEQ ID No:27), the repeated PNSI-14 5' homologous arm sequence, and the recombinant vector obtained by seamless cloning and ligation are detailed in the following documents. Figure 5 .

[0097] Recombinant vector pZQC-P GAP -Pp BIP pZQC-PFLD1 -Pp BIP pZQC-P BIP -Pp BIP With recombinant vector pZQC-P AOX1 -Pp PDI The only difference is that the DNA fragment in SEQ ID No:5 is replaced with the DNA fragments in SEQ ID No:14,15, and17.

[0098] Recombinant vector pZQC-P AOX1 -Pp ERO1 The structure is described as follows: It uses an autonomously replicating sequence (PUC ori, SEQ ID No:25) and bleomycin resistance (BleoR, SEQ ID No:26) as its backbone, with PNSⅢ-10 5' homologous arms (SEQ ID No:23) and PNSⅢ-10 3' homologous arms (SEQ ID No:24) connected to each side, and a P... AOX1 The promoter sequence (SEQ ID No:5) and PpERO1 expression cassette sequence (SEQ ID No:37), URA3 sequence (SEQ ID No:27), the repeating PNSI-14 5' homologous arm sequence, and the recombinant vector obtained by seamless cloning and ligation are detailed in the following documents. Figure 6 .

[0099] Recombinant vector pZQC-P GAP -Pp ERO1 pZQC-P FLD1 -Pp ERO1 pZQC-P ERO1 -Pp ERO1 With recombinant vector pZQC-P AOX1 -Pp ERO1 The only difference is that the DNA fragment in SEQ ID No:5 is replaced with DNA fragments in SEQ ID No:14,15, and18.

[0100] Example 4: Chaperone protein Pp PDI ,Pp BIP and Pp ERO1 Stepwise construction of overexpression strains 1. Obtaining homologous recombination fragments pZQC-P AOX1 -Pp PDI pZQC-P GAP -Pp PDI pZQC-P FLD1 -Pp PDI pZQC-P PDI -Pp PDIpZQC-P AOX1 -Pp BIP pZQC-P GAP -Pp BIP pZQC-P FLD1 -Pp BIP pZQC-P BIP -Pp BIP pZQC-P AOX1 1-Pp ERO1 pZQC-P GAP -Pp ERO1 pZQC-P FLD1 -Pp ERO1 pZQC-P ERO1 -Pp ERO1 Using plasmids as templates, the high-fidelity enzyme Hieff Canace was used to amplify homologous recombination fragments using primers with the 5' and 3' homologous arms of neutral sites PNSⅡ-4, PNSI-14, and PNSⅢ-10 (primer pairs: 12new II-4PDI FP / 11new II-4PDI RP, 7new I-14BIP RP, 13newIII-10ERO1 RP / 14new III-10ERO1 FP, respectively). These fragments were used to construct strains that overexpress chaperone proteins in different combinations.

[0101] 2. Pichia pastoris Pp PDI Pp BIP Pp ERO1 Construction of strains (1) Following the procedure in step 7 of Example 1, Pp was overexpressed again at the neutral site PNSⅡ-4 on the basis of the 9-copy strain. PDI Incubate at 30℃ for 2-3 days. After single clones grow, perform genotyping verification to obtain Pp. PDI Overexpression strains.

[0102] (3) Following the same procedure described above, Pp was overexpressed again at the neutral site PNSI-14. BIP Incubate at 30℃ for 2-3 days. After single clones grow, perform genotyping verification to obtain Pp. BIP Overexpression strains.

[0103] (3) Following the same procedure as above, Pp was overexpressed again at the neutral site PNSⅢ-10. ERO1 Incubate at 30℃ for 2-3 days. After single clones grow, perform genotyping verification to obtain Pp. ERO1 Overexpression strains.

[0104] 3. SDS PAGE detection of secreted proteins (1) Sample preparation: Take 400 μL of fermentation supernatant at each time point, add 100 μL of 5× protein loading buffer (containing 100 mM DTT), mix well, boil for 10 min, centrifuge at 12,000 rpm for 2 min, and take the supernatant for later use.

[0105] (2) Electrophoresis: Prepare a 15% separating gel and a 5% stacking gel (Brazzein has a molecular weight of about 6.5 kDa, so a high-concentration separating gel is chosen to ensure good separation of small molecule proteins). Load 20 μL of sample into each well and electrophore at a constant voltage of 80 V until the bromophenol blue front reaches the bottom of the separating gel.

[0106] (3) Staining and destaining: After electrophoresis, the gel was placed in Coomassie Brilliant Blue R Stain with 250 staining solution (0.25% Coomassie Brilliant Blue, 45% methanol, 10% glacial acetic acid) for 1 hour with shaking, then transfer to destaining solution (10% methanol, 10% glacial acetic acid) until the background is clear.

[0107] (4) Image acquisition and analysis: Images were captured using a gel imaging system, and the target band (at approximately 6.5 kDa) was scanned in grayscale using ImageJ software.

[0108] The results are as follows Figure 11 and Figure 12 As shown, P BIP -Pp BIP P PDI -Pp PDI P GAP -Pp PDI, P FLD -Pp PDI Overexpression of the chaperone protein significantly increased Brazzein expression (>30%).

[0109] Example 4: High-density fermentation in a 5L fermenter 9 copies of Pp were overexpressed. PDI The strain was fermented in a 5L tank. The initial culture medium was BSM (phosphate buffered medium) with a glycerol concentration of 40 g / L. The inoculum size was 10%, the temperature was 30°C, the pH was 5.5 (adjusted with ammonia), and dissolved oxygen (DO) was maintained at 20% through stirring and aeration. 30%. After glycerol depletion, a glycerol feeding phase (50% glycerol, containing PTM1) was initiated, with the flow rate adjusted based on DO feedback. Feeding was stopped once the cell wet weight reached 180 g / L, followed by 30 minutes of starvation, and then methanol induction (100% methanol, containing PTM1) was started, maintaining a methanol concentration of 0.5%. 1.0% (methanol electrode online monitoring). Induction ended after 96 hours. The concentration calculation formula is: concentration after dilution (10-fold dilution) (unit: mg / ml) = (absorbance - 0.0152) / 1.0362.

[0110] At the end of fermentation, the cell wet weight reached 468 g / L, and the Brazzein yield, as determined by BCA analysis, was approximately 5.2 g / L. Figure 14 (and Table 2) demonstrate that the Brazzein engineered strain constructed in this invention has good prospects for industrialization.

[0111] Table 2. BCA concentration results in 5 L fermentation samples

[0112] After ultrafiltration, ion exchange, and lyophilization, the fermentation supernatant was used to determine the molecular weight of the product, which was consistent with the theoretical value. The peptide mass spectrometry identification steps are as follows: 1) Decolorization Clean the cutting plate and blade with 1 mL of ACN. After performing SDS-PAGE on the protein sample, cut the target band into 1 mm wide pieces and place them in EP tubes. Add 500 μL of Cochlear staining destaining solution (50 mM NH4HCO3 / 100% ACN = 1 / 1), and destain until colorless at 37°C and 650 rpm on a constant temperature mixer. Aspirate and discard the destaining solution, wash twice with 500 μL of ACN to dehydrate, and dry in a clean bench for 10 min.

[0113] 2) Reduction and alkylation Add 200 μL of 5 mM DTT solution and incubate at 45°C and 650 rpm for 30 min in a constant temperature mixer to reduce disulfide bonds. Discard the DTT solution, wash twice with 500 μL ACN to dehydrate, and dry in a clean bench for 10 min. Add 200 μL of 11 mM IAA solution (stored protected from light) and place in the dark for 20 min for alkylation to prevent the reopened disulfide bonds from recombinizing. Discard the IAA solution, wash twice with 500 μL ACN to dehydrate, and dry in a clean bench for 10 min.

[0114] 3) In-gel enzymatic hydrolysis Add an appropriate amount of Chymotrypsin enzyme solution to the protein sample, place it in a 4℃ refrigerator for 30 min to fully swell, and finally add Trypsin buffer (200 mM NH4HCO3 / 100% ACN / H2O=2 / 1 / 7) to 100-200 μL. Place it on a constant temperature mixer and incubate at 37℃ and 650 rpm for 16 h.

[0115] 4) Peptide elution Add 20 μL of 10% FA to the protein solution, and terminate the enzymatic digestion by shaking at 37°C and 650 rpm for 10 min on a thermostat. Transfer the solution to a new EP tube. Add 200 μL of peptide elution buffer (100% ACN / 0.2% FA = 1 / 1), and shake at 37°C and 650 rpm for 30 min on a thermostat. Combine the solutions. Repeat the extraction once, combine the eluents, and evaporate the eluent to dryness using a ZLS-1 vacuum centrifuge.

[0116] 5) Mass spectrometry analysis Add 20 μL of 0.1% FA to reconstitute the dried sample, centrifuge at 12000 rpm for 20 min at 4 °C, and transfer the supernatant to a mass spectrometer vial. Perform LC-MS / MS analysis using a Thermo Scientific Orbitrap Eclipse mass spectrometer, and perform library search analysis on the raw mass spectrometry results using PEAKSStudio 12.

[0117] Mass spectrometry results as follows Figure 13 As shown, the peptide coverage is 100%, therefore the molecular weight of the expressed Brazzein protein is consistent with the theory.

[0118] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A recombinant yeast strain, characterized in that, The recombinant yeast strain contains the Brazzein gene, which is derived from... Pentadiplandra brazzeana .

2. The strain according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the Brazzein gene is SEQ ID No:

1.

3. The strain according to claim 1 or 2, characterized in that, The recombinant yeast strain contains 3, 6, or 9 copies of the Brazzein gene.

4. The strain according to any one of claims 1-3, characterized in that, The nucleotide sequence of the Brazzein gene is SEQ ID No:2, SEQ ID No:3, or SEQ ID No:

4.

5. The strain according to any one of claims 1-4, characterized in that, The Brazzein gene is expressed by any of the following promoters: 1) P with nucleotide sequence SEQ ID No:5 AOX1 promoter; 2) P with nucleotide sequence SEQ ID No:6 DAS2 promoter; 3) P with nucleotide sequence SEQ ID No. 7: DAS1 Promoter.

6. The strain according to any one of claims 1-5, characterized in that, The Brazzein gene also incorporates any of the following signal peptides: 1) The α-mating factor signal peptide (α-peptide) with the nucleotide sequence SEQ ID No:8; 2) The nucleotide sequence of the SP0030 peptide is SEQ ID No:

9.

7. The strain according to any one of claims 1-6, characterized in that, The recombinant yeast strain also contains a chaperone protein gene that helps with protein folding; the chaperone protein gene is any one of the following: Pp PDI ,Pp BIP Pp ERP1 Pp AHA1 Pp GPX1 ,Pp HAC1 ,Pp IRE1 ,Pp SEC53 ,Pp YPT6 ,Pp HRD1 ,Pp UBC1 ,Pp BRF2 ,Pp BMH2 ,Pp SED4 ,Pp SLY1 ,Pp SSA1 ,Pp SSA4 ,Pp SSO2 ,Pp WSC4 ,Pp YDJ1 .

8. The strain according to claim 7, characterized in that, The Brazzein protein and the chaperone protein gene are integrated into the recipient yeast strain at the following sites: PNSI-2, PNSI-9, PNSI-13, I-14, PNSⅡ-4, PNSⅡ-5, PNSⅡ-6, PNSⅣ-9, PNSⅢ-4, or PNSⅢ-10.

9. The use of the recombinant yeast strain according to any one of claims 1-8 in the production of Brazzein.

10. A method for producing Brazzein protein, characterized in that, The method uses any one of the recombinant yeast strains described in claims 1-8 for fermentation to obtain the Brazzein protein.

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