A recombinant pichia pastoris for efficiently synthesizing serendipitous protein ii and a construction method and application thereof

Abstract

This invention discloses a recombinant Pichia pastoris for the efficient synthesis of arecoline II, its construction method, and its applications, belonging to the field of yeast synthesis technology. The arecoline II encoding gene, optimized for codon preference in Pichia pastoris, is fused with the encoding sequences of the endogenous Msb2 or Dan4 signal peptides in Pichia pastoris to construct a first expression cassette. Simultaneously, a third expression cassette, encoding a molecular chaperone gene controlled by the AOX1 promoter, is constructed. The first and third expression cassettes are co-transformed into Pichia pastoris host cells, and recombinant Pichia pastoris strains are obtained through screening. Arecoline II is obtained through fermentation and methanol-induced expression. Through the synergistic effect of the endogenous signal peptide and the dual molecular chaperone PDI-Ero1 tandem co-expression strategy, efficient secretory expression and correct folding of arecoline II in Pichia pastoris are achieved, significantly increasing the proportion of active protein and expression yield, and solving problems such as low secretion efficiency, disulfide bond mismatch, and easy protein aggregation and inactivation in existing sweet proteins.

Description

Technical Field

[0001] This invention relates to the field of yeast synthesis technology, and in particular to a recombinant Pichia pastoris for the efficient synthesis of arecoline II, its construction method, and its application. Background Technology

[0002] In recent years, many epidemics such as obesity, diabetes, and dental caries have been linked to excessive sugar intake. The traditional food industry primarily uses natural sugars such as sucrose, glucose, and galactose, or chemically synthesized sweeteners such as sorbitol, as sweeteners. While sugars offer advantages in terms of nutrition and safety, they also have disadvantages such as low sweetness, high calories, and the need for large quantities, easily leading to health problems such as cardiovascular disease, obesity, diabetes, and dental caries. Therefore, given the increasing demand for sweeteners and healthy foods, the development of novel, safe, and non-toxic natural sweeteners has become a research hotspot in the food and pharmaceutical fields.

[0003] Research on natural, nutritional, non-sugar sweeteners primarily focuses on sweet proteins. Natural sweet proteins were initially isolated from the fruits or seeds of tropical African plants. They are named sweet proteins because they possess a sweet taste or the ability to convert sour tastes into sweetness. Once ingested, sweet proteins are digested and broken down into various amino acids. Their absorption and utilization do not depend on insulin, posing no risk of inducing diabetes, obesity, or other diseases. They represent an ideal class of novel sweeteners with broad application prospects, potentially replacing traditional sweeteners in food processing and other fields. Although sweet proteins are considered highly promising "sugar substitute" sweeteners, large-scale commercial use in the food and beverage industries has not yet been achieved. The main limiting factors are twofold: the high cost and low yield of extracting sweet proteins from plant tissues result in high prices (several thousand yuan per kilogram); and most sweet proteins have a certain aftertaste and poor heat tolerance and stability, causing difficulties in transportation and preservation. Therefore, using molecular modification (mutants) and genetic engineering methods to modify and heterologously express sweet proteins is an effective way to improve their yield and application performance.

[0004] Currently, eight sweet taste proteins have been discovered, in the following order of discovery: miraculin, pentadine, monellin, thaumatin, mabinlin, curculin I, brazzein, and neoculin II. Among them, mabinlin was isolated from the plant *Capparis masaikai*. Studies have found five subtypes of mabinlin in mabinlin seeds: Mabinlin I, Mabinlin I-1, Mabinlin II, Mabinlin III, and Mabinlin IV. Of these five subtypes, Mabinlin II exhibits the highest thermal stability, maintaining its sweet taste activity for at least 2 hours at 80°C; while the other four subtypes only maintain their activity for 0.5 hours at 80°C. Better thermal stability makes it more suitable for heat processing in food. Mabinlin II protein has a molecular weight of 10.4 kDa and consists of two chains, A and B. Chain A contains 33 amino acids, and chain B contains 72 amino acids. Mabinlin II protein has high sweetness, good thermal stability, acid resistance, low calories, and is non-toxic, making it an ideal novel sweetener. Therefore, Mabinlin II protein has high application and development value. However, due to various unfavorable factors such as the inability of the original Mabinlin II protein plant to bear fruit when planted in a different location, the high cost of chemical synthesis, and inconsistent sweetness of the product, large-scale production can only be achieved through genetic engineering. However, proteins expressed in prokaryotic expression systems often lack biological activity because prokaryotes themselves lack an effective post-translational protein processing system. Therefore, a eukaryotic expression system is used for the expression of Mabinlin II protein.

[0005] In existing technologies, the expression of arecoside protein II in Pichia pastoris generally suffers from several technical defects: First, the commonly used signal peptides are mostly exogenous universal signal peptides, rather than endogenous Pichia pastoris signal peptides, resulting in poor compatibility with the target protein and directly leading to low secretion efficiency and severe intracellular retention of the arecoside protein. Second, arecoside protein II contains multiple disulfide bonds in its structure, and existing expression systems typically do not co-express molecular chaperones, lacking the auxiliary folding of protein disulfide isomerase PDI and endoplasmic reticulum oxidoreductase Ero1, causing the protein to easily misfold, aggregate, and precipitate, resulting in an extremely low proportion of active protein and poor overall product activity. Furthermore, existing technologies often fail to optimize the target gene for Pichia pastoris codon bias, leading to low gene translation efficiency and further limiting protein expression levels; moreover, the methanol induction concentration lacks precise control, easily causing excessive metabolic burden on the cells and disordered expression systems. Furthermore, traditional construction methods focus only on the expression of a single target protein, without taking into account the stability of the strain and the balance of expression. High copy overexpression can also reduce the genetic stability of the strain. In addition, the lack of suitable high-density fermentation technology makes it difficult to achieve industrial-scale production of recombinant strains, which cannot meet the actual needs of the food, health products, pharmaceutical and cosmetic fields for efficient, stable and highly active preparation of areca catechin II. Summary of the Invention

[0006] Existing methods for expressing arecoside protein II using Pichia pastoris generally suffer from drawbacks such as low secretion efficiency, severe protein misfolding, low proportion of active protein, poor strain stability, and difficulty in large-scale production. These shortcomings fail to meet the practical needs of the food, health product, pharmaceutical, and cosmetic industries for the efficient, stable, and highly active preparation of arecoside protein II. This invention aims to provide a recombinant Pichia pastoris strain for the efficient synthesis of arecoside protein II, its construction method, and its applications.

[0007] To address the shortcomings of existing technologies, this invention provides the following technical solution: In a first aspect, the present invention provides a recombinant Pichia pastoris for the efficient synthesis of arecoline II, wherein the genome of the recombinant Pichia pastoris integrates: The first expression cassette contains a Pichia pastoris endogenous signal peptide coding sequence fused with the coding sequence of arecoside II, wherein the signal peptide is selected from the Pichia pastoris endogenous Msb2 signal peptide or Dan4 signal peptide. The third expression cassette contains the molecular chaperone coding sequence.

[0008] Preferably, the amino acid sequence of the Msb2 signal peptide is shown in SEQ ID NO.1; and the amino acid sequence of the Dan4 signal peptide is shown in SEQ ID NO.2.

[0009] Preferably, the molecular chaperone is a protein disulfide isomerase, or a protein disulfide isomerase and an endoplasmic reticulum oxidoreductase linked together by a self-cleaving peptide.

[0010] Preferably, the self-cleaving peptide is the porcine cyclophosphine virus 2A self-cleaving peptide.

[0011] Preferably, the protease encoding gene is PEP4 and / or YPS1.

[0012] Preferably, the first expression box and / or the third expression box have at least two copies.

[0013] Preferably, the coding sequence of areca catechin II is a sequence optimized by Pichia pastoris codons.

[0014] Preferably, the first expression cassette and / or the third expression cassette further include a promoter and a terminator, wherein the promoter is a methanol-inducible promoter or a constitutive promoter.

[0015] Preferably, the first expression cassette and / or the third expression cassette are integrated into the AOX1 gene site, HIS4 gene site, or rDNA site of Pichia pastoris.

[0016] By fusing the Pichia pastoris codon preference-optimized arecoside glycoprotein II gene with the endogenous Msb2 / Dan4 signal peptide coding sequence, and using the AOX1 promoter to drive the co-expression of PDI or 2A peptide tandem PDI-Ero1 molecular chaperone, we achieved efficient secretory expression and correct folding of arecoside glycoprotein II in Pichia pastoris, significantly increasing the proportion and expression yield of active protein. This solved the technical problems of low secretion efficiency, disulfide bond mismatch, and easy protein aggregation and inactivation in existing heterologous expression systems.

[0017] The Pichia pastoris host cells are GS115, KM71, or X33 strains.

[0018] Preferably, the Pichia pastoris host cell is strain GS115.

[0019] Secondly, the present invention provides a method for constructing Pichia pastoris that efficiently synthesizes arecoside glycoprotein II, comprising introducing the first expression cassette and the third expression cassette into Pichia pastoris host cells and screening to obtain recombinant Pichia pastoris that integrates the expression cassette.

[0020] Preferably, the introduction is carried out by electroporation conversion.

[0021] By introducing the expression cassette into the Pichia pastoris host using electroporation transformation and strictly controlling the methanol volume fraction to 0.5%~1.5% for induction expression, a stable recombinant strain suitable for high-density fermentation was obtained. This technique enabled the efficient and stable large-scale production of areca catechin II, solving the problems of poor genetic stability of recombinant strains, difficulty in controlling induction conditions, and difficulty in adapting to industrial production in the food and pharmaceutical fields in existing methods.

[0022] Preferably, the Pichia pastoris host cell is a strain GS115, a strain KM71, or a strain X33.

[0023] Preferably, the Pichia pastoris host cell is strain GS115.

[0024] Thirdly, the present invention provides the application of the above-mentioned highly efficient Pichia pastoris for synthesizing areca catechin II in the production of areca catechin II.

[0025] Fourthly, the present invention provides a method for producing areca catechin II, comprising culturing the above-mentioned Pichia pastoris that efficiently synthesizes areca catechin II until fermentation is completed, and collecting areca catechin II.

[0026] Preferably, the culture includes high-density fermentation, with the addition of an inducer or the use of a constitutive promoter for sustained expression during fermentation.

[0027] More preferably, the inducer is methanol, and the induction is achieved by maintaining the methanol volume fraction in the fermentation system at 0.5%~1.5%.

[0028] High-density fermentation can significantly increase the wet weight of the cells, and combined with the high-expression strain of this invention, the yield of the target protein can be further amplified.

[0029] Preferably, the areca nut protein II is used in the fields of food, health products, medicine, or cosmetics.

[0030] Compared with the prior art, the present invention achieves the following beneficial effects: This invention provides a highly efficient Pichia pastoris method for synthesizing arecoline II. It utilizes the endogenous Msb2 or Dan4 signal peptide of Pichia pastoris instead of the conventional exogenous signal peptide, resulting in better compatibility with the target protein. This allows for more efficient guidance of arecoline II into the secretory pathway, reducing intracellular retention and significantly increasing the yield of the target protein in the fermentation supernatant. Co-expression of molecular chaperones assists in disulfide bond formation, inhibiting protein aggregation and misfolding. Endogenous proteases in Pichia pastoris degrade heterologously expressed target proteins. Inactivation of these protease-encoding genes reduces the risk of target protein degradation, prolongs protein accumulation time in semi-continuous or continuous fermentation, and improves final yield and product uniformity. The endogenous signal peptide ensures efficient secretion output, the molecular chaperone ensures correct folding of the secreted protein, and protease inactivation ensures that the folded protein is not degraded. The combined use of these three elements achieves a complete chain of "efficient secretion → correct folding → stable accumulation," solving the three major pain points of existing technologies: low yield, poor activity, and easy degradation.

[0031] This invention provides a method for constructing Pichia pastoris for the efficient synthesis of arecoline II. The method simultaneously introduces the secretion module (signal peptide + target gene) and the folding helper module (molecular chaperone) into the host, avoiding the time cost and screening complexity of step-by-step construction. It also allows control over the integration ratio and copy number of the two expression cassettes. The protease-encoding gene is inactivated using knockout or mutation methods, ensuring stable genetic traits in the strain. This eliminates the need for additional protease inhibitors during fermentation, reducing production costs and process complexity. This method is simple to operate, has a short cycle time, and good reproducibility. It can systematically construct engineered strains with the triple advantages of "high secretion, high folding, and low degradation," making it suitable for the mass development of industrial strains.

[0032] The application provided by this invention allows the strain to be used directly as a production tool for the fermentation preparation of areca nut sweet protein II. The strain's inherent high secretion, high folding, and low degradation characteristics are directly translated into high yield, high purity, and high activity in the process. Compared to extraction from the seeds of the natural plant areca nut (extremely low yield and extremely high cost) or the use of prokaryotic expression systems (lacking folding ability and activity), this application scheme achieves large-scale, low-cost, and high-activity production of sweet protein, demonstrating significant industrial practical value.

[0033] The method for producing areca catechin II provided by this invention uses endogenous signal peptide-guided secretory expression. The target protein is mainly found in the fermentation supernatant. The collection steps are simple (centrifugation or filtration is sufficient), eliminating the need for cell disruption, reducing downstream purification costs, minimizing intracellular contamination, and improving product purity and activity recovery rate. Attached Figure Description

[0034] Figure 1 This is a flowchart of the synthesis method of the present invention. Detailed Implementation

[0035] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0036] Pichia pastoris host strains GS115, KM71, and X33 were all purchased from Invitrogan (now Thermo Fisher Scientific). Expression vectors pPIC9K and pPICZα were also purchased from Invitrogan (now Thermo Fisher Scientific). The protein disulfide isomerase PDI gene and the endoplasmic reticulum oxidoreductase Ero1 gene are both endogenous genes of Pichia pastoris, amplified from the genomic DNA of Pichia pastoris GS115 by PCR. The self-splicing peptide sequence of porcine cyclophosphamide virus 2A was obtained through artificial synthesis. The areca catechin II gene was chemically synthesized by GenScript Biotech Co., Ltd. and optimized according to the codon bias of Pichia pastoris.

[0037] I. Experimental Materials and General Methods Unless otherwise specified, the following examples and comparative examples are based on the following general materials and methods: Host strain: Pichia pastoris GS115.

[0038] Target gene: The gene encoding arecoside protein II has been synthesized with full sequence optimization based on the codon preference of Pichia pastoris.

[0039] Signal peptide: The amino acid sequence of the Msb2 signal peptide is shown in SEQ ID NO.1. Amino acid sequence: MKLFKVSSSLFLLLSGVSA; Nucleotide sequence (Pichia pastoris codon optimization): ATGAAGCTTTTCAAAGTTTCAAGTTCACTTTTCCTTCTCCTTTCTGGTGTTTCAGCT (as shown in SEQ ID NO.3). The amino acid sequence of the Dan4 signal peptide is shown in SEQ ID NO.2. Amino acid sequence: MRGPKLLSLLLLGAAAGPAAA; Nucleotide sequence (Pichia pastoris codon optimization): ATGAGAGGACCAAAGCTTCTCTCTCTTCTTCTTCTTGGAGCTGCTGCTGGTCCTGCTGCTGCT (as shown in SEQ ID NO.4) The α-factor signal peptide (used in the comparative example) is a sequence inherent in the Pichia pastoris expression vector.

[0040] Molecular chaperone genes: The gene encoding protein disulfide isomerase (PDI).

[0041] The gene encoding endoplasmic reticulum oxidoreductase (Ero1) is present.

[0042] 2A self-cleaving peptide: Sequence from porcine Chinnarizine virus (P2A). Amino acid sequence of porcine Chinnarizine virus 2A self-cleaving peptide (P2A): ATNFSLLKQAGDVEENPGP (as shown in SEQ ID NO. 5); Nucleotide sequence (Pichia pastoris codon optimization): GCCACAAACTTCAGCCTTCTTAAGCAAGCTGGAGATGTTGAAGAAAACCCAGGACCT (as shown in SEQ ID NO. 6).

[0043] Expression cassette construction: All expression cassettes were transcribed using the AOX1 promoter.

[0044] Conversion method: Electroporation conversion method.

[0045] Fermentation and induction conditions: Fermentation stage: The strain is cultured in BMGY / BMMY or similar fermentation medium to the logarithmic growth phase, reaching the predetermined biomass (OD). 600 ≈10-20).

[0046] Induction phase: Adjust the culture medium and add methanol every 24 hours to a final volume fraction of 0.5%~1.5%, and continue induction for 72-96 hours.

[0047] Detection method: Total protein yield: After separation by SDS-PAGE, the total protein content of the fermentation supernatant was determined by gel scanning or the Bradford method.

[0048] Active protein percentage: The correctness of secondary structure was analyzed using sensory evaluation of sweetness combined with circular dichroism spectroscopy (CD spectroscopy), or quantified using an ELISA method based on sweetness receptor binding activity. Active protein percentage (%) = (Amount of active protein / Total protein in supernatant) × 100%.

[0049] See appendix Figure 1 This invention provides a method for synthesizing Pichia pastoris of areca catechin II, comprising the following steps: S1: The target gene encoding arecoside II is fused with the signal peptide coding sequence selected from the endogenous signal peptide Msb2 or Dan4 of Pichia pastoris to construct the first expression cassette. S2: Construct the third expression cassette of the molecular chaperone-encoding gene controlled by the AOX1 promoter; S3: Introduce the first expression cassette and the third expression cassette into Pichia pastoris host cells; (1) Pick a single colony of Pichia pastoris GS115 and inoculate it into 5 mL of YPD liquid medium. Incubate overnight at 30°C and 200 rpm with shaking.

[0050] (2) Transfer 1% inoculum to 100 mL of YPD liquid medium and incubate at 30 °C and 200 rpm until OD. 600 It is 1.2-1.5.

[0051] (3) Collect the bacterial cells by centrifugation at 4℃ and 5000rpm for 5min, wash twice with pre-cooled sterile deionized water, and wash once with pre-cooled 1M sorbitol.

[0052] (4) The bacterial cells were resuspended in 1 mL of pre-cooled 1 M sorbitol and dispensed into 80 μL tubes.

[0053] (5) Add 5-10 μg of recombinant plasmid (linearized with SalI) to competent cells, incubate on ice for 5 min, and then transfer to a pre-cooled electroporation vessel.

[0054] (6) Electrical parameters: voltage 1.5kV, capacitance 25μF, resistance 200Ω.

[0055] (7) Immediately after electroporation, add 1 mL of pre-cooled 1M sorbitol, mix well, spread on MD solid plates, and incubate at 30°C for 2-3 days.

[0056] S4: Screening to obtain a recombinant Pichia pastoris strain that integrates the expression cassette and efficiently synthesizes arecoline II; (1) The transformants grown on the MD plate were copied onto YPD plates containing different concentrations of G418 (0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL) using a sterile toothpick and incubated at 30°C for 2-3 days.

[0057] (2) Colonies growing on high-concentration G418 plates are multicopy integrons. Yeast genomic DNA was extracted, and the copy number of the target gene was confirmed by real-time PCR (using Pichia pastoris ACT1 gene as an internal reference). The copy number calculation formula is: 2 - (ΔCt target gene - ΔCt internal reference gene) × 2.

[0058] S5: Cultivate the recombinant Pichia pastoris strain to a predetermined biomass in a fermentation medium; (1) Pick a single colony of recombinant Pichia pastoris and inoculate it into 5 mL of BMGY liquid medium. Incubate overnight at 30°C and 250 rpm with shaking.

[0059] (2) According to the initial OD 600 =1.0 was transferred to 50 mL of BMGY medium and incubated at 30 °C and 250 rpm until OD600 was reached. 600 =5-6.

[0060] (3) Collect the bacterial cells by centrifugation at 5000 rpm for 5 min at room temperature, discard the supernatant, and resuspend the bacterial cells in BMMY medium to OD. 600 =1.0.

[0061] (4) Add 50 mL of the above bacterial culture to a 500 mL Erlenmeyer flask, seal the flask with four layers of gauze, and incubate at 30 °C and 250 rpm with shaking. Add methanol every 24 hours to a final volume fraction of 0.5%-1.5% (specific requirements for each example / comparative example). Induce culture for 96 h.

[0062] (5) After fermentation, centrifuge at 4℃ and 8000rpm for 10min and collect the supernatant and cell precipitate respectively.

[0063] S6: Transcriptional expression of the first and third expression cassettes was induced with methanol until fermentation was completed, yielding areca nut protein II.

[0064] In S1, the amino acid sequence of the Pichia pastoris endogenous signal peptide Msb2 signal peptide is shown in SEQ ID NO.1, and the amino acid sequence of the Dan4 signal peptide is shown in SEQ ID NO.2.

[0065] In S1, the target gene encoding areca catechin II is a nucleotide sequence optimized by Pichia pastoris codon preference.

[0066] In S2, the molecular chaperone encoding gene is a protein disulfide isomerase (PDI) encoding gene; or it is a combination of a protein disulfide isomerase (PDI) encoding gene tandemly with a 2A self-cleaving peptide sequence and an endoplasmic reticulum oxidoreductase (Ero1) encoding gene.

[0067] The 2A self-cleaving peptide is the porcine cyclophosphine virus 2A self-cleaving peptide; the third expression cassette can be driven by an inducible AOX1 promoter or a constitutive promoter to achieve constitutive and inducible co-expression of the molecular chaperone.

[0068] In S3, the introduction method is electroporation transformation, and the Pichia pastoris host cell is GS115 strain, KM71 strain, or X33 strain.

[0069] In S6, the methanol induction is performed to induce expression by maintaining the methanol volume fraction in the fermentation system at 0.5%~1.5%.

[0070] The application involves using the recombinant Pichia pastoris strain as the fermentation strain to produce areca catechin II through high-density fermentation. The areca catechin II is used in the food, health product, pharmaceutical, or cosmetic fields. II. Specific Implementation Methods Example 1: (Single copy + Msb2 signal peptide, no molecular chaperone) This embodiment provides a basic method for synthesizing arecoside II. Following the above method, Pichia pastoris is constructed to efficiently synthesize arecoside II. The specific steps are as follows: S1: The Pichia pastoris codon preference-optimized gene encoding arecoside protein II was fused with the Pichia pastoris endogenous Msb2 signal peptide encoding sequence to construct the first expression cassette; wherein, the amino acid sequence of the Msb2 signal peptide is shown in SEQ ID NO. 1.

[0072] S2: This embodiment does not construct molecular chaperone-related expression cassettes.

[0073] S3: The first expression cassette constructed in step S1 was introduced into Pichia pastoris GS115 host cells via electroporation transformation.

[0074] S4: A recombinant Pichia pastoris strain with a single copy integrated into the first expression cassette was obtained after screening for G418 resistance (0.5 mg / mL) and verification by real-time PCR.

[0075] S5: Cultivate the recombinant strain to the predetermined biomass OD in BMGY / BMMY fermentation medium. 600 =5-6.

[0076] S6: Maintain the methanol volume fraction in the fermentation system at 0.5%–1.5% (add methanol every 24 hours to a final concentration of 0.5%) to induce the expression of the target gene. Induce expression for 96 hours until the end of fermentation to obtain areca nut protein II.

[0077] Example 2: (Single-copy Msb2 signal peptide fusion expression + PDI single expression) This embodiment, based on Example 1, introduces a protein disulfide isomerase (PDI) as a molecular chaperone, and includes the following steps: S1: Same as S1 in Example 1, the codon-optimized arecoline II coding gene is fused with the Msb2 signal peptide coding sequence to construct the first expression cassette. The Msb2 signal peptide sequence is shown in SEQ ID NO. 1.

[0078] S2: Construct the third expression cassette (pPIC9K-PDI) of the protein disulfide isomerase PDI gene driven by the AOX1 inducible promoter.

[0079] S3: The first expression cassette and the third expression cassette were mixed at a 1:1 molar ratio and transformed into Pichia pastoris GS115 by electroporation.

[0080] S4: After screening for G418 resistance (1.0 mg / mL) and identification by PCR, a recombinant strain integrating two expression cassettes with a single copy was obtained.

[0081] S5: Fermentation culture to the target biomass, same as in Example 1.

[0082] S6: Same as S6 in Example 1, expressed with 0.5% to 1.5% methanol. After fermentation, areca nut protein II was collected.

[0083] Example 3: (Single-copy Msb2 signal peptide fusion expression + PDI-Ero1 bimolecular chaperone tandem expression) This embodiment, based on Example 2, involves the tandem co-expression of bimolecular chaperone PDI and Ero1, including the following steps: S1: The codon-optimized arecoline II coding gene was fused with the Msb2 signal peptide coding sequence to construct the first expression cassette. The signal peptide sequence is shown in SEQ ID NO. 1, the same as S1 in Example 2.

[0084] S2: Construct a third expression cassette driven by the AOX1 promoter, which encodes the porcine cyclophosphamide virus 2A self-cleaving peptide tandem protein disulfide isomerase (PDI) gene and the Ero1 gene (pPIC9K-PDI-2A-Ero1).

[0085] S3: Mix the first expression cassette and the third expression cassette at a 1:1 molar ratio and electroporate to Pichia pastoris GS115.

[0086] S4: Recombinant strains with single copies of the first and third expression cassettes were obtained through G418 resistance screening (1.0 mg / mL).

[0087] S5: Same as S5 in Example 2, fermentation culture to the predetermined biomass.

[0088] S6: Same as S6 in Example 2, with methanol volume fraction of 0.5% to 1.5% to induce expression and prepare areca nut protein II.

[0089] Example 4: (Dual-copy Msb2 signal peptide fusion expression + PDI-Ero1 bimolecular chaperone tandem expression) This embodiment, based on Embodiment 3, optimizes the copy number of the target gene and includes the following steps: S1: Same as S1 in Example 3, the codon-optimized areca catechin II coding gene is fused with the Msb2 signal peptide coding sequence to construct the first expression cassette, the sequence of which is shown in SEQ ID NO. 1.

[0090] S2: Same as S2 in Example 3, construct a third expression cassette (pPIC9K-PDI-2A-Ero1) driven by the AOX1 promoter and consisting of a 2A peptide linked to PDI and Ero1.

[0091] S3: Mix the first expression cassette and the third expression cassette at a 1:1 molar ratio and electroporate them into Pichia pastoris GS115 host cells.

[0092] S4: Recombinant strains integrating the first expression cassette (2 copies) and the third expression cassette (1 copy) were obtained by G418 gradient screening (2.0 mg / mL) and verified by real-time PCR.

[0093] S5: Same as S5 in Example 3, fermentation culture to the target biomass.

[0094] S6: Same as S6 in Example 3, induced by 0.5% to 1.5% methanol to obtain high yield of areca catechin II, same as in Example 1.

[0095] Comparative Example 1: α-factor signal peptide fusion expression (no molecular chaperone, single copy) The difference between this comparative example and Example 1 is that a different signal peptide was used. The specific steps are as follows: S1: The α-factor signal peptide from the Pichia pastoris vector was used to replace the Msb2 signal peptide and fused with the arecoline II encoding gene to construct an expression cassette. The remaining steps S2-S6 were the same as in Example 1.

[0096] S2: Do not construct a molecular chaperone expression cassette.

[0097] S3: Electroporation to GS115 yielded a single-copy recombinant strain.

[0098] S4~S6: Fermentation and methanol induction conditions are the same as in Example 1.

[0099] Comparative Example 2: Another endogenous Dan4 signal peptide (no molecular chaperone, single copy) The difference between this comparative example and Example 1 is that a different signal peptide was used instead of the Msb2 signal peptide. The specific steps are as follows: S1: The first expression cassette was constructed by replacing the Msb2 signal peptide with the Dan4 signal peptide (as shown in SEQ ID NO. 2) and fusing it with the gene encoding arecoline II.

[0100] S2: Do not construct a molecular chaperone expression cassette.

[0101] S3: Electroconversion GS115, single-copy integration.

[0102] S4~S6: Fermentation and induction conditions are the same as in Example 1.

[0103] Comparative Example 3: Three-copy Msb2 signal peptide (without molecular chaperone) The difference between this comparative example and Example 1 is that a recombinant strain with three copies of the integrated first expression cassette was obtained through screening with a higher concentration of resistance. The specific steps are as follows: S1: The first expression cassette was constructed by fusing the target gene with the Msb2 signal peptide, as in Example 1.

[0104] S2: Do not construct a molecular chaperone expression cassette.

[0105] S3: Electroporation of GS115 was used to screen for recombinant strains with three copies integrated.

[0106] S4~S6: Fermentation and induction conditions are the same as in Example 1.

[0107] Comparative Example 4: Four-copy Msb2 signal peptide (without molecular chaperone) The difference between this comparative example and Example 1 is that a recombinant strain with four copies of the integrated first expression cassette was obtained through screening with a higher concentration of resistance. The remaining steps are exactly the same as in Example 1, and the specific steps are as follows: S1: The first expression cassette was constructed by fusing the target gene with the Msb2 signal peptide.

[0108] S2: Do not construct a molecular chaperone expression cassette.

[0109] S3: Electroporation of GS115 was used to screen and obtain recombinant strains with four copies integrated.

[0110] S4~S6: Fermentation and induction conditions are the same as in Example 1.

[0111] The parameters and results of each embodiment and comparative example are shown in Table 1.

[0112] Table 1: Effects of different construction strategies on the expression of arecoline II

[0113] As shown in Table 1, this invention systematically compared the effects of signal peptide type, molecular chaperone co-expression strategy, and target gene copy number on heterologous expression of arecoside II in Pichia pastoris by setting up multiple sets of examples and comparative examples. The results showed that the Pichia pastoris synthesis method of arecoside II provided by this invention is significantly superior to existing conventional expression technologies in terms of secretion efficiency, protein correct folding efficiency, recombinant strain stability, and total target protein yield. It effectively solves the technical defects of existing technologies, such as low expression level of arecoside II, low proportion of active protein, severe misfolding and protein aggregation, and poor genetic stability of high-expression strains. Compared with the commonly used α-factor signal peptide (Comparative Example 1) and the alternative Dan4 endogenous signal peptide (Comparative Example 2) in existing technologies, the endogenous Msb2 signal peptide of Pichia pastoris selected in this invention has better compatibility with arecoside II. Under the same culture and induction conditions, the target protein secretion is higher, intracellular retention is less, and the proportion of soluble protein is significantly increased. Compared with conventional expression systems without molecular chaperone assistance (Example 1, Comparative Examples 1-4), this invention employs an AOX1 promoter-driven, 2A self-cleaving peptide tandem PDI and Ero1 bimolecular chaperone co-expression strategy, which can simultaneously optimize the endoplasmic reticulum oxidation environment and promote the correct formation of disulfide bonds, significantly reducing protein misfolding and aggregation rates, increasing the proportion of active arecoline protein II from about 40% to over 90%, fundamentally solving the problem of insufficient folding efficiency when expressing exogenous small molecule proteins in Pichia pastoris.

[0114] Regarding gene copy number optimization, simply increasing the target gene copy number cannot continuously improve fermentation performance (Comparative Examples 3 and 4). 3 copies or more will significantly increase the host's metabolic burden, leading to strain growth inhibition, decreased genetic stability, and no significant increase in the proportion of active proteins. The preferred 2-copy integration strategy of this invention can achieve the best balance between target protein yield and physiological load while ensuring the genetic stability of the strain, avoiding the negative effects of high copy number.

[0115] The optimal solution (Example 4) combines the co-expression of Msb2 endogenous signal peptide, PDI-Ero1 dual molecular chaperone, and 2-copy integration. While maintaining stable growth of the strain, it ensures efficient secretion output and maximizes the preservation of the protein's natural activity. The yield of the target protein and the proportion of active protein in the fermentation supernatant both reach the optimal level. It is suitable for industrial high-density fermentation production in the food, health product, pharmaceutical, and cosmetic fields, and has outstanding technical practicality and industrialization advantages.

[0116] The above embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit it. Anyone skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A recombinant Pichia pastoris for efficiently synthesizing sericin II from Sericoderma japonica, characterized in that, The recombinant Pichia pastoris genome integrates: The first expression cassette contains a Pichia pastoris endogenous signal peptide coding sequence fused with the coding sequence of arecoside II, wherein the signal peptide is selected from the Pichia pastoris endogenous Msb2 signal peptide or Dan4 signal peptide. The third expression cassette contains the molecular chaperone coding sequence.

2. The recombinant Pichia pastoris for efficient synthesis of arecoline II according to claim 1, characterized in that, The amino acid sequence of the Msb2 signal peptide is shown in SEQ ID NO.1, and the amino acid sequence of the Dan4 signal peptide is shown in SEQ ID NO.

2.

3. The Pichia pastoris for efficient synthesis of arecoline II according to claim 1, characterized in that, The molecular chaperone is a protein disulfide isomerase, or a protein disulfide isomerase and an endoplasmic reticulum oxidoreductase linked together by a self-cleaving peptide.

4. The recombinant Pichia pastoris for efficient synthesis of arecoline II according to claim 3, characterized in that, The self-cleaving peptide is the porcine chorionic villus virus 2A self-cleaving peptide.

5. The recombinant Pichia pastoris for efficient synthesis of arecoline II according to claim 1, characterized in that, The Pichia pastoris host cells are GS115, KM71, or X33 strains.

6. The recombinant Pichia pastoris for efficient synthesis of arecoline II according to claim 1, characterized in that, The coding sequence for areca catechin II is a sequence optimized using Pichia pastoris codons.

7. The method for constructing recombinant Pichia pastoris for efficient synthesis of arecoline II according to any one of claims 1 to 6, characterized in that, This includes introducing the first expression cassette and the third expression cassette into Pichia pastoris host cells and screening to obtain recombinant Pichia pastoris cells that integrate the expression cassettes.

8. The method for constructing Pichia pastoris for efficient synthesis of arecoline II according to claim 7, characterized in that, The import method is electroporation conversion.

9. The use of Pichia pastoris, as described in any one of claims 1 to 6, for the efficient synthesis of arecoside protein II in the production of arecoside protein II.

10. A method for producing areca catechin II, characterized in that, The method includes culturing Pichia pastoris, which is a highly efficient synthesizer of arecoside protein II according to any one of claims 1 to 6, and collecting arecoside protein II.

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