A thaumatin ii gene expressed in pichia pastoris, expression method and application

Abstract

This invention provides a semathys II gene expressed in Pichia pastoris, its expression method, and its applications. Without altering the amino acid sequence of the mature semathys II protein, this invention comprehensively considers factors such as Pichia pastoris codon preference, codon fitness index, GC content, mRNA secondary structure, unstable sequence deletion, rare codon distribution, and translation rhythm to optimize the nucleic acid sequence encoding the mature semathys II protein using various strategies. Ultimately, an optimized gene, ThaII-m4, suitable for high-secretion expression of semathys II in Pichia pastoris was obtained, and its nucleotide sequence is shown in SEQ ID NO:5. Semathys II prepared by fermentation using Pichia pastoris containing this optimized gene has a high yield and possesses the desired sweetness, making it well-suited for applications in food, beverages, oral care, pharmaceutical excipients, and animal feed.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology, recombinant protein expression and preparation of natural sweet proteins, and specifically relates to a semathys II gene expressed in Pichia pastoris, its expression method and application. Background Technology

[0002] Thaumatin is a type of natural sweet protein derived from the fruit of the African arrowroot. It is characterized by high sweetness, low calories, good safety, and the ability to improve food flavor and mask bitterness and astringency. It has broad application prospects in the fields of food, beverages, oral care, pharmaceutical excipients, and feed attractants.

[0003] Natural sematriol is mainly derived from plant extracts, and its production is limited by the origin of raw materials, the scale of cultivation, the season, and extraction costs, making it difficult to consistently meet the demands of industrial production. Microbial fermentation is a sustainable and scalable method for producing sematriol. Pichia pastoris possesses advantages such as high-density fermentation, secretory expression, a eukaryotic post-translational processing system, and no endotoxin production, making it an important host for the production of recombinant sweet proteins.

[0004] Sematome II is an important member of the semamate family. Its mature protein has a molecular weight of approximately 22 kDa and contains multiple disulfide bonds. The correct folding, secretion efficiency, and extracellular integrity of semamate II significantly influence its sweetness activity. Although various hosts have been developed for expressing semamate proteins, the presence of multiple disulfide bonds in the semamate II molecule can lead to problems during heterologous secretory expression, such as insufficient translation efficiency, increased folding burden, low secretion efficiency, numerous degradation bands, or unstable sweetness activity.

[0005] In heterologous gene expression, different nucleotide sequences encoding the same amino acid sequence may exhibit significantly different expression levels and protein integrity due to factors such as codon bias, codon fitness index, GC content, mRNA secondary structure, rare codon distribution, recessive splicing sites, internal polyA signaling, and translation rhythm. For sweet proteins containing multiple disulfide bonds, simply increasing the codon fitness index may not necessarily achieve optimal secretory expression; excessively rapid translation may increase endoplasmic reticulum folding pressure, leading to misfolding or protein degradation.

[0006] Therefore, it is necessary to design and screen optimized genes for semaside II expression in Pichia pastoris, taking into account the sequence characteristics of semaside II itself and the Pichia pastoris expression system, in order to improve the secretory expression level of semaside II. Summary of the Invention

[0007] Therefore, this invention provides an optimized semaside II gene suitable for expression in Pichia pastoris. The optimized gene was obtained through various codon optimization strategies without altering the amino acid sequence of the mature semaside II protein, and was subsequently screened for expression in Pichia pastoris.

[0008] One of the objectives of this invention is to provide a synonymous optimized gene suitable for efficient expression of sematriene II in Pichia pastoris, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0009] Furthermore, the amino acid sequence of the semathy II protein encoded by the synonymous optimized gene is shown in SEQ ID NO.1, and does not alter the amino acid sequence of the natural mature semathy II protein.

[0010] A second objective of this invention is to provide a recombinant expression vector comprising the aforementioned synonymous optimized gene.

[0011] Furthermore, the recombinant expression vectors include pPIC9K vector, pPICZ vector, pPICZα vector, pAO815 vector, and pGAPZ vector.

[0012] Furthermore, the recombinant expression vector also includes a promoter, a secretion signal peptide coding sequence, and a transcription termination sequence.

[0013] In some embodiments, the promoter includes the AOX1 promoter, the AOX1 core promoter, the GAP promoter, the TEF1 promoter, and the PGK1 promoter; The secretory signal peptide coding sequence includes the α-factor signal peptide coding sequence and the Pichia pastoris endogenous signal peptide coding sequence.

[0014] The third objective of this invention is to provide a recombinant Pichia pastoris strain, wherein the recombinant Pichia pastoris strain includes the above-mentioned recombinant expression vector.

[0015] In some embodiments, the host strain of the recombinant Pichia pastoris strain includes Pichia pastoris GS115, X33, KM71, KM71H, and SMD1168.

[0016] The fourth objective of this invention is to provide a method for preparing sematriline II, wherein the preparation is as follows: inducing fermentation of the above-mentioned recombinant Pichia pastoris strain to induce it to secrete and express sematriline II; collecting the fermentation supernatant; separating and purifying the fermentation supernatant to obtain sematriline II.

[0017] The fifth objective of this invention is to provide the application of sematriol II, prepared by the above method, as a natural sweet protein in food, beverages, oral care, pharmaceutical excipients, and feed.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention optimizes and compares different nucleotide sequences through multiple strategies, so that the optimized gene obtained by screening not only considers the codon fitness index, but also comprehensively considers GC content, mRNA secondary structure, unstable sequence deletion and translation rhythm. Finally, without changing the amino acid sequence of the mature semathys II protein, an optimized semathys II gene suitable for secretory expression in Pichia pastoris is provided.

[0019] (2) The optimized gene obtained by screening in this invention is beneficial to improve the secretion expression level of sematridin II in Pichia pastoris (yield of 1.0~1.5g / L), and the prepared product has the sweetness of natural sematridin II and can be widely used in food, beverage, oral care, pharmaceutical excipients and feed attractant fields. Attached Figure Description

[0020] Figure 1 This refers to the mRNA secondary structure of the four synonymous optimized genes in Example 1 of the present invention; wherein, AD corresponds to ThaII-m1, ThaII-m2, ThaII-m3 and ThaII-m4 in sequence.

[0021] Figure 2 This is a map of the recombinant expression vector in Example 2 of the present invention.

[0022] Figure 3 This is a band diagram of the target protein in the fermentation supernatant of Pichia pastoris containing four different synonymous optimized genes in Example 2 of the present invention; wherein, bands 1-4 correspond to ThaII-m1, ThaII-m2, ThaII-m3 and ThaII-m4 respectively.

[0023] Figure 4 The above describes the expression of semathymidine II in strain ThaII-m4 at different induction time points in Example 3 of this invention; lanes 1-6 represent fermentation supernatant samples after methanol induction for 12h, 24h, 48h, 72h, 96h and 120h, respectively. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0025] Example 1 This invention uses mature semathys II protein as the target protein and provides a multi-strategy optimization method for the semathys II coding sequence, as detailed below: The target protein was selected from mature semathys II derived from African arrowroot. The amino acid sequence of mature semathys II is shown in SEQ ID NO.1: SEQ ID NO.1:ATFEIVNRCSYTVWAAASKGDAALDAGGRQLNSGESWTINVEPGTKGGKIWARTDCYFDDSGRGICRTGDCGGLLQCKRFGRPPTTLAEFSLNQYGKDYIDI SNIKGFNVPMDFSPTTRGCRGVRCAADIVGQCPAKLKAPGGGCNDACTVFQTSEYCCTTGKCGPTEYSRFFKRLCPDAFSYVLDKPTTVTCPGSSNYRVTFCPTAL.

[0026] The nucleotide sequence encoding the mature semathys II protein was optimized without altering the amino acid sequence shown in SEQ ID NO.1. The optimization process comprehensively considered factors such as Pichia pastoris codon bias, codon fitness index, GC content, mRNA secondary structure, deletion of unstable sequences, and distribution of rare codons. The optimization principles included: (1) Codon bias: Based on the codon usage bias of Pichia pastoris, the occurrence of rare codons is reduced to improve translation efficiency.

[0027] (2) Codon fitness index: to make the CAI value of the optimized sequence within the range suitable for Pichia pastoris expression.

[0028] (3) GC content: Control the GC content of the whole sequence and the local GC distribution to avoid high GC regions and continuous AT regions.

[0029] (4) mRNA secondary structure: Reduce the excessive secondary structure in the 5′ translation initiation region to avoid affecting translation initiation.

[0030] (5) Stability: Delete or avoid potentially unstable sequences, recessive splicing sites, internal polyA signals, abnormal repetitive sequences and unfavorable restriction enzyme sites.

[0031] (6) Translation rhythm: For semathymide II containing multiple disulfide bonds, avoid using the highest frequency codons throughout the translation process in some cysteine-rich regions or potentially fold-sensitive regions to reduce folding pressure caused by excessively rapid translation.

[0032] Using the above optimization strategies, four optimized nucleotide sequences encoding the same mature semathy II protein were obtained, named ThaII-m1, ThaII-m2, ThaII-m3, and ThaII-m4, respectively. Among them, ThaII-m1 is optimized for high CAI (Copyability-Induced Algorithm), ThaII-m2 is optimized for GC balance and repetitive sequence avoidance, ThaII-m3 is optimized for translation rhythm, and ThaII-m4 is a multi-target comprehensive optimized sequence.

[0033] The nucleotide sequences of ThaII-m1, ThaII-m2, ThaII-m3 and ThaII-m4 are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0034] SEQ ID NO.2:

[0035] SEQ ID NO.3:GCTACGTTTGAAATTGTAAACAGATGTAGCTATACAGTGTGGGCGGCTGCGAGCAAAGGAGACGCTGCCCTCGATGCGGGAGGTCGTCAATTAAATTCGGGTGAATCTTGGACAATTAATGTAGAACCAGGTACTAAGGGTGGCAAGATTTGGGCTAGAACCGACTGTTACTTTGACGATTCAGGTAGAGGGATATGTCGGACAGGAGATTGCGGCGGTTTATTGCAATGTAAACGTTTCGGTAGACCACCCACAACCCTAGCGGAGTTTTCACTTAACCAATACGGAAAGGACTATATTGATATTTCTAATATTAAGGGATTTAATGTACCGATGGACTTTTCCCCAACTACCAGAGGCTGTCGAGGAGTCAGATGCGCAGCGGACATCGTCGGTCAATGCCCCGCAAAGCTGAAAGCCCCAGGAGGCGGGTGTAATGATGCTTGCACTGTTTTTCAGACATCAGAGTATTGCTGTACAACGGGCAAATGCGGTCCGACAGAATACAGTAGATTCTTTAAGAGACTGTGTCCAGACGCTTTTTCGTATGTTCTTGATAAACCAACCACAGTCACTTGCCCAGGCTCTAGTAACTATCGTGTGACGTTCTGTCCGACAGCTCTT。

[0036] SEQ ID NO.4:GCTACTTTTGAAATCGTGAACCGTTGTTCATACACTGTCTGGGCCGCTGCCTCCAAAGGTGATGCTGCACTGGATGCAGGCGGAAGGCAATTGAACAGTGGGGAGTCTTGGACCATTAATGTTGAGCCAGGGACTAAAGGCGGTAAGATTTGGGCACGTACGGATTGTTATTTCGATGACTCAGGTAGGGGAATTTGTAGGACAGGCGATTGTGGGGGTCTGCTTCAATGTAAAAGATTTGGTAGGCCACCTACCACGTTGGCAGAGTTCTCCTTGAATCAATATGGTAAAGATTATATTGATATCTCAAACATCAAAGGCTTTAATGTCCCAATGGATTTTTCCCCTACTACCAGAGGTTGTAGAGGAGTCCGTTGTGCTGCGGATATTGTCGGACAATGTCCTGCTAAATTGAAAGCTCCTGGTGGGGGTTGTAATGATGCATGCACTGTCTTCCAAACTTCGGAGTATTGCTGCACAACCGGAAAATGCGGTCCAACAGAGTATTCCAGGTTTTTCAAAAGACTTTGCCCAGATGCTTTTTCATACGTATTGGACAAACCAACGACCGTCACATGTCCAGGTTCCTCAAATTATCGTGTAACCTTCTGCCCTACAGCTCTT。

[0037] SEQ ID NO.5:

[0038] Furthermore, the mRNA secondary structures of ThaII-m1, ThaII-m2, ThaII-m3, and ThaII-m4 were predicted and analyzed, and the results are as follows: Figure 1 As shown: Although the above four synonymous optimized genes all encode the same mature semathys II protein, their mRNA secondary structures differ due to variations in codon composition, local GC distribution, and nucleotide arrangement. The stem-loop structure distribution differs between the different synonymous optimized genes in the 5′ region and the full-length mRNA. An overly strong 5′ secondary structure may affect ribosome entry and translation initiation efficiency, while a more complex local stem-loop structure may also affect mRNA stability and translation elongation. Therefore, this invention considers not only the codon fitness index but also the overall impact of mRNA secondary structure on semathys II secretory expression. The base composition and sequence characteristics of the different synonymous optimized genes are shown in Table 1.

[0039] Table 1. Sequence characteristics of different optimized genes

[0040] Therefore, the expression of heterologous genes in Pichia pastoris is a complex process. Even when different nucleotide sequences encode the same mature semathys II protein, differences in codon composition, GC content, and mRNA secondary structure can still lead to variations in the secretory expression levels of the target protein. Figure 1 As shown, the secondary structure of ThaII-m1 mRNA exhibits an overall extended distribution with a relatively long main chain and branching structures and local stem-loops in multiple regions; ThaII-m2 displays a central-radial structure with multiple branches extending outward from the central region, forming a more pronounced concentrated folding pattern; ThaII-m3 has a more distinct continuous main trunk structure with relatively fewer local branches; ThaII-m4 exhibits a long main trunk with multiple branches, forming multiple local stem-loop structures in different regions. These results indicate that although the four synonymous optimized genes encode the same semathyme II mature protein, their mRNA folding conformations differ due to variations in nucleotide composition and arrangement, potentially further affecting mRNA stability, translation process, and the secretory expression level of semathyme II in Pichia pastoris.

[0041] Example 2 This embodiment constructs a semathy II recombinant expression vector and strain based on the synonymous optimized sequence obtained in Example 1, in order to determine which synonymous optimized sequence is most suitable, as detailed below: 2.1 Construction of Recombinant Expression Vectors The four synonymous optimized genes ThaII-m1, ThaII-m2, ThaII-m3, and ThaII-m4 obtained in Example 1 were synthesized. Using the synthesized genes as templates, the coding sequences of ThaII-m1, ThaII-m2, ThaII-m3, and ThaII-m4 were amplified using the corresponding primers (Table 2). Each synonymous optimized gene was amplified using the same PCR reaction system. The reaction system is shown in Table 3, and the PCR amplification procedure is shown in Table 4.

[0042] Table 2. Information on optimized primers for semathymidine II synonymous gene amplification

[0043] Table 3. PCR reaction system for semathymidine II synonymous genes

[0044] Table 4. PCR amplification program for semathymidine II synonymous genes

[0045] After amplification, the PCR amplification products were detected by agarose gel electrophoresis and then purified. The expression vector pPIC9K was double-digested with EcoRI and NotI, and the linearized vector fragment was recovered. ThaII-m1, ThaII-m2, ThaII-m3, and ThaII-m4 were recombined into the EcoRI and NotI-digested expression vector pPIC9K, respectively, to construct the recombinant expression vectors pPIC9K-ThaII-m1, pPIC9K-ThaII-m2, pPIC9K-ThaII-m3, and pPIC9K-ThaII-m4. Figure 2 ).

[0046] Furthermore, in the previous step, the aforementioned semathymidine II synonymous optimized gene can also be ligated into pPICZ, pPICZα, pAO815, pGAPZ, or their derivative expression vectors. The expression cassette can be driven by the AOX1 promoter, AOX1 core promoter, GAP promoter, TEF1 promoter, PGK1 promoter, or their functional variants; the secretion signal peptide can be the α-factor signal peptide, the Pichia pastoris endogenous secretion signal peptide, or their functional variants.

[0047] The constructed recombinant expression vector was transformed into competent E. coli cells, positive clones were screened, plasmids were extracted, and sequencing verification was performed. Sequencing results showed that the inserted fragment sequence was correct and the reading frame was complete.

[0048] 2.2 Construction of the recombinant expression strain of semathymidine II The constructed recombinant expression plasmids pPIC9K-ThaII-m1, pPIC9K-ThaII-m2, pPIC9K-ThaII-m3, and pPIC9K-ThaII-m4 were linearized and transformed into Pichia pastoris GS115 competent cells, allowing them to integrate into the Pichia pastoris chromosome. After transformation, the bacterial culture was plated on selective plates and incubated at 30°C for 2 to 4 days to obtain candidate transformants. Single clones were picked, and colony PCR was performed on the transformants using primer pairs ThaII-m1-F / R, ThaII-m2-F / R, ThaII-m3-F / R, and ThaII-m1-F / R, respectively. A positive result was determined by the amplification of a target band of approximately 0.6 kb, confirming successful integration of the semathy II expression cassette. The colony PCR reaction system is shown in Table 5. The PCR reaction procedure is the same as in Table 4 of section 2.1.

[0049] Table 5. PCR reaction system of recombinant Pichia pastoris colonies

[0050] After screening by colony PCR, recombinant Pichia pastoris strains GS115 / ThaII-m1, GS115 / ThaII-m2, GS115 / ThaII-m3 and GS115 / ThaII-m4 were obtained.

[0051] In addition, the host strain in the above operation can also be X33, KM71, KM71H, SMD1168 or other Pichia pastoris engineered strains.

[0052] 2.3 Comparison of expression levels of different synonymous optimized genes of sematridin II in Pichia pastoris Positive transformants of the recombinant Pichia pastoris were selected and inoculated into 5 mL of BMGY medium, and pre-cultured at 30℃ and 200 rpm for 24 h. Then, they were transferred at a 1% (v / v) inoculation rate to 250 mL culture vessels containing 50 mL of BMGY medium and cultured at 30℃ and 200 rpm for 48 h. After culture, the cells were collected by centrifugation, the supernatant was removed, and the cells were resuspended in BMMY methanol induction medium and induced at 30℃ and 200 rpm. Methanol was added every 24 h during induction to maintain a final methanol concentration of 0.3% to 1.0% in the culture system. After 48 h of induction, the fermentation supernatant was collected by centrifugation, and the expression level of sematriene II secretion was detected by SDS-PAGE (results are shown in [link to results]). Figure 3 ).

[0053] Depend on Figure 3 It was found that the four synonymous optimized genes ThaII-m1, ThaII-m2, ThaII-m3, and ThaII-m4 obtained after optimization using various strategies for sematriline II could all achieve recombinant secretory expression in Pichia pastoris. Among them, the target protein band of the ThaII-m4 expression strain was the most obvious in the fermentation supernatant, and the secretory expression level of sematriline II was superior to that of ThaII-m1, ThaII-m2, and ThaII-m3. Therefore, the sematriline II synonymous optimized gene ThaII-m4 is suitable for efficient secretory expression in Pichia pastoris and can be used as a preferred synonymous optimized gene for subsequent fermenter-level expression validation.

[0054] Example 3 This embodiment is based on the high-efficiency ThaII-m4 gene expression strain of ThaII-m4 obtained in Example 2, and the expression in a fermenter is carried out as follows: The GS115 / ThaII-m4 strain, which showed a high expression level in Example 2, was selected for fermenter expression verification. A single colony was inoculated into 5 mL of YPD liquid medium and cultured at 30°C and 200 rpm for 24 h. Subsequently, it was transferred to 50 mL of YPD liquid medium at a 1% (v / v) inoculation rate and cultured at 30°C and 200 rpm for another 24 h to obtain the fermentation seed culture. The seed culture was then inoculated into the fermentation medium at a rate of 5% to 10% (v / v) for high-density culture. The culture temperature was controlled at 25°C to 30°C, the pH at 4.0 to 6.0, and dissolved oxygen was adjusted by stirring and aeration. After the cells reached the induction stage, semathy II expression was induced using methanol. The induction stage temperature was controlled at 20°C to 28°C, the pH at 3.5 to 6.0, and the induction time was 48 h to 168 h. Samples were taken periodically during fermentation, and the fermentation supernatant was collected by centrifugation. SDS-PAGE was used to detect the expression of semathy II at different induction time points (results are shown in Figure 1). Figure 4 (As shown). Figure 4 Lanes 1 to 6 represent fermentation supernatant samples after methanol induction for 12h, 24h, 48h, 72h, 96h, and 120h, respectively.

[0055] Depend on Figure 4 It was observed that the target band of semaside II in the fermentation supernatant gradually increased with prolonged induction time. Protein spectrometry confirmed that the target band was recombinant semaside II.

[0056] After fermentation, the fermentation broth was collected and purified as follows: (1) The fermentation broth product was separated from the Pichia pastoris cells and the supernatant of the fermentation broth by a tubular centrifuge (14000 rpm, 15 min); (2) A 6KD hollow fiber column was selected, and the supernatant was pumped into the hollow fiber column by a peristaltic pump. The outlet valve was adjusted so that the permeate flow rate was in the range of 1L / h-3L / h. The retaining liquid was collected at this time, and the liquid rich in semathy protein was obtained; (3) The liquid rich in semathy protein was dried (70℃, 24 h) and ground to obtain solid semathy protein powder; finally, a semathy II protein product with a purity of more than 90% and a yield of 1.0-1.5 g / L was obtained.

[0057] Example 4 This embodiment further explores the performance of the semathy II protein product obtained in Example 3, as detailed below: To verify whether the recombinant semaphore II prepared in Example 3 possesses a sweetening function, a sensory sweetness evaluation was performed on the purified semaphore II product. Recombinant semaphore II was serially diluted with deionized water to prepare test samples of different concentrations. Deionized water was used as a negative control, and sucrose solution as a positive control, and samples were randomly numbered. At least 10 healthy evaluators were selected to conduct the evaluation using a sweetness threshold method under blinded conditions. Evaluators started with low-concentration samples, rinsing their mouths with water after each evaluation and waiting a certain time before evaluating the next sample. The sensory evaluation scoring criteria are shown in Table 6.

[0058] Table 6 Sensory Sweetness Evaluation Scoring Criteria

[0059] After the evaluation, the sweetness scores of each sample were tallied, and the average score of at least 10 evaluators was calculated. The results showed that the negative control had no obvious sweetness, the positive control had a identifiable sweetness, and the recombinant semathymidine II sample could be identified as sweet by the evaluators within the corresponding concentration range. Moreover, the sweetness intensity score increased with increasing sample concentration. The sensory sweetness evaluation results are shown in Table 7.

[0060] Table 7 Sensory sweetness evaluation results of recombinant sematriol II

[0061] The results showed that the recombinant semaphore II obtained by secretory expression of the ThaII-m4 synonymous optimized gene in Pichia pastoris possessed the sweet function of semaphore II. This indicates that the synonymous optimized gene can increase the yield of recombinant semaphore II in the Pichia pastoris fermentation system without altering the amino acid sequence of the mature semaphore II protein, and the resulting protein retains the sweet function of semaphore II.

[0062] Based on the above series of studies, this invention successfully obtained a synonymous optimized gene suitable for the efficient secretion and expression of sematriaxone II by Pichia pastoris. Further fermentation using a Pichia pastoris strain containing this synonymous optimized gene yielded sematriaxone II protein with a high yield of 1.0-1.5 g / L, and also possessed the characteristic sweetness of natural sematriaxone II. It can be widely used in food, beverages, oral care, pharmaceutical excipients, and animal feed.

[0063] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0064] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A synonymous optimized gene suitable for efficient expression of sematridin II in Pichia pastoris, characterized in that, The nucleotide sequence of the synonymous optimized gene is shown in SEQ ID NO.

5.

2. The synonymous optimized gene according to claim 1, characterized in that, The amino acid sequence of the semathy II protein encoded by the synonymous optimized gene is shown in SEQ ID NO.

1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector includes the synonymous optimized gene as described in claim 1.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vectors include pPIC9K vector, pPICZ vector, pPICZα vector, pAO815 vector, and pGAPZ vector.

5. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector also includes a promoter, a secretory signal peptide coding sequence, and a transcription termination sequence.

6. The recombinant expression vector according to claim 5, characterized in that, The promoters include AOX1 promoter, AOX1 kernel promoter, GAP promoter, TEF1 promoter, and PGK1 promoter; The secretory signal peptide coding sequence includes the α-factor signal peptide coding sequence and the Pichia pastoris endogenous signal peptide coding sequence.

7. A recombinant Pichia pastoris strain, characterized in that, The recombinant Pichia pastoris strain includes the recombinant expression vector of claim 3.

8. The recombinant Pichia pastoris strain according to claim 7, characterized in that, The host strains of the recombinant Pichia pastoris strain include Pichia pastoris GS115, X33, KM71, KM71H, and SMD1168.

9. A method for preparing sematriol II, characterized in that, The preparation is as follows: the recombinant Pichia pastoris strain of claim 7 is induced to ferment and secrete semathys II; the fermentation supernatant is collected; the fermentation supernatant is separated and purified to obtain semathys II.

10. The application of sematriol II prepared by the method of claim 9 as a natural sweet protein in food, beverages, oral care, pharmaceutical excipients and feed.

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