Engineered OST1 signal peptide for enhancing expression of recombinant protein

Abstract

The invention discloses an engineered OST1 signal peptide which is a mutant formed by replacing or inserting residues VSS at 19-21 sites in a junction region of an H region and a C region of a wild type OST1 signal peptide with an amino acid sequence shown as SEQ ID NO: 1 by acidic residues D and E, and a composite secretion signal peptide formed by the engineered OST1 signal peptide and an alpha-factor Pro section connected to the C terminal of the engineered OST1 signal peptide, the method is used for driving secretory expression of foreign proteins in yeast, can significantly improve the secretory efficiency of high-electric-point proteins, glycoproteins and complex folded proteins, and has popularization and application prospects.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and fermentation engineering technology, specifically relating to an engineered OST1 signal peptide and its use in enhancing the expression of exogenous recombinant proteins in yeast. Background Technology

[0002] The secretory expression of recombinant proteins is a fundamental technology in modern biomanufacturing, food nutrition, functional ingredient production, and biomedicine. With the rapid increase in demand for exogenous proteins such as lactoferrin, glycoproteins, antimicrobial proteins, and immune-related proteins, establishing a safe, efficient, and industrially scalable recombinant protein production system has become a core technological goal for the industry. Among existing expression systems, Pichia pastoris (Komagataella phaffii) is widely used for recombinant protein secretory expression due to its advantages, including high-density fermentation, eukaryotic post-translational processing capabilities, simple fermentation conditions, and applicability to food-grade or nutrient-grade product development.

[0003] Despite this, Pichia pastoris still faces significant challenges in expressing certain structurally complex exogenous proteins with high folding requirements or polysaccharide sites, especially challenging secretory proteins such as lactoferrin. These proteins often exhibit problems such as low secretion levels, easy retention in the endoplasmic reticulum, limited folding efficiency, and degradation after accumulation. Taking lactoferrin as an example, its molecular weight is approximately 80 kDa, it contains multiple N-glycosylation sites and a complex tertiary structure, and it is highly dependent on the secretion pathway, thus becoming one of the typical challenges in improving the secretion system of Pichia pastoris.

[0004] In the secretory expression pathway of eukaryotes, the signal peptide is a crucial control element determining whether exogenous proteins can successfully enter the endoplasmic reticulum. A typical signal peptide comprises three structural regions: the N-region, the H-region, and the C-region, each significantly influencing its overall function. The N-region typically carries a positive charge, facilitating initial recognition by the secretory pathway; the H-region is a strongly hydrophobic core segment that aids in signal peptide insertion into membrane structures; and the C-region, located near the signal peptide's enzyme cleavage site, is a vital sequence region affecting signal peptide processing efficiency, protein entry into the secretory pathway, and subsequent expression levels.

[0005] Currently, commonly used signal peptides in Pichia pastoris include α-factor, KRE1, PHO1, SCW11, SWA2, and OST1, which is derived from yeast itself. While α-factor is widely used, its origin in Saccharomyces cerevisiae presents a potential limitation in food-grade applications due to its host-derived consistency. Furthermore, its performance remains limited when expressing certain target proteins (such as high molecular weight proteins or naturally folded proteins). Therefore, yeast-derived signal peptides that are closer to the host background and offer greater stability are gaining attention. Among these, OST1-pre (the signal peptide of the yeast oligosaccharide transferase complex subunit OST1), as a host-derived signal peptide, possesses advantages such as structural stability and strong adaptability; however, its sequence has not yet been systematically developed and engineered.

[0006] It is worth noting that many structurally complex exogenous proteins or those with specialized functional regions are high isoelectric point (pI) proteins. These proteins typically have a high isoelectric point (pI ≥ 8) and contain a large number of positively charged amino acid residues, making them particularly difficult to express in Pichia pastoris secretory expression. Examples include lactoferrin, small secretory antimicrobial proteins, human immune proteins, and some yeast-derived bioactive peptides. In actual production, high pI proteins are more prone to intracellular retention, decreased processing efficiency, and increased non-specific interactions, making them far more dependent on signal peptides than ordinary proteins. Existing signal peptide replacement strategies generally fail to significantly improve the expression of high pI proteins, and specific optimization strategies for high pI proteins are still lacking in publicly available literature.

[0007] Although signal peptide sequences significantly influence secretion efficiency, current research in signal peptide engineering primarily focuses on: replacing the overall signal peptide sequence from different sources; adjusting the positive charge density of the N-region; enhancing the overall hydrophobicity of the H-region; or using large-scale library screening to find new, highly efficient signal peptides. However, these methods largely lack predictability, stability, or are only effective for specific proteins. For lactoferrin and other difficult-to-secrete proteins, existing systems still have significant limitations in enhancing secretion capacity, particularly lacking a secretion enhancement system that is structurally simple, highly controllable, highly adaptable to the host, and industrially scalable. In summary, there is an urgent need for a novel enhancement strategy that can precisely regulate the key region sequence of the signal peptide and significantly improve the secretion efficiency of high isoelectric point proteins and other structurally complex proteins, to overcome the technical bottlenecks of unstable effects, limited applicability, and lack of host-derived sequence optimization in existing methods. Summary of the Invention

[0008] Existing technical literature has not yet optimized the signal peptide by local micromutation of the C region (or the interface region between the H and C regions), and there is a lack of precise sequence regulation strategies based on the host's own signal peptide. There have been no reports of attempts to use the H-C region junction of the yeast OST1-pre signal peptide as an engineering target, nor have there been any technical solutions for introducing acidic amino acid residues in this region to improve the secretion efficiency of high isoelectric point proteins.

[0009] Based on years of experience in signal peptide development and utilization, we attempted to develop an enhanced secretion system based on the OST1-pre signal peptide. By introducing acidic amino acid residues at the C region or the H region-C region junction of the signal peptide, we constructed a signal peptide variant that significantly improves the secretion efficiency of exogenous proteins. This variant was then fused with the α-factor Pro region and applied to the secretory expression of recombinant proteins in Pichia pastoris. We engineered the natural pre-signal peptide sequence (OST1-pre, amino acid sequence as shown in SEQ ID NO: 1) of the Pichia pastoris oligosaccharide transferase complex subunit OST1. We precisely inserted or replaced 1-2 acidic amino acid residues, including aspartic acid (D) or glutamic acid (E), at the C region near the signal peptide cleavage site or at the junction of the C and H regions, obtaining 40 variants such as OST1-pre+19D (SEQ ID NO: 2), OST1-pre+19ED (SEQ ID NO: 3), OST1-pre+19EED (SEQ ID NO: 4), and OST1+21D (SEQ ID NO: 21). These signal peptide variants were further fused with the α-factor Pro region (SEQ ID NO: 5) to form a complex secretory signal peptide, which drives the expression of the exogenous protein buffalo lactoferrin (wBLF, amino acid sequence as shown in SEQ ID NO: 10, nucleotide sequence of the encoding gene as shown in SEQ ID NO: 13). The fused complex signal peptide structure typically includes a signal peptide region, a Pro region, and a target protein coding sequence, and can be stably expressed by constructing an expression vector and integrating it into the Pichia pastoris genome.After multiple comparative experiments, it was found that the complex signal peptides OST1-pre+19D + Pro (amino acid sequence as shown in SEQ ID NO: 7), OST1-pre+19ED + Pro (amino acid sequence as shown in SEQ ID NO: 8), and OST1-pre+21D + Pro (amino acid sequence as shown in SEQ ID NO: 22), fused with the α-factorPro region of OST1-pre+19D, OST1-pre+19ED + Pro (amino acid sequence as shown in SEQ ID NO: 8), and OST1-pre+21D + Pro (amino acid sequence as shown in SEQ ID NO: 22), significantly promoted the expression and secretion of BLF in Pichia pastoris, with wBLF expression levels increasing by approximately 2.4-fold, 1.5-fold, and 2.3-fold, respectively. However, the complex signal peptides OST1-pre+19EED + Pro (amino acid sequence as shown in SEQ ID NO: 9), OST1-pre+15D + Pro (amino acid sequence as shown in SEQ ID NO: 16), OST1-pre+16D + Pro (amino acid sequence as shown in SEQ ID NO: 17), and OST1-pre+17D + Pro (amino acid sequence as shown in SEQ ID NO: 18) did not show significant changes. However, the effects of SEQ ID NO: 18 and OST1-pre+18D+Pro (amino acid sequence as shown in SEQ ID NO: 19) were not good, even worse than the unmutated wild type. Accordingly, the present invention includes the following technical solutions.

[0010] The first aspect of this invention provides an engineered OST1 signal peptide, which is a mutant of the yeast's own secretory signal peptide OST1 (or OST1-pre) signal peptide. This mutant is a wild-type OST1 signal peptide with the amino acid sequence shown in SEQ ID NO: 1, in which any one, two, or three of the residues VSS at positions 19-21 (H-region and C-region boundary) are replaced or inserted by acidic residues D (aspartic acid) and / or E (glutamic acid). SEQ ID NO: 1 is MRQVWFSWIVGLFLCF FNV SSA (SEQ ID NO: 1).

[0011] Preferably, the amino acid sequences of the above-mentioned OST1 signal peptide mutants are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 21:

[0012] MRQVWFSWIVGLFLCFFNV D SSA (SEQ ID NO: 2) is a mutant of SEQ ID NO: 1 with a D inserted after residue V at position 19. In this paper, it is named OST1+19D, OST1-19D, OST-19D or OST1-pre+19D.

[0013] MRQVWFSWIVGLFLCFFNV ED SSA (SEQ ID NO: 3) is a mutant of SEQ ID NO: 1 with ED inserted after residue V at position 19. In this paper, it is named OST1+19ED, OST1-19ED, OST-19ED or OST1-pre+19ED.

[0014] MRQVWFSWIVGLFLCFFNVSS D A (SEQ ID NO: 21) is a mutant of SEQ ID NO: 1 with D inserted after residue S at position 21. In this paper, it is named OST1+21D, OST1-21D, OST-21D or OST1-pre+21D.

[0015] A second aspect of this invention provides a complex signal peptide, or complex secretion signal peptide, comprising the OST1 signal peptide mutant as described above as a pre-sequence (pre-region) of the complex signal peptide and an α-factor Pro region immediately adjacent to its C-terminus, wherein the amino acid sequence of the α-factor Pro region is shown in SEQ ID NO: 5:

[0016] APVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 5).

[0017] Preferably, the amino acid sequence of the OST1 signal peptide mutant, i.e., the pre-signal peptide sequence (pre-region), is as shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 21, and the corresponding amino acid sequence of the complex (secreted) signal peptide is as shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 22.

[0018] MRQVWFSWIVGLFLCFFNVDSSAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 7), named OST1-pre+19D + Pro;

[0019] MRQVWFSWIVGLFLCFFNVEDSSAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 8), named OST1-pre+19ED+Pro.

[0020] MRQVWFSWIVGLFLCFFNVSSDAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 22), named OST1-pre+21D+Pro.

[0021] A third aspect of the invention provides a gene encoding a complex (secreted) signal peptide as described above.

[0022] Preferably, the gene encoding the complex (secretory) signal peptide SEQ ID NO: 7 is a polynucleotide as shown in the nucleotide sequence SEQ ID NO: 11, or a polynucleotide having 90% or more, preferably 92% or more, preferably 95% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more homology with SEQ ID NO: 11;

[0023] The gene encoding the complex (secretory) signal peptide SEQ ID NO: 8 is a polynucleotide as shown in the nucleotide sequence SEQ ID NO: 12, or a polynucleotide having 90% or more, preferably 92% or more, preferably 95% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more homology with SEQ ID NO: 12;

[0024] The gene encoding the complex (secretory) signal peptide SEQ ID NO: 22 is a polynucleotide of the nucleotide sequence SEQ ID NO: 23, or a polynucleotide having 90% or more, preferably 92% or more, preferably 95% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more homology with SEQ ID NO: 23;

[0025] The fourth aspect of the present invention provides the use of the OST1 signal peptide mutant, the complex signal peptide, and the gene described above in improving the secretion efficiency of exogenous recombinant proteins expressed in yeast.

[0026] The fifth aspect of the present invention provides a DNA molecule, which is an integrated fragment comprising the gene as described above and an upstream promoter such as the AOX1 promoter (nucleotide sequence as shown in SEQ ID NO: 14) or the EM7 promoter, a downstream target protein coding gene, and an AOX1 terminator (nucleotide sequence as shown in SEQ ID NO: 15), which is an expression cassette / expression box of the target protein gene.

[0027] A sixth aspect of the present invention provides a recombinant expression vector suitable for expressing exogenous target proteins, such as bovine lactoferrin (BLF), preferably buffalo lactoferrin coding sequences (wBLF, amino acid sequence as shown in SEQ ID NO: 10, nucleotide sequence of coding gene as shown in SEQ ID NO: 13), in yeast, comprising a fusion protein gene of the gene described above and the target protein coding gene, and an upstream promoter such as the AOX1 promoter.

[0028] In one embodiment, the plasmid vector of the recombinant expression vector is pPICZα, and the yeast is Pichia pastoris (Komagataella phaffii, Pichia pastoris), such as Pichia pastoris GS115 or Saccharomyces cerevisiae (baker's yeast).

[0029] The seventh aspect of the present invention provides a yeast engineered strain, which is a Pichia pastoris engineered strain or a Saccharomyces cerevisiae engineered strain, and the genome integrates the DNA molecules described above. The engineered strain is preferably a transformant of a yeast host strain transformed with the recombinant expression vector described above.

[0030] The eighth aspect of the present invention provides the use of the yeast engineered strains described above in the fermentation production of recombinant proteins such as high isoelectric point proteins, glycoproteins and complex folded proteins such as bovine lactoferrin.

[0031] This invention utilizes acidic residue insertion or substitution mutations at the H-C region junction of the yeast-derived OST1 signal peptide pre-sequence (wild-type OST1-pre, SEQ ID NO: 1) to introduce aspartic acid (D) or glutamic acid (E), constructing variants such as OST1-pre+19D (SEQ ID NO: 2), OST1-pre+19ED (SEQ ID NO: 3), and OST1-pre+21D (SEQ ID NO: 21). These mutant signal peptides are then fused with the α-factor Pro region (SEQ ID NO: 5) to form a complex secretion signal peptide that drives the secretory expression of exogenous proteins. Experimental results show that, compared to wild-type OST1-pre (fused with the same Pro region to form the wild-type complex signal peptide SEQ ID NO: 6), some mutant complex signal peptides significantly increase the secretion of exogenous proteins in engineered Pichia pastoris, for example, lactoferrin expression can be increased by 1.5-2.4 times. The OST1 signal peptide mutant and complex signal peptide of the present invention are suitable for promoting the efficient fermentation production of lactoferrin, glycoproteins and other structurally complex proteins, and have broad prospects for industrial application. Attached Figure Description

[0032] Figure 1 This paper presents a schematic diagram of the structure of the fusion gene expression vector for expressing buffalo lactoferrin (wBLF) in Pichia pastoris constructed according to the present invention. Figure a shows the overall circular structure of the pPICZα-OST-WBLF vector, including the AOX1 promoter, the OST-α-factor secretion signal region (containing the pre region of OST and the Pro region of α-factor), the target protein buffalo lactoferrin coding sequence (wbLF), the AOX1 terminator, the EM7 promoter, the Zeocin resistance gene, and the bacterial origin of replication (ori), among other basic functional elements. Figure b shows a schematic diagram of the linear structure of the expression cassette, including the positions of the signal peptide region and the Pro region, the wbLF target protein coding frame, the relative layout of the AOX1 promoter and terminator, the CYC1 terminator, and the organization of the vector backbone. It also indicates the binding sites of multiple primer sets (such as VER-EAEA-F3, wbLF-VER-F1, AOX-T-VER-F, 3aox-R, Ver-F2, etc.) used to verify the correctness of vector construction. This figure is used to illustrate the overall structural design of the fusion gene expression construct of the present invention, the arrangement of sequence modules, and the spatial relationship of key functional elements in the vector.

[0033] Figure 2The results of plasmid-level PCR identification of the various signal peptide mutant expression cassettes constructed in this invention are shown. The upper figure is an agarose gel electrophoresis image used to verify the sequence correctness of the constructs OST1-pre+19D, OST1-pre+19ED, OST1-pre+19EED, and the control signal peptide sp14-WBLF. The lower figure shows the correspondence between the signal peptide and primer sequences. The target region was amplified using primer pairs paox-ver-f and WBLF-AOX-VER-R. The amplified band sizes generated by each construct were OST1-pre+19D (415bp), OST1-pre+19ED (418bp), OST1-pre+19EED (421bp), and OST1-WBLF (415bp), respectively. In the electrophoresis results, Lanes 1-2 represent the OST1-pre+19D construct, Lanes 3-4 represent the OST1-pre+19ED construct, Lanes 5-6 represent the OST1-pre+19EED construct, and Lanes 7-8 represent the sp14-WBLF construct; M represents the DNA molecular weight standard. All constructs showed band sizes consistent with the theoretical values, indicating correct plasmid construction and accurate sequencing verification.

[0034] Figure 3 The results show the validation of strains after the integration of the buffalo lactoferrin (wbLF) expression cassette into the Pichia pastoris genome using different signal peptides (OST, OST-19D, OST-19ED, OST-19EED). In image a, agarose gel electrophoresis image identified by PCR shows the integrated fragment amplified using specific primers, with a theoretical amplification band size of approximately 1962 bp. Electrophoresis results show that transformants in Lanes 1-15 all exhibited bands consistent with the theoretical values, indicating successful integration of the wbLF expression cassette into the host genome. M represents the molecular weight standard. Image b shows a plate image of the resistance selection culture for positive transformants. Different partitions in the plate correspond to different integrand strain numbers, showing multiple successfully obtained positive Pichia pastoris transformant colonies for subsequent fermentation expression and yield screening.

[0035] Figure 4The results of detection and analysis of buffalo lactoferrin (wbLF) driven by different signal peptides (OST, OST-19D, OST-19ED, OST-19EED) in Pichia pastoris strain are shown. In the figure, a is an SDS-PAGE gel image of the expression product after affinity purification, with transformants 1 and 2 corresponding to each signal peptide construct in the lanes. Target protein bands of approximately 80 kDa were detected under different signal peptide conditions, corresponding to the theoretical molecular weight of lactoferrin. Compared with the control strain, the OST-19D (i.e., OST1-19D) and OST-19ED (i.e., OST1-19ED) groups showed higher band intensities, while the band of the OST-EED (i.e., OST1-19EED) group was significantly weaker. b shows the statistical results of secreted protein concentration obtained based on ELISA or other quantitative methods, showing that the expression levels of the OST-19D and OST-19ED constructs were significantly higher than those of the wild-type OST group, while the expression level of the OST-19EED construct was the lowest. This figure shows that different signal peptides have significant differences in the secretory expression efficiency of lactoferrin, and the sensitivity to acidic mutations in the HC region shows a characteristic expression trend.

[0036] Figure 5 The results of detection and analysis of buffalo lactoferrin (wbLF) driven by different signal peptides (OST, OST-21D, OST-20D, OST-19D, OST-18D, OST-17D, OST-16D, and OST-15D) in Pichia pastoris strain are shown. In the figure, a is the SDS-PAGE gel image of the expression product after affinity purification, with transformants 1 and 2 corresponding to each signal peptide construct in the lanes. Target protein bands of approximately 80 kDa were detected under different signal peptide conditions, corresponding to the theoretical molecular weight of lactoferrin; b is the statistical result of secreted protein concentration obtained based on ELISA or other quantitative methods. The experimental results show that, compared with the unmutated wild-type OST1-pre signal peptide, the introduction of a single acidic residue in the pre region resulted in a significant site-dependent difference in the secretory expression level of buffalo lactoferrin. Specifically, the insertion of aspartic acid residues at positions 19 (OST1-19D) and 21 (OST1-21D) significantly increased the secretory expression level of buffalo lactoferrin, with corresponding protein band intensity and grayscale quantification results being significantly higher than those of wild-type OST1-pre. In contrast, when acidic residues were inserted into other positions in the pre region (such as positions 15–18), the secretory expression level of buffalo lactoferrin did not increase, and some mutants even showed a decreasing trend. This indicates that the promoting effect of acidic residue insertion on signal peptide function is not universal but highly dependent on its specific insertion position within the pre region. Detailed Implementation

[0037] This project aims to address the generally low secretion efficiency of lactoferrin, glycoproteins, and other high isoelectric point proteins in existing Pichia pastoris expression systems. Traditional signal peptide replacement strategies can produce some improvement in certain target proteins, but the overall improvement is limited, and research on the engineering of host-derived signal peptides is relatively scarce. In particular, the yeast OST1-pre signal peptide, as a host-derived sequence, exhibits good stability and adaptability, but fine-tuning strategies for its key regions have not been reported, failing to meet the demand for efficient secretion of high isoelectric point proteins and structurally complex proteins. To provide an enhanced secretion system based on the OST1-pre signal peptide, we constructed a signal peptide variant that significantly improves the secretion efficiency of exogenous proteins by introducing acidic amino acid residues at the C region or the H-C region junction of the signal peptide. This variant was then fused with the α-factor Pro region and applied to the secretory expression of recombinant proteins in Pichia pastoris.

[0038] To achieve the above design, we engineered the natural signal peptide pre-sequence (OST1-pre, SEQ ID NO: 1) of the Pichia pastoris oligosaccharide transferase complex subunit OST1. We precisely inserted or replaced 1-3 acidic amino acid residues, such as aspartic acid (D) or glutamic acid (E), in the C region near the signal peptide cleavage site or at the junction of the C and H regions, obtaining dozens of variants, including OST1-pre+19D (SEQ ID NO: 2), OST1-pre+19ED (SEQ ID NO: 3), OST1-pre+19EED (SEQ ID NO: 4), and OST1+21D (SEQ ID NO: 21). These signal peptide variants were further fused with the α-factor Pro region (SEQ ID NO: 5) to form complex secretory signal peptides such as OST1-pre+19D+Pro (amino acid sequence as shown in SEQ ID NO: 7), OST1-pre+19ED+Pro (amino acid sequence as shown in SEQ ID NO: 8), and OST1-pre+21D+Pro (amino acid sequence as shown in SEQ ID NO: 22), which are used to drive the expression of exogenous proteins. The gene structure after the complex secretory signal peptide is fused with the exogenous protein typically includes a signal peptide region, a Pro region, and a target protein coding sequence. Stable expression of exogenous proteins can be achieved by constructing an expression vector and integrating it into the Pichia pastoris genome.

[0039] Compared with the control strain using the wild-type OST1-pre signal peptide, the signal peptide mutants of this invention exhibit significant advantages in secretory expression. Validated using lactoferrin wBLF as a model protein, under the same expression vector and culture conditions, OST1-pre+19D (SEQ ID NO: 2) increased lactoferrin secretion by approximately 2.4 times, while OST1-pre+19ED (SEQ ID NO: 3) increased it by approximately 1.5 times, and OST1-pre+21D (SEQ ID NO: 21) increased it by approximately 2.3 times. Individual mutants such as the signal peptide variants OST1-pre+19D, OST1-pre+19ED, and OST1-pre+21D demonstrated good stability in multiple independent experiments, making them suitable for high isoelectric point proteins, polysaccharidated proteins, and other structurally complex exogenous proteins. The above-mentioned mutations of the OST1-pre signal peptide are limited to local optimization of individual residues, thus having the advantages of simple construction, high predictability, good adaptability, and high safety, making it particularly suitable for food-grade, nutritional and health-related, and large-scale fermentation production scenarios.

[0040] This invention constructs an enhanced signal peptide system that significantly improves the secretion efficiency of exogenous proteins by precisely inserting or replacing amino acids in key regions of the OST1-pre signal peptide. This system not only enhances the secretion levels of high isoelectric point proteins such as lactoferrin, but also provides a new technical approach for the efficient expression of other structurally complex proteins and glycoproteins, possessing industrial application value.

[0041] In this article, the terms “(exogenous protein expression level) increase”, “enhancement” or “enhancement” used above mean an increase of at least 20% or more compared to the reference level, such as at least 30% or more, at least 50% or more, at least 80% or more, at least about 1, at least about 2, at least about 3, at least about 4 or at least about 5 times compared to the reference level.

[0042] An effective method to achieve co-expression of the OST1-pre signal peptide variant and the exogenous protein in yeast is to use fusion gene expression, that is, to form a fusion gene by combining the signal peptide variant encoding gene, the Pro segment gene, and the downstream exogenous protein gene.

[0043] The specific implementation methods for constructing OST1-pre signal peptide mutants and using them to improve the secretory expression efficiency of exogenous proteins in yeast generally include the following aspects:

[0044] a. Signal peptide sequence design: Based on the characteristics of the signal peptide domain, the junction of the H and C regions of the Pichia pastoris OST1-pre sequence (SEQ ID NO: 1) was modified at a specific site to introduce 1-3 acidic residues, resulting in signal peptide variants such as OST1-pre+19D (SEQ ID NO: 2), OST1-pre+19ED (SEQ ID NO: 3), and OST1-pre+19EED (SEQ ID NO: 4). These mutant pre regions were then linked to the α-factor Pro region (SEQ ID NO: 5) to construct a complex secretory signal peptide sequence.

[0045] b. Fusion gene construction and expression vector assembly: The buffalo lactoferrin wBLF gene (SEQ ID NO: 13) was amplified by PCR, and the signal peptide variant sequence was amplified simultaneously. Homologous arms or suitable restriction endonuclease sites were designed in the primers for subsequent assembly. Using Gibson assembly, overlap-PCR, or enzyme digestion-ligation techniques, the signal peptide variant gene (e.g., SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 23, etc.) was fused with the wBLF coding sequence (SEQ ID NO: 13) to construct a complete open reading frame. Short linker peptides were added as needed to ensure signal peptide transport function. The resulting fusion gene fragment was combined with the AOX1 promoter (SEQ ID NO: 14), α-pro region, AOX1 terminator (SEQ ID NO: 15), and Zeocin resistance selection marker to construct an expression cassette and insert it into the pPICZα vector. The integrity and correctness of the expression vector construction were verified by enzyme digestion and sequencing (see [link to documentation]). Figure 2 (Verification results shown).

[0046] c. Pichia pastoris transformation and positive clone screening: The constructed expression vector was introduced into Pichia pastoris cells, such as GS115, using electroporation, and resistant colonies were screened. Transformants were integrated and validated using PCR; the amplified fragment size should be consistent with the theoretical value (approximately 415-421 bp, depending on the mutation type) (see [link to relevant documentation]). Figure 2 PCR-positive transformed strains were selected and subjected to repeated validation and pick culture on plates (e.g., ...). Figure 3 As shown in b), a stable expression strain library was established.

[0047] d. Induction of Expression and Protein Analysis: Positive strains were inoculated into fermentation medium to induce lactoferrin expression, and samples were taken periodically during the culture process. Protein analysis of the fermentation supernatant was performed by SDS-PAGE; a lactoferrin band was detected at approximately 80 kDa (e.g., ...). Figure 4 As shown in Figure a). The expression level of lactoferrin was quantified using ELISA (as shown in Figure a).Figure 4 (b) The results showed that mutants OST1-pre+19D, OST1-pre+19ED and OST1-pre+21D (SEQ ID NO: 21) could significantly increase lactoferrin secretion; while the promoting effect of OST1-pre+19EED and others was poor, even worse than that of wild-type OST1-pre, indicating that the actual effect of many mutants is difficult to predict.

[0048] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0049] Example

[0050] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0051] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0052] Materials and methods

[0053] In the embodiments, the whole gene synthesis, primer synthesis and sequencing were all completed by Suzhou Genewiz Biotechnology Co., Ltd.

[0054] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual" (4th Edition), edited by M.R. Green and J. Sambrook (USA), translated by He Fuchu, Science Press, Beijing, 2017. Specific experimental conditions can be determined through simple experiments if necessary.

[0055] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0056] The strains used included Pichia pastoris GS115, which was purchased from the Shanghai Institute of Microbiology.

[0057] The PCR amplification primers for some gene fragments in this embodiment are listed in Table 1.

[0058] Table 1. Primers for PCR amplification of gene fragments in this embodiment

[0059] Primer name Sequence (5’-3’) lox71-ResP-F AAGATTAAGTGAGAAtcaaTGATCCCCCACACACCATAGC lox66-ResT-R taccgTTCGTATAATGTATGCTATACGAAGTTATGCAAATTAAAGCCTTCGAGCG Paox1(lox66)-F TTTGCATAACTTCGTATAGCATACATTATACGAAcggtagttggtattgtgaaatagac Paox1-R catcgtttcgaataattagttgtt pre-F AGGCAGGTTTGGTTCTCTTGGATTGT pro-R TCTTCTGTTGTAGTGTTGACTGGAGCAGCAGAAGACACGTTGAAAAAACA pAOX1-a-factor-F ACTAATTATTCGAAACGATGAGGCAGGTTTGGTTCTCTTG afa(wEAM)-R GTTGAGAAATAGTACACCATCTAACGTTCTTTCTTGGAGCtcttttctcgagagatacc ORI-pAOX1-F CTCACGTTAAGGGATTTTGGTCATGAGATCAGATCTAACATCCAAAGACGAAAGGTTG OST-pAOX-R CACAATCCAAGAGAACCAAACCTGCCTCATCGTTTCGAATAATTAGTTGTTTTTTGATC pAOX1-OST-F GATCAAAAAACAACTAATTATTCGAAACGATGAGGCAGGTTTGGTTCTCTTGGATTGTG WBLF-OST-R TAACATTTTTTCTTGGAGCAGCTTCAGCCTCTCTTTTCTCGAGAGATACCCCTTC OST-WBLF-F GGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTGCTCCAAGAAAAAATGTTAGATGGTG tAOX1-WBLF-R AGGCAAATGGCATTCTGACATCCTCTTGATTAGAATCTAGCAAGACCGGTCTTC WBLF-tAOX1-F GAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGC pTEF-tAOX1-R TATGGTGTGTGGGGGATCCGCACAAACGAAGGTCTCACTTAATCTTCTGTACTCTGAAG tAOX1-pTEF-F TTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGCGGATCCCCCACACACCATAG pAOX1-ORI-R AACCTTTCGTCTTTGGATGTTAGATCTGATCTCATGACCAAAATCCCTTAACGTGAG Ost-DF CAACGTGGACTCTTCTGCTGCTCCAGTCAACAC Ost-DR CAGCAGAAGAGTCCACGTTGAAAAAACATAGGAAC Ost-ED-F CAACGTGGAGGACTCTTCTGCTGCTCCAGTCAACAC OST-ED-R CAGAAGAGTCCTCCACGTTGAAAAAACATAGGAACAATCC Ost-EED-F GTTTTTTCAACGTGGAGGAGGACTCTTCTGCTGCTCCAGTCAACAC OST-EED-R AGCAGAAGAGTCCTCCTCCACGTTGAAAAAACATAGGAACAATC

[0060] The suffix "F" in primer names indicates forward; "R" indicates reverse.

[0061] Example 1: Integrating buffalo lactoferrin expression cassettes with different signal peptides into GS115

[0062] Using the HZP-sgRNA plasmid (a gift from Professor Lian Jiachang's group at Zhejiang University; plasmid structure description can be found in the literature DOI: 10.1021 / acssynbio.1c00307) as a template, and lox71-ResP-F / lox66-ResT-R as primers, the zeocin fragment was amplified by PCR. Using the Pichia pastoris genome as a template, and Paox1(lox66)-F / Paox1-R as primers, the Paox1 fragment of the Pichia pastoris AOX1 gene promoter was amplified by PCR. For the signal peptide portion, the engineered OST1-pre (variant) pre-region sequence was amplified using the genetically synthesized OST1-pre+19D, OST1-pre+19ED, and OST1-pre+19EED fragments as templates and pre-designed pre-F / pro-R primers. Simultaneously, the α-pro (α-factor pre-sequence) fragment was amplified by PCR using the pPIC9K plasmid as a template and pAOX1-a-factor-F / afa(wEAM)-R primers. The PCR fragments were purified using a gel extraction kit. One μL of the recovered PCR product fragments Paox1, OST1-pre (variant), and α-pro were mixed as a template, and the Paox1-OST1-pre (variant)-α-pro fusion fragment was amplified by overlap PCR using Paox1(lox66)-F / afa(wEAM)-R primers. Using the codon-optimized buffalo lactoferrin (BLF) gene (SEQ ID NO: 13) as a template, the BLF fragment was amplified by PCR using BLF(wM)-F / BLF-R primers. Using the Pichia pastoris genome as a template, the Taox1 fragment, the terminator of the Pichia pastoris AOX1 gene, was amplified by PCR using Taox1(BLF)-F / Taox1-R primers. One μL of the recovered BLF and Taox1 fragments from the above PCR products were mixed and used as a template for overlap PCR amplification to obtain the BLF-Taox1 fragment. One microliter of each of the recovered PCR product fragments zeocin, Paox1-OST1-pre (variant)-α-pro, and BLF-Taox1 was mixed as a template. Using lox71-ResP-F / Taox1-R as primers, overlap PCR was performed to amplify the fragment finally used for bovine lactoferrin integration. This fragment is an integrated fragment of the engineered signal peptide OST1-pre (variant) and the bovine lactoferrin fusion gene located downstream of it. That is, the lactoferrin recombinant expression integration structure of the present invention: zeocin-Paox1-OST1-pre (variant)-α-pro-BLF-Taox1.

[0063] The preparation process of competent cells of Pichia pastoris is as follows:

[0064] (1) Take out the P. pastoris GS115 glycerol bacteria stored in the -80°C freezer, pick a small amount from the frozen tube and streak it onto YPD antibiotic-free solid medium, incubate at 30°C for 3 days until larger colonies grow, then remove them;

[0065] (2) Pick larger colonies from the plate and inoculate them into 10 mL of YPD medium. Incubate at 30°C and 200 rpm for 1 day.

[0066] (3) Secondary activation: Inoculate 1 mL of bacterial culture obtained in step (2) into 100 mL of YPD medium and culture for 4-5 h to keep the cells highly active.

[0067] (4) Place the culture medium on ice and let it stand. Then dispense it into sterile centrifuge tubes and centrifuge at 4°C and 4,000×g for 5 min. Discard the supernatant. Add 10 mL of sterile water to resuspend the cells and repeat the above operation. Wash with water 3 times in total.

[0068] (5) After washing, add 1 mL of pre-cooled 1 M sorbitol to each tube to resuspend the cells, transfer to a sterile 1.5 mL centrifuge tube, centrifuge at 4°C and 4,000×g for 5 min, and discard the supernatant;

[0069] (6) Add 80 μL of pre-cooled 1 M sorbitol to each tube to resuspend the cells, thus completing the preparation of P. pastoris GS115 competent cells.

[0070] Add the constructed lactoferrin expression integration fragment (5-10 ng) to the prepared competent cells, mix gently, and incubate on ice for 5 min. After the ice incubation, transfer the mixture to a 0.2 cm electroporation cuvette and electroporate at 1,500 V for 5 ms. After electroporation, quickly add 1 mL of ice-cold 1 M sorbitol, mix gently by pipetting, and transfer to a centrifuge tube. Incubate at 30°C in a metal bath for 1-2 h. After incubation, centrifuge at 6,000 × g for 5 min, discard the supernatant, gently resuspend the pellet, spread it on MD plates, and incubate at 30°C for approximately 3 days to obtain positive colonies.

[0071] Transformants were picked from MD plates and subjected to colony PCR. A positive integration strain was identified by amplifying a band of approximately 4.8 kb using lox71-ResP-F / Taox1-R primers. Pichia pastoris engineered strains expressing OST1-pre+19D-BLF, OST1-pre+19ED-BLF, and OST1-pre+19EED-BLF were constructed using the same method.

[0072] Figure 1The results of PCR electrophoresis verification after the OST1-pre+19D-lactoferrin fusion gene was integrated into the Pichia pastoris genome are shown.

[0073] Example 2: Fermentation detection and analysis of bovine lactoferrin expression levels in engineered bacteria and its purification.

[0074] Select the positive transformants and inoculate them into BMGY medium. Incubate at 30°C and 250 rpm for 16-20 h until OD500 reaches the target value. 600 Centrifuge at 4000×g for 10 min, collect the bacterial cells, and resuspend the cells in BMMY medium to OD200. 600 The value was approximately 1. Expression was performed at 28℃, 250 rpm, and pH 6.0. Methanol, the inducer, was added every 24 h until the final concentration was 0.5% v / v. The total induction time was 96 h.

[0075] BMGY medium: yeast extract 10 g / L, peptone 20 g / L, potassium phosphate buffer (pH 6.0) 100 mM, YNB 13.4 g / L, biotin 0.4 mg / L, glycerol 10 mL / L. BMMY medium: yeast extract 10 g / L, peptone 20 g / L, potassium phosphate buffer (pH 6.0) 100 mM, YNB 13.4 g / L, biotin 0.4 mg / L, methanol 5 mL / L.

[0076] The shake-flask fermentation process is as follows:

[0077] (1) Select the positive transformants and incubate them in test tubes with YPD overnight for 24 hours.

[0078] (2) Inoculate into 30 ml of BMGY medium at a ratio of 3% v / v, and incubate at 30℃ and 250 rpm for 16-20 h.

[0079] (3) Centrifuge at 4000×g for 10 min, collect the bacterial cells, and resuspend the cells in 50 ml of BMMY medium until OD. 600 It is around 1.

[0080] (4) Expression was carried out at 30℃, 250 rpm and pH 6.0.

[0081] (5) Add methanol as an inducer every 24 hours until the final concentration is 0.5% v / v.

[0082] (6) The total induction time was 96 h. The endpoint OD600 was about 8-10.

[0083] Buffalo lactoferrin secreted by SDS-PAGE and Western blot was detected and analyzed. Results are shown below. Figure 4 .

[0084] Figure 4 The experimental results showed that, compared with the unmutated wild-type OST1-pre signal peptide, both OST1-pre+19D and OST1-pre+19ED could significantly increase the secretory expression level of bovine lactoferrin, but the secretory expression level of bovine lactoferrin corresponding to the mutant OST1-pre+19EED decreased.

[0085] Example 3: Investigation of aspartic acid (D) mutants inserted at other sites on the OST1-pre signal peptide

[0086] Following the methods described in Examples 1 and 2, mutant strains of Pichia pastoris, namely OST-15D (SEQ ID NO: 16), OST-16D (SEQ ID NO: 17), OST-17D (SEQ ID NO: 18), OST-18D (SEQ ID NO: 19), OST-20D (SEQ ID NO: 20), and OST-21D (SEQ ID NO: 21), were constructed by inserting aspartic acid (D) at positions 15-21 near the C-terminus of wild-type OST1-pre (SEQ ID NO: 1). Fermentation was performed, and the expression level of bovine lactoferrin in the engineered strains was detected and purified.

[0087] Example 4: Analysis of the secretion-enhancing effect of signal peptide variants

[0088] Comparative analysis of ELISA data revealed significant differences in lactoferrin secretion levels among the three expression systems constructed from the three signal peptides. Using wild-type OST1-pre (SEQ ID NO: 1) as a control, the mutant OST1-pre+19D (SEQ ID NO: 2) showed an approximately 1.5-fold increase, and the mutant OST1-pre+19ED (SEQ ID NO: 3) showed an approximately 2.4-fold increase (see...). Figure 4 (See Table 2), the mutant OST-21D (SEQ ID NO: 21) can increase by approximately 2.3 times (see Table 2). Figure 5 (and Table 2).

[0089] Figure 5Experimental results showed that, compared with the unmutated wild-type OST1-pre signal peptide, the secretory expression level of buffalo lactoferrin exhibited a significant site-dependent difference after the introduction of a single acidic residue in the pre region. Specifically, the insertion of aspartic acid residues at positions 19 (OST1-19D) and 21 (OST1-21D) significantly increased the secretory expression level of buffalo lactoferrin, with corresponding protein band intensity and grayscale quantification results significantly higher than those of wild-type OST1-pre. In contrast, when acidic residues were inserted into other positions in the pre region (such as positions 15–18), the secretory expression level of buffalo lactoferrin did not increase, and some mutants even showed a decreasing trend. This indicates that the promoting effect of acidic residue insertion on signal peptide function is not universal but highly dependent on its specific insertion position within the pre region.

[0090] Table 2. Results of buffalo lactoferrin expression level detection in engineered strains

[0091] serial number Buffalo lactoferrin expression level (μg / L) OST-pre (SEQ ID NO: 1) 1200 OST-pre+19D (SEQ ID NO: 2) 4080 OST-pre+19ED(SEQ ID NO: 3) 3000 OST-pre+19EED(SEQ ID NO: 4) 800 OST-15D (SEQ ID NO: 16) 300 OST-16D (SEQ ID NO: 17) 900 OST-17D (SEQ ID NO: 18) 500 OST-18D (SEQ ID NO: 19) 600 OST-20D (SEQ ID NO: 20) 1500 OST-21D (SEQ ID NO: 21) 4000

[0092] The above embodiments are only used to illustrate the technical solutions of the present invention. Without departing from the spirit of the present invention, those skilled in the art can make various modifications or alterations to the present invention on this basis. Equivalent forms of various variations or modifications should also fall within the scope of the present invention.

Claims

1. An OST1 signal peptide mutant, characterized in that, It is a mutant in which any one, two, or three of the residues VSS at positions 19-21 of the wild-type OST1 signal peptide, as shown in SEQ ID NO: 1, are replaced or inserted by acidic residues D (aspartic acid) and / or E (glutamic acid), wherein SEQ ID NO: 1 is MRQVWFSWIVGLFLCFFN VSS A (SEQ IDNO: 1).

2. The OST1 signal peptide mutant as described in claim 1, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 21: MRQVWFSWIVGLFLCFFNV D SSA (SEQ ID NO: 2) is a mutant of SEQ ID NO: 1 with a D inserted after residue V at position 19, named OST1+19D, OST1-19D, OST-19D or OST1-pre+19D; MRQVWFSWIVGLFLCFFNV ED SSA (SEQ ID NO: 3) is a mutant of SEQ ID NO: 1 with ED inserted after residue V at position 19, named OST1+19ED, OST1-19ED, OST-19ED or OST1-pre+19ED; MRQVWFSWIVGLFLCFFNVSS D A (SEQ ID NO: 21) is a mutant of SEQ ID NO: 1 with D inserted after residue S at position 21, named OST1+21D, OST1-21D, OST-21D or OST1-pre+21D.

3. A complex signal peptide, characterized in that, The complex includes the OST1 signal peptide mutant as described in claim 1 or 2 as the pre-signal region and the α-factor Pro region immediately adjacent to its C-terminus, wherein the amino acid sequence of the α-factor Pro region is shown in SEQ ID NO:

5. APVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ IDNO: 5), Preferably, the amino acid sequence of the OST1 signal peptide mutant, i.e., the pre-sequence (pre region) of the complex signal peptide, is as shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 21, and the corresponding amino acid sequence of the complex signal peptide is as shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

22. MRQVWFSWIVGLFLCFFNVDSSAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 7), named OST1-pre+19D + Pro; MRQVWFSWIVGLFLCFFNVEDSSAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 8), named OST1-pre+19ED + Pro; MRQVWFSWIVGLFLCFFNVSSDAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 22), named OST1-pre+21D+Pro.

4. A gene encoding the complex signal peptide as described in claim 3.

5. The gene as described in claim 4, characterized in that, The gene encoding the complex signal peptide SEQ ID NO: 7 is a polynucleotide of the nucleotide sequence SEQ ID NO: 11 or a polynucleotide with more than 90% homology to SEQ ID NO: 11; the gene encoding the complex signal peptide SEQ ID NO: 8 is a polynucleotide of the nucleotide sequence SEQ ID NO: 12 or a polynucleotide with more than 90% homology to SEQ ID NO:

12. The gene encoding the complex signal peptide SEQ ID NO: 22 is a polynucleotide as shown in SEQ ID NO: 23, or a polynucleotide with more than 90% homology to SEQ ID NO:

23.

6. The use of the OST1 signal peptide mutant of claim 2, the complex signal peptide of claim 3, and the gene of claim 4 in improving the secretion efficiency of exogenous recombinant proteins expressed in yeast.

7. A DNA molecule, which is an integrated fragment, characterized in that, It includes the gene as described in claim 5, an upstream promoter, a downstream target protein-coding gene, and a terminator.

8. A recombinant expression vector, characterized in that, This recombinant expression vector is suitable for expressing exogenous target proteins in yeast, comprising a fusion protein gene of the gene as described in claim 5 and the gene encoding the target protein, and an upstream promoter.

9. A type of engineered yeast, which is Pichia pastoris or Saccharomyces cerevisiae, characterized in that, The genome integrates the DNA molecule as described in claim 7, preferably a transformant in a yeast host transformed with the recombinant expression vector as described in claim 8.

10. The application of the engineered yeast strain as described in claim 9 in the fermentation production of recombinant proteins.

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