Phosphatidylethanolamine synthesis regulation to improve exogenous protein expression system, construction method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XINYICUI (SHANGHAI) BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-03-24
- Publication Date
- 2026-06-16
Smart Images
Figure CN122214166A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology, specifically a system for regulating phosphatidylethanolamine synthesis to enhance the expression of exogenous proteins, its construction method, and its application. Background Technology
[0002] Efficient expression and stable secretion of recombinant proteins are core technical challenges in modern biomanufacturing, biopharmaceuticals, and the industrial production of functional proteins. With the continued growth in market demand for lactoferrin, antibody fragments, immunomodulatory proteins, and various high-value-added functional proteins, establishing expression systems that combine high expression levels, fermentation stability, and industrial scalability has become a crucial issue urgently needing to be addressed in the field of industrial biotechnology.
[0003] Among existing eukaryotic expression systems, Pichia pastoris (Komagataella phaffii) is widely used in recombinant protein production due to its high-density fermentation capacity, eukaryotic post-translational modification mechanism, and good industrial scale-up adaptability. However, when expressing high isoelectric point proteins such as lactoferrin or complex folded proteins, problems such as limited expression efficiency, increased secretion burden, or decreased fermentation stability often occur. Existing technologies mainly improve expression levels through promoter enhancement, codon optimization, signal peptide engineering, or carbon flux regulation, but these strategies mostly focus on the transcriptional or translational level, paying insufficient attention to the role of the host cell membrane environment in the secretion process.
[0004] Phosphatidylethanolamine (PE) is an important phospholipid component of the endoplasmic reticulum membrane, playing a crucial role in maintaining membrane structural stability and secretory pathway function. However, current techniques have not systematically investigated the effects of exogenous high isoelectric point protein expression on yeast membrane lipid homeostasis, nor have there been any technical solutions for improving protein expression and secretion performance through engineered regulation of PE biosynthesis. Therefore, constructing novel expression enhancement strategies from the perspective of membrane lipid metabolism regulation is of great significance for overcoming the limitations of traditional expression optimization pathways. Summary of the Invention
[0005] To solve the above problems, the technical solution provided by the present invention is as follows: The present invention discloses a system for regulating phosphatidylethanolamine synthesis to enhance the expression of exogenous proteins. Using yeast host cells as the expression system, the system enhances the expression of key enzymes in the phosphatidylethanolamine biosynthesis pathway or regulates their metabolic flux in the host cells, thereby increasing the content of phosphatidylethanolamine in the cells and the endoplasmic reticulum membrane, thereby improving the expression efficiency of exogenous proteins.
[0006] Preferably, the key enzyme in the phosphatidylethanolamine biosynthesis pathway includes phosphatidylserine decarboxylase.
[0007] Preferably, the phosphatidylserine decarboxylase is PSD1 or PSD2, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO: 2 or SEQ ID NO: 6.
[0008] Preferably, the yeast host cell is Pichia pastoris or Saccharomyces cerevisiae.
[0009] Preferably, the methods for enhancing expression or regulating metabolic flux include one or any combination of overexpression, promoter substitution, multicopy integration, and gene editing.
[0010] Preferably, the exogenous protein is a high isoelectric point protein or a complex folded protein, including lactoferrin, lysozyme, antimicrobial peptides, antibody fragments, or industrial enzymes.
[0011] A method for constructing a system that regulates phosphatidylethanolamine synthesis to enhance exogenous protein expression includes the following steps: (1) Construct a recombinant expression vector that enhances the biosynthesis of phosphatidylethanolamine, wherein the recombinant expression vector comprises a phosphatidylserine decarboxylase PSD1 expression cassette driven by a first promoter and a phosphatidylserine decarboxylase PSD2 expression cassette driven by a second promoter. (2) Transform the recombinant expression vector constructed in step (1) into yeast host cells to obtain engineered bacteria; (3) The expression cassette encoding the target exogenous protein is converted into the engineered bacteria obtained in step (2) to obtain a production strain that simultaneously expresses PSD1, PSD2 and the target exogenous protein; (4) Cultivate the production strain obtained in step (3) to induce expression of the target exogenous protein; (5) Recover the target exogenous protein from the culture.
[0012] Preferably, the first promoter is the GAP promoter and the second promoter is the PGK1 promoter.
[0013] A gene encoding phosphatidylserine decarboxylase, said gene being used to construct the above-described engineered expression system, said gene being as shown in SEQ ID NO: 2 or SEQ ID NO: 6.
[0014] The aforementioned engineered expression system is used in the preparation of high isoelectric point proteins or complex folded proteins.
[0015] Compared with the prior art, the technical solution provided by this invention has the following advantages: This invention discloses a system for regulating phosphatidylethanolamine (PE) synthesis to enhance exogenous protein expression, its construction method, and its application. This invention improves the expression efficiency of exogenous proteins by enhancing the expression of key enzymes in the PE biosynthesis pathway in yeast host cells, particularly by overexpressing phosphatidylserine decarboxylases PSD1 and / or PSD2, thereby increasing the PE content within the cell and in the endoplasmic reticulum membrane. The yeast host cells are preferably Pichia pastoris or Saccharomyces cerevisiae, and the exogenous proteins are preferably high isoelectric point proteins or complex folded proteins such as lactoferrin, lysozyme, antimicrobial peptides, and antibody fragments. This invention also provides a method for constructing this expression system and its application in the preparation of the aforementioned proteins. This invention, starting from the level of membrane lipid metabolism regulation, significantly improves the secretory performance of host cells by restoring or increasing PE levels, resulting in an increase in exogenous protein expression levels of more than two times, demonstrating good versatility and promising industrial application prospects. Attached Figure Description
[0016] Figure 1 A schematic diagram of the dual expression structure constructed for Example 1, in which PSD1 is driven by the GAP promoter and PSD2 is driven by the PGK1 promoter; Figure 2 The colony PCR electrophoresis verification results are for the PSD overexpression module constructed in Example 1 after integration into the Pichia pastoris GS115 genome; Figure 3 This is a graph showing the PCR results of Example 2; Figure 4 The figure shows the results of bovine lactoferrin expression level and purification analysis in Example 3; Figure 5 This is a graph showing the changes in lipid subtype content in Example 4. Detailed Implementation
[0017] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.
[0018] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate for the embodiments of this application described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.
[0019] In this application, the terms "upper," "lower," "left," "right," "front," "rear," "top," "bottom," "inner," "outer," "middle," "vertical," "horizontal," "lateral," and "longitudinal" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. These terms are primarily for the purpose of better describing this application and its embodiments, and are not intended to limit the indicated device, element, or component to having a specific orientation, or to be constructed and operated in a specific orientation.
[0020] Furthermore, in addition to indicating location or positional relationship, some of the aforementioned terms may also have other meanings. For example, the term "above" may also be used in some cases to indicate a certain dependency or connection relationship. Those skilled in the art can understand the specific meaning of these terms in this application based on the specific circumstances.
[0021] Furthermore, the terms "installation," "setup," "equipped with," "connection," "linking," and "socketing" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral structure; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium, or an internal connection between two devices, components, or parts. Those skilled in the art can understand the specific meaning of these terms in this application based on the specific circumstances.
[0022] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.
[0023] The embodiments of this application aim to address the difficulty in simultaneously achieving high expression efficiency and secretory stability for lactoferrin, glycoproteins, and other exogenous proteins with high isoelectric points or complex structures in existing Pichia pastoris expression systems. While traditional yeast expression systems can achieve a certain level of exogenous protein secretion, expression levels are often limited or fermentation stability decreases when expressing high isoelectric point proteins or proteins with a strong secretory burden. Existing technologies primarily focus on transcriptional and translational regulation, such as promoter enhancement, codon optimization, or signal peptide modification, while paying less attention to the role of the host cell membrane lipid environment in the secretion process. Consequently, an industrially scalable membrane lipid-regulated expression platform has not yet been developed.
[0024] During long-term research on yeast expression system optimization and membrane lipid metabolism, we found that expressing Holstein bovine lactoferrin (bLF) in Pichia pastoris significantly reduced the levels of phosphatidylethanolamine (PE) in the host cell as a whole and in endoplasmic reticulum membrane components. Lipidomics analysis further confirmed that this change was particularly pronounced in the separated components of the endoplasmic reticulum membrane. This phenomenon suggests that the expression of exogenous high isoelectric point proteins may be associated with host membrane lipid homeostasis.
[0025] To this end, this invention proposes an engineered expression enhancement system with phosphatidylethanolamine biosynthesis as the core regulatory node. By enhancing phosphatidylserine decarboxylase (PSD1 / PSD2) or strengthening the cytidine diphosphate ethanolamine pathway (also known as the Kennedy pathway) in Pichia pastoris, the proportion of PE in the cell and endoplasmic reticulum membrane is increased, thereby improving the expression and secretion performance of exogenous proteins.
[0026] In this implementation case, using lactoferrin as a model protein, the differences in exogenous protein expression levels between engineered strains that enhanced the PE biosynthesis pathway and control strains without PE pathway regulation were systematically constructed and compared. Experimental results showed that, compared with the control strain, the PE-enhanced strains exhibited significantly increased exogenous protein secretion and expression levels, with an overall increase of approximately 2-fold or more, and demonstrated good reproducibility and stability in multiple independent experiments. These results indicate that regulating PE biosynthesis can mitigate membrane lipid homeostasis disturbances that may occur during exogenous protein expression, thereby improving expression efficiency. This engineering strategy is not dependent on specific protein sequences and can be applied to lactoferrin, lysozyme, antibody fragments, antimicrobial peptides, and other high isoelectric point or complex folded proteins, demonstrating good versatility and industrial scalability potential.
[0027] In this article, the terms “improved level of expression”, “enhancement”, or “enhancement” used above refer to an improvement 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 about 1 or at least about 2 times.
[0028] Effective methods for enhancing the PE pathway include constructing PSD-encoding genes in expression vectors and integrating them into the host genome, or enhancing their expression through multi-copy expression and promoter substitution, thereby achieving stable regulation of membrane lipid metabolism pathways. General implementation methods for constructing engineered PE-driven expression enhancement platforms include the following aspects: a. PSD coding sequence selection and design: Select PSD coding sequences from host or heterologous sources and optimize them for Pichia pastoris expression to ensure that they have phosphatidylserine decarboxylase activity; b. PSD expression cassette construction: The PSD coding sequence is assembled with a promoter and terminator suitable for yeast expression to form a PSD expression cassette, which is then constructed in the expression vector; c. Pichia pastoris transformation and screening of engineered strains: The expression vector was introduced into the Pichia pastoris host by electroporation, positive transformants were screened and identified, and stable engineered strains were established; d. Analysis of exogenous protein integration and expression: Exogenous protein expression cassettes were integrated into the PE-enhanced background strains and cultured under the same fermentation conditions. The expression levels of exogenous proteins were compared and evaluated using protein analysis methods, and changes in the PE ratio were analyzed using lipidomics methods.
[0029] Materials and methods In this embodiment, the full-length gene synthesis, primer synthesis, and sequencing were all performed by Suzhou Kingwise Biotechnology Co., Ltd. The molecular biology experiments in this embodiment included plasmid construction, restriction endonuclease digestion, DNA ligation, competent cell preparation, transformation, selection medium preparation, and related routine operations, mainly following the standard methods in *Molecular Cloning: A Laboratory Manual* (4th Edition) (MR Green, J. Sambrook, translator He Fuchu, Science Press, Beijing, 2017). For procedures not described in detail, conventional methods familiar to those skilled in the art were used. If necessary, reaction conditions could be appropriately optimized through preliminary experiments as needed. PCR amplification experiments were performed according to the instructions of the DNA polymerase kit used or the recommended conditions provided by the template supplier, including reaction system composition, annealing temperature, and cycle program settings. For different primers or template conditions, appropriate optimization could be performed through gradient PCR or other methods to obtain specific amplification products. The host strain used in this embodiment was *Pichia pastoris* GS115, purchased from the Shanghai Institute of Microbiology. Escherichia coli competent cells were used for plasmid amplification, employing strains commonly used in the field. The PCR amplification primers for some gene fragments in this embodiment are listed in Table 1.
[0030] Table 1. Primers for PCR amplification of gene fragments in Example 1
[0031]
[0032] The suffix "F" in primer names indicates forward direction; "R" indicates reverse direction.
[0033] Example 1 The PSD1 / PSD2 dual-expression module was built and integrated into GS115. This embodiment constructs a PSD overexpression module for enhancing phosphatidylethanolamine (PE) biosynthesis and integrates it into the genome of Pichia pastoris (Komagataella phaffii) GS115. Figure 1As shown, the expression structure constructed in this embodiment includes two independent expression modules: a PSD1 expression cassette driven by a GAP promoter and a PSD2 expression cassette driven by a PGK1 promoter. The structure of the PSD1 expression cassette is GAPpromoter–PSD1–AOX1 terminator, and the structure of the PSD2 expression cassette is PGK1 promoter–PSD2–PGK1 terminator. PSD1 and PSD2 encode phosphatidylserine decarboxylase, which enhances the biosynthetic capacity of PE within the host cell.
[0034] In the specific construction process, fragments of the GAP promoter, PSD1 coding sequence, and AOX1 terminator, as well as fragments of the PGK1 promoter, PSD2 coding sequence, and PGK1 terminator, were amplified separately. After agarose gel electrophoresis and purification, the PCR products were sequentially spliced using overlap PCR to form a dual-expression structure: GAP promoter–PSD1–AOX1 terminator–PGK1 promoter–PSD2–PGK1 terminator. The resulting complete expression fragments were used for genome integration.
[0035] The GAP promoter sequence is shown in SEQ ID NO: 1, the PSD1 gene sequence is shown in SEQ ID NO: 2, the PSD1 amino acid sequence is shown in SEQ ID NO: 3, the AOX1 terminator sequence is shown in SEQ ID NO: 4, the PGK1 promoter sequence is shown in SEQ ID NO: 5, the PSD2 gene sequence is shown in SEQ ID NO: 6, the PSD2 amino acid sequence is shown in SEQ ID NO: 7, the PGK1 terminator sequence is shown in SEQ ID NO: 8, the AOX1 promoter sequence is shown in SEQ ID NO: 9, the bovine lactoferrin (bLF) encoding gene is shown in SEQ ID NO: 10, and the full-length amino acid sequence of bovine lactoferrin (bLF) is shown in SEQ ID NO: 11.
[0036] The constructed PSD overexpression integration fragment was transformed into Pichia pastoris GS115 competent cells using electroporation. The electroporation conditions were 1,500 V for 5 ms. After electroporation, pre-cooled 1 M sorbitol was added for recovery, and the cells were incubated at 30°C for 1–2 h before being plated onto selection plates. Transformant colonies were obtained after approximately 3 days of incubation at 30°C.
[0037] Single clones were picked from the screening plate for colony PCR identification. The primers used for identification were pGAP-GS115-F and PE-tAOX1-R, with an expected amplified fragment size of 2468 bp. PCR results are shown below. Figure 2 As shown, a specific band of the theoretical size was observed in the positive transformants, while no corresponding band was detected in the negative control, indicating that the PSD overexpression module had been successfully integrated into the Pichia pastoris GS115 genome. The identified positive strains were used for subsequent construction of exogenous protein expression and functional evaluation.
[0038] Figure 1 This is a schematic diagram of the dual expression structure constructed in this invention, where PSD1 is driven by the GAP promoter and PSD2 is driven by the PGK1 promoter. Figure 1 Figure a shows a schematic diagram of the linear structure of the PSD expression cassette, indicating the relative layout of the PSD expression modules in the vector and their arrangement during genome integration; Figure 1 Figure b represents a schematic diagram of the overall structure of the constructed recombinant expression vector, including basic functional elements such as the promoter, coding sequence, terminator, resistance selection marker, and bacterial origin of replication (ori) for driving the expression of phosphatidylserine decarboxylase (PSD1 / PSD2). This figure is used to illustrate the overall structural design and modular organization of the PE biosynthesis enhancement module in this invention.
[0039] Figure 2 The colony PCR electrophoresis results are for the integration of the PSD overexpression module constructed in this invention into the Pichia pastoris GS115 genome. Using specific primers to amplify the integrated fragment, the agarose gel electrophoresis image identified by PCR showed that transformant 4 had an amplified band of the theoretical size, indicating that the PSD expression module was successfully integrated into the host genome, and a PE-enhanced Pichia pastoris engineered strain was successfully obtained for subsequent expression performance evaluation.
[0040] Example 2 Validation of integration of bovine lactoferrin expression cassette into PSD-overexpressing Pichia pastoris Based on the PSD-overexpressing Pichia pastoris GS115 host obtained in Example 1, this embodiment constructs and integrates a bovine lactoferrin (bLF) expression cassette to obtain an engineered strain with both PSD overexpression and bLF expression capabilities.
[0041] Using a codon-optimized bLF coding sequence as a template, the bLF fragment was obtained by PCR amplification; simultaneously, the AOX1 promoter and terminator fragments were amplified. A bLF expression cassette was constructed by overlap PCR splicing, with the structure: AOX1 promoter–bLF–AOX1 terminator.
[0042] The constructed bLF expression integration fragment was transformed into the PSD-overexpressing GS115 host obtained in Example 1 via electroporation. The electroporation conditions were 1,500 V for 5 ms. After electroporation, 1 M sorbitol was added for resuscitation, and the culture was carried out at 30°C for 1–2 h before being plated onto screening plates to obtain transformant colonies.
[0043] Single clones were picked from the screening plates for colony PCR identification. The primers used for identification were pAOX1-GS115-F and bLF-R, with an expected amplified fragment size of 3366 bp.
[0044] PCR results as follows Figure 3 As shown, a specific band (approximately 3366 bp) of the theoretical size was observed in the positive transformants, indicating that the bLF expression cassette had been successfully integrated into the PSD-overexpressing Pichia pastoris GS115 genome. No corresponding band was detected in the negative control. The identified positively integrated strain was named the bLF-PE engineered strain and used for subsequent expression and functional analysis.
[0045] Example 3 Fermentation detection and purification analysis of bovine lactoferrin expression level in PSD-overexpressing engineered bacteria The bLF-PE-GS115 engineered strain, identified positive by PCR, and the bLF-GS115 control strain without PSD modification were selected and inoculated into BMGY medium. They were cultured at 30℃ and 250 rpm for 16–20 h until the OD600 reached 4–6. The cells were then collected by centrifugation at 4000×g for 10 min, resuspended in BMGY medium until the OD600 was approximately 1, and induced to express expression at 28–30℃, 250 rpm, and pH 6.0. Methanol was added every 24 h until a final concentration of 0.5% (v / v) was reached. The total induction time was 96 h, with a final OD600 of approximately 8–10.
[0046] The BMGY medium consists of: 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, and glycerol 10 mL / L. The BMMY medium consists of: 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, and methanol 5 mL / L.
[0047] After induction, the fermentation supernatant was collected, and cell debris was removed by centrifugation. Secretory bovine lactoferrin was obtained using conventional protein concentration or affinity purification methods. The expression product was detected and analyzed by SDS-PAGE and Western blot, and the results are as follows: Figure 4 As shown. Figure 4 The results showed that, in the PSD overexpression background (bLF-PE-GS115), each independent transformant showed a significantly enhanced bovine lactoferrin band, with a molecular weight consistent with the theoretical value; while the band intensity of the control strain was significantly weaker. Figure 4 b represents the statistical results of expression levels obtained based on band grayscale or ELISA quantitative analysis. The secretory expression level of bovine lactoferrin in the PSD-enhanced strain is about 2–3 times higher than that in the control strain.
[0048] The above results demonstrate that enhancing phosphatidylethanolamine (PE) biosynthesis can significantly improve the secretory expression level of bovine lactoferrin in Pichia pastoris. This engineering strategy can improve the expression efficiency of high isoelectric point or structurally complex glycoproteins by optimizing the secretory environment through regulation of membrane lipid composition without altering the target protein coding sequence, exhibiting good stability and reproducibility.
[0049] Example 4 Lipomics analysis validated the effect of lactoferrin expression on membrane phospholipid composition and PSD regulation. To further elucidate the impact of lactoferrin expression on host membrane lipid homeostasis and to verify at the molecular level the mechanism by which enhanced phosphatidylethanolamine (PE) biosynthesis improves the expression efficiency of high isoelectric point glycoproteins, lipidomics analysis was performed on different engineered strains for comparison. The following strains were selected as experimental subjects: the GS115 control strain (without lactoferrin expression), the GS115-bLF strain (expressing bovine lactoferrin (bLF)), and the GS115-bLF-PSD strain (further overexpressing phosphatidylserine decarboxylase genes (PSD1 / PSD2) in the context of lactoferrin expression). All three groups of strains were fermented under the same culture and induction conditions. After 96 h of induction, the bacterial cells were collected for lipid extraction and detection.
[0050] After washing, the bacterial cells underwent lipid extraction using an organic solvent system. The extracted products were dried and redissolved, and then quantitative lipidomics analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The detection range covered major phospholipid subtypes (including PE, PC, PS, PI, PG, etc.), lysophospholipids (such as LPE, LPC), glycerides (DG, TG), and sphingolipids. Lipid content was expressed as pmol / g dry weight, and quantitative comparisons of different lipid subtypes and their molecular subtypes were performed.
[0051] Results of changes in lipid subtype content as follows Figure 5 As shown in the figure, compared with the GS115 control strain, the total PE content in the GS115-bLF strain expressing lactoferrin was significantly decreased, while PC only showed slight fluctuations or compensatory changes. These results indicate that the expression of high isoelectric point glycoproteins has a significant impact on host membrane phospholipid homeostasis, with PE being the most significantly perturbed lipid class.
[0052] Furthermore, a significant recovery in PE content was observed in the GS115-bLF-PSD strain, with multiple PE subtypes returning to levels close to or higher than the control group, and the PE / PC ratio also significantly increased. Compared with strains expressing only lactoferrin, the recovery of PE content after overexpression of PSD1 / PSD2 was statistically significant. Meanwhile, PC and other lipid classes did not show the same level of systemic change as PE, indicating that PSD regulation mainly affects the PE biosynthetic pathway rather than causing global lipid dysregulation.
[0053] The results of lactoferrin expression detection in the aforementioned examples show that a decrease in PE content corresponds to restricted lactoferrin secretion and expression. However, after restoring PE levels by enhancing PSD-mediated PE biosynthesis, lactoferrin secretion and expression significantly increased. These results indicate a clear correlation between changes in PE levels and the expression efficiency of high isoelectric point glycoproteins.
[0054] This embodiment demonstrates from a lipidomics perspective that the expression of high isoelectric point glycoproteins (PEs) disrupts the PE content in the host cell membrane, while enhancing PE biosynthesis can restore membrane phospholipid homeostasis and improve secretory expression performance. This result provides direct experimental evidence for the membrane lipid engineering strategy proposed in this invention, which aims to enhance the expression efficiency of high isoelectric point glycoproteins by regulating membrane phospholipid composition, particularly by increasing PE levels.
[0055] During their long-term research on yeast expression system optimization and membrane lipid metabolism, the applicant discovered that expressing Holstein bovine lactoferrin (bLF) in Pichia pastoris significantly reduced the levels of phosphatidylethanolamine (PE) in the whole cell and endoplasmic reticulum membrane components. Lipidomics analysis further confirmed that this change was particularly pronounced in the endoplasmic reticulum membrane components, indicating a correlation between the expression of exogenous high isoelectric point proteins and host membrane lipid homeostasis.
[0056] Based on the above findings, this invention significantly increases the proportion of PE in the whole cell and the endoplasmic reticulum membrane by engineering enhancements to the PE biosynthesis pathway, including overexpression of phosphatidylserine decarboxylase (PSD1 / PSD2) or strengthening the CDP-ethanolamine pathway (also known as the Kennedy pathway). Experimental results show that after PE levels are restored or increased, the expression level of bovine lactoferrin (bLF) increases significantly, secretion efficiency is significantly improved, and the stability of the fermentation process is enhanced.
[0057] The above results indicate that the expression of exogenous high isoelectric point proteins may perturb the host membrane lipid composition, and this perturbation can be improved by regulating PE biosynthesis, thereby enhancing protein expression and secretion performance. This invention differs from traditional approaches that optimize only at the transcriptional or translational level; instead, it proposes a novel expression enhancement strategy based on the regulation of membrane lipid metabolism.
[0058] The technical solution provided by this invention uses PE biosynthesis as the core of regulation, and enhances the ability of the yeast host to regulate membrane lipid homeostasis, thereby improving the expression adaptability of high-charge or complex-folded proteins. This strategy does not depend on specific protein sequences, nor does it significantly increase the complexity of the expression system. It can be used in conjunction with glycosylation engineering or signal peptide optimization strategies to construct a multi-dimensional expression optimization system.
[0059] The method is applicable to the large-scale production of lactoferrin, lysozyme, antibody fragments, antimicrobial peptides, and other proteins with high isoelectric points or complex folding, and has broad application prospects in biopharmaceuticals, precision fermentation of functional proteins, special medical foods, and the production of high-value-added industrial enzymes.
[0060] The above-described embodiments are merely illustrative of certain implementations of the present invention, and are described in a relatively specific and detailed manner. However, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements are all within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.
Claims
1. A system for regulating phosphatidylethanolamine synthesis to enhance exogenous protein expression, characterized in that: Using yeast host cells as the expression system, the expression of key enzymes in the phosphatidylethanolamine biosynthesis pathway is enhanced or their metabolic flux is regulated in the host cells, thereby increasing the content of phosphatidylethanolamine in the cells and the endoplasmic reticulum membrane, thus improving the expression efficiency of exogenous proteins.
2. The system for regulating phosphatidylethanolamine synthesis and enhancing exogenous protein expression according to claim 1, characterized in that: The key enzyme in the phosphatidylethanolamine biosynthesis pathway includes phosphatidylserine decarboxylase.
3. The system for regulating phosphatidylethanolamine synthesis and enhancing exogenous protein expression according to claim 2, characterized in that: The phosphatidylserine decarboxylase is PSD1 or PSD2, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO: 2 or SEQ ID NO:
6.
4. The system for regulating phosphatidylethanolamine synthesis and enhancing exogenous protein expression according to claim 1, characterized in that: The yeast host cell is Pichia pastoris or Saccharomyces cerevisiae.
5. The system for regulating phosphatidylethanolamine synthesis and enhancing exogenous protein expression according to claim 1, characterized in that: The methods for enhancing expression or regulating metabolic flux include one or any combination of overexpression, promoter substitution, multicopy integration, and gene editing.
6. The system for regulating phosphatidylethanolamine synthesis and enhancing exogenous protein expression according to claim 1, characterized in that: The exogenous protein is a high isoelectric point protein or a complex folded protein, including lactoferrin, lysozyme, antimicrobial peptides, antibody fragments, or industrial enzymes.
7. A method for constructing a system to enhance exogenous protein expression by regulating phosphatidylethanolamine synthesis, characterized in that, Includes the following steps: (1) Construct a recombinant expression vector that enhances the biosynthesis of phosphatidylethanolamine, wherein the recombinant expression vector comprises a phosphatidylserine decarboxylase PSD1 expression cassette driven by a first promoter and a phosphatidylserine decarboxylase PSD2 expression cassette driven by a second promoter. (2) Transform the recombinant expression vector constructed in step (1) into yeast host cells to obtain engineered bacteria; (3) The expression cassette encoding the target exogenous protein is converted into the engineered bacteria obtained in step (2) to obtain a production strain that simultaneously expresses PSD1, PSD2 and the target exogenous protein; (4) Cultivate the production strain obtained in step (3) to induce expression of the target exogenous protein; (5) Recover the target exogenous protein from the culture.
8. The method for establishing a system for regulating phosphatidylethanolamine synthesis to enhance exogenous protein expression according to claim 7, characterized in that: The first promoter is the GAP promoter, and the second promoter is the PGK1 promoter.
9. A gene encoding phosphatidylserine decarboxylase, characterized in that, The gene is used to construct the engineered expression system as described in any one of claims 1–6, wherein the gene is as shown in SEQ ID NO: 2 or SEQ ID NO:
6.
10. The engineered expression system according to any one of claims 1–6, and its use in the preparation of high isoelectric point proteins or complex folded proteins.